WO1995030743A1 - Variants de proteases - Google Patents

Variants de proteases Download PDF

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Publication number
WO1995030743A1
WO1995030743A1 PCT/DK1995/000180 DK9500180W WO9530743A1 WO 1995030743 A1 WO1995030743 A1 WO 1995030743A1 DK 9500180 W DK9500180 W DK 9500180W WO 9530743 A1 WO9530743 A1 WO 9530743A1
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Prior art keywords
trypsin
protease
amino acid
variant according
residue
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PCT/DK1995/000180
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English (en)
Inventor
Claus Von Der Osten
Henrik Fredholm
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Novo Nordisk A/S
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Priority to AU24451/95A priority Critical patent/AU2445195A/en
Publication of WO1995030743A1 publication Critical patent/WO1995030743A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • 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)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi

Definitions

  • the present invention relates to novel trypsin-like protease variants with improved properties, DNA constructs coding for the expression of said variants, host cells capable of expressing the variants from the DNA constructs, as well as a method of producing the variants by cultivating said host cells.
  • the variants may advantageously be used as constituents in detergent compositions and additives.
  • Trypsin-like proteases i.e. serine-proteases that in structure are similar to trypsin
  • have been extensively described in the literature A J. Greer, "Comparative modelling methods - application to the family of mammalian serineproteases", Proteins, Vol. 7, p. 317-334, 1990; M. A. Phillips & R. J. Fletterick, "Proteases", Curr. Opin. Struct. Biol., Vol. 2, p. 713-720, 1992).
  • Trypsin (Rattus rattus), PDB Code ltrm, S. Sprang et al., "The three-dimensional structure of Asn102 mutant of trypsin", Science, Vol. 235, p. 905, 1987; Trypsin (Bos taurus), PDB Code 2ptn, J. Walter et al., "On the disordered activation domain in trypsinogen", Acta Cryst., Vol. 38B, p. 1462, 1982; Tonin (Rattus rattus), PDB Code lton, M. Fuj inaga et al., "Rat submaxillary gland serine protease, Tonin", J. Mol.
  • Pancreatic Elastase (Sus scrofa), PDB Code 3est, E. Meyer et al., PDB Code 3est, E. Meyer et al., "Structure of native porcine pancreatic elastase at 1.65 Angstroms resolution'', Acta Cryst., Vol. 44B, p. 26, 1988; Neutrophil Elastase (Homo sapiens), PDB Code 1hne, M. A. Navia et al., "Structure of Human Neutrophil Elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84 Angstroms resolution", Proc. Nat. Acad. Sci. USA, Vol. 86, p.
  • the PDB Codes refer to the structural data files deposited at the Brookhaven Protein Data Bank (F. C. Bernstein et al., "The Protein Data Bank: A computer based archival file for macromolecular structures", J. Mol. Biol., Vol. 112, p. 535-542, 1977.
  • WO 89/06270 discloses a trypsin-like protease obtained from a strain of the fungal species Fusarium oxysporum as well as a detergent composition comprising the enzyme. No information as to the amino acid sequence of the enzyme or the three-dimensional structure thereof have been published. SUMMARY OF THE INVENTION
  • the trypsin-like F. oxysporum protease described in WO 89/06270 has now been cloned and sequenced and the three-dimensional structure thereof has been elucidated by X-ray crystallography.
  • This trypsin-like protease has surprisingly been found to belong to the above mentioned trypsin family comprising eight known mammalian and one bacterial trypsin-like protease. From a comparison with the structure of the trypsins belonging to this family it has surprisingly been shown that while the structures are very similar near the active and binding sites and in the core of the protein, there are considerable differences in other regions, particularly in the loops on the surface of the molecule. Furthermore, there is no evidence of any divalent cation binding sites in the F. oxysporum trypsin-like protease.
  • the present invention relates to a variant of a parent trypsin-like Fusarium protease, which
  • iii) is encoded by a DNA sequence which hybridizes with an oligonucleotide probe hybridizing with a DNA sequence encoding the trypsin-like F. oxysporum protease having the amino acid sequence shown in SEQ ID No. 2.
  • trypsin-like Fusarium protease is intended to indicate a trypsin-like protease derived from a fungus of the genus Fusarium, and in particular of the species F. oxysporum, or a functional analogue thereof .
  • oxysporum protease has a three-dimensional structure having a core which is substantially similar to the core of the trypsin-like F .
  • oxysporum protease and which preferably has one or more loop structures corresponding to loops II, IV, VI, IX, X and XIII of the trypsin-like F. oxysporum protease described herein.
  • the term "corresponding" as used about the loop structures is intended to indicate an identity of at least 60% such as 70%, 80%, 90% or up to 100% with the corresponding F. oxysporum protease loop structure(s).
  • the properties characterizing the functional analogue are intended to be understood in an analogous manner to properties i)-iii) listed above and further described below.
  • trypsin-like protease derived from the species F. oxysporum (DSM 2672), the cDNA and amino acid sequences of which are apparent from SEQ ID Nos. 1 and 2, respectively.
  • functional analogues of said protease as defined above e.g. trypsin-like proteases derivable from other organisms such as microorganisms including bacterial and fungal strains, and in particular from other strains of Fusarium spp., may be modified in a manner similar to that described for the trypsin-like F. oxysporum protease described herein.
  • variants of such functional analogous are intended to be considered to be within the scope of the present invention.
  • Examples of other Fusarium strains, which have been found to produce trypsin-like proteases are F. merismoides, F. redolens, F. sambucinum, F. solani and F. verticilloides.
  • the term "variant" is intended to indicate a polypeptide which is derived from a trypsin-like Fusarium protease as defined above and which has one or more of the properties i)-iii) which will be further discussed below.
  • the variant differ from the trypsin-like protease by one or more amino acid residues, which, for instance, may have been added or deleted from either or both of the N-terminal or C-terminal end of the protease, inserted or deleted at one or more sites within the amino acid sequence of the protease, or substituted with one or more amino acid residues within, or at either or both ends of the amino acid sequence of the protease.
  • the present invention relates to a variant of a parent trypsin-like protease comprising at least one of the loop structures of the trypsin-like Fusarium protease.
  • trypsin-like protease is intended to indicate an enzyme having a three-dimensional structure similar to that of the class of trypsins listed in Table 2, below. It will be understood that trypsin as such is considered to be included within this definition.
  • Fig. 1 shows the three-dimensional structure of the Fusarium trypsin-like protease
  • Fig. 3 shows the three-dimensional structure of Trypsin (Bo taurus) (2ptn);
  • Fig. 4 shows the three-dimensional structure of Tonin (Rattus rattus) (1ton);
  • Fig. 5 shows the three-dimensional structure of Kallikrien A (Sus scrofa) (2pka);
  • Fig. 7 shows the three-dimensional structure of
  • Fig. 8 shows the three-dimensional structure of Neutrophil Elastase (Homo sapiens) (1hne);
  • Fig. 9 shows the three-dimensional structure of Mast Cell Proteinanse (Rattus rattus) (3rp2);
  • Fig. 10 shows the three-dimensional structure of the bacterial trypsin-like protease isolated from Trypsin (Streptomyces griseus) (1sgt) .
  • Arg 170 Tyr + Gly 195 Glu or R170Y+G195E representing mutations in positions 170 and 195 substituting tyrosine and glutamic acid for arginine and glycine, respectively.
  • variants of the trypsinlike Fusarium protease of the invention is based on the parent trypsin-like protease derived from the strain of F. oxysporum deposited with the Deutsche Sammlung von Mikroorganismen with the deposit number DSM 2672. It will be understood that functional analogues of said trypsin-like protease as defined above, e.g. other parent trypsin-like Fusarium proteases, may be modified in a similar manner to that described for the trypsin-like F. oxysporum protease, e.g. by modifying similar positions (according to a structural alignment).
  • the trypsin-like F. oxysporum protease shows a reversed Arg/Lys specificity compared to that of bovine trypsin, which means that the trypsin-like F. oxysporum protease is more Arg-active than Lys-active.
  • Fig. 1 The three-dimensional structure of the Fusarium trypsin-like protease is shown in Fig. 1 and that of the mammalian trypsin-like proteases isolated from Trypsin (Rattus rattus) (1trm), Trypsin (Bo taurus) (2ptn), Tonin (Rattus rattus) (1ton), Kallikrien A (Sus scrofa) (2pka), ⁇ -chymotrypsin (Bos taurus) (2gch), Pancreatic Elastase (Sus scrofa) (3est), Neutrophil Elastase (Homo sapiens) (1hne) and Mast Cell Proteinanse (Rattus rattus) (3rp2) in Figs. 2-9, respectively and that of the bacterial trypsin-like protease isolated from Trypsin (Streptomyces ⁇ riseus) (1sgt) in Fig. 10.
  • the variant of the trypsin-like protease of the invention has one or more characteristic properties, some of which will be explained in detail in the following.
  • the variant of the trypsin-like protease i.e. the immunological cross reactivity
  • the antibody which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989.
  • the immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et al., 1989.
  • Property ii) of the variant of the trypsin-like protease of the invention i.e. the homology between the amino acid sequence of the variant and the amino acid sequence shown in SEQ ID No. 2 is intended to indicate the degree of identity between the two sequences indicating a derivation of the first sequence from the second.
  • a polypeptide is considered to be homologous to the trypsin-like protease if a comparison of the respective amino acid sequences reveals an identity of greater than about 60%, such as above 70%, 80% or 85%.
  • Proteases are globular proteins and quite compact due to the considerable amount of folding of the long polypeptide chain.
  • the polypeptide chain essentially consists of the "bac ⁇ kbone” and its "side-groups". As the peptide bond is planar, only rotations around the C ⁇ -N axis and the C a -C' axis are permitted. Rotation around the C ⁇ -N bond of the peptide backbone is denoted by the torsion angle ⁇ (phi), rotation around the C a -C' bond by ⁇ (psi) [vide e.g. Creighton, T.E. (1984);Proteins; W.H. Freeman and Company, New York].
  • angles of rotation are made by assigning the maximum value of +180° (which is identical to -180°) to the maximally extended chain.
  • the N, C a and C' atoms are all "trans" to each other.
  • the angles ⁇ and ⁇ are assigned the value of 0°. Rotation from this position around the bonds so that the atoms viewed behind the rotated bond move "counterclockwise” is assigned negative values by definition, those "clockwise” are assigned positive values.
  • the values of the torsion angles lie within the range -180° to +180°.
  • Proline residues have a reduced degree of rotational freedom around the N-C ⁇ bond compared to other types of amino acids, because the proline sidechain connects back to the amide nitrogen. This connectivity usually restricts the ⁇ -angles of proline residues to a narrow interval around -60°.
  • the equilibrium between the unfolded and folded state of a protein is to a large extent governed by the entropy difference between the two states, and consequently it is envisaged that the trypsin-like Fusarium protease can be stabilized by reducing the number of different conformations that are accessible in the unfolded state.
  • Introduction of proline residues for other residues in the protein sequence generally reduces the entropy of the unfolded state, due to the restricted rotational freedom for proline residues.
  • substitutions to have effect on the stability of the protein, they must be compatible with the structure of the protein in the folded state, that is, the substituted residues must have ⁇ -angles in the folded state that are in the allowed interval for prolines, and the introduced prolines must not cause an energetically unfavourable packing of the protein atoms.
  • trypsin-like Fusarium protease variants are contemplated, in which a naturally occurring amino acid residue (other than proline) of the amino acid sequence of the parent trypsin-like protease has been substituted with a proline residue at one or more positions, at which positions(s) the dihedral angles ⁇ (phi) and ⁇ (psi) constitute values within the intervals [- 90° ⁇ -40° and -180° ⁇ 180°], preferably within the intervals
  • the stabilized trypsin-like Fusarium protease variants according to this embodiment of the invention may be prepared by subjecting the trypsin-like Fusarium protease to analysis for secondary structure, identifying residues in the protease having dihedral angles ⁇ (phi) and ⁇ (psi) confined to the intervals [-90° ⁇ -40° and -180° ⁇ 180°], preferably the inter- vals [-90° ⁇ -40° and 120° ⁇ 180°] or [-90° ⁇ -40° and -50° ⁇ 10°], excluding residues located in regions in which the trypsin-like Fusarium protease is characterized by possessing ⁇ -helical or ⁇ -sheet structure, if a proline residue is not already at the identified position(s), substitution of the naturally occurring amino acid residue with a proline residue at the identified position(s), preferably by site directed mutagenesis of a gene encoding the trypsin-like Fusarium protease,
  • the secondary structure elements are defined on the basis of hydrogen bindings. Cooperative secondary structure is recognized as repeats of the elementary hydrogen-bonding patterns "turn” and “bridge”. Repeating turns are “helices”, repeating bridges are “ladders”, connected ladders are “sheets”.
  • a computer program DSSP (Define Secondary Structure of Proteins), enabling the computation of Kabsch & Sander files and written in standard PASCAL, is available from the Protein Data Bank, Chemistry Dept., Brookhaven National Laboratory, Upton, N.Y. 11973.
  • prolines are generally not compatible with ⁇ -helical and ß-sheet secondary conformations. Due to the same rotational constraint about the C ⁇ -N bond, and due to the requirement that neighbouring amino acids in the chain are not perturbed, the magnitudes of the dihedral angles phi and psi (and in particular phi) are confined to limited intervals for proline residues in polypeptides.
  • the dihedral angles for proline residues in polypeptides are almost exclu ⁇ sively within the intervals [-90° ⁇ -40° and -180° ⁇ 180°], preferably the intervals [-90° ⁇ -40° and 120° ⁇ 180°] or [-90° ⁇ -40° and -50° ⁇ 10°]. In this context, both cis- and trans-proline residues are considered.
  • amino acid residue(s) to be substituted with proline is a hydrophilic or a small hydrophobic amino acid residue, in particular one selected from the amino acid residues A, D, E, K, G, Q, R, S, T, N and V.
  • trypsin-like F. oxysporum protease comprise one or more of the following substitutions:A24P, A49P, V90P, S111P, A124P, A125P, S126P, A132P, S135P, T174P, S175P, S185bP, S185cP, S202P.
  • Stabilization of a given protein may be achieved by covalently binding two regions in the protein that are far apart in sequence but close in space. Such binding may be performed by the introduction of a disulphide-bridge in the protein, i.e. by introducing one or more cysteine residues capable of binding to each other or to other cysteine residues present in the protein.
  • the invention relates to a variant of a trypsin-like Fusarium protease, in which an amino acid residue different from cysteine of the amino acid sequence of the parent trypsin-like protease has been substituted with a cysteine residue in such a manner that the introduced cysteine residue together with another cysteine residue present in the parent protease or introduced therein form a disulphide bridge.
  • Positions in which SS-bridges may be introduced may be identified by comparing the structure of the trypsin-like Fusarium protease with the structures of the homologous trypsins listed in Table 2. By such comparison the following residue pair positions have been identified, between which a disulphide bridge can be introduced:
  • trypsin-like F. oxysporum protease include:
  • the invention consequently further relates to a trypsin-like Fusarium protease variant, in which either or both residues of any of the Asn-Gly sequence appearing in the amino acid sequence of the parent trypsin-like protease is/are deleted or substituted with a residue of a different amino acid.
  • the Asn and/or Gly residue may, for instance, be substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
  • any of the Asn or Gly residues of the Asn-Gly occupying positions 36+38 and/or 217+219 of the parent trypsin-like protease may be deleted or substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
  • a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y may be deleted or substituted with a residue of an amino acid selected from the group consisting of A, Q, S, P, T and Y.
  • SP387 Specific variants of SP387 are:
  • the present invention relates to a trypsin- like Fusarium protease variant, in which one or more amino acid residues present in or constituting a loop structure of the parent trypsin-like protease susceptible to cleavage by a proteolytic enzyme is/are deleted or replaced with one or more amino acid residues so as to obtain a modified loop structure having an improved proteolytic stability.
  • variants according to this embodiment of the invention include a variant, in which loop II of the trypsin-like protease comprising the peptide sequence SRNGGP is substituted with loop II of the trypsin 2ptn isolated from Bos taurus comprising the peptide sequence NSGYH, as follows: S34N+R35S+N36*+G39Y+P40H, and/or loop IV of the trypsin-like protease comprising the peptide sequence VSGYAQSGF is substituted with loop IV of the trypsin 2ptn isolated from Bos taurus comprising the peptide sequence YKSGI , as follows: V59Y+S59a*+G59b*+Y59c*+A60K+Q61S+S62*+F64I, and/or loop IV of the trypsin-like protease comprising the peptide sequence VSGYAQSGF is substituted with loop IV of the trypsin ltrm isolated from Rattus
  • autoproteolysis sites may be removed by changing the amino acids at the autoproteolysis site. Since the trypsin-like F. oxysporum protease cleaves at Lys and Arg residues it is preferred to modify such residues of a parent trypsin-like Fusarium protease having the same or a similar specificity, preferably by substituting with a non or less positively charged amino acid residue.
  • the non or less positively charged amino acid residue may be selected from the group consisting of K, S, V, P, E, D, N, Q, A and G; the amino acid residues K, S, V, or P being particularly preferred.
  • the modification of this parent trypsin-like protease may preferably be made by changing Arg to another amino acid residue (including Lys) or by changing Arg or Lys to a non or less positively charged amino acid as defined above.
  • autoproteolysis can be prevented by changing the amino acid residue occupying the position following the Arg or Lys residue in question to Pro. For instance, this may be done in the positions 90 and/or 123 (according to the structural amino acid numbering defined in Table 1), as follows:
  • the present invention relates to a trypsin-like Fusarium protease variant, in which one or more amino acid residues susceptible to oxidation is/are replaced with another amino acid residue less susceptible to oxidation.
  • the amino acid residue less susceptible to oxidation may for instance be selected from the group consisting of A, E, N, Q, I, L, S and K.
  • the invention relates to a trypsin-like F. oxysporum variant comprising one or more of the following substitutions:
  • the concept is to introduce N-glycosylation sites in loops subject to proteolysis in general and autoproteolysis in particular.
  • the invention relates to a trypsin-like Fusarium protease variant, in which a N-glycosylation site has been introduced at an amino acid residue X located in a loop structure on the surface of the protein subject to proteolysis by changing the sequence segment X-Y-Z to Asn-Y1-Ser or Asn-Y1-Thr, provided that Y1 is different from Gly, so as to confer to the variant an improved proteolysis resistance.
  • residue X The introduction of a N-glycosylation site at residue X will make the segment target for proteins that N-glycosylate the amide nitrogen of the Asn residue introduced.
  • residues X, Y, Y1, Z can be any residue, except Y1 should not be Gly, because this would create an Asn-Gly sequence the introduction of which may result in a less stable variant.
  • Asn in the changed sequence segment to become glycosylated the changed sequence segment must lie on the protein surface such that it can be recognized by glycosylaticn proteins.
  • N-glycosylation site can be introduced at the following positions:
  • the auto- -proteolysis sites cannot be removed by substituting Lys and Arg to other residues where the parent trypsin-like Fusarium protease has low specificity.
  • trypsin-like F. oxysporum variants comprises one or more of the following substitutions:
  • Tyr residues may be modified to other amino acids, preferably a hydrophobic amino acid when the Tyr is burried in the interior of the protein or a hydrophilic amino acid when the Tyr is exposed on the protein surface.
  • Examples of specific trypsin-like F. oxysporum variants comprises on or more of the following substitutions:Y59cN,Q,S,A,F
  • the concept is to alter the pi for the protein such that it approaches the pH of the detergent formulation.
  • the pi can be raised by changing negatively charged or neutral amino acids to positively charged amino acids or by changing positively charged residues to more positively charged residues.
  • the pi can be lowered by changing positively charged or neural amino acids to negatively charged amino acids or by changing negatively charged amino acids to more negatively charged amino acids .
  • the positions suited for substitution should be located on the protein surface. It is preferred that the amino acid substitutions result in a variant protease having a pi just below the pH of the detergent.
  • an amino acid residue located in one or more positions of the parent trypsin-like Fusarium protease and exposed at the surface of the molecule may be substituted: 17, 18, 20, 21, 23, 24, 25, 26, 27, 34, 35, 36, 38, 39, 40, 41, 48, 49, 50, 59a, 59b, 59c, 60, 61, 62, 63, 65, 67, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 86, 87, 88, 89, 90, 91, 93, 94, 95, 98, 99, 100, 101, 109, 110, 111, 113, 114, 115,116, 117, 118, 119, 120, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 143, 144, 145,
  • the invention further relates to a trypsin-like Fusarium protease, in which one or more amino acids around the active site has been substituted with an other amino acid sequence so as to obtain a change in the polarity around the active site.
  • substituting the serine at position 214 in a parent trypsin-like Fusarium protease with another amino acid may make the enzyme more active.
  • An example of a variant of the trypsin-like F. oxysporum protease according to this embodiment of the invention comprises the substitution S214A.
  • the concept is to remove critical or potential
  • N-glycosylation sites near the binding cleft region as they might interfere with binding of substrate For instance, a variant of a trypsin-like F. oxysporum protease in which the N-glycosylation site 223a has been removed may result in an improved substrate binding. More specifically the following substitutions are contemplated: N223aS,G,R,K
  • the present invention relates to a trypsinlike protease variant improved by substituting any of its surface loops near the active site with the corresponding surface loop from the trypsin-like F. oxysporum protease disclosed herein.
  • the loop to be inserted may show a higher homology to that of the trypsin-like F. oxysporum protease, for instance a homology of at least 80%, such as at least 85%, 90% or even at least 95% with that of the corresponding loop structure of the trypsin-like F. oxysporum protease.
  • the loop structure of a trypsin-like protease which "corresponds to" a given loop structure of the trypsin-like F. oxysporum protease may easily be determined by comparison of the three-dimensional structures of the trypsin-like protease in question with that of the trypsin-like F. oxysporum protease.
  • the loop structure to be inserted may either be provided by substituting one or more amino acid residues of the parent loop structure so as to result in the desired modification, or by substituting the entire loop.
  • parent trypsin-like protease to be modified in accordance with this aspect of the invention may be derived from a variety of sources including mammals, vertebrates, insects, microorganism and the like. Examples of mammalian and bacterial trypsin-like proteases are apparent from Table 2 above.
  • any one of the modifications of the amino acid sequence indicated above for the various classes of trypsin-like protease variants may be combmed with any one of the other modifications mentioned above /here appropriate.
  • the DNA sequence encoding a parent trypsin-like protease may be isolated from any cell or microorganism producing the trypsin-like protease in question by various methods, well known in the art.
  • a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the trypsin-like protease to be studied.
  • homologous, labelled oligonucleotide probes may be synthesized and used to identify trypsin-like protease-encoding clones from a genomic library prepared from the organism in question.
  • a labelled oligonucleotide probe containing sequences homologous to a known trypsin-like protease could be used as a probe to identify trypsin-like protease-encoding clones, using hybridization and washing conditions of lower stringency.
  • Yet another method for identifying trypsin-like protease-producing clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming trypsin-like protease-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for the trypsin-like protease thereby allowing clones expressing the trypsin-like protease to be identified.
  • an expression vector such as a plasmid
  • transforming trypsin-like protease-negative bacteria with the resulting genomic DNA library
  • the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., The EMBO J. 3 , 1984,pp. 801-805.
  • phosphoamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA sequence may be of mixed genomic and synthetic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire DNA sequence, in accordance with standard techniques.
  • the DNA sequence may also be prepared bypolymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or R.K. Saiki et al., Science 239, 1988, pp. 487-491.
  • mutations may be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites; mutant nucleotides are inserted during oligonucleotide synthesis.
  • a single-stranded gap of DNA bridging the trypsin-like protease-encodinge sequence, is created in a vector carrying the trypsin-like protease gene.
  • the synthetic nucleotide, bearing the desired mutation is annealed to a homologous portion of the single-stranded DNA.
  • a mutated trypsin-like protease-coding sequence produced by methods described above, or any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • the recombinant expression vector carrying the DNA sequence encoding a trypsin-like protease variant of the invention encoding 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 extrachromo ⁇ somal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, minichromosome or an artificial chromosome.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cellgenome and replicated together with the chromosome(s) into which it has been integrated.
  • the DNA sequence should be operably connected to a suitable promoter sequence.
  • 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 directing the transcription of the DNA sequence encoding a trypsin-like protease variant of the invention, especially in a bacterial host are the promoter of the lac operon of E.
  • coli the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis ⁇ -amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus Amyloliguefaciens ⁇ -amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • useful 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 glu ⁇ coamylase, Rhizomucor miehei lipase, A. orvzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
  • the expression vector of the invention may also comprise a suitable transcription terminator and, ineukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the recombinant protease of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMBl and pIJ702.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • a selectable marker e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise tohygromycin resistance, or the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243.
  • the cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above is advantageously used as a host cell in the recombinant production of a trypsin-like protease variant of the invention.
  • the cell may be transformed with the DNA construct of the invention encoding the variant, conveniently by integrating the DNA construct in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described below in connection with the different types of host cells.
  • the cell of the invention may be a cell of a higher organism such as a mammal or an insect, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
  • a microbial cell e.g. a bacterial or a fungal (including yeast) cell.
  • the yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyces cerevisiae.
  • the filamentous fungus may advantageously belong to a species of Aspergillus, e.g. Aspergillus oryzae orAspergillus niger.
  • Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023.
  • the present invention relates to a method of producing a trypsin-like protease variant of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the protease and recovering the protease from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the protease variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the trypsin-like protease variant secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures including separating the cells from the medium by centrifugation or filtration, and precipi ⁇ tating 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.
  • the protease variant may typically be a component of a detergent composition.
  • it may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molecular 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.
  • a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Other enzyme stabilizers are well known in the art.
  • 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. as powder, granules, paste or liquid.
  • a liquid detergent may be aqueous, typically containing up to 70% water and 0-30% organic solvent, or nonaqueous.
  • the detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic.
  • the detergent will usually contain 0-50% of anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate)
  • anionic surfactant such as linear alkylbenzenesulfonate (LAS), alpha-olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate)
  • AS alcohol ethoxysulfate
  • SAS secondary alkanesulfonates
  • alpha-sulfo fatty acid methyl esters alkyl-or alkenylsuccinic acid, or soap.
  • nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).
  • the detergent composition may additionally comprise one or more other proteases as well as one or more other enzymes conventionally used in detergent compositions, such as an amylase, a lipase, a cutinase, a cellulase, a peroxidase, and/or an oxidase, e.g., a laccase.
  • one or more other proteases as well as one or more other enzymes conventionally used in detergent compositions, such as an amylase, a lipase, a cutinase, a cellulase, a peroxidase, and/or an oxidase, e.g., a laccase.
  • the detergent may contain 1-65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), 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, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).
  • the detergent may also be unbuilt, i.
  • the detergent may comprise one or more polymers.
  • examples are carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), polyethyleneglycol (PEG), poly(vin(l alcohol) (PVA), poly ⁇ carboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers.
  • the enzymes 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 such as, e.g., an aromatic borate ester, and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.
  • 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 such as, e.g., an aromatic borate ester
  • 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, or perfume.
  • fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil-suspending agents, anti-soil-redeposition agents, dyes,bactericides, optical brighteners, or perfume.
  • the pH (measured in aqueous solution at use concentration) will usually be neutral or alkaline, e.g. in the range of 7-11.
  • detergent compositions within the scope of the invention include: 1) A detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising
  • An aqueous liquid detergent composition comprising
  • An aqueous structured liquid detergent composition comprising
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising
  • a detergent composition formulated as a granulate comprising
  • a detergent composition formulated as a granulate comprising
  • An aqueous liquid detergent composition comprising
  • An aqueous liquid detergent composition comprising
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising
  • a detergent composition formulated as a granulate having a bulk density of at least 600 g/l comprising
  • Detergent composition formulated as a nonaqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system (e.g. phosphate), enzyme and alkali.
  • the detergent may also comprise anionic surfactant and/or a bleach system.
  • the protease variant of the invention may be incorporated in concentrations conventionally employed in detergents. It is at present contemplated that, in the detergent composition of the invention, the protease variant may be added in an amount corresponding to 0.00001-1 mg (calculated as pure enzyme protein) of protease variant per liter of wash liquor.
  • ORGANISM Fusarium oxysporum
  • CTGTCTCGCA CTTCTGGTGG TATTACCTCC TCGCTTTCCT CCGTCAGAGT TCACCCTAGC 360

Abstract

La présente invention concerne de nouveaux variants de protéases du type trypsine avec des propriétés améliorées, des ADN de recombinaison codant pour ces variants, des cellules hôtes capables d'exprimer ces variants à partir des ADN de recombinaison, ainsi qu'une méthode pour produire lesdits variants en cultivant les cellules hôtes en question. Les variants obtenus peuvent être avantageusement utilisés dans des compositions et des additifs détergents.
PCT/DK1995/000180 1994-05-04 1995-05-04 Variants de proteases WO1995030743A1 (fr)

Priority Applications (1)

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AU24451/95A AU2445195A (en) 1994-05-04 1995-05-04 Protease variants

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DK50994 1994-05-04
DK0509/94 1994-05-04

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WO1995030743A1 true WO1995030743A1 (fr) 1995-11-16

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6187739B1 (en) 1995-09-21 2001-02-13 Henkel Kommanditgesellschaft Auf Aktien Paste-form washing and cleaning agents
US6248708B1 (en) 1996-09-05 2001-06-19 Henkel-Ecolab Gmbh & Co. Ohg Paste-form detergent containing a mixture of ethoxylated alcohols
US6329333B1 (en) 1997-01-30 2001-12-11 Henkel-Ecolab Gmbh & Co. Ohg Pastelike detergent and cleaning agent
US6380140B1 (en) 1996-04-20 2002-04-30 Henkel Komm.Nditgesellschaft Auf Aktien Enzyme granules containing phosphated starch
US6627592B1 (en) 1998-12-15 2003-09-30 Ecolab Gmbh & Co. Ohg Pasty washing agent
WO2005040372A1 (fr) 2003-10-23 2005-05-06 Novozymes A/S Protease a stabilite amelioree dans les detergents
EP2045321A3 (fr) * 2005-05-27 2009-06-17 Direvo Biotech AG Sérines protéases dotées d'une sensibilité altérée à des substances modulant l'activité
WO2012055954A1 (fr) 2010-10-29 2012-05-03 Ab Enzymes Oy Variantes de protéase à sérine fongique
WO2018077938A1 (fr) * 2016-10-25 2018-05-03 Novozymes A/S Compositions détergentes
CN110269129A (zh) * 2019-07-03 2019-09-24 南京黄教授食品科技有限公司 一种鸭肉源ace抑制肽及其制备方法
CN113637663A (zh) * 2021-08-03 2021-11-12 江南大学 热稳定性提高的胰蛋白酶突变体
RU2783315C2 (ru) * 2018-02-22 2022-11-11 Санофи-Авентис Дойчланд Гмбх Варианты свиного трипсина

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989006270A1 (fr) * 1988-01-07 1989-07-13 Novo-Nordisk A/S Detergent enzymatique
WO1992019729A1 (fr) * 1991-05-01 1992-11-12 Novo Nordisk A/S Enzymes stabilisees et compositions detergentes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989006270A1 (fr) * 1988-01-07 1989-07-13 Novo-Nordisk A/S Detergent enzymatique
WO1992019729A1 (fr) * 1991-05-01 1992-11-12 Novo Nordisk A/S Enzymes stabilisees et compositions detergentes

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6187739B1 (en) 1995-09-21 2001-02-13 Henkel Kommanditgesellschaft Auf Aktien Paste-form washing and cleaning agents
US6380140B1 (en) 1996-04-20 2002-04-30 Henkel Komm.Nditgesellschaft Auf Aktien Enzyme granules containing phosphated starch
US6248708B1 (en) 1996-09-05 2001-06-19 Henkel-Ecolab Gmbh & Co. Ohg Paste-form detergent containing a mixture of ethoxylated alcohols
US6329333B1 (en) 1997-01-30 2001-12-11 Henkel-Ecolab Gmbh & Co. Ohg Pastelike detergent and cleaning agent
US6627592B1 (en) 1998-12-15 2003-09-30 Ecolab Gmbh & Co. Ohg Pasty washing agent
JP2007509615A (ja) * 2003-10-23 2007-04-19 ノボザイムス アクティーゼルスカブ 洗剤中で改良された安定性を有するプロテアーゼ
WO2005040372A1 (fr) 2003-10-23 2005-05-06 Novozymes A/S Protease a stabilite amelioree dans les detergents
JP4880469B2 (ja) * 2003-10-23 2012-02-22 ノボザイムス アクティーゼルスカブ 洗剤中で改良された安定性を有するプロテアーゼ
CN102994486A (zh) * 2003-10-23 2013-03-27 诺维信公司 在洗涤剂中具有改善稳定性的蛋白酶
EP2045321A3 (fr) * 2005-05-27 2009-06-17 Direvo Biotech AG Sérines protéases dotées d'une sensibilité altérée à des substances modulant l'activité
WO2012055954A1 (fr) 2010-10-29 2012-05-03 Ab Enzymes Oy Variantes de protéase à sérine fongique
WO2018077938A1 (fr) * 2016-10-25 2018-05-03 Novozymes A/S Compositions détergentes
RU2783315C2 (ru) * 2018-02-22 2022-11-11 Санофи-Авентис Дойчланд Гмбх Варианты свиного трипсина
CN110269129A (zh) * 2019-07-03 2019-09-24 南京黄教授食品科技有限公司 一种鸭肉源ace抑制肽及其制备方法
CN113637663A (zh) * 2021-08-03 2021-11-12 江南大学 热稳定性提高的胰蛋白酶突变体

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