High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis.
The penicillin G acylase gene (pga) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 (P43 as promoter). The recombinant plasmid was transferred into Bacillus subtilis DB104. Penicillin G acylase production in the B. subtilis transformant was 3-6 u/ml, higher than that of published recombinant strains. Penicillin G acylase production was induced by phenylacetic acid in B. megaterium, whereas the enzyme was produced constitutively in the B. subtilis transformant carrying B. megaterium pga. The recombinant strain showed high stability in antibiotic-free medium for 10 days. Enzyme in crude broth was purified by Al(2)O(3) chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography and the total yield is 79%. The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use.
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China. zhongyi@sunm.shcnc.ac.cn
Sheng
S
Yang
Yan-Hong
YH
Huang
Xiao-Dong
XD
Huang
Shi-Yun
SY
Li
Zhong-Yi
ZY
Yuan