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

C-N水解酶的反应机理和分子进化分析

• 批准号: 11460042
• 负责人: KOBAYASHI Mikihiko
• 金额: $ 9.66万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11460042/
• 关键词: Amidase; Nitrilase; Nitvile; Amide; Isonitvile; Isonitvile hydratase; アシダーゼ; Rhodococcus; 加水分解酵素

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

至于酰胺酶的分析，选择红球菌酰胺酶中的Arg 197残基用于诱变实验，因为它们在酰胺酶家族的共同特征序列中是绝对保守的。通过定点突变，我们构建了两个突变的酰胺酶，其中Argl 97被替换为Lys（R197 K）和Gln（R197 Q）。通过测定突变基因的完整核苷酸序列来确认每个突变体的身份。使用我们为野生型酰胺酶建立的表达系统，在28℃下培养含有每个突变基因的大肠杆菌Escherichia coli Escherichia coli细胞，然后测量从细胞制备的无细胞提取物的上清液中的酰胺酶活性。含有R197 K和R197 Q取代的每种突变酶根本不表现出任何酰胺酶活性。R197 K和R197 Q突变酶的比活性低于检测阈值，即使在反应中使用大量酶时也是如此。 ...更多信息 或12小时。对于异腈水合酶的分析，为了在大肠杆菌中过量生产假单胞菌的异腈水合酶，将编码序列（inhA）插入PET-21 a的Ndel和EcoRI位点之间，产生PET-inhA，其中异腈水合酶基因在T7启动子的控制下。当E.在0.1mMIPTG存在下于28 ℃培养含有PET-mM的大肠杆菌，在无细胞提取物中检测到高水平的异腈水合酶活性（5.23（xmol/min/mg）;与从野生假单胞菌菌株纯化的异腈水合酶的比活性相比，该值对应于31.7%。我们还通过SDS-聚丙烯酰胺凝胶电泳分析了无细胞提取物，并检测到对应于假单胞菌酶亚基的29 kDa蛋白带。通过硫酸铵分级分离和两步柱层析程序将重组细胞中产生的异腈水合酶纯化至均一。该酶的比活力（17.3 μmol/min/mg）和分子量（29 kDa）与假单胞菌酶的理化性质基本一致。少

项目成果

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小林達彦、清水 昌: "スーパー生体触媒ニトリルヒドラターゼ:重金属コバルトとのユニークな関係"蛋白質核酸酵素. 44. 42-50 (1999)

Tatsuhiko Kobayashi、Masaru Shimizu：“超级生物催化剂腈水合酶：与重金属钴的独特关系”《蛋白质核酸酶》44. 42-50 (1999)。

Sakuradani,E., Kobayashi,M .& Shimizu,S.: "△6-Fatty acid desaturase form an arachidonic acid-producing Mortierella fungus: Gene cloning and its heterologous expression in a fungus, Aspergillus"Gene. 238. 445-453 (1999)

Sakuradani, E., Kobayashi, M. & Shimizu, S.：“△6-脂肪酸去饱和酶形成花生四烯酸生产被孢霉真菌：基因克隆及其在真菌曲霉中的异源表达”基因238。445-453。 (1999)

Kobayashi, M., Himizu, S.: "Cobalt proterins"Eur. J. Biochem.. 261. 1-9 (1999)

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Degen, O. et al.: "Selective transport of divalent cations by transition metal permeases : The alcaligenes eatrophus HoxN and the Rhodococcus rhodochrous Nh1F"Arch. Microbiol.. 171. 139-145 (1999)

Degen, O. 等人：“过渡金属渗透酶对二价阳离子的选择性转运：产碱菌 HoxN 和红红球菌 Nh1F”Arch。