基于催化域中loop区结构信息的β-甘露聚糖酶Man1312分子改造

β-Mannanase is one of key hemicellulases which have been used in producing health-care products and processing food, therefore, it is an important objective to develop a kind of mannanase with higher activity and better stability to cater the needs of food heat-treating. Our lab screened and constructed mannanase Man1312 and then its molecular structure was analyzed. To further optimize the activity and stability of Man1312, we intend to engineer the non-active sites in the loop region of Man1312 basing on the relationship of molecular structure and function through the genetic and protein engineering technologies. With the aid of biological software, the gene banks of single-site directed saturation mutagenesis and multi-sites directed saturation mutagenesis will be constructed to obtain the ideal mutants. Additionally, we plan to compare the structure and characteristics between Man1312 and its mutants and try to understand the underlying mechanism of the non-active sites in loop acting on the catalysis and stability of Man1312 and its mutants through CD, DSC and other analytical methods. This project means to provide some theoretical basis for the researches on the relationship of mannanase molecular structure and function and some new ideas for molecular engineering of mannanse. The researches of thermostable enzymes with the potential of scale manufacture have the benefits to promote the green index of industrialization and construct an energy-saving and environment-friendly economy.

β-甘露聚糖酶是重要的半纤维素酶之一，在低聚糖等保健品和食品加工过程中有广泛应用，为使其更适用于食品加工的高温处理过程，获得高活性且热稳定性好的突变酶是甘露聚糖酶研究的目的之一。本项目以课题组自行筛选并构建的甘露聚糖酶Man1312为出发酶，在其结构与功能相关信息的基础上，通过生物学软件的辅助设计，针对Man1312催化域的loop区非活性位点进行定点突变，分别构建单点定点饱和突变库和多点定点饱和突变库，筛选热稳定性更好、酶活力更高的适用于规模化应用的突变酶，并运用圆二色谱法等方法测定酶的动力学稳定性和热动力学稳定性，通过分子对接模拟分析，探索Man1312的loop区非活性中心残基与酶分子稳定性和催化作用的分子机理，为甘露聚糖酶分子结构与功能关系研究及分子定向改造提供新的思路和理论基础。有规模化生产潜力的耐热酶研究有利于提高我国产业化的绿色指数，建设节能减排型经济。

β-甘露聚糖酶是保健品和食品加工过程中重要的半纤维素酶之一，为获得高活性且热稳定性好的甘露聚糖酶，课题组以自行筛选的甘露聚糖酶Man1312为出发酶，在生物学软件的辅助下对Man1312进行了定向突变。本课题的主要结果集中在以下几个方面。1)优化了Man1312编码基因的表达体系，建立了以pHY-p43为表达载体，以Bacillus brevis和Bacillus subtilis WB600为宿主菌的Man1312编码基因表达系统；2)为了进一步提高Man1312编码基因表达系统的效率，建立了以Bacillus brevis的抽提物为基础的无细胞表达体系，使目的基因的表达时间缩短为4个小时，提高了突变库筛选的速度。3)在Man1312的结构与功能信息的基础上，通过生物学软件的辅助设计，针对Man1312催化域的loop区非活性位点进行定点突变，从N146G, S147H, S156P和T157Y四个突变中筛选到热稳定性更好、酶活力更高的突变体，并解析了突变对稳定性贡献的机理；4)在Man1312的N末端进行了定点突变和缺失突变，从中筛选到V3, N7和Q11的缺失突变体因增强了Man1312链末端的刚性，使酶的热稳定性提高了一倍；5)考察了Man1312的α螺旋和β折叠区残基组成，并对该区域残基进行定点突变，筛选到K178R，K207R，N340R和S354R突变体，综合所有位点的突变获得了新型突变酶，其最适作用温度提高了15°C，半衰期提高到80°C，是原始酶的7倍，Tm提高了10.5°C，ΔΔGm提高了5.2 kcal/mol。本项目通过半合理设计的思路开展了甘露聚糖酶Man1312的分子设计工作，为提高酶分子稳定性和活性提供了完整设计思路，并获得了有应用前景的新酶。