Analysis of reaction mechanisums and molecular evolution of C-N hydrolases

As to the analysis of amidase，the Arg197 residue in the Rhodococcus amidase was selected for the mutagenesis experiment because they were absolutely conserved in the common signature sequence of the amidase family。By site-directed mutagenesis，we constructed two mutant amidases，in which Argl97was substituted with Lys(R197K)and Gin(R197Q)。The identity of each mutant was confirmed by determining the complete nucleotide seouence of the mutant gene。The Escherichia coli transformant cells containing each mutant gene were cultured at 28℃using the expression system that we established for the wild-type amidase，and then amidase activities in the supernatants of cell-free extracts prepared from the cells were measured.Each mutant enzyme containing the R197K and R197Q substitution did not exhibit any amidase activity at all.Specific activities of R197K，and R197Q mutant enzymes were less than the detection threshold，even when large amounts of the enzymes were used in the reaction f…More or 12h.These findings indicated that Argl97would be crucial to the catalytic activity，by functioning as an oxyanion hole stabilizing the negatively charged tetrahedral transition state.As to the analysis of isonitrile hydratase，to overproduce isonitrile hydratase of Pseudomonas in Escherichia coli，the coding sequence(InhA)was inserted between the Ndel and EcoRl sites of PET-21 resulting in the h.When E.coli harboring PET-mM was cultured in the presence of 0.1 mM IPTG at28-C，high-level of isonitrile hydratase activity(5.23(xmol/min/mg)was detected in the cell-free extract；this value corresponded to 31.7%compared with the specific activity in the isonitrile hydratase purified from the wild Pseudomonas strain.We also analyzed the cell-free extract by SDS-polyacrylamide gel electrophoresis and detected a29 kDa protein band corresponding to the subunit of the Pseudomonas enzyme.The isonitrile hydratase produced in the recombinant cells was purified to homogeneity through ammonium sulfate fractionation and two-step column procedures。The purified enzyme gave almost the same physico-chemical properties，such as specific activity(17.3µmol/min/mg)and molecular mass(29 kDa，apparently)，as the Pseudomonas enzyme.Less：Less

As to the analysis of amidase, the Arg197 residue in the Rhodococcus amidase was selected for the mutagenesis experiment because they were absolutely conserved in the common signature sequence of the amidase family. By site-directed mutagenesis, we constructed two mutant amidases, in which Argl97 was substituted with Lys (R197K) and Gin (R197Q). The identity of each mutant was confirmed by determining the complete nucleotide seouence of the mutant gene. The Escherichia coli transformant cells containing each mutant gene were cultured at 28℃ using the expression system that we established for the wild-type amidase, and then amidase activities in the supernatants of cell-free extracts prepared from the cells were measured. Each mutant enzyme containing the R197K and R197Q substitution did not exhibit any amidase activity at all. Specific activities of R197K, and R197Q mutant enzymes were less than the detection threshold, even when large amounts of the enzymes were used in the reaction f … More or 12 h. These findings indicated that Argl97 would be crucial to the catalytic activity, by functioning as an oxyanion hole stabilizing the negatively charged tetrahedral transition state.As to the analysis of isonitrile hydratase, to overproduce isonitrile hydratase of Pseudomonas in Escherichia coli, the coding sequence (inhA) was inserted between the Ndel and EcoRl sites of PET-21a, resulting in PET-inhA in which the isonitrile hydratase gene was under the control of the T7 promoter. When E. coli harboring PET-mM was cultured in the presence of 0.1 mM IPTG at 28ーC, high-level of isonitrile hydratase activity (5.23 (xmol/min/mg) was detected in the cell-free extract; this value corresponded to 31.7 % compared with the specific activity in the isonitrile hydratase purified from the wild Pseudomonas strain. We also analyzed the cell-free extract by SDS-polyacrylamide gel electrophoresis and detected a 29 kDa protein band corresponding to the subunit of the Pseudomonas enzyme. The isonitrile hydratase produced in the recombinant cells was purified to homogeneity through ammonium sulfate fractionation and two-step column procedures. The purified enzyme gave almost the same physico-chemical properties, such as specific activity (17.3 μmol/min/mg) and molecular mass (29 kDa, apparently), as the Pseudomonas enzyme. Less