Construction Of Recombinant Pichia Pastoris Producing Glycolate Oxidase And Optimization Of Catalytic Synthesis Of Glyoxylic Acid

Glycolate oxidase is a flavin mononucleotide (FMN)-dependent protein that catalyze the oxidation of glycolic acid to glyoxylic acid, with concomitant reduction of O₂ to H₂O₂.Glyoxylic acid is a fine chemical with diversified applications in perfumery, pharmaceutical, and agrochemical industries. This biocatalytic process, involving the oxidation of glycolic acid by glycolate oxidase and catalase, produces a higher yield of glyoxylic acid and fewer undesirable waste streams than current methods of manufacture. The goal of this work was to synthesize of glyoxylic acid by constructing gene engineering strain. It mainly fouced on construction of recombinant Pichia pastoris producing GO, optimization of fermentation conditions, conditions of synthesis of glyoxylic acid.A cDNA coding for glycolate oxidase was isolated from fresh spinach leaves by RT-PCR. The glycolate oxidase gene was cloned into the pAO815 under the control of AOX1 promoter and integrated into the genome of methylotrophic yeast Pichia pastoris GS115. SDS-PAGE analysis of cell lysate from methanol-induced expression strain demonstrated that the molecular weight of the recombinant GO was about 40 kDa. Fermentation conditions of recombiant Pichia pastoris in shake-flask cultivation were investigated. The maxium activity of GO reached 147 IU/g wet cell when the strains were cultured at 30℃and pH 6.0 with 10 g/L sorbitol and 0.5 g/L methanol as carbon sources.Recombinant Pichia pastoris which expressed glycolate oxidase and endogenous peroxisomal catalase was used as catalyst in synthesis of glyoxylic acid. The reaction conditions on the systhesis of glyoxylic acid by bio-catalyst were examined in this work. Results showed that the yield of glyoxylic acid was 85.0% under the conditions of 0.20 mol/L glycolic acid, 0.01 mmol/L flavin mononuleotide, pH 8.0, air rate of (?), temperature 25℃and reaction time 96 h.