KR100513153B1 - Themostable and Acidophilic Arabinose Isomerase and Process for Preparing Tagatose thereby - Google Patents

Abstract

The present invention relates to a thermophilic and eosinophilic arabinose isomerase and a method for producing tagatose using the same, and more particularly, a novel arabinose isomerase which simultaneously exhibits thermophilic and eosinophilic properties, a nucleic acid molecule encoding the same, A vector comprising the nucleic acid molecule, a transformant comprising the vector, and a method for producing tagatose using the arabinose isomerase. The recombinant arabinose isomerase of the present invention is excellent in eosinophilic and thermophilic properties, can produce tagatose in a high yield under acidic reaction conditions, and can have a minimizing effect of by-products when preparing tagatose under acidic conditions. .

Description

Thermophilic and eosinophilic arabinose isomerase and a method for preparing tagatose using the same {Themostable and Acidophilic Arabinose Isomerase and Process for Preparing Tagatose hence}

The present invention relates to a thermophilic and eosinophilic arabinose isomerase and a method for producing tagatose using the same, and more particularly, a novel arabinose isomerase which simultaneously exhibits thermophilic and eosinophilic properties, a nucleic acid molecule encoding the same, A vector comprising the nucleic acid molecule, a transformant comprising the vector, and a method for producing tagatose using the arabinose isomerase.

In recent years, with increasing interest in health, research on food is being actively conducted. As a result of the study, sugar alcohols, which have been developed as sweeteners that can replace the role of sugar, which can cause not only obesity but also various adult diseases, are being used practically. However, the sugar alcohols are known to have a side effect of diarrhea when ingested a predetermined amount or more, the development of alternative sweeteners without side effects are urgently required.

Among the substances developed as alternative sweeteners with relatively no side effects, tagatose is a keto sugar of galactose, which has the same level of sweetness as fructose (D-fructose), but is not absorbed into the body and does not occur metabolism. It is known that it can be used as a low-calorie sugar substitute sweetener with no side effects. In addition, it is known that it can be used as an intermediate for synthesizing other useful optically active compounds or as an additive and raw material for detergents, cosmetics, and drugs. In particular, it can lower blood glucose levels, and does not require insulin. And prophylactic agents and diets.

Currently, tagatose is mainly prepared from galactose by chemical synthesis (see USP 5,002,612). The preparation method isomerizes galactose using a metal hydroxide as a catalyst in the presence of an inorganic salt, forms an intermediate in the form of a metal hydroxide-tagatose-complex, and then neutralizes with acid to finally form tagatose. It includes the process of producing.

Another method for preparing tagatose is known, in which galactose is converted to tagatose by enzymatic method by conversion of aldose or aldose derivative to ketose or ketose derivative. have. In particular, it is known that arabinose isomerase used to convert L-arabinose to L-ribulose can produce tagatose using galactose as a substrate in vitro . However, since the production yield of galactose to tagatose by arabinose isomerase is very low, about 20%, the conversion from galactose to tagatose has not been carried out on an industrial scale until now. In addition, methods for producing tagatose from cheese or milk have been developed (US Pat. No. 6,057,135), but also were not industrially available due to low yields.

Recently, Professor Yu-Yang Lee's lab (Yonsei University) has developed a superthermophilic enzyme that can produce tagatose in galactose with high yield. The optimum temperature and pH of the enzyme is 85 ° C and 7.0. The browning reaction can be a major problem if the reaction occurs under these conditions, referring to the current industrial high temperature enzymes.

If the browning reaction occurs during the tagatose reaction, there may be various difficulties in the substrate loss and the tagatose purification. In fact, the reaction temperature and pH of glucose isomerase, which convert glucose to fructose, are 55-65 ℃ and 7.5-8.5. This reaction temperature and pH are within the limits due to the browning reaction.

Therefore, there is a need to develop an enzyme capable of producing tagatose in an acidic environment in which reaction conditions are low. To date, thermophilic and eosinophilic arabinose isomerase has not been reported.

Throughout this specification many patent documents and articles are referenced and their citations are indicated. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have diligently researched to develop an enzyme capable of producing tagatose in an acid, and as a result, the cloning of the novel arabinose isomerase and the reaction using the same can be produced in high yield. In addition, it was confirmed that the production of by-products can be greatly reduced, and the present invention was completed.

Accordingly, it is an object of the present invention to provide a novel arabinose isomerase which exhibits both thermophilic and eosinophilic properties.

Another object of the present invention is to provide a nucleic acid molecule encoding the arabinose isomerase of the present invention.

Still another object of the present invention is to provide a vector comprising a nucleic acid molecule encoding the arabinose isomerase of the present invention.

Another object of the present invention is to provide a transformant comprising a vector having a nucleic acid molecule encoding the arabinose isomerase of the present invention.

Another object of the present invention is to provide a method for producing tagatose using the arabinose isomerase of the present invention.

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

According to one aspect of the present invention, the present invention provides arabinose isomerase which simultaneously exhibits thermophilicity and eosinophility.

The arabinose isomerase according to the present invention has both thermophilic and eosinophilic properties, and arabinose isomerase having such characteristics is not known in the art. As used herein, the term "thermophilic" refers to a property of keeping enzyme activity stable at high temperatures, and has the same meaning as thermal stability and heat resistance. The term "acidophilic" means a property that the enzyme activity remains stable at an acidic pH, and has the same meaning as acid resistance.

The arabinose isomerase of the present invention means an enzyme having the ability to form tagatose by catalyzing the isomerization reaction using galactose as a substrate. More preferably, it refers to L-arabinose isomerase having specificity for stereoisomers and having the ability to convert D-galactose to D-tagatose.

The arabinose isomerase of the present invention has the following characteristics: (i) the optimum temperature is 50-80 ° C; (Ii) an optimal pH is 5.0-7.0; (Iii) have a molecular weight of about 56 kDa; And (iii) activity at pH 5 indicates at least 50% of the activity at optimum pH. Preferably, the optimum temperature is 50-70 ° C., the optimum pH is 5.5-6.5, more preferably the optimum temperature is approximately 65 ° C., and the optimum pH is approximately 6.0. On the other hand, the exact molecular weight of the arabinose isomerase of the present invention is about 56,040 Da.

The arabinose isomerase of the present invention is preferably derived from an eosinophilic microorganism, and most preferably, the acidophilic microorganism is derived from Alicyclobacillus acidocaldarius .

According to the most preferred embodiment of the present invention, the arabinose isomerase of the present invention has an amino acid sequence comprising the second sequence of SEQ ID NO. The arabinose isomerase of the present invention is also construed to include an amino acid sequence that exhibits substantial identity to the amino acid sequence described above. The substantial identity is at least 80% when the amino acid sequence of the present invention is aligned with the maximal correspondence with any other sequence, and the aligned sequence is analyzed using algorithms commonly used in the art. Means an amino acid sequence that exhibits homology of, more preferably 90%, and most preferably 98%. Among the arabinose isomerases belonging to the present invention, the amino acid sequence showing substantial identity with the sequence 2nd sequence preferably has lysine and phenylalanine at positions 269 and 338, respectively, of the sequence 2nd sequence. The two amino acids play the most important role in showing eosinophilic properties, as described in the Examples below. The identification of the two amino acids can be used as an important data for the study of acid resistance and heat resistance of the enzyme, and may be usefully used in the pharmaceutical and food industries.

The arabinose isomerase of the present invention can catalyze the isomerization of galactose, preferably D-galactose, to produce tagatose, preferably D-tagatose, and furthermore, because it shows eosinophilic reaction conditions, Can be acidic, has the advantage of minimizing by-products.

According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the arabinose isomerase of the present invention described above.

As used herein, the term “nucleic acid molecule” is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides that are the basic building blocks of nucleic acid molecules are naturally modified nucleotides, as well as modified sugar or base sites. Analogs are also included (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

Most preferably, the nucleic acid molecule of the present invention comprises a nucleotide sequence represented by SEQ ID NO: 1. Nucleic acid molecules of the present invention encoding arabinose isomerase are also construed to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. This substantial identity is at least 80% when the nucleotide sequence of the present invention is aligned with the nucleotide sequence of the present invention to the maximum correspondence, and the aligned sequence is analyzed using algorithms commonly used in the art. A nucleotide sequence that exhibits homology of, more preferably 90%, and most preferably 92.0%.

According to another aspect of the present invention, the present invention provides a vector comprising the nucleic acid molecule of the present invention described above encoding arabinose isomerase.

The vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. It is preferable that the nucleic acid molecule of the present invention is derived from a prokaryotic cell, and the prokaryotic cell is used as a host in consideration of the convenience of culture. Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a p _L ^λ promoter, a trp promoter, a lac promoter, a T7 promoter, etc.) capable of promoting transcription, translation It is common to include ribosomal binding sites and transcriptional / detoxification termination sequences for the initiation of. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14: 399-445 (1980)) can be used as regulatory sites. When a Bacillus bacterium is used as a host cell, a promoter of the toxin protein gene of Bacillus thuringiensis ( Appl. Environ. Microbiol. 64: 3932-3938 (1998); Mol. Gen. Genet. 250: 734-741 (1996) ) Or any promoter expressible in Bacillus may be used as a regulatory site.

On the other hand, vectors that can be used in the present invention include plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pET series and pUC19, etc.), phages (eg, λgt4.λB, λ-, etc., which are often used in the art. Charon, λΔz1 and M13, etc.) or viruses (eg, SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (e.g., metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences to facilitate purification of the arabinose isomerase expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA), and most preferably Is 6 × His because this additional sequence is not antigenic and does not interfere with the folding of the variable regions of the proteins ie heavy and light chains. Because of the additional sequence for this purification, the protein expressed in the host is purified quickly and easily through affinity chromatography.

According to a preferred embodiment of the present invention, the fusion protein expressed by the vector containing the fusion sequence is purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of this enzyme, can be used, and when 6x His is used, a desired scFv can be obtained using a Ni-NTA His-binding resin column (Novagen, USA). Recombinant antibodies can be obtained quickly and easily.

On the other hand, the expression vector of the present invention as an optional marker, and includes antibiotic resistance genes commonly used in the art, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo There are genes resistant to mycin and tetracycline.

According to another aspect of the present invention, the present invention provides a transformant comprising the vector of the present invention described above.

Host cells capable of stable and continuous cloning and expression of the vectors of the present invention are known in the art and can be used with any host cell, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains.

In addition, when transforming a vector of the present invention to eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and the like can be used.

The method of carrying a vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), One method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol. , 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res. , 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, fine injection method (Capecchi, MR, Cell , 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology , 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol. , 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568-9572 (1990)) Can be injected into the host cell.

The vector injected into the host cell can be expressed in the host cell, in which case a large amount of arabinose isomerase is obtained. For example, when the expression vector includes a lac promoter, the host cell may be treated with isopropyl-β-D-thiogalactopyranoside (IPTG) to induce gene expression.

According to another aspect of the invention, the present invention provides a method for producing tagatose comprising the step of contacting the above-described arabinose isomerase of the present invention with galactose under the conditions of pH 6.5 or less.

According to a preferred embodiment of the invention, the process is carried out at pH 4.0 to 7.0 and at a temperature of 40 to 85 ° C., more preferably at pH 5.0 to 6.5 and at a temperature of 60 to 70 ° C., most preferably at about pH 6.0 and It is carried out at a temperature of about 65 ℃.

According to the preparation method of the present invention, it is possible to produce tagatose, preferably D-tagatose, in high yield from the substrate galactose, preferably D-galactose, and furthermore, since the reaction conditions are acidic, It has the advantage of minimizing.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Cloning of arabinose Isomerase Gene

Alicyclobacillus acidocaldarius ATCC 43030 was incubated aerobicly at pH 4 (Alicyclobacillus medium-DSMZ medium: 0.025% CaCl ₂ · 2H ₂ O, 0.05% MgSO ₄ · 7H ₂ O , 0.02% (NH ₄ ) ₂ SO ₄ , 0.3% KH ₂ PO ₄ , 0.2% yeast extract, 0.5% glucose and 1 L of distilled water), centrifugation (8000 × g, 10 minutes) to obtain the cells. Genes were isolated from the cells obtained above, and partially cut with the restriction enzyme Sau 3AI (Takara Biotechnology, Japan) to obtain gene fragments of 12 kb or less. The gene fragment was inserted into a Zap Express Vector (ZAP Express Vector, Stratagene, USA) and packaged to prepare a genomic DNA library of Alicyclobacillus exidocaldarrius ATCC 43030.

By analyzing the nucleotide sequence of a gene encoding a conventional thermophilic or heat-resistant arabinose isomerase, primers araAF: 5'-ATGATCGATCTCAAACAGTATGAG-3 'and primers araAR: 5'-TCATCTTTTTAAAAGTCCCC-3' were prepared and obtained alicyclo. PCR was performed using the primers from all genes of Bacillus exidocaldarius ATCC 43030 to prepare probes for use in DNA-DNA hybridization. The PCR reaction solution used was as follows: 1 μg template (all genes obtained from Alicyclobacillus exidocaldarrius ATCC 43030), 0.25 μl (5 units / μl) TaKaRa Ex Taq ^™ , 5 μl 10X Ex Taq ^™ buffer , 4 μl dNTP mixture (2.5 mM), 0.5 μM (final concentration) araAF and araAR primer, to final 50 μl with sterile distilled water.

A gene containing arabinose isomerase was selected from the gene library by DNA-DNA hybridization using the probe. Conditions for hybridization reactions are described in Sambrook, J. et al., Molecular cloning: a laboratory manual. 3rd ed. The same as described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001). Subsequently, the selected gene was inserted into a pBK-CMV phagemid vector (Stratagene Co., UK) to prepare a vector containing a gene encoding arabinose isomerase of Alicyclobacillus exidocaldarrius ATCC 43030.

The base sequence (SEQ ID NO: 1 sequence) and the amino acid sequence inferred from the gene (SEQ ID NO: 2 sequence) of the arabinose isomerase gene cloned above are the base sequence and amino acid sequence of the known arabinose isomerase gene. Compared with. Table 1 below shows the sequence homology of arabinose isomerase isolated from various strains.

Strain Gene sequence homology (%) % Amino acid sequence homology Geobacillus stearothermophilus 91.0 97.8 Bacillus halodurans 64.3 66.7 Bacillus subtilis 65.0 62.8 Thermotoga neapolitana 61.0 61.9 Thermotoga maritima 60.6 61.5 Escherichia coli 61.9 59.9 Salmonella typhimurium (Salmonella typhimurium) 62.6 59.3 Mycobacterium smegmatis 58.4 54.8 Bifidobacterium longum 47.3 46.2

As shown in Table 1, the arabinose isomerase of the present invention is 91.0% of gene sequence homology and 97.8% amino acid sequence homology, compared to that of the conventional geobacillus stearosis, which is estimated to be arabinose isomerase of Morphilus. Indicated.

Example 2: Preparation of Recombinant Expression Vector and Recombinant Strain

Carried out using a gene encoding the arabinose isomerase of Example 1, in order to mass-expression of the thermophilic arabinose isomerase in E. coli, BamH I, and the expression cut with Hind III vector pET 22b (+) (Novagen, The recombinant gene vector pAAAI was prepared by inserting the enzyme gene (cut by BamH I and Hind III) into the United States of America (see FIG. 1). Next, E. coli BL21 ( E. coli BL21) was transformed with the recombinant expression vector pAAAI to prepare a recombinant strain, which was named " E. coli BL21 / DE3 pAAAI ( E. coli BL21 / DE3 pAAAI)."

Example 3: Expression of Recombinant Arabinos Isomerase

1% (v / v) of the recombinant strain E. coli BL21 / pAAAI prepared in Example 2 was inoculated in LB medium, incubated for 2 and a half hours at 37 ℃, IPTG was added so that the final concentration is 1 mM, Expression of recombinant arabinose isomerase was induced for 4 hours. To measure the activity of the expressed arabinose isomerase, the cultures were centrifuged (8000 × g, 10 minutes) to collect only the cells and resuspended in 10 ml of 100 mM MOPS buffer solution (4-morpholinepropanesulfonic acid, pH 6.0). . Subsequently, the cells are completely disrupted by sonication and then galactose is used as a coenzyme solution. Isomerization reaction was performed. Galactose isomerization is performed by mixing 100 μl of the coenzyme solution with 40 mM galactose as a substrate and a 1 mL enzyme reaction solution containing a cofactor (final concentration 1 mM MnCl ₂ , 1 mM CoCl ₂ ) (100 mM MOPS buffer solution, pH 6.0). ) Was reacted at 70 ° C. for 20 minutes. The resulting product was detected using the cysteine-carbazole-sulfuric acid method (Dische, Z., and E. Borenfreund., A New Spectrophotometric Method for the Detection and Determination of Keto Sugars and Trioses J. Biol. Chem., 192: 583-587 (1951). As a result, it was found that normal galactose isomerization was performed.

Example 4 Pure Separation of Recombinant Arabinos Isomerase

In order to purify the recombinant arabinose isomerase expressed by the method of Example 3, the recombinant strain culture solution was centrifuged (8000 × g, 10 minutes) to collect only the strain, and then sonicated to destroy the cell wall of E. coli, 20,000 Centrifugation at xg for 20 minutes removed the precipitate (cells) and the supernatant was obtained. 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 a supernatant was obtained. Finally, an ion exchange column (Q-Sepharose Fast Flow, Pharmacia, Sweden) was used. Column chromatography was used to purely separate the recombinant arabinose isomerase.

Example 5 Optimal Temperature of Recombinant Arabinos Isomerase

In order to obtain the optimum isomerization reaction temperature of galactose of the purified arabinose isomerase purely isolated in Example 4, the reaction activity of the enzyme according to the reaction temperature was measured using the same method as in Example 3. At this time, the reaction temperature was 40, 45, 50, 55, 60, 65, 70, 75, 80 and 85 ℃, the reaction pH was 6.5, the results of measuring the galactose isomerization activity at each temperature, 65-70 It can be seen that the maximum activity in the vicinity of ℃ (see Figure 2).

Example 6 Reaction Optimal pH of Recombinant Arabinos Isomerase

In order to obtain the optimum pH for the galactose isomerization of the purely isolated recombinant arabinose isomerase in Example 4, the reaction activity of the enzyme according to the reaction pH was measured in the same manner as in Example 3, the reaction temperature is 65 ℃ It was. At this time, the reaction pH is 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, and the results showed that the galactose isomerization activity at each pH showed the maximum activity near pH 6.0. (See FIG. 3).

Example 7: Acid Resistance Study of Recombinant Arabinos Isomerase

In order to determine the pH stability of the recombinant arabinose isomerase of the present invention, the purified arabinose isomerase purely isolated in Example 4 was reacted at pH 4.0, 5.0, 6.0, 6.5, 7.0 and 8.0 at 3, 6, It was left at room temperature for 10, 12, 24, 40 and 48 hours (without addition of a joiner). Subsequently, 1 ml of the enzyme reaction solution was added to proceed the isomerization reaction at 65 ° C., and the residual activity of the recombinant arabinose isomerase was measured in the same manner as in Example 3 (see FIG. 4). As shown in Figure 4, even when left at room temperature for more than 10 hours at pH 5.0, 50% or more residual activity was observed. Thus, it can be seen that the recombinant arabinose isomerase of the present invention has very high acid resistance.

Example 8: Production yield and by-product of tagatose according to the reaction pH

When preparing tagatose from galactose using the recombinant arabinose isomerase of the present invention, the production yield according to the reaction pH was measured. The substrate was reacted with 100 mM galactose instead of 40 mM galactose in the purely separated enzyme reaction solution of Example 4 at 60 ° C. for 24 hours at pH 5.0, 6.0, and 7.0, and the yield of the prepared tagatose was bio-LC. (DIONEX, United States) was used for analysis (see FIG. 5). As shown in Figure 5, the lower the reaction pH was confirmed that less by-products are produced, can be produced in the highest production yield tagatose at a reaction pH 6.0, it can be seen that there are almost no by-products.

Example 9 Investigation of Amino Acid Homology of Arabinos Isomerase and Prediction and Mutation Experiments of Important Amino Acids Related to pH

Alicyclobacillus acidocaldarius ATCC 43030, the acidic bacterium Geobacillus stearothermophilus DSM 22, and the alkaline bacillus Bacillus halodurance DSM 2513 ( Bacillus halodurans DSM) Amino acid homology of arabinose isomerase from (see FIG. 6). In Figure 6, AraAAA, AraABS and AraABH represent arabinose isomerases of Alicyclobacillus ecidocaldarius, Geobacillus stea, Mophilus and Bacillus halodurrans, respectively. Optimal pH for each enzyme reaction is 6.0, 7.0 and 8.0. to be. In particular, in the homology comparison of acidic bacteria (Alycyclobacillus ecidocaldarius) and neutrophils (Giobacillus steamorphose), only 11 (SEQ ID NOs: 156, 176, 212, 227, 232, 235, 247, 269, 274, 338 and 488) differ only in amino acids. When these 11 were again compared with the alkaline bacterium (Bacillus halodurance), the amino acids which were considered to be importantly related to the determination of the optimal pH were sequence 156, 235, based on the amino acid sequence of the alicyclobacillus exidocaldarius arabinose isomerase. 5, 269, 274 and 338. Of these, point mutations were made to the amino acids of SEQ ID NOs: 269 and 338, and the resulting mutants were named "Alycyclobacillus K269E". As a result, it was confirmed that a change in the optimum pH occurred (see FIG. 7).

The point mutations were performed as follows: primers for pAAAI and point mutations 269KE ahF: 5'-AAA GCC TTC CTG GAG GAC GGG AAT-3 'and 269KE ahR: 5'-ATT CCC GTC CTC CAG GAA GGC PCR was performed by preparing TTT-3 '. The PCR consisted of 1 μg template (pAAAI), 1 μl (2.5 units / μl) STRATAGENE Pfu Turbo DNA polymerase, 5 μl 10 × Pfu PCR buffer, 8 μl dNTP mixture (2.5 mM), 0.5 μM (final concentration) 269KE ahF and Mix 269KE ahR primer with sterile distilled water up to 50 μl into a PCR tube and place in a PCR thermocycler (Hybaid, UK) once for 5 minutes at 94 ° C, 30 seconds at 94 ° C, 30 seconds at 55 ° C, These three processes, 7 min at 72 ° C., were performed 30 times and once for 10 min at 72 ° C. as the final elongation reaction. Subsequently, the DNA reaction DNA was treated with dpn I restriction enzyme (STRATAGENE) at 37 ° C. for 30 minutes, mixed with 25 μl 4 M ammonium acetate and 100 μl cold ethanol, and left at −20 ° C. for 30 minutes. Then, centrifuged at 1300 × g for 10 minutes, washed with 70% ethanol and dried at room temperature. The precipitated DNA was dissolved in 5 µl of sterile distilled water, and then E. coli. BL21 (DE3) was transformed and expressed.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described in detail above, the present invention provides a novel arabinose isomerase which simultaneously exhibits thermophilic and eosinophilic properties, a nucleic acid molecule encoding the same, a vector comprising the nucleic acid molecule, a transformant comprising the vector, and the arabinose. Provided is a method for preparing tagatose using isomerase. The recombinant arabinose isomerase of the present invention is excellent in eosinophilic and thermophilic properties, can produce tagatose in a high yield under acidic reaction conditions, and can have a minimizing effect of by-products when preparing tagatose under acidic conditions. .

Figure 1 is a schematic diagram showing a method for producing an expression vector comprising the thermophilic and eosinophilic arabinose isomerase gene of the present invention.

Figure 2 is a graph showing the reaction activity according to the temperature of the thermophilic and eosinophilic arabinose isomerase of the present invention.

Figure 3 is a graph showing the reaction activity according to the pH of the thermophilic and eosinophilic arabinose isomerase of the present invention. The enzyme reaction was carried out at 65 ° C., sodium acetate buffer was used in the range of pH 3.4 to 6.5, and sodium phosphate buffer was used in the range of pH 6.0 to 8.0.

Figure 4 is a graph showing the acid resistance of the thermophilic and eosinophilic arabinose isomerase of the present invention. ◆, ■, ▲, ○, ● and □ indicate pH 4.0, 5.0, 6.0, 6.5, 7.0 and 8.0, respectively.

5 is a graph showing the reaction product according to the pH of the thermophilic and eosinophilic arabinose isomerase of the present invention.

Figure 6 is a diagram showing the homology of thermophilic and eosinophilic arabinose isomerase of the present invention.

7 is a graph showing the optimal pH change of mutants of thermophilic and eosinophilic arabinose isomerase of the present invention. □ and ● represent wild type and variant arabinose isomerase, respectively. Enzyme activity is measured for 20 minutes at pH 6.0-8.0 and 65 ° C. in 40 mM sodium phosphate buffer.

<110> PYUN, Yu-Ryang <120> Themostable and Acidophilic Arabinose Isomerase and Process for Preparing Tagatose hence <130> Pyun-1 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1491 <212> DNA <213> Alicyclobacillus acidocaldarius <220> <221> CDS (222) (1) .. (1491) <223> L-arabinose isomerase <400> 1 atg atg ctg tca tta cgt cct tat gaa ttt tgg ttt gta acg gga agc 48 Met Met Leu Ser Leu Arg Pro Tyr Glu Phe Trp Phe Val Thr Gly Ser 1 5 10 15 cag cac ttg tac gga gaa gaa gca tta aag caa gtt gaa gag cat tca 96 Gln His Leu Tyr Gly Glu Glu Ala Leu Lys Gln Val Glu Glu His Ser 20 25 30 atg atg att gtc aat gag ctg aat caa gat tca gtg ttc ccg ttc cca 144 Met Met Ile Val Asn Glu Leu Asn Gln Asp Ser Val Phe Pro Phe Pro 35 40 45 ctt gtt ttc aaa tca gtt gtc aca acg cca gag gaa att cgg cgc gtt 192 Leu Val Phe Lys Ser Val Val Thr Thr Pro Glu Glu Ile Arg Arg Val 50 55 60 tgc ctt gag gcg aat gcg agc gaa caa tgc gct ggg gtg atc act tgg 240 Cys Leu Glu Ala Asn Ala Ser Glu Gln Cys Ala Gly Val Ile Thr Trp 65 70 75 80 atg cat acg ttc tcg ccg gcg aaa atg tgg att ggc ggc ctt ttg gaa 288 Met His Thr Phe Ser Pro Ala Lys Met Trp Ile Gly Gly Leu Leu Glu 85 90 95 ctg cga aaa ccg tta ttg cat ctt cat act caa ttt aac cgc gat att 336 Leu Arg Lys Pro Leu Leu His Leu His Thr Gln Phe Asn Arg Asp Ile 100 105 110 ccg tgg gac agc atc gat atg gac ttt atg aac tta aac caa tcg gcc 384 Pro Trp Asp Ser Ile Asp Met Asp Phe Met Asn Leu Asn Gln Ser Ala 115 120 125 cac ggc gac cga gaa tac gga ttt atc ggc gca aga atg gga gtc gcc 432 His Gly Asp Arg Glu Tyr Gly Phe Ile Gly Ala Arg Met Gly Val Ala 130 135 140 cga aaa gtg gtg gtc ggg cac tgg gaa gac cca agc gtc cgt gag cgg 480 Arg Lys Val Val Val Gly His Trp Glu Asp Pro Ser Val Arg Glu Arg 145 150 155 160 ctg gcg aag tgg atg cga acg gct gtc gcc ttt gcg gaa agc cgt cat 528 Leu Ala Lys Trp Met Arg Thr Ala Val Ala Phe Ala Glu Ser Arg His 165 170 175 ctc aaa gtc gcc cgt ttt ggc gac aac atg cgt gaa gtg gca gtg act 576 Leu Lys Val Ala Arg Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr 180 185 190 gaa ggg gac aaa gtc gga gcg caa atc caa ttc ggt tgg tcg gtc aat 624 Glu Gly Asp Lys Val Gly Ala Gln Ile Gln Phe Gly Trp Ser Val Asn 195 200 205 ggc tat ggc gtc ggg gat ttg gtt caa tac atc cgc gat gtt tct gaa 672 Gly Tyr Gly Val Gly Asp Leu Val Gln Tyr Ile Arg Asp Val Ser Glu 210 215 220 caa aaa atc aat gaa ctg ctt gag gaa tat gcc gag ctg tat gac att 720 Gln Lys Ile Asn Glu Leu Leu Glu Glu Tyr Ala Glu Leu Tyr Asp Ile 225 230 235 240 gtg ccc gct ggc cgc caa gac ggg ccg gtt cgc gag tcg atc cgt gaa 768 Val Pro Ala Gly Arg Gln Asp Gly Pro Val Arg Glu Ser Ile Arg Glu 245 250 255 cag gcg cgg att gaa ctt gga tta aaa gcc ttc ctg aaa gac ggg aat 816 Gln Ala Arg Ile Glu Leu Gly Leu Lys Ala Phe Leu Lys Asp Gly Asn 260 265 270 ttt gcc gct ttc acg acg acg ttt gag gat ttg cat ggg atg aaa cag 864 Phe Ala Ala Phe Thr Thr Thr Phe Glu Asp Leu His Gly Met Lys Gln 275 280 285 ctc ccr gga ctc gcc gtt cag cgg ctc atg gcg gaa gga tac ggc ttc 912 Leu Xaa Gly Leu Ala Val Gln Arg Leu Met Ala Glu Gly Tyr Gly Phe 290 295 300 ggc ggt gaa ggc gac tgg aaa aca gcc gca ctc gtc cgg ttg atg aag 960 Gly Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Leu Met Lys 305 310 315 320 gtc atg gcg gat ggc aaa gga aca tcg ttc atg gaa gac tac acg tac 1008 Val Met Ala Asp Gly Lys Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr 325 330 335 cac ttt gaa ccg ggc aat gaa atg att ctt ggc gcc cat atg ctc gaa 1056 His Phe Glu Pro Gly Asn Glu Met Ile Leu Gly Ala His Met Leu Glu 340 345 350 gta tgc ccg acg atc gcc gca acc cgg ccg cgc att gaa gtt cat cca 1104 Val Cys Pro Thr Ile Ala Ala Thr Arg Pro Arg Ile Glu Val His Pro 355 360 365 ctg tcg atc ggc gga aaa gaa gat ccg gcc cgt ctt gtg ttt gac ggc 1152 Leu Ser Ile Gly Gly Lys Glu Asp Pro Ala Arg Leu Val Phe Asp Gly 370 375 380 ggc gag ggt gcg gcg gtc aac gcg tca ttg atc gac tta ggg cac cgt 1200 Gly Glu Gly Ala Ala Val Asn Ala Ser Leu Ile Asp Leu Gly His Arg 385 390 395 400 ttc cga ctc atc gtc aat gaa gtc gat gcg gtg aaa ccg gaa cac gac 1248 Phe Arg Leu Ile Val Asn Glu Val Asp Ala Val Lys Pro Glu His Asp 405 410 415 atg ccg aaa tta cca gtc gcc cgc atc tta tgg aag ccg cgt cca tcg 1296 Met Pro Lys Leu Pro Val Ala Arg Ile Leu Trp Lys Pro Arg Pro Ser 420 425 430 ctc cgc gat tca gcc gaa gca tgg att ttg gct ggc ggc gcc cac cat 1344 Leu Arg Asp Ser Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala His His 435 440 445 acg tgc ttc tca ttt gca gtt aca aca gaa caa ctg cag gat ttt gcg 1392 Thr Cys Phe Ser Phe Ala Val Thr Thr Glu Gln Leu Gln Asp Phe Ala 450 455 460 gaa atg gcc ggc atc gaa tgc gtc gtg atc aat gaa cat acg tcc gtc 1440 Glu Met Ala Gly Ile Glu Cys Val Val Ile Asn Glu His Thr Ser Val 465 470 475 480 tcc tca ttc aag aac gaa cta aga tgg aat gaa gta ttt tgg cgg ggg 1488 Ser Ser Phe Lys Asn Glu Leu Arg Trp Asn Glu Val Phe Trp Arg Gly 485 490 495 cgg 1491 Arg <210> 2 <211> 497 <212> PRT <213> Alicyclobacillus acidocaldarius <400> 2 Met Met Leu Ser Leu Arg Pro Tyr Glu Phe Trp Phe Val Thr Gly Ser 1 5 10 15 Gln His Leu Tyr Gly Glu Glu Ala Leu Lys Gln Val Glu Glu His Ser 20 25 30 Met Met Ile Val Asn Glu Leu Asn Gln Asp Ser Val Phe Pro Phe Pro 35 40 45 Leu Val Phe Lys Ser Val Val Thr Thr Pro Glu Glu Ile Arg Arg Val 50 55 60 Cys Leu Glu Ala Asn Ala Ser Glu Gln Cys Ala Gly Val Ile Thr Trp 65 70 75 80 Met His Thr Phe Ser Pro Ala Lys Met Trp Ile Gly Gly Leu Leu Glu 85 90 95 Leu Arg Lys Pro Leu Leu His Leu His Thr Gln Phe Asn Arg Asp Ile 100 105 110 Pro Trp Asp Ser Ile Asp Met Asp Phe Met Asn Leu Asn Gln Ser Ala 115 120 125 His Gly Asp Arg Glu Tyr Gly Phe Ile Gly Ala Arg Met Gly Val Ala 130 135 140 Arg Lys Val Val Val Gly His Trp Glu Asp Pro Ser Val Arg Glu Arg 145 150 155 160 Leu Ala Lys Trp Met Arg Thr Ala Val Ala Phe Ala Glu Ser Arg His 165 170 175 Leu Lys Val Ala Arg Phe Gly Asp Asn Met Arg Glu Val Ala Val Thr 180 185 190 Glu Gly Asp Lys Val Gly Ala Gln Ile Gln Phe Gly Trp Ser Val Asn 195 200 205 Gly Tyr Gly Val Gly Asp Leu Val Gln Tyr Ile Arg Asp Val Ser Glu 210 215 220 Gln Lys Ile Asn Glu Leu Leu Glu Glu Tyr Ala Glu Leu Tyr Asp Ile 225 230 235 240 Val Pro Ala Gly Arg Gln Asp Gly Pro Val Arg Glu Ser Ile Arg Glu 245 250 255 Gln Ala Arg Ile Glu Leu Gly Leu Lys Ala Phe Leu Lys Asp Gly Asn 260 265 270 Phe Ala Ala Phe Thr Thr Thr Phe Glu Asp Leu His Gly Met Lys Gln 275 280 285 Leu Xaa Gly Leu Ala Val Gln Arg Leu Met Ala Glu Gly Tyr Gly Phe 290 295 300 Gly Gly Glu Gly Asp Trp Lys Thr Ala Ala Leu Val Arg Leu Met Lys 305 310 315 320 Val Met Ala Asp Gly Lys Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr 325 330 335 His Phe Glu Pro Gly Asn Glu Met Ile Leu Gly Ala His Met Leu Glu 340 345 350 Val Cys Pro Thr Ile Ala Ala Thr Arg Pro Arg Ile Glu Val His Pro 355 360 365 Leu Ser Ile Gly Gly Lys Glu Asp Pro Ala Arg Leu Val Phe Asp Gly 370 375 380 Gly Glu Gly Ala Ala Val Asn Ala Ser Leu Ile Asp Leu Gly His Arg 385 390 395 400 Phe Arg Leu Ile Val Asn Glu Val Asp Ala Val Lys Pro Glu His Asp 405 410 415 Met Pro Lys Leu Pro Val Ala Arg Ile Leu Trp Lys Pro Arg Pro Ser 420 425 430 Leu Arg Asp Ser Ala Glu Ala Trp Ile Leu Ala Gly Gly Ala His His 435 440 445 Thr Cys Phe Ser Phe Ala Val Thr Thr Glu Gln Leu Gln Asp Phe Ala 450 455 460 Glu Met Ala Gly Ile Glu Cys Val Val Ile Asn Glu His Thr Ser Val 465 470 475 480 Ser Ser Phe Lys Asn Glu Leu Arg Trp Asn Glu Val Phe Trp Arg Gly 485 490 495 Arg

Claims (12)

delete delete delete delete Arabinos isomerase represented by SEQ ID NO: 2 sequence. A nucleic acid molecule encoding the arabinose isomerase of claim 5. The nucleic acid molecule of claim 6, wherein the nucleotide sequence of the nucleic acid molecule is represented by SEQ ID NO: 1. A vector comprising a nucleic acid molecule encoding the arabinose isomerase of claim 6 or 7. Microorganisms transformed with the vector of claim 8. The method for producing tagatose comprising the step of contacting galactose at a condition of pH 4.0 to 7.0 of the arabinose isomerase of claim 5. The method of claim 10, wherein the pH is 4.0 to 7.0 and the reaction temperature is 40 to 85 ° C. The method of claim 11, wherein the pH is 5.0 to 6.5, the reaction temperature is 60 to 70 ℃ manufacturing method of tagatose.