糙皮侧耳以果皮为基质固态发酵产漆酶及其酶学性质的研究

摘要

Different fruit peels were used in solid-state fermentation of Pleurotus ostreatus for laccase production.The fermentation conditions were optimized for laccase enzyme activity and then the laccase from P.ostreatus was purified and studied for its enzymatic properties.The results showed that the highest laccase specific activity (21800 U/g)was obtained when P.ostreatus was inoculated on the solid media composed of shaddock peel,25 mL water and 20 μL Tween 80,and fermented at 30 ℃ for 10 d.Laccase produced under the optimal condition was then purified by ammonium sulfate precipitation,dialysis, polyethylene glycol condensation,and DEAE-cellulose ion exchange column chromatography,and the resultant purified protein was named as Lac h2.The relative molecular weight of Lac h2 was determined around 66000 by SDS-PAGE.The optimal reaction temperature and pH for Lac h2 were 60 ℃and 4.5, respectively.Lac h2 remained stable under 30~50 ℃ and pH 4.5~6.0.Cations Zn2+,Mn2+,Cu2+and Mg2+stimulated Lac h2 activity,whereas Ca2+,Ag+and Fe2+inhibited Lac h2 activity.%以漆酶活性为指标,利用不同果皮为基质对糙皮侧耳(Pleurotus ostreatus)固态发酵产漆酶的条件进行优化并对所产漆酶进行纯化,研究纯化后漆酶的酶学性质.结果表明:以柚皮为培养基质(5 g 柚皮、25 mL水、20 μL吐温-80),30 ℃固态发酵10 d,糙皮侧耳产漆酶的活性最高,比活力为21800 U/g;在该条件下将糙皮侧耳所产漆酶的粗酶液用硫酸铵沉淀、透析、浓缩、DEAE-纤维离子交换柱层析得到漆酶蛋白单一条带, SDS-PAGE检测蛋白相对分子质量约66000;该漆酶命名为Lac h2,其最适反应温度为60 ℃,最适反应 pH值为4.5;在30~50 ℃,pH 4.5~6.0范围内稳定;金属离子 Zn2+、Mn2+、Cu2+和Mg2+对纯化的漆酶 Lac h2的酶活力有促进作用而Ca2+、Ag+和 Fe2+对Lac h2的酶活力有抑制作用.