高产壳聚糖酶菌株筛选鉴定、条件优化及酶学性质分析

Screening,Identification and Conditions Optimization of High Chitosanase Producing Strains,and Analysis of Its Enzymatic Properties

为了筛选出高产壳聚糖酶的菌株,并对其最优产酶条件及酶学性质进行研究。本研究从广西北海养殖虾塘底泥采样,经初筛、复筛选出高产壳聚糖酶菌株,并进行鉴定,再通过单因素实验、Plackett-Burman及正交试验优化产酶工艺,最后对其酶学性质及产物进行研究。结果表明,筛选到1株高产壳聚糖酶的菌株Gxun-5.3.3,鉴定为蜡样芽孢杆菌(Bacillus cereus);菌株Gxun-5.3.3产酶的最优工艺为:麦芽糖5 g/L、酵母粉3 g/L、壳聚糖15 g/L、初始pH6.0、发酵时间84 h,优化后酶活可达62.03±1.18 U/mL,较优化前(39.87±1.24 U/mL)提高了55.58%;酶学性质研究表明,该酶最适反应温度为60℃,最适反应pH为5.0,金属离子中Fe^(2+)、Mn^(2+)对壳聚糖酶有显著(P<0.05)的激活作用,而Al^(3+)、Cu^(2+)则有较强的抑制作用;酶解壳聚糖产物经薄层层析(Thin Layer Chromatography,TLC)测定为聚合度2~4的壳寡糖,表明菌株所产壳寡糖酶为内切型。本研究为B. cereus Gxun-5.3.3壳聚糖酶的生产和壳寡糖的酶法制备提供了理论依据。

To screen and identify high chitosanase producing strains and to optimize the fermentation condition and analyze the enzymatic properties of the target strain.In this study,high chitosanase-producing strain was screened from the sediment of shrimp ponds in Beihai,Guangxi through primary and secondary screening.The strain underwent identification,the enzyme production process of the strain was optimized through single-factor,Plackett-Burman and orthogonal optimization methods.The enzymatic properties and products of chitosanase produced by the strain were studied.The results showed that the strain of Gxun-5.3.3 with high chitosanase production was obtained by screening and was identified as Bacillus cereus.The optimal components for the enzyme-producing medium of B.cereus Gxun-5.3.3 was maltose 5 g/L,yeast powder 3 g/L,chitosan 15 g/L,and the initial pH and fermentation time were 6.0 and 84 h,respectively.Under the optimized conditions,the chitosanase activity reached 62.03±1.18 U/mL,which was 55.58% higher before optimization(39.87±1.24 U/mL).The protease produced by strain Gxun-5.3.3 had maximum activity at 60℃ and optimum pH at 5.0,respectively.Metal ions Fe^(2+)and Mn^(2+)had a significant(P<0.05)activation effect on chitosanase,but Al3+and Cu2+had a strong inhibition.The enzymatic chitosan products were determined by TLC(thin layer chromatography)as chito-oligosaccharides with polymerization degree 2~4,indicating that the chitosanase produced by the strain was acting on endo-oligosaccharides.This study would provide a theoretical basis for the chitosanase production of B.cereus Gxun-5.3.3 and the enzymatic preparation of chito-oligosaccharides.