[ 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 p-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 ea-zyme 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.%[目的]系统研究Bacillus subtilis LC-9所产β-l,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-l,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1 427 μmol/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.
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