巴尔喀什蘑菇子实体漆酶纯化与理化特性及对酚类化合物的降解能力

Purification and Physicochemical Properties of Laccase from Fruiting Bodies of Agaricus balchaschensis

【目的】研究巴尔喀什蘑菇子实体的生物学特性、漆酶理化性质及对酚类化合物的降解能力,为野生巴尔喀什蘑菇的栽培驯化以及栽培料高效利用提供理论依据。【方法】利用纤维素CM-cellulose阳离子交换和Q-Sepharose阴离子交换以及Superdex 75凝胶层析过滤纯化技术,纯化分析该漆酶的降解代谢底物与抗金属离子抑制能力,确定该漆酶活性最适温度与pH值。【结果】获得本漆酶为65 kDa的单亚基蛋白,总纯化倍数为354.58,比活力17.02 U/mg,该酶N端10个氨基酸的序列为SGGPEQNTTA,经过NBCI-BLAST后发现该漆酶与糙皮侧耳、杂色云芝等的漆酶具有同源性。本漆酶底物差异性较大,从强到弱主要为ABTS>二甲氧基酚>甲苯胺。最适pH值为2.2,属中低温活性;最适反应温度为40℃,为中低温活性漆酶。Cu^(2+)对该漆酶催化ABTS有轻微促进作用,Hg^(2+)对其催化ABTS有很强的抑制作用。【结论】巴尔喀什蘑菇子实体漆酶为65 kDa的单亚基蛋白,是一种中低温活性耐酸性漆酶,底物特异性为ABTS>二甲氧基酚>甲苯胺。

【Objective】To determine the physiochemical properties and biological functions of the laccase from the fruiting bodies of wild Agaricus balchaschensis,this study to explore the biological characteristics of the fruiting bodies,the physiochemical properties of the laccase,and the degradation ability of phenolic compoundstheoretical basis for the cultivation domestication of wild A.balchaschensis,as well as the efficient use of cultivating materials.【Methods】Cellulose CM-cellulose cation exchange column,Q-Sepharose anion exchange column,and Superdex 75 gel chromatography filtration and purification technique were used to analyze the degradative metabolic substrate of the laccase and its inhibition ability against metal ions.The optimum temperature and pH value for the activity laccase were determined.【Results】The laccase obtained from the fruiting bodies of A.balchaschensis was a 65 kDa single subunit protein.The total purification multiple was 354.58 and the specific activity was17.02 U/mg.The sequence of the 10 N-terminal amino acids of the enzyme was SGGPEQNTTA.Through NBCI-BLAST,it was found that the laccase was homologous to those of Pleurotus ostreatus and Versicolor Versicolor.The laccase was substrate-specific and varied greatly based on different metabolic substrates.The major metabolic substrates ranked from strong to weak were ABTS>dimethoxyphenol>toluidine.The optimum pH value was 2.2,so the laccase was acid-resistant.The optimum reaction temperature was 40℃,so the laccase was active at medium-low temperatures.Cu^(2+)can slightly promote the laccase to catalyze ABTS,while Hg^(2+)can strongly inhibit its catalysis of ABTS.【Conclusion】The results showed that the laccase from the fruiting bodies of A.balchaschensis was a 65 kDa single subunit protein.