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 journalArticlepeer-review

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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