【作者】 郑瑞娟；

【导师】 刘钟滨；

【作者基本信息】 同济大学 ， 病原生物学， 2006， 硕士

【摘要】 木聚糖酶(Xylanase)，又称β—D—1，4-内切木聚糖酶，是降解木聚糖的关键酶，它能水解木聚糖主链上的β—1，4—糖苷键，将木聚糖降解为低聚糖和木糖，广泛应用于饲料、造纸和食品医药等行业，具有广阔的应用前景。但木聚糖酶的单位产量较低，是限制木聚糖酶应用的主要因素之一。本研究应用同源高效表达策略，在丝状真菌表达系统中构建高产的黑曲霉木聚糖酶基因工程菌。本课题首先从黑曲霉菌(Aspergillus niger)TS1菌株中提取总RNA，以其为模板运用RT-PCR的方法成功克隆出678bp的木聚糖酶xynB基因，将该基因片段插入融合蛋白基因表达载体pYG1.2葡萄糖淀粉酶编码基因的下游，融合之处设计内胰蛋白酶(KEX2)加工位点，构建融合表达质粒pYG1.2—xynB。测序结果表明，该木聚糖酶基因序列与A.niger IBT-90木聚糖酶(GenBank accession AY536639)基因序列具有99.4％的同源性，有4个碱基的差异，仅一个碱基的突变导致氨基酸改变，编码的蛋白质序列同源性达99.5％。随后运用原生质体转化法将pYG1.2—xynB转化尿嘧啶缺陷型宿主黑曲霉菌M54，获得重组菌株。SDS-PAGE结果表明，重组木聚糖酶在转化菌株中获得了高效分泌性表达，其分子量约为21KD。在1％木聚糖为诱导物的培养条件下，重组菌分泌的木聚糖酶酶活有明显提高，最高酶活达507 IU／ml，分别是原始出发菌和宿主菌的6.7倍和3.89倍。对获得的转化子进行连续传代培养结果表明，重组菌生长特性十分稳定，具有良好遗传稳定性。重组菌最佳产酶时间为培养后72h，最适反应pH为5.2，属酸性木聚糖酶；最适反应温度为50℃，在50℃-60℃之间热稳定性较好，60℃保温30min后残余酶活为63.95％。本实验首次运用同源表达的策略，将黑曲霉木聚糖酶基因在同源丝状真菌中表达。研究结果表明，木聚糖酶在重组黑曲霉中获得了高效分泌性表达。重组菌发酵周期短，产酶活性高，具有重要的应用前景，为酸性木聚糖酶的广泛应用奠定了一定基础。更多还原

【Abstract】 Xylan, composed of polysaccharide containingβ-1, 4-linked D-xylopyranosideresidues, is one of the major components of plant cell walls. Endo-β-1,4-xylanase(EC3.2.1.8) degrades the backbone ofβ-1, 4-linkages of xylose.Xylanase is widely applied in animal feed, medicine, food processing and papermanufacturing. However, the low yield of xylanase becomes a limiting factor to theuse of xylanase in various industries. The aim of my study is to construct a high levelxylanase recombinant in filamentous fungal expression system.The cDNA encoding the acidic xylanase from original A. niger TS1 wasamplified by RT-PCR and cloned into the eukaryotic expression vector pYG1.2 underthe control of the glaA promoter. The recombinant plasmids were transformed intopyrG deficient A. niger M54 host strain by protoplast transformation technology. Thishomologous expression system expressed xylanase activity of 507 IU/ml which wasmeasured in shake-flask cultures containing 1％xylan. This activity was 6.7-fold and3.89-fold higher than the activity for the original strain and host strain respectively.The maximum xylanase activity was obtained after culturing for 72 hours and thexylan induced enzymatic activity was 3.94-fold higher compared to non-inducedcondition. The optimum pH of recombinant xylanase was at 5.2,whereas the optimumtemperature was at 50℃and remained more than 63.95％activity even afterincubation at 60℃for 30min.It’s the first time that high secreting expression system was constructed using ahomologous expression process in filamentous fungus. The promising resultsachieved in this study may have a good industrial perspective especially in the animalfeed.更多还原