Skip to main content
SpringerLink
Log in

Mixed Enzymic Reaction—Internal Diffusion Kinetics of Nonuniformly Distributed Immobilized Enzymes

The System Agarose-Micrococcal Endonuclease

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Two types of (CNBr-activated) Agarose-staphylococcal endonuclease derivatives have been prepared, one with the enzyme uniformly distributed in the support, and the other with the enzyme preferentially bound in the most external part of the support particles; the latter were obtained using agarose of very small pores and a high degree of activation. Quantitative enzyme distribution has been determined by scanning fluorescence microscopy. With these insoluble enzyme derivatives, a kinetic study for the hydrolysis of a mononucleotide has been carried out. A simple theoretical model for nonuniformly distributed insoluble enzyme derivatives, which considers only the case of mixed enzymic reaction-internal diffusion kinetics, is proposed. The experimental data agree very well with the predictions of the model.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

asp :

Specific surface area (m2/cm3)

Deff :

Effective diffusion coefficiente of substrate (cm2/s)

d p :

Average pore diameter (nm)

E M :

Amount of enzyme insolublized per unit volume of derivative: average concentration corresponding to a uniform distribution (μM)

E(r) :

Enzyme concentration inside the derivative at a distancer from the center of the particle (μM)

E(ρ):

Enzyme concentration at an adimensional distance ρ from the center of the particle (μM)

f(ρ):

E(ρ)/E M Adimensional enzyme distribution function in the derivative

k′ cat :

Intrinsic catalytic constant for the insolubilized enzyme (min-1)

K′ :

Intrinsic Michaelis constant for the insolubilized enzyme (μM)

r :

Distance from the center of the particle (μm)

R :

Particle radius (μm)

S0 :

Substrate concentration in bulk solution (μM)

S(r) :

Substrate concentration inide the derivative at a distance r from the center of the particle (μM)

v :

Actual reaction rate per unit volume in an insolubilized derivative (μM/min)

Υ:

S(r)/S 0

ρ :

K′ m/S0

ϕ :

R(k′ catE/K′ mDeff)1/ 22. General formulation of Thiele modulus in mixed enzymic reaction-internal diffusion kinetics

ϕ M :

R(k′ cat · EM/K′ mDeff) 21/2. Thiele modulus corresponding to an insolubilized derivative in which the enzyme is homogenously distributed

ϕ(ρ):

R(k ′ cat · E(ρ)/K′Deff)sO58u21/2

ϕsh :

ϕm(1-ρ3 C)-1/2. Thiele modulus corresponding to a distribution of enzyme in a spherical segment

ϕapp :

of a heterogeneously distributed derivative is the Thiele modulus corresponding to an uniformly distributed derivative, which has the same kinetic behavior

η:

Effectiveness factor

η1η0 :

Effectiveness factors corresponding to the limiting cases of first and zero order for enzymic kinetics

ρ:

r/R Adimensional distance from center of particle

ρc = ρsh :

Adimensional distance for which there is no enzyme, in a particle of shell-type nonuniform derivative

ρc :

Adimensional distance for which there is no enzyme, in a linearly distributed derivative

ρeq :

ρc equivalent of nonuniformly insolubilized enzyme, whatever be the type of distribution

ρcq(kin):

ρeq obtained from the fitting of experimental kinetic data to the theoretical model

ρeq(flu):

ρc obtained from scanning fluorescence microscopy┐

References

  1. Bordiert, A., and Buchholz, K. (1979),Bioteclmol. Lett. 1, 15.

    Article  Google Scholar 

  2. Buchholz, K. (1979),Bioteclmol. Lett. 1, 451.

    Article  CAS  Google Scholar 

  3. Guisán, J. M., Fernández, V. M, and Ballesteros, A. (1980), Distribution of Staphylococcal Nuclease Insolubilized on Sepharose, inEnzyme Engineering, vol. 5, Weetall, H. H., and Royer, G. P., eds., Plenum, New York, NY, pp. 435–438.

    Google Scholar 

  4. Buchholz, K., Borchert, A., and Duggal, S. K. (1980), Adsorption of Enzymes in Carriers, inEnzyme Engineering, vol. 5, Weetall, H. H., and Royer, G. P., eds., Plenum, New York, NY, pp. 465–468.

    Google Scholar 

  5. Park, S. H., Lee, S. B., and Ryu, D. D. Y. (1981),Bioteclmol. Bioeng. 23, 2591.

    Article  CAS  Google Scholar 

  6. Juang, H.-D., and Weng, H.-S. (1984)Bioteclmol. Bioeng. 26, 623.

    Article  CAS  Google Scholar 

  7. Borchert, A., and Buchholz, K. (1984)Bioteclmol. Bioeng. 26, 727.

    Article  CAS  Google Scholar 

  8. Kohn, J., and Wilchek, M. (1981),Anal. Biochem. 115, 375.

    Article  CAS  Google Scholar 

  9. Guisán, J. M, Melo, F. V. and Ballesteros, A. (1981),Appl. Biochem. Biotechnol. 6, 37.

    Article  Google Scholar 

  10. Franks, R. G. E. (1972),Modeling and Simulation in Chemical Engineering, John Wiley and Sons, New York, NY.

    Google Scholar 

  11. Aris, R. (1971),Mathematica Theory of Diffusion and Reaction in Permeable Catalysts, vol. 1, Clarendon, Oxford.

    Google Scholar 

  12. Wheeler, A. (1951),Adv. Catal. 3, 249.

    Google Scholar 

  13. Weekman, V. W., and Gorring, R. L. (1965),J. Catalysis 4, 260–270. vol. 44, Mosbach, K., ed. Academic, New York, NY, pp. 19–45.

    Google Scholar 

  14. Porath, J., and Axén, R. (1976), Immobilization of Enzymes to Agar, Agarose and Sephadex Supports, inMethods in Enzymology.

  15. Porath, J., Aspberg, K., Drevin, H., and Axén, R. J. (1973),J. Chromatog. 86, 53.

    Article  CAS  Google Scholar 

  16. Serrano, J. (1981),Ph. D. Thesis, Autonomous University of Madrid, Spain.

    Google Scholar 

  17. Anfinsen, C. B., Cuatrecasas, P., and Taniuchi, H. (1971), Staphylococcal Nuclease, Chemical Properties and Catalysis, inThe Enzymes, vol. 4, Boyer, P. D., ed., Academic, New York, NY, pp. 177–204.

    Google Scholar 

  18. Guisán, J. M., and Ballesteros, A. (1979),J. Solid Phase Biochem. 4, 245.

    Article  Google Scholar 

  19. Cuatrecasas, P., Wilchek, M., and Anfinsen, C. B. (1969),Biochemistry 8, 2277.

    Article  CAS  Google Scholar 

  20. Lash, J., Iwig, M., and Koelsch, R. (1971),Em: J. Biochem. 27, 431.

    Article  Google Scholar 

  21. Ballesteros, A., Guisán, J.M., and Serrano, J. (1982), Influence of the Activation Degree of the Support on the Properties of Agarose-Nuclease, inEnzyme Engineering, vol. 6, Chibata, I., Fukui, A., and Wingard, L. B., Jr., eds., Plenum, New York, NY, pp. 223–224.3, 313.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Instituto de Catálisis, CSIC, 28006, Madrid, Spain

    J. M. Guisan, J. Serrano, F. V. Melo & A. Ballesteros

Authors
  1. J. M. Guisan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Serrano
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F. V. Melo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Ballesteros
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Guisan, J.M., Serrano, J., Melo, F.V. et al. Mixed Enzymic Reaction—Internal Diffusion Kinetics of Nonuniformly Distributed Immobilized Enzymes. Appl Biochem Biotechnol 14, 49–72 (1987). https://doi.org/10.1007/BF02798498

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02798498

Index entries

Search

Navigation