【作者】 王欣；

【导师】 廖祥儒；

【作者基本信息】 江南大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 漆酶(Laccase, EC 1.10.3.2)是一种多酚氧化酶,可以结合多个铜离子,能催化多酚类、芳胺类、羧酸类、生物色素等的氧化,也具有降解木质素的功能。因此,研究其酶的分离、纯化、酶学性质具有一定的理论和现实意义。论文研究了菌株Trametes sp. SYBC-L3固态发酵产漆酶的培养基组成和发酵条件;探讨了漆酶分离纯化方法;分析了漆酶的酶学特性;研究了漆酶在几种染料脱色中的作用;得到如下主要结果:(1)最适固态培养基组成为:豆粕和木屑的比例为1 : 1,麦芽糖浓度为1.0 g/L,NaNO3浓度为0.71 mol/L,Cu2+浓度为0.3 g/L,含水量为65%。最适装样量为40 g/250 mL三角瓶,30℃培养10 d。在最优培养基和培养条件下,酶活最高达91.06 U/g干曲,是优化前(10.37 U/g干曲)的8.78倍。(2)通过硫酸铵分级沉淀、DEAE-cellulose-52离子交换层析、Sephadex G-100凝胶过滤层析三步分离纯化得到电泳纯的漆酶(Lac-T),纯化倍数19.58倍,回收率为18.54%。通过SDS-PAGE实验得出其分子量约为58.7 kDa。以DMP(2, 6-二甲氧基酚)为底物时,在最适温度和最适pH的条件下测得Lac-T的Km为1.32 mmol/L,Vmax为1742.76 U/mg。(3)纯化后的漆酶(Lac-T)最适温度为65℃,在40℃保温1 h仍能保持80%以上的活力,温度稳定性较好;最适pH为3.5,在pH 4.0~9.0的范围内于30℃保温24 h仍能保持80%以上的活力;SO42-、Mg 2+、Cu2+对Lac-T有促进作用;CO32-、K+对Lac-T有轻微的抑制作用,Fe2+的抑制作用较明显。NO3-、Cl-、PO43-、Na+基本不影响Lac-T活力。(4)初步研究了Trametes sp. SYBC-L3固态发酵产漆酶在偶氮染料和蒽醌染料脱色中的作用,用漆酶处理偶氮染料媒介红5G,40 min脱色率达到88%,处理蒽醌类染料弱酸蓝25和酸性蓝129,40 min脱色率分别达到76%和75%,有较好的脱色效果,显示了其在毛纺染料脱色中的潜力。更多还原

【Abstract】 Laccase (EC 1.10.3.2), a type of polyphenol oxidases, can combine a number of copper ions. It can oxidize a wide range of substrates, such as polyphenols, carboxylic acids, biological pigments and several aromatic compounds. It also has the ability to degrade lignin. The study on laccase has a certain degree of theoretical and practical significance.Laccase produced by Trametes sp. SYBC-L3 was studied under solid-state fermentation with different medial and fermenting conditions. Moreover, the purification, characterization and decolorizing role in several dyes of laccase were also investigated. The results were as follows:(1) The optimal solid-state fermentation medium was soybean meal and sawdust with a ratio of 1 : 1, maltose 1.5 g/L, NaNO3 0.71 mol/L, Cu2+ 0.3 g/L, initial medium moisture 65%. The optimized loaded sample on solid-state fermentation condition was 40 g solid-state medium in 250 mL flask, 30℃,incubated for 10 d in dark. The laccase activity reached 91.06 U/g dry substrate with about 8.78 folds of the original fermenting level (10.37 U/g dry substrate).(2) The laccase produced by strain Trametes sp. SYBC-L3 was purified via (NH4)2SO4 fractionation, DEAE-cellulose-52 ion exchange chromatography, and gel filtration with Sephadex G-100 column. Its purity and yield were 19.58 folds and 18.54% of the crude extract, respectively. The molecular mass of the purified laccase (Lac-T) was about 58.7 kDa on SDS-PAGE gel. The Km of Lac-T was 1.32 mmol/L, its Vmax was 1742.76 U/mg protein.(3) The optimal catalytic temperature of laccase (Lac-T) was 65℃, the optimal pH was 3.5. Lac-T was stable under different pH and with relative activity of about 80% at the pH range between 4.0 and 9.0, which indicated that it adapted to a wide range of pH. Lac-T could be activated by SO42-, Mg2+ and Cu2+, and inhibited by CO32- and K+, more significant inhibited by Fe2+. NO3-, Cl-, PO43-, Na+ didn’t affect the activity of Lac-T in a certain extend.(4) The laccase produced by Trametes sp. SYBC-L3 could degrade textile dyes. Eighty eight percent of decolorization was achieved with laccase treatment on Mordant Red 5G, 76% on Weak Acid Blue 25 and 75% on Acid Blue 129 for 40 min, respectively, which indicating that the laccase had the potential ability for decolorization.更多还原