- Email: [email protected]
Engineering of Bacillus subtilis physiological functionalities towards the production of mycosubtilin anteiso-C17
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
New Biotechnology · Volume 29S · September 2012
metabolic orientations towards the production of mycosubtilin with a specific isoform, i.e., the fatty acid moiety anteiso-C17. Indeed, our previous study showed that this peculiar lipopeptide has a strong antifungal activity [2]. This study was carried out under different adverse environmental perturbations such as cold temperature or high osmolarity and with different nitrogen sources added to the culture medium. Several selected genes were found to be knocked-out or overexpressed. The objectives of our work were to physiologically improve microbial metabolic activities status and to further investigate the desired physiological characteristics. Keywords: Anteiso C17; Fatty acids; Lipopeptides; Bacillus subtilis
and intracellular constructs integrated into the P. pastoris X33 genome. Microbial protransglutaminase enzyme was expressed intracellularly and extracellularly in P. pastoris X33 . Expression of protransglutaminase by metabolically engineered P. pastoris X33 cells was observed upon induction 1% methanol at 30◦ C. The production level of protransglutaminase produced by recombinant P. pastoris will be compared with the one produced by recombinant E. coli. http://dx.doi.org/10.1016/j.nbt.2012.08.223 Stream: White-Industrial Biotechnology, Session: Enabling Technologies: Enzyme Technology, Design Methods, Bioinformatics Poster 1.5.01
References [1].Zhang Y, et al. Trends Biotechnol 2009;27:664–72. [2].Fickers, et al. Appl Environ Microbiol 2009;75:4636–40.
http://dx.doi.org/10.1016/j.nbt.2012.08.222 Poster 1.4.19
Purification and characterization of a thermo and acidstable endo-1,3-1,4--glucanase from Bacillus amyliquefaciens YMNg47 Yonca Avcı Duman 1,∗ , Hatice Korkmaz 2 , Burhan Arikan 2 , Sadık Dinc¸er 2 , A. Akın Denizci 3 , Berna Sarıyar Akbulut 4 , Dilek Kazan 4 , Altan Erarslan 1 1
Recombinant transglutaminase production by metabolically engineered pichia pastoris Burcu Gündüz 1 , Remziye Yılmaz 2 , Pınar C ¸ alık 3 1
Middle East Technical University, Department of Biotechnology, Graduate School of Natural and Applied Sciences, 06531, Ankara, Turkey 2 Middle East Technical University, Central Laboratory Biotechnology Unit, 06531 Ankara, Turkey 3 Middle East Technical University, Department of Chemical Engineering, 06531 Ankara, Turkey Transglutaminases are enzymes that catalyze an acyl transfer reaction between a ␥-carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing digestible heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular protransglutaminase producing recombinant Pichia pastoris strains and to increase the protransglutaminase yield by optimization of the fermentation parameters. Microbial protransglutaminase gene from Streptomyces mobaraensis was amplified by PCR and cloned into the PPICZ␣-A expression vector to construct recombinant microbial protransglutaminase gene. ␣-Factor signal sequence was used for secretion of protransglutaminase. Both intracellular and extracellular constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Intracellular (pPICZ␣A::intraTGase) and extracellular (pPICZ␣A::extraTGase) constructs transferred into E. coli and amplified. Then, plasmids isolated from E. coli and linearized by PmeI restriction enzyme. P. pastoris X33 was transfected by linear pPICZ␣A::intraTGase and pPICZ␣A::extraTGase. Extracellular S80
www.elsevier.com/locate/nbt
Kocaeli University, Faculty of Art and Science, Department of Chemistry, Section of Biochemistry, Umuttepe Campus, 41300 I˙zmit-Kocaeli, Turkey 2 C ¸ ukurova University, Faculty of Science and Letters, Department of Biology, Sarıc¸am-Balcalı, Adana, Turkey 3 The Scientific and Technological Research Council of Turkey, Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze Kocaeli, Turkey 4 Marmara University, Faculty of Engineering, Bioengineering Department, Göztepe Campus, 34722 Kadıköy, I˙stanbul, Turkey Microbial endo--glucanases are added to animal feed to degrade and hence overcome the anti-nutritive effects of 1,3-1,4--Dglucans (homopolymers of -D-glucose linked via -1,3 and -1,4 glycosidic linkages) which are abundant in the cereals, particularly barley. Supplementation of barley containing feed with endo-glucanases reduces viscosity of poultry small intestine contents and improves feed conversion efficiencies and weight gain. In this research, YMNg47 strain was isolated from the soils of Tarsus-Mersin located at Southern Anatolia of Turkey and identified as Bacillus amyliquefaciens considering its biochemical and molecular characteristics. Strain was cultivated in CMC containing medium and its endo-1,3-1.4--glucanase was purified (2.7 fold with 22% yield) by DEAE-Sepharose anion-exchange chromatography from culture supernatant following ultrafiltration using 50 and 5 kDa membranes. The molecular mass of the purified enzyme was estimated as 27 kDa by SDS-PAGE. The temperature and pH optimum of enzyme for each of -1,4 and -1,3 hydrolytic activities were found to be 50◦ C and 7.0, respectively. The Km , Vmax and kcat values of enzyme were estimated as 4 × 10−4 mM CMC, 0.93 U/mL/min, 393 min−1 for -1,4 hydrolytic activity at 50◦ C and pH 4.8 (enzyme specific activity: 9.63 U/mg) and 0.26 mg mL−1 barley -glucan, 1.83 U/mL/min, and 763 min−1 for -1,3 hydrolytic activity, at 30◦ C and pH 5.0 (enzyme specific activ-
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
New Biotechnology · Volume 29S · September 2012
metabolic orientations towards the production of mycosubtilin with a specific isoform, i.e., the fatty acid moiety anteiso-C17. Indeed, our previous study showed that this peculiar lipopeptide has a strong antifungal activity [2]. This study was carried out under different adverse environmental perturbations such as cold temperature or high osmolarity and with different nitrogen sources added to the culture medium. Several selected genes were found to be knocked-out or overexpressed. The objectives of our work were to physiologically improve microbial metabolic activities status and to further investigate the desired physiological characteristics. Keywords: Anteiso C17; Fatty acids; Lipopeptides; Bacillus subtilis
and intracellular constructs integrated into the P. pastoris X33 genome. Microbial protransglutaminase enzyme was expressed intracellularly and extracellularly in P. pastoris X33 . Expression of protransglutaminase by metabolically engineered P. pastoris X33 cells was observed upon induction 1% methanol at 30◦ C. The production level of protransglutaminase produced by recombinant P. pastoris will be compared with the one produced by recombinant E. coli. http://dx.doi.org/10.1016/j.nbt.2012.08.223 Stream: White-Industrial Biotechnology, Session: Enabling Technologies: Enzyme Technology, Design Methods, Bioinformatics Poster 1.5.01
References [1].Zhang Y, et al. Trends Biotechnol 2009;27:664–72. [2].Fickers, et al. Appl Environ Microbiol 2009;75:4636–40.
http://dx.doi.org/10.1016/j.nbt.2012.08.222 Poster 1.4.19
Purification and characterization of a thermo and acidstable endo-1,3-1,4--glucanase from Bacillus amyliquefaciens YMNg47 Yonca Avcı Duman 1,∗ , Hatice Korkmaz 2 , Burhan Arikan 2 , Sadık Dinc¸er 2 , A. Akın Denizci 3 , Berna Sarıyar Akbulut 4 , Dilek Kazan 4 , Altan Erarslan 1 1
Recombinant transglutaminase production by metabolically engineered pichia pastoris Burcu Gündüz 1 , Remziye Yılmaz 2 , Pınar C ¸ alık 3 1
Middle East Technical University, Department of Biotechnology, Graduate School of Natural and Applied Sciences, 06531, Ankara, Turkey 2 Middle East Technical University, Central Laboratory Biotechnology Unit, 06531 Ankara, Turkey 3 Middle East Technical University, Department of Chemical Engineering, 06531 Ankara, Turkey Transglutaminases are enzymes that catalyze an acyl transfer reaction between a ␥-carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing digestible heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular protransglutaminase producing recombinant Pichia pastoris strains and to increase the protransglutaminase yield by optimization of the fermentation parameters. Microbial protransglutaminase gene from Streptomyces mobaraensis was amplified by PCR and cloned into the PPICZ␣-A expression vector to construct recombinant microbial protransglutaminase gene. ␣-Factor signal sequence was used for secretion of protransglutaminase. Both intracellular and extracellular constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Intracellular (pPICZ␣A::intraTGase) and extracellular (pPICZ␣A::extraTGase) constructs transferred into E. coli and amplified. Then, plasmids isolated from E. coli and linearized by PmeI restriction enzyme. P. pastoris X33 was transfected by linear pPICZ␣A::intraTGase and pPICZ␣A::extraTGase. Extracellular S80
www.elsevier.com/locate/nbt
Kocaeli University, Faculty of Art and Science, Department of Chemistry, Section of Biochemistry, Umuttepe Campus, 41300 I˙zmit-Kocaeli, Turkey 2 C ¸ ukurova University, Faculty of Science and Letters, Department of Biology, Sarıc¸am-Balcalı, Adana, Turkey 3 The Scientific and Technological Research Council of Turkey, Marmara Research Center, Genetic Engineering and Biotechnology Institute, P.O. Box 21, 41470 Gebze Kocaeli, Turkey 4 Marmara University, Faculty of Engineering, Bioengineering Department, Göztepe Campus, 34722 Kadıköy, I˙stanbul, Turkey Microbial endo--glucanases are added to animal feed to degrade and hence overcome the anti-nutritive effects of 1,3-1,4--Dglucans (homopolymers of -D-glucose linked via -1,3 and -1,4 glycosidic linkages) which are abundant in the cereals, particularly barley. Supplementation of barley containing feed with endo-glucanases reduces viscosity of poultry small intestine contents and improves feed conversion efficiencies and weight gain. In this research, YMNg47 strain was isolated from the soils of Tarsus-Mersin located at Southern Anatolia of Turkey and identified as Bacillus amyliquefaciens considering its biochemical and molecular characteristics. Strain was cultivated in CMC containing medium and its endo-1,3-1.4--glucanase was purified (2.7 fold with 22% yield) by DEAE-Sepharose anion-exchange chromatography from culture supernatant following ultrafiltration using 50 and 5 kDa membranes. The molecular mass of the purified enzyme was estimated as 27 kDa by SDS-PAGE. The temperature and pH optimum of enzyme for each of -1,4 and -1,3 hydrolytic activities were found to be 50◦ C and 7.0, respectively. The Km , Vmax and kcat values of enzyme were estimated as 4 × 10−4 mM CMC, 0.93 U/mL/min, 393 min−1 for -1,4 hydrolytic activity at 50◦ C and pH 4.8 (enzyme specific activity: 9.63 U/mg) and 0.26 mg mL−1 barley -glucan, 1.83 U/mL/min, and 763 min−1 for -1,3 hydrolytic activity, at 30◦ C and pH 5.0 (enzyme specific activ-