Cloning, expression and characterization of two thermostable esterases from Aquifex aeolicus VF5

In this study, two novel esterases were identified in hyperthermophilic bacterium Aquifex aeolicus VF5 based on the typical characteristics of lipolytic enzymes. Two genes, Aaeo1 and Aaeo2 encoded 207 and 226 amino acids respectively, were synthesized after optimization according to the P. pastoris codon preferences. Using sequence alignment and phylogenetic analysis, Aaeo1 and Aaeo2, were classified into the bacterial lipolytic enzyme family VIII and V, respectively. Two genes were cloned and expressed in P. pastoris cells, and the recombinant enzymes were purified from culture supernatants by Ni2+-NTA affinity chromatography and characterized. The optimum temperatures of Aaeo1 and Aaeo2 were 80°C and 85°C, respectively, and both of the optimal pH was 8.0. Aaeo1 had the activity preference both to short and long chain p-nitrophenyl esters and triacylglycerides, and the K m and k cat of the best substrate, p-nitrophenyl butyrate (p-NPC4) were 5.11±0.31mM and 16.12±2.32s−1, respectively. While Aaeo2 only displayed high activity towards short chain p-nitrophenyl esters, p-nitrophenyl butyrate (p-NPC4), and exhibited K m and k cat of 0.79±0.03mM and 3.59±1.15s−1, respectively. The activities of both esterases were inhibited by PMSF, DEPC, and EDTA, but were activated by DTT. The two esterases exhibited certain tolerance to various organic solvents and some detergents. These characteristics make them very promising for the industrial application. Graphical abstract

Source: Journal of Molecular Catalysis B: Enzymatic - January 9, 2017 Category: Biochemistry Source Type: research