Kinetic studies of site-specifically and randomly immobilized alkaline phosphatase on functionalized membranes

Shekhar K. Vishwanath, Cynthia R. Watson, Wei Huang, Leonidas G Bachas, Dibakar Bhattacharyya

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

A comparison of enzyme activities has been made between a site-specifically immobilized and a randomly immobilized bacterial alkaline phosphatase (BAP) on macroporous membranes. An octapeptide tag (FLAG) was attached at the N-terminus of alkaline phosphatase by recombinant DNA techniques (gene fusion) to yield BAP that is modified in a site-directed fashion (SDBAP). The corresponding antibody (antiFLAG) was immobilized on an aldehyde-modified polyethersulfone (MPS) membrane via protein A. Immobilization of SDBAP on this membrane result in a membrane-protein A-antiFLAG-SDBAP linkage. This site-specifically immobilized enzyme demonstrated a relative activity (RA), defined as the ratio of immobilized activity (Vmax) to the corresponding homogeneous enzymatic activity, of 85% as compared with the randomly immobilized BAP which had an RA of 0·8%. BAP, when chemically conjugated to the FLAG peptide and immobilized via anti-FLAG and protein A on the MPS membrane, showed an RA of only 1·9%, demonstrating the effectiveness of site-directed immobilization. SDBAP was also immobilized on the MPS membrane in the absence of protein A. In this case, the RA dropped to 22%, further explaining the effectiveness of ordered immobilizations as compared with random immobilizations. The ratio of immobilized enzyme activity to the activity in the absence of added phosphate inhibitor for the immobilized BAP was three-fold higher than the corresponding homogeneous ratio, showing a reduction in product inhibition for the immobilized enzyme.

Original languageEnglish (US)
Pages (from-to)294-302
Number of pages9
JournalJournal of Chemical Technology and Biotechnology
Volume68
Issue number3
DOIs
StatePublished - 1997
Externally publishedYes

Fingerprint

Phosphatases
phosphatase
Alkaline Phosphatase
Staphylococcal Protein A
membrane
Membranes
Immobilization
kinetics
Immobilized Enzymes
immobilization
Kinetics
Proteins
protein
Enzyme activity
enzyme activity
Membrane Proteins
Enzymes
enzyme
Enzyme inhibition
Recombinant DNA

Keywords

  • Immobilized enzymes
  • Kinetics
  • Polymeric membranes
  • Site-specific modification of enzyme

ASJC Scopus subject areas

  • Chemistry(all)
  • Chemical Engineering(all)
  • Biotechnology
  • Bioengineering

Cite this

Kinetic studies of site-specifically and randomly immobilized alkaline phosphatase on functionalized membranes. / Vishwanath, Shekhar K.; Watson, Cynthia R.; Huang, Wei; Bachas, Leonidas G; Bhattacharyya, Dibakar.

In: Journal of Chemical Technology and Biotechnology, Vol. 68, No. 3, 1997, p. 294-302.

Research output: Contribution to journalArticle

Vishwanath, Shekhar K. ; Watson, Cynthia R. ; Huang, Wei ; Bachas, Leonidas G ; Bhattacharyya, Dibakar. / Kinetic studies of site-specifically and randomly immobilized alkaline phosphatase on functionalized membranes. In: Journal of Chemical Technology and Biotechnology. 1997 ; Vol. 68, No. 3. pp. 294-302.
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