杂色云芝漆酶的分离、纯化和酶学特性研究  

Purification and Properties of Laccases from Coriolus versicolor

作　　者：吴坤[1] 闵航[1] 朱显峰[2] 贾新成[3] 张世敏[3] 喻子牛[4] 

机构地区：[1]浙江大学生命科学学院,浙江杭州310029 [2]河南大学生物工程系,河南开封475001 [3]河南农业大学生物工程学院,河南郑州450002 [4]华中农业大学农业部微生物重点开放实验室,湖北武汉430070

出　　处：《高校化学工程学报》2003年第2期173-179,共7页Journal of Chemical Engineering of Chinese Universities

基　　金：华中农业大学农业部微生物重点开放实验室项目(AML2000-005);国家自然科学基金资助项目(30170030)

摘　　要：杂色云芝发酵液,经超滤、DEAE-Sephadex A250柱层析两步分离、纯化出两型漆酶.以愈创木酚与酶带在适当条件下反应呈棕红色谱带鉴定出漆酶谱带.两型漆酶分别命名为LacA和LacB.经测定两漆酶亚基的分子量分别为66.0kDa和30.7kDa;等电点pI分别为4.79和5.18;LacA的最适作用温度是40℃,LacB为50℃;保温2h, LacA的半失活温度为60℃,LacB为90℃;LacA和LacB催化愈创木酚的酶活最适pH值均为4.0; 二者的稳定pH值也都在2.5～5.5之间.LacA催化氧化愈创木酚的Km和Vmax分别为286.5μmol·L-1和292.4nmol·mL-1·min-1;LacB催化氧化愈创木酚的Km和Vmax分别为813.0μmol·L-1和127.4nmol·mL-1·min-1.Two kinds of laccases from Coriolus versicolor culture were purified to electrophoretic homogeneity by the combination of ultrafiltration and DEAE-Sephadex A-250 ion exchange chromatography. And they were identified by the colorness development of zymogram stained with guaiacol after polyacrylamide gel electrophoresis (PAGE), and named LacA and LacB, respectively. The specific activities of enzymes in purified samples are 9.3-folds and 3.41-folds of those in crude samples with an overall yield of 18.2% for LacA and 0.2% for LacB respectively. The two laccases were enzymologically characterized. The results showed that the molecular weight of LacA and LacB are 66.0 kDa and 30.7kDa, respectively, as determined by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and their isoelectric points(pI) are 4.79 and 5.18 as assayed by polyacrylamide gel isoelectric focusing(PAGIF). Both of these laccases have the same pH (pH 4.0) but a different optimism activity temperature, 40 for LacA and 50 for LacB. The temperature for losing half of the activity (t1/2) within 2 h is 60 for LacA and 90 for LacB. The activities of laccases are relatively stable at pH range of 2.5~5.5. The Km and Vmax of LacA for oxidizing guaiacol are 286.5祄ol-1L-1 and 292.40 mmolL-1min-1 while that of LacB is 813.0祄olL-1 and 127.39mmolL-1min-1 , respectively.

关 键 词：漆酶 杂色云芝 酶的纯化 酶学特性 

分 类 号：Q554.9[生物学—生物化学]