Enzymatic method for synthesizing D-psicose and derivatives thereof from fusion enzyme

Abstract

The invention belongs to the field of enzyme catalysis, and particularly relates to application of a multi-enzyme system / fusion protein in production of D-psicose, a production method of a fusion enzyme and application of the fusion enzyme in production of D-psicose and derivatives thereof from a carbohydrate source. More specifically, the present invention provides a simple, improved and cost-effective method for the production of D-psicose and derivatives thereof from carbohydrate sources from such fusion enzymes.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme catalysis, and in particular relates to use of a fusion enzyme system in the production of D-psicose, a production method of the fusion enzyme, and application of the fusion enzyme in the production of D-psicose. Background Art

[0002] Allulose, also known as D-psicose, is a low-calorie natural sweetener with 70% of the sweetness of sucrose, but only 10% of the calories. Allulose is a naturally occurring monosaccharide that exists in small quantities in wheat and other plants, so it is also classified as a "rare sugar." Rare sugars are a class of monosaccharides and their derivatives that are extremely rare in nature (according to the definition of the International Rare Sugars Society (ISRS) in 2002). In recent years, D-psicose, as a diastereomer of fructose, has attracted widespread attention in the fields of diet, health care, and medicine. The sweetness of D-psicose is 70% of sucrose, but its energy value is only 0.007 kcal / g, and its energy deposition efficiency is only 0.3% of sucrose. Therefore, D-psicose is an ideal low-calorie sweetener that can be used as a sucrose substitute in food applications; D-psicose has been shown to have anti-hypoglycemic effects and can inhibit the activity of liver lipase and intestinal α-glucosidase, thereby reducing abdominal fat deposition. It also has high medical value in the treatment of neurodegenerative diseases and atherosclerotic diseases. D-psicose has many health benefits: close to zero calories; very low glycemic index of only 1; helps regulate blood sugar, etc.

[0003] Traditionally, D-psicose is produced from fructose using D-psicose-3-epimerase as a catalyst. This enzymatic conversion has a low conversion rate of only 32%, and the process requires a simulated moving bed chromatography system to recycle fructose, which increases production costs.

[0004] U.S. Patent 20210254031A1 describes a process for producing D-psicose from starch, sucrose or other carbohydrates using recombinant cells or a multi-enzyme cascade system. The process requires the use of multiple enzymes in a single reactor to convert sucrose into D-psicose. One of the main disadvantages is the use of glucose isomerase to convert fructose into glucose. The efficiency of this conversion step is reportedly only 30%, so separating glucose from the reaction mixture is essential to maintain the forward reaction. In addition, multiple enzymes with different optimal conditions are used in a single reactor, causing some conversions to occur under suboptimal conditions.

[0005] U.S. Patent 11053528B2 discloses a process for producing fructose-6-phosphate from sucrose using multiple enzymes such as sucrose phosphorylase, phosphoglucomutase, glucose phosphomimerase and glucose isomerase. In this process, the efficiency of fructose conversion to glucose is not high, and the conversion efficiency is reported to be only 30%. The process needs to be highly standardized to achieve economical and optimal conversion.

[0006] Therefore, an alternative, cost-effective method is needed to produce psicose in high yield. Ideally, the pathway should incorporate an aggressively favorable chemical reaction in at least one of the steps. In addition, a production process that can be carried out in a single reactor or bioreactor is needed. In addition, a production method that can operate at relatively low phosphate concentrations and has the potential for phosphate recycling would be beneficial. In addition, a psicose production pathway that does not rely on the expensive nicotinamide adenine dinucleotide (NAD(H)) coenzyme in any reaction step would also be advantageous.

[0007] Therefore, the inventors of the present invention have developed a new process for producing D-psicose and its derivatives, which is simple, cost-effective and has a high conversion rate, solving all the problems and limitations in the prior art. Summary of the invention

[0008] The present invention relates to a process for producing D-psicose and its derivatives from sucrose or fructose or glucose derived from starch and / or cellulose. The present invention provides a simple process for producing D-psicose, fructose, allose and allol, wherein the process overcomes the equilibrium limitation of conventional isomerization and epimerization processes and achieves higher conversion efficiency.

[0009] In addition, the present invention also relates to fusion proteins expressing one or more enzymes to convert a carbohydrate source into D-psicose, and processes for producing such fusion proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings illustrate some embodiments of the present invention and are used together with the description to explain the present invention. These drawings are for illustration only and are not limiting. The components in the accompanying drawings are not necessarily drawn to scale, and the emphasis is on clearly illustrating the principles of various aspects of the embodiments.

[0011] FIG. 1 shows a schematic diagram of a process for producing D-psicose and its derivatives from sucrose or fructose or glucose derived from starch and / or cellulose.

[0012] Figure 2 The results of SDS-PAGE analysis of fructokinase are shown.

[0013] Figure 3Shown are the results of SDS-PAGE analysis of psicose-6-phosphate epimerase.

[0014] Figure 4 Shown are the results of SDS-PAGE analysis of psicose-6-phosphate phosphatase.

[0015] Figure 5 Shown are the results of SDS-PAGE analysis of the Psicose-6-phosphate epimerase-Psicose-6-phosphate phosphatase fusion protein. DETAILED DESCRIPTION

[0016] At the beginning of the detailed description, it is understood that the following description only illustrates a specific form of the present invention. However, this specific form is only an exemplary embodiment and is not intended to limit the scope of the present invention in any way. Therefore, this description should be understood as an exemplary embodiment and teaching of the present invention, rather than a restrictive interpretation.

[0017] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In addition, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include the singular form.

[0018] The present invention provides an enzymatic pathway or process for synthesizing psicose with high yield, while greatly reducing the product separation cost and the psicose production cost.

[0019] The inventors of the present invention have developed a novel, improved and simple process for producing D-psicose and its derivatives from carbohydrate sources such as sucrose or fructose or glucose derived from starch and / or cellulose. The inventors of the present invention have designed a simple process for producing D-psicose, fructose, allose and allol, wherein the process overcomes the equilibrium limitations of conventional isomerization and epimerization processes and achieves higher conversion efficiency.

[0020] Compared with conventional processes, the process of the present invention avoids the use of a large amount of enzymes required for producing D-psicose from fructose, and the process does not require the use of expensive ATP and a complex ATP regeneration system. In particular, a variety of complex and expensive separation systems for sugars and other intermediates are avoided. The process of the present invention involves the use of polyphosphate as a phosphate donor to replace ATP or ADP in the body. In order to use polyphosphate as the only phosphate donor, the present invention includes modifying glucokinase or fructokinase to increase its affinity for polyphosphate rather than ATP or ADP.

[0021] The process of the present invention shows a higher conversion rate of raw materials into D-fructose. The raw materials used in the process of the present invention are selected from carbohydrates / polysaccharides, which can be selected from, but not limited to, starch or its derivatives, cellulose or its derivatives, and sucrose. Examples of starch derivatives include amylose, amylopectin, soluble starch, amylopectin, maltodextrin, maltose, and glucose. In certain embodiments of the present invention, the process for preparing psicose involves using an enzyme, such as glucan phosphorylase, amylopectinase, amylase, or a combination of these enzymes, to convert starch into a starch derivative.

[0022] The present invention also provides a method for modifying an enzyme by a gene editing method to improve the thermostability, catalytic activity and substrate affinity of the enzyme, wherein the gene editing method includes but is not limited to point mutation, domain exchange, truncation and amino acid mutation. These modifications are based on bioinformatics analysis of genes and proteins and simulation studies.

[0023] A major aspect of the present invention is to develop fusion proteins expressing one or more enzymes for converting carbohydrates to D-psicose. Preparation of such fusion proteins includes culturing cells engineered to express at least one α-glucan phosphorylase and / or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one phosphogluconate isomerase, at least one psicose-6-phosphate epimerase, at least one psicose-6-phosphate phosphatase, or a combination of at least two (e.g., at least three or at least four) of the above enzymes.

[0024] The fusion proteins prepared by the present invention can be produced by connecting two or more genes or gene fragments encoding different proteins. The translation of the fusion gene produces a single or multiple polypeptides, and its functional properties are derived from each original protein. Multifunctional proteins are single proteins with at least two different activities, wherein the function is the result of the fusion of natural biological functions or engineered enzymes. Other enzymes can also be expressed as single fusion proteins or multifunctional proteins. Therefore, fusion proteins can contain multiple functions of any pathway enzyme described herein.

[0025] Furthermore, the fusion enzyme of the present invention is generated during the cloning / subcloning process of a gene construct, two genes are linked using a fixed linker or a rigid linker, and expressed as a fusion protein under the control of a single promoter.

[0026] Embodiment of the present invention:

[0027] Therefore, one embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein the method comprises the following steps:

[0028] a. converting a carbohydrate source into a mixture of glucose and fructose in the presence of an extracellular enzyme selected from an invertase, an amylase and / or a pullulanase;

[0029] b. Optionally, converting glucose in the mixture of step (a) into fructose by enzymatic conversion in the presence of an isomerase;

[0030] c. converting the fructose obtained in step (b) into fructose-6-phosphate in the presence of fructokinase;

[0031] d. converting the fructose-6-phosphate produced in step (c) into psicose-6-phosphate in the presence of psicose-6-phosphate 3-epimerase;

[0032] e. converting the psicose-6-phosphate obtained in step (d) into D-psicose in the presence of psicose-6-phosphate phosphatase;

[0033] Among them, fructokinase, psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase are expressed as part of a single fusion protein.

[0034] One embodiment of the present invention is to provide an improved and simple method for producing D-psicose and its derivatives, wherein the method comprises the following steps:

[0035] a. converting a carbohydrate source into glucose using an extracellular enzyme selected from an invertase, an amylase and / or a pullulanase;

[0036] b. converting the glucose produced in step (a) into glucose-6-phosphate by glucokinase in the presence of polyphosphate;

[0037] c. converting the glucose-6-phosphate produced in step (b) into fructose-6-phosphate in the presence of phosphoglucose isomerase;

[0038] d. converting the fructose-6-phosphate produced in step (c) into psicose-6-phosphate in the presence of psicose-6-phosphate 3-epimerase;

[0039] e. converting the psicose-6-phosphate obtained in step (d) into D-psicose in the presence of psicose-6-phosphate phosphatase;

[0040] Among them, psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase are expressed as part of a single fusion protein.

[0041] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives from a carbohydrate source, wherein the method comprises the following steps:

[0042] a. converting a carbohydrate source into glucose-1-phosphate in the presence of glycogen / glucan phosphorylase;

[0043] b. converting the glucose-1-phosphate obtained in step (a) into glucose-6-phosphate in the presence of phosphoglucomutase;

[0044] c. converting the glucose-6-phosphate produced in step (b) into fructose-6-phosphate in the presence of phosphoglucose isomerase;

[0045] d. converting the fructose-6-phosphate produced in step (c) into psicose-6-phosphate in the presence of psicose-6-phosphate 3-epimerase;

[0046] e. converting the psicose-6-phosphate obtained in step (d) into D-psicose in the presence of psicose-6-phosphate phosphatase;

[0047] Among them, psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase are expressed as part of a single fusion protein.

[0048] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein the carbohydrate source is selected from sucrose, fructose, or glucose derived from starch and / or cellulose.

[0049] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein the conversion efficiency is between 75% and 95%.

[0050] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein a carbohydrate source is further subjected to acid hydrolysis after enzyme conversion.

[0051] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein a carbohydrate source is further passed through a strong cation exchange resin after being enzymatically converted.

[0052] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein the fusion enzyme is introduced into the reaction mixture in a free form, an immobilized form, or a combination thereof.

[0053] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein the enzymatic conversion is performed in the presence of inorganic polyphosphate or ATP.

[0054] In another important embodiment of the present invention, the derivative of D-psicose is selected from the group consisting of D-allose and allol.

[0055] Another important embodiment of the present invention is to provide a method for producing D-allose, wherein the method comprises converting the obtained D-psicose into D-allose using ribose-5-phosphate isomerase and / or L-rhamnose isomerase.

[0056] Still another important embodiment of the present invention is to provide a method for producing allol, wherein the method comprises converting the obtained D-psicose into allol using ribitol dehydrogenase.

[0057] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein the enzyme is used in a free form, an immobilized form, or a combination thereof.

[0058] One embodiment of the present invention is to provide a method for producing D-psicose and its derivatives, wherein a single enzyme or a multi-enzyme complex having multiple activities is used to produce D-allose or allol.

[0059] Another important embodiment of the present invention is to provide a method for preparing a fusion enzyme, the method comprising the following steps:

[0060] a. preparing a recombinant cell for expressing a recombinant fusion protein encoding fructokinase, psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase;

[0061] b. The recombinant cells were cultured at 37°C and then induced to express the fusion protein at different concentrations of IPTG and a temperature of 20 to 30°C;

[0062] c. Extract and isolate the fusion protein from the culture by cell lysis and separation of the soluble and insoluble fractions.

[0063] Another important embodiment of the present invention is to provide D-psicose and its derivatives obtained by the method of the present invention.

[0064] Another important embodiment of the present invention is to provide D-psicose and its derivatives obtained by the method of the present invention for use as sugar substitutes.

[0065] Advantages

[0066] 1. The process for producing D-psicose and its derivatives is simple.

[0067] 2. The amount of enzyme required to produce D-psicose from fructose is reduced.

[0068] 3. The use of fusion enzymes further reduces the number of process steps, making the process faster than traditional processes.

[0069] 4. The process does not require the use of expensive ATP and complex ATP regeneration systems.

[0070] 5. Avoids the need for complex and expensive simulated moving bed chromatography separation systems.

[0071] 6. Since the reaction equilibrium is overcome, the conversion rate of the raw material into D-psicose is higher.

[0072] 7. The use of a packed bed system for enzymatic conversion avoids protein contamination of the final product.

[0073] 8. The reaction rate of the packed system is high, so the reactor size is small, reducing capital expenditure requirements.

[0074] 9. The multi-step process ensures that each enzyme used in the process works at its optimal level.

[0075] 10. Immobilized enzymes have higher stability, thus minimizing the need for frequent replenishment, which helps reduce operating costs.

[0076] Without in any way limiting the scope of the invention as described above, the invention is further explained by the examples provided below.

[0077] Experimental data:

[0078] Example 1: Expression of fructokinase, psicose-6-phosphate epimerase, and psicose-6-phosphate phosphatase as a single fusion enzyme.

[0079] Enzymes for converting fructose to psicose, including fructokinase, psicose-6-phosphate epimerase, and psicose-6-phosphate phosphatase, were expressed as recombinant proteins from Escherichia coli BL21 cells. Protein expression was controlled by IPTG induction (T7lac promoter). Protein expression was induced at different concentrations of IPTG and 25°C (cultures were grown at 37°C).

[0080] Protein expression in the culture was analyzed by cell lysis and separation of soluble and insoluble fractions. All fractions, including crude lysate (C), soluble fraction (S) and precipitate / insoluble fraction (P), were compared with protein molecular weight standards / ladders (L) and uninduced culture lysate as a negative control (U). Samples were analyzed by 12% polyacrylamide gel electrophoresis under standard conditions.

[0081] Figure 2 , 3and 4 represent the SDS-PAGE analysis results of fructokinase, psicose-6-phosphate epimerase, and psicose-6-phosphate phosphatase, respectively.

[0082] Example 2: Expression of Psicose-6-phosphate epimerase and Psicose-6-phosphate phosphatase as a single fusion enzyme.

[0083] Enzymes for converting fructose to psicose, including psicose-6-phosphate epimerase and psicose-6-phosphate phosphatase, were expressed as recombinant proteins from Escherichia coli BL21 cells as fusion proteins under the control of a single promoter (T7lac promoter) induced by IPTG. The two proteins were connected by a flexible linker to achieve fusion expression of the two. Fusion protein expression was induced at different concentrations of IPTG and 25°C (cultures were grown at 37°C).

[0084] The fusion protein expression in the culture was analyzed by cell lysis and separation of soluble and insoluble fractions. All fractions from different colonies (colony A and B), including crude lysate (C), soluble fraction (S) and precipitate / insoluble fraction (P), were compared with protein molecular weight standards / ladder bands (L) for analysis. Samples were analyzed by 12% polyacrylamide gel electrophoresis under standard conditions.

[0085] Figure 5 Representative results of SDS-PAGE analysis of the Psicose-6-phosphate epimerase and Psicose-6-phosphate phosphatase fusion protein.

[0086] Embodiment 3: D-psicose is produced from a carbohydrate source by using a fusion protein expressing fructokinase, psicose-6-phosphate 3-epimerase, and psicose-6-phosphate phosphatase.

[0087] In a reaction vessel, a carbohydrate source selected from starch or cellulose is converted into a mixture of glucose and fructose in the presence of an extracellular enzyme such as invertase. The obtained glucose is further enzymatically converted into fructose by a reaction catalyzed by glucose isomerase.

[0088] A fusion protein expressing fructokinase, psicose-6-phosphate 3-epimerase, and psicose-6-phosphate phosphatase is added to a reaction mixture containing fructose. In the presence of fructokinase, fructose is converted to fructose-6-phosphate, and then in the presence of psicose-6-phosphate 3-epimerase, fructose-6-phosphate is converted to psicose-6-phosphate. Finally, in the presence of psicose-6-phosphate phosphatase, psicose-6-phosphate is converted to D-psicose.

[0089] Embodiment 4:D-psicose is produced from a carbohydrate source by using a fusion protein expressing psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase.

[0090] In a reaction vessel, a carbohydrate source selected from starch or cellulose is converted into a mixture of glucose in the presence of an exoenzyme such as invertase. The obtained glucose is further enzymatically converted into glucose-6-phosphate by glucokinase in the presence of polyphosphate, and then converted into fructose-6-phosphate in the presence of phosphoglucose isomerase.

[0091] A fusion protein expressing psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase is added to a reaction mixture containing fructose-6-phosphate. In the presence of psicose-6-phosphate 3-epimerase, fructose-6-phosphate is converted to psicose-6-phosphate. Finally, in the presence of psicose-6-phosphate phosphatase, psicose-6-phosphate is converted to D-psicose.

[0092] Embodiment 5: D-psicose is produced from a carbohydrate source by using a fusion protein expressing psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase.

[0093] In a reaction vessel, a carbohydrate source selected from starch or cellulose is converted into a mixture of glucose-1-phosphate in the presence of glycogen / glucan phosphorylase. The glucose-1-phosphate obtained in step (a) is converted into glucose-6-phosphate in the presence of phosphoglucomutase and then into fructose-6-phosphate in the presence of phosphogluconate isomerase.

[0094] A fusion protein expressing psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase is added to a reaction mixture containing fructose-6-phosphate. In the presence of psicose-6-phosphate 3-epimerase, fructose-6-phosphate is converted to psicose-6-phosphate. Finally, in the presence of psicose-6-phosphate phosphatase, psicose-6-phosphate is converted to D-psicose.

[0095] Embodiment 6: Enzymatic synthesis of D-allose from D-psicose

[0096] In a reaction vessel, D-psicose obtained from any one of Examples 3 to 5 is further reacted using an enzyme selected from ribose-5-phosphate isomerase and / or L-rhamnose isomerase to be enzymatically converted into D-allose.

[0097] Embodiment 7: Enzymatic synthesis of allol from D-psicose

[0098] In a reaction vessel, D-psicose obtained from any one of Examples 3 to 5 is further reacted in the presence of ribitol dehydrogenase to be enzymatically converted into allol.

[0099] The foregoing summarizes the features and technical advantages of the present invention to better understand the detailed description of the present invention below. It should be understood by those skilled in the art that the disclosed concepts and specific embodiments can be easily used as a basis for modifying the disclosed methods or achieving the same goals of the present invention.

[0100] It must be noted that, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" used herein include plural forms. Thus, for example, "an expression cassette" includes one or more expression cassettes disclosed herein, and "the method" includes equivalent steps and methods known to those of ordinary skill in the art, which may modify or replace the methods described herein.

[0101] Unless the context requires otherwise, in this specification and the appended claims, the term "comprise" and variations such as "comprises" and "comprising" should be understood to mean including a stated integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps. As used herein, the term "comprises" may be replaced by the term "comprising" or sometimes by the term "having".

[0102] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim elements. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein, any of the terms "comprising," "consisting of," and "consisting essentially of" may be replaced with either of the other two terms.

[0103] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms. The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art. Generally, the terms related to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization techniques described herein are well known and commonly used in the art.

[0104] Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992 and 2002 supplements); Handbook of Biochemistry: Section A Proteins, Vol. 1, 1976, CRC Press; Handbook of Biochemistry: Section A Proteins, Vol. 2, 1976, CRC Press. The nomenclature and laboratory procedures and techniques related to molecular and cellular biology, protein biochemistry, enzymology, and medicinal chemistry described herein are those well known and commonly used in the art.

Claims

1. A method for producing D-psicose and its derivatives from a carbohydrate source, wherein the method comprises the following steps: a. in the presence of an extracellular enzyme selected from an invertase, an amylase and / or a pullulanase, Converts carbohydrate sources into a mixture of glucose and fructose; b. Optionally, converting glucose in the mixture of step (a) into fructose by enzymatic conversion in the presence of an isomerase; c. converting the fructose of step (a) and / or step (b) into fructose-6-phosphate in the presence of fructokinase; d. converting the fructose-6-phosphate produced in step (c) into psicose-6-phosphate in the presence of psicose-6-phosphate 3-epimerase; e. converting the psicose-6-phosphate obtained in step (d) into D-psicose in the presence of psicose-6-phosphate phosphatase; Wherein, the fructokinase, psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase are expressed as a single fusion protein.

2. A method for producing D-psicose and its derivatives from a carbohydrate source, wherein the method comprises the following steps: a. converting a carbohydrate source into glucose using an extracellular enzyme selected from an invertase, an amylase and / or a pullulanase; b. converting the glucose produced in step (a) into glucose-6-phosphate by glucokinase in the presence of polyphosphate; c. converting the glucose-6-phosphate produced in step (b) into fructose-6-phosphate in the presence of phosphoglucose isomerase; d. converting the fructose-6-phosphate produced in step (c) into psicose-6-phosphate in the presence of psicose-6-phosphate 3-epimerase; e. converting the psicose-6-phosphate obtained in step (d) into D-psicose in the presence of psicose-6-phosphate phosphatase; The psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase are expressed as a single fusion protein.

3. A method for producing D-psicose and its derivatives from a carbohydrate source, wherein the method comprises the following steps: a. converting a carbohydrate source into glucose-1-phosphate in the presence of glycogen / glucan phosphorylase; b. converting the glucose-1-phosphate obtained in step (a) into glucose-6-phosphate in the presence of phosphoglucomutase; c. converting the glucose-6-phosphate produced in step (b) into fructose-6-phosphate in the presence of phosphoglucose isomerase; d. converting the fructose-6-phosphate produced in step (c) into psicose-6-phosphate in the presence of psicose-6-phosphate 3-epimerase; e. converting the psicose-6-phosphate obtained in step (d) into D-psicose in the presence of psicose-6-phosphate phosphatase; The psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase are expressed as a single fusion protein.

4. The method of claims 1 to 3, wherein the carbohydrate source is selected from sucrose, fructose or glucose derived from starch and / or cellulose.

5. The method of claims 1 to 3, wherein the fusion enzyme is introduced into the reaction mixture in free form, immobilized form or a combination thereof.

6. The method of claims 1 to 3, wherein the conversion efficiency is between 75% and 95%.

7. The method of claims 1 to 3, wherein the carbohydrate source is further subjected to acid hydrolysis after enzymatic conversion.

8. The method of claims 1 to 3, wherein the carbohydrate source is further passed through a strong cation exchange resin after enzymatic conversion.

9. The method of claim 1, wherein the enzyme reaction is carried out in the presence of inorganic polyphosphate or ATP.

10. The method of claims 1 to 3, wherein the derivative of D-psicose is selected from D-allose and allol.

11. The method of claim 10, further comprising a step of producing D-allose, wherein the method comprises converting D-psicose obtained in step (d) of claims 1 to 3 into D-allose using ribose-5-phosphate isomerase and / or L-rhamnose isomerase.

12. The method of claim 10, further comprising a step of producing allol, wherein the method comprises converting D-psicose obtained in step (d) of claims 1 to 3 into allol using ribitol dehydrogenase.

13. A method for preparing a fusion enzyme, the method comprising the following steps: a. preparing a recombinant cell for expressing a recombinant fusion protein encoding fructokinase, psicose-6-phosphate 3-epimerase and psicose-6-phosphate phosphatase; b. culturing the recombinant cells at 37°C and then inducing the expression of the fusion protein at different concentrations of IPTG and a temperature of 20 to 30°C; c. Extracting and isolating the fusion protein from the culture by cell lysis and separation of the soluble and insoluble fractions.

14. D-psicose and its derivatives produced by the method of claims 1 to 12.

15. D-psicose and its derivatives obtained by the method of claims 1 to 12, for use as a sugar substitute.

Images