Bacillus subtilis LC-9产β-1,3-1,4-葡聚糖酶的酶学性质与催化特性研究

Enzymatic and Catalytic Properties of β-1,3-1,4-glucanase from Bacillus subtilis LC-9

[目的]系统研究Bacillus subtilis LC-9所产β-1,3-1,4-葡聚糖酶的酶学性质,探讨其在催化应用上的可行性。[方法]采用两步阴离子交换层析法对B.subtilis LC-9发酵液中的β-1,3-1,4-葡聚糖酶进行纯化,用SDS-PAGE检测其纯度,研究纯酶的酶学性质及该酶对β-葡聚糖和地衣多糖的降解特性,并采用TLC法检测其水解液中糖的成分。[结果]从自行筛选的糖苷酶产生菌B.subtilis LC-9的发酵液中经两步纯化,得到电泳纯的β-1,3-1,4-葡聚糖酶,其比活力达3 502 U/mg,表观分子量为28 kDa;酶的最适反应pH和温度分别为6.5和45℃,且在45℃下稳定;该酶催化地衣多糖的Km值和Vmax分别为4.13 mg/ml和1 238μmol/min/mg,催化大麦β-葡聚糖的Km值和Vmax分别为3.84 mg/ml和1 427μmol/min/mg;该酶降解大麦β-葡聚糖产生寡聚糖,最终产物主要为三糖和四糖,而降解地衣多糖终产物主要为三糖。[结论]从Bacillus subtilis LC-9发酵液中纯化获得了电泳纯的β-1,3-1,4-葡聚糖酶,性质研究表明该酶是专一性的β-1,3-1,4-葡聚糖酶,有望用于啤酒、饲料等领域。

[Objective] To systematically study the enzymatic properties of purified β-1,3-1,4-glucanase from B.subtilis LC-9,and to explore the possibility of its application in catalysis.[Method] Purification of β-1,3-1,4-glucanase from B.subtilis LC-9 fermentation broth was carried out by a two-step ion exchange chromatography.The purity of the enzyme was identified by SDS-PAGE.Then,the enzymatic characterizations of the purified enzyme were studied.Finally,the products of the barley β-glucan and lichenan hydrolyzed by the enzyme were analyzed by TLC.[Result] The enzyme was purified to homogeneity with a specific activity of 3 502 U/mg.The purified enzyme showed as a single protein band with an apparent molecular mass of 28 kDa.The optimum pH and temperature for the enzyme activity were 6.5 and 45 ℃,respectively.The purified enzyme was stable below 45 ℃.The kinetic parameters Km and Vmax were 4.13 mg/ml,1 238 μmol/min/mg for lichenan and 3.84 mg/ml,1 427 μmol/min/mg for barley β-glucan.The main products were cellubiosyltriose and cellutriosyltraose for barley β-glucan and cellubiosyltriose for lichenan.[Conclusion] A β-1,3-,4-glucanase with high activity from B.subtilis LC-9 was purified.The enzyme exhibited strict specificity for β-1,3-1,4-D-glucans.The characterizations of this enzyme showed that the enzyme was suitable for application to the beer and feed industry.