裂解性多糖单加氧酶的功能机理研究

项目名称： 裂解性多糖单加氧酶的功能机理研究

项目编号： No.31500641

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 于洋

作者单位： 中国科学院天津工业生物技术研究所

项目金额： 20万元

中文摘要： 生物质可能是未来的主要化工原料来源, 主要成分是纤维素。其转化为单糖后，可经生物发酵转化为各种原料和燃料。但是, 现有的纤维素水解酶(CH)对结晶态纤维素活性低，限制了纤维素的利用。裂解性多糖单加氧酶(LPMO)是最近发现的, 能氧化降解纤维素的酶,是CH的有效补充。目前学术界对LPMO的反应机理仍然不清楚，为有效利用和改造LPMO设置了障碍。结构生物学研究揭示了LPMO活性中心有保守的酪氨酸和1-甲基组氨酸（1-MeH），但这些残基的功能还是未知。本研究将使用非天然氨基酸作为电子顺磁共振光谱（EPR）探针，研究酪氨酸在反应过程中的氧化还原变化，并发展1-MeH的插入系统，研究这一独特的翻译后修饰的功能。同时，我们将通过捕捉反应中间体，研究它们的结构和光谱特性。通过这三方面的研究，我们希望解释LPMO这一全新的铜单加氧酶的反应机理，并通过机理研究找到提高酶活并最终应用在生物质转化中。

中文关键词： 金属蛋白；生物质转化；非天然氨基酸；反应中间体；催化

英文摘要： Biomass is probably the main source of commodity chemicals in the future. The main component of biomass is cellulose. After converting to monosaccharide, cellulose can be transformed to various commodity chemicals and fuels through fermentation. However, cellulose hydrolases (CH) have low enzymatic activity toward crystalline cellulose. The low activity is the bottleneck for cellulose utilization. Lytic polysaccharide monooxygenase (LPMO) is a newly found enzyme which could oxidize cellulose and degrade it to smaller molecules. Its activity could complement that of cellulose hydrolases. Although there is growing interest in LPMO, its reaction mechanism remains elusive, which hinders the utilization and engineering of this enzyme. Structure biology studies reveal that a tyrosine and a histidine with 1-methyl modification are conserved, but their roles in the reaction are unknown. This study will use unnatural amino acids as EPR probe to study the redox change of tyrosine during reaction, and develop system for 1-methyl histidine (1-MeH) incorporation to compare the enzymatic activity of LPMO with and without 1-MeH. In the meantime, we will trap reaction intermediates through freeze-quench and stopped flow techniques, and study their structural and spectroscopic properties. Combining those three aspects, we hope to reveal the reaction mechanism of LPMO, a novel class of copper mono-oxygenase. Through study of mechanism, we will find ways to increase the activity and toward industrial application in biomass conversion.

英文关键词： metalloprotein;biomass converstion;unnatural amino acid;reaction intermediate;catalysis