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WO1993020175A1 - Detergent contenant une protease et un inhibiteur de protease, et nouveaux inhibiteurs associes - Google Patents

Detergent contenant une protease et un inhibiteur de protease, et nouveaux inhibiteurs associes Download PDF

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Publication number
WO1993020175A1
WO1993020175A1 PCT/DK1993/000119 DK9300119W WO9320175A1 WO 1993020175 A1 WO1993020175 A1 WO 1993020175A1 DK 9300119 W DK9300119 W DK 9300119W WO 9320175 A1 WO9320175 A1 WO 9320175A1
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WO
WIPO (PCT)
Prior art keywords
inhibitor
protease
subtilisin
pro
composition according
Prior art date
Application number
PCT/DK1993/000119
Other languages
English (en)
Inventor
Torben Halkier
Ib Groth Clausen
Lone Kierstein Nielsen
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Publication of WO1993020175A1 publication Critical patent/WO1993020175A1/fr

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Classifications

    • 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

Definitions

  • subtilisin inhibitors with this improved performance can be derived from known inhibitors by substituting certain amino acids.
  • the novel inhibitors can be produced by known protein engineering methods.
  • the invention also provides a modified subtilisin inhibitor of family VI, as defined above, excluding: Eglin B and C substituted with
  • the invention provides a recombinant DNA molecule comprising a nucleotide sequence coding for a modified subtilisin inhibitor as defined above, a transformed host organism comprising said DNA and a method of producing the modified inhibitor comprising cultivation of the transformed host organism.
  • 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.
  • novel inhibitors provided by the invention may be derived from the known inhibitors of Family VI, described in the above-mentioned references, e.g. from barley subtilisin inhibitor CI-1 or CI-2, potato subtilisin inhibitor (PSI), Eglin B or C r tomato subtilisin inhibitor or Vicia subtilisin inhibitor (VSI).
  • barley subtilisin inhibitor CI-1 or CI-2 potato subtilisin inhibitor (PSI), Eglin B or C r tomato subtilisin inhibitor or Vicia subtilisin inhibitor (VSI).
  • Inhibitors of this family are known to strongly inhibit the subtilisins commonly used in detergents, with inhibitor dissociation constants generally below 10 " ⁇ M. We have found that by using these inhibitors to stabilize a protease in a detergent, the protease is so strongly bound that very little protease activity is released when the detergent is diluted for use in washing, and the protease remains almost completely inactive. We have therefore realized a need for a modified inhibitor with weaker binding to the protease.
  • protease-inhibitor binding can be suitably weakened by substituting the P1 residue with Pro (starting from the reactive site, amino acids positions are numbered P1, P2 etc. in the direction of the N-terminal). This modified inhibitor is resistant to hydrolysis by the protease.
  • the protease-inhibitor binding can be further weakened by a combination of Pro as the P1 residue and one or more of the following amino acid substitutions at the indicated positions.
  • P4 Pro
  • novel inhibitors may be produced by known recombinant DNA techniques. Briefly, a DNA sequence (cDNA or a synthetic gene) encoding a known inhibitor is subjected to mutagenesis in order to replace the codon(s) for the amino acid(s) to be substituted with a new codon (codons) for the desired amino acid substitution(s). This may preferably be carried out by oligonucleotide- directed site-specific mutagenesis in bacteriophage M13 vectors (e.g. M.J. Zoller and M. Smith, Meth. Enzymol. 100 (1983) 468-500), in double-stranded DNA vectors (e.g. Y.
  • M13 vectors e.g. M.J. Zoller and M. Smith, Meth. Enzymol. 100 (1983) 468-500
  • double-stranded DNA vectors e.g. Y.
  • Bacilli including Bacillus alkalophilus, B. amyloliquefaciens, B. brevis, B. lentus, B. licheniformis, B. megaterium, 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.
  • 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.
  • 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. oryzae triose phosphate isomerase.
  • 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 may additionally comprise one or more other enzymes, such as an amylase, lipase, cellulase or peroxidase.
  • bleaching agents or bleach precursors or a system comprising a bleaching agent and/or precursor together with an activator therefor, fabric conditioners, foam boosters, anti-corrosion agents, soil-suspending agents, sequestering agents, anti-soil redeposition agents, stabilizing agents for the enzyme(s), foam depressors, dyes, bactericides, optical brighteners or perfumes.
  • a detergent composition formulated as a detergent powder containing zeolite builder, anionic surfactant, nonionic surfactant, acrylic or equivalent polymer, perborate bleach precursor, amino-containing bleach activator, silicate or other structurant, alkali to adjust to desired pH in use, and neutral inorganic salt.
  • the detergent compositions a)-h) include a protease and a modified subtilisin inhibitor of Family VI as described above, and optionally one or more other enzymes, as indicated above.
  • the invention is particularly applicable to the formulation of 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).
  • 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 barley subtilisin inhibitor and variants hereof according to the invention can be produced biosynthetically in a yeast host expressing a DNA sequence encoding the inhibitor.
  • the DNA sequence encoding the inhibitor can be fused to another DNA-sequence encoding a signal peptide functional in yeast.
  • An 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, efficient secretion of CI-2A inhibitor with the correct N-terminal is achieved. Plasmid construction
  • All expression plasmids are of the C-POT type. Such plasmids are described in EP patent application No. 85303702.6 and are characterized in 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 plasmids furthermore contain the S. cerevisiae triose phosphate isomerase promoter and terminater (PTM and T ⁇ p,).
  • Mutant CI-2A genes were generated using PCR mutagenesis, which was carried out as follows: A primer carrying the mutation flanked by homologous sequences and carrying the introduced Kpnl-site was used together with another primer homologous to sequences in the T ⁇ p, region in a PCR amplification reaction. In that way, fragments were generated which contained the desired mutations. The ends were trimmed with the restriction enzymes Kpnl and Xbal, purified on agarose gels, and cloned into pYACI2 previously digested with the same restriction enzymes. The presence of the mutation was verified by DNA sequencing. The primers used are listed below.
  • Plasmids prepared as described above were transformed into a " S. cerevisiae strain carrying deletions in the TPI gene by selecting for growth on glucose.
  • the transformed yeast strains were grown on YPD medium (Sherman,
  • the lipase expression plasmid is termed p960 and makes use of the A oryzae TAKA amylase promoter for driving the transcription and the Aspergillus niger glucoamylase transcription terminator. 0
  • the plasmid p960 was slightly modified in order to obtain a vector for cloning the inhibitor gene. p960 was digested with Nrul and BamHI restriction enzymes.
  • a pressure filter Zeitz K 250-Neu
  • the filtrate was applied to a Sephadex G25 gelfiltration column equilibrated in 20 mM sodium acetate, pH 4.4.
  • the gelfiltrated protein was adsorbed onto S-Sepharose column material and after washing the column material with 20 mM sodium acetate, pH 4.4, protein was eluted from the material with a 20 mM sodium borate, pH 9.6 buffer (pH 10.2 was used for the basic mutants).
  • the eluate was subjected chromatography on a Q- Sepharose column equilibrated in 20 mM sodium borate, pH 9.6 (10.2).
  • the column was eluted with a linear gradient between 20 mM sodium borate, pH 9.6 (10.2) and the same buffer supplemented with 1M NaCI.
  • Inhibitor-containing fractions were pooled and the buffer was changed to 20 mM sodium acetate, pH 4.4 again using a Sephadex G25 column.
  • the gelfiltrated protein was subjected to chromtography on a S-Sepharose column equilibrated in 20 mM sodium acetate, pH 4.4. Elution of the column was performed with a linear gradient between the equilibration buffer and the equilibration buffer supplemented with 1M NaCI. Finally, inhibitor-containing fractions were collected and used in the subsequent experiments.
  • the protection of lipase from proteolytic degradation in the presence of a protease inhibitor was determined by preparing aqueous solutions of 78 ⁇ M Humicola lanuginosa lipase (Lipolase ® available from Novo Nordisk A/S) with or without 1.9 ⁇ M protease and with or without protease inhibitor (1.9 ⁇ M or 5.6 ⁇ M) in 50 mM Tris-HCI, pH 8.0.
  • the protease used was Savinase ® (available from Novo Nordisk A/S) and the protease inhibitor used was CI-2(M59P).
  • the solutions were stored at room temperature for up to 20 days. Lipase activity was measured before and after storage and expressed as % residual activity.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Detergent Compositions (AREA)

Abstract

Composition détergente comportant une protéase et un inhibiteur modifié de subtilisine appartenant à la famille VI et possédant Pro à titre de reste P1 associé à une ou plusieurs substitutions d'acide aminé comprises parmi les suivantes et situées dans les positions indiquées: P4: Pro; P3: Tyr, Glu, Ala, Arg ou Pro; P2: Arg, Pro, Glu, Val ou Tyr.
PCT/DK1993/000119 1992-03-31 1993-03-31 Detergent contenant une protease et un inhibiteur de protease, et nouveaux inhibiteurs associes WO1993020175A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK0431/92 1992-03-31
DK43192A DK43192D0 (da) 1992-03-31 1992-03-31 Detergentkomposition

Publications (1)

Publication Number Publication Date
WO1993020175A1 true WO1993020175A1 (fr) 1993-10-14

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DK (1) DK43192D0 (fr)
WO (1) WO1993020175A1 (fr)

Cited By (12)

* 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
EP0756619A4 (fr) * 1994-04-22 1997-04-02 Procter & Gamble Compositions de detergent contenant une amylase
WO1998013483A1 (fr) * 1996-09-24 1998-04-02 The Procter & Gamble Company Proteases et leurs variants, sur lesquels sont fondus des inhibiteurs de protease peptidique
US5783546A (en) * 1994-04-22 1998-07-21 Procter & Gamble Company Amylase-containing detergent compositions
WO1998020133A3 (fr) * 1996-11-01 1998-07-23 Pioneer Hi Bred Int Proteines a concentration amelioree en acides amines essentiels
US6579698B1 (en) 1996-09-24 2003-06-17 The Procter & Gamble Company Stabilized proteinaceous protease inhibitors and variants thereof
US6800726B1 (en) 1996-11-01 2004-10-05 Pioneer Hi-Bred International, Inc. Proteins with increased levels of essential amino acids
WO2013054774A1 (fr) * 2011-10-12 2013-04-18 三洋化成工業株式会社 Inhibiteur protéique de protéase, et solution protéique et composition de détergent le contenant
JP2013146227A (ja) * 2012-01-20 2013-08-01 Sanyo Chem Ind Ltd タンパク質溶液及びこれを含む洗剤組成物
US20170137798A1 (en) * 2014-07-04 2017-05-18 Novozymes A/S Subtilase variants and polynucloetides encoding same
US20180355340A1 (en) * 2010-04-15 2018-12-13 The Procter & Gamble Company Automatic dishwashing detergent composition
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
WO1992003529A1 (fr) * 1990-08-24 1992-03-05 Novo Nordisk A/S Composition detergente enzymatique et procede de stabilisation enzymatique
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
WO1992003529A1 (fr) * 1990-08-24 1992-03-05 Novo Nordisk A/S Composition detergente enzymatique et procede de stabilisation enzymatique
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

Cited By (17)

* 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
EP0756619A4 (fr) * 1994-04-22 1997-04-02 Procter & Gamble Compositions de detergent contenant une amylase
US5783546A (en) * 1994-04-22 1998-07-21 Procter & Gamble Company Amylase-containing detergent compositions
WO1998013483A1 (fr) * 1996-09-24 1998-04-02 The Procter & Gamble Company Proteases et leurs variants, sur lesquels sont fondus des inhibiteurs de protease peptidique
US6579698B1 (en) 1996-09-24 2003-06-17 The Procter & Gamble Company Stabilized proteinaceous protease inhibitors and variants thereof
WO1998020133A3 (fr) * 1996-11-01 1998-07-23 Pioneer Hi Bred Int Proteines a concentration amelioree en acides amines essentiels
US6800726B1 (en) 1996-11-01 2004-10-05 Pioneer Hi-Bred International, Inc. Proteins with increased levels of essential amino acids
US7211431B2 (en) 1996-11-01 2007-05-01 Pioneer Hi-Bred International, Inc. Expression cassettes for producing plants with increased levels of essential amino acids
US20180355340A1 (en) * 2010-04-15 2018-12-13 The Procter & Gamble Company Automatic dishwashing detergent composition
WO2013054774A1 (fr) * 2011-10-12 2013-04-18 三洋化成工業株式会社 Inhibiteur protéique de protéase, et solution protéique et composition de détergent le contenant
JPWO2013054774A1 (ja) * 2011-10-12 2015-03-30 三洋化成工業株式会社 タンパク質性プロテアーゼインヒビター並びにこれを含有するタンパク質溶液及び洗剤組成物
EP2767547A4 (fr) * 2011-10-12 2015-04-29 Sanyo Chemical Ind Ltd Inhibiteur protéique de protéase, et solution protéique et composition de détergent le contenant
US9403897B2 (en) 2011-10-12 2016-08-02 Sanyo Chemical Industries, Ltd. Proteinaceous protease inhibitor and protein solution and detergent composition containing it
JP2013146227A (ja) * 2012-01-20 2013-08-01 Sanyo Chem Ind Ltd タンパク質溶液及びこれを含む洗剤組成物
US20170137798A1 (en) * 2014-07-04 2017-05-18 Novozymes A/S Subtilase variants and polynucloetides encoding same
US10550381B2 (en) * 2014-07-04 2020-02-04 Novozymes A/S Variant proteases and amylases having enhanced storage stability
WO2020074517A1 (fr) 2018-10-10 2020-04-16 Novozymes A/S Variants d'inhibiteur de chymotrypsine et utilisation associée

Also Published As

Publication number Publication date
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