Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling.
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 beta-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 alpha 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.
Zhou
Zheng
Z
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.
Zhang
Ai-Hui
AH
Wang
Jing-Ru
JR
Chen
Mao-Lin
ML
Li
Ren-Bao
RB
Yang
Sheng
S
Yuan
Zhong-Yi
ZY
D032621:DNA Shuffling / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D044086:Kluyvera / Q000201:enzymology* / Q000235:genetics; D010399:Penicillin Amidase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011532:Providencia / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D017385:Sequence Homology
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D010399:Penicillin Amidase