SH-Group Introduction to the N-terminal of Subtilisin and Preparation of Immobilized and Dimeric Enzymes

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Abstract
An SH group was introduced at the N-terminal of subtilisin Carlsberg by reacting with 2-iminothiolane·HCl (Traut reagent) in weakly basic media. The obtained subtilisin-SH was coupled with maleimide connected to an amine polymer (poly(allylamine); PAA), both in soluble and insoluble forms, or connected to the N-terminal of another subtilisin molecule. The immobilized subtilisins and subtilisin dimer thus obtained showed higher specificity towards proteineous substrates, although they had lower activities towards a tetrapeptide substrate. Subtilisins immobilized (conjugated) to PAA were more stable against autolytic and thermal disactivation than the intact enzyme.