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| Gene Synthesis, Expression and Property Research of Protein-glutaminase |
| WANG Zheng-hua, ZHU Bei-lin, ZHAO Yun, ZHOU Jie, WU Zi-rong, HUANG Jing |
| School of Life Science, East China Normal University, Shanghai 200241, China |
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Abstract Protein-glutaminase, a novel protein-deamidating enzyme, which has potential for industrial applications. The gene encoding the protein was synthesized using overlap extension PCR and the mature PG gene was cloned into expression vector pET32a(+). The recombinant protein was expressed in Escherichia coli BL21(DE3) as inclusion bodies, and the active PG was obtained after denaturation and renaturation. In order to improve the solubility of PG, the culture was incubated in cold-shocked condition and a chaperone plasmid pTf16-tig was also cloned into pET32a-matPG/BL21(DE3). The results showed that low temperature can improve the solubility of PG slightly. but pTf16-tig was futile. For deamidating activity assay, Cbz-Gln-Gly was used as substrate. The reaction showed that PG can effectively hydrolyzed glutaminyl residues in the Cbz-Gln-Gly and resulting in release of ammonia. The research on enzymatic properties of PG showed that the optimum temperature is 40℃ and the optimal pH is 6.0. The gene encoding protein-glutaminase was synthesized and expressed successfully. The research provided a new idea for heterologous expression of this particular food-enzyme using genetic engineering.
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Received: 15 August 2012
Published: 25 November 2012
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