Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus

Abstract

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86∼90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Aspl98 and His226. The esterase activity test with various acyl chain length of ρ-nitrophenol resulted that Est-AF showed highest specific activity with ρ-nitrophenylbutyrate (PNPC4) and rapidly decrease with ρ-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with ρ-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with ρ-nitrophenylbutyrate (pNPC4) was 80°C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70°C, and 10% of activity still remains after 3 h of incubation at 90°C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 µmole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.

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Correspondence to Yeon-Woo Ryu.

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Kim, SB., Lee, W. & Ryu, YW. Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus . J Microbiol. 46, 100–107 (2008). https://doi.org/10.1007/s12275-007-0185-5

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Keywords

  • esterase
  • extremophile
  • thermostability
  • enantioselectivity
  • Archaeoglobus fulgidus