High Expression of
Penicillin G Acylase Gene from Bacillus megaterium in Bacillus
subtilis
YANG Sheng, HUANG Yan-Hong, HUANG Xiao-Dong, LI Shi-Yun and
YUAN Zhong-Yi*
( Shanghai Institute of Biochemistry, the Chinese Academy of Sciences,
Shanghai 200031, China )
Abstract 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 Al2O3 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.
Key Words Bacillus megaterium； penicillin G acylase； Bacillus subtilis； gene expression
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