小麦芽阿拉伯木聚糖的内源酶降解机理研究

Wheat malts are superior brewing raw materials. Molecular dimension and structure of wheat malt arabinoxylans as well as its degradation products by endogenous xylanase not only have important effect on the viscosity, turbidity, filtration rate， foam properties of beer but also have health care functions such as anti-tumor, enhance immunity and so on. The two important endogenous xylanse are endo-β-（1,4）-D -xylanases and α-L-arabinofuranosidases,which can change the structure, physical and chemical properties of arabinoxylan in wheat malt.This project takes the separation and purification technolgy to acquire the single molecular weight of endo-β-（1,4）-D -xylanases, α-L-arabinofuranosidases and their isoenzyme as a breakthrough point. We analyse the amino acid sequence, three-dimensional conformation, structure domain to reveal the catalytic mechanism, the homology of the isozyme and the composition difference of binding site and structure domains of the purified endogenous xylanase. Meanwhile the substrate specificity, degradation mechanism and of purified enzyme to the water-unextractable arabinoxylans and water-extractable arabinoxylans will also be studied. The key endogenous xyalanses which can transform WUAXs to WEAXs or can change the structure of WEAXs and the key produce polysaccharide structure and viscosity, turbidity properties will be revealed，and the evaluation indices include the molecular size and structure (Dp, A/X, substitution type of arabinose) of produced polysaccharide. The implementations of the research will be of great significance to regulate the reasonable degradation of wheat malt AXs, optimize brewing key process and improve health care function of beer.

小麦芽是优良的啤酒原料，其阿拉伯木聚糖（AXs）及酶解产物分子大小及结构不仅影响啤酒的粘度、浊度、过滤速度、泡沫性能等，还具有抗肿瘤、提高免疫力等保健作用。内切-β-（1,4）-D-木聚糖酶（AXs内切酶）、α-L-阿拉伯呋喃糖苷酶（阿拉伯糖苷酶）是降解小麦芽AXs改变其结构性质的重要内源酶。本项目以分离纯化小麦芽AXs内切酶、阿拉伯糖苷酶及同工酶为切入点，研究纯化酶氨基酸序列、空间构型、结构域组成等，确定同工酶的同源性、作用位点差异性、对小麦芽水不溶（WUAXs）和水溶性（WEAXs）AXs的底物专一性和降解机理。揭示降解小麦芽WUAXs转化为WEAXs；降解WEAXs改变其结构及粘度浊度等性质的关键酶及关键降解产物特征，包括：产物多糖分子量、分子结构（Dp值、A/X值、阿拉伯糖取代型式）。该研究对调控小麦芽AXs的合理降解，优化啤酒酿造关键工艺，提高啤酒的保健作用等意义重大。

小麦芽内切-β-1,4-木聚糖酶（EC 3.2.1.8）是降解小麦（芽）阿拉伯木聚糖，改变其分子大小，改变麦汁及啤酒粘度、浊度、过滤速度等特征的关键酶。本课题经40%-60%硫酸铵沉淀、Q-Sepharose Fast Flow阴离子交换层析、Phenyl-Sepharose 6 F F疏水交换层析、两次Sephacryl S-100 HR凝胶过滤层析获得分子量为27.8kDa，SDA-PAGE电泳条带单一的小麦芽木聚糖内切酶，该酶比活力4.47u/mg，纯化倍数12.08，回收率为1.45%。小麦芽27.8kDa木聚糖内切酶可将分子量为78.9kDa的小麦水溶性木聚糖（WEAX）降为分子量1.35kDa～24.9kDa，使体系粘度下降7.26%。该酶与目前已有氨基酸序列的大麦X-I及其同工酶、X-II及其同工酶、小麦源木聚糖内切酶（AF156977）的分子量存在较大差异，是一种新发现的小麦芽木聚糖内切酶。该酶的发现对后续小麦芽木聚糖内切酶的氨基酸序列及分子机理研究奠定了基础。研究发现14.0kDa蛋白可能含有小麦芽木聚糖内切酶的同工酶，该蛋白的进一步分离纯化以及木聚糖内切酶活力验证拟进一步开展。以上研究结果为小麦芽木聚糖内切酶催化机理、降解机理研究以及小麦木聚糖的合理降解奠定了基础。课题研究成果发表SCI论文3篇，录用SCI论文2篇；授权发明专利2项，受理发明专利1项；培养硕士研究生3名，其中2名已获得硕士学位，1名硕士将于2017年毕业。项目投入经费26万元，支出22.4995万元，剩余经费3.5005万元，剩余经费计划用于本项目研究的后续支出。