ENZYMATIC PRODUCTION OF HEXOSES

Abstract

The present invention relates to methods for producing hexoses from saccharides via enzymatic processes. The methods utilize fructose 6-phosphate and at least one enzymatic step to convert it into a hexose.

Description

ENZYMATIC PRODUCTION OF HEXOSES Cross Reference for Related Requests

[001] This application requests priority to United States Application No. 62 / 470, 605, filed March 13, 2017. United States Application No. 62 / 470, 620, filed March 13, 2017. United States Application No. 62 / 482,148, filed April 5, 2017, and United States Application No. 62 / 480,798, filed April 3, 2017, both of which are incorporated by reference herein in their entirety. Scope of the invention

[002] The invention relates to the preparation of hexose monosaccharides. More specifically, the invention relates to methods of preparing a D-hexose (or hexose) from saccharides (for example: polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose) including a step in that fructose 6-phosphate is converted to hexose by one or more enzymatic steps. Background

[003] Hexoses are monosaccharides with six carbon atoms. These can be classified by functional groups, with aldohexoses having an aldehyde in position 1, and ketohexoses having a ketone in position 2. Aldohexoses (or aldoses) include allose, altrose, glucose, galactose, idose, thallose, and mannose. . Ketohexoses or ketoses include psychose (allulose), fructose, tagatose, and sorbose. Various aspects of these aldohexoses and ketohexoses are mentioned in the following paragraphs.

[004] For example, D-allose (hereinafter allose) is a low-calorie natural sweetener that has 80% of the sweetness of sucrose and is described as a non-caloric sweetener and as a bulking agent. It is a naturally occurring hexose monosaccharide that is present only in small amounts in specific shrubs and algae. Allose has various medical and agricultural benefits, including: cryoprotectants, antioxidants, antihypertensives, immunosuppressants, anti-inflammatory, antitumor and anticancer activities. It also has similar functions to sucrose in drinks and foods. Thus, alose certainly has a variety of applications in the food and beverage industry. However, due to the high prices of allose, its use as a sweetener has been limited.

[005] Currently, allose is mainly produced through the enzymatic isomehzation of Dpsicose (WO 2014069537). Overall, the method is affected by higher raw material costs, costly separation of allose from D-psychose, and low product profits (23%).

[006] Altrose is another artificial aldohexose and a C-3 epimer of mannose. D-Altrose ((2S,3R,4R,5R)2,3,4,5,6-Pentahydroxyhexanal) can be used as a substrate to identify, differentiate and characterize aldose isomerases, such as the L-fucose isomerase of Caldicellulosiruptor saccharolyticus and darabinose isomerase (d-AI) from Bacillus pallidus (B. pallidus) and Klebsiella pneumoniae. Recently, sugar chains, such as oligosaccharides and polysaccharides, which carry out useful functions as a physiologically active substance, have attracted attention in the field of fine chemicals, such as cao / nn / eznz / E / YiAi medicines and agrochemicals. Currently, the objects of research in the sugar chain are restricted to those consisting of monosaccharides present in large quantities in nature and easily available to researchers, such as D-glucose, D-mannose, and D-galactose. However, it is expected that many other monosaccharides that are present in nature will be required in the future in the world of research in the synthesis of sugar chains, performing more useful functions. Under the circumstances, it is highly significant and necessary to develop a method that allows preparing D-altrose, which is a rare and difficult to obtain sugar with high yield while decreasing the number of treatment steps. US Patent No. 5,410,038. [οοη D-gulose is useful, for example, as an excipient, as a chelating agent, as a pharmaceutical intermediate, as a cleaning agent for glass and metals, as a food additive, and as an additive for detergents.

[008] D-galactose (galactose hereinafter) is a natural sweetener that has 33% of the sweetness of sucrose, and is described as a nutritious sweetener. It is a naturally occurring hexose monosaccharide that is present in dairy products, legumes, grains, nuts, tubers, and vegetables. Galactose is used in the baking industry to reduce acidity in foods. It is also used as an energy source to increase endurance in the exercise supplement industry. In the pharmaceutical industry it is an intermediary for various medicines and is also used as a modulator of cellular metabolism to optimize the bioproduction of therapeutic proteins. Additionally, galactose has been shown to be effective as a control agent against plant diseases caused by certain plant pathogens, such as those that affect cucumber, carrot, potato and tomato plants. Due to dietary concerns (e.g., veganism) and health issues (e.g., bovine spongiform encephalopathy, BSE), non-animal sources of galactose are of interest to the industry. Thus, galactose clearly has a variety of applications in the food, beverage, exercise / physical activity, agriculture and pharmaceutical industries. However, due to the high sales prices of galactose, its use has been limited.

[009] Galactose is produced mainly through the hydrolysis of lactose (WO 2005039299A3). This method is less desirable because the raw material and the separation of glucose from galactose is more expensive. Alternatively, galactose can be produced via the hydrolysis of plant blomase (WO 2005001145A1). This method is affected by the costly separation of galactose from multiple other sugars released during biomass hydrolysis (e.g., xylose, arabinose, mannose, glucose, and rhamnose) and low production (4.6% of the dry mass of biomass sources). common is galactose?)

[010] Idosa is not found in nature, but its uranic and iduronic acids are important. It is a component of dermatan sulfate and heparan sulfate, which are glycosaminoglycans.

[011] Talose is an artificial aldohexose that is soluble in water and slightly soluble in methanol. It is a C-2 epimer of galactose and C-4 epimer of mannose. Talose can be used as a substrate to identify, differentiate and characterize Clostridia ribose 5-phosphate isomerase(s).

[012] D-mannose (hereinafter mannose) is a naturally occurring, slightly sweet monosaccharide found in various fruits, vegetables, in plant materials, and even in the human body. Mañosa has multiple health benefits and pharmaceutical applications. For example, CQQ / nn / Q7n7 / e / YiAi can be used to treat carbohydrate-deficient glycoprotein syndrome type 1 b, and more commonly, urinary tract infections. In addition, mannose is a verified prebiotic, does not raise blood glucose levels, and shows anti-inflammatory properties. Additionally, it has been shown to improve body performance in pigs and is a widely used moisturizing agent in skin care products. In this way, mañosa has a variety of applications in the pharmaceutical, cosmetic, beverage, food, dairy, confectionery, and livestock industries. However, due to the high sales prices of mañosa, its use in daily use products has been limited.

[013] Mañosa is mainly produced by extracting plants. Common methods include acid hydrolysis, thermal hydrolysis, enzymatic hydrolysis, microbial fermentation hydrolysis, and mixtures thereof. Less common methods include chemical and biological transformations. In general, these methods are complicated by harsh conditions, high capital expenditures, higher raw material costs, costly separation of mannose from isomerization reactions, and relatively low production yields (15-35%).

[014] D-allulose (also known as D-psychose) (hereinafter psychose) is a low-calorie sweetener, which has 70% of the sweetness of sucrose, but only 10% of the calories. It is a naturally occurring hexose monosaccharide that is present only in small amounts in wheat and other plants. Psychose was approved as a food additive by the Food and Drug Administration (FDA) in 2012, who designated it as safe (GRAS). However, due to the high sales prices of psychose, its use as a sweetener has been limited. Psychose has countless health benefits: it is low in calories (10% sucrose); It has a low glycemic index of 1; It is completely absorbed in the small intestine, but not metabolized and is instead secreted in urine and feces; helps regulate blood sugar by inhibiting alpha amylase, sucrase and maltase; and has a similar function in foods and drinks, like sucrose. Thus, psychose clearly has a variety of applications in the food and beverage industries.

[015] Currently, psychose is produced mainly through the enzymatic isomerization of fructose (WO 2014049373). In general, the method exhibits higher raw material cost, costly separation of psychose from fructose, and relatively low production yields.

[016] Fructose is a simple ketone monosaccharide found in various plants, where it is often linked to glucose to form the disaccharide, sucrose. Commercially, fructose is derived from sugar cane, sugar beets, and corn. The main reason fructose is used commercially in beverages and foods, besides its low cost, is its relative high sweetness. It is the sweetest of all naturally existing carbohydrates. Fructose is also found in the industrialized sweetener, high fructose corn syrup (HFCS), which is produced by enzyme-treated corn syrup, converting glucose into fructose. (https: / / en.wikipedia.org / wik¡ / Fructose#Physical_and_funct¡onal_propert¡es- accessed 3 / 7 / 18).

[017] D-tagatose (hereinafter tagatose) is a low-calorie natural sweetener that has 92% of the sweetness of sucrose, but only 38% of the calories. It is a naturally occurring hexose monosaccharide that is present only in small amounts in fruits, cocoa, and dairy products. The tagatose CQO / nn / eznz / E / YiAi was approved as a food additive by the Food and Drug Administration (FDA) in 2003, which designated it generally as safe (GRAS). However, due to the high sales prices of tagatose, its use as a sweetener has been limited. Tagatose has countless health benefits: it is non-carogenic; low in calories; It has a very low glycemic index of 3; attenuates the glycemic index of glucose by 20%; can lower average blood glucose levels; helps prevent cardiovascular diseases, strokes, and other vascular diseases by raising HDL cholesterol; and is a verified prebiotic and antioxidant. Lu et al., Tagatose, a New Antidiabetic and Obesity Control Drug, Diabetes Obes. Metab. 10(2): 109-34 (2008). Thus, tagatose clearly has a variety of applications in the pharmaceutical, biotechnology, academic, food, beverage, dietary supplement, and grocery industries.

[018] Tagatose is mainly produced through the hydrolysis of lactose by lactase or acid hydrolysis to form D-glucose and D-galactose (WO 2011150556, CN 103025894, US 5002612, US 6057135, and US 8802843). D-galactose is then isomerized to D-tagatose, either chemically by calcium hydroxide under alkaline conditions or enzymatically by L-arabinose isomerase under neutral pH conditions. The final product is isolated by a combination of filtration and ion exchange chromatography. This process is carried out in several tanks or bioreactors. In general, the method has its disadvantages, since the separation of other sugars (for example: D-glucose, D-galactose, and non-hydrolyzed lactose) is expensive and there are low production yields. Various methods are being developed via fermentation of microbial cells, but none has proven to be a convenient alternative because it depends on a high cost of raw materials (for example: galactitol and D-psychose), low production yields, and costly separation. .

[019] Sorbose ((3R,4S,5R)-1,3,4,5,6-pentahydroxyhexane-2-one) is a ketohexose that has a sweetness equivalent to sucrose (common sugar), and is a metabolite vegetable that has been found to occur naturally in small quantities in grapes. It has been determined that D-sorbose is effective as a control agent in plant diseases caused by: Pseudomonas syringae pv. lachrymans and Ralstonia solanacearum. US Patent Application, Publication No. 2016 / 0037768.

[020] There is a need to develop cost-effective synthetic routes for high-yield production of hexoses, such as the aforementioned aldohexoses and aldoketoses, where at least one of the process steps includes an energetically favorable chemical reaction. Furthermore, there is a need for process production, where the process steps can be carried out in a tank or bioreactor and / or where costly separation steps are avoided or eliminated. There is also a need for hexose production processes that can be carried out at a relatively low phosphate concentration, where the phosphate can be recycled, and / or where the process does not require using adenosine triphosphate (ATP) as an added phosphate source. . Hexose production pathways are also needed that do not require using the expensive nicotinamide adenosine dinucleotide coenzyme (NAD(P)(H)) in any of the reaction steps. CQQ / nn / Qznz / B / YiAi BRIEF DESCRIPTION OF THE INVENTION

[021] The inventions described herein generally relate to processes for preparing hexoses from saccharides by enzymatic conversion. The inventions also relate to hexoses prepared by any of the processes described herein.

[022] More specifically, the invention relates to processes for preparing a hexose, selected from allose, mannose, galactose, fructose, altrose, thallose, sorbose, gulose and idose, from a saccharide, wherein the process is It comprises as follows: converting fructose 6-phosphate (F6P) to hexose catalyzed by one or more enzymes selected from a specific isomerase, an epimerase, and a hexose phosphatase and mixtures thereof.

[023] A process of the invention for the production of allose includes converting F6P to psychose 6-phosphate (P6P) catalyzed by psychose 6-phosphate 3-epimerase (P6PE); convert P6P to allose 6-phosphate (A6P) catalyzed by allose 6-phosphate isomerase (A6PI); and convert A6P to allose catalyzed by allose 6-phosphate phosphatase (A6PP).

[024] A process of the invention for the production of mannose includes converting F6P to mannose 6-phosphate (M6P) catalyzed by mannose 6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI); and convert M6P to mannose catalyzed by mannose 6-phosphate phosphatase (M6PP).

[025] A process of the invention for the production of galactose includes converting F6P to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE); convert T6P to galactose 6-phosphate (Gal6P) catalyzed by galactose 6-phosphate isomerase (Gal6PI); and convert Gal6P into galactose catalyzed by galactose 6-phosphate phosphatase (Gal6PP).

[026] A process of the invention for the production of fructose includes converting F6P to fructose catalyzed by fructose 6-phosphate phosphatase (F6PP). [02η A process of the invention for the production of altrose includes converting F6P to P6P catalyzed by P6PE; convert P6P to altrose 6-phosphate (Alt6P) catalyzed by altrose 6-phosphate isomerase (Alt6PI); and convert the AI16P produced to altrose catalyzed by altrose 6-phosphate phosphatase (AH6PP).

[028] A process of the invention for the production of thallose includes converting F6P to T6P catalyzed by F6PE; convert T6P to talose 6-phosphate (Tal6P) catalyzed by talose 6-phosphate isomerase (Tal6PI); and convert Tal6P to talose catalyzed by talose 6-phosphate phosphatase (Tal6PP).

[029] A process of the invention for the production of sorbose includes converting F6P to T6P catalyzed by F6PE; convert T6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE); and convert S6P to sorbose catalyzed by sorbose 6-phosphate phosphatase (S6PP).

[030] A process of the invention for the production of gulose includes converting F6P to T6P catalyzed by F6PE; convert S6P to gulose 6-phosphate (Gul6P) catalyzed by gulose 6-phosphate isomerase (Gul6PI); and convert Gul6P to gulose catalyzed by gulose 6-phosphate phosphatase (Gul6PP).

[031] A process of the invention for the production of gulose includes converting F6P to T6P catalyzed by F6PE; convert T6P to sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE); convert S6P toidose 6-phosphate (I6P) catalyzed byidose 6-phosphate isomerase (I6PI); and convert I6P to idose catalyzed byidose 6-phosphate phosphatase (I6PP). CQQ / nn / Q7n7 / e / YiAi

[032] According to the invention, hexose production processes may include a step of converting glucose 6-phosphate (G6P) to F6P, wherein the step is catalyzed by phosphoglucose isomerase (PGI). The processes may also include the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). Furthermore, according to the invention, the processes could include the step of converting saccharide to G1P, wherein the step is catalyzed by at least one enzyme, and the saccharide is selected from the group consisting of a starch or derivative thereof, cellulose or derivative thereof, and sucrose.

[033] The enzyme or enzymes used in the step of converting a saccharide to G1P in the processes according to the invention may be alpha-glucan phosphorylase (aGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase, and / or cellulose phosphorylase, and mixtures thereof. When the saccharide is starch or derived starch, the derivative may be selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose, and mixtures thereof.

[034] Some processes according to the invention could further include the step of converting starch to a starch derivative, wherein the starch derivative is prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. Additionally, 4-glucanotransferase (4GT) can be added to the processes. 4GT can be used to increase hexose gains by recycling the breakdown products of glucose, maltose, and maltotriose into longer maltooligosaccharides; which can be phosphorolytically cleaved by aGP to produce G1P.

[035] Where the processes use a starch derivative, this derivative may be prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, alpha-amylase or their combinations.

[036] The process according to the inventions may include the step of converting fructose to F6P, wherein the step is catalyzed by at least one enzyme, and, optionally, the step of converting sucrose to fructose, wherein the step It is catalyzed by at least one enzyme. [03η Furthermore, processes for producing a hexose, according to the inventions, may include the step of converting glucose to G6P, wherein the step is catalyzed by at least one enzyme, and, optionally, the step of converting sucrose to glucose , which is catalyzed by at least one enzyme.

[038] According to the invention, the steps in each of the hexose synthesis processes can be carried out at a temperature ranging from 40°C to about 90°C at a pH ranging from 5.0 to about 8.0. They can be done from 8 hours to 48 hours.

[039] The process steps according to the inventions can be carried out only in a bioreactor. The steps are also performed in various bioreactors arranged in series.

[040] The steps of the enzymatic process of the inventions are carried out free of ATP and / or free of NAD(P)(H). The steps can be performed at a phosphate concentration ranging from 0.1 mM to about 150 mM. The phosphate used in the phosphorylation and dephosphorylation steps of the processes according to the inventions can be recycled. At least one of the process steps could include an energetically favorable chemical reaction.

[041] The invention also relates to allose, mannose, galactose, fructose, altrose, thallose, sorbose, gulose and idose produced by these processes. CQO / nn / eznz / E / YiAi

[042] BRIEF DESCRIPTION OF THE FIGURES

[043] FIG 1 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to allose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; P6PE, psychose 6-phosphate 3-epimerase; A6PI, allose 6phosphate isomerase; A6PP, allose 6-phosphate phosphatase.

[044] FIG2 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to mannose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI phosphoglucoisomerase; PGPMI, bifunctional phosphoglucose / phosphomannose isomerase; M6PI, mannose 6-phosphate isomerase; M6PP, mannose 6-phosphate phosphatase.

[045] FIG 3 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to galactose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate isomerase; Gal6PI, galactose 6phosphate isomerase; Gal6PP, galactose 6-phosphate phosphatase.

[046] FIG4 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to galactose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; F6PP, fructose 6-phosphate phosphatase. [04η FIG 5 is a schematic diagram showing an enzymatic pathway that converts sucrose to fructose. The following abbreviations are used: SP, sucrose phosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; F6PP, fructose 6-phosphate phosphatase.

[048] FIG 6 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to altrose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; P6PE, psychose 6-phosphate epimerase; Alt6PI, altrose 6phosphate isomerase; Alt6PI, altrose 6-phosphate phosphatase.

[049] FIG 7 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to thallose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; Tal6PI, talose 6phosphate isomerase; Tal6PP, talose 6-phosphate phosphatase.

[050] FIG 8 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to sorbose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; S6PE, sorbose 6phosphate epimerase; S6PP, sorbose 6-phosphate phosphatase. cao / nn / eznz / E / YiAi

[051] FIG 9 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to glucose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; S6PE, sorbose 6phosphate epimerase; GulGPI, gulose 6-phosphate isomerase, Gul6PP, gulose 6-phosphate phosphatase.

[052] FIG 10 is a schematic diagram showing an enzymatic pathway that converts starch or its derived products to idose. The following abbreviations are used: IA, isoamylase; PA, pullulanase; aGP, alpha-glucan phosphorylase or starch phosphorylase; MP, maltose phosphorylase; PGM, phosphoglucomutase; PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase; F6PE, fructose 6-phosphate epimerase; S6PE, sorbose 6phosphate epimerase; I6PI, idose 6-phosphate isomerase, I6PP, idose 6-phosphate phosphatase.

[053] FIG 11 shows the Gibbs Energy of reaction between intermediates based on the formation of Gibbs Energy for the conversion of glucose 1-phosphate to another hexose. DESCRIPTION OF THE INVENTION

[054] The inventions described herein provide enzymatic routes, or processes, for synthesizing hexoses with high product yield, while greatly decreasing product separation costs and hexose production costs. The hexoses produced in these processes are also described herein.

[055] According to the invention, the processes for preparing a hexose from a saccharide comprise: converting fructose 6-phosphate (F6P) into hexose, catalyzed by one or more enzymes, wherein the hexose is selected from the group consisting in allose, mannose, galactose, fructose, altrose, thallose, sorbose, gulose and idose; and wherein the enzymes are selected from the group consisting of an isomerase, an epimerase and a specific hexose phosphatase, and mixtures thereof.

[056] One of the important advantages of the processes of the invention is that the process steps can be carried out in a single bioreactor or reaction vessel. Alternatively, the steps may also be performed in a plurality of bioreactors, or reaction vessels, that are arranged in series. [05η Phosphate ions produced during the dephosphorylation step can be recycled to the process step where a saccharide is converted to G1P, particularly when all process steps are carried out in a single bioreactor or reaction vessel. The ability to recycle phosphate in the processes described allows the use of non-stoichiometric amounts of phosphate, which keeps reaction phosphate concentrations low. This affects the overall pathway and overall speed of the processes, but does not limit the activity of individual enzymes and allows for overall efficiency of the hexose manufacturing processes.

[058] For example, the reaction phosphate concentrations in each of the processes can vary from about 0.1 mM to about 300 mM, from about 0 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from about 10 mM to about 50 mM. For example, the reaction phosphate concentration in each of the processes can be cao / nn / eznz / E / YiAi approximately 0.1 mM, approximately 0.5 mM, approximately 1 mM, approximately 1.5 mM, approximately 2 mM, approximately 2.5 mM , about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45 mM, about 50 mM or about 55 mM.

[059] The low phosphate concentration results in decreased production costs due to low total phosphate and therefore a lower cost for phosphate removal. It also prevents inhibition of phosphatases by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[060] Furthermore, each of the processes described herein can be performed without added ATP as a phosphate source, i.e., ATP-free. Each of the processes can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the described processes for manufacturing a hexose involves an energetically favorable chemical reaction.

[061] Examples of enzymes used to convert a saccharide to G1P include alpha-glucan phosphorylase (aGP, EC 2.4.1.1), maltose phosphorylase (MP, EC 2.4.1.8), cellodextrin phosphorylase (CDP, EC 2.4.1.49) , cellobiose phosphorylase (CBP, EC 2.4.1.20), cellulose phosphorylase, sucrose phosphorylase (SP, EC 2.4.1.7), and a combination thereof. The choice of enzyme or combination of enzymes depends on the saccharide used in the process.

[062] The saccharides that are used to generate G1P can be polysaccharides, oligosaccharides and / or disaccharides. For example, the saccharide may be starch, one or more starch derivatives, cellulose, one or more cellulose derivatives, sucrose, one or more sucrose derivatives, or a combination thereof.

[063] Starch is the most used energy storage compound in nature and is mainly stored in plant seeds. Natural starch contains linear amylose and branched amylopectin. Examples of starch derivatives include amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, fructose and glucose. Examples of cellulose derivatives include pretreated biomass, regenerated amorphous cellulose, cellodextrin, cellobiose, fructose and glucose. Sucrose derivatives include fructose and glucose.

[064] Methods for preparing F6P from starch and its derivatives, cellulose and its derivatives, and sucrose and its derivatives can be found, for example, in International Patent Application Publication No. WO 2017 / 059278.

[065] Starch derivatives can be prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. Specifically, the enzymatic hydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC. 3.2.1.68), which hydrolyzes α-1,6-glycosidic bonds; pullulanase (PA, EC. 3.2.1.41), which hydrolyzes α-1,6-glycosidic bonds; 4-a-glucanotransferase (4GT, EC. 2.4.1.25), which catalyzes the transglycosylation of short malto oligosaccharides, producing longer malto oligosaccharides; or alpha-amylase (EC 3.2.1.1), which cleaves α-1,4-glycosidic bonds. CQO / nn / eznz / E / YiAi

[066] Furthermore, cellulose derivatives can be prepared by enzymatic hydrolysis of cellulose catalyzed by cellulase mixtures, by acids or by pretreatment of biomass. [Οθη Enzymes used to convert a saccharide to G1P may contain aGP. In this step, when saccharides include starch, G1P is generated from starch by aGP; When saccharides contain soluble starch, amylodextrin or maltodextrin, G1P is produced from soluble starch, amylodextrin or maltodextrin by aGP.

[068] When the saccharides include maltose and the enzymes contain maltose phosphorylase, maltose phosphorylase generates G1P from maltose. If the saccharides include sucrose and the enzymes contain sucrose phosphorylase, sucrose phosphorylase generates G1P from sucrose.

[069] When the saccharides include cellobiose, and the enzymes contain cellobiose phosphorylase, cellobiose phosphorylase can produce G1P from cellobiose.

[070] When the saccharides contain cellodextrins and the enzymes include cellodextrin phosphorylase, G1P can be generated from cellodextrins by cellodextrin phosphorylase.

[071] When converting a saccharide to G1P, when the saccharides include cellulose and the enzymes contain cellulose phosphorylase, cellulose phosphorylase can generate G1P from cellulose.

[072] According to the invention, a hexose can also be produced from fructose. For example, the process involves generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; and convert T6P to tagatose catalyzed by T6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[073] A hexose can be produced from sucrose. The process, for example, provides an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; and convert T6P to tagatose catalyzed by T6PP.

[074] The phosphatase used in the processes of the invention is specific for hexose. For example, allose 6-phosphate is converted to allose by allose 6-phosphate phosphatase; mannose 6-phosphate is converted to mannose by mannose 6-phosphate phosphatase; galactose 6-phosphate is converted to galactose by galactose 6-phosphate phosphatase; Fructose 6-phosphate is converted to fructose by fructose 6-phosphate phosphatase; altrose 6-phosphate is converted to altrose by altrose 6-phosphate phosphatase; thallose 6-phosphate is converted to thallose by thallose 6-phosphate phosphatase; sorbose 6-phosphate is converted to sorbose by sorbose 6-phosphate phosphatase; gulose 6-phosphate is converted to gulose by gulose 6-phosphate phosphatase; and idose 6-phosphate is converted to idose by idose 6-phosphate phosphatase. As used herein, specific means having a higher specific activity for the indicated hexose over other hexoses. For example, allose 6-phosphate phosphatase has a higher specific activity on allose 6-phosphate than, for example, sorbose 6-phosphate or thallose 6-phosphate.

[075] Phosphate ions generated during the hexose dephosphorylation step can be recycled to the step where sucrose is converted to G1P. Additionally, PPFK and polyphosphate can be used to increase hexose yields by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP. CQQ / nn / Q7n7 / B / YIAI

[076] A process to prepare a hexose may include the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and convert fructose to G6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above. G6P can be produced from glucose and sodium polyphosphate by polyphosphate glucokinase. [07η The invention provides processes for converting saccharides, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose and their derivative products, into a hexose. Artificial (non-natural) ATP-free enzymatic pathways can be provided to convert starch, cellulose, sucrose and their derivative products to a hexose using cell-free enzyme cocktails.

[078] As shown above, various enzymes can be used to hydrolyze starch to increase the yield of G1P. Such enzymes include isoamylase, pullulanase and alpha-amylase. Corn starch contains many branches that prevent the action of aGP. Isoamylase can be used to debranch starch, producing linear amylodextrin. Starch pretreated with isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave alpha1,6-glycosidic bonds, allowing more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to hexose and higher solubility.

[079] Maltose phosphorylase (MP) can be used to increase hexose yields by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase hexose yields by recycling the degradation products glucose, maltose and maltotriose into longer malto oligosaccharides; which can be phosphorolytically cleaved by aGP to produce G1P.

[080] Furthermore, cellulose is the most abundant biological resource and is the main component of plant cell walls. Non-food lignocellulosic blomasse contains cellulose, hemicellulose and lignin, as well as other minor components. Pure cellulose, including Avicel (micro crystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, filter paper, etc., can be prepared by different treatments. Partially hydrolyzed cellulosic substrates include water-insoluble cellodextrins whose degree of polymerization is greater than 7, water-soluble cellodextrins with a degree of polymerization of 3-6, cellobiose, glucose and fructose.

[081] Cellulose and its derivative products can be converted to a hexose by following a series of steps. The process provides an in vitro synthetic pathway that involves the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI. In this process, phosphate ions can be recycled by converting cellodextrin and cellobiose to G1P.

[082] Various enzymes can be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose. Such enzymes include endoglucanase and cellobiohydrolase, however, they do not include betaglucosidase (cellobiose). CQO / nn / eznz / E / YiAi

[083] Prior to the hydrolysis of cellulose and the generation of G1P, cellulose and biomass can be treated to increase their reactivity and decrease the degree of polymerization of the cellulose chains. Cellulose and biomass pretreatment methods include dilute acid pretreatment, cellulose solvent-based lignocellulose fractionation, ammonia fiber expansion, aqueous ammonia soaking, ionic liquid treatment, and partially hydrolyzed by using concentrated acids, including hydrochloric acid, sulfuric acid, phosphoric acid and their combinations.

[084] According to the invention, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the processes, thereby increasing the yields of a hexose by phosphorylating the degradation product glucose to G6P.

[085] A hexose can be generated from glucose. Processes for hexose production may include the following steps: generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK) and converting G6P to F6P catalyzed by PGI.

[086] Any biologically compatible buffer solution known in the art can be used in each of the processes of the invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc. The reaction buffer for the processes according to the invention may have a pH ranging between 5.0 and 8.0. Preferably, the pH of the reaction buffer can range from about 6.0 to about 7.3. For example, the pH of the reaction buffer may be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3. [08η The reaction buffer may also contain metal cations. Examples of metal ions include Mg2+ and Zn2+. As is known in the art, suitable salts can be used to introduce the desired metal cation.

[088] In each of the processes of the invention, the reaction temperature at which the process steps are carried out can range between 37 and 95 ° C. Preferably, the steps can be performed at a temperature ranging from about 40°C to about 90°C. The temperature may be, for example, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80 °C, approximately 85 °C, or approximately 90 °C. Preferably, the reaction temperature is about 50°C.

[089] The reaction time of each of the described processes can be adjusted as necessary, and can vary from about 8 hours to about 48 hours. For example, the reaction time may be about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, approximately 36 hours, approximately 38 hours, approximately 40 hours, approximately 42 hours, approximately 44 hours, approximately 46 hours or approximately 48 hours. Preferably, the reaction time is approximately 24 hours.

[090] Typically, the ratios of enzymatic units used in each of the processes described are from 1:1 to 1:1:1:1:1 (depending on the number of steps catalyzed in the process). To optimize product yields, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of intermediates CQQ / nn / Q7n7 / B / YIAI phosphorylated, which will result in increased activity of downstream reactions. In contrast, a ratio of 1:1:1:1:3 can be used to maintain a robust phosphate supply for aGP, which will result in more efficient phosphorolytic cleavage of the alpha-1,4-glycosidic bonds. An enzyme ratio, for example, 3:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of hexose production. For example, a specific enzyme may be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes.

[091] According to the invention, each of the processes can achieve high yields due to the favorable equilibrium constant in the entire reaction. For example, FIG 11 shows the Gibbs Energy of reaction between intermediates based on the formation of Gibbs Energy for the conversion of glucose 1-phosphate to a hexose. Reaction Gibbs Energies were generated using http: / / equilibrator.weizmann.ac.il / . In theory, yields of up to 99% can be achieved if the starting material is completely converted to an intermediate.

[092] The processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose and their derivatives are less expensive raw materials than, for example, lactose. When a hexose is produced from lactose, glucose and other hexoses are separated by chromatography, which involves higher production costs.

[093] Furthermore, the step of dephosphorylation of hexose by a phosphatase according to the invention is an irreversible phosphatase reaction, regardless of the raw material. Therefore, hexose is produced in a very high yield and at the same time effectively minimizes the costs of subsequent product separation.

[094] In some aspects of the invention, phosphatases for converting A6P, M6P, F6P or Gal6P to their respective non-phosphorylated forms use a divalent metal cofactor: preferably magnesium. In other aspects of the invention, the phosphatase contains, but is not limited to, a Rossmann folding domain for catalysis; further contains but is not limited to a C1 or C2 protection domain for substrate specificity; further contains but is not limited to a DxD signature in the first β-strand of the Rossmann fold to coordinate magnesium where the second Asp is a general acid / base catalyst; It also contains but is not limited to a Thr or Ser at the end of the second β-strand of the Rossmann fold that aids the stability of the reaction intermediates; It also contains but is not limited to a Lys at the N terminus of the C terminus of the a helix to the third β strand of the Rossmann fold that aids the stability of the reaction intermediates; further contains but is not limited to a GDxxxD, GDxxxxD, DD or ED signature at the end of the 4th β-strand of the Rossmann fold to coordinate magnesium. These characteristics are known in the art and are mentioned, for example, in Burroughs et al., Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes. J. Mol. Biol. 2006; 361; 1003-1034.

[095] Unlike cell-based manufacturing methods, the invention involves a cell-free preparation of hexoses, has relatively high reaction rates due to the removal of the cao / nn / eznz / E / YiAi cell membrane, which which often slows down the transport of the substrate / product into and out of the cell. It also features a final product free of nutrient / cellular metabolite-rich fermentation media.

[096] Alosa

[097] One embodiment of the invention is a process for preparing allose that includes converting fructose 6-phosphate (F6P) to psychose 6-phosphate (P6P) catalyzed by psychose 6-phosphate 3-epimerase (P6PE), converting P6P to allose 6-phosphate ( A6P) catalyzed by allose 6-phosphate isomerase (A6PI), and convert the produced A6P to allose catalyzed by allose 6-phosphate phosphatase.

[098] Examples of P6PE include, but are not limited to, the following proteins, determined according to UNIPROT identification numbers: D9TQJ4, A0A090IXZ8 and P32719. Of these, D9TQJ4 and A0A090IXZ8 are obtained from thermophilic organisms. P32719 is obtained from a mesophilic organism. P32719 is 53% identical to A0A090IXZ8 and 55% identical to D9TQJ4, and each protein catalyzes the epimerization of F6P to A6P. Furthermore, A0A090IXZ8 is 45% identical to D9TQJ4. On the other hand, other epimerase proteins determined by UNIPROT identification numbers: A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9, and H1XWY2, which have a degree of identity with D9TQJ4 of 45% or less are not catalysts for the epimerization of F6P a A6P. Examples of P6PEs also include any counterpart that has at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity with the Uniprot IDs mentioned above.

[099] Examples of A6PI include, but are not limited to Uniprot ID W4V2C8, with the amino acid sequence set forth in SEQ ID NO: 1; and Uniprot ID Q67LX4, with the amino acid sequence set forth in SEQ ID NO: 2. Uniprot IDs W4V2C8 and Q67LX4 catalyze the A6PI reaction and share a 56% amino acid sequence identity. Therefore, examples of A6PI also include any homologue having at least 55%, preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93% or at least 94%, and even the most preferably at least 96, 97, 98, 99 or 100% amino acid sequence identity with any of the Uniprot IDs mentioned above.

[0100] A6PIs suitable for use in the process to convert P6P to A6P contain a Rossmann fold. A mesophilic A6PI described in the art (Mowbray etal., ID-Ribose-5-Phosphate Isomerase B from Escherichia coli is Also a Functional D-Allose-6-phosphate Isomerase, While the Mycobacterium tuberculosis Enzyme is Not. J. Mol. BioL 2008; 382; 667-679) shares conserved residues with the thermophilic A6PI described in the invention. In some aspects of the invention the isomerase contains but is not limited to a C-terminal His (mesophyll residue 10) to the first strand of the Rossmann fold for phosphate bonding; further contains but is not limited to an Arg (mesophyll residue 133) C terminal to the C terminus of the a helix to the fifth strand of the Rossmann fold also for the phosphate bond; It also contains but is not limited to a His (mesophilic residue 99) in the active site to open the lactone ring; also contains CQO / nn / eznz / E / YiAi but is limited to one Cys (mesophilic residue 66) in the active site to act as the catalytic base; It also contains but is not limited to a Thr (mesophilic residue 68) in the active site to act as a catalytic acid; it further contains but is not limited to a GTG-hydrophobic-G motif near the active site (mesophilic residues 67-71) to stabilize the high-energy intermediates, and further contains but is not limited to an Asn (mesophilic residue 100) near the active site to also stabilize high energy intermediates. An A6PI preferably contains all of these conserved residues.

[0101] Examples of A6PP include, but are not limited to, the following proteins: Uniprot ID S9SDA3, with the amino acid sequence set forth in SEQ ID NO: 3; Q9X0Y1, with the amino acid sequence set forth in SEQ ID NO: 4; I3VT81, with the amino acid sequence set forth in SEQ ID NO: 5; A0A132NF06, with the amino acid sequence set forth in SEQ ID NO: 6; and D1C7G9, with the amino acid sequence set forth in SEQ ID NO: 7. Uniprot IDs S9SDA3 and I3VT81 catalyze the A6PP reaction and share 30% amino acid sequence identity. Therefore, examples of A6PP also include any homologue having at least 30%, preferably at least 35%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, even more preferably at least 75%, most preferably at least 80%, at least 85%, at least 90%, at least 95% or at least 91%, at least 92% , at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99 or 100% amino acid sequence identity with any of the Uniprot IDs mentioned above.

[0102] Preferably, an A6PP for converting A6P to allose, contains a Rossmann fold domain for catalysis, a C1 protection domain, a DxD signature in the first β-strand of the Rossmann fold, a Thr or a Ser al end of the second β-strand of the Rossmann fold, a Lys at the N terminus of the C terminus of the a-helix to the third β-strand of the Rossmann fold, and an ED signature at the end of the 4th β-strand of the Rossmann fold.

[0103] A process for preparing allose according to the invention also includes the step of enzymatically converting glucose 6-phosphate (G6P) to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for preparing allose additionally includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). In further embodiments, the allose production process also includes the step of converting a saccharide to the G1P that catalyzes at least one enzyme.

[0104] Therefore, a process for preparing allose according to the invention may include, for example, the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to P6P via P6PE, (v) convert P6P to A6P via A6PI and (vi) convert A6P to allose via A6PP. An example of the enzymatic process where the saccharide is starch is shown in FIG 1.

[0105] Typically, the ratios of enzymatic units used in the described process are 1:1:1:1:1:1 (aGP:PGM:PGI:P6PE:A6PI:A6PP). To optimize product yields, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in higher activity of cao / nn / eznz / E / YiAi downstream reactions. In contrast, a ratio of 1:1:1:1:1:3 can be used to maintain a robust phosphate supply for aGP, which will result in more efficient phosphorolytic cleavage of the alpha-1,4- bonds. glycosidic. An enzyme ratio, for example, 3:1:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of allose production. For example, a specific enzyme may be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes.

[0106] Phosphate ions produced during the dephosphorylation step of A6P can be recycled in the process step where a saccharide is converted to G1P, particularly when all process steps are carried out in a single bioreactor or reaction vessel. . The ability to recycle phosphate in the processes described allows the use of non-stoichiometric amounts of phosphate, which keeps reaction phosphate concentrations low. This affects the overall pathway and overall speed of the processes, but does not limit the activity of individual enzymes and allows for overall efficiency of allose manufacturing processes.

[0107] For example, the reaction phosphate concentrations in each of the processes can vary from about 0.1 mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from about 10 mM to about 50 mM. For example, the reaction phosphate concentration 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, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM or approximately 55 mM.

[0108] The low phosphate concentration results in decreased production costs due to low total phosphate and therefore a lower cost for phosphate removal. It also prevents inhibition of A6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0109] Furthermore, each of the processes described herein can be performed without added ATP as a phosphate source, i.e., ATP-free. The processes can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the described processes for making allose involves an energetically favorable chemical reaction.

[0110] Allose can also be produced from fructose. For example, the process involves generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to P6P catalyzed by P6PE; convert P6P to A6P catalyzed by A6PI, and convert A6P to allose catalyzed by A6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0111] Allose can also be produced from sucrose. The process provides an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by cao / nn / eznz / E / YiAi PGI; convert F6P to P6P catalyzed by P6PE; convert P6P to A6P catalyzed by A6PI, and convert A6P to allose catalyzed by A6PP.

[0112] Phosphate ions generated when A6P is converted to allose can subsequently be recycled in the step where sucrose is converted to G1P. Furthermore, PPFK and polyphosphate can be used to increase allose yields by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0113] In some embodiments, a process for preparing allose includes the following steps; generate glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, convert glucose to G6P catalyzed by at least one enzyme, generate fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and convert fructose to F6P catalyzed by at least an enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0114] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0115] Various enzymes can be used to hydrolyze starch and thus increase the yield of G1P. Such enzymes include isoamylase, pullulanase and alpha-amylase. Corn starch contains many branches that prevent the action of aGP. Isoamylase can be used to debranch starch, producing linear amylodextrin. Starch pretreated with isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave alpha-1,6-glycosidic bonds, allowing more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to allose and higher solubility.

[0116] Maltose phosphorylase (MP) can be used to increase allose yields by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4glucanotransferase (4GT) can be used to increase allose yields by recycling the degradation products of glucose, maltose, and maltotriose into longer maltooligosaccharides; which can be cleaved phosphorolytically by aGP to produce G1P.

[0117] In certain embodiments, cellulose and its derivative products can be converted to allose through a series of steps. The process offers an in vitro synthetic pathway that includes the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; convert P6P to A6P catalyzed by A6PI, and convert A6P to allose catalyzed by A6PP. In this process, phosphate ions can be recycled through the step of converting cellodextrin and cellobiose to G1P.

[0118] Various enzymes can be used to hydrolyze solid cellulose by water-soluble cellodextrins and cellobiose. Such enzymes include endoglucanase and cellobiohydrolase, not including beta-glucosidase (cellobiase).

[0119] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to this process, when allose yields are increased by phosphorylating the glucose degradation product on G6P. CQQ / nn / Qznz / B / YiAi

[0120] Allose can be produced from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); by converting P6P to A6P catalyzed by A6PI; and convert A6P to allose catalyzed by A6PP.

[0121] The inventive processes for making alose use low-cost starting materials and reduce production costs by reducing costs associated with raw materials and separation products. Starch, cellulose, sucrose and some of their derivatives are more accessible raw materials than, for example, fructose. When allose is produced from psychose, yields are lower than in the present invention, and allose must be separated from psychose through chromatography, which increases production costs.

[0122] Likewise, according to the invention, the step of converting A6P to allose is an irreversible phosphatase reaction, regardless of the raw material. Therefore, allose is produced with very high efficiency, while subsequent product separation costs are effectively minimized.

[0123] Compared to cell-based manufacturing methods, the invention includes a cell-free allose preparation that has relatively high reaction rates, due to the removal of the cell membrane, which often decreases the transport of substrate / product inside and outside the cell. It also has a final product free of nutrient / cellular metabolite-rich fermentation media.

[0124] A particular embodiment of the invention relates to allose produced by the processes described herein for producing allose.

[0125] Because allose has a similar function to sucrose, allose prepared through the processes of the invention could be added to any beverage or food to produce the desired sweetness.

[0126] Allose prepared by the processes described here could also be used to synergize the effect of powerful sweeteners. By combining allose with one or more potent sweeteners, it may be able to effect improvements in sensory characteristics, such as mouthfeel, flavor, and consistency of a sweetened product. The use of low-calorie sweeteners, such as potent sweeteners, in a variety of foods is common in food and beverage formulations. However, many consumers reject products marketed as diet or light versions of products that are artificially sweetened. Over the years, many attempts have been made to improve the flavor delivery of these diet or light products by adding small amounts of carbohydrates. The allose prepared by the processes of the invention would not only be capable of making improvements in the quality of food and beverage formulations, particularly in diet / light beverages, but its use could be synergistic with powerful sweeteners, such that it would be capable of replace significant amounts of potent sweeteners, even when added at minor concentrations below the limit sweetness measure.

[0127] The allose produced by the processes described here could be combined with other sweeteners, such as extracts from the Stevia rebaudiana Bertoni plant for the preparation of low-calorie versions of foods such as ice cream.

[0128] The allose produced by the processes described here could be used in pre-sweetened and ready-to-eat breakfast cereals, and, in other foods where D-allose partially or totally replaces sucrose or other commonly used sugars, such as the frosting. CQQ / nn / Q7n7 / e / YiAi

[0129] The allose produced by the processes described here could be used as part of a sweetener for foods and beverages, in combination with sugars in alcohol, such as erythritol, and nutritious sweeteners with significant caloric content, such as fructose, sucrose, dextrose , maltose, trehalose, rhamnose, corn syrups and fructooligosaccharides.

[0130] The allose produced by the processes described here could also be used as part of a composition that improves the control of diseases in plants.

[0131] Crafty

[0132] One embodiment of the invention is a process for preparing mannose, which includes converting F6P to mannose 6-phosphate (M6P) catalyzed by mannose 6-phosphate isomerase (M6PI); and convert M6P to mannose catalyzed by mannose 6-phosphate phosphatase (M6PP).

[0133] Examples of M6Pls include, but are not limited to the following proteins: Uniprot ID A0A1 M6TLY7, with the amino acid sequence set forth in SEQ ID NO: 8; H1XQS6, with the amino acid sequence set forth in SEQ ID NO: 9; G2Q982, with the amino acid sequence set forth in SEQ ID NO: 10; and F8F1Z8, with the amino acid sequence set forth in SEQ ID NO: 11. Uniprot IDs G2Q982, and F8F1Z8 both carry out the M6PI reaction and share 28% amino acid sequence identity. Therefore, examples of M6Pls also include any of the homologues having at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, most preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, most preferably at least 70%, with more preference of at least 75%, with more preference of at least 80%, with more preference of at least 85%, with more preference of at least 90%, with more preference of at least 91%, with more preference of at less 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, with more preferably at least 98%, more preferably at least 99% or 100% amino acid sequence identity in any of the aforementioned Uniprot IDs.

[134] The M6Pls suitable for use in the process of converting F6P to M6P contain two domains with a base of antiparallel β-strands that resemble the cupin-like fold and a third domain consisting only of a-helices. An M6PI in the matter was structurally qualified (Sagurthi etal. Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism. Acta Cryst. 2009; D65; 724-732) and shares conserved residues with the thermophilic M6Pis described in the invention. In some aspects of the invention, the isomerase contains, but is not limited to containing divalent metal cation, preferably Mg2+ or Zn2+; additionally, but not limited to, containing one Glu and two His residues for use in metal binding (PDB 3H1M residues 134, 99, and 255 respectively); also, but not limited to, containing an Asp and a Lys residue proposed for acid / base catalysis (PDB 3H1M residues 270 and 132 respectively); and, in addition, but not limited to containing Lys, Pro, and Ala residues proposed for phosphate binding (PDB 3H1M residues 132, 133, and 267 respectively). An M6PI preferably contains all of these conserved residues. cao / nn / eznz / E / YiAi

[0135] Examples of M6PPs include, but are not limited to the following proteins: Uniprot ID A0A1A6DSI3, with the amino acid sequence set forth in SEQ ID NO: 12; A0A1M4UN08, with the amino acid sequence set forth in SEQ ID NO: 13; and A0A1N6FCW3, with the amino acid sequence established in SEQ ID NO: 14 Uniprot IDs A0A1A6DSI3 and A0A1N6FCW3, both catalyze the M6PP reaction and share 35% identity of the amino acid sequence. Therefore, examples of M6PPs also include any of the homologues having at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, most preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, most preferably at least 80% , more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, most preferably at at least 94%, more preferably at least 95%, and even more preferably that they present at least 96, 97, 98, 99 or 100% amino acid sequence identity for any of the aforementioned Uniprot IDs.

[0136] Preferably, an M6PP that converts M6P to mannose contains Rossman fold domain for catalysis, a C1 capping domain, characteristic DxD on the 1st β- chain of the Rossman fold, a Thr or Ser at the end of the second β- strand of the Rossman fold, a Lys at the N- end of the C-terminal α-helix for the third β- strand of the Rossman fold, and a characteristic GDxxxD at the end of the fourth β- strand of the Rossman fold .

[0137] According to the invention, a process for preparing mannose also includes the step of enzymatically converting glucose 6-phosphate (G6P) to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for preparing mannose further includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). In additional embodiments, the process for preparing mannose includes the conversion of G6P to F6P to M6P, where the step is catalyzed by phosphoglucose / phosphomannose isomerase (PGPMI). In yet additional embodiments, the mannose production process also includes the step of converting a saccharide to G1P which is catalyzed by at least one enzyme.

[0138] Processes of the invention for the production of craftsmanship use PGPMIs that convert G6P or F6P into M6P. Examples of PGPMIs include, but are not limited to, the following proteins: Uniprot ID D7CPH7, with the amino acid sequence set forth in SEQ ID NO: 15; A0A085L170, with the amino acid sequence set forth in SEQ ID NO: 16; and M1E6Z3, with the amino acid sequence set forth in SEQ ID NO: 17. Both Uniprot IDs A0A085L170 and Μ1E6Z3 catalyze the PGPMI reaction and share 28% amino acid sequence identity. Therefore, examples of PGPMIs also include any of the homologues having at least 25%, more preferably at least 30%, more preferably at least 35%, most preferably at least 40%, even more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, most preferably at least 75% , more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, most preferably at least 93%, with more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, and even more preferably cao / nn / eznz / E / YiAi preferably at least 96, 97, 98, 99 or 100% of amino acid sequence identity for any of the aforementioned Uniprot IDs.

[0139] A PGMPI suitable for use in the process of converting G6P or F6P to M6P contains two Rossman folds. A PGPMI was structurally characterized in the field (Swan et al. A Novel Phosphoglucose Isomerase (PGI) / Phosphomannose Isomerase from the Crenarchaeon Pyrobaculum aerophilum Is a Member of the PGI Superfamily. J. Biol. Chem. 2004: 279; 39838-39845) and shares conserved residues with the thermophilic PGPMIs described in the invention. In some aspects of the invention, the isomerase contains, but is not limited to containing, a GGS motif (PDB1TZB residues 46-48) wherein the Gly residues assist in substrate binding and the Ser residue binds the phosphate; It is also not limited to contain a Hydrophobic SYSG-X-T-X-ET motif (PDB residues 1TZB 87-96) that binds phosphate; Likewise, it is not limited to contain an Arg residue (PDB residue 1TZB 135) that stabilizes high energy intermediates during catalysis; It is also not limited to contain a characteristic EN (PDB residues 1TZB 203-204) where Glu is essential for proton transfer in the active site; It is also not limited to contain a characteristic HN (PDB residues 1TZB 219-220) where His is important for the ring opening / closing of the substrate during catalysis. The functions of the conserved residues are verified in an independent publication (Hansen et al. Bifunctional Phosphoglucose / Phosphomannose Isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum Constitute a Novel Enzyme Family within the Phosphoglucose Isomerase Superfamily. J Biol. Chem. 2004; 279; 2262-2272). A PGPMI preferably contains all these preserved residues.

[0140] Therefore, according to the invention, a process for preparing mannose may, for example, include the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes, ( i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to M6P via mannose 6-phosphate isomerase (M6PI, EC 5.3.1.8), (v) convert G6P to M6P via bifunctional phosphoglucose / phosphomannose isomerase (PGPMI, EC 5.3.1.8 and 5.3.1.9), and ( vi) convert M6P into crafty via M6PP. An example of the process where the saccharide is starch is shown in FIG 2.

[0141] Typically, enzyme unit ratios used in the published process are 1:1:1:1:1 (aGP:PGM:PGI:M6PI:M6PP) or 1:1:1:1 (aGP:PGM :PGPMI:M6PP). To optimize product efficiency, these ratios can be adjusted in a number of combinations. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in increased activity of downstream reactions. On the other hand, a ratio of 1:1:1:1:3 can be used to maintain a robust phosphate distribution for aGP, which will result in more efficient phosphorolytic cleavage of alpha -1,4-glycosidic bonds. An enzyme ratio, for example 3:1:1:1:3, can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of mannose production. For example, a given enzyme could be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes. cao / nn / eznz / E / YiAi

[0142] One of the important advantages of the processes is that the steps can be carried out in a simple bioreactor or a reaction vessel. Alternatively, the steps can be performed in a variety of bioreactors, or reaction vessels, which are arranged in series.

[0143] Phosphate ions produced by dephosphorylation of M6P can then be recycled in the process step in which a saccharide is converted to G1P, mainly when all process steps are performed in a simple bioreactor or reaction vessel. The ability to recycle phosphate in published processes allows nonstoichiometric amounts of phosphate to be used, preserving low phosphate reaction concentrations. This affects the overall pathway and overall rate of processes, but does not limit the activity of individual enzymes and allows for overall efficiency in mannose-making processes.

[0144] For example, phosphate reaction concentrations can range from 0.1 mM to 300 mM, from about 0.1 mM to 150 mM, from 1 mM to 50 mM, preferably from 5 mM to about 50 mM. For example, the concentration of the phosphate reaction 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.

[0145] Therefore, low phosphate concentration results in decreased production costs due to low total phosphate and therefore lower phosphate removal cost. It also prevents inhibition of M6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0146] Likewise, the processes published here can be carried out without added ATP as a phosphate source, that is, free of ATP. The processes can also be carried out without having to add NAD(P)(H), that is, without NAD(P)(H). Other advantages also include the fact that at least one step of the published processes for making mannose involves an energetically favorable reaction.

[0147] Mannose can also be produced from fructose. For example, the process involves the generation of F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to M6P catalyzed by M6PI; and, convert M6P to mannose catalyzed by M6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0148] Mannose can also be produced from sucrose. The process offers an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to M6P catalyzed by M6PI; and, convert M6P into mannose catalyzed by M6PP. In the aforementioned steps, the conversion of G6P to F6P to M6P can optionally be catalyzed by PGPML

[0149] The phosphate ions that are generated when converting M6P to mannose can be recycled in the step of converting sucrose to G1P. Likewise, PPFK and polyphosphate can be used to increase the efficiency of mannose by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose into SP. cao / nn / eznz / E / YiAi

[0150] In some embodiments, the process for making mannose includes the following steps: generating glucose from polysaccharides and oligosaccharides through enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and converting fructose to F6P catalyzed by at least one enzyme. Some examples of polysaccharides and oligosaccharides were listed above.

[0151] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0152] The present publication offers processes for converting saccharides, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose and their derived products into mannose. In certain embodiments, artificial (non-natural) ATP-free enzymatic pathways are offered to convert starch, cellulose, sucrose, and their derivative products to mannose, using cell-free enzyme cocktails.

[0153] As shown above, various enzymes can be used to hydrolyze starch to increase the efficiency of G1P. Such enzymes include isoamylase, pullulanase, and alpha amylase. Corn starch contains many derivatives that prevent the action of aGP. Isoamylase can be used to split starch, obtaining linear amylodextrin. The starch from the pretreated isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulunase cleave alpha-1,6-glycosidic bonds, allowing for more complete degradation of starch by alpha-glucan phosphorylase. Alpha amylase cleaves alpha -1,4-glycosidic bonds, therefore alpha amylase is used to degrade starch into fragments for faster conversion to mannose and increased solubility.

[0154] Maltose phosphorylase (MP) can be used to increase the efficiency of mannose, by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase the efficiency of mannose by recycling the degradation products of glucose, maltose, and maltotriose into longer maltooligosaccharides; which can be phosphorolytically cleaved into aGP to gain G1P.

[0155] In certain embodiments, cellulose and its derivative products can be converted to pulp through a series of steps. The process offers an in vitro synthetic route that involves the following steps: generating G1P from cellodextrin and celloblose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to MSP catalyzed by M6PI; and convert MSP to mannose catalyzed by M6PP. Alternatively, in the upstream pathway the conversion of G6P to F6P and M6P can be catalyzed by PGPML. In this process, phosphate ions can be recycled through the steps of converting cellodextrin and cellobiose to G1P.

[0156] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, thereby increasing the efficiency of mannose by phosphorylating the glucose breakdown product to G6P.

[0157] In other embodiments, mannose may be generated from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); converting G6P to F6P catalyzed by PGI; converting F6P to M6P catalyzed by M6PI; and converting CQQ / nn / Q7n7 / e / YiAi Μ6Ρ in mannose catalyzed by M6PP. Alternatively, the conversion of G6P to F6P and M6P can be catalyzed by PGPML.

[0158] The processes of the invention use low-cost starting materials and reduce production costs by reducing costs associated with raw materials and separation products. Starch, cellulose, sucrose and some of their derivatives are cheaper raw materials than, for example, fructose. When producing mannose from fructose, profits are lower than in the present invention, and mannose must be separated from fructose via chromatography, leading to higher production costs.

[0159] Furthermore, according to the invention, the step of converting M6P to mannose is an irreversible phosphatase reaction, regardless of the raw material. Therefore, mannose is produced with a very high gain, while minimizing effectively the separation costs of the subsequent product.

[0160] Unlike cell-based manufacturing methods, the invention involves a cell-free mannose preparation that has relatively high reaction rates, due to the removal of the cell membrane, which often reduces substrate / substrate transport. product inside and outside the cell. It also has a final product free of nutrient / cellular metabolite-rich fermentation media.

[0161] A particular embodiment of the invention is mannose produced by the processes for producing mannose described herein.

[0162] The craft produced by the processes described here could be used, as discussed above, in a variety of applications in the pharmaceutical, cosmetic, food and beverage, dairy, confectionery, and raw materials industries.

[0163] Furthermore, mannose produced by the processes described herein can be converted to mannitol through hydrogenation. Catalytic hydrogenation of mannose occurs with a stoichiometric gain and results in mannitol. US Patent No. 5,466,795. Mannitol is widely used in the production of sugar-free chewing gum, and in sweet and pharmaceutical excipients. However, the production of high-purity mannose is extremely complicated and expensive. Therefore, mannose produced by the aforementioned processes can be converted to mannitol via catalytic hydrogenation.

[0164] Galactose

[0165] One embodiment of the invention is a process for preparing galactose, which includes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), converting T6P to galactose 6- phosphate (Gal6P) catalyzed by galactose 6- phosphate isomerase (Gal6PI), and convert the Gal6P produced into galactose catalyzed by galactose 6- phosphate phosphatase (Gal6PP).

[0166] Examples of F6PEs include, but are not limited to, the following proteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Therefore, examples of F6Pes also include any of the homologues having at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, most preferably at least 45% , more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, with more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, at cao / nn / eznz / E / YiAi minus 91%, at least 92%, at least 93%, or at at least 94%, and more preferably at least 96, 97, 98, 99 or 100% amino acid sequence identity for any of the aforementioned Uniprot IDs.

[0167] Gal6PI exists as a multimer of two subunits, LacA and LacB. Examples of Gal6Pls include, but are not limited to the following protein subunit pairs (LacA / LacB): Uniprot ID P23494 / P23495, with the amino acid sequences set forth in SEQ ID NO: 18 / SEQ ID NO: 19. Examples of Gal6Pls also include any of the homologues having at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, most preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, most preferably at least 93 %, more preferably at least 94%, more preferably at least 95%, and even more preferably at least 96.97, 98.99 or 100% amino acid sequence identity for the aforementioned Uniprot ID for the LacA subunit and homologs having at least 25%, at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, most preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, most preferably at least 80%, most preferably at least 85%, even more preferably at least 90%, more preferably at least 91%, at least 92%, at least 93%, or at least 94%, and even more preferably at least 96, 97, 98, 99 or 100% amino acid sequence identity for the aforementioned Uniprot ID for the LacB subunit.

[0168] The Gal6Pls appropriate for use in the process to convert T6P to Gal6P contains a heterodimer ('A' and 'Bj that consists of subunits with sandwich-type Rossman αβα folds. The issue of conserved residues is something that is discussed in In some aspects of the invention, the heterodimeric isomerase contains, but is not limited to. to contain Arg130 and Arg134 in 'A' and His9 and Arg39 in 'B' to bind the phosphate group of the substrate; additionally but not limited to containing His96 in A' for the ring opening of the substrate; in A' to stabilize high energy intermediates; and, additionally but not limited to containing Cys65 and Thr67 of 'B' to participate in proton transfer.

[0169] Some examples of Gal6PPs include, but are not limited to Uniprot ID Q8A2F3, with the amino acid sequence set forth in SEQ ID NO: 20. Examples of Gal6PPs also include any of the homologs having at least 25, at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably 95%, at least 91%, at least 92%, at least 93%, or at least 94% and even more preferably at least 96, 97, p8, 99 or 100% amino acid sequence identity to the aforementioned Uniprot ID. cao / nn / eznz / E / YiAi

[0170] Preferably a Gal6PP to convert Gal6P to galactose containing Rossman folds for catalysis, a C2 limited domain, a characteristic DxD in the first β- strand of the Rossman fold, a Thr or Ser at the end of the second β-strand - of the Rosmman fold, and a characteristic GDxxxD at the end of the fourth β- strand of the Rossman fold.

[0171] According to the invention, a process for making galactose also includes the step of enzymatically converting glucose 6-phosphate (G6P) to F6P, and this step is catalyzed through phosphoglucoisomerase (PGI). In other embodiments, the process for making galactose further includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed through phosphoglucomutase (PGM). Even in later representations, the galactose production process also includes the step of converting a saccharide to G1P, which is catalyzed by at least one enzyme.

[0172] Therefore, according to the invention, a process for making galactose may, for example, include the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to T6P via F6PE; (v) convert T6P to Gal6PI (EC 5.3.1.26), and, (vi) convert Gal6P to galactose via Gal6PP. An example of the process where the saccharide is starch is shown in FIG 3.

[0173] Generally, the ratios of enzyme units used in the published process are 1:1:1:1:1:1 (aGP:PGM:PGI:F6PE:Gal6PI:Gal6PP).To optimize product gains, These relationships can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in an increase in the activity of downstream reactions. On the other hand, a ratio of 1:1:1:1:1:3 can be used to maintain a robust phosphate distribution for aGP, which will result in more efficient phosphorolytic cleavage of alpha-1,4 glycosidic bonds. -, An enzyme ratio of, for example, 3:1:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes mentioned above, can be varied to increase the efficiency of galactose production. For example, a given enzyme may be present in an approximate amount of 2x, 3x, 4x, 5x, etc. relative to the amount of other enzymes.

[0174] One of the important advantages of the processes is that their steps can be carried out in a simple bioreactor or in a reaction vessel. Alternatively, the steps can be carried out in a variety of bioreactors, or reaction vessels, which are arranged in series.

[0175] Phosphate ions produced by dephosphorylation of Gal6P can then be recycled in the process step where a saccharide is converted to G1P, especially when all process steps are performed in a simple bioreactor or reaction vessel. In published processes, the ability to recycle phosphate allows nonstoichiometric amounts of phosphate to be used, keeping phosphate reaction concentrations low. This affects the overall pathway and rate of processes, but does not limit the activity of individual enzymes and allows for overall efficiency of galactose manufacturing processes. CQQ / nn / Q7n7 / e / YiAi

[0176] For example, phosphate reaction concentrations can range from 0.1 mM to about 300 mM, from about 0.1 mM to about 150 mM, from 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from 10 mM to about 50 mM. For example, the concentration of the phosphate reaction may be from about 0.1 mM, to about 0.5 mM, to about 1 mM, to about 1.5 mM, to about 2 mM, to about 2.5 mM, to about 5 mM, to about 6 mM, at approximately 7 mM, at approximately 8 mM, at approximately 9 mM, at approximately 10 mM, at approximately 15 mM, at approximately 20 mM, at approximately 25 mM, at approximately 30 mM, at approximately 35 mM, at approximately 40 mM, to about 45 mM, to about 50 mM, or about 55 mM.

[0177] Therefore, low phosphate concentrations result in low production costs, due to low total phosphate, and thus, the cost of phosphate removal is reduced. It also prevents inhibition of Gal6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0178] Likewise, the processes published here can be carried out without added ATP as a phosphate source, that is, free of ATP. The processes can also be carried out without adding NAD(P)(H), i.e., free NAD(P)(H). Other advantages also include the fact that at least one of the steps in the published processes for making galactose involves an energetically favorable reaction.

[0179] Galactose can also be produced from fructose. For example, the process involves generating F6P to extract fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; convert T6P to Gal6P catalyzed by Gal6PI, and, convert Gal6P to galactose catalyzed by Gal6PP. For example, fructose can be produced by an enzymatic conversion of sucrose.

[0180] Galactose can be produced from sucrose. The process offers an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to Gal6P catalyzed by Gal6PI, and, convert Gal6P to galactose catalyzed by Gal6PP.

[0181] The phosphate ions generated when Gal6P is converted to galactose can then be recycled in the step of converting sucrose to G1P. Furthermore, PPFK and polyphosphate can be used to increase galactose gains by producing F6P from fructose generated from the phosphorolytic cleavage of sucrose by SP.

[0182] In some embodiments, a process for making galactose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and, converting fructose to F6P catalyzed by at least one enzyme. Some examples of polysaccharides and oligosaccharides were listed above.

[0183] In other embodiments, G6P is produced from glucose and sodium polyphosphate via polyphosphate glucokinase. cao / nn / eznz / E / YiAi

[0184] The present publication offers processes for converting saccharides, such as polysaccharides and oligosaccharides into starch, cellulose, sucrose, and their derivative products to galactose. In certain embodiments, ATP-free artificial enzymatic pathways are offered to convert starch, cellulose, sucrose, and their derivative products to galactose, using cell-free enzyme cocktails.

[0185] As shown above, various enzymes can be used to hydrolyze starch to increase G1P gain. Such enzymes include isoamylase, pullulunase, and alpha-amylase. Corn starch contains many derivatives that prevent the action of aGP. Isoamylase can be used to split starch, obtaining linear amylodextrin. The starch from the pretreated isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulunase cleave alpha-1,6-glycosidic bonds, allowing for more complete degradation of starch by alpha-glucan phosphorylase. Alpha amylase cleaves alpha -1,4-glycosidic bonds, therefore alpha amylase is used to break down starch into fragments for faster conversion to galactose and increased solubility.

[0186] Maltose phosphorylase (MP) can be used to increase galactose gains by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase galactose gains by recycling the degradation products of glucose, maltose, and maltotriose into longer maltooligosaccharides; which can be cleaved phosphorolytically by aGP to gain G1P.

[0187] In certain embodiments, cellulose and its derivative products can be converted to galactose through a series of steps. The processes offer an in vitro synthetic route that includes the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to Gal6P catalyzed by Gal6PI; and, convert Gal6P to galactose catalyzed by Gal6PP. In this process, phosphate ions can be recycled through the step of converting cellodextrin and cellobiose to G1P.

[0188] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, thereby increasing galactose gains by phosphorylating the glucose breakdown product at G6P.

[0189] In other embodiments, galacotsa can be generated from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK), converting G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to Gal6P catalyzed by Gal6PI; and, convert, Gal6P to galactose catalyzed by Gal6PP.

[0190] Some processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose, and some of their derivatives are more accessible raw materials than, for example, lactose. When galactose is produced from biomass or lactose, profits are lower than in the present invention, and galactose must be separated from other sugars via chromatography, which leads to higher production costs. Furthermore, no animals were used in our procedures. CQQ / nn / Q7n7 / B / YIAI

[0191] According to the invention, the step of converting Gal6P to galactose is an irreversible phosphatase reaction, regardless of the raw material. Therefore, galacotsa is produced at a very high profit while effectively minimizing the subsequent costs of product separation.

[0192] Compared to cell-based manufacturing methods, the invention includes a cell-free galactose preparation that has relatively high reaction rates, due to the removal of the cell membrane, which often decreases the transport of substrate / product inside and outside the cell. It also has a final product free of nutrient / cellular metabolite-rich fermentation media.

[0193] A particular embodiment of the invention is galactose produced by the processes described herein for producing galactose.

[0194] Fructose

[0195] One embodiment of the invention is a process for preparing fructose that includes converting fructose 6phosphate (F6P) to fructose catalyzed by fructose 6-phosphate phosphatase (F6PP).

[0196] A non-limiting example of an F6PP is the Uniprot ID E38CWV3, with the amino acid sequence set forth in SEQ ID NO: 21. Examples of F6PP also include any homologue having at least 25%, at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80% more preferably at least 85%, even more preferably at least 90%, more preferably at least 95%, at least 91%, at least 92% , at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99 or 100% amino acid sequence identity with any of the Uniprot IDs mentioned above.

[0197] Preferably, an F6PP for converting F6P to fructose contains a Rossmann fold domain for catalysis, a C1 protection domain, a DxD signature in the first β-strand of the Rossmann fold, a Thr or a Ser at the end. from the second β-strand of the Rossmann fold, a Lys at the N terminus of the C terminus of the a-helix to the third β-strand of the Rossmann fold, and an ED signature at the end of the 4th β-strand of the Rossmann fold.

[0198] A process for preparing fructose according to the invention also includes the step where glucose 6-phosphate (G6P) is enzymatically converted to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for preparing fructose additionally includes the step where glucose 1-phosphate (G1P) is converted to G6P, where the step is catalyzed by phosphoglucomutase (PGM). In further embodiments, the fructose production process also includes the step where a saccharide is converted to G1P which catalyzes at least one enzyme.

[0199] Therefore, a process for preparing fructose according to the invention may include, for example, the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to fructose using F6PP.

[0200] Typically, the ratios of enzymatic units used in the described process are 1:1:1:1 (aGP:PGM:PGI:F6PP). To optimize product yields, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1 can be used to maximize the cao / nn / eznz / E / YiAi concentration of phosphorylated intermediates, which will result in higher activity of subsequent reactions. In contrast, a ratio of 1:1:1:3 can be used to maintain a robust phosphate supply for aGP, which will result in more efficient phosphorolytic cleavage of the alpha-1,4glycosidic bonds. An enzyme ratio, for example, 3:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of fructose production. For example, a specific enzyme may be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes.

[0201] One of the important advantages of the processes of the invention is that the different steps can be carried out in a single bioreactor or reaction vessel. Alternatively, the steps may also be performed in a plurality of bioreactors, or reaction vessels, that are arranged in series.

[0202] Phosphate ions produced during the dephosphorylation step of F6P can be recycled in the process step where a saccharide is converted to G1P, particularly when all process steps are carried out in a single bioreactor or reaction vessel. . The ability to recycle phosphate in the processes described allows the use of non-stoichiometric amounts of phosphate, which keeps reaction phosphate concentrations low. This affects the overall pathway and overall speed of the processes, but does not limit the activity of individual enzymes and allows for overall efficiency of the fructose manufacturing processes.

[0203] For example, the reaction phosphate concentrations in each of the processes can vary from about 0.1 mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from about 10 mM to about 50 mM. For example, the reaction phosphate concentration 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, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM or approximately 55 mM.

[0204] Therefore, the low phosphate concentration results in decreased production costs due to low total phosphate and thus a lower cost for phosphate removal. It also prevents inhibition of F6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0205] Furthermore, each of the processes described herein can be performed without added ATP as a phosphate source, i.e., ATP-free. The processes can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the described processes for manufacturing fructose involves an energetically favorable chemical reaction.

[0206] Fructose can also be produced from sucrose through an F6P intermediate. The process provides an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphohlase (SP); convert G1P to G6P catalyzed by PGM; cao / nn / eznz / E / YiAi convert G6P to F6P catalyzed by PGI; and convert F6P to fructose catalyzed by F6PP. An example of an enzymatic pathway is provided in FIG 5.

[0207] Phosphate ions that are generated when F6P is converted to fructose can subsequently be recycled at the stage where sucrose is converted to G1P. Furthermore, PPFK and polyphosphate can be used to increase fructose yields by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0208] In some embodiments, a process for preparing fructose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis and convert fructose to F6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0209] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0210] The present document provides processes for converting saccharides, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose and their derivative products, to fructose. In some embodiments, artificial (non-natural) ATP-free enzyme pathways are provided for converting starch, cellulose, sucrose and their derivative products to fructose using cell-free enzyme cocktails.

[0211] As shown above, various enzymes can be used to hydrolyze starch and thus increase the yield of G1P. Such enzymes include isoamylase, pullulanase and alpha-amylase. Corn starch contains many branches that prevent the action of aGP. Isoamylase can be used to debranch starch, producing linear amylodextrin. Starch pretreated with isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave the alpha1,6-glycosidic bonds, allowing for more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to fructose and greater solubility.

[0212] Maltose phosphorylase (MP) can be used to increase fructose yields by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4glucan transferase (4GT) can be used to increase fructose yields by recycling the degradation products glucose, maltose and maltotriose into longer maltooligosaccharides; which can be phosphorolytically cleaved by aGP to produce G1P.

[0213] In some embodiments, cellulose and its derivative products can be converted to fructose by following a series of steps. The process provides an in vitro synthetic pathway that involves the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to fructose catalyzed by F6PP. In this process, phosphate ions can be recycled by converting cellodextrin and cellobiose to G1P. CQQ / nn / Q7n7 / B / YIAI

[0214] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the processes, thereby increasing fructose yields by phosphorylating the breakdown product glucose at G6P.

[0215] In other embodiments, fructose can be generated from glucose. The process includes the steps to generate G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI and convert F6P to fructose catalyzed by F6PP.

[0216] The processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose and some of their derivatives are less expensive raw materials than, for example, lactose. When fructose is produced from biomass or lactose, yields are lower than in the present invention, and fructose must be separated from other sugars by chromatography, leading to higher production costs. Additionally, our process does not involve animals.

[0217] According to the invention, the step of converting F6P into fructose is an irreversible phosphatase reaction, regardless of the raw material. Fructose is therefore produced with a very high yield and at the same time effectively minimizes the costs of subsequent product separation.

[0218] Unlike cell-based manufacturing methods, the invention involves a cell-free fructose preparation, has relatively high reaction rates due to the removal of the cell membrane, which often slows down substrate transport / product in and out of the cell. It also features a final product free of nutrient / cellular metabolite-rich fermentation media.

[0219] A particular embodiment of the invention is fructose produced by the processes described herein for producing fructose.

[0220] Altrose

[0221] One embodiment of the invention is a process for preparing altrose that includes converting fructose 6phosphate (F6P) to psychose 6-phosphate (P6P) catalyzed by psychose 6-phosphate 3-epimerase (P6PE), converting P6P to altrose 6-phosphate ( Alt6P) catalyzed by altrose 6-phosphate isomerase (AH6PI), and convert the produced Alt6P to altrose catalyzed by altrose 6-phosphate phosphatase.

[0222] A process for preparing altrose according to the invention also includes a step where glucose 6-phosphate (G6P) is enzymelically converted to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for preparing altrose additionally includes the step where glucose 1-phosphate (G1P) is converted to G6P, and is catalyzed by phosphoglucomutase (PGM). In further embodiments, the altrose production process also includes the step where a saccharide is converted to G1P which catalyzes at least one enzyme.

[0223] Therefore, a process for preparing altrose according to the invention may include, for example, the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to P6P via P6PE, (v) convert P6P to Alt6P via Alt6PI and (vi) convert Alt6P to altrose via AI16PP. An example of the enzymatic process where the saccharide is starch is shown in FIG 1. CQQ / nn / Q7n7 / B / YIAI

[0224] Typically, the ratios of enzymatic units used in the described process are 1:1:1:1:1:1 (aGP:PGM:PGI:P6PE:Alt6PI:Alt6PP). To optimize product yields, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in increased activity of downstream reactions. In contrast, a ratio of 1:1:1:1:1:3 can be used to maintain a robust phosphate supply for aGP, which will result in more efficient phosphorolytic cleavage of the alpha-1,4- bonds. glycosidic. An enzyme ratio, for example, 3:1:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of altrose production. For example, a specific enzyme may be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes.

[0225] Phosphate ions produced during the dephosphorylation step of Alt6P can be recycled in the process step where a saccharide is converted to G1P, particularly when all process steps are carried out in a single bioreactor or reaction vessel . The ability to recycle phosphate in the processes described allows the use of non-stoichiometric amounts of phosphate, which keeps reaction phosphate concentrations low. This affects the overall pathway and overall speed of the processes, but does not limit the activity of individual enzymes and allows for overall efficiency of the altrose manufacturing processes.

[0226] For example, the reaction phosphate concentrations in each of the processes can vary from about 0.1 mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from about 10 mM to about 50 mM. For example, the reaction phosphate concentration 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, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM or approximately 55 mM.

[0227] Therefore, the low phosphate concentration results in decreased production costs due to low total phosphate and thus a lower cost for phosphate removal. It also prevents inhibition of AH6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0228] Furthermore, each of the processes described herein can be performed without added ATP as a phosphate source, i.e., ATP-free. The process can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the described processes for manufacturing altrose involves an energetically favorable chemical reaction.

[0229] Altrose can also be produced from fructose. For example, the process involves generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to P6P cao / nn / eznz / E / YiAi catalyzed by P6PE; convert P6P to Alt6P catalyzed by Alt6PI, and convert Alt6P to altrose catalyzed by AH6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0230] Altrose can also be produced from sucrose. The process, for example, provides an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; convert P6P to Alt6P catalyzed by AI16PI, and convert Alt6P to altrose catalyzed by AH6PP.

[0231] Phosphate ions generated when Alt6P is converted to altrose can subsequently be recycled in the step where sucrose is converted to G1P. Furthermore, PPFK and polyphosphate can be used to increase altrose yields by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0232] In some embodiments, a process for preparing altrose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and convert fructose to F6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0233] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0234] Various enzymes can be used to hydrolyze starch and thus increase the yield of G1P. Such enzymes include isoamylase, pullulanase and alpha-amylase. Corn starch contains many branches that prevent the action of aGP. Isoamylase can be used to debranch starch, producing linear amylodextrin. Starch pretreated with isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave alpha-1,6-glycosidic bonds, allowing more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to altrose and greater solubility.

[0235] Maltose phosphorylase (MP) can be used to increase altrose yields by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase altrose yields by recycling the degradation products glucose, maltose and maltotriose into longer maltooligosaccharides; which can be phosphorolytically cleaved by aGP to produce G1P.

[0236] In some embodiments, cellulose and its derivative products can be converted to altrose by following a series of steps. The process provides an in vitro synthetic pathway that involves the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; convert P6P to Alt6P catalyzed by AH6P, and convert Alt6P to altrose catalyzed by AH6PP. In this process, phosphate ions can be recycled by converting cellodextrin and cellobiose to G1P. CQQ / nn / Q7n7 / e / YiAi

[0237] Various enzymes can be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose. Such enzymes include endoglucanase and cellobiohydrolase, but do not include beta-glucosidase (cellobiose).

[0238] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the processes, thereby increasing altrose yields by phosphorylating the degradation product glucose at G6P.

[0239] Altrose can be produced from glucose. The process includes the steps to generate G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; convert P6P to Alt6P catalyzed by A6PI and convert Alt6P to altrose catalyzed by Alt6PP.

[0240] The inventive processes for producing altrose utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose and some of their derivatives are less expensive raw materials than, for example, fructose. When altrose is produced from psychose, yields are lower than in the present invention, and altrose must be separated from psychose by chromatography, which leads to higher production costs.

[0241] Also, the step of converting Alt6P to altrose is an irreversible phosphatase reaction, regardless of the raw material. Therefore, altrose is produced at a very high yield and at the same time effectively minimizes the costs of subsequent product separation.

[0242] Unlike cell-based manufacturing methods, the invention involves a cell-free altrose preparation, has relatively high reaction rates due to the removal of the cell membrane, which often slows down substrate transport / product in and out of the cell. It also features a final product free of nutrient / cellular metabolite-rich fermentation media.

[0243] A particular embodiment of the invention is altrose produced by the processes described herein for producing altrose.

[0244] Talose

[0245] One embodiment of the invention is a process for making thallose, which includes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), converting T6P to talose 6- phosphate (Tal6P) catalyzed by talose 6- phosphate isomerase (TalGPI), and convert the Tal6P produced to talose catalyzed by talose 6- phosphate phosphatase (Tal6PP).

[0246] Examples of F6Pes include, but are not limited to the following proteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507, both catalyze the F6PE reaction and share 27% amino acid sequence identity. Therefore, examples of F6Pes also include any of the homologues having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or, at least 99% sequence identity amino acid for any of the aforementioned Uniprot IDs.

[0247] According to the invention, a process for making thallose also includes the step of enzymatically converting glucose 6-phosphate (G6P) to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). CQQ / nn / Q7n7 / e / YiAi In other embodiments, the process for making thallose further includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). Even in later depictions, the thallose production process also includes the step of converting a saccharide to G1P, which is catalyzed by at least one enzyme.

[0248] Therefore, according to the invention, a process for making thallose may, for example, include the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to T6P via F6PE, (v) convert T6P to Tal6P via Tal6PI (EC 5.3.1.26), and (vi) convert Tal6P to thalose, via Tal6PP. An example of the process where the saccharide is starch is shown in FIG 3.

[0249] Typically, enzyme unit ratios used in the published process are 1:1:1:1:1:1 (aGP:PGM:PGI:F6PE:Tal6PI:Tal6PP). To optimize product profits, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in an increase in the activity of downstream reactions. On the other hand, a ratio of 1:1:1:1:1:3 can be used to maintain a robust phosphate distribution for aGP, which will result in more efficient phosphorolytic cleavage of alpha -1,4- glycosidic bonds. An enzyme ratio, for example, of 3:1:1:1:1:3, can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of thallose production. For example, a given enzyme may be present in an amount of about 2x, 3x, 4x, 5x, etc. relative to the amount of other enzymes.

[0250] One of the important advantages of the processes is that their steps can be carried out in a simple bioreactor or reaction vessel. Alternatively, the steps can also be carried out in a variety of bioreactors, reaction vessels, which are arranged in series.

[0251] Phosphate ions produced by dephosphorylation of Tal6P can then be recycled in the process step to convert a saccharide to G1P, especially when all process steps are performed in a simple bioreactor or reaction vessel. The ability to recycle phosphate in published processes allows nonstoichiometric amounts of phosphate to be used, keeping phosphate reaction concentrations low. This affects the overall pathway and overall rate of processes, but does not limit the activity of individual enzymes and allows for overall efficiency in thallose manufacturing processes.

[0252] For example, phosphate reaction concentrations can vary from about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, from about 1 mM to about 50mM, preferably about 5 mM to about from 50mM, or more preferably about 10mM to about 50mM. For example, the concentration of the phosphate reaction 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. cao / nn / eznz / E / YiAi

[0253] Therefore, a low phosphate concentration results in a decrease in production costs due to low total phosphate, and, consequently, a low cost in phosphate removal. It also prevents the inhibition of Tal6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0253] Likewise, the processes published here can be carried out without added ATP as a phosphate source, that is, free of ATP. The processes can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the published processes for making thallose involves an energetically favorable reaction.

[0254] Talose can also be produced from fructose. For example, the process includes generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; convert T6P to Tal6P catalyzed by Tal6PI, and convert Tal6P to thalose catalyzed by Tal6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0256] Talose can be produced from sucrose. The process offers an in vitro synthetic route, which includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6Pen to T6P catalyzed by F6PE; convert T6P to Tal6P catalyzed by Tal6PI, and convert Tal6P to thalose catalyzed by Tal6PP.

[0257] Phosphate ions generated when Tal6P is converted to thallose can be recycled in the step of converting sucrose to G1P. Furthermore, PPFK and polyphosphate can be used to increase thallose gains by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0258] In some embodiments, a process for making thallose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and converting fructose to F6P catalyzed by at least one enzyme. Some examples of polysaccharides and oligosaccharides were listed above.

[0259] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0260] The present publication offers processes for converting saccharides, such as polysaccharides and oligosaccharides, into starch, cellulose, sucrose, and their derivative products for thallose. In certain embodiments, ATP-free artificial enzymatic pathways are provided for converting starch, cellulose, sucrose, and their derivative products to thallose, using cell-free enzyme cocktails.

[0261] As shown above, various enzymes can be used to hydrolyze starch to increase the efficiency of G1P. Such enzymes include isoamylase, pullulunase, and alpha-amylase. Corn starch contains many diversifications that prevent the action of aGP. Isoamylase can be used to debranch starch, resulting in linear amylodextrin. Pretreated isoamylase starch may result in a higher concentration of F6P in the final product. Isoamylase and pullulunase cleave alpha-1,6 glycosidic bonds, allowing for more complete degradation of starch via alpha-glucan phosphorylase. Alpha-amylase divides CQQ / nn / Q7n7 / e / YiAi alpha-1, 4 glycosidic bonds, therefore alpha-amylase is used to degrade starch into fragments, for faster conversion to thallose and increased solubility.

[0262] To increase the efficiency of thallose, maltose phosphorylase (MP) can be used, by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase thallose gains by recycling the degradation products of glucose, maltose, and maltotriose into longer maltooligosaccharides; that can be cleaved phosphorolytically by aGP to gain G1P.

[0263] In certain embodiments, cellulose and its derivative products can be converted to thallose through a series of steps. The process offers an in vitro synthetic route that includes the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to Tal6P catalyzed by Tal6PI, and, convert Tal6P to thalose catalyzed by Tal6PP. In this process, phosphate ions can be recycled through the step of converting cellodextrin and cellobiose into G1P.

[0264] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, thereby increasing thallose gains by phosphorylating the glucose breakdown product to G6P.

[0265] In other embodiments, thallose may be generated from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to Tal6P catalyzed by Tal6PI; and, convert Tal6P to thalose catalyzed by Tal6PP.

[0266] Some processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose and some of their derivatives are more accessible raw materials than, for example, lactose. When producing thallose from biomass or lactose, profits are lower than in the present invention, and thallose must be separated from other sugars via chromatography, resulting in higher production costs. Likewise, animals are not used in our procedures.

[0267] According to the invention, the step of converting Tal6P to thallose is an irreversible phosphatase reaction, regardless of the raw material. Therefore, thallose is produced at a very high profit while effectively minimizing subsequent product separation costs.

[0268] Compared to cell-based manufacturing methods, the invention includes a cell-free thallose preparation, which has relatively high reaction rates, due to the removal of the cell membrane, which often decreases transport. of substrate / product inside and outside the cell. It also has a final product free of nutrient / cellular metabolite-rich fermentation media.

[0269] A particular embodiment of the invention is thallose produced by the processes described herein.

[0270] Sorbose

[0271] One embodiment of the invention is a process for making sorbose, which includes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), converting CQQ / nn / Q7n7 / B / YIAI Τ6Ρ into sorbose 6- phosphate (S6P) catalyzed by sorbose 6- phosphate epimerase (S6PE), and, convert the S6P produced into sorbose catalyzed by sorbose 6- phosphate phosphatase (S6PP).

[0272] Examples of F6Pes include, but are not limited to, the following proteins: Uniprot ID E8N0N6, E4SEH3, 101507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 catalyze the reaction of F6PE and share 27% amino acid sequence identity. Therefore, examples of F6Pes also include any of the homologues having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity for any of the aforementioned Uniprot IDs.

[0273] According to the invention, a process for preparing sorbose also includes the step of enzymatically converting glucose 6-phosphate (G6P) to (F6P), and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for making sorbose further includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). Even in later representations, the sorbose production process also includes the step of converting a saccharide to G1P which is catalyzed by at least one enzyme.

[0274] Therefore, according to the invention, a process for making sorbose may, for example, include the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (i¡) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to T6P via F6PE, (v) convert T6P to S6P via S6PE (EC 5.3.1.26), and (vi) convert S6P to sorbose via S6PP. An example of the process where the saccharide is starch is shown in FIG 3.

[0275] Typically, enzyme unit ratios used in the published process are 1:1:1:1:1:1 (aGP:PGM:PGI:F6PE:S6PE:S6PP). To optimize product profits, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in increased activity of downstream reactions. On the other hand, a ratio of 1:1:1:1:1:3 can be used to maintain a robust phosphate distribution for aGP, which will result in more efficient phosphorolytic cleavage of alpha-1,4 glycosidic bonds. . For example, an enzyme ratio of 3:1:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of sorbose production. For example, a given enzyme may be present in an approximate amount of 2x, 3x, 4x, 5x, etc. relative to the amount of other enzymes.

[0276] One of the important advantages of the processes is that the process steps can be carried out in a simple bioreactor or a reaction vessel. Alternatively, the steps can also be performed in a variety of bioreactors, or reaction vessels that are arranged in series.

[0277] Phosphate ions produced by dephosphorylation of S6P can be recycled in the process step where a saccharide is converted to G1P, especially when all process steps are performed in a simple bioreactor or reaction vessel. The ability to recycle phosphate in the published processes allows non-stoichiometric amounts of phosphate to be used, which keeps the cao / nn / eznz / E / YiAi concentrations of the phosphate reaction low. This affects the total pathway and overall rate of processes, but does not limit the activity of individual enzymes and allows for overall efficiency in sorbose manufacturing processes.

[0278] For example, phosphate reaction concentrations range from about 0.1 mM to about 300 mM, about 0.1 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 concentration of the phosphate reaction 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.

[0279] Therefore, a low phosphate concentration results in a decrease in production costs due to low total phosphate, and, consequently, a lower cost in phosphate removal. Additionally, it prevents inhibition of S6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0280] Likewise, the processes published in this document can be carried out without added ATP as a phosphate source, that is, free of ATP. The processes can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one of the steps in the published processes for making sorbose includes an energetically favorable reaction.

[0281] Sorbose can also be produced from fructose. For example, the process includes generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, and convert S6P to sorbose catalyzed by S6PP. Fructose can be produced, for example, by enzymatic conversion of sucrose.

[0282] Sorbose can be produced from sucrose. The process offers an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1 P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, and convert S6P to sorbose catalyzed by S6PP.

[0283] Phosphate ions that are generated when S6P is converted to sorbose can be recycled in the step of converting sucrose to G1P. Furthermore, PPFK and polyphosphate can be used to increase sorbose efficiency by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0284] In some embodiments, a process for making sorbose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and convert fructose to F6P catalyzed by at least one enzyme. Some examples of polysaccharides and oligosaccharides were listed above.

[0285] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase. cao / nn / eznz / E / YiAi

[0286] The present publication offers processes for converting saccharides, such as polysaccharides and oligosaccharides to starch, cellulose, sucrose, and their derived products to sorbose. In certain embodiments, artificial, ATP-free enzymatic pathways are provided for converting starch, cellulose, sucrose, and their derivative products to sorbose, using cell-free enzyme cocktails.

[0287] As shown above, various enzymes can be used to hydrolyze starch, in order to increase the yield of G1P. Such enzymes include isoamylase, pullulunase, and alpha amylase. Corn starch contains many diversifications that prevent the action of aGP. Isoamylase can be used to debranch starch, resulting in linear amylodextrin. Pretreated isoamylase starch may result in a higher concentration of F6P in the final product. Isoamylase and pullulunase cleave alpha-1,6- glycosidic bonds, allowing more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves the alpha-1,4- glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to sorbose and greater solubility.

[0288] Maltose phosphorylase (MP) can be used to increase the efficiency of sorbose by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase sorbose gains by recycling the degradation products of glucose, maltose, and maltotriose into longer maltooligosaccharides; which can be cleaved phosphorolytically by aGP to produce G1P.

[0289] In certain embodiments, cellulose and its derivative products can be converted to sorbose through a series of steps. The process offers an in vitro synthetic route, which includes the following steps: generating G1P from cellodextrin, cellobiose and free phosphate catalyzed, by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, and, convert S6P to sorbose catalyzed by S6PP. In this process, phosphate ions can be recycled through the step of converting cellodextrin and cellobiose to G1P.

[0290] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) may be added to the process, thereby increasing sorbose gains by phosphorylating the glucose breakdown product to G6P.

[0291] In other embodiments, sorbose can be generated from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE; and, convert S6P to sorbose catalyzed by S6PP.

[0292] Some processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose, and some of their derivatives are more accessible raw materials than, for example, lactose. When sorbose is produced from biomass or lactose, profits are lower than in the present invention, and sorbose must be separated from other sugars via chromatography, leading to higher production costs. Furthermore, our procedures are carried out without using animals. CQQ / nn / Q7n7 / e / YiAi

[0293] According to the invention, the step of converting S6P to sorbose is an irreversible phosphatase reaction, regardless of the raw material. Therefore, sorbose is produced at a very high profit, while effectively minimizing subsequent product separation costs.

[0294] Compared to cell-based manufacturing methods, the invention includes a cell-free preparation of sorbose that has relatively high reaction rates due to the removal of the cell membrane, which often decreases transport. of substrate / product inside and outside the cell. It also has a final product free of nutrient / cellular metabolite-rich fermentation media.

[0295] A particular embodiment of the invention is to produce sorbose by the processes described herein.

[0296] Glutous

[0297] One embodiment of the invention is a process for preparing gulose that includes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), converting T6P to sorbose 6-phosphate ( S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE), convert the S6P produced to gulose 6-phosphate (Gul6P) catalyzed by gulose 6-phosphate isomerase and convert Gul6P to gulose by gulose 6-phosphate phosphatase (Gul6PP) .

[0298] Examples of F6PE include, but are not limited to, the following proteins: Uniprot ID E8N0N6, E4SEH3, 101507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 catalyze the F6PE reaction and share 27% amino acid sequence identity. Therefore, examples of F6PE also include any counterpart that has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least minus 95%. or at least 99% amino acid sequence identity with any of the Uniprot IDs mentioned above.

[0299] A process for preparing glutose according to the invention also includes the step where glucose 6-phosphate (G6P) is enzymatically converted to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for preparing glucose further includes the step where glucose 1-phosphate (G1P) is converted to G6P, where the step is catalyzed by phosphoglucomutase (PGM). In further embodiments, the gluteal production process also includes the step where a saccharide is converted to G1P which catalyzes at least one enzyme.

[0300] Therefore, a process for preparing glutose according to the invention may include, for example, the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (ii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to T6P via F6PE, (v) convert T6P to S6P via S6PE (EC 5.3.1.26), (vi) convert S6P to Gul6P via Gul6PI, and (vii) convert GulP to gulose via by Gul6PP.

[0301] Typically, the ratios of enzymatic units used in the described process are 1:1:1:1:1:1:1 (aGP:PGM:PGI:F6PE:S6PE:Gul6PI:GulPP). To optimize product yields, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in CQQ / nn / Q7n7 / e / YiAi resulted in increased activity of subsequent reactions. Conversely, a ratio of 1:1:1:1:1:1:3 can be used to maintain a robust phosphate supply for aGP, which will result in more efficient phosphorolytic cleavage of alpha-1 bonds, 4-glycosidics. An enzyme ratio, for example, 3:1:1:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, can be varied to increase the efficiency of gluteal production. For example, a specific enzyme may be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes.

[0302] One of the important advantages of the processes of the invention is that the process steps can be carried out in a single bioreactor or reaction vessel. Alternatively, the steps may also be performed in a plurality of bioreactors, or reaction vessels, that are arranged in series.

[0303] Phosphate ions produced during the dephosphorylation step of S6P can be recycled in the process step where a saccharide is converted to G1P, particularly when all process steps are carried out in a single bioreactor or reaction vessel . The ability to recycle phosphate in the processes described allows the use of non-stoichiometric amounts of phosphate, which keeps reaction phosphate concentrations low. This affects the overall pathway and overall speed of the processes, but does not limit the activity of individual enzymes and allows for the overall efficiency of gluteal manufacturing processes.

[0304] For example, the reaction phosphate concentrations in each of the processes can vary from about 0.1 mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from about 10 mM to about 50 mM. For example, the reaction phosphate concentration 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, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM or approximately 55 mM.

[0305] Therefore, the low phosphate concentration results in decreased production costs due to low total phosphate and therefore a lower cost for phosphate removal. It also prevents inhibition of S6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0306] Furthermore, each of the processes described herein can be performed without added ATP as a phosphate source, i.e., ATP-free. The process can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the described processes for manufacturing glutinous involves an energetically favorable reaction.

[0307] Gulose can also be produced from fructose. For example, the process involves generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, convert S6P to Gul6P via GuI6PI, cao / nn / eznz / E / YiAi and convert Gul6P to gulose via Gul6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0308] A gulose can be produced from sucrose. The process provides an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, convert S6P to Gul6P via Gul6PI, and convert Gul6P to gulose via Gul6PP.

[0309] Phosphate ions generated when S6P is converted to sorbose can subsequently be recycled in the step where sucrose is converted to G1P. Furthermore, PPFK and polyphosphate can be used to increase gluteal yields by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0310] In some embodiments, a process for preparing gulose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and convert fructose to F6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0311] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0312] The present document provides processes for converting saccharides, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose and their derived products, to glutose. In some embodiments, artificial (non-natural) ATP-free enzymatic pathways are provided for converting starch, cellulose, sucrose and their derivative products to glutose using cell-free enzyme cocktails.

[0313] As shown above, various enzymes can be used to hydrolyze starch and thus increase the yield of G1P. Such enzymes include isoamylase, pullulanase and alpha-amylase. Corn starch contains many branches that prevent the action of aGP. Isoamylase can be used to debranch starch, producing linear amylodextrin. Starch pretreated with isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave alpha1,6-glycosidic bonds, allowing more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to gulose and greater solubility.

[0314] Maltose phosphorylase (MP) can be used to increase gluteal yields by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase gluteal yields by recycling the degradation products glucose, maltose and maltotriose into longer maltooligosaccharides; which can be phosphorolytically cleaved by aGP to produce G1P.

[0315] In some embodiments, cellulose and its derivative products can be converted to glute by following a series of steps. The process provides an in vitro synthetic pathway that involves the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and CQQ / nn / Q7n7 / e / YiAi cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, and convert S6P to sorbose catalyzed by S6PP. In this process, phosphate ions can be recycled by converting cellodextrin and cellobiose to G1P.

[0316] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the processes, thereby increasing glucose yields by phosphorylating the degradation product glucose to G6P.

[0317] In other embodiments, glucose can be generated from glucose. The process includes the steps to generate G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI, convert F6P to T6P catalyzed by F6PE, convert T6P to S6P catalyzed by S6PE, convert S6P to Gul6P via Gul6PI, and convert Gul6P to glute via Gul6PP.

[0318] The processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose and some of their derivatives are less expensive raw materials than, for example, lactose. When gooseberry is produced from biomass or lactose, yields are lower than in the present invention, and gooseberry must be separated from other sugars by chromatography, leading to higher production costs. Additionally, our process does not involve animals.

[0319] According to the invention, the step of converting S6P to glutose is an irreversible phosphatase reaction, regardless of the raw material. Glucose is therefore produced with a very high yield and at the same time effectively minimizes the costs of subsequent product separation.

[0320] Unlike cell-based manufacturing methods, the invention involves a cell-free gluteal preparation, has relatively high reaction rates due to the removal of the cell membrane, which often slows down substrate transport / product in and out of the cell. It also features a final product free of nutrient / cellular metabolite-rich fermentation media.

[0321] A particular embodiment of the invention is gluttony produced by the processes described herein for producing gluttony.

[0322] Idosa

[0323] One embodiment of the invention is a process for preparing idose that includes converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE), converting T6P to sorbose 6-phosphate ( S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE), convert the produced S6P to idose 6-phosphate (I6P) catalyzed by idose 6-phosphate isomerase, and convert I6P to idose by idose 6-phosphate phosphatase (I6PP).

[0324] Examples of F6PE include, but are not limited to, the following proteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1 and B5YBD7. Uniprot IDs E8N0N6 and I0I507 catalyze the F6PE reaction and share 27% amino acid sequence identity. Therefore, examples of F6PE also include any counterpart that has at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least minus 95%. or so CQQ / nn / Q7n7 / B / YIAI minus 99% amino acid sequence identity with any of the Uniprot IDs mentioned above.

[0325] A process for preparing idose according to the invention also includes the step where glucose 6-phosphate (G6P) is enzymatically converted to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the process for preparing idose additionally includes the step where glucose 1-phosphate (G1P) is converted to G6P, where the step is catalyzed by phosphoglucomutase (PGM). In further embodiments, the idose production process also includes the step where a saccharide is converted to G1P which catalyzes at least one enzyme.

[0326] Therefore, a process for preparing idose according to the invention may include, for example, the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to T6P via F6PE, (v) convert T6P to S6P via S6PE (EC 5.3.1.26), (vi) convert S6P to I6P via I6PI, and (vii) convert I6P toidosa via I6PP.

[0327] Typically, the ratios of enzymatic units used in the described process are 1:1:1:1:1:1:1 (aGP:PGM:PGI:F6PE:S6PE:l6PI:l6PP). To optimize product efficiency, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in increased activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:3 can be used to maintain a robust phosphate supply for aGP, which will result in more efficient phosphorolytic cleavage of alpha-1 bonds, 4-glycosidics. An enzyme ratio, for example, 3:1:1:1:1:1:3 can be used to further increase the reaction rate. Therefore, ratios of enzymes, including other optional enzymes discussed below, can be administered at different concentrations to increase the efficiency of idose production. For example, a specific enzyme may be present in an amount of approximately 2x, 3x, 4x, 5x, etc., relative to the amount of other enzymes.

[0328] One of the important advantages of the processes of the invention is that the process steps can be carried out in a single bioreactor or reaction vessel. Alternatively, the steps may also be performed in a plurality of bioreactors, or reaction vessels, that are arranged in series.

[0329] Phosphate ions produced during the dephosphorylation step of S6P can be recycled in the process step where a saccharide is converted to G1P, particularly when all process steps are carried out in a single bioreactor or reaction vessel . The ability to recycle phosphate in the processes described allows the use of non-stoichiometric amounts of phosphate, which keeps reaction phosphate concentrations low. This affects the overall pathway and overall speed of the processes, but does not limit the activity of individual enzymes and allows for overall efficiency of the idose manufacturing processes.

[0330] For example, the reaction phosphate concentrations in each of the processes can vary from about 0.1 mM to about 300 mM, from about 0.1 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5mM cao / nn / eznz / E / YiAi to about 50mM, or more preferably from about 10mM to about 50mM. For example, the reaction phosphate concentration 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, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM or approximately 55 mM.

[0331] Therefore, the low phosphate concentration results in decreased production costs due to low total phosphate and therefore a lower cost for phosphate removal. It also prevents inhibition of S6PP by high concentrations of free phosphate and decreases the potential for phosphate contamination.

[0332] Furthermore, the processes described herein can be performed without added ATP as a phosphate source, i.e., ATP-free. The process can also be carried out without having to add NAD(P)(H), that is, free of NAD(P)(H). Other advantages also include the fact that at least one step of the processes described for manufacturing idose involves an energetically favorable reaction.

[0333] Idose can also be produced from fructose. For example, the process involves generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, convert S6P to I6P via I6PI, and convert I6P to idose via I6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0334] Idose can be produced from sucrose. The process provides an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, convert S6P to I6P via I6PI, and convert I6P to idose via I6PP.

[0335] Phosphate ions generated when S6P is converted to sorbose can subsequently be recycled in the step where sucrose is converted to G1P. Furthermore, PPFK and polyphosphate can be used to increase the yields of idose by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0336] In some embodiments, a process for preparing idose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and convert fructose to F6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0337] In other embodiments, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0338] The present document provides processes for converting saccharides, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose and their derivative products, to idose. In some representations, artificial (non-natural) ATP-free enzymatic pathways are provided to convert cao / nn / eznz / E / YiAi starch, cellulose, sucrose and their derivative products to idose using cell-free enzyme cocktails.

[0339] As shown above, various enzymes can be used to hydrolyze starch and thus increase the yield of G1P. Such enzymes include isoamylase, pullulanase and alpha-amylase. Corn starch contains many branches that prevent the action of aGP. Isoamylase can be used to debranch starch, producing linear amylodextrin. Starch pretreated with isoamylase may result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave alpha1,6-glycosidic bonds, allowing more complete degradation of starch by alpha-glucan phosphorylase. Alpha-amylase cleaves alpha-1,4-glycosidic bonds, therefore alpha-amylase is used to break down starch into fragments for faster conversion to idose and greater solubility.

[0340] Maltose phosphorylase (MP) can be used to increase the yields of idose by phosphorolytically cleaving the maltose degradation product into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase idose yields by recycling the degradation products glucose, maltose, and maltotriose into longer maltooligosaccharides; which can be phosphorolytically cleaved by aGP to produce G1P.

[0341] In some embodiments, cellulose and its derivative products can be converted to idose by following a series of steps. The process provides an in vitro synthetic pathway that involves the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; convert T6P to S6P catalyzed by S6PE, and convert S6P to sorbose catalyzed by S6PP. In this process, phosphate ions can be recycled by converting cellodextrin and cellobiose to G1P.

[0342] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the processes, thereby increasing the yields of idose by phosphorylating the degradation product glucose to G6P.

[0343] In other embodiments, idose can be generated from glucose. The process includes the steps to generate G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI, convert F6P to T6P catalyzed by F6PE, convert T6P to S6P catalyzed by S6PE, convert S6P to I6P via I6PI, and convert I6P to idose via I6PP.

[0344] The processes of the invention utilize low-cost starting materials and reduce production costs by decreasing costs associated with raw materials and product separation. Starch, cellulose, sucrose and some of their derivatives are less expensive raw materials than, for example, lactose. When idosa is produced from biomass or lactose, yields are lower than in the present invention, and idosa must be separated from other sugars by chromatography, leading to higher production costs. Additionally, our process does not involve animals.

[0345] According to the invention, the step of converting S6P to idose is an irreversible phosphatase reaction, regardless of the raw material. Idosa is therefore produced with a very high yield and at the same time effectively minimizes the costs of subsequent product separation.

[0346] Unlike cell-based manufacturing methods, the invention involves a cell-free preparation of idose, has relatively high reaction rates due to the elimination of the CQQ / nn / Q7n7 / e / YiAi cell membrane, which often slows down the transport of the substrate / product into and out of the cell. It also features a final product free of nutrient / cellular metabolite-rich fermentation media.

[0347] A particular embodiment of the invention is the idose produced by the processes described herein for producing idose.

[0348] Tagatose

[0349] Processes for making tagatose include converting F6P to T6P, catalyzed by an epimerase; and convert T6P into tagatose, catalyzed by a phosphotase.

[0350] Epimerases suitable for use in processes to convert F6P to T6P include F6PEs. Examples of F6Pes include, but are not limited to the following proteins: Uniprot ID E8N0N6, E4SEH3, 101507, H1XRG1, and B5YBD7. Both Uniprot IDs E8N0N6 and I0I507 catalyze the F6PE reaction and share 27% amino acid sequence identity. Therefore, examples of FGPes also include any of the counterparts having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity for any of the aforementioned Uniprot IDs.

[0351] Phosphatases that convert T6P to tagatose (D-tagatose), T6PPs could be used in a process. Examples of T6PPs include, but are not limited to the following proteins: Uniprot ID 029805, D2RHV2 and F2KMK2. Both Uniprot IDs 029805 and F2KMK2 catalyze the F6PE reaction and share 67% amino acid sequence identity. Therefore, examples of T6PPs also include any of the homologues having at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, most preferably at least 85% , even more preferably at least 90%, more preferably at least 95%, and even more preferably at least 96%, 97%, 98%, 99%, or 100% amino acid sequence identity for any of the above mentioned Uniprot IDs.

[0352] A process for making tagatose also includes the step of enzymatically converting glucose 6phosphate (G6P) to F6P, and this step is catalyzed by phosphoglucose isomerase (PGI). The process to make tagatose also includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). Furthermore, the tagatose production process also includes the step of converting a saccharide into G1P, which is catalyzed by at least one enzyme.

[0353] Therefore, a process for making tagatose, for example, includes the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (i) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to T6P via fructose 6-phosphate epimerase (F6PE), and (v) convert T6P to tagatose, via tagatose 6-phosphate phosphatase (T6PP).

[0354] Typically, the ratios of enzyme units used in the process are 1:1:1:1:1 (aGP:PGM:PGI:F6PE:T6PP). To optimize product profits these relationships can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in an increase in CQO / nn / eznz / E / YiAi the activity of descending reactions. On the other hand, a ratio of 1:1:1:1:3 can be used to maintain a robust phosphate distribution for aGP, which will result in more efficient phosphorolytic cleavage of alpha-1,4-glycosidic bonds. An enzyme ratio of, for example, 3:1:1:1:3 can be used to further increase the reaction rate. Therefore, enzyme ratios, including other optional enzymes discussed below, may be varied to increase the efficiency of tagatose production. For example, a given enzyme could be present in an approximate amount of 2x, 3x, 4x, 5x, etc. relative to the amount of other enzymes.

[0355] Tagatose can also be produced from fructose. The process includes, for example, generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to T6P catalyzed by F6PE; and, convert, T6P to tagatose catalyzed by T6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose.

[0356] Tagatose can be produced from sucrose. The process offers an in vitro synthetic route that includes the following enzymatic steps: generating G1P from sucrose and free phosphate catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; and, convert T6P to tagatose catalyzed by T6PP.

[0357] Phosphate ions generated when T6P is converted to tagatose can be recycled in the step of converting sucrose to G1P. Additionally, PPFK and polyphosphate can be used to increase tagatose gains by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0358] A process for making tagatose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and, convert fructose to G6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0359] Cellulose and its derived products can be converted to tagatose through a series of steps. The process includes the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to PGI-catalyzed F6P; convert F6P to T6P catalyzed by F6PE; and, convert T6P to tagatose catalyzed by T6PP. In this process, phosphate ions can be recycled through the step of converting cellodextrin and cellobiose to G1P.

[0360] Tagatose can be generated from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI; convert F6P to T6P catalyzed by F6PE; and, convert T6P to tagatose catalyzed by T6PP.

[0361] Psycho

[0362] The processes for making psychose include converting fructose 6-phosphate (F6P) to psychose 6-phosphate (P6P), catalyzed by an epimerase (e.g., psychose 6-phosphate 3-epimerase, P6PE) and converting the P6P produced into psychose catalyzed by a phosphatase (e.g., psychose 6-phosphate phosphatase, P6PP).

[0363] Examples of P6PEs include, but are not limited to, the following proteins, identified by UNIPROT, identification numbers: D9TQJ4, A0A090IXZ8, and P32719. Both Uniprot IDs A0A090IXZ8 and D9TQJ4 catalyze the P6PE reaction and share 45% amino acid sequence identity. Therefore, CQQ / nn / Q7n7 / e / YiAi examples of P6Pes also include any of the homologues having at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even more preferably at least 96, 97, 98, 99 or 100% for any of the aforementioned Uniprot IDs.

[0364] Examples of P6PPs include, but are not limited to the following proteins: Uniprot ID. A3DC21, Q5LGR4, and Q89ZR1. Both Uniprot IDs A3DC21 and Q89ZR1 catalyze the P6PP reaction and share 45% amino acid sequence identity. Therefore, examples of P6PPs also include any of the homologues having at least 45%, preferably at least 50%, more preferably at least 55%, more preferably at least 60%, most preferably at least 65% , more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even more preferably at least 96, 97, 98, 99 or 100% for any of the aforementioned Uniprot IDs.

[0365] A process for making psychose also includes the step of enzymatically converting glucose 6phosphate (G6P) INTO f6p, and this step is catalyzed by phosphoglucose isomerase (PGI). The process to make psychose also includes the step of converting glucose 1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). Likewise, the psychose production process also includes the step of converting a saccharide into G1P which is catalyzed by at least one enzyme.

[0366] Therefore, a process for making psychose, for example, includes the following steps: (i) converting a saccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii) convert G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (ii) convert G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) convert F6P to P6P via psychose 6-phosphate epimerase (P6PE), and (v) convert P6P to psychose via psychose 6-phosphate phosphatase (P6PP).

[0367] Typically, the ratios of enzyme units used in the process are 1:1:1:1:1 (aGP:PGM:PGI:P6PE:P6PP). To optimize product profits, these ratios can be adjusted in any number of combinations. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will result in an increase in the activity of downstream reactions. On the other hand, a ratio of 1:1:1:1:3 can be used to maintain a robust phosphate distribution for aGP, which will result in more efficient phosphorolytic cleavage of alpha -1,4-glycosidic bonds. An enzyme ratio, for example, 3:1:1:1:3 can be used to further increase the reaction rate. Therefore, the enzyme ratios, including other optional enzymes, discussed below can be varied to increase the efficiency of tagatose production. For example, a given enzyme could be present in an amount of approximately 2x, 3x, 4x, 5x, etc. relative to the amount of other enzymes.

[0368] Psychose can also be produced from fructose. For example, the process includes generating F6P from fructose and polyphosphate catalyzed by polyphosphate fructokinase (PPFK); convert F6P to P6P catalyzed by P6PE; and convert P6P to psychose catalyzed by P6PP. Fructose can be produced, for example, by an enzymatic conversion of sucrose. cao / nn / eznz / E / YiAi

[0369] Psychose can be produced from sucrose. The process includes the following enzymatic steps: generating G1P from sucrose and free phosphate, catalyzed by sucrose phosphorylase (SP); convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; and, convert P6P to psychose catalyzed by P6PP.

[0370] Phosphate ions generated when P6P is converted to psychose can be recycled in the step of converting sucrose to G1P. Furthermore, PPFK and polyphosphate can be used to increase psychose gains by producing F6P from fructose generated by the phosphorolytic cleavage of sucrose by SP.

[0371] A process to make psychose includes the following steps: generating glucose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, converting glucose into G6P catalyzed by at least one enzyme, generating fructose from polysaccharides and oligosaccharides by enzymatic hydrolysis or acid hydrolysis, and, converting fructose to G6P, catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides were listed above.

[0372] Cellulose and its derived products can be converted to psychose through a series of steps. The process includes the following steps: generating G1P from cellodextrin and cellobiose and free phosphate catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; convert G1P to G6P catalyzed by PGM; convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; and, convert P6P to psychose catalyzed by P6PP. In this process, phosphate ions can be recycled through the step of converting cellodextrin and cellobiose to G1P.

[0373] Psychose can be generated from glucose. The process includes the steps of generating G6P from glucose and polyphosphate catalyzed by polyphosphate glucokinase (PPGK); convert G6P to F6P catalyzed by PGI; convert F6P to P6P catalyzed by P6PE; and, convert P6P to psychose catalyzed by P6PP. Examples

[0374] Methods and materials

[0375] Chemicals

[0376] All chemicals, including corn starch, soluble starch, maltodextrins, glucose, and filter paper were of reagent grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA), unless otherwise specified. Restriction enzymes, T4 ligase, and fusion DNA polymerase were purchased from New England Blolabs (Ipswich, MA, USA). Oligonucleotides were synthesized either by Integrated DNA Technologies (Coralville, IA, USA) or Eurofins MWG Operan (Huntsville, AL, USA). Regenerated amorphous cellulose was used in the purification of enzymes, which was prepared in Avicel PH105 (FMC BioPolymer, Philadelphia, PA, USA), through its dissolution and regeneration, as described in: Ye et al., Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose. Appl. Microbiol. Biotechnol. 2011; 92:551 -560. Escheríchia coli Sig10 (Sigma-Aldrich, St. Louis, MO, USA) was used as host cell for DNA manipulation, and E. coli BL21 (DE3) (Sigma-Aldrich, St. Louis, MO, USA) was used. used as a host cell for recombinant expression of proteins. For cell growth and recombinant expression of E. coii proteins, ZYM-5052 medium was used, including either 100 mg L'1 of ampicillin or 50 mg L1kanamycin. Cellulase from Trichoderma reesei (catalog number: C2730) and pullulunase (catalog number: P1067) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and CQQ / nn / Q7n7 / e / YiAi were produced by Novozymes (Franklinton, NC, USA). Maltose phosphorylase (catalog number: M8284) was purchased from Sigma-Aldrich.

[0377] Production and purification of recombinant enzymes

[0378] The E. co / / BL21 (DE3) strain harboring a protein expression plasmid was incubated in a 1-L Erlenmeyer flask, with 100 mL of ZYM-5052 medium, containing either 100 mg L' 1 of ampicillin or 50 mg of L·1kanamycin. Cells were grown at 37°C with rotary shaking at 220 rpm for 16 to 24 hours. Cells were harvested by centrifugation at 12°C and washed once with 20 mM phosphate-buffered saline (pH 7.5) containing 50 mM NaCl and 5 mM MgCl (heat precipitation and cellulose binding module ) or 20 mM phosphate-buffered saline (pH 7.5) containing 300 mM NaCl and 5 mM imidazole (Ni purification). Cell pellets were resuspended in the same buffer and lysed by ultrasound (Fisher Scientific Model 500 Sonic Dismembrator; 5 S pulse and 10 S off, 21 minutes total at 50% magnitude). After centrifugation, the target proteins were purified in the supernatants.

[0379] Three methods were used to purify the various recombinant proteins. His-tagged proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Life Sciences, Marlborough, MA, USA). Fusion proteins containing a cellulose-binding module (CBM) and self-cleavage were purified through high-affinity adsorption on a large surface area where amorphous cellulose was regenerated. Heat precipitation at 70-95°C for 5 to 30 minutes was used to purify hyperthermostable enzymes. The purity of the recombinant proteins was examined on sodium dodecyl sulfate polyacrylamide gel electropheresis. [03801 Enzymes used and their activities in testing

[0381] Alpha-glucan phosphorylase (aGP) from Thermotoga maritime (Uniprot ID G4FEH8) was used. Activity was evaluated in 50 mM sodium phosphate buffer (pH 7.2) containing 1 mM MgCb, and 30 mM maltodextrin at 50°C. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO ) (Vivaproducts, Inc., Littleton, MA, USA). Glucose 1-phosphate (G1P) was measured using a glucose hexokinase / G6PDH assay kit (Sigma Aldrich, Catalog No. GAHK20-1KT) supplemented with 25 U / mL phosphoglucomutase. One unit (U) was described as pmol / min.

[0382] Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (Uniprot ID Q68BJ6) was used. Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCh and 5 mM G1P at 50°C. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The glucose 6-phosphate product (G6P) was determined using a hexokinase / G6PDH assay kit (Sigma Aldrich, Catalog No. GAHK20-1KT).

[0383] Two different sources of phosphoglucoisomerase (PGI) from Clostridium thermocellum (Uniprot ID A3DBX9) and Thermus thermophilus (Uniprot ID Q5SLL6) were used. Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCh and 10 mM G6P at 50°C. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The fructose 6-phosphate product (F6P) was determined using a combined enzyme sample of fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD), where a decrease in absorption at 340 nm indicates a production of F6P. The 200 pL reaction contained 50 mM HEPES (pH 7.2), 5 mM MgCl2, 10 mM G6P, 1.5 mM ATP, 1.5 cao / nn / eznz / E / YiAi mM phosphoenolpyruvate, 200 μΜ NADH, 0.1 U PGI, 5 U PK, and 5 U LD.

[0384] Recombinant cellodextrin phosphorylase and cellobiose phosphorylase from C. thermocellum are described in Ye et al. Spontaneous high-yield production of hydrogen from cellulosic materials and water catalyzed by enzyme cocktails. ChemSusChem 2009; 2:149-152. Their activities were evaluated as described.

[0385] Recombinant polyphosphate glucokinase from Thermobifida fusca YX is described in Liao et al., Onestep purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation withoutATP. Appl. Microbiol. Biotechnol. 2012;93:1109-1117. Their activities were evaluated as described.

[0386] Recombinant isoamylase from Sulfolobus tokodaii is described in Cheng et al., Doubling power output of starch biobattery treated by the most thermostable isoamylase from an archaeon Sulfolobus tokodaii. Scientific Reports2015; 5:13184. Their activities were evaluated as described.

[0387] Recombinant 4-alpha glucanoltransferase from Thermococcus litoralis is described in Jeon et al. 4-aGlucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis. Eur. J. Biochem. 1997; 248:171-178. Their activities were measured as described.

[0388] Sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum (Uniprot ID D9TT09) was used (Verhaeghe et al. The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity. Appl Microbiol Biotechnol. 2014 Aug;98(16) :7027-37). Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10 mM sucrose and 12 mM organic phosphate. Glucose 1-phosphate (G1P) was measured using a glucose hexokinase / G6PDH assay kit supplemented with 25 U / mL phosphoglucomutase as well as alpha-glucan phosphorylase.

[0389] Psychose 6-phosphate 3-epimerase (P6PE) from Thermoanaerobacterium thermosaccharolyticum (Uniprot ID D9TQJ4) was used. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl 2, 500 μΜ C0CI2, 1 U / mL P6PP and 10 mM F6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, psychose 6-phosphate (P6P), was determined using psychose 6-phosphate phosphatase and detecting the release of free phosphate. To detect the release of free phosphate, 500 μΙ of a solution containing 0.1 M zinc acetate and 2 mM ammonium molybdate (pH 5) was added to the 50 μΙ reaction. This was mixed and 125 μΙ of 5% ascorbic acid (pH 5) was added. This solution was mixed and then incubated at 30 °C for 20 min. Absorbance was read at 850 nm to determine free phosphate release. Psychose was then verified by High Performance Liquid Chromatography (HPLC) using an Agilent Hi-Plex H column (sample and control were performed with 5mM H2SO4 at 0.6 mUmin and 65 °C).

[0390] Allose 6-phosphate isomerase (A6PI) from Clostridium thermocellum (Uniprot ID W4V2C8) was used with the amino acid sequence set forth in SEQ ID NO: 1. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl 2, 500 μΜ C0CI2, 1 U / mL P6PE, 1 U / mL A6PP and 10 mM F6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, allose 6-phosphate (P6P), was determined using allose 6-phosphate phosphatase and detecting the release of free phosphate as described for P6PE. Allose was verified via HPLC as was psychose. Another A6PI can be used, such as the A6PI from Symbiobacterium thermophilum (Uniprot ID Q67LX4) with the amino acid sequence set forth in SEQ ID NO: 2. cao / nn / eznz / E / YiAi

[0391] Allose 6-phosphate phosphatase (A6PP) from Rubellimlcrobium thermophilum (Uniprot ID S9SDA3) was used with the amino acid sequence set forth in SEQ ID NO: 3. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl 2, 500 μΜ C0CI2, 1 U / mL P6PE, 1 U / mL A6PI and 10 mM F6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, allose, was determined by detecting free phosphate release as described for P6PE. Allose was verified via HPLC as was psychose. Other A6PPs may be used, such as A6PP from Thermotoga maritime (Uniprot ID Q9X0Y1) with the amino acid sequence set forth in SEQ ID NO: 4, A6PP FROM Thermoanaerobacterium saccharolyticum (Uniprot ID I3VT81) with the amino acid sequence set forth in SEQ ID NO: 5, A6PP from Streptomyces thermoautotrophicus (Uniprot ID A0A132NF06) with the amino acid sequence set forth in SEQ ID NO: 6, and A6PP from Sphaerobacter thermophilus (Uniprot ID D1C7G9) with the amino acid sequence set forth in SEQ ID NO: 7.

[0392] Mannose 6-phosphate isomerase (M6PI) from Pseudonocardia thermophila (Uniprot ID A0A1M6TLY7) was used with the amino acid sequence set forth in SEQ ID NO: 8. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl 2, 1 U / mL PGI, 1 U / mL M6PP and 10 mM F6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, mannose 6-phosphate (M6P), was determined using mannose 6-phosphate phosphatase (M6PP) and detecting free phosphate release as described for P6PE. The crafty was verified through HPLC as was the psychose. Other M6PIs can be used such as M6PI from Caldithrix abyssi (Uniprot ID H1XQS6) with the amino acid sequence set forth in SEQ ID NO: 9, M6PI from Myceliophthora thermophila (Uniprot ID G2Q982) with the amino acid sequence set forth in SEQ ID NO: 10 and M6PI from Treponema caldarium (Uniprot ID F8F1Z8) with the amino acid sequence set forth in SEQ ID NO: 11.

[0393] Mannose 6-phosphate phosphatase (M6PP) from Tepidimonas fonticaldi (Uniprot ID A0A1A6DSI3) was used with the amino acid sequence set forth in SEQ ID NO: 12. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl 2, and 10 mM mannose 6-phosphate at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, mannose, was determined by detecting free phosphate release as described for P6PE. The crafty was verified through HPLC as was the psychose. Other M6PPs such as M6PP from Thermomonas hydrothermalis (Uniprot ID A0A1M4UN08) with the amino acid sequence set forth in SEQ ID NO: 13 and M6PP from Sulfurivirga caldicuralii (Uniprot ID A0A1N6FCW3) with the amino acid sequence set forth in SEQ ID NO: 14 can be used. .

[0394] Bifunctional phosphoglucose / phosphomannose isomerase (PGPMI) from Syntrophothermus lipocalidus (Uniprot ID D7CPH7) was used with the amino acid sequence set forth in SEQ ID NO: 15. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl 2, 1 U / mL PGI, 1 U / mL M6PP and 10 mM G6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, M6P, was determined using M6PP and detecting free phosphate release as described for P6PE. The crafty was verified through HPLC as was the psychose. Other PGPMIs such as PGPMI from Schleiferia thermophila (Uniprot ID A0A085L170) with the amino acid sequence set forth in SEQ ID NO: 16 and PGPMI from Thermodesulfobium narugense (Uniprot ID M1E6Z3) with the amino acid sequence set forth in SEQ ID NO: 17 may be used. . cao / nn / eznz / E / YiAi

[0395] Galactose 6-phosphatase isomerase (Gal6PI) from Lactococcus lactis (Uniprot IDs P23494 and P23495 with the amino acid sequences set forth in SEQ ID NO: 18 and 19, respectively) was used (van Rooijen etal. Molecular Cloning, Characterization, and Nucleotide Sequence of the Tagatose 6-Phosphate Pathway Gene Cluster of the Lactose Operon of Lactococcus Zactis. The activity is measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl, 1 U / mL fructose 6-phosphate 4-epimerase (F6PE), 1 U / mL galactose 6-phosphate phosphatase (GalPP) and 10 mM fructose 6-phosphate at 37°C. The reaction is stopped by filtration of enzyme with a Vivaspin 2 concentrator (10,000 MWCO). The product, galactose 6-phosphate (gal6P), was determined using Gal6PP and detecting the release of free phosphate as described for P6PE. Galactose was verified via HPLC as was psychose.

[0396] Galactose 6-phosphate phosphatase (Gal6PP) from Bacteroides thetaiotaomicron (Uniprot ID Q8A2F3) was used with the amino acid sequence set forth in SEQ ID NO: 20. The activity was measured in 50 mM HEPES buffer (pH 7.2). containing 5 mM MgCl, and 10 mM galactose 6-phosphate at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, galactose, was determined by detecting free phosphate release as described for P6PE. Galactose was verified via HPLC as was psychose.

[0397] Fructose 6-phosphatase phosphatase (F6PP) from Halothermothrix orenii (Uniprot ID B8CWV3) was used with the amino acid sequence set forth in SEQ ID NO: 21. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCb, and 10 mM fructose 6-phosphate at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose, was determined by detecting free phosphate release as described for P6PE. Fructose was verified via HPLC as was psychose.

[0398] Tagatose 6-phosphate phosphatase (T6PP) from Archaeoglobus fugidis (Uniprot ID A0A075WB87) was used. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCH, and 10 mM T6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Tagatose production was determined by detecting free phosphate release as described for F6PE.

[0399] Psychose 6-phosphate phosphatase (P6PP) from Clostridium thermocellum (UNIPROT ID A3DC21) was used. The activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCI2, 80 μΜ CoCI2, 1 U / mL P6PE and 10 mM F6P at 50°C. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, psychose, was determined by detecting free phosphate release as described for P6PE.

[0400] The enzyme units used in each example below can be increased or decreased to adjust the reaction time as desired. For example, if you wanted to perform Example 9 in 8 hours instead of 24 hours, the enzyme units would be increased approximately 3 times. On the contrary, if example 9 were desired to be carried out in 48 hours instead of 24 hours, the enzymatic units could be reduced approximately 2 times. These examples illustrate how the number of enzyme units can be used to increase or decrease the reaction time while keeping the productivity constant.

[0401] All products

[0402] Example 1. cao / nn / eznz / E / YiAi

[0403] To validate the technical feasibility of the enzymatic biosynthesis of fructose 6-phosphate from starch, three enzymes were expressed recombinantly: alpha-glucan phosphorylase from T. maritime (Uniprot ID G4FEH8), phosphoglucomutase from Thermococcus kodadaraensis (Uniprot ID Q68BJ6), and Clostridium thermocellum phosphoglucoisomerase (Uniprot ID A3DBX9). Recombinant proteins were overexpressed in E. coli BL21 (DE3) and purified as described above.

[0404] A 0.20 mL reaction mixture containing 10 g / L soluble starch, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCb, 0.5 mM ZnCb, 0.01 U aGP, 0.01 U was incubated. of PGM and 0.01 U of PGI at 50°C for 24 hours. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD)-coupled enzyme assay where a decrease in absorbance at 340 nm indicates F6P production as described above. The final concentration of F6P after 24 hours was 3.6 g / L.

[0405] Example 2

[0406] The same tests were carried out as in Example 1 (except reaction temperatures) from 40 to 80°C. It was found that 10 g / L of soluble starch produced 0.9 g / L of F6P at 40°C and 3.6 g / L of F6P at 80°C after 40-h reactions. These results suggest that increasing the reaction temperature for this set of enzymes increased the yield of F6P, but increasing the temperature too much may also deteriorate some enzymatic activity.

[0407] Example 3

[0408] It was found that, at 80°C, an enzymatic ratio of aGP:PGM:PGI of approximately 1:1:1 results in rapid generation of F6P. It was observed that the enzymatic ratio did not greatly influence the final concentration of F6P if the reaction time was sufficient. However, the enzyme ratio affects the reaction rates and the total cost of the enzymes used in the system.

[0409] Example 4

[0410] A 0.20 mL reaction mixture containing 10 g / L maltodextrin, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl, 0.5 mM ZnClz, 0.01 U aGP, 0.01 U PGM and 0.01 U of PGI at 50°C for 24 hours. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), was determined using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD)-coupled enzyme assay where a decrease in absorbance at 340 nm indicates F6P production as described above. The final concentration of F6P after 24 hours was 3.6 g / L. [04111 Examples

[0412] To test the production of F6P from Avicel, Sigma cellulase was used to hydrolyze cellulose at 50°C. To remove beta-glucosidase from commercial cellulase, 10 units of filter paper / mL cellulase was mixed with 10 g / L Avicel in an ice water bath for 10 minutes. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. Avicel that was bound to cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C for three days. The cellulose hydrolyzate was mixed with 5 U / ml of cellodextrin phosphorylase, 5 U / L of cellobiose phosphorylase, 5 U / ml of aGP, 5 U / mL of PGM and 5 U / mL of PGI in a cao / nn / buffer. eznz / E / YiAi 100 mM HEPES (pH 7.2) containing 10 mM phosphate, 5 mM MgCl and 0.5 mM eZnCh. The reaction was carried out at 60°C for 72 hours and high concentrations of F6P were found (small amounts of glucose and no cellobiose was found). F6P was detected using the enzyme-coupled assay described above. Glucose was detected using a hexokinase / G6PDH assay kit as described previously. f04131 Example 6

[0414] To increase the efficiency of F6P from Avicel, it was pretreated with concentrated phosphoric acid to produce amorphous cellulose (RAC), as described in Zhang et al. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 2006; 7: 644-648. To remove beta-glucosidase from commercial cellulase, 10 filter paper units / mL cellulase was mixed with 10 g / L RAC in an ice-water bath for 5 minutes. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The RAC that was bound to cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C for 12 h. The RAC hydrolyzate was mixed with 5 U / mL cellodextrin phosphorylase, 5 U / mL cellobiose phosphorylase, 5 U / mL aGP, 5 U / mL PGM, and 5 U / mL PGI in a 100 mM HEPES buffer. (pH 7.2) containing 10 mM phosphate, 5 mM MgCk and 0.5 mM d eZnCb. The reaction was carried out at 60°C for 72 hours. High concentrations of F6P and glucose were recovered because no enzymes were added to convert glucose to F6P. F6P was detected using the enzyme-coupled assay described above. Glucose was detected using a hexokinase / G6PDH assay kit as described previously. [04151 Example 7

[0416] To further increase the F6P efficiency of RAC, polyphosphate glucokinase and polyphosphate were added. To remove beta-glucosidase from commercial cellulase, 10 filter paper units / mL cellulase was mixed with 10 g / L RAC in an ice water bath for 5 minutes. After centrifugation at 4°C, the supernatant containing beta-glucosidase was decanted. The RAC that was bound to cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C, incubated in citrate buffer (pH 4.8) for hydrolysis at 50°C. for 12 hours. The RAC hydrolyzate was mixed with 5 U / mL of polyphosphate glucokinase, 5 U / mL of cellodextrin phosphorylase, 5 U / mL of cellobiose phosphorylase, 5 LJ / mL of aGP, 5 U / mL of PGM and 5 U / mL of PGI in 100 mM HEPES buffer (pH 7.2) containing 50 mM polyphosphate, 10 mM phosphate, 5 mM MgCH, and 0.5 mM ZnCL. The reaction was carried out at 50°C for 72 hours. F6P was found in high concentrations with only trace amounts of glucose now present. F6P was detected using the enzyme-coupled assay described above. Glucose was detected using a hexokinase / G6PDH assay kit as described previously.

[0417] Example 8

[0418] To determine the concentration range of phosphate buffer saline (PBS), a 0.20 mL reaction mixture containing 50 g / L maltodextrin was incubated; 6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline, pH 7.2; 5 mM MgCl2; 0.1 U of aGP; 0.1 U PGM; and 0.1 U of PGI at 50°C for 6 hours. The short duration ensures that the reaction is not completed and therefore CQO / nn / eznz / E / YiAi you can clearly see the differences in efficiency. F6P production was quantified using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK)Zlactate dehydrogenase (LD)-coupled enzyme assay where a decrease in absorbance at 340 nm indicates F6P production. Respectively, a yield of 4.5 g / L, 5.1 g / L, 5.6 g / L, 4.8 g / L, or 4.9 g / L of F6P was obtained for the reactions containing 6.25 mM, 12.5 mM, 25 mM, 37.5 mM , or 50 mM phosphate buffered saline pH 7.2 (Table 1). These results indicate that a concentration of 25 mM PBS pH 7.2 was ideal for these particular reaction conditions. It is important to note that even using 6.25 mM PBS at pH 7.2 results in significant turnover due to phosphate recycling. This shows that the phosphate recycling methods described can keep phosphate levels low even at industrial levels of volumetric productivity (e.g. 200-300 g / L maltodextrin). CQQ / nn / Q7n7 / e / YiAi Table 1 PBS concentration pH 7.2 (mM) g / L of F6P 6.25 4.5 12.5 5.1 25 5.6 37.5 4.8 50 4.9

[0419] Example 9

[0420] To determine the pH range of the cascade reaction, a 0.20 mL reaction mixture containing 50 g / L maltodextrin was incubated; 50 mM phosphate buffered saline pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2 or 7.3; 5 mM MgCl2; 0.02 U of aGP: 0.02 U PGM; and 0.02 U of PGI at 50°C for 16 hours. The units are decreased to ensure that the reaction is not completed and therefore the differences in efficiency can be clearly seen. The production of F6P was quantified as in example 12. Respectively, a yield of 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.1 g / L, 4.4 g / L, 4.1 g / L, 3.8 g was obtained. / L or 4.0 g / L of F6P for reactions containing 50 mM phosphate buffer saline at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2 or 7.3 (Table 2). These results indicate that a pH of 6.8 was ideal for these particular reaction conditions, although the system operates across a wide pH range. Table 2 PBS pH g / L F6P 6.0 4.0 6.2 4.1 6.4 4.2 6.6 4.1 6.8 4.4 7.0 4.1 7.2 3.8 7.3 4.0

[0421] Alosa

[0422] Example 10

[0423] To validate the production of allose from F6P, 10 g / L of F6P was mixed with 1 U / mL of P6PE, 1 U / mL of A6PI and 1 U / mL of A6PP in 50 Mm of HEPES buffer (pH 7.2) containing 5Mm of MgCU and 500 μΜ of C0CI2. The reaction was incubated for 3 hours at 50°C. The conversion of F6P to allose was observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample and control were processed in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0424] Example 11

[0425] To validate the production of allose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 Mm phosphate buffered saline pH 7.2, 5 mM MgCl2, 500 pM was incubated. of C0CI2, 0.05 U of aGP, 0.05 U of PGM, 0.05 U of PGI, 0.05 U of P6PE, 0.05 U of A6PI and 0.05 U of A6PP at 50°C for 24 hours. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Alose was verified by HPLC as described in Example 10.

[0426] Example 12

[0427] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCh and 0.1 g / L isoamylase was incubated at 80 °C for 24 hours. This was used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 Mm phosphate buffered saline pH 7.2, 5 mM MgCl2, 500 pM C0CI2, 0.05 U aGP, 0.05 U PGM, 0.05 U of PGI, 0.05 U of P6PE, 0.05 U of A6PI and 0.05 U of A6PP at 50°C which was incubated for 24 hours. The production of allose was verified as in Example 10.

[0428] Example 13

[0429] To further increase the yields of allose from maltodextrin, 0.05 U of 4glucan transferase (4GT) was added to the reaction described in Example 11.

[0430] 0.2mL of a reaction mixture containing 20 g / L of maltodextrin treated with isoamylase (see example 12), 50 Mm of phosphate buffered saline pH 7.2, 5 mM of MgCU, 500 pM of C0CI2, were used. 0.05 U of aGP, 0.05 U of PGM, 0.05 U of PGI, 0.05 U of P6PE, 0.05 U of A6PI and 0.05 U of A6PP and 0.05 U of 4GT at 50°C which was incubated for 24 hours. The production of allose was verified as in Example 10. [0431 ] Example 14

[0432] To further increase the yields of allose from maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in Example 11.

[0433] Example 15

[0434] To further increase the yields of allose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75Mm of polyphosphate were added to the reaction described in Example 11.

[0435] Example 16

[0436] To produce allose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCU, 500 pM C0CI2, 0.05 was incubated. U of fructose polyphosphate kinase, 0.05 U of P6PE, 0.05 of A6PI and 0.05 U of A6PP at 50°C for 24 hours. Allose production is quantified as in Example 10. cao / nn / eznz / E / YiAi

[0437] Example 17

[0438] To produce allose from fructose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCb, 500 μΜ C0Cl2, 0.05 was incubated. U of glucose polyphosphate kinase, 0.05 U of PGI, 0.05 U of P6PE, 0.05 of A6PI and 0.05 U of A6PP at 50°C for 24 hours. Allose production is quantified as in Example 10.

[0439] Example 18

[0440] To produce allose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgClz, 500 μΜ C0Cl2, 0.05 U of sucrose phosphorylase, 0.05 U of PGM, 0.05 U of PGI, 0.05 U of P6PE, 0.05 U of A6PI and 0.05 U of A6PP at 50°C for 24 hours. Allose production is quantified as in Example 10. [04411 Example 19

[0442] To further increase the yields of allose from sucrose, 75 mM polyphosphate and 0.05 fructokinase polyphosphate are added to the reaction mixture in Example 18. Allose production is quantified as in Example 10.

[0443] Crafty

[0444] Example 20

[0445] To validate the production of mannose from F6P, 10 g / L of F6P was mixed with 1 U / mL of M6PI / PGPMI and 1 U / mL of M6PP in 50 mM HEPES buffer (pH 7.2) that contained 5 mM MgCl2. The reaction was incubated for 3 hours at 50°C. The conversion of F6P to mannose was observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample and control were processed in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0446] Example 21

[0447] To validate the production of mannose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0, 05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (PGI is not needed in the case of PGPMI), and 0.05 U M6PP were incubated at 50°C for 24 hours. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Mannose was verified by HPLC as described in Example 20.

[0448] Example 22

[0449] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2 and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This was used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (no PGP needed in the case of PGPMI), and 0.05 U M6PP were incubated at 50°C for 24 hours. The production of mañosa was verified as in Example 20.

[0450] Example 23

[0451] To further increase the yields of mannose from maltodextrin, 0.05 U of 4glucanoo transferase (4GT) was added to the reaction described in Example 21. CQO / nn / eznz / E / YiAi

[0452] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 22), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (IGP is not needed in the case of PGPMI), 0.05 U of M6PP and 0.05 U of 4GT were incubated at 50°C for 24 hours. The production of mannose was verified as shown in Example 20.

[0453] Example 24

[0454] To further increase the yields of mannose from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction described in Example 21.

[0455] Example 25

[0456] To further increase the yields of mannose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in Example 21.

[0457] Example 26

[0458] To produce mannose from fructose, a reaction mixture containing 10 g / L fructose, with 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U M6PI / PGPMI (no need for IGP in PGPMI), and 0.05 U M6PP is incubated at 50°C for 24 hours. Mannose production is quantified as in Example 20.

[0459] Example 27

[0460] To produce mannose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer, pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U M6PI / PGPMI (no PGI needed in the case of PGPMI), and 0.05 U M6PP are incubated at 50°C for 24 hours. Mannose production is quantified as in Example 20.

[0461] Example 28

[0462] To produce mannose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate-buffered saline pH 7.0, 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (no PGI necessary in the case of PGPMI), and 0.05 U M6PP are incubated at 50°C for 24 hours. Mannose production is quantified as in Example 20.

[0463] Example 29

[0464] To further increase the yields of mannose from sucrose, 75 mM polyphosphate and 0.05 fructokinase polyphosphate are added to the reaction mixture in Example 28. The production of mannose is quantified as in Example 20.

[0465] Galactose

[0466] Example 30

[0467] To validate the production of galactose from F6P, 10 g / L of F6P is mixed with 1 U / mL of Gal6PI and 1 U / mL of Gal6PP in 50 mM HEPES buffer (pH 7.2) containing MgCl2 5mM. The reaction is incubated for 3 hours at 37°C. The conversion of F6P to galactose is observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample and control are run in 5 mM H2SO4 at 0.6 mUmin and 65°C.

[0468] Example 31 cao / nn / eznz / E / YiAi

[0469] To validate the production of galactose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0, 05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI and 0.05 U Gal6PP are incubated at 37°C for 24 hours. The reaction is stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Galactose is verified by HPLC as described in Example 30.

[0470] Example 32

[0471] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2 and 0.1 g / L isoamylase is incubated at 80°C for 24 hours. This is used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI and 0.05 U Gal6PP are incubated at 37°C for 24 hours. Galactose production is verified as in Example 30.

[0472] Example 33

[0473] To further increase galactose yields from maltodextrin, 0.05 U 4-glucan transferase (4GT) is added to the reaction described in Example 31.

[0474] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 12), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI, 0.05 U Gal6PP and 0.05 U 4GT are incubated at 37°C for 24 hours. Galactose production is verified as in Example 30.

[0475] Example 34

[0476] To further increase galactose yields from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction described in Example 31.

[0477] Example 35

[0478] To further increase the yields of galactose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in Example 31.

[0479] Example 36

[0480] To produce galactose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase is incubated. , 0.05 U Gal6PI and 0.05 U Gal6PP. at 37oC for 24 hours. Galactose production is quantified as in Example 30.

[0481] Example 37

[0482] To produce galactose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U Gal6PI and 0.05 U Gal6PP are incubated at 37°C for 24 hours. Galactose production is quantified as in Example 30.

[0483] Example 38

[0484] To produce galactose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate-buffered saline pH 7.0, 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U Gal6PI and 0.05 U Gal6PP are incubated at 37°C for 24 hours. Galactose production is quantified as in Example 30. cao / nn / eznz / E / YiAi

[0485] Example 39

[0486] To further increase the yields of galactose from sucrose, 75 mM polyphosphate and 0.05 fructokinase polyphosphate are added to the reaction mixture in Example 38. Galactose production is quantified as in Example 30.

[0487] Example 40

[0488] To validate galactose production from Gal6P, 10 g / L Gal6P was mixed with 1 U / mL Gal6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated for 1 hour at 50°C. The conversion of Gal6P to galactose is observed in the detection of free phosphate. To detect free release of phosphate, 500 μΙ of a solution containing 0.1 M zinc acetate and 2 mM ammonium molybdate (pH 5) was added to the 50 μΙ reaction. This was mixed and followed by 125 μΙ of 5% ascorbic acid (pH 5). This solution was mixed and then incubated at 30°C for 20 min. Absorbance was read at 850 nm to determine free phosphate release.

[0489] Fructose

[0490] Example 41

[0491] To validate fructose production from F6P, 10 g / L F6P was mixed with 1 LJ / mL F6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated for 3 hours at 50°C. The conversion of F6P to fructose was observed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample and control were processed in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0492] Example 42

[0493] To validate the production of fructose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0, 05 U of aGP, 0.05 U PGM, 0.05 U PGI and 0.05 U F6PP were incubated at 50°C for 24 hours. The reaction was stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Fructose was verified by HPLC as described in Example 41.

[0494] Example 43

[0495] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2 and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This was used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI and 0.05 U F6PP were incubated at 50oC for 24 hours. Fructose production was verified as in Example 41.

[0496] Example 44

[0497] To further increase fructose yields from maltodextrin, 0.05 U of 4-glucan transferase (4GT) was added to the reaction described in Example 42.

[0498] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 12), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PP, and 0.05 U 4GT were incubated at 50°C for 24 hours. Fructose production was verified as in Example 41.

[0499] Example 45 cao / nn / eznz / E / YiAi

[0500] To further increase the fructose yields of maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction described in Example 42.

[0501] Example 46

[0502] To further increase fructose yields from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in Example 42.

[0503] Example 47

[0504] To produce fructose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase is incubated. , PGI 0.05 U, and 0.05 U F6PP. at 50oC for 24 hours. Fructose production is quantified as in Example 41.

[0505] Example 48

[0506] To produce fructose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate-buffered saline pH 7.0, 5 mM MgCl2, 0.05 LJ sucrose phosphorylase, 0 was incubated. .05 PGM, 0.05 U PGI and 0.05 U F6PP at 50oC for 24 hours. Fructose production was quantified as in Example 41.

[0507] Altrose

[0508] Example 49

[0509] To validate the production of altrose from F6P, 10 g / L of F6P is mixed with 1 U / mL of P6PE, 1 U / mL of altrose 6-phosphate isomerase (AH6PI) and 1 U / mL of altrose 6-phosphate phosphatase (AH6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated for 3 hours at 50°C. The conversion of F6P to altrose is observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample and control are run in 5 mM H2SO4 at 0.6 mUmin and 65°C.

[0510] Example 50

[0511] To validate the production of altrose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI and 0.05 U Alt6PP are incubated at 50°C for 24 hours. The reaction is stopped by filtration of an enzyme with a Vivaspin 2 concentrator (10,000 MWCO). Altrose is verified by HPLC as described in Example 49.

[0512] Example 51

[0513] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2 and 0.1 g / L isoamylase is incubated at 80°C for 24 hours. This is used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U Alt6PP are incubated at 50°C for 24 hours. The production of altrose is verified as in Example 49.

[0514] Example 52

[0515] To further increase altrose yields from maltodextrin, 0.05 U 4-glucan transferase (4GT) is added to the reaction described in Example 50. cao / nn / eznz / E / YiAi

[0516] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 50), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, 0.05 U AH6PP and 0.05 U 4GT are incubated at 50°C for 24 hours. The production of altrose is verified as in Example 49.

[0517] Example 53

[0518] To further increase altrose yields from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction described in Example 50.

[0519] Example 54

[0520] To further increase altrose yields from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in Example 50.

[0521] Example 55

[0522] To produce altrose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0 .05 U P6PE, 0.05 U AU6PI and 0.05 U Alt6PP are incubated at 50°C for 24 hours. Altrose production is quantified as in Example 49.

[0523] Example 56

[0524] To produce altrose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0 .05 U PGI, 0.05 U P6PE, 0.05 U AH6PI and 0.05 U AH6PP are incubated at 50°C for 24 hours. Altrose production is quantified as in Example 49.

[0525] Example 57

[0526] To produce altrose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U AH6PP are incubated at 50°C for 24 hours. Altrose production is quantified as in Example 49.

[0527] Example 58

[0528] To further increase altrose yields from sucrose, 75 mM polyphosphate and 0.05 fructokinase polyphosphate are added to the reaction mixture in Example 56. Altrose production is quantified as in Example 49.

[0529] Talose

[0530] Example 59

[0531] To validate the production of thallose from F6P, 10 g / L of F6P is mixed with 1 U / mL of F6PE, 1 U / mL of thallose 6-phosphate isomerase (Tal6PI) and 1 U / mL of thallose. 6-phosphate phosphatase (Tal6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated for 3 hours at 50°C. The conversion of F6P to thallose is observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. Sample and control are run in 5 mM H2SO4 at 0.6 mUrnin and 65°C.

[0532] Example 60 cao / nn / eznz / E / YiAi

[0533] To validate the production of thallose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0, 05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI and 0.05 U Tal6PP are incubated at 50°C for 24 hours. The reaction is stopped by enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Talose is verified by HPLC as described in Example 59.

[0534] Example 61

[0535] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2 and 0.1 g / L isoamylase is incubated at 80°C for 24 hours. This is used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP are incubated at 50°C for 24 hours. The production of thallose is verified as in Example 59.

[0536] Example 62

[0537] To further increase the yields of thallose from maltodextrin, 0.05 U 4-glucan transferase (4GT) is added to the reaction described in Example 60.

[0538] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 60), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, 0.05 U Tal6PP and 0.05 U 4GT are incubated at 50°C for 24 hours. The production of thallose is verified as in Example 59.

[0539] Example 63

[0540] To further increase the yields of thallose from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction described in Example 59.

[0541] Example 64

[0542] To further increase the yields of thallose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in Example 60.

[0543] Example 65

[0544] To produce thallose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U Tal6PI and 0.05 U Tal6PP are incubated at 50°C for 24 hours. Talose production is quantified as in Example 59.

[0545] Example 66

[0546] To produce thallose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0 .05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI and 0.05 U Tal6PP are incubated at 50°C for 24 hours. Talose production is quantified as in Example 59.

[0547] Example 67

[0548] To produce thallose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate-buffered saline pH 7.0, 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP are incubated at 50°C for 24 hours. Talose production is quantified as in Example 59. CQO / nn / eznz / E / YiAi

[0549] Example 68

[0550] To further increase the yields of thallose from sucrose, 75 mM polyphosphate and 0.05 polyphosphate fructokinase are added to the reaction mixture in Example 66. The production of thallose is quantified as in Example 59.

[0551] Sorbose

[0552] Example 69

[0553] To validate the production of sorbose from F6P, 10 g / L of F6P is mixed with 1 U / mL of F6PE, 1 U / mL sorbose 6-phosphate 3-epimerase (S6PE) and 1 U / mL of sorbose 6-phosphate phosphatase (S6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated for 3 hours at 50°C. The conversion of F6P to sorbose is observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The sample and control are processed in 5 mM H2SO4 at 0.6 ml / min and 65°C.

[0554] Example 70

[0555] To verify the production of sorbose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline with pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP at 50°C for 24 hours. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Sorbose was verified via HPLC as described in example 68.

[0556] Example 71

[0557] A reaction mixture containing 200 g / L maltodextran, 10 mM acetate buffer (pH 5.5), 5 mM MgCI2, and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This mixture was used to create another reaction mixture containing 20 g / L maltodextrin isoamylase treated, 50 mM saline buffer with pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP were incubated at 50°C for 24 hours. The production of sorbose was verified in example 69.

[0558] Example 72

[0559] To further increase the sorbose gains from maltodextrin, 0.05 U 4-glucan transferase (4GT) was added to the reaction described in Example 70.

[0560] A 0.2 mL reaction mixture containing 20 g / L isoamylase treated maltodextrin (see Example 70), 50 mM phosphate buffered saline and pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U was incubated. U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U S6PP, and 0.05 U at 50°C for 24 hours. The production of sorbose was verified in example 69. [05611 Example 73

[0562] To further increase the gains of sorbose, from maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in example 70.

[0563] Example 74

[0564] To further increase sorbose yields from maltodextrin, 0.05 U polyphosphate glucokinase and 75 mM polyphosphate is added to the reaction described in Example 69.

[0565] Example 75 CQO / nn / eznz / E / YiAi

[0566] To produce sorbose from fructose, a reaction mixture was incubated at 50°C containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP for 24 hours. The production of sorbose is quantified in example 69.

[0567] Example 76

[0568] To produce sorbose from glucose, a reaction mixture was incubated at 50°C containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgClz, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP for 24 hours. The production of sorbose is quantified in example 69.

[0569] Example 77

[0570] To produce sorbose from sucrose, a reaction mixture was incubated at 50°C containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCk, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP for 24 hours. The production of sorbose is quantified in example 69. [05711 Example 78

[0572] To further increase the gains of sorbose from sucrose, 75 mM polyphosphate and 0.05 fructokinase polyphosphate are added to the reaction mixture in example 76. The production of sorbose is quantified in example 69.

[0573] Glutous

[0574] Example 79

[0575] To verify the production of gulose from F6P, 10 g / L F6P is mixed with 1 U / mL F6PE, 1 U / mL S6PE, 1 U / mL of gulose 6-phosphate isomerase (Gul6PI), and 1 U / mL gulose 6-phosphate phosphatase (Gul6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCk. The reaction was incubated for 3 hours at 50°C. The conversion of F6P to gluteal is viewed via HPLC (Agilent 1100 series), using an Agilent Hi-Plex H-column Refractive Index Detector. Sample and control are run in 5 mM H2SO4 at 0.6 mUmin and 65°C.

[0576] Example 80

[0577] To verify the production of gulose from maltodextrin, a reaction mixture was incubated with 0.20 mL containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline with pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0 05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP at 50°C for 24 hours. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Gulose was verified via HPLC, as described in example 79.

[0578] Example 81

[0579] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCk, and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This is used to create another reaction mixture containing 20 g / L isoamylase treated meltodextrin, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCk, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U cao / nn / eznz / E / YiAi Gul6PI, and 0.05 U Gul6PP. The production of gluttony was verified in example 79.

[0580] Example 82

[0581] To further increase glutose gains from maltodextrin, 0.05 U 4-glucan transferase (4GT) was added to the reaction described in example 80.

[0582] A 0.2 mL reaction mixture was incubated, containing 20 g / L isoamylase treated maltodextrin (see Example 80), 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl·, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, 0.05 U Gul6PP, and 0.05 U 4GT at 50°C for 24 hours. The production of gluttony is verified in example 79.

[0583] Example 83

[0584] To further increase the glutose gains from maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in example 80.

[0585] Example 84

[0586] To further increase glutose gains from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM of polyphosphate were added to the reaction described in example 80.

[0587] Example 85

[0588] To produce gulose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCb, 0.05 was incubated at 50°C for 24 hours. Glucose production was quantified in example 79.

[0589] Example 86

[0590] To produce gulose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgClz, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, was incubated. 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP at 50°C for 24 hours. Glucose production was quantified in example 79. [05911 Example 87

[0592] To produce gulose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCh, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U was incubated PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP at 50°C for 24 hours. Glucose production was quantified in example 79.

[0593] Example 88

[0594] To increase the gains of goulose from sucrose, 75 mM polyphosphate and 0.05 fructokinase polyphosphate were added to the reaction mixture in example 86. The production of goulose was quantified in example 79.

[0595] Idosa

[0596] Example 89

[0597] To verify the production of idose from F6P, 10 g / L F6P was mixed with 1 U / mL F6PE, 1 U / mL S6PE, 1 U / mL idose 6-phosphate isomerase (I6PI), and 1 U / mL idose 6-phosphate phosphatase (I6PP) (I6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCk. The reaction is incubated for 3 hours at 50°C. The conversion of F6P to idose is viewed via HPLC (Agilent 1100 series), using an Agilent Hi-Plex H-column Refractive Index Detector. Sample and control are run in 5 mM H2SO4 at 0.6 mL / min and 65°C. cao / nn / eznz / E / YiAi

[0598] Example 90

[0599] To verify the production ofidose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCk, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP at 50°C for 24 hours. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Idosa was verified via HPLC, as described in example 89. [06001 Example 91

[0601] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCh, and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This was used to create another reaction mixture containing 20 g / L maltodextrin isoamylase treated, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 LJ I6PI, and 0.05 U I6PP at 50°C for 24 hours. The production ofidosa was verified in example 89. [06021 Example 92

[0603] To further increase the gains of idose from maltodextrin, added 0.05 U 4-glucan transferase (4GT) to the reaction described in Example 90.

[0604] A 0.2 mL reaction mixture was incubated containing 20 g / L maltodextrin isoamylase treated (see Example 90), 50 mM phosphate buffered saline pH 7.2, 5 mM MgCb, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, 0.05 U I6PP, and 0.05 U 4GT at 50°C for 24 hours. The production ofidosa was verified in example 89.

[0605] Example 93

[0606] To further increase the gains of idose from maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in Example 90.

[0607] Example 94

[0608] To further increase the efficiency of idose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM of polyphosphate were added to the reaction described in Example 90.

[0609] Example 95

[0610] To produce idose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, was incubated. 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP at 50°C for 24 hours. Idosa production was quantified as in Example 89. [06111 Example 96

[0612] To produce idose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphatekinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP at 50°C for 24 hours. Idosa production was quantified as in Example 89.

[0613] Example 97

[0614] To produce idose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U was incubated PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP at 50°C for 24 hours. The production CQO / nn / eznz / E / YiAi of idose was quantified as in Example 89.

[0615] Example 98

[0616] To further increase the gains of idose from sucrose, 75 mM polyphosphate and 0.05 polyphosphate fructokinase were added to the reaction mixture in Example 96. The production of idose was quantified as in Example 89.

[0617] Tagatose

[0618] Example 99

[0619] To verify the production of tagatose from F6P, 2 g / L F6P was mixed with 1 U / ml fructose 6-phosphate epimerase (F6PE) and 1 U / ml tagatose 6-phosphate phosphatase (T6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated for 16 hours at 50°C. 100% conversion of F6P to tagatose can be observed via HPLC (Agilent 1100 series), using a refractive index detector and an Agilent Hi-Plex H-column. The sample was run in 5 mM H2SO4 at 0.6 mUrnin.

[0620] Example 100

[0621] To verify the production of tagatose from maltodextrin, a reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PP at 50°C for 24 hours. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Tagatose was detected and quantified using an Agilent 1100 series HPLC with refractive index detector and an Agilent HiPlex H-column. The mobile phase was 5 mM H2SO4, which ran at 0.6 mL / min. A gain of 9.2 g / L of tagatose was obtained. This is equivalent to 92% of the theoretical gain, due to the limits of maltodextrin degradation without enzymes, such as isomamylase or 4-glucan transferase. Standards of various concentrations of tagatose were used to quantify our gain.

[0622] Example 101

[0623] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This was used to create another reaction mixture that 20 g / L maltodextrin isoamylase treated, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PP were incubated at 50°C for 24 hours. Tagatose production was quantified as in Example 99. Tagatose gain increased to 16 g / L with maltodextrin pretreatment by isoamylase. This is equivalent to 80% of the theoretical profit. [06241 Example 102

[0625] To further increase the gains of tagatose from maltodextrin, 0.05 U 4-glucan transferase (4GT) was added to the reaction described in Example 100.

[0626] A 0.2 mL reaction mixture was incubated containing 20 g / L maltodextrin isoamylase treated (see example 9), 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U T6PP, and 0.05 U 4GT at 50°C for 24 hours. The production of tagatose was quantified as in example 9. The gain of tagatose increased to 17.7 g / L when 4GT was added to the IA treated maltodextrin. This is equivalent to 88.5% of the theoretical profit. [06271 Example 103 CQO / nn / eznz / E / YiAi

[0628] To investigate on a large scale, a 20 mL reaction mixture containing 50 g / L maltodextrin isoamylase treated (see Example 99), 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 10 was incubated U of aGP, 10 U PGM, 10 U PGI, 10 U F6PE, and 10 U T6PP at 50°C for 24 hours. The production of tagatose was quantified as in example 8. The gain of tagatose was 37.6 g / L on the 20 mL scale and 50 g / L of maltodextrin. This is equivalent to 75% of the theoretical profit. These results indicate that scaling for longer reaction volumes will not result in significant losses in efficiency. [06291 Example 104

[0630] To further increase the efficiency of tagatose from maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in Example 100. [06311 Example 105

[0632] To further increase the gains of tagatose from maltodextrin, 0.05 LJ of polyphysiphate glucokinase and 75 mM of polyphosphate were added to the reaction described in example 99.

[0633] Example 106

[0634] To produce tagatose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphatekinase, 0.05 U was incubated. U F6PE, and 0.05 U T6PP at 50°C for 24 hours. Tagatose production was quantified as in Example 100.

[0635] Example 107

[0636] To produce tagatose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphatekinase, 0.05 U was incubated. U PGI, 0.05 U F6PE, and 0.05 U T6PP at 50°C for 24 hours. Tagatose production was quantified as in example 100. [06371 Example 108

[0638] To produce tagatose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PP at 50°C for 24 hours. Tagatose production was quantified as in Example 100.

[0639] Example 109

[0640] To further increase the efficiency of tagatose from sucrose, mM polyphosphate and 0.05 polyphosphate fructokinase were added to the reaction mixture in Example 15. The production of tagatose was quantified as in Example 100.

[0641] Psycho

[0642] Example 110

[0643] To verify the psychose production of F6P, 2 g / L F6P was mixed with 1 U / ml P6PE and 1 U / ml P6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl and 80 μΜ C0CI2. The reaction was incubated for 6 hours at 50°C. 99% of the conversion of F6P to psychose was seen via HPLC (Agilent 1100 series), using an Agilent Hi-Plex H-column and a refractive index detector. The sample was run in 5 mM H2SO4 at 0.6 mL / min. CQO / nn / eznz / E / YiAi

[0644] Example 111

[0645] To verify the production of psychose from maltodextrin, a reaction mixture containing 0.20 mL 20 g / L maltodextrin, 50 mM phosphate buffered saline pH 7.2, 5 mM MgCl2, 80 μΜ was incubated. CoCI2,0.05 U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE and 0.05 U P6PP at 50°C for 24 hours. The reaction was stopped via enzyme filtration with a Vivaspin 2 concentrator (10,000 MWCO). Psychose was detected and quantified using a Agilent 1100 series HPLC with refractive index detector and an Agilent Hi-Plex H-column. The mobile phase is 5 mM H2SO4, which is run at 0.6 mL / min. Standards of various concentrations of psychose were used to quantify our gain.

[0646] Example 112

[0647] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCE, 80 μΜ C0CI2, and 0.1 g / L isoamylase was incubated at 80°C for 24 hours. This is used to create another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP at 50°C for 24 hours. Psychose production was quantified as in Example 111.

[0648] Example 113

[0649] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 4.5), 5 mM MgCb, and 1:200 dilution of Novozymes D6 pullulunase was incubated at 50°C for 4 hours. . This is used to create another reaction mixture containing 20 g / L pullulanase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCh, 80 μΜ CoCL, 0.05 U aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP at 50°C for 24 hours. Psychose production was quantified as in Example 111.

[0650] Example 114

[0651] To further increase the gains of psychose from maltodextrin, 0.05 U 4-glucan transferase (4GT) was added to the reaction described in Example 111.

[0652] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see example 9), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl, 80 μΜ CoCL, 0.05 was incubated. U of aGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U P6PP, and 0.05 U 4GT at 50°C for 24 hours. Psychose production was quantified as in Example 111.

[0653] Example 115

[0654] To investigate at scale, a 20 mL reaction mixture was incubated, containing 50 g / L isoamylase-treated maltodextrin (see Example 10), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCh , 80 μΜ CoCL, 10 U of aGP, 10 U PGM, 10 U PGI, 10 U P6PE, and 10 U P6PP at 50°C for 24 hours. Psychose production was quantified as in Example 111.

[0655] Example 116

[0656] To further increase the gains of psychose from maltodextrin, 0.05 U of maltose phosphorylase was added to the reaction described in Example 110.

[0657] Example 117

[0658] To further increase the gains of psychose from maltodextrin, 0.05 U of cao / nn / eznz / E / YiAi polyphosphate glucokinase and 75 mM of polyphosphate were added to the reaction described in Example 111.

[0659] Example 118

[0660] To produce psychose from fructose, a reaction mixture containing g / L fructose, 50 mM Tris buffer with pH 7.0, 75 mM polyphosphate, 5 mM MgCl2, 80 μΜ CoCI2, 0.05 U fructose was incubated. polyphosphate kinase, 0.05 U P6PE, and 0.05 U P6PP at 50°C for 24 hours. Psychose production was quantified as in Example 111. [06611 Example 119

[0662] To produce psychose from glucose, a reaction mixture containing g / L glucose, 50 mM Tris buffer with pH 7.0, 75 mM polyphosphate, 5 mM MgClz, 80 μΜ CoClz, 0.05 U was incubated. of glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP at 50°C for 24 hours. Psychose production was quantified as in Example 111.

[0663] Example 120

[0664] To produce psychose from sucrose, a reaction mixture containing g / L sucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl2, 80 μΜ CoCI2, 0.05 U sucrose phosphorylase was incubated. , 0.05 PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP at 50°C for 24 hours. Psychose production was quantified as in Example 111.

[0665] Example 121

[0666] To further increase the gains of psychose from sucrose, 75 mM polyphosphate and 0.05 polyphosphate fructokinase were added to the reaction mixture in Example 20. The production of psychose was quantified as in Example 111.

[0667] The invention includes all substantial representations and variations, as described above and with reference to the examples and figures. Although various embodiments of the invention are set forth herein, adaptations and modifications may be made within the scope of the invention, in accordance with the general knowledge of those skilled in the art.

Claims

1. A process for preparing a hexose from a saccharide, the process including: converting fructose 6-phosphate (F6P) into a hexose catalyzed by one or more enzymes, wherein the hexose is selected from the group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose and idose; and wherein the enzymes are selected from the group consisting of a hexose-specific isomerase, epimerase, and phosphatase, and mixtures thereof.

2. The process according to claim 1, wherein the hexose is allose, and the process includes: converting F6P into psicose 6-phosphate (P6P) catalyzed by psicose 6-phosphate 3-epimerase (P6PE); converting P6P into allose 6-phosphate (A6P) catalyzed by allose 6-phosphate isomerase (A6PI); and converting A6P into allose catalyzed by allose 6-phosphate phosphatase (A6PP).

3. The process according to claim 1, wherein the hexose is mannose, and the process includes: converting F6P into mannose 6-phosphate (M6P) catalyzed by mannose 6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI); and converting M6P into mannose catalyzed by mannose 6-phosphate phosphatase (M6PP).

4. The process according to claim 1, wherein the hexose is galactose, and the process includes: converting F6P into tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE); converting T6P into galactose 6-phosphate (Gal6P) catalyzed by galactose 6-phosphate isomerase (GalGPI); and converting Gal6P into galactose catalyzed by galactose 6-phosphate phosphatase (Gal6PP).

5. The process according to claim 1, wherein the hexose is fructose, and the process includes: converting F6P to fructose catalyzed by fructose 6-phosphate phosphatase (F6PP).

6. The process according to claim 1, wherein the hexose is altrose and the process includes: converting F6P into psicose 6-phosphate (P6P) catalyzed by psicose 6-phosphate 3-epimerase (P6PE); converting P6P into altrose 6-phosphate (Alt6P) catalyzed by altrose 6-phosphate isomerase (Alt6PI); and converting the Alt6P produced into altrose catalyzed by altrose 6-phosphate phosphatase (Alt6PP).

7. The process according to claim 1, wherein the hexose is talose, and the process includes: converting F6P to tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE); converting T6P to talose 6-phosphate (Tal6P) catalyzed by talose 6-phosphate isomerase (Tal6PI); and converting Tal6P to talose catalyzed by talose 6-phosphate phosphatase (Tal6PP). CQQ / nn / Q7n7 / e / YiAi 8. The process according to claim 1, wherein the hexose is talose, and the process includes: converting F6P into tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE); converting T6P into sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE); and converting S6P into sorbose catalyzed by sorbose 6-phosphate phosphatase (S6PP).

9. The process according to claim 1, wherein the hexose is gulose, and the process includes: converting F6P into tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE); converting T6P into sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE); converting S6P into gulose 6-phosphate (Gul6P) catalyzed by gulose 6-phosphate isomerase (Gul6PI); and converting Gul6P into gulose catalyzed by gulose 6-phosphate phosphatase (Gul6PP).

10. The process according to claim 1, wherein the hexose is idose, and the process includes: converting F6P into tagatose 6-phosphate (T6P) catalyzed by fructose 6-phosphate 4-epimerase (F6PE); converting T6P into sorbose 6-phosphate (S6P) catalyzed by sorbose 6-phosphate epimerase (S6PE); converting S6P into idose 6-phosphate (I6P) catalyzed by idose 6-phosphate isomerase (I6PI); and converting I6P into idose catalyzed by idose 6-phosphate phosphatase (I6PP).

11. The process of any of claims 1 to 10 further includes the step of converting glucose 6-phosphate (G6P) to F6P, wherein the step is catalyzed by phosphoglucose isomerase (PGI).

12. The process of claim 11 further includes the step of converting glucose 1-phosphate (G1P) into G6P, wherein the step is catalyzed by phosphoglucomutase (PGM).

13. The process of claim 12 further includes the step of converting a saccharide into G1P, wherein the step is catalyzed by at least one enzyme, wherein the saccharide is selected from a group consisting of a starch or starch derivative, and sucrose.

14. The process of claim 13, wherein at least one enzyme in the step of converting a saccharide into G1P is selected from a group consisting of alpha-glucan phosphorylase (aGP), maltose phosphorylase, and sucrose phosphorylase, and mixtures thereof.

15. The process of claims 13 and 14, wherein the saccharide is starch or a derivative thereof, and is selected from a group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose, and mixtures thereof.

16. The process of claim 15 further includes the step of converting starch into a starch derivative, wherein this derivative is prepared by enzymatic hydrolysis of starch or by acid hydrolysis of starch. CQQ / nn / Q7n7 / e / YiAi 17. The process of claim 15 or 16, wherein 4-glucan transferase (4GT) is added to the process.

18. The process of any of claims 13 to 17, wherein the starch derivative is prepared by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulunase, alpha-amylase, or a combination thereof.

19. The process of claim 2, wherein the A6PI includes an amino acid sequence having at least 55% sequence identity with SEQ ID Nos: 1 or 2, and wherein said A6PI catalyzes the conversion of P6P to A6P.

20. The process of claim 19, wherein the A6PI contains a Rossman fold for catalysis, having a His C-terminal for the first β-strain of the Rossman fold; an Arg C-terminal for the aterminal-C helix for the fifth β-strain of the Rossman fold; a His at the active site; a Cys; a Thr at the active site; a distinctive hydrophobic-G-G GTG near the active site; and an Asn near the active site.

21. The process of any of claims 2 and 19 and 20, wherein the A6PP includes an amino acid sequence having at least 30% sequence identity with any of SEQ ID Nos: 3-7, and wherein said A6PP catalyzes the conversion of A6P to allose.

22. The process of claim 21, wherein the A6PP contains a domain in the Rossman fold, a C1-limited domain, a characteristic DxD in the first β- Rossman fold strain, a Thr or Ser at the end of the second β- Rossman fold strain, a Lys at the N-terminus of the terminal α-helix C for the third β- Rossman fold strain, and a characteristic ED at the end of the fourth Rossman fold strain.

23. The process of claim 3, wherein M6PI includes an amino acid sequence having at least 25% sequence identity with any of SEQ ID Nos: 8-11, and wherein said M6PI catalyzes the conversion of F6P to M6P.

24. The process of claim 23, wherein the M6PI contains two domains with a core of antiparallel β- strains resembling the cupin fold and a third domain consisting only of α- helices, and a divalent metal cation.

25. The process of any of claims 3, 23, or 24, wherein the PGPMI includes an amino acid sequence having at least 25% sequence identity with any of SEQ ID Nos. 15-17, and wherein said PGPMI catalyzes the conversion of F6P to M6P. cao / nn / eznz / E / YiAi 26. The process of claim 25, wherein the PGPMI contains two Rossman folds, a distinctive GGS, a distinctive hydrophobic XTX-ET-, and a distinctive EN where Glu is present for active site proton transfer, and a distinctive HN where HIS is present for substrate ring opening / closing during catalysis.

27. The process of any of claims 3, and 23 to 26, wherein the M6PP includes an amino acid sequence having at least 30% sequence identity with any of the SEQ ID Nos: 12-14, and wherein said M6PP catalyzes the conversion of M6P into mannose.

28. The process of claim 27, wherein the M6PP contains a domain in the Rossman fold for catalysis, a limited domain 01, a characteristic DxD in the first β- Rossman fold strain, a Thr or a Ser at the end of the second β- Rossman fold strain, and a characteristic GDxxxD at the end of the fourth β- Rossman fold strain.

29. The process of claim 5, wherein F6PP includes an amino acid sequence having at least 25% sequence identity with SEQ ID NO: 21, and wherein said F6PP catalyzes the conversion of F6P to fructose.

30. The process of claim 33, wherein the F6PP contains a catalytic domain in the Rossman fold, a C1-bound domain, a characteristic DxD in the first β- Rossman fold strain, a Thr or a Ser at the end of the second Rossman fold strain, a Lys at the N end of the a helix of the C-terminal for the third Rossman fold strain, and a characteristic ED at the end of the fourth β- Rossman fold strain.

31. The process of claim 4, wherein Gal6PI is a multimer of two subunits Lac A and Lac B, wherein said Lac A includes an amino acid sequence having at least 25% sequence identity with SEQ ID No: 18; and wherein said Lac B includes an amino acid sequence having at least 25% sequence identity with SEQ ID No: 19, and wherein Gal6PI catalyzes the conversion of T6P to Gal6P.

32. The process of claim 31, wherein the Gal6PI contains a heterodimer ('A' and B') consisting of sandwich-type Rossman fold subunits αβα, Arg130 and Arg134 in I' and its His9 and Arg39 in B' for binding the substrate phosphate group, His96 in A' for substrate ring opening, Asn97 in A' for stabilizing high-energy intermediates, and Cys65 and Thr67 of B' for participating in proton transfer.

33. The process of any of claims 4, 31, and 32, wherein Gal6PP includes an amino acid sequence having at least 25% sequence identity with SEQ ID NO: 20, and wherein said Gal6PP catalyzes the conversion of Gal6P to galactose. cao / nn / eznz / E / YiAi 34. The process of claim 33, wherein Gal6PP contains a domain in the Rossman fold for catalysis, a C2-limited domain, a characteristic DxD in the first β- Rossman fold strain, a Thr or Ser at the end of the second Rossman fold strain, and a characteristic GDxxxD at the end of the fourth β- Rossman fold strain.

35. The process of claim 14, wherein the hexose is fructose, the saccharide is sucrose, and wherein at least one enzyme is sucrose phosphorylase.

36. The process of any of claims 3 and 23 to 28 further includes the conversion of G6P to F6P catalyzed by PGPMI, and wherein F6P is converted to M6P by PGPMI.

37. The process of any of claims 1 to 36, wherein the process steps are performed at a temperature ranging from approximately 40°C to approximately 70°C, at a pH ranging from approximately 5.0 to approximately 8.0, and / or for approximately 8 to 48 hours.

38. The process of any of claims 1 to 37, wherein the process steps are carried out in a single bioreactor or in a variety of bioreactors arranged in series.

39. The process of any of claims 1 to 38, wherein the process steps are carried out without ATP, without NAD(P)(H), at a phosphate concentration of approximately 0.1 mM to 150 mM, the phosphate is recycled, and / or at least one process step involves an energetically favorable chemical reaction.

40. A hexose prepared by the process of any one of claims 1 to 39, wherein the hexose is selected from a group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, and idose.