Purification And Expression In Yarrowia Lipolytica Of A Chitosanase From Microbacterium Sp.

Chitooligosaccharides(COSs), with various functional properties such as immunity-enhancing, antitumor activity and antimicrobial activity, have become a remarkable resource for development of biomedicine, agriculture, and food industry. Chitosanase, which catalyze the hydrolytic degradation of chitosan, can be used for preparation of functional COSs. The purpose of this study is to investigate the expression of chitosanase gene in yeast to resolve the problem of chitosanase such as high cost and unavailability in bulk quantities. The study will provide a more efficient way for COSs production.Microbacterium sp. that can express chitosanase was isolated previously. Purification, expression in Yarrowia lipolytica and characterization of the recombinant chitosanase are reported in this work.The chitosanase from Microbacterium sp. is purified 9.9 folds by a procedure of ammonium sulfate precipitation, DEAE-sepharose Fast Flow ionexchange and Sephacryl S-100 gel filtration chromatography with a recovery of 20.5%. The molecular weight is estimated to be 33.8 kDa by SDS-PAGE. The reaction products analyzed by LC–MS/MS show that chitosan can be degraded into different lengths of oligosaccharides, chitotetraose and chitopentaose are detected as the major products with some levels of chitobiose and chitotriose. The product such as glucosamine cannot be detected indicating that the mode of chitosanase B is endo–type. The N-terminal amino acid sequence is determined to be ETAGTVDLDAPVQKDT.The primers are designed according to N-terminal amino acid sequence and chitosanase gene registered. A 1000-length fragment encoding the chitosanase gene is obtained using the genomic DNA of Microbacterium sp. as a template. The sequence contains one open reading frame of 801 bp encoding 266 animo acids with a signal peptide of 26 amino acids. The deduced N-terminal amino acid sequence of the chitosanase gene, which is classified into glycoside hydrolase family 46, is completely identical to the result sequenced previously.Yarrowia lipolytica is chosen as the host for chitosanase expression. The amplified fragments are inserted into Sfi I–Kpn I sites of the vector pINA1317, resulting the recombinant plasmid. The expression plasmid is transformed to Y.lipolytica Polh competent cell after linearized with restriction enzyme Not I. The transformants are selected on YNB–N5000 plates without uracil. The colony PCR shows that the chitosanase gene is integrated into the genomic DNA of Y.lipolytica. After positive transformant is grown in PPB medium at 28℃for 120 h, the chitosanase activity in the supernatant of the culture is determined to be 10.4 U/ml, which is about 3.15–fold compared to that of Microbacterium sp.Purification of the recombinant chitosanase is achieved by DEAE–Sepharose Fast Flow ionexchange chromatography. The recombinant protein is demonstrated by western blotting. The molecular mass analyzed by SDS-PAGE is 41 kDa, which is larger than native chitosanase. It's probably that the protein modification is different in Y.lipolytica Polh, such as N-glycosylation, signal peptide cleavage and other modifications of the propreprotein. The optimal temperature for recombinant chitosanase is 45℃and the optimal pH is 5.6 (0.2 M acetate buffer). The chitosanase activity is stable in the pH range of 5.0-7.0 and under 25℃. Na+ has an activating effect on enzyme activity. Ag+(10 mM) and Fe3+ (5 mM, 10 mM) can completely inhibit activity. The apparent Km and Vmax values of the recombinant chitosanase with souble chitoan as substrate are 0.93 mg/ml and 6.15 U/ml, respectively. Chitotriose and chitopentaose are detected as the major products.The chitosanase gene is successfully expressed in Y.lipolytica through the construction of expression vector and engineering strains, making an important step towards the production of chitosanase on industrial scale.