Characterization And Site-directed Mutagenesis Of The Esterases From Alcanivorax Sp. And Aspergillus Fumigatus

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.