HPLC法测定UGT75B1、UGT71B6、UGT71C5酶活性及酶反应动力学分析

HPLC determination of activity of UGT75B1、UGT71B6、UGT71C5 and the kinetic analysis

摘要:

运用PCR扩增获得了属于尿苷二磷酸葡萄糖基转移酶（UGT）家族UGT75B1、UGT71B6、UGT71C5的cDNA序列，构建了三种基因的GST融合表达载体，使用大肠杆菌对三种基因进行原核表达，从裂解液上清中获 得具有活性的目的蛋白.分别以ABA、对氨基苯甲酸作为反应底物，通过高效液相色谱法(HPLC)，测定三种蛋白的酶活力.分析使用的色谱柱为Kromasil C18反相色谱柱（250mm×4.6mm，5μm），使用的流动相是10%-100%水-甲醇或者10%-20%水-乙腈，分别使用醋酸和三氟乙酸调节了pH.使用梯度洗脱的方式对反应样品进行分析.结果发现除了UGT75B1对对氨基苯甲酸表现出很高的活性以外，另外两种蛋白仅表现出了低水平的活性.三种蛋白对ABA都表现出了活性，UGT71B6具有最高的酶活力.对UGT75B1、UGT71B6、UGT71C5三种蛋白对ABA的作用进行了体外酶反应动力学分析，测定出它们的Km值分别为：0.73、0.43和0.45mM.以上结果说明UGT75B1对对氨基苯甲酸的活性明显高于ABA，对氨基苯甲酸是其专一底物.UGT71B6和UGT71C5对ABA都有较高的活性，两者Km值差距不大.

Abstract:

The cDNA sequences of UGT75B1, UGT71B6, UGT71C5 were cloned by PCR method and then were constructed into glutathione S-transferase gene fusion vector PGEX-6P-1. All of the plasmids were transformed into E. coli strain BL21 individually for recombinant protein expression. Recombinant UGTs were purified from supernatant of cell lysates. To investigate the activity of recombinant UGTs, HPLC was employed to determine their activity to catalyze ABA and p-aminobenzoic acid. Analysis by reverse-phase HPLC was carried out using a Kromasil C18 column (250mm×4.6mm, 5μm) and glucose ester was separated by a linear gradient of 10%-100% methanol in H2O (all solutions contained 2.5ml/L of acetic acid and 0.4ml/L of triethylamine) or 10%-20% acetonitrile in H2O (all solutions contained 1ml/L of trifluoroacetic acid). The results showed that only UGT75B1 presented strong p-aminobenzoic acid acylglucosyltransferase activity and the others had lower activity. All of the recombinant UGTs had ABA-GE activity. However, UGT71B6 performed highest activity. Analysis of Michaelis-Menten kinetics of UGT75B1, UGT71B6 and UGT71C5 indicated that their Km was 0.73, 0.43 and 0.45 mM, respectively. In conclusion, our investigations demonstrate that UGT75B1 has higher p-aminobenzoic acid acylglucosyltransferase activity than ABA-GE activity. It seems that p-aminobenzoic acid is the specific substrate of UGT75B1. In comparison, UGT71B6 and UGT71C5 with similar Km perform higher ABA-GE activity.