Expression Of Glycolate Oxidase Gene In Pichia Pastoris

Glycolate oxidase catalyzes the oxidation of glycolate to glyoxylate. Glyoxylate is a potential intermediate, which was widely used in the fields of chemical-engineering, food, medicine, such as vanilline, ethylvanillin, allantion.In this dissertation, the importance and performance of chemical routes production and enzyme-catalyzed production of glyoxylate were discussed. Comparing with other preparation methods, it had great potentiality to produce glyoxylate using glycolate oxidase as catalyst. The characteristic of pichia pastoris host system was also discussed. The construction of recombinant strain with coding glycolate oxidase gene was thought of possessing great practical and academic value. The plasmid of pMD-T-GO and pPIC3.5K were extracted from JM109 strain and then glycolate oxidase gene fragment was cloned into the expression vector (pPIC3.5K). The recombinant plasmids were transformed into E.coli TOP 10Fˊ. The recombinant clones were identified by PCR, digested with SnaB I /Not I and then sequenced.The recombinant plasmid was lineared by Sal I and then transferred into pichia pastoris by electro transformation. Positive clones were selected from media without histidine. The clones with high copies were selected using G418 resistance. With the chromosome genome of the recombinant strain as a template, the feature of integration was examined by PCR. The expression of spinach glycolate oxidase in pichia pastoris appeared to be induced by methanol, the molecular weight of glycolate oxidase was about 39.8kDa by SDS-PAGE and consisted with the data of literature. From 0.8(A)of initial density of the cells, The maximal expression was obtained at 28℃ after inducing for 72hr with the 0.5%(v/v) of methanol. Under optimal conditions, the activity of glycolate oxidase was 40.8IU/g wet cell and 17 timers as high as the host. These suggested that efficient expression of glycolate oxidase gene in the recombinant strains was obtained. The recombinant strain with glycolate oxidase gene was well constructed, which possesses industrial prospect of producing glyoxylate with glycolate oxidase.