Special Issue "Enzymes and Their Biotechnological Applications"

Guest Editor
Prof. Dr. Pabulo Henrique Rampelotto
Interdisciplinary Center for Biotechnology Research, Federal University of Pampa, Antônio Trilha Avenue, P.O.Box 1847, 97300-000, São Gabriel – RS, Brazil
Website: http://www.researcherid.com/rid/B-5752-2009
E-Mail: pabulo@lacesm.ufsm.br

Dear Colleagues,

The development of new enzymes is one of the most thriving branches of biotechnology. Although the applications of enzymes are already well established in some areas, recent advances in modern biotechnology have revolutionized the development of new enzymes. The use of genetic engineering has further improved manufacturing processes and enabled the commercialization of enzymes that could previously not be produced. Protein engineering and the possibility of introducing small changes to proteins are bringing ever more powerful means of analysis to the study of enzyme structure and its biochemical and biophysical properties, which have leading to the rational modification of enzymes to match specific requirements and also the design of new enzymes with novel properties. The developments in bioinformatics and the availability of sequence data have significantly increased the efficiency of identifying genes with biotech potential from nature. Complementary to chemical synthesis, biosynthesis of drug metabolites with mammalian or microbial bioreactors offers certain advantages, and sometime is the only practical route to the desired metabolite. At the same time, new technological developments are stimulating the chemical and pharmaceutical industry to embrace enzyme technology. Altogether, these advances have made it possible to provide tailor-made enzymes displaying new activities and adapted to new process conditions, enabling a further expansion of their use in several branches of biotechnology. This Special Issue focuses on the discovery and development of new enzymes and their applications in different areas of biotechnology. The Special Issue will contain a collection of papers written by authors who are leading experts in the field including selected papers from the 4th International Symposium on Enzymes & Biocatalysis (SEB-2013). We cordially welcome you to join us in this endeavor. The submission of comprehensive reviews or original research articles is most welcome. It is an opportunity to take part in and to influence future trends in one of the fastest growing fields of research.

Professor Pabulo Henrique Rampelotto
Guest Editor

Submission

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• biocatalysis polymerization and polymer synthesis
• biocatalysis synthesis of chiral molecules
• biocatalytic technologies
• enzyme biosensor
• enzyme inhibitors
• environmental enzymology
• enzymatic protein engineering technology
• extremophilic enzymes
• functional genomics and bioinformatics for novel enzymes
• industrial biotechnology
• new enzyme development
• pharmaceutical biotechnology
• structural and functional characterization of enzymes

Type of Paper: Review
Title: Angling for Uniqueness in Enzymatic Preparation of Glycosides
Author: Antonio Trincone
Affiliation: Institute of Biomolecular Chemistry, National Research Council, Via Campi Flegrei, 34, 80078 Pozzuoli, Naples, Italy; E-Mail: antonio.trincone@icb.cnr.it
Abstract: In the early days of biocatalysis, limitations of an enzyme modeled the enzymatic applications; nowadays, enzymes can be engineered to be suitable for the process requirements. However, this is a general bird’s-eye view and cannot be specific for articulated situations found in different class of enzymes or for selected enzymatic processes. As far as the enzymatic preparation of glycosides is concerned, recent scientific literature is crowded with examples of uniqueness related to the features of the biocatalyst (yield, substrate specificity, regioselectivity, resistance to particular reaction condition). The invention of glycosynthases is just one of the aspects that burst forth the research in this field; protein engineering, metagenomics and reaction engineering led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of glycosides. In this review, new examples from last decade are compiled with attention both to cases in which naturally present and genetically inserted characteristic of the catalysts make them attractive for biocatalysis.

Type of Paper: Article
Title: The Effect of Culture Conditions on Lipase Production by Geotrichum Candidum 4013
Authors: Jana Brabcová ^1,2 and Marie Zarevúcka ^2,*
Affiliations: ¹ Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology, Technická 5, 160 28 Prague 6, Czech Republic

² Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo n. 2, 166 10 Prague 6, Czech Republic; E-Mail: zarevucka@uochb.cas.cz (M.Z.)
Abstract: The interest in microbial lipase production has increased in the last decades, because of its large potential in a wide range of industrial applications as additives in food processing, fine chemicals, detergents, waste water treatment, diagnostic, cosmetics, pharmaceuticals and medical. Moreover the lipases from Geotrichum candidum are currently the subject of investigation and industrial interest because it shows a strong preference for unsaturated fatty acids. Till now, the studies on lipolytic enzymes produced by Geotrichum sp. were generally focused on increasing or decreasing of their activity reflecting composition of culture media, but not on effects on the production of particular enzymes. Lipases with different specificities can be produced altering the medium composition. Some components of the culture medium can stimulate lipase production while others can act as repressors. The most common stimulators are long chain fatty acids and organic nitrogen sources, while common repressors are glucose and glycerol. The fungus Geotrichum candidum 4013 produces three extracellular lipases (Lip45kDa, Lip59kDa and Lip61kDa). The effect of culture conditions for maximum individual lipase production by Geotrichum candidum 4013 was studied in connection with composition of culture medium, time and temperature of lipase production. The enzymes were tested for their hydrolytic ability to p-nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols).

Type of Paper: Review
Title: Biocatalytic Synthesis of Chiral Alcohols, Amino acids and Amines for Development of Pharmaceuticals
Author: Ramesh N. Patel
Affiliation: SLRP ASSOCIATES, Consultation in Biotechnology, 572 Cabot Hill Road, Bridgewater, New Jersey, 08807; E-mail: rameshpatelphd@yahoo.com
Abstract: Chirality is a key factor in the safety and efficacy of many pharmaceutical products. The production of single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived there from for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities. Microbial cells and variety of enzymes derived there from can be used for chiral synthesis. Enzymes can be immobilized and reused for many cycles. In addition, enzymes can be over expressed to make biocatalytic processes economically efficient, and enzymes with modified activity can be tailor-made. In this review article, biocatalytic processes are described for the synthesis of chiral alcohols, amino acids and amines for development of pharmaceuticals.

Type of Paper: Review
Title: Quantum Mechanical Modeling of Enzyme Reactions
Author: Gábor Náray-Szabó
Affiliation: Protein Modeling Group HAS/ELTE and Laboratory of Structural Chemistry and Biology, Chemistry Institute, Eötvös Loránd University, Budapest, Hungary; E-mail: naraysza@chem.elte.hu
Abstract: Most enzyme reactions involve formation and cleavage of covalent bonds, while electrostatic effect of distant protein regions, as well as dynamics, may be also crucial. Accordingly, special computational methods are needed to provide an adequate description, which combine quantum mechanics for the reactive region with molecular mechanics and molecular dynamics describing the environment and dynamic effects, respectively. In most cases only energetic aspects can be treated, even if it seems that entropic effects are very important in enzyme reactions. In this review I will give an overview on established computational methods and describe applications to some interesting processes.

Type of Paper: Article
Title: Lipases Immobilisation for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils
Authors: Diana Fattor ¹, Elisabetta De Angelis ², Harumi Veny ³, Luciano Navarini ², Valerio Ferrario ⁴ and Lucia Gardossi ⁴,*
Affiliations: ¹ SPRIN S.p.A., Technology for Sustainable Chemistry, via Flavia 23/1, 34148 Trieste, Italy
² illycaffè S.p.A., via Flavia 110, 34147 Trieste, Italy
³ Department of Chemical Engineering, Faculty of Engineering, University of Malaya
⁴ Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, Piazzale Europa 1, 34127 Trieste, Italy; E-Mail: gardossi@units.it (L.G.)
Abstract: Immobilised lipases were applied to the enzymatic conversion of oils from coffee waste into biodiesel. A computational comparison of the different enzymes allowed the identification of structural features and conformational properties of the enzymes so that tailored protocols were developed for the immobilisation of each lipase. Experimental data indicate that the performance of immobilized lipases depends not only on their structural features but also on the conformational behavior of the lipases placed in different media. The enzymatic synthesis of biodiesel was carried out in mild conditions, with stoichometric amounts of substrates (oil and methanol). Different samples of oils from coffee wastes were tested and the activity and stability of the immobilised lipases were assessed. The results indicate that the oils can be converted quantitatively within hours. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials.

Type of Paper: Article
Title: Pyranose dehydrogenase from Agaricus campestris and Agaricus xanthoderma - characterization and applications in carbohydrate conversions
Authors: Petra Staudigl, Iris Krondorfer, Dietmar Haltrich and Clemens K. Peterbauer*
Affiliation: Food Biotechnology Laboratory, BOKU – University of Natural Resources and Life Sciences, Vienna, Muthgasse 11, 1190 Vienna, Austria;
E-Mail: clemens.peterbauer@boku.ac.at (C.K.P.)
Abstract: Pyranose dehydrogenase (PDH) is a member of the glucose-methanol-choline (GMC) structural family of flavin-dependent oxidoreductases. It is an extracellular monomeric glycoprotein that was first isolated from the edible mushroom Agaricus bisporus and is limited to a rather small group of litter-degrading basidiomycetes. The enzyme is unable to utilize oxygen as electron acceptor using substituted benzoquinones and (organo) metal ions instead. PDH is capable of oxidizing a broad variety of monosaccharides, oligosaccharides and glycosides. Regioselectivity varies between oxidation at C-1, C-2, C-3, depending on substrate, enzyme source and reaction conditions. Double-oxidation at C-2/3 and in rare cases C-3/4 were observed. Valuable sugars like 2-dehydro-D-glucose and 2-dehydro-D-galactose, intermediates for the production of D-fructose from D-glucose and D-tagatose from D-galactose, can be synthesized. In contrast to the closely related enzyme pyranose 2-oxidase (POx), PDHs from several sources are capable of oxidizing α- or β-1→4-linked di- and oligosaccharides, including lactose. PDH from A. xanthoderma mycelial cultures shows preference for C-2 oxidation, producing mainly 2-dehydro-lactose, an intermediate for the production of lactulose, whereas PDH from A. campestris preferentially oxidizes lactose at the C-1 position yielding lactobionic acid. In this work we present the isolation of PDH-encoding genes from A. campestris (Ac) and A. xanthoderma (Ax) and a comparison to other so far isolated PDH- sequences. Secretory overexpression of both enzymes in Pichia pastoris was successful when using their native signal sequences with yields of 371 U/L for AxPDH and 35 U/L for AcPDH. Following a 3-step purification protocol, the enzymes were characterized biochemically and tested for applications in carbohydrate conversion reactions of industrial relevance.

Type of Paper: Review
Title: Biocatalyst Engineering for Improving Catalytic Performance in Neat Organic Solvents
Authors: Munishwar Nath Gupta*, Joyeeta Mukherjee, Priyanka Dubey, Deepika Malhotra
Affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India; Email: munishwar@chemistry.iitd.ernet.in
Abstract: The use of enzymes in nearly anhydrous organic solvent generally results in low initial rates/percentage conversions. The current review focuses on some biocatalyst designs like immobilization on nanomaterials, enzyme precipitated and rinsed with organic solvents (EPROS), crosslinked enzyme crystals (CLEC), crosslinked enzyme aggregates (CLEA), protein coated microcrystals (PCMC) and crosslinked protein coated microcrystals (CLPCMC) which show much better catalytic efficiency in such media as compared to other kinds of biocatalyst preparations. The basic methodology and principle behind these efficient designs is described. The relatively recent results on catalytic promiscuity as seen in low water media as well have also been covered. It is hoped that this will further encourage wider biotechnological applications of enzymes in neat organic solvents.

Type of Paper: Article
Title: Morita-Baylis-Hillman Reaction of 4-Nitrobenzaldehyde with 2-Cyclohexenone Catalysed by Lipases in Aqueous-Organic Co-Solvent Mixtures
Authors: Manali Kapoor , Abir B. Majumder and Munishwar Nath Gupta*
Affiliation: Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. *Email: munishwar@chemistry.iitd.ernet.in
Abstract: Lipases catalyzed the reaction between 4-nitrobenzaldehyde and 2-cyclohexen-1-one in aqueous-DMSO co-solvent mixtures to give Morita-Baylis-Hillman product and aldol product. Among lipases, Burkholderia cepacia lipase gave the best overall conversion of 96% in 50% (v/v) DMSO while Mucor javanicus lipase showed highest stereoselectivity in the formation of the aldol (79% ee) and Morita-Baylis-Hillman product (63% ee) with 30% (v/v) DMSO.

Type of Paper: Review
Title: Biocatalytic Applications of Transglutaminases
Authors: Natalie Rachel1, 2, 3 and Joelle N. Pelletier1, 2, 3, 4 *
Affiliations: 1Département de chimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada; 2CGCC, the Center in Green Chemistry and Catalysis, Montréal, Canada; PROTEO, the Québec Network for Protein Function, Structure and Engineering, Québec, Canada; 4Département de biochimie, Université de Montréal, Montréal, Québec H3C 3J7, Canada; Email: joelle.pelletier@umontreal.ca
Abstract: In nature, transglutaminases catalyze the formation of amide bonds between proteins to form insoluble protein aggregates. They have long been applied in the food and textile industries as a protein cross-linking agent to alter the texture of meat, wool and leather. In recent years, novel applications of transglutaminases in areas ranging from materials sciences to medicine have come to light. There has also been a substantial effort to further investigate the fundamentals of transglutaminases, as there are many characteristics that remain poorly understood. These studies also work towards the goal of developing these enzymes as more efficient tools. Progress in this area includes additional structural information and novel chemical and biological assays. We review these recent biocatalytic achievements in order to illustrate the versatility of transglutaminases.