Heterologous Secretory Expression And Enzymatic Properties Of Chitosanase In Bacillus Subtilis

Chitooligosaccharides(COS),known as chitin oligosaccharides,is an oligomer composed of glucosamine and N-acetylglucosamine by β(1→4)glucoside bond with a polymerization degree of 2-20,molecular weights of under 3200 Da.With characteristics of good water solubility at physiological p H,easy absorption and high biological activity,COS have a variety of functions such as antioxidants,immune enhancements and plant growth promotions.COS are widely used in food industry,agricultures,cosmetics,biomedicines and other fields.COS can be degraded by chitosan.The main production methods are physical,chemical and enzymatic methods.Chitosanase(EC 3.2.1.132)can catalyze the hydrolysis of β(1→4)glycosidic bonds within chitosan molecules to produce COSs with various biological activities.Compared with physical and chemical methods,enzymatic methods overcome the disadvantages of the poor selectivity of chemical methods and the low yields of physical methods.However,the preparation of COS using traditional enzyme methods has shortcomings of complex enzyme purification methods,high production costs and the unclear hydrolysis mechanism.The solution of these problems can provide the theoretical basis and technical support for the industrial production of COS by enzymatic method.In this study,the recombinant plasmid p BE2RS-csn was constructed by linking the Escherichia coli-Bacillus subtilis shuttle vector p BE2 RS with the chitosanase coding gene(csn)cloned in our laboratory.The recombinant CSN from plasmid p BE2RS-csn was heterologously expressed in Bacillus subtilis WB600 cells.Then,the signal peptide of the chitosanase was replaced with a selected signal peptide to improve the secretion and expression efficiency of CSN in Bacillus subtilis.The recombinant BM103 with a high secretion type was constructed.The optimum conditions for the CSN secretion by the recombinant BM103 were further carried out by single-factor and orthogonal tests.The results showed that the optimal culturing condition was an incubation time of 15 h,33℃,and p H6.0.Under this culture condition,the expression of CSN reached 1.458 mg/m L.The SDS-PAGE showed that the molecular weight of recombinant CSN was 29 k Da.Secondly,the enzymatic properties of recombinant CSN expressed in the recombinant strain BM103 were investigated.The results showed that the optimum hydrolysis conditions of the recombinant CSN were as follows: hydrolysis temperature of 49.16℃,the substrate concentration of 1.15 g/L,and p H of 5.19.Under this condition,the enzymatic activity reached 16.161 U/μg.The hydrolysis of recombinant CSN showed that this enzyme was an endonuclease,which only functioned within the chitosans.The β-1,4-glucoside bonds between Glc N residues of chitosans were randomly hydrolyzed,and the final products of the enzymolysis were chitobiose and chitotriose without monosaccharides generation.Finally,the enzymolysis of low molecular weight chitosan was analyzed by molecular simulation to explore the hydrolysis mechanism for chitosan.The binding of chitosans to the substrate differs significantly with different sources of chitosans due to their different crystalline forms.However,the final products of the enzymolysis were consistent.The key binding sites and catalytic sites of enzymatic hydrolysis were also studied.The results indicated that Trp239、Gln181、Asn232、Asp184 in recombinant CSN were the key amino acid sites.In conclusion,the engineered strain constructed in this study could achieve the efficient secretory expression of the recombinant chitosanase without inductions,and the recombinant chitosanases had a high hydrolysis efficiency,which will be an ideal chitosanase for the preparation of high-purity COS with narrow molecular weight distribution.