메타게놈유래의 저온성 에스터라제 EM2L8의 효소적 특성과 이를 활용한 고지혈증 치료제 키랄소재의 생산
Characterization of a Psychrophilic Metagenome Esterase EM2L8 and Production of a Chiral Intermediate for Hyperlipemia Drug
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Esterase EM2L8 gene isolated from deep sea sediment was expressed in Escherichia coli BL21 (DE3) and the esterase activity of the cell-free extract was assayed using p-nitrophenyl butyrate-spectrophotometric method. Its optimum temperature was 40-45℃ and 45% activity of the maximum activity was retained at 15℃. The activation energy at 15-45℃ was calculated to be 4.9 kcal/mol showing that esterase EM2L8 was a typical cold-adapted enzyme. Enzyme activity was maintained for 6 h and 4 weeks at 30℃ and 4℃, respectively. When each ethanol, methanol, and acetone was added to the reaction mixture to 15% concentration, enzyme activity was maintained. In the case of DMSO, enzyme activity was kept up to 40% concentration. (S)-4-Chloro-3-hydroxy butyric acid is a chiral intermediate for the synthesis of Atorvastatin, a hyperlipemia drug. When esterase EM2L8 (40 U) was added to buffer solution (1.2 mL, pH 9.0) containing ethyl-(R,S)-4-chloro-3-hydroxybutyrate (38 mM), it was hydrolyzed into 4-chloro-3-hydroxy butyric acid with a rate of 6.8 μmole/h. The enzyme hydrolyzed (S)-substrate more rapidly than (R)-substrate. When conversion yield was 80%, e.e.s value was 40%. When DMSO was added, hydrolysis rate increased to 10.4 μmole/h. The plots of conversion yield vs e.e.s in the presence or absence of DMSO were almost same, implying that the reaction enantioselectivity was not changed by the addition of DMSO. Taken together, esterase EM2L8 had high activity and stability at low temperatures as well as in various organic solvents/ aqueous solutions. These properties suggested that it could be used as a biocatalyst in the synthesis of useful pharmaceuticals.
Abstract
Esterase EM2L8 gene isolated from deep sea sediment was expressed in Escherichia coli BL21 (DE3) and the esterase activity of the cell-free extract was assayed using p-nitrophenyl butyrate-spectrophotometric method. Its optimum temperature was 40-45℃ and 45% activity of the maximum activity was retained at 15℃. The activation energy at 15-45℃ was calculated to be 4.9 kcal/mol showing that esterase EM2L8 was a typical cold-adapted enzyme. Enzyme activity was maintained for 6 h and 4 weeks at 30℃ and 4℃, respectively. When each ethanol, methanol, and acetone was added to the reaction mixture to 15% concentration, enzyme activity was maintained. In the case of DMSO, enzyme activity was kept up to 40% concentration. (S)-4-Chloro-3-hydroxy butyric acid is a chiral intermediate for the synthesis of Atorvastatin, a hyperlipemia drug. When esterase EM2L8 (40 U) was added to buffer solution (1.2 mL, pH 9.0) containing ethyl-(R,S)-4-chloro-3-hydroxybutyrate (38 mM), it was hydrolyzed into 4-chloro-3-hydroxy butyric acid with a rate of 6.8 μmole/h. The enzyme hydrolyzed (S)-substrate more rapidly than (R)-substrate. When conversion yield was 80%, e.e.s value was 40%. When DMSO was added, hydrolysis rate increased to 10.4 μmole/h. The plots of conversion yield vs e.e.s in the presence or absence of DMSO were almost same, implying that the reaction enantioselectivity was not changed by the addition of DMSO. Taken together, esterase EM2L8 had high activity and stability at low temperatures as well as in various organic solvents/ aqueous solutions. These properties suggested that it could be used as a biocatalyst in the synthesis of useful pharmaceuticals.
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