Matsuda A, Matsuyama K, Yamamoto K, Ichikawa S, Komatsu K
Pharmaceutical Research and Development Department, Asahi Chemical Industry, Co., Ltd., Fuji 416, Japan.
J Bacteriol. 1987 Dec;169(12):5815-20. doi: 10.1128/jb.169.12.5815-5820.1987.
Pseudomonas sp. strain SE83 converts cephalosporin C and 7 beta-(4-carboxybutanamido)cephalosporanic acid (GL-7ACA) to 7-aminocephalosporanic acid (7ACA). A DNA library of this strain was constructed in Escherichia coli and screened for the ability to deacylate GL-7ACA to 7ACA. Apparently, two distinct genes, designated acyI and acyII, were cloned on 4.8- and 6.0-kilobase-pair BglII fragments, respectively. The enzymes encoded by the two genes showed different substrate specificities, and the acyII-encoded enzyme was found to yield 7ACA from cephalosporin C by direct deacylation. Expression of the two genes in E. coli was strongly dependent on a promoter of the vector. The coding regions for acyI and acyII were localized on the 2.5- and 2.8-kilobase-pair fragments, respectively, by subcloning experiments, and high expression of both genes was obtained by placing them under the control of the lacUV5 promoter. The acyII-encoded enzyme was purified and shown to be composed of two nonidentical subunits with molecular weights of 26,000 and 57,000. Maxicell analysis revealed three acyII-specific polypeptides, two of which corresponded to the above subunits. The third polypeptide with a molecular weight of 83,000 was suggested to be the precursor of both subunits.
假单胞菌属菌株SE83可将头孢菌素C和7β-(4-羧基丁酰胺基)头孢烷酸（GL-7ACA）转化为7-氨基头孢烷酸（7ACA）。构建了该菌株在大肠杆菌中的DNA文库，并筛选其将GL-7ACA脱酰基生成7ACA的能力。显然，两个不同的基因，分别命名为acyI和acyII，分别克隆在4.8和6.0千碱基对的BglII片段上。这两个基因编码的酶表现出不同的底物特异性，并且发现acyII编码的酶可通过直接脱酰基作用从头孢菌素C产生7ACA。这两个基因在大肠杆菌中的表达强烈依赖于载体的启动子。通过亚克隆实验，acyI和acyII的编码区分别定位在2.5和2.8千碱基对的片段上，并且通过将它们置于lacUV5启动子的控制下获得了两个基因的高表达。acyII编码的酶被纯化，结果表明它由两个分子量分别为26,000和57,000的不同亚基组成。最大细胞分析揭示了三种acyII特异性多肽，其中两种对应于上述亚基。分子量为83,000的第三种多肽被认为是这两个亚基的前体。
