WO2002016619A1 - Procede de production d'un complexe inhibiteur de proteases - Google Patents

Procede de production d'un complexe inhibiteur de proteases Download PDF

Info

Publication number
WO2002016619A1
WO2002016619A1 PCT/DK2001/000479 DK0100479W WO0216619A1 WO 2002016619 A1 WO2002016619 A1 WO 2002016619A1 DK 0100479 W DK0100479 W DK 0100479W WO 0216619 A1 WO0216619 A1 WO 0216619A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
sequence
inhibitor
complex
subtilisin
Prior art date
Application number
PCT/DK2001/000479
Other languages
English (en)
Inventor
Poul Erik Pedersen
Erwin Ludo Roggen
Original Assignee
Novozymes 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 Novozymes A/S filed Critical Novozymes A/S
Priority to EP01951447A priority Critical patent/EP1313862A1/fr
Priority to US10/344,231 priority patent/US20040038845A1/en
Priority to AU2001272366A priority patent/AU2001272366A1/en
Publication of WO2002016619A1 publication Critical patent/WO2002016619A1/fr

Links

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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

  • This invention relates to a method of improved industrial production of proteases.
  • the proteases are produced in vivo as complexes each comprising two interacting parts, a protease part and a protease inhibitor part.
  • the inhibitor part effectively minimizes proteolytic activity of the protease part of the complex during production and product recovery steps whereafter the complex may be dissociated to recover the pure protease part.
  • the complex may also simply be recovered and used in a relevant application e.g. in a washing detergent where the inhibitor part dissociates from the protease in the dilute detergent during the wash.
  • Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof.
  • proteases such as Alcalase ® (Novo Nordisk), Kannase ® (Novo Nordisk) Savinase ® (Novo Nordisk), or Esperase ® (Novo Nordisk).
  • proteases protein engineered variants of naturally occurring wild type proteases, e.g. Durazym ® (Novo Nordisk), Relase ® (Novo Nordisk), Maxapem ® (Gist-Brocades), Purafect ® (Genencor).
  • Reduced allergenicity is also a property of interest to the protease industry, a number of publications on the mite allergen Der p I, a cystein protease, have stated that the proteolytic activity of this protease is mechanistically linked to the potent allergenicity of house dust mites.
  • protease i) augments the permeability in the bronchial epithelium and ii) may upregulate IgE synthesis by virtue of its ability to specifically cleave the low affinity receptor (CD23) for human IgE (Herbert CA, King CM, Ring PC, Holgate ST, Stewart GA, Thompson PJ, Robinson C (1995) Am J Respir Cell Mol Biol 12: 369-378 - Schulz O, Laing P, Sewell HE, Shakib F (1995) EurJ Immunol 25: 3191-3194).
  • CD23 low affinity receptor
  • the problem to be solved by the present invention is to provide a protease enzyme that can be produced in high yields and may remain stable during storage (long shelf life), that has a satisfyingly high activity in relevant applications such as cleaning detergents, and may have other improved properties e.g. reduced allergenicity or improved thermostability, when compared to a parent protease from which the protease of the invention is derived.
  • the solution is based on that many of the above properties have been found to correlate with the proteolytic activity of the enzyme as such. Simply reducing the proteolytic activity however is not desirable as this also reduces the efficiency of the enzyme in the intended relevant applications.
  • the present invention relies on inhibiting the proteolytic activity of the protease already A7 vivo as the protease is being synthesized, by providing an effective protease inhibitor readily available in close vicinity of the protease immediately after in vivo synthesis.
  • protease complex with the wild type CI-2A inhibitor turned out to be very tightly bound and it did not dissociate in a dilute detergent but rather required higher detergent concentrations, whereas the protease CI-2A(M59P) complex dissociated completely in a dilute detergent (examples below), a finding which corresponds well with our previous observations on the interaction constant, K-, of the respective CI-2A inhibitor variants (supra).
  • the present invention relates to a method for producing a protease-inhibitor complex comprising the steps of: a) constructing a fusion polynucleotide sequence in frame, the sequence comprising a first gene encoding a protease, and a second gene encoding a protease inhibitor; b) introducing the sequence into a host cell; and c) cultivating the host cell, wherein the cell expresses the sequence and produces a non- covalently linked complex of the protease and the inhibitor.
  • the invention relates to a protease-inhibitor complex obtainable by a method as defined in the first aspect.
  • the invention relates to a polynucleotide construct comprising a fusion polynucleotide sequence as defined in the preceding aspect.
  • a polynucleotide construct comprising a fusion polynucleotide sequence as defined in the preceding aspect.
  • host cells comprising a polynucleotide as defined in the previous aspect in order to produce a protein complex as defined in the first aspect.
  • a preferred host cell genus of the industrial enzyme manufacturers is Bacillus, especially cells of the species Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.
  • a fourth aspect of the invention relates to a host cell comprising a polynucleotide construct as defined in the previous aspect, preferably the host cell is of a Bacillus species, and more preferably the host cell is a S. subtilis, B. clausii, or B. licheniformis cell.
  • An integrate part of the invention is that the complex has to be applied under conditions that will dissociate the inhibitor part from the protease part if this was not already done, such conditions are typically found wherever detergents are applied, in cleaning compositions and/or additives of any kind.
  • the invention relates to a detergent composition
  • a detergent composition comprising a protease-inhibitor complex as defined in the first aspect as well as a detergent additive comprising a protease-inhibitor complex as defined in the first aspect in the form of a stabilized liquid or a non-dusting granulate.
  • Subtilisin 309 gene and the sig am y -CI-2A gene were fused as an operon transcribed by the same promoter segment as a polycistronic transcript.
  • subtilase enzyme of the invention may be used in order to obtain a subtilase enzyme of the invention.
  • suitable techniques reference is made to Examples herein (vide infra) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. 5 M. (eds.) "Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990); and WO 96/34946.
  • isolated nucleic acid sequence refers to a nucleic acid
  • isolated molecules which has been isolated and purified and is thus in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules may be those that are separated from their natural environment and include cDNA and genomic clones as well as nucleic acid sequences derived from DNA shuffling experiments or from site-directed mutagenisis experiments. Isolated nucleic acid sequences of the present invention are free of
  • isolated nucleic acid sequence may alternatively be termed “isolated DNA sequence, "cloned nucleic acid sequence” or "cloned DNA sequence”.
  • the term “isolated” indicates that the protein has been removed from its native environment.
  • the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e. "homologous impurities" (see
  • An isolated protein is more than 10% pure, preferably more than 20% pure, more preferably more than 30% pure, as determined by SDS-PAGE. Further it is preferred to provide the protein in a highly purified form, i.e., more than 40% pure, more than 60% pure, more than 80% pure, more preferably more than 95% pure, and most preferably more than 99% pure, as determined by SDS-PAGE.
  • the term "isolated protein" may alternatively be
  • a serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 35 1973 "Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271- 272).
  • the bacterial serine proteases have molecular weights in the 20,000 to 45,000 Dalton range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease.
  • alkaline protease covering a sub-group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753).
  • subtilases A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al. Protein Science 6 (1997) 501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. A subtilisin was previously often defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases. A wide variety of subtilases have been identified, and the amino acid sequence of a number of subtilases has been determined. For a more detailed description of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997).
  • subtilisin 168 subtilisin 168
  • subtilisin BPN subtilisin BPN'
  • subtilisin Carlsberg Alcalase ® , Novo Nordisk A/S
  • subtilisin DY subtilisin DY
  • subtilases I-S2 or high alkaline subtilisins
  • Sub-group I-S2 proteases are described as highly alkaline subtilisins and comprises enzymes such as subtilisin PB92 (BAALKP) (Maxacal ® , Gist-Brocades NV), subtilisin 309 (Savinase ® , Novo Nordisk A/S), subtilisin 147 (BLS147) (Esperase ® , Novo Nordisk A/S), and alkaline elastase YaB (BSEYAB).
  • subtilisin PB92 BAALKP
  • BSS147 subtilisin 147
  • BSEYAB alkaline elastase YaB
  • parent subtilase describes a subtilase defined according to Siezen et al. (1991 and 1997), for further details see description of "Subtilases” above.
  • a parent subtilase may also be a subtilase isolated from a natural source, alternatively the term “parent subtilase” may be termed "wild type subtilase”.
  • modification(s) of a subtilase used herein is defined to include chemical modification of a subtilase as well as genetic manipulation of the DNA encoding a subtilase.
  • the modification(s) can be replacement(s) of the amino acid side chain(s), substitution(s), deletion(s) and/or insertions in or at the amino acid(s) of interest.
  • Gly 195 Glu or G195E A deletion of glycine in the same position is indicated as: Gly 195 * or G195*
  • subtilase variant or mutated subtilase means a subtilase that has been derived from a parent enzyme, the parent gene having been mutated in order to produce a mutant gene from which said mutated subtilase protease is produced when expressed in a suitable host.
  • the mutant gene may also be derived from a parent gene produced by DNA shuffling techniques.
  • the present invention comprises any one or more modifications to the amino acid sequence of the parent subtilase.
  • subtilase variants with different improved properties and a number of those are mentioned in the "Background of the invention” section herein (vide supra).
  • Such combinations comprise the positions: 222 (improve oxidation stability), 218
  • subtilase variant of the invention may advantageously be combined with one or more modification(s) in any of the positions: 27, 36, 57, 76, 97, 101 , 104, 120, 123, 167, 170, 195, 206, 218, 222, 224, 235, 252, 255, 259 and 274.
  • subtilisin 309 and subtilisin BAPB92 variants are considered appropriate for combination:
  • variants comprising any of the variants Y167A+R170S+A194P, V104N+S101G, K27R+V104Y+N123S+T274A, N76D+S103A+V104I, or S101G+S103A+V104I+G159D+ A232V+Q236H+Q245R+N248D+N252K, or Other combinations Of these mutations (V104N, S101G, K27R, V104Y, N123S, T274A, N76D, S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, N252K), in combination with any one or more of the modification(s) mentioned above exhibit improved properties.
  • subtilase variants of the main aspect(s) of the invention are preferably combined with one or more modification(s) in any of the positions 129, ,131, 133 and 194, preferably as 129K, 131 H, 133P, 133D and 194P modifications, and most preferably as P129K, P131H, A133P, A133D and A194P modifications. Any of those modification(s) may give a higher expression level of a subtilase variant of the invention.
  • subtilase genes genes that are well known in the art.
  • general standard procedures for cloning of genes and introducing mutations (random and/or site directed) into said genes may be used in order to obtain a subtilase variant of the invention.
  • suitable techniques reference is made to working examples herein (vide infra) and (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology”. John Wiley and Sons, 1995; Harwood, C. R., and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus”. John Wiley and Sons, 1990); and WO 96/34946.
  • subtilase BPN subtilase BPN'
  • BASBPN subtilase BPN'
  • a frame of reference is defined by aligning an isolated or a parent enzyme with subtilisin BPN' (BASBPN).
  • the GAP routine of the GCG package version 9.1 can be applied (infra) using the same settings.
  • the output from the routine is besides the amino acid alignment the calculation of the "Percent Identity" between the two sequences.
  • the inhibitor of the invention may be derived from the known inhibitors of Family VI e.g. from barley subtilisin inhibitor CI-1 or CI-2A. Inhibitors of this family are known to strongly inhibit the subtilisins commonly used in detergents, with inhibitor constants of interaction generally below 10 "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.
  • Family VI e.g. from barley subtilisin inhibitor CI-1 or CI-2A.
  • Inhibitors of this family are known to strongly inhibit the subtilisins commonly used in detergents, with inhibitor constants of interaction generally below 10 "10 M.
  • protease-inhibitor binding of CI-2A can be suitably weakened by substituting the P1 residue with Pro (M59P) (WO 93/20175; Novo Nordisk) and we've identified a number of other modifications of the barley CI-2A inhibitor resulting in a higher constant of interaction, K- (WO 92/05239 and WO 93/17086; Novo Nordisk, which are incorporated herein by reference).
  • amino acids positions of the inhibitors are numbered P1, P2 etc. in the direction of the N-terminal; and P'1 , P'2 etc. towards the C-terminal according to Schechter and Berger (1967; Biochem Biophys Res Commun. 27:157-162).
  • the following shows the amino acid sequence in the binding region of CI-2A as well as the modifications identified previously which improve properties of the inhibitor for the present invention:
  • P6 Ala, Glu, Tyr, Pro or Lys
  • P5 Gly, Val, Leu, Glu, lie or Pro
  • P4 Val, Pro, Trp, Ser, Glu, Gly, Lys or Arg
  • P3 Tyr, Glu, Ala, Arg, Pro, Ser, Lys, or Trp
  • P2 Ser, Lys, Arg, Pro, Glu, Val, Tyr, Trp, lie, Gly or Ala
  • P1 Arg, Tyr, Pro, Trp, Glu, Val, Ser, Lys, Asp, lie, Gly, or Ala
  • P'1 Gin, Ser, Thr, lie, Lys, Asn, or Pro
  • P'3 Glu, Gin, Asn, Val, Phe, lie, Thr or Tyr.
  • a fusion polynucleotide of the invention has the meaning generally recognized in the art; a polynucleotide sequence that comprises sequences originally encoding two or more parent proteins or variants thereof, where the coding sequences have been fused to form a single open reading frame, they are in frame.
  • additional nucleotides may have been added to the sequences encoding the parent proteins both 5'- and 3'-terminally to form linkers or spacers between or flanking the parent sequences in the sequence of the fusion polynucleotide.
  • N-terminal leader encoding sequences may also have been added such as pro-, pre-pro-, or secretion signals.
  • the present invention also relates to an isolated nucleic acid sequence, which encodes a protease inhibitor complex of the present invention.
  • the techniques used to isolate or clone a nucleic acid sequence encoding a polypeptide include isolation from genomic DNA, preparation from cDNA, or a combination thereof.
  • the cloning of the nucleic acid sequences of the present invention from such genomic DNA can be effected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • Other nucleic acid amplification 5 procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleic acid sequence-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligated activated transcription
  • NASBA nucleic acid sequence-based amplification
  • An isolated nucleic acid sequence can, for example, be obtained by standard cloning procedures used in genetic engineering to relocate the nucleic acid sequence from its natural location to a different site where it will be reproduced.
  • the cloning procedures may involve
  • nucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
  • the degree of identity between two nucleic acid sequences is determined as described above.
  • subtilase of the present invention Modification of a nucleic acid sequence encoding a subtilase of the present invention may be necessary for the synthesis of subtilases substantially similar to the subtilase.
  • substantially similar to the subtilase refers to non-naturally occurring forms of the
  • subtilase 20 subtilase. These subtilases may differ in some engineered way from the subtilase isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like.
  • variants that differ in specific activity, thermostability, pH optimum, or the like.
  • nucleotide substitution see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107.
  • Amino acid residues essential to the activity of the polypeptide encoded by the isolated nucleic acid sequence of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, Science 244: 1081-
  • Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or
  • the present invention also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable host cell.
  • An isolated nucleic acid sequence encoding a protease inhibitor complex of the present invention may be manipulated in a variety of ways to provide for expression of the subtilase. Manipulation of the nucleic acid sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying nucleic acid sequences utilizing recombinant DNA methods are well known in the art.
  • control sequences include all components which are necessary or advantageous for the expression of a subtilase of the present invention.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the subtilase.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control
  • sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a subtilase.
  • control sequence may be an appropriate promoter sequence, a nucleic acid
  • the promoter sequence contains transcriptional control sequences which mediate the expression of the subtilase.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular subtilases either homologous or
  • Suitable promoters for directing the transcription of the nucleic acid constructs of the present invention are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
  • amyM Bacillus amyloliquefaciens alpha- amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the subtilase. Any terminator which is functional in the host cell of choice may be used in the present invention.
  • the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide.
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention.
  • the control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a subtilase and directs the encoded subtilase into the cell's secretory pathway.
  • the 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted subtilase.
  • the 5' end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence.
  • the foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region.
  • the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the subtilase.
  • any signal peptide coding region which directs the expressed subtilase into the secretory pathway of a host cell of choice may be used in the present invention.
  • Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta- lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
  • the control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a subtilase.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding region may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a subtilase, the propeptide region is positioned next to the amino terminus of a subtilase and the signal peptide region is positioned next to the amino terminus of the propeptide region.
  • regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell.
  • regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • a recombinant expression vector comprising a DNA construct encoding the enzyme of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome in part or in its entirety and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the enzyme of the invention is operably linked to additional segments required for transcription of the DNA.
  • the expression vector is derived from plasmid or viral DNA, or may contain elements of both.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the enzyme.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus licheniformis alpha- amylase gene, the Bacillus amyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus pumilus xylosidase gene, or the phage Lambda P R or P L promoters or the E. coli lac, trp or tac promoters.
  • the DNA sequence encoding the enzyme of the invention may also, if necessary, be operably connected to a suitable terminator.
  • the recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, or a gene encoding resistance to e.g. antibiotics like kanamycin, chloramphenicol, erythromycin, tetracycline, spectinomycine, or the like, or resistance to heavy metals or herbicides.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector.
  • the secretory signal sequence is joined to the DNA sequence encoding the enzyme in the correct reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the enzyme.
  • the secretory signal sequence may be that normally associated with the enzyme or may be from a gene encoding another secreted protein.
  • the DNA sequence encoding the present enzyme introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment.
  • homologous is intended to include a DNA sequence encoding an enzyme native to the host organism in question.
  • heterologous is intended to include a DNA sequence not expressed by the host cell in nature.
  • the DNA sequence may be from another organism, or it may be a synthetic sequence.
  • the host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present enzyme and includes bacteria, yeast, fungi and higher eukaryotic cells.
  • bacterial host cells which, on cultivation, are capable of producing the enzyme of the invention are gram-positive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S.
  • the transformation of the bacteria may be effected by protoplast transformation, electroporation, conjugation, or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
  • the enzyme When expressing the enzyme in bacteria such as E. coli, the enzyme may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the enzyme is refolded by diluting the denaturing agent. In the latter case, the enzyme may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the enzyme.
  • the enzyme When expressing the enzyme in gram-positive bacteria such as Bacillus or Streptomyces strains, the enzyme may be retained in the cytoplasm, or may be directed to the extracellular medium by a bacterial secretion sequence. In the latter case, the enzyme may be recovered from the medium as described below.
  • the present invention provides a method of producing an isolated protease and/or protease inhibitor complex according to the invention, wherein a suitable host cell, which has been transformed with a DNA sequence encoding the protease and/or protease inhibitor complex, is cultured under conditions permitting the production of the complex, and the resulting complex or protease is recovered from the culture.
  • homologous impurities mean any impurities (e.g. other polypeptides than the complex or protease of the invention) that originate from the homologous cell, from where the protein of the invention is originally obtained.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question.
  • the expressed subtilase complex may conveniently be secreted into the culture medium and may be recovered therefrom by well- known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • a complex of the invention may be used for a number of industrial applications, in particular within the detergent industry.
  • the present invention also relates to a cleaning or detergent composition, preferably a laundry or dish-wash composition comprising the complex of the invention.
  • cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions.
  • compositions comprising the protease or complex of the invention
  • cleaning and detergent compositions are well described in the art and reference is made to WO 96/34946; WO 97/07202; WO 95/30011 for further description of suitable cleaning and detergent compositions.
  • the enzyme of the invention may be added to and thus become a component of a cleaning or detergent composition.
  • the detergent composition of the invention may for example be formulated as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for hand or machine dishwashing operations.
  • the invention provides a detergent additive comprising the protease or complex of the invention.
  • the detergent additive as well as the detergent composition may comprise one or more other enzymes such as another protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., a laccase, and/or a peroxidase.
  • another protease such as another protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase
  • proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included.
  • the protease may be a serine protease or a metallo 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 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO 89/06270 and WO 94/25583.
  • Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants with substitutions in one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, 252, 255, 259 and 274.
  • Preferred commercially available protease enzymes include Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, and Kannase® (Novo Nordisk A/S), Maxatase®, Maxacal®, Maxapem®, Properase®, Purafect®, Purafect OxP®, FN2®, FN3®, and FN4® (Genencor International Inc.).
  • Suitable lipases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful lipases include lipases from Humlcola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216 or from H. insolens as described in WO 96/13580, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.
  • lipase variants such as those described in WO 92/05249, WO 94/01541 , EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.
  • Preferred commercially available lipase enzymes include Lipolase® and Lipolase
  • Amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, ⁇ -amylases obtained from Bacillus, e.g. a special strain of B. //cA7en/ bm7/s,, described in more detail in GB 1 ,296,839.
  • amylases examples include the variants described in WO 94/02597, WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391 , 408, and 444.
  • Commercially available amylases are Duramyl®, Termamyl®, Fungamyl® and BAN®
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humlcola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humlcola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.
  • cellulases are the alkaline or neutral cellulases having colour care benefits.
  • Examples of such cellulases are cellulases described in EP 0495257, EP 0531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.
  • cellulases include Celluzyme®, and Carezyme® (Novo Nordisk A/S), Clazinase®, and Puradax HA® (Genencor International Inc.), and KAC-500(B)® (Kao Corporation).
  • Peroxidases/Oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • peroxidases include Guardzyme® (Novo Nordisk A/S).
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e. a separate additive or a combined additive, can be formulated e.g. as a granulate, a liquid, a slurry, etc.
  • Preferred detergent additive formulations are granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries. Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid.
  • a liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous.
  • the detergent composition typically comprises one or more surfactants, which may be non-ionic including semi-polar and/or anionic and/or cationic and/or zwitterionic.
  • the surfactants are typically present at a level of from 0.1 % to 60% by weight.
  • the detergent When included therein the detergent will usually contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid or soap. When included therein the detergent will usually contain from about 0.2% to about
  • glucamides N-acyl N-alkyl derivatives of glucosamine
  • the detergent may contain 0-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the detergent may contain a bleaching system which may comprise a H 2 O 2 source such as perborate or percarbonate which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate.
  • a bleaching system may comprise peroxyacids of e.g. the amide, imide, or sulfone type.
  • the enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in e.g. WO 92/19709 and WO 92/19708.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid
  • the detergent may also contain other conventional detergent ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil redeposition agents, dyes, bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes.
  • any enzyme in particular the enzyme of the invention, may be added in an amount corresponding to 0.01-100 mg of enzyme protein per litre of wash liquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor, in particular 0.1-1 mg of enzyme protein per litre of wash liquor.
  • the enzyme of the invention may additionally be incorporated in the detergent formulations disclosed in WO 97/07202 which is hereby incorporated as reference.
  • a method for producing a protease-inhibitor complex comprising the steps of: a) constructing a fusion polynucleotide sequence in frame, the sequence comprising a first gene encoding a protease, and a second gene encoding a protease inhibitor; b) introducing the sequence into a host cell; and c) cultivating the host cell, wherein the cell expresses the sequence and produces a non- covalently linked complex of the protease and the inhibitor.
  • the fusion polynucleotide of the invention comprises two genes fused in frame as described above, and the two genes may be separated by spacing nucleotides, as long as the genes remain in frame and together with the spacing sequence encodes a fusion polypeptide.
  • a preferred embodiment relates to a method of the first aspect, wherein the fusion polynucleotide further comprises a spacer of at least 6 basepairs between the two genes, preferably the size of the spacer is 6 - 300 base pairs, preferably 12 - 150 base pairs, more preferably 21 - 75 base pairs, most preferably 30 - 60 base pairs.
  • the complex can either be recovered and/or purified as such by means known in the art, or the inhibitor part can be dissociated from the complex after having served its purpose of minimizing autoproteolysis of the protease part during production whereupon the protease part can be recovered and/or purified. Consequently a preferred embodiment of the invention relates to the method of the first aspect, wherein step c) is followed by the additional step of: recovering the complex; or dissociating the inhibitor part from the complex and recovering the protease part.
  • subtilases As described above, several classes of subtilases are known in the art, this invention particularly relates to subtilases of the I-S1 and I-S2 classes.
  • a preferred embodiment of the invention relates to the method of the first aspect, wherein the protease is a subtilase I-S1 , I- S2, or a variant thereof.
  • a preferred embodiment relates to the method of the first aspect, wherein the protease is derived from Bacillus and is preferably subtilisin 309, subtilisin 168, subtilisin 147, subtilisin Novo, subtilisin Carisberg, subtilisin BLAP, subtilisin PB92, subtilisin BPN or BPN', or variants thereof.
  • a more preferred embodiment relates to the method of the first aspect, wherein the protease is subtilisin 309 or a variant thereof, preferably the variant comprises one or more of the modifications Y167A, R170S, and A194P.
  • Another preferred embodiment relates to the method of the first aspect, wherein the variant of subtilisin 309 comprises the modifications as follows:
  • a preferred embodiment relates to the method of the first aspect, wherein the second gene encodes a barley chymotrypsin inhibitor, preferably CI-2A (SEQ ID 1) or a variant thereof; preferably the variant of the CI-2A inhibitor has had an amino acid residue at one or more of the positions P6, P5, P4, P3, P2, P1 , P'1 , P'2, or P'3 substituted with another amino acid residue; more preferably the variant of CI-2A comprises one or more of the following amino acid substitutions at the indicated position: P6: Ala, Glu, Tyr, Pro or Lys
  • P5 Gly, Val, Leu, Glu, lie or Pro
  • P3 Tyr, Glu, Ala, Arg, Pro, Ser, Lys, or Trp P2: Ser, Lys, Arg, Pro, Glu, Val, Tyr, Trp, lie, Gly or Ala P1: Arg, Tyr, Trp, Glu, Val, Ser, Lys, Asp, lie, Gly, or Ala P'1 : Gin, Ser, Thr, lie, Lys, Asn, or Pro P'2: Val, Glu, Arg, Pro, Gly or Trp P'3: Glu, Gin, Asn, Val, Phe, lie, Thr or Tyr; and most preferably the variant of CI-2A comprises a praline at position P1 (M59P).
  • An optional part of the fusion polynucleotide sequence of the first aspect is a spacer or linker between the subtilase and the inhibitor encoding parts.
  • a preferred embodiment relates to the method of the first aspect, wherein the spacer encodes a peptide of a size of about 5 - 80 amino acids, preferably about 8 - 40 amino acids, and more preferably about 10 - 30 amino acids; more preferably the spacer encodes a peptide of a size of at least 15 amino acids.
  • a more preferred embodiment relates to the method of the first aspect, wherein the spacer encodes the amino acid sequence HAHAHSVSQEASVTR (SEQ ID 2).
  • Another preferred embodiment relates to the method of the first aspect, wherein the inhibitor is CI-2A(M59P) or a variant thereof, and the spacer encodes a peptide of at least 15 amino acids.
  • a preferred embodiment relates to the method of the first aspect, wherein the protease is subtilisin 309 or a variant thereof, and wherein the inhibitor is CI-2A(M59P) or a variant thereof, and the spacer encodes a peptide of at least 15 amino acids.
  • a most preferred embodiment relates to the method of the first aspect, wherein the fusion polynucleotide sequence comprises a sequence encoding the amino acid sequence shown in SEQ ID 3 or variants thereof.
  • a preferred host cell genus of the industrial enzyme manufacturers is Bacillus, especially cells of the species Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Accordingly a a preferred embodiment relates to a method of the first aspect, wherein the host cell is of a Bacillus species, preferably B. subtilis, B. clausii, or B. licheniformis.
  • allergenicity As mentioned above, one of the properties that the present invention may improve is allergenicity, as the allergenicity of proteases has been correlated with the proteolytic activity.
  • the inhibition of the activity of the protease or the protease-inhibitor complex of the invention may result in much lower allergenicity as determined by an assay.
  • suitable allergenicity assays are exemplified below.
  • a preferred embodiment relates to the protease-inhibitor complex of the second aspect, wherein the allergenicity of the complex is reduced at least 3 times when compared to the allergenicity of the parent protease, preferably at least 10 times reduced, more preferably at least 50 times, even more preferably at least 100 times, still more preferably at least 500 times, yet more preferably at least 1,000 times, more preferably at least 5,000 times, and most preferably at least 10,000 times.
  • the fifth aspect of the invention relates to a detergent composition, as application of proteases or complexes of the invention would be particularly advantageous in such a composition, especially those compositions wherein the protease exhibits a high degree of inhibition in the composition while the degree of inhibition becomes very low upon dilution of the composition for cleaning and/or washing purposes.
  • a preferred embodiment relates to a detergent composition of the fifth aspect, wherein the degree of proteolytic enzyme inhibition in the detergent is at least 60%, preferably at least 70%, more preferably at least 80%, and the degree of proteolytic enzyme inhibition in a 1% detergent composition solution in water is below 10%, preferably below 5%, and most preferably below 2%.
  • Another a preferred embodiment relates to a detergent composition of the fifth aspect, which further comprises Linear Alkylbenzene Sulfonate (LAS).
  • LAS Linear Alkylbenzene Sulfonate
  • Yet another a preferred embodiment relates to a detergent composition of the fourth aspect, wherein the allergenicity of the complex is reduced at least 3 times when compared to the allergenicity of the composition comprising the parent protease, preferably at least 10 times reduced, more preferably at least 50 times, even more preferably at least 100 times, still more preferably at least 500 times, yet more preferably at least 1 ,000 times, more preferably at least 5,000 times, and most preferably at least 10,000 times.
  • a final preferred embodiment relates to a detergent additive of the sixth aspect, wherein the allergenicity of the complex is reduced at least 3 times when compared to the allergenicity of the additive comprising the parent protease, preferably at least 10 times reduced, more preferably at least 50 times, even more preferably at least 100 times, still more preferably at least 500 times, yet more preferably at least 1 ,000 times, more preferably at least 5,000 times, and most preferably at least 10,000 times.
  • proteolytic activity is expressed in Kilo Novo Protease
  • KNPU KNPU Units
  • the activity is determined relatively to an enzyme standard (Savinase®), and the determination is based on the digestion of a dimethyl casein (DMC) solution by the proteolytic enzyme at standard conditions, i.e. 50°C, pH 8.3, 9 min. reaction time, 3 min. measuring time.
  • DMC dimethyl casein
  • a folder AF 220/1 is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
  • the concentration of an inhibitor preparation is estimated from the absorbance measured at 280nm using theoretically calculated extinction coefficients (Gill and von Hippel, 1989, Anal Biochem. 182:319-326) while the exact concentration of active inhibitor is determined using titration with a preparation of subtilisin 309 that has been active site titrated with N-trans-cinnamoyl imidazole (Schonbaum et al., 1961, J Biol Chem. 236:2930-2935; Bender et al., 1966, J Am Chem Soc. 88:5890-5913).
  • Ki values for CI-2A and variants are carried out as follows. In a total volume of 1800 ⁇ l, fixed amounts of subtilisin 309 is incubated in the absence of inhibitor or in the presence of varying amounts of inhibitor. At different time points 90 ⁇ l incubation mixture is assayed for residual enzymatic activity through addition of 10 ⁇ l of a chromogenic substrate in a Cobas Fara automated spectrophotometer. The absorbance at 410nm is measured every five seconds for 250 seconds.
  • the reactions are carried out in 0.1 M Tris-HCI, pH 8.6 @ 25°C and the final concentration of the chromogenic substrate is 1 mM.
  • the [E 0 ] used is between 2x10 "10 M (low K- values) and 1x10 "7 M (high K- values) while [l 0 ] in general is varied from 25% to 250% of [E 0 ].
  • chromogenic substrates Suc-Ala-Ala-Pro-Phe-pNA (inhibitors with low K- values) and Suc-Ala-Ala-Ala-pNA (inhibitors with high Ki values) are used.
  • the gene encoding the alkaline protease subtilisin 309 (Savinase ® ) from B. lentus NCIB 10309 was cloned and inserted in a derivative of pE194 (pPL2002)(Appl Envir Microbiol, 2000, 66(2): 825-827). 10
  • the CI-2A chymotrypsin inhibitor encoding gene of barley and the plasmid carrying the gene translated through an alfa leader sequence were described in US patent No. 5,674,833 (1997).
  • the CI-2A(M59P) chymotrypsin inhibitor was described in WO 92/05239 (Novo Nordisk).
  • amyL promoter region was isolated from a derivative of B. licheniformis ATCC 15 9789.
  • the DNA segments were amplified and joined together mainly by the polymerase chain reaction (PCR) and the sequence overlap extension (SOE) techniques.
  • the stepwise constructions involved a number of PCR primers with convenient flanking tails of either restrictions enzyme sites or overlapping DNA segments for the SOE fusions.
  • the 20 DNA fusion product was inserted in an appropriate vector for inserting the genes in the chromosome of either Bacillus subtilis or B.lentus through recombination between homologous sequences in the host and the incoming plasmid derivative.
  • Construct A was made as outlined herein, however another version "Construct A Pro” was also prepared (similarly to description below), wherein a single amino acid modification was introduced in the CI-2A inhibitor, the methionine in position 59 (or P1) was substituted with a praline (M59P) as previously described in WO 92/05239. 0
  • the CI-2A gene was amplified from a derivative of pYACI2 by PCR: a) For the construction of Expression cassette "A" (fig.1), the CI-2A gene was amplified by the primers pep72 (SEQ ID 4) and pep16 (SEQ ID 5). The N-terminal part was further 5 extended by an additional PCR round with pep63 (SEQ ID 6) and pep16 as primers and the first PCR product as template.
  • the primers pep72 (SEQ ID 4) and pep63 (SEQ ID 6) added the DNA codons for the 15 amino acid spacer region as well as an Mlu1 site in frame with the Mlu1 site of primer pep5 (SEQ ID 7) (below) for amplification of the C-terminal of the Savinase ® gene.
  • Pep16 (SEQ ID 5) added a Bgl2 site to the end of the CI-2A sequence.
  • the CI-2A gene was amplified by the primers pep36 (SEQ ID 8) and pep16 (SEQ ID 5).
  • the pep36 (SEQ ID 8) primer has an overlapping tail to the pep39 (SEQ ID 9) primer for the amplification of the PamyL signal below, c)
  • the CI-2A gene was amplified by the primers pep35 (SEQ ID 10) and pep16 (SEQ ID 5).
  • the pep35 (SEQ ID 10) primer added a BspH1 site in the ATG start region of the CI-2A gene for the later fusion to PamyL.
  • the PamyL segments were amplified with DNA isolated from a derivative of B.licheniformis ATCC 9789 as template: d) For the "B" (fig.1) construct the PamyL region was amplified by the primers pep65
  • pep39 SEQ ID 9
  • the pep65 (SEQ ID 11) primer added an Mlu1 site upstream of the shine dalgarno (SD) of amyl for the right fusion to the amplified C- terminal of the Savinase ® gene.
  • Pep39 (SEQ ID 9) has an overlapping sequence matching the pep36 (SEQ ID 8) tail in the PCR product of step b) above.
  • the PamyL region was amplified by the primers 8805 (SEQ ID 12) and pep39 (SEQ ID 9).
  • the 8805 (SEQ ID 12) primer added a BamHI site upstream of the promoter of amyL for the insertion of the CI-2A behind the Savinase ® gene in pPL2002.
  • the pep39 (SEQ ID 9) primer made an in frame fusion to the amyL signal possible by fusion to the pep36 (SEQ ID 8) tail in the PCR product of step b) above.
  • the Savinase ® segments were amplified with the pPL2002 plasmid as template: f)
  • the C-terminal part of the Savinase ® gene was amplified by the primers pep5 (SEQ ID 7) and 20231 (SEQ ID 13).
  • the pep5 (SEQ ID 7) primer inserted an Mlu1 site in the C-terminal of the coding region for Savinase ® .
  • the terminator region of Savinase ® was amplified using primer 22121 (SEQ ID 14) and 20446 (SEQ ID 15).
  • the primer 22121 (SEQ ID 14) inserted a BamHI site in the Savinase ® end of the terminator.
  • step b) the CI-2A PCR fragment of step b) was digested by Bgl2, and the Savinase ® terminator PCR product of step g) was digested by BamH After ligation of the two fragments, the fused PCR product was amplified by the primers pep39 (SEQ ID 9) and 137393 (SEQ ID 16).
  • amyL CI-2A fusions j) For the "B" construct (fig.1) the amyL PCR fragment in of step d) and the CI-2A PCR fragment of step i) were joined together by SOE using the primers pep65 (SEQ ID 11) and 137393 (SEQ ID 16). k) For the "C” construct (fig.1) the amyL PCR fragment of step e) and the CI-2A PCR fragment of step c) were joined together by SOE using the primers 8805 (SEQ ID 12) and pep16 (SEQ ID 5).
  • Plasmid integration vectors (derivatives of pPL2002 integration vector): o)
  • the final part of the "A" construct (fig.1) was the vector part of the pPL2002 plasmid which was digested by BstX1 and BamHI and ligated to the large fragment (containing the CI-2A part) of the PCR fragment of step m) which was likewise digested by BstX1 and BamHI .
  • the ligation was transformed in parallel with the pE194 plasmid to a B.subtilis (apr, npr) strain selecting for chloramphenicol resistance at 30°C.
  • the right plasmid was identified by restriction and PCR analysis.
  • the sequence of a PCR fragment containing the total C-terminal region of the Savinase ® -CI-2A fusion confirmed the right fusion product and is shown in SEQ ID 17.
  • the final part of the "B" construct (fig.1) was the vector part of the pPL2002 plasmid which was digested by BstX1 and BamHI and ligated to the large fragment (containing the CI-2A part) of the PCR fragment of step n) which was likewise digested by BstX1 and BamHI.
  • the ligation was transformed in parallel with the pE194 plasmid to a B.subtilis (apr, np ⁇ strain selecting for chloramphenicol resistance at 30°C.
  • the right plasmid was identified by restriction and PCR analysis.
  • the sequence of a PCR fragment containing the total C-terminal region of the Savinase ® -CI-2A fusion confirmed the right transcriptional fusion product and is shown in SEQ ID 18.
  • the final part of the "C" construct (fig.1) was the pPL2002 plasmid digested by BamHI and then ligated to the PCR fragment of step k) which was first digested by Bgl2 and
  • the ligation was transformed in parallel with the pE194 plasmid to a B.subtilis (apr, np ⁇ strain selecting for chloramphinicol resistance at 30°C.
  • the right plasmid was identified by restriction and PCR analysis.
  • the sequence of a PCR product containing the PamyL sig-CI-2A fusion confirmed the right fusion product and is shown in SEQ ID 19. r)
  • the final part of the "D" construct (fig.1) was the pPL2002 plasmid digested by BamHI and then ligated to the PCR fragment of step I) which was first digested by Bgl2 and BamHI .
  • the ligation was transformed in parallel with the pE194 plasmid to a B.subtilis (apr, npr) strain selecting for chloramphinicol resistance at 30°C.
  • the right plasmid was identified by restriction and PCR analysis. The sequence of a PCR product containing the PamyL ATG-CI-2A fusion confirmed the right fusion product and is shown in SEQ ID 20.
  • the pPL2002 derivatives in Example 1 were inserted into the chromosome of a protease negative derivative of B.subtilis DN497 (WO91/09129). At first a small part of the Savinase ® gene was inserted in the amyE gene to establish homology, secondly the pPL2002 derivatives containing constructs A, B, C or D of example 1 (fig.1) were inserted in the chromosome by selecting for chloramphenicol. After 4 days of fermentation in shake flasks in complex growth media, the culture broths were analysed for protease and CI-2A content.
  • the CI-2A content was identified through precipitation with antibody IgG raised in rabbits against a CI-2A protein that was isolated from a Yeast transformant known to produce the CI-2A inhibitor.
  • the protease activity was partly inhibited by the co expressed CI-2A protein but could be detected by antibody IgG raised against Savinase ® .
  • the four strains comprising each of the four constructs all produced protein recognized by both Savinase ® -lgG as well as CI-2A-lgG.
  • the CI-2A product of strains comprising constructs A and B was further characterized through SDS PAGE and Western blot analysis -
  • AAPF-pNA in all fractions was measured.
  • the protease test was performed with or without 0.5% linear alkyl benzene sulphonate (LAS) which is known to dissociate the CI-2A Savinase ® complex into an active protease part and a free CI-2A molecule.
  • the protease activity test result confirmed that the major part of the protease was found as the protease inactive protease-CI-2A complex, less than 5% of the normal protease activity could be detected when LAS was not added.
  • a 95% pure fraction containing the Savinase ® CI-2A complex was further analysed by mass spectrometric methods. This molecular weight analysis demonstrated that the protease fraction consist of the Savinase ® molecule with a 5 amino acid HisAlaHisAlaHis tail and that the CI-2A part is degraded to three almost identical molecules. In the degraded CI-2A mixture the N-terminal part of the wild type CI-2A molecule had been removed, and three nearly identical fragments were found: aa 11 to 83, aa 12 to 83, and 15 to 83.
  • Standard wash performance tests were carried out using six different commercial wash detergents to compare the activities of the Savinase ® CI-2A complex and the Savinase ® Cl- 2A(M59P) complex with the commercially available Savinase ® enzyme under standard wash conditions.
  • the Savinase ® CI-2A complex showed no significant performance under these normal washing conditions except a little in Detergent 4 (results not shown), which has a very high LAS content resulting in close to 0.5% final LAS concentration during the wash, a concentration which our results above indicate is high enough to dissociate the CI-2A inhibitor from the protease.
  • Detergent 1 Detergent 2 Detergent 3 Detergent 4 Detergent 5 Detergent 6
  • the detergents used in the assay were 6 different commercially available washing detergents, however a simple model formulation could also be used. pH is adjusted to 10.5 which is within the normal range for a powder detergent. Many compositions of detergents are 5 publicly available and well known to those in the art a simple model detergent (No. 95) is as follows:
  • the complex can be expressed in larger scale, purified by conventional techniques,
  • allergenicity (which is likely, but not necessarily true for a protein with low antibody binding) should be tested in in vivo or in vitro model systems: e.g. in vitro assays for immunogenicity such as assays based on cytokine expression profiles or other
  • animal models for testing allergenicity should be set up to test a limited number of protein variants that show desired characteristics in vitro.
  • Useful animal models include the guinea pig intratracheal model (GPIT) (Ritz, et al. Fund. Appl. Toxicol., 21 , pp. 31-37, 1993), mouse subcutaneous (mouse-SC) (WO 98/30682, Novo Nordisk), the rat intratracheal (rat-IT) (WO 96/17929, Novo Nordisk), and the mouse intranasal (MINT) (Robinson et al., Fund. Appl. Toxicol. 34, pp. 15-24, 1996) models.
  • GPIT guinea pig intratracheal model
  • MINT mouse intranasal
  • the immunogenicity of a complex or protease is measured in animal tests, wherein the animals are immunised with the protein and the immune response is measured. Specifically, it is of interest to determine the allergenicity by repeatedly exposing the animals to the protein by the intratracheal route and following the specific IgG and IgE titers. Alternatively, the mouse intranasal (MINT) test can be used to assess the allergenicity.
  • MINT mouse intranasal
  • the present inventors have demonstrated that the performance in ELISA correlates closely to the immunogenic responses measured in animal tests.
  • the IgE binding capacity of the protein variant must be reduced to at least below 75 %, preferably below 50 %, more preferably below 25 % of the IgE binding capacity of the parent protein as measured by the performance in IgE ELISA, given the value for the IgE binding capacity of the parent protein is set to 100 %.
  • a first asessment of the immunogenicity and/or allergenicity can be made by measuring the antibody binding capacity or antigenicity of the protein using appropriate antibodies. This approach has also been used in the literature (WO 99/47680).
  • the present inventors have demonstrated that the performance in a human lung epithelial cell assay correlates closely to the immunogenic responses measured in animal tests (WO 01/29562).
  • the cytokine profiles produced by the cell after stimulation with allergen must include measarable amounts of interieukin (IL) -6, IL-8, MCP-1 and GM-CSF.
  • mice Twenty subcutaneous (SC) immunisations were performed weekly with 0.05 ml 0.9% (wt/vol) NaCl (control group), or 0,050 ml of a protein dilution ( ⁇ 0,01-0,1 mg/ml). Each group contained 10 female Balb/C mice (about 20 grams) purchased from Bomholdtgaard, Ry, Denmark. Blood samples (0,100 ml) were collected from the eye one week after every second immunisation. Serum was obtained by blood clotting and centrifugation and analysed as indicated below. The results are shown in Table 3 below.
  • a fresh stock solution of cyanuric chloride in acetone (10 mg/ml) is diluted into PBS, while stirring, to a final concentration of 1 mg/ml and immediately aliquoted into CovaLink NH2 plates (100 microliter per well) and incubated for 5 minutes at room temperature. After three washes with PBS, the plates are dryed at 50°C for 30 minutes, sealed with sealing tape, and stored in plastic bags at room temperature for up to 3 weeks.
  • Mouse anti-Rat IgE was diluted 200x in PBS (5 microgram/ml). 100 microliter was added to each well. The plates were coated overnight at 4 °C.
  • Unknown rat sera and a known rat IgE solution were diluted in dilution buffer: Typically 10x, 20x and 40x for the unknown sera, and Vz dilutions for the standard IgE starting from 1 ⁇ g/ml. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3x with washing buffer. The anti-rat IgE (biotin) was diluted 2000x in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3x with washing buffer.
  • Streptavidin was diluted 1000x in dilution buffer. 100 microliter was added to each well. Incubation was for 1 hour at room temperature. Unbound material was removed by washing 3x with 300 microliter washing buffer. OPD (0.6 mg/ml) and H 2 O 2 (0.4 microliter /ml) were dissolved in citrate buffer. 100 microliter was added to each well. Incubation was for 30 minutes at room temperature. The reaction was stopped by addition of 100 microliter H 2 SO 4 . The plates were read at 492 nm with 620 nm as reference.
  • IgG can be performed using anti Rat-lgG and standard rat IgG reagents. Similar determinations of IgG and IgE in mouse serum can be performed using the corresponding species-specific reagents.
  • Human epithelial cells were grown in RPM1 1603 growth medium under serum-free conditions. Typically, 2.5 cm 0 culture wells were used. The cells were stimulated with phosphate buffered saline (PBS) as negative control, lipopolysaccharide (LPS) from PBS.
  • PBS phosphate buffered saline
  • LPS lipopolysaccharide
  • Escherichia coli as positive control, and various amounts of the allergens of interest. Typically, stimulation was with 1, 10 and 100 ug of protein for 0, 1, 2, 4, 6, 16 and 24 hrs. Cytokines present the cell culture medium were quantified by ELISA (R&D Systems). Results are shown in the tables below:
  • Table 2 Quantification of cytokine response of Human epithelial cells after stimulation with the protese TY145 and the protease-inhibitor complex TY145-CI2A, as described above.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention porte sur un procédé de production d'un complexe protéinique consistant à construire une séquence polynucléotidique de fusion in frame, cette séquence comprenant un premier gène codant une protéase, et un second gène codant un inhibiteur de protéase; introduire la séquence dans une cellule hôte; mettre en culture la cellule hôte, la cellule exprimant la séquence et produisant un complexe lié de manière covalente de la protéase et de l'inhibiteur; et récupérer le complexe.
PCT/DK2001/000479 2000-08-21 2001-07-09 Procede de production d'un complexe inhibiteur de proteases WO2002016619A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP01951447A EP1313862A1 (fr) 2000-08-21 2001-07-09 Procede de production d'un complexe inhibiteur de proteases
US10/344,231 US20040038845A1 (en) 2000-08-21 2001-07-09 Method for production of a protease-inhibitor complex
AU2001272366A AU2001272366A1 (en) 2000-08-21 2001-07-09 Method for production of a protease-inhibitor complex

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA200001233 2000-08-21
DKPA200001233 2000-08-21
US22750100P 2000-08-24 2000-08-24
US60/227,501 2000-08-24

Publications (1)

Publication Number Publication Date
WO2002016619A1 true WO2002016619A1 (fr) 2002-02-28

Family

ID=26068862

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2001/000479 WO2002016619A1 (fr) 2000-08-21 2001-07-09 Procede de production d'un complexe inhibiteur de proteases

Country Status (3)

Country Link
EP (1) EP1313862A1 (fr)
AU (1) AU2001272366A1 (fr)
WO (1) WO2002016619A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074419A2 (fr) * 2003-02-18 2004-09-02 Novozymes A/S Compositions detergentes
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
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
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
WO2000001831A2 (fr) * 1998-07-07 2000-01-13 The Procter & Gamble Company Proteases fusionnees avec des variants d'inhibiteur de subtilisine de streptomyces

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
WO2000001831A2 (fr) * 1998-07-07 2000-01-13 The Procter & Gamble Company Proteases fusionnees avec des variants d'inhibiteur de subtilisine de streptomyces

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004074419A2 (fr) * 2003-02-18 2004-09-02 Novozymes A/S Compositions detergentes
WO2004074419A3 (fr) * 2003-02-18 2004-11-11 Novozymes As Compositions detergentes
EP1923455A3 (fr) * 2003-02-18 2009-01-21 Novozymes A/S Compositions détergents
WO2020074517A1 (fr) 2018-10-10 2020-04-16 Novozymes A/S Variants d'inhibiteur de chymotrypsine et utilisation associée
CN113166231A (zh) * 2018-10-10 2021-07-23 诺维信公司 胰凝乳蛋白酶抑制剂变体及其用途

Also Published As

Publication number Publication date
AU2001272366A1 (en) 2002-03-04
EP1313862A1 (fr) 2003-05-28

Similar Documents

Publication Publication Date Title
CA2503965C (fr) Variantes de subtilase
US9732310B2 (en) Subtilase variants
AU771078B2 (en) Subtilase enzymes of the I-S1 and I-S2 sub-groups having an additional amino acid residue in the active site loop region
EP1409659A2 (fr) Variants de subtilase
WO2001068821A2 (fr) Nouvelles enzymes subtilases possedant des performances de lavage ameliorees sur des taches d'oeuf
AU2001239214A1 (en) Novel subtilase enzymes having an improved wash performance on egg stains
US6780629B2 (en) Subtilase enzymes
EP1315806B1 (fr) Procede de criblage de proteases hautement actives et leurs inhibiteurs
EP1183343A1 (fr) Enzymes de subtilase des sous-groupes i-s1 et i-s2 possedant au moins un residu supplementaire d'acide amine entre les positions 125 et 126
US20040038845A1 (en) Method for production of a protease-inhibitor complex
MXPA01011836A (es) Enzimas subtilasas de los sub-grupos i-s1 e i-s2 que tienen al menos un residuo de un aminoacido adicional entre las posiciones 97 y 98.
EP1313862A1 (fr) Procede de production d'un complexe inhibiteur de proteases
EP1183339B1 (fr) Enzymes subtilases des sous-groupes i-s1 et i-s2 possedant au moins un residu d'acide amine additionnel entre les positions 129 et 130
EP1183336B2 (fr) Subtilases des sous- groupes i-s1 et i-s2 comprenant au moins un residu d'acide amine additionnel entre les positions 131 et 132
EP1183335A1 (fr) Enzymes subtilase des sous-groupes i-s1 et i-s2 possedant au moins un acide amine additionnel entre les positions 130 et 131
EP1141257A1 (fr) Enzymes subtilases des sous groupes i-s1 et i-s2 possedant un residu acide amine supplementaire dans une region en boucle de site actif
EP1141260A1 (fr) Enzymes substilases des sous-groupes i-s1 et i-s2 ayant un residu d'acide amine additionnel dans une region boucle de site actif
EP1141261A1 (fr) Enzymes subtilases des sous-groupes i-s1 et i-s2 ayant un residu d'acide amine additionnel dans une region boucle de site actif
EP1183337A1 (fr) Enzymes subtilases des sous-groupes i-s1 et i-s2 renfermant au moins un residu d'acide amine supplementaire entre les positions 132 et 133
EP1183341A1 (fr) Enzymes subtilases des sous groupes i-s1 et i-s2 possedant au moins un residu acide amine supplementaire entre les positions 127 et 128
EP1183340A1 (fr) Enzymes subtilases des sous-groupes i-s1 et i-s2 comprenant au moins un reste d'acide amine additionnel entre les positions 126 et 127
MXPA01006223A (en) Subtilase enzymes of the i-s1 and i-s2 sub-groups having an additional amino acid residue in an active site loop region

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2001951447

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 10344231

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 2001951447

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Ref document number: 2001951447

Country of ref document: EP