马蓝叶β-葡萄糖苷酶的提取分离及酶学性质研究OA北大核心

In this study,the extraction process of β-glucosidase from Baphicacanthus cusia fresh leaves was optimized by single factor experiment.The optimal extraction conditions were as follows:a liquid to sample ratio of 10∶1(mL∶g),extraction with a 50 mmol/L phosphate buffer(pH 6.0)at 4 ℃,homogenization for 60 s,extraction for 1 h,and filtration of the extract.The crude extract then was purified through ammonium sulfate fractionation,ion exchange chromatography and gel filtration chromatography.The specific activity of the purified β-glucosidase was 121.89 U/mg,with a 22.31-fold increase in purity compared to the crude enzyme solution,and an enzyme recovery rate of 20.15％.Moreover,the enzymatic properties of the enzyme were studied.Results showed that the optimal reaction temperature for the enzyme was 50℃and the optimal pH was 6.0.The enzyme presented high thermal stability at 40-50 ℃ and high pH stability at pH of 4.0-7.0.The relative enzyme activity remained above 80％after 20 d at 4℃under sterile conditions.The enzyme with 4-Nitrophenyl-β-D-glucopyranoside(pNPG)as substrate had the Michaelis constant(Km)of 3.40 mmol/L and the maximum reaction rate(Vmax)of 9.27μmol/(L·min).Ca2+,Zn2+,Mg2+,Al3+,ethanol,isopropanol,ascorbic acid,urea,EDTA and oxalate showed no obvious impacts on the enzyme activity;Cu2+,Fe2+,Ag+,methanol,ethyl acetate and sodium 1-dodecanesulfonate(SDS)inhibited the enzyme activity to varying degrees;K+and acetone exhibited activating effect on enzymes.