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| Optimized Prokaryotic Expression of the Recombinant hpgt Gene and Enzyme Kinetic Characteristics |
| WANG Zong-rui, ZHAO Guang-rong |
| Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology of Tianjin University, Tianjin 300072, China |
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Abstract 4-Hydroxyphenylglycine aminotransferase which can synthesize D-phenylglycine transaminase is produced by Pseudomonas. The hpgt gene was synthesized through the codon optimization technology. The recombinant prokaryotic plasmid pCDF-hpgt was obtained. The plasmid was transformed into the competent cell E. coli BL21 (DE3). The recombinant His-HpgT protein was obtained after the optimized expression and purified by nickel chelate affinity chromatography method. The enzyme activity of the forward and reverse reactions was measured and the activity of the forward reaction reached 749mU/mg which was lower than the reverse, 2 257mU/mg. Also the optimized temperature and pH were measured, with the result of 35℃and 8.0. Other kinetic parameters and the mechanism analysis of enzyme characteristics were explained. The enzyme affinity to phenylglycine was higher than the glutamate which obtained by the Michaelis-Menten equation; and the reaction was inhibited by the lower concentration of phenylglyoxylic acid.
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Received: 20 February 2012
Published: 25 May 2012
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