Improving the Specific Synthetic Activity of a Penicillin G Acylase Using DNA Family Shuffling

ZHOU Zheng, ZHANG Ai-Hui, WANG Jing-Ru¹, CHEN Mao-Lin¹, LI Ren-Bao, YANG Sheng¹*, YUAN Zhong-Yi*

( Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China;
¹ Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200032, China )

Abstract Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method. The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9%. The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries. By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E. coli JM109(DE3). Mutants with obvious β-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently. It was shown that the primary structures of selected positives exhibited significant diversity among each library. The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA. It was further proved in this study that the domain of α subunit contributed much more to improve the specific activity of synthesis. Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling.

Key words penicillin G acylase; DNA family shuffling; S/H ratio

*Corresponding author:

YUAN Zhong-Yi: Tel, 86-21-54921246; Fax, 86-21-54921011; e-mail, [email protected]

YANG Sheng: Tel, 86-21-64042090-4720; e-mail, [email protected]