Methods and compositions for treating congenital sucrase-isomaltase deficiency

Abstract

Recombinant mutant invertase enzymes are disclosed. Pharmaceutical compositions comprising invertase enzymes, such as recombinant mutant invertase enzymes and any isomaltase enzymes, are also disclosed. In particular, the enzymes and compositions can be used to treat congenital sucrase-isomaltase deficiency (CSID).

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 388,810, filed on July 13, 2022, and U.S. Provisional Patent Application No. 63 / 388,845, filed on July 13, 2022, and the entire disclosure of each of these applications is hereby incorporated by reference in its entirety for all purposes.

[0002] Reference to Sequence Listing XML This application includes a Sequence Listing submitted electronically in XML format. The Sequence Listing XML is hereby incorporated by reference in this specification. The XML file created on July 12, 2023, is named ANR - 004WO_SL.xml and is 80,804 bytes in size.

[0003] Field of the Invention The present invention generally relates to recombinant mutant invertase (sucrase) enzymes and methods and compositions for treating congenital sucrase - isomaltase deficiency (CSID).

Background Art

[0004] Background Congenital sucrase-isomaltase deficiency (CSID) is a rare autosomal recessive genetic disorder of the small intestine caused by gene mutations in sucrase-isomaltase, an enzyme complex that catalyzes the hydrolysis of dietary sucrose and starch. Ritz et al. GASTROENTEROLOGY 125(6):1678-85. Reduced or absent invertase (sucrase) and / or isomaltase enzyme activity has been found in patients with CSID, and investigation at the subcellular and molecular levels in intestinal biopsy specimens has provided explanations for several phenotypes that differ in protein transport efficiency, processing, and sorting that result in impaired physiological function. Ritz et al. (2003) GASTROENTEROLOGY 125(6):1678-85, Jacob et al. (2000) J. CLIN. INVEST. 106(2):281-7, Alfalah et al. (2009) GASTROENTEROLOGY 136(3):883-92 and Ouwendijk (1996) J. CLIN. INVEST.97(3):633-41.

[0005] Affected subjects may present with osmotic diarrhea, mild steatorrhea, chronic diarrhea, irritability, and vomiting after sucrose consumption. Difficulties in providing appropriate nutrition to these subjects can lead to dehydration, metabolic acidosis, hypercalcemia, growth retardation, and developmental delay. Cohen (2016) MOL. CELL. PEDIATR.3:5. Failure of absorption of dietary disaccharides and starch has implications for the absorption of other nutrients and the hormonal control of gastrointestinal function.

[0006] The prevalence of CSID in the European population is estimated to be 1 in 5,000, although it is higher in the indigenous populations of Alaska, Greenland, and Canada. Marcadier et al. (2015) CANADIAN MED. ASSOC. J. 187(2):102 - 107. There is also evidence that heterozygous carriers experience symptoms of CSID such as chronic diarrhea, abdominal pain, and flatulence. Currently, it is estimated that 2 - 9% of European - Americans may be affected, suggesting that sucrase - isomaltase deficiency is underrecognized. DeJonge et al. (2014) GASTROENTEROLOGY 146:S705.

[0007] Currently, CSID can be treated by oral administration of a solution of sacrosidase (SUCRAID®). However, sacrosidase does not fully replace the enzymatic activity of the endogenous sucrase - isomaltase enzyme complex. The liquid formulation of sacrosidase in 50% glycerol is thermally unstable and unstable under acidic conditions. Consequently, the sacrosidase solution must be discarded after 4 weeks of opening, cannot be used in combination with warm beverages and infant formulas or fruit juices, and has a negative impact on ease of use and patient compliance.

[0008] Although development has been made to date, there remains an ongoing need for new and effective pharmaceutical compositions and treatments for the treatment and management of CSID.

Summary of the Invention

[0009] Summary The present invention is based in part on the development of recombinant mutant invertase (sucrase) enzymes that are active in humans and have higher stability and / or activity than naturally occurring enzymes. In particular, the recombinant mutant enzymes disclosed herein may exhibit improved pH stability, thermal stability, and / or stability to proteolytic digestion compared to the naturally occurring version of the enzyme. Further, the recombinant mutant enzymes disclosed herein may have a higher specific activity than the wild-type invertase (sucrase) enzyme. Additionally, the recombinant mutant enzymes described herein are contemplated to be suitable for oral administration, potentially safer, tolerable, and / or more active than commercially available invertase (sucrase) enzymes, considering their enhanced stability. The present invention is based in part on the development of pharmaceutical compositions, such as solid pharmaceutical compositions comprising an invertase (sucrase) enzyme that is stable at room temperature and any isomaltase enzyme, which enable easier distribution, storage, and administration to a subject compared to compositions that are unstable at room temperature and / or require refrigeration. Recombinant enzymes and compositions can be used, inter alia, to treat congenital sucrase-isomaltase deficiency (CSID).

[0010] Accordingly, in one aspect, the present disclosure relates to a recombinant mutant S. cerevisiae invertase enzyme. In certain embodiments, the invertase has increased activity at acidic pH (e.g., about pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5) relative to the corresponding wild-type invertase. In certain embodiments, the invertase has increased activity at neutral pH (e.g., about pH 7.0) relative to the corresponding wild-type invertase. In certain embodiments, the invertase has increased stability at acidic pH (e.g., about pH 2.5) relative to the corresponding wild-type invertase. In certain embodiments, the invertase has increased thermal stability relative to the corresponding wild-type invertase. In certain embodiments, the invertase has a combination of the above-described characteristics.

[0011] In one aspect, the invertase comprises a substitution of the F residue at the position corresponding to position 172 of SEQ ID NO:2; a substitution of the N residue at the position corresponding to position 429 of SEQ ID NO:2; a substitution of the P residue at the position corresponding to position 479 of SEQ ID NO:2; a substitution of the D residue at the position corresponding to position 122 of SEQ ID NO:2; a substitution of the T residue at the position corresponding to position 140 of SEQ ID NO:2; a substitution of the E residue at the position corresponding to position 156 of SEQ ID NO:2; a substitution of the V residue at the position corresponding to position 178 of SEQ ID NO:2; a substitution of the K residue at the position corresponding to position 259 of SEQ ID NO:2; a substitution of the D residue at the position corresponding to position 272 of SEQ ID NO:2; a substitution of the K residue at the position corresponding to position 277 of SEQ ID NO:2; a substitution of the E residue at the position corresponding to position 285 of SEQ ID NO:2; a substitution of the A residue at the position corresponding to position 378 of SEQ ID NO:2; a substitution of the E residue at the position corresponding to position 382 of SEQ ID NO:2; a substitution of the V residue at the position corresponding to position 399 of SEQ ID NO:2; a substitution of the G residue at the position corresponding to position 431 of SEQ ID NO:2; a substitution of the V residue at the position corresponding to position 477 of SEQ ID NO:2; a substitution of the A residue at the position corresponding to position 501 of SEQ ID NO:2; a substitution of the L residue at the position corresponding to position 549 of SEQ ID NO:2; a substitution of the V residue at the position corresponding to position 581 of SEQ ID NO:2; a substitution of the G residue at the position corresponding to position 586 of SEQ ID NO:2; or a substitution of the N residue at the position corresponding to position 589 of SEQ ID NO:2; or one or more amino acid substitutions selected from the group consisting of any combination of the foregoing substitutions. SEQ ID NO:2 is the amino acid sequence of an exemplary invertase enzyme from Saccharomyces cerevisiae that is wild-type but contains a heterologous FAKS signal sequence. Invertase enzymes containing the foregoing substitutions at the corresponding positions in SEQ ID NO:1 (wild-type invertase enzyme from Saccharomyces cerevisiae containing the native signal sequence) and SEQ ID NO:3 (wild-type invertase enzyme from Saccharomyces cerevisiae without a signal sequence) are also contemplated herein. See Table 1 below.

[0012] In one aspect, the invertase has a substitution of the F residue at the position corresponding to position 102 of SEQ ID NO: 1; a substitution of the N residue at the position corresponding to position 359 of SEQ ID NO: 1; a substitution of the P residue at the position corresponding to position 409 of SEQ ID NO: 1; a substitution of the D residue at the position corresponding to position 52 of SEQ ID NO: 1; a substitution of the T residue at the position corresponding to position 70 of SEQ ID NO: 1; a substitution of the E residue at the position corresponding to position 86 of SEQ ID NO: 1; a substitution of the V residue at the position corresponding to position 108 of SEQ ID NO: 1; a substitution of the K residue at the position corresponding to position 189 of SEQ ID NO: 1; a substitution of the D residue at the position corresponding to position 202 of SEQ ID NO: 1; a substitution of the K residue at the position corresponding to position 207 of SEQ ID NO: 1; a substitution of the E residue at the position corresponding to position 215 of SEQ ID NO: 1; a substitution of the A residue at the position corresponding to position 308 of SEQ ID NO: 1; a substitution of the E residue at the position corresponding to position 312 of SEQ ID NO: 1; a substitution of the V residue at the position corresponding to position 329 of SEQ ID NO: 1; a substitution of the G residue at the position corresponding to position 361 of SEQ ID NO: 1; a substitution of the V residue at the position corresponding to position 407 of SEQ ID NO: 1; a substitution of the A residue at the position corresponding to position 431 of SEQ ID NO: 1; a substitution of the L residue at the position corresponding to position 479 of SEQ ID NO: 1; a substitution of the V residue at the position corresponding to position 511 of SEQ ID NO: 1; a substitution of the G residue at the position corresponding to position 516 of SEQ ID NO: 1; a substitution of the N residue at the position corresponding to position 519 of SEQ ID NO: 1; or a combination of any of the foregoing substitutions.

[0013] In one aspect, the invertase has a substitution of the F residue at the position corresponding to position 83 of SEQ ID NO: 3; a substitution of the N residue at the position corresponding to position 340 of SEQ ID NO: 3; a substitution of the P residue at the position corresponding to position 390 of SEQ ID NO: 3; a substitution of the D residue at the position corresponding to position 33 of SEQ ID NO: 3; a substitution of the T residue at the position corresponding to position 51 of SEQ ID NO: 3; a substitution of the E residue at the position corresponding to position 67 of SEQ ID NO: 3; a substitution of the V residue at the position corresponding to position 89 of SEQ ID NO: 3; a substitution of the K residue at the position corresponding to position 170 of SEQ ID NO: 3; a substitution of the D residue at the position corresponding to position 183 of SEQ ID NO: 3; a substitution of the K residue at the position corresponding to position 188 of SEQ ID NO: 3; a substitution of the E residue at the position corresponding to position 196 of SEQ ID NO: 3; a substitution of the A residue at the position corresponding to position 289 of SEQ ID NO: 3; a substitution of the E residue at the position corresponding to position 293 of SEQ ID NO: 3; a substitution of the V residue at the position corresponding to position 310 of SEQ ID NO: 3; a substitution of the G residue at the position corresponding to position 342 of SEQ ID NO: 3; a substitution of the V residue at the position corresponding to position 388 of SEQ ID NO: 3; a substitution of the A residue at the position corresponding to position 412 of SEQ ID NO: 3; a substitution of the L residue at the position corresponding to position 460 of SEQ ID NO: 3; a substitution of the V residue at the position corresponding to position 492 of SEQ ID NO: 3; a substitution of the G residue at the position corresponding to position 497 of SEQ ID NO: 3; a substitution of the N residue at the position corresponding to position 500 of SEQ ID NO: 3; or a combination of any of the foregoing substitutions.

[0014] In one aspect, in the invertase: the F residue at the position corresponding to position 172 of SEQ ID NO:2 is replaced with Y (F172Y); the N residue at the position corresponding to position 429 of SEQ ID NO:2 is replaced with A (N429A); the P residue at the position corresponding to position 479 of SEQ ID NO:2 is replaced with A (P479A); the D residue at the position corresponding to position 122 of SEQ ID NO:2 is replaced with E (D122E); the T residue at the position corresponding to position 140 of SEQ ID NO:2 is replaced with L (T140L); the E residue at the position corresponding to position 156 of SEQ ID NO:2 is replaced with Q (E156Q); the V residue at the position corresponding to position 178 of SEQ ID NO:2 is replaced with I (V178I); the K residue at the position corresponding to position 259 of SEQ ID NO:2 is replaced with L (K259L); the D residue at the position corresponding to position 272 of SEQ ID NO:2 is replaced with N (D272N); the K residue at the position corresponding to position 277 of SEQ ID NO:2 is replaced with T (K277T); the E residue at the position corresponding to position 285 of SEQ ID NO:2 is replaced with H (E285H); the A residue at the position corresponding to position 378 of SEQ ID NO:2 is replaced with T (A378T); the E residue at the position corresponding to position 382 of SEQ ID NO:2 is replaced with Q (E382Q); the V residue at the position corresponding to position 399 of SEQ ID NO:2 is replaced with A (V399A); the G residue at the position corresponding to position 431 of SEQ ID NO:2 is replaced with R (G431R); the V residue at the position corresponding to position 477 of SEQ ID NO:2 is replaced with D (V477D); the A residue at the position corresponding to position 501 of SEQ ID NO:2 is replaced with N (A501N); the L residue at the position corresponding to position 549 of SEQ ID NO:2 is replaced with I (L549I); the V residue at the position corresponding to position 581 of SEQ ID NO:2 is replaced with I (V581I); the G residue at the position corresponding to position 586 of SEQ ID NO:2 is replaced with S (G586S); or the N residue at the position corresponding to position 589 of SEQ ID NO:2 is replaced with K (N589K); or the invertase comprises any combination of the foregoing substitutions. SEQ ID NO:2 is the amino acid sequence of an exemplary invertase enzyme from Saccharomyces cerevisiae that is wild-type but contains a heterologous FAKS signal sequence.Invertases containing the foregoing substitutions at the corresponding positions in SEQ ID NO:1 (wild-type invertase enzyme from Saccharomyces cerevisiae containing a native signal sequence) and SEQ ID NO:3 (wild-type invertase enzyme from Saccharomyces cerevisiae without a signal sequence) are also contemplated herein.

[0015] In one aspect, in the invertase: the F residue at the position corresponding to position 102 of SEQ ID NO:1 is replaced with Y (F102Y); the N residue at the position corresponding to position 359 of SEQ ID NO:1 is replaced with A (N359A); the P residue at the position corresponding to position 409 of SEQ ID NO:1 is replaced with A (P409A); the D residue at the position corresponding to position 52 of SEQ ID NO:1 is replaced with E (D52E); the A residue at the position corresponding to position 53 of SEQ ID NO:1 is replaced with G (A53G); the T residue at the position corresponding to position 70 of SEQ ID NO:1 is replaced with L (T70L); the E residue at the position corresponding to position 86 of SEQ ID NO:1 is replaced with Q (E86Q); the V residue at the position corresponding to position 108 of SEQ ID NO:1 is replaced with I (V108I); the K residue at the position corresponding to position 189 of SEQ ID NO:1 is replaced with L (K189L); the D residue at the position corresponding to position 202 of SEQ ID NO:1 is replaced with N (D202N); the K residue at the position corresponding to position 207 of SEQ ID NO:1 is replaced with T (K207T); the E residue at the position corresponding to position 215 of SEQ ID NO:1 is replaced with H (E215H); the A residue at the position corresponding to position 308 of SEQ ID NO:1 is replaced with T (A308T); the E residue at the position corresponding to position 312 of SEQ ID NO:1 is replaced with Q (E312Q); the V residue at the position corresponding to position 329 of SEQ ID NO:1 is replaced with A (V329A); the G residue at the position corresponding to position 361 of SEQ ID NO:1 is replaced with R (G361R); the V residue at the position corresponding to position 407 of SEQ ID NO:1 is replaced with D (V407D); the A residue at the position corresponding to position 431 of SEQ ID NO:1 is replaced with N (A431N); the L residue at the position corresponding to position 479 of SEQ ID NO:1 is replaced with I (L479I); the V residue at the position corresponding to position 511 of SEQ ID NO:1 is replaced with I (V511I); the N residue at the position corresponding to position 519 of SEQ ID NO:1 is replaced with K (N519K); or the invertase comprises any combination of the foregoing substitutions.

[0016] In one aspect, in the invertase: the F residue at the position corresponding to position 83 of SEQ ID NO: 3 is replaced with Y (F83Y); the N residue at the position corresponding to position 340 of SEQ ID NO: 3 is replaced with A (N340A); the P residue at the position corresponding to position 390 of SEQ ID NO: 3 is replaced with A (P390A); the D residue at the position corresponding to position 33 of SEQ ID NO: 3 is replaced with E (D33E); the T residue at the position corresponding to position 51 of SEQ ID NO: 3 is replaced with L (T51L); the E residue at the position corresponding to position 67 of SEQ ID NO: 3 is replaced with Q (E67Q); the V residue at the position corresponding to position 89 of SEQ ID NO: 3 is replaced with I (V89I); the K residue at the position corresponding to position 170 of SEQ ID NO: 3 is replaced with L (K170L); the D residue at the position corresponding to position 183 of SEQ ID NO: 3 is replaced with N (D183N); the K residue at the position corresponding to position 188 of SEQ ID NO: 3 is replaced with T (K188T); the E residue at the position corresponding to position 196 of SEQ ID NO: 3 is replaced with H (E196H); the A residue at the position corresponding to position 289 of SEQ ID NO: 3 is replaced with T (A289T); the E residue at the position corresponding to position 293 of SEQ ID NO: 3 is replaced with Q (E293Q); the V residue at the position corresponding to position 310 of SEQ ID NO: 3 is replaced with A (V310A); the G residue at the position corresponding to position 342 of SEQ ID NO: 3 is replaced with R (G342R); the V residue at the position corresponding to position 388 of SEQ ID NO: 3 is replaced with D (V388D); the A residue at the position corresponding to position 412 of SEQ ID NO: 3 is replaced with N (A412N); the L residue at the position corresponding to position 460 of SEQ ID NO: 3 is replaced with I (L460I); the V residue at the position corresponding to position 492 of SEQ ID NO: 3 is replaced with I (V492I); the G residue at the position corresponding to position 497 of SEQ ID NO: 3 is replaced with S (G497S); the N residue at the position corresponding to position 500 of SEQ ID NO: 3 is replaced with K (N500K); or the invertase comprises any combination of the foregoing substitutions.

[0017] In one aspect, the invertase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 mutations relative to the corresponding wild-type invertase.

[0018] In certain embodiments, the invertase comprises the following substitutions at positions corresponding to the amino acids set forth in SEQ ID NO:2, such as substitutions of F172Y, N429A, and P479A; substitutions of E120A, D122E, and P479A; substitutions of K259L, S476N, and K514P; substitutions of T140L, E156Q, and L549I; substitutions of S476A, P479A, and Q597E; substitutions of K277T, E415S, and V581I; substitutions of A123G, T140L, and F143Y; substitutions of N390D, S476N, and K519R; substitutions of E382Q, T573S, and V581I; substitutions of D122E, T470S, and S476N; substitutions of T140Q, P479A, and K514P; substitutions of E120A, V178I, and L549I; substitutions of E382Q, S537D, and N589K; substitutions of T140L, G586S, and F596I; or substitutions of E382Q, L459F, and K519R.

[0019] In certain embodiments, the invertase comprises the following substitutions at positions corresponding to the amino acids set forth in SEQ ID NO:1, such as substitutions of E50A, D52E, and P409A; substitutions of T70L, E86Q, and L479I; substitutions of K189L, S406N, and K444P; substitutions of S406A, P409A, and Q527E; substitutions of K207T, E345S, and V511I; substitutions of A53G, T70L, and F73Y; substitutions of N320D, S406N, and K449R; substitutions of E312Q, T503S, and V511I; substitutions of D52E, T400S, and S406N; substitutions of T70Q, P409A, and K444P; substitutions of F102Y, N359A, and P409A; substitutions of E50A, V108I, and L479I; substitutions of E312Q, S467D, and N519K; substitutions of T70L, G516S, and F526I; or substitutions of E312Q, L389F, and K449R.

[0020] In certain embodiments, the invertase has the following substitutions at positions corresponding to the amino acids set forth in SEQ ID NO: 3, for example: substitutions of E31A, D33E, and P390A; substitutions of T51L, E67Q, and L460I; substitutions of K170L, S387N, and K425P; substitutions of S387A, P390A, and Q508E; substitutions of K188T, E326S, and V492I; substitutions of A34G, T51L, and F54Y; substitutions of N301D, S388N, and K430R; substitutions of E293Q, T484S, and V492I; substitutions of D33E, T381S, and S388N; substitutions of T51L, P390A, and K425P; substitutions of F83Y, N340A, and P390A; substitutions of E31A, V89I, and L460I; substitutions of E293Q, S448D, and N500K; substitutions of T51L, G497S, and F507I; or substitutions of E293Q, L370F, and K430R.

[0021] In certain embodiments, the invertase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of 29, 19 - 28, and 30 - 33, or SEQ ID NOs: 29, 19 - 28, and 30 - 33.

[0022] In certain embodiments, the invertase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 29 or SEQ ID NO: 29.

[0023] In certain embodiments, the invertase comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences of SEQ ID NO: 14, 4 - 13, and 15 - 18, or SEQ ID NOs: 14, 4 - 13, and 15 - 18.

[0024] In one aspect, the present disclosure relates to a recombinant mutant S. cerevisiae invertase enzyme comprising a substitution or combination of substitutions listed in Table 1 or Table 4.

[0025] In certain embodiments, the invertase has a specific activity of at least 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900 or 2,000 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 3.5. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 3.5 compared to the corresponding wild-type invertase.

[0026] In certain embodiments, the invertase has a specific activity of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100 or 2,200 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 5.0. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 5.0 compared to the corresponding wild-type invertase.

[0027] In certain embodiments, the invertase has a specific activity of at least 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700 or 1,800 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 6.0 or 6.2. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 6.0 or 6.2 compared to the corresponding wild-type invertase.

[0028] In certain embodiments, the invertase has a specific activity of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200 or 1,300 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 7.0 or 7.1. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 7.0 or 7.1 compared to the corresponding wild-type invertase.

[0029] In certain embodiments, the invertase retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of its activity after incubation at about pH 2.5 for about 30 minutes. In certain embodiments, the invertase has a stability that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold or 5-fold higher at about pH 2.5 compared to the corresponding wild-type invertase.

[0030] In certain embodiments, the invertase has a Tm of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 °C. In certain embodiments, the invertase has a Tm that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C higher than the corresponding wild-type invertase.

[0031] In certain embodiments, the invertase has higher stability in the presence of pancreatin or pepsin compared to the corresponding wild-type invertase.

[0032] In another aspect, the present disclosure relates to a nucleic acid encoding the invertase described herein. In another aspect, the present disclosure relates to an expression vector comprising the nucleic acid described herein. In another aspect, the present disclosure relates to a cell comprising the expression vector described herein. In one embodiment, the cell is a P. pastoris cell or an S. cerevisiae cell. In another aspect, the present disclosure relates to a method of producing a recombinant mutant S. cerevisiae invertase enzyme, the method comprising growing the host cell described herein under conditions in which the host cell expresses invertase and purifying the invertase.

[0033] In another aspect, the present disclosure relates to a pharmaceutical composition comprising the invertase described herein and a pharmaceutically acceptable carrier and / or excipient. Depending on the circumstances, the invertase can be spray-dried.

[0034] In another aspect, the present disclosure relates to a pharmaceutical composition comprising an invertase enzyme, such as a recombinant mutant invertase described herein; a separate isomaltase enzyme; and a pharmaceutically acceptable carrier and / or excipient. Depending on the circumstances and the pharmaceutical composition for the given purpose, the isomaltase is spray-dried, the invertase is spray-dried, or both the isomaltase and the invertase are spray-dried.

[0035] In certain embodiments of the pharmaceutical compositions disclosed herein, (i) the invertase is a microbial invertase (e.g., from Saccharomyces cerevisiae) or a functional fragment or variant thereof, (ii) the invertase comprises any of the sequences of SEQ ID NOs: 29, 14, 1-13, 15-28, and 30-33 or a functional fragment or variant thereof, (iii) the invertase is a recombinant mutant S. cerevisiae invertase as described herein, (iv) the isomaltase is a microbial isomaltase (e.g., from Saccharomyces cerevisiae or Lactobacillus fermentum or a functional fragment or variant of each thereof), (v) the isomaltase comprises any of SEQ ID NOs: 37-41 or a functional fragment or variant thereof, (vi) the isomaltase comprises the sequence of SEQ ID NO: 47, (vii) the isomaltase has substitutions at positions corresponding to the amino acid sequence set forth in SEQ ID NO: 47, such as substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560V; substitutions of E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L; substitutions of E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L; substitutions of E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D, and F560V; substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L; substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D, and F560V; substitutions of E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V; substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D, and F560L; substitutions of K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560V;Substitutions of K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D, and F560V; Substitutions of T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560L; Substitutions of K115I, A211E, D226S, I421A, A444G, A531D, and F560V; Substitutions of K115I, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L; Substitutions of K115I, A211E, D226S, I421A, A444G, E524Q, A531D, and F560L; Substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L; Substitutions of E93K, K115I, D226S, E310A, I421A, A444G, A531D, and F560L; Substitutions of K115I, D226S, E310A, I421A, A444G, A531D, and F560V; Substitutions of E93K, K115I, D226S, L366M, I421A, A444G, A531D, and F560L; Substitutions of K115I, D226S, I421A, A444G, E524Q, A531D, and F560V;Substitutions of K115I, D226S, E310A, I421A, A444G, E524Q, A531D and F560L; Substitutions of K115I, A211E, D226S, L366M, I421A, A444G, A531D and F560L; Substitutions of K115I, A211E, D226S, E310A, I421A, A444G, A531D and F560L; Substitutions of E93K, K115I, D226S, I421A, A444G, A531D and F560V; Substitutions of E93K, K115I, A211E, D226S, I421A, A444G, A531D and F560L; or Substitutions of K115I, D226S, L366M, I421A, A444G, A531D and F560V, wherein (viii) the isomaltase is a recombinant mutant isomaltase described in U.S. Provisional Patent Application No. 63 / 388,845 filed on July 13, 2022, or (ix) or is a combination of any of the features (i)-(viii). In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 14 or 29 or a functional fragment thereof. In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 49 or 50 or a functional fragment thereof.;

[0036] In certain embodiments, the composition is formulated as an oral dosage form. In certain embodiments, the composition is formulated as a powder, satchel, granule, pellet, micropellet, tablet or mini-tablet. In certain embodiments, the composition is formulated as a powder, satchel or tablet. In certain embodiments, the composition has a shelf life of at least 3, 6, 9, 12, 15, 18, 21, 24, 36, 48, 60, 72, 84, 96, 108 or 120 months at room temperature.

[0037] In another aspect, the present disclosure relates to a method of treating congenital sucrase-isomaltase deficiency (CSID) in a subject in need thereof, the method comprising administering to the subject an enzyme or pharmaceutical composition described herein, such as orally. In certain embodiments, the enzyme or composition is administered to the subject with a meal or snack.

[0038] In another aspect, the present disclosure relates to a method of reducing sucrose and branched (1-6 linked) α-limit dextrin concentrations in a subject, the method comprising administering to the subject an enzyme or pharmaceutical composition described herein, such as orally. In certain embodiments, the method reduces sucrose and branched (1-6 linked) α-limit dextrin concentrations in the subject when measured by a hydrogen breath test, such as a sucrose methane hydrogen breath test or 13 a C-sucrose breath test.

[0039] In another aspect, the present disclosure relates to a method of treating congenital sucrase-isomaltase deficiency (CSID) in a subject in need thereof, the method comprising orally administering to the subject (i) an invertase enzyme and (ii) a separate isomaltase enzyme. In certain embodiments, the invertase is a recombinant mutant S. cerevisiae invertase enzyme described herein. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.

[0040] These and other aspects and features of the invention are set forth in the following detailed description and claims. DETAILED DESCRIPTION

[0041] Detailed Description The present invention is based in part on the discovery of recombinant mutant invertase (sucrase) enzymes that are active in humans and have higher stability and / or activity than naturally occurring enzymes. In particular, the recombinant mutant enzymes of the present invention may exhibit improved pH stability, thermal stability, and / or stability to proteolytic digestion compared to the naturally occurring version of the enzyme. Further, the recombinant mutant enzymes of the present invention may have a higher specific activity than the wild-type invertase (sucrase) enzyme. Additionally, the recombinant mutant enzymes described herein may be suitable for oral administration, potentially safer, more tolerable, and / or more active than commercially available invertase (sucrase) enzymes, considering their enhanced stability. The present invention is also based in part on the discovery of pharmaceutical compositions, such as solid pharmaceutical compositions, comprising invertase (sucrase) enzymes and isomaltase enzymes that are stable at room temperature, enabling easier distribution, storage, and administration to a subject compared to compositions that are unstable at room temperature and / or require refrigeration. In particular, recombinant enzymes and compositions may be used to treat congenital sucrase-isomaltase deficiency (CSID).

[0042] Various features and aspects of the present invention are discussed in detail below. I. Invertase (Sucrase) Enzymes In particular, the present invention provides recombinant mutant invertase (sucrase) enzymes and pharmaceutical compositions comprising recombinant mutant invertase (sucrase) enzymes that are useful for the treatment of disorders such as congenital sucrase-isomaltase deficiency (CSID).

[0043] As used herein, the terms "sucrase" and "invertase" are used interchangeably and refer to an enzyme that can catalyze the hydrolysis of sucrose into fructose and glucose, or a functional fragment thereof. Sucrase and invertase are also referred to as acid invertase, alkaline invertase, β-D-fructofuranosidase, β-fructosidase, β-fructofuranosidase, β-fructofuranoside fructohydrolase, β-fructopyranosidase, β-h-fructosidase, β-invertase, EC 3.2.1.26, exo-β-(2,6)-fructofuranosidase, fructosylinvertase, invertin, glucosucrase, saccharase, and sucrose hydrolase, and unless otherwise indicated, the terms are used interchangeably herein. The term invertase includes variants having one or more amino acid substitutions, deletions or insertions relative to the wild-type invertase sequence and / or fusion proteins or conjugates comprising invertase.

[0044] As used herein, the "functional fragment" of the term invertase refers to a fragment of the full-length naturally occurring invertase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzymatic activity of the corresponding full-length invertase. Invertase enzymatic activity can be assayed by any method known in the art. Exemplary invertase activity assays are described in Bacon (1955) METHODS IN ENZYMOLOGY 1: 258-262, Lever (1972) ANALYTICAL BIOCHEM.47: 273-279, U.S. Patent Application Publication No. 20140250942, the World Wide Web at sigmaaldrich.com / technical-documents / protocols / biology / enzymatic-assay-of-invertase.html and Example 1 herein.

[0045] Exemplary invertase enzymes include invertase enzymes derived from Saccharomyces cerevisiae. The amino acid sequence of an exemplary wild-type invertase enzyme derived from Saccharomyces cerevisiae (including the native signal sequence) is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the exemplary wild-type invertase enzyme derived from Saccharomyces cerevisiae is shown in SEQ ID NO: 35. The amino acid sequence of an exemplary invertase enzyme derived from Saccharomyces cerevisiae (including a heterologous FAKS signal sequence but otherwise wild-type) is shown in SEQ ID NO: 2. The amino acid sequence of an exemplary wild-type invertase enzyme derived from Saccharomyces cerevisiae (without a signal sequence) is shown in SEQ ID NO: 3.

[0046] SEQ ID NO: 1 represents a wild-type S. cerevisiae invertase having a native signal sequence, where the signal sequence is underlined and the mature protein sequence is italicized:

Chemical formula

[0047] SEQ ID NO: 2 represents a wild-type S. cerevisiae invertase having a heterologous FAKS signal sequence, where the signal sequence is underlined and the mature protein sequence is italicized:

Chemical formula

[0048] SEQ ID NO: 3 represents a wild-type S. cerevisiae invertase without a signal sequence:

Chemical formula

[0049] The intended invertase enzyme may include the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; 2 - 10; 3 - 10; 4 - 10; 5 - 10; 6 - 10; 7 - 10; 8 - 10; 9 - 10; 2 - 9; 3 - 9; 4 - 9; 5 - 9; 6 - 9; 7 - 9; 8 - 9; 2 - 8; 3 - 8; 4 - 8; 5 - 8; 6 - 8; 7 - 8; 2 - 6; 3 - 6; 4 - 6; or 5 - 6) mutations, and may include an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0050] Additional exemplary invertase enzymes can be found on the World Wide Web at brenda - enzymes.org / enzyme.php?ecno = 3.2.1.26&onlyTable = Sequence. Additional exemplary invertase enzymes are described in U.S. Patent Application Publication No. 2014 / 0250942.

[0051] In particular, the present invention provides recombinant mutant invertases that are useful, for example, in the treatment of disorders associated with a reduced ability to digest or absorb dietary sucrose and starch.

[0052] In certain embodiments, the invertase comprises (i) increased activity at acidic pH (e.g., about pH 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5) relative to the corresponding wild-type invertase; (ii) increased activity at neutral pH (e.g., about pH 7.0) relative to the corresponding wild-type invertase; (iii) increased stability at acidic pH (e.g., about pH 2.5) relative to the corresponding wild-type invertase; (iv) increased thermal stability relative to the corresponding wild-type invertase; or any combination of features (i), (ii), (iii), or (iv). Exemplary combinations include (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (i), (ii), and (iii); (i), (ii), and (iv); (i), (iii), and (iv); (ii), (iii), and (iv); and (i), (ii), (iii), and (iv).

[0053] In certain embodiments, the recombinant mutant invertase contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; 2 - 10, 3 - 10, 4 - 10, 5 - 10, 6 - 10, 7 - 10, 8 - 10, 9 - 10; 2 - 9, 3 - 9, 4 - 9, 5 - 9, 6 - 9, 7 - 9, 8 - 9; 2 - 8, 3 - 8, 4 - 8, 5 - 8, 6 - 8, 7 - 8; 2 - 6, 3 - 6, 4 - 6, or 5 - 6) mutation at a position corresponding to the wild - type S. cerevisiae invertase of SEQ ID NO:2. In certain embodiments, at least one mutation is a substitution of a residue at a position corresponding to position 122 of SEQ ID NO:2; a substitution of the T residue at a position corresponding to position 140 of SEQ ID NO:2; a substitution of the E residue at a position corresponding to position 156 of SEQ ID NO:2; a substitution of the F residue at a position corresponding to position 172 of SEQ ID NO:2; a substitution of the V residue at a position corresponding to position 178 of SEQ ID NO:2; a substitution of the K residue at a position corresponding to position 259 of SEQ ID NO:2; a substitution of the D residue at a position corresponding to position 272 of SEQ ID NO:2; a substitution of the K residue at a position corresponding to position 277 of SEQ ID NO:2; a substitution of the E residue at a position corresponding to position 285 of SEQ ID NO:2; a substitution of the A residue at a position corresponding to position 378 of SEQ ID NO:2; a substitution of the E residue at a position corresponding to position 382 of SEQ ID NO:2; a substitution of the V residue at a position corresponding to position 399 of SEQ ID NO:2; a substitution of the G residue at a position corresponding to position 431 of SEQ ID NO:2; a substitution of the V residue at a position corresponding to position 477 of SEQ ID NO:2; a substitution of the P residue at a position corresponding to position 479 of SEQ ID NO:2; a substitution of the A residue at a position corresponding to position 501 of SEQ ID NO:2; a substitution of the L residue at a position corresponding to position 549 of SEQ ID NO:2; a substitution of the V residue at a position corresponding to position 581 of SEQ ID NO:2; a substitution of the G residue at a position corresponding to position 586 of SEQ ID NO:2; or a substitution of the N residue at a position corresponding to position 589 of SEQ ID NO:2. In certain embodiments, the recombinant mutant invertase contains any combination of the aforementioned substitutions. SEQ ID NO:2 is the amino acid sequence of an exemplary invertase enzyme from Saccharomyces cerevisiae that is wild - type but contains a heterologous FAKS signal sequence.Invertaases containing the aforementioned substitutions at the corresponding positions in SEQ ID NO:1 (wild-type invertase enzyme from Saccharomyces cerevisiae containing a native signal sequence) and SEQ ID NO:3 (wild-type invertase enzyme from Saccharomyces cerevisiae without a signal sequence) are also contemplated herein.

[0054] In certain embodiments, the invertase comprises a substitution of the D residue at the position corresponding to position 52 of SEQ ID NO:1; a substitution of the T residue at the position corresponding to position 70 of SEQ ID NO:1; a substitution of the E residue at the position corresponding to position 86 of SEQ ID NO:1; a substitution of the F residue at the position corresponding to position 102 of SEQ ID NO:1; a substitution of the V residue at the position corresponding to position 108 of SEQ ID NO:1; a substitution of the K residue at the position corresponding to position 189 of SEQ ID NO:1; a substitution of the D residue at the position corresponding to position 202 of SEQ ID NO:1; a substitution of the K residue at the position corresponding to position 207 of SEQ ID NO:1; a substitution of the E residue at the position corresponding to position 215 of SEQ ID NO:1; a substitution of the A residue at the position corresponding to position 308 of SEQ ID NO:1; a substitution of the E residue at the position corresponding to position 312 of SEQ ID NO:1; a substitution of the V residue at the position corresponding to position 329 of SEQ ID NO:1; a substitution of the G residue at the position corresponding to position 361 of SEQ ID NO:1; a substitution of the V residue at the position corresponding to position 407 of SEQ ID NO:1; a substitution of the P residue at the position corresponding to position 409 of SEQ ID NO:1; a substitution of the A residue at the position corresponding to position 431 of SEQ ID NO:1; a substitution of the L residue at the position corresponding to position 479 of SEQ ID NO:1; a substitution of the V residue at the position corresponding to position 511 of SEQ ID NO:1; a substitution of the G residue at the position corresponding to position 516 of SEQ ID NO:1; or a substitution of the N residue at the position corresponding to position 519 of SEQ ID NO:1. In certain embodiments, the recombinant mutant invertase comprises any combination of the aforementioned substitutions.

[0055] In one aspect, the invertase comprises a substitution of a D residue at a position corresponding to position 33 of SEQ ID NO: 3; a substitution of a T residue at a position corresponding to position 51 of SEQ ID NO: 3; a substitution of an E residue at a position corresponding to position 67 of SEQ ID NO: 3; a substitution of an F residue at a position corresponding to position 83 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 89 of SEQ ID NO: 3; a substitution of a K residue at a position corresponding to position 170 of SEQ ID NO: 3; a substitution of a D residue at a position corresponding to position 183 of SEQ ID NO: 3; a substitution of a K residue at a position corresponding to position 188 of SEQ ID NO: 3; a substitution of an E residue at a position corresponding to position 196 of SEQ ID NO: 3; a substitution of an A residue at a position corresponding to position 289 of SEQ ID NO: 3; a substitution of an E residue at a position corresponding to position 293 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 310 of SEQ ID NO: 3; a substitution of a G residue at a position corresponding to position 342 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 388 of SEQ ID NO: 3; a substitution of a P residue at a position corresponding to position 390 of SEQ ID NO: 3; a substitution of an A residue at a position corresponding to position 412 of SEQ ID NO: 3; a substitution of an L residue at a position corresponding to position 460 of SEQ ID NO: 3; a substitution of a V residue at a position corresponding to position 492 of SEQ ID NO: 3; a substitution of a G residue at a position corresponding to position 497 of SEQ ID NO: 3; or a substitution of an N residue at a position corresponding to position 500 of SEQ ID NO: 3. In one aspect, the recombinant mutant invertase comprises any combination of the aforementioned substitutions.

[0056] In one aspect, the D residue at the position corresponding to position 122 of SEQ ID NO:2 is replaced with E (D122E); the T residue at the position corresponding to position 140 of SEQ ID NO:2 is replaced with L (T140L); the E residue at the position corresponding to position 156 of SEQ ID NO:2 is replaced with Q (E156Q); the F residue at the position corresponding to position 172 of SEQ ID NO:2 is replaced with Y (F172Y); the V residue at the position corresponding to position 178 of SEQ ID NO:2 is replaced with I (V178I); the K residue at the position corresponding to position 259 of SEQ ID NO:2 is replaced with L (K259L); the D residue at the position corresponding to position 272 of SEQ ID NO:2 is replaced with N (D272N); the K residue at the position corresponding to position 277 of SEQ ID NO:2 is replaced with T (K277T); the E residue at the position corresponding to position 285 of SEQ ID NO:2 is replaced with H (E285H); the A residue at the position corresponding to position 378 of SEQ ID NO:2 is replaced with T (A378T); the E residue at the position corresponding to position 382 of SEQ ID NO:2 is replaced with Q (E382Q); the V residue at the position corresponding to position 399 of SEQ ID NO:2 is replaced with A (V399A); the G residue at the position corresponding to position 431 of SEQ ID NO:2 is replaced with R (G431R); the V residue at the position corresponding to position 477 of SEQ ID NO:2 is replaced with D (V477D); the P residue at the position corresponding to position 479 of SEQ ID NO:2 is replaced with A (P479A); the A residue at the position corresponding to position 501 of SEQ ID NO:2 is replaced with N (A501N); the L residue at the position corresponding to position 549 of SEQ ID NO:2 is replaced with I (L549I); the V residue at the position corresponding to position 581 of SEQ ID NO:2 is replaced with I (V581I); the G residue at the position corresponding to position 586 of SEQ ID NO:2 is replaced with S (G586S); or the N residue at the position corresponding to position 589 of SEQ ID NO:2 is replaced with K (N589K); or the invertase comprises any combination of the foregoing substitutions. SEQ ID NO:2 is the amino acid sequence of an exemplary invertase enzyme from Saccharomyces cerevisiae that is wild-type but contains a heterologous FAKS signal sequence.Invertases containing the aforementioned substitutions at the corresponding positions in Accession No.: 1 (wild-type invertase enzyme from Saccharomyces cerevisiae containing a native signal sequence) and Accession No.: 3 (wild-type invertase enzyme from Saccharomyces cerevisiae without a signal sequence) are also contemplated herein.

[0057] In certain embodiments, in the invertase: the D residue at the position corresponding to position 52 of Accession No.: 1 is substituted with E (D52E); the A residue at the position corresponding to position 53 of Accession No.: 1 is substituted with G (A53G); the T residue at the position corresponding to position 70 of Accession No.: 1 is substituted with L (T70L); the E residue at the position corresponding to position 86 of Accession No.: 1 is substituted with Q (E86Q); the F residue at the position corresponding to position 102 of Accession No.: 1 is substituted with Y (F102Y); the V residue at the position corresponding to position 108 of Accession No.: 1 is substituted with I (V108I); the K residue at the position corresponding to position 189 of Accession No.: 1 is substituted with L (K189L); the D residue at the position corresponding to position 202 of Accession No.: 1 is substituted with N (D202N); the K residue at the position corresponding to position 207 of Accession No.: 1 is substituted with T (K207T); the E residue at the position corresponding to position 215 of Accession No.: 1 is substituted with H (E215H); the A residue at the position corresponding to position 308 of Accession No.: 1 is substituted with T (A308T); the E residue at the position corresponding to position 312 of Accession No.: 1 is substituted with Q (E312Q); the V residue at the position corresponding to position 329 of Accession No.: 1 is substituted with A (V329A); the G residue at the position corresponding to position 361 of Accession No.: 1 is substituted with R (G361R); the V residue at the position corresponding to position 407 of Accession No.: 1 is substituted with D (V407D); the P residue at the position corresponding to position 409 of Accession No.: 1 is substituted with A (P409A); the A residue at the position corresponding to position 431 of Accession No.: 1 is substituted with N (A431N); the L residue at the position corresponding to position 479 of Accession No.: 1 is substituted with I (L479I); the V residue at the position corresponding to position 511 of Accession No.: 1 is substituted with I (V511I); the N residue at the position corresponding to position 519 of Accession No.: 1 is substituted with K (N519K); or the invertase contains any combination of the aforementioned substitutions.

[0058] In one aspect, in the invertase: the D residue at the position corresponding to position 33 of SEQ ID NO: 3 is replaced with E (D33E); the T residue at the position corresponding to position 51 of SEQ ID NO: 3 is replaced with L (T51L); the E residue at the position corresponding to position 67 of SEQ ID NO: 3 is replaced with Q (E67Q); the F residue at the position corresponding to position 83 of SEQ ID NO: 3 is replaced with Y (F83Y); the V residue at the position corresponding to position 89 of SEQ ID NO: 3 is replaced with I (V89I); the K residue at the position corresponding to position 170 of SEQ ID NO: 3 is replaced with L (K170L); the D residue at the position corresponding to position 183 of SEQ ID NO: 3 is replaced with N (D183N); the K residue at the position corresponding to position 188 of SEQ ID NO: 3 is replaced with T (K188T); the E residue at the position corresponding to position 196 of SEQ ID NO: 3 is replaced with H (E196H); the A residue at the position corresponding to position 289 of SEQ ID NO: 3 is replaced with T (A289T); the E residue at the position corresponding to position 293 of SEQ ID NO: 3 is replaced with Q (E293Q); the V residue at the position corresponding to position 310 of SEQ ID NO: 3 is replaced with A (V310A); the G residue at the position corresponding to position 342 of SEQ ID NO: 3 is replaced with R (G342R); the V residue at the position corresponding to position 388 of SEQ ID NO: 3 is replaced with D (V388D); the P residue at the position corresponding to position 390 of SEQ ID NO: 3 is replaced with A (P390A); the A residue at the position corresponding to position 412 of SEQ ID NO: 3 is replaced with N (A412N); the L residue at the position corresponding to position 460 of SEQ ID NO: 3 is replaced with I (L460I); the V residue at the position corresponding to position 492 of SEQ ID NO: 3 is replaced with I (V492I); the G residue at the position corresponding to position 497 of SEQ ID NO: 3 is replaced with S (G497S); the N residue at the position corresponding to position 500 of SEQ ID NO: 3 is replaced with K (N500K); or the invertase comprises any combination of the foregoing substitutions.

[0059] In certain embodiments, one or more mutations can be conservative substitutions relative to the wild-type S. cerevisiae invertase of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, where in other certain embodiments, one or more mutations can be non-conservative substitutions relative to the wild-type S. cerevisiae invertase of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. As used herein, the term "conservative substitution" refers to a substitution with an amino acid that is structurally and / or functionally similar.

[0060] In certain embodiments, a given amino acid substitution is by 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 a bulky amino acid based on side chain volume or a smaller amino acid based on side chain volume. Conservative substitutions can also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., the BLOSUM 62 matrix), or the PAM substitution:p matrix (e.g., the PAM 250 matrix). A non-conservative substitution is an amino acid substitution that is not a conservative substitution.

[0061] In certain embodiments, the recombinant mutant invertase enzyme comprises one or more substitutions from Table 1, indicating the positions of the substitutions relative to SEQ ID NO: 1 (wild-type Saccharomyces cerevisiae invertase enzyme, including the native signal sequence), or SEQ ID NO: 2 (Saccharomyces cerevisiae invertase enzyme that includes a heterologous FAKS signal sequence but is otherwise wild-type), or SEQ ID NO: 3 (wild-type Saccharomyces cerevisiae invertase enzyme without a signal sequence).

Table 1

[0062] In certain embodiments, the recombinant mutant invertase comprises at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten or at least eleven) mutation selected from Table 1.

[0063] In certain embodiments, the recombinant mutant invertase comprises at least three substitutions selected from the substitutions of F172Y, N429A and P479A relative to SEQ ID NO:2; the substitutions of E120A, D122E and P479A relative to SEQ ID NO:2; the substitutions of K259L, S476N and K514P relative to SEQ ID NO:2; the substitutions of T140L, E156Q and L549I relative to SEQ ID NO:2; the substitutions of S476A, P479A and Q597E relative to SEQ ID NO:2; the substitutions of K277T, E415S and V581I relative to SEQ ID NO:2; the substitutions of A123G, T140L and F143Y relative to SEQ ID NO:2; the substitutions of N390D, S476N and K519R relative to SEQ ID NO:2; the substitutions of E382Q, T573S and V581I relative to SEQ ID NO:2; the substitutions of D122E, T470S and S476N relative to SEQ ID NO:2; the substitutions of T140Q, P479A and K514P relative to SEQ ID NO:2; the substitutions of E120A, V178I and L549I relative to SEQ ID NO:2; the substitutions of E382Q, S537D and N589K relative to SEQ ID NO:2; the substitutions of T140L, G586S and F596I relative to SEQ ID NO:2; or the substitutions of E382Q, L459F and K519R relative to SEQ ID NO:2. It is understood that the recombinant mutant invertase can include the same group of three substitutions as described above with respect to SEQ ID NO:5, as well as invertases that include the same group of three substitutions with respect to SEQ ID NO:1 or SEQ ID NO:3.

[0064] In one aspect, the invertase comprises the following substitutions at positions corresponding to the amino acids set forth in SEQ ID NO: 1, for example: substitutions of E50A, D52E, and P409A for SEQ ID NO: 1; substitutions of T70L, E86Q, and L479I for SEQ ID NO: 1; substitutions of K189L, S406N, and K444P for SEQ ID NO: 1; substitutions of S406A, P409A, and Q527E for SEQ ID NO: 1; substitutions of K207T, E345S, and V511I for SEQ ID NO: 1; substitutions of A53G, T70L, and F73Y for SEQ ID NO: 1; substitutions of N320D, S406N, and K449R for SEQ ID NO: 1; substitutions of E312Q, T503S, and V511I for SEQ ID NO: 1; substitutions of D52E, T400S, and S406N for SEQ ID NO: 1; substitutions of T70Q, P409A, and K444P for SEQ ID NO: 1; substitutions of F102Y, N359A, and P409A for SEQ ID NO: 1; substitutions of E50A, V108I, and L479I for SEQ ID NO: 1; substitutions of E312Q, S467D, and N519K for SEQ ID NO: 1; substitutions of T70L, G516S, and F526I for SEQ ID NO: 1; or substitutions of E312Q, L389F, and K449R for SEQ ID NO: 1.

[0065] In certain embodiments, the recombinant mutant invertase comprises at least three substitutions selected from the group consisting of: substitutions of E31A, D33E, and P390A relative to SEQ ID NO:3; substitutions of T51L, E67Q, and L460I relative to SEQ ID NO:3; substitutions of K170L, S387N, and K425P relative to SEQ ID NO:3; substitutions of S387A, P390A, and Q508E relative to SEQ ID NO:3; substitutions of K188T, E326S, and V492I relative to SEQ ID NO:3; substitutions of A34G, T51L, and F54Y relative to SEQ ID NO:3; substitutions of N301D, S388N, and K430R relative to SEQ ID NO:3; substitutions of E293Q, T484S, and V492I relative to SEQ ID NO:3; substitutions of D33E, T381S, and S388N relative to SEQ ID NO:3; substitutions of T51L, P390A, and K425P relative to SEQ ID NO:3; substitutions of F83Y, N340A, and P390A relative to SEQ ID NO:3; substitutions of E31A, V89I, and L460I relative to SEQ ID NO:3; substitutions of E293Q, S448D, and N500K relative to SEQ ID NO:3; substitutions of T51L, G497S, and F507I relative to SEQ ID NO:3; or substitutions of E293Q, L370F, and K430R relative to SEQ ID NO:3.

[0066] In certain embodiments, the recombinant mutant invertase comprises substitutions of E120A, D122E, and P479A (relative to SEQ ID NO:2). For example, the recombinant mutant invertase may comprise SEQ ID NO:4, also referred to herein as V6.

[0067] SEQ ID NO:4

Chemical formula

[0068] In certain embodiments, the recombinant mutant invertase comprises substitutions of T140L, E156Q, and L549I relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:5, also referred to herein as V9.

[0069] SEQ ID NO:5 [Chem.]

[0070] In certain embodiments, the recombinant mutant invertase comprises the substitutions S476A, P479A, and Q597E relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:6, also referred to herein as V14.

[0071] SEQ ID NO:6 [Chem.]

[0072] In certain embodiments, the recombinant mutant invertase comprises the substitutions K259L, S476N, and K514P (relative to SEQ ID NO:2). For example, the recombinant mutant invertase may comprise SEQ ID NO:7, also referred to herein as V22.

[0073] SEQ ID NO:7 [Chem.]

[0074] In certain embodiments, the recombinant mutant invertase comprises the substitutions K277T, E415S, and V581I relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:8, also referred to herein as V25.

[0075] SEQ ID NO:8 [Chem.]

[0076] In certain embodiments, the recombinant mutant invertase comprises the substitutions A123G, T140L, and F143Y relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:9, also referred to herein as V32.

[0077] Accession number: 9

Chem.

[0078] In certain embodiments, the recombinant mutant invertase comprises the substitutions N390D, S476N and K519R relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 10, also referred to herein as V38.

[0079] SEQ ID NO: 10

Chem.

[0080] In certain embodiments, the recombinant mutant invertase comprises the substitutions E382Q, T573S and V581I relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 11, also referred to herein as V60.

[0081] SEQ ID NO: 11

Chem.

[0082] In certain embodiments, the recombinant mutant invertase comprises the substitutions D122E, T470S and S476N relative to SEQ ID NO: 2. For example, the recombinant mutant invertase may comprise SEQ ID NO: 12, also referred to herein as V63.

[0083] SEQ ID NO: 12

Chem.

[0084] In certain embodiments, the recombinant mutant invertase comprises the substitutions T140Q, P479A and K514P relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:13, also referred to herein as V70.

[0085] SEQ ID NO:13

Chemical formula

[0086] In certain embodiments, the recombinant mutant invertase comprises the substitutions F172Y, N429A and P479A (relative to SEQ ID NO:2). For example, the recombinant mutant invertase may comprise SEQ ID NO:14, also referred to herein as V78.

[0087] SEQ ID NO:14

Chemical formula

[0088] In certain embodiments, the recombinant mutant invertase comprises the substitutions E120A, V178I and L549I relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:15, also referred to herein as V83.

[0089] SEQ ID NO:15

Chemical formula

[0090] In certain embodiments, the recombinant mutant invertase comprises the substitutions E382Q, S537D and N589K relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:16, also referred to herein as V87.

[0091] SEQ ID NO:16

Chemical formula

[0092] In certain embodiments, the recombinant mutant invertase comprises the substitutions T140L, G586S and F596I relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:17, also referred to herein as V89.

[0093] SEQ ID NO:17

Chemical Structure

[0094] In certain embodiments, the recombinant mutant invertase comprises the substitutions E382Q, L459F and K519R relative to SEQ ID NO:2. For example, the recombinant mutant invertase may comprise SEQ ID NO:18, also referred to herein as V90.

[0095] SEQ ID NO:18

Chemical Structure

[0096] In certain embodiments, the recombinant mutant invertase comprises the substitutions E31A, D33E and P390A (relative to SEQ ID NO:3). For example, the recombinant mutant invertase may comprise SEQ ID NO:19, also referred to herein as V6.

[0097] SEQ ID NO:19

Chemical Structure

[0098] In certain embodiments, the recombinant mutant invertase comprises the substitutions T51L, E67Q and L460I relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:20, also referred to herein as V9.

[0099] SEQ ID NO:20

Chem.

[0100] In one aspect, the recombinant mutant invertase comprises the substitutions S387A, P390A, and Q508E relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 21, also referred to herein as V14.

[0101] SEQ ID NO: 21

Chem.

[0102] In one aspect, the recombinant mutant invertase comprises the substitutions K170L, S387N, and K425P (relative to SEQ ID NO: 3). For example, the recombinant mutant invertase may comprise SEQ ID NO: 22, also referred to herein as V22.

[0103] SEQ ID NO: 22

Chem.

[0104] In one aspect, the recombinant mutant invertase comprises the substitutions K188T, E326S, and V492I relative to SEQ ID NO: 3. For example, the recombinant mutant invertase may comprise SEQ ID NO: 23, also referred to herein as V25.

[0105] SEQ ID NO: 23

Chem.

[0106] In certain embodiments, the recombinant mutant invertase comprises the substitutions A34G, T51L, and F54Y relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:24, also referred to herein as V32.

[0107] SEQ ID NO:24

Chemical formula

[0108] In certain embodiments, the recombinant mutant invertase comprises the substitutions N301D, S388N, and K430R relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:25, also referred to herein as V38.

[0109] SEQ ID NO:25

Chemical formula

[0110] In certain embodiments, the recombinant mutant invertase comprises the substitutions E293Q, T484S, and V492I relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:26, also referred to herein as V60.

[0111] SEQ ID NO:26

Chemical formula

[0112] In certain embodiments, the recombinant mutant invertase comprises the substitutions D33E, T381S, and SN relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:27, also referred to herein as V63.

[0113] SEQ ID NO:27

Chemical formula

[0114] In certain embodiments, the recombinant mutant invertase comprises the substitutions T51Q, P390A, and K425P relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:28, also referred to herein as V70.

[0115] SEQ ID NO:28

Chemical formula

[0116] In certain embodiments, the recombinant mutant invertase comprises the substitutions F83Y, N340A, and P390A (relative to SEQ ID NO:3). For example, the recombinant mutant invertase may comprise SEQ ID NO:29, also referred to herein as V78.

[0117] SEQ ID NO:29

Chemical formula

[0118] In certain embodiments, the recombinant mutant invertase comprises the substitutions E31A, V89I, and L460I relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:30, also referred to herein as V83.

[0119] SEQ ID NO:30

Chemical formula

[0120] In certain embodiments, the recombinant mutant invertase comprises the substitutions E293Q, S448D, and N500K relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:31, also referred to herein as V87.

[0121] SEQ ID NO:31 [Chemical formula]

[0122] In certain embodiments, the recombinant mutant invertase comprises the substitutions T51L, G497S and F507I relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:32, also referred to herein as V89.

[0123] SEQ ID NO:32 [Chemical formula]

[0124] In certain embodiments, the recombinant mutant invertase comprises the substitutions E293Q, L370F and K430R relative to SEQ ID NO:3. For example, the recombinant mutant invertase may comprise SEQ ID NO:33, also referred to herein as V90.

[0125] SEQ ID NO:33 [Chemical formula]

[0126] In certain embodiments, the recombinant mutant invertase comprises a non-wild-type invertase having the F172Y and N429A mutations relative to SEQ ID NO:2 and lacking a signal sequence. For example, the recombinant mutant invertase may comprise Sc_S288C invertase (SEQ ID NO:50) having the F172Y and N429A mutations.

[0127] (SEQ ID NO:50) [Chemical formula]

[0128] In one aspect, the recombinant mutant invertase comprises an amino acid sequence of any one of SEQ ID NOs: 4 to 18 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: 4 to 18.

[0129] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 4. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 11. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12.In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 18.

[0130] In certain embodiments, the recombinant mutant invertase comprises any one of the amino acid sequences of SEQ ID NOs: 19-33, 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: 19-33.

[0131] In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 20. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 21. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 22. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 23. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 24. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 26. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 27.In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 28. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 30, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 31, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 32, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32. In certain embodiments, the recombinant mutant invertase comprises the amino acid sequence of SEQ ID NO: 33, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 33.

[0132] As used herein, percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence after aligning the sequences to introduce gaps, if necessary, to achieve the maximum percent sequence identity. Similarly, percent "identity" between a nucleic acid sequence and a reference sequence is defined as the percentage of nucleotides in the nucleic acid sequence that are identical to the nucleotides in the reference sequence after aligning the sequences to introduce gaps, if necessary, to achieve the maximum percent sequence identity. Alignments for the purpose of determining percent sequence identity (e.g., amino acid sequence identity or nucleic acid sequence identity) can be achieved in a variety of ways within the skill in the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, such as any algorithms needed to achieve a maximum alignment over the entire length of the sequences being compared.

[0133] BLAST (Basic Local Alignment Search Tool) analysis using the algorithms employed 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 the search of sequence similarity. For a discussion of the basic issues in database sequence searches, see Altschul et al., (1994) NATURE GENETICS 6:119-129, which is fully incorporated by reference. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. Search parameters for histogram, description, alignment, expect (i.e., the statistical significance threshold for reporting a match to a database sequence), 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). The four blastn parameters can be adjusted as follows: Q = 10 (gap generation penalty); R = 10 (gap extension penalty); wink = 1 (generate word hits every wink.sup.th position along the query); and gapw = 16 (set the window width within which gapped alignments are generated). Equivalent Blastp parameter settings can be Q = 9; R = 2; wink = 1; and gapw = 32.The search can also be performed using the 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, expect value [real number]: default = 10; -W, word size [integer]: default = 11 for nucleotides / 28 for megablast / 3 for proteins; -y, drop-off (X) for blast extension in bits: default = 20 for blastn / 7 for others; -X, X drop-off value (in bit) for gapped alignment: default = 15 for all programs but not applicable to blastn; and -Z, final X drop-off value (in bit) for gapped alignment: 50 for blastn, 25 for others). ClustalW for pairwise protein alignment can also be used (default parameters may include, for example, Blosum62 matrix and 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 generation penalty) and LEN = 3 (gap extension penalty), and the equivalent settings for protein comparison are GAP = 8 and LEN = 2.

[0134] In certain embodiments, the recombinant mutant invertase has increased stability at acidic pH (e.g., pH 3.0 or 4.0) relative to the corresponding wild-type invertase enzyme. The increased stability at acidic pH can, under certain conditions, allow the recombinant mutant invertase to survive the acidic conditions of the digestive system, particularly the stomach. The increased stability at acidic pH can also, under certain conditions, allow the recombinant mutant invertase to survive in certain foods or beverages, such as fruit juice.

[0135] In certain embodiments, the invertase has a specific activity of at least 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900 or 2,000 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 3.5. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 3.5 compared to the corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100 or 2,200 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 5.0. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 5.0 compared to the corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity of at least 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700 or 1,800 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 6.0 or 6.2. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 6.0 or 6.2 compared to the corresponding wild-type invertase. In certain embodiments, the invertase has a specific activity of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200 or 1,300 μmol of sucrose consumed per minute per milligram of deglycosylated invertase at about pH 7.0 or 7.1. In certain embodiments, the invertase has an activity that is at least 1-fold, 1.5-fold, 2-fold, 2.5-fold or 3-fold higher at about pH 7.0 or 7.1 compared to the corresponding wild-type invertase.In certain embodiments, invertase retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of its activity after incubation at about pH 2.5 for about 30 minutes. In certain embodiments, invertase has at least 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold or 5-fold higher stability at about pH 2.5 compared to the corresponding wild-type invertase.

[0136] Methods for testing the stability and activity of invertase are known in the art and may include, for example, the methods described in Example 1 herein. In certain embodiments, the stability of invertase in low pH is determined by exposing the invertase to a specific pH and monitoring the formation of reducing sugars upon hydrolysis of sucrose using the dinitrosalicylic acid (DNS) colorimetric assay.

[0137] In certain embodiments, the recombinant mutant invertase has increased stability at a higher temperature (e.g., Tm of 56 - 68 °C) compared to the corresponding wild-type invertase enzyme. In certain embodiments, invertase has a Tm of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 °C. In certain embodiments, invertase has a Tm that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C higher than the corresponding wild-type invertase.

[0138] Methods for testing the thermal stability and activity of invertase are known in the art and may include, for example, the methods described in Example 1 herein. In certain embodiments, the stability and activity of invertase at high temperature are determined by treatment of the invertase sample at high temperature prior to assaying the residual activity using the above-described DNS method.

[0139] In certain embodiments, the invertase has increased stability in the presence of a protease (e.g., a serine protease and / or an aspartic protease) relative to the corresponding wild-type invertase enzyme. The increased stability in the presence of a protease can, under certain conditions, allow the recombinant mutant invertase to survive gastric conditions. In certain embodiments, the recombinant mutant invertase has increased stability in the presence of pancreatin or pepsin relative to the corresponding wild-type invertase enzyme.

[0140] In certain embodiments, the disclosed invertase enzyme retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of its activity after incubation with pepsin (e.g., incubation with about 10 mg / ml pepsin in simulated gastric fluid (SGF) at about 37 °C for about 2 hours). In certain embodiments, the disclosed invertase enzyme retains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of its activity after incubation with pancreatin (e.g., incubation with about 3.4 mg / ml pancreatin in simulated intestinal fluid (SIF) at about 37 °C for about 2 hours).

[0141] Methods for testing the proteolytic stability and activity of invertase are known in the art and can include, for example, the methods described in Example 1 herein. In certain embodiments, the stability and activity of invertase in the presence of pepsin or pancreatin are determined by incubation of the invertase with pepsin or pancreatin prior to assaying for residual activity using the DNS method described above.

[0142] II. Isomaltase Enzyme In particular, the present invention provides a pharmaceutical composition optionally further comprising an isomaltase enzyme that is useful for the treatment of disorders such as, for example, congenital sucrase-isomaltase deficiency (CSID).

[0143] As used herein, the term "isomaltase" refers to any enzyme or functional fragment thereof that can catalyze the cleavage of branched (1-6 linked) α-limit dextrin (starch). Isomaltase is also referred to as α-glucosidase, oligo-1,6-glucosidase, and EC 3.2.1.10, and unless otherwise indicated, the term is used interchangeably herein. The term isomaltase includes variants having one or more amino acid substitutions, deletions or insertions relative to the wild-type isomaltase sequence and / or fusion proteins or conjugates comprising isomaltase. As used herein, the "functional fragment" of the term isomaltase refers to a fragment of the full-length naturally occurring isomaltase that retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzyme activity of the corresponding full-length isomaltase. Exemplary isomaltase activity assays are described in Noguchi et al. (2003) J. BIOCHEM. 134(4):543-50 and Schonert et al. (1998) J. BACTERIOL. 180(9):2574-8. Further, since glucose is a product of the enzymatic reaction catalyzed by both invertase and isomaltase, it is understood that assays based on the detection of glucose can be used in certain embodiments to measure the activity of both invertase enzyme and isomaltase enzyme.

[0144] Exemplary isomaltase enzymes include isomaltase enzymes from Saccharomyces cerevisiae. The amino acid sequences of exemplary wild-type isomaltase enzymes from Saccharomyces cerevisiae are shown in SEQ ID NOs: 37-41, and the nucleotide sequences encoding exemplary wild-type isomaltase enzymes from Saccharomyces cerevisiae are shown in SEQ ID NOs: 42-46.

[0145] An exemplary wild-type isomaltase enzyme from L. fermentum is shown in SEQ ID NO: 47.

[0146] Sequence number: 47

Chem.

[0147] Exemplary isomaltase enzyme variants can include amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 47. In certain embodiments, the isomaltase enzyme variant includes at least one amino acid substitution (e.g., substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids) and an amino acid sequence having at least 98%, 98.5%, 99% or 99.5% sequence identity to SEQ ID NO: 47. In certain embodiments, the isomaltase has substitutions at positions corresponding to the amino acid sequence set forth in SEQ ID NO: 47, such as: substitutions of E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D and F560L; substitutions of E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D and F560L; substitutions of E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D and F560V; substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D and F560L; substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D and F560V; substitutions of E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D and F560V; substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D and F560L; substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D and F560V; substitutions of K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D and F560V; substitutions of K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D and F560V; substitutions of E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D and F560V;Substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D and F560V; Substitutions of E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D and F560V; Substitutions of T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D and F560V; Substitutions of E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D and F560V; Substitutions of E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D and F560V; Substitutions of E93K, K115I, D226S, I421A, A444G, E524Q, A531D and F560V; Substitutions of E93K, K115I, D226S, I421A, A444G, E524Q, A531D and F560L; Substitutions of K115I, A211E, D226S, I421A, A444G, A531D and F560V; Substitutions of K115I, D226S, L366M, I421A, A444G, E524Q, A531D and F560L; Substitutions of K115I, A211E, D226S, I421A, A444G, E524Q, A531D and F560L; Substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D and F560L; Substitutions of E93K, K115I, D226S, E310A, I421A, A444G, A531D and F560L; Substitutions of K115I, D226S, E310A, I421A, A444G, A531D and F560V; Substitutions of E93K, K115I, D226S, L366M, I421A, A444G, A531D and F560L; Substitutions of K115I, D226S, I421A, A444G, E524Q, A531D and F560V; Substitutions of K115I, D226S, E310A, I421A, A444G, E524Q, A531D and F560L; Substitutions of K115I, A211E, D226S, L366M, I421A, A444G, A531D and F560L; Substitutions of K115I, A211E, D226S, E310A, I421A, A444G, A531D and F560L;Substitutions of E93K, K115I, D226S, I421A, A444G, A531D and F560V; substitutions of E93K, K115I, A211E, D226S, I421A, A444G, A531D and F560L; or substitutions of K115I, D226S, L366M, I421A, A444G, A531D and F560V.

[0148] Additional exemplary isomaltase enzymes can be found on the World Wide Web at brenda - enzymes.org / enzyme.php?ecno=3.2.1.10&onlyTable=Sequence.

[0149] The isomaltase enzymes contemplated can include the amino acid sequence of any of SEQ ID NOs: 37 - 41, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOs: 42 - 46. Other isomaltase enzymes that can be used in combination with the recombinant invertase enzymes described herein are described in U.S. Provisional Patent Application No. 63 / 388,845, filed on July 13, 2022.

[0150] In certain embodiments, the isomaltase enzyme includes one or more conservative substitutions with respect to the isomaltase enzymes disclosed herein. In other embodiments, the isomaltase enzyme includes one or more non - conservative substitutions with respect to the isomaltase enzymes disclosed herein.

[0151] In certain embodiments, the composition includes (i) an invertase comprising the amino acid sequence of SEQ ID NO: 14 (variant V78 having the FAKS signal sequence) or SEQ ID NO: 29 (variant 78 without a signal sequence) and (ii) one or more isomaltase enzymes described in this Section II.

[0152] In one aspect, the composition comprises (i) an invertase comprising the amino acid sequence of SEQ ID NO: 49 (Sc_S288C invertase having F172Y and N429A mutations relative to SEQ ID NO: 2 and having a wild-type signal sequence) or SEQ ID NO: 50 (Sc_S288C invertase having F172Y and N429A mutations relative to SEQ ID NO: 2 and having no signal sequence) and (ii) one or more isomaltase enzymes described in this Section II.

[0153] III. Enzyme production Methods for producing invertase and / or isomaltase enzymes are known in the art. For example, DNA molecules encoding invertase and / or isomaltase enzymes can be chemically synthesized using the sequence information provided herein. The synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, such as expression control sequences, to generate conventional gene expression constructs encoding the desired invertase and / or isomaltase enzymes.

[0154] Nucleic acids encoding the desired invertase and / or isomaltase enzymes can be incorporated (ligated) into an expression vector and introduced into host cells via conventional transfection or transformation techniques. The transformed host cells can be grown under conditions that cause the host cells to express the gene encoding the invertase and / or isomaltase enzyme.

[0155] An exemplary DNA sequence encoding wild-type S. cerevisiae invertase is shown in SEQ ID NO: 35.

[0156] SEQ ID NO: 35 (wild-type S. cerevisiae invertase DNA sequence):

Chemical formula

[0157] An exemplary DNA sequence encoding wild-type L. fermentum isomaltase is provided in SEQ ID NO: 48.

[0158] Accession number: 48 (wild-type L. fermentum isomaltase DNA sequence):

Chem.

[0159] Nucleic acids encoding recombinant mutant invertase and / or isomaltase can be prepared by mutating the nucleotide sequence encoding wild-type invertase, such as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or wild-type isomaltase, such as SEQ ID NOs: 37-41, using methods known in the art. SEQ ID NOs: 1 and 2 show isomaltase protein sequences containing an N-terminal signal sequence that can be removed after translation. SEQ ID NO: 3 shows the corresponding mature isomaltase protein without an -N-terminal signal sequence. Further, nucleic acids encoding such recombinant invertase and / or isomaltase enzymes can be codon-optimized for expression in heterologous cells, such as E. coli, Saccharomyces cerevisiae or Pichia pastoris cells, using methods known in the art.

[0160] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: E120A, D122E and P479A with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated V6 herein.

[0161] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: T140L, E156Q and L549I with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated V9 herein.

[0162] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: S476A, P479A, and Q597E with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated as V14 herein.

[0163] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: K259L, S476N, and K514P with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated as V22 herein.

[0164] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: K277T, E415S, and V581I with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated as V25 herein.

[0165] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: A123G, T140L, and F143Y with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated as V32 herein.

[0166] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: N390D, S476N, and K519R with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated as V38 herein.

[0167] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: E382Q, T573S, and V581I with respect to SEQ ID NO: 2, for example, the recombinant mutant invertase designated as V60 herein.

[0168] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: D122E, T470S, and S476N with respect to SEQ ID NO:2, for example, the recombinant mutant invertase designated V63 herein.

[0169] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: T140Q, P479A, and K514P with respect to SEQ ID NO:2, for example, the recombinant mutant invertase designated V70 herein.

[0170] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: F172Y, N429A, and P479A with respect to SEQ ID NO:2, for example, the recombinant mutant invertase designated V78 herein.

[0171] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: E120A, V178I, and L549I with respect to SEQ ID NO:2, for example, the recombinant mutant invertase designated V83 herein.

[0172] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: E382Q, S537D, and N589K with respect to SEQ ID NO:2, for example, the recombinant mutant invertase designated V87 herein.

[0173] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: T140L, G586S, and F596I with respect to SEQ ID NO:2, for example, the recombinant mutant invertase designated V89 herein.

[0174] In one aspect, the present disclosure relates to a nucleotide sequence encoding a recombinant mutant invertase comprising the following substitutions: E382Q, L459F and K519R with respect to the recombinant mutant invertase of SEQ ID NO: 2, for example referred to as V90 herein.

[0175] Specific expression and purification conditions will vary depending on the expression system used. For example, when expressing the gene in E. coli, S. cerevisiae or P. pastoris, the gene can be cloned into an expression vector by placing the engineered gene downstream of an appropriate bacterial or yeast promoter, such as Trp or Tac, and a prokaryotic or eukaryotic signal sequence. In yeast, for higher expression, it may be advantageous to introduce the coding gene into the yeast genome (e.g., by homologous recombination). The expressed protein can accumulate in inclusion bodies or refractile bodies or be secreted and recovered by collecting the supernatant when secreted or after cell disruption by French press or sonication. The inclusion bodies are then solubilized and the protein refolded and cleaved by methods known in the art. Secreted proteins can be further purified using methods known in the art, such as ion exchange, affinity chromatography or salt precipitation.

[0176] Invertase and / or isomaltase enzymes can be produced by growing (culturing) host cells transfected with an expression vector encoding such invertase and / or isomaltase enzymes under conditions that allow for the expression of the invertase and / or isomaltase enzymes. After expression, the invertase and / or isomaltase enzymes can be recovered, purified or isolated using techniques known in the art, such as affinity tags like glutathione-S-transferase (GST) and histidine tags.

[0177] Exemplary methods for the recombinant expression and purification of invertase enzyme are described in Mohandesi et al. (2016) BIOTECH 6(2):129. Exemplary methods for the recombinant expression and purification of isomaltase enzyme are described in Deng et al. (2014) FEBS OPEN BIO 4:200-212.

[0178] In certain embodiments, the invertase and / or isomaltase enzyme is dried, for example by spray drying. Pharmaceutical proteins can be dried in many ways, for example by drying using N2, air or an inert gas, vacuum oven drying, lyophilization, washing with a volatile organic solvent followed by evaporation of the solvent, evaporation in a fume hood, tray drying, fluidized bed drying, spray drying, vacuum drying or roller drying, etc., by removal of water, an organic solvent or a liquid polymer.

[0179] By spray drying the invertase and / or isomaltase enzyme, water is separated from the invertase and / or isomaltase enzyme preparation, enabling the continuous production of dry solids in the form of powders, granules or agglomerates from liquid feeds such as emulsions and pumpable suspensions. Spray drying involves the atomization of a liquid feed containing the invertase and / or isomaltase enzyme into droplets and the contact of the droplets with hot air or gas in a drying chamber. The atomization process can be carried out using a two-fluid atomizer that mixes the liquid feedstock with a drying gas, such as compressed air or nitrogen. The operating conditions and dryer design are selected according to the drying characteristics of the invertase and / or isomaltase enzyme and the desired powder quality. Exemplary methods for spray drying enzymes are described in US Patent Application Publication No. 2015 / 0353913. The invertase and isomaltase enzymes can be spray dried separately or together.

[0180] The invertase and / or isomaltase enzymes disclosed are contemplated to be capable of being modified, genetically engineered or chemically conjugated. For example, it is contemplated that the invertase and / or isomaltase enzymes disclosed can be conjugated to effector agents using standard in vitro conjugation chemistries. When the effector agent is a polypeptide, the invertase and / or isomaltase enzyme can be chemically conjugated to the effector or linked to the effector as a fusion protein. Construction of fusion proteins is within the scope of ordinary skill in the art.

[0181] In certain embodiments, depending on the particular dosage form or site of activity, the invertase and / or isomaltase enzymes disclosed can be modified by moieties that improve their stabilization and / or retention in circulation, such as in blood, serum or other tissues. For example, the invertase and / or isomaltase enzymes disclosed can be conjugated to polymers, such as substantially non-antigenic polymers, such as polyalkylene oxides or polyethylene oxides. In certain embodiments, the invertase and / or isomaltase enzymes disclosed are conjugated to water-soluble polymers, such as hydrophilic polyvinyl polymers, such as polyvinyl alcohol or polyvinyl pyrrolidone. Examples of such polymers include polyalkylene oxide homopolymers, such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof and block copolymers thereof. Further useful polymers include polyoxyalkylene, such as polyoxyethylene, polyoxypropylene and block copolymers of polyoxyethylene and polyoxypropylene, polymethacrylate, carbomer and branched or unbranched polysaccharides.

[0182] IV. Pharmaceutical Compositions For therapeutic uses, the invertase and / or isomaltase enzymes described herein are preferably combined with a pharmaceutically acceptable carrier and / or excipient. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications and which provide a reasonable benefit / risk ratio without having excessive toxicity, irritation, allergic response or other problems or complications.

[0183] As used herein, the term "pharmaceutically acceptable carrier" refers to buffers, carriers and excipients which are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications and which provide a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers such as phosphate buffered saline aqueous solutions, water, emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic agents and absorption delaying agents which are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0184] In another aspect, the present disclosure relates to a pharmaceutical composition comprising an invertase enzyme, such as a recombinant mutant invertase described herein; another isomaltase enzyme, and a pharmaceutically acceptable carrier and / or excipient. In certain embodiments, the invertase and / or isomaltase is spray dried.

[0185] In certain embodiments of the pharmaceutical compositions disclosed herein, (i) the invertase is a microbial invertase (e.g., from Saccharomyces cerevisiae) or a functional fragment or variant thereof, (ii) the invertase comprises a sequence of any of SEQ ID NOs: 1 - 33 or a functional fragment or variant thereof, (iii) the invertase is a recombinant mutant S. cerevisiae invertase as described herein, (iv) the isomaltase is a microbial isomaltase (e.g., from S. cerevisiae or L. fermentum or a functional fragment or variant of any of the foregoing), (v) the isomaltase comprises any of SEQ ID NOs: 37 - 41 or a functional fragment or variant thereof, (vi) the isomaltase comprises the sequence of SEQ ID NO: 47, (vii) the isomaltase has substitutions at positions corresponding to the amino acid sequence set forth in SEQ ID NO: 47, such as: substitutions of E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L; substitutions of E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560L; substitutions of E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D, and F560V; substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L; substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D, and F560V; substitutions of E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V; substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D, and F560L; substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560V; substitutions of K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D, and F560V;Substitutions of K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D, and F560V; Substitutions of T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560V; Substitutions of E93K, K115I, D226S, I421A, A444G, E524Q, A531D, and F560L; Substitutions of K115I, A211E, D226S, I421A, A444G, A531D, and F560V; Substitutions of K115I, D226S, L366M, I421A, A444G, E524Q, A531D, and F560L; Substitutions of K115I, A211E, D226S, I421A, A444G, E524Q, A531D, and F560L; Substitutions of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D, and F560L; Substitutions of E93K, K115I, D226S, E310A, I421A, A444G, A531D, and F560L; Substitutions of K115I, D226S, E310A, I421A, A444G, A531D, and F560V; Substitutions of E93K, K115I, D226S, L366M, I421A, A444G, A531D, and F560L; Substitutions of K115I, D226S, I421A, A444G, E524Q, A531D, and F560V;Substitutions of K115I, D226S, E310A, I421A, A444G, E524Q, A531D and F560L; Substitutions of K115I, A211E, D226S, L366M, I421A, A444G, A531D and F560L; Substitutions of K115I, A211E, D226S, E310A, I421A, A444G, A531D and F560L; Substitutions of E93K, K115I, D226S, I421A, A444G, A531D and F560V; Substitutions of E93K, K115I, A211E, D226S, I421A, A444G, A531D and F560L; or Substitutions of K115I, D226S, L366M, I421A, A444G, A531D and F560V, wherein (viii) the isomaltase is a recombinant mutant isomaltase described in U.S. Provisional Application No. 63 / 388,845, filed on July 13, 2022, or (ix) is any combination of features (i)-(viii). In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 14 or 29 or a functional fragment thereof. In certain embodiments, the invertase comprises the amino acid sequence of SEQ ID NO: 49 or 50 or a functional fragment thereof.;

[0186] In certain embodiments, the composition is formulated as an oral dosage form. In certain embodiments, the composition is formulated as a powder, capsule, granule, pellet, micropellet, tablet or mini-tablet. In certain embodiments, the composition is formulated as a powder, capsule or tablet. In certain embodiments, the composition has a shelf life of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months or 120 months at room temperature.

[0187] In certain embodiments, the invertase and / or isomaltase enzyme can be formulated with or together with a pH increasing agent, such as a protein pump inhibitor (PPI), and co-administered (either simultaneously or sequentially), for example, via an intestinal route (e.g., orally), to enhance the stability of the invertase and / or isomaltase enzyme in, for example, an acidic environment, such as in the gastrointestinal tract.

[0188] Proton pump inhibitors are a group of drugs whose main action is a marked and long - lasting reduction in gastric acid production. Proton pump inhibitors act by blocking the hydrogen / potassium adenosine triphosphatase enzyme system (H + / K + ATPase, or more commonly just the gastric proton pump) of the gastric parietal cells. The proton pump is the final step in gastric acid secretion and is the direct cause of the secretion of H + ions into the gastric lumen, 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®).

[0189] The pharmaceutical compositions containing the recombinant invertase and / or isomaltase enzymes disclosed herein may be present in unit dosage forms and may be prepared by any suitable method. The pharmaceutical compositions should be formulated to be compatible with their intended route of administration. The pharmaceutical compositions can be in various forms. For example, these include liquid solutions, dispersions or suspensions, tablets, pills, powders, liposomes and suppositories, such as liquid, semi - solid and solid dosage forms. The preferred form depends on the intended mode of administration and therapeutic application. In one aspect, the composition is formulated as an oral dosage form. The oral dosage form can be formulated, for example, as a powder, capsule, granule, pellet, micropellet, tablet or mini - tablet. In some aspects, the composition is formulated as a powder, capsule or tablet.

[0190] The composition is preferably formulated for enteral (e.g., oral) administration, but such a composition can be administered by a parenteral mode (e.g., intravenous, subcutaneous, intraperitoneal or intramuscular injection). The terms "parenteral administration" and "administered parenterally" as used herein generally mean a mode of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural and substeral injections and infusions.

[0191] The composition can be formulated as a solution, microemulsion, dispersant, liposome or other regular structure suitable for stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating the agents described herein in the required amounts in a suitable solvent, along with one or a combination of the ingredients enumerated above if necessary, and then filtering sterilizing. Generally, dispersants are prepared by incorporating the agents described herein into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients derived from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and lyophilization which yield the powder of the agents described herein and any further desired ingredients from its previously sterile filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, in the case of dispersants by maintaining the required particle size and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as monostearate and gelatin.

[0192] Depending on the mode of administration, e.g., by parenteral administration, it may be desirable to produce a pharmaceutical formulation that is sterile. Sterility can be achieved by any suitable method, e.g., filtration through a sterile filtration membrane. If the composition is lyophilized, filter sterilization can be carried out before or after lyophilization and reconstitution.

[0193] In certain embodiments, the disclosed compositions include a polyionic reagent that can coat, for example, invertase and / or isomaltase enzymes (e.g., the composition includes a polyionic coating). Exemplary polyionic reagents include PSS (poly(sodium 4-styrenesulfonate)), PAA (sodium polyacrylate salt), PMG (poly(methylene-co-guanidine) hydrochloride), DS (dextran sulfate), PMA (poly(methyl acrylate)) or PVS (polyvinyl siloxane).

[0194] In certain embodiments, the disclosed compositions and / or dosages are: (i) from about 750 to about 75,000, from about 750 to about 60,000, from about 750 to about 45,000, from about 750 to about 30,000, from about 750 to about 15,000, from about 750 to about 10,000, from about 750 to about 7500, from about 750 to about 5,000, from about 750 to about 2500, from about 750 to about 1,000, from about 1,000 to about 75,000, from about 1,000 to about 60,000, from about 1,000 to about 45,000, from about 1,000 to about 30,000, from about 1,000 to about 15,000, from about 1,000 to about 10,000, from about 1,000 to about 7,500, from about 1,000 to about 5,000, from about 2,500 to about 75,000, from about 2,500 to about 60,000, from about 2,500 to about 45,000, from about 2,500 to about 30,000, from about 2,500 to about 15,000, from about 2,500 to about 10,000, from about 2,500 to about 7,500, from about 2,500 to about 5,000, from about 5,000 to about 75,000, from about 5,000 to about 60,000, from about 5,000 to about 45,000, from about 5,000 to about 30,000, from about 5,000 to about 15,000, from about 5,000 to about 10,000, from about 5,000 to about 7,500, from about 7,500 to about 75,000, from about 7,500 to about 60,000, from about 7,500 to about 45,000, from about 7,500 to about 30,000, from about 7,500 to about 15,000, from about 7,500 to about 10,000, from about 10,000 to about 75,000, from about 10,000 to about 60,000, from about 10,000 to about 45,000, from about 10,000 to about 30,000, from about 10,000 to about 15,000, from about 15,000 to about 75,000, from about 15,000 to about 60,000, from about 15,000 to about 45,000, from about 15,000 to about 30,000, from about 30,000 to about 75,000, from about 30,000 to about 60,000, from about 30,000 to about 45,000, from about 45,000 to about 75,000, from about 45,000 to about 60,000 or from about 60,000 to about 75,000 international units (I.U.) invertase enzyme; and / or (ii) about 750 to about 75,000, about 750 to about 60,000, about 750 to about 45,000, about 750 to about 30,000, about 750 to about 15,000, about 750 to about 10,000, about 750 to about 7,500, about 750 to about 5,000, about 750 to about 2,500, about 750 to about 1,000, about 1,000 to about 75,000, about 1,000 to about 60,000, about 1,000 to about 45,000, about 1,000 to about 30,000, about 1,000 to about 15,000, about 1,000 to about 10,000, about 1,000 to about 7,500, about 1,000 to about 5,000, about 1,000 to about 2,500, about 2,500 to about 75,000, about 2,500 to about 60,000, about 2,500 to about 45,000, about 2,500 to about 30,000, about 2,500 to about 15,000, about 2,500 to about 10,000, about 2,500 to about 7,500, about 2,500 to about 5,000, about 5,000 to about 75,000, about 5,000 to about 60,000, about 5,000 to about 45,000, about 5,000 to about 30,000, about 5,000 to about 15,000, about 5,000 to about 10,000, about 5,000 to about 7,500, about 7,500 to about 75,000, about 7,500 to about 60,000, about 7,500 to about 45,000, about 7,500 to about 30,000, about 7,500 to about 15,000, about 7,500 to about 10,000, about 10,000 to about 75,000, about 10,000 to about 60,000, about 10,000 to about 45,000, about 10,000 to about 30,000, about 10,000 to about 15,000, about 15,000 to about 75,000, about 15,000 to about 60,000, about 15,000 to about 45,000, about 15,000 to about 30,000, about 30,000 to about 75,000, about 30,000 to about 60,000, about 30,000 to about 45,000, about 45,000 to about 75,000, about 45,000 to about 60,000 or about 60,000 to about 75,000 international units (I.U.) of isomaltase enzyme.

[0195] In certain embodiments, the disclosed compositions and / or dosages are: (i) about 5,000 to about 12,000, about 5,000 to about 11,000, about 5,000 to about 10,000, about 5,000 to about 9,000, about 5,000 to about 8,000, about 5,000 to about 7,000, about 5,000 to about 6,000, about 6,000 to about 12,000, about 6,000 to about 11,000, about 6,000 to about 10,000, about 6,000 to about 9,000, about 6,000 to about 8,000, about 6,000 to about 7,000, about 7,000 to about 12,000, about 7,000 to about 11,000, about 7,000 to about 10,000, about 7,000 to about 9,000, about 7,000 to about 8,000, about 8,000 to about 12,000, about 8,000 to about 11,000, about 8,000 to about 10,000, about 8,000 to about 9,000, about 9,000 to about 12,000, about 9,000 to about 11,000, about 9,000 to about 10,000, about 10,000 to about 12,000, about 10,000 to about 11,000, about 11,000 to about 12,000, about 5,000, about 6,000, about 7,000, about 8,000, about 8,500, about 9,000, about 10,000, about 11,000 or about 12,000 International Units (I.U.(i) an invertase enzyme; and / or (ii) an isomaltase enzyme in an amount of about 5,000 to about 12,000, about 5,000 to about 11,000, about 5,000 to about 10,000, about 5,000 to about 9,000, about 5,000 to about 8,000, about 5,000 to about 7,000, about 5,000 to about 6,000, about 6,000 to about 12,000, about 6,000 to about 11,000, about 6,000 to about 10,000, about 6,000 to about 9,000, about 6,000 to about 8,000, about 6,000 to about 7,000, about 7,000 to about 12,000, about 7,000 to about 11,000, about 7,000 to about 10,000, about 7,000 to about 9,000, about 7,000 to about 8,000, about 8,000 to about 12,000, about 8,000 to about 11,000, about 8,000 to about 10,000, about 8,000 to about 9,000, about 9,000 to about 12,000, about 9,000 to about 11,000, about 9,000 to about 10,000, about 10,000 to about 12,000, about 10,000 to about 11,000, about 11,000 to about 12,000, about 5,000, about 6,000, about 7,000, about 8,000, about 8,500, about 9,000, about 10,000, about 11,000 or about 12,000 International Units (I.U.).

[0196] In certain embodiments, the disclosed invertase and / or isomaltase enzyme or composition is administered to a subject with a meal or a snack. In certain embodiments, the disclosed invertase and / or isomaltase enzyme or composition is administered to a subject with each meal or snack that the subject eats. In certain embodiments, the disclosed invertase and / or isomaltase enzyme or composition is administered to a subject once a week, once every six days, once every five days, once every four days, once every three days, once every two days, once a day, twice a day, three times a day, four times a day, five times a day, six times a day or more than six times a day.

[0197] Depending on the situation, the composition can be formulated as a powder, granule, pellet, micropellet or mini-tablet. The composition can be encapsulated in a capsule, such as a hydroxypropylmethylcellulose (HPMC) capsule, soft gelatin capsule or hard gelatin capsule. Alternatively, the composition can be formulated as a tablet dosage form. Dosage forms, such as granules, tablets, mini-tablets, pellets, micropellets, capsules, can be enteric-coated using, for example, one or more methacrylic acid polymers, such as methyl methacrylate methacrylate copolymers (e.g., Eudragit L and S) and acrylic acid ethyl methacrylate copolymers (Eudragit L30D), hydroxylpropyl methyl acetate cellulose, hydroxylpropyl phthalate methyl succinate cellulose and others.

[0198] In certain embodiments, the composition comprises less than 50%, less than 40%, less than 30%, less than 20%, less than 10% or less than 5% glycerol. In certain embodiments, the composition is free of glycerol.

[0199] In certain embodiments, the disclosed pharmaceutical compositions have a shelf life of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 months, 60 months, 72 months, 84 months, 96 months, 108 months or 120 months at room temperature (e.g., retaining at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of their biological activity). In certain embodiments, the disclosed pharmaceutical compositions have a shelf life of about 3 to about 120 months, about 3 to about 96 months, about 3 to about 72 months, about 3 to about 48 months, about 3 to about 24 months, about 3 to about 21 months, about 3 to about 18 months, about 3 to about 15 months, about 3 to about 12 months, about 3 to about 9 months, about 3 to about 6 months, about 6 to about 120 months, about 6 to about 96 months, about 6 to about 72 months, about 6 to about 48 months, about 6 to about 24 months, about 6 to about 21 months, about 6 to about 18 months, about 6 to about 15 months, about 6 to about 12 months, about 6 to about 9 months, about 9 to about 120 months, about 9 to about 96 months, about 9 to about 72 months, about 9 to about 48 months, about 9 to about 24 months, about 9 to about 21 months, about 9 to about 18 months, about 9 to about 15 months, about 9 to about 12 months, about 12 to about 120 months, about 12 to about 96 months, about 12 to about 72 months, about 12 to about 48 months, about 12 to about 24 months, about 12 to about 21 months, about 12 to about 18 months, about 12 to about 15 months, about 15 to about 120 months, about 15 to about 96 months, about 15 to about 72 months, about 15 to about 48 months, about 15 to about 24 months, about 15 to about 21 months, about 15 to about 18 months, about 18 to about 120 months, about 18 to about 96 months, about 18 to about 72 months, about 18 to about 48 months, about 18 to about 24 months, about 18 to about 21 months, about 21 to about 120 months, about 21 to about 96 months, about 21 to about 72 months, about 21 to about 48 months, about 21 to about 24 months, about 24 to about 120 months, about 24 to about 96 months, about 24 to about 72 months, about 24 to about 48 months, about 48 to about 120 months, about 48 to about 96 months, about 48 to about 72 months, about 72 to about 120 months, about 72 to about 96 months or about 96 to about 120 months at room temperature.In certain embodiments, the disclosed pharmaceutical compositions retain at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of their biological activity after 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks or 4 weeks at 40 °C and 75% relative humidity (RH).

[0200] V. Therapeutic Uses The methods and compositions disclosed herein can be used to treat a disease or disorder. For example, the present disclosure provides a method of treating congenital sucrase-isomaltase deficiency (CSID) in a subject. The method includes administering to the subject (i) an invertase enzyme (e.g., a recombinant mutant invertase enzyme described herein), and / or (ii) an effective amount of an isomaltase enzyme (e.g., by oral administration). For example, the method can include administering a disclosed pharmaceutical composition comprising (i) a spray-dried invertase enzyme (e.g., a recombinant mutant invertase enzyme described herein), and / or (ii) a spray-dried isomaltase enzyme. In certain embodiments, the pharmaceutical composition is administered to the subject with a meal or snack.

[0201] Under certain conditions, the method reduces abdominal pain, distension, and / or nausea in the subject, and as a result, the method reduces the total symptom score (e.g., as measured by abdominal pain, distension, and nausea) for the subject. Further, the present invention enables greater dietary freedom in the subject (e.g., enables the subject to better tolerate certain foods such as grains (e.g., wheat, potatoes, corn, rice, and bread), fruits (e.g., apples, bananas, apricots, and oranges), and / or vegetables (e.g., beets, carrots, and black beans)).

[0202] As used herein, the term "effective amount" refers to the amount of an active agent (e.g., the disclosed invertase and / or isomaltase enzyme) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or route of administration.

[0203] As used herein, "treat," "treating," and "treatment" mean 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) alleviating the disease, i.e., causing regression of the diseased state. As used herein, the terms "subject" and "patient" refer to an organism to be 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.), more preferably humans.

[0204] The present disclosure also provides a method of reducing the amounts of sucrose and branched (1-6 linked) α-limit dextrins in a subject, e.g., a subject having congenital sucrase-isomaltase deficiency (CSID). The method comprises administering to the subject (i) an invertase enzyme (e.g., a recombinant mutant invertase enzyme described herein), and / or (ii) an effective amount of isomaltase (e.g., oral administration). For example, the method can comprise administering a disclosed pharmaceutical composition comprising (i) spray-dried invertase (e.g., a recombinant mutant invertase described herein), and (ii) spray-dried isomaltase. The amounts of sucrose and branched (1-6 linked) α-limit dextrins in the subject can be determined by a hydrogen breath test, e.g., a sucrose methane hydrogen breath test or 13It can refer to the amounts of sucrose and branched (1-6-linked) α-limit dextrin measured by the C-sucrose breath test. The amounts of sucrose and branched (1-6-linked) α-limit dextrin in a subject can refer to the amounts of sucrose and branched (1-6-linked) α-limit dextrin in a body fluid (such as blood, plasma, serum or urine), tissue and / or cells in the subject.

[0205] The methods and compositions described herein can 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 treatments are delivered to a subject during the course of the affliction of a subject having a disorder such that the effects of the treatments overlap at some point in time when there is an effect on the treatment of the patient. In one aspect, there is an overlap in the administration period since the delivery of one treatment is still being carried out when the delivery of the second one is initiated. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other aspects, the delivery of one treatment ends before the delivery of the other treatment begins. In certain aspects in either case, the treatments are more effective for combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen using 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 one aspect, the delivery is such that the reduction of other parameters related to the symptom or disorder is greater than that observed with one treatment delivered in the absence of the others. The effects of the two treatments can be partially additive, wholly additive or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second one is delivered.

[0206] In certain embodiments, the methods or compositions described herein are administered in combination with one or more fructose-degrading enzymes. Exemplary fructose-degrading enzymes include glucose (xylose) isomerase.

[0207] Throughout this specification, when a composition is described as having, including, or comprising specific components, or when a process and method 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 components described, and processes and methods according to the invention that consist essentially of or consist of the process steps described.

[0208] In this application, when an element or component is said to be included in and / or selected from the list of elements or components described, it should be understood that the element or component can be any one of the elements or components described, or can be selected from a group consisting of two or more of the elements or components described.

[0209] Furthermore, the elements and / or features of the compositions or methods described herein, whether explicit or implicit in this specification, can be combined in various ways without departing from the spirit and scope of the invention. For example, when a particular compound is referenced, unless the context indicates otherwise, the compound can be used in various embodiments of the compositions of the invention and / or in the methods of the invention. That is, in this application, embodiments are described and shown in a manner that enables clear and concise application, but it is intended and understood that the embodiments can be variously combined or separated without departing from the teachings and the 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.

[0210] The expression "at least one of" should be understood to individually include each of the things described after said expression and various combinations of two or more of the things described, unless the context and use indicate otherwise. The expression "and / or" with respect to three or more things described should be understood to have the same meaning unless the context indicates otherwise.

[0211] The 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 non-limiting, not excluding, for example, further unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0212] When the term "about" is used before a quantitative value, the present invention includes the specific quantitative value itself as well, unless specifically stated otherwise. As used herein, the term "about" refers to a variation of ±10% from the nominal value, unless otherwise indicated or inferred.

[0213] It should be understood that the order of steps or the order for performing a particular act is not important as long as the present invention remains practicable. Further, two or more steps or acts may be performed simultaneously.

[0214] The use of any and all examples, or exemplary terms herein, such as "such as" or "including", is intended merely to illustrate the present invention better and does not limit the scope of the present invention unless claimed. The terms herein should not be construed to indicate that any unclaimed element is essential to the practice of the present invention.

Examples

[0215] Example The following examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.

[0216] Example 1 - Invertase Selection and Genetic Engineering Modification Invertase Expression This example describes the design of a recombinant mutant S. cerevisiae invertase with improved tolerance to low pH, resistance to digestion by pepsin and pancreatin, improved thermal stability, and high catalytic activity.

[0217] Expression constructs encoding recombinant S. cerevisiae invertase (GenBank: AFN08663.1, SEQ ID NO: 1) were constructed for expression in the yeast expression strains Pichia pastoris and S. cerevisiae. The expression constructs were designed with or without a C-terminal or N-terminal 6x-His affinity tag and tested for the expression and tolerance of the added His tag. Further expression constructs were designed using either the native invertase secretion leader intact (SEQ ID NO: 1) or the substituted alpha mating factor (FAKS) leader (SEQ ID NO: 2). The nucleic acid sequences of each expression construct were optimized for high expression in the corresponding host. The synthesized nucleic acid sequences encoding invertase were cloned into pD1204 (S. cerevisiae) or pD902 (P. pastoris).

[0218] For expression in S. cerevisiae, strain CEN.PK2-1C was chemically transformed using the lithium acetate method (Ito et al. (1983) J. BACTERIOL. 153(1):163-168). Cells carrying the plasmid were selected by plate culture on CM-URA medium (MP Biomedicals, Santa Ana, CA) at 30 °C for 3 days. Three individual colonies for each construct were isolated and used to inoculate 500 μL of CM-URA medium. The inoculated cultures were incubated at 30 °C for 16 h with shaking at 1,000 rpm. Then, 40 μL of the overnight culture was used to inoculate 500 μL of YP induction medium (1% yeast extract, 2% peptone, 0.1 M potassium phosphate, 2% raffinose and 2% galactose). The induction cultures were then incubated at 30 °C for 72 h with shaking at 1,000 rpm. The induction cultures were supplemented with 50 μL of 20% galactose every 24 h after introduction. The supernatant was clarified by centrifugation and secreted invertase expression was analyzed by PAGE (4-12% Bis-tris, MOPS buffered; Invitrogen).

[0219] For expression in P. pastoris, 20 μg of the expression plasmid was linearized by digestion with PmeI. DNA was isolated by precipitation with ethanol and reconstituted in Qiagen EB buffer. For the expression assay, strain BG10 was transformed by electroporation. For protein genetic engineering, strain BG10 was chemically transformed using the lithium acetate method (Ito et al., supra). Cells harboring the plasmid were selected by plating on YPD medium at 30 °C for 3 days using 250 μg / mL of zeocin (Teknova). Three individual colonies were isolated for each construct and 350 μl of BYPG medium containing 250 μg / mL of zeocin was used to inoculate deep well microplates. The inoculated cultures were then incubated at 30 °C for 60 hours with shaking at 1,000 rpm. Then 250 μL of the overnight culture was added to 250 μL of BYPM induction medium (BYP medium supplemented with 20% methanol). The induced cultures were then incubated at 30 °C for 72 hours with shaking at 1,000 rpm. Every 12 hours after incubation, the cultures were supplemented with 50 μL of BYP containing 10% methanol. The supernatant was clarified from the cells by centrifugation and secreted invertase expression was analyzed by PAGE.

[0220] Expression yields were determined by PAGE densitometry after deglycosylation with PNGaseF. Briefly, culture supernatant samples were treated with PNGaseF after denaturation according to the manufacturer's protocol (NEB) and the samples were analyzed by PAGE. The intensity of the full-length deglycosylated invertase band was determined and the protein concentration was estimated using the intensity relative to a BSA standard.

[0221] Expression and yields of S. cerevisiae invertase in P. pastoris were typically better than those in S. cerevisiae.

[0222] Invertase Activity and Stability Assays In the initial stage, when the obtained protein was tested without purification, the specific activity was approximated by assaying the units per amount of deglycosylated protein (1 unit of enzyme consumes 1 μmol of sucrose per minute at 37 °C).

[0223] The activity assay was performed using the dinitrosalicylic acid (DNS) colorimetric quantification method to monitor the formation of reducing sugars during the hydrolysis of sucrose. Briefly, by the DNS method, reducing sugars released by the action of a hydrolase enzyme on a carbohydrate are detected under specific pH and temperature conditions (Bailey (1988) APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 29: 494 - 496). During the reaction with the reducing sugar, DNS is reduced to 3 - amino - 5 - nitrosalicylic acid, and the reaction product can be monitored at 540 nm.

[0224] The culture supernatant was diluted 1 / 20 into a reaction buffer containing 237 mM NaCl and either A) 50 mM glycine, pH 3.5 (buffer A); B) 50 mM sodium acetate, pH 4.7 (buffer B); C) 50 mM MES, pH 5.8 (buffer C); or D) 50 mM sodium phosphate, pH 6.8 (buffer D). The diluted enzyme was then further diluted 10 - fold with 10 mM sucrose in the pre - equilibrated reaction buffer at 37 °C. The progress of the reaction was then estimated by the DNS method. To stop the reaction, 20 μL of the reaction was removed into 30 μL of DNS reagent (10 mg / mL dinitrosalicylic acid, 300 mg / mL tartrate, 0.4 N NaOH), and the mixture was immediately heated to 99 °C for 5 minutes. The 40 μL DNS reaction was diluted with 160 μL of water, and the absorbance was read at 540 nm. The reducing sugars present were determined against a 1:1 glucose:fructose standard.

[0225] The DNS method was used as described above to assay the thermal stability by treating the invertase sample at 56.5 °C for 30 minutes before assaying the residual activity at pH 4.7 (in buffer B as described above).

[0226] The low pH tolerance assay was performed as follows. The culture supernatant containing invertase enzyme was diluted 1 / 20 in 237 mM NaCl, 50 mM glycine, pH 2.8 and pre-incubated at 37 °C for 30 minutes. The pre-incubation was then diluted to 10 mM sucrose in the reaction buffer, finally at pH 4.7, and the activity was assayed using the DNS method as described above.

[0227] The invertase enzyme was treated at 37 °C with soluble pepsin (Sigma-Aldrich, St. Louis, MO) from porcine gastric mucosa at 10 mg / mL in 237 mM NaCl, 50 mM NaOAc, pH 4 to assay the resistance to pepsin. The culture supernatant containing invertase enzyme was diluted 20-fold in 10 mg / mL pepsin and incubated at 37 °C for 2 hours. The pepsin treatment reaction was then diluted 20-fold to 10 mM sucrose in the reaction buffer, finally at pH 4.7, and the residual invertase activity was assayed, and the activity was assayed using the DNS method as described above. Overexpressed S. cerevisiae invertase and most invertase variants showed insignificant degradation under these conditions.

[0228] The invertase was treated at 37 °C with soluble porcine pancreatin (Sigma-Aldrich, St. Louis, MO) at 3.4 mg / mL in 237 mM NaCl, 50 mM MES, pH 6 to assay the resistance to pancreatin. The culture supernatant was diluted 20-fold in 3.4 mg / mL pancreatin and incubated at 37 °C for 2 hours. The pancreatin treatment reaction was then diluted 20-fold to 10 mM sucrose in the reaction buffer, finally at pH 4.7, and the residual invertase activity was assayed, and the activity was assayed using the DNS method as described above.

[0229] Genetic engineering modification of invertase Using the principles of protein engineering, 95 recombinant S. cerevisiae invertase variants (designated V1 - V95), each containing three substitutions relative to the wild - type native protein S. cerevisiae invertase (designated V0), were generated. Table 2 shows specific exemplary amino acid mutations in S. cerevisiae invertase (SEQ ID NO: 2) analyzed in the invertase genetic engineering program. The variants were expressed in P. pastoris together with wild - type (wt) S. cerevisiae invertase and tested as culture supernatants for activity and stability. Using the above - described assays, each variant was tested for seven properties including 1) expression level, 2) activity at pH 3.5, 3) activity at pH 7, 4) thermal stability, 5) stability at pH 2.8, 6) resistance to pepsin, and 7) resistance to pancreatin.

Table 2

[0230] Overall, 94 invertase variants were expressed and tested (one variant did not express) and compared to the wild-type enzyme. Generally, 13 invertase variants showed improvements in pH 2.8 stability, thermal stability, pH 3.5 stability, and pH 7.0 activity relative to the parental invertase, 35 invertases showed mixed properties, and 46 invertase variants showed properties that were worse than the parental invertase. Exemplary results for genetically engineered invertase variants compared to the parental wild-type invertase (S. cerevisiae invertase) are shown in Table 3, and the substitutions present in the various invertase variants (relative to S. cerevisiae invertase (SEQ ID NO:2) listed in Table 3) are shown in Table 4. Table 3 includes the performance of 13 invertase variants (V6, V9, V14, V25, V32, V38, V60, V63, V70, V78, V83, V87, V89, V90) that showed one or more improved properties relative to the parental invertase, and three exemplary invertase variants (V3, V75, and V82) that showed performance worse than the parental invertase.

Table 3

Table 4

[0231] The relative impact of each substitution on each target property was estimated and is shown in Table 5 (amino acid numbers are relative to S. cerevisiae invertase (SEQ ID NO:2)). In particular, the impact of specific amino acid substitutions that showed a significant contribution to at least one of the three major invertase properties: pH 7.0 activity, thermal stability, or low pH survival, relative to wild-type S. cerevisiae invertase (SEQ ID NO:2), is summarized in Table 5.

Table 5

[0232] Further Characterization of Specific Invertase Variants Several invertase variants that exhibit improved properties with respect to activity and / or stability compared to the parental invertase were further characterized. In particular, two tests were performed on independent preparations of the selected variants, and the results are summarized in Tables 6 and 7. Table 6 summarizes the characteristics of three variants compared to the wild-type parental invertase.

Table 6

[0233] Strong improvements in activity, low-pH stability and thermal stability were seen for variants V78 (F172Y, N429A, P479A; SEQ ID NO: 14) and V6 (E120A, D122E, P479A; SEQ ID NO: 4). Both variants V6 and V78 contained the widely beneficial substitution P479A and at least one other substitution that was positive for one or more of the critical properties. V22 (K259L, S476N, K514P; SEQ ID NO: 7) showed an improvement in low-pH stability. V22 contained the K259L substitution that was found to be beneficial for low-pH stability rather than high-pH activity.

[0234] To further compare the enzymes expressed under similar conditions, the Sc_S288C (SEQ ID NO: 36) invertase was expressed in P. pastoris and assayed under the same conditions as the specific ones of the better variants. Additional variants of the Sc_S288C invertase containing two further substitutions (F172Y and N429A) found in V78 were tested to evaluate their effects in the context of the Sc_S288C invertase sequence. The results of the comparison are summarized in Table 7.

Table 7

[0235] The activity profile for Sc_S288C invertase was more comparable to that of the parental S. cerevisiae invertase, although similar differences in low pH and thermal stability were observed. This was not inconsistent with the expected effect of the P479A substitution on invertase activity. Variant V78 showed improved activity against Sc_S288C invertase, although it showed slightly poorer thermal stability.

[0236] Together, these tests identified several substitutions that can be used alone or in combination to positively affect the activity, low pH stability, and thermal stability of S. cerevisiae invertase. Variant V78 shows an overall significant improvement over the parental invertase.

[0237] Incorporation by reference The entire disclosure of each patent and scientific document referenced herein is incorporated by reference for all purposes.

[0238] Equivalents The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive, and the scope of the invention is indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are intended to be embraced within the invention.

[0239] Sequence listing [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Claims

1. Recombinant mutant S-cerevisiae invertase enzyme comprising: (i) increased activity at acidic pH compared to the corresponding wild-type invertase; (ii) increased activity at neutral pH compared to the corresponding wild-type invertase; (iii) increased stability at acidic pH compared to the corresponding wild-type invertase; (iv) increased thermal stability compared to the corresponding wild-type invertase, or a combination of (i), (ii), (iii), or (iv).

2. In invertase, (a) Sequence ID: The F residue at position 172 of 2 is replaced with Y (F172Y); (b) Sequence ID: The N residue at position 429 of 2 is replaced with A (N429A); (c) Sequence ID: The P residue at position 479 of 2 is replaced with A (P479A); (d) Sequence ID: The D residue at position 122 of 2 is replaced with E (D122E); (e) Sequence ID: The T residue at position 140 of 2 is replaced with L (T140L); (f) The E residue at position 156 of sequence number 2 is replaced with Q (E156Q); (g) The V residue at position 178 of sequence number 2 is replaced with I (V178I); (h) The K residue at position 259 of sequence number 2 is replaced with L (K259L); (i) Sequence ID: The D residue at position 272 of 2 is replaced with N (D272N); (j) Sequence ID: The K residue at position 277 of 2 is replaced with T (K277T); (k) The E residue at position 285 of sequence number 2 is replaced with H (E285H); (l) The A residue at position 378 of sequence number 2 is replaced with T (A378T); (m) The E residue at position 382 of sequence number 2 is replaced with Q (E382Q); (n) Sequence ID: The V residue at position 399 of 2 is replaced with A (V399A); (o) The G residue at position 431 of sequence number 2 is replaced with R (G431R); (p) The V residue at position 477 of sequence number 2 is replaced with D (V477D); (q) The A residue at position 501 of sequence number 2 is replaced with N (A501N); (r) The L residue at position 549 of sequence number 2 is replaced with I (L549I); (s) The V residue at position 581 of sequence number 2 is replaced with I (V581I); (t) The G residue at position 586 of sequence number 2 is replaced with S (G586S); or (u) The N residue at position 589 of sequence number 2 is replaced with K (N589K); Alternatively, the invertase according to claim 1, wherein the invertase includes any combination of the substitutions described above.

3. (a) Replacement of F172Y, N429A and P479A; (b) Replacement of E120A, D122E and P479A; (c) Replacement of K259L, S476N and K514P; (d) Replacement of T140L, E156Q and L549I; (e) Replacement of S476A, P479A and Q597E; (f) Replacement of K277T, E415S and V581I; (g) Substitution of A123G, T140L and F143Y; (h) Replacement of N390D, S476N and K519R; (i) Replacement of E382Q, T573S and V581I; (j) Replacement of D122E, T470S and S476N; (k) Replacement of T140Q, P479A and K514P; (l) Replacement of E120A, V178I and L549I; (m) Replacement of E382Q, S537D and N589K; (n) Replacement of T140L, G586S and F596I; or (o) Replacement of E382Q, L459F and K519R The invertase according to claim 2, comprising:

4. The invertase according to claim 1, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 29, 19-28, 30-33, 14, 4-13, 15-18, and 50.

5. (a) Having a specific activity of at least 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900 or 2,000 μmol of sucrose per minute per milligram of deglycosylating invertase at approximately pH 3.5; (b) Having at least 1, 1.5, 2, 2.5, or 3 times higher activity than the corresponding wild-type invertase at approximately pH 3.5; (c) At approximately pH 5.0, having a specific activity of at least 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, or 2,200 μmol of sucrose per minute per milligram of deglycosylating invertase; (d) Compared to the corresponding wild-type invertase, it has at least 1, 1.5, 2, 2.5, or 3 times higher activity at approximately pH 5.0; (e) At approximately pH 6.0 or 6.2, having a specific activity of at least 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700 or 1,800 μmol of sucrose per minute per milligram of deglycosylating invertase; (f) Having at least 1, 1.5, 2, 2.5, or 3 times higher activity compared to the corresponding wild-type invertase at approximately pH 6.0 or 6.2; (g) At approximately pH 7.0 or 7.1, having a specific activity of at least 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200 or 1,300 μmol of sucrose per minute per milligram of deglycosylating invertase; (h) Compared to the corresponding wild-type invertase, it has at least 1, 1.5, 2, 2.5, or 3 times higher activity at approximately pH 7.0 or 7.1; (i) retaining at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the activity after incubation at approximately pH 2.5 for approximately 30 minutes; (j) Compared to the corresponding wild-type invertase, it has at least 1, 1.5, 2, 2.5, 3, 3.5, 4, or 5 times higher stability at approximately pH 2.5; (k) Having a Tm of at least 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68°C; (l) Having a Tm at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10°C higher than the corresponding wild-type invertase; and / or (m) The invertase according to claim 1, which has higher stability in the presence of pancreatin or pepsin compared to the corresponding wild-type invertase.

6. A nucleic acid encoding an invertase according to any one of claims 1 to 5.

7. An expression vector comprising the nucleic acid according to claim 6.

8. A cell comprising the expression vector according to claim 7.

9. a) A step of growing the cells according to claim 8 under conditions in which the host cells express invertase, and b) Process of purifying invertase A method for producing recombinant mutant S-cerevisiae invertase enzyme, including [specific enzyme].

10. A pharmaceutical composition comprising an invertase according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier and / or excipient.

11. A pharmaceutical composition comprising an invertase enzyme, separate isomaltase enzymes, and a pharmaceutically acceptable carrier and / or excipient.

12. (a) Invertase and / or isomaltase are spray-dried and / or (b) The invertase and / or isomaltase is a microbial invertase or microbial isomaltase, respectively, or a functional fragment or variant thereof. The pharmaceutical composition according to claim 11.

13. The pharmaceutical composition according to claim 12, wherein the invertase is a recombinant mutant S-cerevisiae invertase enzyme according to any one of claims 1 to 5.

14. Isomaltase (a) Derived from Saccharomyces cerevisiae; (b) Sequence ID: containing any of 37-41 or a functional fragment or variant thereof; (c) Recombinant Lactobacillus fermentum isomaltase enzyme (SEQ ID NO: 47) or a functional fragment or variant thereof; or (d) i. Replacement of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D and F560V; ii. Replacement of E93K, K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D and F560L; iii. Replacement of E93K, K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D and F560L; iv. Replacement of E93K, K115I, R132K, D226S, E310A, I421A, A444G, A531D and F560V; v. Replacement of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D and F560L; vi. Replacement of E93K, K115I, R132K, D226S, L366M, I421A, A444G, A531D and F560V; vii. Replacement of E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D and F560V; viii. Replacement of K115I, R132K, D226S, E310A, L366M, I421A, A444G, E524Q, A531D and F560L; ix. Replacement of K115I, R132K, D226S, E310A, I421A, A444G, E524Q, A531D and F560V; x. Replacement of K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D and F560V; xi. Replacement of E93K, K115I, R132K, A211E, D226S, I421A, A444G, E524Q, A531D and F560V; xii. Replacement of E93K, K115I, R132K, D226S, L366M, I421A, A444G, E524Q, A531D and F560V; xiii. Replacement of E93K, K115I, R132K, D226S, E275D, I421A, A444G, E524Q, A531D and F560V; xiv. Replacement of T89A, E93K, K115I, R132K, D226S, I421A, A444G, E524Q, A531D and F560V; xv. Replacement of E93K, K115I, R132K, H182R, D226S, I421A, A444G, E524Q, A531D and F560V; xvi. Replacement of E93K, K115I, E122D, R132K, D226S, I421A, A444G, E524Q, A531D and F560V; xvii. Replacement of E93K, K115I, D226S, I421A, A444G, E524Q, A531D and F560V; xviii. Replacement of E93K, K115I, D226S, I421A, A444G, E524Q, A531D and F560L; xix. Replacement of K115I, A211E, D226S, I421A, A444G, A531D and F560V; xx. Replacement of K115I, D226S, L366M, I421A, A444G, E524Q, A531D and F560L; xxi. Replacement of K115I, A211E, D226S, I421A, A444G, E524Q, A531D and F560L; xxii. Replacement of K115I, R132K, D226S, E310A, L366M, I421A, A444G, A531D and F560L; xxiii. Replacement of E93K, K115I, D226S, E310A, I421A, A444G, A531D and F560L; xxiv. Replacement of K115I, D226S, E310A, I421A, A444G, A531D and F560V; xxv. Replacement of E93K, K115I, D226S, L366M, I421A, A444G, A531D and F560L; xxvi. Replacement of K115I, D226S, I421A, A444G, E524Q, A531D and F560V; xxvii. Replacement of K115I, D226S, E310A, I421A, A444G, E524Q, A531D and F560L; xxviii. Replacement of K115I, A211E, D226S, L366M, I421A, A444G, A531D and F560L; xxix. Replacement of K115I, A211E, D226S, E310A, I421A, A444G, A531D and F560L; xxx. Replacement for E93K, K115I, D226S, I421A, A444G, A531D and F560V; xxxi. Replacement of E93K, K115I, A211E, D226S, I421A, A444G, A531D and F560L; or xxxii. Replacement of K115I, D226S, L366M, I421A, A444G, A531D and F560V including, A pharmaceutical composition according to any one of claims 11 to 12.

15. (a) Formulated as a dosage form in the form of a powder, sachet, granules, pellets, micropellets, tablets or minitablets; and / or (b) The pharmaceutical composition according to any one of claims 11 to 12, having a shelf life of at least 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months, 36 months, 48 ​​months, 60 months, 72 months, 84 months, 96 months, 108 months, or 120 months at room temperature.

16. A pharmaceutical composition according to any one of claims 11 to 12 for treating congenital sucrase-isomaltase deficiency (CSID) in a subject requiring treatment for CSID.

17. The pharmaceutical composition according to claim 16, which is administered to a subject together with a meal or snack.

18. A pharmaceutical composition according to any one of claims 11 to 12 for reducing the concentrations of sucrose and branched (1-6 linked) α-limiting dextrin in a subject.

19. The pharmaceutical composition according to claim 16, wherein the subject is a mammal.

20. The pharmaceutical composition according to claim 16, wherein the subject is a human.