NOVEL POLYPHOSPHATE-DEPENDENT GLUCOKINASE AND METHOD FOR PREPARING GLUCOSE 6-PHOSPHATE BY USING SAME

Abstract

The present invention relates to a novel high-temperature active thermoresistant polyphosphate-dependent glucokinase with high thermal stability, a composition including the enzyme, and methods for producing glucose 6-phosphate using the enzyme.

Claims

1. A thermoresistant polyphosphate-dependent glucokinase derived from the genus Anaerolinea.

2. The polyphosphate-dependent glucokinase according to claim 1, wherein the glucokinase is expressed from the DNA sequence set forth in SEQ ID NO. 1.

3. The polyphosphate-dependent glucokinase according to claim 1, wherein the glucokinase has the amino acid sequence set forth in SEQ ID NO. 2.

4. A composition for the production of glucose 6-phosphate comprising a thermoresistant polyphosphate-dependent glucokinase derived from the genus Anaerolinea, glucose, and polyphosphate.

5. The composition according to claim 4, wherein the composition comprises 1% to 3% by weight of the glucose, 1% to 10% by weight of the polyphosphate, and 10 U/ml to 50 U/ml of the polyphosphate-dependent glucokinase based on the total volume of the composition and achieves a conversion yield of at least 70% to glucose 6-phosphate.

6. The composition according to claim 4, wherein the composition comprises 5% to 20% by weight of the glucose, 5% to 12% by weight of the polyphosphate, and 10 U/ml to 50 U/ml of the polyphosphate-dependent glucokinase based on the total volume of the composition and achieves a conversion yield of at least 50% to glucose 6-phosphate.

7. The composition according to claim 5 or 6, further comprising magnesium ions.

8. The composition according to claim 7, wherein the magnesium ions are present at a concentration of 0.2 mM to 20 mM.

9. The composition according to claim 4, wherein glucose 6-phosphate is produced at a temperature of 45 ? C. to 90 ? C. and/or a pH of 4 to 10.

10. A method for producing glucose 6-phosphate from a composition comprising a thermoresistant polyphosphate-dependent glucokinase derived from the genus Anaerolinea, glucose, and polyphosphate.

11. The method according to claim 10, wherein the glucose is prepared by liquefaction or glycosylation of starch or cellulose.

12. The method according to claim 10, wherein the polyphosphate is sodium hexametaphosphate.

13. The method according to claim 10, wherein the glucose 6-phosphate is produced at a temperature of 45 ? C. to 90 ? C. and/or a pH of 4 to 10.

14. The method according to claim 10, wherein the composition further comprises magnesium ions.

15. The method according to claim 10, wherein the polyphosphate-dependent glucokinase is present in an amount of 10 U/ml to 50 U/ml.

16. The method according to claim 10, wherein the glucose is present in an amount of 0.1% to 40% by weight, based on the total weight of the composition.

17. The method according to claim 10, wherein the polyphosphate is present in an amount of 0.5% to 25% by weight, based on the total weight of the composition.

18. A method for producing glucose 6-phosphate from a composition comprising a thermoresistant polyphosphate-dependent glucokinase derived from the genus Anaerolinea, liquefying and saccharifying enzymes, starch, and polyphosphate.

19. The method according to claim 18, wherein the liquefying and saccharifying enzymes is one or more selected from alpha-amylases, glucoamylases and alpha-glycosidases.

20. A recombinant expression vector comprising a gene encoding the polyphosphate-dependent glucokinase according to claim 3.

21. Escherichia coli BL21(DE3)/CJ_at_ppgk(KCCM11814P) transformed with a recombinant expression vector comprising the gene of SEQ ID NO. 1.

22. A method for producing a compound selected from D-glucose 1-phosphate, D-fructose 6-phosphate, D-fructose 1,6-bisphosphate, D-myo-inositol 3-phosphate, D-myo-inositol, D-glucuronate, D-glucosamine 6-phosphate, D-glucosamine, N-acetyl-D-glucosamine 6-phosphate, N-acetyl-D-glucosamine, N-acetyl-D-mannosamine 6-phosphate, N-acetyl-D-mannosamine, N-acetylneuraminic acid (sialic acid), D-mannose 6-phosphate, D-mannose, D-tagatose 6-phosphate, D-tagatose, D-allulose 6-phosphate, D-allulose, D-glyceraldehyde 3-phosphate, and dihydroxyacetone phosphate, the method comprising reacting a thermoresistant polyphosphate-dependent glucokinase derived from the genus Anaerolinea, glucose or starch, polyphosphate, and an additional enzyme.

Description

DESCRIPTION OF DRAWINGS

[0056] FIG. 1 is a flowchart illustrating a method for producing glucose 6-phosphate according to the present invention.

[0057] FIG. 2 shows a reaction scheme for the production of glucose 6-phosphate from glucose and ATP.

[0058] FIG. 3 shows a reaction scheme for the production of glucose 6-phosphate from glucose and polyphosphate.

[0059] FIG. 4 shows SDS-PAGE gel images of a supernatant (CFE) after cell disruption, a size marker (M), and a purified recombinant polyphosphate-dependent glucokinase (PE), which were taken after electrophoresis.

[0060] FIG. 5 shows the pH-dependent activity of a recombinant polyphosphate-dependent glucokinase.

[0061] FIG. 6 shows the temperature-dependent activity of a recombinant polyphosphate-dependent glucokinase.

[0062] FIG. 7 shows the activities of a recombinant polyphosphate-dependent glucokinase in the presence of different kinds of metal ions.

[0063] FIG. 8 shows the activities of a recombinant polyphosphate-dependent glucokinase when heated to different temperatures.

MODE FOR INVENTION

[0064] Glucose is a relatively cheap carbon source and can be mass-produced from starch or cellulose. Glucose is commonly used as a basic raw material in chemical or biological conversion processes for the production of various compounds that are useful in the chemical, pharmaceutical, cosmetic, and food industries.

[0065] However, phosphorylated glucose as a basic raw material in biological processes, particularly enzymatic conversion processes, is currently limited in use due to high price thereof.

[0066] Glucose 6-phosphate is an industrially pivotal metabolite in glucose metabolism and can be used as a basic raw material that can induce very useful reactions based on the use of various metabolic enzymes present in nature (organisms).

[0067] Under these circumstances, the present invention is aimed at providing an enzyme and enzymatic methods for economically producing glucose 6-phosphate, which is a raw material for various industrially useful compounds, from glucose and polyphosphate.

[0068] Using the glucose 6-phosphate produced and the producing method of the present invention can also provide high value-added functional compounds in the pharmaceutical, cosmetic, and food industries that can be prepared by the enzymatic methods.

EXAMPLES

Example 1: Production of Recombinant Expression Vector Including Polyphosphate-Dependent Glucokinase Gene and Transformed Microorganism

[0069] To provide a novel high-temperature active thermoresistant polyphosphate-dependent glucokinase, a polyphosphate-dependent glucokinase gene derived from thermophilic Anaerolinea thermophila was isolated, a recombinant expression vector was constructed, and a transformed microorganism was produced.

[0070] Specifically, gene sequences associated with the enzyme of the present invention were screened from the gene sequences registered in GenBank and only the gene sequence derived from the thermophilic microorganism was selected therefrom. Based on the registered gene sequence (SEQ ID NO. 1) and the amino acid sequence (SEQ ID NO. 2) of Anaerolinea thermophila, a forward primer (SEQ ID NO. 3) and a reverse primer (SEQ ID NO. 4) were designed. The corresponding gene was amplified from Anaerolinea thermophila genomic DNA by polymerase chain reaction (PCR) using the synthesized primers. The amplified polyphosphate-dependent glucokinase gene was inserted into plasmid vector pET21a (Novagen) for expression in E. coli using restriction enzymes NdeI and XhoI to construct a recombinant expression vector, which was named CJ_at_ppgk. CJ_at_ppgk was transfected into strain E. coli BL21(DE3) by a general transformation technique (see Sambrook et al. 1989) to produce a transformed microorganism, which was named E. coli BL21(DE3)/CJ_at_ppgk.

Example 2: Production of Recombinant Polyphosphate-Dependent Glucokinase

[0071] In this example, a recombinant polyphosphate-dependent glucokinase was produced. First, a culture tube containing 5 ml of LB liquid medium was inoculated with E. coli BL21(DE3)/CJ_at_ppgk. The inoculum was cultured in a shaking incubator at 37? C. until an absorbance of 2.0 at 600 nm was reached. The culture broth was added to LB liquid medium in a culture flask, followed by main culture. When the absorbance of the culture at 600 nm reached 2.0, 1 mM IPTG was added to induce the expression and production of a recombinant enzyme. The culture temperature was maintained at 37? C. with stirring at 200 rpm. The culture broth was centrifuged at 8,000?g and 4? C. for 20 min to collect bacterial cells. The collected bacterial cells were washed twice with 50 mM Tris-HCl buffer (pH 7.0) and suspended in the same buffer. Then, cells were disrupted using an ultrasonic homogenizer. The cell lysate was centrifuged at 13,000?g and 4? C. for 20 min and only supernatant of the cell lysate was taken. The recombinant enzyme was purified from the supernatant by His-tag affinity chromatography. The purified recombinant enzyme was dialyzed against 50 mM Tris-HCl buffer (pH 7.0) and was then characterized.

[0072] In FIG. 4, M indicates a size marker, CFE indicates the supernatant after cell disruption, and PE indicates the purified enzyme. The purified recombinant polyphosphate-dependent glucokinase was found to have a molecular weight of about 28 kDa, as determined by SDS-PAGE (FIG. 4).

Example 3: Analysis of Activity of the Recombinant Polyphosphate-Dependent Glucokinase

[0073] In this example, the activity of the recombinant polyphosphate-dependent glucokinase was analyzed. To this end, glucose (4% (w/v)), sodium hexametaphosphate (3% (w/v)), and MgCl.sub.2 (1 mM) were suspended in 50 mM Tris-HCl buffer (pH 7.0) to prepare a reaction composition for analysis of activity. The purified enzyme (0.1 mg/ml) was added to the reaction composition. The reaction was allowed to proceed at 60? C. for 15 min. The reaction product was analyzed by HPLC under the following conditions: Aminex HPX-87C (Bio-rad) column, 80? C., 5 mM H.sub.2SO.sub.4 solution as mobile phase, and flow rate of 0.6 ml/min. Glucose 6-phosphate was detected and analyzed using a Refractive Index Detector.

[0074] The results of analysis revealed the production of glucose 6-phosphate from the reaction product of the purified recombinant enzyme.

Example 4: Analysis of pH-Dependent Activity of the Recombinant Polyphosphate-Dependent Glucokinase

[0075] In this example, the influence of pH on the activity of the inventive enzyme was investigated. To this end, glucose (4% (w/v)), sodium hexametaphosphate (3% (w/v)), and MgCl.sub.2 (1 mM) were suspended in 50 mM buffers of varying pH levels (sodium citrate, pH 4-7; sodium acetate, pH 4-7; Tris-HCl pH 7-10) to prepare reaction compositions for analysis of pH effect. The purified enzyme (0.1 mg/ml) was added to each of the reaction compositions. The reaction was allowed to proceed at 60? C. for 15 min. Thereafter, the production of glucose 6-phosphate was quantitatively analyzed by HPLC.

[0076] The results are shown in FIG. 5. The polyphosphate-dependent glucokinase derived from Anaerolinea thermophila of the present invention showed a maximum activity around pH 4-5, unlike enzymes reported to date. Particularly, the activity of the enzyme was found to be higher in the sodium acetate buffer than in the other buffers in the corresponding pH range. In addition, the activities of the enzyme in the wide pH range of 4-10 were >70% of the maximum activity (FIG. 5).

[0077] The novel characteristic of the polyphosphate-dependent glucokinase of the present invention is acidophilicity and high temperature activity, which enable efficient production of glucose 6-phosphate from starch dextrin when the inventive enzyme is used in combination with a glucoamylase derived from Aspergillus sp. (e.g., commercial glucoamylase AMG 300L (Novozymes) derived from Aspergillus niger). The commercial glucoamylase has an optimum activity at pH 4.5 and 60? C. The inventive enzyme is considered industrially very useful because its activities in the wide pH range of 4-10 are >70% of the maximum activity.

Example 5: Analysis of Temperature-Dependent Activity of the Recombinant Polyphosphate-Dependent Glucokinase

[0078] In this example, the temperature-dependent activity of the recombinant enzyme was analyzed. To this end, glucose (4% (w/v)), sodium hexametaphosphate (3% (w/v)), and MgCl.sub.2 (1 mM) were suspended in 50 mM Tris-HCl buffer (pH 7.0) to prepare a reaction composition for analysis of temperature-dependent activity of the recombinant enzyme. The purified enzyme (0.1 mg/ml) was added to the reaction composition. The reaction was allowed to proceed at 40? C. to 80? C. for 15 min. Thereafter, the production of glucose 6-phosphate was quantitatively analyzed by HPLC.

[0079] The results are shown in FIG. 6. The inventive enzyme showed a maximum activity at around 65-70? C. In addition, the activities of the enzyme in the wide temperature range of 60-80? C. were >95% of the maximum activity (FIG. 6).

[0080] Enzymes derived from Thermobifida fusca are known to be active at and thermoresistant to high temperature among polyphosphate-dependent glucokinases reported to date and were reported to be optimally active at a temperature of 55? C. [see Liao et al. 2012. Appl Microbiol Biotechnol 93:1109-1117].

[0081] Therefore, it can be concluded that the Anaerolinea thermophila-derived polyphosphate-dependent glucokinase of the present invention is more active at high temperature than any polyphosphate-dependent glucokinase reported to date, which is demonstrated by its optimum activity at 65-70? C.

Example 6: Analysis of Activity of the Recombinant Polyphosphate-Dependent Glucokinase Depending on the Kind of Metal Ions

[0082] Polyphosphate-dependent glucokinases reported to date are known to demand metal ions such as Mg.sup.2+, Mn.sup.2+, Co.sup.2+, and Zn.sup.2 + for activity. In this example, the influence of metal ions on the activity of the inventive polyphosphate-dependent glucokinase was investigated. To this end, the inventive enzyme was treated with 10 mM EDTA, followed by dialysis to prepare an enzyme sample. Glucose (2% (w/v)), sodium hexametaphosphate (1.5% (w/v)), and metal ions (NiSO.sub.4, CuSO.sub.4, MnSO.sub.4, CaCl.sub.2, ZnSO.sub.4, MgSO.sub.4, MgCl.sub.2, FeSO.sub.4, NaCl, LiCl, and KCl, 1 mM each) were suspended in 50 mM Tris-HCl buffer (pH 7.0) to prepare reaction compositions. The metal ion-free enzyme sample (0.1 mg/ml) was added to each of the reaction compositions. The reaction was allowed to proceed at 60? C. for 15 min. Thereafter, the production of glucose 6-phosphate was quantitatively analyzed by HPLC. The activity of the enzyme sample untreated with metal ions was compared with the activities of the enzyme samples treated with metal ions.

[0083] As a result, the polyphosphate-dependent glucokinase derived from Anaerolinea thermophila showed demand for the metal ions such as Mg, Mn, Zn, Fe, and Ni for its activity, as shown in FIG. 7. The magnesium ions were more effective than the other metal ions, which was similarly observed in enzymes reported to date. Particularly, the addition of MgSO.sub.4 was found to achieve a maximum activity (FIG. 7).

Example 6: Analysis of Temperature Stability of the Recombinant Polyphosphate-Dependent Glucokinase

[0084] The temperature stability of the inventive polyphosphate-dependent glucokinase was analyzed. To this end, the purified recombinant enzyme (0.2 mg/ml) was heated to temperatures of 55-65? C. for different periods of time, and residual activities were compared and analyzed.

[0085] Glucose (4% (w/v)), sodium hexametaphosphate (3% (w/v)), and MgCl.sub.2 (1 mM) were suspended in 50 mM Tris-HCl buffer (pH 7.0) to prepare a reaction composition. Each of the enzyme sample (0.1 mg/ml) heated to different temperatures was added to the reaction composition for analysis of residual activity. The reaction was allowed to proceed at 60? C. for 15 min. Thereafter, the production of glucose 6-phosphate was quantitatively analyzed by HPLC.

[0086] The results are shown in FIG. 8. A reduction in the activity of the enzyme was not observed at 55? C. for 6 h. The enzyme lost its activity of about 49% after 4 h at 60? C. The activity of the enzyme was maintained at about 62% of its initial value even at 65? C. for 0.5 h (FIG. 8).

[0087] Thermobifida fusca-derived enzymes are known to be more thermoresistant than any polyphosphate-dependent glucokinase reported to date and were reported to lose their activity (by 50%) after heating at 50? C. for 0.25 h. Although Thermobifida fusca-derived enzymes were immobilized for better heat resistance, their activity was reduced to 50% of their initial activity after 2 h [see Liao et al. 2012. Appl Microbiol Biotechnol 93:1109-1117].

[0088] Therefore, it can be concluded that the Anaerolinea thermophila-derived polyphosphate-dependent glucokinase of the present invention is most thermostable of enzymes reported to date because the activity of the inventive enzyme is maintained at about 51% of its initial value even after heating at 60? C. for 4 h.

Example 7: Analysis of Conversion Yields at Different Concentrations of the Substrates

[0089] The conversion yields of glucose 6-phosphate at different concentrations of glucose and sodium hexametaphosphate were analyzed. To this end, glucose (2-15% (w/v)), sodium hexametaphosphate (1.5-11.5% (w/v)), and MgSO.sub.4 (10 mM) were suspended in 50 mM Tris-HCl buffer (pH 7.0) to prepare reaction compositions. The purified enzyme (10-50 U/ml) was added to each of the reaction composition. The reaction was allowed to proceed at 55? C. for 12 h. Thereafter, the production of glucose 6-phosphate was quantitatively analyzed by HPLC.

[0090] As a result, the use of 2% (w/v) glucose and 1.5% (w/v) sodium hexametaphosphate achieved a conversion yield of 81% after reaction for 12 h. The use of 5% (w/v) glucose and 3.5% (w/v) sodium hexametaphosphate achieved a conversion yield of 78% after reaction for 12 h. The use of 10% (w/v) glucose and 7% (w/v) sodium hexametaphosphate achieved a conversion yield of 77% after reaction for 12 h. The use of 15% (w/v) glucose and 10% (w/v) sodium hexametaphosphate achieved a conversion yield of 65% after reaction for 12 h.