Applied Biochemistry and Biotechnology

, Volume 55, Issue 1, pp 55–73 | Cite as

Immobilization of manganese peroxidase fromLentinula edodes on azlactone-functional polymers and generation of Mn3+ by the enzyme-polymer complex

  • Anthony C. .Grabski
  • Patrick L. Coleman
  • Gary J. Drtina
  • Richard R. Burgess
Original Articles


Manganese peroxidase (MnP) purified fromLentinula edodes was covalently immobilized on 3M’s azlactone-functional copolymer, 3M EmphazeTM AB1 Biosupport Medium. Tethered MnP is capable of generating Mn3+ from Mn2+ and H2O2. Mn3+, properly chelated, can be used as a nonspecific oxidant of organopollutants. A variety of conditions designed to maximize coupling efficiency while maintaining Mn3+ -generating catalytic activity were tested. Biochemical characteristics of the MnP enzyme, including amino acid composition, pH and temperature stability, and concentration of its Mn2+ substrate, influenced chemical conditions necessary for the coupling reaction. The physical parameters of immobilization reaction time, protein concentration, ionic conditions, pH, and temperature were examined. Results of these experiments indicated maximum coupling efficiency and enzyme activity were achieved by immobilizing at MnP concentrations < 2 mg/mL for at least 2 h using pH 7.0 buffer containing 1.0M sodium sulfate and 1.0 mM Mn2+. Increasing coupling reaction temperature also improved coupling efficiency. A synthesis of these optimized immobilizations yielded MnP coupling efficiencies of 40–50% with 35% of the coupled protein retaining enzymatic activity. Results of MnP immobilizations on nonporous azlactone-functional dispersion polymers more hydrophobic than Emphaze are also reported, and coupling efficiencies > 65% with 100% of the coupled enzyme active have been measured.

Index Entries

Enzyme immobilization manganese peroxidase Mn3+ azlactone polymers Emphaze biocatalyst 


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  1. 1.
    Vandamme, E. J. (1983),Enzyme Microb. Technol. 5, 403–416.CrossRefGoogle Scholar
  2. 2.
    Meijer, E. M., Boesten, W. H. J., Schoemaker, H. E., and Van Balkan J.A. M. (1985),Biocatal. Org. Synthe. 22, 135–156.Google Scholar
  3. 3.
    Carlson, A., Hill, G. C., and Olson, N. F. (1986),Enzyme Microb. Technol. 8, 642–650.CrossRefGoogle Scholar
  4. 4.
    Erlandsson, P., Marie, I., Hansson, L., Isaksson, R., Pettersson, C., and Pettersson, G.. (1990),J. Am. Chem. Soc. 112, 4573–4576.CrossRefGoogle Scholar
  5. 5.
    Wiseman, A. (1991),Top. Enzyme Ferment. Biotechnol. 7, 264–270.Google Scholar
  6. 6.
    Nannipieri, P. and Bollag, J.-M. (1991)J. Environ. Qual. 20, 510–517.CrossRefGoogle Scholar
  7. 7.
    Sariaslani, F. S. (1989),Crit. Rev. Biotechnol. 9, 171–247.CrossRefGoogle Scholar
  8. 8.
    Bumpus, J. A., Mileski, G., Brock, B., Ashbaugh, W., and Aust, S. D. (1988), US Environ. Prot. Agency, Res. Dev., (Rep) EPA, EPA-600/9-88/021, Land Disposal, Rem. Action, Incineration Treat. Hazard. Waste, 355–370.Google Scholar
  9. 9.
    Paszczynski, A., Huynh, V.-B., and Crawford, R. L. (1986),Arch. Biochem. Biophys. 244, 750–765.CrossRefGoogle Scholar
  10. 10.
    Popp, J. L. and Kirk, T. K. (1991),Arch. Biochem. Biophys. 288, 145–148.CrossRefGoogle Scholar
  11. 11.
    Michel, F. C., Dass, B., Grulke, E. A., and Reddy, C. A. (1991),Appl. Environ. Microbiol. 8, 2368–2375.Google Scholar
  12. 12.
    Forrester, I. T., Grabski, A. C., Kelley, B. D., Strickland, W. N., Leatham, G. F., and Burgess, R. R. (1990),Appl. Microbiol. Biotechnol. 33, 359–365.CrossRefGoogle Scholar
  13. 13.
    Gold, M. H. and Glenn, J. K. (1988),Methods in Enzymol. 161, 258–264.Google Scholar
  14. 14.
    Glenn, J. K., Akileswaran, L., and Gold, M. H. (1986),Arch. Biochem. Biophys. 251, 688–696.CrossRefGoogle Scholar
  15. 15.
    Forrester, I. T., Grabski, A. C., Burgess, R. R., and Leatham, G. F. (1988),Biochem. Biophys. Res. Commun. 157, 992–999.CrossRefGoogle Scholar
  16. 16.
    Wariishi, H., Valli, K., and Gold, M. H. (1989),Biochemistry 28, 6017–6023.CrossRefGoogle Scholar
  17. 17.
    Huynh, V.-B., Paszczynski, A., Olson, P., and Crawford, R. L. (1986),Arch. Biochem. Biophys. 250, 186–196.CrossRefGoogle Scholar
  18. 18.
    Kersten, P. J., Tien, M., Kalyanaraman, B., and Kirk, T. K. (1985),J. Biol. Chem. 260, 2609–2612.Google Scholar
  19. 19.
    Waters, W. A. and Littler, J. S. (1965), inOxidation in Organic Chemistry, vol.54, Wiberg, K. B., ed., Academic, New York, pp. 185–241.Google Scholar
  20. 20.
    Citterio, A., Santini, R., Fiorani, T., and Strologo, S. (1989),J. Org. Chem. 54, 2703–2712.CrossRefGoogle Scholar
  21. 21.
    Citterio, A., Fancelli, D., Finzi, C., and Pesce, L. (1989)J. Org. Chem. 54, 2713–2718.CrossRefGoogle Scholar
  22. 22.
    Snider, B. B., Patricia, J. J., and Kates, S. A. (1988),J. Org. Chem. 53, 2137–2143.CrossRefGoogle Scholar
  23. 23.
    Hammel, K. E., Tardone, P. J., Moen, M. A., and Price, L. A. (1989),Arch. Biochem. Biophys. 270, 404–409.CrossRefGoogle Scholar
  24. 24.
    Bumpus, J. A. and Aust, S. D. (1986), inSolving Hazardous Waste Problems, Exner, J., ed., ACS, Washington, pp. 340–349.Google Scholar
  25. 25.
    Siddique, M. H., St Pierre, C. C., Biswas, N., Bewtra, J. K., and Taylor, K. E. (1993),Water Res. 27, 883–890.CrossRefGoogle Scholar
  26. 26.
    Lin, J. E., Wang, H. Y., and Hickey, R. F. (1991),Biotechnol. Bioeng. 38, 273–279.CrossRefGoogle Scholar
  27. 27.
    Ferrer, I., Dezotti, M., and Duran, N. (1991),Biotechnol Lett. 13, 577–582.CrossRefGoogle Scholar
  28. 28.
    Coleman, P. L., Walker, M. M., Milbrath, D. S., Stauffer, D. M., Rasmussen, J. K., Krepski, L. R., and Heilmann, S. M. (1990)J. Chromatog. 512, 345–363.CrossRefGoogle Scholar
  29. 29.
    Hare, P. E. (1977),Methods Enzymol. 47, 3–18.CrossRefGoogle Scholar
  30. 30.
    Rasmussen, J. K., Heilmann, S. M., Krepski, L. R., Smith, H. K. II, Walker, M. M., Stauffer, D. M., Milbrath, D. S., and Coleman, P. L. (1990),Polym. Prepr. 31, 442–443.Google Scholar
  31. 31.
    Friedrich, O. H., Chun, M., and Sernetz, M. (1980),Biotech. Bioeng. 22, 157–176.CrossRefGoogle Scholar
  32. 32.
    Turkova, J., Blaha, K., Horacek, J., Vajcner, J., Frydrychova, A., and Coupek, J. (1981),J. Chromatogr. 215, 165–180.CrossRefGoogle Scholar
  33. 33.
    Zemanova, L., Turkova, J., Capka, M., Nakhapetyan, L. A., Svek, F., and Kalal, J. (1981),Enzyme Microb. Technol. 3, 229–232.CrossRefGoogle Scholar
  34. 34.
    Smalla, K., Turkova, J., Coupek, J., and Hermann, P. (1988),Biotech. Appl. Biochem. 10, 21–31.Google Scholar
  35. 35.
    Paszczynski, A., Huynh, V.-B., and Crawford, R. L. (1985),FEMS Microbiol. Lett. 29, 37–41.CrossRefGoogle Scholar
  36. 36.
    Glenn, J. K. and Gold, M. H. (1985),Arch. Biochem. Biophys. 242, 329–341.CrossRefGoogle Scholar
  37. 37.
    Smith, P. K., Krohn, R. L., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985),Anal. Biochem. 150, 76–85.CrossRefGoogle Scholar
  38. 38.
    Stich, T. M. (1990),Anal. Biochem. 191, 343–346.CrossRefGoogle Scholar
  39. 39.
    Means, G. and Feeney, R. E. (1971), inChemical Modification of Proteins, Holden-Day, San Francisco, pp. 24–54.Google Scholar
  40. 40.
    Cabrai, J. M. S. and Kennedy, J. F. (1991), inProtein Immobilization Fundamentals and Applications, Taylor, R. F., ed., Marcel Dekker, New York, pp. 73–138.Google Scholar
  41. 41.
    Buchholz, K., Duggal, S. K., and Borchert, A. (1979), inCharacterization of Immobilized Biocatalysts, vol. 84, DECHEMA Monographs, Buchholz, K., ed., Verlag Chemie, Weinheim, pp. 169–181.Google Scholar
  42. 42.
    Trevan, M. D. (1980), inImmobilized Enzymes, John Wiley, New York, pp. 11–55.Google Scholar
  43. 43.
    Royer, G. P. (1979), inImmobilized Enzymes: Preparation and Engineering, Recent Advances, Johnson, J. C., ed., Noyes Data Corp., Park Ridge, NJ, p. 127.Google Scholar
  44. 44.
    Messing, R. A. (1978),Adv. Biochem. Eng. 10, 51.CrossRefGoogle Scholar
  45. 45.
    Chen, L. F. and Tsao, G. T. (1976),Biotechnol. Bioeng. 18, 1507–1516.CrossRefGoogle Scholar
  46. 46.
    Sandwick, R. K. and Schray, K. J. (1987),J. Colloid Interface Sci. 115, 130–138.CrossRefGoogle Scholar
  47. 47.
    Lewis, D. and Whateley, T. L. (1988),Biomaterials 9, 71–75.CrossRefGoogle Scholar
  48. 48.
    Boivin, P., Kobos, R. K., Papa, S. L., and Scouten, W. H. (1991),Biotechnol. Appl. Biochem. 14, 155–169.Google Scholar
  49. 49.
    Sjostrom, E.(1981), inWood Chemistry Fundamentals and Applications, Academic, New York, pp. 68–82.Google Scholar
  50. 50.
    Azuma, J.-I. and Tetsuo, K. (1988),Methods Enzymol. 161, 12–31.Google Scholar
  51. 51.
    Baum, G.(1975),Biotechnol. Bioeng. 17, 253–270.CrossRefGoogle Scholar
  52. 52.
    Martinek, K. and Berezin, I. V. (1977),J. Solid-Phase Biochem. 4, 343–385.Google Scholar

Copyright information

© Humana Press Inc 1995

Authors and Affiliations

  • Anthony C. .Grabski
    • 1
    • 4
  • Patrick L. Coleman
    • 2
    • 4
  • Gary J. Drtina
    • 3
    • 4
  • Richard R. Burgess
    • 1
    • 4
  1. 1.Protein Purification FacilityUniversity of Wisconsin Biotechnology CenterMadison
  2. 2.Biosciences Laboratory
  3. 3.Technology Development Laboratory
  4. 4.3M CenterSt. Paul

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