Characterization of a Psychrophilic Metagenome Esterase EM2L8 and Production of a Chiral Intermediate for Hyperlipemia Drug

메타게놈유래의 저온성 에스터라제 EM2L8의 효소적 특성과 이를 활용한 고지혈증 치료제 키랄소재의 생산

  • Jung, Ji-Hye (Division of Biotechnology, The Catholic University of Korea) ;
  • Choi, Yun-Hee (Division of Biotechnology, The Catholic University of Korea) ;
  • Lee, Jung-Hyun (Marine Biotechnology Research Centre, KORDI) ;
  • Kim, Hyung-Kwoun (Division of Biotechnology, The Catholic University of Korea)
  • Received : 2009.03.19
  • Accepted : 2009.05.25
  • Published : 2009.06.28


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 $40-45^{\circ}C$ and 45% activity of the maximum activity was retained at $15^{\circ}C$. The activation energy at $15-45^{\circ}C$ 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 $30^{\circ}C$ and $4^{\circ}C$, 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 $6.8\;{\mu}mole/h$. 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 $10.4\;{\mu}mole/h$. 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.


Esterase EM2L8;metagenome;cold-adapted enzyme;chiral drug;organic solvent


Supported by : 경기도지역협력연구센터, 가톨릭대학교


  1. Daniel, R. 2004. The soil metagenome-a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 15: 199-204
  2. Ferre, M., F. Martinez-Abarca, and P. N. Golyshin. 2005. Mining genomes and 'metagenomes' for novel catalysts. Curr. Opin. Biotechnol. 16: 588-593
  3. Joseph, B., P. W. Ramteke, and G. Thomas. 2008. Cold active microbial lipases: Some hot issues and recent developments. Biotechnol. adv. 26: 457-470
  4. Choi, Y. H., K. N. Uhm, and H. K. Kim. 2008. Biochemical characterization of Rhodococcus erythropolis N'4 nitrile hydratase acting on 4-chloro-3-hydroxybutyronitrile. J. Molecul. Catal. B: Enzym. 55: 157-163
  5. Gupta, R., N. Gupta, and P. Rathi. 2004. Bacterial lipases: an overview of production, purification and biochemical properties. Appl. Microbiol. Biotechnol. 64: 763-781
  6. Park, H. J., J. H. Jeon, S. G. Kang, J. H. Lee, S. A. Lee, and H. K. Kim. 2007. Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome. Protein Expr Purif. 52: 340-347
  7. Jeon, J. H., J. T. Kim, Y. J. Kim, H. K. Kim, H. S. Lee, S. G. Kang, S. J. Kim, and J. H. Lee. 2009. Cloning and characterization of a new cold-active lipase from a deep-sea sediment metagenome. Appl. Microbiol. Biotechnol. 81: 865-874
  8. Russell, N. 2000. Toward a molecular understanding of cold activity of enzymes from psychrophiles. Extremophiles 4: 83-90
  9. Kulakova, L., A. Galkin, T. Nakayama, T. Nishino, and N. Esaki. 2004. Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of GlyPro substitution near the active site on its catalytic activity and stability. Biochim. Biophys. Acta 1696: 59-65
  10. Ryu, H. S., H. K. Kim, W. C. Choi, M. H. Kim, S. Y. Park, N. S. Han, T. K. Oh, and J. K. Lee. 2006. New cold-adapted lipase from Photobacterium lipolyticum sp. nov. that is closely related to filamentous fungal lipases. Appl Microbiol Biotechnol. 70: 321-326
  11. Demirjian, D. C., F. Moris-Varas, and C. S. Cassidy. 2001. Enzymes from extremophiles. Curr. Opin, Chem. Biol. 5: 144-151
  12. Elend, C., C. Schmeisser, C. Leggewie, P. Babiak, J. D. Carballeira, H. L. Steele, J. L. Reymond, K. E. Jaeger, and W. R. Streit. 2006. Isolation and biochemical characterization of two novel metagenome-derived esterases. Appl. Environ. Microbiol. 72: 3637-3645
  13. Kim, Y. J., G. S. Choi, S. B. Kim, G. S. Yoon, Y. S. Kim, and Y. W. Ryu. 2006. Screening and characterization of a novel esterase from a metagenomic library. Protein Expres. Purif. 45: 315-323
  14. Streit, W. R., R. Daniel, and K. E. Jaeger. 2004. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms. Curr. Opin. Biotechnol. 15: 285-290
  15. Jaeger, K. E. and T. Eggert. 2002. Lipases for biotechnology. Curr. Opin. Biotehnol. 13: 390-397
  16. Park, H. J., K. N. Uhm, and H. K. Kim, 2008. R-Stereoselective amidase from Rhodococcus erythrophlis No.7 acting on 4-chloro-3-hydroxybutyramide. J. Microbiol. Biotechnol. 18: 552-559
  17. Gerday, C., M. Aittaleb, M. Bentahir, J. P. Chessa, P. Claverie, T. Collins, S. D'Amico, J. Dumont, G. Garsoux et al. 2000. Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol. 18: 103-107