KR101063960B1 - Novel mannose-6-phosphate isomerase and variants thereof and uses thereof - Google Patents

Abstract

The present invention relates to a novel mannose-6-phosphate isomerase enzyme, a mutant enzyme thereof, and a method for producing el-ribose using the enzyme, and more particularly, to mannose-6-phosphate isomerization. Recombinant expression vectors comprising the enzyme, its mutant enzyme and its genes, microorganisms transformed therewith, and methods for producing mannose-6-phosphate isomerase or mutants thereof in large quantities, and the mannose-6-phosphate isomerization The present invention relates to a production method for obtaining el-ribose in high yield using an enzyme or a mutant thereof.

Description

Novel mannose 6-phosphate isomerase and mutant etc. and use of the same

The present invention relates to a novel mannose-6-phosphate isomerase enzyme, a mutant enzyme thereof, and a method for producing el-ribose using the enzyme, and more particularly, to mannose-6-phosphate isomerization. Recombinant expression vectors comprising the enzyme, its mutant enzyme and its genes, microorganisms transformed therewith, and methods for producing mannose-6-phosphate isomerase or mutants thereof in large quantities, and the mannose-6-phosphate isomerization The present invention relates to a production method for obtaining el-ribose in high yield using an enzyme or a mutant thereof.

L-ribose is the starting material for the synthesis of many L-type saccharide drugs, the antiviral methyl-L-riboflanoside ("Bezimidavir" TM). ), And the global market for el-ribose and its derivatives was about $ 1.1 billion in 2001.

In addition, non-double oil 1263 double oil 94 (BW1263W94, Glaxo Wellcome), which has recently been developed as a new anti-herpes drug, and L-FMAU, Bukwang & Triangle, which is being developed for the treatment of hepatitis B, etc. As demand is rising as a key intermediate, the development of industrially available manufacturing methods is of interest to many researchers in the field.

L-ribose has been produced by chemical synthesis mainly from L-arabinose, L-xylose, di-glucose, di-galactose, di-ribose or di-manno-1,4-lactone (Akagi, M., et. al ., Chem . Pharm . Bull . ( Tokyo ) 50: 866, 2002; Takahashi, H., et al ., Org. Lett . 4: 2401, 2002; Yun, M., et al ., Tetrahedron Lett . 46: 5903, 2005). However, this chemical synthesis method has several serious problems in its production process.

Indeed, there are risks in the working environment that require high temperatures and pressures, the separation and purification of complex ribose due to the formation of additional sugars after chemical reactions, and the environmental pollution caused by chemical waste generated in this process.

In order to overcome such drawbacks, a method of preparing biological el-ribose from ribitol or el-ribulose has recently been studied.

In addition, using recombinant E. coli containing NDA-dependent mannitol-1-dehydrogenase, 55% conversion yield was obtained from 100 g / L ribitol in 72 hours after fermentation. The productivity of ribose is about 28 times lower than chemical synthesis made from L-Arabinose (Woodyer RN, et. al ., Appl . Environ . Microbiol. 74: 2967, 2008; Jumppanen, J., et al ., US patent 6,140,498).

Meanwhile, El-biological production methods of research ribose is keulribiji Ella pneumoniae (Klebsiella pneumonia) arabinose isomerase derived from Pseudomonas shoe Cherry (Pseudomonas stutzeri ) L-rhamnose isomerase, Streptomyces Ruby Geonosis (Streptomyces rubiginosus) derived from xylose isomerase (D-xylose isomease) and Lactobacillus Cocos Lactis (Lactococcus lactis ) -derived galactose-6-phosphate isomerase, but these enzymes have broad substrate specificity, so they can convert L-riblos to el-ribose, but the conversion rate is Very slow

Recently, the inventors of the Bacillus subtilis ( Bacillus) subtilis ) overcomes the problem of low productivity by converting el-ribulose to el-ribose using mannose-6-phosphate isomerase (Yeom SJ, et. al ., Appl . Environ . Microbiol . 75: 4705, 2009). However, mannose-6-phosphate isomerase derived from Bacillus subtilis is an enzyme derived from mesophilic bacteria and has a limitation in dissolving a large amount of substrate because of low thermal stability and low reaction temperature. Therefore, in order to overcome this, it is urgent to develop economical biological methods that have high L-ribose productivity, high thermal stability, and overcome the limitations of substrate solubility.

In addition, the inventors hot bacterium Bacillus Geo Thermo di NITRY pecan switch (Geobacillus mannose-6-phosphate isomerase from thermodenitrificans was used to convert L-ribulose to L-ribose (Yeom SJ, et. al ., Biotechnol . Lett . 31: 1273, 2009), which also requires higher inactivation enzymes for effective industrialization.

The present invention solves the above problems and the object of the present invention is to provide a novel mannose-6-phosphate isomerase.

Another object of the present invention is to provide a mutant of the mannose-6-phosphate isomerase.

Still another object of the present invention is to provide a method of preparing the mannose-6-phosphate isomerase.

Another object of the present invention is to provide a method for producing high yield of el-ribose.

In order to achieve the above object, the present invention provides a mannose 6-phosphate isomerase described in SEQ ID NO: 1.

The present invention also provides an R142N mutant mannose 6-phosphate isomerase obtained by converting the amino acid of residue 142 of the mannose 6-phosphate isomerase of the present invention from arginine (Arg) to asparagine (Asn).

The present invention also provides a gene encoding the wild type mannose 6-phosphate isomerase of the present invention.

In one embodiment of the present invention, the wild type gene preferably has a nucleotide sequence set forth in SEQ ID NO: 2, but is not limited thereto.

The present invention also provides a gene encoding the mutant mannose 6-phosphate isomerase of the present invention. In one embodiment of the present invention, the gene is preferably a gene having a nucleotide sequence set forth in SEQ ID NO: 3, but is not limited thereto.

The present invention also provides a recombinant expression vector pET 28a (+) / mannose 6-phosphate isomerase comprising the mannose 6-phosphate isomerase gene of the present invention.

In one embodiment of the invention, the recombinant expression vector preferably has a cleavage map described in Figure 8 is not limited thereto.

Also,

a) preparing an expression vector comprising the gene of the present invention;

b) culturing the microorganism transformed with the expression vector;

c) providing a method for producing mannose 6-phosphate isomerase and mutants thereof of the present invention comprising the step of separating and purifying mannose 6-phosphate isomerase from the microorganism.

The present invention also provides a method for producing ribose using the mannose 6-phosphate isomerase or a mutant thereof of the present invention.

In one embodiment of the present invention, the ribose is L-ribose (L-ribose), preferably produced using L-ribulose (L-ribulose) as a substrate, but is not limited thereto.

The present invention also provides a composition for producing ribose comprising the mannose 6-phosphate isomerase of the present invention or a mutant thereof.

Hereinafter, the present invention will be described.

The present inventors have diligently tried to develop a method for producing ribose using mannose-6-phosphate isomerase more effectively, resulting in thermos , a high temperature bacterium. Writing a brush Russ a mannose-6-phosphate isomerase producing a gene in large quantities, and without the generation of by-products in this El environmentally friendly process derived from (Thermus thermophilus) - was identified that can produce a high yield of the ribose, the mannose- 6-phosphate heh mutant enzyme was substituted for specific amino acid sequence with other amino acids of the enzyme of the isomerase has the sseomeoseu The present invention was completed by confirming that it was an el-ribose producing enzyme that surpassed mannose 6-phosphate isomerase derived from Thermus thermophilus .

Hereinafter, the present invention will be described in detail.

Mannose-6-phosphate isomerase of the present invention is characterized by having the amino acid sequence represented by SEQ ID NO: 1. In addition, in the amino acid sequence of SEQ ID NO: 1, at least one variant of deletion, substitution and addition of one or more amino acids is introduced within a range in which the enzyme activity of the present invention indicated by the protein having these amino acid sequences is not impaired. The modified mannose-6-phosphate isomerase is also included in the mannose-6-phosphate isomerase of the present invention, and the most representative example thereof is the amino acid of residue 142 of mannose 6-phosphate isomerase of the present invention. Is an R142N mutant converted to asparagine (Asn).

In the present invention, a mannose 6-phosphate isomerase gene encoding a mannose 6-phosphate isomerase having an amino acid sequence of SEQ ID NO: 1 is included, and the gene sequence thereof is represented by SEQ ID NO: 2. The mutant mannose 6-phosphate isomerase gene encoding the above-described mutant mannose 6-phosphate isomerase obtained by mutating the nucleotide sequences of SEQ ID NO: 1 is also included in the mannose-6-phosphate isomerase gene of the present invention. The most representative example is the gene of SEQ ID NO: 3.

The present invention also includes a recombinant vector containing the mannose-6-phosphate isomerase gene and a transformant transformed with the recombinant vector. The present invention also includes a method for producing mannose-6-phosphate isomerase, wherein the transformant is cultured to separate mannose-6-phosphate isomerase from the obtained culture.

Mannose-6-phosphate isomerase gene according to the present invention is separated from the thermopile sseomeoseu pillar's (Thermus thermophilus) strain. First, the mannose-6-phosphate isomerase gene with sseomeoseu Thermophilus ( Thermus thermophilus ) to obtain chromosomal DNA. Next, the oligonucleotide, and the nucleotide designed as a primer, filler sseomeoseu Thermo switch (Thermus The polymerase chain reaction (PCR) is performed using the chromosomal DNA of the thermophilus strain as a template to partially amplify the mannose-6-phosphate isomerase gene. The PCR amplified fragment thus obtained was Summers Thermophilus ( Thermus thermophilus ), a fragment having a homology close to 100% of the mannose-6-phosphate isomerase gene of the strain, and having a high S / N ratio as a probe when performing colony hybridization, and a hybridization string Facilitate stringency control The PCR amplification fragments are labeled with appropriate reagents, and colony hybridization is performed on the chromosomal DNA library to select mannose-6-phosphate isomerase genes (Current Protocols in Molecular Biology, Vol. 1, p. 603). , 1994).

The DNA fragment containing the mannose-6-phosphate isomerase gene was obtained by recovering the plasmid from the E. coli selected by the above method using alkaline method (Current Protocols in Molecular Biology, Vol. 1, p. 161, 1994). Can be. After determining the nucleotide sequence by the above method, it is possible to obtain the entire gene of the present invention by hybridizing the DNA fragment prepared by digestion by restriction enzymes of the DNA fragment having the nucleotide sequence as a probe. SEQ ID NO: 2 shows the nucleotide sequence of the mannose-6-phosphate isomerase gene of the present invention, and SEQ ID NO: 1 shows the amino acid sequence encoded by the gene.

The transformed microorganism of the present invention is obtained by introducing the recombinant vector of the present invention into a host suitable for the expression vector used when producing the recombinant vector. For example, when a bacterium such as E. coli is used as a host, the recombinant vector according to the present invention is capable of autonomous replication in the host, and at the same time, a DNA containing a promoter, mannose-6-phosphate isomerase gene, and transcription. It is preferable to have a structure required for expression of the termination sequence. Although pET 28a was used as the expression vector used in the present invention, any expression vector satisfying the above requirements can be used.

In the production of mannose-6-phosphate isomerase according to the present invention, a transformant obtained by transforming a host by a recombinant vector having a gene encoding the same is cultured, and a gene product is cultured in a culture (cultured cell or culture supernatant). Phosphorus mannose-6-phosphate isomerase is produced and accumulated and the enzyme is obtained from the culture.

Acquisition and purification of the mannose-6-phosphate isomerase of the present invention is carried out by centrifuging the cells or supernatants from the cultures obtained, followed by cell disruption, affinity chromatography, cation or anion exchange chromatography, or the like. It can carry out by combining.

The present invention provides a novel mannose-6-phosphate isomerase or a recombinant expression vector comprising a mutant thereof and a gene thereof, a microorganism transformed therewith and a method for producing a large amount of mannose-6-phosphate isomerase using them, and By using the mannose-6-phosphate isomerase, there is an effect of providing a production method for obtaining high yield of el-ribose.

Thermos of the present invention Thermo filler's mannose-6-phosphate derived from one (Thermus thermophilus) isomerase or its mutants can produce the ribose of a starting material of pharmaceutical products with high specificity and environmentally friendly way with a high yield, so the production cost EL-ribose is It can be usefully used as a starting material for the synthesis of a drug for a variety of L- form nucleic acid sugar.

Figure 1a shows the enzyme activity according to the metal ion type of mannose-6-phosphate isomerase of the present invention, Figure 1b shows the enzyme activity according to the metal ion concentration.
Figure 2a shows the enzyme activity according to the pH of the mannose-6-phosphate isomerase of the present invention (●: PIPES buffer; ○: EPPS buffer), Figure 2b shows the enzyme activity with temperature.
Figure 3 shows the results of measuring the temperature stability of the mannose-6-phosphate isomerase of the present invention (●: 65 ℃; ■: 70 ℃; ▲: 75 ℃; ○: 80 ℃; and □: 85 ℃).
Figure 4 shows the hourly production of L-ribose according to the present invention.
Figure 5 shows the conversion of ribose by mannose 6-phosphate isomerase (open circle) and mutant enzyme (closed circle) of the present invention at a substrate concentration of 10 Mm.
Figure 6 shows the gene sequence of the mannose 6-phosphate isomerase of the present invention.
Figure 7 shows the gene sequence of the R142N mutant enzyme of the mannose 6-phosphate isomerase of the present invention.
Figure 8 shows a cleavage map of a recombinant expression vector comprising a mannose 6-phosphate isomerase gene of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  One. Mannos Preparation of Recombinant Expression Vectors and Transgenic Microorganisms Containing -6-Phosphate Isomerase Gene

In order to produce a mannose-6-phosphate isomerase of the present invention, the filler's Thermo sseomeoseu (Thermus mannose-6-phosphate isomerase derived from the thermophilus strain was first isolated.

Specifically, the thermos thermophilus KCCM 40897 strain having a gene sequence and amino acid sequence is already selected, and known DNA sequence of mannose-6-phosphate isomerase derived therefrom (Genebank Accession No. AP008226; sequence Based on the number 1), the following primers were devised.

SEQ ID NO: 4 (Forward primer): 5'-TTT CATATG AGGCGGTTGGAGCCCAA-3 '

SEQ ID NO: 5 (reverse primer): 5'-TTT GAATTC ACTCACGCCCCCTCCTT-3 '

The primers were designed with Nde I and EcoR I restriction enzyme cleavage portions, respectively, and amplified the nucleotide sequence of the gene by polymerase chain reaction (PCR).

A large amount of mannose-6-phosphate isomerase gene was inserted into the plasmid vector pET 28 (+) (manufactured by Novagen) using restriction enzymes Nde I and EcoR I and isomerized to pET 28 (+) a / mannose-6-phosphate An enzyme was produced.

The recombinant expression vector thus obtained is transformed into E. coli ER 2566 strain by a conventional transformation method, the transformed microorganism is cultured for the production of L-ribose by adding 20% glycerin (glycerine) solution Frozen before.

Example  2. Mannos Preparation of -6-Phosphate Isomerase

In order to mass produce the mannose-6-phosphate isomerase of the present invention, the frozen E. coli ER 2566 strain was inoculated into a test tube containing 3 ml of LB medium, and the absorbance was 2.0 at 600 nm. The spawn culture was performed with a shake incubator at 37 ° C. until. Then, the seed cultured culture was added to a 2,000 ml flask containing 500 ml of LB medium to carry out the main culture.

In addition, when the absorbance reached 0.6 at 600 nm, 0.1 mM IPTG was added to induce mass expression of mannose-6-phosphate isomerase. At this time, the stirring speed was maintained at 200rpm, the culture temperature was maintained at 37 ℃, after the addition of IPTG was adjusted to the stirring speed 150rpm, culture temperature 16 ℃ and incubated.

In addition, the mannose-6-phosphate isomerase produced by overexpression as described above, the culture medium of the transformed strain was washed twice with 0.85% sodium chloride (NaCl) by centrifugation at 6,000 × g for 30 minutes at 4 ℃. Next, 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole and 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride) were added to disrupt the cell solution with a sonicator. The cell lysate was again centrifuged at 13,000 × g for 20 minutes at 4 ° C., the cell pellet was removed, and only the cell supernatant was obtained for a fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA, USA). Histrap HP adsorption column using His-tag was used as an enzyme solution for the production of L-ribose.

Example  3. Mannos Metal Specificity of -6-Phosphate Isomerase

Example 2 To investigate the specificity for the metal ions of the mannose-6-phosphate isomerase obtained in the activity of the enzyme was treated with 10mM EDTA and metal ion (Mn ^{+ 2,} Zn ^2+, Ba ^{+ 2,} Cu ^{2 +} , Co ^{2 +} , Ca ^{2 +} , Mg ^{2 +} , Ni ^{2 +} , Fe ^{2 +} ) and the reaction was measured as follows.

The enzymatic reaction was performed using 50 mM PIPES (piperazine- N, N' -bis (2-ethane sulfonic acid)) buffer solution (pH 7.0) containing 10 mM L-ribulose, each metal ion and 0.05 unit / ml enzyme. 5 minutes at 75 ° C, and the reaction was stopped again by adding a final concentration of 200 mM hydrogen chloride (HCl).

In the present invention, the enzyme activity was measured using L-ribulose as a substrate, and the enzyme activity (unit) of the enzyme activity was defined as the amount to produce 1 μmol of L-ribose per minute at pH 7.0 and 75 ° C. It was.

In addition, the concentration of ribose and ribulose, and the analysis of other sugars in the determination of enzymatic activity can be determined by using a bio-liquid chromatography (Bio-LC) system equipped with an electrochemical detector and a CarboPacPA column ( Dionex ICS-3000, Sunnylvale, Calif., USA), wherein the Carbopack PA column was passed 200 mM sodium hydroxide (NaOH) at 1 ° C./min at 30 ° C.

As a result, the purified enzyme and the enzyme treated with EDTA had no activity. Among the metal salts tested, copper (Cu ^{2 +} ) was the most effective, and the optimum concentration was 0.5 mM.

From the above results, the mannose-6-phosphate isomerase of the present invention was shown to be affected by 0.5 mM copper metal ion, confirming that it is an enzyme dependent on the metal ion (see FIGS. 1A and 1B).

Example  4. Mannos Of 6-6-phosphate Isomerase

In order to investigate the activity according to the pH and temperature change of the mannose-6-phosphate isomerase obtained in Example 2, the enzyme and the substrate were reacted under various pH and temperature conditions and the enzyme activity was compared.

4-1. Mannos -6-Phosphate Isomerase Activity pH  effect

First, in order to investigate the pH effect on the enzyme activity, 10mM L-ribulose and 0.5mM copper, 50mM PIPES buffer containing 1.5unit / ml enzyme and 10mM L-ribulose, the synthesis was carried out using the enzyme reaction buffer to pH 8.5 in the range of pH 7.5, - 0.5mM copper and 0.05unit / ㎖ the enzyme contains a EPPS ((3-propane sulfonic acid ) N- (2-hydroxyethyl) piperazine- N) Specifically, the reaction was stopped at 75 ° C. for 5 minutes and again added with a final concentration of 200 mM hydrogen chloride.

As a result, the optimum pH was found to be 7.0 (see FIG. 2A).

4-2. Mannos Effect of Temperature on -6-Phosphate Isomerase Activity

To investigate the temperature effect on the activity of the enzyme, the enzyme reaction temperature was ranged from 60 ° C. to 90 ° C. in a 50 mM PIPES buffer at pH 7.0 containing 10 mM L-ribulose, 0.5 mM copper and 0.05 unit / ml enzyme. After each reaction for 20 minutes, 200 mM hydrogen chloride was added to terminate the reaction.

As a result, the optimum temperature was found to be 75 ° C (see FIG. 2B).

4-3. Mannos Investigation of Temperature Stability of --6-phosphate Isomerase

In order to investigate the temperature stability of the enzyme, each enzyme using a 50 mM PIPES buffer, pH 7.0 containing 10 mM L-ribulose, 0.5 mM copper, 0.05 unit / ml enzyme in the temperature range from 65 ℃ to 85 ℃ After the reaction was halved, the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride to measure the activity of mannose-6-phosphate isomerase.

As a result, it was confirmed that the enzymatic activity was reduced by half after elapse of 22 hours at 65 ° C, 10 hours at 70 ° C, 5.5 hours at 75 ° C, 2.2 hours at 80 ° C and 0.3 hours at 85 ° C (see FIG. 3). ).

4-4. Mannos Effect of Substrate Concentration on -6 Phosphate Isomerase Activity

In order to investigate the temperature effect on the activity of the enzyme, pH containing 50, 100, 200 and 300 g / L- ribulose, 0.5 mM copper, 20 unit / ml enzyme in the temperature range from 75 ℃ to 85 ℃ The reaction was stopped for 3 hours using a 50 mM PIPES buffer of 7.0, and then the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride to measure the activity of mannose-6-phosphate isomerase.

As a result, regardless of the concentration of El-ribulose used as a substrate, the conversion yield was about 70%, respectively, at 36, 71, 140, and 211 g / l at 50, 100, 200, and 300 g / l of El-ribulose, respectively. El-Ribose was produced

Example  5. Mannos L- using 6-phosphate isomerase Ribose  production

In order to confirm the productivity of El-ribose using the mannose-6-phosphate isomerase of the present invention, 300 g / L at the temperature (75 ° C) considering the optimal pH 7.0 and the time when the enzyme activity was reduced by half Hourly production of El-ribose was measured using L-ribulose as a substrate. The reaction solution used was 50 mM PIPES buffer at pH 7.0 containing 300 g / L L-ribulose, 0.5 mM copper, 25 units / ml enzyme.

As a result, after 2.5 hours of reaction, 213 g / L of L-ribose was produced in 300 g / L of L-ribulose, which showed 85.2 g / L of productivity and 71% conversion yield per hour (see FIG. 4).

These results show the highest productivity of ribose production to date, and the chemical synthesis method (23% conversion yield and 20 g / l / hour productivity from L-Arabinose using molybdic acid) has been shown. J., J. Nurmi, and O. Pastinen. October 2000. Process for the continuous production of high purity of L-ribose.US patent 6,140,498) or using mannose-6-phosphate isomerase from Bacillus subtilis. Yield of 213 g / l of L-ribose after 3 hours of reaction from 300 g / l of L-ribulose showed 71 g / l of productivity per hour (Yoem et al ., Appl . Environ. Microbiol . 75.4705-4710).

Example  6: Mannos  Preparation of Recombinant Expression Vectors and Transgenic Microorganisms Comprising 6-Phosphate Isomerase Gene and Corresponding Mutations

For the production of mannose 6-phosphate isomerase, the mannose 6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain it was first separated.

Specifically, gene sequence and the amino acid sequence's sseomeoseu Thermo filler that has already been specified (Thermus thermophilus) DNA base sequence of the known KCCM of mannose 6-phosphate isomerase derived from screening the 40 879 strains, and from which the (Genebank Accession Number AP008226; Based on SEQ ID NO 1), the following primers were designed.

SEQ ID NO: 6 (Forward primer): TTT CATATG AGGCGGTTGGAGCCCAA

SEQ ID NO: 7 (reverse primer): TTT GAATTC ACTCACGCCCCCTCCTT

The primers were designed with Nde I and EcoR I restriction enzyme cleavage sites, respectively. PCR was performed using the primers to amplify the nucleotide sequence of the gene. Mannose 6-phosphate isomerase genes obtained in large quantities are restriction enzymes Nde I and EcoR. I was inserted into the plasmid vector pET 28 (+) (Novagen) to prepare pET 28 (+) a / mannose 6-phosphate isomerase.

Primers were designed to construct mutant vectors of mannose 6-phosphate isomerase (see Table 1). Mutation was induced using the primers and the Quick-Change kit (Stratagene, Beverly, MA) to construct a pET 28 (+) a / mannose 6-phosphate isomerase mutant vector.

The recombinant expression vector thus obtained was transformed into E. coli ER 2566 strain by a conventional transformation method. In addition, the transformed microorganism was stored frozen before the culture for the production of ribose by adding a 20% glycerin (glycerine) solution.

Table 1 shows primers for constructing mutant vectors of mannose 6-phosphate isomerase.

Example  7: Mannos  Preparation of 6-Phosphate Isomerase and Mutant Enzymes

In order to mass produce mannose 6-phosphate isomerase and mutant enzyme, the recombinant E. coli ER 2566 strain prepared in Example 6 was inoculated into a test tube containing 3 ml of LB medium and inoculated at 600 nm. The spawn cultivation was performed by shaking incubator at 37 degreeC until absorbance became 2.0. The seed cultured culture was then added to a 2,000 ml flask containing 500 ml of LB medium to carry out the main culture. In addition, when the absorbance at 600 nm became 0.6, 0.1 mM IPTG was added to induce mass expression of mannose 6-phosphate isomerase. The stirring rate during the process was adjusted to 200rpm, the culture temperature was maintained at 37 ℃, and after the addition of IPTG, the stirring rate was adjusted to 150rpm, the culture temperature was adjusted to 16 ℃.

In addition, the mannose 6-phosphate isomerase and the mutant enzyme produced as overexpressed as described above were centrifuged at 6,000 × g for 30 minutes at 6,000 × g and twice with 0.85% sodium chloride (NaCl). After washing, the cell solution was crushed by adding 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride). The cell lysate was again centrifuged at 13,000 × g for 20 minutes at 4 ° C., the cell pellet was removed, and only the cell supernatant was obtained, followed by a fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA, USA). ) Was equipped with a HisTrap HP adsorption column using a heat-tag and separated as an enzyme solution used for ribose production.

Example  8: Mannos  L- of 6-phosphate isomerase and mutant enzyme In Ribuloose  For specific activity  And kinetic parameter

Experiments were performed to measure and compare the specific activity of mannose 6-phosphate isomerase and mutant enzymes against el-ribulose.

The enzyme reaction was using 10 mM Li fire agarose, 0.5 mM Cu ^{2 +} a 50 mM PIPES metal ions are contained in a buffer solution (pH 7.0) performed on the 75 ℃ for 5 minutes, again adding a final concentration of 200 mM chloride susoeul The reaction was stopped. In the present invention, the enzyme activity was measured using ribose as a substrate, and the enzyme activity (unit) of the enzyme activity was defined as the amount to produce 1 nmole of ribose per minute at pH 7.0 and 75 ℃ to facilitate the comparative analysis. In addition, the analysis of ribose and ribulose concentrations and other sugars in the measurement of enzyme activity was carried out using a bio-liquid chromatography (Bio-LC) system equipped with an electrochemical detector and a CarboPacPA column. Dionex ICS-3000, Sunnylvale, CA). At this time, the CarboPacPA column was allowed to pass 200 mM sodium hydroxide at 30 ° C. at a rate of 1 ml / min.

As a result, it was confirmed that the activity was increased by 1.4-fold when L- ribulose substrate as compared to wild enzyme in the R142N mutant enzyme. As a result of the kinetic experiments, it was confirmed that R142N mutant enzyme was 1.5 times improved to 579 mM ^-1 s ^{- 1} compared to wild enzyme having a catalytic efficiency of 374 mM ^-1 s ^{- 1} . It was confirmed that this is the highest level of enzyme reaction of el ribose to el ribose to date (see Table 2).

Table 2 shows the specific activity and kinetic parameters of mannose 6-phosphate isomerase and its mutant enzymes for el-ribulose.

Example  9: Mannos  EL using 6-phosphate isomerase and mutant enzyme Libuloseurobu Ter El Ribose  Conversion comparison

In order to develop a method for producing ribose using mannose 6-phosphate isomerase and a mutant enzyme, 10 mM ribulose was prepared at a temperature (65 ° C) considering the optimum pH 7.0 and the time when the enzyme activity was reduced by half. The hourly yield of ribose was measured.

As a result, 70 minutes after the reaction, the conversion ratio of ribulose to ribose at 10 mM was found to be 51% for wild enzyme and 64% for R142N mutant enzyme (see FIG. 5). The highest productivity of ribose production so far is Summers. Thermophilus ( Thermus thermophilus ) was derived from mannose 6-phosphate isomerase, but the mutant R142N mutant enzyme showed a higher activity. This enzyme is reported mutations R142N the biological el-sseomeoseu with the highest productivity and production levels in ribose production It is shown to be an el-ribose producing enzyme that surpasses mannose 6-phosphate isomerase derived from Thermus thermophilus .

The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (12)

delete R142N mutant mannose 6-phosphate isomerase wherein amino acid of residue 142 of mannose 6-phosphate isomerase of amino acid sequence of SEQ ID NO: 1 is converted from arginine (Arg) to asparagine (Asn). delete delete The gene encoding the mutant mannose 6-phosphate isomerase of claim 2. The gene according to claim 5, wherein the gene has a nucleotide sequence set forth in SEQ ID NO: 3. A recombinant expression vector comprising the mannose 6-phosphate isomerase gene of claim 5 or 6. 8. The recombinant expression vector of claim 7, wherein said recombinant expression vector has a cleavage map as described in FIG. A wild type mannose 6-phosphate isomerized by preparing an R142N mutant mannose 6-phosphate isomerase obtained by converting the amino acid of residue 142 of mannose 6-phosphate isomerase of the amino acid sequence of SEQ ID NO: 1 from arginine (Arg) to asparagine (Asn) How to improve the el ribose productivity of the enzyme. A method of producing ribose using mannose 6-phosphate isomerase of claim 2. The method of claim 10, wherein the ribose is L-ribose and is produced using L-ribulose as a substrate. A composition for producing ribose comprising the mannose 6-phosphate isomerase of claim 2.

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