Improving the activity of immobilized subtilisin by site-directed attachment through a genetically engineered affinity tag

Jianquan Wang, D. Bhattacharyya, L. G. Bachas

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

An octapeptide affinity tag, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (termed FLAG), was genetically fused to the C-terminus of subtilisin BPN' (SBT) from Bacillus amyloliquefaciens. The fusion protein SBT-FLAG was immobilized to nonporous polystyrene and silica beads both in a site-directed and a random fashion. Site-directed immobilization was achieved by employing the interaction between protein A and a monoclonal antibody specific for the FLAG peptide, while random immobilization was obtained by using glutaraldehyde as a cross-linking reagent. The activity of the immobilized enzymes was compared. It was found that the site-directed subtilisin had higher catalytic efficiency, kcat/KM, which was more than 7-fold of that of the randomly immobilized enzyme. It was also noted that the site-directly immobilized enzyme had superior storage stability over the homogeneous enzyme.

Original languageEnglish (US)
Pages (from-to)280-285
Number of pages6
JournalFresenius' Journal of Analytical Chemistry
Volume369
Issue number3
DOIs
StatePublished - Jan 1 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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