云南高寒环境低温甘露聚糖酶的克隆及低温活性机理研究

As the second most abundant hemicellulose in nature, mannans and heteromannans are widely distributed in animal feeds and reduce the digestion and absorption of nutrition. Effective breakdown of mannans and heteromannans at low temperatures (<30℃) mainly needs low-temperature-active endo-1,4- β -mannanases. The potential of low-temperature-active endo-1,4-β-mannanases include applications in low-temperature environments and biotechnological processes, such as aquaculture where low temperatures are required. Research on the mechanism of enzymatic adaptation to low temperatures is also important to improve endo-1,4-β-mannanases (gaining low-temperature activity) based on rational engineering. However, low-temperature-active endo-1,4-β-mannanases have rarely been reported, not to mention their catalytic mechanisms at low temperatures. Novel low-temperature-adapted microorganisms and low-temperature-active endo-1,4-β-mannanases, which have been forced to adapt to alpine environments in Yunnan province, are important to investigate. In this study, low-temperature-active endo-1,4-β-mannanases will be cloned from alpine environments in Yunnan province using both the classical culture-dependent and the culture-independent methods, called m-TAIL-PCR isolating functional genes directly from metagenome. Further amino acid residues and fragments mutageneses will be used to reveal the low-temperature-active mechanism of endo-1,4-β-mannanases. In a word, this study will contribute greatly to the study of low-temperature-active endo-1,4-β-mannanases in the following two aspects: screening novel low-temperature-active endo-1,4-β-mannanases with potential application in aquaculture from alpine environments in Yunnan province and revealing low-temperature-active mechanism of endo-1,4-β-mannanases.

甘露聚糖是自然界中半纤维素的第二大组分，广泛存在于饲料原料中，降低饲料营养物质的消化利用率。低温甘露聚糖酶在低温环境下（<30℃）高效水解甘露聚糖，可应用于低温环境和低温下的食品发酵和加工过程（如水产饲用酶制剂）。对低温甘露聚糖酶低温催化机理的研究可用于指导酶的分子改良，使不具低温活性的酶具有低温活性，扩展酶的应用范围。然而，国内外对低温甘露聚糖酶的报导极少，对其低温活性机理则尚未报导。云南高寒环境极可能孕育了新颖的低温微生物和低温甘露聚糖酶，具有重要的开发价值。本项目结合微生物的可培养法和直接克隆宏基因组中目的基因的m-TAIL-PCR方法，克隆云南高寒环境中的低温甘露聚糖酶，再通过点突变和肽段突变的方法揭示其低温活性机理。该项目对挖掘云南高寒环境中蕴藏的具有水产饲用潜力的新型低温甘露聚糖酶和揭示低温甘露聚糖酶的低温活性机理具有重要意义。

甘露聚糖酶是甘露聚糖降解过程的关键酶。国家发展和改革委员会发布2016年新修订的《战略性新兴产业重点产品和服务指导目录》中，甘露聚糖酶是重点研发产品之一。本项目挖掘新型低温甘露聚糖酶，并对低温甘露聚糖酶进行分子改造和低温甘露聚糖酶的应用潜能进行研究，以期达到经济效益、节能减排和环境保护等多方面的共赢。. 本项目对云南3种特殊生境来源微生物的甘露聚糖酶进行研究，揭示GH26家族甘露聚糖酶ManAGN25的新型低温活性及其分子适应特征。相比中高温甘露聚糖酶，ManAGN25的催化位点附近位点增加的氨基酸残基构成了增多或增长的α-螺旋和无规则卷曲，且整个蛋白的总可及表面积和总结构容积均增大。基于相关适应特征，对GH26家族甘露聚糖酶ManAJB13的温度特性进行理性设计，成功获得热活性和热稳定性改良的低温甘露聚糖酶。由此可见，GH26甘露聚糖酶ManAJB13的N端loop区是影响其热活性和热稳定性的关键区域之一，为GH26家族低温甘露聚糖酶的分子改造提供理论指导；揭示了GH5家族甘露聚糖酶Man5HJ14的分子特性及其在洗涤工业中的应用潜能，为耐表面活性剂甘露聚糖酶的低温活性改造提出了新的研究方向；揭示了不同甘露聚糖酶酶法制备甘露寡糖的差异及其益生潜能，为低温甘露聚糖酶的应用提供思路。. 项目主持人张蕊作为第一作者公开发表2篇SCI收录论文和1篇CSCD收录论文，作为通信作者指导学生公开发表学术论文3篇。