Abstract
A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 × His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria–Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap™ FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS–PAGE analysis. Detected by 4700 MALDI-TOF–TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.
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Acknowledgments
The project was supported by The National Natural Science Foundation of China (Nos. 31172449, 41306165), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China Postdoctoral Science Foundation (No. 2011M501169), and The National High Technology Research and Development Program of China (No. 2012AA10A401).
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Sun, Y., Zhang, J. & Wang, S. Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli . Indian J Microbiol 55, 194–199 (2015). https://doi.org/10.1007/s12088-014-0505-5
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DOI: https://doi.org/10.1007/s12088-014-0505-5