Site-directed and random immobilization of subtilisin on functionalized membranes: Activity determination in aqueous and organic media

S. Viswanath, J. Wang, Leonidas G Bachas, D. A. Butterfield, D. Bhattacharyya

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Kinetic comparisons have been made between a randomly immobilized and a site-specifically immobilized subtilisin BPN' on microfiltration membranes of varying hydrophilicities in both aqueous and organic media. Site-directed mutagenesis was employed to introduce a single cysteine into the amino acid sequence of subtilisin at a location away from the active site. Immobilization of this mutant enzyme was then carried out using the single cysteine residue to orient the active site of the enzyme away from the membrane surface. Kinetic comparison of the immobilized mutant enzyme with the randomly immobilized wild-type enzyme in aqueous media showed an activity enhancement on both hydrophilic silica-containing and hydrophobic poly(ether)sulfone membranes. Higher loading efficiencies were observed for the site-directed enzyme on immobilization. Optimal enzyme loading values were calculated for the randomly immobilized enzyme. An enhancement of activity was also observed for the site-directed immobilized systems using nearly anhydrous hexane as the solvent.

Original languageEnglish (US)
Pages (from-to)608-616
Number of pages9
JournalBiotechnology and Bioengineering
Volume60
Issue number5
DOIs
StatePublished - Dec 5 1998
Externally publishedYes

Fingerprint

Subtilisin
Immobilization
Immobilized Enzymes
Enzymes
Membranes
Cysteine
Catalytic Domain
Subtilisins
Sulfones
Hexanes
Site-Directed Mutagenesis
Hydrophobic and Hydrophilic Interactions
Silicon Dioxide
Ether
Amino Acid Sequence
Mutagenesis
Kinetics
Microfiltration
Hydrophilicity
Enzyme kinetics

Keywords

  • Membrane
  • Oriented immobilization
  • Site-directed mutagenesis
  • Subtilisin

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology

Cite this

Site-directed and random immobilization of subtilisin on functionalized membranes : Activity determination in aqueous and organic media. / Viswanath, S.; Wang, J.; Bachas, Leonidas G; Butterfield, D. A.; Bhattacharyya, D.

In: Biotechnology and Bioengineering, Vol. 60, No. 5, 05.12.1998, p. 608-616.

Research output: Contribution to journalArticle

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