大兴安岭多年冻土来源的低温木糖苷酶AX543冷适应机制的研究

Cold active xylosidase has been widely used in the food industries because of its high catalytic efficiency at low temperature. Thus, the revelation of xylosidase psychrophilic mechanism is helpful to understand its structure and function, and can be used to molecules modify other xylosidases. However, cold active xylosidases have rarely been reported, not to mention their catalytic mechanism at low temperature. Previously, we obtained a cold active xylosidase AX543 from Greater Khingan Mountains permafrost soil. AX543 displayed its maximum temperature at 25℃, lower than most of the other reported cold active xylosidases, and remained higher relative activity at lower temperature. Based on the sequence alignment, homology modeling and structure comparison analysis, we had found that AX543 contains five different loop regions, less salt-bridge and hydrogen bond, and more glycine and less arginine and proline, which were inferred to be related to the low temperature activity of AX543. In this study, the psychrophilic mechanism of xylosidase AX543 will be studied through loops replacement, truncation, glycine mutation and oligomeric glycine replacement, salt-bridge and hydrogen bond rebuilt, and site-directed mutation of the key amino acids. The completion of our work will offer us theoretical guidance for the modification of other mesophilic or thermophilic xylosidases to get the cold active counterparts.

低温木糖苷酶因能在低温下高效水解木聚糖而在低温食品工业中具有巨大的应用潜力，低温木糖苷酶的冷适应机制研究不仅有助于拓展对木糖苷酶结构与功能的了解，并可指导分子改良获得高效低温木糖苷酶，解决当前木糖苷酶低温活力差的问题。然而，目前国内外发现的低温木糖苷酶数量极少，更未见对其冷适应机制的报道。项目申请人从大兴安岭多年冻土中获得一个新的木糖苷酶AX543，与其它低温木糖苷酶相比，AX543具有更低的最适温度（25℃）和更高的低温催化活性。进一步分析发现loop区的差异、盐桥和氢键的减少以及精氨酸、甘氨酸和脯氨酸含量的变化可能与其嗜冷特性相关。本项目拟结合序列比对、同源建模、分子动力学模拟和虚拟突变等方法，利用替换、截短、甘氨酸扫描和定点突变等技术对差异loop区、盐桥、氢键以及关键氨基酸进行突变研究，探明木糖苷酶AX543的冷适应分子机理，为木糖苷酶的冷适应性改造提供理论依据。

低温木糖苷酶可以在低温下高效水解木聚糖，所以在低温食品工业中具有巨大的应用潜力。低温木糖苷酶的冷适应机制研究不仅有助于拓展对木糖苷酶结构与功能的了解，并可指导分子改良获得高效低温木糖苷酶，从而解决当前木糖苷酶低温活力差的问题。然而目前国内外发现的低温木糖苷酶数量极少，更未见对其冷适应机制的报道。项目申请人发现低温木糖苷酶AX543与其它低温木糖苷酶相比具有更低的最适温度和更高的低温催化活性，具有良好的应用前景，所以探讨其冷适应分子机制具有非常重大的科研意义和应用价值。本研究首先通过对木糖苷酶AX543和中高温酶通过多序列比对和结构比对、氨基酸组成、分子作用力等分析，结果发现4处差异显著的loop结构、减少的氢键和盐键数量以及差异甘氨酸、脯氨酸和精氨酸位点可能是导致AX543冷适应性相关的重要原因。然后对大量突变体的筛选，结果发现在4个loop区中loop2突变体未改变最适温度，但显著提升热稳定性和比活力，其热稳定性提升主要是由于增加了氢键数量而导致了整体结构的稳定性；在与氢键盐键相关的氨基酸位点中，发现Q201R突变体显著提升了热稳定性和比活力，仍保持原最适温度，同时突变后增加了整体结构的氢键数量；在关键甘氨酸和脯氨酸的中发现G110S突变体同样也提升了热稳定性和比活力，未改变最适温度。最后进一步研究影响最适温度的关键因素，发现A1和A4序列模块的替换可以分别将低温木糖苷酶的最适温度提升10℃和15℃。终上所述，研究发现loop2结构、G110位点和Q201位点是影响低温木糖苷酶的热稳定性的关键因素，它们主要通过增加氢键数量来提升低温木糖苷酶AX543热稳定性，并获得3个热稳定性显著提升的木糖苷酶突变体；同时发现A1和A4序列模块是导致低温木糖苷酶具有超低最适反应温度的关键因素。本研究探明了低温木糖苷酶AX543的冷适应机制，为未来理性改造获得高效低温木糖苷酶及其他工业酶提供坚实的理论依据和技术支撑。