果胶多聚半乳糖醛酸酶适酸分子机理研究

Polygalacturonase (PG; EC, 3.2.1.15) is the enzyme that effectively degrades the complexes formed by polygalacturonic acid. Acidic PG is an important industrial enzyme in fruit juice processing. Under natural conditions, acidic PG can reduce the viscosity and improve the clarity of juice. The objective of this study is to explore the molecular mechanisms of acid adaptation of PGs based on the relationship of structure and function and to provide the theoretical basis for optimization of PGs used for juice. An acidic PG (PG63) and a neutral-basic PG (PGKle) have been selected that share high sequence identity and have similar tertiary structures. PG63 has a pH optimum of 3.5 and is stable only at acidic pH range. PGKle shows maximal activity at pH 6.0 but retains stable at pH range of 3.0 to 12.0. Different mutant libraries will be constructed through directed evolution combined with rational design, DNA shuffling based on the conserved DNA sequences, reassembly of function modules, site-directed mutagenesis, and random combination of mutants. Further enzyme characterization will be used to screen mutants and consequently to identify the critical region, function module and key residues for acid adaptation. Integrated analysis of the results will give an insight into the molecular mechanisms of acid adaptation of acidic PG. The information will be of importance in PG improvement for application in fruit juice industries.

多聚半乳糖醛酸酶（Polygalacturonase, EC 3.2.1.15, 以下简称PG）能有效降解多聚半乳糖醛酸复合物，酸性PG能在果汁天然酸性环境下使果汁降粘澄清，是果汁加工用主要酶种。开展酸性PG酸适应性的分子机理研究，将为果汁酶的酸碱性质改性提供理论依据。本研究的核心材料为我们分离到的一个酸性PG（PG63）和一个中碱性PG（PGKle），它们氨基酸序列相似性较高，三维结构高度一致，但pH特性有显著差异。PG63最适pH3.5，仅在酸性范围内保持活性；而PGKle最适pH6.0，但在pH3-12之间有活性。通过定向进化和理性设计，基于同源保守片段DNA改组、功能模块重装配、定点突变及突变随机组合等手段形成突变文库，确定影响酶适酸性质的关键区域、结构功能模块和关键位点，分析酸性PG的适酸机理，为进一步改造果汁工业用PG的pH性质提供理论基础。

本课题以果胶多聚半乳糖醛酸酶（PG酶）PG63和PG8fn为核心材料研究PG酶的适酸性分子机制。实验成功获得并解析PG8fn的晶体结构，进一步通过三维结构比对分析、功能模块组合、分子动力学分析及突变体试验验证，确定了酶分子的中间催化区域是影响酶适酸性质的主要模块，能造成最适pH变化幅度高达2个单位的改性；而N端和C端区域模块对酸碱性质的调整起到辅助作用，模块替换也能造成1.0—1.5个单位的最适pH酸性偏移，说明酶分子的最适pH作用环境是一个整体结构效应。进一步突环区的突变体研究精确定位 T3 loop 1区域对决定PG酶分子的适酸性起着决定性作用，替换后最适pH酸改性幅度达2个单位。对不同候选位点的突变体筛选及研究确定了两个关键氨基酸位点：T90位点空间位阻决定其在相同酸碱环境中不同残基所带电荷性质影响酶分子与底物的结合程度，其碱性氨基酸突变能使最适pH偏向酸性1个单位且有利于酶在酸性条件下提高催化效率；而位于酶分子催化口袋中的关键位点N94在酶分子与底物形成复合物过程中由于氢键的形成导致突环区柔性的变化从而改变催化位点与底物分子的距离，进而影响催化效率，此位点在不同酸碱度下对底物的识别和稳定起着重要的作用。总体上本研究通过不同的理性设计及突变体验证确定了核心材料酶分子中不同结构层面中与适酸性质高度相关的关键功能模块、突环区及氨基酸位点，初步解析了PG酶适酸性的分子机理，为后期工业用多聚半乳糖醛酸酶酸碱性质改良提供了相关的依据。本课题实施已完成预期的研究论文及专利任务指标。