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。

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.