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Sample records for model lipase enzymes

  1. Enzymes used in detergents: Lipases | Hasan | African Journal of ...

    African Journals Online (AJOL)

    This review describes the applications of microbial lipases in detergents. Enzymes can reduce the environmental load of detergent products as the chemicals used in conventional detergents are reduced; they are biodegradable, non-toxic and leave no harmful residues. Besides lipases, other enzymes are widely used in ...

  2. Beneficial effects of adding lipase enzyme to broiler diet

    International Nuclear Information System (INIS)

    Elbarkouky, E.M.A.

    2005-01-01

    A total number of 300 Ross broiler chicks were obtained from commercial hatchery at one day of age. The chicks were divided into three groups (50 males and 50 females in each). The first and second groups were supplemented with 3000 and 2000 lU/kg diet of lipase enzyme, respectively, while the third group served as control and fed on basal diet. Birds fed on diets that supplemented with lipase enzyme showed significant increase in body weight and dry matter intake, as well as fats and protein content dry matters. The serum lipase activity showed significant increase in treated groups compared to the control. Non-significant changes were determined in serum total lipids, T3, T4 and ash content. Birds supplemented with lipase showed significant decrease in cholesterol concentration. It could be concluded that birds fed diets containing 2000 or 3000 lU/kg diet of lipase enzyme exhibited improvement in broiler performance

  3. 21 CFR 184.1420 - Lipase enzyme preparation derived from Rhizopus niveus.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Lipase enzyme preparation derived from Rhizopus... GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1420 Lipase enzyme preparation derived from Rhizopus niveus. (a) Lipase enzyme preparation contains lipase enzyme (CAS Reg. No...

  4. Gastric lipase: localization of the enzyme in the stomach

    International Nuclear Information System (INIS)

    DeNigris, S.J.; Hamosh, M.; Hamosh, P.; Kasbekar, D.K.

    1986-01-01

    Isolated gastric glands prepared from human and rabbit stomach secrete lipase in response to secretagogues. They have investigated the localization of this enzyme in three species (rabbit, baboon, guinea pig). Gastric mucosa was sampled from the cardia (C), fundus-smooth (FS), fundus-ruggae (FR) and the antral area (A). Lipase activity was measured in mucosal homogenates using 3 H-triolein as substrate and is expressed in units (U) = nmols free fatty acid released/min/mg wet weight. The localization of lipase is compared with that of pepsin (measured by hydrolysis of 2% hemoglobin at pH 1.8 and expressed in I.U.). Lipase is localized in a well defined area in the rabbit and is diffusely distributed in both guinea pig and baboon. The distribution of lipase and pepsin containing cells differs in all three species. The cellular origin of gastric lipase remains to be determined

  5. Comparison of lipases for in vitro models of gastric digestion

    DEFF Research Database (Denmark)

    Sassene, P J; Fanø, M; Mu, H

    2016-01-01

    The aim of this study was to find a lipase suitable as a surrogate for Human Gastric Lipase (HGL), since the development of predictive gastrointestinal lipolysis models are hampered by the lack of a lipase with similar digestive properties as HGL. Three potential surrogates for HGL; Rhizopus Oryz...

  6. Kinetic model of biodiesel production using immobilized lipase Candida antarctica lipase B

    DEFF Research Database (Denmark)

    Fedosov, Sergey; Brask, Jesper; Pedersen, Anders K.

    2013-01-01

    We have designed a kinetic model of biodiesel production using Novozym 435 (Nz435) with immobilized Candida antarctica lipase B (CALB) as a catalyst. The scheme assumed reversibility of all reaction steps and imitated phase effects by introducing various molecular species of water and methanol....... Conversion to 95% requires 1.5–6 days of incubation depending on the temperature, enzyme concentration, glycerol inhibition, etc. Other substrates, free fatty acids (F), diglycerides (D) and monoglycerides (M), are utilized much faster (1–2h). This means that waste oil is a better feedstock for CALB...... in waste oil before the conventional alkaline conversion. Up to 30-fold reduction of F-content can be achieved in 1–2h, and the residual enzyme (if any) does not survive the following alkaline treatment....

  7. Application of alkaline thermo-stable lipase(s) enzyme produced from irradiated microbial isolate in the field of detergent technology

    International Nuclear Information System (INIS)

    Ahmed, O.E.A.M.S

    2010-01-01

    Due to continuous demand for manufacture of high quality, low coast industrial detergents containing lipolytic enzymes and due to continuous accumulation of enviro-agro-industrial wastes which are good and suitable conditions for growth and reproduction of pathogenic microorganisms, our study aims at isolating thermoalkalophilic lipase producer microorganisms from enviro-agro-industrial wastes and selection of the most potent isolate for studying physiological conditions controlling enzyme formation also purification characterization and some applications on purified and crude enzyme as bio-detergent. Some environmental and industrial wastes were collected from different places. The industrial wastes include, cotton seed, soyabean, sun flower, lin seed and olive oil wastes. Environmental wastes include poultry and fish wastes, all these wastes were dried at 70 degree C, grounded and used for isolation of microorganisms and lipase(s) production.Nine thermoalkalophilic bacterial isolates were isolated from enviro-agro-industrial wastes at ph 11.5 and 70 degree C. They were purified and screening for their ability of thermoalkalo-stable lipase(s) formation, this is followed by examining the effect of different nutritional media and exposure of bacterial isolates to different doses of gamma irradiation and the influence of these radiation on lipase(s) productivity by these isolates. From the results it was found that.1- The most potent lipase(s) forming bacterial isolates were isolates number B 2 and B 3 which cultivated on medium A amended with fish-wastes as being the best nutritional medium for enzyme formation. 2-Bacterial isolate B 2 finally was selected as being the most potent lipase(s) forming bacterial isolate cultivated on fish-wastes and yeast extract (in tap water) and identified according to key's of Bergey Manual of Systematic Bacteriology (1984) as being Bacillus brevis B 2 .The optimum culture conditions for maximum biosynthesis of extracellular lipase(s

  8. Kinetic Model For Triglyceride Hydrolysis Using Lipase:Review

    Directory of Open Access Journals (Sweden)

    Heri Hermansyah

    2010-10-01

    Full Text Available Triglyceride hydrolysis using lipase has been proposed as a novel method to produce raw materials in food and cosmetic industries such as diacylglycerol, monoacylglycerol, glycerol and fatty acid. In order to design a reactor for utilizing this reaction on industrial scale, constructing a kinetic model is important. Since the substrates are oil and water, the hydrolysis takes place at oil-water interface. Furthermore, the triglyceride has three ester bonds, so that the hydrolysis stepwise proceeds. Thus, the reaction mechanism is very complicated. The difference between the interfacial and bulk concentrations of the enzyme, substrates and products, and the interfacial enzymatic reaction mechanism should be considered in the model.

  9. Adipose Triglyceride Lipase, Not Hormone-Sensitive Lipase, Is the Primary Lipolytic Enzyme in Fasting Elephant Seals (Mirounga angustirostris).

    Science.gov (United States)

    Fowler, Melinda A; Costa, Daniel P; Crocker, Daniel E; Shen, Wen-Jun; Kraemer, Fredric B

    2015-01-01

    Little is known about the mechanisms that allow capital breeders to rapidly mobilize large amounts of body reserves. Northern elephant seals (Mirounga angustirostris) utilize fat reserves for maternal metabolism and to create high fat milk for the pup. Hormone-sensitive lipase (HSL) has been hypothesized to be an important lipolytic enzyme in fasting seals, but the activity of HSL and adipose triglyceride lipase (ATGL) has not been quantified in fasting adult seals, nor has their relationship to milk lipid content been assessed. Blubber and milk samples were obtained from 18 early lactation and 19 late lactation females, as well as blubber from five early and five late molting female seals. Blubber lipolytic activity was assessed with radiometric assays. HSL activity was negligible in seal blubber at all fasting stages. Total triglyceride lipase activity was stable among early and late lactation and early molt but increased in late molting seals. Relative abundance of ATGL protein increased across fasting, but neither activity nor relative protein levels were related to circulating nonesterified fatty acids or milk lipid content, suggesting the possibility of other regulatory pathways between lipolytic activity and milk lipid content. These results demonstrate that HSL is not the primary lipolytic enzyme in fasting adult female seals and that ATGL contributes more to lipolysis than HSL.

  10. Enzyme mediated silicon-oxygen bond formation; the use of Rhizopus oryzae lipase, lysozyme and phytase under mild conditions.

    Science.gov (United States)

    Abbate, Vincenzo; Bassindale, Alan R; Brandstadt, Kurt F; Lawson, Rachel; Taylor, Peter G

    2010-10-21

    The potential for expanding the variety of enzymic methods for siloxane bond formation is explored. Three enzymes, Rhizopus oryzae lipase (ROL), lysozyme and phytase are reported to catalyse the condensation of the model compound, trimethylsilanol, formed in situ from trimethylethoxysilane, to produce hexamethyldisiloxane in aqueous media at 25 °C and pH 7. Thermal denaturation and reactant inhibition experiments were conducted to better understand the catalytic role of these enzyme candidates. It was found that enzyme activities were significantly reduced following thermal treatment, suggesting a potential key-role of the enzyme active sites in the catalysis. Similarly, residue-specific modification of the key-amino acids believed to participate in the ROL catalysis also had a significant effect on the silicon bio-catalysis, indicating that the catalytic triad of the lipase may be involved during the enzyme-mediated formation of the silicon-oxygen bond. E. coli phytase was found to be particularly effective at catalysing the condensation of trimethylsilanol in a predominantly organic medium consisting of 95% acetonitrile and 5% water. Whereas the use of enzymes in silicon chemistry is still very much a developing and frontier activity, the results presented herein give some grounds for optimism that the variety of enzyme mediated reactions will continue to increase and may one day become a routine element in the portfolio of the synthetic silicon chemist.

  11. AKTIVITAS HIDROLISIS ENZIM LIPASE DARI KENTOS KELAPA TERHADAP MINYAK KELAPA Hidrolysis Activity of Lipase Enzyme from Coconut Houstorium for Coconut Oil

    Directory of Open Access Journals (Sweden)

    Mohammad Su’i

    2012-05-01

    Full Text Available This research was aimed to study hydrolysis conditions of houstorium lipases enzyme using coconut oil as substrate. Hydrolysis conditions studied were substrate (coconut oil concentration, enzyme substrate ratio, duration of hydro- lysis and effect of stirring to hydrolysis. The results show  that lipase of coconut houstorium may be optimally used at a coconut oil concentration of 10 %, enzyme to substrate ratio of 3 : 10 (v/v and hydrolysis for 60 minutes with stirring. ABSTRAK Penelitian ini mempelajari kondisi hidrolisis minyak kelapa yang optimum menggunakan enzim lipase dari kentos kelapa. Kondisi hidrolisis yang dipelajari meliputi konsentrasi substrat optium, perbandingan enzim : substrat dan lama hidrolisis yang optimum serta pengaruh pengadukan selama hidrolisis. Hasil penelitian menunjukkan bahwa, hidrolisis minyak kelapa menggunakan enzim lipase kentos kelapa menghasilkan asam lemak bebas paling banyak pada kon- sentrasi substrat (minyak kelapa 10 %, perbandingan enzim : substrat yaitu 3 : 10 (v/v, lama hidroloisa 60 menit dan dilakukan pengadukan selama hidrolisis.

  12. Relevant pH and lipase for in vitro models of gastric digestion.

    Science.gov (United States)

    Sams, Laura; Paume, Julie; Giallo, Jacqueline; Carrière, Frédéric

    2016-01-01

    The development of in vitro digestion models relies on the availability of in vivo data such as digestive enzyme levels and pH values recorded in the course of meal digestion. The variations of these parameters along the GI tract are important for designing dynamic digestion models but also static models for which the choice of representative conditions of the gastric and intestinal conditions is critical. Simulating gastric digestion with a static model and a single set of parameters is particularly challenging because the variations in pH and enzyme concentration occurring in the stomach are much broader than those occurring in the small intestine. A review of the literature on this topic reveals that most models of gastric digestion use very low pH values that are not representative of the fed conditions. This is illustrated here by showing the variations in gastric pH as a function of meal gastric emptying instead of time. This representation highlights those pH values that are the most relevant for testing meal digestion in the stomach. Gastric lipolysis is still largely ignored or is performed with microbial lipases. In vivo data on gastric lipase and lipolysis have however been collected in humans and dogs during test meals. The biochemical characterization of gastric lipase has shown that this enzyme is rather unique among lipases: (i) stability and activity in the pH range 2 to 7 with an optimum at pH 4-5.4; (ii) high tensioactivity that allows resistance to bile salts and penetration into phospholipid layers covering TAG droplets; (iii) sn-3 stereospecificity for TAG hydrolysis; and (iv) resistance to pepsin. Most of these properties have been known for more than two decades and should provide a rational basis for the replacement of gastric lipase by other lipases when gastric lipase is not available.

  13. Enzymic resolution of 2-substituted cyclohexanols through lipase-mediated esterification

    Czech Academy of Sciences Publication Activity Database

    Wimmer, Zdeněk; Skouridou, V.; Zarevúcka, Marie; Šaman, David; Kolisis, F. N.

    2004-01-01

    Roč. 15, - (2004), s. 3911-3917 ISSN 0957-4166 R&D Projects: GA MŠk ME 692 Institutional research plan: CEZ:AV0Z4055905 Keywords : enzymic resolution * 2-substituted cyclohexanol s * lipase-mediated esterification Subject RIV: CC - Organic Chemistry Impact factor: 2.386, year: 2004

  14. Modelling and Optimization Studies on a Novel Lipase Production by Staphylococcus arlettae through Submerged Fermentation

    Directory of Open Access Journals (Sweden)

    Mamta Chauhan

    2013-01-01

    Full Text Available Microbial enzymes from extremophilic regions such as hot spring serve as an important source of various stable and valuable industrial enzymes. The present paper encompasses the modeling and optimization approach for production of halophilic, solvent, tolerant, and alkaline lipase from Staphylococcus arlettae through response surface methodology integrated nature inspired genetic algorithm. Response surface model based on central composite design has been developed by considering the individual and interaction effects of fermentation conditions on lipase production through submerged fermentation. The validated input space of response surface model (with R2 value of 96.6% has been utilized for optimization through genetic algorithm. An optimum lipase yield of 6.5 U/mL has been obtained using binary coded genetic algorithm predicted conditions of 9.39% inoculum with the oil concentration of 10.285% in 2.99 hrs using pH of 7.32 at 38.8°C. This outcome could contribute to introducing this extremophilic lipase (halophilic, solvent, and tolerant to industrial biotechnology sector and will be a probable choice for different food, detergent, chemical, and pharmaceutical industries. The present work also demonstrated the feasibility of statistical design tools integration with computational tools for optimization of fermentation conditions for maximum lipase production.

  15. Enzyme promiscuity in the hormone-sensitive lipase family of proteins.

    Science.gov (United States)

    Giuseppe, Manco; Luigia, Merone; Elena, Porzio; Yan, Feng; Luigi, Mandrich

    2012-02-01

    The number of enzymes endowed with the capacity to catalyse other reactions than the main, physiological one, a feature that has been called promiscuity, is increasing at a fast pace. Promiscuity is a highly pervasive phenomenon that is present at each level of life complexity. For enzymes, promiscuity encompasses interesting aspects related to their physiological role, evolution and biotechnological applications. Herein, at first we will describe some general aspects of enzyme promiscuity and then we will report some examples from the α/β hydrolase superfamily of proteins, with particular emphasis to the hormone-sensitive lipase family.

  16. Transesterification using the cross-linked enzyme aggregate of Photobacterium lipolyticum lipase M37.

    Science.gov (United States)

    Han, Jin Yee; Kim, Hyung Kwoun

    2011-11-01

    Biodiesel is methyl and ethyl esters of long-chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have occasionally been used for the production of this biofuel. Recently, biodiesel production using immobilized lipase has received increased attention. Through enhanced stability and reusability, immobilized lipase can contribute to the reduction of the costs inherent to biodiesel production. In this study, methanol-tolerant lipase M37 from Photobacterium lipolyticum was immobilized using the cross-linked enzyme aggregate (CLEA) method. Lipase M37 has a high lysine content (9.7%) in its protein sequence. Most lysine residues are located evenly over the surface of the protein, except for the lid structure region, which makes the CLEA preparation yield quite high (~93%). CLEA M37 evidences an optimal temperature of 30oC, and an optimal pH of 9-10. It was stable up to 50°C and in a pH range of 4.0-11.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentrations of methanol, ethanol, 1-propanol, and nbutanol. That is, their activities were maintained at solvent concentrations above 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. Additionally, CLEA M37 generated biodiesel via both 2-step methanol feeding procedures. Considering its physical stability and reusability, CLEA M37 may potentially be used as a catalyst in organic synthesis, including the biodiesel production reaction.

  17. Bacterial lipases

    OpenAIRE

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    1994-01-01

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase rea...

  18. [State of Fungal Lipases of Rhizopus microsporus, Penicillium sp. and Oospora lactis in Border Layers Water-Solid Phase and Factors Affecting Catalytic Properties of Enzymes].

    Science.gov (United States)

    Khasanov, Kh T; Davranov, K; Rakhimov, M M

    2015-01-01

    We demonstrated that a change in the catalytic activity of fungal lipases synthesized by Rhizopus microsporus, Penicillium sp. and Oospora lactis and their ability to absorb on different sorbents depended on the nature of groups on the solid phase surface in the model systems water: lipid and water: solid phase. Thus, the stability of Penicillium sp. lipases increased 85% in the presence ofsorsilen or DEAE-cellulose, and 55% of their initial activity respectively was preserved. In the presence of silica gel and CM-cellulose, a decreased rate of lipid hydrolysis by Pseudomonas sp. enzymes was observed in water medium, and the hydrolysis rate increased by 2.4 and 1.5 times respectively in the presence of aminoaerosil and polykefamid. In an aqueous-alcohol medium, aminoaerosil and polykefamid decreased the rate of substrate hydrolysis by more than 30 times. The addition of aerosil to aqueous and aqueous-alcohol media resulted in an increase in the hydrolysis rate by 1.2-1.3 times. Sorsilen stabilized Penicillium sp. lipase activity at 40, 45, 50 and 55 degrees C. Either stabilization or inactivation of lipases was observed depending on the pH of the medium and the nature of chemical groups localized on the surface of solid phase. The synthetizing activity of lipases also changed depending on the conditions.

  19. Immobilization of Lipases on Alkyl Silane Modified Magnetic Nanoparticles: Effect of Alkyl Chain Length on Enzyme Activity

    Science.gov (United States)

    Wang, Jiqian; Meng, Gang; Tao, Kai; Feng, Min; Zhao, Xiubo; Li, Zhen; Xu, Hai; Xia, Daohong; Lu, Jian R.

    2012-01-01

    Background Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification. Methodology/Principal Findings Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe3O4 were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining. Conclusions/Significance The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization

  20. Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity.

    Directory of Open Access Journals (Sweden)

    Jiqian Wang

    Full Text Available BACKGROUND: Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification. METHODOLOGY/PRINCIPAL FINDINGS: Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18 modified Fe(3O(4 were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining. CONCLUSIONS/SIGNIFICANCE: The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for

  1. Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity.

    Science.gov (United States)

    Wang, Jiqian; Meng, Gang; Tao, Kai; Feng, Min; Zhao, Xiubo; Li, Zhen; Xu, Hai; Xia, Daohong; Lu, Jian R

    2012-01-01

    Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification. Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe(3)O(4) were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining. The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.

  2. Molecular modelling studies of substrate binding to the lipase from Rhizomucor miehei

    Science.gov (United States)

    Yagnik, Asutosh T.; Littlechild, Jennifer A.; Turner, Nicholas J.

    1997-05-01

    Lipase enzymes have found increasingly widespread use, especially in biotransformation reactions in organic synthesis. Due to their efficiency and high enantioselectivity, they can be employed in a variety of reactions to carry out asymmetric hydrolyses, esterifications and transesterifications. However, the reasons for their stereospecificity have not been fully correlated with the enzyme structure. Employing molecular modelling techniques and existing experimental data, a transesterification reaction using Rhizomucor miehei lipase was studied. The results indicate that the major controlling factor for this reaction is hydrophobic in nature, providing support for previous literature hypotheses. In addition, computational experiments suggest that the origin of enantioselectivity is the formation of essential hydrogen bonds in and around the catalytic triad of active site residues. Only one enantiomer of the substrate is able to form these hydrogen bonds during the formation of the first tetrahedral transition state.

  3. Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone.

    Science.gov (United States)

    Sivalingam, G; Madras, Giridhar

    2004-01-01

    Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.

  4. Screening for Extracellular Lipase Enzymes with Transesterification Capacity in Mucoromycotina Strains

    Directory of Open Access Journals (Sweden)

    Alexandra Kotogán

    2014-01-01

    Full Text Available In this study, 169 zygomycetes fungal strains including some cold-tolerant isolates were screened for their extracellular lipolytic activity towards tributyrin. Nineteen of them were outstanding in their enzyme production as they developed the largest lipolytic halo around the colonies in plate tests. Mortierella alpina, M. echinosphaera, Mucor corticolus, Rhizomucor miehei, Rhizopus oryzae, Rh. stolonifer, Umbelopsis autotrophica, U. isabellina, U. ramanniana var. angulispora and U. versiformis were selected for further studies to characterise their lipolytic enzyme production in detail. In these assays, effect of Tween 80 and palm, soybean, sunflower, olive, extra virgin olive, wheat germ, corn germ, sesame seed, pumpkin seed and cottonseed oils on the enzyme activities was investigated, and wheat bran-based submerged and solid-state fermentations were also tested. Tween 80 and olive oil proved to be efficient inductors for lipolytic enzyme production, which was also enhanced when wheat bran was used as support. Addition of mineral salts and olive oil to the solid fermentation medium resulted in at least 1.5-fold increment in the enzyme activities of the crude extracts. Organic synthesis was also assayed by the selected lipases, in which enzymes from the fungi R. miehei, Rh. stolonifer and M. echinosphaera gave the best yields during transesterification reactions between p-nitrophenyl palmitate and ethanol.

  5. The metagenome-derived enzymes LipS and LipT increase the diversity of known lipases.

    Directory of Open Access Journals (Sweden)

    Jennifer Chow

    Full Text Available Triacylglycerol lipases (EC 3.1.1.3 catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75 °C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70 °C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70 °C. LipS had an optimum temperature at 70 °C and LipT at 75 °C. Both enzymes catalyzed hydrolysis of long-chain (C(12 and C(14 fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R-ibuprofen-phenyl ester with an enantiomeric excess (ee of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70 °C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure.

  6. Modeling the methanolysis of triglyceride catalyzed by immobilized lipase in a continuous-flow packed-bed reactor

    International Nuclear Information System (INIS)

    Tran, Dang-Thuan; Lin, Yi-Jan; Chen, Ching-Lung; Chang, Jo-Shu

    2014-01-01

    Highlights: • A Burkholderia lipase was immobilized on alkyl-grafted celite carriers. • Celite-alkyl-lipase catalyzed the methanolysis of triglyceride in packed-bed reactor. • The kinetics of the enzymatic transesterification follows Ping Pong Bi Bi mechanism. • Models were developed to discuss the mass transfer and enzyme kinetics in the PBR. - Abstract: A Burkholderia lipase was immobilized on celite grafted with long alkyl groups. The immobilized lipase-catalyzed methanolysis of sunflower oil in a packed-bed reactor (PBR) follows the Ping Pong Bi Bi mechanism. The external mass transfer and enzymatic reaction that simultaneously occurred in the PBR were investigated via the mathematical models. The overall biodiesel production in the PBR was verified to work in an enzymatic reaction-limited regime. Triglyceride conversion and biodiesel yield were higher under a lower reactant feeding rate, while a larger amount of biocatalyst would be required to achieve the designated conversion rate if a higher reactant feeding rate was employed. The PBR can achieve nearly complete conversion of triglyceride at a biocatalyst bed height of 60 cm (ca. 29 g biocatalyst) and a flow rate of 0.1 ml min −1 , whereas the biodiesel yield was lower than 67%, probably due to the positional specificity of Burkholderia lipase and the accumulation of glycerol

  7. Association between alcoholism and the gene encoding the endocannabinoid synthesizing enzyme diacylglycerol lipase alpha in the Japanese population.

    Science.gov (United States)

    Ishiguro, Hiroki; Higuchi, Susumu; Arinami, Tadao; Onaivi, Emmanuel S

    2017-10-12

    The endocannabinoid system has been recognized to be involved in neuropsychiatric diseases. 2-arachidonoyl glycerol (2-AG) is one of the two main endocannabinoids, and their regulation could play roles in disorders under environmental influence. This study investigated the involvement of the 2-AG biosynthesizing enzyme diacylglycerol lipase alpha (DAGLA) in the pathogenesis of alcoholism. We investigated a possible association between alcoholism and single nucleotide polymorphisms (SNPs) of the human DAGLA gene in the Japanese population. To discern any environmental influences on Dagla function in an animal study, the Dagla gene expression in the brain from stressed model mice was analyzed. The SNPs, including missense polymorphism Pro899Leu in the DAGLA gene, showed associations with alcoholism in the Japanese population. Dagla expression in mice was found to be influenced by chronic mild stress and by the acquisition of alcohol preference. Our findings indicated the involvement of DAGLA in alcoholism, possibly by its genetic dysfunction and also by the influence of stress. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Modeling high-intensity pulsed electric field inactivation of a lipase from Pseudomonas fluorescens.

    Science.gov (United States)

    Soliva-Fortuny, R; Bendicho-Porta, S; Martín-Belloso, O

    2006-11-01

    The inactivation kinetics of a lipase from Pseudomonas fluorescens (EC 3.1.1.3.) were studied in a simulated skim milk ultrafiltrate treated with high-intensity pulsed electric fields. Samples were subjected to electric field intensities ranging from 16.4 to 27.4 kV/cm for up to 314.5 micros, thus achieving a maximum inactivation of 62.1%. The suitability of describing experimental data using mechanistic first-order kinetics and an empirical model based on the Weibull distribution function is discussed. In addition, different mathematical expressions relating the residual activity values to field strength and treatment time are supplied. A first-order fractional conversion model predicted residual activity with good accuracy (A(f) = 1.018). A mechanistic insight of the model kinetics was that experimental values were the consequence of different structural organizations of the enzyme, with uneven resistance to the pulsed electric field treatments. The Weibull model was also useful in predicting the energy density necessary to achieve lipase inactivation.

  9. Techno-economic analysis of lipase enzyme production from agro-industry waste with solid state fermentation method

    Science.gov (United States)

    Hidayatullah, I. M.; Arbianti, R.; Utami, T. S.; Suci, M.; Sahlan, M.; Wijanarko, A.; Gozan, M.; Hermansyah, H.

    2018-03-01

    Needs for this kind of catalyst derived from biological raw materials (biocatalysts) has increased along with development of products based on eco-friendly. To achieve the needs of biocatalyst (enzyme), large production is necessary. This study aimed to get the best conditions and design equipment to produce lipase enzyme based on solid state fermentation using SuperPro Designer v9.0. Several equipment such as Tray Bioreactor, Mixing Tank 1, Filter Press, centrifuge, Mixing Tank 2, and a dryer have been improved during the simulation. Economic analysis in the form of NPV, IRR, Payback Period, and the Benefit Cost Ratio was evaluated respectively. The result showed that production of 10 kg enzyme with NPV Rp112.796.147.423,00; IRR 54.20%; Payback Period 1.95 years; and Benefit Cost Ratio of 3.36 was more advantageous.

  10. Production of Biodiesel from High Acid Value Waste Cooking Oil Using an Optimized Lipase Enzyme/Acid-Catalyzed Hybrid Process

    Directory of Open Access Journals (Sweden)

    N. Saifuddin

    2009-01-01

    Full Text Available The present study is aimed at developing an enzymatic/acid-catalyzed hybrid process for biodiesel production using waste cooking oil with high acid value (poor quality as feedstock. Tuned enzyme was prepared using a rapid drying technique of microwave dehydration (time required around 15 minutes. Further enhancement was achieved by three phase partitioning (TPP method. The results on the lipase enzyme which was subjected to pH tuning and TPP, indicated remarkable increase in the initial rate of transesterification by 3.8 times. Microwave irradiation was found to increase the initial reaction rates by further 1.6 times, hence giving a combined increase in activity of about 5.4 times. The optimized enzyme was used for hydrolysis and 88% of the oil taken initially was hydrolyzed by the lipase. The hydrolysate was further used in acid-catalyzed esterification for biodiesel production. By using a feedstock to methanol molar ratio of 1:15 and a sulphuric acid concentration of 2.5%, a biodiesel conversion of 88% was obtained at 50 °C for an hour reaction time. This hybrid process may open a way for biodiesel production using unrefined and used oil with high acid value as feedstock.

  11. Template-based modeling of a psychrophilic lipase: conformational changes, novel structural features and its application in predicting the enantioselectivity of lipase catalyzed transesterification of secondary alcohols.

    Science.gov (United States)

    Xu, Tao; Gao, Bei; Zhang, Lujia; Lin, Jingpin; Wang, Xuedong; Wei, Dongzhi

    2010-12-01

    In order to fully explore the structure-function relationship of a Proteus lipase (LipK107) that was screened from the soil in our previous study, we have modeled the three-dimensional (3-D) structures of the enzyme in its active and inactive conformations on the basis of crystal structures of Burkholderia glumae and Pseudomonas aeruginosa lipases in the present study. Both homology models suggested that LipK107 possessed a catalytic triad (Ser79-Asp232-H254), an oxyanion hole (Leu13 and Gln80) which was used to stabilize the reaction tetrahedral intermediates, and a lid substructure that controlled the access of the substrate to the active site. The existence of the lid was further verified by carrying out the interfacial activation experiment. The conformational change of LipK107 which was caused by lid opening action was predicted by superimposing the two theoretical models for the first time. Finally, both 3-D structures were used to predict the enantioselectivity of LipK107 when the enzyme was used to catalyze the resolution of racemic 1-phenylethanol. Lid-open model of LipK107 identified the R-enantiomer as the preferred enantiomer, while lid-closed mode showed that the S-enantiomer was more favored. However, only the lid-open conformational model could led to predictions that agreed with the following the experimental result of real biocatalysis reaction of 1-phenylethanol. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  12. In silico modeling of lipase H | Jabeen | African Journal of ...

    African Journals Online (AJOL)

    LAH 2 is a type of autosomal recessive hypotrichosis that affect hairs, eyebrows, scalp and eyelashes. Mutations in Lipase H gene result in LAH 2. Changes that result from mutation on physiochemical properties, post-translational modifications, functional sites, secondary structure and tertiary structure lipase H gene (LIPH) ...

  13. Application of lipase immobilized on the biocompatible ternary blend polymer matrix for synthesis of citronellyl acetate in non-aqueous media: kinetic modelling study.

    Science.gov (United States)

    Badgujar, Kirtikumar C; Bhanage, Bhalchandra M

    2014-04-10

    This work reports the use of new support for immobilization of lipase Burkholderia cepacia (BCL) matrix made up of polylactic acid (PLA), chitosan (CH), and polyvinyl alcohol (PVA). Initially lipase from various microbial sources and immobilization support composition was screened to obtain a robust biocatalyst. Among various biocatalysts preparation, the PLA:PVA:CH:BCL (1:6:1:2) was worked as a robust biocatalyst for the citronellyl acetate synthesis. Various reaction parameters were studied in detail to obtain the suitable reaction conditions for model citronellyl acetate synthesis reaction. Various kinetic parameters such as r(max), K(i(citronellol)), K(m(citronellol)), K(m(vinyl acetate)) were determined using non-linear regression analysis for the ternary complex as well as bi-bi ping-pong mechanism. The experimental results and kinetic study showed that citronellyl acetate synthesis catalyzed by immobilized lipase BCL followed the ternary complex mechanism with inhibition by alcohol (citronellol). The energy of activation for citronellyl acetate synthesis was found to be lower for immobilized lipase (8.9 kcal/mol) than aggregated lipase (14.8 kcal/mol) enzyme. The developed biocatalyst showed four to fivefold higher catalytic activity and excellent recyclability (up to six cycles) than the aggregated lipase. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Immobilization of lipases on hydrophobic supports involves the open form of the enzyme.

    Science.gov (United States)

    Manoel, Evelin A; Dos Santos, José C S; Freire, Denise M G; Rueda, Nazzoly; Fernandez-Lafuente, Roberto

    2015-04-01

    The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength. The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Chitosan–Collagen Coated Magnetic Nanoparticles for Lipase Immobilization—New Type of “Enzyme Friendly” Polymer Shell Crosslinking with Squaric Acid

    Directory of Open Access Journals (Sweden)

    Marta Ziegler-Borowska

    2017-01-01

    Full Text Available This article presents a novel route for crosslinking a polysaccharide and polysaccharide/protein shell coated on magnetic nanoparticles (MNPs surface via condensation reaction with squaric acid (SqA. The syntheses of four new types of collagen-, chitosan-, and chitosan–collagen coated magnetic nanoparticles as supports for enzyme immobilization have been done. Structure and morphology of prepared new materials were characterized by attenuated total reflectance Fourier-transform infrared (ATR-FTIR, XRD, and TEM analysis. Next, the immobilization of lipase from Candida rugosa was performed on the nanoparticles surface via N-(3-dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC/N-hydroxy-succinimide (NHS mechanism. The best results of lipase activity recovery and specific activities were observed for nanoparticles with polymer shell crosslinked via a novel procedure with squaric acid. The specific activity for lipase immobilized on materials crosslinked with SqA (52 U/mg lipase was about 2-fold higher than for enzyme immobilized on MNPs with glutaraldehyde addition (26 U/mg lipase. Moreover, a little hyperactivation of lipase immobilized on nanoparticles with SqA was observed (104% and 112%.

  16. Enzyme-catalyzed degradation of biodegradable polymers derived from trimethylene carbonate and glycolide by lipases from Candida antarctica and Hog pancreas.

    Science.gov (United States)

    Liu, Feng; Yang, Jian; Fan, Zhongyong; Li, Suming; Kasperczyk, Janusz; Dobrzynski, Piotr

    2012-01-01

    Enzyme-catalyzed degradation of poly(trimethylene carbonate) homo-polymer (PTMC) and poly(trimethylene carbonate-co-glycolide) co-polymer (PTGA) was investigated in the presence of lipases from Candida antarctica and Hog pancreas. Degradation was monitored by gravimetry, size-exclusion chromatography (SEC), nuclear magnetic resonance (NMR), tensiometry and environmental scanning electron microscopy (ESEM). PTMC can be rapidly degraded by Candida antarctica lipase with 98% mass loss after 9 days, while degradation by Hog pancreas lipase leads to 27% mass loss. Introduction of 16% glycolide units in PTMC chains strongly affects the enzymatic degradation. Hog pancreas lipase becomes more effective to PTGA co-polymer with a mass loss of 58% after 9 days, while Candida antarctica lipase seems not able to degrade PTGA. Bimodal molecular weight distributions are observed during enzymatic degradation of both PTMC and PTGA, which can be assigned to the fact that the surface is largely degraded while the internal part remains intact. The composition of the PTGA co-polymer remains constant, and ESEM shows that the polymers are homogeneously eroded during enzymatic degradation. Contact angle measurements confirm the enzymatic degradation mechanism, i.e., enzyme adsorption on the polymer surface followed by enzyme-catalyzed chain cleavage.

  17. Atomistic Model for the Polyamide Formation from beta-Lactam Catalyzed by Candida antarctica Lipase B

    NARCIS (Netherlands)

    Baum, Iris; Elsaesser, Brigitta; Schwab, Leendert W.; Loos, Katja; Fels, Gregor; Elsässer, Brigitta

    Candida antarctica lipase B (CALB) is an established biocatalyst for a variety of transesterification, amidation, and polymerization. reactions. In contrast to polyesters, poly amides are not yet generally accessible via enzymatic polymerization. In this regard, an enzyme-catalyzed ring-opening

  18. Enzyme catalysis in organic solvents: influence of water content, solvent composition and temperature on Candida rugosa lipase catalyzed transesterification.

    Science.gov (United States)

    Herbst, Daniela; Peper, Stephanie; Niemeyer, Bernd

    2012-12-31

    In the present study the influence of water content, solvent composition and reaction temperature on the transesterification of 1-phenylpropan-2-ol catalyzed by Candida rugosa lipase was examined. Reactions were carried out in different mixtures of hexane and tetrahydrofurane. The studies showed that an increasing water content of the organic solvent results in an increasing enzyme activity and a decreasing enantiomeric excess. Furthermore, a significant influence of the solvent hydrophilicity both on the enzyme activity and on the enantiomeric excess was found. An increase in solvent hydrophilicity leads to a decrease of enzyme activity and an increase of the enantiomeric excess. This indicates that the enzyme becomes more selective with decreasing flexibility. Similar effects were found by variation of the reaction temperature. Taken together, the decrease in conversion and the increase in selectivity with increasing solvent hydrophilicity are induced by the different water contents on the enzyme surface and not by the solvent itself. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

    Science.gov (United States)

    2014-01-01

    Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production

  20. Enhancement of Lipase Enzyme Activity in Non-Aqueous Media through a Rapid Three Phase Partitioning and Microwave Irradiation

    Directory of Open Access Journals (Sweden)

    N. Saifuddin

    2008-01-01

    Full Text Available Three phase partitioning is fast developing as a novel bio-separation strategy with a wide range of applications including enzyme stability and enhancement of its catalytic activity. pH tuning of enzyme is now well known for use in non-aqueous systems. Tuned enzyme was prepared using a rapid drying technique of microwave dehydration (time required around 15 minutes. Further enhancement was achieved by three phase partitioning (TPP method. With optimal condition of ammonium sulphate and t-butanol, the protein appeared as an interfacial precipitate between upper t-butanol and lower aqueous phases. In this study we report the results on the lipase which has been subjected to pH tuning and TPP, which clearly indicate the remarkable increase in the initial rate of transesterification by 3.8 times. Microwave irradiation was found to increase the initial reaction rates by further 1.6 times, hence giving a combined increase in activity of about 5.4 times. Hence it is shown that microwave irradiation can be used in conjunction with other strategies (like pH tuning and TPP for enhancing initial reaction rates.

  1. Lipase from Aspergillus niger obtained from mangaba residue fermentation: biochemical characterization of free and immobilized enzymes on a sol-gel matrix

    Directory of Open Access Journals (Sweden)

    Elis Augusta Leite dos Santos

    2017-02-01

    Full Text Available In this study, mangaba residue (seeds was used as a substrate for Aspergillus niger lipase production by solid-state fermentation. The partially purified enzyme was efficiently immobilized in a sol-gel matrix by covalent bonding with an immobilization yield of 91.2%. The immobilized biocatalyst and free lipase had an optimum pH of 2.0 and 5.0, respectively. However, greater stability was obtained at pH 4.0 and 7.0, respectively. The biocatalysts showed stability at the optimum temperature of 55°C, where the residual activity was above 87% after 240 min., of incubation. The lower deactivation constant (kd and higher half-life of the immobilized biocatalyst indicated greater thermal stability than those obtained with the free enzyme. The Michaelis Constant (Km (77 and 115 mM for free and immobilized lipase, respectively and maximum reaction rate (Vmax (1250 and 714 U mg-1 for free and immobilized lipase, respectively indicated that the immobilization process reduced enzyme-substrate affinity. Regarding the operational stability, the biocatalyst showed relative activity above 50% until seven cycles of reuse in olive oil hydrolysis. This novel biocatalyst obtained from a tropical fruit residue showed biochemical characteristics that support its application in future biocatalysis studies.

  2. Functional expression of a novel alkaline-adapted lipase of Bacillus amyloliquefaciens from stinky tofu brine and development of immobilized enzyme for biodiesel production.

    Science.gov (United States)

    Cai, Xianghai; Ma, Jing; Wei, Dong-Zhi; Lin, Jin-Ping; Wei, Wei

    2014-11-01

    Using enrichment procedures, a lipolytic strain was isolated from a stinky tofu brine and was identified as Bacillus amyloliquefaciens (named B. amyloliquefaciens Nsic-8) by morphological, physiological, biochemical tests and 16S rDNA sequence analysis. Meanwhile, the key enzyme gene (named lip BA) involved in ester metabolism was obtained from Nsic-8 with the assistance of homology analysis. The novel gene has an open reading frame of 645 bp, and encodes a 214-amino-acid lipase (LipBA). The deduced amino acid sequence shows the highest identity with the lipase from B. amyloliquefaciens IT-45 (NCBI database) and belongs to the family of triacylglycerol lipase (EC 3.1.1.3). The lipase gene was expressed in Escherichia coli BL21(DE3) using plasmid pET-28a. The enzyme activity and specific activity were 250 ± 16 U/ml and 1750 ± 153 U/mg, respectively. The optimum pH and temperature of the recombinant enzyme were 9.0 and 40 °C respectively. LipBA showed much higher stability under alkaline conditions and was stable at pH 7.0-11.0. The Km and Vmax values of purified LipBA using 4-nitrophenyl palmitate as the substrate were 1.04 ± 0.06 mM and 119.05 ± 7.16 μmol/(ml min), respectively. After purification, recombinant lipase was immobilized with the optimal conditions (immobilization time 3 h at 30 °C, with 92 % enzyme recovery) and the immobilized enzyme was applied in biodiesel production. This is the first report of the lipase activity and lipase gene obtained from B. amyloliquefaciens (including wild strain and recombinant strain) and the recombinant LipBA with the detailed enzymatic properties. Also the preliminary study of the transesterification shows the potential value in biodiesel production applications.

  3. Yarrowia lipolytica Lipase 2 Is Stable and Highly Active in Test Meals and Increases Fat Absorption in an Animal Model of Pancreatic Exocrine Insufficiency.

    Science.gov (United States)

    Aloulou, Ahmed; Schué, Mathieu; Puccinelli, Delphine; Milano, Stéphane; Delchambre, Chantal; Leblond, Yves; Laugier, René; Carrière, Frédéric

    2015-12-01

    Pancreatic exocrine insufficiency (PEI) reduces pancreatic secretion of digestive enzymes, including lipases. Oral pancreatic enzyme replacement therapy (PERT) with pancreatin produces unsatisfactory results. The lipase 2 produced by the yeast Yarrowia lipolytica (YLLIP2; GenBank: AJ012632) might be used in PERT. We investigated its ability to digest triglycerides in a test meal and its efficacy in reducing fecal fat in an animal model of PEI. YLLIP2 was produced by genetically engineered Y lipolytica and purified from culture media. YLLIP2 or other gastric (LIPF) and pancreatic (PNLIPD) lipases were added to a meal paste containing dietary triglycerides, at a range of pH values (pH 2-7), with and without pepsin or human bile and incubated at 37°C. We collected samples at various time points and measured lipase activities and stabilities. To create an animal model of PEI, steatorrhea was induced by embolization of the exocrine pancreas gland and pancreatic duct ligation in minipigs. The animals were given YLLIP2 (1, 4, 8, 40, or 80 mg/d) or pancreatin (100,000 US Pharmacopeia lipase units/d, controls) for 9 days. We then collected stool samples, measured fat levels, and calculated coefficient of fat absorption (CFA) values. YLLIP2 was highly stable and poorly degraded by pepsin, and had the highest activity of all lipases tested on meal triglyceride at pH 4-7 (pH 6 with bile: 94 ± 34 U/mg; pH 4 without bile: 43 ± 13 U/mg). Only gastric lipase was active and stable at pH 3, whereas YLLIP2 was sensitive to pepsin hydrolysis after pH inactivation. From in vitro test meal experiments, the lipase activity of YLLIP2 (10 mg) was estimated to be equivalent to that of pancreatin (1200 mg; 100,000 US Pharmacopeia units) at pH 6. In PEI minipigs, CFA values increased from 60.1% ± 9.3% before surgery to 90.5% ± 3.2% after administration of 1200 mg pancreatin (P meal triglycerides in a large pH range, with and without bile. Oral administration of milligram amounts of

  4. Mathematical modeling of lipase and protease production by Penicillium restrictum in a batch fermenter.

    Science.gov (United States)

    Freire, D M; Sant'Anna, G L; Alves, T L

    1999-01-01

    This work presents a mathematical model that describes time course variations of extracellular lipase and protease activities for the batch fermentation of the fungus Penicillium restrictum, a new and promising strain isolated from soil and wastes of a Brazilian babassu coconut oil industry. The fermentation process was modeled by an unstructured model, which considered the following dependent variables: cells, fat acid, dissolved oxygen concentrations, lipase and protease activities, and cell lysate concentration. The last variable represents the amount of cells that has been lysed by the shear stress and natural cell death. Proteases released to the medium, as consequence of this process, enhance lipase inactivation. The model is able to predict the effects of some operation variables such as air flow rate and agitation speed. The mathematical model was validated against batch-fermentation data obtained under several operating conditions. Because substrate concentration has antagonistic effects on lipase activity, a typical optimization scheme should be developed in order to minimize these deleterious effects while maximizing lipase activity.

  5. Lingual and gastric lipases.

    Science.gov (United States)

    Hamosh, M

    1990-01-01

    The 1973 discovery of lingual lipase, which is secreted by lingual serous glands and hydrolyzes medium- and long-chain triglycerides in the stomach, has renewed interest in the gastric phase of fat digestion. In humans, lipase is present in the serous (von Ebner) glands of the tongue, where it is localized in zymogen granules. In the stomach, the highest lipase activity is in the body. By immunocytochemistry, gastric lipase is confined to the chief cells of the fundic mucosa and is colocalized with pepsin. Human lipase purified from lingual serous glands or gastric juice has a MW of 45k to 51K but tends to aggregate (MW 270-300K and 500K) and is highly hydrophobic. Secretion of gastric lipase appears to be stimulated by at least two receptor mechanisms. It has been suggested that the products of gastric lipolysis maintain the sterility of the gastrointestinal tract. These enzymes are essential for the digestion of milk fat in the newborn because, contrary to other digestive lipases (pancreatic or milk digestive lipase), lingual and gastric lipases can penetrate into the milk fat globule and initiate the digestive process. Lingual and gastric lipase activity has been found in subjects with cystic fibrosis and appears to continue in the upper small intestine in these patients, perhaps replacing some of the missing pancreatic lipase. It is possible that lingual and gastric lipase supplements would be more effective in preventing steatorrhea in these patients than are the pancreatic enzyme supplements now given. The same therapeutic utility might be obtained in patients with alcoholic pancreatic insufficiency.

  6. [Industrial application of lipases].

    Science.gov (United States)

    Bancerz, Renata

    2017-01-01

    The ability of lipases to perform specific reactions of transformation (biotransformation) makes these enzymes a useful tool used in many syntheses, for example: in the production of detergents, cosmetics, biosurfactants, in the oil-chemical, paper, dairy, food or pharmaceutical industries. Lipases are ubiquitous enzymes but only lipases produced by microorganisms are important for industrial applications due to their wide variety of properties such as stability in organic solvents, action under mild conditions, high substrate specificity and region- and enantioselectivity, as well as the relatively simple methods of their production in fermentors and recovery from the culture medium. This paper reviews the latest achievements in the production of lipases in the submerged fermentation and solid state fermentation using waste products from the agricultural industry. In addition, new applications of lipases were described, including those for the synthesis of biopolymers and biodiesel and for the production of enantiomeric pharmaceuticals, agrochemicals and flavoring compounds.

  7. Kinetics of lipase-catalyzed esterification in organic media : correct model and solvent effects on parameters

    NARCIS (Netherlands)

    Janssen, A.E.M.; Sjursnes, B.J.; Vakunov, A.V.; Halling, P.J.

    1999-01-01

    The Ping-Pong model (incl. alcohol inhibition) is not the correct model to describe the kinetics of a lipase-catalyzed esterification reaction. The first product, water, is always present at the start of the reaction. This leads to an equation with one extra parameter. This new equation fits our

  8. Lipase enzymes on graphene oxide support for high-efficiency biocatalysis

    Czech Academy of Sciences Publication Activity Database

    Hermanová, S.; Zarevúcka, Marie; Bouša, D.; Mikulics, M.; Sofer, Z.

    2016-01-01

    Roč. 5, Dec (2016), s. 200-208 ISSN 2352-9407 R&D Projects: GA ČR(CZ) GA15-09001S Institutional support: RVO:61388963 Keywords : biocatalysis * graphene oxide * enzyme * acylglycerols Subject RIV: CC - Organic Chemistry

  9. Atomistic Model for the Polyamide Formation from β-Lactam Catalyzed by Candida Antarctica Lipase B

    Energy Technology Data Exchange (ETDEWEB)

    Baum, Iris; Elsasser, Brigitta M.; Schwab, Leendert; Loos, Katja; Fels, Gregor

    2011-04-01

    Candida antarctica lipase B (CALB) is an established biocatalyst for a variety of transesterification, amidation, and polymerization reactions. In contrast to polyesters, polyamides are not yet generally accessible via enzymatic polymerization. In this regard, an enzyme-catalyzed ring-opening polymerization of {beta}-lactam (2-azetidinone) using CALB is the first example of an enzymatic polyamide formation yielding unbranched poly({beta}-alanine), nylon 3. The performance of this polymerization, however, is poor, considering the maximum chain length of 18 monomer units with an average length of 8, and the molecular basis of the reaction so far is not understood. We have employed molecular modeling techniques using docking tools, molecular dynamics, and QM/MM procedures to gain insight into the mechanistic details of the various reaction steps involved. As a result, we propose a catalytic cycle for the oligomerization of {beta}-lactam that rationalizes the activation of the monomer, the chain elongation by additional {beta}-lactam molecules, and the termination of the polymer chain. In addition, the processes leading to a premature chain termination are studied. Particularly, the QM/MM calculation enables an atomistic description of all eight steps involved in the catalytic cycle, which features an in situ-generated {beta}-alanine as the elongating monomer and which is compatible with the experimental findings.

  10. Comparative evaluation of the efficacy of ginger and orlistat on obesity management, pancreatic lipase and liver peroxisomal catalase enzyme in male albino rats.

    Science.gov (United States)

    Mahmoud, R H; Elnour, W A

    2013-01-01

    Obesity is a disease involving body weight gain. Several synthetic drugs of better efficacy are being introduced in the modern system of medicine. Orlistat is a pharmacological agent promoting weight loss in obese subjects via inhibiting of gastric and pancreatic lipase. Ginger (Zingiber officinale Roscoe, Zingiberacae) is one of the most commonly used spices around the world; it has long been used in traditional medicine as a cure for some diseases. To evaluate the effect of ginger and orlistat on rats fed high fat diet. Forty male Albino rats were either not treated (control), or fed high fat diet, or fed high fat diet with dietary orlistat supplementation (200 mg/kg diet), or fed high fat diet supplemented with 5% ginger powder. After four weeks of treatment, final body weight and food intake were determined. Blood samples were collected, lipid parameters, total bilirubin, pancreatic lipase were determined. Liver peroxisomes were isolated from rat livers and peroxisomal catalase activity was determined. Treatment with both ginger and orlistat had significant effect in reducing body weight, besides, supplementing diet with orlistat increase food intake. Both ginger and orlistat had the ability to reduce lipid profile, ginger had great effect in increasing HDL-cholesterol than orlistat. When compared to the control group, ginger treatment did not alter either total bilirubin or pancreatic lipase activity while orlistat clearly reduced their concentration. Orlistat supplementation induced a significant reduction in peroxisomal catalase level, while ginger has been reported to interfere with enzyme activity increasing its level. Ginger has a great ability to reduce body weight without inhibiting pancreatic lipase level, or affecting bilirubin concentration, with positive effect on increasing peroxisomal catalase level and HDL-cholesterol.

  11. Extracellular Lipase and Protease Production from a Model Drinking Water Bacterial Community Is Functionally Robust to Absence of Individual Members.

    Directory of Open Access Journals (Sweden)

    Graham G Willsey

    Full Text Available Bacteria secrete enzymes into the extracellular space to hydrolyze macromolecules into constituents that can be imported for microbial nutrition. In bacterial communities, these enzymes and their resultant products can be modeled as community property. Our goal was to investigate the impact of individual community member absence on the resulting community production of exoenzymes (extracellular enzymes involved in lipid and protein hydrolysis. Our model community contained nine bacteria isolated from the potable water system of the International Space Station. Bacteria were grown in static conditions individually, all together, or in all combinations of eight species and exoproduct production was measured by colorimetric or fluorometric reagents to assess short chain and long chain lipases, choline-specific phospholipases C, and proteases. The exoenzyme production of each species grown alone varied widely, however, the enzyme activity levels of the mixed communities were functionally robust to absence of any single species, with the exception of phospholipase C production in one community. For phospholipase C, absence of Chryseobacterium gleum led to increased choline-specific phospholipase C production, correlated with increased growth of Burkholderia cepacia and Sphingomonas sanguinis. Because each individual species produced different enzyme activity levels in isolation, we calculated an expected activity value for each bacterial mixture using input levels or known final composition. This analysis suggested that robustness of each exoenzyme activity is not solely mediated by community composition, but possibly influenced by bacterial communication, which is known to regulate such pathways in many bacteria. We conclude that in this simplified model of a drinking water bacterial community, community structure imposes constraints on production and/or secretion of exoenzymes to generate a level appropriate to exploit a given nutrient environment.

  12. Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds.

    Science.gov (United States)

    Zhang, Meng; Yu, Xiao-Wei; Swapna, G V T; Xiao, Rong; Zheng, Haiyan; Sha, Chong; Xu, Yan; Montelione, Gaetano T

    2016-07-13

    In order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (δ1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce (13)C, (15)N-enriched and even (2)H,(13)C, (15)N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (δ1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (δ1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds. The 33 kDa protein-Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of (13)C, (15)N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli. These data demonstrate efficient production of (2)H, (13)C, (15)N

  13. Synthetic and Natural Lipase Inhibitors.

    Science.gov (United States)

    Białecka-Florjańczyk, Ewa; Fabiszewska, Agata Urszula; Krzyczkowska, Jolanta; Kuryłowicz, Alina

    2016-06-30

    Lipases are enzymes that catalyse the hydrolysis of ester bonds of triglycerides ranging among biocatalysts of considerable physiological significance and industrial potential. Better understanding of the catalytic functions and achieving the possibility to control the biocatalysis process, in particular exploring some activators and inhibitors of lipases, seems to be crucial in the context of novel applications. The lipase activity is a function of interfacial composition: the enzyme can be there activated as well as denaturated or deactivated and the interface is an appropriate site for modulating lipolysis. Lipase inhibitor, interacts directly with the enzyme and inhibits lipase action. Alternatively, some compounds can postpone the lipolytic reaction via adsorption to the interphase or to the substrate molecules. The aim of this review is to summarise the current knowledge concerning human, animal and microbial lipase inhibitors, which were grouped into two categories: synthetic lipase inhibitors (including phosphonates, boronic acids and fats analogues) and natural compounds (including β-lactones and some botanical foodstuffs - plant extracts and plant metabolites, mainly polyphenols and saponins as well as peptides and some dietary fibers). The topics discussed include also inhibition issues from the viewpoint of obesity treatment. Among natural compounds able to inhibit lipase activity are β-lactones including orlistat. Orlistat is the only registered drug for obesity treatment in many countries, especially pancreatic lipase which is responsible for the hydrolysis of over 80% of total dietary fats. Its effectiveness in obesity treatment was also described.

  14. Multi-enzyme Process Modeling

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia

    The subject of this thesis is to develop a methodological framework that can systematically guide mathematical model building for better understanding of multi-enzyme processes. In this way, opportunities for process improvements can be identified by analyzing simulations of either existing...... features of the process and provides the information required to structure the process model by using a step-by-step procedure with the required tools and methods. In this way, this framework increases efficiency of the model development process with respect to time and resources needed (fast and effective...... in the scientific literature. Reliable mathematical models of such multi-catalytic schemes can exploit the potential benefit of these processes. In this way, the best outcome of the process can be obtained understanding the types of modification that are required for process optimization. An effective evaluation...

  15. Role of adipose triglyceride lipase (PNPLA2) in protection from hepatic inflammation in mouse models of steatohepatitis and endotoxemia.

    Science.gov (United States)

    Jha, Pooja; Claudel, Thierry; Baghdasaryan, Anna; Mueller, Michaela; Halilbasic, Emina; Das, Suman K; Lass, Achim; Zimmermann, Robert; Zechner, Rudolf; Hoefler, Gerald; Trauner, Michael

    2014-03-01

    Hepatic inflammation is a key feature of progressive liver disease. Alterations of fatty acid (FA) metabolism and signaling may play an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH). Moreover, FAs activate peroxisome proliferator-activated receptor α (PPARα) as a key transcriptional regulator of hepatic FA metabolism and inflammation. Since adipose triglyceride lipase (ATGL/PNPLA2) is the key enzyme for intracellular hydrolysis of stored triglycerides and determines FA signaling through PPARα, we explored the role of ATGL in hepatic inflammation in mouse models of NASH and endotoxemia. Mice lacking ATGL or hormone-sensitive lipase (HSL) were challenged with a methionine-choline-deficient (MCD) diet as a nutritional model of NASH or lipopolysaccharide (LPS) as a model of acute hepatic inflammation. We further tested whether a PPARα agonist (fenofibrate) treatment improves the hepatic phenotype in MCD- or LPS-challenged ATGL-knockout (KO) mice. MCD-fed ATGL-KO mice, although partially protected from peripheral lipolysis, showed exacerbated hepatic steatosis and inflammation. Moreover, ATGL-KO mice challenged by LPS showed enhanced hepatic inflammation, increased mortality, and torpor, findings which were attributed to impaired PPARα DNA binding activity due to reduced FABP1 protein levels, resulting in impaired nuclear FA import. Notably, liganding PPARα through fenofibrate attenuated hepatic inflammation in both MCD-fed and LPS-treated ATGL-KO mice. In contrast, mice lacking HSL had a phenotype similar to the WT mice on MCD and LPS challenge. These findings unravel a novel protective role of ATGL against hepatic inflammation which could have important implications for metabolic and inflammatory liver diseases. © 2014 by the American Association for the Study of Liver Diseases.

  16. Lipase-catalyzed reactions in organic media: competition and applications.

    Science.gov (United States)

    Deleuze, H; Langrand, G; Millet, H; Baratti, J; Buono, G; Triantaphylides, C

    1987-01-05

    Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) have been used in organic media for the catalysis of reactions such as hydrolysis, esterification and transesterification. In these conditions it was confirmed that all reactions proceed through an acyl enzyme intermediate in two successive steps: acyl enzyme formation and solvolysis. The competition between two acyl acceptors (acyl donors) for reaction with a donor (acceptor) is described for the first time. A kinetic model is proposed using a competitive factor which is in good accordance with experimental results. The model was used successfully for the prediction of alcohol (acid) separations and resolutions by lipases.

  17. NRSA enzyme decomposition model data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme activities measured at more than 2000 US streams and rivers. These enzyme data were then used to predict organic matter decomposition and microbial...

  18. Evaluation of medium components by Plackett-Burman statistical design for lipase production by Candida rugosa and kinetic modeling.

    Science.gov (United States)

    Rajendran, Aravindan; Palanisamy, Anbumathi; Thangavelu, Viruthagiri

    2008-03-01

    Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3.6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL(-1) was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30 degrees C, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, alpha and beta, it was found that the lipase production by Candida rugosa is growth associated.

  19. Design and implementation of two-dimensional polymer adsorption models: evaluating the stability of Candida antarctica lipase B/solid-support interfaces by QCM-D.

    Science.gov (United States)

    Orski, Sara V; Kundu, Santanu; Gross, Richard; Beers, Kathryn L

    2013-02-11

    A two-dimensional model of a solid-supported enzyme catalyst bead is fabricated on a quartz crystal microbalance with dissipation monitoring (QCM-D) sensor to measure in situ interfacial stability and mechanical properties of Candida antarctica Lipase B (CAL B) under varied conditions relating to ring-opening polymerization. The model was fabricated using a dual photochemical approach, where poly(methyl methacrylate) (PMMA) thin films were cross-linked by a photoactive benzophenone monolayer and blended cross-linking agent. This process produces two-dimensional, homogeneous, rigid PMMA layers, which mimic commercial acrylic resins in a QCM-D experiment. Adsorption of CAL B to PMMA in QCM-D under varied buffer ionic strengths produces a viscoelastic enzyme surface that becomes more rigid as ionic strength increases. The rigid CAL B/PMMA interface demonstrates up to 20% desorption of enzyme with increasing trace water content. Increased polycaprolactone (PCL) binding at the enzyme surface was also observed, indicating greater PCL affinity for a more hydrated enzyme surface. The enzyme layer destabilized with increasing temperature, yielding near complete reversible catalyst desorption in the model.

  20. Enzymatic Production of FAME Biodiesel with Soluble Lipases

    DEFF Research Database (Denmark)

    T. Gundersen, Maria; Heltborg, Carsten Kirstejn; Yang, V

    Biodiesel is a viable alternative to fossil fuels, and biocatalysis is gaining interest as a greener process. We focus on converting oils to Fatty Acid Methyl Ester (FAME) using soluble lipases, which offer an advantage compared to immobilized enzymes by cost efficiency and ease of implementation...... the defined operating space concerning: temperature, water content, initial methanol concentration and enzyme content. The identified optimum range was experimentally evaluated, and model findings were confirmed. Another barrier in lipase use in biodiesel production is the higher melting point (m...

  1. Mechanistic Modelling of Biodiesel Production using a Liquid Lipase Formulation

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Hofmann, Björn; Silva, Vanessa T. L.

    2014-01-01

    , with respect to the industrial production of biodiesel. The developed kinetic model, coupled with a mass balance of the system, was fitted to and validated on experimental results for the fed-batch transesterification of rapeseed oil. The confidence intervals of the parameter estimates, along...... that constrains the amount of methanol in the reactor was computed and the predictions experimentally validated. Monte-Carlo simulations were then used to characterize the effect of the parameter uncertainty on the model outputs, giving a biodiesel yield, based on the mass of oil, of 90.8 ± 0.55 mass %. © 2014...

  2. Continuous Production of Lipase-Catalyzed Biodiesel in a Packed-Bed Reactor: Optimization and Enzyme Reuse Study

    Directory of Open Access Journals (Sweden)

    Hsiao-Ching Chen

    2011-01-01

    Full Text Available An optimal continuous production of biodiesel by methanolysis of soybean oil in a packed-bed reactor was developed using immobilized lipase (Novozym 435 as a catalyst in a tert-butanol solvent system. Response surface methodology (RSM and Box-Behnken design were employed to evaluate the effects of reaction temperature, flow rate, and substrate molar ratio on the molar conversion of biodiesel. The results showed that flow rate and temperature have significant effects on the percentage of molar conversion. On the basis of ridge max analysis, the optimum conditions were as follows: flow rate 0.1 mL/min, temperature 52.1∘C, and substrate molar ratio 1 : 4. The predicted and experimental values of molar conversion were 83.31±2.07% and 82.81±.98%, respectively. Furthermore, the continuous process over 30 days showed no appreciable decrease in the molar conversion. The paper demonstrates the applicability of using immobilized lipase and a packed-bed reactor for continuous biodiesel synthesis.

  3. Properties of Immobilized Candida antarctica Lipase B on Highly Macroporous Copolymer

    International Nuclear Information System (INIS)

    Handayani, N.; Achmad, S.; Wahyuningrum, D.

    2011-01-01

    In spite of their excellent catalytic properties, enzymes should be improved before their implementation both in industrial and laboratorium scales. Immobilization of enzyme is one of the ways to improve their properties. Candida antarctica lipase B (Cal-B) has been reported in numerous publications to be a particularly useful enzyme catalizing in many type of reaction including regio- and enantio- synthesis. For this case, cross-linking of immobilized Cal-B with 1,2,7,8 diepoxy octane is one of methods that proved significantly more stable from denaturation by heat, organic solvents, and proteolysis than lyophilized powder or soluble enzymes. More over, the aim of this procedure is to improve the activity and reusability of lipase. Enzyme kinetics test was carried out by transesterification reaction between 4-nitrophenyl acetate (pNPA) and methanol by varying substrate concentrations, and the result is immobilized enzymes follows the Michaelis-Menten models and their activity is match with previous experiment. Based on the V max values, the immobilized enzymes showed higher activity than the free enzyme. Cross-linking of immobilized lipase indicate that cross-linking by lower concentration of cross-linker, FIC (immobilized lipase that was incubated for 24 h) gave the highest activity and cross-linking by higher concentration of cross-linker, PIC (immobilized lipase that was incubated for 2 h) gives the highest activity. However, pore size and saturation level influenced their activity. (author)

  4. Bacterial lipases

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Ransac, Stéphane; Dijkstra, Bauke W.; Colson, Charles; Heuvel, Margreet van; Misset, Onno

    Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation,

  5. Bile salt-stimulated lipase: an animal model for human lactation

    International Nuclear Information System (INIS)

    Hamosh, M.; Freed, L.M.; York, C.M.; Sturman, J.A.; Hamosh, P.

    1986-01-01

    To date, bile salt-stimulated lipase (BSSL), an important digestive enzyme for the newborn, has only been described in the milk of primates - human and gorilla. The authors report the presence of BSSL in milks of dog and cat. Serial collections from two dogs (day 1-49) and cats (day 5-57) were analyzed for BSSL activity using a 3 H-triolein emulsion as substrate. Comparable analyses of pooled, term human milk were made for comparison. BSSL activity in individual dog milks (x = 32.0; range: 4.8-107.4 U/ml) was similar, while that in cat milk (x = 6.6; range: 2.2-16.9 U/ml) was lower than in human milk (x = 37.0; range: 10-80 U/ml; n = 35). Longitudinal patterns for BSSL differed depending upon the enzyme source. Dog, cat and human milk BSSL all showed a neutral to alkaline pH optimum (pH 7.0-8.4), stability at low pH, and 95-100% inhibition (at concentrations of 0.6 mM) by NaCl and eserine. BSSL activity from all sources had an obligate requirement for primary bile salts. These data are the first to show BSSL activity in milk from mammals other than human and gorilla. Presence of BSSL in nonprimate milk will permit the careful study of BSSL biology in the mammary gland as well as its role in neonatal fat digestion

  6. trans-10,cis-12 Conjugated linoleic acid inhibits lipoprotein lipase but increases the activity of lipogenic enzymes in adipose tissue from hamsters fed an atherogenic diet.

    Science.gov (United States)

    Zabala, Amaia; Churruca, Itziar; Fernández-Quintela, Alfredo; Rodríguez, Víctor M; Macarulla, M Teresa; Martínez, J Alfredo; Portillo, María P

    2006-06-01

    The aim of the present work was to investigate the effects of trans-10,cis-12 conjugated linoleic acid (CLA) on the activity and expression of lipogenic enzymes and lipoprotein lipase (LPL), as well as on the expression of transcriptional factors controlling these enzymes, in adipose tissue from hamsters, and to evaluate the involvement of these changes in the body fat-reducing effect of this CLA isomer. Thirty male hamsters were divided into three groups and fed atherogenic diets supplemented with 0 (linoleic group), 5 or 10 g trans-10,cis-12 CLA/kg diet, for 6 weeks. Body and adipose tissue weights, food intake and serum insulin were measured. Total and heparin-releasable LPL and lipogenic enzyme activities (acetyl-CoA carboxylase (ACC); fatty acid synthase (FAS); glucose-6-phosphate dehydrogenase (G6PDH); and malic enzyme (ME)) were assessed. ACC, FAS, LPL, sterol regulatory element-binding proteins (SREBP-1a), SREBP-1c and PPARgamma mRNA levels were also determined by real-time PCR. CLA did not modify food intake, body weight and serum insulin level. CLA feeding reduced adipose tissue weight, LPL activity and expression, and increased lipogenic enzyme activities, despite a significant reduction in ACC and FAS mRNA levels. The expression of the three transcriptional factors analysed (SREBP-1a, SREBP-1c and PPARgamma) was also reduced. These results appear to provide a framework for partially understanding the reduction in body fat induced by CLA. Inhibition of LPL activity seems to be an important mechanism underlying body fat reduction in hamsters. Further research is needed to better characterize the effects of CLA on lipogenesis and the role of these effects in CLA action.

  7. [Structure and Activity of Fungal Lipases in Bile Salt Solutions].

    Science.gov (United States)

    Bogdanova, L R; Bakirova, D R; Valiullina, Yu A; Idiyatullin, B Z; Faizullin, D A; Zueva, O S; Zuev, Yu F

    2016-01-01

    The changes in structure and catalytic properties of fungal lipases (Candida rugosa, Rhizomucor miehei, Mucor javanicus) were investigated in micellar solutions of bile salts that differ in hydrophilic-lypophilic balance and reaction medium properties. The methods of circular dichroism and tryptophan fluorescence were applied to estimate the changes in peptide structure within complexes with bile salt micelles. Bile salts do not exert a significant influence on the structure of the enzymes under study: in Rh. miehei and M. javanicus lipases the alpha helix content slightly decreased, the influence of bile salts on the C. rugosa structure was not revealed. Despite negligible structural modifications in the enzymes, in bile salt solutions a considerable change in their catalytic properties was observed: an abrupt decrease in catalytic effectiveness. Substrate-bile salts micelles complex formation was demonstrated by the NMR self-diffusion method. The model of a regulation of fungal lipase activity was proposed.

  8. Molecular Modeling of Enzyme Dynamics Towards Understanding Solvent Effects

    DEFF Research Database (Denmark)

    Wedberg, Nils Hejle Rasmus Ingemar

    ) in water and organic solvents. The effects of solvent on structural and dynamical enzyme properties are studied, and special attention is given to how enzyme properties in organic solvents are affected by the hydration level, which is shown to be related to the water activity. In experimental studies...... of enzyme kinetics in non-aqueous media, it has been a fruitful approach to fix the enzyme hydration level by controlling the water activity of the medium. In this work, a protocol is therefore developed for determining the water activity in non-aqueous protein simulations. The method relies on determining......This thesis describes the development of a molecular simulation methodology to study properties of enzymes in non-aqueous media at fixed thermodynamic water activities. The methodology is applied in a molecular dynamics study of the industrially important enzyme Candida antarctica lipase B (CALB...

  9. Comparative analyses of lipoprotein lipase, hepatic lipase, and endothelial lipase, and their binding properties with known inhibitors.

    Directory of Open Access Journals (Sweden)

    Ziyun Wang

    Full Text Available The triglyceride lipase gene subfamily plays a central role in lipid and lipoprotein metabolism. There are three members of this subfamily: lipoprotein lipase, hepatic lipase, and endothelial lipase. Although these lipases are implicated in the pathophysiology of hyperlipidemia and atherosclerosis, their structures have not been fully solved. In the current study, we established homology models of these three lipases, and carried out analysis of their activity sites. In addition, we investigated the kinetic characteristics for the catalytic residues using a molecular dynamics simulation strategy. To elucidate the molecular interactions and determine potential key residues involved in the binding to lipase inhibitors, we analyzed the binding pockets and binding poses of known inhibitors of the three lipases. We identified the spatial consensus catalytic triad "Ser-Asp-His", a characteristic motif in all three lipases. Furthermore, we found that the spatial characteristics of the binding pockets of the lipase molecules play a key role in ligand recognition, binding poses, and affinities. To the best of our knowledge, this is the first report that systematically builds homology models of all the triglyceride lipase gene subfamily members. Our data provide novel insights into the molecular structures of lipases and their structure-function relationship, and thus provides groundwork for functional probe design towards lipase-based therapeutic inhibitors for the treatment of hyperlipidemia and atherosclerosis.

  10. Characterization of alkaline lipase from Fusarium oxysporum and the effect of different surfactants and detergents on the enzyme activity Caracterização de lipase alcalina de Fusarium oxysporum e o efeito de diferentes surfactantes e detergentes sobre a atividade enzimática

    Directory of Open Access Journals (Sweden)

    Janaina Nicanuzia dos Prazeres

    2006-12-01

    Full Text Available Nowadays, there is a tendency of use of low temperature in laundry cleaning for both environmental and economical reasons, which makes the use of enzymes in detergent products indispensable. Since lipases are efficient catalyst both in solution and at the water-liquid interface, they are potentially suitable for lipid stain removal applications in industrial laundry and household detergents. The effect of different commercial detergents and surfactants on enzymatic activity of lipase from Fusarium oxysporum was observed through p-nitrophenylpalmitate (pNPP assay. The enzyme was compatible with various ionic and non-ionic surfactants as well as commercial detergents. Lipase activity was strongly inhibited by Sodium Dodecyl Sulphate (SDS, but not by Triton X-100 and Triton X-114. The best assay conditions observed for this lipase were pH 8.0 and 50ºC. The enzyme was stable at alkaline pH and remained 93% of residual activity during 1 h incubation at 60ºC. The highest lipase activity was measured with triglycerides of middle and long chain fatty acids (C8-C18. This enzyme showed a variable specifity/hydrolytic activity towards various fats and oils. All these properties and its resistance towards various surfactants and tolerance to commercial detergents make this lipase a potential additive for detergent formulation.Atualmente, há uma tendência de utilizar baixas temperaturas em limpeza de lavanderia por razões ambientais e econômicas, situações estas que tornam indispensáveis o uso de enzimas em produtos detergentes. Devido às lipases serem excelentes catalisadoras em soluções e em interface água-óleo, elas são potencialmente apropriadas para aplicação na remoção de manchas de gorduras em lavanderia industrial e detergentes domésticos. O efeito de diferentes detergentes comerciais e surfactantes sobre a atividade enzimática da lipase de Fusarium oxysporum foi observado através de ensaios com p-nitrofenilpalmitato (pNPP. A

  11. Lipases as Tools in the Synthesis of Prodrugs from Racemic 9-(2,3-Dihydroxypropyladenine

    Directory of Open Access Journals (Sweden)

    Marcela Krečmerová

    2012-11-01

    Full Text Available Lipases from Geotrichum candidum 4013 (extracellular lipase and cell-bound lipase were immobilized by adsorption on chitosan beads. The enzyme preparations were tested in the synthesis of ester prodrugs from racemic 9-(2,3-dihydroxypropyladenine in dimethylformamide with different vinyl esters (acetate, butyrate, decanoate, laurate, palmitate. The transesterification activities of these immobilized enzymes were compared with commercially available lipases (lipase from hog pancreas, Aspergillus niger, Candida antarctica, Pseudomonas fluorescens. Lipase from Candida antarctica was found to be the most efficient enzyme regarding chemical yield of the desired products, while transesterification by lipase from Aspergillus niger resulted in lower yields.

  12. Magnetic Cross-Linked Enzyme Aggregates (mCLEAs) of Candida antarctica Lipase: An Efficient and Stable Biocatalyst for Biodiesel Synthesis

    Science.gov (United States)

    Cruz-Izquierdo, Álvaro; Picó, Enrique A.; López, Carmen; Serra, Juan L.; Llama, María J.

    2014-01-01

    Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs) of Candida antarctica lipase B (CALB) which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with –NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization) and the magnetic character, and thus the final product (mCLEAs) are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil) conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available. PMID:25551445

  13. Magnetic Cross-Linked Enzyme Aggregates (mCLEAs of Candida antarctica lipase: an efficient and stable biocatalyst for biodiesel synthesis.

    Directory of Open Access Journals (Sweden)

    Álvaro Cruz-Izquierdo

    Full Text Available Enzyme-catalyzed production of biodiesel is the object of extensive research due to the global shortage of fossil fuels and increased environmental concerns. Herein we report the preparation and main characteristics of a novel biocatalyst consisting of Cross-Linked Enzyme Aggregates (CLEAs of Candida antarctica lipase B (CALB which are covalently bound to magnetic nanoparticles, and tackle its use for the synthesis of biodiesel from non-edible vegetable and waste frying oils. For this purpose, insolubilized CALB was covalently cross-linked to magnetic nanoparticles of magnetite which the surface was functionalized with -NH2 groups. The resulting biocatalyst combines the relevant catalytic properties of CLEAs (as great stability and feasibility for their reutilization and the magnetic character, and thus the final product (mCLEAs are superparamagnetic particles of a robust catalyst which is more stable than the free enzyme, easily recoverable from the reaction medium and reusable for new catalytic cycles. We have studied the main properties of this biocatalyst and we have assessed its utility to catalyze transesterification reactions to obtain biodiesel from non-edible vegetable oils including unrefined soybean, jatropha and cameline, as well as waste frying oil. Using 1% mCLEAs (w/w of oil conversions near 80% were routinely obtained at 30°C after 24 h of reaction, this value rising to 92% after 72 h. Moreover, the magnetic biocatalyst can be easily recovered from the reaction mixture and reused for at least ten consecutive cycles of 24 h without apparent loss of activity. The obtained results suggest that mCLEAs prepared from CALB can become a powerful biocatalyst for application at industrial scale with better performance than those currently available.

  14. Production of a Solvent, Detergent, and Thermotolerant Lipase by a Newly Isolated Acinetobacter sp. in Submerged and Solid-State Fermentations

    Directory of Open Access Journals (Sweden)

    Anahita Khoramnia

    2011-01-01

    Full Text Available The lipase production ability of a newly isolated Acinetobacter sp. in submerged (SmF and solid-state (SSF fermentations was evaluated. The results demonstrated this strain as one of the rare bacterium, which is able to grow and produce lipase in SSF even more than SmF. Coconut oil cake as a cheap agroindustrial residue was employed as the solid substrate. The lipase production was optimized in both media using artificial neural network. Multilayer normal and full feed forward backpropagation networks were selected to build predictive models to optimize the culture parameters for lipase production in SmF and SSF systems, respectively. The produced models for both systems showed high predictive accuracy where the obtained conditions were close together. The produced enzyme was characterized as a thermotolerant lipase, although the organism was mesophile. The optimum temperature for the enzyme activity was 45°C where 63% of its activity remained at 70°C after 2 h. This lipase remained active after 24 h in a broad range of pH (6–11. The lipase demonstrated strong solvent and detergent tolerance potentials. Therefore, this inexpensive lipase production for such a potent and industrially valuable lipase is promising and of considerable commercial interest for biotechnological applications.

  15. A stochastic model of enzyme kinetics

    Science.gov (United States)

    Stefanini, Marianne; Newman, Timothy; McKane, Alan

    2003-10-01

    Enzyme kinetics is generally modeled by deterministic rate equations, and in the simplest case leads to the well-known Michaelis-Menten equation. It is plausible that stochastic effects will play an important role at low enzyme concentrations. We have addressed this by constructing a simple stochastic model which can be exactly solved in the steady-state. Throughout a wide range of parameter values Michaelis-Menten dynamics is replaced by a new and simple theoretical result.

  16. Lipase production from a novel thermophilic Bacillus sp.: application of Plackett-Burman design for evaluating culture conditions affecting enzyme formation.

    Science.gov (United States)

    Abdel-Fattah, Yasser R; Soliman, Nadia A; Gaballa, Ahmed A; Sabry, Soraya A; El-Diwany, Ahmed I

    2002-01-01

    A novel thermophilic Bacillus sp. capable of producing lipase was locally isolated. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Bacillus thermoleovorans. Plackett-Burman experimental design was used to evaluate cultural conditions affecting lipase production process. Fifteen variables and four dummy variables were examined in the experimental design. Tween 80, temperature, olive oil, aeration, beef extract and inoculum age were found to be the highest positive significant variables affecting lipase activity, whereas pH and calcium chloride were the highest negative significant variables. Moreover, Tween 80, temperature and olive oil positively affected lipase specific activity. On the other hand, gum arabic, inoculum size and calcium chloride had the highest negative effect on lipase specific activity. This study would improve the further optimization steps on the bioprocess development track.

  17. Crocus cancellatus subsp. damascenus stigmas: chemical profile, and inhibition of α-amylase, α-glucosidase and lipase, key enzymes related to type 2 diabetes and obesity.

    Science.gov (United States)

    Loizzo, Monica R; Marrelli, Mariangela; Pugliese, Alessandro; Conforti, Filomena; Nadjafi, Farsad; Menichini, Francesco; Tundis, Rosa

    2016-01-01

    Spices are appreciated for their medicinal properties besides their use as food adjuncts to enhance the sensory quality of food. In this study, Crocus cancellatus subsp. damascenus was investigated for its antioxidant activities employing different in vitro systems. Stigma extract demonstrated a radical scavenging activity against both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals with IC50 values of 34.6 and 21.6 µg/mL and a good ferric reducing ability (53.9 µM Fe(II)/g). In order to clarify the potential functional properties of this spice, the carbohydrate-hydrolysing enzymes and pancreatic lipase inhibitory properties were investigated. Crocus cancellatus subsp. damascenus extract inhibited α-amylase and α-glucosidase with IC50 values of 57.1 and 68.6 µg/mL, respectively. The bioactivity was discussed in terms of phytochemicals content. The obtained results may be of interest from a functional point of view or as food additive and to promote the revalorization of this species.

  18. Task-specific enhancement of hippocampus-dependent learning in mice deficient in monoacylglycerol lipase, the major hydrolyzing enzyme of the endocannabinoid 2-arachidonoylglycerol

    Directory of Open Access Journals (Sweden)

    Yasushi eKishimoto

    2015-06-01

    Full Text Available Growing evidence indicates that the endocannabinoid system is important for the acquisition and/or extinction of learning and memory. However, it is unclear which endocannabinoid(s play(s a crucial role in these cognitive functions, especially memory extinction. To elucidate the physiological role of 2-arachidonoylglycerol (2-AG, a major endocannabinoid, in behavioral and cognitive functions, we conducted a comprehensive behavioral test battery in knockout (KO mice deficient in monoacylglycerol lipase (MGL, the major hydrolyzing enzyme of 2-AG. We found age-dependent increases in spontaneous physical activity in MGL KO mice. Next, we tested the MGL KO mice using 5 hippocampus-dependent learning paradigms (i.e., Morris water maze [MWM], contextual fear conditioning, novel object recognition test, trace eyeblink conditioning, and water-finding test. In the MWM, MGL KO mice showed normal acquisition of reference memory, but exhibited significantly faster extinction of the learned behavior. Moreover, they showed faster memory acquisition on the reversal-learning task of the MWM. In contrast, in the contextual fear conditioning, MGL KO mice tended to show slower memory extinction. In the novel object recognition and water-finding tests, MGL KO mice exhibited enhanced memory acquisition. Trace eyeblink conditioning was not altered in MGL KO mice throughout the acquisition and extinction phases. These results indicate that 2-AG signaling is important for hippocampus-dependent learning and memory, but its contribution is highly task-dependent.

  19. LIPASES PRODUCED BY YEASTS: POWERFUL BIOCATALYSTS FOR INDUSTRIAL PURPOSES

    Directory of Open Access Journals (Sweden)

    Luiza Lux Lock

    2007-12-01

    Full Text Available The term “lipolytic enzymes” refers to the lipases and carboxylic ester hydrolases. Lipase production is widespread among yeasts, butfew are capable of producing lipases with interesting characteristics and in sufficient amounts to be industrially useful. The literatureconcerning lipases produced by Candida rugosa, Yarrowia (Candida lipolytica, Candida antarctica and other emerging lipaseproducingyeasts is reviewed. The use of recombinant lipases is discussed, with emphasis on the utilization of heterologous expressionsystems and design of chimeras. Finally, the three approaches that aim the improvement of lipase production or the modification of thesubstrate selectivity of the enzyme (medium engineering, biocatalyst engineering, and protein engineering are discussed.

  20. Neutrophil chemotaxis by Propionibacterium acnes lipase and its inhibition.

    Science.gov (United States)

    Lee, W L; Shalita, A R; Suntharalingam, K; Fikrig, S M

    1982-01-01

    The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10(-4) mM) completely inhibited lipase activity as well as polymorphonuclear leukocyte chemotaxis generated by lipase. Tetracycline hydrochloride and erythromycin base at concentrations of 10(-1) mM and 1 mM, respectively, caused 100% inhibition of PMN migration toward lipase or zymosan-activated serum. The inhibiting activity of the antibiotics was directed against cells independently of any effect on lipase. Chemotaxis by P. acnes lipase suggests a wider role for this enzyme in the inflammatory process and the pathogenesis of acne vulgaris.

  1. Mutation induced enhanced biosynthesis of lipase | Bapiraju ...

    African Journals Online (AJOL)

    The purpose of the present investigation is to enhance production of biomedically important enzyme lipase by subjecting the indigenous lipase producing strain Rhizopus sp. BTS-24 to improvement by natural selection and random mutagenesis (UV and N-methyl-N'-nitro-N-nitroso guanidine, NTG). The isolation of mutants ...

  2. Modelling Fungal Fermentations for Enzyme Production

    DEFF Research Database (Denmark)

    Albæk, Mads Orla; Gernaey, Krist; Hansen, Morten S.

    We have developed a process model of fungal fed-batch fermentations for enzyme production. In these processes, oxygen transfer rate is limiting and controls the substrate feeding rate. The model has been shown to describe cultivations of both Aspergillus oryzae and Trichoderma reesei strains in 550...

  3. Lipases in Medicine: An Overview.

    Science.gov (United States)

    Loli, Heni; Narwal, Sunil Kumar; Saun, Nitin Kumar; Gupta, Reena

    2015-01-01

    Lipases are part of the family of hydrolases that act on carboxylic ester bonds. They are involved in catalyzing the hydrolysis of triglycerides (TG) into chylomicrons and very low density lipoprotein (VLDL) particles. Uses of lipases are evolving rapidly and currently they are reported to show high potential in medicine. Intensive study and investigations have led researchers to explore lipases for their use in substitution therapy, where in enzyme deficiency during diseased conditions is compensated by their external administration. In our body, they are used to break down fats present in food so that they can be absorbed in the intestine and deficiency of lipases leads to malabsorption of fats and fat-soluble vitamins. Lipases help a person who has cystic fibrosis, Alzheimer's disease, atherosclerosis and act as a candidate target for cancer prevention and therapy. They act as diagnostic tool and their presence or increasing levels can indicate certain infection or disease. Obesity causes metabolic disease and is a serious health problem around the world. Thus inhibiting digestive lipase to reduce fat absorption has become the main pharmacological approach to the treatment of obesity in recent years.

  4. Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y.

    Science.gov (United States)

    Masomian, Malihe; Jasni, Azmiza Syawani; Rahman, Raja Noor Zaliha Raja Abd; Salleh, Abu Bakar; Basri, Mahiran

    2017-12-20

    A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Evaluation of Canine Pancreas-Specific Lipase Activity, Lipase Activity, and Trypsin-Like Immunoreactivity in an Experimental Model of Acute Kidney Injury in Dogs.

    Science.gov (United States)

    Hulsebosch, S E; Palm, C A; Segev, G; Cowgill, L D; Kass, P H; Marks, S L

    2016-01-01

    Diagnosis of pancreatitis in dogs is complicated by extrapancreatic disorders that can alter the results of laboratory tests. Extrapancreatic disorders can also affect the diagnosis of exocrine pancreatic insufficiency (EPI). The effects of acute kidney injury (AKI) on pancreas-specific lipase activity (Spec cPL(®) Test), serum lipase activity and trypsin-like immunoreactivity (TLI) in dogs have not been evaluated. Serum Spec cPL, lipase activity, and TLI concentrations will increase secondary to decreased kidney function. Five purpose-bred dogs. Experimental prospective study. Gentamicin was used to induce AKI in 5 purpose-bred dogs. Serum samples were collected for measurement of creatinine, Spec cPL, lipase activity and TLI over 60 days, during both induction of, and recovery from, AKI. All dogs developed and recovered from AKI. Six of 52 (12%) serum Spec cPL concentrations were increased (2 in the equivocal zone and 4 consistent with pancreatitis) in 2 of 5 (40%) dogs. Two of 51 (4%) serum lipase activity values were increased in 2 of 5 dogs. Serum TLI was increased above the reference range in 17 of 50 (34%) samples in 3 of 5 dogs. For all biomarkers, there was no consistent correlation with increases in serum creatinine concentration. Decreased renal excretion during experimental AKI did not cause consistent and correlated increases in serum Spec cPL, lipase activity, or TLI in this cohort of dogs. Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  6. Use of Protease and Lipase Enzymes by Different Methods to Accelerate Kaşar Cheese Ripening 1. The Effect on Cheese Physical and Chemical Properties (Turkish with English Abstract

    Directory of Open Access Journals (Sweden)

    2015-02-01

    Full Text Available In this study, pasteurized cow milk was used for Kaşar cheese processing. One lipase (Palatase M, one protease (Neutrase, and combination of both enzymes were added to milk which was processed into cheese. Three different enzymes with the levels (based on raw milk amount of 0.0001% Palatese M. 0.004% Neutrase and 0.0001% Palatase M + 0.004% Neutrase were evaluated for physical and chemical properties at 2nd, 30th, 60th and 90th day of the ripening periods. Enzyme added cheese samples were compared with the Control-I (from fresh milk and Control-II (from pasteurized and starter added cheese samples. The 1% starter composed of Lactococcus lactis and Streptococcus cremoris (1:1 was added into the milk. Experiment was set up according to random block design with factorial arrangement, and the analysis were carried out in duplicate. Cheese yields of all the cheese samples produced with the enzyme addition were lower than the control groups. But, 0.0001% Palatase M(Mik-L added with microencapsulation technique gave the higher cheese yield which is closer to the control groups. With the enzyme addition, dry-matter; fat, protein, salt, acidity, fat in dry-matter, salt in dry-matter and the ripening degree; with the ripening period dry-matter, fat, protein, ash, salt, acidity, salt in dry-matter and the ripening degree gave statistically (P<0.01 significant results. It was determined that the interaction of enzyme addition x ripened period had an significant influence on dry-matter, fat, protein, acidity, fat in dry-matter and ripening degree of the cheeses produced in this research (P<0.01. The best ripened condition was achieved by adding lipase+protease enzymes together into milk with direct technique (Di-L+P. The same ripened state in the cheeses was reached in 30-60 days ripened period with the direct incorporation of lipase + protease enzymes together (Di+L+P or protease only (Di-P, but in 90 days in control groups containing no enzymes.

  7. Seeing & Feeling How Enzymes Work Using Tangible Models

    Science.gov (United States)

    Lau, Kwok-chi

    2013-01-01

    This article presents a tangible model used to help students tackle some misconceptions about enzyme actions, particularly the induced-fit model, enzyme-substrate complementarity, and enzyme inhibition. The model can simulate how substrates induce a change in the shape of the active site and the role of attraction force during enzyme-substrate…

  8. Organic Solvent Tolerant Lipases and Applications

    Directory of Open Access Journals (Sweden)

    Shivika Sharma

    2014-01-01

    Full Text Available Lipases are a group of enzymes naturally endowed with the property of performing reactions in aqueous as well as organic solvents. The esterification reactions using lipase(s could be performed in water-restricted organic media as organic solvent(s not only improve(s the solubility of substrate and reactant in reaction mixture but also permit(s the reaction in the reverse direction, and often it is easy to recover the product in organic phase in two-phase equilibrium systems. The use of organic solvent tolerant lipase in organic media has exhibited many advantages: increased activity and stability, regiospecificity and stereoselectivity, higher solubility of substrate, ease of products recovery, and ability to shift the reaction equilibrium toward synthetic direction. Therefore the search for organic solvent tolerant enzymes has been an extensive area of research. A variety of fatty acid esters are now being produced commercially using immobilized lipase in nonaqueous solvents. This review describes the organic tolerance and industrial application of lipases. The main emphasis is to study the nature of organic solvent tolerant lipases. Also, the potential industrial applications that make lipases the biocatalysts of choice for the present and future have been presented.

  9. Improving activity and enantioselectivity of lipase via immobilization on macroporous resin for resolution of racemic 1- phenylethanol in non-aqueous medium

    Science.gov (United States)

    2013-01-01

    Background Burkholderia cepacia lipase (BCL) has been proved to be capable of resolution reactions. However, its free form usually exhibits low stability, bad resistance and no reusability, which restrict its further industrial applications. Therefore, it is of great importance to improve the catalytic performance of free lipase in non-aqueous medium. Results In this work, macroporous resin NKA (MPR-NKA) was utilized as support for lipase immobilization. Racemic transesterification of 1-phenylethanol with vinyl acetate was chosen as model reaction. Compared with its free form, the enzyme activity and enantioselectivity (ees) of the immobilized lipase have been significantly enhanced. The immobilized BCL exhibited a satisfactory thermostability over a wide range of temperature (from 10 to 65°C) and an excellent catalytic efficiency. After being used for more than 30 successive batches, the immobilized lipase still kept most of its activity. In comparison with other immobilized lipases, the immobilized BCL also exhibits better catalytic efficiency, which indicates a significant potential in industrial applications. Conclusion The results of this study have proved that MPR-NKA was an excellent support for immobilization of lipase via the methods of N2 adsorption–desorption, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier transform-infrared spectroscopy (FT-IR). The improvement of enzyme activity and ees for the immobilized lipase was closely correlated with the alteration of its secondary structure. This information may contribute to a better understanding of the mechanism of immobilization and enzymatic biotransformation in non-aqueous medium. PMID:24168516

  10. Hepatic lipase activity is increased in non-alcoholic fatty liver disease beyond insulin resistance.

    Science.gov (United States)

    Miksztowicz, V; Lucero, D; Zago, V; Cacciagiú, L; Lopez, G; Gonzalez Ballerga, E; Sordá, J; Fassio, E; Schreier, L; Berg, G

    2012-09-01

    Hepatic lipase is a lipolytic enzyme mostly synthesized and localized at the surface of liver sinusoidal capillaries, which hydrolyses triglycerides and phospholipids of intermediate density, large low density (LDL) and high density lipoproteins. Hepatic lipase activity is increased in insulin resistant states. Non-alcoholic fatty liver disease (NAFLD) is characterized by insulin resistance. However, at present, no data are available regarding the behaviour of hepatic lipase with regard to the degree of hepatic steatosis. Our aim was to evaluate hepatic lipase activity in NAFLD patients and its relationship to the severity of hepatic steatosis. We studied 48 patients with NAFLD (diagnosed by ultrasonography and confirmed by liver biopsy) and 30 controls. Steatosis was semi-quantitatively assessed and considered as mild or grade 1, moderate or grade 2 and severe or grade 3. hepatic lipase activity, lipid and lipoprotein profile (including intermediate density lipoproteins and dense LDL), adiponectin, insulin, glucose and high sensitivity C-reactive protein were measured. Homeostasis model assessment for insulin resistance (HOMA) index was calculated. Patients with hepatic steatosis presented with higher hepatic lipase activity, HOMA and dense LDL and lower levels of adiponectin, high density lipoproteins, cholesterol and apoA-I. Hepatic lipase activity positively correlated significantly with the severity of hepatic steatosis. Hepatic lipase correlated with a more atherogenic profile and persisted higher in patients even after corrected for age, gender, body mass index, HOMA and adiponectin. The higher hepatic lipase activity in NAFLD patients contributes to a more atherogenic profile linked to increased cardiovascular risk, beyond the insulin resistance and the reduction in adiponectin. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Cloning, Phylogenetic Analysis and 3D Modeling of a Putative Lysosomal Acid Lipase from the Camel, Camelus dromedarius

    Directory of Open Access Journals (Sweden)

    Farid Shokry Ataya

    2012-08-01

    Full Text Available Acid lipase belongs to a family of enzymes that is mainly present in lysosomes of different organs and the stomach. It is characterized by its capacity to withstand acidic conditions while maintaining high lipolytic activity. We cloned for the first time the full coding sequence of camel’s lysosomal acid lipase, cLIPA using RT-PCR technique (Genbank accession numbers JF803951 and AEG75815, for the nucleotide and aminoacid sequences respectively. The cDNA sequencing revealed an open reading frame of 1,197 nucleotides that encodes a protein of 399 aminoacids which was similar to that from other related mammalian species. Bioinformatic analysis was used to determine the aminoacid sequence, 3D structure and phylogeny of cLIPA. Bioinformatics analysis suggested the molecular weight of the translated protein to be 45.57 kDa, which could be decreased to 43.16 kDa after the removal of a signal peptide comprising the first 21 aminoacids. The deduced cLIPA sequences exhibited high identity with Equus caballus (86%, Numascus leucogenys (85%, Homo sapiens (84%, Sus scrofa (84%, Bos taurus (82% and Ovis aries (81%. cLIPA shows high aminoacid sequence identity with human and dog-gastric lipases (58%, and 59% respectively which makes it relevant to build a 3D structure model for cLIPA. The comparison confirms the presence of the catalytic triad and the oxyanion hole in cLIPA. Phylogenetic analysis revealed that camel cLIPA is grouped with monkey, human, pig, cow and goat. The level of expression of cLIPA in five camel tissues was examined using Real Time-PCR. The highest level of cLIPA transcript was found in the camel testis (162%, followed by spleen (129%, liver (100%, kidney (20.5% and lung (17.4%.

  12. Immobilization of lipase and keratinase on functionalized SBA-15 nanostructured materials

    Science.gov (United States)

    Le, Hy G.; Vu, Tuan A.; Tran, Hoa T. K.; Dang, Phuong T.

    2013-12-01

    SBA-15 nanostructured materials were synthesized via hydrothermal treatment and were functionalized with 3- aminopropyltriethoxysilane (APTES). The obtained samples were characterized by different techniques such as XRD, BET, TEM, IR and DTA. After functionalization, it showed that these nanostrucrured materials still maintained the hexagonal pore structure of the parent SBA-15. The model enzyms chosen in this study were lipase and keratinase. Lipase was a biocatalyst for hydrolyzation of long chain triglycerides or methyl esters of long chain alcohols and fatty acids; keratinase is a proteolytic enzyme that catalyzes the cleavage of keratin. The functionalized SBA-15 materials were used to immobilize lipase and keratinase, exhibiting higher activity than that of the unfunctionalized pure silica SBA-15 ones. This might be due to the enhancing of surface hydrophobicity upon functionalization. The surface functionalization of the nanostructured silicas with organic groups can favor the interaction between enzyme and the supports and consequently increasing the operational stability of the immobilized enzymes. The loading of lipase on functionalized SBA-15 materials was higher than that of keratinase. This might be rationalized by the difference in size of enzyms.

  13. Structural characterization of MAPLE deposited lipase biofilm

    Energy Technology Data Exchange (ETDEWEB)

    Aronne, Antonio [Department of Chemical Engineering, Materials and Industrial Production, Università degli Studi di Napoli Federico II, Piazzale V. Tecchio 80, 80125 Napoli (Italy); Ausanio, Giovanni; Bloisi, Francesco [CNR-SPIN and Department of Physics, Università degli Studi di Napoli Federico II, Piazzale V. Tecchio 80, 80125 Napoli (Italy); Calabria, Raffaela [Istituto Motori-CNR, via G. Marconi 8, 80125 Napoli (Italy); Califano, Valeria, E-mail: v.califano@im.cnr.it [Istituto Motori-CNR, via G. Marconi 8, 80125 Napoli (Italy); Fanelli, Esther [Department of Chemical Engineering, Materials and Industrial Production, Università degli Studi di Napoli Federico II, Piazzale V. Tecchio 80, 80125 Napoli (Italy); Massoli, Patrizio [Istituto Motori-CNR, via G. Marconi 8, 80125 Napoli (Italy); Vicari, Luciano R.M. [CNR-SPIN and Department of Physics, Università degli Studi di Napoli Federico II, Piazzale V. Tecchio 80, 80125 Napoli (Italy)

    2014-11-30

    Highlights: • Lipase from Candida Rugosa was deposited by Matrix Assisted Pulsed Laser Evaporation (MAPLE) on KBr pellets, mica and glass substrate. • The deposited film was characterized morphologically and structurally by optical microscopy, SEM and FTIR analysis. • Results of characterization underlined a phenomenon of aggregation taking place. • The aggregation phenomenon was reversible since lipase showed activity in the transesterification reaction between soybean oil and isopropyl alcohol once detached from the substrate. - Abstract: Lipases (triacylglycerol ester hydrolases) are enzymes used in several industrial applications. Enzymes immobilization can be used to address key issues limiting widespread application at industrial level. Immobilization efficiency is related to the ability to preserve the native conformation of the enzyme. MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique, a laser deposition procedure for treating organic/polymeric/biomaterials, was applied for the deposition of lipase enzyme in an ice matrix, using near infrared laser radiation. Microscopy analysis showed that the deposition occurred in micrometric and submicrometric clusters with a wide size distribution. AFM imaging showed that inter-cluster regions are uniformly covered with smaller aggregates of nanometric size. Fourier transform infrared spectroscopy was used for both recognizing the deposited material and analyzing its secondary structure. Results showed that the protein underwent reversible self-association during the deposition process. Actually, preliminary tests of MAPLE deposited lipase used for soybean oil transesterification with isopropyl alcohol followed by gas chromatography–mass spectrometry gave results consistent with undamaged deposition of lipase.

  14. Seed lipases: sources, applications and properties - a review

    Directory of Open Access Journals (Sweden)

    M. Barros

    2010-03-01

    Full Text Available This paper provides an overview regarding the main aspects of seed lipases, such as the reactions catalyzed, physiological functions, specificities, sources and applications. Lipases are ubiquitous in nature and are produced by several plants, animals and microorganisms. These enzymes exhibit several very interesting features, such as low cost and easy purification, which make their commercial exploitation as industrial enzymes a potentially attractive alternative. The applications of lipases in food, detergents, oils and fats, medicines and fine chemistry, effluent treatment, biodiesel production and in the cellulose pulp industry, as well as the main sources of oilseed and cereal seed lipases, are reviewed.

  15. Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction

    Directory of Open Access Journals (Sweden)

    Jingjing Sun

    2015-01-01

    Full Text Available To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification, we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12. The free lipase lost most activity (35.3% and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h. Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7% after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition.

  16. Immobilization of Yarrowia lipolytica Lipase on Macroporous Resin Using Different Methods: Characterization of the Biocatalysts in Hydrolysis Reaction

    Science.gov (United States)

    Sun, Jingjing; Chen, Yiling; Sheng, Jun; Sun, Mi

    2015-01-01

    To improve the reusability and organic solvent tolerance of microbial lipase and expand the application of lipase (hydrolysis, esterification, and transesterification), we immobilized marine microbial lipase using different methods and determined the properties of immobilized lipases. Considering the activity and cost of immobilized lipase, the concentration of lipase was fixed at 2 mg/mL. The optimal temperature of immobilized lipases was 40°C and 5°C higher than free lipase. The activities of immobilized lipases were much higher than free lipase at alkaline pH (more than 50% at pH 12). The free lipase lost most activity (35.3%) and immobilized lipases retained more than 46.4% of their initial activity after 3 h heat treatment at 70°C. At alkaline pH, immobilized lipases were more stable than free lipase (more than 60% residue activity at pH 11 for 3 h). Immobilized lipases retained 80% of their activity after 5 cycles and increased enzyme activity (more than 108.7%) after 3 h treatment in tert-butanol. Immobilization of lipase which improved reusability of lipase and provided a chance to expand the application of marine microbial lipase in organic system expanded the application range of lipase to catalyze hydrolysis and esterification in harsh condition. PMID:26240816

  17. Kinetics of Enantiomerically Enriched Synthesis of Solketal Esters Using Native and SBA-15 supported P. Fluorescens Lipase

    Directory of Open Access Journals (Sweden)

    Zniszczoł Aurelia

    2017-06-01

    Full Text Available The studies showed that alkaline lipase from Pseudomonas fluorescens enables an irreversible transesterification of vinyl esters to give enantiomeric excess (eeR of about 80% using vinyl butyrate as acyl donor and diisopropyl ether as a solvent, at partially optimized conditions. For the native lipase the process was adequately described by a five-parameter Ping-Pong Bi Bi model for both enantiomers plus expression accounting for the formation of enzyme-acyl donor complex, but for the same lipase supported on mesoporous materials of SBA-15-Oc type, R-product inhibition also had to be taken into account. The use of hydrophobic support increased by more than two-fold the rate of the S-solketal conversion but even more that of R-solketal. Thus the immobilization of lipase had very positive effect on the process kinetics but decreased its enantioselectivity.

  18. Engineering Lipases: walking the fine line between activity and stability

    Science.gov (United States)

    Dasetty, Siva; Blenner, Mark A.; Sarupria, Sapna

    2017-11-01

    Lipases are enzymes that hydrolyze lipids and have several industrial applications. There is a tremendous effort in engineering the activity, specificity and stability of lipases to render them functional in a variety of environmental conditions. In this review, we discuss the recent experimental and simulation studies focused on engineering lipases. Experimentally, mutagenesis studies have demonstrated that the activity, stability, and specificity of lipases can be modulated by mutations. It has been particularly challenging however, to elucidate the underlying mechanisms through which these mutations affect the lipase properties. We summarize results from experiments and molecular simulations highlighting the emerging picture to this end. We end the review with suggestions for future research which underscores the delicate balance of various facets in the lipase that affect their activity and stability necessitating the consideration of the enzyme as a network of interactions.

  19. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  20. Enhancing activity and thermostability of lipase A from Serratia marcescens by site-directed mutagenesis.

    Science.gov (United States)

    Mohammadi, Mohsen; Sepehrizadeh, Zargham; Ebrahim-Habibi, Azadeh; Shahverdi, Ahmad Reza; Faramarzi, Mohammad Ali; Setayesh, Neda

    2016-11-01

    Lipases as significant biocatalysts had been widely employed to catalyze various chemical reactions such as ester hydrolysis, ester synthesis, and transesterification. Improving the activity and thermostability of enzymes is desirable for industrial applications. The lipase of Serratia marcescens belonging to family I.3 lipase has a very important pharmaceutical application in production of chiral precursors. In the present study, to achieve improved lipase activity and thermostability, using computational predictions of protein, four mutant lipases of SML (MutG2P, MutG59P, Mut H279K and MutL613WA614P) were constructed by site-directed mutagenesis. The recombinant mutant proteins were over-expressed in E. coli and purified by affinity chromatography on the Ni-NTA system. Circular dichroism spectroscopy, differential scanning calorimetry and kinetic parameters (Km and kcat) were determined. Our results have shown that the secondary structure of all lipases was approximately similar to one another. The MutG2P and MutG59P were more stable than wild type by approximately 2.3 and 2.9 in T 1/2 , respectively. The catalytic efficiency (kcat/Km) of MutH279K was enhanced by 2-fold as compared with the wild type (p<0.05). These results indicate that using protein modeling program and creating mutation, can enhance lipase activity and/or thermostability of SML and it also could be used for improving other properties of enzyme to the desired requirements as well as further mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Hidrolisis Minyak Kelapa oleh Enzim Lipase dari Kentos Kelapa

    OpenAIRE

    Su'i, Moh.

    2012-01-01

    Hydrolysis of Coconut Oil by Lipase Enzyme from Coconut Houstorium ABSTRACT Penelitian ini mempelajari jenis dan jumlah asam lemak yang dihasilkan dari hidrolisis minyak kelapa menggunakan enzim lipase dari kentos kelapa. Minyak kelapa dihidrolisis oleh enzim lipase kentos selama 30, 60, 9, dan 120 menit. Selanjutnya jenis asam lemak yang dihasilkan dan jumlahnya dianalisa. Hasil penelitian menunjukkan bahwa, hidrolisis selama 90 menit menghasilkan asam lemak paling tinggi yaitu sebes...

  2. Modeling of an immobilized lipase tubular reactor for the production of glycerol and fatty acids from oils; Modelado de un reactor tubular de lipasas inmovilizadas para la produccion de glicerol y acidos grasos a partir de aceites

    Energy Technology Data Exchange (ETDEWEB)

    Oddone, S.; Grasselli, M.; Cuellas, A.

    2010-07-01

    Advances in the design of a bioreactor in the fats and oils industry have permitted the hydrolysis of triglycerides in mild conditions and improved productivity while avoiding the formation of unwanted byproducts. The present work develops a mathematical model that describes the hydrolytic activity of a tubular reactor with immobilized lipases for the production of glycerol and fatty acids from the oil trade. Runge Kuttas numerical method of high order has been applied, considering that there is no accumulation of the substratum in the surface of the membrane, where the enzyme is. At the same time, different equations based on the kinetic model of Michaelis Mentens and the Ping-Pong bi-bi mechanism were examined. Experimental data in discontinuous systems are the basis for the development of the quantitative mathematical model that was used to simulate the process computationally. The obtained results allow for optimizing both the operative variables and the economic aspects of industrial processes. (Author)

  3. Molecular characterization of a Galactomyces geotrichum lipase, another member of the cholinesterase/lipase family.

    Science.gov (United States)

    Phillips, A; Pretorius, G H; van Rensburg, H G

    1995-10-25

    Geotrichum candidum secretes several lipase isoenzymes, differing in their selectively towards esters of long chain fatty acids with a cis-9 double bond. One group shows an absolute selectively towards these fatty acid esters, the other group has a more relaxed specificity and will also hydrolyze other long chain fatty acid esters. Galactomyces geotrichum secrets a lipase that has the same specificity as the latter group. The corresponding lipase gene was cloned from Galactomyces geotrichum. From an alignment of our enzymes' primary structure with those of different strains of Geotrichum candidum, remarkable conservation is evident and it is not yet possible to identify residues/structures responsible for differences in fatty acid specificity. Comparison of the GCL/GGL family with a variety of lipases from other sources, indicated that they are more related to mammalian than microbial lipases.

  4. Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus

    DEFF Research Database (Denmark)

    Prathumpai, Wai; Flitter, S.J.; Mcintyre, Mhairi

    2004-01-01

    Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on differe...... shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall....

  5. TLC bioautographic method for detecting lipase inhibitors.

    Science.gov (United States)

    Hassan, Abdel Moniem Sadek

    2012-01-01

    Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC bioautographic assay has previously been established for the detection of acetylcholinesterase inhibitors but not for lipases. Development of a TLC bioautographic method for detecting lipase inhibitors in plant extracts. After migration of the plant extracts, the TLC plate was sprayed with α-naphtyl acetate and enzyme solutions before incubation at 37°C for 20 min. Finally, the solution of Fast Blue B salt was sprayed onto the TLC plate giving a purple background colouration. Lipase inhibitors were visualised as white spots on the TLC plates. Orlistat (a known lipase inhibitor) inhibited lipase down to 0.01 µg. Methanolic extracts of Camellia sinensis (L.) kuntz and Rosmarinus officinalis L after migration on TLC gave enzymatic inhibition when applied in amounts of 82 and 56 µg, respectively. On the other hand the methanolic extract of Morus alba leaves did not exhibit any lipase inhibitory activity. The screening test was able to detect lipase inhibition by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2011 John Wiley & Sons, Ltd.

  6. Study the effect of F17S mutation on the chimeric Bacillus thermocatenulatus lipase

    Directory of Open Access Journals (Sweden)

    Seyed Hossein Khaleghinejad

    2016-06-01

    Full Text Available Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3 are one of the highest value commercial enzymes as they have potential applications in biotechnology for detergents, food, pharmaceuticals, leather, textiles, cosmetics, and paper industries; and are currently receiving considerable attention because of their potential applications in biotechnology. Bacillus thermocatenulatus Lipase 2 (BTL2 is one of the most important research targets, because of its potential industrial applications. In this study, the effect of substitution Phe17 with Ser in mutated BTL2 lipase, which conserved pentapeptide (112Ala-His-Ser-Gln-Gly116 was replaced with similar sequences (207Gly-Glu-Ser-Ala-Gly211 of Candida rugosa lipase (CLR at the nucleophilic elbow region. Docking results confirmed the mutated lipase to be better than the chimeric lipase. So, cloning was conducted, and the mutated and chimeric btl2 genes were expressed in Escherichia coli, and then the enzymes were purified by anion exchange chromatography. The mutation increased lipase lipolytic activity against most of the applied substrates, with the exception of tributyrin when compared with chimeric lipase. Further, the mutated lipase exhibited higher activity than the chimeric lipase at all temperatures. Optimum pH of the mutated lipase was obtained at pH 9.5, which was more than the chimeric one. Enzyme activity of the mutated lipase in the presence of organic solvents, detergents, and metal ions was also improved than the chimeric lipase.

  7. A lumped model of the lipase catalyzed hydrolysis of sardine oil to maximize polyunsaturated fatty acids content in acylglycerols.

    Science.gov (United States)

    Morales-Medina, R; Munio, M; Guadix, A; Guadix, E M; Camacho, F

    2018-02-01

    The aim of this work was to produce diacylglycerols (DAG) and monoacylglycerols (MAG) with a high content of polyunsaturated fatty acids (PUFA). Rhizomucor miehei lipase mediated-hydrolysis of sardine oil was conducted at several water activities. The system was mechanistically modeled to predict the time evolution of the concentration of triacylglycerols, DAG, MAG and free fatty acids (FFA) and the concentration of saturated, mono- and polyunsaturated fatty acids. The release of the first fatty acid from the triacylglycerol was independent on the unsaturation degree. Contrary, the hydrolysis of the second one was highly affected by the degree of unsaturation, PUFA being the fatty acids that showed the highest resistance to hydrolysis. MAG percentage was maximum (7mol%) at lower water activities, while DAG content was favored at higher water activities (35mol%), achieving a 2-fold concentration of DHA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Classification of EC 3.1.1.3 bacterial true lipases using phylogenetic ...

    African Journals Online (AJOL)

    Lipases play an important role in lipid metabolism and are produced by a variety of species. All lipases are members of the á/â hydrolase fold super-family. Also, lipases share a conserved active site signature, the Gly-Xaa-Ser-Xaa-Gly motif. To obtain an overview of this industrially and very important class of enzymes and ...

  9. Optimization of lipase production by Staphylococcus sp. Lp12

    African Journals Online (AJOL)

    USER

    2010-02-08

    Feb 8, 2010 ... of the genera Pseudomonas, Bacillus, Staphylococcus,. Achromobacter have been cloned and characterized. Bacterial lipases are mostly inducible enzymes and require some form of oil, fatty acid, fatty acid alcohol or fatty acid ester and surfactants for induction (Immanuel et al., 2008). Lipase biosynthesis ...

  10. Comparison of Enzymes / Non-Enzymes Proteins Classification Models Based on 3D, Composition, Sequences and Topological Indices

    OpenAIRE

    Munteanu, Cristian Robert

    2014-01-01

    Comparison of Enzymes / Non-Enzymes Proteins Classification Models Based on 3D, Composition, Sequences and Topological Indices, German Conference on Bioinformatics (GCB), Potsdam, Germany (September, 2007)

  11. State of the art and prospective of lipase-catalyzed transesterification reaction for biodiesel production

    International Nuclear Information System (INIS)

    Amini, Zeynab; Ilham, Zul; Ong, Hwai Chyuan; Mazaheri, Hoora; Chen, Wei-Hsin

    2017-01-01

    Highlights: • Enzymatic transesterification process is less energy intensive and robust. • Nano-materials are promising immobilization supports for lipase. • Packed-bed reactors are appropriate for scale-up use. • Potential recombinant, whole cell and recombinant whole cell lipases were enlisted. • Genetic engineering is a promising prospect in biodiesel area. - Abstract: The world demand for fuel as energy sources have arisen the need for generating alternatives such as biofuel. Biodiesel is a renewable fuel used particularly in diesel engines. Currently, biodiesel is mainly produced through transesterification reactions catalyzed by chemical catalysts, which produces higher fatty acid alkyl esters in shorter reaction time. Although extensive investigations on enzymatic transesterification by downstream processing were carried out, enzymatic transesterification has yet to be used in scale-up since commercial lipases are chiefly limited to the cost as well as long reaction time. While numerous lipases were studied and proven to have the high catalytic capacity, still enzymatic reaction requires more investigation. To fill this gap, finding optimal conditions for the reaction such as alcohol and oil choice, water content, reaction time and temperature through proper reaction modelling and simulations as well as the appropriate design and use of reactors for large scale production are crucial issues that need to be accurately addressed. Furthermore, lipase concentration, alternative lipase resources through whole cell technology and genetic engineering, recent immobilizing materials including nanoparticles, and the capacity of enzyme to be reused are important criteria to be neatly investigated. The present work reviews the current biodiesel feedstock, catalysis, general and novel immobilizing materials, bioreactors for enzymatic transesterification, potential lipase resources, intensification technics, and process modelling for enzymatic

  12. A lipase-responsive vehicle using amphipathic polymer synthesized with the lipase as catalyst.

    Science.gov (United States)

    Ge, Jun; Lu, Diannan; Yang, Cheng; Liu, Zheng

    2011-03-16

    We describe an enzyme-responsive polymeric vehicle, which is of great interest in controlled drug delivery, biosensing, and other related areas. The polymer synthesized using lipase as catalyst in DMSO has a favorable molecular structure that is quickly hydrolyzed by lipase in aqueous phase, and allows a fast release of encapsulated molecules. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Production of structured lipids in a packed-bed reactor with Thermomyces lanuginosa lipase

    DEFF Research Database (Denmark)

    Xu, Xuebing; Porsgaard, Trine; Zhang, Hong

    2002-01-01

    Lipase-catalyzed interesterification between fish oil and medium-chain TAG has been investigated in a packed-bed reactor with a commercially immobilized enzyme. The enzyme, a Thermomyces lanuginosa lipase immobilized on silica by granulation (Lipozyme TL IM; Novozymes A/S, Bagsvaerd, Denmark), ha...... without adjustment of water content or activity of the column and the substrate mixture.......Lipase-catalyzed interesterification between fish oil and medium-chain TAG has been investigated in a packed-bed reactor with a commercially immobilized enzyme. The enzyme, a Thermomyces lanuginosa lipase immobilized on silica by granulation (Lipozyme TL IM; Novozymes A/S, Bagsvaerd, Denmark), has...

  14. Pancreatic lipase

    African Journals Online (AJOL)

    use

    2011-12-05

    Dec 5, 2011 ... bean ”glyoxysomes” (Ricinus communis) have been characterized and have respectively a molecular weight of 65 and 62 kDa (Lin and Huang, 1984; Maeshima and. Beevers, 1985). However, and despite several studies, our knowledge about these plant enzymes is still in-. *Corresponding author.

  15. Beauveria bassiana Lipase A expressed in Komagataella (Pichia pastoris with potential for biodiesel catalysis

    Directory of Open Access Journals (Sweden)

    Ana Claudia Vici

    2015-10-01

    Full Text Available Lipases (EC 3.1.1.3 comprise a biotechnologically important group of enzymes because they are able to catalyze both hydrolysis and synthesis reactions, depending on the amount of water in the system. One of the most interesting application of lipase is in the biofuel industry for biodiesel production by oil and ethanol (or methanol transesterification. Entomopathogenic fungi, which are potential source of lipases, are still poorly explored in biotechnological processes. The present work reports the heterologous expression and biochemical characterization of a novel Beauveria bassiana lipase with potential for biodiesel production. The His-tagged B. bassiana lipase A (BbLA was produced in Komagataella pastoris in Buffered Methanol Medium (BMM induced with 1% methanol at 30 °C. Purified BbLA was activated with 0.05% Triton X-100 and presented optimum activity at pH 6.0 and 50°C. N-glycosylation of the recombinant BbLA accounts for 31.5% of its molecular weight. Circular dichroism and molecular modeling confirmed a structure composed of α-helix and β-sheet, similar to α/β hydrolases. Immobilized BbLA was able to promote transesterification reactions in fish oil, demonstrating potential for biodiesel production. BbLA was successfully produced in Komagataella pastoris and shows potential use for biodiesel production by the ethanolysis reaction.

  16. Modeling metabolic response to changes of enzyme amount in ...

    African Journals Online (AJOL)

    Based on the work of Hynne et al. (2001), in an in silico model of glycolysis, Saccharomyces cerevisiae is established by introducing an enzyme amount multiple factor (.) into the kinetic equations. The model is aimed to predict the metabolic response to the change of enzyme amount. With the help of .α, the amounts of ...

  17. Optimization of Fermentation Medium for Extracellular Lipase Production from Aspergillus niger Using Response Surface Methodology.

    Science.gov (United States)

    Jia, Jia; Yang, Xiaofeng; Wu, Zhiliang; Zhang, Qian; Lin, Zhi; Guo, Hongtao; Lin, Carol Sze Ki; Wang, Jianying; Wang, Yunshan

    2015-01-01

    Lipase produced by Aspergillus niger is widely used in various industries. In this study, extracellular lipase production from an industrial producing strain of A. niger was improved by medium optimization. The secondary carbon source, nitrogen source, and lipid were found to be the three most influential factors for lipase production by single-factor experiments. According to the statistical approach, the optimum values of three most influential parameters were determined: 10.5 g/L corn starch, 35.4 g/L soybean meal, and 10.9 g/L soybean oil. Using this optimum medium, the best lipase activity was obtained at 2,171 U/mL, which was 16.4% higher than using the initial medium. All these results confirmed the validity of the model. Furthermore, results of the Box-Behnken Design and quadratic models analysis indicated that the carbon to nitrogen (C/N) ratio significantly influenced the enzyme production, which also suggested that more attention should be paid to the C/N ratio for the optimization of enzyme production.

  18. Optimization of Fermentation Medium for Extracellular Lipase Production from Aspergillus niger Using Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Jia Jia

    2015-01-01

    Full Text Available Lipase produced by Aspergillus niger is widely used in various industries. In this study, extracellular lipase production from an industrial producing strain of A. niger was improved by medium optimization. The secondary carbon source, nitrogen source, and lipid were found to be the three most influential factors for lipase production by single-factor experiments. According to the statistical approach, the optimum values of three most influential parameters were determined: 10.5 g/L corn starch, 35.4 g/L soybean meal, and 10.9 g/L soybean oil. Using this optimum medium, the best lipase activity was obtained at 2,171 U/mL, which was 16.4% higher than using the initial medium. All these results confirmed the validity of the model. Furthermore, results of the Box-Behnken Design and quadratic models analysis indicated that the carbon to nitrogen (C/N ratio significantly influenced the enzyme production, which also suggested that more attention should be paid to the C/N ratio for the optimization of enzyme production.

  19. Mechanism of acetaldehyde-induced deactivation of microbial lipases

    Directory of Open Access Journals (Sweden)

    Jaeger Karl E

    2011-02-01

    Full Text Available Abstract Background Microbial lipases represent the most important class of biocatalysts used for a wealth of applications in organic synthesis. An often applied reaction is the lipase-catalyzed transesterification of vinyl esters and alcohols resulting in the formation of acetaldehyde which is known to deactivate microbial lipases, presumably by structural changes caused by initial Schiff-base formation at solvent accessible lysine residues. Previous studies showed that several lipases were sensitive toward acetaldehyde deactivation whereas others were insensitive; however, a general explanation of the acetaldehyde-induced inactivation mechanism is missing. Results Based on five microbial lipases from Candida rugosa, Rhizopus oryzae, Pseudomonas fluorescens and Bacillus subtilis we demonstrate that the protonation state of lysine ε-amino groups is decisive for their sensitivity toward acetaldehyde. Analysis of the diverse modification products of Bacillus subtilis lipases in the presence of acetaldehyde revealed several stable products such as α,β-unsaturated polyenals, which result from base and/or amino acid catalyzed aldol condensation of acetaldehyde. Our studies indicate that these products induce the formation of stable Michael-adducts at solvent-accessible amino acids and thus lead to enzyme deactivation. Further, our results indicate Schiff-base formation with acetaldehyde to be involved in crosslinking of lipase molecules. Conclusions Differences in stability observed with various commercially available microbial lipases most probably result from different purification procedures carried out by the respective manufacturers. We observed that the pH of the buffer used prior to lyophilization of the enzyme sample is of utmost importance. The mechanism of acetaldehyde-induced deactivation of microbial lipases involves the generation of α,β-unsaturated polyenals from acetaldehyde which subsequently form stable Michael-adducts with the

  20. Antioxidant property and [Formula: see text]-glucosidase, [Formula: see text]-amylase and lipase inhibiting activities of Flacourtia inermis fruits: characterization of malic acid as an inhibitor of the enzymes.

    Science.gov (United States)

    Alakolanga, A G A W; Kumar, N Savitri; Jayasinghe, Lalith; Fujimoto, Yoshinori

    2015-12-01

    Flacourtia inermis Roxb. (Flacourtiaceae), is a moderate sized tree cultivated in Sri Lanka for its fruits known as Lovi. The current study was undertaken to study the biological activity of extracts of the fruits in an attempt to increase the value of the under exploited fruit crops. Fruits of F. inermis were found to be rich in phenolics and anthocyanins. Polyphenol content of the fruits was determined to be 1.28 g gallic acid equivalents per 100 g of fresh fruit and anthocyanin content was estimated as 108 mg cyanidin-3-glucoside equivalents per 100 g of fresh fruits. The EtOAc extract showed moderate antioxidant activity in the DPPH radical scavenging assay with IC50 value of 66.2 ppm. The EtOAc and MeOH extracts of the fruits also exhibited inhibitory activities toward α-glucosidase, α-amylase and lipase enzymes with IC50values ranging from 549 to 710 ppm, 1021 to 1949 ppm and 1290 to 2096 ppm, respectively. The active principle for the enzyme inhibition was isolated through activity-guided fractionation and was characterized as (S)-malic acid. The results of this study indicate that F. inermis fruits have the potential to be used in health foods and in nutritional supplements.

  1. Endothelial lipase is a major determinant of HDL level

    Energy Technology Data Exchange (ETDEWEB)

    Ishida, Tatsuro; Choi, Sungshin; Kundu, Ramendra K.; Hirata, Ken-Ichi; Rubin, Edward M.; Cooper, Allen D.; Quertermous, Thomas

    2003-01-30

    For the past three decades, epidemiologic studies have consistently demonstrated an inverse relationship between plasma HDL cholesterol (HDL-C) concentrations and coronary heart disease (CHD). Population-based studies have provided compelling evidence that low HDL-C levels are a risk factor for CHD, and several clinical interventions that increased plasma levels of HDL-C were associated with a reduction in CHD risk. These findings have stimulated extensive investigation into the determinants of plasma HDL-C levels. Turnover studies using radiolabeled apolipoprotein A-I, the major protein component of HDL, suggest that plasma HDL-C concentrations are highly correlated with the rate of clearance of apolipoprotein AI. However, the metabolic mechanisms by which HDL are catabolized have not been fully defined. Previous studies in humans with genetic deficiency of cholesteryl ester transfer protein, and in mice lacking the scavenger receptor BI (SR-BI), have demonstrated that these proteins participate in the removal of cholesterol from HDL, while observations in individuals with mutations in hepatic lipase indicate that this enzyme hydrolyzes HDL triglycerides. In this issue of the JCI, reports from laboratories of Tom Quertermous and Dan Rader now indicate that endothelial lipase (LIPG), a newly identified member of the lipase family, catalyzes the hydrolysis of HDL phospholipids and facilitates the clearance of HDL from the circulation. Endothelial lipase was initially cloned by both of these laboratories using entirely different strategies. Quertermous and his colleagues identified endothelial lipase as a transcript that was upregulated in cultured human umbilical vein endothelial cells undergoing tube formation, whereas the Rader group cloned endothelial lipase as a transcript that was upregulated in the human macrophage-like cell line THP-1 exposed to oxidized LDL. Database searches revealed that endothelial lipase shows strong sequence similarity to lipoprotein

  2. Purification of lipases.

    Science.gov (United States)

    Taipa, M A; Aires-Barros, M R; Cabral, J M

    1992-11-01

    Interest on lipases from different sources (microorganisms, animals and plants) has markedly increased in the last decade due to the potential applications of lipases in industry and in medicine. Microbial and mammalian lipases have been purified to homogeneity, allowing the successful determination of their primary aminoacid sequence and, more recently, of the three-dimensional structure. The X-ray studies of pure lipases will enable the establishment of the structure-function relationships and contribute for a better understanding of the kinetic mechanisms of lipase action on hydrolysis, synthesis and group exchange of esters. This article reviews the separation and purification techniques that were used in the recovery of microbial, mammalian and plant lipases. Several purification procedures are analysed taking into account the sequence of the methods and the number of times each method is used. Novel purification methods based on liquid-liquid extraction, membrane processes and immunopurification are also reviewed.

  3. Structural characterization of MAPLE deposited lipase biofilm

    Science.gov (United States)

    Aronne, Antonio; Ausanio, Giovanni; Bloisi, Francesco; Calabria, Raffaela; Califano, Valeria; Fanelli, Esther; Massoli, Patrizio; Vicari, Luciano R. M.

    2014-11-01

    Lipases (triacylglycerol ester hydrolases) are enzymes used in several industrial applications. Enzymes immobilization can be used to address key issues limiting widespread application at industrial level. Immobilization efficiency is related to the ability to preserve the native conformation of the enzyme. MAPLE (Matrix Assisted Pulsed Laser Evaporation) technique, a laser deposition procedure for treating organic/polymeric/biomaterials, was applied for the deposition of lipase enzyme in an ice matrix, using near infrared laser radiation. Microscopy analysis showed that the deposition occurred in micrometric and submicrometric clusters with a wide size distribution. AFM imaging showed that inter-cluster regions are uniformly covered with smaller aggregates of nanometric size. Fourier transform infrared spectroscopy was used for both recognizing the deposited material and analyzing its secondary structure. Results showed that the protein underwent reversible self-association during the deposition process. Actually, preliminary tests of MAPLE deposited lipase used for soybean oil transesterification with isopropyl alcohol followed by gas chromatography-mass spectrometry gave results consistent with undamaged deposition of lipase.

  4. Characterization of Cross-Linked Lipase Aggregates

    DEFF Research Database (Denmark)

    Prabhavathi Devi, Bethala Lakshmi Anu; Guo, Zheng; Xu, Xuebing

    2009-01-01

    Commercially available microbial lipases from different sources were immobilized as cross-linked enzyme aggregates (CLEAs) using different precipitants and glutaraldehyde as cross-linkers. These CLEAs were assayed based on esterification between lauric acid and n-propanol in solvent-free systems...

  5. Combining solvent engineering and thermodynamic modeling to enhance selectivity during monoglyceride synthesis by lipase-catalyzed esterification.

    Science.gov (United States)

    Bellot, J C.; Choisnard, L; Castillo, E; Marty, A

    2001-03-08

    Monoglyceride synthesis by Rhyzomucor miehei lipase was investigated via direct esterification between glycerol (adsorbed onto silica gel) and oleic acid in organic solvents. The main difficulty is to avoid the unwanted production of di- and tri-glycerides. It was demonstrated that an increase in solvent polarity, using mixtures of n-hexane and 2-methyl-2-butanol (2M2B), improves drastically the selectivity toward monoglyceride formation. In pure n-hexane, the monoglyceride represents only 6 molar % of the total products at the thermodynamic equilibrium (34 and 60% for di- and tri-glyceride respectively). Use of an equivolume mixture of n-hexane/2M2B enables a product mixture to be obtained containing 94% of monoglyceride at equilibrium (2.4 and 0% for di- and tri-glyceride respectively). This positive effect is counterbalanced by a decrease both in initial velocities and in substrate conversion at thermodynamic equilibrium.A modeling, able to predict the three thermodynamic equilibria governing the 3 consecutive reactions, based on activity coefficient calculations using the UNIFAC model, is proposed. It takes into account both the partition of water between solvent and immobilized catalyst, and the partition of glycerol between solvent and silica gel. A good correlation with experimental data obtained in n-hexane/2M2B mixtures was observed.

  6. Enzymes/non-enzymes classification model complexity based on composition, sequence, 3D and topological indices.

    Science.gov (United States)

    Munteanu, Cristian Robert; González-Díaz, Humberto; Magalhães, Alexandre L

    2008-09-21

    The huge amount of new proteins that need a fast enzymatic activity characterization creates demands of protein QSAR theoretical models. The protein parameters that can be used for an enzyme/non-enzyme classification includes the simpler indices such as composition, sequence and connectivity, also called topological indices (TIs) and the computationally expensive 3D descriptors. A comparison of the 3D versus lower dimension indices has not been reported with respect to the power of discrimination of proteins according to enzyme action. A set of 966 proteins (enzymes and non-enzymes) whose structural characteristics are provided by PDB/DSSP files was analyzed with Python/Biopython scripts, STATISTICA and Weka. The list of indices includes, but it is not restricted to pure composition indices (residue fractions), DSSP secondary structure protein composition and 3D indices (surface and access). We also used mixed indices such as composition-sequence indices (Chou's pseudo-amino acid compositions or coupling numbers), 3D-composition (surface fractions) and DSSP secondary structure amino acid composition/propensities (obtained with our Prot-2S Web tool). In addition, we extend and test for the first time several classic TIs for the Randic's protein sequence Star graphs using our Sequence to Star Graph (S2SG) Python application. All the indices were processed with general discriminant analysis models (GDA), neural networks (NN) and machine learning (ML) methods and the results are presented versus complexity, average of Shannon's information entropy (Sh) and data/method type. This study compares for the first time all these classes of indices to assess the ratios between model accuracy and indices/model complexity in enzyme/non-enzyme discrimination. The use of different methods and complexity of data shows that one cannot establish a direct relation between the complexity and the accuracy of the model.

  7. La lipase de Candida rugosa : caractérisation biochimique

    Directory of Open Access Journals (Sweden)

    Mtibaa Hounaida

    2002-01-01

    Full Text Available Les lipases ou triacylglycérols hydrolases (EC 3.1.1.3 sont des enzymes qui agissent en milieu hétérogène. Ces enzymes catalysent l’hydrolyse des liaisons esters des triacylglycérols à l’interface huile/eau [1]. Leur particularité vient du fait que ces enzymes sont plus actives sur les lipides qui sont sous forme agrégée [2]. Les lipases sont présentes dans la plupart des tissus animaux et végétaux ainsi que chez les microorganismes qui constituent une source importante de production de lipases à grande échelle. À ce jour, de nombreuses lipases de microorganismes ont été purifiées et caractérisées et certaines d’entres elles ont été cristallisées (lipase de Pseudomonas glumae [3], lipase de Rhizomucor miehei [4], lipase Geotrichum candidum [5], lipase de Candida rugosa [6].... La lipase de Candida cylindracea (qui est l’ancien nom de Candida rugosa a été cristallisée en présence et en l’absence d’inhibiteurs [7]. Il s’agit d’une alpha/beta hydrolase comprenant 11 brins beta entourés par 8 hélices alpha [6]. La triade catalytique est cachée sous un flap constitué de 26 résidus d’aminoacides. Dans le présent travail, nous avons cherché à étudier quelques caractéristiques biochimiques de la lipase de Candida rugosa (CRL qui a été purifiée dans notre laboratoire à partir de la poudre commercialisée.

  8. Lipase biocatalysis for useful biodegradable products

    Energy Technology Data Exchange (ETDEWEB)

    Linko, Y.Y.; Wang, Zhuo Lin; Uosukainen, E.; Seppaelae, J. [Helsinki Univ. of Technology, Espoo (Finland); Laemsae, M. [Raisio Group Oil Milling Industry, Raisio (Finland)

    1996-12-31

    It was shown that lipases can be used as biocatalysts in the production of useful biodegradable compounds such as 1-butyl oleate by direct esterification of butanol and oleic acid to decrease viscosity of biodiesel in winter use. By enzymic transesterification, a mixture of 2-ethyl-1-hexyl esters from rapeseed oil fatty acids can be obtained in good yields for use as a solvent, and of trimethylolpropane esters for use as a lubricant. Finally, it was demonstrated that polyesters with a mass average molar mass in excess of 75,000 g mol{sup -}1 can be obtained by esterification or transesterification by using lipase as biocatalyst. (author) (3 refs.)

  9. The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes.

    Science.gov (United States)

    Rengachari, Srinivasan; Bezerra, Gustavo A; Riegler-Berket, Lina; Gruber, Christian C; Sturm, Christian; Taschler, Ulrike; Boeszoermenyi, Andras; Dreveny, Ingrid; Zimmermann, Robert; Gruber, Karl; Oberer, Monika

    2012-07-01

    Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2Å and in complex with phenylmethylsulfonyl fluoride at 1.8Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Lipase immobilization and production of fatty acid methyl esters from canola oil using immobilized lipase

    International Nuclear Information System (INIS)

    Yuecel, Yasin; Demir, Cevdet; Dizge, Nadir; Keskinler, Buelent

    2011-01-01

    Lipase enzyme from Aspergillus oryzae (EC 3.1.1.3) was immobilized onto a micro porous polymeric matrix which contains aldehyde functional groups and methyl esters of long chain fatty acids (biodiesel) were synthesized by transesterification of crude canola oil using immobilized lipase. Micro porous polymeric matrix was synthesized from styrene-divinylbenzene (STY-DVB) copolymers by using high internal phase emulsion technique and two different lipases, Lipozyme TL-100L ® and Novozym 388 ® , were used for immobilization by both physical adsorption and covalent attachment. Biodiesel production was carried out with semi-continuous operation. Methanol was added into the reactor by three successive additions of 1:4 M equivalent of methanol to avoid enzyme inhibition. The transesterification reaction conditions were as follows: oil/alcohol molar ratio 1:4; temperature 40 o C and total reaction time 6 h. Lipozyme TL-100L ® lipase provided the highest yield of fatty acid methyl esters as 92%. Operational stability was determined with immobilized lipase and it indicated that a small enzyme deactivation occurred after used repeatedly for 10 consecutive batches with each of 24 h. Since the process is yet effective and enzyme does not leak out from the polymer, the method can be proposed for industrial applications. -- Research highlights: → Lipozyme TL-100L and Novozym 388 were immobilized onto micro porous polymeric matrix by both physical adsorption and covalent linking. → Immobilized enzymes were used for synthesis of fatty acid methyl esters by transesterification of canola oil and methanol using semi-continuous operation system. → According to chromatographic analysis, Lipase Lipozyme TL-100L resulted in the highest yield of methyl ester as 92%.

  11. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

    Energy Technology Data Exchange (ETDEWEB)

    Noureddini, H.; Gao, X.; Philkana, R.S. [Nebraska-Lincoln Univ., NE (United States). Dept. of Chemical Engineering

    2005-05-01

    Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 {sup o}C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 {sup o}C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1 h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when subjected to repeated uses. (author)

  12. Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil.

    Science.gov (United States)

    Noureddini, H; Gao, X; Philkana, R S

    2005-05-01

    Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.

  13. Properties Of Lipase (Ec 3.1.1.3) From Different Varieties Of Maize ...

    African Journals Online (AJOL)

    This highactivity was correlated with high speciicity of corn lipase on linoleic acid. Thermal inactivation studies showed that the enzyme was stable up to 50oC and showed rapid inactivation above this temperature. Its optimum temperature was 50oC and the optimum pH, 8. Keywords: Lipase, Enzymes, Maize, Thermal ...

  14. Interfacial Polymerization of Dopamine in a Pickering Emulsion: Synthesis of Cross-Linkable Colloidosomes and Enzyme Immobilization at Oil/Water Interfaces.

    Science.gov (United States)

    Qu, Yanning; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

    2015-07-15

    Colloidosomes are promising carriers for immobilizing enzyme for catalytic purposes in aqueous/organic media. However, they often suffer from one or more problems regarding catalytic performance, stability, and recyclability. Here, we report a novel approach for the synthesis of cross-linkable colloidosomes by the selective polymerization of dopamine at oil/water interfaces in a Pickering emulsion. An efficient enzyme immobilization method was further developed by covalently bonding enzymes to the polydopamine (PDA) layer along with the formation of such colloidosomes with lipase as a model enzyme. In this enzyme system, the PDA layer served as a cross-linking layer and enzyme support for simultaneously enhancing the colloidosomes' stability and improving surface availability of the enzymes for catalytic reaction. It was found that the specific activity of lipases immobilized on the colloidosome shells was 8 and 1.4 times higher than that of free lipase and encapsulated lipase positioned in the aqueous cores of colloidosomes, respectively. Moreover, the immobilized lipases demonstrated excellent operational stability and recyclability, retaining 86.6% of enzyme activity after 15 cycles. It is therefore reasonable to expect that this novel approach for enzyme immobilization has great potential to serve as an important technique for the construction of biocatalytic systems.

  15. Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

    Science.gov (United States)

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

    2011-02-17

    Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

  16. Covalent immobilization of lipases on monodisperse magnetic microspheres modified with PAMAM-dendrimer

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Weiwei [Lanzhou University, State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology (China); Zhang, Yimei [Suzhou Research Academy of North China Electric Power University (China); Hou, Chen; Pan, Duo; He, Jianjun; Zhu, Hao, E-mail: zhuhao07@lzu.edu.cn [Lanzhou University, State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Institute of Biochemical Engineering and Environmental Technology (China)

    2016-02-15

    This paper reported an immobilization of Candida rugosa lipase (CRL) onto PAMAM-dendrimer-grafted magnetic nanoparticles synthesized by a modified solvothermal reduction method. The dendritic magnetic nanoparticles were amply characterized by several instrumental measurements, and the CRL was covalently anchored on the three generation supports with glutaraldehyde as coupling reagent. The amount of immobilized enzyme was up to 150 mg/g support and the factors related with the enzyme activity were investigated. The immobilization of lipase improved their performance in wider ranges of pH and temperature. The immobilized lipase exhibited excellent thermal stability and reusability in comparison with free enzyme and can be reused 10 cycles with the enzymatic activity remained above 90 %. The properties of lipase improved obviously after being immobilized on the dendritic supports. The inactive immobilized lipase could be regenerated with glutaraldehyde and Cu{sup 2+}, respectively. This synthetic strategy was facile and eco-friendly for applications in lipase immobilization.

  17. Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme based decomposition models

    Directory of Open Access Journals (Sweden)

    Daryl L Moorhead

    2013-08-01

    Full Text Available We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013 conducted 14-day laboratory incubations of sugar maple (Acer saccharum or white oak (Quercus alba leaves, mixed with sand (0.4% organic C content or loam (4.1% organic C. They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG, β-N-acetyl-glucosaminidase (NAG, and acid phosphatase (AP activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG versus BG/AP represent relative microbial investments in C (length, and N and P (angle acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles > 45˚. Reductions in vector angles to < 45˚ for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies.

  18. OPTIMASI ISOLASI LIPASE INDIGENOUS BIJI KAKAO (Theobroma cacao L. The Optimizing of Isolation of Cocoa Bean Indogenous Lipase (Theobroma cacao L.

    Directory of Open Access Journals (Sweden)

    I D. G. Mayun Permana

    2012-05-01

    Full Text Available The aim of the research is to optimize the isolation method of cocoa bean lipase. The research is held by determining the position of lipase on cocoa bean, varying extraction medium and isolation process. The result shows that the lipase of cocoa bean is   cytosolic enzyme. The defatting process do not increase the lipase activity. Polyphenols inhibit the lipase activity, so that removal of the polyphenol will increase the activity. Blocking the polyphenol with polyvinilpolypirrolidone (PVPP will also increase the activity.The optimum consentration of PVPP is 8 %. The lipase activity will reach the highest when homogenized for 10 menit at 10,000 rpm. The best medium extraction for lipase isolation is 0.15 M phosphate buffer pH 7.5 containing sucrose 0.6 M and CaCl  1.0 mM.   ABSTRAK Penelitian ini bertujuan untuk mengoptimasi isolasi lipase indigenous biji kakao. Optimasi diawali dengan menentukan keberadaan lipase kemudian optimasi medium ekstraksi dan proses ekstraksi. Hasil penelitian menunjukkan bahwa lipase berada dalam sitosol. Penghilangan lemak tidak meningkatkan aktivitas lipase. Senyawa polifenol menghambat aktivitas lipase dan penghilangan polifenol dapat meningkatkan aktivitas lipase. Polyvinilpolypirrolidone (PVPP dapat menghambat polifenol sehingga dapat meningkatkan aktivitas lipase. Konsentrasi PVPP optimum adalah 8 % dari berat biji kakao. Proses homogenisasi optimum diperoleh dalam waktu 10 menit pada kecepatan 10.000 rpm. Medium ekstraksi untuk isolasi lipase biji kakao terbaik adalah bufer fosfat 0,15 M  dan pH 7,5 yang mengandung sukrosa 0,6 M dan 1,0 mM CaCl .

  19. Bacterial lipases for biotechnological applications

    NARCIS (Netherlands)

    Jaeger, Karl-Erich; Schneidinger, Bernd; Rosenau, Frank; Werner, Michael; Lang, Dietmar; Dijkstra, Bauke W.; Schimossek, Klaus; Zonta, Albin; Reetz, Manfred T.

    1997-01-01

    Lipase genes originating from the Gram-negative bacteria Serrutiu marcescens and Pseudomonus urruginosa were cloned. S. marcescens lipase was overexpressed in Escherichia coli yielding inclusion bodies which were purified and finally refolded to give enzymatically active lipase. The lipase operon of

  20. Lipases as biocatalysts for biodiesel production

    Directory of Open Access Journals (Sweden)

    Ognjanović Nevena D.

    2010-01-01

    Full Text Available Lipases can be used for a variety of biotechnological applications: synthesis of fine chemicals, therapeutics, agrochemicals, cosmetics, flavors, biopolymers and biodiesel. Biodiesel is an alternative fuel for diesel engines that is environmentally acceptable. Conventionally, biodiesel is produced by transesterification of triglycerides and short chain alcohols in the presence of an acid or an alkaline catalyst. There are several problems associated with this kind of production that can be resolved by using lipase as the biocatalyst. The usage of lipases has several advantages over the conventional chemical methods. It is considered as less energy intensive and environmentally friendly. However, there are two main obstacles associated with the effective utilization of lipases in the production of biodiesel. The main one is the cost of the enzyme and its poor stability in the presence of excess alcohol. Several strategies are proposed to overcome these drawbacks: immobilization of lipases, stepwise addition of alcohol, and the usage of novel acyl acceptors and the usage of whole cell biocatalysts.

  1. Monitoring changes in plasma levels of pancreatic and intestinal enzymes in a model of pancreatic exocrine insufficiency--induced by pancreatic duct-ligation--in young pigs.

    Science.gov (United States)

    Lozinska, Liudmyla; Prykhodko, Olena; Sureda, Ester Arévalo; Szwiec, Katarzyna; Podgurniak, Pawel; Pierzynowski, Stefan; Weström, Björn

    2015-03-01

    Plasma levels of pancreatic and intestinal enzymes were measured after pancreatic duct ligation (PDL) to monitor pancreatic exocrine insufficiency (PEI) in a model using young pigs. Five, 6 week-old pigs (10.9±0.2kg), underwent PDL while age-matched, un-operated pigs were used as controls. Plasma levels of immunoreactive cationic trypsinogen (IRCT), amylase, lipase, and diamine oxidase (DAO) activities were analyzed for 48 days after PDL, including 1 week of oral pancreatic enzyme supplementation (PES) with Creon(®). PDL resulted in an arrested body growth and a rapid surge of pancreatic enzymes (IRCT, amylase and lipase) into the plasma. Nine days after PDL, the plasma levels of these pancreatic enzymes had decreased. IRCT then remained below the level in un-operated pigs while amylase only fell below control at 25 days. The intestinally derived marker DAO and plasma protein levels were unaffected by PDL but DAO decreased slightly with time in PEI pigs. One-week of oral PES restored body growth, but had little effect on pancreatic enzyme plasma levels, except for a tendency towards increased DAO. The study showed that PEI developed within 1-2 weeks after PDL and that only IRCT is a reliable plasma enzyme marker for this. The reduced plasma DAO indicated that PEI also affected the intestines, while PES therapy restored growth of the PDL pigs and slightly increased plasma DAO, suggesting an improved intestinal function. Copyright © 2015 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  2. Frozen Microemulsions for MAPLE Immobilization of Lipase

    Directory of Open Access Journals (Sweden)

    Valeria Califano

    2017-12-01

    Full Text Available Candida rugosa lipase (CRL was deposited by matrix assisted pulsed laser evaporation (MAPLE in order to immobilize the enzyme with a preserved native conformation, which ensures its catalytic functionality. For this purpose, the composition of the MAPLE target was optimized by adding the oil phase pentane to a water solution of the amino acid 3-(3,4-dihydroxyphenyl-2-methyl-l-alanine (m-DOPA, giving a target formed by a frozen water-lipase-pentane microemulsion. Fourier transform infrared (FTIR spectroscopy and atomic force microscopy (AFM were used to investigate the structure of MAPLE deposited lipase films. FTIR deconvolution of amide I band indicated a reduction of unfolding and aggregation, i.e., a better preserved lipase secondary structure in the sample deposited from the frozen microemulsion target. AFM images highlighted the absence of big aggregates on the surface of the sample. The functionality of the immobilized enzyme to promote transesterification was determined by thin layer chromatography, resulting in a modified specificity.

  3. Sequential Detection of Thermophilic Lipase and Protease by Zymography.

    Science.gov (United States)

    Kurz, Liliana; Hernández, Zully; Contreras, Lellys M; Wilkesman, Jeff

    2017-01-01

    Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copolymerized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside time and resource saving.

  4. Use of stable emulsion to improve stability, activity, and enantioselectivity of lipase immobilized in a membrane reactor.

    Science.gov (United States)

    Giorno, L; Li, N; Drioli, E

    2003-12-20

    The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity. Copyright 2003 Wiley Periodicals, Inc.

  5. MOLECULAR CLONING AND CHARACTERIZATION OF NOVEL THERMOSTABLE LIPASE FROM SHEWANELLA PUTREFACIENS AND USING ENZYMATIC BIODIESEL PRODUCTION

    Directory of Open Access Journals (Sweden)

    Fahri Akbas

    2015-02-01

    Full Text Available A novel thermostable lipase from Shewanella putrefaciens was identified, expressed in Escherichia coli, characterized and used in biodiesel production. Enzyme characterization was carried out by enzyme assay, SDS-PAGE and other biochemical reactions. The recombinant lipase was found to have a molecular mass of 29 kDa and exhibited lipase activity when Tween 80 was used as the substrate. The purified enzyme showed maximum activity at pH 5.0 and at 80°C. The recombinant lipase was used for the transesterification of canola oil and waste oil. The enzyme retains 50% of its activity at 90°C for 30 minutes. It is also able to retain 20% of its activity even at 100 °C for 20 minutes. These properties of the obtained new recombinant thermostable lipase make it promising as a biocatalyst for industrial processes.

  6. Integrating microbial physiology and enzyme traits in the quality model

    Science.gov (United States)

    Sainte-Marie, Julien; Barrandon, Matthieu; Martin, Francis; Saint-André, Laurent; Derrien, Delphine

    2017-04-01

    Microbe activity plays an undisputable role in soil carbon storage and there have been many calls to integrate microbial ecology in soil carbon (C) models. With regard to this challenge, a few trait-based microbial models of C dynamics have emerged during the past decade. They parameterize specific traits related to decomposer physiology (substrate use efficiency, growth and mortality rates...) and enzyme properties (enzyme production rate, catalytic properties of enzymes…). But these models are built on the premise that organic matter (OM) can be represented as one single entity or are divided into a few pools, while organic matter exists as a continuum of many different compounds spanning from intact plant molecules to highly oxidised microbial metabolites. In addition, a given molecule may also exist in different forms, depending on its stage of polymerization or on its interactions with other organic compounds or mineral phases of the soil. Here we develop a general theoretical model relating the evolution of soil organic matter, as a continuum of progressively decomposing compounds, with decomposer activity and enzyme traits. The model is based on the notion of quality developed by Agren and Bosatta (1998), which is a measure of molecule accessibility to degradation. The model integrates three major processes: OM depolymerisation by enzyme action, OM assimilation and OM biotransformation. For any enzyme, the model reports the quality range where this enzyme selectively operates and how the initial quality distribution of the OM subset evolves into another distribution of qualities under the enzyme action. The model also defines the quality range where the OM can be uptaken and assimilated by microbes. It finally describes how the quality of the assimilated molecules is transformed into another quality distribution, corresponding to the decomposer metabolites signature. Upon decomposer death, these metabolites return to the substrate. We explore here the how

  7. The immobilization of lipase on PVDF-co-HFP membrane

    Science.gov (United States)

    Kayhan, Naciye; Eyüpoǧlu, Volkan; Adem, Şevki

    2016-04-01

    Lipase is an enzyme having a lot of different industrial applications such as biodiesel production, biopolymer synthesis, enantiopure pharmaceutical productions, agrochemicals, etc. Its immobilized form on different substances is more conventional and useful than its free form. Supporting material was prepared using PVDF-co-HFP in laboratory conditions and attached 1,4-diaminobutane (DA) and epichlorohydrin (EPI) ligands to the membrane to immobilize lipase enzyme. The immobilization conditions such as enzyme amount, pH, the concentration of salt, thermal stability and activity were stabilized for our experimental setup. Then, biochemical characterizations were performed on immobilized lipase PVDF-co-HFP regarding optimal pH activity, temperature and thermal stability. Also, the desorption ratios of immobilized enzyme in two different pathway were investigated to confirm immobilization stability for 24 hours.

  8. Monoacylglycerol Lipase Is a Therapeutic Target for Alzheimer's Disease

    Directory of Open Access Journals (Sweden)

    Rongqing Chen

    2012-11-01

    Full Text Available Alzheimer's disease (AD is the most common cause of dementia among older people. There are no effective medications currently available to prevent and treat AD and halt disease progression. Monoacylglycerol lipase (MAGL is the primary enzyme metabolizing the endocannabinoid 2-arachidonoylglycerol in the brain. We show here that inactivation of MAGL robustly suppressed production and accumulation of β-amyloid (Aβ associated with reduced expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1 in a mouse model of AD. MAGL inhibition also prevented neuroinflammation, decreased neurodegeneration, maintained integrity of hippocampal synaptic structure and function, and improved long-term synaptic plasticity, spatial learning, and memory in AD animals. Although the molecular mechanisms underlying the beneficial effects produced by MAGL inhibition remain to be determined, our results suggest that MAGL, which regulates endocannabinoid and prostaglandin signaling, contributes to pathogenesis and neuropathology of AD, and thus is a promising therapeutic target for the prevention and treatment of AD.

  9. Synthetic applications of immobilized lipases in polymers

    International Nuclear Information System (INIS)

    Dalla-Vecchia, Roberto; Nascimento, Maria da Graca; Soldi, Valdir

    2004-01-01

    The application of biocatalysis is a promising field related to new technologies for organic synthesis. The development of immobilization techniques is very important due to the multiple or repetitive use of a single batch of enzymes and the ability to stop the reaction rapidly, at any stage, by removing the enzymes. In most cases, after immobilization, enzymes and microorganisms maintain or even increase their activity and stability. This work presents an overview of the common methods for lipase immobilization in polymers and applications of these systems to obtain compounds of synthetic interest. (author)

  10. O emprego de lipases como agentes de resolução cinética de enantiômeros em síntese orgânica: aspectos gerais sobre a influência do solvente

    Directory of Open Access Journals (Sweden)

    Costa Valentim Emilio Uberti

    1999-01-01

    Full Text Available In organic synthesis, lipases are the most frequently used biocatalysts. They are efficient stereoselective catalysts in the kinetic resolution of a wide variety of chiral compounds. The discovery that enzymes possess catalytic activity in organic solvents has made it possible to address the question of reaction medium influence on enzymatic specificity. Perhaps the most exciting and significant development in this emerging area is the discovery that enzyme specificity, in particular enantioselectivity, can be affected by changing from one organic solvent to another. This article discusses the scope and possible mechanistic models of this phenomenon in hydrolases, specially lipases, as well as directions of future research in the area.

  11. Synthesis Biodiesel from Palm Oil Through Interesterification Using Imobilized Lipase Enzym as Catalyst: The Effect of Amount of Biocatalyst, Mole Ratio of Reactan, Temperature to Yield

    Directory of Open Access Journals (Sweden)

    Melina Widyawati

    2014-10-01

    Full Text Available Biodiesel usually synthesized by transesterification of triglyceride and alcohol by addition of acid or base catalyst so there is could produce a waste of chemical process. Alternative process is by using biocatalyst such as enzyme to synthesize biodiesel without chemical process waste. In this research, synthesis of biodiesel from Crude Palm Oil (CPO that through the process of degumming and methyl acetate as acyl donor has been investigated with using Lipozyme as biocatalyst. Variables in this research are amount of biocatalyst, mole ratio of reactant, and temperature, and its respond to the yield conversion of biodiesel that presented by using Response Surface Methodology (RSM. Yield raging from 15% - 68% were achived during 10 hours reaction time. The results showed that the most influential variable is amount of biocatalyst.

  12. Enzymatic production of biodiesel from canola oil using immobilized lipase

    Energy Technology Data Exchange (ETDEWEB)

    Dizge, Nadir; Keskinler, Buelent [Department of Environmental Engineering, Gebze Institute of Technology, Gebze 41400 (Turkey)

    2008-12-15

    In the present work, a novel method for immobilization of lipase within hydrophilic polyurethane foams using polyglutaraldehyde was developed for the immobilization of Thermomyces lanuginosus lipase to produce biodiesel with canola oil and methanol. The enzyme optimum conditions were not affected by immobilization and the optimum pH for free and immobilized enzyme were 6, resulting in 80% immobilization yield. Using the immobilized lipase T. lanuginosus, the effects of enzyme loading, oil/alcohol molar ratio, water concentration, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 20 g of refined canola oil were: 430 {mu}g lipase, 1:6 oil/methanol molar ratio, 0.1 g water and 40 C for the reactions with methanol. Maximum methyl esters yield was 90% of which enzymatic activity remained after 10 batches, when tert-butanol was adopted to remove by-product glycerol during repeated use of the lipase. The immobilized lipase proved to be stable and lost little activity when was subjected to repeated uses. (author)

  13. Karakterisasi ekstrak kasar lipase Rhizopus stolonifer UICC 137

    Directory of Open Access Journals (Sweden)

    Sri Sumiarsih

    2001-12-01

    Full Text Available There is an increasing commercial interest in enzymatic production of biologically active component, because there are a number of well-known advantages compared to chemical synthesis. One of the most valuable synthetic features of enzyme is their ability to discriminate between enantiomers of racemic substrates. Lipase have become of great interest to the chemical industries wing their usefulness in both hydrolytic and synthesis reactions. The aim of this work was to study the production of lipase by Rhizopus stolonifer UICC 137, and determine the crude lipase preparation characteristics. The lipolytic activity was determined by titrimetric method toward oil-arabic gum emultion as a substrate. The strain produced lipase at appreciable lipolytic when cultivated for 72 hours in medium containing 3% glucose and 1% olive oil. Our data suggest that the strain produced lipase since the exponential phase of its growth. Lipase with optimum lipolytic activity was obtained at late stationary phase. The optimum condition for lipolytic activity measurement were pH of 7.5 and temperature 37oC, the crude enzyme had a specific activity 20.2 unit/ mg protein, the Vmax was 15.1 mol/ min and KM was 12.5 mg/ ml. The crude enzyme retained 79.9%, 68.0% and 52.6% of its lipolytic activity, when incubated for 90 minutes at temperature of 40, 50, and 60oC respectively.

  14. Lipase applications in oil hydrolysis with a case study on castor oil: a review.

    Science.gov (United States)

    Goswami, Debajyoti; Basu, Jayanta Kumar; De, Sirshendu

    2013-03-01

    Lipase (triacylglycerol acylhydrolase) is a unique enzyme which can catalyze various types of reactions such as hydrolysis, esterification, alcoholysis etc. In particular, hydrolysis of vegetable oil with lipase as a catalyst is widely studied. Free lipase, lipase immobilized on suitable support, lipase encapsulated in a reverse micelle and lipase immobilized on a suitable membrane to be used in membrane reactor are the most common ways of employing lipase in oil hydrolysis. Castor oil is a unique vegetable oil as it contains high amounts (90%) of a hydroxy monounsaturated fatty acid named ricinoleic acid. This industrially important acid can be obtained by hydrolysis of castor oil. Different conventional hydrolysis processes have certain disadvantages which can be avoided by a lipase-catalyzed process. The degree of hydrolysis varies widely for different lipases depending on the operating range of process variables such as temperature, pH and enzyme loading. Immobilization of lipase on a suitable support can enhance hydrolysis by suppressing thermal inactivation and estolide formation. The presence of metal ions also affects lipase-catalyzed hydrolysis of castor oil. Even a particular ion has different effects on the activity of different lipases. Hydrophobic organic solvents perform better than hydrophilic solvents during the reaction. Sonication considerably increases hydrolysis in case of lipolase. The effects of additives on the same lipase vary with their types. Nonionic surfactants enhance hydrolysis whereas cationic and anionic surfactants decrease it. A single variable optimization method is used to obtain optimum conditions. In order to eliminate its disadvantages, a statistical optimization method is used in recent studies. Statistical optimization shows that interactions between any two of the following pH, enzyme concentration and buffer concentration become significant in presence of a nonionic surfactant named Span 80.

  15. Structure of product-bound SMG1 lipase: active site gating implications.

    Science.gov (United States)

    Guo, Shaohua; Xu, Jinxin; Pavlidis, Ioannis V; Lan, Dongming; Bornscheuer, Uwe T; Liu, Jinsong; Wang, Yonghua

    2015-12-01

    Monoacylglycerol and diacylglycerol lipases are industrially interesting enzymes, due to the health benefits that arise from the consumption of diglycerides compared to the traditional triglyceride oils. Most lipases possess an α-helix (lid) directly over the catalytic pocket which regulates the activity of the enzyme. Generally, lipases exist in active and inactive conformations, depending on the positioning of this lid subdomain. However, lipase SMG1, a monoacylglycerol and diacylglycerol specific lipase, has an atypical activation mechanism. In the present study we were able to prove by crystallography, in silico analysis and activity tests that only two positions, residues 102 and 278, are responsible for a gating mechanism that regulates the active and inactive states of the lipase, and that no significant structural changes take place during activation except for oxyanion hole formation. The elucidation of the gating effect provided data enabling the rational design of improved lipases with 6-fold increase in the hydrolytic activity toward diacylglycerols, just by providing additional substrate stabilization with a single mutation (F278N or F278T). Due to the conservation of F278 among the monoacylglycerol and diacylglycerol lipases in the Rhizomucor miehei lipase-like family, the gating mechanism described herein might represent a general mechanism applicable to other monoacylglycerol and diacylglycerol lipases as well. Database: Structural data are available in the Protein Data Bank under the accession numbers 4ZRE (F278D mutant) and 4ZRD (F278N mutant). © 2015 FEBS.

  16. Evolutionary history of versatile-lipases from Agaricales through reconstruction of ancestral structures.

    Science.gov (United States)

    Barriuso, Jorge; Martínez, María Jesús

    2017-01-03

    Fungal "Versatile carboxylic ester hydrolases" are enzymes with great biotechnological interest. Here we carried out a bioinformatic screening to find these proteins in genomes from Agaricales, by means of searching for conserved motifs, sequence and phylogenetic analysis, and three-dimensional modeling. Moreover, we reconstructed the molecular evolution of these enzymes along the time by inferring and analyzing the sequence of ancestral intermediate forms. The properties of the ancestral candidates are discussed on the basis of their three-dimensional structural models, the hydrophobicity of the lid, and the substrate binding intramolecular tunnel, revealing all of them featured properties of these enzymes. The evolutionary history of the putative lipases revealed an increase on the length and hydrophobicity of the lid region, as well as in the size of the substrate binding pocket, during evolution time. These facts suggest the enzymes' specialization towards certain substrates and their subsequent loss of promiscuity. These results bring to light the presence of different pools of lipases in fungi with different habitats and life styles. Despite the consistency of the data gathered from reconstruction of ancestral sequences, the heterologous expression of some of these candidates would be essential to corroborate enzymes' activities.

  17. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was

  18. Bio F1B hamster: a unique animal model with reduced lipoprotein lipase activity to investigate nutrient mediated regulation of lipoprotein metabolism

    Directory of Open Access Journals (Sweden)

    Cornish Marion L

    2007-12-01

    lipoprotein lipase activity and mRNA levels contribute to varied response of these hamsters to dietary fat, highlighting the importance of genetic background in the regulation of lipid and lipoprotein metabolism by dietary fats. Bio F1B hamster may prove to be an important animal model to investigate nutrient mediated regulation of metabolic parameters under lipoprotein lipase deficiency.

  19. Modeling metabolic response to changes of enzyme amount in ...

    African Journals Online (AJOL)

    Jane

    2010-10-11

    Oct 11, 2010 ... In this work, we first introduced the enzyme parameter (ɑ) into the kinetic equations and consequently established an in silico glycolysis model of Saccharomyces cerevisiae in XML format (Figure 1), based on the work of Hynn et al. (2001). Equation 1 shows how the ɑis introduced into the kinetic equation.

  20. Modelling non-redox enzymes: Anaerobic and aerobic acetylene ...

    Indian Academy of Sciences (India)

    Administrator

    Modelling non-redox enzymes: Anaerobic and aerobic acetylene hydratase. SABYASACHI SARKAR. Department of Chemistry, Indian Institute of Technology, Kanpur 208 016,. India. Acetaldehyde is the first metabolite produced during acetylene degradation by bacteria either aerobically or anaerobically. Conversion of ...

  1. Microbial lipases with interest in biotechnology and infectious diseases: isolation, characterization and inhibition by natural substances

    OpenAIRE

    Ruiz Rueda, Cristian

    2005-01-01

    Lipases are carboxylic ester hydrolases which act on acylglycerols to liberate fatty acids and glycerol. These enzymes are currently attracting an enormous attention because they are among the most versatile and widely used enzymes in biotechnological applications and due to their unique properties. Moreover, these enzymes and their inhibitors have a high pharmacological interest because some microbial lipases can act as virulence factors in several infectious diseases. Therefore, the general...

  2. Síntese do butirato de n-butila empregando lipase microbiana imobilizada em copolímero de estireno-divinilbenzeno Synthesis of butyl butyrate by microbial lipase immobilized onto styrene-divinylbenzene copolymer

    Directory of Open Access Journals (Sweden)

    Pedro Carlos de Oliveira

    2000-10-01

    Full Text Available This work investigates the reaction parameters of an immobilized lipase in the esterification reaction of n-butanol and butyric acid. Microbial lipase from Candida rugosa was immobilized onto styrene-divinylbenzene copolymer (STY-DVB and subsequently introduced in an organic medium containing substrates in appropriate concentrations. Heptane was selected as solvent on the basis of its compatibility with the resin and the enzyme. The influence of molar ratio of acid to alcohol, amount of immobilized lipase and temperature on the butyl butyrate formation was determined. The results were compared with those achieved with free lipase and Lipozyme (commercially immobilized lipase under the same operational conditions.

  3. Lipase from a Brazilian strain of Penicillium citrinum.

    Science.gov (United States)

    Pimentel, M C; Krieger, N; Coelho, L C; Fontana, J O; Melo, E H; Ledingham, W M; Lima Filho, J L

    1994-10-01

    A lipases (glycerol ester hydrolases E. C. 3.1.1.3) from a brazilian strain of Penicillium citrinum has been investigated. When the microorganism was cultured in the simple medium (1.0% olive oil and 0.5% yeast extract), using olive oil in as carbon source in the inocula, the enzyme extracted showed maximum activity (409 IU/mL). In addition, decrease of yeast extract concentration also reduces the lipase activity. Nevertheless, when yeast extract was replaced by ammonium sulfate, no activity was detected. Purification by precipitation with ammonium sulfate showed best activity in the 40-60% fraction. The optimum temperature for enzyme activity was found in the range of 34-37 degrees C. However, after 30 min at 60 degrees C, the enzyme was completely inactivated. The enzyme showed optimum at pH 8.0. The dried concentrated fraction (after dialysis and lyophilization) maintained its lipase activity at room temperature (28 degrees C) for 8 mo. This result in lipase stability suggests an application of lipases from P. citrinum in detergents and other products that require a high stability at room temperature.

  4. Biodiesel production from microalgae oil catalyzed by a recombinant lipase.

    Science.gov (United States)

    Huang, Jinjin; Xia, Ji; Jiang, Wei; Li, Ying; Li, Jilun

    2015-03-01

    A recombinant Rhizomucor miehei lipase was constructed and expressed in Pichia pastoris. The target enzyme was termed Lipase GH2 and it can be used as a free enzyme for catalytic conversion of microalgae oil mixed with methanol or ethanol for biodiesel production in an n-hexane solvent system. Conversion rates of two major types of biodiesel, fatty acid methyl ester (FAME) and fatty acid ethyl ester (FAEE), reached maximal values (>90%) after 24h. The process of FAME production is generally more simple and economical than that of FAEE production, even though the two processes show similar conversion rates. In spite of the damaging effect of ethanol on enzyme activity, we successfully obtained ethyl ester by the enzymatic method. Our findings indicate that Lipase GH2 is a useful catalyst for conversion of microalgae oil to FAME or FAEE, and this system provides efficiency and reduced costs in biodiesel production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Infrared Analysis Of Enzymes Adsorbed Onto Model Surfaces

    Science.gov (United States)

    Story, Gloria M.; Rauch, Deborah S.; Brode, Philip F.; Marcott, Curtis A.

    1989-12-01

    The adsorption of the enzymes, subtilisin BPN' and lysozyme, onto model surfaces was examined using attenuated total reflectance (ATR) infrared (IR) spectroscopy. Using a cylindrical internal reflection (CIRcle) cell with a Germanium (Ge) internal reflection element (IRE), model hydrophilic surfaces were made by plasma cleaning the IRE and model hydrophobic surfaces were made by precoating the IRE with a thin film of polystyrene. Gas chromatography (GC)-IR data collection software was used to monitor adsorption kinetics during the first five minutes after injection of the enzyme into the CIRcle cell. It was found that for both lysozyme and BPN', most of the enzyme that was going to adsorb onto the model surface did so within ten seconds after injection. Nearly an order-of-magnitude more BPN' adsorbed on the hydrophobic Ge surface than the hydrophilic one, while lysozyme adsorbed somewhat more strongly to the hydrophilic Ge surface. Overnight, the lysozyme layer continued to increase in thickness, while BPN' maintained its initial coverage. The appearance of carboxylate bands in some of the adsorbed BPN' spectra suggests the occurrence of peptide bond hydrolysis. A Au/Pd coating on the CIRcle cell o-rings had a significant effect on the adsorption of BPN'. (This coating was applied in an attempt to eliminate interfering Teflon absorption bands.) An apparent electrochemical reaction occurred, involving BPN', Ge, Au/Pd, and the salt solution used to stabilize BPN'. The result of this reaction was enhanced adsorption of the enzyme around the coated o-rings, etching of the Ge IRE at the o-ring site, and some autolysis of the enzyme. No such reaction was observed with lysozyme.

  6. Highly regioselective synthesis of undecylenic acid esters of purine nucleosides catalyzed by Candida antarctica lipase B.

    Science.gov (United States)

    Gao, Wen-Li; Li, Ning; Zong, Min-Hua

    2011-11-01

    Regioselective undecylenoylation of purine nucleosides as potential dual prodrugs was achieved by Candida antarctica lipase B using adenosine as a model reactant. The optimum organic solvent, molar ratio of vinyl ester to nucleoside, enzyme dosage, reaction temperature and molecular sieve amount were anhydrous THF, 5:1, 20 U/ml, 45°C and 75 mg/ml, respectively. Under the optimum conditions, the initial reaction rate, yield and 5'-regioselectivity were 1.1 mM/h, 90% and >99%, respectively. The enzymatic acylation of various nucleosides furnished the desired 5'-ester derivatives with the yields of 60-95% and 5'-regioselectivities of >99%. In addition, the lipase displayed excellent operational stability in THF, and retained 96% of its initial activity after reused for five batches.

  7. Lipase nanogel catalyzed transesterification in anhydrous dimethyl sulfoxide.

    Science.gov (United States)

    Ge, Jun; Lu, Diannan; Wang, Jun; Liu, Zheng

    2009-06-08

    The present work showed that Candida rugosa lipase, which is inactive in anhydrous dimethyl sulfoxide (DMSO), has been granted its original catalytic activity and greatly enhanced stability when encapsulated into a polyacrylamide nanogel. The molecular simulation and structural analysis suggested that the polyacrylamide nanogel shielded the extraction of essential water and maintained the native configuration of encapsulated lipase in anhydrous DMSO at an elevated temperature. The electron and fluorescence microscopy showed that the lipase nanogel would be well dispersed in anhydrous DMSO where its native counterpart aggregated. The encapsulated lipase behaved as a stable catalyst for transesterification between dextran and vinyl decanoate in anhydrous DMSO at 60 degrees C for 240 h and yielded a dextran-based polymeric surfactant with regioselectivity toward the C-2 hydroxyl group in the glucopyranosyl unit of dextran. All these indicated a high potential of enzyme nanogel for nonaqueous biocatalysis.

  8. Extraction of lipase and protease and characterization of activated sludge from pulp and paper industry.

    Science.gov (United States)

    Karn, Santosh Kr; Kumar, Pradeep; Pan, Xiangliang

    2013-01-01

    Hydrolytic enzymes released by the microorganisms in activated sludge are responsible for the organic matter degradation there; however, the optimal extraction procedure of this valuable resource has not been well established until now. In this study protease and lipase were extracted from activated sludge using ultrasound disintegration combined with a nonionic detergent (Triton X-100) and cation-exchange resin (CER) in combination for the extraction of protease and lipase. It was observed that the concentration of 0.1% and 1% Triton X-100 has a strong influence for the extraction of lipase and protease respectively. Closer study of the enzyme extraction process is essential for different enzymes from activated sludge process.

  9. Role of spacer length in interaction between novel gemini imidazolium surfactants and Rhizopus oryzae lipase.

    Science.gov (United States)

    Adak, Sunita; Datta, Sougata; Bhattacharya, Santanu; Banerjee, Rintu

    2015-11-01

    An insight into the effects of new ionic liquid-type gemini imidazolium cationic surfactants on the structure and function of the lipases is of prime importance for their potential application. Changes in the activity, stability and structure of Rhizopus oryzae lipase in the presence of novel gemini surfactants, [C16-3-C16im]Br2 and [C16-12-C16im]Br2 were probed in the present study. Surfactant with shorter spacer length, [C16-3-C16im]Br2 was found to be better in improving the hydrolytic activity and thermal stability of the lipase. For both the surfactants, activation was concentration dependent. CD spectroscopy results showed a decrease in α-helix and an increase in β-sheet content in the presence of these surfactants. A higher structural change observed in presence of [C16-12-C16im]Br2 correlated with lower enzyme activity. Isothermal titration calorimetric studies showed the binding to be spontaneous in nature based on sequential two site binding model. The forces involved in binding were found to differ for the two surfactants proving that the spacer length is an important factor which governs the interaction. These surfactants could be used as promising components both in enzyme modification and media engineering for attaining the desired goals in biocatalytic reactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Activity and Spatial Distribution of Candida antarctica Lipase B Immobilized on Macroporous Organic Polymeric Adsorbents

    DEFF Research Database (Denmark)

    Nielsen, Anne Veller Friis; Andric, Pavle; Munk Nielsen, Per

    2014-01-01

    A systematic study of the influence of carrier particle size (500 − 850 μ m) and enzyme load (26 200 − 66 100 lipase activity units (LU)/g dry carrier) on the content and activity of Candida antarctica lipase B (CALB) immobilized by adsorption onto macroporous poly(methyl methacrylate) (PMM...

  11. Immobilization of lipases in PSS/PEO blends and applications in esters synthesis

    International Nuclear Information System (INIS)

    Vecchia, Roberto D.; Nascimento, Maria G.; Soldi, Valdir

    2001-01-01

    Various lipases were immobilized in PSS/PEO blends and used as bio catalysts in the esterification reaction of lauric acid with n-pentanol, in hexane as a solvent for 24 h at 35 deg C. The best results in the ester conversion, were obtained by using lipase from Rhryzopus oryzae immobilized in PSS/PEO 80:20 blend. The data are in agreement with DSC and TGA values, which showed that these systems (blend/lipase) were very stable with low mass loss. No product was obtained by using lipase FAP-15 immobilized in PSS film , showing the strong influence of the polymer on enzyme activity. (author)

  12. Maximization of Intracellular Lipase Production in a Lipase-Overproducing Mutant Derivative of Rhizopus oligosporus DGM 31: A Kinetic Study

    Directory of Open Access Journals (Sweden)

    Tehreema Iftikhar

    2008-01-01

    Full Text Available Regulation and maximization of lipase production in a mutant derivative of R. oligosporus has been investigated using different substrates, inoculum sizes, pH of the medium, temperature, and nitrogen sources in shake flask experiments and batch fermentation in a fermentor. The production of intracellular lipase was improved 3 times following medium optimization involving one-at-a-time approach and aeration in the fermentor. Interestingly, intracellular lipase was poorly induced by oils, instead its production was induced by sugars, mainly starch, lactose, sucrose, xylose, glucose and glycerol. Dependent variables studied were cell mass, lipase activity, lipase yield, lipase specific and volumetric rate of formation. It was confirmed that lipase production in the derepressed mutant is sufficiently uncoupled from catabolite repression. The results of average specific productivities at various temperatures worked out according to the Arrhenius equation revealed that mutation decreased the magnitude of enthalpy and entropy demand in the inactivation equilibrium during product formation, suggesting that mutation made the metabolic network of the organism thermally more stable. The highest magnitudes of volumetric productivity (QP=490 IU/(L·h and other product attributes of lipase formation occurring on optimized medium in the fermentor are greater than the values reported by other workers. The purified enzyme is monomeric in nature and exhibits stability up to 80 °C and pH=6.0–8.0. Activation energy, enthalpy and entropy of catalysis at 50 °C, and magnitudes of Gibbs free energy for substrate binding, transition state stabilization and melting point indicated that this lipase is highly thermostable.

  13. OPTIMASI KONDISI PURIFIKASI PARSIAL LIPASE Aspergillus niger 65I6 SOLID STATE FERMENTATION (SSF) PADA MEDIUM BUNGKIL JARAK SEBAGAI SUMBER BELAJAR PENGANTAR MIKROBIOLOGI

    OpenAIRE

    Maulina Nurikasari

    2012-01-01

    Extracellular lipase is an enzyme that catalyzes the hydrolysis reaction of triglycerides into free fatty acids and glycerol and synthesis reaction of esters. Lipase can be produced by microbes include bacteria, fungi and yeasts. Lipase by Aspergillus niger 65I6 produced in the medium of Jatropha cake using solid-state fermentation has a different character of the lipase produced by other microorganisms. The aims of this research were to determine the optimum condition partially purified lipa...

  14. Biochemical Properties of a New Cold-Active Mono- and Diacylglycerol Lipase from Marine Member Janibacter sp. Strain HTCC2649

    Directory of Open Access Journals (Sweden)

    Dongjuan Yuan

    2014-06-01

    Full Text Available Mono- and di-acylglycerol lipase has been applied to industrial usage in oil modification for its special substrate selectivity. Until now, the reported mono- and di-acylglycerol lipases from microorganism are limited, and there is no report on the mono- and di-acylglycerol lipase from bacteria. A predicted lipase (named MAJ1 from marine Janibacter sp. strain HTCC2649 was purified and biochemical characterized. MAJ1 was clustered in the family I.7 of esterase/lipase. The optimum activity of the purified MAJ1 occurred at pH 7.0 and 30 °C. The enzyme retained 50% of the optimum activity at 5 °C, indicating that MAJ1 is a cold-active lipase. The enzyme activity was stable in the presence of various metal ions, and inhibited in EDTA. MAJ1 was resistant to detergents. MAJ1 preferentially hydrolyzed mono- and di-acylglycerols, but did not show activity to triacylglycerols of camellia oil substrates. Further, MAJ1 is low homologous to that of the reported fungal diacylglycerol lipases, including Malassezia globosa lipase 1 (SMG1, Penicillium camembertii lipase U-150 (PCL, and Aspergillus oryzae lipase (AOL. Thus, we identified a novel cold-active bacterial lipase with a sn-1/3 preference towards mono- and di-acylglycerides for the first time. Moreover, it has the potential, in oil modification, for special substrate selectivity.

  15. Modeling cutinase enzyme regulation in polyethylene terepthalate plastic biodegradation

    Energy Technology Data Exchange (ETDEWEB)

    Apri, M., E-mail: m.apri@math.itb.ac.id; Silmi, M. [Department of Mathematics, Institut Teknologi Bandung, Jalan Ganeca 10 Bandung, 40132 (Indonesia); Heryanto, T. E.; Moeis, M. R. [School of Life Sciences and Technology, Institut Teknologi Bandung, Jalan Ganeca 10 Bandung, 40132 (Indonesia)

    2016-04-06

    PET (Polyethylene terephthalate) is a plastic material that is commonly used in our daily life. The high production of PET and others plastics that can be up to three hundred million tons per year, is not matched by its degradation rate and hence leads to environmental pollution. To overcome this problem, we develop a biodegradation system. This system utilizes LC Cutinase enzyme produced by engineered escherichia coli bacteria to degrade PET. To make the system works efficaciously, it is important to understand the mechanism underlying its enzyme regulation. Therefore, we construct a mathematical model to describe the regulation of LC Cutinase production. The stability of the model is analyzed. We show that the designated biodegradation system can give an oscillatory behavior that is very important to control the amount of inclusion body (the miss-folded proteins that reduce the efficiency of the biodegradation system).

  16. Modeling cutinase enzyme regulation in polyethylene terepthalate plastic biodegradation

    Science.gov (United States)

    Apri, M.; Silmi, M.; Heryanto, T. E.; Moeis, M. R.

    2016-04-01

    PET (Polyethylene terephthalate) is a plastic material that is commonly used in our daily life. The high production of PET and others plastics that can be up to three hundred million tons per year, is not matched by its degradation rate and hence leads to environmental pollution. To overcome this problem, we develop a biodegradation system. This system utilizes LC Cutinase enzyme produced by engineered escherichia coli bacteria to degrade PET. To make the system works efficaciously, it is important to understand the mechanism underlying its enzyme regulation. Therefore, we construct a mathematical model to describe the regulation of LC Cutinase production. The stability of the model is analyzed. We show that the designated biodegradation system can give an oscillatory behavior that is very important to control the amount of inclusion body (the miss-folded proteins that reduce the efficiency of the biodegradation system).

  17. Modeling cutinase enzyme regulation in polyethylene terepthalate plastic biodegradation

    International Nuclear Information System (INIS)

    Apri, M.; Silmi, M.; Heryanto, T. E.; Moeis, M. R.

    2016-01-01

    PET (Polyethylene terephthalate) is a plastic material that is commonly used in our daily life. The high production of PET and others plastics that can be up to three hundred million tons per year, is not matched by its degradation rate and hence leads to environmental pollution. To overcome this problem, we develop a biodegradation system. This system utilizes LC Cutinase enzyme produced by engineered escherichia coli bacteria to degrade PET. To make the system works efficaciously, it is important to understand the mechanism underlying its enzyme regulation. Therefore, we construct a mathematical model to describe the regulation of LC Cutinase production. The stability of the model is analyzed. We show that the designated biodegradation system can give an oscillatory behavior that is very important to control the amount of inclusion body (the miss-folded proteins that reduce the efficiency of the biodegradation system).

  18. Lipases from the genus Rhizopus: Characteristics, expression, protein engineering and application.

    Science.gov (United States)

    Yu, Xiao-Wei; Xu, Yan; Xiao, Rong

    2016-10-01

    Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Developing an Enzyme Mediated Soil Organic Carbon Decomposition Model

    Science.gov (United States)

    Mayes, M. A.; Post, W. M.; Wang, G.; Jagadamma, S.; Steinweg, J. M.; Schadt, C. W.

    2012-12-01

    We developed the Microbial-ENzyme-mediated Decomposition (MEND) model in order to mechanistically model the decomposition of soil organic carbon (C). This presentation is an overview of the concept and development of the model and of the design of complementary lab-scale experiments. The model divides soil C into five pools of particulate, mineral-associated, dissolved, microbial, and enzyme organic C (Wang et al. 2012). There are three input types - cellulose, lignin, and dissolved C. Decomposition is mediated via microbial extracellular enzymes using the Michaelis-Menten equation, resulting in the production of a common pool of dissolved organic C. Parameters for the Michaelis-Menten equation are obtained through a literature review (Wang and Post, 2012a). The dissolved C is taken up by microbial biomass and proportioned according to microbial maintenance and growth, which were recalculated according to Wang and Post (2012b). The model allows dissolved C to undergo adsorption and desorption reactions with the mineral-associated C, which was also parameterized based upon a literature review and complementary laboratory experiments. In the lab, four 14C-labeled substrates (cellulose, fatty acid, glucose, and lignin-like) were incubated with either the particulate C pool, the mineral-associated C pool, or to bulk soils. The rate of decomposition was measured via the production of 14CO2 over time, along with incorporation into microbial biomass, production of dissolved C, and estimation of sorbed C. We performed steady-state and dynamic simulations and sensitivity analyses under temperature increases of 1-5°C for a period of 100 y. Simulations indicated an initial decrease in soil organic C consisting of both cellulose and lignin pools. Over longer time intervals (> 6 y), however, a shrinking microbial population, a concomitant decrease in enzyme production, and a decrease in microbial carbon use efficiency together decreased CO2 production and resulted in greater

  20. Isolation of a lipase-producing Trichosporon spp and enzyme extraction by two-phase aqueous system Isolamento de Trichosporon spp produtor de lipase e extração enzimática pelo sistema bifásico aquoso

    Directory of Open Access Journals (Sweden)

    Juliana A. Santos

    2007-03-01

    Full Text Available A lipase-producing yeast strain isolated from crude cheese and identified as Trichosporon spp produced 7.3 U/mL (59.3 U/µg after 72h of cultivation. Lipase showed optimum activity at pH 7.0-8.0 and 45-50ºC. Extraction by the two-phase aqueous system (PEG-phosphate salts showed an elevated recuperation (99.8% of enzymatic activity in the PEG phase.Uma levedura produtora de lipase isolada de queijo coalho e identificada como Trichosporon spp produziu 7,3 U/mL (59,3 U/µg após 72h de cultivo. A lipase mostrou atividade ótima em pH 7,0-8,0 e temperatura ótima entre 45-50ºC. Extração pelo sistema PEG - sais de fosfato apresentou 99,8% de recuperação da atividade enzimática na fase PEG.

  1. Application of lipase technology for transesterification of fatty acid ester

    Directory of Open Access Journals (Sweden)

    JOKO SULISTYO

    2005-07-01

    Full Text Available We have reported the potency of microbial extracellular enzyme for synthesis of fatty acid ester. Further investigation was aimed to study capacity of the enzyme on bioprocess of crude palm oil by transesterification of saturated fatty acid to fatty acid ester. We have studied some lipases from culture filtrate of Candida rugosa FM-9301, Bacillus subtilis FM-9101 and Pseudomonas aerogenes FM-9201, which were preincubated in a medium containing olive oil as inducers, using a shaker under conditions that allowed for lipase production at pH 4.5-6.5 and room temperature for 5 days. Those strains shown different activities during the hydrolysis of substrates, which resulted in decreasing or increasing free fatty acids those, were liberated from media containing crude palm oil and organic solvents. The optimal transesterification condition was at temperature of 45-50C and at pH 4.5 for C. rugosa and pH 6.0 to 7.0 for P. aerogenes and B. subtilis. Under the enzyme concentration of 50% (v/v, the transesterification was rapidly occurred, while at the concentration of 20% (v/v the enzymatically biosynthesis required longer incubation period. The substrates incubated with C. rugosa lipase exhibited higher linoleic and linolenic acid (7.16 and 2.15%, respectively, than that of B. subtilis lipase (4.85% and 1.43%, respectively, while P. aerogenes lipase (3.73% and 1.11%, respectively.

  2. HIDROLISIS MINYAK KELAPA OLEH ENZIM LIPASE DARI KENTOS KELAPA

    Directory of Open Access Journals (Sweden)

    Moh. Su'i

    2012-05-01

    Full Text Available Hydrolysis of Coconut Oil by Lipase Enzyme from Coconut Houstorium ABSTRACT Penelitian ini mempelajari jenis dan jumlah asam lemak yang dihasilkan dari hidrolisis minyak kelapa menggunakan enzim lipase dari kentos kelapa. Minyak kelapa dihidrolisis oleh enzim lipase kentos selama 30, 60, 9, dan 120 menit. Selanjutnya jenis asam lemak yang dihasilkan dan jumlahnya dianalisa. Hasil penelitian menunjukkan bahwa, hidrolisis selama 90 menit menghasilkan asam lemak paling tinggi yaitu sebesar 40,20 %. Jenis asam lemak yang paling banyak dihidrolisis adalah asam laurat sebesar 21,23 %, kemudian asam miristat 7,62 %, dan asam kaprat 3,06 % dari total asam lemak dalam minyak kelapa. ABSTRACT This research learned the kind and amount of fatty acid that was produced from coconut oil hydrolyzed by lipase enzyme from coconut houstorium. Coconut oil was hydrolyzed by lipase enzyme from coconut houstorium for 30, 60, 90, and 120 minutes. The kind and amount of fatty acid obtained from hidrolization were measured. The result showed that hidrolization for 90 minutes produced the highest fatty acid 40,20 %. The highest fatty acid was lauric acid with 21,229 %, furthermore miristic acid 7,615 %, and capric acid 3,062 %.

  3. Model for Quantitative Evaluation of Enzyme Replacement Treatment

    Directory of Open Access Journals (Sweden)

    Radeva B.

    2009-12-01

    Full Text Available Gaucher disease is the most frequent lysosomal disorder. Its enzyme replacement treatment was the new progress of modern biotechnology, successfully used in the last years. The evaluation of optimal dose of each patient is important due to health and economical reasons. The enzyme replacement is the most expensive treatment. It must be held continuously and without interruption. Since 2001, the enzyme replacement therapy with Cerezyme*Genzyme was formally introduced in Bulgaria, but after some time it was interrupted for 1-2 months. The dose of the patients was not optimal. The aim of our work is to find a mathematical model for quantitative evaluation of ERT of Gaucher disease. The model applies a kind of software called "Statistika 6" via the input of the individual data of 5-year-old children having the Gaucher disease treated with Cerezyme. The output results of the model gave possibilities for quantitative evaluation of the individual trends in the development of the disease of each child and its correlation. On the basis of this results, we might recommend suitable changes in ERT.

  4. Application of lipases to regiospecific interesterification of exotic oils from an Amazonian area.

    Science.gov (United States)

    Speranza, Paula; Ribeiro, Ana Paula Badan; Macedo, Gabriela Alves

    2016-01-20

    Enzymatic interesterification may favor the development of lipid fractions from Amazonian oils with greater application potential. In this study, the Amazonian buriti oil and murumuru fat were subjected to enzymatic interesterification using two lipases in three different enzyme systems: one with a commercial lipase from Thermomyces lanuginosa, a second with the lipase produced by Rhizopus sp., and a third with a mixture of both lipases. The three enzyme systems were able to catalyze the reaction, but the enzymes showed different specificities. The commercial lipase was specific for unsaturated fatty acids, whereas the Rhizopus sp. lipase was specific for both unsaturated fatty acids and the positions sn -1 and sn -3 of the fatty acid on the triacylglycerol. The mixture of both lipases showed no synergistic effect: the results were intermediate between the two enzymes applied alone. Interesterification reduced the levels of trisaturated and triunsaturated triacylglycerols and increased the levels of diunsaturated-monosaturated and monounsaturated-disaturated triacylglycerols. The thermal melting behavior indicated the formation of a single endothermic region with more homogeneous triacylglycerols. The content of the bioactive β-carotene was preserved after the interesterification reaction with all three-enzyme systems. The interesterified lipids obtained, because of the characteristics of the oils, may be applied to the formulation of cosmetics and pharmaceuticals. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. OPTIMASI DAN PEMEKATAN LIPASE Bacillus halodurans CM1

    Directory of Open Access Journals (Sweden)

    Arina Aisyah

    2017-08-01

    Full Text Available Abstrak Lipase diketahui memiliki peranan penting dalam bidang industri. Produksi lipase dapat dihasilkan oleh kapang, khamir, dan bakteri. Penelitian bertujuan untuk meningkatkan aktivitas lipase yang dihasilkan oleh Bacillus halodurans CM1. Aktivitas lipase dapat ditingkatkan dengan optimasi komposisi media, mutasi bakteri dengan radiasi gamma dan N-methyl-N’-nitro-N-nitrosoguanidine (NTG. Enzim yang dihasilkan dipekatkan dengan metode stirred-cell ultrafiltration (UF-ammonium sulfat dan UF-Polyethylene glycol (PEG. Uji aktivitas dilakukan pada tujuh media yang berbeda untuk mendapatkan media produksi. Delapan variabel komposisi media dioptimasi dengan rancangan Plackett-Burman. Bakteri dimutasi dengan radiasi gamma dosis 0,1–0,4 kGy dan NTG 0,05–0,15 mg/mL dengan waktu inkubasi 1–3 jam. Hasil penelitian menunjukkan bahwa media produksi yang digunakan berdasarkan optimasi media dan komposisi media Plackett-Burman adalah media dasar Bora & Bora yang mengandung 0,5% palm oil (PO dan 0,09% CaCl2. Aktivitas lipase optimal diproduksi oleh bakteri hasil mutasi dengan NTG 0,1 mg/mL yang diinkubasi selama 3 jam. Pemekatan enzim UF-ammonium sulfat dan UF-PEG mampu meningkatkan aktivitas enzim lipase sebesar 18,44%.  Abstract Lipase is known to have an important role in the industrial field. Lipase can be produced by molds, yeasts, and bacteria. The research aimed to increase the activity of lipase produced by Bacillus halodurans CM1. Lipase activity can be improved by optimization of the composition of the media, the mutation of bacteria with gamma radiation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG. The enzyme was concentrated by stirred-cell ultrafiltration method (UF-ammonium sulfate and UF-Polyethylene glycol (PEG. The activity test was performed on seven different media to get production media. The eight variables of the media composition were optimized by Plackett-Burman design. The bacteria were subject to mutation by using 0.1–0

  6. HIDROLISIS MINYAK KELAPA DENGAN LIPASE TERIMOBILISASI ZEOLIT PADA PEMBUATAN PERISA ALAMI

    Directory of Open Access Journals (Sweden)

    Dwina Moentamaria

    2016-12-01

    Full Text Available Free Fatty acid resulting from hydrolisis of various types of oil enzymatically has been great interest recently to save energy, in other hand that the product are environmentally friendly. Lipases as biocatalysts for synthesis reactions will be dissolved with the product, making difficult their reuse. Efficiency can be done with the use of enzyme immobilization, which can be used for repeated reaction. The products of free fatty acids from coconut oil by hydrolysis of lipase can be used as a natural substrate for making flavor that can be consumed and safe for health. The effect of free lipase and immobilization of lipase on hydrolisis were studied. Reaction time of hydrolisis was varied as 30, 60, 90, 120 and 150 minutes. The variation of concentration of lipase addition was 4, 5, 6, 7 and 8 % . The types of treatment were used in this research free lipase and the immobilized lipase. The results shows that the highest conversion on hydrolisis of coconut oil by using free lipase treatment was performed by 6 % of lipase addition with reaction time 60 minutes that are 52,31%. While, the highest conversion on hydrolisis of coconut oil by using the immobilized lipase was shown by 8% of lipase addition with reaction time 120 minutes that is 56,01%. The results of the hydrolysis process in the form of fatty acid was used as the base material esterification process resulting ester product (natural flavor. Ester yield was produced by free lipase esterification was 28,21 and 32,14 % in immobilized lipase esterification.

  7. Strategies to Characterize Fungal Lipases for Applications in Medicine and Dairy Industry

    Directory of Open Access Journals (Sweden)

    Subash C. B. Gopinath

    2013-01-01

    Full Text Available Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications.

  8. Les lipases sont des hydrolases atypiques : principales caractéristiques et applications

    Directory of Open Access Journals (Sweden)

    Fickers P.

    2008-01-01

    Full Text Available ipases are atypical hydrolases: principal characteristics and applications. Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous solution or as biocatalysts in organic synthesis. As hydrolases, they are responsible of the triglycerids catabolism into fatty acids and glycerol. In many organisms, this reaction plays a major role in the fat and lipid metabolism. In addition, lipases are also able to hydrolyse phospholipids and cholesterol esters. In organic solvent, lipases could catalyse reactions such as esterifications, acidolysis or alcoolysis with enantio-, regio- and chimioselectivity. Lipases form a mixed class of enzyme due to their animal, vegetal or microbial origins. All those properties led to the development of many applications in the food and chemical industries but also in the medical and therapeutic field.

  9. Strategies to Characterize Fungal Lipases for Applications in Medicine and Dairy Industry

    Science.gov (United States)

    Gopinath, Subash C. B.; Anbu, Periasamy; Lakshmipriya, Thangavel; Hilda, Azariah

    2013-01-01

    Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications. PMID:23865040

  10. Lipase-catalyzed biodiesel synthesis with different acyl acceptors

    Directory of Open Access Journals (Sweden)

    Ognjanović Nevena D.

    2008-01-01

    Full Text Available Biodiesel is an alternative fuel for diesel engine that is environmentally acceptable. Conventionally, biodiesel is produced by transesterification of triglycerides and short alcohols in the presence of an acid or an alkaline catalyst. There are several problems associated with this kind of production that can be resolved by using lipase as the biocatalyst. The aim of the present work was to investigate novel acyl acceptors for biodiesel production. 2-Propanol and n-butanol have a less negative effect on lipase stability, and they also improve low temperature properties of the fuel. However, excess alcohol leads to inactivation of the enzyme, and glycerol, a major byproduct, can block the immobilized enzyme, resulting in low enzymatic activity. This problem was solved by using methyl acetate as acyl acceptor. Triacetylglycerol is produced instead of glycerol, and it has no negative effect on the activity of the lipase.

  11. Les lipases immobilisées et leurs applications

    Directory of Open Access Journals (Sweden)

    Thonart P.

    2008-01-01

    Full Text Available Immobilized lipases and their applications. Lipases are able to catalyse the hydrolysis of glyceridic esters in aqueous media and the synthesis of esters in non-aqueous media. They are thus able to catalyse numerous reactions of industrial interest. Whether it is by inclusion, by adsorption or by covalent link, the immobilisation of lipases aims at conferring them a good stability that enables a reuse of the enzymes after a reaction and the development of continuous processes. The reactions of triglycerides hydrolysis constitute main applications for immobilised lipases, however their use in different types of esterification reactions has also arose: there exist processes involving reactions of transesterification, of interesterification or of esters synthesis. The production of structured lipids by interesterification is one example. Although the reaction conditions dissent from those of hydrolysis, the same lipases have been used in both cases. A lipase specifically adapted for esterification though would be a highly capable tool: a series of strategies is in progress in order to reach this goal.

  12. Corn steep liquor as a nutrition adjunct for the production of Aspergillus niger lipase and hydrolysis of oils thereof.

    Science.gov (United States)

    Edwinoliver, N G; Thirunavukarasu, K; Purushothaman, S; Rose, C; Gowthaman, M K; Kamini, N R

    2009-11-25

    Corn steep liquor (CSL) has been used as a nutrition adjunct for the production of an extracellular lipase from Aspergillus niger, which has immense importance as an additive in laundry detergent formulations. A five-level four-factorial central composite design was chosen to determine the optimal medium components with four critical variables, namely, CSL, NH4H2PO4, Na2HPO4, and sesame oil, that were found to be influential for lipase production by the classical one-factor-at-a-time method. The model suggested that all of the factors chosen had a significant impact on lipase production, and the optimum values of the influential parameters were CSL, 2.0%, w/v; NH4H2PO4, 0.05%, w/v; Na2HPO4, 0.75%, w/v; and sesame oil, 2.0%, w/v, with an activity of 26.7 U/mL at 48 h and 30 degrees C, which was 2.16-fold higher than the initial activity (12 U/mL) obtained by the conventional one-factor-at-a-time method. Furthermore, the enzyme has good potential for the hydrolysis of vegetable oils and fish oils, and a hydrolytic ratio of 88.73% was obtained with palm oil at 48 h. The utilization of CSL and sesame oil for lipase production from A. niger makes the process green, because both are renewable substrates and economically viable at an industrial scale.

  13. Molecular and enzymatic characterization of alkaline lipase from Bacillus amyloliquefaciens E1PA isolated from lipid-rich food waste.

    Science.gov (United States)

    Saengsanga, Thanakorn; Siripornadulsil, Wilailak; Siripornadulsil, Surasak

    2016-01-01

    Bacillus amyloliquefaciens E1PA is a lipase-producing strain that was originally isolated from lipid-rich food waste, and the production of its lipase was found to be induced by vegetable oils. The E1PA lipase was successfully expressed and secreted in a heterologous Escherichia coli host and was ultimately purified. The conserved pentapeptide motif Ala-His-Ser-Met-Gly was observed at positions 108-112. The purified recombinant lipase was stable over a pH range of 4.0-11.0 at 40 °C and exhibited maximal activity at pH 10. The recombinant E1PA lipase hydrolyzed a wide range of acyl esters (C4-C18). However, the highest activity (3.5 units mg(-1)) was observed when the p-nitrophenyl ester of myristate (C14) was used as a substrate. Compared to the lipases produced by Bacillus spp., the E1PA lipase displayed a structural molecular mass excluding the leader sequence (19.22 kDa) and a pI (9.82) that were similar to those reported for B. amyloliquefaciens lipases and lipase subfamily I.4 but that were quite distinct from those of lipase subfamily I.5 (approximately 43 kDa, pI 6). These results suggested that Bacillus lipases are closely related. Although the recombinant E1PA lipase digested only certain oils, the wild-type E1PA lipase degraded a variety of oils, including blended and re-used cooking oils. The recombinant and wild-type forms of the E1PA lipase were able to digest heterogeneous lipid-rich food waste at similar levels; this result suggests that this lipase can function even when it solely consists of its structural enzyme component. The enzyme exhibited lipid hydrolysis ability as either an intracellular domain of the recombinant protein or an extracellular domain secreted by the E1PA strain. However, the recombinant lipase showed higher activity than the wild-type E1PA lipase, indicating that the recombinant protein from E. coli possessed effective lipase activity. Thus, the inducible alkaline E1PA lipase exhibited the ability to act on a broad spectrum

  14. Proteinuria and lipoprotein lipase activity in Miniature schnauzer dogs with and without hypertriglyceridemia

    OpenAIRE

    Furrow, E.; Jaeger, J.Q.; Parker, V.J.; Hinchcliff, K.W.; Johnson, S.E.; Murdoch, S.J.; de Boer, I.H.; Sherding, R.G.; Brunzell, J.D.

    2016-01-01

    Spontaneous hyperlipidemia in rats causes glomerular disease. Idiopathic hypertriglyceridemia (HTG) is prevalent in Miniature schnauzers, but its relationship with proteinuria is unknown. Decreased activity of major lipid metabolism enzymes, lipoprotein lipase (LPL) and hepatic lipase (HL), may play a role in the cyclic relationship between hyperlipidemia and proteinuria. These enzymes have also not been previously investigated in Miniature shnauzers. The aims of this study were to determine ...

  15. Models for enzyme superactivity in aqueous solutions of surfactants.

    OpenAIRE

    Viparelli, P; Alfani, F; Cantarella, M

    1999-01-01

    Theoretical models are developed here for enzymic activity in the presence of direct micellar aggregates. An approach similar to that of Bru et al. [Bru, Sánchez-Ferrer and Garcia-Carmona (1989) Biochem. J. 259, 355-361] for reverse micelles has been adopted. The system is considered to consist of three pseudo-phases: free water, bound water and surfactant tails. The substrate concentration in each pseudo-phase is related to the total substrate concentration in the reaction medium. In the abs...

  16. Enhancement of the performance of covalently immobilized lipase ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-06-07

    Jun 7, 2010 ... FT-IR, fourier transform infrared. the enzyme towards organic media (Xie et al., ... characterized by FT-IR spectroscopy (Zeiss, Specord M 80) using. KBr method. Covalent immobilization of lipase in the ... and kcat, interpreted from the Lineweaver-Burk plots, were shown in Table 1. The data indicate that the ...

  17. Solvent free lipase catalyzed synthesis of butyl caprylate

    Indian Academy of Sciences (India)

    To achieve maximum conversion theoptimum parameters thus established include; temperature 60 ◦C, mole ratio of caprylic acid and butanol as 1:2, lipase loading 2% (w/v), 250 rpm speed of agitation and 4g of molecular sieves. The immobilized enzyme wasalso recycled and reused for 7 cycles with only 30% loss from its ...

  18. Selection and optimization of extracellular lipase production using ...

    African Journals Online (AJOL)

    Pedro

    2014-01-22

    Jan 22, 2014 ... tion, alcoholysis, acidolysis and aminolysis (Joseph et al.,. 2008). Lipases represent an important group of enzymes with various applications in the food, chemical and pharmaceutical industry (Salihu et al., 2012), besides having great interest for the treatment of effluents with high lipid content (Dharmsthiti ...

  19. Solvent free lipase catalyzed synthesis of butyl caprylate

    Indian Academy of Sciences (India)

    MEERA T SOSE

    2017-11-10

    Nov 10, 2017 ... C, mole ratio of caprylic acid and butanol as 1:2, lipase loading 2% (w/v), 250 rpm speed of agitation and 4g of molecular sieves. The immobilized enzyme was also recycled and reused for 7 cycles with only 30% loss from its initial activity. The thermodynamic parameters at different temperatures were also ...

  20. Recombinant Lipases and Phospholipases and Their Use as Biocatalysts for Industrial Applications

    Science.gov (United States)

    Borrelli, Grazia M.; Trono, Daniela

    2015-01-01

    Lipases and phospholipases are interfacial enzymes that hydrolyze hydrophobic ester linkages of triacylglycerols and phospholipids, respectively. In addition to their role as esterases, these enzymes catalyze a plethora of other reactions; indeed, lipases also catalyze esterification, transesterification and interesterification reactions, and phospholipases also show acyltransferase, transacylase and transphosphatidylation activities. Thus, lipases and phospholipases represent versatile biocatalysts that are widely used in various industrial applications, such as for biodiesels, food, nutraceuticals, oil degumming and detergents; minor applications also include bioremediation, agriculture, cosmetics, leather and paper industries. These enzymes are ubiquitous in most living organisms, across animals, plants, yeasts, fungi and bacteria. For their greater availability and their ease of production, microbial lipases and phospholipases are preferred to those derived from animals and plants. Nevertheless, traditional purification strategies from microbe cultures have a number of disadvantages, which include non-reproducibility and low yields. Moreover, native microbial enzymes are not always suitable for biocatalytic processes. The development of molecular techniques for the production of recombinant heterologous proteins in a host system has overcome these constraints, as this allows high-level protein expression and production of new redesigned enzymes with improved catalytic properties. These can meet the requirements of specific industrial process better than the native enzymes. The purpose of this review is to give an overview of the structural and functional features of lipases and phospholipases, to describe the recent advances in optimization of the production of recombinant lipases and phospholipases, and to summarize the information available relating to their major applications in industrial processes. PMID:26340621

  1. Gene cloning, expression, and characterization of the Bacillus amyloliquefaciens PS35 lipase

    Directory of Open Access Journals (Sweden)

    Palanisamy Kanmani

    2015-01-01

    Full Text Available AbstractLipases are enzymes of immense industrial relevance, and, therefore, are being intensely investigated. In an attempt to characterize lipases at molecular level from novel sources, a lipase gene from Bacillus amyloliquefaciens PS35 was cloned, heterologously expressed in Escherichia coli DH5α cells and sequenced. It showed up to 98% homology with other lipase sequences in the NCBI database. The recombinant enzyme was then purified from E. coli culture, resulting in a 19.41-fold purification with 9.7% yield. It displayed a preference for long-chain para-nitrophenyl esters, a characteristic that is typical of true lipases. Its optimum pH and temperature were determined to be 8.0 and 40 °C, respectively. The half-lives were 2.0, 1.0 and 0.5 h at 50 °C, 60 °C and 70 °C, respectively. The metal ions K+and Fe3+ enhanced the enzyme activity. The enzyme displayed substantial residual activity in the presence of various tested chemical modifiers, and interestingly, the organic solvents, such as n-hexane and toluene, also favored the enzyme activity. Thus, this study involves characterization of B. amyloliquefaciens lipase at molecular level. The key outcomes are novelty of the bacterial source and purification of the enzyme with desirable properties for industrial applications.

  2. Crystal structure of a triacylglycerol lipase from Penicillium expansum at 1.3 A determined by sulfur SAD

    Energy Technology Data Exchange (ETDEWEB)

    Bian, Chuanbing; Yuan, Cai; Chen, Liqing; Meehan, Edward J.; Jiang, Longguang; Huang, Zixiang; Lin, Lin; Huang, Mingdong; (UAH); (Fujian); (Chinese Aca. Sci.)

    2010-04-05

    Triacylglycerol lipases (EC 3.1.1.3) are present in many different organisms including animals, plants, and microbes. Lipases catalyze the hydrolysis of long-chain triglycerides into fatty acids and glycerol at the interface between the water insoluble substrate and the aqueous phase. Lipases can also catalyze the reverse esterification reaction to form glycerides under certain conditions. Lipases of microbial origin are of considerable commercial interest for wide variety of biotechnological applications in industries, including detergent, food, cosmetic, pharmaceutical, fine chemicals, and biodiesel. Nowadays, microbial lipases have become one of the most important industrial enzymes. PEL (Penicillium expansum lipase) is a fungal lipase from Penicillium expansum strain PF898 isolated from Chinese soil that has been subjected to several generations of mutagenesis to increase its enzymatic activity. PEL belongs to the triacylglycerol lipases family, and its catalytic characteristics have been studied. The enzyme has been used in Chinese laundry detergent industry for several years (http://www.leveking.com). However, the poor thermal stability of the enzyme limits its application. To further study and improve this enzyme, PEL was cloned and sequenced. Furthermore, it was overexpressed in Pichia pastoris. PEL contains GHSLG sequence, which is the lipase consensus sequence Gly-X1-Ser-X2-Gly, but has a low amino acid sequence identities to other lipases. The most similar lipases are Rhizomucor miehei (PML) and Rhizopus niveus (PNL) with a 21% and 20% sequence identities to PEL, respectively. Interestingly, the similarity of PEL with the known esterases is somewhat higher with 24% sequence identity to feruloyl esterase A. Here, we report the 1.3 {angstrom} resolution crystal structure of PEL determined by sulfur SAD phasing. This structure not only presents a new lipase structure at high resolution, but also provides a structural platform to analyze the published

  3. Temporal expression profiling of lipase during germination and rice caryopsis development.

    Science.gov (United States)

    Vijayakumar, K R; Gowda, Lalitha R

    2012-08-01

    Lipolytic enzymes play an important role in plant growth and development. In order to identify their functional roles, the temporal expression profiling of lipase was carried out during rice seed germination, growth and development of caryopsis. Changes in specific activities during germination revealed that the lipolytic activity increased significantly until the end of germination. As the lipase activity increased, two different lipase species were observed, which were designated as Lipase-I and Lipase-II based on their relative mobility. Lipase-II was active during germination. Lipase-I was responsible for lipid mobilization, a requirement for the growth of root and shoot. In comparison with the endosperm, the lipolytic activity in roots was three fold higher. During rice caryopsis development, the lipolytic activity increased gradually from initial panicle development and reached maximum as the grain dried to harvest maturity. Quantitative real-time PCR analysis revealed that the Lipase-II was a stage specific expressing gene during reproductive growth. The transcript level of Lipase-II reached maximum with completion of germination, then decreased and remained stable during post-germinative growth. During caryopsis development, Lipase-II is predominantly expressed in the developing seeds. The transcript abundance increased gradually during initial stages of development and reached a maximum until seed maturation. The results implicate that the dynamic changes in the enzyme activity of the two isoforms of lipase and gene expression patterns are associated with the energy reserve mobilization during seed germination and reproductive growth. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  4. Enzymatic transesterification of soybean oil by using immobilized lipase on magnetic nano-particles

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Wenlei; Ma, Ning [School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001 (China)

    2010-06-15

    Lipase was covalently immobilized onto magnetic Fe{sub 3}O{sub 4} nano-particles by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as an activating agent, and the bound lipase was used to catalyze the transesterification of vegetable oils with methanol to produce fatty acid methyl esters. The binding of lipase to magnetic particles was confirmed by enzyme assays, transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectra. It was determined that the immobilized lipase exhibited better resistance to temperature and pH inactivation in comparison to free lipase. Using the immobilized lipase, the major parameters affecting the transesterification reaction, such as the alcohol/oil molar ratio, enzyme loading and free fatty acid present in reactants were investigated to obtain the optimum reaction condition. The conversion of soybean oil to methyl esters reached over 90% in the three-step transesterification when 40% immobilized lipase was used. Moreover, the lipase catalyst could be used for 3 times without significant decrease of the activity. (author)

  5. Rhizomucor miehei triglyceride lipase is processed and secreted from transformed Aspergillus oryzae.

    Science.gov (United States)

    Huge-Jensen, B; Andreasen, F; Christensen, T; Christensen, M; Thim, L; Boel, E

    1989-09-01

    The cDNA encoding the precursor of the Rhizomucor miehei triglyceride lipase was inserted in an Aspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of the Aspergillus oryzae alpha-amylase gene promoter and the Aspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced into Aspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted from A. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the native Rhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted from Aspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.

  6. Evaluation of a new lipase from Staphylococcus sp. for detergent additive capability.

    Science.gov (United States)

    Chauhan, Mamta; Chauhan, Rajinder Singh; Garlapati, Vijay Kumar

    2013-01-01

    Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase.

  7. Evaluation of a New Lipase from Staphylococcus sp. for Detergent Additive Capability

    Directory of Open Access Journals (Sweden)

    Mamta Chauhan

    2013-01-01

    Full Text Available Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62% from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase.

  8. Evaluation of a New Lipase from Staphylococcus sp. for Detergent Additive Capability

    Science.gov (United States)

    Chauhan, Mamta; Chauhan, Rajinder Singh; Garlapati, Vijay Kumar

    2013-01-01

    Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase. PMID:24106703

  9. Biodiesel production by transesterification using immobilized lipase.

    Science.gov (United States)

    Narwal, Sunil Kumar; Gupta, Reena

    2013-04-01

    Biodiesel can be produced by transesterification of vegetable or waste oil catalysed by lipases. Biodiesel is an alternative energy source to conventional fuel. It combines environmental friendliness with biodegradability, low toxicity and renewability. Biodiesel transesterification reactions can be broadly classified into two categories: chemical and enzymatic. The production of biodiesel using the enzymatic route eliminates the reactions catalysed under acid or alkali conditions by yielding product of very high purity. The modification of lipases can improve their stability, activity and tolerance to alcohol. The cost of lipases and the relatively slower reaction rate remain the major obstacles for enzymatic production of biodiesel. However, this problem can be solved by immobilizing the enzyme on a suitable matrix or support, which increases the chances of re-usability. The main factors affecting biodiesel production are composition of fatty acids, catalyst, solvents, molar ratio of alcohol and oil, temperature, water content, type of alcohol and reactor configuration. Optimization of these parameters is necessary to reduce the cost of biodiesel production.

  10. Characterization of lipase in reversed micelles formulated by Cibacron Blue F-3GA modified Span 85

    DEFF Research Database (Denmark)

    Zhang, Dong Hao; Guo, Zheng; Sun, Yan

    2007-01-01

    as a model reaction. The micellar hydrodynamic radius results reflected, to some extent, the redistribution of surfactant and water after enzyme addition, and the correlation between surfactant formulation, water content (W0), micellar size, and enzyme activity. An adequate modification density of CB......Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil...... was found to be important for the reversed micelles to retain enough hydration capacity and achieve high enzyme activity. Compared with the results in AOT-based reversed micelles, CRL in this micellar system exhibited a different activity behavior versus W0. The optimal pH and temperature...

  11. Effects of high intensity pulsed electric field and thermal treatments on a lipase from Pseudomonas fluorescens.

    Science.gov (United States)

    Bendicho, S; Estela, C; Giner, J; Barbosa-Cánovas, G V; Martin, O

    2002-01-01

    Milk and dairy products may contain microorganisms capable of secreting lipases that cause sensory defects and technological problems in the dairy industry. In this study, the effects of thermal and high-intensity pulsed electric field (HIPEF) treatments on an extracellular lipase from Pseudomonas fluorescens, suspended in a simulated skim milk ultrafiltrate (SMUF) have been evaluated. Heat treatments applied were up to 30 min from 50 to 90 degrees C. HIPEF treatments were carried out using pilot plant facilities in a batch or continuous flow mode, where treatment chambers consisted of parallel and coaxial configuration, respectively. Samples were subjected to up to 80 pulses at electric field intensities ranging from 16.4 to 37.3 kV/cm. This resulted in a lipase that was quite resistant to heat and also to HIPEF. High (75 degrees C-15 s) and low pasteurization treatments (63 degrees C-30 min) led to inactivations of 5 and 20%, respectively. Using the batch-mode HIPEF equipment, a 62.1% maximum activity depletion was achieved after 80 pulses at 27.4 kV/cm. However, when HIPEF treatments were applied in the continuous flow mode, an inactivation rate of just 13% was achieved, after applying 80 pulses at 37.3 kV/cm and 3.5 Hz. The results of both heat and HIPEF treatments on enzyme inactivation were adjusted with good agreement to a first-order kinetic model (R2 > 62.3%).

  12. Comparison of immunoreactive serum trypsinogen and lipase in Cystic Fibrosis

    International Nuclear Information System (INIS)

    Lloyd-Still, J.D.; Weiss, S.; Wessel, H.; Fong, L.; Conway, J.J.

    1984-01-01

    The incidence of Cystic Fibrosis (CF) is 1 in 2,000. Early detection and treatment of CF may necessitate newborn screening with a reliable and cost-effective test. Serum immunoreactive trypsinogen (IRT) an enzyme produced by the pancreas, is detectable by radioimmunoassay (RIA) techniques. Recently, it has been shown that IRT is elevated in CF infants for the first few months of life and levels become subnormal as pancreatic insufficiency progresses. Other enzymes produced by the pancreas, such as lipase, are also elevated during this time. The author's earlier work confirmed previous reports of elevated IRT levels in CF infants. The development of a new RIA for lipase (nuclipase) has enabled comparison of these 2 pancreatic enzymes in C.F. Serum IRT and lipase determinations were performed on 2 groups of CF patients; infants under 1 year of age, and children between 1 and 18 years of age. Control populations of the same age groups were included. The results showed that both trypsin (161 +- 92 ng/ml, range 20 to 400) and lipase (167 +- 151 ng/ml, range 29 to 500) are elevated in CF in the majority of infants. Control infants had values of IRT ranging from 20 to 29.5 ng/ml and lipase values ranging from 23 to 34 ng/ml. IRT becomes subnormal in most CF patients by 8 years of age as pancreatic function insufficiency increases. Lipase levels and IRT levels correlate well in infancy, but IRT is a more sensitive indicator of pancreatic insufficiency in older patients with CF

  13. Isolation and identification of a cold-adapted lipase producing strain ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-05-03

    May 3, 2010 ... The optimal pH and temperature of the enzyme was 10.0 and 30°C, respectively. About ... of temperature and pH value in the lipase activity and .... H2S. -. Inositol. +. Gelatin. +. D-mannitol. +. D-fructose. +. +, Positive; - negative. Enzyme purification. The enzyme was purified about 1.5-fold with a yield of.

  14. Elevated Amylase and Lipase Levels in the Neurosurgery Intensive Care Unit

    Directory of Open Access Journals (Sweden)

    Cheng-Chia Lee

    2010-01-01

    Conclusion: Various neurosurgery events and diagnoses may lead to different degrees of serum pancreatic enzyme elevation. Patients with elevated pancreatic enzyme levels have a higher mortality rate than those with normal enzyme levels. We believe that abdominal CT should be indicated for patients if their amylase levels are more than 3-fold the upper normal limit and lipase levels are more than 5-fold.

  15. Harmful effect of detergents on lipase.

    Science.gov (United States)

    Fatima, Sadaf; Ajmal, Rehan; Badr, Gamal; Khan, Rizwan H

    2014-11-01

    In order to study effects of detergents at molecular level, we have done activity measurements of wheat germ lipase in increasing concentration of some commercial detergents. Conformational changes in protein structure using circular dichroism and fluorescence spectroscopy were studied in increasing concentration of sodium dodecyl sulfate. Our study proves that detergents may lead to loss of enzymatic activity and structure of plant enzymes. Since detergents are common source of pollution in water bodies and the water from these resources can be used in fields, our study may prove helpful in creating awareness about harmful action of detergents.

  16. New alkaline lipase from Rhizomucor variabilis: Biochemical properties and stability in the presence of microbial EPS.

    Science.gov (United States)

    Bancerz, Renata; Osińska-Jaroszuk, Monika; Jaszek, Magdalena; Janusz, Grzegorz; Stefaniuk, Dawid; Sulej, Justyna; Janczarek, Monika; Jarosz-Wilkołazka, Anna; Rogalski, Jerzy

    2016-01-01

    A new strain of Rhizomucor variabilis producing an active extracellular lipase was identified and characterized in the present studies. The culture conditions were optimized and the highest lipase production amounting to 136 U/mL was achieved after 4 days of cultivation. The optimum pH (5.5) and temperature (28 °C) were determined as the best conditions for R. variabilis lipase production. The isolated enzyme preparation exhibited maximum activity at 40 °C and pH 8.0. Lipase from R. variabilis was stable up to 50 °C during 2 H retaining 80% of its initial activity. The enzyme was highly stable in the pH range of 7.0-9.0. Moreover, the addition of naturally obtained exopolysaccharides (EPS) significantly enhanced lipase activity. The presence of EPS derived from Ganoderma applanatum and Rhizobium leguminosarum enhanced the lipase activity, which was 22% and 31%, respectively, higher than that in the control experiments. Simultaneously, the pH activity profiles remained unchanged. The Michaelis-Menten constant and the turnover number of the enzyme for p-nitrophenyl palmitate in the standard assay conditions were estimated at a level of 0.631 mM and 0.674 Sec(-1) . In conclusion, the results obtained in this work present a newly isolated lipase preparation stabilized with EPS or without modification as a very effective tool for industrial application. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  17. IDENTIFIKASI POTENSI ENZIM LIPASE DAN SELULASE PADA SAMPAH KULIT BUAH HASIL FERMENTASI

    Directory of Open Access Journals (Sweden)

    La Ode Sumarlin

    2013-12-01

    Full Text Available Fermentation is one of bioconversion to produce profitable anaerobic microbes and to produce various enzymes. Lipases and cellulases are widely used enzymes so far. Cellulases play an important role in bioconversion of organic waste cellulosic materials to glucose, single cell proteins, animal feed, and ethanol. Lipases can also degrade fatty ester bond. Therefore, both enzymes are potential to be used in industry as well as in households. Fermentation of fruit peel waste is an attempt to produce cellulase and lipase that can be carried out in a simple way. Cellulase as says was performed using DNS (3.5-dinitrosalicylic acid and acid-base titration for analysis of lipase using cooking oil as the substrate. The results showed that the highest cellulase activity was obtained from watermelon rind mixed with citrus fruit peel of 0.036 U/mL, and mixed of banana peel and citrus fruit, which was 0.035 U/mL. The optimum lipase activity was at 30 oC, pH 7, and reaction time of 60 minutes. The highest lipase activity (1.36 U/mL was obtained from mixture of watermelon and orange rind. Thus, the fruit peel waste is potential to produce cellulase and lipase by fermentation .

  18. Partial purification and characterization of lipase (EC 3.1.1.3) from Propionibacterium acnes.

    Science.gov (United States)

    Ingham, E; Holland, K T; Gowland, G; Cunliffe, W J

    1981-06-01

    Lipase from Propionibacterium acnes has been purified 4800-fold from crude culture supernatant. The purified enzyme preparation had no assayable protease, hyaluronate lyase or acid phosphatase activities. The molecular weight of the lipase was 46,770 as determined by gel filtration. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a major protein component (mol. wt 41,190) together with two minor protein components (mol. wt 67,000 and 125,900). The lipase had a pH optimum of 6.8, was most stable in the pH range 5.0 to 6.0 and was completely inactivated after 30 min at 60 degrees C. The lipase hydrolysed trilaurin, triolein, trimyristin and tripalmitin at decreasing rates and did not exhibit phospholipase activity. Analysis of the reaction products from the hydrolysis of triolein by P. acnes lipase did not demonstrate an accumulation of 2-monoolein which suggested that the enzyme did not exhibit a positional specificity for the 1-position of the triacylglycerol. Crude lipase preparations contained an aggregated high molecular weight form of the enzyme which was eluted with the void volume from Sephadex G-200. This aggregated form was dissociated to produce the lower molecular weight lipase species by subsequent dialysis and elution from Sephadex G-200 using buffer with a higher ionic strength.

  19. HIDROLISIS ENZIMATIS STEARIN SAWIT MENJADI MONOGLISERIDA OLEH LIPASE DARI Rhizomucor miehei DAN PANKREAS (Enzymatic Hydrolysis of Palm Stearin to Produce Monoglyceride by Lipase from Rhizomucor miehei and Pancreatic

    Directory of Open Access Journals (Sweden)

    Steivie Karaouw

    2013-06-01

    Full Text Available The objectives of the research were to evaluate the effect of the pH, ratio of substrate:phospate buffer, and reaction time on the enzymatic hydrolysis of palm stearin to obtain monoglyceride by R. miehei and pancreatic lipases. Hydrolysis was evaluated at various pH (6.0; 6.5; 7.0; 7.5 dan 8.0. Enzymatic hydrolysis reactions were held at various ratio of substrate:phospate buffer (10:1, 10:2, 10:3, 10:4, 10:5, 10:6 and duration time of 6, 12, 18, 24 hours by R. miehei lipase and 24, 30, 36, 42, 48 hours by pancreatic lipase. Enzymatic hydrolysis reaction was carried out in waterbath shaker 80 stroke/minute, at 40oC with R.miehei lipase and 37oC with pancreatic lipase. The hydrolysis products were monitored using TLC with petroleum ether:diethyl ether:acetic acid=60:40:1 as developing solvent on silica gel F254 20×20 cm plate. The results showed that optimum pH for both R. miehei and pancreatic lipases were 6.5 and their activities were 332.25 unit/g enzyme amobile and 228.04 unit/g enzyme, respectively. The highest monoglyceride fraction was obtained from ratio substrate:phospate buffer 10:1 at 18 hours of incubation by Rhizomucor miehei lipase (21,59% and ratio substrate:phospate buffer 10:4 at 42 hours of incubation by pancreatic lipase (40,45%. Keywords: Hydrolysis, palm stearin, monoglyceride, lipase, Rhizomucor miehei, pancreatic   ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh pH, rasio substrat:buffer fosfat dan waktu hidrolisis terhadap produksi monogliserida 2-monopalmitin secara enzimatis menggunakan lipase dari Rhizomucor miehei dan lipase pankreas. Hidrolisis dilakukan pada pH (6,0; 6,5; 7,0; 7,5 dan 8,0, dengan rasio substrat:buffer fosfat (10:1, 10:2, 10:3, 10:4, 10:5 dan 10:6 dan waktu hidrolisis (6, 12, 18 dan 24 jam menggunakan lipase dari R. miehei dan (6, 12, 18, 24, 30, 36, 42 dan 48 jam menggunakan lipase pankreas. Reaksi hidrolisis berlangsung dalam shaker waterbath 80 stroke/menit, pada suhu 40oC untuk

  20. Anaerobic biodegradability of dairy wastewater pretreated with porcine pancreas lipase

    Directory of Open Access Journals (Sweden)

    Adriano Aguiar Mendes

    2010-12-01

    Full Text Available Lipids-rich wastewater was partial hydrolyzed with porcine pancreas lipase and the efficiency of the enzymatic pretreatment was verified by the comparative biodegradability tests (crude and treated wastewater. Alternatively, simultaneous run was carried out in which hydrolysis and digestion was performed in the same reactor. Wastewater from dairy industries and low cost lipase preparation at two concentrations (0.05 and 0.5% w.v-1 were used. All the samples pretreated with enzyme showed a positive effect on organic matter removal (Chemical Oxygen Demand-COD and formation of methane. The best results were obtained when hydrolysis and biodegradation were performed simultaneously, attaining high COD and color removal independent of the lipase concentration. The enzymatic treatment considerably improved the anaerobic operational conditions and the effluent quality (lower content of suspended solids and less turbidity. Thus, the use of enzymes such as lipase seemed to be a very promising alternative for treating the wastewaters having high fat and grease contents, such as those from the dairy industry.O presente trabalho teve como objetivo o pré-tratamento de efluente da indústria de laticínios na hidrólise de lipídeos, empregando lipase de fonte de células animais de baixo custo disponível comercialmente (pâncreas de porco na formação de gás metano por biodegradabilidade anaeróbia empregando diferentes concentrações de lipase (0,05 e 0,5 % w.v-1. A utilização de lipase no pré-tratamento do efluente acelerou a hidrólise de lipídeos e, conseqüentemente, auxiliou o tratamento biológico resultando na redução da matéria orgânica em termos de Demanda Química de Oxigênio (DQO, cor e sólidos em suspensão como lipídeos. Os melhores resultados em termos de remoção de DQO e cor foram obtidos quando a hidrólise e biodigestão foram realizadas simultaneamente, independente da concentração de lipase empregada. Estes resultados

  1. Fungal Morphology in Industrial Enzyme Production - Modelling and Monitoring

    DEFF Research Database (Denmark)

    Quintanilla, D.; Hagemann, T.; Hansen, K.

    2015-01-01

    Filamentous fungi are widely used in the biotechnology industry for the production of industrial enzymes. Thus, considerable work has been done with the purpose of characterizing these processes. The ultimate goal of these efforts is to be able to control and predict fermentation performance......, and on the way the data is interpreted-i.e. which models were applied. The main filamentous fungi used in industrial fermentation are introduced, ranging from Trichoderma reesei to Aspergillus species. Due to the fact that secondary metabolites, like antibiotics, are not to be considered bulk products, organisms...... that spans across strains and scales, as most studies indeed conclude that their respective results might be strain specific and not necessarily valid across scales....

  2. New Tailor-Made Alkyl-Aldehyde Bifunctional Supports for Lipase Immobilization

    Directory of Open Access Journals (Sweden)

    Robson Carlos Alnoch

    2016-11-01

    Full Text Available Immobilized and stabilized lipases are important biocatalytic tools. In this paper, different tailor-made bifunctional supports were prepared for the immobilization of a new metagenomic lipase (LipC12. The new supports contained hydrophobic groups (different alkyl groups to promote interfacial adsorption of the lipase and aldehyde groups to react covalently with the amino groups of side chains of the adsorbed lipase. The best catalyst was 3.5-fold more active and 5000-fold more stable than the soluble enzyme. It was successfully used in the regioselective deacetylation of peracetylated d-glucal. The PEGylated immobilized lipase showed high regioselectivity, producing high yields of the C-3 monodeacetylated product at pH 5.0 and 4 °C.

  3. Statistical optimization for lipase production from solid waste of vegetable oil industry.

    Science.gov (United States)

    Sahoo, Rajesh Kumar; Kumar, Mohit; Mohanty, Swati; Sawyer, Matthew; Rahman, Pattanathu K S M; Sukla, Lala Behari; Subudhi, Enketeswara

    2018-04-21

    The production of biofuel using thermostable bacterial lipase from hot spring bacteria out of low-cost agricultural residue olive oil cake is reported in the present paper. Using a lipase enzyme from Bacillus licheniformis, a 66.5% yield of methyl esters was obtained. Optimum parameters were determined, with maximum production of lipase at a pH of 8.2, temperature 50.8°C, moisture content of 55.7%, and biosurfactant content of 1.693 mg. The contour plots and 3D surface responses depict the significant interaction of pH and moisture content with biosurfactant during lipase production. Chromatographic analysis of the lipase transesterification product was methyl esters, from kitchen waste oil under optimized conditions, generated methyl palmitate, methyl stearate, methyl oleate, and methyl linoleate.

  4. Screening of supports for immobilization of commercial porcine pancreatic lipase

    Directory of Open Access Journals (Sweden)

    Robison Scherer

    2011-12-01

    Full Text Available The aim of this work is to report the performance of different supports for the immobilization of commercial porcine pancreatic lipase. The immobilization tests were carried out in several types of Accurel, activated alumina, kaolin, montmorillonite, ion exchange resins and zeolites. The characterization of the supports showed differences in terms of specific area and morphology. The characteristics of the supports influenced the amount of enzyme adsorbed, yield of immobilization and esterification activity of the resulting immobilized catalyst. The clays KSF and natural and pillared montmorillonites presented potential for use as support for lipase immobilization in terms of yield and esterification activity. Yields of immobilization of 76.32 and 52.01% were achieved for clays KSF and natural montmorillonite, respectively. Esterification activities of 754.03, 595.51, 591.88 and 515.71 U.g-1 were obtained for lipases immobilized in Accurel MP-100, Amberlite XAD-2, mordenite and pillared montmorillonite, respectively.

  5. Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils

    OpenAIRE

    Ferrario, Valerio; Veny, Harumi; Angelis, Elisabetta De; Navarini, Luciano; Ebert, Cynthia; Gardossi, Lucia

    2013-01-01

    Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to ...

  6. Synthesis and characterization of chitosan/TiO2 composite beads for improving stability of porcine pancreatic lipase.

    Science.gov (United States)

    Deveci, Ilyas; Doğaç, Yasemin Ispirli; Teke, Mustafa; Mercimek, Bedrettin

    2015-01-01

    The purpose of the present work is improving stability properties of porcine pancreatic lipase (triacylglycerol lipase, E.C.3.1.1.3) by immobilization on chitosan/TiO2 composite beads. The immobilization parameters were initial enzyme concentration (0.5-2 mg/ml), adsorption time (5-25 min), and glutaraldehyde concentration (1-4 % v/v). The optimum temperature (20-60 °C), optimum pH (3.0-10.0), kinetic parameters, thermal stability (4-70 °C), pH stability (4.0-9.0), and reusability (9 times) were investigated for characterization of immobilized lipase system. The optimum temperatures of free and immobilized lipase were 30 °C. The temperature profile of the immobilized lipase was spread over a large area. The optimum pH values for the free lipase and immobilized lipase were found to be 6.5 and 7.5, respectively. The thermal stability of immobilized lipase was evaluated, and it maintained 45 % activity at 70 °C. But, at this temperature, soluble lipase protected only 15 % activity. Also, the structural characterization of chitosan/TiO2 composite beads was analyzed with scanning electron microscope (SEM), X-ray diffraction (XRD), thermal gravimetric analysis (TGA), and attenuated total reflection Fourier transform infrared spectroscopy analysis (ATR-FTIR). The significance of this study is improving of stability properties of lipase for the industrial usage especially production of biodiesel and dairy products.

  7. Growth and extracellular enzyme production by microorganisms ...

    African Journals Online (AJOL)

    Ugba', an indigenous Nigerian fermented food condiment. The isolated microorganisms were screened for amylase, protease and lipase production, the activity and specific activity of the enzymes were also determined. The effect of pH and ...

  8. growth and extracellular enzyme production by microorganisms

    African Journals Online (AJOL)

    Okorie

    2013-06-26

    Ugba', an indigenous. Nigerian fermented food condiment. The isolated microorganisms were screened for amylase, protease and lipase production, the activity and specific activity of the enzymes were also determined.

  9. Lipase-catalyzed Baeyer-Villiger Oxidation ofcellulose-derived Levoglucosenone into(S-gamma-hydroxymethyl-□alpha,beta□-butenolide:Optimization by Response SurfaceMethodology

    Directory of Open Access Journals (Sweden)

    Andreia eTeixeira

    2016-04-01

    Full Text Available Cellulose-derived levoglucosenone (LGO has been efficiently converted into pure (S-gamma-hydroxymethyl-alpha,beta-butenolide (HBO, a chemical platform suited for the synthesis of drugs, flavors and antiviral agents. This process involves two-steps: a lipase-catalyzed Baeyer-Villager oxidation of LGO followed by an acid hydrolysis of the reaction mixture to provide pure HBO. Response surface methodology (RSM, based on central composite face-centered (CCF design, was employed to evaluate the factors effecting the enzyme-catalyzed reaction: pka of solid buffer (7.2 - 9.6, LGO concentration (0.5 - 1 M and enzyme loading (55 - 285 PLU.mmol-1. Enzyme loading and pka of solid buffer were found to be important factors to the reaction efficiency (as measured by the conversion of LGO while only the later had significant effects on the enzyme recyclability (as measured by the enzyme residual activity. LGO concentration influences both responses by its interaction with the enzyme loading and pka of solid buffer. The optimal conditions which allow to convert at least 80% of LGO in 2 hours at 40 °C and reuse the enzyme for a subsequent cycle were found to be: solid buffer pka = 7.5, [LGO] = 0.50 M and 113 PLU.mmol-1 for the lipase. A good agreement between experimental and predicted values was obtained and the validity of the model confirmed (p-value 0.05.

  10. Lipase-catalyzed Baeyer-Villiger Oxidation of cellulose-derived Levoglucosenone into (S)-gamma-hydroxymethyl-□alpha,beta□-butenolide: Optimization by Response Surface Methodology

    Science.gov (United States)

    Teixeira, Andreia; Flourat, Amandine; Peru, Aurelien; Brunissen, Fanny; Allais, Florent

    2016-04-01

    Cellulose-derived levoglucosenone (LGO) has been efficiently converted into pure (S)-gamma-hydroxymethyl-alpha,beta-butenolide (HBO), a chemical platform suited for the synthesis of drugs, flavors and antiviral agents. This process involves two-steps: a lipase-catalyzed Baeyer-Villager oxidation of LGO followed by an acid hydrolysis of the reaction mixture to provide pure HBO. Response surface methodology (RSM), based on central composite face-centered (CCF) design, was employed to evaluate the factors effecting the enzyme-catalyzed reaction: pka of solid buffer (7.2 - 9.6), LGO concentration (0.5 - 1 M) and enzyme loading (55 - 285 PLU.mmol-1). Enzyme loading and pka of solid buffer were found to be important factors to the reaction efficiency (as measured by the conversion of LGO) while only the later had significant effects on the enzyme recyclability (as measured by the enzyme residual activity). LGO concentration influences both responses by its interaction with the enzyme loading and pka of solid buffer. The optimal conditions which allow to convert at least 80% of LGO in 2 hours at 40 °C and reuse the enzyme for a subsequent cycle were found to be: solid buffer pka = 7.5, [LGO] = 0.50 M and 113 PLU.mmol-1 for the lipase. A good agreement between experimental and predicted values was obtained and the validity of the model confirmed (p-value 0.05).

  11. Lipase production by recombinant strains of Aspergillus niger expressing a lipase-encoding gene from Thermomyces lanuginosus.

    Science.gov (United States)

    Prathumpai, Wai; Flitter, Simon J; McIntyre, Mhairi; Nielsen, Jens

    2004-11-01

    Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.

  12. Immobilization of Organic Solvent-Tolerant Lipase from Pseudomonas mendocina M-37 with Potential Synthetic Activities

    Directory of Open Access Journals (Sweden)

    Praveen Dahiya

    2014-01-01

    Full Text Available A thermostable solvent-tolerant lipase was isolated from Pseudomonas mendocina M-37. The lipase production medium was optimized for cost-effective production. Olive oil as a carbon source, and glycine as a nitrogen source were selected as the best for maximum lipase production. Medium optimization led to 3.75-fold increase in the lipase production. The extracellular lipase was purified 42.2-fold to homogeneity by precipitation using polyethyleneglycol, ultrafiltration and hydrophobic interaction chromatography. Its molecular mass, determined with sodium dodecyl sulphate polyacrylamide gel electrophoresis, was 32 kDa. The enzyme was further immobilized on microcrystalline cellulose. The lipase showed an optimal water activity of 0.53 for both, acidolysis and interesterification reactions. Six- to sevenfold increase in synthetic activity of immobilized lipase was observed when interesterification activity of 0.139 IU/mg and transesterification activity of 0.181 IU/mg, respectively, were obtained. This is the first report on Pseudomonas mendocina lipase with synthetic activity immobilized on microcrystalline cellulose.

  13. Silk-Cocoon Matrix Immobilized Lipase Catalyzed Transesterification of Sunflower Oil for Production of Biodiesel

    Directory of Open Access Journals (Sweden)

    Sushovan Chatterjee

    2014-01-01

    Full Text Available Biodiesel from sunflower oil using lipase chemically immobilized on silk-cocoon matrix in a packed-bed bioreactor was investigated. The immobilization was demonstrated by field-emission scanning electron microscopy and activity study. The lipase loading was 738.74 U (~0.01 g lipase powder/g-lipase-immobilized matrix. The Km (Michaelis-Menten constant of the free and the immobilized lipase was 451.26 μM and 257.26 μM, respectively. Low Km value of the immobilized lipase is attributed to the hydrophobic nature of the matrix that facilitated the substrate diffusion to the enzyme surface. The biodiesel yield of 81.62% was obtained at 48 hours reaction time, 6 : 1 methanol : oil ratio (v/v, and 30°C. The immobilized lipase showed high operational stability at 30°C. The substrate conversion was only marginally decreased till third cycle (each of 48 hours duration of the reaction since less than even 5% of the original activity was decreased in each of the second and third cycle. The findings demonstrated the potential of the silk-cocoon as lipase immobilization matrix for industrial production of biodiesel.

  14. Microbial lipase mediated by health beneficial modification of cholesterol and flavors in food products: A review.

    Science.gov (United States)

    Sharma, Ranjana; Sharma, Nivedita

    2017-06-14

    The tremendous need of lipase in varied applications in biotechnological increases its economical value in food and allied industries. Lipase has an impressive number of applications viz. enhancements of flavor in food products (Cheese, butter, alcoholic beverages, milk chocolate and diet control food stuffs), detergent industry in removing oil, grease stain, organic chemical processing, textile industry, oleochemical industry, cosmetic industry and also as therapeutic agents in pharmaceutical industries. This communication extends the frontier of lipase catalyzed benefits to human body by lowering serum cholesterol and enhancement of flavor in different food products. Among all, multiple innovations going on in the field of lipase applications are widening its scope in food industries consistently. Therefore in the present work an effort has been made to explore the utilization of lipase in the field of food product enhancement. Supplementation of food products with lipase results in modification of its physical, chemical and biochemical properties by enhancing its therapeutic activity. Lipases are the most important enzymes used in food industries. They are utilized as industrial catalysts for lipid hydrolysis. Because of lipases hydrolysis nature it is widely exploited to catalyze lipids or fats in different food products and enhancement of food flavors. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. The production and characterization of a new active lipase from Acremonium alcalophilum using a plant bioreactor.

    Science.gov (United States)

    Pereira, Eridan Orlando; Tsang, Adrian; McAllister, Tim A; Menassa, Rima

    2013-01-01

    Microorganisms are the most proficient decomposers in nature, using secreted enzymes in the hydrolysis of lignocellulose. As such, they present the most abundant source for discovery of new enzymes. Acremonium alcalophilum is the only known cellulolytic fungus that thrives in alkaline conditions and can be cultured readily in the laboratory. Its optimal conditions for growth are 30°C and pH 9.0-9.2. The genome sequence of Acremonium alcalophilum has revealed a large number of genes encoding biomass-degrading enzymes. Among these enzymes, lipases are interesting because of several industrial applications including biofuels, detergent, food processing and textile industries. We identified a lipA gene in the genome sequence of Acremonium alcalophilum, encoding a protein with a predicted lipase domain with weak sequence identity to characterized enzymes. Unusually, the predicted lipase displays ≈ 30% amino acid sequence identity to both feruloyl esterase and lipase of Aspergillus niger. LipA, when transiently produced in Nicotiana benthamiana, accumulated to over 9% of total soluble protein. Plant-produced recombinant LipA is active towards p-nitrophenol esters of various carbon chain lengths with peak activity on medium-chain fatty acid (C8). The enzyme is also highly active on xylose tetra-acetate and oat spelt xylan. These results suggests that LipA is a novel lipolytic enzyme that possesses both lipase and acetylxylan esterase activity. We determined that LipA is a glycoprotein with pH and temperature optima at 8.0 and 40°C, respectively. Besides being the first heterologous expression and characterization of a gene coding for a lipase from A. alcalophilum, this report shows that LipA is very versatile exhibiting both acetylxylan esterase and lipase activities potentially useful for diverse industry sectors, and that tobacco is a suitable bioreactor for producing fungal proteins.

  16. Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

    Directory of Open Access Journals (Sweden)

    Bijay Kumar Sethi

    2016-03-01

    Full Text Available Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

  17. Purification and characterization of extracellular lipase by geotrichum candidum of dairy origin

    International Nuclear Information System (INIS)

    Muhammad, A.; Ali, N.; Imran, M.; Bokhari, S.A.I.; Mukhtar, H.

    2017-01-01

    In the present study, thermostable lipase from Geotrichum candidum UCMA 91(ATCC 204307) was purified and characterized. Lipase produced after optimization of the various cultural and physico-chemical conditions was purified to homogeneity by two step methods of purification: ammonium sulfate precipitation and column chromatography. The enzyme was purified by 60% ammonium sulfate precipitation and lipase activity of 5.77U mg/sup -1/ was attained. Then, Sephadex G-75 was used for gel filtration chromatography and 62.36 fold purification was achieved. Molecular mass of lipase was estimated to be 59 KDa by using SDS-PAGE. It is also determined from the study that lipase showed stability at varying range of pH (5-12) and thermo stability (15-65 degree C). The lipase was completely inhibited by EDTA (3.98%) confirming it as a metalloprotease, whereas the enzyme was found to be stable in various organic solvents. The results demonstrate that lipase hydrolyzes vegetable oils, which validates its technological relevance for use in the dairy, pharmaceuticals and bakery industry. (author)

  18. Synthetic activity enhancement of membrane-bound lipase from Rhizopus chinensis by pretreatment with isooctane.

    Science.gov (United States)

    Wang, Dong; Xu, Yan; Teng, Yun

    2007-05-01

    The cell-bound lipase from Rhizopus chinensis CCTCC M201021 with high catalysis ability for ester synthesis was located as a membrane-bound lipase by the treatments of Yatalase firstly. In order to improve its synthetic activity in non-aqueous phase, the pretreatments of this enzyme with various organic solvents were investigated. The pretreatment with isooctane improved evidently the lipase synthetic activity, resulting in about 139% in relative synthetic activity and 115% in activity recovery. The morphological changes of mycelia caused by organic solvent pretreatments could influence the exposure of the membrane-bound enzyme from mycelia and the exhibition of the lipase activity. The pretreatment conditions with isooctane and acetone were further investigated, and the optimum effect was obtained by the isooctane pretreatment at 4 degrees C for 1 h, resulting in 156% in relative synthetic activity and 126% in activity recovery. When the pretreated lipases were employed as catalysts for the esterification production of ethyl hexanoate in heptane, higher initial reaction rate and higher final molar conversion were obtained using the lipase pretreated with isooctane, compared with the untreated lyophilized one. This result suggested that the pretreatment of the membrane-bound lipase with isooctane could be an effective method to substitute the lyophilization for preparing biocatalysts used in non-aqueous phase reactions.

  19. Valorization of Palm Oil Industrial Waste as Feedstock for Lipase Production.

    Science.gov (United States)

    Silveira, Erick A; Tardioli, Paulo W; Farinas, Cristiane S

    2016-06-01

    The use of residues from the industrial processing of palm oil as carbon source and inducer for microbial lipase production can be a way to add value to such residues and to contribute to reduced enzyme costs. The aim of this work was to investigate the feasibility of using palm oil industrial waste as feedstock for lipase production in different cultivation systems. Evaluation was made of lipase production by a selected strain of Aspergillus niger cultivated under solid-state (SSF) and submerged fermentation (SmF). Lipase activity levels up to 15.41 IU/mL were achieved under SSF. The effects of pH and temperature on the lipase activity of the SSF extract were evaluated using statistical design methodology, and maximum activities were obtained between pH 4.0 and 6.5 and at temperatures between 37 and 55 °C. This lipase presented good thermal stability up to 60 °C and higher specificity towards long carbon chain substrates. The results demonstrate the potential application of palm oil industrial residues for lipase production and contribute to the technological advances needed to develop processes for industrial enzymes production.

  20. Marine Fungal and Bacterial Isolates for Lipase Production: A Comparative Study.

    Science.gov (United States)

    Patnala, H S; Kabilan, U; Gopalakrishnan, L; Rao, R M D; Kumar, D S

    Lipases, belonging to the class of enzymes called hydrolases, can catalyze triglycerides to fatty acids and glycerol. They are produced by microbes of plant and animal origin, and also by marine organisms. As marine microorganisms thrive in extreme conditions, lipases isolated from their origin possess characteristics of extremozymes, retain its activity in extreme conditions and can catalyze few chemical reactions which are impossible otherwise relative to the lipase produced from terrestrial microorganisms. Lipases are useful in many industries like detergent, food, leather, pharmaceutical, diary, etc. Few commercial enzymes have been developed and the use of them in certain industries like dairy, soaps are proved to be beneficial. There are few research papers reporting the production of lipase from marine bacteria and fungi. Lipase production involves two types of fermentation processes-solid-state fermentation (SSF) and submerged fermentation (SmF). Although SmF process is used conventionally, SSF process produces lipase in higher amounts. The production is also influenced by the composition of the medium, physiochemical parameters like temperature, pH, carbon, and nitrogen sources. © 2016 Elsevier Inc. All rights reserved.

  1. A newly high alkaline lipase: an ideal choice for application in detergent formulations

    Directory of Open Access Journals (Sweden)

    Cherif Slim

    2011-11-01

    Full Text Available Abstract Background Bacterial lipases received much attention for their substrate specificity and their ability to function in extreme environments (pH, temperature.... Many staphylococci produced lipases which were released into the culture medium. Reports of thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described. Results A newly soil-isolated Staphylococcus sp. strain ESW secretes an induced lipase in the culture medium. The effects of temperature, pH and various components in a detergent on the activity and stability of Staphylococcus sp. lipase (SL1 were studied in a preliminary evaluation for use in detergent formulation solutions. The enzyme was highly active over a wide range of pH from 9.0 to 13.0, with an optimum at pH 12.0. The relative activity at pH 13.0 was about 60% of that obtained at pH 12.0. It exhibited maximal activity at 60°C. This novel lipase, showed extreme stability towards non-ionic and anionic surfactants after pre-incubation for 1 h at 40°C, and relative stability towards oxidizing agents. Additionally, the crude enzyme showed excellent stability and compatibility with various commercial solid and liquid detergents. Conclusions These properties added to the high activity in high alkaline pH make this novel lipase an ideal choice for application in detergent formulations.

  2. Chemoenzymatic dynamic kinetic resolution of primary amines using a recyclable palladium nanoparticle catalyst together with lipases.

    Science.gov (United States)

    Gustafson, Karl P J; Lihammar, Richard; Verho, Oscar; Engström, Karin; Bäckvall, Jan-E

    2014-05-02

    A catalyst consisting of palladium nanoparticles supported on amino-functionalized siliceous mesocellular foam (Pd-AmP-MCF) was used in chemoenzymatic dynamic kinetic resolution (DKR) to convert primary amines to amides in high yields and excellent ee's. The efficiency of the nanocatalyst at temperatures below 70 °C enables reaction conditions that are more suitable for enzymes. In the present study, this is exemplified by subjecting 1-phenylethylamine (1a) and analogous benzylic amines to DKR reactions using two commercially available lipases, Novozyme-435 (Candida antartica Lipase B) and Amano Lipase PS-C1 (lipase from Burkholderia cepacia) as biocatalysts. The latter enzyme has not previously been used in the DKR of amines because of its low stability at temperatures over 60 °C. The viability of the heterogeneous Pd-AmP-MCF was further demonstrated in a recycling study, which shows that the catalyst can be reused up to five times.

  3. A model of extracellular enzymes in free-living microbes: Which strategy pays off?

    DEFF Research Database (Denmark)

    Traving, Sachia J; Thygesen, Uffe Høgsbro; Riemann, Lasse

    2015-01-01

    An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes...... must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy...

  4. Usage of immobilized porcine pancreas lipase in the hydrolysis of roselle (Hibiscus sabdariffa L.) seed oil

    Science.gov (United States)

    Ai, Chau Tran Diem; Linh, Vo Thi Hong; Yen, Tran Thi Ngoc; Nguyen, Nguyen Thi; Hoa, Phan Ngoc

    2017-09-01

    This study focused on the comparison among the usage of immobilized porcine pancreas lipase (PPL) on different hydrotalcite carriers (uncalcined and calcined hydrotalcite - like compound Mg /Al) and free lipase as the catalysts to hydrolyze of roselle (Hibiscus sabdariffa L.) seed oil. The reaction conditions were investigated including the ratio of oil to buffer, ratio of enzyme to substrate, the temperature of the hydrolysis, pH. The calcined hydrotalcite showed a higher lipase immobilization yield and a better reusability than the uncalcined hydrotalcite (87.15% and 86.78%, respectively).

  5. Effect of water content and temperature on Carica papaya lipase catalyzed esterification and transesterification reactions

    Directory of Open Access Journals (Sweden)

    Turon Fabrice

    2003-09-01

    Full Text Available Temperature and water activity (a w of the reaction medium are two factors that govern enzyme reactions. We studied the influence of these two parameters on the esterification and transesterification activity of Carica papaya lipase in water and solvent free reactions. It was found that over the course of reaction the catalytic activity of C. papaya lipase was dependent on these factors. The best lipase activity for both reactions was at a temperature of 55°C and water activity of 0.22, which corresponds to 2 g of water per 100 g of C. papaya latex.

  6. Burkholderia cepacia lipase is a promising biocatalyst for biofuel production.

    Science.gov (United States)

    Sasso, Francesco; Natalello, Antonino; Castoldi, Simone; Lotti, Marina; Santambrogio, Carlo; Grandori, Rita

    2016-07-01

    Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Dual bioimprinting of Thermomyces lanuginosus lipase for synthesis of biodiesel

    Directory of Open Access Journals (Sweden)

    Joyeeta Mukherjee

    2016-06-01

    Full Text Available Use of biodiesel as an alternative to non-renewable sources of energy has become an attractive option in recent years. The enzymatic synthesis of biodiesel by transesterification of fats/oils with an alcohol is a much more sustainable route than the chemical method. However, cost effectiveness of the enzymatic route is a major barrier in its commercialization. In this work, a high activity biocatalyst design of Thermomyces lanuginosus lipase is made by dually bioimprinting it with substrate and a surfactant (which is believed to open up the lid covering the active site of the lipase during precipitation of the lipase in organic solvent. When the lipase was bioimprinted with only the surfactants, 28 U of the enzyme/g of oil could yield 99% biodiesel from soybean oil in about 4 h. However, when dually bioimprinted even very low enzyme load 1.4 U/g of oil, yielded 99% biodiesel within 48 h.

  8. One-pot synthesis of (R)-1-(pyridin-4-yl)ethyl acetate using tandem catalyst prepared by co-immobilization of palladium and lipase on mesoporous foam: Optimization and kinetic modeling.

    Science.gov (United States)

    Magadum, Deepali B; Yadav, Ganapati D

    2017-12-01

    The synthesis of (R)-1-(pyridin-4-yl)ethyl acetate was achieved over tandem palladium-lipase catalyst with 100% selectivity using 4-acetyl pyridine as a reactant. The 2% w/w palladium and lipase catalyst was successfully co-immobilized in the microenvironment of the mesocellular foam and characterized by various techniques. The palladium metal from catalyst hydrogenated 4-acetyl pyridine to form 1-(pyridin-4-yl)ethanol. The generated intermediate product then underwent kinetic resolution over lipase and selectively gave (R)-1-(pyridin-4- yl)ethyl acetate. The catalytic conditions were then studied for optimal performance of both steps. The reaction conditions were optimized to 50 °C and toluene as a solvent. Both chemical and enzymatic kinetic models of the reaction were developed for a given set of reaction conditions and kinetic parameters were predicted. At optimal conditions, the obtained selectivity of intermediate (1-(pyridin-4-yl)ethanol) was 51.38%. The final product yield of ((R)-1-(pyridin-4-yl)ethyl acetate) was 48.62%. © 2017 Wiley Periodicals, Inc.

  9. Purification and characterization of lipase by Bacillus methylotrophicus PS3 under submerged fermentation and its application in detergent industry

    Directory of Open Access Journals (Sweden)

    Pushpinder Sharma

    2017-12-01

    Full Text Available Lipase production bacterial isolate was isolated from soil of service station and identified as Bacillus methylotrophicus PS3 by 16SrRNA with accession number |LN999829.1|. Lipase enzyme was purified by sequential methods of ammonium sulfate precipitation and Sephadex G-100 gel column chromatography. The molecular weight of purified enzyme was 31.40 kDa on SDS-PAGE. This purification procedure resulted in 2.90-fold purification of lipase with a 24.10% final yield. The purified lipase presented maximal hydrolytic activity at a temperature of 55 °C, and pH of 7.0. Lipase activity was stimulated by Triton X-100 and SDS with Mg2+ and Ca2+ metals employ a positive effect and outlast its stable in organic solvent i.e. methanol and ethanol.

  10. Enhanced Promiscuity of Lipase-Inorganic Nanocrystal Composites in the Epoxidation of Fatty Acids in Organic Media.

    Science.gov (United States)

    Hua, Xiufu; Xing, Yi; Zhang, Xuan

    2016-06-29

    In the present study, Candida antarctica lipase B (CALB) was encapsulated in inorganic nanocrystal composites with flower-like shapes, retaining 92% of its catalytic activity compared to that of native lipase. Surprisingly, CALB-inorganic crystal nanoflowers exhibited promiscuous activity at levels 25- and 4-fold higher than those of native lipase and the commercial immobilized lipase Novozym 435, respectively, as demonstrated by the chemoenzymatic epoxidation of fatty acids conducted in organic media. To the best of our knowledge, we showed for the first time that the promiscuity of enzymes can be significantly improved by enzyme immobilization, suggesting that the enzyme-inorganic nanocrystal composites are a very promising type of immobilized enzyme that can be used to address the challenge of the extremely low efficiency of enzymatic promiscuity.

  11. Lipase-catalyzed highly enantioselective kinetic resolution of boron-containing chiral alcohols.

    Science.gov (United States)

    Andrade, Leandro H; Barcellos, Thiago

    2009-07-16

    The first application of enzymes as catalysts to obtain optically pure boron compounds is described. The kinetic resolution of boron-containing chiral alcohols via enantioselective transesterification catalyzed by lipases was studied. Aromatic, allylic, and aliphatic secondary alcohols containing a boronate ester or boronic acid group were resolved by lipase from Candida antartica (CALB), and excellent E values (E > 200) and high enantiomeric excesses (up to >99%) of both remaining substrates and acetylated product were obtained.

  12. Catalytic properties of a lipase from Photobacterium lipolyticum for biodiesel production containing a high methanol concentration.

    Science.gov (United States)

    Yang, Kyung Seok; Sohn, Jung-Hoon; Kim, Hyung Kwoun

    2009-06-01

    Biodiesel, an alternative fuel, is generated via the transesterification reaction of vegetable oil or animal oil with alcohol. Currently, many reports have noted that microbial lipases might be utilized for the production of biodiesel. Among them, immobilized Candida antarctica lipase B (Novozym435) is frequently utilized for its biocatalytic efficiency and availability. However, as the enzyme is unstable in a medium containing high concentrations of methanol, a multi-stepwise methanol supply is required for the efficient production of biodiesel. Photobacterium lipolyticum lipase (M37) was determined to be quite stable in a medium containing a high concentration of methanol. The enzyme activity was maintained for longer than 48 h without any loss at a methanol concentration of 10%. In an effort to evaluate enzyme performance in the production of biodiesel, we have compared M37 lipase and Novozym435 in the biodiesel production reaction using fresh or waste oil and methanol. In the 3-stepwise methanol feeding method generally conducted for Novozym435 in biodiesel production, the M37 lipase showed a similar or superior conversion yield to Novozym435. However, the M37 lipase evidenced significantly higher conversion yields in the 2 and 1 step methanol feeding reactions. Particularly in the 1 step process using 10% of methanol where almost no conversion was detected by Novozym435, the biodiesel yield achieved with M37 lipase reached a level of up to 70% of the possible maximum yield. Consequently, this methanol-tolerant lipase, M37, has been shown to be a suitable enzyme for use in the biodiesel production process.

  13. Enhancement of sequential zymography technique for the detection of thermophilic lipases and proteases.

    Science.gov (United States)

    Wilkesman, Jeff; Hernández, Zully; Fernández, Marleny; Contreras, Lellys M; Kurz, Liliana

    2014-05-01

    Analysis of lipases and proteases present in cell-free fractions of thermophilic Bacillus sp. cultures were performed in an enhanced sequential zymography method. After the PAGE run, the gel was electrotransferred to another polyacrylamide gel containing a mixture of glycerol tributyrate, olive oil and gelatin. After transference, this substrate-mix gel was incubated for lipase detection, until bands appeared, and later stained with CBB for protease detection. Assets are, besides detecting two enzymes on a single gel, time and material saving.

  14. Medium-chain versus long-chain triacylglycerol emulsion hydrolysis by lipoprotein lipase and hepatic lipase: Implications for the mechanisms of lipase action

    International Nuclear Information System (INIS)

    Deckelbaum, R.J.; Hamilton, J.A.; Butbul, E.; Gutman, A.; Moser, A.; Bengtsson-Olivecrona, G.; Olivecrona, T.; Carpentier, Y.A.

    1990-01-01

    To explore how enzyme affinities and enzyme activities regulate hydrolysis of water-insoluble substrates, the authors compared hydrolysis of phospholipid-stabilized emulsions of medium-chain (MCT) versus long-chain triacylglycerols (LCT). Because substrate solubility at the emulsion surface might modulate rates of hydrolysis, the ability of egg yolk phosphatidylcholine to solubilize MCT was examined by NMR spectroscopy. Chemical shift measurements showed that 11 mol % of [ 13 C]carbonyl enriched trioctanoin was incorporated into phospholipid vesicles as a surface component. Line widths of trioctanoin surface peaks were half that of LCT, and relaxation times, T 1 , were also shorter for trioctanoin, showing greater mobility for MCT in phospholipid. In assessing the effects of these differences in solubility on lipolysis, they found that both purified bovine milk lipoprotein lipase and human hepatic lipase hydrolyzed MCT at rates at least 2-fold higher than for LCT. Differences in affinity were also demonstrated in mixed incubations where increasing amounts of LCT emulsion resulted in decreased hydrolysis of MCT emulsions. These results suggest that despite lower enzyme affinity for MCT emulsions, shorter chain triacylglycerols are more readily hydrolyzed by lipoprotein and hepatic lipases than long-chain triacylglycerols because of greater MCT solubility and mobility at the emulsion-water interface

  15. Study on optimization of process parameters for enhancing the multi-hydrolytic enzyme activity in garbage enzyme produced from preconsumer organic waste.

    Science.gov (United States)

    Arun, C; Sivashanmugam, P

    2017-02-01

    The garbage enzymes produced from preconsumer organic waste containing multi hydrolytic enzyme activity which helps to solubilize the waste activated sludge. The continuous production of garbage enzyme and its scaling up process need a globe optimized condition. In present study the effect of fruit peel composition and sonication time on enzyme activity were investigated. Garbage enzyme produced from 6g pineapple peels: 4g citrus peels pre-treated with ultrasound for 20min shows higher hydrolytic enzymes activity. Simultaneously statistical optimization tools were used to model garbage enzyme production with higher activity of amylase, lipase and protease. The maximum activity of amylase, lipase and protease were predicted to be 56.409, 44.039, 74.990U/ml respectively at optimal conditions (pH (6), temperature (37°C), agitation (218 RPM) and fermentation duration (3days)). These optimized conditions can be successfully used for large scale production of garbage enzyme with higher hydrolytic enzyme activity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Enzyme kinetics modeling as a tool to optimize food industry: a pragmatic approach based on amylolytic enzymes.

    Science.gov (United States)

    Galanakis, Charis M; Patsioura, Anna; Gekas, Vassilis

    2015-01-01

    Modeling is an important tool in the food industry since it is able to simplify explanation of phenomena and optimize processes that cover a broad field from manufacture to byproducts treatment. The goal of the current article is to explore the development of enzyme kinetic models and their evolution over the last decades. For this reason, corresponding simulations were classified in deterministic, empirical, and stochastic models, prior investigating limitations, corrections, and industrial applications in each case. The ultimate goal is to provide an answer to a major problem: how can we develop an intermediate complexity model that achieves satisfactorily representation of the main phenomena with a limited number of parameters?

  17. Biotechnological Utilisation of Fusel Oil, A Food Industry By-Product - A Kinetic Model on Enzymatic Esterification of i-Amyl Alcohol and Oleic Acid by Candida antarctica Lipase B

    Directory of Open Access Journals (Sweden)

    László Gubicza

    2008-01-01

    Full Text Available Fusel oil is a by-product of distilleries, its main component is i-amyl alcohol, which can form ester compounds. Esterification of oleic acid and i-amyl alcohol by Candida antarctica lipase B (Novozym 435 preparation in n-heptane solvent was studied in this work. Ping-pong bi-bi mechanism (inhibition phenomena taken into account was applied as a complex kinetic model. The parameters of the model were determined by numerical methods. It was found that four-parameter model fitted well with the experimental results and described properly the enzymatic reaction.

  18. Enhanced catalytic activity of lipase encapsulated in PCL nanofibers.

    Science.gov (United States)

    Song, Jie; Kahveci, Derya; Chen, Menglin; Guo, Zheng; Xie, Erqing; Xu, Xuebing; Besenbacher, Flemming; Dong, Mingdong

    2012-04-10

    Use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Enzyme immobilization as an effective strategy for improving the enzyme activity has emerged from developments especially in nanoscience and nanotechnology. Here, lipase from Burkholderia cepacia (LBC), as an example of the luxuriant enzymes, was successfully encapsulated in polycaprolactone (PCL) nanofibers, proven by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Evaluated in both organic and aqueous medium, the activation factor of the encapsulated enzymes in the hydrolysis reaction was generally higher than that in the transesterification reaction. Enhanced catalytic activities were found when 5-20 w/w % of LBC was loaded. The effect of different solvents pretreatment on the activity of immobilized LBC was also investigated. The highest activation factor was found up to 14 for the sample containing acetone-treated LBC/PCL (10 w/w %). The encapsulated lipase reserved 50% of its original activity after the 10th run in the transesterification reaction in hexane medium. The mechanism of activation of lipase catalytic ability based on active PCL nanofiberous matrix is proposed. © 2012 American Chemical Society

  19. Response surface analysis for the production of an enantioselective lipase from Aspergillus niger by solid-state fermentation.

    Science.gov (United States)

    Contesini, Fabiano Jares; da Silva, Vania Castriani Fernades; Maciel, Rafael Ferreira; de Lima, Rosemary Joana; Barros, Francisco Fábio Cavalcante; de Oliveira Carvalho, Patrícia

    2009-10-01

    The lipase produced by the Aspergillus niger strain AC-54 has been widely studied due to its enantioselectivity for racemic mixtures. This study aimed to optimize the production of this enzyme using statistical methodology. Initially a Plackett-Burman (PB) design was used to evaluate the effects of the culture medium components and the culture conditions. Twelve factors were screened: water content, glucose, yeast extract, peptone, olive oil, temperature, NaH(2)P0(4), KH(2)P0(4), MgS0(4)-7H(2)0, CaCl(2), NaCI, and MnS0(4). The screening showed that the significant factors were water content, glucose, yeast extract, peptone, NaH(2)P0(4), and KH(2)P0(4), which were optimized using response surface methodology (RSM) and a mathematical model obtained to explain the behavioral process. The best lipase activity was attained using the following conditions: water content (20%), glucose (4.8%), yeast extract (4.0%), and NaH2P04 (4.0%). The predicted lipase activity was 33.03 U/ml and the experimental data confirmed the validity of the model. The enzymatic activity was expressed as micromoles of oleic acid released per minute of reaction (micromol/min).

  20. Structure and Function of Lipase

    DEFF Research Database (Denmark)

    Skjold-Jørgensen, Jakob

    investigations structural movements associated with lipaseactivation at medium to high solvent polarities. The findings strengthen and expand on the understandingof the lid’s role in the activation of lipases, and have created interesting perspectives in designing futurelipases with faster activation, controlled.......e. the waterlipidinterface. For Thermomyces lanuginosus lipase (TlL) and related lipases, activation of the enzymeinvolves a rearrangement of a structural domain, called the “lid”, which covers the active site inhomogenous aqueous solution. At the water-lipid interface, the lid is displaced from the active site andmoves...... towards an open conformation enabling the substrate to gain access, thus initiating catalysis.Lipases have been studied for decades and their functional features have drawn much attention withinindustrial applications since their first discovery. However, given that their molecular action takes placeat...

  1. Biodiesel production with immobilized lipase: A review.

    Science.gov (United States)

    Tan, Tianwei; Lu, Jike; Nie, Kaili; Deng, Li; Wang, Fang

    2010-01-01

    Fatty acid alkyl esters, also called biodiesel, are environmentally friendly and show great potential as an alternative liquid fuel. Biodiesel is produced by transesterification of oils or fats with chemical catalysts or lipase. Immobilized lipase as the biocatalyst draws high attention because that process is "greener". This article reviews the current status of biodiesel production with immobilized lipase, including various lipases, immobilization methods, various feedstocks, lipase inactivation caused by short chain alcohols and large scale industrialization. Adsorption is still the most widely employed method for lipase immobilization. There are two kinds of lipase used most frequently especially for large scale industrialization. One is Candida antartica lipase immobilized on acrylic resin, and the other is Candida sp. 99-125 lipase immobilized on inexpensive textile membranes. However, to further reduce the cost of biodiesel production, new immobilization techniques with higher activity and stability still need to be explored. Copyright 2010 Elsevier Inc. All rights reserved.

  2. Characterization of Aspergillus Niger 65i6 lipase from solid-state fermentation using Jatropha seed cake medium

    OpenAIRE

    Hidayat, Chusnul; Darmasiwi, Sari; Nurikasari, Maulina; Cahyanto, Muhammad Nur

    2017-01-01

    Jatropha curcas seed cake contains a high amount of protein, and consequently has very high potentialas a medium for lipase production. The objective of this research was to characterize lipase from Aspergillusniger 6516, which was produced by solid-state fermentation on Jatropha curcas seed cake as the medium. The effects of pH and temperature on enzyme activity were evaluated, along with substrate specifcity and enzyme stability. Fermentation was performed at a water concentration of 63% an...

  3. Enzymatic activity of a novel halotolerant lipase from Haloarcula hispanica 2TK2

    Directory of Open Access Journals (Sweden)

    Ozgen Melis

    2016-06-01

    Full Text Available A strain of Haloarcula hispanica isolated from Tuzkoy salt mine, Turkey exhibited extracellular lipolytic activity. Important parameters such as carbon sources and salt concentration for lipase production were investigated. Optimal conditions for the enzyme production from Haloarcula hispanica 2TK2 were determined. It was observed that the lipolytic activity of Haloarcula hispanica was stimulated by some of the carbon sources. The high lipase acitivity values were obtained in the presence of 2% (v/v walnut oil (6.16 U/ml, 1% (v/v fish oil (5.07 U/ml, 1% (v/v olive oil (4.52 U/ml and 1% (w/v stearic acid (4.88 U/ml at 4M NaCl concentration. Lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Optimal temperature and pH values were determined as 45°C and 8.0, respectively. Lipase activity decreased with the increasing salt concentration, but 85% activity of the enzyme was maintained at 5M NaCl concentration. The enzyme preserved 41% of its relative activity at 90°C. The partially purified lipase maintained its activity in the presence of surfactants such as Triton X-100 and SDS. Therefore, the lipase which is an extremozyme may have potential applications especially in detergent industry.

  4. Effect of immobilized lipase supplementation of diets on the performance of the Japanese quails

    International Nuclear Information System (INIS)

    Abu-Taleb, A.M.; Ezzat, I.E.; Saleh, M.

    2004-01-01

    In the present study, lipase was immobilized onto two different supports, agarose and gelatin. Some physico-chemical properties of the free and immobilized lipase such as optimum temperature, optimum ph and storage stability were studied. Storage of the enzymes for 2 months showed that the free enzyme lost its activity, while the immobilized on the gelatin showed better resistance towards ph and temperature variations than that immobilized onto agarose. Four experiments were conducted to test the effect of the immobilized lipase supplementation on the productive performance of the Japanese quails. During the first 3 weeks, the addition of lipase to poultry diets caused an increase in the body weight gain of birds than the enzyme-free diet. An obvious improvement in quail day egg production during the laying period was observed with the groups fed on a diet supplemented with 3000 and 2000 I U of immobilized lipase per kilogram feed. Blood cholesterol was not affected with lipase addition, while total lipids were significantly increased. Significant reduction was also observed in thyroid hormones (T 3 and T 4 ) as compared with the control group

  5. Effects of methanol on lipases: molecular, kinetic and process issues in the production of biodiesel.

    Science.gov (United States)

    Lotti, Marina; Pleiss, Jürgen; Valero, Francisco; Ferrer, Pau

    2015-01-01

    The biotechnological production of biodiesel is based on transesterification/esterification reactions between a source of fatty acids and a short-chain alcohol, usually methanol, catalysed by enzymes belonging to the class known as lipases. Several lipases used in industrial processes, although stable in the presence of other organic solvents, are inactivated by methanol at or below the concentration optimal for biodiesel production, making it necessary to use stepwise methanol feeding or pre-treatment of the enzyme. In this review article we focus on what is currently know about methanol inactivation of lipases, a phenomenon which is not common to all lipase enzymes, with the goal of improving the biocatalytic process. We suggest that different mechanisms can lead to inactivation of different lipases, in particular substrate inhibition and protein unfolding. Attempts to improve the performances of methanol sensitive lipases by mutagenesis as well as process engineering approaches are also summarized. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. From Structure to Catalysis: Recent Developments in the Biotechnological Applications of Lipases

    Directory of Open Access Journals (Sweden)

    Cristiane D. Anobom

    2014-01-01

    Full Text Available Microbial lipases are highly appreciated as biocatalysts due to their peculiar characteristics such as the ability to utilize a wide range of substrates, high activity and stability in organic solvents, and regio- and/or enantioselectivity. These enzymes are currently being applied in a variety of biotechnological processes, including detergent preparation, cosmetics and paper production, food processing, biodiesel and biopolymer synthesis, and the biocatalytic resolution of pharmaceutical derivatives, esters, and amino acids. However, in certain segments of industry, the use of lipases is still limited by their high cost. Thus, there is a great interest in obtaining low-cost, highly active, and stable lipases that can be applied in several different industrial branches. Currently, the design of specific enzymes for each type of process has been used as an important tool to address the limitations of natural enzymes. Nowadays, it is possible to “order” a “customized” enzyme that has ideal properties for the development of the desired bioprocess. This review aims to compile recent advances in the biotechnological application of lipases focusing on various methods of enzyme improvement, such as protein engineering (directed evolution and rational design, as well as the use of structural data for rational modification of lipases in order to create higher active and selective biocatalysts.

  7. Properties of Immobilized Candida antarctica Lipase B on Highly Macroporous Copolymer

    NARCIS (Netherlands)

    Handayani, Nurrahmi; Miletic, Nemanja; Loos, Katja; Achmad, Sadijah; Wahyuningrum, Deana

    In spite of their excellent catalytic properties, enzymes should be improved before their implementation both in industrial and laboratorium scales. Immobilization of enzyme is one of the ways to improve their properties. Candida antarctica lipase B (Cal-B) has been reported in numerous publications

  8. Quarternary structure and enzymological properties of the different hormone-sensitive lipase (HSL) isoforms

    DEFF Research Database (Denmark)

    Krintel, Christian; Klint, Cecilia; Lindvall, Håkan

    2010-01-01

    Hormone-sensitive lipase (HSL) is a key enzyme in the mobilization of energy in the form of fatty acids from intracellular stores of neutral lipids. The enzyme has been shown to exist in different isoforms with different molecular masses (84 kDa, 89 kDa and 117 kDa) expressed in a tissue-dependen...

  9. Improved triglyceride transesterification by circular permuted Candida antarctica lipase B.

    Science.gov (United States)

    Yu, Ying; Lutz, Stefan

    2010-01-01

    Lipases represent a versatile class of biocatalysts with numerous potential applications in industry including the production of biodiesel via enzyme-catalyzed transesterification. In this article, we have investigated the performance of cp283, a variant of Candida antarctica lipase B (CALB) engineered by circular permutation, with a series of esters, as well as pure and complex triglycerides. In comparison with wild-type CALB, the permutated enzyme showed consistently higher catalytic activity (2.6- to 9-fold) for trans and interesterification of the different substrates with 1-butanol and ethyl acetate as acyl acceptors. Differences in the observed rates for wild-type CALB and cp283 are believe to be related to changes in the rate-determining step of the catalytic cycle as a result of circular permutation.

  10. Covalent immobilization of Pseudomonas cepacia lipase on semiconducting materials

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez, Renny Edwin [Microelectronics and MEMS Laboratory, Department of Electrical Engineering, Indian Institute of Technology Madras, Chennai (India)], E-mail: rennyedwin@gmail.com; Bhattacharya, Enakshi [Microelectronics and MEMS Laboratory, Department of Electrical Engineering, Indian Institute of Technology Madras, Chennai (India)], E-mail: enakshi@ee.iitm.ac.in; Chadha, Anju [Department of Biotechnology, National Centre for Catalysis Research, Indian Institute of Technology Madras, Chennai (India)], E-mail: anjuc@iitm.ac.in

    2008-05-30

    Lipase from Pseudomonas cepacia was covalently immobilized on crystalline silicon, porous silicon and silicon nitride surfaces. The various stages of immobilization were characterized using FTIR (Fourier transform infrared) spectroscopy. The surface topography of the enzyme immobilized surfaces was investigated using scanning electron microscopy (SEM). The quantity of the immobilized active enzyme was estimated by the para-nitrophenyl palmitate (pNPP) assay. The immobilized lipase was used for triglyceride hydrolysis and the acid produced was detected by a pH sensitive silicon nitride surface as a shift in the C-V (capacitance-voltage) characteristics of an electrolyte-insulator-semiconductor capacitor (EISCAP) thus validating the immobilization method for use as a biosensor.

  11. Metal-coordinated phenoxyl radicals: Models for the enzyme ...

    Indian Academy of Sciences (India)

    Administrator

    In recent years, studies on metal coordinated phenoxyl radicals have gained importance due to their biochemical significance. Such radicals are involved in the action of various enzymes like galactose oxidase, ribonucleotide reductase etc. The present report deals with the synthesis and characterization of new ligand ...

  12. Heterologous Expression and Purification of a Heat-Tolerant Staphylococcus xylosus Lipase.

    Science.gov (United States)

    Brod, Fábio Cristiano Angonesi; Pelisser, Márcia Regina; Bertoldo, Jean Borges; Vernal, Javier; Bloch, Carlos; Terenzi, Hernán; Arisi, Ana Carolina Maisonnave

    2010-02-01

    Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42 degrees C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC(2), pNPC(4), pNPC(10), pNPC(12), pNPC(14), pNPC(16), pNPC(18)). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95 degrees C, 77% of the initial activity was retained.

  13. Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

    Energy Technology Data Exchange (ETDEWEB)

    Cabot, M.C.

    1984-08-30

    Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56/sup 0/C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83-86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2-1.0 ..mu..M range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the (/sup 14/C)dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of (/sup 14/)dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never before documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols. 44 references, 3 figures, 1 table.

  14. Optimization of extracellular thermophilic highly alkaline lipase from thermophilic bacillus sp isolated from hotspring of Arunachal Pradesh, India

    Science.gov (United States)

    Bora, Limpon; Bora, Minakshi

    2012-01-01

    Studies on lipase production were carried out with a bacterial strain (Bacillus sp LBN 2) isolated from soil sample of hotspring of Arunachal Pradesh, India. The cells were cultivated in a mineral medium with maximum production at 1% groundnut oil. The optimum temperature and initial medium pH for lipase production by the organism were 500C and 9.0 respectively. The molecular mass was found to be 33KDa by SDS PAGE. The optimal pH and temperature for activity were 10 and 600C respectively. The enzyme was found to be stable in the pH range of 8–11 with 90% retention of activity at pH 11. The enzyme retained 90% activity at 600C and 70% of activity at 700C for 1h. The lipase was found to be stable in acetone followed by ethanol. The present findings suggested the enzyme to be thermophilic alkaline lipase. PMID:24031801

  15. Magnetically responsive enzyme powders

    International Nuclear Information System (INIS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-01-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction

  16. Modelling of different enzyme productions by solid-state fermentation on several agro-industrial residues.

    Science.gov (United States)

    Diaz, Ana Belen; Blandino, Ana; Webb, Colin; Caro, Ildefonso

    2016-11-01

    A simple kinetic model, with only three fitting parameters, for several enzyme productions in Petri dishes by solid-state fermentation is proposed in this paper, which may be a valuable tool for simulation of this type of processes. Basically, the model is able to predict temporal fungal enzyme production by solid-state fermentation on complex substrates, maximum enzyme activity expected and time at which these maxima are reached. In this work, several fermentations in solid state were performed in Petri dishes, using four filamentous fungi grown on different agro-industrial residues, measuring xylanase, exo-polygalacturonase, cellulose and laccase activities over time. Regression coefficients after fitting experimental data to the proposed model turned out to be quite high in all cases. In fact, these results are very interesting considering, on the one hand, the simplicity of the model and, on the other hand, that enzyme activities correspond to different enzymes, produced by different fungi on different substrates.

  17. Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

    Directory of Open Access Journals (Sweden)

    Ana Caroline Lustosa de Melo Carvalho

    2015-12-01

    Full Text Available Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015, concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs and their intermediates in which the key step involves the action of a lipase.

  18. Recent Advances in Lipase-Mediated Preparation of Pharmaceuticals and Their Intermediates

    Science.gov (United States)

    Carvalho, Ana Caroline Lustosa de Melo; Fonseca, Thiago de Sousa; de Mattos, Marcos Carlos; de Oliveira, Maria da Conceição Ferreira; de Lemos, Telma Leda Gomes; Molinari, Francesco; Romano, Diego; Serra, Immacolata

    2015-01-01

    Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the resolution process of racemates, without the use of cofactors. Moreover, this class of enzymes has generally excellent stability in the presence of organic solvents, facilitating the solubility of the organic substrate to be modified. Further improvements and new applications have been achieved in the syntheses of biologically active compounds catalyzed by lipases. This review critically reports and discusses examples from recent literature (2007 to mid-2015), concerning the synthesis of enantiomerically pure active pharmaceutical ingredients (APIs) and their intermediates in which the key step involves the action of a lipase. PMID:26690428

  19. Enzymatic properties of lipase and characteristics production by Lactobacillus delbrueckii subsp. bulgaricus.

    Science.gov (United States)

    el-Sawah, M M; Sherief, A A; Bayoumy, S M

    1995-01-01

    Some properties of an extracellular lipase produced by Lactobacillus delbrueckii subsp. bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl2 to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40 degrees C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35 degrees C gave the highest lipolytic activity.

  20. Synthesis activity-based zymography for detection of lipases and esterases.

    Science.gov (United States)

    Kwon, Min-A; Kim, Hyun Suk; Hahm, Dae-Hyun; Song, Jae Kwang

    2011-04-01

    A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.

  1. PRODUCTION OF MEDIUM-CHAIN ACYLGLYCEROLS BY LIPASE ESTERIFICATION IN PACKED BED REACTOR: PROCESS OPTIMIZATION BY RESPONSE SURFACE METHODOLOGY

    Directory of Open Access Journals (Sweden)

    ZANARIAH MOHD DOM

    2014-06-01

    Full Text Available Medium-chain acylglycerols (or glycerides are formed of mono-, di- and triacylglycerol classes. In this study, an alternative method to produce MCA from esterifying palm oil fatty acid distillate (PFAD with the presence of oil palm mesocarp lipase (OPML which is a plant-sourced lipase and PFAD is also cheap by-product is developed in a packed bed reactor. The production of medium-chain acylglycerols (MCA by lipase-catalysed esterification of palm oil fatty acid distillate with glycerol are optimize in order to determine the factors that have significant effects on the reaction condition and high yield of MCA. Response surface methodology (RSM was applied to optimize the reaction conditions. The reaction conditions, namely, the reaction time (30-240 min, enzyme load (0.5-1.5 kg, silica gel load (0.2-1.0 kg, and solvent amount (200-600 vol/wt. Reaction time, enzyme loading and solvent amount strongly effect MCA synthesis (p0.05 influence on MCA yield. Best-fitting models were successfully established for MCA yield (R 2 =0.9133. The optimum MCA yield were 75% from the predicted value and 75.4% from the experimental data for 6 kg enzyme loading, a reaction time of 135min and a solvent amount of 350 vol/wt at 65ºC reaction temperature. Verification of experimental results under optimized reaction conditions were conducted, and the results agreed well with the predicted range. Esterification products (mono-, di- and triacylglycerol from the PBR were identified using Thin Layer Chromatography method. The chromatograms showed the successful fractionation of esterified products in this alternative method of process esterification.

  2. Characterization and spray drying of lipase produced by the endophytic fungus Cercospora kikuchii

    Directory of Open Access Journals (Sweden)

    T. A. Costa-Silva

    2014-12-01

    Full Text Available A lipase from the endophytic fungus Cercospora kikuchii was purified, biochemically characterized and the effects of spray drying on stabilization of the purified enzyme were studied. The lipase was purified 9.31-fold with recovery of 26.6% and specific activity of 223.6 U/mg. The optimum pH and temperature were 4.6 and 35 ºC, respectively, while the Vmax was 10.28 µmol/min.mg-1 protein and Km 0.0324 mM. All the metal ions tested enhanced the enzyme activity. The lipase retained almost 100% activity in the presence of strong oxidants and was also resistant to Triton X, Tween 80 and 20 and SDS, as well as to proteases. The purified lipase was spray dried and kept until 85.2% of enzymatic activity. At least 70% of the enzymatic activity was maintained for spray dried purified lipase during the storage period. The lipase produced by Cercospora kikuchii has properties useful for industrial application and showed adequate stabilization and retention of its enzymatic activity after spray drying.

  3. Effect of culture conditions on lipase production by Fusarium solani in batch fermentation.

    Science.gov (United States)

    Maia, M M; Heasley, A; Camargo de Morais, M M; Melo, E H; Morais, M A; Ledingham, W M; Lima Filho, J L

    2001-01-01

    Lipase (Glycerol ester hydrolase EC 3.1.1.3.) from a Brazilian strain of Fusarium solani FSI has been investigated. The effect of different carbon sources and trace elements added to basal medium was observed with the aim of improving enzyme production. Lipase specific activity was highest (0.45 U mg(-1)) for sesame oil. When this medium was supplemented with trace elements using olive oil, corn oil and sesame oil the lipase specific activity increased to 0.86, 1.89 and 1.64 U mg(-1), respectively, after 96 h cultivation without any considerable biomass increase. The Km of this lipase using pNPP (p-nitrophenylpalmitate) as substrate, was 1.8 mM with a Vmax of 1.7 micromol min(-1) mg protein(-1). Lipase activity increased in the presence of increasing concentrations of hexane and toluene. In contrast, incubation of this enzyme with water-soluble solvents decreased its activity after 10% concentration (v/v) of the solvent. The lipase activity was stable below 35 degrees C but above this temperature activity losses were observed.

  4. Selective production of ricinoleic acid by hydrolysis of castor oil using lipase immobilized in N-polyisopropylacrylamide gel; Ripaze koteika N-poriisopuropiruakuriruamidogeru wo mochiita himashiyu no kasuibunaki ni yoru rishinorusan no sentakuteki seisei

    Energy Technology Data Exchange (ETDEWEB)

    Goto, M.; Hatanaka, C.; Haraguchi, T. [Kitakyushu National College of Technology, Fukuoka (Japan)

    2000-05-10

    Lipase from Candida cylindracea or Rhizopus was immobilized in gel beads prepared by copolymerization of N-isopropylacrylamide, N-N'-methylenebisacrylamide, and acrylamide. The hydrolysis reaction of castor oil was carried out at 37 degree C by using immobilized lipase or free lipase. The optimal condition of immobilization of lipase and the productivity of ricinoleic acid is investigated. It is found that thermal inactivation of enzyme was suppressed and the formation of by-products such as estolide decreases by immobilization into the gel compared with free lipase. (author)

  5. Utilization of coconut oil cake for the production of lipase using Bacillus coagulans VKL1.

    Science.gov (United States)

    Gowthami, Palanisamy; Muthukumar, Karuppan; Velan, Manickam

    2015-01-01

    The overproduction of enzymes was performed by manipulating the medium components. In our study, solvent-tolerant thermophilic lipase-producing Bacillus coagulans was isolated from soil samples and a stepwise optimization strategy was employed to increase the lipase production using coconut oil cake basal medium. In the first step, the influence of pH, temperature, carbon source, nitrogen source and inducers on lipase activity was investigated by the One-Factor-At-A-Time (OFAT) method. In the second step, the three significant factors resulted from OFAT were optimized by the statistical approach (CCD).The optimum values of olive oil (0.5%), Tween 80 (0.6%) and FeSO4 (0.05%) was found to be responsible for a 3.2-fold increase in the lipase production identified by Central Composite Design.

  6. Monoglycerides and Diglycerides Synthesis in a Solvent-Free System by Lipase-Catalyzed Glycerolysis

    Science.gov (United States)

    Fregolente, Patricia Bogalhos Lucente; Fregolente, Leonardo Vasconcelos; Pinto, Gláucia Maria F.; Batistella, Benedito César; Wolf-Maciel, Maria Regina; Filho, Rubens Maciel

    Five lipases were screened (Thermomyces lanuginosus free and immobilized forms, Candida antarctica B, Candida rugosa, Aspergillus niger, and Rhizomucor miehei) to study their ability to produce monoglycerides (MG) and diglycerides (DG) through enzymatic glycerolysis of soybean oil. Lipase from C. antarctica was further studied to verify the enzyme load (wt% of oil mass), the molar ratio glycerol/oil, and the water content (wt% of glycerol) on the glycerolysis reaction. The best DG and MG productions were in the range 45-48% and 28-30% (w/w, based on the total oil), respectively. Using immobilized lipases, the amount of free fatty acids (FFA) produced was about 5%. However, the amount of FFA produced when using free lipases, with 3.5% extra water in the system, is equivalent to the MG yield, about 23%. The extra water content provides a competition between hydrolysis and glycerolysis reactions, increasing the FFA production.

  7. Ecological screening of lipolytic cultures and process optimization for extracellular lipase production from fungal hyperproducer

    International Nuclear Information System (INIS)

    Iftikhar, T.; Niaz, M.; Anwer, M.; Abbas, S.Q.; Saleem, M.; Jabeen, R.

    2011-01-01

    Present investigation describes the biosynthesis of extracellular lipases by various local fungal strains isolated from various lipid rich habitats of Faisalabad. The isolated cultures of Aspergillus niger, Penicillium chrysogenum, Rhizopus microsporus, Mucor mucedo, Alternaria alternata, Trichophyton sp., Fusarium semitectum, E (un-identified), Curvularia sp., Aspergillus flavus, G (un-identified), F (Mucor sp.) and H (Synnematous) were identified and screened for the extracelluler lipases production. Different environmental parameters such as pH, temperature, inoculum size, amount of substrate and incubation time were optimized for the selected hyper producer. It was found that maximum production of lipases by Trichophyton sp., was obtained after 48 h of batch fermentation. Similarly, the diluent pH of 7.0 and incubation temperature of 30 deg. C were found optimum for enzyme production by the microorganism. The maximum production of lipases during the course of present studies was 65.20 +- 1.13a U/g. (author)

  8. MALDI imaging of enzymatic degradation of glycerides by lipase on textile surface

    DEFF Research Database (Denmark)

    Hall-Andersen, Jonatan; Kaasgaard, Svend G; Janfelt, Christian

    2018-01-01

    Most modern laundry detergents contain enzymes such as proteases, amylases, and lipases for more efficient removal of stains containing proteins, carbohydrates, and lipids during wash at low temperature. The function of the lipases is to hydrolyse the hydrophobic triglycerides from fats and oils...... to the more hydrophilic lipids diglycerides, monoglycerides and free fatty acids. Here, we use MALDI imaging to study the effect of enzymatic degradation of triglycerides by lipases directly on the textile surface. Textile samples were created by using swatches of different textile blends, adding a lipid...... stain and simulating washing cycles using well-defined detergents with lipase concentrations ranging between 0 and 0.5ppm. After washing, the textile swatches as well as cryo-sections of the swatches were imaged using MALDI imaging in positive ion mode at pixel sizes of 15-75μm. Similar samples were...

  9. Formation of polyol-fatty acid esters by lipases in reverse micellar media.

    Science.gov (United States)

    Hayes, D G; Gulari, E

    1992-06-05

    The synthesis of polyol-fatty acid esters has strong implications in such industries as foods, cosmetics, and polymers. We have investigated these esterification reactions employing the polyols ethylene glycol, 2-monoglyceride, and sugars and their derivatives with the biocatalyst lipase in water/AOT/isooctane reverse micellar media. For the first reaction, 50-60% conversion was achieved and product selectivity toward the monoester over the diester shown possible by employing lipase from Rhizopus delemar. A simple kinetic model based on the formation of acyl-enzyme intermediate accurately predicted the effect of polyol concentration but not the effect of fatty acid or water concentration probably due to the model exclusion of partitioning effects. The success of this reaction in reverse micellar media is due greatly to its capacity to solubilize large quantities of glycol despite the media's overall hydrophobicity. The second reaction, investigated for its potential for production of "mixed" glycerides, also achieved about 50% conversion but had only a small portion of triglyceride in its product distribution. Also, isomerization of the 2-monoglyceride to 1-monoglyceride, followed by hydrolysis of the latter, unfortunately occurred to a significant extent. Attempts at esterification with hexoses and their derivatives such as glucose and mannitol produced no conversion.

  10. A model of extracellular enzymes in free-living microbes: which strategy pays off?

    Science.gov (United States)

    Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

    2015-11-01

    An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Phosphonium alkyl PEG sulfate ionic liquids as coating materials for activation of Burkholderia cepacia lipase.

    Science.gov (United States)

    Matsubara, Yui; Kadotani, Shiho; Nishihara, Takashi; Hikino, Yoshichika; Fukaya, Yukinobu; Nokami, Toshiki; Itoh, Toshiyuki

    2015-12-01

    Lipases are among the most widely used enzymes applicable for various substrates; however, the slow reactions or poor enantioselective reactions are sometimes obtained. To develop ionic liquid type activating agents for lipase, four types of phosphonium cetyl(PEG)10 sulfate ionic liquids have been synthesized and used as coating materials of Burkholderia cepacia lipase (Lipase PS) through the lyophilization process. Tributyl ([2-methoxy]ethoxymethyl)phosphonium cetyl(PEG)10 sulfate ([P444MEM ][C16 (PEG)10 SO4 ]) (PL1) worked best among them, and PL1-coated lipase PS displayed high reactivity in transesterification of broad types of secondary alcohols using vinyl acetate as an acylating reagent with perfect enantioselectivity (E > 200). The substrate preference of PL1-PS differs from that of commercial lipase PS or [bdmim] [C16 (PEG)10 SO4 ]-coated lipase (IL1-PS); PL1-PS displayed excellent enantioselectivity in the reaction of 2-chloro-1-phenylethanol with E > 200, though insufficient E values were recorded for lipase PS (E = 12) and IL1-PS (E = 123) for this alcohol. PL1-PS also showed perfect enantioselectivity (E > 200) for the reaction of 1-(pyridin-2-yl)ethanol, while IL1-PS showed E = 130 for this compound. We further succeeded in demonstrating the recyclable use of PL1-PS five times in tributyl(3-methoxypropyl)phosphonium bis(trifluoromethylsulfonyl)amide ([P444PM ][Tf2 N]) as a solvent. Since PL1-PS is easily applicable to 10-20 gram-scaled reactions, it is expected that the IL-coated enzyme might be useful for practical preparation of a wide variety of chiral secondary alcohols. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.

    Science.gov (United States)

    Dhouib, R; Laroche-Traineau, J; Shaha, R; Lapaillerie, D; Solier, E; Rualès, J; Pina, M; Villeneuve, P; Carrière, F; Bonneu, M; Arondel, V

    2011-01-01

    Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex. © 2010 The Authors Journal compilation © 2010 FEBS.

  13. Magnetically responsive enzyme powders

    Science.gov (United States)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (-20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties.

  14. Encapsulation in a sol-gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue.

    Science.gov (United States)

    Zubiolo, Claudia; Santos, Rafaela Cristiane Andrade; Carvalho, Nayara Bezerra; Soares, Cleide Mara Faria; Lima, Alvaro Silva; de Aquino Santana, Luciana Cristina Lins

    2014-09-01

    Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol-gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t 1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis-Menten equation. The K m values indicated that the encapsulation process reduced enzyme-substrate affinity and the V max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.

  15. The Purification and Characterization of Lipases from Lasiodiplodia theobromae, and Their Immobilization and Use for Biodiesel Production from Coconut Oil.

    Science.gov (United States)

    Venkatesagowda, Balaji; Ponugupaty, Ebenezer; Barbosa-Dekker, Aneli M; Dekker, Robert F H

    2017-12-18

    The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)-purified 25.41-fold, recovery of 47.1%-and lipase B (32,000 Da)-purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca 2+ , exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5-10.0 and 20-80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.

  16. Comparative genomic analysis of the Lipase3 gene family in five plant species reveals distinct evolutionary origins.

    Science.gov (United States)

    Wang, Dan; Zhang, Lin; Hu, JunFeng; Gao, Dianshuai; Liu, Xin; Sha, Yan

    2018-04-01

    Lipases are physiologically important and ubiquitous enzymes that share a conserved domain and are classified into eight different families based on their amino acid sequences and fundamental biological properties. The Lipase3 family of lipases was reported to possess a canonical fold typical of α/β hydrolases and a typical catalytic triad, suggesting a distinct evolutionary origin for this family. Genes in the Lipase3 family do not have the same functions, but maintain the conserved Lipase3 domain. There have been extensive studies of Lipase3 structures and functions, but little is known about their evolutionary histories. In this study, all lipases within five plant species were identified, and their phylogenetic relationships and genetic properties were analyzed and used to group them into distinct evolutionary families. Each identified lipase family contained at least one dicot and monocot Lipase3 protein, indicating that the gene family was established before the split of dicots and monocots. Similar intron/exon numbers and predicted protein sequence lengths were found within individual groups. Twenty-four tandem Lipase3 gene duplications were identified, implying that the distinctive function of Lipase3 genes appears to be a consequence of translocation and neofunctionalization after gene duplication. The functional genes EDS1, PAD4, and SAG101 that are reportedly involved in pathogen response were all located in the same group. The nucleotide diversity (Dxy) and the ratio of nonsynonymous to synonymous nucleotide substitutions rates (Ka/Ks) of the three genes were significantly greater than the average across the genomes. We further observed evidence for selection maintaining diversity on three genes in the Toll-Interleukin-1 receptor type of nucleotide binding/leucine-rich repeat immune receptor (TIR-NBS LRR) immunity-response signaling pathway, indicating that they could be vulnerable to pathogen effectors.

  17. Desempenho de diferentes lipases imobilizadas na síntese de biodiesel de óleo de palma = Performance of different immobilized lipases in palm oil biodiesel synthesis

    Directory of Open Access Journals (Sweden)

    Grazielle dos Santos Silva

    2011-04-01

    Full Text Available O presente trabalho teve como objetivo determinar as condicoes otimizadas da sintese enzimatica de biodiesel, a partir do oleo de palma e etanol, empregando diferentes lipases imobilizadas (lipase de Pseudomonas fluorescens imobilizada em SiO2-PVA e lipase de Candida antartica imobilizada em resina acrilica - Novozym„µ 435 em meio isento de solvente. Uma matriz de planejamento fatorial foi utilizada para avaliar a influencia da temperatura (42 ¡V 58„aC e a razao molar entre etanol e oleo de palma (6:1 ¡V 18:1 no rendimento detransesterificacao alcancado para cada preparacao de lipase. Os efeitos principais foram ajustados por analise de regressao multipla a modelos lineares e o rendimento maximo foi obtido quando o sistema operacional foi operado a 42„aC com substratos contendo etanol eoleo de palma na razao molar de 18:1. Os modelos matematicos que representam o rendimento global da reacao para cada lipase imobilizada foram considerados adequados para descrever os resultados experimentais.Optimized conditions for palm oil and ethanol enzymatic biodiesel synthesis were determined with different immobilized lipases SiO2-PVA-immobilized lipase from Pseudomonas fluorescens and acrylic resin-immobilized lipase, NovozymR435, from Candida antartica, in solvent-free medium. A full factorial design assessed the influence oftemperature (42 ¡V 58¢XC and ethanol: palm oil (6:1 ¡V 18:1 molar ratio on the transesterification yield. Main effects were adjusted by multiple regression analysis to linear models and the maximum transesterification yield was obtained at 42¢XC and 18:1 ethanol:palm oil molar ratio. Mathematical models featuring total yield for each immobilized lipase were suitable to describe the experimental results.

  18. Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii

    Directory of Open Access Journals (Sweden)

    Marita G. Pereira

    2017-09-01

    Full Text Available Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr, glyoxyl-agarose (GX, MANAE-agarose activated with glutaraldehyde (GA and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr, at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea, cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA/docosahexaenoic acid (DHA ratio than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of

  19. GDSL lipases modulate immunity through lipid homeostasis in rice.

    Directory of Open Access Journals (Sweden)

    Mingjun Gao

    2017-11-01

    Full Text Available Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG and digalactosyldiacylglycerol (DGDG. We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity.

  20. GDSL lipases modulate immunity through lipid homeostasis in rice.

    Science.gov (United States)

    Gao, Mingjun; Yin, Xin; Yang, Weibing; Lam, Sin Man; Tong, Xiaohong; Liu, Jiyun; Wang, Xin; Li, Qun; Shui, Guanghou; He, Zuhua

    2017-11-01

    Lipids and lipid metabolites play important roles in plant-microbe interactions. Despite the extensive studies of lipases in lipid homeostasis and seed oil biosynthesis, the involvement of lipases in plant immunity remains largely unknown. In particular, GDSL esterases/lipases, characterized by the conserved GDSL motif, are a subfamily of lipolytic enzymes with broad substrate specificity. Here, we functionally identified two GDSL lipases, OsGLIP1 and OsGLIP2, in rice immune responses. Expression of OsGLIP1 and OsGLIP2 was suppressed by pathogen infection and salicylic acid (SA) treatment. OsGLIP1 was mainly expressed in leaf and leaf sheath, while OsGLIP2 showed high expression in elongating internodes. Biochemical assay demonstrated that OsGLIP1 and OsGLIP2 are functional lipases that could hydrolyze lipid substrates. Simultaneous down-regulation of OsGLIP1 and OsGLIP2 increased plant resistance to both bacterial and fungal pathogens, whereas disease resistance in OsGLIP1 and OsGLIP2 overexpression plants was significantly compromised, suggesting that both genes act as negative regulators of disease resistance. OsGLIP1 and OsGLIP2 proteins mainly localize to lipid droplets and the endoplasmic reticulum (ER) membrane. The proper cellular localization of OsGLIP proteins is indispensable for their functions in immunity. Comprehensive lipid profiling analysis indicated that the alteration of OsGLIP gene expression was associated with substantial changes of the levels of lipid species including monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). We show that MGDG and DGDG feeding could attenuate disease resistance. Taken together, our study indicates that OsGLIP1 and OsGLIP2 negatively regulate rice defense by modulating lipid metabolism, thus providing new insights into the function of lipids in plant immunity.

  1. Extracellular lipase of Aspergillus niger NRRL3; production, partial purification and properties.

    Science.gov (United States)

    Adham, Nehad Z; Ahmed, E M

    2009-03-01

    Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO(4).7H(2)O, 0.05% KCl, 0.2% K(2)HPO(4) and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50-60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg(2+) and K(+), whereas Ca(2+) and Mn(2+) greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.

  2. Study of new feruloyl esterases to understand lipase evolution: the case of Bacillus flexus.

    Science.gov (United States)

    Sánchez-González, Mónica; Blanco-Gámez, Allan; Parra-Saldívar, Roberto; Mateos-Díaz, Juan Carlos; Estrada-Alvarado, María Isabel

    2012-01-01

    Recently, the crystal structure of the feruloyl esterase A from Aspergillus niger (AnFaeA) was elucidated. This enzyme displays an α/β hydrolase fold and a catalytic triad similar to that found in fungal lipases (30-37% identity). Surprisingly, AnFaeA showed an overall fold similarity with the Rhizomucor miehei and other related fungal lipases. All these data strongly suggest that the ancestral function (lipase) had shifted, with molecular adaptation leading to a novel enzyme (type-A feruloyl esterase). The discovery of new feruloyl esterases could lead to get insight into the evolutionary pathways of these enzymes and into new possibilities of directed evolution of lipases. In this chapter, the production of Bacillus flexus NJY2 feruloyl esterases is described. Unlike the previously described feruloyl esterases, which mostly belong to eukaryotes (mainly fungus), this unique feruloyl esterases from a prokaryotic alkaliphile microorganism could be the starting point for new discoveries on lipase and feruloyl esterase evolutionary relationships.

  3. Bioconjugation of lipase and cholesterol oxidase with graphene or graphene oxide

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Rubens A.; Souza, Michele L.; Bloisi, Georgia D.; Corio, Paolo; Petri, Denise F. S., E-mail: dfsp@iq.usp.br [Universidade de São Paulo, Instituto de Química (Brazil)

    2015-04-15

    The catalytic behavior of lipase and cholesterol oxidase (ChOx) in the absence and in the presence of graphene (G) or graphene oxide (GO) was investigated at 24 ± 1 °C and pH 6.5. GO flat sheets (0.5–2 μm) were ∼2-nm thick, while G formed aggregates. The maximum reaction velocity (V{sub max}) values and turnover numbers (k{sub cat}) determined for reactions catalyzed by physical mixtures of lipase (at 0.01 g l{sup −1}) or ChOx (at 0.03 g l{sup −1}) and G (0.012 g l{sup −1}) increased six-fold or doubled, respectively, in comparison to neat enzymes. Circular dichroism (CD) and photoluminescence (PL) spectroscopic measurements revealed the preservation of native secondary structures of enzymes and bioconjugation driven by hydrophobic interaction and energy transfer (redshift) between lipase or ChOx and G, corroborating with the enhanced catalytic behavior. On the other hand, the interactions between GO, which has hydrophilic moieties on the basal plane, and ChOx caused enzyme deactivation, as evidenced by the absence of typical CD signal. At low GO concentration (<0.012 g l{sup −1}), bioconjugates of lipases with GO led to V{sub max} and k{sub cat} values four-fold lower than their counterparts with G, but the GO hydrophilic groups probably favored the affinity for the substrate, because the Michaelis constant (K{sub m}) values decreased in comparison to that of neat lipase. Upon increasing the GO concentration, lipases lost secondary structure and the typical lipase PL bands disappeared.

  4. Biocatalytic potential of lipase from Staphylococcus sp. MS1 for transesterification of jatropha oil into fatty acid methyl esters.

    Science.gov (United States)

    Sharma, Monika; Singh, Shelley Sardul; Maan, Pratibha; Sharma, Rohit

    2014-11-01

    An extracellular lipase producing isolate Staphylococcus sp. MS1 was optimized for lipase production and its biocatalytic potential was assessed. Medium with tributyrin (0.25 %) and without any exogenous inorganic nitrogen source was found to be optimum for lipase production from Staphylococcus sp. MS1. The optimum pH and temperature for lipase production were found to be pH 7 and 37 °C respectively, showing lipase activity of 37.91 U. It showed good lipase production at pH 6-8. The lipase was found to be stable in organic solvents like hexane and petroleum ether, showing 98 and 88 % residual activity respectively. The biotransformation using the concentrated enzyme in petroleum ether resulted in the synthesis of fatty acid methyl esters like methyl oleate, methyl palmitate and methyl stearate. Thus, the lipase under study has got the potential to bring about transesterification of oils into methyl esters which can be exploited for various biotechnological applications.

  5. Polyethyleneimine-modified superparamagnetic Fe{sub 3}O{sub 4} nanoparticles for lipase immobilization: Characterization and application

    Energy Technology Data Exchange (ETDEWEB)

    Khoobi, Mehdi; Motevalizadeh, Seyed Farshad [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411 (Iran, Islamic Republic of); Asadgol, Zahra [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 1417614411 (Iran, Islamic Republic of); Forootanfar, Hamid [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Shafiee, Abbas, E-mail: ashafiee@ams.ac.ir [Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411 (Iran, Islamic Republic of); Faramarzi, Mohammad Ali, E-mail: faramarz@tums.ac.ir [Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran 1417614411 (Iran, Islamic Republic of)

    2015-01-15

    Magnetically separable nanospheres consisting of polyethyleneimine (PEI) and succinated PEI grafted on silica coated magnetite (Fe{sub 3}O{sub 4}) were prepared and characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray diffraction, vibrating sample magnetometer, scanning electron microscopy and transmission electron microscopy. The prepared magnetic nanoparticles were then applied for physical adsorption or covalent attachment of Thermomyces lanuginosa lipase (TLL) via glutaraldehyde or hexamethylene diisocyanate. The reusability, storage, pH and thermal stabilities of the immobilized enzymes compared to that of free lipase were examined. The obtained results showed that the immobilized lipase on MNPs@PEI-GLU was the best biocatalyst which retained 80% of its initial activity after 12 cycles of application. The immobilized lipase on the selected support (MNPs@PEI-GLU) was also applied for the synthesis of ethyl valerate. Following 24 h incubation of the immobilized lipase on the selected support in n-hexane and solvent free media, the esterification percentages were 72.9% and 28.9%, respectively. - Graphical abstract: A schematic of the preparation of PEI- and succinated PEI-grafted Fe{sub 3}O{sub 4} MNPs (MNPs@PEI) and the immobilization of lipase by covalent bonding and adsorption. - Highlights: • Functionalized polyethylenimine-grafted magnetic nanoparticles were synthesized. • The prepared supports were fully characterized by various analysis methods. • Lipase was immobilized on the nanostructures by adsorption and covalent attachment. • Immobilized lipase produced ethyl valerate in solvent free medium.

  6. Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution

    Directory of Open Access Journals (Sweden)

    Li Pin Lee

    2015-01-01

    Full Text Available Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.

  7. Identification and characterization of a new true lipase isolated through metagenomic approach

    Directory of Open Access Journals (Sweden)

    de Souza Emanuel M

    2011-07-01

    Full Text Available Abstract Background Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1 and lipases (triacylglycerol lipases, EC 3.1.1.3. In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant. Results In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of Yersinia enterocolitica subsp. palearctica Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v. Conclusions The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range

  8. EFSA CEF Panel (EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids), 2014. Scientific Opinion on lipase from a genetically modified strain of Aspergillus oryzae (strain NZYM-FL)

    DEFF Research Database (Denmark)

    Poulsen, Morten; Hallas-Møller, Torben; Binderup, Mona-Lise

    in a sufficiently high Margin of Exposure. The allergenicity was evaluated by searching for similarity of the amino acid sequence to those of known allergens. The Panel considered that the likelihood of food allergic reactions to the enzyme is low and therefore does not raise safety concern. Based on the genetic...

  9. Lipase catalysed biodiesel synthesis with integrated glycerol separation in continuously operated microchips connected in series.

    Science.gov (United States)

    Šalić, Anita; Tušek, Ana Jurinjak; Sander, Aleksandra; Zelić, Bruno

    2018-02-05

    Although the application of microreactors in different processes has been extensively explored in recent decades, microreactors continue to be underexplored in the context of the enzyme-catalysed process for biodiesel production. Due to their numerous advantages, microreactors could become the next step in the development of a biodiesel production process characterised by sustainability, cost-effectiveness and energy efficiency. In this investigation, biodiesel production was catalysed by lipase from Thermomyces lanuginosus (Lipolase L100). Edible sunflower oil was used as a model substrate in order to investigate the process. After optimal process conditions had been determined, waste-cooking oil was used for biodiesel production to make the production process more sustainable. Three different substrate-feeding strategies were investigated and finally an optimal strategy was proposed. In all the investigated systems, fatty acids methyl esters (FAME) content was higher than 95% and obtained in a significantly shorter time (less than 2 h) compared to the batch process in which biodiesel production was catalysed by lipase (C = 95%, t = 96 h). After the optimal biodiesel production system had been proposed, an integrated system with two microchips connected in series was developed. The first microchip was used for biodiesel production and the second for simultaneous purification i.e. glycerol separation. Finally, purified biodiesel was produced with glycerol content below the detection limit. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Dependency of water concentration on ethanolysis of trioleoylglycerol by lipases

    DEFF Research Database (Denmark)

    Piyatheerawong, W.; Iwasaki, Y; Xu, Xuebing

    2004-01-01

    molecules tightly to retain its catalytic activity even in dry ethanol (a water-depriving solvent). It is suggested that some structural features, such as a carbohydrate molecule, a lid-like domain, or very tight bonding of the essential water molecule(s), might be involved in this very unique property.......The effects of water concentration on ethanolysis of trioleoylglycerol catalyzed by four different lipases were studied. The target product of the ethanolysis was 2-monooleoylglycerol (2-MO). Novozym 435 (a commercially available preparation of immobilized Candida antarctica lipase B, CALB...... in other two different preparations of CALB; i.e., free CALB powder and silica-immobilized CALB as well as Novozym 435. Thus, it was confirmed that no extra water requirement of CALB was an intrinsic property of CALB itself. No extra water requirement of CALB implies that this enzyme is able to keep water...

  11. Regioselective Alcoholysis of Silychristin Acetates Catalyzed by Lipases

    Directory of Open Access Journals (Sweden)

    Eva Vavříková

    2015-05-01

    Full Text Available A panel of lipases was screened for the selective acetylation and alcoholysis of silychristin and silychristin peracetate, respectively. Acetylation at primary alcoholic group (C-22 of silychristin was accomplished by lipase PS (Pseudomonas cepacia immobilized on diatomite using vinyl acetate as an acetyl donor, whereas selective deacetylation of 22-O-acetyl silychristin was accomplished by Novozym 435 in methyl tert-butyl ether/ n-butanol. Both of these reactions occurred without diastereomeric discrimination of silychristin A and B. Both of these enzymes were found to be capable to regioselective deacetylation of hexaacetyl silychristin to afford penta-, tetra- and tri-acetyl derivatives, which could be obtained as pure synthons for further selective modifications of the parent molecule.

  12. Preparation of detergent-lipase complexes utilizing water-soluble amphiphiles in single aqueous phase and catalysis of transesterifications in homogeneous organic solvents.

    Science.gov (United States)

    Mine, Y; Fukunaga, K; Maruoka, N; Nakao, K; Sugimura, Y

    2000-01-01

    A novel method of preparing detergent-enzyme complexes that can be employed in organic media was developed utilizing newly synthesized water-soluble nonionic gemini-type detergents, N,N-bis(3-D-gluconamidopropyl)-3-(dialkyl-L-glutamatecarbonyl)propanamides (BIG2CnCA: n = 10,12,14,16,18) and N,N-bis(3-D-lactonamidopropyl)-3-(dialkyl-L-glutamatecarbonyl)propanamides (BIL2CnCA: n = 16,18), and nonionic twin-headed detergents, N,N-bis(3-D-gluconamidopropyl)alkanamides (BIG1Cn: n = 12,14,16,18,delta9). This method simply entails mixing a selected enzyme with an appropriate detergent in an aqueous solution followed by lyophilization, and it offers the advantages of enhanced enzymatic activity in organic solvents and eliminates both enzyme loss and the necessity for an organic solvent in the preparation stage. Using various modified lipases originating from Aspergillus niger (Lipase A), Candida rugosa (Lipase C), Pseudomonas cepacia (Lipase P), and porcine pancreas (PPL), prepared using the novel method and detergents, including conventional synthesized nonionic detergents such as dialkyl N-D-glucona-L-glutamates (2CnGE: n = 12,18delta9) and octanoyl-N-methylglucamide (MEGA-8), enantioselective transesterifications of 6-methyl-5-hepten-2-ol (sulcatol) and 2,2-dimethyl-1,3-dioxolane-4-methanol (solketal) with a vinyl or isopropenyl carboxylate were carried out in an organic solvent. The modified lipase activity was influenced by both the lipases and the structure of the detergents. The value for the hydrophile-lipophile balance (HLB) of the detergent provided a means of correlating the structure and the obtained modified lipase activity. For detergents of the same class with a HLB value of approximately 9 and 12, the highest activity was obtained for Lipase A and Lipase P, and Lipase C and PPL, respectively. Among detergents of the same HLB value tested, the gemini-type detergents possessing the most bulky head and tail were most effective as a modifier for lipases of all

  13. Characterization of smallest active monomeric lipase from novel rhizopus strain: application in transesterification.

    Science.gov (United States)

    Kantak, Jayshree B; Prabhune, Asmita A

    2012-04-01

    An extracellular lipase-producing fungus was isolated from oil-rich soil. This fungus belongs to the genus Rhizopus and clades with Rhizopus oryzae. Lipase was purified to homogeneity from this novel fungal source using ammonium sulphate precipitation followed by Q-Sepharose chromatography. The extracellular lipase was purified 8.6-fold, and enzymatic properties were studied. The molecular mass of the purified enzyme was estimated to be 17 kD by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and 16.25 kD by matrix-assisted laser desorption ionization/time-of-flight analysis. The native molecular mass was estimated to be 17.5 kD by gel filtration, indicating the protein to be monomer. The optimum pH and temperature for the enzyme catalysis were 7.0 °C and 40 °C, respectively. Enzyme was stable in pH range 6.0-7.0 and retains 95-100% activity when incubated at 50 °C for 1 h. The pI of the purified lipase was 4.2. Enzyme was stable in the organic solvents such as ethanol, hexane and methanol for 2 h. Purified enzyme was used for transesterification of oleic acid in the presence of ethanol for production of oleic acid ethyl ester with a conversion efficiency of 66% after 24 h at 30 °C.

  14. Conformation Analysis of T1 Lipase on Alcohols Solvent using Molecular Dynamics Simulation

    Science.gov (United States)

    Putri, A. M.; Sumaryada, T.; Wahyudi, S. T.

    2017-07-01

    Biodiesel usually is produced commercially via a transesterification reaction of vegetable oil with alcohol and alkali catalyst. The alkali catalyst has some drawbacks, such as the soap formation during the reaction. T1 Lipase enzyme had been known as a thermostable biocatalyst which is able to produce biodiesel through a cleaner process. In this paper the performance of T1 lipase enzyme as catalyst for transesterification reaction in pure ethanol, methanol, and water solvents were studied using a Molecular Dynamics (MD) Simulation at temperature of 300 K for 10 nanoseconds. The results have shown that in general the conformation of T1 lipase enzyme in methanol is more dynamics as shown by the value of root mean square deviation (RMSD), root mean squared fluctuation (RMSF), and radius of gyration. The highest solvent accessible surface area (SASA) total was also found in methanol due to the contribution of non-polar amino acid in the interior of the protein. Analysis of MD simulation has also revealed that the enzyme structure tend to be more rigid in ethanol environment. The analysis of electrostatic interactions have shown that Glu359-Arg270 salt-bridge pair might hold the key of thermostability of T1 lipase enzyme as shown by its strong and stable binding in all three solvents.

  15. Enzyme adsorption at solid-liquid interfaces

    NARCIS (Netherlands)

    Duinhoven, S.

    1992-01-01

    Enzymes are proteins with the capacity of catalysing various reactions. Nowadays two types of enzymes, proteases and lipases, are available for use in detergent formulations for household and industrial laundry washing. Proteases are capable of catalysing the hydrolysis of proteins while

  16. Expression of a Deschampsia antarctica Desv. Polypeptide with Lipase Activity in a Pichia pastoris Vector

    Directory of Open Access Journals (Sweden)

    Claudia Rabert

    2014-02-01

    Full Text Available The current study isolated and characterized the Lip3F9 polypeptide sequence of Deschampsia antarctica Desv. (GeneBank Accession Number JX846628, which was found to be comprised of 291 base pairs and was, moreover, expressed in Pichia pastoris X-33 cells. The enzyme was secreted after 24 h of P. pastoris culture incubation and through induction with methanol. The expressed protein showed maximum lipase activity (35 U/L with an optimal temperature of 37 °C. The lipase-expressed enzyme lost 50% of its specific activity at 25 °C, a behavior characteristic of a psychrotolerant enzyme. Recombinant enzyme activity was measured in the presence of ionic and non-ionic detergents, and a decrease in enzyme activity was detected for all concentrations of ionic and non-ionic detergents assessed.

  17. Purification and Properties of a Glycerol Ester Hydrolase (Lipase) from Propionibacterium shermanii

    Science.gov (United States)

    Oterholm, Anders; Ordal, Z. John; Witter, Lloyd D.

    1970-01-01

    An intracellular glycerol ester hydrolase (lipase) from Propionibacterium shermanii was recovered from cell-free extracts and purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography on diethylaminoethylcellulose. Maximum enzyme activity was observed at pH 7.2 and 47 C when an emulsion of tributyrin was used as substrate. The enzyme was stable between pH 5.5 and 8. Heating the enzyme solution at 45 C for 10 min resulted in a 75% decrease in activity. Maximum rate of hydrolysis of triglycerides was observed on tripropionin, followed in order by tributyrin, tricaproin, and tricaprylin. The lipase was strongly inhibited by mercury and arsenicals, but specific sulfhydryl reagents had little or no inhibiting effect on the enzyme activity. The enzyme also showed some esterase activity, but the hydrolysis of substrates in solution was small as compared to the hydrolysis of substrates in emulsion. PMID:5456938

  18. Dry entrapment of enzymes by epoxy or polyester resins hardened on different solid supports.

    Science.gov (United States)

    Barig, Susann; Funke, Andreas; Merseburg, Andrea; Schnitzlein, Klaus; Stahmann, K-Peter

    2014-06-10

    Embedding of enzymes was performed with epoxy or polyester resin by mixing in a dried enzyme preparation before polymerization was started. This fast and low-cost immobilization method produced enzymatically active layers on different solid supports. As model enzymes the well-characterized Thermomyces lanuginosus lipase and a new threonine aldolase from Ashbya gossypii were used. It was shown that T. lanuginosus lipase recombinantly expressed in Aspergillus oryzae is a monomeric enzyme with a molecular mass of 34kDa, while A. gossypii threonine aldolase expressed in Escherichia coli is a pyridoxal-5'-phosphate binding homotetramer with a mass of 180kDa. The enzymes were used freeze dried, in four different preparations: freely diffusing, adsorbed on octyl sepharose, as well as cross-linked enzyme aggregates or as suspensions in organic solvent. They were mixed with standard two-component resins and prepared as layers on solid supports made of different materials e.g. metal, glass, polyester. Polymerization led to encapsulated enzyme preparations showing activities comparable to literature values. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Pre-treatment of high oil and grease pet food industrial wastewaters using immobilized lipase hydrolyzation.

    Science.gov (United States)

    Jeganathan, Jeganaesan; Bassi, Amarjeet; Nakhla, George

    2006-09-01

    Wastewaters generating from pet food industries contain high concentration of oil and grease (O&G), which is difficult to treat through conventional biological treatment systems. In this study, the hydrolysis of O&G originating from pet food industrial wastewater was evaluated. Candida rugosa lipase was immobilized in calcium alginate beads and applied in the hydrolysis experiment. Results showed that approximately 50% of the O&G was hydrolyzed due to the enzyme activity. A significant increment in COD and VFA production was also observed. The immobilized lipase activity was confirmed with p-nitrophenyl palmitate (pNPP) before and after O&G hydrolysis. During the 3-day experiment, approximately 65% of the beads were recovered and after the hydrolysis, approximately 70% of the enzyme activity remained in the beads. This study shows the potential of immobilized lipase as a pre-treatment step in biological treatment of pet food manufacturing wastewater.

  20. Pre-treatment of high oil and grease pet food industrial wastewaters using immobilized lipase hydrolyzation

    International Nuclear Information System (INIS)

    Jeganathan, Jeganaesan; Bassi, Amarjeet; Nakhla, George

    2006-01-01

    Wastewaters generating from pet food industries contain high concentration of oil and grease (O and G), which is difficult to treat through conventional biological treatment systems. In this study, the hydrolysis of O and G originating from pet food industrial wastewater was evaluated. Candida rugosa lipase was immobilized in calcium alginate beads and applied in the hydrolysis experiment. Results showed that approximately 50% of the O and G was hydrolyzed due to the enzyme activity. A significant increment in COD and VFA production was also observed. The immobilized lipase activity was confirmed with p-nitrophenyl palmitate (pNPP) before and after O and G hydrolysis. During the 3-day experiment, approximately 65% of the beads were recovered and after the hydrolysis, approximately 70% of the enzyme activity remained in the beads. This study shows the potential of immobilized lipase as a pre-treatment step in biological treatment of pet food manufacturing wastewater

  1. Ultrasonic enhancement of lipase-catalysed transesterification for biodiesel synthesis.

    Science.gov (United States)

    Bhangu, Sukhvir Kaur; Gupta, Shweta; Ashokkumar, Muthupandian

    2017-01-01

    The production of biodiesel was carried out from canola oil and methanol catalysed by lipase from Candida rugosa under different ultrasonic experimental conditions using horn (20kHz) and plate (22, 44, 98 and 300kHz) transducers. The effects of experimental conditions such as horn tip diameter, ultrasonic power, ultrasonic frequency and enzyme concentrations on biodiesel yield were investigated. The results showed that the application of ultrasound decreased the reaction time from 22-24h to 1.5h with the use of 3.5cm ultrasonic horn, an applied power of 40W, methanol to oil molar ratio of 5:1 and enzyme concentration of 0.23wt/wt% of oil. Low intensity ultrasound is efficient and a promising tool for the enzyme catalysed biodiesel synthesis as higher intensities tend to inactivate the enzyme and reduce its efficiency. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Highly efficient preparation of lipophilic hydroxycinnamates by solvent-free lipase-catalyzed transesterification.

    Science.gov (United States)

    Weitkamp, Petra; Vosmann, Klaus; Weber, Nikolaus

    2006-09-20

    Various medium- or long-chain alkyl cinnamates and hydroxycinnamates, including oleyl p-coumarate as well as palmityl and oleyl ferulates, were prepared in high yield by lipase-catalyzed transesterification of an equimolar mixture of a short-chain alkyl cinnamate and a fatty alcohol such as lauryl, palmityl, and oleyl alcohol under partial vacuum at moderate temperature in the absence of solvents and drying agents in direct contact with the reaction mixture. Immobilized lipase B from Candida antarctica was the most effective biocatalyst for the various transesterification reactions. Transesterification activity of this enzyme was up to 56-fold higher than esterification activity for the preparation of medium- and long-chain alkyl ferulates. The relative transesterification activities found for C. antarctica lipase were of the following order: hydrocinnamate > cinnamate > 4-hydroxyhydrocinnamate > 3-methoxycinnamate > 2-methoxycinnamate approximately 4-methoxycinnamate approximately 3-hydroxycinnamate > hydrocaffeate approximately 4-hydroxycinnamate > ferulate > 2-hydroxycinnamate > caffeate approximately sinapate. With respect to the position of the hydroxy substituents at the phenyl moiety, the transesterification activity of C. antarctica lipase B increased in the order meta > para > ortho. The immobilized lipases from Rhizomucor miehei and Thermomyces lanuginosus demonstrated moderate and low transesterification activity, respectively. Compounds with inverse chemical structure, that is, 3-phenylpropyl alkanoates such as 3-(4-hydroxyphenyl)propyl oleate and 3-(3,4-dimethoxyphenyl)propyl oleate, were obtained by C. antarctica lipase-catalyzed transesterification of fatty acid methyl esters with the corresponding 3-phenylpropan-1-ols in high yield, as well.

  3. A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents.

    Science.gov (United States)

    Solanki, Kusum; Gupta, Munishwar Nath

    2011-05-15

    Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals), EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of modified lipase gave highest rate (1970 nmol min(-1)mg(-1)) as compared to unmodified pH tuned lipase (128 nmol min(-1) mg(-1)). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest initial rate of 0.72 and 7.2 nmol min(-1) mg(-1), respectively. Unmodified pH tuned lipase showed no activity at all in DMF with both 1% and 5% (v/v) water content. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Production of Thermoalkaliphilic Lipase from Geobacillus thermoleovorans DA2 and Application in Leather Industry

    Directory of Open Access Journals (Sweden)

    Deyaa M. Abol Fotouh

    2016-01-01

    Full Text Available Thermophilic and alkaliphilic lipases are meeting a growing global attention as their increased importance in several industrial fields. Over 23 bacterial strains, novel strain with high lipolytic activity was isolated from Southern Sinai, Egypt, and it was identified as Geobacillus thermoleovorans DA2 using 16S rRNA as well as morphological and biochemical features. The lipase was produced in presence of fatty restaurant wastes as an inducing substrate. The optimized conditions for lipase production were recorded to be temperature 60°C, pH 10, and incubation time for 48 hrs. Enzymatic production increased when the organism was grown in a medium containing galactose as carbon source and ammonium phosphate as nitrogen source at concentrations of 1 and 0.5% (w/v, respectively. Moreover, the optimum conditions for lipase production such as substrate concentration, inoculum size, and agitation rate were found to be 10% (w/v, 4% (v/v, and 120 rpm, respectively. The TA lipase with Triton X-100 had the best degreasing agent by lowering the total lipid content to 2.6% as compared to kerosene (7.5% or the sole crude enzyme (8.9%. It can be concluded that the chemical leather process can be substituted with TA lipase for boosting the quality of leather and reducing the environmental hazards.

  5. Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

    Directory of Open Access Journals (Sweden)

    P. Gururaj

    Full Text Available ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v inoculum, 2% (v/v castor oil (inducer, and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

  6. Rational evolution of the unusual Y-type oxyanion hole of Rhodococcus sp. CR53 lipase LipR.

    Science.gov (United States)

    Infanzón, Belén; Sotelo, Pablo H; Martínez, Josefina; Diaz, Pilar

    2018-01-01

    Rhodococcus sp CR-53 lipase LipR was the first characterized member of bacterial lipase family X. Interestingly, LipR displays some similarity with α/β-hydrolases of the C. antartica lipase A (CAL-A)-like superfamily (abH38), bearing a Y-type oxyanion hole, never found before among bacterial lipases. In order to explore this unusual Y-type oxyanion hole, and to improve LipR performance, two modification strategies based on site directed or saturation mutagenesis were addressed. Initially, a small library of mutants was designed to convert LipR Y-type oxyanion hole (YDS) into one closer to those most frequently found in bacteria (GGG(X)). However, activity was completely lost in all mutants obtained, indicating that the Y-type oxyanion hole of LipR is required for activity. A second approach was addressed to modify the two main oxyanion hole residues Tyr 110 and Asp 111 , previously described for CAL-A as the most relevant amino acids involved in stabilization of the enzyme-substrate complex. A saturation mutagenesis library was prepared for each residue (Tyr 110 and Asp 111 ), and activity of the resulting variants was assayed on different chain length substrates. No functional LipR variants could be obtained when Tyr 110 was replaced by any other amino acids, indicating that this is a crucial residue for catalysis. However, among the Asp 111 variants obtained, LipR D111G produced a functional enzyme. Interestingly, this LipR-YGS variant showed less activity than wild type LipR on short- or mid- chain substrates but displayed a 5.6-fold increased activity on long chain length substrates. Analysis of the 3D model and in silico docking studies of this enzyme variant suggest that substitution of Asp by Gly produces a wider entrance tunnel that would allow for a better and tight accommodation of larger substrates, thus justifying the experimental results obtained. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Detection of enzyme activity in decontaminated spices of industrial use

    International Nuclear Information System (INIS)

    Müller, R.; Theobald, R.

    1995-01-01

    A range of decontaminated spices of industrial use have been examinated for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material [de

  8. Production of thermostable multiple enzymes from Bacillus amyloliquefaciens KUB29.

    Science.gov (United States)

    Devaraj, Kubendran; Aathika, Salma; Periyasamy, Karthik; Manickam Periyaraman, Premkumar; Palaniyandi, Shanmugam; Subramanian, Sivanesan

    2018-01-16

    A strain of Bacillus amyloliquefaciens KUB29 was identified by 16S ribosomal RNA sequencing (Genbank: MF772779.1). Production of thermostable protease, amylase and lipase were done by the isolated strain. The produced enzymes were partially purified by ammonium precipitation followed by dialysis process. Protease and lipase enzymes are effectively used in bio-oil extraction from proteinaceous sample followed by transesterification to produce methyl ester. Amylase enzyme is widely used in food and laundry industry. The produced enzymes are active at thermophilic condition of 55 °C. Use of these enzymes in biofuel production process will make the process cleaner and greener.

  9. Transesterification Synthesis of Chloramphenicol Esters with the Lipase from Bacillus amyloliquefaciens

    Directory of Open Access Journals (Sweden)

    Fengying Dong

    2017-09-01

    Full Text Available This work presents a synthetic route to produce chloramphenicol esters by taking advantage the high enantio- and regio-selectivity of lipases. A series of chloramphenicol esters were synthesized using chloramphenicol, acyl donors of different carbon chain length and lipase LipBA (lipase cloned from Bacillus amyloliquefaciens. Among acyl donors with different carbon chain lengths, vinyl propionate was found to be the best. The influences of different organic solvents, reaction temperature, reaction time, enzyme loading and water content on the synthesis of the chloramphenicol esters were studied. The synthesis of chloramphenicol propionate (0.25 M with 4.0 g L−1 of LipBA loading gave a conversion of ~98% and a purity of ~99% within 8 h at 50 °C in 1,4-dioxane as solvent. The optimum mole ratio of vinyl propionate to chloramphenicol was increased to 5:1. This is the first report of B. amyloliquefaciens lipase being used in chloramphenicol ester synthesis and a detailed study of the synthesis of chloramphenicol propionate using this reaction. The high enzyme activity and selectivity make lipase LipBA an attractive catalyst for green chemical synthesis of molecules with complex structures.

  10. Mixed reverse micelles facilitated downstream processing of lipase involving water-oil-water liquid emulsion membrane.

    Science.gov (United States)

    Bhowal, Saibal; Priyanka, B S; Rastogi, Navin K

    2014-01-01

    Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water-oil-water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0-fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1-17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins. © 2014 American Institute of Chemical Engineers.

  11. Transesterification Synthesis of Chloramphenicol Esters with the Lipase from Bacillus amyloliquefaciens.

    Science.gov (United States)

    Dong, Fengying; Li, Lingmeng; Lin, Lin; He, Dannong; Chen, Jingwen; Wei, Wei; Wei, Dongzhi

    2017-09-19

    This work presents a synthetic route to produce chloramphenicol esters by taking advantage the high enantio- and regio-selectivity of lipases. A series of chloramphenicol esters were synthesized using chloramphenicol, acyl donors of different carbon chain length and lipase Lip BA (lipase cloned from Bacillus amyloliquefaciens ). Among acyl donors with different carbon chain lengths, vinyl propionate was found to be the best. The influences of different organic solvents, reaction temperature, reaction time, enzyme loading and water content on the synthesis of the chloramphenicol esters were studied. The synthesis of chloramphenicol propionate (0.25 M) with 4.0 g L -1 of Lip BA loading gave a conversion of ~98% and a purity of ~99% within 8 h at 50 °C in 1,4-dioxane as solvent. The optimum mole ratio of vinyl propionate to chloramphenicol was increased to 5:1. This is the first report of B. amyloliquefaciens lipase being used in chloramphenicol ester synthesis and a detailed study of the synthesis of chloramphenicol propionate using this reaction. The high enzyme activity and selectivity make lipase Lip BA an attractive catalyst for green chemical synthesis of molecules with complex structures.

  12. Bioprospecting of Thermostable Cellulolytic Enzymes through Modeling and Virtual Screening Method

    Directory of Open Access Journals (Sweden)

    R. Navanietha Krishnaraj

    2017-04-01

    Full Text Available Cellulolytic enzymes are promising candidates for the use of cellulose in any bioprocess operations and for the disposal of the cellulosic wastes in an environmentally benign manner. Cellulases from thermophiles have the advantage of hydrolyzing cellulose at wider range of operating conditions unlike the normal enzymes. Herein we report the modeled structures of cellulolytic enzymes (endoglucanase, cellobiohydrolase and ß-glucosidase from a thermophilic bacterium,Clostridium thermocellumand their validation using Root Mean Square Deviation (RMSD and Ramachandran plot analyses. Further, the molecular interactions of the modeled enzyme with cellulose were analyzed using molecular docking technique. The results of molecular docking showed that the endoglucanase, cellobiohydrolase and ß-glucosidase had the binding affinities of -10.7, -9.0 and -10.8 kcal/mol, respectively. A correlation between the binding affinity of the endoglucanase with cellulose and the enzyme activity was also demonstrated. The results showed that the binding affinities of cellulases with cellulose could be used as a tool to assess the hydrolytic activity of cellulases. The results obtained could be used in virtual screening of cellulolytic enzymes based on the molecular interactions with the substrate, and aid in developing systems biology models of thermophiles for industrial biotechnology applications.

  13. Intermittent Fasting Alleviates the Increase of Lipoprotein Lipase Expression in Brain of a Mouse Model of Alzheimer's Disease: Possibly Mediated by β-hydroxybutyrate

    Directory of Open Access Journals (Sweden)

    Jingzhu Zhang

    2018-01-01

    Full Text Available Intermittent fasting has been demonstrated to protect against Alzheimer's disease (AD, however, the mechanism is unclear. Histone acetylation and lipoprotein lipase (LPL are involved in AD progression. Importantly, LPL has been documented to be regulated by histone deacetylases (HDACs inhibitors (increase histone acetylation level in adipocyte and mesenchymal stem cells, or by fasting in adipose and muscle tissues. In brain, however, whether histone acetylation or fasting regulates LPL expression is unknown. This study was designed to demonstrate intermittent fasting may protect against AD through increasing β-hydroxybutyrate, a HDACs inhibitor, to regulate LPL. We also investigated microRNA-29a expression associating with regulation of LPL and histone acetylation. The results showed LPL mRNA expression was increased and microRNA-29a expression was decreased in the cerebral cortex of AD model mice (APP/PS1, which were alleviated by intermittent fasting. No significant differences were found in the total expression of LPL protein (brain-derived and located in capillary endothelial cells from peripheral tissues in the cerebral cortex of APP/PS1 mice. Further study indicated that LPL located in capillary endothelial cells was decreased in the cerebral cortex of APP/PS1 mice, which was alleviated by intermittent fasting. LPL and microRNA-29a expression were separately increased and down-regulated in 2 μM Aβ25−35-exposed SH-SY5Y cells, but respectively decreased and up-regulated in 10 μM Aβ25−35-exposed cells, which were all reversed by β-hydroxybutyrate. The increase of HDAC2/3 expression and the decrease of acetylated H3K9 and H4K12 levels were alleviated in APP/PS1 mice by intermittent fasting treatment, as well in 2 or 10 μM Aβ25−35-exposed cells by β-hydroxybutyrate treatment. These findings above suggested the results from APP/PS1 mice were consistent with those from cells treated with 2 μM Aβ25−35. Interestingly, LPL

  14. Intermittent Fasting Alleviates the Increase of Lipoprotein Lipase Expression in Brain of a Mouse Model of Alzheimer's Disease: Possibly Mediated by β-hydroxybutyrate.

    Science.gov (United States)

    Zhang, Jingzhu; Li, Xinhui; Ren, Yahao; Zhao, Yue; Xing, Aiping; Jiang, Congmin; Chen, Yanqiu; An, Li

    2018-01-01

    Intermittent fasting has been demonstrated to protect against Alzheimer's disease (AD), however, the mechanism is unclear. Histone acetylation and lipoprotein lipase (LPL) are involved in AD progression. Importantly, LPL has been documented to be regulated by histone deacetylases (HDACs) inhibitors (increase histone acetylation level) in adipocyte and mesenchymal stem cells, or by fasting in adipose and muscle tissues. In brain, however, whether histone acetylation or fasting regulates LPL expression is unknown. This study was designed to demonstrate intermittent fasting may protect against AD through increasing β-hydroxybutyrate, a HDACs inhibitor, to regulate LPL. We also investigated microRNA-29a expression associating with regulation of LPL and histone acetylation. The results showed LPL mRNA expression was increased and microRNA-29a expression was decreased in the cerebral cortex of AD model mice (APP/PS1), which were alleviated by intermittent fasting. No significant differences were found in the total expression of LPL protein (brain-derived and located in capillary endothelial cells from peripheral tissues) in the cerebral cortex of APP/PS1 mice. Further study indicated that LPL located in capillary endothelial cells was decreased in the cerebral cortex of APP/PS1 mice, which was alleviated by intermittent fasting. LPL and microRNA-29a expression were separately increased and down-regulated in 2 μM Aβ 25-35 -exposed SH-SY5Y cells, but respectively decreased and up-regulated in 10 μM Aβ 25-35 -exposed cells, which were all reversed by β-hydroxybutyrate. The increase of HDAC2/3 expression and the decrease of acetylated H3K9 and H4K12 levels were alleviated in APP/PS1 mice by intermittent fasting treatment, as well in 2 or 10 μM Aβ 25-35 -exposed cells by β-hydroxybutyrate treatment. These findings above suggested the results from APP/PS1 mice were consistent with those from cells treated with 2 μM Aβ 25-35 . Interestingly, LPL expression was

  15. Preparation of a crosslinked bioimprinted lipase for enrichment of polyunsaturated fatty acids from fish processing waste.

    Science.gov (United States)

    Yan, Jinyong; Li, Lifan; Tang, Qianli; Jiang, Manzhou; Jiang, Shenzhou

    2010-10-01

    Geotrichum sp. lipase modified with a combined method composed of crosslinking and bioimprinting was employed to selectively hydrolyze waste fish oil for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in glycerides. Crosslinked polymerization by monomer (polyethylene glycol 400 dimethyl acrylate), crosslinker (trimethylolpropane trimethylacrylate), and photoinitiator (benzoin methyl ether) coupled to bioimprinting using palmitic acid as imprint molecule, resulted in much more effective enzyme preparation used in aqueous hydrolysis reaction. Since the crosslinked polymerization modification maintained bioimprinted property and gave good dispersion of enzyme in reaction mixture, the crosslinked bioimprinted enzyme exhibited higher hydrolysis temperature, enhanced specific activity, shorter hydrolysis time, and better operational stability compared to free lipase. Crude fish oil was treated at 45 degrees C with this crosslinked bioimprinted lipase for 8 h, and 46% hydrolysis degree resulted in the production of glycerides containing 41% of EPA and DHA (EPA+DHA), achieving 85.7% recovery of initial EPA and DHA. The results suggested that bioimprinted enzymes did not lose their induced property in aqueous environment when prepared according to the described crosslinking-bioimprinting method. It could also be seen that the crosslinked bioimprinted lipase was effective in producing glycerides that contained a higher concentration of polyunsaturated fatty acid with better yield.

  16. New efficient recombinant expression system to engineer Candida antarctica lipase B.

    Science.gov (United States)

    Emond, Stéphane; Montanier, Cédric; Nicaud, Jean-Marc; Marty, Alain; Monsan, Pierre; André, Isabelle; Remaud-Siméon, Magali

    2010-04-01

    Here, we report the use of Yarrowia lipolytica as a versatile expression host for developing protein engineering approaches to modify the properties of Candida antarctica lipase B. A reliable screening protocol was defined and validated using a saturation mutagenesis library, yielding mutants displaying higher catalytic efficiencies than the wild-type enzyme.

  17. Continuous Production of Structured Phospholipids in a Packed Red Reactor with Lipase from Thermomyces lanuginosa

    DEFF Research Database (Denmark)

    Vikbjerg, Anders Falk; Peng, Lifeng; Mu, Huiling

    2005-01-01

    The possibilities of producing structured phospholipids by lipase-catalyzed acidolysis between soybean phospholipids and caprylic acid were examined in continuous packed bed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercial...

  18. A comparative study of the lipase yield by solid state and submerged ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-01-05

    Jan 5, 2009 ... Lipase extraction from solid state fermentation. After the completion of 96 h of fermentation the enzyme was ex- tracted and the activity determined as follows: The biomass with substrate was agitated with 50 ml of distilled water on a magnetic stirrer for 30 min. The contents were filtered through muslin cloth.

  19. Hindiii and S447x polymorphisms of lipoprotein lipase gene and ...

    African Journals Online (AJOL)

    Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Lipoprotein lipase is a key enzyme in lipoprotein metabolism and its gene is a major candidate gene for coronary heart disease. The aim of the present work was to study the association of HindIII and S447X polymorphisms in lipoprotein ...

  20. Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor

    NARCIS (Netherlands)

    Dröge, M.J; Ruggeberg, C.J.; van der Sloot, Almer Martinus; Schimmel, J.; Dijkstra, Durk; Verhaert, R.M D; Reetz, M.T.; Quax, Wim; Droge, MJ; Dijkstra, DS

    2003-01-01

    Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties

  1. Substrate specificity, regioselectivity and hydrolytic activity of lipases activated from Geotrichum sp

    Czech Academy of Sciences Publication Activity Database

    Stránský, Karel; Zarevúcka, Marie; Kejík, Z.; Wimmer, Zdeněk; Macková, M.; Demnerová, K.

    2007-01-01

    Roč. 34, č. 3 (2007), s. 209-216 ISSN 1369-703X R&D Projects: GA ČR GA203/04/0120 Institutional research plan: CEZ:AV0Z40550506 Keywords : biocatalysis * enzyme activity * gas chromatography * lipase * blackcurrant oil Subject RIV: CC - Organic Chemistry Impact factor: 1.872, year: 2007

  2. Lipoprotein Lipase mRNA expression in different tissues of farm ...

    African Journals Online (AJOL)

    Lipoprotein lipase (LPL) controls triacylglycerol partitioning between adipose tissues and muscles, so it is important enzyme for fattening of animals .The present work was planned to clarify the use of polymerase chain reaction (PCR) for detection of LPL mRNA expression in different tissues representing internal organs of ...

  3. The dynamic response of a fungal lipase in the presence of charged surfactants

    DEFF Research Database (Denmark)

    Peters, Günther H.J.

    2002-01-01

    Lipases are efficient catalysts for lipolytic reactions and require a lipid interface for optimal activity. To study the effect of small charged lipid aggregates on the behavior of these enzymes, we have performed molecular dynamics simulations on five different systems. The simulations carried o...

  4. Regulation and role of hormone-sensitive lipase in rat skeletal muscle

    DEFF Research Database (Denmark)

    Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

    2004-01-01

    -sensitive lipase (HSL). This enzyme is known to be rate limiting for intracellular TG hydrolysis in adipose tissue. The presence of HSL has been demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying. The content of HSL varies between...

  5. A comparative study of the lipase yield by solid state and submerged ...

    African Journals Online (AJOL)

    A comparative study of lipase enzyme yields by solid state fermentation (SSF) and submerged fermentation (SmF) was performed here. Three fungal colonies were isolated from biopharmaceutical oil waste collected from “Oushadhi” (The Pharmaceutical Corporation (IM) Kerala Ltd). The pure colonies were then used as ...

  6. A Novel Low-Temperature Alkaline Lipase from Acinetobacter johnsonii LP28 Suitable for Detergent Formulation

    Directory of Open Access Journals (Sweden)

    Hai Kuan Wang

    2011-01-01

    Full Text Available A strain LP28 that produces alkaline and low-temperature lipase was isolated from the soil collected from the Bay of Bohai, PR China and identified as Acinetobacter johnsonii using 16S rDNA sequencing. The lipase was purified to homogeneity by centrifugation, followed by ammonium sulphate precipitation, dialysis, ion exchange chromatography on cellulose DE-52 and gel filtration chromatography on Sephadex G-75. The enzyme was purified about 34-fold with a final yield of 13 % and the relative molecular mass of the enzyme was determined to be 53 kDa by SDS-PAGE. The purified enzyme exhibited maximum activity at 30 °C and pH=9.0, and retained 94.53 % of its maximum activity at 20 °C. The enzyme was stable at 50 °C and retained 80.9 % of its original activity for 30 min. It was also highly stable in a pH range of 8.0–11.0. The enzyme hydrolyzed a wide range of oils and showed a high level of lipase activity in hydrolyzing tributyrin. The enzyme activity was promoted in the presence of Na+, Ca2+, K+, Mg2+ and sodium citrate. Ba2+, Mn2+, Cr3+ and Co2+ did not affect the enzyme activity, whereas the presence of Al3+, Cu2+, Fe2+, Fe3+, Zn2+ and EDTA reduced the enzyme activity. Regarding the stability of detergent process, the enzyme was highly stable in the presence of various oxidizing agents, some commercial detergents and alkaline protease, and its activity was also promoted by most of the surfactants, viz. Tween 20, Tween 80, sodium cholate, sodium taurocholate and saponin. For these characteristics, the lipase from Acinetobacter johnsonii LP28 showed good potential as an additive in laundry detergent formulation.

  7. Enhanced biocatalytic esterification with lipase-immobilized chitosan/graphene oxide beads.

    Directory of Open Access Journals (Sweden)

    Siaw Cheng Lau

    Full Text Available In this work, lipase from Candida rugosa was immobilized onto chitosan/graphene oxide beads. This was to provide an enzyme-immobilizing carrier with excellent enzyme immobilization activity for an enzyme group requiring hydrophilicity on the immobilizing carrier. In addition, this work involved a process for the preparation of an enzymatically active product insoluble in a reaction medium consisting of lauric acid and oleyl alcohol as reactants and hexane as a solvent. This product enabled the stability of the enzyme under the working conditions and allowed the enzyme to be readily isolated from the support. In particular, this meant that an enzymatic reaction could be stopped by the simple mechanical separation of the "insoluble" enzyme from the reaction medium. Chitosan was incorporated with graphene oxide because the latter was able to enhance the physical strength of the chitosan beads by its superior mechanical integrity and low thermal conductivity. The X-ray diffraction pattern showed that the graphene oxide was successfully embedded within the structure of the chitosan. Further, the lipase incorporation on the beads was confirmed by a thermo-gravimetric analysis. The lipase immobilization on the beads involved the functionalization with coupling agents, N-hydroxysulfosuccinimide sodium (NHS and 1-ethyl-(3-dimethylaminopropyl carbodiimide (EDC, and it possessed a high enzyme activity of 64 U. The overall esterification conversion of the prepared product was 78% at 60 °C, and it attained conversions of 98% and 88% with commercially available lipozyme and novozyme, respectively, under similar experimental conditions.

  8. Wood mimetic hydrogel beads for enzyme immobilization.

    Science.gov (United States)

    Park, Saerom; Kim, Sung Hee; Won, Keehoon; Choi, Joon Weon; Kim, Yong Hwan; Kim, Hyung Joo; Yang, Yung-Hun; Lee, Sang Hyun

    2015-01-22

    Wood component-based composite hydrogels have potential applications in biomedical fields owing to their low cost, biodegradability, and biocompatibility. The controllable properties of wood mimetic composites containing three major wood components are useful for enzyme immobilization. Here, lipase from Candida rugosa was entrapped in wood mimetic beads containing cellulose, xylan, and lignin by dissolving wood components with lipase in [Emim][Ac], followed by reconstitution. Lipase entrapped in cellulose/xylan/lignin beads in a 5:3:2 ratio showed the highest activity; this ratio is very similar to that in natural wood. The lipase entrapped in various wood mimetic beads showed increased thermal and pH stability. The half-life times of lipase entrapped in cellulose/alkali lignin hydrogel were 31- and 82-times higher than those of free lipase during incubation under denaturing conditions of high temperature and low pH, respectively. Owing to their biocompatibility, biodegradability, and controllable properties, wood mimetic hydrogel beads can be used to immobilize various enzymes for applications in the biomedical, bioelectronic, and biocatalytic fields. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Isolation and characterization of novel thermophilic lipase-secreting bacteria

    Directory of Open Access Journals (Sweden)

    Mohammed Rabbani

    2013-12-01

    Full Text Available The purpose of the present study was to screen and identify the lipase-producing microorganisms from various regions of Iran. Samples collected from hot spring, Persian Gulf, desert area and oil-contaminated soil, were analyzed for thermophilic extracellular-lipase producing organisms. Six strains with high activity on rhodamine B plates were selected for chemical identification and further study. Among these isolated bacteria, four strains show higher activity in pH-Stat method at 55 °C. These strains were identified by PCR amplification of 16s rRNA genes using universal primers. Fermentation increased the activity up to 50%. The growth medium, designed for lipase production, increased the activity up to 4.55 folds. The crude supernatant of ZR-5 after fermentation and separation the cells, was lyophilized and the activity was measured. Total activity of this strain was 12 kU/g that shows its potential for industrial uses. Further study is required for purification of enzyme and calculation its specific activity. Immobilization is another approach should be considered.

  10. Properties of protease and lipase from whole and individual organ of viscera from three tuna species

    Directory of Open Access Journals (Sweden)

    Thiraratana Prachumratana

    2008-04-01

    Full Text Available Properties of visceral enzymes from yellowfin tuna (Thunnus albacares, skipjack tuna (Katsuwonus pelamis and tonggol tuna (Thunnus albacares were studied. The crude enzymes from viscera of yellowfin tuna and skipjack tuna exhibited the highest activities at pH 10.0 whereas it was at pH 9.0 for those from viscera of tonggol tuna. The enzymes were the most stable at their optima pH after 120 min incubation. The optimum temperatures for protease and lipase activities were at 50oC and 60oC, respectively but the extracted enzymes were more stable in the temperature range of 37- 40oC. The protease and lipase from spleen were the most thermostable with the half-life of 120 and 90 min at 60oC incubation, respectively. Protease activity from spleen accounted for 45.6% of the total protease activity of the whole tuna viscera.

  11. Immobilization of a Plant Lipase from Pachira aquatica in Alginate and Alginate/PVA Beads

    Directory of Open Access Journals (Sweden)

    Bárbara M. Bonine

    2014-01-01

    Full Text Available This study reports the immobilization of a new lipase isolated from oleaginous seeds of Pachira aquatica, using beads of calcium alginate (Alg and poly(vinyl alcohol (PVA. We evaluated the morphology, number of cycles of reuse, optimum temperature, and temperature stability of both immobilization methods compared to the free enzyme. The immobilized enzymes were more stable than the free enzyme, keeping 60% of the original activity after 4 h at 50°C. The immobilized lipase was reused several times, with activity decreasing to approximately 50% after 5 cycles. Both the free and immobilized enzymes were found to be optimally active between 30 and 40°C.

  12. Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

    Science.gov (United States)

    Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

    2013-12-01

    Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed. © 2013 FEBS.

  13. Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

    Science.gov (United States)

    2014-01-01

    Background There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite®545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI.3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I.3, requiring a refolding step, was poorly immobilized on all supports tested (best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes. PMID:24755191

  14. Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

    DEFF Research Database (Denmark)

    Terp, G E; Christensen, I T; Jørgensen, Flemming Steen

    2000-01-01

    to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions......Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach...... differences between the eight enzyme-substrate complexes were studied with particular emphasis on the active site, and possible sites for obtaining selectivity among the MMP's are discussed. Differences in the P1' pocket are well-documented and have been extensively exploited in inhibitor design. The present...

  15. Vibrational spectroscopy modeling of a drug in molecular solvents and enzymes

    Science.gov (United States)

    Devereux, Christian J.; Fulfer, Kristen D.; Zhang, Xiaoliu; Kuroda, Daniel G.

    2017-09-01

    Modeling of drugs in enzymes is of immensurable value to many areas of science. We present a theoretical study on the vibrational spectroscopy of Rilpivirine, a HIV reverse transcriptase inhibitor, in conventional solvents and in clinically relevant enzymes. The study is based on vibrational spectroscopy modeling of the drug using molecular dynamics simulations, DFT frequency maps, and theory. The modeling of the infrared lineshape shows good agreement with experimental data for the drug in molecular solvents where the local environment motions define the vibrational band lineshape. On the other hand, the theoretical description of the drug in the different enzymes does not match previous experimental findings indicating that the utilized methodology might not apply to heterogeneous environments. Our findings show that the lack of reproducibility might be associated with the development of the frequency map which does not contain all of the possible interactions observed in such systems.

  16. Production of Biodiesel Using Immobilized Lipase and the Characterization of Different Co-Immobilizing Agents and Immobilization Methods

    Directory of Open Access Journals (Sweden)

    Kang Zhao

    2016-08-01

    Full Text Available Lipase from Candida sp. 99–125 is widely employed to catalyzed transesterification and can be used for biodiesel production. In this study, the lipase was immobilized by combined adsorption and entrapment to catalyze biodiesel production from waste cooking oil (WCO via transesterification, and investigating co-immobilizing agents as additives according to the enzyme activity. The addition of the mixed co-immobilizing agents has positive effects on the activities of the immobilized lipase. Three different immobilizing methods were compared by the conversion ratio of biodiesel and structured by Atom Force Microscopy (AFM and Scanning Electron Microscopy (SEM, respectively. It was found that entrapment followed by adsorption was the best method. The effect of the co-immobilizing agent amount, lipase dosage, water content, and reuse ability of the immobilized lipase was investigated. By comparison with previous research, this immobilized lipase showed good reuse ability: the conversion ratio excesses 70% after 10 subsequent reactions, in particular, was better than Novozym435 and TLIM on waste cooking oil for one unit of lipase.

  17. A coarse-grained model for synergistic action of multiple enzymes on cellulose

    Directory of Open Access Journals (Sweden)

    Asztalos Andrea

    2012-08-01

    Full Text Available Abstract Background Degradation of cellulose to glucose requires the cooperative action of three classes of enzymes, collectively known as cellulases. Endoglucanases randomly bind to cellulose surfaces and generate new chain ends by hydrolyzing β-1,4-D-glycosidic bonds. Exoglucanases bind to free chain ends and hydrolyze glycosidic bonds in a processive manner releasing cellobiose units. Then, β-glucosidases hydrolyze soluble cellobiose to glucose. Optimal synergistic action of these enzymes is essential for efficient digestion of cellulose. Experiments show that as hydrolysis proceeds and the cellulose substrate becomes more heterogeneous, the overall degradation slows down. As catalysis occurs on the surface of crystalline cellulose, several factors affect the overall hydrolysis. Therefore, spatial models of cellulose degradation must capture effects such as enzyme crowding and surface heterogeneity, which have been shown to lead to a reduction in hydrolysis rates. Results We present a coarse-grained stochastic model for capturing the key events associated with the enzymatic degradation of cellulose at the mesoscopic level. This functional model accounts for the mobility and action of a single cellulase enzyme as well as the synergy of multiple endo- and exo-cellulases on a cellulose surface. The quantitative description of cellulose degradation is calculated on a spatial model by including free and bound states of both endo- and exo-cellulases with explicit reactive surface terms (e.g., hydrogen bond breaking, covalent bond cleavages and corresponding reaction rates. The dynamical evolution of the system is simulated by including physical interactions between cellulases and cellulose. Conclusions Our coarse-grained model reproduces the qualitative behavior of endoglucanases and exoglucanases by accounting for the spatial heterogeneity of the cellulose surface as well as other spatial factors such as enzyme crowding. Importantly, it captures

  18. Lipase biofilm deposited by Matrix Assisted Pulsed Laser Evaporation technique

    Science.gov (United States)

    Aronne, Antonio; Bloisi, Francesco; Calabria, Raffaela; Califano, Valeria; Depero, Laura E.; Fanelli, Esther; Federici, Stefania; Massoli, Patrizio; Vicari, Luciano R. M.

    2015-05-01

    Lipase is an enzyme that finds application in biodiesel production and for detection of esters and triglycerides in biosensors. Matrix Assisted Pulsed Laser Evaporation (MAPLE), a technique derived from Pulsed Laser Deposition (PLD) for deposition of undamaged biomolecules or polymers, is characterized by the use of a frozen target obtained from a solution/suspension of the guest material (to be deposited) in a volatile matrix (solvent). The presence of the solvent avoids or at least reduces the potential damage of guest molecules by laser radiation but only the guest material reaches the substrate in an essentially solvent-free deposition. MAPLE can be used for enzymes immobilization, essential for industrial application, allowing the development of continuous processes, an easier separation of products, the reuse of the catalyst and, in some cases, enhancing enzyme properties (pH, temperature stability, etc.) and catalytic activity in non-aqueous media. Here we show that MAPLE technique can be used to deposit undamaged lipase and that the complex structure (due to droplets generated during extraction from target) of the deposited material can be controlled by changing the laser beam fluence.

  19. Characteristics of a Monoacylglycerol Lipase Isolated from Pseudomonas sp. LP7315 -Hydrolysis and Synthesis of Monoglycerides

    OpenAIRE

    Sakiyama, Takaharu; Yoshimi, Tsuyoshi; Miyake, Akira; Umeoka, Midori; Tanaka, Atsushi; Ozaki, Sho; Nakanishi, Kazuhiro

    2001-01-01

    A monoacylglycerol lipase (MGL) was purified from Pseudomonas sp. LP7315 by ammonium sulfate precipitation, anion-exchange chromatography, and preparative electrophoresis. The purified enzyme was homogeneous on an SDS-polyacrylamide gel with a molecular mass of 59 kDa. Itshydrolytic activity was confirmed to be specific for monoglycerides: the enzyme did not hydrolyze diandtriglycerides. MGL was found to be stable even after l-h incubation at 65℃. The hydrolytic activity depended not only on ...

  20. Production and characterization of an extracellular lipase from Candida guilliermondii

    OpenAIRE

    Oliveira,Anne Caroline Defranceschi; Fernandes,Maria Luiza; Mariano,André Bellin

    2014-01-01

    Extracellular lipases from the endophytic yeast Candida guilliermondii isolated from castor leaves (Ricinus communis L.) were produced using low-cost raw materials such as agro-industrial residues and applying them in the esterification of oleic acid for evaluating their potential use in biodiesel production. After partial purification using ammonium sulfate, the enzyme was characterized and presented higher activity (26.8 ± 1.5 U mL-1) in the presence of 5 mmol L-1 NaCl at 30 ºC and pH ...

  1. Physical immobilization of 60 kDa chaperonin linked lipase from pseudomonas aeruginosa BN-1

    International Nuclear Information System (INIS)

    Syed, M.N.; Mehmood, S.; Bashir, A.; Ashraf, F.

    2012-01-01

    Abstract: The 60 kDa chaperone linked lipase from Pseudomonas aeruginosa was subjected to physical adsorption on silica 60 and acrylic beads. It was found that higher enzyme loading was achieved on silica gel than acrylic bead. The half life of immobilized enzyme was greater compared to the free enzyme. The adsorption of the enzyme onto a solid phase also resulted in increased thermo and solvent stability. It was observed that soluble enzyme showed maximum stability at 70 degree C while immobilized enzyme showed stability up to 80 degree C for 45 minutes. The stability of immobilized enzyme increased up to 48 hours from 24 hours against different organic solvent at 1.0 M concentration. It was noted that enzyme immobilized on acrylic beads have greater reusability compared to silica immobilized enzyme. (author)

  2. Adsorption of Candida rugosa lipase at water-polymer interface: The case of poly( DL)lactide

    Science.gov (United States)

    Kamel, Gihan; Bordi, Federico; Chronopoulou, Laura; Lupi, Stefano; Palocci, Cleofe; Sennato, Simona; Verdes, Pedro V.

    2011-12-01

    Insights into the interactions between biological macromolecules and polymeric surfaces are of great interest because of potential uses in developing biotechnologies. In this study we focused on the adsorption of a model lipolytic enzyme, Candida rugosa lipase (CRL), on poly-(D,L)-lactic acid (PDLLA) polymer with the aim to gain deeper insights into the interactions between the enzyme and the carrier. Such studies are of particular relevance in order to establish the optimal conditions for enzyme immobilization and its applications. We employed two different approaches; by analyzing the influence of adsorbed CRL molecules on the thermodynamic behavior of Langmuir films of PDLLA deposited at air-water interface, we gained interesting information on the molecular interactions between the protein and the polymer. Successively, by a systematic analysis of the adsorption of CRL on PDLLA nanoparticles, we showed that the adsorption of a model lipase, CRL, on PDLLA is described in terms of a Langmuir-type adsorption behavior. In this model, only monomolecular adsorption takes place (i.e. only a single layer of the protein adsorbs on the support) and the interactions between adsorbed molecules and surface are short ranged. Moreover, both the adsorption and desorption are activated processes, and the heat of adsorption (the difference between the activation energy for adsorption and desorption) is independent from the surface coverage of the adsorbing species. Finally, we obtained an estimate of the number of molecules of the protein adsorbed per surface unit on the particles, a parameter of a practical relevance for applications in biocatalysis, and a semi-quantitative estimate of the energies (heat of adsorption) involved in the adsorption process.

  3. Contribution of diacylglycerol lipase β to pain after surgery

    Directory of Open Access Journals (Sweden)

    Luk J

    2018-03-01

    Full Text Available Jennifer Luk,1 Yong Lu,1 Amanda Ackermann,1 Xiaoxue Peng,1 Diane Bogdan,1 Michelino Puopolo,1 David E Komatsu,2 Simon Tong,3 Iwao Ojima,3,4 Mario J Rebecchi,1 Martin Kaczocha1,4 1Department of Anesthesiology, 2Department of Orthopedics, 3Department of Chemistry, 4Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY, USA Background: Metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG yields arachidonic acid (AA, the precursor to proalgesic eicosanoids including prostaglandin E2 (PGE2. Diacylglycerol lipase β (DAGLβ is an enzyme that synthesizes 2-AG and its inhibition reduces eicosanoid levels and produces antinociceptive effects in models of inflammatory pain. Here we test whether inhibition of DAGLβ produces antinociceptive effects in a model of postoperative pain.Methods: Rats were administered the selective DAGLβ inhibitor KT109 or vehicle and underwent plantar incision. Postsurgical pain/disability was examined using evoked (mechanical hyperalgesia, functional (incapacitance/weight bearing, and functional/spontaneous (locomotion modalities.Results: Activity-based protein profiling confirmed that KT109 inhibited DAGLβ in the lumbar spinal cord (LSC and brain, confirming that it is a systemically active DAGLβ inhibitor. Treatment with KT109 reduced basal 2-AG, AA, and PGE2 levels in the liver but not the brain, indicating that DAGLβ activity does not significantly contribute to basal PGE2 production within the central nervous system. Plantar incision elevated the levels of 2-AG and PGE2 in the LSC. Although KT109 did not alter postsurgical 2-AG levels in the LSC, it slightly reduced PGE2 levels. In contrast, the clinically efficacious cyclooxygenase inhibitor ketoprofen completely suppressed PGE2 levels in the LSC. Similarly, KT109 had no significant effect upon postsurgical 2-AG, AA, or PGE2 levels at the incision site, while ketoprofen abolished PGE2 production at this location. KT109

  4. Immobilization of Lipase on Single Walled Carbon Nanotubes in Ionic Liquid

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Han Ki; Lee, Jae Kwan; Kim, Mahn Joo [Pohang University of Science and Technology, Pohang (Korea, Republic of); Lee, Cheol Jin [Korea University, Seoul (Korea, Republic of)

    2010-03-15

    A lipase from Pseudomonas cepacia was immobilized onto single walled carbon nanotubes (SWNTs) in two different ways in each of two solvent systems (buffer and ionic liquid). The most efficient immobilization was achieved in ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate, BMIM-BF4). In this procedure, carbon nanotubes were first functionalized noncovalently with 1-pyrenebutyric acid N-hydroxysuccinimide ester and then subject to the coupling reaction with the lipase in ionic liquid. The resulting immobilized enzyme displayed the highest activity in the transesterification of 1-phenylethyl alcohol in the presence of vinyl acetate in toluene.

  5. Lipase production by Penicillium restrictum using solid waste of industrial babassu oil production as substrate.

    Science.gov (United States)

    Palma, M B; Pinto, A L; Gombert, A K; Seitz, K H; Kivatinitz, S C; Castilho, L R; Freire, D M

    2000-01-01

    Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, olive oil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.

  6. Investigations of the efficiency of enzyme production technologies using modelling tools

    DEFF Research Database (Denmark)

    Albæk, Mads Orla; Gernaey, Krist; Hansen, Morten Skov

    fermentations of the filamentous fungus Trichoderma reesei in 550litre pilot scale stirred tank reactors for a range of process conditions. Based on the experimental data a process model has been created, which satisfactory simulates the effect of the changing process conditions: Aeration rate, agitation speed......Growing markets and new innovative applications of industrial enzymes leads to increased interest in efficient production of these products. Most industrial enzymes are currently produced in traditional stirred tank reactors in submerged fed batch culture. The limiting parameter in such processes...... and head space pressure. Examples of simulation results and outlines of the future uses of the model will be given....

  7. Purification and characterization of solvent tolerant lipase from Bacillus sp. for methyl ester production from algal oil.

    Science.gov (United States)

    Sivaramakrishnan, Ramachandran; Incharoensakdi, Aran

    2016-05-01

    Lipase from Bacillus sp. isolated from the oil contaminated soil was purified by ammonium sulphate precipitation and ion-exchange chromatography with a 5.1-fold purification and 10.5% yield. SDS-PAGE analysis of the enzyme revealed the molecular mass of 24 kDa. The optimum pH and temperature for lipase activity were 6.5 and 37°C, respectively. The isolated lipase was stimulated by pretreatment with methanol and ethanol as well as by divalent metal ions Ca(2+), Mg(2+) and Mn(2+). The enzyme showed high activity towards oleic rich oils. The enzyme immobilized on celite could retain 90% lipase activity after eight cycles. Transesterification of Botryococcus sp. oil using the immobilized enzyme for 40 h resulted in 80% yield of fatty acid methyl esters which had good properties for use as biodiesel. Overall results suggested that the solvent tolerant Bacillus lipase can be a potential biocatalyst for methyl ester production. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. A new alkaline lipase obtained from the metagenome of marine sponge Ircinia sp.

    Science.gov (United States)

    Su, Jing; Zhang, Fengli; Sun, Wei; Karuppiah, Valliappan; Zhang, Guangya; Li, Zhiyong; Jiang, Qun

    2015-07-01

    Microorganisms associated with marine sponges are potential resources for marine enzymes. In this study, culture-independent metagenomic approach was used to isolate lipases from the complex microbiome of the sponge Ircinia sp. obtained from the South China Sea. A metagenomic library was constructed, containing 6568 clones, and functional screening on 1 % tributyrin agar resulted in the identification of a positive lipase clone (35F4). Following sequence analysis 35F4 clone was found to contain a putative lipase gene lipA. Sequence analysis of the predicted amino acid sequence of LipA revealed that it is a member of subfamily I.1 of lipases, with 63 % amino acid similarity to the lactonizing lipase from Aeromonas veronii (WP_021231793). Based on the predicted secondary structure, LipA was predicted to be an alkaline enzyme by sequence/structure analysis. Heterologous expression of lipA in E. coli BL21 (DE3) was performed and the characterization of the recombinant enzyme LipA showed that it is an alkaline enzyme with high tolerance to organic solvents. The isolated lipase LipA was active in the broad alkaline range, with the highest activity at pH 9.0, and had a high level of stability over a pH range of 7.0-12.0. The activity of LipA was increased in the presence of 5 mM Ca(2+) and some organic solvents, e.g. methanol, acetone and isopropanol. The optimum temperature for the activity of LipA is 40 °C and the molecular weight of LipA was determined to be ~30 kDa by SDS-PAGE. LipA is an alkaline lipase and shows good tolerance to some organic solvents, which make it of potential utility in the detergent industry and enzyme mediated organic synthesis. The result of this study has broadened the diversity of known lipolytic genes and demonstrated that marine sponges are an important source for new enzymes.

  9. A Kinetic Modelling of Enzyme Inhibitions in the Central Metabolism of Yeast Cells

    Science.gov (United States)

    Kasbawati; Kalondeng, A.; Aris, N.; Erawaty, N.; Azis, M. I.

    2018-03-01

    Metabolic regulation plays an important role in the metabolic engineering of a cellular process. It is conducted to improve the productivity of a microbial process by identifying the important regulatory nodes of a metabolic pathway such as fermentation pathway. Regulation of enzymes involved in a particular pathway can be held to improve the productivity of the system. In the central metabolism of yeast cell, some enzymes are known as regulating enzymes that can be inhibited to increase the production of ethanol. In this research we study the kinetic modelling of the enzymes in the central pathway of yeast metabolism by taking into consideration the enzyme inhibition effects to the ethanol production. The existence of positive steady state solution and the stability of the system are also analysed to study the property and dynamical behaviour of the system. One stable steady state of the system is produced if some conditions are fulfilled. The conditions concern to the restriction of the maximum reactions of the enzymes in the pyruvate and acetaldehyde branch points. There exists a certain time of fermentation reaction at which a maximum and a minimum ethanol productions are attained after regulating the system. Optimal ethanol concentration is also produced for a certain initial concentration of inhibitor.

  10. Protein engineering by random mutagenesis and structure-guided consensus of Geobacillus stearothermophilus Lipase T6 for enhanced stability in methanol.

    Science.gov (United States)

    Dror, Adi; Shemesh, Einav; Dayan, Natali; Fishman, Ayelet

    2014-02-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production.

  11. Protein Engineering by Random Mutagenesis and Structure-Guided Consensus of Geobacillus stearothermophilus Lipase T6 for Enhanced Stability in Methanol

    Science.gov (United States)

    Dror, Adi; Shemesh, Einav; Dayan, Natali

    2014-01-01

    The abilities of enzymes to catalyze reactions in nonnatural environments of organic solvents have opened new opportunities for enzyme-based industrial processes. However, the main drawback of such processes is that most enzymes have a limited stability in polar organic solvents. In this study, we employed protein engineering methods to generate a lipase for enhanced stability in methanol, which is important for biodiesel production. Two protein engineering approaches, random mutagenesis (error-prone PCR) and structure-guided consensus, were applied in parallel on an unexplored lipase gene from Geobacillus stearothermophilus T6. A high-throughput colorimetric screening assay was used to evaluate lipase activity after an incubation period in high methanol concentrations. Both protein engineering approaches were successful in producing variants with elevated half-life values in 70% methanol. The best variant of the random mutagenesis library, Q185L, exhibited 23-fold-improved stability, yet its methanolysis activity was decreased by one-half compared to the wild type. The best variant from the consensus library, H86Y/A269T, exhibited 66-fold-improved stability in methanol along with elevated thermostability (+4.3°C) and a 2-fold-higher fatty acid methyl ester yield from soybean oil. Based on in silico modeling, we suggest that the Q185L substitution facilitates a closed lid conformation that limits access for both the methanol and substrate excess into the active site. The enhanced stability of H86Y/A269T was a result of formation of new hydrogen bonds. These improved characteristics make this variant a potential biocatalyst for biodiesel production. PMID:24362426

  12. Factors screening to statistical experimental design of racemic atenolol kinetic resolution via transesterification reaction in organic solvent using free Pseudomonas fluorescens lipase.

    Science.gov (United States)

    Agustian, Joni; Kamaruddin, Azlina Harun; Aboul-Enein, Hassan Y

    2017-07-01

    As the (R)-enantiomer of racemic atenolol has no β-blocking activity and no lack of side effects, switching from the racemate to the (S)-atenolol is more favorable. Transesterification of racemic atenolol using free enzymes investigated as a resource to resolve the racemate via this method is limited. Screenings of enzyme, medium, and acetyl donor were conducted first to give Pseudomonas fluorescens lipase, tetrahydrofuran, and vinyl acetate. A statistical design of the experiment was then developed using Central Composite Design on some operational factors, which resulted in the conversions of 11.70-61.91% and substrate enantiomeric excess (ee) of 7.31-100%. The quadratic models are acceptable with R 2 of 95.13% (conversion) and 89.63% (ee). The predicted values match the observed values reasonably well. Temperature, agitation speed, and substrate molar ratio factor have low effects on conversion and ee, but enzyme loading affects the responses highly. The interaction of temperature-agitation speed and temperature-substrate molar ratio show significant effects on conversion, while temperature-agitation speed, temperature-substrate molar ratio, and agitation speed-substrate molar ratio affect ee highly. Optimum conditions for the use of Pseudomonas fluorescens lipase, tetrahydrofuran, and vinyl acetate were found at 45°C, 175 rpm, 2000 U, and 1:3.6 substrate molar ratio. © 2017 Wiley Periodicals, Inc.

  13. Transesterification of waste cooking oil by an organic solvent-tolerant alkaline lipase from Streptomyces sp. CS273.

    Science.gov (United States)

    Mander, Poonam; Yoo, Hah-Young; Kim, Seung Wook; Choi, Yun Hee; Cho, Seung Sik; Yoo, Jin Cheol

    2014-02-01

    The aim of this present study was to produce a microbial enzyme that can potentially be utilized for the enzymatic transesterification of waste cooking oil. To that end, an extracellular lipase was isolated and purified from the culture broth of Streptomyces sp. CS273. The molecular mass of purified lipase was estimated to be 36.55 kDa by SDS PAGE. The optimum lipolytic activity was obtained at alkaline pH 8.0 to 8.5 and temperature 40 °C, while the enzyme was stable in the pH range 7.0 ∼ 9.0 and at temperature ≤40 °C. The lipase showed highest hydrolytic activity towards p-nitrophenyl myristate (C14). The lipase activity was enhanced by several salts and detergents including NaCl, MnSo₄, and deoxy cholic acid, while phenylmethylsulfonyl fluoride at concentration 10 mM inhibited the activity. The lipase showed tolerance towards different organic solvents including ethanol and methanol which are commonly used in transesterification reactions to displace alcohol from triglycerides (ester) contained in renewable resources to yield fatty acid alkyl esters known as biodiesel. Applicability of the lipase in transesterification of waste cooking oil was confirmed by gas chromatography mass spectrometry analysis.

  14. Two-step synthesis of fatty acid ethyl ester from soybean oil catalyzed by Yarrowia lipolytica lipase

    Directory of Open Access Journals (Sweden)

    Chen Jinnan

    2011-03-01

    Full Text Available Abstract Background Enzymatic biodiesel production by transesterification in solvent media has been investigated intensively, but glycerol, as a by-product, could block the immobilized enzyme and excess n-hexane, as a solution aid, would reduce the productivity of the enzyme. Esterification, a solvent-free and no-glycerol-release system for biodiesel production, has been developed, and two-step catalysis of soybean oil, hydrolysis followed by esterification, with Yarrowia lipolytica lipase is reported in this paper. Results First, soybean oil was hydrolyzed at 40°C by 100 U of lipase broth per 1 g of oil with approximately 30% to 60% (vol/vol water. The free fatty acid (FFA distilled from this hydrolysis mixture was used for the esterification of FFA to fatty acid ethyl ester by immobilized lipase. A mixture of 2.82 g of FFA and equimolar ethanol (addition in three steps were shaken at 30°C with 18 U of lipase per 1 gram of FFA. The degree of esterification reached 85% after 3 hours. The lipase membranes were taken out, dehydrated and subjected to fresh esterification so that over 82% of esterification was maintained, even though the esterification was repeated every 3 hours for 25 batches. Conclusion The two-step enzymatic process without glycerol released and solvent-free demonstrated higher efficiency and safety than enzymatic transesterification, which seems very promising for lipase-catalyzed, large-scale production of biodiesel, especially from high acid value waste oil.

  15. Lipase catalyzed interesterification of Amazonian patauá oil and palm stearin for preparation of specific-structured oils.

    Science.gov (United States)

    Speranza, Paula; Ribeiro, Ana Paula Badan; Macedo, Gabriela Alves

    2015-12-01

    This study showed that enzymatic interesterification of Amazonian oils could be an important tool in order to produce new oils with physicochemical properties that improve the applications of these raw materials. Structured oils of Amazonian patauá oil and palm stearin using two lipases were produced in three different enzymatic systems: first, a crude lipase from the fungus Rhizopus sp (a microorganism isolated in our laboratory); second, a commercial lipase; and third, to check any synergistic effect, a mixture of both lipases (Rhizopus sp and commercial). The lipase from Rhizopus sp was specific in the incorporation of oleic acid at the sn-1,3 positions of the triacylglycerol, resulting in an oil richer in saturated fatty acid in the sn-2 position. This enzyme, produced by solid-state fermentation, even though crude, was fatty acid and positional specific and able to operate at low concentration (2.5 %, w/w). In the second enzyme system, the commercial lipase from Thermomyces lanuginosus was not specific in the tested conditions; there was no change in the distribution of saturated and unsaturated fatty acids in the three positions of the triacylglycerol profile, there was only a replacement by the type of fatty acid at the same position. In the third enzyme system, the mixture of both lipases shows no synergic effect. The structured oils retained the concentration of bioactive α- and γ- tocopherol in the three enzyme systems. Triacylglycerol classes and Thermal behavior tests indicated the formation of more homogeneous triacylglycerols, especially the mono and di-unsaturated.

  16. Autodisplay for the co-expression of lipase and foldase on the surface of E. coli: washing with designer bugs

    Science.gov (United States)

    2014-01-01

    Background Lipases including the lipase from Burkholderia cepacia are in a main focus in biotechnology research since many years because of their manifold possibilities for application in industrial processes. The application of Burkholderia cepacia lipase for these processes appears complicated because of the need for support by a chaperone, the lipase specific foldase. Purification and reconstitution protocols therefore interfere with an economic implementation of such enzymes in industry. Autodisplay is a convenient method to express a variety of passenger proteins on the surface of E. coli. This method makes subsequent purification steps to obtain the protein of interest unnecessary. If enzymes are used as passengers, the corresponding cells can simply be applied as whole cell biocatalysts. Furthermore, enzymes surface displayed in this manner often acquire stabilization by anchoring within the outer membrane of E. coli. Results The lipase and its chaperone foldase from B. cepacia were co-expressed on the surface of E. coli via autodisplay. The whole cell biocatalyst obtained thereby exhibited an enzymatic activity of 2.73 mU mL-1 towards the substrate p-nitrophenyl palmitate when applied in an OD578 =1. Outer membrane fractions prepared from the same culture volume showed a lipase activity of 4.01 mU mL-1. The lipase-whole cell biocatalyst as well as outer membrane preparations thereof were used in a standardized laundry test, usually adopted to determine the power of washing agents. In this test, the lipase whole cell biocatalyst and the membrane preparation derived thereof exhibited the same lipolytic activity as the purified lipase from B. cepacia and a lipase preparation which is already applied in commercial washing agents. Conclusions Co-expression of both the lipase and its chaperone foldase on the surface of E. coli yields a lipid degrading whole cell biocatalyst. Therefore the chaperone supported folding process, absolutely required for the lipolytic

  17. Development of a microtiter plate-based assay for the detection of lipase-catalyzed transesterifications in organic solvents.

    Science.gov (United States)

    Pöhnlein, Martin; Finkbeiner, Tim; Syldatk, Christoph; Hausmann, Rudolf

    2015-03-01

    A microtiter plate-based assay was developed to evaluate the ability of lipases to perform transesterifications when employed in different organic solvents. A 4-nitrophenol assay was carried out employing seven different lipase formulations and two fatty acid methyl esters with different chain lengths in a total of six organic solvents with logP values approximately between 1 and -1. This assay delivered results within comparatively short times measured by a color reaction and thus facilitates the choice of an enzyme-solvent combination for the synthesis of glycolipids. To validate the findings, glycolipid syntheses were performed using the same lipase formulation in the same solvents. When comparing the results obtained using the microtiter plate-based assay to the results of the glycolipid syntheses using the same lipases and solvents, matching results were obtained.

  18. Otimização da esterificação de ácido hexanóico com n-butanol empregando lipase (Termomyces lanuginosus imobilizada em gelatina Optimization of n-butyl hexanoate synthesis applying lipase immobilized (Termomyces Lanuginosus in gelatin

    Directory of Open Access Journals (Sweden)

    Everton Skoronski

    2013-01-01

    Full Text Available The application of Lipozyme (Termomyces lanuginosus immobilized in gelatin gel in aliphatic ester synthesis was investigated taking the esterification of hexanoic acid with n-butanol as a model reaction. Conditions were optimized by factorial design and the highest conversion was obtained under the following conditions: molar ratio alcohol: acid of 2:1, reaction time of 48 h and biocatalyst weight of 7.0 g. Under these conditions the esterification yield was around 98 %. The operational stability of the immobilized lipase was assessed and results showed that after 12 batch runs, the enzyme showed no significant loss of activity.

  19. Lipase in milk, curd and cheese

    NARCIS (Netherlands)

    Geurts, T.J.; Lettink, F.J.; Wouters, J.T.M.

    2003-01-01

    Presence of lipase in milk, curd, whey and cheese was studied. A small amount of the product was added to a large volume of lipase-free whole milk that had been made sensitive to lipolysis by homogenization. Increase of the acidity of the fat in the mixture, determined after incubation, was

  20. Enhanced lipase production by mutation induced Aspergillus ...

    African Journals Online (AJOL)

    ... the HNO2 mutant (AHN3) and 217% higher than the UV mutant (AUV3) and 276% higher lipase activity than the parent strain. The results indicated that UV, HNO2 and NTG treatment were effective physical and chemical mutagenic agents for strain improvement of Aspergillus japonicus for enhanced lipase productivity.

  1. Selectivity of lipases for estolides synthesis

    NARCIS (Netherlands)

    Todea, Anamaria; Frissen, A.E.; Otten, Linda G.; Arends, I.; Peter, F.; Boeriu, C.G.

    2015-01-01

    Lipase-catalyzed synthesis of estolides starting from different saturated (C16 16OH, C18 12OH) and unsaturated (C18:1 9 cis 12-OH) hydroxy-fatty acids was investigated. For this reason, the catalytic efficiency of several native and immobilized lipases in different organic reaction media at

  2. Effect of ethyleneoxide groups of anionic surfactants on lipase activity.

    Science.gov (United States)

    Magalhães, Solange S; Alves, Luís; Sebastião, Marco; Medronho, Bruno; Almeida, Zaida L; Faria, Tiago Q; Brito, Rui M M; Moreno, Maria J; Antunes, Filipe E

    2016-09-01

    The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant-enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE 0 S) and of sodium lauryl ether sulfate with two EO units (SLE 2 S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE 2 S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276-1282, 2016. © 2016 American Institute of Chemical Engineers.

  3. Cloning and characterization of newly isolated lipase from Enterobacter sp. Bn12.

    Science.gov (United States)

    Farrokh, Parisa; Yakhchali, Bagher; Karkhane, Ali Asghar

    2014-01-01

    A mesophilic Enterobacter sp. Bn12 producing an alkaline thermostable lipase was isolated from soil in Tehran, Iran. The lipase gene (ELBn12) was identified from a genomic library. Sequence analysis of the DNA fragment revealed an open reading frame of 879 bp encoding a lipase with a molecular mass of 31.3 kDa. The deduced amino acid sequence showed 96% identity with a lipase of Enterobacter sp. Ag1 and the identity of their DNA sequences was 88.9%. ELBn12 belongs to the lipase subfamily I.1 and its catalytic triad consists of Ser82, Asp237 and His259. The lipase was expressed in Escherichia coli (BL21) pLysS and partially purified by anion exchange chromatography. The maximum activity of ELBn12 was obtained at temperature of 60 °C and pH 8.0 towards tricaprylin (C8) and its specific activity was around 2900 U/mg. ELBn12 was stable within a broad pH range from 6.0 to 11.0. The enzyme showed high stability in both polar and nonpolar organic solvents at 50% (v/v). The lipase activity was enhanced in the presence of 10 mM of Ca(2+), Mg(2+) and K(+), while heavy metals (Fe(3+) and Zn(2+)) had strong inhibitory effect. ELBn12 showed high activity in the presence of 1% (w/v) nonionic surfactants, however ionic surfactants inhibited the lipolytic activity. ELBn12 characteristics show that it has a potential to be used in various industrial processes.

  4. Effect of prolonged refrigeration on the lipid profile, lipase activity, and oxidative status of human milk.

    Science.gov (United States)

    Bertino, Enrico; Giribaldi, Marzia; Baro, Cristina; Giancotti, Valeria; Pazzi, Marco; Peila, Chiara; Tonetto, Paola; Arslanoglu, Sertac; Moro, Guido E; Cavallarin, Laura; Gastaldi, Daniela

    2013-04-01

    The study was aimed at evaluating the effect of prolonged refrigeration of fresh human milk (HM) on its fatty acid profile, free fatty acid content, lipase activities, and oxidative status. HM from mothers of preterm newborns was collected, pooled, and placed in the neonatal intensive care unit (NICU) refrigerator. Pooled milk was aliquoted and analyzed within 3 hours of collection, and after 24, 48, 72, and 96 hours of storage. The milk samples were analyzed for pH, total and free fatty acid profile, lipase activity at room temperature and at 4°C, lipase activity at room temperature in presence of sodium cholate (bile salt-dependent lipase), total antioxidant capacity, thiobarbituric acid reactive species, malondialdehyde, and conjugated diene concentration. The experiment was replicated in 3 independent trials. Prolonged refrigeration did not affect the fatty acid composition of breast milk, and preserved both its overall oxidative status and the activity of HM lipolytic enzymes. In particular, bile salt-dependent lipase activity, long-chain polyunsaturated fatty acids, and medium-chain saturated fatty acid concentrations were unaffected for up to 96 hours of refrigerated storage. Prolonged refrigeration of fresh HM for 96 hours maintained its overall lipid composition. The limited lipolysis during storage should be ascribed to the activity of lipoprotein lipase, responsible for the decrease in pH. Our study demonstrates that infants who receive expressed milk stored for up to 96 hours receive essentially the same supply of fatty acids and active lipases as do infants fed directly at the breast.

  5. Facile fabrication of a stable and recyclable lipase@amine-functionalized ZIF-8 nanoparticles for esters hydrolysis and transesterification

    Science.gov (United States)

    Cheong, Ling-Zhi; Wei, Yayu; Wang, Hongbin; Wang, Zhiying; Su, Xiurong; Shen, Cai

    2017-08-01

    Zeolitic imidazolate frameworks (ZIF) represent one of the metal organic frameworks (MOF) with high potential for enzyme immobilization due to their exceptional chemical and thermal stability, negligible cytotoxicity, and easy synthesis under mild biocompatible conditions. Amine-functionalized ZIF-8 (An-ZIF-8) are capable of forming multipoint attachment via hydrogen bonding with lipase which will immobilize and further enhance stabilization of lipase. In addition, increased hydrophilicity of An-ZIF-8 will increase partitioning of An-ZIF-8 immobilized lipase at the aqueous/organic interface which enable lipase to expose its active site and retain its catalytic activity at its highest. Present study reports the use of ZIF-8 and An-ZIF-8 nanoparticles as carrier for Burkholderia cepacia lipase (BCL), compares the ester hydrolysis and transesterification activities of immobilized lipase with those of free lipase, and evaluates the reusability and recovery rate of the immobilized lipase. An-ZIF-8 nanoparticles (average 130.42 ± 0.55 nm) were facilely synthesized via mixing ZIF-8 nanoparticles with ammonia hydroxide solution. Despite having similar characteristics of high crystallinity and forming cuboid-like particles, An-ZIF-8 demonstrated significantly ( P < 0.05) lower Brunauer-Emmett-Teller (BET) surface area and higher thermal stability than ZIF-8. BCL were successfully immobilized on ZIF-8 (BCL@ZIF-8) and An-ZIF-8 (BCL@An-ZIF-8) nanoparticles with an average lipase loading rate of 8 mg/g MOF. The immobilized BCL demonstrated no significant differences in terms of esters hydrolysis and transesterification activities with those of free BCL. BCL@An-ZIF-8 demonstrated superior catalytic stability in comparison to BCL@ZIF-8 with retainment of more than 80% of its initial hydrolysis and transesterification activity for at least 10 repeated runs. In addition, more than 80% of the BCL@An-ZIF-8 can be easily recovered during each cycle of the reusability test through

  6. Lipase-Catalyzed Kinetic Resolution of Novel Antifungal N-Substituted Benzimidazole Derivatives.

    Science.gov (United States)

    Łukowska-Chojnacka, Edyta; Staniszewska, Monika; Bondaryk, Małgorzata; Maurin, Jan K; Bretner, Maria

    2016-04-01

    A series of new N-substituted benzimidazole derivatives was synthesized and their antifungal activity against Candida albicans was evaluated. The chemical step included synthesis of appropriate ketones containing benzimidazole ring, reduction of ketones to the racemic alcohols, and acetylation of alcohols to the esters. All benzimidazole derivatives were obtained with satisfactory yields and in relatively short times. All synthesized compounds exhibit significant antifungal activity against Candida albicans 900028 ATCC (% cell inhibition at 0.25 μg concentration > 98%). Additionally, racemic mixtures of alcohols were separated by lipase-catalyzed kinetic resolution. In the enzymatic step a transesterification reaction was applied and the influence of a lipase type and solvent on the enantioselectivity of the reaction was studied. The most selective enzymes were Novozyme SP 435 and lipase Amano AK from Pseudomonas fluorescens (E > 100). © 2016 Wiley Periodicals, Inc.

  7. Cinnamyl acetate synthesis by lipase-catalyzed transesterification in a solvent-free system.

    Science.gov (United States)

    Geng, Bo; Wang, Mengfan; Qi, Wei; Su, Rongxin; He, Zhimin

    2012-01-01

    Cinnamyl acetate was synthesized using immobilized lipase through transesterification between ethyl acetate and cinnamyl alcohol. In the solvent-free system, ethyl acetate acted as not only the acyl donor but also as the solvent of cinnamyl alcohol. Conversion (90.06%) was achieved after 3 H when transesterification was carried out at ethyl acetate/cinnamyl alcohol 15:1, 2.67 g L of lipase (Novozym 435) loading, and 40°C. Excellent stability and reusability of the enzyme resulted from the moderate reaction system. Kinetic studies showed that the Michaelis constants for ethyl acetate and cinnamyl alcohol and the inhibition constant of cinnamyl alcohol were 2.241, 206.82, and 0.461 mmol L⁻¹, respectively, which indicated that the reaction complied with the Ping-Pong Bi-Bi mechanism, with the inhibition of cinnamyl alcohol on lipase. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  8. Evaluation of inorganic matrixes as supports for immobilization of microbial lipase

    Directory of Open Access Journals (Sweden)

    Castro H.F.

    2000-01-01

    Full Text Available Candida rugosa was immobilized by physical adsorption on several inorganic supports using hexane as coupling medium. The enzymatic activities of the different derivatives were determined by both hydrolysis of olive oil and esterification of n-butanol with butyric acid. The results were compared to previous data obtained by using a controlled porous silica matrix. The goal was to contribute in searching inexpensive supports for optimum lipase performance. All supports examined exhibited good properties for binding the enzyme lipase. Zirconium phosphate was the best support, giving the highest percentage of protein fixation (86% and the highest retention of lipase activity after immobilization (34%. The operational stability performance for niobium oxide derivative was improved by previously activated the support with silane and glutaraldehyde. Thermal stabilities were also examined by thermal gravimetric analysis (TG.

  9. Surface modification of magnetite nanoparticles using gluconic acid and their application in immobilized lipase.

    Science.gov (United States)

    Sui, Ying; Cui, Yu; Nie, Yong; Xia, Guang-Ming; Sun, Guo-Xin; Han, Jing-Tian

    2012-05-01

    Superparamagnetic magnetite nanoparticles (SMN) were surface-modified with gluconic acid (GLA) to improve their hydrophilicity and bio-affinity. Gluconic acid was successfully coated on the surface of magnetite nanoparticles and characterized using Fourier transform infrared spectroscopy (FT-IR). With water-soluble carbodiimide (EDC) as the coupling reagent, lipase was successfully immobilized onto the hydroxyl-functionalized magnetic nanoparticles. The immobilized lipase had better resistance to temperature and pH inactivation in comparison to the free form and hence widened the reaction pH and temperature range. Thermostability and storage stability of the enzyme improved upon covalent immobilization. Immobilized lipase showed higher activity after recycling when compared to the free one and could be recovered by magnetic separation. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. A Numerical Procedure for Model Identifiability Analysis Applied to Enzyme Kinetics

    DEFF Research Database (Denmark)

    Daele, Timothy, Van; Van Hoey, Stijn; Gernaey, Krist

    2015-01-01

    The proper calibration of models describing enzyme kinetics can be quite challenging. In the literature, different procedures are available to calibrate these enzymatic models in an efficient way. However, in most cases the model structure is already decided on prior to the actual calibration...... and Pronzato (1997) and which can be easily set up for any type of model. In this paper the proposed approach is applied to the forward reaction rate of the enzyme kinetics proposed by Shin and Kim(1998). Structural identifiability analysis showed that no local structural model problems were occurring......) identifiability problems. By using the presented approach it is possible to detect potential identifiability problems and avoid pointless calibration (and experimental!) effort....

  11. The molecular basis for lipase stereoselectivity.

    Science.gov (United States)

    Chen, Hui; Meng, Xiao; Xu, Xiaoqing; Liu, Wenbo; Li, Shengying

    2018-04-01

    Lipases are among the most applied biocatalysts in organic synthesis to catalyze the kinetic resolution of a wide range of racemic substrates to yield optically pure compounds. Due to the rapidly increased demands for optically pure compounds, deep understanding of the molecular basis for lipase stereoselectivity and how to obtain lipases with excellent asymmetric selectivity have become one of primary research goals in this field. This review is focused on the molecular factors that have impacts on the stereoselectivity of lipases including the steric complementarity between the lipase topological structure and its substrate, the regional structural flexibility, the hydrogen bonds between the residues around the catalytic site and the tetrahedral intermediates, and the electrostatic interactions between surface residues. Moreover, the synergistic effects of these structural factors on the catalytic properties including stereoselectivity, activity, and stability are also discussed.

  12. A model-based framework for design of intensified enzyme-based processes

    DEFF Research Database (Denmark)

    Román-Martinez, Alicia

    This thesis presents a generic and systematic model-based framework to design intensified enzyme-based processes. The development of the presented methodology was motivated by the needs of the bio-based industry for a more systematic approach to achieve intensification in its production plants wi...

  13. Trend to equilibrium for a reaction-diffusion system modelling reversible enzyme reaction

    OpenAIRE

    Elias, Jan

    2016-01-01

    20 pages; A spatio-temporal evolution of chemicals appearing in a reversible enzyme reaction and modelled by a four component reaction-diffusion system with the reaction terms obtained by the law of mass action is considered. The large time behaviour of the system is studied by means of entropy methods.

  14. Trend to Equilibrium for a Reaction-Diffusion System Modelling Reversible Enzyme Reaction.

    Science.gov (United States)

    Eliaš, Ján

    2018-01-01

    A spatio-temporal evolution of chemicals appearing in a reversible enzyme reaction and modelled by a four-component reaction-diffusion system with the reaction terms obtained by the law of mass action is considered. The large time behaviour of the system is studied by means of entropy methods.

  15. Anti- and Pro-Lipase Activity of Selected Medicinal, Herbal and Aquatic Plants, and Structure Elucidation of an Anti-Lipase Compound

    Directory of Open Access Journals (Sweden)

    Muhammad Abubakar Ado

    2013-11-01

    Full Text Available Plants that help in slowing down the digestion of triacylglycerols (TAGs in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98 medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents. Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4% exhibited moderate inhibition (41%–80% and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack I.C Nielsen L. (jering, Cynometra cauliflora (nam-nam and Aleurites moluccana (L. Willd (candle nut/buah keras had the highest (100% anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis, activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.

  16. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    Science.gov (United States)

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  17. Structural basis of phospholipase activity of Staphylococcus hyicus lipase

    NARCIS (Netherlands)

    Tiesinga, Jan J. W.; van Pouderoyen, Gertie; Nardini, Marco; Ransac, Stephane; Dijkstra, Bauke W.

    2007-01-01

    Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A, activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 angstrom resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule.

  18. Kinetic and thermodynamic characterization of lipase produced by Cellulomonas flavigena UNP3.

    Science.gov (United States)

    Prajapati, Vimal; Patel, Honey; Trivedi, Ujjval; Patel, Kamlesh

    2014-09-01

    Lipase of Cellulomonas flavigena UNP3 was purified by two-step purification process comprising ammonium sulfate precipitation followed by gel permeation chromatography (GPC). The recovery of lipase after GPC was found to be 1.70% with 20.98-fold increase in specific activity. The molecular weight of lipase protein was found to be 45.2 kDa by SDS-PAGE. Activation energy for p-nitrophenol palmitate (pNPP) hydrolysis was 26.45 kJ mol(-1) , while temperature quotient (Q10 ) was found to be 1.64. The enzyme was found to be stable over wide pH range and thermally stable at 30-40 °C up to 60 min of incubation while exhibited maximum activity at 30 °C with pH 7.0. Vmax , Km , and Kcat for pNPP were found to be 666.71 U ml(-1) , 1.33 mM (pNPP) and 433 min(-1) , respectively. Activation energy for irreversible inactivation Ea(d) of lipase was 64.32 kJ mol(-1) . Thermodynamic parameters of irreversible inactivation of lipase and pNPP hydrolysis were also determined. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Fungal endophytes associated with Viola odorata Linn. as bioresource for pancreatic lipase inhibitors.

    Science.gov (United States)

    Katoch, M; Paul, A; Singh, G; Sridhar, S N C

    2017-08-03

    As per the recent statistical reports of World Health Organisation (WHO), 13% of total global population is obese. Orlistat remains to be the only drug approved for the long term treatment of obesity. Recent findings highlighted severe adverse effects of orlistat that included hepatotoxicity, gall stones, kidney stones and acute pancreatitis. Therefore, search for new drug is required. The investigations based on endophytic natural products would prove pivotal in the global fight against this health issue. Obesity is associated with lipid metabolism involving pancreatic lipase enzyme. The inhibition of pancreatic lipase is demonstrated by using the extracts of endophytes isolated from Viola odorata Linn. In addition, endophytes were identified using ITS based rDNA sequencing. Present study involves the isolation and identification of 27 endophytes from V. odorata. All the endophytes were evaluated for lipase inhibitory activities. The extracts of seven endophytes exhibited lipase inhibitory activity with IC 50  endophytic community with potent lipase inhibitory activity. VOLF4 is the potential endophyte. The extract of VOLF4 can be used to develop the potential drug to treat obesity.

  20. Evaluation of a novel thermo-alkaline Staphylococcus aureus lipase for application in detergent formulations

    Directory of Open Access Journals (Sweden)

    Abir Ben Bacha

    2018-03-01

    Full Text Available An extracellular lipase of a newly isolated S. aureus strain ALA1 (SAL4 was purified from the optimized culture medium. The SAL4 specific activity determined at 60 °C and pH 12 by using olive oil emulsion or TC4, reached 7215 U/mg and 2484 U/mg, respectively. The 38 NH2-terminal amino acid sequence of the purified enzyme starting with two extra amino acid residues (LK was similar to known staphylococcal lipase sequences. This novel lipase maintained almost 100% and 75% of its full activity in a pH range of 4.0–12 after a 24 h incubation or after 0.5 h treatment at 70 °C, respectively. Interestingly, SAL4 displayed appreciable stability toward oxidizing agents, anionic and non-ionic surfactants in addition to its compatibility with several commercial detergents. Overall, these interesting characteristics make this new lipase promising for its application in detergent industry. Keywords: Staphylococcus aureus lipase, Purification, Characterization, Thermo-alkaline, Detergent-stable

  1. Malassezia globosa MgMDL2 lipase: Crystal structure and rational modification of substrate specificity.

    Science.gov (United States)

    Lan, Dongming; Xu, Huan; Xu, Jinxin; Dubin, Grzegorz; Liu, Jinsong; Iqbal Khan, Faez; Wang, Yonghua

    2017-06-24

    Lipases play an important role in physiological metabolism and diseases, and also have multiple industrial applications. Rational modification of lipase specificity may increase the commercial utility of this group of enzymes, but is hindered by insufficient mechanistic understanding. Here, we report the 2.0 Å resolution crystal structure of a mono- and di-acylglycerols lipase from Malassezia globosa (MgMDL2). Interestingly, residues Phe278 and Glu282 were found to involve in substrate recognition because mutation on each residue led to convert MgMDL2 to a triacylglycerol (TAG) lipase. The Phe278Ala and Glu282Ala mutants also acquired ability to synthesize TAGs by esterification of glycerol and fatty acids. By in silicon analysis, steric hindrance of these residues seemed to be key factors for the altered substrate specificity. Our work may shed light on understanding the unique substrate selectivity mechanism of mono- and di-acylglycerols lipases, and provide a new insight for engineering biocatalysts with desired catalytic behaviors for biotechnological application. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. ENZYMATIC PRODUCTION OF ETHYL OLEATE ESTER USING A LIPASE FROM CANDIDA ANTARCTICA B

    Directory of Open Access Journals (Sweden)

    N. Sampaio Neta

    2012-05-01

    Full Text Available Lipases are biocatalysts of great importance in different areas, being able to catalyze reactions in aqueous or organic media. Furthermore, these enzymes are capable of using several substrates being stable in a wide range of pH and temperatures. Lipases promote the esterification between fatty acids and ethanol producing oleate esters. The aim of this work is to produce ethyl oleate ester by enzymatic esterification of oleic acid with ethanol. A lipase from Candida antarctica type B was used at a temperature of 55 °C. The reaction was conducted using oleic acid, sodium sulfate anhydrous, lipase and ethanol, with a ratio of oleic acid (0.03 mol or 10 ml, lipase (0.1 mol or 0.01 g, sodium sulfate anhydrous (5 g and ethanol 99 % (100 ml. Several reaction times were studied, namely 48, 72, 96 and 120 hours. Nuclear Magnetic Resonance (1H and 13C and Infrared spectra confirmed the production of ethyl oleate ester for the studied conditions. The highest ethyl oleate production yield was obtained for 96 hours reaction time. Ethyl oleate esters have been reported to possess interesting applications in several industrial fields, such as food, aromatics, cosmetics, detergents, flavors and pharmaceuticals.

  3. Smart conjugated polymer nanocarrier for healthy weight loss by negative feedback regulation of lipase activity

    Science.gov (United States)

    Chen, Yu-Lei; Zhu, Sha; Zhang, Lei; Feng, Pei-Jian; Yao, Xi-Kuang; Qian, Cheng-Gen; Zhang, Can; Jiang, Xi-Qun; Shen, Qun-Dong

    2016-02-01

    Healthy weight loss represents a real challenge when obesity is increasing in prevalence. Herein, we report a conjugated polymer nanocarrier for smart deactivation of lipase and thus balancing calorie intake. After oral administration, the nanocarrier is sensitive to lipase in the digestive tract and releases orlistat, which deactivates the enzyme and inhibits fat digestion. It also creates negative feedback to control the release of itself. The nanocarrier smartly regulates activity of the lipase cyclically varied between high and low levels. In spite of high fat diet intervention, obese mice receiving a single dose of the nanocarrier lose weight over eight days, whereas a control group continues the tendency to gain weight. Daily intragastric administration of the nanocarrier leads to lower weight of livers or fat pads, smaller adipocyte size, and lower total cholesterol level than that of the control group. Near-infrared fluorescence of the nanocarrier reveals its biodistribution.Healthy weight loss represents a real challenge when obesity is increasing in prevalence. Herein, we report a conjugated polymer nanocarrier for smart deactivation of lipase and thus balancing calorie intake. After oral administration, the nanocarrier is sensitive to lipase in the digestive tract and releases orlistat, which deactivates the enzyme and inhibits fat digestion. It also creates negative feedback to control the release of itself. The nanocarrier smartly regulates activity of the lipase cyclically varied between high and low levels. In spite of high fat diet intervention, obese mice receiving a single dose of the nanocarrier lose weight over eight days, whereas a control group continues the tendency to gain weight. Daily intragastric administration of the nanocarrier leads to lower weight of livers or fat pads, smaller adipocyte size, and lower total cholesterol level than that of the control group. Near-infrared fluorescence of the nanocarrier reveals its biodistribution

  4. Rational enhancement of enzyme performance in organic solvents. Final technical report, 1992--1996

    Energy Technology Data Exchange (ETDEWEB)

    Klibanov, A.M.

    1996-12-31

    This research focused on the following: the dependence of enzymatic activity of several model hydrolases in nonaqueous solvents; control of substrate selectivity of the protease subtilisin Carlsberg by the solvent; control of catalytic activity and enantioselectivity of this enzyme in organic solvents by immobilization support; lipase-catalyzed acylation of sugars in anhydrous hydrophobic media; the possibility of accelerating enzymatic processes in organic solvents by certain cosolvents; whether lipase catalysis in organic solvents can be enhanced by introducing interfaces in the in the reaction medium; the structure of proteins suspended in organic solvents; improving enzymatic enantioselectivity in organic solvents; analyzing the plunge in enzymatic activity upon replacing water with organic solvents; and the structural basis for the phenomenon of molecular memory of imprinted proteins in organic solvents.

  5. VLDL hydrolysis by hepatic lipase regulates PPARδ transcriptional responses.

    Directory of Open Access Journals (Sweden)

    Jonathan D Brown

    Full Text Available PPARs (α,γ,δ are a family of ligand-activated transcription factors that regulate energy balance, including lipid metabolism. Despite these critical functions, the integration between specific pathways of lipid metabolism and distinct PPAR responses remains obscure. Previous work has revealed that lipolytic pathways can activate PPARs. Whether hepatic lipase (HL, an enzyme that regulates VLDL and HDL catabolism, participates in PPAR responses is unknown.Using PPAR ligand binding domain transactivation assays, we found that HL interacted with triglyceride-rich VLDL (>HDL≫LDL, IDL to activate PPARδ preferentially over PPARα or PPARγ, an effect dependent on HL catalytic activity. In cell free ligand displacement assays, VLDL hydrolysis by HL activated PPARδ in a VLDL-concentration dependent manner. Extended further, VLDL stimulation of HL-expressing HUVECs and FAO hepatoma cells increased mRNA expression of canonical PPARδ target genes, including adipocyte differentiation related protein (ADRP, angiopoietin like protein 4 and pyruvate dehydrogenase kinase-4. HL/VLDL regulated ADRP through a PPRE in the promoter region of this gene. In vivo, adenoviral-mediated hepatic HL expression in C57BL/6 mice increased hepatic ADRP mRNA levels by 30%. In ob/ob mice, a model with higher triglycerides than C57BL/6 mice, HL overexpression increased ADRP expression by 70%, demonstrating the importance of triglyceride substrate for HL-mediated PPARδ activation. Global metabolite profiling identified HL/VLDL released fatty acids including oleic acid and palmitoleic acid that were capable of recapitulating PPARδ activation and ADRP gene regulation in vitro.These data define a novel pathway involving HL hydrolysis of VLDL that activates PPARδ through generation of specific monounsaturated fatty acids. These data also demonstrate how integrating cell biology with metabolomic approaches provides insight into specific lipid mediators and pathways of lipid

  6. A Systems Biology Framework for Modeling Metabolic Enzyme Inhibition of Mycobacterium Tuberculosis

    Science.gov (United States)

    2009-09-15

    parameters, and group IV included those that, by definition , were directly determined once the other parameters were defined. During the sensitivity...III parameters were assumed to be related to the parameters of the first two groups; and group IV parameters are, by definition , determined once the...population growth model are described below. sAMS inhibition model sAMS inhibits the enzyme salicyl -AMP ligase (MbtA; encoded by the gene Rv2384) that

  7. Purification and characterization of a cold-adapted lipase from Oceanobacillus strain PT-11.

    Directory of Open Access Journals (Sweden)

    Tian Jiewei

    Full Text Available We isolated a moderately halophilic lipase-producing bacterium from the saline soil. Based on the morphological, physiological, chemotaxonomic and phylogenetic analysis, the isolate PT-11 was postulated to be a novel species identified as Oceanobacillus rekensis PT-11. The lipase was purified 2.50-fold by Q-Sepharose FF and SP-Sepharose FF chromatography and its molecular mass was estimated to be 23.5 kDa by SDS-PAGE. It was highly active over the broad temperature ranging from 10 to 35°C and showed up to 80% of the maximum activity at 10°C indicating the lipase to be a typical cold-adapted enzyme. The enzyme activity was slightly enhanced by Na+, Li+ and K+. Incubation with detergents, such as Tween-20 and Tween-80, slightly inhibited the enzyme activity; while Triton X-100decreased the enzyme activity. The enzyme was fairly stable in the presence of long-chain alcohols but was highly denatured in hydrophilic solvents such as acetone or short-chain alcohols (C1-C3.

  8. Immobilization of Candida antarctica Lipase B by Adsorption to Green Coconut Fiber

    Science.gov (United States)

    Brígida, Ana I. S.; Pinheiro, Álvaro D. T.; Ferreira, Andrea L. O.; Gonçalves, Luciana R. B.

    An agroindustrial residue, green coconut fiber, was evaluated as support for immobilization of Candida antarctica type B (CALB) lipase by physical adsorption. The influence of several parameters, such as contact time, amount of enzyme offered to immobilization, and pH of lipase solution was analyzed to select a suitable immobilization protocol. Kinetic constants of soluble and immobilized lipases were assayed. Thermal and operational stability of the immobilized enzyme, obtained after 2 h of contact between coconut fiber and enzyme solution, containing 40 U/ml in 25 mM sodium phosphate buffer pH 7, were determined. CALB immobilization by adsorption on coconut fiber promoted an increase in thermal stability at 50 and 60 °C, as half-lives (t 1/2) of the immobilized enzyme were, respectively, 2- and 92-fold higher than the ones for soluble enzyme. Furthermore, operational stabilities of methyl butyrate hydrolysis and butyl butyrate synthesis were evaluated. After the third cycle of methyl butyrate hydrolysis, it retained less than 50% of the initial activity, while Novozyme 435 retained more than 70% after the tenth cycle. However, in the synthesis of butyl butyrate, CALB immobilized on coconut fiber showed a good operational stability when compared to Novozyme 435, retaining 80% of its initial activity after the sixth cycle of reaction.

  9. Mixed substrate solid state fermentation for production and extraction of lipase from Aspergillus niger MTCC 2594.

    Science.gov (United States)

    Mala, John Geraldine Sandana; Edwinoliver, Nesamony Gnanadhas; Kamini, Numbi Ramudu; Puvanakrishnan, Rengarajulu

    2007-08-01

    A novel mixed substrate solid-state fermentation (SSF) process has been developed for Aspergillus niger MTCC 2594 using wheat bran (WB) and gingelly oil cake (GOC) and the results showed that addition of GOC to WB (WB : GOC, 3 : 1, w/w) increased the lipase activity by 36.0% and the activity was 384.3+/-4.5 U/g dry substrate at 30 degrees C and 72 h. Scale up of lipase production to 100 g and 1 kg tray-level batch fermentation resulted in 95.0% and 84.0% of enzyme activities respectively at 72 h. A three-stage multiple contact counter-current extraction yielded 97% enzyme recovery with a contact time of 60 min. However, extraction by simple percolation and plug-flow methods resulted in decreased enzyme recoveries. The mixed substrate SSF process has resulted in a significant increase in specific activity (58.9%) when compared to a submerged fermentation (SmF) system. Furthermore, an efficient process of extraction has been standardized with this process. Use of GOC along with WB as potential raw materials for enzyme production could be of great commercial significance. This is the first report on the production and extraction of lipase from Aspergillus niger using mixed solid substrates, WB and GOC, which are potential raw materials for the production of enzymes and other value-added products.

  10. Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3.

    Science.gov (United States)

    Kumar, Satyendra; Kikon, Khyodano; Upadhyay, Ashutosh; Kanwar, Shamsher S; Gupta, Reena

    2005-05-01

    A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.

  11. Enzyme Informatics

    Science.gov (United States)

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  12. Study of Antiobesity Effect through Inhibition of Pancreatic Lipase Activity of Diospyros kaki Fruit and Citrus unshiu Peel

    Directory of Open Access Journals (Sweden)

    Gyo-Nam Kim

    2016-01-01

    Full Text Available Pancreatic lipase is the enzyme responsible for digestion and absorption of triglycerides, being its inhibition one of the widest studied methods used to determine the potential activity of natural products to inhibit dietary fat absorption. Decrease of energy intake from dietary fat through inhibition of this enzyme may be an excellent strategy to prevent and treat obesity. The inhibitory activity on pancreatic lipase enzyme of Diospyros kaki fruit and Citrus unshiu peel mixture extract (PCM was evaluated in vitro and its antiobesity effects were studied based on the serum lipid parameters analysis from high-fat diet- (HFD- fed mice in vivo. PCM was orally administered at a dose of 50 and 200 mg/kg body weight for 6 weeks. In addition, the activity of pancreatic lipase was assessed using orlistat (positive control. PCM exhibited inhibitory effect on lipase activity with IC50 value of 507.01 μg/mL. Moreover, serum triacylglycerol, total cholesterol levels, and visceral fat weight were significantly reduced compared to HFD control mice in PCM 200 mg/kg-treated mice (p<0.05. These results suggest that PCM administration may be a novel potential antiobesity agent for reduction of fat absorption via inhibition of pancreatic lipase.

  13. Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation.

    Science.gov (United States)

    Gutarra, Melissa L E; Godoy, Mateus G; Maugeri, Francisco; Rodrigues, Maria Isabel; Freire, Denise M G; Castilho, Leda R

    2009-11-01

    The production of a lipase by a wild-type Brazilian strain of Penicillium simplicissimum in solid-state fermentation of babassu cake, an abundant residue of the oil industry, was studied. The enzyme production reached about 90 U/g in 72 h, with a specific activity of 4.5 U/mg of total proteins. The crude lipase showed high activities at 35-60 degrees C and pH 4.0-6.0, with a maximum activity at 50 degrees C and pH 4.0-5.0. Enzyme stability was enhanced at pH 5.0 and 6.0, with a maximum half-life of 5.02 h at 50 degrees C and pH 5.0. Thus, this lipase shows a thermophilic and thermostable behavior, what is not common among lipases from mesophilic filamentous fungi. The crude enzyme catalysed the hydrolysis of triglycerides and p-nitrophenyl esters (C4:0-C18:0), preferably acting on substrates with medium-chain fatty acids. This non-purified lipase in addition to interesting properties showed a reduced production cost making feasible its applicability in many fields.

  14. Purification and Immobilization of a Novel Enantioselective Lipase from Tsukamurella tyrosinosolvents for Efficient Resolution of Ethyl 2-(2-oxopyrrolidin-1-yl) Butyrate.

    Science.gov (United States)

    Huang, Jin; Yan, Ren; He, Jun-Yao; Wang, Pu

    2016-10-01

    A highly enantioselective lipase from Tsukamurella tyrosinosolvents E105 was purified via ultrasonic extraction, precipitation, and chromatographic steps. The enzyme was purified about 38-fold with the recovery yield of 9 % and was confirmed as a dimer protein consisting of two identical subunits with a molecular mass of 24 kDa. The purified lipase was used to catalyze resolution of racemic ethyl 2-(2-oxopyrrolidin-1-yl) butyrate to (S)-2-(2-oxopyrrolidin-1-yl) butyric acid. The maximum activity of such lipase was obtained at pH 7.5, 35 °C, and the highest relative activity (156.80 %) was observed in the presence of 0.5 mM Co 2+ . Subsequently, the lipase was encapsulated within a mixture of 3 % sodium alginate and 0.8 % carrageenan, and then cross-linked with 0.6 % glutaraldehyde to enhance its biocatalytic capability and stability. Comparing with 36.9 % product yield and 97.5 % product ee of free lipase, the highest product yield of 46.3 % and ee of 98.5 % for immobilized lipase were achieved with the presence of 20 mM substrate. In addition, the reusability of immobilized lipase was also investigated, which could maintain 63.7 % of its initial conversion yield after seven repeated batch reactions. Thus, the evaluated enantioselective lipase in this work has a good potential for further industrial application.

  15. Concentration, characterization and application of lipases from Sporidiobolus pararoseus strain

    Science.gov (United States)

    Smaniotto, Alessandra; Skovronski, Aline; Rigo, Elisandra; Tsai, Siu Mui; Durrer, Ademir; Foltran, Lillian Liva; Paroul, Natália; Di Luccio, Marco; Oliveira, J. Vladimir; de Oliveira, Débora; Treichel, Helen

    2014-01-01

    Lipases produced by a newly isolated Sporidiobolus pararoseus strain have potential catalytic ability for esterification reactions. After production, the enzymatic extracts (conventional crude and precipitated, ‘CC’ and ‘CP’, and industrial crude and precipitated, ‘IC’ e ‘IP’) were partially characterized. The enzymes presented, in general, higher specificity for short chain alcohols and fatty acids. The precipitated extract showed a good thermal stability, higher than that for crude enzymatic extracts. The ‘CC’ and ‘CP’ enzymes presented high activities after exposure to pH 6.5 and 40 °C. On the other hand, the ‘IC’ and ‘IP’ extracts kept their activities in a wide range of pH memory but presented preference for higher reaction temperatures. Preliminary studies of application of the crude lipase extract in the enzymatic production of geranyl propionate using geraniol and propionic acid as substrates in solvent-free system led to a reaction conversion of 42 ± 1.5%. PMID:24948948

  16. Neutrophil chemotaxis by Propionibacterium acnes lipase and its inhibition.

    OpenAIRE

    Lee, W L; Shalita, A R; Suntharalingam, K; Fikrig, S M

    1982-01-01

    The chemoattraction of Propionibacterium acnes lipase for neutrophils and the effect of lipase inhibitor and two antibiotic agents on the chemotaxis were evaluated. Of the various fractions tested, partially purified lipase (fraction 2c) was the most active cytotaxin produced by P. acnes. Serum mediators were not required for the generation of chemotaxis by lipase in vitro. Diisopropyl phosphofluoridate at low concentration (10(-4) mM) completely inhibited lipase activity as well as polymorph...

  17. Tuberculosis alters pancreatic enzymes in the absence of pancreatitis

    Directory of Open Access Journals (Sweden)

    Modisa S. Motswaledi

    2014-10-01

    Objective: To compare serum lipase and amylase activity levels in patients with tuberculosis (TB against those of healthy controls. Methods: Serum lipase and amylase activity levels were measured in 99 patients and 143 healthy controls using the Vitros 250 Chemistry analyser. Reference ranges for serum lipase and amylase were 23–300 U/L and 30–110 U/L, respectively. Results: Lipase was higher in patients with MTB than in controls (81.5 IU/L versus 66.5 IU/L, p = 0.006. Similarly, amylase was higher in the MTB patient group (76 IU/L versus 60 IU/L, p < 0.001. The Pearson correlation coefficient for lipase versus amylase (R was higher in the controls (R = 0.351, p < 0.0001 compared with MTB patients (R = 0.217, p = 0.035. Amongst MTB patients, lipase activity correlated positively with erythrocyte sedimentation rate (ESR (R = 0.263, p = 0.013, but not with haemoglobin concentration or treatment duration. A weak inverse correlation was noted between ESR and treatment duration (R = -0.222, p = 0.028. Conclusion: Pancreatic enzyme levels differ between MTB patients and normal controls; however, this difference still lies within the normal range. The concomitant increase of lipase with ESR, an inflammatory marker, could conceivably suggest a causal relationship. Further research is necessary to characterise MTB-derived enzymes for diagnostic and therapeutic utility.

  18. Lysosomal acid lipase deficiency in rats: Lipid analyses and lipase activities in liver and spleen

    International Nuclear Information System (INIS)

    Kuriyama, M.; Yoshida, H.; Suzuki, M.; Fujiyama, J.; Igata, A.

    1990-01-01

    We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for [14C]triolein, [14C]cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans

  19. Kinetics of enzymatic transesterification and thermal deactivation using immobilized Burkholderia lipase as catalyst.

    Science.gov (United States)

    Tran, Dang-Thuan; Chang, Jo-Shu

    2014-03-01

    The most effective way of enzymatic synthesis of biodiesel is through lipase-catalyzed transesterification, while its performance and economic feasibility should still be improved. In this study, lipase produced by an isolated Burkholderia sp. was immobilized on microsize Celite materials functionally modified with long alkyl groups. The specific activity of the immobilized lipase was 1,154 U/g. The methanolysis of olive oil catalyzed by the immobilized lipase obeyed Ping Pong Bi Bi model with an estimated V max, K m,TG, K m,M and K i,M value of 0.61 mol/(L min), 7.93 mol/L, 1.01 mol/L, and 0.24 mol/L, respectively. The activation energy of the enzymatic reaction is estimated as 15.51 kJ/mol. The immobilized lipase exhibits high thermal stability with thermal deactivation energy of 83 kJ/mol and a long half-life. The enthalpy, Gibb's free energy, and entropy of the immobilized lipase were in the range of 80.02-80.35 kJ/mol, 88.35-90.13 kJ/mol, and -28.22 to -25.11 J/(mol K), respectively.

  20. Computational study of a model system of enzyme-mediated [4+2] cycloaddition reaction.

    Science.gov (United States)

    Gordeev, Evgeniy G; Ananikov, Valentine P

    2015-01-01

    A possible mechanistic pathway related to an enzyme-catalyzed [4+2] cycloaddition reaction was studied by theoretical calculations at density functional (B3LYP, O3LYP, M062X) and semiempirical levels (PM6-DH2, PM6) performed on a model system. The calculations were carried out for the key [4+2] cycloaddition step considering enzyme-catalyzed biosynthesis of Spinosyn A in a model reaction, where a reliable example of a biological Diels-Alder reaction was reported experimentally. In the present study it was demonstrated that the [4+2] cycloaddition reaction may benefit from moving along the energetically balanced reaction coordinate, which enabled the catalytic rate enhancement of the [4+2] cycloaddition pathway involving a single transition state. Modeling of such a system with coordination of three amino acids indicated a reliable decrease of activation energy by ~18.0 kcal/mol as compared to a non-catalytic transformation.

  1. Computational study of a model system of enzyme-mediated [4+2] cycloaddition reaction.

    Directory of Open Access Journals (Sweden)

    Evgeniy G Gordeev

    Full Text Available A possible mechanistic pathway related to an enzyme-catalyzed [4+2] cycloaddition reaction was studied by theoretical calculations at density functional (B3LYP, O3LYP, M062X and semiempirical levels (PM6-DH2, PM6 performed on a model system. The calculations were carried out for the key [4+2] cycloaddition step considering enzyme-catalyzed biosynthesis of Spinosyn A in a model reaction, where a reliable example of a biological Diels-Alder reaction was reported experimentally. In the present study it was demonstrated that the [4+2] cycloaddition reaction may benefit from moving along the energetically balanced reaction coordinate, which enabled the catalytic rate enhancement of the [4+2] cycloaddition pathway involving a single transition state. Modeling of such a system with coordination of three amino acids indicated a reliable decrease of activation energy by ~18.0 kcal/mol as compared to a non-catalytic transformation.

  2. Improved Performance of Magnetic Cross-Linked Lipase Aggregates by Interfacial Activation: A Robust and Magnetically Recyclable Biocatalyst for Transesterification of Jatropha Oil

    Directory of Open Access Journals (Sweden)

    Weiwei Zhang

    2017-12-01

    Full Text Available Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called “interfacial activation”. As interfacial activation can lead to a significant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML. Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after five instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability.

  3. Improved Performance of Magnetic Cross-Linked Lipase Aggregates by Interfacial Activation: A Robust and Magnetically Recyclable Biocatalyst for Transesterification of Jatropha Oil.

    Science.gov (United States)

    Zhang, Weiwei; Yang, Huixia; Liu, Wanyi; Wang, Na; Yu, Xiaoqi

    2017-12-07

    Lipases are the most widely employed enzymes in commercial industries. The catalytic mechanism of most lipases involves a step called "interfacial activation". As interfacial activation can lead to a significant increase in catalytic activity, it is of profound importance in developing lipase immobilization methods. To obtain a potential biocatalyst for industrial biodiesel production, an effective strategy for enhancement of catalytic activity and stability of immobilized lipase was developed. This was performed through the combination of interfacial activation with hybrid magnetic cross-linked lipase aggregates. This biocatalyst was investigated for the immobilization of lipase from Rhizomucor miehei (RML). Under the optimal conditions, the activity recovery of the surfactant-activated magnetic RML cross-linked enzyme aggregates (CLEAs) was as high as 2058%, with a 20-fold improvement over the free RML. Moreover, the immobilized RML showed excellent catalytic performance for the biodiesel reaction at a yield of 93%, and more importantly, could be easily separated from the reaction mixture by simple magnetic decantation, and retained more than 84% of its initial activities after five instances of reuse. This study provides a new and versatile approach for designing and fabricating immobilized lipase with high activation and stability.

  4. A systems biology framework for modeling metabolic enzyme inhibition of Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Reifman Jaques

    2009-09-01

    Full Text Available Abstract Background Because metabolism is fundamental in sustaining microbial life, drugs that target pathogen-specific metabolic enzymes and pathways can be very effective. In particular, the metabolic challenges faced by intracellular pathogens, such as Mycobacterium tuberculosis, residing in the infected host provide novel opportunities for therapeutic intervention. Results We developed a mathematical framework to simulate the effects on the growth of a pathogen when enzymes in its metabolic pathways are inhibited. Combining detailed models of enzyme kinetics, a complete metabolic network description as modeled by flux balance analysis, and a dynamic cell population growth model, we quantitatively modeled and predicted the dose-response of the 3-nitropropionate inhibitor on the growth of M. tuberculosis in a medium whose carbon source was restricted to fatty acids, and that of the 5'-O-(N-salicylsulfamoyl adenosine inhibitor in a medium with low-iron concentration. Conclusion The predicted results quantitatively reproduced the experimentally measured dose-response curves, ranging over three orders of magnitude in inhibitor concentration. Thus, by allowing for detailed specifications of the underlying enzymatic kinetics, metabolic reactions/constraints, and growth media, our model captured the essential chemical and biological factors that determine the effects of drug inhibition on in vitro growth of M. tuberculosis cells.

  5. Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: preparation, optimization, characterization, and application for enantioselective resolution reactions.

    Science.gov (United States)

    Kartal, Funda; Kilinc, Ali

    2012-07-01

    Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80-pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)-1-phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R-S)-1-phenylethanol with vinylacetate. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

  6. Toward the Establishment of Standardized In Vitro Tests for Lipid-Based Formulations. 5. Lipolysis of Representative Formulations by Gastric Lipase

    DEFF Research Database (Denmark)

    Bakala-N'Goma, Jean-Claude; Williams, Hywel D.; Sassene, Philip J.

    2015-01-01

    Purpose Lipid-based formulations (LBF) are substrates for digestive lipases and digestion can significantly alter their properties and potential to support drug absorption. LBFs have been widely examined for their behaviour in the presence of pancreatic enzymes. Here, the impact of gastric lipase...... on the digestion of representative formulations from the Lipid Formulation Classification System has been investigated. Methods The pHstat technique was used to measure the lipolysis by recombinant dog gastric lipase (rDGL) of eight LBFs containing either medium (MC) or long (LC) chain triglycerides and a range...

  7. Student Collaboration in a Series of Integrated Experiments to Study Enzyme Reactor Modeling with Immobilized Cell-Based Invertase

    Science.gov (United States)

    Taipa, M. A^ngela; Azevedo, Ana M.; Grilo, Anto´nio L.; Couto, Pedro T.; Ferreira, Filipe A. G.; Fortuna, Ana R. M.; Pinto, Ine^s F.; Santos, Rafael M.; Santos, Susana B.

    2015-01-01

    An integrative laboratory study addressing fundamentals of enzyme catalysis and their application to reactors operation and modeling is presented. Invertase, a ß-fructofuranosidase that catalyses the hydrolysis of sucrose, is used as the model enzyme at optimal conditions (pH 4.5 and 45 °C). The experimental work involves 3 h of laboratory time…

  8. Lipase immobilization for catalytic applications obtained using fumed silica deposited with MAPLE technique

    Science.gov (United States)

    Bloisi, Francesco; Califano, Valeria; Perretta, Giuseppe; Nasti, Libera; Aronne, Antonio; Di Girolamo, Rocco; Auriemma, Finizia; De Rosa, Claudio; Vicari, Luciano R. M.

    2016-06-01

    Lipases are enzymes used for catalyzing reactions of acylglycerides in biodiesel production from lipids, where enzyme immobilization on a substrate is required. Silica nanoparticles in different morphologies and configurations are currently used in conjunction with biological molecules for drug delivery and catalysis applications, but up to date their use for triglycerides has been limited by the large size of long-chain lipid molecules. Matrix assisted pulsed laser evaporation (MAPLE), a laser deposition technique using a frozen solution/suspension as a target, is widely used for deposition of biomaterials and other delicate molecules. We have carried out a MAPLE deposition starting from a frozen mixture containing fumed silica and lipase in water. Deposition parameters were chosen in order to increase surface roughness and to promote the formation of complex structures. Both the target (a frozen thickened mixture of nanoparticles/catalyst in water) and the deposition configuration (a small target to substrate distance) are unusual and have been adopted in order to increase surface contact of catalyst and to facilitate access to long-chain molecules. The resulting innovative film morphology (fumed silica/lipase cluster level aggregation) and the lipase functionality (for catalytic biodiesel production) have been studied by FESEM, FTIR and transesterification tests.

  9. Hypocholesterolemic Effect and In Vitro Pancreatic Lipase Inhibitory Activity of an Opuntia ficus-indica Extract

    Directory of Open Access Journals (Sweden)

    Eduardo Padilla-Camberos

    2015-01-01

    Full Text Available Cholesterol control is fundamental for prevention of cardiovascular disorders. In this work, the hypocholesterolemic activity of an aqueous Opuntia ficus-indica extract (AOE was tested in triton-induced mice. The inhibitory activity on pancreatic lipase enzyme was evaluated in vitro by the same extract. Furthermore, polyphenol content of the extract was evaluated. Hypercholesterolemia was induced in three groups of mice by intraperitoneal administration of Triton WR-1339. After induction of hypercholesterolemia, the groups were treated with an AOE (500 mg/kg and saline solution and the positive control group with orlistat, respectively. Cholesterol levels were measured 24 h later in peripheral blood. The levels of blood cholesterol after administration of AOE significantly decreased compared to negative control. The inhibitory activity of AOE on pancreatic lipase enzyme was evaluated at concentrations from 60 to 1000 μg/mL. The AOE inhibited the pancreatic lipase with an IC50 = 588.5 μg/mL. The AOE had a high content of polyphenolic compounds. These results show that AOE is able to prevent hypercholesterolemia by pancreatic lipase inhibition, in part due to its polyphenolic compounds.

  10. Resolution of Racemic Acids, Esters and Amines by Candida rugosa Lipase in Slightly Hydrated Organic Media

    Directory of Open Access Journals (Sweden)

    Andrés R. Alcántara

    2004-01-01

    Full Text Available Commercial crude lipase from Candida rugosa is widely used as a biocatalyst in the resolution of racemic mixtures and in organic synthesis in slightly hydrated organic solvents. In many cases, reproducible results are not obtained when the same crude lipase is used, but from different suppliers of lots, this being due to the presence of different isoenzymes. The current work addresses this problem and strategies to overcome it. The yeast Candida rugosa ATCC 14380 was cultivated in a minimal culture medium, using different substances as inducers and carbon sources. The percentage of inducer that gave the optimum productivity of extracellular lipases was determined. Lyophilized extracellular enzymes were characterized by SDS-PAGE electrophoresis and isoelectric focusing (IEF. Depending on the nature of the carbon source, different isoenzymes were produced in various proportions. These samples were partially purified by different methodologies, including dialysis, adsorption chromatography and precipitation with ammonium sulfate or organic solvents. These characterizations allowed us to explain the relative catalytic activity of different samples, showing that in biocatalysis enzymes should not be treated simply as a »white magic powder« that can solve all the challenges in organic synthesis. Heptyl oleate synthesis, alcoxycarbonylation of amines and hydrolysis of the ester of ketoprofen are excellent reaction tests for the evaluation of lipase samples as biocatalysts.

  11. Optimization of Lipase Synthesis by Mucor racemosus - Production in a Triple Impeller Bioreactor

    Directory of Open Access Journals (Sweden)

    Nadia, N.

    2010-01-01

    Full Text Available Fungal cultures were screened for their ability to produce extracellular lipase through different incubation periods, in spite of some Aspergillus niger gave highest lipolytic activities, Mucor racemosus was selected in this study due to its non-pathogenicity. Optimum lipase activity was obtained after 3 days incubation period at 35 °C using submerged culture technique. Using a modified complex medium (3% peptone; 0.2% KH2PO4; 0.05% KCl; 0.05% MgSO4.7H2O; 1% glucose; 1% olive oil and 0.01 M barium acetate as additives; pH was adjusted to 7.0 using 0.2 M phosphate buffer stimulated M. racemosus lipase by 114.3%. The production of lipolytic enzyme by M. racemosus was up-scaled to 5 L stirred-tank bioreactor. Under optimized conditions, lipase and specific enzyme activities were improved by 2 folds (1211 U/mL and 28 U/mg protein as compared to shake flask studies.

  12. m-DOPA addition in MAPLE immobilization of lipase for biosensor applications

    Directory of Open Access Journals (Sweden)

    Valeria Califano

    2015-12-01

    Full Text Available Matrix Assisted Pulsed Laser Evaporation (MAPLE is a thin film deposition technique which uses a pulsed laser beam impinging, inside a high vacuum chamber, on a frozen target containing the guest molecules in a volatile matrix to induce fast “evaporation” of the matrix, and ejection of the guest molecules. Lipase, an enzyme acting as a catalyst in hydrolysis of lipids, is widely used in biosensors for detection of triglycerides in blood serum. A key action to this purpose is lipase immobilization on a substrate. In a recent paper, we have shown that MAPLE technique is able to deposit lipase on a substrate in an active form. Here we show that addition to the guest/matrix target of a small amount of m-DOPA (3-(3,4-dihydroxyphenyl-2-methyl-l-alanine in order to improve adhesion and protect lipase secondary structure, also allows the lowering the laser pulse energy required for matrix evaporation and therefore the risk of damaging the enzyme.

  13. Hypocholesterolemic Effect and In Vitro Pancreatic Lipase Inhibitory Activity of an Opuntia ficus-indica Extract

    Science.gov (United States)

    Flores-Fernandez, Jose Miguel; Fernandez-Flores, Ofelia; Gutierrez-Mercado, Yanet; Carmona-de la Luz, Joel; Sandoval-Salas, Fabiola; Mendez-Carreto, Carlos

    2015-01-01

    Cholesterol control is fundamental for prevention of cardiovascular disorders. In this work, the hypocholesterolemic activity of an aqueous Opuntia ficus-indica extract (AOE) was tested in triton-induced mice. The inhibitory activity on pancreatic lipase enzyme was evaluated in vitro by the same extract. Furthermore, polyphenol content of the extract was evaluated. Hypercholesterolemia was induced in three groups of mice by intraperitoneal administration of Triton WR-1339. After induction of hypercholesterolemia, the groups were treated with an AOE (500 mg/kg) and saline solution and the positive control group with orlistat, respectively. Cholesterol levels were measured 24 h later in peripheral blood. The levels of blood cholesterol after administration of AOE significantly decreased compared to negative control. The inhibitory activity of AOE on pancreatic lipase enzyme was evaluated at concentrations from 60 to 1000 μg/mL. The AOE inhibited the pancreatic lipase with an IC50 = 588.5 μg/mL. The AOE had a high content of polyphenolic compounds. These results show that AOE is able to prevent hypercholesterolemia by pancreatic lipase inhibition, in part due to its polyphenolic compounds. PMID:26078966

  14. Production of extracellular lipase by the phytopathogenic fungus Fusarium solani FS1 Produção de lipase extracelular pelo fungo fitopatogênico Fusarium solani FS1

    Directory of Open Access Journals (Sweden)

    Maria de Mascena Diniz Maia

    1999-12-01

    Full Text Available A Brazilian strain of Fusarium solani was tested for extracellular lipase production in peptone-olive oil medium. The fungus produced 10,500 U.l-1 of lipase after 72 hours of cultivation at 25oC in shake-flask at 120 rpm in a medium containing 3% (w/v peptone plus 0.5% (v/v olive oil. Glucose (1% w/v was found to inhibit the inductive effect of olive oil. Peptone concentrations below 3% (w/v resulted in a reduced lipase production while increased olive oil concentration (above 0.5% did not further stimulate lipase production. The optimum lipase activity was achieved at pH 8.6 and 30oC and a good enzyme stability (80% activity retention was observed at pH ranging from 7.6 to 8.6, and the activity rapidly dropped at temperatures above 50oC. Lipase activity was stimulated by the addition of n-hexane to the culture medium supernatants, in contrast to incubation with water-soluble solvents.

  15. Application of a sensitive collection heuristic for very large protein families: Evolutionary relationship between adipose triglyceride lipase (ATGL and classic mammalian lipases

    Directory of Open Access Journals (Sweden)

    Berezovsky Igor

    2006-03-01

    Full Text Available Abstract Background Manually finding subtle yet statistically significant links to distantly related homologues becomes practically impossible for very populated protein families due to the sheer number of similarity searches to be invoked and analyzed. The unclear evolutionary relationship between classical mammalian lipases and the recently discovered human adipose triglyceride lipase (ATGL; a patatin family member is an exemplary case for such a problem. Results We describe an unsupervised, sensitive sequence segment collection heuristic suitable for assembling very large protein families. It is based on fan-like expanding, iterative database searches. To prevent inclusion of unrelated hits, additional criteria are introduced: minimal alignment length and overlap with starting sequence segments, finding starting sequences in reciprocal searches, automated filtering for compositional bias and repetitive patterns. This heuristic was implemented as FAMILYSEARCHER in the ANNIE sequence analysis environment and applied to search for protein links between the classical lipase family and the patatin-like group. Conclusion The FAMILYSEARCHER is an efficient tool for tracing distant evolutionary relationships involving large protein families. Although classical lipases and ATGL have no obvious sequence similarity and differ with regard to fold and catalytic mechanism, homology links detected with FAMILYSEARCHER show that they are evolutionarily related. The conserved sequence parts can be narrowed down to an ancestral core module consisting of three β-strands, one α-helix and a turn containing the typical nucleophilic serine. Moreover, this ancestral module also appears in numerous enzymes with various substrate specificities, but that critically rely on nucleophilic attack mechanisms.

  16. Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol using extracellular lipases from Bacillus subtilis.

    Science.gov (United States)

    Detry, Jean; Rosenbaum, Thorsten; Lütz, Stephan; Hahn, Doris; Jaeger, Karl-Erich; Müller, Michael; Eggert, Thorsten

    2006-10-01

    Two extracellular lipases from Bacillus subtilis, B. subtilis lipase A and lipase B, have been expressed in the heterologous host Escherichia coli, biochemically characterized and used for the kinetic resolution of (rac)-trans-1,2-diacetoxycyclohexane. Both enzymes were selectively acting on the (R,R)-enantiomer of the racemic substrate, highly specifically hydrolyzing only one of the two ester groups present, thus allowing the preparation of enantiopure (R,R)- and (S,S)-cyclohexane-trans-1,2-diol. The reaction conditions for the use of purified enzyme and crude cell lyophilizate were optimized and reactions in batch and repetitive batch modes were carried out on a preparative scale to yield enantiopure product (>99% enantiomeric excess).

  17. Optimization of lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids by response surface methodology.

    Science.gov (United States)

    Ha, Sung Ho; Van Anh, Tran; Koo, Yoon-Mo

    2013-06-01

    Lipase-catalyzed caffeic acid phenethyl ester (CAPE) synthesis in ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]), was investigated in this study. The effects of several reaction conditions, including reaction time, reaction temperature, substrate molar ratio of phenethyl alcohol to caffeic acid (CA), and weight ratio of enzyme to CA, on CAPE yield were examined. In a single parameter study, the highest CAPE yield in [Emim][Tf(2)N] was obtained at 70 °C with a substrate molar ratio of 30:1 and weight ratio of enzyme to CA of 15:1. Based on these results, response surface methodology (RSM) with a 3-level-4-factor central composite rotatable design (CCRD) was adopted to evaluate enzymatic synthesis of CAPE in [Emim][Tf(2)N]. The four major factors were reaction time (36-60 h), reaction temperature (65-75 °C), substrate molar ratio of phenethyl alcohol to CA (20:1-40:1), and weight ratio of enzyme to CA (10:1-20:1). A quadratic equation model was used to analyze the experimental data at a 95 % confidence level (p ratio of phenethyl alcohol to CA (27.1:1), and weight ratio of enzyme to CA (17.8:1)] established by our statistical method, whereas the experimental conversion yield was 96.6 ± 2 %.

  18. Six-membered cyclic carbonates from trimethylolpropane: Lipase-mediated synthesis in a flow reactor and in silico evaluation of the reaction.

    Science.gov (United States)

    Bornadel, Amin; Ismail, Mohamed; Sayed, Mahmoud; Hatti-Kaul, Rajni; Pyo, Sang-Hyun

    2017-03-01

    Six-membered cyclic carbonates with hydroxyl and methoxycarbonyloxy functional groups were prepared by transesterification of trimethylolpropane (TMP) with dimethylcarbonate (DMC) by solvent-free lipase-mediated flow reaction followed by thermal cyclization. The flow reaction efficiency was evaluated using different configurations of reactor consisting of packed beds of Novozym®435 (immobilized Candida antarctica lipase B-CalB-a.k.a. N435) and molecular sieves, flowrate, and biocatalyst loads. The mixed column of the biocatalyst and molecular sieves, allowing rapid and efficient removal of the by-product-methanol-was the most efficient setup. Higher conversion (81.6%) in the flow reaction compared to batch process (72%) was obtained using same amount of N435 (20% (w/w) N435:TMP) at 12 h, and the undesirable dimer and oligomer formation were suppressed. Moreover, the product was recovered easily without extra separation steps, and the biocatalyst and the molecular sieves remained intact for subsequent regeneration and recycling. The reaction of CalB with DMC and the primary transesterification product, monocarbonated TMP, respectively, as acyl donors was evaluated by in silico modeling and empirically to determine the role of the enzyme in the formation of cyclic carbonates and other side products. DMC was shown to be the preferred acyl donor, suggesting that TMP and its carbonated derivatives serve only as acyl acceptors in the lipase-catalyzed reaction. Subsequent cyclization to cyclic carbonate is catalyzed at increased temperature and not by the enzyme. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:375-382, 2017. © 2016 American Institute of Chemical Engineers.

  19. Optimization of the production conditions of the lipase produced by Bacillus cereus from rice flour through Plackett-Burman Design (PBD) and response surface methodology (RSM).

    Science.gov (United States)

    Vasiee, Alireza; Behbahani, Behrooz Alizadeh; Yazdi, Farideh Tabatabaei; Moradi, Samira

    2016-12-01

    In this study, the screening of lipase positive bacteria from rice flour was carried out by Rhodamin B agar plate method. Bacillus cereus was identified by 16S rDNA method. Screening of the appropriate variables and optimization of the lipase production was performed using Plackett-Burman design (PBD) and response surface methodology (RSM). Among the isolated bacteria, an aerobic Bacillus cereus strain was recognized as the best lipase-producing bacteria (177.3 ± 20 U/ml). Given the results, the optimal enzyme production conditions were achieved with coriander seed extract (CSE)/yeast extract ratio of 16.9 w/w, olive oil (OO) and MgCl 2 concentration of 2.37 g/L and 24.23 mM, respectively. In these conditions, the lipase activity (LA) was predicted 343 U/mL that was approximately close to the predicted value (324 U/mL), which was increased 1.83 fold LA compared with the non-optimized lipase. The kinetic parameters of V max and K m for the lipase were measured 0.367 μM/min.mL and 5.3 mM, respectively. The lipase producing Bacillus cereus was isolated and RSM was used for the optimization of enzyme production. The CSE/yeast extract ratio of 16.9 w/w, OO concentration of 2.37 g/L and MgCl 2 concentration of 24.23 mM, were found to be the optimal conditions of the enzyme production process. LA at optimal enzyme production conditions was observed 1.83 times more than the non-optimal conditions. Ultimately, it can be concluded that the isolated B. cereus from rice flour is a proper source of lipase. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. High-level expression and biochemical characterization of a novel cold-active lipase from Rhizomucor endophyticus.

    Science.gov (United States)

    Duan, Xiaojie; Zheng, Mingming; Liu, Yu; Jiang, Zhengqiang; Yang, Shaoqing

    2016-12-01

    To identify novel cold-active lipases from fungal sources and improve their production by heterologous expression in Pichia pastoris. A novel cold-active lipase gene (ReLipB) from Rhizomucor endophyticus was cloned. ReLipB was expressed at a high level in Pichia pastoris using high cell-density fermentation in a 5-l fermentor with the highest lipase activity of 1395 U/ml. The recombinant lipase (RelipB) was purified and biochemically characterized. ReLipB was most active at pH 7.5 and 25 °C. It was stable from pH 4.5-9.0. It exhibited broad substrate specificity towards p-nitrophenyl (pNP) esters (C 2 -C 16 ) and triacylglycerols (C 2 -C 12 ), showing the highest specific activities towards pNP laurate (231 U/mg) and tricaprylin (1840 U/mg), respectively. In addition, the enzyme displayed excellent stability with high concentrations of organic solvents including cyclohexane, n-hexane, n-heptane, isooctane and petroleum ester and surfactants. A novel cold-active lipase from Rhizomucor endophyticus was identified, expressed at a high level and biochemically characterized. The high yield and unique enzymatic properties make this lipase of some potential for industrial applications.

  1. High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization

    Science.gov (United States)

    Wang, Jian-Rong; Li, Yang-Yuan; Xu, Shu-De; Li, Peng; Liu, Jing-Shan; Liu, Dan-Ni

    2014-01-01

    A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL) was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL) was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca2+ and inhibited by Hg2+ and Ag+. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0) and triglyceride-Trilaurin (C12:0). PMID:24368519

  2. High-Level Expression of Pro-Form Lipase from Rhizopus oryzae in Pichia pastoris and Its Purification and Characterization

    Directory of Open Access Journals (Sweden)

    Jian-Rong Wang

    2013-12-01

    Full Text Available A gene encoding Rhizopus oryzae lipase containing prosequence (ProROL was cloned into the pPICZαA and electrotransformed into the Pichia pastoris X-33 strain. The lipase was functionally expressed and secreted in Pichia pastoris with a molecular weight of 35 kDa. The maximum lipase activity of recombinant lipase (rProROL was 21,000 U/mL, which was obtained in a fed-batch cultivation after 168 h induction with methanol in a 50-L bioreactor. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut off membrane and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. The optimum pH and temperature of the rProROL were pH 9.0 and 40 °C, respectively. The lipase was stable from pH 4.0 to 9.0 and from 25 to 55 °C. The enzyme activity was enhanced by Ca2+ and inhibited by Hg2+ and Ag+. The lipase showed high activity toward triglyceride-Tripalmitin (C16:0 and triglyceride-Trilaurin (C12:0.

  3. Production and Characterization of Lipases by Two New Isolates of Aspergillus through Solid-State and Submerged Fermentation

    Science.gov (United States)

    Colla, Luciane Maria; Ficanha, Aline M. M.; Rizzardi, Juliana; Bertolin, Telma Elita; Reinehr, Christian Oliveira; Costa, Jorge Alberto Vieira

    2015-01-01

    Due to the numerous applications of lipases in industry, there is a need to study their characteristics, because lipases obtained from different sources may present different properties. The aim of this work was to accomplish the partial characterization of lipases obtained through submerged fermentation and solid-state fermentation by two species of Aspergillus. Fungal strains were isolated from a diesel-contaminated soil and selected as good lipases producers. Lipases obtained through submerged fermentation presented optimal activities at 37°C and pH 7.2 and those obtained through solid-state fermentation at 35°C and pH 6.0. The enzymes produced by submerged fermentation were more temperature-stable than those obtained by solid-state fermentation, presenting 72% of residual activity after one hour of exposition at 90°C. Lipases obtained through submerged fermentation had 80% of stability in acidic pH and those obtained through solid-state fermentation had stability greater than 60% in alkaline pH. PMID:26180809

  4. Kinetic study on the enzymatic esterification of octanoic acid and hexanol by immobilized Candida antarctica lipase B

    DEFF Research Database (Denmark)

    Lopresto, Catia Giovanna; Calabro, Vincenza; Woodley, John M.

    2014-01-01

    and pharmaceutical products. The reaction is performed in n-decane as the solvent, to improve enzyme stability and to increase the reaction yield. The influence of substrate concentration on hexyl octanoate synthesis is investigated over a wide range up to 2 M. The observed bi-substrate inhibition pattern follows...... for the examined reaction system – and are essential to extend analysis to a large-scale process and for a wide range of operating conditions. The progress of the reaction is also observed and the kinetic model is validated by fitting experimental progress curves with two different concentrations of biocatalyst......tThis study investigates reaction kinetics of the esterification of octanoic acid and hexanol into hexyloctanoate, catalyzed by an immobilized Candida antarctica lipase (Novozym®435). The product is considered natural and used as a fresh vegetable and fruity flavour additive in food, cosmetic...

  5. Effect of Vitamin B5 on Liver Enzyme Levels in Bile Duct Ligation Cholestatic Rat Model

    OpenAIRE

    Fatemeh Sadat Emami; Akram Eidi; Pejman Mortazavi; Ahmad Asghari

    2017-01-01

    Background and Objectives: Accumulation of toxic bile salts in a bile duct ligation (BDL) animal model plays a pivotal role in the induction of liver fibrosis. Vitamin B5 is an essential nutrient, which acts as a cofactor in many detoxification system enzymes. In the present research, the antifibrotic effect of vitamin B5 was investigated on liver cholestasis induced by BDL in rats. Methods: In this experimental study, 72 male Wistar rats were divided into 9 groups: Control, sham-operat...

  6. Aplicações sintéticas de lipases imobilizadas em polímeros

    Directory of Open Access Journals (Sweden)

    Dalla-Vecchia Roberto

    2004-01-01

    Full Text Available The application of biocatalysis is a promising field related to new technologies for organic synthesis. The development of immobilization techniques is very important due to the multiple or repetitive use of a single batch of enzymes and the ability to stop the reaction rapidly, at any stage, by removing the enzymes. In most cases, after immobilization, enzymes and microorganisms maintain or even increase their activity and stability. This work presents an overview of the common methods for lipase immobilization in polymers and applications of these systems to obtain compounds of synthetic interest.

  7. Enzymes used in detergents: Lipases | Hasan | African Journal of ...

    African Journals Online (AJOL)

    African Journal of Biotechnology. Journal Home · ABOUT THIS JOURNAL · Advanced Search · Current Issue · Archives · Journal Home > Vol 9, No 31 (2010) >. Log in or Register to get access to full text downloads. Username, Password, Remember me, or Register · Download this PDF file. The PDF file you selected should ...

  8. Binding orientation and interaction of bile salt in its ternary complex with pancreatic lipase-colipase system.

    Science.gov (United States)

    Haque, Neshatul; Prakash Prabhu, N

    2018-05-23

    The interfacial activity of pancreatic lipases (PL) depends on the presence of colipase and bile salt. The activity of PL is inhibited by micellar concentrations of bile salt which can be restored by the addition of colipase. Though the formation of 1:1:1 tertiary complex by lipase-colipase-bile salt micelle is well accepted, the residue-level interactions between lipase-colipase and bile salt are yet to be clearly understood. Molecular dynamic simulations of lipase-colipase complex, lipase and colipase were performed in the presence of a model bile salt, sodium taurocholate (NaTC), at its near-CMC and supra-micellar concentrations. From the interactions obtained from the molecular dynamic simulations, the ternary complex was modelled and compared with earlier reports. The analysis suggested that a micelle of NaTC consisting of nine monomers was formed at the concave groove between lipase and colipase chain and it mainly interacted with the fourth finger of colipase. This complex was mainly stabilized by van der Waals interactions. Interestingly, the C-terminal domain of lipase which holds the colipase did not show any significant role in formation or stabilization of NaTC micelle. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Dihydrofolate reductase as a model for studies of enzyme dynamics and catalysis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Amnon Kohen

    2015-12-01

    Full Text Available Dihydrofolate reductase from Escherichia coli (ecDHFR serves as a model system for investigating the role of protein dynamics in enzyme catalysis. We discuss calculations predicting a network of dynamic motions that is coupled to the chemical step catalyzed by this enzyme. Kinetic studies testing these predictions are presented, and their potential use in better understanding the role of these dynamics in enzyme catalysis is considered. The cumulative results implicate motions across the entire protein in catalysis.

  10. Reatividade in vitro de lipase submetida a diferentes tratamentos tecnológicos Reactivity in vitro of the lipase submitted to different technological treatments

    Directory of Open Access Journals (Sweden)

    V.P.A. Ferreira

    2005-06-01

    Full Text Available Analisou-se a atividade enzimática in vitro de uma lipase em diferentes condições de temperatura (simulando o processo de peletização e pH (acidez gástrica e na presença de íons minerais e metálicos. Para avaliar a estabilidade térmica, a enzima foi incubada a 80(0C por quatro tempos (0, 5, 10 e 15 minutos. O efeito do pH foi estudado submetendo-se a enzima a quatro valores de pH (7,3, 2,0, 3,2 e 5,0. Para avaliar o efeito da lipase na presença de íons minerais e metálicos, incubou-se a enzima em diferentes soluções contendo cálcio, cobre, cobalto, manganês e ferro. Frente às diferentes condições, a lipase conservou atividade parcial, mas não pôde ser considerada uma enzima ideal em função do efeito inibidor promovido pelo pH ácido e pelas soluções iônicas testadas.The in vitro catalytic activity of a lipase was assessed in different conditions of temperature (simulating that of pelleted ration manufacturing, pH (gastric acidity, and its stability in the presence of metallic and mineral ions. To assess the thermal stability, the enzyme was incubated for 0, 5, 10 or 15 minutes at 80ºC. The effect of pH was studied by submitting the lipase to pH values of 7.3, 2.0, 3.2 or 5.0. In order to evaluate the catalytic activity in the presence of metallic and mineral ions, the enzyme was incubated in solutions containing calcium, copper, cobalt, manganese and iron. In the above mentioned conditions, the lipase partially preserved its activity, but, could not be considered an ideal enzyme due to its inhibition by the acidic pH and the ionic solutions tested.

  11. Vanadium(V) complexes in enzyme systems: aqueous chemistry, inhibition and molecular modeling in inhibitor design.

    Science.gov (United States)

    Bhattacharyya, S; Tracey, A S

    2001-05-01

    Vanadate in aqueous solution is known to influence a number of enzyme-catalyzed reactions. Such effects are well known to carry over to living systems where numerous responses to the influence of vanadium have been well-documented; perhaps the most studied being the insulin-mimetic effect. Studies of the aqueous chemistry of vanadate provide an insight into the mechanisms by which vanadate affects enzyme systems and suggests methods for the elucidation of specific types of responses. Studies of the corresponding enzymes provide complementary information that suggests model vanadate systems be studied and provides clues as to functional groups that might be utilized in the development of selective enzyme inhibition. The insulin-mimetic effect is thought by many workers to originate in the effectiveness of vanadium as an inhibitor of protein tyrosine phosphatase (PTPase) activity. One, or more PTPases regulate the phosphotyrosine levels of the insulin receptor kinase domain. Appropriate ligands allow modification of the reactivity and function of vanadate. For instance, although the complex, ((CH(3))(2)NO)(2)V(O)OH, is not quite as good an inhibitor of PTPase activity as is vanadate, it is much more effective in cell cultures for increasing glucose transport and glycogen synthesis. Studies of the chemistry of this complex provide an explanation of the efficacy of this compound as a PTPase inhibitor that is supported by computer modeling studies. Computer calculations using X-ray data of known PTPases as a basis for homology modeling then suggests functionality that needs to be addressed in developing selective PTPase inhibitors.

  12. Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris.

    Science.gov (United States)

    Qiao, Hanzhen; Zhang, Wenfei; Guan, Wutai; Chen, Fang; Zhang, Shihai; Deng, Zixiao

    2017-10-01

    Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0-9.0) and temperature range (20 °C-45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg 2+ , Ca 2+ , Mn 2+ and Cu 2+ and inhibited by Fe 2+ , Fe 3+ , Zn 2+ and Co 2+ . This lipase may prove useful to the detergent industry and in organic synthesis reactions. Copyright © 2017. Published by Elsevier Inc.

  13. Hydrolysis characteristics of bovine milk fat and monoacid triglycerides mediated by pregastric lipase from goats and kids.

    Science.gov (United States)

    Lai, D T; MacKenzie, A D; O'Connor, C J; Turner, K W

    1997-10-01

    Commercial extracts from oro-pharyngeal tissues of goats and kids have been used as the source of pregastric lipase and have been processed to yield partially purified samples of the primary pregastric lipase. The activity of these lipases against tributyrylglycerol has been determined over a range of pH and temperatures. Optimum pH conditions for pregastric lipase ranged from pH 5.6 to 6.5 for goats and from pH 5.5 to 6.2 for kids, respectively; the optimum temperature ranged from 43 to 60 degrees C. Optima for kid lipase extended slightly below pH 5.5 and higher than 60 degrees C; which were the limits of the test conditions. The enzymes were also used as catalysts for the hydrolysis of monoacid triglycerides (C4:0 to C12:0) at 40 degrees C and pH 6.5; activity was maximum against tributyrylglycerol (C4:0). Values for the Michaelis-Menten constant, increased as carbon chain length of the carboxylic moiety on the triglycerides increased, but values were identical for pregastric lipases of both goats and kids. Anhydrous milk fat was hydrolyzed by the commercial extracts of pregastric lipases of goats and kids, and the resulting profiles for free fatty acids were very similar to one another and to the corresponding profile for a commercial sample of Parmesan cheese. There appear to be no significant differences in activity between the enzyme preparations from goats and kids.

  14. Physicochemical Properties and Enzymes Activity Studies in a ...

    African Journals Online (AJOL)

    Soil Physicochemical properties and enzyme concentration were evaluated in soil from a refined-oil contaminated community in Isiukwuato, Abia State three years after the spill. The soil enzymes examined were urease, lipase, oxidase, alkaline and acid phosphatases. Results show a significant (P< 0.05) decrease in the ...

  15. Digestive enzymes in the alimentary canal of Clarias anguillaris ...

    African Journals Online (AJOL)

    Three groups of digestive enzymes were observed in the alimentary canals of the fingerlings and adults of Clarias anguillaris. The enzymes were carbohydrases including amylase, sucrase, lactase and maltase, proteases including peptase, tryptase and peptidase, and lipase. The activities of the protease were found to be ...

  16. Sempervivum davisii: phytochemical composition, antioxidant and lipase-inhibitory activities.

    Science.gov (United States)

    Uzun, Yusuf; Dalar, Abdullah; Konczak, Izabela

    2017-12-01

    Sempervivum davisii Muirhead (Crassulaceae) is a traditional medicinal herb from Eastern Anatolia. To date the composition of phytochemicals and physiological properties of this herb were not subjected to any research. This study identifies compounds in S. davisii hydrophilic extracts and evaluates their potential biological properties. Ethanol-based lyophilized extracts were obtained from aerial parts of plant (10 g of ground dry plant material in 200 mL of acidified aqueous ethanol, shaken for 2 h at 22 °C with supernatant collected and freeze-dried under vacuum). Phytochemical composition was investigated by liquid chromatography mass spectrometry (LC-MS/MS, phenolics) and gas chromatography mass spectrometry (GC-MS, volatiles). Phenolic compounds were quantified by high-performance liquid chromatography (HPLC) and the Folin-Ciocalteu assay. Subsequently, antioxidant capacity [ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) assays] and enzyme inhibitory properties (isolated porcine pancreatic lipase) of the extracts were determined. Polyphenolic compounds were the main constituents of lyophilized extracts, among which kaempferol glycosides and quercetin hexoside dominated. The extracts exhibited potent antioxidant (FRAP values of 1925.2-5973.3 μM Fe 2+ /g DW; ORAC values of 1858.5-4208.7 μM Trolox Eq./g DW) and moderate lipase inhibitory (IC 50 : 11.6-2.96 mg/mL) activities. Volatile compounds (nonanal, dehydroxylinalool oxide isomers, 2-decenal, 2-undecenal, 2,6-di-tetr-butylphenol) were also found. Phenolic compounds with the dominating kaempferol and quercetin derivatives are the sources of potent antioxidant properties of S. davisii hydrophilic extracts. The extracts exhibit moderate inhibitory properties towards isolated pancreatic lipase.

  17. OPTIMASI KONDISI PURIFIKASI PARSIAL LIPASE Aspergillus niger 65I6 SOLID STATE FERMENTATION (SSF PADA MEDIUM BUNGKIL JARAK SEBAGAI SUMBER BELAJAR PENGANTAR MIKROBIOLOGI

    Directory of Open Access Journals (Sweden)

    Maulina Nurikasari

    2012-11-01

    Full Text Available Extracellular lipase is an enzyme that catalyzes the hydrolysis reaction of triglycerides into free fatty acids and glycerol and synthesis reaction of esters. Lipase can be produced by microbes include bacteria, fungi and yeasts. Lipase by Aspergillus niger 65I6 produced in the medium of Jatropha cake using solid-state fermentation has a different character of the lipase produced by other microorganisms. The aims of this research were to determine the optimum condition partially purified lipase by Aspergillus niger 65I6. The methods of this research were conducted in 2 steps: (1 production of lipase, (2 partial purification using ion exchange chromatography, (3 SDS-PAGE. The results of this research showed that partial purification lipase by Aspergillus niger 65I6 using anion exchange chromatography esterification activity of 45.58 U/ml with specific activity of 421.97 U/mg adsorbed at pH 8 and 0.5 M NaCl elution. Partially purified lipase by Aspergillus niger 65I6 was 86.11% yield and purification factor 12,12 and a molecular mass of Lipase by Aspergillus niger at 19 kDa estimated by SDS-PAGE. Found of this research can be explained for the development study of microbiology concept and practical contribution about procedure research simple design. Students understand about technique and condition of partial purification.   Kata kunci:         Lipase, Aspergillus niger, Solid-State Fermentation, Purifikasi, SDS-PAGE

  18. Agroindustrial Wastes as Alternative for Lipase Production by Candida viswanathii under Solid-State Cultivation: Purification, Biochemical Properties, and Its Potential for Poultry Fat Hydrolysis

    Directory of Open Access Journals (Sweden)

    Alex Fernando de Almeida

    2016-01-01

    Full Text Available The aims of this work were to establish improved conditions for lipase production by Candida viswanathii using agroindustrial wastes in solid-state cultivation and to purify and evaluate the application of this enzyme for poultry fat hydrolysis. Mixed wheat bran plus spent barley grain (1 : 1, w/w supplemented with 25.0% (w/w olive oil increased the lipase production to 322.4%, compared to the initial conditions. When olive oil was replaced by poultry fat, the highest lipase production found at 40% (w/w was 31.43 U/gds. By selecting, yeast extract supplementation (3.5%, w/w, cultivation temperature (30°C, and substrate moisture (40%, w/v, lipase production reached 157.33 U/gds. Lipase was purified by hydrophobic interaction chromatography, presenting a molecular weight of 18.5 kDa as determined by SDS-PAGE. The crude and purified enzyme showed optimum activity at pH 5.0 and 50°C and at pH 5.5 and 45°C, respectively. The estimated half-life at 50°C was of 23.5 h for crude lipase and 6.7 h at 40°C for purified lipase. Lipase presented high activity and stability in many organic solvents. Poultry fat hydrolysis was maximum at pH 4.0, reaching initial hydrolysis rate of 33.17 mmol/L/min. Thus, C. viswanathii lipase can be successfully produced by an economic and sustainable process and advantageously applied for poultry fat hydrolysis without an additional acidification step to recover the released fatty acids.

  19. The effect of thermodynamic properties of solvent mixtures explains the difference between methanol and ethanol in C.antarctica lipase B catalyzed alcoholysis.

    Science.gov (United States)

    Sasso, Francesco; Kulschewski, Tobias; Secundo, Francesco; Lotti, Marina; Pleiss, Jürgen

    2015-11-20

    Kinetic modelling, molecular modelling, and experimental determination of the initial reaction velocity of lipase-catalyzed alcoholysis were combined to study the effect of the alcohol substrate to catalytic activity. The model system consisted of methanol or ethanol at varying concentrations, vinyl acetate as ester substrate 15.2% (v/v), toluene as organic solvent, water at a controlled thermodynamic activity of 0.09, and C. antarctica lipase B as enzyme. For both alcohol substrates, the initial reaction velocity increased sharply at low concentrations and reached a maximum at 0.7% (v/v) for methanol and 2% (v/v) for ethanol. For higher concentrations, the reaction rate decreased to a level of 74% and 60% of the peak value, respectively, due to substrate inhibition. The concentration dependency was described by a kinetic model, including a ping-pong bi-bi mechanism and competitive inhibition by the alcohol, and confirmed previous observations that methanol is more efficiently inhibiting the enzyme than ethanol. However, if the initial reaction velocity was expressed in terms of thermodynamic activity of the two alcohol substrates, the maximum of initial reaction velocity was similar for methanol (a MeOH(max)=0.19) and ethanol (a EtOH(max)=0.21). This was confirmed by molecular modelling which resulted in similar KM (0.22 and 0.19) and Ki values (0.44 and 0.49) for methanol and ethanol, respectively, if expressed in thermodynamic activities. Thus, the experimentally observed difference between methanol and ethanol is not due to differences in interaction with the enzyme but is a consequence of the thermodynamics of the substrate-solvent mixture. For low concentrations in toluene, the activity coefficient of methanol is 40% higher than the activity coefficient of ethanol (γ MeOH=8.5, γ EtOH=6.1). Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Biodiesel production from sunflower, soybean, and waste cooking oils by transesterification using lipase immobilized onto a novel microporous polymer.

    Science.gov (United States)

    Dizge, Nadir; Aydiner, Coskun; Imer, Derya Y; Bayramoglu, Mahmut; Tanriseven, Aziz; Keskinler, Bülent

    2009-03-01

    This study aims at carrying out lipase-catalyzed synthesis of fatty acid methyl esters (biodiesel) from various vegetable oils using lipase immobilized onto a novel microporous polymeric matrix (MPPM) as a low-cost biocatalyst. The research is focused on three aspects of the process: (a) MPPM synthesis (monolithic, bead, and powder forms), (b) microporous polymeric biocatalyst (MPPB) preparation by immobilization of lipase onto MPPM, and (c) biodiesel production by MPPB. Experimental planning of each step of the study was separately carried out in accordance with design of experiment (DoE) based on Taguchi methodology. Microporous polymeric matrix (MPPM) containing aldehyde functional group was synthesized by polyHIPE technique using styrene, divinylbenzene, and polyglutaraldehyde. Thermomyces lanuginosus lipase was covalently attached onto MPPM with 80%, 85%, and 89% immobilization efficiencies using bead, powder, and monolithic forms, respectively. Immobilized enzymes were successfully used for the production of biodiesel using sunflower, soybean, and waste cooking oils. It was shown that immobilized enzymes retain their activities during 10 repeated batch reactions at 25 degrees C, each lasting 24h. Since the developed novel method is simple yet effective, it could have a potential to be used industrially for the production of chemicals requiring immobilized lipases.

  1. Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

    Science.gov (United States)

    De Yan, Hong; Zhang, Yin Jun; Liu, Hong Cai; Zheng, Jian Yong; Wang, Zhao

    2013-01-01

    p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  2. Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

    Science.gov (United States)

    Dutra, Julio C. V.; da Terzi, Selma C.; Bevilaqua, Juliana Vaz; Damaso, Mônica C. T.; Couri, Sônia; Langone, Marta A. P.; Senna, Lilian F.

    The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.

  3. 3D structure prediction of lignolytic enzymes lignin peroxidase and manganese peroxidase based on homology modelling

    Directory of Open Access Journals (Sweden)

    SWAPNIL K. KALE

    2016-04-01

    Full Text Available Lignolytic enzymes have great biotechnological value in biopulping, biobleaching, and bioremediation. Manganese peroxidase (EC 1:11:1:13 and lignin peroxidase (EC 1:11:1:14 are extracellular and hem-containing peroxidases that catalyze H2O2-dependent oxidation of lignin. Because of their ability to catalyse oxidation of a wide range of organic compounds and even some inorganic compounds, they got tremendous industrial importance. In this study, 3D structure of lignin and manganese peroxidase has been predicted on the basis of homology modeling using Swiss PDB workspace. The physicochemical properties like molecular weight, isoelectric point, Grand average of hydropathy, instability and aliphatic index of the target enzymes were performed using Protparam. The predicted secondary structure of MnP has 18 helices and 6 strands, while LiP has 20 helices and 4 strands. Generated 3D structure was visualized in Pymol. The generated model for MnP and LiP has Z-score Qmean of 0.01 and -0.71, respectively. The predicted models were validated through Ramachandran Plot, which indicated that 96.1 and 95.5% of the residues are in most favored regions for MnP and LiP respectively. The quality of predicted models were assessed and confirmed by VERIFY 3D, PROCHECK and ERRAT. The modeled structure of MnP and LiP were submitted to the Protein Model Database.

  4. Two-step biocatalytic process using lipase and whole cell catalysts for biodiesel production from unrefined jatropha oil.

    Science.gov (United States)

    Zhou, Gui-xiong; Chen, Guan-yi; Yan, Bei-bei

    2015-10-01

    To avoid lipase deactivation by methanol in the enzymatic transesterification process, a two-step biocatalytic process for biodiesel production from unrefined jatropha oil was developed. Unrefined jatropha oil was first hydrolyzed to free fatty acids (FFAs) by the commercial enzyme Candida rugosa lipase. The maximum yield achieved of FFAs 90.3% at 40 °C, water/oil ratio 0.75:1 (v/v), lipase content 2% (w/w) after 8 h reaction. After hydrolysis, the FFAs were separated and converted to biodiesel by using Rhizopus oryzae IFO4697 cells immobilized within biomass support particles as a whole-cell biocatalyst. Molecular sieves (3 Å) were added to the esterification reaction mixture to remove the byproduct water. The maximum fatty acid methyl ester yield reached 88.6% at 35 °C, molar ratio of methanol to FFAs 1.2:1, molecular sieves (3 Å) content 60% (w/w) after 42 h. In addition, both C. rugosa lipase and R. oryzae whole cell catalyst in the process showed excellent reusability, retaining 89 and 79% yields, respectively, even after six batches of reactions. This novel process, combining the advantages of enzyme and whole cell catalysts, saved the consumption of commercial enzyme and avoid enzyme deactivation by methanol.

  5. Immobilization of Aspergillus niger F7-02 Lipase in Polysaccharide Hydrogel Beads of Irvingia gabonensis Matrix.

    Science.gov (United States)

    Kareem, Safaradeen Olateju; Adio, Olayinka Quadri; Osho, Michael Bamitale

    2014-01-01

    The potential of polysaccharide Irvingia gabonensis matrix as enzyme immobilization support was investigated. Lipase of Aspergillus niger F7-02 was immobilized by entrapment using glutaraldehyde as the cross-linking agent and stabilized in ethanolic-formaldehyde solution. The pH and temperature stability and activity yield of the immobilized enzyme were determined. Such parameters as enzyme load, bead size, number of beads, and bead reusability were also optimized. Adequate gel strength to form stabilized beads was achieved at 15.52% (w/v) Irvingia gabonensis powder, 15% (v/v) partially purified lipase, 2.5% (v/v) glutaraldehyde, and 3 : 1 (v/v) ethanolic-formaldehyde solution. There was 3.93-fold purification when the crude enzyme was partially purified in two-step purification using Imarsil and activated charcoal. Optimum lipase activity 75.3 Ug(-1) was achieved in 50 mL test solution containing 15 beads of 7 mm bead size. Relative activity 80% was retained at eight repeated cycles. The immobilization process gave activity yield of 59.1% with specific activity of 12.3 Umg(-1) and stabilized at optimum pH 4.5 and temperature 55°C. Thus the effectiveness and cost-efficiency of I. gabonensis as a polymer matrix for lipase immobilization have been established.

  6. Kinetic Resolutions and Enantioselective Transformations of 5-(Acyloxy)pyrrolinones Using Candida antarctica Lipase B : Synthetic and Structural Aspects

    NARCIS (Netherlands)

    Cuiper, A.D; Kouwijzer, M.L C E; Grootenhuis, P.D J; Kellogg, R.M; Feringa, B.L.

    1999-01-01

    Various 5-(acyloxy)pyrrolinones have been prepared in enantiomerically pure form by means of an enzymatic resolution or an asymmetric transformation. Either enantiomer is obtained using the same enzyme, Candida antarctica lipase B, by modification of the procedure from transesterification to

  7. Lipases as Tools in the Synthesis of Prodrugs from Racemic 9-(2,3-Dihydroxypropyl)adenine

    Czech Academy of Sciences Publication Activity Database

    Brabcová, Jana; Blažek, Jiří; Janská, Lucie; Krečmerová, Marcela; Zarevúcka, Marie

    2012-01-01

    Roč. 17, č. 12 (2012), s. 13813-13824 ISSN 1420-3049 Grant - others:AV ČR(CZ) M200551203 Institutional support: RVO:61388963 Keywords : lipase * transesterification * prodrug * immobilization of enzymes Subject RIV: CC - Organic Chemistry Impact factor: 2.428, year: 2012

  8. Lipases and proteinases in milk : occurrence, heat inactivation, and their importance for the keeping quality of milk products

    NARCIS (Netherlands)

    Driessen, F.M.

    1983-01-01

    The occurrence and heat inactivation of native and bacterial lipases and proteinases in milk were studied.

    Production of these enzymes by Gram-negative psychrotrophic bacteria in milk was found to take place towards the end of exponential growth and in the stationary growth

  9. Immobilization of Aspergillus niger F7-02 Lipase in Polysaccharide Hydrogel Beads of Irvingia gabonensis Matrix

    Directory of Open Access Journals (Sweden)

    Safaradeen Olateju Kareem

    2014-01-01

    Full Text Available The potential of polysaccharide Irvingia gabonensis matrix as enzyme immobilization support was investigated. Lipase of Aspergillus niger F7-02 was immobilized by entrapment using glutaraldehyde as the cross-linking agent and stabilized in ethanolic-formaldehyde solution. The pH and temperature stability and activity yield of the immobilized enzyme were determined. Such parameters as enzyme load, bead size, number of beads, and bead reusability were also optimized. Adequate gel strength to form stabilized beads was achieved at 15.52% (w/v Irvingia gabonensis powder, 15% (v/v partially purified lipase, 2.5% (v/v glutaraldehyde, and 3 : 1 (v/v ethanolic-formaldehyde solution. There was 3.93-fold purification when the crude enzyme was partially purified in two-step purification using Imarsil and activated charcoal. Optimum lipase activity 75.3 Ug−1 was achieved in 50 mL test solution containing 15 beads of 7 mm bead size. Relative activity 80% was retained at eight repeated cycles. The immobilization process gave activity yield of 59.1% with specific activity of 12.3 Umg−1 and stabilized at optimum pH 4.5 and temperature 55°C. Thus the effectiveness and cost-efficiency of I. gabonensis as a polymer matrix for lipase immobilization have been established.

  10. Otimização da esterificação de ácido hexanóico com n-butanol empregando lipase (Termomyces lanuginosus imobilizada em gelatina

    Directory of Open Access Journals (Sweden)

    Everton Skoronski

    2013-01-01

    Full Text Available The application of Lipozyme (Termomyces lanuginosus immobilized in gelatin gel in aliphatic ester synthesis was investigated taking the esterification of hexanoic acid with n-butanol as a model reaction. Conditions were optimized by factorial design and the highest conversion was obtained under the following conditions: molar ratio alcohol: acid of 2:1, reaction time of 48 h and biocatalyst weight of 7.0 g. Under these conditions the esterification yield was around 98 %. The operational stability of the immobilized lipase was assessed and results showed that after 12 batch runs, the enzyme showed no significant loss of activity.

  11. Combining regio- and enantioselectivity of lipases for the preparation of (R)-4-chloro-2-butanol.

    Science.gov (United States)

    Méndez, Jonh J; Oromi, Mireia; Cervero, Maria; Balcells, Mercè; Torres, Mercè; Canela, Ramon

    2007-01-01

    Preparation of 98% ee (R)-4-chloro-2-butanol was carried out by the enzymatic hydrolysis of chlorohydrin esters, using fungal resting cells and commercial enzymes. Hydrolyzes were carried out using lipases from Candida antarctica (Novozym 435), C. rugosa, Rhizomucor miehei (Lipozyme IM), Burkolia cepacia, and resting cells of Rhizopus oryzae and Aspergillus flavus. The influence of the enzyme, the solvent, the temperature, and the alkyl chain length on the selectivity of hydrolyzes of isomeric mixtures of chlorohydrin esters is described. Regioselectivity was higher than 95% for some of the tested lipases. Novozym 435 allowed preparation of the (R)-4-chloro-2-butanol after 15 min of reaction at 30-40 degrees C. (c) 2006 Wiley-Liss, Inc.

  12. Effect of reaction temperature on biodiesel production from waste cooking oil using lipase as biocatalyst

    Science.gov (United States)

    Istiningrum, Reni Banowati; Aprianto, Toni; Pamungkas, Febria Lutfi Udin

    2017-12-01

    This study aims to determine the effect of temperature on conversion of biodiesel from waste cooking oil enzymatically using lipase extracted from rice bran. The feedstock was simulated waste cooking oil and lipase enzyme was extracted with buffer pH variation. The enzyme activity was titrimetrically determined and the optimum pH buffer was used to study the effect of temperature on the transesterification reaction. Temperature effects were assessed in the range of 45-60 °C and the content of methyl esters in biodiesel was determined by GC-MS. The reaction temperature significantly influences the transesterification reaction with optimum biodiesel conversion occurred at 55 °C with methyl ester content of 81.19%. The methyl ester composition in the resulting biodiesel is methyl palmitate, methyl oleate and methyl stearate.

  13. Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae.

    Science.gov (United States)

    Taipa, M A; Liebeton, K; Costa, J V; Cabral, J M; Jaeger, K E

    1995-06-06

    Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.

  14. Lipoprotein lipase and endothelial lipase in human testis and in germ cell neoplasms

    DEFF Research Database (Denmark)

    Nielsen, J E; Lindegaard, M L; Friis-Hansen, L

    2009-01-01

    The aim of this study was to investigate endothelial lipase (EL, LIPG) and lipoprotein lipase (LPL) mRNA and protein expression in normal human testis and testicular germ cell tumours (GCT). Both EL and LPL were expressed in normal seminiferous tubules and in the interstitial compartment. EL mRNA...

  15. Self-assembly of amphiphilic janus particles into monolayer capsules for enhanced enzyme catalysis in organic media.

    Science.gov (United States)

    Cao, Wei; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

    2015-01-14

    Encapsulation of enzymes during the creation of an emulsion is a simple and efficient route for enhancing enzyme catalysis in organic media. Herein, we report a capsule with a shell comprising a monolayer of silica Janus particles (JPs) (referred to as a monolayer capsule) and a Pickering emulsion for the encapsulation of enzyme molecules for catalysis purposes in organic media using amphiphilic silica JPs as building blocks. We demonstrate that the JP capsules had a monolayer shell consisting of closely packed silica JPs (270 nm). The capsules were on average 5-50 μm in diameter. The stability of the JP capsules (Pickering emulsion) was investigated with the use of homogeneous silica nanoparticles as a control. The results show that the emulsion stabilized via amphiphilic silica JPs presented no obvious changes in physical appearance after 15 days, indicating the high stability of the emulsions and JP capsules. Furthermore, the lipase from Candida sp. was chosen as a model enzyme for encapsulation within the JP capsules during their formation. The catalytic performance of lipase was evaluated according to the esterification of 1-hexanol with hexanoic acid. It was found that the specific activity of the encapsulated enzymes (28.7 U mL(-1)) was more than 5.6 times higher than that of free enzymes in a biphasic system (5.1 U mL(-1)). The enzyme activity was further increased by varying the volume ratio of water to oil and the JPs loadings. The enzyme-loaded capsule also exhibited high stability during the reaction process and good recyclability. In particular, the jellification of agarose in the JP capsules further enhanced their operating stability. We believe that the monolayer structure of the JP capsules, together with their high stability, rendered the capsules to be ideal enzyme carriers and microreactors for enzyme catalysis in organic media because they created a large interfacial area and had low mass transfer resistance through the monolayer shell.

  16. Enhanced thermostability of silica-immobilized lipase from Bacillus coagulans BTS-3 and synthesis of ethyl propionate.

    Science.gov (United States)

    Kumar, Satyendra; Pahujani, Shweta; Ola, R P; Kanwar, S S; Gupta, Reena

    2006-06-01

    A lipase from the thermophilic isolate Bacillus coagulans BTS-3 was produced and purified. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The purified lipase was immobilized on silica and its binding efficiency was found to be 60%. The enzyme took 60 min to bind maximally onto the support. The pH and temperature optima of immobilized lipase were same as those of the free enzyme, i.e. 8.5 and 55 degrees C, respectively. The immobilized enzyme had shown marked thermostability on the elevated temperatures of 55, 60, 65 and 70 degrees C. The immobilized enzyme was reused for eigth cycles as it retained almost 80% of its activity. The catalytic activity of immobilized enzyme was enhanced in n-hexane and ethanol. The immobilized enzyme when used for esterification of ethanol and propionic acid showed 96% conversion in n-hexane in 12 h at 55 degrees C.

  17. Thermodynamics of information processing based on enzyme kinetics: An exactly solvable model of an information pump

    Science.gov (United States)

    Cao, Yuansheng; Gong, Zongping; Quan, H. T.

    2015-06-01

    Motivated by the recent proposed models of the information engine [Proc. Natl. Acad. Sci. USA 109, 11641 (2012), 10.1073/pnas.1204263109] and the information refrigerator [Phys. Rev. Lett. 111, 030602 (2013), 10.1103/PhysRevLett.111.030602], we propose a minimal model of the information pump and the information eraser based on enzyme kinetics. This device can either pump molecules against the chemical potential gradient by consuming the information to be encoded in the bit stream or (partially) erase the information initially encoded in the bit stream by consuming the Gibbs free energy. The dynamics of this model is solved exactly, and the "phase diagram" of the operation regimes is determined. The efficiency and the power of the information machine is analyzed. The validity of the second law of thermodynamics within our model is clarified. Our model offers a simple paradigm for the investigating of the thermodynamics of information processing involving the chemical potential in small systems.

  18. Characterization of cellulase, hemicellulase and lipase and its use in deinking of laser printed paper

    Directory of Open Access Journals (Sweden)

    Che Omar, I.

    2013-01-01

    Full Text Available Aims: It is recognized that laser printed paper are difficult to deink using conventional method. This had lead to the suggestion of enzymatic approach to overcome the problem encountered by commonly employed deinking techniques. The present study aimed to investigate 7 commercially available enzymes for their suitability use in deinking of laser printed paper. Methodology and results: 3 cellulases, hemicellulases, xylanase and 2 lipases were used in enzymatic deinking of laser-printed wastepaper. Cellulase A “Amano”3 (C, Hemicellulase (H and lipase (L were selected for used in deinking because they possess either highest activity or broad pH stability compared to others enzymes. Different combination of enzymes was carried out to evaluate their effectiveness in deinking process. CH enzymes sequence was determined to be the most effective sequence in toner removal with 1.90% of brightness increment. However, only 0.95% of brightnessincrement was gained by enzyme sequence L. Highest deinking efficiency obtained was not proportional to the highest total reducing sugar produced. Conclusion, significance and impact of study: Enzyme (cellulase and hemicellulase can be used to de-ink laserprintedwastepaper, which are difficult to be deinked by conventional chemical deinking process. Thus, enzyme deinking has high possibility as alternative method to current chemical deinking process which is not environmental friendly.

  19. Solvent-Free Synthesis of Flavour Esters through Immobilized Lipase Mediated Transesterification

    OpenAIRE

    Garlapati, Vijay Kumar; Banerjee, Rintu

    2013-01-01

    The synthesis of methyl butyrate and octyl acetate through immobilized Rhizopus oryzae NRRL 3562 lipase mediated transesterification was studied under solvent-free conditions. The effect of different transesterification variables, namely, molarity of alcohol, reaction time, temperature, agitation, addition of water, and enzyme amount on molar conversion (%) was investigated. A maximum molar conversion of 70.42% and 92.35% was obtained in a reaction time of 14 and 12 h with the transesterifica...

  20. Influence of a lipid interface on protein dynamics in a fungal lipase

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Bywater, R. P.

    2001-01-01

    Lipases catalyze lipolytic reactions and for optimal activity they require a lipid interface. To study the effect of a lipid aggregate on the behavior of the enzyme at the interfacial plane and how the aggregate influences an attached substrate or product molecule in time and space, we have...... forms strong hydrogen bonds with Ser-82, Ser-144, and Trp-88, suggesting that the formation of hydrogen bonds may be an important contribution to the mechanism by which product inhibition might take place....

  1. Biodiesel production by using lipase immobilized onto novel silica-based hybrid foams

    Energy Technology Data Exchange (ETDEWEB)

    Brun, Nicolas [Centre de Recherche Paul Pascal, Pessac (France); Institut des Sciences Moleculaires, Talence (France); Garcia, Annick Babeau; Oestreicher, Victor; Durand, Fabien; Backov, Renal [Centre de Recherche Paul Pascal, Pessac (France); Deleuze, Herve [Institut des Sciences Moleculaires, Talence (France); Laurent, Guillaume; Sanchez, Clement [Laboratoire de Chimie de la Matiere Condensee, Paris (France)

    2010-07-01

    The covalent immobilization of crude lipases within silica-based macroporous frameworks have been performed by combining sol-gel process, concentrated direct emulsion, lyotropic mesophase and post-synthesis functionalizations. The assynthesized open cell hybrid monoliths exhibit high macroscopic porosity, around 90%, providing interconnected scaffold while reducing the diffusion low kinetic issue. The entrapment of enzymes in such foams deals with a high stability over esterification of fatty acids, hydrolysis of triglycerides (not shown herein) and biodiesel production by transesterification. (orig.)

  2. Continuous Production of Structured Phospholipids in a Packed Red Reactor with Lipase from Thermomyces lanuginosa

    DEFF Research Database (Denmark)

    Vikbjerg, Anders Falk; Peng, Lifeng; Mu, Huiling

    2005-01-01

    The possibilities of producing structured phospholipids by lipase-catalyzed acidolysis between soybean phospholipids and caprylic acid were examined in continuous packed bed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent-free system with the commercial...... was favored by high substrate ratio between acyl donor and phospholipids, longer residence time, and higher reaction temperature. Under certain conditions, an incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent....

  3. New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

    Science.gov (United States)

    Melo, Ricardo Rodrigues de; Alnoch, Robson Carlos; Vilela, Adriana Ferreira Lopes; Souza, Emanuel Maltempi de; Krieger, Nadia; Ruller, Roberto; Sato, Hélia Harumi; Mateo, Cesar

    2017-06-29

    Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system ( E. coli extract) and four enzymes ( Candida rugosa lipase, metagenomic lipase, β-galactosidase and β-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

  4. Covalently immobilized lipase on aminoalkyl-, carboxy- and hydroxy-multi-wall carbon nanotubes in the enantioselective synthesis of Solketal esters.

    Science.gov (United States)

    Zniszczoł, Aurelia; Herman, Artur P; Szymańska, Katarzyna; Mrowiec-Białoń, Julita; Walczak, Krzysztof Z; Jarzębski, Andrzej; Boncel, Sławomir

    2016-06-01

    Aiming at the preparation of efficient, stable on storage and recyclable nanobiocatalysts for enantioselective transesterification, alkaline lipase from Pseudomonas fluorescens was covalently immobilized (up to 8.5wt.%) on functionalized multi-wall carbon nanotubes (f-MWCNTs). f-MWCNTs were synthesized via: (a) (2+1)-cycloaddition of a nitrene to the C-sp(2) nanotube walls (3.2mmolg(-1), a novel synthetic approach) and, (b) oxidative treatments, i.e. Fenton reagent (3.5mmolg(-1)) and nitrating mixture (2.5mmolg(-1)), yielding aminoalkyl-, hydroxyl- and carboxyl-MWCNTs, respectively. Amino- and epoxy- functionalized mesoporous silica (f-SBA-15) were used as the reference supports. Transesterification of vinyl n-butyrate by racemic Solketal with a chromatographically (GC) traced kinetics was selected as the model reaction. The studies revealed that different chemical functionalization of morphologically identical nanotube supports led to various enzyme loadings, catalytic activities and enantioselectivities. MWCNT-NH2-based nanobiocatalyst was found to be the most active composite among all of the tested systems (yield 20%, t=0.5h, 1321Ug(-1)), i.e. 12 times more active than the native enzyme. In turn, lipase immobilized on MWCNT-COOH emerged as the most enantioselective system (ex aequo with SBA-NH2) (eeR=74%, t=0.5h at yield of 3-5%). The activity of the MWCNT-NH2-based nanobiocatalyst after 8 cycles of transesterification dropped to 60% of its initial value, whereas for SBA-NH2-based composite remained unchanged. Importantly, stability on storage was fully maintained for all MWCNT-based nanobiocatalysts or even 'extra-enhanced' for MWCNT-OH. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. A glimpse into the specialization history of the lipases/acyltransferases family of CpLIP2.

    Science.gov (United States)

    Jan, Anne-Hélène; Dubreucq, Eric; Drone, Jullien; Subileau, Maeva

    2017-09-01

    Lipases/acyltransferases homologous to CpLIP2 from Candida parapsilosis belong to the α/β hydrolase superfamily as lipase A from Moesziomyces antarcticus (Candida antarctica), and constitute a consistent phylogenetic subgroup with at least 56% identity. Lipases/acyltransferases share the phenotypic characteristic of a high acyltransfer activity even in aqueous media with very high water thermodynamic activity. Previous mutagenesis and evolution strategies have given insights into the role of key residues and protein subdomains in the reaction and substrate specificities of these enzymes. However, multiple mutations are often deleterious for the activity and the identification of all the residues that historically led to the function is complicated. A new complementary approach to elucidate structural determinant was conducted in this study, based on the resurrection of ancestral proteins to understand how the evolution led to the present properties of the biocatalysts. By doing so, the comparison with the extant proteins can lead to the identification of key residues involved in the enzymes' specialization. Using Ancestral Sequence Reconstruction, we have generated a putative ancestral lipases/acyltransferases, PaleoLAc. This enzyme shares a high level of identity with CpLIP2 but has a different catalytic behavior. PaleoLAc allowed the identification of putative key residues involved in acyltransfer ability and supports the hypothesis that this exceptional property within the lipases/acyltransferases family is linked to a cluster of residues in the vicinity of the active site. As a representative of the ancestral origin of the diversity of the catalytic behaviors observed in modern lipases/acyltransferases, PaleoLAc constitutes a powerful tool for further engineering toward targeted specialization. Copyright © 2017. Published by Elsevier B.V.

  6. Fungal Screening on Olive Oil for Extracellular Triacylglycerol Lipases: Selection of a Trichoderma harzianum Strain and Genome Wide Search for the Genes

    Directory of Open Access Journals (Sweden)

    Miguel Angel Canseco-Pérez

    2018-01-01

    Full Text Available A lipolytic screening with fungal strains isolated from lignocellulosic waste collected in banana plantation dumps was carried out. A Trichoderma harzianum strain (B13-1 showed good extracellular lipolytic activity (205 UmL−1. Subsequently, functional screening of the lipolytic activity on Rhodamine B enriched with olive oil as the only carbon source was performed. The successful growth of the strain allows us to suggest that a true lipase is responsible for the lipolytic activity in the B13-1 strain. In order to identify the gene(s encoding the protein responsible for the lipolytic activity, in silico identification and characterization of triacylglycerol lipases from T. harzianum is reported for the first time. A survey in the genome of this fungus retrieved 50 lipases; however, bioinformatic analyses and putative functional descriptions in different databases allowed us to choose seven lipases as candidates. Suitability of the bioinformatic screening to select the candidates was confirmed by reverse transcription polymerase chain reaction (RT-PCR. The gene codifying 526309 was expressed when the fungus grew in a medium with olive oil as carbon source. This protein shares homology with commercial lipases, making it a candidate for further applications. The success in identifying a lipase gene inducible with olive oil and the suitability of the functional screening and bioinformatic survey carried out herein, support the premise that the strategy can be used in other microorganisms with sequenced genomes to search for true lipases, or other enzymes belonging to large protein families.

  7. Microbial lipase obtained from the fermentation of pumpkin seeds: immobilization potential of hydrophobic matrices

    Directory of Open Access Journals (Sweden)

    Rafaela Cristiane Andrade Santos

    2014-04-01

    Full Text Available Immobilization potential of lipase from Aspergillus niger on sol-gel matrix was evaluated by physical adsorption and covalent binding and the biochemical characterization of free and immobilized enzyme was performed. Lipase was produced by solid state fermentation of pumpkin seed flour with 30% moisture, at 30°C for 120h. The enzyme was pre-purified with ammonium sulfate and immobilized in the sol-gel matrix by covalent attachment and physical adsorption. A higher yield of immobilization (81.88% was obtained in the latter. The free enzyme presented higher hydrolytic activity with pH 4.0, at 37°C; moreover, it was more stable with pH between 6.0 and 7.0, at 35°C. The immobilized lipase showed maximum hydrolytic activity with pH 11.0, at 50°C; it was more stable with pH 11.0, at 37°C. Parameters Km and Vmax were best determined by Hanes-Woolf linearization.

  8. A new lipase isolated from oleaginous seeds from Pachira aquatica (Bombacaceae).

    Science.gov (United States)

    Polizelli, Patricia Peres; Facchini, Fernanda Dell Antonio; Cabral, Hamilton; Bonilla-Rodriguez, Gustavo Orlando

    2008-09-01

    A new lipase from seeds of Pachira aquatica was purified to homogeneity by SDS-PAGE obtaining an enzyme with a molecular weight of approximately 55 kDa. The purified lipase exhibited maximum activity at 40 degrees C and pH 8.0, for an incubation time of 90 min. Concerning temperature stability, at the range from 4 to 50 degrees C, it retained approximately 47% of its original activity for 3 h. The enzyme activity increased in the presence of Ca(++) and Mg(++), but was inhibited by Hg(++), Mn(++), Zn(++), Al(+++) and various oxidizing and reducing agents. The lipase was highly stable in the presence of organic solvents, and its activity was stimulated by methanol. The values of K(m) and V(max) were 1.65 mM and 37.3 micromol mL(-1) min(-1), respectively, using p-nitrophenylacetate as substrate. The enzyme showed preference for esters of long-chain fatty acids, but demonstrated significant activity against a wide range of substrates.

  9. Efficient lipase-catalyzed Knoevenagel condensation: utilization of biocatalytic promiscuity for synthesis of benzylidene-indolin-2-ones.

    Science.gov (United States)

    Ding, Yan; Xiang, Xinran; Gu, Mengjie; Xu, Haoran; Huang, He; Hu, Yi

    2016-01-01

    Based on the screening of biocatalysts and reaction conditions including solvent, water content, temperature, enzyme loading, and reaction time, lipase from porcine pancreas (PPL) showed the prominent promiscuity for the Knoevenagel condensation between 1,3-dihydroindol-2-one heterocycle and aromatic aldehydes. Under the optimized procedure, both electron-withdrawing and electron-donating substituent of aldehydes substrates could react efficiently, and benzylidene-indolin-2-ones were obtained in excellent yields (75.0-96.6%). Benzylidene-indolin-2-ones derivatives were efficiently synthesized by the Knoevenagel condensation between various aromatic aldehydes and 1,3-dihydroindol-2-one catalyzed by lipase from porcine pancreas with excellent yields obtained.

  10. Enzymatic transesterification of soybean oil with ethanol using lipases immobilized on highly crystalline PVA microspheres

    International Nuclear Information System (INIS)

    Bergamasco, Juliana; Araujo, Marcelo V. de; Vasconcellos, Adriano de; Luizon Filho, Roberto A.; Hatanaka, Rafael R.; Giotto, Marcus V.; Aranda, Donato A.G.; Nery, José G.

    2013-01-01

    Polyvinyl alcohol (PVA) microspheres with different degree of crystallinity were used as solid supports for Rhizomucor miehei lipase immobilization, and the enzyme-PVA complexes were used as biocatalysts for the transesterification of soybean oil to fatty acid ethyl esters (FAEE). The amounts of immobilized enzyme on the polymeric supports were similar for both the amorphous microspheres (PVA4) and the high crystalline microspheres (PVA25). However, the enzymatic activity of the immobilized enzymes was depended on the crystallinity degree of the PVA microspheres: enzymes immobilized on the PVA4 microspheres have shown low enzymatic activity (6.13 U mg −1 ), in comparison with enzymes immobilized on the high crystalline PVA25 microspheres (149.15 U mg −1 ). A synergistic effect was observed for the enzyme-PVA25 complex during the transesterification reaction of soybean oil to FAEE: transesterification reactions with free enzyme with the equivalent amount of enzyme that were immobilized onto the PVA25 microspheres (5.4 U) have yielded only 20% of FAEE, reactions with the pure highly crystalline microsphere PVA25 have not yielded FAEE, however reactions with the enzyme-PVA25 complexes have yielded 66.3% of FAEE. This synergistic effect of an immobilized enzyme on a polymeric support has not been observed before for transesterification reaction of triacylglycerides into FAEE. Based on ATR-FTIR, 23 Na- and 13 C-NMR-MAS spectroscopic data and the interaction of the polymeric network intermolecular hydrogen bonds with the lipases residual amino acids a possible explanation for this synergistic effect is provided. Highlights: • Rhizomucor miehei lipase was immobilized on PVA microspheres (PVA4, PVA12, PVA25). • Polymer-enzyme complex was characterized by XDR, SEM, ATR-FTIR, 13 C-CPMAS-NMR, 23 Na-MAS-NMR. • Polymer-enzymes (PVA12 and PVA25) enzymes yielded considerable amount of ethyl esters. • Synergistic effect was observed for the polymer-enzyme complexes

  11. Production of fatty acid butyl esters using the low cost naturally immobilized Carica papaya lipase.

    Science.gov (United States)

    Su, Erzheng; Wei, Dongzhi

    2014-07-09

    In this work, the low cost naturally immobilized Carica papaya lipase (CPL) was investigated for production of fatty acid butyl esters (FABE) to fulfill the aim of reducing the lipase cost in the enzymatic butyl-biodiesel process. The CPL showed specificities to different alcohol acyl acceptors. Alcohols with more than three carbon atoms did not have negative effects on the CPL activity. The CPL catalyzed butanolysis for FABE production was systematically investigated. The reaction solvent, alcohol/oil molar ratio, enzyme amount, reaction temperature, and water activity all affected the butanolysis process. Under the optimized conditions, the highest conversion of 96% could be attained in 24 h. These optimal conditions were further applied to CPL catalyzed butanolysis of other vegetable oils. All of them showed very high conversion. The CPL packed-bed reactor was further developed, and could be operated continuously for more than 150 h. All of these results showed that the low cost Carica papaya lipase can be used as a promising lipase for biodiesel production.

  12. The interactions between lipase and pyridinium ligands investigated by electrochemical and spectrophotometric methods

    Directory of Open Access Journals (Sweden)

    Simona Patriche

    2016-04-01

    Full Text Available The interaction between pyridinium ligands derived from 4,4’-bipyridine (N,N’-bis(p-bromophenacyl-4,4’-bipyridinium dibromide – Lr and (N,N’-bis(p-bromophenacyl-1,2-bis (4-pyridyl ethane dibromide – Lm with lipase enzyme was evaluated. The stability of the pyridinium ligands, having an essential role in biological systems, in 0.1 M KNO3 as supporting electrolyte is influenced by the lipase concentration added. The pH and conductometry measurements in aqueous solution suggest a rapid ionic exchange process. The behavior of pyridinium ligands in the presence of lipase is investigated by cyclic voltammetry and UV/Vis spectroscopy, which indicated bindings and changes from the interaction between them. The voltammograms recorded on the glassy carbon electrode showed a more intense electronic transfer for the Lr interaction with lipase compared to Lm, which is due to the absence of mobile ethylene groups from Lr structure.

  13. Immobilization of Beauveria bassiana Lipase on Silica Gel by Physical Adsorption

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    Vanessa Hitomi Sugahara

    2014-12-01

    Full Text Available Extracellular lipase from Beauveria bassianastrain CG481 was immobilized by using thirteen different immobilization protocols. Silica gel was chosen as the most suitable adsorbent with 94.8% of activity yield. The adsorption on silica gel did not change the optimum pH (8.5 and temperature (45ºC values of the free lipase (FL for lipolytic activity, and it showed higher activities in extreme conditions (pH 9.0 to 10.5, 60ºC. The lipase immobilized on silica gel (ILS showed enhanced stability at pH 7.0 after 120 h incubation (69.0% when compared to FL (33.3%. The thermal stability was also enhanced by immobilization at 60ºC in aqueous (64.6% and organic medium (95.1%, while FL showed only 40.6% of residual activity in aqueous medium and exhibited no activity for esterification reaction in n-heptane. The treatment of ILS with 0.8 M NaCl prevented lipase desorption while Triton X-100 (0.1% resulted the enzyme leakage. The ILS was reused for four times for esterification reaction with 80.8% of initial activity.

  14. Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

    Science.gov (United States)

    Schinke, Claudia; Germani, José C

    2012-03-01

    Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

  15. Biodiesel Production by Aspergillus niger Lipase Immobilized on Barium Ferrite Magnetic Nanoparticles.

    Science.gov (United States)

    El-Batal, Ahmed I; Farrag, Ayman A; Elsayed, Mohamed A; El-Khawaga, Ahmed M

    2016-05-12

    In this study, Aspergillus niger ADM110 fungi was gamma irradiated to produce lipase enzyme and then immobilized onto magnetic barium ferrite nanoparticles (BFN) for biodiesel production. BFN were prepared by the citrate sol-gel auto-combustion method and characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and scanning electron microscopy with energy dispersive analysis of X-ray (SEM/EDAX) analysis. The activities of free and immobilized lipase were measured at various pH and temperature values. The results indicate that BFN-Lipase (5%) can be reused in biodiesel production without any treatment with 17% loss of activity after five cycles and 66% loss in activity in the sixth cycle. The optimum reaction conditions for biodiesel production from waste cooking oil (WCO) using lipase immobilized onto BFN as a catalyst were 45 °C, 4 h and 400 rpm. Acid values of WCO and fatty acid methyl esters (FAMEs) were 1.90 and 0.182 (mg KOH/g oil), respectively. The measured flash point, calorific value and cetane number were 188 °C, 43.1 MJ/Kg and 59.5, respectively. The cloud point (-3 °C), pour point (-9 °C), water content (0.091%) and sulfur content (0.050%), were estimated as well.

  16. Influence of protein solution in nucleation and optimized formulation for the growth of ARM lipase crystal

    Science.gov (United States)

    Rahman, Raja Noor Zaliha Raja Abd; Masomian, Malihe; Leow, Adam Thean Chor; Ali, Mohd Shukuri Mohamad

    2015-09-01

    ARM lipase is a thermostable and organic solvent tolerant enzyme which was highly purified prior to crystallization. The His-tagged ARM lipase was purified with immobilized metal affinity chromatography followed by anion-exchange chromatography. The effect of different salt concentrations on stability, solubility and crystal nucleation of the protein was studied. The highly purified and homogeneous ARM lipase with protein concentration of 2 mg/mL was successfully crystallized by a sitting drop, vapor diffusion method with the use of 0.1 M MES monohydrate pH 6.5 and 12% (v/v) polyethylene glycol (PEG) 20000 as precipitant. The crystallization conditions were optimized by changing the pH and concentration of the precipitant. The optimum crystallization condition was 2 mg/mL ARM lipase in 0.1 M Tris-HCl, 0.15 M NaCl, pH 8.0 protein solution, crystallized using 0.1 M Tris-HCl, pH 8.0 and 12% (v/v) PEG 20000 as precipitant.

  17. Biodiesel Production by Aspergillus niger Lipase Immobilized on Barium Ferrite Magnetic Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ahmed I. El-Batal

    2016-05-01

    Full Text Available In this study, Aspergillus niger ADM110 fungi was gamma irradiated to produce lipase enzyme and then immobilized onto magnetic barium ferrite nanoparticles (BFN for biodiesel production. BFN were prepared by the citrate sol-gel auto-combustion method and characterized by transmission electron microscopy (TEM, X-ray diffraction (XRD, Fourier transform infrared (FTIR and scanning electron microscopy with energy dispersive analysis of X-ray (SEM/EDAX analysis. The activities of free and immobilized lipase were measured at various pH and temperature values. The results indicate that BFN–Lipase (5% can be reused in biodiesel production without any treatment with 17% loss of activity after five cycles and 66% loss in activity in the sixth cycle. The optimum reaction conditions for biodiesel production from waste cooking oil (WCO using lipase immobilized onto BFN as a catalyst were 45 °C, 4 h and 400 rpm. Acid values of WCO and fatty acid methyl esters (FAMEs were 1.90 and 0.182 (mg KOH/g oil, respectively. The measured flash point, calorific value and cetane number were 188 °C, 43.1 MJ/Kg and 59.5, respectively. The cloud point (−3 °C, pour point (−9 °C, water content (0.091% and sulfur content (0.050%, were estimated as well.

  18. Methods for Creating and Animating a Computer Model Depicting the Structure and Function of the Sarcoplasmic Reticulum Calcium ATPase Enzyme.

    Science.gov (United States)

    Chen, Alice Y.; McKee, Nancy

    1999-01-01

    Describes the developmental process used to visualize the calcium ATPase enzyme of the sarcoplasmic reticulum which involves evaluating scientific information, consulting scientists, model making, storyboarding, and creating and editing in a computer medium. (Author/CCM)

  19. Towards A Deeper Understanding of the Interfacial Adsorption of Enzyme Molecules in Gigaporous Polymeric Microspheres

    Directory of Open Access Journals (Sweden)

    Weichen Wang

    2016-04-01

    Full Text Available Compared with the one immobilized in the conventional mesoporous microspheres, the enzyme immobilized in gigaporous microspheres showed much higher activity and better stability. To gain a deeper understanding, we herein selected lipase as a prototype to comparatively analyze the adsorption behavior of lipase at interfaces in gigaporous and mesoporous polystyrene microspheres at very low lipase concentration, and further compared with the adsorption on a completely flat surface (a chip. Owing to the limited space of narrow pores, lipase molecules were inclined to be adsorbed as a monolayer in mesoporous microspheres. During this process, the interaction between lipase molecules and the interface was stronger, which could result in the structural change of lipase molecular and compromised specific activity. In addition to monolayer adsorption, more multilayer adsorption of enzyme molecules also occurred in gigaporous microspheres. Besides the adsorption state, the pore curvature also affected the lipase adsorption. Due to the multilayer adsorption, the excellent mass transfer properties for the substrate and the product in the large pores, and the small pore curvature, lipase immobilized in gigaporous microspheres showed better behaviors.

  20. The angiotensin converting enzyme inhibitor captopril protects nigrostriatal dopamine neurons in animal models of parkinsonism.

    Science.gov (United States)

    Sonsalla, Patricia K; Coleman, Christal; Wong, Lai-Yoong; Harris, Suzan L; Richardson, Jason R; Gadad, Bharathi S; Li, Wenhao; German, Dwight C

    2013-12-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by a prominent loss of nigrostriatal dopamine (DA) neurons with an accompanying neuroinflammation. The peptide angiotensin II (AngII) plays a role in oxidative-stress induced disorders and is thought to mediate its detrimental actions via activation of AngII AT1 receptors. The brain renin-angiotensin system is implicated in neurodegenerative disorders including PD. Blockade of the angiotensin converting enzyme or AT1 receptors provides protection in acute animal models of parkinsonism. We demonstrate here that treatment of mice with the angiotensin converting enzyme inhibitor captopril protects the striatum from acutely administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrine (MPTP), and that chronic captopril protects the nigral DA cell bodies from degeneration in a progressive rat model of parkinsonism created by the chronic intracerebral infusion of 1-methyl-4-phenylpyridinium (MPP+). The accompanying activation of microglia in the substantia nigra of MPP+-treated rats was reduced by the chronic captopril treatment. These findings indicate that captopril is neuroprotective for nigrostriatal DA neurons in both acute and chronic rodent PD models. Targeting the brain AngII pathway may be a feasible approach to slowing neurodegeneration in PD. © 2013.