Site-directed and random enzyme immobilization on functionalized membranes: kinetic studies and models

S. Vishwanath, D. Bhattacharyya, W. Huang, L. G. Bachas

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

A comparison of Michaelis-Menten kinetic parameters, Km and Vmax has been made between a randomly immobilized and a site-specifically immobilized β-galactosidase on macroporous membranes. A biotinylated β-galactosidase conjugate (SDBG), was prepared by posttranslational modification of a recombinant fusion protein in E. coli. This conjugate had biotin attached at a specific location on a polypeptide tag fused to the N-terminus of β-galactosidase. Avidin, which has a very strong interaction with biotin, was immobilized on a pre-activated aldehyde modified polysulfone (MPS) membrane; both, commercial biotin-labeled β-galactosidase and the enzyme conjugate mentioned above, (SDBG) were immobilized on this membrane separately. The immobilized β-galactosidase showed a dramatic drop in activity for the directly, randomly immobilized case; a relative activity (RA) of 1.8% compared to the RA of SDBG which was 87.7%. The RA of the commercial biotin-labeled β-galactosidase, immobilized through an avidin-biotin complex as a spacer was 12.6% compared to a corresponding RA of SDBG of 25%. Thus, site-directed immobilization of β-galactosidase offers significant advantages over random immobilization. The diffusion-reaction process which occurs inside the pores of a membrane was modeled to extract intrinsic data from the experiments performed. The values of the effectiveness factor for directly attached SDBG were closely matched with the values of xk x, the reaction-limited reactor length.

Original languageEnglish (US)
Pages (from-to)1-13
Number of pages13
JournalJournal of Membrane Science
Volume108
Issue number1-2
DOIs
StatePublished - Dec 15 1995
Externally publishedYes

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Keywords

  • Immobilized enzymes
  • Kinetics
  • Modeling
  • Polysulfone membranes
  • Site-directed modification of enzyme

ASJC Scopus subject areas

  • Biochemistry
  • Materials Science(all)
  • Physical and Theoretical Chemistry
  • Filtration and Separation

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