高比活耐热1,3-1,4-β-葡聚糖酶的耐酸性改造及其耐酸机制研究

1,3-1,4-β-glucanase can efficiently hydrolyze the high-molecular-weight β-glucans into oligosaccharides, thus playing important roles in food, animal feed and biofuels industries. However, most existed β-glucanases are unstable in acidic condition, which severely limited their application in industries. The acidic tolerance of β-glucanase has been far from being fully explored. Moreover, the change of residues in the catalytic center and substrate binding region of β-glucanase which favor the enzyme acidic tolerance ability will severely damage the enzymatic activity, thus negatively influence the application of β-glucanase in industries. The present project will use a previously constructed highly active and thermostable β-glucanase as the research object. After resolving the crystal structure of the β-glucanase, the key residue sites in the acidic tolerance of β-glucanase are to be predicted through amino acid sequence/crystal structures comparison and molecular dynamics simulation. Subsequently, the relation between the acidic tolerance ability and catalytic activity of β-glucanase will be summarized through analyzing the effect of the key residue sites in enzyme acidic tolerance ability and catalytic activity using molecular dynamics simulation and genetic engineering methods. Finally, a highly acidic tolerant β-glucanase mutant will be rationally constructed by site-directed mutagenesis and saturation mutagenesis and the molecular basis of acidic tolerance of β-glucanase will be revealed.

1,3-1,4-β-葡聚糖酶在食品、饲料和生物质能源等行业有重要应用价值，然而现有的大多数β-葡聚糖酶耐酸性较差，严重影响了酶在工业上的应用。目前对β-葡聚糖酶耐酸性机制的了解仍不透彻，同时发现针对β-葡聚糖酶催化活性中心及底物结合区域部分氨基酸的改变在提高酶耐酸性的同时会影响酶的催化性质，严重限制了酶的应用价值。本项目拟以前期构建的高比活耐热β-葡聚糖酶为研究对象，解析该酶晶体结构，并通过不同耐酸性β-葡聚糖酶氨基酸序列/三维结构比对分析和分子动力学模拟，推测β-葡聚糖酶耐酸性相关结构特点和关键氨基酸位点；在此基础上通过生物信息学和基因工程手段分析关键氨基酸位点在β-葡聚糖酶耐酸性中的功能，总结酶耐酸性与催化性质之间的关系；在此基础上理性设计分子改造策略在高比活耐热β-葡聚糖酶基础上构建耐酸酶突变体，从蛋白质结构角度阐明β-葡聚糖酶耐酸性分子机制，为推动β-葡聚糖酶的工业化应用奠定基础。

1,3-1,4-β-葡聚糖酶在食品、饲料等行业有重要应用价值，然而现有大多数β-葡聚糖酶耐酸性较差，严重影响了酶在工业上的应用。目前对β-葡聚糖酶耐酸性机制的了解仍不透彻，同时发现针对β-葡聚糖酶催化活性中心及底物结合区域部分氨基酸的改变在提高酶耐酸性的同时会影响酶的催化性质，严重限制了酶的应用价值。本项目以特基拉芽孢杆菌来源β-葡聚糖酶为研究对象，通过氨基酸序列和结构比对分析总结了不同耐酸性β-葡聚糖酶氨基酸序列和蛋白质结构差异及其与酶耐酸性之间的关系；进一步通过pH依赖分子动力学模拟解析了β-葡聚糖酶在中性和不同酸性pH条件下的结构变化轨迹，重点研究了酶催化活性中心和底物结合区域氨基酸与酶耐酸性的相关性。基于上述分析，通过定点突变共构建了30个酶单突变体和12个组合突变体，最终得到了一株酶五突变体Q1E/I133L/G12N/D22N/T187A，其最适pH值和野生酶相比降低了0.5个pH，在pH4.5、pH5.0和pH5.5的半衰期和野生酶相比分别提高了232.8%、137.6%和134.5%。此外，酶五突变体的最适温度和野生酶相比提高了5℃，且在pH5.0、pH5.5和pH6.0的kcat/Km值和野生酶相比分别提高了13.65%、9.33%和12.01%。由此可见，五个突变的同时引入成功实现了在提升β-葡聚糖酶耐酸性的基础上同时提高酶催化活性。进一步通过分子动力学模拟和酶结构分析，发现酸性环境会导致β-葡聚糖酶结构部分展开，而碱性环境会导致β-葡聚糖酶蛋白进行聚集。在酸性环境中，β-葡聚糖酶N端和C端柔性较高，通过氢键或其他作用力降低酶N/C端柔性后可明显提升酶在酸性环境的稳定性；此外，耐酸性更强的β-葡聚糖酶突变体其蛋白表面含有更多的负电荷，有助于提升酶耐酸性；最后，将得到的酶突变体在麦汁协定糖化过程中进行了初步应用，发现其对于麦汁过滤速度的提升及黏度的下降均具有良好的效果，其应用于使用低β-葡聚糖含量麦芽和高β-葡聚糖含量麦芽的麦汁协定糖化过程中能使麦汁黏度分别降低16.53%和25.17%，而过滤时间分别减少36.32%和44.59%，效果不逊于进口市售β-葡聚糖酶，说明该酶具有一定的市场应用前景。