Allulose production

Abstract

Provided herein are compositions and methods relating to improved production of allulose by converting fructose to allulose using a allulose or psicose-3-epimerase from Brachyspira suanatina in the presence of base NaOH at 50 or 100mg / L.

Description

ALLULOSE PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 534,719, filed August 25, 2023, which is incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled NB42187WOPCT_SequenceListing.xml, created on August 19, 2024, which is 38,673 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] Provided herein are methods and compositions relating to improved production of allulose.BACKGROUND

[0004] Allulose, also known as D-allulose and D-psicose, is a rare, naturally occurring low calorie sugar having a sweetness profile like that of sucrose. As such, allulose is an attractive alternative to higher calorie sweeteners, such as sucrose, fructose, and glucose. Allulose is a C-3 epimer of D-fructose, and may thus be produced, e.g., commercially, by conversion of D-fructose to allulose by enzymes such as epimerases.

[0005] Epimerases capable of converting D-fructose to allulose have been found to have a variety of properties, e.g., temperature, pH, and metal cofactor requirements, that can impact their enzymatic activity. While epimerases having desirable properties for commercial production of allulose, e.g., stable at high temperature and low pH with a reduced need for supplemented metal cofactors, are typically used to maximize allulose yield, the process for allulose production itself may play a role in yield.

[0006] Thus, there remains a need for methods optimized to improve allulose production. The methods and compositions provided herein address these and other needs in the art.SUMMARY OF THE INVENTION

[0007] In an aspect is provided a method for producing allulose, including contacting a substrate containing fructose with an epimerase, wherein the contacting occurs in the presence of a base present at a concentration of at least 5 mg / L. In an aspect is provided a method for producing allulose, including contacting a substrate containing fructose with an epimerase, wherein the contacting occurs under conditions comprising a pH in a range of about 5 to about 10. In someembodiments, the contacting occurs in the presence of a base. In some embodiments, the base is present at a concentration of at least 5 mg / L. In some embodiments, the base is present at a concentration in a range of 20 to 500 mg / L. In some embodiments, the base is sodium hydroxide. In some embodiments, the base is continuously added to maintain the pH in the range of about 5 to about 10. In some embodiments, the conditions further include a metal cofactor. In some embodiments, the metal cofactor is magnesium, cobalt, manganese, or any combination thereof. In some embodiments, the conditions further include a temperature in a range of about 50°C to about 90°C. In some embodiments, the epimerase is soluble. In some embodiments, the epimerase comprises an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 2. In some embodiments, the contacting occurs in a reactor. In some embodiments, the reactor is a tank, vessel, or column. In some embodiments, the substrate containing fructose is a fructose syrup or is produced by: (i) contacting a substrate containing glucose with a glucose isomerase prior to contacting the epimerase; or (ii) contacting a substrate containing glucose with a glucose isomerase at the same time as contacting the epimerase. In some embodiments, the method includes purifying the produced allulose.

[0008] In an aspect is provided a composition for producing allulose, including: i) an epimerase; ii) a substrate comprising fructose; and iii) a base at a concentration of at least 5 mg / L. In some embodiments, the base is sodium hydroxide. In some embodiments, the epimerase is soluble. In some embodiments, the epimerase comprises an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 2. In some embodiments, the substrate comprises glucose. In some embodiments, the composition further includes a glucose isomerase. In some embodiments, the composition is contained in a reactor. In some embodiments, the reactor is a tank, vessel, or column.

[0009] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.DETAILED DESCRIPTION

[0010] Allulose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose, that is rarely found in nature. Allulose has similar physical properties to those of sucrose, such as bulk, mouthfeel, browning capability, gelling, and freezing point, and its sweetness is estimated to be about 70% of the sweetness of sucrose. The energy value of allulose, however, is approximately 0.3% of that of sucrose. In addition to having low caloric value, allulose may have beneficial physiological effects, such as blood glucose suppression, reactive oxygen species scavenging, and neuroprotection among others. These properties have made allulose an attractive substitute for higher calorie sweeteners, e.g., sucrose, fructose, and glucose.

[0011] Since allulose is naturally present in only small quantities in certain foods, there exists a need for methods to efficiently and effectively produce allulose. The bio-conversion of D-fructose to D-allulose by epimerases, for example D-tagatose 3-epimerases (DT3E) and D-allulose 3- epimerases, is one such method of producing allulose. Epimerases capable of performing this conversion are known to have varying properties, such as temperature, pH, and metal cofactor requirements, that can impact enzymatic activity and efficiency. For example, most of the epimerases that have been identified as capable of performing this conversion show a dependence on manganese, cobalt, and / or magnesium as a cofactor to be active and optimal temperature and pH ranges for activity between 40°C and 70°C and 7.0 to 9.0 pH, respectively. For commercial production, it is preferable to use higher temperatures, e.g., about or greater than 50°C, to shift the thermodynamic equilibrium in favor of converting fructose to allulose, thereby increasing the ratio of allulose to fructose. It is also preferable to use an acidic pH, in particular at elevated temperatures, to reduce non-enzymatic browning of the sugars, e.g., via the Maillard reaction. The use of acidic pH in the production process provides additional advantages such as microbial control, for example by reducing microbial growth. In addition, lower pH prevents sugars from reacting with enzyme side chains, e.g., fructosylation.

[0012] As described herein, it was surprisingly found that controlling the pH of the reaction to produce allulose by adding a base, improved allulose yield by increasing the amount of allulose produced by the enzyme. See, Examples. An advantage of this result is that less enzyme may be used to produce the same amount of allulose, or the same amount of enzyme may produce more allulose than conditions without a controlled pH. These advantages may offer environmental and / or cost-saving benefits to commercial producers. The methods and compositions provided herein harness these surprising findings.

[0013] The methods and compositions described herein increase allulose yield and offer an opportunity to reduce the amount of enzyme (epimerase) needed for allulose production. The use of the methods and composition described herein may assist in diversifying the sweetener product portfolio associated with com processing by adding a natural low-calorie sweetener and bulking agent to the traditional sweeteners derived from corn starch (e.g., corn syrup, high fructose corn syrup (HFCS), glucose, and fructose).

[0014] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. The reader will appreciate that statements made in one section may apply toother sections. Any terms defined may be more fully defined by reference to the specification as a whole.

[0015] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications, and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.Definitions

[0016] Definitions of terms may appear throughout the specification. It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” include “at least one” and “one or more.”

[0018] The terms "comprising", "comprises," and "comprised of” as used herein are synonymous with "including," "includes," "containing," "contains," and grammatical variants thereof, and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The terms "comprising," "comprises," "comprised of,” "including," "includes," "containing," "contains," and grammatical variants thereof also include the term "consisting of”.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] The term “starch” refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (CeHwOsjx, wherein X can be any number. The term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, com, rye, rice, sorghum, brans, cassava, millet, milo, potato, sweet potato, and tapioca. The term “starch” includes granular starch. The term “granular starch” refers to raw, i.e., uncooked starch, e.g., starch that has not been subject to gelatinization.

[0021] The terms, “wild-type,” “parental,” or “reference,” with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, ordeletion at one or more amino acid positions. Similarly, the terms “wild-type,” “parental,” or “reference,” with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.

[0022] Reference to the wild-type polypeptide is understood to include the mature form of the polypeptide. A “mature” polypeptide or variant, thereof, is one in which a signal sequence is absent, for example, cleaved from an immature form of the polypeptide during or following expression of the polypeptide.

[0023] The term “variant,” with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally- occurring or man-made substitutions, insertions, or deletions of an amino acid. Similarly, the term “variant,” with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. A variant may include two or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, substitutions, deletions, and / or insertions compared to the wild-type, parental, or reference polypeptide or polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.

[0024] In the case of the epimerases described herein, “activity” refers to epimerase activity, which can be measured as described herein. It should be appreciated that epimerases operate bidirectionally as an equilibrium conversion reaction to interconvert fructose to allulose. In some embodiments, the activity includes or is the conversion of fructose to allulose. In some embodiments, the activity includes or is the conversion of allulose to fructose. Estimates of activity may be determined by assays designed to assess fructose formation from allulose, e.g., by colorimetric assay, and / or allulose formation from fructose, e.g., by high-performance liquid chromatography (HPLC). In some embodiments, the activity is referred to as a residual activity. As used herein, “residual activity” includes or is the activity of an epimerase following a challenge, e.g., elevated temperature challenge and / or pH challenge, compared to the activity of an unchallenged epimerase, which serves as a baseline for comparison, or is epimerase activity determined in a specific state, e.g., an immobilized state, compared to the activity of an epimerase in a different state (e.g., solubilized), which serves as a baseline. Residual activity may be expressed as a percentage or fraction of the baseline activity (e.g., baseline activity is equal to 100% or 1). Methods for determining activity of an enzyme are various and known in the art.

[0025] The term “recombinant,” when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and / or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from a native sequence by one or more amino acids and / or are fused with heterologous sequences. A vector comprising a nucleic acid encoding an epimerase may be referred to as a recombinant vector.

[0026] The terms “recovered,” “isolated,” and “separated,” refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component. In some embodiments, the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component is naturally associated as found in nature. In some embodiments, the at least one other material or component is at least one other material or component with which the compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component is associated with under experimental or production conditions and / or systems. For example, an “isolated” polypeptide includes, but is not limited to, a polypeptide removed from a culture broth containing a heterologous host cell expressing the polypeptide.

[0027] The term “purified” refers to material (e.g., an isolated compound, polypeptide, polynucleotide, or other specified material or component) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure.

[0028] The term “enriched” refers to material (e.g., an isolated compound, polypeptide, polynucleotide, or other specified material or component) that is in about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 80% pure.

[0029] As used herein, "derived from" encompasses "originated from," "obtained from," or "isolated from."

[0030] The terms “thermal stability,” “thermostable,” and “thermostability,” with reference to an enzyme, refer to the ability of the enzyme to retain activity at elevated temperatures or after exposure to an elevated temperature. Methods for determining thermostability are various and known in the art. In some cases, thermostability of an enzyme, such as an epimerase enzyme, may be measured by its half-life (tl / 2) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions. The half-life may be calculated by measuring residualepimerase activity following exposure to an elevated temperature. In some cases, thermostability is determined by measuring epimerase activity following exposure to an elevated temperature and comparing the measured activity against a baseline activity, where the baseline activity is measured from an epimerase that was not exposed to an elevated temperature. The value resulting from the comparison may be referred to as a residual activity.

[0031] An enzyme’ s pH range refers to the range of pH values under which the enzyme exhibits activity. The pH range where an enzyme demonstrates activity may be referred to as the “pH activity profile” of the enzyme. The terms “pH stable” and “pH stability,” with reference to an enzyme, relate to the ability of the enzyme to retain activity at a pH or after exposure to a pH. Methods for determining a pH profile and pH stability of an enzyme are various and known in the art. In some cases, the pH profile of an enzyme is determined by measuring the activity of the epimerase across a range of pHs. In this case, the minimum and maximum activity levels may be determined to produce a dose response curve or standard curve. In some cases, pH stability is determined by measuring epimerase activity following exposure to a pH and comparing the measured activity against a baseline activity, where the baseline activity is measured from an epimerase that was not exposed to the pH. The value resulting from the comparison may be referred to as a residual activity.

[0032] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino- to-carboxy terminal orientation (i.e., N— >C).

[0033] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may contain chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0034] “Hybridization” refers to the process by which one strand of nucleic acid forms a duplex with, i.e., base pairs with, a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65 °C and 0.1X SSC (where IX SSC = 0.15 M NaCl, 0.015 M Na3 citrate, pH 7.0). Hybridized, duplex nucleic acids are characterized by a melting temperature (Tm), whereone half of the hybridized nucleic acids are unpaired with the complementary strand. Mismatched nucleotides within the duplex lower the Tm.

[0035] The terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

[0036] The term “introduced” in the context of inserting a nucleic acid sequence into a cell, encompasses, but is not limited to, “transfection”, “transformation” and “transduction,” as known in the art. Exemplary methods for introducing polynucleotides or polypeptides by transformation into a host cell, include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods (such as induced competence using chemical (e.g. divalent cations such as CaCh), mechanical (electroporation) means, or methods such as those described in published international applications WO 2018 / 114983 and WO 2010 / 149721, which are incorporated herein by reference in their entireties), ballistic particle acceleration (particle bombardment), direct gene transfer, viral-mediated introduction, cellpenetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery. Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by conjugation, which is a specific method of natural DNA exchange requiring physical cell-to-cell contact. Introducing a nucleic acid, construct, plasmid, or vector into a host cell may be carried out by transduction, which is the introduction of DNA via a virus (e.g., phage) infection which is also a natural method of DNA exchange. Generally, such methods involve incorporating a polynucleotide within a viral DNA or RNA molecule.

[0037] A “host cell” is an organism into which an expression vector, phage, virus, or other nucleic acid sequence including a polynucleotide encoding a polypeptide of interest (e.g., an epimerase) has been introduced. Exemplary host cells are microorganism cells (e.g., bacteria, filamentous fungi, and yeast), mammalian cells, and plant cells capable of expressing the polypeptide of interest. The term “host cell” includes protoplasts created from cells.

[0038] The term “heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0039] The term “endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0040] The term “expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.

[0041] A “selective marker” or “selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and / or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

[0042] A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[0043] An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0044] The term “operably linked” means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.

[0045] A “signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0046] The term “specific activity” refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0047] “A cultured cell material comprising an epimerase” or similar language, refers to a cell lysate or supernatant (including media) that includes an epimerase as a component. The cell material may be from a heterologous host cell that is grown in culture for the purpose of producing the epimerase.

[0048] “Percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues or nucleotides identical to those in a specified reference sequence, when aligned using e.g., the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:Gap opening penalty: 10.0Gap extension penalty: 0.05Protein weight matrix: BLOSUM seriesDNA weight matrix: IUBDelay divergent sequences %: 40Gap separation distance: 8DNA transitions weight: 0.50List hydrophilic residues: GPSNDQEKRUse negative matrix: OFFToggle Residue specific penalties: ONToggle hydrophilic penalties: ONToggle end gap separation penalty OFF.

[0049] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included.

[0050] The term “degree of polymerization” (DP) refers to the number (n) of anhydroglucopyranose units in a given saccharide. Examples of DPI are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose. The term “DE,” or “dextrose equivalent,” is defined as the percentage of reducing sugar, i.e., D-glucose, as a fraction of total carbohydrate in a syrup.

[0051] The term “dry solids content” (ds) refers to the total solids of a slurry in a dry weight percent basis. The term “slurry” refers to an aqueous mixture containing insoluble solids.

[0052] The phrase “simultaneous saccharification and fermentation (SSF)” refers to a process in the production of biochemicals in which a microbial organism, such as an ethanologenic microorganism, and at least one enzyme, such as an amylase, are present during the same process step. SSF includes the contemporaneous hydrolysis of starch substrates (granular, liquefied, or solubilized) to saccharides, including glucose, and the fermentation of the saccharides into alcohol or other biochemical or biomaterial in the same reactor vessel.

[0053] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or bothof the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0054] Numerical values and ranges may be presented herein with the numerical value being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context. All values and ranges implicitly include the term “about” unless the context clearly dictates otherwise.I. METHODS OF PRODUCING ALLULOSE

[0055] Provided herein are methods of producing allulose using enzymes, such as epimerases and compositions containing epimerases, under conditions that increase allulose yield. In some embodiments, the conditions allow for less enzyme to be used to produce the same amount of allulose. In some embodiments, the conditions result in the enzyme (epimerase) producing more allulose compared to control conditions.

[0056] In aspects are provided methods for producing allulose, including contacting a substrate containing fructose with an epimerase, where the contacting occurs in the presence of a base present at a concentration of at least 5 mg / L. In aspects are provided methods for producing allulose including contacting a substrate containing fructose with an enzyme, where the contacting occurs under conditions including a pH in a range of about 5 to about 10. In some embodiments, the methods for producing allulose include contacting fructose, e.g., fructose syrup, with an enzyme, where the contacting occurs under conditions including a pH in a range of about 5 to about 10. In some embodiments, the enzyme is an epimerase. In some embodiments, the method includes contacting a substrate containing fructose with an epimerase, where the contacting occurs under conditions including a pH in a range of about 5 to about 10. In some embodiments, the method includes contacting fructose with an epimerase, where the contacting occurs under conditions including a pH in a range of about 5 to about 10. In some embodiments, the epimerase is an epimerase described in Section I-C below. The contacted fructose or substrate containing fructose and the epimerase may be referred to herein as a reaction mixture.

[0057] In some embodiments, contacting the substrate containing fructose and the epimerase under conditions including a pH in the range of 5 to 10 increases the production of allulose. In some embodiments, the increase in allulose produced is at least about 1%, 5%, 10%, 15%, 20%,25%, 50%, 75%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or more compared to a control condition. In some embodiments, the increase in allulose produced is at least about 10% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 50% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 100% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 200% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 300% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 400% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 500% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 400% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 600% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is in the range of about 10% to about 900% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is in the range of about 10% to about 600% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is in the range of about 50% to about 600% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is in the range of about 100% to about 600% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is in the range of about 100% to about 500% compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or more compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 2-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 3 -fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 4-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 5-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about6-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 7-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 8-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 9-fold compared to the amount of allulose produced under a control condition. In some embodiments, the increase in allulose produced is or is at least about 10-fold compared to the amount of allulose produced under a control condition. In some embodiments, the control condition is where the pH is outside of the range of 5 to 10. In some embodiments, the control condition is where a base is not present in the reaction mixture. In some embodiments, the control condition is where a base is not present in the reaction mixture at a concentration described herein, e.g., Section I-B.A. Substrate

[0058] In some embodiments, the substrate containing fructose is a syrup. In some embodiments, the substrate containing fructose is or is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% fructose. In some embodiments, the substrate containing fructose is a fructose syrup. In some embodiments, the substrate containing fructose contains glucose and / or other sugars. In some embodiments, the substrate containing fructose is 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% glucose. In some embodiments, when the substrate containing fructose contains glucose, the substrate containing fructose may be contacted with glucose isomerase to convert the glucose to fructose.

[0059] In some embodiments, the substrate containing fructose is produced as part of a carbohydrate production process. In some embodiments, the methods for producing allulose provided herein are implemented as part of a carbohydrate production process. In some embodiments, the carbohydrate production process is a production process implemented at a biorefinery.

[0060] Those of general skill in the art are aware of available methods that may be used to prepare fructose and substrates containing fructose for use in the methods disclosed herein. Methods of preparation generally include process steps such as milling / grinding, liquefaction, saccharification, and isomerization for converting biomass to a sugar (e.g., a syrup).

[0061] Fructose and substrates containing fructose may be obtained from tubers, roots, stems, legumes, cereals or whole grain by processing derived starches. In some embodiments, the starch, and subsequently the fructose or substrate containing fructose, may be obtained from com, cobs, sugar cane, sugar beets, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum,rice, peas, bean, banana, or potatoes. In some embodiments, the fructose or substrate containing fructose is obtained from processing starch from corn or cobs.

[0062] Starch from a grain may be ground or whole and may include solids, such as com kernels, bran and / or cobs. The starch may also be highly refined raw starch or feedstock from starch refinery processes. Various starches, fructose, and substrates containing fructose also are commercially available.

[0063] The starch may be a crude starch from milled whole grain, which contains non-starch fractions, e.g., germ residues and fibers. Milling may comprise either wet milling or dry milling or grinding. In wet milling, whole grain is soaked in water or dilute acid to separate the grain into its component parts, e.g. , starch, protein, germ, oil, kernel fibers. Wet milling efficiently separates the germ and meal (i.e., starch granules and protein) and is suitable for production of syrups.

[0064] In dry milling or grinding, whole kernels are ground into a fine powder and often processed without fractionating the grain into its component parts. In some cases, oils and / or fiber from the kernels are recovered. Dry ground grain thus will comprise significant amounts of non-starch carbohydrate compounds, in addition to starch. Dry grinding of the starch substrate can be used for production of ethanol and other biochemicals.

[0065] Liquefaction refers to a process by which starch is converted to less viscous and shorter chain dextrins. Generally, this process involves gelatinization of starch simultaneously with or followed by the addition of an a-amylase, although additional liquefaction-inducing enzymes optionally may be added. The starch substrate is generally slurried with water. The starch slurry may contain starch as a weight percent of dry solids of about 10-55%, about 20-45%, about 30- 45%, about 30-40%, or about 30-35%. The a-amylase typically used for this application is thermally stable. The a-amylase is usually supplied, for example, at about 1500 units per kg dry matter of starch. To optimize a-amylase stability and activity, the pH of the slurry typically is adjusted to about pH 4.5-6.5 and about 1 mM of calcium (about 40 ppm free calcium ions) can also be added, depending upon the properties of the amylase used. Bacterial a-amylase remaining in the slurry following liquefaction may be deactivated via a number of methods, including lowering the pH in a subsequent reaction step or by removing calcium from the slurry in cases where the enzyme is dependent upon calcium.

[0066] The slurry of starch plus a-amylase may be pumped continuously through a jet cooker, which is steam heated to a temperature in the range of about 105°C to 110°C. Gelatinization occurs rapidly under these conditions, and the enzymatic activity, combined with the significant shear forces, begins the hydrolysis of the starch substrate. The residence time in the jet cooker is brief, e.g., anywhere in the range of about 4 to about 12 minutes. The partly gelatinized starch may bepassed into a series of holding tubes maintained at 105-110°C and held for 5-8 min. to complete the gelatinization process (“primary liquefaction”). Hydrolysis to the required DE is completed in holding tanks at 85 -95 °C or higher temperatures for about 1 to 2 hours (“secondary liquefaction”). The slurry is then allowed to cool to room temperature. This cooling step can be 30 minutes to 180 minutes, e.g., 90 minutes to 120 minutes. The liquefied starch typically is in the form of a slurry having a dry solids content (w / w) of about 10-50%; about 10-45%; about 15-40%; about 20-40%; about 25-40%; or about 25-35%.

[0067] Liquefied starch can be saccharified into a syrup rich in lower DP (e.g., DPI + DP2) saccharides, using glucoamylases, optionally in the presence of another enzyme(s). Exemplary DPI saccharides include glucose and fructose, and DP2 saccharides include, for example, maltose and sucrose. Depending on the enzymes used, syrups may contain a weight percent of DP2 of the total oligosaccharides in the saccharified starch exceeding 30%, e.g. , 45% - 65% or 55% - 65%. The weight percent of (DPI + DP2) in the saccharified starch may exceed about 70%, e.g., 75% - 85% or 80% - 85%.

[0068] In some embodiments, an isomerization step may be used to modify the composition of lower DP in the syrup. In some embodiments, enzymes may be used to increase the amount of fructose or DPI saccharides capable of being converted to fructose, e.g., glucose, in the syrup. However, any method of increasing the DPI content of syrup is contemplated as useful for the methods of converting D-fructose to allulose provided herein since DPI saccharides, such as glucose and fructose, can be converted either indirectly or directly to allulose. For example, a syrup may be contacted with the epimerases described herein allowing for the direct conversion of fructose present in the syrup to allulose. In some cases, the syrup may be contacted with a glucose isomerase enzyme to convert glucose present in the syrup to fructose, which can in turn be converted to allulose by contact with the provided epimerases.

[0069] In some embodiments, the substrate containing fructose is a syrup. In some embodiments, the syrup is high fructose corn syrup (HFCS). In some embodiments, the substrate containing fructose is a syrup that does not contain fructose, but contains saccharides, e.g., DPI saccharides, capable of being converted to fructose. In some embodiments, the substrate containing fructose is a syrup including DPI saccharides that may be converted to fructose. In some embodiments, the substrate containing fructose is a syrup containing fructose and DPI saccharides that may be converted to fructose. In some embodiments, the DPI saccharides is glucose. In some embodiments, the conversion of glucose to fructose is accomplished by enzymes, e.g., glucose isomerases.

[0070] The method of producing allulose from a substrate containing fructose, e.g., a syrup, may proceed by contacting an epimerase protein described herein with the fructose or the substrate containing fructose under the conditions described herein, see Section I-B. In this way, the conversion to allulose is improved. In some embodiments, when the substrate containing fructose includes only or further includes glucose, the substrate may be contacted or also contacted with a glucose isomerase to convert the glucose to fructose. Suitable isomerases for conversion of glucose to fructose include, but are not limited to, SWEETZYME® IT, IT Extra, T (Novozymes A / S); G-ZYME® IMGI, and G-ZYME® G993, KETOMAX®, G-ZYME® G993, G-ZYME® G993 liquid; GENSWEET® IGI (SA, HF, VHF, MAX); and GENSWEET® SGI. In some cases, the fructose may be further isolated or purified to increase the percentage of fructose. In some embodiments, the mixture may be purified to contain about or at least 95% fructose. The substrate containing fructose obtained from the converted glucose, may be contacted with an epimerase under the conditions described herein to convert the fructose to allulose. In some embodiments, the substrate is contacted with the glucose isomerase and subsequently contacted with the epimerase under the conditions described herein. In some embodiments, the substrate is contacted with the glucose isomerase and the epimerase simultaneously under the conditions described herein. It should be appreciated that in some cases a metal cofactor may be added to facilitate the activity of the glucose isomerase.

[0071] In some embodiments, the epimerase with which the fructose or substrate containing fructose is contacted is in soluble form. In some embodiments, the epimerase with which the fructose or substrate containing fructose is contacted is immobilized on a matrix. In some embodiments, for example when the substrate containing fructose is contacted with a glucose isomerase, the glucose isomerase is immobilized on a matrix. In some embodiments, the matrix is a granule. In some embodiments, the matrix is an ion exchange resin.

[0072] In some embodiments, the epimerase is immobilized on a first matrix and the glucose isomerase is immobilized on a second matrix. In some embodiments, the first matrix and the second matrix are made of different material. In some embodiments, the first matrix and the second matrix are made of the same material. In some embodiments, the first matrix and second matrix are granules. In some embodiments, the first matrix and second matrix are ion exchange resins. In some embodiments, the epimerase and the glucose isomerase are co-immobilized on a matrix. In some embodiments, the matrix is a granule. In some embodiments, the matrix is an ion exchange resin.

[0073] In some embodiments, the epimerase is in a soluble form and present in a reactor. In some embodiments, contacting the epimerase with the substrate containing fructose occurs by addingthe substrate to the reactor. In some embodiments, the reactor is a column, a tank, or a vessel. In some embodiments, the reactor is a column. Non- limiting examples of columns contemplated for use herein include fixed-bed columns and fluidized bed columns. In some embodiments, the substrate is allowed to pass through the column and is collected. In some embodiments, the reactor is a tank or vessel. Non-limiting examples of tanks and vessels contemplated for use herein include fluidized bed tanks, stirred tanks, and stirred vessels. In some embodiments, the substrate is collected from the tank or reactor following contact with the epimerase.

[0074] It should be understood that the number and configuration of reactors depends on the composition of the substrate and whether sequential or simultaneous contacting of the substrate with the enzymes is preferred.

[0075] In some embodiments, the substrate contacted with the epimerase is collected from the reactor. For example, if using a column, the substrate may be added at one end of the column, allowed to pass through the matrix having the immobilized protein, and collected at the other end. If using a tank or vessel, in some embodiments, the substrate is collected from the tank or vessel. In some embodiments, the collected substrate contains allulose. In some embodiments, the allulose is purified from the collected substrate. Purification of the allulose may include one or more steps. In some embodiments, a step in the purification process includes removing a base.B. Conditions

[0076] In some embodiments, the conditions under which the contacting of the fructose containing substrate and the enzyme (epimerase) occur to increase the yield of allulose production involve control of the pH of the reaction. In some embodiments, the conditions involve control of the metal cofactor concentration in the reaction. In some embodiments, the conditions involve control of the temperature of the reaction. In some embodiments, the conditions involve the control of pH and metal cofactor of the reaction. In some embodiments, the conditions involve the control of pH, metal cofactor, and temperature of the reaction.

[0077] In some embodiments, the contacting occurs in the presence of a base. In some embodiments, the base is a corrosive or highly corrosive base. In some embodiments, the base is sodium hydroxide. In some embodiments, the base is added to the substrate containing fructose. For example, the base may be added to the substrate containing fructose prior to contacting the substrate with the epimerase. In some embodiments, the base is added to the substrate containing fructose no more than 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute before contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the substrate containing fructose no more than 30 minutes before contacting the substrate containing fructosewith the enzyme. In some embodiments, the base is added to the substrate containing fructose no more than 15 minutes before contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the substrate containing fructose no more than 10 minutes before contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the substrate containing fructose no more than 5 minutes before contacting the substrate containing fructose with the enzyme.

[0078] In some embodiments, the base is added to the reaction mixture (the substrate containing fructose and the epimerase). In some embodiments, the base is added at the beginning of the reaction. For example, in some embodiments, the base is added at the same time or shortly after the substrate containing fructose and the epimerase are put in contact, e.g., the reaction mixture is formed. In some embodiments, the base is added to the reaction mixture no more than 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute after contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the reaction mixture no more than 30 minutes after contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the reaction mixture no more than 15 minutes after contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the reaction mixture no more than 10 minutes after contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added to the reaction mixture no more than 5 minutes after contacting the substrate containing fructose with the enzyme. In some embodiments, the base is added once to the substrate containing fructose, e.g., prior to forming the reaction mixture. In some embodiments, the base is added once or at least once during the reaction, for example, at the same time or shortly after the substrate containing fructose and the epimerase are put in contact, e.g., as described herein. In some embodiments, the base is added more than once. For example, in some embodiments, the base may be added to the substrate containing fructose prior to contacting the substrate with the epimerase (e.g., forming the reaction mixture) and then added again to the reaction mixture. In some cases where the base is added to the substrate before contacting with the epimerase, the base may be added immediately or shortly after the substrate containing fructose and the epimerase are put in contact and then added at intervals or continuously throughout the duration of the reaction. In some embodiments, the base may be added immediately or shortly after the substrate containing fructose and the epimerase are put in contact and then added at intervals or continuously throughout the duration of the reaction. In some embodiments, the base is added, for example to the substrate or the reaction mixture, to bring the reaction mixture to a pH between about 5 and about 10. In some embodiments, the base is added, for example tothe substrate or the reaction mixture, to bring the reaction mixture to a pH between about 5 and about 9. In some embodiments, the base is added, for example to the substrate or the reaction mixture, to bring the reaction mixture to a pH between about 5 and about 8. In some embodiments, the base is added at intervals or continuously throughout the duration of the reaction to bring the reaction mixture to a pH between about 5 and about 10. In some embodiments, the base is added at intervals or continuously throughout the duration of the reaction to bring the reaction mixture to a pH between about 5 and about 9. In some embodiments, the base is added at intervals or continuously throughout the duration of the reaction to bring the reaction mixture to a pH between about 5 and about 8. In some embodiments, the pH of the reaction is maintained at a pH between about 5 and about 10 by adding, as described herein, a base to the substrate containing fructose prior to the contacting or to the reaction mixture. In some embodiments, the pH of the reaction is maintained at a pH between about 5 and about 9 by adding, as described herein, a base to the substrate containing fructose prior to the contacting or to the reaction mixture. In some embodiments, the pH of the reaction is maintained at a pH between about 5 and about 8 by adding, as described herein, a base to the substrate containing fructose prior to the contacting or to the reaction mixture.

[0079] In some embodiments, the concentration of base added, e.g., as described herein, is at least 5 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 10 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 25 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 50 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 75 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 100 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 125 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 150 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 200 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 300 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 400 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is at least 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 400 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 300 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 200 mg / L. In some embodiments,the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 150 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 125 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 100 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 75 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 50 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 25 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 5 mg / L to 10 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 10 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 25 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 50 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 75 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 100 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 125 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 150 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 200 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 300 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 400 mg / L to 500 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 20 mg / L to 150 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 25 mg / L to 125 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 50 mg / L to 125 mg / L. In some embodiments, the concentration of base added, e.g., as described herein, is in a range of 50 mg / L to 100 mg / L. It should be appreciated that the addition of the base may occur as described above, for example, once, at intervals, or continuously, and to the syrup, epimerase, or the reaction mixture. The amount of the base added will depend on whether the addition is once, at intervals, or continuously. In some embodiments, the amount of base added is determined by the pH to be achieved and / or maintained in the reaction, e.g., a pH in the range of 5 to 10. In some embodiments, the amount of base added is determined by the pH to be achieved and / or maintained in the reaction, e.g., a pH in the range of 5 to 10, and the method of adding, e.g., once, at intervals, or continuously.

[0080] In some embodiments, the contacting occurs under conditions where no metal cofactor is added. For example, in some cases, the epimerase does not require more metal cofactor than what is naturally present in the reaction. In some embodiments, the contacting occurs under conditions where a metal cofactor is present. In some embodiments, the contacting further occurs under conditions where a metal cofactor is present. In some embodiments, the metal cofactor is present at a concentration of less than about 0.5 ppm. In some embodiments, the metal cofactor is present at a concentration of less than about 0. 1 ppm. In some embodiments, the metal cofactor is present at a concentration of less than about 0.05 ppm. In some embodiments, the metal cofactor is present at a concentration of about or at least about 0.5 ppm. In some embodiments, the metal cofactor is present at a concentration of at least 0.5 ppm. In some embodiments, the metal cofactor is present at a concentration of greater than 0.5 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 100 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 75 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 50 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 25 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 15 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 10 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 5 ppm. In some embodiments, the metal cofactor is present at a concentration in the range of about 0.5 ppm to about 1 ppm.

[0081] In some embodiments, the concentration of the metal cofactor described herein exists without the need to supplement the metal cofactor. In some embodiments, the metal cofactor is added to the epimerase. In some embodiments, the metal cofactor is added to the substrate containing fructose. In some embodiments, the metal cofactor is added to the substrate containing fructose no more than 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute before contacting the substrate containing fructose with the enzyme. In some embodiments, the metal cofactor is added to the reaction mixture. In some embodiments, the metal cofactor is added at the beginning of the reaction. For example, in some embodiments, the metal cofactor is added at the same time or shortly, e.g., as described above, after the substrate containing fructose and the epimerase are put in contact, e.g., the reaction mixture is formed. In some embodiments, the metal cofactor is added once or at least once during the reaction, for example, at the same time or shortly after the substrate containing fructose and the epimerase are put in contact. In some embodiments, the metal cofactoris added more than once during the reaction. For example, in some embodiments, the base may be added at the same time or shortly after the substrate containing fructose and the epimerase are put in contact and then added at intervals or continuously throughout the duration of the reaction. In some embodiments, the metal cofactor is added to bring or maintain the metal cofactor at a concentration described herein. In some embodiments, the metal cofactor is added at intervals or continuously throughout the duration of the reaction to bring or maintain the metal cofactor at a concentration described herein.

[0082] In some embodiments, the metal cofactor is an ion. In some embodiments, the metal cofactor is a salt. In some embodiments, the metal cofactor is magnesium, manganese, cobalt or any combination thereof. In some embodiments, the metal cofactor is magnesium or a salt thereof. In some embodiments, the metal cofactor is manganese or a salt thereof. In some embodiments, the metal cofactor is cobalt or a salt thereof.

[0083] In some embodiments, the contacting occurs under conditions including a temperature in the range of about 50°C to about 90°C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 50°C to about 85°C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 50°C to about 80°C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 50°C to about 75°C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 50 °C to about 70 °C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 60°C to about 70°C. In some embodiments, the contacting occurs under conditions including a temperature of about 50°C. In some embodiments, the contacting occurs under conditions including a temperature of about 55 °C. In some embodiments, the contacting occurs under conditions including a temperature of about 60 °C. In some embodiments, the contacting occurs under conditions including a temperature of about 65 °C. In some embodiments, the contacting occurs under conditions including a temperature of about 70°C. In some embodiments, the contacting occurs under conditions including a temperature of about 75 °C. It is also possible for the contacting to occur at an initial temperature, e.g., 50°C, when the enzymes are first contacted with the substrate containing fructose, and ramped to higher temperatures, e.g., up to 70°C, for subsequent contacting with the substrate containing fructose.C. Epimerases

[0084] Enzymes contemplated for use in the methods described herein include epimerases capable of converting fructose to allulose. In some embodiments, the epimerases provided are D-allulose 3-epimerases and homologs found in microorganisms, e.g., bacteria. In some embodiments, theepimerases have an increased thermal stability, an increased pH stability or activity, and / or do not require or require less added metal cofactor compared to other D-allulose 3-epimerase homologs. Methods of determining thermostability, pH stability and activity, and metal cofactor requirements are known in the art. Nonlimiting examples of suitable epimerases are described in International PCT publication WO2023 / 114814, which is incorporated by reference in its entirety.

[0085] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:2, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:2, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:2, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:2, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:2, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:2.

[0086] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:1.

[0087] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:4, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:4, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:4, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:4, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:4, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:4.

[0088] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:3.

[0089] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:6, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:6, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:6, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:6, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:6, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:6.

[0090] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:5.

[0091] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO: 8, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:8, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO: 8, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:8, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:8, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:8.

[0092] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:7.

[0093] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO: 10, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 10, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO: 10, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO: 10, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:10, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO: 10.

[0094] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:9.

[0095] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO: 12, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 12, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO: 12, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO: 12, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:12, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO: 12.

[0096] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO: 11.

[0097] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO: 14, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 14, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO: 14, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO: 14, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:14, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO: 14.

[0098] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO: 13.

[0099] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:16, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 16, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO: 16, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO: 16, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:16, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO: 16.

[0100] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO: 15.

[0101] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO: 18, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 18, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO: 18, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO: 18, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO: 18, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO: 18.

[0102] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO: 17.

[0103] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:20, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:20, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:20, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:20, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:20, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:20.T1

[0104] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO: 19.

[0105] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:22, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:22, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:22, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:22, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:22, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:22.

[0106] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:21.

[0107] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:24, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:24, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:24, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:24, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:24, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:24.

[0108] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:23.

[0109] In some embodiments, the epimerase includes or is an amino acid sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:26, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:26, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:26, and where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 90% sequence identity to the sequence set forth by SEQ ID NO:26, where the protein has epimerase activity. In some embodiments, the epimerase includes or is an amino acid sequence having at least 95% sequence identity to the sequence set forth by SEQ ID NO:26, where the protein has epimerase activity. In some embodiments, the epimerase includes or is the amino acid sequence set forth by SEQ ID NO:26.

[0110] In some embodiments, the nucleic acid molecule is or contains a nucleic acid sequence encoding an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by of SEQ ID NO:25.EXEMPLARY EMBODIMENTS

[0111] Among the provided embodiments are:1. A method for producing allulose, comprising contacting a substrate comprising fructose with an epimerase, wherein the contacting occurs in the presence of a base present at a concentration of at least 5 mg / L.2. A method for producing allulose, comprising contacting a substrate comprising fructose with an epimerase, wherein the contacting occurs under conditions comprising a pH in a range of about 5 to about 10.3. The method of embodiment 2, wherein the contacting occurs in the presence of a base.4. The method of embodiment 3, wherein the base is present at a concentration of at least 5 mg / L.5. The method of any one of embodiments 1, 3, or 4, wherein the base is present at a concentration in a range of 20 to 500 mg / L.6. The method of any one of embodiments 1, 3, 4, or 5, wherein the base is sodium hydroxide.7. The method of any one of embodiments f or 3-6, wherein the base is continuously added to maintain the pH in the range of about 5 to about 10.8. The method of any one of embodiments 1-7, wherein the conditions further comprise a metal cofactor.9. The method of embodiment 8, wherein the metal cofactor is magnesium, cobalt, manganese, or any combination thereof.10. The method of any one of embodiments 1-9, wherein the conditions further comprise a temperature in a range of about 50°C to about 90 °C.11. The method of any one of embodiments 1-10, wherein the epimerase is soluble.12. The method of any one of embodiments 1-11, wherein the epimerase comprises an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 2.13. The method of any one of embodiments 1-12, wherein the contacting occurs in a reactor.14. The method of embodiment 13, wherein the reactor is a tank, vessel, or column.15. The method of any one of embodiments 1-14, wherein the substrate comprising fructose is a fructose syrup or is produced by: (i) contacting a substrate comprising glucose with a glucose isomerase prior to contacting the epimerase; or (ii) contacting a substrate comprising glucose with a glucose isomerase at the same time as contacting the epimerase.16. The method of any one of embodiments 1-15, comprising purifying the produced allulose.17. A composition for producing allulose comprising: i) an epimerase; ii) a substrate comprising fructose; and iii) a base at a concentration of at least 5 mg / L.18. The composition of embodiment 17, wherein the base is sodium hydroxide.19. The composition of embodiment 17 or embodiment 18, wherein the epimerase is soluble.20. The composition of embodiment 19, wherein the epimerase comprises an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO: 2.21. The composition of any one of embodiments 17-20, wherein the substrate comprises glucose.22. The composition of any one of embodiments 17-21, further comprising a glucose isomerase.23. The composition of any one of embodiments 17-22, wherein the composition is comprised in a reactor, optionally wherein the reactor is a tank, vessel, or column.IL EXAMPLES

[0112] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Expression and production of D-allulose 3-epimerases

[0113] Amino acid and nucleic acid sequences of an exemplary D-allulose 3-epimerase are shown in Table El. The expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif, 55:40- 52, 2007) was employed for the expression of the exemplary epimerase in Bacillus subtilis. The plasmid contained an aprE promoter followed by a codon-optimized nucleotide sequence encoding the protein sequence of the target gene. The corresponding protein sequence (SEQ ID NO: 2) and codon-optimized gene sequence (SEQ ID NO: 1) are shown in Table El.

[0114] Competent B. subtilis cells were transformed and plated on Luria Agar plates supplemented with 5 ppm chloramphenicol. Colonies were picked and subjected to fermentation in a 250ml shake flask with MBD medium (a MOPS based defined medium, supplemented with additional 5mM CaCh). Supernatants from these cultures were used to confirm the protein expression by SDS-PAGE analysis and assay for enzyme activity.

[0115] D-allulose 3-epimerase proteins were produced by liquid fermentation of B. subtilis. The inoculum was grown in a seed flask containing LB medium. A production medium including minerals (e.g., potassium sulfate, magnesium sulfate, ferrous sulfate, citric acid), one or more carbon sources (e.g., glucose, soy flour), and a complex nitrogen source was used to produce theexemplary epimerases. The production media was pH controlled and cells were fed according to oxygen uptake rates. The D-allulose 3-epimerase protein accumulated in the broth / cells.

[0116] Various parameters were monitored, including, but not limited to: CER (carbon dioxide evolution rate), OUR (oxygen uptake rate), pH, DO (dissolved oxygen), OD (optical density).Example 2: Effect of base addition on enzymatic conversion of fructose to allulose

[0117] An ultrafiltration concentrate (UFC) prepared from lysed production broth containing D-allulose 3-epimerase (see Example 1) was dosed into a fructose syrup with or without the addition of sodium hydroxide.

[0118] The syrup contained crystalline fructose (Spectrum Chemicals, catalog no. F1092) dissolved in Milli-Q Ultrapure Water supplemented with 100 ppm sodium metabisulfite (Spectrum Chemicals, catalog no. SO181). Syrup was then supplemented with sodium hydroxide at different concentrations (JT Baker, fisher scientific catalog number 02-004-148).

[0119] D-allulose 3-epimerase containing UFC was dosed into syrup on a weight per weight basis and the resulting mixture incubated in a water bath at 55 °C for 21 hours. The maximum dose of enzyme used was the same for each condition and is shown as “1” in Table E2 below. The lower doses of enzyme are shown relative to the maximum dose and were also identical across conditions. Conversion of fructose to allulose was measured by HPLC on an Agilent 1200 series equipped with a Rezex RCM-Monosaccharide Ca+2 (8%) LC Column 150 x 7.8 mm (Phenomenex catalog number OOF-0130-K0). The allulose peak integration area was divided by the integration area of the fructose and allulose peaks added together.Table E2: Epimerase conversion of fructose in the presence or absence of sodium hydroxide (NaOH).

[0120] The presence of base at any concentration increased the conversion of fructose to allulose by at least 4-fold, regardless of enzyme dose, compared to the conversion in the absence of sodium hydroxide.

[0121] These results indicate the presence of a base improves the ability of epimerases to convert fructose into allulose.Example 3: Expression and production of exemplary D-allulose 3-epimerases

[0122] Amino acid and nucleic acid sequences of exemplary D-allulose 3-epimerases are shown in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26 and SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25, respectively. The expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif, 55:40-52, 2007) may be employed for the expression of the exemplary epimerases in Bacillus subtilis. The plasmid may contain an aprE promoter followed by a codon- optimized nucleotide sequence encoding the protein sequence of the target gene.

[0123] Competent B. subtilis cells will be transformed and plated on Luria Agar plates supplemented with 5 ppm chloramphenicol. Colonies will be picked and subjected to fermentation in a 250ml shake flask with MBD medium (a MOPS based defined medium, supplemented with additional 5mM CaCh). Supernatants from these cultures will be used to confirm the protein expression by SDS-PAGE analysis and assay for enzyme activity.

[0124] The exemplary D-allulose 3-epimerase proteins will be produced by liquid fermentation of B. subtilis. The inoculum will be grown in a seed flask containing LB medium. A production medium including minerals (e.g., potassium sulfate, magnesium sulfate, ferrous sulfate, citric acid), one or more carbon sources (e.g., glucose, soy flour), and a complex nitrogen source will be used to produce the exemplary epimerases. The production media will be pH controlled and cells will be fed according to oxygen uptake rates. The D-allulose 3-epimerase protein will accumulate in the broth / cells.

[0125] Various parameters will be monitored, including, but not limited to: CER (carbon dioxide evolution rate), OUR (oxygen uptake rate), pH, DO (dissolved oxygen), OD (optical density).Example 4: Effect of base addition on enzymatic conversion of fructose to allulose by exemplary epimerases

[0126] An ultrafiltration concentrate (UFC) prepared from lysed production broth containing exemplary D-allulose 3-epimerases (see Example 3) will be dosed into a fructose syrup with or without the addition of sodium hydroxide.

[0127] The syrup will contain crystalline fructose (Spectrum Chemicals, catalog no. F1092) dissolved in Milli-Q Ultrapure Water supplemented with 100 ppm sodium metabisulfite (Spectrum Chemicals, catalog no. SO 181). Syrup will then be supplemented with sodium hydroxide at different concentrations (JT Baker, fisher scientific catalog number 02-004-148).

[0128] D-allulose 3-epimerase containing UFC will be dosed into syrup on a weight per weight basis and the resulting mixture incubated in a water bath at 55 °C for 21 hours. The maximum dose of enzyme will be the same for each condition and shown as “ 1 The lower doses of enzyme will be shown relative to the maximum dose and will be identical across conditions. Conversion of fructose to allulose will be measured by HPLC on an Agilent 1200 series equipped with a Rezex RCM-Monosaccharide Ca+2 (8%) LC Column 150 x 7.8 mm (Phenomenex catalog number OOF-0130-K0). The allulose peak integration area will be divided by the integration area of the fructose and allulose peaks added together.

[0129] The presence of base at any concentration is expected to increase the conversion of fructose to allulose compared to the conversion in the absence of sodium hydroxide.

[0130] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. Although the invention may be described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for producing allulose, comprising contacting a substrate comprising fructose with an epimerase, wherein the contacting occurs in the presence of a base present at a concentration of at least 5 mg / L.

2. A method for producing allulose, comprising contacting a substrate comprising fructose with an epimerase, wherein the contacting occurs under conditions comprising a pH in a range of about 5 to about 10.

3. The method of claim 2, wherein the contacting occurs in the presence of a base.

4. The method of claim 3, wherein the base is present at a concentration of at least 5 mg / L.

5. The method of any one of claims 1, 3, or 4, wherein the base is present at a concentration in a range of 20 to 500 mg / L.

6. The method of any one of claims 1, 3, 4, or 5, wherein the base is sodium hydroxide.

7. The method of any one of claims 1 or 3-6, wherein the base is continuously added to maintain the pH in the range of about 5 to about 10.

8. The method of any one of claims 1-7, wherein the conditions further comprise a metal cofactor.

9. The method of claim 8, wherein the metal cofactor is magnesium, cobalt, manganese, or any combination thereof.

10. The method of any one of claims 1-9, wherein the conditions further comprise a temperature in a range of about 50°C to about 90 °C.

11. The method of any one of claims 1-10, wherein the epimerase is soluble.

12. The method of any one of claims 1-11, wherein the substrate comprising fructose is a fructose syrup or is produced by:(i) contacting a substrate comprising glucose with a glucose isomerase prior to contacting the epimerase; or(ii) contacting a substrate comprising glucose with a glucose isomerase at the same time as contacting the epimerase.

13. The method of any one of claims 1-12, comprising purifying the produced allulose.

14. A composition for producing allulose comprising: i) an epimerase; ii) a substrate comprising fructose; and iii) a base at a concentration of at least 5 mg / L.

15. The composition of claim 14, wherein the base is sodium hydroxide.

16. The composition of claim 14 or claim 15, wherein the epimerase is soluble.

17. The composition of any one of claims 14-16, wherein the substrate comprises glucose.

18. The composition of any one of claims 14-17, further comprising a glucose isomerase.

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