【作者】 张珊珊；

【导师】 刘子铎；

【作者基本信息】 华中农业大学 ， 微生物学， 2014， 硕士

【摘要】 酯酶,即羧酸酯酶(carboxylesterases, EC3.1.1.1),普遍存在于微生物、植物以及动物中。从广义上来说,酯酶是催化酯水解的酶的总称；狭义上来说,酯酶则是催化短链脂肪酸酯降解的一类脂肪酶,在农业生产、医药、洗涤剂、生物柴油的合成等方面具有广泛的应用。本研究以食烷菌总DNA为模板,克隆获得一酯酶基因estB。序列比对结果显示,EstB具有很低的同源性,最高仅有45%,因此可判定其为一种新型的酯酶。此外,EstB包含有存在于大多数酯酶及脂肪酶结构中的催化三联体Ser211-Trp353-Gln385和保守五肽Gly209-X-Ser211-X-Gly213。在酶学性质方面,EstB的最适温度为20℃C,最适pH为8.5,动力学常数Km,kcat以及kcat/Km分别是0.15mM,0.54×103s-1and3.6x103s-1mM-1。与近年来已报道的酯酶相比,EstB不仅表现出极其罕见的冷活性：在0-20℃C温度范围内活性几乎保持不变,0℃C下依然具有97%左右活性；而且具有良好的耐有机溶剂能力：在浓度高达70%的有机溶剂中保温30min后,仍然剩余70%-110%左右的活性。EstB所表现出的冷活性以及耐有机溶剂能力使其在需要极端条件下进行的工业过程中具有广泛被应用的潜力。以烟曲霉为目标菌株,得到一酯酶蛋白EstQ。 EstQ的最适温度为40℃C,最适pH为9.0。为了提高酶的热稳定性,以理性设计为基础,通过定点突变技术构建了三个突变体,分别为A134T,V160T和A134T/V160T,并对其酶学性质进行研究。研究结果显示,A134T和V160T与WT具有相似的温度变化曲线,最适温度均为40℃, A134T/V160T的最适温度则提高至450C。突变体与WT均具有相似的最适pH和pH稳定性曲线。与WT相比,突变体的热稳定性均有不同程度的提高：在45℃C保温30min后,剩余50%-76%的活性,与WT相比,提高20-40%左右。WT, A134T及V160T在50℃C的T1/2分别为5min,10min及15min。突变体A134T/V160T的T1/2则超过了实验时间120min,与WT相比,提高了24倍左右。然而氨基酸的替换却导致EstQ对底物pNPC4的亲和力(1/Km)以及催化效率(kcat/Km)的降低。为了探究突变位点对酶热稳定性影响的机理,本研究对WT以及A134T/V160T的同源结构进行了模拟。根据模拟结果可推断出,EstQ热稳定性的提高可能是由于突变位点的疏水性氨基酸(Ala134, Va1160)被亲水性氨基酸Thr替换后,其与溶液之间以及周围亲疏区域之间形成了更有利于蛋白质稳定的相互作用。除此之外,Thr160与Asn157以及Thr134与水分子之间形成的氢键,也被认为有利于EstQ热稳定性的提高。这项研究结果为酶的体外进化提供了有用的参考依据。更多还原

【Abstract】 Esterases, namely the so-called carboxylesterases (EC3.1.1.1), are widely spread in animals, plants as well as the microorganism. Broadly speaking, all the enzymes catalyzing the ester hydrosis reaction are classfied as esterases. While, to some extent, esterases could be also recognised as lipases as they can catalyze the degradation of fatty acid esters with short chain. Esterases were suggested as potential catalysts used in medicine, detergents, and the production of biodiesel et al.On one hand, a novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax sp. and overexpressed in E. coli DE3(BL21). The sequence alignment results indicated that EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (45%) from Rhodococcus jostii RHA1, suggesting its novelty. EstB also had a catalytic triad (Ser211-Trp353-Gln385) and the classical consensus motif Gly209-X-Ser2"-X-Gly213conserved in most lipases and esterases. EstB, displaying its optimal activity around pH8.5and20℃, was identified to be extremely cold-adaptative retaining more than97%activity between0and20℃. The values of kinetic parameters on p-NP caproate (Km, kCat and Kcat/Km) were0.15mM,0.54×103s-1and3.6×103s-1mM-1, respectively. In addition, compared to most of the reported esterases, EstB showed remarkable stability in several studied organic solvents of high concentrations up to70%with the retention of70%-110%activity. The cold-activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.On the other hand, an esterase, EstQ, was obtained from Aspergillus fumigatus. The optimal activity of EstQ was found to be around pH9.0and40℃. In order to obtain more thermostable esterases, three mutants were constructed by site-directed mutagenesis based on rational design and characterized for further research. Compared to WT, A134T and V160T displaying the similar temperature curve, A134T/V160T exhibited a5℃higher optimal temperature. Similar optimal pH and pH stability curves were also found in WT and the mutants. All the mutants displayed favorable effects on thermostability and the retained activity determined after pre-incubation for30min at45℃were53-76%, about 20-40%higher than that of the WT. Besides that, the half-life of WT, A134T and V160T at50℃were5min,10min and15min, respectively. And a longer T1/2was found in A134T/V160T, more than24times than that of the WT. With an increase in Km of the mutants, a decrease in catalytic efficiency kcat/Km was observed in mutant V160T and A134T/V160T against p-nitrophenyl butyrate. Homology models of WT and A134T/V160T were built to understand the structure-function relationship. The analysis results showed that the improved thermostability may be due to the favorable interaction between the hydrophilic amino acid Thr and solvent as well as the surrounding areas. Besides, the additional hydrogen bonds formed between Thr160and Asn157, as well as Thr134and the water molecules were also believed to display favorble effects on the improvement of thermostability. This study provides useful reference for enzyme evolution in vitro.更多还原