Immobilized enzyme composition for hexose production

Abstract

To provide an immobilized enzyme composition for hexose production or an improved process for hexose production. [Solution] The present invention relates to an immobilized enzyme composition for the preparation of hexoses. Examples of hexoses include tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, and inositol. The present invention also relates to an enzymatic process for preparing hexoses from sugars by contacting starch derivatives with the immobilized enzyme composition of the present invention.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Application No. 62 / 875,321, filed on July 17, 2019, and U.S. Application No. 62 / 924,323, filed on October 22, 2019, the entireties of which are incorporated herein by reference.

[0002] The present invention relates to the preparation of hexose monosaccharides using an immobilized enzyme composition. More specifically, the present invention relates to a method for preparing D - hexose (or hexose) from saccharides (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D - glucose, and D - fructose), including the step of converting fructose 6 - phosphate to hexose by one or more enzyme steps catalyzed by an immobilized enzyme.

Background Art

[0003] Hexose is a monosaccharide having six carbon atoms. Examples of hexose include tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gulose, idose, and inositol. Hexose is used in many industries and clearly has various uses in the pharmaceutical industry, biotechnology, as well as the food and beverage industry. Hexose can be prepared from saccharides (e.g., monosaccharides, oligosaccharides, starch, starch derivatives, cellulose, etc.) using an enzymatic process. Solution - based enzymatic processes are described in Published PCT Applications Nos. 2018 / 169957, 2017 / 059278, and 2018 / 112139 (which are incorporated herein by reference).

[0004] The commercial development of enzyme processes is essential to increasing the number of industrial processes that utilize green chemistry and thus reduce the environmental impact of synthesis. While many enzyme processes have been developed for commercial production (e.g., PCT applications 2018 / 169957, 2017 / 059278, and 2018 / 112139), there are hurdles to commercialization related to the cost of using the enzyme, especially when these processes are for low-cost products (such as alternative sweeteners). A common solution to this problem is to immobilize the enzyme on a support (e.g., WO2016 / 160573). Immobilization allows the enzyme to be reused between batches or used in continuous processes. The ability to reuse the enzyme can significantly reduce the cost of using the enzyme per kilogram of product and may be essential for commercial viability. Many methods exist for enzyme immobilization, and there are no established rules regarding which method should be preferred for a particular process (Datta et al., Enzyme immobilization: an overview on techniques and support materials. 3 Biotech (2013), 3:1-9). Therefore, a unique solution must be developed for each process.

[0005] In commercial processes, enzymes can be adsorbed onto insoluble organic or inorganic supports, commonly used to enhance their functionality, as is known in the art. These include polymeric supports such as agarose, methacrylate, polystyrene, phenol-formaldehyde, or dextran, as well as inorganic supports such as glass, metal, or carbon-based materials. These materials are often produced with a large surface area-to-volume ratio and specialized surfaces to facilitate the attachment and activity of immobilized enzymes. Enzymes can attach to these solid supports via covalent, ionic, or hydrophobic interactions. Enzymes can also attach to other protein or peptide sequences (most often polyhistidine sequences) that have affinity for the solid support via genetically engineered interactions, such as covalent fusion. Enzymes may attach directly to a surface or surface coating, or to other proteins already present on the surface or surface coating. Enzymes can all be immobilized on a single support, on individual supports, or on a combination of two supports (e.g., two enzymes per support, then mixing those supports). These variations can be mixed uniformly or within a predetermined layer to optimize turnover in a continuous reactor. These enzymes may be mixed uniformly or within a predetermined layer or zone to optimize turnover. For example, the reactor start may have a layer of aGP to ensure a high initial G1P increase. The enzymes may all be immobilized on a single carrier bead, each on an individual carrier bead, or as a group of enzymes on a carrier bead. Similarly, enzymes may be immobilized on a specific carrier or multiple carriers within a single process using one or more immobilization methodologies.

[0006] There is a need for an improved process for producing hexoses that enables scalable and reusable enzyme compositions while achieving high yields of the desired hexoses. [Overview of the Initiative]

[0007] The present invention relates to an immobilized enzyme composition for the preparation of hexoses. Examples of hexoses include tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, and inositol. The immobilized enzyme composition of the present invention comprises, essentially consists of, or consists of, at least two, at least three, at least four, at least five, at least six carriers, at least seven, or at least eight of the following enzymes immobilized on at least one carrier or mixture of carriers: a) α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), and optionally, 1,4-glucantransferase (4-GT), and b) An enzyme selected from the following combinations of enzymes: (i) Phosphoglucoisomerase (PGI), fructose-6-phosphate epimerase (F6PE), and tagatose-6-phosphate phosphatase (T6PP) for preparing tagatose, (ii) Phosphoglucoisomerase (PGI), picose-6-phosphate epimerase (P6PE), and picose-6-phosphate phosphatase (P6PP) for preparing allulose, (iii) Phosphoglucoisomerase (PGI), P6PE, allose-6-phosphate isomerase (A6PI), and allose-6-phosphate phosphatase (A6PP) for preparing allose, (iv) For the preparation of mannose, phosphoglucoisomerase (PGI), mannose-6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI), and mannose-6-phosphate phosphatase (M6PP), (v) For the preparation of galactose, phosphoglucoisomerase (PGI), F6PE, galactose 6-phosphate isomerase (Gal6PI), and galactose 6-phosphate phosphatase (Gal6PP), (vi) PGI and fructose 6-phosphate phosphatase (F6PP) for the preparation of fructose, (vii) PGI, P6PE, altrose 6-phosphate isomerase (Alt6PI), and altrose 6-phosphate phosphatase (Alt6PP) for the preparation of altrose, (viii) PGI, F6PE, talose 6-phosphate isomerase (Tal6PI), and talose 6-phosphate phosphatase (Tal6PP) for preparing talose, (ix) PGI, F6PE, sorbose 6-phosphate epimerase (S6PE), and sorbose 6-phosphate phosphatase (S6PP) for preparing sorbose. (x) PGI, F6PE, S6PE, growth 6-phosphate isomerase (Gul6PI), and growth 6-phosphate phosphatase (Gul6PP) for preparing growth, (xi) PGI, F6PE, S6PE, idose 6-phosphate isomerase (I6PI), and idose 6-phosphate phosphatase (I6PP) for preparing idose, (xii) Inositol 3-phosphate synthase (IPS) and inositol monophosphatase (IMP) for preparing inositol.

[0008] In the immobilized enzyme composition of the present invention, the weight (w / w) percentage of each enzyme relative to the total weight of the enzymes is in the range of 0.1% to 40%.

[0009] Furthermore, the present invention relates to an enzymatic process for preparing hexoses from sugars by contacting a starch derivative with the immobilized enzyme composition of the present invention under suitable reaction conditions to convert the starch derivative into a hexose. [Brief explanation of the drawing]

[0010] [Figure 1A] This graph shows the amount of enzyme in an immobilized enzyme composition optimized for tagatose production. [Figure 1B]This graph shows the amount of enzyme in the immobilized enzyme composition. Because the enzymes have equivalent activity units within the cascade, their amounts are normalized based on the observed reaction rates of each enzyme to T6PP activity. [Figure 1C] This graph shows the amount of enzyme in the immobilized enzyme composition, with a w / w ratio of 1:1:1:1:1:1. [Figure 2] This graph shows the relationship between enzyme loading (total enzyme weight / carrier weight w / w%) and the activity of an immobilized enzyme composition optimized for tagatose production in DUOLITE® A568. The results shown are based on the relative enzyme cascade rate to a 5% loaded carrier. [Modes for carrying out the invention]

[0011] The following description discloses the present invention according to embodiments relating to the production and use of enzymes immobilized on a carrier ("immobilized enzyme composition") in processes for converting starch and starch derivatives and sugars into hexose monosaccharides (e.g., tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, growth, idose, and inositol). These processes can generally be described as enzymatic reactions that use free phosphate (without ATP) to produce phosphorylated intermediates from starch, starch derivatives, or sugars. The free phosphate is released in a highly energetically favorable final step to produce the desired hexose (e.g., tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, growth, idose, or inositol). Next, additional starch, starch derivatives, or sugars are converted into phosphorylation intermediates so that the phosphate can be reused and the process can be repeated. This makes it possible to utilize non-stoichiometric amounts of phosphate, reduces the cost of using phosphate in the process, and limits the level of phosphate in the waste.

[0012] Embodiments of the present invention include compositions of at least two, at least three, at least four, at least five, at least six carriers, at least seven, or at least eight enzymes immobilized on carriers, each catalyzing a reaction in an enzymatic process for converting starch, starch derivatives, and / or sugars to hexoses. The immobilized enzyme compositions of the present invention offer several advantages over their use in free solution, including longer activity duration (due to protection of the structural features of the protein), reuse in multiple cycles, and elimination of the need to remove downstream enzymes. Furthermore, the immobilization of enzymes on a solid surface can function in any of stirred-tank reactors, packed-bed reactors, or rotary-bed reactors, allowing for flexibility in scale-up.

[0013] The enzymes contained in the immobilized enzyme compositions of the present invention catalyze at least two, at least three, at least four, at least five, at least six carriers, at least seven, or at least eight reactions involved in the stepwise conversion of starch, starch derivatives, or sugars to hexoses. The following patent publications, all of which are disclosed herein in their entirety, disclose enzymatic processes (i.e., enzymatic reaction cascades) for producing hexoses in solution: Published PCT applications 2018 / 169957, 2017 / 059278, and 2018 / 112139. The immobilized enzyme compositions of the present invention may, but are not limited to, any of the enzymes and combinations of enzymes disclosed in these references.

[0014] Some immobilized enzyme compositions of the present invention comprise combinations of enzymes that catalyze reactions, which are common among processes that produce different hexoses (e.g., reaction steps leading to the conversion of glucose 6-phosphate (G6P) to fructose 6-phosphate (F6P)). The enzymes that catalyze these common reaction steps are sometimes called "core enzymes". Thus, in some immobilized enzyme compositions of the present invention, the immobilized enzyme composition comprises at least a core enzyme, α-glucan phosphorylase (αGP) that converts sugar to glucose 1-phosphate (G1P), and phosphoglucomutase (PGM) that converts G1P to glucose 6-phosphate (G6P). The enzymes in the immobilized enzyme compositions of the present invention catalyze additional reaction steps for converting G6P to various hexose products and may be co-immobilized with the core enzyme or contained in separate immobilized enzyme compositions.

[0015] Typically, the core enzyme is combined in an immobilized composition with one or more enzymes used to produce tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, or inositol.Therefore, some immobilized enzyme compositions according to the present invention also include, in addition to αGP and PGM: (i) for preparing tagatose, phosphoglucoisomerase (PGI), fructose-6-phosphate epimerase (F6PE), and tagatose-6-phosphate phosphatase (T6PP); (ii) for preparing allulose, PGI, picose-6-phosphate epimerase (P6PE), and picose-6-phosphate phosphatase (P6PP); (iii) for preparing allose, PGI, P6PE, and allose-6-phosphate isomerase. (iv) PGI, mannose-6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI) and mannose-6-phosphate phosphatase (M6PP) for preparing mannose, (v) PGI, F6PE, galactose-6-phosphate isomerase (Gal6PI) and galactose-6-phosphate phosphatase (Gal6PP) for preparing galactose, (vi) PGI for preparing fructose (vii) PGI, P6PE, altrose 6-phosphate isomerase (Alt6PI), and altrose 6-phosphate phosphatase (Alt6PP) for preparing altrose, (viii) PGI, F6PE, talose 6-phosphate isomerase (Tal6PI), and talose 6-phosphate phosphatase (Tal6PP) for preparing talose, (ix) PGI, F6PE, sorbose 6-phosphate epimerase (S6PE), and sorbose (x) PGI, F6PE, S6PE, growth 6-phosphate isomerase (Gul6PI), and growth 6-phosphate phosphatase (Gul6PP) for preparing growth, (xi) PGI, F6PE, S6PE, idose 6-phosphate isomerase (I6PI), and idose 6-phosphate phosphatase (I6PP) for preparing idose, and (xii) inositol 3-phosphate synthase (IPS) and inositol monophosphatase (IMP) for preparing inositol.Each combination of the core enzyme and enzyme compositions (i) to (xii) is a distinct embodiment of the present invention.

[0016] The above immobilized enzyme composition may optionally include 4-glucantransferase (4GT). 4GT can be used to increase the yield of hexoses by reusing the degradation products glucose, maltose, and maltotriose into longer maltooligosaccharides, which can be cleaved phosphorylated by αGP to produce G1P.

[0017] The relative weight ratios of enzymes in the immobilized enzyme composition of the present invention may range from 1:1000 to 1000:1, 1:100 to 100:1, or 1:50 to 50:1 when comparing any two enzymes in the immobilized composition. The enzyme ratios can be varied to increase the efficiency of hexose production, including other optional enzymes considered below. For example, a particular enzyme may be present in amounts approximately 2, 3, 4, 5, or 10 times greater than that of another enzyme.

[0018] The relative weight / weight ratio between enzymes in the immobilized enzyme composition of the present invention can be optimized to increase process performance and / or hexose yield. In this regard, the weight (w / w)% of each enzyme in the immobilized enzyme composition relative to the total weight of the enzymes ranges from 0.1% to 70%. For example, some of the immobilized enzyme compositions of the present invention include 10 - 30% (αGP), 10 - 30% (PGM), and 0.1 - 10% (PGI) (if present) and 0.1 - 10% (4GT) (if present). For example, the immobilized enzyme composition of the present invention can be used for the production of tagatose, and the weight (w / w)% of each enzyme relative to the total weight of the enzymes is 10 - 30% (αGP), 0 - 10% (4GT), 10 - 30% (PGM), 0.1 - 10% (PGI), 15 - 35% (F6PE), and T6PP (25 - 45%), and the total weight of the enzymes in the composition is 100 w / w% relative to the total weight of the enzymes. When used for the production of tagatose, some of the immobilized enzyme compositions of the present invention include 19% αGP, 3% 4GT, 17% PGM, 3% PGI, 23% F6PE, and 35% T6PP, and the weight % of each enzyme is relative to the total weight of the enzymes in the immobilized enzyme composition of the present invention, and the total weight of the enzymes in the composition is 100 w / w% relative to the total weight of the enzymes. In other examples, the immobilized enzyme composition of the present invention is used for the production of allulose, and the weight (w / w)% of each enzyme relative to the total weight of the enzymes is 10 - 30% (αGP), 0 - 10% (4GT), 10 - 30% (PGM), 0.1 - 10% (PGI), 0.1 - 10% (P6PE), and 45 - 65% (P6PP), and the total weight of the enzymes in the composition is 100 w / w% relative to the total weight of the enzymes. When used for the production of allulose, some of the immobilized enzyme compositions of the present invention include 20% αGP, 3% 4GT, 16.5% PGM, 3% PGI, 3% P6PE, and 54% P6PP, and the weight % of each enzyme is relative to the total weight of the enzymes in the immobilized enzyme composition of the present invention, and the total weight of the enzymes in the composition is 100 w / w% relative to the total weight of the enzymes.

[0019] The enzymes contained in the immobilized compositions of the present invention are generally referred to based on the reactions they catalyze (i.e., by specificity and function), but enzymes are usually identified by amino acid sequences (e.g., database identification numbers such as sequence numbers, UniProt IDs), amino acid sequence identity / similarity to enzymes of known function, nucleotide sequences, or nucleotide sequence identity / similarity to enzymes of known function. Enzymes known in the art and used for preparing hexoses can be used in the immobilized enzyme compositions of the present invention, and this includes immobilized enzyme compositions that can be used to generate tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gulose, idose, and inositol. Exemplary enzymes known in the art can be used in the immobilized enzyme compositions of the present invention and are identified in the following related patent documents. The disclosures of the enzymes in the listed patents are specifically incorporated herein by reference. [Table 1]

[0020] Thus, in certain immobilized enzyme compositions, Table 1 provides UniProt IDs and sequence numbers, and identifies the amino acid sequences of the following non-limiting example enzymes that can be included in the immobilized enzyme compositions of the present invention: αGP, PGM, PGI, F6PE, T6PP, 4-GT, P6PE, and P6PP. [Table 2]

[0021] The amino acid sequences of enzymes included in the immobilized enzyme compositions of the present invention may include enzymes that can be modified for any reason, such as to improve activity, stability (i.e., half-life), or yield. Examples of such modified enzymes include enzyme fragments, amino acid substitutions, and chimeric proteins. Modified enzymes include variants of any enzyme disclosed herein. A variant may include amino acid substitutions of one or more amino acid residues. A variant may include 15 or fewer, 12 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer conserved amino acid substitutions compared to a naturally occurring enzyme, and / or 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer non-conserved amino acid substitutions compared to a naturally occurring enzyme, or 1 or fewer non-conserved amino acid substitutions. A conserved amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). The variant enzymes of the present invention may include amino acid analogs and amino acid substitutions by amino acids, as described herein.

[0022] The enzymes contained in the enzyme composition of the present invention may be (i) enzymes that share at least 35% sequence identity with the amino acid sequence of the enzymes disclosed herein, and (ii) enzymes that can catalyze the same reaction as a specific disclosed enzyme having the specificity required for the process. Therefore, the enzymes in the composition of the present invention may share at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 6 The present invention relates to enzymes having amino acid sequences with sequence identity of 5%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. As used herein, the term “sequence identity” refers to the similarity between two or more amino acid sequences or nucleic acid sequences. Sequence identity is typically measured in terms of the percentage of identity (or similarity or homology) between amino acid sequences, with a higher percentage indicating greater similarity between the sequences being compared.

[0023] As described above, the enzyme compositions of the present invention are immobilized on at least one carrier. The carrier may also be generally referred to herein as “carrier material,” “carrier resin,” or “carrier beads.” In the immobilized enzyme compositions of the present invention, one or more enzymes can be immobilized using multiple different carriers. The carrier in the immobilized enzyme compositions of the present invention is not necessarily limited to a specific material, but in some immobilized enzyme compositions of the present invention, the carrier is a weakly basic ion exchange resin which may optionally consist of phenol-formaldehyde (i.e., phenol-formaldehyde polycondensate). In yet another immobilized enzyme composition of the present invention, the carrier material is based on controlled pore glass (CPG) particles or hybrid CPG particles (WO2015 / 115993A1). In some immobilized enzyme compositions of the present invention, the carrier is functionalized by containing a tertiary amine group, while in others, a secondary amine group provides the functionality. In some immobilized enzyme compositions of the present invention, the carrier resin is functionalized with a group used to chelate metals such as iron or zinc, for example. Chelate metal groups enable high-affinity binding of molecules via appropriate binding groups, such as histidine (His) tags. Examples of such functional groups are found in WO2015 / 115993A1 and Cassimjee et al. A general protein purification and immobilization method on controlled porosity glass: biocatalytic applications. Chem.Commun., 2014, 50, 9134, and include, but are not limited to, 2,4-dihydroxybenzyl residues.

[0024] The carriers in some of the immobilized enzyme compositions of the present invention have two or more of the characteristics of the various carriers listed above. For example, in some of the immobilized enzyme compositions of the present invention, for use in processes that convert starch and starch derivatives and sugars into hexose monosaccharides (including processes that produce tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, or inositol), the enzyme composition of any of the immobilized enzyme compositions described herein is immobilized on a weakly basic anion exchange resin, which may or may not consist of a phenol-formaldehyde polycondensate and may or may not be functionalized with a tertiary amine group. A carrier resin having the above characteristics is commercially available as DUOLITE® A568. In other immobilized enzyme compositions of the present invention, any of the enzyme compositions of any of the immobilized enzyme compositions described herein, for use in processes that convert starch and starch derivatives and sugars into hexose monosaccharides (including processes that produce tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, or inositol), is immobilized on a weakly basic anion exchange resin, which may or may not be composed of a phenol-formaldehyde polycondensate and may or may not be functionalized with a secondary amine group. A carrier resin having the above characteristics is commercially available as DUOLITE® PWA7. In yet another immobilized enzyme composition of the present invention, any of the enzyme compositions of any of the immobilized enzyme compositions described herein for use in processes that convert starch and starch derivatives and sugars into hexose monosaccharides (including processes that produce tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, or inositol) is immobilized on a His tag affinity resin, which may or may not consist of a CPG or hybrid CPG particle material carrier, and may or may not chelate iron or zinc.The carrier resin having the above characteristics is commercially available as EziG(trademark) Opal.

[0025] Generally, the total weight of the enzyme in the immobilized enzyme composition of the present invention is in the range of 2.5% to 12.5% ​​relative to the weight (w / w) of the carrier. Therefore, for use in processes that convert starch and starch derivatives and sugars into hexose monosaccharides (including processes that produce tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, or inositol), the total weight of the enzyme composition of any of the immobilized enzyme compositions described herein may be about 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, or any w / w% in between, relative to the weight (w / w) of the carrier. For example, in some immobilized enzyme compositions used to produce tagatose, the total weight of the enzyme (αGP, PGM, PGI, F6PE, and T6PP) or (αGP, 4GT, PGM, PGI, F6PE, and T6PP) is 5%, while the total weight of the enzyme (αGP, PGM, PGI, P6PE, and P6PP) or (αGP, 4GT, PGM, PGI, P6PE, and P6PP) is 6.5%.

[0026] The present invention also relates to an enzymatic process for preparing hexoses from sugars, comprising the step of contacting a starch derivative with the immobilized enzyme composition of the present invention under suitable reaction conditions to convert the starch derivative into a hexose. In the enzymatic process of the present invention for preparing hexoses, at least two, at least three, at least four, at least five, at least six carriers, at least seven, or at least eight enzymes of the process may be immobilized on the same carrier or on multiple carriers. For example, in the enzyme process of the present invention, the enzyme may be immobilized on the same support, or the immobilized enzyme may be distributed among at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight supports (which may be any combination of the same type of support or different supports), and the immobilization method includes a weakly basic anion exchange resin support, a phenol formaldehyde polycondensate support, a support containing a tertiary amine functional group (e.g., DUOLITE® A568), a support containing a secondary amine functional group (e.g., DUOLITE® PWA7), a support containing a His tag affinity resin, a support containing controlled porous glass (CPG) particles, and a support functionalized with a chelate metal (including a support in which the chelate metal is iron or zinc (e.g., EziG® Opal)).

[0027] Methods for immobilizing enzymes on any of the carriers described herein are known in the art and include appropriate buffers and reaction conditions for binding the enzymes to the carrier resin. See, for example, WO2016 / 160573 (which is incorporated herein by reference in its entirety).

[0028] The process for converting starch and starch derivatives and sugars to hexoses using the immobilized enzyme composition of the present invention can be carried out under the same temperature, buffer, and reaction time parameters as those used for producing hexoses using unimmobilized enzymes in solution. For example, the immobilized enzyme composition of the present invention can be used to produce tagatose, psicose, fructose, allose, mannose, galactose, altrose, talose, sorbose, gross, idose, or inositol from starch and starch derivatives and sugars under the reaction conditions described in published PCT applications 2018 / 169957, 2017 / 059278, and 2018 / 112139. The multiple catalytic reaction steps in the process of producing hexoses using the immobilized enzyme composition of the present invention can be carried out in a single bioreactor, or in multiple bioreactors or reaction vessels arranged in series. Alternatively, these steps can be carried out in multiple bioreactors or reaction vessels arranged in series or in parallel. All of the aforementioned processes can be performed in batch mode or continuous mode. A "one-pot" process in a single bioreactor is preferred.

[0029] The steps in the enzyme process of the present invention can be carried out at temperatures in the range of about 35°C to about 90°C, about 40°C to about 70°C, about 50°C to about 60°C, or about 55°C, and at pH in the range of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.5. These can be carried out for about 0.5 hours to about 48 hours, about 4 hours to 24 hours, or about 8 hours to 12 hours. The steps in the enzyme process of the present invention may be carried out without ATP and / or without NAD(P)(H). The steps can be carried out with phosphate concentrations in the range of about 0.1 mM to about 150 mM. The phosphate used in the phosphorylation and dephosphorylation steps of the process according to the present invention can be reused in the enzyme cascade reaction. The process of the present invention can be carried out in a packed column or in a slurry.

[0030] For example, the reaction phosphoric acid concentration in each process may range from about 0.1 mM to about 300 mM, about 0 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the reaction phosphoric acid concentration in each process may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0031] Lower phosphate concentrations result in lower total phosphate concentrations, which reduces production costs and, consequently, the cost of removing phosphate. Furthermore, inhibition of process enzymes by high concentrations of free phosphate is prevented, reducing the potential for phosphate contamination.

[0032] Furthermore, each of the processes disclosed herein can be carried out without adding ATP as a source of phosphate (i.e., ATP-free). Each process can also be carried out without adding NAD(P)(H) (i.e., NAD(P)(H)-free). Another advantage is that at least one step of the disclosed process for producing hexoses involves a very energetically favorable chemical reaction, which is essential for high yields. The very energetically favorable chemical reactions for the processes of the present invention have at least two, at least three, or at least four equilibrium constants (K). eq ) has.

[0033] As the first step in the process of the present invention, starch derivatives can be prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. See, for example, WO2017 / 059278. For example, enzymatic hydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC.3.2.1.68) which hydrolyzes α-1,6-glucosidic bonds, pullulanase (PA, EC.3.2.1.41) which hydrolyzes α-1,6-glucosidic bonds, 4-α-glucanotransferase (4GT, EC.2.4.1.25) which catalyzes the transglycosylation of short maltooligosaccharides to produce longer maltooligosaccharides, or α-amylase (EC.3.2.1.1) which cleaves α-1,4-glucosidic bonds. Furthermore, cellulose derivatives can be prepared by enzymatic hydrolysis of cellulose, catalyzed by a cellulase mixture, by acid, or by biomass pretreatment. [Examples]

[0034] Example 1. Evaluation of enzyme carriers. Enzyme carriers were evaluated for their effect on the relative reaction rate of the enzyme composition for producing tagatose, as well as on the stability of the enzyme. With these objectives in mind, the following enzyme carrier materials were evaluated: four brands of phenol-formaldehyde matrix resins (DUOLITE® A568, DUOLITE® A561, DUOLITE® PWA7, AmberLite® FPA54), two brands of polystyrene resins (Lifetech® ECR1640 and LifeTech® ECR1504), two brands of polymethyl acrylate resins (Lifetech® ECR8309M and Chromolite® D6154), and one brand of controlled porous glass resin (EziG Opal®).

[0035] DUOLITE® A568 (DuPont) ion exchange resin is a highly porous, granular, weakly basic anion exchange resin with a cross-linked phenol-formaldehyde polycondensation system. Its hydrophilicity and controlled pore size distribution make it ideally suited for use as an enzyme carrier in many bioprocess applications. The ionic strength, pore volume, pore size, and particle size of DUOLITE® A568 are designed for optimal immobilization of enzymes used in the starch and fat (and other) industries.

[0036] DUOLITE® A561 ion exchange resin is a weakly basic anion exchanger made from phenol formaldehyde having a tertiary amine functional group. Its broad specifications are similar to those of DUOLITE® A568 resin, but DUOLITE® A561 has a different bead form.

[0037] DUOLITE® PWA7 is a weakly basic anion exchanger with amine functionality and a salt form.

[0038] AMBERLITE® FPA54 ion exchange resin is a highly porous, weakly basic anion exchange resin with a cross-linked phenol-formaldehyde matrix. AMBERLITE® FPA54's low swelling properties provide excellent osmotic and physical stability, resulting in less product loss and longer product life compared to conventional styrene-based resins in food processing and bioprocess applications. The hydrophilic, phenolic porous matrix of AMBERLITE® FPA54 enables the reversible adsorption of high molecular weight organic chromosomes frequently found in solutions of natural and fermentation products. AMBERLITE® FPA54 exhibits high selectivity for sulfuric acid and phosphoric acid, and therefore, due to its excellent osmotic stability, is ideal for processing both citric acid and lactic acid derived from fermentation, especially when used over extended periods.

[0039] Lifetech® ECR1640 is a copolymer of divinylbenzene (DVB) and styrene functionalized with quaternary amines. It is used for enzyme immobilization via ionic interactions between ionizable surface amino acids (Lys, Arg, His, Asp, Glu) and tertiary amines on the polymer. It is particularly suitable for immobilizing enzymes with pI in the range of 3–5, such as many glycosidases. Key features of Lifetech® ECR1640 include the possibility of resin regeneration, pH adjustment before immobilization, and a large particle size suitable for column applications. It is available in DVB / styrene, quaternary amine, 300–1200 microns, pH stability 1–14, wet feed (66–72% water), volume 0.85 equivalents / l, and in Cl form.

[0040] Lifetech® ECR1504 is a copolymer of divinylbenzene (DVB) and styrene functionalized with tertiary amines. It is used for enzyme immobilization via ionic interactions between ionizable surface amino acids (Lys, Arg, His, Asp, Glu) and the tertiary amines on the polymer. It is particularly suitable for immobilizing enzymes with pI in the range of 3–5, such as many glycosidases. Key features of Lifetech® ECR1504 include the possibility of resin regeneration, pH adjustment before immobilization, and a large particle size suitable for column applications. It is a DVB / styrene copolymer with tertiary amines, particle size 300–1200 microns, pH stability 1–14, wet feed (53–62% water), and a capacity of 1.3 equivalents / l of free base.

[0041] Lifetech® ECR8309M is a hydrophilic, highly porous methacrylate polymer functionalized with amino groups on short spacers (C2).

[0042] Chromolite® D6154 is a macroporous polymethacrylate, a material functionalized for specific affinity bonding to polyHis tags. The functional groups are iminodiacetic acid, Na. + It is a form.

[0043] EziG® Opal is made from controlled porous glass (CPG) particles and has a hydrophilic surface. This material has a narrow pore size distribution and is typically produced with a pore size of approximately 500 Å. A mass loading of 15–60% active oxygen is expected. Other variations of EziG® exist and were tested (Amber and Coral), but they were not optimal compared to Opal.

[0044] An enzyme composition containing each of the following enzymes in the following weight / weight (w / w)% relative to the total enzyme weight was immobilized on the respective carriers described above: 19% α-glucan phosphorylase (αGP, UNIPROT ID G8NCC0, SEQ ID NO: 1), 17% phosphoglucomutase (PGM, UNIPROT ID A0A0P6YKY9, SEQ ID NO: 2), 3% phosphoglucoisomerase (PGI, UNIPROT ID Q5SLL6, SEQ ID NO: 3), 23% fructose-6-phosphate epimerase (F6PE, UNIPROT ID A0A0P6XN50, SEQ ID NO: 4), 35% tagatose-6-phosphate phosphatase (T6PP, UNIPROT ID D1C7G9, SEQ ID NO: 5), and 3% 1,4-glucantransferase (4-GT, UNIPROT ID E8MXP8, SEQ ID NO: 6). All immobilized enzyme preparations had an enzyme-total weight / carrier ratio of 5%. See Table 2. The aforementioned enzyme ratios were previously optimized for tagatose production using the immobilization method described below. [Table 3]

[0045] Before attaching the enzymes in the composition to each carrier, the carriers were equilibrated with 2 equivalents of water, followed by 3 equivalents of immobilization buffer (pH 7.2) (5 mM Na2PO4, 5 mM MgSO4, 0.25 mM MnCl2). The enzymes were suspended in the immobilization buffer to form the enzyme composition (preferably 5-10 g / L of enzyme), which was then added to the carriers to prepare a slurry. The absorbance of the enzyme composition and carrier slurry was measured at 280 nm during incubation at room temperature on an 800 rpm orbital shaker to track the adsorption of the enzymes to the carriers until more than 95% of the soluble enzyme was no longer suspended in the solution. This took approximately 6 hours for a 5% (w / w, enzyme / carrier) loaded sample. The supernatant was removed, and the immobilized carrier was washed with reaction buffer (pH 7.2) (25 mM Na2PO4, 4 mM Na2SO3, 2.5 mM MgSO4, 0.25 mM MnCl2) to remove all remaining soluble enzymes. Each slurry sample was mixed with an equal volume of 2x concentrated feed solution (320 g / L maltodextrin dextrose equivalent 5 (DE5) pH 7.2, 25 mM Na2PO4, 4 mM Na2SO3, 2.5 mM MgSO4, 0.25 mM MnCl2), and the final maltodextrin substrate concentration was adjusted to 160 g / L under reaction conditions. The maltodextrin-carrier mixture was shaken overnight at 800–1500 rpm in a 2.0 mL microfuse tube in an Eppendorf thermomixer F2.0 at either 50°C for DUOLITE® A568, Lifetech® ECR1640, LifeTech® ECR1504, Lifetech® ECR8309M, Chromolite® D6154, and EziG® Opal composition-carrier combinations, or at 55°C for DUOLITE® A561, DUOLITE® PWA7, and AmberLite® FPA54 composition-carrier combinations. This reaction was continued for 15–18 hours. The resulting product was developed on a HiPlex H ligand exchange column (Agilent) using an Agilent 1100 series HPLC system with an in-line refractive index detector (0.6 mL / min at 65°C with 5 mM H2SO4 mobile phase).The concentration of tagatose was determined by comparing the peak area of ​​the sample with that of a known tagatose standard solution. The cascade activity rate (total enzyme, μmol / min / mg, tagatose produced) of the immobilized enzyme composition was calculated for each carrier. The cascade activity rate of each carrier-composition preparation is reported in Table 1, compared with the cascade activity rate of the Duolite A568 carrier-composition preparation. The remaining maltodextrin and tagatose were washed away from each immobilized enzyme preparation by five washes with at least three volume equivalents of reaction buffer, and the preparations were equilibrated for reuse. The immobilized enzyme preparations were reused by adding a 2x concentrated maltodextrin supply solution as before. The reaction rate was calculated and plotted after each subsequent use to confirm the operating half-life of the immobilized catalyst. The half-life was determined by measuring the cascade rate (μmol / min / mg) on ​​day 0 and subsequent days until less than half of the activity (compared to day 0) was consistently lost. In Table 3, the designation n / d indicates that a half-life of less than 10% was found, or that the cascade activity was less than 50% of that of Duolite A568. [Table 4]

[0046] Example 2. Effect of enzyme ratio on cascade rate. To evaluate the effect of changing the ratio of various immobilized enzymes on tagatose formation, the amounts of αGP, PGM, PGI, F6PE, T6PP, and 4-GT immobilized on DUOLITE® A568 were varied in two immobilized composition preparations compared to a DUOLITE® A568 immobilized composition prepared using the enzyme ratios described in Example 1 and Table 1 ("Immobilized Composition of Example 1"). Their activity rates were evaluated against the performance of the immobilized composition of Example 1. In one of the immobilized compositions of various concentrations, the amount of enzyme was based on the observed rate of each enzyme relative to T6PP in solution, as shown in Table 4 (Figure 1B), with an equal amount of enzyme activity (μmol / min) added for each enzyme. The activity rate of this immobilized composition was 74% of the optimal ratio. Other immobilized compositions of various concentrations contained equal amounts of each enzyme by weight (Figure 1C). The activity rate of this immobilized composition was 85% of the optimal ratio. The reaction was carried out, and the conversion rate was measured in μmol / min / mg total enzyme as described in Example 1. [Table 5]

[0047] Example 3. Effect of enzyme distribution on one or more carriers. The process of producing tagatose from maltodextrin using a DUOLITE® A568 immobilized composition (19% αGP, 17% PGM, 3% PGI, 23% F6PE, 35% T6PP, and 3% 4-GT) prepared in a single immobilization reaction as described in Example 1 was compared to the activity rate of a process carried out using: (1) DUOLITE® A568 immobilized composition of core-producing enzymes (αGP, PGM, PGI, and 4GT) (1) and (2) a mixture of DUOLITE® A568 immobilized compositions of tagatose-specific enzymes (F6PE and T6PP), and (3) six distinct immobilized enzyme preparations: DUOLITE® A568 immobilized αGP, DUOLITE® A568 immobilized PGM, DUOLITE® A568 immobilized PGI, DUOLITE® A568 immobilized 4GT, DUOLITE® A568 immobilized F6PE, and DUOLITE® A568 immobilized T6PP. All enzymes in (1) and (2) were present in the same proportions as the referenced DUOLITE® A568 immobilized compositions, and all immobilization was carried out using a 5% w / w ratio (enzyme / carrier) and buffer / reaction conditions as described in Example 1. The results are shown in Table 5. [Table 6]

[0048] Example 4. Effects of enzyme loading. Enzymes under different loading conditions (g enzyme / g carrier, ranging from 2.5% to 12.5% ​​at 2.5% intervals) were immobilized on Duolite A568 as described in Example 1 and in the ratios listed in Table 2. However, unlike Example 1, immobilization was continued for 16 hours for each sample instead of 6 hours, allowing more time for enzyme binding to be completed at higher loading conditions. The soluble enzyme was added to at least three sample volumes of reaction buffer pH 7.2 (25 mM Na2PO4). 4、The sample was washed five times with a solution containing 4 mM Na₂SO₃, 2.5 mM MgSO₄, and 0.25 mM MnCl₂. The sample was reacted with a maltodextrin supply solution for 16 hours, and the conversion rate was measured in μmol / min / mg of total enzyme as in Example 1. The relative enzyme cascade rate to the 5% loaded sample is plotted as a function of loading in Figure 2. The loading response, along with the cost of the enzyme and support, allows for the design of the most cost-effective catalyst.

[0049] Example 5. Preparation of allulose using immobilized enzyme compositions. As shown in Table 6, enzyme compositions containing the following enzymes in three different ratios: αGP, PGM, PGI, psicose 6-phosphate 3-epimerase (P6PE), psicose 6-phosphate phosphatase (P6PP), and 4-GT, were immobilized on DUOLITE® A568 in their respective immobilization reactions. A comparative analysis of the three immobilized composition preparations for producing allulose from maltodextrin was performed. [Table 7]

[0050] To prepare the immobilization cocktail, DUOLITE® A568 was pretreated in a 1% glutaraldehyde (GA) aqueous solution in an end-over-end rotator at room temperature for 2 hours. The GA was removed by washing five times with water and twice with the immobilization buffer (10 mM sodium phosphate buffer (pH 7.2), 5 mM MgSO4, and 80 μM CoCl2). The enzyme solution (Table 5) was then added to the GA-pretreated carrier after the final washing step (the supernatant was discarded). The enzyme solution consisted of 5 g / L of enzyme in the reaction buffer (10 mM sodium phosphate buffer (pH 7.2), 5 mM MgSO4, 5 mM NaSO3, and 80 μM CoCl2). The enzyme and carrier solutions were incubated on an orbital shaker set to 800 rpm at room temperature for 16 hours to immobilize the enzyme mixture onto the carrier. The total percentage loading was 6.5% (mg of enzyme per 1 mg of carrier). The supernatant was washed six times with reaction buffer to remove any remaining unbound enzyme. The final supernatant was removed, and 150 g / L of maltodextrin, pre-dissolved in reaction buffer, was added. The maltodextrin-carrier mixture was shaken overnight (15-16 hours) at 800-1500 rpm at 55°C in a 2.0 mL microfuse tube of an Eppendorf thermomixer F2.0. The resulting product was developed on a SupelCogel Pb column (Sigma Aldrich) using an Agilent 1100 series HPLC system with an in-line refractive index detector (0.6 mL / min at 80°C with ultrapure water mobile phase). Allulose concentration was determined by comparing the sample peak area with that of a known allulose standard solution, and the enzyme cascade specific activity was calculated. The allulose-producing reaction product was washed away from each immobilized preparation by four washes with reaction buffer, and the immobilized preparations were equilibrated for reuse. The relative specific activity results for each sample composition are shown in Table 7 below. [Table 8]

[0051] Example 6. Preparation of allose from maltodextrin using an immobilized enzyme composition. Allose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, 4GT, and P6PE, A6PI, and A6PP.

[0052] Example 7. Preparation of fructose from maltodextrin using an immobilized enzyme composition. Fructose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, 4GT, and F6PP.

[0053] Example 8. Preparation of mannose from maltodextrin using an immobilized enzyme composition. Fructose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, 4GT, and M6PI or PGPMI and M6PP.

[0054] Example 9. Preparation of galactose from maltodextrin using an immobilized enzyme composition. Galactose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, F6PE, 4GT, Gal6PI, and Gal6P.

[0055] Example 10. Preparation of altrose from maltodextrin using an immobilized enzyme composition. Altrose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, P6PE, Alt6PI, and Alt6PP.

[0056] Example 11. Preparation of talose from maltodextrin using an immobilized enzyme composition. Talose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, F6PE, Tal6PI, and Tal6PP.

[0057] Example 12. Preparation of sorbose from maltodextrin using an immobilized enzyme composition. Sorbose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, F6PE, S6PE, and S6PP.

[0058] Example 13. Preparation of growth from maltodextrin using an immobilized enzyme composition. Growth is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, F6PE, S6PE, Gul6PI, and Gul6PP.

[0059] Example 15. Preparation of idose from maltodextrin using an immobilized enzyme composition. Idose is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, PGI, F6PE, S6PE, I6PI, and I6PP.

[0060] Example 16. Preparation of inositol from maltodextrin using an immobilized enzyme composition. Inositol is produced from maltodextrin using an immobilized enzyme composition containing αGP, PGM, 4GT, IPS, and IMP.

Claims

1. An immobilized enzyme composition for the preparation of hexoses, comprising at least two, at least three, at least four, at least five, at least six carriers, at least seven, or at least eight of the following enzymes immobilized on at least one carrier or mixture of carriers: a) α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), and optionally, 1,4-glucantransferase (4-GT), b) An enzyme selected from the following combinations of enzymes: (i) Phosphoglucoisomerase (PGI), fructose-6-phosphate epimerase (F6PE), and tagatose-6-phosphate phosphatase (T6PP) for preparing tagatose, (ii) Phosphoglucoisomerase (PGI), picose-6-phosphate epimerase (P6PE), and picose-6-phosphate phosphatase (P6PP) for preparing allulose, (iii) For preparing allose, phosphoglucoisomerase (PGI), P6PE, allose-6-phosphate isomerase (A6PI), and allose-6-phosphate phosphatase (A6PP), (iv) For the preparation of mannose, phosphoglucoisomerase (PGI), mannose-6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI), and mannose-6-phosphate phosphatase (M6PP), (v) Phosphoglucoisomerase (PGI), F6PE, galactose 6-phosphate isomerase (Gal6PI), and galactose 6-phosphate phosphatase (Gal6PP) for preparing galactose. (vi) PGI and fructose 6-phosphate phosphatase (F6PP) for preparing fructose. (vii) PGI, P6PE, altrose 6-phosphate isomerase (Alt6PI), and altrose 6-phosphate phosphatase (Alt6PP) for preparing altrose. (viiii) PGI, F6PE, talose 6-phosphate isomerase (Tal6PI), and talose 6-phosphate phosphatase (Tal6PP) for preparing talose, (ix) PGI, F6PE, sorbose 6-phosphate epimerase (S6PE), and sorbose 6-phosphate phosphatase (S6PP) for preparing sorbose. (x) PGI, F6PE, S6PE, growth 6-phosphate isomerase (Gul6PI), and growth 6-phosphate phosphatase (Gul6PP) for preparing growth. (xi) PGI, F6PE, S6PE, idose 6-phosphate isomerase (I6PI), and idose 6-phosphate phosphatase (I6PP) for preparing idose, (xi) Inositol 3-phosphate synthase (IPS) and inositol monophosphatase (IMP) for preparing inositol.

2. The immobilized enzyme composition according to claim 1, wherein the weight (w / w) percentage of each enzyme relative to the total weight of the enzymes is in the range of 0.1% to 40%.

3. The immobilized enzyme composition according to claim 1 or 2, comprising 10-30% (αGP), 0-10% (4GT), 10-30% (PGM), and, if present, 0.1-10% (PGI).

4. An immobilized enzyme composition according to any one of claim 3, further comprising PGI, F6PE, and T6PP for preparing tagatose.

5. The immobilized enzyme composition according to claim 4, wherein the weight (w / w) percentage of each enzyme relative to the total weight of the enzymes is 10-30% (αGP), 0-10% (4GT), 10-30% (PGM), 0.1-10% (PGI), 15-35% (F6PE), and T6PP (25-45%), and the total weight of the enzymes in the composition is 100 w / w% of the total weight of the enzymes.

6. The αGP comprises the amino acid sequence of SEQ ID NO: 1, or a fragment thereof. The 4-GT comprises the amino acid sequence of SEQ ID NO: 6, or a fragment thereof. The PGM comprises the amino acid sequence of SEQ ID NO: 2, or a fragment thereof. The PGI comprises the amino acid sequence of SEQ ID NO: 3, or a fragment thereof. The F6PE comprises the amino acid sequence of SEQ ID NO: 4, or a fragment thereof. The immobilized enzyme composition according to claim 5, wherein the T6PP comprises the amino acid sequence of Sequence ID No. 5 or a fragment thereof.

7. An immobilized enzyme composition according to any one of claim 3, further comprising PGI, P6PE, and P6PP for preparing allulose.

8. The immobilized enzyme composition according to claim 7, wherein the weight (w / w)% of each enzyme relative to the total weight of the enzymes is 10-30% (αGP), 0-10% (4GT), 10-30% (PGM), 0.1-10% (PGI), 0.1-10% (P6PE), and (45-65%) P6PP, and the total weight of the enzymes in the composition is 100 w / w% of the total weight of the enzymes.

9. The αGP comprises the amino acid sequence of SEQ ID NO: 1, or a fragment thereof. The aforementioned 4-GT contains the amino acid sequence of SEQ ID NO: 6, The PGM comprises the amino acid sequence of SEQ ID NO: 2, The PGI comprises the amino acid sequence of SEQ ID NO: 3, The P6PE contains the amino acid sequence of SEQ ID NO: 7, The immobilized enzyme composition according to claim 8, wherein the P6PP comprises the amino acid sequence of SEQ ID NO:

8.

10. The immobilized enzyme composition according to any one of claims 1 to 9, wherein the total weight of the enzyme (weight / weight) relative to the weight of the carrier is 2.5% to 12.5%.

11. The immobilized enzyme composition according to any one of claims 1 to 10, wherein the carrier is a weakly basic anion exchange resin.

12. The immobilized enzyme composition according to claim 11, wherein the carrier comprises a phenol-formaldehyde polycondensate.

13. The immobilized enzyme composition according to claim 11 or 12, wherein the carrier comprises a tertiary amine functional group, and the composition is optionally DUOLITE® A568.

14. The immobilized enzyme composition according to claim 11 or 12, wherein the carrier comprises a secondary amine functional group, and the composition is optionally DUOLITE™ PWA7.

15. The immobilized enzyme composition according to any one of claims 1 to 10, wherein the carrier comprises a His tag affinity resin.

16. The immobilized enzyme composition according to any one of claims 15, wherein the carrier comprises controlled porous glass (CPG) particles.

17. The immobilized enzyme composition according to claim 15 or 16, wherein the carrier is functionalized with a chelate metal.

18. The immobilized enzyme composition according to claim 18, wherein the chelate metal is iron or zinc, or the carrier is optionally EziG(trademark)Opal.

19. An enzymatic process for preparing a hexose from a sugar, comprising the step of contacting a starch derivative with an immobilized enzyme composition according to any one of claims 1 to 18 under suitable reaction conditions to convert the starch derivative to a hexose.

20. The process according to claim 19, wherein the steps of the process are carried out at a temperature in the range of about 40°C to about 85°C, at a pH in the range of about 5.0 to about 8.0, and / or for about 0.5 hours to about 48 hours.

21. The process according to claim 19 or 20, wherein the steps of the process are carried out in a single bioreactor, a plurality of bioreactors arranged in series, or a plurality of bioreactors arranged in parallel.

22. The process according to any one of claims 19 to 21, wherein the steps of the process are carried out at a phosphate concentration of about 0.1 mM to about 150 mM, without ATP and without NAD(P)(H), the phosphate is reused in an enzyme cascade reaction, and / or at least one step of the process involves a highly energetically favorable chemical reaction.

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