黑曲霉α-L-鼠李糖苷酶的热稳定性、酶活性和底物适应性协同分子进化研究

The improvement of the defects of native enzymes is an important approach to get excellent enzymes for food industry. However,the stability and activity trade rule brings difficulties to obtain an enzyme which has good stability, high activity and desirable substrate adaptability. Thus, optimization of enzyme stability, activity and substrate adaptability is the critical problem in scientific for preparing an excellent industrial enzyme. The enzyme α-L-rhamnosidase which has many important applications in food industry, e.g., fruit and vegetable processing and wine brewing, is a new number in the glycoside hydrolase family 78. The structure-function relationship is unclear currently. Therefore, it is difficult to rationally design its stability, activity and substrate adaptability. Moreover, traditional irrational designs and semi-rational designs that allow improving an enzyme property without aware of the structural basis, can not achieve the optimization of the multi-properties of an enzyme, limiting the effective development of enzymes with practical values. In this context, a new modified semi-rational design method is proposed and applied to improve the thermal stability of the α-L-rhamnosidase from Aspergillus niger, as well maintain its high activity and good substrate adaptability. A directed evolutionary technique is, firstly, used to explore effective regions for promoting the thermal stability, followed by saturation mutagenesis to create different mutation types at each target mutagenic site. Thereafter, the enzyme mutants are analyzed in their structure and main properties to resolve the rules that the structural changes in the effective area determine the main properties, i.e., thermal stability, enzyme activity and substrate adaptability. Based on this rule, the enzyme structures are going to be simulated and screened regarding improving the thermal stability and avoiding a significant decrease in the enzyme activity and substrate adaptability. This study is expected to illustrate the structural mechanism for improving the stability and keeping desirable activities towards common substrates existing in food processing materials, which set up the foundation to practice this enzyme in the food industry. Furthermore, the project is also expected to develop a new procedure that introduces the relationship of structure changes affecting multi properties in the modification region, which, provide a new method to optimize the stability, activity and substrate adaptabilty of enzymes for food industry. This study is very important regarding its scientific novelty and practical significance.

对酶稳定性、酶活性和底物适应性进行协同改造是设计优良食品酶制剂需要解决的科学难题。α-L-鼠李糖苷酶是一种重要的食品酶，其结构、功能关系尚不明确，难以进行理性设计改造，而传统非理性、半理性方法难以实现酶多方面性质的协同进化。本项目构建改良的半理性方法对黑曲霉α-L-鼠李糖苷酶进行改造，提高其热稳定性并协同进化保持其酶活性和底物适应性。借助定向进化方法寻找该酶的热稳定性改造有效区域，基于饱和突变获得各区域的不同突变体，鉴定它们结构和酶学性质，解析各区域内结构变化对热稳定性、酶活性和底物适应性的作用规律；以此为基础模拟、筛选酶学性能协同进化的结构特征，从而提高其热稳定性并保持其酶活性和底物适应性。项目研究有望阐明提高α-L-鼠李糖苷酶的热稳定性并保持其对常见食品底物良好作用活性的结构机理，为实现其在食品工业中应用奠定基础，也为协同进化设计其它食品用酶提供新方法，具有重要的理论价值和实践意义。

运用协同分子改造思路，对黑曲霉α-L-鼠李糖苷酶进行定向改造和设计，获得热稳定性、酶活性和底物适应性符合食品加工过程应用要求的α-L-鼠李糖苷酶。研究内容分为5个方面，分别为：黑曲霉α-L-鼠李糖苷酶的异源表达体系构建、酶热稳定性改造有效区域分析、酶微观结构变化与酶学性质关系分子模拟、酶热稳定性/酶活性和底物适应性协同进化分子模拟设计、协同进化α-L-鼠李糖苷酶应用性能分析。.首先，建立了大肠杆菌、酿酒酵母和毕赤酵母等表达黑曲霉α-L-鼠李糖苷酶基因的技术体系，其中毕赤酵母表达的α-L-鼠李糖苷酶活最高，确定为本项目研究的表达系统。其次，建立黑曲霉α-L-鼠李糖苷酶易错PCR突变库，从中筛选得到了1株热稳定性提高的突变体及4株热稳定性降低的突变体。进一步通过定点突变、分子模拟分析发现，在α-L-鼠李糖苷酶529位进行突变，增加了氢键和疏水作用力，可保持提高热稳定性、酶活力且保持底物适应性；在573、631位蛋白质表面的氨基酸β-折叠与loop 区连接处引进芳香族氨基酸，增加cation-π相互作用，可以使酶稳定性提高，并保持相应的酶活力及底物适应性；将406、440、573位赖氨酸突变为精氨酸后，增加了氢键作用力和cation-π相互作用，从而使其热稳定性有显著的提高，且保持了相应的酶活力及底物适应性。此外，用热稳定性酶变体在果汁加工预热过程中对柑橘类果汁进行处理，可将柚皮苷的含量降低至苦味阈值以下，而野生型酶处理后柚皮苷含量还远高于苦味阈值。用酶对西红柿和苹果汁进行处理，2-Pentenal、2-Hexenal、(Z)-2-Heptenal、1-Octen-3-one等香气成分大幅度提高。.以上研究阐明了协同进化改造α-L-鼠李糖苷酶热稳定性、酶活性和底物适应性的结构机理，同时获得了热稳定性好、酶活力较高、底物适应性较广的进化酶，为实现该酶在食品工业中的应用奠定了基础。