粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

Purification and characterization of a laccase from the mutated strain SAH-12 of Trametes gallica

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液(LM3)中至少分泌3种漆酶同工酶(Lac1、Lac2、Lac3)。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃(pH4.0)以下和pH1.5～5.0(28℃)范围内稳定。金属离子Fe2+、Ag+、Hg2+和Cr3+与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe2+和DTT完全抑制酶活,而Cu2+对酶有明显激活作用,Mn2+、Zn2+对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶(1U/mL)处理苹果汁5h,其多酚含量可降低40%。

Laccase high-yielding strain SAH-12 of Trametes gallica was obtained by UV mutagenesis. Three isoenzymes of laccase （viz. Lacl, Lac2 and Lac3） were obtained in the culture solution of SAH-12, among which Lacl of electrophoretic homogeneity was purified by the steps of ammonium sulfate precipitation, dialysis and Sephadex G-75 chromatography. Purification of about 6.54 fold was achieved with an overall yield of 59.7%. The molecular weight of purified Lacl was estimated to be about 61.5kDa by SDS-PAGE. It was a glycoprotein with carbohydrate content of 11.6%. The isoelectric point was 4.40 （room temperature）. The optimum temperature and pH of the Lacl activity were 60℃ and 2.6, respectively in catalytic reaction of oxidizing ABTS Michaelis constant of the enzyme for ABTS was 25.0μtmol/L. The enzyme activity was stable under 40℃ and pH4.0, and within pH range of 1.5-5.0 under 28℃. The activity of Lacl was enhanced by Cu2^＋, whereas it was inhibited by the metal ions Fe2^＋, Ag^＋, Hg^2＋ and Cr^3＋, and inhibitors DTT, SDS, EDTA and DMSO. The activities of Lacl were completely inhibited by Fe^2＋ and DTT, while Mn^2＋ and Zn^2＋ did not strongly effect on laccase activity. Lacl was not only able to decolorize some synthetic dyes efficiently, but also be able to digest polyphenol in apple juice. The polyphenol of apple juice decreased 40% by Lac 1 at 40℃ for 5h.