耐热丝状真菌可生产具有重要工业应用价值的耐热酶蛋白和代谢产物，为目前亟需开发的重要微生物资源之一；耐热脂肪酶在高温条件下可稳定发挥其催化活性，适用于多个工业领域，但目前可工业应用的耐热脂肪酶来源较少，难以满足相关工业工程中不同加工底物及过程的需要。为丰富耐热丝状真菌微生物资源，获得其所产耐热性能优良的新型脂肪酶，本项目结合合作双方丰富的微生物资源，筛选并建立耐热丝状真菌库；快速筛查耐热丝状真菌脂肪酶的酶学性质及底物特异性，挖掘耐热性能优秀的新型脂肪酶；进一步通过分子生物学技术结合生物信息学手段，揭示新型脂肪酶的空间构象与酶学特性之间的关系。由此，本项目通过对耐热丝状真菌的筛选及其耐热脂肪酶的挖掘，为真菌资源的开发和应用提供了更为丰富的生物材料，并阐明支撑新型耐热脂肪酶酶学特性的分子基础，为理性改造脂肪酶提供理论依据，为脂肪酶的应用提供理论指导，为工业加工过程开发新型的耐热脂肪酶奠定基础。

Thermophilic filamentous fungi are excellent producers of industrial enzymes with high thermal stability and their potential for broad industrial applications has attracted increasing research interest. Thermostable enzymes are best suited to harsh conditions of many industrial processes due to their overall inherent stability and high reaction rates at elevated temperatures. Themostable lipases (EC 3.1.1.3) are of particular interest with numerous potential applications in diverse industries because of their substrate specificity, region-specificity, chiral-selectivity and resistance to chemical denaturants. However, the limited number of available commercial thermostable lipases and their poorly understood catalytic mechanisms have hampered the wide applications of this class of enzymes. This project aims at exploring the rich fungal resources of China and South Africa to prospect the thermophilic filamentous fungi from diverse habitats and to create a bank of the collected thermophilic strains. Novel target thermostable lipases will be screened by high-throughput methods; molecular techniques and bioinformatics will be employed to probe the relationship between the structures and enzymatic properties of the discovered lipases. The completion of this project is expected to: (1) provide invaluable culture collection of thermophilic filamentous fungi; (2) lay a foundation for rational design and engineering of thermostable lipases for key industrial applications through a better understanding of the molecular basis underlying the thermostability and catalytic properties of thermostable fungal lipases.

耐热丝状真菌可生产具有重要工业应用价值的耐热酶蛋白和代谢产物，为目前亟需开发的重要微生物资源之一；耐热脂肪酶在高温条件下可稳定发挥其催化活性，适用于多个工业领域，但目前可工业应用的耐热脂肪酶来源较少，难以满足相关工业工程中不同加工底物及过程的需要。本项目结合合作双方丰富的微生物资源，通过在南非及国内收集样品，规模筛选嗜热嗜碱丝状真菌，建立了包括22个属、40个种的嗜热、嗜碱丝状真菌菌种库。从建立的菌种库丝状真菌中，筛选获得了耐热的疏棉状嗜热丝孢菌和耶氏解酯酵母脂肪酶（TLL和LipY2）及耐碱的圆弧青霉脂肪酶（PCL）。以TLL、LipY2及PCL为研究对象，利用定点突变、易错PCR等技术对其进行改造，获得耐热性能提升的TLL突变体STLL（S146A）、耐酸性能提升的LipY2突变体M36（K36G/K39T）、M63（R63G）、M36-63（K36G/K39T/R63G）、及耐热性能提升的PCL突变体PCLM（L41P、G47I和L41P/G47I）。同时，分析获得上述突变体酶学性质、底物特异性、动力学等酶学特性信息。利用Swiss-model服务器对TLL、LipY2及PCL和各突变体同源建模，采用Swiss PDB viewer 和PyMol分析突变氨基酸及位点对TLL、LipY2及PCL分子催化位点、底物结合位点、二级结构及酶蛋白整体构象的影响和突变体中疏水作用、静电场力、氢键和盐桥的相关信息，结合酶学特性信息，分析表明疏水作用、氢键、脯氨酸结构等对酶的活性和稳定性有重要影响。由此，本项目通过对耐热丝状真菌的筛选及其耐热脂肪酶的挖掘，为真菌资源的开发和应用提供了更为丰富的生物材料，并阐明支撑新型耐热脂肪酶酶学特性的分子基础，为理性改造脂肪酶提供理论依据，为脂肪酶的应用提供理论指导，为工业加工过程开发新型的耐热脂肪酶奠定基础。