A PROCESS FOR THE PREPARATION OF LIPASE

Abstract: A process for producing an active lipase enzyme in vitro, comprising mixing an inactive or partly active lipase enzyme with a chaperone molecule and subjecting the mixture to denaturation followed by renaturation to produce the active lipase enzyme.

Description: A PROCESS FOR THE PREPARATION OF LIPASE FIELD OF INVENTION The present invention relates to processes for preparing an active lipase enzyme |π vitro, a DNA construct encoding the lipase, a recombinant vector including the DNA construct, as well as a host cell transformed with the vector. BACKGROUND OF THE INVENTION Upases are enzymes which catalyze the hydrolysis of ester bonds in triglycerides resulting in the formation of diglycerides, monoglycerides, glycerin and free fatty acids. Some lipases also catalyze other reactions involving ester bonds such as synthesis of ester bonds or transesterification reactions. Lipases are produced by a wide variety of different organisms. Microbial lipases in particular are of considerable practical utility for a variety of purposes where lipolysis of fats is desired, e.g. in the food industry and in detergents. One particular lipase which has been found to be particularly advantageous for inclusion in a detergent for the removal of fatty stains or soiling from fabrics is a lipa¬ se produced by strains of Pseudomonas cepacia. In EP 214 761 (to Novo Industri A/S), this lipase is disclosed as a lipase which is active at a temperature below 60°C, which is important as most present-day fabrics are washed at temperatures below 60°C. Another important Pseudomonas cepacia lipase for use as a detergent additive is the one disclosed in WO 89/01032 (to Novo Industri A/S) as a positionally non¬ specific lipase, i.e. one which is able to react with all three fatty acyl groups of a trigiyceride. In order to facilitate Pseudomonas cepacia lipase production, it may be advantageous to employ recombinant DNA techniques, for instance in order to optimize lipase expression by introducing a stronger promoter from which the DN sequence encoding the enzyme is expressed or by introducing more efficien ribosome binding sites or signal peptide coding sequences, or in order to select host organism for the production of the enzyme which is easier to cultivate (e.g. i 5 terms of its being a standard production organism such as E. coli or the like) o which results in higher lipase yields. However, as described below, such approaches will sometimes fail to yield the expected results, e.g. in cases where one or more genes in addition to the structural gene coding for the protein in question, play some part in the production of the gene product (examples of such genes are the Bacillus sac and iep genes, and genes required for the production of Klebsiella pullulanase and E. coli hemolysin). The cloning of a lipase gene from another Pseudomonas species, Pseudomonas frag], is known from, e.g., S. Aoyama et al. (1988) and W. Kugimiya et al. (1986). However, the lipase produced by P.fraαi differs from that of P. cepacia in its amino acid sequence, and in these publications, there is no indication that one or more additional genes may be required in order to achieve a significant lipase production in a host organism. EP 331 376 discloses a recombinant DNA encoding a Pseudomonas cepacia lipase as well as a protein participating in the production of the lipase. WO 90/00908 discloses the production of a Pseudomonas cepacia lipase in heterologous host cells by means of a polypeptide expressed in the host cell, which polypeptide acts as a modulator of lipase production. SUMMARY OF THE INVENTION It has surprisingly been found possible to increase the yield of an active lipase enzyme produced by a recombinant host cell when the lipase recovered from the cells is subjected to denaturation followed by renaturation in the presence of a chaperone molecule. Accordingly, the present invention relates to a process for preparing an active lipase enzyme in vitro, the process comprising (a) culturing a host cell transformed with a DNA sequence encoding a lipase enzyme under suitable conditions to produce the lipase enzyme in inactive or partly active form, recovering the lipase enzyme from the culture, and subjecting the recovered lipase enzyme to denaturation, (b) mixing the denaturated lipase enzyme with a chaperone molecule, and (c) subjecting the mixture of step (b) to renaturation to produce the active lipase enzyme. Alternatively, the invention relates to a process for preparing an active lipase enzyme jn vitro, the process comprising (a) culturing a host cell transformed with a DNA sequence encoding a lipase enzyme under suitable conditions to produce the lipase enzyme in inactive or partly active form and recovering the lipase enzyme from the culture, (b) mixing the recovered lipase enzyme with a chaperone molecule, and (c) subjecting the mixture of step (b) to denaturation followed by renaturation to produce the active lipase enzyme. In a further embodiment, the invention relates to a process for preparing an active lipase enzyme jn vitro, the process comprising (a) culturing a host cell transformed with a DNA sequence encoding a lipase enzym and with a DNA sequence encoding a chaperone molecule under suitable condition to produce the lipase enzyme in inactive or partly active form and recovering a lipas enzyme chaperone molecule mixture from the culture, and (b) subjecting the mixture of step (a) to denaturation followed by renaturation to produce the active lipase enzyme, optionally with addition of a further amount of chaperone molecule to the mixture. As indicated above, the present invention is based on the finding that the natural conformation of a protein or protein complex is not always only determined by the amino acid sequence of the protein. Thus, in some cases, accessory proteins, termed chaperone molecules, are required to mediate the formation of the correct tertiary structure of another protein or protein complex, but are not themselves components of the final functional structure (Ellis et al., 1991). In the present context, the term "chaperone molecule" is intended to indicate such accessory proteins, i.e. a protein involved in facilitating other polypeptides in maintaining the unfolded state, enabling their correct transmembrane targeting or folding and oligomeric assembly, and in disassembly of protein complexes (cf. R.J. Ellis and S.M. Hemmingsen (1989), J.E. Rothman (1989), Morimoto et al.(1990)). Although, in general, a covalent modification of the target protein or protein complex has not been observed by the action of a chaperone molecule it cannot be excluded that the chaperone molecule to be used in the processes of the invention is capable of causing such covalent modification. Accordingly, the term "chaperone molecule" as used herein is intended to include a chaperone molecule causing non-covalent as well as covalent modification of the lipase enzyme. In another aspect, the present invention relates to a DNA construct comprising a first DNA sequence encoding a lipase enzyme fused to a second DNA sequence encoding a chaperone molecule in such a way that the lipase enzyme and chaperone molecule or functional part thereof are expressed as a single fusio protein on culturing a suitable host cell transformed with the DNA construct. DETAILED DISCLOSURE OF THE INVENTION The chaperone molecule to be added to the recovered and optionally denaturate lipase enzyme in step b) above is advantagously produced by a process comprisin culturing a host cell transformed with a DNA sequence encoding a chaperon molecule under suitable conditions to produce the chaperone molecule and recovering the chaperone molecule from the culture. Furthermore, when the chaperone molecule is to be added to a denaturated lipase enzyme it may be advantageous that the chaperone molecule itself is denaturated. Accordingly, the process of the invention may comprise a further step in which the chaperone molecule is subjected to a denaturation treatment before it is mixed with the denatured lipase enzyme in step b). In the present context, "partly active form" as used about the lipase enzyme is intended to indicate that the lipase has some, but not full activity as determined by an activity measurement, e.g. by titration using a pH stat as later described. Less than full activity is taken to mean, that the partly active lipase preparation has a lower specific activity than a corresponding preparation of fully active lipase protein, the two preparations containing the same total amount of lipase protein. The denaturation of the inactive or partly active lipase enzyme and optionally the chaperone molecule, which may be performed separately or on a mixture of the two components according to the processes of the invention may be carried out in a manner known per se. For instance, the denaturation may be obtained by subjecting the mixture to the action of a denaturating agent (e.g. 8M urea) and subsequently removing this agent, e.g. by dialysis. Preferred lipases for production by the process of the invention are lipases derived from a Pseudomonas sp. or a Chromobacter sp. In particular, the lipase enzyme may be a Pseudomonas cepacia. Pseudomonas frag], Pseudomonas gladioli Pseudomonas fluorescens, Pseudomonas stutzeri. Pseudomonas alcaliqenes Pseudomonas pseudoalcaliαenes. Pseudomonas putida. Pseudomonas qlumae (e.g as described in EP 407225), Pseudomonas aeruqinosa or Chromobacter viscosu 5 lipase, or a derivative of said lipase enzyme. In particular, the lipase enzyme is on derived from a strain of Pseudomonas cepacia. e.g. a strain deposited in the Deut sche Sammlung von Mikroorganismen in connection with the invention disclosed in EP 21 761, with the deposit numbers DSM 3333-3337 and DSM 3401, as well as the strain deposited in the Deutsche Sammlung von Mikroorganismen in connection with the invention disclosed in WO 89/01032, with the deposit number DSM 3959. In the present context, the term "derivative" is intended to indicate a protein with lipolytic activity which is derived from the native lipase by suitably modifying the DNA sequence coding for the native lipase, resulting in the addition of one or more amino acids to either or both the C- and N-terminal end of the native protein, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids 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 acids at one or more sites in the native amino acid sequence. Such modifications of DNA coding for native proteins are well known and widely practised in the art. Typically, the amino acid sequence of the derivative will be homologous to that of the native lipase protein, e.g. exhibiting a substantial degree of homology, or the derivative will react with an antibody raised against the native lipase. The host cell used in the process of the invention may be any suitable bacterium which, on cultivation, produces large amounts of the lipase. Examples of suitable bacteria may be grampositive bacteria such as Bacillus subtilis. Bacillus licheniformis. Bacillus lentus. Bacillus brevis. Bacillus stearothermophilus. Bacillus alkalophilus. Bacillus amyloliαuefaciens. Bacillus coaαulans. Bacillus circulans. Bacillus lautus or Streptomvces lividans. It has been found that E. coli is able to produce a high yield, i.e. at least 5% of the total cellular protein, of (intracellular) lipase, and is therefore a preferred host organism. In E. coli, the lipase enzyme is typically produced in the form of inclusion bodies. The transformation of the bacteria may for instance be ef fected by protoplast transformation or by using competent cells in a manner know per se. Another suitable bacterial host cell is a cell of a Pseudomonas spp. such a Pseudomonas cepacia. Pseudomonas frag], Pseudomonas gladioli. Pseudomona fluorescens. Pseudomonas stutzeri. Pseudomonas alcaliqenes. Pseudomona pseudoalcalioenes. Pseudomonas putida. Pseudomonas olumae or Pseudomona aeruginosa. Alternatively, the host cell may be a fungus, i.e. a cell of a yeast or of a filamentou fungus. The yeast host cell may, for instance, be a cell of the genus Saccharomvces such as S ^ cerevisiae. The filamentous fungus host organism may conveniently be one which has previously been used as a host for producing recombinant proteins, e.g. a strain of Asperoillus sp., such as A. niger. ± nidulans or Δ_. orvzae. The techniques used to transform a fungal host cell and obtain expression of the recombinant protein may suitably be as described in EP 238 023. For expression of the protein in the host cell the DNA sequence encoding the protein may be preceded by a promoter. The promoter may be any DNA sequence exhibiting a strong transcriptional activity in the host cell of choice and may be derived from a gene encoding an extracelluar or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme. Other sequences involved in expression of the protein include termination and polyadenylation sequences as well as ribosome binding sites and may suitably be derived from the same sources as the promoter. In the process of the invention, the DNA sequence encoding the lipase enzyme and/or the chaperone molecule is advantagously preceded by the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α- amylase gene, Bacillus amyloliguefaciens α-amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters, the phage T7 gene 10 promoter or the E coli lac promoter. The DNA sequence encoding the lipase enzyme and/or the chaperone molecule may b preceded by a ribosome binding site of the Bacillus stearothermophilus maltogeni amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α amylase gene, Bacillus subtilis alcaline protease gene, Bacillus pumilus xylosidas 5 gene, phage T7 gene 10 or E cgK lac gene. According to the invention, the chaperone molecule is advantageously Pseudomonas lipase modulator protein, preferably selected from the grou consisting of the Pseudomonas cepacia lipase modulator (disclosed in WO 90/00908), the Pseudomonas glumae lipase modulator, and the Pseudomonas aerooinosa lipase modulator, or a derivative of any such lipase modulator. In the present context, a derivative of a lipase modulator is to be understood in the same manner as indicated above in connection with derivatives of the lipase enzyme, i.e. a protein with chaperone activity which is derived from the native lipase by suitably modifying the DNA sequence coding for the native lipase modulator as discussed above. The chaperone activity of the derivative may, for instance, be determined by analysing the capability of the derivative in producing an active lipase enzyme as explained herein. In a preferred embodiment of the process according to the invention, the lipase enzyme is a Pseudomonas cepacia lipase or a derivative thereof, and the chaperone molecule is a Pseudomonas cepacia lipase modulator (or a derivative thereof) (both disclosed in WO 90/00908). The DNA construct of the invention comprising the DNA sequence encoding the lipase enzyme and/or the chaperone molecule may be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library of an appropriate organism, and screening for DNA sequences coding for all or part of the lipase or the chaperone by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., 1989). The DNA construct of the invention may also be prepared synthetically b established standard methods, e.g. the phosphoamidite method described by S.L Beaucage et al. (1981), Matthes et al. (1984). According to the phosphoamidit method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer purified, ligated, and cloned in an appropriate vector. Finally, the DNA construct may be of mixed synthetic and genomic, mixed syntheti and cDNA or mixed genomic and cDNA origin prepared by ligating fragments o synthetic, genomic or cDNA origin (as appropriate), the fragments correspondin to various parts of the entire DNA construct, in accordance with standard techniques. In the DNA construct of the invention, the DNA sequence encoding the lipas enzyme may be one derived from a Pseudomonas sp. or a Chromobacter sp. Fo instance, the first DNA sequence may be one encoding a Pseudomonas cepacia. Pseudomonas frag], Pseudomonas gladioli. Pseudomonas fluorescens. Pseudomonas stutzeri. Pseudomonas alcalioenes. Pseudomonas pseudoalcalioenes. Pseudomonas putida. Pseudomonas glumae. Pseudomonas aeruginosa or Chromobacter viscosum lipase, or a derivative of said lipase enzyme. The second DNA sequence may be one encoding a Pseudomonas cepacia lipase modulator, a Pseudomonas glumae lipase modulator, a Pseudomonas aerooinosa lipase modulator, or another Pseudomonas lipase modulator protein or a derivative of any of these modulators. Most preferably, the first DNA sequence encodes a Pseudomonas cepacia lipase or a derivative thereof, and the second DNA sequence encodes a Pseudomonas cepacia lipase modulator or a derivative thereof, as described in WO 90/00908 incorporated herein by reference. A particularly preferred DNA construct is one which has the sequence shown in the SEQ ID No. 5 appended hereto. The sequence may be modified in accordance with conventional practise. Examples of suitable modifications of the DNA sequence are nucleotide substitutions which do not give rise to another amino acid sequence of the lipase or lipase modulator, but which may correspond to the codon usage of the host organism into which the DNA construct is introduced 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 either the lipase or the lipase modulator. Other examples of possible modifications are insertion 5 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 wfthin the sequence. In a further aspect, the invention relates to a recombinant expression vector comprising a DNA construct as described above. The expression vector carrying the DNA sequence encoding the lipase and/or chaperone molecule may be any vector which is capable of replicating autonomously in a given host organism, typically a plasmϊd or bacteriophage. In the vector, the DNA sequence encoding the lipase and/or chaperone molecule should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell and may be derived from genes encoding proteins either homologous or heterologous to the host organism. The promoter is preferably the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α-amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambda P B or P L promoters, the phage T7 gene 10 promoter or the E coli lac promoter. The vector may also comprise a selectable marker, e.g. a gene whose product confers antibiotic resistance, " such as ampicillin, chloramphenicol or tetracyclin resistance, or the da genes from B.subtilis or B.licheniformis. In a still further aspect, the present invention relates to a process for preparing a lipase in active form, the process comprising culturing a host cell transformed with the DNA construct described above under suitable conditions to produce the lipase, and recovering the lipase from the culture, optionally followed by denaturation and renaturation of the lipase/chaperone fusion protein. The medium used to cultivate the cells may be any conventional medium suitable for growing bacteria. The lipase may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells to recover an intracellular product, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like. While the use of chaperone molecules in the preparation of active lipase enzyme is considered to be of particular importance in connection with recombinant DNA techniques (as explained above), it is believed that the presence of a chaperone molecule is critical in connection with any denaturation and renaturation treatment in which an active lipase enzyme, as described herein, is desired, irrespectively of the production method of the lipase enzyme. Thus, for instance in connection with a lipase enzyme produced by conventional fermentation of a naturally-occurring or non-genetically engineered organism, which is to be subjected to a denaturation treatment, it is believed that the presence of a chaperone molecule in the subsequent renuturation treatment is critical. Accordingly, in a further general aspect the invention relates to a process for denaturating and renaturating a lipase enzyme, the process comprising (a) subjecting a lipase enzyme to a denaturation treatment, (b) mixing the denaturated lipase enzyme obtained in step (a) with a chaperone molecule which has optionally been subjected to a denaturation treatment, and (c) subjecting the mixture of step (b) to renaturation to produce the active lipase enzyme. Alternatively, the denaturation/renaturation treatment of the lipase enzyme may carried out by a) mixing a lipase enzyme to be subjected to the denaturation and renaturatio treatment with a chaperone molecule, and b) subjecting the mixture of step a) to denaturation followed by renaturation so a to produce an active lipase enzyme. DESCRIPTION OF THE DRAWINGS The present invention is described in the following with reference to the appende drawings, in which Fig. 1 illustrates the plasmid pAHE2; Fig. 2 illustrates the plasmid pAHEδ; Fig. 3 illustrates the plasmid pAHEIO; Fig. 4 illustrates the plasmid pCBE6; Fig. 5 illustrates the plasmid pCBE7; Fig. 6 illustrates the plasmid pCBE12; Fig. 7 illustrates the plasmid pCBE18; Fig. 8 illustrates the plasmid pCBE19; Fig. 9 shows the result of a SDS-PAGE analysis of proteins expressed in E. coli JA221 performed as described in Example 3, in which lanes: A & S molecular weight markers; 45, 36, 24 and 20 B pJW2 (vector) C pSJ150 (original lipase construct in pUC) D pAHE2 30°C 1.0 hours(hr) E pAHE2 42°C 1.0 hrs F pAHE2 42°C 1.5 hrs G pAHE2 42°C 2.0 hrs H, M & R purified lipase from P. cepacia I pAHE10 30°C 1.0 hrs J pAHE10 42°C 1.0 hrs K pAHE10 42°C 1.5 hrs L pAHE2 42°C 2.0 hrs N pCBE6 -IPTG 1.0 hrs 0 pCBE6 +IPTG 1.0 hrs P pCBE6 +IPTG 1.5 hrs Q pCBE6 +IPTG 2.0 hrs Fig. 10 shows the result of an immunoblot analysis performed as described in Example 3 using Lip antibody, in which lanes: N O P R pAHE2 + pAHE10,42°C 1.5 hrs; Fig. 11 illustrates the cellular localization of UmA in P. cepacia as determined by an immunoblot analysis of lipase (Fig. 11a) and Lim (Fig. 11b) induced with oleyl alcohol in intracellular (cytoplasm and inner cell membrane), periplasmic and extracellular fractions of P. cepacia (Example 7). Equal amounts of protein were loaded in each lane corresponding to 0.01, 17 and 21%, respectively. In (a) (Lipase immunoblot) the lanes contained: A purified lipase from P. cepacia B intracellular fraction C periplasmic fraction D extracellular fraction, in (b) (Urn immunoblot) the lanes contained: A lim expressed from pJ38 B intracellular fraction C periplasmic fraction D extracelluar fraction; Fig. 12 illustrates the plasmid pAHE19; Fig. 13 illustrates the plasmid pAHE22; Fig. 14 illustrates the plasmid pAHE16; Fig. 15 illustrates the plasmid pAHE23; and Fig. 16 illustrates the plasmid pCBF1-6. The present invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed. MATERIALS AND METHODS Bacterial Strains E. coli TG1 supE hsd-5 thi-(lac-proAB) F'[traD36proAB+laclq lacZ-M15) (Gibson, 1984) E. coli JA221 (Clarke and Carbon, 1978) E. coli BL21 (DE3) B strain lysogen, placUV5-T7 RNApol (IPTG inducible) (Studier, 1990) Plasmids pSJ150 - Jorgensen et al. (1991) pJW2 - Wang et al. (1990) pET3a - Rosenberg et al. (1987) pT7-7 - Obtained from Stan Tabor, Dept. of Biol. Chem., Harvard Medical School pLysE and pLysS - pACYC184::T7 lysozyme from ptet and opposite orientation (Studier, 1990). General Methods Standard DNA manipulations were performed essentially as described in Sambrook et al. (1989). Restriction enzymes, T4 DNA ' ligase, DNA Polymerase I (Klenow fragment) were obtained from Boehringer Mannheim or Promega and used as recommended by the supplier. Chicken egg white lysozyme was obtained from Sigma. Preparation of plasmids and transformation of E. coli was carried out as described in Sambrook et al. (1989). SDS-polyacrylamide gels were prepared, electrophoresed and stained as describe in Sambrook et al. (1989). Protein molecular weight markers were purchased from Sigma. ϋ ' pase analysis Upase activity was detected on plates containing either glycerol tributyrate or an olive oil emulsion and brilliant green. Upase activity was measured by a pH-stat method using glycerol tributyrate as substrate. 1 LU (lipase unit) is the amount of enzyme which liberates 1 umole titratable butyric acid per minute under the following conditions: A lipase screening assay was performed in microtiter dishes according to the following procedure: First an emulsification reagent is made up: 17.9 g NaCI + 0.41 g KH 2 P0 4 + 6.0 g Gum Arabic + 540 ml glycerol is brought to a final volume of 1000 ml with demineralized water. The lipase assay reagent is made up as follows: 12.5 ml of the above emulsification reagent + 3.75 ml of glycerol tributyrate + 0.25 ml Brilliant green (40 mg/ml in H 2 0) + 50 ml of 10 mM Tris pH 9.0 is emulsified for 1 min in an Ultra Turrax emulsifier. The actual assay is performed by mixing 100 μl of this assay reagent with 100 μ\ o the lipase sample. The color development is compared to that of a known lipas sample. The brilliant green plates consisted of 15 ml LB agar, with a top layer of 3 ml LB aga containing 0.3 ml of a 10% olive oil emulsion and 0J ml of a 40 mg/ml solution o brilliant green in distilled water. The olive oil emulsion was olive oil 10 ml + Gu Arabic 1 g + deionised water 90 ml, mixed using an Ultra Turrax emulsifier. Induction of E. coli cultures for high level expression (a) pJW2 based plasmids Overnight cultures were grown at 30°C in an orbital shaker at 250 rpm, diluted 1 :10 and grown for 3-4 hrs at 30°C until an A600 of 0.5 was reached. One of a set o duplicate cultures was then shifted to 42°C for induction of protein synthesis Samples from both cultures were taken at time = 0, 30, 60, 90 and 120 minutes afte induction. (b) pET3a/oT7-7 based plasmids Overnight cultures were grown at 37°C in an orbital shaker at 250 rpm, diluted 1 :50 and grown at 37°C until an A600 of 0.5 was reached. IPTG was then added to a final concentration of 1.5 mM to one of a set of duplicate cultures. Rifampicin may also be added to the induced culture 15 min after IPTG addition to a final concentration of 100 μg/ml. Samples from both cultures were taken at time = 0, 30, 60, 90 and 120 minutes after induction. Treatment and preparation of protein samples Samples were immediately cooled to 0°C. Cells were harvested by centrifugation in a microfuge at 12,000 g for 5 min at 4°C. Cell pellets were resuspended in Laemmli sample buffer and frozen at -20°C. Proteins were precipitated from the supernatants by addition of an equal volume of acetone and leaving on ice for 30 min followed by centrifugation for 15 min at 12,000 g at 4°C. The precipitated proteins were resuspended in Laemmli sample buffer and frozen at -20°C. Analysis of proteins by SDS-poiyacrylamide gel electrophoresis Protein samples were boiled for 5 min prior to loading on 12% SDS-polyacrylamid gels. The gels were electrophoresed in Tris-glycine until the dye front reached th gel end and were then stained in 0.25% coomassie brilliant blue and destained i 5 10% acetic acid. Western analysis The method used was equivalent to that of Towbin et al. (1979). Protein sample were electrophoresed in 12% SDS-polyacrylamide gels and electrophoretically transf erred to nitrocellulose. Filters were preincubated with blocking buffer and then in cubated with anti-lipase antibody. Alkaline phosphatase-conjugated goatanti-rabbi IgG (Sigma) was then incubated with the filter. NitroBlue Tetrazolium an 5-bromo-4-chloro-indolyl phosphate (both from Sigma) were used to visualize th proteins. Method for Cell Lysis: adapted from Marston (1987) Overnight cultures were diluted 1:100 into 100 mis LB medium and grown to an OD600 value of 0.5. Induction with heat in the case of pAHE2 and pAHEIO and IPTG in the case of pCBE6 was carried out for 2 hours. The cultures were then centrifuged at 500 g for 15 minutes at 4°C. The supernatant was removed and the pellet was weighed. For each gram (wet weight) of E coli cells. 3 ml of lysis buffer was added and the pellet was resuspended. Lysis Buffer contained 50 mM Tris.CI, 1mM EDTA, lOO mM NaCI. For each gram of cells, 8 ml of a 50 mM PMSF stock and 80 μl of lysozyme (1 Omg/- ml) was added and stirred for 20 minutes. 4 mg of deoxycholic acid was added per gram of cells while stirring continuously. The lysate was placed at 37°C and stirred with a glass rod. When the lysate became viscous 20 μl of DNAasel (1mg/ml) was added per gram of cells. The lysate was placed at room temperature until it was no longer viscous (approx 30 minutes). The cell lysate was then stored at 4°C until re¬ quired. Purification and washing of inclusion bodies: (Marston et al 1984) The cell lysate was centrifuged at 12000 g for 15 minutes at 4°C. The supernatant was decanted and the pellet was resuspended in 9 volumes of lysis buffer containing 0.5% Triton and 10mM EDTA(pH8.0). Storage at room temperature for 5 minutes was followed by centrifugation at 12000 g for 15 minutes at 4°C. The supernatant was decanted and set aside and the pellet was resuspended in 100 μl of H 2 0. 10 μl samples were removed from the supernatant and the resuspended pellet and were mixed with 10 μl of 2X SDS gel loading buffer and analyzed by SDS-polyacry¬ lamide gel electrophoresis to determine if most of the protein of interest is in the pellet. The lipase screening assay was also carried out to determine if most of the lipase activity of pAHE2 was in the pellet fraction. Solubilization of inclusion bodies and renaturation: (adapted from Marston et al., (1984)) 100 l of lysis buffer containing 0J mM PMSF (added fresh), 8M Urea (deionized), and 0J M beta-mercaptoethanol was added to the washed pellet and stored for 1 hour at room temperature. The lipase activity screening assay was carried out in order to observe whether the lipase activity had disappeared due to the protein being totally denatured. 50 μl of each of the denatured protein samples were placed in dialysis tubing. The protein samples were dialysed in a buffer of 50 mM KH2P04 (pH10.7),1 mM EDTA (pH8.0), 50 mM NaCI and which also contained 8 M urea and 0J M BME. The pH was maintained at 10.7 with KOH. Initial dialysis in 8 M urea was carried out overnight. Further dialyses using a lower concentration of urea (ie 6 M; 4 M; 2 M; 0 M) were carried out in the above buffer in the absence of BME. The pH in these dialysis reactions was maintained at 8.0 using HCI. Dialysis in each of the different concentrations of urea was allowed to take place for 6 hours. As a control experiment duplicate samples were dialysed exactly as above except in the absence of urea and BME. The renatured samples were removed from the dialysis tubing and lipase activity of the samples was examined on glycerol tributyrate and brilliant green plates. The lipase activity was also examined using the microtitre lipase screening assay. Recovery of proteins from SDS-polyacrylamide gels The procedure for recovering proteins from SDS-polyacrylamide gels was describe by Hager and Burgess (1980). Protein samples were electrophoresed on a SDS-polyacrylamide gel. After electrophoresis, the gel was removed into a tray rinsed with water and stained for 10 minutes with ice-cold 250 mM KCl and 1 m DTT. The gel was destained in cold water containing 1 mM DTT and the protein band of interest was cut out. (A portion of the gel was stained with coomassie blue to stain molecular weight standards to ensure the correct band was cut out). The gel band was crushed through a 1 ml syringe with G18 needle and 1 ml of elutϊon buffer containing 0.1% SDS, 50 mM Tris/HCl (pH7.9), 0J mM EDTA, 5 mM DTT, and 200 mM NaCI, was added. The protein was allowed to elute for at least 1 hour at 25°C with occasional agitation. The mixture was centrifuged briefly to pellet the crumbled gel. The protein was precipitated from the supernatant by adding 4 volumes of cold acetone and incubating at -70°C for 20 min. The precipitated protein was centrifuged and the pellet was allowed to dry. Generation of anti-U ' mA anti-sera in rabbits In order to raise antibodies against LimA, three rabbits were injected with purified LimA protein according to a schedule similar to that described by Vaitukaitis, (1981). The LimA protein was purified from preparative SDS-polyacrylamide gels. On day 1 , Urn in 10 mM sodium phosphate, pH 7.2 was mixed with complete Freunds adjuvant and injected intradermally at 10 sites in the back of each rabbit. On day 21 a booster injection, mixed with incomplete Freunds adjuvant, was injected intramuscularly into the leg of each rabbit. An identical booster was injected in the same way on day 31 of the schedule. A test bleed revealed that anti-Urn antibodies had been generated. Media LB per litre : 10 g bacto-tryptone 5 g bacto-yeast extract 10 g NaCI Plates contained 2% agar. EXAMPLES EXAMPLE 1 Constructions for high level UpA expression (a) lipA + limA : PAHE2 and PAHE8 Plasmid pAHE2 (Fig. 1) was constructed by subcloning the 2.264 kb Nsil fragment encoding both lipA+limA from pSJ150 (Jorgensen et al.,1991) into Ndel-digested pJW2 (Wang et al., 1990). Ligations were transformed into E. coli TG1 at 30°C and plasmid DNA mini-preparations were used to identify plasmids with insert in the correct orientation. pAHE2 is one of a few correct constructs obtained. In pAHE2 the initiation codon of the lipase gene is immediately downstream from the phage T7 ribosome binding site. E57 is E. coli strain JA221 transformed with pAHE2. When E57 is induced for high level protein production, lipase protein can be identified on SDS-polyacrylamide gels (and by western analysis) and lipase activity can be detected on brilliant green and glycerol tributyrate plates and by the microtitre assay and by pH-stat method. Plasmid pAHE8 (Fig. 2) was constructed by subcloning the 2.264kb Ndel fragment encoding lipA + limA from pAHE2 into the expression vector pET3a digested with Ndel. Ugations were transformed into E coli strain TG1 and plasmid DNA wa prepared from transformants to identify the correct plasmids. E73 is E coli strain BL21 (DE3) pLysS transformed with pAHE8. When E73 i induced for high level protein production, lipase protein (approx. MW 34kD) can b seen on SDS-polyacrylamide gels and lipase activity is detected on brilliant green and glycerol tributyrate plates and by the microtitre assay. (b) lipA without limA : pAHEIO Plasmid pAHEIO (Fig. 3) is a derivative of pAHE2 from which 2/3rds of the coding region of the limA gene was deleted. Plasmid pAHE10 was constructed by digesting pAHE2 with the restriction enzymes Clal and Notl, followed by treatment with Mung Bean Nuclease. DNA of the size 6.3 kb was excised as a band from an agarose gel stained with ethidium bromide. The DNA was purified using a Gene Clean kit, ligated and transformed into competent E. coli TG1. Plasmid DNA mini-preparations were used to identify the correct clones. E68 is E. coli JA221 transformed with pAHEIO. When E68 is induced for high level protein production, lipase protein, in comparable quantities to that from pAHE2, can be seen on SDS-polyacrylamide gels and by western analysis, but no lipase activity can be detected. EXAMPLE 2 Constructions for high level UmA expression: pCBE6, pCBE7, pCBEl 2, pCBEl 8 and pCBE19 Plasmid pCBE6 (Fig. 4), from which limA alone is expressed, was constructed by subcloning the 1.17 kb Clal-Sphl fragment from pSJ150 as a blunt-ended fragment into the expression vector pET3a digested with Ndel and treated with Klenow to generate blunt ends. E. coli strain E102 is strain BL21 (DE3)pLysS transformed with ' pCBE6. Plasmid pCBE7 (Fig. 5) was constructed by subcloning the same 1.2 kb Clal-Sphl fragment into the expression vector pT7-7. E103 is BL21 (DE3) pLysS transformed with pCBE7. Upon induction of E102 and E103, a protein of molecular weight approx. 32 kD was observed. Plasmid pCBE12 (Fig. 6) was constructed by subcloning the Bglll-BamHI fragment of pCBE7 into pACYC177. Lim expressed from pCBE12 in trans to lipase expressed from pSJ518 (described in Fig. 3 of WO 90/00908) and pAHEIO gives rise to lipase activity. Plasmids pCBE18 and 19 (Fig. 7 and 8) were constructed by subcloning the Bglll-BamHI fragment of pCBE7 into the Bell site of pLysS. Lim expressed from pCBE18 and 19 in trans to lipase from pAHEIO gives rise to lipase activity. EXAMPLE 3 Growth/induction experiments to obtain high level protein expression Cultures were induced to produce high levels of both LipA and LimA as follows. E coll strains E57 (JA221/pAHE2) and E68 (JA221 /pAHEIO) were grown overnight at 30°C at 250 rpm. The cultures were diluted 1 :100 and grown at 30°C at 250 rpm until the A600 was 0.5. The cultures were then shifted to grow at 42°C. E. coli strain E102 (BL21 (DE3)pLysS/ pCBE6) was grown overnight at 37°C, diluted 1 :100 and grown until A600 was 0.5. IPTG was then added to the culture to a final concentration of 1.5mM. Fifteen minutes later rifampicin was added to a final concentration of 100 μg/ml. The culture was incubated at 37°C and shaken at 250 rpm throughout the growth. Samples were taken from each culture at 60, 90 and 120 minutes after induction. Samples were analysed by SDS-polyacrylamide gel electrophoresis and by western analysis using anti-serum raised against lipase, see Fig. 9 and 10. Lipase protein was observed to be induced in strains containing either pAHE2 or pAHEIO, in equivalent amounts. The lipase for the majority about 95% is larger than the matur lipase isolated from Pseudomonas cepacia and the size is consistent with tha expected for the complete prelipase. Samples were also assayed for lipase activity by the microtitre assay. Lipase activity was observed from cultures of E57 (pAHE2, 5 lipA + limA), but none was observed from cultures of E68 (pAHEIO, lipA only) or E102 (pCBE6, limA). T e results demonstrate that lipase protein is made in equivalent amounts in the presence and in the absence of the limA gene, but no lipase activity is detected in the absence of limA. Active lipase is only produced when the limA gene is also present. EXAMPLE 4 Purification of Up and Urn Overnight cultures of JA221/pAHE2; JA221 /pAHEIO; BL21/pLysS/pCBE6 and BL21 were diluted 1:100 into 100 mis LB medium and grown to an OD600 value of 0.5. Induction with heat in the case of pAHE2 and pAHEIO and IPTG in the case of pCBE6 was carried out for 2 hours. The cultures were then centrifuged at 500 g for 15 minutes at 4°C. Cells were lysed by the method described. The Lim protein was found to be in the soluble fraction of the cell lysate. The lipase was found to reside in inclusion bodies. Inclusion bodies were prepared by a method adapted from Marston et al. (1984). Pure lipase and Lim proteins were prepared after SDS-PAGE of inclusion bodies and soluble lysate fractions, respectively, by a method described by Hager and Burgess (1980). Samples were separated by 12% SDS-PAGE. After electrophoresis, the gel was soaked in transfer buffer (10 mM CAPS, 10% methanol, pH11) for 20 min. The gel was electroblotted onto Problott which had been pretreated in 100% methanol (5-10 sec) and destilled water (2 x 1 min) and then soaked in transfer buffer. Eiectroblotting was carried out at 200 mA for 3 hours in transfer buffer at room temperature. The blot was removed from the sandwich, rinsed in destilled water an then in methanol (5-10 sec). The blot was then stained in Amido Black (0.1% w/v in 1% (v/v) acetic acid, 40% (v/v) methanol for 1 min. The blot was rinsed in frequen changes af destilled water, air dried and stored at -20°C between layers of filte paper. The bands corresponding to LimA and prelipase were excised an sequenced directly on an Applied Biosystems 477A protein sequencer. The following sequences were obtained: LimA (pCBE6): TARGGRAPL-RRAWYGAVG (SEQ ID NO 6) preLipA (pAHE2) : ARTMRSRWAGAVA-AMSIA (SEQ ID NO 7) preLipA (pAHEIO): ARTMRSRWAGAV--AM-IA (SEQ ID NO 8) Except for the fact that the N-terminal methionine has been cleaved off, th sequences are as expected for LimA and for preLipA as deduced from the DN sequence. Gel-purified LimA protein was used to raise polyclonal antibodies as described in the methods section. The purity of the proteins was checked by further SDS-PAGE analysis. EXAMPLE 5 Denaturation and renaturation of pAHE2 and pAHEIO UpA Denaturation/renaturation experiments were carried out with UpA from either pAHE2 or pAHEIO (present in inclusion bodies) together with LimA (pCBE6 and present in the cell lysate). 60 μl of inclusion bodies was mixed with varying volumes of pCBE6 cell lysate (0 - 30 μl) and solubiiized together in 8 M urea. Renaturation was carried out by dialysis against decreasing concentrations of urea. Experiments were carried out in the presence and absence of 5% glycerol. The lipase activity was measured using the pH stat. The results of this experiment (shown below) indicate that increasing percentages of lipase activity may be recovered by denaturing/renaturing in the presence of increasing quantities of LimA, and that the presence of glycerol does not affect the recovery of lipase activity. Results for pAHE2 and pAHEIO LipA Inclusion bodies undiluted; LimA from lysate Initial lipase activity measured for pAHE2(pJW2::lipA/limA) =7.5 LU (volume 60 μl) Initial lipase activity measured for pAHE10(pJW2::lipA) =0 LU Denaturation and renaturation Sample No of LU No of LU No of LU denatured recovered recovered (-Glycerol) (+ Glycerol) EXAMPLE 6 Denaturation and renaturation of pAHE2 and pAHEIO UpA For this set of experiments the inclusion body fractions were diluted 1 :10. Samples of pAHE2 and pAHEIO (Lip) inclusion bodies and pCBE6 (Lim) cell lysate were analysed by SDS-polyacrylamide gel electrophoresis. The Biorad protein assay was used to quantify the amount of proteins used in the denaturation/renaturation experiments. 10 μl of pAHE2 inclusion bodies was determined to contain 10 μg of protein; 10 μl of pAHEIO inclusion bodies to contain 8 μg of protein; and 10 μl of pCBE6 lysate to contain 25 μg total protein. The results of this experiment (shown below) indicate that increasing the quantity of UmA in denaturation/renaturation reactions results in initially increasing recoveries of lipase activity. Addition of further quantities of LimA to the reactions is inhibitory. Results for pAHE2 and pAHEIO Upases Inclusion bodies diluted 1:10; LimA from lysate Sample dialysed No of lipase units recovered. pAHE2 10 μl 0.4 pAHE2 " +10μl LimA 5.6 pAHE2 " +15 uμ UmA 5.7 pAHE2 " +20 μl UmA 4.5 pAHE2 " +30 μl UmA 9.5 pAHE2 " +40 μl LimA 3.2 pAHEIO 10 μl 0.0 pAHEIO " +10μl UmA 2.5 pAHEIO " +15μl UmA 7.8 pAHEIO " +20 μl UmA 7.3 pAHEIO " +30 μl UmA 3.5 pAHEIO " +40 μI UmA 0.5 EXAMPLE 7 Upase and prelipase from Pseudomonas cepacia DSM 3959 P. cepacia DSM 3959 (the strain from which lipA and limA were cloned) was induced to produce lipase by growth in LB media supplemented with 80 g/l oleyl alcohol, and extracellular, periplasmic and intracellular fractions were isolated as described by Neu and Heppel (1965). Immunoblots (Fig. 11) and lipase activity analysis showed that the extracellular fraction contained only active, mature lipase, whereas the intracellular fraction contained only inactive prelipase. When LimA was provided as an extract of E coH BL21 (DE3) pLysS pCBE6, mature, extracellular lipase form P. cepacia could be reactivated quantitatively after 10 denaturation/renaturation only in the presence of LimA. No lipase activity was observed when intracellular prelipase was used in a similar experiment with or without LimA, as is apparent from the following table: Extracellular fraction (mature lipase) The activation of lipase but not prelipase from P ^ cepacia by LimA during renaturation experiments is consistent with the results using lipase proteins produced in E. coli. The E. coli lipase samples are composed of approximately 5% mature lipase and 95% prelipase. The amount of lipase activity observed before denaturation (pAHE2only) and after renaturation (ρAHE2and pAHEIO) is equivalent to approximately 5% of that expected from the total amount of lipase protein seen on SDS-PAGE. EXAMPLE 8 Constructions for expression of mature UpA protein Plasmids from which the lipase UpA was expressed in a form lacking the signal peptide, i.e. as mature lipase, were constructed. These are pAHE19 (Fig. 12), which contains a modified version of the lipA gene encoding the mature lipase followed by the limA gene, and pAHE22 (Fig. 13), which encodes the mature lipase without limA. They were constructed as follows: The following DNA fragment was synthesized (standard methods): <MluI><HindIII 5*-CGCGTAAGCTTCACATTGAAAGGGGAGGAGAATCATGGCC- 3'- ATTCGAAGTGTAACTTTCCCCTCCTCTTAGTACCGG- <MluI> GCTGGCTACGCGGCGA -3• (SEQ ID NO 9) CGACCGATGCGCCGCTGCGC-5' (SEQ ID NO 10) This DNA fragment basically ' contains the Bacillus licheniformis amyL ribosome binding site and start codon located in front of the sequence encoding the amino acids AAGYAA (SEQ ID NO 11) from the N-terminal of the mature LipA protein. This DNA fragment was ligated into Mlul digested pSJ420 (identical to pSJ416 described in WO 90/00908, and pSJ838 was isolated as a plasmid in which the Hindlll site in the synthetic DNA fragment was located proximal to the amyL promoter on pSJ420. pSJ838 carries the amyL promoter, amyL RBS, the amyL signal peptide fused to the first 6 codons of lipA, amyL RBS, mature lipA, and limA. By in vivo recombination from pSJ838 (essentially as described in Jørgensen et al., 1990) plasmid pSJ897 was obtained, which contains the amyL promoter, amyL RBS, and mature lipA followed by limA. immediately upstream from the amyL RBS on pSJ897 is the recognition sequence for restriction enzyme Ndel. pAHE19 was constructed by ligation of the 4.9 kb Ndel-EcoRI fragment of pJW2, the 0.46 kb Ndel-BamHI fragment of pSJ897, and the 2.07 kb BamHI-EcoRI fragment of pSJ150. pAHE22 was constructed from pAHE19 by Clal + Notl digestion and ligation following exonuclease S1 treatment to make ends blunt. Upon induction, lipase activity was observed from E. coli JA221 containing pAHE19, but not from E. coli JA221 containing pAHE22. EXAMPLE 9 Denaturation and renaturation of mature UpA with and without UmA Mature lipase was provided as the soluble fraction following induction, harvest, and lysis of cells as described in methods, and was used in a denaturation/renaturation experiment in the presence and absence of a LimA containing cell lysate, as de¬ scribed in methods. The results of this experiment (table below) shows that mature LipA produced in E. coli only in the presence of LimA can be renatured to give active lipase enzyme. pAHE19: mature UpA, LimA. pAHE22: mature UpA. pAHE19 (100 l) PAHE19 (100 μl) + UmA (10 μl) pAHE19 (100 μl) + UmA (20 μl) pAHE19 (100 μl) + UmA (30 μl) pAHE19 (100 μl) + non-LimA lysate (30 μl) pAHE22 (100 μl) pAHE22 (100 μl) + UmA (10 μl) pAHE22 (100 μl) + UmA (20 μl) pAHE22 (100 μl) + UmA (30 μl) pAHE22 (100 μl) + non-UmA lysate (30 μl) EXAMPLE 10 Cloning and sequence of lipD and iimD from P. cepacia DSM 3401 Another isolate of P. cepacia. DSM 3401 also called strain 75-10A, produce a lipase with similarity to LipA from DSM 3959. Cloning and sequencing of the lipase encoding DNA from DSM 3401 (using standard methods as described in WO 90/00908) revealed two genes, hereafter referred to as lipD and IimD with a relatively high homology to lipA and limA. Due to the extreme GC content of the DNA, the sequence was difficult to determine, and there still remains some undetermined basepairs - at 4 positions in the lipD gene, and at two positions in the IimD gene. The IimD start codon is positioned three basepairs downstream from the lipD stop codon, as is the case for lipA and limA. The DNA sequence encoding lipD is given in SEQ ID NO 1 , and the corresponding protein sequence in SEQ ID NO 3. The DNA sequence encoding IimD is given in SEQ ID NO 2, and the corresponding protein sequence in SEQ ID NO 4. From an alignment study of the amino acid sequences of LipA and LipD it appeared that there are 22 differences in the mature part of the enzyme, in addition to 5 positions where the LipD aminoacid sequence could not be deduced. From an alignment study of the amino acid sequences of LimA and UmD it appeared that there are 32 differences, in addition to 2 positions where the LimD amino acid sequence could not be deduced. Based on these studies it is clear that LipD and LipA as well as LimD and LimA are homologous, but still different lipase and lipase modulator proteins. It was therefore of interest to see if LimA could activate LipD in a denaturation/renaturation experiment. EXAMPLE 11 Denaturation/renaturation experiments with UpD purified from P. cepacia LipD from strain 7510-A (=DSM3401) was provided as a partially purified protein from DSM3401 , using standard protein purification methods. Denaturation/renaturation experiments were carried out using the lipase from strai 7510-A in the presence and absence of LimA. 18 LU were denatured and renature with varying ratios of LimA and the recovered lipase activity was measured. Upas activity was measured on the pH-stat. As control, the experiments were also carrie 5 out in the absence of urea. The results obtained show that when the 7510-A lipase was denatured and renatured in the absence of LimA effectively no lipase activity was recovered. When increasing levels of LimA were added to the 7510-A lipase increasing levels of lipase activity were observed. However, the percentages of lipase activity recovered were 10 still very low. Results for 7510-A lipase 13,000 LU/ml stock used; LimA from lysate Sample dialysed No of LU denatured 15 75 0A 15μI 18 7510A " + 5 μI LimA 18 7510A " +10μl UmA 18 7510A " +15 il LimA 18 20 7510A " +30 μl UmA 18 An explanation for the low recovery of lipase activity was later found. It turned out that the lipase in this particular preparation was degraded during denaturation/renaturation, so the experiment was repeated with a new, partly purified 5 preparation of lipase from DSM3401 and LimA from induced BL21 (DE3) pLysS pCBE6. The following results show that UmA is well able to activate UpD. LipD (10 μl) + LipD (10 μl) + 5 μl LimA LipD (10 μl) + 10 μl LimA LipD (10 μl) + 20 μl LimA LipD (10 μl) + 30 μl LimA LipD (10 μl) + 40 μl LimA EXAMPLE 12 Constructions for UpD expression The lipA sequence contains a Mlul site at a position corresponding to 7 amino acids into the mature LipA sequence and a Clal site following the lipA stop codon. The lipD sequence contains Mlul and Clal sites at the equivalent positions. Therefore, plasmids could easily be constructed based on the former lipA expression vectors by insertion of the lipD Mlul-Clal fragment in stead of the lipA Mlul-Clal fragment to allow expression of a hybrid protein, in which only the first 7 amino acids in the mature protein are from LipA, whereas the rest of the mature protein is from LipD. pAHE16 (Fig. 14) expresses the LipA-LipD hybrid lipase together with LimA, and was constructed from pAHE2 by replacement of the Mlul-Clal fragment as described above. pAHE23 (Fig. 15) expresses only the LipA-LipD hybrid lipase, and no Lim protein. It was constructed from pAHE16 by deletion of the Clal-Notl fragment of LimA. EXAMPLE 13 Denaturation/renaturation of the iipA-UpD hybrid lipase The UpA-UpD hybrid protein expressed from pAHE16 and pAHE23 was used in a denaturation/renaturation experiment with and without LimA added as described in methods. The lipase sample used in this experiment was the soluble fraction following lysis of the induced E. coli JA221 cells containing either pAHE16 or pAHE23 (although the vast majority of the lipase protein upon induction resides in inclusion bodies, the soluble fraction still contains some lipase, and there seem to be relatively more mature lipase in the soluble fraction than in the inclusion bodies). UmA was obtained from an induced culture of BL21 (DE3) pLysS pCBE6. The results presented below show that LimA is able to activate the LipA-UpD hybrid lipase in a denaturation/renaturation experiment. LU before LU after denaturation renaturation pAHE16 (30 μl) + 0.5 0.5 pAHE16 (30 μl) + LimA (10 μl) 0.5 0.75 pAHE16 (30 μl) + LimA (20 μl) 0.5 0.75 pAHE16 (30 μl) + LimA (30 μl) 0.5 0.75 pAHE16 (30 μl) + UmA (40 μl) 0.5 1.0 pAHE16 (30 μl) + non-UmA cell lysate 0.5 0.5 (40 μl) pAHE23 (30 μl) + 0.0 0.0 pAHE23 (30 μl) + LimA (10 μl) 0.0 0.75 pAHE23 (30 μl) + UmA (20 μl) 0.0 0.75 pAHE23 (30 μl) + LimA (30 μl) 0.0 1.0 pAHE23 (30 μl) + LimA (40 μl) 0.0 1.0 pAHE23 (30 μl) + non-LimA cell lysate 0.0 0.0 (40 μl) EXAMPLE 14 Construction of lip-lim fusion Plasmid pSJ721 is a deletion derivative of pSJ377 in which 15 bp have been deleted at the Clal site, deleting the lipA stop codon, and the codons for the first three amino acids of lim (Jørgensen et al., 1991). pSJ721 thus encodes a Lim protein which is fused to the eight amino acids following the SphI site at the C-terminal of lipA. The 838 bp Sphl-Notl fragment of pSJ721 (which includes the C-terminal of lip fused to most of lim) and the 915 bp Mlul-Sphl fragment of pSJ150 (which includes most of the lipase coding sequence) were ligated with pAHEδ digested with Mlul and Notl, such that a lip-lim fusion plasmid was generated. Transformants were screened for loss of the Clal site which is normally present at the beginning of the limA gene and which is absent in pSJ721 , and should be absent in lip-lim fusion plasmids. A number of plasmids were deemed to be correct from restriction enzyme analysis. Six of these were named pCBF1-6 (Fig. 16). E. coli BL21 (DE3) pLysS was transformed with each of the fusion plasmids and transformants were grown and induced with IPTG. Plating on tributyrine plates and analysis of activity in broth samples revealed that these plasmids gave rise to lipase activity upon induction. Protein samples from both cells and broth were analysed on SDS-polyacrylamide gels and by western analysis using anti-lipase anti-serum. Only a lipase protein identical in size to the unprocessed lipase produced by pAHE2 or pAHEδ was detected. No proteins of the higher molecular weight expected were observed, even in samples taken early in time course induction experiments. Per¬ haps the artificial lip-lim fusion is particularly susceptible to proteolytic cleavage at the junction of the proteins. References Clarke.L and Carbon, J. (1978) J.MoLBiol. 120, 517-534. Ellis, R . & van der Vies, S.M. (1991), Annual Review of Biochemistry 60, 321-347. Hager.DA and Burgess.R.R. (1980) Elution of proteins from sodium dodecyl sul- phate-polyacrylamide gels, removal of sodium dodecyl sulphate, and renaturation of enzymatic activity: results with sigma subunit of Escheria coli RNA polymerase, wheat germ DNA topoisomerase and other enzymes. Anal. Biochem. 109, 76 -86. Jørgensen.S., Skov, K.W. and Diderichsen.B. (1991) Cloning, sequence and expression of a lipase gene from Pseudomonas cepacia: lipase production in heterologous hosts requires two Pseudomomnas genes. J.Bacteriol. 173, 559-567. Marston, F.A.O. (1937) The purification of eukaryotic polypeptides expressed in E. coli. In DNA Cloning: A practical approach (ed D.M. Glover), vol.3, p.59. IRL Press, Oxford. Marston, F.A.O., P.A. Lowe, M.T. Doel, J.M Schoemaker. 1934. Purification of calf prochymosin synthetized in E. coli. BioTechnology 2:800. RosenbergAH., Chui.D.S., Uu,S.-W., Dunn.J.J., and Studier.F.W. (1987) Vectors for selective expression of cloned DNAs by T7 RNA Polymerase. Gene 56 125-135. Sambrook ,J., Fritsch.E.F. and Maniatis.T. (19δ9) Molecular Cloning: a laboratory manual. 2nd edition Vaϊtukaitis. (1981) Methods in Enzymology 21, 46-52 Wang,H., McConnell,D.J.andO.Mahony,D.J. (1990)Anefficienttemperature-inducibl vector incorporating the T7 gene 10 translation initiation leader region. Nucleic Acid Res. 1δ, 1070. Neu, H.C., Heppel, LA (1965), J.Biol.Chem. 240, 3685-3692. Jørgensen, P.L, Hansen, C.K. Poulsen, G.B., Diderichsen, B. (1990), Gene, 96, 37- 41. S. Aoyama et al., FEBS Letters 242 (1), December 1986, pp. 36-40 W. Kugimiya et al.. Biochem. Biophvs. Res. Comm. 141 (1). November 26, 1986, pp. 185-190 R.J. Ellis and S.M. Hemmingsen, Trends Biochem. Sci. 14. 1989, pp. 339-342; J.E. Rothman, CeH 59, 1989, pp. 591-601 R. Morimoto et al. in Stress Proteins in Biology and Medicine. R. Morimoto et al., eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1990, pp. 1-36) Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, 1989 S.L Beaucage and M.H. Caruthers, Tetrahedron Letters 22. 1981 , pp. 1859-1669, Matthes et al., EMBO Journal 3. 1984, pp. 801 -δ05 In the Sequence Usting below SEQ ID NO 1 is the nucleotide sequence of lipD; SEQ ID NO 2 is the nucleotide sequence of IimD; SEQ ID NO 3 is the amino acid sequence of UpD. The sequence is that of the prelipase, the first amino acid residue of the mature lipase is A (amino acid residue 45). SEQ ID NO 4 is the amino acid sequence of IimD; SEQ ID NO 5 is the nucleotide sequence of the lip-lim fusion gene; SEQ ID NO 6-8 are the peptide fragments discussed in Example 4; and SEQ ID NO 9-11 are the nucleotide and amino acid sequences shown in Example 8 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: NOVO NORDISK A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DENMARK (F) POSTAL CODE (ZIP): DK-2880 (G) TELEPHONE: +4544448888 (H) TELEFAX: +4544493256 (I) TELEX: 37304 (ii) TITLE OF INVENTION: A process for the preparation of an active lipase (iii) NUMBER OF SEQUENCES: 11 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: WO PCT/DK91/00402 (B) FILING DATE: 20-DEC-1991 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1092 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM 3401 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATGGCCAGAT CGATGCGTTC CAGGGTGGTG GCAGGGGCAG TGGCATGCGC GATGAGCGTC 60 GCGCCGTTCG CGGGGGCGAC CGCGGTGATG ACGCTTGCGA CGACGCACGC GGCGATGGCG 120 GCGACCGCGC CCGCCGACGA CTACGCGACG ACGCGTTATC CGATCATCCT CGTGCACGGG 180 CTCACGGGTA CCGACAAGTA CGCGGGCGTG CTCGAGTACT GGTACGGCAT CCAGGAAGAC 240 CTGCAGCAGC ATGGCGCGAC CGTCTACGTC GCGAACCTGT CGGGCTTCCA GAGCGACGAC 300 GGGCCGAACG GGCGCGGCGA ACAGTTGCTC GCGTACGTGA AGACGGTGCT CGCGGCGACG 360 GGCGCGACCA AGGTCAATCT CGTCGGCCAC NCGCAGGGCG GGCTCACGTC GCGTTACGTT 420 GCGGCTGTCG CGCCCGATCT CGTCGCGTCG GTGACGACGA TCGGCACGCC GCATCGTGCN 480 NCCGAGTTCG CCGACTTCGT GCAGGGCGTG CTCGCATACG ATCCGACCGG GCTTTCGTCA 540 TCGGTGATCG CGGCGTTCGT CAATGTGTTC GGAATCCTGA CGAGCAGCAG CCACAACACG 600 AACCAGGACG CACTCGCGTC GCTGAAGACG CTGACGACCG CCCAGGCCGC CGCGTACAAC 660 CAGAACTATC CGAGCGCGGG CCTCGGTGCG CCGGGCAGTT GCCAGACCGG CNNNCCGACG 720 GAAACCGTGC GGTNCAACAC GCATCTGCTG TATTCGTGGG CCGGCACGGC GATCCAGCCG 780 ACGCTCTCCG TGTTCGGTGT CACGGGCGCG ACGGACACGA GCACCATTCC GCTCGTCGAT 840 CCGGCGAACG CGCTCGACCC GTCGACGCTT GCGCTGTTCG GCACGGGCAC GGTGATGATC 900 AACCGCGGCT CGGGCCCGAA CGACGGGCTC GTATCGAAGT GCAGCGCGCT GTACGGCCAG 960 GTGCTGAGCA CGAGCTACAA GTGGAACCAT ATCGACGAGA TCAACCAGTT GCTCGGCGTG 1020 CGCGGCGCGA ATGCGGAAGA TCCCGTCGCG GTGATCCGCA CGCATGCGAA CCGGCTGAAG 1080 CTGGCGGGCG TG 1092 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1032 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: l near (ii) MOLECULE TYPE: DNA (genomic) (ii ) HYPOTHETICAL: NO (i i) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM 3401 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGGCGGCAC GTGAAGGGCG CGCGCCGCTG GCGCGGCGCG CTGCAGTCTA CGGTGTCGTG 60 GGGCTGGCGG CGATCGCCGG CGTCGCGATG TGGAGCGGGG CGGGATGGCA TCGCGGTACG 120 GGTAGCGTCG GCGAAGCGCC CGATGCGGCG GCAGTGGGCG GCGTGGCTGC GGCACCGCCG 180 CAGGCGGCCG TGCCGGCGAG CGCGGGCCTG CCGTCGTCGC TGGCCGGCTC CAGCGCGCCG 240 CGCGTGCCGC TCGATGCGGG CGGCCATCTC GCGAAGGTGC GCGCGGTGCG CGATTTCTTC 300 GACTACTGCC TGACCGCGCA GAGCGACCTC AGTGCGGCCG CGCTCGATGC ACTCGTCGTG 360 CGCGAGATTG CCGCGCAGCT CGACGGCACG GCGGCGCAGG CCGAGGCGCT CGACGTGTGG 420 CATCGCTATC GTGCGTATCT CGACGCGCTC GCGAAACTGC GCGATGCCGG CGCGGTCGAC 480 AAGTCCGACC TGGGCGCGCT GCAGCTCGCG CTCGACCAGC GCGCATCGAT CGCGTATCGC 540 ACGCTCGGCG ACTGGAGCCA GCCGTTCTTC GGCGCGGAGC AGTGGCGGCA GCGCTACGAT 600 CTCGCGCGGC TGAAGATCGC GCAGGATCGC ACGCTGACCG ATGCGCAGAA GGCCGAACGG 660 CTCGCGGCGC TGCAGCAACA GATGCCGGCC GACGAACGCG CGGCTCAGCA GGCGGTCGAC 720 CGGCAGCGGG CCGCGATCGA CCAGAGTCCG NAGTTGCAGA AGAGCGGGAC GACGCCCGAT 780 GCGATGCGCG CGCAACTGAC GCAGACGCTC GGGCCCGAGG CCGCCGCGCG CGTCGGCCAG 840 ATGCAGCAGG ACGACGCATC GTGGCAGAGN CGCTACGCGG ACTATGCGGC GCAGCGCGCG 900 CAGATCGAGT CGGCCGGCCT GTCGCCGCAG GGCCGCGACG CGCAGATCGC CGCACTGCGG 960 CAGCGCGTGT TCACGAAGCC CGGCGAAGCC GTGCGCGCGG CGTCGCTCGA TCGCGGGGCG 1020 GGCAGCGCGC AG 1032 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 364 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM 3401 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Ala Arg Ser Met Arg Ser Arg Val Val Ala Gly Al a Val Ala Cys 1 5 10 15 Al Me Ser Val Ala Pro Phe Ala Gly Ala Thr Ala Val Met Thr Leu 20 25 30 Ala Thr Thr His Ala Ala Met Ala Ala Thr Ala Pro Ala Asp Asp Tyr 35 40 45 Ala Thr Thr Arg Tyr Pro He He Leu Val His Gly Leu Thr Gly Thr 50 55 60 Asp Lys Tyr Ala Gly Val Leu Glu Tyr Trp Tyr Gly He Gin Glu Asp 65 70 75 80 Leu Gin Gin His Gly Ala Thr Val Tyr Val Ala Asn Leu Ser Gly Phe 85 90 95 Gin Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gin Leu Leu Ala Tyr 100 105 110 Val Lys Thr Val Leu Al Ala Thr Gly Ala Thr Lys Val Asn Leu Val 115 120 125 Gly His Xaa Gin Gly Gly Leu Thr Ser Arg Tyr Val Ala Ala Val Ala 130 135 140 Pro Asp Leu Val Ala Ser Val Thr Thr He Gly Thr Pro His Arg Xaa 145 150 155 160 Xaa Glu Phe Ala Asp Phe Val Gin Gly Val Leu Al a Tyr Asp Pro Thr 165 170 175 Gly Leu Ser Ser Ser Val He Ala Ala Phe Val Asn Val Phe Gly He 180 185 190 Leu Thr Ser Ser Ser His Asn Thr Asn Gin Asp Ala Leu Ala Ser Leu 195 200 205 Lys Thr Leu Thr Thr Ala Gin Ala Ala Ala Tyr Asn Gin Asn Tyr Pro 210 215 220 Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gin Thr Gly Xaa Pro Thr 225 230 235 240 Glu Thr Val Arg Xaa Asn Thr His Leu Leu Tyr Ser Trp Ala Gly Thr 245 250 255 Ala He Gin Pro Thr Leu Ser Val Phe Gly Val Thr Gly Ala Thr Asp 260 265 270 Thr Ser Thr He Pro Leu Val Asp Pro Ala Asn Ala Leu Asp Pro Ser 275 280 285 Thr Leu Ala Leu Phe Gly Thr Gly Thr Val Met He Asn Arg Gly Ser 290 295 300 Gly Pro Asn Asp Gly Leu Val Ser Lys Cys Ser Ala Leu Tyr Gly Gin 305 310 315 320 Val Leu S&r Thr Ser Tyr Lys Trp Asn His He Asp Glu He Asn Gin 325 330 335 Leu Leu Gly Val Arg Gly Ala Asn Ala Glu Asp Pro Val Ala Val He 340 345 350 Arg Thr His Ala Asn Arg Leu Lys Leu Ala Gly Val 355 360 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 344 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM 3401 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Ala Ala Arg Glu Gly Arg Ala Pro Leu Ala Arg Arg Ala Ala Val 1 5 10 15 Tyr Gly Val Val Gly Leu Ala Ala He Ala Gly Val Ala Met Trp Ser 20 25 30 Gly Ala Gly Trp His Arg Gly Thr Gly Ser Val Gly Glu Ala Pro Asp 35 40 45 Ala Ala Ala Val Gly Gly Val Ala Ala Ala Pro Pro Gin Ala Ala Val 50 55 60 Pro Ala Ser Ala Gly Leu Pro Ser Ser Leu Ala Gly Ser Ser Ala Pro 65 70 75 80 Arg Val Pro Leu Asp Al a Gly Gly His Leu Al a Lys Val Arg Al a Val 85 90 95 Arg Asp Phe Phe Asp Tyr Cys Leu Thr Ala Gin Ser Asp Leu Ser Ala 100 105 110 Al a Ala Leu Asp Ala Leu Val Val Arg Glu He Ala Al a Gin Leu Asp 115 120 125 Gly Thr Ala Ala Gin Ala Glu Ala Leu Asp Val Trp His Arg Tyr Arg 130 135 140 Ala Tyr Leu Asp Ala Leu Ala Lys Leu Arg Asp Ala Gly Ala Val Asp 145 150 155 160 Lys Ser Asp Leu Gly Ala Leu Gin Leu Al a Leu Asp Gin Arg Ala Ser 165 170 175 He Ala Tyr Arg Thr Leu Gly Asp Trp Ser Gin Pro Phe Phe Gly Ala 180 185 190 Glu Gin Trp Arg Gin Arg Tyr Asp Leu Ala Arg Leu Lys He Ala Gin 195 200 205 Asp Arg Thr Leu Thr Asp Ala Gin Lys Ala Glu Arg Leu Ala Ala Leu 210 215 220 Gin Gin Gin Met Pro Ala Asp Glu Arg Ala Ala Gin Gin Ala Val Asp 225 230 235 240 Arg Gin Arg Ala Ala He Asp Gin Ser Pro Xaa Leu Gin Lys Ser Gly 245 250 255 Thr Thr Pro Asp Al a Met Arg Ala Gin Leu Thr Gin Thr Leu Gly Pro 260 265 270 Glu Ala Ala Ala Arg Val Gly Gin Met Gin Gin Asp Asp Ala Ser Trp 275 280 285 Gin Xaa Arg Tyr Ala Asp Tyr Al a Ala Gin Arg Ala Gin He Glu Ser 290 295 300 Ala Gly Leu Ser Pro Gin Gly Arg Asp Ala Gin He Ala Ala Leu Arg 305 310 315 320 Gin Arg Val Phe Thr Lys Pro Gly Gl u Al a Val Arg Al a Al a Ser Leu 325 330 335 Asp Arg Gly Al a Gly Ser Al a Gin 340 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2118 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATGGCCAGGA CGATGCGTTC CAGGGTGGTG GCAGGGGCAG TGGCATGCGC GATGAGCATC 60 GCGCCGTTCG CGGGGACGAC CGCGGTGATG ACGCTCGCGA CGACGCACGC GGCAATGGCG 120 GCCACCGCGC CCGCCGCTGG CTACGCGGCG ACGCGTTACC CGATCATCCT CGTGCACGGG 180 CTCTCGGGTA CCGACAAGTA CGCCGGCGTG CTCGAGTATT GGTACGGCAT CCAGGAGGAC 240 CTGCAACAGA ACGGTGCGAC CGTCTACGTC GCGAACCTGT CGGGTTTCCA GAGCGACGAC 300 GGCCCGAACG GGCGCGGCGA ACAGTTGCTC GCTTACGTGA AGACGGTGCT CGCGGCGACG 360 GGGGCGACCA AGGTCAATCT CGTCGGTCAC AGCCAGGGCG GCCTCTCGTC GCGCTATGTT 420 GCTGCCGTCG CGCCCGATCT CGTTGCGTCG GTGACGACGA TCGGCCCAGC CGATCGCGGC 480 TCCGAATTCG CCGACTTCGT GCAGGACGTG CTCGCGTACG ATCCGACCGG GCTTTCGTCA 540 TCGGTGATCG CCGCGTTCGT CAATGTGTTC GGGATCCTGA CGAGCAGCAG CCACAACACC 600 AACCAGGACG CGCTCGCCGC ACTGCAGACG CTGACCACCG CACGGGCCGC CACGTACAAC 660 CAGAACTATC CGAGCGCGGG CCTGGGTGCG CCGGGCAGTT GCCAGACCGG TGCGCCGACC 720 GAAACCGTCG GCGGCAACAC GCACCTGCTG TATTCGTGGG CCGGCACGGC GATCCAGCCG 780 ACGCTCTCCG TGTTCGGCGT CACGGGCGCG ACGGACACGA GCACCCTTCC GCTCGTCGAT 840 CCGGCGAACG TGCTCGACCT GTCGACGCTC GCGCTGTTCG GCACCGGCAC GGTGATGATC 900 AACCGCGGCT CCGGGCAGAA CGACGGGCTC GTGTCGAAGT GCAGTGCGCT GTACGGCAAG 960 GTGCTGAGCA CGAGCTACAA GTGGAACCAC CTCGACGAGA TCAACCAGCT GCTCGGCGTG 1020 CGCGGCGCGT ATGCGGAAGA TCCCGTCGCG GTGATCCGCA CGCATGCGAA CCGGCTGAAG 1080 CTGGCGGGGG CACGAGGAGG ACGCGCGCCG CTGGCGCGCC GCGCCGTGGT CTATGGTGCC 1140 GTGGGGCTGG CGGCGATTGC CGGCGTGGCG ATGTGGAGCG GCGCGGGCCG GCATGGCGGG 1200 ACGGGCGCAT CCGGCGAGCC GCCGGATGCG TCGGCGGCAC GCGGACCGGC TGCCGCACCG 1260 CCGCAGGCCG CCGTGCCGGC AAGCACGAGC CTGCCGCCGT CGCTCGCCGG CTCCAGCGCG 1320 CCCCGCTTGC CGCTCGATGC CGGCGGCCAT CTCGCGAAGG CGCGCGCGGT GCGGGATTTC 1380 πCGACTACT GCCTGACCGC GCAGAGCGAC CTGAGTGCGG CCGGGCTCGA TGCGTTCGTC 1440 ATGCGCGAGA TTGCCGCACA GCTCGACGGG ACCGTTGCGC AGGCCGAGGC GCTCGACGTG 1500 TGGCACCGGT ATCGCGCGTA TCTCGACGCA CTCGCGAAAT TGCGCGATGC CGGCGCGGTC 1560 GACAAGTCGG ACCTGGGTGC ATTGCAGCTC GCGCTCGACC AGCGCGCGTC GATCGCGTAC 1620 CGGTGGCTCG GCGACTGGAG CCAGCCGTTC TTCGGTGCGG AGCAATGGCG GCAGCGCTAC 1680 GACCTCGCGC GGCTGAAGAT CGCGCAGGAC CCCGCGCTGA CGGATGCGCA GAAGGCCGAA 1740 CGGCTCGCGG CGCTCGAACA GCAGATGCCG GCCGACGAAC GCGCCGCGCA GCAGCGCGTC 1800 GACCGGCAGC GCGCGGCGAT CGACCAGATC GCGCAATTGC AGAAGAGCGG GGCGACGCCC 1860 GATGCGATGC GCGCACAACT GACGCAGACG CTCGGCCCCG AAGCGGCCGC GCGCGTCGCG 1920 CAGATGCAGC AGGACGACGC ATCGTGGCAG AGGCGCTACG CGGACTACGC GGCGCAGCGT 1980 GCGCAGATCG AGTCGGCCGG CCTGTCGCCG CAGGATCGCG ACGCGCAGAT CGCCGCGCTG 2040 CGGCAGCGCG TGTTTACGAA GCCCGGCGAA GCCGTGCGCG CGGCATCGCT CGATCGCGGG 2100 GCG.GGCAGCG CGCGGTAA 2118 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRAh'DEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE.-, peptide (i i) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM3959 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Thr Ala Arg Gly Gly Arg Ala Pro Leu Arg Arg Ala Val Val Tyr Gly 1 5 10 15 Ala Val Gly (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM3959 (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Al a Arg Thr Met Arg Ser Arg Val Val Al a Gly Al a Val Al a Al a Met 1 5 10 15 Ser He Al a (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM3959 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Ala Arg Thr Met Arg Ser Arg Val Val Ala Gly Al a Val Al a Met He 1 5 10 15 Ala (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGCGTAAGCT TCACATTGAA AGGGGAGGAG AATCATGGCC GCTGGCTACG CGGCGA 56 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 56 base airs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear . ) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CGCGTCGCCG CGTAGCCAGC GGCCATGATT CTCCTCCCCT TTCAATGTGA AGCTTA 56 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Pseudomonas cepacia (B) STRAIN: DSM3959 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Ala Ala Gly Tyr Ala Ala 1 5

Claims: CLAIMS 1. A process for preparing an active lipase enzyme In vitro, the process comprisin (a) culturing a host cell transformed with a DNA sequence encoding a lipase enzyme under suitable conditions to produce the lipase enzyme in inactive or partly active form, recovering the lipase enzyme from the culture, and subjecting the recovered lipase enzyme to denaturation, (b) mixing the denatured lipase enzyme obtained in step (a) with a chaperone molecule, and (c) subjecting the mixture of step (b) to renaturation to produce the active lipase enzyme. 2. A process for preparing an active lipase enzyme in vitro, the process comprising (a) culturing a host cell transformed with a DNA sequence encoding a lipase enzyme under suitable conditions to produce the lipase enzyme in inactive or partly active form and recovering the lipase enzyme from the culture, (b) mixing the recovered lipase enzyme with a chaperone molecule, and (c) subjecting the mixture of step (b) to denaturation followed by renaturation to produce the active lipase enzyme. 3. A process according to claim 1 or 2, in which the chaperone molecule is produced by culturing a host cell transformed with a DNA sequence encoding the chaperone molecule under suitable conditions to produce the chaperone molecule and recovering the chaperone molecule from the culture. 4. A process according to any of claims 1 - 3, in which the chaperone molecule is subjected to a denaturation treatment before being added to the denaturated lipase in step (b). 5. A process for preparing an active lipase enzyme jn vitro, the process comprising (a) culturing a host cell transformed with a DNA sequence encoding a lipase enzyme and with a DNA sequence encoding a chaperone molecule under suitable conditions to produce the lipase enzyme in inactive or partly active form and recovering a lipase enzyme chaperone molecule mixture from the culture, and (b) subjecting the mixture of step (a) to denaturation followed by renaturation to produce the active lipase enzyme, optionally with addition of a further amount of chaperone molecule to the mixture. 6. A process according to any of claims 1 - 5, wherein the lipase enzyme is one derived from a Pseudomonas sp. or a Chromobacter sp. 7. A process according to claim 6, wherein the lipase enzyme is a Pseudomonas cepacia. Pseudomonas fragi. Pseudomonas gladioli. Pseudomonas fluorescens. Pseudomonas stutzeri. Pseudomonas alcaligenes. Pseudomonas pseudoalcalioenes. Pseudomonas putida. Pseudomonas glumae. Pseudomonas aeruginosa or Chromobacter viscosum lipase, or a derivative of said lipase enzyme. 8. A process according to any of claims 1 - 7, wherein the host cell for the production of the lipase enzyme is Escherichia coli. 9. A process according to claim 8, wherein the DNA sequence encoding the lipase enzyme is preceded by the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α- amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters, the phage T7 gene 10 promote or the E coil lac promoter. 10. A process according to claim 8, wherein the DNA sequence encoding the lipas enzyme is preceded by a ribosome binding site of of the Bacillus stearothermophilus 5 maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α-amylase gene, Bacillus subtilis alcaline protease gene, Bacillus pumilus xylosidase gene, phage T7 gene 10 or E_ coH lac gene. 11. A process according to any of claims 1 - 10, wherein the lipase enzyme is produced intracellularly in a high yield. 10 12. A process according to claim 11 , wherein the lipase enzyme is produced in the form of inclusion bodies. 13. A process according to any of claims 1 - 5, wherein the chaperone molecule is a Pseudomonas lipase modulator or a derivative thereof. 14. A process according to claim 13, in wherein the chaperone molecule is selected 15 from the group consisting of Pseudomonas cepacia lipase modulator, the Pseudomonas glumae lipase modulator, the Pseudomonas aeroginosa lipase modulator or a derivative thereof. 15. A process according to claim 3, wherein the host cell for the production of the chaperone molecule is E. coli: 20 16. A process according to claim 15, wherein the DNA sequence encoding the chaperone molecule is preceded by the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α-amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage Lambda P R or P L promoters, the 5 phage T7 gene 10 promoter or the E cgli lac promoter. 17. A process according to claim 15, wherein the DNA sequence encoding the chaperone molecule is preceded by a ribosome binding site of of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α-amylase gene, Bacillus subtilis alcaline protease gene, 5 Bacillus pumilus xylosidase gene, phage T7 gene 10 or E cgJl lac gene. 18. A process according to any of claims 13 - 17, wherein the chaperone molecule is produced intracellularly in a high yield. 19. A process according to any of claims 1 - 18, wherein the lipase enzyme is a Pseudomonas cepacia lipase, and wherein the chaperone molecule is a 10 Pseudomonas cepacia lipase modulator. 20. A DNA construct comprising a first DNA sequence encoding a lipase enzyme fused to a second DNA sequence encoding a chaperone molecule in such a way that the lipase enzyme and chaperone molecule or functional part thereof are expressed as a single fusion protein on culturing a suitable host cell transformed 15 with the DNA construct. 21. A DNA construct according to claim 20, wherein the first DNA sequence encoding the lipase enzyme is one derived from a Pseudomonas sp. or a Chromobacter sp. 22. A DNA construct according to claim 21 , wherein the first DNA sequence is one 20 encoding a Pseudomonas cepacia. Pseudomonas fragi. Pseudomonas gladioli. Pseudomonas fluorescens. Pseudomonas stutzeri. Pseudomonas alcalioenes. Pseudomonas pseudoalcalioenes. Pseudomonas putida. Pseudomonas glumae. Pseudomonas aeruoinosa or Chromobacter viscosum lipase, or a derivative of said lipase enzyme. 25 23. A DNA construct according to claim 20, wherein the second DNA sequence is one encoding a Pseudomonas lipase modulator or a derivative thereof. 24. A DNA construct according to claim 23, wherein the second DNA sequence is one encoding a Pseudomonas cepacia lipase modulator, a Pseudomonas glumae lipase modulator, a Pseudomonas aeroginosa lipase modulator or a derivative thereof. 5 25. A DNA construct according to any of claims 20 - 24, wherein the first DNA sequence encodes a Pseudomonas cepacia lipase or a derivative thereof, and wherein the second DNA sequence encodes a Pseudomonas cepacia lipase modulator or a derivative thereof. 26. A DNA construct according to claim 25 which has the sequence shown in SEQ 10 ID No. 7 appended hereto. 27. A recombinant expression vector comprising a DNA construct according to any of claims 20-26. 28. A host cell transformed with a DNA construct according to any of claims 20-26 or with a recombinant expression vector according to claim 27. 15 29. A host cell according to claim 28 which is a cell of a strain of E. coli. 30. A host cell according to claim 29, wherein the DNA construct is preceded by the promoter of the Bacillus stearothermophilus maltogenic amylase gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α-amylase gene, Bacillus subtilis alcaline protease gene, or Bacillus pumilus xylosidase gene, or by the phage 0 Lambda P R or P L promoters, the phage T7 gene 10 promoter or the E cgH lac promoter. 31. A host cell according to claim 29, wherein the DNA construct is preceded by th ribosome binding site of of the Bacillus stearothermophilus maltogenic amylas gene, Bacillus licheniformis α-amylase gene, Bacillus amyloliguefaciens α-amylas gene, Bacillus subtilis alcaline protease gene, Bacillus pumilus xylosidase gene, phage T7 gene 10 or E coli lac gene. 32. A process for preparing a lipase in active form, the process comprising culturing a host cell according to any of claims 28 - 31 under suitable conditions to produce the lipase, and recovering the lipase from the culture. 33. A process for denaturating and renaturating a lipase enzyme, the process comprising (a) subjecting a lipase enzyme to a denaturation treatment, (b) mixing the denaturated lipase enzyme obtained in step (a) with a chaperone molecule, which optionally has been denaturated, and (c) subjecting the mixture of step (b) to renaturation to produce the active lipase enzyme. 34. A process for denaturating and renaturating a lipase enzyme, the process comprising a) mixing a lipase enzyme to be subjected to a denaturation and renaturation treatment with a chaperone molecule, and b) subjecting the mixture of step a) to denaturation followed by renaturation so as to produce an active lipase enzyme.

Inventors: BUCKLEY CATHERINE M, DIDERICHSEN BOERGE KRAG, HOBSON AUDREY, JOERGENSEN STEEN TROELS, MCCONNELL DAVID J, BUCKLEY, CATHERINE, M., DIDERICHSEN, BOERGE, KRAG, HOBSON, AUDREY, JOERGENSEN, STEEN, TROELS, MCCONNELL, DAVID, J.
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1992
Main classification: C12N9
IPC codes: C07K14, C12N1, C12N9, C12N15, C12P19, C12R1