Kobayashi M, Komeda H, Nagasawa T, Nishiyama M, Horinouchi S, Beppu T, Yamada H, Shimizu S
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Eur J Biochem. 1993 Oct 1;217(1):327-36. doi: 10.1111/j.1432-1033.1993.tb18250.x.
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, 23-33] 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 Km 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.
从红平红球菌J1 [小林，M.等人（1991年）《生物化学与生物物理学报》1129, 23 - 33] 中克隆得到含有低分子量腈水合酶（L - NHase）基因的质粒pNHJ20L的9.4 - kb插入片段，用各种限制性内切酶进行消化，将修剪后的片段插入pUC18或pUC19中。发现位于L - NHase基因下游1.9 - kb处的一个1.96 - kb的EcoRI - SphI区域对于大肠杆菌中酰胺酶活性的表达至关重要；红平红球菌J1中酰胺酶和NHase的基因排列与包括N - 774在内的红球菌属物种以及绿针假单胞菌B23中的不同。核苷酸测定序列表明，该酰胺酶由515个氨基酸组成（54626 Da），推导的酰胺酶氨基酸序列与包括N - 774和绿针假单胞菌B23在内的红球菌属物种的酰胺酶以及丁香假单胞菌的吲哚 - 3 - 乙酰胺水解酶具有高度相似性。在lac启动子控制下，起始密码子上游核苷酸序列经修饰的酰胺酶基因在大肠杆菌中表达量占总可溶性蛋白的8%。以苯甲酰胺为底物时，大肠杆菌无细胞提取物中酰胺酶活性水平为0.468单位/毫克。该酰胺酶从大肠杆菌转化体提取物中纯化至同质，总回收率为30.4%。通过高效液相色谱法估计该酶的分子量约为110 kDa，且该酶由两个分子量相同（55 kDa）的亚基组成。该酶作用于脂肪族酰胺如丙酰胺，也作用于芳香族酰胺如苯甲酰胺。丙酰胺和苯甲酰胺的表观Km值分别为0.48 mM和0.15 mM。该酰胺酶对2 - 苯丙酰胺的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至羟胺以产生相应的异羟肟酸。
