KR20140111093A - L-arabinose isomerase variants with improved conversion activity and method for production of d-tagatose using them - Google Patents

Abstract

The present invention relates to development of Thermotoganeapolitana DSM5068 derived L-arabinose isomerase variant. Moreover, the present invention relates to a method for producing D-tagatose from D-galactose by using the enzyme and coryneform microorganism.

Description

TECHNICAL FIELD [0001] The present invention relates to an L-arabinose isomerase variant having improved conversion activity and a method for producing D-tagatose using the same. BACKGROUND ART [0002]

The present invention relates to an L-arabinose isomerase mutant derived from Thermotoga neapolitana DSM 5068, which has improved conversion activity to D-galactose by applying protein engineering, And a method for producing D-tagatose from D-galactose using these microorganisms.

D-Tagatose is a monosaccharide that gives off sweetness of about 90% of sugar while having characteristics such as low calorie and non-chitosity, and can be used as a health sweetener free from problems of various adult diseases caused by existing sweeteners.

D-Tagatose has been attracting attention as a sugar substitute sweetener due to these characteristics, and is known to have a high market potential in the food market. However, since tagatose is a rare sugar contained in a small amount in dairy products or some plants, it is not commonly found in the natural world. Therefore, in order to be used as a low-calorie functional sweetener, a technique capable of mass production must be developed.

D-tagatose was produced by Arla Foods in 2003 from a D-galactose-catalyzed Ca (OH) _2- catalyzed chemical isomerization method, and was designated as "Gaio-tagatose"'Was released under the brand name. However, it is known that the chemical isomerization method is superior in the conversion yield of isomerization, but it is difficult to recover and purify and the process is complicated, and the yield is lower than that of the enzymatic isomerization method in consideration of the overall yield of the process.

L-arabinose isomerase (EC 5.3.1.5) is an enzyme that catalyzes isomerization to convert L-arabinose to L-ribulose. It is also known that L-arabinose isomerase not only inherently L-arabinose but also structurally similar substrate D-galactose is isomerized to D-tagatose.

The most important factor that can contribute to productivity improvement in the process of producing D-tagatose from D-galactose by using L-arabinose isomerase is that it has good reactivity through improvement of isomerization enzyme, To develop enzymes. Increasing productivity has played a crucial role in maximizing profits by cost reduction and success or failure of business, necessitating the continuous improvement of arabinose isomerase.

Thermophilic microorganisms, such as Thermotoga , Neapolitanus DSM 5068 ( Thermotoga neapolitana DSM 5068) is highly thermostable, but it needs to be further improved in order to obtain an economical productivity of the level of glucose isomerase.

In general, methods for producing mutant enzymes in order to enhance enzyme activity or to impart activity to a new substrate include large-scale random mutagenesis and rational design, . Randomly generated mutations can be applied without specific information on the enzyme of interest, and they are used extensively, but screening systems must be available to detect mutated enzymes in very large numbers of individuals. On the other hand, the rational design of enzymes can only produce a limited number of mutant enzymes, so no special screening system is needed. However, the catalytic mechanism of the target enzyme, the binding properties of the enzyme or the determinants of substrate specificity You should know the details.

The applicant of the present invention has found that, based on the molecular modeling technique of protein engineering and the analysis of the enzyme reaction mechanism, the enzymatic reaction mechanism of Thermotoga neapolitana DSM 5068 ( Thermotoga neapolitana Arabinose isomerase) of the L-arabinose isomerase (DSM 5068), thereby enhancing the substrate specificity of the L-arabinose isomerase to D-galactose.

It is an object of the present invention to provide a method for the preparation of Thermotoga < RTI ID = 0.0 > neapolitana DSM 5068) and a gene sequence encoding the same.

Another object of the present invention is to provide a recombinant vector comprising the above gene sequence and a microorganism of the genus Corynebacterium transformed with the recombinant vector.

Yet another object of the present invention is to provide a method for producing D-tagatose from D-galactose using the Arabidopsis isomerase mutant or a culture of the transformed microorganism or the transformed microorganism.

In order to achieve the above object, the present invention relates to a thermo- sensitive material, galactose in which D-galactose, in which the 469th leucine of arabinose isomerase from neapolitana DSM 5068 is replaced with proline and the 275th amino acid is replaced with an amino acid other than phenylalanine, An improved activity of converting an arabinose isomerase enzyme and a gene sequence encoding the same.

The present invention also provides a recombinant vector comprising the gene base sequence and a Corynebacterium sp. Microorganism transformed with the recombinant vector.

The present invention also provides a method for producing D-tagatose from D-galactose using the arabinose isomerase mutant or the transformed microorganism or a culture of the transformed microorganism.

The productivity of D-tagatose is improved by using the novel Arabidopsis isomerase mutant of the present invention or the genus Corynebacterium strain transformed with the gene sequence encoding the same, thereby being useful for cost reduction and reduction of infrastructure investment cost will be.

Fig. 1 is a diagram showing the structure of the active site of the L-arabinose isomerase derived from the thermostable neapolitan or the manganese ion as a co-factor.
FIG. 2 is a diagram showing sites where L-arabinose and D-galactose bind to active sites of L-arabinose isomerase and functional groups that interact with each other. Galactose 6 and the residue 275 of phenylalanine cause steric hindrance to each other.
FIG. 3 shows the result of a saturated mutagenesis of the 275th residue of the L-arabinose isomerase and the relative activity of the mutants selected by the cysteine-carbazole method through a color reaction. A number of variants with higher activity than the control group were identified.
FIG. 4 is a diagram for predicting the structures of the selected mutants F275V / L469P, F275M / L469P, and F275I / L469P by a molecular modeling technique.
Fig. 5 is an SDS-PAGE analysis of L-arabinose isomerase mutants derived from purified and purified serotonin neapolitan.
FIG. 6 is a graph showing the relative activity of each temperature in order to confirm the optimal temperature of the selected mutants.
FIG. 7 is a graph showing the thermal stability of selected mutants at 95.degree. C. over time. FIG.
FIG. 8 is a graph showing the relative activity at various concentrations in order to confirm the manganese dependency of the selected mutants.

In one embodiment, the present invention is directed to a method for the production of a medicament, such as a Thermotoga < RTI ID = 0.0 > neapolitana DSM 5068) and a gene sequence encoding the same.

In a preferred embodiment of the present invention, the present invention relates to a pharmaceutical composition comprising the 279th amino acid of the arabinose isomerase derived from Thermotoga neapolitana DSM 5068 (DSM 5068) substituted with an amino acid other than phenylalanine and the 469th leucine leucine is replaced with proline, an Arabinos isomerase mutant having improved activity of converting D-galactose into D-tagatose, and a gene sequence encoding the same.

In the present invention, the term "arabinose isomerase that converts D-galactose to D-tagatose" means an enzyme that catalyzes the isomerization reaction using D-galactose as a substrate to produce D-tagatose.

It is preferable that the 275th amino acid of the arabinose isomerase is substituted with an amino acid having a nonpolar aliphatic side chain.

As used herein, the "amino acids with nonpolar aliphatic side chains" means alanine, valine, isoleucine, leucine, methionine and proline.

Preferably, the 275th amino acid of the arabinose isomerase is substituted with any amino acid selected from the group consisting of valine, methionine, and isoleucine.

It is also preferred that the arabinose isomerase variant of the present invention is further substituted with proline at the 469th leucine of the isomerase.

As used herein, the term "substitution " means to make a mutation by replacing an amino acid at a specific position with another amino acid. Appropriate mutagenesis methods can be any method available to those skilled in the art for this purpose. In particular, there are the saturated mutagenesis method, the random mutagenesis method and the site-directed mutagenesis method (Evolutionary molecular engineering based on RNA replication, Pure Appl. Chem. 1984, 56 : 967-978; Mutants generated by the insertion of random oligonucleotides into the active-site of the beta-lactamase gene (Proc. Natl. Acad. Sci. USA, 1986, 83: 7405-7409 , Biochemistry 1989, 28: 5703-5707).

Preferably, the 275th amino acid in the arabinose isomerase variant of the present invention is substituted by the saturation mutagenesis method, and the 469th leucine is substituted by site directed mutagenesis.

In one embodiment, the present invention relates to a method for producing a recombinant vector comprising the steps of: (a) culturing the wild-type arabinose isomerase (the amino acid sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 6) A mutant showing a certain amount of improved enzyme characteristics and its genetic information were obtained. As a result of the comprehensive analysis, it was confirmed that the change of the amino acid sequence of the C-terminal portion of the arabinose isomerase tends to affect the enzyme activity.

In addition, the amino acid at the C-terminal region constituting the active site of wild-type arabinose isomerase and arabinose isomerase mutant was confirmed by molecular modeling. As a result, the mutant was changed to proline at the 469th leucine residue of the wild-type arabinose isomerase, so that the 18th beta-sheet of the protein disappeared and the angle of the backbone changed, We confirmed that the tertiary structural position of the alpha-helix moved toward the protein body and changed the structure of the protein.

Based on the above results, the 469th leucine of the wild-type arabinose isomerase was substituted with proline using a site-directed mutagenesis method to obtain a mutant (L469P) (amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: ). ≪ / RTI > As a result of measuring the activity after culturing these mutants, it was confirmed that the mutant exhibited high activity against galactose, which is a substrate, compared with the wild type arabinose isomerase.

In one embodiment of the present invention, in order to obtain an enzyme having improved conversion activity in the Arabinos isomerase variant (L469P), major residues of the substrate binding portion and the active portion of the enzyme are selected and the reaction mechanism is estimated to saturate the finally selected 275th amino acid Mutations were further introduced using the mutation method, and screened to select mutants having improved conversion activity.

Sequence analysis showed that the 275th amino acid was substituted with valine (L469P / F275V), methionine (L469P / F275M) and isoleucine (L469P / F275I), respectively. These three mutants were transformed into microorganisms of the genus Corynebacterium and cultured to be isomerized. As a result, it was confirmed that all three mutants exhibited improved activity compared to mutant L469P in which 469th leucine was substituted with proline.

To characterize the three variants, arabinose isomerase was isolated from the cultured Corynebacterium sp. Microorganism under the above conditions. The purified protein showed arabinose isomerase activity against D-galactose, and the molecular weight was also confirmed to agree with SDS PAGE.

Optimal temperature, thermal stability, metal ion availability and enzyme activity were measured using the purified mutant enzymes. All of the three variants were found to have the highest activity at 75 ° C, but the thermal stability was somewhat reduced and the metal ion availability was not significantly different. The activity of arabinose isomerase was about 5.5 times (F275V / L469P) , 5 times (F275M / L469P) and 3.9 times (F275I / L469P), respectively.

In one embodiment, the invention provides a nucleotide sequence encoding said Arabidopsis enzyme variant.

The gene sequence may be any one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10.

The arabinose isomerase variant of the present invention is preferably 80% or more, more preferably 80% or more, more preferably 80% or more, more preferably 80% or more, more preferably 80% or more as long as it can maintain or enhance the activity of the arabinose isomerase of the arabinose isomerase variant of the present invention , More preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more homology, and most preferably, the gene described in SEQ ID NOS: 8 to 10 Have a base sequence.

As used herein, the term "homology" refers to the identity between two amino acid sequences and is intended to be within the scope of one of ordinary skill in the art, using BLAST 2.0 to calculate parameters such as score, identity, Can be determined in a well-known manner.

In addition, the polynucleotide of the present invention can be hybridized with the polynucleotide of SEQ ID NOS: 8 to 10 or a probe derived from the polynucleotide under 'stringent conditions', and encoding a normally functioning Arabinos isomerase variant Lt; / RTI >

As used herein, the term "stringent conditions" means conditions that allow specific hybridization between polynucleotides. For example, hybridization buffer (3.5> SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5 mM NaH ₂ PO ₄ (pH 7), 0.5% SDS, 2 mM EDTA) (Molecular Cloning, A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) or Current Protocols in Molecular Biology (FM Ausubel et al., Editors, John Wiley & Sons, Inc., New York). Here, SSC is 0.15 M sodium chloride / 0.15 M sodium citrate at pH 7. After hybridization, the membrane carrying the DNA is washed with 2 > SSC at room temperature and then with 0.1 to 0.5> SSC / 0.1 x SDS at a temperature of 68 [deg.] C.

In one embodiment, the present invention also provides an isomerizing enzyme variant having any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: Specifically, the mutant refers to an arabinose isomerase variant in which the 275th amino acid is substituted with any one amino acid selected from the group consisting of valine, methionine, and isoleucine, and at the same time, the 469th amino acid is substituted with proline.

However, the amino acid sequence of the enzyme exhibiting the activity of the polypeptide may differ depending on the species or strain of the microorganism. Therefore, the present invention is not limited thereto. That is, as long as the arabinose isomerase activity can be maintained or enhanced, the mutant of the present invention may comprise one or several amino acid residues at one or more positions other than the 275th and 469th amino acids of the amino acid sequence of SEQ ID NOS: A mutant or an artificial variant encoding a polypeptide having an amino acid sequence including substitution, deletion, insertion or addition of an amino acid, and the like.

Here, "several" is different depending on the position and kind in the three-dimensional structure of the amino acid residue of the protein. Specifically, it is 2 to 20, preferably 2 to 10, more preferably 2 to 5 .

In addition, substitution, deletion, insertion, addition, or inversion of such amino acids may be carried out on naturally occurring mutants or artificial variants, such as those based on differences in the species or species of microorganisms containing the arabinose isomerase activity And the like.

In one embodiment, the invention also provides a recombinant vector in which the polynucleotide is operably linked.

As used herein, the term "vector" means a DNA product containing a nucleotide sequence of a polynucleotide encoding the desired protein operably linked to a suitable regulatory sequence so as to be capable of expressing the protein of interest in a suitable host. The regulatory sequence includes a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. The vector may be transcribed into a suitable host, then replicated or functional, independent of the host genome, and integrated into the genome itself.

The vector used in the present invention is not particularly limited as long as it is replicable in a host, and any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages.

The vector used in the present invention is a vector capable of inserting a polynucleotide encoding a target protein into a host cell by transformation into a host cell, preferably a shuttle vector capable of self-replication in both E. coli and coryneform bacteria pECCG112 vector (Kor.Jour.Microbiol., July 1991, p149-154, Nogado), but is not limited thereto.

In addition, a polynucleotide encoding an intrinsic target protein in a chromosome can be replaced with a novel polynucleotide through a vector for insertion of an intracellular chromosome. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, homologous recombination.

Since the vector of the present invention can be inserted into a chromosome by homologous recombination, it may further include a selection marker for confirming whether the chromosome is inserted. The selection marker may be a vector for selecting, That is, markers that give a selectable phenotype such as drug resistance, nutritional requirement, tolerance to cytotoxic agent, or surface protein expression can be used for confirming insertion of a target polynucleotide. In the environment treated with the selective agent, only the cells expressing the selection marker survive or express different phenotypes, so that the transformed cells can be selected.

In one embodiment, the present invention also provides a microorganism of the genus Corynebacterium transformed with said recombinant vector.

Herein, the term "transformation" means introducing a vector comprising a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotides include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, so long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as far as it is capable of being introduced into a host cell and expressed. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct containing all the elements necessary for its expression. The expression cassette typically includes a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell.

The microorganism of the present invention includes both prokaryotic microorganisms and eukaryotic microorganisms as long as it is capable of expressing the above-mentioned isomerizing enzyme mutants. For example, Escherichia spp., Erwinia spp., Serratia spp., Providencia spp., Corynebacterium spp. And Brevibacterium spp. Microbial strains belonging to the genus Escherichia. Preferably, the microorganism of the present invention is a microorganism belonging to the genus Corynebacterium, more preferably a microorganism belonging to the genus Corynebacterium glutamicum ).

In a preferred embodiment of the present invention, Corynebacterium glutamicum having L-amino acid producing ability was transformed with a vector having the nucleotide sequences of SEQ ID NOS: 8, 9 and 10, and the strains thus prepared were respectively transformed into Corynebacterium glue Corynebacterium glutamicum) pFIS-1-TNAI- 2, pFIS-1-TNAI-3, pFIS-1-TNAI-4 naming, and a February 14, 2013, Seodaemun-gu, Seoul, Republic of Korea with one Hongje such material to the address 361-221 And deposited at the Korean Microorganism Conservation Center, Korean Society of Infectious Diseases, respectively, under the accession numbers KCCM 11378P, KCCM 11379P and KCCM 11380 P. In one embodiment, the present invention also provides a culture of the Corynebacterium sp.

The culture may be a stock solution containing the microorganism cells, or may be a microorganism obtained by removing or concentrating the culture supernatant. The composition of the culture liquid may additionally contain not only components required for culturing a normal Corynebacterium sp. Microorganism but also components that act synergistically to the growth of Corynebacterium sp. Microorganisms. Can be readily selected by those skilled in the art. In addition, the culture may be in a liquid state or a dry state, and the drying method may be, but not limited to, air drying, natural drying, spray drying and freeze drying.

The cultivation of the Corynebacterium sp. Microorganism of the present invention can be carried out by a conventional method. Specifically, the microorganism can be inoculated in a culture medium containing a part or all of glucose or sugar as a carbon source. The culturing may be performed according to a suitable culture medium and culture conditions known in the art. Such a culturing process can be easily adjusted according to the strain selected by those skilled in the art. Examples of the culture method include, but are not limited to, batch, continuous, and fed-batch cultivation. The medium used for the culture should suitably meet the requirements of the particular strain.

Specifically, the medium used in the present invention may be a saccharide containing glucose or glucose as a main carbon source, and a molasses containing a large amount of raw sugar as a carbon source. Other appropriate carbon sources may be variously used, Use purified glucose. Examples of nitrogen sources that may be used include inorganic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and soybean wheat and inorganic sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, And preferably, peptone is used. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts as a source. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and suitable precursors and the like may be included. These media or precursors may be added to the culture either batchwise or continuously.

Specifically, the incubation temperature is usually 27 ° C to 37 ° C, preferably 30 ° C to 37 ° C, and the incubation period can be continued as long as the desired protein is expressed, preferably 10 to 100 hours.

The present invention relates to an Arabinos isomerase mutant having an activity of converting the D-galactose into D-tagatose, a microorganism belonging to the genus Corynebacterium or a microorganism of the genus Corynebacterium Galactose is reacted with a substance providing at least one metal ion selected from the group consisting of manganese ion, magnesium ion and zinc ion to produce D-tagatose in the presence of any one selected from the group consisting of Wherein the method comprises the steps of:

The microorganism or culture of the genus Corynebacterium can be treated with a surfactant, lysozyme, and xylene, preferably 0.1% POESA, in order to make the microorganism or culture in the microorganism or culture in a state capable of inflow into the substrate, .

The solution containing D-galactose of the present invention may be selected from the group consisting of purified D-galactose, D-galactose from biomass, and D-galactose obtained through hydrolysis of lactose, but is not limited thereto.

The arabinose isomerase may be selected from the group consisting of manganese ion, magnesium ion and zinc ion, but is not limited thereto. The arabinose isomerase may be selected from the group consisting of isomerase Can be all the metal ions capable of bonding and isomerizing. Specifically, the manganese ion providing material may include manganese chloride; Materials providing magnesium ions include magnesium chloride; And zinc chloride, but are not limited thereto.

The reaction solution for producing D-tagatose contains a buffer system for maintaining a pH such as a Tris buffer, a phosphate buffer, and preferably a Tris buffer having a pH of 6.5 to 7.5. Manganese chloride, magnesium chloride or zinc chloride include 0.1 mM to 10 mM, preferably 1 mM to 5 mM. Galactose is added in an amount of 1 g / L to 300 g / L, preferably 18 g / L to 300 g / L, and the reaction temperature is 60 ° C to 95 ° C, preferably 70 ° C to 80 ° C, Preferably 72 ° C to 78 ° C, to produce D-tagatose.

Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art that these embodiments are for further illustrating the present invention and that the scope of the present invention is not limited to these embodiments in accordance with the gist of the present invention.

<Examples>

Example  1: for the production of enzymes with improved conversion activity Thamoto Neapolitanah  Derived Arabinose  Isomerase protein modelling  And analogy of major amino acid variations

Thermotoga neapolitana ). Since the tertiary structure of the wild-type arabinose isomerase derived from DSM 5068 is not known yet, the tertiary structure has already been revealed, and Escherichia coli coli) was subjected to three-dimensional structure predicted by molecular modeling techniques to the protein arabinose isomerase as a template. A comparative modeling technique was used for predicting the tertiary structure and a structural model was obtained using an APM module (Tripos, USA) in Trimos' molecular modeling package.

The comparative modeling technique is the most widely used method for predicting protein tertiary structure. If the desired protein amino acid sequence is similar to that of other proteins whose tertiary structure is known, the tertiary structure of the desired protein can be readily predicted using this method, and the prediction accuracy is very high in this case.

The structure of the E. coli-derived arabinose isomerase was determined in the form of a trimer registered in the protein data bank (PDB) as 2HXG.pdb.

As a result of the modeling, the wild-type arabinose isomerase (amino acid sequence; SEQ ID NO: 1, nucleotide sequence; SEQ ID NO: 6) derived from Thermotania neapolitanus DSM 5068 contains not only the arybynos isomerase and the base sequence of the Escherichia coli, The tertiary structure was also predicted to be very similar. In the case of the arabinose isomerase of Escherichia coli, several studies have revealed information about the substrate binding site, the cofactor manganese ion (Mn ^{2 +} ) binding site, and the main amino acids constituting it (Manjasetty & Chance, J. Mol. Biol., 2006. 360: 297-309). Based on this, the major residues of Arabomonas isomerase derived from Thermo-Neapolitanus DSM 5068 were deduced.

Sequence alignment, molecular docking simulation, and reaction mechanism analysis were performed for major amino acid residue analysis. Protein sequence analysis was performed using the clustalW algorithm ( //www.ebi.ac.uk/Tools ) based on the sequence information of 10 L-arabinose isomerase and Thermotoga neapoltanabinos isomerase / msa / clustalw2 / ) .

As a result of the sequencing analysis, the arabinose isomerase derived from Thermotaina napolata DSM 5068 had a very well conserved sequence of the metal binding moiety and the active moiety as well as other isomerizing enzymes. The isomerization enzyme derived from Thermomonas neapolitanus DSM 5068 is an active site including E302 (302th glutamic acid), E329 (329th glutamic acid), H346 (346th histidine), H445 (445th histidine) amino acid residue and manganese ion active site (FIG. 1), and the amino acid residues E302, E329, H346 and H445 affect manganese ion binding. In particular, the E302 and E329 residues are predicted as the most important factor promoting the isomerization reaction (Manjasetty & Chance, J. Mol. Biol., 2006. 360: 297-309).

It is assumed that the substrate selectivity is determined by the size, shape, and characteristics of the amino acid residues constituting the active site of the Arabomonas isomerase enzyme derived from Thermotaurus neapolitan. Important residues for substrate recognition were selected through molecular docking simulation (SurflexDock; Tripos, USA) using the original substrate L-arabinose. Also, the most suitable D-galactose binding site was selected through a reaction mechanism analysis (Adrian J. Mulholland, Drug Discov. Today. 2005. 10 (20): 1393-402). Based on this, amino acid residues which are highly likely to interfere with the binding of the active portion of arabinose isomerase and D-galactose were selected.

The amino acid at position 275 has an aromatic side chain and is made up of phenylalanine, which is relatively large in size and low in flexibility. It is predicted that this part will cause steric hindrance with the sixth carbon of D-galactose (Fig. 2). Since L-arabinose exhibits almost the same structure except that one carbon is smaller than D-galactose in pentose, substituting 275 amino acids with other kinds of amino acids makes D-galactose of the arabinose isomerase And the reactivity of the reaction mixture was greatly increased.

It is an amino acid that can replace phenylalanine. It has a similar polarity, is relatively small in size, and has high flexibility. It minimizes the repulsion of D-galactose with carbon number 6, while reducing the effect on the entire structure of arabinose isomerase The expected valine, methionine, and isoleucine were selected.

They are expected to be structurally free and substitute for phenylalanine, since they are nonpolar aliphatic chains compared to phenylalanine containing aromatics.

As a result of predicting the structure of a mutant (amino acid; SEQ ID NOS: 3 to 5, nucleic acid; SEQ ID NOS: 8 to 10) in which a point mutation was generated by a molecular modeling technique, the above mutations affect the entire structure of the arabinose isomerase The impact was expected to be insignificant.

In addition, a mutant exhibiting a certain amount of improved enzyme characteristics was obtained through random enzyme mutagenesis using the wild-type arabinose isomerase from Saemoto naeapitalana DSM 5068 and its genetic information. As a result of the comprehensive analysis, it was confirmed that the change of the amino acid sequence of the C-terminal portion of the arabinose isomerase tends to affect the enzyme activity.

The molecular structure of the C-terminal region of Arabinose isomerase was greatly changed by molecular prediction. As a result, it was found that by substituting proline for the 469th amino acid of the wild-type arabinose isomerase, the arabinose isomerization It was expected that the enzyme could greatly increase the reactivity to D-galactose.

Example  2: Designed Arabinose  Isomerase Mutant  Produce

(1) amino acid residue 469 Louis Xin  Replace with proline

The 469th leucine of the wild-type arabinose isomerase from S. thermophila DSM 5069 was replaced with proline by site-directed mutagenesis using specific primers.

The N-terminal primer (SEQ ID NO: 13) and the C-terminal primer (SEQ ID NO: 14), which are oligonucleotides of two complementary nucleotide sequences with mutations, were used as primers. Plasmid DNA was used as a template to amplify and synthesize plasmids with new mutations in vitro. The wild type DNA was then cleaved with Dpn I restriction enzyme. That is, the wild-type DNA is used as a template but as the DNA isolated from E. coli cut by Dpn I cutting recognizes G ^m6 ATC, not synthesized in vitro mutant DNA is cut.

This mutant strain was transformed into E. coli DH5alpha, and the mutation was confirmed by analyzing the nucleotide sequence of the mutant gene. This mutant was transformed into the final expression strain Corynebacterium &lt; RTI ID = 0.0 &gt; glutamicum ATCC 13032) to produce a recombinant strain, which was named L469P. Thereafter, this was used as a control.

(2) substituting the amino acid other than phenylalanine for the 259th amino acid

In order to induce additional mutations in the prepared variant L469P, a saturated mutagenesis technique was performed using a pair of primers containing a mutation in a vector in which the isomerizing enzyme was cloned.

Designed primer pairs were designed so that the 275th amino acid codon was replaced by NNS (N: A, T, G or C, S: G or C) and the variants obtained by this method could contain all 20 amino acids SEQ ID NO: 11 and SEQ ID NO: 12). These mutants can be obtained as a single colony through transformation, and their activity can be analyzed by selecting them to confirm the change of activity according to the 20 amino acid mutations at the corresponding positions.

Specifically, to construct a library containing 20 kinds of amino acids, PCR was carried out by adding a forward and reverse primer with a pECCG117-CJ1-TNAI_L469P plasmid (Korean Patent Laid-Open No. 10-2010-0016948) at a concentration of 100 μl / Polymerase Chain Reaction). The PCR reaction was carried out under the conditions shown in Tables 1 and 2 below. The library obtained through the PCR reaction was transformed into E. coli K12 DH5a strain to form a colony. Since the used plasmid can be cloned and expressed in both E. coli and coryneform bacteria, it was expressed in E. coli and the activity test was carried out as it was.

The isomerization enzyme expressed from the 110 colonies thus obtained was transformed into cysteine-carbazole method (Dische, Z., and E. Borenfreund, A New Spectrophotometric Method for Detection and Determination of Keto Sugars and Trioses, J. Mol. Biol. Chem., 192: 583-587, 1951). As a result, a large number of clones with higher activity than the control (L469P) could be identified (FIG. 3).

10 colonies which were most highly active were selected and their sequences were sequenced to confirm their respective sequences. As a result, it was confirmed that valine, methionine, isoruicin (amino acid; SEQ ID NO: 3 to 5, 10), indicating that the activity was improved. In addition, since no mutation was found except amino acid 275, it was confirmed that the phenylalanine residue at position 275 had a role of inhibiting the reactivity of D-galactose as assumed.

Again, the structure of the mutants was predicted by a molecular modeling technique (FIG. 4) to see how the mutation would affect the isomerization enzyme. According to the structural prediction results, it was confirmed that all three kinds of mutants were substituted for the positions of phenylalanine without significant changes in the secondary and tertiary structures. Thus, it could be expected that the reactivity to D-galactose was increased without significant changes in thermal stability and other production indicators.

By transforming the mutant strain in the final expression strain Corynebacterium glutamicum 13032 ACTC after producing the recombinant strain, respectively, "Corynebacterium glutamicum (Corynebacterium glutamicum pFIS-1-TNAI-2, pFIS-1-TNAI-3 and pFIS-1-TNAI-4 ", which were deposited on February 14, 2013 at 361-221 Hongse- KCCM11378P, KCCM11379P and KCCM11380P, respectively, at the Korean Microorganism Conservation Center, which is an international depository institution of the Korean Society of Microbiology.

Saturated mutagenesis PCR Reaction solution composition Addition amount (μl) PCR buffer (pfu-ultra) 10X 5 dNTP (2.5 mM) 5 pCJ1-TNAI_L469P One TNAI275_F primer One TNAI275_R primer One pfu-ultra One DDW Up to 50 μl

PCR reaction conditions step Temperature time cycle Initial denaturation 95 ℃ 5 minutes One denaturalization 95 ℃ 45 seconds
18 Annealing 60 ° C 45 seconds kidney 68 ° C 18 minutes Final height 72 ℃ 10 minutes One

Example  3: Corynebacterium  In the genus Microorganism Arabinose  Isomerase Mutant  Expression

In order to measure the degree of activity enhancement of the selected mutants and the applicability to actual D-tagatose production, the three kinds of mutants were expressed in microorganisms of the genus Corynebacterium, and the productivity and production related indicators were studied .

The three kinds of mutants selected in Example 2 were transformed into wild type Corynebacterium glutamicum (ATCC 13032) to prepare a recombinant strain. These were cultured in a medium containing 50 μg / ml kanamycin (20 g / L of glucose, 10 g / L of polypeptone, 10 g / L of yeast extract, 10 g / L of ammonium sulfate, 5.2 g / L of KH ₂ PO ₄ , K ₂ HPO at _{4 10.7g / L, MgSO 4 0.5g} / L, Urea 1.5g / L, D-Biotin 1.8mg / L, Thiamine 9mg / L, Ca-Panthothenic 9mg / L, Niacinamide 60mg / L) at 30 ℃ 20 sigan To induce the expression of recombinant arabinose isomerase variants.

To measure the activity of the expressed arabinose isomerase, the culture was centrifuged at 8000 g-force for 10 minutes, and the cells were collected and resuspended in 50 mM Tris-HCl (pH 7.5) buffer solution. The suspended cells were treated with 0.1% POESA at room temperature for 1 hour to weaken the cell wall. The cells were recovered by centrifugation again under the above conditions and resuspended in a mixed solution of 300 g / L D-galactose, 5 mM manganese chloride, and 50 mM Tris-HCl (pH 7.5) so as to be 4% (w / v). The reaction was carried out at 75 ° C for 1 hour. HPLC analysis (WATERS HPLC, EMPOWER system, WATERS SugarPak ID 6.5-L300 mm column, 2414 Refractive Index Detector) was performed for determination of D-galactose and D-tagatose.

One hour reaction, the control group L469P produced 35 g / L of D-tagatose, while the mutants F275V / L469P and F275I / L469P were 121 g / L · h, 117 g / L · h and F275I / L469P, And productivity of 101 g / L · h was confirmed.

Example  4: Corine  Expressed in bacteria Arabinose  Isomerase Mutant  Separation purification and activity measurement

A recombinant strain including three kinds of F275V / L469P, F275M / L469P, TNAI-F275I / L469P, TNAI-F275I / L469P mutant and L469P mutant with activity was seeded and cultured in a 2 L flask under the same conditions as in Example 3 Respectively. In order to determine the expression of arabinose isomerase, the activity was measured under the same conditions as in Example 3 using a part of the culture medium.

Cells obtained from the culture were resuspended in a buffer solution of 20 mM Tris-HCl (pH 7.5) containing 0.1 mM manganese chloride and cultured in a high-pressure cell crusher T-series 4.0 kW (T-series 4.0 kW: Constant systems, UK) Lt; / RTI &gt; For removal of endogenous proteins except arabinose isomerase, heat treatment was carried out at 75 ° C for 20 minutes. The heat-treated cell debris was removed by centrifugation at 8,000 g-force for 10 minutes, and then centrifuged at 60,000 g-force (BECKMAN COULTER Optima L-80 XP Ultracentrifuge) to remove cell debris and lipid components. As much as possible.

The cell extract thus obtained was purified using anion exchange chromatography (Mono Q ^TM 10/100 GL, GE Healthcare). After pre-equilibration with binding solution [binding solution, 50 mM NaCl, 0.1 mM manganese chloride, 20 mM Tris-HCl (pH 7.5)], excess cell lysate was bound and eluted solution [1 M NaCl, 0.1 mM manganese chloride, 20 mM Tris-HCl (pH 7.5)]. The fraction showing activity against the substrate D-galactose was selected through cystein-carbazole-sulfuric acid method and analyzed by SDS-PAGE.

As a result, it was confirmed that the purified protein had a molecular weight of about 56 kDa, which is consistent with the molecular weight of arabinose isomerase originating from the genus Neapolitania actually known. One of the best purified fractions was selected by SDS-PAGE and high concentration of NaCl was removed using a desalting column (PD-10 Desalting column, GE Healthcare). The finally obtained purified enzyme was confirmed by SDS-PAGE (Fig. 5). The purified proteins were quantitated using the Bradford assay and BSA (Bovine Serum Albumin) was used as the standard protein.

Example  5: Arabinose  Isomerase Mutant  Characterization study

It was confirmed that the three kinds of arabinose isomerase mutants prepared in the above examples exhibited remarkably enhanced activity compared to the mutant substituted with proline at position 469 of lucsin. Based on these results, experiments were conducted on the parameters related to the reaction conditions affecting the production of D-tagatose.

<5-1> Study on optimum temperature

Serotoga napolatanas arabinose isomerase is an enzyme of highly thermophilic fungi and has relatively high thermal stability and optimum temperature. The temperature conditions suitable for producing D-tagatose from D-galactose are between 55 ° C and 75 ° C, and at lower temperatures there is a problem due to contamination of heterologous bacteria, and the stability of produced D-tagatose at high temperatures . The existing wild-type arabinose isomerase or the control L469P has an optimal reaction temperature of 85 ° C, which is relatively low at a temperature at which the production process can be applied.

In order to investigate the optimal temperature of the mutant arabinose isomerase isolated and purified in Example 4, a purified enzyme was added to 100 mM D-galactose substrate and 50 mM Tris-HCl buffer solution containing 1 mM manganese chloride (MnCl ₂ ) (pH 7.5) at 60 ° C to 90 ° C at 5 ° C intervals.

The activity measurement was measured by the cysteine-carbazole method. As shown in FIG. 6, the optimum reaction temperature of the three mutant arabinose isomerases was 75 ° C, which was shifted by 10 ° C compared to L469P. As a result, it is confirmed that the activity of the isomerase is generally high in the process applicable temperature range, so that the operation range of the production process is broadly formed.

In addition, it can be guessed that the characteristics of the phenylalanine residue, amino acid 275, affect the optimum reaction temperature. It can be assumed that the phenylalanine 275 in the reaction of D-galactose, not the original substrate, is sufficient to ensure that the steric hindrance to D-galactose is minimized. As the temperature increases, the molecular motions of the phenyl residue and its vicinal moieties of phenylalanine become active, which may reduce the 6-carbon and steric hindrance of D-galactose. At the same time, it can be predicted that an optimal temperature will be formed within a range that does not greatly affect the tertiary or quaternary structure of the protein. Therefore, there may be a change in the optimum temperature in mutants in which the steric hindrance is fundamentally reduced by the 275 mutations. However, further analysis and experimentation is necessary to scientifically demonstrate this.

<5-2> Study on thermal stability

To investigate the thermal stability of mutant arabinose isomerase, enzyme purified at a concentration of 20 μg / ml in 50 mM Tris-HCl (pH 7.5) and 1 mM manganese chloride solution was added and incubated at 95 ° C. for 180 minutes in a constant temperature bath. The enzymatic reaction was performed with the enzyme solution sampled over time to measure the residual activity of the enzyme.

As a result of the thermal stability analysis, the arabinose isomerase mutants showed a decrease in the residual activity with time at 95 ° C as shown in Fig. 7, but the difference in thermal stability between the mutants was not large. In order to obtain more quantitative data, half-life of the activity was measured. As a result, it was confirmed that L469P had a half-life of about 3 hours under the above conditions and that the half-life of the 275 variants was about 2 hours 3). Relative thermal stability of 275 mutants was relatively low, but it is considered to be enough to offset the increase in activity and lowering the process temperature.

Half-life of the arabinose isomerase measured at 95 ° C Mutant Half-life (minutes) L469P 185 F275V / L469P 122 F275M / L469P 126 F275I / L469P 134

<5-3> Study on the change of activity by metal ion effect

Since many enzymes require metal ions for catalysis, changes in enzyme activity by metal ions were examined using purified enzymes in order to confirm the dependency of the arabinose isomerase mutant of the present invention on the metal ion in the thermal stability .

In order to investigate the change in enzyme activity according to the concentration by addition of the enzyme tablet in 100mM D- galactose substrate, 75 ℃ in 5mM manganese chloride in a concentration from 1mM (MnCl ₂₎ with 50mM Tris-HCl (pH 7.5) containing a solution , For 10 minutes (FIG. 8). As a result, all of the mutants showed similar activity within the range of the experiment, and it was found that the effect of manganese ions on isomerase activity was not significant depending on the urea concentration essential for enzyme activity.

<5-4> Study on enzyme activity

In order to investigate the rate of enzyme reaction, the specific activity of the arabinose isomerase mutant at a reaction temperature of 75 ° C was determined. 1 mM enzyme activity was measured by adding 1 mM manganese chloride and 100 mM D-galactose at pH 7.5 and 75 ° C. As a result of inactivation for 10 min under these conditions, the 275 mutants showed about 5.5 times (F275V), 5 times (F275M) and 3.9 times (F275I) higher activity than L469P, respectively (Table 4).

Inactivity of mutants L469P F275V / L469P F275M / L469P F275I / L469P Inactivity (U / mg) 2.4 13.1 12.1 9.3

Korea Microorganism Conservation Center (overseas) KCCM11378P 20130214 Korea Microorganism Conservation Center (overseas) KCCM11379P 20130214 Korea Microorganism Conservation Center (overseas) KCCM11380P 20130214

<110> CJ Cheiljedang Corporation <120> L-arabinose isomerase variants with improved conversion activity          and method for production of D-tagatose using them <130> PA12-0444 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 496 <212> PRT <213> Thermotoga neapolitana <400> 1 Met Ile Asp Leu Lys Gln Tyr Glu Phe Trp Phe Leu Val Gly Ser Gln   1 5 10 15 Tyr Leu Tyr Gly Leu Glu Thr Leu Lys Lys Val Glu Gln Gln Ala Ser              20 25 30 Arg Ile Val Glu Ala Leu Asn Asn Asp Pro Ile Phe Pro Ser Lys Ile          35 40 45 Val Leu Lys Pro Val Leu Lys Asn Ser Ala Glu Ile Arg Glu Ile Phe      50 55 60 Glu Lys Ala Asn Ala Glu Pro Lys Cys Ala Gly Val Ile Val Trp Met  65 70 75 80 His Thr Phe Ser Pro Ser Lys Met Trp Ile Arg Gly Leu Ser Ile Asn                  85 90 95 Lys Lys Pro Leu Leu His Leu His Thr Gln Tyr Asn Arg Glu Ile Pro             100 105 110 Trp Asp Thr Ile Asp Met Asp Tyr Met Asn Leu Asn Gln Ser Ala His         115 120 125 Gly Asp Arg Glu His Gly Phe Ile His Ala Arg Met Met Arg Leu Pro Arg     130 135 140 Lys Val Val Gly His Trp Glu Asp Arg Glu Val Arg Glu Lys Ile 145 150 155 160 Ala Lys Trp Met Arg Val Ala Cys Ala Ile Gln Asp Gly Arg Thr Gly                 165 170 175 Gln Ile Val Arg Phe Gly Asp Asn Met Arg Glu Val Ala Ser Thr Glu             180 185 190 Gly Asp Lys Val Glu Ala Gln Ile Lys Leu Gly Trp Ser Ile Asn Thr         195 200 205 Trp Gly Val Gly Glu Leu Ala Glu Arg Val Lys Ala Val Pro Glu Asn     210 215 220 Glu Val Glu Glu Leu Leu Lys Glu Tyr Lys Glu Arg Tyr Ile Met Pro 225 230 235 240 Glu Asp Glu Tyr Ser Leu Lys Ala Ile Arg Glu Gln Ala Lys Met Glu                 245 250 255 Ile Ala Leu Arg Glu Phe Leu Lys Glu Lys Asn Ala Ile Ala Phe Thr             260 265 270 Thr Thr Phe Glu Asp Leu His Asp Leu Pro Gln Leu Pro Gly Leu Ala         275 280 285 Val Gln Arg Leu Met Glu Glu Gly Tyr Gly Phe Gly Ala Glu Gly Asp     290 295 300 Trp Lys Ala Ala Gly Leu Val Arg Ala Leu Lys Val Met Gly Ala Gly 305 310 315 320 Leu Pro Gly Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Leu Thr                 325 330 335 Pro Gly Asn Glu Leu Val Leu Gly Ala His Met Leu Glu Val Cys Pro             340 345 350 Thr Ile Ala Lys Glu Lys Pro Arg Ile Glu Val His Pro Leu Ser Ile         355 360 365 Gly Gly Lys Ala Asp Pro Ala Arg Leu Val Phe Asp Gly Gln Glu Gly     370 375 380 Pro Ala Val Asn Ala Ser Ile Val Asp Met Gly Asn Arg Phe Arg Leu 385 390 395 400 Val Val Asn Arg Val Leu Ser Val Pro Ile Glu Arg Lys Met Pro Lys                 405 410 415 Leu Pro Thr Ala Arg Val Leu Trp Lys Pro Leu Pro Asp Phe Lys Arg             420 425 430 Ala Thr Ala Trp Ile Leu Ala Gly Gly Ser His His Thr Ala Phe         435 440 445 Ser Thr Ala Val Asp Val Glu Tyr Leu Ile Asp Trp Ala Glu Ala Leu     450 455 460 Glu Ile Glu Tyr Leu Val Ile Asp Glu Asn Leu Asp Leu Glu Asn Phe 465 470 475 480 Lys Lys Glu Leu Arg Trp Asn Glu Leu Tyr Trp Gly Leu Leu Lys Arg                 485 490 495 <210> 2 <211> 496 <212> PRT <213> Thermotoga neapolitana <400> 2 Met Ile Asp Leu Lys Gln Tyr Glu Phe Trp Phe Leu Val Gly Ser Gln   1 5 10 15 Tyr Leu Tyr Gly Leu Glu Thr Leu Lys Lys Val Glu Gln Gln Ala Ser              20 25 30 Arg Ile Val Glu Ala Leu Asn Asn Asp Pro Ile Phe Pro Ser Lys Ile          35 40 45 Val Leu Lys Pro Val Leu Lys Asn Ser Ala Glu Ile Arg Glu Ile Phe      50 55 60 Glu Lys Ala Asn Ala Glu Pro Lys Cys Ala Gly Val Ile Val Trp Met  65 70 75 80 His Thr Phe Ser Pro Ser Lys Met Trp Ile Arg Gly Leu Ser Ile Asn                  85 90 95 Lys Lys Pro Leu Leu His Leu His Thr Gln Tyr Asn Arg Glu Ile Pro             100 105 110 Trp Asp Thr Ile Asp Met Asp Tyr Met Asn Leu Asn Gln Ser Ala His         115 120 125 Gly Asp Arg Glu His Gly Phe Ile His Ala Arg Met Met Arg Leu Pro Arg     130 135 140 Lys Val Val Gly His Trp Glu Asp Arg Glu Val Arg Glu Lys Ile 145 150 155 160 Ala Lys Trp Met Arg Val Ala Cys Ala Ile Gln Asp Gly Arg Thr Gly                 165 170 175 Gln Ile Val Arg Phe Gly Asp Asn Met Arg Glu Val Ala Ser Thr Glu             180 185 190 Gly Asp Lys Val Glu Ala Gln Ile Lys Leu Gly Trp Ser Ile Asn Thr         195 200 205 Trp Gly Val Gly Glu Leu Ala Glu Arg Val Lys Ala Val Pro Glu Asn     210 215 220 Glu Val Glu Glu Leu Leu Lys Glu Tyr Lys Glu Arg Tyr Ile Met Pro 225 230 235 240 Glu Asp Glu Tyr Ser Leu Lys Ala Ile Arg Glu Gln Ala Lys Met Glu                 245 250 255 Ile Ala Leu Arg Glu Phe Leu Lys Glu Lys Asn Ala Ile Ala Phe Thr             260 265 270 Thr Thr Phe Glu Asp Leu His Asp Leu Pro Gln Leu Pro Gly Leu Ala         275 280 285 Val Gln Arg Leu Met Glu Glu Gly Tyr Gly Phe Gly Ala Glu Gly Asp     290 295 300 Trp Lys Ala Ala Gly Leu Val Arg Ala Leu Lys Val Met Gly Ala Gly 305 310 315 320 Leu Pro Gly Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Leu Thr                 325 330 335 Pro Gly Asn Glu Leu Val Leu Gly Ala His Met Leu Glu Val Cys Pro             340 345 350 Thr Ile Ala Lys Glu Lys Pro Arg Ile Glu Val His Pro Leu Ser Ile         355 360 365 Gly Gly Lys Ala Asp Pro Ala Arg Leu Val Phe Asp Gly Gln Glu Gly     370 375 380 Pro Ala Val Asn Ala Ser Ile Val Asp Met Gly Asn Arg Phe Arg Leu 385 390 395 400 Val Val Asn Arg Val Leu Ser Val Pro Ile Glu Arg Lys Met Pro Lys                 405 410 415 Leu Pro Thr Ala Arg Val Leu Trp Lys Pro Leu Pro Asp Phe Lys Arg             420 425 430 Ala Thr Ala Trp Ile Leu Ala Gly Gly Ser His His Thr Ala Phe         435 440 445 Ser Thr Ala Val Asp Val Glu Tyr Leu Ile Asp Trp Ala Glu Ala Leu     450 455 460 Glu Ile Glu Tyr Pro Val Ile Asp Glu Asn Leu Asp Leu Glu Asn Phe 465 470 475 480 Lys Lys Glu Leu Arg Trp Asn Glu Leu Tyr Trp Gly Leu Leu Lys Arg                 485 490 495 <210> 3 <211> 496 <212> PRT <213> Thermotoga neapolitana <400> 3 Met Ile Asp Leu Lys Gln Tyr Glu Phe Trp Phe Leu Val Gly Ser Gln   1 5 10 15 Tyr Leu Tyr Gly Leu Glu Thr Leu Lys Lys Val Glu Gln Gln Ala Ser              20 25 30 Arg Ile Val Glu Ala Leu Asn Asn Asp Pro Ile Phe Pro Ser Lys Ile          35 40 45 Val Leu Lys Pro Val Leu Lys Asn Ser Ala Glu Ile Arg Glu Ile Phe      50 55 60 Glu Lys Ala Asn Ala Glu Pro Lys Cys Ala Gly Val Ile Val Trp Met  65 70 75 80 His Thr Phe Ser Pro Ser Lys Met Trp Ile Arg Gly Leu Ser Ile Asn                  85 90 95 Lys Lys Pro Leu Leu His Leu His Thr Gln Tyr Asn Arg Glu Ile Pro             100 105 110 Trp Asp Thr Ile Asp Met Asp Tyr Met Asn Leu Asn Gln Ser Ala His         115 120 125 Gly Asp Arg Glu His Gly Phe Ile His Ala Arg Met Met Arg Leu Pro Arg     130 135 140 Lys Val Val Gly His Trp Glu Asp Arg Glu Val Arg Glu Lys Ile 145 150 155 160 Ala Lys Trp Met Arg Val Ala Cys Ala Ile Gln Asp Gly Arg Thr Gly                 165 170 175 Gln Ile Val Arg Phe Gly Asp Asn Met Arg Glu Val Ala Ser Thr Glu             180 185 190 Gly Asp Lys Val Glu Ala Gln Ile Lys Leu Gly Trp Ser Ile Asn Thr         195 200 205 Trp Gly Val Gly Glu Leu Ala Glu Arg Val Lys Ala Val Pro Glu Asn     210 215 220 Glu Val Glu Glu Leu Leu Lys Glu Tyr Lys Glu Arg Tyr Ile Met Pro 225 230 235 240 Glu Asp Glu Tyr Ser Leu Lys Ala Ile Arg Glu Gln Ala Lys Met Glu                 245 250 255 Ile Ala Leu Arg Glu Phe Leu Lys Glu Lys Asn Ala Ile Ala Phe Thr             260 265 270 Thr Thr Val Glu Asp Leu His Asp Leu Pro Gln Leu Pro Gly Leu Ala         275 280 285 Val Gln Arg Leu Met Glu Glu Gly Tyr Gly Phe Gly Ala Glu Gly Asp     290 295 300 Trp Lys Ala Ala Gly Leu Val Arg Ala Leu Lys Val Met Gly Ala Gly 305 310 315 320 Leu Pro Gly Gly Thr Ser Phe Met Glu Asp Tyr Thr Tyr His Leu Thr                 325 330 335 Pro Gly Asn Glu Leu Val Leu Gly Ala His Met Leu Glu Val Cys Pro             340 345 350 Thr Ile Ala Lys Glu Lys Pro Arg Ile Glu Val His Pro Leu Ser Ile         355 360 365 Gly Gly Lys Ala Asp Pro Ala Arg Leu Val Phe Asp Gly Gln Glu Gly     370 375 380 Pro Ala Val Asn Ala Ser Ile Val Asp Met Gly Asn Arg Phe Arg Leu 385 390 395 400 Val Val Asn Arg Val Leu Ser Val Pro Ile Glu Arg Lys Met Pro Lys                 405 410 415 Leu Pro Thr Ala Arg Val Leu Trp Lys Pro Leu Pro Asp Phe Lys Arg             420 425 430 Ala Thr Ala Trp Ile Leu Ala Gly Gly Ser His His Thr Ala Phe         435 440 445 Ser Thr Ala Val Asp Val Glu Tyr Leu Ile Asp Trp Ala Glu Ala Leu     450 455 460 Glu Ile Glu Tyr Pro Val Ile Asp Glu Asn Leu Asp Leu Glu Asn Phe 465 470 475 480 Lys Lys Glu Leu Arg Trp Asn Glu Leu Tyr Trp Gly Leu Leu Lys Arg                 485 490 495

Claims (10)

The 275th amino acid of the amino acid sequence represented by SEQ ID NO: 1 is substituted with an amino acid other than phenylalanine, the 469th amino acid is substituted with proline, and the activity of converting D-galactose into D-tagatose is improved An arabinose isomerase variant. The method according to claim 1,
Wherein the 275th amino acid is substituted with any one amino acid selected from the group consisting of valine, methionine, and isoleucine. 10. A polynucleotide characterized by encoding the arabinose isomerase variant of claim 1. The method of claim 3,
Wherein the polynucleotide has the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. A microorganism of the genus Corynebacterium transformed with a recombinant vector comprising the polynucleotide of claim 3. 6. The method of claim 5,
Wherein the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum. 6. The method of claim 5,
The genus Corynebacterium microorganisms Corynebacterium glutamicum (Corynebacterium glutamicum ) pFIS-1-TNAI-2 (KCCM11378P), pFIS-1-TNAI-3 (KCCM11379P) or pFIS-1-TNAI-4 (KCCM11380P). A method for producing D-tagatose comprising culturing a microorganism belonging to the genus Corynebacterium according to any one of claims 5 to 7. 9. The method of claim 8,
Wherein the solution containing D-galactose is reacted with a substance providing a metal ion selected from the group consisting of manganese chloride, magnesium chloride and zinc chloride in the presence of the microorganism of the genus Corynebacterium. &Lt; / RTI &gt; 9. The method of claim 8,
Wherein the manganese chloride is added in a concentration of 0.1 mM to 10 mM.

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