桂西北野生真菌新型纤维素酶结构及性质研究

Lignocellulose is the most abundant and renewable biological carbon resource on earth, the rate of saccharification of cellulase hydrolysis of cellulose is low，which is the common technical problems of enzymatic hydrolysis and microbial fermentation for cellulosic ethanol production. Screening of new cellulose degradation species, new cellulase is a potential breakthrough of the utilization of natural cellulose. Here we report the isolation of a new cellulase-producing Fusarium chlamydosporum HML 278 and Aspergillus oryzae HML366 from the soil beneath the rotten wood in virgin forest in the northwest of guangxi province. For the first time, we report here the isolation of F.chlamydosporum HML278 producing complete cellulase system, results from thin layer chromatography showed that the β-glucosidases of F. chlamydosporum have relatively high transglycosylation activity, could direct conversion of cellulose to ethanol. A. oryzae HML366 can produce novel β-glucoside and xylanase. Bagasse, rich sources, low price, is a potential resource for biofuel industry. This project is planned to purify cellulase from the newly isolated fungus, Time-of-flight mass spectrometry coupled with zymogram analysis analysis of novel cellulase，Determined amino acid sequences and extended target genes for molecular genetics research. Multiple sequences alignmen of gene and amino acid, protein homology modeling, determined the secondary structure of fusion proteins by circular dichroism spectrum (CD), discuss the relationship between protein structure and function, Study the mechanism of synergistic enzymatic cellulose degradation by the novel cellulose. A. oryzae HML366 producing β-glucosidase with high transglycosylation activity, which enables the enzymatic synthesis of gentiobiose，gentiobiose can induce F.chlamydosporum HML278 produce cellulase, significantly improved the overall enzyme vitality of mixed fermentation cellulase system.Simplified process for bagasse cellulose pretreatment, provide an experimental basis for the comprehensive utilization of lignocellulose.

纤维素是最丰富的可再生资源，纤维素酶水解糖化率低是纤维素综合应用共性技术难题，筛选高效降解纤维素的新菌种、新酶提高水解糖化率是纤维素应用的一个突破点。申请人从桂西北原始森林取样筛选获得蕴含丰富新酶资源的产纤维素酶菌种，首次报道厚垣镰孢霉产高活性耐热纤维素酶系，所产β-葡萄糖苷酶具有转苷活性，具有将纤维素糖化后直接转化为乙醇的潜能；首次报道米曲霉产高转苷活性β-葡萄糖苷酶，可合成龙胆二糖诱导其他真菌产纤维素酶。本项目计划以来源丰富的甘蔗渣为发酵材料，运用非变性电泳结合层析方法方法系统地对厚垣镰孢霉及米曲霉纤维素酶进行纯化，新酶飞行质谱鉴定或PCR扩展目的基因，序列比对、同源建模、圆二色谱测定纯化酶二级结构结合酶学性质研究解析新酶结构及功能关系，研究新酶协同降解纤维素机理；两菌混合发酵研究龙胆二糖诱导垣镰孢霉产纤维素酶条件，优化纤维素酶系组成提高糖化率，为纤维素的综合利用提供实验依据。

认真按计划开展厚垣镰孢霉HML278葡聚糖外切酶、葡聚糖内切酶和木聚糖酶的纯化，米曲霉HML366葡聚糖外切酶、葡聚糖内切酶的纯化及酶学特性研究； 纯化酶结构及功能研究。研究新纤维素酶协同作用降解纤维素材料的机理；龙胆二糖诱导厚垣镰孢霉HML278产纤维素酶机制研究，厚垣镰孢霉HML278和米曲霉HML366混合发酵研究，提高纤维素水解糖化率，课题研究为木质纤维素应用提供实验依据。. 1.完成米曲霉 HML366一种同时具有葡聚糖外切酶和葡聚糖内切酶功能的新酶纯化及酶学性质研究，厚垣镰孢霉新的耐热β-葡萄糖苷酶一步纯化及酶学性质研究，发表两篇SCI论文，注明是国家自然科学基金支持。.（1）One-step purification of two novel hermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum ML278 and their characterization ，AMB Express，2020.（2）Purification and Enzymatic Properties of a Difunctional Glycoside Hydrolase from Aspergillus oryzae HML366 ，Indian Journal of Microbiology，2020.2.课题研究相关3个专利已经授权： .（1）一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法（专利号：2017107099308）.（2）一种厚垣镰孢霉分离纯化获得β-葡萄糖苷酶的方法（专利号：2017103635397）.（3）一种米曲霉发酵酶液的提取物及其应用（专利号：2016100468824）.3.发表3篇中文核心论文，注明是国家自然科学基金支持。.[1]何海燕,付跃,秦文芳,张玉兰,甘小富,覃拥灵.原始森林高产纤维素酶刺器腐霉的筛选鉴定[J].食品研究与开发,2019.[2]何海燕,陆秀青,秦文芳,何雪颖,韦秋艳,覃拥灵.预处理及表面活性剂对米曲霉固体发酵产纤维素酶影响的研究[J].中国饲料,2019.[3]何海燕,付越,秦文芳,柳雨珠,何麟,覃拥灵.高产碱性纤维素酶丝状真菌筛选及产酶研究[J]..饲料研究,2019