| Glucose oxidase has been widely applied in the fields of food,medicine and biologyetc. In this research paper, the gene encoding glucose oxidase from Aspergillus niger A9had been cloned and expressed in Pichia pastoris.In this study, the DNA fragment encoding Aspergillus niger A9 Glucose Oxidasewas amplified by PCR usingA.niger A9 genomic DNA as template with primers designedaccording to published GOD gene sequences. Then the product of PCR was inserted intovector pMD18-T. The sequence analysis showed that the cloned DNA had a length of 1743 bp, encoding a protein of 581 amino acids.(GenBank Accession number: DQ836361).Electroporation conditions for Pichia pastoris were investigated. By changing parameters,amethod of high efficient electro transformation for plasmid pPIC9K/GOD to Pichiapastoris was established. Transforments were obtained by screening on MD and MMplates and PCR technique. The expression products of GOD gene were analyzed bySDS-PAGE and activity of GOD. The results indicated that the GOD gene fromAspergillus niger A9 was successfully transformed and over expressed in Pichia pastoris.The expression level of flask ferment supernatant induced for 168 hours achieved17.35 U/mL and 659.56μg/mL, Measured by SDS-PAGE, the molecular weight ofmonomer was 90,000 Dalton. The glycosylation of purified glucose oxidase was 33.4％based on sulfuric acid-phenol method. The specificity for substrate of glucose oxidase isbetter, and for glucose the Km is 38.04 mmol/L, Vmax is 37.88μmol/min which is testedby dual-reciprocal graph. The product is active under pH 5.0～8.0, while it reaches thehighest activity under pH6.8, the best temperature for activity is 38℃. The activity isalmost unchanged between 20～50℃, but dropped much in 60℃. Metallic ions hadinfluence on glucose oxidase. The glucose oxidase was inhibited strongly by Ag+,Hg2+,NaHSO3 and was inhibited a little by Cu2+,Fe2+, but it was actived by Ca2+,Fe3+,EDTAand SDS in some extent.
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