WO2010130622A1

WO2010130622A1 - Enzyme composition for the treatment of prancreatic insufficiency

Info

Publication number: WO2010130622A1
Application number: PCT/EP2010/056169
Authority: WO
Inventors: Luppo Edens; Willem Bijleveld
Original assignee: Dsm Ip Assets B.V.
Priority date: 2009-05-11
Filing date: 2010-05-06
Publication date: 2010-11-18

Abstract

The present invention provides a lipolytic enzyme or a composition comprising a lipolytic enzyme - which lipolytic enzyme is enzymatically active at a neutral to acid pH, for example at pH 7, 6, 5, 4, 3 or 2; - which the lipolytic enzyme has activity towards triacylglycerol lipids; - which the lipolytic enzyme has activity towards phospholipids; - which the lipolytic enzyme has activity towards lysophospholipids; and - which the lipolytic enzyme has activity towards galactolipids, for use as medicament.

Description

ENZYME COMPOSITION FOR THE TREATMENT OF PANCREATIC

INSUFFICIENCY

Field of the invention

The present invention relates to a composition comprising an enzyme having lipolytic activity (or lipase) and the use of the lipolytic enzyme and the composition for treating patients suffering from pancreatic insufficiency and cystic fibrosis.

Background of the invention

Food compositions typically comprise proteins, carbohydrates, hemicelluloses, fats and phospholipids. Upon their consumption each one of these components is mechanically and enzymatically degraded to render units small enough to be absorbed via the intestinal wall into the blood. To facilitate this enzymatic degradation process, the gastrointestinal tract is divided into different compartments.

After ingestion food is diminuated by chewing, mixed with saliva and, after swallowing, it reaches the stomach where it is mechanically mixed with acid and proteolytic and lipolytic enzyme activities. Typical residence times of solid food in the stomach range from one to two hours. During this period the diminuated food is acidified by gastric acid produced by the parietal cells present in the gastric mucosa. Despite these rather long residence times, proteolysis (by the gastric protease pepsin) and lipolysis (by the lingual/gastric lipase) in the stomach are rather limited. Althought there exist great individual variations, food flowing from the stomach into the duodenum typically shows pH values between 2 and 3.

Occasional opening of the pyloris allows the acidified and partly hydrolysed food to flow into the small intestine. In the first part of the small intestine i.e. in the duodenum, pancreatic juice as well as bile are added. The pancreatic juice contains bicarbonate to neutralize the acid stomach contents and a large number of digestive enzymes for further degrading proteins, carbohydrates and lipids.

The intestinal degradation and uptake of especially dietary lipids is a complex process. All relevant lipolytic activities required to hydrolyse the various dietary lipids have to be present as well as the proper emulsifying agents. Moreover, the pH plays an important role in both hydrolysis and emulsification. In the duodenum and beyond, bile, a mixture of bile acids and phosphatidyl choline secreted by the liver, acts as an emulsifier to improve the water solubility of the lipid hydrolysis products. After the duodenum, the digest reaches the jejunum. Together with the duodenum, the jejunum presents the major food absorption site in the gastrointestinal tract.

The last part of the small intestine is formed by the ileum, after which the digest enters the large intestine (colon). In the colon, there is an intensive fermentation but there is no appreciable absorption of food components. Because of the impact of the pancreatic enzymes on the metabolization of various food components, pancreatic failure is a life-threatening event. Diseases such as chronic pancreatitis and cystic fibrosis can lead to pancreatic failure. Chronic pancreatitis is an ongoing inflammatory disorder associated with the loss of exocrine and endocrine pancreatic tissue. The loss of exocrine tissue leads to substantially decreased enzyme secretions and the loss of endocrine pancreatic tissue can lead to diabetes mellitus. Cystic fibrosis is a disorder in which a single gene mutation results in defective sodium, chloride and water transport in the epithelial cells of the respiratory, hepato-biliary, gastro-intestinal and reproductive tracts. In 85% of the cystic fibrosis patients pancreatic insufficiency occurs. The reason for this is that the pancreas of such patients produces such thick secretions that the digestive enzymes cannot pass through the pancreatic ducts and do not reach the intestines. Similar to the situation in pancreatitis patients, the digestion of fats, proteins and carbohydrates becomes incomplete This is not only due to a shortage of digestive enzymes in the intestinal lumen of such patients, but also to the fact that bicarbonate secretion, required for neutralizing the acid stomach contents, is affected. Obviously the inability to properly hydrolyse the food consumed results in malabsorbtion, malnutrition and serious weight loss. Moreover abdominal cramping, diarrhoea and bloating are regularly reported. Noteworthy is that the production of gastric acid and gastric enzymes is not affected in persons suffering from pancreatic enzyme insufficiency or cystic fibrosis.

For decades oral administration of porcine pancreatin (a preparation incorporating the major pancreatic enzyme products) is a standard therapy to aid patients suffering from chronic pancreatitis and cystic fibrosis. However, pancreatin is taken orally so that its enzymatic content, destined to work under the near neutral conditions of the intestine, is first exposed to the acidic and proteolytic conditions in the stomach. To protect the pancreatic enzymes against such harsh surroundings, so-called enteric coatings are applied. Enteric coatings stay intact under acidic conditions of the stomach, but gradually dissolve under the near neutral pH conditions of the duodenum so that the enzymic contents are released. Unfortunately, enteric coatings are expensive and come with a number of shortcomings. The prior art addresses several of these and other shortcomings of the classical pancreatic preparations a.o. by introducing enzyme preparations derived from various microorganisms.

Although multiple lipolytic compositions for treating pancreatic enzymatic insufficiency are commercially available or known, all of them have one or more drawbacks. Examples of such drawbacks are the large amounts that have to be consumed to reach an acceptable efficacy, an instability in the stomach so that expensive and voluminous enteric coatings are required and a limited substrate specificity so that the oral lipolytic enzyme cannot degrade all dietary lipids. Additionally many of the prior art lipases or coated lipases cannot cope with the acid conditions that can occur in the proximal intestine of patients suffering from pancreatitis or cystic fibrosis.

Summary of the invention

It is an object of the present invention to provide a lipolytic enzyme (or lipase) or a composition comprising said lipolytic enzyme for use as a medicament and which solves at least one of the above-mentioned problems that are associated with oral lipase administration to pancreatitis and cystic fibrosis patients.

The lipolytic enzyme used according to the present invention - is enzymatically active at neutral or acid pH, for example at pH 7, 6, 5, 4, 3 or 2, and - has hydrolytic activity towards triacylglycerol lipids, phospholipids, lysophospholipids and galactolipids.

The present invention relates to a lipolytic enzyme for use as a medicament or a composition comprising said lipolytic enzyme, said lipolytic enzyme has one or more of the following additional properties: - the lipolytic enzyme has an acid pH optimum;

- the lipolytic enzyme is enzymatically stable at acid pH, for example at pH 6, 5, 4, 3 or 2;

- the lipolytic enzyme is gastric acid resistant; - the lipolytic enzyme is active in the presence of pepsin;

- the lipolytic enzyme is enzymatically stable in the presence of pepsin;

- the lipolytic enzyme is resistant to pepsin; and/or

- the lipolytic enzyme has no activity towards triacylglycerol lipids under assay conditions (pH 9) of United States Pharmacopeia (USP) procedure. In a preferred embodiment, said lipolytic enzyme is of microbial origin, more preferably of fungal origin, even more preferably from Aspergillus species. The lipolytic enzyme used according to the present invention has the following properties: the lipolytic enzyme has activity towards triacylglycerol lipids; - the lipolytic enzyme has activity towards phospholipids; the lipolytic enzyme has activity towards lysophospholipids; and - the lipolytic enzyme has activity towards galactolipids.

So the lipolytic enzyme used according to the invention or present in the composition of the invention has at least four hydrolytic activities. According to another aspect of the invention, the composition of the invention comprises also a phospholipase A2, a protease and/or an amylase. Preferably the phospholipase A2, the protease and/or amylase is of non-animal origin or non- animal derived preferably is of microbial origin or produced in a microbial host. The present invention also relates to the use of the lipolytic enzyme or a composition comprising the lipolytic enzyme as a medicament, preferably a medicament to treat digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis. Additionally the lipolytic preparation according to the invention is helpful as a digestive aid or dietary ingredient to diminish or to lower gastrointestinal discomfort. The present invention provides a composition incorporating a lipolytic enzyme having triacylglycerol lipase activity as well as phospholipase, lysophospholipase and galactolipase activity. Moreover, the lipolytic enzyme according to the invention is active under acid conditions and is preferably enzymatically stable and active in the presence of pepsin so that an enteric coating to survive the stomach passage is not required. According to another aspect of the invention the lipolytic enzyme or the composition according to the invention is not only active in the stomach but also in the duodenum. According to a further aspect of the invention the phospholipase activity present in the lipolytic enzyme can compensate for a limited emulsifying capacity, for example the limited emulsifying capacity that results from substantially reduced bile acid levels in the intestinal lumen. Such reduced bile acid levels can be the result of a decreased secretion and/or a precipitation of the bile acid due to low pH value in the intestinal tract of pancreatitis and cystic fibrosis patients. Because said lipolytic enzyme is active in the acid surroundings of the stomach and in the duodenum, smaller amounts of said lipolytic enzyme can be used and hence smaller pharmaceutical products (such as but not limited to tablets) can be consumed by a patient in need thereof, for example for compensation of pancreatic insufficiency. The nature of said lipolytic enzyme is such that, in conjunction with the gastric lipase naturally present in the stomach, it can degrade the larger part of the dietary lipids into components that are readily absorbable by the cells of the intestinal lining. According to the present invention the lipolytic enzyme is preferably combined with a phospholipase A2, a protease and/or an amylase. Preferably the phospholipase A2, the protease and/or amylase is of non- animal origin or is non-animal derived preferably is of microbial origin or produced in a microbial host. Preferably the phospholipase A2, the protease and/or amlysae have acid pH optima for its use as a medicament.

Detailed description of the invention According to one aspect of the present invention an enzyme composition is disclosed that has one or more advantages over the prior art. Such advantages can be an improved lipolytic activity at acidic pH, an (improved) lipolytic activity in both the stomach and the duodenum, a lipolytic activity with an improved stability upon a low pH treatment, a lipolytic activity with an improved stability against proteases including pepsin, an improved lipolytic activity with respect to activity towards triacylglycerol lipids, phospholipids, lysophospholipids and/or galactolipids, an improved lipolytic activity for in-vivo use, an improved lipolytic activity at temperatures between 36 and 38 degrees Celsius, lower dosages of the lipolytic enzyme or avoiding (expensive) coatings of the composition of the invention, the advantage being relative to one or more lipolytic enzymes used or known for the treatment of digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis as decribed in the prior art.

The present invention also relates to the use of the present lipolytic enzyme as a medicament (or pharmaceutical) or dietary supplement, and to a composition comprising the lipolytic enzyme for use as a medicament or dietary supplement, and to the preparartion of a medicament or dietary supplement, comprising the lipolytic enzyme.

This lipolytic enzyme used according to the present invention - is enzymatically active at low pH, for example at pH 6, 5, 4, 3 or 2 and

- has hydrolytic activity towards triacylglycerol lipids (according to EC3.1.1.3), towards phospholipids (according to EC3.1.1.32 or according to EC3.1.1.4), towards lysophospholipids (according to EC3.1.1.5) and towards galactolipids (according to EC3.1.1.26). Surprising is that despite these many advantages, the lipase according the invention will be inactive in the lipase assay specified in the United States Pharmacopeia (USP) procedure for determining the activity of various pancreatin preparations at pH 9.0.

The lipolytic activity towards triacyl-glycerol lipids is determined in an assay with the chromogenic substrate p-nitrophenyl palmitate as outlined in the Materials & Methods section. Preferably the lipolytic activity towards galactolipids is determined using digalactosyl diglyceride as a substrate according to the procedure outlined in the Materials & Methods section. Preferably the lipolytic activities towards triacyl-glycerol under various pH conditions are determined in a mixture of olive oil and gum arabic as outlined in the Materials & Methods section. Preferably the lipolytic activity (either A1 or A2) towards phospholipids is determined spectrophotometrically by using 1 ,2-dimercaptodioctanoyl- phosphatidylcholine as a substrate according to the procedure outlined in the Materials & Methods section. Preferably the lysolipolytic activity towards phospholipids is determined in an assay in which lysophosphatidyl choline is used as the substrate according to the procedure outlined in the Materials & Methods section.

By enzymatically stable at low pH (or acid stable) is meant that the lipase retains its hydrolytic activity towards triacyl-glycerol at pH 3.0 and in the presence of the proteolytic enzyme pepsine under conditions further specified in Example 7 of the present application.

By an acidic pH optimum is meant an pH optimum which is between pH 2 and pH 7, preferably between an pH between pH 3 and pH 6, more preferably below pH 5.0. Preferably the determination of pH optimum is done (indicated using the relevant (triacylglycerol) assay outlined in the Materials & Methods section. Instead of the pH value indicated by this method, the measurement is carried out) at several pH values (between pH 2 and pH 7) using olive oil plus gum arabic as a substrate and an appropriate buffer system as specified in Example 2 of the present application. Malfunctioning of the pancreas leads to substantially decreased enzyme secretions resulting in an incomplete digestion of fats, proteins and carbohydrates and, longer term, in malnutrition. In the latter aspect especially the problematic fat digestion forms a serious problem as dietary lipids provide a major part of our energy. Typical (US) daily fat consumption is 96 grams of which triglycerides represent by far the greatest quantity. The triglycerides contain predominantly long chain, water insoluble fatty acids (14 to 20 carbons). The daily intake of other lipids like cholesterol, cholesteryl esters, galactolipids and phospholipids is not more than a few grams per day. However, the presence of these compounds can be of particular relevance for improving oil emulsification. To properly digest the various water insoluble, fatty components, a healthy pancreas secretes a large array of lipolytic activities such as:

• pancreatic triglyceride lipase in conjunction with its co-lipase,

• pancreatic lipase-related protein 2 (PLRP2),

• carboxyl ester lipase, • lysophospholipase and

• phospholipase A2.

Each one of these lipolytic activities plays its own role in hydrolysing the various dietary fats such as triacylglycerols (a glycerol backbone with three esterified fatty acids in outer (sn-1 and sn-3) and the middle (sn-2) position), galactolipids (a glycerol backbone with two esterified fatty acids in an outer (sn-1 ) and middle (sn- 2) position, while the third hydroxyl group is bound to sugar residues such as a galactose), phospholipids (a glycerol backbone with two esterified fatty acids in the outer (sn-1 ) and the middle (sn-2) position, while the third hydroxyl group of the glycerol is esterified with phosphoric acid which, in turn, is esterified to for example an amino alcohol like ethanolamine or choline) and lysophospholipids (phospholipids missing one or both of its two O-acyl chains). Phospholipase A2 is of particular relevance for the emulsification process as it selectively removes the esterified fatty acid from the sn-2 position of phospholipids. As a result the water solubility of the "lyso"-phospholipid formed increases hereby improving lipid emulsification as a whole.

The various lipolytic activities involved in the hydrolysis of these substrate molecules are included in the internationally recognized schemes for the classification and nomenclature of all enzymes from IUMB. The IUMB text can be found at the internet site: http://www.chem.qmw/ac.uk/iubmb/enzyme/EC3/4/1 1/. In this system enzymes are defined by the fact that they catalyze a single reaction implying that different proteins are all described as the same enzyme, and that a protein catalysing more than one reaction is treated as more than one enzyme. The system categorises the various lipolytic activities as discussed in the present text under the heading "carboxylic ester hydrolases" (EC3.1.1 ). Triacylglycerol lipase (EC3.1.1.3) hydrolyzes a triacylglycerol to yield diacylglycerol plus a carboxylate, lysophospholipase (EC 3.1.1.5) hydrolyzes lysophospholipids, phospholipase A1 (EC3.1.1.32) cleaves phospholipids by removing the fatty acid attached to the 1-position and phospholipase A2 (EC3.1.1.4) cleaves phospholipids by removing the fatty acid attached to the 2-position. Galacto- lipases (EC3.1.1.26) hydrolyze 1 ,2-diacyl-3-beta-D-galactosyl-sn-glycerol to yield 3-beta-D-galactosyl-sn-glycerol plus two carboxylates.

The lipid hydrolysis products formed in the mammalian digestive tract are predominantly mono- and di-acylglycerols, fatty acids, lysophosphatidylcholine and cholesterol. All these hydrolysis products are poorly water soluble. However, for an efficient uptake by the intestinal epithelial cells, a good water solubility of these hydrolysis products is of paramount importance. In healthy individuals this is accomplished by the bile secreted by the liver. The bile emulsifies the oil droplets to form micelles. In the form of such micelles, the lipid hydrolysis products are 100 to 1000 times more water-soluble thus facilitating their uptake and subsequent metabolisation. Unfortunately the malfunctioning of the pancreas in pancreatitis or cystic fibrosis patients results not only in an inadequate secretion of digestive enzymes (and sometimes in sub-optimal bile levels) in the intestinal lumen, it also affects bicarbonate production. In healthy individuals the acidified and partly hydrolysed stomach content is neutralized in the duodenum by the bicarbonate produced by the pancreas. Under the resulting near neutral pH conditions, the pancreatic enzymes are optimally active. However, in patients suffering from pancreatitis or cystic fibrosis, gastric secretions can be enhanced and pancreatic bicarbonate production can be reduced so that these patients may suffer from an acid duodenum. Such an acid duodenum has major consequences, especially for lipid hydrolysis and emulsification of the resulting cleavage products because:

- the effect of the inadequate levels of pancreatic enzymes is augmented by pH values that are sub-optimal for these enzymes, - the effect of the inadequate levels of bile is augmented by the fact that the bile can easily precipitate under such acid conditions so that a proper emulsification of the fat hydrolysis products cannot take place

- the major part of the fatty acids enzymatically released from the dietary lipids become protonated and lipophilic under such very acid conditions so that they cannot migrate to the micellar phase.

According to one aspect of the present invention, the present lipolytic enzyme or a composition comprising this enzyme is capable to compensate for these shortcomings by the special features of the lipolytic enzyme or a composition comprising this enzyme. These features enable the lipase according to the invention to be active in the stomach and in an acid or a near neutral duodenum. Furthermore, the lipolytic enzyme according to the invention can hydrolyse triacylglycerol lipids responsible for generating the major part of our energy and, at the same time, improve the emulsification process by an in-situ generation of emulsifying agents such as lysophospholipids and galactosylglycerols. Said lysophospholipids are obtained from dietary phospholipids by the phospholipase A1 activity of the lipase or lipolytic enzyme, said galactosylglycerols are obtained from dietary galactolipids by the galactolipase activity according to the present invention. Converting dietary phospholipids into "lyso"phospholipids that are desirable for improving fat emulsification under acid conditions, can be accomplished by adding an active non-animal derived phospholipase A2 (pancreas phospholipase A2 produced in Aspergillus niger see US Gras Notification GRN 183) to the present lipolytic enzyme or to a composition comprising the present enzyme. Optionally fat digestion under acid conditions can be improved by combining the the present lipolytic enzyme or to a composition comprising the present enzyme with dietary emulsifiers. Examples of suitable emulsifiers include phospholipids, "lyso"phospholipids or detergents such as, for example, the non-ionic Triton X-100 or Tween-80 or the anionics sodium cholate, sodium deoxycholate or sodium taurodeoxycholate. The lipase or lipolytic enzyme used according to the present invention may be in an isolated form. As defined herein an isolated polypeptide is an endogenously produced or a recombinant polypeptide which is isolated from other polypeptides, and is typically at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, still more preferably at least 90% pure, or most preferably at least 95% pure, as determined by SDS-PAGE. The polypeptide may be isolated by centrifugation, filtration (for example utrafiltration) or chromatographic methods, or any other technique known in the art for obtaining purified proteins from crude solutions. It will be understood that the polypeptide may be mixed with carriers or diluents which do not interfere with the intended purpose of the polypeptide, and thus the polypeptide in this form will still be regarded as isolated. It will generally comprise the polypeptide in a preparation in which more than 10%, for example more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%, by weight of the proteins in the preparation is a polypeptide for use of the process of the present invention.

The composition according to the invention is preferably a composition for (at least in part) improving the digestion and absorption of the lipid materials present in the food. This may relate to in vivo digestion and/or absorption i.e. in a mammal, or to in vitro digestion and/or absorption i.e. in a model system. In its simplest form the latter model system can be an in vitro system held for fixed periods at a pH value mimicking the pH value of either the stomach or the duodenum of a pancreatitis or cystic fibrosis patient. The presence of the gastric endoprotease pepsin is mimicked by simply adding purified pepsin to the incubation mixture. In a more sophisticated approach, the passage of food through the gastrointestinal tract can be simulated in dynamic gastrointestinal in vitro models. In such models the successive dynamic processes in the stomach and in the small intestine are simulated using validated procedures (Minekus et al, ATLA 1995, 23, 197-209; Larsson et al, J Sci Food Agic 1997, 74, 99-106). As will be explained in more detail later on, a composition according to the invention preferably comprises at least one lipolytic enzyme wherein said lipolytic enzyme is preferably not degraded by pepsin under the conditions specified. To establish whether said at least one lipase is active in the presence of pepsin at pH 3.0 the skilled person preferably preincubates said lipase with pepsin (hereby simulating the situation in the stomach) according to the procedure specified in Example 7 of the present application. Subsequently the thus treated lipase is incubated with the triolein substrate according to the assay procedure at pH 5.0 also specified in Example 7 of the present application. The lipolytic enzyme according to the invention is preferably of microbial origin, more preferably of fungal origin such as from Aspergillus species. Rhizopus Javanicus lipase as mentioned in WO96/38170 can as far as we know not produced in a self-cloned production host. Moreover the pH optimum of Rhizopus javanicus was found to be pH 5.5. More preferably the lipolytic enzyme is secreted by the microorganism into the fermentation broth. After fermentation, the secreted enzyme is recovered using known methods. Briefly, from the fermented liquid the biomass is filtered off and the resulting liquid is then concentrated by ultrafiltration. Optionally its salt content can be lowered by diafiltration. Optionally the diafiltered Iquid can be subjected to chromatography to further increase the purity of the enzyme product. The final enzyme concentrate can be stabilized and used as such or can be dried to obtain an enzyme powder or granulate.

The lipolytic enzyme of the present invention and/or additional enzymes to be used in the composition or to the methods of the present invention may be in any form suitable for the use, e.g., in the form of a liquid, in particular a stabilized liquid, Nutritionally acceptable stabilizers include as sugar, sugar alcohol, or another polyol, and/or lactic acid or other organic acids. Or it can be in a dry form, which is preferred for use according to the present invention. To that end the concentrated liquid is then preferably spray dried. Alternatively the lipolytic enzyme or one or more of the other enzymes present may be in the form of a protected enzyme such described in WO01/11974 and WO02/26044, or in a crystal form which enhances the stability to the acidic pH of the stomach and/or its resistance to proteolytic degradation. Enzyme crystals can be obtained via methods well described in prior art. The powder resulting from these different treatments can be used, optionally after mixing with the necessary excipients, for the ultimate dosage form, composition or formulation such as a pill, granulate, capsule, for example a soft gel capsule, or tablet production. Granulates and agglomerated powders may be prepared by conventional methods, e.g., by spraying the lipolytic enzyme according to the invention onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g., a salt (such as NaCI or sodium sulphate), sugar (such as sucrose or lactose), sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy.

A pill, granulate, capsule, for example a soft gel capsule, or tablet as described above preferably further comprises at least one excipient as an inactive ingredient, the excipient being selected from, for example, a filler, a flow agent, a colorant, a flavoring, a dissolving agent, and any combination thereof. The excipient may comprise up to 95 weight percent of the final dosage form. Furthermore, the composition of the invention can include or be used in combination with buffering agents that will raise the intestinal pH value. A composition as described is preferably a pharmaceutical composition suitable for oral administering, i.e. the invention preferably provides an oral composition, i.e. a composition suitable for oral intake. As a consequence, the lipolytic enzyme described in general above is preferably present in a "non-enteric coated" dosage form, composition or formulation like a pill, granulate, capsule or tablet. According to another aspect of the invention the lipolytic enzyme or the composition comprising the lipolytic enzyme is protected by an enteric coating. A composition according to the invention, such as a dosage form, composition or formulation, may further comprise other enzymes, Compositions comprising a lipase, an amylase and a protease are well known and are already (commercially) used in the treatment of pancreatic enzyme insufficiency. For example, one commercial preparation comprises protease: amylase: lipases = 337: 94: 442 USP/mg (USP = United States Pharmacopae units). Another preparation comprises Proteases 45,000 NFU, Amylases 67,500 NFU/tablet, Upases 7050 NFU/tablet (NFU = National Formulary Unit). Preferably said composition comprises a protease and/or an amylase with acid pH optima. All enzymes (i.e. proteases and carbohydrases with either acid or near neutral pH optima) are preferably produced and incorporated in the dosage form, composition or formulation, in isolated state. Isolated enzymes can be obtained for example from plants or by overexpression of the enzyme in a suitable modified or transformed host micro-organism. One advantage of plant or microbial enzymes over animal derived enzymes is their Kosher status.

The lipolytic enzyme according to the invention and/or additional enzymes may be contained in slow-release formulations. Methods for preparing slow- release formulations are well known in the art. The lipolytic enzyme according to the invention and/or additional enzymes may also be contained in formulations protected by an enteric coating. Techniques for doing so are well known in the prior art. Both solvent and aqueous processing techniques are available; see for example publications by Colorcon, West Point, PA or visit http://www.pharma- excipiente.com/aqueous-enteric-coating-polymer.html.

A composition as described is very useful in the treatment of pancreatic enzyme insufficiency. The invention therefore also provides the use of the lipolytic enzyme according to the invention for the manufacture of a medicament for the treatment of digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis. In a preferred embodiment, said lipolytic enzyme is present in a pill, granulate, capsule or tablet.

In another preferred embodiment, the composition of the invention is useful in methods for treating pancreatic enzyme insufficiency in a mammal subject, including humans as well as pets. The invention thus provides a method for treating malabsorption in a mammal comprising administering to said mammal a therapeutically effective amount of a composition comprising at least one lipolytic enzyme which is active at acid pH .

In one of its embodiments, the composition of the invention is administered to a subject in need thereof at the time of or during a, preferably each, meal, snack or shot, in one or more oral dosage form, composition or formulation such as pill(s), granulate(s), capsule(s) or tablet(s). As already described the preferred oral dosage form, composition or formulation such as pills, granulates, capsules or tablet are advantageously combined with an oral dosage form, composition or formulation comprising additional enzymes such as an amylase, and/or a protease.

The invention will be explained in more detail in the following detailed description which does not limit the invention. The present invention provides a lipolytic enzyme or a composition comprising a lipolytic enzyme which is suitable as medicament (pharmaceutical) or dietary supplement. The invention provides there to polynucleotides encoding lipolytic enzymes and use thereof in the composition of the invention.

The polynucleotide according to the invention comprises a nucleotide sequence selected from:

(a) the nucleotide sequence as set out in SEQ ID NO: 1 having at least 60, 70, 80 or 90% homology to the nucleotide sequence of SEQ ID NO: 1 ;

(b) a nucleotide sequence which hybridizes with a polynucleotide being the complement of SEQ ID NO: 1 and wherein said sequence is at least 60, 70, 80 or 90% homologous to the nucleotide sequence of SEQ ID NO: 1 ;

(c) a nucleotide sequence encoding the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 having at least 60, 70, 80 or 90% homology to the mature polypeptide in the amino acid sequence of SEQ ID NO: 2; (d) a sequence which is degenerate as a result of the degeneracy of the genetic code to a sequence as defined in any one of (a), (b), (c);

(e) a nucleotide sequence which is the complement of a nucleotide sequence as defined in (a), (b), (c), (d).

SEQ ID NO: 1 and SEQ ID NO: 2 correspond to the sequences mentioned in WO 2004/018660 referred to as NBE 031 (hereinafter LPY). In particular, the invention provides for polynucleotides having a nucleotide sequence that hybridizes preferably under high stringent conditions with a polynucleotide being the complement of SEQ ID NO: 1 and wherein said sequence is at least 60, 70, 80 or 90% homologous to the nucleotide sequence of SEQ ID NO: 1. Consequently, the invention provides polynucleotides that are at least 90%, preferably at least 91%, more preferably at least 92%, 93%, 94%,

95%, even more preferably at least 96%, 97%, 98% or 99% homologous to the sequence according to SEQ ID NO: 1.

In one embodiment such isolated polynucleotide can be obtained synthetically, e.g. by solid phase synthesis or by other methods known to the person skilled in the art.

In another embodiment the invention provides a lipolytic enzyme gene according to SEQ ID NO: 1 or functional equivalents that are still coding for the active enzyme. Preferably the polynucleotide according to the invention is a DNA sequence.

The invention also relates to vectors comprising a polynucleotide sequence according to the invention and primers, probes and fragments that may be used to amplify or detect the DNA according to the invention.

In a further preferred embodiment, a vector is provided wherein the polynucleotide sequence according to the invention is operably linked with at least one regulatory sequence allowing for expression of the polynucleotide sequence in a suitable host cell. Preferably said suitable host cell is a filamentous fungus, more preferably Aspergillus species. Suitable strains belong to Aspergillus niger, oryzae or nidulans. Preferably the host cell is Aspergillus niger.

The invention also relates to recombinantly produced host cells that contain polynucleotides according to the invention.

The invention also provides methods for preparing polynucleotides and vectors according to the invention.

In another embodiment, the invention provides recombinant host cells wherein the expression of a polynucleotide according to the invention is significantly increased or wherein the production level of lipolytic activity is significantly improved. In another embodiment the invention provides for a recombinantly produced host cell that contains heterologous or homologous DNA according to the invention and wherein the cell is capable of producing a functional lipolytic enzyme according to the invention, i.e. it is capable of expressing or preferably over-expressing a polynucleotide encoding for the lipolytic enzyme according to the invention, for example an Aspergillus strain comprising an increased copy number of a gene according to the invention.

In yet another aspect of the invention, an isolated polypeptide having lipolytic acitivity is provided. The polypeptides according to the invention comprises preferably an amino acid sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof having an amino acid sequence at least 60, 70, 80 or 90% homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2.

In one embodiment the invention also relates to an isolated polypeptide having lipolytic activity which is a functional equivalent of the mature polypeptide in the amino acid sequence of SEQ ID NO: 2, which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% homologous to said mature polypeptide.

Fusion proteins comprising a polypeptide according to the invention are also within the scope of the invention. The invention also provides methods of making the polypeptides according to the invention.

The invention also relates to the use of the lipolytic enzyme according to the invention in any industrial process as described herein, more particular as medicament or pharmaceutical

Polynucleotides

The present invention provides in a first aspect an isolated polynucleotide which comprises a nucleotide sequence selected from:

(a) a nucleotide sequence as set out in SEQ ID NO: 1 having at least 60, 70, 80 or 90% homology to the nucleotide sequence of SEQ ID NO: 1 ;

(c) a nucleotide sequence encoding the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 having at least 60, 70, 80 or 90% homology to the mature polypeptide in the amino acid sequence of SEQ ID NO: 2;

(d) a sequence which is degenerate as a result of the degeneracy of the genetic code to a sequence as defined in any one of (a), (b), (c); (e) a nucleotide sequence which is the complement of a nucleotide sequence as defined in (a), (b), (c), (d).

In one embodiment, the present invention provides polynucleotides encoding lipolytic enzymes, having an amino acid sequence corresponding to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 or functional equivalents having at least 60, 70, 80 or 90% homology to the amino acid sequence corresponding to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2.

In the context of the present invention "mature polypeptide" is defined herein as a polypeptide having lipolytic activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. The process of maturation may depend on the particular expression vector used, the expression host and the production process. A "nucleotide sequence encoding the mature polypeptide" is defined herein as the polynucleotide sequence which codes for the mature polypeptide. In another embodiment the invention relates to an isolated polynucleotide encoding an isolated polypeptide having lipolytic activity which is a functional equivalent of the mature polypeptide in the amino acid sequence of SEQ ID NO:2, which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% homologous to said mature polypeptide.

The invention provides polynucleotide sequences comprising the gene encoding the lipolytic enzyme as well as its coding sequence. Accordingly, the invention relates to an isolated polynucleotide comprising the nucleotide sequence according to SEQ ID NO: 1 or to variants such as functional equivalents thereof having at least 60, 70, 80 or 90% homology to SEQ ID NO: 1.

In particular, the invention relates to an isolated polynucleotide comprising a nucleotide sequence which hybridises, preferably under stringent conditions, more preferably under highly stringent conditions, to the complement of a polynucleotide according to SEQ ID NO: 1 and wherein preferably said sequence is at least 60, 70, 80 or 90% homologous to the nucleotide sequence of SEQ ID NO: 1.

More specifically, the invention relates to an isolated polynucleotide comprising or consisting essentially of a nucleotide sequence according to SEQ ID NO: 1. Such isolated polynucleotide may be obtained by synthesis with methods known to the person skilled in the art.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding a protein, e.g. a lipolytic enzyme. A gene may include coding sequences, non-coding sequences, introns and regulatory sequences. Moreover, a gene refers to an isolated nucleic acid molecule or polynucleotide as defined herein.

A nucleic acid molecule of the present invention, such as a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 or a functional equivalent thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of the nucleic acid sequence of SEQ ID NO: 1 as a hybridization probe, nucleic acid molecules according to the invention can be isolated using standard hybridization and cloning techniques (e. g., as described in Sambrook, J., Fritsh, E. F., and

Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor

Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQ

ID NO: 1 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained in SEQ ID NO: 1.

A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to or hybridisable to the complement of the nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence according to SEQ ID NO: 1. The sequence of SEQ ID NO: 1 encodes the polypeptide according to SEQ ID NO: 2 and the lipolytic enzyme according to the mature polypeptide in SEQ ID NO: 2. The lipolytic enzyme according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 is indicated as L01. The nucleotide sequence according to SEQ ID NO: 1 is indicated as DNA L01.

In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO: 1 or a functional equivalent of these nucleotide sequences.

A nucleic acid molecule which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex.

One aspect of the invention pertains to isolated nucleic acid molecules that encode a polypeptide of the invention or a variant, such as a functional equivalent thereof, for example a biologically active fragment or domain, as well as nucleic acid molecules sufficient for use as hybridisation probes to identify nucleic acid molecules encoding a polypeptide of the invention and fragments of such nucleic acid molecules suitable for use as PCR primers for the amplification or mutation of nucleic acid molecules.

An "isolated polynucleotide" or "isolated nucleic acid" is a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promotor) sequences that are immediately contiguous to the coding sequence. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state.

As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a nucleic acid molecule according to the invention, e.g., the coding strand of a nucleic acid molecule according to the invention.

Also included within the scope of the invention are the complement strands of the polynucleotides according to the invention.

Nucleic acid fragments, probes and primers

A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence according to SEQ ID NO: 1 , for example a fragment which can be used as a probe or primer or a fragment encoding a portion of a the protein according to the invention. The nucleotide sequence according to the invention allows for the generation of probes and primers designed for use in identifying and/or cloning functional equivalents of the protein according to the invention having at least 60, 70, 80 or 90% homology to the protein according to SEQ ID NO: 2. The probe/primer typically comprises substantially purified oligonucleotide which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, preferably about 22 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence according to the invention.

Probes based on the nucleotide sequences according to the invention, more preferably based on SEQ ID NO: 1 can be used to detect transcripts or genomic sequences encoding the same or homologous proteins for instance in organisms. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can also be used as part of a diagnostic test kit for identifying cells which express a protein according to the invention.

Identity & homology

The terms "homology" or "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent homology of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/based or amino acids. The identity is the percentage of identical matches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The skilled person will be aware of the fact that several different computer programs are available to align two sequences and determine the homology between two sequences (Kruskal, J. B. (1983) An overview of squence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The percent identity between two amino acid sequences or between two nucleotide sequences may be determined using the Needleman and Wunsch algorithm for the alignment of two sequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453). Both aminoacid sequences and nucleotide sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purpose of this invention the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. LongdenJ. and Bleasby,A. Trends in Genetics 16, (6) pp276 — 277, http://emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. The optional parameters used are a gap-open penalty of 10 and a gap extension penalty of 0.5. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms. After alignment by the program NEEDLE as described above the percentage of identity between a query sequence and a sequence of the invention is calculated as follows: Number of corresponding positions in the alignment showing an identical aminoacid or identical nucleotide in both sequences devided by the total length of the alignment after substraction of the total number of gaps in the alignment. The identity defined as herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled in the output of the program as "longest- identity".

The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (199O) J. MoI. Biol. 215:403— 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

Hybridisation

As used herein, the term "hybridizing" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about

60%, 65%, 80%, 85%, 90%, preferably at least 93%, more preferably at least 95% and most preferably at least 98% homologous to each other typically remain hybridized to the complement of each other.

A preferred, non-limiting example of such hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45⁰C, followed by one or more washes in 1 X SSC, 0.1 % SDS at 5O⁰C, preferably at 55⁰C, preferably at 6O⁰C and even more preferably at 65⁰C. Highly stringent conditions include, for example, hybridizing at 68⁰C in 5x

SSC/5x Denhardt's solution / 1.0% SDS and washing in 0.2x SSC/0.1% SDS at room temperature. Alternatively, washing may be performed at 42⁰C.

The skilled artisan will know which conditions to apply for stringent and highly stringent hybridisation conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.). Of course, a polynucleotide which hybridizes only to a poly A sequence

(such as the 3' terminal poly(A) tract of mRNAs), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-standed cDNA clone).

Obtaining full length DNA from other organisms

In a typical approach, cDNA libraries constructed from other organisms, e.g. filamentous fungi, in particular from the species Fusarium can be screened.

For example, Fusarium strains can be screened for homologous polynucleotides with respect to SEQ ID NO:1 , by Northern blot analysis. Upon detection of transcripts homologous to polynucleotides according to the invention, cDNA libraries can be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art. Alternatively, a total genomic DNA library can be screened using a probe hybridisable to a polynucleotide according to the invention.

Homologous gene sequences can be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.

The template for the reaction can be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention. The PCR product can be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new nucleic acid sequence according to the invention, or a functional equivalent thereof.

The PCR fragment can then be used to isolate a full-length cDNA clone by a variety of known methods. For example, the amplified fragment can be labeled and used to screen a bacteriophage or cosmid cDNA library. Alternatively, the labeled fragment can be used to screen a genomic library.

PCR technology also can be used to isolate full-length cDNA sequences from other organisms. For example, RNA can be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction can be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.

The resulting RNA/DNA hybrid can then be "tailed" (e.g., with guanines) using a standard terminal transferase reaction, the hybrid can be digested with RNase H, and second strand synthesis can then be primed (e.g., with a poly-C primer). Thus, cDNA sequences upstream of the amplified fragment can easily be isolated. For a review of useful cloning strategies, see e.g., Sambrook et al., supra; and Ausubel et al., supra.

Vectors Another aspect of the invention pertains to vectors, including cloning and expression vectors, comprising a polynucleotide sequence according to the invention encoding a polypeptide having lipolytic acitivity or a functional equivalent thereof according to the invention. The invention also pertains to methods of growing, transforming or transfecting such vectors in a suitable host cell, for example under conditions in which expression of a polypeptide of the invention occurs. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

Polynucleotides of the invention can be incorporated into a recombinant replicable vector, for example a cloning or expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are described below.

The vector into which the expression cassette or polynucleotide of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. A vector according to the invention may be an autonomously replicating vector, i. e. a vector which exists as an extra-chromosomal 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 chromosome (s) into which it has been integrated.

One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably herein as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as cosmid, viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) and phage vectors which serve equivalent functions.

Vectors according to the invention may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

A vector of the invention may comprise two or more, for example three, four or five, polynucleotides of the invention, for example for overexpression.

The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.

Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell), i.e. the term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences or the sequences are arranged so that they function in concert for their intended purpose, for example transcription initiates at a promoter and proceeds through the DNA sequence encoding the polypeptide.

The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

The term regulatory sequences includes those sequences which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in a certain host cell (e.g. tissue-specific regulatory sequences).

A vector or expression construct for a given host cell may thus comprise the following elements operably linked to each other in a consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the polypeptide of the first invention: (1 ) a promoter sequence capable of directing transcription of the nucleotide sequence encoding the polypeptide in the given host cell ; (2) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into a culture medium; (3) a DNA sequence of the invention encoding a mature and preferably active form of a polypeptide having having lipolytic activity according to the invention; and preferably also (4) a transcription termination region (terminator) capable of terminating transcription downstream of the nucleotide sequence encoding the polypeptide.

Downstream of the nucleotide sequence according to the invention there may be a 3' untranslated region containing one or more transcription termination sites (e. g. a terminator). The origin of the terminator is less critical. The terminator can, for example, be native to the DNA sequence encoding the polypeptide. However, preferably a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell (in which the nucleotide sequence encoding the polypeptide is to be expressed). In the transcribed region, a ribosome binding site for translation may be present. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

Enhanced expression of the polynucleotide of the invention may also be achieved by the selection of heterologous regulatory regions, e. g. promoter, secretion leader and/or terminator regions, which may serve to increase expression and, if desired, secretion levels of the protein of interest from the expression hostand/or to provide for the inducible control of the expression of a polypeptide of the invention.

It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. the polypeptide having lipolytic activity according to the invention, mutant forms the polypeptide, fragments, variants or functional equivalents thereof, fusion proteins, etc.). The recombinant expression vectors of the invention can be designed for expression of the polypeptides according to the invention in prokaryotic or eukaryotic cells. For example, the polypeptides according to the invention can be produced in bacterial cells such as E. coli and Bacilli, insect cells (using baculovirus expression vectors), fungal cells, yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. For most filamentous fungi and yeast, the vector or expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants. However, for certain yeasts also suitable episomal vectors are available into which the expression construct can be incorporated for stable and high level expression, examples thereof include vectors derived from the 2μ and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMA1 from Aspergillus). In case the expression constructs are integrated in the host cells genome, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene.

Accordingly, expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episome, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.

The nucleotide insert should be operatively linked to an appropriate promoter. Aside from the promoter native to the gene encoding the polypeptide of the invention, other promoters may be used to direct expression of the polypeptide of the invention. The promoter may be selected for its efficiency in directing the expression of the polypeptide of the invention in the desired expression host. Examples of promoters which may be useful in the invention include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled person. In a specific embodiment, promoters are preferred that are capable of directing a high expression level of the polypeptides according to the invention in a fungus or yeast. Such promoters are known in the art.

A variety of promoters can be used that are capable of directing transcription in the host cells of the invention. Preferably the promoter sequence is derived from a highly expressed gene. Examples of preferred highly expressed genes from which promoters are preferably derived and/or which are comprised in preferred predetermined target loci for integration of expression constructs, include but are not limited to genes encoding glycolytic enzymes such as triose- phosphate isomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK or PKI), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, proteases, xylanases, cellobiohydrolases,β-galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins. Specific examples of suitable highly expressed genes include e. g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively, the glucoamylase {glaA) genes from A. niger and A. awamori, the A. oryzae TAKA-amylase gene, the A. nidulans gpdA gene and the T. reesei cellobiohydrolase genes.

Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (x/nA), phytase, ATP-synthetase, subunit 9 (o//C), triose phosphate isomerase (tpi), alcohol dehydrogenase [AdhA), a-amylase {amy), amyloglucosidase (AG-from the glaA gene), acetamidase {amdS) and glyceraldehyde-3-phosphate dehydrogenase {gpd) promoters.

Examples of strong yeast promoters are those obtainable from the genes for alcohol dehydrogenase, lactase, 3-phosphoglycerate kinase andtriosephosphate isomerase.

Examples of strong bacterial promoters are the α-amylase and SPo2 promoters as well as promoters from extracellular protease genes.

Promoters suitable for plant cells include nopaline synthase {nos), octopine synthase (ocs), mannopine synthase (mas), ribulose small subunit (rubisco ssu), histone, rice actin, phaseolin, cauliflower mosaic virus (CMV) 35S and 19S and circovirus promoters.

All of the above-mentioned promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to RNA which comprise sequences homologous to eukaryotic genomic sequences or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of a host cell.

The vector may contain a polynucleotide of the invention oriented in an antisense direction to provide for the production of antisense RNA. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art- recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-percipitation, DEAE- dextran-mediated transfection, transduction, infection, lipofection, cationic lipidmediated transfection or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2^nd,ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al., Basic Methods in Molecular Biology (1986) and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include, but are not limited to, those which confer resistance to drugs or which complement a defect in the host cell. They include e. g. versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdS, niaD, facA genes or cDNAs from A. nidulans, A. oryzae or A. niger), or genes providing resistance to antibiotics like G418, hygromycin, bleomycin, kanamycin, methotrexate, phleomycin orbenomyl resistance (benA). Alternatively, specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e. g.URA3 (from S. cerevisiae or analogous genes from other yeasts), pyrG or pyrA (from A. nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC. In a preferred embodiment the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes.

Other markers include ATP synthetase, subunit 9 (oliC), orotidine-5'- phosphatedecarboxylase (pvrA), the bacterial G418 resistance gene (this may also be used in yeast, but not in fungi), the ampicillin resistance gene (£. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for β- glucuronidase (GUS). Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

Expression of proteins in prokaryotes is often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, e.g. to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.

As indicated, the expression vectors will preferably contain selectable markers. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracyline or ampicillin resistance for culturing in E. coli and other bacteria. Representative examples of appropriate host include bacterial cells, such as E. coli, Streptomyces Salmonella typhimurium and certain Bacillus species; fungal cells such as Aspergillus species, for example A. niger, A. oryzae and A. nidulans, such as yeast such as Kluyveromyces, for example K. lactis and/or Puchia, for example P. pastoris; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS and Bowes melanoma; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art. Vectors preferred for use in bacteria are for example disclosed in W0-A1-

2004/074468, which are hereby enclosed by reference. Other suitable vectors will be readily apparent to the skilled artisan.

Known bacterial promotors suitable for use in the present invention include the promoters disclosed in W0-A1 -2004/074468, which are hereby enclosed by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretation signal may be incorporated into the expressed gene. The signals may be endogenous to the polypeptide or they may be heterologous signals. The polypeptide according to the invention may be produced in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification.

Polypeptides according to the invention

The invention provides an isolated polypeptide having lipolytic activity comprising:

(a) the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof having an amino acid sequence at least 60, 70, 80 or 90% homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2; (b) the mature polypeptide in the amino acid sequence encoded by a polynucleotide according to the invention.

Therefore the invention provides an isolated polypeptide having lipolytic activity comprising the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2, and an amino acid sequence obtainable by expressing the polynucleotide of SEQ ID NO: 1 in an appropriate host. Also, a peptide or polypeptide being a functional equivalent and being at least 60, 70, 80 or 90% homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 is comprised within the present invention. In another embodiment the invention also relates to an isolated polypeptide having lipolytic activity which is a functional equivalent of the mature polypeptide in the amino acid sequence of SEQ ID NO: 2, which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% homologous to said mature polypeptide.

The above polypeptides are collectively comprised in the term "polypeptides according to the invention".

The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" (or protein) is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2^nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989),

By "isolated" polypeptide or protein is intended a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins produced in host cells are considered isolated for the purpose of the invention as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

As is known to the person skilled in the art it is possible that the N-termini of SEQ ID NO: 2 or of the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 might be heterogeneous as well as the C-terminus of SEQ ID NO: 2 or of the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2, due to processing errors during maturation. In particular such processing errors might occur upon overexpression of the polypeptide. In addition, exo-protease activity might give rise to heterogeneity. The extent to which heterogeneity occurs depends also on the host and fermentation protocols that are used. Such C-terminual processing artefacts might lead to shorter polypeptides or longer polypeptides as indicated with SEQ ID NO: 2 or with the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2. As a result of such errors the N-terminus might also be heterogeneous. In a further embodiment, the invention provides an isolated polynucleotide encoding at least one functional domain of a polypeptide according to SEQ ID NO: 2 or of the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 which contain additional residues and start at position -1 , or -2, or -3 etc. Alternatively, it might lack certain residues and as a consequence start at position 2, or 3, or 4 etc. Also additional residues may be present at the C-terminus. Alternatively, the C-terminus might lack certain residues.

The lipolytic enzyme according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art (Protein Purification Protocols, Methods in Molecular Biology series by Paul Cutler, Humana Press, 2004).

Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

Polypeptide fragments

The invention also features biologically active fragments of the polypeptides according to the invention. Biologically active fragments of a polypeptide of the invention include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the protein according to the invention (e.g., the mature polypeptide in the amino acid sequence of SEQ ID NO: 2), which include fewer amino acids than the full length protein but which exhibit at least one biological activity of the corresponding full-length protein, preferably which exhibit lipolytic activity. Typically, biologically active fragments comprise a domain or motif with at least one activity of the protein according to the invention. A biologically active fragment of a protein of the invention can be a polypeptide which is, for example, 5, 10, 15, 20, 25, or more amino acids in length shorter than the mature polypeptide in SEQ ID NO: 2, and which has at least 60, 70, 80 or 90% homology to the mature polypeptide in SEQ ID NO: 2. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the biological activities of the native form of a polypeptide of the invention.

The invention also features nucleic acid fragments which encode the above biologically active fragments of the protein according to the invention.

Fusion proteins The polypeptides according to the invention or functional equivalents thereof, e.g., biologically active portions thereof, can be operably linked to a polypeptide not according to the invention (e.g., heterologous amino acid sequences) to form fusion proteins. A "polypeptide not according to the invention" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the protein according to the invention. Such "non- polypeptide not according to the invention" can be derived from the same or a different organism. Within a fusion protein the polypeptide according to the invention can correspond to all or a biologically active fragment of the lipolytic enzyme according to the invention. In a preferred embodiment, a fusion protein comprises at least two biologically active portions of the protein according to the invention. Within the fusion protein, the term "operably linked" is intended to indicate that the polypeptide according to the invention and the polypeptide not according to the invention are fused in-frame to each other. The polypeptide not according to the invention can be fused to the N-terminus or C-terminus of the polypeptide.

For example, in one embodiment, the fusion protein is a fusion protein in which the amino acid sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of the recombinant protein according to the invention. In another embodiment, the fusion protein according to the invention is a protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and yeast host cells), expression and/or secretion of the protein according to the invention can be increased through use of a hetereologous signal sequence.

In another example, the gp67 secretory sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretory sequences of melittin and human placental alkaline phosphatase (Stratagene; La JoIIa, California). In yet another example, useful prokarytic heterologous signal sequences include the phoA secretory signal (Sambrook et al., supra) and the protein A secretory signal (Pharmacia Biotech; Piscataway, New Jersey).

A signal sequence can be used to facilitate secretion and isolation of a protein or polypeptide of the invention. Signal sequences are typically characterized by a core of hydrophobic amino acids, which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by known methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence, which facilitates purification, such as with a GST domain. Thus, for instance, the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purificaton of the fusion protein. The HA tag is another peptide useful for purification which corresponds to an epitope derived of influenza hemaglutinin protein, which has been described by Wilson et al., Cell 37:767 (1984), for instance.

Preferably, a fusion protein according to the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger- ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers, which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g, a GST polypeptide). A nucleic acid encoding for a polypeptide according to the invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the protein according to the invention.

Functional equivalents

The terms "functional equivalents" and "functional variants" are used interchangeably herein.

Functional equivalents of the polynucleotide according to the invention are isolated polynucleotides having at least 60%, 65%, 70%, 75%, 80%, 85%, preferably at least 60, 70, 80 or 90% homology to the nucleotide sequence of SEQ ID NO: 1 and that encodes a polypeptide that exhibits at least a particular function of the lipolytic enzyme according to the invention, preferably a polypeptide having lipolytic activity. A functional equivalent of a polypeptide according to the invention is a polypeptide having at least 60%, 65%, 70%, 75%, 80%, 85%, preferably at least 90% homology to the mature polypeptide in the amino acid sequence of SEQ ID NO: 2 and that exhibits at least one function of a lipolytic enzyme according to the invention, preferably which exhibits lipolytic activity. Functional equivalents as mentioned herewith also encompass biologically active fragments having lipolytic activity as described above.

Functional equivalents of the polypeptide according to the invention may contain substitutions of one or more amino acids of the mature polypeptide of the amino acid sequence according to SEQ ID NO: 2 or substitutions, insertions or deletions of amino acids which do not affect the particular functionality of the enzyme. Accordingly, a functionally neutral amino acid substitution is a susbtitution in the mature polypeptide of the amino acid sequence according to SEQ ID NO: 2 that does not substantially alters its particular functionality. For example, amino acid residues that are conserved among the proteins of the present invention are predicted to be particularly unamenable to alteration. Furthermore, amino acids conserved among the proteins according to the present invention and other lipolytic enzymes are not likely to be amenable to alteration.

Functional equivalents of the polynucleotides according to the invention may typically contain silent mutations or mutations that do not alter the biological function of the encoded polypeptide. Accordingly, the invention provides nucleic acid molecules encoding polypeptides according to the invention that contain changes in amino acid residues that are not essential for a particular biological activity. Such proteins differ in amino acid sequence from the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 and yet retain at least one biological activity thereof, preferably they retain the lipolytic activity. In one embodiment a functional equivalent of the polynucleotide according to the invention comprises a nucleotide sequence encoding a polypeptide according to the invention, wherein the polypeptide comprises a substantially homologous amino acid sequence of at least about 60%, 65%, 70%, 75%, 80%, 85%, preferably at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2.

A functional equivalent of the polynucleotide according to the invention encoding a polypeptide according to the invention will comprise a polynucleotide sequence which is at least about 60%, 65%, 70%, 75%, 80%, 85%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence according to SEQ ID NO 1.

An isolated polynucleotide encoding a protein homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 can be created by introducing one or more nucleotide substitutions, additions or deletions into the coding nucleotide sequences according to SEQ ID NO: 1 such that one or more amino acid substitutions, deletions or insertions are introduced into the encoded protein. Such mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. The polynucleotides according to the invention may be optimized in their codon use, preferably according to the methods described in WO2006/077258 and/or WO2008/000632. WO2008/000632 addresses codon- pair optimization. Codon-pair optimisation is a method wherein the nucleotide sequences encoding a polypeptide are modified with respect to their codon- usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the encoded polypeptide. Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence. Nucleic acid molecules corresponding to variants (e.g. natural allelic variants) and homologues of the polynucleotides according to the invention can be isolated based on their homology to the nucleic acids disclosed herein using the cDNAs disclosed herein or a suitable fragment thereof, as a hybridisation probe according to standard hybridisation techniques preferably under highly stringent hybridisation conditions.

In another aspect of the invention, improved proteins are provided. Improved proteins are proteins wherein at least one biological activity is improved if compared with the biological activity of the polypeptide having amino acid sequence according to SEQ ID NO: 2. Such proteins may be obtained by randomly introducing mutations along all or part of the coding sequence SEQ ID NO: 1 , such as by saturation mutagenesis, and the resulting mutants can be expressed recombinantly and screened for biological activity. For instance, the art provides for standard assays for measuring the enzymatic activity of lipolytic enzymes and thus improved proteins may easily be selected. In a preferred embodiment the polypeptide is at least 60, 70, 80 or 90% homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 and retains at least one biological activity of a mature polypeptide in the amino acid sequence according to SEQ ID NO: 2, preferably it retains the lipolytic activity and yet differs in amino acid sequence due to natural variation or mutagenesis as described above.

In a further preferred embodiment, the protein according to the invention has an amino acid sequence encoded by an isolated nucleic acid fragment which hybridizes with a polynucleotide being the complement of SEQ ID NO: 1 and wherein said nucleotide sequence is at least 60, 70, 80 or 90% homologous to the nucleotide sequence of SEQ ID NO: 1 , preferably under highly stringent hybridisation conditions.

Accordingly, the protein according to the invention is preferably a protein which comprises an amino acid sequence at least about 90%, 91% 92% 93% 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO 2 and retains at least one functional activity of the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2.

Functional equivalents of a protein according to the invention can also be identified e.g. by screening combinatorial libraries of mutants, e.g. truncation mutants, of the protein of the invention for lipolytic enzyme activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display). There are a variety of methods that can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; ltakura et al. (1984) Annu. Rev. Biochem. 53:323; ltakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 1 1 :477).

In addition, libraries of fragments of the coding sequence of a polypeptide of the invention can be used to generate a variegated population of polypeptides for screening a subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations of truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811- 7815; Delgrave et al. (1993) Protein Engineering 6(3): 327-331 ). Fragments of a polynucleotide according to the invention may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids according to the invention irrespective of whether they encode functional or non-functional polypeptides can be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a lipolytic activity according to the invention include, inter alia, (1 ) isolating the gene encoding the protein, or allelic variants thereof from a cDNA library; (2) in situ hybridization (e.g. FISH) to metaphase chromosomal spreads to provide precise chromosomal location of the gene as described in Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern blot analysis for detecting expression of mRNA in specific tissues and/or cells and 4) probes and primers that can be used as a diagnostic tool to analyse the presence of a nucleic acid hybridisable to the probe in a given biological (e.g. tissue) sample.

Also encompassed by the invention is a method of obtaining a functional equivalent of a gene according to the invention. Such a method entails obtaining a labelled probe that includes an isolated nucleic acid which encodes all or a portion of the protein sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 or a variant of any of them; screening a nucleic acid fragment library with the labelled probe under conditions that allow hybridisation of the probe to nucleic acid fragments in the library, thereby forming nucleic acid duplexes, and preparing a full-length gene sequence from the nucleic acid fragments in any labelled duplex to obtain a gene related to the gene according to the invention.

Host cells

In another embodiment, the invention features cells, e.g., transformed host cells or recombinant host cells comprising a polynucleotide according to the invention or comprising a vector according to the invention.

A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a nucleic acid according to the invention. Both prokaryotic and eukaryotic cells are included, e.g., bacteria, fungi, yeast, and the like. Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines. A number of vectors suitable for stable transfection of mammalian cells are available to the public, methods for constructing such cell lines are also publicly known, e.g., in Ausubel et al. (supra). Especially preferred are cells from filamentous fungi, in particular Aspergillus species such as Aspergillus niger or oryzae or awamori.

A host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.

Various host cells have characteristic and specific mechanisms for post- translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those of skill in the art of molecular biology and/or microbiology can be chosen to ensure the desired and correct modification and processing of the foreign protein produced. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such host cells are well known in the art. If desired, a cell as described above may be used to in the preparation of a polypeptide according to the invention. Such a method typically comprises cultivating a recombinant host cell (e. g. transformed or transfected with an expression vector as described above) under conditions to provide for expression (by the vector) of a coding sequence encoding the polypeptide, and optionally recovering, more preferably recovering and purifying the produced polypeptide from the cell or culture medium. Polynucleotides of the invention can be incorporated into a recombinant replicable vector, e. g. an expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector. The vector may be recovered from the host cell. Preferably the polypeptide is produced as a secreted protein in which case the nucleotide sequence encoding a mature form of the polypeptide in the expression construct is operably linked to a nucleotide sequence encoding a signal sequence. Preferably the signal sequence is native (homologous) to the nucleotide sequence encoding the polypeptide. Alternatively the signal sequence is foreign (heterologous) to the nucleotide sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the nucleotide sequence according to the invention is expressed. Examples of suitable signal sequences for yeast host cells are the signal sequences derived from yeast a-factor genes. Similarly, a suitable signal sequence for filamentous fungal host cells is e. g. a signal sequence derived from a filamentous fungal amyloglucosidase (AG) gene, e. g. the A. niger glak gene. This may be used in combination with the amyloglucosidase (also called (gluco) amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences may also be used with the context of the present invention. Preferred heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (g/aA-both 18 and 24 amino acid versions e. g. from Aspergillus), the α-factor gene (yeasts e. g. Saccharomyces and Kluyveromyces) or the α-amylase gene {Bacillus).

The vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide. The invention thus provides host cells transformed or transfected with or comprising a polynucleotide or vector of the invention. Preferably the polynucleotide is carried in a vector for the replication and expression of the polynucleotide. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.

A heterologous host may also be chosen wherein the polypeptide of the invention is produced in a form which is substantially free of enzymatic activities that might interfere with the applications, e.g. free from starch degrading, cellulose-degrading or hemicellulose degrading enzymes. This may be achieved by choosing a host which does not normally produce such enzymes.

The invention encompasses processes for the production of the polypeptide of the invention by means of recombinant expression of a DNA sequence encoding the polypeptide. For this purpose the DNA sequence of the invention can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the polypeptide in a suitable homologous or heterologous host cell. A homologous host cell is a host cell which is of the same species or which is a variant within the same species as the species from which the DNA sequence is derived. Suitable host cells are preferably prokaryotic microorganisms such as bacteria, or more preferably eukaryotic organisms, for example fungi, such as yeasts or filamentous fungi, or plant cells. In general, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from yeasts, or in some cases are not processed properly (e. g. hyperglycosylation in yeast). In these instances, a fungal host organism should be selected.

The host cell may over-express the polypeptide, and techniques for engineering over-expression are well known. The host may thus have two or more copies of the encoding polynucleotide (and the vector may thus have two or more copies accordingly).

Therefore in one embodiment of the invention the recombinant host cell according to the invention is capable of expressing or overexpressing a polynucleotide or vector according to the invention. According to the present invention, the production of the polypeptide of the invention can be effected by the culturing of a host cell according to the invention, which have been transformed with one or more polynucleotides of the present invention, in a conventional nutrient fermentation medium.

The recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available which are conducive to the expression the DNA sequence encoding the polypeptide. After reaching the desired cell density or titre of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures. The fermentation medium can comprise a known culture medium containing a carbon source (e. g. glucose, maltose, molasses, etc.), a nitrogen source (e. g. ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e. g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e. g. phosphate, magnesium, potassium, zinc, iron, etc.).

The selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the expression construct. Such media are known to those skilled in the art. The medium may, if desired, contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms.

The fermentation can be performed over a period of 0.5-30 days. It may be a batch, continuous or fed-batch process, suitably at a temperature in the range of, for example, from about 0 to 45⁰C and/or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions are a temperature in the range of from about 20 to about 37⁰C and/or at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of the expression host and the protein to be produced.

After fermentation, if necessary, the cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped or after removal of the cells, the polypeptide of the invention may then be recovered and, if desired, purified and isolated by conventional means.

Legends to the Figures Figure 1 : The substrate preference of lipolytic enzyme LPY assayed using p- nitrophenyl-acetate, -butyrate, -decanoate and -palmitate.

Figure 2: The pH optimum of lipolytic enzyme LPY assayed using p-nitrophenyl palmitate as the substrate Figure 3: SDS-PAGE analysis of the lipase according to the invention after incubation for 90 minutes at 37 degrees C with and without pepsin at pH 4.0.

Lane 1 : molecular weight markers (Invitrogen); lane 2: pepsin (Sigma) in the low concentration; lane 3: whey protein isolate (Bipro-Davisco) as such (after incubation); lane 4: whey protein isolate after incubation with pepsin in the low concentration; lane 5: whey protein isolate after incubation with pepsin in the high concentration; lane 6: lipase LPY as such (after incubation); lane 7: lipase LPY after incubation with pepsin in the low concentration; lane 8: lipase LPY after incubation with pepsin in the high concentration; lane 9: pepsin (Sigma) in the high concentration; lane 10: molecular weight markers (Invitrogen). Figure 4: The pH optimum of lipolytic enzyme LPY assayed using olive oil in combination with gum arabic as a substrate

Materials & Methods

Triacyl-glycerol lipase activity assay using olive oil and gum arabic.

Lipase activity assays often use a commercially available mix of triolein, high concentrations of Triton X-100 and NaCI ("Lipase substrate" from Fluka; art. no. 62314). Although the stable emulsion foms an ideal substrate for various lipolytic enzymes, it does not necessarily reflect the substrate conditions in human gastrointestinal tract. In order to imitate the in vivo situation of lipid hydrolysis in the stomach as closely as possible, an activity assay was developed in which olive oil was used as a natural substrate and gum arabic as an emulsion stabilizer. Similar to the activity test with triolein, in the olive oil assay enzymatic activity is measured by quantitating the free fatty acids produced. The olive oil emulsion was prepared by adding 50 ml of a 1.0% (w/v) aqueous solution of gum arabic (Reagent Grade from Sigma) to 1.0 g of olive oil, which was then mixed at 10.000 rpm for 3 minutes in an Ultra Turrax. To 500 μl of the emulsion 50 μl of a 0.1 M aqueous solution of calcium chloride and 250 μl of the desired aqueous buffer solution (0.2 mol/l) were added. The total mixture was then pre-heated in a water bath at 37^°C for about 10 minutes, then 100 μl of the enzyme solution was added, the mixture was vortexed for 30 seconds and then incubated for 10 minutes . The reaction was stopped by adding 100 μl of 1 N aqueous solution of hydrochloric acid. To dissolve the fatty acids liberated during the reaction, 2.0 ml of a 5.0% (w/v) aqueous solution of Triton X-100 was added and mixed again. The amount of free fatty acids contained in 10 μl of the reaction mixture was determined with the NEFA kit (HR series NEFA-HR(2); Wako ChemicalsGmbH, Neuss, Germany). The analysis was performed with the analyzer KONELAB-ARENA 30 (ThermoScientific, Breda, The Netherlands). The enzyme activity that produces 1 μmol of fatty acids per minute of the enzyme reaction was defined as 1 unit.

Other lipase assays used

Measuring lipolytic activity using the triolein "Fluka" assay

Basically this assay is identical to the olive oil/gum arabic assay but in this case the emulsion formed is stabilized by using a high concentration of the emulsifier Triton-X100. In this test starting point is the commercially available lipase substrate provided by Fluka. This emulsion contains triolein (4.5 millimol/l), Triton X-100 (13%w/v) and NaCI (1 mol/l). To 500 μl of this triolein emulsion 50 μl of a 0.1 M aqueous solution of calcium chloride and 250 μl of the desired aqueous buffer solution (0.2 mol/l) were added. similar to the procedure described for the olive oil/gum arabic assay. After a pre-heating at 37^°C, again 100 μl of the enzyme solution was added and incubated as described. As described earlier, the amount of free fatty acids contained in 10 μl of the reaction mixture was determined with the NEFA kit. The analysis was performed with the analyzer KONELAB-ARENA 30. The enzyme activity that produces 1 μmol of fatty acids per minute of the enzyme reaction was defined as 1 unit.

Measuring lipolytic activity using p-nitrophenyl palmitate (pNPP) as a substrate The lipolytic activity towards long chain triacylglycerol was determined in an assay with the chromogenic substrate p-nitrophenyl palmitate (pNPP). The substrate (Sigma N2752) was dissolved in 2-propanol (4 mg/mL). While vigorously stirring 5 ml. of this solution was drop wise added to 45 mL 100 millimol/l acetate buffer pH 4.0 containing 1 % Triton X-100. At time t=0, 50 μl_ of sample was mixed with 1 mL substrate solution. After 7.5 minutes incubation at 37 ⁰C, the reaction was stopped by adding 1 mL of 2% TRIS solution also containing 1% Triton X-100 and the change in absorption was measured at 405 nm against a sample blank. The activity is expressed in pNPP units/ml. One pNPP Unit is the amount of enzyme that liberates 1 micromole pNP per minute at 37 ⁰C. Measuring Ivsophospholipase activity The lysophospholipase activity of the enzyme according to the invention was determined in an assay in which lysophosphatidyl choline (Sigma L-4129) was used as the substrate. To that end lysophosphatidyl choline was dissolved in a concentration of 1 mg/mL in 100 millimol/l acetate buffer pH 4.5 in presence of 5 millimol/l Ca²⁺ and 0.2% Triton X-100. The enzyme (50 μL) was incubated with 1 mL substrate at 37 ⁰C for 30 minutes. Subsequently the reaction was stopped by freezing the sample in liquid nitrogen.

The sample was dried via lyophilisation and subsequently solved in deuterated chloroform (CDCI₂) containing triethyl phosphate as internal standard. The conversion of the substrate is determined with ³¹P-NMR. The activity is expressed in U/ml. One lysophospholipase unit is the amount of enzyme that liberates 1 micromole glycerophosphocholine per minute at pH 4.5 and 37 ⁰C. Measuring phospholipase activity

Phospholipase activity (either A1 , A2 or B) was determined spectrophotometrically by using 1 ,2-dimercaptodioctanoyl-phosphatidylcholine (Syncom B.V., Groningen, The Netherlands) as a substrate. Phospholipase hydrolyses the thioester bond at the 1 position (PLA1 ), at the 2 position (PLA2) or at both positions (PLB) thereby liberating octamoic acid and 1 , 2-dimercapto-mono-octanoyl-phosphatidylcholine and 1 , 2-dimercapto-phosphatidylcholine. The liberated thiol groups are titrated in a subsequent reaction with 4,4'-dithiopyridine to form 4-thiopyridone. The latter compound is in tautomeric equilibrium with 4-mercaptopyridine that absorbs at 334 nm. The reaction is carried out in 100 millimol/l acetate buffer pH 4.0 also containing 0.48 millimol/l 1 ,2-dimercaptodioctanoyl-phosphatidylcholine, 0.32 millimol/l 4,4'-dithiopyridine and 0.2 % Triton-X-100 at 37°C. The reaction was followed by measuring the absorbance at 334 nm in time. The change of absorbance per minute (in the linear part of the curve) was used as measure for the activity. The activity is expressed in PLU/ml. One PLU is the amount of enzyme that liberates 1 nanomole 4-mercaptopyridine per minute at pH 4.0 and 37 ⁰C. Note that the PLU is based upon the release of nanomoles whereas the unit definitions for activities towards triacylglycerol, lysophospholipids and galactolipids is based upon the release of micromoles. To compensate for this difference, phospholipase activities used for comparing the relative activities of an enzyme towards the different substrates are expressed in kPLU's. Thousand PLU's is one kPLU. Measuring galactolipase activities Galactolipase activity at pH 4.5 was determined using digalactosyl diglyceride (Lipid Products, Redhill, UK) as a substrate. To that end digalactosyl diglyceride was suspended in a concentration of 1 mg/mL in 100 millimol/l acetate buffer pH 4.5 with 0.2 % Triton X-100 in presence of 5 millimol/l Ca²⁺ The enzyme (50 μL) was incubated with 1 mL of substrate at 37 ⁰C for 30 minutes. Subsequently the reaction was stopped by freezing the sample in liquid nitrogen. The sample was dried via lyophilisation and subsequently dissolved in methanol^ containing p- nitrotoluene as internal standard. The conversion of the substrate was determined with ¹H-NMR The activity was expressed in U/ml; one unit is the amount of enzyme that liberates 1 micromole free fatty acid per minute at pH 4.5 and 37 ⁰C. Measuring pepsin resistancy of the lipase according to the invention by judging physical integrity using SDS-PAGE

The pepsin resistancy of a lipase can be established by judging its physical integrity according to the following test. The chromatographically pure lipase is diluted with demiwater to a concentration of 5 mg protein/ml and the pH of the solution is adjusted to 4.0 using diluted HCI. The diluted enzyme is then mixed 1 :1 with 8 mg/ml of pepsin (Sigma, product nr P6887 having 4230 units/mg protein, 86%) in 10 millimol/l acetate buffer (pH 4.0). The mixture is then incubated for 90 minutes at 37 degrees C. The lipase solution adjusted to pH 4.0 and diluted with 10 millimol/l acetate buffer without pepsin is co-incubated and serves as a reference.

To confirm the efficacy of the pepsin treatment, a whey protein isolate ("Bipro" from Davisco, Foods International, Inc. (Le Seuer, MN) reference sample is incubated under similar conditions. After incubation SDS-PAGE analysis (see below) is carried out to confirm the integrity of the lipase molecule and the disappearance of the alpha-lactalbumin band.

Integrity of the lipase band as judged by a band intensity of at least 50% compared to the band intensity of the "reference" band after incubation with pepsin followed by SDS-PAGE under the above specified conditions, implies "pepsin resistancy" of the lipase according to the invention. To obtain quantitative information on band intensities, first a digital image (OptiGo, IsogenLife Science: Ijsselstein, The Netherlands) was obtained from the gels which was then analysed using Totallab, TL100 (Nonlinear Dynamics LTD, www.nonlinear.com) image analysis software.

Determining pH optima of the lipase according to the invention . In first instance the pH optimum of the triacylglycerol lipolytic activity of the purified lipase according to the invention was determined using p-nitrophenyl palmitate (pNPP) as a substrate according to the pNPP lipase assay procedure detailed in the Materials & Methods section. In this tests the pH values desired were established using a 100 millimol/l Na-acetate buffer for pH values between 3-6 and a 100 millimol/l Tris-HCI buffer for pH values 6.5-9. In order to refine our insights into the behaviour of the lipase according to the invention under in vivo conditions, in second instance the pH optimum of the LPY enzyme was determined using the activity assay with olive oil and gum arabic.

The latter procedure is described in detail in Example 2 of the present application.

Protein determinations

Protein amounts were determined by the biuret method using Bioquant reagens (Merck, Darmstadt) and using BSA (Sigma) as a reference.

SDS-PAGE

All materials used for SDS-PAGE and staining were purchased from Invitrogen (Carlsbad, CA, US). SDS-PAGE was performed using 10% BT gels and MES buffers. Samples were pre-treated with LDS sample buffer and reducing agent. SDS-PAGE and sample treatment was performed according to manufactures' instructions. Staining was performed using Simply Blue Safe Stain (Collodial Coomassie G250). Example 1

Lipases which share a hydrolytic activity towards triacylglycerol, phospholipids and galactolipids

WO 2004/018660 relates to novel lipolytic enzymes from Aspergillus niger and describes the overproduction, purification and characterization of ten of these newly identified enzymes. In Example 3 of WO 2004/018660 the lipolytic enzyme activities of each one of these enzymes towards a triacylglycerol, a phospholipid and a galactolipid was determined. The results obtained are shown in Table 1 underneath. The data clearly demonstrate a wide array of lipolytic activities among the various new enzymes. However, only two out of the ten enzymes, i.e. NBE031 and NBE033, share a hydrolytic activity towards triacylglycerol, phospholipids and galactolipids hereby illustrating that an activity towards such a range of lipid substrates by a single enzyme is not very common.

As mentioned before, triacyl glycerides containing long chain, water insoluble fatty acids represent by far the greatest quantity in the human diet. According to the data in Table 1 , lipase NBE031 is much more active towards such substrates than lipase NBE033. Therefore, we decided to focus on lipase NBE031 and to rename this enzyme as lipase LPY. Example 2

Lipase LPY has an acidic pH optimum tested on pNPP and olive oil as a substrate

Lipase LPY (NBE031 ) from Aspergillus niger was overproduced using Aspergillus niger as a host cell according to procedures detailed in WO 2004/018660. The cell-free broth was first filtered and then concentrated on a Pellicon 2 "mini" ultrafiltration system using a Biomax 1Ox Millipore filter. The concentrated enzyme solution was then purified by column chromatography over Q-sepharose XK. The enzyme was applied in a 20 millimol/l Na-citrate buffer pH 6.4, washed with the same buffer and then eluted using a gradient starting with 20 millimol/l Na-citrate buffer pH 6.4 to 20 millimol/l Na-citrate buffer pH 6.4 with 1 mol/l of NaCI. Peak fractions were identified by SDS-PAGE followed by staining as detailed in the Materials & Methods section and then pooled. The pH optimum of the triacylglycerol lipolytic activity of the purified lipase LPY was in first instance established using the synthetic p-nitrophenyl palmitate

(pNPP) as a substrate according to the pNPP lipase assay procedure detailed in the Materials & Methods section. The pH values desired were established using a 100 millimol/l Na-acetate buffer for pH values between 3-6 and a 100 millimol/l Tris-HCI buffer for pH values 6.5-9. According to the data obtained and graphically depicted in Figure 2, the lipolytic enzyme according to the invention is active between pH 2.5 to 6.3. and its maximal activity is deployed between pH 4 and 5.

In order to refine our insights into the behaviour of the lipase according to the invention under in vivo conditions, the pH optimum of the LPY enzyme was also determined using the activity assay with olive oil and gum arabic. The experimental details of this test using a natural substrate are described in the Materials & Methods section under "Triacyl-glycerol lipase assay using olive oil and gum Arabic". In this experiment the pH values desired were established using a 200 millimol/l glycine / hydrochloric acid buffer for pH 2.5 - 3.5, a 200 millimol/l acetic acid / sodium acetate buffer for pH 3.6 - 5.5, a 200 millimol/l MES / sodium hydroxide buffer for pH 6.0 - 6.5, a 200 millimol/l MOPS / sodium hydroxide buffer for pH 6.5 - 7.5 and a 200 millimol/l TRIS / hydrochloric acid buffer for pH 8.0 - 9.0. The data obtained in the the latter test are depicted in Figure 4 and confirm the very acid pH optimum of the enzyme. A minor difference with the data obtained on the synthetic substrate is that using olive oil as the substrate the enzyme appears to be active up to a pH of 7.0.

Example 3 Lipase LPY preferentially cleaves long chain fatty acids

To identify the substrate preference for enzyme LPY, a solution of the enzyme that was isolated and purified as described in Example 2 was contacted with different chromogenic ester compounds. Stock solutions of these esters compounds were made in water with 2.5% Triton X-100. The 200 microliter mixtures in a Na-citrate buffer pH 4.0 incorporated 1 millimol/l of either the p- nitrophenyl-acetate, -butyrate, -decanoate and -palmitate substrate pre-heated at 37 degrees C. Incubation with the enzyme added took place for 15 minutes. The reaction with pNP-acetate was terminated by adding 800 microliter of 50 millimol/l Na2 CO3, the other reactions were terminated by adding 800 microliter of 250 millimol/l Na2 CO3. Quantification of the enzymatic conversion was done by a spectrophotometric measurement at 405 nm. The results, shown in Figure 2, clearly demonstrate that lipase LPY has a preference for hydrolysing ester bonds involving long chain fatty acids.

Example 4

Lipase LPY is active towards towards triacylglycerol lipids, galactolipids and phospholipids

A healthy pancreas produces a large array of lipases that can degrade the dietary triacylglycerol lipids, phospholipids and galactolipids. Therefore, a lipase used to compensate pancreatic insufficiencies should be able to hydrolyse all these different substrate molecules at 37 degrees C and preferably at an acid pH. The hydrolytic capabilities of the lipase according to the invention under acid conditions (pH 4.0-4.5) was tested in the present Example by incubation with a number of substrates according to the assays specified in the Materials & Methods section.

A solution of lipase LPY that was isolated and purified as described in Example 2 of the present application, was subjected to the lipolytic tests specified in the Materials & Methods section. From the units/ ml enzyme liquid measured in each test (kPLU's for phospholipase activities- see Materials & Methods), the activities relative to the activity towards triacylglycerol (i.e. p-nitrophenyl palmitate - pNPP) were calculated and specified in Table 2 underneath. The data obtained clearly show that under the acid conditions used, LPY exhibits hydrolytic activity against triacylglycerol lipids, phospholipids, lysophospholipids and galactolipids.

Table 2: The relative lipolytic activities of enzyme LPY.

Example 5 The lipase according to the invention is not degraded by pepsin under simulated stomach conditions at pH 4.0 according to SDS-PAGE

Ideally enzymes that are applicable for treatment of pancreatitis should be resistant to pepsin during stomach passage so that the use of enteric coatings can be prevented. Upon swallowing, the food together with the orally applied enzymes reaches the stomach where they become mechanically mixed with gastric acid, the endogeneous proteolytic enzyme ("pepsin") and the endogeneous lipolytic enzyme ("gastric/lingual lipase"). Typical residence times of solid food in the stomach range from one to two hours. Although in an empty stomach pH values as low as 2 can be measured, the acidity in a stomach filled with food is higher and depends on the amount and the nature of the food ingested.

To evaluate the compatability of the oral lipase according to the invention with the acidic and highly proteolytic gastric conditions existing in the stomach, the following in vitro experiment was carried out. Lipase LPY was purified according to the procedure outlined in Example 2 and diluted with demiwater to a concentration of 5 mg protein/ml. The pH was adjusted to 4.0 using diluted HCI. The diluted enzyme was then mixed 1 :1 with either a solution of 0.26 mg/ml of pepsin (Sigma, product nr P6887 having 4230 units/mg protein, 86%) in 10 millimol/l acetate buffer (pH 4.0) yielding the "low pepsin" dosage, or with a solution of 8 mg/ml of pepsin (Sigma, product nr P6887 having 4230 units/mg protein, 86%) in 10 millimol/l acetate buffer (pH 4.0) yielding the "high pepsin" dosage. To confirm the efficacy of the pepsin treatments used, reference samples were prepared in which whey protein isolate ("Bipro" from Davisco, Foods International, Inc., Le Seuer, MN) was incubated under similar pH 4 and "low"and "high" pepsin conditions. Whereas the alpha-lactalbumin moiety of the whey protein is known to be susceptible to pepsin under pH 4.0 conditions, the beta- lactoglobulin fraction is known to be stable. Lipase LPY adjusted to pH 4.0 and diluted in the 10 millimol/l acetate buffer (pH 4.0) but without any pepsin addition was co-incubated and served as another reference. Enzyme incubation was for 90 minutes at 37 degrees C and immediately thereafter samples of the various incubations were subjected to SDS-PAGE according to the method detailed in the Materials & Methods section. The integrity of the lipase according to the invention after incubation with the two pepsin concentrations at pH 4.0 is clearly illustrated in Figure 3. In the same Figure the complete degradation of the alpha-lactalbumin moiety of whey protein isolate under both the "low" and the "high" pepsin incubation conditions is visible illustrating the efficacy of the pepsin treatment used.

Example 6 Tabletting the lipase according to the invention Starting from the powdered enzyme, an enzyme containing tablet suitable for oral intake can be prepared according to the following protocol. To 2.80 g Polyplasdone XL10 (Crospovidone), add 180.56 g Avicel pH 302 microcrystalline cellulose and push through a 1 mm sieve. Then add 95.24 g of enzyme powder and mix for 10 minutes with a tumbler mixer. Add 1.4 g Mg-stearate and mix again for 2 min. The resulting tablet mixture is then compressed to tablets on a single punch press:

Tablet press: Comprex Il

Punch: oblong, 22 mm x 9 mm

Compression force: 20 klM The tablets obtained weigh approximately 1400 milligrams and incorporate 480 mg of the powdered enzyme.

Example 7 The lipase according to the invention remains enzymatically active upon incubation under simulated stomach conditions at pH 3.0

According to the experiments described in Example 5, the lipase according to the invention remains physically intact after an incubation for 90 minutes at 37 degrees C under "high" pepsin conditions at pH 4.0. Hannibal and Rune (European Journal of Clinical Investigation (1983), 13, 455-460) observed that despite great individual variations, human gastric pH values are typically below 3.0 except for the first 40-60 minutes after a meal when the gastric pH is around 4. The implication is that in order to guarantee that a non-enteric coated lipase canl be active in the stomach and will retain its full activity after stomach passage, in vitro testing criteria should be more stringent than the conditions used in Example 5. To that end the current Example describes the survival of lipolytic activity of the lipase according to the invention at pH 3 and under the "high" pepsin concentrations used in Example 5. Basically the experiment was carried out according to the procedure detailed in Example 5,. However, in the present experiment the pH was maintained at 3.0 and only the "high" pepsin concentration was applied. Furthermore the "Lipolase®" enzyme from Thermomyces lanuginosus (Sigma-Aldrich) was included in the test as a reference. As described in WO2006/136159 this enzyme is seen as an excellent non-animal derived alternative to replace the lipolytic activity of pancreatine.

Both the enzyme according to the invention as well as the Lipolase enzyme were diluted with demiwater to a concentration of approx. 0.2 mg protein/ml. After the 90 minutes of incubation at 37 degrees C, the pH was raised to 5.0 by adding acetic acid / sodium acetate buffer and residual lipolytic activity was assayed with the "Triolein assay" as specified in the Materials & Methods section.

According to the results obtained and shown in Table 3, the lipolytic activity of the known "Lipolase" enzyme is completely lost at pH 3 and in the presence of pepsin. In contrast with this, the enzymatic activity of the lipase according to the invention, lipase LPY, is not significantly affected by such harsh conditions. Therefore, this result further supports the results described in Example 5 and emphasizes the unique character of enzyme LPY.

In combination with the pH-activity data shown in Figure 4, the data obtained thus confirm that the non-enteric coated lipase according to the invention will be active in the stomach and can continue its enzymatic activity in the duodenum and beyond.

Table 3: Residual enzymatic activity of lipases LPY and Lipolase after pepsin incubation at pH 3

Claims

1. A lipolytic enzyme or a composition comprising a lipolytic enzyme

- which lipolytic enzyme is enzymatically active at a neutral to acid pH, for example at pH 7, 6, 5, 4, 3 or 2; - which lipolytic enzyme has activity towards triacylglycerol lipids;

- which lipolytic enzyme has activity towards phospholipids;

- which lipolytic enzyme has activity towards lysophospholipids; and

- which lipolytic enzyme has activity towards galactolipids, and for use as medicament.

2. A lipolytic enzyme or a composition comprising a lipolytic enzyme according to claim 1 which has one or more of the following additional properties:

- the lipolytic enzyme has an acid pH optimum;

- the lipolytic enzyme is gastric acid resistant;

- the lipolytic enzyme is active in the presence of pepsin;

- the lipolytic enzyme is enzymatically stable in the presence of pepsin;

-the lipolytic enzyme has no activity towards triacylglycerol lipids under assay conditions (pH 9) of United States Pharmacopeia (USP) procedure and/or

- the lipolytic enzyme is resistant to pepsin.

3. A lipolytic enzyme or a composition comprising a lipolytic enzyme according to claim 1 or 2 whereby the lipolytic enzyme is of microbial origin, more preferably is of fungal origin.

4. A lipolytic enzyme or a composition comprising a lipolytic enzyme according to any one of claims 1 to 3 for use as medicament to treat digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis.

5. A lipolytic enzyme or a composition comprising a lipolytic enzyme according to any one of claims 1 to 4 wherein the lipolytic enzyme is an isolated polypeptide comprising:

(a) an amino acid sequence according to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 having an amino acid sequence at least 60, 70, 80 or 90% homologous to the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2;

(b) an amino acid sequence according to the mature polypeptide in the amino acid encoded by a polynucleotide polynucleotide which comprises:

(a) the nucleotide sequence as set out in SEQ ID NO: 1 or a functional equivalent thereof having at least 60, 70, 80 or 90% homology to the nucleotide sequence of SEQ ID NO: 1 ;

(b) a nucleotide sequence which hybridizes with a polynucleotide being the complement of SEQ ID NO: 1 and wherein said nucleotide sequence is at least 60, 70, 80 or 90% homologous to the nucleotide sequence of SEQ ID NO: 1 ;

(c) a nucleotide sequence encoding the mature polypeptide in the amino acid sequence according to SEQ ID NO: 2 having at least 60, 70, 80 or 90% homology to the mature polypeptide in the amino acid sequence of

SEQ ID NO: 2;

(d) a sequence which is degenerate as a result of the degeneracy of the genetic code to a sequence as defined in any one of (a), (b), (c);

6. A composition comprising a lipolytic enzyme according to any one of claims 1 to

5 which further comprises a protease and/or amylase.

7. A composition comprising a lipolytic enzyme according to any one of claims 1 to

6 which is in oral dosage form.

8. A lipolytic enzyme according to any one of claims 1 to 5 for use in the preparation of a medicament, for the treatment of digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis.

9. Use of a lipolytic enzyme or a composition comprising a lipolytic enzyme

- which lipolytic enzyme is enzymatically active at a neutral to acid pH, for example at pH 7, 6, 5, 4, 3 or 2; - which the lipolytic enzyme has activity towards triacylglycerol lipids;

- which the lipolytic enzyme has activity towards phospholipids;

- which the lipolytic enzyme has activity towards lysophospholipids; and

- which the lipolytic enzyme has activity towards galactolipids, as medicament preferably for the treatment of digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis.

10. A medicament treatment preferably a treatment of digestive disorders, pancreatic insufficiency, pancreatitis or cystic fibrosis which comprises the administering of a lipolytic enzyme or a composition comprising a lipolytic enzyme according to any one of claims 1 to 8.