【目的】提高10β-去乙酰巴卡亭Ⅲ乙酰氧基转移酶(DBAT)活力，从而更高效地体外酶促合成巴卡亭Ⅲ。【方法】通过DBAT与其天然底物乙酰CoA的计算机分子模拟对接，选定C165W、N300I、F160C 3个氨基酸残基进行单点定点突变。突变基因经大肠埃希菌重组表达，突变重组酶特性经体外酶促反应测定。【结果】3种突变重组酶的相对分子质量为67 000；最适反应温度均为32.5℃，与野生酶DBAT一致；DBATF160C与DBAT的最适反应pH为7.5，略高于DBATC165W和DBATN300I的最适反应pH (7.0)；DBATC165W、DBATF160C和DBATN300I的比活力与DBAT相比分别提高了61.5%、59.6%和19.2%，催化效率分别提高了55.4%、35.1%和2.9%，3种突变酶的米式常数(Km)和最大反应速率(vmax)均高于DBAT。【结论】疏水突变导致突变酶的比活力相比野生型均有显著提高，研究结果为体外高效酶促合成紫杉醇化学半合成的直接前体物巴卡亭Ⅲ奠定了基础。
【Objective】 To increase the activity of 10-deacetyl baccatin Ⅲ-10β-O-acetyl transferase (DBAT) and the efficiency of in vitro enzymatic synthesis of baccatin Ⅲ.【Method】 Computer simulation of molecular docking between DBAT and its natural substrate acetyl CoA was performed. Three amino acid residues including C165W, N300I and F160C were chosen for site-directed mutation. The recombinant mutants were expressed in Escherichia coli and their enzymatic properties were determined by reactions in vitro.【Result】 The relative molecular mass of each of the three mutant enzymes was 67 000, and the optimum reaction temperature was 32.5 ℃ which was consistant with that of the wild type enzyme (DBAT). The optimum reaction pH of DBATF160C or DBAT was 7.5, slightly higher than that of DBATC165W or DBATN300I (pH 7.0). Compared with DBAT, DBATC165W, DBATF160C and DBATN300I had 61.5%, 59.6% and 19.2% higher specific activities, 55.4%、35.1% and 2.9% higher catalytic efficiency, as well as higher Km and vmax.【Conclusion】 Hydrophobic mutations lead to significantly increased specific activities for mutant enzymes compared with the wild type enzyme. This study provides a basis for efficient in vitro enzymatic synthesis of baccatin Ⅲ, a direct precursor for chemical semi-synthesis of anti-cancer drug paclitaxel.