Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain.
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.
Matsuda
A
A
Pharmaceutical Research and Development Department, Asahi Chemical Industry, Co., Ltd., Fuji 416, Japan.
Matsuyama
K
K
Yamamoto
K
K
Ichikawa
S
S
Komatsu
K
K
D000215:Acylation; D000581:Amidohydrolases / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009693:Nucleic Acid Hybridization; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D012988:Soil Microbiology; D013379:Substrate Specificity
D004269:DNA, Bacterial; D002265:Carboxylic Ester Hydrolases; C019861:cephalosporin-C deacetylase; D004262:DNA Restriction Enzymes; D000581:Amidohydrolases; D010399:Penicillin Amidase