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糖基水解酶Lxy-l p1-1和Lxy-l p1-2是天然药物活性物质与功能国家重点实验室,卫生部天然药物生物合成重点实验室克隆自真菌香菇的重组蛋白,具有β-木糖苷酶/β-葡萄糖苷酶双重活性,能专一性水解脱除7-木糖-10-去乙酰紫杉醇等的木糖基,生成的C-7位羟基化产物可以作为前体半合成重要的抗肿瘤药物紫杉醇或其类似物。前期工作显示:Lxy-l p1-1和Lxy-lp1-2的序列同源性高达97%,但后者的催化活性是前者的2倍以上;Lxy-l p1-2水解生色底物PNP-Xyl的最适温度为50℃,最适pH为4.0。为系统地观察两酶的催化特性,在对天然药物活性物质与功能国家重点实验室,卫生部天然药物生物合成重点实验室构建的工程酵母进行培养、诱导7 d后冷冻干燥菌体。将冷干的菌体破碎得到蛋白粗提物,再经过Ni亲和色谱和HPLC凝胶色谱法制备和纯化得到Lxy-l p1-1和Lxy-l p1-2重组蛋白。以生色底物PNP-Xyl和PNP-Glc及7-木糖紫杉烷底物7-木糖-10-去乙酰紫杉醇(XDT)与重组酶进行反应,重点测定Lxy-l p1-2水解XDT的最适温度和最适pH,并在最适条件下测定重组酶对不同底物的动力学参数。试验结果表明:Lxy-l p1-2水解底物XDT的最适温度为45℃,最适pH为4.5;动力学测定结果显示:Lxy-l p1-1和Lxy-l p1-2的β-葡萄糖苷酶活性均显著高于其β-木糖苷酶活性,这与之前的研究结果相一致;Lxy-l p1-1对于底物PNP-Xyl,PNP-Glc和XDT的kcat/Km值分别低于Lxy-lp1-2对于上述3种底物的kcat/Km值(0.77 s-1×mM-1,1.65 s-1×mM-1和0.30 s-1×mM-1vs.1.67 s-1×mM-1,2.85 s-1×mM-1和1.07 s-1×mM-1)。
The glycosyl hydrolases Lxy-l p1-1 and Lxy-l p1-2 are the key proteins of natural medicine active substances and functions, the key laboratories of natural medicine biosynthesis of Ministry of Health, cloned from fungi mushrooms, Glycosidase / β-glucosidase dual activity, the specific hydrolysis of xylosyl 7-xylose-10-deacetyltaxol, etc., the resulting hydroxy-C-7 product can be used as a precursor of semi-synthetic important Of the antitumor drug paclitaxel or its analogs. The preliminary work showed that the sequence homology of Lxy-1p1-1 and Lxy-1p1-2 was as high as 97%, but the latter was more than twice that of the former; Lxy-1p1-2 hydrolyzed chromogenic substrate PNP The optimum temperature of -Xyl was 50 ° C and the optimum pH was 4.0. In order to systematically observe the catalytic properties of the two enzymes, engineering yeast constructed by the State Key Laboratory of Active Pharmaceutical Ingredients and Functioning of Natural Medicine and Key Laboratory of Biopharmaceutical Synthesis of Natural Medicines of Ministry of Health were cultured and lyophilized for 7 days. The cold dried bacterial cells were broken to obtain the protein crude extract, which was then prepared and purified by Ni affinity chromatography and HPLC gel chromatography to obtain Lxy-l p1-1 and Lxy-l p1-2 recombinant proteins. The chromogenic substrates PNP-Xyl and PNP-Glc and the 7-xylosyl taxane substrate 7-xylose-10-deacetyltaxol (XDT) were reacted with the recombinase to focus on the hydrolysis of Lxy-lp1-2 XDT optimum temperature and pH optimum, and under the optimum conditions for the determination of recombinant enzyme kinetic parameters of different substrates. The results showed that the optimal temperature for XDT hydrolysis of Lxy-l p1-2 was 45 ℃, and the optimum pH was 4.5. Glucosidase activity was significantly higher than its β-xylosidase activity, which is consistent with the previous study; kcat / Km values of Lxy-l p1-1 for the substrate PNP-Xyl, PNP-Glc and XDT were low The kcat / Km values (0.77 s-1 × mM-1, 1.65 s-1 × mM-1 and 0.30 s-1 × mM-1 vs. 1.67 s-1 × mM-1, 2.85 s-1 x mM-1 and 1.07 s-1 x mM-1).