Optimized Expression Of The Recombinant HpgT Gene And Enzyme Kinetic Characteristics

4-Hydroxyphenylglycine aminotransferase which can synthesize D-phenylglycinetransaminase is produced by Pseudomonas. Domestic HpgT research has few studies，so the catalytic activity of this enzyme in vitro, the catalytic function and the enzymekinetics of HpgT which measured in this paper, are very important to the applicationsof the enzyme in industry.First, the hpgt gene was synthesized through the codon optimization technologyand the shuttle vector pPIC9K-hpgt of E.coli and P. pastoris was constructed. Afterthat, the shuttle vector pPIC9K-hpgt was integrated into the P. pastoris GS115’schromosome so as to realize secretory expression. HpgT was produced fromrecombinant strains fermented in the shaking flask and meadured.Then the recombinant prokaryotic plasmid pCDF-hpgt was obtained. The plasmidwas transformed into the competent cell E. coli BL21(DE3). The recombinantHis-HpgT protein was obtained after the optimized expression and purified by nickelchelate affinity chromatography method. The enzyme activity of the forward andreverse reactions was measured and the activity of the forward reaction reached749mU/mg which was lower than the reverse,2257mU/mg. Also the optimizedtemperature and pH were measured, with the result of35℃and8.0. Other kineticparameters and the mechanism analysis of enzyme characteristics were explained. Theenzyme affinity to phenylglycine was higher than the glutamate which obtained by theMichaelis-Menten equation; and the reaction was inhibited by lower concentration ofphenylglyoxylic acid.At last, this protein structure was analysised and small-scale mutations on hpgtwas researched, on the basis of optimized expression and enzyme kineticcharacteristics of hpgt gene from Pseudomonas stutzeri. This can supply sometheoretical basis.