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| Cloning and Characterization of Bacillus Licheniformis Glutamyl Endopeptidase |
| ZHU Bei-lin, ZHOU Jie, WANG Zheng-hua, ZHAO Yun, HUANG Jing, WU Zi-rong |
| School of Life Science, East China Normal University, Shanghai 200241, China |
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Abstract The glutamyl endopeptidase gene was amplified by PCR with genomic DNA of Bacillus1icheniformis ATCCl4580 as template, and expressed in Escherichia coli BL21 (DE3) by yielding hybrid plasmid pET28a-GE. Induced with IPTG, the E.coli BL21(DE3) harboring pET28a-GE successfully expressed the glutamyl endopeptidase as inclusion bodies. In order to improve the solubility, the gene was expressed with thioredoxin protein or coexpressed with a chaperone plasmid pTf16-tig,or the culture was incubated at low temperature.. The results showed that the chaperone could improve the solubility of recombinant glutamyl endopeptidase, while thioredoxin and low temperature was futile. The measurement of enzyme activity with Benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide(Z-Phe-Leu-Glu-pNA)as substrate demonstrated that the recombinant glutamyl endopeptidase can effectively hydrolyze the alpha- carboxyl of glutamic acid residue, releasing p-nitroanilide. The optimum temperature and pH for the glutamyl endopeptidase is 42℃, 8.0, respectively, and Mn ion can improve the enzyme activity.
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Received: 26 November 2012
Published: 25 March 2013
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