巨大芽孢杆菌ATCC 14945青霉素G酰化酶基因在大肠杆菌和枯草芽孢杆菌中的表达。

Expression of penicillin G acylase gene from Bacillus megaterium ATCC 14945 in Escherichia coli and Bacillus subtilis.

作者信息

Kang J H, Hwang Y, Yoo O J

机构信息

Department of Biological Science and Engineering, Korea Advanced Institute of Science and Technology, Seoul.

出版信息

J Biotechnol. 1991 Feb;17(2):99-108. doi: 10.1016/0168-1656(91)90001-c.

DOI:10.1016/0168-1656(91)90001-c

PMID:1367491

Abstract

Penicillin G acylase gene from Bacillus megaterium ATCC 14945 has been isolated. Recombinant Escherichia coli clones were screened for clear halo forming activity on the lawn of Staphylococcus aureus ATCC 6538P using the enzymatic acylating reaction of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and D-(alpha)-phenylglycine methylester. The gene was contained within a 2.8 kb DNA fragment and expressed efficiently when transferred from E. coli to Bacillus subtilis. A twenty times greater amount of enzyme was produced in B. subtilis transformant than that in B. megaterium. The purified enzyme from subcloned B. subtilis showed that the native enzyme consisted of two identical subunits, each with a molecular weight of 57,000. The enzyme was able to react on various cephalosporins, i.e., cephalothin, cefamandole, cephaloridine, cephaloglycin, cephalexin and cephradine.

摘要

已从巨大芽孢杆菌ATCC 14945中分离出青霉素G酰化酶基因。利用7-氨基去乙酰氧基头孢烷酸（7-ADCA）和D-（α）-苯甘氨酸甲酯的酶促酰化反应，在金黄色葡萄球菌ATCC 6538P菌苔上筛选重组大肠杆菌克隆的清晰晕圈形成活性。该基因包含在一个2.8 kb的DNA片段中，当从大肠杆菌转移到枯草芽孢杆菌时能高效表达。枯草芽孢杆菌转化子产生的酶量比巨大芽孢杆菌多20倍。从亚克隆的枯草芽孢杆菌中纯化得到的酶表明，天然酶由两个相同的亚基组成，每个亚基的分子量为57,000。该酶能够作用于多种头孢菌素，即头孢噻吩、头孢孟多、头孢匹林、头孢甘氨酸、头孢氨苄和头孢拉定。