有机溶剂毒害生物催化剂是制约非水酶学发展的技术瓶颈，从微生物中探寻天然耐受有机溶剂的脂肪酶则是实现此突破行之有效的措施，而已有研究却忽略了内生菌这一潜在的酶资源库。项目针对已分离的葡萄内生菌，以有机溶剂和油脂为选择压力，定向筛选1-2株有机溶剂高耐受性产脂肪酶目标菌株，确定其分类学地位；掌握有机溶剂对目标菌的毒性作用规律，建立菌株的有机溶剂耐受谱，从细胞生理水平揭示其耐受有机溶剂的适应性机制；阐明目标菌产脂肪酶的规律，提高产酶能力，建立分批发酵产酶动力学模型；最后，分离纯化目标菌分泌的脂肪酶，测其分子量和N端序列，表征其酶学性质，明确酶的特异性。项目为深入了解菌株耐受有机溶剂的分子机制，揭示脂肪酶的结构、构效关系、作用机理、基因表达调控等奠定基础，对于拓宽有机溶剂极端耐受菌来源，获取新颖耐有机溶剂酶类，开发全细胞生物催化、生物修复环境污染物、催化不对称合成反应、酶法合成生物柴油等意义重大。

Organic solvents which are toxic to bio-catalysts have become a bottleneck of developing non-aqueous enzymology . Exploring for organic solvent tolerant lipase from natural microbes is an effective way to achieve this breakthrough, however, many researchers neglected the endophytes which are a great potential enzyme repository. This program is to directionally screen one or two strains producing high organic solvents tolerant lipase from grape endophytes by using the organic solvents and oils as selection pressure and determine their taxonomic status, and to reveal the laws of organic solvents being toxic to the targeted strains and establish an organic solvent tolerant strains spectrum, and to clarify the mechanism of the strains being tolerant to organic solvents further to highlight the mechanism of cellular adaption to organic solvents and elucidate the laws of lipase production further to improve the lipase production and establish batch fermentation enzyme kinetics model.Finally, the program is to separate and purify the lipases, and determine the molecular weight of the enzymes by N-terminal sequencing method, and to characterize the targeted enzymes. This research will establish a good foundation to further reveal the molecular mechanisms of the lipases being high tolerant to organic solvents and to elucidate structure-activity relationship,mechanism of action, regulation of gene expression. This research is significant to broaden sources of organic solvent tolerant microbes and to obtain novel organic solvent tolerant enzymes, and to develop whole-cell bio-catalysis, bioremediation of environmental pollutants , catalytic asymmetric synthesis and enzymatic synthesis of bio-diesel.