Screening,Identification And Enzymatic Properties Of Bacillus Sp.CLT07 Producing Acid Resistant Chitosan Enzyme

Up to date,chitosan oligosaccharide is the only positively charged oligosaccharide.It is widely used in food industry,agriculture,cosmetics industry,medicine and other fields because of its low relative molecular weight,good water solubility,non-toxic,natural pollution-free,regulating human immunity,lipid-lowering and anti-cancer activities.Chitosan enzyme is a target enzyme for hydrolyzing chitosan to produce chitosan oligosaccharide.Compared with chemical method,enzymatic method has less environmental pollution and mild reaction conditions,and the relative molecular weight distribution of products during degradation is controllable.However,at present,chitosan hydrolysis catalyzed by chitosan enzyme is mostly carried out under acidic conditions,and the short half-life of chitosan enzyme under this condition has become the bottleneck restricting the preparation of chitosan oligosaccharides by enzymatic method.Therefore,this study screened the strains producing acid resistant chitosanase from the sea mud samples of Haizhou Bay,Lianyungang,identified the strains,optimized the fermentation conditions,and studied the purification and enzymatic properties of the enzyme.CLT07 strain with high chitosanase activity was screened from the mud samples of Haizhou Bay,Lianyungang.The chitosanase producing strain was identified by morphology,physiology and biochemistry and 16Sr DNA analysis.It showed that it belonged to bacillus,and strain CLT07was identified as Bacillus pseudococcides.Single factor experiments and the response-surface methodology were used to optimize the conditions of chitosanase production by Bacillus sp.CLT07.The results showed that the optimal culture media of strain Bacillus sp.CLT07 was glucose 1.1%,（NH₄）₂SO₄ 0.92%and colloidal chitosan concentration 1.5%.The optimum culture conditions were as follows:temperature 40℃,fermentation time 48 h,inoculation amount 2%,initial p H4.0 Through optimization,the enzyme activity of Bacillus sp.CLT07 chitosanase increased to 4.01 U/ml,which was 3.5 times higher than that before optimization.The chitosanase of strain Bacillus sp.CLT07 as puriifed by ammonium sulfate salting out,ion exchange chromatography and gel filtration chromatography.The recovery of chitosanase of BCsn07 was increased by 14.82 times,the yield was 25.69%,and the molecular weight was about36 k Da.Some enzymatic properties,acid hydrolysis ability and antifungal activity of the chitosanase were analyzed.The results showed that the optimum reaction temperature and p H value of BCsn07 chitosanase were 45℃and p H 3.0.Fe²⁺and Ca²⁺in metal ions can promote the enzyme activity,and it is inhibited by Na⁺,CO²⁺and EDTA;The results of substrate specificity showed that it only had certain hydrolysis ability to chitosan;Thin layer chromatography analysis showed that the main product was DP5-6 chitosan oligosaccharide,and there was no monosaccharide product.It belonged to an endogenous chitosan enzyme.The acid resistance test of the chitosanase showed that it had strong hydrolysis activity under the condition of p H≤3.0,and the colloidal chitosan was hydrolyzed within 4.5 h,which had acid resistance;In the bacteriostatic test,it was found that the addition amount of pure enzyme solution was 200μL had inhibition on Fusarium wilt.