Purification, immobilization and kinetic characterization of G-x-S-x-G esterase with short chain fatty acid specificity from Lysinibacillus fusiformis AU01

Abstract

Abstract An esterase from Lysinibacillus fusiformis AU01 was purified to homogeneity with specific activity of 1.1 × 105 U/mg and 52.93% recovery. From SDS-PAGE and MS analysis the molecular mass of the esterase was found to be 29 kDa. Amino acid sequence alignments revealed the presence of classical Gly-His-Ser-Met-Gly pentapeptide (G-X-S-X-G motif). Complete loss of enzyme activity in the presence of PMSF confirmed the presence of active site serine residue. The AU01 esterase was found to be resistant to proteolytic cleavage by its native protease. The purified esterase was immobilized onto celite545 with 3-aminopropyltriethoxysilane as linker molecule. The free and immobilized esterase showed maximum activity at 40 and 45 °C respectively at pH 9.0. Investigating thermal denaturation kinetics and thermodynamic parameters revealed that the immobilization improved the thermal stability of esterase by twofold increase in the half-life and D-value. The free and immobilized esterase showed similar stability profile in the presence of organic solvents. Immobilized esterase retained about 60% of their initial activities after 10 days incubation at 25 °C and retained 70% of its initial activity after 4 reuses. The immobilized enzyme showed higher kcat value when compared to free enzyme. Among the tested substrates, the catalytic efficiency kcat/KM of free (52.40 s−1 mM−1) and immobilized enzyme (47.93 s−1 mM−1) was found to be highest for shorter chain length substrates pNPC4 and pNPC5. This enzyme may be developed as potential candidate for selectively enriching short-chain fatty acid flavour esters.