WO1993017086A1

WO1993017086A1 - A detergent containing a protease and a protease inhibitor and novel inhibitors for use therein

Info

Publication number: WO1993017086A1
Application number: PCT/DK1993/000063
Authority: WO
Inventors: Lone Kierstein Nielsen; Torben Halkier; Ib Groth Clausen
Original assignee: Novo Nordisk A/S
Priority date: 1992-02-25
Filing date: 1993-02-23
Publication date: 1993-09-02
Also published as: DK23692D0

Abstract

Detergent composition comprising a protease and, as a protease inhibitor, plasminostreptin or a variant thereof, wherein the ratio of the dissociation constant to the protease concentration is in the range of from 0.006 to 6.

Description

A DETERGENT CONTAINING A PROTEASE AND A PROTEASE INHIBITOR AND NOVEL INHIBITORS FOR USE THEREIN

FIELD OF INVENTION

The present invention relates to an improved detergent composition comprising a protease (particularly a subtilisin) and a reversible protease inhibitor, as well as to a detergent additive comprising such a protease and inhibitor. The invention also relates to novel inhibitor variants for use in said detergent, a DNA construct comprising a nucleotide sequence coding for the inhibitor variant, a host cell transformed with the DNA construct and a method of producing the modified inhibitor.

BACKGROUND OF THE INVENTION

Proteases, especially subtilisins, are widely used as ingredients in commercial detergents. A major problem in formulating protease-containing detergents, especially liquid detergents, is that of ensuring enzyme stability during storage.

The prior art has dealt extensively with improving the storage stability. For example, JP-A 62-269689 demonstrates improvement of the stability of a protease (e.g. a subtilisin) in a liquid detergent by incorporation of a protease inhibitor of protein type. As stated in this publication, the protease inhibitor should ideally show essentially no inhibiting effect under dilute washing conditions, i.e. when the detergent is in use.

As described in PCT/DK91/00279, it has been found that in the known detergents containing protease and inhibitor, the protease is almost totally inhibited under dilute washing conditions. It was also found that by a suitable choice of inhibitor for a given protease, it is possible to essentially avoid inhibition at the dilute conditions of washing, while still achieving effective enzyme stabilization in the detergent during storage. Essentially, it was found that the ratio of the dissociation constant to the protease concentration should be in the range from 0.006 to 6, or that the dissociation constant should be in the range from 0.05 to 50 μM. SUMMARY OF THE INVENTION

The present invention relates to a detergent composition comprising a protease and, as a protease inhibitor, plasminostreptin or a variant thereof, wherein the ratio of the dissociation constant to the protease concentration is in the range of from 0.006 to 6.

Plasminostreptin is a known protease inhibitor belonging to Family III of subtilisin inhibitors. The inhibitor has been isolated from cultures of Streptomyces antifibrinolyticus (Kakinuma et al., J. Biol. Chem. 253, 1978, pp. 1529-1537). Preferred variants of plasminostreptin are those which exhibit a weaker binding to the protease than the native inhibitor.

In another aspect, the invention relates to a detergent additive comprising comprising a protease in the form of a stabilised liquid or a non-dusting granulate and, as a protease inhibitor, plasminostreptin or a variant thereof, characterized by a dissociation constant in the range of 0.05-50 μM.

In a further aspect, the invention relates to a novel plasminostreptin variant wherein an amino acid residue at one or more of the positions P6, P5, P4, P3, P2, P1, P'1, P'2, another amino acid residue.

In a still further aspect, the invention relates to a DNA construct comprising a nucleotide sequence coding for a plasminostreptin variant as indicated above, a host cell transformed with the DNA construct, as well as a method of producing a plasminostreptin variant of the invention by said host cell under conditions permitting the production of the variant, and recovering the variant from the culture. DETAILED DESCRIPTION OF THE INVENTION

Protease

The protease used in the invention is preferably of microbial origin. It may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, e.g. subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (both described in WO 89/06279) and mutant subtilisins such as those described in WO 89/06279 and DK 0541/90. Examples of commercial Bacillus subtilisins are Alcalase^®, Savinase^® and Esperase^®, all products of Novo Nordisk A/S. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270.

The amount of protease in the detergent will typically be 0.2-40 μM, especially 1-20 μM (generally 5-1000 mg/l, especially 20-500 mg/1) of the pure enzyme protein.

Inhibitor

According to the invention, the inhibitor is chosen for a given detergent (protease type and concentration etc.) so that the dissociation constant (K_d) is high enough to allow adequate release of protease when the detergent is diluted with water, yet the dissociation constant is low enough to allow efficient inhibition in the concentrated detergent during storage. K_d is commonly defined for a given protease and a given inhibitor in a given system as the equilibrium constant

K_d = [E] * [I] / [El]

where the square brackets indicate molar concentration of free enzyme (E), free inhibitor (I) and enzyme-inhibitor complex (El), respectively.

The ratio of the dissociation constant to the protease concentration is preferably from 0.06 to 6. The dissociation constant is preferably from 1 to 10 μM (i.e. 10^-6-10^-5 M).

The amount of inhibitor is preferably such that the molar ratio of inhibitor reactive site to protease active site is above 0.6, preferably 1-10.

Novel inhibitor

The novel inhibitor of the invention is derived from a known subtilisin inhibitor of Family III - plasminostreptin. Plasminostreptin has been purified from cultures of Streptomyces antifibrinolyticus (Kakinuma et al. (1978) J. Biol. Chem. 253, 1529-1537). Plasminostreptin is known to inhibit the subtilisins commonly used in detergents with inhibitor dissociation constants around 10^-8 M. We have found that using this inhibitor to stabilize a protease in a detergent, the protease is often so strongly bound to the inhibitor that little protease activity is released when the detergent is diluted for use in washing. A plasminostreptin variant exhibiting a weaker binding to the protease is. therefore preferred for use in detergents.

Variants of subtilisin inhibitors of Family III are known (Kojima et al. (1991), J. Biochem. (Tokyo) 109, 377-382; Kojima et al. (1990), Protein Engineering 3, 527-530), but their use in detergents and the resulting advantages have not been disclosed or suggested.

The following shows a comparison of the amino acid sequences in the binding region of two of the Family III members - Streptomyces subtilisin inhibitor (SSI) and plasminostreptin (PLST) (Kakinuma et al. in Protein Protease Inhibitor - The Case of Streptomyces Subtilisin Inhibitor (SSI) (1985), pp.363-391). Starting from the reactive site bond, amino acid positions are numbered P1, P2 etc. in the direction of the N-terminus and P'1, P'2 etc. towards the C-terminus.

P8 P7 P6 P5 P4 P3 P2 PI P'1 P'2 P'3 P'4 P'5 PLST Arg-Gly-Asp-Val-Ala-Cys-Thr-Lys-Gln-Phe-Asp-Pro-Val

SSI Gly-Glu-Asp-Val-Met-Cys-Pro-Met-Val-Tyr-Asp-Pro-Val

The two inhibitors show some conserved features but also differences in the binding region. We have found that the protease-inhibitor binding can be suitably weakened by substituting one or more of these amino acids, e.g. with one that is not represented at that position, i.e. with one that has a different side chain length and/or is differently charged from those represented. The novel inhibitor variants have been found to be resistant to hydrolysis by the protease.

In particular, the plasminostreptin variant of the invention is one comprising one or more of the following amino acid residues at the indicated position:

P6: Tyr, Pro or Lys,

P5: Gly, Ile, Pro, Glu,

P4: Gly, Pro, Lys, Glu,

P3: Ser,

P2: Ile, Glu, Tyr, Gly or Pro, P1: Pro , Asp, Ile, Gly or Met,

P' 1: Asn, Thr, Glu,

P ' 2 : Pro, Gly, Glu,

P ' 3 : Ile, Thr, Glu.

In vitro mutaqenesis of the plasminostreptin gene.

The amino acid sequence of the plasminostreptin inhibitor is known (cf. Sugino et al., J. Biol. CHem. 253, 1978, pp. 1546-1555). Therefore it is possible to construct a synthetic gene encoding the inhibitor by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3. 1984, pp. 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

Different approaches may be used for introducing mutations into the plasminostreptin gene.

One of these methods is oligonucleotide site-directed mutagenesis which is described by Zoller & Smith, DNA, Vol.

3, No. 6, 479-488 (1984). The plasminostreptin gene may be inserted into a circular M13 bacteriophage vector. To the single-stranded genome, a chemically synthesized complementary DNA-strand is annealed. This DNA-strand contains the mutation to be introduced flanked by sequences complementary to plasminostreptin sequences on the circular

DNA. In vitro, the primer is then extended in the entire length of the circular genome biochemically using Klenow polymerase. When transformed in E.coli, the heteroduplex will give rise to double-stranded DNA with the desired sequence from which a fragment can be isolated and re-inserted into the expression plasmid. Another method which may be employed is described in Nelson & Long, Analytical Biochemistry, 180, 147-151 (1989). It involves the 3-step generation of a PCR (polymerase chain reaction) fragment containing the desired mutation introduced by using a chemically synthesized DNA-strand as one of the primers in the PCR-reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation can be isolated by cleavage with restriction enzymes and re-inserted into the expression plasmid. In a further method, usually termed "cassette mutagenesis", a segment between two restriction sites of the plasminostreptin-encoding region may be replaced by a synthetic DNA linker containing the desired mutation. Part of the plasminostreptin sequence may then be replaced by the linker sequence to construct a DNA sequence coding for the plasminostreptin variant in question.

Expression of plasminostreptin and variants

Various species of Bacilli, including Bacillus alkalophilus, B. amyloliquefaciens, B. brevis, B. lentus, B. licheniformis, B. meqaterium, B. stearothermophilus, and B. subtilis, are known to secrete proteins efficiently. In many cases this has also been shown to be the case for heterologous proteins. Since expression of a secreted protease inhibitor has the potential advantage of facilitating purification, it is obviously interesting to attempt to express the inhibitor as a secreted product from a Bacillus strain. This could for instance be accomplished by combining the structural part of the inhibitor with the promoter and signal peptide of a well expressed and secreted Bacillus enzyme as for instance the maltogenic amylase from B. stearothermophilus (Diderichsen, B. and Christiansen, L. Cloning of a maltogenic alpha-amylase from Bacillus stearothermophilus, FEMS Microbiol. Lett. 56:53-60. 1988) or the alpha- amylase from B. licheniformis (Jørgensen, P.L., C.K. Hansen, G.B. Poulsen and B. Diderich sen. In vivo genetic engineering: Homologous recombination as a tool for plasmid construction, GENE 96: 37-41, 1990). This may be accomplished in many ways as known by people skilled in the art. One way is to use in vivo genetic engineering (Jørgensen et al. 1990, op. cit.). The advantage of this method is that it easily generates a perfect fusion between signal peptide and mature inhibitor which according to well documented rules for signal peptide processing would be expected to give the correct N-terminal amino acid residue of the inhibitor.

The plasminostreptin inhibitor or a variant thereof according to the invention may also be produced in a suitable yeast host. To this end, the DNA sequence encoding plasminostreptin or a variant thereof may be provided on a recombinant expression vector. In the vector, the DNA sequence encoding plasminostreptin or a variant thereof of the invention should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the yeast host and may be derived from genes encoding proteins either homologous or heterologous to yeast. The promoter and may be derived from a gene encoding a protein, intracellular or extracellular, such as an amylase, a protease or a glycolytic enzyme.

Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J.

Biol. Chem. 255, 1980, pp. 12073-12080; Alber and Kawasaki,

J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of

Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (US 4, 599, 311) or ADH2- 4c (Russell et al., Nature 304, 1983, pp. 652-654) promoters.

Other suitable promoters are those derived from the GAPDH

(glyceraldehyde 3-phosphate dehydrogenase) and PGK

(phosphoglycerate kinase) genes from Saccharomyces cerevisiae. The DNA sequence encoding plasminostreptin or a variant thereof may also be operably connected to a suitable terminator, such as the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters. The recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An examples of such a sequence the yeast plasmid 2μ replication genes REP 1-3 and origin of replication.

To achieve secretion to the growth medium, the DNA sequence encoding the inhibitor can be fused to another DNA-sequence encoding a signal peptide functional in yeast.. One example hereof is the Saccharomyces cerevisiae MFα-1 leader sequence (Kurjan & Herskowitz, Cell 30, 933-943 (1982). A preferred construction uses the DNA sequence encoding the entire 85 aminoacid MFα-1 leader sequence including the dibasic site LysArg. In that way, an efficient secretion of the plasminostreptin inhibitor with the correct N-terminal is achieved.

The procedures used to ligate the DNA sequences coding for the inhibitor of the invention, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

The yeast organism used as the host cell according to the invention may be any yeast organism which, on cultivation, produces large quantities of plasminostreptin or a variant thereof. Examples of suitable yeast organisms are strains of the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe or Saccharomyces uvarum. The transformation of yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se.

In another method of producing plasminostreptin inhibitor and variants hereof, a filamentous fungus is used as the host organism. The filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant proteins, e.g. a strain of Aspergillus sp., such as A. niger, A. nidulans or A. oryzae. The use of A. oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.

For expression of the plasminostreptin inhibitor or variant thereof in Aspergillus, the DNA sequence encoding plasminostreptin or the variant is preceeded by a promoter. The promoter may be any DNA sequence exhibiting a strong transcriptional activity in Aspergillus and may be derived from a gene encoding an extracellular or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.

Examples of suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease or A. orvzae triose phosphate isomerase. In particular when the host organism is A. oryzae, a preferred promoter for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong transcriptional activity in A. oryzae. The sequence of the TAKA amylase promoter appears from EP 238 023.

Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. To ensure secretion of the inhibitor or variants hereof from the host cell, the DNA sequence encoding the inhibitor may be preceded by a signal sequence which may be a naturally occurring signal sequence or a functional part thereof or a synthetic sequence providing secretion of the protein from the cell. In particular, the signal sequence may be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or proteinase, or a gene encoding a Humicola cellulase, xylanase or lipase.

Detergent composition

The detergent composition of the invention may be in any convenient form, e.g. powder, granules or liquid. A liquid detergent may be aqueous, typically containing up to 70% water and 0-20% organic solvent.

The detergent composition comprises a surfactant which may be anionic, non-ionic, cationic, amphoteric or a mixture of these types. The composition will usually contain 5-30% anionic surfactant such as linear alkyl benzene sulphonate (LAS), alpha-olefin sulphonate (AOS), alkyl sulphate (AS), alcohol ethoxy sulphate (AES) or alkali metal salts of natural fatty acids. It may also contain 3-20% non-ionic surfactant such as nonyl phenol ethoxylate or alcohol ethoxylate. The detergent composition may additionally comprise one or more other enzymes, such as an amylase, lipase, cellulase or peroxidase.

The pH (measured in aqueous detergent solution) will usually be neutral or alkaline, e.g. 7-10. The detergent composition may contain 1-40% of a detergent builder such as zeolite, phosphate, phosphonate, citrate, NTA, EDTA, DTPA, alkenyl succinic anhydride or silicate, or it may be unbuilt (i.e. essentially free of a detergent builder). It may also contain other conventional detergent ingredients, e.g. fabric conditioners, foam boosters, bactericides, optical brighteners and perfumes. Specific examples of detergent compositions according to the invention may be obtained from the compositions disclosed in WO 89/04361, DK 5111/89 or PCT/DK91/00243 by incorporating protease and inhibitor according to the invention. PCT/DK91/00243 is incorporated herein by reference. The invention is particularly applicable to the formulation of liquid detergents with pronounced enzyme stability problems, e.g those containing oxidizing agents. Such detergents typically contain 1-40%, especially 5-20% oxidizing agent. They may be granular detergents containing granules of a perborate or percarbonate and separate granules containing enzyme and inhibitor according to the invention, or they may be aqueous or non-aqueous liquid detergents containing hydrogen peroxide, a perborate or a percarbonate (see e.g. EP 378,261, EP 378,262, EP 294,904, EP 368,575). Detergent additive

The protease and inhibitor may be included in the detergent of the invention by separate addition or by adding the combined additive provided by the invention. The additive will usually contain 0.2-8 mM protease (0.5-20%) and have an inhibitor/protease ratio as described above.

The detergent additive may be in liquid form for incorporation in a liquid detergent. A liquid additive may contain 20-90% propylene glycol; 0.5-3% (as Ca) of a soluble calcium salt; 0-10% glycerol; minor amounts of short-chain fatty acids and carbohydrate; and water up to 100%. The detergent additive may further comprise one or more other enzymes, such as an amylase, lipase, cellulase or peroxidase.

The invention is further described in the following examples which are not in any way intended to limit the scope of the invention as claimed.

Example 1: Expression of plasminostreptin inhibitor in Saccharomyces cerevisiae

Construction of a synthetic plasminostreptin gene

A synthetic gene encoding plasminostreptin was constructed by the successive cloning of 6 DNA linkers each encoding part of the gene.

In the first cloning step an EcoRI/XbaI linker carrying the plasminostreptin sequence from EcoRI to BssHII and extended with an Xbal-site was inserted into pUC19 digested with EcoRI and Xbal. Into the resulting plasmid between BssHII and Xbal a BssHII/XbaI linker carrying the plasminostreptin sequence from BssHII to KpnI and extended with an Xbal-site was inserted. Steps three to six being cloning of KpnI/XbaI-, SacII/XbaI-, BstEII/XbaI- and MluI/XbaI linkers were carried out in a manner similar to steps one and two.

The final DNA sequence and the deduced aminoacid sequence of the EcoRI/XbaI insert is shown below. The sequence makes use of preferred yeast codons, except for the positions where a restriction site is positioned.

Plasmid construction The expression plasmid is of the C-POT type. Such a plasmid is described in EP patent application No. 85303702.6 and is characterized by containing the S. pombe triose phosphate isomerase gene (POT) for the purpose of plasmid stabilization. A plasmid containing the POT-gene is available from a deposited E.coli strain (ATCC 39685). The plasmid furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminator (P_TPI and T_TPI). It is identical to pLaC200 described in WO 89/02463, except for the region defined by the EcoRI/XbaI restriction fragment encoding a signal/leader/insulin precursor sequence. In the present example, this region is replaced by a fragment encoding the MFα-1 leader fused to the plasminostreptin sequence shown above. The sequence of the entire fragment appears from SEQ ID No. 1. Cloning of the MFα-1 leader is described by Kurjan & Herskowitz (supra). Modifications and assembly of the two sequences were carried out using entirely standard techniques. A map of the expression plasmid pYAPLMS is shown in Fig. 1.

Expression of plasminostreptin in yeast The plasmid prepared as described above was transformed into a S. cerevisiae strain carrying deletions in the TPI gene by selecting for growth on glucose.

The transformed yeast strain was grown on YPD medium (Sherman, F. et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory 1981). 100 ml medium in shake-flasks was inoculated with individual transformants and shaken at 30ºC for approx. 48 hours after which the inhibitor could be purified from the medium.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Novo Nordisk A/S

(B) STREET: NOVO Alle

(C) CITY: Bagsvaerd

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2880

(G) TELEPHONE: +45 4444 8888

(H) TELEFAX: +45 4449 3256

(ii) TITLE OF INVENTION: A Detergent Containing a Protease and a

Protease Inhibitor and Novel Inhibitors for Use therein

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE OERACTERISTICS:

(A) LENGTH: 678 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: synthetic

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 77..658

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 77. .331

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 332. .658 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAATTCCATT CAAGAATAGT TCAAACAAGA AGATTACAAA CTATCAATTT CATACACAAT 60

ATAAACGATT AAAAGA ATG AGA TTT CCT TCA ATT TTT ACT GCA GTT TTA 109

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu

-85 -80 -75

TTC GCA GCA TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA 157 Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu

-70 -65 -60

GAT GAA ACG GCA CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TCA GAT 205 Asp Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp

-55 -50 -45

TTA GAA GGG GAT TTC GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA 253 Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr

-40 -35 -30

AAT AAC GGG TTA TTG TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT 301 Asn Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala

-25 -20 -15

AAA GAA GAA GGG GTA TCT TTG GAT AAA AGA GGT TTG TAC GCT CCA TCT 349 Lys Glu Glu Gly Val Ser Leu Asp Lys Arg Gly Leu Tyr Ala Pro Ser

-10 -5 1 5

GCT TTG GTT TTG ACT ATG GGT CAC GGT AAC TCT GCT GCT ACT GTT AAC 397 Ala Leu Val Leu Thr Met Gly His Gly Asn Ser Ala Ala Thr Val Asn

10 15 20

CCA GAG OGC GCT GTT ACT TTG AAC TGT GCT CCA ACT GCT TCT GGT ACC 445 Pro Glu Arg Ala Val Thr Leu Asn Cys Ala Pro Thr Ala Ser Gly Thr

25 30 35

CAC CCA GCT GCT TTG CAA GCT TGT GCT GAA TTG AGA GGT GCT GGT GGT 493 His Pro Ala Ala Leu Gln Ala Cys Ala Glu Leu Arg Gly Ala Gly Gly

40 45 50

GAC TTC GAC GCT TTG ACT GTC CGC GGT GAC GTT GCT TGT ACT AAG CAA 541 Asp Phe Asp Ala Leu Thr Val Arg Gly Asp Val Ala Cys Thr Lys Gln

55 60 65 70

TTC GAC CCA GTT GTG GTT ACC GTT GAC GGT GTT TGG CAA GGT AAA CGC 589 Phe Asp Pro Val Val Val Thr Val Asp Gly Val Trp Gln Gly Lys Arg

75 80 85

GTT TCT TAC GAA AGA ACT TTC GCT AAC GAA TGT GTT AAG AAC TCT TAC 637 Val Ser Tyr Glu Arg Thr Phe Ala Asn Glu Cys Val Lys Asn Ser Tyr

90 95 100

GGT ATG ACT GTT TTC ACT TTC TAGTGATAGC AAGCTCTAGA 678

Gly Met Thr Val Phe Thr Phe

105 (2) INPOEMATICTT FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser -85 -80 -75 -70

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

-65 -60 -55 Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

-50 -45 -40

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

-35 -30 -25

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val -20 -15 -10

Ser Leu Asp Lys Arg Gly Leu Tyr Ala Pro Ser Ala Leu Val Leu Thr -5 1 5 10

Met Gly His Gly Asn Ser Ala Ala Thr Val Asn Pro Glu Arg Ala Val

15 20 25

Thr Leu Asn Cys Ala Pro Thr Ala Ser Gly Thr His Pro Ala Ala Leu

30 35 40

Gln Ala Cys Ala Glu Leu Arg Gly Ala Gly Gly Asp Phe Asp Ala Leu 45 50 55

Thr Val Arg Gly Asp Val Ala Cys Thr Lys Gln Phe Asp Pro Val Val 60 65 70 75

Val Thr Val Asp Gly Val Trp Gln Gly Lys Arg Val Ser Tyr Glu Arg

80 85 90

Thr Phe Ala Asn Glu Cys Val Lys Asn Ser Tyr Gly Met Thr Val Phe

95 100 105

Thr Phe

Claims

1. A detergent composition comprising a protease and, as a protease inhibitor, plasminostreptin or a variant thereof, wherein the ratio of the dissociation constant to the protease concentration is in the range of from 0.006 to 6.

2. A composition according to claim 1, wherein said ratio is in the range of from 0.06 to 6.

3. A detergent composition according to claim 1, characterized by an dissociation constant in the range 0.05-50 μM.

4. A composition according to claim 3 , wherein said constant is in the range of 1-10 μM.

5. A composition according to any of claims 1 - 4, wherein the protease is a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.

6. A composition according to claim 5, wherein the alkaline microbial protease is a subtilisin.

7. A composition according to any of claims 1-6, wherein an amino acid residue at one or more of the positions P6, P5, P4, P3, P2, P1, P'1, P'2, P'3 of the plasminostreptin sequence is substituted with another amino acid residue.

8. A detergent composition according to claim 7, wherein the plasminostreptin variant comprises one or more of the following amino acid residues at the indicated position:

P6: Tyr, Pro or Lys,

P5: Gly, Ile, Pro, Glu,

P4: Gly, Pro, Lys, Glu,

P3: Ser,

P2: Ile, Glu, Tyr, Gly or Pro, P1: Pro, Asp, Ile, Gly or Met,

P' 1: Asn, Thr, Glu,

P' 2 : Pro, Gly, Glu,

P' 3 : Ile, Thr, Glu.

9. A composition according to any of claims 1-7, wherein the subtilisin is derived from Bacillus and is preferably subtilisin Novo, subtilisin Carlsberg, BPN', subtilisin 309, subtilisin 147 or subtilisin 168.

10. A composition according to any of the preceding claims, wherein the molar ratio of inhibitor reactive site to protease active site is more than 0.6, preferably 1-10.

11. A composition according to any of the preceding claims, wherein the amount of protease is 0.2-40 μM, preferably 1-20 μM.

12. A composition according to any of the preceding claims, additionally comprising another enzyme, in particular an amylase, lipase, cellulase or peroxidase.

13. A composition according to any of the preceding claims, which is in the form of an aqueous liquid.

14. A composition according to any of claims 1-13, wherein the degree of protease inhibition in the detergent is at least 60%, and the degree of protease inhibition in a 1% detergent solution in water is below 10%.

15. A detergent additive comprising comprising a protease in the form of a stabilised liquid or a non-dusting granulate and, as a protease inhibitor, plasminostreptin or a variant thereof, characterized by a dissociation constant in the range of 0.05-50 μM.

16. A detergent additive according to claim 15, wherein an amino acid residue at one or more of the positions P6, P5, P4, P3, P2, P1, P'1, P'2, P'3 of the plasminostreptin sequence is substituted with another amino acid residue.

17. A detergent additive according to claim 16, wherein the plasminostreptin variant comprises one or more of the following amino acid residues at the indicated position:

P6: Tyr, Pro or Lys,

P5: Gly, Ile, Pro, Glu,

P4: Gly, Pro, Lys, Glu,

P3: Ser,

P2: Ile, Glu, Tyr, Gly or Pro,

P1: Pro, Asp, Ile, Gly or Met,

P'1: Asn, Thr, Glu,

P'2: Pro, Gly, Glu,

P'3: Ile, Thr, Glu.

18. A detergent additive according to claim 15, wherein the protease is a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.

19. A detergent additive according to claim 18, wherein the alkaline microbial protease is a subtilisin.

20. A detergent additive according to claim 19, wherein the subtilisin is derived from Bacillus and is preferably subtilisin Novo, subtilisin Carlsberg, BPN', subtilisin 309, subtilisin 147 or subtilisin 168.

21. A detergent additive according to any of claims 15-20, additionally comprising another enzyme, in particular an amylase, lipase, cellulase or peroxidase.

22. A plasminostreptin variant wherein an amino acid residue at one or more of the positions P6, P5, P4, P3, P2, P1, P'1, P'2, P'3 of the plasminostreptin sequence is substituted with another amino acid residue.

23. A plasminostreptin variant according to claim 22 comprising one or more of the following amino acid residues at the indicated position:

P6: Tyr, Pro or Lys,

P5: Gly, Ile, Pro, Glu,

P4: Gly, Pro, Lys, Glu,

P3: Ser,

P2: Ile, Glu, Tyr, Gly or Pro,

P1: Pro, Asp, Ile, Gly or Met,

P'1: Asn, Thr, Glu,

P'2: Pro, Gly, Glu,

P'3: Ile, Thr, Glu.

24. A DNA construct comprising a nucleotide sequence coding for a plasminostreptin variant according to claim 22 or 23.

25. A host cell transformed with a DNA construct according to claim 24.

26. A method of producing a plasminostreptin variant according to claim 22 or 23, the method comprising culturing a host cell according to claim 25 under conditions permitting the production of the variant, and recovering the variant from the culture.