纤维素酶的活性和产物耐受性协同进化机制研究

Cellulases are the third largest industrial enzyme in the world, but are limited by low activity and low product tolerance. The theory about reducing binding energies of products and cellulases can guide enzymes towards evolution of high product tolerance, but decreases activity of enzymes; it is not enough for sporadically scattered information of cellulases with high activity and high product tolerance to elucidate the synergetic evolution mechanism of activity and product tolerance. Therefore, many cellulases with high activity and high glucose tolerance should be excavated, and it is theoretically meaningful to study the synergetic evolution mechanism of activity and product tolerance on the relationship between structure and function. In the previous study, β-glucosidase (BGL, belonging to cellulase) from Penicillium oxalicum 16 had an optimum temperature of 70°C, indicating that it had potential application values, but was inhibited by high concentrations of glucose. BGL from P.oxalicum 16 will be regarded as the object of the study, and multiple rounds of error prone PCR, multiple rounds of DNA shuffling and site saturation mutagenesis coupled with a high-throughput screening method will be used to find new enzymes with high activity and high glucose tolerance; homology modeling, molecular docking and molecular dynamics simulation will be used to construct and analyze 3D structures of glucose-cellulase, and the most relevant region or key sites related with activity and product tolerance of cellulase will be revealed by structure information combined with enzymatic properties and enzyme kinetic parameters.

纤维素酶是全球第三大工业用酶，却受低活性和低产物耐受性的限制。减弱产物和纤维素酶结合能的理论，可指导酶朝着高产物耐受性方向进化，但降低了酶活性；零星分散的有高产物耐受性的高活性纤维素酶信息，不足以阐明活性和产物耐受性协同进化机制。因此，发掘更多有高产物耐受性的高活性纤维素酶，从结构与功能关系上，研究活性和产物耐受性协同进化机制具有理论意义。前期研究表明，草酸青霉16的β-葡萄糖苷酶（BGL，属纤维素酶）最适温度可达70℃，具有潜在应用价值，但受高浓度葡萄糖抑制。本项目拟以草酸青霉16的BGL为研究对象，采用多轮易错PCR、多轮DNA改组和定点饱和突变技术，耦合高通量筛选方法，发现有高葡萄糖耐受性的高活性新酶；采用同源建模、分子对接和分子动力学模拟技术，构建、分析葡萄糖-纤维素酶3D结构，再结合酶学性质及酶反应动力学参数，揭示与纤维素酶的活性和产物耐受性协同进化最相关的区域或关键位点。

纤维素酶是全球第三大工业用酶，却受低活性和低产物耐受性的限制。本研究以草酸青霉16纤维素酶的β-葡萄糖苷酶为研究对象，通过定向进化和高通量筛选方法，获得兼具高活性和产物耐受性的突变酶；采用同源建模、分子对接和分子动力学模拟技术，构建、分析3D结构，再结合酶学性质及酶反应动力学参数，揭示与纤维素酶的活性和产物耐受性协同进化最相关的区域或关键位点。我们鉴定了两个突变体，一个是存在三处突变（M280T/V484L/D589E）的L-6C，一个是存在三处突变（G414S/D421V/T441S）的Y-1-B1。L-6C的葡萄糖抑制强度降低了3.63倍（也就是产物耐受性提高了），活性提高了22％，L-6C和纤维二糖的结合能略有下降。Y-1-B1的最适催化温度降低了20°C，活性却提高了1.7倍，葡萄糖抑制强度降低了1.1倍。我们进行了分子对接和分子动力学模拟（MD），利用分子力学/泊松玻耳兹曼表面积（MM/PBSA）方法计算了结合自由能，计算表明L-6C减少了与pNPG的结合自由能，这与实验数据一致。MD模拟显示，Y-1-B1亲核残基Asp261与pNPG的糖苷中心具有更紧密的接触，表明该突变体更有利于催化，也更有利于产物的脱离。将这两个突变体应用到生物乙醇生产中，与原始酶相比，生物乙醇产率分别提高了22%和10%，因此，这两个突变体在生物乙醇生产中具有潜在的应用价值。本研究主要成果有：共发表了5篇SCI论文，其中2篇为二区TOP文章；1个授权专利，2个申请专利；培养了5个硕士研究生，其中2个研究生已经毕业；参加了全国生物工程学会年会、华东六省一市生物化学与分子生物学学会年会和江西省生物化学与分子生物学学会年会；因此，我们超额圆满地完成了本研究的各项任务。