烟管菌漆酶的分离纯化及部分酶学性质研究  

Purification and characterization of laccase from Bjerkandera adusta WZFF.W-Y11

作　　者：王剑锋[1] 王璋[2] 饶军[1] 李江[1] 白涛[1] 

机构地区：[1]东华理工大学生物系,抚州344000 [2]中国食品发酵工业研究院

出　　处：《菌物学报》2008年第2期297-308,共12页Mycosystema

摘　　要：烟管菌Bjerkandera adustaWZFF.W-Y11漆酶粗酶液经过丙酮分级沉淀、DEAE-Cellulose离子交换层析、Sephadex G-100凝胶过滤,得到了三种电泳纯的漆酶同工酶,总酶活回收率达到65.5%,其中LacA平均纯化了29.2倍,LacB纯化了5.1倍,LacC纯化了18.5倍;三种同工酶的分子量分别为LacA:68.7kDa、LacB:80.2kDa、LacC:77.2kDa。LacA、LacC氧化愈创木酚的Km大于氧化ABTS的Km,最适作用温度在45-70℃,最适反应pH3.0-5.5,65℃时LacC比LacA稳定,LacC在pH3.5-6.0稳定,LacA在pH5.0-9.0稳定,Al3+对LacA、LacC的酶活有促进作用,Cl-、Fe3+、Hg2+抑制LacA、LacC的酶活,Cu2+抑制LacC的酶活,而对LacA的活性没有明显影响。Three kinds of laccases from Bjerkandera adusta WZFF.W-Y11 were purified to electrophoretic homogeneity by the combination of acetone fractionation, DEAE-Cellulose ion exchange, and Sephadex G-100 chromatography. They were identified by the color development of zymogram stained with guaiacol afterpolyacrylamide gel electrophoresis （PAGE）, and named LacA, LacB and LacC, respectively. The enzymes were purified 29.2-folds, 5.1-folds and 18.5-folds respectively from crude samples with a total recovery yield of 65.5%. The laccases were enzymologically characterized and the results showed that the molecular weight of LacA, LacB and LacC were 68.TkDa, 80.2kDa, and 77.2kDa, respectively, as determined by SDS-polyacrylamide gel electrophoresis （SDS-PAGE）. The Km of both LacA and LacC for oxidizing guaiacol were higher than that for oxidizing ABTS. Their optimum pH and temperature were 3.0-5.5 and 45℃-70℃, respectively. At 65℃, LacC showed a better stability than LacA. The activity of LacA was relatively stable at pH range of 5.0-9.0, and LacC at pH range of 3.5-6.0. Various ions had different effects on laccase activity, which was enhanced by Al^3＋, but strongly inhibited by Hg^2＋, Fe^3＋ and C1. Cu^2＋ had no effect on LacA activity but inhibited LacC activity.

关 键 词：同工酶 丙酮分级沉淀 凝胶过滤 愈创木酚 

分 类 号：Q814[生物学—生物工程]