Codon Optimization And High Level Expression In Pichia Pastoris Of Glucose Oxidase

Glucose oxidase(GOD)is a nuturally produced enzyme by many microorganisms.It Catalyses the oxidationβ-D-glucose to D-gluconolactone and hydrogen peroxide.A glucose oxidase gene has been cloned from Penicillium notatum, the gene of glucose oxsidase was cloned into pPIC9 and was successfully expressed in Pichia pastoris.However the expression level was low.This study analyses the characteristic of GOD.Many rare codons were found in it which maybe result in the low expression level. According to the bias in codon choice of the high expression gene in pichia pastoris,and the GC% of GOD. the gene of Glucose Oxidase from penicillium notatum was optimized and artificially synthesized without changing the amino acid sequence,272 nucleic acids were changed which relate to 96 amino acids. Then the modified gene(GOD-M) was inserted to the pastoris pichia expression vector pPIC-9,then it was introduced to pastoris pichia GS115.fifteen high activity strains were screed by the colouration plate and two highest activity strains GOD-M33# and GOD-M40# were obtained.Arfter induced by methanol for 120h at 30℃in fermenter.The activity of glucose oxidase of GOD-M33# and GOD-M40# achieved 365U/ml and 348U/ml,which were 2.5 and 2.35 times of the original strain.SDS-PAGE revealed that the molecular weight of the submits of glucose oxidase was about 67 kDa. The glucose oxidase has a tempertrue optimum of 35℃and pH optimum of 6.2,and are active at pH range from 3.0 to 7.0. the acitivities of glucose oxidase was above 90% between pH 4.0-7.0, and 62% at pH 3.0.After inclubation at 50℃for 2h,the residual activities of glucose oxsidase were 67%. in our study,most metal ions did not affect glucose oxidase activity. The activity of glucose oxidase was significantly inhibited in the presence of 1 mmol/L SDS.the Km,Vmax values of purified god using the substratesβ-D-glucose were 83.21 mmol/L,2170μmol/min/mg at the optimum condition.