Immobilization of Escherichia coli cells expressing 4-oxalocrotonate tautomerase for improved biotransformation of β-nitrostyrene


The enzyme 4-oxalocrotonate tautomerase (4-OT) encoded by the xylH gene is a part of the degradation pathway of aromatic compounds in Pseudomonas putida mt-2. 4-OT was described to catalyze Michael-type addition of acetaldehyde to β-nitrostyrene, and the whole cell system based on recombinantly expressed 4-OT has been developed previously. In this study biocatalytic process based on Escherichia coli whole cells expressing 4-OT was significantly improved using immobilization and ex situ product recovery strategies. Whole cell immobilization in alginate beads was applied in biocatalytic production of 4-nitro-3-phenyl-butanal from β-nitrostyrene and acetaldehyde. Immobilized biocatalyst showed wider pH activity range and could tolerate twofold higher initial concentrations of substrate in comparison to the free whole cell biocatalyst. Beads retained their initial activity over 10 consecutive biotransformations of the model reaction and remained suitable for the repetitive use with 85 % of the initial activity after two months of storage. Bioprocess was further improved by utilizing Amberlite XAD-2 hydrophobic resin for the product recovery. With this modification, the amount of organic solvent was reduced 40-fold in comparison to previously reported method making this biocatalytic process greener.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3


  1. 1.

    Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194

    CAS  Article  Google Scholar 

  2. 2.

    Drauz K, Gröger H, May O (2012) Enzyme catalysis in organic synthesis, Vol. 3, Wiley-VCH

  3. 3.

    Buchholz K, Kasche V, Bornscheuer UT (2012) Biocatalysts and Enzyme Technology second edition ed., Wiley-VCH

  4. 4.

    Bickerstaff G (2000) Immobilization of biocatalysts, 4 ed., Humana Press, New Jersey

    Google Scholar 

  5. 5.

    Lalonde J, Margolin A (2002) Immobilization of enzymes, vol 1, 2nd edn. Wiley-VCH, Weinheim

    Google Scholar 

  6. 6.

    Pedersen S, Christensen M (2000) Immobilized biocatalysts, 2nd edn. Academic Press, Amsterdam

    Google Scholar 

  7. 7.

    End N, Schöning K-U (2004) Immobilized biocatalysts in industrial research and production, vol 242. Springer, Heidelberg

    Google Scholar 

  8. 8.

    Meyer H-P, Birch JR (1999) Production with bacteria and mammalian cells—some experience. Chimia 53:562–565

    CAS  Google Scholar 

  9. 9.

    Park JK, Chang HN (2000) Microencapsulation of microbial cells. Biotechnol Adv 18:303–319

    CAS  Article  Google Scholar 

  10. 10.

    Zhang Y-Z, Prabhu P, Lee J-K (2010) Alginate immobilization of recombinant Escherichia coli whole cells harboring L-arabinose isomerase for L-ribulose production. Bioprocess Biosyst Eng 33:741–748

    CAS  Article  Google Scholar 

  11. 11.

    Li H, Yang T, Gong J-S, Xiong L, Lu Z-M, Li H, Shi J-S, Xu Z-H (2015) Improving the catalytic potential and substrate tolerance of Gibberella intermedia nitrilase by whole-cell immobilization. Bioprocess Biosyst Eng 38:189–197

    CAS  Article  Google Scholar 

  12. 12.

    Kaleem I, Shen H, Lv B, Wei B, Rasool A, Li L (2014) Efficient biosynthesis of glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) in water-miscible ionic liquid by immobilized whole cells of Penicillium purpurogenum Li-3 in alginate gel. Chem Eng Sci 106:136–143

    CAS  Article  Google Scholar 

  13. 13.

    Harayama S, Rekik M, Ngai KL, Ornston LN (1989) Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida. J Bacteriol 171:6251–6258

    CAS  Google Scholar 

  14. 14.

    Zandvoort E, Geertsema EM, Quax WJ, Poelarends GJ (2012) Enhancement of the promiscuous aldolase and dehydration activities of 4-oxalocrotonate tautomerase by protein engineering. ChemBioChem 13:1274–1277

    CAS  Article  Google Scholar 

  15. 15.

    Zandvoort E, Geertsema EM, Baas BJ, Quax WJ, Poelarends GJ (2012) An unexpected promiscuous activity of 4-oxalocrotonate tautomerase: the cis-trans isomerisation of nitrostyrene. ChemBioChem 13:1869–1873

    CAS  Article  Google Scholar 

  16. 16.

    Milhazes N, Calheiros R, Marques MPM, Garrido J, Cordeiro MNDS, Rodrigues C, Quinteira S, Novais C, Peixe L, Borges F (2006) β-Nitrostyrene derivatives as potential antibacterial agents: a structure-property-activity relationship study. Bioorg Med Chem 14:4078–4088

    CAS  Article  Google Scholar 

  17. 17.

    Munoz DS, Hoyos P, Hernaiz MJ, Alcantara AR, Sanchez-Montero JM (2012) Industrial biotransformations in the synthesis of building blocks leading to enantiopure drugs. Biores Technol 115:196–207

    Article  Google Scholar 

  18. 18.

    Narancic T, Radivojevic J, Jovanovic P, Francuski D, Bigovic M, Maslak V, Savic V, Vasiljevic B, O’Connor KE, Nikodinovic-Runic J (2013) Highly efficient Michael-type addition of acetaldehyde to β-nitrostyrenes by whole resting cells of Escherichia coli expressing 4-oxalocrotonate tautomerase. Biores Technol 142:462–468

    CAS  Article  Google Scholar 

  19. 19.

    Radivojevic J, Minovska G, Senerovic L, O’Connor K, Jovanovic P, Savic V, Tokic-Vujosevic Z, Nikodinovic-Runic J, Maslak V (2014) Synthesis of γ-nitroaldehydes containing quaternary carbon in the a-position using a 4-oxalocrotonate tautomerase whole-cell biocatalyst. RSC Advances 4:60502–60510

    CAS  Article  Google Scholar 

  20. 20.

    Sambrook J, Russell WD (2001) Molecular cloning a laboratory manual, vol 3, 3rd edn. Cold Spring Harbour Laboratory Press, Cold Spring Harbour

    Google Scholar 

  21. 21.

    Zandvoort E, Geertsema EM, Baas BJ, Quax WJ, Poelarends GJ (2012) Bridging between organocatalysis and biocatalysis: asymmetric addition of acetaldehyde to ss-nitrostyrenes catalyzed by a promiscuous proline-based tautomerase. Angew Chem Int Ed 51:1240–1243

    CAS  Article  Google Scholar 

  22. 22.

    Miao Y, Geertsema EM, Tepper PG, Zandvoort E, Poelarends GJ (2013) Promiscuous catalysis of asymmetric Michael-type additions of linear aldehydes to β-nitrostyrene by the proline-based enzyme 4- oxalocrotonate tautomerase. ChemBioChem 14:191–194

    CAS  Article  Google Scholar 

  23. 23.

    He Y-C, Liu F, Zhang D-P, Gao S, Li Z-Q, Tao Z-C, Ma C-L (2015) Biotransformation of 1,3-propanediol cyclic sulfate and its derivatives to diols by toluene-permeabilized cells of Bacillus sp. CCZU11-1. Appl Biochem Biotechnol. doi:10.1007/s12010-12014-11457-12012

    Google Scholar 

  24. 24.

    Bornscheuer U, Buchholz K (2005) Highlights in biocatalysis—historical landmarks and current trends. Eng Life Sci 5:309–323

    CAS  Article  Google Scholar 

  25. 25.

    Chen XH, Wang XT, Lou WY, Li Y, Wu H, Zong MH, Smith TJ, Chen XD (2012) Immobilization of Acetobacter sp. CCTCC M209061 for efficient asymmetric reduction of ketones and biocatalyst recycling. Microb Cell Factories 11:119

    Article  Google Scholar 

  26. 26.

    Ban K, Hama S, Nishizuka K, Kaieda M, Matsumoto T, Kondo A, Noda H, Fukuda H (2002) Repeated use of whole-cell biocatalysts immobilized within biomass support particles for biodiesel fuel production. J Mol Cat B Enzymatic 17:157–165

    CAS  Article  Google Scholar 

  27. 27.

    Zajkoska P, Rosenberg M, Heath R, Malone KJ, Stloukal R, Turner NJ, Rebroš M (2015) Immobilised whole-cell recombinant monoamine oxidase biocatalysis. Applied Micro Biotechnol 99:1229–1236

    CAS  Article  Google Scholar 

  28. 28.

    Stark D, Von Stockar U (2003) In situ product removal (ISPR) in whole cell biotechnology during the last twenty years. Adv Biochem Eng Biotechnol 80:149–175

    CAS  Google Scholar 

  29. 29.

    Woodley JM, Bisschops M, Straathof AJJ, Ottens M (2008) Future directions for in situ product removal (ISPR). J Chem Technol Biotechnol 83:121–123

    CAS  Article  Google Scholar 

  30. 30.

    Sardari RR, Dishisha T, Pyo SH, Hatti-Kaul R (2013) Biotransformation of glycerol to 3-hydroxypropionaldehyde: improved production by in situ complexation with bisulfite in a fed-batch mode and separation on anion exchanger. J Biotechnol 168:534–542

    CAS  Article  Google Scholar 

  31. 31.

    Whittall J, Sutton P (eds) (2010) Practical methods for biocatalysis and biotransformations. John Wiley & Sons Ltd, Chichester

    Google Scholar 

  32. 32.

    Held M, Schmid A, Kohler HP, Suske W, Witholt B, Wubbolts MG (1999) An integrated process for the production of toxic catechols from toxic phenols based on a designer biocatalyst. Biotechnol Bioeng 62:641–648

    CAS  Article  Google Scholar 

  33. 33.

    Wang P, He J-Y, Yin J-F (2015) Enhanced biocatalytic production of l-cysteine by Pseudomonas sp. B-3 with in situ product removal using ion-exchange resin. Bioprocess Biosyst Eng 38:421–428

    CAS  Article  Google Scholar 

Download references


This work was supported by Ministry of Education and Science, Republic of Serbia Grant 173048 and 172049. Authors acknowledge COST Action CM1303.

Author information



Corresponding author

Correspondence to Jasmina Nikodinovic-Runic.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 542 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Djokic, L., Spasic, J., Jeremic, S. et al. Immobilization of Escherichia coli cells expressing 4-oxalocrotonate tautomerase for improved biotransformation of β-nitrostyrene. Bioprocess Biosyst Eng 38, 2389–2395 (2015).

Download citation


  • Immobilization
  • Whole cell
  • Biotransformation
  • 4-Oxalocrotonate tautomerase
  • Michael-type addition
  • Alginate