特异性几丁质酶的催化机制、几丁寡糖的高效制备及功能

Chitin is one of the most abundant renewable biomass on earth, only after cellulose, hence its high efficient utilization is of great importance. The hydrolytic products, N-acetyl chitooligosaccharides (COSs), can be used in various industries, such as functional food, biochemistry product, medicine, exhibiting great application potential and broad development prospects. So far, many reports concerning conversion of chitosan (deacetylated chintin) into COSs are availavle. However, there are relatively few reports on conversion of chintin into N-acetyl COSs, some drawbacks in conversion of chitin to N-acetyl COSs still exist for industrial applications till now, such as low yield, environmental pollution. In addition, the biological functions of the individual ingredients of N-acetyl COSs have not been studied systematically and intensively. The present proposal will focus on the high efficient utilization of chitin resource. Firstly, the specific chitinases which can hydrolyze chitin to yield mainly N-acetyl COSs will be isolated and intensively investigated. Then various chitin degradation methods will be studied with the enzymatic hydrolysis of the chitin. Finanly, the individual ingredients in N-acetyl COSs will be separated and studied for their biological functions, and the underlying mechanisms of biological functions will be further investigated on the basis of their relationship between structures and functions. Thus, the project will not only provide therotical basis for the conversion of chitin resources, but also promote the development and application of N-acetyl COSs in industries.

几丁质是自然界中丰富的可再生生物质资源，含量仅次于纤维素，其高效利用具有重要的现实意义。几丁质的水解产物几丁寡糖在保健食品、生物化工、医药等领域具有重要的应用价值和广阔的发展前景。目前，脱乙酰几丁质转化为壳寡糖的研究报道很多；而几丁质转化几丁寡糖的研究报道较少，制备工艺还存在一些不足，如生产效率低、环境污染严重等，此外，几丁寡糖单一组分的生物活性及作用机制研究还不够系统、深入。在前期研究多种新型几丁质酶和成功制备得到几丁单糖和二糖的基础上，本项目拟继续围绕几丁质的高效利用开展相关工作。首先发掘特异性几丁质酶并研究其酶学性质和催化机制；其次基于几丁质酶水解法，耦合预处理等方法研究几丁质的高效、绿色降解方法；最后研究几丁寡糖单一组分的高效分离制备及其生物活性，探明不同单一组分几丁寡糖的构效关系及其作用机制。本项目实施不仅对几丁质资源转化具有重要的理论意义，还将促进几丁寡糖的工业开发和利用。

几丁质酶在几丁质生物转化中起重要作用，但由于特异性几丁质酶生产成本高且催化机制尚不清楚，很难大规模应用于工业生产。几丁寡糖是几丁质水解产物，在食品、化工、药品和农业等领域具有很大的应用潜力。然而，几丁寡糖的高效制备困难，几丁寡糖的功能尚未系统和深入研究。.在前期研究的基础上，将米黑根毛霉几丁质酶基因（RmChi44）在大肠杆菌中异源表达，纯化得到重组几丁质酶（RmChi44）并研究生化性质。RmChi44水解胶体几丁质产生几丁二糖。该酶还具有β-N-乙酰氨基葡糖苷酶活性，可将聚合度2-5的几丁寡糖水解为单糖。其次，研究了巴伦葛兹类芽孢杆菌的几丁质酶PbChi67的纯化和性质，发现PbChi67水解胶体几丁质的主要产物为几丁二糖和单糖。从桑式链霉菌克隆一个几丁质酶基因（SsChi28）并在大肠杆菌中表达。SsChi28水解胶体几丁质主要产物为几丁二糖占总糖的73%。然后，以2型糖尿病（T2D）模型小鼠为对象，探究了几丁二糖对糖尿病相关代谢紊乱的影响及其对MAPK信号通路的调控机制。结果表明，与阿卡波糖组相比，几丁二糖组可以显著改善T2D模型小鼠的高血糖、高血脂和体重异常等状态。另外，以动脉粥样硬化模型载脂蛋白E基因敲除（Apo E-/-）小鼠为对象，研究了几丁二糖对动脉粥样硬化病变的影响，几丁二糖可以通过调血脂代谢及抑制体内炎性反应等途径起到防治动脉粥样硬化的作用。此外，还研究了高效制备壳寡糖，将枯草芽孢杆菌壳聚糖酶基因（BsCsn46）克隆转化至巴斯德毕赤酵母中进行高效表达，壳聚糖酶BsCsn46水解壳聚糖生成聚合度 2-4壳寡糖的得率高达90.9％。.特异性几丁质酶的发掘与催化机制研究，以及酶法制备几丁寡糖，不仅对几丁质的生物转化具有重要的理论意义，还将促进几丁寡糖的生产和应用。研究还发现几丁二糖可改善T2D相关代谢紊乱和防治动脉粥样硬化功能，有作为新型功能食品原料的潜力。

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