Genetically engineered lipase enzymes, their production and uses

Abstract

Provided are genetically engineered lipase enzymes, methods for making such genetically engineered lipases, dosage forms containing such genetically engineered lipases, and methods of using such genetically engineered lipases to treat diseases or disorders associated with reduced ability to digest and / or absorb triglycerides (fats).

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to co-pending U.S. Provisional Patent Application No. 63 / 250,403, filed September 30, 2021, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present invention relates generally to genetically engineered lipase enzymes, methods for making such genetically engineered lipases, dosage forms containing such genetically engineered lipases, and methods of using such genetically engineered lipases to treat diseases or disorders associated with a reduced ability to digest and / or absorb triglycerides (fats). [Background technology]

[0003] background Long-chain triglycerides (fats) are the most abundant and important source of dietary lipids. Their digestion and absorption depend on a complex interplay between pancreatic lipase, co-lipases, bile acids, transit time through the body, sites of absorption, and dietary contents. Pancreatic lipase hydrolyzes triglyceride molecules to produce two fatty acid molecules and a 2-monoacylglycerol molecule. For this, lipase binds to the oil-water interface of triglyceride-containing droplets. Once liberated, the long-chain free fatty acid and the 2-monoacylglycerol molecule are absorbed in the small intestine and delivered to plasma and tissues, where they are also used for energy. Thus, there is a limited time for lipase to digest the fat and facilitate its absorption in the small intestine.

[0004] Although humans typically produce an adequate supply of pancreatic lipase to digest triglycerides, there are certain diseases and disorders that significantly affect this complex physiological balance. Malabsorption syndromes are a series of life-threatening conditions that affect one or more of the steps of triglyceride hydrolysis or absorption. Fat malabsorption can be caused by (1) impaired secretion of pancreatic enzymes, usually associated with exocrine pancreatic insufficiency (EPI); (2) improvements in gastric, duodenal, hepatic, biliary or gallbladder physiology, manifested as (a) altered gastric secretion, (b) disturbed gastrointestinal transit, motility, mixing, emptying, and / or (c) critical loss of intestinal mucosal function due to mucosal damage. Diseases affecting the pancreas, such as cystic fibrosis (CF), chronic pancreatitis (CP) and pancreatic cancer, can result in fat malabsorption leading to malnutrition.

[0005] The current state of the art uses porcine derived products (PERT), such as pancrelipase and pancreatin, which are known to have several limitations, such as loss of activity from acid denaturation and proteolytic degradation during passage through the gastrointestinal (GI) tract (Lankisch et al. (1993) DIGESTION 54:148-155; Thiruvengadam et al. EP (1988) AM. J. PHYSIOL. 255:G476-G481; Guarner et al. (1993) GUT 34:708-712). Porcine derived enzymes are extracted from pig pancreases at the slaughterhouse and may contain certain impurities such as poorly characterized proteins, porcine viruses and other biological substances. Medical-grade products based on porcine extracts (e.g., pancrelipase) have a significant limitation in that the lipase is inactivated by the low pH of the stomach and by proteolytic degradation (DiMagno et al. (1977) N. ENGL. J. MED. 296(23):1318-22.). To prevent inactivation, current preparations are often enteric coated using phthalates to prevent release of the contents of the preparation until the pH reaches 5.5 (Creon®, Prescribing Information, Pharmacokinetics; Katherine E. Kelley et al. (2012) ENVIRON. HEALTH PERSPECT. 120(3): 379-384). There have been many attempts to treat fat malabsorption using bacterial or fungal lipases that have failed due to the complex nature of fat digestion and absorption and the inherent instability of lipases due to acid denaturation, proteolytic degradation or unfolding, and bile salt inhibition. Manufacturers' attempts to improve stability (viability) using enteric coating or chemical stabilization techniques have resulted in a mismatch in lipase availability and lipid substrates necessary for proper fat digestion and absorption in the small intestine. Furthermore, despite chronic use, current PERT is ineffective because clinical nutritional goals are not being met, particularly in adults and young children with cystic fibrosis (CF).The insufficient hydrolysis of current PERT can result in reduced caloric intake, poor weight control and significant levels of undesirable GI symptoms that dramatically affect quality of life. Furthermore, due to the insufficient stability of the lipase in PERT, there are no liquid compatible formulations available for infants, children and adults who are unable to swallow pills.

[0006] A stable lipase that can be active immediately without the need for enteric coating or other techniques that can interfere with solubility provides the ability for lipase to have a longer time to interact with fat substrates, allowing for further substrate hydrolysis and absorption. In people with EPI, pancreatic and duodenal bicarbonate secretion is insufficient to neutralize the gastric acid load. Therefore, duodenal pH is typically lower in subjects with CF compared to healthy subjects. Thus, CF patients may have a significantly longer postprandial period during which duodenal pH is less than 4. As a result, this extended time that hyperacidity exists pushes out the time that lipase enzymes in the subject are available to digest fat, which can delay fat digestion and absorption, and can skip a significant portion of the duodenum, resulting in steatorrhea and significant undesirable GI symptoms. The delayed small intestinal buffering capacity in EPI subjects seems to support the idea that delayed dissolution of enteric coated products due to insufficient solubility may be a contributing factor to poor fat absorption (Gelfond et al. (2017) CLINICAL AND TRANSLATIONAL GASTROENTEROLOGY (2017) 8, e81).

[0007] The absorption of long-chain triglycerides first requires the enzymatic action of pancreatic lipase, whereas medium-chain triglycerides, due to their shorter chain length, can be absorbed through the intestinal lumen by the action of gastric lipase. Although all fats provide caloric benefits, they have different effects on physiological functions (St-Ogne et al. (2002) JOURNAL OF NUTRITION 132(3):329-332). Although both long-chain triglycerides and medium-chain triglycerides provide calories, only long-chain triglycerides in the form of long-chain polyunsaturated fatty acids (e.g., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) provide structural components of membranes and biological mediators involved in the control of many physiological functions. Furthermore, when substituted for long-chain triglycerides, medium-chain triglycerides have been shown to increase energy expenditure and satiety, leading to reduced overall caloric intake and reduced body fat mass. Thus, proper digestion and absorption of long-chain lipids is important for good health.

[0008] Despite the efforts made to date in treating disorders associated with a reduced ability to digest and / or absorb triglycerides (fat), there remains an ongoing need for new and effective therapies to treat such disorders. Summary of the Invention

[0009] Summary of the Invention The present invention is based in part on the development of engineered lipase enzymes optimized to provide enhanced activity in the gastrointestinal tract as well as reduced susceptibility to proteolytic degradation and increased resistance to acidic pH levels. The engineered lipase enzymes can hydrolyze physiologically relevant fats (triglycerides) at pH ranges early in the digestive process, e.g., during transit through the stomach where a low pH environment exists (e.g., in the range of 60-120 minutes), which then facilitates rapid absorption of the resulting fatty acids during transit through the small intestine over a short period of time, e.g., in the range of 2-4 hours. Furthermore, it is contemplated that the recombinant enzymes described herein may be suitable for oral administration given their enhanced viability, and therefore potentially safer and more tolerable than commercially available PERT enzymes. The terms "stability" and "viability" are used interchangeably herein to refer to the ability of a lipase to maintain functional activity, e.g., enzymatic activity, under given conditions, e.g., conditions encountered in the gastrointestinal tract of a primate subject. Measuring stability / viability can be done using any of the methods described herein, such as assessing the ability of the lipase to break down lipid triglycerides into monoglycerides and free fatty acids. Engineered lipase enzymes can be used to treat diseases or disorders associated with a reduced ability to digest or absorb fats (triglycerides).

[0010] In one aspect, the disclosure relates to recombinant variant microbial lipase enzymes (e.g., variant Burkholderia cepacia lipases) that comprise one or more of the following characteristics: (i) increased stability at acidic pH (e.g., pH 3.0 or 4.0) relative to the corresponding wild-type microbial lipase enzyme; (ii) increased stability in the presence of a protease (e.g., a serine protease and / or an aspartic protease) relative to the corresponding wild-type microbial lipase enzyme; or (iii) at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the enzymatic activity of the corresponding wild-type microbial lipase enzyme, such as characteristics (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii), and (i), (ii) and (iii).

[0011] In one embodiment, the lipase is: (a) Substitution of a residue at the position corresponding to position 39 of wild-type Burkholderia cepacia lipase; (b) substitution of a residue at the position corresponding to position 79 of wild-type B. cepacia lipase; (c) substitution of a residue at a position corresponding to position 102 of wild-type B. cepacia lipase; (d) substitution of a residue at a position corresponding to position 125 of wild-type B. cepacia lipase; (e) substitution of a residue at the position corresponding to position 128 of wild-type B. cepacia lipase; (f) substitution of a residue at the position corresponding to position 137 of wild-type B. cepacia lipase; (g) substitution of a residue at a position corresponding to position 138 of wild-type B. cepacia lipase; (h) substitution of a residue at a position corresponding to position 153 of wild-type B. cepacia lipase; (i) substitution of a residue at the position corresponding to position 154 of wild-type B. cepacia lipase; (j) substitution of a residue at a position corresponding to position 161 of wild-type B. cepacia lipase; (k) substitution of a residue at a position corresponding to position 170 of wild-type B. cepacia lipase; (l) substitution of a residue at a position corresponding to position 221 of wild-type B. cepacia lipase; (m) substitution of a residue at a position corresponding to position 227 of wild-type B. cepacia lipase; (n) substitution of a residue at a position corresponding to position 240 of wild-type B. cepacia lipase; (o) substitution of a residue at a position corresponding to position 249 of wild-type B. cepacia lipase; (p) substitution of a residue at a position corresponding to position 250 of wild-type B. cepacia lipase; (q) substitution of a residue at a position corresponding to position 260 of wild-type B. cepacia lipase; (r) substitution of a residue at the position corresponding to position 266 in wild-type B. cepacia lipase; (s) substitution of a residue at a position corresponding to position 281 of wild-type B. cepacia lipase; (t) substitution of a residue at a position corresponding to position 300 of wild-type B. cepacia lipase; or any combination of the foregoing substitutions.

[0012] In one embodiment: (a) The residue at the position corresponding to position 39 of wild-type B. cepacia lipase is substituted with R; (b) the residue at the position corresponding to position 79 of wild-type B. cepacia lipase is substituted with Q; (c) the residue at the position corresponding to position 102 of wild-type B. cepacia lipase is substituted with Q; (d) the residue at the position corresponding to position 125 of wild-type B. cepacia lipase is substituted with S; (e) the residue at the position corresponding to position 128 of wild-type B. cepacia lipase is substituted with N; (f) the residue at the position corresponding to position 137 of wild-type B. cepacia lipase is substituted with A; (g) the residue at the position corresponding to position 138 of wild-type B. cepacia lipase is substituted with I; (h) the residue at the position corresponding to position 153 of wild-type B. cepacia lipase is substituted with N; (i) the residue at the position corresponding to position 154 of wild-type B. cepacia lipase is substituted with H; (j) the residue at the position corresponding to position 161 of wild-type B. cepacia lipase is substituted with A; (k) the residue at the position corresponding to position 170 of wild-type B. cepacia lipase is substituted with S; (l) the residue at the position corresponding to position 221 of wild-type B. cepacia lipase is substituted with L; (m) the residue at the position corresponding to position 227 of wild-type B. cepacia lipase is substituted with K; (n) the residue at the position corresponding to position 240 of wild-type B. cepacia lipase is substituted with V; (o) the residue at the position corresponding to position 249 of wild-type B. cepacia lipase is substituted with L; (p) the residue at the position corresponding to position 250 of wild-type B. cepacia lipase is substituted with A; (q) the residue at the position corresponding to position 260 of wild-type B. cepacia lipase is substituted with A; (r) the residue at the position corresponding to position 266 of wild-type B. cepacia lipase is substituted with L; (s) the residue at the position corresponding to position 281 of wild-type B. cepacia lipase is substituted with A; (t) whether the residue at the position corresponding to position 300 in wild-type B. cepacia lipase is substituted with Y; Or the lipase includes any combination of the foregoing substitutions.

[0013] In one embodiment, the lipase is: (a) Substitution of a Q residue at the position corresponding to position 39 in wild-type B. cepacia lipase (Q39); (b) substitution of a T residue at the position corresponding to position 79 in wild-type B. cepacia lipase (T79); (c) substitution of a D residue at the position corresponding to position 102 in wild-type B. cepacia lipase (D102); (d) substitution of a G residue at the position corresponding to position 125 in wild-type B. cepacia lipase (G125); (e) substitution of an A residue at the position corresponding to position 128 in wild-type B. cepacia lipase (A128); (f) substitution of a T residue at the position corresponding to position 137 in wild-type B. cepacia lipase (T137); (g) substitution of a V residue at the position corresponding to position 138 in wild-type B. cepacia lipase (V138); (h) substitution of an S residue at the position corresponding to position 153 in wild-type B. cepacia lipase (S153); (i) substitution of an N residue at the position corresponding to position 154 in wild-type B. cepacia lipase (N154); (j) substitution of an L residue at the position corresponding to position 161 in wild-type B. cepacia lipase (L161); (k) substitution of an A residue at the position corresponding to position 170 in wild-type B. cepacia lipase (A170); (l) substitution of an F residue at the position corresponding to position 221 in wild-type B. cepacia lipase (F221); (m) substitution of a T residue at the position corresponding to position 227 in wild-type B. cepacia lipase (T227); (n) substitution of an A residue at the position corresponding to position 240 in wild-type B. cepacia lipase (A240); (o) substitution of an F residue at the position corresponding to position 249 in wild-type B. cepacia lipase (F249); (p) substitution of a G residue at the position corresponding to position 250 in wild-type B. cepacia lipase (G250); (q) substitution of an S residue at the position corresponding to position 260 in wild-type B. cepacia lipase (S260); (r) substitution of a V residue at the position corresponding to position 266 in wild-type B. cepacia lipase (V266); (s) substitution of an S residue at the position corresponding to position 281 in wild-type B. cepacia lipase (S281); (t) substitution of an N residue at the position corresponding to position 300 in wild-type B. cepacia lipase (N300); or any combination of the foregoing substitutions.

[0014] In one embodiment: (a) The Q residue at the position corresponding to position 39 in wild-type B. cepacia lipase was replaced with R (Q39R); (b) the T residue at the position corresponding to position 79 in wild-type B. cepacia lipase was replaced with Q (T79Q); (c) the D residue at the position corresponding to position 102 in wild-type B. cepacia lipase was replaced with Q (D102Q); (d) the G residue at the position corresponding to position 125 in wild-type B. cepacia lipase was replaced with S (G125S); (e) the A residue at the position corresponding to position 128 in wild-type B. cepacia lipase was replaced with N (A128N); (f) the T residue at the position corresponding to position 137 in wild-type B. cepacia lipase was replaced with A (T137A); (g) the V residue at the position corresponding to position 138 in wild-type B. cepacia lipase was replaced with I (V138I); (h) the S residue at the position corresponding to position 153 in wild-type B. cepacia lipase was replaced with N (S153N); (i) the N residue at the position corresponding to position 154 in wild-type B. cepacia lipase was replaced with H (N154H); (j) the L residue at the position corresponding to position 161 in wild-type B. cepacia lipase was replaced with A (L161A); (k) the A residue at the position corresponding to position 170 in wild-type B. cepacia lipase was replaced with S (A170S); (l) the F residue at the position corresponding to position 221 in wild-type B. cepacia lipase was replaced with L (F221L); (m) the T residue at the position corresponding to position 227 in wild-type B. cepacia lipase was replaced with K (T227K); (n) the A residue at the position corresponding to position 240 in wild-type B. cepacia lipase was replaced with V (A240V); (o) the F residue at the position corresponding to position 249 in wild-type B. cepacia lipase was replaced with L (F249L); (p) the G residue at the position corresponding to position 250 in wild-type B. cepacia lipase was replaced with A (G250A); (q) the S residue at the position corresponding to position 260 in wild-type B. cepacia lipase was replaced with A (S260A); (r) the V residue at the position corresponding to position 266 in wild-type B. cepacia lipase was replaced with L (V266L); (s) the S residue at the position corresponding to position 281 in wild-type B. cepacia lipase was replaced with A (S281A); (t) the N residue at the position corresponding to position 300 in wild-type B. cepacia lipase is replaced with Y (N300Y); Or the lipase includes any combination of the foregoing substitutions.

[0015] In some embodiments, the lipase comprises multiple substitutions, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more different substitutions. For example, the lipase may comprise 3 substitutions. Alternatively, the lipase may comprise 4 substitutions. Alternatively, the lipase may comprise 5 substitutions. Alternatively, the lipase may comprise 6 substitutions. Alternatively, the lipase may comprise 7 substitutions. Alternatively, the lipase may comprise 8 substitutions. Alternatively, the lipase may comprise 9 substitutions. Alternatively, the lipase may comprise 10 substitutions. Alternatively, the lipase may comprise 11 substitutions. Alternatively, the lipase may comprise 12 substitutions.

[0016] In one embodiment, the lipase is: (a) D102Q, N154H and F221L substitutions; (b) D102Q, G125S, N154H, F221L, V266L, and N300Y substitutions; (c) T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, F249L, V266L, and N300Y substitutions; (d) T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, V266L, S281A, and N300Y substitutions; (e) T79Q, D102Q, G125S, S153N, N154H, F221L, T227K, V266L, S281A, and N300Y substitutions; (f) T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, G250A, V266L, and N300Y substitutions; (g) T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, S281A, and N300Y substitutions; (h) T79Q, D102Q, G125S, N154H, F221L, T227K, F249L, V266L, S281A, and N300Y substitutions; (i) D102Q, G125S, T137A, S153N, N154H, F221L, T227K, F249L, V266L and N300Y substitutions; (j) D102Q, G125S, T137A, S153N, N154H, F221L, T227K, G250A, V266L, and N300Y substitutions; (k) Substitutions of D102Q, G125S, T137A, N154H, F221L, T227K, G250A, V266L, S281A, and N300Y; (l) Substitution of D102Q, G125S, S153N, N154H, F221L, T227K, F249L, G250A, V266L, and N300Y; or (m) Contains the substitutions D102Q, G125S, S153N, N154H, F221L, T227K, F249L, V266L, S281A, and N300Y.

[0017] In some embodiments, the lipase is an α / β-hydrolase lipase, and the lipase may include an active site that includes a serine-histidine-aspartic acid triad. Alternatively or additionally, the lipase may include a hydrophobic lid that opens to allow binding and / or hydrolysis of triglycerides, e.g., with a chain length longer than 8 carbons. Alternatively or additionally, in some embodiments, the lipase includes a calcium binding site, where the lipase is stabilized when calcium binds to the calcium binding site. Alternatively or additionally, in some embodiments, the lipase includes an oxyanion hole, where the oxyanion hole stabilizes a negatively charged intermediate generated during fatty acid bond hydrolysis.

[0018] In some embodiments, the lipase is a fungal lipase or a bacterial lipase. In some embodiments, the lipase is a family I bacterial lipase, such as a I.1, I.2 or I.3 subfamily bacterial lipase, such as a I.1 or I.2 subfamily bacterial lipase. In some embodiments, the lipase is a I.2 subfamily bacterial lipase.

[0019] In some embodiments, the lipase is a Burkholderia, Pseudomonas or Chromobacterium lipase. In some embodiments, the lipase is a Burkholderia cepacia (B. cepacia), Bacillus subtilis, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas luteola or Chromobacterium viscosum lipase. In some embodiments, the lipase is a Burkholderia cepacia lipase.

[0020] In certain embodiments, the lipase comprises an S residue at a position corresponding to position 87 in wild-type B. cepacia (S87), a D residue at a position corresponding to position 264 in wild-type B. cepacia (D264), and an H residue at a position corresponding to position 286 in wild-type B. cepacia (H286). These amino acids are conserved between lipase subfamilies I.1 and I.2 (see FIG. 3).

[0021] In one embodiment, the lipase comprises the amino acid sequence of any of SEQ ID NOs:2-14 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs:2-14.

[0022] In some embodiments, the lipase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 mutations relative to a corresponding wild-type microbial lipase.

[0023] In another aspect, the disclosure relates to recombinant variant microbial lipase enzymes comprising a substitution or combination of substitutions listed in Table 1 or Table 2.

[0024] In some embodiments, the lipases disclosed herein have a half-life in the presence of a serine protease of at least 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, or 200 minutes. Alternatively or additionally, in some embodiments, the lipase has at least 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater stability in the presence of a serine protease (e.g., Aspergillus melleus protease) compared to the corresponding wild-type lipase.

[0025] In some embodiments, the lipase has a half-life of at least 50 minutes, 75 minutes, 100 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, or 150 minutes at about pH 3.0. Alternatively or additionally, in some embodiments, the lipase has at least 1.5 times, 2 times, 2.5 times, or 3 times greater stability at about pH 3.0 compared to the corresponding wild-type lipase.

[0026] In some embodiments, the lipase has a half-life in the presence of an aspartic protease (e.g., at pH 3.6) of at least 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 200 minutes, 225 minutes, 230 minutes, or 235 minutes. Alternatively or additionally, in some embodiments, the lipase has at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold greater stability in the presence of an aspartic protease (e.g., at pH 3.6) compared to the corresponding wild-type lipase. In some embodiments, the aspartic protease is pepsin.

[0027] In some embodiments, the lipase has a half-life of at least 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, or 200 minutes in the presence of pancreatin. Alternatively or additionally, in some embodiments, the lipase has at least 0.5 times, 1 times, 1.5 times, 2 times, 2.5 times, or 3 times greater stability in the presence of pancreatin compared to the corresponding wild-type lipase. Alternatively or additionally, in some embodiments, the lipase has at least 0.5 times, 1 times, 1.5 times, 2 times, 2.5 times, or 3 times greater activity at about pH 3.0 compared to the corresponding wild-type lipase.

[0028] In certain embodiments, the lipase has a specific activity at pH 3.0 on a long chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long chain triglyceride substrates set forth in Table 3) of at least 300, 400, 500, 600, 700, 800, 900, or 1,000 μmol fatty acid (FA) produced / min / mg of lipase. In certain embodiments, the lipase has a specific activity of at least 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or 2,000 μmol fatty acid (FA) produced / min / mg of lipase at pH 4.0, pH 5.0, or pH 6.0 on a long chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long chain triglyceride substrates set forth in Table 3). In certain embodiments, the lipase has a specific activity at pH 7.0 on a long chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long chain triglyceride substrates set forth in Table 3) of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or 2,000 μmol fatty acid (FA) produced / min / mg of lipase.

[0029] In some embodiments, the lipase preferentially hydrolyzes the sn-1 and sn-3 positions on triglycerides, and / or the lipase enzymatic activity (e.g., specific activity) is not inhibited by bile acid residues, and / or the lipase does not require a co-lipase. In some embodiments, the lipase is not cross-linked and / or crystallized.

[0030] In some embodiments, the lipase remains sufficiently active at a pH ranging from 3.5 to 7.0 to hydrolyze long-chain polyunsaturated fats (LCPUFAs), such as DHA and EPA, or long-chain triglycerides, such as oleic acid or triolein, in the gastrointestinal tract of a subject, hi some embodiments, the lipase is at least 2, 10, 100, or 1000 times more active than pancrelipase when tested under the same conditions.

[0031] In some embodiments, greater than 50%, 60%, 70%, 80% or 90% of the lipase remains active for 60-120 minutes in the fed state stomach of the subject, hi some embodiments, the lipase digests greater than 20%, 30%, 40% or 50% of ingested fat in the stomach of the subject into fatty acids and monoglycerides.

[0032] In some embodiments, greater than 50%, 60%, 70%, 80% or 90% of the lipase remains active for 240-360 minutes through the small intestine of the subject, hi some embodiments, the lipase digests greater than 50%, 60%, 70%, 80% or 90% of ingested fat in the small intestine of the subject into fatty acids and monoglycerides.

[0033] In some embodiments, lipase increases the absorption of long chain unsaturated fatty acids in the plasma of a subject within 30, 45, 60, 90 or 120 minutes by more than 25%, 35%, 50%, 100% or 200% relative to the same subject when not administered lipase or to a similar subject when not administered lipase. In some embodiments, lipase increases the absorption of fat-soluble vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K). In some embodiments, lipase increases the absorption of choline.

[0034] In another aspect, the disclosure relates to a nucleic acid encoding a lipase described herein.

[0035] In another aspect, the present disclosure relates to an expression vector comprising the nucleic acid sequence described herein. In some embodiments, the nucleic acid sequence encoding the recombinant variant lipase is codon-optimized for expression in a heterologous cell. In some embodiments, the heterologous cell is a Bacillus subtilis, Bacillus oryzae, Pseudomonas fluorescens, Chromobacterium viscosum, Pseudomonas luteola, Pseudomonas fragi or Escherichia coli cell.

[0036] In another aspect, the present disclosure relates to a cell comprising the expression vector described herein. In some embodiments, the cell is a B. cepacia, Pseudomonas glumae, Pseudomonas fluorescens, Chromobacterium viscosum, Pseudomonas luteola, Pseudomonas fragilis, or Escherichia coli cell.

[0037] In certain aspects, the disclosure relates to a method of making a recombinant variant microbial lipase enzyme, the method comprising growing a cell described herein under conditions such that the host cell expresses the recombinant variant microbial lipase enzyme and purifying the recombinant variant microbial lipase enzyme.

[0038] In another aspect, the present disclosure relates to a pharmaceutical composition comprising the lipase described herein and a pharma- ceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition further comprises a microbial protease and / or a microbial amylase. In some embodiments, the protease is A. melleus protease and / or the amylase is Aspergillus amylase. In some embodiments, the composition is formulated as an oral dosage form. In some embodiments, the composition is formulated as a powder, granule, pellet, micropellet, liquid or tablet. In some embodiments, the composition is encapsulated or formulated as a tablet dosage form. In some embodiments, the composition does not include an enteric coating.

[0039] In another aspect, the present disclosure relates to a method of treating a disease or disorder associated with a reduced ability to digest or absorb lipids resulting in increased amounts of undigested lipid in a subject in need of such treatment, the method comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby treating the disease or disorder in the subject.

[0040] In another aspect, the present disclosure relates to a method of treating lipid maldigestion or malabsorption in a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby treating the disease or disorder in the subject.

[0041] In some embodiments, the subject has a physiological condition that exhibits low levels of pancreatic enzyme secretion or affects fat hydrolysis or fat absorption (e.g., reduced gastric, duodenal, hepatic, biliary or gallbladder function); reduced gastrointestinal transit, motility, mixing, emptying; or reduced intestinal mucosal function (e.g., induced by mucosal injury) resulting in fat maldigestion or fat malabsorption or fatty acid deficiency.

[0042] In certain embodiments, the lipid maldigestion or malabsorption is associated with a disease or disorder selected from exocrine pancreatic insufficiency (EPI), malabsorption syndromes, cystic fibrosis, chronic pancreatitis, acute pancreatitis, Shwachman-Diamond syndrome, fatty acid disorders, familial lipoprotein lipase deficiency, Johanson-Blizzard syndrome, Zollinger-Ellison syndrome, Pearson bone marrow syndrome, short bowel syndrome, liver disease, primary biliary atresia, cholestasis, celiac disease, fatty liver disease, pancreatitis, diabetes, aging, cancer of the pancreas, stomach, small intestine, colon, rectum / anus, liver, hepatic, gallbladder or esophagus, cachexia, or a gastrointestinal disorder (e.g., Crohn's disease, irritable bowel syndrome or ulcerative colitis), stomach, small intestine, liver, gallbladder or pancreas surgical intervention.

[0043] In another aspect, the present disclosure relates to a method of improving fatty acid absorption in a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby improving fatty acid absorption in the subject.

[0044] In another aspect, the present disclosure relates to a method for increasing the amount of fatty acids in plasma, red blood cells or tissues of a subject in need thereof, the method comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby increasing the amount of fatty acids in the subject.

[0045] In another aspect, the disclosure relates to a method of increasing the ratio of omega-3 to omega-6 fatty acids in plasma, red blood cells or tissues of a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby increasing the amount of fatty acids in the subject.

[0046] In another aspect, the disclosure relates to a method for reducing the amount of fatty acids in the stool of a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby reducing the amount of fatty acids in the stool of the subject.

[0047] In some embodiments, the fatty acid is a long chain polyunsaturated fatty acid (LCPUFA). In some embodiments, the fatty acid is an omega-3 fatty acid. In some embodiments, the omega-3 fatty acid is DHA, EPA or DPA. In some embodiments, the subject is administered less than 400, 600, 800 or 1,000 mg of lipase or pharmaceutical composition per day. In some embodiments, the lipase or pharmaceutical composition is administered in combination with a nutritional formulation that includes fat-soluble vitamins (e.g., vitamins A, D, E or K), acid blockers or triglycerides.

[0048] In certain embodiments, the subject is a mammal, such as a human.

[0049] These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief description of the drawings]

[0050] BRIEF DESCRIPTION OF THE DRAWINGS The invention may be more fully understood with reference to the following drawings. [Figure 1A] 1A shows a schematic of an exemplary lipase in which, in the absence of long-chain triglycerides, the active site is protected from the environment due to the interaction of the lid and the subdomain, and is believed to exist in a closed conformation in which the lid covers the active site cleft and the subdomain covers the lid. In the presence of long-chain triglycerides, the conformational change of the lipase is believed to produce an open conformation in which the lid and the subdomain open to expose the active site cleft. Structural studies suggest that the hydrophobic lipid binding site is exposed by the retraction or opening movement of the lid from the active site at the oil-water interface. [Figure 1B]FIG. 1B shows a space-filling model of the three-dimensional structure of an exemplary lipase from Burkholderia cepacia in both the closed, inactive and open, active conformations. [Diagram 2] Figure 2 shows a ribbon model of lipase from Burkholderia cepacia in which amino acids 118–159 define the lid, amino acids 214–261 define the lid-facing subdomain, residues 262–320 contain α-helix 11, and amino acids 160–213 contain α-helix 7. The amino acids that contribute to the catalytic triad (i.e., serine 87, aspartic acid 264, and histidine 286) are shown. [Diagram 3] FIG. 3 illustrates a phylogenetic tree of family I bacterial lipases and their classification into six subfamilies (designated I.1 to I.6). [Figure 4]FIG. 4 shows Pseudomonas aeruginosa PAO1 (family I.1, SEQ ID NO: 29), Pseudomonas fluorescens (family I.1, SEQ ID NO: 30), Burkholderia cepacia (family I.2, SEQ ID NO: 1), Burkholderia glumae (family I.2, SEQ ID NO: 31), Chromobacterium viscosum (family I.2, SEQ ID NO: 32), Pseudomonas luteola (family I.2, SEQ ID NO: 33), Pseudomonas fluorescens ABA 72135 (family I.1, SEQ ID NO:34), Pseudomonas fluorescens AEV60646 (family I.1, SEQ ID NO:35), Pseudomonas sp. WP-015093259 (family I.3, SEQ ID NO:36), Pseudomonas fragilis CAA32193 (family I.1, SEQ ID NO:37), Pseudomonas fragilis CAC07191 (family I.1, SEQ ID NO:38), Pseudomonas stutzeri (SEQ ID NO:41) and Pseudomonas mendocina LipA (SEQ ID NO:42). The amino acid residues that make up the catalytic triad (active site) and calcium binding site are indicated in the figure (boxed and shaded). Substitutions made in the final round of lipase engineering (see Example 7) are shown relative to the wild-type B. cepacia sequence (no box or shading). [Diagram 5] Figure 5 shows a sequence alignment showing the conservation of residues between Burkholderia cepacia (family I.2, SEQ ID NO:1), Burkholderia glumae (family I.2, SEQ ID NO:31), Chromobacterium viscosum (family I.2, SEQ ID NO:32) and Pseudomonas luteola (family I.2, SEQ ID NO:40), identifying the conserved amino acids that make up the oxyanion hole, lid, subdomain, catalytic triad and calcium binding site. The positions of the amino acid substitutions made in the final round of lipase engineering (see Example 7) relative to the wild-type B. cepacia sequence are shown in boxes with dark outlines. [Figure 6]FIG. 6 shows a three-dimensional model of B. cepacia lipase showing the arrangement of the catalytic lid, oxyanion hole, catalytic triad, calcium domain and the location of the top variant substitutions. [Figure 7] FIG. 7 is a schematic diagram of B. cepacia lipase showing the arrangement of the active site / catalytic triad (stars), calcium sites (circles), last round amino acid substitutions (triangles), oxyanion hole, lid and lid facing subdomains (various shading). [Figure 8] FIG. 8 is a schematic diagram for an exemplary three-step reaction for free fatty acid detection. [Figure 9] Figure 9 is a flow diagram of the pH viability assay. Lipase solutions are pretreated by incubation at specific pH for a series of time points and then assayed for lipase activity with 4-nitrophenyl palmitate (p-NPP) to detect a colorimetric response at 405 nm and report the pH stability over time for each pH. [Figure 10] FIG. 10 illustrates the mechanism of hydrolysis of p-NPP (colorless) to 4-nitrophenolate (pNP, yellow) by lipase. [Figure 11] Figure 11 is a flow diagram of the pepsin viability assay. Lipase solutions are pre-treated by incubation with pepsin for a series of time points and then assayed for lipase activity with 4-nitrophenyl palmitate (p-NPP), detecting a colorimetric response at 405 nm to report the half-life over time for pepsin. [Figure 12] Figure 12 is a flow diagram of the A. melleus protease (oryzin) viability assay. Lipase solutions are pre-treated by incubation with oryzin for a series of time points and then assayed for lipase activity with 4-nitrophenyl palmitate (p-NPP), detecting a colorimetric response at 405 nm and reporting the half-life over time for oryzin. [Figure 13]FIG. 13 is a graph showing the effect of the indicated lipase mutations on stability in the presence of A. melleus protease, stability at low pH, stability in the presence of pepsin / SGF, activity at pH 4, and activity in the presence of bile salts at pH 7. [Figure 14] 14 is a graph showing the stability or activity of engineered mutants against B. cepacia V290 lipase variants. Conditions tested were stability in the presence of A. melleus protease (t1 / 2), stability at low pH (t1 / 2), stability in the presence of pepsin / SGF (t1 / 2) and activity at pH 4 (U / mg). [Figure 15] 15 is a graph showing the half-life of the top 11 B. cepacia lipase variants under the conditions indicated. Three controls were used: (1) wild-type (WT) B. cepacia lipase, (2) V130 (a top variant from an early round), and (3) V290 (a top variant from one of the later rounds). [Figure 16] 16 is a graph showing lipase engineering viability improvement for the top three B. cepacia lipase variants, V325, V366 and V318, under the conditions indicated (proteolytic stability, stability at low pH and stability in the presence of pepsin). Three controls were used: (1) wild-type (WT) B. cepacia lipase, (2) V130 variant and (3) V290 variant. The Y-axis indicates time in minutes. [Figure 17] 17 is a graph showing the percentage of lipase surviving A. melleus protease treatment at different time points (5, 30, 60, 120, 180 and 240 min). The graph shows the top three B. cepacia lipase variants, V325, V366 and V318, as well as three controls (wild type (WT) B. cepacia lipase, V130 variant, V290 variant). [Figure 18]FIG. 18 is a graph showing the percentage of lipase surviving pH 3.0 treatment at different time points (5, 30, 60 and 120 minutes) for the top three B. cepacia lipase variants, V325, V366 and V318 and three controls (wild type (WT) B. cepacia lipase, V130 variant, V290 variant). [Figure 19] FIG. 19 is a graph showing the percentage of lipase surviving pepsin treatment at pH 3.58 (typical fed stomach) at different time points (5, 30, 60 and 120 min) for the top three B. cepacia lipase variants V325, V366 and V318 and three controls (wild type (WT) B. cepacia lipase, V130 variant, V290 variant). [Figure 20] Figures 20A and 20B are graphs showing the activity per serving (free fatty acid releasing DHA oil) of wild type lipase, top three variants (V318, V325 and V336) and pancrelipase at 40 mg (Figure 20A) and 80 mg (Figure 20B). [Figure 21] FIG. 21 is a schematic diagram of the treatment group design for the dose-finding study for V325 in the EPI pig model described in Experiment 1 of Example 9. [Figure 22] FIG. 22 is a graph showing the AUC and Cmax for the free fatty acids DHA and EPA in plasma of animals administered an omega-3 triglyceride substrate and the indicated doses of V325 or no enzyme ("NE"). [Diagram 23] FIG. 23 is a graph showing the mean AUC24 over time calculated from the AUC data provided in FIG. [Figure 24] FIG. 24 is a graph showing baseline subtracted Cmax calculated from the Cmax data provided in FIG. [Diagram 25] FIG. 25 is a graph showing the AUC and Cmax for total fatty acids in plasma of animals administered the substrate and the indicated doses of V325 or no enzyme ("NE"). [Figure 26]FIG. 26 is a graph showing the mean AUC24 over time calculated from the AUC data provided in FIG. [Figure 27] FIG. 27 is a graph showing baseline subtracted Cmax calculated from the Cmax data provided in FIG. [Figure 28] FIG. 28 is a schematic diagram of the treatment group design for the evaluation of the activity and stability of V325 in the EPI pig model described in Experiment 2 of Example 9. [Figure 29] Figure 29A is a graph showing that the AUC and Cmax for DHA + EPA in plasma of animals administered omega-3 triglyceride substrate and the indicated doses of V325 were significantly higher than animals administered Creon® or no enzyme ("NE"). Figure 29B is a graph showing the baseline-subtracted AUC average over time for time points 6, 8, 12 and 24, calculated from the AUC data provided in Figure 29A. [Diagram 30] Figure 30A is a graph showing that the AUC and Cmax for total fatty acids in plasma of animals administered the substrate and the indicated doses of V325 were significantly higher than animals administered Creon® or no enzyme ("NE"). Figure 30B is a graph showing the baseline-subtracted AUC average over time for time points 6, 8, 12 and 24, calculated from the AUC data provided in Figure 30A. [Diagram 31] Figure 31A is a graph showing the AUC for free fatty acid release over time in different compartments of the gastrointestinal tract (stomach, duodenum, ileum) for animals administered V325 or Creon®. Figure 31B is a graph showing the average AUC over time calculated from the AUC data provided in Figure 31A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Detailed Description The present invention is based in part on the development of engineered lipase enzymes optimized to provide enhanced viability and activity in the gastrointestinal tract, as well as reduced susceptibility to proteolytic degradation and increased tolerance to acidic pH levels. Engineered lipase enzymes can hydrolyze physiologically relevant fatty triglycerides (long chain polyunsaturated fatty acids (LCPUFA) and dietary long chain triglycerides) in the pH range early in the digestive process, for example during passage through the stomach where a low pH environment exists, which then facilitates rapid absorption of the resulting fatty acids during transit through the small intestine. Furthermore, it is contemplated that the recombinant enzymes described herein may be suitable for oral administration given their enhanced stability, and thus potentially safer and more tolerable than commercially available PERT enzymes. Engineered lipase enzymes may be used to treat diseases or disorders associated with reduced ability to digest or absorb fats (triglycerides).

[0052] Various features and aspects of the invention are discussed in greater detail below.

[0053] I. Lipase Typically, lipase enzymes hydrolyze dietary fats (triglycerides) to produce two fatty acid molecules and a monoacylglycerol molecule. Most lipases are members of the α / β hydrolase fold superfamily, one of the largest groups of structurally related but functionally diverse enzymes. The three-dimensional structures of most lipases share a common folding motif known as the α / β hydrolase fold.

[0054] Hydrolytic lipase enzymes that hydrolyze carboxy ester bonds in lipids, i.e., carboxylesterases and true lipases, are collectively referred to as lipolytic enzymes. Carboxyl esterases (esterases) usually hydrolyze water-soluble esters, while true lipases (lipases) can also hydrolyze water-insoluble substrates (Verger (1997) TRENDS IN BIOTECHNOLOGY 15(1):P32-38; Ali et al. (2012) Lipases and Phospholipases, New York, USA, Humana Press, p. 31-51). The longer the fatty acid chain in a triglyceride, the less water-soluble the triglyceride becomes. As a result, enzymes that hydrolyze long-chain triglycerides are referred to as lipases, and those that hydrolyze tributyrin (short-chain C4 fatty acid) are referred to as esterases (Jaeger et al. (1994) FEMS MICROBIOL REV 15:29-63). Long-chain triglycerides are primarily ingested in the human diet, while short-chain fatty acids are typically by-products of carbohydrate metabolism by anaerobic bacteria in the colon. A property of true lipases (also referred to herein as lipases) that distinguishes lipases from esterases is their enhanced activity at oil-water interfaces, a phenomenon referred to as "interfacial activation" (Schrag et al. (1991) NATURE 351(6329):761-764).

[0055] Lipases contain an active site cleft that is structurally conserved and covered by a flexible, amphipathic α-helix that functions as a "lid" covering the active site cleft depending on the surrounding conditions. When the lid is closed, the active site is protected from the environment and inaccessible to triglyceride substrates. Figure 1A shows a schematic of an exemplary lipase in which, in the absence of long-chain triglycerides, the active site is protected from the environment due to the interaction of the lid and the subdomain, and is thought to exist in a closed conformation in which the lid covers the active site cleft and the subdomain covers the lid. However, in the presence of long-chain triglycerides, a conformational change in the lipase produces an open conformation in which the lid and the subdomain open to expose the active site cleft. Structural examination suggests that the hydrophobic lipid binding site is exposed by the retraction or opening movement of the lid from the active site at the oil-water interface.

[0056] FIG. 1B shows a space-filling model of the three-dimensional structure of an exemplary lipase from Burkholderia cepacia in both the closed, inactive and open, active conformations. In the inactive conformation, the lid covers the active site. In the active conformation, the lid and subdomain (also called the facing lid) move to expose the indicated active site cleft, which contains three amino acid residues (serine, histidine, and aspartic acid) that are conserved among many lipases (Brenner (1988) NATURE 334:528-530; Brady et al. (1990) NATURE 343(6260):767-70; Schrag et al. (1991) supra).

[0057] Figure 2 shows a ribbon model of lipase from B. cepacia in which amino acids 118-159 define the lid, amino acids 214-261 define the lid-facing subdomain, residues 262-320 contain α-helix 11, and amino acids 160-213 contain α-helix 7. The amino acids that contribute to the catalytic triad (i.e., serine 87, aspartic acid 264, and histidine 286) are shown.

[0058] Bacterial lipases have been classified into eight families (Families I-VIII) based on differences in amino acid sequences and biological properties. Among them, Family I, shown in FIG. 3, is the largest group and is further subdivided into six subfamilies (designated I.1-I.6), of which I.1, I.2 and I.3 are representative Gram-negative bacterial lipases. Family I lipases are highly conserved, and the activity of lipases in this family relies on the presence of a catalytic active site formed by three conserved amino acids, namely serine, histidine and aspartic acid. (Nardini et al. (2000) J. BIOL. CHEM. 275(40):31219-31225; Kim et al. (1997) STRUCTURE 5(2):173-185.)

[0059] FIG. 4 shows the results of the chromosome 111 expression of Pseudomonas aeruginosa PAO1 (family I.1, SEQ ID NO: 29), Pseudomonas fluorescens (family I.1, SEQ ID NO: 30), Burkholderia cepacia (family I.2, SEQ ID NO: 1), Burkholderia glumae (family I.2, SEQ ID NO: 31), Chromobacterium viscosum (family I.2, SEQ ID NO: 32), Pseudomonas luteola (family I.2, SEQ ID NO: 33), Pseudomonas fluorescens ABA72135 (family I.1, SEQ ID NO: 34), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 35), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 36), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 37), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 38), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 39), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 40), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 41), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 42), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: 43), Pseudomonas fluorescens BAC72135 (family I.1, SEQ ID NO: Figure 1 shows a sequence alignment showing the conservation of amino acids between the lipase sequences of Monas fluorescens AEV60646 (family I.1, SEQ ID NO:35), Pseudomonas sp. WP-015093259 (family I.3, SEQ ID NO:36), Pseudomonas fragilis CAA32193 (family I.1, SEQ ID NO:37), Pseudomonas fragilis CAC07191 (family I.1, SEQ ID NO:38), Pseudomonas stutzeri (SEQ ID NO:41) and Pseudomonas mendocina LipA (SEQ ID NO:42). The amino acid residues that make up the catalytic triad (active site; dark shading) and calcium binding site (light shading) are shown in the figure. Substitutions made in lipase engineering (see Example 7) are shown relative to the wild-type B. cepacia sequence (unshaded boxes). Figure 5 shows a sequence alignment showing the conservation of residues between Burkholderia cepacia (family I.2), Pseudomonas glumae (family I.2, SEQ ID NO:31), Chromobacterium viscosum (family I.2, SEQ ID NO:32) and Pseudomonas luteola (family I.2, SEQ ID NO:40), identifying the conserved amino acids that make up the oxyanion hole, lid, subdomain, catalytic triad and calcium binding site. The placement of the amino acid substitutions made in the lipase engineering (see Example 7) relative to the wild-type B. cepacia sequence is shown in the figure (boxes with dark outlines).

[0060] Family I.2 includes lipases from the gram-negative bacterium Burkholderia cepacia (also known as Pseudomonas cepacia lipase). B. cepacia lipase (1) has high activity towards long-chain polyunsaturated fatty acids such as DHA, (ii) has a broad level of activity over the physiologically relevant pH range in the gastrointestinal tract, (iii) is active with and without minerals such as bile salts or calcium, (iv) does not require a co-lipase for catalytic activity, and (v) has a higher level of activity towards the sn-1 and sn-3 regions of triglycerides to catalyze the hydrolysis of triglycerides to yield two fatty acids and 2-monoglycerides, thereby providing a good starting point for engineering the enzyme to mimic human pancreatic lipase. B. cepacia lipase contains approximately 320 amino acid residues, has a predicted molecular weight of approximately 33 kDa, and has structural features that are conserved among lipases. In particular, B. cepacia lipase contains an active site cleft that contains a catalytic triad (conserved serine, histidine and aspartic acid residues) and a lid that opens to expose the active site to allow the entry of triglycerides for hydrolysis or closes to close the active site. Other conserved features of lipases include an oxyanion hole and a calcium ion binding site. The conservation of these structural features among family I.1, family I.2 and family I.3 ​​lipases suggests that these lipases share the same mechanism of catalysis and interfacial activation (Kim et al. (1997) supra; Nardini et al. (2000) supra; Barbe et al. (2009) PROTEINS 77:509-523; Schrag et al. (1991) supra). Without wishing to be bound by theory, it is contemplated that interfacial activation of lipases occurs primarily through a conformational change in the lipase that exposes the active site and provides a hydrophobic surface for interaction with triglyceride substrates. Crystallographic and biochemical studies have demonstrated that the mechanism of hydrolysis by lipases is similar to that of serine proteases.In both cases, the oxyanion generated during hydrolysis is thought to be located in a so-called "oxyanion hole" when the lipase is in the open lid conformation (Kim et al. (1997), supra).

[0061] As discussed in more detail below, the B. cepacia lipase was subjected to rounds of mutagenesis as discussed in Examples 1, 6 and 7 to result in several amino acid substitutions that improve one or more properties of the B. cepacia lipase to achieve specific design objectives, such as producing a lipase with one or more of the following: (i) pH activity in the range of pH 3.0 to pH 7.0; (ii) high substrate specificity and activity towards certain long chain polyunsaturated fatty acids (e.g., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and long chain triglycerides (e.g., oleic acid in olive oil); (iii) no requirement for cofactors (e.g., colipase or prolipase); (iv) reduced or independent of bile salts or minerals for activity; (v) high specific activity; (vi) thermostable, e.g., in a temperature range of 35-40° C.; and (vii) proteolytic stability.

[0062] Initially, 57 amino acid substitutions that enhanced pH range and proteolytic survivability were identified (see Example 1). Sixteen substitutions were kept and additional substitutions were made, resulting in combinations of substitution properties that enhanced proteolytic and pH survivability (see Example 6). Finally, various combinations of the initially identified 18 substitutions were tested to identify specific combinations of substitutions that enhanced pH stability (survivability against gastric acid at pH 3.0) and proteolytic stability (survivability against pepsin at pH 3.6 and Aspergillus melleus protease at pH 6.4) (see Example 7). Based on these tests, specific amino acid substitutions and combinations of such substitutions that enhanced one or more properties of the lipase were found to be located in the lid, subdomains and oxyanion hole of the lipase, as shown in the sequence alignment in FIG. 5, the three-dimensional ribbon model of the enzyme shown in FIG. 6, or a schematic representation of the enzyme (FIG. 7).

[0063] II. Recombinant mutant lipases In particular, the present invention provides recombinant variant lipases that are useful for treating disorders associated with a reduced ability to digest or absorb lipids, e.g., resulting in increased amounts of undigested lipid in a subject, e.g., disorders in which the subject exhibits low levels of pancreatic enzyme secretion or has a physiological condition affecting fat hydrolysis or fat absorption (e.g., reduced gastric, duodenal, hepatic, biliary or gallbladder function; reduced gastrointestinal transit, motility, mixing, emptying; or reduced intestinal mucosal function (e.g., induced by mucosal injury). In certain embodiments, the lipase is (i) a corresponding wild-type (ii) increased stability at acidic pH (e.g., pH 3.0 or 4.0) relative to the corresponding wild-type microbial lipase enzyme; (iii) increased stability in the presence of a protease (e.g., a serine protease and / or an aspartic protease) relative to the corresponding wild-type microbial lipase enzyme; (iii) activity for a length of time sufficient to transit the GI tract (e.g., a half-life of about 75 to 225 minutes); or (iv) at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the enzymatic activity of the corresponding wild-type microbial lipase enzyme.

[0064] In some embodiments, the lipase is an α / β-hydrolase lipase and may optionally or additionally include a serine-histidine-aspartic acid activity triad. In some embodiments, the lipase includes a hydrophobic lid that opens to allow binding and / or hydrolysis of lipids. The hydrophobic lid may open sufficiently to allow binding and / or hydrolysis of triglycerides having chain lengths greater than 8 carbons.

[0065] In some embodiments, the lipase comprises a calcium binding site, where when calcium binds to the calcium binding site, the lipase is stabilized. In some embodiments, the lipase comprises an oxyanion hole, where the oxyanion hole stabilizes the negatively charged intermediate generated during fatty acid bond hydrolysis. In some embodiments, the lipase is a fungal lipase or a bacterial lipase. In some embodiments, the lipase is a family I bacterial lipase, and can be an I.1, I.2 or I.3 subfamily bacterial lipase, such as an I.1 or I.2 subfamily bacterial lipase, or can be an I.2 subfamily bacterial lipase.

[0066] In some embodiments, the lipase is a Burkholderia, Pseudomonas or Chromobacterium lipase. In some embodiments, the lipase is a B. cepacia, Pseudomonas glumae, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas luteola or Chromobacterium viscosum lipase. In some embodiments, the lipase is a B. cepacia lipase.

[0067] In one embodiment, the lipase comprises an S residue at a position corresponding to position 87 in wild-type B. cepacia (S87), a D residue at a position corresponding to position 264 in wild-type B. cepacia (D264), and an H residue at a position corresponding to position 286 in wild-type B. cepacia (H286), exhibiting amino acids conserved between lipase subfamilies I.1 and I.2 (see FIG. 4).

[0068] Unless otherwise stated, wild-type B. cepacia lipase as used herein refers to a B. cepacia lipase having the amino acid sequence of SEQ ID NO:1 or a functional fragment thereof that digests long-chain triglyceride substrates into fatty acids.

[0069] SEQ ID NO:1 (wild type B. cepacia lipase): [Table 1]

[0070] As used herein, the term "functional fragment" is understood to be a protein fragment of a lipase that has at least 50%, 60%, 70%, 80%, 90%, 95% or 98% of the activity of the corresponding full-length lipase to digest long-chain triglyceride substrates into fatty acids.

[0071] In some embodiments, the lipase is not cross-linked and / or crystallized.

[0072] In one aspect, the present invention provides a method for the preparation of a lipase comprising administering to a subject a lipase having at least one of the following amino acids at the corresponding position of a wild-type B. cepacia lipase of SEQ ID NO: 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, e.g., 2-13, 3-13, 4-13, 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, 12-13, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 11-12, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, 10-11, 2-10, and wherein at least one of the mutations is a substitution of a residue at a position corresponding to position 39 of wild-type B. cepacia lipase; a substitution of a residue at a position corresponding to position 79 of wild-type B. cepacia lipase; a substitution of a residue at a position corresponding to position 102 of wild-type B. cepacia lipase; a substitution of a residue at a position corresponding to position 110 of wild-type B. cepacia lipase; a substitution of a residue at a position corresponding to position 120 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 125 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 128 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 137 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 138 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 153 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 154 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 161 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 170 of wild-type B. cepacia lipase. substitution of a residue at a position corresponding to position 221 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 227 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 240 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 249 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 250 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 260 of wild-type B. cepacia lipase; substitution of a residue at a position corresponding to position 266 of wild-type B. cepacia lipase;a substitution of a residue at a position corresponding to position 281 of wild-type B. cepacia lipase; a substitution of a residue at a position corresponding to position 300 of wild-type B. cepacia lipase; or any combination of the foregoing substitutions.

[0073] In some embodiments, the residue at the position corresponding to position 39 of the wild-type B. cepacia lipase is substituted with R, H, or K; the residue at the position corresponding to position 79 of the wild-type B. cepacia lipase is substituted with Q, N, or C; the residue at the position corresponding to position 102 of the wild-type B. cepacia lipase is substituted with Q, N, or C; the residue at the position corresponding to position 125 of the wild-type B. cepacia lipase is substituted with N, C, Q, S, or T; the residue at the position corresponding to position 128 of the wild-type B. cepacia lipase is substituted with N, C, Q, S, or T; the residue at position corresponding to position 137 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; the residue at position corresponding to position 138 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; the residue at position corresponding to position 153 of wild-type B. cepacia lipase is substituted with N, C, Q, S, or T; the residue at position corresponding to position 154 of wild-type B. cepacia lipase is substituted with R, H, or K; the residue at position corresponding to position 161 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; the residue at position corresponding to position 162 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; The residue at the position corresponding to position 70 is substituted with N, C, Q, S, or T; the residue at the position corresponding to position 221 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; the residue at the position corresponding to position 227 of wild-type B. cepacia lipase is substituted with R, H, or K; the residue at the position corresponding to position 240 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; the residue at the position corresponding to position 249 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V; the residue at the position corresponding to position 250 of wild-type B. cepacia lipase is substituted with A, I, L, M, or V. the residue at a position corresponding to position 260 of wild-type B. cepacia lipase is substituted with A, I, L, M or V; the residue at a position corresponding to position 266 of wild-type B. cepacia lipase is substituted with A, I, L, M or V; the residue at a position corresponding to position 281 of wild-type B. cepacia lipase is substituted with A, I, L, M or V; the residue at a position corresponding to position 300 of wild-type B. cepacia lipase is substituted with F, W or Y; or the lipase comprises any combination of the foregoing substitutions.

[0074] In one embodiment, the residue at position corresponding to position 39 of the wild-type B. cepacia lipase is substituted with R; the residue at position corresponding to position 79 of the wild-type B. cepacia lipase is substituted with Q; the residue at position corresponding to position 102 of the wild-type B. cepacia lipase is substituted with Q; the residue at position corresponding to position 125 of the wild-type B. cepacia lipase is substituted with S; the residue at position corresponding to position 128 of the wild-type B. cepacia lipase is substituted with N; The residue at the position corresponding to position 137 of wild-type B. cepacia lipase is substituted with A; the residue at the position corresponding to position 138 of wild-type B. cepacia lipase is substituted with I; the residue at the position corresponding to position 153 of wild-type B. cepacia lipase is substituted with N; the residue at the position corresponding to position 154 of wild-type B. cepacia lipase is substituted with H; the residue at the position corresponding to position 161 of wild-type B. cepacia lipase is substituted with A; the residue at position corresponding to position 170 of wild-type B. cepacia lipase is substituted with S; the residue at position corresponding to position 221 of wild-type B. cepacia lipase is substituted with L; the residue at position corresponding to position 227 of wild-type B. cepacia lipase is substituted with K; the residue at position corresponding to position 240 of wild-type B. cepacia lipase is substituted with V; the residue at position corresponding to position 249 of wild-type B. cepacia lipase is substituted with L; and the residue at position corresponding to position 250 of wild-type B. cepacia lipase is substituted with the residue at a position corresponding to position 260 of wild-type B. cepacia lipase is substituted with A; the residue at a position corresponding to position 266 of wild-type B. cepacia lipase is substituted with L; the residue at a position corresponding to position 281 of wild-type B. cepacia lipase is substituted with A; the residue at a position corresponding to position 300 of wild-type B. cepacia lipase is substituted with Y; or the lipase comprises any combination of the foregoing substitutions.

[0075] In some embodiments, the lipase comprises a substitution of a Q residue at a position corresponding to position 39 of wild-type B. cepacia lipase (Q39); a substitution of a T residue at a position corresponding to position 79 of wild-type B. cepacia lipase (T79); a substitution of a D residue at a position corresponding to position 102 of wild-type B. cepacia lipase (D102); a substitution of a G residue at a position corresponding to position 125 of wild-type B. cepacia lipase (G125); a substitution of an A residue at a position corresponding to position 128 of wild-type B. cepacia lipase (A12 8); substitution of a T residue at the position corresponding to position 137 of wild-type B. cepacia lipase (T137); substitution of a V residue at the position corresponding to position 138 of wild-type B. cepacia lipase (V138); substitution of an S residue at the position corresponding to position 153 of wild-type B. cepacia lipase (S153); substitution of an N residue at the position corresponding to position 154 of wild-type B. cepacia lipase (N154); substitution of an L residue at the position corresponding to position 161 of wild-type B. cepacia lipase (L161); substitution of an A residue at the position corresponding to position 170 of wild-type B. cepacia lipase (A170); substitution of an F residue at the position corresponding to position 221 of wild-type B. cepacia lipase (F221); substitution of a T residue at the position corresponding to position 227 of wild-type B. cepacia lipase (T227); substitution of an A residue at the position corresponding to position 240 of wild-type B. cepacia lipase (A240); substitution of an F residue at the position corresponding to position 249 of wild-type B. cepacia lipase (F249); substitution of a G residue at a position corresponding to position 250 of wild-type B. cepacia lipase (G250); substitution of an S residue at a position corresponding to position 260 of wild-type B. cepacia lipase (S260); substitution of a V residue at a position corresponding to position 266 of wild-type B. cepacia lipase (V266); substitution of an S residue at a position corresponding to position 281 of wild-type B. cepacia lipase (S281); substitution of an N residue at a position corresponding to position 300 of wild-type B. cepacia lipase (N300); or any combination of the foregoing substitutions.

[0076] In some embodiments, the Q residue at position corresponding to position 39 of the wild-type B. cepacia lipase is substituted with R (Q39R); the T residue at position corresponding to position 79 of the wild-type B. cepacia lipase is substituted with Q (T79Q); the D residue at position corresponding to position 102 of the wild-type B. cepacia lipase is substituted with Q (D102Q); the G residue at position corresponding to position 125 of the wild-type B. cepacia lipase is substituted with S (G125S); the A residue at position corresponding to position 128 of the wild-type B. cepacia lipase is substituted with N (A128N); The T residue at position corresponding to position 137 of wild-type B. cepacia lipase is substituted with A (T137A); the V residue at position corresponding to position 138 of wild-type B. cepacia lipase is substituted with I (V138I); the S residue at position corresponding to position 153 of wild-type B. cepacia lipase is substituted with N (S153N); the N residue at position corresponding to position 154 of wild-type B. cepacia lipase is substituted with H (N154H); the L residue at position corresponding to position 161 of wild-type B. cepacia lipase is substituted with A (L161A); The A residue at position corresponding to position 170 of wild-type B. cepacia lipase is substituted with S (A170S); the F residue at position corresponding to position 221 of wild-type B. cepacia lipase is substituted with L (F221L); the T residue at position corresponding to position 227 of wild-type B. cepacia lipase is substituted with K (T227K); the A residue at position corresponding to position 240 of wild-type B. cepacia lipase is substituted with V (A240V); the F residue at position corresponding to position 249 of wild-type B. cepacia lipase is substituted with L (F249L); the G residue at a position corresponding to position 260 of wild-type B. cepacia lipase is substituted with A (G250A); the S residue at a position corresponding to position 266 of wild-type B. cepacia lipase is substituted with A (S260A); the V residue at a position corresponding to position 266 of wild-type B. cepacia lipase is substituted with L (V266L); the S residue at a position corresponding to position 281 of wild-type B. cepacia lipase is substituted with A (S281A); the N residue at a position corresponding to position 300 of wild-type B. cepacia lipase is substituted with Y (N300Y); or the lipase comprises any combination of the foregoing substitutions.

[0077] In certain embodiments, the one or more mutations may be conservative substitutions relative to the wild-type B. cepacia lipase of SEQ ID NO: 1, and in certain other embodiments, the one or more mutations may be non-conservative substitutions relative to the wild-type B. cepacia lipase of SEQ ID NO: 1. As used herein, the term "conservative substitution" refers to a substitution with a structurally similar amino acid.

[0078] In certain embodiments, substitution of a given amino acid is with a hydrophobic amino acid (e.g., A, I, L, M, or V), a positively charged amino acid (e.g., K, R, or H), a negatively charged amino acid (e.g., D or E), a polar neutral amino acid (e.g., N, C, Q, S, or T), an aromatic amino acid (e.g., F, Y, or W), or an amino acid that is bulky based on side chain volume or smaller based on side chain volume. Amino acids are indicated by the single letter code.

[0079] Conservative substitutions can also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, BLOSUM substitution matrices (e.g., BLOSUM 62 matrix) or PAM substitution:p matrices (e.g., PAM 250 matrix). Non-conservative substitutions are amino acid substitutions that are not conservative substitutions.

[0080] In one aspect, the recombinant variant lipase enzyme comprises one or more substitutions from Table 1, where the positions of the substitutions are shown relative to wild-type B. cepacia (eg, SEQ ID NO:1). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0081] In another aspect, the present invention provides recombinant variant lipases comprising at least one mutation(s) (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 different mutations). In certain embodiments, the present invention provides recombinant variant lipases comprising at least one mutation(s) selected from Table 1 (e.g., at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or at least 11 different mutations). In certain embodiments, the one or more mutations may be conservative substitutions relative to the wild-type B. cepacia lipase of SEQ ID NO:1, and in certain other embodiments, the one or more mutations may be non-conservative substitutions relative to the wild-type B. cepacia lipase of SEQ ID NO:1.

[0082] In another aspect, the recombinant variant lipase comprises up to 11 substitutions listed in a given row of Table 2, where the position of the substitution is indicated relative to wild-type B. cepacia (eg, SEQ ID NO:1). [Table 3-1] [Table 3-2]

[0083] In certain embodiments, in any of the foregoing recombinant variant lipases, the lipase comprises the following substitutions: (i) D102Q, N154H, and F221L; (ii) T79Q, V266L, and L287V; (iii) L91M, V220A, and V266L; (iv) G125D, D159N, and F249L; (v) Q39A, T137A, and F249L; (vi) D102Q, G125S, N154H, F221L , V266L and N300Y; (vii) D102Q, T137A, F221L, E35S, G250A and V305I; (viii) D102Q, N154H, L161A, F221L, S281A and I218A; (ix) L91M, D102Q, A128N, N154H, F221L and Q177A; or (x) D102Q, S153N, N154H, F221L, Q39R and T92S.

[0084]

[0023] In certain embodiments, in any of the foregoing recombinant variant lipases, the lipase comprises one or more of the following substitutions: (i) D102Q, N154H and F221L; (ii) D102Q, G125S, N154H, F221L, V266L and N300Y; (iii) T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, F249L, V266L and N300Y; (iv) T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, F249L, V266L and N300Y; 27K, V266L, S281A and N300Y;(v) T79Q, D102Q, G125S, S153N, N154H, F221L, T227K, V266L, S281A and N300Y;(vi) T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, G250A, V266L and N300Y;(vii) T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, S281A and N3 (viii) T79Q, D102Q, G125S, N154H, F221L, T227K, F249L, V266L, S281A and N300Y; (ix) D102Q, G125S, T137A, S153N, N154H, F221L, T227K, F249L, V266L and N300Y; (x) D102Q, G125S, T137A, S153N, N154H, F221L, T227K, G250A, V266L and N300Y; (xi) D102Q, G12 (xii) D102Q, G125S, S153N, N154H, F221L, T227K, F249L, G250A, V266L, S281A and N300Y; or (xiii) D102Q, G125S, S153N, N154H, F221L, T227K, F249L, V266L, S281A and N300Y, either alone or in combination with other substitutions.

[0085] The present invention further relates to a recombinant variant lipase comprising the following substitutions: D102Q, N154H and F221L, such as the following amino acid sequence: [Table 4] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V130.

[0086] The present invention further provides a recombinant variant lipase comprising the following substitutions: D102Q, G125S, N154H, F221L, V266L and N300Y, such as the following amino acid sequence: [Table 5] The present invention provides a recombinant variant B. cepacia lipase, such as the recombinant variant lipase designated herein as V290, comprising:

[0087] The present invention further provides a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, F249L, V266L, N300Y, such as the following amino acid sequence: [Table 6] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V309.

[0088] The present invention further provides a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, V266L, S281A and N300Y, such as the following amino acid sequence: [Table 7] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V311.

[0089] The present invention further provides a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, S153N, N154H, F221L, T227K, V266L, S281A and N300Y, such as the following amino acid sequence: [Table 8] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V317.

[0090] The present invention further provides a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, N300Y and G250A, such as the following amino acid sequence: [Table 9] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V318.

[0091] The present invention further provides a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, S281A and N300Y, such as the following amino acid sequence: [Table 10] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V319.

[0092] The present invention further provides a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, N154H, F221L, T227K, F249L, V266L, S281A and N300Y, such as the following amino acid sequence: [Table 11] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V322.

[0093] The present invention further provides a recombinant variant lipase comprising the following substitutions: D102Q, G125S, T137A, S153N, N154H, F221L, T227K, F249L, V266L and N300Y, such as the following amino acid sequence: [Table 12] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V325.

[0094] The present invention further provides a recombinant variant lipase comprising the following substitutions: D102Q, G125S, T137A, S153N, N154H, F221L, T227K, V266L and N300Y, such as the following amino acid sequence: [Table 13] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V326.

[0095] The present invention further provides a recombinant variant lipase comprising the following substitutions: D102Q, G125S, T137A, N154H, F221L, T227K, G250A, V266L, S281A and N300Y, such as the following amino acid sequence: [Table 14] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V333.

[0096] The present invention further provides a recombinant variant lipase comprising the following substitutions: D102Q, G125S, S153N, N154H, F221L, T227K, F249L, G250A V266L and N300Y, such as the following amino acid sequence: [Table 15] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V335.

[0097] The present invention further provides a recombinant variant lipase comprising the following substitutions: D102Q, G125S, S153N, N154H, F221L, T227K, F249L, V266L, S281A and N300Y, such as the following amino acid sequence: [Table 16] The present invention provides a recombinant variant B. cepacia lipase comprising, for example, the recombinant variant lipase designated herein as V336.

[0098] In one embodiment, the lipase comprises the amino acid sequence of any one of SEQ ID NOs:2-14 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs:2-14.

[0099] Sequence identity can be determined in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. BLAST (Basic Local Alignment Search Tool) analysis using the algorithm used by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al., (1990) PROC. NATL. ACAD. SCI. USA 87:2264-2268; Altschul, (1993) J. MOL. EVOL. 36, 290-300; Altschul et al., (1997) NUCLEIC ACIDS RES.25:3389-3402, incorporated by reference) is adjusted for searching sequence similarity. For a discussion of basic issues in searching sequence databases, see Altschul et al., (1994) NATURE GENETICS 6:119-129, fully incorporated by reference. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. Search parameters for histogram, description, alignment, expect (i.e., the statistical significance threshold for reporting matches to database sequences), cutoff, matrix and filter are at default settings. The default scoring matrix used by blastp, blastx, tblastn and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) PROC. NATL. ACAD. SCI. USA 89:10915-10919, fully incorporated by reference).Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every wink.sup.th position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). Equivalent Blastp parameter settings can be Q=9; R=2; wink=1; and gapw=32. Searches may also be performed using NCBI (National Center for Biotechnology Information) BLAST Advanced Option parameters (e.g.: -G, cost for open gap [integer]: default = 5 for nucleotides / 11 for proteins; -E, cost for extension gap [integer]: default = 2 for nucleotides / 1 for proteins; -q, penalty for nucleotide mismatch [integer]: default = -3; -r, reward for nucleotide match [integer]: default = 1; -e, expectation value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, dropoff for blast extension in bits (X): default = 20 for blastn / 7 for others; -X, X dropoff value for gapped alignments (in bits): default = 15 for all programs but not applicable to blastn; and -Z, final X dropoff value for gapped alignments (in bits): 50 for blastn, 25 for others). ClustalW for pairwise protein alignments may also be used (default parameters may include, for example, the Blosum62 matrix and a Gap Opening Penalty=10 and Gap Extension Penalty=0.1).Optimal comparison between sequences, available in the GCG package version 10.0, uses DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty); equivalent settings for protein comparisons are GAP=8 and LEN=2.

[0100] a. Recombinant variant lipase with increased stability at low pH In certain embodiments, the recombinant variant lipase has increased stability at acidic pH (e.g., pH 3.0 or 4.0) relative to the corresponding wild-type lipase enzyme. Increased stability at acidic pH allows the recombinant variant lipase to survive the acidic conditions of the digestive system, particularly the stomach. The normal pre-meal stomach pH varies from about 1.5 to about 3.5, and the post-meal pH increases to about 5. During the meal interval, there is a slow but continuous emptying of stomach contents though the pyloric valve, by which time the chyme is below about pH 4 and more than 60-90% of the meal has been transferred to the duodenum. Wild-type lipase from B. cepacia (e.g., SEQ ID NO:1) has good survivability up to pH 4. However, there may be short periods during which the lipase may be subjected to pH levels below pH 4.0. Therefore, it may be desirable for the recombinant variant lipase to exhibit improved stability up to about pH 3.0-3.5. In some embodiments, the recombinant variant lipase may be taken with food, so enhanced stability at the very low pH of the fasted stomach may not be necessary.

[0101] In some embodiments, the lipase has a half-life at about pH 3.0 of at least about 35 minutes, at least about 50 minutes, at least about 75 minutes, at least about 100 minutes, at least about 125 minutes, at least about 130 minutes, at least about 135 minutes, at least about 140 minutes, at least about 145 minutes, or at least about 150 minutes. For example, in certain embodiments, the lipase has a half-life of about 50 minutes to about 200 minutes, e.g., about 50 minutes to about 100 minutes, about 50 minutes to about 150 minutes, about 50 minutes to about 175 minutes, about 50 minutes to about 200 minutes, about 75 minutes to about 100 minutes, about 75 minutes to about 150 minutes, about 75 minutes to about 175 minutes, about 75 minutes to about 200 minutes, about 100 minutes to about 150 minutes, about 100 minutes to about 175 minutes, about 100 minutes to about 200 minutes, about 150 minutes to about 175 minutes, or about 150 minutes to about 200 minutes.

[0102] In some embodiments, the lipase has at least 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater stability at about pH 3.0 compared to the corresponding wild-type lipase. For example, the lipase may have about 1.5-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 3.5-fold, about 2-fold to about 2.5-fold, about 2-fold to about 3-fold, about 2-fold to about 3.5-fold, about 2.5-fold to about 3-fold, about 2.5-fold to about 3.5-fold, or about 3-fold to about 3.5-fold greater stability at about pH 3.0 compared to the corresponding wild-type lipase.

[0103] Methods for testing the stability of lipases are known in the art and may include, for example, the method described herein in Example 3. In one embodiment, the stability of a lipase at low pH is determined by exposing the lipase to a particular pH (e.g., pH 3.0), adding p-NPP (p-nitrophenyl palmitate), and detecting the presence or amount of the p-NPP cleavage product p-nitrophenolate by a colorimetric assay.

[0104] b. Recombinant variant lipase with increased stability in the presence of proteases In some embodiments, the lipase has increased stability in the presence of proteases (e.g., serine and / or aspartic acid proteases) relative to the corresponding wild-type microbial lipase enzyme. In some embodiments, the recombinant variant lipases described herein are designed to be readily available in the stomach, where they are exposed to proteolytic enzymes, such as pepsin and other proteases. The increased stability in the presence of proteases allows the recombinant variant lipase to survive the harsh conditions of the stomach.

[0105] In some embodiments, the engineered lipase has increased stability in the presence of aspartic acid (e.g., pepsin) relative to the corresponding wild-type lipase. Pepsin has maximum activity at low pH levels (pH 1.5-4). Thus, in some embodiments, the engineered lipase also has increased stability at low pH (e.g., pH 3.8).

[0106] In certain embodiments, the lipase has a half-life in the presence of an aspartic protease, such as pepsin, of at least about 50 minutes, at least about 75 minutes, at least about 100 minutes, at least about 125 minutes, at least about 150 minutes, at least about 175 minutes, at least about 200 minutes, at least about 225 minutes, at least about 230 minutes, or at least about 235 minutes. In one embodiment, the lipase is reacted in the presence of an aspartic protease such as pepsin for about 75 minutes to about 100 minutes, about 75 minutes to about 125 minutes, about 75 minutes to about 150 minutes, about 75 minutes to about 175 minutes, about 75 minutes to about 200 minutes, about 75 minutes to about 225 minutes, about 75 minutes to about 230 minutes, about 75 minutes to about 235 minutes, about 75 minutes to about 250 minutes, about 100 minutes to about 125 minutes, about 100 minutes to about 150 minutes, about 100 minutes to about 175 minutes, about 100 minutes to about 200 minutes, about 100 minutes to about 225 minutes, about 100 minutes to about 230 minutes, about 100 minutes to about 100 minutes 235 minutes, 100 minutes to 250 minutes, 125 minutes to 150 minutes, 125 minutes to 175 minutes, 125 minutes to 200 minutes, 125 minutes to 225 minutes, 125 minutes to 230 minutes, 125 minutes 235 minutes, 125 minutes to 250 minutes, 150 minutes to 175 minutes, 150 minutes to 200 minutes, 150 minutes to 225 minutes, 150 minutes to 230 minutes, 150 minutes 235 minutes, 150 minutes to 250 minutes, 175 minutes to 200 minutes, 175 minutes to 225 minutes, 175 minutes to 230 minutes, 175 minutes It has a half-life of about to about 235 minutes, about 175 minutes to about 250 minutes, about 200 minutes to about 225 minutes, about 200 minutes to about 230 minutes, about 200 minutes about to about 235 minutes, about 200 minutes to about 250 minutes, about 225 minutes to about 230 minutes, about 225 minutes about to about 235 minutes, about 225 minutes to about 250 minutes, or about 235 minutes to 250 minutes. In some embodiments, pepsin is present at a low pH (e.g., pH 3.6) typical of the stomach in a fed state.

[0107] Methods for testing the stability of lipases in the presence of aspartic proteases such as pepsin are known in the art and may include, for example, the method described herein in Example 4. In one embodiment, the stability of lipases in the presence of aspartic proteases is determined by exposing the lipase to a protease (e.g., pepsin), inactivating the pepsin, adding p-NPP (p-nitrophenyl palmitate), and detecting the presence or amount of the p-NPP cleavage product p-nitrophenolate by a colorimetric assay.

[0108] In certain embodiments, the lipase has at least 1.5, 2, 2.5, 3, 3.5, or 4 times greater stability in the presence of an aspartic protease, such as pepsin (e.g., pH 3.6), compared to the corresponding wild-type lipase. In certain embodiments, the lipase has about 1.5-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 3.5-fold, about 1.5-fold to about 4-fold, about 2-fold to about 2.5-fold, about 2-fold to about 3-fold, about 2-fold to about 3.5-fold, about 2-fold to about 4-fold, about 2.5-fold to about 3-fold, about 2.5-fold to about 3.5-fold, about 2.5-fold to about 4-fold, about 3-fold to about 3.5-fold, about 3-fold to about 3.5-fold, or about 3.5-fold to about 4-fold greater stability in the presence of an aspartic protease such as pepsin (e.g., pH 3.6) compared to a corresponding wild-type lipase.

[0109] Further, in some embodiments, the engineered lipase is delivered in combination with a protease for protein digestion and an amylase for starch digestion. Thus, in some embodiments, the lipase is exposed to a protease from A. melleus for co-administration. A. melleus protease is a serine protease with maximum activity at pH 7-pH 8 and 50% higher activity in the pH range of pH 5-pH 11. Unlike mammalian proteases such as trypsin and chymotrypsin, which cleave proteins only after specific amino acids, A. melleus protease (also called SAP or oryzin) cleaves proteins down to small oligomers and individual amino acids. Since the recombinant variant lipase described herein is expected to remain in the presence of A. melleus protease for 3-6 hours (the transit time from the stomach in a fed state through the small intestine), in some embodiments, the engineered lipase is resistant to degradation by this protease.

[0110] In certain embodiments, the lipase has a half-life of at least 50 minutes, 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, or 200 minutes in the presence of a serine protease, such as an A. melleus protease. For example, the lipase may have a half-life of about 75 minutes to about 100 minutes, about 75 minutes to about 125 minutes, about 75 minutes to about 150 minutes, about 75 minutes to about 175 minutes, about 75 minutes to about 200 minutes, about 75 minutes to about 225 minutes, about 100 minutes to about 125 minutes, about 100 minutes to about 150 minutes, about 100 minutes to about 175 minutes, about 100 minutes to about The half-life may be about 200 minutes, about 100 minutes to about 225 minutes, about 125 minutes to about 150 minutes, about 125 minutes to about 175 minutes, about 125 minutes to about 200 minutes, about 125 minutes to about 225 minutes, about 150 minutes to about 175 minutes, about 150 minutes to about 200 minutes, about 150 minutes to about 225 minutes, about 175 minutes to about 200 minutes, about 175 minutes to about 225 minutes, or about 200 minutes to about 225 minutes.

[0111] In certain embodiments, the lipase has at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold greater stability in the presence of a serine protease, such as an A. melleus protease, compared to the corresponding wild-type lipase. In certain embodiments, the lipase has about 1.5-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 3.5-fold, about 1.5-fold to about 4-fold, about 2-fold to about 2.5-fold, about 2-fold to about 3-fold, about 2-fold to about 3.5-fold, about 2-fold to about 4-fold, about 2.5-fold to about 3-fold, about 2.5-fold to about 3.5-fold, about 2.5-fold to about 4-fold, about 3-fold to about 3.5-fold, about 3-fold to about 4-fold, or about 3.5-fold to about 4-fold greater stability in the presence of a serine protease, such as an A. melleus protease, compared to a corresponding wild-type lipase.

[0112] Methods for testing the stability of lipases in the presence of aspartic proteases such as pepsin are known in the art and may include, for example, the method described herein in Example 5. In one embodiment, the stability of lipases in the presence of aspartic proteases is determined by exposing the lipase to a protease (e.g., pepsin), inactivating the pepsin, and then adding p-NPP (p-nitrophenyl palmitate) and detecting the presence or amount of the p-NPP cleavage product p-nitrophenolate by a colorimetric assay.

[0113] In some embodiments, recombinant variant lipase is administered in combination with pancreatin. Pancreatin contains up to 20 different enzymes, with three main enzyme classes as active ingredients: amylase, lipase and protease. Pancreatin proteases include trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B. Pancreatin and pancreatin-based preparations, such as pancrelipase, are currently used to manage exocrine pancreatic insufficiency. Thus, in some embodiments, lipase is exposed to proteases in pancreatin for co-administration. Therefore, engineered lipase can be resistant to degradation by pancreatin.

[0114] In some embodiments, the lipase has a half-life of at least 50 minutes, 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, or 200 minutes in the presence of pancreatin. For example, the lipase has a half-life of about 75 minutes to about 100 minutes, about 75 minutes to about 125 minutes, about 75 minutes to about 150 minutes, about 75 minutes to about 175 minutes, about 75 minutes to about 200 minutes, about 75 minutes to about 225 minutes, about 100 minutes to about 125 minutes, about 100 minutes to about 150 minutes, about 100 minutes to about 175 minutes, about 100 minutes to about 200 minutes, about 10 minutes to about 2 ... It may have a half-life of 0 minutes to about 225 minutes, about 125 minutes to about 150 minutes, about 125 minutes to about 175 minutes, about 125 minutes to about 200 minutes, about 125 minutes to about 225 minutes, about 150 minutes to about 175 minutes, about 150 minutes to about 200 minutes, about 150 minutes to about 225 minutes, about 175 minutes to about 200 minutes, about 175 minutes to about 225 minutes, or about 200 minutes to about 225 minutes.

[0115] In some embodiments, the lipase has at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold higher stability in the presence of pancreatin compared to the corresponding wild-type lipase. In some embodiments, the lipase has about 1.5-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 3.5-fold, about 1.5-fold to about 4-fold, about 2-fold to about 2.5-fold, about 2-fold to about 3-fold, about 2-fold to about 3.5-fold, about 2-fold to about 4-fold, about 2.5-fold to about 3-fold, about 2.5-fold to about 3.5-fold, about 2.5-fold to about 4-fold, about 3-fold to about 3.5-fold, about 3-fold to about 4-fold, or about 3.5-fold to about 4-fold higher stability in the presence of pancreatin compared to the corresponding wild-type lipase.

[0116] The method for testing the stability of lipase in the presence of pancreatin is known in the art.In one embodiment, the stability of lipase in the presence of pancreatin is determined by exposing lipase to pancreatin, optionally inactivating protease in pancreatin, then adding p-NPP (p-nitrophenyl palmitate) and detecting the amount of p-NPP cleavage product p-nitrophenolate by colorimetric assay.Stability is determined by comparing the amount of p-NPP cleavage to the control sample of lipase that is not treated with pancreatin.

[0117] c. The recombinant variant lipase may have increased lipase activity Dietary lipids, including long-chain polyunsaturated fats (LCPUFA), such as DHA, EPA, and AA, are primarily in the form of long-chain triglycerides. Long-chain triglycerides are made of three long-chain fatty acids attached to a glycerol molecule via ester bonds. Absorption of long-chain triglycerides by the body first requires the enzymatic action of lipases, such as pancreatic lipase, which hydrolyzes the triglycerides and breaks them down into one sn-2 monoglyceride and two free fatty acids. The term "free fatty acids", i.e., fatty acids that are not attached to other molecules (e.g., the glycerol backbone), is used to refer to the by-products of fat digestion. The terms "digestion" and "hydrolysis" are used interchangeably to refer to the enzymatic action of lipases to break down lipid triglycerides into monoglycerides and free fatty acids. The hydrolysis products monoglycerides and free fatty acids are then used as energy and absorbed into enterocytes, largely by passive diffusion. Once free fatty acids and monoglycerides are absorbed, they are transported to the liver and ultimately to tissues throughout the body for a variety of physiological purposes.

[0118] Additionally, the length of the fatty acid chain and the number of carbon-carbon double bonds can affect fat absorption. Dietary fatty acids found in food are long-chain fatty acids with at least 12 carbons, e.g., 16, 18 or 20 carbons, known as C16, C18 and C20 long-chain fatty acids. Medium-chain fatty acids with 12 or fewer carbons, e.g., 8 and 12 carbons, known as C8 and C12, are not generally found in food (except coconut) and are therefore less important for digestion and absorption in humans. Short-chain fatty acids with a few carbons or less, e.g., 2, 3 and 4 carbons, known as C2, C3 and C4, are the major anions found in stool but not in food. Short-chain fatty acids result from digestion by bacteria in the colon.

[0119] Although all fats provide caloric benefits, they have different effects on physiological functions. Short-chain triglycerides (SCT) and medium-chain triglycerides (MCT) are absorbed directly by the villi of the intestinal mucosa. MCTs can be readily absorbed due to their short chain length and residual activity of gastric lipase even in patients with impaired pancreatic output or pancreatic insufficiency. Long-chain triglycerides (LCT) are not directly absorbed, but instead must first be hydrolyzed by pancreatic lipase to free fatty acids and monoglycerides, which are then absorbed in the small intestine. Once the free fatty acids and monoglycerides are absorbed, they are transported to the liver and ultimately to tissues throughout the body for various physiological purposes. Although both LCTs and MCTs provide calories, only LCTs, specifically LCPUFAs, provide structural components of biological mediators associated with the control of membranes and many physiological functions. When substituted for LCTs, MCTs have been shown to increase energy expenditure and satiety, resulting in reduced overall caloric intake and reduced body fat mass. This makes MCTs a poor long-term energy source for patients with impaired pancreatic output or pancreatic insufficiency.

[0120] In certain embodiments, the recombinant variant lipases described herein have at least 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater activity (e.g., at about pH 3.0) than the corresponding wild-type lipase. In certain embodiments, the lipase has about 1.5-fold to about 2-fold, about 1.5-fold to about 2.5-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 3.5-fold, about 2-fold to about 2.5-fold, about 2-fold to about 3-fold, about 2-fold to about 3.5-fold, about 2.5-fold to about 3-fold, about 2.5-fold to about 3.5-fold, or about 3-fold to about 3.5-fold greater activity (e.g., at about pH 3.0) than the corresponding wild-type lipase.

[0121] In some embodiments, the lipase preferentially hydrolyzes the sn-1 and sn-3 positions on triglycerides. In some embodiments, the lipase enzyme activity (e.g., specific activity) is not inhibited by bile salts. In some embodiments, the lipase does not require a co-lipase.

[0122] In some embodiments, the lipase has a specific activity of at least 300, 400, 500, 600, 700, 800, 900, or 1,000 μmol fatty acid (FA) produced / min / mg lipase at pH 3.0 on a long-chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long-chain triglyceride substrates set forth in Table 3). For a long-chain triglyceride substrate comprising DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long-chain triglyceride substrates described in Table 3), at pH 3.0, about 300 to about 400, about 300 to about 500, about 300 to about 600, about 300 to about 700, about 300 to about 800, about 300 to about 900, about 300 to about 1,000, about 300 to about 1,100, about 400 to about 500, about 400 to about 600, about 400 to about 700, about 400 to about 800, about 400 to about 900, about 400 to about 1,000, about 400 to about 1,100, about 500 to about 60 The specific activity of the lipase is 0, about 500 to about 700, about 500 to about 800, about 500 to about 900, about 500 to about 1,000, about 500 to about 1,100, about 600 to about 700, about 600 to about 800, about 600 to about 900, about 600 to about 1,000, about 600 to about 1,100, about 700 to about 800, about 700 to about 900, about 700 to about 1,000, about 700 to about 1,100, about 800 to about 900, about 800 to about 1,000, about 800 to about 1,100, about 900 to about 1,000, about 900 to about 1,100, or about 1,000 to about 1,100 μmol fatty acid (FA) production / min / mg of lipase.

[0123] In certain embodiments, the lipase has a specific activity of at least 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or 2,000 μmol fatty acid (FA) produced / min / mg of lipase at pH 4.0, pH 5.0, or pH 6.0 on a long chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long chain triglyceride substrates set forth in Table 3).In certain embodiments, the lipase has a catalysis of about 600 to about 700, about 600 to about 800, about 600 to about 900, about 600 to about 1,000, about 600 to about 1,100, about 600 to about 1,200, about 600 to about 1,300, about 600 to about 1,400, about 600 to about 1,500, or about 600 to about 1,600 at pH 4.0, pH 5.0, or pH 6.0 for a long-chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long-chain triglyceride substrates described in Table 3). , about 600 to about 2,000, about 700 to about 800, about 700 to about 900, about 700 to about 1,000, about 700 to about 1,100, about 700 to about 1,200, about 700 to about 1,300, about 700 to about 1,400, about 700 to about 1,500, about 700 to about 2,000, about 800 to about 900, about 800 to about 1,000, about 800 to about 1,100, about 800 to about 1,200, about 800 to about 1,300, about 800 to about 1,400, about 800 to about 1,500, about 800 to about 2, 000, about 900 to about 1,000, about 900 to about 1,100, about 900 to about 1,200, about 900 to about 1,300, about 900 to about 1,400, about 900 to about 1,500, about 900 to about 2,000, about 1,000 to about 1,100, about 1,000 to about 1,200, about 1,000 to about 1,300, about 1,000 to about 1,400, about 1,000 to about 1,500, about 1,000 to about 2,000, about 1,100 to about 1,200, about 1,100 to about 1,300, about 1,100 about 1,400, about 1,100 to about 1,500, about 1,100 to about 2,000, about 1,200 to about 1,300, about 1,200 to about 1,400, about 1,200 to about 1,500, about 1,200 to about 2,000, about 1,300 to about 1,400, about 1,300 to about 1,500, about 1,300 to about 2,000, about 1,400 to about 1,500, about 1,400 to about 2,000, or about 1,500 to 2,000 μmol fatty acid (FA) production / min / mg of lipase.

[0124] In some embodiments, the lipase has a specific activity of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or 2,000 μmol fatty acid (FA) produced / min / mg lipase at pH 7.0 on a long chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long chain triglyceride substrates set forth in Table 3). For a long-chain triglyceride substrate comprising DHA triglyceride and 22% oleic acid triglyceride or triolein (e.g., exemplary long-chain triglyceride substrates described in Table 3), the glycerol concentrations at pH 7.0 are about 1,000 to about 1,100, about 1,000 to about 1,200, about 1,000 to about 1,300, about 1,000 to about 1,400, about 1,000 to about 1,500, about 1,000 to about 2,000, about 1,100 to about 1,200, about 1,100 to about 1,300, about 1,100 to about 1,400, about 1,200 to about 1,500, about 1,300 to about 1,600, about 1,400 to about 1,800, about 1,500 to about 2,000, about 1,600 to about 1,800, about 1,700 to about 1,900, about 1,800 to about 2,000, about 1,900 to about 2,000, about 1,100 to about 1,200, about 1,100 to about 1,300, about 1,100 to about 1,400, about 1,200 to about 1,500, about 1,300 to about 1,600, about 1,400 to about 1,800, about 1,500 to about 2,000, about 1,600 to about 1,800, about 1,700 to about 2,000, about 1,800 to about 2,000, about 1,900 to about The specific activity is 0 to about 1,400, about 1,100 to about 1,500, about 1,100 to about 2,000, about 1,200 to about 1,300, about 1,200 to about 1,400, about 1,200 to about 1,500, about 1,200 to about 2,000, about 1,300 to about 1,400, about 1,300 to about 1,500, about 1,300 to about 2,000, about 1,400 to about 1,500, about 1,400 to about 2,000, or about 1,500 to 2,000 μmol fatty acid (FA) production / min / mg of lipase.

[0125] In certain embodiments, the lipase has at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the enzymatic activity of the corresponding wild-type microbial lipase enzyme. In certain embodiments, the lipase has about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 96%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 60% to about 100%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 70% to about 100%, about 80% to about 85%, about 80% to about 90% of the enzymatic activity of the corresponding wild-type microbial lipase enzyme. %, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 85% to about 100%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 90% to about 100%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 95% to about 100%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 96% to about 100%, about 97% to about 98%, about 97% to about 99%, about 97% to about 100%, about 98% to about 99%, about 98% to about 100%, or about 99% to about 100%.

[0126] In some embodiments, the lipase remains sufficiently active to hydrolyze long chain polyunsaturated fatty acids (LCPUFAs), such as DHA and EPA, or long chain triglycerides, such as oleic acid or triolein, in the gastrointestinal tract of a subject at a pH ranging from 3.5 to 7.0, hi some embodiments, the lipase is at least 2, 10, 100 or 1000 times more active than pancrelipase when tested under the same conditions.

[0127] In some embodiments, greater than 50%, 60%, 70%, 80% or 90% of the lipase remains active for 60-120 minutes in the fed state stomach of the subject. In some embodiments, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, or about 70% to about 90%, or about 80% to about 90% of the lipase remains active for 60-120 minutes in the fed state stomach of the subject.

[0128] In some embodiments, the lipase digests greater than 20%, 30%, 40% or 50% of ingested fat in the subject's stomach into fatty acids and monoglycerides. In some embodiments, the lipase digests about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 30% to about 40%, about 30% to about 50%, or about 40% to about 50% of ingested fat in the subject's stomach into fatty acids and monoglycerides.

[0129] In some embodiments, greater than 50%, 60%, 70%, 80% or 90% of the lipase remains active for about 240 to about 360 minutes through the small intestine of the subject. In some embodiments, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, or about 70% to about 90%, or about 80% to about 90% of the lipase remains active for about 240 to about 360 minutes through the small intestine of the subject.

[0130] In some embodiments, it may be desirable for the activity of lipase to be reduced in the large intestine.Thus, in some embodiments, lipase has reduced activity in the large intestine after 10 hours, 12 hours or 18 hours.In some embodiments, lipase can digest less than 50%, less than 60%, or less than 70%, or less than 80%, or less than 90% of the fat remaining in the large intestine.

[0131] In some embodiments, the lipase digests greater than 50%, 60%, 70%, 80% or 90% of ingested fat in the small intestine of the subject into fatty acids and monoglycerides. In some embodiments, the lipase digests about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, or about 70% to about 90%, or about 80% to about 90% of ingested fat in the small intestine of the subject into fatty acids and monoglycerides.

[0132] In some embodiments, lipase increases the absorption of long chain unsaturated fatty acids in the plasma of a subject by more than 25%, 35%, 50%, 100% or 200% within 30, 45, 60, 90 or 120 minutes relative to the same subject not receiving lipase or to a similar subject not receiving lipase. In some embodiments, lipase increases the absorption of fat-soluble vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K). In some embodiments, lipase increases the absorption of choline.

[0133] Lipase fat hydrolysis can be assayed using any method known in the art.For example, modified quantitative colorimetric assay (Abcam® Free Fatty Acid Quantitation Kit) can be used to measure the amount of free fatty acid using a given lipid substrate.More complex fats (e.g., fats with longer chain length and more double bonds) are more difficult for lipase to hydrolyze into free fatty acid and monoglyceride.One such complex fat DHA is a relevant surrogate for overall fat hydrolysis or digestion because it has a longer carbon chain and more double bonds than other fats or LCPUFAs, and is therefore more difficult to hydrolyze.Therefore, assaying DHA hydrolysis is a useful surrogate for the ability of lipase to digest all triglycerides.Throughout the course of experiments, the substrate DHA from oil is in the form of triglyceride, and the product measured by the method is DHA free fatty acid form.

[0134] In one embodiment, lipase activity can be measured using a DHA hydrolysis assay using an oil containing about 37% DHA triglyceride and about 22% oleic acid triglyceride, with the remainder mostly made up of myristic acid triglyceride, palmitate triglyceride, stearic acid triglyceride, and lauric acid triglyceride, and palmitoleic acid triglyceride. (NuCheck, Elysian Minn.) The major components of such DHA triglyceride oil are shown in Table 3. [Table 17]

[0135] Oleic acid triglyceride is a fat substrate with three fatty acids (18 carbons) attached to a glycerol backbone and contains one double bond. Oleic acid triglyceride is a common dietary fat and occurs in olive oil at a percentage of about 55% to 83%. Oleic acid triglyceride is hydrolyzed by pancreatic lipase to form two oleic acid fatty acids and an sn-2 monoglyceride. Similar to DHA triglyceride, oleic acid triglyceride can act as a substitute for overall dietary fat hydrolysis.

[0136] Triolein is a refined form of oleic acid in triglyceride form. Because olive oil varies from lot to lot, the use of olive oil in hydrolysis assays may produce inconsistent measurements. Therefore, triolein may be used to evaluate the ability of lipase to hydrolyze oleic acid in triglyceride form, and may provide more consistent results compared to olive oil.

[0137] In one embodiment, a lipase potency assay is used to measure the release of fatty acids from triglycerides by a four-step process: 1) hydrolysis of triglycerides at pH 6 to release free fatty acids (FFAs), 2) conjugation of FFAs to coenzyme A, 3) oxidation of the FFA-coenzyme A complex to generate hydrogen peroxide, and 4) detection of peroxides using a colorimetric oxidation dye. The amount of colorimetric dye produced is proportional to the amount of FFA released by the lipase, and the specific activity of the lipase is defined as the amount of enzyme required to convert 1 μmole of substrate per minute. The assay is described in more detail in Example 2 herein.

[0138] It is contemplated that the disclosed recombinant variant lipase can be modified, genetically engineered or chemically conjugated.For example, it is contemplated that the disclosed recombinant variant lipase can be conjugated to an effector agent using standard in vitro conjugation chemistry.When the effector agent is a polypeptide, the lipase can be chemically conjugated to the effector or linked to the effector as a fusion protein.The construction of a fusion protein is within the ordinary skill of the art.

[0139] III. Lipase Production The method for producing the lipase enzyme of the present invention is known in the art.For example, the DNA molecule encoding lipase can be chemically synthesized using the sequence information provided herein.The synthetic DNA molecule can be ligated to other suitable nucleotide sequences, such as expression control sequences, to produce a conventional gene expression construct that encodes the desired lipase.

[0140] The nucleic acid encoding the desired lipase can be incorporated (ligated) into an expression vector, which can be introduced into a host cell by conventional transfection or transformation techniques. The transformed host cell can be grown under conditions that cause the host cell to express the gene encoding the lipase enzyme.

[0141] Nucleic acids encoding the recombinant variant lipases of the invention can be made by mutating a nucleotide sequence encoding a wild-type B. cepacia lipase, such as SEQ ID NO: 1 disclosed herein, using methods known in the art. Furthermore, in certain embodiments, nucleic acids encoding the recombinant variant B. cepacia lipases of the invention can be codon-optimized for expression in a heterologous cell, such as a B. cepacia cell, a Burkholderia glumae cell, a Pseudomonas fluorescens cell, a Chromobacterium viscosum cell, a Pseudomonas luteola cell, a Pseudomonas fragilis cell, or an Escherichia coli cell, using methods known in the art.

[0142] In some embodiments, the disclosure relates to a cell comprising an expression vector described herein, wherein the cell is a B. cepacia, Pseudomonas glumae, Pseudomonas fluorescens, Chromobacterium viscosum, Pseudomonas luteola, Pseudomonas fragilis, or Escherichia coli cell.

[0143] In one embodiment, the present disclosure relates to exemplary nucleotide sequences encoding recombinant variant lipases. In some embodiments, the nucleotide sequences encoding recombinant variant lipases contain nucleotide substitutions compared to wild-type lipases, such as wild-type B. cepacia lipase. Wild-type nucleic acids encoding B. cepacia lipase are known in the art, and include, for example, the following sequence, SEQ ID NO:39: [Table 18] Includes.

[0144] In one aspect, the present disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, N154H and F221L, such as the nucleotide sequence encoding the recombinant variant B. cepacia lipase designated herein as V130.

[0145] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, G125S, N154H, F221L, V266L and N300Y, e.g., a nucleotide sequence encoding a recombinant variant B. cepacia lipase designated herein as V290.

[0146] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, T137A, N154H, F221L, F249L, V266L, N300Y, and T227K, such as a nucleotide sequence encoding a recombinant variant lipase designated herein as V309.

[0147] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, T137A, N154H, F221L, V266L, S281A, N300Y, and T227K, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V311.

[0148] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, S153N, N154H, F221L, V266L, S281A, N300Y, and T227K, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V317.

[0149] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, N300Y, and G250A, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V318.

[0150] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, S281A and N300Y, such as a nucleotide sequence encoding a recombinant variant lipase designated herein as V319.

[0151] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: T79Q, D102Q, G125S, N154H, F221L, F249L, V266L, S281A, N300Y, and T227K, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V322.

[0152] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, G125S, T137A, S153N, N154H, F221L, F249L, V266L, N300Y, and T227K, such as a nucleotide sequence encoding a recombinant variant lipase designated herein as V325.

[0153] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, G125S, T137A, S153N, N154H, F221L, V266L, N300Y, T227K, and G250A, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V326.

[0154] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, G125S, T137A, N154H, F221L, V266L, S281A, N300Y, T227K, and G250A, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V333.

[0155] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, G125S, S153N, N154H, F221L, F249L, V266L, N300Y, T227K, and G250A, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V335.

[0156] In one aspect, the disclosure relates to an exemplary nucleotide sequence encoding a recombinant variant lipase comprising the following substitutions: D102Q, G125S, S153N, N154H, F221L, F249L, V266L, S281A, N300Y, and T227K, such as the nucleotide sequence encoding the recombinant variant lipase designated herein as V336.

[0157] Specific expression and purification conditions vary according to the expression system used.For example, if gene is expressed in E. coli, gene can be cloned into expression vector by placing engineered gene downstream from suitable bacterial promoter and prokaryotic signal sequence.Expressed secretory protein is targeted to accumulate in periplasmic space, where protein is harvested by osmotic shock or cell disruption by French press or sonication.Refractile bodies are then solubilized, and protein is refolded and cleaved by methods known in the art.

[0158] Lipase can be produced by growing (culturing) a host cell transfected with an expression vector encoding such lipase under conditions that allow the expression of the lipase. After expression, lipase can be harvested and purified or isolated using techniques known in the art, such as affinity tags such as glutathione-S-transferase (GST) and histidine tags. An exemplary expression and purification protocol for lipase is described in Liu et al. (2011) APPL. MICROBIOL. BIOTECHNOL.92(3):529-37.

[0159] IV. Pharmaceutical Compositions and Dosages For therapeutic use, the recombinant lipase described herein is preferably combined with a pharma- ceutically acceptable carrier and / or excipient. The term "pharma-ceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are within the bounds of sound medical judgment and suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0160] The term "pharmacologically acceptable carrier" as used herein refers to buffers, carriers and excipients suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions) and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020). Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharma-ceutically active substances is known in the art.

[0161] In certain embodiments, the lipase may be formulated or co-administered (either simultaneously or sequentially) with a pH-increasing agent, e.g., by an enteral route (e.g., orally), e.g., to increase the stability of the lipase in an acidic environment, e.g., in the gastrointestinal tract.

[0162] Proton pump inhibitors are a group of drugs whose primary action is a significant and long-lasting reduction in gastric acid production. Proton pump inhibitors inhibit the hydrogen / potassium adenosine triphosphatase enzyme system (H + / K + It works by blocking the gastric ATPase or more commonly just the gastric proton pump. The proton pump is the final step in gastric acid secretion and transports H into the gastric lumen. + It is directly responsible for the secretion of ions, making it an ideal target for inhibiting acid secretion. Examples of proton pump inhibitors include: omeprazole (trade names: LOSEC®, PRILOSEC®, ZEGERID®); lansoprazole (trade names: PREVACID®, ZOTON®, INHIBITOL®); esomeprazole (trade name: NEXIUM®) and pantoprazole (trade names: PROTONIX®, SOMAC®, PANTOLOC®).

[0163] In some embodiments, the lipase can be formulated or co-administered (either simultaneously or sequentially) with, for example, a microbial protease and / or a microbial amylase. The amylase hydrolyzes the α-1,4-glucosidic bonds of starch, glycogen and polysaccharides to produce a mixture of maltose and glucose. In some embodiments, the protease is A. melleus protease and / or the amylase is Aspergillus oryzae amylase. In some embodiments, the composition is formulated as an oral dosage form. In some embodiments, the composition is formulated as a powder, granule, pellet, micropellet, liquid or tablet. In some embodiments, the composition is encapsulated or formulated as a tablet dosage form. In some embodiments, the composition does not include an enteric coating.

[0164] The pharmaceutical composition comprising the recombinant lipase disclosed herein may be in unit dosage form and may be prepared in any suitable manner. The pharmaceutical composition should be formulated to be compatible with its intended route of administration, for example, oral administration. The pharmaceutical composition may be in a variety of forms. For example, these include liquid, semi-solid and solid dosage forms, such as liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

[0165] The composition can be formulated as a solution, microemulsion, dispersion, liposome or other ordered structure suitable for stable storage at high concentration.Sterile solution can be prepared by incorporating the agent described herein in the required amount in a suitable solvent with one or combination of the components listed above, if necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the agent described herein into a sterile vehicle that contains a basic dispersion medium and other necessary components from those listed above.In the case of sterile powder for preparing sterile solution, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the agent described herein and any additional desired components from its solution that has been previously sterile filtered.The proper fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0166] The amount of lipase administered to a subject depends on several variables, such as the dietary content and the amount or type of fat ingested, as well as the age, weight, sex, health status, or disease or disorder associated with reduced ability to digest and / or absorb triglycerides that a given subject may have. Exemplary doses may include less than 400, 600, 800, or 1,000 mg of lipase or pharmaceutical composition per day. The total units of lipase per meal may be about 10,000, 20,000, 50,000, 100,000, 200,000, 400,000, or more.

[0167] V. Therapeutic uses The present invention provides a method for treating a disease or disorder associated with an increased amount of undigested lipids in a subject. In some embodiments, the disease or disorder is associated with an increased amount of undigested lipids in the gastrointestinal tract of the subject. The method includes administering to the subject an effective amount of the disclosed recombinant lipase, either alone or in combination with another therapeutic agent, to treat the disease or disorder in the subject. The term "effective amount" as used herein refers to an amount of an active agent (e.g., a recombinant lipase of the present invention) sufficient to produce a beneficial or desired result, such as improved uptake of fatty acids in plasma and tissues or reduced undigested fat in the small intestine. An effective amount can be administered in one or more administrations, applications, or doses, and is not intended to be limited to a particular formulation or route of administration.

[0168] In certain embodiments, the methods include orally administering to the subject an effective amount of the disclosed recombinant lipase, either alone or in combination with another therapeutic agent, to treat the disease or disorder in the subject.

[0169] As used herein, "treat", "treating" and "treatment" refer to the treatment of a disease in a subject, e.g., a human. This includes (a) inhibiting the disease, i.e., preventing its onset; and (b) relieving the disease, i.e., causing a regression of the disease state. The term "treating" can also include improving the symptoms of the disease in a subject. As used herein, the terms "subject" and "patient" refer to an organism that is treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably, humans.

[0170] Examples of diseases or disorders associated with increased amounts of undigested lipids include those in which a subject exhibits low levels of pancreatic enzyme secretion or has a physiological condition affecting fat hydrolysis or fat absorption (e.g., reduced gastric, duodenal, hepatic, biliary or gallbladder function); reduced gastrointestinal transit, motility, mixing, emptying; or reduced intestinal mucosal function (e.g., induced by mucosal injury) resulting in fat maldigestion or fat malabsorption or fatty acid deficiency. For example, such diseases and disorders may include exocrine pancreatic insufficiency (EPI), malabsorption syndromes, cystic fibrosis, chronic pancreatitis, acute pancreatitis, Shwachman-Diamond syndrome, fatty acid disorders, familial lipoprotein lipase deficiency, Johanson-Blizzard syndrome, Zollinger-Ellison syndrome, Pearson bone marrow syndrome, short bowel syndrome, liver disease, primary biliary atresia, cholestasis, celiac disease, fatty liver disease, pancreatitis, diabetes, aging, cancer of the pancreas, stomach, small intestine, colon, rectum / anus, liver, hepatic, gallbladder or esophagus, cachexia, or gastrointestinal disorders (e.g., Crohn's disease, irritable bowel syndrome or ulcerative colitis), surgical intervention of the stomach, small intestine, liver, gallbladder and pancreas.Other subjects suitable for treatment with the methods and compositions described herein are infants and those in critical care who have an increased likelihood of exhibiting lipid indigestion or malabsorption.

[0171] In another aspect, the present disclosure relates to a method of improving fatty acid absorption in a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby improving fatty acid absorption in the subject.

[0172] In another aspect, the present disclosure relates to a method for increasing the amount of fatty acids in plasma, red blood cells or tissue of a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein to increase the amount of fatty acids in the subject.

[0173] In another aspect, the disclosure relates to a method of increasing the ratio of omega-3 to omega-6 fatty acids in plasma, red blood cells or tissue of a subject in need thereof, comprising administering to the subject an effective amount of a lipase or pharmaceutical composition described herein, thereby increasing the amount of fatty acids in the subject.

[0174] In another embodiment, the present disclosure relates to a method for reducing the amount of fatty acid in the stool of a subject in need of reducing the amount of fatty acid in stool, comprising administering to the subject an effective amount of lipase or pharmaceutical composition as described herein, thereby reducing the amount of fatty acid in the stool of the subject.In some embodiments, the fatty acid is a long chain polyunsaturated fatty acid (LCPUFA).In some embodiments, the fatty acid is an omega-3 fatty acid.In some embodiments, the omega-3 fatty acid is DHA, EPA or DPA.

[0175] In some embodiments, a subject is administered less than 400, 600, 800, or 1,000 mg of lipase or pharmaceutical composition per day. The total units of lipase per meal can be about 10,000, 20,000, 50,000, 100,000, 200,000, 400,000, or more.

[0176] In certain embodiments, the lipase or pharmaceutical composition is administered in combination with a nutritional formulation containing fat-soluble vitamins (e.g., vitamins A, D, E, or K), acid blockers, or triglycerides.

[0177] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0178] The methods and compositions described herein may be used alone or in combination with other therapeutic agents and / or modalities. The term "administered in combination," as used herein, is understood to mean that two (or more) different therapies are delivered to a subject during the course of the subject's affliction with a disorder, such that the effects of the therapies on the patient overlap at some point. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so there is an overlap in the administration period. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other begins. In some embodiments of either case, the treatments are more effective because of the combined administration. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be seen if the second treatment were administered in the absence of the first treatment, or a similar situation is seen with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than that observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, totally additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second is delivered.

[0179] Throughout this description, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited process steps.

[0180] In this application, when an element or component is said to be included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0181] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the present invention. For example, when reference is made to a particular compound, the compound can be used in various aspects of the compositions of the present invention and / or in the methods of the present invention, unless otherwise understood from the context. That is, in this application, the embodiments are described and shown in a manner that allows for clear and concise application as described and illustrated, but it is intended and understood that the embodiments can be variously combined or separated without departing from the present teachings and invention(s). For example, it is understood that all features described and shown herein can be applicable to all aspects of the invention(s) described and shown herein.

[0182] The phrase "at least one of" should be understood to include each of the recited items following the phrase and various combinations of two or more of the recited items individually, unless otherwise understood from the context and application. The phrase "and / or" with respect to three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0183] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood to be open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless specifically stated or understood from the context to the contrary.

[0184] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0185] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be performed simultaneously.

[0186] The use of any and all examples or exemplary terms herein, such as "such as" or "including," is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless and until claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0187] Working Example The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0188] Example 1 - Lipase engineering lipase selection This example describes the design of a recombinant mutant Burkholderia cepacia lipase that has improved stability against the harsh conditions of the fed-state stomach (low pH and pepsin) and against proteolytic degradation over the length of the gastrointestinal (GI) tract and gastric transit time through the small intestine, while maintaining high levels of activity against physiologically relevant fats over a pH range of 3.5-7.

[0189] The goal of lipase engineering is to develop the following characteristics: Inherent stability against the harsh conditions of the fed state stomach (low pH and pepsin) and against proteolytic degradation along the length of the GI tract through the small intestine, while maintaining high levels of activity over a pH range of 3.0-7; Improved stability against proteolysis without loss of activity in the relevant pH range; · Activity and viability to proteolytic degradation at the relevant pH and over the length of the GI tract of interest (stomach, duodenum, jejunum, proximal ileum); Maintaining a high activity profile (units / mg) compared to wild-type lipase; and Stabilization to initiate digestion of fats in the stomach into absorbable fatty acids and monoglycerides The aim of the present invention was to design a lipase enzyme that has one or more of the following properties:

[0190] The benchmark physiological residence time of fat for humans with exocrine pancreatic insufficiency (EPI) was used as a standard for targeting lipase engineering. Specifically, for humans with EPI, (1) transit time through the low pH environment in the stomach is about 60-90 minutes, (2) transit time through gastric proteases (e.g., pepsin) is about 90-120 minutes depending on the contents of the meal consumed, and (3) transit time through the small intestine is about 240-360 minutes.

[0191] Lipase characteristics for consideration in genetic engineering include: Lipase activity at physiologically relevant pH conditions of the gastrointestinal tract (pH 3.5-7) without the need for enteric coating; · Ability to digest biologically relevant fats, including long-chain polyunsaturated triglycerides (e.g. DHA) and trioleic acid (pure oleic acid triglyceride; the major component of olive oil and common triglycerides in the standard human diet); Lipase solubility at physiologically relevant pH conditions of the gastrointestinal tract. ·Co-lipase not required for activity; ·Not inhibited by bile salts; Hydrolysis preference for sn-1 and sn-3 positions on triglycerides versus sn-2. · Viability at low pH; · Survivability against pepsin in the stomach; · the viability of the lipase against proteolytic degradation in the particular A. melleus protease with which it is co-formulated; and -Heat stability at 37℃ (body temperature) were mentioned.

[0192] A number of lipases were screened and evaluated for activity at physiologically relevant pH conditions (pH 4.0-7.0) with and without bile salts. Exemplary lipases are shown in Figure 3, and sequence alignments of exemplary lipases are shown in Figures 4 and 5.

[0193] The base (i.e., starting) lipase used for mutational analysis was a microbial I.2 class lipase enzyme from B. cepacia, having the amino acid sequence of SEQ ID NO:1. From the lipase enzymes, I.2 lipase was selected and tested against a wide range of fats (triglycerides), including the most difficult to digest fats, e.g., omega-3 fats (DHA and EPA triglycerides). I.2 class lipases were selected for mutational analysis because these lipases exhibit (i) high activity against long-chain polyunsaturated fatty acids (LCPUFAs), e.g., DHA, (ii) a broad level of activity in the physiologically relevant pH range (pH 3.5-7), and (iii) high activity with and without bile salts.

[0194] Lipase genetic engineering In general, it is an iterative process: 1. Identification of key properties of proteins and development of robust high-throughput analytical methods to test each key property 2. Making changes to the amino acid sequence of a protein though site-directed mutagenesis to generate an array of variants 3. Expression and testing of each variant against each analytical method and grade of how each change affected protein performance 4. Selection of top variants for advancement to further testing that meet predefined testing objectives. have been used in protein engineering to select and improve protein properties.

[0195] At the end of step 4 - the best performing variant is used as the "parent" or "base" sequence for the next round and steps 2-4 are repeated until the variants created have the desired properties.

[0196] Because low pH and proteolytic degradation often occur on the surface of proteins, three-dimensional molecular modeling of B. cepacia lipase identified amino acids on the surface of the protein. Without wishing to be bound by theory, it is believed that low pH and proteolytic degradation on the protein surface may result in improper folding of the lid or subdomain, which may reduce the activity of the enzyme.

[0197] B. cepacia lipase (SEQ ID NO:1) is a member of the I.2 subfamily of bacterial lipases. Each lipase within this subfamily is structurally related and has several common features. The I.1 and I.2 families of lipases also share a high degree of related amino acid sequence similarity, share several structural features such as a serine-histidine-aspartic acid active domain, a calcium binding site, a lid and subdomains that serve to protect the active site, disulfide bridges, and require a foldase (lipase-specific foldase ("Lif")) to ensure correct folding.

[0198] The amino acid substitutions considered for mutagenesis were derived from evaluation across many I.2 lipases. It is contemplated that the improved product properties for B. cepacia lipase should be applicable to other members of family I lipases such as subgroups I.1, I.2, and I.3. To illustrate this approach, a portion of the bacterial lipase phylogenetic tree is shown in FIG. 3, and a sequence alignment of selected I.1, I.2, and I.3 bacterial lipases is shown in FIG. 4.

[0199] Lipase genetic engineering A number of mutant B. cepacia lipases were designed, each with up to three amino acid substitutions relative to the wild-type sequence. In the first round, each mutant DNA sequence contained up to three amino acid changes (so-called substitutions) and, when expressed, gave rise to a protein with the three amino acid changes, a so-called variant.

[0200] The top selected distinct amino acid substitutions in B. cepacia lipase are listed in Table 4. [Table 19]

[0201] A list of the top amino acid combinations from one of the first rounds is shown in Table 5. [Table 20]

[0202] Briefly, DNA fragments encoding mutant B. cepacia lipases were cloned into expression vectors and all constructs were confirmed by gene sequencing. The lipase enzyme was expressed in the periplasmic space of E. coli. Osmotic shock was used to disrupt the outer cell membrane and harvest the lipase.

[0203] The recombinant mutant B. cepacia lipase was tested for lipase activity, pH viability, pepsin protease stability and A. melleus protease proteolytic stability as described in Example 2, Example 3, Example 4 and Example 5, respectively.

[0204] Analysis of recombinant mutant B. cepacia lipases from the lipase engineering campaign revealed that variant V130 had the best net positive effect across the conditions tested. V130 contained three substitutions (D102Q, N154H and F221L). The V130 variant substitutions were further modified in subsequent rounds that engineered additional B. cepacia lipases. An additional 13 substitutions were selected for use given their effect on one or more of enzyme activity, pH viability, pepsin protease stability and A. melleus protease proteolytic stability. The substitutions carried forward are shown in Table 4 above.

[0205] Example 2 - Lipase Activity Assay The goal of the lipase engineering campaign was to generate variant lipases that are active over a broad range of dietary conditions from pH 4 to pH 7, are not inhibited by bile salts, are soluble in this pH range, and are stable at 37° C. This example describes a lipase assay to determine whether the lipase variants may be active in the part of the gastrointestinal tract where hydrolysis and nutrient absorption occurs.

[0206] Because LCPUFAs are very difficult to digest given their chain length and double bonds, lipase activity was tested on LCPUFA-triglycerides and, in addition, on oleic acid, the major constituent of olive oil and a high component of the standard human diet.

[0207] In contrast to mammalian lipases, B. cepacia lipase does not require a co-lipase for catalytic activity and has been shown to be stable in the presence and absence of bile salts. B. cepacia lipase catalyzes the hydrolysis of triglycerides to fatty acids and monoglycerides with a higher level of activity towards the sn-1 and sn-3 regions of triglycerides, thereby having a similar function to the human pancreatic enzyme.

[0208] LCPUFA are triglycerides commonly found in fish oils. The preference for hydrolytic activity in the sn-1 and sn-3 regions is consistent with human pancreatic lipase, which allows fat to be digested into two fatty acids and one monoglyceride for absorption into plasma and incorporation into intestinal cells and tissues.

[0209] The turnover of a given substrate (eg, fat) is driven by the enzyme activity (units / mg surviving degradation) and the interaction of the enzyme with the substrate at the relevant pH level.

[0210] To ensure that the lipase was not inhibited by bile salts, activity assays were also performed in the presence of 8 mM bile salts at pH 7. This pH was chosen because bile salts are not appreciably soluble at pH ≦6. The bile salts used and their relative ratios are shown in Table 6. [Table 21]

[0211] Lipase fat digestion occurs at the oil / water interface, and to model this, lipase activity assays were performed using physiologically relevant substrates and oil-water emulsions were created. To ensure that the assay could distinguish improvements in engineered lipases, long-chain fats were used as substrates because they are more difficult to digest. Also, LCPUFA deficiencies, especially DHA and EPA, were shown in subjects with EPI, CF and / or malabsorption.

[0212] For activity assays, substrates highly enriched in both DHA triglyceride and oleic acid triglyceride were selected.

[0213] DHA is a triglyceride in which each fatty acid has 22 carbons and contains 6 double bonds, and is one of the longest chain fatty acids commonly encountered in diet.In addition, omega-3 fatty acids, such as DHA and EPA, are structural components of membranes and biological mediators related to the control of various physiological functions, so these fatty acids play an important role in the composition, development and function of cardiac, hepatic and nervous tissues, as well as in the control of inflammation and the immune system.LCPUFA and omega-3 fatty acids, especially DHA and EPA, have been shown to be deficient in subjects with EPI, CF and / or malabsorption.

[0214] Oleic acid is another fat substrate that is a common dietary fat, constituting approximately 55% to 83% of olive oil. Given the variability in olive oil composition, triolein, a synthetic fat similar to olive oil, was selected for use in the activity assays as it provides more consistent results. Each fatty acid in triolein is oleic acid (containing 18 carbons and one unsaturated bond).

[0215] The DHA oil selected for use in the activity assay was derived from algae and contained approximately 37% DHA triglycerides and approximately 22% oleic acid triglycerides (Nu-chek, Elysian, MN), with the remainder consisting mostly of myristic acid triglycerides and palmitate triglycerides. The amounts of the major components are shown in Table 3.

[0216] In the assay, the substrate was emulsified in a buffer at a specific pH and lipase was added to the substrate. After a fixed incubation time (allowing the lipase to digest triglycerides to form soluble free fatty acids), the lipase was heat inactivated. An aliquot was removed and a fatty acid quantification kit was used to tag the fatty acids with coenzyme A. The tagged fatty acids were then quantified by either colorimetric or fluorometric signal. SDS-PAGE was used to establish the concentration of each lipase and combined with the assay data to provide specific activity.

[0217] Given that the wild-type B. cepacia lipase has potent activity towards DHA over the pH range of interest (pH 4.0-7.0) and is not inhibited by bile salts, the goal of the engineering campaign was to ensure that changes made to improve the viability aspect of the enzyme did not reduce the activity level and render the lipase inhibited by bile salts. Thus, the goals of the lipase engineering were to: High activity in the pH range of 3.0-7, ensuring that the enzyme is able to digest fats from the stomach to the distal jejunum in a fed state; · Ensuring that lipase is not inhibited by bile salts at pH 7.0; Ensuring that the lipase is stable at 37°C; and Ensure that the lipase is soluble in the desired pH range (pH 3.0-7) Focused on.

[0218] Further, each variant produced by the lipase genetic engineering process can be produced by the following method: · Using DHA triglyceride oil substrate at pH 4 and 37°C; using a DHA triglyceride oil substrate at pH 7 and 37°C; and Using DHA triglyceride oil substrate with 8 mM bile salts at pH 7 and 37°C Activity was tested.

[0219] Briefly, DHA oil substrate was emulsified in water and stabilized with gum arabic to form a stable emulsion. The emulsion was then added to an appropriate volume of a specific pH buffer to adjust the pH. After 15 minutes, the reaction was stopped by inactivating the lipase with heat. The fatty acids produced were quantified using a commercially available free fatty acid assay kit (e.g., ABCAM, UK, Free Fatty Acid Assay Kit). The final reaction produced a colorimetrically detectable response.

[0220] The activities of the substitutions evaluated showed a strong correlation between activity at pH 4 and activity at pH 7, indicating that amino acid changes that affect activity at one pH also affect activity at the other pH. These substitutions in Table 1 (e.g., S153N, L287V, I232L, Y129N, V143A, A128N, N154H, F249L) have the potential to improve activity in the important pH ranges of interest and were prioritized for further engineering.

[0221] Example 3 - pH Viability Assay This example describes an assay to determine lipase viability in the low pH conditions of the stomach.

[0222] The goal of the lipase engineering campaign was to create a lipase that can survive the acidic conditions of the digestive system, particularly the stomach. The pH of gastric aspirates from children with CF ranges from about 2 to higher than 5. The pre-meal pH is low (about pH 2), but soon after a meal is consumed the pH rapidly increases to above pH 5 and then slowly decreases to pH 2 over about 120 minutes. During the meal-state interval, there is a slow but continuous emptying of the stomach contents through the pyloric valve, by which time the chyme has a pH below 4 and more than 60-90% of the meal has passed into the duodenum. The wild-type (starter) lipase from B. cepacia has good survivability down to pH 4. However, there may be times when the lipase can be subjected to pH levels below pH 4.0. Therefore, one goal of lipase engineering was to improve survivability down to pH 3.0-3.5. Since lipase is ingested by the patient with food, survivability at the very low pH of the fasted stomach is of less concern, however there is a risk of inactivation associated with any lipase remaining in the stomach in the late fed state if the pH drops below 4.

[0223] Therefore, a lipase with a half-life of at least 60-90 minutes at pH 3.0-3.5 is desirable. This ensures that less than half of the lipase is inactivated by low acid during passage through the stomach. Since wild-type B. cepacia lipase has a half-life of 40-50 minutes, the aim was to achieve an improvement of 50-100%.

[0224] To test the viability of each lipase engineered lipase, a high-throughput microtiter plate assay was developed to assess lipase viability at low pH and 37° C. In this pH range (pH 3.2-3.5), wild-type lipase has a half-life of 40-50 minutes, making the method sufficiently sensitive to be able to distinguish the effects of amino acid substitutions and associated improvements in lipase viability.

[0225] A flow chart describing the acid viability assay is shown in Figure 9. Briefly, lipase was added to the buffer at the assay pH (pH 3.0-pH 3.3) for 30-120 min at 37°C. At each time interval, aliquots were withdrawn and the pH neutralized. The activity of each aliquot was measured using the synthetic substrate p-nitrophenyl palmitate (p-NPP), which is cleaved by lipase to form p-nitrophenol, which is quantified by either a colorimetric or fluorometric signal. The data was then compared to a control that was not exposed to acid, and the data was analyzed to establish the half-life. A description of the method is provided below in part (a).

[0226] As lipase engineering progresses, it is expected that variants will have improved survival. If improvements make it difficult to distinguish between variants within the time frame of the method, the pH was lowered to increase stringency and aid in the differentiation of variants. Thus, the top variants were tested using the following method for pH viability: pH about 3.0-3.5 or less, 37°C for 2 hours, p-NPP substrate, allowing differentiation between variants.

[0227] pH viability assay using p-NPP fluorimetric detection In each well of a 96-well plate, periplasmic samples containing each lipase were added simultaneously in 2x concentrated buffer set at the specific assay pH. For the viability assay, the time of addition was considered as T=0. At the specified time points, aliquots were withdrawn and transferred to daughter plates. The reaction volume was diluted 1:9 into stop / indicator buffer for a 1 / 10 dilution. The pH shift prevented any acid-mediated degradation. Viable lipase started to hydrolyze the p-NPP colorimetric substrate. The reaction of p-NPP (p-nitrophenyl palmitate) with lipase had a strong colorimetric response at 405 nm, yielding measurable palmitate and para-nitrophenolate. The mechanism of p-NPP hydrolysis by lipase reaction is shown in Figure 10. The daughter plates were read consecutively in kinetic mode at 405 nm. Each successive time point of the experiment had less viable lipase and the kinetic curve had a shallower slope. The slopes of each time point were used to establish the half-life of each lipase variant. The variants were run alongside assay controls that included a WT-lipase control (expressed in E. coli) and a control of commercially purified WT lipase (Amano Enzyme, Nagoya, Japan).

[0228] From a viability standpoint, it was desirable that improved pH viability not adversely affect performance against proteases and vice versa.

[0229] Example 4 - Pepsin viability assay This example describes an assay to determine lipase proteolytic viability in pepsin conditions.

[0230] In some embodiments, the engineered lipase survives with pepsin present in the stomach. This may not be an issue for standard medical products based on pancrelipase, as the enzyme has an enteric coating that prevents it from being exposed to pepsin in the stomach. In contrast, the engineered B. cepacia lipase described herein is designed in some embodiments to be readily available in the stomach and exposed to pepsin. Pepsin is an aspartic protease with maximum activity at low pH levels (pH 1.5-4). Thus, the engineered B. cepacia lipase was evaluated for the effect of pepsin on lipase viability.

[0231] To test the viability of each lipase variant, a high throughput microtiter plate assay was developed to assess lipase viability using pepsin at approximately pH 4 and 37°C. The starting amount of pepsin added was based on the USP chapter on Simulated Gastric Fluid (SGF) Test Solution, which suggests a pepsin concentration of 3.2 mg / mL. Pepsin was added to the lipase solution to form a solution that was 3.2 mg / mL for pepsin and 0.01 mg / mL for lipase. As lipase engineering progressed, the amount of pepsin was increased to force differentiation without extending the assay time. Preliminary engineering used 19 mg / mL (6x USP) and later versions used 32 mg / mL (10x USP). This condition can be more severe than the normal conditions of the stomach because there are no background proteins present and thus no proteins other than lipase for pepsin to attack. Thus, the method was able to distinguish the effect of amino acid substitutions on the viability of lipase variants.

[0232] In the assay, lipase was added to a buffer containing pepsin at pH 4. At each time interval, an aliquot was withdrawn and the pH was neutralized to inactivate the pepsin. The activity of each aliquot was measured using the synthetic substrate p-nitrophenyl palmitate (p-NPP), which is cleaved by lipase to form p-nitrophenol, which is quantified by either a colorimetric or fluorometric signal. The data was then compared to a control not exposed to pepsin, and the data was analyzed to establish lipase viability, expressed as a half-life.

[0233] The goal of the engineered lipase was to ensure that the lipase at 0.01 mg / mL had a half-life of at least 90-120 minutes at pH 4 with 32 mg / mL pepsin. Given the excess amount of pepsin present, this goal ensured that minimal lipase was not inactivated by pepsin upon passage through the stomach. Wild-type B. cepacia lipase has a half-life of 50-70 minutes, so a minimum of 50-125% improvement was desired.

[0234] Each variant generated during the lipase engineering process was tested for pH viability by adding 0.01 mg / mL lipase to 32 mg / mL pepsin at pH 3.5. Detection using p-NPP substrate as detailed below was performed at 37° C. for 30 minutes.

[0235] The top variants were tested for pH viability by adding 0.01 mg / mL lipase to 32 mg / mL pepsin at pH 3.5. Detection using p-NPP substrate was performed at 37° C. for 2 hours, as described in detail below.

[0236] As lipase engineering progresses, variants are expected to have improved survival. If, within the time frame of the method, improvements make it difficult to distinguish between variants, the pH was lowered to increase the action of pepsin and aid in distinguishing between variants.

[0237] Pepsin viability assay using p-NPP fluorimetric detection A flow chart showing the pepsin viability assay is shown in FIG.

[0238] In each well of a 96-well plate, samples of each lipase-containing periplasm were added simultaneously to 2x concentrated buffer set at a specific assay pH containing pepsin at a specific concentration. At specific time points, aliquots were withdrawn and transferred to daughter plates. The reaction volume was diluted 1:9 into stop / indicator buffer for a 1 / 10 dilution. Viable lipase initiated hydrolysis of the p-NPP colorimetric substrate. Reaction of p-NPP (p-nitrophenyl palmitate) with lipase yielded palmitate and para-nitrophenolate with a strong colorimetric response at 405 nm. This reaction is shown in Figure 10. Each successive time point in the experiment had less viable lipase and the kinetic curve had a shallower slope. The slope of each time point was used to establish the half-life of each lipase variant. The engineered lipase variants were run alongside assay controls, including a wild-type lipase control (expressed in E. coli) and a control of purified wild-type lipase (Amano Enzyme, Nagoya, Japan).

[0239] Exemplary results for pepsin stability for various mutations and variants are shown, for example, in Figures 13-16 and 19.

[0240] Example 5 - Proteolytic (A. melleus protease) viability assay This example describes an assay to determine lipase viability under A. melleus protease conditions.

[0241] The goal for lipase engineering was improved lipase survivability in the presence of proteases present in the stomach and small intestine. Engineered lipase can be delivered in combination with proteases and amylases for protein and starch digestion, respectively. Thus, lipase can be exposed to proteases from A. melleus for co-administration. A. melleus protease is a serine protease with maximum activity at pH 7-pH 8 and higher than 50% activity in the pH range of pH 5-pH 11. Unlike mammalian proteases such as trypsin and chymotrypsin, which cleave proteins only after specific amino acids, A. melleus protease (also called SAP or oryzin) cleaves proteins non-specifically, down to small oligomers and individual amino acids. Thus, A. melleus protease provides representative harsh conditions for evaluating engineered lipase survivability against pancreatic proteases. If selected for combination use, the engineered lipase is expected to be in the presence of A. melleus protease for 3-6 hours (the transit time from the stomach through the small intestine in a fed state), and therefore it is desirable for the engineered lipase to be resistant to degradation by this protease.

[0242] To test the viability of each lipase engineered lipase, a high-throughput microtiter plate assay was developed to assess lipase viability using A. melleus protease at pH 6-7 and 37°C. This pH was chosen because it is in the range of maximum A. melleus protease proteolytic activity and represents the typical pH found in the small intestine. Initial testing was performed with 3.3 mg / mL protease with 0.01 mg lipase at pH 6.0. This amount of added protease was chosen to allow for differentiation in the experimental time frame requirement of less than 1 hour. For this in vitro assay, the ratio of protease to lipase was at least 100-fold higher than in the formulation expected for co-administration. The conditions may be more severe than the normal conditions of the stomach because there are no background proteins present and thus no proteins for the protease to attack other than lipase. Thus, the method could differentiate the effect of amino acid substitutions on the viability of lipase variants.

[0243] In the assay described in more detail below (see "A. melleus protease viability assay using p-NPP fluorimetric detection"), lipase was added to a buffer containing the protease at pH 6.0. At each time interval, aliquots were withdrawn and the pH was neutralized to inactivate the protease. The activity of each aliquot was measured using the synthetic substrate p-nitrophenyl palmitate (p-NPP), which is cleaved by the lipase to form p-nitrophenol, which is quantified by either a colorimetric or fluorimetric signal. Data was then compared to a control not exposed to the protease, and the data was analyzed to establish the lipase half-life.

[0244] Conditions in subsequent experiments as part of the final analysis were set to model those of the fed intestine for normal (non-pancreatic insufficient) test subjects in the more realistic case: in these cases, the ratio was 0.33 mg / mL protease with 0.01 mg lipase and 10 mg / mL casein at pH 6.0.

[0245] Initial experiments under these conditions showed improved viability.

[0246] A. melleus protease viability assay using p-NPP fluorimetric detection An overview of the A. melleus viability assay is shown in FIG.

[0247] In each well of a 96-well plate, samples of each lipase-containing periplasm were added simultaneously to 2x concentrated buffer set at a specific assay pH (centered at pH 6) containing A. melleus protease at a specific concentration. Final concentrations of A. melleus protease ranged from 1.6 mg / mL to 3.3 mg / mL. The time of addition was considered T=0 for the viability assay. Aliquots were withdrawn at specific time points, transferred to daughter plates, and diluted directly into the indicated buffer as described for the p-NPP assay above. Viable lipase began to hydrolyze the p-NPP colorimetric substrate. The reaction of p-NPP (p-nitrophenyl palmitate) with lipase yields palmitate and para-nitrophenolate, which have a strong colorimetric response at 405 nm. This reaction is shown in Figure 11. The daughter plates were read continuously in kinetic mode at 405 nm. Because oryzin was still active, the kinetic curve curved over time (as more lipase was inactivated by continuing proteolysis). Each successive time point in the experiment had less viable lipase, and the kinetic curve had a shallower initial slope. The slope of each time point was used to establish each lipase variant half-life. The engineered lipase variants were run along with assay controls, including a WT-lipase control (expressed in E. coli) and a control of commercially available purified WT lipase (Amano Enzyme, Nagoya, Japan).

[0248] The data for each variant was analyzed to determine the effect of each individual substitution on survival probability, expressed as half-life. The data correlated well with the model predictions (p-values ​​less than 0.01), indicating that the model was highly predictive of observed survival half-life.

[0249] Example 6 - Further Lipase Engineering This example illustrates further steps in the B. cepacia lipase engineering process.

[0250] Information about which substitutions improved each desired property and which substitutions were detrimental to each desired property was taken into account in modeling to predict novel amino acid substitutions that were suggested for further evaluation.

[0251] The top mutant B. cepacia lipase variants are shown in Table 7. [Table 22]

[0252] The variants contained 2 to 6 additional amino acid substitutions relative to the top ones contained in the V130 base variant. Each variant lipase was represented and evaluated for each test in the assay (stability at low pH, stability in the presence of proteases, etc.). Each novel substitution was present in five different variants across the array, which provided sufficient replicates to allow the use of multivariate statistical deconvolution tools to identify which amino acid substitution was responsible for the improvement. Unless otherwise indicated, mutant design, expression, purification, and assays of pH, pepsin, and A. melleus protease stability were all performed as described above.

[0253] Viability in the presence of low acid conditions pH 3.2 and in the presence of A. melleus protease was tested in the same manner as previously performed.

[0254] Although the wild-type B. cepacia lipase showed some resistance to pepsin, one goal of the engineering was to improve its survivability against pepsin to 90-120 min. Initially, at 19 mg / mL pepsin and pH 4.0, the variants were indistinguishable. After some engineering, to increase stringency, the concentration of pepsin was increased to 32 mg / mL and the pH of the load was reduced to 3.8 to increase the action of pepsin and aid in the discrimination of beneficial amino acid substitutions.

[0255] The top substitutions were used based on a combination of desirable properties (low pH survival, survival against aspartic and serine proteases).

[0256] Analysis of the recombinant mutant B. cepacia lipase from the lipase engineering campaign identified variant V290, which contains six substitutions (D102Q, N154H, F221L, V266L, G125S and N300Y). This variant had the best net positive effect, and the six substitutions together were found to work well, so the V290 variant substitutions were moved forward to form the base sequence. As a result, the mutant B. cepacia lipase enzymes containing these six substitutions were used as parents in the design of further B. cepacia lipases. Additionally, 12 additional substitutions were selected for inclusion based on the strength of improvement shown. The substitutions are shown in Table 8, based on the strength of improvement in all parameters of interest. [Table 23]

[0257] Table 9 shows the top B. cepacia lipase amino acid substitutions for pH viability. [Table 24]

[0258] Table 10 shows the top B. cepacia lipase amino acid substitutions for serine protease viability. [Table 25]

[0259] Table 11 shows the top B. cepacia lipase amino acid substitutions for pepsin viability. [Table 26]

[0260] Exemplary data for specific amino acid substitutions evaluated for inclusion are shown in FIG. 13. Stability in the presence of A. melleus protease, stability at low pH, stability in the presence of pepsin / SGF, activity at pH 4, and activity at pH 7 in the presence of bile salts were evaluated for each amino acid substitution using the multivariate statistical deconvolution tool described above. As shown, certain mutations caused positive changes in stability under certain conditions but not others. Some mutations caused decreased activity but increased stability (see, e.g., V266L). Also, some mutations caused decreases in all variables tested (see, e.g., N157I), and in most cases such mutations did not progress through the selection process. However, various amino acids were ultimately selected for further testing in combinations.

[0261] Example 7 - Final lipase engineering In this example, the engineering of B. cepacia lipase using the best variants identified above is presented.

[0262] Unlike earlier rounds where novel substitutions were introduced, the aim of the final round was to recombine all the best performing amino acid substitutions from previous rounds in different configurations to achieve additive, synergistic or potentiating improvements in product properties. In the final round, 46 variants were designated V301-V346. Each final variant contained 8-11 amino acid substitutions (approximately 3% change compared to the starting wild-type B. cepacia lipase sequence). A complete list of the final variants is shown in Table 12. [Table 27-1] [Table 27-2]

[0263] Unless otherwise indicated, mutant design, expression, purification, and pH, pepsin, and A. melleus protease viability assays were all performed essentially similarly to those described above, however, the goal of the studies was to evaluate and select the best final variants for further analysis.

[0264] The top performing variants were selected based on their overall ability to show improved viability against A. melleus proteases, pepsin and low pH (pH 3.0) while maintaining or even improving activity at pH 4.0, pH 7.0 and with bile salts, pH 7.0. In the first round of testing, all 46 variants were screened and the results were used to restrict the pool to the top 11 variants based on performance for each of the key properties. These data are presented in Figure 14, highlighting the top 11 variants.

[0265] The top variants were then screened again to initially confirm the results and allow for more statistical power. In addition to the top 11 variants, three controls were evaluated: (1) wild-type B. cepacia lipase expressed in E. coli, (2) V130, one of the top variants from the first round, and (3) V290, one of the top variants from a later round. The top variants contained a total of 10 amino acid substitutions relative to the wild-type B. cepacia lipase, which has 320 amino acids, accounting for 97% homology with the wild-type B. cepacia lipase. These controls allowed us to show the performance of each variant against the output of earlier variants in the lipase engineering process to facilitate visualization of the improvements and understand the relative improvements seen.

[0266] Eleven variants showed superior survival in both low acid conditions (pH 3.2) and against pepsin (pH 3.8). Thus, the pH of the acid challenge was lowered to 3.04 to increase the action of the acid, and the pH of the pepsin challenge was lowered to 3.58 to aid in differentiation of the variants. A summary of the corresponding viability data is shown in Figure 15. The results show that there is a clear progression of improvement from the wild-type lipase through the performance of V130-V290 for each of the top eleven variants, and with the exception of only V311 for the A. melleus protease, all of the half-lives were higher than V290 for all eleven variants under all conditions tested. The improved viability of the variant lipases at low pH is confirmed by the increased activity at pH 3.

[0267] Example 8 - Selection of superior engineered B. cepacia lipase This example shows the selection of the top two B. cepacia lipase variants using a two-axis approach for selection, where the top two variants in terms of viability were carried forward as well as the best variant in terms of activity.

[0268] The dataset used to select the top two variants in terms of viability is shown in Table 13. [Table 28]

[0269] For each of the three viability properties, there was a progression of improvement from wild type to the top variant from the first round (V130) and finally to the top candidates from the final round (V325 and V336). The longer half-life goal of 150-180 minutes for A. melleus protease survival at pH 6 was achieved by the final round of modification. The final variant survived longer than 190 minutes at pH 6. The half-life goal of 60-90 minutes survival at pH 3.5 was also achieved. The final variant survived longer than 150 minutes at a more stringent pH of 3.04. The half-life goal of 90-120 minutes for pepsin survival at pH 4 was also achieved. The final variant survived longer than 235 minutes at a more stringent pH of 3.58. Exemplary data and goals (dotted lines) are provided in FIG. 16.

[0270] The improvement factors for each property are listed in Table 14, which shows the viability improvement factors for the four B. cepacia lipase variants compared to the wild type (WT) enzyme. [Table 29]

[0271] The A. melleus protease improvement factor in the top variants was 3.6-fold more resistant than wild type. The low pH improvement factor at pH 3.04 in the top variants was 3.2-3.3-fold more resistant than wild type. The pepsin improvement factor at pH 3.58 in the top variants was 3.4-4.3-fold more resistant than wild type. The percentage of lipase surviving at a series of time points for each viability test is shown in the chart below. A. melleus protease viability at pH 6 is shown in Figure 17 and Table 15, where the top variants (V325 and V336) were compared to the wild type lipase as well as a top variant from the first round (V130) and a top variant from one of the subsequent rounds (V290). [Table 30]

[0272] Low pH viability at pH 3.0 is shown in FIG.

[0273] Table 16 shows the percentage of lipase surviving at pH 3.0 over a range of time points, where the top variants (V325 and V336) were compared to the wild-type lipase as well as the top variants (V130) and (V290). [Table 31]

[0274] Pepsin viability at pH 3.58 is shown in FIG.

[0275] Table 17 shows the percentage of lipase surviving in the presence of pepsin at pH 3.58 over a range of time points, where the top variants (V325 and V336) were compared to the wild-type lipase as well as the top variants (V130) and (V290). [Table 32]

[0276] Two of the top variants, V325 and V336, were tested head-to-head on the same assay plate against wild-type lipase and three different concentrations of pancrelipase. The resulting data are shown in Figures 20A and 20B. In this chart, the activity of 40 mg and 80 mg of each lipase variant is shown along with the activity of four capsules of pancrelipase (4x300 mg - 1,200 mg pancrelipase in total).

[0277] In the critical pH range of 4-7, each of the top variants (V325 and V336) has a specific activity that is either comparable to or shows a modest improvement over the wild-type specific activity. This ensures that each of these candidates can digest fat from the stomach to the end of the jejunum and proximal ileum in a fed state. None of the top variants (V325 and V336) were inhibited by bile salts. In the critical pH range of 4-7 and the binge eating criteria, 80 mg of wild-type lipase or all of the variants had specific activities at least 10 times higher than that of 4 capsules of pancrelipase. At pH 4, pancrelipase had almost no activity. Considering that the literature reports that porcine pancreatic preparations, e.g., pancrelipase, are degraded by acid, this result was not surprising since pancrelipase requires an enteric coating at pH 5.5 to survive the passage through the stomach. The greatest activity increase in the variants was observed at low pH (pH 3) (data not shown). These improvements are not the result of a true improvement in the catalytic efficiency of the lipase, but instead a significant increase in viability.

[0278] This example shows that the engineered lipase variants remain stable to proteolysis, resist the low acidity and harsh conditions of the stomach, and remain highly active in patients deficient in physiologically relevant fat (DHA). The top engineered lipases met all targets and maintained high activity. The increased viability of the engineered lipases is believed to maximize fat hydrolysis, improve performance, and make the lipases readily active to treat patients with fat hydrolysis deficiencies, such as those who have not responded to standard medical treatments.

[0279] Example 9: Dosing test for lipase variant V325 This example describes a dosing study to support dose selection for use in clinical trials with patients with exocrine pancreatic insufficiency (EPI) or malabsorption.

[0280] The dosing studies used a porcine model for EPI, an established surgical model of pancreatic insufficiency used to test macronutrient uptake and evaluate different preparations of orally administered pancreatic enzymes (Donaldson et al. (2009) ADV. MED. SCI. 54(1):7-13; Pierzynowska et al. (2018) ARCH. MED. SCI. 14(2):407-414; Freedman et al. (2004) N. ENGL. J. MED. 350(6):560-9, Abello et al. (1989) PANCREAS 4(5):556-64).

[0281] The EPI pig model was chosen because humans and pigs share many similarities functionally and developmentally with regard to gastrointestinal tract, urogenital structures, and brain and pancreas development (Gonzalez et al. (2015) TRANSL RES. 166(1):12-27; Luu et al. (2020) BMC GASTROENTEROL 20:403). Comparison of the daily allowance of vitamins and minerals in the human diet and the daily nutritional needs of pigs reveals similarities between the two species. The EPI pig model appears to be well adapted to evaluate native porcine enzymes (pancreatin, pancrelipase) and their role in exocrine pancreatic insufficiency. The EPI pig model has also been adapted to test the efficacy of microbial-derived enzymes (Grujic et al. (2015) "The Long Term Positive Effect of G-Tube Feeding with an In-Line Enzyme Cartridge (EFIC) on the Tissue Levels of DHA and EPA in Pig Model of Exocrine Pancreatic Insufficiency (EPI)", PEDIATRIC PULMONOLOGY 50:405-406).

[0282] Exocrine pancreatic insufficiency in pigs is achieved by ligation of the accessory exocrine pancreatic duct, which serves as the main pancreatic duct draining pancreatic juice into the duodenum. Surgical ligation dramatically reduces the levels of digestive enzymes released into the duodenum, resulting in reduced digestion and absorption of fats, proteins and carbohydrates. Duodenal pH is also reduced, similar to that in humans with EPI, resulting in another negative effect on enzyme activity in the intestinal lumen. (Martin et al. (2014) "A novel point-of-care lipase (ALCT-460) increases fat hydrolysis and omega 3 fat absorption in pics with exocrine pancreatic insufficiency," JOURNAL OF CYSTIC FIBROSIS 13(Supplement 2): S58;Martin et al. (2014) "Increased Total Fat and Long Chain Polyunsaturated Fatty Acid Absorption in Pigs with Exocrine Pancreatic Insufficiency Fed a Formula Pre-Hydrolyzed with a Novel Point-of Care Lipase (ALCT-460), PEDIATRIC PULMONOLOGY 49:408). Increased acidity in the small intestine may also cause micelle formation and bile acid precipitation affecting lipid absorption. All of these observations are consistent with results observed in humans with EPI (Corring et al. (1977) J. NUTR. 107(7):1216-21, Lankisch (1993) DIGESTION 54 Suppl 2:21-9).

[0283] The EPI pig model was used to evaluate specific measures of macronutrient absorption by assessing the by-products of digestion (e.g., fatty acids and monoglycerides for lipase) and their uptake in plasma and tissues (erythrocytes, enterocytes). Evidence from the EPI pig model provides substantial support for the safety, efficacy and stability of V325 lipase in combination with proteases and amylases.

[0284] Experimental design Eighteen (n=18) juvenile pigs were surgically operated on to induce EPI. The study treatment period included 12 (n=12) juvenile EPI pigs. The onset of EPI was confirmed by growth cessation and steatorrhea. The 12 EPI pigs included in the treatment period were selected based on the degree of steatorrhea and weight. The pigs weighed approximately 10±2 kg each. The pigs were fed a diet of 4% of body weight, with approximately 1% of body weight during the morning meal and approximately 3% of body weight during the afternoon meal.

[0285] The study included two study periods. Study 1 was a dosing trial to support dose selection for human patients with EPI or malabsorption. Experiment 2 continued with a selection of six pigs from experiment 1. Experiment 2 evaluated the release characteristics and activity of lipase, protease and amylase as measured by assessing the by-products of digestion in the chyme analyzed by placing cannulas in the stomach, duodenum and proximal ileum.

[0286] Experiment 1 - Protocol The study design included five blocks, each running over three days to facilitate testing of varying V325 lipase doses given together with protease and amylase with a standard human diet to assess absorption. A schematic of the treatment group design is provided in FIG. 21. On days 1, 4, 7, 10 and 13, pigs received V325 lipase and selected substrates (4 g DHA and EPA triglycerides ("DHA+EPA"), 10 g whey ("W") and 20 g potato starch ("PS")) as indicated. Blood was collected 24 hours later (i.e., days 2, 5, 8, 11 and 14), with a second blood collection 48 hours later (i.e., days 3, 6, 9, 12 and 15). For group 1, dose 1 was given on day 1, dose 2 on day 4, dose 3 on day 10, and dose 4 on day 13. For group 2, dose 4 was given on day 1, dose 3 on day 4, dose 2 on day 10, and dose 1 on day 13. Days 3, 6, 9, and 12 represented washout days after which the next treatment was started. The primary objective was to evaluate the safety, dosage, and performance of V325 lipase in combination with protease and amylase.

[0287] The Substrate Absorption Challenge Test (SACT) assessed clinical biomarkers of absorption directly related to by-products of hydrolysis for individual substrates of fat, protein and starch in a well-controlled and standardized test environment. Absorption was assessed using variable doses of V325 lipase. SACT can provide a measurement of intraluminal (small intestine: duodenum, jejunum, ileum) enzyme activity and a direct assessment of nutrient absorption through the gastrointestinal lumen. In SACT, V325 lipase was orally administered with a fixed amount of food (see above) and substrate (4g DHA and EPA triglycerides), and the product(s) of the lipolysis reaction (e.g. DHA and EPA fatty acids, with a total of 24 fatty acids) during absorption was monitored in the blood.

[0288] Triglycerides of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were used as SACT loading substrates for measurement of lipase activity, since they are the most difficult dietary fats to digest and absorb due to their chain length and number of double bonds (Burdge et al. (2005) REPROD. NUTR. DEV. 45:581-597; Hussein et al. (2005) JOURNAL OF LIPID RESEARCH 46:269-280). DHA and EPA are clinically significant fatty acids, biologically relevant, and important for growth and development. The use of DHA and EPA triglycerides as substrates in SACT to assess in vivo lipase activity (lipolysis) allows the measurement of their degradation products (DHA and EPA fatty acids) in blood over a 24-hour period using a validated gas chromatography-flame ionization detector (GC-FID) method (OmegaQuant, South Dakota). Furthermore, because endogenous conversion of essential fatty acids to EPA and DHA is excessively limited, these fatty acids are unique direct absorption biomarkers for measuring the efficacy of exogenously administered V325 lipase. The DHA / EPA loading test directly assesses the ability of exogenously administered lipase to digest long-chain polyunsaturated fatty acids (LCPUFAs), and the stringent test of lipolysis and absorption readout over time provide reliable pharmacokinetic measurements, e.g., C for DHA and EPA. max , T max and AUC. Fatty acids, specifically DHA and EPA in plasma and red blood cells, are strongly correlated with dietary fat intake and are biomarkers for overall fat absorption in people with CF.

[0289] SACT was performed by administering a pill containing omega-3 triglycerides derived from fish oil (approximately 4 g DHA and EPA) to pigs and drawing small amounts of blood 6-8 times over a 24 hour period, as shown in Table 18 (see also, e.g., Freedman et al. (2004) N. ENGL. J. MED. 350(6):560-9). [Table 33]

[0290] Fat absorption in plasma was measured over a 24-hour period as measured by area under the curve (AUC) and peak concentration (C max ) or time to peak concentration (T max ) was determined. Thus, the changes for V325 lipase (or comparator) were measured in a standardized manner. In addition to EPA (20:5n-3) and DHA (22:6n-3), the following 22 fatty acids (by classification) were also measured: a) Saturation (14:0, 16:0, 18:0, 20:0, 22:0 24:0), b) monounsaturated (16:1, 18:1, 20:1, 24:1), c) trans unsaturated (16:1, 18:1, 18:2), d) n-6 polyunsaturated (18:2, 18:3, 20:2, 20:3, 20:4, 22:4, 22:5), and e) n-3 polyunsaturated (18:3, 22:5) was measured.

[0291] The sum of these 24 fatty acids constitutes the total fatty acid content of the blood, and each individual fatty acid can be expressed as a percentage of the total or as a concentration (e.g., μg / mL). As shown in Figure 21, each SACT period provided an assessment of plasma uptake corresponding to each V325 lipase dose compared to a period without enzyme. There were five (5) SACT periods during this experiment: four (4) lipase doses, 20 mg, 40 mg, 80 mg, 120 mg, and a period without enzyme. [Table 34]

[0292] Results Experiment 1 As shown in FIG. 22, V325 lipase showed significantly higher AUC and C compared to the control (no enzyme ("NE") + substrate). max indicates Tmax The AUC over time was approximately 4 hours. Elevated V325 lipase (40mg, 80mg, 120mg) demonstrated significantly higher DHA+EPA uptake over 24 hours compared to control (40mg p=0.02, 80mg p=0.04, 120mg p=0.03). 24 The averages are shown in Figure 23 (40mg = 42%, 80mg = 83%, 120mg = 63%). As shown in Figure 24, baseline subtracted C max was significantly higher for the 40 mg, 80 mg, and 120 mg doses of V325 lipase compared to no enzyme (p=0.02, 0.0006, 0.009, respectively).

[0293] A similar response was observed when total fatty acids (FA) (n=24 total fatty acids) were evaluated. As shown in FIG. 25, V325 lipase AUC and C max was significantly higher when compared to the control (no enzyme + substrate). Increasing V325 lipase doses (20 mg, 40 mg, 80 mg, 120 mg) showed higher uptake of total fatty acids over 8 hours compared to the control (Figure 25). AUC and C max was approximately 2-3 fold higher at the higher V325 doses (80 mg, 120 mg) when compared to the control (no enzyme). The AUC for the V325 doses (80 mg, 120 mg) was significantly increased when compared to no enzyme (p=0.006, 0.02). AUC over time for total fatty acids 24 The averages are shown in Figure 26 (40 mg = 42%, 80 mg = 83%, 120 mg = 63%). As shown in Figure 27, the C for V325 doses (80 mg, 120 mg) max was significantly increased when compared to no enzyme (p=0.003, 0.006). Similar results were seen for the common dietary fats oleic, palmitic, stearic and elaidic acids (OPSE); saturated fatty acids; and beneficial fatty acids (DHA+EPA+docosapentaenoic acid (DPA)) (data not shown).

[0294] Experiment 2 - Protocol Both chyme and plasma absorption markers were evaluated in experiment 2. Tests for plasma absorption were performed as in experiment 1.

[0295] Chyme was used to assess enzyme activity as measured by the release of oleic acid, and plasma biomarkers of fatty acids were used to assess end products of triglyceride digestion. Oleic acid is a common dietary fat, the major part of olive oil, and has been used historically in the lipase USP method. Chyme is a thick semi-fluid mass of partially digested food that passes from the stomach to the duodenum and through the small intestine. Chyme is a highly relevant dietary substrate to assess lipase activity, stability and performance as measured by the release of lipid degradation by-products (e.g. oleic fatty acids) including natural postprandial conditions (e.g. pH, dietary contents, bile salts, micronutrient interactions).

[0296] During this experiment, a commercial porcine enzyme product (Creon® pancrelipase) was also used as a comparator. Figure 28 provides a schematic diagram of the experimental protocol. There were four (4) test periods: two V325 lipase doses (80 mg, 120 mg), Creon® 50,000 Units, and an enzyme-free period. Since the EPI model was developed and optimized from the outset for the evaluation of porcine extracts (pancreatin, pancrelipase), it was expected that the EPI pigs would show improved performance since Creon® pancrelipase contains native porcine enzyme. The maximum human recommended for humans with cystic fibrosis for Creon® was used as a comparator (2,500 kg body weight).

[0297] Chyme was harvested at various locations and time points in the gastrointestinal tract as shown in Table 20. [Table 35]

[0298] Lipase activity was measured by measuring lipid degradation in chyme, specifically the release of oleic acid (c:18:1 n-9) (μM / min / mL chyme). Oleic acid release was assessed using a colorimetric assay (Lipase Detection Kit ab102524, Abcam, UK) at a physiologically relevant pH of 6.0.

[0299] Results Experiment 2 Plasma: As shown in FIG. 29A, V325 lipase resulted in significantly higher AUC and C of DHA+EPA when compared to Creon® (pancrelipase) and control (NE: no enzyme). max V325 lipase showed higher overall AUC and C than Creon® or the control. max With earlier uptake T max As shown in Figure 29B, V325 showed a 20-30% increase in DHA+EPA mean change in AUC when compared to Creon®. These changes in response were consistent from 6 to 24 hours. Similar results were observed for total fatty acids (see Figures 30A and 30B).

[0300] V325 Activity and Stability: As shown in Figure 31, V325 lipase showed significantly higher and faster release of fatty acid (oleic acid) in each GI compartment tested (stomach, duodenum, ileum) compared to Creon®. Additionally and importantly, V325 lipase doses showed similar fatty acid release profiles in each compartment demonstrating the stability of V325 lipase in vivo. V325 lipase begins fat digestion immediately in the stomach and duodenum, with sustained activity across the GI compartments of interest confirming enzyme engineered stability in a physiological environment. Understanding where fats are cleaved by lipase informs how they are absorbed and dose selection. Evaluating enzyme activity in chyme allows for evaluation of activity in natural postprandial conditions (e.g. pH, meal contents, bile salts, micronutrient interactions). Lipase Cmax was similar in each compartment tested, supporting the stability of V325 lipase.

[0301] Faster release and absorption, as shown by V325, allows for more physiological absorption in the upper small intestine. Since the major absorption transporters are believed to be located in the upper small intestine, slow enzyme release or poor stability would not allow for physiological absorption. Furthermore, chyme viscosity increases significantly between the duodenum and ileum. Thus, faster lipid breakdown as shown by V325 allows for higher mixing and improved performance.

[0302] This example shows that administration of V325 lipase results in significantly higher fatty acid (oleic acid) release at pH 6.0 compared to Creon® 50,000. Additionally, the faster release of fatty acids by V325 lipase and its stability across key compartments of the stomach and small intestine are evidence that V325 lipase provides improved performance compared to not only the no enzyme control, but also the medical standard (Creon®).

[0303] Incorporation by Reference The entire disclosures of each of the patent and scientific literature referenced herein are incorporated by reference for all purposes.

[0304] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the foregoing embodiments are to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. (i) increased stability at acidic pH (e.g., pH 3.0 or 4.0) relative to the corresponding wild-type lipase enzyme; (ii) increased stability in the presence of a protease (e.g., a serine protease and / or an aspartic acid protease) relative to the corresponding wild-type lipase enzyme; or (iii) at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the enzymatic activity of the corresponding wild-type lipase enzyme. wherein the recombinant variant lipase enzyme has at least 90% sequence identity to SEQ ID NO:

2.

2. (I) The lipase (a) substitution of a residue at a position corresponding to position 102 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with Q; (b) substitution of a residue at a position corresponding to position 125 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with S; (c) substitution of a residue at a position corresponding to position 137 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with A; (d) substitution of a residue at a position corresponding to position 153 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with N; (e) substitution of a residue at a position corresponding to position 154 of wild-type B. cepacia lipase, wherein optionally, the residue is replaced with H; (f) substitution of a residue at a position corresponding to position 221 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with L; (g) substitution of a residue at a position corresponding to position 227 of wild-type B. cepacia lipase, wherein optionally, the residue is replaced with K; (h) substitution of a residue at a position corresponding to position 249 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with L; (i) substitution of a residue at a position corresponding to position 266 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with L; (j) substitution of a residue at a position corresponding to position 300 of wild-type B. cepacia lipase, wherein optionally, the residue is replaced with Y; (k) a substitution of a residue at a position corresponding to position 39 of a wild-type Burkholderia cepacia lipase, wherein optionally, the residue is substituted with R; (l) substitution of a residue at a position corresponding to position 79 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with Q; (m) a substitution of a residue at a position corresponding to position 128 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with N; (n) substitution of a residue at a position corresponding to position 138 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with I; (o) substitution of a residue at a position corresponding to position 161 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with A; (p) a substitution of a residue at a position corresponding to position 170 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with S; (q) substitution of a residue at a position corresponding to position 240 of wild-type B. cepacia lipase, wherein optionally, the residue is replaced with V; (r) substitution of a residue at a position corresponding to position 250 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with A; (s) a substitution of a residue at a position corresponding to position 260 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with A; (t) substitution of a residue at a position corresponding to position 281 of wild-type B. cepacia lipase, wherein optionally, the residue is substituted with A; or any combination of the above substitutions Including, (II) Lipase (a) substitution of a D residue at the position corresponding to position 102 of wild-type B. cepacia lipase (D102), wherein optionally, the D residue is substituted with a Q (D102Q); (b) a substitution of a G residue at the position corresponding to position 125 of wild-type B. cepacia lipase (G125), wherein optionally, the G residue is substituted with S (G125S); (c) a substitution of a T residue at the position corresponding to position 137 of wild-type B. cepacia lipase (T137), wherein optionally, the T residue is substituted with an A (T137A); (d) a substitution of an S residue at the position corresponding to position 153 of wild-type B. cepacia lipase (S153), wherein optionally, the S residue is substituted with N (S153N); (e) a substitution of an N residue at a position corresponding to position 154 of wild-type B. cepacia lipase (N154), wherein optionally, the N residue is substituted with H (N154H); (f) a substitution of an F residue at a position corresponding to position 221 of wild-type B. cepacia lipase (F221), wherein optionally, the F residue is substituted with an L (F221L); (g) a substitution of a T residue at a position corresponding to position 227 of wild-type B. cepacia lipase (T227), wherein optionally, the T residue is substituted with a K (T227K); (h) a substitution of an F residue at a position corresponding to position 249 of wild-type B. cepacia lipase (F249), wherein optionally, the F residue is substituted with an L (F249L); (i) a substitution of a V residue at a position corresponding to position 266 of wild-type B. cepacia lipase (V266), wherein optionally, the V residue is substituted with an L (V266L); (j) a substitution of an N residue at a position corresponding to position 300 of wild-type B. cepacia lipase (N300), wherein optionally, the N residue is substituted with Y (N300Y); (k) a substitution of a Q residue at a position corresponding to position 39 of wild-type Burkholderia cepacia lipase (Q39), wherein optionally, the Q residue is substituted with R (Q39R); (l) a substitution of a T residue at the position corresponding to position 79 of wild-type B. cepacia lipase (T79), wherein optionally, the T residue is substituted with a Q (T79Q); (m) a substitution of an A residue at a position corresponding to position 128 of wild-type B. cepacia lipase (A128), wherein optionally, the A residue is substituted with N (A128N); (n) a substitution of a V residue at a position corresponding to position 138 of wild-type B. cepacia lipase (V138), wherein optionally, the V residue is substituted with I (V138I); (o) a substitution of an L residue at a position corresponding to position 161 of wild-type B. cepacia lipase (L161), wherein optionally, the L residue is substituted with A (L161A); (p) a substitution of an A residue at a position corresponding to position 170 of wild-type B. cepacia lipase (A170), wherein optionally, the A residue is substituted with S (A170S); (q) a substitution of an A residue at a position corresponding to position 240 of wild-type B. cepacia lipase (A240), wherein optionally, the A residue is substituted with a V (A240V); (r) a substitution of a G residue at the position corresponding to position 250 of wild-type B. cepacia lipase (G250), wherein optionally, the G residue is substituted with an A (G250A); (s) a substitution of an S residue at a position corresponding to position 260 of wild-type B. cepacia lipase (S260), wherein optionally, the S residue is substituted with an A (S260A); (t) a substitution of an S residue at the position corresponding to position 281 of wild-type B. cepacia lipase (S281), wherein optionally, the S residue is substituted with A (S281A); or any combination of the above substitutions Including, (III) the lipase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations relative to the corresponding wild-type lipase, and optionally, the lipase comprises: (a) D102Q, N154H, and F221L substitutions; (b) D102Q, G125S, N154H, F221L, V266L, and N300Y substitutions; (c) T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, F249L, V266L, and N300Y substitutions; (d) T79Q, D102Q, G125S, T137A, N154H, F221L, T227K, V266L, S281A, and N300Y substitutions; (e) T79Q, D102Q, G125S, S153N, N154H, F221L, T227K, V266L, S281A, and N300Y substitutions; (f) T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, G250A, V266L, and N300Y substitutions; (g) T79Q, D102Q, G125S, S153N, N154H, F221L, F249L, V266L, S281A, and N300Y substitutions; (h) T79Q, D102Q, G125S, N154H, F221L, T227K, F249L, V266L, S281A, and N300Y substitutions; (i) D102Q, G125S, T137A, S153N, N154H, F221L, T227K, F249L, V266L, and N300Y substitutions; (j) D102Q, G125S, T137A, S153N, N154H, F221L, T227K, G250A, V266L, and N300Y substitutions; (k) D102Q, G125S, T137A, N154H, F221L, T227K, G250A, V266L, S281A, and N300Y substitutions; (l) Substitutions of D102Q, G125S, S153N, N154H, F221L, T227K, F249L, G250A, V266L, and N300Y; or (m) Replacement for D102Q, G125S, S153N, N154H, F221L, T227K, F249L, V266L, S281A and N300Y and / or The lipase according to claim 1, wherein the lipase (IV) is an α / β-hydrolase lipase.

3. (a) The lipase comprises a serine-histidine-aspartic acid active triad. (b) the lipase comprises a hydrophobic lid that opens to allow binding and / or hydrolysis of lipids, and optionally the hydrophobic lid opens sufficiently to allow binding and / or hydrolysis of triglycerides having a chain length greater than 8 carbons; (c) the lipase contains a calcium binding site and is stabilized when calcium binds to the calcium binding site; (d) the lipase contains an oxyanion hole, which stabilizes a negatively charged intermediate produced during fatty acid bond hydrolysis; (e) the lipase comprises an S residue at position corresponding to position 87 in wild-type B. cepacia (S87), a D residue at position corresponding to position 264 in wild-type B. cepacia (D264), and an H residue at position corresponding to position 286 in wild-type B. cepacia (H286); and / or (f) The lipase according to claim 1 or 2, wherein the lipase comprises an amino acid sequence of any one of SEQ ID NOs: 2 to 14 or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 2 to 14.

4. (I) The lipase (a) has a half-life in the presence of a serine protease of at least 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, or 200 minutes; and / or (b) at least 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater stability in the presence of a serine protease compared to a corresponding wild-type lipase, optionally wherein the serine protease is an Aspergillus melleus protease; (II) Lipase (a) has a half-life of at least 50 minutes, 75 minutes, 100 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, or 150 minutes at about pH 3.0; and / or (b) have at least 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater stability at about pH 3.0 compared to the corresponding wild-type lipase; (III) Lipase (a) has a half-life in the presence of aspartic protease of at least 50 minutes, 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 200 minutes, 225 minutes, 230 minutes, or 235 minutes; and / or (b) at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, or 4-fold greater stability in the presence of an aspartic protease compared to the corresponding wild-type lipase, optionally wherein the aspartic protease is pepsin; and / or (IV) Lipase (a) has a half-life in the presence of pancreatin of at least 50 minutes, 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, or 200 minutes; and / or (b) the lipase according to any one of claims 1 to 3, having at least 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater stability in the presence of pancreatin compared to the corresponding wild-type lipase. (a) the lipase has at least 0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, or 3-fold greater activity at about pH 3.0 compared to the corresponding wild-type lipase; (b) the lipase has a specific activity of at least 300, 400, 500, 600, 700, 800, 900, or 1,000 μmol fatty acid (FA) produced / min / mg lipase at pH 3.0 on a long-chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein; (c) the lipase has a specific activity of at least 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or 2,000 μmol fatty acid (FA) produced / min / mg lipase at pH 4.0, pH 5.0, or pH 6.0 on a long-chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein; (d) the lipase has a specific activity of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, or 2,000 μmol fatty acid (FA) produced / min / mg lipase at pH 7.0 on a long-chain triglyceride substrate comprising 37% DHA triglyceride and 22% oleic acid triglyceride or triolein; (e) lipase preferentially hydrolyzes the sn-1 and sn-3 positions on triglycerides; (f) lipase enzyme activity (e.g., specific activity) is not inhibited by bile salts; (g) the lipase does not require a co-lipase; (h) the lipase is not cross-linked and / or crystallized; (i) the lipase remains sufficiently active at a pH in the range of 3.5 to 7.0 to hydrolyze long-chain polyunsaturated fatty acids (LCPUFAs), such as DHA and EPA, or long-chain triglycerides, such as oleic acid or triolein, in the gastrointestinal tract of the subject, and is optionally at least 2-fold, 10-fold, 100-fold, or 1,000-fold more active than pancrelipase when tested under the same conditions; (j) greater than 50%, 60%, 70%, 80%, or 90% of the lipase remains active for 60 to 120 minutes in the subject's fed-state stomach; (k) the lipase digests more than 20%, more than 30%, more than 40%, or more than 50% of ingested fat in the subject's stomach into fatty acids and monoglycerides; (l) greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the lipase remains active throughout the small intestine of the subject for about 240 to about 360 minutes; (m) lipase digests greater than 50%, 60%, 70%, 80%, or 90% of ingested fat into fatty acids and monoglycerides in the small intestine of the subject; (n) the lipase increases the absorption of long-chain unsaturated fatty acids into the plasma of the subject by more than 25%, more than 35%, more than 50%, more than 100%, or more than 200% within 30, 45, 60, 90, or 120 minutes relative to the same subject when not administered the lipase or to a similar subject when not administered the lipase; (o) lipase increases the absorption of fat-soluble vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K); and / or (p) Lipase increases choline absorption. The lipase according to any one of claims 1 to 4.

6. A nucleic acid encoding the lipase according to any one of claims 1 to 5.

7. 7. An expression vector comprising the nucleic acid sequence of claim 6, wherein optionally the nucleic acid sequence encoding the recombinant variant lipase is codon-optimized for expression in a heterologous cell, and optionally the heterologous cell is a cell of Burkholderia cepacia, Burkholderia glumae, Pseudomonas fluorescens, Chromobacterium viscosum, Pseudomonas luteola, Pseudomonas fragilis or Escherichia coli.

8. 8. A host cell comprising the expression vector of claim 7, which is a cell of Burkholderia cepacia, Pseudomonas glumae, Pseudomonas fluorescens, Chromobacterium viscosum, Pseudomonas luteola, or Escherichia coli.

9. 10. A method for producing a recombinant mutant Burkholderia cepacia lipase enzyme, comprising growing the cell of claim 8 under conditions such that the host cell expresses the recombinant mutant Burkholderia cepacia lipase enzyme, and purifying the recombinant mutant Burkholderia cepacia lipase enzyme.

10. A pharmaceutical composition comprising the lipase according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier and / or excipient, optionally comprising: (a) further comprising a microbial protease and / or a microbial amylase, optionally wherein the protease is an Aspergillus melleus protease and / or the amylase is an Aspergillus amylase; (b) formulated as an oral dosage form; (c) formulated as a powder, granules, pellets, micropellets, liquid, or tablet; (d) encapsulated or formulated in tablet form; and / or (e) A pharmaceutical composition that does not include an enteric coating.

11. A lipase or pharmaceutical composition for use in treating a subject in need of treatment for a disease or disorder associated with a reduced ability to digest or absorb lipids resulting in increased amounts of undigested lipids, wherein the subject is administered an effective amount of the lipase or pharmaceutical composition, said lipase or pharmaceutical composition being as described in any one of claims 1 to 5 or said pharmaceutical composition being as described in claim 10.

12. 1. A lipase or pharmaceutical composition for use in treating lipid maldigestion or malabsorption in a subject in need thereof, wherein the subject is administered an effective amount of the lipase or pharmaceutical composition, and optionally (a) the subject exhibits low levels of pancreatic enzyme secretion or has a physiological condition affecting fat hydrolysis or fat absorption (e.g., reduced gastric, duodenal, hepatic, biliary, or gallbladder function); reduced gastrointestinal transit, motility, mixing, or emptying; or reduced intestinal mucosal function (e.g., induced by mucosal injury) resulting in fat maldigestion or fat malabsorption or fatty acid deficiency; and / or 11. The lipase of any one of claims 1 to 5 or the pharmaceutical composition of claim 10, wherein the lipid maldigestion or malabsorption is associated with a disease or disorder selected from exocrine pancreatic insufficiency (EPI), malabsorption syndromes, cystic fibrosis, chronic pancreatitis, acute pancreatitis, Shwachman-Diamond syndrome, fatty acid disorders, familial lipoprotein lipase deficiency, Johanson-Blizzard syndrome, Zollinger-Ellison syndrome, Pearson bone marrow syndrome, short bowel syndrome, liver disease, primary biliary atresia, cholestasis, celiac disease, fatty liver disease, pancreatitis, diabetes, aging, cancer of the pancreas, stomach, small intestine, colon, rectum / anus, liver, hepatic, gallbladder, or esophagus, cachexia, or a gastrointestinal disorder (e.g., Crohn's disease, irritable bowel syndrome, or ulcerative colitis), or surgical intervention of the stomach, small intestine, liver, gallbladder, or pancreas.

13. (a) For use in a method for improving fatty acid absorption in a subject in need thereof, comprising: (b) for use in a method for increasing the amount of a fatty acid in plasma, red blood cells, or tissue in a subject in need thereof; (c) for use in a method for increasing the ratio of omega-3 to omega-6 fatty acids in plasma, red blood cells, or tissue in a subject in need thereof; or (d) A lipase or pharmaceutical composition for use in a method for reducing the amount of fatty acids in the stool of a subject in need thereof, wherein the subject is administered an effective amount of the lipase or pharmaceutical composition.

14. (a) a fatty acid (i) long-chain polyunsaturated fatty acids (LCPUFAs), and / or (ii)ω-3 fatty acids and optionally the omega-3 fatty acid is DHA, EPA or DPA. (b) the subject is administered less than 400, 600, 800, or 1,000 mg of lipase or pharmaceutical composition per day; (c) the lipase or pharmaceutical composition is administered in combination with a nutritional formulation containing a fat-soluble vitamin (e.g., vitamin A, D, E, or K), an acid blocker, or a triglyceride; (d) the subject is a mammal, and / or (e) The lipase or pharmaceutical composition according to any one of claims 11 to 13, wherein the subject is a human.

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