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An NAPDH-dependent oxidoreductase from Saccharomyces carlsbergensis as key enzyme in the synthesis of γ-lactones
New Biotechnology · Volume 29S · September 2012
have not been used for the detailed characterization of scalable bioconversion systems involving suspensions of immobilized biocatalysts, and the effect of different operational conditions has not been assessed. Further work is being currently performed, to further establish the reproducibility of data throughout scales and biocatalyst stability. http://dx.doi.org/10.1016/j.nbt.2012.08.219 Poster 1.4.16 An NAPDH-dependent oxidoreductase from Saccharomyces carlsbergensis as key enzyme in the synthesis of ␥-lactones Enriqueta Martinez-Rojas 1 , Leif-Alexander Garbe 2 1
Research and Teaching Institute for Brewing in Berlin (VLB), Berlin, Germany 2 Technical University of Berlin, Berlin, Germany ␣,-Unsaturated carbonyl compounds are industrially important substances used as starting materials of several products, including plastics resins, dyes and pharmaceuticals. Because of the increasing demand for natural products, biotechnological processes for the production of lactones have been developed. Generally, these processes involve the bioconversion of a hydroxy fatty acid to ␥- or ␦-lactones. In 2004 Garbe et al. have postulated the formation pathway of 4R-␥-dodecalactone from 9S,10S-dihydroxyoctadecanoic acid in Saccharomyces cerevisiae, with 4-Oxo-2E-dodecenoic acid as intermediate. The latter was reduced by an enone reductase into 4-oxo-dodecanoic acid, further to 4R-hydroxy-dodecanoic acid and finally cycled to 4R-␥-dodecanolactone. This study explored the biochemical performance of a purified enone reductase from Saccharomyces carlsbergensis and its role in the synthesis of enatiopure lactone. The enzyme was isolated and purified to homogeneity from 250 M 1-octen-3-one grown S. carlsbergensis. The NADPHdependent oxidoreductase has a high activity toward different ␣,-unsaturated carbonyl compound. In addition, unsaturated alcohols are transformed into saturated ketones and alcohols. 13S-Hydroxy-9Z,11E-octadecadienoate was transformed into 13oxo-9Z-octadeceoate and subsequently into ␦-decalactone by yeast cells. (±)-4-Hydroxy-2E-decenoic acid ethyl ester was finally reduced to ␥-decalactone. The result of this study have laid foundation for a more thorough understanding of how the yeast cells cope with oxidative stress, this has implication for industrial fields, since ␣,-unsaturated carbonyl compounds play a major role as raw material for synthesis of fine chemicals. http://dx.doi.org/10.1016/j.nbt.2012.08.220
Poster 1.4.17 GluB20-2 protein with a 1,3--glucanase activity synthesis in bioreactor scale Halina Zasłona ∗ , Anna Trusek-Hołownia Division of Chemical and Biochemical Processes, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze 27, 50˙ St. Wyspianskiego ´ 370 Wrocław, Poland In the XXI century, when the progressing deficit of non-renewable resources has become a problem on the world scale, the greater reliance began to put in the alternative source of carbon and energy – specially in the lignocellulosic materials. Lignocellulose is mainly plant biomass which is easy to receive. For that reason this kind of resources seems to be a very attractive substrate for bioconversion and that is why it has still more and more followers. The most often used enzyme in enzymatic hydrolysis of the lignocellulosic materials is mixture of cellulase and hemicellulase. Cellulase is usually mixture of egzo--1,4-glucanase, endo--1,4glucanase and -glucanase. Hemicellulase is a mixture of different enzymes selected in dependence of lignocellulosic materials, which can be consist of diverse hemicelluloses. Some interesting specificities was identified in Escherichia coli genetically modified. In assigned condition, E. coli produces GluB20-2 – protein with a 1,3--glucanase activity. It is used to hydrolyze bonds in -glucan. This protein was produced in the scale bioreactors. The influence of oxygen concentration, the intensity of mixing, the initial concentration of carbon and nitrogen sources on the amount of enzyme synthesized was tested. An increase in the intensity of biocatalyst production was observed in semi-continuous system. A method for initial purification of the enzyme in a semi-industrial scale was elaborated. The resulting preparation can be directly used in the reactions of hydrolysis of the lignocellulosic materials. http://dx.doi.org/10.1016/j.nbt.2012.08.221 Poster 1.4.18 Engineering of Bacillus subtilis physiological functionalities towards the production of mycosubtilin anteiso-C17 N.E. Chihib, J. Guy, F. Coucheney, J.S. Guez, F. Coutte, M. Béchet, P. Jacques ∗ ProBioGEM, Laboratoire de Procédés Biologiques, Génie Enzymatique et Microbien, UPRES EA 1026, Polytech’Lille, IUT A, Université Lille 1 Sciences et Technologies, F-59655 Villeneuve d’Ascq Cedex, France The engineering of bacterial physiological functionalities can be realized by different ways such as strain adaptation and genetic modification. The main physiological characteristics for industrial applications are related to microbial metabolic capability, insensitivity of pathway key enzymes to end-product inhibition or feedback repression, robustness under adverse environmental perturbations, tolerance of high concentration substrates or metabolites, and fitness throughout the entire production processes [1]. Mycosubtilin is synthesized by a hybrid polyketide synthase/nonribosomal synthetase by some strains of Bacillus subtilis. In the present work, our investigations aimed to find out selective www.elsevier.com/locate/nbt S79
have not been used for the detailed characterization of scalable bioconversion systems involving suspensions of immobilized biocatalysts, and the effect of different operational conditions has not been assessed. Further work is being currently performed, to further establish the reproducibility of data throughout scales and biocatalyst stability. http://dx.doi.org/10.1016/j.nbt.2012.08.219 Poster 1.4.16 An NAPDH-dependent oxidoreductase from Saccharomyces carlsbergensis as key enzyme in the synthesis of ␥-lactones Enriqueta Martinez-Rojas 1 , Leif-Alexander Garbe 2 1
Research and Teaching Institute for Brewing in Berlin (VLB), Berlin, Germany 2 Technical University of Berlin, Berlin, Germany ␣,-Unsaturated carbonyl compounds are industrially important substances used as starting materials of several products, including plastics resins, dyes and pharmaceuticals. Because of the increasing demand for natural products, biotechnological processes for the production of lactones have been developed. Generally, these processes involve the bioconversion of a hydroxy fatty acid to ␥- or ␦-lactones. In 2004 Garbe et al. have postulated the formation pathway of 4R-␥-dodecalactone from 9S,10S-dihydroxyoctadecanoic acid in Saccharomyces cerevisiae, with 4-Oxo-2E-dodecenoic acid as intermediate. The latter was reduced by an enone reductase into 4-oxo-dodecanoic acid, further to 4R-hydroxy-dodecanoic acid and finally cycled to 4R-␥-dodecanolactone. This study explored the biochemical performance of a purified enone reductase from Saccharomyces carlsbergensis and its role in the synthesis of enatiopure lactone. The enzyme was isolated and purified to homogeneity from 250 M 1-octen-3-one grown S. carlsbergensis. The NADPHdependent oxidoreductase has a high activity toward different ␣,-unsaturated carbonyl compound. In addition, unsaturated alcohols are transformed into saturated ketones and alcohols. 13S-Hydroxy-9Z,11E-octadecadienoate was transformed into 13oxo-9Z-octadeceoate and subsequently into ␦-decalactone by yeast cells. (±)-4-Hydroxy-2E-decenoic acid ethyl ester was finally reduced to ␥-decalactone. The result of this study have laid foundation for a more thorough understanding of how the yeast cells cope with oxidative stress, this has implication for industrial fields, since ␣,-unsaturated carbonyl compounds play a major role as raw material for synthesis of fine chemicals. http://dx.doi.org/10.1016/j.nbt.2012.08.220
Poster 1.4.17 GluB20-2 protein with a 1,3--glucanase activity synthesis in bioreactor scale Halina Zasłona ∗ , Anna Trusek-Hołownia Division of Chemical and Biochemical Processes, Faculty of Chemistry, Wroclaw University of Technology, Wybrzeze 27, 50˙ St. Wyspianskiego ´ 370 Wrocław, Poland In the XXI century, when the progressing deficit of non-renewable resources has become a problem on the world scale, the greater reliance began to put in the alternative source of carbon and energy – specially in the lignocellulosic materials. Lignocellulose is mainly plant biomass which is easy to receive. For that reason this kind of resources seems to be a very attractive substrate for bioconversion and that is why it has still more and more followers. The most often used enzyme in enzymatic hydrolysis of the lignocellulosic materials is mixture of cellulase and hemicellulase. Cellulase is usually mixture of egzo--1,4-glucanase, endo--1,4glucanase and -glucanase. Hemicellulase is a mixture of different enzymes selected in dependence of lignocellulosic materials, which can be consist of diverse hemicelluloses. Some interesting specificities was identified in Escherichia coli genetically modified. In assigned condition, E. coli produces GluB20-2 – protein with a 1,3--glucanase activity. It is used to hydrolyze bonds in -glucan. This protein was produced in the scale bioreactors. The influence of oxygen concentration, the intensity of mixing, the initial concentration of carbon and nitrogen sources on the amount of enzyme synthesized was tested. An increase in the intensity of biocatalyst production was observed in semi-continuous system. A method for initial purification of the enzyme in a semi-industrial scale was elaborated. The resulting preparation can be directly used in the reactions of hydrolysis of the lignocellulosic materials. http://dx.doi.org/10.1016/j.nbt.2012.08.221 Poster 1.4.18 Engineering of Bacillus subtilis physiological functionalities towards the production of mycosubtilin anteiso-C17 N.E. Chihib, J. Guy, F. Coucheney, J.S. Guez, F. Coutte, M. Béchet, P. Jacques ∗ ProBioGEM, Laboratoire de Procédés Biologiques, Génie Enzymatique et Microbien, UPRES EA 1026, Polytech’Lille, IUT A, Université Lille 1 Sciences et Technologies, F-59655 Villeneuve d’Ascq Cedex, France The engineering of bacterial physiological functionalities can be realized by different ways such as strain adaptation and genetic modification. The main physiological characteristics for industrial applications are related to microbial metabolic capability, insensitivity of pathway key enzymes to end-product inhibition or feedback repression, robustness under adverse environmental perturbations, tolerance of high concentration substrates or metabolites, and fitness throughout the entire production processes [1]. Mycosubtilin is synthesized by a hybrid polyketide synthase/nonribosomal synthetase by some strains of Bacillus subtilis. In the present work, our investigations aimed to find out selective www.elsevier.com/locate/nbt S79