Enzymatic Production Of 7-Aminocephalosporanic Acid

High-level expression of D-amino acid oxidase (DAO) has been reported in Pichia pastor is by integrating the DAO gene under the control of the alcohol oxidase promoter (PAOX1). However, the time taken to reach peak product concentration is usually long (～43 h), and cultivation requires tight regulation of methanol feeding. In this paper, we describe the expression of His-tagged DAO (HDAO) in P. pastoris using the glyceraldehydes-3-phosphate dehydrogenase promoter (PGAP). The maximal level of HDAO expression using the PGAP integrant is attained in 13 h and is equal to that obtained using the PAOX1 integrant in 43 h. We also explored the possibility of secreting HDAO in P. pastoris. In-frame fusion of Saccharomyces cerevisiaeα-factor secretion signal under a PGAP or PAOX1 resulted in low-level secretion of active HDAO, which was not of practical use. The intracellularly expressed HDAO under PGAP was purified by agar-based affinity support and then immobilized on Am-berzyme oxirane resin. The immobilized HDAO, with specific activity of 75 U/g (wet weight), could be recycled more than 14 times without significant loss of activity. The data suggest that intracellular production of HDAO under PGAP, followed by affinity purification and immobilization on oxirane resin, may serve as an effective process for the manufacture of immobilized DAO for industrial application.D-amino acid oxidase belongs to flavoprotein. The aeration of O₂ or increase of pressure in the tank was a means of increasing the oxygen concentration in order to improve the catalytic performance of several oxidases. The growth of host cells and production of targeted products would be increased by Vitreoscilla sp. Hemoglobin under limited oxygen conditions. It was reported that the D-amino acid oxidase of R. gracilis fused with hemoglobin was expressed in E. coli and the catalytic efficiency of immobilized fused protein against CPC was 12.5 fold higher than that of the control. We inferred that hemoglobin would also act as oxygen carrier in vitro and improve the catalytic performance of D-amino acid oxidase. The activities of immobilized D-amino acid oxidase increased three fold and two fold, respectively, after addition of one gram of pure hemoglobin and three hundred milligram of immobilized hemoglo- bin during its conversion of Cephalosporin C. After addition of 1.0 mg of pure hemoglobin, Hemoglobin would improve catalytic performance of flavooxidase when dissolved oxygen became one of limited factors.GL-7-ACA acylase (GLA) is an enzyme that converts GL-7-ACA to 7-ACA, a starting material for semi-synthetic cephalosporin antibiotics. Four his-tagged GLAs were constructed in this work different in their N-terminal amino acid residues. Among them the highest activity of GLA reached 3800 U/l in 19 h for the recombi-nant strain BL21 (DE3)/pET28GA01. The immobilized GLA on TALON affinity support, agar-EPI-IDA-Co²⁺ and FP-IDA-Ni²⁺ was evaluated by one-step purification and immobilization, and the highest immobilized activity was 163 U/g for TALON affinity support. But more robust matrix made FP-IDA-Ni²⁺ suitable for the industrial application. The activities of four immobilized GLAs on FP-IDA-Ni²⁺ ranged from 66.7 U/g to 80 U/g. Immobilized GLA showed good stability at pH value below 11.0 and at temperature up to 30°C. To the consecutive conversion of GL-7-ACA, immobilized GLA was recycled twenty-one times without significant loss of activity, and the average yield rate of 7-ACA reached 90%. These results indicated that intracellu-lar production of his-tagged GLA in E. coli, followed by one-step affinity purification and immobilization on FP-IDA-Ni²⁺ resins, might serve as an effective process for the industrial application.