Subtilisin is a proteolytic enzyme that has considerable utility in food processing and laundry applications for degrading proteinaceous materials. In addition to these applications, subtilisin is also capable of promoting a wide variety of chemical conversions, such as peptide synthesis; the resolution of racemic alcohols, esters and amines; the regioselective acylation of polyhydroxy-compounds such as glycols, steroids and sugars, and the like. Many of these chemical reactions must be carried out in organic media in order to be practically useful. While useful to some extent for conducting such reactions in organic media, subtilisins exhibit relatively low levels of enzymatic activity in organic media. This relative lack of activity in organic solvents severely limits the commercial and industrial applications of subtilisin enzymes in chemical synthesis. In view of this, it would be desirable to be able to produce modified subtilisins which have improved activities in organic media.
Subtilisin has found considerable utility in industrial and commercial applications [see, for example, U.S. Pat. No. 3,623,957 and J. Millet, J. Appl. Bact. 33:207 (1970)]. For example, subtilisins and other proteases are commonly used in detergents for the removal of protein-based stains. They also are used in food processing to accommodate the proteinaceous substances present in the food preparations to their desired impact on the composition.
Subtilisins have also been employed in organic synthesis to catalyze a wide variety of chemical reactions in organic media. For example, the resolution of racemic alcohols employing hydrolases is reviewed by Klibanov in Accts. Chem. Res. Vol. 23:114 (1990). The use of subtilisin for the resolution of racemic amines in anhydrous organic solvent is described by Kitaguchi et al., in J. Am. Chem. Soc. Vol. 111:3094 (1989).
Regioselective acylation of 5.alpha.-androstane-3.beta., 17.beta.-diol, promoted by subtilisin in anhydrous acetone, has been described by Riva and Klibanov in J. Am. Chem. Soc. Vol. 110:3291 (1988). Similarly, subtilisin has been used for the regioselective acylation of the primary hydroxyls of unprotected mono- and oligosaccharides in anhydrous dimethylformamide and pyridine [see Riva et al, J. Am. Chem. Soc. Vol. 110:584 (1988)].
Subtilisin has also been used for the synthesis of peptides in organic solvents [see, for example, Ferjancic et al., Appl. Microbiol. Technol. Vol. 32:65 (1990)]. In addition to solubilizing the reactants, organic solvent alters the relative amidase and esterase activities of the enzyme, providing a catalyst that is well-suited to peptide synthesis by aminolysis [see, for example, Wong and Wang, Experientia 47:1123 (1991)]. The ability of enzymes such as subtilisin to discriminate between optical isomers of a substrate can also be altered by carrying out such reactions in organic solvents. For example, D-amino acid-containing peptides can be prepared using subtilisin in anhydrous tert-amyl alcohol [see Margolin et al., J. Am. Chem. Soc. Vol. 109:7885 (1987)].
Enzymes having characteristics which vary from available stock are required. In particular, enzymes having enhanced catalytic activity and/or stability in non-aqueous media will be useful in extending the range of processes for which such enzymes can be employed. Other characteristics which one may wish to vary relative to available stock include enzyme shelf life and an enzyme's ability to withstand exposure to high temperatures. Because many industrial processes are conducted at temperatures that are above the stability range of many enzymes, highly stable proteases not only will be advantageous to certain industries such as detergent and hide dehairing, that already require stable proteases, but may be useful in industries that currently use chemical means to carry out the reactions described earlier: peptide synthesis, resolution of racemic compounds, acylation reactions, and the like.
Chemical modification of enzymes is known. Such modifications have been carried out primarily to improve the stability of the target enzyme. For example, see Svendsen, I., Carlsberg Res. Commun. 41(5): 237-291 (1976). The ability of chemical modification to impart improved catalytic activity (rather than stability), however, has not been reported. While chemical modification of active-site residues of the serine protease chymotrypsin results in dramatic change in the ratio of esterase-to-amidase activities (Wong and Wang, supra), the modifications drastically reduce the reaction rates.
Chemical modification methods, moreover, suffer from numerous disadvantages, e.g., being dependent upon the presence of amino acid residues convenient for such modification. In addition, these methods are frequently nonspecific in that all accessible residues with common side chains are modified, and such methods are not capable of reaching sterically and/or electronically inaccessible amino acid residues without further processing (e.g., denaturation; once denatured, it is generally not possible to fully reinstitute enzyme activity). To the extent that such methods have the objective of replacing one amino acid residue side chain for another side chain or equivalent functionality, then mutagenesis promises to supplant such methods.
Substantial work has been done to develop variants of subtilisin which exhibit useful new properties, including increased thermostability [Pantoliano et al., Biochemistry Vol. 28:7205 (1989); Pantoliano et al., Biochemistry Vol. 26:2077 (1987); Takagi et al., J. Biol. Chem. Vol. 265:6874 (1990)], to increase the ratio of esterase to amidase activity for peptide synthesis [Abrahmsen et al., Biochemistry Vol. 30:4151 (1991)], to alter the pH dependence of the catalytic activity [Thomas et al., Nature Vol. 318:375 (1985); Wells and Estell, TIBS Vol. 13:291 (1988)], to increase resistance to chemical oxidation [Estell et al., J. Biol. Chem. Vol. 260:6518 (1985)], to increase amidase activity [Takagi et al., J. Biol. Chem. Vol. 263:19592 (1988)], and to alter substrate specificity [Wells et al., Proc. Natl. Acad. Sci. USA Vol. 84:5167 (1987); Carter and Wells, Science Vol. 237:394 (1987)].
Modifying the activity/stability/pH-activity profiles of subtilisins (especially in organic media) would be desirable in making these enzymes more widely applicable in a wide variety of processes. For example, enhancing the enzymatic activity of subtilisins in organic media will make it possible to use such enzymes in reactions which are preferably conducted in organic media, such as, for example, peptide synthesis, and the like.
Mutations of proteases such as subtilisins will hopefully provide a variety of different proteases having modified properties such as improved K.sub.m, k.sub.cat, K.sub.m /k.sub.cat ratio and substrate specificity. These mutations would then allow such enzymes to be tailored for the particular medium to be employed, or the substrate which is anticipated to be present, for example in peptide synthesis, or for hydrolytic processes such as laundry uses.