Polyphosphate-dependent glucokinase and method for producing glucose 6-phosphate using same

Abstract

The present invention relates to a novel thermophilic and thermoresistant polyphosphate-dependent glucokinase having excellent stability, a composition comprising the same, and a method for producing glucose 6-phosphate using the same.

Claims

1. A method for producing glucose 6-phosphate comprising contacting a thermoresistant polyphosphate-dependent glucokinase having the amino acid sequence of SEQ ID NO:2 with glucose and polyphosphate at a temperature of 40° C. to 55° C.

2. The method according to claim 1, wherein the glucose is prepared by liquefaction or glycosylation of starch or cellulose.

3. The method according to claim 1, wherein the polyphosphate is sodium hexametaphosphate.

4. The method according to claim 1, wherein glucose 6-phosphate is produced at a temperature of 40° C. to 60° C. and/or a pH of 7 to 9.

5. The method according to claim 1, wherein the composition further comprises magnesium ions.

6. The method according to claim 1, wherein the polyphosphate-dependent glucokinase is present in an amount of 0.1 mg/ml to 0.5 mg/ml.

7. The method according to claim 1, wherein the glucose is present in an amount of 1% to 30% by weight, based on the total volume of the composition.

8. The method according to claim 1, wherein the polyphosphate is present in an amount of 1% to 10% by weight, based on the total volume of the composition.

9. The method according to claim 1, wherein the glucose is prepared by contacting a diastatic enzyme with starch, wherein the diastatic enzyme is selected from alpha-amylases, glucoamylases, alpha-glycosidases, and mixtures thereof.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a flowchart illustrating a method for producing glucose 6-phosphate according to the present invention.

(2) FIG. 2 shows a reaction scheme for the production of glucose 6-phosphate from ATP and glucose.

(3) FIG. 3 shows a reaction scheme for the production of glucose 6-phosphate from polyphosphate and glucose.

(4) FIG. 4 shows SDS-PAGE gel images of a size marker (M), a crude extract (CE) after cell destruction, an enzyme whose gene expression was not induced as a negative control (Ne), and a purified enzyme (PE) after gene expression, which were taken after electrophoresis.

(5) FIG. 5 shows the pH-dependent activity of a recombinant polyphosphate-dependent glucokinase.

(6) FIG. 6 shows the temperature-dependent activity of a recombinant polyphosphate-dependent glucokinase.

(7) FIG. 7 shows the activities of a recombinant polyphosphate-dependent glucokinase in the presence of different kinds of metal ions.

(8) FIG. 8 shows the activities of a polyphosphate-dependent glucokinase at different concentrations of MgCl.sub.2.

(9) FIG. 9 shows the activities of a polyphosphate-dependent glucokinase when heated to different temperatures.

MODE FOR INVENTION

(10) Glucose is a relatively cheap carbon source and can be mass-produced from starch or cellulose. Glucose is currently 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.

(11) 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.

(12) 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).

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

(14) The present invention is also aimed at providing high value-added functional compounds in the pharmaceutical, cosmetic, and food industries that can be prepared from glucose 6-phosphate produced by the enzymatic methods.

EXAMPLES

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

(15) The Dg_ppgk gene of a polyphosphate-dependent glucokinase derived from thermophilic Deinococcus geothermalis, was isolated.

(16) Specifically, gene sequences associated with the novel thermophilic and thermoresistant enzyme according to the present invention were primarily screened from the gene sequences registered in Genbank and only the gene sequence derived from the thermophilic microorganism was finally selected therefrom. The registered gene sequence (SEQ ID NO. 1) and the amino acid sequence (SEQ ID NO. 2) of Deinococcus geothermalis were analyzed to design a forward primer (SEQ ID NO. 3) and a reverse primer (SEQ ID NO. 4). The corresponding gene was amplified from the Deinococcus geothermalis 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 E. coli expression using restriction enzymes NdeI and XhoI to construct a recombinant expression vector (pET21a-Dg_ppgk). The recombinant expression vector was transformed into strain E. coli BL21(DE3) by a general transformation technique (see Sambrook et al. 1989) to produce a transformed microorganism.

Example 2: Production of Polyphosphate-Dependent Glucokinase

(17) In this example, a recombinant polyphosphate-dependent glucokinase was produced. First, a culture tube containing 5 ml of LB liquid medium was inoculated with the transformed microorganism. 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 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.

(18) The recombinant enzyme produced by overexpression was purified by the following procedure. First, the culture broth of the transformed strain was centrifuged at 8,000×g and 4° C. for 20 min and washed twice with 50 mM Tris-Cl buffer (pH 7.5). Then, cells were disrupted using an ultrasonic homogenizer. The cell lysate was centrifuged at 13,000×g and 4° C. for 20 min. The supernatant was collected and purified by His-tag affinity chromatography. The purified recombinant enzyme was dialyzed against buffer for activity measurement (50 mM Tris-Cl, pH 7.5) and was then characterized.

(19) In FIG. 4, M indicates a size marker, CE indicates the crude extract after cell destruction, Ne indicates the enzyme whose gene expression was not induced (negative control), and PE indicates the purified enzyme after gene expression. The purified polyphosphate-dependent glucokinase was found to have a molecular weight of ˜30 kDa, as determined by SDS-PAGE (FIG. 4).

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

(20) The purified recombinant polyphosphate-dependent glucokinase derived from the gene constructed based on the gene sequencing result of the sequence registered in Genbank was analyzed for activity.

(21) Glucose (2% (w/v)), MgCl.sub.2 (10 mM), sodium hexametaphosphate (1.5% (w/v)), and the purified enzyme (0.3 mg/ml) were mixed together in 50 mM Tris-HCl (pH 7.5) to prepare a reaction composition. The enzymatic reaction was allowed to proceed at 40° C. for 30 min, followed by HPLC. The HPLC conditions were as follows: 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.

(22) As a result, the gene-derived recombinant protein was detected to be active.

Example 4: Analysis of Influences of pH and Temperature on the Activity of the Recombinant Polyphosphate-Dependent Glucokinase

4-1. Analysis of pH Influence

(23) The influence of pH on the enzyme activity was investigated. To this end, glucose (2% (w/v)), MgCl.sub.2 (10 mM), sodium hexametaphosphate (1.5% (w/v)), and the purified enzyme (0.3 mg/ml) were mixed together in 50 mM buffers (sodium acetate, pH 4-6; potassium phosphate, pH 6-8; Tris-HCl, pH 7-8) to prepare reaction compositions. The enzymatic reaction was allowed to proceed at 40° C. for 30 min, followed by HPLC. The results are shown in FIG. 5. The enzyme showed a maximum activity around pH 8. The activity of the enzyme was found to be higher in the Tris-HCl buffer than in the other buffers (FIG. 5).

4-2. Analysis of Temperature Influence

(24) Changes in the activity of the enzyme according to temperature variation were investigated. To this end, glucose (2% (w/v)), MgCl.sub.2 (10 mM), sodium hexametaphosphate (1.5% (w/v)), and the purified enzyme (0.3 mg/ml) were mixed together in 50 mM Tris-HCl (pH 7.5) to prepare a reaction composition. The enzymatic reaction was allowed to proceed at different temperatures of 30° C. to 65° C. for 30 min, followed by HPLC.

(25) The highest activity of the enzyme was observed at 50° C. (FIG. 6).

Example 5: Analysis of Demand of the Recombinant Polyphosphate-Dependent Glucokinase for Metal Ions

(26) The polyphosphate-dependent glucokinases reported to date are known to demand metal ions. 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.

(27) Glucose (2% (w/v)), 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, KCl, and CoCl.sub.2, 5 mM each), sodium hexametaphosphate (1.5% (w/v)), and the metal ion-free enzyme sample (0.3 mg/ml) were mixed together in 50 mM Tris-HCl (pH 7.5) to prepare reaction compositions. The enzymatic reaction was allowed to proceed at 50° C. for 30 min, followed by HPLC.

(28) The enzyme sample before treatment with metal ions was used as a control. The activity of the control was compared with the activities of the reaction compositions after treatment with metal ions. The magnesium salts were more effective for the production of glucose 6-phosphate by the polyphosphate-dependent glucokinase derived from Deinococcus geothermalis than the other metal salts analyzed, as shown in FIG. 7. Particularly, the addition of MgCl.sub.2 was confirmed to exhibit the maximum activity. Experiments were conducted at different concentrations of MgCl.sub.2. As a result, a higher activity was observed at a concentration of 10 mM (FIG. 8).

Example 6: Temperature Stability

(29) The temperature stability of the polyphosphate-dependent glucokinase was investigated. To this end, the enzyme was heated to different temperatures of 40-70° C. for 6 h, and residual activities were measured, compared, and analyzed.

(30) Glucose (2% (w/v)), MgCl.sub.2 (10 mM), sodium hexametaphosphate (1.5% (w/v)), and the purified enzyme (0.3 mg/ml) were mixed together in Tris-HCl (50 mM, pH 7.5) to prepare a reaction composition. The enzymatic reaction was allowed to proceed at 50° C. for 30 min, followed by HPLC.

(31) The results are shown in FIG. 9. The activity of the enzyme was reduced to half of its initial value after 6 h at 40° C. and after 1 h at 50° C.

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

(32) The conversion yields of glucose 6-phosphate at different concentrations of the substrates were analyzed to investigate the productivity of glucose 6-phosphate using the inventive purified enzyme.

(33) Glucose (3% and 15% (w/v)), MgCl.sub.2 (10 mM), sodium hexametaphosphate (1.5% and 7.5% (w/v)), and the purified enzyme (0.3 mg/ml) were mixed together in Tris-HCl (50 mM, pH 8.0) to prepare reaction compositions. The enzymatic reaction was allowed to proceed at 50° C. for different periods of time, followed by HPLC.

(34) As a result, the use of 3% (w/v) glucose and 1.5% (w/v) sodium hexametaphosphate achieved a conversion yield of 88.5% after reaction for 0.8 h. The use of 15% (w/v) glucose and 7.5% (w/v) sodium hexametaphosphate achieved a conversion yield of 74.2% after reaction for 24 h.