MAMMALIAN PANCREATIC LIPASE AND VARIANT THEREOF

Abstract: A guinea pig 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 as well as certain variants of the Human Pancreatic Lipase (HPL). The GPL and GPL and HPL variants may be prepared by recombinant DNA techniques and may be used as constituents in a detergent composition or as a digestive enzyme.

Description: MAMMALIAN PANCREATIC LIPASE AND VARIANT THEREOF FIELD OF INVENTION 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. BACKGROUND OF THE INVENTION 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) . The purification of 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. DESCRIPTION OF THE INVENTION 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 . Accordingly, in one aspect, 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. In the present context, 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. The term "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. De Caro et al., Eur. J. Biochem. 158, 1987, pp. 601-607). Thus, if one wishes to prepare a smaller GPL molecule with decreased esterase ac¬ tivity, but retained lipase activity, GPL may be C-terminally truncated. A preferred GPL variant of this type is GPL W ι 9 . As indicated above, it has surprisingly been found that 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. In particular, the insertional substitution comprises the following amino acid sequence Lys Ala Asn Leu Gin Lys Asn lie Leu Ser Gin lie Val Asp lie Asp Gly lie Trp Glu Ala Gly Thr Arg Phe Val Ala or a fragment thereof capable of functioning as a loop struc- ture covering the active site in the inactive state of the enzyme. In a preferred embodiment, 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 (corresponding to the loop structure found in HPL) . The construction of this GPL variant is described in Example 6. Furthermore, it has been found that either Cys 103 or Cysι 05 of native GPL may be non-disulfide linked. The free -SH group pre¬ sent 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 GPL variant, wherein Cys 103 or Cys 105 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 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. It should be noted that, according to the invention, 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. In another aspect, the present invention relates to a recom- binant DNA molecule comprising a DNA sequence encoding GPL, or an enzymatically active variant thereof. In particular, 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. Examples of suitable modifications of the DNA sequence are 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) . Other examples of possible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence and deletion of one or more nucleotides at either end of or within the sequence. Specific modifications of the DNA sequence are those which, when expressed, give rise to any one of the GPL variants described above. 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. In this case, 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. Alternatively, 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. 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 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. In a further aspect, the present invention relates to certain variants of human pancreatic lipase (HPL) . Thus, in one embodiment, 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. For example, 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) . In Example 5 the construction of a HPL variant with no loop structure is described. In another embodiment, 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 . Similarly to GPL, 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. It should be noted that, according to the invention, 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. Another type of 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. These recombinant lipase variants are expected to provide interesting new activity profiles combining some of the enzymatic properties exerted by HPL and GPL, respectively, in their native state. In the present context the terms "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. In a further aspect 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. Furthermore, 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. For this purpose, 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. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosσmal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, 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. In the vector, 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 (Palmiter et al. , Science 222. 1983, pp. 809-814) or the adenovirus 2 major late promoter. Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Che . 255, 1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1. 1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering: of Microorganisms for Chemicals (Hollaender et al, eds.J, Plenum Press, New York, 1982), or the TPI1 (US 4, 599, 311) or ADH2-4C (Russell et al., Nature 304. 1983, pp. 652-654) promoters. Promoters suitable for use in filamentous fungi are described below. 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. An examples of such a sequence (when the host cell is a mammalian cell) 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. The procedures used to ligate the DNA sequences coding for the mammalian pancreatic lipase, the promoter and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al.. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). 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. 725; Corsaro and Pearson, Somatic Cell Genetics 1_, 1981, p. 603, Graham and van der Eb, Virology 52. 1973, p. 456; and Neumann et al., EMBO J. 1. 1982, pp. 841-845. Also 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) . Alternatively, fungal cells (including yeast cells) or bac- terial cells may be used as host cells. Examples of suitable yeast cells include cells of Saccharomyces spp. or Schizosac- charomyces spp. , in particular strains of Saccharomyces cerevisiae. Examples of bacterial cells include cells of the genus Bacillus, especially B. subtilis. An example of 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) . While intracellular expression may be advantageous in some respects, e.g. when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. In order to obtain extracellular expression, 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. While any of the above explained expression systems may be suitable for the production of mammalian pancreatic Upases the presently preferred process for the production of a mammalian pancreatic lipase is by use of a filamentous fungus as a host cell. Accordingly, in a.. still further aspect, the present invention relates to.a process for the extracellular production of a mammalian pancreatic lipase in a filamentous fungus, the process comprising (a) providing a recombinant DNA vector which comprises a DNA sequence encoding a mammalian pancreatic lipase, and a DNA sequence encoding a preregion permitting secretion of the expressed mammalian pancreatic lipase into the culture medium, (b) transforming a suitable filamentous fungus with the recombinant DNA vector of step (a) , and (c) culturing the transformed filamentous fungus in a suitable culture medium under conditions conducive to the production of the mammalian pancreatic lipase. The term "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. It is evident that 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. Examples of suitable promoters are those derived from the gene encoding A^. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A_j_ niger neutral α-amylase, ^ niger acid stable α- 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. The use of A ^ . oryzae in the production of recombinant proteins is extensively described in, e.g. EP 238 023. In particular when the host organism is A_j_ orvzae. 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. In the process of the present invention, 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) . Thus, if argB is chosen as the selection marker, an ArgB " mutant strain of Aj. orvzae (which does not express the ArgB gene) must be used as the host organism. On the other hand, 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. Thus, the preregion may be derived from a gene coding for a secreted protein derived from any source. In particular, 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 α-amylase, A_j_ niger acid- stable α-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. In a preferred embodiment of the process of the invention, 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. In a still further aspect, the invention relates to 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. Typical examples of 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. However, according to the invention, it has been found most advantageous for the stability and activity of the enzyme to include an alkyl phosphate or AEO or a combination thereof as the surfactant. 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 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. However, it has been found that the content of lipase in these preparations is too low to ensure an adequate enzymatic degradation of triglycerides in the diet so as to result in a normal lipid absorption from the gastrointestinal tract. It has previously been suggested to use microbial lipases as digestive aids (DE 16 42 654 and GB 1 442 677) . The use of recombinant human gastric lipase for treating lipase deficiency is suggested in WO 86/01532. It is, however, assumed to be an advantage to use 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. The present invention is further illustrated in the appended figures in which 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 (GPLι_ 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, and Fig. 9 illustrates the procedure used for constructing the in vitro recombined hybrids described in Example 13. The present invention is further illustrated in the following examples which are not intended to limit, in any way, the scope 5 of the invention as claimed. Example 1 Purification and partial seguencing of native GPL Guinea pig pancreatic lipase (GPL) was purified to homogeneity essentially as previously described (Fauvel J. et al. Biochim. 10 Biophys. Acta 663 (1981) 446-456) . Approx. 0.6 nmol of purified GPL was subjected to automatic Ed an degradation using an Applied Biosystems 470 A gas phase sequencer as previously described (Thim, L. et al FEBS Lett. .212 (1987) 307-312. The result from this sequence analysis is given in Table 1. Approx. 151.4 nmol of GPL was dissolved in 100 μl of 0.2M TRIS • HC1 buffer, Ph = 7.5 and 57 mg guanidine hydrochloride was added. The lipase was reduced by the addition of 0.25 mg dithiothreitol and incubation at room temperature for 16 h. The resulting protein was derivatized by the addition of 1 μl 4- 0 vinyl pyridine and incubation at room temperature for 1 h. The reaction was stopped by the addition of 1.0 mg dithiothreitol and the pyridyl ethylated GPL (PE-GPL) was purified by reverse phase HPLC on a Vydac C-4, 4.6 x 250 mm, column. The column was equilibrated and eluted with a linear gradient mixture of 5 acetonitrile in 0.1% trifluoroacetic acid. PE-GPL eluted at approx. 44% of acetonitrile. The purified PE-GPL was lyophilized and redissolved in 200 μl of 50 mM sodium phosphate buffer, pH = 8.0 and digested by the addition of 0.5 μg trypsin and incubation at 37°C for 24 h. 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. Table 1 Amino acid se uence anal sis of native GPL Table 2 Amino acid sequence analysis of tryptic fragment of PE-GPL Example 2 Construction of a cDNA library from guinea pig pancreas Total RNA was extracted from homogenized guinea pig pancreas according to the procedure described by Chirgwin et al. (Biochemistry (1979) JL8., 5294-5299) . Messenger RNA was obtained by two cycles of affinity chromatography on oligo (dT) cellulose as described by Aviv and Leder ( Proc. Natl. Acad. Aci. USA (1972) 69., 1408-1412) . A cDNA library was constructed starting from 3.5 μg 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 (Stratagene) according to the high-transformation-efficiency procedure described by Hanahan et al (1983) J.Mol.Biol. 166, 557-580. Approximately 10.000 recombinant clones were obtained. Example 3 Isolation and characterization of a cDNA clone encoding guine pig pancreatic lipase (GPL) : Recombinant clones obtained as described in Example 2 wer transferred to Millipore nitrocellulose filters and immobilize as described in "Molecular Cloning" Ed. Sambrook et al. (1989) 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. The oligonucleotides which were synthesized on an Applie Biosystems, Inc. DNA synthesizer were of the following sequenc and composition: #1853: 5'GA(TC) GA(AG) AA(AG) CC(ATGC) TGG GC3' (degeneracy=32) corresponding to the sequence Asp-Glu-Lys-Pro-Trp-(Ala) of the N-terminal fragment. and #1854: 5' Ca(AG) GA(AG) AA(TC) GT(ATGC) GA(AG) CA3 (degeneracy=64) corresponding to the sequence Gln-Glu-Asn-Val-Glu-(Gln) of fragment#49. 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 wer 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". 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. Example 4 Isolation of a cDNA clone encoding a human pancreatic lipase fHPL^ : A cDNA clone coding for the human pancreatic lipase (HPL) was obtained by PCR technology based on the sequence of the mature HPL (i.e. minus the signal peptide) published by Winkler et al. (1990) Nature 343. 771-774. Starting from 3 μg of human placenta mRNA, a first strand cDNA was synthesized using conditions specified by the manufacturer of the Superscript reverse transcriptase (BRL) . The reaction was carried out at 42°C for 45 min. Subsequently a tenth of this material was used as a template in a PCR reaction using the following oligonucleotides as primers: AH#25: CCTGGATCCGCCATG AA(AG) GA(AG) GT(ATGC) TA(TC) GA (degeneracy = 64) including the sequence corresponding to Lys-Glu-Val-Cys-Thy-(Glu) of the six most N-terminal amino acids of the mature HPL protein, and AH#26: CCAGGATCCTCA AG(AG)CA (ATGC)GG (ATGC)GT (ATGC)A(AG) (ATGC)GT (Degeneracy = 1024) including the sequence corresponding to Cys-Pro-Thr-Leu-Thr of the five most C-terminal amino acids of HPL: The conditions of the PCR reaction were as follows: a 100 μl 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 μM 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. Example 5 Construction of human pancreatic lipase mutant fHPL(- ' )') with no loop structure: The HPL mutant designated 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. Example 6 Construction of guinea pig pancreatic lipase mutant fGPLf+i containing a loop structure: In the GPL mutant designated 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) : 5'half: AH#65 and AH#131 3 , half: AH#130 and AH#66. Subsequently, 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. Subsequently, 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). Example 7 Construction of a truncated guinea pig pancreatic lipase (GPL L319 ) : 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) . In view of the extensive homology exhibited by the mammalian pancreatic lipases, it was decided to synthesize a recombinant truncated form of the guinea pig pancreatic lipase, express it in Aspergillus and subsequently characterize the enzymatic behaviour of the truncated GPL protein. The truncated form of the GPL protein was designated GPLι. 319 as the phenylalanine residue of this protein corresponding to the cleavage site in the porcine protein constitutes residue 319 of the mature GPL protein. 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. Example 8 Expression of guinea pig pancreatic lipase in A. oryzae. An expression plasmid harbouring the guinea pig pancreatic lipase (GPL) gene was constructed from 3 elements, as outlined in Fig. 4. The synthetic oligonucleotide linker NOR 2460/2465 contains the N-terminal part of the GPL signal sequence to be connected to the TAKA promoter sequence at the BamHI site. The BamHl-Ball linker sequence is as follows: NOR2460, 2465 : 5' GATCCGCTCGGCATGATGCTGTTT 3' GCGAGCCGTACTACGACAAA GCGTGGACCATCGGCCTTCTCCTGCTGG 3' 52bp CGCACCTGGTAGCCGGAAGAGGACGACC 5' 48bp 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) . We used a modification of the amdS gene 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. Example 9 Purification of rGPL from an Aspergillus oryzae culture 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. After freeze-drying, 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. In all purification steps, PMSF was added to inhibit the alcaline protease produced by A^ oryzae. and dithiothreitol was added to keep the free -SH group of the lipase reduced. Example 10 Expression of pancreatic lipases in a baculovirus system It is contemplated that all relevant pancreatic lipase constructs of the HPL and GPL series may be expressed in a baculovirus system. In this 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. 1986, The Molecular Biology of the Baculoviruses, Current Topics in Microbiology and Immunology, Vol 131, Springer-Verlag, Berlin.) Initially the HPL/GPL genes are cloned into the transfer vector pVL941 (Phar ingen) into which appropriate restriction sites have been introduced. Alternatively, 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. Subsequently 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. 16, 1630-1637, resulting in selection of a single clone of recombinant virus. Following amplification of the virus, Sf9 cells (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852- 1776 Accession number CRL1711) 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". Approximately four days post-infection - at which time the 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. Example 11 Characterization of rGPL Specific activity The specific activity of rGPL purified as described in example 9 above, was determined using tributyrin and olive oil, respectively, as the substrate. 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. 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. The specific activity of rGPL was determined to be as follows: Tributyrin assay: 1630 LU/ g protein Olive oil assay: 12 μmol free fatty acid/min./mg protein It should be noted that 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. When the kinetics of rGPL and Humicola lanuginosa lipase were compared in the olive oil assay, it was found that the activity of the H_i_ lanuginosa lipase was greatly reduced after about 1 minute. This reduction in activity is assumed to be caused by accumulation of liberated fatty acids at the oil/water interface resulting in a conformational change from active to inactive. rGPL, on the other hand, appeared to tolerate a far higher concentration of fatty acids at the oil/water interface than the H. lanuginosa lipase (cf. Fig. 5) . pH and temperature optimum When determined in the tributyrin assay, 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) . Activity in the presence of surfactants 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: α-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; Dobanol 25-7) Alkyl phosphate (Berol 522) It appears from Fig. 8 that 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%. Stability in the presence of detergents The stability of rGPL was determined in model detergent systems by means of the tributyrin assay. 3 μg/ml rGPL was incubated in 1 ml detergent solution at 30°C for 20 minutes at pH 9.0 in the absence of DTT and at a water hardness of 0°dH. Samples were taken at t=0.5 and t=20 minutes. The activities shown in Table 3 below are given as the activity at t=20 relative to the activity at t=0.5. Table 3 10 It appears from the table that LAS and AOS are strongly inactivating, while AEOS and AEO are weakly inactivating at high concentrations (1 g/1) , and alkyl phosphate does not have any inactivating effect in the model systems. Example 12 Isolation of in vivo Recombined Hybrids of GPL and HPL The 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. The following arrangement in the Aspergillus expression vector pHD 414: 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 Isolation of in vitro Recombined Hybrid of GPL and HPL The 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. Alternatively, separate HPL and GPL DNA sequences may be used for the construction of .in vitro recombinants. The procedure involves 2 cycles of PCR which are illustrated in Fig. 9. 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. It will be understood that primer 1 and primer 4, respectively, may also be situated outside the genes, e.g. in the promoter region and terminator region, respectively. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Novo Nordisk A/S (ii) TITLE OF INVENTION: Mammalian Pancreatic Lipase and Variants thereof (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Novo Nordisk A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: Denmark (F) ZIP: DK-2880 (v) 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 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (Viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Sørensen, Lise Abildgaard (C) REFERENCE/DOCKET NUMBER: 3625.204-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: +45 4444 8888 (B) TELEFAX: +45 4449 3256 (C) TELEX: 37304 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1384 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (Vi) ORIGINAL SOURCE: (A) ORGANISM: guinea pig (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 17..1375 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CTCGAGTCAG CTOGGC ATG ATG CTG TTT GCG TGG ACC ATC GGC CTT CTC 49 Met Met Leu Phe Ala Tcp Thr He Gly Leu Leu 1 5 10 CTG CTG GCC ACT GTC AGA GGA GCG GAA GTC TGC TAT TCA CAC CTT GGC 97 Leu Leu Ala Thr Val Arg Gly Ala Glu Val Cys Tyr Ser His Leu Gly 15 20 25 TGC TTC TCA GAT GAA AAA CCA TGG GCA GGA ACC TCT CAG CGA CCT ATA 145 cys Phe Ser Asp Glu Lys Pro Trp Ala Gly Thr Ser Gin Arg Pro lie 30 35 40 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 TAC ACA AAT GAA AAC CAG AAT AGC TAC CAA TIG ATC AGG GCC ACT GAC 241 Tyr Thr Asn Glu Asn Gin Asn Ser Tyr Gin Leu lie Thr Ala Thr Asp 60 65 70 75 ATA GCC ACC ATC AAG GCT TCC AAC TTC AAC CTG AAC CGC AAG ACA CGC 289 lie Ala Thr lie Lys Ala Ser Asn Phe Asn Leu Asn Arg Lys Thr Arg 80 85 90 TIC ATT ATA CAT GGC TTC ACG GAC AGT GGA GAG AAC AGC TGG CTA TCA 337 Phe lie He His Gly Phe Thr Asp Ser Gly Glu Asn Ser Trp Leu Ser 95 100 105 GAC ATG TGC AAG AAT ATG TTC CAA GTG GAG AAG GTG AAC TGC ATC TCT 385 Asp Mat Cys Lys Asn Met Phe Gin Val Glu Lys Val Asn Cys lie Cys HO H5 120 GTG GAC TGG AAG.GGG GGA TCT AAG GCC CAA TAC AGC CAA GCG TCT CAG 433 Val Asp Trp Lys Gly Gly Ser Lys Ala Gin Tyr Ser Gin Ala Ser Gin 125 130 135 AAT ATT OST GOT GTG GGC GCA GAG GTC GCT TAC TTG GTG CAA GTG CTT 481 Asn He Arg Val Val Gly Ala Glu Val Ala Tyr Leu Val Gin Val Leu 140 145 150 155 TOG ACG AGC CTG AAT TAC GCC CCT GAG AAT GEA CAC ATC ATC GGC CAC 529 Ser Thr Ser Leu Asn Tyr Ala Pro Glu Asn Val His He lie Gly His 160 165 170 ACT T G GGA GCG CAC ACT GCC GGG GAG GCG GGC AAG AGG CTG AAT GGC 577 Ser Leu Gly Ala His Thr Ala Gly Glu Ala Gly Lys Arg Leu Asn Gly 175 180 185 CTC GTG GGC AGG ATC ACA GGG CTG GAT CCA GCA GAG CCA TAC TTC CAG 625 Leu Val Gly Arg He Thr Gly Leu Asp Pro Ala Glu Pro Tyr Phe Gin 190 195 200 GAC ACG CCC GAG GAG GIT CGG CTG GAT CCA TCT GAT GCC AAG TTT CTG 673 Asp Thr Pro Glu Glu Val Arg Leu Asp Pro Ser Asp Ala Lys Phe Val 205 210 215 GAT GTG ATT CAC ACA GATAΪTTCCCXT.AΪΑCTTOCCTCCTTGGCTTTT 721 Asp Val He His Thr Asp He Ser Pro He Leu Pro Ser Leu Gly Phe 220 225 230 235 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 AAA GAC ATG CCT GGA TCT AAA ACA GGA ATC TCT TGC AAC CAC CAC CGG 817 Lys Asp Met Pro Gly Cys Lys Thr Gly He Ser Cys Asn His His Arg 255 260 265 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 GGC TAC CCC TCT GCC TCC TAC GAC GAG TTC CAG GAG ACT GGC TCT TTC 913 Gly Tyr Pro Cys Ala Ser Tyr Asp Glu Phe Gin Glu Ser Gly Cys Phe 285 290 295 CCT TCT CCA GCT AAA GGA TCT CCA AAA ATG GGG CAC TTT GCA GAC CAA 961 Pro Cys Pro Ala Lys Gly cys Pro Lys Met Gly His Phe Ala Asp Gin 300 305 310 315 TAT COG GGA AAA ACC AAT GCT GTG GAA CAA ACC TTT TTC CEA AAC ACA 1009 Tyr Pro Gly Lys Thr Asn Ala Val Glu Gin Thr Phe Phe Leu Asn Thr 320 325 330 GGA GCG ACT GAT AAC TTT ACT 0CT TGG AGG TAT AAG GTA ACT CTC ACA 1057 Gly Ala Ser Asp Asn Phe Thr Arg Trp Arg Tyr Lys Val Thr Val Thr 335 340 345 CEA TCT GGA GAA AAG GAT OCA AGC GGA AAC ATC AAT GTT GCT TTG TTG 1105 Leu Ser Gly Glu Lys Asp Pro Ser Gly Asn He Asn Val Ala Leu Leu 350 355 360 GGA AAG AAC GGA AAC TCA GCA CAA TAT CAG GTT TTC AAG GGG ACC CTC 1153 Gly Lys Asn Gly Asn Ser Ala Gin Tyr Gin Val Phe Lys Gly Thr Leu 365 370 375 AAA CCA GAT GCC ACT TAT ACT AAT AGC ATT GAT GTG GAG CTC AAT CTT 1201 Lys Pro Asp Ala Ser Tyr Thr Asn Ser lie Asp Val Glu Leu Asn Val 380 385 390 395 GGA ACA ATT CAG AAG CTT ACA TTC CTC TGG AAG AGA ACT GGG ATA ACT 1249 Gly Thr lie Gin Lys Val Thr Hie Leu Trp Lys Arg Ser Gly He Ser 400 405 410 CTC TCC AAG CCC AAA ATG GGG GCT TCC CGA ATC ACC GTG CAG AGC GCT 1297 Val Ser Lys Pro Lys Met Gly Ala Ser Arg lie Thr Val Gin Ser Gly 415 420 425 AAA GAT GGG ACC AAG TAT AAT TTT TGC AGC AGC GAC ATT CTG CAA GAA 1345 Lys Asp Gly Thr Lys Tyr Asn Hie cys Ser Ser Asp lie Val Gin Glu 430 435 440 AAT CTT GAA CAG ACC CTT TCC CCT TCT TAATGACTOG AG 1384 Asn Val Glu Gin Thr Leu Ser Pro Cys 445 450 (2) INFORMATICaT FOR SEQ ID NO:2: (i) SEQUENCE ( "" HARACTERISTTCS: (A) LENGTH: 452 amino acids (B) TYPE: amino acid (D) TOPOIOGY: linear (ii) MDLECDEE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Met Leu Phe Ala Trp Thr He Gly Leu Leu Leu Leu Ala Thr Val 1 5 10 15 Arg Gly Ala Glu Val Cys Tyr Ser His Leu Gly Cys Phe Ser Asp Glu 20 25 30 Lys Pro Trp Ala Gly Thr Ser Gin Arg Pro He Lys Ser Leu Pro Ser 35 40 45 Asp Pro Lys Lys lie Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn 50 55 60 Gin Asn Ser Tyr Gin Leu lie Thr Ala Thr Asp lie Ala Thr He Lys 65 70 75 80 Ala Ser Asn Hie Asn Leu Asn Arg Lys Thr Arg Hie He He His Gly 85 90 95 Phe Thr Asp Ser Gly Glu Asn Ser Trp Leu Ser Asp Met Cys Lys Asn 100 105 110 Met Phe Gin Val Glu Lys Val Asn Cys He Cys Val Asp Trp Lys Gly 115 120 125 Gly Ser Lys Ala Gin Tyr Ser Gin Ala Ser Gin Asn He Arg Val Val 130 135 140 Gly Ala Glu Val Ala Tyr Leu Val Gin Val Leu Ser Thr Ser Leu Asn 145 150 155 160 Tyr Ala Pro Glu Asn Val His He He Gly His Ser Leu Gly Ala His 165 170 175 Thr Ala Gly Glu Ala Gly Lys Arg Leu Asn Gly Leu Val Gly Arg He 180 185 190 Thr Gly Leu Asp Pro Ala Glu Pro Tyr Phe Gin Asp Thr Pro Glu Glu 195 200 205 Val Arg Leu Asp Pro Ser Asp Ala Lys Phe Val Asp Val He His Thr 210 215 220 Asp He Ser Pro He Leu Pro Ser Leu Gly Hie Gly Met Ser Gin Lys 225 230 235 240 Val Gly His Met Asp Phe Phe Pro Asn Gly Gly Lys Asp Met Pro Gly 245 250 255 Cys Lys Thr Gly He Ser Cys Asn His His Arg Ser He Glu Tyr Tyr 260 265 270 His Ser Ser He Leu Asn Pro Glu Gly Phe Leu Gly Tyr Pro Cys Ala 275 280 285 Ser Tyr Asp Glu Hie Gin Glu Ser Gly Cys Phe Pro Cys Pro Ala Lys 290 295 300 Gly Cys Pro Lys Met Gly His Hie Ala Asp Gin Tyr Pro Gly Lys Thr 305 310 315 320 Asn Ala Val Glu Gin Thr Phe Phe Leu Asn Thr Gly Ala Ser Asp Asn 325 330 335 Phe Thr Arg Trp Arg Tyr Lys Val Thr Val Thr Leu Ser Gly Glu Lys 340 345 350 Asp Pro Ser Gly Asn He Asn Val Ala Leu Leu Gly Lys Asn Gly Asn 355 360 365 Ser Ala Gin Tyr Gin Val Phe Lys Gly Thr Leu Lys Pro Asp Ala Ser 370 375 380 Tyr Thr Asn Ser He Asp Val Glu Leu Asn Val Gly Thr lie Gin Lys 385 390 395 400 Val Thr Phe Leu Trp Lys Arg Ser Gly lie Ser Val Ser Lys Pro Lys 405 410 415 Met Gly Ala Ser Arg He Thr Val Gin Ser Gly Lys Asp Gly Thr Lys 420 425 430 Tyr Asn Hie Cys Ser Ser Asp lie Val Gin Glu Asn Val Glu Gin Thr 435 440 445 Leu Ser Pro cys 450 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1404 base pairs (B) TYPE: nucleic acid (C) S * TRANDEDNESS: single (D) TDPOIOGY: linear (ϋ) MDLECULE TYPE: CDNA (Vi) ORIGINAL SOURCE: (A) ORGANISM: human (ix) FEATURE: (A) NAME/KEY: CDS (B) IOCATICN: 22..1371 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCTAGAACTA GTGGATCOGC C ATG AAG GAG GTG TCT TAC GAA AGA CTC GGC 51 Met Lys Glu Val Cys Tyr Glu Arg Leu Gly 1 5 10 TGC TTC ACT GAT GAC TCC CCA TGG TCA GGA ATT ACG GAA AGA CCC CTC 99 Cys Ihe Ser Asp Asp Ser Pro Trp Ser Gly He Thr Glu Arg Pro Leu 15 20 25 CAT ATA TTG CCT TGG TCT CCA AAA GAT GTC AAC ACC CGC TTC CTC CTA 147 His He Leu Pro Trp Ser Pro Lys Asp Val Asn Thr Arg Hie Leu Leu 30 35 40 TAT ACT AAT GAG AAC CCA AAC AAC TTT CAA GAA GTT GCC GCA GAT TCA 195 Tyr Thr Asn Glu Asn Pro Asn Asn Hie Gin Glu Val Ala Ala Asp Ser 45 50 55 TCA AGC ATC ACT GGC TCC AAT TTC AAA ACA AAT AGA AAA ACT OGC TTT 243 Ser Ser He Ser Gly Ser Asn Phe Lys Thr Asn Arg Lys Thr Arg Phe 60 65 70 ATT ATT CAT GGA TTC ATA GAC AAG GGA GAA GAA AAC TGG CTG GCC AAT 291 He He His Gly Phe He Asp Lys Gly Glu Glu Asn Trp Leu Ala Asn 75 80 85 90 GTG TGC AAG AAT CTG TTC AAG GTG GAA ACT GTG AAC TCT ATC TCT GIG 339 Val Cys Lys Asn Leu Hie Lys Val Glu Ser Val Asn Cys He Cys Val 95 100 105 GAC TGG AAA GCT GGC TCC CGA ACT GGA TAC ACA CAA GCC TOG CAG AAC 387 Asp Trp Lys Gly Gly Ser Arg Thr Gly Tyr Thr Gin Ala Ser Gin Asn 110 115 120 ATC AGG ATC GTG GGA GCA GAA GTG GCA TAT TTT GTT GAA TTT CTT CAG 435 He Arg He Val Gly Ala Glu Val Ala Tyr Hie Val Glu Phe Leu Gin 125 130 135 TOG GCG TTC GCT TAC TCA CCT TCC AAC GTG CAT GTC ATT GGC CAC AGC 483 Ser Ala Hie Gly Tyr Ser Pro Ser Asn Val His Val He Gly His Ser 140 145 150 CTG GCT GCC CAC GCT GCT GGG GAG GCT GGA AGG AGA ACC AAT GGG ACC 531 Leu Gly Ala His Ala Ala Gly Glu Ala Gly Arg Arg Thr Asn Gly Thr 155 160 165 170 AIT GGA CGC ATC ACA GGG TIG GAC CCA GCA GAA CCT TGC TTT CAG GGC 579 He Gly Arg He Thr Gly Leu Asp Pro Ala Glu Pro Cys Phe Gin Gly 175 180 185 ACA CCT G TTA GTC CGA TTG GAC CCC AGC GAT GCC AAA TTT CTG GAT 627 Thr Pro Glu Leu Val Arg Leu Asp Pro Ser Asp Ala Lys Phe Val Asp 190 195 200 CTA ATT CAC ACG GAT GCT GCC CCC ATA GTC CCC AAT TTG GGG TTT GGA 675 Val He His Thr Asp Gly Ala Pro He Val Pro Asn Leu Gly Phe Gly 205 210 215 ATG AGC CAA GTC GTG GGC CAC CTA GAT TTC TTT CCA AAT GGA GGA CTG 723 Met Ser Gin Val Val Gly His Leu Asp Hie Phe Pro Asn Gly Gly Val 220 225 230 GAA ATG CCT GGA TCT AAA AAG AAC ATT CTC TCT CAG A T GTG GAC ATA 771 Glu Met Pro Gly Cys Lys Lys Asn He Leu Ser Gin He Val Asp He 235 240 245 250 GAC GGA ATC TGG G GGG ACT CGA GAC TTT GCG GCC TCT AAT CAC TTA 819 Asp Gly He Trp Glu Gly Thr Arg Asp Phe Ala Ala Cys Asn His Leu 255 260 265 AGA AGC TAC AAA TAT TAC ACT GAT AGC ATC GTC AAC CCT GAT GGC TIT 867 Arg Ser Tyr Lys Tyr Tyr Thr Asp Ser He Val Asn Pro Asp Gly Phe 270 275 280 GCT GGA TTC CCC TCT GCC TCT TAC AAC CTC TTC ACT GCA AAC AAG TCT 915 Ala Gly Hie Pro Cys Ala Ser Tyr Asn Val Phe Thr Ala Asn Lys Cys 285 290 295 TTC CCT TCT CCA ACT GGA GGC TGC CCA CAG ATG GCT CAC TAT GCT GAT 963 Hie Pro Cys Pro Ser Gly Gly Cys Pro Gin Met Gly His Tyr Ala Asp 300 305 310 AGA TAT CCT GGG AAA ACA AAT GAT GTG GGC CAG AAA TTT TAT CTA GAC 1011 Arg Tyr Pro Gly Lys Thr Asn Asp Val Gly Gin Lys Phe Tyr Leu Asp 315 320 325 330 ACT GCT GAT GCC ACT AAT TTT GCA CCT TGG AGG TAT AAG CTA TCT CTC 1059 Thr Gly Asp Ala Ser Asn Phe Ala Arg Trp Arg Tyr Lys Val Ser Val 335 340 345 ACA CTG TCT GGA AAA AAG GTT ACA GGA CAC ATA CTA CTT TCT TTG TTC 1107 Thr Leu Ser Gly Lys Lys Val Thr Gly His lie Leu Val Ser Leu Phe 350 355 360 GGA AAT AAA GGA AAC TCT AAG CAG TAT GAA ATT TTC AAG GGC ACT CTC 1155 Gly Asn Lys Gly Asn Ser Lys Gin Tyr Glu He Phe Lys Gly Thr Leu 365 370 375 AAA CCA GAT ACT ACT CAT TCC AAT GAA TTT GAC TCA GAT GTG GAT GTT 1203 Lys Pro Asp Ser Thr His Ser Asn Glu Hie Asp Ser Asp Val Asp Val 380 385 390 GGG GAC TTG CAG ATG CTT AAA TTT ATT TGG TAT AAC AAT GTG ATC AAC 1251 Gly Asp Leu Gin Met Val Lys Hie He Trp Tyr Asn Asn Val He Asn 395 400 405 410 CCA ACT TTA CCT AGA GTG GGA GCA TCC AAG ATT ATA GTG GAG ACA AAT 1299 Pro Thr Leu Pro Arg Val Gly Ala Ser Lys lie He Val Glu Thr Asn 415 420 425 GTT GGA AAA CAG TTC AAC TTC TCT ACT CCA GAA ACC GTC AGG GAG GAA 1347 Val Gly Lys Gin Hie Asn Hie Cys Ser Pro Glu Thr Val Arg Glu Glu 430 435 440 GTT CTG CTC ACG CTG ACA CCC TGC TGAGGATCCC COGGGCTGCA GGAAITOGAT 1401 Val Leu Leu Thr Leu Thr Pro Cys 445 450 ATC 1404 (2) INPORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTΕRISTTCS: (A) LENGTH: 450 amino acids (B) TYPE: amino acid (D) TOFOIDGY: linear (ii) MDLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Lys Glu Val Cys Tyr Glu Arg L≥u Gly Cys Phe Ser Asp Asp Ser 1 5 10 15 Pro Trp Ser Gly He Thr Glu Arg Pro Leu His He Leu Pro Trp Ser 20 25 30 Pro Lys Asp Val Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn Pro 35 40 45 Asn Asn Phe Gin Glu Val Ala Ala Asp Ser Ser Ser He Ser Gly Ser 50 55 60 Asn Phe Lys Thr Asn Arg Lys Thr Arg Phe He He His Gly Hie He 65 70 75 80 Asp Lys Gly Glu Glu Asn Trp Leu Ala Asn Val Cys Lys Asn Leu Phe 85 90 95 Lys Val Glu Ser Val Asn Cys He Cys Val Asp Trp Lys Gly Gly Ser 100 105 110 Arg Thr Gly Tyr Thr Gin Ala Ser Gin Asn He Arg He Val Gly Ala 115 120 125 Glu Val Ala Tyr Hie Val Glu Phe Leu Gin Ser Ala Phe Gly Tyr Ser 130 135 140 Pro Ser Asn Val His Val He Gly His Ser Leu Gly Ala His Ala Ala 145 150 155 160 Gly Glu Ala Gly Arg Arg Thr Asn Gly Thr He Gly Arg He Thr Gly 165 170 175 Leu Asp Pro Ala Glu Pro cys Phe Gin Gly Thr Pro Glu Leu Val Arg 180 185 190 Leu Asp Pro Ser Asp Ala Lys Phe Val Asp Val He His Thr Asp Gly 195 200 205 Ala Pro He Val Pro Asn Leu Gly Phe Gly Met Ser Gin Val Val Gly 210 215 220 His Leu Asp Hie Phe Pro Asn Gly Gly Val Glu Met Pro Gly Cys Lys 225 230 235 240 Lys Asn lie Leu Ser Gin lie Val Asp lie Asp Gly He Trp Glu Gly 245 250 255 Thr Arg Asp Phe Ala Ala Cys Asn His Leu Arg Ser Tyr Lys Tyr Tyr 260 265 270 Thr Asp Ser He Val Asn Pro Asp Gly Phe Ala Gly Hie Pro Cys Ala 275 280 285 Ser Tyr Asn Val Hie Thr Ala Asn Lys Cys Hie Pro Cys Pro Ser Gly 290 295 300 Gly Cys Pro Gin Met Gly His Tyr Ala Asp Arg Tyr Pro Gly Lys Thr 305 310 315 320 Asn Asp Val Gly Gin Lys Phe Tyr Leu Asp Thr Gly Asp Ala Ser Asn 325 330 335 Phe Ala Arg Trp Arg Tyr Lys Val Ser Val Thr Leu Ser Gly Lys Lys 340 345 350 Val Thr Gly His He Leu Val Ser Leu Hie Gly Asn Lys Gly Asn Ser 355 360 365 Lys Gin Tyr Glu lie Phe Lys Gly Thr Leu Lys Pro Asp Ser Thr His 370 375 380 Ser Asn Glu Phe Asp Ser Asp Val Asp Val Gly Asp Leu Gin Met Val 385 390 395 400 Lys Hie He Trp Tyr Asn Asn Val He Asn Pro Thr Leu Pro Arg Val 405 410 415 Gly Ala Ser Lys He He Val Glu Thr Asn Val Gly Lys Gin Phe Asn 420 425 430 Phe cys Ser Pro Glu Thr Val Arg Glu Glu Val Leu Leu Thr Leu Thr 435 440 445 Pro cys 450

Claims: CLAIMS 1. 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. 2. A GPL according to claim 1, which is C-terminally truncated. 3. GPL L319 according to claim 2. 4. A GPL according to claim 1 or 2, which comprises an insertional substitution between amino acid 239 and amino acid 245 of the native GPL sequence. 5. A GPL according to claim 4, wherein the insertional substitution comprises the following amino acid sequence Lys Ala Asn Leu Gin Lys Asn lie Leu Ser Gin lie Val Asp lie Asp Gly lie Trp Glu Ala Gly Thr Arg Asp Phe Val Ala or a subsequence thereof. 6. A GPL according to claim 5, wherein the insert 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 7. A GPL according to any of claims 1-6, wherein Cys 103 or Cys j05 is replaced by another amino acid residue. 8. A GPL according to claim 7, wherein Cysι ∞ or Cys 105 is replaced by Thr or Ser. 9. A GPL according to any of claims 1-8, wherein Ser 154 is replaced by Thr and/or Asp I78 is replaced by Glu. 10. A recombinant DNA molecule comprising a DNA sequence encoding guinea pig pancreatic lipase (GPL) or an enzymatically active variant thereof. 11. A recombinant DNA molecule according to claim 10, wherein the DNA sequence is as shown in the appended Sequence Listing ID No. 1, or a modification of said sequence encoding GPL or an enzymatically active variant thereof. 12. A recombinant DNA molecule according to claim 10, wherein the DNA sequence encodes a C-terminally truncated GPL. 13. A recombinant DNA molecule according to claim 12, wherein the DNA seguence encodes GPL^^. 14. A recombinant DNA molecule according to claim 11, which comprises an insertional substitution between nucleotides 771 and 787 of the coding sequence of GPL. 15. A recombinant DNA molecule according to claim 14, wherein the insertional substitution encodes the following amino acid sequence Lys Ala Asn Leu Gin Lys Asn lie Leu Ser Gin lie Val Asp lie Asp Gly lie Trp Glu Ala Gly Thr Arg Asp Phe Val Ala or a fragment thereof. 16. A recombinant DNA molecule according to claim 15, wherein the insertional substitution comprises the following nucleotide sequence AAA AAG AAC ATC TTG TCT CAA ATC GTT GAC ATC GAC GGT ATC TGG GAA GGT ACC AGA GAC TTC GCT GCT 17. A recombinant DNA molecule according to claim 11, which comprises a mutation in the codon specifying Cys 103 or Cysι 05 so that it codes for another amino acid residue, in particular Ser or Thr. 18. A human pancreatic lipase (HPL) variant which is deleted of one or more amino acids in the sequence between and Cys 262 . 19. A HPL variant according to claim 18, wherein the sequence between and Cys 262 is replaced by the sequence Lys-Thr-Gly- Ile-Ser. 20. A HPL variant comprising a c-terminal truncation. 21. HPL j . 336 according to claim 21. 22. A HPL variant according to claim 20 or 21, which is deleted of one or more amino acids in the sequence between and 23. A HPL variant wherein Cys^ or Cγs m is replaced by another amino acid residue. 24. A HPL variant according to claim 23, wherein Cys 102 or Cys 1M is replaced by Thr or Ser. 25. A HPL variant according to any of claims 18-24, wherein c y s ιo 2 or c Y s ιo 4 i s replaced by another amino acid residue. 26. A HPL variant according to claim 25, wherein Cys J02 or Cys 1M is replaced by Thr or Ser. 27. A HPL variant according to any of claims 18-26, wherein Ser 153 is replaced by Thr and/or Asp 177 is replaced by Glu. 28. An enzymatically active recombinant lipase variant which comprises at least one GPL fragment and at least one HPL fragment. 29. A DNA sequence encoding any of the lipase variants claimed in claims 19-27 or 28. 30. A process for the extracellular production of a mammalian pancreatic lipase in a filamentous fungus, the process comprising (a) providing a recombinant DNA vector which comprises a DNA sequence encoding a mammalian pancreatic lipase and a DNA sequence encoding a preregion permitting secretion of the expressed mammalian pancreatic lipase into the culture medium, (b) transforming a suitable filamentous fungus with the recombinant DNA vector of step (a) , and (c) culturing the transformed filamentous fungus in a suitable culture medium under conditions conducive to the production of the mammalian pancreatic lipase. 31. A process according to claim 30, wherein the filamentous fungus is a strain of Aspergillus sp. 32. A process according to claim 31, wherein the Aspergillus sp. is Aspergillus oryzae. 33. A process according to claim 30, wherein the preregion is derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase, a gene encoding a Humicola cellulase or xylanase, or a gene encoding a mammalian pancreatic lipase. 34. A process according to claim 33, wherein the preregion is derived from the gene encoding Aj_ oryzae TAKA amylase, A^. niger neutral α-amylase, A^. niger acid-stable α-amylase, A_;_ niger glucoamylase, EL. insolens cellulase or xylanase, or the gene encoding guinea pig, human, canine or porcine pancreatic lipase. 35. A process according to any of claims 30-34, wherein the mammalian pancreatic lipase is guinea pig pancreatic lipase or human pancreatic lipase. 36. A process according to claim 35, wherein the guinea pig pancreatic lipase is one encoded by a recombinant DNA molecule according to any of claims 10-17. 37. A process according to claim 35, wherein the human pancreatic lipase is a HPL variant according to any of claims 18-27 or a recombinant lipase variant according to claim 28. 38. A detergent additive comprising a GPL according to any of claims 1-9, a HPL variant according to any of claims 18-27 or a recombinant lipase variant according to claim 28, preferably in the form of a non-dusting granulate, stabilized, or protected enzyme. 39. A detergent additive according to claim 38 which contains 0.2-200 mg enzyme protein per gram of the additive. 40. A detergent additive according to claim 38, which further comprises another enzyme such as a protease, amylase, cellulase or peroxidase. 41. A detergent composition comprising a GPL according to any of claims 1-9, a HPL variant according to any of claims 18-27 or a recombinant lipase variant according to claim 28. 42. A detergent composition according to claim 41, which further comprises another enzyme such as a protease, amylase, cellulase or peroxidase. 43. A detergent composition according to claim 41 or 42, which as surfactant comprises an alkyl phosphate. 44. A GPL according to any of claims 1-9 for use as a digestive enzyme. 45. A HPL variant according to any of claims 18-27 for use as a digestive enzyme. 46. A recombinant lipase variant according to claim 28 for use as a digestive enzyme.

Inventors: GORMSEN ERIK, HJORTH SIV ANNEGRETHE, THIM LARS, VERGER ROBERT, WOLDIKE HELLE FABRICIUS, GORMSEN, ERIK, HJORTH, SIV, ANNEGRETHE, THIM, LARS, VERGER, ROBERT, WOLDIKE, HELLE, FABRICIUS
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: C12N15
IPC codes: C11D3, C12N9, C12N15