基于酸性矿山废水宏基因组的新型嗜酸β-甘露聚糖酶的挖掘及嗜酸机理研究

Acid Mine Drainages (AMD) are typically featured by extreme acidity and high concentrations of toxic metals and sulfate, and they represent an extreme environment for microorganisms. These acidophilic microorganisms have unique gene types, special physiological mechanisms and metabolites, which provide important sources for extracting acidophilic enzymes. Only few enzymes have been identified from AMD by traditional isolation and culture methods, while a large portion of uncultured microorganisms thrived in AMD are uncharacterized. Endo-β-mannanases are industrial glycoside hydrolases that randomly cleave the β-1,4-linkage in mannans. Acidophilic β-mannanases were the most widely applied but there is few research on the acidophilic mechanisms of them. In this research, function/sequence-based screening of an AMD-derived metagenomic library and high-throughput metagenome sequencing were combined to screen acidophilic β-mannanases. Genes encoding β-mannanase were cloned and expressed in Escherichia Coli or Pichia Pastoris, as well as their biochemical characterizations were analyzed. Finally, bioinformatic, mutational and homology model studies were carried out to elucidate the acid adaptation mechanisms of acidophilic β-mannanases. This study may provide a novel strategy to modify the acid and/or alkali properties of the related glycosidases, and also provide valuable experiences of exploitation acidophilic enzymes from extreme environments.

酸性矿山废水（AMD）是生物地球化学循环最活跃地区之一，pH值极低，是研究极端微生物的典型环境。酸性矿山废水特殊生境下的微生物往往具有独特的代谢途径和适应机制，极可能产生结构复杂多样、生物活性广泛的代谢产物，是嗜酸酶的重要来源，且在基因资源的开发和应用方面具有独特优势。目前从该环境中分离嗜酸酶的研究主要基于纯培养技术，大量未培养微生物中的嗜酸酶尚未得到充分挖掘和鉴定。β‑甘露聚糖酶是能水解甘露聚糖的工业酶，其中嗜酸β‑甘露聚糖的应用最为广泛，但是目前少有对β-甘露聚糖酶嗜酸机理的研究。本项目拟耦合宏基因组文库构建和宏基因组测序挖掘酸性矿坑水中的β-甘露聚糖酶基因，对酶基因进行异源表达和酶学特性分析，结合生物信息学、突变和同源建模等方法解析嗜酸β-甘露聚糖酶的酸适应机制。其研究成果不仅可为相关糖苷水解酶的酸碱性质改造提供指导，而且对极端生境中酶资源的挖掘与利用有重要的借鉴意义。

β-甘露聚糖酶是一种重要的水解甘露糖苷键的半纤维素酶，广泛应用于饲料、能源和食品等领域。然而GH113家族β-甘露聚糖酶成员数量少，耐酸性差，限制了其在饲料等领域的应用。本研究以酸性矿山废水为对象，利用基于序列的宏基因组方法，发现了两个新型高活性GH113家族β-甘露聚糖酶基因（man113Ali8和man113Ali9）。这两个甘露聚糖酶与来自Alicyclobacillus sp.A4的GH113家族β-甘露聚糖酶有最高的一致性（68%），在pH6.0和50℃下显示出最高的活性，能够有效水解多种甘露聚糖底物，并对甘露四糖到甘露六糖表现出转糖苷活性，具有广阔的应用前景。利用定向进化对Man113Ali9进行酸适应性机制研究。通过多轮筛选，获得兼具高活性和良好酸稳定性的突变体。野生酶在pH4.0条件下处理1h后完全失活，突变体Y97F-N119S-V246M-F252Y的残留活性达53%，其中第246位甲硫氨酸和第252位的酪氨酸的替换是造成突变体酶学性质改变的主要原因。分析发现突变体中引入的新的氢键和范德华力的增强有利于β-甘露聚糖酶酸稳定性的提高。通过分子动力学模拟发现，突变体在酸性条件下的结构位移更小，蛋白质结构更紧致，在No.238-242区域氨基酸柔性的降低可能有助于底物结合。本研究对于从极端环境挖掘新型生物活性物质有重要借鉴意义，丰富了对糖苷水解酶耐酸机制的认识，为相关酶种的酸碱性质改造提供了理论依据。