文献类型：期刊文献

中文题名：马蓝叶β-葡萄糖苷酶的提取分离及酶学性质研究

英文题名：Extraction,Seperation and Characterization ofβ-Glucosidase From Leaves of Baphicacanthus cusia

作者：杨潇然[1,2] 周昊[1,2] 薛兴颖[1,2] 商士斌[1,2] 王成章[1,2]

第一作者：杨潇然

机构：[1]中国林业科学研究院林产化学工业研究所,江苏省生物质能源与材料重点实验室,国家林业和草原局林产化学工程重点实验室,林木生物质低碳高效利用国家工程研究中心,江苏南京210042;[2]南京林业大学江苏省林业资源高效加工利用协同创新中心,江苏南京210037

年份：2025

卷号：45

期号：3

起止页码：79-86

中文期刊名：林产化学与工业

外文期刊名：Chemistry and Industry of Forest Products

收录：;北大核心:【北大核心2023】；

基金：中国林科院中央级公益性科研院所基本科研业务费专项资金(CAFYBB2022XB002);贵州省科技支撑项目(2022MAAJMNNNX21916)。

语种：中文

中文关键词：马蓝叶;β-葡萄糖苷酶;提取;分离;酶学性质

外文关键词：leaves of Baphicacanthus cusia;β-glucosidase;extraction;separation;enzymatic properties

分类号：TQ35

摘要：从新鲜马蓝叶提取β-葡萄糖苷酶,通过单因素试验优化提取工艺,最佳提取条件为液料比10∶1(mL∶g)、提取溶剂为4℃的pH值为6.0的50 mmol/L磷酸缓冲液、匀浆时间60 s、浸提时间1 h,过滤后得到粗酶液;之后通过硫酸铵分级沉淀、离子交换层析和凝胶过滤层析纯化得到β-葡萄糖苷酶,纯化后该酶比活力为121.89 U/mg,与粗酶液相比,纯度提高了22.31倍,酶回收率为20.15%。对该酶的酶学性质进行了研究,结果表明:该酶的最适反应温度为50℃,在40~50℃条件下具有较好的热稳定性;在4℃无菌条件下保存20 d,相对酶活力仍保持在80%以上;最适pH值为6.0,在pH值为4.0~7.0体系内稳定性较好;测得以4-硝基苯基-β-D-吡喃葡萄糖苷(pNPG)为底物的酶的米氏常数(Km)为3.40 mmol/L,最大反应速度(Vmax)为9.27μmol/(L·min);Ca^(2+)、Zn^(2+)、Mg^(2+)、Al^(3+)、乙醇、异丙醇、抗坏血酸、尿素、乙二胺四乙酸(EDTA)和草酸对酶活无明显影响;Cu^(2+)、Fe^(2+)、Ag^(+)、甲醇、乙酸乙酯和十二烷基磺酸钠(SDS)均会不同程度地抑制酶的活性,K+和丙酮对酶活有一定促进作用。
In this study,the extraction process ofβ-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 purifiedβ-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).Ca^(2+),Zn^(2+),Mg^(2+),Al^(3+),ethanol,isopropanol,ascorbic acid,urea,EDTA and oxalate showed no obvious impacts on the enzyme activity;Cu^(2+),Fe^(2+),Ag^(+),methanol,ethyl acetate and sodium 1-dodecanesulfonate(SDS)inhibited the enzyme activity to varying degrees;K+and acetone exhibited activating effect on enzymes.