嗜酸普鲁兰芽孢杆菌普鲁兰酶耐酸能力分子结构解析及耐酸机理研究

Pullulanase is a key starch-debranching enzyme in the saccharification process, which is usually carried out at low pH (4.2-4.5). Acid resistance is an important industrial application property. At present, we still have no acid resistant pullulanase with independent intellectual property rights. To clarify the molecular structure and the mechanism for acid resistance has important significance to guide the development of acid resistant pullulanase. In this project, we plan to select the amino acid sites responsible for the acid resistance of Bacillus acidopullulyticus pullulanase based on the combination of structural biology, bioinformatics and homologous sequence alignment analysis. These sites are mutated according to rational design results using site directed mutagenesis and random mutation methods. The differences of enzyme properties between wide-type enzyme and mutants are compared. The effect of amino acid mutation on the acidity coefficient (pKa value) of catalytic group is also analyzed. The sequence and the structure features associated with the acid resistant can be identified based on the comprehensive analysis of the results of a number of experiments. The relationship between sequence, structure change and pH value variety will be established. The project aims for providing the deep understanding of acid resistance mechanism and the methodological reference of molecular modification.

普鲁兰酶是淀粉糖化脱支过程中的关键酶制剂，该过程需要在低pH下进行，耐酸能力是其重要的工业应用属性，但目前国内还没有自主产权的耐酸性普鲁兰酶。因此阐明耐酸性普鲁兰酶的序列、结构特征及耐酸机制，对指导开发自主产权普鲁兰酶的耐酸性具有重要意义。本项目以嗜酸普鲁兰芽孢杆菌普鲁兰酶（BaPul）为研究对象，依据结构生物学、生物信息学及同源多序列比对结果，分析BaPul催化基团所在微环境的序列、结构特征，甄选可能参与调控BaPul耐酸能力的氨基酸进行全新的分子设计构建突变体。考察突变体在不同pH条件下的相对酶活力和稳定性，分析氨基酸突变对催化基团酸度系数（pKa值）的影响。综合分析多项实验结果，鉴定与耐酸能力相关的关键氨基酸及空间结构特征并建立序列、结构变化与pH值变化之间的关联。项目旨在为深入理解普鲁兰酶耐酸性分子机制及耐酸能力分子改造提供科学依据。

项目以Bacillus acidopullulyticus普鲁兰酶为研究对象，建立多种方法甄选可能参与调控耐酸能力的氨基酸，进行全新的分子设计构建嗜酸性突变体。重点研究了基于氢键的关键氨基酸识别方法进行的酶蛋白嗜酸性改造。考察突变体在不同pH条件下的相对酶活力和稳定性，分析氨基酸突变对催化基团酸度系数的影响。突变体L627R的最适pH 4.0，酸性条件下的稳定性优于野生型，且在pH 4.0时的相对酶活是野生型的117%。动力学结果表明催化效率提高的主因是突变体具有较高的底物亲和性。采用突变体进行淀粉糖化实验时，葡萄糖浓度最高可达97.4%，糖化时间仅需40小时，较野生型减少了10小时。突变体催化基团酸度系数较野生型下调一个单位。采用淀粉酶家族中的另一重要成员α-淀粉酶验证本研究策略可行性，多个突变体的最适pH酸向偏移。研究结果表明基于氢键的关键氨基酸识别方法可用于淀粉酶嗜酸性改造，该究结果为深入理解淀粉酶耐酸性分子机制提供了科学依据。