KMID : 0880220080460010100
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Journal of Microbiology 2008 Volume.46 No. 1 p.100 ~ p.107
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Cloning and Characterization of Thermostable Esterase from Archaeoglobus fulgidus
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Kim Seung-Bum
Lee Won-Kyu Ryu Yeon-Woo
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Abstract
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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, Asp198 and His226. The esterase activity test with various acyl chain length of ¥ñ-nitrophenol resulted that Est-AF showed highest specific activity with ¥ñ-nitrophenylbutyrate (pNPC4) and rapidly decrease with ¥ñ-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 ¥ñ-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with ¥ñ-nitrophenylbutyrate (pNPC4) was 80¡ÆC. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70¡ÆC, and 10% of activity still remains after 3 h of incubation at 90¡ÆC. 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 ¥ìmole/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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KEYWORD
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esterase, extremophile, thermostability, enantioselectivity, Archaeoglobus fulgidus
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