- Email: [email protected]
Effect on enantioselectivity of esterase, Est25, by addition of organic solvent and surfactants
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
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