title: Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli.
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.
authors: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, 59 Cangwu Road, Lianyungang, 222005 China ; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071 China ; Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, 59 Cangwu Road, Lianyungang, 222005 China.
authors: Yuying
authors: Y
authors: Sun
authors: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071 China.
authors: Jiquan
authors: J
authors: Zhang
authors: Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Huaihai Institute of Technology, 59 Cangwu Road, Lianyungang, 222005 China ; Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, 59 Cangwu Road, Lianyungang, 222005 China.
authors: Shujun
authors: S
authors: Wang
keywords: Chitosanase; Heterologous; Microbacterium sp. OU01; Recombinant expression vector; Secretion