WO1993000426A1 - Lipase pancreatique de mammifere et sa variante - Google Patents

Lipase pancreatique de mammifere et sa variante Download PDF

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Publication number
WO1993000426A1
WO1993000426A1 PCT/DK1992/000200 DK9200200W WO9300426A1 WO 1993000426 A1 WO1993000426 A1 WO 1993000426A1 DK 9200200 W DK9200200 W DK 9200200W WO 9300426 A1 WO9300426 A1 WO 9300426A1
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WIPO (PCT)
Prior art keywords
gpl
ser
gly
thr
hpl
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PCT/DK1992/000200
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English (en)
Inventor
Siv Annegrethe Hjorth
Helle Fabricius Woldike
Lars Thim
Erik Gormsen
Robert Verger
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Novo Nordisk A/S
Centre National De La Recherche Scientifique (C.N.R.S)
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Priority to EP92914609A priority Critical patent/EP0599859A1/fr
Publication of WO1993000426A1 publication Critical patent/WO1993000426A1/fr

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    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to mammalian pancreatic lipase and variants thereof, a DNA sequence encoding the lipase, a method of producing mammalian pancreatic lipases and lipase variants, and their use as detergent enzymes and digestive enzymes.
  • the DNA sequence encoding human pancreatic lipase, and the tertiary structure of the enzyme appears from F.K. Winkler et al.. Nature 343. 1990, pp. 771-774.
  • One of the outstanding features of the enzyme is the presence of a surface loop structure between Cys 238 and Cys 262 which covers the active site when the lipase is in inactive form, and which changes its conformation when the lipase is activated so as to make the active serine accessible to a lipid substrate (cf. also Brady et al.. Nature 343. 1990, pp. 767-770, describing a similar structure of a microbial lipase) .
  • lipase from guinea pig pancreas is desc- ribed by J. Fauvel et al., Biochim. Biophvs. Acta 663. 1981, pp. 446-456. It is noted that, apart from lipase activity, guinea pig pancreatic lipase also has phospholipase activity.
  • Guinea pig pancreatic lipase has now been cloned and sequenced at the DNA and amino acid level. Although the guinea pig lipase has a high degree of sequence homology to the human pancreatic enzyme, it was surprisingly found that the guinea pig lipase lacks a loop structure corresponding to that covering the active site of the human lipase. It is therefore envisaged that the guinea pig lipase has certain properties which differ from those of the human lipase such as a different substrate specificit .
  • the present invention relates to a guinea pig pancreatic lipase (GPL) essentially free from other guinea pig proteins which has the amino acid sequence shown in the appended Sequence Listing ID No. 2, or an enzymatically active variant thereof.
  • GPL pancreatic lipase
  • the term "variant" is intended to indicate a polypeptide which is derived from the native lipase protein by suitably modifying the DNA sequence coding for the native lipase, resulting in the addition of one or more amino acid residues to either or both the N- and C-terminal end of the native enzyme, substitution of one or more amino acid residues at one or more different sites in the amino acid sequence, deletion of one or more amino acid residues at either or both ends of the native protein or at one or more sites in the amino acid sequence, or insertion of one or more amino acid residues at one or more sites in the amino acid sequence.
  • variant is also intended to include naturally * occurring variants of the enzyme which are homologous to the amino acid sequence shown herein, e.g. pancreatic lipases of other mammalian species such as porcine, bovine, canine, feline or coypu pancreatic lipase, or enzymatically active derivatives thereof.
  • the term "enzymatically active" as used about the GPL variant is intended to indicate that the variant should have at least one enzymatic activity and normally more, especially one or more of the enzymatic activities of the GPL. It has been found that, apart from lipase and phospholipase activity, the native GPL also exhibits esterase activity and presumably protease activity. Any of the enzymatic activities of the GPL, and thus of a GPL variant as defined herein, may be determined using assays known in the art for the enzymatic activity in question. The region of the enzyme which gives rise to at least some of the esterase activity is presumed to be localized to the C- terminal end of the lipase (cf. J.D.
  • GPL may be C-terminally truncated.
  • a preferred GPL variant of this type is GPL W ⁇ 9 .
  • GPL lacks the loop structure which, in HPL, covers the active site of the enzyme when the HPL • is in the inactive state. More specifically, the GPL of the invention has a sequence of five amino acids (Lys-Thr-Gly-Ile-Ser) between the amino acids 239 and 245 rather than the longer loop structure known from HPL. It may therefore be possible to prepare a GPL variant with different enzymatic properties than the native enzyme, said variant comprising an insertional substitution between amino acid 239 and amino acid 245 of the native GPL sequence.
  • the insertional substitution comprises the following amino acid sequence
  • the insertional substitution comprises the amino acid sequence
  • Lys Lys Asn lie Leu Ser Gin lie Val Asp lie Asp Gly lie Trp Glu Gly Thr Arg Asp Phe Ala Ala
  • a still further GPL variant of the invention is one in which an amino acid of the catalytic triad Ser-His-Asp is replaced by another residue, in particular in which Ser 154 is replaced by Thr and/or Asp 17g is replaced by Glu.
  • any one of the modifications of the native GPL protein described above may be combined with one or more of the other modifications, resulting in a GPL variant which exhibits two or more of the features noted above.
  • the present invention relates to a recom- binant DNA molecule comprising a DNA sequence encoding GPL, or an enzymatically active variant thereof.
  • the invention relates to a recombinant DNA molecule with the DNA sequence shown in the appended Sequence Listing ID No. 1, or a modification of said sequence encoding GPL or an enzymatically active variant thereof.
  • nucleotide substitutions which do not give rise to another amino acid sequence of the lipase but which may correspond to the codon usage of the host organism into which the recombinant DNA molecule is introduced (i.e. modifications which, when expressed, results in GPL with the amino acid sequence shown in the appended Sequence Listing ID no. 2) , or nucleotide substitutions which do give rise to a different amino acid sequence and therefore, possibly, a different polypeptide structure without, however, impairing the properties of the lipase (i.e. modifications which, when expressed, results in enzymatically active GPL variants as defined herein) .
  • a DNA sequence encoding the present enzyme or an enzymatically active variant thereof may, for instance, be isolated by establishing a guinea pig 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 enzyme or by selecting for clones expressing the appropriate enzyme activity, or by selecting for clones producing a protein which is reactive with an antibody raised against the native GPL.
  • a genomic or cDNA sequence encoding the lipase may be modified at a site corresponding to the site(s) at which it is desired to introduce amino acid substitutions, e.g. by site- directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures.
  • the DNA sequence encoding the enzyme or an enzymatically active variant thereof may be prepared synthe ⁇ tically by established standard methods, e.g. the phosphoami ⁇ dite method described by S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22., 1981, pp. 1859-1869, or the method described by Matthes et al.. The EMBO J. 3_, 1984, pp. 801-805.
  • 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 synthe ⁇ tic, 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 recombinant DNA molecule, in accordance with standard techniques.
  • the recombinant DNA molecule may also be prepared by 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.
  • the present invention relates to certain variants of human pancreatic lipase (HPL) .
  • HPL human pancreatic lipase
  • the invention relates to a HPL variant which is deleted of one or more amino acids in the sequence between Cys ⁇ and Cys 262 , in other words, in which the loop structure described by Winkler et al. , op. cit. has been altered.
  • the entire sequence constituting the loop structure may be deleted or replaced by the sequence Lys-Thr-Gly-Ile-Ser (i.e. the corresponding "loop" sequence of GPL) .
  • Example 5 the construction of a HPL variant with no loop structure is described.
  • the HPL variant may be one which is C- terminally truncated for the preparation of a HPL variant with decreased esterase activity, as noted above for the C- terminally truncated GPL variant.
  • a particularly preferred variant of this type is HPLj. 336 .
  • native HPL contains a non-disulfide linked cystine residue at either Cys ⁇ or Cys 10 .
  • the free -SH group present on the cystine residue is sensitive to oxidation, which may be detrimental to the storage stability of the enzyme or its performance as a detergent enzyme. It may therefore be an advantage to provide a HPL variant, wherein Cys I02 or Cys 1M is replaced by another amino acid residue.
  • Such an amino acid residue is preferably an uncharged and non-bulky residue, and may preferably be selected from Ser or Thr.
  • a still further HPL variant of the invention is one in which an amino acid of the catalytic triad Ser-His-Asp is replaced by another residue, in particular in which Ser ⁇ is replaced by Thr and/or Asp is replaced by Glu.
  • any one of the modifications of the native HPL protein described above may be combined with one or more of the other modifications, resulting in a HPL variant which exhibits two or more of the features noted above.
  • lipase variants of the present invention is an enzymatically active recombinant lipase which comprises at least one GPL fragment and at least one HPL fragment, i.e. a hybrid lipase.
  • GPL fragment and HPL fragment are intended to indicate any fragment of GPL and HPL, respectively, which is sufficiently large to be recognized as such, for instance a fragment comprising at least 10 amino acid residues.
  • the recombinant variants may be prepared by in vivo or in vitro recombination, e.g. as explained in Example 12 and 13.
  • the present invention relates to a DNA sequence encoding any of the lipase variants disclosed herein.
  • a DNA sequence encoding a recombinant lipase variant as described above may be prepared by in vitro or in vivo recom ⁇ bination as described in Examples 12 and 13 from DNA encoding GPL or HPL or variants thereof.
  • the DNA sequence encoding a recombinant lipase variant may be prepared synthe- tically, e.g. as described above.
  • the GPL and GPL and HPL variants of the invention are suitably prepared on the basis of recombinant DNA procedures known in the art using eukaryotic or prokaryotic expression systems, examples of which are a mammalian expression system, an insect expression system, a fungal (including yeast) expression system and an bacterial expression system.
  • eukaryotic or prokaryotic expression systems examples of which are a mammalian expression system, an insect expression system, a fungal (including yeast) expression system and an bacterial expression system.
  • a sui ⁇ table host cell harbouring a recombinant expression vector carrying a DNA sequence encoding the enzyme is cultured under conditions conducive to the production of the enzyme.
  • the recombinant expression vector into which the DNA sequence is inserted may be any vector which may conveniently be subjec ⁇ ted to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromos ⁇ mal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosom (s) into which it has been integrated.
  • the DNA sequence encoding the enzyme should be operably connected to a suitable promoter sequence.
  • the promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the enzyme in mammalian cells are the SV 40 promoter (Subramani et al., Mol. Cell Biol. l, 1981, pp. 854-864) , the MT-1 (metallothionein gene) promoter
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes
  • the DNA sequence encoding the GPL or a GPL or HPL variant of the invention may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPI1 terminator (Alber and Kawasaki, op. cit.) .
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb region) , transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs) .
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell -in question.
  • a DNA sequence enabling the vector to replicate in the host cell -in question.
  • An examples of such a sequence is the SV 40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the host cell into which the expression vector is introduced may be any cell which is capable of producing the mammalian pancreatic lipase an example of which is a eukaryotic cell, in particular a mammalian cell.
  • Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159. 1982, pp. 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 , 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79. 1982, pp. 422-426; Wigler et al. , Cell 14, 1978, p.
  • insect cells may be used as host cells for the expression of the mammalian pancreatic lipase, for instance, using the principles described by Summers, M.D. and Smith, G.E., 1987, A Manual of Methods for Baculovirus Vectors and the Insect Cell Culture Procedures, Texas Agricultural Experiment Station & Texas A&M University, College Station Texas 77843-2475) .
  • fungal cells including yeast cells
  • bac- terial cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosac- charomyces spp. , in particular strains of Saccharomyces cerevisiae.
  • bacterial cells include cells of the genus Bacillus, especially B. subtilis.
  • suitable fungal cells i.e. a filamentous fungus, will be dealt with in detail below.
  • the medium used to culture the cells may be any conventional medium suitable for growing the cells in question, such as a serum-containing or serum-free medium containing appropriate supplements (for mammalian cells) .
  • Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) .
  • the expression vector should normally further comprise a DNA sequence encoding a preregion permitting secretion of the expressed mammalian pancreatic lipase into the culture medium.
  • the mammalian pancreatic lipase may be recovered from the medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • purification a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.
  • the present invention relates to.a process for the extracellular production of a mammalian pancreatic lipase in a filamentous fungus, the process comprising
  • step (b) transforming a suitable filamentous fungus with the recombinant DNA vector of step (a) , and
  • filamentous fungus is intended to include fungi belonging to the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes or fungi imperfecti, including Hyphomycetes such as the genera Aspergillus, Trichoderma , Penicillium. Fusariu or Humicola.
  • the recombinant DNA vector further comprises DNA sequences encoding functions permitting gene expression, and where appropriate a DNA sequence coding for a suitable marker for the selection of transformants.
  • DNA sequences encoding functions permitting gene expression typically comprise a promoter, transcription initiation sites, and transcription termination and polyadenylation functions.
  • the promoter which may be preceded by upstream activating sequences and enhancer sequences as known in the art may be any DNA sequence exhibiting a strong transcriptional activity in filamentous fungi and may be derived from a gene encoding an extracellular or intracellular protein such as an amylase, a glucoamylase, 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_j_ niger neutral ⁇ -amylase, ⁇ niger acid stable ⁇ - a ylase, A_j_ niger glucoamylase, Rhizomucor miehei lipase, ⁇ . oryzae alkaline protease or A . orvzae triose phosphate isomerase.
  • the filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recom ⁇ binant proteins, e.g. a strain of Aspergillus sp., such as ⁇ . niger. A. nidulans or A ⁇ . oryzae.
  • a strain of Aspergillus sp. such as ⁇ . 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.
  • 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 . 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 adapted from the methods of transforming A. nidulans described in, for instance, Yelton et al. , Proc. Natl. Acad. Sci. USA 81. 1984, pp. 1470-1474, or EP 215 594, or from the methods of transforming A ⁇ . niger described in, for instance Buxton et al., Gene 37. 1985, pp. 207-215 or US 4,885,249, or from the method of transforming A_j_ orvzae described in EP 238 5023.
  • the host cell may be transformed with a vector comprising a DNA sequence coding for a selection marker which is capable of being incorporated in the genome of the host organism on transformation, but which is either not expressed by the host before transformation or o expressed in amounts which are not sufficient to permit growth under selective conditions. Transformants can then be selected and isolated from non-transformants " on the basis of the incorporated selection marker.
  • Suitable selection markers are derived from the A_j_ nidulans or 5 Aj_ niger argB gene, the A ⁇ . nidulans trpC gene, the A_j_ nidulans amdS gene, the Neurospora crassa pyr4 or DHFR genes, or the A. niger or A_j_ oryzae niaD gene.
  • Preferred selection markers for use in the present invention are derived from the A ⁇ . nidulans or A_j_ niger a dS or argB 0 genes.
  • Wild-type Aj. oryzae strains are usually ArgB + (which means that the argB gene is expressed in A ⁇ . orvzae) .
  • an ArgB " mutant strain of Aj. orvzae (which does not express the ArgB gene) must be used as the host organism.
  • the amdS gene may 5 be used as the selection marker in wild-type A_j_ oryzae strains which do not express this gene in sufficient amounts to permit growth under selective conditions.
  • the preregion provided on the vector to ensure efficient direction of the expressed product into the secretory pathway 0 of the host cell may be a naturally occurring signal or leader peptide or a functional part thereof or a synthetic sequence providing secretion of the protein from the cell.
  • the preregion may be derived from a gene coding for a secreted protein derived from any source.
  • the preregion may be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, a gene encoding a Coprinus sp. peroxidase, a gene encoding a Humicola cellulase or xylanase, or a gene encoding a mammalian pancreatic lipase.
  • the preregion is preferably derived from the gene encoding A. oryzae TAKA amylase, A ⁇ niger neutral ⁇ -amylase, A_j_ niger acid- stable ⁇ -amylase, Aj_ niger glucoamylase, or a Coprinus macrorhizus or cinereus peroxidase, or H_j_ insolens cellulase or xylanase, or the gene encoding guinea pig, human, canine or porcine pancreatic lipase.
  • the DNA sequences coding for the lipase, preregion, promoter and terminator may be inserted in a vector containing the selection marker, or it may be inserted in a separate vector for introduction into the host cell.
  • the vector or vectors may be linear or closed circular molecules.
  • two vectors are used, one carrying the DNA sequence coding for the selection marker, and the other carrying the DNA sequences encoding the lipase, the preregion and the functions permitting gene expression.
  • the mammalian pancreatic lipase producible by the process of the invention may, in principle, be derived from any mammalian source, and may thus for instance be of bovine, porcine, canine, coypu, human or guinea pig origin.
  • Guinea pig lipase may, in particular, be one encoded by the recombinant DNA molecule of the present invention, as described above.
  • Human pancreatic lipase is preferably a HPL variant according to the invention, as described above.
  • the medium used to culture the transformed host cells may be any conventional medium suitable for growing filamentous fungi.
  • the transformants are usually stable and may be cultured in the absence of selection pressure. However, if the transformants are found to be unstable, the selection marker introduced into the cells may be used for selection.
  • the mature lipase 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 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 comprising GPL or a GPL or a HPL variant according to the invention, 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 0.2-200 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 protease, cellulase, peroxidase or amylase, conventionally included in detergent additives.
  • the invention relates to a detergent composition
  • a detergent composition comprising GPL or a GPL or a HPL variant of the invention.
  • 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.
  • anionic surfactants are linear alkyl benzene sulfonates (LAS) , alpha olefin sulfonates (AOS) , alkyl sulphate (AS) , alcohol ethoxy sulfates (AES) , alcohol ethoxylates (AEO) and alkali metal salts of natural fatty acids.
  • LAS linear alkyl benzene sulfonates
  • AOS alpha olefin sulfonates
  • AS alkyl sulphate
  • AES alcohol ethoxy sulfates
  • AEO alcohol e
  • 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 other Upases, proteases, cellulases or amylases may be included the detergent compositions of the invention, either separately or in a combined additive as described above.
  • the present invention further relates to a GPL or a HPL variant of the invention for use as a digestive enzyme.
  • the oral substitution of pancreatic lipase is of vital importance in the treatment of patients suffering from severe exocrine pancreatic insufficiency, such as cystic fibrosis and chronic pancreatitis, which leads to malabsorption and steatorrhoea.
  • Conventional enzyme preparations for this purpose are mainly based on enzyme extracts of porcine pancreas and also contains other enzymes, primarily proteases and amylases.
  • pancreatic lipase as a digestive aid in case of patients with pancreatic insufficiency as these patients produce normal levels of gastric lipase and as the pancreatic lipase is active in an intestinal rather than gastric environment.
  • Fig. 1 illustrates the construction of the human pancreatic lipase mutant (HPL(-)) with no loop structure further explained in Example 5,
  • Fig. 2 illustrates the construction of the guinea pig pancrea ⁇ tic lipase mutant (GPL(+)) containing a loop structure further explained in Example 6,
  • Fig. 3 illustrates the oligonucleotide sequences used for the construction of the truncated guinea pig pancreatic lipase (GPL ⁇ _ 319 ) further explained in Example 7,
  • Fig. 4 illustrates the construction of the expression plasmid harbouring the guinea pig pancreatic lipase gene which is described in Example 8,
  • Fig. 5 is a graph illustrating the activity of recombinant guinea pig lipase as compared to that of a H.lanuginosa lipase in the olive oil assay further described in Example 11,
  • Figs. 6 and 7 illustrates the pH and temperature profile, respectively, of the recombinant guinea pig lipase in a tributyrin assay further discussed in Example 11,
  • Fig. 8 illustrates the activity of recombinant guinea pig lipase in the presence of the surfactants listed in Example 11, as determined in the olive oil assay
  • Fig. 9 illustrates the procedure used for constructing the in vitro recombined hybrids described in Example 13.
  • Guinea pig pancreatic lipase (GPL) was purified to homogeneity essentially as previously described (Fauvel J. et al. Biochim.
  • the 0 protein digest was submitted to peptide mapping using the HPLC- syste described above.
  • Peptide fragments were isolated and subjected to amino acid sequence analysis as described above. The sequencing results of 3 of these fragments are given in Table 2.
  • a cDNA library was constructed starting from 3.5 ⁇ g of mRNA using a slightly modified version of Okyama and Berg protocol (Okayama and Berg (1982) Mol. Cell. Biology 2., 161-170) eliminating poly(dA) purification of oligo(dT) tailed primer fragment.
  • the cloning vectors used were as follows:
  • the vector primer fragment was pCDVl-PL8 linearized by KpnI.
  • the plasmid pCDVl-PL8 is a derivative of the pCDVl-PL plasmid as originally described by Noma et al (1986) Nature 319. 640-646.
  • the modification of pCDVL-PLl consists of insertion of a NotI site immediately adjacent to the KpnI thus permitting linearization of the plasmid 3 ' of the cDNA insert.
  • the linker fragment consisted of an oligo(dG)-tailed HindlH-SacI fragment of the pSP62-K2 plasmid (Noma et al (1986) Nature 319i, 640-646) .
  • the cDNA library was transformed into competent SCSI cells
  • Hybridization probes were chosen according to the amino aci sequence data resulting from microsequencing (cf. Example 1) o two fragments of the purified guinea pig pancreatic lipase.
  • oligonucleotides which were synthesized on an Applie Biosystems, Inc. DNA synthesizer were of the following sequenc and composition:
  • Duplicate filters were prepared from the primary plating of th cDNA library and each set of filters were then hybridized usin polynucleotide kinase 32 P labelled ("Molecular Cloning" Ed Sambrook et al. 1989) oligonucleotides. The initial screenin was carried out using the #1853 probe. Hybridization was carrie out at 45°C in 6 x SSC, 5 x Denhardt's and 0.5% SDS for 4 hours Filters were washed briefly at 45°C. Twenty clones were identified and picked as potential GPL cDNA clones.
  • a secondar screening using oligonucleotides #1853 as well as #1854 as probes identified a total of four cDNA clones as putative GPL clone by the criteria of showing positive hybridization to both probes.
  • Plasmid DNA was prepared from each of the putative GPL clones and subjected to DNA sequencing by the Sanger dideoxy- termination-method (Sanger et al. PNAS (1977) 74., 5463-5467). Sequencing identified all four clones as true GPL clones, three of these being full-length cDNA clones of independent origin (i.e. the sequence of the cDNA inserts diverge at the very ends) .
  • the identity of cDNA clones was established on the basis of the peptide sequence resulting from microsequencing of the purified protein and on the basis of showing a high degree of homology to other known mammalian pancreatic lipases with the one major exception of a distinct discrepancy in the central part of the gene.
  • This region encompasses the "loop structure" of the human pancreatic lipase.
  • the loop structure of the human pancreatic lipase (HPL) forms what appears from X-ray crystallography to constitute a separate structural domain, delineated by two cystein residues (Winkler et al. (1990) Nature 343. 771-774) .
  • the discrepancy in the sequence of the guinea pig pancreatic lipase (GPL) relative to that of the human HPL protein corresponds precisely to a substitution of this 23 amino acid loop structure by a short 5 amino acid "mini-loop".
  • GPL#1 One of the GPL clones, designated GPL#1, was sequenced in its entire length.
  • the cDNA insert of GPL#1 is 1424 bp long encoding an open reading frame of 452 amino acids, 18 of which corresponds to a signal peptide. All four GPL cDNA clones were sequenced across the central homology-gap-region (the "mini- loop" region) and demonstrated to be identical.
  • HPL human pancreatic lipase
  • the conditions of the PCR reaction were as follows: a 100 ⁇ l reaction volume of buffer composition as recommended by the manufacturer (Perkin-Elmer Cetus AmpliTaq Kit) contained first strand material, dNTPs to a final concentration of 200 ⁇ M and 100 pmoles of each oligonucleotide primer. The reaction was run for 30 cycles at temperature levels of 98°C, 55°C and 72°C respectively. The resulting PCR fragment of approximately 1400 bp was subsequently subcloned as a BamHI fragment into the pBSKII vector. A clone resulting from this experiment, designated HPL#1, was shown by sequencing to encode a 431 amino acid protein that matched the published sequence of HPL. The DNA sequence and derived amino acid sequence is shown in the appended Sequence Listing ID No. 3.
  • HPL(-) carries an internal deletion relative to the native form which corresponds exactly to the region non-homologous to the guinea pig enzyme, i.e. the loop structure.
  • the details of the substitution are presented in the diagram of figure 1.
  • the mutant form of HPL was synthesized by PCR. First the two halves of the gene, the 5' end and the 3' end respectively, were synthesized in separate PCR reactions using the native HPL (HPL#1) gene as a template and oligonucleotide primers as indicated in figure 1. Subsequently these two fragments were used as templates in a second PCR ligation reaction using the oligonucleotides corresponding to the most 5' respectively 3 ' end of the gene as primers. The resulting fragment was subsequently cloned into the pBSKII (Stratagene) vector and sequenced throughout. An internal Ncol-Aflll fragment was finally exchanged with the corresponding Ncol-AfIII fragment of the native HPL gene as present in the Aspergillus expression plasmid pHD414 described in the European Patent Application No. 91610022.5.
  • GPL(+) amino acids 239 to 245 of the mature protein were replaced with a 23 amino acid sequence corresponding to amino acids 238 to 262 of the HPL protein. This region encompasses the loop structure. The details of this substitution are outlined in figure 2.
  • the GPL(+) mutant was synthesized in two consecutive PCR reactions. Initially the 5' and 3 ' halves of the GPL(+) gene were synthesized in separate PCR reactions using the following combination of oligonucleotides (the sequence appears from figure 2) :
  • the 5' half and the 3 ' half of the GPL(+) gene were combined by PCR ligation using oligonucleotides AH#65 and AH#66 as primers.
  • the Hindlll-Sall fragment resulting from this reaction was subcloned into the pBSKII vector and verified by sequencing.
  • the GPL (+) Hindlll-Sall fragment was subcloned into the Hindlll, Sail sites of the Aspergillus expression vector pHD414 (described in European Patent Application No. 91610022.5).
  • Partial proteolytic cleavage by chymotrypsin of the porcine pancreatic lipase has demonstrated an ester-hydrolysing activity as being present in the extreme C-terminal part of the protein, viz. 336-449. It was, however, not possible to obtain the N- terminal fragment of the lipase (1-335) in intact form so as to analyze the enzymatic properties of this part of the protein (cf. De Caro et al. (1986) Eur. J. Biochem. 158, 601-607) .
  • the truncated form of the GPL was synthesized by PCR using the original GPL cDNA (GPL#1) as a template.
  • the oligonucleotides AH#65 and AH#80 were used as primers for the PCR.
  • the sequence of the oligonucleotides appears from figure 3.
  • Oligonucleotide AH#65 corresponds to the 5' end of the GPL gene and includes a Sail site.
  • the oligonucleotide AH#80 corresponds to the GPL sequence including the codon for the phenylalanine residue number 319 immediately followed by a (premature) translational stop signal and a Hindlll site.
  • the 1100 bp fragment resulting from the PCR reaction outlined above was subsequently cloned into pBSII using Sall-Hindlll, checked by sequencing in its entity and finally cloned into the Sall-Hindlll sites of the Aspergillus expression vector pHD414.
  • GPL pancreatic lipase
  • the Ball-Sail fragment of -1.5 kb from GPL#1 (cf. example 3) has the entire GPL sequence including the signal sequence. This fragment was inserted into the BamHI-Xhol-cut expression vector pToC68 which is described in detail in International Patent Application No. PCT/DK91/00123. As controlling elements it has the TAKA amylase promoter from A.oryzae and the AMG terminator region from A.niger.
  • the GPL expression vector pHW713 was transformed into A.oryzae A1560-T40, a protease-deficient derivative of A.orvzae IFO 4177, using the procedure described in EP 238 023. Selection on acetamide was performed by cotransformation with pToC 186 harbouring the amdS gene from A.nidulans as a 2.7 kb Xbal fragment (Corrick et al. (1987) , Gene 53., 63-71) on a pUC 19 vector (Yannisch-Perron et al. (1985) , Gene 3_3_, 103-119) .
  • amdS gene was modified with two up-promoter mutations, amdI9 (Hynes et al. (1988), Mol. Cell. Biol. 8., 2589- 2596) and amdI66 (Katz et al., (1990), Mol. Gen. Genet. 220. 373-376) .
  • Transformants were grown in YPD medium (Sherman et al.. Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) for 3-4 days and analysed for new protein species in the supernatant by SDS-PAGE. GPL appeared as a band with an apparent Mw of 48 kD. The calculated Mw of GPL is 47.7 kD. The expected level of esterase activity was found using p-nitrophenyl laurate as substrate.
  • the pH of a supernatant from a culture of an A ⁇ . oryzae transformant according to example 8 was adjusted to 7 by adding NaOH.
  • the supernatant was subsequently applied to an anion exchange HPLC column (Si 300 Polyol CM, available from Serva, FRG) and subjected to high performance liquid chromatography (HPLC) followed by step-wise elution with 50 mM morpholinopropane sulfonic acid (M0PS)/1M NaCl/25% isopropanol as the eluent and desalination of active fractions on a Sephadex G-25 (Pharmacia, Sweden) column by means of 10 mM ammonium bicarbonate.
  • M0PS morpholinopropane sulfonic acid
  • the active fractions were subjected to size exclusion chromatography on a TSK G2000 SWG HPLC column using a 50 mM MOPS, pH 7.2, buffer, followed by desalination of active fractions on a Sephadex G-25 column by means of 10 mM ammonium bicarbonate.
  • the active fractions were pooled and freeze-dried.
  • pancreatic lipases in a baculovirus system
  • pancreatic lipase constructs of the HPL and GPL series may be expressed in a baculovirus system.
  • expression of the gene of interest is driven by the autographa califo nica multiple nuclear polyhedrosis virus (AcMNPV) polyhedrin promoter, which in nature is responsible for a very high level expression of the polyhedrin protein, comprising up to 50% of total cellular protein at the end of the lytic cycle (Doerfler, W. , Bohm, P. Eds.
  • HPL/GPL genes are cloned into the transfer vector pVL941 (Phar ingen) into which appropriate restriction sites have been introduced.
  • transfer vector pVL941 Phar ingen
  • appropriate HPL/GPL gene inserts are produced by PCR-directed mutagenesis aimed at introducing restriction sites at 5' and 3' ends of the respective genes.
  • the transfer plasmid is cotransfected with linearized BaculoGoldTM baculovirus DNA (Pharmingen) , and due to a lethal deletion residing in the baculovirus DNA which is complemented by the transfer plasmid, nearly all of the released viruses contain integrated cDNA.
  • a plaque assay is subsequently performed substantially as described by Volkman, L.E. and Summers, M.D. (1975) , "Nuclear pyhedrosis virus detection: relative capabilities of clones developed from Trichoplusia ni ovarian cell line TN-368 to serve as indicator cells in a plaque assay", J.Virol.
  • Sf9 cells American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852- 1776 Accession number CRL1711
  • Sf9 cells are infected at an infection multiplicity of 1 substantially in accordance with the procedures described by Burand, J.P., Summers, M.D. and Smith, G.E. (1980), "Transfection with baculovirus DNA”.
  • concentration of recombinant protein peaks - medium is collected and the protein is purified by a procedure as described in example 9 for expression of HPL/GPL in Aspergillus.
  • the tributyrin assay was carried out as described in Novo Nordisk Analysis Methods #AF95/5 (available from Novo Nordisk A/S on request) , using tributyrin emulsified in gum arabic as the substrate and pH-stat titration at pH 7.0 and 30°C. Unless otherwise indicated, 0.05 M DTT (dithiothreitol) was added to the substrate immediately prior to the start of the reaction.
  • the olive oil assay was carried out by pH-stat titration at pH 9.0 and 30°C.
  • the substrate used was a substrate powder consisting of PVC particles (Pevicon PE 712, available from Kema Nord) coated with olive oil to an oil content of 16.7% w/w.
  • PVC particles Pevicon PE 712, available from Kema Nord
  • To 1 g of substrate powder was added 20.5 ml buffer (70 mM Na 2 S0 4/ 2.5 mM Tris) and heated to 30°C, after which the substrate powder was dispersed by means of ultrasound for 15 seconds. Unless otherwise indicated, 0.05 mM DTT was added immediately prior to the start of the reaction.
  • Tributyrin assay 1630 LU/ g protein
  • Olive oil assay 12 ⁇ mol free fatty acid/min./mg protein
  • the olive oil assay is carried out in the absence of Ca 2+ , which accounts for the difference in specific activity determined in the tributyrin and olive oil assays.
  • the pH optimum of the rGPL was determined to be about 8.0, and the temperature optimum about 30°C (cf. Figs. 6 and 7) .
  • rGPL The activity of rGPL was determined in the olive oil assay in the presence of the following surfactants:
  • LAS linear alkyl benzene sulphonate (Nansa 1169/P)
  • AOS ⁇ -olefinsulphonate (C l4 _ n )
  • AEOS alcohol ethylene glycol ether sulphate (C 12 . 15 ,3 EO;
  • Dobanol 25-3S/60 AEO: alcohol ethoxylate (C 12 . 15 ,7 EO;
  • rGPL is completely inhibited at a given concentration of the surfactant in question. It further appears that one surfactant, alkyl phosphate, results in a significantly lower degree of inhibition than the other surfactants studied. At the surfactant concentration usually employed in detergents (about 0.1-1 g/1) , rGPL retains an activity between 40 and 100%.
  • HPL and GPL genes are so homologous that they will recombine to some extent in vivo, if they are placed as neighbours in a DNA construct and transformed into E. coli.
  • Example 13 provides for analysis of hybrids from E. coli on the DNA level after cleavage at the unique restriction site between the two genes and re-transformation in E. coli to eliminate the non- recombined majority of plasmids. Potentially interesting hybrids are then transformed into A. oryzae for expression and analysis of enzyme properties by a transformation method known in the art, e.g. using the procedure described in Example 8 above.
  • Example 13
  • tandem configuration of GPL and HPL genes described in example 12 may form the basis for creating specific hybrids of the two genes in vitro.
  • separate HPL and GPL DNA sequences may be used for the construction of .in vitro recombinants.
  • PCR fragments produced in the first cycle are purified before the second cycle.
  • the fusion products are cloned after cleavage at unique restriction sites at some distance from the primers 1 and 4, which can be used in all constructions, while primers 2 and 3 vary according to the wanted recombination site.
  • the primers are oligonucleotides prepared on the basis of corresponding parts of the GPL and/or HPL DNA sequences.
  • primer 1 and primer 4 may also be situated outside the genes, e.g. in the promoter region and terminator region, respectively.
  • AAG TCA CIT OCC TOG GAC CCC
  • AAG AAG ATC AAC ACT AGG TTT CTT CTG 193 Lys Ser Leu Pro Ser Asp Pro Lys Lys He Asn Thr Arg Phe Leu Leu 45 50 55
  • GGA ATG AGC CAA AAG GTG GGC CAT ATG GAT TTC TTT CCA AAT GGA GGA 769 Gly Met Ser Gin Lys Val Gly His Met Asp Hie Phe Pro Asn Gly Gly 240 245 250
  • AGC ATC GAG TAC TAT CAC AGC AGC ATC CTC AAC CCC GAA GGC TTC CTG 865 Ser He Glu Tyr Tyr His Ser Ser He Leu Asn Pro Glu Gly Phe Leu 270 275 280
  • Asp Pro Lys Lys lie Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn 50 55 60
  • Lys Asn lie Leu Ser Gin lie Val Asp lie Asp Gly He Trp Glu Gly 245 250 255
  • Lys Gin Tyr Glu lie Phe Lys Gly Thr Leu Lys Pro Asp Ser Thr His 370 375 380

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Abstract

Lipase de cochon d'Inde (GPL) essentiellement exempte d'autres protéines de conchon d'Inde, dont la séquence aminoacide est présentée dans la liste de séquence ID no. 2 ou sa variante présentant une efficacité enzymatique, ainsi que certaines variantes de la lipase pancréatique humaine (HPL). On peut préparer les variantes de GPL et HPL au moyen de technique d'ADN recombinant et les utiliser en tant que constituants d'une composition détergente ou en tant qu'enzyme digestif.
PCT/DK1992/000200 1991-06-25 1992-06-25 Lipase pancreatique de mammifere et sa variante WO1993000426A1 (fr)

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FR2720756A1 (fr) * 1994-06-02 1995-12-08 Rhone Poulenc Rorer Sa Virus recombinants, préparation et utilisation en thérapie génique.
WO1997043379A1 (fr) * 1996-05-15 1997-11-20 The Procter & Gamble Company Compositions detergentes comprenant une enzyme lipolytique specifique et un dispersant a base de savon de chaux
FR2754827A1 (fr) * 1996-10-17 1998-04-24 Biocem Lipases pancreatiques et/ou colipases recombinantes et polypeptides dervies produits par les plantes, leurs procedes d'obtention et leurs utilisations
WO2001018218A1 (fr) * 1999-09-09 2001-03-15 Iogen Bio-Products Corporation Expression de proteines dans des champignons modifies par genie genetique
US6814962B1 (en) 1994-06-02 2004-11-09 Aventis Pharma S.A. Recombinant viruses and their use for treatment of atherosclerosis and other forms of coronary artery disease and method, reagent, and kit for evaluating susceptibility to same
US6939704B1 (en) 1998-03-10 2005-09-06 Iogen Energy Corporation Enhanced expression of proteins in genetically modified fungi
WO2009068501A1 (fr) * 2007-11-28 2009-06-04 Henkel Ag & Co. Kgaa Détergents contenant des enzymes stabilisées
AU2010246342A1 (en) * 2003-03-06 2010-12-09 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US8071350B2 (en) 2002-10-31 2011-12-06 Verenium Corporation Amylases, nucleic acids encoding them and methods for making and using them
US8334118B2 (en) 2001-02-21 2012-12-18 Verenium Corporation Enzymes having alpha amylase activity and methods of making and using them
US8338131B2 (en) 2001-02-21 2012-12-25 Verenium Corporation Amylases and methods for use in starch processing
US8343747B2 (en) 2006-12-21 2013-01-01 Verenium Corporation Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them

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EP0238023A2 (fr) * 1986-03-17 1987-09-23 Novo Nordisk A/S Procédé de production de produits protéiniques dans aspergillus oryzae et promoteur à utiliser dans aspergillus
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FR2720756A1 (fr) * 1994-06-02 1995-12-08 Rhone Poulenc Rorer Sa Virus recombinants, préparation et utilisation en thérapie génique.
WO1995033840A1 (fr) * 1994-06-02 1995-12-14 Rhone-Poulenc Rorer S.A. Virus recombinants, preparation et utilisation en therapie genique
US6814962B1 (en) 1994-06-02 2004-11-09 Aventis Pharma S.A. Recombinant viruses and their use for treatment of atherosclerosis and other forms of coronary artery disease and method, reagent, and kit for evaluating susceptibility to same
WO1997043379A1 (fr) * 1996-05-15 1997-11-20 The Procter & Gamble Company Compositions detergentes comprenant une enzyme lipolytique specifique et un dispersant a base de savon de chaux
FR2754827A1 (fr) * 1996-10-17 1998-04-24 Biocem Lipases pancreatiques et/ou colipases recombinantes et polypeptides dervies produits par les plantes, leurs procedes d'obtention et leurs utilisations
WO1998017807A1 (fr) * 1996-10-17 1998-04-30 Meristem Therapeutics S.A. Lipases pancreatiques et/ou colipases recombinantes et polypeptides derives produits par les plantes, leurs procedes d'obtention et leurs utilisations
US6939704B1 (en) 1998-03-10 2005-09-06 Iogen Energy Corporation Enhanced expression of proteins in genetically modified fungi
WO2001018218A1 (fr) * 1999-09-09 2001-03-15 Iogen Bio-Products Corporation Expression de proteines dans des champignons modifies par genie genetique
US8334118B2 (en) 2001-02-21 2012-12-18 Verenium Corporation Enzymes having alpha amylase activity and methods of making and using them
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US10047350B2 (en) 2001-02-21 2018-08-14 Basf Enzymes Llc Enzymes having alpha amylase activity and methods of making and using them
US9701950B2 (en) 2001-02-21 2017-07-11 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US9062295B2 (en) 2001-02-21 2015-06-23 Basf Enzymes Llc Recombinant polypeptides
US10100293B2 (en) 2002-10-31 2018-10-16 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US8071350B2 (en) 2002-10-31 2011-12-06 Verenium Corporation Amylases, nucleic acids encoding them and methods for making and using them
US9249400B2 (en) 2002-10-31 2016-02-02 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US10793844B2 (en) 2002-10-31 2020-10-06 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US8551754B2 (en) 2002-10-31 2013-10-08 Verenium Corporation Amylases, nucleic acids encoding them and methods for making and using them
AU2010246342C1 (en) * 2003-03-06 2017-11-30 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
AU2010246342B2 (en) * 2003-03-06 2013-03-28 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US8222035B2 (en) 2003-03-06 2012-07-17 Verenium Corporation Amylases, nucleic acids encoding them and methods for making and using them
AU2010246342A1 (en) * 2003-03-06 2010-12-09 Basf Enzymes Llc Amylases, nucleic acids encoding them and methods for making and using them
US8343747B2 (en) 2006-12-21 2013-01-01 Verenium Corporation Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
US10100299B2 (en) 2006-12-21 2018-10-16 Basf Enzymes Llc Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
US10883098B2 (en) 2006-12-21 2021-01-05 Basf Enzymes Llc Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them
US8466098B2 (en) 2007-11-28 2013-06-18 Henkel Ag & Co. Kgaa Washing agent having stabilized enzymes
WO2009068501A1 (fr) * 2007-11-28 2009-06-04 Henkel Ag & Co. Kgaa Détergents contenant des enzymes stabilisées

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