Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis.
The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.
Zhou
Zheng
Z
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.
Zhou
Li-Ping
LP
Chen
Mei-Juan
MJ
Zhang
Yan-Liang
YL
Li
Ren-Bao
RB
Yang
Sheng
S
Yuan
Zhong-Yi
ZY
D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013045:Species Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010399:Penicillin Amidase