以葡聚糖填料作为载体，采用共价结合法对嗜热嗜碱土芽孢杆菌T1脂肪酶进行固定化，在获得固定化酶制备的最佳方案的基础上进一步考察固定化酶的催化特性。在单因素实验中，2-碘酰基苯甲酸终浓度1.23%、固定化温度50 ℃、固定化时间2 h的条件下效果最好，酶活力达到50.25 U/mg基质。利用该固定化酶催化橄榄油和甲醇进行醇解反应生成脂肪酸甲醇酯，60 h后得率为14.08%。同时，对固定化脂肪酶的热稳定性以及有机溶剂耐受性进行了研究，结果发现固定化T1脂肪酶在60 ℃孵育7 h后依然保持70%以上的酶活性；且除甲醇、乙醇外，固定化T1脂肪酶在经过正丁醇、正丙醇、异丙醇处理后酶活力能够增加到120%~140%。以上催化特性研究的结果表明，该固定化酶具有催化甲醇生成生物柴油的潜力，具有良好的热稳定性和有机溶剂耐受性，是一种具有良好工业应用前景的脂肪酶。

Using glucan as carrier, thermoalkaliphilic T1 lipase from Geobacillus sp. strain T1 was immobilized via covalent binding. The optimized conditions for enzyme preparation were established and catalytic properties of the immobilized T1 lipase were determined. In a single-factor experiment, specific activity of immobilized T1 lipase reached 50.25 U/mg matrix by using optimal conditions including 1.23% 2-iodoxy benzoate, immobilization temperature of 50 ℃ and time for 2 h. Immobilized T1 lipase was used to catalyze the alcoholysis of olive oil and methanol, yielding 14.08% fatty acid methyl esters after 60 h. Meanwhile, the thermostability and organic solvent tolerance of the immobilized T1 lipase were also studied. The results indicated that the immobilized T1 lipase could maintain more than 70% of its activity after a 7-h incubation at 60 ℃, and the activity increased to 120% to 140% after treatment with n-butanol, n-propanol, and isopropanol, in addition to methanol and ethanol. These results demonstrate that immobilized T1 lipase has a potential to catalyze the reaction involving methanol to produce biodiesel and exhibits good thermostability and organic solvent tolerance; therefore, it has potential for industrial application.

广东省自然科学基金博士启动项目（S2012040007734 ）；中央高校基本科研业务费专项资金项目（2014ZM0062）