EP0667915A1 - Proteine protease-stable - Google Patents

Proteine protease-stable

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Publication number
EP0667915A1
EP0667915A1 EP92924587A EP92924587A EP0667915A1 EP 0667915 A1 EP0667915 A1 EP 0667915A1 EP 92924587 A EP92924587 A EP 92924587A EP 92924587 A EP92924587 A EP 92924587A EP 0667915 A1 EP0667915 A1 EP 0667915A1
Authority
EP
European Patent Office
Prior art keywords
seq
lipase
protein according
amino acid
type
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.)
Withdrawn
Application number
EP92924587A
Other languages
German (de)
English (en)
Inventor
Allan Svendsen
Ib Groth Clausen
Shamkant Anant Pathar
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0667915A1 publication Critical patent/EP0667915A1/fr
Withdrawn legal-status Critical Current

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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

Definitions

  • 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.
  • 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 5 et al., BioEssavs 8. (2), 1988, pp.
  • loops 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, deletions or sequence changes) among homologous proteins typically resides in the 0 loop structures.
  • most loops have five or less amino acid residues, and the majority of these have 4 or 5 residues.
  • 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.
  • the present invention relates to a protein with improved stability against proteolytic degradation, wherein one or more protease labile amino acid segments are substituted by protease non-labile amino acid segment(s) .
  • 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 /3-sheet) . Such amino acid segments are often found in loop regions connecting such regular structures.
  • proteolytic enzyme proteolytic enzyme
  • proteolytic non-labile is used to indicate an amino acid segment which is more slowly, or not at all, degraded by a proteolytic enzyme.
  • Non-labile 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 in the molecule. It has furthermore been found that different proteolytic enzymes preferentially attack different amino acid segments so that a segment which is non-labile in the presence 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 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 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 sequencing after isolation of the peptide fragments by HPLC (cf. K.L.
  • Non-labile segments which may also be derived from loop regions, are then fitted into a computer graphic 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 possible to substitute one or more amino acid residues within the segment to ensure a better fit.
  • amino acids amino acids:
  • 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 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 may be any protein which is frequently brought into contact with proteases when used and which is consequently subject to loss or substantial reduction of activity due to proteolytic cleavage.
  • the protein may be an enzyme, in particular a detergent enzyme which is frequently used together with a protease.
  • enzymes are an amylase, a cellulase, a peroxidase, a xylanase and a protease.
  • 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.
  • the parent 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.
  • the parent lipase may be selected from yeast, e.g. Candida , lipases, bacterial, e.g. Pseudomonas, lipases or fungal, e.g. Humicola or Rhizomucor, lipases.
  • the parent lipase is a Humicola lanuginosa lipase, in particular the lipase produced by H . lanuginosa strain DSM 4106 (cf. EP 258 068) .
  • the protease labile 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.
  • 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 (SEQ 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 ID No. 15), SRGV (SEQ ID No. 16), LDTG (SEQ ID No. 17), YYAA (SEQ ID No.
  • the segment DYGN may be substituted by a segment 0 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.
  • segment EGID may be 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).
  • one or more amino acid residues in the segment REFG (SEQ ID No. 1) or DYGN (SEQ ID No. 2) may be substituted by any amino acid residue capable of making the lipase less protease labile.
  • amino acid residues are proline and arginine.
  • 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 by Pro or Arg.
  • 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 cDNA or genomic library and screening for positive clones by 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 5 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.
  • 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. 2, 1984, pp.
  • oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
  • the DNA sequence may be of mixed genomic and 0 synthetic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire DNA construct, in accordance with standard techniques.
  • the DNA construct may also be prepared by 5 polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or R.K. Saiki et al., Science 239, 1988, pp. 487-491.
  • DNA sequence produced by methods described above, or any alternative methods known in the art, 0 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.
  • control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
  • nucleotides encoding a "signal sequence” may be inserted prior to the protein-coding sequence.
  • 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., 1978, Proc. Natl. Acad. " Sci. U.S.A. 75:3727- 103731) and the tac promoter (DeBoer, et al. , 1983, Proc. Natl. Acad. Sci. U.S.A. 8 ⁇ :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 5 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.
  • suitable yeast cells include cells of Saccharomyces spp. , such as S . cerevisiae or a methylotrophic yeast from the 0 genera Hansenula , such as Hansenula polymorpha , or Pichia such as Pichia pastoris .
  • suitable bacterial cells include cells of Bacillus spp., such as cells of B . subtilis, B . licheniformis or B . lentus.
  • the host cell is transformed by an 5 expression vector carrying the DNA sequence.
  • 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 it is cleaved from the product upon secretion.
  • control sequences as defined above is intended to include a signal sequence, when is present.
  • a filamentous fungus is used as the host organism.
  • the filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant proteins, e.g. a strain of Aspergillus sp. , such as A. niger, A . nidulans or A . oryzae .
  • a strain of Aspergillus sp. such as A. niger, A . nidulans or A . oryzae .
  • the use of A . oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023.
  • the DNA sequence coding for the protein variant is preceded by a promoter.
  • the promoter may be any DNA sequence exhibiting a strong transcriptional activity in Aspergillus and may be derived from a gene encoding an extracelluar or intracellular protein such as an amylase, a glucoa ylase, a protease, a lipase, a cellulase or a glycolytic enzyme.
  • suitable promoters are those derived from the gene encoding A . oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A . niger neutral ⁇ -amylase, A . niger acid stable ⁇ - amylase, A . niger glucoamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease or A . oryzae triose phosphate isomerase.
  • a preferred promoter for use in the process of the present invention is the A. oryzae TAKA amylase promoter as it exhibits a strong transcriptional activity in A . oryzae .
  • the sequence of the TAKA amylase promoter appears from EP 238 023.
  • Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the techniques used to transform a fungal host cell may suitably be as described in EP 238 023.
  • the DNA sequence encoding the protein may be preceded by a signal sequence which may be a naturally occurring signal sequence or a functional part thereof or a synthetic sequence providing secretion of the protein from the cell.
  • the signal sequence may be derived from a gene encoding an 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 ⁇ - 10 amylase or A. niger glucoamylase.
  • the medium used to culture the transformed host cells may be any 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 5 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 0 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 5 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.
  • a detergent additive 5 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
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods. Other enzyme stabilizers are well known in the art.
  • Protected enzymes may be prepared according to the method disclosed in EP 238 216.
  • the detergent 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 other enzymes, such as a cellulase, peroxidase or amylase, conventionally included in detergent additives.
  • the invention relates to a detergent composition
  • a detergent composition comprising a lipase protein of the invention and a protease.
  • Detergent compositions of the invention additionally comprise surfactants which may be of the anionic, non-ionic, cationic, amphoteric, or zwitterionic type as well as mixtures of these surfactant classes.
  • Suitable surfactants are linear alkyl benzene sulfonates (LAS) , alpha olefin sulfonates (AOS) , alcohol ethoxy sulfates (AEOS) , alcohol "ethoxylates (AEO) , alkyl sulphates (AS) , alkyl polyglycosides (APG) and alkali metal salts of natural fatty acids.
  • LAS linear alkyl benzene sulfonates
  • AOS alpha olefin sulfonates
  • AEOS alcohol ethoxy sulfates
  • AEO alcohol "ethoxylates
  • AS alkyl sulphates
  • APG alkyl polyglycosides
  • alkali metal salts of natural fatty acids 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, 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 be stabilized in a liquid detergent by inclusion of enzyme stabilizers as indicated above.
  • 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 detergent compositions of the invention, either separately or in a combined additive as described above.
  • Fig. 1 shows a restriction map of-plasmid pAOl
  • 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 mutagenesis by PCR.
  • Fig. 5 shows the protease stability of variant lipases of the invention as compared to that of the wild type H. lanuginosa lipase.
  • the cloning of the Humicola lanuginosa lipase and the express- 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- ding region.
  • the modifications were made in the following way: p960 was digested with Nrul and BamHI restriction enzymes. Between these two sites the BamHI/Nhel fragment from plasmid pBR322, in which the Nhel fragment was filled in with Klenow polymerase, was cloned, thereby creating plasmid pAOl (Fig. 1) , which contains unique BamHI and Nhel sites. Between these 5 unique sites BamHI/Xbal fragments from p960 was cloned to give pAHL (Fig. 2) .
  • the circular plasmid pAHL was linearized with the restriction enzyme SphI in the following 50 ⁇ l reaction mixture: 50 mN NaCl, 10 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , 1 mM dithiothreitol, 1 ⁇ g plasmid and 2 units of SphI.
  • the digestion was carried out for 2 hours at 37°C.
  • 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 centrifugation and drying of the pellet, the linearized DNA was dissolved in 50 ⁇ l of H-,0 and the concentration estimated on an agarose gel.
  • Helper 1 and helper 2 are complementary to sequences outside the coding region, and can thus be used in combination with any mutagenisation primer in the construction of a mutant sequence. All 3 steps were carried out in the following buffer contai ⁇ ning: 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.001% gelatin, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP, 0.2 mM TTP, 2.5 units Taq polymerase.
  • step 1 100 pmol primer A, 100-pmol primer B and
  • 1 fmol linearized plasmid were added to a total of 100 ⁇ l reac ⁇ tion mixture and 15 cycles consisting of 2 minutes at 95°C, 2 minutes at 37°C and 3 minutes at 72°C were carried out.
  • step 2 The concentration of the PCR product was estimated on an agaro- 10 se gel. Then, step 2 was carried out. 0.6 pmol step 1 product and 1 fmol linearized plasmid were contained in a total of 100 ⁇ l of the previously mentioned buffer and 1 cycle consisting of 5 minutes at 95°C, 2 minutes at 37°C and 10 minutes at 72°C was carried out.
  • step 2 15
  • 100 pmol primer C and 100 pmol primer D are added (1 ⁇ l of each) and 20 cycles consisting of 2 minutes at 95°C, 2 minutes at 37°C and 3 minutes at 72°C were carried out.
  • This manipulation constituted step 3 in the mutagenisation procedure.
  • step 3 The product from step 3 was isolated from an agarose gel and re-dissolved in 20 ⁇ l of H 2 0. Then, it was digested with the re ⁇ striction enzymes BamHI and BstXI in a total volume of 50 ⁇ l with the following composition: 100 mM NaCl, 50 mM Tris-HCl, pH
  • the expression plasmid pAHL was cleaved with BamHI and BstXI 0 under the conditions described above and the large fragment was isolated from an agarose gel.
  • the mutated frag ⁇ ment isolated above was ligated and the ligation mix was used 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 5 chain termination procedure developed by Sanger.
  • the plasmid was named pAHLS(209-212) GASG and is identical to pAHL, except for the substituted codons.
  • G212R by substituting the glycine (G) residue in position 212 0 with an arginine (R) residue;
  • G212P by substituting the glycine (G) residue in position 212 with a proline (P) residue;
  • E210R by substituting the glutamic acid (E) residue of position 210 with an arginine (R) residue.
  • TCTGC-3' (SEQ ID No. 53) pAHLY164R 5'-CACGTCGATATCGCGACCATTTCCACG-3' (SEQ ID NO. 54) pAHLG212R 5'-GAATGGCTGTATCTAAATTCGCGCG-3' (SEQ ID No. 55) pAHLG212P 5'-GAATGGCTGTATGGAAATTCGCGCG-3' (SEQ ID No. 56)
  • PAHLE210R 5'-GTAACCGAATCTGCGCGGCGGG-3' (SEQ ID No. 57)
  • the plasmids described above were transformed into A. oryzae IFO 4177 by cotransformation with p3SR2 containing the amdS gene from A. nidulans as described in the transformation pro ⁇ cedure given in the methods section above.
  • Protoplasts prepared 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 lipase activity described below.
  • a substrate for lipase was prepared by emulsifying glycerine 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 1 Centrifuge the fermentation supernatant, discard the 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 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. 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 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 S and pH to 7. Apply the pool on High performance Q Sepharose (Pharmacia) column pre- equilibrated with 50 m tris -acetate buffer pH 7. Elute the bound enzyme with linear salt gradient.
  • High performance Q Sepharose Pharmacia
  • the variant lipases Subst. (162-165)PRLP, Subst. (209-212)YPRS, Subst. (209-212)GASG, G212R and G212P were purified as described in Example 4 above and each diluted with 0.1M Tris-puffer, pH 9.0. 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 1:5. The final lipase concentration was lmg/ml (except for the variant G212P, for which it was 0.8 mg/ml) . The reaction was carried out at 22°C. At appropiate times, probes were taken from the reaction mixture and immediately subjected to the above described lipase activity assay.
  • SavinaseTM concentration of lOOmg/ml in 50% mono-propylene glycol
  • 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.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOIiSCULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • liYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

Une protéine avec une stabilité contre la dégradation protéolytique améliorée est décrite. Dans cette protéine, un ou plusieurs segments d'acide aminé de protéase labile sont substitués par un/des segment(s) d'acide aminé de protéase non labile. De manière avantageuse, la protéine à stabiliser est une enzyme, par exemple d'origine microbienne telle qu'une lipase, par exemple une lipase dérivée d'une souche de l'Humicola, par exemple l'Humicola lanuginosa, ou le Rhinomucor, par exemple le Rhinomucor miehei. Les protéines stabilisées peuvent être produites par les techniques de recombination de l'ADN et peuvent avantageusement être utilisées pour des détergents.
EP92924587A 1991-11-26 1992-11-26 Proteine protease-stable Withdrawn EP0667915A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
WOPCT/DK91/00350 1991-11-26
DK9100350 1991-11-26
PCT/DK1992/000351 WO1993011254A1 (fr) 1991-11-26 1992-11-26 Proteine protease-stable

Publications (1)

Publication Number Publication Date
EP0667915A1 true EP0667915A1 (fr) 1995-08-23

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EP92924587A Withdrawn EP0667915A1 (fr) 1991-11-26 1992-11-26 Proteine protease-stable

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EP (1) EP0667915A1 (fr)
JP (1) JPH07504807A (fr)
BR (1) BR9206815A (fr)
CA (1) CA2124316A1 (fr)
FI (1) FI942467A (fr)
WO (1) WO1993011254A1 (fr)

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DK154292D0 (da) * 1992-12-23 1992-12-23 Novo Nordisk As Nyt enzym
WO2002095076A2 (fr) * 2001-05-23 2002-11-28 Kabushiki Kaisha Toyota Chuo Kenkyusho Polypeptides modifies presentant une resistance a la protease et/ou une sensibilite a la protease
CN103181400B (zh) 2004-09-10 2016-08-10 诺维信北美公司 防止、去除、减少或破坏生物膜的方法
JP2011513539A (ja) * 2008-02-29 2011-04-28 ザ プロクター アンド ギャンブル カンパニー リパーゼを含む洗剤組成物
US20140147895A1 (en) 2011-07-22 2014-05-29 Novozymes A/S Processes for Pretreating Cellulosic Material and Improving Hydrolysis Thereof

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GB8915658D0 (en) * 1989-07-07 1989-08-23 Unilever Plc Enzymes,their production and use

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BR9206815A (pt) 1995-10-31
CA2124316A1 (fr) 1993-06-10
JPH07504807A (ja) 1995-06-01
WO1993011254A1 (fr) 1993-06-10
FI942467A0 (fi) 1994-05-26
FI942467A (fi) 1994-05-26

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