WO1993017086A1 - Detergent contenant une protease, un inhibiteur de protease, et nouveaux inhibiteurs destines a etre utilises dans le detergent - Google Patents

Detergent contenant une protease, un inhibiteur de protease, et nouveaux inhibiteurs destines a etre utilises dans le detergent Download PDF

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Publication number
WO1993017086A1
WO1993017086A1 PCT/DK1993/000063 DK9300063W WO9317086A1 WO 1993017086 A1 WO1993017086 A1 WO 1993017086A1 DK 9300063 W DK9300063 W DK 9300063W WO 9317086 A1 WO9317086 A1 WO 9317086A1
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WIPO (PCT)
Prior art keywords
protease
glu
pro
gly
plasminostreptin
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PCT/DK1993/000063
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English (en)
Inventor
Lone Kierstein Nielsen
Torben Halkier
Ib Groth Clausen
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Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Publication of WO1993017086A1 publication Critical patent/WO1993017086A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors

Definitions

  • 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.
  • protease-containing detergents especially liquid detergents
  • a major problem in formulating protease-containing detergents, especially liquid detergents, is that of ensuring enzyme stability during storage.
  • 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.
  • a protease inhibitor e.g. a subtilisin
  • the protease inhibitor should ideally show essentially no inhibiting effect under dilute washing conditions, i.e. when the detergent is in use.
  • the present invention relates to a detergent composition
  • 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.
  • 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 ⁇ M.
  • 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.
  • 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.
  • 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.
  • 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.
  • Bacillus subtilisins examples include Alcalase ® , Savinase ® and Esperase ® , all products of Novo Nordisk A/S.
  • trypsin-like proteases examples include 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 ⁇ M, especially 1-20 ⁇ M (generally 5-1000 mg/l, especially 20-500 mg/1) of the pure enzyme protein.
  • 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
  • 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 ⁇ M (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.
  • 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.
  • 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 two inhibitors show some conserved features but also differences in the binding region.
  • 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.
  • 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,
  • P2 Ile, Glu, Tyr, Gly or Pro
  • P1 Pro , Asp, Ile, Gly or Met
  • 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.
  • oligonucleotide site-directed mutagenesis which is described by Zoller & Smith, DNA, Vol.
  • the plasminostreptin gene may be inserted into a circular M13 bacteriophage vector.
  • 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
  • the primer is then extended in the entire length of the circular genome biochemically using Klenow polymerase.
  • 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.
  • a DNA fragment carrying the mutation can be isolated by cleavage with restriction enzymes and re-inserted into the expression plasmid.
  • 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.
  • 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.
  • B. stearothermophilus Diderichsen, B. and Christiansen, L. Cloning of a maltogenic alpha-amylase from Bacillus stearothermophilus, FEMS Microbiol. Lett. 56:53-60. 1988
  • the alpha- amylase from B. licheniformis J ⁇
  • the plasminostreptin inhibitor or a variant thereof according to the invention may also be produced in a suitable yeast host.
  • the DNA sequence encoding plasminostreptin or a variant thereof may be provided on a recombinant expression 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.
  • 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 2 ⁇ replication genes REP 1-3 and origin of replication.
  • the DNA sequence encoding the inhibitor can be fused to another DNA-sequence encoding a signal peptide functional in yeast.
  • yeast One example hereof is the Saccharomyces cerevisiae MF ⁇ -1 leader sequence (Kurjan & Herskowitz, Cell 30, 933-943 (1982).
  • a preferred construction uses the DNA sequence encoding the entire 85 aminoacid MF ⁇ -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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease or A. orvzae triose phosphate isomerase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 may be granular detergents containing granules of a perborate or percarbonate and separate
  • 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.
  • Example 1 Expression of plasminostreptin inhibitor in Saccharomyces cerevisiae
  • a synthetic gene encoding plasminostreptin was constructed by the successive cloning of 6 DNA linkers each encoding part of the gene.
  • 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.
  • 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.
  • POT S. pombe triose phosphate isomerase gene
  • 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 ).
  • 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.

Abstract

L'invention se rapporte à une composition détergente comprenant une protéase et, comme inhibiteur de protéase, la plasminostreptine ou une variante de celle-ci. Le rapport entre la constante de dissociation et la concentration de protéase varie de 0.006 à 6.
PCT/DK1993/000063 1992-02-25 1993-02-23 Detergent contenant une protease, un inhibiteur de protease, et nouveaux inhibiteurs destines a etre utilises dans le detergent WO1993017086A1 (fr)

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DK0236/92 1992-02-25
DK23692A DK23692D0 (da) 1992-02-25 1992-02-25 Detergentkomposition

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5527487A (en) * 1991-11-27 1996-06-18 Novo Nordisk A/S Enzymatic detergent composition and method for enzyme stabilization
WO1998037910A1 (fr) * 1997-03-02 1998-09-03 Emory University Inhibiteurs peptidiques de propeptide/prohormone convertases
US6579698B1 (en) 1996-09-24 2003-06-17 The Procter & Gamble Company Stabilized proteinaceous protease inhibitors and variants thereof
WO2009095425A1 (fr) * 2008-02-01 2009-08-06 Novozymes A/S Composition d'enzymes liquide
WO2020074517A1 (fr) 2018-10-10 2020-04-16 Novozymes A/S Variants d'inhibiteur de chymotrypsine et utilisation associée

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5039446A (en) * 1988-07-01 1991-08-13 Genencor International, Inc. Liquid detergent with stabilized enzyme
EP0473502A1 (fr) * 1990-08-24 1992-03-04 Shiseido Company Limited Composition de nettoyage pour la prévention et la diminution de l'irritation de la peau
WO1992005239A1 (fr) * 1990-09-18 1992-04-02 Novo Nordisk A/S Detergent contenant une protease ainsi qu'un inhibiteur et nouveaux inhibiteurs destines a etre utilises dans ce detergent

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5039446A (en) * 1988-07-01 1991-08-13 Genencor International, Inc. Liquid detergent with stabilized enzyme
EP0473502A1 (fr) * 1990-08-24 1992-03-04 Shiseido Company Limited Composition de nettoyage pour la prévention et la diminution de l'irritation de la peau
WO1992005239A1 (fr) * 1990-09-18 1992-04-02 Novo Nordisk A/S Detergent contenant une protease ainsi qu'un inhibiteur et nouveaux inhibiteurs destines a etre utilises dans ce detergent

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Volume 88, No. 18, 1 May 1978, (Columbus, Ohio, USA), SUGINO HIROMU et al., "Plasminostreptin, a Protein Proteinase Inhibitor Produced by Streptomyces Antifibrinolyticus. II. Determination of the Reactive Site for Proteinases", page 189, The Abstract No. 132571p; & J. BIOL. CHEM. 1978, 253(5), *
PATENT ABSTRACTS OF JAPAN, Vol. 12, No. 155, C-494; & JP,A,62 269 689 (SHOWA DENO K.K. et al.), 24 November 1987. *
SWISSPROT., 21 Jul 1986, Accession No. P0100 7, Plasminostreptin. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 253, No. 5, March 1978, ATSUSHI KAKINUMA et al., "Plasminostreptin, a Protein Proteinase Inhibitor Produced by Streptomyces Antifibrinolyticus". *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5527487A (en) * 1991-11-27 1996-06-18 Novo Nordisk A/S Enzymatic detergent composition and method for enzyme stabilization
US6579698B1 (en) 1996-09-24 2003-06-17 The Procter & Gamble Company Stabilized proteinaceous protease inhibitors and variants thereof
WO1998037910A1 (fr) * 1997-03-02 1998-09-03 Emory University Inhibiteurs peptidiques de propeptide/prohormone convertases
WO2009095425A1 (fr) * 2008-02-01 2009-08-06 Novozymes A/S Composition d'enzymes liquide
WO2020074517A1 (fr) 2018-10-10 2020-04-16 Novozymes A/S Variants d'inhibiteur de chymotrypsine et utilisation associée

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