基于同源/非同源结构置换的木聚糖酶-葡聚糖酶结构元件功能解析

Degrading non-starch polysaccride (NSP) anti-nutritional factors, a highly active Aspergillus niger GH11 xylanase (X) is important in food industry. However, the X activity has a low stabiity, and the challenge is also faced by all food using enzyme. The X employs a β jelly-roll structure, and shares 69% structural similarity with an extremely stable Thermotoga maritima GH12 glucanase (G). Stability difference arises from their different structural elements, and function elucidation of structural elements is a key point for property-engineering of the X and the G. Based on structural analysis of non- and homologous regions between the two enzymes, the project intends to: 1) elucidate functions of structural elements relating to stability, activity, and substrate-binding by respectively replacing helix-C mega-element, disassembled into helix and C-terminal element, further disassembled into basic element of the X with homologous structural element of the G, 2) produce stable, specific, and highly active xylanase, 3) investigate evolutionary process from X to G by replacing the specific thumb of X with specific elements of the G, 4) determine the T.maritima GH10 xylanase carbohydrate-binding module C2 as an evolutionary intermediate from X to G by respective transplantation with specific elements of the X and the G to endue it with glucanase and xylanase activity. From view-point of structural replacement, the project elucidates functions of structural elements relating to enzyme properties, evolutionary process from X to G, and “element assembly” engineering theory that produce expectation properties and evolve enzyme quickly by assembling related structural elements.

高活性黑曲霉GH11木聚糖酶X降解非淀粉多糖类抗营养因子在食品工业有重要作用，但其稳定性不足，是所有食品加工用酶面临的挑战。X 为β jelly-roll结构，与其69%结构同源的海栖热袍菌GH12葡聚糖酶G极其稳定，稳定性差异源于G/X结构元件不同，元件功能解析是酶学性质改造的关键。本项目基于同源结构比对，以G中 helix-C端大元件、拆分为helix/C端小元件、进而拆分为单个元件分别置换X同源结构、以G特异性元件置换X特异性thumb结构，解析各结构元件与稳定性、底物特异性、活性的构效关系，得到稳定-特异-高活木聚糖酶，探讨X/G进化过程。在木聚糖酶底物结合结构域C2中分别植入G/X特异元件赋予其葡聚糖/木聚糖降解活性，确立C2为X/G进化中间体假说。项目从结构置换角度探讨酶学性质相关结构元件的功能、构建组装相应元件改造酶性质、加速酶分子进化的“元件组装”理性改造理论。

基于Thermotaga maritima葡聚糖酶Cel12B、Aspergillus.niger GH11木聚糖酶Xyn、Thermotaga maritima碳水化合物结合模块CBM9_2（C2）结构比对，项目通过将Thumb引入葡聚糖酶和C2构建不同突变体，探讨了Xyn的特异性Thumb结构对底物特异性作用。首先通过序列比对确定Thumb中关键氨基酸（Aa）位点从而进行单点突变，突变体E119Q、T123E、S126T降低了Xyn对底物km值、T129D突变体增加了km值，表明这些位点是Xyn与底物结合的关键性Aa位点，其中E119Q、T123E、S126T提高了对底物亲合力。进而在葡聚糖酶中引入Thumb结构后，HPLC检测发现突变体降解木聚糖底物后产物中木二糖、木三糖和木四糖比例增加，表明Thumb结构对产物性质发挥关键作用，进一步单点、双点和三点叠加突变Thumb结构关键Aa位点（原Thumb的E119Q、T123E、S126T），突变体E177Q、T181E、S184T木聚糖/CMC底物活性比值与突变前比值一致、而双点和三点叠加突变后对木聚糖/CMC底物活性比值增加5-7倍，表明突双点和三点变体增加了木聚糖活性、降低了CMC水解活性，且双点和三点Aa突变对木聚糖底物活性叠加作用更加明显，表明底物催化活性是单点突变叠加效果产生的累积作用。对于无底物催化活性的C2，引入Thumb结构后产生C79T突变体，结果表明C7T产生从无到有的木聚糖降解活性。本研究基于结构分析，在葡聚糖酶Cel12B和C2中分别引入Thumb结构、并对其中三个关键Aa进行单点和叠加突变，提高了对木聚糖底物亲和力和水解活性，在保持空间结构基础上进化为具有木聚糖酶活性的酶分子，为底物特异性和宽泛性改造提供了新方向，为酶工程领域提供了新的酶分子稳定性改良方法和模式，在理论上探讨了在保持结构基础上从一种底物进化到另一种底物的催化功能。