产纤维素酶工程菌株的构建及其在醇化烟叶中的应用

Construction of Cellulase Producing Engineering Strains and the Application in Aged Tobacco Leaves

烟叶中过高的纤维素含量使烟叶组织容易破碎,影响加工过程中烟叶的可塑性,使烟叶杂气变重等。为了获得产纤维素酶的优良菌株,实现醇化烟叶纤维素的有效降解,利用同源重组法成功构建了10株纤维素降解的枯草芽孢杆菌(Bacillus subtilis)工程菌株。通过刚果红平板筛选、羧甲基纤维素钠酶活、滤纸酶活及滤纸崩解率等检测,共筛选出C36、CM、KF和GH54株产纤维素酶能力较强的重组菌株。将醇化烟叶作为底物进行纤维素降解,发现重组菌株CM的产纤维酶效率最高,其羧甲基纤维素钠酶活和滤纸酶活分别为39.55和23.52 U·mL-1。结果表明,构建的重组菌株能够利用醇化烟叶中的纤维素产生纤维素酶,可为工业生产中醇化烟叶纤维素降解提供理论支撑。

High cellulose content make the tissue of tobacco leaves broken easily,affect the plasticity of tobacco leaves during processing,and make the heavy impurity of tobacco leaves.In order to obtain excellent cellulase producing strains and achieve effective degradation of cellulose in aged tobacco leaves,ten Bacillus subtilis engineering strains of cellulose-degrading were suc‐cessfully constructed by homologous recombination method.Four recombinant strains C36,CM,KF and GH5 with strong cellu‐lase production capacity were screened by the Congo red plate method,carboxymethyl cellulose sodium enzyme activity assay,filter paper enzyme activity assay and filter paper disintegration rate detection.When aged tobacco leaves were used as substa‐rate,the recombinant strain CM showed the highest cellulase production efficiency,and its carboxymethyl cellulose sodium en‐zyme activity was 39.55 U·mL-1 and filter paper enzyme activity was 23.52 U·mL-1,respectively.The results indicated that the recombinant strains could utilize cellulose in aged tobacco leaves to produce cellulase,which could provide theoretical support for cellulose degradation of aged tobacco leaves in industrial production.