AMIDASE COUPLED WITH LOW-MOLECULAR-MASS NITRILE HYDRATASE FROM RHODOCOCCUS-RHODOCHROUS J1 - SEQUENCING AND EXPRESSION OF THE GENE AND PURIFICATION AND CHARACTERIZATION OF THE GENE-PRODUCT
AMIDASE COUPLED WITH LOW-MOLECULAR-MASS NITRILE HYDRATASE FROM RHODOCOCCUS-RHODOCHROUS J1 - SEQUENCING AND EXPRESSION OF THE GENE AND PURIFICATION AND CHARACTERIZATION OF THE GENE-PRODUCT
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DOI：
10.1111/j.1432-1033.1993.tb18250.x
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发表时间：
1993-10-01
期刊：
影响因子：
--
通讯作者：
SHIMIZU, S
中科院分区：
文献类型：
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作者：
KOBAYASHI, M;KOMEDA, H;SHIMIZU, S
从紫红红球菌（Rhodococcus rhodochrous）J1 [小林，M.等人（1991）Biochim. Biophys.用各种限制酶消化Acta 1129，23-331，并将修剪的片段插入pUC 18或pUC 19。发现位于L-NHase基因下游1.9kb处的1.96kb EcoRI-SphI区域对于大肠杆菌中酰胺酶活性的表达是必需的; Rhodochrous J1与包括N-774和绿针假单胞菌B23在内的红球菌属物种中的那些不同。核苷酸序列测定表明，该酰胺酶由515个氨基酸组成氨基酸序列分析表明，该酰胺酶基因与红球菌属N-774和绿针假单胞菌P. chlororaphis B23的酰胺酶基因和沙瓦斯坦假单胞菌的吲哚-3-乙酰胺水解酶基因具有较高的相似性，起始密码子上游核苷酸序列的修饰使该酰胺酶基因在大肠杆菌中的表达量为8%。可溶性总蛋白的含量占E. coli中表达。对E.以苯甲酰胺为底物，对大肠杆菌的最低抑菌浓度为0.468单位/mg。该酰胺酶从E.回收率为30.4%。通过HPLC估计的酶的分子量为约110 kDa，并且该酶由分子量相同的两个亚基（55 kDa）组成。该酶作用于脂族酰胺如丙酰胺，也作用于芳族酰胺如苯甲酰胺。丙酰胺和苯甲酰胺的表观K（m）值分别为0.48 mM和0.15 mM。该酰胺酶对2-苯基丙酰胺的S-对映体具有高度特异性，但不能识别2-氯丙酰胺的构型。它还催化酰基从酰胺转移到羟胺以产生相应的异羟肟酸盐。
The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-331 was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-determined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi.The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent K(m) values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.