A newly isolated Klebsiella pneumoniae producing a thermostable stereo-selective esterase for production of D-β-acetylthioisobutyric acid

D-β-Acetylthioisobutyric acid 생산을 위한 내열성 광학선택적 esterase 활성 Klebsiella pneumoniae의 분리

Chung, Yong-Joon

  • Received : 2019.05.23
  • Accepted : 2019.06.04
  • Published : 2019.06.30


The synthesis of captopril as an important chiral drug in commerce needs expensive resolution process of racemic mixture. Microorganisms, producing a thermostable esterase that catalyzes the stereo-selective hydrolysis of methyl DL-${\beta}$-acetylthioisobutyrate (DL-ester) to D-${\beta}$-acetylthioisobutyric acid (DAT) were screened from soils. Among the strains tested, strain No CJ-317 and strain No CJ-187 with highest activity were selected as the best DAT producer. The newly isolated microorganisms were identified respectively, as Klebsiella pneumoniae and Pseudomonas putida. The cell activity of esterase from K. pneumoniae CJ-317 and P. putida CJ-187 were showed an optimal reaction activity at $75^{\circ}C$ and $60^{\circ}C$, respectively. Also the cell activity of K. pneumoniae CJ-317 was stable up to $80^{\circ}C$ for 1 h, while that of P. putida CJ-187 was not over $60^{\circ}C$. By varying the concentration of DAT in the reaction mixture, the cell activity of P. putida CJ-187 showed about 55% and 80% of product inhibition in the presence of 2.5% (w/v) and 5.0% of DAT respectively. K. pneumoniae CJ-317 had less product inhibition than P. putida CJ-187 by about 35% and 44% at the same concentrations respectively. The esterase of newly isolated K. pneumoniae CJ-317 could be useful for the stereo-selective hydrolysis of DL-ester to DAT.


Klebsiella pneumoniae;D-${\beta}$-acetylthioisobutyric acid (DAT);esterase;stereo-selective;thermostable


  1. Chirumamilla RR, Marchant R, and Nigam P. 2001. Captopril and its synthesis from chiral intermediates. J. Chem. Technol. Biotechnol. 76, 123-127.
  2. Cohen N, Eichel WF, Lopresti RJ, Neucom C, and Saucy G. 1976. Synthetic studies on (2R, 4'R, 8'R)-$\alpha$-tocopherol, an approach utilizing side chain synthons of microbiological origin. J. Org. Chem. 41, 3505-3511.
  3. Cushman DW, Cheung HS, Sabo EF, and Ondetti MA. 1977. Design of potent competitive inhibitors of angiotensin converting enzyme: carboxy alkanoyl and mercaptoalkanoyl amino acid. Biochemistry 16, 5484-5491.
  4. Gokul B, Lee JH, Song KB, Panda T, Rhee SK, and Kim CH. 2000. Screening of microorganisms producing esterase for the production of (R)-$\beta$-Acetylmercaptoisobutyric acid from methyl (R,S)-$\beta$-acetylmercaptoisobutyrate. Biotechnol. Bioprocess Eng. 5, 57-60.
  5. Hasegawa J, Ogura M, Kanema H, Kawaharada H, and Watanabe K. 1981. Stereoselective conversion of isobutyric acid to $\beta$-hydroxyisobutyric acid by microorganism. J. Ferment. Technol. 59, 203-208.
  6. Honda K, Kataoka M, and Shimizu S. 2002. Enzymatic preparation of D-$\beta$-acetylthioisobutyric acid and cetraxate hydrochloride using a stereo- and/or regioselective hydrolase, 3,4-dihydrocoumarin hydrolase from Acinetobacter calcoaceticus. Appl. Microbiol. Biotechnol. 60, 288-292.
  7. Lee JH, Gokul B, Song KB, Rhee SK, and Kim CH. 2000. Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-$\beta$-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001. J. Biosci. Bioeng. 90, 684-687.
  8. Ondetti MA and Cushman J. 1981. Inhibition of renin-angiotensin system. A new approach to the theory of hypertension. J. Med. Chem. 24, 355-361.
  9. Ondetti MA, Rubin B, and Cushman DW. 1977. Design of specific inhibitors of angiotensin converting enzyme: new class of orally active antihypertensive agents. Science 196, 441-444.
  10. Ozaki E, Sakimae A, and Numazawa R. 1994. Cloning and expression of Pseudomonas putida gene in Escherichia coli and its use in enzymatic production of D-$\beta$-acetylthioisobutyric acid. Biosci. Biotechnol. Biochem. 58, 1745-1776.
  11. Ozaki E, Sakimae A, and Numazawa R. 1995. Nucleotide sequence of the gene for a thermostable esterase from Pseudomonas putida MR-2068. Biosci. Biotechnol. Biochem. 59, 1204-1207.
  12. Romano D, Bonomi F, Mattos MC, Fonseca TS, Oliveira MCF, and Molinari F. 2015. Esterases as stereoselective biocatalysts. Biotechnol. Adv. 33, 547-565.
  13. Sakimae A, Hosoi A, Kobayashi YH, Ousuga N, Numazawa R, Watanabe I, and Ohnishi H. 1992a. Screening of microorganisms producing D-$\beta$-acetylthioisobutyric acid from methyl D,L-$\beta$-acetylthioisobutyrate. Biosci. Biotechnol. Biochem. 56, 1252-1256.
  14. Sakimae A, Kobayashi Y, Ousuga N, Numazawa R, and Ohnishi H. 1993a. Chemical racemization of methyl L-$\beta$-acetylthioisobutyrate. Biosci. Biotechnol. Biochem. 57, 17-19.
  15. Sakimae A, Numazawa R, and Ohnishi H. 1992b. A newly isolated microorganism producing D-$\beta$-acetylthioisobutyric acid from methyl D,L-$\beta$-acetylthioisobutyrate. Biosci. Biotechnol. Biochem. 56, 1341.
  16. Sakimae A, Ozaki E, Toyama H, Ousuga N, Numazawa R, Muraoka I, Hamada E, and Ohnishi H. 1993b. Process conditions for production of D-$\beta$-acetylthioisobutyric acid from methyl D,L-$\beta$-acetylthioisobutyrate with the cells of Pseudomonas putida MR-2068. Biosci. Biotechnol. Biochem. 57, 782-786.
  17. Shimazaki M, Hasegawa J, Kan K, Nomura K, Nose Y, Kondo H, Ohashi T, and Watanabe K. 1982. Synthesis of captopril starting from an optically active $\beta$-hydroxy acid. Chem. Pharm. Bull. 30, 3139-3146.
  18. Shaw SY, Chen YJ, Ou JJ, and Ho L. 2006. Enzymatic resolution of methyl DL-$\beta$-acetylthioisobutyrate and DL-$\beta$-acetylthioisobutyramide using a stereoselective esterase from Pseudomonas putida IFO12996. J. Mol. Catal. B Enzym. 38, 163-170.