新型木聚糖酶的发掘、性质及应用基础研究

Xylanases (EC 3.2.1.8) is an important type of hemicellulose degrading enzymes, catalyzing the hydrolysis of xylan to produce xylooligosaccharides (XOSs) and small amount of xyloses. Xylanases have been one of the research focuses in enzyme industry in recent years due to their great application potential in different fields, such as food, animal feed, paper and pulping, and bio-energy. Though a number of xylanases have been reported, only several xylanases have been commercially avaiable, with the producers limited in several strains from Aspergillus and Trichoderma genus. In addition, the characterization of the enzymes are not satisfied, especially those xylanases which can specifically hydrolyze xylan to yield XOSs with relatively higher degree of polymerization (DP ≥ 4) are in scarcity, which can’t meet the actual requirements in industrial applications. We have investigated more than 10 kinds of xylanases and the industrilization of XOSs. Based on the previous research achievements, the proposal will continue to isolate novel xylanase producing microbes, clone and efficiently express xylanase genes, purify and characterize various novel xylanases, investigate the catalytic mechanisms of the xylanases and apply the xylanases in the production of flour based products and XOSs with higher DP in food industry. Thus, the project will not only help to enhance the number of xylanase sources, broaden the xylanaes’ application fields, but also provide theoretical basis for the application of xylanases in food industries.

木聚糖酶是一类重要的半纤维素水解酶，能够催化水解木聚糖生成低聚木糖和木糖。木聚糖酶由于在食品、饲料、造纸、能源等领域中显示了重要的应用价值和发展潜力，目前已成为酶制剂研究的热点之一。虽然木聚糖酶的相关研究较多，但是迄今已商品化木聚糖酶品种仍然较少，仅局限于曲霉属和木霉属少数菌株，且酶学特性单一，尤其缺少一些特异性木聚糖酶，如能够水解木聚糖产生高聚合度木寡糖（DP ≥ 4）的木聚糖酶。在实验室研究10余种木聚糖酶和低聚木糖产业化的基础上，本项目拟采用高通量筛选法继续从自然界发掘高产不同水解酶家族新型木聚糖酶的微生物菌株，克隆和高效表达新型木聚糖酶基因，研究新型木聚糖酶的酶学性质，阐述新型木聚糖酶的催化机理，进一步探讨木聚糖酶在面制品及高聚合度低聚木糖等食品工业中的应用潜力。项目的实施不仅有助于丰富木聚糖酶资源，拓宽木聚糖酶应用领域，也为木聚糖酶在食品工业中的应用提供依据。

木聚糖酶是一类重要的半纤维素水解酶，能够催化水解木聚糖生成低聚木糖和木糖。木聚糖酶由于在食品、饲料、造纸、能源等领域中显示了重要的应用价值和发展潜力，目前已成为酶制剂研究的热点之一。虽然木聚糖酶的相关研究较多，但是迄今已商品化的木聚糖酶品种仍然较少，仅局限于曲霉属和木霉属少数菌株，且酶学特性单一，尤其缺少一些特异性的木聚糖酶。本项目重点围绕高产不同水解酶家族新型木聚糖酶的微生物菌株的高通量筛选、新型木聚糖酶的克隆和高效表达以及应用基础等开展了较为系统的研究工作。取得了系列创新型成果：1）从巴伦葛兹类芽孢杆菌中克隆木聚糖酶基因PbXyn10A并在大肠杆菌中成功表达，利用该酶水解经电解水蒸煮喷爆预处理的玉米芯可制备以木二糖-木四糖为主的低聚木糖，低聚木糖转化率可达75%；2）从铜绿拟青霉中克隆GH10家族还原端外切木聚糖酶PaXyn10并表达于毕赤酵母中，经5-L发酵罐高密发酵144 h，胞外酶活力可达22000 U/mL，利用该酶水解山楂核，可制备富含乙酰基的低聚木糖，转化率可高达66.8%；3）从毛壳霉CQ31中成功扩增GH10家族木聚糖酶基因CsXynB，将其异源表达于毕赤酵母，5-L发酵罐中高密度发酵酶活力可达10000 U/mL，该酶耐热性良好，在食品和饲料加工中具有一定的优势；4）从毛壳霉（Chaetomium sp.）CQ31中成功扩增GH11家族木聚糖酶CsXynE，并在毕赤酵母中表达，酶活力达到19000 U/mL。5）从巴伦葛兹类芽孢杆菌中克隆还原端外切木聚糖酶基因PbRex8并在大肠杆菌中成功表达，该酶最适pH为5.5，是目前报道最耐酸的还原端外切木聚糖酶。项目的研究成果不仅有助于丰富木聚糖酶资源，拓宽木聚糖酶应用领域，而且也可以为木聚糖酶在食品工业中的应用提供依据，尤其是在功能性低聚木糖的酶法制备中具有重要的应用价值。