Expression, Purification And Immobilization Of Recombinant D-amino Acid Oxidase

7-aminocephalosporanic acid(7-ACA)is a key intermediate in the production of semi-synthetic-cephalosporin antibiotics, the DAAO from Trigonopsis variabilis has considerable biotechnological importance because it is used for the enzymatic conversion CPC to 7-ACA. This paper concentrates on the engineering production. The crude extract from the recombinant strain pLHB-3 was purified in a single step by affinity resin—FP-IDA-Co, at the temperature is 30℃,the enzyme activity is detected by 5%CPC, we can get the DAAO activity is 2900U, the activity of immobilization enzyme is 142U/g,The conversion ratio reached up to 98% at 30min with 4% CPC as substrate. . The immobilized DAO could be recycled more than 10 times without significant activity loss. The data suggests that intracellular production of DAO followed by affinity purification and immobilization on oxirane resin may sever as an effective process for manufacture of immobilized DAO for industrial application.In this study we constructed an recombinant expression gene by fusing Vitreoscilla Hemoglobin(VHb) gene with TVDAAO to determine whether bacterial hemoglobine can be used as an oxygen donor for immobilizing flavoenzyme. We designed the primers of VHb gene with an additional NcoI restriction sites, amplified VHb gene by PCR and cloned it into plasmid pLHB-3, the sequence is confirmed by DNA sequencing.. The recombinant plasmid was then transformed into E.coli we can detected the DAAO activity of the recombinant cells after IPTG inducement. But the recombinant cells shows no differences in DAAO activity compared with the control that do not fusion with VHB.A gene of DAAO was obtained by isolating total RNA from Rhodotorula gracilis and then amplified by reverse transcription(RT)-PCR.Comparing its nucleotide sequence with other DAAO genes from Rhodotorula gracilis reported in literature, considerable homology(more than 99%) was observed The DAAO gene digested with EcoR I and Pst I ,and then was inserted into a prokaryotic expression vector pRSET. By colony-PCR method screening, a recombinant plasmid pRSET-DAAO was obtained and then transformed into the expression host BL21(DE3).lt was induced by 1mM IPTG, The result was, when the induction conditions such as the temperature was 30°C,the initial bacterial concentration was OD₆00=1.0, the induction time was 5h, we could get the DAAO activity amount to 390U/ml.