基于特殊多肽序列的新型SOD嗜热机制解析及耐高温工业酶改造

SOD is an important barrier for the living organisms to defense the oxidative damage. It is one of the earliest and most widely used enzymes for industrial application. Traditional industrial SODs are most mesophilic enzymes, which have poor thermotolerance and stress resistance. SODs extracted from natural source have not yet been widely applied in the industrial applications because of the inconvenience in the process of separation and reusing of thermophilic enzymes, and the high cost of the enzymes. Meanwhile, the existing biological or chemical modification methods have limitations in the industrial application because of the unclear mechanisms or the defect of tedious operation. Therefore, it is significant and has application value to explore new thermophilic mechanisms and develop easy ways for the thermostability improvement of enzymes. NG80-2 is a crude oil-degrading thermophilic bacterium isolated from a deep-subsurface oil reservoir, which encodes a special thermophilic Fe/Mn-SOD. In the present study, we demonstrated that the N-terminal peptide of Fe/Mn-SOD from NG80-2 determines its thermostability and found that this type of N-terminal peptide only exists in Geobacillus and Paenibacillus with a considerable difference between them. On the basis of the study above, the methods of bioinformatics, protein crystal structure analysis, genetic recombination and gene manipulation will be applied to reveal the two novel thermophilic mechanisms of special polypeptide sequences and uncover the important role that the N-terminal peptide plays in thermostability. Based on this, the special peptide will be applied to modify and enhance the thermostability of mesophilic SODs (especially the traditional industrial SODs). The outcome of this study is expected to provide a new strategy for the thermostability improvement of mesophilic SODs and lay the foundation for the upgrade of traditional SODs and the development of new thermostable SODs. Furthermore, the project provides a new thought for the thermostability improvement of other enzymes.

SOD是生物防御氧化损伤的重要屏障，也是最早和最广泛应用的工业酶之一。传统工业SOD多为常温酶，其耐高温和抗逆性差。天然嗜热SOD在应用中有局限性，同时，现有人工改造手段因嗜热机制不明或方法繁琐，不具操作性。探寻新型嗜热机制、开发简便易行耐高温改造方法，具有重要意义和应用价值。前期研究表明，极端环境菌NG80-2编码特殊的嗜热SOD，其嗜热性源于一段特殊N端多肽。该N端多肽仅存在于Geobacillus属和Paenibacillus属，具一定共性特征但不尽相同。本项目在此基础上，采用生物信息学、蛋白晶体结构解析、基因重组和基因操作等研究方法，开展基于特殊多肽序列的两种新型嗜热机制的解析及差异研究，揭示N端多肽在空间结构上对蛋白热稳定性的重要作用。在此基础上，利用特殊多肽进行常温SOD（尤其传统工业SOD）的耐热性改造和高效表达，为传统SOD的升级换代和开发新型高效耐高温工业SOD奠定基础。

SOD是重要的工业酶并广泛应用于医药、食品、日用化工和农业等领域。探寻新型嗜热机制、开发耐高温超稳定SOD工业酶，具有重要意义和应用价值。前期研究发现，极端环境菌Geobacillus属和Paenibacillus属中编码的SOD存在一系列独立于功能域之外的特殊N端多肽序列，且序列特征不尽相同。本项目在此基础上，综合采用基因操作、酶学分析、生物信息学的方法对这些特殊N端多肽的作用机制进行了全面系统地研究，揭示了其在空间结构上对蛋白稳定性的重要作用。主要包括：1）首次阐明了Geobacillus属SOD的新型蛋白嗜热机制。这种机制主要源于N端多肽中存在的1-2个高度相似的repeat序列。具有2个repeat序列的N端可提高SOD的嗜热性（即耐高温性，最适温度可从30℃提升至70℃以上），而含1个repeat序列的N端更倾向于提高SOD的热稳定性。此外，N端序列大大增加了SOD对抑制剂、变性剂和去污剂的抵抗力。2）发现了Paenibacillus属SOD 的N端特殊多肽可通过金属转移机制提高蛋白活性和稳定性。该N端多肽中包含一种特殊的金属离子结合域（DUF2935），可特异性结合Fe/Mn/Mg金属离子并通过转移的方式为SOD的催化中心提供金属离子，从而提高SOD的活性和稳定性。我们推测这可能是一种独立的SOD稳定性机制。3）开展了基于上述两种N端多肽的耐热性和稳定性机制的差异和叠加效应研究，初步证实两者存在叠加效应，可对SOD的稳定性进一步提高。4）实现了N端多肽对不同类型（包括常温和天然嗜热）SOD的改造，获得了5个新型高效重组耐高温SOD工业酶（包括目前已知最为耐热的超稳定SOD）。上述研究结果首次揭示了一种全新的蛋白热稳定性机制，具有显著的科学意义。同时，与其它已报道机制相比，该机制提供了一种更为简便易行的耐高温酶改造方法，所改造获得的超稳定SOD工业酶具有更高的应用价值。研究成果共发表学术论文8篇（标注资助），包括Scientific reports、Food chemistry等SCI收录国际学术期刊5篇，总影响因子15.65；申请国家发明专利4项（2项已授权）。