半理性设计改造南极假丝酵母脂肪酶B催化合成氯乙酰胺

Semi-rational Design Modifies Candida antactica Lipase B to Catalyze Chloroacetamide Synthesis

首次报道了固定化CAL-B突变体通过直接酰化的方式催化生成氯乙酰胺。氯乙酰胺在医药工业上主要用于合成长效磺胺B(SMPZ)和酚妥拉明(phentolamine),利用南极假丝酵母脂肪酶B合成氯乙酰胺具有较好的应用前景。针对南极假丝酵母脂肪酶B,采用半理性设计的策略并进行迭代突变,获得酶活提高的突变体Lv-01(A281L/W104A/T159A/I189A/V190A)。与野生型相比,比活性(U/mg)提高了1.76倍,进一步通过固定化提升氯乙酰胺生成效率63.95%。通过Discovery Studio进行分子对接分析了突变体Lv-01相比于野生型催化效率提高的原因,S105、H224与底物氯乙酸的氢氧根脱氢的氧离子相连,由原本的范德瓦耳斯力变成氢键和静电相互作用,促使氯乙酸与铵根离子结合脱去一分子水形成氯乙酰胺。

The catalytic generation of chloroacetamide by direct acylation of an immobilized Candida antarctica lipase B(CAL-B)mutant was reported for the first time.Chloroacetamide is mainly used for the synthesis of long-acting sulfonamide B(SMPZ)and phentolamine in the pharmaceutical industry,and the synthesis of chloroacetamide using CAL-B has good application prospects.In this work,a semi-rational design strategy and iterative mutation were used to obtain the mutant Lv-01(A281L/W104A/T159A/I189A/V190A)with improved enzyme activity for S.pseudomallei lipase B.The mutant was characterized by a high specific activity(A281L/W104A/T159A/I189A/V190A)compared with the wild type.The specific activity(U/mg)was increased by 1.76-fold compared with the wild type,and the chloroacetamide generation efficiency was further increased by 63.95%by immobilization.The reason for the increased catalytic efficiency of the mutant Lv-01 compared with the wild type was analyzed by molecular docking in Discovery Studio.S105 and H224 were bonded to the oxygen ions dehydrogenated from the hydroxyl radical of the substrate chloroacetic acid,and the original van der Waals forces were converted to hydrogen bonding and electrostatic interactions,which resulted in the combination of chloroacetic acid with ammonium ions to remove a molecule of water to form chloroacetamide.