Dried body of immobilized enzyme capable of enduring drying, and manufacturing method and preservation method therefor

Abstract

[Problem] To provide a ketose 3-epimerase or a protein having the same activity in a mode in which an effect thereof can be maintained over a long period of time even under severe preservation conditions. [Solution] A dried body of an immobilized enzyme or of an immobilized protein having said enzymatic activity, the dried body being characterized in that the enzyme or protein having said enzymatic activity is a ketose 3-epimerase or a protein having the ketose 3-epimerase activity, and that the immobilized enzyme or immobilized protein having said enzymatic activity prior to drying is the immobilized enzyme or immobilized protein having said enzymatic activity, in which the temperature (optimal temperature) indicating the maximum activity has further improved than that prior to the immobilization, and the dried body being capable of enduring drying. A manufacturing method for immobilizing the enzyme or the protein, manufacturing the immobilized enzyme or immobilized protein having said enzymatic activity, in which the temperature (optimal temperature) indicating the maximum activity has further improved than that prior to the immobilization, and then drying the same. The enzyme or the protein are supported by an ion exchange resin, organic polymer carrier, and / or inorganic carrier and dried.

Description

A dried immobilized enzyme that can withstand drying, and its manufacturing method and storage method

[0001] The present invention relates to an immobilized enzyme or a dried form of an immobilized protein having ketose 3-epimerase activity that is an enzyme that can withstand drying, such as a ketose 3-epimerase (e.g., D-allulose 3-epimerase) that retains transport resistance and suppresses a decrease in activity, or a protein having ketose 3-epimerase activity, as well as methods for producing and storing the same.

[0002] Conventional technology produces pure rare sugar D-allulose (D-psicose) using ketose 3-epimerase (e.g., D-allulose 3-epimerase) from D-fructose as a raw material. The rare sugar D-allulose can be produced by treating D-fructose with epimerase to create a mixture of D-fructose and D-allulose, which can then be separated and purified to produce powdered crystalline D-allulose. This D-allulose has uses such as a sweetener and pharmaceutical, and is being mass-produced and sold worldwide. Because D-allulose has zero energy and various functional properties, it is being sold primarily as a sweetener. Furthermore, the climate change issue facing humanity in recent years has become a specific challenge known as "decarbonization," and is becoming increasingly important in the food and beverage industry, where starch, a product of carbon dioxide assimilation in plants, is involved. The development of globally competitive starch-based rare sugar products is awaited. One approach is to reduce raw material costs, which account for the majority of production costs, and to facilitate smooth introduction into the global market, by introducing rare sugars into inexpensive isomerized sugar, a mixed sugar containing D-glucose and D-fructose, for which a global-scale industry already exists, as consumption of high-fructose sugar increased to 14 million tons in the United States in 1999. For example, in search of a sweetener that contains the three sugars D-glucose, D-fructose, and D-allulose and has the functionality of D-allulose, one possibility is to move toward global-scale mass production methods (new manufacturing processes).

[0003] High-fructose corn syrup (HFCS), an inexpensive raw material, was developed in Japan in the 1960s and subsequently commercial production began in the United States. The initial HFCS42 (42% fructose) was not widely consumed due to its insufficient sweetness. However, when the sweeter HFCS55 (55% fructose) was developed in the 1970s, major beverage manufacturers began using it as a sugar substitute due to its lower price compared to sugar. Subsequently, HFCS55 consumption increased with the expansion of soft drink consumption, and by 1999, U.S. high-fructose corn syrup consumption had grown to 14 million tons. Today, the U.S. high-fructose corn syrup market is already mature, and demand is considered saturated. HFCS42 production has declined due to declining domestic consumption, and net imports (imports minus exports) are negligible. Recently, the decline in consumption has slowed, but there are no signs of a reversal or an upward trend at present. Domestic consumption of HFCS55 in the U.S. is also on a downward trend, but thanks to export demand to Mexico, a certain level of stable production volume has been maintained. However, as with HFCS42, there are no signs of a reversal of domestic consumption growth, and in addition, Mexico's high-fructose sugar market has recently reached saturation, so it will be difficult to continue the current pattern of increasing exports to cover the decline in domestic demand and ultimately stabilizing production.

[0004] The costs required for the production of high-fructose corn syrup include raw material (corn), labor, and fuel costs. The so-called "production cost" is calculated by subtracting the revenues from by-products (gluten meal, gluten feed, and corn oil) obtained during the high-fructose corn syrup production process. While the production cost of HFCS55 is, on average, $50-$60 (¥6,150-¥7,380) per ton higher than that of HFCS42, raw material costs are a major factor in the fluctuations in production costs for both HFCS42 and HFCS55. While labor, fuel, and electricity costs have remained stable, the cost of high-fructose corn syrup production has fluctuated significantly in recent years, peaking in 2012 and then dropping by approximately 30% over the following two years due to volatile corn prices.

[0005] D-Allulose, also known as D-psicose, is an epimer of D-fructose and has a sweetness level of about 70% that of sugar, similar to that of D-fructose in terms of sweetness quality. However, unlike D-fructose, D-allulose is hardly metabolized upon absorption in the body, has almost no calories, and has been shown to reduce abdominal fat by inhibiting the activity of lipid synthesis enzymes. It has been reported that D-allulose can be used as a low-calorie sweetener (Patent Document 1) and as a sweetener effective for weight loss (Non-Patent Documents 1 and 2). Patent Document 2 focuses on the blood sugar elevation inhibitory effect of D-allulose and describes its use in health foods, foods and beverages for diabetics, and foods and beverages for weight loss.

[0006] D-tagatose has a sweetness of about 90% of sugar and an energy content of 2 kcal / g, and is used as a low-calorie sweetener. It is also known to have the effect of increasing HDL cholesterol and suppressing body fat accumulation (Patent Document 3), and like D-allulose, it has the effect of suppressing postprandial blood glucose elevation.

[0007] Currently, methods using isomerases are used to produce these rare sugars, a result of the discovery of a useful enzymatic reaction. One type of isomerase, ketose 3-epimerase, can use multiple ketoses as substrates, and the name of the ketose with the highest epimerization activity at the 3-position among the substrate ketoses is sometimes used. For example, an enzyme with the highest epimerization activity at the 3-position for D-tagatose is sometimes called D-tagatose 3-epimerase. By utilizing this D-tagatose 3-epimerase (DTE), a technology has been established for producing the rare sugar D-allulose from D-fructose.

[0008] For example, Non-Patent Document 3 discloses a D-ketose 3-epimerase derived from Pseudomonas cichorii ST-24 strain, and describes that D-allulose can be produced from D-fructose by using this enzyme. However, as this enzyme is also known as D-tagatose 3-epimerase, it has the highest epimerization activity at the 3-position of D-tagatose and a relatively weak effect on D-fructose.

[0009] Furthermore, Patent Document 4 reports a method for producing D-psicose (D-allulose) using D-psicose 3-epimerase derived from Agrobacterium tumefaciens, and Patent Document 5 reports a method for producing D-allulose using ketose 3-epimerase derived from Arthrobacter globiformis. Currently, Arthrobacter globiformis is used in production sites, but for industrial-scale mass production of D-allulose and D-tagatose, a more active enzyme is required. In this context, a novel ketose 3-epimerase has been discovered that is derived from a microorganism that is approved for use in food production and is considered non-toxic, has high activity, and is heat-resistant, and is capable of catalyzing the isomerization of D-fructose to D-allulose with high activity (Patent Document 6).

[0010] Patent No. 4473980 Patent No. 5421512 Patent No. 5749880 Patent No. 4648975 Patent No. 5997693 WO2020 / 105709

[0011] Asia Pac.J.Clin.Nutr.(2001).10,233-237Asia Pac.J.Clin.Nutr.(2004)13,127Biosci.Biotech.Biochem.(1993)57,1037-1039

[0012] In the production of isomerized sugar, raw material costs often account for the majority of production costs, and while the benefits of improved enzyme catalytic performance do not account for a significant portion, if competition becomes global, the business scale will become very large, and the benefits of improved enzyme catalytic performance will be significant. Based on the heat resistance of the ketose 3-epimerase described in Patent Document 6, its use is expected to accelerate on a global scale, increasing the possibility of establishing a catalyst business or licensing business for the global supply of this enzyme. As the business scale expands and production bases increase around the world, highly active enzyme-catalyzed ketose 3-epimerase will be required, and it is highly likely that cross-border transportation from specialized enzyme catalyst departments will begin. In this case, transporting the enzyme in the form of an immobilized enzyme is optimal so that it can be used directly at the raw material injection port of the resin column upon arrival at the production site. Conventional immobilized enzyme storage methods, which typically involve immersing the immobilized enzyme in an appropriate buffer solution at a low temperature (2-15°C) but not freezing, or adding additives to prevent activity loss, will be adopted. However, such a form creates new problems. Long-distance transport poses several problems, including the need to transport water, the need for specialized temperature control vendors, the storage space required, and the need for tight packaging to prevent water leakage, all of which need urgent resolution. At the same time, the buffer solution itself is likely to be a suitable culture medium for microorganisms, and mold contamination could destroy the immobilized enzyme itself. Furthermore, recent changes in container transport routes between China and the United States have caused shipping costs from Japan to skyrocket, further increasing the importance of the issue of transportation costs. Dried immobilized enzymes clearly offer advantages in solving the issues of (a) price, (b) transit time, and (c) temperature control, which are factors in selecting a transportation method. Furthermore, while Japan is a standard container transport route between China and the United States, due to the long loading times, it has become common for enzymes to be shipped by air from the United States to China, thereby increasing shipping costs from Japan. Given this container shortage, it is necessary to achieve efficient transportation while maintaining convenience.

[0013] On the other hand, the scientific principle that "drying" also makes something "stabilized" so that it can maintain its effectiveness for a long period of time even under harsh storage conditions is true. For complex structures, the enzyme's three-dimensional structure in solution does not change significantly, and enzymatic activity can be maintained. However, there is concern that drying can significantly change the enzyme's structure, resulting in a significant decrease in enzyme activity. It is often considered that the (freeze) drying process itself can be a cause of deterioration, and it is necessary to find an answer as to whether this principle is applicable to enzyme-catalyzed ketose 3-epimerase, especially to immobilized enzymes.

[0014] The present invention aims to provide a globally competitive, cost-effective method for mass-producing, on a global scale, a sweetener that contains three sugars, D-glucose, D-fructose, and D-allulose, and has the functionality of D-allulose and a taste similar to that of sugar, by combining the use of, for example, "inexpensive isomerized sugar or invert sugar, which is a sugar mixture containing D-glucose and D-fructose," which reduces raw material costs, which account for the majority of the production costs, with the shortening of the multi-step process by producing the product without using simulated moving bed chromatography, and the use of a low-cost enzyme catalyst, in a form that retains its effectiveness for a long period of time even under harsh storage conditions, i.e., a low-cost enzyme catalyst derived from the high durability (lifespan) of the enzyme, as a "drying-resistant enzyme."

[0015] The present invention discloses the provision of a ketose 3-epimerase (e.g., D-allulose 3-epimerase) or a protein having ketose 3-epimerase activity in the form of a dried immobilized enzyme or immobilized protein that retains its activity for extended periods even under harsh storage conditions. Immobilized enzymes themselves utilize biomolecules, and prolonged use inevitably leads to a decline in activity. Water, as a solvent, is a medium that uniformly disperses various substances, but it also allows the enzyme catalyst to undergo physical and chemical changes (deterioration). In a dried state, even if unstable substances coexist, the enzyme catalyst remains solidified in the same manner as the enzyme catalyst, thereby restricting the movement of substances (molecules) involved in degradation, enabling long-term storage. While drying can be considered a stabilizing process, the dehydration process of drying can destabilize enzyme molecules that are originally stable in the presence of water molecules due to the loss of hydrogen bonds. Furthermore, for complex structures such as enzyme catalysts, the (freeze)-drying process itself can often be a factor that is thought to cause denaturation of the complex structure. Naturally, if activity declines, the immobilized enzyme must be replaced, resulting in significant economic losses. To address these issues, if immobilized ketose 3-epimerase (e.g., D-allulose 3-epimerase) supported on ion exchange resins could be stored in a dried state, which would be an effective solution to transportation costs, preventing contamination due to liquid leakage, and reducing microbial contamination. However, the business using ketose 3-epimerase (e.g., D-allulose 3-epimerase) is still in its infancy and is not yet large-scale, and the drying issues associated with immobilized enzymes have not yet been recognized. While preparations are underway to increase production bases around the world, there has been no incentive to address the drying issue, and thus no immobilized dried ketose 3-epimerase (e.g., D-allulose 3-epimerase) has yet been put to practical use. With regard to ketose 3-epimerase (for example, D-allulose 3-epimerase), it was unclear until trials were carried out whether an immobilized dried product could be obtained that retained transport resistance and suppressed decline in activity.The present inventors believed that the first step would be to fully understand the problem of drying in the form of an immobilized enzyme, and conducted extensive research and experiments. As a result, they found that in the case of immobilized enzymes, drying, a dehydration procedure, can cause enzyme molecules, which are originally stable in the presence of water molecules, to become unstable due to the loss of hydrogen bonds; however, by selecting an appropriate carrier, hydrogen bonds can be formed between the enzyme molecules and the carrier molecules, allowing the enzyme to exist stably even at a low water content. In other words, if the immobilized enzyme can be made stable at the same temperature or higher, with activity at least comparable to that before immobilization, then the problem can be solved, and the enzyme (ketose 3-epimerase) or a protein having the enzyme activity (a protein having ketose 3-epimerase activity) can be made into a "dried product that does not require washing before use, retains transport resistance, and is inhibited from losing activity," thereby completing the present invention.

[0016] The present invention relates to a dried body of an immobilized enzyme or an immobilized protein having the enzymatic activity of the enzyme that can withstand drying, as described in (1) to (9) below. (1) A dried body of an immobilized enzyme or a protein having the enzymatic activity, characterized in that the enzyme or protein having the enzymatic activity is a ketose 3-epimerase or a protein having ketose 3-epimerase activity, and that the temperature at which the immobilized enzyme or protein having the enzymatic activity exhibits maximum activity (optimal temperature) is improved before drying compared to before immobilization. (2) A dried body of an immobilized enzyme or an immobilized protein having the enzymatic activity of the enzyme that can withstand drying described in (1) above, which does not contain any additives for maintaining the enzymatic activity and whose activity does not decrease for at least six months. (3) A dried product of an immobilized enzyme that is a desiccation-resistant enzyme or an immobilized protein having the enzymatic activity according to (1) or (2) above, wherein the dried product is a dried product of the enzyme or protein having the enzymatic activity supported on a carrier that is an ion exchange resin, an organic polymer carrier, and / or an inorganic carrier. (4) A dried product of an immobilized enzyme that is a desiccation-resistant enzyme or an immobilized protein having the enzymatic activity according to any of (1) to (3) above, wherein the dried product does not require washing before use, retains transport resistance, and is inhibited from decreasing in activity for at least six months. (5) A dried product of an immobilized enzyme that is a desiccation-resistant enzyme or an immobilized protein having the enzymatic activity according to any of (1) to (4) above, wherein the dried product is a freeze-dried product or a product dried in an incubator at 30°C. (6) A dried product of an immobilized enzyme or an immobilized protein having the enzyme activity, which is an enzyme that can withstand drying according to any one of (1) to (5) above, characterized in that the enzyme or protein having the enzyme activity is selected from the following enzyme (i) and protein having the enzyme activity (ii):(i) A ketose 3-epimerase derived from a microorganism belonging to the genus Arthrobacter, which has a molecular mass of 36 kDa as measured by SDS-PAGE and has the substrate specificity of (A) and (B) below and the physicochemical properties of (C) and (D) below. (A) It has the activity of epimerizing the 3-position of D- or L-ketose to produce the corresponding D- or L-ketose. (B) It has the highest epimerization activity at the 3-position of D-allulose among D- or L-ketohexoses. (C) The optimum reaction pH is 6 to 8. (D) The optimum reaction temperature is 60°C. (ii) A protein described in (a), (b), (c), or (d) below. (a) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. (b) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO: 1, wherein the amino acid sequence does not contain a substitution of any of the amino acids at H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO: 1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and which has the ketose 3-epimerase activity of (A) and (B) above, and wherein the ratio of the ketose 3-epimerase activities at reaction temperatures of 70°C and 50°C (T70 / T50) is 1.55 or more. (c) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO: 1, wherein the amino acid sequence does not contain a substitution of any of the amino acids at H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO: 1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and which has the ketose 3-epimerase activity of (A) and (B) above and has a residual activity of 31% or more after incubation at 60°C for 1 hour.(d) A protein consisting of an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, which has an amino acid substitution at at least one site selected from H56, T59, A60, P77, L94, L97, D101, A109, A128, V129, V132, S144, S167, P172, E199, A200, K202, and S218 in the amino acid sequence represented by SEQ ID NO: 1, and which has the ketose 3-epimerase activity of (A) and (B) above, and which has a higher ketose 3-epimerase activity ratio (T70 / T50) at reaction temperatures of 70°C to 50°C or higher residual activity after incubation at 60°C for 1 hour than the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. (7) A dried product of the immobilized enzyme or immobilized protein having the enzymatic activity described in any one of (1) to (6) above, wherein the immobilized enzyme or protein having the enzymatic activity is a crude enzyme present in a bacterial cell disruption product or a partially purified enzyme from a crude enzyme solution, which is immobilized on a carrier. (8) A dried product of the immobilized enzyme or protein having the enzymatic activity described in any one of (1) to (7) above, wherein the ketose 3-epimerase has substrate specificity in the order of D-allulose, D-tagatose, L-tagatose, D-fructose, and L-allulose. (9) A dried product of the immobilized enzyme or immobilized protein having the enzymatic activity described in any one of (6) to (8) above, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter histidinolovorans.

[0017] The present invention also encompasses the following methods (10) to (18) for producing a dried product of an immobilized enzyme that is a drying-resistant enzyme or an immobilized protein having the activity of the enzyme: (10) A method for producing a dried product of an immobilized enzyme that is a drying-resistant enzyme or an immobilized protein having the activity of the enzyme, comprising immobilizing the enzyme ketose 3-epimerase or the protein having ketose 3-epimerase activity to produce an immobilized enzyme or protein having the activity of the enzyme that has an improved temperature (optimum temperature) at which it shows maximum activity compared to before immobilization, and then drying the immobilized enzyme or protein. (11) A method for producing a dried product of an immobilized enzyme that is a drying-resistant enzyme or an immobilized protein having the activity of the enzyme described in (10) above, comprising supporting and immobilizing the preenzyme ketose 3-epimerase or the protein having ketose 3-epimerase activity on a carrier that is an ion exchange resin, an organic polymer carrier, and / or an inorganic carrier. (12) A method for producing a dried body of the immobilized enzyme or protein that can withstand drying according to (11) or (12) above, which is dried without adding any additives for maintaining the enzyme activity. (13) A method for producing a dried body of the immobilized enzyme that is an enzyme that can withstand drying according to any of (10) to (12) above, or an immobilized protein having the activity of the enzyme, which is an enzyme that can withstand drying according to any of (10) to (12) above, which is dried by freeze-drying or drying in an incubator at 30° C. (14) A method for producing a dried body of the immobilized enzyme that is an enzyme that can withstand drying according to any of (10) to (13) above, which is characterized in that the proenzyme ketose 3-epimerase or the protein having ketose 3-epimerase activity is selected from the following enzyme (i) and protein (ii) having the enzyme activity: (i) A ketose 3-epimerase derived from a microorganism belonging to the genus Arthrobacter, which has a molecular mass of 36 kDa as measured by SDS-PAGE and has the substrate specificity of (A) and (B) below and the physicochemical properties of (C) and (D) below: (A) The ketose 3-epimerase has the activity of epimerizing the 3-position of a D- or L-ketose to produce the corresponding D- or L-ketose.(B) Among D- or L-ketohexoses, it has the highest epimerization activity at position 3 for D-allulose. (C) The optimum reaction pH is 6 to 8. (D) The optimum reaction temperature is 60°C. (ii) A protein described in (a), (b), (c), or (d) below. (a) A protein consisting of the amino acid sequence represented by SEQ ID NO: 1. (b) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO: 1, wherein the amino acid sequence does not contain a substitution of any of the amino acids at H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO: 1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and which has the ketose 3-epimerase activity of (A) and (B) above, and wherein the ratio of the ketose 3-epimerase activities at reaction temperatures of 70°C and 50°C (T70 / T50) is 1.55 or more. (c) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO: 1, wherein the amino acid sequence does not contain a substitution of any of the amino acids at H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO: 1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, and which has the ketose 3-epimerase activity of (A) and (B) above and has a residual activity of 31% or more after incubation at 60°C for 1 hour.(d) A protein consisting of an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, which has an amino acid substitution at at least one site selected from H56, T59, A60, P77, L94, L97, D101, A109, A128, V129, V132, S144, S167, P172, E199, A200, K202, and S218 in the amino acid sequence represented by SEQ ID NO: 1, and which has the ketose 3-epimerase activity of (A) and (B) above, and which has a higher ketose 3-epimerase activity ratio (T70 / T50) at reaction temperatures of 70°C to 50°C or higher residual activity after incubation at 60°C for 1 hour than the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. (15) A method for producing a dried product of the immobilized enzyme or immobilized protein having the enzymatic activity of any one of (10) to (14) above, which comprises immobilizing a crude enzyme present in a disrupted bacterial cell product or a partially purified enzyme from a crude enzyme solution on a carrier. (16) A method for producing a dried product of the immobilized enzyme or immobilized protein having the enzymatic activity of any one of (10) to (15) above, which is an enzyme capable of withstanding desiccation, wherein the ketose 3-epimerase has substrate specificity in the order of D-allulose, D-tagatose, L-tagatose, D-fructose, and L-allulose. (17) A method for producing a dried product of the immobilized enzyme or immobilized protein having the enzymatic activity of any one of (14) to (16) above, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter histidinolovorans. (18) A method for producing a dried product of an immobilized enzyme or an immobilized protein having the enzyme activity, which is an enzyme that can withstand desiccation according to any one of (14) to (17), wherein the microorganism belonging to the genus Arthrobacter is the Arthrobacter histidinolovorans Y586-1 strain deposited at the Patent Microorganisms Depositary Center under accession number NITE BP-02813.

[0018] The present invention also provides methods for preserving a dried immobilized enzyme or a dried immobilized protein having the enzyme activity described below in (19) and (20), which are enzymes that can withstand desiccation and maintain transport resistance while suppressing a decrease in activity. (19) A method for preserving a dried immobilized enzyme or a dried immobilized protein having the enzyme activity described above in any of (1) to (9), characterized in that the preservation is carried out either at refrigeration or at room temperature. (20) A method for preserving a dried immobilized enzyme or a dried immobilized protein having the enzyme activity described above in (19), which is an enzyme that can withstand desiccation and which is sealed in a sealable container made of paper, plastic, and metal.

[0019] The present invention provides a ketose 3-epimerase (e.g., D-allulose 3-epimerase) or a protein having ketose 3-epimerase activity in a form that retains its activity for an extended period of time, even under harsh storage conditions. This enables the establishment of a catalyst business or licensing business for the global supply of the enzyme, and by combining the cost of raw materials, which accounts for the majority of production costs, with the use of, for example, "inexpensive isomerized sugar or invert sugar, which is a sugar mixture containing D-glucose and D-fructose," shortening the multi-step process by producing the product without using simulated moving bed chromatography, and the use of a low-cost enzyme catalyst due to the long durability (lifespan) of the enzyme, a method for the global mass production of a sweetener containing three sugars, D-glucose, D-fructose, and D-allulose, with the functionality of D-allulose and a taste similar to that of sugar, at a sufficiently price-competitive price, can be spread worldwide.

[0020] 1 is a diagram showing the effect of temperature (optimum temperature) on immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes. 2 is a diagram showing the effect of temperature (thermal stability) on immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes. 3 is a diagram showing the effect of pH (optimum pH) on immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes. 4 is a diagram showing the effect of pH (pH stability) on immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes. This figure shows a table comparing the properties (optimum temperature, temperature stability, and optimum pH) of D-allulose 3-epimerase (purified enzyme) derived from Y586-1 (parent strain), site-directed mutant enzymes of D-allulose 3-epimerase derived from Y586-1 (purified enzyme), and immobilized site-directed mutant enzymes of D-allulose 3-epimerase derived from Y586-1 (partially purified enzyme). This figure also shows a method for drying the immobilized enzyme. Figure (1) shows a comparison of the activity of the untreated and dried enzymes (dried at 30°C and stored at room temperature, dried at 30°C and stored in a refrigerator (4°C), freeze-dried and stored at room temperature, and freeze-dried and stored in a refrigerator (4°C)) on days 0 and 192 of storage. (2) shows the relative activity of the dried enzyme (drying at 30°C → storage at room temperature, drying at 30°C → storage in a refrigerator (4°C), freeze-drying → storage at room temperature, freeze-drying → storage in a refrigerator (4°C))) with the activity of the untreated enzyme taken as 100%. (1) shows a comparison of the activity of the untreated and dried enzyme (drying at 30°C → storage at room temperature, drying at 30°C → storage in a refrigerator (4°C), freeze-drying → storage at room temperature, freeze-drying → storage in a refrigerator (4°C))) on days 0 and 224 of storage. (2) shows the relative activity of the dried enzyme (drying at 30°C → storage at room temperature, drying at 30°C → storage in a refrigerator (4°C), freeze-drying → storage at room temperature, freeze-drying → storage in a refrigerator (4°C)) with the activity of the untreated enzyme taken as 100%.

[0021] <Enzymes or proteins with the enzyme activity> Currently, D-psicose and D-allose are rare sugars that can be mass-produced. One of the English names for D-psicose is "D-allulose," but hereafter, D-allulose will be used in the present invention. D-Allulose is the D-isomer of allulose, which is classified as a ketohexose, and is a hexose. When considering isomerases from the perspective of rare sugar production, the enzyme ketose 3-epimerase plays a role as a key enzyme. Isomerase is a general term for a group of enzymes that catalyze reactions that convert biomolecules into isomers, and is also called isomerase. The name "isomerase" includes a wide group of enzymes that catalyze "reactions that do not change the chemical formula", such as aldose isomerase, an enzyme that links monosaccharides aldose and ketose, and ketose 3-epimerase, an enzyme that links ketose and ketose. Among isomerases that catalyze the interconversion of optical isomers, an enzyme that isomerizes one of the multiple asymmetric points (stereostructures) in a substrate molecule is called an epimerase. Since sugars have many asymmetric points, isomerizing enzymes are generally called epimerases. In the case of monosaccharides, epimerases are enzymes that move the position of -OH bonded to C to the opposite side, and are "reactions that do not change the chemical formula C ６ H １２ O ６ It catalyzes a "reaction in which the molecule does not change." Therefore, it is an enzyme that belongs to the isomerase family. Isomerases are superior to epimerases. That is, the enzyme "isomerase" is well known in the industry, and examples include ketose 3-epimerase, D-xylose isomerase of aldose isomerase, and glucose isomerase. For example, aldose isomerase catalyzes "transfer of hydrogen within a molecule," and C １ and C ２It promotes an oxidation-reduction reaction between D-tagatose and D-sorbose. D-tagatose 3-epimerase (DTE), a type of ketose 3-epimerase, is an enzyme discovered in the microorganism Pseudomonas cichorii, which was isolated from the campus of the Faculty of Agriculture, Kagawa University in 1991. D-tagatose 3-epimerase reverses the orientation of the hydroxyl group at the third carbon position of D-tagatose, converting it to D-sorbose. This reaction is a reversible equilibrium reaction, with the equilibrium ratio of D-tagatose to D-sorbose being 20:80. Furthermore, this enzyme exhibits broad substrate specificity, acting not only on D-tagatose and D-sorbose, but also on all six-carbon ketoses, such as D-fructose and D-allulose (equilibrium ratio 70:30), L-tagatose and L-sorbose (equilibrium ratio 27:73), and L-allulose and L-fructose (equilibrium ratio 24:76), and is also capable of catalyzing the epimerization of five-carbon ketoses, such as D-xylulose and D-ribulose (equilibrium ratio 85:15) and L-xylulose and L-ribulose (equilibrium ratio 70:30).The discovery of this enzyme has enabled the production of numerous rare sugars at Kagawa University and has become a driving force behind research into rare sugar-producing enzymes.

[0022] <An immobilized enzyme or dried protein capable of withstanding drying, in which the enzyme or protein having the enzyme activity is a ketose 3-epimerase or a protein having ketose 3-epimerase activity> First, the ketose 3-epimerase or the protein having ketose 3-epimerase activity will be described. In the present invention, the protein having ketose 3-epimerase activity preferably refers to an amino acid substitution mutant of the ketose 3-epimerase derived from the Y586-1 strain, and may be abbreviated as a site-directed mutant enzyme of the Y586-1-derived ketose 3-epimerase. Among rare sugars, D-allulose is the one that has begun to be mass-produced and sold on a global scale, and is produced using a ketose 3-epimerase (e.g., D-allulose 3-epimerase) from D-fructose as a raw material. Therefore, D-allulose 3-epimerase (DAE), which is used in the production of D-allulose, will be described as an enzyme to be dried. This D-allulose 3-epimerase (DAE) may be of any origin or have any classification name, as long as it is an enzyme that isomerizes D-fructose to D-allulose.

[0023] For example, the Arthrobacter globiformis M30 strain, which was discovered to be derived from a non-toxic microorganism approved for use in food production, has high activity and heat resistance, and is capable of catalyzing the isomerization of D-fructose to D-allulose with high activity (Patent Document 5). The M30 strain was originally deposited on June 22, 2011, with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number NITE BP-1111, and was transferred to a deposit under the Budapest Treaty on May 2, 2012. Another preferred example is the ketose 3-epimerase derived from the Arthrobacter histidinolovorans Y586-1 strain (Patent Document 6). The Y586-1 strain was internationally deposited with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (NIET), located at Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, on November 7, 2018, under the accession number NITE BP-02813.

[0024] (Strain Y586-1) The present inventors focused on bacterial species that are approved for use in foods not only in Japan but also in Europe and the United States and are listed as having little toxicity. They collected soil samples from various locations, isolated microorganisms from the soil, and continued to search for microorganisms having ketose 3-epimerase activity. As a result, among the many isolated strains, they found a microorganism belonging to the genus Arthrobacter that produces a novel ketose 3-epimerase. This Arthrobacter histidinolovorans strain Y586-1, a microorganism of the genus Arthrobacter, produces a novel ketose 3-epimerase that is highly active and highly thermostable. The ketose 3-epimerase derived from the Y586-1 strain acts on free D- or L-ketoses or D- or L-ketopentoses to epimerize the 3-position of these ketoses, easily producing the corresponding D- or L-ketoses or D- or L-ketoses. This reaction paves the way for the mass production of D-allulose using various ketoses, particularly D-fructose, as a raw material. In particular, compared to conventional ketose 3-epimerases, the total activity obtained from the culture medium is approximately three times higher than that of the control enzyme, making mass production of D-allulose possible using the enzyme derived from the Y586-1 strain. Furthermore, because the enzyme has a high optimum temperature of 70°C and is a highly thermostable enzyme, a higher D-allulose production yield can be achieved when the reaction is carried out at an optimum temperature of around 60 to 70°C. Furthermore, the amino acid substitution mutants of the ketose 3-epimerase derived from strain Y586-1 contain many mutants that are more thermostable than the unmutated enzyme. Based on their high activity and high thermostability, they were selected for immobilization as a dry enzyme for global use.

[0025] This novel microbial ketose 3-epimerase acts on the 3-position of D- or L-ketose and catalyzes the epimerization to the corresponding D- or L-ketose. Among D- or L-ketohexoses, it has the highest epimerization activity at the 3-position for D-allulose, making it suitable for producing D-allulose from D-fructose.

[0026] Specifically, the ketose 3-epimerase selected for the dried immobilized enzyme is preferably a ketose 3-epimerase that can be isolated from microorganisms belonging to the genus Arthrobacter, and has the distinctive properties of high enzyme production per culture medium and thermostability. Microorganisms of the genus Arthrobacter are safe microorganisms that are included on the Japanese food additive list as sources of enzymes such as α-amylase, isomaltodextranase, invertase, urease, glucanase, α-glucosyltransferase, and fructosyltransferase, as well as producers of trehalose and vitamin K (menaquinone). An advantage of using this enzyme derived from microorganisms of the genus Arthrobacter in the food industry is the safety of the microorganisms.

[0027] Ketose is a chemical term used to classify carbohydrates by their structure. It is a general term for monosaccharides that contain one keto group (ketonic carbonyl group) within the chain structure, and has the molecular formula C ｎ H ２ｎ O ｎ (n≧3). A typical ketose is fructose, but the term also includes pentoses. In the present invention, ketose refers to ketose in a broad sense, including pentoses, and the term ketohexose refers to a hexose ketohexose having a ketose structure. Specifically, these include fructose, allulose, tagatose, and sorbose, and D- or L-ketose refers to the D- or L-isomers thereof. Therefore, the ketose 3-epimerase selected as the target of the dried immobilized enzyme of the present invention has the activity of epimerizing the 3-position of D- or L-ketose to produce the corresponding D- or L-ketose, and specifically, it can catalyze the interconversion between D- or L-fructose and D- or L-allulose, and between D- or L-tagatose and D- or L-sorbose. Furthermore, it can also act on all other ketoses.

[0028] The ketose 3-epimerase derived from the Y586-1 strain has approximately twice the activity of the enzyme produced per culture medium compared to the ketose 3-epimerase derived from Arthrobacter globiformis described in Patent Document 5, and its use enables efficient mass production of D-allulose. Furthermore, the enzyme has a high optimum temperature of 70°C, and even at 80°C, its relative activity is 59.4 (approximately 60%), with the activity at the optimum temperature of 70°C being taken as 100, demonstrating high thermostability. Furthermore, the residual activity after incubation at 60°C for 1 hour was 49.4%, demonstrating thermostability, enabling the production of a larger amount of product in reactions at the optimum temperature of 70°C. The excellent thermostability of this enzyme can be further enhanced by creating amino acid substitution mutants of the enzyme. Specifically, by introducing mutations into the ketose 3-epimerase gene derived from the Y586-1 strain and substituting the corresponding amino acid residues with other amino acid residues by site-directed mutagenesis to prepare mutants with various amino acid substitutions, it is possible to obtain enzymes with higher thermostability than the wild-type enzyme without amino acid substitutions, and these mutants can be used. These mutants have thermostability, evaluated using two indices: the ratio of ketose 3-epimerase activity at reaction temperatures of 70°C and 50°C (T70 / T50), and the residual activity after incubation at 60°C for 1 hour. This has enabled an industrially practical, low-cost production method based on total productivity derived from the high durability (lifespan) of the D-allulose 3-epimerase used, resulting from the high thermostability of the enzyme. Furthermore, various ketose 3-epimerase mutants were prepared by base substitution using site-directed mutagenesis. From among these, those at positions that would yield amino acid substitution mutants with higher thermostability than the wild-type were selected, taking into consideration the general technical knowledge of enzymes that thermostability is an important property for extending the enzyme's lifespan. Of these, 20 mutant enzymes out of 37 were obtained that exceeded the DAE (1.78) of the parent wild-type recombinant enzyme strain Y586-1. The amino acid substitution mutant listed at position 8 in Table 10 in the specification of the international application (Patent Document 6) can be used. This mutant is a protein consisting of (1) the name of the mutant sequence and (2) the amino acid sequence represented by SEQ ID NO: 1.(1) Y586DAE_V129IA200VS218N (2) Amino acid sequence represented by SEQ ID NO: 1 MKIGCHGLVWTGRFDAEGIRYSVQKTKEAGFDLIEFPLMDPFSFDVATAKSALAEHGLTASASLGLSEATDVSSEDPAIVKAGEELLNRALDVLAELGATDFCGVIYSAMKKYMEPATEQGLSNSKAAIGRVVDRAAGLGINVSLEVVNRYETNVLNTGRQALSYLSDVKRPNLGVHLDTYHMNIEESDMFSPVLDTAEVLKYVHIGESHRGYLGTGNVDFDNFFKALGRIGYDGPVVFESFSSAVVAPDLSRMLGIWRNLWADNEELGAHANAFIRDKLTAIKTIELH By using such a thermostable mutant, high thermostability and high productivity result in total productivity that is derived from the high durability (lifespan) of the enzyme rather than activity. Using this ketose 3-epimerase or a protein having ketose 3-epimerase activity, a potential substrate ketose can be epimerized at its 3-position to convert it into the corresponding ketose, thereby producing the corresponding ketose. Because this ketose 3-epimerase or a protein having ketose 3-epimerase activity has high epimerization activity at the 3-position of D-allulose or D-fructose, it enables efficient mass production of D-allulose, a rare sugar, from D-fructose, a potential substrate. In either case, these enzymes may be purified enzymes or microorganisms that produce the enzymes.

[0029] <Immobilization of Enzymes> Next, we will explain immobilized enzymes or proteins, in which the enzyme or protein having the enzyme activity is a ketose 3-epimerase or a protein having ketose 3-epimerase activity. Furthermore, the ketose 3-epimerase selected as the target of the dried immobilized enzyme of the present invention can be obtained as a highly active immobilized enzyme by various immobilization methods, and the use of the immobilized enzyme enables continuous, large-scale epimerization reactions. Industrial immobilization enables mass production of the target ketose. Hereinafter, the term "enzyme" may also be used to refer to proteins with enzymatic activity. The enzyme is supported on a carrier such as an ion exchange resin, an organic polymer carrier, and / or an inorganic carrier, and dried. The immobilized enzyme itself utilizes a biomolecule, and a decrease in activity is unavoidable with long-term use. However, it has been found that by stabilizing the immobilized enzyme itself, the decrease in activity does not occur even when dried. These enzymes may be crude enzyme solutions or purified enzymes, or they may be enzyme-producing microorganisms or proteins with enzymatic activity. A crude enzyme solution, a purified enzyme, a microorganism producing the enzyme, or a protein having enzymatic activity can be immobilized and used. One embodiment of a method for immobilizing ketose 3-epimerase is immobilization using a crude enzyme solution. A crude enzyme solution containing ketose 3-epimerase obtained by ultrasonicating a bacterial cell suspension is added to an ion exchange resin or the like, and the ketose 3-epimerase can be immobilized by binding at low temperature. Extracting ketose 3-epimerase from bacterial cells requires disrupting the bacterial cell walls, which can be achieved by ultrasonication or enzyme treatment with lysozyme or the like. It has been found that a large amount of active ketose 3-epimerase is contained in the crude enzyme solution obtained by ultrasonication. The immobilized enzyme can be obtained using known immobilization methods, such as carrier binding, crosslinking, gel entrapment, and microencapsulation, and the carrier may be any carrier. In carrier binding, the enzyme can be bound (supported) to a carrier using physical adsorption, ionic bonding, covalent bonding, or the like.In the examples, the enzyme was immobilized using a site-directed mutant of ketose 3-epimerase derived from Arthrobacter histidinolovorans Y586-1 strain (parent strain). In the examples, a partially purified enzyme obtained by heat treatment of the crude enzyme was used for immobilization. However, the properties of the immobilized enzyme remain unchanged whether the crude enzyme or the purified enzyme is used, and the present invention is not limited to the examples. The use of an immobilized enzyme enables continuous, large-scale epimerization reactions, and the use of a thermostable enzyme derived from Arthrobacter histidinolovorans extends the enzyme's lifespan. While any known support can be used to immobilize the enzyme, it is most practical to immobilize the enzyme on an appropriate substrate such as an ion exchange resin or synthetic adsorbent. Ion exchange resins, such as basic anion exchange resins, can be either strongly basic or weakly basic. Examples of strongly basic anion exchange resins include HPA25L (manufactured by Mitsubishi Chemical Corporation) and IRA904CL (manufactured by Organo Corporation). Examples of weakly basic anion exchange resins include WA30 (manufactured by Mitsubishi Chemical Corporation), FPA54, and FPA95 (manufactured by Organo Corporation). In the examples, an anion exchange resin (A111S, manufactured by Purolite) was used for immobilization. Enzyme isomerization can be performed by immobilizing the enzyme, and stable, easily usable immobilized enzymes can be obtained using various immobilization methods. The use of immobilized enzymes enables continuous, large-scale isomerization and isomerization reactions. For example, immobilized enzymes with an activity of 1,000 U / g wet weight resin are used. A crude enzyme solution is collected from the solution of disrupted bacterial cells, and the crude enzyme solution is passed through a column filled with ion exchange resin or a synthetic adsorbent at a low temperature (4°C) to bind the crude enzyme protein to the ion exchange resin. The resin is then washed with purified water to obtain an immobilized enzyme. This provides an immobilization system that can withstand continuous production in terms of stability (activity retention) that is completely satisfactory for commercial production. Furthermore, it goes without saying that immobilized proteins obtained by immobilizing enzymes expressed in E. coli may also be used, similar to the crude and purified enzymes.The resulting immobilized enzyme can be dried and stored for a long period of time, allowing for continuous, large-scale isomerization (epimerization) reactions. Since the immobilized ketose 3-epimerase can be easily eluted after its isomerization ability is lost, regeneration of the immobilization carrier can be performed very easily, reducing production costs. The use of a heat-resistant enzyme extends its lifespan, further reducing production costs. Even after drying, storage, transportation, etc., the enzyme can be easily restored to its pre-drying state and can be used in the same way as immediately after immobilization.

[0030] <Confirmation that the immobilized enzyme or protein having the enzyme activity has an improved temperature (optimum temperature) at which it exhibits maximum activity compared to before immobilization> Immobilization reduces the activity compared to before immobilization. However, immobilization provides stability that can withstand an increase in the optimum temperature and prolonged heat treatment. The enzyme ketose 3-epimerase or the protein having the protein ketose 3-epimerase activity is immobilized to produce an immobilized enzyme or protein having the enzyme activity with an improved temperature (optimum temperature) at which it exhibits maximum activity compared to before immobilization, and then the enzyme is dried. Before drying, it was confirmed through experiments that the immobilized enzyme or protein having the enzyme activity has an improved temperature (optimum temperature) at which it exhibits maximum activity compared to before immobilization. The enzymatic activity of the immobilized enzyme for this confirmation can be measured as follows: The enzymatic activity of the immobilized enzyme is measured by measuring the amount of D-fructose produced when the enzyme is reacted with D-allulose as a substrate. First, the reaction mixture was composed of 100 mg of immobilized enzyme resin, Tris-HCl buffer (pH 7.5) (final concentration 50 mM), and 400 μl of D-allulose (final concentration 100 mM). The reaction was allowed to proceed for 10-30 minutes in a 30°C thermostatic water bath, and then immediately terminated by boiling at 100°C for 2 minutes. The reaction solution was then cooled to room temperature and desalted using an ion exchange resin (a mixed resin of 200CT and IRA67 (both manufactured by Organo Corporation)). The resulting solution was then filtered to prepare an analytical sample. Analysis was performed by measuring the peak area of ​​the D-fructose produced using high-performance liquid chromatography (column: GL-C611 (Hitachi), temperature: 60°C, eluent: 0.1 mM NaOH, flow rate: 1.0 ml / min, detector: RID-20A (Shimadzu)). The enzyme activity of the various supports used for enzyme immobilization was compared to select the optimal support. To confirm the influence of the carrier used for enzyme immobilization, the enzyme activity of the present enzyme immobilized on each of the following carriers was compared: HPA25L, WA30, IRA904CL, FPA54, FPA95, and XAD7HP. The relative activity was calculated by setting the peak area of ​​the sample with the largest peak area among the comparative samples as 100.The relative activity of various immobilized enzymes is examined and taken into consideration when selecting the immobilized enzyme with the highest activity. The greater the amount of crude enzyme immobilized, the higher the relative activity of each resin. The enzyme activity of immobilized enzymes is also compared depending on temperature. Reactions are carried out at various temperatures between 30 and 80°C, and the relative activity of each immobilized enzyme carrier is measured. Mg. ２＋ Whether or not ions are added, the enzyme immobilized using the synthetic adsorbent XAD7HP has a high relative activity at room temperature, while the enzymes immobilized using the anion exchange resins HPA25L, IRA904CL, and WA30 have a high relative activity at high temperatures. This shows that the immobilization carrier should be selected depending on the reaction temperature.

[0031] <Dried Form> Next, we will explain a dried form of an immobilized enzyme or protein, in which the enzyme or protein having the enzyme activity is a ketose 3-epimerase or a protein having ketose 3-epimerase activity. This is a freeze-dried form or a form dried in an incubator at 30°C. If the immobilized enzyme is stable, it can maintain its activity in both freeze-drying and 30°C incubator drying. In the dried form of an immobilized enzyme, the immobilization carrier is dried in the same way as the enzyme catalyst, so its movement is restricted, allowing for long-term storage. The dehydration process of drying can destabilize enzyme molecules, which are originally stable in the presence of water molecules, due to the loss of hydrogen bonds. However, in the presence of an appropriate carrier, the enzyme molecules and carrier molecules hydrogen bond, allowing them to exist stably even at low water contents. We have also found that, despite the complex structure of the enzyme catalyst, in the immobilized form, the (freeze) drying process itself does not cause denaturation. It has been found that ketose 3-epimerase (e.g., D-allulose 3-epimerase) itself can be stored in a dried state as an immobilized enzyme supported on an ion exchange resin or the like. Experiments have shown that when the enzyme is stabilized at the immobilized stage, with activity at the same level as before immobilization at the same or higher temperatures, the enzyme (ketose 3-epimerase) or a protein having the enzyme activity (protein having ketose 3-epimerase activity) can be dried to a "dried form that does not require washing before use, retains transport resistance, and is inhibited from losing activity."

[0032] The dried enzymes have been stored both refrigerated and at room temperature, with no significant loss of activity observed under either condition. Therefore, refrigeration is not necessary when shipping the dried enzymes. Because the dried immobilized enzymes contain no moisture, they can be transported in either paper or plastic containers. Plastic containers are used for storage in experiments. Because no buffers or additives are required, the cost of transporting the same amount of immobilized enzyme is reduced compared to methods that require buffers or additives. Furthermore, because the enzymes are simply dried, there is no need to remove additives (i.e., wash the immobilized enzymes) before use. Factors that determine the method of transportation include: 1) price, 2) transit time (several days to several months for ocean transport, 4–7 days for air transport), and 3) temperature control (if temperature control is required to maintain quality, specialized companies must be contracted; otherwise, temperatures can reach 70°C on ships crossing the equator, and container yards at ports and transit points can be subject to high temperatures depending on the region and season). Dried immobilized enzymes offer an advantageous solution to these problems. Dried enzymes are only viable if the enzyme can withstand drying.

[0033] Specific examples of the present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

[0034] (Drying of immobilized enzyme) <Cultivation of recombinant E. coli> Recombinant E. coli that highly expresses a mutant enzyme in which three site-specific mutations (V129I, A200V, S218N) were added to the Y586-1-derived D-allulose 3-epimerase was cultured, and the site-specific mutant enzyme was expressed by adding IPTG to a final concentration of 1 mM. After cultivation, the recombinant E. coli was collected by centrifugation.

[0035] <Preparation of Partially Purified Enzyme> The resulting recombinant E. coli was suspended in 20 mM Tris-HCl buffer (pH 7.5), and the cells were disrupted using a bead mill. After treatment with the bead mill, the mixture was centrifuged to precipitate the cell debris, and the supernatant containing the site-directed mutant enzyme was collected as a crude enzyme solution. To precipitate nucleic acids, manganese chloride was added to the crude enzyme solution to a concentration of 10 mM, and the mixture was heat-treated at 60°C for 10 minutes. Proteins other than the target enzyme were thermally denatured and removed together with the nucleic acids by centrifugation. This yielded a partially purified enzyme.

[0036] <Preparation of immobilized enzyme> The obtained partially purified enzyme was added to an anion exchange resin (A111S) previously equilibrated with 20 mM Tris-HCl buffer (pH 7.5) to prepare an immobilized enzyme. The partially purified enzyme was added at 200 U per 1 g of carrier and the mixture was stirred appropriately to immobilize the enzyme on the carrier. After immobilization, the immobilized enzyme was washed with 20 mM Tris-HCl buffer (pH 7.5) to prepare the immobilized enzyme.

[0037] <Drying of immobilized enzyme> The immobilized enzyme was dried at 30°C and by freeze-drying. The immobilized enzyme was first wiped dry with Kimwipes and then placed in a container for drying. For drying at 30°C, the enzyme was left standing in an incubator for 24 to 48 hours to remove moisture. For freeze-drying, the enzyme was pre-frozen at -80°C and then freeze-dried for 24 to 48 hours to remove moisture.

[0038] <Storage of dried immobilized enzyme> The dried immobilized enzyme was stored at room temperature and 4°C.

[0039] <Measurement of activity of dried immobilized enzyme> An appropriate amount of stored dried immobilized enzyme was weighed out, and 700 μL of 100 mM Tris-HCl buffer (pH 7.5) was added. 700 μL of 200 mM D-allulose was added as a substrate to initiate the enzyme reaction. The reaction was carried out at 60°C for 10 minutes. After the reaction, the supernatant was collected and allowed to stand at 100°C for 3 minutes, then filtered through a desalting filter. The sugar composition of the reaction solution was analyzed using HCl to determine the activity.

[0040] The activity of immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes was examined over a 10-minute reaction. The immobilized enzymes, in which Y586-1-derived ketose 3-epimerase site-directed mutant enzymes were immobilized, were reacted at various temperatures for 10 minutes, and the activity was measured. D-allulose was used as the substrate at a final concentration of 100 mM. Tris-HCl buffer (pH 7.5) was used to achieve a final concentration of 50 mM. As shown in Figure 1, the activity at 80°C and 90°C was comparable, indicating that the maximum activity was achieved at a higher temperature than that of the non-immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes.

[0041] Immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes were heat-treated at various temperatures for 60 minutes and then cooled on ice. The reaction was then carried out at 60°C for 10 minutes, and the residual activity was measured. D-allulose was used as the substrate at a final concentration of 100 mM. Tris-HCl buffer (pH 7.5) was used to a final concentration of 50 mM. As shown in Figure 2, no decrease in activity was observed up to 80°C, demonstrating higher thermal stability than the non-immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzyme. While the non-immobilized enzyme lost its enzymatic activity even after 10 minutes of heat treatment, the immobilized enzyme maintained its activity even after 60 minutes of heat treatment, demonstrating its high stability.

[0042] The activity of the immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzyme was investigated using buffer solutions of different pH values ​​at 60°C for 10 minutes. D-allulose was used as the substrate at a final concentration of 100 mM. Various buffer solutions were used to achieve a final concentration of 50 mM. As a result, as shown in Figure 3, the pH at which maximum activity was observed was pH 6.0 (MES buffer).

[0043] The pH stability of immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes was investigated. The immobilized enzymes, which contained immobilized Y586-1-derived ketose 3-epimerase site-directed mutant enzymes, were left in 100 mM buffer solutions for 24 hours. The buffer solution was then removed, and the enzyme was incubated with MES buffer (pH 6.0) at a final concentration of 50 mM for 10 minutes at 60°C. The activity was then measured. D-allulose was used as the substrate at a final concentration of 100 mM. As shown in Figure 4, the enzyme exhibited high activity and stability at pH 5.0 to pH 9.0.

[0044] 5 is a table comparing the properties (optimum temperature, temperature stability, and optimum pH) of D-allulose 3-epimerase (purified enzyme) derived from Y586-1 (parent strain), site-directed mutant D-allulose 3-epimerase (purified enzyme) derived from Y586-1, and immobilized site-directed mutant D-allulose 3-epimerase (partially purified enzyme) derived from Y586-1. It can be seen that immobilization improves the optimum temperature and temperature stability, and shifts the optimum pH to the acidic side.

[0045] Figure 6 shows the drying method for immobilized enzymes. Immobilized enzymes were prepared by adding partially purified enzymes to 1 g of carrier at 200 U by heat treatment at 60°C for 10 minutes. The immobilized enzymes were dried in an incubator at 30°C or freeze-dried. The immobilized enzymes dried under each condition were stored at room temperature or 4°C.

[0046] The enzyme activity was measured for the immobilized enzyme before drying and for the immobilized enzyme dried under various conditions (stored at room temperature and 4°C). The enzyme activity was measured after a 10-minute reaction at 60°C. D-allulose was used as the substrate at a final concentration of 100 mM. Tris-HCl buffer (pH 7.5) was used to obtain a final buffer concentration of 50 mM. Figure 7(1) shows a comparison of the activity of the untreated and dried enzyme (30°C dried → room temperature storage, 30°C dried → refrigerated (4°C) storage, lyophilized → room temperature storage, lyophilized → refrigerated (4°C) storage) on days 0 and 192 of storage. No activity was observed in any of the samples. Figure 7(2) shows the relative activity of the dried enzyme (30°C dried → room temperature storage, 30°C dried → refrigerated (4°C) storage, lyophilized → room temperature storage, lyophilized → refrigerated (4°C) storage), with the untreated activity set at 100%. There was also no decrease in activity. The storage period was further extended by one month, and Figure 8 (1) shows a comparison of the activity of the untreated and dried enzyme (dried at 30°C → stored at room temperature, dried at 30°C → stored in a refrigerator (4°C), freeze-dried → stored at room temperature, freeze-dried → stored in a refrigerator (4°C)) on days 0 and 224 of storage. The activity remained unchanged in all samples. Figure 8 (2) shows the relative activity of the dried enzyme (dried at 30°C → stored at room temperature, dried at 30°C → stored in a refrigerator (4°C), freeze-dried → stored at room temperature, freeze-dried → stored in a refrigerator (4°C)) with the activity of the untreated enzyme taken as 100%. The activity also remained unchanged.

[0047] The highly active and thermostable ketose 3-epimerase produced by the Arthrobacter histidinolovorans Y586-1 strain, which has a high optimum temperature of 70°C and is therefore a highly thermostable enzyme, results in a higher D-allulose production yield when reacted at an optimum temperature of around 60 to 70°C. After immobilization, the highly active and thermostable ketose 3-epimerase and its site-specific mutant enzymes can be transported in a dried form, which is expected to pave the way for increasing the number of production bases around the world for the mass production of D-allulose using D-fructose as a raw material.

Claims

1. A dry body of an immobilized enzyme or an immobilized protein having the enzyme activity, The enzyme or the protein having the enzyme activity is a ketose 3-epimerase or a protein having ketose 3-epimerase activity, and is selected from the following enzyme (i) and protein (ii) having the enzyme activity: the temperature at which the immobilized enzyme or the immobilized protein having the enzyme activity exhibits maximum activity (optimum temperature) is improved before drying compared to before immobilization; And, The dried product does not contain any additives to maintain the enzyme activity, and the activity does not decrease for at least six months.

1. A dried product of an immobilized enzyme which is an enzyme that can withstand drying or an immobilized protein having said enzyme activity, Note (i) A ketose 3-epimerase derived from a microorganism belonging to the genus Arthrobacter, the ketose 3-epimerase having a molecular mass of 36 kDa as measured by SDS-PAGE and having the substrate specificity of the following (A) and (B) and the physicochemical properties of the following (C) and (D). (A) having the activity of epimerizing the 3-position of D- or L-ketose to produce the corresponding D- or L-ketose; (B) Among D- or L-ketohexoses, D-allulose has the highest epimerization activity at position 3. (C) The optimum pH for the reaction is 6 to 8. (D) The optimum reaction temperature is 60°C. (ii) A protein described in the following (a), (b), (c) or (d): (a) A protein consisting of the amino acid sequence represented by SEQ ID NO:

1. (b) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO:1, wherein the amino acid sequence does not substitute any amino acid at any of the positions H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO:1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO:1, and which has the ketose 3-epimerase activity of (A) and (B) above, and has a ketose 3-epimerase activity ratio (T70 / T50) at reaction temperatures of 70°C and 50°C of 1.55 or more. (c) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO:1, which does not have any amino acid substitutions at any of the amino acids H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO:1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO:1, which has the ketose 3-epimerase activity of (A) and (B) above, and has a residual activity of 31% or more after incubation at 60°C for 1 hour. (d) a protein consisting of an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO:1, which has an amino acid substitution at at least one site selected from H56, T59, A60, P77, L94, L97, D101, A109, A128, V129, V132, S144, S167, P172, E199, A200, K202, and S218 in the amino acid sequence represented by SEQ ID NO:1, and which has the ketose 3-epimerase activity of (A) and (B) above, and which has a higher ketose 3-epimerase activity ratio (T70 / T50) at reaction temperatures of 70° C. and 50° C. or a higher residual activity after incubation at 60° C. for 1 hour than the protein consisting of the amino acid sequence represented by SEQ ID NO:

1.

2. 2. A dried body of an immobilized enzyme or an immobilized protein having the enzyme activity, which is an enzyme that can withstand drying, as described in claim 1, wherein the dried body is a dried body of an enzyme or a protein having the enzyme activity supported on a carrier which is an ion exchange resin, an organic polymer carrier, and / or an inorganic carrier.

3. 3. A dried product of an immobilized enzyme that is an enzyme that can withstand drying according to claim 1 or 2, or an immobilized protein having said enzyme activity, which is a dried product that does not require washing before use, retains resistance to transportation, and is inhibited from decreasing in activity for at least six months or more.

4. A dried product of an immobilized enzyme or an immobilized protein having said enzyme activity, which is an enzyme capable of withstanding drying as described in any one of claims 1 to 3, wherein the dried product is a freeze-dried product or a product dried in an incubator at 30°C.

5. A dried form of an immobilized enzyme or immobilized protein having the enzyme activity, which is an enzyme that can withstand drying, as described in any one of claims 1 to 4, wherein the immobilized enzyme or immobilized protein having the enzyme activity is a crude enzyme present in a crushed bacterial cell material, or a partially purified enzyme from a crude enzyme solution, which is immobilized on a carrier.

6. The immobilized enzyme that is an enzyme that can withstand drying according to any one of claims 1 to 5, or a dried product of the immobilized protein having the enzyme activity, wherein the ketose 3-epimerase has substrate specificity in the order of D-allulose, D-tagatose, L-tagatose, D-fructose, and L-allulose.

7. 7. The immobilized enzyme or the dried product of the immobilized protein having the enzyme activity according to claim 1, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter histidinolovorans.

8. A method for producing a dried product of an immobilized enzyme or an immobilized protein having ketose 3-epimerase activity, which is an enzyme that can withstand drying, characterized in that an enzyme ketose 3-epimerase selected from the following enzyme (i) and a protein having said enzyme activity (ii) or said protein having ketose 3-epimerase activity is immobilized to produce an immobilized enzyme or protein having said enzyme activity that has an improved temperature (optimum temperature) at which it shows maximum activity compared to before immobilization, and then drying is performed without adding an additive for maintaining the enzyme activity. Note (i) A ketose 3-epimerase derived from a microorganism belonging to the genus Arthrobacter, the ketose 3-epimerase having a molecular mass of 36 kDa as measured by SDS-PAGE and having the substrate specificity of the following (A) and (B) and the physicochemical properties of the following (C) and (D). (A) having the activity of epimerizing the 3-position of D- or L-ketose to produce the corresponding D- or L-ketose; (B) Among D- or L-ketohexoses, D-allulose has the highest epimerization activity at position 3. (C) The optimum pH for the reaction is 6 to 8. (D) The optimum reaction temperature is 60°C. (ii) A protein described in the following (a), (b), (c) or (d): (a) A protein consisting of the amino acid sequence represented by SEQ ID NO:

1. (b) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO:1, wherein the amino acid sequence does not substitute any amino acid at any of the positions H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO:1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO:1, and which has the ketose 3-epimerase activity of (A) and (B) above, and has a ketose 3-epimerase activity ratio (T70 / T50) at reaction temperatures of 70°C and 50°C of 1.55 or more. (c) A protein consisting of an amino acid sequence having 75% or more identity with the amino acid sequence represented by SEQ ID NO:1, which does not have any amino acid substitutions at any of the amino acids H6, S63, M110, E146, E152, D179, H182, H205, R211, and E240 in the amino acid sequence represented by SEQ ID NO:1, and which maintains the three-dimensional structure of a protein consisting of the amino acid sequence represented by SEQ ID NO:1, which has the ketose 3-epimerase activity of (A) and (B) above, and has a residual activity of 31% or more after incubation at 60°C for 1 hour. (d) a protein consisting of an amino acid substitution mutant of a protein consisting of the amino acid sequence represented by SEQ ID NO:1, which has an amino acid substitution at at least one site selected from H56, T59, A60, P77, L94, L97, D101, A109, A128, V129, V132, S144, S167, P172, E199, A200, K202, and S218 in the amino acid sequence represented by SEQ ID NO:1, and which has the ketose 3-epimerase activity of (A) and (B) above, and which has a higher ketose 3-epimerase activity ratio (T70 / T50) at reaction temperatures of 70° C. and 50° C. or a higher residual activity after incubation at 60° C. for 1 hour than the protein consisting of the amino acid sequence represented by SEQ ID NO:

1.

9. The method for producing a dry product of an immobilized enzyme that is an enzyme that can withstand drying or an immobilized protein having the enzyme activity according to claim 8, wherein the enzyme ketose 3-epimerase or the protein having ketose 3-epimerase activity is supported and immobilized on a carrier that is an ion exchange resin, an organic polymer carrier, and / or an inorganic carrier.

10. A method for producing a dry product of an immobilized enzyme or an immobilized protein having said enzyme activity, which is an enzyme capable of withstanding drying according to claim 8 or 9, comprising drying by freeze-drying or drying in an incubator at 30°C.

11. A method for producing a dried product of an immobilized enzyme or an immobilized protein having the enzyme activity, which is an enzyme that can withstand drying, as described in any one of claims 8 to 10, comprising immobilizing a crude enzyme present in a crushed bacterial cell product or a partially purified enzyme from a crude enzyme solution on a carrier.

12. The method for producing a dried product of an immobilized enzyme or protein capable of withstanding drying according to any one of claims 8 to 11, wherein the substrate specificity of the ketose 3-epimerase is in the order of D-allulose, D-tagatose, L-tagatose, D-fructose, and L-allulose.

13. A method for producing a dried product of an immobilized enzyme or an immobilized protein having the enzyme activity, which is an enzyme capable of withstanding desiccation, according to any one of claims 8 to 12, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter histidinolovorans.

14. A method for producing a dried product of an immobilized enzyme or an immobilized protein having the enzyme activity according to any one of claims 8 to 13, wherein the microorganism belonging to the genus Arthrobacter is the Arthrobacter histidinolovorans Y586-1 strain deposited at the Patent Microorganisms Depositary Center under the accession number NITE BP-02813.

15. 8. A method for preserving a dried body of an immobilized enzyme or an immobilized protein having said enzyme activity, which is an enzyme that can withstand desiccation as described in any one of claims 1 to 7, characterized in that the method is carried out either at refrigeration or at room temperature.

16. A method for preserving a dried form of an immobilized enzyme which is an enzyme capable of withstanding desiccation as described in claim 15 or an immobilized protein having said enzyme activity, which is enclosed in a sealable container made of paper, plastic or metal.