| Summary: | 碩士 === 大同大學 === 生物工程研究所 === 91 === D-amino acid oxidase (DAO, EC1.4.3.3) is a key enzyme for producing 7-aminocephalosporanic acid, the precursor of synthetic cephalosporins. Pichia pastoris is a methylotrophic yeast capable of metabolizing methanol as its sole carbon source. Recently, it has been also develped into an efficent eukaryotic heterologous expression system.
In this study, a DAO cDNA gene from yeast Rhodosporidium toruloides was transformed into P. pastoris for the heterologous expression. To optomize the DAO expression, the expression vector, inducible pPIC3.5K or constitutive pGAPZA in combination with P. pastoris strain KM71 or GS115 as expression host were used. The better DAO expression was obtain from the constitutive KM71 cultures. The DAO activity peak of ~2470 U/L was obtained 22 hours after culturing with an initial yeast concentration of OD600=0.192. The highest DAO activity from inducible KM71 cultures was ~1212 U/L obtained after 11-hour induction by 2% methanol. The specific activities of recombinant DAO from inducible and constitutive cultures were 85 and 109 U/mg, respectively. To maximize the DAO recovery, the efficiency of four cell disruption methods for protein purification was evaluated. The treatment of Y-PERTM yeast protein extraction reagent appeared better than sonication, pressure disruption or vortexing.
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