【作者】 王旭； 赵勇； 程备久； 潘迎捷； 陶芳；

【Author】 Wang Xu;Zhao Yong;Cheng Beijiu;Pan Yingjie;Tao Fang;College of Food Science and Technology,Shanghai Ocean University;College of Life and Science,Anhui Agricultural University;Laboratory of Quality & Safety Risk Assessment for Aquatic Product on Storage and Preservation(Shanghai) Ministry of Agriculture;

【机构】 上海海洋大学食品学院； 安徽农业大学生命科学学院； 农业部水产品贮藏保鲜质量安全风险评估实验室；

【摘要】 纤维素是自然界中数量最大的可再生性资源,它的降解依赖于纤维素酶系。内切葡聚糖酶EGI是纤维素酶系中的主要组份,由催化结构域、连接肽和结合结构域组成。本研究以里氏木霉、拟康氏木霉和长枝木霉的egl基因为模板,通过DNA改组技术构建突变体库,并对酵母重组分泌的表达突变体筛选。结果表明:对3种木霉成熟肽编码序列进行DNA改组,构建突变体库,通过活性筛选获得高活性突变体,命名为New8。诱导培养96h达到活性最高1.97 U/mL,是3种木霉的野生型EGI平均酶活的2倍;最适温度为50%,最适pH为5.6。SDS-PAGE电泳显示New8亚基蛋白分子量比理论分子量偏大。New8 eg1序列与里氏木霉,拟康氏木霉和长枝木霉的相似度分别为94%,95%,96%,有114个重组位点和4个突变位点。在4个突变位点中,V10A和F192L未引起New8 EGI疏水性改变,G100S和S318G引起亲水性改变。蛋白质三级结构预测结果表明,突变位点S318G在催化结构域的活性中心附近。这一研究将为今后纤维素酶系中其它酶类(CBH、BG)的基因改造,乃至构筑高效的纤维素降解体系奠定良好的前期基础。更多还原

【Abstract】 Cellulose is the most abundant renewable bioresource produced in the biosphere and enzymatic degradation of cellulose is achieved by the synergistic action cellulase enzyme groups.Endoglucanases I,the important component of cellulase complex,is composed of catalytic domain,linker and cellulose binding domain.We constructed and screened Saccharomyces cerevisiae mutant library by DNA shuffling of three wild eg1 genes from Trichoderma reesei,Hypocrea pseudokoningiiand Trichoderma longibrachiatum.The results showed that the mutant library was construced by DNA shuffling of three wild eg1 genes encoding the mature peptide.After screening,variant with high activity was obtained and named New8.Extracellular enzyme activity of New8 approached the highest level(1.97U/mL) when cultured for 96 h,which is about 2-fold than the average of the wild EGI.The optimum temperature and pH were 50℃ and 5.6respectively.The expression was confirmed by a protein band in the SDS-PAGE,which is a little larger than predicted EGI.The gene New8eg1,about 1320 bp long,was subcloned and sequencing.It is 94%,95%and 96%identical to T.reesei、 T.longibrachiatum and H.pseudokoningiiegl.Analysis of the sequences indicated that there were 114 recombination sites,and four extra mutations had been introduced,caused amino acid substitution.V10 A and F192 L didn’t change the hydrophobicity.G100 S and S318 G changed the hydrophilicity.S3I8 G located near the activity center in the catalytic domain by tertiary structure prediction.This system should facilitate the improvement of other cellulase(CBH and BG) and lay the foundation for constructing efficient cellulose degradation system.更多还原