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.
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