Novel Lipase Enzyme

Abstract

The present invention relates to a lipase enzyme having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, or a functional fragment thereof encompassing position Q55, with the proviso that at position Q55 there is a substitution of an amino acid with a basic side chain at neutral pH (Figure 1).

Description

[Technical Field]

[0001] The present application relates to novel lipase enzymes. [Background technology]

[0002] Lipid indigestion and digestive disorders are becoming increasingly prevalent in general medicine and in the field of internal medicine. These digestive disorders, although more or less severe, are often the result of a significant deficiency of so-called pancreatic enzymes. Under healthy conditions, these enzymes are synthesized in the pancreas by highly specialized cells, known as acinar cells, and secreted into the duodenum by exocytosis via the acinar lumen and the main pancreatic duct. The pancreatic secretion volume is approximately 2 liters per day. In addition to lipase, pancreatic secretions also contain enzymes for digesting proteins (trypsin, chymotrypsin, and carboxypeptidase) and carbohydrates (α-amylase). Pancreatic enzyme secretion is precisely regulated by endogenous mechanisms mediated by hormones such as gastrin, secretin, and pancreozymin. Many factors can disrupt this control system, resulting in reduced pancreatic enzyme secretion or complete loss of pancreatic exocrine function. This subsequently results in chyme indigestion in the small intestine, leading to digestive disorders. This disease of the digestive tract, also known as exocrine pancreatic insufficiency (EPI), can be caused by different factors. In addition to drug-induced gastrointestinal disorders, pancreatic insufficiency can also be caused by chronic atrophic gastritis and chronic pancreatitis, which are often caused by alcohol consumption, surgical disorders (e.g., Billroth I and II procedures, vagotomy, pancreatectomy), and cystic fibrosis. In any case, chronic digestive disorders are a sociomedical and economic concern, as they often do not manifest symptoms and shorten life expectancy.

[0003] Pancreatic digestive disorders, especially EPI, cause many problems for patients, including diarrhea, constipation, feeling of exhaustion, upper abdominal complaints, and weight loss.

[0004] To avoid the malnutrition-related morbidity and mortality, enzyme replacement therapy is initiated as soon as pancreatic indigestion or EPI is diagnosed, regardless of its cause and symptoms. This means that the missing enzymes, primarily lipase, protease, and amylase, must be provided from an exogenous source. Treatment primarily involves oral intake of enzymes during meals, which then pass through the stomach and into the small intestine, where they digest chyme and thereby compensate for the lack of endogenous pancreatic enzymes.

[0005] Pancreatic enzyme replacement therapy (PERT), based on the supplementation / replacement of key enzymes, lipase and protease, is often used to treat digestive disorders resulting from pancreatic enzyme deficiency. Regarding PERT, a wide variety of enzyme preparations are already commercially available. These are based in part on porcine pancreatic enzymes, such as COMBIZYM®, FESTAL®, PANKREON®, KREON®, PANZYTRAT®, METEOZYM®, or ENZYM-LEFAX N®. Preparations containing pancreatic enzymes, so-called pancreatic enzyme preparations or PEPs, are mostly obtained from porcine carcasses, e.g., the pancreas. The end product of the preparation process is pancreatin. PEPs, consisting of porcine lipase, amylase, and protease, are used in patients with cystic fibrosis, chronic pancreatitis, and EPI secondary to pancreatic resection.

[0006] Pig-derived PEP cannot be used by patients suffering from digestive disorders due to porcine protein allergies. Furthermore, pigs are considered natural reservoirs of human pathogenic influenza viruses and numerous other porcine viruses, and contamination of pancreatin with such viruses cannot be ruled out. In other words, pancreatic tissue, which represents slaughter waste, may exhibit high levels of viral contamination if not further processed. As a result of their natural origin, pancreatic tissue, pancreatin, and PEP may also be contaminated with porcine viruses. It should be emphasized that the International Conference on Harmonization (ICH) has set very high standards in its guideline ICH Topic Q5A(R1), requiring maximum assurance that products are free of viral contamination. The US FDA's Center for Drug Evaluation and Research (CDER) has already required aggressive risk mitigation strategies for lipase-containing PEPs, such as Creon.

[0007] This is due to the risk of PEP contamination with porcine parvovirus and porcine circovirus, as well as the risk from many porcine viruses that are already known to be pathogenic for humans.

[0008] For these reasons, a more well-defined, less risky lipase enzyme is desirable for pancreatic enzyme replacement therapy.

[0009] Additionally, each formulation used must contain a sufficient amount of enzyme, which must also be provided in an enteric-coated formulation, have a small particle size, and be fully bioavailable in the gastrointestinal tract.

[0010] In fact, patients' daily doses can be substantial: starting doses range from 50,000 to 75,000 units of lipase with meals and 25,000 units with snacks.

[0011] To alleviate this burden and improve patient compliance, it would be desirable to provide lipases with higher activity.

[0012] Another challenge is that commercially available lipase and PERT products are often not specifically adapted to the environmental conditions of the human small intestine, such as pH and bile acid concentration and composition, the latter two parameters that can differ significantly between humans and, for example, pigs.

[0013] Therefore, one object of the present invention is to provide better treatment options for patients suffering from lipid indigestion or lipid digestion disorders, such as, for example, pancreatic insufficiency (EPI).

[0014] Another object of the present invention is to provide an alternative treatment to conventional pancreatic enzyme replacement therapy (PERT).

[0015] These and other objects are achieved by the methods and means set forth in the independent claims. The dependent claims relate to specific embodiments. Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention provides modified lipase enzymes. The general advantages of the present invention and its features are described in detail below. [Brief explanation of the drawings]

[0017] [Figure 1] Comparison of the activity of wild-type (WT) and Q55K mutant with 3 mM 86339 taurocholate.

[0018] [Figure 2] Comparison of activity between WT and Q55K mutant using 7.5 mM EPS0900000 taurocholate.

[0019] [Figure 3]Changes in specific lipase activity of purified WT- and Q55K mutant lipases with increasing mixed bile salt concentrations and 140 mM NaCl.

[0020] [Figure 4] Bile salt concentrations in the human small intestine. Adapted from Northfield et al. McColl, 1973. In all sections except the lower ileum, the total bile acid concentration is greater than 4 μM / ml (equivalent to greater than 4 mM).

[0021] [Figure 5] pH distribution in the human small intestine. Adapted from Koziolek et al., 2015. SBTTnorm= is the normalized small intestinal transit time (SBTT) between the time of gastric emptying and the time of colonic arrival. This also means that the lipase of the present invention exhibits higher activity compared to WT lipase under conditions that reflect the in vivo situation.

[0022] [Figure 6] Vector map of the pMA vector used. The target gene (Tt00320120), origin of replication (Ori), ampicillin resistance (AmpR), and different restriction sites (PsiI, etc.) are indicated. pMA was derived from the pMX system.

[0023] [Figure 7] This is a vector map of the pAX vector used, showing the expression cassette (promoter: MTT1, target gene: Ttherm_00320120, terminator: BTU2), three resistance sequences: chloramphenicol (CmR), ampicillin (AmpR), a codon-harmonized paromycin resistance sequence (ha_NeoR), two Cre / loxP flanks (loxP), three replication origins (colE1: E. coli, rDNA ori: T. thermophila), and various restriction sites (EcoRV, etc.). The backbone is a pUC119 vector. DETAILED DESCRIPTION OF THE INVENTION

[0024] Before describing the present invention in detail, it is to be understood that the present invention is not limited to the specific component parts of the described devices or to the process steps of the described methods, as such devices and methods may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural references unless the context clearly dictates otherwise. Furthermore, when a range of parameters bounded by numerical values ​​is given, it should be understood that the range is deemed to include those limits.

[0025] Furthermore, it should be understood that the embodiments disclosed herein are not meant to be understood as separate, unrelated embodiments. Features discussed in one embodiment are meant to be disclosed in relation to other embodiments shown herein. In one case, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, those skilled in the art will understand that this does not necessarily mean that the feature is not disclosed in the other embodiments. Those skilled in the art will understand that while it is the intent of the present application to disclose the feature in other embodiments as well, this has not been done merely for the sake of clarity and to keep the description manageable.

[0026] Furthermore, the contents of the prior art documents referred to herein are incorporated by reference. This refers in particular to prior art documents that disclose standard or conventional methods. In that case, incorporation by reference is intended to provide a fully enabling disclosure and to avoid lengthy repetition.

[0027] According to a first aspect of the present invention, there is provided a lipase enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, but with a substitution at position Q55 of an amino acid having a basic side chain at neutral pH.

[0028] It is understood that the lipase sequence according to SEQ ID NO: 1 lacks a signal peptide (= lead peptide). One suitable signal peptide is disclosed as SEQ ID NO: 9, which has a length of 16 amino acid residues.

[0029] All amino acid positions referred to in this application are counted in SEQ ID NO: 1. However, in the case of a lipase having a signal peptide (= lead peptide) at the N-terminus, such as SEQ ID NO: 9, additional N-terminal amino acid residues of the signal peptide must be taken into consideration.

[0030] As used herein, the term "amino acid having a basic side chain at neutral pH" includes, for example, amino acids having an NH group in the side chain that is positively charged at neutral pH.

[0031] In one embodiment, the substitution is with an amino acid selected from the group consisting of lysine (K), arginine (R) and histidine (H).

[0032] Alternatively, the lipase enzyme comprises a functional fragment of SEQ ID NO: 1, or a sequence having at least 90% sequence identity to SEQ ID NO: 1, the fragment encompassing position Q55 and having the substitutions described.

[0033] Such a fragment may be, for example, a sequence having AA residues 21-256 of SEQ ID NO:1, or a sequence having at least 90% sequence identity with SEQ ID NO:1 with the substitution of Q55.

[0034] The term "functional" means that such fragments retain lipase activity, especially under the pH and bile salt conditions defined herein.

[0035] The fragments may preferably have a minimum length of ≥ 100 amino acid residues (AA), more preferably ≥ 110 AA, ≥ 120 AA, ≥ 130 AA, ≥ 140 AA, ≥ 150 AA, ≥ 160 AA, ≥ 170 AA, ≥ 180 AA, ≥ 190 AA, ≥ 200 AA, ≥ 210 AA, ≥ 220 AA, ≥ 230 AA, ≥ 240 AA, ≥ 250 AA, ≥ 260 AA, ≥ 270 AA, and most preferably ≥ 280 AA.

[0036] The fragments may preferably have a maximum length of <280 AA, more preferably <270 AA, <260 AA, <250 AA, <240 AA, <230 AA, <220 AA, <210 AA, <200 AA, <190 AA, <180 AA, <170 AA, <160 AA, <150 AA, <140 AA, <130 AA, <120 AA, <110 AA, and most preferably <100 AA.

[0037] SEQ ID NO: 1 is the amino acid sequence of a lipase from the ciliate Tetrahymena thermophila, designated TTHERM_00320120 (UniProtKB-Q237S4(Q237S4_TETTS)). Its use in the treatment of pancreatic insufficiency has been described in Brock et al., 2016.

[0038] The inventors have surprisingly shown that the claimed lipase enzyme has increased lipolytic activity in media having a pH of 5.5 or higher, in media having a bile salt concentration of 2.5 mM or higher, and / or in media containing a mixture of two or more bile acids, compared to a lipase enzyme having the amino acid sequence of SEQ ID NO: 1.

[0039] These conditions reflect the in vivo situation in the human small intestine.

[0040] In this specification, the terms bile salts and bile acids are used interchangeably. Bile acids are steroid acids found in abundance in mammalian bile. In humans, taurocholic acid and glycocholic acid (derivatives of cholic acid), as well as taurochenodeoxycholic acid and glycochenodeoxycholic acid (derivatives of chenodeoxycholic acid), are the major bile salts in bile, with concentrations approximately equal. Their 7α-dehydroxylated derivatives, conjugate salts deoxycholic acid and lithocholic acid, also exist. Cholic acid, chenodeoxycholic acid, and deoxycholic acid derivatives account for more than 90% of human bile acids. Bile acids account for approximately 80% of the organic compounds in bile. The main function of bile acids is to emulsify fats and oils as surfactants, forming micelles with the hydrophobic side facing the fat and the hydrophilic side facing outward, thereby enabling the digestion of edible fats and oils. The hydrophilic side is negatively charged, which prevents the bile-coated lipid droplets from re-aggregating into larger lipid particles. Duodenal micelles typically have a diameter of 14-33 μm.

[0041] The dispersion of dietary fats into micelles greatly increases the surface area for the activity of pancreatic lipase, which actually digests triglycerides, allowing them to reach the fat nucleus through the bile salt gap.

[0042] In some embodiments, a lipase according to the invention has ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98, or most preferably ≧99% sequence identity to SEQ ID NO:1, except that the lipase enzyme has a substitution at position Q55.

[0043] In some embodiments, a lipase according to the present invention has ≧91%, ≧92%, ≧93%, ≧94%, ≧95%, ≧96%, ≧97%, ≧98, or most preferably ≧99% sequence identity to any one of SEQ ID NO:3 (“Q55K”), SEQ ID NO:4 (“Q55R”), or SEQ ID NO:5 (“Q55H”).

[0044] In some embodiments, a lipase according to the present invention is identical to SEQ ID NO:1, except that the lipase enzyme has a substitution at position Q55. Thus, in some embodiments, the lipase is identical to SEQ ID NO:1, except that the lipase enzyme has a substitution Q55K (SEQ ID NO:3). In some embodiments, the lipase is identical to SEQ ID NO:1, except that the lipase enzyme has a substitution Q55R (SEQ ID NO:4). In some embodiments, the lipase is identical to SEQ ID NO:1, except that the lipase enzyme has a substitution Q55H (SEQ ID NO:5).

[0045] The substitution at position Q55 replaces the aliphatic, neutrally charged amino acid glutamine (Q) with a positively charged lysine (K), arginine (R) or histidine (H).

[0046] In general, most lipases have a catalytic triad consisting of aspartate, histidine, and serine in the active center. Aspartate activates histidine by abstracting a proton from it. The catalytically active histidine then abstracts a proton from serine, making the serine residue more nucleophilic. The latter can attack the carbonyl carbon of the substrate ester located in the active center, and this ester forms part of the enzyme's lipid substrate.

[0047] We have shown that position Q55 is only 15 Å from the serine residue of the catalytic triad, and without being bound by theory, it is possible that the substitution of the neutral Q55 with a positive amino acid residue is indeed responsible for the observed increase in activity.

[0048] In one embodiment, a lipase according to the invention has at least 90% sequence identity to, or is a fragment of, SEQ ID NO: 1, as described elsewhere herein, and has a Q55 substitution that retains a catalytic triad comprising aspartic acid (D), histidine (H), and serine (S). In one embodiment, the catalytic triad comprises the following amino acid residues according to SEQ ID NO: 1: S140, D199, H256.

[0049] In one embodiment, a lipase according to the invention has at least 90% sequence identity with, or is a fragment of, SEQ ID NO: 1, as described elsewhere herein, followed by a Q55 substitution, and optionally, followed by preservation of the catalytic triad as described elsewhere herein, while retaining an oxyanion pocket that stabilizes the intermediate product, said pocket being formed primarily by the following amino acid residues according to SEQ ID NO: 1: Tyr21 and Thr76 (and optionally S140 and H256).

[0050] Other lipases having at least 90% sequence identity with SEQ ID NO: 1 are the lipases set forth in SEQ ID NOs: 10 to 12 (with a Q55K mutation), SEQ ID NOs: 13 to 15 (with a Q55R mutation), and SEQ ID NOs: 16 to 18 (with a Q55H mutation).

[0051] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (e.g., a polypeptide) that does not contain additions or deletions due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0052] The terms "identical" or percent "identity," with respect to two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity over a specified region, or, if not specified, the entire reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region, measured using one of the sequence comparison algorithms described below, or by manual alignment and visual inspection.

[0053] The BLAST algorithm is one of the algorithms suitable for determining sequence identity. Another algorithm for determining sequence identity is the Clustal Omega algorithm.

[0054] The disclosure provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein. In some cases, identity exists over a region at least about 15, 25, or 50 nucleotides in length, or more preferably over a region 100 to 500 or 1000 or more nucleotides in length, or over the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity can exist over a region at least 5, 10, 15, or 20 amino acids in length, in some cases at least about 25, 30, 35, 40, 50, 75, or 100 amino acids in length, in some cases at least about 150, 200, or 250 amino acids in length, or over the entire length of the reference sequence. With respect to shorter amino acid sequences, e.g., sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted according to conservative substitutions as defined herein.

[0055] According to one embodiment, the lipase enzyme has the substitution Q55K.

[0056] According to another embodiment, the lipase enzyme has a lipolytic activity of at least 30,000 U / g.

[0057] In the context of the present disclosure, the terms "lipase activity" and "lipolytic activity" are used interchangeably. Lipase activity (lipolytic activity) can be measured using a modification of the colorimetric assay of Nixon and Chan (1979), as discussed elsewhere herein.

[0058] According to another embodiment, the lipase enzyme comprises at least one further conservative amino acid substitution in addition to the substitution at position Q55 compared to the amino acid according to SEQ ID NO:1.

[0059] In this context, "conservative amino acid substitutions" have less impact on lipase function than non-conservative substitutions. Amino acids can be classified in a variety of ways, but they are often divided into six major groups based on their structure and the general chemical properties of the R group. In some embodiments, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include: Basic side chains (lysine, arginine, histidine, etc.), acidic side chains (aspartic acid, glutamic acid, etc.), Uncharged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.), Nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.), β-branched side chains (threonine, valine, isoleucine, etc.), Contains aromatic side chains (tyrosine, phenylalanine, tryptophan, histidine, etc.).

[0060] Other conservative amino acid substitutions can also occur across amino acid side chain families, such as substituting aspartic acid for asparagine to modify the charge of the peptide. Conservative changes can also include the substitution of chemically homologous non-natural amino acids (i.e., a synthetic non-natural hydrophobic amino acid for leucine, a synthetic non-natural aromatic amino acid for tryptophan).

[0061] SEQ ID NOs: 6-8 show variants of the wild-type sequence with two conservative amino acid substitutions: V70I / V152I; V71I / L207I or V119I / Y168F, respectively. The Q55 mutation defined herein can also be achieved in these wild-type variants.

[0062] According to another embodiment, the lipase enzyme has increased lipolytic activity in a medium at pH > 5.5 compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:1.

[0063] In one embodiment, such increased lipolytic activity is present at a pH between ≧5.5 and ≦11, between ≧6 and ≦10, or between ≧6.5 and ≦9, compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:1.

[0064] According to another embodiment, the lipase enzyme has improved lipolytic activity in a medium having a total bile salt concentration of ≧2.5 mM compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:1.

[0065] In one embodiment, such increased lipolytic activity is present in a medium having a total bile salt concentration of between ≧2.5 mM and ≦15 mM compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:1.

[0066] In different embodiments, the bile salt is selected from the group consisting of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and lithocholic acid.

[0067] According to another embodiment, the lipase enzyme has increased lipolytic activity in a medium containing a mixture of two or more different bile salts compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:1.

[0068] In different embodiments, the two or more different bile salts are selected from the group consisting of cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and lithocholic acid.

[0069] In all three cases, activity can be increased by ≧5%, ≧10%, ≧15%, ≧20%, ≧25%, ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, ≧90%, ≧100%, ≧150%, or ≧200%. Lipase activity (lipolytic activity) can be measured using a modified colorimetric assay of Nixon and Chan (1979), as discussed elsewhere herein.

[0070] According to another aspect of the present invention, there is provided a nucleic acid encoding a lipase enzyme according to any one of the preceding embodiments. Such a nucleic acid can be, for example, mRNA or cDNA. Also provided is a suitable vector comprising such a nucleic acid.

[0071] According to another embodiment, there is provided a use of the above lipase enzyme (for the manufacture of a medicament) for the treatment of a human or animal subject diagnosed with, suffering from, or at risk of developing a lipid indigestion, digestive disorder, and / or inflammatory condition, or for the prevention of such a condition.

[0072] According to one embodiment, the digestive disorder is exocrine pancreatic insufficiency, which may be caused by, among other things, cystic fibrosis, pancreatic duct obstruction, or pancreatectomy.

[0073] According to another embodiment, the inflammatory condition is chronic inflammation of the pancreas (pancreatitis) or inflammatory bowel disease.

[0074] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the lipase enzyme described above, and optionally one or more pharmaceutically acceptable excipients.

[0075] According to another aspect of the present invention, there is provided a combination comprising (i) a lipase enzyme as described above or a pharmaceutical composition as described above, and (ii) one or more therapeutically active compounds.

[0076] According to another aspect of the present invention, there is provided a method for treating or preventing lipid indigestion, digestive disorders, and / or inflammatory conditions, the method comprising administering to a human or animal subject a therapeutically sufficient amount of (i) a lipase enzyme, (ii) a pharmaceutical composition, or (iii) a combination of the above. According to another aspect of the present invention, there is provided a therapeutic kit of parts, comprising: a) (i) a lipase enzyme, (ii) a pharmaceutical composition, or (iii) a combination according to the above; b) a device for administering the composition, composition or combination, and c) Instructions for use of the kit. Such devices are, for example, capsules, tablets, syringes, inhalers, and the like.

[0077] In another aspect of the present invention, there is provided a method for producing a lipase enzyme comprising the steps of: a) expressing a lipase enzyme in an expression host from the order Ciliata; and b) purifying the lipase enzyme expressed in step a).

[0078] According to one embodiment, the method comprises, prior to step a), a step of transforming the ciliate with a vector encoding said lipase enzyme. Suitable vectors are disclosed elsewhere herein.

[0079] Methods for the transformation of ciliates that can be used in the context of the present invention consist in particular of microinjection, electroporation and particle bombardment and are described, for example, in Tondravi & Yao (1986), Gaertig & Gorovsky (1992) and Cassidy-Hanley et al. (1997).

[0080] Methods for transformation and heterologous protein expression have been described for a few protists (WO 00 / 58483 and WO 00 / 46381). The generation of mitotically stable transformants of the ciliate Tetrahymena thermophila can be achieved after transfection of either the somatic macronucleus or the generative micronucleus by microinjection, electroporation, or biolistic bombardment.

[0081] Selection of transformants can be performed using different selection markers, such as neomycin resistance (Weide et al. 2006, BMC), and by integrating heterologous genes by homologous DNA recombination, stable thymidine-auxotrophic Tetrahymena cells can be obtained (Weide et al. 2006, BMC). Blasticidin S (Weide et al. 2007, BMC) or paclitaxel (WO 00 / 46381) resistance has also been considered.

[0082] Suitable promoters for lipase expression in ciliates are disclosed, for example, in commonly assigned US2008261290A1, the contents of which are incorporated herein by reference, which discloses heat-inducible promoters and metallothionein promoters that can also be used for the purposes of the present invention.

[0083] According to a further embodiment, the expression host is from the genus Tetrahymena.

[0084] According to a further embodiment, the expression host is Tetrahymena thermophila. [Example]

[0085] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0086] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'. Materials and Methods Culture medium LB (Lysogeny Broth) medium 10 g / L casein peptone 5 g / L yeast extract 5 g / L NaCl Distilled water, pH 7.5 LB agar medium 15g / L Agar-agar in LB medium Possibly 25 μg / mL kanamycin or 100 μg / mL ampicillin Cre-Lox-LB agar plates 7% (w / v) sucrose 30 μg / mL chloramphenicol 100 μg / mL ampicillin 86% (v / v) LB with 15g / L agar Super Optimal Broth (SOC) medium, Invitrogen GmbH, Karlsruhe, Germany Dryls(1x) 1.5 mM Trisodium Citrate-Dihydrate 1mM NaH2PO4·monohydrate 1 mM Na2HPO4 1.5 mM CaCl2 MW 1010 10 g / L malt extract 10 g / L Wheat Peptone E1 5 g / L yeast extract 2 g / L glucose monohydrate 1mL / L Ferrous Sulfate Chelate Solution MW1515 15 g / L malt extract 15 g / L Wheat Peptone E1 5 g / L yeast extract 2 g / L glucose monohydrate 1mL / L Ferrous Sulfate Chelate Solution Paromomycin concentration for fermentation: 224 μg / ml paromomycin Paromomycin concentration for mutant adaptation: 196 μg / ml paromomycin

[0087] buffer As buffers, typical buffers used in hydrophobic interaction chromatography are applied, for example as disclosed in Djogo et al. 1999, the contents of which are incorporated herein by reference. Machine Column JPEG0007741827000001.jpg133170

[0088] 1. Mutation of expression vector The MTT1_TTHERM_00320120 gene (UniProtKB - Q237S4, the amino acid sequence of which is shown herein as SEQ ID NO: 1) in the pAM vector was transfected with the QuikChange method (QUIKCHANGE TM Mutagenesis was performed using the Site-Directed Mutagenesis Kit from Stratagene, Catalog #200518, described by Loke et al. (2001). Two primers with point mutations were generated to generate amino acid substitutions. After mutation in the pMA vector, the fragment was ligated into the pDL_S2 vector and then inserted into the shuttle vector pAX_ha_neo using the Cre-dependent recombinase system. For details of each vector, see Figure 6 and Figure 7.

[0089] 2. Cultivation of Tetrahymena and transformation of expression plasmids (biolistic bombardment) In this experiment, Tetrahymena thermophila strains 1868 / 4 and 1868 / 7 were transformed as described by Cassidy-Hanley et al. (1997). Cultures were grown in 1 L bioreactors in MW1515 medium (15 g / L malt extract, 15 g / L wheat peptone E1, 5 g / L yeast extract, 1 ml / L ferrous sulfate / chelate solution, 2 g / L glucose monohydrate) for 50 hours. Approximately 24 hours after inoculation, the addition of CdCl2 to a final concentration of 10 μg / mL at mid-log phase confirmed induction of MTT1 promoter-driven expression. Correct expression was confirmed by Coomassie-stained SDS-Page and WB analysis using a polyclonal antibody.

[0090] 3. Purification To measure the specific activity in several assays, the lipase was gradually purified in multiple steps. The supernatant was concentrated and buffer exchanged by diafiltration. After 20-fold concentration, it was diluted with phosphate buffer and bound to a hydrophobic interaction chromatography (HIC) column. After washing the column, elution was performed in one step by gradually decreasing the ammonium sulfate concentration to 0%. The eluted protein was collected and used for further purification on a Superdex75 Increase 10 / 300 GL size exclusion chromatography (SEC) column.

[0091] 4. Measurement of Lipase Activity For lipase activity (lipolytic activity), a modified version of the colorimetric assay from Nixon and Chan (1979) was used as described in Brock et al. (2016), the contents of which are incorporated herein by reference. Various bile acids and defined bile salt mixtures were tested in the experiments. 3 mM taurocholate was used for standards and samples, while 7.5 mM taurocholate (BRP) and the mixed bile salt solution (MBS) described by Gargouri et al. (1986) were used for samples only. pH was adjusted with 54 mM phosphate buffer, pH 6-7.5, containing a final concentration of 140 mM NaCl.

[0092] Example 1 Figure 1. Comparison of activity between wild-type (WT) and Q55K mutant 86339 with 3 mM taurocholate. The initial 3 mM taurocholic acid 86339 (Sigma) used was 97% pure, and the normalized volumetric activity of WT-0120 lipase measured by WB assay differed only from that of the Q55K-mutant lipase at pH 4 to pH 8. The concentration of taurocholic acid did not reach the critical micelle concentration (CMC), and did not begin to inhibit activity at basic pH.

[0093] Figure 2: Comparison of activity between WT and Q55K mutant with 7.5 mM EPS0900000 taurocholate. The other taurocholic acid, EPS0900000 BRP (LGC), recommended in the European Pharmacopoeia, lacks a purity certificate and is brownish in color, resulting in a completely different activity profile across pH values. When measured at a concentration of 7.5 mM, both WT and Q55K lipases exhibited very similar activity up to pH 6, after which the activity of WT decreased, whereas Q55K activity maintained a high level from pH 6 onward and maintained higher activity than WT at pH 7 and 8.

[0094] Without being bound by theory, this effect may be due to a second impurity of taurocholic acid, which means that there are other bile acids with much lower CMCs than taurocholic acid. These bile acids are able to inhibit the activity of WT lipase significantly at basic pH values.

[0095] In fact, media containing a mixture of bile acids better reflect the in vivo situation than media consisting of pure taurocholate alone.

[0096] Figure 3: Changes in specific lipase activity of purified WT- and Q55K mutant lipases with increasing mixed bile salt concentrations and 140 mM NaCl. Purified WT and Q55K lipases were analyzed at MBS concentrations similar to in vivo conditions, and a strong effect was found at pH 7. Both lipases showed an initial increase in specific activity that correlated with MBS concentration, with WT lipase already starting at a high activity. The activity of Q55K lipase exceeded that of WT only after MBS concentrations reached 2.5 to 5.0 mM, indicating that the Q55K mutant has high tolerance to high bile salt concentrations at neutral to basic pH.

[0097] Figure 4: Bile salt concentrations in the human small intestine. Adapted from Northfield et al. McColl, 1973. In all sections except the lower ileum, the total bile salt concentration is above 4 μM / ml (equivalent to above 4 mM), which means that the lipase according to the invention exhibits higher activity compared to WT lipase under conditions that reflect the in vivo situation.

[0098] Figure 5: pH distribution in the human small intestine. Adapted from Koziolek et al., 2015. SBTT norm= is the normalized small intestinal transit time (SBTT) between gastric emptying and colonic arrival, and this means that the lipase of the present invention exhibits higher activity compared to WT lipase under conditions that reflect the in vivo situation.

[0099] References: JPEG0007741827000002.jpg201170JPEG0007741827000003.jpg67170

[0100] array The following sequences form part of the disclosure of this application. A WIPO ST 25 compatible electronic sequence listing is also provided with this application. For the avoidance of doubt, in the event of any discrepancy between a sequence in the table below and a sequence in the electronic sequence listing, the sequence in the table shall be deemed correct. JPEG0007741827000004.jpg222170JPEG0007741827000005.jpg207170JPEG0007741827000006.jpg146170

Claims

1. A lipase enzyme comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 1, or a functional fragment thereof encompassing position Q55, with the proviso that position Q55 has a substitution of an amino acid having a basic side chain at neutral pH.

2. 2. The lipase enzyme of claim 1, wherein the substitution is with an amino acid selected from the group consisting of lysine (K), arginine (R), and histidine (H).

3. 3. The lipase enzyme of claim 1 or 2 having the substitution Q55K.

4. 4. The lipase enzyme of any one of claims 1 to 3, having a lipolytic activity of at least 30,000 U / g.

5. A lipase enzyme according to any one of claims 1 to 4, which comprises at least one further conservative amino acid substitution in addition to the substitution at position Q55 compared to the amino acid sequence set forth in SEQ ID NO:

1.

6. 6. The lipase enzyme according to claim 1, wherein the lipase enzyme has improved lipolytic activity in a medium having a pH in the range of 6<pH≦9 and a total bile salt concentration of ≧5 mM, compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:

1.

7. The lipase enzyme according to any one of claims 1 to 6, characterized in that it has improved lipolytic activity in a medium containing a mixture of two or more different bile acids, compared to a lipase enzyme having the amino acid sequence of SEQ ID NO:

1.

8. A nucleic acid encoding a lipase enzyme described in any one of claims 1 to 7.

9. Use of a lipase enzyme according to any one of claims 1 to 7 in the manufacture of a medicament for the treatment of a human or animal subject diagnosed with, suffering from or at risk of developing a lipid indigestion, digestive disorder and / or inflammatory condition, or for the prevention of such a condition.

10. 10. The use according to claim 9, wherein the digestive disorder is exocrine pancreatic insufficiency.

11. A pharmaceutical composition comprising the lipase enzyme of any one of claims 1 to 7 and one or more pharmaceutically acceptable excipients.

12. A combination comprising (i) a lipase enzyme according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 11 and (ii) one or more therapeutically active compounds.

13. A pharmaceutical for treating or preventing lipid indigestion, digestive disorders or inflammatory conditions, comprising: (i) a lipase enzyme according to any one of claims 1 to 7; (ii) a pharmaceutical composition according to claim 11; or (iii) a combination according to claim 12.

14. a) (i) a lipase enzyme according to any one of claims 1 to 7, (ii) a pharmaceutical composition according to claim 11, or (iii) a combination according to claim 12, b) a device for administering said lipase enzyme, pharmaceutical composition or combination, and c) Instructions for use A treatment kit comprising:

15. a) expressing a lipase enzyme in an expression host from the order Ciliata; b) purifying the lipase enzyme expressed in step a).

16. The method of claim 15, further comprising, prior to step a), transforming the ciliate with a vector encoding the lipase enzyme.

17. 17. The method of claim 15 or 16, wherein the expression host is of the genus Tetrahymena.

18. The method according to any one of claims 15 to 17, wherein the expression host is Tetrahymena thermophila.

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