Sample records for biobleaching

  1. Producing a True Lignin Depolymerase for Biobleaching Softwood Kraft Pulp

    Simo Sarkanen


    This project constituted an intensive effort devoted to producing, from the white-rot fungus Tramets Cingulata, a lignin degrading enzyme (lignin depolymerase) that is directly able to biobleach or delignify softwood kraft pulp brownstock. To this end, the solutions in which T. cingulata was grown contained dissolved kraft lignin which fulfilled two functions; it behaved as a lignin deploymerase substrate and it also appeared to act as an inducer of enzyme expression. However, the lignin depolymerase isoenzymes (and other extracellular T. cingulata enzymes) interacted very strongly with both the kraft lignin components and the fungal hypae, so the isolating these proteins from the culture solutions proved to be unexpectedly difficult. Even after extensive experimentation with a variety of protein purification techniques, only one approach appeared to be capable of purifying lignin depolymerases to homogeneity. Unfortunately the procedure was extremely laborious; it involved the iso electric focusing of concentrated buffer-exchanged culture solutions followed by electro-elution of the desired protein bands from the appropriate polyacrylamide gel segments

  2. Biobleaching chemistry of laccase-mediator systems on high-lignin-content kraft pulps

    Chakar, F.S.; Ragauskas, A.J.


    A high-lignin-content softwood kraft pulp was reacted with laccase in the presence of 1-hydroxybenzotriazole (HBT), N-acetyl-N-phenylhydroxylamine (NHA), and violuric acid (VA). The biodelignification response with violuric acid was superior to both 1-hydroxybenzotriazole and N-acetyl-N-phenylhydroxylamine. NMR analysis of residual lignins isolated before and after the biobleaching treatments revealed that the latter material was highly oxidized and that the magnitude of structural changes was most pronounced with the laccase - violuric acid biobleaching system. An increase in the content of carboxylic acid groups and a decrease in methoxyl groups were noted with all three laccase-mediator systems. The oxidation biobleaching pathway is directed primarily towards noncondensed C5 phenolic lignin functional structures for all three laccase-mediated systems. The laccase - violuric acid system was also reactive towards C5-condensed phenolic lignin structures. (author)

  3. Biobleaching of wheat straw-rich soda pulp with alkalophilic laccase from gamma-proteobacterium JB: optimization of process parameters using response surface methodology.

    Singh, Gursharan; Ahuja, Naveen; Batish, Mona; Capalash, Neena; Sharma, Prince


    An alkalophilic laccase from gamma-proteobacterium JB was applied to wheat straw-rich soda pulp to check its bleaching potential by using response surface methodology based on central composite design. The design was employed by selecting laccase units, ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) concentration and pH as model factors. The results of second order factorial design experiments showed that all three independent variables had significant effect on brightness and kappa number of laccase-treated pulp. Optimum conditions for biobleaching of pulp with laccase preparation (specific activity, 65 nkat mg(-1) protein) were 20 nkat g(-1) of pulp, 2mM ABTS and pH 8.0 which enhanced brightness by 5.89% and reduced kappa number by 21.1% within 4h of incubation at 55 degrees C, without further alkaline extraction of pulp. Tear index (8%) and burst index (18%) also improved for laccase-treated pulp as compared to control raw pulp. Treatment of chemically (CEH1H2) bleached pulp with laccase showed significant effect on release of chromophores, hydrophobic and reducing compounds. Laccase-prebleaching of raw pulp reduced the use of hypochlorite by 10% to achieve brightness of resultant hand sheets similar to the fully chemically bleached pulp.

  4. Application of a novel alkali-tolerant thermostable DyP-type peroxidase from Saccharomonospora viridis DSM 43017 in biobleaching of eucalyptus kraft pulp.

    Wangning Yu

    Full Text Available Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50-80°C. It also showed broad pH adaptability (>35% activity at pH 4.0-9.0 and alkali-tolerance (>80% activity after incubation at pH 5-10 for 1 h at 37°C, and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0. SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.

  5. Application of thermoalkalophilic xylanase from Arthrobacter sp. MTCC 5214 in biobleaching of kraft pulp

    Khandeparker, R.; Bhosle, N.B.

    released by enzyme treatment showed a characteristic peak at 280 nm indicating the presence of lignin in the released coloring matter. Enzymatic prebleaching of kraft pulp showed 20 % reduction in kappa number of the pulp without much change in viscosity...

  6. Xylanases of marine fungi of potential use for biobleaching of paper pulp

    Raghukumar, C.; Muraleedharan, U.; Gaud, V.R.; Mishra, R.

    isolates obtained from marine habitat showed alkaline xylanase activity. The crude enzyme from NIOCC isolate # 3 (Aspergillus niger) with high xylanase activity, cellulase-free and unique properties containing 580 U L-1 of xylanase, could bring about...


    Ana Maria Queijeiro López

    Full Text Available Lignin is present in plant cell secondary wall, associated to carbohydrates preventing their efficient hydrolysis, and cellulose pulp manufacture basically consists in breaking down the middle lamella of plant cells, individualizing fibers such as cellulose from the other biopolymers. Different levels of lignocellulose are found in plant residues and they can be decomposed by extracellular fungal lignin modifying enzymes, used as a tool to reduce waste materials in contaminated soils and effluents. In the paper mill industries, for instance, they are a suitable or complementary alternative to the traditional methods of pulping/bleaching, contributing to improve paper strength as well as to reduce the pitch content, the quantity of chemicals and the consume of electrical energy. The aim of this review was to describe the fungal degradation of lignocellulosic like-material, the non-specific enzymatic aspects of the attack of wood and agricultural wastes, the fungal ability for biosorption and bioconversion, and its applications in the pulp/paper industry and bioremediation.

  8. Production and characterization of thermostable xylanase from ...



    Feb 20, 2013 ... produced from Trichoderma (Huitron et al., 2008). Only a ... around their colonies against a red background, were selected and .... Resistance to antibiotic ..... Xylanases of marine fungi potential use for biobleaching of paper.

  9. Phoenix dactylifera L.



    May 31, 2010 ... their use for flavoring foods, beverages and medication. (Vayalil, 2002). Minerals are .... Potassium is vital to cellular integrity and fluid balance as it plays an important role in .... mechanical pulp for bio-bleaching. Food Chem.

  10. African Journal of Biotechnology - Vol 11, No 22 (2012)

    In vitro regeneration of selected commercial Tanzanian open pollinated maize varieties · EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT ... Biobleaching of wheat straw-rich-soda pulp by the application of alkalophilic and thermophilic mannanase from Streptomyces sp. PG-08-3 · EMAIL FREE ...

  11. Optimizing Bacillus circulans Xue-113168 for biofertilizer production ...

    In this study, Bacillus circulans Xue-113168 biofertilizer was produced through solid state fermentation processes using food waste and feldspar. Results confirmed that solid state fermentation has considerable advantages compared to complex process (solid-state and bio-bleach). The control of pH, temperature, and ...

  12. Biopulping of sugarcane bagasse and decolorization of kraft liquor by the laccase produced by Klebsiella aerogenes NCIM 2098

    Jha H.


    Full Text Available Aims: Laccase, a copper-containing enzyme, oxidizes variety of aromatic compounds. Since laccase is essential for lignin degradation, it can be used for lignin removal in the pulp and paper industry (biopulping. Laccase is also employed as a dechlorinating agent (biobleaching, along with the removal of phenolic and other aromatic pollutants. In the present investigation it was aimed to employ the laccase produced by the bacterium Klebsiella aerogenes along with the bacterium itself in biopulping of sugarcane bagasse and biobleaching of kraft liquor effluent. Methodology and results: A laccase was isolated from the bacterium K. aerogenes, purified to homogeneity and characterized. The enzyme was purified by conventional techniques following salt precipitation, ion exchange chromatography, and affinity chromatography on Con A sepharose. The purified laccase was found to be monomeric glycoprotein with a Mr of 64 kDa when measured by Sephadex G-200 gel chromatography and SDS-PAGE. The Vmax and Km of laccase towards the substrate guaiacol was determined. The optimum pH of the laccase was found to be 5.0. biopulping and biobleaching activities were determined by TAPPI standard methods. Treatment of sugarcane baggase by K. aerogenes also significantly reduced lignin content of the bagasse. Conclusion, significance and impact of study: The bacterium K. aerogenes and a laccase produced by it were used separately for biopulping of sugarcane bagasse and biobleaching of kraft liquor effluent. Treatment with both brought significant reduction in lignin content and kappa number of the pulp. The handsheets prepared from the treated pulp showed improved brightness without affecting the strength properties of paper. The bacterium and the laccase efficiently decolorized the kraft liquor proving to have biobleaching potential.

  13. Bacterial laccase: recent update on production, properties and industrial applications.

    Chauhan, Prakram Singh; Goradia, Bindi; Saxena, Arunika


    Laccases (benzenediol: oxygen oxidoreductase, EC are multi-copper enzymes which catalyze the oxidation of a wide range of phenolic and non-phenolic aromatic compounds in the presence or absence of a mediator. Till date, laccases have mostly been isolated from fungi and plants, whereas laccase from bacteria has not been well studied. Bacterial laccases have several unique properties that are not characteristics of fungal laccases such as stability at high temperature and high pH. Bacteria produce these enzymes either extracellularly or intracellularly and their activity is in a wide range of temperature and pH. It has application in pulp biobleaching, bioremediation, textile dye decolorization, pollutant degradation, biosensors, etc. Hence, comprehensive information including sources, production conditions, characterization, cloning and biotechnological applications is needed for the effective understanding and application of these enzymes at the industrial level. The present review provides exhaustive information of bacterial laccases reported till date.

  14. Utilization of deoiled Jatropha curcas seed cake for production of xylanase from thermophilic Scytalidium thermophilum.

    Joshi, Chetna; Khare, S K


    Jatropha curcas is a major biodiesel crop. Large amount of deoiled cake is generated as by-product during biodiesel production from its seeds. Deoiled J. curcas seed cake was assessed as substrate for the production of xylanase from thermophilic fungus Scytalidium thermophilum by solid-state fermentation. The seed cake was efficiently utilized by S. thermophilum for its growth during which it produced good amount of heat stable extracellular xylanase. The solid-state fermentation conditions were optimized for maximum xylanase production. Under the optimized conditions viz. deoiled seed cake supplemented with 1% oat-spelt xylan, adjusted to pH 9.0, moisture content 1:3 w/v, inoculated with 1×10(6) spores per 5 g cake and incubated at 45 °C, 1455 U xylanase/g deoiled seed cake was obtained. The xylanase was useful in biobleaching of paper pulp. Solid-state fermentation of deoiled cake appears a potentially viable approach for its effective utilization. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. Engineering Thermostable Microbial Xylanases Toward its Industrial Applications.

    Kumar, Vishal; Dangi, Arun Kumar; Shukla, Pratyoosh


    Xylanases are one of the important hydrolytic enzymes which hydrolyze the β-1, 4 xylosidic linkage of the backbone of the xylan polymeric chain which consists of xylose subunits. Xylanases are mainly found in plant cell walls and are produced by several kinds of microorganisms such as fungi, bacteria, yeast, and some protozoans. The fungi are considered as most potent xylanase producers than that of yeast and bacteria. There is a broad series of industrial applications for the thermostable xylanase as an industrial enzyme. Thermostable xylanases have been used in a number of industries such as paper and pulp industry, biofuel industry, food and feed industry, textile industry, etc. The present review explores xylanase-substrate interactions using gene-editing tools toward the comprehension in improvement in industrial stability of xylanases. The various protein-engineering and metabolic-engineering methods have also been explored to improve operational stability of xylanase. Thermostable xylanases have also been used for improvement in animal feed nutritional value. Furthermore, they have been used directly in bakery and breweries, including a major use in paper and pulp industry as a biobleaching agent. This present review envisages some of such applications of thermostable xylanases for their bioengineering.

  16. Statistical Optimization of Conditions for Decolorization of Synthetic Dyes by Cordyceps militaris MTCC 3936 Using RSM

    Baljinder Kaur


    Full Text Available In the present study, the biobleaching potential of white rot fungus Cordyceps militaris MTCC3936 was investigated. For preliminary screening, decolorization properties of C. militaris were comparatively studied using whole cells in agar-based and liquid culture systems. Preliminary investigation in liquid culture systems revealed 100% decolorization achieved within 3 days of incubation for reactive yellow 18, 6 days for reactive red 31, 7 days for reactive black 8, and 11 days for reactive green 19 and reactive red 74. RSM was further used to study the effect of three independent variables such as pH, incubation time, and concentration of dye on decolorization properties of cell free supernatant of C. militaris. RSM based statistical analysis revealed that dye decolorization by cell free supernatants of C. militaris is more efficient than whole cell based system. The optimized conditions for decolorization of synthetic dyes were identified as dye concentration of 300 ppm, incubation time of 48 h, and optimal pH value as 5.5, except for reactive red 31 (for which the model was nonsignificant. The maximum dye decolorizations achieved under optimized conditions for reactive yellow 18, reactive green 19, reactive red 74, and reactive black 8 were 73.07, 65.36, 55.37, and 68.59%, respectively.

  17. Amenability of Acacia and Eucalyptus Hardwood Pulps to Elemental Chlorine-Free Bleaching: Application and Efficacy of Microbial Xylanase

    Avdhesh Kumar Gangwar


    Full Text Available This study outlines the results of a biobleaching study of acacia (A. mangium and eucalyptus (E. globulus hardwood kraft pulps with commercial xylanase (Optimase CX 72 L. The comparative study was carried out using an elemental chlorine-free (ECF bleaching sequence (D0EPD1D2 after the enzyme (X stage. The enzyme treatment resulted in improved optical properties with a reduction in bleach chemical consumption. At an equivalent bleach chemical consumption, a brightness gain of 2.1 and 1.7 units and a whiteness gain of 2.7 and 2.3 units were observed with xylanase treatment in acacia and eucalyptus pulps, respectively. In ECF bleaching using the D0EPD1D2 sequence, a final brightness was achieved to the extent of 90% ISO and 89% ISO for acacia and eucalyptus, respectively, at an equivalent charge of bleach chemicals. The post-color (PC number was also reduced by up to 45% for both hardwood pulps compared with the control. The bleachability of acacia was observed to be significantly higher than that of eucalyptus. In addition, a 17.0% and 23.0% reduction in chlorine dioxide and sodium hydroxide, respectively, were obtained for both hardwood pulps after xylanase pre-bleaching, thus indicating an environmentally friendly approach to the process.


    Vasanta V. Thakur,


    Full Text Available The biobleaching efficiency of xylanase and laccase enzymes was studied on kraft pulps from wood and nonwood based raw materials employed in the Indian paper industry. Treatment of these pulps with xylanase enzyme could result in improved properties, showing 2.0% ISO gain in pulp brightness and/or reducing the demand of chlorine-based bleach chemicals by up to 15% with simultaneous reduction of 20 to 25% in AOX generation in bleach effluents. Further, mill-scale trial results revealed that enzymatic prebleaching can be successfully employed with xylanases to reach the same bleach boosting efficacy. Laccase bleaching was also studied on hardwood pulp at a pH around 8.0, where most of the pulp mills in India are operating, in contrast to earlier studies on laccase enzyme bleaching, which were conducted at acidic pHs, i.e. 4.0 to 5.0. In case of laccase bleaching, interesting results were found wherein a bleach-boosting effect was observed even at pH 8.0. Further studies carried out with HOBT as mediator in comparison to the commonly used and expensive ABTS laccase mediator system (LMS resulted in improvement of the bleaching efficiency with reduction in demand of chlorine dioxide by more than 35%. Potential for further reduction was indicated by the brightness gain, when compared with a control using the DE(pD bleach sequence.

  19. Isolation, Purification, and Characterization of Xylanase Produced by a New Species of Bacillus in Solid State Fermentation

    Rajashri D. Kamble


    Full Text Available A thermoalkalophilic new species of Bacillus, similar to Bacillus arseniciselenatis DSM 15340, produced extracellular xylanase under solid state fermentation when wheat bran is used as carbon source. The extracellular xylanase was isolated by ammonium sulfate (80% precipitation and purified using ion exchange chromatography. The molecular weight of xylanase was ~29.8 ;kDa. The optimum temperature and pH for the enzyme activity were 50°C and pH 8.0. The enzyme was active on birchwood xylan and little active on p-nitrophenyl xylopyranoside but not on Avicel, CMC, cellobiose, and starch, showing its absolute substrate specificity. For birchwood xylan, the enzyme gave a Km 5.26 ;mg/mL and Vmax 277.7 ;μmol/min/mg, respectively. In addition, the xylanase was also capable of producing high-quality xylo-oligosaccharides, which indicated its application potential not only in pulp biobleaching processes but also in the nutraceutical industry.

  20. Laccase engineering: from rational design to directed evolution.

    Mate, Diana M; Alcalde, Miguel


    Laccases are multicopper oxidoreductases considered by many in the biotechonology field as the ultimate "green catalysts". This is mainly due to their broad substrate specificity and relative autonomy (they use molecular oxygen from air as an electron acceptor and they only produce water as by-product), making them suitable for a wide array of applications: biofuel production, bioremediation, organic synthesis, pulp biobleaching, textiles, the beverage and food industries, biosensor and biofuel cell development. Since the beginning of the 21st century, specific features of bacterial and fungal laccases have been exhaustively adapted in order to reach the industrial demands for high catalytic activity and stability in conjunction with reduced production cost. Among the goals established for laccase engineering, heterologous functional expression, improved activity and thermostability, tolerance to non-natural media (organic solvents, ionic liquids, physiological fluids) and resistance to different types of inhibitors are all challenges that have been met, while obtaining a more comprehensive understanding of laccase structure-function relationships. In this review we examine the most significant advances in this exciting research area in which rational, semi-rational and directed evolution approaches have been employed to ultimately convert laccases into high value-added biocatalysts. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. [Biological treatments for contaminated soils: hydrocarbon contamination. Fungal applications in bioremediation treatment].

    Martín Moreno, Carmen; González Becerra, Aldo; Blanco Santos, María José


    Bioremediation is a spontaneous or controlled process in which biological, mainly microbiological, methods are used to degrade or transform contaminants to non or less toxic products, reducing the environmental pollution. The most important parameters to define a contaminated site are: biodegradability, contaminant distribution, lixiviation grade, chemical reactivity of the contaminants, soil type and properties, oxygen availability and occurrence of inhibitory substances. Biological treatments of organic contaminations are based on the degradative abilities of the microorganisms. Therefore the knowledge on the physiology and ecology of the biological species or consortia involved as well as the characteristics of the polluted sites are decisive factors to select an adequate biorremediation protocol. Basidiomycetes which cause white rot decay of wood are able to degrade lignin and a variety of environmentally persistent pollutants. Thus, white rot fungi and their enzymes are thought to be useful not only in some industrial process like biopulping and biobleaching but also in bioremediation. This paper provides a review of different aspects of bioremediation technologies and recent advances on ligninolytic metabolism research.

  2. Performance of an alkalophilic and halotolerant laccase from gamma-proteobacterium JB in the presence of industrial pollutants.

    Singh, Gursharan; Sharma, Prince; Capalash, Neena


    An alkalophilic and halotolerant laccase from gamma-proteobacterium JB catalyzed in high concentrations of organic solvents and various salts. The enzyme retained 80-100% activity in 10% concentration of dimethylsulfoxide (DMSO), ethanol, acetone or methanol; 100, 85 and 50% activity in 20 mM MgCl(2), 5.0 mM MnCl(2) and 0.1 mM CuCl(2); 140, 120 and 110% activity in 5.0 mM MnSO(4), 10 mM MgSO(4) and 1mM CaSO(4), respectively. Sodium halides inhibited the enzyme in the order: F(-)> Br(-)> I(-)> Cl(-). In 0.5 M NaCl, pH 6.0, laccase was approximately 60% active. Decolorization of indigo carmine by laccase at pH 9.0 was not inhibited even in the presence of 0.5 M NaCl. Release of chromophoric, reducing and hydrophobic compounds during biobleaching of straw rich-soda pulp by laccase was not inhibited when the enzyme was applied in the presence of 1 M NaCl at pH 8.0. Laccase retained 50% residual activity even when incubated with 5% calcium hypochlorite for 30 min.

  3. FY 1999 report on the results of the project on the industrial science technology R and D. Development of utilization technology of biological resources such as bioconsortium system (Development of the petroleum substituting fuel production technology using living organisms); 1999 nendo sangyo gijutsu kenkyu kaihatsu jigyo. Fukugou seibutsukei tou seibutsu shigen riyo gijutsu kaihatsu (Seibutsu riyo sekiyu daitai nenryo seizo gijutsu no kaihatsu) seika hokokusho



    For the purpose of developing the technology for producing and degrading useful substances using bioconsortia, study was made of the handling technology of bioconsortia, the basic element technology, etc., and the FY 1999 results were reported. In the study of the high-grade utilization technology of lignocellulose/the components, an sample was obtained in which the effect of the bacteria culture supernatant treatment was recognized in the biobleaching by co-treatment of the bacteria culture filtrate - MnP. As to the search for control factor of lignin degrading enzyme and the utilization technology, it was found out that bisphenol A was efficiently degraded by a combination of laccase and mediator production bacteria. Concerning the utilization technology of plant symbiotic bacteria, classification/identification have been finished of approximately 60% of the stored bacteria. In the study of the production technology of the petroleum substituting useful resource, a system was constructed in which immature embryos were used for callus induction and regeneration of plantlets, and plants were regenerated at high frequency via the formation of adventitious embryos. By this, the culture cell with high propagation ability was obtained. (NEDO)

  4. Aspects microbiologiques de la production par fermentation solide des endo-beta-1,4-xylanases de moisissures : le cas de Penicillium canescens

    Assamoi AA.


    Full Text Available Microbial aspects of endo-β-1,4-xylanase production in solid-state fermentation by Penicillia: the case of Penicillium canescens. Production of xylanases by Penicillium canescens 10-10c is the research object in Walloon Center of Industrial Biology. Previous works used submerged or liquid fermentation. The actual works are oriented more and more towards solid fermentation from agricultural or agro-alimentary residues. In addition to the valorization of these residues, solid-state fermentation reaches an increasingly significant interest in various other fields like the biological breakdown of the solid residues, the bioremediation of the organic pollutants in the grounds and the reduction of the air pollution by the biofiltration. Xylanase is an industrial enzyme used in general in extraction and clarification processes. P. canescens can produce an activity of it, particularly in its balanced forms of xylanases, beta-xylosidase and arabinosidase, and not contaminated by cellulolytic and amylolytic activities. It is a hyper producing strain of xylanase. The production rate is one of the highest in literature (535 and 9,632 U.g-1 in Erlenmeyer flasks, in submerged and solid state fermentation, respectively. The biobleaching activity of the cellulose pulp by the purified enzyme is higher than a commercial preparation of xylanases from Trichoderma longibrachiatum used industrially. It has a complete hydrolysis degree of 40% (on glucuronoxylan and 35% (on arabinoxylan at 55°C and at pH of 5.9. These characteristics lead to many industrial applications of this enzyme. That is why the optimization of its production by the solid-state fermentation at the laboratory scale in order to define a policy for the industrial transposition later is carried out. This article presents a summary of the scientific literature on this subject.

  5. Redesigning pH optimum of Geobacillus sp. TF16 endoxylanase through in silico designed DNA swapping strategy.

    Uzuner, Ugur; Canakci, Sabriye; Bektas, Kadriye Inan; Sapmaz, Merve Tuncel; Belduz, Ali Osman


    Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  6. Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation.

    Dwivedi, Pallavi; Vivekanand, V; Pareek, Nidhi; Sharma, Amit; Singh, Rajesh P


    Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

    Akhtar MN


    Full Text Available Abstract Background Acetyl-xylan esterase (AXE, EC hydrolyses acetate group from the linear chain of xylopyranose residues bound by β-1,4-linkage. The enzyme finds commercial applications in bio-bleaching of wood pulp, treating animal feed to increase digestibility, processing food to increase clarification and converting lignocellulosics to feedstock and fuel. In the present study, we report on the production of an extracellular AXE from Penicillium notatum NRRL-1249 by solid state fermentation (SSF. Results Wheat bran at a level of 10 g (with 4 cm bed height was optimized as the basal substrate for AXE production. An increase in enzyme activity was observed when 7.5 ml of mineral salt solution (MSS containing 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4.7H2O, 0.3% NaNO3, 0.001% FeSO4.2H2O and 0.1% (v/w Tween-80 as an initial moisture content was used. Various nitrogen sources including ammonium sulphate, urea, peptone and yeast extract were compared for enzyme production. Maximal enzyme activity of 760 U/g was accomplished which was found to be highly significant (p ≤ 0.05. A noticeable enhancement in enzyme activity was observed when the process parameters including incubation period (48 h, initial pH (5, 0.2% (w/w urea as nitrogen source and 0.5% (v/w Tween-80 as a stimulator were further optimized using a 2-factorial Plackett-Burman design. Conclusion From the results it is clear that an overall improvement of more than 35% in terms of net enzyme activity was achieved compared to previously reported studies. This is perhaps the first report dealing with the use of P. notatum for AXE production under batch culture SSF. The Plackett-Burman model terms were found highly significant (HS, suggesting the potential commercial utility of the culture used (df = 3, LSD = 0.126.

  8. Improvement for enhanced xylanase production by Cellulosimicrobium cellulans CKMX1 using central composite design of response surface methodology.

    Walia, Abhishek; Mehta, Preeti; Guleria, Shiwani; Shirkot, Chand Karan


    The effects of yeast extract (X 1 ), NH 4 NO 3 (X 2 ), peptone (X 3 ), urea (X 4 ), CMC (X 5 ), Tween 20 (X 6 ), MgSO 4 (X 7 ), and CaCO 3 (X 8 ) on production of xylanase from Cellulosimicrobium cellulans CKMX1 were optimized by statistical analysis using response surface methodology (RSM). The RSM was used to optimize xylanase production by implementing the Central composite design. Statistical analysis of the results showed that the linear, interaction and quadric terms of these variables had significant effects. However, only the linear effect of X 4 , X 5 , interaction effect of X 1 X 7 , X 1 X 8 , X 2 X 3 , X 2 X 8 , X 3 X 6 , X 3 X 8 , X 4 X 6 , X 4 X 7 , X 5 X 7 , X 5 X 8 and quadratic effect of X 3 2 , X 5 2 and X 7 2 found to be insignificant terms in the quadratic model and had no response at significant level. The minimum and maximum xylanase production obtained was 331.50 U/g DBP and 1027.65 U/g DBP, respectively. The highest xylanase activity was obtained from Run No. 30, which consisted of yeast extract (X 1 ), 1.00 g (%); NH 4 NO 3 (X 2 ), 0.20 g (%); peptone (X 3 ), 1.00 g (%); urea (X 4 ), 10 mg (%); CMC (X 5 ), 1.00 g (%); Tween 20 (X 6 ), 0.02 mL (%); CaCO 3 (X 7 ), 0.50 g (%) and MgSO 4 (X 8 ), 9.0 g (%). The optimization resulted in 3.1-fold increase of xylanase production, compared with the lowest xylanase production of 331.50 U/g DBP after 72 h of incubation in stationary flask experiment. Application of cellulase-free xylanase in pulp biobleaching from C. cellulans CKMX1 under C-E P -D sequence has been shown to bring about a 12.5 % reduction of chlorine, decrease of 0.8 kappa points (40 %), and gain in brightness was 1.42 % ISO points in 0.5 % enzyme treated pulp as compared to control.

  9. Computational analysis and low-scale constitutive expression of laccases synthetic genes GlLCC1 from Ganoderma lucidum and POXA 1B from Pleurotus ostreatus in Pichia pastoris.

    Claudia M Rivera-Hoyos

    Full Text Available Lacasses are multicopper oxidases that can catalyze aromatic and non-aromatic compounds concomitantly with reduction of molecular oxygen to water. Fungal laccases have generated a growing interest due to their biotechnological potential applications, such as lignocellulosic material delignification, biopulping and biobleaching, wastewater treatment, and transformation of toxic organic pollutants. In this work we selected fungal genes encoding for laccase enzymes GlLCC1 in Ganoderma lucidum and POXA 1B in Pleurotus ostreatus. These genes were optimized for codon use, GC content, and regions generating secondary structures. Laccase proposed computational models, and their interaction with ABTS [2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid] substrate was evaluated by molecular docking. Synthetic genes were cloned under the control of Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase (GAP constitutive promoter. P. pastoris X-33 was transformed with pGAPZαA-LaccGluc-Stop and pGAPZαA-LaccPost-Stop constructs. Optimization reduced GC content by 47 and 49% for LaccGluc-Stop and LaccPost-Stop genes, respectively. A codon adaptation index of 0.84 was obtained for both genes. 3D structure analysis using SuperPose revealed LaccGluc-Stop is similar to the laccase crystallographic structure 1GYC of Trametes versicolor. Interaction analysis of the 3D models validated through ABTS, demonstrated higher substrate affinity for LaccPost-Stop, in agreement with our experimental results with enzymatic activities of 451.08 ± 6.46 UL-1 compared to activities of 0.13 ± 0.028 UL-1 for LaccGluc-Stop. This study demonstrated that G. lucidum GlLCC1 and P. ostreatus POXA 1B gene optimization resulted in constitutive gene expression under GAP promoter and α-factor leader in P. pastoris. These are important findings in light of recombinant enzyme expression system utility for environmentally friendly designed expression systems, because of the wide range

  10. Mill Designed Bio bleaching Technologies

    Institute of Paper Science Technology


    A key finding of this research program was that Laccase Mediator Systems (LMS) treatments on high-kappa kraft could be successfully accomplished providing substantial delignification (i.e., > 50%) without detrimental impact on viscosity and significantly improved yield properties. The efficiency of the LMS was evident since most of the lignin from the pulp was removed in less than one hour at 45 degrees C. Of the mediators investigated, violuric acid was the most effective vis-a-vis delignification. A comparative study between oxygen delignification and violuric acid revealed that under relatively mild conditions, a single or a double LMS{sub VA} treatment is comparable to a single or a double O stage. Of great notability was the retention of end viscosity of LMS{sub VA} treated pulps with respect to the end viscosity of oxygen treated pulps. These pulps could then be bleached to full brightness values employing conventional ECF bleaching technologies and the final pulp physical properties were equal and/or better than those bleached in a conventional ECF manner employing an aggressively O or OO stage initially. Spectral analyses of residual lignins isolated after LMS treated high-kappa kraft pulps revealed that similar to HBT, VA and NHA preferentially attack phenolic lignin moieties. In addition, a substantial decrease in aliphatic hydroxyl groups was also noted, suggesting side chain oxidation. In all cases, an increase in carboxylic acid was observed. Of notable importance was the different selectivity of NHA, VA and HBT towards lignin functional groups, despite the common N-OH moiety. C-5 condensed phenolic lignin groups were overall resistant to an LMS{sub NHA, HBT} treatments but to a lesser extent to an LMS{sub VA}. The inactiveness of these condensed lignin moieties was not observed when low-kappa kraft pulps were biobleached, suggesting that the LMS chemistry is influenced by the extent of delignification. We have also demonstrated that the current

  11. Advanced Recombinant Manganese Peroxidase for Biosynthesis of Lignin Bioproducts, Phase I Final Report, STTR Grant #: DE-SC0007503.

    Beatty, Christopher; Kitner, Joshua; Lajoie, Curtis; McClain, Sean; Potochnik, Steve


    The core purpose of this Phase I STTR was to evaluate the feasibility of a new method of producing a recombinant version of manganese peroxidase (MnP) enzyme. MnP is a potentially valuable enzyme for producing high value lignin products and also for industrial de-coloring operations such as biobleaching of pulp and color removal from textile dye effluents. This lignin-modifying enzyme is produced in small amounts by the native host, a white rot fungus. Previous work by Oregon State University developed a secreted recombinant version of the enzyme in the yeast Pichia pastoris. Unfortunately, the expression is barely moderate and the enzyme is heavily glycosylated, which inhibits purification. In this work, the gene for the enzyme is given a tag which targets production of the enzyme to the peroxisome. This is a promising approach since this location is also where heme and hydrogen peroxide are sequestered, which are both necessary cofactors for MnP. More than ten recombinant strains were constructed, verified, and expressed in the Pichia system. Constitutive (GAP) and methanol-induced promoters (AOX) were tried for peroxisomal targeted, cytosolic, and secreted versions of MnP. Only the secreted strains showed activity. The amount of expression was not significantly changed. The degree of glycosylation was lessened using the AOX (methanol) promotoer, but the resulting enzyme was still not able to be purified using immobilized metal affinity chromatography. Additional work beyond the scope of the defined Phase I project was undertaken to construct, verify, and express Pichia strains that mutated the MnP glycosylation sites to inhibit this process. These strains did not show significant activity. The cause is not known, but it is possible that these sites are important to the structure of the enzyme. Also beyond the scope proposed for our Phase I STTR, the team collaborated with AbSci, a startup with a new E. coli based expression system focused on the production of

  12. Structure and Biochemestry of Laccases from the Lignin-Degrading Basidiomycete, Ganoderma lucidum

    C.A.Reddy, PI


    G. lucidum is one of the most important and widely distributed ligninolytic white rot fungi from habitats such as forest soils, agricultural soils, and tropical mangrove ecosystems and produce laccases as an important family of lignin modifying enzymes. Biochemically, laccases are blue multi copper oxidases that couple four electron reduction of molecular oxygen to water. There is a growing interest in the use of laccases for a variety of industrial applications such as bio-pulping and biobleaching as well as in their ability to detoxify a wide variety of toxic environmental pollutants. These key oxidative enzymes are found in all the three domains of life: Eukaryota. Prokarya, and Archaea. Ganoderma lucidum (strain no.103561) produces laccase with some of the highest activity (17,000 micro katals per mg of protein) reported for any laccases to date. Our results showed that this organism produces at least 11 different isoforms of laccase based on variation in mol. weight and/or PI. Our Studies showed that the presence of copper in the medium yields 15- to 20-fold greater levels of enzyme by G. lucidum. Dialysation of extra cellular fluid of G. lucidum against 10mM sodium tartrate (pH5.5) gave an additional 15 to 17 fold stimulation of activity with an observed specific activity of 17,000 {micro}katals/mg protein. Dialysis against acetate buffer gave five fold increase in activity while dialysis against glycine showed inhibition of activity. Purification by FPLC and preparative gel electrophoresis gave purified fractions that resolved into eleven isoforms as separated by isoelectric focusing, and the PI,s were 4.7, 4.6, 4.5, 4.3, 4.2, 4.1, 3.8, 3.7, 3.5, 3.4 and 3.3. Genomic clones of laccase were isolated using G. lucidum DNA as a template and using inverse PCR and forward/reverse primers corresponding to the sequences of the conserved copper binding region in the N-terminal domain of one of the laccases of this organism. Inverse PCR amplication of HindIII digested