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Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method

Abstract

To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E app). The E app closely matches the value of true enantioselectivity (E true) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.

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References

  1. 1.

    Agranat I, Caner H, Caldwell J (2002) Putting chirality to work: the strategy of chiral switches. Nat Rev Drug Discov 1:753–768

  2. 2.

    Dewanti AR, Mitra B (2003) A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida. Biochemistry 42:12893–12901

  3. 3.

    Dewanti AR, Xu Y, Mitra B (2004) Role of glycine 81 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate specificity and oxidase activity. Biochemistry 43:10692–10700

  4. 4.

    Hummel W, Kula MR (1989) Dehydrogenases for the synthesis of chiral compounds. Eur J Biochem 184:1–13

  5. 5.

    Wada Y, Iwai S, Tamura Y, Ando T, Shinoda T, Arai K, Taguchi H (2008) A new family of d-2-hydroxyacid dehydrogenases that comprises d-mandelate dehydrogenases and 2-ketopantoate reductases. Biosci Biotechnol Biochem 72:1087–1094

  6. 6.

    Wohlgemuth R (2011) Biocatalysis—key to sustainable industrial chemistry. Curr Opin Biotechnol 21:713–724

  7. 7.

    Voss CV, Gruber CC, Kroutil W (2008) Deracemization of secondary alcohols through a concurrent tandem biocatalytic oxidation and reduction. Angew Chem Int Edit 47:741–745

  8. 8.

    Takahashi E, Nakamichi K, Furui M (1995) R-(−)-mandelic acid production from racemic mandelic acids using Pseudomonas polycolor IFO 3918 and Micrococcus freudenreichii FERM-P 13221. J Ferment Bioeng 80:247–250

  9. 9.

    Schrittwieser JH, Sattler J, Resch V, Mutti FG, Kroutil W (2011) Recent biocatalytic oxidation–reduction cascades. Curr Opin Chem Biol 15:249–256

  10. 10.

    Tsuchiya S, Miyamoto K, Ohta H (1992) Highly efficient conversion of (R)-mandelic acid to its (R)-(−)-enantiomer by combination of enzyme-mediated oxidation and reduction. Biotechnol Lett 14:1137–1142

  11. 11.

    Miyamoto K, Ohta H (1992) Enantioselective oxidation of mandelic acid using a phenylmalonate metabolizing pathway of a soil bacterium: Alcaligenes bronchisepticus KU 1201. Biotechnol Lett 14:363–366

  12. 12.

    Takahashi E, Nakamichi K, Furui M, Mori T (1995) R-(−)-mandelic acid production from racemic mandelic acids by Pseudomonas polycolor with asymmetric degrading activity. J Ferment Bioeng 79:439–442

  13. 13.

    He Y-C, Xu J-H, Pan J, Ouyang L-M, Xu Y (2008) Preparation of (R)-(−)-mandelic acid and its derivatives from racemates by enantioselective degradation with a newly isolated bacterial strain Alcaligenes sp. ECU0401. Bioproc Biosyst Eng 31:445–451

  14. 14.

    Huang H-R, Xu J-H, Xu Y, Pan J, Liu X (2005) Preparation of (S)-mandelic acids by enantioselective degradation of racemates with a new isolate Pseudomonas putida ECU1009. Tetrahedron Asymmetry 16:2113–2117

  15. 15.

    Navarro-Gonzalez R, Negron-Mendoza A, Albarran G (1991) Analysis of keto acids as their methyl esters of 2,4-dinitrophenylhydrazone derivatives by gas chromatography and gas chromatography-mass spectrometry. J Chromatogr A 587:247–254

  16. 16.

    Fuchs M, Engel J, Campos M, Matejec R, Henrich M, Harbach H, Wolff M, Weismüller K, Menges T, Heidt M, Welters I, Krüll M, Hempelmann G, Mühling J (2009) Intracellular alpha-keto acid quantification by fluorescence-HPLC. Amino Acids 36:1–11

  17. 17.

    Chen CS, Fujimoto Y, Girdaukas G, Sih CJ (1982) Quantitative analyses of biochemical kinetic resolutions of enantiomers. J Am Chem Soc 104:7294–7299

  18. 18.

    Li GY, Huang KL, Jiang YR, Ding P (2007) Analysis of the content of benzoylformic acid in mixture by colorimetry. J Anal Sci 23:734–736

  19. 19.

    Pollard DJ, Woodley JM (2007) Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol 25:66–73

  20. 20.

    Baumann M, Stürmer R, Bornscheuer UT (2001) A high-throughput-screening method for the identification of active and enantioselective hydrolases. Angew Chem Int Edit 40:4201–4204

  21. 21.

    Wang B, Tang XL, Ren GF, Liu J, Yu HW (2009) A new high-throughput screening method for determining active and enantioselective hydrolases. Biochem Eng J 46:345–349

  22. 22.

    Hwang BY, Kim BG (2004) High-throughput screening method for the identification of active and enantioselective omega-transaminases. Enzyme Microb Technol 34:429–436

  23. 23.

    Lin Z-J, Zheng R-C, Lei L-H, Zheng Y-G, Shen Y-C (2011) Ferrous and ferric ions-based high-throughput screening strategy for nitrile hydratase and amidase. J Microbiol Methods 85:214–220

  24. 24.

    Zheng R-C, Zheng Y-G, Shen Y-C (2007) A screening system for active and enantioselective amidase based on its acyl transfer activity. Appl Microbiol Biotechnol 74:256–262

  25. 25.

    Saczk AA, Okumura LL, Firmino de Oliveira M, Boldrin Zanoni MV, Ramos Stradiotto N (2005) Rapid and sensitive method for the determination of acetaldehyde in fuel ethanol by high-performance liquid chromatography with UV–Vis detection. Anal Bioanal Chem 381:1619–1624

  26. 26.

    Medvedovici A, Albu F, Farca A, David V (2004) Validated HPLC determination of 2-[(dimethylamino)methyl]cyclohexanone, an impurity in Tramadol, using a precolumn derivatisation reaction with 2,4-dinitrophenylhydrazine. J Pharm Biomed Anal 34:67–74

  27. 27.

    Demirjian DC, Shah P, Moris-Varas F (1999) Screening for novel enzymes. Top Curr Chem 200:1–29

  28. 28.

    Moris-Varas F, Shah A, Aikens J, Nadkarni NP, Rozzell JD, Demirjian DC (1999) Visualization of enzyme-catalyzed reactions using pH indicators: rapid screening of hydrolase libraries and estimation of the enantioselectivity. Bioorg Med Chem 7:2183–2188

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Acknowledgments

This work was supported by the Fund of the National High Technology Research and Development Program of China (863 Program) (No. 2011AA02A210), the Major Basic Research Development Program of China (973 Project) (No. 2009CB724704) and Natural Science Foundation of Zhejiang Province (No. Z4090612 and No. Y4080334).

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Correspondence to Yu-Guo Zheng.

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Xue, Y., Wang, W., Wang, Y. et al. Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method. Bioprocess Biosyst Eng 35, 1515–1522 (2012). https://doi.org/10.1007/s00449-012-0741-1

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Keywords

  • High-throughput screening
  • α-Hydroxyacid
  • α-Hydroxyacid dehydrogenase
  • Enantioselectivity
  • Pseudomonas aeruginosa