松杉灵芝漆酶的分离纯化及酶学性质研究

Isolation,Purification and Characterization of Laccase from Ganodermatsugae

采用硫酸铵分级沉淀、透析和DEAE-sepharose阴离子交换柱层析对松杉灵芝漆酶进行分离纯化并研究其部分酶学性质。结果表明,纯化漆酶比活力为55.438 7 U/mg,回收率为31.7%,相对分子质量约为52 000。该酶催化底物ABTS的最适反应温度和最适反应pH值分别为40℃和4.0,在30~40℃及pH 4.0~5.0范围内具有良好的稳定性。以ABTS为底物的米氏常数K_m为0.909 mmol/L,vmax为454.5 mmol/(Lmin)。K^+和Fe^(3+)对漆酶具有明显的激活作用;NH_4^+、Cu^(2+)对漆酶活性影响不大;Fe^(2+)能完全抑制漆酶的活性。

To the best of author＇ s knowledge,this is the first report of isolation,purification and characterization of laccase from Ganodermatsugae. In the present study,the laccase from Ganodermatsugae was isolated and purified by ammonium sulfate fractional precipitation,dialysis and DEAE sepharose anion exchange chromatography,and its characterization was determinated. The results showed that the enzymatic specific activity of about 55. 438 7 U/mg was reached with an overall yield of 31. 7%. SDS-PAGE analysis displayed a single band and the molecular weight of this laccase was estimated to be about 52 k. The author performed the NativePAGE analysis of this crude or purified laccase where it manifested only one enzyme. The optimal reaction temperature and pH towards ABTS as substrate were 40 ° C and 4. 0; furthermore the enzyme activity exhibited excellent stability at pH 4. 0 ~ 5. 0 and30 ~ 40 ° C respectively. A kinetic study revealed that ABTS was a suitable substrate with a K_mof 0. 909 mmol/L and a vmaxof454. 5 mmol/（Lmin）. K~＋and Fe^（3＋）had the most effective actiavation on laccase while Fe^（2＋）could inhibit enzyme activity completely. In addition,NH_4~＋、Cu^（2＋）had no obvious effect on laccase activity. The above enzymatic characterization indicated that this laccase is highly stable at the temperature and pH. Moreover,it could work in the presence of various metal ions. These enzymatic properties made it an ideal candidate for potential applications in food and beverage industry. The author＇ s work will pave the ways for the study of the function of the laccase.