KR20110092881A - A novel isomerase and l- rhamnulose production using the same enzyme - Google Patents

Abstract

PURPOSE: A novel isomerase is provided to make optimal reaction condition for producing L-rhamnulose and to produce a large amount of L-rhamnulose. CONSTITUTION: A novel isomerase contains monosaccharide having amino acid sequences of sequence number 4. The monosaccharide is selected from rhamnose, mannose, talose, and gulose. The enzyme is derived from Bacillus halodurance of heat resistance.

Description

A novel isomerase and L-rhamnulose production using the same enzyme}

The present invention relates to a novel isomerase and a method for preparing L-ramnulos using the same, and more particularly, to an isomerase exhibiting heat resistance, a nucleic acid molecule encoding the same, a vector comprising the nucleic acid molecule, and a vector comprising the vector. The present invention relates to a method for preparing L-Ranulose using a transformant and the L-Ranose Isomerase.

In recent years, the use of L-carbohydrates and nucleoside derivatives derived therefrom is greatly increasing in the medical field, in particular, several modified nucleosides have shown considerable potential as useful antiviral agents. L-Ranulose is a rare saccharide, combined with D-cycose, L-galactose, L-fructose, D-tagatose and L-xylulose in the food industry and in the synthesis of many other antiviral agents. Is an important hexose. L-nucleosides are high potential candidates that can be used as therapeutic materials because they have high stability against nucleases and the like in the body when compared to D-nucleosides.

Despite the importance and usefulness as above, unlike D-Ranulose, L-Ranulose is not only present in nature in a small amount, but a method for producing L-Ranulose is known, but requires relatively high cost. . Therefore, there is a need for a method that can produce L-ramnulose commercially at low cost.

A rare sugar production study using rhamose isomerase (Leang K and Izumori K (2004) J. Biosci. Bioeng. 97 (6): 383-388; Menavuvu BT and Izumori K (2006) J. Biosci Bioeng.101) (4): 340-345), but has disadvantages such as low yield and difficulty of mass synthesis (Bhuiyan SH and Izumori K. (1999) J. Biosci. Bioeng. 88 (5): 567-70). In addition, the above method is a series of chemical reactions, which require a long time, require relatively expensive chemicals, produce unnecessary by-products, and require a lot of labor. Therefore, there is a need for a method of biochemically producing L-Ranulose from L-Ranose using microorganisms and enzymes thereof. In addition, the enzymatic production method for producing L-ramnulose using such a biocatalyst is expected to overcome the above disadvantages.

In the present invention, L-Ranulose by presenting the optimum reaction conditions for the production of L-Ranulose by using a rhamnose isomerase capable of producing L-Ranulose from L-Ranose and such enzymes To provide a method for mass production at low cost with high yield.

The present invention solves the above problems, and the first object of the present invention to be devised by the necessity to provide an isomerase.

It is a second object of the present invention to provide a gene encoding the isomerase.

A third object of the present invention is to provide a recombinant expression vector containing the gene of the isomerase.

It is a fourth object of the present invention to provide all the transforming strains including the transformed recombinant E. coli.

A fifth object of the present invention is to provide a recombinant catalytic enzyme using the transformed recombinant E. coli.

A sixth object of the present invention is to provide a method for producing L-ramnulos from rhamnose from L-rhamnoose using the enzyme.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

In order to achieve the above object, the present invention provides a specific isomerase for a monosaccharide selected from the group consisting of monosaccharides, preferably rhamnose, mannose, taloose, and cellulose. The isomerase of the present invention is SEQ ID NO: 4 Characterized by having an amino acid sequence represented by. In addition, in the amino acid sequence of SEQ ID NO: 4, a mutation in which one or more amino acid deletions, substitutions, and additions of at least one variant of one or more amino acids is introduced within a range in which isomerase activity indicated by a protein having these amino acid sequences is not impaired. Isomerase is also included in the isomerase which concerns on this invention.

In addition, the present invention includes an isomerase gene encoding an isomerase having an amino acid sequence of SEQ ID NO: 4, and the gene sequence includes those represented by SEQ ID NO: 3. In addition, the mutant isomerase encoding the mutant isomerase obtained by mutating these nucleotide sequences of SEQ ID NO: 3 is also included in the isomerase gene of the present invention.

In addition, the present invention includes a recombinant vector containing the isomerase gene of the present invention and a transformant transformed by the recombinant vector. The present invention also includes a method for producing the isomerase of the present invention, wherein the isomerase of the present invention is isolated from the culture obtained by culturing the transformant.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The isomerase gene of the present invention is isolated from the cells of Bacillus halodurans. First, chromosomal DNA is obtained from a strain having the isomerase gene of the present invention.

Next, a polymerase chain reaction (PCR) is carried out using the designed oligonucleotide as a primer, and the chromosomal DNA of the Bacillus halodurans strain as a template to partially amplify the isomerase gene. The PCR amplified fragment thus obtained was a fragment having 100% homology to the isomerase gene of the Bacillus halodurans strain, and at the same time, a high S / N ratio can be expected as a probe for colony hybridization. Facilitate stringency control of hybridization. The PCR amplification fragment is labeled with a suitable reagent, and colony hybridization is performed on the chromosomal DNA library to select isomerase genes (Current Protocols in Molecular Biology, Vol. 1, p. 603, 1994).

DNA fragments containing L-Ranose isomerase gene can be obtained by recovering the plasmid from the E. coli selected by the above method using alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). have. After determining the nucleotide sequence by the above method, it is possible to obtain the entire gene of the present invention by hybridizing the DNA fragment prepared by digestion by restriction enzymes of the DNA fragment having the nucleotide sequence as a probe. SEQ ID NO: 3 shows the nucleotide sequence of the isomerase gene of the present invention, SEQ ID NO: 4 shows the amino acid sequence encoded by the gene.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector. For example, when a bacterium such as E. coli is used as a host, the recombinant vector according to the present invention can autonomously replicate in the host, and at the same time, a promoter, DNA containing the isomerase gene of the present invention, and a transcription sequence It is preferable to have what is necessary for expression of these. As the expression vector used in the present invention, pQE80L vector was used, but any expression vector satisfying the above requirements can be used.

In the production of the isomerase according to the present invention, a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same is cultured, and a isomerase which is a gene product is produced in culture (cultured cell or culture supernatant). By accumulating and obtaining enzyme from the culture.

Acquisition and purification of the isomerase of the present invention can be carried out by centrifuging the cells or supernatant from the obtained culture, and then, individually or in combination, cell disruption, affinity chromatography, cation or anion exchange chromatography. .

The present inventors cloned the gene of L-Rhamnose isomerase from Bacillus halodurans to develop an enzyme capable of producing L-Ranulose with high yield. It was confirmed that the recombinant strain in which the gene was inserted could not only prepare L-ranulose with high yield from L-rhamnose, but also greatly reduce the production of by-products, and completed the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention clones a gene encoding L-Rhamnose isomerase from a gene of Bacillus halodurans to produce an industrially useful thermophilic or heat-resistant L-Rhamnose isomerase, and the base sequence of the electric gene and inferred therefrom. Analyze the amino acid sequence.

L-Ranose isomerase of the present invention is an enzyme that forms L-Ranulose by catalyzing the isomerization reaction using L-Ranose as a substrate, and more preferably has L-Ranose as a specificity for stereoisomers. L-rhamnose isomerase with the ability to convert to L-Ranulose.

L-Rhamose isomerase of the present invention has the following characteristics: (iii) the optimum temperature is 60-75 ° C; (Ii) an optimum pH of 6.0-10.0; (Iii) have a molecular weight of about 48 kDa; (Ⅳ) and increase in the Mn ^{+ 2} or Co ^{2 +} activity when present,

Preferably, the L-rhamose isomerase of the present invention has an optimum temperature of 70-75 ° C., an optimal pH of 6.5-10.0, more preferably an optimum temperature of 70 ° C., and an optimum pH of about 7.0.

L- ramno agarose isomerase of the present invention is active upon 1mM Mn ^{+ 2} or Co ^{+ 2} present respectively increased 100-fold and 93-fold.

The K _m value of the Bacillus halodurans-derived L-rhamose isomerase obtained in the present invention is about 369 mM, k _cat value is about 8971 min ⁻¹ , and k _cat / K _m value is 17 min ⁻¹ mM ⁻¹ .

The L-rhamnose isomerase of the present invention exhibits specific activity to L-rhamnose, L-mannose, L-talose and D-glucose having hydroxyl functional groups at the carbon of the second and third sites of the substrate. The enzyme of the present invention having such substrate specificity will be very specific and will be usefully applied for the production of L-ramnulos from sugar mixtures.

According to another aspect of the present invention, the present invention provides a gene sequence encoding the aforementioned L-rhamnose isomerase.

The present invention also provides a method for producing L-Ranulose from L-Ranose as a substrate using L-Ranose Isomerase.

L-Ranulose produced by the L-Rhamnose isomerase of the present invention is an important core grenade that constitutes the backbone in the synthesis of L-ribonucleosides, L-oligoribonucleosides and many other therapeutic agents. It is a party. The nucleosides derived here have shown considerable potential as useful antiviral agents. Therefore, the conversion of L-Ranulose from L-Rannose will play an important role as a basic step for the production of L-Ranulose.

1 is a view showing a method for producing an expression vector comprising the L-rhamnose isomerase gene derived from the Bacillus halodurans strain.
2 is a SDS-PAGE gel photograph of L-Rhamnose isomerase derived from Bacillus halodurans strain.
Figure 3 (a) is a graph showing the reaction activity according to the pH of L- rhamnose isomerase derived from the Bacillus halodurans strain.
Figure 3 (b) is a graph showing the reaction activity according to the temperature of L- rhamnose isomerase derived from the Bacillus halodurans strain.
Figure 4 is a graph showing the enzyme activity according to the L- rhamnose substrate concentration of L-rhamnose isomerase derived from Bacillus halodurans strain.
Figure 5 (a) is a diagram showing the appropriate enzyme concentration of L- rhamnose isomerase in the method for producing L-ramnulos using the coenzyme solution obtained from the Bacillus halodurans strain.
Figure 5 (b) is a diagram showing the results of synthesizing L-ramnulos using the crude enzyme solution obtained from the Bacillus halodurans strain.
Figure 5 (c) is a diagram showing the production of L-ramnulos using the crude enzyme solution obtained from the Bacillus halodurans strain.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  1: Gene of L-Rhamnose Isomerase Cloning

Bacillus halodurans (ATCC BAA-125) was incubated at 37 ℃ and centrifuged (8000g, 10 minutes) to obtain the cells. Isolate genomic DNA from the cells obtained previously, using the base sequence of the gene encoding the Bacillus halodurance isomerase primers BHLI F-5 GCG GAT CCA TGA GCA TGA AAA GTC A-3 and BHLI R-5 TTA AGC TTA TGG CGC TGG AGC AGC-3 (SEQ ID NO: 2) was prepared and PCR was performed. The nucleotide sequence was analyzed by inserting a PCR product, that is, a gene containing L-rhamnose isomerase amplified in Bacillus halodurans into the pGEM T-easy vector (SEQ ID NO: 3).

Example  2: Preparation of Recombinant Expression Vectors and Recombinant Strains

BamHI and Hind III sites of the expression vector pQE80L (Qiagen, USA) to express a large amount of heat-resistant L-rhamnose isomerase in Escherichia coli using a gene encoding L-rhamnose isomerase according to Example 1 After inserting the enzyme gene into E. coli BL21 (DE3) (Novagen Madison, WI) was transformed (Fig. 1).

Example  3: Expression and Pure Separation of Recombinant L-Ranose Isomerase

The recombinant strain prepared in Example 2 was inoculated in LB medium and incubated at 37 ° C. for 24 hours to confirm the protein expressed in the SDS-PAGE gel (FIG. 2).

In order to purify the recombinant L-rhamose isomerase expressed by the method of Example 3, the recombinant strain culture solution was centrifuged (8000 × g, 10 minutes) to collect only the cells, and then subjected to sonication to disrupt the cell wall of Escherichia coli. , Centrifuged at 20,000 × g for 20 minutes to remove the precipitate (cells) to obtain a supernatant. Subsequently, the supernatant was heat-treated at 70 ° C. for 15 minutes, centrifuged at 20,000 × g for 20 minutes to remove precipitates, and then the supernatant was obtained. Finally, column chromatography using a Ni-NTA column (Qiagen, USA) was performed. By doing so, the recombinant L-rhamose isomerase was purely separated (FIG. 2).

Example  4: Characterization of L-Ranose Isomerase

The physical and chemical properties of the L-rhamnose isomerase isolated in Example 4 were investigated, and L-rhamnose (Sigma, St. Louis, MO) was used as a substrate.

Example  4-1: Thermal stability  And optimum temperature

To determine the thermostability of the isomerization activity of L-Ranose isomerase, the enzyme was present in 1 mM Mn ²⁺ at 50, 60, 70 or 80 ° C. for 0-2 hours in 50 mM maleate buffer (pH 7) or While reacting in the absence, samples were taken at regular intervals and the remaining activity was measured. The residual activity of the enzyme was measured by a cysteine-carbazole-sulfuric acid assay by taking a sample. Bacillus L- ramno of the present invention obtained from the halo Durance agarose isomerase exhibited stability exhibit greater than 90% activity for more than 15 hours in the presence of Mn ^{+ 2} in 60 ℃. On the other hand, L-rhamnose isomerase of the present invention was the optimum temperature was formed at 70 ℃ (Fig. 3a).

Example  4-2: Optimal pH

The determination of the optimal pH was carried out at 70 ° C. using the following buffer (50 mM): sodium acetate / acetic acid buffer (pH 4.0-5.0), malate buffer (pH 5.0-7.0) and Tris-HCl buffer (pH 7.0- 10.0). A sample was taken and enzyme activity was measured by the method of Example 2. As can be seen in Figure 3, the optimum pH of L-rhamnose isomerase was 7 at 70 ℃, more than 50% of the highest level of activity was maintained at pH 5.0 ~ 10.0 (Fig. 3b).

Example  4-3: Effect of Metal Ions

Pure purified enzyme was treated with EDTA at a final concentration of 10 mM and after standing for 24 hours or more, dialyzed with 50 mM maleate buffer and used as an enzyme solution for this experiment. Preincubate the enzyme in 50 mM malate buffer for 5 min with MgCl ₂ , MnCl ₂ , CoCl ₂ , ZnCl ₂ , CaCl ₂ , FeSO ₄ , CuSO ₄ , KCl, HgCl ₂ , or BaCl _{2 at a} final concentration of 1 mM The remaining activity of the enzyme was measured. The effect on the L-rhamnose isomerase activity of various metals at 1 mM concentration is shown in Table 1. Mn ^{+ 2} or Co ^{+ 2} addition is increased 100-fold and 93-fold activity relative to the control group, respectively.

Table 1 shows the effect of various metals on L-rhamnose isomerase activity.

Example  4-4: Kinetic Parameters of L-Rhamnose Isomerase

70 ℃ to the L- ramno agarose (5-500mM) in various concentrations as a substrate, pH 7 (50mM maleate buffer) were reacted in the enzyme in the presence of 1mM Mn ^{+ 2} for 5 minutes, copper through a non-linear regression analysis Mechanical parameters were measured (FIG. 4). The K _cat value for L-Rhamnose of L-Rhamnose isomerase derived from Bacillus halodurans was determined to be 8971 min ⁻¹ , and the value of k _cat / K _m was 17 min ⁻¹ mM ⁻¹ . This corresponds to the conversion rate and catalytic activity of high L-rhamose among the enzymes reported to date.

Example  5: L- Using Novel L-Ranose Isomerase Ramnulos  Production method

Example  5-1: L- Ramnulos  Optimum temperature for production

Production of L-Ranulose was performed under the following conditions using L-Ranose Isomerase purely separated in Example 3. In the production method of L-Ranulose of the present invention using L-Ranose Isomerase, the ratio of L-Ranose and L-Ranulose was confirmed while changing the temperature.

As microbial production medium, LB was used. As enzyme production medium, glycerol 10g L ^-1 , peptone 1g L ^-1 , yeast extract 30g L ^-1 , potassium diphosphate 0.14g L ^-1 , sodium monophosphate 1g L ^-1 Configured medium was used. When the absorbance at 600 nm was 0.6, 0.1 mM ITPG was added to induce enzyme production. Stirring speed during the process was 200 rpm, the culture temperature was maintained at 37 ℃.

Bacterial culture and enzyme purification method was carried out as performed in Example 3, 1g L ^-1 of substrate solution and allowed to react for enzyme and substrate at a temperature of 25, 40, 55, 70, 75 ℃ in the presence of 1mM Mn ^{+ 2.} As shown in Table 2, the yield was excellent when the reaction temperature was maintained at 60-75 ℃, especially the production of L- lanulose at 70 ℃ was the highest. Therefore, it can be seen that the optimum temperature during the reaction of L-rhamnose isomerase and substrate in the L-ramnulose production method of the present invention is between 60-75 ℃.

Table 2 is a table showing the production yield of L-ramnulose according to the reaction temperature.

Example  5-2: L- Ramnulos  Optimum enzyme concentration for production

In the production method of L-Ranulose of the present invention using a novel L-Ranose isomerase derived from Bacillus halodurans, the appropriate enzyme concentration of L-Ranose isomerase was confirmed, and the results are shown in FIG. 5 (a). It was.

As shown in FIG. 5 (a), the titre enzyme concentration of L-rhamnose isomerase was 10000 units ml ⁻¹ in L-ramnulos production.

Example  5-3: L- Ramnulos  Optimal initial substrate concentration for production

In the L-Ranulose production method of the present invention using L-Ranose isomerase, the production amount of L-Ranulose was confirmed while changing the concentration of the initial substrate. Bacterial culture and enzyme purification were carried out as in Example 3, in the presence of 1mM Mn ^{+ 2} concentration of the substrate, L- ramno agarose as 10-500g L ^-1 were measured by agarose amount L- ramnyul. The results are shown in Figure 5 (b).

As shown in FIG. 5 (b), when L-rhamnose, which is a substrate in L-Ranulose production, was 10g L ⁻¹ , the conversion ratio of L-Ranulose to L-Rannoose was 55%, When the substrate L-rhamose was set to 500 g L ^-1 , the conversion ratio of L-rhamnose to L-ramnulose was 51%. Therefore, in consideration of the final concentration, the concentration of the initial substrate was 300g L ^-1 in the method of producing L-ranulose of the present invention.

Example  5-4: Bacillus Halodurance  L- under Optimum Conditions Using Derived Novel L-Ranose Isomerase Ramnulos  production

The isomerization reaction was carried out under the following optimum conditions for maximum L-ranulose production using the novel L-Ranose isomerase. Substrate solution of 500g L ^-1, L- ramno agarose was carried out in the production of trehalose under the experimental isomerase 10000 units ml ^-1, 70 ℃, the presence of 1mM Mn ^{+ 2} to L- ramnyul, the result is Fig. 5 (c) Shown in

As shown in Figure 5 (c), the productivity of L- ramno agarose was 264.5 L ^-1 h ^-1, the conversion of L- ramno agarose from L- ramno trehalose is 56%, the production levels 281.3g L ^{- 1.} These yields and final concentrations are very good values for L-ranulose production from L-rhamnose. Therefore, the production method of L- lanululose of the present invention enables the production of L- lanululose with excellent yield and high concentration compared to the conventional method.

<110> Konkuk University Industrial Cooperation Corp. <120> A novel isomerase and L- rhamnulose production using the same          enzyme <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gcggatccat gagcatgaaa agtca 25 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ttaagcttat ggcgctggag cagc 24 <210> 3 <211> 1257 <212> DNA <213> Bacillus halodurans <400> 3 atgagcatga aaagtcaatt tgagcgagcg aaaatagaat atggacaatg gggcattgac 60 gtagaggaag cacttgagcg gttgaagcaa gttccaatat cgatccattg ctggcaagga 120 gacgatgtag gcggcttcga attgagcaaa ggcgagttgt ctggcgggat tgatgtgacg 180 ggagattatc cagggaaagc gaccacccct gaagagttac ggatggactt agaaaaagcg 240 ctgtcgctga tcccgggaaa gcatcgcgtt aatttgcatg cgatttatgc agaaacggac 300 ggtaaagtgg tggagcggga tcaactcgaa ccgaggcatt ttgaaaagtg ggtccgttgg 360 gcgaaaaggc atgggctagg gctagatttt aacccgacgt tgttttctca tgaaaaagcg 420 aaagacggac tgacccttgc tcacccagac caagcgatcc gtcagttttg gatcgaccat 480 tgtatcgcta gtcgaaagat tggtgagtat tttgggaaag agctcgagac cccgtgttta 540 acgaacattt ggattcctga tggttacaaa gatacaccga gtgatcgctt gacccctcga 600 aaacgattaa aggaatcgct ggatcaaatt tttgctgctg agatcaatga agcgtacaat 660 cttgatgcgg tggaaagcaa gctgtttggc atcggctctg aatcatatgt cgtcggctcc 720 cacgagtttt atttaagcta tgccttgaaa aatgataagc tctgcttact cgatacaggc 780 cattaccacc cgactgaaac ggtctctaat aaaatttccg cgatgctcct ttttcatgat 840 aagctcgctt tacacgtctc ccgaccggtt cgctgggata gcgatcacgt cgtcacgttt 900 gatgatgagc tgagggaaat tgccttggaa attgtgcgaa acgatgctct tgatcgcgtc 960 ctgatcggat tggatttctt tgatgcgagc attaatcgaa tcgcggcgtg gacgattgga 1020 acgcgcaatg tgattaaagc attgttgttc gctatgctca tcccgcacaa acagttaaaa 1080 gagtggcagg agacaggaga ttatacgaga cggttggcag tgctagaaga atttaaaacg 1140 tatccgctgg gcgcgatatg gaatgaatat tgcgaaagga tgaatgtgcc cattaaagag 1200 gagtggctca aggagattgc gatttatgag aaagaagtgc tgctccagcg ccattag 1257 <210> 4 <211> 418 <212> PRT <213> Bacillus halodurans <400> 4 Met Ser Met Lys Ser Gln Phe Glu Arg Ala Lys Ile Glu Tyr Gly Gln   1 5 10 15 Trp Gly Ile Asp Val Glu Glu Ala Leu Glu Arg Leu Lys Gln Val Pro              20 25 30 Ile Ser Ile His Cys Trp Gln Gly Asp Asp Val Gly Gly Phe Glu Leu          35 40 45 Ser Lys Gly Glu Leu Ser Gly Gly Ile Asp Val Thr Gly Asp Tyr Pro      50 55 60 Gly Lys Ala Thr Thr Pro Glu Glu Leu Arg Met Asp Leu Glu Lys Ala  65 70 75 80 Leu Ser Leu Ile Pro Gly Lys His Arg Val Asn Leu His Ala Ile Tyr                  85 90 95 Ala Glu Thr Asp Gly Lys Val Val Glu Arg Asp Gln Leu Glu Pro Arg             100 105 110 His Phe Glu Lys Trp Val Arg Trp Ala Lys Arg His Gly Leu Gly Leu         115 120 125 Asp Phe Asn Pro Thr Leu Phe Ser His Glu Lys Ala Lys Asp Gly Leu     130 135 140 Thr Leu Ala His Pro Asp Gln Ala Ile Arg Gln Phe Trp Ile Asp His 145 150 155 160 Cys Ile Ala Ser Arg Lys Ile Gly Glu Tyr Phe Gly Lys Glu Leu Glu                 165 170 175 Thr Pro Cys Leu Thr Asn Ile Trp Ile Pro Asp Gly Tyr Lys Asp Thr             180 185 190 Pro Ser Asp Arg Leu Thr Pro Arg Lys Arg Leu Lys Glu Ser Leu Asp         195 200 205 Gln Ile Phe Ala Ala Glu Ile Asn Glu Ala Tyr Asn Leu Asp Ala Val     210 215 220 Glu Ser Lys Leu Phe Gly Ile Gly Ser Glu Ser Tyr Val Val Gly Ser 225 230 235 240 His Glu Phe Tyr Leu Ser Tyr Ala Leu Lys Asn Asp Lys Leu Cys Leu                 245 250 255 Leu Asp Thr Gly His Tyr His Pro Thr Glu Thr Val Ser Asn Lys Ile             260 265 270 Ser Ala Met Leu Leu Phe His Asp Lys Leu Ala Leu His Val Ser Arg         275 280 285 Pro Val Arg Trp Asp Ser Asp His Val Val Thr Phe Asp Asp Glu Leu     290 295 300 Arg Glu Ile Ala Leu Glu Ile Val Arg Asn Asp Ala Leu Asp Arg Val 305 310 315 320 Leu Ile Gly Leu Asp Phe Phe Asp Ala Ser Ile Asn Arg Ile Ala Ala                 325 330 335 Trp Thr Ile Gly Thr Arg Asn Val Ile Lys Ala Leu Leu Phe Ala Met             340 345 350 Leu Ile Pro His Lys Gln Leu Lys Glu Trp Gln Glu Thr Gly Asp Tyr         355 360 365 Thr Arg Arg Leu Ala Val Leu Glu Glu Phe Lys Thr Tyr Pro Leu Gly     370 375 380 Ala Ile Trp Asn Glu Tyr Cys Glu Arg Met Asn Val Pro Ile Lys Glu 385 390 395 400 Glu Trp Leu Lys Glu Ile Ala Ile Tyr Glu Lys Glu Val Leu Leu Gln                 405 410 415 Arg his        

Claims (9)

Isomerase specific for a monosaccharide having the amino acid sequence of SEQ ID NO: 4. According to claim 1, wherein the monosaccharide isomerase, characterized in that the monosaccharide selected from the group consisting of rhamnose, mannose, talose and cellulose. The isomerase of claim 1, wherein the enzyme is derived from Bacillus halodurans. The isomerase of claim 1, wherein the enzyme is heat resistant. Gene encoding the enzyme of any one of claims 1 to 4. The gene according to claim 5, wherein the gene has a nucleotide sequence of SEQ ID NO. A method for producing an isomerase by culturing a strain transformed with a recombinant expression vector comprising an isomerase gene having a nucleotide sequence of SEQ ID NO: 3 derived from Bacillus halodurans. A method for producing L-Ranulose from L-Ranose using the Rhamnose isomerase of any one of claims 1 to 4. The method of claim 8, wherein the method produces L-Ranulose from L-Ranose with an optimal temperature of 60-75 ° C. and an optimal pH of 6.0-10.0.

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