Lecitase ultra: A phospholipase with great potential in biocatalysis

Lecitase Ultra is a chimera produced by the fusion of the genes of the lipase from Thermomyces lanuginosus and the phospholipase A1 from Fusarium oxysporum. The enzyme was first designed for the enzymatic degumming of oils, as that problem was not fully resolved before. It is commercialized only as an enzyme solution by Novo Nordisk A/S. This review shows the main uses of this promising enzyme. Starting from the original degumming use, the enzyme has found applications in many other food modification applications, like production of structured phospholipids (e.g., derivatives of phosphatidylcholine), tuning the properties of flour, etc. Moreover, the enzyme has been used in fine chemistry (resolution of racemic mixtures), in the production of aromas and fragrances, polymers modification, etc. Some papers show the use of the enzyme in biodiesel production. Moreover, we present the different technologies applied to obtain a suitable immobilized biocatalyst, remarking the immobilization via interfacial activation and how heterofunctional acyl supports may solve some of the limitations. Immobilized enzyme physical and chemical modifications have also been presented. Finally, Lecitase Ultra has been one of the model enzymes in a new strategy to coimmobilize lipases and other less stable enzymes.We gratefully recognize the financial support from MINECO from Spanish Government (project number CTQ2017-86170-R), Colciencias, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX, Convocatoria Ecosistema Científico – Colombia Científica. Fondo Francisco José de Caldas, Contrato RC-FP44842-212-2018, Colciencias (Colombia, project number FP 44842-076-2016), Generalitat Valenciana (PROMETEO/2018/076), FAPERGS (project number 17/2551-0000939-8), FUNCAP (project number BP3-0139-00005.01.00/18) and CONACYT (Mexico, project number CB-2016-01, 286992)

Immobilization of Proteins in Poly-Styrene-Divinylbenzene Matrices: Functional Properties and Applications

Supports based on poly-styrene-divinylbenzene (PSD) are commercially available since a long time ago. However, they are not commonly used as enzyme immobilization matrices. The main reason for this lies in the negative effect of the very hydrophobic surface on enzyme stability that produces the instantaneous enzyme inactivation in many instances. However, they have recently regained some impact in enzyme immobilization. They are easy to modify, and have been prepared with different modifiers. We will pay special attention to the coating of these supports with ionic liquids, which permits to have the ionic liquid phase anchored to the solid and modulate the enzyme properties without risk of losing these expensive and potentially toxic compounds. Thus, this review will present the covalent or physical immobilization of enzymes on PSD supports, submitted to different modifications. Moreover, lipases immobilized via interfacial activation on some naked PSD supports have shown some unexpected improvement in their catalytic properties, with uses in reactions like hydrolysis, esterification or transesterification.We gratefully recognize the support from the Spanish Government, CTQ2013-41507-R and CNPq (Brazil). The predoctoral fellowships for Ms. García-Galán (Spanish Government), Mr K. Hernandez (I3P-CSIC) and Mr dos Santos (CNPq, Brazil) are also recognized. ). Á. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovacion for a Ramon y Cajal fellowship (RyC-2009-03813)

Polyethylenimine: a very useful ionic polymer in the design of immobilized enzyme biocatalysts

This review discusses the possible roles of polyethylenimine (PEI) in the design of improved immobilized biocatalysts from diverse perspectives. This includes their use to activate supports and immobilize enzymes via ion exchange, as well as to improve immobilized enzymes by coating with PEI. PEI is a polymer containing primary, secondary and tertiary amino groups, having a strong anion exchange capacity under a broad range of conditions, and the capability to chemically react with different moieties on either an enzyme or a support. Also, as a multifunctional polymer, it has been modified stepwise to introduce different functionalities into the same polymer. This polymer (in combination with other anionic ones) permits the generation of “saline” environments around enzyme molecules, improving enzyme stability in the presence of hydrophobic compounds. The use of PEI as a physical glue useful to crosslink enzyme subunits in multimeric enzymes, monomeric enzymes immobilized via physical interactions or production of enzyme multilayers will be specially emphasized as new open avenues for enzyme coimmobilization. The coimmobilization of enzymes and cofactors using PEI may become one of the future developments allowed through an adequate use of this polymer and new pathways towards the design of enzyme combi-catalysts for their use in cascade reactions. Some unexplored but suggested uses derived from the properties of PEI are also proposed in the review, like the use of the buffering power of this multifunctional polymer to avoid pH gradients inside biocatalyst particles. Thus, although PEI has been a largely popular polymer in biocatalyst design, it looks like a long and in some cases almost unexplored road lies ahead.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) project number CTQ2013-41507-R, CNPq (process 403505/2013-5). A. B. M. thanks MINECO, Generalitat Valenciana and FEDER (CTQ2015-66080-R MINECO/FEDER and PROMETEOII/2014/010) for financial support

甘味受容体における呈味調節物質作用モデルの構築及びその検証

学位の種別: 課程博士審査委員会委員 : (主査)東京大学准教授 三坂 巧, 東京大学教授 伏信 進矢, 東京大学特任教授 朝倉 富子, 東京大学准教授 永田 宏次, 東京大学准教授 寺田 透University of Tokyo(東京大学

Novozym 435: the “perfect” lipase immobilized biocatalyst?

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on immobilization via interfacial activation of lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene. N435 is perhaps the most widely used commercial biocatalyst in both academy and industry. Here, we review some of the success stories of N435 (in chemistry, energy and lipid manipulation), but we focus on some of the problems that the use of this biocatalyst may generate. Some of these problems are just based on the mechanism of immobilization (interfacial activation) that may facilitate enzyme desorption under certain conditions. Other problems are specific to the support: mechanical fragility, moderate hydrophilicity that permits the accumulation of hydrophilic compounds (e.g., water or glycerin) and the most critical one, support dissolution in some organic media. Finally, some solutions (N435 coating with silicone, enzyme physical or chemical crosslinking, and use of alternative supports) are proposed. However, the N435 history, even with these problems, may continue in the coming future due to its very good properties if some simpler alternative biocatalysts are not developed.We gratefully recognize the financial support from MINECO from the Spanish Government (project number CTQ2017-86170-R, Colciencias, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX, Convocatoria Ecosistema Científico – Colombia Científica. Fondo Francisco José de Caldas, Contrato RC-FP44842-212-2018 and Colciencias (Colombia) (project number FP44842-076-2016), Generalitat Valenciana (PROMETEO/2018/076), FAPERGS (project number 17/2551-0000939-8), CONICET (R. Argentina), FUNCAP (project number BP3-0139-00005.01.00/18) and ANPCyT (PICT 2015-0932 and PICT CABBIO 4687)

Biotechnological relevance of the lipase A from Candida antarctica

This review intends to present some of the latest studies on the lipase A from Candida antarctica (CALA). This lipase is among the most stable ones and has some capability to attack the sn-2 position of triglycerides. This makes it a very interesting lipase, especially considering that it is commercially available. The cloning and production of the enzyme together with some structural facts and applications will be discussed in this review. Special focus will be put on the immobilization of the enzyme. The use of the commercially available crosslinked enzyme aggregates of this enzyme will be explained, together with the use of the enzyme in some new trends in enzyme immobilization, such as bio-imprinting of the open form of CALA by detergents and the fixation of the open structure, the design of heterofunctional supports able to take full advantage of the immobilization via interfacial activation but preventing enzyme release, or the design of strategies for the preparation of multiple layers of lipase enzymes (using just CALA or combining CALA with other lipases).ABM (RTI2018-095291-B-I00, MINECO/FEDER) and RFL (project number CTQ2017-86170-R). Thank MICINN for financial support, ABM also thanks Generalitat Valenciana (PROMETEOII/2018/076). JJVO thanks to CONACYT (Mexico) for the financial support to the basic science project number, CB-2016-01, 286,992. JCSS thanks to Brazilian Agencies for Scientific and Technological Development, Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), project number BP3-0139-00005.01.00/18 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Project number 422942/2016-2. The continous support supply of lipases from R. Martinez (Novozym Spain) is gratefully recognized

Liquid lipase preparations designed for industrial production of biodiesel. Is it really an optimal solution?

There are growing demands by the biodiesel companies to get cheap lipase formulation, preferably in liquid forms, which can give an answer to their needs. Thus, many commercial or home-produced enzymes are being used in this process in free form. Among these enzymes, Eversa is a liquid lipase formulation derived from the lipase from Thermomyces lanuginosus designed for the enzymatic biodiesel production. The potential of this promising enzyme is hereby reported. However, the specificity of lipases is a problem in biodiesel production. That way the concept of an optimal lipase for biodiesel production is not appropriate, being preferable the use of concept of combi-lipases, using Eversa as a component of the lipase mixture. The application of Eversa for the production of biodiesel through the transesterification, esterification and hydroesterification of oils and fats with different free fatty acids content is thoroughly discussed. Finally, even though Eversa was launched to be used as liquid biocatalyst in the production of biodiesel, this work will discuss how the immobilization of this enzyme through different strategies enhances its performance. That is, a properly designed immobilized Eversa biocatalyst may be more expensive, but it may have some advantages that overcome this drawback.RFL thanks the support by Spanish Ministerio de Ciencia e Innovación (grant number CTQ2017-86170-R). JCSS thanks to Brazilian Agencies for Scientific and Technological Development: Fundaçao Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP, project number BP3-0139-00005.01.00/18), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, project numbers 422942/2016-2 and 311062/2019-9). PWT thanks to Brazilian Agencies for Scientific and Technological Development: Sao Paulo Research Foundation (FAPESP, grant#2016/10636-8), CNPq (project numbers 405889/2016-0 and 308212/2017-7), and Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. RRCM thanks Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The continous support supply of lipases from R. Martinez (Novozym Spain) is gratefully recognized