毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

Synthesis of glucose laurate monoester catalyzed by Candida antarctica lipase B-displaying Pichia pastoris whole-cells

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。

We developed a new enzymatic-catalyzing producing process of glucose laurate monoester.In the process we used Candida antarctica lipase B-displaying Pichia pastoris whole-cells as biocatalyst,glucose as the acyl acceptor and lauric acid as the acyl donor.The product glucose laurate monoester was purified by silica gel column chromatography and preparative liquid chromatography,and identified by liquid chromatography-mass spectrometry.Then we optimized the process from various aspects,such as solvent composition, ratio of dmethyl sulfoxide to 2-Methyl-2-butanol （V/V）, catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were： glucose 0.5 mmol/L, lauric acid 1.0 mmol/L, ratio of 2-Methyl-2-butanol to Dmethyl sulfoxide is 7：3 in 5 mL volume, temperature 60℃, the best initial water activity of whole-cells biocatalyst is 0.11. The maximum glucose conversion could be 48.7% after 72 h.