烟管菌漆酶的分离纯化及部分酶学性质研究

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。

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

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 after polyacrylamide 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 68.7kDa, 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 Al3+, but strongly inhibited by Hg2+, Fe3+ and Cl-. Cu2+ had no effect on LacA activity but inhibited LacC activity.