Cloning and characterization of thermostable esterase from Archaeoglobus fulgidus.
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 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-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 rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees 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 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.
Kim
Seung-Bum
SB
Department of Molecular Science and Technology, College of Engineering, Ajou University, Suwon 443-749, Republic of Korea.
Lee
Wonkyu
W
Ryu
Yeon-Woo
YW
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics; D043182:Carboxylesterase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000494:pharmacology; D009930:Organic Chemicals / Q000494:pharmacology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013501:Surface-Active Agents / Q000494:pharmacology; D013696:Temperature
D019843:Archaeal Proteins; D009596:Nitrophenols; D009930:Organic Chemicals; D013501:Surface-Active Agents; D043182:Carboxylesterase; C024836:4-nitrophenol