纤维素酶的最适温度进化机制及其应用基础研究

Cellulases have been applied in industries and are limited by some characteristics such as high optimum temperatures and high energy consumption for application, and it is feasible that cellulases which acquire high activities at room temperature are produced by molecular modification. The most theories used to explain the thermal stability mechanism of cellulases can't directly clarify the evolutional mechanism of cellulase optimum temperatures at present, and therefore it is theoretically meaningful to study the evolutional mechanism of cellulase optimum temperatures. In our previous study, two cellulases of Phaeosphaeria sp. LH21 have optimum pH of 8 and are stable at pH3-10, indicating that they will have potential application values, but their optimum temperatures are 60℃ and 65℃, respectively. In the study, amino acid sequence exchange, error-prone PCR and DNA shuffling will jointly modify the two cellulases to generate cellulases with high activities and with optimum temperatures of 20-40℃, and several new enzymes which can be applied in textile, paper and detergent industries will be screened from the cellulases; the relationship between optimum temperatures of cellulases and every amino acid of primary structures will be revealed by stepwise regression analysis; homology modeling coupled with molecular dynamics simulations will be used to construct and analyze protein 3D models, which will help to find regions associated with optimum temperatures of cellulases; then site-directed saturation mutagenesis based on semi-rational design will help to mine critical sites related to optimum temperatures of cellulases.

纤维素酶在工业上已广泛应用，却受最适温度高、应用能耗高等特点的限制，而采用分子改造是获得常温下高活性纤维素酶的一种有效途径。目前，解释纤维素酶热稳定性机制的多数理论尚不能直接阐明纤维素酶最适温度进化机制，因此，研究纤维素酶最适温度进化机制具有理论意义。前期研究发现，Phaeosphaeria sp. LH21 两个纤维素酶最适pH均为8、pH3-10均稳定，具有潜在应用价值，但最适温度为60℃和65℃。本项目拟对它们联合采用氨基酸序列互换、易错PCR和DNA改组技术，获得最适温度20-40℃的高活性纤维素酶，找到几种能应用在纺织、造纸、洗涤剂方面的新酶；拟采用逐步回归分析，阐明纤维素酶一级结构中每种氨基酸与酶最适温度之间的关系；拟采用同源建模技术耦合分子动力学模拟，构建和分析蛋白3D模型，发现与酶最适温度相关的区域；再基于半理性设计，进行定点饱和突变，挖掘与酶最适温度相关的关键位点。

纤维素酶在工业上已广泛应用，却受最适温度高、应用能耗高等特点的限制，而采用分子改造是获得常温下高活性纤维素酶的一种有效途径。目前，解释纤维素酶热稳定性机制的多数理论尚不能直接阐明纤维素酶最适温度进化机制，因此，研究纤维素酶最适温度进化机制具有理论意义。前期研究发现，Phaeosphaeria sp. LH21 两个纤维素酶最适pH均为8、pH3-10均稳定，具有潜在应用价值，但最适温度为60℃和65℃。具体研究内容如下：1）确定了分子改造方法及其反应条件；2）建立了纤维素酶高通量的筛选和酶活测定方法；3）建立了简单高效的纯化体系，表征了酶学性质，构建了蛋白3D模型，确定了与最适温度相关的区域、关键位点; 4）从目的突变酶中找到几种能用于工业的新酶。研究结果表明，经过氨基酸序列交换、易错PCR、高通量筛选后，与纤维素酶最适温度相关的位点是11、13、14、16、19、22、46、112、122和133；通过纤维素酶的3D结构和分子动力学模拟的比较分析可知，纤维素酶最适温度的改变不是仅仅由于1种分子作用力的改变所引起的，它是由疏水区相互作用力、盐桥、氢键、侧链氨基酸大小、脯氨酸限制了环的构象等的协同进化共同促进了纤维素酶最适温度的进化。本项目为探索纤维素酶最适温度的改造提供了新的思路，因此，具有重要的理论意义和应用潜力。本项目在运行期间，培养了2名硕士，发表与纤维素酶相关研究的论文3篇，申请专利2项，圆满地完成了项目预期目标。