Enzymes and especially proteases have for more than 20 years been used in detergent and cleaning compositions for removing or facilitating the removal of unwanted proteinaceous soil, etc. Commercially most important among the enzymes used for these purposes are proteases, especially subtilisin proteases.
Although proteases have been used in the detergent industry for more than 20 years, it is still not exactly known which physical or chemical characteristics are responsible for good washing results. The currently used proteases have been found by isolating proteases from nature and testing them in detergent formulations.
Serine proteases are known as a class of enzymes, including subtilisins, which catalyse the hydrolysis of peptide bonds, and which are characterised by an essential serine residue at the active site (White, Handler and Smith, "Principles of Biochemistry", 5th edition, McGraw-Hill Book Co, New York, 1973, pp 271-272).
The known serine proteases have molecular weights in the 25,000 to 30,000 range. They are inhibited by diisopropyl-fluorophosphonate, but in contrast to metalloproteases, are resistant to ethylenediamine-tetra-acetic acid (EDTA) (although they are stabilised at high temperatures by calcium ion). They hydrolyse simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. The alternative term, alkaline protease, reflects the high pH optimum of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest, 1977, Bacteriological Rev. 41:711-753).
A subtilisin is a serine protease produced by Gram-positive bacteria or fungi. A wide variety of subtilisins have been identified, and the amino acid sequences of at least eight subtilisins have been determined. These include six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesentericopeptidase (Kurihara et al., 1972, J. Biol. Chem. 247:5629-5631; Stahl and Ferrari, 1984, J. Bacteriol. 159:811-819, Jacobs et al., 1985, Nucl. Acids. Res. 13:8913-8926; Nedkov et al., 1985, Biol. Chem. Hoppe-Seyler 366:421-430, Svendsen et al., 1986, FEBS Lett. 196:228-232), and two fungal subtilisins, subtilisin thermitase from Thermoactinomyces vulgaris (Meloun et al., 1985, FEBS Lett. 1983:195-200) and proteinase K from Tritirachium album (Jany and Mayer, 1985, Biol. Chem. Hoppe-Seyler 366:584-492).
Subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisin have been determined which delineate the binding of substrate, transition state, products, three different protease inhibitors, and define the structural consequences for natural variation (Kraut, 1977, Ann. Rev. Biochem. 46:331-358).
Random and site-directed mutations of the subtilisin gene have both arisen from knowledge of the physical and chemical properties of the enzyme and contributed information relating to subtilisin's catalytic activity, substrate specificity, tertiary structure, etc. (Wells et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84; 1219-1223; Wells et al., 1986, Phil. Trans. R. Soc. Lond. A. 317:415-423: Hwang and Warshel, 1987, Biochem. 26:2669-2673; Rao et al., 1987, Nature 328:551-554).
The technique of site-directed mutagenesis of the subtilisin gene has attracted much attention, and various mutations are described in the following patent applications and patents:
EP 0 130 756 (Genentech) (corresponds to U.S. Pat. No. 4,760,025 (Genencor)) relates to site specific or randomly generated mutations in "carbonyl hydrolases" and subsequent screening of the mutated enzymes for various properties, such as Kcat/Km ratio, pH-activity profile, and oxidation stability. Apart from revealing that site-specific mutation is feasible, and that mutation of subtilisin BPN' in certain specified positions, i.e. -1Tyr, 32Asp, 135Asn, 104Tyr, 222Met, 166Gly, 64His, 169Gly, 189Phe, 33Ser, 221Ser, 217Tyr, 156Glu or 152Ala, provide for enzymes exhibiting altered properties, this application does not contribute to solving the problem of deciding where to introduce mutations in order to obtain enzymes with desired properties.
EP 0 214 435 (Henkel) relates to cloning and expression of subtilisin Carlsberg and two mutants thereof (but gives no reason to mutate 158Asp to 158Ser and 161Ser to 161Asp).
International Patent Application WO 87/04461 (Amgen) describes reducing the number of Asn-Gly sequences present in the parent enzyme to obtain mutated enzymes exhibiting improved pH and heat stabilities, with emphasis on removing, mutating, or modifying the 109Asn and the 218Asn residues in subtilisin BPN'.
International Patent Application WO 87/05050 (Genex) discloses random mutation and subsequent screening of a large number of mutants of subtilisin BPN' for improved properties, and describes mutations in positions 218Asn, 131Gly, 254Thr, 166Gly, 116Ala, 188Ser, 126Leu, and 53Ser.
EP 0 251 446 (Genentech) describes how homology considerations at both primary and tertiary structural levels may be applied to identify equivalent amino acid residues whether conserved or not. This information together with the authors' knowledge of the tertiary structure of subtilisin BPN' led the authors to select a number of positions susceptible to mutation with an expectation of obtaining mutants with altered properties. The positions so identified are: 124Met, 222Met, 104Tyr, 152Ala, 156Glu, 166Gly, 169Gly, 189Phe, 217Tyr. Also 155Asn, 21Tyr, 22Thr, 24Ser, 32Asp, 33Ser, 36Asp, 46Gly, 48Ala, 49Ser, 50Met, 77Asn, 87Ser, 94Lys, 95Val, 96Leu, 107Ile, 110Gly, 170Lys, 171Tyr, 172Pro, 197Asp, 199Met, 204Ser, 213Lys, and 221Ser are identified as having influence on various properties of the enzyme, and a number of mutations are exemplified to support these suggestions. In addition to single mutations in these positions the authors also performed a number of multiple mutations. Further the authors identify 215Gly, 67His, 126Leu, 135Leu, and amino acid residues within the segments 97-103, 126-129, 213-215, and 152-172 as having interest, but mutations in these positions are not exemplified.
EP 0 260 105 (Genencor) describes modification of certain properties in enzymes containing a catalytic triad by selecting an amino acid residue within about 15 A from the catalytic triad and replace the selected amino acid residue with another residue. Enzymes of the subtilisin type described in the present specification are specifically mentioned as belonging to the class of enzymes containing a catalytic triad. In subtilisins positions 222 and 217 are indicated as preferred positions for replacement.
Also, it has been shown by Thomas, Russell, and Fersht (Nature (1985) 318, 375-376) that changing 99Asp into 99Ser in subtilisin BPN' changes the pH dependency of the 803-813) the same authors also discuss the substitution of 156Ser in place of 156Glu. Both these mutations are within a distance of about 15 A from the active 64His.
In Nature 328, 496-500 (1987) Russell and Fersht discuss the results of their experiments and present rules for changing pH-activity profiles by mutating an enzyme to obtain changes in surface charge.
At present the following subtilisin proteases are well-known and many of them are marketed in large quantities in many countries of the world, especially for detergent uses:
Subtilisin BPN' or Novo, available from e.g. Sigma, St Louis, USA; PA0 Subtilisin Carlsberg, marketed by Novo Nordisk A/S (Denmark) as ALCALASE (TM) and by IBIS (Holland) as MAXATASE (TM); PA0 A Bacillus lentus subtilisin, marketed by Novo Nordisk A/S (Denmark) as SAVINASE (TM); PA0 SAVINASE (TM) analogues, such as MAXACAL (TM) marketed by IBIS and OPTICLEAN (TM) marketed by Miles Kali Chemie (Germany); PA0 A Bacillus lentus subtilisin, marketed by Novo Nordisk A/S (Denmark) as ESPERASE (TM); PA0 KAZUSASE (TM) marketed by Showa Denko (Japan). PA0 A=Ala=Alanine PA0 V=Val=Valine PA0 L=Leu=Leucine PA0 I=Ile=Isoleucine PA0 P=Pro=Proline PA0 F=Phe=Phenylalanine PA0 W=Trp=Tryptophan PA0 M=Met=Methionine PA0 G=Gly=Glycine PA0 S=Ser=Serine PA0 T=Thr=Threonine PA0 C=Cys=Cysteine PA0 Y=Tyr=Tyrosine PA0 N=Asn=Asparagine PA0 Q=Gln=Glutamine PA0 D=Asp=Aspartic acid PA0 E=Glu=Glutamic acid PA0 K=Lys=Lysine PA0 R=Arg=Arginine PA0 H=His=Histidine PA0 B=Asx=Asp or Asn PA0 Z=Glx=Glu or Gln PA0 H17Q, K27R, H39S, E54D, Y91F, K94R, H120D, H120N, Y167E, Y167F, Y171V, Y192E, Y192F, Y209F, Y214T, H226S, K235L, K235R, K237R, K251E, K251N, Y263F. The mutations E54D and K94R should normally be introduced together. PA0 a--K27R; PA0 b--H17Q+K27R+H39S; PA0 c--E54D+Y91F+K94R; PA0 d--E54D+Y91F+K94R+H120D; PA0 e--E54D+Y91F+K94R+H120N; PA0 f--Y167F+Y171V+Y192F+Y209F+Y214T; PA0 g--K235L+K237R+K251E+Y263F; PA0 h--K235L+K237R+K251N+Y263F; PA0 i--H226S+K235L+K237R+K251N+Y263F; PA0 k--H226S+K235L+K237R+K251E+Y263F; PA0 g'--K235R+K237R+K251E+Y263F; PA0 h'--K235R+K237R+K251N+Y263F; PA0 i'--H226S+K235R+K237R+K251N+Y263F; PA0 k'--H226S+K235R+K237R+K251E+Y263F; PA0 A: K27R; PA0 B: K235R+K237R+K251E+Y263F; PA0 C: E54D+Y91F+K94R: PA0 D: K27R+E54D+Y91F+K94R+Y209F+Y214T+K235R+K237R+K251E+Y236F; PA0 E: K27R+E54D+Y91F+K94R+Y167F+Y171V+Y192F+Y209F+Y214T+K235R+K237R+K251E+Y263F (all Y, K changed); PA0 F: as mutant E with two further mutations adding the charge of a D residue and one adding the charge of an E residue PA0 G: as mutant F with further mutation of histidines at positions 17, 39, 120, 226 to neutral residues PA0 H: as mutant G with the N-terminal chemically modified (blocked) to give a neutral group or a group having a pK.sub.a outside the range from 7 to 12. PA0 l) An enzymatic detergent composition formulated to give a wash liquor ionic strength of 0.03 or less, e.g. 0.02 or less, when used at a rate corresponding to 0.4-0.8 g/l surfactant. PA0 m) An enzymatic detergent composition formulated to give a wash liquor ionic strength of 0.01 or more, e.g. 0.02 or more, when used at a rate corresponding to 0.4-0.8 g/l surfactant.
To be effective, however, such enzymes must not only exhibit activity under washing conditions, but must also be compatible with other detergent components during production and storage.
For example, subtilisins may be used in combination with other enzymes active against other substrates, and the selected subtilisin should possess stability towards such enzymes, and also the selected subtilisin preferably should not digest the other enzymes. Also, the chosen subtilisin should be resistant to the action from other components in the detergent formulation, such as bleaching agents, oxidising agents, etc., in particular an enzyme to be used in a detergent formulation should be stable with respect to the oxidizing power, calcium binding properties, detergency, and pH conditions rendered by the non-enzymatic components in the detergent during storage and in the wash liquor during wash. The ability of the enzyme to remain stable in the wash liquor is often referred to as its washing ability or washability.
Naturally occurring subtilisins have been found to possess properties which are highly variable in relation to their washing power or ability under variations in parameters such as pH and ionic strength. Several of the above marketed detergent proteases, indeed, have a better performance than those marketed about 20 years ago, but for optimal performance each enzyme has its own specific conditions regarding formulation and wash conditions, e.g. pH, temperature, ionic strength (I), active system, builders, etc.
As a consequence it is found that an enzyme possessing desirable properties at low pH and low I may be less attractive at more alkaline conditions or vice versa.
Furthermore, it is desirable to produce and use enzymes which are relatively resistant to changes in pH of wash liquors which occur during washing processes.
It is possible now to construct enzymes having desired amino acid sequences, and as indicated above a fair amount of research has been devoted to designing subtilisins with altered properties. Among the proposals, the technique of producing and screening a large number of mutated enzymes as described in EP 0 130 756 (Genentech) (US Pat. No. 4,760,025 (Genencor)) and International patent application WO 87/05050 (Genex) corresponds to the classical method of isolating native enzymes and screening them for their properties, but is more efficient.
Since a subtilisin protease typically comprises about 275 amino acid residues each capable of being 1 out of 20 possible naturally occurring amino acids, one very serious drawback in that procedure is the very large number of mutations generated that has to be submitted to a preliminary screening prior to further testing of selected mutants showing interesting characteristics at the first screening, since no guidance is indicated in determining which amino acid residues to mutate in order to obtain a desired enzyme with improved properties for the use in question, such as, in this case formulating detergent compositions exhibiting improved washing ability under specified conditions of the wash liquor.
A procedure as outlined in these patent applications will consequently only be slightly better than the traditional random mutation procedures which have been known for years.
The other known techniques relates to changing specific properties, such as hydrolysis rate (EP 0 260 105 (Genencor)) and pH-activity profile (Thomas, Russell, and Fersht, supra). None of these publications relates to changing the wash performance or `washability` of enzymes.
Indeed, no relationship has yet been identified in the art between such well defined properties of an enzyme and the wash performance or `washability` of an enzyme.
In International Patent Application No. WO 89/06279 (PCT/DK 88/00002) (Novo Industri A/S) it is proposed to use the concept of homology comparison to determine which amino acids should be changed and which amino acids should be introduced in order to obtain a desired change in washability.
(Unpublished) European patent application 90306952.4 (Unilever) describes the production and use of mutant subtilisin proteases with altered pI, and detergent compositions containing them.
A remaining problem seems to be that although much research has been directed at revealing the mechanism of protease enzyme action, still only little is known about the factors in structure and amino acid residue combinations that determine the properties of enzymes in relation to their wash performance.
Consequently there still exists a need for further improvement and tailoring of protease enzymes to wash systems, as well as a better understanding of the mechanism of protease action in the practical use of cleaning or detergent compositions.