A New Method Of Preparation Of Highly Deacetylated Chito-oligosaccharides With Chitosanase And Chitin Deacetylase

Chitosan is the only positive and natural biopolymer on the earth, and is a kind of alkiline polysaccharose. Chitosan is the deacetylated product of chitin and hard to dissolve in water, the prices of which are raised with the increase of deacetylaed degree of chitin. Chito-oligosaccharides are the products of chitosan degradation and possess special functional properties such as inhibiting the growth of microorganisms, acting as immunopotentiating effectors exerting antitumor activity and modulating the serum lipids. In present report, functional chito-oligosaccharides are mianly the mixture of differently deacetylated degree of chito-oligosaccharides, whereas the activity and fuction of chito-oligosaccharides with highly deacetylated degree are reported little.In traditional way, chito-oligosaccharides is obtained by chitosanase degradation highly deacetylated chitosan, which is prepared using the method of concentrated alkali and pyrolysis, but this method leads to environmental contamination and energy consumption. Therefore, in this study, we used Pchia pastoris expression system to express a kind of chitosanase(CSN-P) from Aspergillus fumigatus and chitin deacetylase(CLCD) from Colletotrichum lindemuthianume, which can convert lowly deacetylated chitosan into chito-oligosaccharides, and then remove the acetyl groups of chito-oligosaccharide to prepare highly deacetylated chito-oligosaccharides. Using this method, we can construct an energy-saving, ecological, efficient way of preparing highly and homogeneously acetylated chito-oligosaccharides, which passes the way for the functional reseach of highly acetylated chito-oligosaccharides.In present study, the sequence of endo-chitosanase gene from Aspergillus fumigatus was modified basing on preferred codons of P. pastoris and synthesized by overlapping PCR in vitro followrd by secretorily expressing in P. pastoris. The results showed that the yield of CSN-P can reach about3mg/ml with high density fermentation, and the enzyme activity is about25000U/ml. The optimal pH and temperature of CSN-P are about6.5and55-65℃, respectively. The activity of CSN-P is completely inhibited by adding of0.1%SDS and10mM Fe3+, however, CSN-P shows stablility in the presence of urea, which remians70%of residual activities in10M urea. More importantly, the secretory CSN-P is highly thermostable, and the half-lives of CSN-P are120min at80℃,60min at90℃and30min at100℃. CSN-P exhibited approximately90%of the initial activity after incubated at80℃for60min,90℃for30min and100℃for15min, which is more stable to heat compared with authigenic chitosanase from Aspergillus fumigatus. Furthermore, glycoprotein staining assay, site-directed mutagenesis and mass spectrometry analysis proved CSN-P is O-glycosylated, which may be the reason why CSN-P is thermostable. Hydrolysis of50%deacetylated chitosan was performed, showing that CSN-P can degrade lowly deacetylated chitoasan efficiently. After24h hydrolysis reaction, mass spectrometer and TLC analysis showed main hydrolysis products of chitosan composed of chitotriose to chitohexaose along with their derivatives, bearing various number of acetyl groups. These mixture was treated with CLCD and the results indicated that CLCD can remove acetyl groups of chito-oligosaccharides. All results of the above suggest that using CSN-P and CLCD, we can produce highly and homogeneously acetylated chito-oligosaccharides.