| 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 |
| 投稿时间：2006-07-30 修订日期：2007-07-16 |
| 中文关键词:合成染料，脱色，苹果汁，多酚 |
| 英文关键词:synthetic dyes, decolorization, apple juice, polyphenol |
| 基金项目:四川省教育厅重点科研项目（No.2002A009，No.2004A026）；四川省科学技术厅应用基础项目（No.04JY029-032）；四川农业大学青年科技创新基金（No.00731200） |
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| 中文摘要: |
| 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pI 4.40，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%。 |
| 英文摘要: |
| Laccase high-yielding strain SAH-12 of Trametes gallica was obtained by UV mutagenesis. Three isoenzymes of laccase (viz. Lac1, Lac2 and Lac3) were obtained in the culture solution of SAH-12, among which Lac1 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 Lac1 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 Lac1 activity were 60℃ and 2.6, respectively in catalytic reaction of oxidizing ABTS. Michaelis constant of the enzyme for ABTS was 25.0μmol/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 Lac1 was enhanced by Cu2+, whereas it was inhibited by the metal ions Fe2+, Ag+, Hg2+ and Cr3+, and inhibitors DTT, SDS, EDTA and DMSO. The activities of Lac1 were completely inhibited by Fe2+ and DTT, while Mn2+ and Zn2+ did not strongly effect on laccase activity. Lac1 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 Lac1 at 40℃ for 5h. |
| 黄乾明,杨婉身,陈华萍,张寒飞,刘一江,谢君. 粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究[J]. 菌物学报, 2007, 26(4): 539-548 |
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