- Email: [email protected]
Effects of catalytic site mutations on active expression of phage fused penicillin acylase with intein-mediated post-translational autoprocessing
S206
Abstracts / Journal of Biotechnology 136S (2008) S201–S211
III5-P-006 Highly productive cell-free protein synthesis system using dual energy sources Tae-Wan Kim 1,∗
Kim 1 , Ho-Cheol
Kim 1 , In-Seok
Oh 2 , Dong-Myung
1
Department of Fine Chemical Engineering and Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea 2 School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea E-mail address: [email protected] (D.-M. Kim). A stable supply of ATP is one of major factors affecting the efficiency of cell-free protein synthesis reactions (Kim and Swartz, 1999). In most cases, the regeneration of ATP is conducted through the substrate-level phosphorylation reactions using a secondary energy source carrying high-energy phosphate bonds (Spirin, 2004). However, consumption of the secondary energy source causes accumulation of inorganic phosphate, which can halt protein synthesis by the sequestration of magnesium ions in the reaction mixture (Kim et al., 2006). In this study, we developed a novel energy regeneration system using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate and glucose are used simultaneously, the phosphate ion released from the creatine phosphate is subsequently used in the glycolytic pathway for the utilization of the glucose, thereby enhancing the ATP supply and retarding the phosphate accumulation. As a result, protein synthesis was prolonged up to 3 h, producing substantially 2–3-fold enhanced amounts of proteins. We expect that the developed cell-free protein synthesis system will serve as an attractive and viable option for protein expression for many post-genomic development programs.
Kim, D.M., Swartz, J.R., 1999. Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol. Bioeng. 66, 180–188. Kim, T.W., Kim, D.M., Choi, C.Y., 2006. Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system. J. Biotechnol. 124, 373–380. Spirin, A.S., 2004. High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol. 22, 538–545.
doi:10.1016/j.jbiotec.2008.07.434 III5-P-008 Construction and identification of recombinant adenovirus mediated human cytomegalovirus gB gene in HEK293 cells Chao Jiang 1,∗
Yong
Chen 1,∗,a ,
Li
Gong 2 ,
Min
Guo 1 ,
Ping
Li 1 ,
Lin
1 Lanzhou Institute of Biological Products (LIBP), The China National Biotec Group (CNBG), Lanzhou, Gansu, China 2 Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, Gansu, China
E-mail addresses: [email protected] (Y. Chen), [email protected] (L. Jiang). Human cytomegalovirus (HCMV), double-stranded DNA virus, its infection rate of more than 60% in the general population. The pneumonia rooted from HCMV is the most important complica-
a
References Alba, R., Bosch, A., Chillon, M., 2005. Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther. (12 Suppl. 1), s18–s27. Barajas, M., Mazzolini, G., Genove, G., 2001. Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin-l2. Hepatology 33 (1), 52–61. Britt, W.J., Alford, C.A., 1996. Cytomegalovirus. Infields Virol., ed3. Plostine, S.A., 1999. Cytomegalovirus vaccine. Am. Heart J. 138 (52), 484–487. Stone, D., Ni, S., Li, Z.-Y., Gaggar, A., DiPaolo, N., Feng, Q., Sandig, V., Lieber, A., 2005. Development and assessment of human adenovirus type II as a gene transfer vector. J. Virol. 79 (8), 5090–5104.
doi:10.1016/j.jbiotec.2008.07.435 III5-P-009
Reference
Ma 1,a ,
tions among the immunosuppressed patients, mortality rate can be as high as 80% to 90%. The pregnant women infected HCMV may induce fetal growth retardation and deformities. The gB protein, a kind of virus glycoprotein, as the crucial role in the adsorption and membrane fusion between cytomegalovirus and the host, can induce the anti-virus antibody. The epitopes triggering humoral immune were found in the gB protein. Adenovirus vector loading the P53 gene has been applied for the treatment of head and neck cancer. In our study, we hope gB gene can be expressed by the adenovirus vector. The recombinant plasmid named pAd-Easy-1/gB was constructed. The liposome was used to co-transfect the pAd-Easy-1/gB into HEK293 cells. HCMV-gB positive recombinant adenovirus was screened by plaque and PCR assays. Observed the expression of the marker protein-Green fluorescence protein (GFP) with fluorescence microscope to screen the recombinant virus. The plaque assay was used to determine the virus titter, 2 × 106 PFU/ml was observed in our study. In conclusion, the adenovirus carrying the gB gene was constructed, and the virus-like particles were produced. It was the foundation for recombinant adenovirus-HCMV combined vaccine.
These authors equally contributed to this work.
Effects of catalytic site mutations on active expression of phage fused penicillin acylase with intein-mediated post-translational autoprocessing Yi-Feng Shi 1,∗ , Patrice Soumillion 2 , Mitsuyoshi Ueda 3 1
Department of Biotechnology, Dalian Polytechnic University, Dalian 116034, China 2 Unit de Biochimie, Institute of Life Science, Universite Catholique de Louvain, B1348 Louvain-la-Neuve, Belgium 3 Department of Biomacromolecular Chemistry, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan E-mail address: [email protected] (Y.-F. Shi).
Penicillin G acylase (EC 3.5.1.11) is 86-kDa large heterodimeric protein comprising two peptide A 23-kDa and B 62-kDa, produced by intein-mediated auto-splicing of a 92-kDa precursor. Directed evolution of penicillin acylase using phage display technology for extending its novel specificity is an interesting topic both of industry and academic because penicillin G acylase was potentially employed in the preparation of a wide range of semi-syntheticlactam antibiotics from acyl side-chain precursors and -lactam nucleus. We fused the penicillin acylase to fd phage coat protein III and used pIII secretion signal sequence instead of penicillin acylase. Western blotting and enzyme activity assay were performed to demonstrate penicillin acylase has been functionally displayed on phage surface. Owing to the intimate association of enzyme activity and prescursor processing in penicillin acylase, substitutions of enzyme residues to make a phage library should be careful not to lead to processing defects. By site-directed mutagenesis, we
Abstracts / Journal of Biotechnology 136S (2008) S201–S211
have then identified some active mutations of catalytic residue Ser B1 and Asn B241 on phage fused penicillin acylase which showed post-translational maturation.
S207
Sørensen, H.P., Mortensen, K.K., 2005. Advanced genetic strategies for recombinant expression in Escherichia coli. J. Biotechnol. 115, 113–128. Yang, Q., Xu, X.M., Li, M., Yuan, X.D., Su, Z.G., Janson, J.C., An, L.J., 2002. Cloning and expression of defibrase cDNA from the venom of Gloydius shedaoensis. Biotechnol. Lett. 24, 135–138.
References doi:10.1016/j.jbiotec.2008.07.437 Duggleby, H.J., Tolley, S.P., Hill, C.P., Dodson, E.J., Dodson, G., Moody, P.C.E., 1995. Penicillin acylase has a single amino-acid catalytic amino-acid catalytic center. Nature 373, 264–268. McVey, C.E., Walsh, M.A., Dodson, G.G., Wilson, K.S., Brannigan, J.A., 2001. Crystal structure of penicillin acylase enzyme-substrate complex: structural insight into the catalytic mechanism. J. Mol. Biol. 313, 139–150. Verhaert, R.M.D., Duin, J.V., Quax, W.J., 1999. Processing and functional display of the 86 kDa penicillin G acylase on the surface of phage fd. Biochem. J. 342, 415–422.
III5-P-011 Effect on enantioselectivity of esterase, Est25, by addition of organic solvent and surfactants Seung-Bum Kim, Won Kyu Lee, Doo Hun Kim, Yoen-Woo Ryu ∗
doi:10.1016/j.jbiotec.2008.07.436
Department of Science and Technology, Ajou University, Suwon, South Korea
III5-P-010
E-mail address: [email protected] (Y.-W. Ryu).
Inhibitory effect of transition metal ions on a recombinant thrombin-like enzyme gloshedobin from Gloydius shedaoensis, in Escherichia coli Jianqiang Xu 1 , Qing Yang 1,∗ , Jun Yang 1 , Hua Yang 1 , Wimal Ubhayasekera 2 , Sherry L. Mowbray 2 , Jan-Christer Janson 3 1
Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China 2 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Uppsala 751 24, Sweden 3 Department of Surface Biotechnology, Uppsala University, Uppsala Biomedical Center, P.O. Box 577, Uppsala 751 23, Sweden E-mail address: [email protected] (Q. Yang).
Snake venom proteolytic enzymes attract special interest due to their effects on hemostatic system and abilities in clinical treatment of thrombosis diseases (Castro et al., 2004). In this study, inhibitory effect of transition metal ions on a recombinant snake venom thrombin-like enzyme termed gloshedobin (Yang et al., 2002) from Escherichia coli cytosol was investigated (Sørensen and Mortensen, 2005). Its amidolytic activity toward N-␣-benzoyl-dl-arginine p-nitroanilide (BApNA) was greatly inhibited by 1 mM of transition metal ions by the order: Fe2+ > Cu2+ ≈ Zn2+ > Hg2+ Ni2+ . And its fibrinogenolytic activity toward bovine fibrinogen was inhibited by divalent transition metal ions by the order: Cu2+ > Ni2+ > Zn2+ > Co2+ . Other ions such as Ca2+ and Mg2+ have almost no effect on either activity. These results showed its unique enzymatic properties compared with other thrombin-like enzymes, and might provide additional insights for increasing interests and concerns in applying recombinant fibrinogenolytic enzymes in the prevention and the clinical treatment of thrombosis and in turn to search for new defibrinogenating drugs. Acknowledgements Financial support by 973 Project (2003CB114400), 863 Project (2003AA2Z3520), the Fok Ying Tung Education Foundation (101072) and the National Natural Science Foundation of China (20536010, 20576016 and 20676021) is greatly acknowledged. S.L. Mowbray and W. Ubhayasekera were supported by a grant from the Swedish National Research Council. References Castro, H.C., Zingali, R.B., Albuquerque, M.G., Pujol-Luz, M., Rodrigues, C.R., 2004. Snake venom thrombin-like enzymes: from reptilase to now. Cell. Mol. Life Sci. 61, 843–856.
Enantioselective enzymes, especially for those that catalyze hydrolysis reaction, are useful catalysts in production of pure enantiomers for pharmaceutical, agrochemical and bioactive materials. Esterases are among the group of enzymes considered as potential catalysts in industrial process. Ketoprofen is one of nonsteroidal anti-inflammatory drug. It is generally alleged that the (S)-ketoprofen has the higher pharmacological effect compare with the racemic mixture of profens (Sheldon, 1993). Because of their abundance and great versatility in mediated reactions, esterases have recently been considered as a possible candidate for the chiral resolution of ketoprofen. Esterase-mediated chiral resolution of racemic compound has been ubiquitously found in a variety of biomolecules and synthetic chemicals (Bornscheuer and Kazlauskas, 1999). Est25, novel esterase from a metagenomic library, efficiently hydrolyzed (R,S)-ketoprofen ethyl ester which was significantly improved by the addition of emulsifier surfactants, but showed no significant enantioselectivity (Kim et al., 2006). In the process of (R,S)-ketoprofen ethyl ester hydrolysis, enantioselectivity of Est25 toward S-ketoprofen ethyl ester was remarkably increased when (R,S)-ketoprofen ethyl ester dissolved in ethanol then surfactant such as triton X-100 was added separately, instead (R,S)-ketoprofen ethyl ester was dissolved in the buffer containing surfactant. When percentage of ethanol is increased enantioselectivity of Est25 was also increased. Ethanol may act as an inhibitor of R-ketoprofen ethyl ester. Effects of other organic solvents and surfactants on enantioselectivity are investigated and results will be discussed. References Bornscheuer, U.T., Kazlauskas, R.J., 1999. Hydrolases in Organic Synthesis- Regioand Stereoselective Biotransformations. Wiley–VCH, Weinheim, Germany. Kim, Y.J., Choi, G.S., Kim, S.B., Yoon, G.S., Kim, Y.S., Ryu, Y.W., 2006. Screening and characterization of a novel esterase from a metagenomic library. Protein Expression and Purification 45, 315–323. Sheldon, R.A., 1993. Chirotechnology: Industrial Synthesis of Optically Active Compounds. Marcel Dekker, New York.
doi:10.1016/j.jbiotec.2008.07.438
Abstracts / Journal of Biotechnology 136S (2008) S201–S211
III5-P-006 Highly productive cell-free protein synthesis system using dual energy sources Tae-Wan Kim 1,∗
Kim 1 , Ho-Cheol
Kim 1 , In-Seok
Oh 2 , Dong-Myung
1
Department of Fine Chemical Engineering and Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea 2 School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-742, Republic of Korea E-mail address: [email protected] (D.-M. Kim). A stable supply of ATP is one of major factors affecting the efficiency of cell-free protein synthesis reactions (Kim and Swartz, 1999). In most cases, the regeneration of ATP is conducted through the substrate-level phosphorylation reactions using a secondary energy source carrying high-energy phosphate bonds (Spirin, 2004). However, consumption of the secondary energy source causes accumulation of inorganic phosphate, which can halt protein synthesis by the sequestration of magnesium ions in the reaction mixture (Kim et al., 2006). In this study, we developed a novel energy regeneration system using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate and glucose are used simultaneously, the phosphate ion released from the creatine phosphate is subsequently used in the glycolytic pathway for the utilization of the glucose, thereby enhancing the ATP supply and retarding the phosphate accumulation. As a result, protein synthesis was prolonged up to 3 h, producing substantially 2–3-fold enhanced amounts of proteins. We expect that the developed cell-free protein synthesis system will serve as an attractive and viable option for protein expression for many post-genomic development programs.
Kim, D.M., Swartz, J.R., 1999. Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol. Bioeng. 66, 180–188. Kim, T.W., Kim, D.M., Choi, C.Y., 2006. Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system. J. Biotechnol. 124, 373–380. Spirin, A.S., 2004. High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotechnol. 22, 538–545.
doi:10.1016/j.jbiotec.2008.07.434 III5-P-008 Construction and identification of recombinant adenovirus mediated human cytomegalovirus gB gene in HEK293 cells Chao Jiang 1,∗
Yong
Chen 1,∗,a ,
Li
Gong 2 ,
Min
Guo 1 ,
Ping
Li 1 ,
Lin
1 Lanzhou Institute of Biological Products (LIBP), The China National Biotec Group (CNBG), Lanzhou, Gansu, China 2 Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, Gansu, China
E-mail addresses: [email protected] (Y. Chen), [email protected] (L. Jiang). Human cytomegalovirus (HCMV), double-stranded DNA virus, its infection rate of more than 60% in the general population. The pneumonia rooted from HCMV is the most important complica-
a
References Alba, R., Bosch, A., Chillon, M., 2005. Gutless adenovirus: last-generation adenovirus for gene therapy. Gene Ther. (12 Suppl. 1), s18–s27. Barajas, M., Mazzolini, G., Genove, G., 2001. Gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin-l2. Hepatology 33 (1), 52–61. Britt, W.J., Alford, C.A., 1996. Cytomegalovirus. Infields Virol., ed3. Plostine, S.A., 1999. Cytomegalovirus vaccine. Am. Heart J. 138 (52), 484–487. Stone, D., Ni, S., Li, Z.-Y., Gaggar, A., DiPaolo, N., Feng, Q., Sandig, V., Lieber, A., 2005. Development and assessment of human adenovirus type II as a gene transfer vector. J. Virol. 79 (8), 5090–5104.
doi:10.1016/j.jbiotec.2008.07.435 III5-P-009
Reference
Ma 1,a ,
tions among the immunosuppressed patients, mortality rate can be as high as 80% to 90%. The pregnant women infected HCMV may induce fetal growth retardation and deformities. The gB protein, a kind of virus glycoprotein, as the crucial role in the adsorption and membrane fusion between cytomegalovirus and the host, can induce the anti-virus antibody. The epitopes triggering humoral immune were found in the gB protein. Adenovirus vector loading the P53 gene has been applied for the treatment of head and neck cancer. In our study, we hope gB gene can be expressed by the adenovirus vector. The recombinant plasmid named pAd-Easy-1/gB was constructed. The liposome was used to co-transfect the pAd-Easy-1/gB into HEK293 cells. HCMV-gB positive recombinant adenovirus was screened by plaque and PCR assays. Observed the expression of the marker protein-Green fluorescence protein (GFP) with fluorescence microscope to screen the recombinant virus. The plaque assay was used to determine the virus titter, 2 × 106 PFU/ml was observed in our study. In conclusion, the adenovirus carrying the gB gene was constructed, and the virus-like particles were produced. It was the foundation for recombinant adenovirus-HCMV combined vaccine.
These authors equally contributed to this work.
Effects of catalytic site mutations on active expression of phage fused penicillin acylase with intein-mediated post-translational autoprocessing Yi-Feng Shi 1,∗ , Patrice Soumillion 2 , Mitsuyoshi Ueda 3 1
Department of Biotechnology, Dalian Polytechnic University, Dalian 116034, China 2 Unit de Biochimie, Institute of Life Science, Universite Catholique de Louvain, B1348 Louvain-la-Neuve, Belgium 3 Department of Biomacromolecular Chemistry, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan E-mail address: [email protected] (Y.-F. Shi).
Penicillin G acylase (EC 3.5.1.11) is 86-kDa large heterodimeric protein comprising two peptide A 23-kDa and B 62-kDa, produced by intein-mediated auto-splicing of a 92-kDa precursor. Directed evolution of penicillin acylase using phage display technology for extending its novel specificity is an interesting topic both of industry and academic because penicillin G acylase was potentially employed in the preparation of a wide range of semi-syntheticlactam antibiotics from acyl side-chain precursors and -lactam nucleus. We fused the penicillin acylase to fd phage coat protein III and used pIII secretion signal sequence instead of penicillin acylase. Western blotting and enzyme activity assay were performed to demonstrate penicillin acylase has been functionally displayed on phage surface. Owing to the intimate association of enzyme activity and prescursor processing in penicillin acylase, substitutions of enzyme residues to make a phage library should be careful not to lead to processing defects. By site-directed mutagenesis, we
Abstracts / Journal of Biotechnology 136S (2008) S201–S211
have then identified some active mutations of catalytic residue Ser B1 and Asn B241 on phage fused penicillin acylase which showed post-translational maturation.
S207
Sørensen, H.P., Mortensen, K.K., 2005. Advanced genetic strategies for recombinant expression in Escherichia coli. J. Biotechnol. 115, 113–128. Yang, Q., Xu, X.M., Li, M., Yuan, X.D., Su, Z.G., Janson, J.C., An, L.J., 2002. Cloning and expression of defibrase cDNA from the venom of Gloydius shedaoensis. Biotechnol. Lett. 24, 135–138.
References doi:10.1016/j.jbiotec.2008.07.437 Duggleby, H.J., Tolley, S.P., Hill, C.P., Dodson, E.J., Dodson, G., Moody, P.C.E., 1995. Penicillin acylase has a single amino-acid catalytic amino-acid catalytic center. Nature 373, 264–268. McVey, C.E., Walsh, M.A., Dodson, G.G., Wilson, K.S., Brannigan, J.A., 2001. Crystal structure of penicillin acylase enzyme-substrate complex: structural insight into the catalytic mechanism. J. Mol. Biol. 313, 139–150. Verhaert, R.M.D., Duin, J.V., Quax, W.J., 1999. Processing and functional display of the 86 kDa penicillin G acylase on the surface of phage fd. Biochem. J. 342, 415–422.
III5-P-011 Effect on enantioselectivity of esterase, Est25, by addition of organic solvent and surfactants Seung-Bum Kim, Won Kyu Lee, Doo Hun Kim, Yoen-Woo Ryu ∗
doi:10.1016/j.jbiotec.2008.07.436
Department of Science and Technology, Ajou University, Suwon, South Korea
III5-P-010
E-mail address: [email protected] (Y.-W. Ryu).
Inhibitory effect of transition metal ions on a recombinant thrombin-like enzyme gloshedobin from Gloydius shedaoensis, in Escherichia coli Jianqiang Xu 1 , Qing Yang 1,∗ , Jun Yang 1 , Hua Yang 1 , Wimal Ubhayasekera 2 , Sherry L. Mowbray 2 , Jan-Christer Janson 3 1
Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China 2 Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Uppsala 751 24, Sweden 3 Department of Surface Biotechnology, Uppsala University, Uppsala Biomedical Center, P.O. Box 577, Uppsala 751 23, Sweden E-mail address: [email protected] (Q. Yang).
Snake venom proteolytic enzymes attract special interest due to their effects on hemostatic system and abilities in clinical treatment of thrombosis diseases (Castro et al., 2004). In this study, inhibitory effect of transition metal ions on a recombinant snake venom thrombin-like enzyme termed gloshedobin (Yang et al., 2002) from Escherichia coli cytosol was investigated (Sørensen and Mortensen, 2005). Its amidolytic activity toward N-␣-benzoyl-dl-arginine p-nitroanilide (BApNA) was greatly inhibited by 1 mM of transition metal ions by the order: Fe2+ > Cu2+ ≈ Zn2+ > Hg2+ Ni2+ . And its fibrinogenolytic activity toward bovine fibrinogen was inhibited by divalent transition metal ions by the order: Cu2+ > Ni2+ > Zn2+ > Co2+ . Other ions such as Ca2+ and Mg2+ have almost no effect on either activity. These results showed its unique enzymatic properties compared with other thrombin-like enzymes, and might provide additional insights for increasing interests and concerns in applying recombinant fibrinogenolytic enzymes in the prevention and the clinical treatment of thrombosis and in turn to search for new defibrinogenating drugs. Acknowledgements Financial support by 973 Project (2003CB114400), 863 Project (2003AA2Z3520), the Fok Ying Tung Education Foundation (101072) and the National Natural Science Foundation of China (20536010, 20576016 and 20676021) is greatly acknowledged. S.L. Mowbray and W. Ubhayasekera were supported by a grant from the Swedish National Research Council. References Castro, H.C., Zingali, R.B., Albuquerque, M.G., Pujol-Luz, M., Rodrigues, C.R., 2004. Snake venom thrombin-like enzymes: from reptilase to now. Cell. Mol. Life Sci. 61, 843–856.
Enantioselective enzymes, especially for those that catalyze hydrolysis reaction, are useful catalysts in production of pure enantiomers for pharmaceutical, agrochemical and bioactive materials. Esterases are among the group of enzymes considered as potential catalysts in industrial process. Ketoprofen is one of nonsteroidal anti-inflammatory drug. It is generally alleged that the (S)-ketoprofen has the higher pharmacological effect compare with the racemic mixture of profens (Sheldon, 1993). Because of their abundance and great versatility in mediated reactions, esterases have recently been considered as a possible candidate for the chiral resolution of ketoprofen. Esterase-mediated chiral resolution of racemic compound has been ubiquitously found in a variety of biomolecules and synthetic chemicals (Bornscheuer and Kazlauskas, 1999). Est25, novel esterase from a metagenomic library, efficiently hydrolyzed (R,S)-ketoprofen ethyl ester which was significantly improved by the addition of emulsifier surfactants, but showed no significant enantioselectivity (Kim et al., 2006). In the process of (R,S)-ketoprofen ethyl ester hydrolysis, enantioselectivity of Est25 toward S-ketoprofen ethyl ester was remarkably increased when (R,S)-ketoprofen ethyl ester dissolved in ethanol then surfactant such as triton X-100 was added separately, instead (R,S)-ketoprofen ethyl ester was dissolved in the buffer containing surfactant. When percentage of ethanol is increased enantioselectivity of Est25 was also increased. Ethanol may act as an inhibitor of R-ketoprofen ethyl ester. Effects of other organic solvents and surfactants on enantioselectivity are investigated and results will be discussed. References Bornscheuer, U.T., Kazlauskas, R.J., 1999. Hydrolases in Organic Synthesis- Regioand Stereoselective Biotransformations. Wiley–VCH, Weinheim, Germany. Kim, Y.J., Choi, G.S., Kim, S.B., Yoon, G.S., Kim, Y.S., Ryu, Y.W., 2006. Screening and characterization of a novel esterase from a metagenomic library. Protein Expression and Purification 45, 315–323. Sheldon, R.A., 1993. Chirotechnology: Industrial Synthesis of Optically Active Compounds. Marcel Dekker, New York.
doi:10.1016/j.jbiotec.2008.07.438