KR20230058757A - Method for producing D-allose from D-allulose using L-rhamnose isomerase from Paenibacillus baekrokdamisoli - Google Patents

Abstract

The present invention relates to a producing method of D-allose from D-allulose using an L-rhamnose isomerase derived from Paenibacillus baekrokdamisoli. According to the producing method of the present invention, allose can be mass-produced from allulose safely and with high efficiency by using a microbial-derived L-rhamnose isomerase. The produced allulose can be very useful in various fields, not only in low-calorie sweetener industries but also as a material for health foods, medicine, and cosmetics. The producing method of D-allose is provided which comprises a step of reacting D-allulose and an L-rhamnose isomerase derived from Paenibacillus baekrokdamisoli.

Description

Method for producing D-allose from D-allulose using L-rhamnose isomerase from Paenibacillus baekrokdamisoli}

The present invention relates to a method for producing D-allose from D-allulose using L-rhamnose isomerase derived from Fenibacillus whiterhodamisoly.

The market for alternative sweeteners to replace sugar and fructose, which have been pointed out as causes of obesity, has recently been continuously growing. However, alternative sweeteners (oligosaccharides, sugar alcohols, similar sweeteners, etc.) have differences in physical properties from those of conventional sugar or fructose, and thus are limited in use.

Meanwhile, in Japan, as syrup containing rare sugars (about 15%) has gained sensational popularity, saccharide sweeteners that can replace sugar are widely used in the fields of syrups, beverages, confectionery, baking, and soy sauce, mainly by large companies. there is. As the 3rd carbon epimer of glucose (D-glucose), D-allose, known as a rare monosaccharide that is an isomer of allulose, is one of the rare natural saccharides that exist in extremely small amounts in nature. It is one, and it has sweetness, but its calories are very low compared to sugar, and it is expected to be a material that can receive very high attention as an alternative sweetener in the future. In addition, allose is used as an anti-cancer substance because it has the property of inhibiting the proliferation of cancer cells, and because it has the function of inhibiting organ damage due to ischemic action, it can also be used as an organ preservation solution. In addition, allose has been reported to inhibit the formation of segmented neutrophils as well as the reduction of platelets, and thus is one of the rare saccharides currently receiving medical attention. Although allose has such a high useful value, it is necessary to secure allose in a sufficient amount in order to effectively supply it to the field of medicine because only a very small amount exists in nature. Also, until now, allose has been mainly produced only by chemical synthesis. However, the chemical synthesis method of allose has several serious problems in the production process. In fact, problems in the working environment requiring high temperature and high pressure, generation of additional saccharide after chemical reaction, complex separation and purification process of allose, and environmental pollution due to chemical waste generated in this process are caused.

In order to solve these problems, a biological production method for obtaining allose in high yield using an enzyme expressed by microorganisms has been studied as an environment-friendly method capable of efficiently producing sugar with high specificity. Specifically, Pseudomonas stutzeri, Bacillus pallidus , Thermoanaerobacterium saccharolyticum , Caldicellulosiruptor saccharolyticus , and Bacillus subtilis ( It has been reported that L-rhamnose isomerase derived from Bacillus subtilis can be used for an allose production reaction.

As a technology related to the production method of allose, Korean Patent No. 1998477 discloses an L-rhamnose isomerase variant derived from Clostridium stuccolarium and a method for producing allose from allulose using the same, and Korean Patent No. No. 1841267 discloses a ribose-5-phosphate isomerase, a method for producing the same, and a method for producing allose using the same. There is no disclosure of a method for producing D-allose from

The present invention has been derived from the above needs, and the present invention consists of the amino acid sequence of SEQ ID NO: 1, Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) Using L-rhamnose isomerase derived from L-rhamnose isomerase, D-allose can be effectively produced from D-allulose under the conditions of a reaction temperature of 55 to 65 ° C and a reaction pH of 7.5 to 8.5. By confirming that there is, the present invention was completed.

In order to solve the above problems, the present invention is D- allulose (D-allulose) and Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) It provides a method for producing D-allose comprising the step of reacting the derived L-rhamnose isomerase.

In addition, the present invention consists of the amino acid sequence of SEQ ID NO: 1, Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) Provided is a composition for producing D-allose containing a derived L-rhamnose isomerase.

The present invention relates to a method for producing D-allose from D-allulose using L-rhamnose isomerase derived from Panibacillus baekrhamnose isomerase, and the method of the present invention relates to a microorganism-derived L-rhamnose isomerase It is possible to mass-produce allulose from allulose safely and with high efficiency by using, and the allulose produced in this way can be used very usefully in various fields as a material for health food, medicine, and cosmetics as well as the low-calorie sweetener industry. .

1 is Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) This is a PCR result for amplifying the derived L-rhamnose isomerase gene fragment. M: Size marker.
Figure 2 is Panibacillus baekrokudamisolii This is the result of confirming the L-rhamnose isomerase gene fragment from the recombinant vector pET-PBRI containing the derived L-rhamnose isomerase and pET-28a(+) was prepared and transformed into E. coli. M: size marker, 1: recombinant vector pET-PBRI containing L-rhamnose isomerase and pET-28a(+).
Figure 3 is the result of confirming the expression of the recombinant L-rhamnose isomerase derived from Fanibacillus whiterhodamisolii. M: size marker, 1: unpurified recombinant L-rhamnose isomerase, 2: purified L-rhamnose isomerase.
4 is a result of comparing the optimum concentration of Mn ²⁺ , which is the optimum metal ion, of the recombinant L-rhamnose isomerase derived from Fennibacillus baekrhodamisoli in the production of D-allose.
5 is a result of comparing the activities of recombinant L-rhamnose isomerase derived from Fanibacillus whiterhodamisolyi according to pH in the production of D-allose.
6 is a result of comparing the activity of recombinant L-rhamnose isomerase derived from Fanibacillus whiterhodamisolyi according to temperature in the production of D-allose.
7 is a result of confirming the temperature stability of the recombinant L-rhamnose isomerase derived from Fanibacillus whiterhodamisolii in the production of D-allose.
8 is a result of confirming the effect of the concentration of D-allulose on the production of D-allose by the recombinant L-rhamnose isomerase derived from Fenibacillus whiterhodamisolii in the production of D-allose.
FIG. 9 shows the results of confirming the effect of the concentration of the recombinant L-rhamnose isomerase derived from Fennibacillus whiterhodamisoly on the production of D-allose from D-allulose in the production of D-allose.
10 is a result of confirming the optimal reaction time of D-allulose and recombinant L-rhamnose isomerase derived from Fenibacillus whiterhodamisoly in the production of D-allose. A: Conversion rate of allulose from allulose with reaction time, B: D-allose production with reaction time.

In order to achieve the object of the present invention, the present invention is D- allulose (D-allulose) and Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) It provides a method for producing D-allose, including the step of reacting the derived L-rhamnose isomerase.

In the method for producing D-allose of the present invention, the L-rhamnose isomerase is characterized in that it consists of the amino acid sequence of SEQ ID NO: 1. The scope of the L-rhamnose isomerase according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 1 and functional equivalents of the protein. "Functional equivalent" means at least 80% or more, more preferably 90% or more, still more preferably 95% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. It refers to a protein that exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 1.

The optimal enzyme concentration of the L-rhamnose isomerase may be 90 to 110 U/ml, preferably 104 U/ml, but is not limited thereto.

In addition, the reaction temperature may be 55 to 65 ° C, the reaction pH may be 7.5 to 8.5, preferably the reaction temperature may be 60 ° C, and the reaction pH may be 8.0, but is not limited thereto.

In addition, the enzymatic reaction may be performed under 0.3-2mM Mn ²⁺ conditions, preferably under 0.5mM Mn ²⁺ conditions, but is not limited thereto.

In addition, the concentration of the D-allulose may be 10% (w / v) to 60% (w / v), preferably the concentration of the D- allulose is 45% (w / v) to It may be 55% (w/v), more preferably 50% (w/v), but is not limited thereto.

In addition, the reaction may be carried out for 1 hour to 6 hours, preferably the reaction may be carried out for 2 hours to 5 hours, and more preferably for 3 hours, but is not limited thereto.

The present invention also consists of the amino acid sequence of SEQ ID NO: 1, Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) Provided is a composition for producing D-allose containing a derived L-rhamnose isomerase.

In the composition for producing D-allose of the present invention, the substrate of the L-rhamnose isomerase may be D-allulose, but is not limited thereto.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited thereto.

Example 1. Fanibacillus baekrhodamisolii ( Paenibacillus baekrokdamisoli ) PCR amplification of L-rhamnose isomerase gene (WP_125653350.1) derived from KCTC 33723

Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) PCR conditions for amplifying the derived L-rhamnose isomerase gene were preheated at 95° C. for 5 minutes; Denaturation 95° C. for 1 minute; annealing 55° C. for 30 seconds; and extension (extension) 72 ℃, 1 minute was repeated a total of 30 times, the PCR amplified fragment was confirmed by electrophoresis on a 1.5% agarose gel (agarose gel). As a result, it was confirmed that the L-rhamnose isomerase gene (1.2Kb) was expressed (FIG. 1).

Example 2. Fanibacillus baekrondamisoli ( Paenibacillus baekrokdamisoli ) Cloning of L-rhamnose isomerase gene (WP_125653350.1) fragment derived from KCTC 33723 with pET-28a(+) vector

After treating 20 μl of pET-28a(+) plasmid with restriction enzymes Nde I and Xho I at 37°C for 4 hours, injecting 1 μl of CIAP (calf intestinal alkaline phosphatase) to prevent self-ligation, the same temperature was further treated for 1 hour. And the secured pET-28a(+) vector and L-rhamnose isomerase For ligation of the fragment, pET-28a(+) vector, L-rhamnose isomerase fragment and T4 DNA ligase (ELPIS, Deajeon, Korea) were reacted at 16°C for 14 hours, and L-rhamnose A pET-PBRI vector containing isomerase was constructed. Then, host cells, E. coli BL21 (DE3) and the pET-PBRI vector were transformed using a MicroPulser ^TM electroporator (MicroPulser electroporator, California, USA) and LB containing kanamycin (40 μg/ml). After smearing on a solid medium, the culture was smeared at 37 ° C. for 24 hours. Then, colonies on the medium ^were isolated and cultured in 20 ml of LB liquid medium containing kanamycin (40 μg/ml) at 37° C. for 24 hours. Vectors were extracted. After treating the extracted pET-PBRI vector with restriction enzymes Nde I and Xho I at 37°C for 4 hours, the L-rhamnose isomerase gene fragment (1.2 Kb) was confirmed on a 1.5% agarose gel (FIG. 2). ).

Example 3. Fanibacillus baekrondamisoli ( Paenibacillus baekrokdamisoli ) Expression and confirmation of KCTC 33723-derived recombinant L-rhamnose isomerase (L-RhI)

Germany)에 주입 후 50mM EPPS 버퍼(pH 8.0)로 세척하고 재조합 L-RhI의 발현 및 정제를 확인하기 위해 SDS-PAGE(Sodium dodecyl sulfate polyacrylamide gel eletropohoresis) 상에서 분석을 수행하였다. 이후에, 쿠마시 브릴리언트 블루 R250(Coomassie Brilliant Blue R250)에 1시간 동안 침지하여 단백질을 염색하고 탈염색(destaining) 버퍼로 탈색 후 발현된 재조합 L-RhI(46kDa)를 확인하였다(도 3).The L-rhamnose isomerase in E. coli BL21 strain transformed with the pET-PBRI vector containing the L-rhamnose isomerase derived from Panibacillus white rhodamisoli carried out in Example 2 (hereinafter referred to as 'recombinant L-RhI') In order to express the gene, inoculate 4 ml of LB liquid medium containing kanamycin (40 μg / ml) and pre-incubate at 37 ° C. for 24 hours at 150 rpm using a shaking incubator (HANBEAK, Bucheon, Korea), It was subcultured in 500 ml of LB liquid medium of the same composition. Then, when OD ₆₀₀ reached around 0.6, 0.1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added, and expression of L-RhI was induced at 16°C and 100 rpm for 16 hours. To purify the expressed L-RhI, the culture medium was centrifuged (6,000 x g , 4° C., 30 min) to remove the supernatant and resuspended in 200 mM EPPS buffer (pH 7.5). After repeating this process twice, the recovered cells were resuspended in a lysis buffer (50 mM KH ₂ PO ₄ , 300 mM NaCl, pH 8.0), disrupted using an ultrasonicator (VCX 130, SONICS, California, USA), and centrifuged. (10,000 x g , 4° C., 30 min) and recovered as a supernatant (recombinant L-RhI). For the purification of expressed recombinant L-RhI, a Bio-Scale™ Mini Profinity™ IMAC cartridge column (Bio-Rad, California, USA) was used to purify recombinant L-RhI. The column purification method is as follows; First, after equilibrium with 10 ml (2 ml/min) of lysis buffer (pH 8.0), 10 ml (1 ml/min) of recombinant L-RhI was flowed on the column. Next, wash buffer (50 mM KH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole) 10 ml (2 ml/min) and lysis buffer (50 mM KH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole) 10 ml (1 ml) /min) was sequentially flowed to elute the recombinant L-RhI enzyme. And to remove the imidazole component of the purified recombinant L-RhI, 10,000 MW VIVASPIN20 (sartorius, G

Germany), washed with 50 mM EPPS buffer (pH 8.0), and analyzed on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel eletropohoresis) to confirm the expression and purification of recombinant L-RhI. Thereafter, the protein was stained by soaking in Coomassie Brilliant Blue R250 for 1 hour, and after destaining with a destaining buffer, the expressed recombinant L-RhI (46 kDa) was confirmed (FIG. 3).

Example 4. Confirmation of optimal metal ion and metal ion concentration conditions for recombinant L-rhamnose isomerase (L-RhI)

In order to confirm the optimal activity of recombinant L-rhamnose isomerase for the production of D-allose from D-allulose, activity according to metal ion and metal ion concentration conditions The change in was measured, The amount of enzyme used with L-RhI is 0.6 U/ml concentration. Prior to analysis, metal ions in recombinant L-rhamnose isomerase were completely removed using EDTA, and then 0.5 mM Co ²⁺ , Mn ²⁺ , Ni ²⁺ , Ba ²⁺ , Fe ² corresponding to divalent metal ions were added. ⁺ , Cu ²⁺ , Mg ²⁺ and Enzymatic activity was measured by adding Zn ²⁺ metal ions, respectively, and enzyme activity was measured in the range of 0.1 to 3 mM to confirm the optimal metal ion concentration. As a result, as shown in Table 1 below, the metal ion showing the maximum activity was Mn ²⁺ , and the optimum concentration was confirmed to be 0.5 mM (FIG. 4).

Optimal metal ion and its optimal concentration of L-rhamnose isomerase Metal ion (0.5 mM) Relative activity (%) None 0 EDTA 0 Mn ²⁺ 100 ±2 Co ²⁺ 62±0.8 Mg ²⁺ 39 ±1 Ni ²⁺ 13 ±1 Zn ²⁺ 9.3 ±2 Ba ²⁺ 0 Fe ²⁺ 0 Cu ²⁺ 0

Example 5. Confirmation of pH, temperature and temperature stability for optimal activity of recombinant L-rhamnose isomerase

In order to measure the activity of recombinant L-rhamnose isomerase, it is converted from D-allulose using an electrochemical detector from Bio-LC Systems (Dionex, Sunnyvale, CA) and a CarboPac PA I column. D-allose was analyzed. In order to confirm the optimal activity of recombinant L-rhamnose isomerase for the production of D-allose from D-allulose, the change in activity according to pH and temperature conditions was measured, and the amount of enzyme used in the present invention was 0.6 U /ml is the concentration. pH 7 to 8.5 was titrated with an EPPS buffer, pH 8.5 to 10.5 with a glycine-NaOH buffer, and the temperature was measured in the range of 30 to 80 °C. As a result, as shown in FIGS. 5 and 6, the optimum pH was 8.0 and the optimum temperature was confirmed to be 60°C. In addition, as a result of measuring the temperature stability of the recombinant L-rhamnose isomerase in the range of 50 to 75 ° C, the temperature stability was maintained at a very high temperature up to 65 ° C, and the temperature stability rapidly decreased from 70 ° C (Fig. 7). ), when the half-life of the recombinant L-rhamnose isomerase activity of the present invention (half-life, the time when the enzyme activity is 50%) was measured, 417 hours at 50 ° C, 57 hours at 55 ° C, 60 ° C and It was confirmed to have 50% activity at 65 ° C. for 27 hours and 20 hours, respectively (Table 2). Through these results, it was demonstrated that the recombinant L-rhamnose isomerase of the present invention has excellent temperature stability by maintaining activity for a long time up to 65 ° C.

Half-life of recombinant L-rhamnose isomerase activity Temperature (℃) half-life (hr) formula 50 417 y=-0.0020x+100.1015 55 57 y=-0.0148x+100.5835 60 27 y=-0.0309x+100.8472 65 20 y=-0.0425x+100.6490 70 3.3 y=-0.2712x+104.4802 75 0.2 y=-3.3100x+88.9

Example 6. Confirmation of substrate concentration, enzyme concentration and reaction time for the production of D-allose from D-allulose using recombinant L-rhamnose isomerase

In order to confirm the maximum concentration of D-allulose, which is a substrate for producing D-allulose, in the concentration range of 10 to 50% (w / v) (progressing to 50% due to the viscosity of sugar) D-allulose 170 U/ml of recombinant L-rhamnose isomerase was reacted with agarose at 60° C. for 6 hours under conditions of 50 mM EPPS buffer (pH 8.0) and 0.5 mM Mn ²⁺ .

As a result, it was confirmed that the maximum substrate concentration for the production of D-allose was 50% (w/v) of D-allulose (FIG. 8). To determine the optimal concentration of recombinant L-rhamnose isomerase for the production of D-allulose from D-allulose, 50% (w/v) D-allulose and 1 to 170 U/ml of enzyme was reacted, and the reaction conditions were reacted for 6 hours at 60° C. under 50mM EPPS buffer (pH 8.0) and 0.5mM Mn ²⁺ conditions. As a result, the optimum enzyme concentration for D-allose production was confirmed to be 104 U/ml (FIG. 9).

In addition, in order to confirm the optimal enzyme-substrate reaction time for the production of D-allulose from D-allulose, 50% (w / v) D-allulose was reacted in the range of 10 to 360 minutes, Reaction conditions were reacted at 60 ℃ for 6 hours under the conditions of 50mM EPPS buffer (pH 8.0) and 0.5mM Mn ²⁺ . As a result, the optimal reaction time for the production of D-allose was confirmed to be 3 hours (FIG. 10A), and at this reaction time, D-allose was produced at a conversion rate of about 25% from D-allulose (FIG. 10B ).

<110> DAEGU CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> Method for producing D-allose from D-allulose using L-rhamnose isomerase from Paenibacillus baekrokdamisoli <130> PN21217 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 420 <212> PRT <213> Paenibacillus baekrokdamisoli <400> 1 Met Asp Gln Lys Val Val Asn Asn Tyr Glu Ala Ala Lys Glu Leu Tyr 1 5 10 15 Ala Gln His Gly Ile Asp Val Asp Gln Val Leu Asp Arg Leu Asn Thr 20 25 30 Ile Lys Ile Ser Met His Cys Trp Gln Gly Asp Asp Val Arg Gly Phe 35 40 45 Leu Asn Lys Asp Gln Ala Leu Thr Gly Gly Ile Ser Val Thr Gly Asn 50 55 60 Tyr Pro Gly Ala Ala Thr Thr Pro Ala Glu Leu Arg Ala Asp Leu Glu 65 70 75 80 Lys Ala Phe Ser Leu Ile Pro Gly Lys His Lys Val Asn Leu His Ala 85 90 95 Ile Tyr Ala Asp Thr Asp Glu Val Val Glu Leu Asp Lys Ile Glu Pro 100 105 110 Lys His Tyr Gln Lys Trp Val Asp Trp Ser Lys Glu Gln Gly Leu Gly 115 120 125 Leu Asp Phe Asn Pro Thr Cys Phe Ser His Glu Lys Ser Lys Asp Gly 130 135 140 Phe Thr Leu Ser His Pro Asp Pro Glu Ile Arg Arg Phe Trp Ile Asp 145 150 155 160 His Cys Lys Ala Ser Arg Lys Ile Gly Ala Tyr Phe Gly Glu Gln Leu 165 170 175 Gly Gln Thr Cys Val Thr Asn Val Trp Val Pro Asp Gly Phe Lys Asp 180 185 190 Val Pro Ala Asp Arg Met Ala Pro Arg Gln Arg Leu Lys Asp Ala Leu 195 200 205 Asp Gln Val Phe Ala Glu Glu Ile Asn Pro Ala His Asn Leu Asp Ala 210 215 220 Ile Glu Ser Lys Leu Phe Gly Ile Gly Ser Glu Ala Tyr Val Val Gly 225 230 235 240 Ser His Glu Phe Tyr Met Gly Tyr Gly Ile Arg Asn Asn Lys Leu Ile 245 250 255 Cys Leu Asp Ala Gly His Phe His Pro Thr Glu Val Ile Ser Asn Lys 260 265 270 Leu Ser Ser Leu Ala Leu Phe Thr Asp Gly Ile Leu Leu His Val Ser 275 280 285 Arg Pro Met Arg Trp Asp Ser Asp His Val Val Thr Leu Asp Asp Glu 290 295 300 Leu Ile Asp Ile Gly Arg Glu Leu Val Arg Gly Asp Leu Leu Asp Lys 305 310 315 320 Thr His Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Val Ala 325 330 335 Ala Trp Val Ile Gly Thr Arg Asn Thr Ile Lys Ala Leu Leu Arg Gly 340 345 350 Met Leu Asp Pro Ile Glu Ala Leu Lys Ala Ala Glu Leu Glu Gly Asp 355 360 365 Tyr Thr Thr Arg Leu Ala Leu Thr Glu Glu Phe Lys Ser Tyr Pro Phe 370 375 380 Gly Ala Val Trp Asp Tyr Tyr Cys Ala Gln Gln Gly Val Pro Val Arg 385 390 395 400 Glu Asp Trp Leu Ala Val Val Lys Asp Tyr Glu Lys Asn Val Leu Leu 405 410 415 Gln Arg Gly Val 420

Claims (7)

D-allulose and Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) A method for producing D-allose comprising the step of reacting the derived L-rhamnose isomerase. The method of claim 1, wherein the L-rhamnose isomerase is composed of the amino acid sequence of SEQ ID NO: 1. The method for producing D-allose according to claim 1, wherein the reaction temperature is 55 to 65 °C and the reaction pH is 7.5 to 8.5. The method for producing D-allose according to claim 1, wherein the concentration of D-allulose is 10% (w/v) to 60% (w/v). The method of claim 1, wherein the reaction is performed using 90 to 110 U/ml of L-rhamnose isomerase having the amino acid sequence of SEQ ID NO: 1, and the reaction temperature is 55 to 65° C. under 0.3 to 2 mM Mn ²⁺ conditions. , The reaction pH is 7.5 to 8.5, the concentration of D-allulose is 45% (w / v) to 55% (w / v), D-allose, characterized in that carried out for 2 hours to 5 hours production method. Consisting of the amino acid sequence of SEQ ID NO: 1, Panibacillus baekrokdamisoli ( Paenibacillus baekrokdamisoli ) A composition for the production of D-allose containing a derived L-rhamnose isomerase. The composition for producing allose according to claim 6, wherein the substrate of the L-rhamnose isomerase is D-allulose.

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