KR101895914B1 - Preparation method of D-allose using a novel L-rhamnose isomerase - Google Patents

Abstract

The present invention relates to a novel pharmaceutical composition for inhibiting proliferation of cancer cells by L-rhamnose isomerase derived from Clostridium stercorarium and for inhibiting organ damage due to ischemic action And a method for mass production of the rare saccharide Alross at a high yield. The production method of the present invention is characterized in that alouls is produced by reacting alulose with an L-rhamnose isomerase protein derived from a Clostridium sulphate cell having D-allose isomerization activity.
The method for producing an alos production method of the present invention is environmentally friendly by using an enzyme and produces only a high specificity of alosin without producing additional saccharides other than the desired saccharide and can be used as an efficient method for mass production of alos.

Description

Preparation method of D-allose using a novel L-rhamnose isomerase < RTI ID = 0.0 >

The present invention relates to a novel L-rhamnose, and more particularly, to a method for producing aloses by converting an allylus with an L-rhamnose isomerase protein derived from Clostridium stercorarium .

Usually, alos is an epimer of glucose (D-glucose) 3 and is known as a rare sugar monosaccharide as an isomer of alulose. Such an inhibition of the proliferation of cancer cells can be used as an anticancer substance, and it has a function of suppressing organs damage due to ischemic action and can be used as a long-term preservation liquid, and inhibition of segmented neutrophil leukocyte formation, It is a rare saccharide that has a lot of attention because it has the functionality to inhibit the decrease and can be medically applied. (Hossain et al., J. Hepatobiliary Pancreat Surg. 10: 218225, 2003. Hossain et al., J. Hepatobiliary Pancreat Surg. 11: 181189, 2004 .; Hossain et al., J. Biosci. Bioeng. 101: 369371, 2006)

Because of the high availability in the pharmaceutical industry, there is a very small amount of ALROS in the natural world. Therefore, it is necessary to secure enough ALROS in order to effectively apply ALROS. In general, aloses have been produced by chemical methods. These chemical production methods have various serious problems such as the danger of work environment requiring high temperature and high pressure, the separation and purification process of complex alcohol due to the addition of sugar after chemical reaction, and the problem of environmental pollution caused by chemical waste .

In order to solve these problems, a biological production method has been developed in which an enzyme produced by a microorganism is used as a method for efficiently producing an objective sugar with high environmental specificity and high specificity and a high yield of aloses is obtained . Pseudomonas stutzeri and its derived L-rhamnose isomerase have been reported as specific biological production methods (Leang et al., Biochim Biophys Acta. 1674: 68-77, 2004). However, there is a problem in that alos production by the Rum Nos-isomerization enzyme derived from Pseudomonas shukerii produces a large amount of altrose besides alos. (Park et al., Biotechnol Lett. 29: 13871391, 2007), ribose-5-phosphate isomerase has been reported to produce alos by using ribose-5-phosphate isomerase -Phosphate isomerase has low activity. Therefore, it is necessary to develop enzymes that can produce high yields of aloses in a short time without the production of altrose.

 Therefore, the present inventors attempted to isolate an enzyme capable of producing a high-yield aldolase, which has a high enzyme activity on alulose without producing a by-product, altrose. For reference, the L-rhamnose isomerase has the highest activity for converting rhamnose into rhamnulose with metal dependency, but has activity against several rare saccharides due to its wide substrate specificity, Has already been reported to have the activity to convert Ross to Alloz.

In view of this fact, it has been proved that al-Rhos can be produced from alurulose, which is a substrate, by using the el-rhamnose isomerase derived from Clostridium stearicallium. We constructed a large-scale expression vector to produce large quantities of enzymes using E. coli to produce alos without any byproducts and to provide a biological method for mass production of aloses by examining optimal reaction conditions (temperature, pH, substrate concentration, etc.) Thereby successfully completing the present invention.

An object of the present invention is to provide a method for producing aloses by reacting an el-rhamnose isomerase protein comprising the amino acid sequence of SEQ ID NO: 1 with alulose.

In order to achieve the above object, the present invention provides a method for producing alos by reacting an el-rhamnose isomerase protein composed of the amino acid sequence of SEQ ID NO: 1 with alulose.

In the present invention, the "el-lambda isomerase" is known to be an enzyme that converts rhamnose to rhamnulose, but it has been known that it has an activity capable of simultaneously carrying out an isomerization reaction against several rare saccharides with a wide substrate specificity. Considering the function of this enzyme, we confirmed its applicability to the production of the rare saccharide, and found that it has an activity of converting the high concentration of the allylose to the high yield by using the above-mentioned L-rhamnose isomerase It was first demonstrated in the invention.

For the purpose of the present invention, the above-mentioned L-rhamnose isomerase may be an L-rhamnose isomerase derived from Clostridium stercorarium , more preferably an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 But is not limited to, a l-lambda isomerase comprising a gene consisting of the nucleotide sequence described.

The L-rhamnose isomerase has not only the amino acid sequence shown in SEQ ID NO: 1 but also an amino acid sequence having 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, , Preferably not less than 98%, and most preferably not less than 99%, as long as it is a protein having an activity of L-rhamnose capable of substantially converting alu- lose to alos, It is obvious that a protein variant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added is also included within the scope of the present invention if it is an amino acid sequence having substantially the same or a corresponding biological activity as that of L-rhamnose .

The similarity is intended to indicate the similarity with the amino acid sequence of the wild type protein or the nucleic acid sequence encoding the wild type protein and includes the sequence having the same sequence as the amino acid sequence or the nucleic acid sequence of the present invention above . Comparison of these similarities can be performed using a visual program or a comparison program that is easy to purchase.

As the recombinant vector, any vector may be used as long as it is a plasmid vector used in the art for gene recombination. More preferably, pET28a (+) plasmid is used, but is not limited thereto.

As the above-mentioned transformed microorganism, any microorganism may be used as long as it is a microorganism used in the art as a system for over-expressing a desired protein by transformation with a recombinant vector. Specifically, it is more preferable to use Escherichia coli BL21 (DE3) strain But is not limited thereto.

The concentration of the alulose of the present invention can be from 25% (w / v) to 75% (w / v), preferably from 40% (w / v) to 60% (w / v) And preferably 50% (w / v).

Metal ions may be added to the conversion reaction of the present invention. The metal ion may be any one or more selected from the group consisting of Mn ²⁺ , Ni ²⁺ and Co ²⁺ , preferably Mn ²⁺ or Ni ²⁺ , and most preferably Mn ²⁺ .

The reaction of the present invention can be carried out at pH 6 to pH 8.5, preferably at pH 6.5 to pH 8.0, and most preferably at pH 7.

The reaction time of the present invention can be from 1 hour to 6 hours, most preferably from 2 hours to 5 hours, and most preferably 3 hours.

As mentioned above, the production method of aloses in the present invention is environmentally friendly, unlike a chemical production method, because it uses enzymes over-expressed from microorganisms, and produces aloses by biological methods, Yield of aloses.

FIG. 1 is a graph showing the activity (A) of an L-rhamnose isomerase according to pH and the activity (B) of an L-lambda isomerase according to a temperature,
FIG. 2 is a table showing the conversion of alulose to alose by time in the absence of addition sugars in the production of aloses by the el-rhamnose isomerase.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing a recombinant microorganism, which comprises culturing a microorganism transformed with an expression vector containing the gene of L-rhamnose isomerase derived from Clostridium stearicaria, and expressing a large amount of the L-lambda isomerase, In a high yield.

The above-mentioned .lambda.-Rhamnose isomerase used in the present invention is known as an enzyme that converts rhamnose to rhamnolose, but it is known that it has an activity of being capable of simultaneously carrying out an isomerization reaction against several rare saccharides with a wide substrate specificity. Considering the function of the present enzyme, the applicability to the production of the rare saccharin alos was confirmed, and it was confirmed that the enzyme has an activity of converting the high concentration of the allylose to the high yield by using the el-rhamnose isomerase It was first demonstrated in the invention.

In the present invention, a large amount of the L-rhamnose isomerase gene is replicated from Clostridium stearicaria containing the gene of L-rhamnose isomerase through polymerase chain reaction (PCR) Into an expression vector to prepare a recombinant vector containing the L-rhamnose isomerase gene, and then culturing a strain transformed with the appropriate microorganism as the recombinant vector to overexpress the enzyme.

The method of producing an ally of the present invention comprises a step of obtaining an isomerization reaction of an allylose, which is a substrate, into an allose by using an el-rhamnose isomerase which has been treated with a metal ion.

The expression vector used for cloning of the L-rhamnose isomerase gene in the method of producing an allosylase of the present invention may be any vector that has been used for gene recombination including pET-24a, Escherichia coli ER2566 is preferably used as the strain to be transformed, but any strain may be used as long as it is a strain capable of producing a protein having an activity by over-expressing a desired gene after transformation with a gene recombinant vector.

More specifically, in the present invention, Escherichia coli ER2566 strain was used as a recombinant strain for obtaining the L-rhamnose isomerase, and LB was used as a microorganism producing medium. The frozen Escherichia coli ER2566 strain was inoculated into a test tube containing 3 ml of LB medium and cultured in a shaking incubator at 37 ° C. until the absorbance at 600 nm reached 2.0. The culture broth was added to 450 ml of LB medium , And the mixture was incubated until the absorbance at 600 nm reached 0.6. Then, 1 mM ITPG was added to induce the overexpressed enzyme. At this time, the stirring speed during the above procedure was 200 rpm, The incubation temperature is maintained at 37 캜, and after the addition of 1 mM IPTG, the agitation speed is 150 rpm, and the incubation temperature is maintained at 16 캜, which is preferable for mass production of L-rhamnose isomerase.

The over-expressing L-rhamnose isomerase was centrifuged at 6,000 × g for 30 minutes at 4 ° C., washed twice with 0.85% sodium chloride (NaCl) and resuspended in lysis buffer pH 8.0), the cell solution was disrupted with a sonicator, and the cell lysate was centrifuged at 13,000 x g for 20 minutes at 4 DEG C to remove the precipitate, and the supernatant was applied to a Histrapaplip column His trap HP column was used to obtain the purified enzyme. The purified enzyme was dialyzed against 50 mM PIPES piperazine-N, N'-bis (2-ethanesulfonic acid), pH 7.0. The enzyme was treated with EDTA, a metal chelating agent, and Mn ²⁺ , which had the highest activity among various metals, was used as an enzyme solution for the production of aloses.

The method of producing an alos according to the present invention is environmentally friendly because it uses an enzyme obtained from a microorganism, and in producing alros only in a high yield without producing a by-product alross from aluloses, The complexity of the purification process can be reduced. The mass produced ALROS in this way can be usefully used in industry, especially medicine industry.

In the present invention, the enzyme activity was measured by using alulose as a substrate. The reaction was carried out in a 50 mM phosphate buffer solution containing 10 mM of allylose at pH 7.0 and 70 ° C for 10 minutes, followed by the addition of 2 M hydrochloric acid solution. I stopped. To facilitate comparative analysis of enzyme activity, one unit of enzyme was defined as the amount that produced 1 μmol of alanine at pH 7.0 and 70 ° C per minute.

In addition, the analysis of the alulose and alanine concentrations for enzyme activity measurement was performed by bio-liquid chromatography (Bio-LC) equipped with an electrochemical detector and a CarboPac PA1 column. At this time, in the case of the Carbopeakpeari 1 column, it was flowed at a rate of 1 ml / min at 30 ° C so as to pass through 96% of distilled water and 4% of 200 mM sodium hydroxide solution.

Hereinafter, the present invention will be described in more detail by way of specific examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

  Example 1. Mass production of L-rhamnose isomerase

Primers were designed on the basis of Dnae (DNA) base sequence and amplified by PCR amplification. Then, restriction enzymes Nhe I and Xho I were added to the E. coli And inserted into a vector T-blunt vector [Stratagene]. Next, the Rael-Rhamnose isomerase-containing fragment recovered using the restriction enzymes Nhe I and Xho I was treated with alkaline phosphatase and inserted into the Nhe I and Xho I sites of the expression vector pET-24a, A vector pET-24a / l-lambda isomerization enzyme was prepared and transformed into Escherichia coli ER2566 by a conventional transformation method. Recombinant E. coli was frozen at 80 ° C in 20% glycerine solution before incubation for overexpression of the enzyme.

In order to mass-produce the L-rhamnose isomerase, a frozen E. coli ER2566 strain transformed with the above enzyme was inoculated into a test tube containing 3 ml of LB medium, and the absorbance at 600 nm was 2.0 The seed culture was performed in a shaking incubator at 37 ° C, and the culture medium was added to a 1000 ml flask containing 450 ml of LB medium and cultured. At this time, when the absorbance at 600 nm was 0.6, ITPG at a concentration of 0.1 mM was added to induce overexpression of the L-rhamnose isomerase. At this time, the stirring speed was 200 rpm and the incubation temperature was maintained at 37 캜. After the addition of IPTG, the agitation speed was 150 rpm and the culturing temperature was 16 캜.

Example 2. Metal specificity of the L-rhamnose isomerase

To investigate the metal salt effect, the enzyme activity was determined by adding 10 mM EDTA and then adding 1 mM metal ions (Mn ²⁺ , Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ ) And the enzyme activity was measured. At this time, the reaction was carried out in 50 mM Tris-HCl buffer containing 10 mM of allylose and 0.075 mg / ml of enzyme at pH 7.5 and 65 ° C for 2 hours, and 2 M hydrochloric acid solution was added to the reaction volume And the reaction was terminated.

As shown in Table 1, the L-lambda isomerase was found to be a metal ion-dependent enzyme, and the L-rhamnose isomerase exhibited the highest activity in manganese ion.

metal Relative activity (%) Not treated 5.45 Mn ²⁺ 100.00 Ni ²⁺ 98.17 Co ²⁺ 77.56 Cu ²⁺ 3.78 Zn ²⁺ 1.87

Example 3: Activity according to pH and temperature change of L-rhamnose isomerase

In order to confirm the activity of the above-mentioned L-rhamnose isomerase according to pH and temperature changes, enzymes and substrates were reacted at various pH and temperature to compare enzyme activities. In order to investigate the pH effect, first, 10 mM allylose and 50 mM MES (2- (N-morpholino) ethanesulfonic acid) containing 0.075 mg / And 50 mM PIPES (piperazine-N, N'-bis (2-ethanesulfonic acid)) containing 10 mM of allylose and 0.075 mg / 50 mM EPPS buffer solution containing 10 mM of allylose and 0.075 mg / ml of enzyme was added to 50 mM EPPS (3- [4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid ) Were reacted in the range of pH 7.5 to 8.5. The enzyme reaction was carried out at 70 ° C for 10 minutes, and the reaction was terminated at a volume corresponding to 10% of the reaction volume of 2M hydrochloric acid solution. The activity of the enzyme was measured and the results are shown in FIG. 1 (A).

To investigate the temperature effect, the reaction was carried out for 10 minutes at a temperature of 60 ° C to 80 ° C in a 50 mM pH 7.0 Peps buffer containing 10 mM of allylose and 0.075 mg / ml of enzyme, and 2 M hydrochloric acid The reaction was terminated by adding a volume corresponding to 10% of the reaction volume, and the activity of the enzyme was measured. The results are shown in FIG. 1 (B).

As a result, optimum pH and temperature were 7.0 and 75 ℃, respectively.

In the graph (A) showing the activity of the L-rhamnose isomerase according to the pH in FIG. 1,? Represents the Mies buffer solution,? Represents the Phepe buffer solution,? Represents the PBS buffer solution Line.

Example 4. Safety against temperature of L-rhamnose isomerase

In order to confirm the safety of the above-mentioned L-rhamnose isomerase with respect to temperature, the enzyme was allowed to stand at a temperature of 65 ° C to 85 ° C and reacted with the allylose using an enzyme which was allowed to stand at a time unit at each temperature, . At this time, a 50 mM Peps buffer solution containing 0.075 mg / ml of enzyme was allowed to react at a temperature of 75 ° C. for 10 minutes with 10 mM alu- rulose, The reaction was completed by adding a volume corresponding to 10% of the reaction volume, and the activity of the enzyme was measured. The results are shown in FIG. Since the half-life is 29.4 hours at 65 占 폚, 10.9 hours at 70 占 폚, 1.7 hours at 75 占 폚 and 10 minutes at 80 占 폚, the optimum temperature for producing the allyl by the el-rhamnose isomerase is 5 hours The maximum temperature at which the residual activity can be maintained above 50% was determined as 70 ° C.

In Fig. 2,? Represents 65 占 폚,? Represents 70 占 폚,? Represents 75 占 폚, and? Represents 80 占 폚.

Example 5: Production of aloses by L-rhamnose isomerase

The reaction was carried out at a temperature of 70 ° C for 5 hours in a buffer solution of 500 g / l of allylose, pH 7.0, containing 9 mg / ml of enzyme, and 50 mM of Pipes buffer for the production of the high concentration of the present invention. In order to confirm the production state of aloses by time, the reaction was terminated by adding a 2 M hydrochloric acid solution to the solution which had been reacted for a predetermined time by adding a volume corresponding to 10% of the reaction volume, and the production of aloses was confirmed by Bio-LC . Table 2 shows the production of aloses according to the reaction time.

  Reaction time (hour)  Alross (g / l) 0 0 0.5 51.5 One 82.5 2 125 3 131 4.5 150 5 165

As a result, 165 g / l of alos was produced at a culture time of 6 hours, which corresponds to a conversion yield of 33% and a productivity of 33 g / ℓ-h. This is 1.2 times higher than the reported productivity of 27.5 g / ℓ-h. In addition, the enzymatic activity of the previously reported al-rhamnose isomerase from Thermotagamaritima on alulose is 1.1 μmol / min / mg, which is the highest level reported in the literature. The enzyme activity of L-rhamnose from Clostridium stearicallium is 2.0 μmol / min / mg, which is higher than that of L-rhamnose.

Alross is produced as a by-product in the production of aloses from aluloses using the Rum Nos-isomerase derived from Pseudomonas schlegeli, which is known as the only production enzyme of Al-Ross. However, in the production of al- rous by the use of the rhamnose isomerase In the production of ALROS, it was confirmed that only ALROS produces high selectivity without the production of by-product ALTROS. Without the production of such by-product, altrose, the purification process is reduced during the separation and purification process of the alum from the reaction solution, thereby improving the yield. In the production of aloses from aluloses using the rhamnose isomerase, the production of alochmans without the production of altros was confirmed by bio-liquid chromatography with an electrochemical detector and CarboPac PAI 1 Respectively.

<110> KONKUK UNIVERSITY INDUSTRIAL COOPERATION CORP <120> Preparation method of D-allose using a novel L-rhamnose isomerase <130> 1061544 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 425 <212> PRT <213> Clostridium stercorarium <400> 1 Met Tyr Leu Tyr Asn Lys Asp Arg Ile Glu Gln Asp Tyr Lys Ala Ala   1 5 10 15 Lys Glu Ile Tyr Ala Ala Tyr Gly Val Asp Thr Asp Arg Val Met Glu              20 25 30 Glu Met Lys Thr Ile Pro Val Ser Ile His Cys Trp Gln Gly Asp Asp          35 40 45 Val Val Gly Phe Glu Asn Ala Gly Gly Thr Ser Ser Gly Gly Ile Met      50 55 60 Ala Thr Gly Asn Tyr Pro Gly Arg Ala Arg Asn Gly Asp Glu Leu Arg  65 70 75 80 Gln Asp Met Asp Lys Ala Leu Ser Leu Ile Pro Gly Lys His Arg Val                  85 90 95 Asn Ile His Ala Met Tyr Ala Glu Thr Gly Gly Lys Lys Val Asp Arg             100 105 110 Asp Glu Ile Thr Val Glu His Phe Gln Arg Trp Ile Asp Trp Ala Arg         115 120 125 Glu Lys Gly Ile Gly Ile Asp Phe Asn Pro Thr Phe Phe Ala His Pro     130 135 140 Leu Ala Asp Ser Gly Tyr Thr Leu Ser Asn Pro Asp Glu Lys Ile Arg 145 150 155 160 Lys Phe Trp Ile Glu His Ala Lys Arg Ser Ser Glu Ile Ala Ala Ala                 165 170 175 Ile Gly Lys Ala Leu Asn Ser Pro Cys Val Asn Asn Ile Trp Ile Pro             180 185 190 Asp Gly Ser Lys Asp Leu Pro Ala Asp Arg Ile Thr Lys Arg Gln Ile         195 200 205 Leu Lys Glu Ala Leu Asp Glu Ile Leu Asp Lys Lys Tyr Asp Arg Asn     210 215 220 Phe Leu Val Asp Ser Val Glu Ser Lys Leu Phe Gly Ile Gly Ser Glu 225 230 235 240 Ser Tyr Val Val Gly Ser His Glu Phe Tyr Met Gly Tyr Val Met Ser                 245 250 255 Arg Asp Asp Val Ile Leu Cys Leu Asp Ala Gly His Phe His Pro Thr             260 265 270 Glu Thr Ile Ala Asp Lys Ile Ser Ser Ile Leu Ala Phe Lys Asp Glu         275 280 285 Leu Leu His Val Ser Arg Gly Val Arg Trp Asp Ser Asp His Val     290 295 300 Val Ile Phe Asn Asp Asp Thr Leu Ala Ile Ala Gln Glu Ile Lys Arg 305 310 315 320 Cys Asp Ala Tyr Arg Lys Val His Ile Ala Leu Asp Phe Phe Asp Ala                 325 330 335 Ser Ile Asn Arg Ile Thr Ala Trp Val Thr Gly Thr Arg Ala Thr Leu             340 345 350 Lys Ala Ile Met Tyr Ser Leu Leu Glu Pro Thr His Leu Leu Val Glu         355 360 365 Ala Glu Lys Asp Gly Asn Leu Gly Asn Arg Leu Ala Leu Met Glu Glu     370 375 380 Phe Lys Ala Leu Pro Phe Gly Ala Val Trp Asn Lys Tyr Cys Ala Glu 385 390 395 400 Asn Asn Val Val Gly Ser Ala Trp Leu Glu Glu Val Arg Lys Tyr                 405 410 415 Glu Glu Glu Val Leu Ser Leu Arg Lys             420 425 <210> 2 <211> 1278 <212> DNA <213> Clostridium stercorarium <400> 2 atgtatttat acaacaagga cagaattgag caggactaca aagccgcaaa ggaaatatac 60 gcagcatatg gtgttgatac cgacagggtt atggaagaaa tgaaaacaat cccggtttca 120 attcactgct ggcagggcga tgacgttgta gggtttgaaa atgcgggcgg aacgtccagc 180 ggcggaataa tggccaccgg aaattacccc ggcagggcgc gtaacggcga tgagttaaga 240 caggacatgg acaaagcgtt gagtttaatc cccgggaaac acagggttaa tattcatgcg 300 atgtatgcag agaccggcgg taaaaaagtg gatcgggacg aaataaccgt tgaacatttt 360 cagcgctgga tagactgggc aagggaaaaa ggtatcggaa tagatttcaa tcccacgttt 420 tttgcacatc ctctcgccga ttcaggttat actctttcaa atcccgatga aaaaatacgt 480 aaattctgga tagaacatgc caaacgttca agagaaattg ccgcggcaat aggtaaggcg 540 ttaaattcac cttgcgtgaa caatatatgg attcctgacg gttcaaagga tttgcctgcc 600 gacaggataa caaaacggca aatattgaaa gaagctcttg atgaaatact ggataagaaa 660 tacgacagaa actttcttgt cgattcagtg gaatcaaagc ttttcggcat cggttccgaa 720 agctatgttg taggttccca cgaattctat atgggatatg tgatgagccg tgacgatgta 780 attctttgcc ttgatgccgg acatttccat cccaccgaaa caatagccga caaaatttct 840 tcaattctgg ccttcaagga tgaactgctg ctgcatgtga gccgcggggt acgctgggac 900 agcgaccatg tggttatatt caatgacgat actttggcga tagcgcagga gattaagaga 960 tgcgacgcat accggaaggt tcacattgcc ctcgatttct ttgatgcgtc aataaacaga 1020 atcaccgcat gggtaaccgg aacaagggca acactgaaag ctattatgta ttcattgctt 1080 gagcccacac acctgctggt tgaagcggaa aaagacggta atctcggaaa caggcttgcg 1140 ctgatggaag aatttaaggc gctgcctttt ggtgcggtat ggaacaaata ctgtgctgaa 1200 aacaatgtac ctgttggatc ggcatggctg gaagaagtga ggaaatacga ggaagaagtg 1260 ctgtccttaa gaaagtga 1278

Claims (7)

Comprising the step of reacting an el-rhamnose isomerase protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and alulose in the presence of a metal ion at a pH of 6 to 8.5 and a temperature of 55 to 80 DEG C ,
^Wherein the metal ion is at least one selected from the group consisting of Mn ²⁺ , Ni ²⁺ and Co ²⁺ ,
Characterized in that the by-product, D-altrose, is not produced. The method of claim 1, wherein the concentration of the alulose is 25% (w / v) to 75% (w / v). delete delete delete delete The method of claim 1, wherein the reaction is performed for 1 to 6 hours.

Images