KR101008556B1 - Method for Production of L?ribose - Google Patents

Abstract

Remove the ribose relates to a production method of the (L-ribose), and more particularly Bacillus subtilis (Bacillus subtilis) bacteria from mannose-6-phosphate isomerase (mannose 6-phosphate isomerase) gene - the invention el The present invention relates to a method for producing high yield of el-ribose by reacting the produced mannose-6-phosphate isomerase with L-ribulose.

According to the present invention, by using recombinant mannose-6-phosphate isomerase derived from Bacillus subtilis, it is possible to produce L-ribose, which is a raw material of pharmaceuticals, with high specificity and environmentally friendly.

L-ribose, Bacillus subtilis, mannose-6-phosphate isomerase

Description

Method of Production of L-Ribose {Method for Production of L-ribose}

The invention el-ribose relates to a production method of the (L-ribose), and more particularly Bacillus subtilis (Bacillus subtilis) High yield of L-ribose was produced by reacting mannose-6-phosphate isomerase gene produced by separating mannose 6-phosphate isomerase gene from L-ribulose and L-ribulose. It is about how to.

L-ribose is the starting material for the synthesis of sugars of many L-type nucleic acids, and is used for the synthesis of antiviral methyl-L-riboflanoside ("Bezimidavir" ^TM ). As a result, the world market for el-ribose and its derivatives was about $ 1.1 billion in 2001, and recently, a non-double oil 1263 double oil 94 (BW1263W94, Glaxo Wellcome) and a non-hepatitis B drug, which are currently being developed as a new anti-herpes drug (Antiherpes) As the core intermediates of L-FMAU, Bukwang & Triangle, etc., which are being developed by the company, the demand is soaring, the development of industrially available manufacturing method 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, chemical synthesis methods have several serious problems in their production process. Indeed, there are risks in the working environment that require high temperatures and pressures, complex el-ribose separation and purification due to the formation of additional sugars after chemical reactions, and environmental pollution from the chemical waste produced 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. Recently, recombinant E. coli containing NDA-dependent mannitol-1-dehydrogenase was used to obtain 55% conversion yield at 72 hours of fermentation from 100 g / L ribitol, but -The productivity of ribose is about 28 times lower than the 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 ).

Specifically, the biological production method of El-ribose was studied as follows. Keulribiji Ella pneumoniae (Klebsiella pneumonia) derived from arabinose isomerase, Pseudomonas shoe cherry (Pseudomonas stutzeri) derived rhamnose isomerase (L-rhamnose isomerase), Streptomyces ruby Geonosis (Streptomyces rubiginosus) derived from xylose isomerase ( D-xylose isomease) and Lactococcus lactis Cocos (Lactococcus lactis) derived from galactose-6-phosphate isomerase (galactose-6-phosphate isomerase) is a method has a wide range of substrate specificity el-a ribul Ross el-ribose, but could be converted to The conversion speed is very slow.

El-lee fire seuneun biochemical El-know from ribose Novak other derived El (Acinetobacter sp.) - ribose isomerase, yiseri Escherichia coli (Escherichia coli) originated el-arabinose mutated isomerase, evil Tino Playa Ness smile Wu N-Sys (Actinoplanes missouriensis) derived from xylose isomerase mutant and Cornell la see below barley (Cohnella laevoribosii) It has been converted using the derived di-rixos isomerase. However, the enzymatic method so far has a problem of very low productivity. Therefore, there is an urgent need in the art to develop an economical biological method with high productivity of el-ribose.

Therefore, the present inventors have made efforts to develop a method for producing rare monosaccharides more effectively. As a result, the mannose 6-phosphate isomerase found in various microorganisms is derived from L-ribulose. confirming that can produce the following first to ribose, Bacillus subtilis Construction of recombinant expression vector and a transformed microbial conversion which comprises a mannose-6-phosphate isomerase gene derived from (Bacillus subtilis) and mannose using these -6 -Production of phosphate isomerase in large quantities and this environmentally friendly process to determine the optimum reaction conditions to obtain only high yields of ribose without the production of by-products and completed the present invention.

After all, the object of the present invention is to react the mannose-6-phosphate isomerase and L-ribulose produced by separating the mannose-6-phosphate isomerase gene from the Bacillus subtilis bacteria, and the L-ribulose (L-ribulose). -ribose) provides a way to produce high yields.

In order to achieve the above object, the present invention comprises the steps of (a) separating the mannose-6-phosphate isomerase gene from Bacillus subtilis bacteria, and inserting it into an expression vector; (b) transforming the expression vector into Escherichia coli and culturing; (c) isolating and purifying mannose-6-phosphate isomerase from the E. coli; And (d) reacting the mannose-6-phosphate isomerase with el-ribulose as a substrate.

In the present invention, the mannose-6-phosphate isomerase gene may be characterized in that SEQ ID NO: 1.

In the present invention, the expression vector may be characterized in that the FT-28A (plus) (pET-28a (+)).

In the present invention, the el-ribulose may be characterized in that the concentration is added to the range of 100 g / L to 400 g / L.

In the present invention, the reaction is a state in which any one or more metal ions selected from the group consisting of manganese, aluminum, zinc, barium, copper, cobalt, calcium, magnesium, nickel and iron in the range of 0.1 mM to 2 mM It may be characterized in that, and preferably the metal ion is characterized in that the cobalt.

In the present invention, the reaction may be characterized in that the pH ranges from 6.5 to 9.0.

In the present invention, the enzymatic reaction may be characterized in that the temperature is made in the range of 30 ℃ to 45 ℃.

The present invention provides a method for producing el-ribose in high yield by reacting mannose-6-phosphate isomerase and El-ribulose prepared by separating mannose-6-phosphate isomerase gene from Bacillus subtilis. It works.

According to the present invention, by using recombinant mannose-6-phosphate isomerase derived from Bacillus subtilis, it is possible to produce el-ribose, which is a raw material of pharmaceuticals with high specificity and eco-friendliness, and the el-ribose is various L-forms. As a starting material for the synthesis of pharmaceutical products per sugar nucleic acid can be usefully used in the manufacture of pharmaceuticals.

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

The method for producing L-ribose of the present invention comprises the steps of: (a) separating mannose-6-phosphate isomerase gene from Bacillus subtilis bacteria and inserting it into an expression vector; (b) transforming the expression vector into Escherichia coli and culturing; (c) isolating and purifying mannose-6-phosphate isomerase from the E. coli; And (d) reacting the mannose-6-phosphate isomerase with el-ribulose as a substrate.

If the recombinant expression vector can be used in the production of ribose in the production of any of the vectors used in the genetic recombination method, including the expression vector FT-28A (plus) can be used, the microorganism transformed with the recombinant expression vector As E. coli ER2566 (ER2566) is preferably used, any strain that can be transformed with a recombinant expression vector to overexpress a desired gene and produce an active enzyme protein can be used.

The mannose-6-phosphate isomerase used in the present invention is a metal ion dependent enzyme that is affected for metals that enhance enzyme activity. Therefore, the enzymatic reaction may be performed in a state in which at least one metal ion selected from the group consisting of manganese, aluminum, zinc, barium, copper, cobalt, calcium, magnesium, nickel and iron is treated in the range of 0.1 mM to 2 mM. Preferably, the metal ion may be characterized in that the cobalt.

In addition, the ribose is preferably synthesized using ribulose as a substrate, more preferably using el-ribulose, the substrate concentration is preferably in the range of 100 g / L to 400 g / L and 300 g / It is more preferable that it is a range inside and outside L. In addition, the enzyme reaction is preferably made in the range of pH 6.5 to pH 9.0, more preferably in the range of pH 7.5. In addition, it is preferable that the said enzyme reaction is made in the range of 30 degreeC-45 degreeC, and it is more preferable that it is made in the range of about 40 degreeC of temperature. In addition, the said enzyme reaction time can also be adjusted suitably according to a conventional method.

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

Example 1 Preparation of Recombinant Expression Vectors and Transgenic Microorganisms Comprising Mannose-6-Phosphate Isomerase Gene

To prepare mannose-6-phosphate isomerase, mannose-6-phosphate isomerase derived from the Bacillus subtilis strain was first isolated.

Specifically, Bacillus subtilis HTC (238CC strains) , in which the gene sequence and the amino acid sequence are already specified, were selected, and a known DNA sequence of mannose-6-phosphate isomerase derived therefrom (Genebank Accession Number AF324506). Based on Sequence Listing 1), the following primers were designed.

SEQ ID NO: 2 (Forward primer): 5'-TTT CATATG ACGCATCCTTTATT-3 '

SEQ ID NO: 3 (Reverse primer): 5'-TTT CTCGAG TTAAGGATGAGATATCA-3 '

The primers were designed with Nde I and Xho I restriction enzyme cleavage sites, respectively. Polymerase chain reaction (PCR) was performed using the primers to amplify the nucleotide sequence of the gene. The mannose-6-phosphate isomerase gene obtained in large quantities was inserted into the plasmid vector Piti-28A (Plus) (Novagen) using restriction enzymes Endi and XII sources for Piti-28A (Plus) / Mannose-6-phosphate isomerase was produced.

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

Example 2 Preparation of Mannose-6-Phosphate Isomerase

In order to mass-produce mannose-6-phosphate isomerase, the recombinant E. coli 2566 strain prepared in Example 1, which was prepared in Example 1 above, was inoculated into a test tube containing 3 ml of LB medium and 600 nm. The spawn culture was performed with a shake incubator at 37 ° C. until the absorbance reached 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 Iptage (IPTG) was added to induce mass expression of mannose 6-phosphate isomerase. The stirring speed during the process was adjusted to 200 rpm, the culture temperature was maintained at 37 ℃, after adding the ipitage, the stirring speed was adjusted to 150 rpm, the culture temperature was adjusted to 16 ℃.

In addition, the mannose-6-phosphate isomerase produced as overexpressed as described above, the culture medium of the transformed strain was centrifuged at 6,000 × g for 30 minutes at 4 ℃, washed twice with 0.85% sodium chloride (NaCl). The cell solution was then disrupted with a sonicator 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 for a fast protein liquid chromatography system (Bio-Rad Laboratories, Hercules, CA, USA). ) Was equipped with a HisTrap HP adsorption column using a His-tag, and separated as an enzyme solution used for the production of L-ribose.

Example 3: Investigation of metal specificity of mannose-6-phosphate isomerase

In order to investigate the specificity of the mannose-6-phosphate isomerase of the present invention for metal ions, the enzyme activity was treated with 10 mM IDTA (EDTA) and the metal ions (Mn ²⁺ , Al ³⁺ , Zn ²⁺ , Ba ²⁺ , Cu ²⁺ , Co ²⁺ , Ca ²⁺ , Mg ²⁺ , Ni ²⁺ and Fe ²⁺ ) were added to the reaction and the activity was measured as follows. The enzymatic reaction was carried out at 40 ° C. for 20 minutes using 10 mM L-ribose, 50 mM EPPS buffer (pH 7.5) containing each metal ion and 1.5 unit / ml enzyme, and finally The reaction was stopped by adding a concentration of 200 mM hydrogen chloride.

Enzyme activity was measured using El-ribose as a substrate, and one unit of enzyme activity was defined as the amount producing 1 μmole of El-ribulose per minute at pH 7.5 and 40 ° C. for 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, the purified enzyme and the enzyme treated with IDT were inactive. Of metal salts from Bacillus subtilis Cobalt (Co ²⁺ ) was most effective for ribose isomerization by mannose-6-phosphate isomerase, and the optimal concentration was 0.5 mM. Mannose-6-phosphate isomerase was shown to be affected by 0.5 mM cobalt metal ion, and it was confirmed that the mannose 6-phosphate isomerase of the present invention is an enzyme dependent on metal ions, and its activity is shown in FIGS. 1A and 1B. Shown in

Example 3: Effect of pH and Temperature on Mannose 6-Phosphate Isomerase Activity

The activity of the mannose-6-phosphate isomerase isolated in Example 2 was changed according to pH and temperature. Enzymes and substrates were reacted under various pH and temperature conditions and enzyme activities were compared.

3-1: Effect of pH on Mannose-6-phosphate Isomerase

To investigate the pH effect on the enzyme, 50 mM pipese piperazine- N , N' -bis (2-ethane sulfonic acid, PIPES) containing 10 mM ribose, 0.5 mM cobalt, 1.5 unit / ml enzyme as substrate Use 50 mM N- (2-hyroxyethyl) piperazine- N- (3-propane sulfonic acid, EPPS) buffer containing 10 mM L-ribose, 0.5 mM cobalt and 1.5 units / ml enzyme The enzymatic reaction was carried out in the range of pH 7.5 to 8.5. Specifically, the enzymatic reaction was performed at 40 ° C. for 20 minutes, and the reaction was stopped again by adding a final concentration of 200 mM hydrogen chloride, as shown in FIG. 2A. Likewise, the optimum pH was found to be 7.5.

3-2: Effect of temperature on mannose-6-phosphate isomerase

In order to investigate the temperature effect on the enzyme, the enzyme reaction was carried out using a 50 mM EPPS buffer at pH 7.5 containing 10 mM ribose, 0.5 mM cobalt and 1.5 unit / ml enzyme, ranging from 20 to 50 ° C. The reaction was carried out for minutes. The reaction was then stopped by adding a final concentration of 200 mM hydrogen chloride. As a result, as shown in FIG. 2B, it was found that the optimum temperature was 40 ° C.

Figure 2a is shown by examining the enzyme activity according to the pH of the mannose-6-phosphate isomerase of the present invention, Figure 2b is shown by examining the enzyme activity according to the temperature of the mannose-6-phosphate isomerase of the present invention. . In the activity of mannose-6-phosphate isomerase according to pH in FIG. 2A,? Represents the pipe buffer solution and? Represents the PIPS buffer solution.

3-3: Temperature Stability Study of Mannose-6-Phosphate Isomerase

To investigate the temperature stability of mannose-6-phosphate isomerase, 50 mM EPPS buffer at pH 7.5 with 10 mM L-ribose, 0.5 mM cobalt, 1.5 unit / ml enzyme in the temperature range of 25 ° C. to 50 ° C. The solution was used to advance the reaction until the time when each enzyme activity was reduced by half. After the reaction was completed, the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride, and the activity of mannose-6-phosphate isomerase enzyme was measured.

Figure 3 shows the results of measuring the temperature stability of the mannose-6-phosphate isomerase of the present invention. In the graph of FIG. 3, temperature notation is represented by 25 ° C. (°), 30 ° C. (°), 35 ° C. (°), 40 ° C. (°), 45 ° C. (°), and 50 ° C. (°), respectively.

As a result, as shown in Figure 3, the enzyme activity at 461 hours at 30 ℃, 325 hours at 30 ℃, 236 hours at 35 ℃, 111 hours at 40 ℃, 56 hours at 45 ℃ and 10 hours at 50 ℃ This can be seen to be reduced by half.

Example 4 Production of El-Ribose Using Mannose-6-Phosphate Isomerase

To develop a method for producing L-ribose using mannose-6-phosphate isomerase, 300 g / L L at a temperature (40 ° C.) considering the optimum pH 7.5 of the enzyme identified above and the time when the enzyme activity was reduced by half The hourly yield of L-ribose was measured with ribulose.

FIG. 4 is a graph showing the amount of ribose production by mannose-6-phosphate isomerase of the present invention at an concentration of 300 g / L el-ribulose as a substrate, after 3 hours from 300 g / L el-ribulose. 213 g / L ribose was obtained, yielding 71 g L ⁻¹ h ⁻¹ productivity and 71% conversion yield. Therefore, it could be confirmed that the production of 213 g / L L-ribose in 3 hours.

To date, the highest productivity of El-ribose has been produced. Chemical synthesis using Molybdic acid yielded 23% conversion and 20 g L ^-1 h ^-1 productivity from El-Arabinose (Jumppanen, J., J.). Nurmi, and O. Pastinen.October 2000. Process for the continuous production of high purity of L-ribose.US patent 6,140,498.

In another comparison, 52 g / L L- after 72 hours of incubation from 100 g / L ribitol using recombinant E. coli containing NAD-dependent mannitol-1-dehydrogenase. Ribose was obtained and showed a productivity of 0.72 g L ^-1 h ^-1 (Woodyer, R., N. Wymer, F. Racine, S. Khan, and B. Saha. 2008. Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst.Appl.Environ.Microbiol. 74: 2967-2975).

This is the highest productivity and production concentration in the reported biological el-ribose production. Productivity and production concentration of mannose 6-phosphate isomerase derived from Bacillus subtilis were determined in recombinant E. coli containing NDA-dependent mannitol-1-dehydrogenase. The results show that 98 times and 4 times higher than the obtained results, the el-ribose production enzyme method obtained in the present invention is much better than the fermentation method and chemical synthesis method in productivity, production concentration and ease of purification.

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.

Figure 1 shows the comparison of the enzyme activity according to the inorganic salt type (1a) and inorganic salt concentration (1b) of the mannose-6-phosphate isomerase of the present invention.

Figure 2 shows the comparison of the enzyme activity according to the pH (2a) and temperature (2b) of the mannose-6-phosphate isomerase of the present invention.

Figure 3 shows the stability measurement results for the temperature of mannose-6-phosphate isomerase.

Figure 4 shows the amount of ribose production by mannose-6-phosphate isomerase of the present invention at a substrate concentration of 300 g / L.

<110> Konkuk University Industrial Cooperation Corp. <120> Method for Production of L-ribose <130> P08-E349 <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 948 <212> DNA <213> Bacillus subtilis <400> 1 atgacgcatc ctttattttt agagcctgtc tttaaagaaa gactatgggg agggacgaag 60 cttcgtgacg cttttggcta cgcaataccc tcacaaaaaa caggtgagtg ctgggccgtt 120 tctgcacatg cccatggctc gtcgtctgta aaaaatggcc cgctggcagg aaagacactt 180 gatcaagtat ggaaagatca tccagagata ttcgggtttc cggatggtaa ggtgtttccg 240 ctgctggtaa agctgctgga cgccaatatg gatctctccg tgcaagtcca tcctgatgat 300 gattatgcaa aactgcacga aaatggcgac cttggtaaaa cggagtgctg gtatatcatt 360 gattgcaaag atgacgccga actaattttg ggacatcatg caagcacaaa ggaagagttc 420 aaacaacgaa tagaaagcgg tgattggaac gggctgctga ggcgaatcaa aatcaagcca 480 ggagatttct tttatgtgcc aagcggtaca ctccatgctt tatgtaaggg aacccttgtc 540 cttgaaatcc agcaaaactc tgatacaaca tatcgcgtat acgattatga ccgctgtaat 600 gaccagggcc aaaaaagaac tcttcatata gaaaaagcca tggaagtcat aacgataccg 660 catatcgata aagtgcatac accggaagta aaagaagttg gtaacgctga gatcattgtt 720 tatgtgcaat cagattattt ctcagtgtac aaatggaaga ttagcggccg agctgctttt 780 ccttcatatc aaacctattt gctggggagt gttctgagcg gatcaggacg aatcataaat 840 aatggtattc agtatgaatg caatgcaggc tcacacttta ttctgcctgc gcattttgga 900 gaatttacaa tagaaggaac atgtgaattc atgatatctc atccttaa 948 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tttcatatga cgcatccttt att 23 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tttctcgagt taaggatgag atatca 26  

Claims (8)

Production method of L-ribose comprising the following steps: (a) Bacillus subtilis (Bacillus subtilis) bacteria from mannose-6-phosphate isomerase (mannose-6-phosphate isomerase) a step of separating the gene inserted into the expression vector; (b) transforming the expression vector into Escherichia coli and culturing; (c) isolating and purifying mannose-6-phosphate isomerase from the E. coli; And (d) reacting the mannose-6-phosphate isomerase with L-ribulose as a substrate. The method of claim 1, wherein the mannose-6-phosphate isomerase gene is SEQ ID NO: 1. Method of producing L-ribose (L-ribose). The method of claim 1, wherein the expression vector is P-28A (plus) (pET-28a (+)) characterized in that the production method of L-ribose (L-ribose). According to claim 1, wherein the L-ribulose (L-ribulose) is a method for producing L-ribose (L-ribose), characterized in that the concentration is added in the range of 100 g / L to 400 g / L. The method of claim 1, wherein the reaction is performed by treating any one or more metal ions selected from the group consisting of manganese, aluminum, zinc, barium, copper, cobalt, calcium, magnesium, nickel, and iron in the range of 0.1 mM to 2 mM. Production method of L-ribose, characterized in that made in. The method of claim 1 or 5, wherein the metal ion is cobalt. The method of claim 1, wherein the reaction is in the range of pH 6.5 to pH 9.0 production method of L-ribose (L-ribose). According to claim 1, wherein the enzymatic reaction is a method for producing L-ribose (L-ribose), characterized in that the temperature is made in the range of 30 ℃ to 45 ℃.

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