WorldWideScience

Sample records for biocatalysis

  1. Biocatalysis

    DEFF Research Database (Denmark)

    Lange, L.; Parmar, V.; Meyer, A.

    2017-01-01

    Biocatalysis is important for addressing global challenges: climate change, substitution of fossils, feeding a growing population—basically because bioprocessing in food, feed, and nonfood industries improves resource efficiency, getting more out of the raw biomaterials. Microbial enzymes are the......, producing less waste water. Enzymes and biocatalysis are key elements in sustainable production of biobased products in the new bioeconomy era....

  2. Biocatalysis for Biobased Chemicals

    Science.gov (United States)

    de Regil, Rubén; Sandoval, Georgina

    2013-01-01

    The design and development of greener processes that are safe and friendly is an irreversible trend that is driven by sustainable and economic issues. The use of Biocatalysis as part of a manufacturing process fits well in this trend as enzymes are themselves biodegradable, require mild conditions to work and are highly specific and well suited to carry out complex reactions in a simple way. The growth of computational capabilities in the last decades has allowed Biocatalysis to develop sophisticated tools to understand better enzymatic phenomena and to have the power to control not only process conditions but also the enzyme’s own nature. Nowadays, Biocatalysis is behind some important products in the pharmaceutical, cosmetic, food and bulk chemicals industry. In this review we want to present some of the most representative examples of industrial chemicals produced in vitro through enzymatic catalysis. PMID:24970192

  3. Biodegradable products by lipase biocatalysis.

    Science.gov (United States)

    Linko, Y Y; Lämsä, M; Wu, X; Uosukainen, E; Seppälä, J; Linko, P

    1998-11-18

    The interest in the applications of biocatalysis in organic syntheses has rapidly increased. In this context, lipases have recently become one of the most studied groups of enzymes. We have demonstrated that lipases can be used as biocatalyst in the production of useful biodegradable compounds. A number of examples are given. 1-Butyl oleate was produced by direct esterification of butanol and oleic acid to decrease the viscosity of biodiesel in winter use. Enzymic alcoholysis of vegetable oils without additional organic solvent has been little investigated. We have shown that a mixture of 2-ethyl-1-hexyl esters can be obtained in a good yield by enzymic transesterification from rapeseed oil fatty acids for use as a solvent. Trimethylolpropane esters were also similarly synthesized as lubricants. Finally, the discovery that lipases can also catalyze ester syntheses and transesterification reactions in organic solvent systems has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. In direct polyesterification of 1,4-butanediol and sebacic acid, polyesters with a mass average molar mass of the order of 56,000 g mol-1 or higher, and a maximum molar mass of about 130,000 g mol-1 were also obtained by using lipase as biocatalyst. Finally, we have demonstrated that also aromatic polyesters can be synthesized by lipase biocatalysis, a higher than 50,000 g mol-1 mass average molar mass of poly(1,6-hexanediyl isophthalate) as an example.

  4. The limits to biocatalysis: pushing the envelope.

    Science.gov (United States)

    Sheldon, Roger A; Brady, Dean

    2018-06-12

    In the period 1985 to 1995 applications of biocatalysis, driven by the need for more sustainable manufacture of chemicals and catalytic, (enantio)selective methods for the synthesis of pharmaceutical intermediates, largely involved the available hydrolases. This was followed, in the next two decades, by revolutionary developments in protein engineering and directed evolution for the optimisation of enzyme function and performance that totally changed the biocatalysis landscape. In the same period, metabolic engineering and synthetic biology revolutionised the use of whole cell biocatalysis in the synthesis of commodity chemicals by fermentation. In particular, developments in the enzymatic enantioselective synthesis of chiral alcohols and amines are highlighted. Progress in enzyme immobilisation facilitated applications under harsh industrial conditions, such as in organic solvents. The emergence of biocatalytic or chemoenzymatic cascade processes, often with co-immobilised enzymes, has enabled telescoping of multi-step processes. Discovering and inventing new biocatalytic processes, based on (meta)genomic sequencing, evolving enzyme promiscuity, chemomimetic biocatalysis, artificial metalloenzymes, and the introduction of non-canonical amino acids into proteins, are pushing back the limits of biocatalysis function. Finally, the integral role of biocatalysis in developing a biobased carbon-neutral economy is discussed.

  5. Multi-enzyme catalyzed processes: Next generation biocatalysis

    DEFF Research Database (Denmark)

    Andrade Santacoloma, Paloma de Gracia; Sin, Gürkan; Gernaey, Krist

    2011-01-01

    Biocatalysis has been attracting increasing interest in recent years. Nevertheless, most studies concerning biocatalysis have been carried out using single enzymes (soluble or immobilized). Currently, multiple enzyme mixtures are attractive for the production of many compounds at an industrial...

  6. Role of Biocatalysis in Sustainable Chemistry

    DEFF Research Database (Denmark)

    Sheldon, Roger A; Woodley, John M.

    2018-01-01

    Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled...... successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost...

  7. Green polymer chemistry: biocatalysis and biomaterials

    Science.gov (United States)

    This overview briefly surveys the practice of green chemistry in polymer science. Eight related themes can be discerned from the current research activities: 1) biocatalysis, 2) bio-based building blocks and agricultural products, 3) degradable polymers, 4) recycling of polymer products and catalys...

  8. Role of Biocatalysis in Sustainable Chemistry.

    Science.gov (United States)

    Sheldon, Roger A; Woodley, John M

    2018-01-24

    Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme can improve its stability and enable its reuse multiple times, resulting in better performance and commercial viability. Consequently, biocatalysis is being widely applied in the production of pharmaceuticals and some commodity chemicals. Moreover, its broader application will be further stimulated in the future by the emerging biobased economy.

  9. Enzyme immobilisation in biocatalysis : Why, what and how

    NARCIS (Netherlands)

    Sheldon, R.A.; Van Pelt, S.

    2013-01-01

    In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling

  10. New opportunities for biocatalysis: making pharmaceutical processes greener

    DEFF Research Database (Denmark)

    Woodley, John

    2008-01-01

    The pharmaceutical industry requires synthetic routes to be environmentally compatible as well as to fulfill the demands of process economics and product specification and to continually reduce development times. Biocatalysis has the potential to deliver 'greener' chemical syntheses, and in this ......The pharmaceutical industry requires synthetic routes to be environmentally compatible as well as to fulfill the demands of process economics and product specification and to continually reduce development times. Biocatalysis has the potential to deliver 'greener' chemical syntheses...... of biocatalysis for making pharmaceutical processes greener....

  11. Enzymes from Higher Eukaryotes for Industrial Biocatalysis

    Directory of Open Access Journals (Sweden)

    Zhibin Liu

    2004-01-01

    Full Text Available The industrial production of fine chemicals, feed and food ingredients, pharmaceuticals, agrochemicals and their respective intermediates relies on an increasing application of biocatalysis, i.e. on enzyme or whole-cell catalyzed conversions of molecules. Simple procedures for discovery, cloning and over-expression as well as fast growth favour fungi, yeasts and especially bacteria as sources of biocatalysts. Higher eukaryotes also harbour an almost unlimited number of potential biocatalysts, although to date the limited supply of enzymes, the high heterogeneity of enzyme preparations and the hazard of infectious contaminants keep some interesting candidates out of reach for industrial bioprocesses. In the past only a few animal and plant enzymes from agricultural waste materials were employed in food processing. The use of bacterial expression strains or non-conventional yeasts for the heterologous production of efficient eukaryotic enzymes can overcome the bottleneck in enzyme supply and provide sufficient amounts of homogenous enzyme preparations for reliable and economically feasible applications at large scale. Ideal enzymatic processes represent an environmentally friendly, »near-to-completion« conversion of (mostly non-natural substrates to pure products. Recent developments demonstrate the commercial feasibility of large-scale biocatalytic processes employing enzymes from higher eukaryotes (e.g. plants, animals and also their usefulness in some small-scale industrial applications.

  12. Thermophilic archaeal enzymes and applications in biocatalysis.

    Science.gov (United States)

    Littlechild, Jennifer A

    2011-01-01

    Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.

  13. ECUT (Energy Conversion and Utilization Technologies) program: Biocatalysis project

    Science.gov (United States)

    Baresi, Larry

    1989-03-01

    The Annual Report presents the fiscal year (FY) 1988 research activities and accomplishments, for the Biocatalysis Project of the U.S. Department of Energy, Energy Conversion and Utilization Technologies (ECUT) Division. The ECUT Biocatalysis Project is managed by the Jet Propulsion Laboratory, California Institute of Technology. The Biocatalysis Project is a mission-oriented, applied research and exploratory development activity directed toward resolution of the major generic technical barriers that impede the development of biologically catalyzed commercial chemical production. The approach toward achieving project objectives involves an integrated participation of universities, industrial companies and government research laboratories. The Project's technical activities were organized into three work elements: (1) The Molecular Modeling and Applied Genetics work element includes research on modeling of biological systems, developing rigorous methods for the prediction of three-dimensional (tertiary) protein structure from the amino acid sequence (primary structure) for designing new biocatalysis, defining kinetic models of biocatalyst reactivity, and developing genetically engineered solutions to the generic technical barriers that preclude widespread application of biocatalysis. (2) The Bioprocess Engineering work element supports efforts in novel bioreactor concepts that are likely to lead to substantially higher levels of reactor productivity, product yields and lower separation energetics. Results of work within this work element will be used to establish the technical feasibility of critical bioprocess monitoring and control subsystems. (3) The Bioprocess Design and Assessment work element attempts to develop procedures (via user-friendly computer software) for assessing the energy-economics of biocatalyzed chemical production processes, and initiation of technology transfer for advanced bioprocesses.

  14. Integrating protein engineering with process design for biocatalysis

    DEFF Research Database (Denmark)

    Woodley, John M.

    2017-01-01

    Biocatalysis uses enzymes for chemical synthesis and production, offering selective, safe and sustainable catalysis. While today the majority of applications are in the pharmaceutical sector, new opportunities are arising every day in other industry sectors, where production costs become a more...... important driver. In the early applications of the technology, it was necessary to design processes to match the properties of the biocatalyst. With the advent of protein engineering, organic chemists started to develop and improve enzymes to suit their needs. Likewise in industry, although not widespread......, a new paradigm was already implemented several years ago to engineer enzymes to suit process needs. Today, a new era is entered, where the effectiveness with which such integrated protein and process engineering is achieved becomes critical to implementation. In this paper, the development of a tool...

  15. Progress in biocatalysis with immobilized viable whole cells: systems development, reaction engineering and applications

    Czech Academy of Sciences Publication Activity Database

    Polakovič, M.; Švitel, J.; Bučko, M.; Filip, J.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 39, č. 5 (2017), s. 667-683 ISSN 0141-5492 Institutional support: RVO:68081731 Keywords : biocatalysis * immobilization methods * immobilized whole-cell biocatalyst * multienzyme cascade reactions * process economics * reaction engineering Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 1.730, year: 2016

  16. 8th International Congress on Biocatalysis (Biocat2016), Hamburg, Germany, 28 August–1 September, 2016

    OpenAIRE

    Skander Elleuche; Petra Esselun; Ralf Grote; Barbara Klippel; Anna Krüger; Andreas Liese; Margit Ostrop; Christian Schäfers; Carola Schröder; Garabed Antranikian

    2016-01-01

    The “8th International Congress on Biocatalysis (biocat2016)” is part of a biennial conference series. Biocatalysis is a topic based on the edge of biology and chemistry, which brings together scientists from the life sciences, engineers and computer scientists. This international conference serves as a platform to meet researchers from all over the world, to find collaboration partners for future projects and to gain novel insights into modern topics and techniques. Biocat covers the most ex...

  17. Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase.

    Science.gov (United States)

    Blank, Lars M; Ionidis, Georgios; Ebert, Birgitta E; Bühler, Bruno; Schmid, Andreas

    2008-10-01

    A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of

  18. Biodiesel production from triolein and short chain alcohols through biocatalysis.

    Science.gov (United States)

    Salis, Andrea; Pinna, Marcella; Monduzzi, Maura; Solinas, Vincenzo

    2005-09-29

    Oleic acid alkyl esters (biodiesel) were synthesised by biocatalysis in solvent-free conditions. Different commercial immobilised lipases, namely Candida antarctica B, Rizhomucor miehei, and Pseudomonas cepacia, were tested towards the reaction between triolein and butanol to produce butyl oleate. Pseudomonas cepacia lipase resulted to be the most active enzyme reaching 100% of conversion after 6h. Different operative conditions such as reaction temperature, water activity, and reagent stoichiometric ratio were investigated and optimised. These conditions were then used to investigate the effect of linear and branched short chain alcohols. Methanol and 2-butanol were the worst alcohols: the former, probably, due to its low miscibility with the oil and the latter because secondary alcohols usually are less reactive than primary alcohols. Conversely, linear and branched primary alcohols with short alkyl chains (C(2)--C(4)) showed high reaction rate and conversion. A mixture of linear and branched short chain alcohols that mimics the residual of ethanol distillation (fusel oil) was successfully used for oleic acid ester synthesis. These compounds are important in biodiesel mixtures since they improve low temperature properties.

  19. Biocatalysis in organic chemistry and biotechnology: past, present, and future.

    Science.gov (United States)

    Reetz, Manfred T

    2013-08-28

    Enzymes as catalysts in synthetic organic chemistry gained importance in the latter half of the 20th century, but nevertheless suffered from two major limitations. First, many enzymes were not accessible in large enough quantities for practical applications. The advent of recombinant DNA technology changed this dramatically in the late 1970s. Second, many enzymes showed a narrow substrate scope, often poor stereo- and/or regioselectivity and/or insufficient stability under operating conditions. With the development of directed evolution beginning in the 1990s and continuing to the present day, all of these problems can be addressed and generally solved. The present Perspective focuses on these and other developments which have popularized enzymes as part of the toolkit of synthetic organic chemists and biotechnologists. Included is a discussion of the scope and limitation of cascade reactions using enzyme mixtures in vitro and of metabolic engineering of pathways in cells as factories for the production of simple compounds such as biofuels and complex natural products. Future trends and problems are also highlighted, as is the discussion concerning biocatalysis versus nonbiological catalysis in synthetic organic chemistry. This Perspective does not constitute a comprehensive review, and therefore the author apologizes to those researchers whose work is not specifically treated here.

  20. Predicting when precipitation-driven synthesis is feasible : application to biocatalysis

    NARCIS (Netherlands)

    Ulijn, R.V.; Janssen, A.E.M.; Moore, B.D.; Halling, P.J.

    2001-01-01

    Precipitation-driven synthesis offers the possibility of obtaining high reaction yields using very low volume reactors and is finding increasing applications in biocatalysis. Here, a model that allows straightforward prediction of when such a precipitation-driven reaction will be thermodynamically

  1. Using Biocatalysis to Integrate Organic Chemistry into a Molecular Biology Laboratory Course

    Science.gov (United States)

    Beers, Mande; Archer, Crystal; Feske, Brent D.; Mateer, Scott C.

    2012-01-01

    Current cutting-edge biomedical investigation requires that the researcher have an operational understanding of several diverse disciplines. Biocatalysis is a field of science that operates at the crossroads of organic chemistry, biochemistry, microbiology, and molecular biology, and provides an excellent model for interdisciplinary research. We…

  2. On the (un)greenness of biocatalysis : Some challenging figures and some promising options

    NARCIS (Netherlands)

    Dominguez de Maria, P; Hollmann, F.

    2015-01-01

    Biocatalysis is generally regarded as a “green” technology. This statement is justified by the mild reaction conditions, the use of aqueous reaction media—with water as the paradigm of green solvents—, and the renewable nature of the biocatalysts. However, researchers making these statements

  3. More efficient redox biocatalysis by utilising 1,4-butanediol as a ‘smart cosubstrate’

    NARCIS (Netherlands)

    Kara, S.; Spickermann, D.; Schrittwieser, J.H.; Leggewie, C.; Van Berkel, W.J.H.; Arendsa, I.W.C.E.; Hollmann, F.

    2012-01-01

    1,4-Butanediol is shown to be an efficient cosubstrate to promote NAD(P)H-dependent redox biocatalysis. The thermodynamically and kinetically inert lactone coproduct makes the regeneration reaction irreversible. Thereby not only the molar surplus of cosubstrate is dramatically reduced but also

  4. More efficient redox biocatalysis by utilizing 1,4-butanediol as a ‘smart cosubstrate'

    NARCIS (Netherlands)

    Kara, S.; Spickermann, D.; Schrittwieser, J.H.; Leggewie, C.; Berkel, van W.J.H.; Arends, I.W.C.E.; Hollmann, F.

    2013-01-01

    1,4-Butanediol is shown to be an efficient cosubstrate to promote NAD(P)H-dependent redox biocatalysis. The thermodynamically and kinetically inert lactone coproduct makes the regeneration reaction irreversible. Thereby not only the molar surplus of cosubstrate is dramatically reduced but also

  5. 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

  6. 8th International Congress on Biocatalysis (Biocat2016), Hamburg, Germany, 28 August - 1 September, 2016 : meeting report

    OpenAIRE

    Elleuche, Skander; Esselun, Petra; Grote, Ralf; Klippel, Barbara; Krüger, Anna; Liese, Andreas; Ostrop, Margit; Schäfers, Christian; Schröder, Carola; Antranikian, Garabed

    2016-01-01

    The “8th International Congress on Biocatalysis (biocat2016)” is part of a biennial conference series. Biocatalysis is a topic based on the edge of biology and chemistry, which brings together scientists from the life sciences, engineers and computer scientists. This international conference serves as a platform to meet researchers from all over the world, to find collaboration partners for future projects and to gain novel insights into modern topics and techniques. Biocat covers the most...

  7. Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale.

    Science.gov (United States)

    Wachtmeister, Jochen; Rother, Dörte

    2016-12-01

    Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Applications of biocatalysis in fragrance chemistry: the enantiomers of alpha-, beta-, and gamma-irones.

    Science.gov (United States)

    Brenna, Elisabetta; Fuganti, Claudio; Serra, Stefano

    2008-11-01

    The history of iris extracts, and of the isolation and enzyme-mediated synthesis of their odoriferous principle, the "irones", will be used to describe the improvement brought about by chemistry and biocatalysis in the development of natural fragrances. In particular, this tutorial review will discuss how the progress in the field of enzyme chemistry allowed the optimisation of accelerated procedures for the preparation of natural irone extracts, and the synthesis of all the ten isomers of irone, starting from commercial irone alpha.

  9. 8th International Congress on Biocatalysis (Biocat2016, Hamburg, Germany, 28 August–1 September, 2016

    Directory of Open Access Journals (Sweden)

    Skander Elleuche

    2016-10-01

    Full Text Available The “8th International Congress on Biocatalysis (biocat2016” is part of a biennial conference series. Biocatalysis is a topic based on the edge of biology and chemistry, which brings together scientists from the life sciences, engineers and computer scientists. This international conference serves as a platform to meet researchers from all over the world, to find collaboration partners for future projects and to gain novel insights into modern topics and techniques. Biocat covers the most exciting aspects and the latest developments in biocatalysis, including enzyme discovery, evolution and application, bioprocess engineering, cascade reaction systems and nanobiotechnology. In 2016, we welcomed 367 expert delegates in the respective fields. Established and young scientists from academia and the industry presented 51 lectures, 37 lightning talks and 234 posters. In addition, the biocat award, which is among the most prestigious awards in the field of biotechnology, has been awarded for the sixth time in the categories “Science in academia”, “Lifetime achievement” and “Industry”.

  10. Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone.

    Science.gov (United States)

    Rich, Joseph O; Budde, Cheryl L; McConeghey, Luke D; Cotterill, Ian C; Mozhaev, Vadim V; Singh, Sheo B; Goetz, Michael A; Zhao, Annie; Michels, Peter C; Khmelnitsky, Yuri L

    2009-06-01

    Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.

  11. On the (ungreenness of Biocatalysis: Some challenging figures and some promising options

    Directory of Open Access Journals (Sweden)

    Pablo eDomínguez de María

    2015-11-01

    Full Text Available Biocatalysis is generally regarded as a ‘green’ technology. This statement is justified by the mild reaction conditions, the use of aqueous reaction media – with water as the paradigm of green solvents –, and the renewable nature of the biocatalysts. However, researchers making these statements frequently do not take into account the entire picture of their processes. Aspects like water consumption, wastewater production, titers and metrics of the (diluted? biocatalytic processes are important as well. With those figures at hand, many biocatalytic reactions do not appear so green anymore. This article critically discusses some common wrong assumptions given for biocatalytic approaches, with regard to their environmental impact and actual greenness. Some promising biocatalytic approaches, such as the use of biphasic systems involving biogenic solvents, deep-eutectic-solvents (and biogenic ionic liquids, water-free media, solvent-free processes, are briefly introduced, showing that enzyme catalysis can actually be a robust sustainable alternative for chemical processes.

  12. Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts.

    Science.gov (United States)

    Prier, Christopher K; Arnold, Frances H

    2015-11-11

    Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.

  13. Expression of a Dianthus flavonoid glucosyltransferase in Saccharomyces cerevisiae for whole-cell biocatalysis.

    Science.gov (United States)

    Werner, Sean R; Morgan, John A

    2009-07-15

    Glycosyltransferases are promising biocatalysts for the synthesis of small molecule glycosides. In this study, Saccharomyces cerevisiae expressing a flavonoid glucosyltransferase (GT) from Dianthus caryophyllus (carnation) was investigated as a whole-cell biocatalyst. Two yeast expression systems were compared using the flavonoid naringenin as a model substrate. Under in vitro conditions, naringenin-7-O-glucoside was formed and a higher specific glucosyl transfer activity was found using a galactose inducible expression system compared to a constitutive expression system. However, S. cerevisiae expressing the GT constitutively was significantly more productive than the galactose inducible system under in vivo conditions. Interestingly, the glycosides were recovered directly from the culture broth and did not accumulate intracellularly. A previously uncharacterized naringenin glycoside formed using the D. caryophyllus GT was identified as naringenin-4'-O-glucoside. It was found that S. cerevisiae cells hydrolyze naringenin-7-O-glucoside during whole-cell biocatalysis, resulting in a low final glycoside titer. When phloretin was added as a substrate to the yeast strain expressing the GT constitutively, the natural product phlorizin was formed. This study demonstrates S. cerevisiae is a promising whole-cell biocatalyst host for the production of valuable glycosides.

  14. The Protagonism of Biocatalysis in Green Chemistry and Its Environmental Benefits

    Directory of Open Access Journals (Sweden)

    Viridiana Santana Ferreira-Leitão

    2017-01-01

    Full Text Available The establishment of a bioeconomy era requires not only a change of production pattern, but also a deep modernization of the production processes through the implementation of novel methodologies in current industrial units, where waste materials and byproducts can be utilized as starting materials in the production of commodities such as biofuels and other high added value chemicals. The utilization of renewable raw resources and residues from the agro-industries, and their exploitation through various uses and applications through technologies, particularly solid-state fermentation (SSF, are the main focus of this review. The advocacy for biocatalysis in green chemistry and the environmental benefits of bioproduction are very clear, although this kind of industrial process is still an exception and not the rule. Potential and industrial products, such as biocatalysts, animal feed, fermentation medium, biofuels (biodiesel, lignocelulose ethanol, CH4, and H2, pharmaceuticals and chemicals are dealt with in this paper. The focus is the utilization of renewable resources and the important role of enzymatic process to support a sustainable green chemical industry.

  15. Thermophilic enzymes and their applications in biocatalysis: a robust aldo-keto reductase.

    Science.gov (United States)

    Willies, Simon; Isupov, Misha; Littlechild, Jennifer

    2010-09-01

    Extremophiles are providing a good source of novel robust enzymes for use in biocatalysis for the synthesis of new drugs. This is particularly true for the enzymes from thermophilic organisms which are more robust than their mesophilic counterparts to the conditions required for industrial bio-processes. This paper describes a new aldo-keto reductase enzyme from a thermophilic eubacteria, Thermotoga maritima which can be used for the production of primary alcohols. The enzyme has been cloned and over-expressed in Escherichia coli and has been purified and subjected to full biochemical characterization. The aldo-keto reductase can be used for production of primary alcohols using substrates including benzaldehyde, 1,2,3,6-tetrahydrobenzaldehyde and para-anisaldehyde. It is stable up to 80 degrees C, retaining over 60% activity for 5 hours at this temperature. The enzyme at pH 6.5 showed a preference for the forward, carbonyl reduction. The enzyme showed moderate stability with organic solvents, and retained 70% activity in 20% (v/v) isopropanol or DMSO. These properties are favourable for its potential industrial applications.

  16. Identification and use of an alkane transporter plug-in for application in biocatalysis and whole-cell biosensing of alkanes

    DEFF Research Database (Denmark)

    Grant, Chris; Deszcz, Dawid; Wei, Yu-Chia

    2014-01-01

    Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially rele...

  17. Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises.

    Science.gov (United States)

    Gong, Jin-Song; Shi, Jin-Song; Lu, Zhen-Ming; Li, Heng; Zhou, Zhe-Min; Xu, Zheng-Hong

    2017-02-01

    Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.

  18. Polyethylenimine-mediated synthetic insertion of gold nanoparticles into mesoporous silica nanoparticles for drug loading and biocatalysis.

    Science.gov (United States)

    Pandey, Prem C; Pandey, Govind; Narayan, Roger J

    2017-03-27

    Mesoporous silica nanoparticles (MSNPs) have been used as an efficient and safe carrier for drug delivery and biocatalysis. The surface modification of MSNPs using suitable reagents may provide a robust framework in which two or more components can be incorporated to give multifunctional capabilities (e.g., synthesis of noble metal nanoparticles within mesoporous architecture along with loading of a bioactive molecule). In this study, the authors reported on a new synthetic route for the synthesis of gold nanoparticles (AuNPs) within (1) unmodified MSNPs and (2) 3-trihydroxysilylpropyl methylphosphonate-modified MSNPs. A cationic polymer, polyethylenimine (PEI), and formaldehyde were used to mediate synthetic incorporation of AuNPs within MSNPs. The AuNPs incorporated within the mesoporous matrix were characterized by transmission electron microscopy, energy dispersive x-ray analysis, and high-resolution scanning electron microscopy. PEI in the presence of formaldehyde enabled synthetic incorporation of AuNPs in both unmodified and modified MSNPs. The use of unmodified MSNPs was associated with an increase in the polycrystalline structure of the AuNPs within the MSNPs. The AuNPs within modified MSNPs showed better catalytic activity than those within unmodified MSNPs. MSNPs with an average size of 200 nm and with a pore size of 4-6 nm were used for synthetic insertion of AuNPs. It was found that the PEI coating enabled AuNPs synthesis within the mesopores in the presence of formaldehyde or tetrahydrofuran hydroperoxide at a temperature between 10 and 25 °C or at 60 °C in the absence of organic reducing agents. The as-made AuNP-inserted MSNPs exhibited enhanced catalytic activity. For example, these materials enabled rapid catalytic oxidation of the o-dianisidine substrate to produce a colored solution in proportion to the amount of H 2 O 2 generated as a function of glucose oxidase-catalyzed oxidation of glucose; a linear concentration range from 80 to

  19. Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

    Science.gov (United States)

    Adalbjörnsson, Björn V; Toogood, Helen S; Fryszkowska, Anna; Pudney, Christopher R; Jowitt, Thomas A; Leys, David; Scrutton, Nigel S

    2010-01-25

    We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.

  20. ECUT: Energy Conversion and Utilization Technologies program. Industry, university and research interest in the US Department of Energy ECUT biocatalysis research activity

    Science.gov (United States)

    Wilcox, R. E.

    1983-01-01

    The results of a Research Opportunity Notice (RON) disseminated by the Jet Propulsion Laboratory for the U.S. Department of Energy Conversion and Utilization Technologies (ECUT) Program's Biocatalysis Research Activity are presented. The RON was issued in late April of 1983 and solicited expressions of interest from petrochemical and chemical companies, bioengineering firms, biochemical engineering consultants, private research laboratories, and universities for participating in a federal research program to investigate potential applications of biotechnology in producing chemicals. The RON results indicate that broad interest exists within the nation's industry, universities, and research institutes for the Activity and its planned research and development program.

  1. Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization.

    Science.gov (United States)

    Schrewe, Manfred; Julsing, Mattijs K; Lange, Kerstin; Czarnotta, Eik; Schmid, Andreas; Bühler, Bruno

    2014-09-01

    The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step. In two-liquid phase biotransformations, the product formation pattern was found to be controlled by DAME availability. Supplying DAME as bulk organic phase led to accumulation of the terminal alcohol as the predominant product. Limiting DAME availability via application of bis(2-ethylhexyl)phthalate (BEHP) as organic carrier solvent enabled almost exclusive acid accumulation. Furthermore, utilization of BEHP enhanced catalyst stability by reducing toxic effects of substrate and products. A further shift towards the overoxidized products was achieved by co-expression of the gene encoding the alcohol dehydrogenase AlkJ, which was shown to catalyze efficient and irreversible alcohol to aldehyde oxidation in vivo. With DAME as organic phase, the aldehyde accumulated as main product using resting cells containing AlkBGT, AlkL, as well as AlkJ. This study highlights the versatility of whole-cell biocatalysis for synthesis of industrially relevant bifunctional building blocks and demonstrates how integrated reaction and catalyst engineering can be implemented to control product formation patterns in biocatalytic multistep reactions. © 2014 Wiley Periodicals, Inc.

  2. Enzymatic reduction of 4-(dimethylamino)benzaldehyde with carrot bits (Daucus carota): a simple experiment for understanding biocatalysis; Reducao enzimatica do 4-(dimetilamino)benzaldeido com pedacos de cenoura (Daucus carota): um experimento simples na compreensao da biocatalise

    Energy Technology Data Exchange (ETDEWEB)

    Omori, Alvaro Takeo; Portas, Viviane Barbosa; Oliveira, Camila de Souza de, E-mail: alvaro.omori@ufabc.edu.br [Centro de Ciencias Naturais e Humanas, Universidade Federal do ABC, SP (Brazil)

    2012-07-01

    The present paper describes a simple, low-costly and environmentally friendly procedure for reduction of 4-(dimethylamino)benzaldehyde using carrot bits in water. This interdisciplinary experiment can be used to introduce the concepts of biocatalysis and green chemistry to undergraduate students. (author)

  3. Miniaturization in Biocatalysis

    Directory of Open Access Journals (Sweden)

    Pedro Fernandes

    2010-03-01

    Full Text Available The use of biocatalysts for the production of both consumer goods and building blocks for chemical synthesis is consistently gaining relevance. A significant contribution for recent advances towards further implementation of enzymes and whole cells is related to the developments in miniature reactor technology and insights into flow behavior. Due to the high level of parallelization and reduced requirements of chemicals, intensive screening of biocatalysts and process variables has become more feasible and reproducibility of the bioconversion processes has been substantially improved. The present work aims to provide an overview of the applications of miniaturized reactors in bioconversion processes, considering multi-well plates and microfluidic devices, update information on the engineering characterization of the hardware used, and present perspective developments in this area of research.

  4. Generic DART-MS platform for monitoring the on-demand continuous-flow production of pharmaceuticals: Advancing the quantitative protocol for caffeates in microfluidic biocatalysis.

    Science.gov (United States)

    Xu, Yan; Zhang, Dong-Yang; Meng, Xiang-Yun; Liu, Xi; Sheng, Sheng; Wu, Guo-Hua; Wang, Jun; Wu, Fu-An

    2017-04-15

    Today, continuous processing is regarded as an effective on-demand production technique of pharmaceuticals. Homemade microreactors packed with immobilized lipase under continuous-flow conditions were first applied to tailor the production of high-value caffeic acid phenethyl ester (CAPE) from methyl caffeate (MC) and 2-phenylethanol (PE) in cyclohexane via transesterification; however, this method is challenging due to the lack of a rapid platform for monitoring caffeates in microfluidic biocatalysis. The reactants were directly analyzed using Direct Analysis in Real Time Mass Spectrometry (DART-MS), and the corresponding ionization parameters were investigated. Special ions produced from MC (parent ion m/z 192.87 and product ion m/z 133.44) and CAPE (parent ion m/z 282.93 and product ion m/z 178.87) were determined using DART-MS 2 in the negative ion mode. The peak areas of the select reaction monitoring (SRM) signals were calculated to develop the standard curves for quantitative analyses of the concentration. Reasonable linear regression equations of MC and CAPE were obtained in the range of 3.125-50.000mg/L, with linear coefficients (R 2 ) of 0.9515 and 0.9973, limits of detection (LOD) of 0.005 and 0.003mg/L, limits of quantification (LOQ) of 0.02 and 0.01mg/L, and recovery ranges of 92.50-97.11% and 90.11-97.60%, respectively. The results using DART-MS 2 were in good agreement with those using conventional High-Performance Liquid Chromatography with a UV detector (HPLC-UV) and were successfully applied to monitor the kinetics constants and mass transfer coefficients in a continuous-flow packed bed microreactor. Thus, the DART-MS 2 method is an efficient tool for analyzing caffeates in microfluidic biocatalysis with limited sample preparation and short operating time. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. [Polyketone Reaction in Biosynthetic Pathways of 2, 3, 5, 4'-Tetrahydroxy Stilhene-2-O-β-D-glucoside in Polygonum multiflorum by Biocatalysis].

    Science.gov (United States)

    Lei, Lei; Xia, Wan-xia; Shao, Li; Zhao, Shu-jin

    2015-10-01

    2, 3, 5, 4'-Tetrahydroxy stilbene-2-O-β-D-glucoside (THSG), the active ingredient of Polygonum multiflorum, its polyketone reaction in the biosynthesis pathways was studied by biocatalysis method. The substrates 4-coumaroyl-CoA and malonyl-CoA were catalyzed in vitro by the crude enzyme extracted from Polygonum multiflorum callus, then the products were verified by HPLC and LC-MS methods. And the crude enzyme was analyzed by ammonium sulfate precipitation method and SDS-PAGE. HPLC chromatogram showed the same retention time of both the product and resveratrol standards; LC-MS spectra showed that the m/z of product was 227, which was consistent with resveratrol standards under the mode of negative ion; Ammonium sulfate (AS) precipitation method showed AS of 40% - 70% had catalytic activity,and 50% - 60% was the optimum; SDS-PAGE showed protein bands were obviously different among different AS concentration between 20% - 80%, the protein band of 42 kDa was found in AS of 50% - 60%, which had the same molecular weight with stilbene synthase. The product of polyketone reaction in the biosynthesis of THSG is resveratrol rather than THSG, so it is speculated that THSG is the conversion product of resveratrol instead of the direct product of the polyketone reaction.

  6. Deacetylation biocatalysis and elicitation by immobilized Penicillium canescens in Astragalus membranaceus hairy root cultures: towards the enhanced and sustainable production of astragaloside IV.

    Science.gov (United States)

    Gai, Qing-Yan; Jiao, Jiao; Luo, Meng; Wang, Wei; Yao, Li-Ping; Fu, Yu-Jie

    2017-03-01

    A novel biotechnology approach by combining deacetylation biocatalysis with elicitation of immobilized Penicillium canescens (IPC) in Astragalus membranaceus hairy root cultures (AMHRCs) was proposed for the elevated production of astragaloside IV (AG IV). The highest AG IV accumulation was achieved in 36-day-old AMHRCs co-cultured with IPC for 60 h, which resulted in the enhanced production of AG IV by 14.59-fold in comparison with that in control (0.193 ± 0.007 mg/g DW). Meanwhile, AG IV precursors were almost transformed to AG IV by IPC deacetylation. Moreover, expression of genes involved in AG IV biosynthetic pathway was significantly up-regulated in response to IPC elicitation. Also, FTIR and SEM showed that cell wall lignification was enhanced following IPC treatment and root surface was likely to be IPC deacetylation site. Overall, dual roles of IPC (biocatalyst and elicitor) offered an effective and sustainable way for the mass production of AG IV in AMHRCs. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  7. Biocatalysis conversion of methanol to methane in an upflow anaerobic sludge blanket (UASB) reactor: Long-term performance and inherent deficiencies.

    Science.gov (United States)

    Lu, Xueqin; Zhen, Guangyin; Chen, Mo; Kubota, Kengo; Li, Yu-You

    2015-12-01

    Long-term performance of methanol biocatalysis conversion in a lab-scale UASB reactor was evaluated. Properties of granules were traced to examine the impact of methanol on granulation. Methanolic wastewater could be stably treated during initial 240d with the highest biogas production rate of 18.6 ± 5.7 L/Ld at OLR 48 g-COD/Ld. However, the reactor subsequently showed severe granule disintegration, inducing granule washout and process upsets. Some steps (e.g. increasing influent Ca(2+) concentration, etc.) were taken to prevent rising dispersion, but no clear improvement was observed. Further characterizations in granules revealed that several biotic/abiotic factors all caused the dispersion: (1) depletion of extracellular polymeric substances (EPS) and imbalance of protein/polysaccharide ratio in EPS; (2) restricted formation of hard core and weak Ca-EPS bridge effect due to insufficient calcium supply; and (3) simplification of species with the methanol acclimation. More efforts are required to solve the technical deficiencies observed in methanolic wastewater treatment. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase.

    Science.gov (United States)

    Thornburg, Chelsea K; Walter, Tyler; Walker, Kevin D

    2017-11-07

    In this study, we demonstrate an enzyme cascade reaction using a benzoate CoA ligase (BadA), a modified nonribosomal peptide synthase (PheAT), a phenylpropanoyltransferase (BAPT), and a benzoyltransferase (NDTNBT) to produce an anticancer paclitaxel analogue and its precursor from the commercially available biosynthetic intermediate baccatin III. BAPT and NDTNBT are acyltransferases on the biosynthetic pathway to the antineoplastic drug paclitaxel in Taxus plants. For this study, we addressed the recalcitrant expression of BAPT by expressing it as a soluble maltose binding protein fusion (MBP-BAPT). Further, the preparative-scale in vitro biocatalysis of phenylisoserinyl CoA using PheAT enabled thorough kinetic analysis of MBP-BAPT, for the first time, with the cosubstrate baccatin III. The turnover rate of MBP-BAPT was calculated for the product N-debenzoylpaclitaxel, a key intermediate to various bioactive paclitaxel analogues. MBP-BAPT also converted, albeit more slowly, 10-deacetylbaccatin III to N-deacyldocetaxel, a precursor of the pharmaceutical docetaxel. With PheAT available to make phenylisoserinyl CoA and kinetic characterization of MBP-BAPT, we used Michaelis-Menten parameters of the four enzymes to adjust catalyst and substrate loads in a 200-μL one-pot reaction. This multienzyme network produced a paclitaxel analogue N-debenzoyl-N-(2-furoyl)paclitaxel (230 ng) that is more cytotoxic than paclitaxel against certain macrophage cell types. Also in this pilot reaction, the versatile N-debenzoylpaclitaxel intermediate was made at an amount 20-fold greater than the N-(2-furoyl) product. This reaction network has great potential for optimization to scale-up production and is attractive in its regioselective O- and N-acylation steps that remove protecting group manipulations used in paclitaxel analogue synthesis.

  9. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Directory of Open Access Journals (Sweden)

    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  10. Thermodynamic Calculations for Systems Biocatalysis

    DEFF Research Database (Denmark)

    Abu, Rohana; Gundersen, Maria T.; Woodley, John M.

    2015-01-01

    the transamination of a pro-chiral ketone into a chiral amine (interesting in many pharmaceutical applications). Here, the products are often less energetically stable than the reactants, meaning that the reaction may be thermodynamically unfavourable. As in nature, such thermodynamically-challenged reactions can...... on the basis of kinetics. However, many of the most interesting non-natural chemical reactions which could potentially be catalysed by enzymes, are thermodynamically unfavourable and are thus limited by the equilibrium position of the reaction. A good example is the enzyme ω-transaminase, which catalyses...... be altered by coupling with other reactions. For instance, in the case of ω-transaminase, such a coupling could be with alanine dehydrogenase. Herein, the aim of this work is to identify thermodynamic bottlenecks within a multi-enzyme process, using group contribution method to calculate the Gibbs free...

  11. Amides in Nature and Biocatalysis

    NARCIS (Netherlands)

    Pitzer, J.; Steiner, K.

    2016-01-01

    Amides are widespread in biologically active compounds with a broad range of applications in biotechnology, agriculture and medicine. Therefore, as alternative to chemical synthesis the biocatalytic amide synthesis is a very interesting field of research. As usual, Nature can serve as guide in the

  12. Amides in Nature and Biocatalysis.

    Science.gov (United States)

    Pitzer, Julia; Steiner, Kerstin

    2016-10-10

    Amides are widespread in biologically active compounds with a broad range of applications in biotechnology, agriculture and medicine. Therefore, as alternative to chemical synthesis the biocatalytic amide synthesis is a very interesting field of research. As usual, Nature can serve as guide in the quest for novel biocatalysts. Several mechanisms for carboxylate activation involving mainly acyl-adenylate, acyl-phosphate or acyl-enzyme intermediates have been discovered, but also completely different pathways to amides are found. In addition to ribosomes, selected enzymes of almost all main enzyme classes are able to synthesize amides. In this review we give an overview about amide synthesis in Nature, as well as biotechnological applications of these enzymes. Moreover, several examples of biocatalytic amide synthesis are given. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Enzyme immobilization and biocatalysis of polysiloxanes

    Science.gov (United States)

    Poojari, Yadagiri

    Lipases have been proven to be versatile and efficient biocatalysts which can be used in a broad variety of esterification, transesterification, and ester hydrolysis reactions. Due to the high chemo-, regio-, and stereo-selectivity and the mild conditions of lipase-catalyzed reactions, the vast potential of these biocatalysts for use in industrial applications has been increasingly recognized. Polysiloxanes (silicones) are well known for their unique physico-chemical properties and can be prepared in the form of fluids, elastomers, gels and resins for a wide variety of applications. However, the enzymatic synthesis of silicone polyesters and copolymers is largely unexplored. In the present investigations, an immobilized Candida antarctica lipase B (CALB) on macroporous acrylic resin beads (Novozym-435 RTM) has been successfully employed as a catalyst to synthesize silicone polyesters and copolymers under mild reaction conditions. The silicone aliphatic polyesters and the poly(dimethylsiloxane)--poly(ethylene glycol) (PDMS-PEG) copolymers were synthesized in the bulk (without using a solvent), while the silicone aromatic polyesters, the silicone aromatic polyamides and the poly(epsilon-caprolactone)--poly(dimethylsiloxane)--poly(epsilon-caprolactone) (PCL-PDMS-PCL) triblock copolymers were synthesized in toluene. The synthesized silicone polyesters and copolymers were characterized by Gel Permeation Chromatography (GPC), Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD). This dissertation also describes a methodology for physical immobilization of the enzyme pepsin from Porcine stomach mucosa in silicone elastomers utilizing condensation-cure room temperature vulcanization (RTV) of silanol-terminated poly(dimethylsiloxane) (PDMS). The activity and the stability of free pepsin and pepsin immobilized in silicone elastomers were studied with respect to pH, temperature, cross-link density, organic solvents and storage time using a hemoglobin assay. A notable finding was that free pepsin had zero activity in neutral buffer solution (pH 7) after incubation for 5 hours, while pepsin immobilized in the silicone elastomers was found to retain more than 70% of its maximum normalized activity. These results demonstrate that cross-linked poly(dimethylsiloxane) (PDMS) is a promising support material for the physical entrapment of hydrolytic enzymes such as pepsin. The Novozym-435 has been widely employed as a biocatalyst for esterification and transesterification of a variety of organic compounds including synthesis of polyesters and polylactones due to its high catalytic-efficiency and high thermal stability in organic media. However, the Novozym-435 was found to have poor mechanical stability and the enzyme was found to leach out from the resin into the organic media. In the present research work, efforts were made to solve the above two problems by chemical immobilization of CALB on surface modified porous silica gel particles. The surface of the porous silica gel particles was silanized using (gamma-Aminopropyl)triethoxysilane and then the CALB was chemically crosslinked onto the surface of the silica gel particles using glutaraldehyde. Although the thermal stability of the CALB immobilized silica gel particles was found to be lower compared to that of Novozym-435. The CALB immobilized silica gel particles showed higher enzymatic activity and higher mechanical stability compared to that of Novozym-435.

  14. Solvent-tolerant bacteria in biocatalysis.

    NARCIS (Netherlands)

    Bont, de J.A.M.

    1998-01-01

    The toxicity of fine chemicals to the producer organism is a problem in several biotechnological production processes. In several instances, an organic phase can be used to extract the toxic product from the aqueous phase during a fermentation. With the discovery of solvent-tolerant bacteria, more

  15. Application of biocatalysis in synthetic chemistry

    CSIR Research Space (South Africa)

    Brady, D

    2006-11-01

    Full Text Available 20 25 30 35 40 45 50 0 1 2 3 4 5 6 Time (h) C o n v e r s i o n ( % ) 10 units 20 Units 30 Units Slide 13 © CSIR 2006 www.csir.co.za Thermostable lipase – ESL 001 73.50 76.62 79.75 82.88 86...

  16. Ferrofluid modified Saccharomyces cerevisiae cells for biocatalysis

    Czech Academy of Sciences Publication Activity Database

    Šafaříková, Miroslava; Maděrová, Zdeňka; Šafařík, Ivo

    2009-01-01

    Roč. 42, - (2009), s. 521-524 ISSN 0963-9969 R&D Projects: GA MPO 2A-1TP1/094; GA MŠk(CZ) OC 157 Institutional research plan: CEZ:AV0Z60870520 Keywords : Saccharomyces cerevisiae * magnetic fluid * hydrogen peroxide Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.414, year: 2009

  17. Computer-Aided Solvent Screening for Biocatalysis

    DEFF Research Database (Denmark)

    Abildskov, Jens; Leeuwen, M.B. van; Boeriu, C.G.

    2013-01-01

    constrained properties related to chemical reaction equilibrium, substrate and product solubility, water solubility, boiling points, toxicity and others. Two examples are provided, covering the screening of solvents for lipase-catalyzed transesterification of octanol and inulin with vinyl laurate....... Esterification of acrylic acid with octanol is also addressed. Solvents are screened and candidates identified, confirming existing experimental results. Although the examples involve lipases, the method is quite general, so there seems to be no preclusion against application to other biocatalysts....

  18. 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.)

  19. 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)

    1997-12-31

    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.)

  20. Towards improved artificial lungs through biocatalysis.

    Science.gov (United States)

    Kaar, Joel L; Oh, Heung-Il; Russell, Alan J; Federspiel, William J

    2007-07-01

    Inefficient CO(2) removal due to limited diffusion represents a significant barrier in the development of artificial lungs and respiratory assist devices, which use hollow fiber membranes (HFMs) as the blood-gas interface and can require large blood-contacting membrane area. To offset the underlying diffusional challenge, "bioactive" HFMs that facilitate CO(2) diffusion were prepared via covalent immobilization of carbonic anhydrase (CA), an enzyme which catalyzes the conversion of bicarbonate in blood to CO(2), onto the surface of plasma-modified conventional HFMs. This study examines the impact of enzyme attachment on the diffusional properties and the rate of CO(2) removal of the bioactive membranes. Plasma deposition of surface reactive hydroxyls, to which CA could be attached, did not change gas permeance of the HFMs or generate membrane defects, as determined by scanning electron microscopy, when low plasma discharge power and short exposure times were employed. Cyanogen bromide activation of the surface hydroxyls and subsequent modification with CA resulted in near monolayer enzyme coverage (88%) on the membrane. The effect of increased plasma discharge power and exposure time on enzyme loading was negligible while gas permeance studies showed enzyme attachment did not impede CO(2) or O(2) diffusion. Furthermore, when employed in a model respiratory assist device, the bioactive membranes improved CO(2) removal rates by as much as 75% from physiological bicarbonate solutions with no enzyme leaching. These results demonstrate the potential of bioactive HFMs with immobilized CA to enhance CO(2) exchange in respiratory devices.

  1. The application of reaction engineering to biocatalysis

    DEFF Research Database (Denmark)

    Ringborg, Rolf Hoffmeyer; Woodley, John

    2016-01-01

    outline the benefits of reaction engineering in this development process, with particular emphasis of reaction kinetics. Future research needs to focus on rapid methods to collect such data at sufficient accuracy that it can be used forthe effective design of new biocatalytic processes....

  2. Oxygen transfer rates and requirements in oxidative biocatalysis

    DEFF Research Database (Denmark)

    Pedersen, Asbjørn Toftgaard; Rehn, Gustav; Woodley, John

    2015-01-01

    Biocatalytic oxidation reactions offer several important benefits such as regio- and stereoselectivity, avoiding the use of toxic metal based catalysts and replacing oxidizing reagents by allowing the use of oxygen. However, the development of biocatalytic oxidation processes is a complex task......-up is relatively straight forward (Gabelman and Hwang, 1999), and membrane contactors are implemented for various industrial applications (Klaassen et al., 2005)....

  3. Biocatalysis with Sol-Gel Encapsulated Acid Phosphatase

    Science.gov (United States)

    Kulkarni, Suhasini; Tran, Vu; Ho, Maggie K.-M.; Phan, Chieu; Chin, Elizabeth; Wemmer, Zeke; Sommerhalter, Monika

    2010-01-01

    This experiment was performed in an upper-level undergraduate biochemistry laboratory course. Students learned how to immobilize an enzyme in a sol-gel matrix and how to perform and evaluate enzyme-activity measurements. The enzyme acid phosphatase (APase) from wheat germ was encapsulated in sol-gel beads that were prepared from the precursor…

  4. Recent developments in biocatalysis in multiphasic ionic liquid reaction systems.

    Science.gov (United States)

    Meyer, Lars-Erik; von Langermann, Jan; Kragl, Udo

    2018-06-01

    Ionic liquids are well known and frequently used 'designer solvents' for biocatalytic reactions. This review highlights recent achievements in the field of multiphasic ionic liquid-based reaction concepts. It covers classical biphasic systems including supported ionic liquid phases, thermo-regulated multi-component solvent systems (TMS) and polymerized ionic liquids. These powerful concepts combine unique reaction conditions with a high potential for future applications on a laboratory and industrial scale. The presence of a multiphasic system simplifies downstream processing due to the distribution of the catalyst and reactants in different phases.

  5. Enzyme-Embedded, Microstructural Reactors for Industrial Biocatalysis

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Sarah E. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Knipe, J. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Oakdale, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Stolaroff, J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-10-04

    In this project we explored enzyme-catalyzed methane conversion to methanol. Industrial biological approaches to methane conversion using whole organisms are predicted to be more energy efficient than chemical approaches, but are limited by mass transfer of the gas phase reactants, methane and oxygen, to the organisms. We demonstrated that 3D printing the enzyme particulate Methane Mono Oxygenase (pMMO) embedded in a polymer can improve the kinetics of methane to methanol conversion. This improvement was likely due to the ability to increase the surface area of the catalytic material using 3D printing. We also demonstrated the first continuous use of pMMO in a flow-through reactor. In order to understand the fundamental kinetic properties of pMMO, we conducted an in-depth study of pMMO kinetics using analytical tools developed in our lab. Finally, we developed a new copolymer system that allowed tuning of the gas permeability of the biocatalytic material.

  6. Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

    Directory of Open Access Journals (Sweden)

    Gong Jin-Song

    2012-10-01

    Full Text Available Abstract Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications.

  7. Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

    Science.gov (United States)

    2012-01-01

    Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications. PMID:23106943

  8. Engineering protein scaffolds for protein separation, biocatalysis and nanotechnology applications

    Science.gov (United States)

    Liu, Fang

    Globally, there is growing appreciation for developing a sustainable economy that uses eco-efficient bio-processes. Biotechnology provides an increasing range of tools for industry to help reduce cost and improve environmental performance. Inspired by the naturally evolved machineries of protein scaffolds and their binding ligands, synthetic protein scaffolds were engineered based on cohesin-dockerin interactions and metal chelating peptides to tackle the challenges and make improvements in three specific areas: (1) protein purification, (2) biofuel cells, and (3) nanomaterial synthesis. The first objective was to develop efficient and cost-effective non-chromatographic purification processes to purify recombinant proteins in an effort to meet the dramatically growing market of protein drugs. In our design, the target protein was genetically fused with a dockerin domain from Clostridium thermocellum and direct purification and recovery was achieved using thermo-responsive elastin-like polypeptide (ELP) scaffold containing the cohesin domain from the same species. By exploiting the highly specific interaction between the dockerin and cohesin domain and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as endoglucanase CelA, chloramphenicol acetyl transferase (CAT) and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single purification step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins by another cycle of thermal precipitation. The purification cost can be further reduced by regenerating and recycling the ELP-cohesin capturing scaffolds. However, due to the high binding affinity between cohesin and dockerin domains, the bound dockerin-intein tag cannot be completely disassociated from ELP-cohesin scaffold after binding. Therefore, a truncated dockerin with the calcium-coordinating function impaired was used in replace of the original full length dockerin domain. The truncated dockerin domain maintained its functionality as an effective affinity tag, and efficient EDTA mediated dissociation of the bound dockerin-intein tag was also realized. The regenerated ELP capturing scaffold was reused for additional purification cycles without any decrease in efficiency. The second objective was to assemble biocatalysts for biofuel cells. Three beta-NAD dependent dehydrogenases, alcohol dehydrogenase (ADH), formaldehyde dehydrogenase (FALDH) and formate dehydrogenase (FDH), were site-specifically co-localized onto the scaffolds displayed on the yeast surface based on the high-affinity interactions between three orthogonal cohesin/dockerin pairs. The assembled multi-enzyme cascades, which can completely convert methanol to CO2, showed improved production yield compared with that of the non-complexed enzyme mixture, indicating efficient substrate channeling among the three enzymes. This strategy can be easily extended to other complex cascade reactions for enzymatic fuel cell applications. To further explore the role of biotechnology toward environmental sustainability, Escherichia coli was engineered to express phytochelatin synthase, which converted glutathione into the metal-binding peptide phytochelatin (PC). PCs served as peptide scaffolds and mediated synthesis of CdS nanocrystals. This approach may be generalized to guide the in vitro self-assembly of a wide range of nanocrystals with different compositions and sizes.

  9. Continuous Membrane-Based Screening System for Biocatalysis

    Directory of Open Access Journals (Sweden)

    Matthias Kraume

    2011-02-01

    Full Text Available The use of membrane reactors for enzymatic and co-factor regenerating reactions offers versatile advantages such as higher conversion rates and space-time-yields and is therefore often applied in industry. However, currently available screening and kinetics characterization systems are based on batch and fed-batch operated reactors and were developed for whole cell biotransformations rather than for enzymatic catalysis. Therefore, the data obtained from such systems has only limited transferability for continuous membrane reactors. The aim of this study is to evaluate and to improve a novel screening and characterization system based on the membrane reactor concept using the enzymatic hydrolysis of cellulose as a model reaction. Important aspects for the applicability of the developed system such as long-term stability and reproducibility of continuous experiments were very high. The concept used for flow control and fouling suppression allowed control of the residence time with a high degree of precision (±1% accuracy in a long-term study (>100 h.

  10. Biocatalysis of azidolysis of epoxides: Computational evidences on ...

    Indian Academy of Sciences (India)

    Active site model and crystal structure data reveal that the Tyr145 and Ser132 form weak hydrogen bonds with ... been computed using PCM model with water as solvent. (ε = 78.39). ... of p-nitro styrene oxide in HheC pocket (PDB ID: 1ZMT).

  11. Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis.

    Science.gov (United States)

    Kell, Douglas B; Swainston, Neil; Pir, Pınar; Oliver, Stephen G

    2015-04-01

    Because they mainly do not involve chemical changes, membrane transporters have been a Cinderella subject in the biotechnology of small molecule production, but this is a serious oversight. Influx transporters contribute significantly to the flux towards product, and efflux transporters ensure the accumulation of product in the much greater extracellular space of fermentors. Programmes for improving biotechnological processes might therefore give greater consideration to transporters than may have been commonplace. Strategies for identifying important transporters include expression profiling, genome-wide knockout studies, stress-based selection, and the use of inhibitors. In addition, modern methods of directed evolution and synthetic biology, especially those effecting changes in energy coupling, offer huge opportunities for increasing the flux towards extracellular product formation by transporter engineering. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Engineering Non-Heme Mono- and Dioxygenases for Biocatalysis

    Directory of Open Access Journals (Sweden)

    Adi Dror

    2012-09-01

    Full Text Available Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.

  13. Biocatalysis and bacterial cells; problems and prospects for biohydrogen

    International Nuclear Information System (INIS)

    Hallenbeck, P.C.; Viger, J.-F.

    2000-01-01

    Biological hydrogen production, studied in a number of model and small-scale systems over the last twenty-five years, has been advanced as a potential technology for producing clean energy. However, the rates and efficiencies of hydrogen production by any system fall far short of economic feasibility at present. A number of different systems have been proposed and studied including direct biophotolytic processes and two stage systems. Direct biophotolytic processes, though inherently attractive, suffer from the perhaps insurmountable barriers of the oxygen sensitivity of the enzymes involved and intrinsic limitations in light conversion efficiencies. Fermentative processes, using either biomass obtained in a first stage light conversion process or perhaps more attractively, various waste streams, present an interesting yet largely unexplored avenue for the biological production of hydrogen. Much is presently known about the molecular biology and biochemistry of the hydrogen producing enzymes, reductant generating systems, and physiology of many hydrogen producing organisms. The potential of metabolic engineering for redirecting electron flux to hydrogen production is discussed. Some of the relevant details of hydrogen evolving systems are reviewed in the hopes of identifying potentially limiting factors and therefore indicating directions for future research aimed at increasing production rates and conversion efficiencies to economically feasible levels. (author)

  14. The role of biocatalysis in the asymmetric synthesis of alkaloids

    NARCIS (Netherlands)

    Schrittwieser, J.H.; Resch, V.

    2013-01-01

    Alkaloids are not only one of the most intensively studied classes of natural products, their wide spectrum of pharmacological activities also makes them indispensable drug ingredients in both traditional and modern medicine. Among the methods for their production, biotechnological approaches are

  15. Characterization of a continuous agitated cell reactor for oxygen dependent biocatalysis

    DEFF Research Database (Denmark)

    Pedersen, Asbjørn Toftgaard; Teresa de Melo Machado Simoes Carvalho, Ana; Sutherland, Euan

    2017-01-01

    Biocatalytic oxidation reactions employing molecular oxygen as the electron acceptor are difficult to conduct in a continuous flow reactor because of the requirement for high oxygen transfer rates. In this paper, the oxidation of glucose to glucono-1,5-lactone by glucose oxidase was used as a model...

  16. The Protagonism of Biocatalysis in Green Chemistry and Its Environmental Benefits

    OpenAIRE

    Viridiana Santana Ferreira-Leitão; Magali Christe Cammarota; Erika Cristina Gonçalves Aguieiras; Lívian Ribeiro Vasconcelos de Sá; Roberto Fernandez-Lafuente; Denise Maria Guimarães Freire

    2017-01-01

    The establishment of a bioeconomy era requires not only a change of production pattern, but also a deep modernization of the production processes through the implementation of novel methodologies in current industrial units, where waste materials and byproducts can be utilized as starting materials in the production of commodities such as biofuels and other high added value chemicals. The utilization of renewable raw resources and residues from the agro-industries, and their exploitation thro...

  17. Characterization of a continuous agitated cell reactor for oxygen dependent biocatalysis.

    Science.gov (United States)

    Toftgaard Pedersen, Asbjørn; de Carvalho, Teresa Melo; Sutherland, Euan; Rehn, Gustav; Ashe, Robert; Woodley, John M

    2017-06-01

    Biocatalytic oxidation reactions employing molecular oxygen as the electron acceptor are difficult to conduct in a continuous flow reactor because of the requirement for high oxygen transfer rates. In this paper, the oxidation of glucose to glucono-1,5-lactone by glucose oxidase was used as a model reaction to study a novel continuous agitated cell reactor (ACR). The ACR consists of ten cells interconnected by small channels. An agitator is placed in each cell, which mixes the content of the cell when the reactor body is shaken by lateral movement. Based on tracer experiments, a hydrodynamic model for the ACR was developed. The model consisted of ten tanks-in-series with back-mixing occurring within and between each cell. The back-mixing was a necessary addition to the model in order to explain the observed phenomenon that the ACR behaved as two continuous stirred tank reactors (CSTRs) at low flow rates, while it at high flow rates behaved as the expected ten CSTRs in series. The performance of the ACR was evaluated by comparing the steady state conversion at varying residence times with the conversion observed in a stirred batch reactor of comparable size. It was found that the ACR could more than double the overall reaction rate, which was solely due to an increased oxygen transfer rate in the ACR caused by the intense mixing as a result of the spring agitators. The volumetric oxygen transfer coefficient, k L a, was estimated to be 344 h -1 in the 100 mL ACR, opposed to only 104 h -1 in a batch reactor of comparable working volume. Interestingly, the large deviation from plug flow behavior seen in the tracer experiments was found to have little influence on the conversion in the ACR, since both a plug flow reactor (PFR) model and the backflow cell model described the data sufficiently well. Biotechnol. Bioeng. 2017;114: 1222-1230. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  18. μ-structured devices as tools for screening process intensification in biocatalysis

    DEFF Research Database (Denmark)

    Bodla, Vijaya Krishna; Woodley, John

    different modules can be developed at microscale. Such configurations enable effective screening and rapid process development of biocatalytic reactions assuring economic viability and shorter time to market for pharmaceutical products. Thus the work presented in this thesis is based on the application......Biocatalytic processes have been emerging as potential replacements of traditional chemical synthesis in many industrial relevant production processes. However the implementation of new biocatalytic processes can be a very challenging procedure which requires both biocatalyst and process screening....... Such knowledge is subsequently applied to design customized reactor configurations. It has been demonstrated that this knowledge can be crucial for the choice and design of reactors. The second part focuses on developing μ-scale modules for rapid screening and integrating process units. The increase...

  19. Application of mechanistic models to fermentation and biocatalysis for next-generation processes

    DEFF Research Database (Denmark)

    Gernaey, Krist; Eliasson Lantz, Anna; Tufvesson, Pär

    2010-01-01

    of variables required for measurement, control and process design. In the near future, mechanistic models with a higher degree of detail will play key roles in the development of efficient next-generation fermentation and biocatalytic processes. Moreover, mechanistic models will be used increasingly......Mechanistic models are based on deterministic principles, and recently, interest in them has grown substantially. Herein we present an overview of mechanistic models and their applications in biotechnology, including future perspectives. Model utility is highlighted with respect to selection...

  20. BIOSYNTHESIS, CHARACTERIZATION AND APPLICATION OF TIO2 NANOPARTICLES IN BIOCATALYSIS AND PROTEIN FOLDING

    Directory of Open Access Journals (Sweden)

    Razi Ahmad,

    2013-08-01

    Full Text Available The nano-TiO2 was synthesized using Lactobacillus sp. and characterized by XRD and TEM. The X-ray diffraction showed that TiO2 nanoparticles were crystalline in nature. TEM images revealed that these particles are irregular in shape with an average particle size of 50–100 nm. The biosynthesized nanoparticles were used for the immobilization and refolding of thermally inactivated alpha amylase enzyme. The enzyme after adsorption on TiO2 nanoparticles retained 71% of enzyme activity. The immobilized enzyme was found to be thermally more stable as compared to the free enzyme. When the enzyme was heated to 60°C for 60 min the free enzyme loses all of its activity whereas the adsorbed enzyme retained 82% of its activity.The adsorbed/immobilized protein could be reused five times without any loss in enzyme activity. The operational stability data also shows that after immobilization the stability of alpha amylase increases. To study the nanoparticles-protein interaction, alpha amylase enzyme was inactivated by heating at 60°C for 1 hour. The thermally inactivated alpha amylase when incubated with the biosynthesized TiO2 nanoparticles regains nearly 65% activity after 2.0 hour. Thus TiO2 nanoparticles assist in refolding of the enzyme.

  1. A microfluidic toolbox for the development of in-situ product removal strategies in biocatalysis

    DEFF Research Database (Denmark)

    Heintz, Søren; Mitic, Aleksandar; Ringborg, Rolf Hoffmeyer

    2016-01-01

    A microfluidic toolbox for accelerated development of biocatalytic processes has great potential. This is especially the case for the development of advanced biocatalytic process concepts, where reactors and product separation methods are closely linked together to intensify the process performan...

  2. Biocatalysis for the application of CO2 as a chemical feedstock

    Directory of Open Access Journals (Sweden)

    Apostolos Alissandratos

    2015-12-01

    Full Text Available Biocatalysts, capable of efficiently transforming CO2 into other more reduced forms of carbon, offer sustainable alternatives to current oxidative technologies that rely on diminishing natural fossil-fuel deposits. Enzymes that catalyse CO2 fixation steps in carbon assimilation pathways are promising catalysts for the sustainable transformation of this safe and renewable feedstock into central metabolites. These may be further converted into a wide range of fuels and commodity chemicals, through the multitude of known enzymatic reactions. The required reducing equivalents for the net carbon reductions may be drawn from solar energy, electricity or chemical oxidation, and delivered in vitro or through cellular mechanisms, while enzyme catalysis lowers the activation barriers of the CO2 transformations to make them more energy efficient. The development of technologies that treat CO2-transforming enzymes and other cellular components as modules that may be assembled into synthetic reaction circuits will facilitate the use of CO2 as a renewable chemical feedstock, greatly enabling a sustainable carbon bio-economy.

  3. Exploiting Catalytic Promiscuity for Biocatalysis : Carbon-Carbon Bond Formation by a Proline-Based Tautomerase

    NARCIS (Netherlands)

    Miao, Yufeng

    2015-01-01

    Een belangrijk thema bij het ontwikkelen van nieuwe biokatalysatoren is katalytische promiscuïteit, waarbij een enzym alternatieve reacties katalyseert naast de reactie die biologisch relevant is. Promiscue enzymactiviteiten zijn een veelbelovende bron van synthetisch bruikbare katalytische

  4. Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase.

    Science.gov (United States)

    Munawar, Nayla; Engel, Paul C

    2013-01-01

    Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting L-methionine, L-norleucine and L-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.

  5. COLLABORATIVE RESEARCH AND DEVELOPMENT CONTRACT. Delivery Order 0038: Microbial Biotechnology and Biocatalysis

    Science.gov (United States)

    2010-04-01

    Insights into Bacillus Spore Coat Assembly. Eby, D.M., and Johnson, G.R. Air Force Research Laboratory Materials Directorate, Biotechnology Group...integration with the HVAC intake to building. The sensitivity and detection limits demonstrate the potential f the system as a proof of concept, but

  6. Nitrile-converting enzymes: an eco-friendly tool for industrial biocatalysis.

    Science.gov (United States)

    Ramteke, Pramod W; Maurice, Navodita G; Joseph, Babu; Wadher, Bharat J

    2013-01-01

    Nitriles are organic compounds bearing a − C ≡ N group; they are frequently known to occur naturally in both fauna and flora and are also synthesized chemically. They have wide applicability in the fields of medicine, industry, and environmental monitoring. However, the majority of nitrile compounds are considered to be lethal, mutagenic, and carcinogenic in nature and are known to cause potential health problems such as nausea, bronchial irritation, respiratory distress, convulsions, coma, and skeletal deformities in humans. Nitrile-converting enzymes, which are extracted from microorganisms, are commonly termed nitrilases and have drawn the attention of researchers all over the world to combat the toxicity of nitrile compounds. The present review focuses on the utility of nitrile-converting enzymes, sources, classification, structure, properties, and applications, as well as the future perspective on nitrilases. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  7. Cellulase assisted synthesis of nano-silver and gold: Application as immobilization matrix for biocatalysis.

    Science.gov (United States)

    Mishra, Abhijeet; Sardar, Meryam

    2015-01-01

    In the present study, we report in vitro synthesis of silver and gold nanoparticles (NPs) using cellulase enzyme in a single step reaction. Synthesized nanoparticles were characterized by UV-VIS spectroscopy, Dynamic Light Spectroscopy (DLS), Transmission Electron Microscopy (TEM), Energy-dispersive X-ray Spectroscopy (EDX), X-ray Diffraction (XRD), Circular Dichroism (CD) and Fourier Transform Infrared Spectroscopy (FTIR). UV-visible studies shows absorption band at 415nm and 520nm for silver and gold NPs respectively due to surface plasmon resonance. Sizes of NPs as shown by TEM are 5-25nm for silver and 5-20nm for gold. XRD peaks confirmed about phase purity and crystallinity of silver and gold NPs. FTIR data shows presence of amide I peak on both the NPs. The cellulase assisted synthesized NPs were further exploited as immobilization matrix for cellulase enzyme. Thermal stability analysis reveals that the immobilized cellulase on synthesized NPs retained 77-80% activity as compared to free enzyme. While reusability data suggests immobilized cellulase can be efficiently used up to sixth cycles with minimum loss of enzyme activity. The secondary structural analysis of cellulase enzyme during the synthesis of NPs and also after immobilization of cellulase on these NPs was carried out by CD spectroscopy. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism

    DEFF Research Database (Denmark)

    Nikel, Pablo I.; de Lorenzo, Víctor

    2018-01-01

    are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed...... biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial...... stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally...

  9. Alpha chymotrypsin coated clusters of Fe3O4 nanoparticles for biocatalysis in low water media

    Directory of Open Access Journals (Sweden)

    Mukherjee Joyeeta

    2012-11-01

    Full Text Available Abstract Background Enzymes in low water containing non aqueous media are useful for organic synthesis. For example, hydrolases in such media can be used for synthetic purposes. Initial work in this area was carried out with lyophilized powders of enzymes. These were found to have poor activity. Drying (removing bulk water by precipitation turned out to be a better approach. As enzymes in such media are heterogeneous catalysts, spreading these precipitates over a large surface gave even better results. In this context, nanoparticles with their better surface to volume ratio provide obvious advantage. Magnetic nanoparticles have an added advantage of easy separation after the reaction. Keeping this in view, alpha chymotrypsin solution in water was precipitated over a stirred population of Fe3O4 nanoparticles in n-propanol. This led to alpha chymotrypsin activity coated over clusters of Fe3O4 nanoparticles. These preparations were found to have quite high transesterification activity in low water containing n-octane. Results Precipitation of alpha chymotrypsin over a stirred suspension of Fe3O4 nanoparticles (3.6 nm diameter led to the formation of enzyme coated clusters of nanoparticles (ECCNs. These clusters were also magnetic and their hydrodynamic diameter ranged from 1.2- 2.6 microns (as measured by dynamic light scattering. Transmission electron microscopy (TEM, showed that these clusters had highly irregular shapes. Transesterification assay of various clusters in anhydrous n-octane led to optimization of concentration of nanoparticles in suspension during precipitation. Optimized design of enzyme coated magnetic clusters of nanoparticles (ECCN 3 showed the highest initial rate of 465 nmol min-1 mg-1protein which was about 9 times higher as compared to the simple precipitates with an initial rate of 52 nmol min-1 mg-1 protein. Circular Dichroism (CD(with a spinning cell accessory showed that secondary structure content of the alpha Chymotrypsin in ECCN 3 [15% α-helix, 37% β-sheet and 48% random coil] was identical to the simple precipitates of alpha chymotrypsin. Conclusion A strategy for obtaining a high activity preparation of alpha chymotrypsin for application in low water media is described. Such high activity biocatalysts are useful in organic synthesis.

  10. Challenges and prospects of xylitol production with whole cell bio-catalysis: A review.

    Science.gov (United States)

    Dasgupta, Diptarka; Bandhu, Sheetal; Adhikari, Dilip K; Ghosh, Debashish

    2017-04-01

    Xylitol, as an alternative low calorie sweetener is well accepted in formulations of various confectioneries and healthcare products. Worldwide it is industrially produced by catalytic hydrogenation of pure d-xylose solution under high temperature and pressure. Biotechnological xylitol production is a potentially attractive replacement for chemical process, as it occurs under much milder process conditions and can be based on sugar mixtures derived from low-cost industrial and agri-waste. However, microbial fermentation route of xylitol production is not so far practiced industrially. This review highlights the challenges and prospects of biotechnological xylitol production considering possible genetic modifications of fermenting microorganisms and various aspects of industrial bioprocessing and product downstreaming. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. Asymmetric synthesis of synthetic alkaloids by a tandem biocatalysis/Ugi/Pictet-Spengler-type

    NARCIS (Netherlands)

    Znabet, A.; Zonneveld, J.; Janssen, E.; de Kanter, F.J.J.; Helliwell, M.; Turner, N.J.; Ruijter, E.; Orru, R.V.A.

    2010-01-01

    We have combined the biocatalytic desymmetrization of 3,4-cis-substituted meso-pyrrolidines with an Ugi-type multicomponent reaction followed in situ by a Pictet-Spengler-type cyclization reaction sequence for the rapid asymmetric synthesis of alkaloid-like polycyclic compounds. © The Royal Society

  12. Molecular imprinting of enzymes with water-insoluble ligands for nonaqueous biocatalysis.

    Science.gov (United States)

    Rich, Joseph O; Mozhaev, Vadim V; Dordick, Jonathan S; Clark, Douglas S; Khmelnitsky, Yuri L

    2002-05-15

    Attaining higher levels of catalytic activity of enzymes in organic solvents is one of the major challenges in nonaqueous enzymology. One of the most successful strategies for enhancing enzyme activity in organic solvents involves tuning the enzyme active site by molecular imprinting with substrates or their analogues. Unfortunately, numerous imprinters of potential importance are poorly soluble in water, which significantly limits the utility of this method. In the present study, we have developed strategies that overcome this limitation of the molecular-imprinting technique and that thus expand its applicability beyond water-soluble ligands. The solubility problem can be addressed either by converting the ligands into a water-soluble form or by adding relatively high concentrations of organic cosolvents, such as tert-butyl alcohol and 1,4-dioxane, to increase their solubility in the lyophilization medium. We have succeeded in applying both of these strategies to produce imprinted thermolysin, subtilisin, and lipase TL possessing up to 26-fold higher catalytic activity in the acylation of paclitaxel and 17beta-estradiol compared to nonimprinted enzymes. Furthermore, we have demonstrated for the first time that molecular imprinting and salt activation, applied in combination, produce a strong additive activation effect (up to 110-fold), suggesting different mechanisms of action involved in these enzyme activation techniques.

  13. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism.

    Science.gov (United States)

    Nikel, Pablo I; de Lorenzo, Víctor

    2018-05-16

    The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochemical stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biological systems. Much of the biotechnological interest on P. putida stems from the distinct architecture of its central carbon metabolism. The native biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metabolism) for making complex, new-to-Nature molecules. A number of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of novel chemical elements into the extant biochemistry, as well as genomic stability and scaling-up issues. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Whole-Cell Biocatalysis for Producing Ginsenoside Rd from Rb1 Using Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Ku, Seockmo; You, Hyun Ju; Park, Myeong Soo; Ji, Geun Eog

    2016-07-28

    Ginsenosides are the major active ingredients in ginseng used for human therapeutic plant medicines. One of the most well-known probiotic bacteria among the various strains on the functional food market is Lactobacillus rhamnosus GG. Biocatalytic methods using probiotic enzymes for producing deglycosylated ginsenosides such as Rd have a growing significance in the functional food industry. The addition of 2% cellobiose (w/v) to glucose-free de Man-Rogosa-Sharpe broths notably induced β-glucosidase production from L. rhamnosus GG. Enzyme production and activity were optimized at a pH, temperature, and cellobiose concentration of 6.0, 40°C, and 2% (w/v), respectively. Under these controlled conditions, β-glucosidase production in L. rhamnosus GG was enhanced by 25-fold. Additionally, whole-cell homogenates showed the highest β-glucosidase activity when compared with disrupted cell suspensions; the cell disruption step significantly decreased the β-glucosidase activity. Based on the optimized enzyme conditions, whole-cell L. rhamnosus GG was successfully used to convert ginsenoside Rb1 into Rd.

  15. Introducing an In Situ Capping Strategy in Systems Biocatalysis To Access 6-Aminohexanoic acid

    DEFF Research Database (Denmark)

    Sattler, Johann H.; Fuchs, Michael; Mutti, Francesco G.

    2014-01-01

    The combination of two cofactor self-sufficient biocatalytic cascade modules allowed the successful transformation of cyclohexanol into the nylon-6 monomer 6- aminohexanoic acid at the expense of only oxygen and ammonia. A hitherto unprecedented carboxylic acid capping strategy was introduced to ...

  16. Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations.

    Science.gov (United States)

    Heintz, Søren; Börner, Tim; Ringborg, Rolf H; Rehn, Gustav; Grey, Carl; Nordblad, Mathias; Krühne, Ulrich; Gernaey, Krist V; Adlercreutz, Patrick; Woodley, John M

    2017-03-01

    An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid-liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 g MPPA  · L -1 , a purity up to 70% g MPPA  · g tot -1 and a recovery in the range of 80% mol · mol -1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600-609. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Enzymes in lipid modification: From classical biocatalysis with commercial enzymes to advanced protein engineering tools

    Directory of Open Access Journals (Sweden)

    Bornscheuer Uwe T.

    2013-01-01

    Full Text Available In this review, the application of enzymes, especially lipases, for the modification of fats and oils is covered. This includes the lipase-catalyzed selective production of structured triglycerides and the isolation or incorporation of specific fatty acids. Protein engineering methods to modify lipases on a molecular level were used to alter the fatty acid chain-length and ‘‘trans over cis’’ selectivity of lipase A from Candida antarctica. Furthermore, an enzymatic cascade reaction to remove 3-monochloropropanediol and the identification of a phospholipase C for degumming are briefly covered.

  18. Microreactors and CFD as Tools for Biocatalysis Reactor Design: A case study

    DEFF Research Database (Denmark)

    Bodla, Vijaya Krishna; Seerup, R.; Krühne, Ulrich

    2013-01-01

    Microreactors have been used for acquiring process data while consuming significantly lower amounts of expensive reagents. In this article, the combination of microreactor technology and computational fluid dynamics (CFD) is shown to contribute significantly towards understanding the diffusional ...... with similar dimensions to the ones tested here can be used as a screening tool for screening biocatalyst and process alternatives....

  19. Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

    Energy Technology Data Exchange (ETDEWEB)

    Mark Eiteman

    2007-07-31

    This report describes results toward developing a process to sequester CO{sub 2} centered on the enzymes PEP carboxylase and pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The study reports on strain development and process development. In the area of strain development, knockouts in genes which divert carbon from the enzymatic steps involved in CO{sub 2} consumption were completed, and were shown not to affect significantly the rate of CO{sub 2} sequestration and succinic acid generation. Furthermore, the pyc gene encoding for pyruvate carboxylase proved to be unstable when integrated onto the chromosome. In the area of process development, an optimal medium, pH and base counterion were obtained, leading to a sequestration rate as great as 800 mg/Lh. Detailed studies of gas phase composition demonstrated that CO{sub 2} composition has a significant affect on CO{sub 2} sequestration, while the presence of 'toxic' compounds in the gas, including NO{sub 2}, CO and SO{sub 2} did not have a detrimental effect on sequestration. Some results on prolonging the rate of sequestration indicate that enzyme activities decrease with time, suggesting methods to prolong enzyme activity may benefit the overall process.

  20. Development of in-situ product removal strategies in biocatalysis applying scaled-down unit operations

    DEFF Research Database (Denmark)

    Heintz, Søren; Börner, Tim; Ringborg, Rolf Hoffmeyer

    2017-01-01

    different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit...... operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi...

  1. Merging Iron Catalysis and Biocatalysis-Iron Carbonyl Complexes as Efficient Hydrogen Autotransfer Catalysts in Dynamic Kinetic Resolutions

    KAUST Repository

    El-Sepelgy, Osama

    2016-09-29

    A dual catalytic iron/lipase system has been developed and applied in the dynamic kinetic resolution of benzylic and aliphatic secondary alcohols. A detailed study of the Knölker-type iron complexes demonstrated the hydrogen autotransfer of alcohols to proceed under mild reaction conditions and allowed the combination with the enzymatic resolution. Different racemic alcohols were efficiently converted to chiral acetates in good yields and with excellent enantioselectivities. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

  2. Potencial de biocatálise enantiosseletiva de lipases microbianas Potential of enantioselective biocatalysis by microbial lipases

    Directory of Open Access Journals (Sweden)

    Patrícia de O. Carvalho

    2005-08-01

    Full Text Available Microbial lipases have a great potential for commercial applications due to their stability, selectivity and broad substrate specificity because many non-natural acids, alcohols or amines can be used as the substrate. Three microbial lipases isolated from Brazilian soil samples (Aspergillus niger; Geotrichum candidum; Penicillium solitum were compared in terms of their stability and as biocatalysts in the enantioselective esterification using racemic substrates in organic medium. The lipase from Aspergillus niger showed the highest activity (18.2 U/mL and was highly thermostable, retaining 90% and 60% activity at 50 ºC and 60 ºC after 1 hour, respectively. In organic medium, this lipase provided the best results in terms of enantiomeric excess of the (S-active acid (ee = 6.1% and conversion value (c = 20% in the esterification of (R,S-ibuprofen with 1-propanol in isooctane. The esterification reaction of the racemic mixture of (R,S-2-octanol with decanoic acid proceeded with high enantioselectivity when lipase from Aspergillus niger (E = 13.2 and commercial lipase from Candida antarctica (E = 20 were employed.

  3. Biotechnology and genetic engineering in the new drug development. Part III. Biocatalysis, metabolic engineering and molecular modelling.

    Science.gov (United States)

    Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

    2013-01-01

    Industrial biotechnology has been defined as the use and application of biotechnology for the sustainable processing and production of chemicals, materials and fuels. It makes use of biocatalysts such as microbial communities, whole-cell microorganisms or purified enzymes. In the review these processes are described. Drug design is an iterative process which begins when a chemist identifies a compound that displays an interesting biological profile and ends when both the activity profile and the chemical synthesis of the new chemical entity are optimized. Traditional approaches to drug discovery rely on a stepwise synthesis and screening program for large numbers of compounds to optimize activity profiles. Over the past ten to twenty years, scientists have used computer models of new chemical entities to help define activity profiles, geometries and relativities. This article introduces inter alia the concepts of molecular modelling and contains references for further reading.

  4. Merging Iron Catalysis and Biocatalysis-Iron Carbonyl Complexes as Efficient Hydrogen Autotransfer Catalysts in Dynamic Kinetic Resolutions

    KAUST Repository

    El-Sepelgy, Osama; Alandini, Nurtalya; Rueping, Magnus

    2016-01-01

    A dual catalytic iron/lipase system has been developed and applied in the dynamic kinetic resolution of benzylic and aliphatic secondary alcohols. A detailed study of the Knölker-type iron complexes demonstrated the hydrogen autotransfer of alcohols to proceed under mild reaction conditions and allowed the combination with the enzymatic resolution. Different racemic alcohols were efficiently converted to chiral acetates in good yields and with excellent enantioselectivities. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

  5. A resettable and reprogrammable keypad lock based on electrochromic Prussian blue films and biocatalysis of immobilized glucose oxidase in a bipolar electrode system.

    Science.gov (United States)

    Yu, Xue; Liang, Jiying; Yang, Tiangang; Gong, Mengjie; Xi, Dongman; Liu, Hongyun

    2018-01-15

    Herein, a resettable and reprogrammable biomolecular keypad lock on the basis of a closed bipolar electrode (BPE) system was established. In this system, one ITO electrode with immobilized chitosan (CS) and glucose oxidase (GOD), designated as CS-GOD, acted as one pole of BPE in the sensing cell; another ITO with electrodeposited Prussian blue (PB) films as the other pole in the reporting cell. The addition of ascorbic acid (AA) in the sensing cell with driving voltage (V tot ) at +2.5V would make the PB films become Prussian white (PW) in the reporting cell, accompanied by the color change from blue to nearly transparent. On the other hand, with the help of oxygen, the addition of glucose in the sensing cell with V tot at -1.5V would induce PW back to PB. The change of color and the corresponding UV-vis absorbance at 700nm for the PB/PW films in the reporting cell could be reversibly switched by changing the solute in the sensing cell between AA and glucose and then switching V tot between +2.5 and -1.5V. Based on these, a keypad lock was developed with AA, glucose and V tot as 3 inputs, and the color change of the PB/PW films as the output. This keypad lock system combined enzymatic catalysis with bipolar electrochemistry, demonstrating some unique advantages such as good reprogrammability, easy resettability and visual readout by naked eye. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Stereochemical determination and bioactivity assessment of {(S)}-(+)-curcuphenol dimers isolated from the marine sponge Didiscus aceratus and synthesized through laccase biocatalysis

    DEFF Research Database (Denmark)

    Lassen, Peter Rygaard

    2005-01-01

    Electrospray ionization mass spectrometry-guided isolation of extracts from Didiscus aceratus led to the discovery of several new derivatives of the bioactive bisabolene-type sponge metabolite (S)-(+)-curcuphenol (1). The compounds obtained by this method included a mixture of known (2) and new (3...

  7. Enzymatic conversion of CO2 to CH3OH via reverse dehydrogenase cascade biocatalysis: Quantitative comparison of efficiencies of immobilized enzyme systems

    DEFF Research Database (Denmark)

    Marpani, Fauziah Binti; Pinelo, Manuel; Meyer, Anne S.

    2017-01-01

    A designed biocatalytic cascade system based on reverse enzymatic catalysis by formate dehydrogenase (EC 1.2.1.2), formaldehyde dehydrogenase (EC 1.2.1.46), and alcohol dehydrogenase (EC 1.1.1.1) can convert carbon dioxide (CO2) to methanol (CH3OH) via formation of formic acid (CHOOH......) and formaldehyde (CHOH) during equimolar cofactor oxidation of NADH to NAD+. This reaction is appealing because it represents a double gain: (1) reduction of CO2 and (2) an alternative to fossil fuel based production of CH3OH. The present review evaluates the efficiency of different immobilized enzyme systems...

  8. Integration of Enzymes in Polyaniline-Sensitized 3D Inverse Opal TiO2 Architectures for Light-Driven Biocatalysis and Light-to-Current Conversion.

    Science.gov (United States)

    Riedel, Marc; Lisdat, Fred

    2018-01-10

    Inspired by natural photosynthesis, coupling of artificial light-sensitive entities with biocatalysts in a biohybrid format can result in advanced photobioelectronic systems. Herein, we report on the integration of sulfonated polyanilines (PMSA1) and PQQ-dependent glucose dehydrogenase (PQQ-GDH) into inverse opal TiO 2 (IO-TiO 2 ) electrodes. While PMSA1 introduces sensitivity for visible light into the biohybrid architecture and ensures the efficient wiring between the IO-TiO 2 electrode and the biocatalytic entity, PQQ-GDH provides the catalytic activity for the glucose oxidation and therefore feeds the light-driven reaction with electrons for an enhanced light-to-current conversion. Here, the IO-TiO 2 electrodes with pores of around 650 nm provide a suitable interface and morphology needed for the stable and functional assembly of polymer and enzyme. The IO-TiO 2 electrodes have been prepared by a template approach applying spin coating, allowing an easy scalability of the electrode height and surface area. The successful integration of the polymer and the enzyme is confirmed by the generation of an anodic photocurrent, showing an enhanced magnitude with increasing glucose concentrations. Compared to flat and nanostructured TiO 2 electrodes, the three-layered IO-TiO 2 electrodes give access to a 24-fold and 29-fold higher glucose-dependent photocurrent due to the higher polymer and enzyme loading in IO films. The three-dimensional IO-TiO 2 |PMSA1|PQQ-GDH architecture reaches maximum photocurrent densities of 44.7 ± 6.5 μA cm -2 at low potentials in the presence of glucose (for a three TiO 2 layer arrangement). The onset potential for the light-driven substrate oxidation is found to be at -0.315 V vs Ag/AgCl (1 M KCl) under illumination with 100 mW cm -2 , which is more negative than the redox potential of the enzyme. The results demonstrate the advantageous properties of IO-TiO 2 |PMSA1|PQQ-GDH biohybrid architectures for the light-driven glucose conversion with improved performance.

  9. Evaluation of parallel milliliter-scale stirred-tank bioreactors for the study of biphasic whole-cell biocatalysis with ionic liquids.

    Science.gov (United States)

    Dennewald, Danielle; Hortsch, Ralf; Weuster-Botz, Dirk

    2012-01-01

    As clear structure-activity relationships are still rare for ionic liquids, preliminary experiments are necessary for the process development of biphasic whole-cell processes involving these solvents. To reduce the time investment and the material costs, the process development of such biphasic reaction systems would profit from a small-scale high-throughput platform. Exemplarily, the reduction of 2-octanone to (R)-2-octanol by a recombinant Escherichia coli in a biphasic ionic liquid/water system was studied in a miniaturized stirred-tank bioreactor system allowing the parallel operation of up to 48 reactors at the mL-scale. The results were compared to those obtained in a 20-fold larger stirred-tank reactor. The maximum local energy dissipation was evaluated at the larger scale and compared to the data available for the small-scale reactors, to verify if similar mass transfer could be obtained at both scales. Thereafter, the reaction kinetics and final conversions reached in different reactions setups were analysed. The results were in good agreement between both scales for varying ionic liquids and for ionic liquid volume fractions up to 40%. The parallel bioreactor system can thus be used for the process development of the majority of biphasic reaction systems involving ionic liquids, reducing the time and resource investment during the process development of this type of applications. Copyright © 2011. Published by Elsevier B.V.

  10. Novel Lipases: Expression and Improvement for Applied Biocatalysis = Nuevas lipasas: expresión y mejoras para biocatálisis aplicada

    OpenAIRE

    Infanzón Ramos, Belén

    2017-01-01

    This thesis is focused in the identification and improvement of lipases for biotechnological application. The importance of lipases is increasing in several industries. However, the commercial use of lipases is still a drawback in the economics of the lipase-based industrial applications. There are many tools for improving and adapting the enzyme properties to the desired requirements of a process that could lead lipase catalysis through a cost-effective process. In this context, the main obj...

  11. Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

    NARCIS (Netherlands)

    van Bloois, Edwin; Winter, Remko T.; Janssen, Dick B.; Fraaije, Marco W.

    Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially

  12. Novel Lipases: Expression and Improvement for Applied Biocatalysis = Nuevas lipasas: expresión y mejoras para biocatálisis aplicadatalysi Novel Lipases: Expression and Improvement for Applied Biocatalysis = Nuevas lipasas: expresión y mejoras para biocatálisis aplicada

    OpenAIRE

    Infanzón Ramos, Belén

    2017-01-01

    [eng] This thesis is focused in the identification and improvement of lipases for biotechnological application. The importance of lipases is increasing in several industries. However, the commercial use of lipases is still a drawback in the economics of the lipase-based industrial applications. There are many tools for improving and adapting the enzyme properties to the desired requirements of a process that could lead lipase catalysis through a cost-effective process. In this context, the m...

  13. Considerations for implementation of novel enzyme-based processes

    DEFF Research Database (Denmark)

    Deslauriers, Maria Gundersen

    Biocatalysis is the use of enzymes to catalyze chemical reactions. It is an established synthesisroute in chemical synthesis, alongside conventional chemistry. Biocatalysis is often applied due to excellent regio and stereoselectivity, in addition to its environmentally benign properties....... This thesis aims at increasing the potential use of industrial biocatalysis, both in terms of broadening its current use and expanding it to new applications. This academic study is carried out through two case studies. These two case studies were chosen because they represent each end of the spectra...... learned from these two case studies justify general conclusions for biocatalysis, irrespective of their application. The work in this thesis therefore contributes, not only to industrial biocatalysis in these two areas, but also increases the understanding of biocatalysis as a whole....

  14. Advances in the Process Development of Biocatalytic Processes

    DEFF Research Database (Denmark)

    Tufvesson, Pär; Lima Ramos, Joana; Al-Haque, Naweed

    2013-01-01

    Biocatalysis is already established in chemical synthesis on an industrial scale, in particular in the pharmaceutical sector. However, the wider implementation of biocatalysis is currently hindered by the extensive effort required to develop a competitive process. In order that resources spent...

  15. Amine-functionalized magnetic nanoparticles as robust support for ...

    Indian Academy of Sciences (India)

    their surface properties via introduction of functional groups holds great prospect in the field of ... Biomaterials; enzyme activity; enzyme biocatalysis; nanoparticles; surface properties. 1. .... lyzer (Pyris Diamond TG-DTA) with a heating rate. 8.

  16. Current state and perspectives of penicillin G acylase-based biocatalyses

    Czech Academy of Sciences Publication Activity Database

    Marešová, Helena; Plačková, Martina; Grulich, Michal; Kyslík, Pavel

    2014-01-01

    Roč. 98, č. 7 (2014), s. 2867-2879 ISSN 0175-7598 Institutional support: RVO:61388971 Keywords : Penicillin G acylase * beta lactam biocatalysis * Enantioselectivity Subject RIV: EE - Microbiology, Virology Impact factor: 3.337, year: 2014

  17. Optimisation of stabilised carboxylesterase NP for enantioselective hydrolysis of naproxen methyl ester

    CSIR Research Space (South Africa)

    Steenkamp, Lucia H

    2008-12-01

    Full Text Available Although the enantioselective kinetic resolution of ester racemates of the non-steroidal antiinflammatory drug naproxen ([2-(6-methoxy-2-naphthyl) propionic acid]) is a common demonstration for biocatalysis, the enantiomeric excess of the reactions...

  18. 1399-IJBCS-Article-Mohamed Isa

    African Journals Online (AJOL)

    USER

    SHESTCO), Abuja, Nigeria. *Corresponding author ... Chitin was synthesized from Nigerian brown shrimps by a chemical process involving demineralization and deproteinisation. ... biomedical, biocatalysis and waste water treatment processes (Li ...

  19. Bioprospecting Archaea: Focus on Extreme Halophiles

    KAUST Repository

    Antunes, André ; Simõ es, Marta F.; Grö tzinger, Stefan W.; Eppinger, Jö rg; Braganç a, Judith; Bajic, Vladimir B.

    2016-01-01

    knowledge, and (c) utilization of Archaea in biotechnology. They are increasingly employed in fields as diverse as biocatalysis, biocomputing, bioplastic production, bioremediation, bioengineering, food, pharmaceuticals, and nutraceuticals. This chapter

  20. Parallel Synthesis and Biocatalytic Amplification of Marine-Inspired Libraries: An Integrated Approach Toward Discovering New Chemotherapeutics

    Science.gov (United States)

    2007-09-01

    synthesis and biocatalysis. We will use a combination of highly efficient chemistry and biocatalysis to prepare a library of small organic molecules whose...potential for the synthesis of diverse alkaloids . KEY RESEARCH ACCOMPLISHMENTS OF THIS REPORTING PERIOD Cyclopentenone libraries were screened for...growth and proliferation of cancer cells. The newer approach is to use the tools of chemical synthesis to create large collections (“libraries”)

  1. Engineering Ultrastable Protein Filaments into 2D and 3D Templates for Advanced Nanomaterials: A New Dimension in Materials Design

    Science.gov (United States)

    2013-08-16

    Littlechild,   Thermophilic  archaeal   enzymes  and  applications  in  biocatalysis,  Biochem  Soc  Trans  39   (2011...spatially arrange thermostable enzymes for high- temperature biocatalysis[7][8] and as biotemplates for fabrication and patterning of inorganic...upon which to position hyperthermostable enzymes that have optimal activities at or above 100°C  [8], which are temperatures beyond the stability of

  2. Biocatálisis y biotecnología

    OpenAIRE

    Arroyo, Miguel; Acebal, Carmen; de la Mata, Isabel

    2014-01-01

    Biocatalysis has emerged as a rich field within Biotechnology, enabling the application of enzymes in a wide range of industries ranging from pharmaceuticals and fine chemicals to food and energy. This striking development of Biocatalysis is due to novel technologies such as bioinformatics, high-throughput screening (HTS), directed evolution, as well as other well-established techniques such as enzyme immobilization and protein engineering or medium engineering. Sustainable manufacturing is a...

  3. Enzymatic Inverse Opal Hydrogel Particles for Biocatalyst.

    Science.gov (United States)

    Wang, Huan; Gu, Hongcheng; Chen, Zhuoyue; Shang, Luoran; Zhao, Ze; Gu, Zhongze; Zhao, Yuanjin

    2017-04-19

    Enzymatic carriers have a demonstrated value for chemical reactions and industrial applications. Here, we present a novel kind of inverse opal hydrogel particles as the enzymatic carriers. The particles were negatively replicated from spherical colloidal crystal templates by using magnetic nanoparticles tagged acrylamide hydrogel. Thus, they were endowed with the features of monodispersity, small volume, complete penetrating structure, and controllable motion, which are all beneficial for improving the efficiency of biocatalysis. In addition, due to the ordered porous nanostructure, the inverse opal hydrogel particles were imparted with unique photonic band gaps (PBGs) and vivid structural colors for encoding varieties of immobilized enzymes and for constructing a multienzymes biocatalysis system. These features of the inverse opal hydrogel particles indicate that they are ideal enzymatic carriers for biocatalysis.

  4. Model visualization for evaluation of biocatalytic processes

    DEFF Research Database (Denmark)

    Law, HEM; Lewis, DJ; McRobbie, I

    2008-01-01

    Biocatalysis offers great potential as an additional, and in some cases as an alternative, synthetic tool for organic chemists, especially as a route to introduce chirality. However, the implementation of scalable biocatalytic processes nearly always requires the introduction of process and/or bi......,S-EDDS), a biodegradable chelant, and is characterised by the use of model visualization using `windows of operation"....

  5. 88 Nigerian Journal of Chemical Research Vol. 22, No. 2, 2017 ...

    African Journals Online (AJOL)

    kyari

    suggest that; it would make a good alternative fuel for diesel engines. Key words: ..... AD. 2.09. 1. 2.09. 0.43. 0.5175. BC. 98.00. 1. 98.00. 19.93. < 0.0001. BD. 138.33. 1 .... Minas [online], 78: 90 – 97. 13. ... Biocatalysis: towards ever greener.

  6. Combinatorial One-Pot Synthesis of Poly-N-acetyllactosamine Oligosaccharides with Leloir-Glycosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Rech, C.; Rosencrantz, R. R.; Křenek, Karel; Pelantová, Helena; Bojarová, Pavla; Roemer, Ch. E.; Hanisch, F.-G.; Křen, Vladimír; Elling, L.

    2011-01-01

    Roč. 353, č. 13 (2011), s. 2492-2500 ISSN 1615-4150 R&D Projects: GA MŠk OC09045 Institutional research plan: CEZ:AV0Z50200510 Keywords : combinatorial chemistry * biocatalysis * carbohydrates Subject RIV: CC - Organic Chemistry Impact factor: 6.048, year: 2011

  7. Structure, function and operational stability of peroxidases

    NARCIS (Netherlands)

    Haandel, van M.J.H.

    2000-01-01

    This PhD project was started in 1995 and was supported by the dutch Ministry of Economic Affairs through the programme "IOP Catalysis". The main goal of "IOP catalysis" is to obtain clean and more efficient technologies, to improve the quality of the Dutch fine chemistry. Biocatalysis

  8. Heterologous Expression of Peroxidases : Chapter 12

    NARCIS (Netherlands)

    Christien Lokman; S. de Weert

    2010-01-01

    This monograph describes many applications of peroxidase-based biocatalysis in the biotechnology industry. The need for such a book emerges from the considerable amount of new data regarding the phylogeny, reaction mechanisms, thermodynamic characterization and structural features of fungal and

  9. News: Green Chemistry & Technology

    Science.gov (United States)

    A series of 21 articles focused on different features of green chemistry in a recent issue of Chemical Reviews. Topics extended over a wide range to include the design of sustainable synthetic processes to biocatalysis. A selection of perspectives follows as part of this colu

  10. Guidelines and cost analysis for catalyst production in biocatalytic processes

    DEFF Research Database (Denmark)

    Tufvesson, Pär; Lima Ramos, Joana; Nordblad, Mathias

    2011-01-01

    Biocatalysis is an emerging area of technology, and to date few reports have documented the economics of such processes. As it is a relatively new technology, many processes do not immediately fulfill the economic requirements for commercial operation. Hence, early-stage economic assessment could...

  11. Laccase-catalyzed dimerization of glycosylated lignols

    Czech Academy of Sciences Publication Activity Database

    Bassanini, I.; Gavezzotti, P.; Monti, D.; Krejzová, Jana; Křen, Vladimír; Riva, S.

    2016-01-01

    Roč. 134, SI (2016), s. 295-301 ISSN 1381-1177 R&D Projects: GA MŠk(CZ) LD15085 Institutional support: RVO:61388971 Keywords : Biocatalysis * Biooxidation * Laccase Subject RIV: CE - Biochemistry Impact factor: 2.269, year: 2016

  12. Lipase-Catalyzed Hydrolysis of Blackcurrant Oil in Supercritical Carbon Dioxide

    Czech Academy of Sciences Publication Activity Database

    Sovová, Helena; Zarevúcka, Marie

    2003-01-01

    Roč. 58, č. 11 (2003), s. 2339-2350 ISSN 0009-2509 R&D Projects: GA ČR GA203/99/1457 Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z4072921 Keywords : supercritical fluid * biocatalysis * blackcurrant oil Subject RIV: CI - Industrial Chemistry, Chemical Engineering Impact factor: 1.562, year: 2003

  13. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences. Dhurairajan senthilnathan. Articles written in Journal of Chemical Sciences. Volume 123 Issue 3 May 2011 pp 279-290. Biocatalysis of azidolysis of epoxides: Computational evidences on the role of halohydrin dehalogenase (HheC) · Dhurairajan senthilnathan ...

  14. Aplikace cíleně modifikovaných enzymů v biosyntézách beta-laktamových antibiotik

    OpenAIRE

    Schneiderová, Michaela

    2012-01-01

    Nowadays beta-lactam antibiotics are widely used as bacteriostatic agents. The chemical synthesis of antibiotics or its derivatives could be replaced with biocatalysis. This work deals with basics of industrial synthesis beta-lactam antibiotics and, mainly, with used enzymes. This work acquainted with methodes used in enzyme modifications and improving, so they could fit the best for the industrial syntheses.

  15. Protein engineering of enzymes for process applications

    DEFF Research Database (Denmark)

    Woodley, John M

    2013-01-01

    opportunities will be targeted on modification to enable process application. This article discusses the challenges involved in enzyme modification focused on process requirements, such as the need to fulfill reaction thermodynamics, specific activity under the required conditions, kinetics at required...... concentrations, and stability. Finally, future research directions are discussed, including the integration of biocatalysis with neighboring chemical steps....

  16. Progress in emerging techniques for characterization of immobilized viable whole-cell biocatalysts

    Czech Academy of Sciences Publication Activity Database

    Bučko, M.; Vikartovská, A.; Schenkmayerová, A.; Tkáč, J.; Filip, J.; Chorvát Jr., D.; Neděla, Vilém; Ansorge-Schumacher, M.B.; Gemeiner, P.

    2017-01-01

    Roč. 71, č. 11 (2017), s. 2309-2324 ISSN 0366-6352 Institutional support: RVO:68081731 Keywords : bioelectrocatalysis * imaging techniques * immobilized whole-cell biocatalyst * multienzyme cascade reactions * online kinetics Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 1.258, year: 2016

  17. [The rise of enzyme engineering in China].

    Science.gov (United States)

    Li, Gaoxiang

    2015-06-01

    Enzyme engineering is an important part of the modern biotechnology. Industrial biocatalysis is considered the third wave of biotechnology following pharmaceutical and agricultural waves. In 25 years, China has made a mighty advances in enzyme engineering research. This review focuses on enzyme genomics, enzyme proteomics, biosynthesis, microbial conversion and biosensors in the Chinese enzyme engineering symposiums and advances in enzyme preparation industry in China.

  18. 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

  19. Towards Keratan Sulfate - Chemoenzymatic Cascade Synthesis of Sulfated N-Acetyllactosamine (LacNAc) Glycan Oligomers

    Czech Academy of Sciences Publication Activity Database

    Lange, B.; Šimonová, Anna; Fischoeder, T.; Pelantová, Helena; Křen, Vladimír; Elling, L.

    2016-01-01

    Roč. 358, č. 4 (2016), s. 584-596 ISSN 1615-4150 R&D Projects: GA MŠk(CZ) LD13042; GA ČR GC15-02578J Institutional support: RVO:61388971 Keywords : biocatalysis * carbohydrates * glycoconjugates Subject RIV: CE - Biochemistry Impact factor: 5.646, year: 2016

  20. Polycyclic Ketone Monooxygenase from the Thermophilic Fungus Thermothelomyces thermophila : A Structurally Distinct Biocatalyst for Bulky Substrates

    NARCIS (Netherlands)

    Fürst, Maximilian J L J; Savino, Simone; Dudek, Hanna M; Gómez Castellanos, J Rúben; Gutiérrez de Souza, Cora; Rovida, Stefano; Fraaije, Marco W; Mattevi, Andrea

    2017-01-01

    Regio- and stereoselective Baeyer-Villiger oxidations are difficult to achieve by classical chemical means, particularly when large, functionalized molecules are to be converted. Biocatalysis using flavin-containing Baeyer-Villiger monooxygenases (BVMOs) is a well-established tool to address these

  1. DOE Laboratory Catalysis Research Symposium - Abstracts

    Energy Technology Data Exchange (ETDEWEB)

    Dunham, T.

    1999-02-01

    The conference consisted of two sessions with the following subtopics: (1) Heterogeneous Session: Novel Catalytic Materials; Photocatalysis; Novel Processing Conditions; Metals and Sulfides; Nuclear Magnetic Resonance; Metal Oxides and Partial Oxidation; Electrocatalysis; and Automotive Catalysis. (2) Homogeneous Catalysis: H-Transfer and Alkane Functionalization; Biocatalysis; Oxidation and Photocatalysis; and Novel Medical, Methods, and Catalyzed Reactions.

  2. The effect of cultivation media and washing whole-cell biocatalysts on monoamine oxidase catalyzed oxidative desymmetrization of 3-azabicyclo[3,3,0]octane

    DEFF Research Database (Denmark)

    Ramesh, Hemalata; Zajkoska, Petra; Rebros, Martin

    2016-01-01

    It is well known that washing whole-cells containing enzyme activities after fermentation, but prior to biocatalysis can improve their activity in the subsequent reaction. In this paper, we quantify the impact of both the fermentation media and cell washing on the performance of whole-cell biocat...

  3. Not so monofunctional-a case of thermostable Thermobifida fusca catalase with peroxidase activity

    NARCIS (Netherlands)

    Lončar, Nikola; Fraaije, Marco W

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and

  4. Development of a two-step “green” synthesis for (-)-ambafuran production

    CSIR Research Space (South Africa)

    Steenkamp, Lucia H

    2010-09-01

    Full Text Available The main objective of the development of a two-step “green” synthesis for (-)-ambafuran production is to find an alternative synthesis of (-) Ambrox from sclareol, to use a bioconversion or biocatalysis route, and that it results in a natural...

  5. Exploitation of a Library of alfa-Galactosidases for the Synthesis of Building Blocks for Glycopolymers

    Czech Academy of Sciences Publication Activity Database

    Casali, M.; Tarantini, L.; Riva, S.; Huňková, Zdenka; Weignerová, Lenka; Křen, Vladimír

    2002-01-01

    Roč. 77, č. 1 (2002), s. 105-110 ISSN 0006-3592 R&D Projects: GA ČR GA303/99/1382; GA ČR GA203/01/1018 Keywords : glycosidases * biocatalysis * enzymatic library Subject RIV: EE - Microbiology, Virology Impact factor: 2.211, year: 2002

  6. Development of advanced biorefinery concepts using magnetically responsive materials

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Pospišková, K.; Baldíková, E.; Šafaříková, Miroslava

    2016-01-01

    Roč. 116, DEC (2016), s. 17-26 ISSN 1369-703X R&D Projects: GA ČR GA13-13709S Institutional support: RVO:67179843 Keywords : saccharomyces-cerevisiae cells * solid acid catalysts * Separation * Biocatalysis * Immobilization * Bioconversion * Magnetic particles * Magnetic enzymes and cells Subject RIV: EH - Ecology, Behaviour Impact factor: 2.892, year: 2016

  7. Development of advanced biorefinery concepts using magnetically responsive materials

    Czech Academy of Sciences Publication Activity Database

    Šafařík, Ivo; Pospíšková, K.; Baldíková, E.; Šafaříková, Miroslava

    2016-01-01

    Roč. 116, SI (2016), s. 17-26 ISSN 1369-703X Institutional support: RVO:60077344 Keywords : separation * biocatalysis * immobilization * bioconversion * magnetic particles * magnetic enzymes and cells Subject RIV: GM - Food Processing Impact factor: 2.892, year: 2016

  8. .I.Georichum candidum./I. lipase: activation and its enantioselectivity towards xenobiotic substrates

    Czech Academy of Sciences Publication Activity Database

    Kejík, Z.; Zarevúcka, Marie; Wimmer, Zdeněk; Demnerová, K.

    2003-01-01

    Roč. 97, č. 6 (2003), s. 390 ISSN 0009-2770. [International Symposium on Biocatalysis and Biotransformations /6./. 28.06.2003-03.07.2003, Olomouc] R&D Projects: GA MŠk OC D13.10 Institutional research plan: CEZ:AV0Z4055905 Keywords : .I.Georichum candidum./I. lipase Subject RIV: CC - Organic Chemistry

  9. Immobilisation and characterisation of biocatalytic co-factor recycling enzymes, glucose dehydrogenase and NADH oxidase, on aldehyde functional ReSynTM polymer microspheres

    CSIR Research Space (South Africa)

    Twala, BV

    2012-03-01

    Full Text Available The use of enzymes in industrial applications is limited by their instability, cost and difficulty in their recovery and re-use. Immobilisation is a technique which has been shown to alleviate these limitations in biocatalysis. Here we describe...

  10. Energy conversion and utilization technologies

    International Nuclear Information System (INIS)

    1988-01-01

    The DOE Energy Conversion and Utilization Technologies (ECUT) Program continues its efforts to expand the generic knowledge base in emerging technological areas that support energy conservation initiatives by both the DOE end-use sector programs and US private industry. ECUT addresses specific problems associated with the efficiency limits and capabilities to use alternative fuels in energy conversion and end-use. Research is aimed at understanding and improving techniques, processes, and materials that push the thermodynamic efficiency of energy conversion and usage beyond the state of the art. Research programs cover the following areas: combustion, thermal sciences, materials, catalysis and biocatalysis, and tribology. Six sections describe the status of direct contact heat exchange; the ECUT biocatalysis project; a computerized tribology information system; ceramic surface modification; simulation of internal combustion engine processes; and materials-by-design. These six sections have been indexed separately for inclusion on the database. (CK)

  11. Process considerations for protein engineering of ω-Transaminase

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson; Schwarze, Daniel; Tufvesson, Pär

    Over the past decades, the use of biocatalysis in the chemical and pharmaceutical industry has significantly increased. In parallel and contributing to this trend, many enzymes have been discovered and isolated from different biological sources. This has broadened the scope of biocatalysis...... and nowadays allows the green regio- and enantio-selective synthesis of many compounds, potentially with less time and energy demand and avoiding the use of toxic reagents. The technology therefore has many advantages over classical chemical synthesis to prepare fine chemical and pharmaceutical intermediates...... are important building blocks for the chemical and pharmaceutical industries. On the other hand, there are a number of challenges associated with the use of this enzyme for instance substrate and product inhibition, and a potentially unfavorable equilibrium. In the present work it was investigated how changes...

  12. Enzymatic processes in alternative reaction media: a mini review

    Directory of Open Access Journals (Sweden)

    Mansour Ghaffari-Moghaddam

    2015-08-01

    Full Text Available Biocatalysis is a growing field in the production of fine chemicals and will most probably increase its share in the future. Enzymatic reactions are carried out under mild conditions, i.e., non-toxic solvents, low temperature and pressure, which eliminates most environmental drawbacks associated with conventional production methods. The superiority of chemo-, regio- and enantioselectivity of enzymes exhibit significant advantages over conventional catalysts for production of fine chemicals, flavors, fragrances, agrochemicals and pharmaceuticals. Enzymes can function both in aqueous and non-aqueous solvents. As a result of the growing scientific and industrial interest towards green chemistry, green solvent systems, which are mainly water, supercritical fluids, ionic liquids, fluorinated solvents, and solvent-free systems have become more popular in biocatalysis. However, the activity and selectivity of an enzyme is heavily dependent on solvent properties. In this review, various green solvents were classified and some of their influential features on enzyme activity were discussed.

  13. Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase

    OpenAIRE

    Karuppiah, Vijaykumar; Ranaghan, Kara E.; Leferink, Nicole G. H.; Johannissen, Linus O.; Shanmugam, Muralidharan; Ní Cheallaigh, Aisling; Bennett, Nathan J.; Kearsey, Lewis J.; Takano, Eriko; Gardiner, John M.; van der Kamp, Marc W.; Hay, Sam; Mulholland, Adrian J.; Leys, David; Scrutton, Nigel S.

    2017-01-01

    Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS)...

  14. [Biotechnological potential of methylotrophic bacteria: a review of current status and future prospects].

    Science.gov (United States)

    Trotsenko, Iu A; Doronina, N V; Khmelenina, V N

    2005-01-01

    Major results of the authors' findings on the implementation of biotechnological potential of aerobic methylobacteria and methanotrophs for obtaining forage proteins, biopolymers (polybutyrate and polysaccharides), enzymes (oxidoreductases), and bioprotectors (ectoin), as well as for degrading toxic C1 and Cn compounds have been reviewed. Unique features of the structural and functional organization of the metabolism of extremophilic (tolerant) methylotrophs are discussed, with a view for their prospective use in various fields of modern biotechnology, including biocatalysis and nanotechnology.

  15. Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

    OpenAIRE

    Rami Gherib; Hisham M. Dokainish; James W. Gauld

    2013-01-01

    Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a pleth...

  16. Molecular catalysts structure and functional design

    CERN Document Server

    Gade, Lutz H

    2014-01-01

    Highlighting the key aspects and latest advances in the rapidly developing field of molecular catalysis, this book covers new strategies to investigate reaction mechanisms, the enhancement of the catalysts' selectivity and efficiency, as well as the rational design of well-defined molecular catalysts. The interdisciplinary author team with an excellent reputation within the community discusses experimental and theoretical studies, along with examples of improved catalysts, and their application in organic synthesis, biocatalysis, and supported organometallic catalysis. As a result, readers wil

  17. Process technology for the application of d-amino acid oxidases in pharmaceutical intermediate manufacturing

    DEFF Research Database (Denmark)

    Tindal, Stuart; Carr, Reuben; Archer, Ian V. J.

    2011-01-01

    Recent advances in biocatalysis have seen increased interest in the use of D-amino acid oxidase to synthesize optically pure amino acids. However, the creation of a genuine oxidase based platform technology will require suitable process technology as well as an understanding of the challenges...... and opportunities of a wider portfolio of synthetic targets. In this article we address some of the recent progress in process technology to enable the future development of a generic platform technology....

  18. Biocatalytic production of mandelic acid and analogues: a review and comparison with chemical processes

    Czech Academy of Sciences Publication Activity Database

    Martínková, Ludmila; Křen, Vladimír

    2018-01-01

    Roč. 102, č. 9 (2018), s. 3893-3900 ISSN 0175-7598 R&D Projects: GA ČR(CZ) GA18-00184S; GA MŠk LTC17009 Institutional support: RVO:61388971 Keywords : Mandelic acid * Nitrilase * Lipase Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 3.420, year: 2016

  19. European Congress on Biotechnology (4th) Held in Amsterdam, The Netherlands, on June 1987

    Science.gov (United States)

    1988-02-19

    car be used and thus higher Nmaraso Fumarate enzyme loadings can be achieved with a Glucose isomerase High- fructose corn syrup large effectiveness...is only after most of the the following reactions: glucose + oxy- glucose has been metabolized that aerobic gen + glucose oxidasa + water - gluconic...are obtained by mixing water solutions of two water -soluble * Biocatalysis polymers. Both phases have a high water * Animal cell cultures content and do

  20. A new strategy for aromatic ring alkylation in cylindrocyclophane biosynthesis.

    Science.gov (United States)

    Nakamura, Hitomi; Schultz, Erica E; Balskus, Emily P

    2017-08-01

    Alkylation of aromatic rings with alkyl halides is an important transformation in organic synthesis, yet an enzymatic equivalent is unknown. Here, we report that cylindrocyclophane biosynthesis in Cylindrospermum licheniforme ATCC 29412 involves chlorination of an unactivated carbon center by a novel halogenase, followed by a previously uncharacterized enzymatic dimerization reaction featuring sequential, stereospecific alkylations of resorcinol aromatic rings. Discovery of the enzymatic machinery underlying this unique biosynthetic carbon-carbon bond formation has implications for biocatalysis and metabolic engineering.

  1. Simplification of lipase design in the enzymatic kinetic resolution of amines by saturation transfer difference NMR

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Marcio S.; Pietrobom, Daniel, E-mail: s.marcio@ufabc.edu.br [Universidade Federal do ABC (CCNH/UFABC), Santo André, SP (Brazil). Centro de Ciências Naturais e Humanas

    2016-07-01

    In this work, we demonstrate a nuclear magnetic resonance (NMR) method for racemic amide and lipase interaction as a first-pass design method in the enzymatic kinetic resolution of amines. As a novel adaptation of commonly used protein-ligand screening NMR methodologies, this approach relies upon a lipase-amide interaction wherein the time-consuming is reduced drastically and new insights are produced during the development of biocatalysis reactions. (author)

  2. Biphasic whole-cell synthesis of R-2-octanol with recycling of the ionic liquid

    OpenAIRE

    Dennewald, Danielle

    2011-01-01

    Whole-cell biocatalysis in biphasic systems permits the synthesis of inhibiting chiral alcohols if appropriate non-water miscible ionic liquids are used. Taking the asymmetric reduction of 2-octanone to R-2-octanol by a recombinant Escherichia coli as a model reaction, a detailed characterisation of the biocatalytic reaction was performed with [HMPL][NTF] as ionic liquid. This made the asymmetric synthesis of R-2-octanol in a simple batch at a conversion > 99 % and at an enantiomeric excess >...

  3. A methodology for development of biocatalytic processes

    OpenAIRE

    Lima Ramos, Joana; Woodley, John; Tufvesson, Pär

    2013-01-01

    The potential advantages displayed by biocatalytic processes for organic synthesis (such as exquisite selectivity under mild operating conditions), have prompted the increasing number of processes running on a commercial scale. However, biocatalysis is still a fairly underutilised technology. As a relatively new technology biocatalytic processes often do not immediately fulfil the required process metrics that are key for an economically and/or environmentally competitive process at an indust...

  4. Set-Up and Validation of a Dynamic Solid/Gas Bioreactor

    KAUST Repository

    Lloyd-Randol, Jennifer D.

    2012-05-01

    The limited availability of fossil resourses mandates the development of new energy vectors, which is one of the Grand Challenges of the 21st Century [1]. Biocatalytic energy conversion is a promising solution to meet the increased energy demand of industrialized societies. Applications of biocatalysis in the gas-phase are so far limited to production of fine chemicals and pharmaceuticals. However, this technology has the potential for large scale biocatalytic applications [2], e.g. for the formation of novel energy carriers. The so-called solid/gas biocatalysis is defined as the application of a biocatalyst immobilized on solid-phase support acting on gaseous substrates [3]. This process combines the advantages of bio-catalysis (green chemistry, mild reaction conditions, high specicity & selectivity) and heterogeneous dynamic gas-phase processes (low diffusion limitation, high conversion, simple scale-up). This work presents the modifications of a PID Microactivity Reference reactor in order to make it suitable for solid/gas biocatalysis. The reactor design requirements are based on previously published laboratory scale solid/gas systems with a feed of saturated vapors [4]. These vapors are produced in saturation flasks, which were designed and optimized during this project. Other modifications included relocation of the gas mixing chamber, redesigning the location and heating mechanism for the reactor tube, and heating of the outlet gas line. The modified reactor system was verified based on the Candida antarctica lipase B catalyzed transesterication of ethyl acetate with 1-hexanol to hexyl acetate and ethanol and results were compared to liquid-phase model reactions. Products were analyzed on line by a gas chromatograph with a flame ionization detector. C. antarc- tica physisorbed on silica particles produced a 50% conversion of hexanol at 40 C in the gas-phase. A commercial immobilized lipase from Iris Biotech produced 99% and 97% conversions of hexanol in

  5. Hydrolysis of nitriles by soil bacteria: variation with soil origin.

    Science.gov (United States)

    Rapheeha, O K L; Roux-van der Merwe, M P; Badenhorst, J; Chhiba, V; Bode, M L; Mathiba, K; Brady, D

    2017-03-01

    The aim of this study was to explore bacterial soil diversity for nitrile biocatalysts, in particular, those for hydrolysis of β-substituted nitriles, to the corresponding carboxamides and acids that may be incorporated into peptidomimetics. To achieve this, we needed to compare the efficiency of isolation methods and determine the influence of land use and geographical origin of the soil sample. Nitrile-utilizing bacteria were isolated from various soil environments across a 1000 km long transect of South Africa, including agricultural soil, a gold mine tailing dam and uncultivated soil. The substrate profile of these isolates was determined through element-limited growth studies on seven different aliphatic or aromatic nitriles. A subset of these organisms expressing broad substrate ranges was evaluated for their ability to hydrolyse β-substituted nitriles (3-amino-3-phenylpropionitrile and 3-hydroxy-4-phenoxybutyronitrile) and the active organisms were found to be Rhodococcus erythropolis from uncultivated soil and Rhodococcus rhodochrous from agricultural soils. The capacity for hydrolysis of β-substituted nitriles appears to reside almost exclusively in Rhodococci. Land use has a much greater effect on the biocatalysis substrate profile than geographical location. Enzymes are typically substrate specific in their catalytic reactions, and this means that a wide diversity of enzymes is required to provide a comprehensive biocatalysis toolbox. This paper shows that the microbial diversity of nitrile hydrolysis activity can be targeted according to land utilization. Nitrile biocatalysis is a green chemical method for the enzymatic production of amides and carboxylic acids that has industrial applications, such as in the synthesis of acrylamide and nicotinamide. The biocatalysts discovered in this study may be applied to the synthesis of peptidomimetics which are an important class of therapeutic compounds. © 2016 The Society for Applied Microbiology.

  6. Discovery of a diazo-forming enzyme in cremeomycin biosynthesis.

    Science.gov (United States)

    Waldman, Abraham J; Balskus, Emily P

    2018-05-17

    The molecular architectures and potent bioactivities of diazo-containing natural products have attracted the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes involved in diazo group construction have not been identified. Here, we show the ATP-dependent enzyme CreM installs the diazo group in cremeomycin via late-stage N-N bond formation using nitrite. This finding should inspire efforts to use diazo-forming enzymes in biocatalysis and synthetic biology and enable genome-based discovery of new diazo-containing metabolites.

  7. Next-Generation Catalysis for Renewables: Combining Enzymatic with Inorganic Heterogeneous Catalysis for Bulk Chemical Production

    DEFF Research Database (Denmark)

    Vennestrøm, Peter Nicolai Ravnborg; Christensen, C.H.; Pedersen, S.

    2010-01-01

    chemical platform under different conditions than those conventionally employed. Indeed, new process and catalyst concepts need to be established. Both enzymatic catalysis (biocatalysis) and heterogeneous inorganic catalysis are likely to play a major role and, potentially, be combined. One type...... of combination involves one-pot cascade catalysis with active sites from bio- and inorganic catalysts. In this article the emphasis is placed specifically on oxidase systems involving the coproduction of hydrogen peroxide, which can be used to create new in situ collaborative oxidation reactions for bulk...

  8. Studies on whole cell fluorescence-based screening for epoxide hydrolases and Baeyer-Villiger monooxygenases

    International Nuclear Information System (INIS)

    Bicalho, Beatriz; Chen, Lu S.; Marsaioli, Anita J.; Grognux, Johann; Reymond, Jean-Louis

    2004-01-01

    Biocatalysis reactions were performed on microtiter plates (200 μL) aiming at the utilization of fluorogenic substrates (100 μmol L -1 ) for rapid whole cell screening for epoxide hydrolases (EHs) and Baeyer-Villiger monooxygenases (BVMOs). A final protocol was achieved for EHs, with 3 new enzymatic sources being detected (Agrobacterium tumefaciens, Pichia stipitis, Trichosporom cutaneum). The fluorogenic assay for BVMO did not work as expected. However, an approach to possible variables involved (aeration; pH) provided the first detection of a BVMO activity in T. cutaneum. (author)

  9. Aplicações de enzimas na síntese e na modificação de polímeros

    Directory of Open Access Journals (Sweden)

    Marcos de Campos Cavalcanti de Albuquerque

    2014-01-01

    Full Text Available Enzymes are biological catalysts that offer great potential for use in the synthesis and modification of polymers, being more specific and greener than chemical catalysts. In this work, enzymes from the classes of hydrolases (lipase, cutinase and protease and of oxidoreductases (horseradish peroxidase, manganese peroxidase and laccase were identified as the main biocatalysts responsible for the synthesis of polymers. Biocatalysis can potentially be part of the life cycle of several polymers, including polyesters, polyurethanes, polycarbonates, polyamides, functionalized polysaccharides and polystyrene, allowing the synthesis of specialty macromolecules for fine applications and with higher added-value than commodity polymers.

  10. Biotransformação de limoneno: uma revisão das principais rotas metabólicas Biotransformation of limonene: a review of the main metabolic pathways

    Directory of Open Access Journals (Sweden)

    Mário Roberto Maróstica Júnior

    2007-04-01

    Full Text Available There is considerable progress in the study of the biotransformation of limonene. Extensive research on the biotransformation of limonene has resulted in the elucidation of new metabolic pathways. Natural flavors can be produced via biotransformation, satisfying consumer demand for natural products. This review presents some elements concerning the biotransformation of limonene with emphasis on the metabolic pathways. Some comments are also made on problems related to biocatalysis as well as on the application of some compounds originating from the biotransformation of the inexpensive limonene.

  11. Biocatálisis y biotecnología

    Directory of Open Access Journals (Sweden)

    Arroyo, Miguel

    2014-08-01

    Full Text Available Biocatalysis has emerged as a rich field within Biotechnology, enabling the application of enzymes in a wide range of industries ranging from pharmaceuticals and fine chemicals to food and energy. This striking development of Biocatalysis is due to novel technologies such as bioinformatics, high-throughput screening (HTS, directed evolution, as well as other well-established techniques such as enzyme immobilization and protein engineering or medium engineering. Sustainable manufacturing is a major driver of Biocatalysis, which will provide many real challenges and opportunities for the future. In this article, some of the main methods in enzyme technology have been reviewed, as well as several biotechnological applications of enzymes in industry. Finally, a brief overview of the situation of Biocatalysis in both Spanish academia and industry has also been reported.La Biocatálisis ha surgido como un área de gran riqueza dentro de la Biotecnología, y ha permitido la aplicación de las enzimas en un amplio número de industrias dedicadas a la fabricación de fármacos y otros compuestos químicos, así como alimentos o biocombustibles. Este sorprendente desarrollo de la Biocatálisis se debe a nuevas tecnologías como la bioinformática, el cribado de alta resolución, la evolución dirigida, así como otras técnicas arraigadas como la inmovilización de enzimas y la ingeniería de proteínas o del medio de reacción. La fabricación sostenible de productos de consumo es uno de los objetivos principales de la Biocatálisis, y supondrá muchos desafíos y oportunidades en el futuro. En este artículo, se han revisado algunos de los principales métodos empleados en tecnología enzimática, así como varios ejemplos de aplicaciones de las enzimas en la industria. Finalmente, se indica un breve comentario sobre la situación actual de los grupos de investigación y empresas dedicados a la Biocatálisis en España.

  12. Polyelectrolyte Complex Beads by Novel Two-Step Process for Improved Performance of Viable Whole-Cell Baeyer-Villiger Monoxygenase by Immobilization

    Czech Academy of Sciences Publication Activity Database

    Krajčovič, T.; Bučko, M.; Vikartovská, A.; Lacík, I.; Uhelská, L.; Chorvát, D.; Neděla, Vilém; Tihlaříková, Eva; Gericke, M.; Heinze, T.; Gemeiner, P.

    2017-01-01

    Roč. 7, č. 11 (2017), s. 353-364 ISSN 2073-4344 Institutional support: RVO:68081731 Keywords : polyelectrolyte complex beads * environmental scanning electron microscopy * confocal laser scanning microscopy * Baeyer-Villiger biooxidation * cyclohexanone monoxygenase * immobilization * viable whole-cell biocatalyst Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering OBOR OECD: Bioprocessing technologies (industrial processes relying on biological agents to drive the process) biocatalysis, fermentation Impact factor: 3.082, year: 2016 http://www.mdpi.com/2073-4344/7/11/353

  13. Laboratory investigation of microbiologically influenced corrosion of C1018 carbon steel by nitrate reducing bacterium Bacillus licheniformis

    International Nuclear Information System (INIS)

    Xu, Dake; Li, Yingchao; Song, Fengmei; Gu, Tingyue

    2013-01-01

    Nitrate injection is used to suppress reservoir souring in oil and gas fields caused by Sulfate Reducing Bacteria (SRB) through promotion of nitrate respiration by Nitrate Reducing Bacteria (NRB). However, it is not well publicized that nitrate reduction by NRB can cause Microbiologically Influenced Corrosion (MIC) because nitrate reduction coupled with iron oxidation is thermodynamically favorable. NRB benefits bioenergetically from this redox reaction under biocatalysis. This work showed that the Bacillus licheniformis biofilm, when grown as an NRB biofilm, caused a 14.5 μm maximum pit depth and 0.89 mg/cm 2 normalized weight loss against C1018 carbon steel in one-week lab tests

  14. Protein crystallography and site-direct mutagenesis analysis of the poly(ethylene terephthalate) hydrolase PETase from Ideonella sakaiensis.

    Science.gov (United States)

    Liu, Bing; He, Lihui; Wang, Liping; Li, Tao; Li, Changcheng; Liu, Huayi; Luo, Yunzi; Bao, Rui

    2018-03-30

    Compared with traditional recycle strategies, biodegradation provides a sustainable solution for poly (ethylene terephthalate) (PET) wastes disposal. PETase, a newly identified enzyme from Ideonella sakaiensis, has high efficiency and specificity towards PET, which provides a prominent prospect on PET degradation. Based on the biochemical analysis, we propose that the wide substrate-binding pocket is critical for its excellent property on crystallized PET hydrolysis. Structure-guided site-directed mutagenesis exhibited improvement in PETase catalytic efficiency, providing valuable insight on how the molecular engineering of PETase can optimize its application in biocatalysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. 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...

  16. Global transcriptional response of solvent-sensitive and solvent-tolerant Pseudomonas putida strains exposed to toluene

    DEFF Research Database (Denmark)

    Molina-Santiago, Carlos; Udaondo, Zulema; Gómez Lozano, María

    2017-01-01

    for the degradation and synthesis of a wide range of chemicals. For the use of these microbes in bioremediation and biocatalysis, it is critical to understand the mechanisms underlying these phenotypic differences. In this study, RNA-seq analysis compared the short- and long-term responses of the toluene-sensitive KT...... pathways, using toluene as source of energy. Among the unique genes encoded by DOT-T1E is a 70kb island composed of genes of unknown function induced in response to toluene....

  17. Immobilization of Escherichia coli containing ω‐transaminase activity in LentiKats®

    DEFF Research Database (Denmark)

    Cárdenas‐Fernández, Max; Lima Afonso Neto, Watson; López, Carmen

    2012-01-01

    Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized...... whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole...

  18. Trehalose Analogues: Latest Insights in Properties and Biocatalytic Production

    Directory of Open Access Journals (Sweden)

    Maarten Walmagh

    2015-06-01

    Full Text Available Trehalose (α-d-glucopyranosyl α-d-glucopyranoside is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals, medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (α-d-glucopyranosyl α-d-galactopyranoside or galactotrehalose (α-d-galactopyranosyl α-d-galactopyranoside, offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. “Greener” alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis.

  19. Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting.

    Science.gov (United States)

    Daiha, Karina de Godoy; Angeli, Renata; de Oliveira, Sabrina Dias; Almeida, Rodrigo Volcan

    2015-01-01

    The great potential of lipases is known since 1930 when the work of J. B. S. Haldane was published. After eighty-five years of studies and developments, are lipases still important biocatalysts? For answering this question the present work investigated the technological development of four important industrial sectors where lipases are applied: production of detergent formulations; organic synthesis, focusing on kinetic resolution, production of biodiesel, and production of food and feed products. The analysis was made based on research publications and patent applications, working as scientific and technological indicators, respectively. Their evolution, interaction, the major players of each sector and the main subject matters disclosed in patent documents were discussed. Applying the concept of technology life cycle, S-curves were built by plotting cumulative patent data over time to monitor the attractiveness of each technology for investment. The results lead to a conclusion that the use of lipases as biocatalysts is still a relevant topic for the industrial sector, but developments are still needed for lipase biocatalysis to reach its full potential, which are expected to be achieved within the third, and present, wave of biocatalysis.

  20. From water-in-oil to oil-in-water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems.

    Science.gov (United States)

    Santos, Luísa D F; Coutinho, João A P; Ventura, Sónia P M

    2015-01-01

    Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water-in-oil or oil-in-water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil-in-water emulsions), the imidazolium-based IL acts as an enhancer of the lipase catalytic capacity, super-activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers.

  1. Metabolic engineering is key to a sustainable chemical industry.

    Science.gov (United States)

    Murphy, Annabel C

    2011-08-01

    The depletion of fossil fuel stocks will prohibit their use as the main feedstock of future industrial processes. Biocatalysis is being increasingly used to reduce fossil fuel reliance and to improve the sustainability, efficiency and cost of chemical production. Even with their current small market share, biocatalyzed processes already generate approximately US$50 billion and it has been estimated that they could be used to produce up to 20% of fine chemicals by 2020. Until the advent of molecular biological technologies, the compounds that were readily accessible from renewable biomass were restricted to naturally-occurring metabolites. However, metabolic engineering has considerably broadened the range of compounds now accessible, providing access to compounds that cannot be otherwise reliably sourced, as well as replacing established chemical processes. This review presents the case for continued efforts to promote the adoption of biocatalyzed processes, highlighting successful examples of industrial chemical production from biomass and/or via biocatalyzed processes. A selection of emerging technologies that may further extend the potential and sustainability of biocatalysis are also presented. As the field matures, metabolic engineering will be increasingly crucial in maintaining our quality of life into a future where our current resources and feedstocks cannot be relied upon.

  2. Mechanistic modeling of biocorrosion caused by biofilms of sulfate reducing bacteria and acid producing bacteria.

    Science.gov (United States)

    Xu, Dake; Li, Yingchao; Gu, Tingyue

    2016-08-01

    Biocorrosion is also known as microbiologically influenced corrosion (MIC). Most anaerobic MIC cases can be classified into two major types. Type I MIC involves non-oxygen oxidants such as sulfate and nitrate that require biocatalysis for their reduction in the cytoplasm of microbes such as sulfate reducing bacteria (SRB) and nitrate reducing bacteria (NRB). This means that the extracellular electrons from the oxidation of metal such as iron must be transported across cell walls into the cytoplasm. Type II MIC involves oxidants such as protons that are secreted by microbes such as acid producing bacteria (APB). The biofilms in this case supply the locally high concentrations of oxidants that are corrosive without biocatalysis. This work describes a mechanistic model that is based on the biocatalytic cathodic sulfate reduction (BCSR) theory. The model utilizes charge transfer and mass transfer concepts to describe the SRB biocorrosion process. The model also includes a mechanism to describe APB attack based on the local acidic pH at a pit bottom. A pitting prediction software package has been created based on the mechanisms. It predicts long-term pitting rates and worst-case scenarios after calibration using SRB short-term pit depth data. Various parameters can be investigated through computer simulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Enzyme Stability and Activity in Non-Aqueous Reaction Systems: A Mini Review

    Directory of Open Access Journals (Sweden)

    Shihui Wang

    2016-02-01

    Full Text Available Enormous interest in biocatalysis in non-aqueous phase has recently been triggered due to the merits of good enantioselectivity, reverse thermodynamic equilibrium, and no water-dependent side reactions. It has been demonstrated that enzyme has high activity and stability in non-aqueous media, and the variation of enzyme activity is attributed to its conformational modifications. This review comprehensively addresses the stability and activity of the intact enzymes in various non-aqueous systems, such as organic solvents, ionic liquids, sub-/super-critical fluids and their combined mixtures. It has been revealed that critical factors such as Log P, functional groups and the molecular structures of the solvents define the microenvironment surrounding the enzyme molecule and affect enzyme tertiary and secondary structure, influencing enzyme catalytic properties. Therefore, it is of high importance for biocatalysis in non-aqueous media to elucidate the links between the microenvironment surrounding enzyme surface and its stability and activity. In fact, a better understanding of the correlation between different non-aqueous environments and enzyme structure, stability and activity can contribute to identifying the most suitable reaction medium for a given biotransformation.

  4. Efficient production of (R-2-hydroxy-4-phenylbutyric acid by using a coupled reconstructed D-lactate dehydrogenase and formate dehydrogenase system.

    Directory of Open Access Journals (Sweden)

    Binbin Sheng

    Full Text Available (R-2-hydroxy-4-phenylbutyric acid [(R-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R-HPBA synthetic processes remain unsatisfactory.The Y52L/F299Y mutant of NAD-dependent D-lactate dehydrogenase (D-nLDH in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA. The mutant D-nLDHY52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3 to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R-HPBA from OPBA. The biocatalysis conditions were then optimized.Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R-HPBA in 90 min. Given its high product enantiomeric excess (>99% and productivity (47.9 mM h(-1, the constructed coupling biocatalysis system is a promising alternative for (R-HPBA production.

  5. Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

    Science.gov (United States)

    Hartley, Carol J; French, Nigel G; Scoble, Judith A; Williams, Charlotte C; Churches, Quentin I; Frazer, Andrew R; Taylor, Matthew C; Coia, Greg; Simpson, Gregory; Turner, Nicholas J; Scott, Colin

    2017-01-01

    Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP) is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

  6. Co-production of bio-ethanol, xylonic acid and slow-release nitrogen fertilizer from low-cost straw pulping solid residue.

    Science.gov (United States)

    Huang, Chen; Ragauskas, Arthur J; Wu, Xinxing; Huang, Yang; Zhou, Xuelian; He, Juan; Huang, Caoxing; Lai, Chenhuan; Li, Xin; Yong, Qiang

    2018-02-01

    A novel bio-refinery sequence yielding varieties of co-products was developed using straw pulping solid residue. This process utilizes neutral sulfite pretreatment which under optimal conditions (160 °C and 3% (w/v) sulfite charge) provides 64.3% delignification while retaining 90% of cellulose and 67.3% of xylan. The pretreated solids exhibited excellent enzymatic digestibility, with saccharification yields of 86.9% and 81.1% for cellulose and xylan, respectively. After pretreatment, the process of semi-simultaneous saccharification and fermentation (S-SSF) and bio-catalysis was investigated. The results revealed that decreased ethanol yields were achieved when solid loading increased from 5% to 30%. An acceptable ethanol yield of 76.8% was obtained at 20% solid loading. After fermentation, bio-catalysis of xylose remaining in fermentation broth resulted in near 100% xylonic acid (XA) yield at varied solid loadings. To complete the co-product portfolio, oxidation ammoniation of the dissolved lignin successfully transformed it into biodegradable slow-release nitrogen fertilizer with excellent agricultural properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Sugar analog synthesis by in vitro biocatalytic cascade: A comparison of alternative enzyme complements for dihydroxyacetone phosphate production as a precursor to rare chiral sugar synthesis.

    Directory of Open Access Journals (Sweden)

    Carol J Hartley

    Full Text Available Carbon-carbon bond formation is one of the most challenging reactions in synthetic organic chemistry, and aldol reactions catalysed by dihydroxyacetone phosphate-dependent aldolases provide a powerful biocatalytic tool for combining C-C bond formation with the generation of two new stereo-centres, with access to all four possible stereoisomers of a compound. Dihydroxyacetone phosphate (DHAP is unstable so the provision of DHAP for DHAP-dependent aldolases in biocatalytic processes remains complicated. Our research has investigated the efficiency of several different enzymatic cascades for the conversion of glycerol to DHAP, including characterising new candidate enzymes for some of the reaction steps. The most efficient cascade for DHAP production, comprising a one-pot four-enzyme reaction with glycerol kinase, acetate kinase, glycerophosphate oxidase and catalase, was coupled with a DHAP-dependent fructose-1,6-biphosphate aldolase enzyme to demonstrate the production of several rare chiral sugars. The limitation of batch biocatalysis for these reactions and the potential for improvement using kinetic modelling and flow biocatalysis systems is discussed.

  8. Industrial high pressure applications. Processes, equipment and safety

    Energy Technology Data Exchange (ETDEWEB)

    Eggers, Rudolf (ed.) [Technische Univ. Hamburg-Harburg, Hamburg (Germany). Inst. fuer Thermische Verfahrenstechnik

    2012-07-01

    Industrial high pressure processes open the door to many reactions that are not possible under 'normal' conditions. These are to be found in such different areas as polymerization, catalytic reactions, separations, oil and gas recovery, food processing, biocatalysis and more. The most famous high pressure process is the so-called Haber-Bosch process used for fertilizers and which was awarded a Nobel prize. Following an introduction on historical development, the current state, and future trends, this timely and comprehensive publication goes on to describe different industrial processes, including methanol and other catalytic syntheses, polymerization and renewable energy processes, before covering safety and equipment issues. With its excellent choice of industrial contributions, this handbook offers high quality information not found elsewhere, making it invaluable reading for a broad and interdisciplinary audience.

  9. Theoretical pKa prediction of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc

    Science.gov (United States)

    Vipperla, Bhavaniprasad; Griffiths, Thomas M.; Wang, Xingyong; Yu, Haibo

    2017-01-01

    The pKa value of the α-phosphate moiety of uridine 5‧-diphosphate-GlcNAc (UDP-GlcNAc) has been successfully calculated using density functional theory methods in conjunction with the Polarizable Continuum Models. Theoretical methods were benchmarked over a dataset comprising of alkyl phosphates. B3LYP/6-31+G(d,p) calculations using SMD solvation model provide excellent agreement with the experimental data. The predicted pKa for UDP-GlcNAc is consistent with most recent NMR studies but much higher than what it has long been thought to be. The importance of this study is evident that the predicted pKa for UDP-GlcNAc supports its potential role as a catalytic base in the substrate-assisted biocatalysis.

  10. Oxygen Dependent Biocatalytic Processes

    DEFF Research Database (Denmark)

    Pedersen, Asbjørn Toftgaard

    Enzyme catalysts have the potential to improve both the process economics and the environ-mental profile of many oxidation reactions especially in the fine- and specialty-chemical industry, due to their exquisite ability to perform stereo-, regio- and chemo-selective oxida-tions at ambient...... to aldehydes and ketones, oxyfunctionalization of C-H bonds, and epoxidation of C-C double bonds. Although oxygen dependent biocatalysis offers many possibilities, there are numerous chal-lenges to be overcome before an enzyme can be implemented in an industrial process. These challenges requires the combined...... far below their potential maximum catalytic rate at industrially relevant oxygen concentrations. Detailed knowledge of the en-zyme kinetics are therefore required in order to determine the best operating conditions and design oxygen supply to minimize processing costs. This is enabled...

  11. Transformações biológicas: contribuições e perspectivas Biological transformations: contributions and perspectives

    Directory of Open Access Journals (Sweden)

    Luciana Gonzaga de Oliveira

    2009-01-01

    Full Text Available In a moment that amazingly advances are being reached on the development of technologies to obtain high value chemical compounds as polymers, fine chemicals, pharmaceutical industry intermediates and chemical entities, we cannot refuse that a meaningful progress is due to the maturing in knowledge of biological transformations. Biocatalysis and biotransformations are being widespread applied to generate processes and products with incredible success. In this review article we present the main contributions of biotechnology and biological catalytic processes to Chemistry, the most important evolution steps on enzymatic transformations, how it has being applied and which are the perspectives to academic and industrial environments. We also would like to stimulate the community to step out research in biotechnology applicable to chemical and pharmaceutical industries, trying to achieve what we believe to be the ideal layout: integrating chemical transformations, enzymatic conversions and fermentation processes.

  12. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    , A. Bouchu, G. Descotes, Y. Queneau, Tetrahedron: Lett. 2000, 41, 3597. [7]           Q. Wu, N. Wang, Y.M. Xiao, D.S. Lu, X.F. Lin, Carbohydr. Res., 2004, 339, 2059. [8]           H.G. Park, H.N. Chang, Biotechnol. Lett. 2000, 22, 39 [9]           S. Riva, M. Nonini, G. Ottolina, B. Danieli......, Carbohydr. Res., 1998, 314, 259. [10]         N.R. Pedersen, R. Wimmer, R. Matthiesen, L.H. Pedersen, A. Gessesse, Tetrahedron: Asymmetry 2003, 14, 667. [11]         L. H. Pedersen, S. Ritthitham and M. Kristensen (2008) in Modern Biocatalysis Eds W. D. Fessner and T. Anthonsen, Wiley-VCH in press...

  13. Biotransformation of (-)-(1R,4S)-Menthone and (+)-(1S,4R)-Menthone by the Common Cutworm Spodoptera litura Larvae.

    Science.gov (United States)

    Marumoto, Shinsuke; Okuno, Yoshiharu; Hagiwara, Yuki; Miyazawa, Mitsuo

    2017-08-01

    Using biotransformation as a biocatalytic process has the advantage of being able to proceed under mild conditions and with high regio- and enantioselectivity. This study investigated the biotransformation of (-)-(1R,4S)-menthone (1) and (+)-(1S,4R)-menthone (2) by Spodoptera litura larvae. Compound 1 was converted to (-)-(1R,4S)-7-hydroxymenthone (1-1), (+)-(1R,3S,4S)-7-hydroxyneomenthol (1-2) and (-)-(1R,4S,8R)-p-menth-3-one-9-oic acid (1-3). The metabolism of substrate 2 generated three enantiomers of the above metabolites, designated as 2-1 to 2-3, respectively. The C-9 position of (-)-menthone and (+)-menthone was oxidized to carboxylic acid by S. litura, which is a metabolic pathway not observed in any other example of biocatalysis.

  14. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    Science.gov (United States)

    de Visser, Sam P.; Stillman, Martin J.

    2016-01-01

    In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1). This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties. PMID:27070578

  15. Challenging Density Functional Theory Calculations with Hemes and Porphyrins

    Directory of Open Access Journals (Sweden)

    Sam P. de Visser

    2016-04-01

    Full Text Available In this paper we review recent advances in computational chemistry and specifically focus on the chemical description of heme proteins and synthetic porphyrins that act as both mimics of natural processes and technological uses. These are challenging biochemical systems involved in electron transfer as well as biocatalysis processes. In recent years computational tools have improved considerably and now can reproduce experimental spectroscopic and reactivity studies within a reasonable error margin (several kcal·mol−1. This paper gives recent examples from our groups, where we investigated heme and synthetic metal-porphyrin systems. The four case studies highlight how computational modelling can correctly reproduce experimental product distributions, predicted reactivity trends and guide interpretation of electronic structures of complex systems. The case studies focus on the calculations of a variety of spectroscopic features of porphyrins and show how computational modelling gives important insight that explains the experimental spectra and can lead to the design of porphyrins with tuned properties.

  16. New Sustainable Model of Biorefineries: Biofactories and Challenges of Integrating Bio- and Solar Refineries.

    Science.gov (United States)

    Abate, Salvatore; Lanzafame, Paola; Perathoner, Siglinda; Centi, Gabriele

    2015-09-07

    The new scenario for sustainable (low-carbon) chemical and energy production drives the development of new biorefinery concepts (indicated as biofactories) with chemical production at the core, but flexible and small-scale production. An important element is also the integration of solar energy and CO2 use within biobased production. This concept paper, after shortly introducing the motivation and recent trends in this area, particularly at the industrial scale, and some of the possible models (olefin and intermediate/high-added-value chemicals production), discusses the opportunities and needs for research to address the challenge of integrating bio- and solar refineries. Aspects discussed regard the use of microalgae and CO2 valorization in biorefineries/biofactories by chemo- or biocatalysis, including possibilities for their synergetic cooperation and symbiosis, as well as integration within the agroenergy value chain. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Effects of PEGylation on biomimetic synthesis of magnetoferritin nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Caiyun, E-mail: ycy@mail.iggcas.ac.cn; Cao, Changqian, E-mail: changqiancao@mail.iggcas.ac.cn; Cai, Yao, E-mail: caiyao@mail.iggcas.ac.cn; Xu, Huangtao, E-mail: xuhuangtao@mail.iggcas.ac.cn; Zhang, Tongwei, E-mail: ztw@mail.iggcas.ac.cn; Pan, Yongxin, E-mail: yxpan@mail.iggcas.ac.cn [Institute of Geology and Geophysics, Chinese Academy of Sciences, Key Laboratory of Earth and Planetary Physics (China)

    2017-03-15

    Recent studies have demonstrated that ferrimagnetic magnetoferritin nanoparticles are a promising novel magnetic nanomaterial in biomedical applications, including biocatalysis, imaging, diagnostics, and tumor therapy. Here we investigated the PEGylation of human H-ferritin (HFn) proteins and the possible influence on biomimetic synthesis of magnetoferritin nanoparticles. The outer surface of HFn proteins was chemically modified with different PEG molecular weights (PEG10K and PEG20K) and different modification ratios (HFn subunit:PEG20K = 1:1, 1:2, 1:4). The PEGylated HFn proteins were used for biomimetic synthesis of ferrimagnetic magnetoferritin nanoparticles. We found that, compared with magnetoferritin using non-PEGylated HFn protein templates, the synthesized magnetoferritin using the PEGylated HFn protein templates possessed larger magnetite cores, higher magnetization and relaxivity values, and improved thermal stability. These results suggest that the PEGylation of H-ferritin may improve the biomineralization of magnetoferritin nanoparticles and enhance their biomedical applications.

  18. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  19. Process technology for multi-enzymatic reaction systems

    DEFF Research Database (Denmark)

    Xue, Rui; Woodley, John M.

    2012-01-01

    In recent years, biocatalysis has started to provide an important green tool in synthetic organic chemistry. Currently, the idea of using multi-enzymatic systems for industrial production of chemical compounds becomes increasingly attractive. Recent examples demonstrate the potential of enzymatic...... synthesis and fermentation as an alternative to chemical-catalysis for the production of pharmaceuticals and fine chemicals. In particular, the use of multiple enzymes is of special interest. However, many challenges remain in the scale-up of a multi-enzymatic system. This review summarizes and discusses...... the technology options and strategies that are available for the development of multi-enzymatic processes. Some engineering tools, including kinetic models and operating windows, for developing and evaluating such processes are also introduced....

  20. Atomistic Simulations of Thermophoretic Motion of water Nanodroplets in Carbon Nanotubes

    DEFF Research Database (Denmark)

    Zambrano, Harvey A; Walther, Jens Honore; Koumoutsakos, Petros

    2008-01-01

    Open-ended nanotubes offer unique possibilities as fluid conduits with applications ranging from molecule separation devices in biocatalysis to encapsulation media for drug storage and delivery. Liquids and solids in nanochannels may be driven by electrophoresis, osmosis, gradients in the surface......-1925 for thermodiffusion in gases. Ibbs found that the coefficient of thermal diffusion is more sensitive than any of the other transport coefficients to the nature of the intermolecular forces.1 Thus, a complete understanding of the thermal diffusion could provide a powerful means of investigation of forces between...... molecules. Although the theoretical explanation of thermodiffusion for molecules in liquids is still under debate, the investigation of its practical usability is motivated by potential applications in nanotechnology. Hence, thermodiffusion was recently used as the driving mechanism in artificially...

  1. Magnetically recoverable nanocatalysts

    KAUST Repository

    Polshettiwar, Vivek

    2011-05-11

    A broad overview on magnetically recoverable nanocatalysts is presented and the use of magnetic nanomaterials as catalysts is discussed. Magnetic materials are used as organocatalysts and their applications range to challenging reactions, such as hydroformylation and olefin metathesis. Magnetic nanomaterials are also being used in environmental applications, such as for photo- and biocatalysis and for the adsorption and removal of pollutants from air and water. These materials show great promise as enantioselective catalysts, which are used extensively for the synthesis of medicines, drugs, and other bioactive molecules. By functionalizing these materials using chiral ligands, a series of chiral nanocatalysts can be designed, offering great potential to reuse these otherwise expensive catalyst systems. Characterization of magnetic catalysts is often a challenging task, and NMR characterization of these catalysts is difficult because the magnetic nature of the materials interferes with the magnetic field of the spectrometer.

  2. Renewable biofuels bioconversion of lignocellulosic biomass by microbial community

    CERN Document Server

    Rana, Vandana

    2017-01-01

    This book offers a complete introduction for novices to understand key concepts of biocatalysis and how to produce in-house enzymes that can be used for low-cost biofuels production. The authors discuss the challenges involved in the commercialization of the biofuel industry, given the expense of commercial enzymes used for lignocellulose conversion. They describe the limitations in the process, such as complexity of lignocellulose structure, different microbial communities’ actions and interactions for degrading the recalcitrant structure of lignocellulosic materials, hydrolysis mechanism and potential for bio refinery. Readers will gain understanding of the key concepts of microbial catalysis of lignocellulosic biomass, process complexities and selection of microbes for catalysis or genetic engineering to improve the production of bioethanol or biofuel.

  3. Process Technology for Immobilized Lipasecatalyzed Reactions

    DEFF Research Database (Denmark)

    Xu, Yuan

    Biocatalysis has attracted significant attention recently, mainly due to its high selectivity and potential benefits for sustainability. Applications can be found in biorefineries, turning biomass into energy and chemicals, and also for products in the food and pharmaceutical industries. However......, most applications remain in the production of high-value fine chemicals, primarily because of the expense of introducing new technology. In particular lipasecatalyzed synthesis has already achieved efficient operations for high-value products and more interesting now is to establish opportunities...... for low-value products. In order to guide the industrial implementation of immobilized-lipase catalyzed reactions, especially for highvolume low-value products, a methodological framework for dealing with the technical and scientific challenges and establishing an efficient process via targeted scale...

  4. Process engineering tools to guide implementation and scale-up of transaminase cascades

    DEFF Research Database (Denmark)

    Tufvesson, Pär; Janes, Kresimir; Lima Ramos, Joana

    Biocatalysis is gaining ground in the pharmaceutical and fine chemical industry as a selective and potentially green technology to help synthesize industrially interesting products. In particular in the last decade the application of transaminases (E.C. 2.6.1.X ) has gained particular attention...... properties and values. A major challenge in the transaminase catalysed synthesis of chiral amines is the unfavourable equilibrium position [1]. There are several solutions to such equilibrium problems, including the use of in-situ product removal (ISPR) and cascade reactions to degrade or recycle the co......-product formed. Such techniques, especially those using cascades can be a great tool to overcome the thermodynamic hurdle, but also present some new challenges with respect to compatibility of reaction conditions, recycling of co-factors and last but not least, the added cost of the cascade system components [2...

  5. The nanobiotechnology handbook

    CERN Document Server

    Xie, Yubing

    2012-01-01

    A thorough overview of nanobiotechnology and its place in advances in applied science and engineering, The Nanobiotechnology Handbook combines contributions from physics, bioorganic and bioinorganic chemistry, molecular and cellular biology, materials science, and medicine as well as from mechanical, electrical, chemical, and biomedical engineering to address the full scope of current and future developments. World-class experts discuss the role of nanobiotechnology in bioanalysis, biomolecular and biomedical nanotechnology, biosensors, biocatalysis and biofuel, and education and workforce development. It includes a companion CD that contains all figures in the book. The book begins with discussions of biomimetic nanotechnology, including a comprehensive overview of DNA nanostructure and DNA-inspired nanotechnology, aptamer-functionalized nanomaterials as artificial antibodies, artificial enzymes, molecular motors, and RNA structures and RNA-inspired nanotechnology. It shows how nanotechnology can be inspired...

  6. Ultrasonic treatment of Viscozyme Cassava C preparation for improving cellulase activity

    Science.gov (United States)

    Tra, Tran Thi Thu; Vu, Huynh Minh; Man, Le Van Viet

    2017-09-01

    In this study, the effects of ultrasonic treatment on the cellulolytic activity of Viscozyme Cassava C preparation were investigated. The biocatalyst was treated with ultrasound at different enzyme concentrations (from 0.02 to 19.50 mg protein/mL), ultrasonic powers (from 0 to 12 W/mL) and times (from 0 to 120 seconds). The highest cellulase activity was achieved when the enzyme preparation was ultrasonicated at 7.3 W/mL for 40 sec, under which the cellulase activity increased by 18.1% over the control. The optimal pH and temperature of the sonicated and unsonicated biocatalysts were statistically similar. However, the half-life value of the sonicated preparation at 4 °C was 24.5% higher than that of the unsonicated preparation. This result indicated that ultrasonic treatment of the enzyme preparation could reduce its amount used in biocatalysis.

  7. Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions.

    Science.gov (United States)

    Liu, Jiaheng; Li, Huiling; Zhao, Guangrong; Caiyin, Qinggele; Qiao, Jianjun

    2018-05-01

    NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

  8. Biotransformation of 20(R)-panaxatriol by Mucor racemosus and the anti-hepatic fibrosis activity of some products.

    Science.gov (United States)

    Chen, Guangtong; Li, Jie; Yan, Sensen; Lin, Haijun; Wu, Juanjuan; Zhai, Xuguang; Song, Yan; Li, Jianlin

    2017-08-01

    Biocatalysis of 20(R)-panaxatriol (PT) was performed by the fungus Mucor racemosus. Six metabolites (1-6) including five new compounds were obtained, and their structures were elucidated as 20(R),25-epoxy-12β,24β-dihydroxydammaran-3,6-dione (2), 20(R),25-epoxy-12β,22β-dihydroxydammaran-3,6-dione (3), 20(R),25-epoxy-23β-hydroxydammaran-3,6,12-trione (4), 20(R),25-epoxy-12β,23α- dihydroxydammaran-3,6-dione (5), and 20(R),25-epoxy-12β-hydroxydammaran-3,6,23-trione (6) by spectroscopic analysis. Pharmacological studies revealed that compounds 2, 3 and 5 exhibited significant antihepatic fibrosis activity, while 4 and 6 showed cytotoxicity against HSC-T6 cells.

  9. Oxide nanomaterials: synthetic developments, mechanistic studies, and technological innovations.

    Science.gov (United States)

    Patzke, Greta R; Zhou, Ying; Kontic, Roman; Conrad, Franziska

    2011-01-24

    Oxide nanomaterials are indispensable for nanotechnological innovations, because they combine an infinite variety of structural motifs and properties with manifold morphological features. Given that new oxide materials are almost reported on a daily basis, considerable synthetic and technological work remains to be done to fully exploit this ever increasing family of compounds for innovative nano-applications. This calls for reliable and scalable preparative approaches to oxide nanomaterials and their development remains a challenge for many complex nanostructured oxides. Oxide nanomaterials with special physicochemical features and unusual morphologies are still difficult to access by classic synthetic pathways. The limitless options for creating nano-oxide building blocks open up new technological perspectives with the potential to revolutionize areas ranging from data processing to biocatalysis. Oxide nanotechnology of the 21st century thus needs a strong interplay of preparative creativity, analytical skills, and new ideas for synergistic implementations. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Biodecontamination of concrete

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, R.D. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1995-12-31

    This paper describes the development and results of a demonstration for a continuous bioprocess for mixed waste treatment. A key element of the process is a unique microbial strain, which tolerates high levels of aromatic solvents and surfactants. This microorganism is the biocatalysis of the continuous flow system designed for processing stored liquid scintillation wastes. During the past year, a process demonstration has been conducted on commercial formulation of liquid scintillation cocktails (LSQ). Based on data obtained from this demonstration, the Ohio Environmental Protection Agency granted the Mound Applied Technologies Laboratory a treatability permit allowing the limited processing of actual mixed waste. Since August 1994, the system has been successfully processing stored {open_quotes}hot{close_quotes} LSC waste. This paper discusses the bioprocess, rates of processing, effluent, and implications of bioprocessing for mixed waste management.

  11. NREL Advancements in Methane Conversion Lead to Cleaner Air, Useful Products

    Energy Technology Data Exchange (ETDEWEB)

    2016-06-01

    Researchers at NREL leveraged the recent on-site development of gas fermentation capabilities and novel genetic tools to directly convert methane to lactic acid using an engineered methanotrophic bacterium. The results provide proof-of-concept data for a gas-to-liquids bioprocess that concurrently produces fuels and chemicals from methane. NREL researchers developed genetic tools to express heterologous genes in methanotrophic organisms, which have historically been difficult to genetically engineer. Using these tools, researchers demonstrated microbial conversion of methane to lactate, a high-volume biochemical precursor predominantly utilized for the production of bioplastics. Methane biocatalysis offers a means to concurrently liquefy and upgrade natural gas and renewable biogas, enabling their utilization in conventional transportation and industrial manufacturing infrastructure. Producing chemicals and fuels from methane expands the suite of products currently generated from biorefineries, municipalities, and agricultural operations, with the potential to increase revenue and significantly reduce greenhouse gas emissions.

  12. Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

    Science.gov (United States)

    Carvalho, Alexandra T P; Dourado, Daniel F A R; Skvortsov, Timofey; de Abreu, Miguel; Ferguson, Lyndsey J; Quinn, Derek J; Moody, Thomas S; Huang, Meilan

    2017-10-11

    Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

  13. Enzymes - important players in green chemistry

    Directory of Open Access Journals (Sweden)

    Agata Tarczykowska

    2017-09-01

    Full Text Available Green chemistry has become a worldwide approach that leads to sustainable growth through application and development of its principles. A lot of work has to be put into designing new processes comprising of materials which do not emit pollutants to the atmosphere. Inventing new safer methods and finding less harmful products can be challenging. Enzymes are a great hope of scientists in the field of green chemistry. Enzymes as catalysts require mild conditions therefore it is a great way of saving resources such as energy or water. Processes with the use of enzymes have become more feasible by being more cost effective and eco friendly. Taking into account the benefits of green chemistry, enzyme biocatalysis has quickly replaced traditional chemical processes in several fields, and this substitution is going to reach even more areas because of new emerging technologies in enzyme engineering.

  14. Magnetically recoverable nanocatalysts

    KAUST Repository

    Polshettiwar, Vivek; Luque, Rafael L.; Fihri, Aziz; Zhu, Haibo; Bouhrara, Mohamed; Basset, Jean-Marie

    2011-01-01

    A broad overview on magnetically recoverable nanocatalysts is presented and the use of magnetic nanomaterials as catalysts is discussed. Magnetic materials are used as organocatalysts and their applications range to challenging reactions, such as hydroformylation and olefin metathesis. Magnetic nanomaterials are also being used in environmental applications, such as for photo- and biocatalysis and for the adsorption and removal of pollutants from air and water. These materials show great promise as enantioselective catalysts, which are used extensively for the synthesis of medicines, drugs, and other bioactive molecules. By functionalizing these materials using chiral ligands, a series of chiral nanocatalysts can be designed, offering great potential to reuse these otherwise expensive catalyst systems. Characterization of magnetic catalysts is often a challenging task, and NMR characterization of these catalysts is difficult because the magnetic nature of the materials interferes with the magnetic field of the spectrometer.

  15. Marine Microorganisms as Source of Stereoselective Esterases and Ketoreductases: Kinetic Resolution of a Prostaglandin Intermediate

    KAUST Repository

    De Vitis, Valerio; Guidi, Benedetta; Contente, Martina Letizia; Granato, Tiziana Mariarita; Conti, Paola; Molinari, Francesco; Crotti, Elena; Mapelli, Francesca; Borin, Sara S.; Daffonchio, Daniele; Romano, Diego

    2014-01-01

    A screening among bacterial strains isolated from water-brine interface of the deep hypersaline anoxic basins (DHABs) of the Eastern Mediterranean was carried out for the biocatalytical resolution of racemic propyl ester of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (R,S)-1, a key intermediate for the synthesis of d-cloprostenol. Bacillus horneckiae 15A gave highly stereoselective reduction of (R,S)-1, whereas Halomonas aquamarina 9B enantioselectively hydrolysed (R,S)-1; in both cases, enantiomerically pure unreacted (R)-1 could be easily recovered and purified at molar conversion below 57–58 %, showing the potential of DHAB extremophile microbiome and marine-derived enzymes in stereoselective biocatalysis.

  16. Archaeal Enzymes and Applications in Industrial Biocatalysts.

    Science.gov (United States)

    Littlechild, Jennifer A

    2015-01-01

    Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.

  17. Process Technology for Immobilized LipaseProcess Technology for Immobilized Lipase-catalyzed

    DEFF Research Database (Denmark)

    Xu, Yuan

    Biocatalysis has attracted significant attention recently, mainly due to its high selectivity and potential benefits for sustainability. Applications can be found in biorefineries, turning biomass into energy and chemicals, and also for products in the food and pharmaceutical industries. However......, most applications remain in the production of high-value fine chemicals, primarily because of the expense of introducing new technology. In particular lipasecatalyzed synthesis has already achieved efficient operations for high-value products and more interesting now is to establish opportunities......-down experimental work is described in this thesis. The methodology uses economic targets to test options characterized via a set of tools. In order to validate the methodology, two processes based on immobilized lipase-catalysis have been studied: transesterification and esterification of vegetable oils...

  18. Immobilisation of ω-transaminase for industrial application: Screening and characterisation of commercial ready to use enzyme carriers

    DEFF Research Database (Denmark)

    Lima Afonso Neto, Watson; Schürmann, Martin; Panella, Lavinia

    2015-01-01

    Despite of the advantages that enzyme immobilisation can bring to industrial biocatalysis, its utilisation is still limited to a small number of enzymes and processes. Transaminase catalysed processes are a good example where immobilisation can be of major importance and even decisive for economi...... and possibility to store the biocatalyst for more than 70 days (at room temperature) were obtained as result of the immobilisation on the selected supports.......)-selective ω-transaminases. These carriers allowed the re-use of the immobilised enzyme for 8 cycles of 24 h each, under relevant process conditions, corresponding to approximately 250 h of operation, with more than 50% of the initial activity retained. Likewise the stability towards higher temperatures...

  19. Process Evaluation Tools for Enzymatic Cascades Welcome Message

    DEFF Research Database (Denmark)

    Abu, Rohana

    improvement and implementation. Hence, the goal of this thesis is to evaluate the process concepts in enzymatic cascades in a systematic manner, using tools such as thermodynamic and kinetic analysis. Three relevant case studies have been used to exemplify the approach. In the first case study, thermodynamic......Biocatalysis is attracting significant attention from both academic and industrial scientists due to the excellent capability of enzyme to catalyse selective reactions. Recently, much interest has been shown in the application of enzymatic cascades as a useful tool in organic synthesis......, the kinetics can be controlled in a highly efficient way to achieve a sufficiently favourable conversion to a given target product. This is exemplified in the second case study, in the kinetic modelling of the formation of 2-ketoglutarate from glucoronate, the second case study. This cascade consists of 4...

  20. From dirt to industrial applications: Pseudomonas putida as a Synthetic Biology chassis for hosting harsh biochemical reactions.

    Science.gov (United States)

    Nikel, Pablo I; Chavarría, Max; Danchin, Antoine; de Lorenzo, Víctor

    2016-10-01

    The soil bacterium Pseudomonas putida is endowed with a central carbon metabolic network capable of fulfilling high demands of reducing power. This situation arises from a unique metabolic architecture that encompasses the partial recycling of triose phosphates to hexose phosphates-the so-called EDEMP cycle. In this article, the value of P. putida as a bacterial chassis of choice for contemporary, industrially-oriented metabolic engineering is addressed. The biochemical properties that make this bacterium adequate for hosting biotransformations involving redox reactions as well as toxic compounds and intermediates are discussed. Finally, novel developments and open questions in the continuous quest for an optimal microbial cell factory are presented at the light of current and future needs in the area of biocatalysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. What are the Limitations of Enzymes in Synthetic Organic Chemistry?

    Science.gov (United States)

    Reetz, Manfred T

    2016-12-01

    Enzymes have been used in organic chemistry and biotechnology for 100 years, but their widespread application has been prevented by a number of limitations, including the often-observed limited thermostability, narrow substrate scope, and low or wrong stereo- and/or regioselectivity. Directed evolution provides a means to address and generally solve these problems, especially since recent methodology development has made this protein engineering method faster, more efficient, and more reliable than in the past. This Darwinian approach to asymmetric catalysis has led to a number of industrial applications. Metabolic-pathway engineering, mutasynthesis, and fermentation are likewise enzyme-based techniques that enrich chemistry. This account outlines the scope, and particularly, the limitations, of biocatalysis. The complementary nature of enzymes and man-made catalysts is emphasized. © 2016 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Fossil energy biotechnology: A research needs assessment. Final report

    Energy Technology Data Exchange (ETDEWEB)

    1993-11-01

    The Office of Program Analysis of the US Department of Energy commissioned this study to evaluate and prioritize research needs in fossil energy biotechnology. The objectives were to identify research initiatives in biotechnology that offer timely and strategic options for the more efficient and effective uses of the Nation`s fossil resource base, particularly the early identification of new and novel applications of biotechnology for the use or conversion of domestic fossil fuels. Fossil energy biotechnology consists of a number of diverse and distinct technologies, all related by the common denominator -- biocatalysis. The expert panel organized 14 technical subjects into three interrelated biotechnology programs: (1) upgrading the fuel value of fossil fuels; (2) bioconversion of fossil feedstocks and refined products to added value chemicals; and, (3) the development of environmental management strategies to minimize and mitigate the release of toxic and hazardous petrochemical wastes.

  3. Marine Microorganisms as Source of Stereoselective Esterases and Ketoreductases: Kinetic Resolution of a Prostaglandin Intermediate

    KAUST Repository

    De Vitis, Valerio

    2014-09-30

    A screening among bacterial strains isolated from water-brine interface of the deep hypersaline anoxic basins (DHABs) of the Eastern Mediterranean was carried out for the biocatalytical resolution of racemic propyl ester of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (R,S)-1, a key intermediate for the synthesis of d-cloprostenol. Bacillus horneckiae 15A gave highly stereoselective reduction of (R,S)-1, whereas Halomonas aquamarina 9B enantioselectively hydrolysed (R,S)-1; in both cases, enantiomerically pure unreacted (R)-1 could be easily recovered and purified at molar conversion below 57–58 %, showing the potential of DHAB extremophile microbiome and marine-derived enzymes in stereoselective biocatalysis.

  4. Solvent-Free Lipase-Catalyzed Synthesis of Technical-Grade Sugar Esters and Evaluation of Their Physicochemical and Bioactive Properties

    Directory of Open Access Journals (Sweden)

    Ran Ye

    2016-05-01

    Full Text Available Technical-grade oleic acid esters of sucrose and fructose were prepared using solvent-free biocatalysis at 65 °C, without any downstream purification applied, and their physicochemical and bioactivity-related properties were evaluated and compared to a commercially available sucrose laurate emulsifier. To increase the conversion of sucrose and fructose oleate, prepared previously using solvent-free lipase-catalyzed esterification catalyzed by Rhizomucor miehei lipase (81% and 83% ester, respectively, the enzymatic reaction conditions was continued using CaSO4 to control the reactor’s air headspace and a lipase (from Candida antarctica B with a hydrophobic immobilization matrix to provide an ultralow water activity, and high-pressure homogenation, to form metastable suspensions of 2.0–3.3 micron sized saccharide particles in liquid-phase reaction media. These measures led to increased ester content of 89% and 96% for reactions involving sucrose and fructose, respectively. The monoester content among the esters decreased from 90% to <70% due to differences in regioselectivity between the lipases. The resultant technical-grade sucrose and fructose lowered the surface tension to <30 mN/m, and possessed excellent emulsification capability and stability over 36 h using hexadecane and dodecane as oils, comparable to that of sucrose laurate and Tween® 80. The technical-grade sugar esters, particularly fructose oleate, more effectively inhibited gram-positive foodborne pathogens (Lactobacillus plantarum, Pediococcus pentosaceus and Bacillus subtilis. Furthermore, all three sugar esters displayed antitumor activity, particularly the two sucrose esters. This study demonstrates the importance of controlling the biocatalysts’ water activity to achieve high conversion, the impact of a lipase’s regioselectivity in dictating product distribution, and the use of solvent-free biocatalysis to important biobased surfactants useful in foods, cosmetics

  5. Magnetic biocatalysts and their uses to obtain biodiesel and biosurfactants.

    Science.gov (United States)

    López, Carmen; Cruz-Izquierdo, Alvaro; Picó, Enrique A; García-Bárcena, Teresa; Villarroel, Noelia; Llama, María J; Serra, Juan L

    2014-01-01

    Nanobiocatalysis, as the synergistic combination of nanotechnology and biocatalysis, is rapidly emerging as a new frontier of biotechnology. The use of immobilized enzymes in industrial applications often presents advantages over their soluble counterparts, mainly in view of stability, reusability and simpler operational processing. Because of their singular properties, such as biocompatibility, large and modifiable surface and easy recovery, iron oxide magnetic nanoparticles (MNPs) are attractive super-paramagnetic materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field. Cross-linked enzyme aggregates (CLEAs) have several benefits in the context of industrial applications since they can be cheaply and easily prepared from unpurified enzyme extracts and show improved storage and operational stability against denaturation by heat and organic solvents. In this work, by using the aforementioned advantages of MNPs of magnetite and CLEAs, we prepared two robust magnetically-separable types of nanobiocatalysts by binding either soluble enzyme onto the surface of MNPs functionalized with amino groups or by cross-linking aggregates of enzyme among them and to MNPs to obtain magnetic CLEAs. For this purpose the lipase B of Candida antarctica (CALB) was used. The hydrolytic and biosynthetic activities of the resulting magnetic nanobiocatalysts were assessed in aqueous and organic media. Thus, the hydrolysis of triglycerides and the transesterification reactions to synthesize biodiesel and biosurfactants were studied using magnetic CLEAs of CALB. The efficiency and easy performance of this magnetic biocatalysis validates this proof of concept and sets the basis for the application of magnetic CLEAs at industrial scale.

  6. Enzyme and microbial technology for synthesis of bioactive oligosaccharides: an update.

    Science.gov (United States)

    Chen, Rachel

    2018-04-01

    Oligosaccharides, in either free or bound forms, play crucial roles in a wide range of biological processes. Increasing appreciation of their roles in cellular communication, interaction, pathogenesis, and prebiotic functions has stimulated tremendous interests in their synthesis. Pure and structurally defined oligosaccharides are essential for fundamental studies. On the other hand, for those with near term medical and nutraceutical applications, their large-scale synthesis is necessary. Unfortunately, oligosaccharides are notoriously difficult in their synthesis, and their enormous diverse structures leave a vast gap between what have been synthesized in laboratory and those present in various biological systems. While enzymes and microbes are nature's catalysts for oligosaccharides, their effective use is not without challenges. Using examples of galactose-containing oligosaccharides, this review analyzes the pros and cons of these two forms of biocatalysts and provides an updated view on the status of biocatalysis in this important field. Over the past few years, a large number of novel galactosidases were discovered and/or engineered for improved synthesis via transglycosylation. The use of salvage pathway for regeneration of uridine diphosphate (UDP)-galactose has made the use of Leloir glycosyltransferases simpler and more efficient. The recent success of large-scale synthesis of 2' fucosyllactose heralded the power of whole-cell biocatalysis as a scalable technology. While it still lags behind enzyme catalysis in terms of the number of oligosaccharides synthesized, an acceleration in the use of this form of biocatalyst is expected as rapid advances in synthetic biology have made the engineering of whole cell biocatalysts less arduous and less time consuming.

  7. Neutrons for Catalysis: A Workshop on Neutron Scattering Techniques for Studies in Catalysis

    International Nuclear Information System (INIS)

    Overbury, Steven H.; Coates, Leighton; Herwig, Kenneth W.; Kidder, Michelle

    2011-01-01

    This report summarizes the Workshop on Neutron Scattering Techniques for Studies in Catalysis, held at the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory (ORNL) on September 16 and 17, 2010. The goal of the Workshop was to bring experts in heterogeneous catalysis and biocatalysis together with neutron scattering experimenters to identify ways to attack new problems, especially Grand Challenge problems in catalysis, using neutron scattering. The Workshop locale was motivated by the neutron capabilities at ORNL, including the High Flux Isotope Reactor (HFIR) and the new and developing instrumentation at the SNS. Approximately 90 researchers met for 1 1/2 days with oral presentations and breakout sessions. Oral presentations were divided into five topical sessions aimed at a discussion of Grand Challenge problems in catalysis, dynamics studies, structure characterization, biocatalysis, and computational methods. Eleven internationally known invited experts spoke in these sessions. The Workshop was intended both to educate catalyst experts about the methods and possibilities of neutron methods and to educate the neutron community about the methods and scientific challenges in catalysis. Above all, it was intended to inspire new research ideas among the attendees. All attendees were asked to participate in one or more of three breakout sessions to share ideas and propose new experiments that could be performed using the ORNL neutron facilities. The Workshop was expected to lead to proposals for beam time at either the HFIR or the SNS; therefore, it was expected that each breakout session would identify a few experiments or proof-of-principle experiments and a leader who would pursue a proposal after the Workshop. Also, a refereed review article will be submitted to a prominent journal to present research and ideas illustrating the benefits and possibilities of neutron methods for catalysis research.

  8. Neutrons for Catalysis: A Workshop on Neutron Scattering Techniques for Studies in Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Overbury, Steven {Steve} H [ORNL; Coates, Leighton [ORNL; Herwig, Kenneth W [ORNL; Kidder, Michelle [ORNL

    2011-10-01

    This report summarizes the Workshop on Neutron Scattering Techniques for Studies in Catalysis, held at the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory (ORNL) on September 16 and 17, 2010. The goal of the Workshop was to bring experts in heterogeneous catalysis and biocatalysis together with neutron scattering experimenters to identify ways to attack new problems, especially Grand Challenge problems in catalysis, using neutron scattering. The Workshop locale was motivated by the neutron capabilities at ORNL, including the High Flux Isotope Reactor (HFIR) and the new and developing instrumentation at the SNS. Approximately 90 researchers met for 1 1/2 days with oral presentations and breakout sessions. Oral presentations were divided into five topical sessions aimed at a discussion of Grand Challenge problems in catalysis, dynamics studies, structure characterization, biocatalysis, and computational methods. Eleven internationally known invited experts spoke in these sessions. The Workshop was intended both to educate catalyst experts about the methods and possibilities of neutron methods and to educate the neutron community about the methods and scientific challenges in catalysis. Above all, it was intended to inspire new research ideas among the attendees. All attendees were asked to participate in one or more of three breakout sessions to share ideas and propose new experiments that could be performed using the ORNL neutron facilities. The Workshop was expected to lead to proposals for beam time at either the HFIR or the SNS; therefore, it was expected that each breakout session would identify a few experiments or proof-of-principle experiments and a leader who would pursue a proposal after the Workshop. Also, a refereed review article will be submitted to a prominent journal to present research and ideas illustrating the benefits and possibilities of neutron methods for catalysis research.

  9. Dendritic platforms for biomimicry and biotechnological applications.

    Science.gov (United States)

    Nagpal, Kalpana; Mohan, Anand; Thakur, Sourav; Kumar, Pradeep

    2018-02-15

    Dendrimers, commonly referred to as polymeric trees, offer endless opportunities for biotechnological and biomedical applications. By controlling the type, length, and molecular weight of the core, branches and end groups, respectively, the chemical functionality and topology of dendrimeric archetypes can be customized which further can be applied to achieve required solubility, biodegradability, diagnosis and other applications. Given the physicochemical variability of the dendrimers and their hybrids, this review attempts to discuss a full spectrum of recent advances and strides made by these "perfectly designed structures". An extensive biotech/biomimicry application profiling of dendrimers is provided with focus on complex archetypical designs such as protein biomimicry (angiogenic inhibitors, regenerative hydroxyapatite and collagen) and biotechnology applications. In terms of biotechnological advances, dendrimers have provided distinctive advantages in the fields of biocatalysis, microbicides, artificial lights, mitochondrial function modulation, vaccines, tissue regeneration and repair, antigen carriers and even biosensors. In addition, this review provides overview of the extensive chemo-functionalization opportunities available with dendrimers which makes them a perfect candidate for forming drug conjugates, protein hybrids, bio mimics, lipidic derivatives, metal deposits and nanoconjugates thereby making them the most multifunctional platforms for diverse biotechnological applications.

  10. Estimating Hydrogen Production Potential in Biorefineries Using Microbial Electrolysis Cell Technology

    Energy Technology Data Exchange (ETDEWEB)

    Borole, Abhijeet P [ORNL; Mielenz, Jonathan R [ORNL

    2011-01-01

    Microbial electrolysis cells (MECs) are devices that use a hybrid biocatalysis-electrolysis process for production of hydrogen from organic matter. Future biofuel and bioproducts industries are expected to generate significant volumes of waste streams containing easily degradable organic matter. The emerging MEC technology has potential to derive added- value from these waste streams via production of hydrogen. Biorefinery process streams, particularly the stillage or distillation bottoms contain underutilized sugars as well as fermentation and pretreatment byproducts. In a lignocellulosic biorefinery designed for producing 70 million gallons of ethanol per year, up to 7200 m3/hr of hydrogen can be generated. The hydrogen can either be used as an energy source or a chemical reagent for upgrading and other reactions. The energy content of the hydrogen generated is sufficient to meet 57% of the distillation energy needs. We also report on the potential for hydrogen production in existing corn mills and sugar-based biorefineries. Removal of the organics from stillage has potential to facilitate water recycle. Pretreatment and fermentation byproducts generated in lignocellulosic biorefinery processes can accumulate to highly inhibitory levels in the process streams, if water is recycled. The byproducts of concern including sugar- and lignin- degradation products such as furans and phenolics can also be converted to hydrogen in MECs. We evaluate hydrogen production from various inhibitory byproducts generated during pretreatment of various types of biomass. Finally, the research needs for development of the MEC technology and aspects particularly relevant to the biorefineries are discussed.

  11. Metabolic engineering in methanotrophic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Kalyuzhnaya, MG; Puri, AW; Lidstrom, ME

    2015-05-01

    Methane, as natural gas or biogas, is the least expensive source of carbon for (bio)chemical synthesis. Scalable biological upgrading of this simple alkane to chemicals and fuels can bring new sustainable solutions to a number of industries with large environmental footprints, such as natural gas/petroleum production, landfills, wastewater treatment, and livestock. Microbial biocatalysis with methane as a feedstock has been pursued off and on for almost a half century, with little enduring success. Today, biological engineering and systems biology provide new opportunities for metabolic system modulation and give new optimism to the concept of a methane-based bio-industry. Here we present an overview of the most recent advances pertaining to metabolic engineering of microbial methane utilization. Some ideas concerning metabolic improvements for production of acetyl-CoA and pyruvate, two main precursors for bioconversion, are presented. We also discuss main gaps in the current knowledge of aerobic methane utilization, which must be solved in order to release the full potential of methane-based biosystems. (C) 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  12. Hydrolase stabilization via entanglement in poly(propylene sulfide) nanoparticles: stability towards reactive oxygen species

    International Nuclear Information System (INIS)

    Allen, Brett L; Johnson, Jermaine D; Walker, Jeremy P

    2012-01-01

    In the advancement of green syntheses and sustainable reactions, enzymatic biocatalysis offers extremely high reaction rates and selectivity that goes far beyond the reach of chemical catalysts; however, these enzymes suffer from typical environmental constraints, e.g. operational temperature, pH and tolerance to oxidative environments. A common hydrolase enzyme, diisopropylfluorophosphatase (DFPase, EC 3.1.8.2), has demonstrated a pronounced efficacy for the hydrolysis of a variety of substrates for potential toxin remediation, but suffers from the aforementioned limitations. As a means to enhance DFPase’s stability in oxidative environments, enzymatic covalent immobilization within the polymeric matrix of poly(propylene sulfide) (PPS) nanoparticles was performed. By modifying the enzyme’s exposed lysine residues via thiolation, DFPase is utilized as a comonomer/crosslinker in a mild emulsion polymerization. The resultant polymeric polysulfide shell acts as a ‘sacrificial barrier’ by first oxidizing to polysulfoxides and polysulfones, rendering DFPase in an active state. DFPase–PPS nanoparticles thus retain activity upon exposure to as high as 50 parts per million (ppm) of hypochlorous acid (HOCl), while native DFPase is observed as inactive at 500 parts per billion (ppb). This trend is also confirmed by enzyme-generated (chloroperoxidase (CPO), EC 1.11.1.10) reactive oxygen species (ROS) including both HOCl (3 ppm) and ClO 2 (100 ppm). (paper)

  13. Predicting novel substrates for enzymes with minimal experimental effort with active learning.

    Science.gov (United States)

    Pertusi, Dante A; Moura, Matthew E; Jeffryes, James G; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E J

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of ~80% using ~33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  14. Binding mechanism and dynamic conformational change of C subunit of PKA with different pathways.

    Science.gov (United States)

    Chu, Wen-Ting; Chu, Xiakun; Wang, Jin

    2017-09-19

    The catalytic subunit of PKA (PKAc) exhibits three major conformational states (open, intermediate, and closed) during the biocatalysis process. Both ATP and substrate/inhibitor can effectively induce the conformational changes of PKAc from open to closed states. Aiming to explore the mechanism of this allosteric regulation, we developed a coarse-grained model and analyzed the dynamics of conformational changes of PKAc during binding by performing molecular dynamics simulations for apo PKAc, binary PKAc (PKAc with ATP, PKAc with PKI), and ternary PKAc (PKAc with ATP and PKI). Our results suggest a mixed binding mechanism of induced fit and conformational selection, with the induced fit dominant. The ligands can drive the movements of Gly-rich loop as well as some regions distal to the active site in PKAc and stabilize them at complex state. In addition, there are two parallel pathways (pathway with PKAc-ATP as an intermediate and pathway PKAc-PKI as an intermediate) during the transition from open to closed states. By molecular dynamics simulations and rate constant analyses, we find that the pathway through PKAc-ATP intermediate is the main binding route from open to closed state because of the fact that the bound PKI will hamper ATP from successful binding and significantly increase the barrier for the second binding subprocess. These findings will provide fundamental insights of the mechanisms of PKAc conformational change upon binding.

  15. Noncovalent functionalization of graphene by CdS nanohybrids for electrochemical applications

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Li [Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); Qi, Wei, E-mail: qiwei@tju.edu.cn [Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072 (China); Su, Rongxin [Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072 (China); Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072 (China); He, Zhimin [Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072 (China); State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072 (China); Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072 (China)

    2014-10-01

    Graphene–CdS (GR–CdS) nanocomposites were synthesized via a noncovalent functionalization process. To retain the intrinsic electronic and mechanical properties of graphene, the pristine graphene was firstly modified with 1-aminopyrene based on a strong π–π bond between the pyrenyl groups and the carbon rings of the graphene. Then the CdS nanocrystals were uniformly grown on the amino-graphene. The GR–CdS nanocomposites were characterized by UV–vis spectroscopy and scanning electron microscopy. A glucose biosensor was then fabricated based on the as-prepared GR–CdS nanocomposite by immobilizing glucose oxidase (GOD) in a chitosan thin film on a glassy carbon electrode. Direct electron transfer between GOD and the electrode was achieved and the biosensor showed good electrocatalytic activity with glucose ranging from 0.5 to 7.5 mM and a sensitivity of 45.4 μA mM{sup −1} cm{sup −2}. This work provided a simple and nondestructive functionalization strategy to fabricate graphene-based hybrid nanomaterials and it is expected that this composite film may find more potential applications in biosensors and biocatalysis. - Highlights: • A simple noncovalent approach to synthesize graphene–CdS (GR–CdS) nanocomposites • Direct electrochemistry of glucose oxidase based on synergistic effect of GR–CdS • A sensitive glucose biosensor was fabricated based on the GR–CdS hybrids.

  16. Noncovalent functionalization of graphene by CdS nanohybrids for electrochemical applications

    International Nuclear Information System (INIS)

    Wang, Li; Qi, Wei; Su, Rongxin; He, Zhimin

    2014-01-01

    Graphene–CdS (GR–CdS) nanocomposites were synthesized via a noncovalent functionalization process. To retain the intrinsic electronic and mechanical properties of graphene, the pristine graphene was firstly modified with 1-aminopyrene based on a strong π–π bond between the pyrenyl groups and the carbon rings of the graphene. Then the CdS nanocrystals were uniformly grown on the amino-graphene. The GR–CdS nanocomposites were characterized by UV–vis spectroscopy and scanning electron microscopy. A glucose biosensor was then fabricated based on the as-prepared GR–CdS nanocomposite by immobilizing glucose oxidase (GOD) in a chitosan thin film on a glassy carbon electrode. Direct electron transfer between GOD and the electrode was achieved and the biosensor showed good electrocatalytic activity with glucose ranging from 0.5 to 7.5 mM and a sensitivity of 45.4 μA mM −1 cm −2 . This work provided a simple and nondestructive functionalization strategy to fabricate graphene-based hybrid nanomaterials and it is expected that this composite film may find more potential applications in biosensors and biocatalysis. - Highlights: • A simple noncovalent approach to synthesize graphene–CdS (GR–CdS) nanocomposites • Direct electrochemistry of glucose oxidase based on synergistic effect of GR–CdS • A sensitive glucose biosensor was fabricated based on the GR–CdS hybrids

  17. Microbial Biotransformation to Obtain New Antifungals

    Science.gov (United States)

    Bianchini, Luiz F.; Arruda, Maria F. C.; Vieira, Sergio R.; Campelo, Patrícia M. S.; Grégio, Ana M. T.; Rosa, Edvaldo A. R.

    2015-01-01

    Antifungal drugs belong to few chemical groups and such low diversity limits the therapeutic choices. The urgent need of innovative options has pushed researchers to search new bioactive molecules. Literature regarding the last 15 years reveals that different research groups have used different approaches to achieve such goal. However, the discovery of molecules with different mechanisms of action still demands considerable time and efforts. This review was conceived to present how Pharmaceutical Biotechnology might contribute to the discovery of molecules with antifungal properties by microbial biotransformation procedures. Authors present some aspects of (1) microbial biotransformation of herbal medicines and food; (2) possibility of major and minor molecular amendments in existing molecules by biocatalysis; (3) methodological improvements in processes involving whole cells and immobilized enzymes; (4) potential of endophytic fungi to produce antimicrobials by bioconversions; and (5) in silico research driving to the improvement of molecules. All these issues belong to a new conception of transformation procedures, so-called “green chemistry,” which aims the highest possible efficiency with reduced production of waste and the smallest environmental impact. PMID:26733974

  18. Halloysite Clay Nanotubes for Enzyme Immobilization.

    Science.gov (United States)

    Tully, Joshua; Yendluri, Raghuvara; Lvov, Yuri

    2016-02-08

    Halloysite clay is an aluminosilicate nanotube formed by rolling flat sheets of kaolinite clay. They have a 15 nm lumen, 50-70 nm external diameter, length of 0.5-1 μm, and different inside/outside chemistry. Due to these nanoscale properties, they are used for loading, storage, and controlled release of active chemical agents, including anticorrosions, biocides, and drugs. We studied the immobilization in halloysite of laccase, glucose oxidase, and lipase. Overall, negatively charged proteins taken above their isoelectric points were mostly loaded into the positively charged tube's lumen. Typical tube loading with proteins was 6-7 wt % from which one-third was released in 5-10 h and the other two-thirds remained, providing enhanced biocatalysis in nanoconfined conditions. Immobilized lipase showed enhanced stability at acidic pH, and the optimum pH shifted to more alkaline pH. Immobilized laccase was more stable with respect to time, and immobilized glucose oxidase showed retention of enzymatic activity up to 70 °C, whereas the native sample was inactive.

  19. Protein engineering and its applications in food industry.

    Science.gov (United States)

    Kapoor, Swati; Rafiq, Aasima; Sharma, Savita

    2017-07-24

    Protein engineering is a young discipline that has been branched out from the field of genetic engineering. Protein engineering is based on the available knowledge about the proteins structure/function(s), tools/instruments, software, bioinformatics database, available cloned gene, knowledge about available protein, vectors, recombinant strains and other materials that could lead to change in the protein backbone. Protein produced properly from genetic engineering process means a protein that is able to fold correctly and to do particular function(s) efficiently even after being subjected to engineering practices. Protein is modified through its gene or chemically. However, modification of protein through gene is easier. There is no specific limitation of Protein Engineering tools; any technique that can lead to change the protein constituent of amino acid and result in the modification of protein structure/function is in the frame of Protein Engineering. Meanwhile, there are some common tools used to reach a specific target. More active industrial and pharmaceutical based proteins have been invented by the field of Protein Engineering to introduce new function as well as to change its interaction with surrounding environment. A variety of protein engineering applications have been reported in the literature. These applications range from biocatalysis for food and industry to environmental, medical and nanobiotechnology applications. Successful combinations of various protein engineering methods had led to successful results in food industries and have created a scope to maintain the quality of finished product after processing.

  20. Efficient production of D-tagatose using a food-grade surface display system.

    Science.gov (United States)

    Liu, Yi; Li, Sha; Xu, Hong; Wu, Lingtian; Xu, Zheng; Liu, Jing; Feng, Xiaohai

    2014-07-16

    D-tagatose, a functional sweetener, is commonly transformed from D-galactose by L-arabinose isomerase (L-AI). In this study, a novel type of biocatalyst, L-AI from Lactobacillus fermentum CGMCC2921 displayed on the spore surface of Bacillus subtilis 168, was developed for producing D-tagatose. The anchored L-AI, exhibiting the relatively high bioactivity, suggested that the surface display system using CotX as the anchoring protein was successfully constructed. The stability of the anchored L-AI was significantly improved. Specifically, the consolidation of thermal stability representing 87% of relative activity was retained even at 80 °C for 30 min, which remarkably favored the production of D-tagatose. Under the optimal conditions, the robust spores can convert 75% D-galactose (100 g/L) into D-tagatose after 24 h, and the conversion rate remained at 56% at the third cycle. Therefore, this biocatalysis system, which could express the target enzyme on the food-grade vector, was an alternative method for the value-added production of D-tagatose.

  1. Bioengineered 2'-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines.

    Science.gov (United States)

    Weichert, Stefan; Jennewein, Stefan; Hüfner, Eric; Weiss, Christel; Borkowski, Julia; Putze, Johannes; Schroten, Horst

    2013-10-01

    Human milk oligosaccharides help to prevent infectious diseases in breastfed infants. Larger scale testing, particularly in animal models and human clinical studies, is still limited due to shortened availability of more complex oligosaccharides. The purpose of this study was to evaluate 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) synthesized by whole-cell biocatalysis for their biological activity in vitro. Therefore, we have tested these oligosaccharides for their inhibitory potential of pathogen adhesion in two different human epithelial cell lines. 2'-FL could inhibit adhesion of Campylobacter jejuni, enteropathogenic Escherichia coli, Salmonella enterica serovar fyris, and Pseudomonas aeruginosa to the intestinal human cell line Caco-2 (reduction of 26%, 18%, 12%, and 17%, respectively), as could be shown for 3-FL (enteropathogenic E coli 29%, P aeruginosa 26%). Furthermore, adherence of P aeruginosa to the human respiratory epithelial cell line A549 was significantly inhibited by 2'-FL and 3-FL (reduction of 24% and 23%, respectively). These results confirm the biological and functional activity of biotechnologically synthesized human milk oligosaccharides. Mass-tailored human milk oligosaccharides could be used in the future to supplement infant formula ingredients or as preventatives to reduce the impact of infectious diseases. © 2013 Elsevier Inc. All rights reserved.

  2. 1,2-Dielaidoylphosphocholine/1,2-dimyristoylphosphoglycerol supported phospholipid bilayer formation in calcium and calcium-free buffer

    International Nuclear Information System (INIS)

    Evans, Kervin O.

    2012-01-01

    Phospholipid membranes are useful in the field of biocatalysis because a supported phospholipid membrane can create a biomimetic platform where biocatalytic processes can readily occur. In this work, supported bilayer formation from sonicated phospholipid vesicles containing 1,2-dielaidoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] was studied using a quartz crystal microbalance with dissipation monitoring and an atomic force microscope. The molar percentages of DEPC and DMPG were varied to determine the effect of overall lipid composition on supported bilayer formation. This work also explored the effect that calcium ion concentration had on supported bilayer formation. Results show that vesicles with up to 50 mol% dimyristoylphosphoglycerol can form a supported bilayer without the presence of calcium ions; however, supported bilayer formation in calcium buffer was inhibited as the anionic (negatively charged) lipid concentration increased. Data suggest that supported phospholipid bilayer formation in the absence of Ca 2+ from vesicles containing negatively charged lipids is specific to phosphatidylglycerol. - Highlights: ► SPB formation of DEPC vesicles containing 0 to 50 mol% DMPG monitored using QCM-D. ► Ca 2+ inhibited SPB formation of DEPC vesicles containing 30 to 50 mol% DMPG. ► Vesicles containing DMPG at 0 to 50 mol% formed SPB in buffer free of Ca 2+ .

  3. Single-step production of the simvastatin precursor monacolin J by engineering of an industrial strain of Aspergillus terreus.

    Science.gov (United States)

    Huang, Xuenian; Liang, Yajing; Yang, Yong; Lu, Xuefeng

    2017-07-01

    Monacolin J is a key precursor for the synthesis of simvastatin (Zocor), an important drug for treating hypercholesterolemia. Industrially, monacolin J is manufactured through alkaline hydrolysis of lovastatin, a fungal polyketide produced by Aspergillus terreus. Multistep chemical processes for the conversion of lovastatin to simvastatin are laborious, cost expensive and environmentally unfriendly. A biocatalysis process for monacolin J conversion to simvastatin has been developed. However, direct bioproduction of monacolin J has not yet been achieved. Here, we identified a lovastatin hydrolase from Penicillium chrysogenum, which displays a 232-fold higher catalytic efficiency for the in vitro hydrolysis of lovastatin compared to a previously patented hydrolase, but no activity for simvastatin. Furthermore, we showed that an industrial A. terreus strain heterologously expressing this lovastatin hydrolase can produce monacolin J through single-step fermentation with high efficiency, approximately 95% of the biosynthesized lovastatin was hydrolyzed to monacolin J. Our results demonstrate a simple and green technical route for the production of monacolin J, which makes complete bioproduction of the cholesterol-lowering drug simvastatin feasible and promising. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  4. Magnetic biocatalysts and their uses to obtain bioproducts

    Science.gov (United States)

    López, Carmen; Cruz-Izquierdo, Álvaro; Picó, Enrique; García-Bárcena, Teresa; Villarroel, Noelia; Llama, María; Serra, Juan

    2014-08-01

    Nanobiocatalysis, as the synergistic combination of nanotechnology and biocatalysis, is rapidly emerging as a new frontier of biotechnology. The use of immobilized enzymes in industrial applications often presents advantages over their soluble counterparts, mainly in view of stability, reusability and simpler operational processing. Because of their singular properties, such as biocompatibility, large and modifiable surface and easy recovery, iron oxide magnetic nanoparticles (MNPs) are attractive super-paramagnetic materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field. Cross-linked enzyme aggregates (CLEAs) have several benefits in the context of industrial applications since they can be cheaply and easily prepared from unpurified enzyme extracts and show improved storage and operational stability against denaturation by heat and organic solvents. In this work, by using the aforementioned advantages of MNPs of magnetite and CLEAs, we prepared two robust magnetically-separable types of nanobiocatalysts by binding either soluble enzyme onto the surface of MNPs functionalized with amino groups or by cross-linking aggregates of enzyme among them and to MNPs to obtain magnetic CLEAs. For this purpose the lipase B of Candida antarctica (CALB) was used. The hydrolytic and biosynthetic activities of the resulting magnetic nanobiocatalysts were assessed in aqueous and organic media and compared between them and to those showed by the corresponding soluble enzyme. Thus, the hydrolysis of triglycerides or the transesterification reactions to synthesize biodiesel and biosurfactants were studied using magnetic CLEAs of CALB.

  5. An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.

    Science.gov (United States)

    Zuo, Ran; Zhang, Yi; Huguet-Tapia, Jose C; Mehta, Mishal; Dedic, Evelina; Bruner, Steven D; Loria, Rosemary; Ding, Yousong

    2016-05-01

    Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Recent advances in exploiting ionic liquids for biomolecules: Solubility, stability and applications.

    Science.gov (United States)

    Sivapragasam, Magaret; Moniruzzaman, Muhammad; Goto, Masahiro

    2016-08-01

    The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) - potentially attractive "green" and "designer" solvents - rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule-based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein-, enzyme-, and DNA-based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule-friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL-based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule-based applications. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Koushik; Balog, Eva Rose M.; Sista, Prakash; Williams, Darrick J.; Martinez, Jennifer S., E-mail: jenm@lanl.gov, E-mail: rcrocha@lanl.gov; Rocha, Reginaldo C., E-mail: jenm@lanl.gov, E-mail: rcrocha@lanl.gov [Center for Integrated Nanotechnologies, Materials Physics and Applications Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States); Kelly, Daniel [Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States)

    2014-02-01

    We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP) as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca{sup 2+} or Cu{sup 2+}, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon the temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.

  8. Temperature-dependent morphology of hybrid nanoflowers from elastin-like polypeptides

    Directory of Open Access Journals (Sweden)

    Koushik Ghosh

    2014-02-01

    Full Text Available We report a method for creating hybrid organic-inorganic “nanoflowers” using calcium or copper ions as the inorganic component and a recombinantly expressed elastin-like polypeptide (ELP as the organic component. Polypeptides provide binding sites for the dynamic coordination with metal ions, and then such noncovalent complexes become nucleation sites for primary crystals of metal phosphates. We have shown that the interaction between the stimuli-responsive ELP and Ca2+ or Cu2+, in the presence of phosphate, leads to the growth of micrometer-sized particles featuring nanoscale patterns shaped like flower petals. The morphology of these flower-like composite structures is dependent upon the temperature of growth and has been characterized by scanning electron microscopy. The composition of nanoflowers has also been analyzed by energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The temperature-dependent morphologies of these hybrid nanostructures, which arise from the controllable phase transition of ELPs, hold potential for morphological control of biomaterials in emerging applications such as tissue engineering and biocatalysis.

  9. Phospholipid-sepiolite biomimetic interfaces for the immobilization of enzymes.

    Science.gov (United States)

    Wicklein, Bernd; Darder, Margarita; Aranda, Pilar; Ruiz-Hitzky, Eduardo

    2011-11-01

    Biomimetic interfaces based on phosphatidylcholine (PC) assembled to the natural silicate sepiolite were prepared for the stable immobilization of the urease and cholesterol oxidase enzymes. This is an important issue in practical advanced applications such as biocatalysis or biosensing. The supported lipid bilayer (BL-PC), prepared from PC adsorption, was used for immobilization of enzymes and the resulting biomimetic systems were compared to several other supported layers including a lipid monolayer (ML-PC), a mixed phosphatidylcholine/octyl-galactoside layer (PC-OGal), a cetyltrimethylammonium monolayer (CTA), and also to the bare sepiolite surface. Interfacial characteristics of these layers were investigated with a focus on layer packing density, hydrophilicity/hydrophobicity, and surface charge, which are being considered as key points for enzyme immobilization and stabilization of their biological activity. Cytoplasmic urease and membrane-bound cholesterol oxidase, which served as model enzymes, were immobilized on the different PC-based hybrid materials to probe their biomimetic character. Enzymatic activity was assessed by cyclic voltammetry and UV-vis spectrophotometry. The resulting enzyme/bio-organoclay hybrids were applied as active phase of a voltammetric urea biosensor and cholesterol bioreactor, respectively. Urease supported on sepiolite/BL-PC proved to maintain its enzymatic activity over several months while immobilized cholesterol oxidase demonstrated high reusability as biocatalyst. The results emphasize the good preservation of bioactivity due to the accommodation of the enzymatic system within the biomimetic lipid interface on sepiolite.

  10. Tailored functionalization of iron oxide nanoparticles for MRI, drug delivery, magnetic separation and immobilization of biosubstances.

    Science.gov (United States)

    Hola, Katerina; Markova, Zdenka; Zoppellaro, Giorgio; Tucek, Jiri; Zboril, Radek

    2015-11-01

    In this critical review, we outline various covalent and non-covalent approaches for the functionalization of iron oxide nanoparticles (IONPs). Tuning the surface chemistry and design of magnetic nanoparticles are described in relation to their applicability in advanced medical technologies and biotechnologies including magnetic resonance imaging (MRI) contrast agents, targeted drug delivery, magnetic separations and immobilizations of proteins, enzymes, antibodies, targeting agents and other biosubstances. We review synthetic strategies for the controlled preparation of IONPs modified with frequently used functional groups including amine, carboxyl and hydroxyl groups as well as the preparation of IONPs functionalized with other species, e.g., epoxy, thiol, alkane, azide, and alkyne groups. Three main coupling strategies for linking IONPs with active agents are presented: (i) chemical modification of amine groups on the surface of IONPs, (ii) chemical modification of bioactive substances (e.g. with fluorescent dyes), and (iii) the activation of carboxyl groups mainly for enzyme immobilization. Applications for drug delivery using click chemistry linking or biodegradable bonds are compared to non-covalent methods based on polymer modified condensed magnetic nanoclusters. Among many challenges, we highlight the specific surface engineering allowing both therapeutic and diagnostic applications (theranostics) of IONPs and magnetic/metallic hybrid nanostructures possessing a huge potential in biocatalysis, green chemistry, magnetic bioseparations and bioimaging. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Magnetic biocatalysts and their uses to obtain bioproducts

    Directory of Open Access Journals (Sweden)

    Carmen eLópez

    2014-08-01

    Full Text Available Nanobiocatalysis, as the synergistic combination of nanotechnology and biocatalysis, is rapidly emerging as a new frontier of biotechnology. The use of immobilized enzymes in industrial applications often presents advantages over their soluble counterparts, mainly in view of stability, reusability and simpler operational processing. Because of their singular properties, such as biocompatibility, large and modifiable surface and easy recovery, iron oxide magnetic nanoparticles (MNPs are attractive super-paramagnetic materials that serve as a support for enzyme immobilization and facilitate separations by applying an external magnetic field. Cross-linked enzyme aggregates (CLEAs have several benefits in the context of industrial applications since they can be cheaply and easily prepared from unpurified enzyme extracts and show improved storage and operational stability against denaturation by heat and organic solvents. In this work, by using the aforementioned advantages of MNPs of magnetite and CLEAs, we prepared two robust magnetically-separable types of nanobiocatalysts by binding either soluble enzyme onto the surface of MNPs functionalized with amino groups or by cross-linking aggregates of enzyme among them and to MNPs to obtain magnetic CLEAs. For this purpose the lipase B of Candida antarctica (CALB was used. The hydrolytic and biosynthetic activities of the resulting magnetic nanobiocatalysts were assessed in aqueous and organic media and compared between them and to those showed by the corresponding soluble enzyme. Thus, the hydrolysis of triglycerides or the transesterification reactions to synthesize biodiesel and biosurfactants were studied using magnetic CLEAs of CALB.

  12. Glycolysis of poly(3-hydroxybutyrate) via enzyme catalysis; Glicolise do poli(3-hidroxibutirato) por via enzimatica

    Energy Technology Data Exchange (ETDEWEB)

    Paula, Everton Luiz de, E-mail: everton2804@gmail.com [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Departamento de Quimica; Campos, Tiago Ferreira; Mano, Valdir [Universidade Federal de Sao Joao del-Rei (UFSJ), MG (Brazil). Departamento de Ciencias Naturais

    2014-05-15

    Poly(3-hydroxybutyrate), PHB, is a polymer with broad potential applications because of its biodegradability and biocompatibility. However, its high crystallinity is a limiting factor for many applications. To overcome this drawback, one strategy currently employed involves the reduction of the molecular weight of PHB with the concomitant formation of end-functionalized chains, such as those obtained via glycolysis. The glycolysis of PHB can be catalyzed by acid, base, or organometallic compounds. However, to our knowledge, there are no reports regarding PHB glycolysis catalyzed enzymatically. Among the major types of enzymes used in biocatalysis, the lipases stand out because they have the ability to catalyze reactions in both aqueous and organic media. Thus, in this study, we performed the enzymatic glycolysis of PHB using the lipase Amano PS (Pseudomonas cepacia) with ethane-1,2-diol (ethylene glycol) as the functionalizing agent. The results indicated that the glycolysis was successful and afforded hydroxyl-terminated oligomeric PHB polyols. Nuclear magnetic resonance spectra of the products showed characteristic signals for the terminal hydroxyl groups of the polyols, while thermogravimetric and differential scanning calorimetry analyses confirmed an increase in the thermal stability and a decrease in the crystallinity of the polyols compared with the starting PHB polymer, which were both attributed to the reduction in the molecular weight due to glycolysis. (author)

  13. Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

    Science.gov (United States)

    Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

    2014-01-01

    Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

  14. A preliminary investigation on the interaction between sol-gel immobilized glucose oxidase and freely diffusing glucose by means of two-photon microscopy

    Science.gov (United States)

    Delfino, I.; Portaccio, M.; De Rosa, M.; Lepore, M.

    2013-02-01

    To study immobilized protein interactions with dissolved substrates is a very important topic both from a fundamental and technological standpoint. In the present report we illustrate the preliminary results obtained on sol-gel immobilized glucose oxidase (GOD) using a standard de-scanned two-photon microscope based on a modified confocal scanhead with internal detectors and a Ti:sapphire laser as a source. Data acquisition conditions were preliminary defined using functionalized beads of different dimensions. Various sol-gel supports were then investigated by monitoring endogeneous fluorescence due to the flavoadenine (FAD) molecules, present in GOD. Linear absorption and fluorescence spectroscopy along with Fourier Transform Infrared microscopy were employed for a full-optical characterization of the samples. The results show that GOD immobilization processes can be successfully monitored in some cases and also the interaction with glucose could be studied by this approach. This assessment holds potentials to better understand the characteristic of immobilized enzymes biocatalysis and to develop new biosensing schemes.

  15. Biocatalytic synthesis and polymerization via ROMP of new biobased phenolic monomers: a greener process towards sustainable antioxidant polymers

    Science.gov (United States)

    Diot-Néant, Florian; Migeot, Loïs; Hollande, Louis; Reano, Felix A.; Domenek, Sandra; Allais, Florent

    2017-12-01

    Antioxidant norbornene-based monomers bearing biobased sterically hindered phenols (SHP) - NDF (norbornene dihydroferulate) and NDS (norbornene dihydrosinapate) - have been successfully prepared through biocatalysis from naturally occurring ferulic and sinapic acids, respectively, in presence of Candida antarctica Lipase B (Cal-B). The ring opening metathesis polymerization (ROMP) of these monomers was investigated according to ruthenium catalyst type (GI) vs. (HGII) and monomer to catalyst molar ratio ([M]/[C]). The co-polymerization of antioxidant functionalized monomer (NDF or NDS) and non-active norbornene (N) has also been performed in order to adjust the number of SHP groups present per weight unit and tune the antioxidant activity of the copolymers. The polydispersity of the resulting copolymers was readily improved by a simple acetone wash to provide antioxidant polymers with well-defined structures. After hydrogenation with p-toluenesulfonylhydrazine (p-TSH), the radical scavenging ability of the resulting saturated polymers was evaluated using α,α-diphenyl-β-picrylhydrazyl (DPPH) analysis. Results demonstrated that polymers bearing sinapic acid SHP exhibited higher antiradical activity than the polymer bearing ferulic acid SHP. In addition it was also shown that only a small SHP content was needed in the copolymers to exhibit a potent antioxidant activity.

  16. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

    2010-01-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  17. From gene to biorefinery: microbial β-etherases as promising biocatalysts for lignin valorization

    Directory of Open Access Journals (Sweden)

    Pere ePicart

    2015-09-01

    Full Text Available The set-up of biorefineries for the valorization of lignocellulosic biomass will be core in the future to reach sustainability targets. In this area, biomass-degrading enzymes are attracting significant research interest for their potential in the production of chemicals and biofuels from renewable feedstock. Gluthatione-dependent β-etherases are emerging enzymes for the biocatalytic depolymerization of lignin, a heterogeneous aromatic polymer abundant in Nature. They selectively catalyze the reductive cleavage of β-O-4 aryl-ether bonds which account for 45-60% of linkages present in lignin. Hence, application of β-etherases in lignin depolymerization would enable a specific lignin breakdown, selectively yielding (valuable low-molecular-mass aromatics. Albeit β-etherases have been biochemically known for decades, only very recently novel β-etherases have been identified and thoroughly characterized for lignin valorization, expanding the enzyme toolbox for efficient β-O-4 aryl-ether bond cleavage. Given their emerging importance and potential, this mini-review discusses recent developments in the field of β-etherase biocatalysis covering all aspects from enzyme identification to biocatalytic applications with real lignin samples.

  18. From gene to biorefinery: microbial β-etherases as promising biocatalysts for lignin valorization.

    Science.gov (United States)

    Picart, Pere; de María, Pablo Domínguez; Schallmey, Anett

    2015-01-01

    The set-up of biorefineries for the valorization of lignocellulosic biomass will be core in the future to reach sustainability targets. In this area, biomass-degrading enzymes are attracting significant research interest for their potential in the production of chemicals and biofuels from renewable feedstock. Glutathione-dependent β-etherases are emerging enzymes for the biocatalytic depolymerization of lignin, a heterogeneous aromatic polymer abundant in nature. They selectively catalyze the reductive cleavage of β-O-4 aryl-ether bonds which account for 45-60% of linkages present in lignin. Hence, application of β-etherases in lignin depolymerization would enable a specific lignin breakdown, selectively yielding (valuable) low-molecular-mass aromatics. Albeit β-etherases have been biochemically known for decades, only very recently novel β-etherases have been identified and thoroughly characterized for lignin valorization, expanding the enzyme toolbox for efficient β-O-4 aryl-ether bond cleavage. Given their emerging importance and potential, this mini-review discusses recent developments in the field of β-etherase biocatalysis covering all aspects from enzyme identification to biocatalytic applications with real lignin samples.

  19. Nanodiamonds as pH-switchable oxidation and reduction catalysts with enzyme-like activities for immunoassay and antioxidant applications.

    Science.gov (United States)

    Chen, T M; Tian, X M; Huang, L; Xiao, J; Yang, G W

    2017-10-19

    Nanodiamonds (NDs) have recently become a focus of interest from the viewpoints of both science and technology. Their intriguing properties make them suitable as biologically active substrates, in biosensor applications as well as diagnostic and therapeutic biomedical imaging probes. Here, we demonstrate that NDs, as oxidation and reduction catalysts, possess intrinsic enzyme mimetic properties of oxidase, peroxidase and catalase, and these behaviors can be switched by modulating the pH value. NDs not only catalyze the reduction of oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) at acidic pH, but also catalyze the dismutation decomposition of H 2 O 2 to produce O 2 at alkaline pH. It was proposed that the molecular mechanism of their peroxidase-like activity is electron-transfer acceleration, the source of which is likely derived from oxygen containing functional groups on their surface. Based on the color reaction, a nanodiamond-based enzyme linked immunosorbent assay (ELISA) was established for the detection of immunoglobulin G (IgG). Surprisingly, NDs display an excellent antioxidant activity due to the protective effect against H 2 O 2 -induced cellular oxidative damage. These findings make NDs a promising enzyme mimetic candidate and expand their applications in biocatalysis, bioassays and nano-biomedicine.

  20. Electrocatalytic hydrogen evolution under densely buffered neutral pH conditions

    KAUST Repository

    Shinagawa, Tatsuya

    2015-08-18

    Under buffered neutral pH conditions, solute concentrations drastically influence the hydrogen evolution reaction (HER). The iR-free HER performance as a function of solute concentration was found to exhibit a volcano-shaped trend in sodium phosphate solution at pH 5, with the maximum occurring at 2 M. A detailed microkinetic model that includes calculated activity coefficients, solution resistance, and mass-transport parameters accurately describes the measured values, clarifying that the overall HER performance is predominantly governed by mass-transport of slow phosphate ions (weak acid). In the HER at the optimum concentration of approximately 2 M sodium phosphate at pH 5, our theoretical model predicts that the concentration overpotential accounts for more than half of the required overpotential. The substantial concentration overpotential would originate from the electrolyte property, suggesting that the proper electrolyte engineering will result in an improved apparent HER performances. The significance of concentration overpotential shown in the study is critical in the advancement of electrocatalysis, biocatalysis, and photocatalysis.

  1. On-Demand Production of Flow-Reactor Cartridges by 3D Printing of Thermostable Enzymes.

    Science.gov (United States)

    Maier, Manfred; Radtke, Carsten P; Hubbuch, Jürgen; Niemeyer, Christof M; Rabe, Kersten S

    2018-05-04

    The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three-dimensional (3D) printing techniques offer high-throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose-based inks for a simple temperature-controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D-printed parts for a continuous, two-step sequential biotransformation in a fluidic setup. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Bioprospecting Archaea: Focus on Extreme Halophiles

    KAUST Repository

    Antunes, André

    2016-12-12

    In 1990, Woese et al. divided the Tree of Life into three separate domains: Eukarya, Bacteria, and Archaea. Archaea were originally perceived as little more than “odd bacteria” restricted to extreme environmental niches, but later discoveries challenged this assumption. Members of this domain populate a variety of unexpected environments (e.g. soils, seawater, and human bodies), and we currently witness ongoing massive expansions of the archaeal branch of the Tree of Life. Archaea are now recognized as major players in the biosphere and constitute a significant fraction of the earth’s biomass, yet they remain underexplored. An ongoing surge in exploration efforts is leading to an increase in the (a) number of isolated strains, (b) associated knowledge, and (c) utilization of Archaea in biotechnology. They are increasingly employed in fields as diverse as biocatalysis, biocomputing, bioplastic production, bioremediation, bioengineering, food, pharmaceuticals, and nutraceuticals. This chapter provides a general overview on bioprospecting Archaea, with a particular focus on extreme halophiles. We explore aspects such as diversity, ecology, screening techniques and biotechnology. Current and future trends in mining for applications are discussed.

  3. Enzymes as modular catalysts for redox half-reactions in H2-powered chemical synthesis: from biology to technology.

    Science.gov (United States)

    Reeve, Holly A; Ash, Philip A; Park, HyunSeo; Huang, Ailun; Posidias, Michalis; Tomlinson, Chloe; Lenz, Oliver; Vincent, Kylie A

    2017-01-15

    The present study considers the ways in which redox enzyme modules are coupled in living cells for linking reductive and oxidative half-reactions, and then reviews examples in which this concept can be exploited technologically in applications of coupled enzyme pairs. We discuss many examples in which enzymes are interfaced with electronically conductive particles to build up heterogeneous catalytic systems in an approach which could be termed synthetic biochemistry We focus on reactions involving the H + /H 2 redox couple catalysed by NiFe hydrogenase moieties in conjunction with other biocatalysed reactions to assemble systems directed towards synthesis of specialised chemicals, chemical building blocks or bio-derived fuel molecules. We review our work in which this approach is applied in designing enzyme-modified particles for H 2 -driven recycling of the nicotinamide cofactor NADH to provide a clean cofactor source for applications of NADH-dependent enzymes in chemical synthesis, presenting a combination of published and new work on these systems. We also consider related photobiocatalytic approaches for light-driven production of chemicals or H 2 as a fuel. We emphasise the techniques available for understanding detailed catalytic properties of the enzymes responsible for individual redox half-reactions, and the importance of a fundamental understanding of the enzyme characteristics in enabling effective applications of redox biocatalysis. © 2017 The Author(s).

  4. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

    Science.gov (United States)

    Payne, Karl Ap; Quezada, Carolina P; Fisher, Karl; Dunstan, Mark S; Collins, Fraser A; Sjuts, Hanno; Levy, Colin; Hay, Sam; Rigby, Stephen Ej; Leys, David

    2015-01-22

    Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

  5. Extraction and Application of Laccases from Shimeji Mushrooms (Pleurotus ostreatus Residues in Decolourisation of Reactive Dyes and a Comparative Study Using Commercial Laccase from Aspergillus oryzae

    Directory of Open Access Journals (Sweden)

    Ricardo Sposina S. Teixeira

    2010-01-01

    Full Text Available Oxidases are able to degrade organic pollutants; however, high costs associated with biocatalysts production still hinder their use in environmental biocatalysis. Our study compared the action of a commercial laccase from Aspergillus oryzae and a rich extract from Pleurotus ostreatus cultivation residues in decolourisation of reactive dyes: Drimaren Blue X-3LR (DMBLR, Drimaren Blue X-BLN (DMBBLN, Drimaren Rubinol X-3LR (DMR, and Drimaren Blue C-R (RBBR. The colour removal was evaluated by considering dye concentration, reaction time, absence or presence of the mediator ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, and the source of laccase. The presence of ABTS was essential for decolourisation of DMR (80–90%, 1 h and RBBR (80–90%, 24 h with both laccases. The use of ABTS was not necessary in reactions containing DMBLR (85–97%, 1 h and DMBBLN (63–84%, 24 h. The decolourisation of DMBBLN by commercial laccase showed levels near 60% while the crude extract presented 80% in 24 h.

  6. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    Energy Technology Data Exchange (ETDEWEB)

    Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: zydi@mail.hust.edu.c [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)

    2010-07-15

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  7. Versatile de novo enzyme activity in capsid proteins from an engineered M13 bacteriophage library.

    Science.gov (United States)

    Casey, John P; Barbero, Roberto J; Heldman, Nimrod; Belcher, Angela M

    2014-11-26

    Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, we developed a biochemical platform for engineering de novo semisynthetic enzymes, functionally modular and widely stable, based on the M13 bacteriophage. The hydrolytic bacteriophage described in this paper catalyzes a range of carboxylic esters, is active from 25 to 80 °C, and demonstrates greater efficiency in DMSO than in water. The platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work will tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality.

  8. Protein design for pathway engineering.

    Science.gov (United States)

    Eriksen, Dawn T; Lian, Jiazhang; Zhao, Huimin

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Carbohydrates in sustainable development I. Renewable resources for chemistry and biotechnology

    Energy Technology Data Exchange (ETDEWEB)

    Rauter, Amelia, P. [Lisboa Univ. (Portugal). Dept. Quimica e Bioquimica; Vogel, Pierre [Swiss Institute of Technology (EPFL), Lausanne (Switzerland). Lab. of Glycochemistry and Asymmetric Synthesis; Queneau, Yves (eds.) [Lyon Univ. Villeurbanne (France). Inst. de Chimie et Biochimie

    2010-07-01

    Sucrose: A Prospering and Sustainable Organic Raw Material, By S. Peters, T. Rose, and M. Moser; Sucrose-Utilizing Transglucosidases for Biocatalysis, By I. Andre, G. Potocki-Veronese, S. Morel, P. Monsan, and M. Remaud-Simeon; Difructose Dianhydrides (DFAs) and DFA-Enriched Products as Functional Foods, By C. Ortiz Mellet and J. M. Garcia Fernandez; Development of Agriculture Left-Overs: Fine Organic Chemicals from Wheat Hemicellulose-Derived Pentoses, By F. Martel, B. Estrine, R. Plantier-Royon, N. Hoffmann, and C. Portella; Cellulose and Derivatives from Wood and Fibers as Renewable Sources of Raw-Materials, By J.A. Figueiredo, M.I. Ismael, C.M.S. Anjo, and A.P. Duarte; Olive Pomace, a Source for Valuable Arabinan-Rich Pectic Polysaccharides, By M. A. Coimbra, S. M. Cardoso, and J. A. Lopes-da-Silva; Oligomannuronates from Seaweeds as Renewable Sources for the Development of Green Surfactants,By T. Benvegnu and J.-F. Sassi; From Natural Polysaccharides to Materials for Catalysis, Adsorption, and Remediation, By F. Quignard, F. Di Renzo, and E. Guibal. (orig.)

  10. In vitro metabolic engineering for the salvage synthesis of NAD(.).

    Science.gov (United States)

    Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

    2016-05-01

    Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  11. High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).

    Science.gov (United States)

    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival

    2016-09-02

    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP(+) to NAD(+). Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD(+), and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP(+) to NAD(+). This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

  12. The Biocatalytic Potential of Extremophiles and Extremozymes

    Directory of Open Access Journals (Sweden)

    Walter Steiner

    2004-01-01

    Full Text Available Extremophiles are bizarre microorganisms that can grow and thrive in extreme environments, which were formerly considered too hostile to support life. The extreme conditions may be high or low temperature, high or low pH, high salinity, high metal concentrations, very low nutrient content, very low water activity, high radiation, high pressure and low oxygen tension. Some extremophiles are subject to multiple stress conditions. Extremophiles are structurally adapted at the molecular level to withstand these harsh conditions. The biocatalysts, called extremozymes, produced by these microorganisms, are proteins that function under extreme conditions. Due to their extreme stability, extremozymes offer new opportunities for biocatalysis and biotransformation. Examples of extremozymes include cellulases, amylases, xylanases, proteases, pectinases, keratinases, lipases, esterases, catalases, peroxidases and phytases, which have great potential for application in various biotechnological processes. Currently, only 1–2 % of the microorganisms on the Earth have been commercially exploited and amongst these there are only a few examples of extremophiles. However, the renewed interest that is currently emerging as a result of new developments in the cultivation and production of extremophiles and success in the cloning and expression of their genes in mesophilic hosts will increase the biocatalytic applications of extremozymes.

  13. Two New Preyssler-Type Polyoxometalate-Based Coordination Polymers and Their Application in Horseradish Peroxidase Immobilization.

    Science.gov (United States)

    Du, Jing; Cao, Mei-Da; Feng, Shu-Li; Su, Fang; Sang, Xiao-Jing; Zhang, Lan-Cui; You, Wan-Sheng; Yang, Mei; Zhu, Zai-Ming

    2017-10-17

    Enzyme immobilization is of increasing importance for biocatalysis, for which good supports are critical. Herein, two new Preyssler-type polyoxometalate (POM)-based coordination polymers, namely, {[Cu(H 2 biim) 2 ][{Cu(H 2 biim) 2 (μ-H 2 O)} 2 Cu(H 2 biim)(H 2 O) 2 ]H[({Cu(H 2 biim)(H 2 O) 2 } 0.5 ) 2 ((μ-C 3 HN 2 Cl 2 ){Cu(H 2 biim)} 2 ){Z(H 2 O)P 5 W 30 O 110 }]⋅x H 2 O} n (1: Z=Na, x=9; 2: Z=Ag, x=10; H 2 biim=2,2'-biimidazole) were designed and synthesized. Compounds 1 and 2 exhibit the same skeletons, which contain multiple Cu II complex fragments and penta-supported {ZP 5 W 30 } (Z=Na, Ag) clusters. They were first employed to immobilize horseradish peroxidase (HRP). Results show that compounds 1 and 2 are good supports for HRP immobilization, and exhibit higher enzyme loading, lower loading times, and excellent reusability. The immobilized HRP (HRP/1 or HRP/2) was further applied to detect H 2 O 2 , and good sensitivity, wide linear range, low detection limit, and fast response were achieved. This work shows that POM-based hybrid materials are a new kind of promising support for enzyme immobilization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Review on Bifidobacterium bifidum BGN4: Functionality and Nutraceutical Applications as a Probiotic Microorganism

    Directory of Open Access Journals (Sweden)

    Seockmo Ku

    2016-09-01

    Full Text Available Bifidobacterium bifidum BGN4 is a probiotic strain that has been used as a major ingredient to produce nutraceutical products and as a dairy starter since 2000. The various bio-functional effects and potential for industrial application of B. bifidum BGN4 has been characterized and proven by in vitro (i.e., phytochemical bio-catalysis, cell adhesion and anti-carcinogenic effects on cell lines, and immunomodulatory effects on immune cells, in vivo (i.e., suppressed allergic responses in mouse model and anti-inflammatory bowel disease, and clinical studies (eczema in infants and adults with irritable bowel syndrome. Recently, the investigation of the genome sequencing was finished and this data potentially clarifies the biochemical characteristics of B. bifidum BGN4 that possibly illustrate its nutraceutical functionality. However, further systematic research should be continued to gain insight for academic and industrial applications so that the use of B. bifidum BGN4 could be expanded to result in greater benefit. This review deals with multiple studies on B. bifidum BGN4 to offer a greater understanding as a probiotic microorganism available in functional food ingredients. In particular, this work considers the potential for commercial application, physiological characterization and exploitation of B. bifidum BGN4 as a whole.

  15. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165

    Directory of Open Access Journals (Sweden)

    Deepthy Alex

    2014-01-01

    Full Text Available Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a Km and Vmax of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol.

  16. Chemical databases evaluated by order theoretical tools.

    Science.gov (United States)

    Voigt, Kristina; Brüggemann, Rainer; Pudenz, Stefan

    2004-10-01

    Data on environmental chemicals are urgently needed to comply with the future chemicals policy in the European Union. The availability of data on parameters and chemicals can be evaluated by chemometrical and environmetrical methods. Different mathematical and statistical methods are taken into account in this paper. The emphasis is set on a new, discrete mathematical method called METEOR (method of evaluation by order theory). Application of the Hasse diagram technique (HDT) of the complete data-matrix comprising 12 objects (databases) x 27 attributes (parameters + chemicals) reveals that ECOTOX (ECO), environmental fate database (EFD) and extoxnet (EXT)--also called multi-database databases--are best. Most single databases which are specialised are found in a minimal position in the Hasse diagram; these are biocatalysis/biodegradation database (BID), pesticide database (PES) and UmweltInfo (UMW). The aggregation of environmental parameters and chemicals (equal weight) leads to a slimmer data-matrix on the attribute side. However, no significant differences are found in the "best" and "worst" objects. The whole approach indicates a rather bad situation in terms of the availability of data on existing chemicals and hence an alarming signal concerning the new and existing chemicals policies of the EEC.

  17. Optimization of oligomeric enzyme activity in ionic liquids using Rhodotorula glutinis yeast phenylalanine ammonia lyase.

    Science.gov (United States)

    Barron, Christiaan C; Sponagle, Brandon J D; Arivalagan, Pugazhendhi; D'Cunha, Godwin B

    2017-01-01

    Phenylalanine ammonia lyase (E.C.4.3.1.24, PAL) activity of Rhodotorula glutinis yeast has been demonstrated in four commonly used ionic liquids. PAL forward reaction was carried out in 1-butyl-3-methylimidazolium methyl sulfate ([BMIM][MeSO 4 ]), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF 4 ]), 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF 6 ]) and 1-butyl-3-methylimidazolium lactate ([BMIM][lactate]). Our experiments have revealed that PAL is catalytically active in ionic liquids and the enzyme activity in ([BMIM][PF 6 ]) is comparable to that obtained in aqueous buffer medium. Different conditions were optimized for maximal PAL forward activity including time of incubation (30.0min) L -phenylalanine substrate concentration (30.0mM), nature of buffer (50.0mM Tris-HCl), pH (9.0), temperature (37°C), and speed of agitation (100 rev min -1 ). Under these optimized conditions, about 83% conversion of substrate to product was obtained for the PAL forward reaction that was determined using UV spectroscopy at 290nm. PAL reverse reaction in ([BMIM][PF 6 ]) was determined spectrophotometrically at 520nm; and about 59% substrate conversion was obtained. This data provides further knowledge in enzyme biocatalysis in non-aqueous media, and may be of importance when studying the function of other oligomeric/multimeric proteins and enzymes in ionic liquids. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Simultaneous photocatalytic and microbial degradation of dye-containing wastewater by a novel g-C3N4-P25/photosynthetic bacteria composite.

    Directory of Open Access Journals (Sweden)

    Xinying Zhang

    Full Text Available Azo dyes are very resistant to light-induced fading and biodegradation. Existing advanced oxidative pre-treatment methods based on the generation of non-selective radicals cannot efficiently remove these dyes from wastewater streams, and post-treatment oxidative dye removal is problematic because it may leave many byproducts with unknown toxicity profiles in the outgoing water, or cause expensive complete mineralization. These problems could potentially be overcome by combining photocatalysis and biodegradation. A novel visible-light-responsive hybrid dye removal agent featuring both photocatalysts (g-C3N4-P25 and photosynthetic bacteria encapsulated in calcium alginate beads was prepared by self-assembly. This system achieved a removal efficiency of 94% for the dye reactive brilliant red X-3b and also reduced the COD of synthetic wastewater samples by 84.7%, successfully decolorized synthetic dye-contaminated wastewater and reduced its COD, demonstrating the advantages of combining photocatalysis and biocatalysis for wastewater purification. The composite apparently degrades X-3b by initially converting the dye into aniline and phenol derivatives whose aryl moieties are then attacked by free radicals to form alkyl derivatives, preventing the accumulation of aromatic hydrocarbons that might suppress microbial activity. These alkyl intermediates are finally degraded by the photosynthetic bacteria.

  19. State-of-the-art protein engineering approaches using biological macromolecules: A review from immobilization to implementation view point.

    Science.gov (United States)

    Bilal, Muhammad; Iqbal, Hafiz M N; Guo, Shuqi; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

    2018-03-01

    Over the past years, technological and scientific advances have proven biocatalysis as a sustainable alternative than traditional chemical catalysis including organo- or metallocatalysis. In this context, immobilization based approaches represent simple but effective routes for engineering enzyme catalysts with higher activities than wild-type derivatives. Many enzymes including oxidoreductases have been engineered by rational and directed evolution, to realize the catalytic activity, enantioselectivity, and stability attributes which are essential for their biotechnological exploitation. Induce yet stable activity in enzyme catalysis offer new insights and motivation to engineer efficient catalysts for practical and commercial purposes. It has now become possible to envisage substrate accessibility to the catalytic site of the enzyme by current computational capabilities that reduce the experimental work related to the enzyme selection, screening, and engineering. Herein, state-of-the-art protein engineering approaches for improving enzymatic activities including chemical modification, directed evolution, and rational design or their combination methods are discussed. The emphasis is also given to the applications of the resulting tailored catalysts ranging from fine chemicals to novel pharmaceutical compounds that use biocatalysts as a vital step. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

    Science.gov (United States)

    Ravikumar, Yuvaraj; Nadarajan, Saravanan Prabhu; Hyeon Yoo, Tae; Lee, Chong-Soon; Yun, Hyungdon

    2015-12-01

    The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Some like it hot, some like it cold: Temperature dependent biotechnological applications and improvements in extremophilic enzymes.

    Science.gov (United States)

    Siddiqui, Khawar Sohail

    2015-12-01

    The full biotechnological exploitation of enzymes is still hampered by their low activity, low stability and high cost. Temperature-dependent catalytic properties of enzymes are a key to efficient and cost-effective translation to commercial applications. Organisms adapted to temperature extremes are a rich source of enzymes with broad ranging thermal properties which, if isolated, characterized and their structure-function-stability relationship elucidated, could underpin a variety of technologies. Enzymes from thermally-adapted organisms such as psychrophiles (low-temperature) and thermophiles (high-temperature) are a vast natural resource that is already under scrutiny for their biotechnological potential. However, psychrophilic and thermophilic enzymes show an activity-stability trade-off that necessitates the use of various genetic and chemical modifications to further improve their properties to suit various industrial applications. This review describes in detail the properties and biotechnological applications of both cold-adapted and thermophilic enzymes. Furthermore, the review critically examines ways to improve their value for biotechnology, concluding by proposing an integrated approach involving thermally-adapted, genetically and magnetically modified enzymes to make biocatalysis more efficient and cost-effective. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

    Directory of Open Access Journals (Sweden)

    Anika eRiedel

    2015-10-01

    Full Text Available Ene-reductases are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ene-reductase OYERo2 was found within a set of 14 putative Old Yellow Enzymes (OYEs obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37 °C. OYERo2 showed highest specific activities (4550 U mg-1 on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R-products with excellent optical purity (ee > 99%. OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32 °C from 28 min to 87 min and improved the tolerance towards organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

  3. Systematic comparison of co-expression of multiple recombinant thermophilic enzymes in Escherichia coli BL21(DE3).

    Science.gov (United States)

    Chen, Hui; Huang, Rui; Zhang, Y-H Percival

    2017-06-01

    The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).

  4. 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.

  5. The hydroxynitrile lyase from almond: crystal structure and mechanistical studies

    International Nuclear Information System (INIS)

    Dreveny, Ingrid

    2001-09-01

    Cyanogenesis is a defense process of several thousand plant species. Hydroxynitrile lyase (HNL), a key enzyme of this process, cleaves a cyanohydrin precursor into hydrocyanic acid and the corresponding aldehyde or ketone. The reverse reaction constitutes an important tool in industrial biocatalysis. Different classes of hydroxynitrile lyases have convergently evolved from FAD-dependent oxidoreductases, α/β hydrolases and alcohol dehydrogenases. The FAD-dependent hydroxynitrile lyases (FAD-HNLs) carry a flavin cofactor whose redox properties appear to be unimportant for catalysis. The high resolution crystal structure of the hydroxynitrile lyase from almond (Prunus amygdalus), PaHNL1, has been determined and constitutes the first 3D structure of an FAD-HNL. The overall fold and the architecture of the active site region showed that PaHNL1 belongs to the glucose-methanol-choline-oxidoreductase family, with closest structural similarity to glucose oxidase. There is strong evidence from the sequence and the reaction product that FAD-dependent hydroxynitrile lyases have evolved from an aryl alcohol oxidizing precursor. Structures of PaHNL1 in complex with its natural substrate mandelonitrile and the competitive inhibitor benzyl alcohol provided insight into the residues involved in catalysis and a mechanism without participation of the cofactor could be suggested. Although the catalytic residues differ between the α/β-hydrolase-type HNLs and PaHNL1, common general features relevant for hydroxynitrile lyase activity could be proposed. (author)

  6. Bioprocessing of a stored mixed liquid waste

    Energy Technology Data Exchange (ETDEWEB)

    Wolfram, J.H.; Rogers, R.D. [Idaho National Engineering Lab., Idaho Falls, ID (United States); Finney, R. [Mound Applied Technologies, Miamisburg, OH (United States)] [and others

    1995-12-31

    This paper describes the development and results of a demonstration for a continuous bioprocess for mixed waste treatment. A key element of the process is an unique microbial strain which tolerates high levels of aromatic solvents and surfactants. This microorganism is the biocatalysis of the continuous flow system designed for the processing of stored liquid scintillation wastes. During the past year a process demonstration has been conducted on commercial formulation of liquid scintillation cocktails (LSC). Based on data obtained from this demonstration, the Ohio EPA granted the Mound Applied Technologies Lab a treatability permit allowing the limited processing of actual mixed waste. Since August 1994, the system has been successfully processing stored, {open_quotes}hot{close_quotes} LSC waste. The initial LSC waste fed into the system contained 11% pseudocumene and detectable quantities of plutonium. Another treated waste stream contained pseudocumene and tritium. Data from this initial work shows that the hazardous organic solvent, and pseudocumene have been removed due to processing, leaving the aqueous low level radioactive waste. Results to date have shown that living cells are not affected by the dissolved plutonium and that 95% of the plutonium was sorbed to the biomass. This paper discusses the bioprocess, rates of processing, effluent, and the implications of bioprocessing for mixed waste management.

  7. Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest.

    Science.gov (United States)

    Mechri, Sondes; Ben Elhoul Berrouina, Mouna; Omrane Benmrad, Maroua; Zaraî Jaouadi, Nadia; Rekik, Hatem; Moujehed, Emna; Chebbi, Alif; Sayadi, Sami; Chamkha, Mohamed; Bejar, Samir; Jaouadi, Bassem

    2017-01-01

    The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH 2 -terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    Science.gov (United States)

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Improvement of AD Biosynthesis Response to Enhanced Oxygen Transfer by Oxygen Vectors in Mycobacterium neoaurum TCCC 11979.

    Science.gov (United States)

    Su, Liqiu; Shen, Yanbing; Gao, Tian; Luo, Jianmei; Wang, Min

    2017-08-01

    In steroid biotransformation, soybean oil can improve the productivity of steroids by increasing substrate solubility and strengthen the cell membrane permeability. However, little is known of its role as oxygen carrier and its mechanism of promoting the steroid biotransformation. In this work, soybean oil used as oxygen vector for the enhancement of androst-4-ene-3,17-dione (AD) production by Mycobacterium neoaurum TCCC 11979 (MNR) was investigated. Upon the addition of 16% (v/v) soybean oil, the volumetric oxygen transfer coefficient (K L a) value increased by 44%, and the peak molar yield of AD (55.76%) was achieved. Analysis of intracellular cofactor levels showed high NAD + , ATP level, and a low NADH/NAD + ratio. Meanwhile, the two key enzymes of the tricarboxylic acid (TCA) cycle, namely, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, were upregulated after incubation with soybean oil. These enhancements induced by the increasing of oxygen supply showed positive effects on phytosterol (PS) bioconversion. Results could contribute to the understanding of effects of soybean oil as oxygen vector on steroid biotransformation and provided a convenient method for enhancing the efficiency of aerobic steroid biocatalysis.

  10. Not so monofunctional--a case of thermostable Thermobifida fusca catalase with peroxidase activity.

    Science.gov (United States)

    Lončar, Nikola; Fraaije, Marco W

    2015-03-01

    Thermobifida fusca is a mesothermophilic organism known for its ability to degrade plant biomass and other organics, and it was demonstrated that it represents a rich resource of genes encoding for potent enzymes for biocatalysis. The thermostable catalase from T. fusca has been cloned and overexpressed in Escherichia coli with a yield of 400 mg/L. Heat treatment of disrupted cells at 60 °C for 1 h resulted in enzyme preparation of high purity; hence, no chromatography steps are needed for large-scale production. Except for catalyzing the dismutation of hydrogen peroxide, TfuCat was also found to catalyze oxidations of phenolic compounds. The catalase activity was comparable to other described catalases while peroxidase activity was quite remarkable with a k obs of nearly 1000 s(-1) for catechol. Site directed mutagenesis was used to alter the ratio of peroxidase/catalase activity. Resistance to inhibition by classic catalase inhibitors and an apparent melting temperature of 74 °C classifies this enzyme as a robust biocatalyst. As such, it could compete with other commercially available catalases while the relatively high peroxidase activity also offers new biocatalytic possibilities.

  11. Biological synthesis of nanoparticles in biofilms.

    Science.gov (United States)

    Tanzil, Abid H; Sultana, Sujala T; Saunders, Steven R; Shi, Liang; Marsili, Enrico; Beyenal, Haluk

    2016-12-01

    The biological synthesis of nanoparticles (NPs) by bacteria and biofilms via extracellular redox reactions has received attention because of the minimization of harmful chemicals, low cost, and ease of culturing and downstream processing. Bioreduction mechanisms vary across bacteria and growth conditions, which leads to various sizes and shapes of biosynthesized NPs. NP synthesis in biofilms offers additional advantages, such as higher biomass concentrations and larger surface areas, which can lead to more efficient and scalable biosynthesis. Although biofilms have been used to produce NPs, the mechanistic details of NP formation are not well understood. In this review, we identify three critical areas of research and development needed to advance our understanding of NP production by biofilms: 1) synthesis, 2) mechanism and 3) stabilization. Advancement in these areas could result in the biosynthesis of NPs that are suitable for practical applications, especially in drug delivery and biocatalysis. Specifically, the current status of methods and mechanisms of nanoparticle synthesis and surface stabilization using planktonic bacteria and biofilms is discussed. We conclude that the use of biofilms to synthesize and stabilize NPs is underappreciated and could provide a new direction in biofilm-based NP production. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

    Directory of Open Access Journals (Sweden)

    Rami Gherib

    2013-12-01

    Full Text Available Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis.

  13. Catalytically-active inclusion bodies-Carrier-free protein immobilizates for application in biotechnology and biomedicine.

    Science.gov (United States)

    Krauss, Ulrich; Jäger, Vera D; Diener, Martin; Pohl, Martina; Jaeger, Karl-Erich

    2017-09-20

    Bacterial inclusion bodies (IBs) consist of unfolded protein aggregates and represent inactive waste products often accumulating during heterologous overexpression of recombinant genes in Escherichia coli. This general misconception has been challenged in recent years by the discovery that IBs, apart from misfolded polypeptides, can also contain substantial amounts of active and thus correctly or native-like folded protein. The corresponding catalytically-active inclusion bodies (CatIBs) can be regarded as a biologically-active sub-micrometer sized biomaterial or naturally-produced carrier-free protein immobilizate. Fusion of polypeptide (protein) tags can induce CatIB formation paving the way towards the wider application of CatIBs in synthetic chemistry, biocatalysis and biomedicine. In the present review we summarize the history of CatIBs, present the molecular-biological tools that are available to induce CatIB formation, and highlight potential lines of application. In the second part findings regarding the formation, architecture, and structure of (Cat)IBs are summarized. Finally, an overview is presented about the available bioinformatic tools that potentially allow for the prediction of aggregation and thus (Cat)IB formation. This review aims at demonstrating the potential of CatIBs for biotechnology and hopefully contributes to a wider acceptance of this promising, yet not widely utilized, protein preparation. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A water-forming NADH oxidase from Lactobacillus pentosus and its potential application in the regeneration of synthetic biomimetic cofactors

    Directory of Open Access Journals (Sweden)

    Claudia eNowak

    2015-09-01

    Full Text Available The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox. Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13 % FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyse the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as a by-product.

  15. Glicólise do poli(3-hidroxibutirato por via enzimática

    Directory of Open Access Journals (Sweden)

    Everton Luiz de Paul

    2014-06-01

    Full Text Available Poly(3-hydroxybutyrate, PHB, is a polymer with broad potential applications because of its biodegradability and biocompatibility. However, its high crystallinity is a limiting factor for many applications. To overcome this drawback, one strategy currently employed involves the reduction of the molecular weight of PHB with the concomitant formation of end-functionalized chains, such as those obtained via glycolysis. The glycolysis of PHB can be catalyzed by acid, base, or organometallic compounds. However, to our knowledge, there are no reports regarding PHB glycolysis catalyzed enzymatically. Among the major types of enzymes used in biocatalysis, the lipases stand out because they have the ability to catalyze reactions in both aqueous and organic media. Thus, in this study, we performed the enzymatic glycolysis of PHB using the lipase Amano PS (Pseudomonas cepacia with ethane-1,2-diol (ethylene glycol as the functionalizing agent. The results indicated that the glycolysis was successful and afforded hydroxyl-terminated oligomeric PHB polyols. Nuclear magnetic resonance spectra of the products showed characteristic signals for the terminal hydroxyl groups of the polyols, while thermogravimetric and differential scanning calorimetry analyses confirmed an increase in the thermal stability and a decrease in the crystallinity of the polyols compared with the starting PHB polymer, which were both attributed to the reduction in the molecular weight due to glycolysis.

  16. Glycolysis of poly(3-hydroxybutyrate) via enzyme catalysis

    International Nuclear Information System (INIS)

    Paula, Everton Luiz de; Campos, Tiago Ferreira; Mano, Valdir

    2014-01-01

    Poly(3-hydroxybutyrate), PHB, is a polymer with broad potential applications because of its biodegradability and biocompatibility. However, its high crystallinity is a limiting factor for many applications. To overcome this drawback, one strategy currently employed involves the reduction of the molecular weight of PHB with the concomitant formation of end-functionalized chains, such as those obtained via glycolysis. The glycolysis of PHB can be catalyzed by acid, base, or organometallic compounds. However, to our knowledge, there are no reports regarding PHB glycolysis catalyzed enzymatically. Among the major types of enzymes used in biocatalysis, the lipases stand out because they have the ability to catalyze reactions in both aqueous and organic media. Thus, in this study, we performed the enzymatic glycolysis of PHB using the lipase Amano PS (Pseudomonas cepacia) with ethane-1,2-diol (ethylene glycol) as the functionalizing agent. The results indicated that the glycolysis was successful and afforded hydroxyl-terminated oligomeric PHB polyols. Nuclear magnetic resonance spectra of the products showed characteristic signals for the terminal hydroxyl groups of the polyols, while thermogravimetric and differential scanning calorimetry analyses confirmed an increase in the thermal stability and a decrease in the crystallinity of the polyols compared with the starting PHB polymer, which were both attributed to the reduction in the molecular weight due to glycolysis. (author)

  17. Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris.

    Science.gov (United States)

    Krainer, Florian W; Gerstmann, Michaela A; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton

    2016-09-10

    Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. An updated view on horseradish peroxidases: recombinant production and biotechnological applications.

    Science.gov (United States)

    Krainer, Florian W; Glieder, Anton

    2015-02-01

    Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge-the efficient recombinant production of horseradish peroxidase enzymes.

  19. Development of individual semiconductor nanowire for bioelectrochemical device at low overpotential conditions

    Energy Technology Data Exchange (ETDEWEB)

    Crespilho, Frank N.; Lanfredi, Alexandre J.C. [Universidade Federal do ABC (UFABC), Santo Andre 09210-170 (Brazil); Leite, Edson R.; Chiquito, Adenilson J. [Universidade Federal do Sao Carlos (UFSCar), Sao Carlos, SP (Brazil)

    2009-09-15

    In this work we report the bioelectrochemical study using an individual indium tin oxide (ITO) nanowire (ITO-NW) electrode modified with glucose oxidase enzyme (GOx), in which the enzymatic activity and the biocatalytic activity was evaluated. The main objective is to show that at low overpotential condition, semiconductor NW can be used as an electron donor during biocatalytic process. We demonstrate the possibility of immobilizing an ITO-NW electrode on gold contacts deposited on top of a microchip (oxidized Si wafer). A protective polymer layer containing an aperture over the sample area was photolithographically deposited over the microchip to isolate the metallic contacts. For H{sub 2}O{sub 2} reduction during the biocatalysis at ITO-NWs surface, with {eta} << 50 mV, normal linear behavior is not observed and an exponential current is evident, similar to n-p semiconductor junction behavior. These results can open new tools for studying redox enzymes at the single-molecule level, and the device described here is very promising as a candidate for further exploration in bioelectrochemical devices, such as biofuel cells and biosensors. (author)

  20. Electrocatalytic hydrogen evolution under densely buffered neutral pH conditions

    KAUST Repository

    Shinagawa, Tatsuya; Takanabe, Kazuhiro

    2015-01-01

    Under buffered neutral pH conditions, solute concentrations drastically influence the hydrogen evolution reaction (HER). The iR-free HER performance as a function of solute concentration was found to exhibit a volcano-shaped trend in sodium phosphate solution at pH 5, with the maximum occurring at 2 M. A detailed microkinetic model that includes calculated activity coefficients, solution resistance, and mass-transport parameters accurately describes the measured values, clarifying that the overall HER performance is predominantly governed by mass-transport of slow phosphate ions (weak acid). In the HER at the optimum concentration of approximately 2 M sodium phosphate at pH 5, our theoretical model predicts that the concentration overpotential accounts for more than half of the required overpotential. The substantial concentration overpotential would originate from the electrolyte property, suggesting that the proper electrolyte engineering will result in an improved apparent HER performances. The significance of concentration overpotential shown in the study is critical in the advancement of electrocatalysis, biocatalysis, and photocatalysis.

  1. Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

    Directory of Open Access Journals (Sweden)

    Villaverde Antonio

    2012-06-01

    Full Text Available Abstract A growing number of insights on the biology of bacterial inclusion bodies (IBs have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70, enzymes (catalase and dihydrofolate reductase, grow factors (leukemia inhibitory factor, LIF and structural proteins (the cytoskeleton keratin 14 have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills, for their extra- or intra-cellular release in medicine and cosmetics.

  2. Predicting novel substrates for enzymes with minimal experimental effort with active learning

    Energy Technology Data Exchange (ETDEWEB)

    Pertusi, Dante A.; Moura, Matthew E.; Jeffryes, James G.; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E. J.

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of similar to 80% using similar to 33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways.

  3. White rot fungi and advanced combined biotechnology with nanomaterials: promising tools for endocrine-disrupting compounds biotransformation.

    Science.gov (United States)

    Huang, Danlian; Guo, Xueying; Peng, Zhiwei; Zeng, Guangming; Xu, Piao; Gong, Xiaomin; Deng, Rui; Xue, Wenjing; Wang, Rongzhong; Yi, Huan; Liu, Caihong

    2017-10-29

    Endocrine-disrupting compounds (EDCs) can interfere with endocrine systems and bio-accumulate through the food chain and even decrease biodiversity in contaminated areas. This review discusses a critical overview of recent research progress in the biotransformation of EDCs (including polychlorinated biphenyl and nonylphenol, and suspected EDCs such as heavy metals and sulfonamide antibiotics) by white rot fungi (WRF) based on techniques with an emphasis on summarizing and analyzing fungal molecular, metabolic and genetic mechanisms. Not only intracellular metabolism which seems to perform essential roles in the ability of WRF to transform EDCs, but also advanced applications are deeply discussed. This review mainly reveals the removal pathway of heavy metal and antibiotic pollutants because the single pollution almost did not exist in a real environment while the combined pollution has become more serious and close to people's life. The trends in WRF technology and its related advanced applications which use the combined technology, including biocatalysis of WRF and adsorption of nanomaterials, to degrade EDCs have also been introduced. Furthermore, challenges and future research needs EDCs biotransformation by WRF are also discussed. This research, referring to metabolic mechanisms and the combined technology of WRF with nanomaterials, undoubtedly contributes to the applications of biotechnology. This review will be of great benefit to an understanding of the trends in biotechnology for the removal of EDCs.

  4. Development of an in vivo glucosylation platform by coupling production to growth: Production of phenolic glucosides by a glycosyltransferase of Vitis vinifera.

    Science.gov (United States)

    De Bruyn, Frederik; De Paepe, Brecht; Maertens, Jo; Beauprez, Joeri; De Cocker, Pieter; Mincke, Stein; Stevens, Christian; De Mey, Marjan

    2015-08-01

    Glycosylation of small molecules can significantly alter their properties such as solubility, stability, and/or bioactivity, making glycosides attractive and highly demanded compounds. Consequently, many biotechnological glycosylation approaches have been developed, with enzymatic synthesis and whole-cell biocatalysis as the most prominent techniques. However, most processes still suffer from low yields, production rates and inefficient UDP-sugar formation. To this end, a novel metabolic engineering strategy is presented for the in vivo glucosylation of small molecules in Escherichia coli W. This strategy focuses on the introduction of an alternative sucrose metabolism using sucrose phosphorylase for the direct and efficient generation of glucose 1-phosphate as precursor for UDP-glucose formation and fructose, which serves as a carbon source for growth. By targeted gene deletions, a split metabolism is created whereby glucose 1-phosphate is rerouted from the glycolysis to product formation (i.e., glucosylation). Further, the production pathway was enhanced by increasing and preserving the intracellular UDP-glucose pool. Expression of a versatile glucosyltransferase from Vitis vinifera (VvGT2) enabled the strain to efficiently produce 14 glucose esters of various hydroxycinnamates and hydroxybenzoates with conversion yields up to 100%. To our knowledge, this fast growing (and simultaneously producing) E. coli mutant is the first versatile host described for the glucosylation of phenolic acids in a fermentative way using only sucrose as a cheap and sustainable carbon source. © 2015 Wiley Periodicals, Inc.

  5. Composite system based on biomolecules-functionalized multiwalled carbon nanotube and ionic liquid: Electrochemistry and electrocatalysis of tryptophane

    International Nuclear Information System (INIS)

    Li Li; Bu Caihong; Zhang Yijun; Du Jie; Lu Xiaoquan; Liu Xiuhui

    2011-01-01

    The combination of biomolecules-functionalized multiwalled carbon nanotube (MWNTs) and ionic liquid (IL) yields nanostructured biointerfaces, formed a novel kind of structurally uniform and bioelectrocatalytic activity material. Rutin was chosen as a model biomolecules to investigate the composite system. The MWNTs–Rutin–IL composite film was characterized by different methods including thermogravimetric analysis (TGA), UV–vis spectra, electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscope (SECM). A pair of well-defined quasi reversible redox peaks of rutin was obtained at the MWNTs–Rutin–IL composite film modified glassy carbon electrode (GCE) by direct electron transfer between the rutin and the GCE electrode. Dramatically enhanced biocatalytic and electrocatalytic activity was exemplified at the MWNTs–Rutin–IL/GCE electrode by the oxidized of tryptophane. The oxidation peak currents of tryptophane in such modified electrode increased linearly with the concentrations of tryptophane in the range from 8 × 10 −8 to 2 × 10 −5 mol L −1 with a detection limit of 3.0 × 10 −8 mol L −1 . The unique composite material based on biomolecules-functionalized carbon nanotube and ionic liquid have wide potential applications in direct electrochemistry, biosensors, and biocatalysis.

  6. Industrial Applications of Enzymes: Recent Advances, Techniques, and Outlooks

    Directory of Open Access Journals (Sweden)

    Jordan Chapman

    2018-06-01

    Full Text Available Enzymes as industrial biocatalysts offer numerous advantages over traditional chemical processes with respect to sustainability and process efficiency. Enzyme catalysis has been scaled up for commercial processes in the pharmaceutical, food and beverage industries, although further enhancements in stability and biocatalyst functionality are required for optimal biocatalytic processes in the energy sector for biofuel production and in natural gas conversion. The technical barriers associated with the implementation of immobilized enzymes suggest that a multidisciplinary approach is necessary for the development of immobilized biocatalysts applicable in such industrial-scale processes. Specifically, the overlap of technical expertise in enzyme immobilization, protein and process engineering will define the next generation of immobilized biocatalysts and the successful scale-up of their induced processes. This review discusses how biocatalysis has been successfully deployed, how enzyme immobilization can improve industrial processes, as well as focuses on the analysis tools critical for the multi-scale implementation of enzyme immobilization for increased product yield at maximum market profitability and minimum logistical burden on the environment and user.

  7. Past, present, and future industrial biotechnology in China.

    Science.gov (United States)

    Li, Zhenjiang; Ji, Xiaojun; Kan, Suli; Qiao, Hongqun; Jiang, Min; Lu, Dingqiang; Wang, Jun; Huang, He; Jia, Honghua; Ouyuang, Pingkai; Ying, Hanjie

    2010-01-01

    Fossil resources, i.e. concentrated carbon from biomass, have been irrecoverably exhausted through modern industrial civilization in the last two hundred years. Serious consequences including crises in resources, environment and energy, as well as the pressing need for direct and indirect exploitation of solar energy, pose challenges to the science and technology community of today. Bioenergy, bulk chemicals, and biomaterials could be produced from renewable biomass in a biorefinery via biocatalysis. These sustainable industries will match the global mass cycle, creating a new form of civilization with new industries and agriculture driven by solar energy. Industrial biotechnology is the dynamo of a bioeconomy, leading to a new protocol for production of energy, bulk chemicals, and materials. This new mode of innovation will place the industry at center stage supported by universities and research institutes. Creativity in industrial biotechnology will be promoted and China will successfully follow the road to green modernization. China's rapid economic development and its traditional capacity in fermentation will place it in an advantageous position in the industrial biotechnology revolution. The development and current status of industrial biotechnology in China are summarized herein.

  8. Immobilization of horseradish peroxidase on ZnO nanowires/macroporous SiO2 composites for the complete decolorization of anthraquinone dyes.

    Science.gov (United States)

    Sun, Huaiyan; Jin, Xinyu; Jiang, Feng; Zhang, Ruifeng

    2018-03-01

    A zinc oxide (ZnO) nanowires/macroporous silicon dioxide composite was used as support to immobilize horseradish peroxidase (HRP) simply by in situ cross-linking method. As cross-linker was adsorbed on the surface of ZnO nanowires, the cross-linked HRP was quite different from the traditional cross-linking enzyme aggregates on both structure and catalytic performance. Among three epoxy compounds, diethylene glycol diglycidyl ether (DDE) was the best cross-linker, with which the loading amount of HRP with pI of 5.3 reached as high as 118.1 mg/g and specific activity was up to 14.9 U/mg-support. The mass loss of HRP cross-linked with DDE was negligible during 50-H leaching at 4 °C, and the thermal stability of the immobilized HRP was also quite good. The catalytic performance of immobilized HRP to decolorize anthraquinone dye was explored by using Reactive Blue 19 (RB 19) and Acid Violet 109 (AV 109) as model substrates. The results indicated that the immobilized HRP exhibited high decolorization efficiency and good reusability. The decolorization efficiency reached 94.3% and 95.9% for AV 109 and RB 19 within the first 30 Min, respectively. A complete decolorization of these two dyes has been realized within 2-3 H by using this new biocatalysis system. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  9. Amyloglucosidase enzymatic reactivity inside lipid vesicles

    Directory of Open Access Journals (Sweden)

    Kim Jin-Woo

    2007-10-01

    Full Text Available Abstract Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG (EC 3.2.1.3 from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC multilamellar vesicles (MLVs and large unilamellar vesicles (LUVs was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.

  10. Magnetic separations: From steel plants to biotechnology

    Energy Technology Data Exchange (ETDEWEB)

    Cafer T. Yavuz; Arjun Prakash; J.T. Mayo; Vicki L. Colvin [Rice University, Houston, TX (United States). Department of Chemistry

    2009-05-15

    Magnetic separations have for decades been essential processes in diverse industries ranging from steel production to coal desulfurization. In such settings magnetic fields are used in continuous flow processes as filters to remove magnetic impurities. High gradient magnetic separation (HGMS) has found even broader use in wastewater treatment and food processing. Batch scale magnetic separations are also relevant in industry, particularly biotechnology where fixed magnetic separators are used to purify complex mixtures for protein isolation, cell separation, drug delivery, and biocatalysis. In this review, we introduce the basic concepts behind magnetic separations and summarize a few examples of its large scale application. HGMS systems and batch systems for magnetic separations have been developed largely in parallel by different communities. However, in this work we compare and contrast each approach so that investigators can approach both key areas. Finally, we discuss how new advances in magnetic materials, particularly on the nanoscale, as well as magnetic filter design offer new opportunities for industries that have challenging separation problems.

  11. Flexibility Matters: Cooperative Active Sites in Covalent Organic Framework and Threaded Ionic Polymer.

    Science.gov (United States)

    Sun, Qi; Aguila, Briana; Perman, Jason; Nguyen, Nicholas; Ma, Shengqian

    2016-12-07

    The combination of two or more reactive centers working in concert on a substrate to facilitate the reaction is now considered state of the art in catalysis, yet there still remains a tremendous challenge. Few heterogeneous systems of this sort have been exploited, as the active sites spatially separated within the rigid framework are usually difficult to cooperate. It is now shown that this roadblock can be surpassed. The underlying principle of the strategy presented here is the integration of catalytic components with excellent flexibility and porous heterogeneous catalysts, as demonstrated by the placement of linear ionic polymers in close proximity to surface Lewis acid active sites anchored on the walls of a covalent organic framework (COF). Using the cycloaddition of the epoxides and CO 2 as a model reaction, dramatic activity improvements have been achieved for the composite catalysts in relation to the individual catalytic component. Furthermore, they also clearly outperform the benchmark catalytic systems formed by the combination of the molecular organocatalysts and heterogeneous Lewis acid catalysts, while affording additional recyclability. The extraordinary flexibility and enriched concentration of the catalytically active moieties on linear polymers facilitate the concerted catalysis, thus leading to superior catalytic performance. This work therefore uncovers an entirely new strategy for designing bifunctional catalysts with double-activation behavior and opens a new avenue in the design of multicapable systems that mimic biocatalysis.

  12. An investigation of the mimetic enzyme activity of two-dimensional Pd-based nanostructures

    Science.gov (United States)

    Wei, Jingping; Chen, Xiaolan; Shi, Saige; Mo, Shiguang; Zheng, Nanfeng

    2015-11-01

    In this work, we investigated the mimetic enzyme activity of two-dimensional (2D) Pd-based nanostructures (e.g. Pd nanosheets, Pd@Au and Pd@Pt nanoplates) and found that they possess intrinsic peroxidase-, oxidase- and catalase-like activities. These nanostructures were able to activate hydrogen peroxide or dissolved oxygen for catalyzing the oxidation of organic substrates, and decompose hydrogen peroxide to generate oxygen. More systematic investigations revealed that the peroxidase-like activities of these Pd-based nanomaterials were highly structure- and composition-dependent. Among them, Pd@Pt nanoplates displayed the highest peroxidase-like activity. Based on these findings, Pd-based nanostructures were applied for the colorimetric detection of H2O2 and glucose, and also the electro-catalytic reduction of H2O2. This work offers a promising prospect for the application of 2D noble metal nanostructures in biocatalysis.In this work, we investigated the mimetic enzyme activity of two-dimensional (2D) Pd-based nanostructures (e.g. Pd nanosheets, Pd@Au and Pd@Pt nanoplates) and found that they possess intrinsic peroxidase-, oxidase- and catalase-like activities. These nanostructures were able to activate hydrogen peroxide or dissolved oxygen for catalyzing the oxidation of organic substrates, and decompose hydrogen peroxide to generate oxygen. More systematic investigations revealed that the peroxidase-like activities of these Pd-based nanomaterials were highly structure- and composition-dependent. Among them, Pd@Pt nanoplates displayed the highest peroxidase-like activity. Based on these findings, Pd-based nanostructures were applied for the colorimetric detection of H2O2 and glucose, and also the electro-catalytic reduction of H2O2. This work offers a promising prospect for the application of 2D noble metal nanostructures in biocatalysis. Electronic supplementary information (ESI) available: TEM images, EDX and dispersion stability of Pd-based nanomaterials

  13. La catalyse enzymatique en milieu organique Enzymatic Catalysis in Organic Media

    Directory of Open Access Journals (Sweden)

    Monot F.

    2006-11-01

    Full Text Available L'environnement naturel des enzymes étant de nature aqueuse, leurs applications industrielles se sont en général limitées à des réactions mettant en jeu des substrats solubles dans l'eau. Depuis quelques années, la possibilité de faire fonctionner des enzymes dans des milieux hydrophobes (hydrocarbures, solvants organiques a été mise en évidence, engendrant ainsi de nombreux travaux visant, d'une part à mieux comprendre les mécanismes permettant aux enzymes de rester actives dans un tel environnement et, d'autre part à explorer les nouvelles applications envisageables. Les produits pétroliers constituant par excellence le domaine des molécules hydrophobes, le présent article se propose de faire le point sur ces deux aspects, compréhension et intérêt de la catalyse enzymatique en milieu organique. Nous détaillerons ainsi les vues actuelles sur le fonctionnement des enzymes dans des solvants organiques, les différents modes de mise en oeuvre possibles et, à travers une revue de leurs applications potentielles, les principaux systèmes enzymatiques utilisés. The extension of enzymatic catalysis, classically carried out in aqueous media, to organic media can be first ascribed to the possibility of using substrates that are poorly soluble or insoluble in water. In biphasic media consisting of an aqueous phase containing the enzyme in solution and of a non water-miscible organic solvent, the enzyme is kept in a suitable aqueous environment. A variant biphasic system consists in creating reverse micelles by the addition of a surfactant in order to increase the interfacial area and thus to improve the transfers between the aqueous phase where the enzyme is located and the organic phase. In these two cases, the partition coefficient of the different reactants plays a crucial role by governing the rates and yields of reaction. Microaqueous media constitute a new system for biocatalysis in organic media. In this case, a solid enzyme

  14. Characterization of structure and activity of garlic peroxidase (POX(1B)).

    Science.gov (United States)

    El Ichi, Sarra; Miodek, Anna; Sauriat-Dorizon, Hélène; Mahy, Jean-Pierre; Henry, Céline; Marzouki, Mohamed Nejib; Korri-Youssoufi, Hafsa

    2011-01-01

    Structural characterization and study of the activity of new POX(1B) protein from garlic which has a high peroxidase activity and can be used as a biosensor for the detection of hydrogen peroxide and phenolic compounds were performed and compared with the findings for other heme peroxidases. The structure-function relationship was investigated by analysis of the spectroscopic properties and correlated to the structure determined by a new generation of high-performance hybrid mass spectrometers. The reactivity of the enzyme was analyzed by studies of the redox activity toward various ligands and the reactivity with various substrates. We demonstrated that, in the case of garlic peroxidase, the heme group is pentacoordinated, and has an histidine as a proximal ligand. POX(1B) exhibited a high affinity for hydrogen peroxide as well as various reducing cosubstrates. In addition, high enzyme specificity was demonstrated. The k(cat) and K(M) values were 411 and 400 mM(-1) s(-1) for 3,3',5,5'-tetramethylbenzidine and 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), respectively. Furthermore, the reduction of nitro compounds in the presence of POX(1B) was demonstrated by iron(II) nitrosoalkane complex assay. In addition, POX(1B) showed a great potential for application for drug metabolism since its ability to react with 1-nitrohexane in the presence of sodium dithionite was demonstrated by the appearance of a characteristic Soret band at 411 nm. The high catalytic efficiency obtained in the case of the new garlic peroxidase (POX(1B)) is suitable for the monitoring of different analytes and biocatalysis.

  15. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

    Directory of Open Access Journals (Sweden)

    Anzhang Li

    Full Text Available Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m = 0.82 mM, k(cat/K(m = 16.43 mM(-1 · s(-1. DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

  16. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

    Science.gov (United States)

    Li, Anzhang; Shao, Zongze

    2014-01-01

    Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m) = 0.82 mM, k(cat)/K(m) = 16.43 mM(-1) · s(-1)). DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

  17. A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

    Science.gov (United States)

    Buchholz, Klaus

    2016-05-01

    Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that

  18. Replacing Escherichia coli NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a NADP-dependent enzyme from Clostridium acetobutylicum facilitates NADPH dependent pathways.

    Science.gov (United States)

    Martínez, Irene; Zhu, Jiangfeng; Lin, Henry; Bennett, George N; San, Ka-Yiu

    2008-11-01

    Reactions requiring reducing equivalents, NAD(P)H, are of enormous importance for the synthesis of industrially valuable compounds such as carotenoids, polymers, antibiotics and chiral alcohols among others. The use of whole-cell biocatalysis can reduce process cost by acting as catalyst and cofactor regenerator at the same time; however, product yields might be limited by cofactor availability within the cell. Thus, our study focussed on the genetic manipulation of a whole-cell system by modifying metabolic pathways and enzymes to improve the overall production process. In the present work, we genetically engineered an Escherichia coli strain to increase NADPH availability to improve the productivity of products that require NADPH in its biosynthesis. The approach involved an alteration of the glycolysis step where glyceraldehyde-3-phosphate (GAP) is oxidized to 1,3 bisphophoglycerate (1,3-BPG). This reaction is catalyzed by NAD-dependent endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoded by the gapA gene. We constructed a recombinant E. coli strain by replacing the native NAD-dependent gapA gene with a NADP-dependent GAPDH from Clostridium acetobutylicum, encoded by the gene gapC. The beauty of this approach is that the recombinant E. coli strain produces 2 mol of NADPH, instead of NADH, per mole of glucose consumed. Metabolic flux analysis showed that the flux through the pentose phosphate (PP) pathway, one of the main pathways that produce NADPH, was reduced significantly in the recombinant strain when compared to that of the parent strain. The effectiveness of the NADPH enhancing system was tested using the production of lycopene and epsilon-caprolactone as model systems using two different background strains. The recombinant strains, with increased NADPH availability, consistently showed significant higher productivity than the parent strains.

  19. Redox cycles of vitamin E: Hydrolysis and ascorbic acid dependent reduction of 8a-(alkyldioxy)tocopherones

    International Nuclear Information System (INIS)

    Liebler, D.C.; Kaysen, K.L.; Kennedy, T.A.

    1989-01-01

    Oxidation of the biological antioxidant α-tocopherol (vitamin E; TH) by peroxyl radicals yields 8a-(alkyldioxy)tocopherones, which either may hydrolyze to α-tocopheryl quinone (TQ) or may be reduced by ascorbic acid to regenerate TH. To define the chemistry of this putative two-electron TH redox cycle, we studied the hydrolysis and reduction of 8a-[(2,4-dimethyl-1-nitrilopent-2-yl)dioxyl]tocopherone (1) in acetonitrile/buffer mixtures and in phospholipid liposomes. TQ formation in acetonitrile/buffer mixtures, which was monitored spectrophotometrically, declined with increasing pH and could not be detected above pH 4. The rate of TQ formation from 1 first increased with time and then decreased in a first-order terminal phase. Rearrangement of 8a-hydroxy-α-tocopherone (2) to TQ displayed first-order kinetics identical with the terminal phase for TQ formation from 1. Both rate constants increased with decreasing pH. Hydrolysis of 1 in acetonitrile/H 2 18 O yielded [ 18 O]TQ. These observations suggest that 1 loses the 8a-(alkyldioxy) moiety to produce the tocopherone cation (T + ), which hydrolyzes to 2, the TQ-forming intermediate. Incubation of either 1 or 2 with ascorbic acid in acetonitrile/buffer yielded TH. Reduction of both 1 and 2 decreased with increasing pH. In phosphatidylcholine liposomes at pH 7, approximately 10% of the T + generated from 1 was reduced to TH by 5 mM ascorbic acid. The results collectively demonstrate that T + is the ascorbic acid reducible intermediate in a two-electron TH redox cycle, a process that probably would require biocatalysis to proceed in biological membranes

  20. Surface display for metabolic engineering of industrially important acetic acid bacteria

    Directory of Open Access Journals (Sweden)

    Marshal Blank

    2018-04-01

    Full Text Available Acetic acid bacteria have unique metabolic characteristics that suit them for a variety of biotechnological applications. They possess an arsenal of membrane-bound dehydrogenases in the periplasmic space that are capable of regiospecific and enantioselective partial oxidations of sugars, alcohols, and polyols. The resulting products are deposited directly into the medium where they are easily recovered for use as pharmaceutical precursors, industrial chemicals, food additives, and consumer products. Expression of extracytoplasmic enzymes to augment the oxidative capabilities of acetic acid bacteria is desired but is challenging due to the already crowded inner membrane. To this end, an original surface display system was developed to express recombinant enzymes at the outer membrane of the model acetic acid bacterium Gluconobacter oxydans. Outer membrane porin F (OprF was used to deliver alkaline phosphatase (PhoA to the cell surface. Constitutive high-strength p264 and moderate-strength p452 promoters were used to direct expression of the surface display system. This system was demonstrated for biocatalysis in whole-cell assays with the p264 promoter having a twofold increase in PhoA activity compared to the p452 promoter. Proteolytic cleavage of PhoA from the cell surface confirmed proper delivery to the outer membrane. Furthermore, a linker library was constructed to optimize surface display. A rigid (EAAAK1 linker led to the greatest improvement, increasing PhoA activity by 69%. This surface display system could be used both to extend the capabilities of acetic acid bacteria in current biotechnological processes, and to broaden the potential of these microbes in the production of value-added products.

  1. Stabilization of enzymes in ionic liquids via modification of enzyme charge.

    Science.gov (United States)

    Nordwald, Erik M; Kaar, Joel L

    2013-09-01

    Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

  2. Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions.

    Science.gov (United States)

    Greening, Chris; Ahmed, F Hafna; Mohamed, A Elaaf; Lee, Brendon M; Pandey, Gunjan; Warden, Andrew C; Scott, Colin; Oakeshott, John G; Taylor, Matthew C; Jackson, Colin J

    2016-06-01

    5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (Fo) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F420 is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F420 in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F420 in methanogenic archaea in processes such as substrate oxidation, C1 pathways, respiration, and oxygen detoxification. We also describe how two F420-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid by Mycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of Fo and F420 are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis. Copyright © 2016, American Society for

  3. Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development.

    Science.gov (United States)

    Schmölzer, Katharina; Gutmann, Alexander; Diricks, Margo; Desmet, Tom; Nidetzky, Bernd

    2016-01-01

    Sucrose synthase (SuSy, EC 2.4.1.13) is a glycosyltransferase (GT) long known from plants and more recently discovered in bacteria. The enzyme catalyzes the reversible transfer of a glucosyl moiety between fructose and a nucleoside diphosphate (NDP) (sucrose+NDP↔NDP-glucose+fructose). The equilibrium for sucrose conversion is pH dependent, and pH values between 5.5 and 7.5 promote NDP-glucose formation. The conversion of a bulk chemical to high-priced NDP-glucose in a one-step reaction provides the key aspect for industrial interest. NDP-sugars are important as such and as key intermediates for glycosylation reactions by highly selective Leloir GTs. SuSy has gained renewed interest as industrially attractive biocatalyst, due to substantial scientific progresses achieved in the last few years. These include biochemical characterization of bacterial SuSys, overproduction of recombinant SuSys, structural information useful for design of tailor-made catalysts, and development of one-pot SuSy-GT cascade reactions for production of several relevant glycosides. These advances could pave the way for the application of Leloir GTs to be used in cost-effective processes. This review provides a framework for application requirements, focusing on catalytic properties, heterologous enzyme production and reaction engineering. The potential of SuSy biocatalysis will be presented based on various biotechnological applications: NDP-sugar synthesis; sucrose analog synthesis; glycoside synthesis by SuSy-GT cascade reactions. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Final Technical Report - Consolidating Biomass Pretreatment with Saccharification by Resolving the Spatial Control Mechanisms of Fungi

    Energy Technology Data Exchange (ETDEWEB)

    Schilling, Jonathan [Univ. of Minnesota, Minneapolis, MN (United States)

    2017-07-06

    Consolidated bioprocessing (CBP) of lignocellulose combines enzymatic sugar release (saccharification) with fermentation, but pretreatments remain separate and costly. In nature, lignocellulose-degrading brown rot fungi consolidate pretreatment and saccharification, likely using spatial gradients to partition these incompatible reactions. With the field of biocatalysis maturing, reaction partitioning is increasingly reproducible for commercial use. Therefore, my goal was to resolve the reaction partitioning mechanisms of brown rot fungi so that they can be applied to bioconversion of lignocellulosic feedstocks. Brown rot fungi consolidate oxidative pretreatments with saccharification and are a focus for biomass refining because 1) they attain >99% sugar yield without destroying lignin, 2) they use a simplified cellulase suite that lacks exoglucanase, and 3) their non-enzymatic pretreatment is facilitative and may be accelerated. Specifically, I hypothesized that during brown rot, oxidative pretreatments occur ahead of enzymatic saccharification, spatially, and the fungus partitions these reactions using gradients in pH, lignin reactivity, and plant cell wall porosity. In fact, we found three key results during these experiments for this work: 1) Brown rot fungi have an inducible cellulase system, unlike previous descriptions of a constitutive mechanism. 2) The induction of cellulases is delayed until there is repression of oxidatively-linked genes, allowing the brown rot fungi to coordinate two incompatible reactions (oxidative pretreatment with enzymatic saccharification, to release wood sugars) in the same pieces of wood. 3) This transition is mediated by the same wood sugar, cellobiose, released by the oxidative pretreatment step. Collectively, these findings have been published in excellent journal outlets and have been presented at conferences around the United States, and they offer clear targets for gene discovery en route to making biofuels and biochemicals

  5. Synthesis of 1,3-di(4-amino-1-pyridinium)propane ionic liquid functionalized graphene nanosheets and its application in direct electrochemistry of hemoglobin

    International Nuclear Information System (INIS)

    Li, Rui; Liu, Changxian; Ma, Ming; Wang, Zhengguo; Zhan, Guoqing; Li, Buhai; Wang, Xian; Fang, Huaifang; Zhang, Huijuan; Li, Chunya

    2013-01-01

    Highlights: ► 1,3-Di(4-amino-1-pyridinium)propane tetrafluoroborate ionic liquid was successfully synthesized. ► Ionic liquid modified graphene nanosheets were successfully prepared and fully characterized. ► Hb was immobilized on the as-prepared graphene–ionic liquid nanosheets. ► Direct electrochemistry of Hb was succeeded. ► Biocatalysis of Hb towards H 2 O 2 was demonstrated, and was used in H 2 O 2 determination. -- Abstract: 1,3-Di(4-amino-1-pyridinium)propane tetrafluoroborate (DAPPT) ionic liquid was successfully synthesized, and was used as a modifier to functionalize graphene nanosheets through covalent binding of amino groups and epoxy groups in an alkaline solution. The as-prepared graphene-DAPPT nanosheets (Gr-DAPPT) were confirmed with transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), UV/vis and FTIR spectroscopy. A biocompatible platform based on Gr-DAPPT was constructed for the immobilization of hemoglobin (Hb) through a cross-linking step with chitosan and glutaraldehyde. The direct electron transfer and bioelectrocatalytic reaction of Hb immobilized on Gr-DAPPT surface were achieved. A pair of reversible redox peaks of hemoglobin was observed, and bioelectrocatalytic activity toward the reduction of H 2 O 2 was also demonstrated, displaying a potential application for the fabrication of novel biosensors to sense H 2 O 2 . Such results indicated that Gr-DAPPT based interface would be a promising platform for biomacromolecular immobilization and biosensor preparation

  6. New insight into protein-nanomaterial interactions with UV-visible spectroscopy and chemometrics: human serum albumin and silver nanoparticles.

    Science.gov (United States)

    Wang, Yong; Ni, Yongnian

    2014-01-21

    In recent years, great efforts have focused on the exploration and fabrication of protein nanoconjugates due to potential applications in many fields including bioanalytical science, biosensors, biocatalysis, biofuel cells and bio-based nanodevices. An important aspect of our understanding of protein nanoconjugates is to quantitatively understand how proteins interact with nanomaterials. In this report, human serum albumin (HSA) and citrate-coated silver nanoparticles (AgNPs) are selected as a case study of protein-nanomaterial interactions. UV-visible spectroscopy together with multivariate curve resolution by alternating least squares (MCR-ALS) algorithm is first exploited for the detailed study of AgNPs-HSA interactions. Introduction of the chemometrics tool allows extracting the kinetic profiles, spectra and distribution diagrams of two major absorbing pure species (AgNPs and AgNPs-HSA conjugate). These resolved profiles are then analysed to give the thermodynamic, kinetic and structural information of HSA binding to AgNPs. Transmission electron microscopy, circular dichroism spectroscopy and Fourier transform infrared spectroscopy are used to further characterize the complex system. Moreover, a sensitive spectroscopic biosensor for HSA is fabricated with the MCR-ALS resolved concentration of absorbing pure species. It is found that the linear range for the HSA nanosensor was from 1.9 nM to 45.0 nM with a detection limit of 0.9 nM. It is believed that the proposed method will play an important role in the fabrication and optimization of a robust nanobiosensor or cross-reactive sensors array for the detection and identification of biocomponents.

  7. Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition – a facile method for encapsulation of diverse cell types in silica matrices

    Energy Technology Data Exchange (ETDEWEB)

    Johnston, Robert [New Mexico Inst. of Mining and Technology, Socorro, NM (United States). Materials Engineering Dept.; Rogelj, Snezna [New Mexico Inst. of Mining and Technology, Socorro, NM (United States). Biology Dept.; Harper, Jason C. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States). Bioenergy and Biodefense Technologies Dept.; Tartis, Michaelann [New Mexico Inst. of Mining and Technology, Socorro, NM (United States). Materials and Chemical Engineering Dept.

    2014-12-12

    In nature, cells perform a variety of complex functions such as sensing, catalysis, and energy conversion which hold great potential for biotechnological device construction. However, cellular sensitivity to ex vivo environments necessitates development of bio–nano interfaces which allow integration of cells into devices and maintain their desired functionality. In order to develop such an interface, the use of a novel Sol-Generating Chemical Vapor into Liquid (SG-CViL) deposition process for whole cell encapsulation in silica was explored. In SG-CViL, the high vapor pressure of tetramethyl orthosilicate (TMOS) is utilized to deliver silica into an aqueous medium, creating a silica sol. Cells are then mixed with the resulting silica sol, facilitating encapsulation of cells in silica while minimizing cell contact with the cytotoxic products of silica generating reactions (i.e. methanol), and reduce exposure of cells to compressive stresses induced from silica condensation reactions. Using SG-CVIL, Saccharomyces cerevisiae (S. cerevisiae) engineered with an inducible beta galactosidase system were encapsulated in silica solids and remained both viable and responsive 29 days post encapsulation. By tuning SG-CViL parameters, thin layer silica deposition on mammalian HeLa and U87 human cancer cells was also achieved. Thus, the ability to encapsulate various cell types in either a multi cell (S. cerevisiae) or a thin layer (HeLa and U87 cells) fashion shows the promise of SG-CViL as an encapsulation strategy for generating cell–silica constructs with diverse functions for incorporation into devices for sensing, bioelectronics, biocatalysis, and biofuel applications.

  8. An experimental modeling of trinomial bioengineering- crp, rDNA, and transporter engineering within single cell factory for maximizing two-phase bioreduction.

    Science.gov (United States)

    Basak, Souvik; Ghosh, Sumanta Kumar; Punetha, Vinay Deep; Aphale, Ashish N; Patra, Prabir K; Sahoo, Nanda Gopal

    2017-02-01

    A carbonyl reductase (cr) gene from Candida glabrata CBS138 has been heterologously expressed in cofactor regenerating E. coli host to convert Ethyl-4-chloro-3-oxobutanoate (COBE) into Ethyl-4-chloro-3-hydroxybutanoate (CHBE). The CR enzyme exhibited marked velocity at substrate concentration as high as 363mM with highest turnover number (112.77±3.95s -1 ). Solitary recombineering of such catalytic cell reproduced CHBE 161.04g/L per g of dry cell weight (DCW). Introduction of combinatorially engineered crp (crp*, F136I) into this heterologous E. coli host yielded CHBE 477.54g/L/gDCW. Furthermore, using nerolidol as exogenous cell transporter, the CHBE productivity has been towered to 710.88g/L/gDCW. The CHBE production has thus been upscaled to 8-12 times than those reported so far. qRT-PCR studies revealed that both membrane efflux channels such as acrAB as well as ROS scavenger genes such as ahpCF have been activated by engineering crp. Moreover, membrane protecting genes such as manXYZ together with solvent extrusion associated genes such as glpC have been upregulated inside mutant host. Although numerous proteins have been investigated to convert COBE to CHBE; this is the first approach to use engineering triad involving crp engineering, recombinant DNA engineering and transporter engineering together for improving cell performance during two-phase biocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium.

    Science.gov (United States)

    Gerber, Adrian; Kleser, Michael; Biedendieck, Rebekka; Bernhardt, Rita; Hannemann, Frank

    2015-07-29

    Cholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis. CYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed. We established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism's PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the

  10. Nitrile Metabolizing Yeasts

    Science.gov (United States)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  11. Polyphosphonate induced coacervation of chitosan: Encapsulation of proteins/enzymes and their biosensing

    International Nuclear Information System (INIS)

    Liu, Hailing; Cui, Yanyun; Li, Pan; Zhou, Yiming; Chen, Yu; Tang, Yawen; Lu, Tianhong

    2013-01-01

    find extensive applications in biocatalysis, biosensors, bioelectronics and biofuel cells

  12. Polyphosphonate induced coacervation of chitosan: Encapsulation of proteins/enzymes and their biosensing

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Hailing; Cui, Yanyun; Li, Pan; Zhou, Yiming; Chen, Yu, E-mail: ndchenyu@yahoo.cn; Tang, Yawen; Lu, Tianhong

    2013-05-07

    would certainly find extensive applications in biocatalysis, biosensors, bioelectronics and biofuel cells.

  13. The Molecular Mechanism of the Catalase-like Activity in Horseradish Peroxidase.

    Science.gov (United States)

    Campomanes, Pablo; Rothlisberger, Ursula; Alfonso-Prieto, Mercedes; Rovira, Carme

    2015-09-02

    Horseradish peroxidase (HRP) is one of the most relevant peroxidase enzymes, used extensively in immunochemistry and biocatalysis applications. Unlike the closely related catalase enzymes, it exhibits a low activity to disproportionate hydrogen peroxide (H2O2). The origin of this disparity remains unknown due to the lack of atomistic information on the catalase-like reaction in HRP. Using QM(DFT)/MM metadynamics simulations, we uncover the mechanism for reduction of the HRP Compound I intermediate by H2O2 at atomic detail. The reaction begins with a hydrogen atom transfer, forming a peroxyl radical and a Compound II-like species. Reorientation of the peroxyl radical in the active site, concomitant with the transfer of the second hydrogen atom, is the rate-limiting step, with a computed free energy barrier (18.7 kcal/mol, ∼ 6 kcal/mol higher than the one obtained for catalase) in good agreement with experiments. Our simulations reveal the crucial role played by the distal pocket residues in accommodating H2O2, enabling formation of a Compound II-like intermediate, similar to catalases. However, out of the two pathways for Compound II reduction found in catalases, only one is operative in HRP. Moreover, the hydrogen bond network in the distal side of HRP compensates less efficiently than in catalases for the energetic cost required to reorient the peroxyl radical at the rate-determining step. The distal Arg and a water molecule in the "wet" active site of HRP have a substantial impact on the reaction barrier, compared to the "dry" active site in catalase. Therefore, the lower catalase-like efficiency of heme peroxidases compared to catalases can be directly attributed to the different distal pocket architecture, providing hints to engineer peroxidases with a higher rate of H2O2 disproportionation.

  14. A novel amperometric alcohol biosensor developed in a 3rd generation bioelectrode platform using peroxidase coupled ferrocene activated alcohol oxidase as biorecognition system.

    Science.gov (United States)

    Chinnadayyala, Somasekhar R; Kakoti, Ankana; Santhosh, Mallesh; Goswami, Pranab

    2014-05-15

    Alcohol oxidase (AOx) with a two-fold increase in efficiency (Kcat/Km) was achieved by physical entrapment of the activator ferrocene in the protein matrix through a simple microwave based partial unfolding technique and was used to develop a 3rd generation biosensor for improved detection of alcohol in liquid samples. The ferrocene molecules were stably entrapped in the AOx protein matrix in a molar ratio of ~3:1 through electrostatic interaction with the Trp residues involved in the functional activity of the enzyme as demonstrated by advanced analytical techniques. The sensor was fabricated by immobilizing ferrocene entrapped alcohol oxidase (FcAOx) and sol-gel chitosan film coated horseradish peroxidase (HRP) on a multi-walled carbon nanotube (MWCNT) modified glassy carbon electrode through layer-by-layer technique. The bioelectrode reactions involved the formation of H2O2 by FcAOx biocatalysis of substrate alcohol followed by HRP-catalyzed reduction of the liberated H2O2 through MWCNT supported direct electron transfer mechanism. The amperometric biosensor exhibited a linear response to alcohol in the range of 5.0 × 10(-6) to 30 × 10(-4)mol L(-1) with a detection limit of 2.3 × 10(-6) mol L(-1), and a sensitivity of 150 µA mM(-1) cm(-2). The biosensor response was steady for 28 successive measurements completed in a period of 5h and retained ~90% of the original response even after four weeks when stored at 4 °C. The biosensor was successfully applied for the determination of alcohol in commercial samples and its performance was validated by comparing with the data obtained by GC analyses of the samples. © 2013 Published by Elsevier B.V.

  15. Green chemistry at work

    Energy Technology Data Exchange (ETDEWEB)

    Frost, J. [Michigan State Univ., East Lansing, MI (United States)

    1994-12-31

    The 1.7 billion pounds of benzene produced each year in the US provide one measure of its utility. At the same time, there are a number of environmental reasons for avoiding the use of benzene in chemical manufacture. Perhaps most compelling: benzene is a potent carcinogen. Scrutiny of many of the chemicals derived from benzene reveals that each molecule contains at least one oxygen atom while benzene completely lacks oxygen atoms. Introduction of oxygen to make up for this lack can require processes that are environmentally problematic. One of the steps used to introduce oxygen atoms during manufacture of adipic acid, a component of Nylon 66, is responsible for 10% of the annual global increase in atmospheric nitrous oxide. This by-product is a causative agent of atmospheric ozone depletion and has been implicated in global warming. With support from EPA and the National Science Foundation, alternative manufacturing processes are being explored. By these new methods, chemicals usually created from benzene are made instead from nontoxic glucose, a component of table sugar. Unlike benzene, glucose is obtained from such renewable resources as plant starch and cellulose. ``Green`` manufacturing routes ideally should lead to chemicals that are economically competitive with chemicals produced by traditional methods. For two chemicals of roughly comparable cost, the consumer or producer can then be realistically expected to choose in favor of the chemical produced by a ``green`` process. Projections indicate that catechol and hydroquinone can be biocatalytically produced from glucose at a cost competitive with current market prices. Synthesis of chemicals from glucose using biocatalysis offers the premise of achieving fundamental environmental improvement while increasing the demand for agricultural products. In the final analysis, what is good for the environment can also be good for American agriculture.

  16. 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.

  17. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    Science.gov (United States)

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  18. New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

    Directory of Open Access Journals (Sweden)

    Rocha-Martín Javier

    2011-11-01

    Full Text Available Abstract Background The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds. Results The NADH-oxidase (NOX presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C. The hyperactivated form presented a high specific activity (37.5 U/mg at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme. The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols. Conclusion We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.

  19. Heterologous Protein Expression in Pichia pastoris: Latest Research Progress and Applications.

    Science.gov (United States)

    Juturu, Veeresh; Wu, Jin Chuan

    2018-01-04

    Pichia pastoris is a well-known platform strain for heterologous protein expression. Over the past five years, different strategies to improve the efficiency of recombinant protein expression by this yeast strain have been developed; these include a patent-free protein expression kit, construction of the P. pastoris CBS7435Ku70 platform strain with its high efficiency in site-specific recombination of plasmid DNA into the genomic DNA, the design of synthetic promoters and their variants by combining different core promoters with multiple putative transcription factors, the generation of mutant GAP promoter variants with various promoter strengths, codon optimization, engineering the α-factor signal sequence by replacing the native glutamic acid at the Kex2 cleavage site with the other 19 natural amino acids and the addition of mammalian signal sequence to the yeast signal sequence, and the co-expression of single chaperones, multiple chaperones or helper proteins that aid in recombinant protein folding. Publically available high-quality genome data from multiple strains of P. pastoris GS115, DSMZ 70382, and CBS7435 and the continuous development of yeast expression kits have successfully promoted the metabolic engineering of this strain to produce carotenoids, xanthophylls, nootkatone, ricinoleic acid, dammarenediol-II, and hyaluronic acid. The cell-surface display of enzymes has obviously increased enzyme stability, and high-level intracellular expression of acyl-CoA and ethanol O-acyltransferase, lipase and d-amino acid oxidase has opened up applications in whole-cell biocatalysis for producing flavor molecules and biodiesel, as well as the deracemization of racemic amino acids. High-level expression of various food-grade enzymes, cellulases, and hemicellulases for applications in the food, feed and biorefinery industries is in its infancy and needs strengthening. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Tailoring crystallinity and configuration of silica nanotubes by electron irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, Tomitsugu, E-mail: taguchi.tomitsugu@jaea.go.jp; Yamaguchi, Kenji

    2015-05-01

    Highlights: •Single-crystal SiO{sub 2} nanotubes were successfully synthesized for the first time. •The single-crystal SiO{sub 2} was α-crystobalite. •Desired area of single-crystal nanotube can be changed to amorphous by electron irradiation. •The configuration of nanotube can be controlled using the focused electron irradiation technique. -- Abstract: SiO{sub 2} nanotubes show potential in applications such as nanoscale electronic and optical devices, bioseparation, biocatalysis, and nanomedicine. As-grown SiO{sub 2} nanotubes in the previous studies always have an amorphous wall, and here we demonstrate the successful synthesis of single-crystal nanotubes for the first time by the heat treatment of SiC nanotubes at 1300 °C for 10 h under low-vacuum conditions. According to TEM observations, the single-crystal SiO{sub 2} was α-cristobalite. We also demonstrate that single-crystal SiO{sub 2} nanotubes can be transformed into amorphous SiO{sub 2} nanotubes by electron beam irradiation. Moreover, we synthesized a crystalline/amorphous SiO{sub 2} composite nanotube, in which crystalline and amorphous SiO{sub 2} coexisted in different localized regions. In addition, for biomedical applications such as drug delivery systems, controlling the configuration of the open end, the diameter, and capsulation of SiO{sub 2} nanotubes is crucial. We can also obturate, capsulate, and cut a SiO{sub 2} nanotube, as well as modify the inner diameter of the nanotube at a specific, nanometer-sized region using the focused electron beam irradiation technique.

  1. Enzyme electrode configurations : for application in biofuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang Xiaoju

    2012-07-01

    The conducting polymer, poly (3,4-ethylenedioxythiophene) (PEDOT) film is a suitable matrix material for the enzymes, due to its outstanding properties, specifically, high electrical conductivity and excellent inherent environmental stability. The counter ions for PEDOT have a significant effect on the structural features and morphology of the polymer film produced by electropolymerization. Different carbon-based materials, ranging from paper-like carbon ink paper or carbon paper to reticulated vitreous carbon foam (RVC foam), were explored as substrate materials for PEDOT film generation by electropolymerization. The immobilization of Trametes hirsuta laccase (ThL) in the PEDOT film was facilitated via in situ entrapment during electropolymerization. When 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonate) (ABTS{sup 2-}) was used as the mediator, the immobilized ThL exhibited catalytic activity for the reduction of O{sub 2} to water. The amount of ThL in the PEDOT matrix is tunable by controlling the manufacturing parameters, including the charge density used for the electropolymerization of the EDOT monomer and the ThL concentration in the electropolymerization electrolyte. The use of a porous material, e.g., RVC foam, as the PEDOT supporting template was tested to improve the current density per unit area/volume generated by biocathodes. These RVC foam-based biocathodes produced a large current density, reaching 1 mA/cm{sup 3} at 0.45 V when 19.5 {mu}g/ml of ThL was used in the electropolymerization electrolyte. In addition, direct electron transfer (DET) type biocatalysis was accomplished for ThL by immobilizing ThL into a fine-tuned dual-layer-architecture of PEDOT films. In a PEDOTNO{sub 3}/ ThL/PEDOT-PSS enzyme electrode, the reduction of O{sub 2} to water was catalyzed by ThL with the T1 Cu site as the primary electron acceptor. The fabrication parameters included different combinations of PEDOT films, ThL loadings, and thicknesses of both PEDOT layers

  2. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    via a subsequent 4-h cell culturing. Conclusions This study demonstrates, for the first time, the methodology by which the engineered P. putida with surface-immobilized laccase was successfully used as regenerable biocatalyst for biodegrading synthetic dyes, thereby opening new perspectives in the use of biocatalysis in industrial dye biotreatment.

  3. Light induced expression of β-glucosidase in Escherichia coli with autolysis of cell.

    Science.gov (United States)

    Chang, Fei; Zhang, Xianbing; Pan, Yu; Lu, Youxue; Fang, Wei; Fang, Zemin; Xiao, Yazhong

    2017-11-07

    β-Glucosidase has attracted substantial attention in the scientific community because of its pivotal role in cellulose degradation, glycoside transformation and many other industrial processes. However, the tedious and costly expression and purification procedures have severely thwarted the industrial applications of β-glucosidase. Thus development of new strategies to express β-glucosidases with cost-effective and simple procedure to meet the increasing demands on enzymes for biocatalysis is of paramount importance. Light activated cassette YF1/FixJ and the SRRz lysis system were successfully constructed to produce Bgl1A(A24S/F297Y), a mutant β-glucosidase tolerant to both glucose and ethanol. By optimizing the parameters for light induction, Bgl1A(A24S/F297Y) activity reached 33.22 ± 2.0 U/mL and 249.92 ± 12.25 U/mL in 250-mL flask and 3-L fermentation tank, respectively, comparable to the controls of 34.02 ± 1.96 U/mL and 322.21 ± 10.16 U/mL under similar culture conditions with IPTG induction. To further simplify the production of our target protein, the SRRz lysis gene cassette from bacteriophage Lambda was introduced to trigger cell autolysis. As high as 84.53 ± 6.79% and 77.21 ± 4.79% of the total β-glucosidase were released into the lysate after cell autolysis in 250 mL flasks and 3-L scale fermentation with lactose as inducer of SRRz. In order to reduce the cost of protein purification, a cellulose-binding module (CBM) from Clostridium thermocellum was fused into the C-terminal of Bgl1A(A24S/F297Y) and cellulose was used as an economic material to adsorb the fusion enzyme from the lysate. The yield of the fusion protein could reach 92.20 ± 2.27% after one-hour adsorption at 25 °C. We have developed an efficient and inexpensive way to produce β-glucosidase for potential industrial applications by using the combination of light induction, cell autolysis, and CBM purification strategy.

  4. Protic ionic liquid as additive on lipase immobilization using silica sol-gel.

    Science.gov (United States)

    de Souza, Ranyere Lucena; de Faria, Emanuelle Lima Pache; Figueiredo, Renan Tavares; Freitas, Lisiane dos Santos; Iglesias, Miguel; Mattedi, Silvana; Zanin, Gisella Maria; dos Santos, Onélia Aparecida Andreo; Coutinho, João A P; Lima, Álvaro Silva; Soares, Cleide Mara Faria

    2013-03-05

    Ionic liquids (ILs) have evolved as a new type of non-aqueous solvents for biocatalysis, mainly due to their unique and tunable physical properties. A number of recent review papers have described a variety of enzymatic reactions conducted in IL solutions, on the other hand, to improve the enzyme's activity and stability in ILs; major methods being explored include the enzyme immobilization (on solid support, sol-gel, etc.), protic ionic liquids used as an additive process. The immobilization of the lipase from Burkholderia cepacia by the sol-gel technique using protic ionic liquids (PIL) as additives to protect against inactivation of the lipase due to release of alcohol and shrinkage of the gel during the sol-gel process was investigated in this study. The influence of various factors such as the length of the alkyl chain of protic ionic liquids (monoethanolamine-based) and a concentration range between 0.5 and 3.0% (w/v) were evaluated. The resulting hydrophobic matrices and immobilized lipases were characterised with regard to specific surface area, adsorption-desorption isotherms, pore volume (V(p)) and size (d(p)) according to nitrogen adsorption and scanning electron microscopy (SEM), physico-chemical properties (thermogravimetric - TG, differential scanning calorimetry - DSC and Fourier transform infrared spectroscopy - FTIR) and the potential for ethyl ester and emulsifier production. The total activity yields (Y(a)) for matrices of immobilized lipase employing protic ionic liquids as additives always resulted in higher values compared with the sample absent the protic ionic liquids, which represents 35-fold increase in recovery of enzymatic activity using the more hydrophobic protic ionic liquids. Compared with arrays of the immobilized biocatalyst without additive, in general, the immobilized biocatalyst in the presence of protic ionic liquids showed increased values of surface area (143-245 m(2) g(-1)) and pore size (19-38 Å). Immobilization with

  5. Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1).

    Science.gov (United States)

    de la Torre, Andrea; Metivier, Aisha; Chu, Frances; Laurens, Lieve M L; Beck, David A C; Pienkos, Philip T; Lidstrom, Mary E; Kalyuzhnaya, Marina G

    2015-11-25

    Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration. A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1-0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion

  6. Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

    Science.gov (United States)

    Currin, Andrew; Swainston, Neil; Day, Philip J.

    2015-01-01

    The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (K d) and catalytic (k cat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving k cat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole

  7. Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras.

    Science.gov (United States)

    Degregorio, Danilo; D'Avino, Serena; Castrignanò, Silvia; Di Nardo, Giovanna; Sadeghi, Sheila J; Catucci, Gianluca; Gilardi, Gianfranco

    2017-01-01

    Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a V max increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.

  8. Engineering Escherichia coli Nicotinic Acid Mononucleotide Adenylyltransferase for Fully Active Amidated NAD Biosynthesis.

    Science.gov (United States)

    Wang, Xueying; Zhou, Yongjin J; Wang, Lei; Liu, Wujun; Liu, Yuxue; Peng, Chang; Zhao, Zongbao K

    2017-07-01

    NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli , NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering. IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich

  9. Rhamnolipids--next generation surfactants?

    Science.gov (United States)

    Müller, Markus Michael; Kügler, Johannes H; Henkel, Marius; Gerlitzki, Melanie; Hörmann, Barbara; Pöhnlein, Martin; Syldatk, Christoph; Hausmann, Rudolf

    2012-12-31

    combine a holistic X-omics strategy with metabolic engineering to achieve the next step in rhamnolipid production based on non-food renewable resources. This review discusses different approaches towards optimization of rhamnolipid production and enhancement of product spectra. The optimization of rhamnolipid production with P. aeruginosa strains, screening methods for new non-pathogenic natural rhamnolipid producers and recombinant rhamnolipid production are examined. Finally, biocatalysis with rhamnolipids for the synthesis of l-rhamnose, β-hydroxyfatty acids, and tailor-made surfactants is discussed. Biosurfactants are still in the phase of initial commercialization. However, for next generation development of rhamnolipid production processes and next generation biosurfactants there are still considerable obstacles to be surmounted, which are discussed here. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

    Science.gov (United States)

    Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

    2016-11-14

    the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

  11. A molecular dynamics study on the transport of a charged biomolecule in a polymeric adsorbent medium and its adsorption onto a charged ligand.

    Science.gov (United States)

    Riccardi, E; Wang, J-C; Liapis, A I

    2010-08-28

    findings have significant practical implications to the design and construction of polymeric porous adsorbent media for effective bioseparations and to the synthesis and operation of processes employed in the separation of biomolecules. The modeling and analysis methods presented in this work could also be suitable for the study of biocatalysis where an enzyme is immobilized on the surface of the pores of a porous medium.

  12. Role of L-alanine for redox self-sufficient amination of alcohols.

    Science.gov (United States)

    Klatte, Stephanie; Wendisch, Volker F

    2015-01-23

    In white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578-5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed. The enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-ta Cv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily

  13. Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes

    International Nuclear Information System (INIS)

    Key, Hanna M.; Dydio, Paweł; Liu, Zhennan

    2017-01-01

    Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C-H bond. Here, we postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, > 70:1 dr, > 75% yield, and ~10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Altogether, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and

  14. Engineering a more sustainable world through catalysis and green chemistry.

    Science.gov (United States)

    Sheldon, Roger A

    2016-03-01

    The grand challenge facing the chemical and allied industries in the twenty-first century is the transition to greener, more sustainable manufacturing processes that efficiently use raw materials, eliminate waste and avoid the use of toxic and hazardous materials. It requires a paradigm shift from traditional concepts of process efficiency, focusing on chemical yield, to one that assigns economic value to replacing fossil resources with renewable raw materials, eliminating waste and avoiding the use of toxic and/or hazardous substances. The need for a greening of chemicals manufacture is readily apparent from a consideration of the amounts of waste generated per kilogram of product (the E factors) in various segments of the chemical industry. A primary source of this waste is the use of antiquated 'stoichiometric' technologies and a major challenge is to develop green, catalytic alternatives. Another grand challenge for the twenty-first century, driven by the pressing need for climate change mitigation, is the transition from an unsustainable economy based on fossil resources--oil, coal and natural gas--to a sustainable one based on renewable biomass. In this context, the valorization of waste biomass, which is currently incinerated or goes to landfill, is particularly attractive. The bio-based economy involves cross-disciplinary research at the interface of biotechnology and chemical engineering, focusing on the development of green, chemo- and biocatalytic technologies for waste biomass conversion to biofuels, chemicals and bio-based materials. Biocatalysis has many benefits to offer in this respect. The catalyst is derived from renewable biomass and is biodegradable. Processes are performed under mild conditions and generally produce less waste and are more energy efficient than conventional ones. Thanks to modern advances in biotechnology 'tailor-made' enzymes can be economically produced on a large scale. However, for economic viability it is generally

  15. Molecular Energy and Environmental Science: A Workshop Sponsored by The National Science Foundation and The Department of Energy May 26-27, 1999 in Rosemont, Illinois

    Energy Technology Data Exchange (ETDEWEB)

    Stair, Peter C [Northwestern Univ., Evanston, IL (United States); DeSimone, Joseph M. [University of North Carolina Chapel Hill; Frost, John W. [Michigan State Univ., East Lansing, MI (United States)

    1999-05-26

    research centers and 40 collaborative research groups are needed at a total annual funding level of $30 Million. Technological Challenges 1. Energy 2. Chemical Synthesis & Processing 3. Clean Air 4. Clean Water 5. Clean Earth Enabling Research Areas A. Materials Synthesis & Nanoscience B. Metallo-Enzymes & Metal Chelators C. Interfacial Science, Corrosion & Separations D. Catalysis/Biocatalysis E. Alternative Solvents F. Waste Treatment G. Supporting Capabilities & Technologies The body of this report provides examples of the Technological Challenges and Enabling Research Areas relevant to energy and the environment. The report

  16. Are ionic liquids extremophiles cell wall breakers? Esther Gutiérrez, M. Ángeles Sanromán, Ana Rodríguez, Francisco J. Deive * Department of Chemical Engineering, University of Vigo, 36310, Vigo, Spain * Corresponding author: Tel.: +34986818723; E-mail address: deive@uvigo.es

    Directory of Open Access Journals (Sweden)

    Esther Gutiérrez

    2014-06-01

    Full Text Available The increasing interest in the development of more competitive biotechnological processes is demanding the development of new downstream strategies to maximize product recovery and foster the economic feasibility and robustness of any desired process. From a biotechnological point of view, lipase production is considered one of the three most important bioprocesses in terms of enzyme sales. During the last years, lipolytic enzymes applications have been broaden to sectors ranging from the petrochemical, pharmaceutical, food and paper to waste management industries, as a result of a close collaboration between academics and industry (Houde et al., 2004. The interest on triacylglycerol hydrolases or lipases (EC 3.1.1.3 lies in the fact that they play a crucial role in biocatalysis of a plethora of chemical reactions, such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis. Their reputation is built largely on their distinctive features, namely, they are quite stable and active in organic solvents, they do not require cofactors, they exhibit a high degree of chemo-, enantio- and regioselectivity, and they possess a wide range of substrate specificity. These features make these enzymes trade to be a well-known billion dollar business (Jaeger and Reetz, 1998; Hasan et al., 2006. However, there are concerns related to the stability of these enzymes at the operating conditions usually employed in biocatalysis. This problem can be circumvented by using extremozymes, whose naturally developed resistance to drastic reaction conditions (like resistance to denaturalization by chemical agents and by extreme values of temperature, pH and salinity turns out to be their main appeal. One of the main limitations observed for the industrial implementation of the processes to produce this kind of enzymes lies in the high costs of downstream operations which represent more than 50-80% of the total processing cost. Surprisingly

  17. Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin.

    Science.gov (United States)

    Simon, Oliver; Klaiber, Iris; Huber, Armin; Pfannstiel, Jens

    2014-09-23

    Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a

  18. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    Science.gov (United States)

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  19. Cell-in-Shell Hybrids: Chemical Nanoencapsulation of Individual Cells.

    Science.gov (United States)

    Park, Ji Hun; Hong, Daewha; Lee, Juno; Choi, Insung S

    2016-05-17

    , bacterial endospores. Bioinspired silicification and phenolics-based coatings are, so far, the main approaches to the formation of cytoprotective cell-in-shell hybrids, because they ensure cell viability during encapsulations and also generate durable nanoshells on cell surfaces. The resulting cell-in-shell hybrids extrinsically possess enhanced resistance to external aggressors, and more intriguingly, the encapsulation alters their metabolic activity, exemplified by retarded or suppressed cell cycle progression. In addition, recent developments in the field have further advanced the synthetic tools available to the stage of chemical sporulation and germination of mammalian cells, where cytoprotective shells are formed on labile mammalian cells and broken apart on demand. For example, individual HeLa cells are coated with a metal-organic complex of ferric ion and tannic acid, and cellular adherence and proliferation are controlled by the programmed shell formation and degradation. Based on these demonstrations, the (degradable) cell-in-shell hybrids are anticipated to find their applications in various biomedical and bionanotechnological areas, such as cytotherapeutics, high-throughput screening, sensors, and biocatalysis, as well as providing a versatile research platform for single-cell biology.

  20. High power density yeast catalyzed microbial fuel cells

    Science.gov (United States)

    Ganguli, Rahul

    Microbial fuel cells leverage whole cell biocatalysis to convert the energy stored in energy-rich renewable biomolecules such as sugar, directly to electrical energy at high efficiencies. Advantages of the process include ambient temperature operation, operation in natural streams such as wastewater without the need to clean electrodes, minimal balance-of-plant requirements compared to conventional fuel cells, and environmentally friendly operation. These make the technology very attractive as portable power sources and waste-to-energy converters. The principal problem facing the technology is the low power densities compared to other conventional portable power sources such as batteries and traditional fuel cells. In this work we examined the yeast catalyzed microbial fuel cell and developed methods to increase the power density from such fuel cells. A combination of cyclic voltammetry and optical absorption measurements were used to establish significant adsorption of electron mediators by the microbes. Mediator adsorption was demonstrated to be an important limitation in achieving high power densities in yeast-catalyzed microbial fuel cells. Specifically, the power densities are low for the length of time mediator adsorption continues to occur. Once the mediator adsorption stops, the power densities increase. Rotating disk chronoamperometry was used to extract reaction rate information, and a simple kinetic expression was developed for the current observed in the anodic half-cell. Since the rate expression showed that the current was directly related to microbe concentration close to the electrode, methods to increase cell mass attached to the anode was investigated. Electrically biased electrodes were demonstrated to develop biofilm-like layers of the Baker's yeast with a high concentration of cells directly connected to the electrode. The increased cell mass did increase the power density 2 times compared to a non biofilm fuel cell, but the power density

  1. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

    Directory of Open Access Journals (Sweden)

    Mädje Katharina

    2012-01-01

    Full Text Available Abstract Background Enzymatic NADH or NADPH-dependent reduction is a widely applied approach for the synthesis of optically active organic compounds. The overall biocatalytic conversion usually involves in situ regeneration of the expensive NAD(PH. Oxidation of formate to carbon dioxide, catalyzed by formate dehydrogenase (EC 1.2.1.2; FDH, presents an almost ideal process solution for coenzyme regeneration that has been well established for NADH. Because isolated FDH is relatively unstable under a range of process conditions, whole cells often constitute the preferred form of the biocatalyst, combining the advantage of enzyme protection in the cellular environment with ease of enzyme production. However, the most prominent FDH used in biotransformations, the enzyme from the yeast Candida boidinii, is usually expressed in limiting amounts of activity in the prime host for whole cell biocatalysis, Escherichia coli. We therefore performed expression engineering with the aim of enhancing FDH activity in an E. coli ketoreductase catalyst. The benefit resulting from improved NADH regeneration capacity is demonstrated in two transformations of technological relevance: xylose conversion into xylitol, and synthesis of (S-1-(2-chlorophenylethanol from o-chloroacetophenone. Results As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3 that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by

  2. Center for Catalysis at Iowa State University

    Energy Technology Data Exchange (ETDEWEB)

    Kraus, George A.

    2006-10-17

    The overall objective of this proposal is to enable Iowa State University to establish a Center that enjoys world-class stature and eventually enhances the economy through the transfer of innovation from the laboratory to the marketplace. The funds have been used to support experimental proposals from interdisciplinary research teams in areas related to catalysis and green chemistry. Specific focus areas included: • Catalytic conversion of renewable natural resources to industrial materials • Development of new catalysts for the oxidation or reduction of commodity chemicals • Use of enzymes and microorganisms in biocatalysis • Development of new, environmentally friendly reactions of industrial importance These focus areas intersect with barriers from the MYTP draft document. Specifically, section 2.4.3.1 Processing and Conversion has a list of bulleted items under Improved Chemical Conversions that includes new hydrogenation catalysts, milder oxidation catalysts, new catalysts for dehydration and selective bond cleavage catalysts. Specifically, the four sections are: 1. Catalyst development (7.4.12.A) 2. Conversion of glycerol (7.4.12.B) 3. Conversion of biodiesel (7.4.12.C) 4. Glucose from starch (7.4.12.D) All funded projects are part of a soybean or corn biorefinery. Two funded projects that have made significant progress toward goals of the MYTP draft document are: Catalysts to convert feedstocks with high fatty acid content to biodiesel (Kraus, Lin, Verkade) and Conversion of Glycerol into 1,3-Propanediol (Lin, Kraus). Currently, biodiesel is prepared using homogeneous base catalysis. However, as producers look for feedstocks other than soybean oil, such as waste restaurant oils and rendered animal fats, they have observed a large amount of free fatty acids contained in the feedstocks. Free fatty acids cannot be converted into biodiesel using homogeneous base-mediated processes. The CCAT catalyst system offers an integrated and cooperative catalytic

  3. From Unnatural Amino Acid Incorporation to Artificial Metalloenzymes

    KAUST Repository

    Makki, Arwa A.

    2016-12-04

    Studies and development of artificial metalloenzymes have developed into vibrant areas of research. It is expected that artificial metalloenzymes will be able to combine the best of enzymatic and homogenous catalysis, that is, a broad catalytic scope, high selectivity and activity under mild, aqueous conditions. Artificial metalloenzyme consist of a host protein and a newly introduced artificial metal center. The host protein merely functions as ligand controlling selectivity and augmenting reactivity, while the metal center determines the reactivity. Potential applications range from catalytic production of fine chemicals and feedstock to electron transfer utilization (e.g. fuel cells, water splitting) and medical research (e.g. metabolic screening). Particularly modern asymmetric synthesis is expected to benefit from a successful combination of the power of biocatalysis (substrate conversion via multi-step or cascade reactions, potentially immortal catalyst, unparalleled selectivity and optimization by evolutionary methods) with the versatility and mechanism based optimization methods of homogeneous catalysis. However, so far systems are either limited in structural diversity (biotin-avidin technology) or fail to deliver the selectivities expected (covalent approaches). This thesis explores a novel strategy based on the site-selective incorporation of unnatural, metal binding amino acids into a host protein. The unnatural amino acids can either serve directly as metal binding centers can be used as anchoring points for artificial metallo-cofactors. The identification expression, purification and modification of a suitable protein scaffolds is fundamental to successfully develop this field. Chapter 2 and 3 detail a rational approach leading to a highly engineered host protein. Starting with fluorescent proteins, which combine high thermal and pH stability, high expression yields, and fluorescence for ease of quantification and monitoring an efficient and fast

  4. Especially for High School Teachers

    Science.gov (United States)

    Howell, J. Emory

    1999-12-01

    -Minute Experiment. Block scheduling has brought an end to the 50-minute period in many classrooms, but the experiment is valid and potentially useful in providing experience with real-world samples. Write Now! With the coming of December days are shorter and nights are longer, and for many readers in the United States and Canada winter weather has set in. If you have been thinking about writing an article for JCE perhaps now is a good time to be doing it. I would like to call your attention to four feature columns designed especially for high school teachers: Chemical Principles Revisited Cary Kilner, Editor Exeter High School, 7 Salmon Street, Newmarket, NH 03857 Phone: 603/659-6825; Fax: 603/772-8287; email: CaryPQ@aol.com Interdisciplinary Connections Mark Alber, Editor Darlington School, 1014 Cave Spring Road, Rome, GA 30161 Phone: 706/236-0442; Fax: 706/236-0443; email: malber@darlington.rome.ga.us Second Year and Advanced Placement Chemistry John Fischer, Editor Ashwaubenon High School, 2391 Ridge Road, Green Bay, WI 54304 Phone: 414/492-2955 ext 2020; email: fischer@netnet.net View from My Classroom David Byrum, Editor Flowing Wells High School, 3301 E. Ft. Lowell Rd., Tucson, AZ 85716 Phone: 520/795-2928; email: DavidB1032@aol.com The titles are descriptive of the content sought for each feature, whose mission statement can be found at the JCE Web site, jchemed.chem.wisc.edu. Click on "Features" in the left-hand frame on your screen. All these editors will be happy to discuss your ideas for an article. Secondary School Feature Articles JCE Classroom Activity #22: Colors to Dye for: Preparation of Natural Dyes, p 1688A Applications of Biocatalysis to Industrial Processes, by John T. Sime, p 1658