低温外切菊粉酶的低温适应机理研究

Given the abundance of inulin in many plants, inulin is considered to be a prominent renewable carbohydrate source. Exo-inulinases are capable of efficiently hydrolyzing inulin to produce fructose yields as high as 90–95%, which is rapidly emerging as an important ingredient in the food, pharmaceutical, and energy industries. The method is environmentally friendly and includes only single-step process. Interestingly, over 75% of Earth’s biosphere is classified as permanently cold, and the largest proportion of biomass on Earth is generated at low temperatures. Effective hydrolysis of inulin at low temperatures requires low-temperature-active exo-inulinases. The use of low-temperature-active exo-inulinases at the industrial level is important because (1) low-temperature activity is required in various potential industrial applications such as brewing, making pickles, washing, and inulin processing at low temperatures, and (2) biotechnological processes performed at low temperatures reduce energy consumption and help avoid microbial development and fermentation, product denaturation, and alterations in the ingredient and product quality. Basic studies on exo-inulinases can give insights into mechanisms for adaptation to low temperatures and improve performances of these enzymes. However, low-temperature-active exo-inulinase has rarely been reported, not to mention mechanisms for adaptation to low temperatures. In previous studies, we have identified and characterized two low-temperature-active exo-inulinases, designated InuAGN25 and InuAMN8, which showed a potential for use in the production of fructose at low temperatures. We have also characterized a mesophilic exo-inulinase InuAJB13. In the study, mechanisms for adaptation to low temperatures of exo-inulinases will be revealed by amino acid residues and fragments mutageneses of InuAGN25, InuAMN8, and InuAJB13 based on the strategies of rational engineering and DNA shuffling. This study will provide theoretical basis for exo-inulinases improvement.

菊粉是一种来源丰富的可再生资源。外切菊粉酶可一步水解菊粉，获得高达90–95%的果糖。果糖在食品、医药和能源工业中都具有重要的应用价值。地球上低温的地域超过地球总面积的75%。低温外切菊粉酶可应用于低温环境要求下的生物技术领域，如酒的发酵、食品的腌制、去污、低温下的菊粉加工等，具有重要的开发价值。低温下的处理还可防止微生物污染、营养损失和食品品质降低，且可降低能耗。对低温外切菊粉酶低温适应机理的研究可用于指导酶的分子改良，使不具低温活性的酶具有低温活性，扩展酶的应用范围。然而，除了本研究工作基础外，国内外尚未报导低温外切菊粉酶及其低温适应机理。本项目拟以已获取的具有应用潜力的两个低温外切菊粉酶为主要材料，并以已获取的一个中温外切菊粉酶为辅助材料，基于分子动力学模拟辅助的理性设计和DNA改组的方法，通过突变等实验，揭示外切菊粉酶的低温适应机理，最终为外切菊粉酶的分子改良提供相应的理论依据。

菊粉是一种来源丰富的可再生资源。外切菊粉酶可一步水解菊粉，获得高达90–95%的果糖。果糖在食品、医药和能源工业中都具有重要的应用价值。地球上低温的地域超过地球总面积的80%。低温外切菊粉酶可应用于低温环境要求下的生物技术领域，如酒的发酵、食品的腌制、去污、低温下的菊粉加工等，具有重要的开发价值。低温下的处理还可防止微生物污染、营养损失和食品品质降低，且可降低能耗。对低温外切菊粉酶低温适应机理的研究可用于指导酶的分子改良，使不具低温活性的酶具有低温活性，扩展酶的应用范围。然而，到目前为止，已发表的大部分论文从底物结合位点及其相关作用方面对外切菊粉酶的催化机理进行研究，但是，对外切菊粉酶的热适应性机理的研究报导仍然很少。. 本项目以2个外切菊粉酶为对象进行研究，成功获得了低温活性提高的突变体，也获得了热稳定性提高的突变体。另外，由于蛋白存在活性-稳定性间的跷跷板现象，很少有研究能同时提高酶在低温下的活性和热稳定性，本项目实施期间成功获得了低温活性和热稳定性同时提高的外切菊粉酶突变体。. 本项目的实施以有力的实验数据为依据、在一定程度上揭示了外切菊粉酶的低温适应机理：外切菊粉酶的低温适应性在高级结构上与酶的N端、C端、loop区有关，这些区域受α-螺旋的构成、离子键和阳离子–π键的作用，当α-螺旋的占比上升、离子键（特别是离子键网）和阳离子–π键数量升高时，这些区域的柔性降低，能有效地在高温下维持酶的结构，避免结构坍塌，从而维持酶的稳定性，但如果催化口袋中的loop区柔性降低，则会导致酶催化时所需的能障升高，最终降低酶在低温下的活性。. 在本项目开展较顺利的基础上，项目实施期间还研究并揭示了其它3种酶的潜在催化适应性机理，其中，包括2种低温酶的潜在低温适应机理。.项目主持人周峻沛作为第一作者或通讯作者，将获得本项目资助的研究成果发表于SCI收录杂志，共计9篇，获得授权发明专利2项。