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Use of oxidoreductive enzymes in sequential biotransformations
New Biotechnology · Volume 25S · September 2009
ABSTRACTS
for loadings higher than 10% in AlPO4-Sepiolite. FFA content increased with lipase load. Thermal oxidation was negligible. For all the biocatalysts tested, the catalytic activity increased with the aw. A 100% SFC35◦ C reduction was observed after six hours of reaction, at aw of 1. However, the estimated half-life time of the lipase in AlPO4-Sepiolite was only about 53 hours, when used in consecutive batches.
2.3.011 Use of oxidoreductive enzymes in sequential biotransformations P. Di Gennaro 1,∗ , F. Orsini 2 , S. Bernasconi 2 , G. Sello 2 1
2
University Milano-Bicocca, Milano, Italy University Milano, Milano, Italy
doi:10.1016/j.nbt.2009.06.392
E-mail address: [email protected] (M. Guillén). One of the most important groups of biocatalysts for biotechnological applications is formed by lipolytic enzymes. Lipases of Rhizopus sp. are an example of interesting lipolytic enzymes because of their high regioselectivity, a property that grants them an essential function in reactions of modification, synthesis and hydrolysis of esters. On the other hand, the methylotrophic yeast Pichia pastoris is widely used nowadays as a host to produce recombinant proteins from different sources, due to its high productivity and simplicity of expression and fermentation system. The present work is based on the characterization of the recombinant Rhizopus oryzae lipase, produced by P. pastoris, versus the lipase produced by the native organism (supplied from Amano). The optimal pH and temperature of both lipases were determined. Furthermore, a study of the substrate specificity was performed using p-nitrophenol esters, and the molecular weight of the active lipases was determined putting in to practice an electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gel and a zymogram with 4-methylumbelliferone (MUF) derivative as substrate. Because of the lower stability of the recombinant lipase compared with the native one in aqueous solution, two different kinds of supports, polypropylene powder EP100 and Eupergit C© , were tested to immobilize the enzyme with the aim of improving it. The study of the stability of both enzymes was performed by means of the Box-Hunter experiment design. In addition, EP100 adsorption isotherms were determined for both, protein concentration and lipolytic activity, and the results show significant differences between recombinant and native lipase.
Biocatalysis is both an alternative to previous procedures and an improvement for applications and results. Several interesting enzymatic activities are of microbial source, because microorganisms are easy to manipulate and to exploit as protein producers. In addition to the single biocatalytic transformation it is as well as important the analysis of the possibility to perform sequential transformations, exploiting the peculiarity that several enzymatic reactions show, that is the ability of occurring in similar conditions, concerning temperature, pH and solvent and above all their chemoselectivity. To this end, some activities that could potentially allow the preparation of special compounds have been selected. The selection of the activities has been directed both by their availability and by the nature of the wanted compound. More precisely, four cell catalysts have been selected. All of them show oxidoreductive activities, but with very different selectivity. Among the biocatalysts the recombinant strains E. coli JM109 (pTAB19), that expresses a monooxygenase activity (SMO) from P. fluorescens ST, E. coli JM109 (pVL2028), that expresses a dehydrogenase activity (NDDH) from P. fluorescens N3, and P. putida PAW340 (pKPT-SMOTouR + pVL3128), expressing both the activities, and a wild-type strain E. coli that expresses a reductase activity (ECAKR), have been selected. Consequently, the available transformations are: epoxidation of double bonds, oxidation of alcohols reduction of carbonyls. The study has concerned the test of the single activities on different substrates and the analysis of some reaction sequences (mainly formed by two steps). Special attention should be paid to the use of biocatalysts expressing two activities. Here, it is fundamental to regulate with care the expression time; in fact, it can happen that the product of one activity is incompatible with the second activity. Our bifunctionalized catalysts express their enzymes under the control of specific promoters. Prepared compounds were: 1-(3-hydroxymethyl-phenyl)ethanol, 3-oxyranyl-benzoic acid, 3-phenyl-oxirane-2-caboxylic acid, 1-(3-phenyl-oxyranyl)-ethanone, 1-(3-phenyl-oxyranyl)ethanol; all of them have high enantiomeric purity. The results allowed the validation of the approach potential, the high flexibility of the biocatalysts and the possibility of producing many compounds, using simple and efficient procedures.
doi:10.1016/j.nbt.2009.06.393
doi:10.1016/j.nbt.2009.06.394
2.3.010 Biochemical characterization and studies of adsorption and immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris M. Guillén ∗ , M.D. Benaiges, F. Valero Department of Chemical Engineering, ETSE, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
S112
www.elsevier.com/locate/nbt
ABSTRACTS
for loadings higher than 10% in AlPO4-Sepiolite. FFA content increased with lipase load. Thermal oxidation was negligible. For all the biocatalysts tested, the catalytic activity increased with the aw. A 100% SFC35◦ C reduction was observed after six hours of reaction, at aw of 1. However, the estimated half-life time of the lipase in AlPO4-Sepiolite was only about 53 hours, when used in consecutive batches.
2.3.011 Use of oxidoreductive enzymes in sequential biotransformations P. Di Gennaro 1,∗ , F. Orsini 2 , S. Bernasconi 2 , G. Sello 2 1
2
University Milano-Bicocca, Milano, Italy University Milano, Milano, Italy
doi:10.1016/j.nbt.2009.06.392
E-mail address: [email protected] (M. Guillén). One of the most important groups of biocatalysts for biotechnological applications is formed by lipolytic enzymes. Lipases of Rhizopus sp. are an example of interesting lipolytic enzymes because of their high regioselectivity, a property that grants them an essential function in reactions of modification, synthesis and hydrolysis of esters. On the other hand, the methylotrophic yeast Pichia pastoris is widely used nowadays as a host to produce recombinant proteins from different sources, due to its high productivity and simplicity of expression and fermentation system. The present work is based on the characterization of the recombinant Rhizopus oryzae lipase, produced by P. pastoris, versus the lipase produced by the native organism (supplied from Amano). The optimal pH and temperature of both lipases were determined. Furthermore, a study of the substrate specificity was performed using p-nitrophenol esters, and the molecular weight of the active lipases was determined putting in to practice an electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gel and a zymogram with 4-methylumbelliferone (MUF) derivative as substrate. Because of the lower stability of the recombinant lipase compared with the native one in aqueous solution, two different kinds of supports, polypropylene powder EP100 and Eupergit C© , were tested to immobilize the enzyme with the aim of improving it. The study of the stability of both enzymes was performed by means of the Box-Hunter experiment design. In addition, EP100 adsorption isotherms were determined for both, protein concentration and lipolytic activity, and the results show significant differences between recombinant and native lipase.
Biocatalysis is both an alternative to previous procedures and an improvement for applications and results. Several interesting enzymatic activities are of microbial source, because microorganisms are easy to manipulate and to exploit as protein producers. In addition to the single biocatalytic transformation it is as well as important the analysis of the possibility to perform sequential transformations, exploiting the peculiarity that several enzymatic reactions show, that is the ability of occurring in similar conditions, concerning temperature, pH and solvent and above all their chemoselectivity. To this end, some activities that could potentially allow the preparation of special compounds have been selected. The selection of the activities has been directed both by their availability and by the nature of the wanted compound. More precisely, four cell catalysts have been selected. All of them show oxidoreductive activities, but with very different selectivity. Among the biocatalysts the recombinant strains E. coli JM109 (pTAB19), that expresses a monooxygenase activity (SMO) from P. fluorescens ST, E. coli JM109 (pVL2028), that expresses a dehydrogenase activity (NDDH) from P. fluorescens N3, and P. putida PAW340 (pKPT-SMOTouR + pVL3128), expressing both the activities, and a wild-type strain E. coli that expresses a reductase activity (ECAKR), have been selected. Consequently, the available transformations are: epoxidation of double bonds, oxidation of alcohols reduction of carbonyls. The study has concerned the test of the single activities on different substrates and the analysis of some reaction sequences (mainly formed by two steps). Special attention should be paid to the use of biocatalysts expressing two activities. Here, it is fundamental to regulate with care the expression time; in fact, it can happen that the product of one activity is incompatible with the second activity. Our bifunctionalized catalysts express their enzymes under the control of specific promoters. Prepared compounds were: 1-(3-hydroxymethyl-phenyl)ethanol, 3-oxyranyl-benzoic acid, 3-phenyl-oxirane-2-caboxylic acid, 1-(3-phenyl-oxyranyl)-ethanone, 1-(3-phenyl-oxyranyl)ethanol; all of them have high enantiomeric purity. The results allowed the validation of the approach potential, the high flexibility of the biocatalysts and the possibility of producing many compounds, using simple and efficient procedures.
doi:10.1016/j.nbt.2009.06.393
doi:10.1016/j.nbt.2009.06.394
2.3.010 Biochemical characterization and studies of adsorption and immobilization of recombinant Rhizopus oryzae lipase expressed in Pichia pastoris M. Guillén ∗ , M.D. Benaiges, F. Valero Department of Chemical Engineering, ETSE, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
S112
www.elsevier.com/locate/nbt