Molecular Cloning, Over-expression, Kinetic and Structural Properties of Purified Recombinant Family VII Carboxyl esterases

Abstract

Carboxyl esterases (EC 3.1.1.1) are α/β hydrolases which act on acylglycerols to liberate fatty acids and glycerol. They are amongst the most sort-after biocatalysts due to the potential for application in various industries i.e. food, dairy, pharmaceutical, detergents, textile, biodiesel, and cosmetic industries, a factor that drives the continued identification and kinetic characterization for these enzymes. The carboxyl esterase (CEST) genes from Geobacillus kaustophilus (Est179), B. licheniformis (Est96) and B. pumilus (Est95) were expressed in Escherichia coli. For the purified CESTs, temperature optima assays showed that Est95 and 96 were optimally active at 45 o C while the optimum activity for Est179 was recorded between 45 o C and 55 o C. Furthermore, Est95 and 96 retained more than 95% of activity at temperature ranges between 25-40 ° C while, Est179 retained about 80-100% activity between 25-60 o C. The CESTS were also subjected to substrate specificity assays which revealed that the enzymes showed high affinity for p-nitrophenyl butyrate (ρNP-C4) as demonstrated by the low K m obtained in comparison to other test substrates. However, the ratio for the specificity constants (k cat /K M) revealed ρNP-C3 (propionate) was a preferred substrate for Est95, while ρNP-C2 (acetate) and ρNP-C4 were preferred substrates for Est96 and Est179, respectively. Circular Dichroism (CD) spectroscopy-assisted secondary structure predictions recorded spectra that were consistent with a α-helical content for all three proteins. Intrinsic fluorescence spectroscopy-based comparative analysis of protein tertiary structure revealed a difference in the conformation for the 3 proteins, a possible explanation for the differences in the kinetic properties. Therefore, this study constitutes a report on three family VII CESTs that are optimally active at near-neutral to alkaline pH (6.5-9), mesotable to moderately thermostable and with a preference for short-chain acyl esters. The outcomes of this study forms the basis for future experiments aimed at evaluating CESTs properties for potential application in specific industries.