EP0667915A1

EP0667915A1 - Protease-stable proteins

Info

Publication number: EP0667915A1
Application number: EP92924587A
Authority: EP
Inventors: Allan Svendsen; Ib Groth Clausen; Shamkant Anant Pathar
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1991-11-26
Filing date: 1992-11-26
Publication date: 1995-08-23
Also published as: BR9206815A; WO1993011254A1; CA2124316A1; JPH07504807A; FI942467A0; FI942467A

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.

Description

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 INVENTION

10 Apart from regular α-helices and β-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 of 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 0 together. E.J. Milner-White and R. Poet, TIBS 12. 1987, pp. 189-192, describe the structure of different types of loops, primarily β-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. 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, deletions or sequence changes) among homologous proteins typically resides in the 0 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. 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 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 α-helix or a /3-sheet) . Such amino acid segments are often found in loop regions connecting such regular structures. The term "protease 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.

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 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. Stone et al., "Enzymatic digestion of proteins and HPLC peptide isolation" in A Practical Guide to Protein and Peptide Purification for Microsequencing, 1989) . Such identified primary cleavage sites may then be analyzed by means 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 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 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. In the preisent description and claims, the following abbreviations are used: Amino acids:

In describing lipase variants according to the invention, the following nomenclature is used for ease of reference:

Original amino acid(s) :position(s) :substituted amino acid(s)

According to this nomenclature, for instance the substitution of tyrosine for arginine in position 164 is shown as:

Y164R 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 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 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 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. 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 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. Candida , lipases, bacterial, e.g. Pseudomonas, lipases or fungal, 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 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. 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 (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. 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 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. 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); and the segment EGID (SEQ ID No. 3) 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).

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 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 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 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.

10 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. 2, 1984, pp.

15 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, 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.

According to the invention, 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. To permit the secretion of the expressed protein, nucleotides 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 β-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.

Examples of 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 . 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 5 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 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 method of producing the protein of the 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 proteins, e.g. 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.

For expression of the protein in Aspergillus , 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.

Examples of suitable promoters are those derived from the gene encoding A . oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A . niger neutral α-amylase, A . niger acid stable α- amylase, A . niger glucoamylase, Rhizomucor miehei lipase, A . oryzae alkaline protease or A . oryzae triose phosphate isomerase.

In particular when the host organism is A . oryzae , 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.

To ensure secretion of the protein from the host cell, 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. 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 α-amylase, A. niger acid-stable α- 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. 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 methods known in the art. 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.

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 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) , 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, 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. 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 detergent compositions of the invention, either separately or in a combined additive as described above. BRIEF 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 pAOl;

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 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 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 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) .

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,

10 180, 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 iracloth and washed with 200 ml of 0.6 M MgS0₄. The mycelium was suspended

25 in 15 ml of 1.2 M MgS0₄, 10 mM NaH₂P0₄, 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 37°C until a large number 0 of protoplasts was visible in a sample inspected under the microscope. The suspension was filtered through miracloth, the filtrate transferred to a sterile tube and overlaid with 5 ml of 0.6 M sorbitol, 100 mM Tris-HCl, pH 7.0. Centrifugation was performed for 15 min. at 1000 g and the protoplasts were collected from the top of the MgS0₄ cushion. 2 volumes of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH 7.5, 10 mM- CaCl₂) 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 protoplasts were resuspended in 0.2-1 ml of STC.

100 μl of protoplast suspension was mixed with 5-25 μg of p3SR2 (an A. nidulans amdS gene carrying plasmid. described in Hynes et al., Mol. and Cel. Biol., Vol. 3, No. 8, 1430-1439, Aug. 1983) in 10 μl of STC. The mixture was left at room temperature 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 2 ml of 1.2 M sorbitol. 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 37°C spores 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. EXAMPLES

EXAMPLE 1

Construction of a plasmid expressing the Subst. (209-212)GASG variant of Humicola lanuginosa lipase

Linearization of plasmid pAHL The circular plasmid pAHL was linearized with the restriction enzyme SphI in the following 50 μl reaction mixture: 50 mN NaCl, 10 mM Tris-HCl, pH 7.9, 10 mM MgCl₂, 1 mM dithiothreitol, 1 μg 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 μl of H-,0 and the concentration estimated on an agarose gel.

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 1 (=B) 5'-GGTCATCCAGTCACTGAGACCCTCTACCTATTAAA- TCGGC-3' (SEQ ID No. 49)

PCR Helper 2 (=C) 5 '-CCATGGCTTTCACGGTGTCT-3 ' (SEQ ID No. 50) PCR Handle (=D) 5'-GGTCATCCAGTCACTGAGAC-3 ' (SEQ ID No. 51)

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 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 1, 100 pmol primer A, 100-pmol primer B and

1 fmol linearized plasmid were added to a total of 100 μl 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.

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 μl 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.

15 To the step 2 reaction mixture, 100 pmol primer C and 100 pmol primer D are added (1 μl 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.

20 Isolation of mutated restriction fragment:

The product from step 3 was isolated from an agarose gel and re-dissolved in 20 μl of H₂0. Then, it was digested with the re¬ striction enzymes BamHI and BstXI in a total volume of 50 μl with the following composition: 100 mM NaCl, 50 mM Tris-HCl, pH

257.9, 10 mM MgCl₂, 1 mM DTT, 10 units of BamHI and 10 units of BstXI. Incubation was carried out at 37°C for 2 hours. The 733 bp BamHI/BstXI fragment was isolated from an agarose gel.

Ligation to expression vector pAHL:

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. To this vector, 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.

EXAMPLE 2

Construction of plasmids expressing other variants of Humicola ιo lipase

Using the same method as described in example l, the following mutants were constructed:

Subst. (209-212) YPRS by substituting amino acid residues 209-212 with the fragment YPRS;

15. Subst. (162-165)PRLP by substituting amino acid residues 162-165 with the fragment PRLP;

Y164R by substituting the tyrosine (Y) residue in position 164 with an arginine (R) residue;

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; and

E210R by substituting the glutamic acid (E) residue of position 210 with an arginine (R) residue.

5 The plasmid names (pAHL followed by the mutant names indicated above) and primers used for the modifications are listed below. Plasmid name Primer A sequence pAHLS(209-212)YPRS 5'-TTTGATCCAGTACTCTGGGCTAGAATGGCTGTAAGATCT- TGGGTACGGCGGGAGTCTAGG-3 ^r (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) pAHLG212P 5'-GAATGGCTGTATGGAAATTCGCGCG-3' (SEQ ID No. 56)

PAHLE210R 5'-GTAACCGAATCTGCGCGGCGGG-3' (SEQ ID No. 57)

EXAMPLE 3

Expression of lipase variants in A. oryzae

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 μg 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.

The best transformant was selected for further studies and grown in a l 1 shake flask on 200 ml FG4 medium (3% soy meal, 3% maltodextrin, 1% peptone, pH adjusted to 7.0 with 4 M NaOH) for 4 days at 30°C. EXAMPLE 4

Purification of lipase variants of the invention

Assay for lipase activity:

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.

EXAMPLE 5

Protease stability of variant proteins

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 Savinase™ (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.

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.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT:

(A) NAME: NOVO NORDISK A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd

(E) COUNTRY: DENMARK

(F) POSTAL CODE (ZIP) : DK-2880

(G) TELEPHONE: +45 44448888 (H) TELEFAX: +45 4449 3256

(I) TELEX: 37304

(ii) TITLE OF INVENTION: Protease-Stable Proteins (iii) NUMBER OF SEQUENCES: 57 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: WO PCT/DK91/00350

(B) FILING DATE: 26-NOV-1991

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Arg Glu Phe Gly 1 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Asp Tyr Gly Asn

1

(2) INFORMATION FOR SEQ ID NO: 3 : (i) SEQUENCE OiARACTERISTTCS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) røLECULE TYPE: peptide

(iii) HYPCHHEETCAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Glu Gly lie Asp 1

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gly Ala Ser Gly

1

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Gly Ala Ala Gly

1

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOIECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Gly Ala Arg Gly 1

(2) INIΌRMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) 13YPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) ERAC-MENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Tyr Pro Gly Ser 1

(2) INFORMATION FOR SEQ 3D NO: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Tyr Pro Arg Ser

1

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: His Asn Arg Gly 1 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Tyr Thr Gly Asn 1

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: lie Ser Ser Glu 1

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTT-SENSE: NO (v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 12: Asn Asn Ala Gly

1

(2) INFORMATION FOR SEQ 3D NO: 13 : (i) SEQUENCE CHARACTERISTICS:

(A) -LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MDI^ECULE TYPE: peptide

(iϋ) HYPOTHETICAL: NO

(ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 13:

Ser Phe lie Asn

1

(2) INFORMATION FOR SEQ 3D NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) 1-lYPOTHETTCAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 14: Asp Gin Asn Gly 1

(2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: Ala Ser Phe Ser 1

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Ser Arg Gly Val

1

(2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTTCS: (A) lENGTH: 4 amino acids

(B) TYPE: aπiino acid

(C) STRANDEDNESS: single

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: Leu Asp Thr Gly 1 (2) INFORMATION FOR SEQ 3D NO: 18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ϋi) HYPOTHETICAL: NO ( i) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 18:

Tyr Tyr Ala Ala

1

(2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS:

(A) IENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iϋ) HYPCTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 19: lie Asn Asp lie 1

(2) INFORMATION FOR SEQ 3D NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) lYDI.-ECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: Trp Tyr Phe Gly

1

(2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: Ser lie Glu Asn 1

(2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MDLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Gly Ser Thr Tyr 1

(2) DEFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) R^POTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal ( i) SEQUENCE DESCRIPTION: SEQ 3D NO: 23: Asp Ser Thr Asn 1

(2) INFORMATION FOR SEQ 3D NO: 24: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (ϋi) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 24: Pro Asp Leu Arg 1

(2) INFORMATION FOR SEQ 3D NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAQYIENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 25: leu Asp Thr Gly 1 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Gly Asn Arg Tyr 1

(2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTT-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: Ser Gly Val Met 1

(2) INTORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 28: Arg Tyr Pro Ser

1

(2) INFORMATION FOR SEQ 3D NO: 29: - (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 29:

Asn Gly leu Val

1

(2) INFORMATION FOR SEQ 3D NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(v) FRAlGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: Ser Phe Ser lie 1

(2) INFORMATION FOR SEQ 3D NO: 31: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iii) ANTT-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: Leu Gly Ser Pro 1

(2) INFORMATION FOR SEQ ID NO: 32: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOIiSCULE TYPE: peptide (iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Arg Ala Ser Phe 1

(2) INFORMATION FOR SEQ ID NO: 33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: Val Pro Trp Gly 1 (2) INFORMATION FOR SEQ 3D NO: 34: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) KDLECULE TYPE: peptide

(ϋi) HYPCTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 34:

Pro Asp Leu Asn

1

(2) INFORMATION FOR SEQ 3D NO: 35: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single ^' (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAQYIENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 35: Ser Phe Val Pro 1

(2) INFORMATION FOR SEQ 3D NO: 36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: peptide

(iii) 1-iYPOTHEIICAL: NO (ϋi) ANTT-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Pro Asp Tyr Arg

1

(2) INPOI?MATION FOR SEQ ID NO: 37: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOIECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAQYENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Pro Arg Leu Pro

1

(2) INFORMATION FOR SEQ ID NO: 38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Thr Val Leu Pro 1

(2) INFORMATION FOR SEQ ID NO: 39: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 aπiino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: peptide

(iϋ) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal ( i) SEQUENCE DESCRIPTION: SEQ 3D NO: 39: lie Gly Thr Cys 1

(2) INFORMATION FOR SEQ 3D NO: 40: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal ( i) SEQUENCE DESCRIPTION: SEQ 3D NO: 40:

Thr Gly Gly Thr

1

(2) INPORMATION FOR SEQ 3D NO: 41: (i) SEQUENCE OΪAEIACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 41: Thr Asn Lys Leu 1 (2) INFORMATION FOR SEQ ID NO: 42: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

(iii) ANΠ-SENSE: NO (v) FRAGMENT TYPE: internal

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Val Gly Asp Val 1

(2) INFORMATION FOR SEQ ID NO: 43: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: He Gly Val Leu 1

(2) INFORMATION FOR SEQ ID NO: 44: (i) SEQUENCE CHARACTERISTICS: (A) IENGTH: 4 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTTON: SEQ 3D NO: 44: Gly Ser Thr Tyr

1

(2) IDOTΌRMATION FOR SEQ 3D NO: 45: (i) SEQUENCE CHARACTERISTICS:

(A) IENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPCuΗETICAL: NO

(iii) ANTI-SENSE: NO

(v) FRAQYENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 45: Arg Tyr Ala Asn 1

(2) INFORMATION FOR SEQ 3D NO: 46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAQYENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 46: Pro Asn He Pro 1

(2) 32-IFORMATION FOR SEQ 3D NO: 47: (i) SEQUENCE CHARACTERISTICS:

(A) IENGTH: 4 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(ϋi) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: Thr Leu Val Pro 1

(2) INFORMATION FOR SEQ ID NO: 48: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) liYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:

TTTGATCCAG TACTCTGGGC TAGAATGGCT GTAACCAGAA GCACCOGGOG GGAGTCTAGG 60

(2) INFORMATION FOR SEQ ID NO: 49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iϋ) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: GGTCATCCAG TCACTGAGAC CCTCTACCTA TTAAATOGGC 40 (2) INFORMATION FOR SEQ 3D NO: 50: (i) SEQUENCE OiARACIΕRISTTCS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iϋ) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 50: ccaTGGcriT CΆCGGTGTCΓ 20

(2) INFORMATION FOR SEQ 3D NO: 51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE: cDNA (ϋi) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 51: GGTCATCCAG TCACTGAGAC 20

(2) INFORMATION FOR SEQ 3D NO: 52: (i) SEQUENCE CHARACTERISTICS:

(A) .LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iϋ) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52 : TTTGATCCAG TACTCTGGGC TAGAATGGCT GT GATCTT GGGTACGGCG GGAGTCTAGG 60

(2) INPORMATION FOR SEQ ID NO: 53 : (i) SEQUENCE CHARACTERISTICS: (A) ILENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear ( ) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTT-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: TGAAAACAOG TCGATTGGCA ATCTTGGTCC ACGCAGGTCT GC 42

(2) INPORMATION FOR SEQ ID NO: 54: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs ^" (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iϋ) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:

CACGTCGATA TCGCGACCAT TTCCACG 27

(2) INPORMATION FOR SEQ ID NO: 55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear ( ) MOLECULE TYPE: cDNA

(iϋ) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAQYENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 55: GAATGGCTGT ATCTAAATrC GCGCG 25

5 (2) INFORMATION FOR SEQ 3D NO: 56: (i) SEQUENCE CHARACTERISTICS:

(A) .LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (ϋi) HYPOTHETICAL: NO (ϋi) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ 3D NO: 56: GAATGGCTGT ATGGAAATTC GCGCG 25

(2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iϋ) HYPOTHETICAL: NO (iϋ) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCKE-TTGN: SEQ 3D NO: 57: GTAACCGAAT CTGCOOGGCG GG 22

Claims

1. 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

5 segment(s) .

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.

104. 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.

157. 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- 0 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.

10. A lipase protein according to claim 9, wherein the segment 5 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 5 (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

10 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

15 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

20 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.

25 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.

3016. A lipase protein according to claim 5, wherein the parenr 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.

519. 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 10 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 .

15 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_-_

20 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.

2525. 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.