Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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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 (Department of Functional Food and Biotechnology, School of Medical Science, Jeonju University)
  • 정용준 (전주대학교 의과학대학 바이오기능성식품학과)
  • Received : 2019.05.23
  • Accepted : 2019.06.04
  • Published : 2019.06.30
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Abstract

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.

라세믹체 형태의 전구물질 methyl DL-${\beta}$-acetylthioisobutyrate (DL-ester)로부터 captopril 합성과정의 중간물질로 알려져 있는 D-${\beta}$-acetylthioisobutyric acid (DAT)를 효율적으로 제조하기 위해 광학선택적 esterase활성을 가진 신규 미생물을 탐색하였고 활성이 우수한 균주 CJ-317과 균주 CJ-187을 선별하고 동정한 결과, 각각 Klebsiella pneumoniae와 Pseudomonas putida로 동정하였다. 두 균주가 생산하는 esterase의 최적 반응온도와 내열성을 조사한 결과, P. putida CJ-187와 K. pneumoniae CJ-317의 최적활성은 각각 $60^{\circ}C$와 $75^{\circ}C$이었으며 또한 P. putida CJ-187의 경우, $60^{\circ}C$까지 안정한 반면 K. pneumoniae CJ-317은 $80^{\circ}C$에서도 1시간 동안 안정된 내열성 효소의 특성을 보였다. DAT에 의한 최종 산물의 활성 저해도에 있어서도 P. putida CJ-187은 2.5%와 5%의 DAT에 대해 각각 55%와 80%의 저해활성을 보인 반면 K. pneumoniae CJ-317는 각각 35%와 44%의 낮은 저해활성을 보임으로서 K. pneumoniae CJ-317은 captopril 합성의 중간체인 DAT 제조과정에 유용하게 활용할 수 있는 우수한 내열성 광학선택적 esterase 활성을 가지는 신규 균주임을 확인하였다.

Keywords

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Fig. 1. Enzymatic resolution of D-β-acetylthioisobutyric acid (DAT) from DL-β-acetylthioisobutyrate (DL-ester) for synthesis of captopril.

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Fig. 2. Phylogenic tree based on 16S rRNA sequence of Klebsiella pneumoniae CJ-317.

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Fig. 3. Residual esterase activity of the K. pneumoniae CJ-317 (○) and P. putida CJ-187 (●).

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Fig. 4. Effects of reaction temperature on the esterase activity of K. pneumoniae CJ-317 (○) and P. putida CJ-187 (●).

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Fig. 5. Effects of the concentration of D-β-acetylthioisobutyric acid (DAT) on the esterase activity of K. pneumoniae CJ-317 (○) and P. putida CJ-187 (●).

Table 1. Morphological and physiological characteristics of isolates

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Table 2. Thermostability of esterases by various strains

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