CA2124316A1 - Protease-stable proteins - Google Patents
Protease-stable proteinsInfo
- Publication number
- CA2124316A1 CA2124316A1 CA002124316A CA2124316A CA2124316A1 CA 2124316 A1 CA2124316 A1 CA 2124316A1 CA 002124316 A CA002124316 A CA 002124316A CA 2124316 A CA2124316 A CA 2124316A CA 2124316 A1 CA2124316 A1 CA 2124316A1
- Authority
- CA
- Canada
- Prior art keywords
- seq
- lipase
- protein
- amino acid
- protease
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
Classifications
-
- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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
- C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
Abstract
A protein with improved stability against proteolytic degradation is provided, wherein one or more protease labile amino acid segments are substituted by protease non-labile amino acid segment(s). The protein to be stabilized is advantageously an enzyme, e.g.
of microbial origin, such as a lipase, for instance a lipase derived from a strain of Humicola, e.g. H. lanuginosa, or Rhizomucor, e.g. R. miehei. The stabilized protein may be produced by recombinant DNA techniques and may advantageously be used for detergent purposes.
of microbial origin, such as a lipase, for instance a lipase derived from a strain of Humicola, e.g. H. lanuginosa, or Rhizomucor, e.g. R. miehei. The stabilized protein may be produced by recombinant DNA techniques and may advantageously be used for detergent purposes.
Description
WO93/11254 ~ ~ ~ 3 1 i~ PCT/DK92/0035l PROTEASE-STABLE PROTEINS
FIELD OF INVENTION
The present invention relates to a protein with improved stability against proteolytic degradation, a DNA sequence 5 encoding the protein, an expression vector and cell including the DNA sequence, a method of producing the protein, as well as a detergent additive and composition incorporating a specific class of protein of the invention.
BACKGROUND OF THE IIIVENTION
o Apart from regular ~-helices and ~-sheets, the secondary structure of proteins has been found to comprise non-regular structures which are most commonly termed loops. Several attempts to define such loop structures more precisely have been published. Thus, J. Leszczynski and G.D. Rose, Science 15.234, 1986, pp. 849-855, have identified loops on most o~ the 67 proteins they examined. These loops are characterised as continuous segments of 6-16 amino acid residues located on the surface of the protein molecule, typically containing one or more reverse turns in order to bring the ends of the segments 20 together. E.J. Milner-White and R. Poet, TIBS 12, 1987, pp.
189-192, describe the structure of different types of loops, primarily ~-turns or hairpins which are classified in four different classes according to their hydrogen bond arrangements and which may have a length from 1 to 8 residues. J.M. Thornton 25 et al., BioEssavs 8 (2), 1988, pp. 63-68, define loops as segments which connect the regular secondary protein structures. The loops often form binding and recognition sites, and any variability (such as insertions, delet-ons or sequence changes) among homologous proteins typically resides in the ~o loop structures. According to Thornton et al., most loops have five or less amino acid residues, and the majority of these have 4 or 5 residues.
WO93/11254 - PCT/DK92/003~1 l fi 2 As mentioned above, the various loop structures are typically present on the surface of proteins. They are therefore prone to degradation by proteolytic degradation which usually has an adverse effect on protein activity. It is an object of the 5 present invention to provide proteins which are less prone to attack by proteolytic enzymes.
SUMMARY OF THE INVENTION
The present invention relates to a protein with improved stability against proteolytic degradation, wherein one or more l0 protease labile amino acid segments are substituted by protease non-labile amino acid segment(s~.
In the present context, the term "amino acid segment" is intended to indicate a sequence of consecutive amino acid residues typically comprising two, three, four, five or more amino acid residues, which may be located anywhere in the protein molecule, but which typically does not form part of a regular secondary structure of a protein (i.e~ an ~-helix or a ~-sheet). Such amino acid segments are often found in loop regions connecting such regular structures. The term "protease 20 labile" is used to indicate an amino acid segment which is preferentially subject to degradation by a proteolytic enzyme, while the term "protease non-labile" is used to indicate an amino acid segment which is more slowly, or not at all, degraded by a proteolytic enzyme.
25 According to the invention, it has been found that certain amino acid segments are less liable to be degraded by proteolytic enzymes, due to the amino acids present in the segment and their contacts (such as hydrogen bonds, van der Waals contacts and ionic interactions) with other amino acids 30 in the molecule. It has furthermore been found that different proteolytic enzymes preferentially attack different amino acid segmenls so that a segment which is non-labile in the presence WO93/11254 ,~/~ PCT/DK92/003~1 ~ u~J
of one protease may be labile in the presence of another protease. Non-labile amino acid segments may be identified by the following method:
Amino acid segments of a specific protein which are labile to s a particular protease are identified by incubating the protein with that protease for a period of time sufficient to provide cleavage of the protein into smaller peptide fragments. Each combination of protein, and protease will, under the same reaction conditions, result in the same pattern of peptides lO generated by proteolytic cleavage (a so-called peptide map).
The progression of the proteolytic degradation of the protein may be analysed by varying the incubation time. If the incubation time is kept very brief, primary cleavage sites in the protein may be identified by N-terminal amino acid 15 sequencing after isolation of the peptide fragments by HPLC
(cf. K.L. Stone et al., "Enzymatic digestion of proteins and HPLC peptide isolation" in A Practical Guide to Protein and Pe~tide Purification for Microsequencinq, 1989). Such identified prlmary cleavage sites may then be analyzed by means 20 of computer graphics, where the cleavage sites are highlighted on the known three-dimensional structure of the protein. As indicated above, such cleavage sites are located on the surface of the protein (at least in non-proteolytic proteins), especially in loop regions. Amino acid segments on the same and 25 other proteins (including the protease itself) which are assumed not to be labile to the protease when tested under similar conditions are compiled by searching an appropriate database. Such non-labile segments, which may also be derived from loop regions, are then fitted into a computer graphic 30 model of the protein and evaluated for appropriate sequence, three-dimensional structure and contacts with surrounding amino acid residues in the protein. If contacts between amino acid residues in the substituent amino acid segment and the protein sequence in which it has been introduced are not optimal, it is ~s possible to substitute one or more amino acid residues within the segment to ensure a better fit.
W093/11254 PCT/DK92/003~}
In the present description and claims, the following abbreviations are used:
Amino acids:
A = Ala = Alanine 5 V = Val = Valine L = Leu = Leucine I = Ile = Isoleucine P = Pro - Proline F = Phe = Phenylalanine lo W = Trp = Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser = Serine T = Thr = Threonine 15 C = Cys = Cysteine Y = Tyr = Tyrosine : N = Asn = Asparagine Q = Gln = Glutamine D = Asp = Aspartic Acid 20 E = Glu = Glutamic Acid K = Lys = Lysine ~ ~ R = Arg = Arginine :~ ; H = His = Histidine In describing lipase variants according to the invention, the 2s following nomenclature is usèd for ease of reference:
Original amino acid(sJ:position(s):substituted amino acid(s) According to this nomenclature, for instance the substitution of tyrosine for arginine in position 164 is shown as:
30 A substitution of an amino acid segment, for instance the : substitution of amino acids 209-212 for the segment YPRS (SEQ
ID No. 8) is shown as:
subst.(209-212)YPRS
WO 93/11254 ~ ~ 6 PCI/DK92/003~1 ..., .~ _ DETAILED DISCLOSURE OF THE INVENTION
In particular, the protease non-labile amino acid segment may be derived from a lipase or protease or any other protein in which a suitable non-labile amino acid segment has been 5 identified as described above, or it may be a synthetic segment constructed in accordance with the principles outlined above.
The protein according to the invention, provided with non-labile loop sequence(s) may be any protein which is frequently brought into contact with proteases when used and which is lo consequently subject to loss or substantial reduction of activity due to proteolytic cleavage. Thus, the protein may be an enzyme, in particular a detergent enzyme which is frequently used together with a protease. Examples of such enzymes are an amylase, a cellulase, a peroxidase, a xylanase and a protease.
;~:15 In particular the enzyme may be a lipase as it has previously been recognised that lipases are prone to proteolytic degradation for which reason it is problematic to include both lipases ~ and proteases in detergent compositions (cf. for ~;instance Wo 89/04361 and EP 407 225). Although the parent 20 lipase may be derived from a variety of sources such as mammalian lipases, e.g. pancreatic, gastric, hepatic or lipoprotein lipases, it is generally preferred that it is a microbial lipase. As such, the parent lipase may be selected from yeast, e.g. Candid~, lipases, bacterial, e.g. Pseudomonas, 25 lipases or fungalj e.g. Humicola or Rhizomucor, lipases.
In a preferred embodiment of the lipase protein of the invention, the parent lipase is a Humicola lanuginosa lipase, in particular the lipase produced by H. lanuginosa strain DSM
4106 (cf. EP 258 068). In this embodiment, the protease labile 30 amino acid segment to be substituted is preferably REFG (SEQ ID
No. 1) at positions 209-212 of the lipase molecule, DYGN (SEQ
ID No. 2) at positions 162-165 of the lipase molecule, or EGID
(SEQ ID No. 3) at positions 239-242 of the lipase molecule.
WO93/11254 PCT/DK92/003~1 ~ ~h~ 6 In particular, the segment REFG may be substituted by a segment selected from the group consisting of GASG (SEQ ID No. 4), GAAG
(SEQ ID No. 5), GARG (SEQ ID No. 6~, YPGS (SEQ ID No. 7), YPRS
(SEQ ID No. 8), HNRG tSEQ ID No. 9), YTGN (SEQ ID No. 10), ISSE
5 (SEQ ID No. 11), NNAG (SEQ ID No. 12), SFIN (SEQ ID No. 13), DQNG (SEQ ID No. 14~, ASFS (SEQ ~D No. 15), SRGV (SEQ ID No.
16), LDTG (SEQ ID No. 17), YYAA (SEQ ID No. 18), INDI (SEQ ID
No. 19), WYFG (SEQ ID No. 20), and SIEN (SEQ ID No. 21) ; the segment DYGN (SEQ ID No. 2) may be substituted by a segment lO selected from the group consisting of GSTY (SEQ ID No. 22), DSTN (SEQ ID No. 23), PDLR (SEQ ID No. 24), LDTG (SEQ ID No.
25), GNRY (SEQ ID No. 26), SGVM (SEQ ID No. 27) , RYPS (SEQ ID
No. 28), NGLV (SEQ ID No. 29), SFSI (SEQ ID No. 30), LGSP (SEQ
ID No. 31), RASF (SEQ ID No. 32), VPWG (SEQ ID No. 33), PDLN
15 (SEQ ID No. 34), SFVP (SEQ ID No. 35), PDYR (SEQ ID No. 36), PRLP (SEQ ID No. 37), TVLP (SEQ ID No. 38), IGTC (SEQ ID No.
39), TGGT (SEQ ID No. 40), TNKL (SEQ ID No. 41), and VGDV tSEQ
ID No. 42); and the segment EGID (SEQ ID No. 3) may be substituted by a segment selected from the group consisting of 20 IGVL (SEQ ID No. 43), GSTY ~SEQ ID No. 44), RYAN ~SEQ ID No.
: 45), PNIP (SEQ ID No. 46), and TLVP (SEQ ID No. 47).
By an alternative procedure to produce a protease non-labile amino acid segment, one or more amino acid residues in the ; segment REFG (SEQ ID No. 1) or DYGN (SEQ ID No. 2) may be : 25 substituted by any amino acid residue capable of making the lipase less protease labile. Examples of such amino acid residues are proline and arginine. In particular, Arg 209, Glu 210, Phe 211 or Gly 212 may be substituted by Pro or Arg, and/or Asp 162, Tyr 163, Gly 164 or Asn 165 may be substituted 30 by Pro or Arg.
In another aspect, the present invention relates to a DNA
construct comprising a DNA sequence encoding a protein of the invention. A DNA sequence encoding the present protein may, for instance, be isolated by initially establishing an appropriate 35 cDNA or genomic library and screening for positive clones by WV93/112~4 ' PCT/DK92/003~1 i ) 1 J~
conventional procedures such as by hybridization to oligonucleotide probes synthesized on the basis of the full or partial amino acid sequence of the protein. The genomic or cDNA
sequence encoding the protein may then be modified at a site s corresponding to the site(s) at which it is desired to introduce substituent amino acid segments, e.g. by site-directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence in accordance with well-known procedures.
lo Alternatively, the DNA sequence'encoding the protein 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.
15 801 805. According to the phospho~midite method, oligonucleotides are synthesized, e.g. in an automatic DNA
synthesizer, purified, anneaIed, ligated and cloned in appropriate vectors.
Finally, the DNA sequence may be of mixed genomic and 20 syntheticj mixed synthetic and cDNA or mixed genomic and cDNA
origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments ~orresponding to various parts of the entire D~A construct, in accordance with standard techniques. The DNA construct may also be prepared by ~' 25 polymerase chain reaction using specific primers, for ins~ance as described in Us 4,683,202 or R.K. Saiki et al., Science 239, ~988, pp. 487-491.
According to the invention, DNA sequence produced by methods described above, or any alternative methods known in the art, 30 may be inserted into a recombinant 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.
To permit the secretion of the expressed protein, nucleotides , ..~ '3 '--encoding a "signal sequence" may be inserted prior to the protein-coding sequence. For expression under the direction of control sequences, a target gene to be treated according to the invention is operably linked to the control sequences in the 5 proper reading frame. Promoter sequences that can be in-corporated into plasmid vectors, and which can support the transcription of the mutant protein gene, include but are not limited to the prokaryotic ~-lactamase promoter (Villa-Kamaroff, et al., I978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-lo 3731) and the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Further references can also be found in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94.
The host cell used for the production of the present protein 15 may be a higher eukaryotic cell such as an insect cell or a prokaryotic or a eukaryotic microorganism such as a bacterium or a fungus, including yeast and filamentous fungus.
Examples of suitable yeast cells include cells of Saccharomyces spp., such as S. cerevisiae or a methylotrophic yeast from the 20 genera Hansenula, such as Hansenula polymorpha, or Pichia such as Pichia pastoris. Examples of suitable bacterial cells include cells of Bacillus spp., such as cells of B. subtilis, B. licheniformis or B. lentus.
According to one embodiment-the host cell is transformed by an 25 expression vector carrying the DNA sequence. If expression is to take place in a secreting microorganism such as B. subtilis a signal sequence may follow the translation initiation signal and precede the DNA sequence of interest. The signal sequence ::
acts to transport the expression product to the cell wall where 30 it is cleaved from the product upon secretion. The term "control sequences" as defined above is intended to include a signal sequence, when is present.
In a currently preferred melhod of producing the protein of the WO93/11254 ~ 3~ PCT/DK92/003~1 invention, a filamentous fungus is used as the host organism.
The filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant pro~eins, e.g. a strain of Aspergillus sp., such as s A. ni~er, A. nidulans or A. oryzae. The use of A. oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.
For expression of the protein in Aspergillus, the DNA sequence coding for the protein variant is preceded by a promoter. The lO promoter may be any DNA sequence exhibiting a strong transcriptional activity in Aspergillus and may be derived from a gene encoding an e~tracelluar or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.
;: ~
15 Examples of suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ~-amylase, A. niger acid stable ~-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A.
oryzae alkaline protease or A. oryzae triose phosphate 20 isomerase.
In particular when the host organism is A. oryzae, a preferred promote~ for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong transcriptional actlvlty in A. oryzae. The sequence of the TAKA
25 amylase promoter appears from EP 238 023.
Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
; The techniques used to transform a fungal host cell may suitably be as described in EP 238 023.
30 To ensure secreiion of the protein from the host cell, the DNA
sequence encoding the protein may be preceded by a signal 2~4 PCT/DK92/003~1 sequence which may be a naturally occurring signai s~oquence or a functional part thereof or a synthetic se~uence providing secretion of the protein from the cell. In particular, the signal sequence may be derived from a gene encoding an 5 Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, or a gene encoding a Humicola cellulase, xylanase or lipase. The signal sequence is preferably derived from the gene encoding A. oryzae TAKA
amylase, A. niger neutral ~-amylase, A. niger acid-stable ~-lO amylase or A. niger glucoamylase.
The medium used to culture the transformed host cells may beany conventional medium suitable for growing Aspergillus cells.
The transformants are usually stable and may be cultured in the absence of selection pressure. However, if the transformants 15 are found to be unstable, a selection marker introduced into the cells may be used for selection.
The mature protein 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 ; 20 centrifugation or filtration, and 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.
The present invention also relates to a detergent additive 25 comprising a lipase protein according to the invention and a protease, preferably in the form of a non-dusting granulate, stabilized liquid or protected enzyme. Non-dusting granulates may be produced e.g. according to US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by 30 methods known in the art. Liguid 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. Other enzyme stabilizers are well ~.nown in the art. Protected enzymes may be prepared WO 93J1 12S4 ~ n PCT/DK92/00351 according to the method disclosed in EP 238 216.
The detergent additive may suitably contain 0.02-200 mg of enzyme protein per gram of the additive. It will be understood that the detergent additive may further include one or more 5 other enzymes, such as a cellulase, peroxidase or amylase~
conventionally included in detergent additives.
In a still further aspect, the invention relates to a detergent composition comprising a lipase protein of the invention and a protease. Detergent compositions of the invention additionally 10 comprise surfactants which may be of the anionic, non-ionic, cationic, amphoteric, or zwitterionic type as well as mixtures of these surfactant classes. Typical examples of suitable surfactants are linear alkyl benzene sulfonates (LAS), alpha olefin sulfonates (AOS), alcohol ethoxy sulfates (AEOS), 15 alcohol ethoxylates (AEO), alkyl sulphates (AS), alkyl ~, polyglycosides (APG) and alkali metal salts of natural fatty acids.
Detergent compositions of the invention may contain other detergent ingredients known in the art as e.g. builders, 20 bleaching agents, bleach activators, anti-corrosion agents, sequestering agents, anti soil-redeposition agents, perfumes, enzyme stabilizers, etc.
. ~
The detergent composition of the invention may be formulated in any convenient form, e.g. as a powder or liquid. The enzyme may 2s be stabilized in a liquid detergent by inclusion of enzyme stabilizers as indicated above. Usually, the pH of a solution of the detergent composition of the invention will be 7-12 and in some instances 7.0-10.5. Other detergent enzymes such as cellulases, peroxidases or amylases may be included the 30 detergent compositions of the invention, either separately or in a combined additive as described above.
WO93/112~4 PCT/DK92/003~1 ,;~ l2 ~RIEF DESCRIPTION OF THE DRAWINGS
The present invention is described in the following with reference to the appended drawings, in which Fig. 1 shows a restriction map of plasmid pAO1;
5 Fig. 2 shows a restriction map of plasmid pAHL;
Fig. 3 is a schematic representation of the preparation of plasmids encoding lipase variants by polymerase chain reaction (PCR);
Fig. 4 is a schematic representation of the three-step 0 mutagenesis by PCR; and Fig. 5 shows the protease stability of variant lipases of the invention as compared to that of the wild type H. lanuginosa lipase. ~
The invention is further illustrated in the following examples 15 which are not in any way intended to limit the scope of the invention as claimed.
METHODS `
.
Expression of Humicola lanuginosa lipase in Aspergillus oryzae The cloning of the ~umicola lanuginosa lipase and the express-20 ion and characterization thereof in Aspergillus oryzae is desc-ribed in European patent application No. 305 216. The expres-sion plasmid used was named p960.
The expression plasmid used in this application is identical to p960, except for minor modifications just 3' to the lipase co-2s ding region. The modifications were made in the following way:p960 was digested with NruI and BamHI restriction enzymes.
WO93/11254 ~ . PCT/DK92/003~1 ~ s, Between these two sites the BamHI/NheI fragment from plasmid pBR322, in which the NheI fragment was filled in with Klenow polymerase, was cloned, thereby creating plasmid pAO1 (Fig. 1), which contains unique BamHI and NheI sites. Between these s unique sites BamHI/XbaI fragments from p960 was cloned to give pAHL (Fig. 2).
Site directed in vitro mutagenisation of the lipase gene The approach used for introducing mutations into the lipase gene is described by Nelson & Long, Analytical Biochemistry, lo 180, 147-151 (1989). It involves the 3-step generation of a PCR
(polymerase chain reaction) fragment containing the desired mutation introduced by using a chemically synthesized DNA-strand as one of the primers in the PCR-reactions. From the PCR
generated fragment, a DNA fragment carrying the mutation can be 15 isolated by cleavage with restriction enzymes and re-inserted into the expression plasmid. This method is thoroughly descri-bed in example 1. In Fig. 3 and 4 the method is further outlined.
Transformation of Aspergillus oryzae (general procedure) 20 100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) was inoculated with spores of A . oryzae and incubated with shaking for about 24 hours. The mycelium was harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgS04. The mycelium was suspended 2s in 15 ml of 1.2 M MgS04, 10 mM NaH2PO~, pH 5.8. The suspension was cooled on ice and 1 ml of buffer containing 120 mg of Novo-zym~ 234, batch 1687 was added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma type H25) was added and incubation with gentle agi-tation continued for 1.5-2.5 hours at 37C until a large number ,o of protoplasts was visible in a sample inspected under the microscope.
WO93/l125~ , j,;" ~ ,,"~ 14 PCT/DK92/003~1 The suspension was filtered through miracloth, the filtrate transferred to a sterile tube and overlaid with 5 ml of 0.6 M
sorbitol, 100 m~'Tris-HCl, pH 7Ø Centrifugation was performed for 15 min. at 1000 g and the protoplasts were collected from s the top of the MgS04 cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris-HC1, pH 7.5, 10 mM CaCl~) were added to the protoplast suspension and the mixture was centrifugated for 5 min. at 1000 g. The protoplast pellet was resuspended in 3 ml of STC and repelleted. This step was repeated. Finally, the lo protoplasts were resuspended in 0.2-l ml of STC.
100 ~l of protoplast suspension was mixed with 5-25 ~g of p3SR2 (an A. ni~ulans amdS gene carrying plasmid described in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. ~, 1430-1439, Aug.
1983) in lo ~l of STC. The mixture was left at room temperature 15 for 25 min. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl2 and 10 mM Tris-HCl, pH 7.5 was added and carefully mixed (twice) and finally 0.85 ml of the same solution was added and careful-ly mixed. The mixture was left at room temperature for 25 min., spun at 2.500 g for 15 min. and the pellet was resuspended in 20 2 ml of 1.2 M sorbltol. After one more sedimentation the pro-toplasts were spread on minimal plates (Cove, Biochem. Biophys.
Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH = 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit back-ground growth. After incubation for 4-7 days at 37C spores 25 were picked, suspended in sterile water and spread for single colonies. This procedure was repeated and spores of a single colony after the second reisolation were stored as a defined transformant.
~:
WO93/112~4 PCT/DK92/003;1 ~ f EXAMPLES
Construction of a plasmid expressing the Subst.t209-2l2)GASG
variant of Humicola lanuginosa lipase 5 Linearization of Plasmid pAHL
The circular plasmid pAHL was linearized with the restriction enzyme SphI in the following 50 ~l reaction mixture: 50 mM
NaCl, lO mM Tris-HCl, pH 7.9, lO mM MgCl~, 1 mM dithiothreitol, l ~g plasmid and 2 units of SphI. The digestion was carried out lO for 2 hours at 37C. The reaction mixture was extracted with phenol (equilibrated with Tris-HCl, pH 7.5) and precipitated by adding 2 volumes of ice-cold 96% ethanol. After ~entrifugation and drying of the pellet, the linearized DNA was dissolved in 50 ~l of H.O and the concentration estimated on an agarose gel.
15 3-step PCR mutaqenesis As shown in Fig. 5, the 3-step mutagenisation involves the use of four primers:
Mutagenisation primer (=A): 5'-TTTGATCCAGTACTCTGGGCTA-GAATGGCTGTAACCAGAAGCACCCGGCGGGA-GTCTAGG-3'(SEQ ID No. 48) ~;
PCR Helper I (=B): 5'-GGTCATCCAGTCACTGAGACCCTCTACCTATTAAA-TCGGC-3'(SEQ ID No. 49) PCR Helper 2 ~=C) : 5'-CCATGGCTTTCACGGTGTCT-3' (SEQ ID No. 50) 25 PCR Handle (=D): 5'-GGTCATCCAGTCACTGAGAC-3' (SEQ ID No. 5l) Helper l and helper 2 are complementary to sequences outside the coding region, and can thus be used in comblnation with any mutagenisation primer in the construction of a mutant sequence.
All 3 steps were carried out in the following buffer contai-ning: lO mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001%
gelatin, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM TTP, 2.5 units Taq polymerase.
5 In step l, lO0 pmol primer A, lOO pmol primer B and l fmol linearized plasmid were added to a total of lO0 ~l reac-tion mixture and 15 cycles consisting of 2 minutes at 95C, 2 minutes at 37C and 3 minutes at 72C were carried out.
The concentration of the PCR product was estimated on an agaro-lO se gel. Then, step 2 was carried out. 0.6 pmol step l productand l fmol linearized plasmid were contained in a total of lO0 ~l of the previously mentioned buffer and l cycle consisting of 5 minutes at 95C, 2 minutes at 37C and lO minutes at 72C was carried out.
lS To the step 2 reaction mixture, lO0 pmol primer C and lO0 pmol primer D are added (l ~l of each) and 20 cycles consisting of 2 minutes at 95C, 2 minutes at 37C and 3 minutes at 72C were carried out. This manipulation constituted step 3 in the mutagenisaticn procedure.
20 Isolation of mutated restriction fraament:
The product from step 3 was isolated from an agarose gel and re-dissolved in 20 ~l of H20. Thenj it was digested with the re-striction enzymes BamHI and BstXI in a total volume of 50 ~l with the following composition: lO0 mM NaCl, 50 mM Tris-HCl, pH
25 7.9, lO mM MgCl2, 1 mM DTT, lO units of BamHI and lO units of BstXI. Incubation was carried out at 37C for 2 hours. The 733 bp BamHI/BstXI fragment was isolated from an agarose gel.
Liqation to ex~ression vector pAHL:
The expression plasmid pAHL was cleaved with BamHI and BstXI
30 under the conditions described above and the large fragment was isolated from an agarose gel. To this vector, the mutated frag-ment isolated above was ligated and the ligation mix was used WO 93/112~4 , PCrJDK92/003~1 r~
to transform E.coli. The presence and orientation of the frag-ment was verified by cleavage of a plasmid preparation from a transformant with restriction enzymes. Sequence analysis was carried out on the double-stranded plasmid using the dideoxy s chain termination procedure developed by Sanger. The plasmid was named pAHLS(209-212)GASG and is identical to pAHL, except for the substituted codons.
Construction of plasmids expressing other variants of Humicola 10 lipase Using the same method as described in example 1, the following mutants were constructed:
Subst.(209-212)YPRS by substituting amino acid residues 209-212 with the fragment YPRS;
15 Subst.(162-16S)PRI,P by substituting amino acid residues 162-165 with the fragment PRLP;
: ~ `
Y164R by substituting the tyrosine (Y) residue in position 164 with an a~ginine (R) residue;
:
G212R by substituting the glycine (G) residue in position 212 20 with an arginine (R) residue;
G212P by substituting the glycine (G) residue in position 212 with a proline (P) residue; and E210R by substituting the glutamic acid (E) residue of position 21Q with an arginine (R) residue.
.
2s The plasmid names (pAHL followed by the mutant names indicated above) and primers used for the modifications are listed below.
W093/l12~4 ~ ~ PCT/DK92/003~l ~ ~ 18 Plasmid name Primer A sequence pAHLS(209-212)YPRS 5'-TTTGATCCAGTACTCTGGGCTAGAATGGCTGTAAGATCT-TGGGTACGGCGGGAGTCTAGG-3' (SEQ ID No. 52) pAHLS(162~165)PRLP 5'-TGAAAACACGTCGATTGGCAATCTTGGTCCACGCAGG-TCTGC-3' (SEQ ID No. 53) pAHLY164R 5'-CACGTCGATATCGCGACCATTTCCACG-3' (SEQ ID No. 54) pAHLG212R 5'-GAATGGCTGTATCTAAATTCGCGCG-3' (SEQ ID No. 55) lo pAHLG212P 5'-GAATGGCTGTATGGAAATTCGCGCG-3' ~SEQ ID No. 56) PAHLE210R 5'-GTAACCGAATCTGCGCGGCGGG-3' (SEQ ID No.
57) lS Expression of lipase ~ariants in A. oryzae The plasmids descri~ed above were transformed into A. oryzae IFO 4177 by cotransformation with p3SR2 containing the amdS
gene from A. nidul ans as described in the transformation pro-cedure given in the methods section above. Protoplasts prepared 20 as described were incubated with a mixture of equal amounts of expression plasmid and p3SR2, approximately 5 ~g of each were used. Transformants which could use acetamide as a sole nitro-gen source were reisolated twice. After growth on YPD for three days, culture supernatants were analyzed using the assay for 25 lipase activity described below.
The best transformant was selected for further studies and grown in a 1 l shake flask on 200 ml FG4 medium t3% soy meal, 3% maltodextrin, 1% peptone, pH adjusted to 7.0 with 4 M NaOH) for 4 days at 300C.
FIELD OF INVENTION
The present invention relates to a protein with improved stability against proteolytic degradation, a DNA sequence 5 encoding the protein, an expression vector and cell including the DNA sequence, a method of producing the protein, as well as a detergent additive and composition incorporating a specific class of protein of the invention.
BACKGROUND OF THE IIIVENTION
o Apart from regular ~-helices and ~-sheets, the secondary structure of proteins has been found to comprise non-regular structures which are most commonly termed loops. Several attempts to define such loop structures more precisely have been published. Thus, J. Leszczynski and G.D. Rose, Science 15.234, 1986, pp. 849-855, have identified loops on most o~ the 67 proteins they examined. These loops are characterised as continuous segments of 6-16 amino acid residues located on the surface of the protein molecule, typically containing one or more reverse turns in order to bring the ends of the segments 20 together. E.J. Milner-White and R. Poet, TIBS 12, 1987, pp.
189-192, describe the structure of different types of loops, primarily ~-turns or hairpins which are classified in four different classes according to their hydrogen bond arrangements and which may have a length from 1 to 8 residues. J.M. Thornton 25 et al., BioEssavs 8 (2), 1988, pp. 63-68, define loops as segments which connect the regular secondary protein structures. The loops often form binding and recognition sites, and any variability (such as insertions, delet-ons or sequence changes) among homologous proteins typically resides in the ~o loop structures. According to Thornton et al., most loops have five or less amino acid residues, and the majority of these have 4 or 5 residues.
WO93/11254 - PCT/DK92/003~1 l fi 2 As mentioned above, the various loop structures are typically present on the surface of proteins. They are therefore prone to degradation by proteolytic degradation which usually has an adverse effect on protein activity. It is an object of the 5 present invention to provide proteins which are less prone to attack by proteolytic enzymes.
SUMMARY OF THE INVENTION
The present invention relates to a protein with improved stability against proteolytic degradation, wherein one or more l0 protease labile amino acid segments are substituted by protease non-labile amino acid segment(s~.
In the present context, the term "amino acid segment" is intended to indicate a sequence of consecutive amino acid residues typically comprising two, three, four, five or more amino acid residues, which may be located anywhere in the protein molecule, but which typically does not form part of a regular secondary structure of a protein (i.e~ an ~-helix or a ~-sheet). Such amino acid segments are often found in loop regions connecting such regular structures. The term "protease 20 labile" is used to indicate an amino acid segment which is preferentially subject to degradation by a proteolytic enzyme, while the term "protease non-labile" is used to indicate an amino acid segment which is more slowly, or not at all, degraded by a proteolytic enzyme.
25 According to the invention, it has been found that certain amino acid segments are less liable to be degraded by proteolytic enzymes, due to the amino acids present in the segment and their contacts (such as hydrogen bonds, van der Waals contacts and ionic interactions) with other amino acids 30 in the molecule. It has furthermore been found that different proteolytic enzymes preferentially attack different amino acid segmenls so that a segment which is non-labile in the presence WO93/11254 ,~/~ PCT/DK92/003~1 ~ u~J
of one protease may be labile in the presence of another protease. Non-labile amino acid segments may be identified by the following method:
Amino acid segments of a specific protein which are labile to s a particular protease are identified by incubating the protein with that protease for a period of time sufficient to provide cleavage of the protein into smaller peptide fragments. Each combination of protein, and protease will, under the same reaction conditions, result in the same pattern of peptides lO generated by proteolytic cleavage (a so-called peptide map).
The progression of the proteolytic degradation of the protein may be analysed by varying the incubation time. If the incubation time is kept very brief, primary cleavage sites in the protein may be identified by N-terminal amino acid 15 sequencing after isolation of the peptide fragments by HPLC
(cf. K.L. Stone et al., "Enzymatic digestion of proteins and HPLC peptide isolation" in A Practical Guide to Protein and Pe~tide Purification for Microsequencinq, 1989). Such identified prlmary cleavage sites may then be analyzed by means 20 of computer graphics, where the cleavage sites are highlighted on the known three-dimensional structure of the protein. As indicated above, such cleavage sites are located on the surface of the protein (at least in non-proteolytic proteins), especially in loop regions. Amino acid segments on the same and 25 other proteins (including the protease itself) which are assumed not to be labile to the protease when tested under similar conditions are compiled by searching an appropriate database. Such non-labile segments, which may also be derived from loop regions, are then fitted into a computer graphic 30 model of the protein and evaluated for appropriate sequence, three-dimensional structure and contacts with surrounding amino acid residues in the protein. If contacts between amino acid residues in the substituent amino acid segment and the protein sequence in which it has been introduced are not optimal, it is ~s possible to substitute one or more amino acid residues within the segment to ensure a better fit.
W093/11254 PCT/DK92/003~}
In the present description and claims, the following abbreviations are used:
Amino acids:
A = Ala = Alanine 5 V = Val = Valine L = Leu = Leucine I = Ile = Isoleucine P = Pro - Proline F = Phe = Phenylalanine lo W = Trp = Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser = Serine T = Thr = Threonine 15 C = Cys = Cysteine Y = Tyr = Tyrosine : N = Asn = Asparagine Q = Gln = Glutamine D = Asp = Aspartic Acid 20 E = Glu = Glutamic Acid K = Lys = Lysine ~ ~ R = Arg = Arginine :~ ; H = His = Histidine In describing lipase variants according to the invention, the 2s following nomenclature is usèd for ease of reference:
Original amino acid(sJ:position(s):substituted amino acid(s) According to this nomenclature, for instance the substitution of tyrosine for arginine in position 164 is shown as:
30 A substitution of an amino acid segment, for instance the : substitution of amino acids 209-212 for the segment YPRS (SEQ
ID No. 8) is shown as:
subst.(209-212)YPRS
WO 93/11254 ~ ~ 6 PCI/DK92/003~1 ..., .~ _ DETAILED DISCLOSURE OF THE INVENTION
In particular, the protease non-labile amino acid segment may be derived from a lipase or protease or any other protein in which a suitable non-labile amino acid segment has been 5 identified as described above, or it may be a synthetic segment constructed in accordance with the principles outlined above.
The protein according to the invention, provided with non-labile loop sequence(s) may be any protein which is frequently brought into contact with proteases when used and which is lo consequently subject to loss or substantial reduction of activity due to proteolytic cleavage. Thus, the protein may be an enzyme, in particular a detergent enzyme which is frequently used together with a protease. Examples of such enzymes are an amylase, a cellulase, a peroxidase, a xylanase and a protease.
;~:15 In particular the enzyme may be a lipase as it has previously been recognised that lipases are prone to proteolytic degradation for which reason it is problematic to include both lipases ~ and proteases in detergent compositions (cf. for ~;instance Wo 89/04361 and EP 407 225). Although the parent 20 lipase may be derived from a variety of sources such as mammalian lipases, e.g. pancreatic, gastric, hepatic or lipoprotein lipases, it is generally preferred that it is a microbial lipase. As such, the parent lipase may be selected from yeast, e.g. Candid~, lipases, bacterial, e.g. Pseudomonas, 25 lipases or fungalj e.g. Humicola or Rhizomucor, lipases.
In a preferred embodiment of the lipase protein of the invention, the parent lipase is a Humicola lanuginosa lipase, in particular the lipase produced by H. lanuginosa strain DSM
4106 (cf. EP 258 068). In this embodiment, the protease labile 30 amino acid segment to be substituted is preferably REFG (SEQ ID
No. 1) at positions 209-212 of the lipase molecule, DYGN (SEQ
ID No. 2) at positions 162-165 of the lipase molecule, or EGID
(SEQ ID No. 3) at positions 239-242 of the lipase molecule.
WO93/11254 PCT/DK92/003~1 ~ ~h~ 6 In particular, the segment REFG may be substituted by a segment selected from the group consisting of GASG (SEQ ID No. 4), GAAG
(SEQ ID No. 5), GARG (SEQ ID No. 6~, YPGS (SEQ ID No. 7), YPRS
(SEQ ID No. 8), HNRG tSEQ ID No. 9), YTGN (SEQ ID No. 10), ISSE
5 (SEQ ID No. 11), NNAG (SEQ ID No. 12), SFIN (SEQ ID No. 13), DQNG (SEQ ID No. 14~, ASFS (SEQ ~D No. 15), SRGV (SEQ ID No.
16), LDTG (SEQ ID No. 17), YYAA (SEQ ID No. 18), INDI (SEQ ID
No. 19), WYFG (SEQ ID No. 20), and SIEN (SEQ ID No. 21) ; the segment DYGN (SEQ ID No. 2) may be substituted by a segment lO selected from the group consisting of GSTY (SEQ ID No. 22), DSTN (SEQ ID No. 23), PDLR (SEQ ID No. 24), LDTG (SEQ ID No.
25), GNRY (SEQ ID No. 26), SGVM (SEQ ID No. 27) , RYPS (SEQ ID
No. 28), NGLV (SEQ ID No. 29), SFSI (SEQ ID No. 30), LGSP (SEQ
ID No. 31), RASF (SEQ ID No. 32), VPWG (SEQ ID No. 33), PDLN
15 (SEQ ID No. 34), SFVP (SEQ ID No. 35), PDYR (SEQ ID No. 36), PRLP (SEQ ID No. 37), TVLP (SEQ ID No. 38), IGTC (SEQ ID No.
39), TGGT (SEQ ID No. 40), TNKL (SEQ ID No. 41), and VGDV tSEQ
ID No. 42); and the segment EGID (SEQ ID No. 3) may be substituted by a segment selected from the group consisting of 20 IGVL (SEQ ID No. 43), GSTY ~SEQ ID No. 44), RYAN ~SEQ ID No.
: 45), PNIP (SEQ ID No. 46), and TLVP (SEQ ID No. 47).
By an alternative procedure to produce a protease non-labile amino acid segment, one or more amino acid residues in the ; segment REFG (SEQ ID No. 1) or DYGN (SEQ ID No. 2) may be : 25 substituted by any amino acid residue capable of making the lipase less protease labile. Examples of such amino acid residues are proline and arginine. In particular, Arg 209, Glu 210, Phe 211 or Gly 212 may be substituted by Pro or Arg, and/or Asp 162, Tyr 163, Gly 164 or Asn 165 may be substituted 30 by Pro or Arg.
In another aspect, the present invention relates to a DNA
construct comprising a DNA sequence encoding a protein of the invention. A DNA sequence encoding the present protein may, for instance, be isolated by initially establishing an appropriate 35 cDNA or genomic library and screening for positive clones by WV93/112~4 ' PCT/DK92/003~1 i ) 1 J~
conventional procedures such as by hybridization to oligonucleotide probes synthesized on the basis of the full or partial amino acid sequence of the protein. The genomic or cDNA
sequence encoding the protein may then be modified at a site s corresponding to the site(s) at which it is desired to introduce substituent amino acid segments, e.g. by site-directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence in accordance with well-known procedures.
lo Alternatively, the DNA sequence'encoding the protein 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.
15 801 805. According to the phospho~midite method, oligonucleotides are synthesized, e.g. in an automatic DNA
synthesizer, purified, anneaIed, ligated and cloned in appropriate vectors.
Finally, the DNA sequence may be of mixed genomic and 20 syntheticj mixed synthetic and cDNA or mixed genomic and cDNA
origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments ~orresponding to various parts of the entire D~A construct, in accordance with standard techniques. The DNA construct may also be prepared by ~' 25 polymerase chain reaction using specific primers, for ins~ance as described in Us 4,683,202 or R.K. Saiki et al., Science 239, ~988, pp. 487-491.
According to the invention, DNA sequence produced by methods described above, or any alternative methods known in the art, 30 may be inserted into a recombinant 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.
To permit the secretion of the expressed protein, nucleotides , ..~ '3 '--encoding a "signal sequence" may be inserted prior to the protein-coding sequence. For expression under the direction of control sequences, a target gene to be treated according to the invention is operably linked to the control sequences in the 5 proper reading frame. Promoter sequences that can be in-corporated into plasmid vectors, and which can support the transcription of the mutant protein gene, include but are not limited to the prokaryotic ~-lactamase promoter (Villa-Kamaroff, et al., I978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-lo 3731) and the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Further references can also be found in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94.
The host cell used for the production of the present protein 15 may be a higher eukaryotic cell such as an insect cell or a prokaryotic or a eukaryotic microorganism such as a bacterium or a fungus, including yeast and filamentous fungus.
Examples of suitable yeast cells include cells of Saccharomyces spp., such as S. cerevisiae or a methylotrophic yeast from the 20 genera Hansenula, such as Hansenula polymorpha, or Pichia such as Pichia pastoris. Examples of suitable bacterial cells include cells of Bacillus spp., such as cells of B. subtilis, B. licheniformis or B. lentus.
According to one embodiment-the host cell is transformed by an 25 expression vector carrying the DNA sequence. If expression is to take place in a secreting microorganism such as B. subtilis a signal sequence may follow the translation initiation signal and precede the DNA sequence of interest. The signal sequence ::
acts to transport the expression product to the cell wall where 30 it is cleaved from the product upon secretion. The term "control sequences" as defined above is intended to include a signal sequence, when is present.
In a currently preferred melhod of producing the protein of the WO93/11254 ~ 3~ PCT/DK92/003~1 invention, a filamentous fungus is used as the host organism.
The filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant pro~eins, e.g. a strain of Aspergillus sp., such as s A. ni~er, A. nidulans or A. oryzae. The use of A. oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.
For expression of the protein in Aspergillus, the DNA sequence coding for the protein variant is preceded by a promoter. The lO promoter may be any DNA sequence exhibiting a strong transcriptional activity in Aspergillus and may be derived from a gene encoding an e~tracelluar or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.
;: ~
15 Examples of suitable promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral ~-amylase, A. niger acid stable ~-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A.
oryzae alkaline protease or A. oryzae triose phosphate 20 isomerase.
In particular when the host organism is A. oryzae, a preferred promote~ for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong transcriptional actlvlty in A. oryzae. The sequence of the TAKA
25 amylase promoter appears from EP 238 023.
Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
; The techniques used to transform a fungal host cell may suitably be as described in EP 238 023.
30 To ensure secreiion of the protein from the host cell, the DNA
sequence encoding the protein may be preceded by a signal 2~4 PCT/DK92/003~1 sequence which may be a naturally occurring signai s~oquence or a functional part thereof or a synthetic se~uence providing secretion of the protein from the cell. In particular, the signal sequence may be derived from a gene encoding an 5 Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, or a gene encoding a Humicola cellulase, xylanase or lipase. The signal sequence is preferably derived from the gene encoding A. oryzae TAKA
amylase, A. niger neutral ~-amylase, A. niger acid-stable ~-lO amylase or A. niger glucoamylase.
The medium used to culture the transformed host cells may beany conventional medium suitable for growing Aspergillus cells.
The transformants are usually stable and may be cultured in the absence of selection pressure. However, if the transformants 15 are found to be unstable, a selection marker introduced into the cells may be used for selection.
The mature protein 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 ; 20 centrifugation or filtration, and 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.
The present invention also relates to a detergent additive 25 comprising a lipase protein according to the invention and a protease, preferably in the form of a non-dusting granulate, stabilized liquid or protected enzyme. Non-dusting granulates may be produced e.g. according to US 4,106,991 and 4,661,452 (both to Novo Industri A/S) and may optionally be coated by 30 methods known in the art. Liguid 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. Other enzyme stabilizers are well ~.nown in the art. Protected enzymes may be prepared WO 93J1 12S4 ~ n PCT/DK92/00351 according to the method disclosed in EP 238 216.
The detergent additive may suitably contain 0.02-200 mg of enzyme protein per gram of the additive. It will be understood that the detergent additive may further include one or more 5 other enzymes, such as a cellulase, peroxidase or amylase~
conventionally included in detergent additives.
In a still further aspect, the invention relates to a detergent composition comprising a lipase protein of the invention and a protease. Detergent compositions of the invention additionally 10 comprise surfactants which may be of the anionic, non-ionic, cationic, amphoteric, or zwitterionic type as well as mixtures of these surfactant classes. Typical examples of suitable surfactants are linear alkyl benzene sulfonates (LAS), alpha olefin sulfonates (AOS), alcohol ethoxy sulfates (AEOS), 15 alcohol ethoxylates (AEO), alkyl sulphates (AS), alkyl ~, polyglycosides (APG) and alkali metal salts of natural fatty acids.
Detergent compositions of the invention may contain other detergent ingredients known in the art as e.g. builders, 20 bleaching agents, bleach activators, anti-corrosion agents, sequestering agents, anti soil-redeposition agents, perfumes, enzyme stabilizers, etc.
. ~
The detergent composition of the invention may be formulated in any convenient form, e.g. as a powder or liquid. The enzyme may 2s be stabilized in a liquid detergent by inclusion of enzyme stabilizers as indicated above. Usually, the pH of a solution of the detergent composition of the invention will be 7-12 and in some instances 7.0-10.5. Other detergent enzymes such as cellulases, peroxidases or amylases may be included the 30 detergent compositions of the invention, either separately or in a combined additive as described above.
WO93/112~4 PCT/DK92/003~1 ,;~ l2 ~RIEF DESCRIPTION OF THE DRAWINGS
The present invention is described in the following with reference to the appended drawings, in which Fig. 1 shows a restriction map of plasmid pAO1;
5 Fig. 2 shows a restriction map of plasmid pAHL;
Fig. 3 is a schematic representation of the preparation of plasmids encoding lipase variants by polymerase chain reaction (PCR);
Fig. 4 is a schematic representation of the three-step 0 mutagenesis by PCR; and Fig. 5 shows the protease stability of variant lipases of the invention as compared to that of the wild type H. lanuginosa lipase. ~
The invention is further illustrated in the following examples 15 which are not in any way intended to limit the scope of the invention as claimed.
METHODS `
.
Expression of Humicola lanuginosa lipase in Aspergillus oryzae The cloning of the ~umicola lanuginosa lipase and the express-20 ion and characterization thereof in Aspergillus oryzae is desc-ribed in European patent application No. 305 216. The expres-sion plasmid used was named p960.
The expression plasmid used in this application is identical to p960, except for minor modifications just 3' to the lipase co-2s ding region. The modifications were made in the following way:p960 was digested with NruI and BamHI restriction enzymes.
WO93/11254 ~ . PCT/DK92/003~1 ~ s, Between these two sites the BamHI/NheI fragment from plasmid pBR322, in which the NheI fragment was filled in with Klenow polymerase, was cloned, thereby creating plasmid pAO1 (Fig. 1), which contains unique BamHI and NheI sites. Between these s unique sites BamHI/XbaI fragments from p960 was cloned to give pAHL (Fig. 2).
Site directed in vitro mutagenisation of the lipase gene The approach used for introducing mutations into the lipase gene is described by Nelson & Long, Analytical Biochemistry, lo 180, 147-151 (1989). It involves the 3-step generation of a PCR
(polymerase chain reaction) fragment containing the desired mutation introduced by using a chemically synthesized DNA-strand as one of the primers in the PCR-reactions. From the PCR
generated fragment, a DNA fragment carrying the mutation can be 15 isolated by cleavage with restriction enzymes and re-inserted into the expression plasmid. This method is thoroughly descri-bed in example 1. In Fig. 3 and 4 the method is further outlined.
Transformation of Aspergillus oryzae (general procedure) 20 100 ml of YPD (Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) was inoculated with spores of A . oryzae and incubated with shaking for about 24 hours. The mycelium was harvested by filtration through miracloth and washed with 200 ml of 0.6 M MgS04. The mycelium was suspended 2s in 15 ml of 1.2 M MgS04, 10 mM NaH2PO~, pH 5.8. The suspension was cooled on ice and 1 ml of buffer containing 120 mg of Novo-zym~ 234, batch 1687 was added. After 5 min., 1 ml of 12 mg/ml BSA (Sigma type H25) was added and incubation with gentle agi-tation continued for 1.5-2.5 hours at 37C until a large number ,o of protoplasts was visible in a sample inspected under the microscope.
WO93/l125~ , j,;" ~ ,,"~ 14 PCT/DK92/003~1 The suspension was filtered through miracloth, the filtrate transferred to a sterile tube and overlaid with 5 ml of 0.6 M
sorbitol, 100 m~'Tris-HCl, pH 7Ø Centrifugation was performed for 15 min. at 1000 g and the protoplasts were collected from s the top of the MgS04 cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris-HC1, pH 7.5, 10 mM CaCl~) were added to the protoplast suspension and the mixture was centrifugated for 5 min. at 1000 g. The protoplast pellet was resuspended in 3 ml of STC and repelleted. This step was repeated. Finally, the lo protoplasts were resuspended in 0.2-l ml of STC.
100 ~l of protoplast suspension was mixed with 5-25 ~g of p3SR2 (an A. ni~ulans amdS gene carrying plasmid described in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. ~, 1430-1439, Aug.
1983) in lo ~l of STC. The mixture was left at room temperature 15 for 25 min. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl2 and 10 mM Tris-HCl, pH 7.5 was added and carefully mixed (twice) and finally 0.85 ml of the same solution was added and careful-ly mixed. The mixture was left at room temperature for 25 min., spun at 2.500 g for 15 min. and the pellet was resuspended in 20 2 ml of 1.2 M sorbltol. After one more sedimentation the pro-toplasts were spread on minimal plates (Cove, Biochem. Biophys.
Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH = 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit back-ground growth. After incubation for 4-7 days at 37C spores 25 were picked, suspended in sterile water and spread for single colonies. This procedure was repeated and spores of a single colony after the second reisolation were stored as a defined transformant.
~:
WO93/112~4 PCT/DK92/003;1 ~ f EXAMPLES
Construction of a plasmid expressing the Subst.t209-2l2)GASG
variant of Humicola lanuginosa lipase 5 Linearization of Plasmid pAHL
The circular plasmid pAHL was linearized with the restriction enzyme SphI in the following 50 ~l reaction mixture: 50 mM
NaCl, lO mM Tris-HCl, pH 7.9, lO mM MgCl~, 1 mM dithiothreitol, l ~g plasmid and 2 units of SphI. The digestion was carried out lO for 2 hours at 37C. The reaction mixture was extracted with phenol (equilibrated with Tris-HCl, pH 7.5) and precipitated by adding 2 volumes of ice-cold 96% ethanol. After ~entrifugation and drying of the pellet, the linearized DNA was dissolved in 50 ~l of H.O and the concentration estimated on an agarose gel.
15 3-step PCR mutaqenesis As shown in Fig. 5, the 3-step mutagenisation involves the use of four primers:
Mutagenisation primer (=A): 5'-TTTGATCCAGTACTCTGGGCTA-GAATGGCTGTAACCAGAAGCACCCGGCGGGA-GTCTAGG-3'(SEQ ID No. 48) ~;
PCR Helper I (=B): 5'-GGTCATCCAGTCACTGAGACCCTCTACCTATTAAA-TCGGC-3'(SEQ ID No. 49) PCR Helper 2 ~=C) : 5'-CCATGGCTTTCACGGTGTCT-3' (SEQ ID No. 50) 25 PCR Handle (=D): 5'-GGTCATCCAGTCACTGAGAC-3' (SEQ ID No. 5l) Helper l and helper 2 are complementary to sequences outside the coding region, and can thus be used in comblnation with any mutagenisation primer in the construction of a mutant sequence.
All 3 steps were carried out in the following buffer contai-ning: lO mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001%
gelatin, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM TTP, 2.5 units Taq polymerase.
5 In step l, lO0 pmol primer A, lOO pmol primer B and l fmol linearized plasmid were added to a total of lO0 ~l reac-tion mixture and 15 cycles consisting of 2 minutes at 95C, 2 minutes at 37C and 3 minutes at 72C were carried out.
The concentration of the PCR product was estimated on an agaro-lO se gel. Then, step 2 was carried out. 0.6 pmol step l productand l fmol linearized plasmid were contained in a total of lO0 ~l of the previously mentioned buffer and l cycle consisting of 5 minutes at 95C, 2 minutes at 37C and lO minutes at 72C was carried out.
lS To the step 2 reaction mixture, lO0 pmol primer C and lO0 pmol primer D are added (l ~l of each) and 20 cycles consisting of 2 minutes at 95C, 2 minutes at 37C and 3 minutes at 72C were carried out. This manipulation constituted step 3 in the mutagenisaticn procedure.
20 Isolation of mutated restriction fraament:
The product from step 3 was isolated from an agarose gel and re-dissolved in 20 ~l of H20. Thenj it was digested with the re-striction enzymes BamHI and BstXI in a total volume of 50 ~l with the following composition: lO0 mM NaCl, 50 mM Tris-HCl, pH
25 7.9, lO mM MgCl2, 1 mM DTT, lO units of BamHI and lO units of BstXI. Incubation was carried out at 37C for 2 hours. The 733 bp BamHI/BstXI fragment was isolated from an agarose gel.
Liqation to ex~ression vector pAHL:
The expression plasmid pAHL was cleaved with BamHI and BstXI
30 under the conditions described above and the large fragment was isolated from an agarose gel. To this vector, the mutated frag-ment isolated above was ligated and the ligation mix was used WO 93/112~4 , PCrJDK92/003~1 r~
to transform E.coli. The presence and orientation of the frag-ment was verified by cleavage of a plasmid preparation from a transformant with restriction enzymes. Sequence analysis was carried out on the double-stranded plasmid using the dideoxy s chain termination procedure developed by Sanger. The plasmid was named pAHLS(209-212)GASG and is identical to pAHL, except for the substituted codons.
Construction of plasmids expressing other variants of Humicola 10 lipase Using the same method as described in example 1, the following mutants were constructed:
Subst.(209-212)YPRS by substituting amino acid residues 209-212 with the fragment YPRS;
15 Subst.(162-16S)PRI,P by substituting amino acid residues 162-165 with the fragment PRLP;
: ~ `
Y164R by substituting the tyrosine (Y) residue in position 164 with an a~ginine (R) residue;
:
G212R by substituting the glycine (G) residue in position 212 20 with an arginine (R) residue;
G212P by substituting the glycine (G) residue in position 212 with a proline (P) residue; and E210R by substituting the glutamic acid (E) residue of position 21Q with an arginine (R) residue.
.
2s The plasmid names (pAHL followed by the mutant names indicated above) and primers used for the modifications are listed below.
W093/l12~4 ~ ~ PCT/DK92/003~l ~ ~ 18 Plasmid name Primer A sequence pAHLS(209-212)YPRS 5'-TTTGATCCAGTACTCTGGGCTAGAATGGCTGTAAGATCT-TGGGTACGGCGGGAGTCTAGG-3' (SEQ ID No. 52) pAHLS(162~165)PRLP 5'-TGAAAACACGTCGATTGGCAATCTTGGTCCACGCAGG-TCTGC-3' (SEQ ID No. 53) pAHLY164R 5'-CACGTCGATATCGCGACCATTTCCACG-3' (SEQ ID No. 54) pAHLG212R 5'-GAATGGCTGTATCTAAATTCGCGCG-3' (SEQ ID No. 55) lo pAHLG212P 5'-GAATGGCTGTATGGAAATTCGCGCG-3' ~SEQ ID No. 56) PAHLE210R 5'-GTAACCGAATCTGCGCGGCGGG-3' (SEQ ID No.
57) lS Expression of lipase ~ariants in A. oryzae The plasmids descri~ed above were transformed into A. oryzae IFO 4177 by cotransformation with p3SR2 containing the amdS
gene from A. nidul ans as described in the transformation pro-cedure given in the methods section above. Protoplasts prepared 20 as described were incubated with a mixture of equal amounts of expression plasmid and p3SR2, approximately 5 ~g of each were used. Transformants which could use acetamide as a sole nitro-gen source were reisolated twice. After growth on YPD for three days, culture supernatants were analyzed using the assay for 25 lipase activity described below.
The best transformant was selected for further studies and grown in a 1 l shake flask on 200 ml FG4 medium t3% soy meal, 3% maltodextrin, 1% peptone, pH adjusted to 7.0 with 4 M NaOH) for 4 days at 300C.
, s Purification of lipase variants of the invention Assav for lipase activity:
A substrate for lipase was prepared by emulsifying glycerine s tributyrat (MERCK) using gum-arabic as emulsifier.
Lipase activity was assayed at pH 7 using pH stat method. One unit of lipase activity (LU/mg) was defined as the amount needed to liberate one micromole fatty acid per minute.
Step l:- Centrifuge the fermentation supernatant, discard the 0 precipitate. Adjust the pH of the supernatant to 7 and add gradually an equal volume of cold 96 ~ ethanol. Allow the mixture to stand for 30 minutes in an ice bath. Centrifuge and discard the precipitate.
Step 2:- Ion exchange chromatography. Filter the supernatant 15 and apply on DEAE-fast flow (Pharmacia TM) column equilibrated with 50 mM tris-acetate buffer pH 7. Wash the column with the same buffer till absorption at 280 nm is lower than 0.05 OD.
Elute the bound enzymatic activity with linear salt gradient in the same buffer (0 to 0.5 M NaCl ) using five column volumes.
20 Pool the fractions containing enzymatic activity.
Step 3:- Hydrophobic chromatography. Adjust the molarity of the pool containing enzymatic activity to 0.8 M by adding solid Ammonium acetate. Apply the enzyme on TSK gel Butyl- Toyopearl 650 C column (available from Tosoh Corporation Japan) which 25 was pre-equilibrated with 0.8 M ammonium acetate. Wash the unbound material with 0.8 M ammonium acetate and elute the bound material with distilled water.
Step 4:- Pool containing lipase activity is diluted with water to adjust conductance to 2 mS and pH to 7. Apply the pool on 30 High performance Q Sepharose (Pharmacia) column pre-equilibrated with 50 mM tris -aceta~e buffer pH 7. Elute the ) s ,~, -f 31 i PCT/DK92/00351 bound enzyme with linear salt gradient.
EXAMPLE S
Protea~e stability of variant proteins The variant lipases Subst.(162-165)PRLP, Subst.(209-212)YPRS, 5 Subst.(209-212)GASG, G212R and G212P were purified as described in Example 4 above and each diluted with O.lM Tris-puffer, pH
9Ø Subsequently, a protease solution containing SavinaseTM
(concentration of lOOmg/ml in 50% mono-propylene glycol (MPG), 1% Boric acid) was added in a proportion of lipase: protease of o 1:5. The final lipase concentration was lmg/ml (except for the variant G212P, for ~hich it was 0.8 mg/ml). The reaction was carried out at 22C. At appropiate times, probes were taken from the reaction mixture and immediately subjected to the above described lipase activity assay.
15 In Fig. 5 the residual activity in per cent of the starting material is shown versus incubation time, for the wild type Humicola lanuginosa lipase (wt) and the variant lipases.
W O 93/11254 ~ PCT/DK92tO03~1 SEQUENCE LISTING
(1) GENERAL IMFORM~IION:
(i) APPLICANT:
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S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DENM~RK
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(D) SOFTW~RE: PatentIn Release #l-r Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: WO PCT/DK9l/00350 . (8) ~ G DATE: 26-NOV-l99l (2) INFORMATION FOR SEQ ID NO: l:
:: (i) SEQUENCE CHARACTERISIICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: s mgle (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: peptide (iii) HYPCrHETICAL: NO
(iii) ANrI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEO ID NO: l:
Arg Glu Phe Gly W O 93/112~4 J ~ PCT/DK92/003~1 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL3GY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FR~GMENT TYPE: internal (xi) SE2UEN OE DESCRIPTION: SEQ ID NO: 2:
Asp Tyr Gly Asn (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUEN OE CHARACTERISTICS:
tA) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRAMDEDNESS: single (D) T~POLOGY: linP~r ::; 20 (ii) MOLECULE TYPE: peptide : (iiij HYPOTHEIICAL: NO
: (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 3:
Glu Gly Ile Asp (2) INFORM~TION FOR SEQ ID NO: 4:
(i) SE~UEN OE CH~RACTERISIICS:
~A) LENGTH: 4 amlno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLCGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOrHETICAL: NO
(iii) ANII-S~SE: NO
W O 93/lt254 PCT/DK92/003~1 s~
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Ala Ser Gly 5 (2) INF3RM~TION FOR SEQ ID NO: 5:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGIH: 4 amlno acids ~B) TYPE: amuno acid (C) STRAMDEDNESS: single (D) TOPOL0GY: linear (ii) MOL~CULE TYPE: peptide (iii~ EhFCT~E~ICAL: NO
~iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQVENCE DESCRlPqlON: SEQ ID NO: 5:
Gly Ala Ala Gly (2) INFORM~TION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
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(iii) ANII-SENSE: NO
(v) F~AGMENT TYFE: internal (xi) S~2UEt~OE DESCRIPIION: S~)Q ID NO: 6:
:; Gly Ala An~ Gly (2) INF~TION F~R SE;2 ID NO: 7:
( i ) SEQU~NOE CHARAC~ERISIICS:
(A) IEN~I: 4 amino acids (B) TYPE: alTLino acid W O 93/ll2~ PCT/DK92/003Sl t~ 'f 24 (C) STRANDEDNESS: single (D) TOPOLCGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HYPOn9E~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEO ID NO: 7:
Tyr Pro Gly Ser 10 (2) INFORM~TION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACIERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL0GY: linear (ii) MOLECULE TYPE: peptide (iii) HhECn~ETICAL: NO
.. . .
(lll) ~ -SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DES~ IPTION: SEQ ID NO: 8:
Tyr Pro Arg Ser : 1 (2~ INFORM~TION FOR SEQ ID NO: 9:
: : (i) SEQUEN OE CHARACTERISTICS::
(A) LENGTH: 4 amlno acids ~B) TYPE:~amino acld (C) STRANDE~SS: single (D) TOPOL,OGY:~linear (ii~ MOLECULE TYPE: peptide (iii~ HYEC~ETIC~L: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPIION: SEQ ID NO: 9:
His Asn Arg Gly _5 W O 93/112~ PCT/DK92/003~1 . 2 (2) INFORMATION FVR SEQ ID NO: l0:
(i) SEQUENCE CHARACIERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRAMDEDNESS: s m~le (D) TOPOL~GY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOqHE~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
Tyr Thr Gly Asn (2) INFORM~TION FOR SEQ ID NO: ll:
(i) SEQUEN OE CHARACTERISllCS:
(A) LENGTH: 4 amino acids (B) TYPE: amlno acid (C) STRANDEDNESS: single (D) ~POL~GY: linear (ii) MO ~ TYPE: peptide :: (iii) HYPOI~ETqCAL: NO
~; (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal ~xi) SEQUEN OE DESCRIPIION: SEQ ID NO: ll:
Ile Ser Ser Glu ~2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amlno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCrHErICAL: NO
3, (iii) ANTI-SENSE: NO
W O 93/1l254 P~CT/DK92/003~1 c ~
. 26 (v) FRAGMENT TYPE: internal txl) SEQUENCE DESCRIPqION: SE~ ID NO: 12:
Asn Asn Ala Gly 5 (2) INFORM~IION FOR SEQ ID NO: 13:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCrHETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: lnternal (xi) SEQVEN OE DESCRIPTION: SEO ID NO: 13:
~ S~er Phe Ile Asn ; 1 (2) INFORM~TION FOR SEQ ID NO: 14:
(i) SEQUENOE CHARACTE~ISIICS:
(A) LENGTH: 4 am mo aci~ds : (B) 'TYPE: amino acid : : (C) SIRANDEDNESS: s mgle (D) TOPOLOGY: linear (ii) MOT~CULE TYPE: peptide 2s (iii) HYPOqlE~ICAL: NO
(iii) ANII-SENSE: NO
(v) FRAEMENr TYPE: internal (xi) SEQUENCE DESCRIPIION: SEQ ID NO: 14:
Asp Gln Asn Gly (2) INFORM~TION FOR SEQ ID NO: 15:
(i) SEQUEN OE CHARACTERISTICS:
tA) LENGT~: 4 amlno acids (B) TYPE: amino acid W O 93/112~4 PCT/DK92/003~1 -. " "'~
27 ,~ ,~
(C) STRAMDEDNESS: single (D) TOPOL0GY: linear (ii) MOLECVLE TYPE: peptide (iii) HYPOI~ETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENr TYPE: internal (xi) SEQUENCE DESCRIPqION: SEQ ID NO: 15:
Ala Ser Phe Ser 10 (2) IMFORMATION FOR SEQ ID NO: 16:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid ~C) STRAMDEDNESS: single (D) ~OPOL0GY: linear :~ (ii) MO ~ E TYPE: peptide (iii) HYPOIHE~ICAL: NO
: (iii) ANII-SENSE: NO
(v) FRA ~ T TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Ser Arg Gly Val (2) INFO~M~TION FOR SEQ ID NO: 17:
: : (i) SEQUEN æ CHARA ~ SIICS:
2s (A) LENGTH: 4 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOL~GY: linear (ii) MOLECULE TYPE: peptide (iii) ~YP0qWEIICAL: NO
(iii) ANII-SENSE: NO
(v) FRAG~ENT TYPE: internal (xi) SE~3~EN OE DESCRI~lION: SEQ ID NO: 17:
~ Leu Asp Thr Gly W O 93/11254 ~ PCT/DK92/003~1 ' ' V -' ~2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUEN OE CHARACTERISIICS:
(A) LENGTH: 4 a~ino acids (B) TYPE: amino acid s (C) STRANDEDNESS: single (D) TOPOLDGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCqHETqCAL: NO
~ ii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 18:
Tyr Tyr Ala Ala (2~ INFORM~TION FOR SEQ ID NO: l9:
(i) SEQUEN OE CHARACTERISTICS:
~A) LENGTH: 4 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: slngl~
(D) TOPOLOGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAG~ENT TYPE: inte~nal (xi) SEQUENCE DESCRIP5ION: SEO ID NO: l9:
Ile Asn Asp Ile (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amino acids : 30 (B) TYPE: amino acid (Cj STRANDEDNESS: single (D) TOPOLLGY: linear (ii) MOLECULE TYPE: pe.ptide (iii) HYPCIHErICAL: NO
(iii) ANTI-SENSE: NO
W O 93/1125~ PCT/DK92/003~1 .
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPIION: SEO ID NO: 20:
Trp Tyr Phe Gly 5 (2) INFORM~TION FOR SEQ ID NO: 21:
(i) SEQVEN OE CHARACIERI~llCS:
(A) LENGTH: 4 amuno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) EYPOqHE~ICAL: NO
(iil) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (Xi) SEOUENCE DESCRIPTION: SEQ ID NO: 21:
Ser Ile Glu Asn : :~ (2) INFORM~TION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARA ~ STICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDED~ESS: single (D) TOPOLOGY linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHE~ICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (Xl) SEQVEN OE DES ~ PTION: SEQ ID NO: 22:
Gly Ser Thr Tyr (2) ~FORMATION FO~ SEQ ID NO: 23:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amlno acid W O 93/1125~ PCT/DK92/003~1 ~ ; 30 (C) STRANDEDNESS: single (D) TOPOLLGY: 11nOE
(ii) MO~ECULE TYPE: peptide (iii) HYFCq~E~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEQ ID NO: 23:
Asp Ser Thr Asn 10 (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEOUEN OE CHAR~.CTERISTICS:
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(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: int ~ 1 (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 24:
Pro Asp Leu Arg (2) INFORMATION FOR SEO ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amuno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL~GY:~linear (ii) MOLECULE TYPE: peptide (iii) HYPOnHE~qC~L: NO
(iii) ANTI-SENSE: NO
(v) F2AGMENT T~PE: intern21 (Xl) SEQUEN OE DESCRIPTION: SEQ ID NO: 25:
Leu Asp Thr Gly W O 93tll254 PCT/DK92/00351 . . .
, v (2) INFO~MATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISllCS:
(A) LENGTH: 4 amino acids (B) TYPE: amlno acid (C) STRANDEDNESS: single (D) TOPOLCGY: linear (ii) ~OLECULE TYPE: peptide (iii) HYFCq~E~lCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 26:
Gly Asn Arg Tyr ~
(2) IMFORM~TION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids : ~ (B) TYPE: amino acid (C) SIhANDEDNESS: single (D) TOPOL0GY: linear (ii) MOLECULE TYPE: peptide : : (iii) HYFCn~ET~CAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) S~QUENOE DESCRIPTION: SEQ ID NO: 27: -Ser Gly Val Met (2) IMFOgM~TION F0R SEQ ID NO: 28:
(i) SEQUEN OE CHARACTEgISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCIHErIC~L: NO
(iii) ANTI-SENSE: NO
W O 93/11254 '~ ?~ 3 ~ P~T/DK92/00351 (v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEQ ID NO: 28:
Arg ~r Pro S~
5 (2) INF~RM~IION FOR SEO ID NO: 29:
(i) SEQUEN OE CH~RACTERIS'IICS:
(A) LENG~H: 4 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) IOP~LOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYFC~ET1CAL: NO
(iii) ANII-SENSE: NO
(v) FRA~MENT TYPE: internal (xi) SEOUENCE DESCRIPTION: SEO ID NO: 29:
: Asn Gly Ieu Val (2) INFORM~TION FOR S ~ ID NO: 30:
(i) SEQUENCE ~ CTERISTICS:
(A) IENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sLr~le (D) ~:; linear (ii) MOLECULE TYPE: peptide 2s ~iii) HYE~r~q~C~L: NO
: (iii) ANTI-SENSE::NO
(v) ~GME~T TYP--.: internal ` (xi) SEOUENCE DESCRIPTION: SEQ ID NO: 30:
Ser Phe Ser Ile :::
~:: (2j INFORMATION FOR SE~O ID NO: 31:
( i ) S~3QUENCE a~C~ISllCS:
(A) LENGIH: 4 amino acids (B) TYPE: amino acid WO93/11254 ~ I r~ PCT/DK92/00351 (C) STRANDEDNESS: single (D) TOPOLOGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HypcrHErIcAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUENOE DESCRIPIION: SEQ ID NO: 31:
Leu Gly Ser Pro 10 (2) INFORM~TION FOR SEO ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) SIRANDEDNESS: single (D) TOPOLOGY: linOE
(ii) MOLECULE TYPE:: peptide iii) HYPOqHErICAL: NO
(iii) ANII-SENSE: NO
(v) FR~GMENr TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
:
Ary Ala Ser Phe (2) INFORM~TION FOR SEQ ID NO: 33: ~-(i) SEQUEN OE STICS:
2s (A) LENGTH: 4 amino acids (B) TYPE: amino acid c) srRANDEDNEss: single (D) TOPOL3GY: linear (ii) MOLECULE TYPE: peptide 30 (iii) HYP~qHEIICAL: NO
(iii) ANTI-SENSE: NO
(v) F~AGMENT TYPE: internal (xi) SEO~UEN OE DESCRIPTION: SEQ ID NO: 33:
Val Pro Trp Gly ~: 35 .
~ " " " ~""~ ? j~
W O 93/11254 PCT/DK92/003~1 ~ 34 (2) INFORM~TION FOR SEQ ID NO: 34:
(i) SEQUEN OE CHARACTERISIICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOI~E~ICAL: NO
(iii) ~NTI-SENSE: NO
(v) FR~GMENT TYPE: m ternal (xi) SEQUEN OE DESCRIPIION: SEQ ID NO: 34:
Pro Asp Leu Asn ,. .
(2) INFORM~TION FOR SEQ ID NO: 35:
(i) SEQUEN OE CHARACTERISTICS:
(A) ~ENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLLGY: linear (ii) MOLECULE TYPE:~peptide ~: tiii) HyFoo~EIIcAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 35: :
Ser Phe Val Pro (2) INFO~M~TION FOR SEQ ID NO: 36:
(i) SEOUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLCGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCIH~TqC~L: NO
(iii) ANrI-SENSE: NO
WO93/112~4 ,~ . PCI/DK92/003~1 -.'`, s ~ r~
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEQ ID NO: 36:
Pro Asp Tyr Arg s (2) INFORM~TION F3R SEO ID NO: 37:
~i) SEQUEN OE CHARACIERISTICS:
(A) ~ENGIH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL0GY: l mear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
~iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 37:
Pro Arg Leu Pro (2) IMFO ~ TION FOR SEQ ID~NO: 38:
) SEQUEN OE CHARACTERISTICS:
(A) T.FNG~: 4 amlno acids tB) TYPE: amlno acid tc) S ~ EDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE~DESCRIPTION: SEQ ID NO: 38:
Thr Val Leu Pro t2) INFORMATION F~R SEO ID NO: 39:
ti~ SE~UENCE CHARACTERISTICS:
(A) LENGT~: ~s a~lno acios (B) TYPE: am~no acid W O 93/112~4 PCT/DK92/003~1 J 3~ q ~C) STRAMDEDNESS: single (D3 IOPQLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYiOq~E~ICAL: NO
S (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 39:
Ile Gly Thr Cys :
l0 (2) INFORM~TION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 4 amino acids (B~ TYPE: amlno acid (C) STRANDEDNESS: single (D) TOPOL0GY: linear (ii) M~ ~ TYPE: peptide (iii) HYPCrHE~ICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGNENT TYPE: internal (xi) SEQUEN OE DESC~IPTION: SEO ID NO: 40:
Thr Gly Gly Thr ~`~ (2) INF3 ~ TION FOR SEQ ID NO: 41:
(i) SEQ~ENOE CH~RACTERlSTICS:
2s (A) LENGTH: 4 amino;acids ; ~ (B) TYPE: amino acid (C) STRAND~NESS: single (D) TOPOL~GY: linear (ii) MOLECULE TYPE: peptide : 30 (iii) HYPCIHETIC~L: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPqION: SEQ ID NO: 41:
Thr ~sn Lys Leu :~ 35 W O 93~112~ PCT/DK92/003~1 (2) INFORM~TION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amlno acids (B) TYPE: amlno acid (C) STRA~DEDNESS: single (D) TOPOL5GY: line~r (ii) MOLECULE TYPE: peptide (iii) HYPCIHE~ICAL: NO
(iii) ANII-SENSE: NO
0 (v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 42:
Val Gly Asp Val (2) INFOgM~TION FOR SEQ ID NO: 43:
lS (i) Sl~UENCE ~CIERISllCS:
:~ (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sLngle (D) TOPOL0GY: l mear (ii) MOLECULE TYPE: peptide :: (iii) HYP0qHE~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SE2UENCE DESCRIPTION: SEO ID NO: 43:
~ ~25 Ile Gly Val Leu :~ 1 ,'.
(2) INFORM~TION FOR SEQ ID NO: 44:
: (i) SEQUENCE CH~RACTERISTICS:
(A) T.FNGTH: 4 amino acids 0 (B) TYPE: amlno acid (C) STR~NDED1~ESS: single (D) ~OPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HypcrHE~Ic~L: NO
~ 35 (iii) ANII-SENSE~: NO
:
W O 93~11254 PC~r/D K92/00351 ~ 6 3~
, ~' (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPIION: SE~ ID NO: 44:
Gly Ser Thr Tyr s (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUEN OE CH~RACIERISTICS:
(A) LENGIH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOqEETICAL: NO
(iil) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal : 15 (xi) SEQUEN OE DESCRIPqION: SEQ ID NO: 45:
Arg Tyr Ala Asn (Z) INFORM~TION FOR SEQ ID NO: 46:
(i) SEQUEN OE CHARAC5ERISTICS:
(A):LENGTH: 4 amlno acids (B) TYPE: amino a~cld : (C) STRANDEDNESS: single (Dj: TOPOL0GY: 1LnOE
(ii) MCLECULE TYPE: peptide : 2s (iii) HYPOqHErICAL: NO
~: (iii) ANTI-SENSE: NO :
(v) FRAGMENT TYPE: internaI
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 46:
:~ Pro Asn Ile Pro ~ : 30 :: (2) Irn~F~$~TION F0R SEQ ID NO: 47:
(i) SEOUENCE CHUiRAC1~3RISTICS:
(A) ~ NG~H: 4 anLLno acids (B) I~PE: amino acid W O 93/11254 ,;f ~ PCT/DK92/003~1 (C) STRANDEDNESS: s mgle (D) TOPOLCGY: linear (ii) MOLECVLE TYPE: peptide (iii) EYFCn~E~lCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: mternal ~xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 47:
Thr Leu Val Pro 10 (2) IMFORMATION FOR SEO ID NO: 48:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOL~GY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DES ~ ON: SEO ID NO: 48:
TTCA1CCAG TACTCTGGGC TAGAATGGCT GTA~CCAGAA GCACCCGGCG GGAGTCTAGG 60 (2) INFCRM~TION FOR SEQ ID NO: 49:
(i) SEQVEN OE CH~RACTERISTICS:
~(A) LENGTH: 40 base pairs : 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: s mgle (D) TOPOLOGY: linear : (iij MOLECULE TYPE: cDNA
(iii) HYP3q~ETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 49:
GGTCAT~C~G TCACIGAGAC CCTCTACCTA TTAA~.TCGGC 40 WO 93t11254 PCI/DK92/003~1 4 o (2) INF~lION FOR S~Q ID NO: 50:
(i) SEQ~ENOE CH~RACrERIS~CS:
(A) LENGrH: 20 base pairs (B) TYPE: nucleic acid s (C) SIRANDEDNESS: sir~le (D) l~POLOGY: linear ~ii) ~OLE;C[~IE TY~E: cDNA
(iii) HYFaI~ICAL: NO
(iii) ANl~-SENSE: NO
(vj ~RAt~NT TYPE: internal (xi) SEQUENOE DESCRIPTION: SEQ ID NO: 50:
CCAI~GCFIT CACGGI~I~r 20 (2) INF~RMP,TION FOR S~Q ID NO: 51:
(i) SE~VENOE CH~CI~ISIICS:
(A) T~NGrH: 20 base pairs (B) TYPE: nucleic acid (C) S~EDNESS: single (D) IOPO~GY: linear (ii) ~OLEC[~LE TYPE: cDNA
(iii) H~r~llcAL: NO
(iii) ANTI-SENSE: NO
~: (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCNPTION: SEQ ID NO: 51:
GGI~I~G TCACrGAG~C 20 25 (2) INFO~ION FOR S~Q ID NO: 52:
(i) SEQUENCE CH~ rERISTICS:
(A) LENGrH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~o (D) TOPOLOGY: linP~r (ii) MOLECULE TYPE: cDNA
(iii) H~l~CAL: NO
(iii) ANTI-SENSE: NO
(vj ~?AG~lT TYPE: internal W O 93/11254 PCT/DK92/~Q3~1 (xi) SEQUENCE DESCRIPIION: SEQ ID NO: 52:
T11GATCC~G TACTCT3GGC TAGAATGGCT GIAAGAICIT GGGTACGGCG GGAGTCI~GG 60 (2) IMFORM~ION FOR SEQ ID NO: 53:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~iii) HYPOnEETqCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 53:
~ CACG TCGATTGGCA ATCTTGGTCC ACGCAGGTCT GC 42 l5 (2) INFORMATION FOR SEO ID NO: 54:
(i) SEQUEN OE CHARACTERISTlCS:
(A) LEN ~ : 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear : (ii) MOLECULE TYPE: cDNA
(iii) HYPCrEETICAL: NO
: (iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal ; 25 (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 54:
CACGTOGArA TCGCGACCAT TrCCACG 27 (2) INFORM~TION F~R SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 25 base pairs (B) TYPE: nucleic acid (C) STRANDED~ ~ S: single (D) TOPOLOGY: lin~r (ii) MOLECULE rfPE: cD~.
(iii) Hypor~ErIcAL: ~-10 W O 93/11254 PCT/DK9~/003~1 ~ . 42 (iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
s (2) INFORM~TION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERIs~lCS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: si~gle ~D) TOPOL~GY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOrHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 56:
G~AIGGCI~T ATGGAAATTC GCGCG 25 (2) INFO~M~TION FOR SE~ ID NO: 57:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B3 TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~; (ii) MOLEC~IE TYPE: cDNA :
(iii) HYPC~TICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGr~T TYPE: internal (xi) SEQUENOE DESCRIPIION: SEQ ID NO: 57:
GI~CCGAAT C~oeGCG GG 22
A substrate for lipase was prepared by emulsifying glycerine s tributyrat (MERCK) using gum-arabic as emulsifier.
Lipase activity was assayed at pH 7 using pH stat method. One unit of lipase activity (LU/mg) was defined as the amount needed to liberate one micromole fatty acid per minute.
Step l:- Centrifuge the fermentation supernatant, discard the 0 precipitate. Adjust the pH of the supernatant to 7 and add gradually an equal volume of cold 96 ~ ethanol. Allow the mixture to stand for 30 minutes in an ice bath. Centrifuge and discard the precipitate.
Step 2:- Ion exchange chromatography. Filter the supernatant 15 and apply on DEAE-fast flow (Pharmacia TM) column equilibrated with 50 mM tris-acetate buffer pH 7. Wash the column with the same buffer till absorption at 280 nm is lower than 0.05 OD.
Elute the bound enzymatic activity with linear salt gradient in the same buffer (0 to 0.5 M NaCl ) using five column volumes.
20 Pool the fractions containing enzymatic activity.
Step 3:- Hydrophobic chromatography. Adjust the molarity of the pool containing enzymatic activity to 0.8 M by adding solid Ammonium acetate. Apply the enzyme on TSK gel Butyl- Toyopearl 650 C column (available from Tosoh Corporation Japan) which 25 was pre-equilibrated with 0.8 M ammonium acetate. Wash the unbound material with 0.8 M ammonium acetate and elute the bound material with distilled water.
Step 4:- Pool containing lipase activity is diluted with water to adjust conductance to 2 mS and pH to 7. Apply the pool on 30 High performance Q Sepharose (Pharmacia) column pre-equilibrated with 50 mM tris -aceta~e buffer pH 7. Elute the ) s ,~, -f 31 i PCT/DK92/00351 bound enzyme with linear salt gradient.
EXAMPLE S
Protea~e stability of variant proteins The variant lipases Subst.(162-165)PRLP, Subst.(209-212)YPRS, 5 Subst.(209-212)GASG, G212R and G212P were purified as described in Example 4 above and each diluted with O.lM Tris-puffer, pH
9Ø Subsequently, a protease solution containing SavinaseTM
(concentration of lOOmg/ml in 50% mono-propylene glycol (MPG), 1% Boric acid) was added in a proportion of lipase: protease of o 1:5. The final lipase concentration was lmg/ml (except for the variant G212P, for ~hich it was 0.8 mg/ml). The reaction was carried out at 22C. At appropiate times, probes were taken from the reaction mixture and immediately subjected to the above described lipase activity assay.
15 In Fig. 5 the residual activity in per cent of the starting material is shown versus incubation time, for the wild type Humicola lanuginosa lipase (wt) and the variant lipases.
W O 93/11254 ~ PCT/DK92tO03~1 SEQUENCE LISTING
(1) GENERAL IMFORM~IION:
(i) APPLICANT:
(A) NAME: NOVO NORDISK A/S
S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DENM~RK
(F) POSTAL OODE (ZIP): DK-2880 (G) TELEPHONE: +45 44448888 (H) TELEFAX: +45 444g 3256 (I) TELEX: 37304 (il) TITrl~ OF ~ENTIO~: Protease-Stable Proteins (iii) NUMBER OF SE~UEN OES: 57 (iv) COMPU}ER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) CQMPUTER: IBM PC ccmpatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTW~RE: PatentIn Release #l-r Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: WO PCT/DK9l/00350 . (8) ~ G DATE: 26-NOV-l99l (2) INFORMATION FOR SEQ ID NO: l:
:: (i) SEQUENCE CHARACTERISIICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: s mgle (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: peptide (iii) HYPCrHETICAL: NO
(iii) ANrI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEO ID NO: l:
Arg Glu Phe Gly W O 93/112~4 J ~ PCT/DK92/003~1 (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL3GY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FR~GMENT TYPE: internal (xi) SE2UEN OE DESCRIPTION: SEQ ID NO: 2:
Asp Tyr Gly Asn (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUEN OE CHARACTERISTICS:
tA) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRAMDEDNESS: single (D) T~POLOGY: linP~r ::; 20 (ii) MOLECULE TYPE: peptide : (iiij HYPOTHEIICAL: NO
: (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 3:
Glu Gly Ile Asp (2) INFORM~TION FOR SEQ ID NO: 4:
(i) SE~UEN OE CH~RACTERISIICS:
~A) LENGTH: 4 amlno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLCGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOrHETICAL: NO
(iii) ANII-S~SE: NO
W O 93/lt254 PCT/DK92/003~1 s~
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Ala Ser Gly 5 (2) INF3RM~TION FOR SEQ ID NO: 5:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGIH: 4 amlno acids ~B) TYPE: amuno acid (C) STRAMDEDNESS: single (D) TOPOL0GY: linear (ii) MOL~CULE TYPE: peptide (iii~ EhFCT~E~ICAL: NO
~iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQVENCE DESCRlPqlON: SEQ ID NO: 5:
Gly Ala Ala Gly (2) INFORM~TION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) 1~: l~near (ii) ffl)LE~lE TYPE: peptide (iii) E[~r~C~: NO
(iii) ANII-SENSE: NO
(v) F~AGMENT TYFE: internal (xi) S~2UEt~OE DESCRIPIION: S~)Q ID NO: 6:
:; Gly Ala An~ Gly (2) INF~TION F~R SE;2 ID NO: 7:
( i ) SEQU~NOE CHARAC~ERISIICS:
(A) IEN~I: 4 amino acids (B) TYPE: alTLino acid W O 93/ll2~ PCT/DK92/003Sl t~ 'f 24 (C) STRANDEDNESS: single (D) TOPOLCGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HYPOn9E~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEO ID NO: 7:
Tyr Pro Gly Ser 10 (2) INFORM~TION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACIERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL0GY: linear (ii) MOLECULE TYPE: peptide (iii) HhECn~ETICAL: NO
.. . .
(lll) ~ -SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DES~ IPTION: SEQ ID NO: 8:
Tyr Pro Arg Ser : 1 (2~ INFORM~TION FOR SEQ ID NO: 9:
: : (i) SEQUEN OE CHARACTERISTICS::
(A) LENGTH: 4 amlno acids ~B) TYPE:~amino acld (C) STRANDE~SS: single (D) TOPOL,OGY:~linear (ii~ MOLECULE TYPE: peptide (iii~ HYEC~ETIC~L: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPIION: SEQ ID NO: 9:
His Asn Arg Gly _5 W O 93/112~ PCT/DK92/003~1 . 2 (2) INFORMATION FVR SEQ ID NO: l0:
(i) SEQUENCE CHARACIERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRAMDEDNESS: s m~le (D) TOPOL~GY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOqHE~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
Tyr Thr Gly Asn (2) INFORM~TION FOR SEQ ID NO: ll:
(i) SEQUEN OE CHARACTERISllCS:
(A) LENGTH: 4 amino acids (B) TYPE: amlno acid (C) STRANDEDNESS: single (D) ~POL~GY: linear (ii) MO ~ TYPE: peptide :: (iii) HYPOI~ETqCAL: NO
~; (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal ~xi) SEQUEN OE DESCRIPIION: SEQ ID NO: ll:
Ile Ser Ser Glu ~2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amlno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCrHErICAL: NO
3, (iii) ANTI-SENSE: NO
W O 93/1l254 P~CT/DK92/003~1 c ~
. 26 (v) FRAGMENT TYPE: internal txl) SEQUENCE DESCRIPqION: SE~ ID NO: 12:
Asn Asn Ala Gly 5 (2) INFORM~IION FOR SEQ ID NO: 13:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCrHETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: lnternal (xi) SEQVEN OE DESCRIPTION: SEO ID NO: 13:
~ S~er Phe Ile Asn ; 1 (2) INFORM~TION FOR SEQ ID NO: 14:
(i) SEQUENOE CHARACTE~ISIICS:
(A) LENGTH: 4 am mo aci~ds : (B) 'TYPE: amino acid : : (C) SIRANDEDNESS: s mgle (D) TOPOLOGY: linear (ii) MOT~CULE TYPE: peptide 2s (iii) HYPOqlE~ICAL: NO
(iii) ANII-SENSE: NO
(v) FRAEMENr TYPE: internal (xi) SEQUENCE DESCRIPIION: SEQ ID NO: 14:
Asp Gln Asn Gly (2) INFORM~TION FOR SEQ ID NO: 15:
(i) SEQUEN OE CHARACTERISTICS:
tA) LENGT~: 4 amlno acids (B) TYPE: amino acid W O 93/112~4 PCT/DK92/003~1 -. " "'~
27 ,~ ,~
(C) STRAMDEDNESS: single (D) TOPOL0GY: linear (ii) MOLECVLE TYPE: peptide (iii) HYPOI~ETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENr TYPE: internal (xi) SEQUENCE DESCRIPqION: SEQ ID NO: 15:
Ala Ser Phe Ser 10 (2) IMFORMATION FOR SEQ ID NO: 16:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid ~C) STRAMDEDNESS: single (D) ~OPOL0GY: linear :~ (ii) MO ~ E TYPE: peptide (iii) HYPOIHE~ICAL: NO
: (iii) ANII-SENSE: NO
(v) FRA ~ T TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Ser Arg Gly Val (2) INFO~M~TION FOR SEQ ID NO: 17:
: : (i) SEQUEN æ CHARA ~ SIICS:
2s (A) LENGTH: 4 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOL~GY: linear (ii) MOLECULE TYPE: peptide (iii) ~YP0qWEIICAL: NO
(iii) ANII-SENSE: NO
(v) FRAG~ENT TYPE: internal (xi) SE~3~EN OE DESCRI~lION: SEQ ID NO: 17:
~ Leu Asp Thr Gly W O 93/11254 ~ PCT/DK92/003~1 ' ' V -' ~2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUEN OE CHARACTERISIICS:
(A) LENGTH: 4 a~ino acids (B) TYPE: amino acid s (C) STRANDEDNESS: single (D) TOPOLDGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCqHETqCAL: NO
~ ii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 18:
Tyr Tyr Ala Ala (2~ INFORM~TION FOR SEQ ID NO: l9:
(i) SEQUEN OE CHARACTERISTICS:
~A) LENGTH: 4 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: slngl~
(D) TOPOLOGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAG~ENT TYPE: inte~nal (xi) SEQUENCE DESCRIP5ION: SEO ID NO: l9:
Ile Asn Asp Ile (2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amino acids : 30 (B) TYPE: amino acid (Cj STRANDEDNESS: single (D) TOPOLLGY: linear (ii) MOLECULE TYPE: pe.ptide (iii) HYPCIHErICAL: NO
(iii) ANTI-SENSE: NO
W O 93/1125~ PCT/DK92/003~1 .
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPIION: SEO ID NO: 20:
Trp Tyr Phe Gly 5 (2) INFORM~TION FOR SEQ ID NO: 21:
(i) SEQVEN OE CHARACIERI~llCS:
(A) LENGTH: 4 amuno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) EYPOqHE~ICAL: NO
(iil) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (Xi) SEOUENCE DESCRIPTION: SEQ ID NO: 21:
Ser Ile Glu Asn : :~ (2) INFORM~TION FOR SEQ ID NO: 22:
(i) SEQUENCE CHARA ~ STICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDED~ESS: single (D) TOPOLOGY linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHE~ICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (Xl) SEQVEN OE DES ~ PTION: SEQ ID NO: 22:
Gly Ser Thr Tyr (2) ~FORMATION FO~ SEQ ID NO: 23:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amlno acid W O 93/1125~ PCT/DK92/003~1 ~ ; 30 (C) STRANDEDNESS: single (D) TOPOLLGY: 11nOE
(ii) MO~ECULE TYPE: peptide (iii) HYFCq~E~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEQ ID NO: 23:
Asp Ser Thr Asn 10 (2) INFORMATION FOR SEQ ID NO: 24:
(i) SEOUEN OE CHAR~.CTERISTICS:
(A) LENGTH: 4 amlno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLCGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HYPrT~ETqCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: int ~ 1 (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 24:
Pro Asp Leu Arg (2) INFORMATION FOR SEO ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amuno acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL~GY:~linear (ii) MOLECULE TYPE: peptide (iii) HYPOnHE~qC~L: NO
(iii) ANTI-SENSE: NO
(v) F2AGMENT T~PE: intern21 (Xl) SEQUEN OE DESCRIPTION: SEQ ID NO: 25:
Leu Asp Thr Gly W O 93tll254 PCT/DK92/00351 . . .
, v (2) INFO~MATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISllCS:
(A) LENGTH: 4 amino acids (B) TYPE: amlno acid (C) STRANDEDNESS: single (D) TOPOLCGY: linear (ii) ~OLECULE TYPE: peptide (iii) HYFCq~E~lCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 26:
Gly Asn Arg Tyr ~
(2) IMFORM~TION FOR SEQ ID NO: 27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids : ~ (B) TYPE: amino acid (C) SIhANDEDNESS: single (D) TOPOL0GY: linear (ii) MOLECULE TYPE: peptide : : (iii) HYFCn~ET~CAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: mternal (xi) S~QUENOE DESCRIPTION: SEQ ID NO: 27: -Ser Gly Val Met (2) IMFOgM~TION F0R SEQ ID NO: 28:
(i) SEQUEN OE CHARACTEgISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCIHErIC~L: NO
(iii) ANTI-SENSE: NO
W O 93/11254 '~ ?~ 3 ~ P~T/DK92/00351 (v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEQ ID NO: 28:
Arg ~r Pro S~
5 (2) INF~RM~IION FOR SEO ID NO: 29:
(i) SEQUEN OE CH~RACTERIS'IICS:
(A) LENG~H: 4 amino acids (B) TYPE: amino acid ~C) STRANDEDNESS: single (D) IOP~LOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYFC~ET1CAL: NO
(iii) ANII-SENSE: NO
(v) FRA~MENT TYPE: internal (xi) SEOUENCE DESCRIPTION: SEO ID NO: 29:
: Asn Gly Ieu Val (2) INFORM~TION FOR S ~ ID NO: 30:
(i) SEQUENCE ~ CTERISTICS:
(A) IENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sLr~le (D) ~:; linear (ii) MOLECULE TYPE: peptide 2s ~iii) HYE~r~q~C~L: NO
: (iii) ANTI-SENSE::NO
(v) ~GME~T TYP--.: internal ` (xi) SEOUENCE DESCRIPTION: SEQ ID NO: 30:
Ser Phe Ser Ile :::
~:: (2j INFORMATION FOR SE~O ID NO: 31:
( i ) S~3QUENCE a~C~ISllCS:
(A) LENGIH: 4 amino acids (B) TYPE: amino acid WO93/11254 ~ I r~ PCT/DK92/00351 (C) STRANDEDNESS: single (D) TOPOLOGY: lin~r (ii) MOLECULE TYPE: peptide (iii) HypcrHErIcAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEOUENOE DESCRIPIION: SEQ ID NO: 31:
Leu Gly Ser Pro 10 (2) INFORM~TION FOR SEO ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) SIRANDEDNESS: single (D) TOPOLOGY: linOE
(ii) MOLECULE TYPE:: peptide iii) HYPOqHErICAL: NO
(iii) ANII-SENSE: NO
(v) FR~GMENr TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
:
Ary Ala Ser Phe (2) INFORM~TION FOR SEQ ID NO: 33: ~-(i) SEQUEN OE STICS:
2s (A) LENGTH: 4 amino acids (B) TYPE: amino acid c) srRANDEDNEss: single (D) TOPOL3GY: linear (ii) MOLECULE TYPE: peptide 30 (iii) HYP~qHEIICAL: NO
(iii) ANTI-SENSE: NO
(v) F~AGMENT TYPE: internal (xi) SEO~UEN OE DESCRIPTION: SEQ ID NO: 33:
Val Pro Trp Gly ~: 35 .
~ " " " ~""~ ? j~
W O 93/11254 PCT/DK92/003~1 ~ 34 (2) INFORM~TION FOR SEQ ID NO: 34:
(i) SEQUEN OE CHARACTERISIICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOI~E~ICAL: NO
(iii) ~NTI-SENSE: NO
(v) FR~GMENT TYPE: m ternal (xi) SEQUEN OE DESCRIPIION: SEQ ID NO: 34:
Pro Asp Leu Asn ,. .
(2) INFORM~TION FOR SEQ ID NO: 35:
(i) SEQUEN OE CHARACTERISTICS:
(A) ~ENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLLGY: linear (ii) MOLECULE TYPE:~peptide ~: tiii) HyFoo~EIIcAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 35: :
Ser Phe Val Pro (2) INFO~M~TION FOR SEQ ID NO: 36:
(i) SEOUEN OE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLCGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCIH~TqC~L: NO
(iii) ANrI-SENSE: NO
WO93/112~4 ,~ . PCI/DK92/003~1 -.'`, s ~ r~
(v) FRAGMENT TYPE: internal (xi) SEOUEN OE DESCRIPTION: SEQ ID NO: 36:
Pro Asp Tyr Arg s (2) INFORM~TION F3R SEO ID NO: 37:
~i) SEQUEN OE CHARACIERISTICS:
(A) ~ENGIH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOL0GY: l mear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
~iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 37:
Pro Arg Leu Pro (2) IMFO ~ TION FOR SEQ ID~NO: 38:
) SEQUEN OE CHARACTERISTICS:
(A) T.FNG~: 4 amlno acids tB) TYPE: amlno acid tc) S ~ EDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPCTHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE~DESCRIPTION: SEQ ID NO: 38:
Thr Val Leu Pro t2) INFORMATION F~R SEO ID NO: 39:
ti~ SE~UENCE CHARACTERISTICS:
(A) LENGT~: ~s a~lno acios (B) TYPE: am~no acid W O 93/112~4 PCT/DK92/003~1 J 3~ q ~C) STRAMDEDNESS: single (D3 IOPQLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYiOq~E~ICAL: NO
S (iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 39:
Ile Gly Thr Cys :
l0 (2) INFORM~TION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH 4 amino acids (B~ TYPE: amlno acid (C) STRANDEDNESS: single (D) TOPOL0GY: linear (ii) M~ ~ TYPE: peptide (iii) HYPCrHE~ICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGNENT TYPE: internal (xi) SEQUEN OE DESC~IPTION: SEO ID NO: 40:
Thr Gly Gly Thr ~`~ (2) INF3 ~ TION FOR SEQ ID NO: 41:
(i) SEQ~ENOE CH~RACTERlSTICS:
2s (A) LENGTH: 4 amino;acids ; ~ (B) TYPE: amino acid (C) STRAND~NESS: single (D) TOPOL~GY: linear (ii) MOLECULE TYPE: peptide : 30 (iii) HYPCIHETIC~L: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPqION: SEQ ID NO: 41:
Thr ~sn Lys Leu :~ 35 W O 93~112~ PCT/DK92/003~1 (2) INFORM~TION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amlno acids (B) TYPE: amlno acid (C) STRA~DEDNESS: single (D) TOPOL5GY: line~r (ii) MOLECULE TYPE: peptide (iii) HYPCIHE~ICAL: NO
(iii) ANII-SENSE: NO
0 (v) FRAGMENT TYPE: mternal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 42:
Val Gly Asp Val (2) INFOgM~TION FOR SEQ ID NO: 43:
lS (i) Sl~UENCE ~CIERISllCS:
:~ (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sLngle (D) TOPOL0GY: l mear (ii) MOLECULE TYPE: peptide :: (iii) HYP0qHE~ICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SE2UENCE DESCRIPTION: SEO ID NO: 43:
~ ~25 Ile Gly Val Leu :~ 1 ,'.
(2) INFORM~TION FOR SEQ ID NO: 44:
: (i) SEQUENCE CH~RACTERISTICS:
(A) T.FNGTH: 4 amino acids 0 (B) TYPE: amlno acid (C) STR~NDED1~ESS: single (D) ~OPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HypcrHE~Ic~L: NO
~ 35 (iii) ANII-SENSE~: NO
:
W O 93~11254 PC~r/D K92/00351 ~ 6 3~
, ~' (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPIION: SE~ ID NO: 44:
Gly Ser Thr Tyr s (2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUEN OE CH~RACIERISTICS:
(A) LENGIH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOqEETICAL: NO
(iil) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal : 15 (xi) SEQUEN OE DESCRIPqION: SEQ ID NO: 45:
Arg Tyr Ala Asn (Z) INFORM~TION FOR SEQ ID NO: 46:
(i) SEQUEN OE CHARAC5ERISTICS:
(A):LENGTH: 4 amlno acids (B) TYPE: amino a~cld : (C) STRANDEDNESS: single (Dj: TOPOL0GY: 1LnOE
(ii) MCLECULE TYPE: peptide : 2s (iii) HYPOqHErICAL: NO
~: (iii) ANTI-SENSE: NO :
(v) FRAGMENT TYPE: internaI
(xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 46:
:~ Pro Asn Ile Pro ~ : 30 :: (2) Irn~F~$~TION F0R SEQ ID NO: 47:
(i) SEOUENCE CHUiRAC1~3RISTICS:
(A) ~ NG~H: 4 anLLno acids (B) I~PE: amino acid W O 93/11254 ,;f ~ PCT/DK92/003~1 (C) STRANDEDNESS: s mgle (D) TOPOLCGY: linear (ii) MOLECVLE TYPE: peptide (iii) EYFCn~E~lCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: mternal ~xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 47:
Thr Leu Val Pro 10 (2) IMFORMATION FOR SEO ID NO: 48:
(i) SEOUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOL~GY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DES ~ ON: SEO ID NO: 48:
TTCA1CCAG TACTCTGGGC TAGAATGGCT GTA~CCAGAA GCACCCGGCG GGAGTCTAGG 60 (2) INFCRM~TION FOR SEQ ID NO: 49:
(i) SEQVEN OE CH~RACTERISTICS:
~(A) LENGTH: 40 base pairs : 25 (B) TYPE: nucleic acid (C) STRANDEDNESS: s mgle (D) TOPOLOGY: linear : (iij MOLECULE TYPE: cDNA
(iii) HYP3q~ETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 49:
GGTCAT~C~G TCACIGAGAC CCTCTACCTA TTAA~.TCGGC 40 WO 93t11254 PCI/DK92/003~1 4 o (2) INF~lION FOR S~Q ID NO: 50:
(i) SEQ~ENOE CH~RACrERIS~CS:
(A) LENGrH: 20 base pairs (B) TYPE: nucleic acid s (C) SIRANDEDNESS: sir~le (D) l~POLOGY: linear ~ii) ~OLE;C[~IE TY~E: cDNA
(iii) HYFaI~ICAL: NO
(iii) ANl~-SENSE: NO
(vj ~RAt~NT TYPE: internal (xi) SEQUENOE DESCRIPTION: SEQ ID NO: 50:
CCAI~GCFIT CACGGI~I~r 20 (2) INF~RMP,TION FOR S~Q ID NO: 51:
(i) SE~VENOE CH~CI~ISIICS:
(A) T~NGrH: 20 base pairs (B) TYPE: nucleic acid (C) S~EDNESS: single (D) IOPO~GY: linear (ii) ~OLEC[~LE TYPE: cDNA
(iii) H~r~llcAL: NO
(iii) ANTI-SENSE: NO
~: (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCNPTION: SEQ ID NO: 51:
GGI~I~G TCACrGAG~C 20 25 (2) INFO~ION FOR S~Q ID NO: 52:
(i) SEQUENCE CH~ rERISTICS:
(A) LENGrH: 60 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single ~o (D) TOPOLOGY: linP~r (ii) MOLECULE TYPE: cDNA
(iii) H~l~CAL: NO
(iii) ANTI-SENSE: NO
(vj ~?AG~lT TYPE: internal W O 93/11254 PCT/DK92/~Q3~1 (xi) SEQUENCE DESCRIPIION: SEQ ID NO: 52:
T11GATCC~G TACTCT3GGC TAGAATGGCT GIAAGAICIT GGGTACGGCG GGAGTCI~GG 60 (2) IMFORM~ION FOR SEQ ID NO: 53:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
~iii) HYPOnEETqCAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 53:
~ CACG TCGATTGGCA ATCTTGGTCC ACGCAGGTCT GC 42 l5 (2) INFORMATION FOR SEO ID NO: 54:
(i) SEQUEN OE CHARACTERISTlCS:
(A) LEN ~ : 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear : (ii) MOLECULE TYPE: cDNA
(iii) HYPCrEETICAL: NO
: (iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal ; 25 (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 54:
CACGTOGArA TCGCGACCAT TrCCACG 27 (2) INFORM~TION F~R SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 25 base pairs (B) TYPE: nucleic acid (C) STRANDED~ ~ S: single (D) TOPOLOGY: lin~r (ii) MOLECULE rfPE: cD~.
(iii) Hypor~ErIcAL: ~-10 W O 93/11254 PCT/DK9~/003~1 ~ . 42 (iii) ANII-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
s (2) INFORM~TION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERIs~lCS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: si~gle ~D) TOPOL~GY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOrHETICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUEN OE DESCRIPTION: SEQ ID NO: 56:
G~AIGGCI~T ATGGAAATTC GCGCG 25 (2) INFO~M~TION FOR SE~ ID NO: 57:
(i) SEQUEN OE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B3 TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~; (ii) MOLEC~IE TYPE: cDNA :
(iii) HYPC~TICAL: NO
(iii) ANTI-SENSE: NO
(v) FRAGr~T TYPE: internal (xi) SEQUENOE DESCRIPIION: SEQ ID NO: 57:
GI~CCGAAT C~oeGCG GG 22
Claims (31)
1. A protein with improved stability against proteolytic degradation, wherein one or more protease labile amino acid segments located on the surface of the protein are substituted by protease non-labile amino acid segment(s), which in a computer graphic model of the protein have been evaluated for appropriate sequence, three-dimensional structure and contacts with surrounding amino acid residues in the protein.
2. A protein according to claim 1, which is an enzyme.
3. A protein according to claim 1 or 2, wherein the protease non-labile amino acid segment is derived from a lipase or protease, or wherein it is a synthetic segment.
4. A protein according to claim 2 or 3, which is a lipase.
5. A lipase protein according to claim 4, wherein the parent lipase is a microbial lipase.
6. A lipase protein according to claim 5, wherein the parent lipase is a fungal lipase.
7. A lipase protein according to claim 6, wherein the parent lipase is a strain of Humicola or Rhizomucor.
8. A lipase protein according to claim 7, wherein the parent lipase is a Humicola lanuginosa lipase.
9. A lipase protein according to claim 8, wherein the protease-labile amino acid segment is REFG (SEQ ID No. 1) at positions 209-212 of the lipase molecule, DYGN (SEQ ID No. 2) at positions 162-165 of the lipase molecule, or EGID (SEQ ID No.
3) at positions 239-242 of the lipase molecule.
3) at positions 239-242 of the lipase molecule.
10. A lipase protein according to claim 9, wherein the segment REFG (SEQ ID No. 1) is substituted by a segment selected from the group consisting of GASG (SEQ ID No. 4), GAAG (SEQ ID No.
5), GARG (SEQ ID No. 6), YPGS (SEQ ID No. 7), YPRS (SEQ ID No.
8), HNRG (SEQ ID No. 9), YTGN (SEQ ID No. 10), ISSE (SEQ ID No.
11), NNAG (SEQ ID No. 12), SFIN (SEQ ID No. 13), DQNG (SEQ ID
No. 14), ASFS (SEQ ID No. 15), SRGV (SEQ ID No. 16), LDTG (SEQ
ID No. 17), YYAA (SEQ ID No. 18), INDI (SEQ ID No. 19), WYFG
(SEQ ID No. 20), and SIEN (SEQ ID No. 21).
5), GARG (SEQ ID No. 6), YPGS (SEQ ID No. 7), YPRS (SEQ ID No.
8), HNRG (SEQ ID No. 9), YTGN (SEQ ID No. 10), ISSE (SEQ ID No.
11), NNAG (SEQ ID No. 12), SFIN (SEQ ID No. 13), DQNG (SEQ ID
No. 14), ASFS (SEQ ID No. 15), SRGV (SEQ ID No. 16), LDTG (SEQ
ID No. 17), YYAA (SEQ ID No. 18), INDI (SEQ ID No. 19), WYFG
(SEQ ID No. 20), and SIEN (SEQ ID No. 21).
11. A lipase protein according to claim 9, wherein the segment DYGN (SEQ ID No. 2) is substituted by a segment selected from the group consisting of GSTY (SEQ ID No. 22), DSTN (SEQ ID No.
23), PDLR (SEQ ID No. 24), LDTG (SEQ ID No. 25), GNRY (SEQ ID
No. 26), SGVM (SEQ ID No. 27) , RYPS (SEQ ID No. 28), NGLV (SEQ
ID No. 29), SFSI (SEQ ID No. 30), LGSP (SEQ ID No. 31), RASF
(SEQ ID No. 32), VPWG (SEQ ID No. 33), PDLN (SEQ ID No. 34), SFVP (SEQ ID No. 35), PDYR (SEQ ID No. 36), PRLP (SEQ ID No.
37), TVLP (SEQ ID No. 38), IGTC (SEQ ID No. 39), TGGT (SEQ ID
No. 40), TNKL (SEQ ID No. 41), and VGDV (SEQ ID No. 42).
23), PDLR (SEQ ID No. 24), LDTG (SEQ ID No. 25), GNRY (SEQ ID
No. 26), SGVM (SEQ ID No. 27) , RYPS (SEQ ID No. 28), NGLV (SEQ
ID No. 29), SFSI (SEQ ID No. 30), LGSP (SEQ ID No. 31), RASF
(SEQ ID No. 32), VPWG (SEQ ID No. 33), PDLN (SEQ ID No. 34), SFVP (SEQ ID No. 35), PDYR (SEQ ID No. 36), PRLP (SEQ ID No.
37), TVLP (SEQ ID No. 38), IGTC (SEQ ID No. 39), TGGT (SEQ ID
No. 40), TNKL (SEQ ID No. 41), and VGDV (SEQ ID No. 42).
12. A lipase protein according to claim 9, wherein the segment EGID (SEQ ID No. 3) is substituted by a segment selected from the group consisting of IGVL (SEQ ID No. 43), GSTY (SEQ ID No.
44), RYAN (SEQ ID No. 45), PNIP (SEQ ID No. 46), and TLVP (SEQ
ID No. 47).
44), RYAN (SEQ ID No. 45), PNIP (SEQ ID No. 46), and TLVP (SEQ
ID No. 47).
13. A lipase protein according to claim 9, wherein, to produce a protease non-labile amino acid segment, one or more amino acid residues in the segment REFG (SEQ ID No. 1) or DYGN (SEQ
ID No. 2) are substituted by proline or arginine.
ID No. 2) are substituted by proline or arginine.
14. A lipase protein according to claim 13, wherein Gly 212 is substituted by Pro or Arg, and/or wherein Gly 164 is substituted by Pro or Arg.
15. A lipase protein according to claim 7, wherein the parent lipase is a Rhizomucor miehei lipase.
16. A lipase protein according to claim 5, wherein the parent lipase is a yeast lipase.
17. A lipase protein according to claim 16, wherein the yeast lipase is a Candida lipase.
18. A lipase protein according to claim 5, wherein the parent lipase is a bacterial lipase.
19. A lipase protein according to claim 18, wherein the parent lipase is derived from a strain of Pseudomonas.
20. A DNA construct comprising a DNA sequence encoding a protein according to any of claims 1-19.
21. A recombinant expression vector which carries a DNA
construct according to claim 20.
construct according to claim 20.
22. A cell which is transformed with a DNA construct according to claim 20 or a vector according to claim 21.
23. A cell according to claim 22 which is a fungal cell, e.g.
belonging to the genus Aspergillus, such as A. niger, A.
oryzae, or A. nidulans; a yeast cell, e.g. belonging to a strain of Saccharomyces, such as S. cerevisiae, or a methylo-trophic yeast from the genera Hansenula, such as H. polymorpha, or Pichia, such as P. pastoris; or a bacterial cell, e.g.
belonging to a strain of Bacillus, such as B. subtilis, B.
licheniformis or B. lentus.
belonging to the genus Aspergillus, such as A. niger, A.
oryzae, or A. nidulans; a yeast cell, e.g. belonging to a strain of Saccharomyces, such as S. cerevisiae, or a methylo-trophic yeast from the genera Hansenula, such as H. polymorpha, or Pichia, such as P. pastoris; or a bacterial cell, e.g.
belonging to a strain of Bacillus, such as B. subtilis, B.
licheniformis or B. lentus.
24. A method of producing a protein according to any of claims 1-19, wherein a cell according to claim 22 or 23 is cultured under conditions permitting the production of the protein and the protein is subsequently recovered from the culture.
25. A detergent additive comprising a lipase protein according to any of claims 4-19 as well as a protease, optionally in the form of a non-dusting granulate, stabilised liquid or protected enzyme.
26. A detergent additive according to claim 25, which contains 0.02-200 mg of enzyme protein/g of the additive.
27. A detergent additive according to claim 25 or 26 which additionally comprises another enzyme such as an amylase, peroxidase and/or cellulase.
28. A detergent composition comprising a lipase protein according to any of claims 4-19 as well as a protease.
29. A detergent composition according to claim 28 which additionally comprises another enzyme such as an amylase, peroxidase and/or cellulase.
30. A detergent composition according to claim 28 or 29, which is in liquid form.
31. A method of constructing a protein with improved stability against proteolytic degradation, wherein one or more protease labile amino acid segments located on the surface of the protein are substituted by protease non-labile amino acid segment(s), which method comprises, a) identifying labile amino acid segments located on the surface of the protein by (i) incubating the protein with a protease for a period of time sufficient to provide cleavage of the protein into smaller peptide fragments, (ii) isolating the peptide fragments resulting from step (i) by HPLC, (iii) identifying primary cleavage sites in the protein by N-terminal amino acid sequencing of the peptide fragments isolated in step (ii), b) compiling amino acid segments on the protein to be stabilized or on other proteins which are assumed not to be labile to the protease when tested under similar conditions by searching an appropriate database, c) fitting the thus compiled non-labile amino acid segment(s) into a computer graphic model of the protein and evaluating for appropriate sequence, three-dimensional structure and contacts with surrounding amino acid residues in the protein, and optionally, if contacts between amino acid residues in the substituent amino acid segment and the protein sequence in which it has been introduced are not optimal, substituting one or more amino acid residues within the segment to ensure a better fit.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| WOPCT/DK91/00350 | 1991-11-26 | ||
| DK9100350 | 1991-11-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2124316A1 true CA2124316A1 (en) | 1993-06-10 |
Family
ID=8153700
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002124316A Abandoned CA2124316A1 (en) | 1991-11-26 | 1992-11-26 | Protease-stable proteins |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0667915A1 (en) |
| JP (1) | JPH07504807A (en) |
| BR (1) | BR9206815A (en) |
| CA (1) | CA2124316A1 (en) |
| FI (1) | FI942467A (en) |
| WO (1) | WO1993011254A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK154292D0 (en) * | 1992-12-23 | 1992-12-23 | Novo Nordisk As | NEW ENZYM |
| WO2002095076A2 (en) * | 2001-05-23 | 2002-11-28 | Kabushiki Kaisha Toyota Chuo Kenkyusho | Modified polypeptides having protease-resistance and/or protease-sensitivity |
| WO2006031554A2 (en) | 2004-09-10 | 2006-03-23 | Novozymes North America, Inc. | Methods for preventing, removing, reducing, or disrupting biofilm |
| EP2247721A2 (en) * | 2008-02-29 | 2010-11-10 | The Procter & Gamble Company | Detergent composition comprising lipase |
| WO2013016115A1 (en) | 2011-07-22 | 2013-01-31 | Novozymes North America, Inc. | Processes for pretreating cellulosic material and improving hydrolysis thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3854249T2 (en) * | 1987-08-28 | 1996-02-29 | Novo Nordisk As | Recombinant Humicola Lipase and Process for the Production of Recombinant Humicola Lipases. |
| GB8915658D0 (en) * | 1989-07-07 | 1989-08-23 | Unilever Plc | Enzymes,their production and use |
-
1992
- 1992-11-26 WO PCT/DK1992/000351 patent/WO1993011254A1/en not_active Application Discontinuation
- 1992-11-26 CA CA002124316A patent/CA2124316A1/en not_active Abandoned
- 1992-11-26 JP JP5509724A patent/JPH07504807A/en active Pending
- 1992-11-26 BR BR9206815A patent/BR9206815A/en not_active Application Discontinuation
- 1992-11-26 EP EP92924587A patent/EP0667915A1/en not_active Withdrawn
-
1994
- 1994-05-26 FI FI942467A patent/FI942467A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1993011254A1 (en) | 1993-06-10 |
| FI942467A0 (en) | 1994-05-26 |
| EP0667915A1 (en) | 1995-08-23 |
| FI942467A (en) | 1994-05-26 |
| JPH07504807A (en) | 1995-06-01 |
| BR9206815A (en) | 1995-10-31 |
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Effective date: 19981126 |