Subtilisin alkaline stability

Cunningham, B.C., Wells, J.A. Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure. Prot. Eng. 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

Subtilisins are a family of serine proteases, the most important members of which are subtilisin Carlsberg (from Bacillus licheniformis) and subtilisin BPN (from Bacillus amyloliquefaciens)luoK Both enzymes are alkaline proteases with a pH optimum of 6-9. Because of their industrial importance, both subtilisin Carlsberg and subtilisin BPP have been studied intensively and are produced on a large scale. The crystal structures of both subtilisins have been determined1821. Directed evolution and site-directed mutagenesis and chemical modification of subtilisin were carried out in order to influence the stability, activity and enantioselectivity of the enzyme, in particular in organic solvents11111. As in the case of other enzymes,... 

Last but not least, it should be mentioned that a couple of peptidases have industrial importance. In particular, since subtilisins have a broad substrate specificity and are highly stable at neutral and alkaline pH they are of considerable industrial interest as protein-degrading additives to detergents. These reasons combined with their large data base make subtilisins attractive for protein engineering. Extensive engineering studies have been carried out on the Bacillus subtilins and more than 500 site-directed mutants have been produced to alter specific enzyme properties, such as pH profile, thermal stability or substrate specificity (see e. g. references[37 391). 

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