The Molecular Rebuilding Of Penicillin G Acylase And The Expression Of Recombinant Sars Cornavirus Proteins

Industry of β-lactam antibiotics production is undergoing a huge reformation as the biocatalyzed processes are replacing the chemical synthesis processes. Penicillin G acylase (EC 3.5.1.11, PGA) now plays a central role in the production of β-lactam antibiotics. In view of drawbacks of the conventional chemical processes, such as the high toxicity of some reagents and the requirement for a lot of reaction steps, enzymes with higher synthetic capability facilitate applications in β-lactam antibiotics production. Penicillin G acylases(PGAs) that have been sequenced are those from the gram-negative bacteria family members Alcaligenes faecalis (Af), Escherichia coli (Ec), Kluyvera citrophila (Kc) and Providencia rettgeri (Pr) and from the gram-positive bacteria Bacillus megaterium (Bm) and Arthrobacter viscosus. Besides having similar substrate preferences, these enzymes are evolutionarily related. Their nucleotide sequences and amino acid sequences are mostly conserved.First, by shuffling PGA genes derived from the Escherichia coli, Kluevera citrophila and Providencia rettgeri, which shared DNA identity ranged from 62.5% to 96.9%, five interspecies fusion libraries were created. Mutants with enzyme activity were selected from the plates and then their ratios of cephalexin synthesis to hydrolysis (S/H) were determined subsequently. As a result, the best mutant possesses 40% higher synthetic activity than the wild type PrPGA while primary structures of selected positives exhibit significant diversity existing among each library.Then a novel family shuffling and a conventional family shuffling method were separately used in the rebuilding of five PGAs(AfPGA, EcPGA,...