基于耐酸、耐热及蛋白酶抗性的新型木聚糖酶发掘与分子改造

Xylanases is an important kind of hemicellulose-degrading enzymes, which are widely used in feed, food, paper and pulping industries. Though many xylanases have been reported previously, special xylanases suitable for feed production with acid-resistant, thermostabe and protease tolerant properties are still less. Some novel xylanases have been identified from several new glycoside hydrolase (GH) families, including GH families 5, 7, 8, 30 and 43, but their enzymatic properties and action mechanisms are not all clear. Hence, in order to fulfill actual demand in feed industry, on the basis of our previous study on 8 thermostable xylanases, the proposal will focus on the identification of novel xylanase from various GH families; the enzymatic characterizations and action mechanisms of novel xylanases will also be investigated; In addition, the properties such as acid-resistance, thermostability and protease tolerance of novel xylanases will be modified with the aim of improving their application adaptability. Thus, the project will not only provide more theoretical information for enzymology, but also will have a great influence on the development of feed industry.

木聚糖酶是一类重要的半纤维素水解酶，广泛应用于饲料、食品、制浆造纸等行业。目前已有许多木聚糖酶的报道，但是仍然缺乏适用于饲料工业的具有耐酸、耐热和蛋白酶抗性等特性的特异性木聚糖酶。已报道的木聚糖酶大多属于糖苷水解酶10和11家族，近年来又发现了一些新糖苷水解酶家族（5、7、8、30和43）的木聚糖酶，其中部分家族木聚糖酶的酶学性质、结构信息、催化机制和应用适应性仍不明确。因此，基于饲料工业的实际需求，在前期研究8种耐热木聚糖酶的基础上，本项目拟继续发掘不同糖苷水解酶家族和适合饲料工业应用的新型木聚糖酶，研究新酶的酶学特性和催化作用机制；同时采用蛋白质工程的方法改造获得的新型木聚糖酶，提高其耐酸、耐热和蛋白酶抗性等特性，提升其应用适应性。本项目的实施不仅具有重要的酶学理论意义，而且相关研究成果将有助于促进我国饲料行业的绿色、健康发展。

本项目的主要研究内容为基于饲料工业应用研究新型木聚糖酶的发掘、分子改造和作用机制。从四株微生物中挖掘四个新型耐酸、耐热和具有蛋白酶抗性的木聚糖酶，分别为巴伦葛兹类芽孢杆菌来源PbRex8、铜绿拟青霉来源的PaXyn10A、海栖热袍菌来源的mTmXynB和嗜热毛壳菌CQ31来源的CsXyn11A。项目从巴伦葛兹类芽孢杆菌中发掘一个新家族木聚糖酶，PbRex8为新型GH8家族木聚糖酶，可从还原端将木寡糖全部水解为木糖。PbRex8晶体结构的解析和催化作用机制的阐明为拓展该家族酶在饲料等工业的应用奠定了理论基础。山楂籽作为一种农业副产物，开发其高附加值益生元具有重要意义。基于山楂籽木聚糖结构发掘得到一个耐酸、耐热及水解特性优良的木聚糖酶PaXyn10A，并开发了山楂籽乙酰化低聚木糖生产新工艺，转化率达66.7%。针对谷物中小麦阿拉伯木聚糖底物项目选育出CsXyn11A和mTmXynB，CsXyn11A在面制品中展现良好的应用潜力，分子改造后的mTmXynB大幅度提升了该酶在啤酒领域的应用潜力。本项目发掘了四种优良木聚糖酶，系统研究了它们的高效表达、酶学性质、催化机制和应用，对饲料、食品等工业的绿色发展具有理论意义和科学价值。