Robert M. Caldwell

Altering the proteolytic activity of subtilisin through protein engineering.

The utility of protein engineering in redesigning the structure of a protein to tailor its functional properties has been firmly established. In particular, the Bacillus serine protease subtilisin has proven to be a useful model protein for examining the use of systematic structural modification to incorporate novel functional properties into an enzyme.1.2 The list of properties that have been altered in subtilisin via such modification includes oxidative ~tability,”~ thermal ~tability,~ alkaline pH stability,h stability in organic ~olvent ,~ substrate specificity in aqueous nucleophile specificity,l2.l3 and pH activity profile.14 In addition to demonstrating the versatility of protein engineering, these studies have also provided valuable insight into the expected consequences of protein structure modification. For example, it is now recognized that while amino acid substitutions generally lead to only slight structural perturbations, these minor changes in structure can cause significant changes in protein function. Furthermore, it is apparent from several studies with subtilisin that multiple amino acid substitutions may additively affect a particular functional property. Provided with this extensive data base of structure-function relationships in subtilisin, thc cngineering of subtilisin for altered proteolytic activity is now being attempted. Increasing the proteolytic activity of subtilisin could boost the enzyme’s effectiveness as an additive to household laundry detergents. Subtilisin sold for use in laundry detergents accounts for the largest share of the worldwide industrial enzyme market with sales estimated for 1991 at

Non-human carbonyl hydrolase mutants, DNA sequences and vectors encoding same and hosts transformed with said vectors

200 million. Furthermore, the utility of subtilisin for peptide synthesis in aqueous systems can be enhanced by decreasing the enzyme’s proteolytic activity. This would alleviate the problem of low synthesis yields obtained due to proteolysis of the peptide product.