WO2011049409A9 - Mannose 6-phosphate isomerase, mutants thereof, and use thereof - Google Patents

Abstract

The present invention relates to a novel mannose 6-phosphate isomerase enzyme, mutants thereof, and a method of manufacturing L-ribose with such mutant enzymes. More particularly, the invention relates to mannose 6-phosphate isomerase enzymes and variants thereof, recombinant expression vectors containing expression genes thereof, microorganisms transformed to express such genes, manufacturing methods for producing large quantities of mannose 6-phosphate isomerase enzyme and its mutants, and production methods for high yields of L-ribose made with the mannose 6-phosphate isomerase mutant enzymes.

Description

Novel mannose-6-phosphate isomerase, its mutants and uses thereof

The present invention relates to a novel mannose 6-phosphate isomerase, a mutant enzyme and a method for producing el-ribose using the enzyme, and more particularly mannose-6-phosphate isomerization. Recombinant expression vectors comprising the enzyme, the mutant enzyme and the corresponding gene, microorganisms transformed therewith, and methods for producing the mannose-6-phosphate isomerase or its mutants in large quantities using the same and the mannose-6-phosphate The present invention relates to a production method for obtaining el-ribose in high yield using an isomerase 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 ND-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 the chemical synthesis made from El-Arabinose (Woodyer RN, et al., Appl. Environ. Microbiol. 74: 2967, 2008; Jumppanen, J., et al., US patent 6,140 , 498). ).

Meanwhile, El-biological production research method is keulribiji Ella pneumoniae (Klebsiella pneumonia) derived arabinose isomerase, Pseudomonas shoe Cherry (Pseudomonas stutzeri) derived from rhamnose isomerase (L-rhamnose isomerase) of ribose, Streptomyces Ruby Geonosis (Streptomyces rubiginosus) derived from xylose isomerase (D-xylose isomease) and Lactococcus lactis Cocos (Lactococcus lactis) derived from galactose-6-phosphate isomerase, but using the enzyme (galactose-6-phosphate isomerase) , the enzyme They have a broad substrate specificity that can convert El-Riblos to El-Ribose, but their conversion is very slow.

Recently, the present inventors have overcome the problem of low productivity by converting el-ribulose to el-ribose using mannose-6-phosphate isomerase derived from Bacillus subtilis (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.

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 novel mannose-6-phosphate isomerase.

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

Still another object of the present invention is to provide a method of preparing a mutant of 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 used in the production of L-ribose.

In a preferred embodiment of the invention, the mannose-6-phosphate isomerase is it not limited thereto preferably one derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) or geo Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) strains .

In a preferred embodiment of the present invention, the isomerization enzyme preferably has an amino acid sequence of SEQ ID NO: 1 or 2, but induces one or more mutations in these sequences to have mannose-6-phosphate isomerase activity of the present invention. All mutant enzymes are included in the scope of the present invention,

Examples of these mutant enzymes include, but are not limited to:

a) a mutant obtained by converting an amino acid of residue 142 of mannose 6-phosphate isomerase of SEQ ID NO: 1 from arginine (Arg) to asparagine (Asn); b) a mannose 6-phosphate isomerase of SEQ ID NO: 2; The amino acids of residues 21, 74, and 134 of 6-phosphate isomerase were respectively converted to glutamic acid (E), threonine (T), and arginine (R) in lysine (K), asparagine (N), and methionine (M), respectively. C) the amino acids of residues 67 and 238 of mannose 6-phosphate isomerase as set forth in SEQ ID NO: 2 in glycamic acid (E) and threonine (T), respectively; (G), a mutant converted to isoleucine (I); d) the amino acid of residue 124 of mannose 6-phosphate isomerase as set forth in SEQ ID NO: 2 in the lysine (K) Mutants converted to R); e) mannose 6-phosphate isomers as set forth in SEQ ID NO: 2 Enzyme (mannose 6-phosphate isomerase) 129 that converts the number of amino acid residues from the leucine (L) to phenylalanine (F) or tyrosine (Y) mutants; f) The amino acid of residue 90 of mannose 6-phosphate isomerase as set forth in SEQ ID NO: 2 is substituted with alanine (A), aspartic acid (D), histidine (H) in asparagine (N) or A mannose 6-phosphate isomerase mutant selected from the group consisting of mutants converted to leucine (L); and g) a combination of a single point mutation of any one or more of b) to f), wherein the original amino acid b A mutant wherein the mutation is carried out at two or more corresponding residues converted to the corresponding mutant amino acids of any one or more of) through f).

In another embodiment of the present invention, the mutant of g) comprises an alanine (N) as the amino acid of residue 90 of the mannose 6-phosphate isomerase of SEQ ID NO: 2 Preferred is, but is not limited to, a mannose 6-phosphate isomerase mutant converted to A) and the amino acid of residue 129 converted from leucine (L) to phenylalanine (F).

The present invention also provides a gene encoding the enzyme of the present invention.

In one embodiment of the invention, the gene preferably has a nucleotide sequence of any one of SEQ ID NO: 3 or 4, but having a homology of 80% or more with these nucleotide sequences in consideration of the degeneracy of the genetic code, etc. All genes having functional mannose-6-phosphate isomerase activity or functional fragments thereof for the purpose of the present invention are also included in the scope of the present invention, and examples thereof include, but are not limited to, the nucleotide sequences set forth in SEQ ID NOs: 5 to 12. No.

The present invention also provides a recombinant expression vector comprising a mannose-6-phosphate isomerase gene having a nucleotide sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 12.

In one embodiment of the present invention, the recombinant expression vector is preferably an expression vector pET 28 (+) a / mannose-6-phosphate isomerase or pTrc 99a / mannose-6-phosphate isomerase, but is not limited thereto.

In addition, the present invention a) culturing the microorganism transformed with the expression vector of the present invention;

b) providing a method of preparing mannose 6-phosphate isomerase or a mutant enzyme thereof of the present invention comprising the step of separating mannose 6-phosphate isomerase from the microorganism.

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

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

The mannose-6-phosphate isomerase gene of the present invention is isolated from Thermos thermophilus or Geobacilli thermodinitripicans strain. First, chromosomal DNA is obtained from a Thermos thermophilus or Geobacillus thermodinitripicans strain with a mannose-6-phosphate isomerase gene. Next, a polymerase chain reaction (PCR) is carried out using the designed oligonucleotide as a primer, the chromosomal DNA of the Geobacillus thermodinitiphipicans strain as a template, and partially amplify the mannose-6-phosphate isomerase gene. do. The PCR amplification fragment thus obtained is a fragment having a homology close to 100% to the mannose-6-phosphate isomerase gene of the Thermos thermophilus or Geobacilli thermodinititripicans strain, and colony hybridi. A high S / N ratio can be expected as a probe at the time of aging, and the stringency control of hybridization is facilitated. 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.

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. As the expression vector used in the present invention, pET 28 (+) a or pTrc 99a was used, but any expression vector satisfying the above requirements can be used.

Production of the mannose-6-phosphate isomerase mutant according to the present invention comprises culturing a transformant obtained by transforming a host with a recombinant vector having a gene encoding the same, and culturing the transformant into a culture (cultured cell or culture supernatant). It is performed by generating and accumulating mannose-6-phosphate isomerase, which is a gene product, and acquiring the enzyme 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 mannose-6-phosphate isomerase or a mutant thereof and a recombinant expression vector comprising the corresponding gene, a microorganism transformed therewith and a method for producing a large amount of mannose-6-phosphate isomerase using these, and the mannose It is effective to provide a production method for obtaining high yield of L-ribose using -6-phosphate isomerase.

The mannose-6-phosphate isomerase of the present invention can produce a high yield of ribose, which is a raw material of medicines by a high specificity and environmentally friendly method, and the produced L-ribose starts the synthesis of medicines per sugar of various L-type nucleic acids. It can be usefully used as a substance.

Figure 1 shows the enzymatic activity of the metal ion species of mannose-6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain of the present invention, Figure 2 shows the enzyme activity of the metal ion concentration .

Figure 3 shows the enzyme activities of the pH of the mannose-6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strains of the invention (●: PIPES buffer; ○: EPPS buffer), 4 temperature It shows the enzyme activity according to.

5 is sseomeoseu Thermo filler scan shows the temperature stability of the measurement results of the mannose-6-phosphate isomerase derived from (Thermus thermophilus) strain (● of the present invention: 65 ℃; ■: 70 ℃ ; ▲: 75 ℃; ○ : 80 ° C. and □: 85 ° C.).

Figure 6 is a mannose-6-phosphate isomerase of El derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain according to the invention shows the hourly production of ribose.

Figure 7 shows the conversion of ribose by a mannose-6-phosphate isomerase and the mutant enzyme (closed circle) derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strains of the invention in a substrate concentration 10 Mm.

Figure 8 shows a gene sequence of the mannose-6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain of the present invention.

Figure 9 shows the gene sequence of the mutant enzyme R142N of mannose-6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain of the present invention.

Figure 10 shows the cleavage map of a recombinant expression vector containing the gene of the mannose-6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain of the present invention.

Figure 11 shows in comparison an enzyme activity according to the kind of the inorganic salt Geo Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) a mannose-6-phosphate isomerase derived from a strain of the present invention.

12 shows in comparison an enzyme activity according to the optimum concentration of the inorganic salt Geo Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) a mannose-6-phosphate isomerase derived from a strain of the present invention.

Figure 13 shows in comparison an enzyme activity according to the pH of geo Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) a mannose-6-phosphate isomerase derived from a strain of the present invention.

Figure 14 illustrates in comparison the enzyme activity according to the temperature of the thermopile di Gio Bacillus NITRY pecan switch (Geobacillus thermodenitrificans) a mannose-6-phosphate isomerase derived from a strain of the present invention.

Figure 15 shows the stability measurement in accordance with the temperature of the thermopile di Gio Bacillus NITRY pecan switch (Geobacillus thermodenitrificans) a mannose-6-phosphate isomerase derived from a strain of the present invention.

Figure 16 shows the production of ribose by a mannose-6-phosphate isomerase derived from Bacillus Geo Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) strains of the invention in a substrate concentration 300g / ℓ.

Figure 17-21 are geo Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) Mutant 1 (18), which are mutant enzymes of the mannose-6-phosphate isomerase (Fig. 17) derived from a strain of the present invention, Mutant 2 (FIG. 19), the gene sequences of Mutant 3 (FIG. 20), and Mutant 4 (FIG. 21) are shown.

22 is an illustration showing a cleavage map of the expression vector containing the gene of the mannose-6-phosphate isomerase derived from Bacillus Geo Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) strain.

23-24 show the relative activities of the point mutants compared to the wild type of amino acid residues 90 (FIG. 23) and 129 (FIG. 24) substitution point mutants.

Figure 25 shows the relative activity of amino acids residues 90 and 129 of the mannose-6-phosphate isomerase of the present invention compared to the wild type of single and double mutant enzymes.

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 being limited by these examples.

Example 1 Preparation of Recombinant Expression Vector and Transgenic Microorganism Comprising Mannose-6-Phosphate Isomerase Gene

1-1: Thermos Thermofiller ( Thermus thermophilus ) Mannose-6-phosphate isomerase derived from strain

In order to produce a mannose-6-phosphate isomerase of the present invention, a mannose-6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) strain it was first separated.

Specifically, the Thermos Thermophilus KCCM 40897 strain, in which the gene sequence and the amino acid sequence are already specified, is selected, and a known DNA sequence (Genebank Accession No. AP008226) of mannose-6-phosphate isomerase derived therefrom is selected. On the basis of the following primers were devised.

SEQ ID NO: 13 (Forward primer): 5'-TTT CATATG AGGCGGTTGGAGCCCAA-3 '

SEQ ID NO: 14 (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.

1-2: Geo Bacillus Thermodini Tripicans ( Geobacillus thermodenitrificans ) Mannose-6-phosphate isomerase derived from strain

Mannose-6-phosphate for the production of isomerase, and separating the geo Bacillus Thermo di NITRY pecan's one mannose-6-phosphate isomerase derived from (Geobacillus thermodenitrificans) strains before.

Specifically, gene sequence and amino acid sequence of the chipping chamber Russ Thermo di NITRY already specified screened pecan's strain and (Dae-Heoun Baek, Yujin Lee , Hong-Sig Sin, and Deok-Kun (2004) J Microbiol. Biotechnol. 14: 312-316) and the following primers were devised based on the known DNA sequence (Genebank Accession Number CP000557) of mannose-6-phosphate isomerase derived therefrom.

Mannose-6-phosphate isomerase

SEQ ID NO: 15 (Forward primer): 5'-TTT GAATTC ATGCATCAAGAACCGATTTTTC-3 '

SEQ ID NO: 16 (Reverse primer): 5'-TTT AAGCTT TTATTTGCTTGTCCGTGG-3 '

The primers of the mannose-6-phosphate isomerase gene were designed with EcoR I and Hind III restriction enzyme cleavage portions. PCR was performed using the primers to amplify the nucleotide sequence of the gene. A large amount of mannose-6-phosphate isomerase gene was inserted into the plasmid vector pTRC 99a (Novagen) using each restriction enzyme to prepare pTRC 99a / mannose-6-phosphate isomerase.

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.

Example 2 Preparation of Mannose-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 Investigation of Metal Specificity of Mannose-6-Phosphate Isomerase

In order to investigate the specificity of mannose-6-phosphate isomerase obtained in Example 2 for metal ions, the activity of the enzyme was treated with 10 mM EDTA, and metal ions (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.

Enzyme 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.Thermos Thermofiller(Thermus thermophilus) Mannose-6-phosphate isomerase derived from²⁺) Was the most effective, and the optimal concentration was 0.5mM.Geobacillus Thermodinitripicans (Geobacillus)of thermodenitrificans) Ribose isomerase by mannose-6-phosphate isomerase is cobalt (Co²⁺) Was the most effective, and the optimum concentration of the metal salt for enzyme was 1 mM.

From the above results, the mannose-6-phosphate isomerase of the present invention was shown to be affected by copper or cobalt metal ions, confirming that it is an enzyme dependent on metal ions (see FIGS. 1, 2, 11, and 12).

Example 4 Investigation of Activity of Mannose-6-Phosphate Isomerase

In order to investigate the activity of the mannose-6-phosphate isomerase obtained in Example 1 according to the pH and temperature change, the enzyme and the substrate were reacted under various pH and temperature conditions and the enzyme activity was compared.

4-1. PH effect on mannose-6-phosphate isomerase activity

First, to investigate the pH effect on the enzyme activity, 10 mM L-ribulose and 0.5 mM copper (or 1 mM cobalt; for geobacillus), 1.5 unit (or 2 unit: for geobacillus) / ml as substrates 50 mM PIPES buffer with enzyme and EPPS (N- (2-hydroxyethyl) with 10 mM L-ribulose, 0.5 mM copper (or 1 mM cobalt; for Geobacillus) and 0.05 unit / ml enzyme) Enzymatic reaction is carried out using a piperazine- N- (3-propane sulfonic acid)) buffer from pH 7.5 to pH 8.5. Specifically, the reaction is stopped at 75 ° C. for 5 minutes and stopped again by adding a final concentration of 200 mM hydrogen chloride. I was.

As a result, the optimum pH was found to be 7.0 (see FIGS. 3 and 13).

4-2. Effect of temperature on mannose-6-phosphate isomerase activity

In order to investigate the effect of temperature on the activity of the enzyme, the enzyme reaction temperature was ranged from 60 ° C. to 90 ° C. in a range of 10 mM L-ribulose, 0.5 mM copper (or 1 mM cobalt; for geobacillus), 1.5 units (or 2). unit: in case of Geobacillus) / ml The reaction was carried out for 20 minutes using 50 mM Pipes (PIPES) buffer containing the enzyme, and the reaction was stopped by adding a final concentration of 200 mM hydrogen chloride.

As a result, the optimum temperature was found to be 70 ° C. for Geobacillus and 75 ° C. for Thermos, respectively (see FIGS. 4 and 14).

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

In order to investigate the temperature stability of the enzyme, a pH containing 10 mM L-ribulose, 0.5 mM copper (or 1 mM cobalt; in the case of Geobacillus), 0.05 unit / ml enzyme in the temperature range of 65 to 85 ° C The reaction was carried out using 50 mM PIPES buffer of 7.0 until the time when the enzyme activity was reduced by half, and then the reaction was stopped by adding the final concentration of 200 mM hydrogen chloride to measure the activity of mannose-6-phosphate isomerase.

As a result, in the temperature 65 ℃ 22 hours, in 70 ℃ 10 hours, 75 ℃ 5.5 hours, the elapsed time 0.3 hours 2.2 hours and 85 ℃ in 80 ℃ sseomeoseu Thermo filler's mannose derived from (Thermus thermophilus) strain 6-phosphate isomerase activity was found to be reduced by half (see FIG. 5).

In addition, as shown in FIG. 15, the geobacillus thermodynic tripicans after 338 hours at 65 ℃, 73 hours at 65 ℃, 27 hours at 70 ℃, 17 hours at 75 ℃, 6.2 hours at 80 ℃ Geobacillus thermodenitrificans The enzyme activity was reduced by half.

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

In order to investigate the effect of temperature on the activity of the enzyme, 50, 100, 200 and 300 g / L- ribulose, 0.5 mM copper (or 1 mM cobalt; Geobacillus case) in the temperature range from 75 ℃ to 85 ℃ ), And the reaction was stopped for 3 hours using 50 mM PIPES buffer containing pH 7.0 containing 20 units / ml enzyme, 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, the filler's Thermo sseomeoseu (Thermus thermophilus) for the mannose-6-phosphate isomerase derived from a strain El was used as substrate - regardless of the re-fire agarose concentration of any image shows a conversion yield of about 70% 50, 100, 200 And 36, 71, 140, 211 g / l of el-ribose, respectively, at 300 g / l of el-ribulose.

Example 5 Production of El-Ribose Using Mannose-6-Phosphate Isomerase

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.

For enzymes derived from the Thermos thermophilus strain, 213 g / l of L-ribose was produced in 300 g / l of L-ribulose after 2.5 hours of reaction, yielding 85.2 g / l of productivity and 71% of the time. Conversion yield is shown (see FIG. 6).

Further Geo Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) if the derived enzyme, after reaction 2.2 hours is 300 g / ℓ Li fire agarose in ribose of 210 g / ℓ of the production productivity per hour, 87.5 g / ℓ and 70 % Conversion yield is shown (see FIG. 16).

To date, the highest productivity of ribose production has been achieved by chemical synthesis using Molybdic acid, which yields 23% conversion from L-Arabinose and 20 g / l per hour. Productivity was shown (Jumppanen, J., J. Nurmi, and O. Pastinen. October 2000. Process for the continuous production of high purity of L-ribose. U.S. patent 6,140,498.).

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

6-1: Thermos Thermophilus ( Thermus thermophilus ) Mannose 6-phosphate isomerase mutant derived from strain

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 sseomeoseu Thermo filler's (Thermus thermophilus) DNA base sequence (Genebank Accession Number AP008226) of the known mannose 6-phosphate isomerase, evaluate whether KCCM 40879 strain, and derived therefrom with a gene sequence and amino acid sequence has already been specified Based on the primer (primer) was devised.

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. A large amount of mannose 6-phosphate isomerase gene was inserted into plasmid vector pET 28 (+) (Novagen) using restriction enzymes Nde I and EcoR I to prepare pET 28 (+) a / mannose 6-phosphate isomerase. It was.

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

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

6-2: Obacillus Thermodini Tripicans ( Geobacillus thermodenitrificans ) Mannose 6-phosphate isomerase mutant derived from strain

If five Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) PCR mutagenesis kit ( ClonTech Laboratories, Palo Alto. CA, USA) is random in a mannose-6-phosphate isomerase derived from a strain to mutagenic (Random mutagenesis) PTrc99a / mannose-6-phosphate isomerase mutant vector was prepared.

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.

Example 7 Preparation of Mannose 6-Phosphate Isomerase and Mutant Enzymes

7-1: Thermos Thermophilus ( Thermus thermophilus ) Mannose 6-phosphate isomerase mutant derived from strain

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.

7-2: Obacillus Thermodini Tripicans ( Geobacillus thermodenitrificans ) Mannose 6-phosphate isomerase mutant derived from strain

If five Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) In order to mass-produce a mannose-6-phosphate isomerase mutant derived from the strain, produced in Example 5, and stored frozen The recombinant E. coli strain ER 2566 to LB The seed was inoculated into a test tube containing 3 ml of medium, and the seed culture was performed with a shake incubator at 37 ° C. until absorbance was 2.0 at 600 nm. 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 speed during the process was adjusted to 200rpm, the culture temperature was maintained at 37 ℃, and after incubating for 5 hours under the same conditions after the addition of the IP.

In addition, the mannose-6-phosphate isomerase mutant produced by overexpression as described above was 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 with a sonicator by adding 50 mM PIPES (pH 7.0) buffer solution and 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride). The cell lysate was heat-treated at 70 ° C. for 10 minutes and then again centrifuged at 13,000 × g at 4 ° C. for 20 minutes, cell pellets were removed, and only cell supernatant was obtained to obtain a fast protein liquid chromatography system. Rad Laboratories, Hercules, Calif., USA) was equipped with an anion resin Hi Trap ^TM HP adsorption column and separated as an enzyme solution used for the production of L-ribose.

Example 8 Specific Activity and Kinetic Parameters of Mannose 6-Phosphate Isomerase and Mutant Enzyme for L-Ribulose

8-1: Thermos Thermophilus ( Thermus thermophilus ) Mannose 6-phosphate isomerase mutant derived from strain

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

The enzymatic reaction was performed at 75 ° C. for 5 minutes using 50 mM PIPES buffer solution (pH 7.0) containing 10 mM ribulose, 0.5 mM Cu ²⁺ metal ions, followed by addition of 200 mM hydrogen chloride. 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 the R142N mutant enzyme was 579 mM ^-1 s ^{-1, which is} 1.5 times higher than the 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

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

8-2: Obacillus Thermodini Tripicans ( Geobacillus thermodenitrificans ) Mannose 6-phosphate isomerase mutant derived from strain

If five Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) derived from a strain mannose-6-phosphate isomerase and El of the mutant-enzyme activity for the re-fire agarose was carried out the measurement and the comparison experiments.

The enzymatic reaction was performed for 5 minutes at 70 ° C. using 50 mM PIPES buffer solution (pH 7.0) containing 10 mM ribulose, 1 mM Co ²⁺ metal ions, followed by addition of 200 mM hydrogen chloride. The reaction was stopped. In the present invention, the enzyme activity was measured using L-ribulose as a substrate, and the enzyme activity of 1 unit (unit) of the enzyme activity was defined as the amount to produce 1 nmole of ribose per minute at pH 7.0 and 70 ℃, comparative analysis It was smooth. 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 activity was increased by 1.2-1.4 times when erythrose was used as a substrate in comparison with wild enzyme in four mutant enzymes. At this time, it was confirmed that the specific activity of wild enzyme for elibulose was 504 U / mg (see Table 1). As a result of comparing the DNA base sequence and amino acid sequence of the mutant enzyme with the wild enzyme, it was confirmed that 1 to 3 points of mutation were induced. This is not to show a five Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans), but mannose-6-phosphate isomerase strain derived, mutant enzyme has higher activity hence showing the highest productivity of the production of ribose to the present I could confirm it. It biological El with a mutant enzyme report shows that the enzyme produces ribose - if five Bacillus Thermo di NITRY pecan switch (Geobacillus thermodenitrificans) a mannose-6-phosphate isomerase El surpassing derived from having the highest productivity in the production of ribose .

TABLE 3

Enzyme	Mutation point	Relative activity (%)
Wild	None		100
Mutant 1	K21E, N74T, M134R	121
Mutant 2	E67G, T238I	132
Mutant 3	K124R, L129F	131
Mutant 4	N90D	125

Table 3 shows the enzymatic activity of mannose-6-phosphate isomerase and mutant enzymes against el-ribulose.

8-3: Obacillus Thermodini Tripicans ( Geobacillus thermodenitrificans Other mannose 6-phosphate isomerase mutants derived from strain

The two most active residues were screened by screening mannose 6-phosphate isomerase derived from the strain of Geobacillus thermodenitrificans. The residues with the highest activity were selected by substitution with different amino acids.

Specifically, the N90 residue and the L129 residue are converted into amino acids having different properties, and then point mutations are performed with the following N90A, N90D, N90E, N90H, N90K, N90L, N90Y, L129A, L129F, L129H, L129W, and L129Y. The activity test was compared with mannose-6-phosphate isomerase derived from wild type Geobacillus thermodinitripicans strain.

The screening test of this experiment was performed by ketose assay using error prone PCR using Clontech Diversify PCR Random Mutagenesis Kit. The resulting mutants were converted to one residue each using a QuikChange Site-Directed Mutagenesis Kit from Stratagene, replaced with another amino acid, or subjected to two mutations. In addition, the activity was compared to the reaction with the existing MPi. The reaction was performed with 10 mmol / L L-ribulose with 0.5 mg / ml of enzyme containing co-factor 1 mM Co ²⁺ in 50 mmole / L PIPES buffer. The specific activity was measured by comparing the reaction at 70 minutes for 10 minutes (see FIGS. 23 and 24).

8-4: Analysis of Two Point Mutants and Productivity of Mannose-6-Phosphate Isomerase Derived from Geobacillus Thermodinitripicans Strain

Among the highly active residues obtained by screening for mannose-6-phosphate isomerase derived from the Geobacillus thermodinitripicans strain, the two most active residues were double-mutated and compared for their activities.

Specifically, doble mutations of N90A and L129F together confirmed higher activity (see FIG. 25).

Example 9 Comparison of El Ribose to El Ribose Conversion Using Mannose 6-Phosphate Isomerase and Mutant Enzymes

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, it was confirmed that 70 minutes after the reaction, the conversion rate from ribulose to ribose was 51% for wild enzyme and 64% for R142N mutant enzyme (see FIG. 7). This can be confirmed to show it's sseomeoseu Thermo filler (Thermus thermophilus) The origin of the mannose 6-phosphate isomerase, but, mutant enzymes, R142N mutant enzyme is higher activities hence showing the highest productivity of the production of ribose to the present there was. Biological El with a mutant enzyme R142N report shows that the enzyme produces ribose-El that surpassed the mannose 6-phosphate isomerase derived from Thermococcus sseomeoseu pillar's (Thermus thermophilus) with the highest productivity and production levels in ribose production.

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

1. Mannose-6-phosphate isomerase used for the production of L-ribose.
2. The method of claim 1, wherein the mannose-6-phosphate isomerase is sseomeoseu Thermo filler's (Thermus thermophilus) or geo Bacillus Thermo di NITRY pecan's mannose-6-phosphate isomerase, characterized in that derived from the strain (Geobacillus thermodenitrificans) enzyme.
3. The mannose-6-phosphate isomerase of claim 1, wherein the isomerase has an amino acid sequence represented by SEQ ID NO: 1 or 2.
4. A gene encoding the enzyme of any one of claims 1 to 3.
5. The gene of claim 4, wherein the gene has a nucleotide sequence of SEQ ID NO: 3 or 4. 6.
6. Recombinant expression vector comprising a mannose-6-phosphate isomerase gene having the nucleotide sequence of any one of SEQ ID NO: 3 to SEQ ID NO: 12.
7. The method of claim 6,

   The recombinant expression vector is a recombinant expression vector pET 28 (+) a / mannose-6-phosphate isomerase or pTrc 99a / mannose-6- phosphate isomerase.
8. a) culturing the microorganism transformed with the expression vector of claim 6;

   b) A method for preparing mannose 6-phosphate isomerase according to any one of claims 1 to 3, comprising the step of separating mannose 6-phosphate isomerase from the microorganism.
9. A method for producing L-ribose using the mannose-6-phosphate isomerase of any one of claims 1 to 3.
10. a) a mutant obtained by converting an amino acid of residue 142 of mannose 6-phosphate isomerase of SEQ ID NO: 1 from arginine (Arg) to asparagine (Asn);

    b) the amino acids of residues 21, 74 and 134 of mannose 6-phosphate isomerase of SEQ ID NO: 2 in lysine (K), asparagine (N) and methionine (M), respectively Mutants converted to glutamic acid (E), threonine (T), and arginine (R), respectively;

    c) The amino acids of residues 67 and 238 of mannose 6-phosphate isomerase as set forth in SEQ ID NO: 2 were respectively converted into glycine (G) and isoleucine in glutamic acid (E) and threonine (T), respectively. Mutants converted to I);

    d) a mutant obtained by converting the amino acid of residue 124 of mannose 6-phosphate isomerase described in SEQ ID NO: 2 from lysine (K) to arginine (R);

    e) a mutant in which amino acid at residue 129 of mannose 6-phosphate isomerase described in SEQ ID NO: 2 is converted from leucine (L) to phenylalanine (F) or tyrosine (Y);

    f) The amino acid of residue 90 of mannose 6-phosphate isomerase as set forth in SEQ ID NO: 2 is substituted with alanine (A), aspartic acid (D), histidine (H) in asparagine (N) or Mannose 6-phosphate isomerase mutants selected from the group consisting of mutants converted to leucine (L); and

    g) a combination of single point mutations of any one or more of b) to f) wherein mutations have been carried out at two or more corresponding residues wherein the original amino acid has been converted from the two or more corresponding residues to the corresponding mutant amino acids of any one or more of b) to f) Mutant

    At least one mannose 6-phosphate isomerase mutant enzyme selected from the group consisting of:
11. A gene encoding the mannose 6-phosphate isomerase mutant of claim 10.
12. The gene of claim 11, wherein the gene has a nucleotide sequence set forth in SEQ ID NO: 2 to SEQ ID NO: 12.
13. The mutant of g) converts the amino acid of residue 90 of mannose 6-phosphate isomerase as set forth in SEQ ID NO: 2 from asparagine (N) to alanine (A). And mannose 6-phosphate isomerase mutant enzyme wherein amino acid of residue 129 is converted from leucine (L) to phenylalanine (F).
14. A composition for producing ribose comprising the mannose 6-phosphate isomerase mutant of claim 10.
15. A method for producing el-ribose using the mannose-6-phosphate isomerase mutant of claim 10.

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