米曲霉(Aspergillus oryzae)沪酿3.042内切型纤维素酶的分离纯化与鉴定

为了解米曲霉纤维素酶在物料分解过程中的作用,从米曲霉沪酿3.042成曲中提取粗酶液,利用DNS法分析了粗酶液中外切酶、内切酶及β-葡萄糖苷酶的活力.用刚果红染色法确定了沪酿3.042成曲粗酶液中含有4种内切型纤维素酶,分别称为Cx酶Ⅰ,Cx酶Ⅱ,Cx酶Ⅲ和Cx酶Ⅳ.经DEAE-Cellulose-52离子交换层析,0.15,0.20,0.25,0.30mol/L NaCl洗脱峰均测得内切型纤维素酶活性,制备电泳纯化得到4个内切型纤维素酶组分,SDS-PAGE结果显示,Cx酶Ⅰ,Cx酶Ⅱ,Cx酶Ⅲ为单体酶,分子质量分别为31 800,34 000,26 000u,Cx酶Ⅳ含有4个亚基,分子质量分别为14 000,23 600,26 000,33 500u.粗酶液中内切型纤维素酶的最适反应温度为50℃,最适反应pH值为4.0.

Purification and identification of endocellulases from Aspergillus oryzae Huniang 3.042

The crude enzyme solution from Aspergillus oryzae Huniang 3.042 mature Koji was extracted for the purpose of revealing the roles of cellulases in the process of material decomposing,and the activity of exocellulase,endocellulase and β-glucosidase was analyzed by DNS method.Four Cx enzymes were identified by Congo red staining,called as Cx enzyme Ⅰ,Cx enzyme Ⅱ,Cx enzyme Ⅲ and Cx enzyme Ⅳ respectively in this article.DEAE-Cellulose-52 ion exchange chromatography was used to primarily separate endocellulases in the crude enzyme solution,it was found that the elution peak of 0.15,0.20,0.25,0.30 mol/L NaCl contained the active components.Then,four purified Cx enzymes were obtained by preparative electrophoresis.First three Cx enzymes were monomeric enzyme and their molecular mass were 31 800,34 000,26 000 u respectively determined by SDS-PAGE,the last one contained four subunits,and their molecular mass were 14 000,23 600,26 000,33 500 u respectivly.The optimal temperature of endocellulases in the crude enzyme solution was 50 ℃,and the optimal pH was 4.0.