白腐菌Panus conchatus产漆酶的诱导、纯化及酶学特性研究  

Purification and Characterization of Copper Induced Laccase from White-rot Fungus Panus Conchatus

作　　者：张丽[1] 付时雨[1] 傅恺[1] 刘浩[1] 李兵云[1] 

机构地区：[1]华南理工大学制浆造纸工程国家重点实验室,广东广州510640

出　　处：《造纸科学与技术》2013年第2期24-29,36,共7页Paper Science & Technology

基　　金：国家自然科学基金(编号30771689;31170549);国家973计划项目(编号2010CB732206)资助项目

摘　　要：铜离子对贝壳状革耳菌(Panus conchatus)胞外漆酶有明显的诱导作用,其最适诱导浓度为3mmol/L。在培养基中添加铜离子,发酵14 d后漆酶活性提高11倍,达196 IU/mL。发酵液经硫酸铵盐析,离子交换色谱和凝胶过滤色谱等分离纯化后,漆酶的比活力为912IU/mg,纯化倍数为6.77,酶活得率为74%;SDS-PAGE和Native-PAGE显示出单条蛋白条带,其分子量约是65kDa;以ABTS为底物时Km为0.0057mmol/L;反应最适温度和pH分别为60℃和2.5;漆酶在4℃和pH 8.0时具有较高的稳定性;K+,Na+,Cu2+,Mg2+等金属离子和乙醇,乙腈对酶活影响较小;而SDS、半胱氨酸和NaN3等对酶活影响较大,几乎完全抑制漆酶活性。Laccase production by Panus conchatus reached the highest enzyme activity 196 IU/mL at 14th day under induction of 3 mmol/L Cu2＋ . The enzyme was purified by ammonium sulfate precipitation, anion-exchange chroma- tography and size-exclusion chromatography. Purification of about 6.77-fold was achieved with an over yield of 74 % and a specific activity of 912 IU/mg. A single laccase band by Native-PAGE was found with a molecular mass of 65 kDa by SDS - PAGE. The Michaelis constant of the enzyme for ABTS was 0. 0057 mmol/L, the optimal temper- ature and pH for the enzyme activity are 60 ℃ and 2.5. The laccase was stable under 4 ℃ and pH 8.0, and resist- ant to metal ions such as K＋ , Na＋ , Cu2＋ , Mg2＋ and other chemicals such as ethanol and acetonitrile, but is com- pletely inhibited by SDS, cysteine and NaN3.

关 键 词：铜离子 贝壳状革耳菌 漆酶 纯化 酶学性质 

分 类 号：TK6[动力工程及工程热物理—生物能]