Compositions and methods for producing allulose

Abstract

Provided herein are compositions and methods relating to epimerase enzymes for converting fructose to allulose.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from International Application No. PCT / CN2021 / 137842, filed December 14, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference The contents of the electronic submission of the Sequence Listing, named "NB42012WOPCT2_SequenceListing.xml", was created on December 13, 2022, is 50KB in size, and is incorporated by reference herein in its entirety.

[0003] Provided herein are compositions and methods relating to epimerase enzymes for converting fructose to allulose. [Background technology]

[0004] Allulose, also known as D-allulose and D-psicose, is a rare naturally occurring low-calorie sugar with a sweetness profile similar to that of sucrose, making it a desirable alternative to high-calorie sweeteners such as sucrose, fructose, and glucose. Allulose is the C-3 epimer of D-fructose and therefore can be produced commercially, for example, by the conversion of D-fructose to allulose by an enzyme such as an epimerase.

[0005] Epimerases capable of converting D-fructose to allulose have been found to have various characteristics that can affect their enzymatic activity, such as temperature, pH and metal cofactor requirements.Epimerases that are stable at high temperature and low pH and have reduced need for supplemental metal cofactors are particularly valuable for increasing the yield and quality of allulose during commercial production.

[0006] Thus, there is a need for an epimerase that can convert D-fructose to allulose at low pH and high temperature and has a reduced need for added metal cofactors. The compositions and methods provided herein address these and other needs in the art. Summary of the Invention [Problem to be solved by the invention]

[0007] Provided herein is a protein having epimerase activity comprising an amino acid sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. Provided herein is a protein having epimerase activity comprising an amino acid sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In some embodiments, the amino acid sequence has at least 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the amino acid sequence has at least 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In some aspects, the protein is immobilized on a matrix. In some embodiments, the matrix is ​​a granule or an ion exchange resin. [Means for solving the problem]

[0008] In one aspect, a nucleic acid molecule is provided that comprises a nucleic acid sequence encoding a protein described herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that i) encodes an amino acid sequence having at least 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; ii) has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18; or iii) hybridizes under stringent conditions to a nucleic acid sequence having a sequence complementary to the sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that: i) encodes an amino acid sequence having at least 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23; ii) has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28; or iii) hybridizes under stringent conditions to a nucleic acid sequence having a sequence complementary to the sequence shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28. In some embodiments, the nucleic acid molecule comprises a heterologous regulatory sequence. In some embodiments, the heterologous regulatory sequence is a promoter sequence.

[0009] In one aspect, a vector is provided that comprises a nucleic acid sequence described herein. In one aspect, a host cell is provided that comprises a nucleic acid molecule described herein or a vector described herein. In some embodiments, the host cell is a yeast, a bacterium, a mammalian cell, or a plant cell. In some embodiments, the host cell is a Bacillus spp. In some embodiments, the host cell is a Bacillus subtilis.

[0010] In one aspect, there is provided a cultured cell material comprising the proteins described herein and / or the host cells described herein. In one aspect, there is provided allulose produced by the proteins described herein.

[0011] In one aspect, a composition for producing allulose is provided, the composition comprising: i) a protein as described herein; ii) a substrate containing fructose. In some aspects, the protein is immobilized on a matrix. In some embodiments, the substrate comprises glucose. In some embodiments, the composition further comprises glucose isomerase immobilized on the matrix. In some embodiments, the protein and glucose isomerase are co-immobilized on the same matrix or immobilized on different matrices. In some embodiments, the composition is comprised in a reactor.

[0012] In one aspect, a use of the protein described herein for producing allulose is provided. In one aspect, a method of producing allulose is provided, comprising contacting a protein described herein with a substrate comprising fructose. In some embodiments, the contacting is performed under conditions comprising a temperature in the range of about 50° C. to about 90° C. In some embodiments, the contacting is performed under conditions comprising a pH in the range of about 4.5 to about 8. In some embodiments, the contacting is performed under conditions in which no metal cofactor is added or in which an amount of metal cofactor is added that is less than the metal cofactor concentration required for epimerase activity. In some embodiments, the protein is soluble and is contained in a reactor, and the contacting is performed by adding a substrate containing fructose to the reactor. In some embodiments, the protein is immobilized on a matrix contained in a reactor, and the contacting is performed by adding a substrate containing fructose to the reactor. In some embodiments, the fructose-containing substrate is produced by (i) contacting a substrate containing glucose with glucose isomerase prior to contacting the protein; or (ii) contacting a substrate containing glucose with glucose isomerase simultaneously with contacting the protein. In some embodiments, the method includes purifying the produced allulose.

[0013] In one aspect, a kit is provided that includes: (i) a protein described herein, a nucleic acid molecule described herein, a vector described herein, a host cell described herein, and / or a cell culture material described herein; and (ii) instructions for use.

[0014] Each of the aspects and embodiments described herein can be used together unless expressly or specifically excluded from the context of that embodiment or aspect. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Provided herein are compositions and methods related to epimerase enzymes, also referred to as epimerases, for converting D-fructose (D-fructose and fructose are used interchangeably herein) to allulose.

[0016] Allulose is a hexoketose monosaccharide sweetener that is the C-3 epimer of D-fructose, which is rarely found in nature. Allulose has similar physical properties to sucrose, such as bulk, texture, browning ability, gelation, and freezing point, and its sweetness is estimated to be about 70% of that of sucrose. However, the energy value of allulose is about 0.3% of that of sucrose. In addition to having a low caloric value, allulose may have beneficial physiological effects such as blood glucose suppression, reactive oxygen species scavenging, and neuroprotection, among others. These properties make allulose an attractive alternative to high-calorie sweeteners, such as sucrose, fructose, and glucose.

[0017] Since allulose naturally occurs in small amounts in certain foods, there is a need for methods to efficiently and effectively produce allulose. Bioconversion of D-fructose to D-allulose by epimerases, such as D-tagatose 3-epimerase (DT3E) and D-allulose 3-epimerase, is one such method to produce allulose. However, epimerases capable of performing this conversion, most of which are of bacterial origin, are known to have various properties that may affect enzyme activity and efficiency, such as temperature, pH and metal cofactor requirements. For example, most of the epimerases identified as capable of performing this conversion show a dependency on manganese, cobalt and / or magnesium as cofactors, with activity and optimal temperature and pH ranges for activity of 40°C-70°C and 7.0-9.0 pH, respectively. For commercial production, it is preferred to use higher temperatures, e.g., temperatures above about 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 preferred to use an acidic pH, especially at high temperatures, to reduce nonenzymatic browning of sugars, e.g., via the Maillard reaction. The use of high temperatures and acidic pH in the production process provides additional benefits, such as microbial control, e.g., by reducing microbial growth. Furthermore, for commercial production, it is beneficial to use enzymes that do not require the addition (supplementation) or require reduced amounts of metal cofactors added for activity, in order to eliminate additional steps in the production process and / or reduce production costs. As described herein, D-allulose 3-epimerase homologs have been identified that can convert D-fructose to allulose and have such desirable temperature, pH and / or metal cofactor properties for commercial production. See Examples. The compositions and methods provided herein take advantage of these surprising discoveries.

[0018] The conversion of D-fructose to D-allulose using compositions, such as epimerases and / or immobilized compositions thereof, and the methods described herein provide a means to produce allulose under favorable commercial conditions for epimerase activity and sugar stability. The use of the compositions and methods described herein can help diversify the sweetener product portfolio associated with corn processing by adding natural low-calorie sweeteners and bulking agents to traditional sweeteners derived from corn starch (e.g., corn syrup, high fructose corn syrup (HFCS), glucose and fructose).

[0019] The headings provided herein are not intended to limit the various aspects or embodiments of the present disclosure, which can be understood by reference to the specification as a whole. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. The reader will understand that statements made in one section may apply to other sections. Any terms defined may be more fully defined by reference to the specification as a whole.

[0020] 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 was individually incorporated by reference. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims. To the extent that a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0021] definition Definitions of terms may appear throughout the specification. It is to be understood that the present disclosure is not limited to the specific embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting.

[0022] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" includes "at least one" as well as "one or more."

[0023] As used herein, the terms "comprising," "including," and "composed of" are synonymous with "including," "comprises," or "containing," "containing" and grammatical variations thereof, and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "including," "comprises," "comprising," "comprising," "containing," "containing," "containing" and grammatical variations thereof also include the term "consisting of."

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0025] The term "starch" means a compound of the formula (CH 10 O5)x (wherein X can be any number) and is composed of complex polysaccharide carbohydrates of plants composed of amylose and amylopectin. The term includes plant-based materials such as grains, cereals, grasses, tubers and roots, more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, bran, 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 pregelatinized.

[0026] The terms "wild-type," "parent," or "reference" with respect to a polypeptide refer to a naturally occurring polypeptide that does not contain an artificial substitution, insertion, or deletion at one or more amino acid positions. Similarly, the terms "wild-type," "parent," or "reference" with respect to a polynucleotide refer to a naturally occurring polynucleotide that does not contain artificial nucleoside changes. However, it should be noted that a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to naturally occurring polynucleotides, but encompasses any polynucleotide that encodes a wild-type, parent, or reference polypeptide.

[0027] Reference to a wild-type polypeptide is understood to include the mature form of the polypeptide. A "mature" polypeptide or variant thereof is one in which the signal sequence is absent, e.g., one that is cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

[0028] The term "variant" with respect to a polypeptide refers to a polypeptide that differs from a particular wild-type, parent, or reference polypeptide in that it contains one or more naturally occurring or artificial substitutions, insertions, or deletions of amino acids. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parent, or reference polynucleotide. A variant may contain more than one mutation, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, substitutions, deletions, and / or insertions, compared to a wild-type, parent, or reference polypeptide or polynucleotide. The identity of the wild-type, parent, or reference polypeptide or polynucleotide will be clear from the context.

[0029] In the case of epimerases described herein, "activity" refers to epimerase activity, which can be measured as described herein. It should be understood that epimerases act in both directions as an equilibrium conversion reaction that interconverts fructose to allulose. In some embodiments, activity includes or is the conversion of fructose to allulose. In some embodiments, activity includes or is the conversion of allulose to fructose. Estimates of activity can be determined by an assay designed to assess fructose formation from allulose, such as a colorimetric assay, and / or by an assay designed to assess allulose formation from fructose, such as high performance liquid chromatography (HPLC). In some embodiments, activity is referred to as residual activity. As used herein, "residual activity" includes or is the activity of an epimerase after stimulation, such as high temperature stimulation and / or pH stimulation, compared to the activity of an unstimulated epimerase, which serves as a baseline for comparison, and is the epimerase activity determined in a particular state, such as immobilized state, compared to the activity of the epimerase in a different state (e.g., solubilized), which serves as a baseline. Residual activity can be expressed as a percentage or fraction of baseline activity (e.g., baseline activity is equal to 100% or 1). Methods for determining the activity of an enzyme are various and known in the art.

[0030] The term "recombinant" when used with respect to a subject cell, nucleic acid, protein, or vector indicates that the subject has been modified from its native state. Thus, for example, a recombinant cell expresses a gene not found in the native (non-recombinant) form of the cell, or expresses a native gene at a different level or under different conditions than found in nature. A recombinant nucleic acid differs from the native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence, e.g., a heterologous promoter in an expression vector. A recombinant protein may differ from the native sequence by one or more amino acids and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding an epimerase may be referred to as a recombinant vector.

[0031] The terms "recovered," "isolated," and "separated" refer to a compound, a protein (polypeptide), a cell, a nucleic acid, an amino acid, or other specific material or component that has been 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 specific 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 specific material or component is associated under experimental or production conditions and / or systems. For example, an "isolated" polypeptide includes, but is not limited to, a polypeptide that has been removed from a culture broth containing a heterologous host cell that expresses the polypeptide.

[0032] The term "purified" refers to a 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.

[0033] The term "enriched" refers to a substance (e.g., an isolated compound, polypeptide, polynucleotide, or other specified material or component) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 80% pure.

[0034] As used herein, "derived" includes "originating from," "obtained from," or "isolated from."

[0035] The terms "thermostable", "thermostable" and "thermostable" with respect to an enzyme refer to the ability of the enzyme to retain activity at or after exposure to high temperatures. Methods for determining thermostability are varied and known in the art. In some cases, the thermostability of an enzyme, such as an epimerase enzyme, can be measured by its half-life (t1 / 2), given in minutes, hours or days, during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring the residual epimerase activity after exposure to high temperatures. In some cases, thermostability is determined by measuring the epimerase activity after exposure to high temperatures and comparing the measured activity to a baseline activity, which is measured from an epimerase that was not exposed to high temperatures. The value resulting from the comparison can be referred to as the residual activity.

[0036] The "pH range" of an enzyme refers to the range of pH values ​​over which the enzyme is active. The pH range over which an enzyme is active may be referred to as the "pH activity profile" of the enzyme. The terms "pH stability" and "pH stability" of an enzyme refer to the ability of an enzyme to retain activity after exposure to a pH or pH. Methods for determining the 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 over a range of pH. In this case, minimum and maximum activity levels may be determined to generate a dose-response curve or a standard curve. In some cases, the pH stability is determined by measuring the epimerase activity after exposure to pH and comparing the measured activity to a baseline activity, the baseline activity being measured from an epimerase that was not exposed to pH. The value resulting from the comparison may be referred to as the residual activity.

[0037] The term "amino acid sequence" is synonymous with, and is used interchangeably with, the terms "polypeptide," "protein," and "peptide." Such amino acid sequences, if they exhibit activity, may be referred to as "enzymes." Conventional single-letter or three-letter codes for amino acid residues are used, and amino acid sequences are presented in the standard amino-carboxy terminal orientation (i.e., N→C).

[0038] The term "nucleic acid" includes DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids can be single-stranded or double-stranded and can include chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid, and the present compositions and methods include nucleotide sequences that code for a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5' to 3' orientation.

[0039] "Hybridization" refers to the process in which one strand of nucleic acid forms a duplex with a complementary strand, i.e., base pairs with the complementary strand, as occurs during blot hybridization and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65°C and 0.1xSSC (where 1xSSC=0.15M NaCl, 0.015M Na3 citrate, pH 7.0). Hybridized duplex nucleic acids are characterized by a melting temperature (Tm), with one-half of the hybridized nucleic acid unpaired with the complementary strand. Mismatched nucleotides in the duplex lower the Tm.

[0040] The terms "transformed," "stably transformed," and "transgenic" as used with respect to a cell mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence that is integrated into its genome or maintained as an episome that is maintained over multiple generations.

[0041] The term "introduced" in the context of inserting a nucleic acid sequence into a cell includes, but is not limited to, "transfection," "transformation," and "transduction" as known in the art. Exemplary methods for introducing a polynucleotide or polypeptide into a host cell by transformation include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods (e.g., chemical (e.g., divalent cations such as CaCl2), mechanical (electroporation) means, or inducibility using methods such as those described in published International Patent 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, virus-mediated transfer, cell-penetrating peptides or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery. Introducing a nucleic acid, construct, plasmid, or vector into a host cell can be performed by conjugation, a specific method of natural DNA exchange that requires physical cell-cell contact. Introduction of a nucleic acid, construct, plasmid or vector into a host cell can be by transduction, which is the introduction of DNA via viral (e.g., phage) infection, which is also a natural method of DNA exchange. Generally, such methods involve incorporating a polynucleotide into a viral DNA or RNA molecule.

[0042] A "host cell" is an organism into which an expression vector, phage, virus, or other nucleic acid sequence containing a polynucleotide encoding a polypeptide of interest (e.g., an epimerase) has been introduced. Exemplary host cells are microbial cells (e.g., bacteria, filamentous fungi, and yeast), mammalian cells, and plant cells capable of expressing a polypeptide of interest. The term "host cell" includes protoplasts made from cells.

[0043] The term "heterologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0044] The term "endogenous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally within a host cell.

[0045] The term "expression" refers to the process by which a polypeptide is produced based on a nucleic acid sequence, which process includes both transcription and translation.

[0046] "Selection marker" or "selectable marker" refers to a gene that can be expressed in a host to facilitate selection of host cells that harbor the gene. Examples of selectable markers include, but are not limited to, antibacterial agents (e.g., hygromycin, bleomycin, or chloramphenicol) and / or genes that confer a metabolic advantage, such as a nutritional advantage, to the host cell.

[0047] "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, etc.

[0048] "Expression vector" refers to a DNA construct containing a DNA sequence encoding a polypeptide of interest, the coding sequence of which 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 for transcription, an optional operator sequence for controlling transcription, a sequence encoding suitable ribosome binding sites on the mRNA, an enhancer and sequences for controlling the termination of transcription and translation.

[0049] The term "operably linked" means that the particular components are in a relationship (including, but not limited to, juxtaposition) that permits them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0050] A "signal sequence" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[0051] 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 specified conditions. Specific activity is generally expressed as units (U) / mg of protein.

[0052] "Cultured cell material containing epimerase" or similar phrases refers to a cell lysate or supernatant (including culture medium) that contains the epimerase as a component. The cell material can be derived from a heterologous host cell grown in culture for the purpose of producing the epimerase.

[0053] "Percent sequence identity" means that a particular sequence has at least a specified percentage of identical amino acid residues or nucleotides to those in a particular reference sequence, when aligned, for example, using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are as follows: Gap Start Penalty: 10.0 Gap extension penalty: 0.05 Protein Weight Matrix: BLOSUM Series DNA Weight Matrix: IUB Delayed divergence sequence%:40 Gap Separation Distance: 8 DNA transfer weight: 0.50 List of hydrophilic residues: GPSNDQEKR Use negative matrix: OFF Toggle residue-specific penalty: ON Toggle Hydrophilic Penalty: ON Toggle End Gap Separation Penalty OFF.

[0054] Deletions are counted as non-identical residues compared to the reference sequence, including deletions occurring at either end.

[0055] The term "degree of polymerization" (DP) refers to the number (n) of anhydro-glucopyranose units in a given sugar. Examples of DP1 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 sugars, i.e., D-glucose, as a percentage of the total carbohydrates in a syrup.

[0056] The term "dry solids" (ds) refers to the total solids of the slurry on a dry weight percent basis. The term "slurry" refers to an aqueous mixture containing insoluble solids.

[0057] The phrase "simultaneous saccharification and fermentation (SSF)" refers to a process in the production of biochemicals in which a microorganism, e.g., an ethanologenic microorganism, and at least one enzyme, e.g., an amylase, are present in the same process step. SSF includes the simultaneous hydrolysis of a starch substrate (granular, liquefied, or solubilized) to sugars, including glucose, and the fermentation of the sugars to alcohol or other biochemicals or biological materials in the same reaction vessel.

[0058] Where a range of values ​​is given, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed to the first decimal place of the lower limit. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either limit is included, neither is included, or both are included in the smaller range, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0059] Numerical values ​​and ranges may be presented herein with the term "about" before the numerical value. The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close or approximately close to the number it precedes. In determining whether a number is close to or close to a specifically recited number, a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented may be a close or close number that is not recited. For example, the term "about" in relation to a numerical value refers to a range of -10% to +10% of the numerical value, unless the term is clearly defined otherwise in the context. All values ​​and ranges implicitly include the term "about" unless the context clearly indicates otherwise.

[0060] I. Epimerase Provided herein is an epimerase and a composition containing the epimerase that is useful for converting D-fructose to D-allulose.The epimerase described herein has functional properties that allow the enzyme to function under favorable conditions for converting D-fructose to allulose, such as stable sugar and equilibrium conversion conditions.Furthermore, as further described in Section II, the epimerase provided herein can be used to produce allulose from the effluent of existing procedures for producing fructose from starch.

[0061] In some aspects, the epimerases provided herein have increased thermostability, pH stability or activity compared to other D-allulose 3-epimerase homologs, and / or have magnesium (Mg 2+ D-allulose 3-epimerase homologues found in microorganisms, such as bacteria, that do not or only rarely require the addition of metal cofactors such as D-allulose 3-epimerase. See, e.g., the Examples. Methods for determining thermostability, pH stability and activity, as well as metal cofactor requirements, are known in the art and are also described in the Examples below (see Section V).

[0062] The epimerases described herein may be thermostable at elevated temperatures. In some embodiments, the epimerases described herein exhibit epimerase activity (converting fructose to allulose) at temperatures where the equilibrium is shifted resulting in a higher allulose conversion yield. In some embodiments, the epimerase is thermostable at a temperature of at least 40°C. In some embodiments, the epimerase is thermostable at a temperature of at least 50°C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 40°C to about 90°C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 50°C to about 90°C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 50°C to about 85°C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 50°C to about 80°C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 50°C to about 75°C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 50° C. to about 70° C. In some embodiments, the epimerase is thermostable at a temperature ranging from about 60° C. to about 70° C. In some embodiments, the epimerase is thermostable at a temperature of about 60° C. In some embodiments, the epimerase is thermostable at a temperature of about 70° C. In some embodiments, the epimerase is thermostable at a temperature of about 80° C. In some embodiments, the epimerase is thermostable at a temperature of about 85° C. In some embodiments, the epimerase is thermostable at a temperature of about 90° C. In some embodiments, at a temperature ranging from about 50° C. to about 90° C., the epimerase retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its baseline activity. In some embodiments, at a temperature ranging from about 50° C. to about 85° C., the epimerase retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its baseline activity.In some embodiments, the epimerase retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its baseline activity at a temperature in the range of about 50° C. to about 80° C. In some embodiments, the epimerase retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its baseline activity at a temperature in the range of about 60° C. to about 80° C. In some embodiments, at a temperature in the range of about 60° C. to about 75° C., the epimerase retains at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the baseline activity. In some embodiments, at a temperature in the range of about 60° C. to about 70° C., the epimerase retains at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the baseline activity. In some embodiments, at a temperature in the range of about 60° C. to about 70° C., the epimerase retains about 50% to 100% of the baseline activity. In some embodiments, at a temperature in the range of about 60° C. to about 70° C., the epimerase retains about 75% to 100% of the baseline activity. In some embodiments, at a temperature in the range of about 60° C. to about 70° C., the epimerase retains about 80% to 100% of the baseline activity. In some embodiments, at a temperature in the range of about 60° C. to about 70° C., the epimerase retains about 90% to 100% of the baseline activity. The baseline activity can be an activity determined for an epimerase that has not been exposed to a temperature (e.g., an elevated temperature) as described in this paragraph. In some embodiments, the baseline activity is an activity determined for an epimerase at a temperature of about 50° C. In some embodiments, the retained activity is sufficient to convert fructose to allulose.

[0063] Thermostability may be determined as the residual activity measured after the epimerase is exposed (e.g., incubated) at an elevated temperature for a particular period of time. In some embodiments, the epimerase retains residual activity after incubation at a temperature ranging from about 60° C. to about 70° C. for a period of about 5 minutes to about 120 minutes. In some embodiments, the residual activity retained after incubation is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the baseline activity. In some embodiments, the residual activity retained after incubation is at least or about 50% of the baseline activity. In some embodiments, the residual activity retained after incubation is at least or about 60% of the baseline activity. In some embodiments, the residual activity retained after incubation is at least or about 70% of the baseline activity. In some embodiments, the residual activity retained after incubation is at least or about 80% of the baseline activity. In some embodiments, the residual activity retained after incubation is at least or about 90% of the baseline activity. In some embodiments, the epimerase retains about 50%-100% of the baseline activity. In some embodiments, the epimerase retains about 75%-100% of the baseline activity. In some embodiments, the epimerase retains about 80%-100% of the baseline activity. In some embodiments, the epimerase retains about 90%-100% of the baseline activity. The baseline activity can be an activity determined for an epimerase that has not been subjected to an elevated temperature, such as the temperatures described above and in the previous paragraph. In some embodiments, the baseline activity is an activity determined for the epimerase at a temperature of about 50° C.

[0064] The epimerase for use described herein may have a desirable pH stability. For example, the epimerase described herein may have a pH stability in a pH range in which fructose and / or allulose are stable. In some embodiments, the epimerase is pH stable in a pH range that reduces non-enzymatic browning of sugars via the Maillard reaction. For example, the pH may be in a range in which the Maillard reaction proceeds at a slower rate compared to a neutral or basic pH, such as 7, 7.5, 8.5, 9, 9.5, or 10 pH. In some embodiments, the epimerase has a pH activity profile that is in or overlaps with a pH range in which the Maillard reaction reduces non-enzymatic browning of sugars via the Maillard reaction. In some embodiments, the epimerase is pH stable in a pH range in which the Maillard reaction reduces non-enzymatic browning of sugars via the Maillard reaction at a given temperature. In some embodiments, the epimerase has a pH activity profile that is in or overlaps with a pH range in which the Maillard reaction reduces non-enzymatic browning of sugars via the Maillard reaction at a given temperature. In some embodiments, the pH range may be a range that reduces the rate of the Maillard reaction at a particular temperature compared to a neutral or basic pH, such as 7, 7.5, 8.5, 9, 9.5, or 10 pH at the same temperature. In some embodiments, the epimerase is stable at a pH in the range of about 4 to about 10. In some embodiments, the epimerase is stable at a pH in the range of about 4.5 to about 10. In some embodiments, the epimerase is stable at a pH in the range of about 4.5 to about 9. In some embodiments, the epimerase is stable at a pH in the range of about 4.5 to about 8. In some embodiments, the epimerase is stable at a pH in the range of about 5 to about 8. In some embodiments, the epimerase is stable at a pH in the range of about 5 to about 7. In some embodiments, the epimerase is stable at a pH in the range of about 5.5 to about 6.5. The pH range in which the epimerase is stable may also be referred to herein as a pH range. For example, the pH ranges described above may be referred to as pH ranges.

[0065] In some embodiments, the epimerase retains an activity level of at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level of at least or about 25% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level of at least or about 45% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level of at least or about 50% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level of at least or about 60% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level of at least or about 70% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level of at least or about 75% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level in the range of about 50% to about 100% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level in the range of about 75% to about 100% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level in the range of about 80% to about 100% of the maximum activity level across the pH range. In some embodiments, the epimerase retains an activity level in the range of about 90% to about 100% of the maximum activity level across the pH range. In some embodiments, the pH range is the pH range described in the previous paragraph. In some embodiments, the pH range is about 4.5 to about 10. In some embodiments, the pH range is about 4.5 to about 9. In some embodiments, the pH range is about 4.5 to about 8. In some embodiments, the pH range is about 4.5 to about 7.5. In some embodiments, the pH range is from about 4.5 to about 7. In some embodiments, the pH range is from about 4.5 to about 6.5.In some embodiments, the pH range is about 4.5 to about 6. In some embodiments, the pH range is about 5 to about 6.5. In some embodiments, the pH range is about 5.5 to about 6.5. The maximum activity level can be determined by measuring the activity of the epimerase over a range of pH, e.g., to characterize a dose-response curve or a standard curve, to find the minimum and maximum activity levels. In some embodiments, the retained activity is sufficient to convert fructose to allulose.

[0066] The epimerases for use described herein may have reduced or no need for metal cofactor supplementation for activity. In some embodiments, the epimerases described herein retain the activity observed in the presence of a metal cofactor, for example, when the metal cofactor is intentionally added, when no metal cofactor is added or when a reduced amount of the metal cofactor is added. Thus, in some embodiments, the epimerases described herein do not require or require small amounts of metal cofactor to be supplemented in order for the epimerase to be active. In some embodiments, the epimerases described herein use existing metal cofactors present in the production process for activity. In some embodiments, the production process is a process for producing a substrate containing fructose (see, for example, Section II-A). In some embodiments, the epimerase retains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains at least or about 50% of the baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains at least or about 60% of the activity in the absence of added metal cofactor. In some embodiments, the epimerase retains at least or about 70% of the baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains at least or about 75% of the baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains at least or about 80% of its baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains at least or about 90% of its baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains about 50%-100% of its baseline activity in the absence of added metal cofactor.In some embodiments, the epimerase retains about 75% to 100% of the baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains about 80% to 100% of the baseline activity in the absence of added metal cofactor. In some embodiments, the epimerase retains about 90% to 100% of the baseline activity in the absence of added metal cofactor. In some embodiments, the baseline activity is the activity measured in the presence of a metal cofactor in a concentration range of about 0.1 to about 2 mM, about 0.1 to about 1 mM, about 0.1 to about 0.5 mM, about 0.1 to about 0.25 mM, or about 0.1 to about 0.15 mM. In some embodiments, the epimerase retains a percentage of the activity described in this paragraph and / or herein when an amount of metal cofactor is added that is less than the concentration of metal cofactor present for the baseline activity. In some embodiments, the reduced amount of metal cofactor is at a concentration less than the concentration of metal cofactor present for the baseline activity. In some embodiments, the reduced amount is at least or about 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% lower than the concentration of metal cofactor present for the baseline activity. In some embodiments, the reduced amount is in the range of about 20% to about 90% lower than the concentration of metal cofactor present for the baseline activity. In some embodiments, the reduced amount is in the range of about 30% to about 90% lower than the concentration of metal cofactor present for the baseline activity. In some embodiments, the reduced amount is in the range of about 40% to about 90% lower than the concentration of metal cofactor present for the baseline activity. In some embodiments, the reduced amount is in the range of about 50% to about 90% lower than the concentration of metal cofactor present for the baseline activity. In some embodiments, the retained activity is sufficient to convert fructose to allulose. In some embodiments, the metal cofactor is an ion. In some embodiments, the metal cofactor is magnesium. In some embodiments, the metal cofactor is in the form of a salt, hi some embodiments, the metal cofactor is a magnesium salt.

[0067] Epimerases for use as described herein may have any one or more of thermostability, pH stability, and / or reduced or no requirement for added metal cofactors as described above and herein. In some embodiments, the epimerase is thermostable, pH stable, and has reduced or no dependency on added metal cofactors. In some embodiments, the epimerase is thermostable. In some embodiments, the epimerase is pH stable. In some embodiments, the epimerase has reduced or no requirement for added metal cofactors.

[0068] The epimerases described herein having the characteristics described above and herein may be described as proteins, nucleic acid molecules, part of a vector, or as compositions. Also provided herein are methods for producing such epimerases.

[0069] A. Protein In one aspect, epimerase enzyme is provided, which is a protein having epimerase activity and can convert D-fructose to allulose.In some embodiments, the protein is D-allulose 3-epimerase.In some embodiments, the epimerase has one or more of the thermostability, pH stability, or metal cofactor requirement attributes described herein.

[0070] In some embodiments, the protein, i.e., the epimerase protein, comprises 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, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 70% sequence identity to the sequence set forth by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80% sequence identity to the sequence set forth by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 90% sequence identity to a sequence set forth by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, and the protein has epimerase activity.

[0071] In some embodiments, the protein, i.e., the epimerase protein, comprises 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 in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 90% sequence identity to a sequence set forth by SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23, and the protein has epimerase activity.

[0072] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 2, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:2.

[0073] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 3, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 3. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:3.

[0074] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 4, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 4. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:4.

[0075] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 5, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 5. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:5.

[0076] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:6, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:6. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:6. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:6.

[0077] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 7, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:7.

[0078] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 8, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:8.

[0079] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 9, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:9.

[0080] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 19. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:19.

[0081] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 20, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 20. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 20. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:20.

[0082] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 21, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 21. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 21. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:21.

[0083] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 22, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 22. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 22. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:22.

[0084] In some embodiments, the protein comprises or is an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 23, and the protein has epimerase activity. In some embodiments, the protein comprises or is an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 23. In some embodiments, the protein comprises or is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 23. In some embodiments, the protein comprises or is the amino acid sequence set forth in SEQ ID NO:23.

[0085] In some embodiments, the protein (i.e., the epimerase protein) may have any number of conservative amino acid substitutions well recognized in the art. The epimerase may be "precursor," "immature," or "full-length," in which case it includes a signal sequence, or it may be "mature," in which case it lacks a signal sequence. The mature form of the protein is generally the most useful. The epimerase may also be truncated to remove the N- or C-terminus, or extended to include additional N- or C-terminal residues, so long as the resulting protein retains activity.

[0086] B. Nucleic acid molecules In another aspect, a nucleic acid molecule is provided that is or comprises a nucleic acid sequence encoding an epimerase. The nucleic acid sequence may encode a particular epimerase described herein or an epimerase having a particular degree of amino acid sequence identity to a particular epimerase.

[0087] In some embodiments, the nucleic acid molecule is or comprises 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 SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the nucleic acid molecule is or comprises 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 SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 2. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 3. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 4. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence as set forth in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as set forth in SEQ ID NO: 5. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 6. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 7.In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 8, or a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 9, or a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 9. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 19, or a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 19. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 20, or a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 20. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 21, or a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 21. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID NO: 22, or a nucleic acid sequence encoding an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 22.In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence that encodes an amino acid sequence set forth in SEQ ID NO: 23, or a nucleic acid sequence that encodes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 23. It will be understood that due to the degeneracy of the genetic code, multiple nucleic acids may encode the same polypeptide.

[0088] In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) an epimerase protein having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) an epimerase protein having at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) an epimerase protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid encoding (or complementary to a nucleic acid encoding) an epimerase protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23.

[0089] In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth by SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 11, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 11. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 12, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 13, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 14, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 14. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 15, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 16, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 16.In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 17, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 17. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 18, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 18. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 24, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 24. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 25, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 25. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 26, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 26. In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO: 27, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 27.In some embodiments, the nucleic acid molecule is or comprises a nucleic acid sequence having a sequence set forth in SEQ ID NO:28, or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:28.

[0090] In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid sequence set forth in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18, or a nucleic acid complementary to these nucleic acid sequences. In some embodiments, the nucleic acid hybridizes under stringent conditions to a nucleic acid sequence set forth in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28, or a nucleic acid complementary to these nucleic acid sequences.

[0091] The nucleic acid molecule can encode a "full-length" ("fl" or "FL") epimerase that includes the signal sequence, only the mature form of the epimerase lacking the signal sequence, or a truncated form of the epimerase lacking the N-terminus or C-terminus of the mature form.

[0092] The nucleic acid molecule encoding the epimerase can be operably linked to various promoters and regulatory elements to drive expression, for example, when present in a host cell. Exemplary promoters are from B. licheniformis, B. subtilis, and Streptomyces. In some embodiments, the promoter is the aprE promoter. Such nucleic acid molecules can also be linked to other coding sequences, for example, to encode chimeric polypeptides. In some embodiments, a nucleic acid sequence encoding an epimerase described herein operably linked to a heterologous promoter and / or regulatory elements is referred to as a recombinant nucleic acid sequence.

[0093] In some embodiments, the nucleic acid molecules described herein may be present in a vector. The vector may be any vector into which a nucleic acid molecule can be inserted, introduced into a host cell, and optionally replicated in the host cell. In some embodiments, the vector may be referred to as an expression vector, meaning that the coding nucleic acid sequence contained in the vector is capable of in vivo or in vitro expression. The choice of vector, e.g., a plasmid, cosmid, virus, or phage vector, often depends on the host cell into which it is introduced. In some embodiments, the vector is a plasmid.

[0094] In some cases, the vector may contain one or more selectable marker genes, such as genes that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

[0095] C. Epimerase production The epimerases described herein can be produced in host cells using methods well known in the art, for example, by secretion or intracellular expression. Appropriate assays can be used to monitor epimerase activity in samples from host cell cultures, such as medium or cell samples (e.g., lysed cell samples). Fructose and / or allulose concentrations can be determined using high performance liquid chromatography (HPLC) or other means known in the art, such as colorimetric assays. In some embodiments, production of epimerase occurs by liquid fermentation of host cells. See, for example, Example 2.

[0096] Separation, isolation, and purification techniques are well known in the art, and conventional methods can be used to extract the epimerase from the host cell and / or culture medium. In some embodiments, the host cell is used to produce a cultured cell material that contains the epimerase. In some embodiments, the cultured cell material is a cell lysate or supernatant that contains the epimerase.

[0097] In some aspects, a host cell is provided that contains a nucleic acid molecule or vector described in Section IB. In some embodiments, the host cell is a yeast, a bacterium, a mammalian cell, or a plant cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is a bacterium. In some embodiments, the host cell is a Bacillus species. In some embodiments, the host cell is B. subtilus or B. lichenifomis. In some embodiments, the host cell is B. subtilus.

[0098] D. Epimerase Compositions In some embodiments, the epimase protein provided herein is in soluble form. In some embodiments, the soluble protein can be used in a reactor, such as a column, vessel, or tank reactor, to convert fructose, which is added to the reactor as part of a liquid substrate, into allulose. In some embodiments, the soluble protein is included in a composition that includes other proteins or enzymes, such as glucose isomerase. In some embodiments, the soluble protein is included in a composition that includes other components, such as metal ion cofactors.

[0099] In some embodiments, the epimerase protein described herein is immobilized on a matrix.Immobilizing epimerase on a matrix is ​​advantageous for extending the useful life of the enzyme.In some cases, immobilization allows the protein to be used in an industrial-scale process for the commercial production of allulose.For example, the matrix on which the protein is immobilized can be used in a reactor, such as a column, vessel, or tank reactor, to convert fructose, which is added to the reactor as part of a liquid substrate, into allulose.

[0100] In some embodiments, the produced epimerase is isolated and / or purified and immobilized on a matrix, e.g., as described in Sections I-C. In some embodiments, the host cells expressing the epimerase are immobilized on a matrix. For example, the host cells for epimerase production described in Section IC are immobilized on a matrix. In some embodiments, the broth containing the epimerase and the lysed host cells used to produce the expressed epimerase is immobilized on a matrix. In some embodiments, the cultured cell material containing the epimerase is immobilized on a matrix. The matrix may be contacted with the isolated and / or purified epimerase, the host cells expressing the epimerase, the broth, and / or the cultured cell material such that at least the epimerase is immobilized on the matrix. In some embodiments, the matrix on which the epimerase is immobilized is insoluble.

[0101] Exemplary matrices include, but are not limited to, granules, beads, ion exchange resins, and polymer encapsulation. Non-limiting examples of matrices contemplated herein as suitable supports include weak base polystyrene resins, weak base (-N(R)2) phenol-formaldehyde resins, strong base (-N(R)3) polystyrene resins, and / or various enzyme adsorbents such as DEAE-Sephadex, DEAE-Glycophase, QAE-Glycophase, DEAE Bio-Gel A, CM Bio-Gel A, Selectacel DEAE-cellulose, Granular DEAE-cellulose, DEAE Sephacel, DEAE-Cellulose Beads, Controlled Pore Glass, Controlled Pore Aluminia, Titania, Zirconia (Corning Glass), Bentonite, calcium carbonate, etc.

[0102] In some embodiments, the epimerase is immobilized on a matrix, for example, as described in U.S. Pat. Nos. 3,796,634; 4,355,105; 4,713,333; 5,177,005; 5,437,993; 5,811,280; 5,916,789; and 7,297,510. In some embodiments, the epimerase is immobilized in a granule. In some embodiments, the granule is a colloidal particle. The granule may include colloidal silica, activated carbon, hydroxyapatite, alumina C gamma, bentonite, diatomaceous earth, or a combination thereof. In some embodiments, the granule contains polyethyleneimine (PEI). In some embodiments, the granule includes polyethyleneimine (PEI) and glutaraldehyde. In some embodiments, the epimerase is immobilized in a granule. In some embodiments, the epimerase is immobilized on a resin. In some embodiments, the epimerase is immobilized on an ion exchange resin. In some embodiments, the epimerase is immobilized on a matrix by weakly basic ion exchange (i.e., electrostatic interactions based on the charge of the protein and the charge of a matrix, such as a resin). Non-limiting examples of ion exchange resins include, for example, DuoLite™ and Amberlite™, as described herein. In some embodiments, the epimerase is immobilized by non-specific binding to porous regions of a matrix, such as a resin.

[0103] In one aspect, a conjugate is provided that includes an epimerase and a matrix. A conjugate is a molecule that is composed of two or more substructures that are linked together through a linking group to form a single structure. The linking can be achieved by linking the subunits through a linking group. In some embodiments, the conjugate is formed by an amino group present in the epimerase reacting with an amine-reactive material present on the matrix, such as glutaraldehyde. In some embodiments, the conjugate is a matrix on which the epimerase is immobilized. In some embodiments, the conjugate is a granule, glass bead, ion exchange resin, or polymer encapsulation on which the epimerase is immobilized. In some embodiments, the conjugate is a granule on which the epimerase is immobilized. In some embodiments, the conjugate is a resin on which the epimerase is immobilized, such as an ion exchange resin. In some embodiments, the conjugate is insoluble.

[0104] II. Methods for Producing Allulose Also provided herein are methods for producing allulose using the epimerases and compositions containing the epimerases disclosed herein. In some aspects, the methods include contacting an epimerase protein, such as a protein described in Section I, with fructose or a substrate containing fructose. In some embodiments, the substrate containing fructose is, for example, a syrup, as described in Section II-A-2 below. In some embodiments, the contacting is performed under conditions in which, for example, the fructose and allulose present in the substrate are stable. For example, the contacting can be performed at a temperature and / or pH that prevents or reduces the Maillard reaction, thus promoting sugar browning and / or allulose conversion. In some embodiments, the contacting is performed under conditions that promote epimerase activity. For example, the contacting can be performed at a temperature, pH, and / or concentration of metal cofactor that promotes epimerase activity.

[0105] As described in Section I above, the epimerase provided herein may have a temperature range, pH range and / or concentration of metal cofactor range in which activity or amount is maintained.Similarly, fructose and allulose may have a temperature range and / or pH range that promotes conversion to allulose, and the sugar is stable.Thus, in some cases, contact occurs under conditions, such as temperature, pH, metal cofactor content, where equilibrium is favorable for the formation of allulose and the stability of the sugar and the activity of the overlapping epimerase.

[0106] 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 in the range of about 50° C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 55° C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 60° C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 65° C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 70° C. In some embodiments, the contacting occurs under conditions including a temperature in the range of about 75° C. Contacting can occur at an initial temperature, e.g., 50° C., when the enzyme is first contacted with fructose or a substrate containing fructose, and can be increased to a higher temperature, e.g., 70° C., for subsequent contacts with fructose or a substrate containing fructose. For example, if the enzyme is used over weeks and / or months, either continuously or intermittently as required by the production process, the temperature conditions for contacting can be increased incrementally over time.

[0107] In some embodiments, the contacting is performed under conditions including a pH in the range of about 4 to about 10. In some embodiments, the contacting is performed under conditions including a pH in the range of about 4 to about 9. In some embodiments, the contacting is performed under conditions including a pH in the range of about 4 to about 8. In some embodiments, the contacting is performed under conditions including a pH in the range of about 4.5 to about 8. In some embodiments, the contacting is performed under conditions including a pH in the range of about 5 to about 8. In some embodiments, the contacting is performed under conditions including a pH in the range of about 5 to about 7.5. In some embodiments, the contacting is performed under conditions including a pH in the range of about 5 to about 7. In some embodiments, the contacting is performed under conditions including a pH in the range of about 5 to about 6.5. In some embodiments, the contacting is performed under conditions including a pH in the range of about 5 to about 6. In some embodiments, the contacting is performed under conditions including a pH of about 5. In some embodiments, the contacting is performed under conditions including a pH of about 5. In some embodiments, the contacting is performed under conditions including a pH of about 6. In some embodiments, the contacting is performed under conditions including a pH of about 6. In some embodiments, the contacting is performed under conditions including a pH of about 6. In some embodiments, the contacting is performed under conditions including a pH of about 7. In some embodiments, the contacting is carried out under conditions comprising a pH of about 7.5. It is also possible to contact the enzyme at an initial pH, e.g., 5, when it is first contacted with fructose or a substrate containing fructose, and then increase to a more basic pH, e.g., up to 10, for subsequent contact with fructose or a substrate containing fructose. For example, if the enzyme is used continuously or intermittently as required by the production process over weeks and / or months, the pH conditions for contacting can be increased incrementally (become more basic) over time.

[0108] In some embodiments, the contacting is performed under conditions where the metal cofactor is added (supplemented) to reach a concentration in the range of about 0 to about 2 mM. In some embodiments, the contacting is performed under conditions where the metal cofactor is added (supplemented) to reach a concentration in the range of about 0 to about 1 mM. In some embodiments, the contacting is performed under conditions where the metal cofactor is added (supplemented) to reach a concentration in the range of about 0 to about 0.5 mM. In some embodiments, the contacting is performed under conditions where the metal cofactor is added (supplemented) to reach a concentration in the range of about 0 to about 0.25 mM. In some embodiments, the contacting is performed under conditions where the metal cofactor is added (supplemented) to reach a concentration in the range of about 0 to about 0.15 mM. In some embodiments, the contacting is performed under conditions where the metal cofactor is added (supplemented) to reach a concentration in the range of about 1 mM to about 2 mM. In some embodiments, the metal cofactor is at a concentration in the range of about 1 to about 2 mM when the metal cofactor is in the form of a salt. In some embodiments, the contacting is performed under conditions including a metal cofactor at a concentration ranging from about 0.15 to about 0.25 mM. In some embodiments, if the metal cofactor is not in salt form, e.g., if it is present as an ion, the metal cofactor is at a concentration ranging from about 0.15 to about 0.25 mM. In any case where a metal cofactor is added, the amount of metal cofactor added is less than the target concentration. In some embodiments, the contacting is performed under conditions where no metal cofactor is added (not supplemented). In some embodiments, the concentration of metal cofactor is present without the need to supplement the metal cofactor. In some embodiments, the cofactor is an ion. In some embodiments, the cofactor is a salt. In some embodiments, the metal cofactor is magnesium or a salt thereof.

[0109] A. Fructose, fructose-containing substrates, and its conversion to allulose In some embodiments, fructose or a substrate containing fructose is produced as part of a carbohydrate production process. In some embodiments, the method of producing allulose provided herein is carried out as part of a carbohydrate production process. In some embodiments, the carbohydrate production process is a production process carried out in a biorefinery.

[0110] Those skilled in the art will clearly recognize available methods that can be used to prepare starch substrates for use in the processes disclosed herein. Preparation methods generally include process steps such as grinding / shredding, liquefaction, saccharification, and isomerization to convert biomass into sugars (e.g., syrup).

[0111] 1. Crushing Fructose and fructose-containing substrates can be obtained from tubers, roots, stems, legumes, cereals or whole grains by processing the starch obtained. In some embodiments, starch and then fructose or fructose-containing substrates can be obtained from corn, cobs, sugar cane, sugar beet, wheat, barley, rye, triticale, milo, sago, millet, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes. In some embodiments, fructose or fructose-containing substrates are obtained from processing starch from corn or cobs.

[0112] Starch from grains may be milled or whole and may include solids such as corn kernels, bran and / or cobs. The starch may also be highly refined raw starch or a feedstock from a starch refining process. A variety of starches and fructose are also commercially available.

[0113] The starch may be crude starch from milled whole grains containing non-starch fractions such as germ residues and fiber. Milling may involve either wet milling or dry milling or grinding. In wet milling, the whole grain is soaked in water or dilute acid to separate the grain into its component parts such as starch, protein, germ, oil, grain fiber. Wet milling efficiently separates the germ and meal (i.e., starch granules and protein) and is particularly suitable for the production of syrups.

[0114] In dry milling or grinding, whole kernels are broken down into fine flour and often processed without fractionating the kernel into its component parts. In some cases, oil and / or fiber are recovered from the kernel. Thus, dry milled grains contain significant amounts of non-starch carbohydrate compounds in addition to starch. Dry milling of starch substrates can be used for the production of ethanol and other biochemicals.

[0115] 2. Liquefaction Liquefaction refers to the process by which starch is converted to less viscous short chain dextrins. Generally, this process involves pregelatinization of starch simultaneously or following the addition of α-amylase, although additional liquefaction-inducing enzymes may be optionally 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 α-amylases typically used for this application are heat stable. The α-amylase is typically supplied at about 1500 units per kg dry matter of starch, for example. To optimize the stability and activity of the α-amylase, the pH of the slurry is typically adjusted to about pH 4.5-6.5, and about 1 mM calcium (about 40 ppm free calcium ions) may also be added depending on the characteristics of the amylase used. Bacterial α-amylase remaining in the slurry after liquefaction can be inactivated by several methods, including lowering the pH in a subsequent reaction step or, if the enzyme is calcium dependent, removing calcium from the slurry.

[0116] The starch + α-amylase slurry may be continuously pumped through a steam heated jet cooker to a temperature in the range of about 105°C to 110°C. Gelation occurs rapidly under these conditions, and the enzyme activity, combined with significant shear forces, initiates hydrolysis of the starch substrate. The residence time in the jet cooker is short, for example, anywhere in the range of about 4 to about 12 minutes. The partially pregelatinized starch may be passed through a series of holding tubes maintained at 105 to 110°C and held for 5 to 8 minutes to complete the pregelatinization process ("primary liquefaction"). Hydrolysis to the required DE is completed in a holding tank at a temperature of 85 to 95°C or higher for about 1 to 2 hours ("secondary liquefaction"). The slurry is then cooled to room temperature. This cooling step may be 30 to 180 minutes, for example 90 to 120 minutes. The liquefied starch is typically 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%.

[0117] 3. Glycation and isomerization Liquefied starch can be saccharified using glucoamylase, optionally in the presence of another enzyme, into a syrup rich in low DP (e.g., DP1+DP2) sugars. Exemplary DP1 sugars include glucose and fructose, and DP2 sugars include, for example, maltose and sucrose. Depending on the enzyme used, the syrup may contain a weight percentage of DP2 of the total oligosaccharides in the saccharified starch of more than 30%, such as 45%-65% or 55%-65%. The weight percentage of (DP1+DP2) in the saccharified starch may be greater than about 70%, such as 75%-85% or 80%-85%.

[0118] In some embodiments, an isomerization step can be used to modify the low DP composition in the syrup. In some embodiments, an enzyme can be used to increase the amount of fructose or DP1 sugars, such as glucose, that can be converted to fructose in the syrup. However, since DP1 sugars, such as glucose and fructose, can be indirectly or directly converted to allulose, any method that increases the DP1 content of the syrup is believed to be useful in the method of converting D-fructose to allulose provided herein. For example, the syrup can be contacted with an epimerase described herein to directly convert the fructose present in the syrup to allulose. In some cases, the syrup can be contacted with a glucose isomerase enzyme to convert the glucose present in the syrup to fructose, which can then be converted to allulose by contact with the epimerase provided.

[0119] In some embodiments, the fructose-containing substrate is a syrup. In some embodiments, the syrup is high fructose corn syrup (HFCS). In some embodiments, the fructose-containing substrate is a syrup that does not contain fructose but contains a sugar that can be converted to fructose, such as a DP1 sugar. In some embodiments, the fructose-containing substrate is a syrup that includes a DP1 sugar that can be converted to fructose. In some embodiments, the fructose-containing substrate is a syrup that contains fructose and a DP1 sugar that can be converted to fructose. In some embodiments, the DP1 sugar is glucose. In some embodiments, the conversion of glucose to fructose is accomplished by an enzyme, such as glucose isomerase.

[0120] The method of producing allulose from fructose or a fructose-containing substrate, such as syrup, can proceed by contacting the epimase protein described herein with fructose or a fructose-containing substrate. This converts the fructose or fructose contained in the substrate to allulose. In some embodiments, when the fructose-containing substrate contains only or further contains glucose, the substrate can be contacted or contacted with glucose isomerase to convert the glucose to fructose. Suitable isomerases for the 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. After isomerization of glucose, the mixture typically contains about 40-45% fructose, for example 42% fructose. In some cases, the mixture can be further isolated or purified to increase the percentage of fructose. In some embodiments, the mixture can be purified to contain about or at least 95% fructose. A fructose-containing substrate obtained from the converted glucose can be contacted with an epimerase described herein to convert the fructose to allulose. In some embodiments, the substrate is contacted with glucose isomerase and subsequently with the epimerase. In some embodiments, the substrate is contacted with glucose isomerase and epimerase simultaneously. It should be understood that in some cases, a metal cofactor may be added to promote the activity of glucose isomerase.

[0121] In some embodiments, the epimerase contacted by the fructose or fructose-containing substrate is in soluble form. In some embodiments, the epimerase contacted by the fructose or fructose-containing substrate is immobilized on a matrix. See, e.g., Section ID. In some embodiments, for example, when the fructose-containing substrate is contacted with glucose isomerase, the glucose isomerase is immobilized on a matrix. In some embodiments, the matrix is ​​granular. In some embodiments, the matrix is ​​an ion exchange resin.

[0122] In some embodiments, the epimerase is immobilized in a first matrix and the glucose isomerase is immobilized in a second matrix. In some embodiments, the first matrix and the second matrix are made of different materials. In some embodiments, the first matrix and the second matrix are made of the same material. In some embodiments, the first matrix and the second matrix are granules. In some embodiments, the first matrix and the second matrix are ion exchange resins. In some embodiments, the epimerase and glucose isomerase are co-immobilized on a matrix. In some embodiments, the matrix is ​​granules. In some embodiments, the matrix is ​​an ion exchange resin.

[0123] Various advantages are associated with the use of immobilized proteins, including, but not limited to, longer protein usage periods and avoidance of steps to inactivate or remove the protein from the product when the immobilized protein is contained in a reactor that allows the substrate to contact the immobilized protein and be recovered. Thus, in some embodiments, the protein, e.g., epimerase and / or glucose isomerase, is immobilized on, e.g., a packed or loaded matrix present in the reactor. In some aspects, the protein, e.g., epimerase and / or glucose isomerase, is in a soluble form and present in the reactor. In some embodiments, contacting the protein with the substrate containing fructose is performed by adding the substrate to the reactor. In some embodiments, the reactor is a column, tank, or 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 passed through the column and 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 recovered from the tank or reactor after contact with the immobilized protein.

[0124] In some embodiments, the reactor comprises a matrix in which the epimerase is immobilized. In some embodiments, the reactor comprises a matrix in which the glucose isomerase is immobilized. In some embodiments, the reactor comprises a first matrix in which the epimerase is immobilized and a second matrix in which the glucose isomerase is immobilized. In some embodiments, the reactor comprises a matrix in which the epimerase and the glucose isomerase are co-immobilized. It should be understood that the number and configuration of the reactors will depend on the composition of the substrate and whether sequential or simultaneous contact of the substrate with the protein is preferred.

[0125] In some embodiments, the substrate contacted with the protein is collected from the reactor. For example, when using a column, the substrate can be added to one end of the column, passed through the matrix with immobilized protein, and collected at the other end. In some embodiments, the collected substrate contains fructose, which can be optionally purified and passed through another reactor containing a matrix with immobilized epimerase to promote conversion to allulose. In some embodiments, the collected substrate contains allulose. In some embodiments, allulose is purified from the collected substrate.

[0126] III. Kit Also provided are kits that include compositions, e.g., epimerase proteins, immobilized epimerase proteins, nucleic acid sequences, vectors, host cells, and / or cultured cell materials that include an epimerase described herein, and may further include instructions regarding methods of using the compositions, such as the uses described herein. In some embodiments, the kits include an epimerase protein described herein. In some embodiments, the kits include a polynucleotide that encodes an epimerase described herein. In some embodiments, the kits include a vector that encodes an epimerase described herein. In some embodiments, the kits include cell culture materials that include an epimerase described herein. In some embodiments, the kits include an epimerase immobilized on a matrix described herein. In some embodiments, the kits include a conjugate described herein. The kits may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, columns (e.g., reactor columns), containers (e.g., reactor vessels), and inserts with instructions for use.

[0127] IV. Illustrative Embodiments Among the embodiments provided are the following: 1. A protein comprising an amino acid sequence having at least 70% sequence identity to the sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9, and having epimerase activity. 2. The protein of embodiment 1, wherein the amino acid sequence has at least 80%, 90%, 95%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. 3. The protein according to embodiment 1 or embodiment 2, which is immobilized on a matrix. 4. The protein of embodiment 3, wherein the matrix is ​​a granule or an ion exchange resin. 5. A nucleic acid molecule comprising a nucleic acid sequence encoding a protein according to any one of embodiments 1 to 4. 6. The nucleic acid molecule according to embodiment 5, comprising a nucleic acid sequence that: i) encodes an amino acid sequence having at least 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; ii) has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity with the sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18; or iii) hybridizes under stringent conditions to a nucleic acid sequence having a sequence complementary to the sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:18. 7. The nucleic acid molecule according to embodiment 5 or embodiment 6, comprising a heterologous regulatory sequence, optionally a promoter sequence. 8. A vector comprising a nucleic acid molecule according to any one of embodiments 5 to 7. 9. A host cell comprising a nucleic acid molecule according to any one of embodiments 5 to 7 or a vector according to embodiment 8. 10. The host cell of embodiment 9, which is a yeast, bacterial, mammalian cell or plant cell. 11. The host cell of embodiment 9 or claim 10, which is a Bacillus subtilis. 12. A cultured cell material comprising a protein according to embodiment 1 or embodiment 2 and / or a host cell according to any one of embodiments 9 to 11. 13. Allulose produced by a protein according to any one of embodiments 1 to 4. 14. A composition for producing allulose, comprising: i) a protein described in any one of embodiments 1 to 4; and ii) a substrate comprising fructose. 15. The composition of embodiment 14, wherein the protein is immobilized on a matrix. 16. The composition of embodiment 14 or embodiment 15, wherein the substrate comprises glucose. 17. The composition according to any one of embodiments 14 to 16, further comprising glucose isomerase immobilized on the matrix. 18. The composition according to any one of embodiments 14 to 17, wherein the protein and glucose isomerase are co-immobilized on the same matrix or on different matrices. 19. The composition of any one of embodiments 14 to 18, contained in a reactor. 20. Use of a protein according to any one of embodiments 1 to 4 for producing allulose. 21. A method for producing allulose, comprising contacting a protein according to any one of embodiments 1 to 4 with a substrate containing fructose. 22. The method of embodiment 21, wherein the contacting is carried out under conditions comprising a temperature in the range of about 50°C to about 90°C. 23. The method of embodiment 21 or embodiment 22, wherein the contacting is carried out under conditions comprising a pH in the range of about 4.5 to about 8. 24. The method of any one of embodiments 21 to 23, wherein the contacting is performed under conditions in which no metal cofactor is added or in which the metal cofactor is added in an amount that is less than the metal cofactor concentration required for epimerase activity. 25. The method according to any one of embodiments 21 to 24, wherein the protein is immobilized in a matrix contained in the reactor, and the contacting is carried out by adding a substrate comprising fructose to the reactor. 26. The method of any one of embodiments 21 to 25, wherein the fructose-containing substrate is produced by (i) contacting the glucose-containing substrate with glucose isomerase prior to contacting the protein; or (ii) contacting the glucose-containing substrate with glucose isomerase simultaneously with contacting the protein. 27. The method according to any one of embodiments 21 to 26, comprising purifying the allulose produced. 28. A kit comprising: (i) a protein according to any one of embodiments 1 to 4, a nucleic acid molecule according to any one of embodiments 5 to 7, a vector according to embodiment 8, a host cell according to any one of embodiments 9 to 11, and / or a cell culture material according to embodiment 12; and (ii) instructions for use. EXAMPLES

[0128] V. Working Examples The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0129] Example 1: Expression of D-allulose 3-epimerase The amino acid sequences of D-allulose 3-epimerase were found in the NCBI database (accession numbers are shown in Table E1). The expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif, 55:40-52, 2007) was used for expression of all listed exemplary epimerases in Bacillus subtilis. The plasmid contained the aprE promoter followed by a codon-optimized nucleotide sequence encoding the protein sequence of the target gene. The corresponding protein sequences (SEQ ID NOs: 1-9, 19-23) and codon-optimized gene sequences (SEQ ID NOs: 10-18, 24-28) are shown in Table E1.

[0130] 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 250 ml shake flasks containing MBD medium (a MOPS-based defined medium supplemented with an additional 5 mM CaCl2). Supernatants from these cultures were used to confirm protein expression by SDS-PAGE analysis and enzyme activity assays. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

[0131] Example 2: Production of D-allulose 3-epimerase in Bacillus subtilis This example demonstrates the production of D-allulose 3-epimerase protein in liquid fermentation of B. subtilis.

[0132] The inoculum was grown in a seed flask containing LB medium. A production medium containing 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 the exemplary epimerase. The production medium was pH adjusted and the cells were fed according to the oxygen uptake rate. The D-allulose 3-epimerase protein accumulated in the broth / cells.

[0133] Various parameters were monitored during the run, including but not limited to: CER (carbon dioxide evolution rate), OUR (oxygen uptake rate), pH, DO (dissolved oxygen), OD (optical density).

[0134] Example 3: Evaluation of D-allulose 3-epimerase specific activity and metal cofactor requirements The specific activity of the exemplary D-allulose 3-epimerase described in Example 1, produced according to the method described in Example 2, was assayed based on the release of allulose from fructose using an HPLC method. The dependence of activity on the magnesium cofactor was also evaluated.

[0135] Substrate solution was prepared by mixing 5 mL of fructose (200 mM in milliq water), 0.5 mL of 1 M pH 7.5 Tris-HCl buffer, 4.5 mL of milliq water, and 20 μL of 0.5 M MgSO4 (or 20 μL of milliq water for the group without ion addition) in a 15 mL conical tube. Serial dilutions of epimerase samples were prepared in milliq water. Each epimerase sample (10 μL) was transferred to a new microtiter plate (Agilent 5042-1385, PP) containing 90 μL of substrate solution that was preincubated at 50 °C for 5 min. Incubation was performed at 50 °C for 15 min with shaking (650 rpm) in an iEMS incubator (ThermoFisher). Reactions were quenched by adding 100 μL of 100 mM pH 3.5 Na-acetate buffer. The quenched reaction mixture was diluted 2-fold with milliq water and filtered for allulose analysis by HPLC using an Agilent 1200 series system equipped with a Shodex SP0810 HPLC column. An allulose standard curve was generated and used to calculate allulose release from the epimerase reaction.

[0136] Specific activity was calculated using the following formula: Specific activity (units / mg) = slope (dose-response curve) ÷ 15 × 1000 (where 1 unit = 1 μmol allulose / min).

[0137] Table E2 shows the specific activity of the epimerases tested in the presence and absence of MgSO4. As shown in Table E2, the control epimerase AglEpi showed significant ion dependency, while the exemplary epimerase samples BsuEpi1, CbaEpi17 and CbaEpi18 showed ion independence and maintained their activity without added ions. The exemplary epimerases OspEpi5, OspEpi10, OspEpi3 and DspEpi3 maintained at least 60% specific activity in the absence of ions, demonstrating reduced ion dependency. [Table 9]

[0138] Example 4: pH activity profile of D-allulose 3-epimerase The pH activity profile (pH 3 to 10) of D-allulose 3-epimerase samples was analyzed using fructose (100 mM) as substrate. Substrate solution was prepared by mixing 1 mL of fructose (200 mM in milliq water), 0.04 mL of 1 M glycine / sodium acetate / HEPES (pH varying from 3 to 10), 0.96 mL of milliq water, and 4 μL of 0.5 M MgSO4. Enzyme working solutions were prepared in water at specific doses (dose-response curves showing signals within the linear range). All incubations were performed at 50 °C for 15 min with shaking (650 rpm) in an iEMS incubator (ThermoFisher). The reaction was quenched by adding 100 μL of 100 mM pH 3.5 Na acetate buffer. Released allulose was measured using an HPLC method with a similar procedure as described in Example 3. The enzyme activity at each pH was reported as a relative activity compared to the enzyme activity at the optimum pH where the enzyme showed maximum activity. The pH profiles of the epimerases are shown in Table E3. OsiEpi1 retained >50% of its activity at pH 5, while the control epimerase, AglEpi, retained minimal activity under the same pH conditions. [Table 10]

[0139] Example 5: Thermostability of D-allulose 3-epimerase The thermal stability of D-allulose 3-epimerase was evaluated by pre-incubating the enzyme working solution (500 ppm) at 70°C for 0 (control), 5, 15, 30, 60, and 120 min, respectively. The epimerase residual activity was then measured by incubating 10 μL of the above enzyme working solution with 90 μL of 100 mM fructose at pH 7.5 and 50°C for 15 min with shaking (650 rpm) in an iEMS incubator (ThermoFisher). The reaction was quenched by adding 100 μL of 100 mM pH 3.5 Na-acetate buffer. Allulose release was measured using an HPLC method in a similar procedure as described in Example 3. As shown in Table E4, BsuEpi1, NdeEpi1, MspEpi3, and MspEpi4 retained more than 70% of their activity at 70°C after 60 min of pre-incubation. MspEpi3 retained more than 90% of its activity after 120 min of preincubation. The control epimerase, AglEpi, lost its activity after 30 min of preincubation at 70°C. [Table 11]

[0140] Example 6: Temperature profile of D-allulose 3-epimerase The temperature profile of an exemplary D-allulose 3-epimerase was analyzed using allulose (100 mM) as a substrate. The substrate solution was prepared by mixing 5 mL of allulose (200 mM in milliq water), 0.5 mL of 1 M pH 7.5 Tris-HCl buffer, 4.5 mL of milliq water, and 20 μL of 0.5 M MgSO4 in a 15 mL conical tube. The enzyme working solution was prepared in water at a dose that showed a signal within the linear range according to the dose-response curve. All incubations were performed at temperatures between 40 °C and 90 °C, respectively, for 10 min with shaking (650 rpm) in an iEMS incubator (ThermoFisher). The reaction was quenched by adding 100 μL of 100 mM pH 3.5 Na-Acetate buffer. Fructose release was measured using the D-Fructose / D-Glucose Measurement Kit (K-FRUGL) from Megazyme. The enzyme activity at each temperature was reported as a relative activity compared to the enzyme activity at the optimum temperature. The temperature profiles of the epimerases are shown in Table E5. MspEpi3 showed an optimum temperature at 80°C, which was 15°C higher than the control epimerase AglEpi. LphEpi1, MspEpi4, NdeEpi1 and BsuEpi1 maintained more than 80% of their activity at 80°C. NdeEpi1 maintained 96% activity at 90°C, while AglEpi showed only 50% and 18% of its activity at 80°C and 90°C, respectively. [Table 12]

[0141] Example 7: Conversion of fructose to allulose by immobilized D-allulose 3-epimerase The exemplary epimerases described in Example 1 and produced according to Example 2 were evaluated for their ability to convert fructose to allulose when immobilized on a matrix.

[0142] An exemplary epimerase present in the lysed production broth (see Example 2) was immobilized into granules using known cross-linking methods (U.S. Pat. No. 4,355,105). Briefly, the lysed production broth was added to a slurry containing bentonite (Cholino, Patagonia, Argentina; P / N F30), Celite 505™ (Imerys), polyethyleneimine (Epomin P-1050, Nippon Shokubai), and a 5% glutaraldehyde solution (Sigma Aldrich) with the pH adjusted to 7.3-7.6. A second addition of polyethyleneimine and 5% glutaraldehyde was then added to the slurry and the pH adjusted to 8.3-8.5. The insoluble immobilized solids were filtered, extruded, and then dried using a fluid bed coater (FL-1 fluid bed coater, Freund Vector Corporation).

[0143] To measure the activity of the immobilized epimerase, the granules (0.3 g) with immobilized epimerase were prewashed (3-4 times) in 50 ml DI water for 30 min in a 50 ml conical tube. The wet granules were packed into a 1 cm × 5 cm stainless steel HPLC column, and the column was placed online into an Agilent LC column compartment.

[0144] Heated 35 mM NaPO4, pH 7.5 and 5% fructose was pumped through the column at a rate of 0.2 ml / min and the column section was maintained at 50° C. throughout the experiment. Allulose conversion efficiency was determined by HPLC over a 14 day period and the conversion activity on days 7 and 14 relative to day 1 (day 1=100%) is shown in Table E6 below.

[0145] As seen in Table E6, the conversion activity of the immobilized exemplary epimerases, CbaEpi17 and Cb8Epi, was maintained for at least 14 days. However, the activity of the control immobilized epimerase showed a decrease of about 4% on day 7 compared to day 1, and a further decrease on day 14 compared to day 1. [Table 13]

[0146] These data suggest that the immobilized exemplary epimerase is capable of maintaining enzymatic activity over time.

[0147] Example 8: Conversion of fructose to allulose by immobilized D-allulose 3-epimerase under stress conditions The ability of the exemplary epimerase described in Example 1 and produced according to Example 2 to convert fructose to allulose when immobilized on a matrix was evaluated under various temperature and pH stress conditions and compared to the soluble enzyme.

[0148] Matrix-immobilized enzyme was prepared from the lysed production broth according to Example 7. The soluble enzyme was isolated from the lysed production broth (see Example 2) using known purification methods. Briefly, the soluble components of the lysed production broth were loaded onto a Phenyl Sepharose FF column equilibrated with 20 mM Tris pH 7.0 and 1 M ammonium sulfate. The column was washed with equilibration buffer and eluted with 20 mM Tris pH 7.0. The epimerase-containing fraction was then loaded onto a Q-Sepharose column equilibrated with 20 mM Tris pH 7.0 and 150 mM NaCl. The column was washed with equilibration buffer and eluted with 20 mM Tris pH 7.0 and 1 M NaCl. The epimerase-containing fraction was then concentrated using a Sartorius Vivaflow 200 Crossflow Cassette equipped with a 10 kDa MWCO membrane. The purified epimerase was formulated with 12 mM Tris pH 7.0, 90 mM NaCl and 40% glycerol.

[0149] To evaluate activity under various conditions, 8 μg of soluble enzyme or 5 mg of immobilized enzyme granules were loaded into a well of a microtiter plate and combined with 200 μL of 50% fructose syrup containing 1 mM MgSO4, 100 mM NaCl, 1 mM sodium metabisulfite, and either 20 mM sodium phosphate buffer (pH 7.2) or sodium acetate buffer (pH 5.0). Prior to adding the syrup, the enzyme was subjected to stress conditions at either 20° C. or 60° C. in either sodium phosphate buffer (pH 7.2) or sodium acetate buffer (pH 5.0) for 1 hour. The epimerase was then incubated with fructose syrup at 50° C. for 1 hour to measure epimerase activity. Activity was determined by fructose conversion measured by HPLC. Conversion compared to control conditions (20° C., pH 7.2) is shown in Table E7 for both soluble and immobilized exemplary enzyme Cb8Epi. [Table 14]

[0150] These data show that the soluble enzyme retains activity at pH 7.2 and 20° C., and the immobilized enzyme retains activity under both heat and low pH stress conditions.

[0151] The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such variations can be made without departing from the true scope and spirit of the present disclosure, and are intended to fall within the scope of the present disclosure. Although the present invention may be described in connection with certain 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 that are obvious to those skilled in the art of molecular biology or related fields are intended to be included within the scope of the following claims.

Claims

1. A protein comprising an amino acid sequence having at least 70% sequence identity to the sequence shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23, and having epimerase activity.

2. 2. The protein of claim 1, wherein the amino acid sequence has at least 80%, 90%, 95%, 99% or 100% sequence identity to the sequence represented by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:

23.

3. 3. The protein of claim 1 or claim 2, which is immobilized on a matrix.

4. The protein of claim 3 , wherein the matrix is ​​a granule or an ion exchange resin.

5. A nucleic acid molecule comprising a nucleic acid sequence encoding the protein according to any one of claims 1 to 4.

6. 6. The nucleic acid molecule of claim 5 : i) encoding an amino acid sequence having at least 90%, 95%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 or SEQ ID NO:23; ii) has at least 70%, 80%, 90%, 95%, 99% or 100% sequence identity to a sequence represented by SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28; or iii) hybridizes under stringent conditions to a nucleic acid sequence having a sequence complementary to the sequence shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:

28. A nucleic acid molecule comprising a nucleic acid sequence.

7. 7. The nucleic acid molecule of claim 5 or claim 6, comprising a heterologous regulatory sequence, optionally a promoter sequence.

8. A vector comprising the nucleic acid molecule according to any one of claims 5 to 7.

9. A host cell comprising a nucleic acid molecule according to any one of claims 5 to 7 or a vector according to claim 8.

10. The host cell of claim 9 which is a yeast, bacterial, mammalian cell or plant cell.

11. 11. The host cell of claim 9 or claim 10, which is a Bacillus spp.

12. A cultured cell material comprising a protein according to claim 1 or claim 2 and / or a host cell according to any one of claims 9 to 11.

13. Allulose produced by the protein according to any one of claims 1 to 4.

14. 1. A composition for producing allulose, comprising: i) a protein according to any one of claims 1 to 4; and ii) Fructose-containing substrate A composition comprising:

15. The composition of claim 14 , wherein the protein is immobilized on a matrix.

16. 16. The composition of claim 14 or 15, wherein the substrate comprises glucose.

17. The composition of any one of claims 14 to 16, further comprising glucose isomerase immobilized on the matrix.

18. The composition according to any one of claims 14 to 17, wherein the protein and glucose isomerase are co-immobilized on the same matrix or immobilized on different matrices.

19. The composition according to any one of claims 14 to 18, contained in a reactor.

20. Use of the protein according to any one of claims 1 to 4 for producing allulose.

21. A method for producing allulose, comprising contacting the protein according to any one of claims 1 to 4 with a substrate containing fructose.

22. 22. The method of claim 21, wherein the contacting is carried out under conditions comprising a temperature in the range of about 50°C to about 90°C.

23. 23. The method of claim 21 or claim 22, wherein the contacting is carried out under conditions comprising a pH in the range of about 4.5 to about 8.

24. 24. The method of any one of claims 21 to 23, wherein the contacting is performed under conditions in which no metal cofactor is added or in which a metal cofactor is added in an amount that is less than the metal cofactor concentration required for epimerase activity.

25. 25. The method of any one of claims 21 to 24, wherein the protein is soluble and is contained in a reactor and / or the protein is immobilized on a matrix contained in a reactor, and wherein the contacting is performed by adding the substrate comprising fructose to the reactor.

26. The substrate comprising fructose is (i) contacting a substrate containing glucose with glucose isomerase prior to contacting the protein; or (ii) contacting the protein and simultaneously contacting a substrate containing glucose with glucose isomerase. The method according to any one of claims 21 to 25, wherein the nucleic acid is produced by

27. The method of any one of claims 21 to 26, comprising purifying the allulose produced.

28. (i) a protein according to any one of claims 1 to 4, a nucleic acid molecule according to any one of claims 5 to 7, a vector according to claim 8, a host cell according to any one of claims 9 to 11, and / or a cell culture material according to claim 12; and (ii) Instructions for use Including the kit.

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