β-淀粉酶的分离纯化及酶学性质研究  

A Study of Isolation and Purification of β-Amylase and Its Enzymatic Properties

作　　者：陶敏 杨浩[1] 白亚娟 郭小路 唐菁[1] 傅玉凡[1] 唐云明[1] TAO Min;YANG Hao;BAI Ya-juan;GUO Xiao-lu;TANG Jing;FU Yu-fan;TANG Yun-ming(School of Life Sciences, Southwest University, Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education, Chongqing Sweet Potato Engineering Research Center, Chongqing 400775, China;Fengdu Ecological Environmental Monitoring Station, Fengdu , Chongqing 408299, China)

机构地区：[1]西南大学生命科学学院/重庆市甘薯工程研究中心/三峡库区教育部重点实验室,重庆400715 [2]丰都县生态环境监测站,重庆408299

出　　处：《西南大学学报（自然科学版）》2018年第6期1-8,共8页Journal of Southwest University(Natural Science Edition)

基　　金：重庆市科委项目(cstc2015shms-ztzx80002)

摘　　要：以"渝薯17"为实验材料,从淀粉生产废水中分离纯化β-淀粉酶.去皮、1∶10(m/V)匀浆抽提,经过乙醇分级沉淀、DEAE-Sepharose离子交换层析和Superdex-200凝胶过滤层析获得电泳纯β-淀粉酶并对其酶学性质进行了研究.结果表明:该酶的比活力高达333.15U/mg,显著高于市售枯草杆菌来源酶活(50U/mg);纯化倍数9.93倍,回收率66.60%;亚基分子量约为55.12kD,全酶分子量约为223.54kD;最适温度50℃,最适pH值6.2;50℃以下,pH值6~8具较好的稳定性.在最适条件下以可溶性淀粉为底物,测得Km为0.001 36g/mL,V_(max)为0.112mg/mL·min;Mn^(2+),Pb^(2+),Li^+,Zn^(2+),K^+,Cu^(2+),草酸,SDS对该酶有不同程度的抑制作用,Co^(2+)有激活作用,有机物作用不明显.β-Amylase was extracted and purified from the starch production wastewater with the sweet potato variety＂Yushu 17＂as the raw material.After peeling,1∶10（m/V）homogenizing extraction,fractional precipitation with ethanol,DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration chromatography,electrophoretically pureβ-amylase was obtained.Then its enzymatic properties were studied.The results showed that the specific activity ofβ-amylase obtained in this study（333.15 U/mg）was significantly higher than that of commercially available sources of Bacillus subtilis（50 U/mg）;its purification fold was 9.93 and its recovery rate was 66.6%;its subunit molecular weight was about 55.12 kD and its enzyme molecular weight was about 223.54 kD;and its optimum temperature was 50℃ and its optimum pH was6.2.Thisβ-amylase showed good stability with a temperature of〈50℃ and a pH of 6～8.Measured under the optimal conditions with soluble starch as the substrate,it had a Km of 0.001 36 g/mL and a Vmax of0.112 mg/mL·min.In addition,Mn2＋,Pb2＋,Li＋,Zn2＋,K＋,Cu2＋,oxalic acid and SDS inhibited,in different extents,thisβ-amylase,Co2＋activated it,and organic matter had no obvious effect on it.

关 键 词：甘薯 Β-淀粉酶 分离纯化 酶学性质 

分 类 号：Q946.5[生物学—植物学]