A Comparative Study of the Structural and Kinetic Properties of Papain Immobilized on Modified Polysulfone and Modified Cellulose Acetate Membranes

  • Jinbo Lee
  • Dibakar Bhattacharyya
  • Alan D. Butterfield


In order to determine if the chemical nature of the polymeric membrane were a factor in the conformation of bound enzymes, electron paramagnetic resonance (EPR) spin labeling techniques using a short, active-site specific spin label have been employed to study the properties of a model enzyme, papain, immobilized on fully-hydrated modifiied polysulfone membranes, hydrophobic, or modified cellulose acetate membranes, hydrophilic. The properties of the immobilized enzymes are compared with that of the free enzyme in solution. Both polysulfone and cellulose acetate membranes provide some stability for papain, but in general hydrophoilic cellulose acetate membranes give papain properties more similar to those of papain in aqueous solution.

This study reports the development of an effective method to acquire EPR spectra of fullyhydrated, spin labeled enzymes immobilized on polymeric membranes. All these findings indicate that the EPR spin labeling technique provides a powerful method for studying immobilized enzyme systems. Furthermore, understanding the dependence of the properties of the immobilized enzyme on the characteristics of the polymeric membrane may be helpful in the selection of support matrices for particular applications of mnembrane-based enzyme bioreactors, affnity membranes, and biosensors.


Election Paramogmeter Resonance Election Paramogmeter Resonance Spectrum Election Paramogmeter Resonance Signal Spin Label Guanidine Hydrochloride 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    D.A. Butterfield, J. Lee, S. Ganapathi, and D. Bhattacharyya, “Biofunctional membranes IV. Active site structure and stability of an immobilized enzyme, papain, on modified polysulfone membranes studied by electron paramagnetic resonance and kinetics,” J. Membr. Sci.,91:47(1994).CrossRefGoogle Scholar
  2. 2.
    D.A. Butterfield, and J. Lee, “Active site structure and stability of the thiol protease, papain, studied by electron paramagnetic resonance employing a methanethiosulfonate spin label,” Arch. Biochem Biophys., 310:167 (1994).PubMedCrossRefGoogle Scholar
  3. 3.
    P. Zhuang, and D.A. Butterfield, “Structural and enzymatic characterization of papain immobilized onto vinyl alcohol/vinyl butyral copolymer membrane,” J. Membr. Sci., 66:247 (1992).CrossRefGoogle Scholar
  4. 4.
    P. Zhuang, and D.A. Butterfield, “Spin labeling and kinetic studies of a membrane immobilized proteolytic enzyme,” Biotech. Prog., 8:204 (1992).CrossRefGoogle Scholar
  5. 5.
    P. Zhuang, and D.A. Butterfield, “Optimization of covalently coupling enzymes to polymeric membranes,” J. Appl. Polym. Sci., 47:1329 (1993).CrossRefGoogle Scholar
  6. 6.
    J. Lee, M.S. Thesis (University of Kentucky), 1993.Google Scholar
  7. 7.
    O.H. Lowry, N.J. Rosebrough, A.L. Farr, and R.J. Randall, “Protein measurement with the folin phenol reagent,” J. Biol. Chem., 193:265 (1951).PubMedGoogle Scholar
  8. 8.
    L.J. Berliner, “Spin labeling in enzymology: spin labeled enzymes and proteins,” Meth. Enzymol., 49:418 (1978).PubMedCrossRefGoogle Scholar
  9. 9.
    D.A. Butterfield, “Principles of biofunctional membrane structures: alteration in the physical state of one side of the membrane by modulation of the physical state of the opposite side of the membrane,” J. Membr. Sci., 53:3 (1990).CrossRefGoogle Scholar
  10. 10.
    S. Ganapathi, D.A. Butterfield and D. Bhattacharyya, “Flat sheet and hollow fiber membrane bioreactors: A study of the kinetics and active site conformational changes of immobilized papain including sorption studies of reaction constituents,” J. Chem. Technol. Biotechnol., in press (1995).Google Scholar
  11. 11.
    D.S. Clark, and J.E. Bailey, “Characterization of heterogeneous immobilized enzyme subpopulations using EPR spectroscopy,” Biotechnol. Bioeng., 26:231 (1984).PubMedCrossRefGoogle Scholar
  12. 12.
    D.S. Clark, and J.E. Bailey, “Kinetics and EPR spectroscopy studies of immobilized chymotrypsin deactivation,” Ann. N.. Y.. Acad Sci., 434:31 (1984).PubMedCrossRefGoogle Scholar
  13. 13.
    G.A. Marg, G.L. Millhauser, P.S. Skerker, and D.S. Clark, “Application of EPR methods in studies of immobilized enzyme systems,” Ann. N.. Y.. Acad. Sci., 469:253 (1986).PubMedCrossRefGoogle Scholar
  14. 14.
    J.E. Bailey, and D.S. Clark, “Electron paramagnetic resonance studies of immobilized chymotrypsin,” Meths. Enzymol., 135:502 (1987).CrossRefGoogle Scholar
  15. 15.
    D.S. Clark, P.S. Skerker, E.J. Fernadez, and RB. Jagoda, “Spectroscopic studies of structure-function relationships in free and immobilized alcohol dehydrogenase,” Ann. N..Y. Acadi Sci., 506:117 (1987).CrossRefGoogle Scholar
  16. 16.
    Y. Song, G.E. Means, X. Wan, and L.J. Berliner, “A spin label study of immobilized enzyme spectral subpopulations,” Biotechnol. Bioneng., 40:306 (1992).CrossRefGoogle Scholar
  17. 17.
    R. Uibrich, A. Schellenberger, and W. Damerau, “Studies on the thermal inactivation of immobilized enzymes,” Biotechnol. Bioeng., 28:511 (1986).CrossRefGoogle Scholar
  18. 18.
    T. Asakura, H. Yoshimizu, and M. Kakizaki, “An ESR study of spin-labeld silk fibroin membranes and spin-labeled glucose oxidase immobilized in silk fibroin membranes. Biotechnol. Bioeng.,” 35:511 (1990).PubMedCrossRefGoogle Scholar
  19. 19.
    H. Yoshimizu and T. Asakura, “Preparation and Characterization of siIk fibroin powder and its application to enzyme immobilization,” J. Applied Polymer Sci., 40:127 (1990).CrossRefGoogle Scholar
  20. 20.
    S.D. Cordt, K. Vanhoof, J. Hu, G. Maesmans, M. Hendrickx, and P. Tobback, “Thermostabiltiy of soluble and immobilized α- amylase from bacillus licheniformis,” Biotechnol. and Bioeng., 40:396 (1992).CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1996

Authors and Affiliations

  • Jinbo Lee
    • 1
  • Dibakar Bhattacharyya
    • 2
    • 3
  • Alan D. Butterfield
    • 1
    • 3
  1. 1.Departments of ChemistryUniversity of KentuckyLexingtonUSA
  2. 2.Departments of Chemical and Materials EngineeringUniversity of KentuckyLexingtonUSA
  3. 3.Center of Membrane SciencesUniversity of KentuckyLexingtonUSA

Personalised recommendations