嗜热地衣芽孢杆菌SR01葡聚糖酶基因的原核表达及酶学性质研究

Expression and biochemical characterization of aglucanase from thermophilic Bacillus licheniformis SR01

从一株嗜热地衣芽孢杆菌SR01中克隆了β-葡聚糖酶的编码基因,并通过原核表达对重组酶的酶学性质进行了研究。结果显示,该酶编码基因ORF包含741bp,编码246个氨基酸,理论分子量为28.03ku,等电点为6.42。在温度30℃、浓度0.05mmol/L条件下IPTG诱导4h,重组蛋白得到显著表达。酶的最适反应温度、pH值分别为55℃、pH6.0~7.0;在温度40~90℃、pH5.0~10.0条件下具有良好的稳定性。Cu2＋、Fe2＋、Ca2＋、Ba2＋、Mn2＋、EDTA对该酶有不同程度的抑制作用,K＋、Na＋对该酶起轻微的激活作用。该酶对体外模拟胃肠环境具有较好的耐受性,在模拟胃液中放置90min仍有80%左右的活性,胰液对该酶有一定的激活作用。

A gene（741bp）encoding anβ-glucanase of 246 amino acids was cloned from the thermophilic Bacillus licheniformis SR01.The enzymatic properties of the recombinant enzyme were also studied by prokaryotic expression.The protein was expressed with an estimated molecular mass of about 28.03 ku by SDS-PAGE and it showed a pI of 6.42.Under the condition of 0.05mmol/L IPTG induction for 4hat 30℃,the recombinant protein was expressed significantly.The recombinant glucanase displayed its maximum activity at 55℃ and pH6.0~7.0.It was very stable below 40~90℃ at pH5.0~10.0.The enzyme activity was inhibited by Cu2＋、Fe2＋、Ca2＋、Ba2＋、Mn2＋and EDTA,while K＋、Na＋could activate the activity slightly.Under the condition of simulated stomach and intestine in vitro,the enzyme retained 80%activity afte 90 min,which showed better tolerance,and the pancreatic juice had certain activation.