基于红藻kappa-卡拉胶工业废渣绿色利用的海洋卡拉胶酶资源筛选及性能优化

对海洋红藻kappa-卡拉胶工业废渣进行绿色利用具有重要经济及环境生态价值。kappa-卡拉胶酶是促进该废渣利用的关键因素，但已知的kappa-卡拉胶酶耐碱性和耐热性达不到应用要求，影响了该废渣利用效率。针对该问题，本项目收集kappa-卡拉胶酶资源信息，以耐碱性为主要指标并兼顾耐热性筛选初始酶，运用非理性和半理性手段对初始酶的耐碱性和耐热性进行改良；借助X-ray晶体衍射和理论计算研究酶的结构特征，根据蛋白质功能区理论分析热点突变位点、热点氨基酸和分子作用力对耐碱性和耐热性影响的关联性规律，以此为依据协同改造酶的耐碱性和耐热性，并评价其对废渣中过滤介质、废碱和卡拉胶的回收利用效果。预期阐明kappa-卡拉胶酶的热点氨基酸突变方向、氢键、疏水作用等与耐碱性和耐热性变化的关联规律，建立协同改造该酶的耐碱性和耐热性的方法，为kappa-卡拉胶工业废渣绿色利用奠定酶学基础。

Red algae are important marine biological resources. The green utilization of the waste from red algae kappa-carrageenan industry is of significance for both industry and environment. kappa-Carrageenase plays a critical role in utilizing the waste. The waste mainly comprises with filter medium, kappa-carrageen and NaOH, which requires enzymes used in the treatment have desirable alkali-resistance ability and thermal stability. However, the kappa-carrageenases currently do not fit the tough environment in treating the kappa-carrageenan waste, which remarked lowers the efficiency of utilization of kappa-carrageen waste. To solve this problem, the project plans to collect kappa-carrageenase resources that are evolved from marine, which will be screened to get an initial enzyme using alkali-resistance ability as the main index and thermal stability as the secondary indicator. Subsequently, the initial enzyme will be submitted to respectively improve alkali-resistance ability and thermal stability using irrational and semi-rational means. Thirdly, the enzyme will be analyzed in the structural characteristic using X-ray crystal diffraction and theoretical calculation; and the protein functional-region theory will be applied to correlatively simulate the relationship how mutation sites, key amino acids and molecular forces determine alkali-resistance ability and thermal stability. Lastly, the enzyme will be co-evolved in its alkaline- resistance ability and thermal stability, based on rational design with the structural characteristics and protein functional-regions in relation to the alkaline-resistance ability and thermal stability, and by the aid of virtual screening surface charge and refolding free energy. In addition, the resultant co-evolved enzyme will be investigated in the ability to recover filtration medium, waste alkali and oligo-carrageenan from the waste. Through this study, it is expected to elucidate the correlation how hot sites, key amino acids, hydrogen bonds, hydrophobic action and other forces determine the alkali-resistance ability and thermal stability of the kappa-carrageenase; and develop an effective method to co-evolve alkali-resistance ability and thermal stability of the kappa-carrageenase. The expected results will shed a light on the green utilization of kappa-carrageenan industrial waste, and provide a general reference for the green utilization of marine algae by mean of marine biotechnology.