, Volume 6, Issue 3, pp 191–201 | Cite as

Immobilisation of the D-2-haloacid dehalogenase fromPseudomonas putida strain AJ1/23

  • Karen Parker
  • John Colby


A variety of procedures were used to immobilise D-2-haloacid dehalogenase. Natural polymer supports were insufficiently robust to withstand degradation by high concentrations of 2-chloropropionate. The best results were obtained with enzyme covalently attached to controlled-pore glass via a diazo linkage. The immobilisation procedure was optimised with respect to enzyme loading, pH, temperature and the presence of substrate during attachment. Immobilisation significantly modified the kinetics of the enzyme, in particular improving its temperature stability and ability to withstand mildly alkaline conditions where it is most active. The performance of the immobilised preparation in batch and plug-flow bioreactors was assessed. Biocatalyst half-life in plug-flow reactors was better than in batch bioreactors whereas effectiveness factors, although concentration dependent in the batch reactor, were similar at least with 200 mM D,L-2-CPA as substrate.

Key words

Chiral biotransformation chloropropionic acid dehalogenase enzyme immobilisation haloacid dehalogenase 


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  1. Adamich M, Voss HF and Dennis EA (1978) Cobra venom phospholipase A2 immobilized to porous glass beads. Archives of Biochemistry and Biophysics 189 (2): 417–423Google Scholar
  2. Carleysmith SW, Dunnill P & Lilly MD (1980) Kinetic behavior of immobilized penicillin acylase. Biotechnology and Bioengineering XXII: 735–756Google Scholar
  3. Fadda MB, Dessi MR, Rinaldi A & Satta G (1989) Sandy alumina as substrate for economic and highly efficient immobilization of b-glucosidase. Biotechnology and Bioengineering 33 (Communications to the editor): 777–779Google Scholar
  4. Garcia III A, Oh S & Engler CR (1989) Cellulase immobilization on Fe3O4 and characterization. Biotechnology and Bioengineering 33: 321–326Google Scholar
  5. Johansson A-C & Mosbach K (1974) Acrylic copolymers as matrices for the immobilization of enzymes. II. The effect of a hydrophobic microenvironment on enzyme reactions studied with alcohol dehydrogenase immobilized to different acrylic copolymers. Biochimica et Biophysica Acta 370: 348–353Google Scholar
  6. Kierstan M & Bucke C (1977) The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Biotechnology and Bioengineering XIX: 387–397Google Scholar
  7. Larson E, Howlett B & Jagendorf A (1986) Artificial reductant enhancement of the Lowry method for protein determination. Analytical Biochemistry 155: 243–248Google Scholar
  8. Mosbach K (Ed) (1976) Immobilized Enzymes. Methods in Enzymology XLIV. Academic Press Inc, New YorkGoogle Scholar
  9. Naoi M, Naoi M & Yagi K (1978) Immobilized D-amino acid oxidase. Biochimica et Biophysica Acta 523: 19–26Google Scholar
  10. Pifferi PG, Tramontini M & Malacarne A (1989) Immobilization of endo-polygalacturonase from Aspergillus niger on various types of macromolecular supports. Biotechnology and Bioengineering 33: 1258–1266Google Scholar
  11. Smith J, Harrison K, Colby J & Taylor SC (1989) Determination of D-2-halopropionate dehaslogenase activity fromPseudomonas putida strain AJ1/23 by ion chromatography. FEMS Microbiology Letters 57: 71–4Google Scholar
  12. Smith J, Harrison K & Colby J (1990) Purification and characterisation of D-2-haloacid dehalogenase fromPseudomonas putida strain AJ1/23. Journal of General Microbiology 136: 881–6Google Scholar
  13. Srere PA & Uyeda K (1976) In: Mosbach K (Ed) Methods in Enzymology 44: 11–19, Academic Press Inc, New YorkGoogle Scholar
  14. Wang DIC, Cooney CL, Demain AL, Dunnill P, Humphrey AE & Lilly MD (Eds) (1979) Fermentation & Enzyme Technology. John Willey & Sons, New YorkGoogle Scholar
  15. Weetall H (1976) In: Mosbach K (Ed) Methods in Enzymology 44: 134–148. Academic Press Inc, New YorkGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Karen Parker
    • 1
  • John Colby
    • 1
  1. 1.School of Health SciencesUniversity of SunderlandSunderlandUK

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