基于羰基还原的热解生物油生物催化提质机理研究

Bio-oil production from fast pyrolysis of biomass is an important approach to the high-valued utilization of biomass. But many studies suggested that the application of bio-oil had been greatly restrained due to its high oxygen content and poor thermal stability. Therefore, product upgrading is needed for its commercial application. To date the main method of bio-oil upgrading includes catalytic hydrogenation and catalytic pyrolysis. During these processes, the higher temperature would lead to the coke occurred because of condensation of activated oxygen-containing functional groups in bio-oil. Based on this, we proposed a new way that converting unstable aldehydes and ketones from bio-oil into mixed alcohols by carbonyl reduction using biocatalyst. This method could make them possible to improve the bio-oil stability under low temperature and achieve the improvement of bio-oil quality. According to the proposed method, this project focuses on the substrate specificity, structure-activity relationship and the increase of the solvent tolerance of carbonyl reductase. Through genome bioinformatics analysis, molecular directed evolution and biotransformation experiment, combining with molecular docking simulation and density functional theory calculation, a suitable carbonyl reductase for bio-oil is acquired and synergy mechanism coupled mass-transfer and reaction is revealed in the bio-oil reduction process. Our work would provide the theoretical basis for the bio-oil upgrading under low temperature based on bio-carbonyl reduction.

生物质快速热解制备生物油是目前生物质高值化利用的重要途径，但生物油氧含量高、热稳定差等缺陷严重制约其应用，需要进行品质提升。目前生物油提质方法主要有催化加氢和催化裂解，由于过程温度较高会引起生物油中活泼含氧基团受热聚合结焦。基于此，本项目提出通过生物催化羰基还原反应在常温下将生物油中最不稳定的醛酮类物质转化为混合醇的新途径，该途径可望通过低温还原提高生物油稳定性，实现油品低温提质。围绕上述途径，项目通过基因组生物信息学分析、分子定向进化和生物转化实验，结合生物分子底物对接模拟和密度泛函理论计算，开展羰基还原酶底物特异性筛选、构效关系研究及溶剂耐受性改造，揭示生物油生物还原过程物质传质和反应耦合调控规律，建立基于生物羰基还原的生物油生物催化提质的理论基础。

针对生物质快速热解生物油氧含量高问题，以较低温度催化加氢避免高温引起生物油中活泼含氧基团受热聚合结焦为目标，深入研究利用微生物催化羰基还原实现在常温下将生物油中最不稳定的醛酮类物质转化为混合醇的新途径。在分析热解生物油中组成分布基础上，确定生物油典型羰基组分为糠醛，甲基糠醛、香草醛、3-羟基-2-丁酮（乙偶姻）和环戊酮；采用糠醛作为模化物进行筛选生物还原菌株，比较菌株对糠醛的分解途径和还原效率，确定凝结芽孢杆菌B.coagulans NL01为目标菌株；采用密度泛函理论方法计算酮醛类化合物利用NADH及NADPH为氢供体羰基还原生成醇类物质的焓变，理论上证实生物油中各种醛酮底物的生物催化还原的可行，提出NADH为辅酶的羰基还原酶更有利于生物油还原；通过基因组探矿建立了包括26个假定蛋白B.coagulansNL01羰基还原酶元件库，并结合酶与底物分子对接底物特异性模拟预测和分子克隆表达实验研究其中11 个羰基还原酶的活力性质和底物特异性以期筛选适于生物油底物谱和生物还原能力强的羰基还原酶。研究发现，SDR-1和SDR-8具有较为广泛的底物谱，前者对芳香醛、呋喃醛和酮类均具有活力，后者对脂肪醛和芳香类醛酮具有活力；以天然筛选获得的羰基还原酶为模板，研究其溶剂耐受规律，以生物油为筛选压力构建SDR-1突变文库开展定向进化改造进一步提高羰基还原酶对生物油的适应性，突变酶对不同醛酮底物活力提高120-223%；采用羰基还原酶构建羰基还原反应，开展羰基模型化合物和实际生物油的生物转化研究，设计和优化生物还原反应体系和条件，研究生物油转化前后醛酮类物质转化规律，解析生物油生物还原的关键因素和调控机制。首次实现和证实可利用微生物在双相体系还原低浓度生物油轻油，转换后生物油中糠醛，2-丁酮，丁二酮等酮醛类物质消失，出现了相应醇类物质。该途径的研究和建立可望通过低温还原提高生物油稳定性，实现油品低温提质，建立基于生物羰基还原的生物油生物催化提质的理论基础。