KR101015343B1 - L-arabinose isomerase variant having improved activity - Google Patents

Abstract

The present invention is applied to the protein engineering technology, the arabinose isomerase variants with improved enzyme activity by changing the protein three-dimensional structure of the wild type arabinose isomerase derived from Thermotoga neapolitana DSM 5068 It relates to the manufacture of. More specifically, the non-polar side chain (non-polar side chain) located at the end of the wild type arabinose isomerase The present invention relates to the production of arabinose isomerase variants with improved enzymatic activity compared to the wild type arabinose isomerase by replacing leucine (leucine) located at the 469th hydrophobic amino acid with proline.

Arabinose isomerase, variant, three-dimensional structure, molecular modeling, proline, galactose, tagatose

Description

L-ARABINOSE ISOMERASE VARIANT HAVING IMPROVED ACTIVITY

The present invention is applied to protein engineering technology to change the three-dimensional structure of the wild type arabinose isomerase derived from Thermotoga neapolitana DSM 5068, compared to the wild type arabinose isomerase It relates to the preparation of arabinose isomerase variants with improved enzyme activity.

Tagatose is a sweet sugar that exists in nature, and 10% of the aqueous solution of tagatose gives a sweet taste of 90% to sugar. Its shape also has the form of white crystalline powder that does not smell like sugar and does not contribute to calories because it is hardly metabolized upon absorption in the body. In addition, sugar alcohols, which are most commonly used as sugar substitute sweeteners, may induce diarrhea when consumed in a predetermined amount or more, whereas tagatose has an advantage of not having such side effects. Therefore, tagatose has characteristics such as low calorie, non-cavity, and overcoming the limitations of diarrhea-induced limit, and is a health sweetener that can be safely consumed by adults and children with adult diseases such as obesity and diabetes, resulting from excessive intake of sugar, etc. It is also expected to contribute to the prevention of various social structural diseases.

Tagatose is attracting attention as a sugar substitute sweetener because of its properties, and it is known as a material with great market potential in the food market. However, since tagatose is not commonly present in nature but is a rare sugar contained in dairy products or some plants, a technique capable of mass production has to be developed to be used as a low-calorie functional sweetener.

Tagatose has been produced since 2003 by Arla Foods, a chemical isomerization method of Ca (OH) ₂ from D-galactose, and is known as 'Gaio-tagatose'. We are pioneering the market under the brand name. However, the chemical isomerization method is excellent in terms of isomerization conversion yield, but is difficult to recover and purify and the process is complicated. Therefore, the yield is expected to be lower than that of enzymatic isomerization in consideration of overall process yield.

L-arabinose isomerase (EC 5.3.1.5) is an enzyme that converts arabinose to L-ribulose in vivo, but in vitro Converts D-galactose, a structurally similar substrate, to D-tagatose. Thus, the use of highly active L-arabinose isomerase can produce tagatose in a much more efficient process than the chemical isomerization method described above.

Thermomicroorganism Neapolitana DSM 5068 ( thermotoga which is high temperature microorganism Arabinose isomerase derived from neapolitana DSM 5068) has excellent heat safety, but needs to be further improved to secure economic productivity at the level of glucose isomerase.

In general, a method of preparing a mutant enzyme to enhance the activity of an enzyme or to impart activity on a new substrate is divided into random mutagenesis and rational design. There is this. Random mutations are widely used because they can be applied without specific information about the enzymes of interest, but a screening system should be available for investigating the mutations of a large number of individuals. On the other hand, the improvement of enzymes by rational design generates only a limited number of mutant enzymes, and thus does not require a special screening system. You should know in detail.

Sugar converting enzymes such as arabinose isomerase, an enzyme that converts galactose to tagatose, are generally difficult to apply to systems capable of screening modified enzymes at high speed. There is a point that the improvement is practically not practical.

Therefore, the present inventors applied computer-based protein engineering technology so that arabinose isomerase enzyme, which has potential for tagatose production, has industrial productivity, so that Thermomotoga DSM 5068 ( Thermotoga Through the structure of arabinose isomerase derived from neapolitana DSM 5068), a foundation for designing arabinose isomerase having desired characteristics is provided, and the protein three-dimensional structure of the wild type arabinose isomerase is changed. The present invention has been accomplished by designing and improving arabinose isomerase to have improved activity against galactose substrates.

Another object of the present invention is to provide a gene encoding the arabinose isomerase variant, a recombinant vector containing the gene, and a microorganism transformed by the recombinant vector.

Another object of the present invention is to provide a method for producing tagatose from galactose, characterized in that using the arabinose isomerase variant and the transformed microorganism.

In order to achieve the above object, the present invention is a thermomotor Neapolitana DSM 5068 ( Thermotoga The 469th leucine of wild type arabinose isomerase derived from neapolitana DSM 5068) is replaced with proline to provide arabinose isomerase variants with improved enzyme activity.

The present invention also provides a gene encoding the arabinose isomerase variant, a recombinant vector containing the gene and a microorganism transformed by the recombinant vector.

The present invention also provides a method for producing tagatose from galactose, characterized in that using the arabinose isomerase variant and the transformed microorganism.

By isomerizing galactose as a substrate using the recombinant arabinose isomerase variant of the present invention, it can be usefully used to greatly increase the productivity of tagatose and greatly reduce the production cost.

The present invention provides arabinos isomerase variants with improved enzymatic activity by applying protein engineering techniques to the protein tertiary structure of wild type arabinose isomerase derived from Thermotoga neapolitana DSM 5068. .

Protein engineering technology uses existing protein structures to induce substitution of specific gene fragments, which is a technology that applies the evolution of protein in nature. The evolution of proteins with new functions in nature has been reported to produce superfamily proteins of various functions using existing protein frameworks rather than de novo occurrences.

In a preferred embodiment of the invention, the thermomoto has a nonpolar side chain located at the end of the Neapolitana or DSM 5068-derived wild type arabinose isomerase By replacing the 469th leucine in the hydrophobic amino acid with proline, an arabinose isomerase variant with improved enzyme activity compared to the wild type enzyme was prepared.

The preparation of the arabinose isomerase variant according to the present invention is as follows.

(1) Step 1: Selection of amino acids to cause mutation

The three-dimensional structure of the wild type arabinose isomerase derived from Thermomotor Neapolita or DSM 5068 is not yet known. In order to prepare a new variant of the wild type arabinose isomerase derived from Neapolitana, the thermomotor, whose tertiary structure has not been identified, is replaced with arabinose isomerase derived from Neapolitana. The structure obtained by X-ray crystallography of north isomerase was used. The structure of the E. coli-derived arabinose isomerase is a structure determined in the form of a trimer registered as 2HXG.pdb in a protein data bank (PDB).

The most commonly used method for the prediction of protein tertiary structure is the comparative modeling method. If the tertiary structure of the desired protein amino acid sequence is very similar to that of other known proteins, the method can be used to easily predict the tertiary structure of the desired protein, in which case the prediction accuracy is very high.

The comparative model method includes a fragment-based model and a constraint-based model. In the present invention, a method based on constraint conditions was used. First, the distance between amino acids or atoms of the template protein was measured, and then the distance was used as a restraint to predict the tertiary structure of the desired protein. An exemplary method of the constraint based method is "MODELLER".

As the structure file of the template protein for this experiment, 2HXG.pdb, which is a structure file of E. coli-derived arabinose isomerase, is used. Since the C-monomer was removed from the trimer structure obtained from the 2HXG.pdb, the structure file was obtained leaving only the A-monomer and the B-monomer. At this time, since the C-monomer was removed only in the text of the pdb file, the structure of the A-monomer and the B-monomer was not changed at all.

To run the modeler, you need to change 2HXG.pdb file to 2HXG.atm file. Thereafter, the amino acid sequences of the template protein and the target protein are paired and arranged at the European Bioinformatics Institute (EBI). Based on these two files, the modeler is run to create a tertiary structure of the target protein, similar to the template protein.

The wild type arabinose isomerase, which is the subject of the present invention predicted by the above method, has been found to exist as a tetramer. However, since the active site of the protein can not be identified by the monomer, the study was conducted in the dimer (dimmer) structure of the protein.

Using a wild-type arabinose isomerase derived from Neapolitana as a template, Thermomoto obtained a variant and genetic information showing a certain amount of improved enzyme properties through a multi-year randomized enzyme mutation method. As a result, it was confirmed that the change in the amino acid sequence of the C-terminal portion of the enzyme tends to affect the increase of enzyme activity.

The amino acid of the C-terminal region constituting the active site of the wild type arabinose isomerase was confirmed through molecular modeling. As a result, the 469th leucine residue of the wild-type arabinose isomerase changes to proline, causing the final beta-sheet of the protein to disappear and the backbone angle to be distorted. The final alpha-helix protein core was moved to alter the structure of the protein.

In a preferred embodiment of the present invention, Thermomoto made the monomeric tertiary structure of the wild type arabinose isomerase and arabinose isomerase variant proteins derived from Neapolitana or DSM 5068 using a Modeler program. The structure is shown in FIG. In FIG. 1, the 469th leucine (L469) and the 469th proline (P469) can be expressed in wild-type arabinose isomerase (TNAI_Wild) and arabinose isomerase variant (TNAI_L469P), respectively, to confirm the position of amino acids present in the protein. .

As a result, the wild type arabinose isomerase maintained the intact 18th beta screen structure, but the arabinose isomerase variant was confirmed that the beta screen structure was lost in the part containing the 469th proline residue. This is shown in FIG. 2.

To see the overall structural differences between the wild type arabinose isomerase and arabinose isomerase variant proteins, the degree of similarity between the root mean square deviation (RMSD) of the two proteins and the three-dimensional structure of the two optimally overlapping proteins is examined. To numerically represent the square-average-distance between corresponding pairs of atoms. As a result, it showed an RMSD of 1.0, and found that the overall structure between the wild-type arabinose isomerase and arabinose isomerase variant proteins is very similar (FIG. 3).

In addition, in order to see the secondary structure between wild-type arabinose isomerase and arabinose isomerase variant proteins, secondary structure of the proteins was predicted by Kabsch-Sander method in InsightII (molecular modeling software), which is shown in Table 1.

As shown in Table 1, comparing the 18th beta folding structure of the wild-type arabinose isomerase (TNAI_Wild) and arabinose isomerase mutant (TNAI_L469P), arabinose isomerase mutant (TNAI_L469P) is the 469th residue leucine first part It is not formed as a folding structure. As mentioned in FIG. 2, it can be seen that the 469th leucine was substituted with proline, thereby affecting the secondary structure change of the arabinose isomerase variant protein.

Secondary Structure Comparison of Wild-type Arabinos Isomerase (TNAI_Wild) and Arabinos Isomerase Variants (TNAI_L469P) Kabsch-Sander
Wild type Arabinos  Isomerase
( TNAI _ wild )
Arabinos  Isomerase Mutant
( TNAI _ L469P )
Alpha Helix Structure Beta screen structure Alpha Helix Structure Beta screen structure One 3-6 8-13 3-6 8-14 2 21-40 48-50 25-38 47-54 3 57-69 73-79 57-69 76-79 4 91-95 100-104 86-89 100-105 5 118-122 145-148 118-121 145-149 6 126-129 177-181 126-129 177-181 7 131-140 205-209 131-140 205-209 8 154-174 270-272 151-169 270-273 9 195-201 298-300 171-175 298-301 10 211-220 326-334 197-201 326-334 11 223-236 341-345 211-220 341-345 12 252-266 360-361 223-235 360-361 13 287-295 376-377 252-264 376-377 14 305-317 384-393 286-295 384-393 15 430-439 398-407 305-317 398-407 16 454-464 422-426 430-439 422-426 17 477-491 445-449 454-463 445-448 18 467-471 477-491 470-471

Figure 4 shows that the beta screen structure disappears as the 469th leucine residue of wild-type arabinose isomerase is changed to proline, and the main chain is misaligned. As the 18th beta screen structure containing the 469th leucine residue of the arabinose isomerase variant disappears and the backbone is distorted, the tertiary structural position of the 17th alpha helix following the 18th beta screen structure is lost. Moving toward the body predicted that the structure behind the arabinose isomerase variant residues would have changed.

The final alpha helical motion predicted above is not known exactly as the role of arabinose isomerase variant by monomer structure alone, so that each of the proteins of wild type arabinose isomerase and arabinose isomerase variants is dimerized. I made a structure and looked at it structurally. Similarity was confirmed by superimposing each of the wild type arabinose isomerase and arabinose isomerase variant proteins to the ECAI dimer obtained from 2HXG.pdb. The overall dimer structure is shown in FIG. 5.

As the 469th leucine residue was changed to proline, it was possible that the structure after the 469th proline might have moved more inward, so that the carbonyl carbon of the mutated arabinose isomerase variant residues and the preceding The distance between the 480th phenylalanine (F) and the 484th leucine carbonyl carbon was measured. If the distance measured in the arabinose isomerase variant is closer than the distance measured in the wild type arabinose isomerase, it can be determined that there is movement of the 18th alpha helix as mentioned above. As shown in Figure 6, wild-type arabinose isomerase showed a distance of 9.08 Å and 10.49 와 with the two previous residues, and arabinose isomerase variants showed a distance of 8.69 Å and 10.06 Å, respectively. As a result, arabinose isomerase variants appeared shorter at these two distances than wild type arabinose isomerase. It is clearly thought to indicate that protein mutations occurred and moved toward the protein body.

To further confirm this, the distance was measured including other peripheral residues. The crystal structure of 2HXG.pdb, a template of the arabinose isomerase, has a structure up to the 493th leucine. The wild-type arabinose isomerase and arabinose isomerase proteins, which are modeled as examples, also have the correct structure up to L493. Therefore, the distance between the last residue, L493, the 469th leucine residue and the 469th proline residue, was measured, respectively.

As a result, as shown in FIG. 7, the distance between the wild type arabinose isomerase and the L493 residue was 20.18 ,, and the distance between the arabinose isomerase variant and the L493 residue was 19.70 Å, and the 17th alpha of the mutant arabinose isomerase protein was shown. It was confirmed that the spiral moved toward the protein body.

Finally, dimer structure was used to confirm this. Looking at the dimeric structure of wild type arabinose isomerase and arabinose isomerase variants (FIG. 5), it can be seen that the last alpha helices of the A- and B-monomers face each other. Thus, by measuring the distance between the L493 residues of this dimer structure, if the two distances are farther away from the wild-type arabinose isomerase than the protein of the arabinose isomerase variant, it is clear that the last 17th alpha helix x has moved towards the protein body. Could be.

As a result, the wild type arabinose isomerase was 13.92Å and the arabinose isomerase variant was 14.81Å. Therefore, the mutation of the L469 residue of the wild-type arabinose isomerase into the P469 residue of the arabinose isomerase variant caused the final alpha helix to move toward the protein body, which is believed to alter the structure of the protein core. .

As a result, the substitution of leucine, the 469th amino acid of the wild-type arabinose isomerase, with proline resulted in a significant change in the chain structure of the C-terminal region of the arabinose isomerase by molecular prediction. Although only one amino acid was substituted, the molecular predictive structure itself showed a significant change.

(2) Step 2: Induce Mutation for Amino Acid at Specific Location

To prepare arabinose isomerase variants with high activity against galactose substrates, the 469th leucine, the position selected in the first step, was replaced with proline using a site-directed mutagenesis method. .

(3) Step 3: Confirmation of the activity of the improved arabinose isomerase variant

The sequencing of the variant prepared in the second step was confirmed whether the substitution with proline at the 469th position. As a result of culturing colonies containing these variants and measuring their activity, it was confirmed that the variants showed higher activity against galactose as a substrate than wild-type arabinose isomerase.

The present invention also provides a gene encoding the arabinose isomerase variant, a recombinant vector containing the gene and a microorganism transformed by the recombinant vector.

Gene encoding the arabinose isomerase variant of the present invention is Thermomotoga neapolitana or DSM 5068 ( Thermotoga neapolitana DSM 5068), preferably the gene encoding the arabinose isomerase variant in which the 469th amino acid leucine is substituted with proline. More preferably, the gene has the amino acid sequence of SEQ ID NO.

The vector usable in the present invention is not particularly limited and may be a known expression vector. Preferably, a vector including the gene may be used.

The present invention provides a microorganism prepared by transforming the microorganism of the genus Corynebacterium with the recombinant vector. Preferably, the transformed microorganism is Corynebacterium glutamicum ( Corynebacterium glutamicum ) ATCC 13032, the microorganism of the genus Corynebacterium prepared Corynebacterium glutamicum ( Corynebacterium glutamicum ) CJ-1-TANI_L469P (Accession No .: KCCM10955P).

The present invention can also prepare tagatose from galactose using the arabinose isomerase variant and the transformed microorganism.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example  1: 469th amino acid Leucine  By substitution Arabinos  Preparation of Isomerase Variants ( Cloning )

The present inventors predicted the structure of the wild type arabinose isomerase based on the structure of the arabinose isomerase derived from E. coli, which is similar to E. coli. In addition, it was confirmed that the 469th leucine of the wild-type arabinose isomerase using site-directed mutagenesis using a specific primer was confirmed that the high activity when the leucine amino acid located at 469th is replaced with proline. By proline.

N-terminal primer (SEQ ID NO: 1) and C-terminal primer (SEQ ID NO: 2), which are oligonucleotides of two complementary base sequences with mutations, were used as primers. Plasmids with new mutations were amplified and synthesized in vitro using the plasmid DNA as a template, and the original wild type DNA was digested with Dpn I restriction enzyme and removed. That is, the wild type DNA used as a template is cleaved by Dpn I, which recognizes and cleaves G ^m6 ATC as DNA isolated from Escherichia coli, but does not cleave the mutated DNA synthesized in vitro.

SEQ ID NO: 5'- GGA TCC CAT ACT CCT TTC TCA ACA GAG-3 '

SEQ ID NO: 5'- CGC TGT TGA GAA AGG AGT ATG ATG GGA TCC-3 '

The strain was transformed into E. coli DH5alpha to obtain a mutant strain, and then the nucleotide sequence of the mutant gene was analyzed to confirm that the mutation occurred. The mutant strain was finally expressed as Corynebacterium glutamicum ATCC 13032 ( Corynebacterium). glutamicum ATCC 13032) to produce a recombinant strain, and then "corynebacterium glutamicum ( Corynebacterium glutamicum ) CJ-1-TNAI_L469P ", which was assigned to KCCM10955P on June 30, 2008 to the Korea Microbiological Conservation Center, an international depository organization located at 361-221, Hongje 1-dong, Seodaemun-gu, Seoul, Korea. Deposited.

Example  2: In the Corynebacterium Arabinos  Isomerase Mutant  Expression

Recombinant strain Corynebacterium glutamicum CJ-1-TNAI_L469P (Accession Number KCCM10955P) prepared in Example 1 AC medium containing 10% g / ml Kanamycin (Kanamycin) (Polypepton 10g / L, Bacto-yeast extract 5g / L, (NH4) 2SO4 5g / L, KH ₂ PO ₄ 4g / L, K ₂ HPO ₄ 8g / L, Urea 1.5g / L, MgSO ₄ -7H ₂ O 0.5g / L, L-Cystein 0.5g / L, d-Biotin 0.2mg / L, Tiamine-HCl 1.5mg / L, Ca-Panthothenic 2mg / L, Niacinamide 3mg / L) incubated at 30 ° C for 20 hours to induce the expression of recombinant arabinose isomerase variants .

In order to measure the activity of the expressed arabinose isomerase, the culture medium was centrifuged at 8000 × g for 10 minutes, and the cells were collected, and then resuspended in 50 mM Tris-HCl (pH 7.5) buffer solution. Suspended cells were subjected to sonication of cells by sonication and galactose isomerization using the supernatant as a coenzyme solution. Galactose isomerization was performed by mixing 100 μl of enzyme solution containing 100 mM galactose as a substrate and 1 ml of reaction buffer solution (50 mM Tris-HCl, pH 7.5).

At this time, a final concentration of 1 mM manganese chloride (MnCl ₂ ) was added to the reaction solution. The prepared coenzyme reaction solution was reacted at 75 ° C. for 20 minutes, and the activity of the coenzyme was measured by the cystein-carbazol-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). Protein quantification in the coenzyme reaction solution was carried out using the BCA method (Bicinchoninic acid; Sigma Chemical Co., USA) using a reaction in which bicinconinate (BCA) forms a complex with copper ions and develops purple color, and as a result, galactose isomerization It was confirmed that the reaction product tagatose was normally produced.

Example  3: recombination Arabinos  Isomerase Mutant  Purification and purification of pure protein

Corynebacterium glutamicum CJ-1-TNAI_L469P, a recombinant strain whose activity was confirmed in Example 2, was used as an AC medium containing kanamycin at a concentration of 10 μg / ml, which is an optimal medium condition (Polypepton 10g / L, Bacto-yeast extract 5g / L, (NH ₄ ) ₂ SO ₄ 5g / L, KH ₂ PO ₄ 4g / L, K ₂ HPO ₄ 8g / L, Urea 1.5g / L, MgSO ₄ -7H ₂ O 0.5g / L, L-Cystein 0.5g / L, d-Biotin 0.2mg / L, Tiamine-HCl 1.5mg / L, Ca-Panthothenic 2mg / L, Niacinamide 3mg / L) and 50g / in 5L fermenter (Biotron Co., Korea) l sucrose, 10 g / l polypeptone, 10 g / l yeast extract, 2.5 g / l sodium chloride, 0.5 g / l L-cysteine, 10 g / l (NH ₄ ) ₂ SO ₄ , 1.2 g / l KH ₂ PO ₄ , d- biotin, thiamine -HCl, Ca- pantothenate, niacinamide _{(niacinamide), MnSO 4 -5H 2} O, FeSO 4 -7H 2 O, ZnSO 4 -7H 2 O, CuSO 4 -5H 2 O, MgSO 4 -7H Inoculated at an initial concentration of OD600 = 0.1 in 3 L medium consisting of ₂ O and incubated.

Incubation at 30 ° C. for 20 hours induced expression of recombinant arabinose isomerase. To measure the activity of the expressed arabinose isomerase, the cells were collected by centrifuging the culture at 8000xg for 10 minutes, and then in a 50 mM Tris-HCl pH 7.5 buffer solution containing 5 mM manganese chloride (MnCl ₂ ). Resuspend.

The suspended cells were cell disrupted using a high pressure cell crusher T-series 4.0kW (T-series 4.0kW: Constant systems, UK). In order to remove host proteins except arabinose isomerase, heat treatment was performed at 75 ° C. for 20 minutes. The heat-treated cell debris was removed by centrifugation at 9000 rpm for 20 minutes, and then filtered through a 0.2 μm filter to separate the expressed heat-resistant arabinose isomerase.

The recombinant enzyme solution thus separated was purified and purified by ion exchange resin chromatography (Q Sepharose Fast Flow, Amersham Bioscience, U.S.A). 20 mM Tris-HCl (pH 7.5) was used after pre-equilibration with a binding solution. Elution was used to add 0.5M NaCl to the above binding solution.

As a result of purification by concentrating the culture solution about 30 times, many impurities were found in the heat treatment step, but the final enzyme solution passed through the Q Sepharose Fast Flow (anion exchange column) was relatively free of impurities. After selecting the fraction showing activity for the substrate galactose through the cysteine-carbazol-sulfuric acid method, it was analyzed by SDS-PAGE as shown in FIG.

As a result, it was confirmed that the purified protein had a molecular weight of about 56 kDa, which indicates that the actually known thermomoto was consistent with the molecular weight of the arabinose isomerase derived from Neapolitana genus. Dialysis with 20mM Tris-HCl buffer (pH 7.5), solution to select one of the finest fractions via SDS-PAGE and remove high concentration of NaCl: MWCO = 8,000 (Spectra / Por membrane, SPECTRUM Lab. Inc.) .)). Finally obtained enzyme solution was confirmed through SDS-PAGE (Fig. 8).

Example  4 : Variant Arabinos  Biochemical Characterization of Isomerase

<4-1> Study on optimum temperature

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

The activity was measured by the cysteine-carbazol-sulfuric acid method. As a result of the enzyme activity according to the temperature, the mutant arabinose isomerase was 85 ° C. as shown in FIG. It can be seen that there is no big difference with. This seems to be due to the increased thermal stability of the variant arabinose isomerase.

<4-2> Study on thermal stability

In order to investigate the thermostability of the mutant arabinose isomerase, 0.05 mg of purified enzyme was added and the temperature was changed at 70 ° C. to 95 ° C. at 5 ° C. intervals and incubated in a constant temperature water bath for 180 minutes. In addition, the thermal stability of co-factor (manganese chloride (MnCl ₂ ) 1mM) was confirmed. Enzyme reaction was performed with the enzyme solution sampled by time to measure the remaining activity of the enzyme.

As a result of thermal stability, wild-type arabinose isomerase showed that the enzyme was relatively stable up to 80 ° C. when manganese chloride was added as shown in FIG. 10, but at higher temperatures, the stability of the enzyme decreased as the temperature increased. The arabinose isomerase variant showed stable enzyme up to 90 ° C.

Since the arabinose isomerase variant is a thermophilic microorganism derived from Neapolitana, a thermophilic microorganism, half-life according to the presence or absence of manganese chloride was measured. When manganese chloride was added, the enzyme activity was very stable up to 90 ° C, and the enzyme half-life at 95 ° C was measured to be about 6 hours.However, if manganese chloride was not added, the enzyme half-life was about 8 hours and 30 minutes even at 85 ° C. It was found to have. Thus, this arabinose isomerase variant It was found that the enzymatic stability according to the addition of the metal showed a great effect, it was confirmed that the metal dependency is high (Table 2).

Half-life of arabinose isomerase at different temperatures (t _{1/2 /} min) Cofactor
1 mM MnCl ₂ Sample 60 ℃ 65 ℃ 70 ℃ 75 ℃ 80 ℃ 85 ℃ 90 ° C 95 ℃ (+) Wild ND ^a 395.6 135.1 36.5 34 (+) 469P ND ^a ND ^a ND ^a ND ^a 327.7 (-) 469P 1235.5 994.2 1635.3 546.3 491.5

ND ^a : Not detected (very stable)

<4-3> Study on activity change by metal ion effect

Since many enzymes require metal ions for catalysis, to investigate the dependence of metal ions on the thermal stability of the arabinose isomerase variant of the present invention, we investigated the change of enzyme activity by metal ions using purified enzyme. .

First, all metal ions in the enzyme were removed using 10 mM EDTA, and then purified enzyme was added to 100 mM galactose substrate to obtain an optimal temperature in 50 mM Tris-HCl buffer containing pH of 1 mM manganese chloride (MnCl ₂ ). The experiment was performed at 85 ° C. for 30 minutes. 1 mM addition of various metal ions confirmed the change of enzyme activity (FIG. 12). Mn ^{2 +} and Co ^{2 +} metal ions with a relatively high enzyme activity were Co ^{2 +} .

<4-4> Study on the effect of metal ion by concentration

Purified enzyme was added to 100 mM galactose substrate to investigate the change of enzymatic activity according to the concentration of Mn ^{2 +} and Co ^{2 +} , in 50 mM Tris-HCl buffer containing pH of 1 mM manganese chloride (MnCl ₂ ). The experiment was performed at 85 ° C. for 30 minutes at the optimum temperature. Various metal ions were added from 0mM to 10mM concentration to confirm the effect of metal ion concentration. For the wild-type enzyme and mutant enzymes as a result of FIG 13, Mn ^{2 +} is a look the same activity at 1mM or more concentrations, in the case of Co ^{2 +} was the maximum activity at 2mM.

<4-5> Study on optimum pH

The activity of arabinose isomerase variants was measured by varying the pH from 4 to 9 to investigate the optimal pH. The reaction temperature was experimented for 30 minutes in various pH solutions containing 1mM manganese chloride in 100mM galactose substrate at 85 ℃ the optimum temperature. The buffer solutions used were as follows: 50 mM Sodium acetate (pH 4-5), 50 mM Sodium phosphate (pH 6-7), 50 mM Tris-Cl (pH 7-8), 50 mM Glycin-NaOH (pH 9).

As shown in FIG. 14, the optimal pH of the wild-type arabinose isomerase was 7.0, but the optimal pH of the enhanced arabinose isomerase variant was increased to pH 7.4.

<4-6> Study on Enzyme Reaction Rate

In order to investigate the enzyme kinetics, kinetic parameters of arabinose isomerase variants were obtained under the reaction temperature of 75 ° C. (Table 3). As a result of examining the substrate affinity K _m value, wild type arabinose isomerase was 139.4 mM and the arabinose isomerase variant was 186.1 mM, showing no significant difference. The enzyme reaction rate V _max was 4.26U / mg for wild-type arabinose isomerase, but arabinose isomerase variant was increased to 9.5U / mg. The enzyme catalytic efficiency k _cat / K _m was 1.71 mM ^-1 min ^{- 1} for wild-type arabinose isomerase, whereas the arabinose isomerase variant was 7.8 mM ^-1 min ^-1 .

This means that arabinose isomerase variants have increased tagatose production capacity. In conclusion, the arabinose isomerase variant, in which the 469th leucine residue of wild-type arabinose isomerase was changed to proline, changed the structure of the protein core part because the last alpha helix moved toward the protein body, and compared to wild-type arabinose isomerase. The activity against (K _cat / K _m ) was 5 times higher.

Catalytic Properties of Wild-type Arabinos Isomerase and Arabinos Isomerase Variants characteristic Arabinose isomerase Temp _opt
(° C) pH _opt t _{One /2}
( min ) v _max       (U / mg ) K ^m                 ( mM ) k _cat Of K _m           ( mM ^-One min ^-One ) Wild type 80-85 7.0-7.5 ND ^a 4.26 139.4 1.71 Mutant 85-90 6.5-7.0 ND ^a 9.6 ± 0.26 68.5 ± 1.95 7.8 ± 0.01

ND ^a : Not detected (very stable)

1 is a thermomotor Neapolitana DSM 5068 ( Thermotoga This study compares the tertiary structure of wild type arabinose isomerase derived from neapolitana DSM 5068) and arabinose isomerase variant in which the 469th lysine (L469) residue was changed to proline (P469).

FIG. 2 shows the L469 site of wild-type arabinose isomerase (TNAI_Wild) and the P469 site of arabinose isomerase variant (TNAI_L469P).

3 is the RMSD of TNAI wild and TNAI_L469P.

Figure 4 is a result of comparing the structure of the wild type arabinose isomerase derived from Thermomotor Neapolitana or DSM 5068 and the arabinose isomerase variant in which the L469 residue is changed to P469.

5 is a dimer structure.

FIG. 6 is a result of comparing the distance between the L469 residue of wild-type arabinose isomerase and the peripheral residue of the arabinose isomerase variant changed to P469.

7 shows the result of calculating the distance between the wild type enzyme and the surrounding residues of the mutant enzyme TNAI_L469P .

8 shows SDS-PAGE results of wild-type arabinose and arabinose isomerase variants obtained by pure separation. (Lane 1, left) wild type arabinose isomerase, (lane 2, right) arabinose isomerase variant

Figure 9 is a graph showing the enzyme activity according to the temperature of the wild type enzyme and the substitution mutant enzyme 469 leucine (Leucine, L) changed to proline (Proline, P) according to the present invention.

10 is a graph showing the thermal stability of both proteins in the presence of 1 mM manganese chloride (MnCl ₂ ). (A) Wild type arabinose isomerase, (B) arabinose isomerase variant

11 is a graph showing the thermal stability of the substitution mutase 469 leucine (Leucine, L) changed to proline (P) according to the present invention.

12 is a graph showing the change in activity of wild-type arabinose isomerase and arabinose isomerase variants by metal ion effect. (A) Wild-type arabinose isomerase, (B) variant arabinose isomerase

FIG. 13 is a graph illustrating an optimum concentration of manganese and cobalt two metal ions having increased enzymatic activity in Example <4-3>. (A) wild-type arabinose isomerase (B) arabinose isomerase variant

14 is a graph showing the optimum pH of wild-type arabinose isomerase and arabinose isomerase variants.

<110> CJ Cheiljedang Corporation <120> L-arabinose isomerase variant having improved activity <130> PA08-0162 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggatcccata ctcctttctc aacagag 27 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cgctgttgag aaaggagtat gatgggatcc 30 <210> 3 <211> 496 <212> PRT <213> Corynebacterium glutamicum <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 Arg Leu Pro Arg     130 135 140 Lys Val 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 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 (6)

The amino acid sequence of SEQ ID NO: 3, wherein leucine, the 469th amino acid of the C-terminal region of the wild type arabinose isomerase derived from Thermotoga neapolitana DSM 5068, is replaced with proline, improving the enzyme activity. Arabinose isomerase variants encoded with a branched gene. delete Paragraph (1) SEQ ID NO: 3 of the four amino acids transformed with a recombinant vector Corey bacterium comprising a gene having a sequence Solarium glutamicum (Corynebacterium glutamicum). The method of claim 3, The transformed Corynebacterium glutamicum is Corynebacterium glutamicum ( Corynebacterium glutamicum ) Corynebacterium glutamicum characterized in that CJ-1-TANI_L469P (Accession Number: KCCM10955P). A method for producing tagatose from galactose using the arabinose isomerase variant of claim 1. A method for producing tagatose from galactose using the Corynebacterium glutamicum of claim 3.

Images