云南莴笋尖酪氨酸酶分离纯化及酶学性质  

Isolation,Purification and Partial Characterization of Tyrosinase from Yunnan-Grown Lettuce Tip

作　　者：廖海君 李蕊伽[1] 陶敏[1] 白亚娟[1] 唐菁[1] 唐云明[1] LIAO Haijun;LI Ruijia;TAO Min;BAI Yajuan;TANG Jing;TANG Yunming(Key Laboratory of Eco-Environments in Three Gorges Reservoir Region, Ministry of Education Chongqing Sweet Potato Engineering Research Center, School of Life Science, Southwest University, Chongqing 400715, 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,三峡库区生态环境教育部重点实验室,重庆400715

出　　处：《食品科学》2017年第8期30-36,共7页Food Science

基　　金：中央高校基本科研业务费专项(XDJK2016C110)

摘　　要：为获得更多成本廉价且易得的酪氨酸酶来源,为云南莴笋尖酪氨酸酶抑制剂的研究提供参考,选取云南莴笋尖为实验材料,通过匀浆、缓冲液抽提、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析和Superdex-200凝胶过滤层析等方法纯化,获得电泳纯的酪氨酸酶,并对纯化后的酪氨酸酶进行部分酶学性质研究。结果表明,云南莴笋尖中酪氨酸酶的比活力为20.62 U/mg,分子质量约为995.40 k D,亚基分子质量约为65.23 k D。最适反应温度和最适反应p H值分别为60℃和8.0,在p H 6.0~8.0及20~40℃范围内较稳定。最适条件下,以左旋多巴为底物的K_m值为1.567 mmol/L,vmax为0.037 6 μmol/(min·L)。乙醇、异丙醇、正丁醇对该酶活性有不同程度的抑制作用;甲醇、Co^(2+)、Mn^(2+)、Pb^(2+)、Ag^+、Cd^(2+)、Ba^(2+)、Ca^(2+)对该酶活性有不同程度的激活作用,Co^(2+)、Mn^(2+)对该酶具有显著的激活作用,低浓度的Zn^(2+)、Cu^(2+)、草酸、抗坏血酸对该酶有激活作用,高浓度时有抑制作用。尿素、EDTA、Li^+、K^+、Mg^(2+)对该酶活性影响较小。To find a new cheap and easily available source of tyrosinase and to provide an experimental basis for further research on inhibitors of tyrosinase from lettuce tip,electrophoretically pure tyrosinase was obtained from Yunnan-grown lettuce tip through consecutive homogenization,buffer extraction,ammonium sulfate fractionation,DEAE-Sepharose fast flow chromatography and Superdex-200prep grade chromatography.The results showed that the specificity activity of the purified enzyme was20.62U/mg.The relative molecular weight of the tyrosinase was approximately995.40kD,in which the subunit molecular mass was roughly65.23kD.The enzymatic properties showed that the optimum temperature and pH for the tyrosinase were60℃and8.0,respectively.The enzyme was stable in the pH range of6.0-8.0and in the temperature range of20-40℃.Its apparent Km and vmax were1.567mmol/L and0.0376μmol/(min·L),respectively.The tyrosinase activity was slightly affected by EDTA,urea,K+,Mg2+and Li+.The enzyme activity could be inhibited by ethanol,isopropanol,n-butanol,but activated by methanol,Co2+,Mn2+,Pb2+,Ag+,Cd2+,Ba2+,and Ca2+,and it could be obviously activated by Co2+,Mn2+.Ascorbic acid,Zn2+,Cu2+,and oxalic acid enhanced the enzyme activity at low concentrations but inhibited it at high concentrations.

关 键 词：云南莴笋尖 酪氨酸酶 分离纯化 酶学性质 

分 类 号：Q946.5[生物学—植物学]