有机溶剂高耐受性脂肪酶菌株的定向筛选、细胞适应性机制及酶学表征

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.

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。

从微生物中寻找天然耐有机溶剂脂肪酶是突破非水酶学发展中有机溶剂毒害生物催化剂这一技术瓶颈的有效措施。.本项目首先针对已分离的73株葡萄内生菌，通过罗丹明B平板初筛、有机溶剂复筛及遗传稳定性实验，获得一株有机溶剂高耐受性脂肪酶产生菌C2J6，经形态和分子生物学鉴定，确定该菌为黑曲霉（Aspergillus niger）。.其次，探寻了黑曲霉C2J6耐有机溶剂的规律，发现菌株具有广泛的有机溶剂耐受性，能够在醇类、酮类、烷烃、苯环类、酯类、醚类等多种有机溶剂中生长和产酶，但菌体宏观形态、菌体量和酶活有所不同。为揭示菌株在有机溶剂中生长和产酶的细胞适应性机制，对其在Log P值差异显著的环己酮和异丙醇中的细胞微观结构及完整性进行了研究，发现差异显著：异丙醇中的细胞形态结构和细胞膜完整性较好，细胞表面疏水性整体分布在70%-90%，胞外核苷酸含量最多不超过100 μg/mL，异丙醇浓度在1%-2%时，有利于菌体的生长繁殖，菌体对其利用率高达90%，降解产物主要是丙醇，细胞膜中饱和脂肪酸含量增加，不饱和脂肪酸含量降低。而在不同浓度环己酮中，菌株的细胞表面疏水性则降至15%，胞外核苷酸超过100 μg/mL，菌体对其利用率不足13%，降解产物主要是环戊酮，脂肪酸种类和含量变化明显。表明菌体为了抵御有机溶剂的毒害作用，会通过改变细胞结构和组成加强对有机溶剂的适应能力。.第三，优化了菌株产脂肪酶的培养基和培养条件，酶活力提高到60.0u/mL。菌体生长符合Logistic 方程，脂肪酶的合成符合Leudeking-Piret 方程。.最后，表征了所产脂肪酶的结构和性质。鉴定该酶为G9M5R3，理论分子量31.567 kDa，等电点4.28。N-端测序得到了6个氨基酸序列。该酶最适反应温度为40℃，最适反应pH7.0，在35-50℃、pH6.0-7.0时较稳定，在多种有机溶剂中表现出活性。以 p-NPP 为底物的酶动力学研究发现其Km为3.15mmol/L， Vmax为17.92µmol/min•mL。.项目为深入了解菌株耐受有机溶剂的分子机制，解析脂肪酶的基因表达调控及酶的改性和修饰等奠定基础，对于非水相酶催化、环境污染物治理、生物柴油合成等意义重大。