Preparation Of Immobilizated Alcaligenes Faecalis Penicillin G Acylase And Its Kinetic Resolution Of DL-phenylalanine & Derivatives

Penicillin acylase(E.C 3.5.1.11) has been widely used for the production of some semi-synthetic beta-lactam antibiotics. It is notable about penicillin G acylase from Alcaligenes faecalis (Af-PGA) for outstanding stability. In this work, the extraction, immobilization and application in DL-phenylalanine resolution of Af-PGA were studied. The work could be divided into three sections.The first part, Af-PGA was extracted from the cells.5%(v/v) butyl acetate could release 92.0% of Af-PGA from the E. coli cells at 25℃. The specific activity of the resulting Af-PGA extracting solution was 1 time higher than that of Af-PGA solution prepared by ultrasonic treatment.The second part, Af-PGA was immobilized on both amino-support and epoxy-support. By optimizing the immobilizing conditions, we prepared the immobilized Af-PGA successfully, which was better than the commercial enzyme IPA750 in terms of activity. The optimal temperature and pH of immobilized enzyme were 65℃and 8.0 respectively, and the temperature and pH stabilities of Af-PGA were improved through immobilization.The third part, enantioselective acylation of phenylalanine with immobilized Af-PGA was expored. The optimal acylation reaction was carried out at pH 10,35℃with a molar ratio of 2.5:1 (phenylacetamide to phenylalanine) and enzyme amount of 1IU/ml. And D-phenylalanine was purified by crystallization. These resulted in a D-phenylalanine yield of about 65.2% and D- phenylalanine enantiomeric excess of 97.2%. A series of phenylalanine derivatives were also resoluted under the same conditions and positive results were achieved. In terms of enantiomeric excess, the phenylalanines with electron-withdrawing group substitution were better than those with electron-donating group substitution.