Improving the Activity of Immobilized Subtilisin by Site-Specific Attachment to Surfaces

Wei Huang, Jianquan Wang, Dibakar Bhattacharyya, Leonidas G. Bachas

Research output: Contribution to journalArticle

72 Scopus citations

Abstract

Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors. The protease subtilisin BPN′ was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties. To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis. This cysteine residue was designed to be away from the active site of the enzyme. The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports. This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase. Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.

Original languageEnglish (US)
Pages (from-to)4601-4607
Number of pages7
JournalAnalytical Chemistry
Volume69
Issue number22
DOIs
StatePublished - Nov 15 1997
Externally publishedYes

ASJC Scopus subject areas

  • Analytical Chemistry

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