KR20060059622A - A producing method of fructose-6-phosphate using thermostable enzymes - Google Patents

Abstract

The present invention relates to a method for preparing 6-fructose 6-phosphate which is a kind of phosphosugar from starch by using a heat-resistant enzyme. More specifically, the reaction is performed using various heat-resistant enzymes such as starch phosphorylase, phosphoglucomutase, glucose 6-phosphate isomerase, and pullulanase. The present invention relates to a method of forming a resin, separating using an ion exchange resin, or the like, and purifying using crystallization or the like.

6-fructose 6-phosphate, 1-glucose 1-phosphate, 6-glucose 6-phosphate, glucan phosphatase, starch phosphorylase, phosphoglucomutase, Glucose 6-phosphate isomerase, pullulanase, heat resistant enzyme

Description

A production method of fructose-6-phosphate using thermostable enzymes}             

1 is a schematic diagram of the route for preparing 6-phosphate fructose from saccharides,

The enzymes used in each step

G1P production in starch: starch kinase (a)

G1P production from glycogen: glycogen kinase (b)

G1P production from sugar: sugar kinase (c)

G1P Production in Maltose: Maltose Kinase (d)

G6P Production in Glucose: Glucose Kinase (e)

F6P Production in Fructose: Fructose Kinase (f)

G6P Production from G1P: Glucose-Transferase (g)

F6P production in G6P: 6-glucose isomerase (h).

Figure 2 is a graph showing the change in absorbance and glucose concentration of the cells produced by batch culture of recombinant E. coli,

3 is a graph showing the change in absorbance and glucose concentration of the cells produced by fed-batch culture of recombinant E. coli,

Figure 4 is a graph showing the change of each reactant, product concentration in the process of bio conversion from 1-phosphate glucose (G1P) to 6-phosphate fructose (F6P) using a heat resistant enzyme.

Fructose 6-phosphate is a sugar compound phosphorylated at position 6 of fructose and is widely present in plant cells or animal tissues. Especially in plant cells, 6-phosphate fructose is an important intermediate of glycolysis, gluconeogenesis, pentasaccharide phosphate pathway, and the Calvin cycle. The substance is also known to exist in equilibrium with glucose 6-phosphate in animal tissues. It generally exists as D-form isomers and is abbreviated as F6P.

Synthesis of 6-phosphate fructose in living organisms is produced by the action of isoglucoisomerase on 6-phosphate glucose in the second stage of glycolysis. The next step in glycolysis is 6-phosphate fructose with ATP to produce 1,6-diphosphate fructose (Fructose 1,6-biphosphate) and ADP with the help of an enzyme called phosphofructokinase.

According to the literature, 6-phosphate fructose has potential for various uses. Particularly, according to the 1992 document EP0506772 (WO9109604), the possibility that phosphosugar, in which carbohydrates are combined with phosphoric acid, can be used in various fields such as diabetes and inflammation. However, no specific mechanism of action or clinical trial data is available.

Enzyme stability and high productivity are very important for industrial use of bioconversion using enzymes. In this sense, the recombinant heat-resistant enzyme has a high industrial value. Recently, genome information of Thermus Sp., A representative microorganism among industrially available microorganisms, has been published ( Nature Biotech. 22: 547-553 (2004)), further increasing its value as a useful genetic resource. However, this information has been discovered recently, and there are no reports on the massive bioconversion of phosphate sugars using heat-resistant enzymes. One of the notable researches on the bioconversion of phosphate sugar using a non-heat resistant enzyme is a bioconversion study of 1-phosphate glucose.

In the present invention, in order to produce 6-phosphate fructose (F6P) from starch in detail, bioconversion from starch to 1-phosphate glucose (G1P), internal bioconversion from 1-phosphate glucose (G1P) to 6-phosphate glucose (G6P) And three stages: bioconversion from 6-phosphate glucose (G6P) to 6-phosphate fructose (F6P). According to the conventional reports, first, the production of 1-phosphate glucose (G1P) using an enzyme is derived from the sugar ( ruuconostock mesenteroides ) that synthesizes fructose (fructose) and 1-phosphate glucose from sugar (sucrose) Sucrose phosphorylase has been reported [ Agric. Biol. Chem. 557: 1805-1810 (1991)], glucan kinase that synthesizes 1-phosphate glucose from highly polymerizable polysaccharides such as alpha-glucan or αtoglucan, maltodextrins, dextrin, starch, and the like. -glucan-phosphorylases have been reported. In particular, Kayane et al . Of the European patent have reported that it is possible to bioconvert up to 60% of 1-phosphate glucose using starch kinase from starch [EP 0305981A2 (1989), Recent developments in the chemistry of natural carbon compounds Springer Berlin (1971). There is also a report on the development of a continuous production process of 1-phosphate glucose using starch kinase immobilized on a carrier [ J. Biotechnol. 36: 11-17 (1994).

Meanwhile, 6-phosphate glucose (G6P) must be internally converted from mono-phosphate glucose, which is catalyzed by phosphoglucomutases (PGM) in the consumption and synthesis of sugar. This enzyme is expressed in many different forms of proteins by genetic recombination [ Plant Physiol. 117: 997-1006 (1998), Microbiol. 143: 855-865 (1997).

To produce 6-phosphate fructose, 6-phosphate glucose must be bioconverted by 6-phosphoglucose isomerase. The enzyme is also derived from plants such as bananas, and microorganisms such as Aeropyrum pernix and Thermoplasma acidophilum have also been reported [ Biosci. Biotechnol. Biochem. 65: 2174-2180 (2001), J. Biologic. Chem. 279: 2262-2272 (2004). In addition to the points mentioned, a typical production route of 6-phosphate fructose (F6P) using an industrial substrate is shown in detail in FIG. 1.

Thermus sp. Bacteria are thermophilic bacteria that grow at high temperatures, generally growing at 70-75 ° C, and some species growing at 85 ° C (Brock, TD (1986) Thermophiles, pp 27-). 28, John Wiley Sons, Inc., New York. Thus, bacteria in Dermus are a good source of enzymes for activity at high temperatures, and several types of heat-resistant enzymes have been reported to be separated from bacteria in Dermus. However, although carbohydrate-related enzymes are useful in the field of biotransformation, there are not many studies on biotransformation using enzymes isolated from the bacteria of Dermus (Taguchi, H. et al. J. Biochem) . ., Tokyo (1982) 91, 1343-1348; Hoide, S. et al J. Biochem, Tokyo, (1991) 109, 6-7;.... Park, JH et al Eur J. Biochem (1993). 241, 135-140; Ono, M. et al. J. Biochem. (1990) 107, 21-26).

It is an object of the present invention to produce high efficiency 6-phosphate fructose from starch which is a relatively inexpensive raw material.

In the present invention, genes of various heat-resistant enzymes derived from the Thermus strain were transferred to E. coli, and then fermented to E. coli in large quantities, and 6-phosphate fructose was produced from starch using the enzyme produced therein. In addition, the 6-phosphate fructose thus produced was efficiently isolated and purified.

The present invention is a reaction temperature using a heat resistant starch phosphorylase derived from the genus Thermus sp., Phosphoglucomutase, 6-glucose 6-phosphate isomerase It relates to a process for producing 6-phosphate fructose from water-soluble or insoluble starch at 40-80 ° C.

The present invention also relates to a method for producing 6-phosphate fructose from starch by adding pullulanase derived from Thermus sp.

The present invention also relates to a method for producing 6-phosphate fructose from starch, characterized in that the ratio of the glucose phosphate transferase: 6-glucose isomerization enzyme is 1: 1 to 1: 1.5. The yield of 6-phosphate fructose was high when the ratio of the two enzymes was in the above range.

The present invention also relates to a process for producing 6-phosphate fructose from starch, characterized in that the reaction temperature is 60 to 70 ° C. When the temperature is 60 ~ 70 ℃, the solubility of the starch, which is a substrate increases, and the effect of inactivating other proteins is excellent.

The present invention further relates to a method for producing 6-phosphate fructose from starch, wherein the concentration of the insoluble starch is 0.01 g / L to 20 g / L. If this range is exceeded, G1P productivity is lower than that of the previous quarter, which is undesirable in terms of industrial utilization.

In addition, the present invention is characterized by adding the separation, purification step after the 6-phosphate fructose manufacturing process.

An embodiment of the present invention will be described in detail as follows. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the following examples.

Example 1 Fed-Batch Culture of Recombinant Escherichia Coli for Enzyme Production

For the large-scale fermentation of E. coli grafted with the glucan phosphorylase gene derived from the Thermus caldophilus strain, a working volume of 2l and a stirring speed of 700rpm in a 5l fermentor The culture was carried out with an air flow rate of 1 vvm, a culture temperature of 37 ° C., and an inoculation amount of 10%. When batch culture was performed, the highest cell mass remained at absorbance (OD 600) of 14 (FIG. 2), but when fed-batch culture, the peak cell mass increased to absorbance 45 (FIG. 3). At this time, the final concentration of IPTG, an inducer added to produce enzyme, was 5 mM.

In addition, phosphoglucomutase and phosphoglucose isomerase, which are recombinant enzymes derived from Thermus caldophilus , used in the present invention, are also fermented in the same manner as above to obtain similar results. ( J. Ind. Microbiol. Biot. (2000) 24, 89-93).

Example 2 Preparation of 1-Glucose Glucose from Starch

Glucose monophosphate (G1P) was prepared from soluble starch using α-glucan phosphorylase. Recombinant glucan kinase from Thermus sp. Was characterized as a single band 90kDa on SDS-PAGE and showed maximum activity at optimal pH 7.0, 70 ° C. Since the enzyme is smoothly reacted at a high temperature (70 ° C. or more), it is possible to increase the solubility of starch having low solubility at room temperature, thereby enabling a high concentration of substrate reaction. The reaction was initiated by adding the enzyme 9.4 U / g-starch at 5% soluble starch and 1M phosphate buffer (pH 7.0, 70 ° C.) by weight, yielding 164 mM G1P. The production of G1P by the amount of enzyme added between 13-50U per g of substrate starch was examined over time. The final product concentration was similarly high, but the concentration of substrate starch influenced the initial reaction rate. Considering the prevention of aging of starch during the reaction, it is necessary to control the optimum reaction time by adjusting the amount of enzyme addition (Table 1). Temperature conditions were carried out at 70 ° C.

Enzyme amount (U / g starch) G1P Production (mM) 1h 4h 8h 72h 13 - 20 60 141.6 25 45.1 91.7 100 152.2 50 55.5 95 109.8 158.7

On the other hand, when reacting with industrial insoluble starch, high concentration of starch could not be used as a substrate due to solubility limitation. When industrial 2% insoluble starch was dissolved in 300mM phosphate buffer solution, reaction with 37 U / g-starch of enzyme yielded 15.3mM G1P. Comparing G1P production by concentration of insoluble starch is shown in Table 2 below. Temperature conditions were performed at 70 ° C.

Insoluble Starch (g / ℓ) 300 mM sodium phosphate buffer (pH 7.0) 1-phosphate glucose (g / ℓ) Productivity (Ys / g) * One 0.5 (0.2) 0.17 5 10 (3.3) 0.67 10 10.8 (3.6) 0.36 20 15.3 (5.1) 0.26

* Ys / g = 1-phosphate glucose concentration (g / l) / starch concentration (g / l)

As substrate concentration increased, 1-phosphate glucose (G1P) production tended to increase. In addition, considering the productivity, the reaction at low concentrations is efficient, but considering the G1P price ratio of the starch price, the substrate concentration of the titration line is variable.

Next, a 200 L scale reaction was performed in a 500 L reactor for mass production of G1P. G1P produced as a result of 16 hours of reaction at 70 ° C using 1.2% soluble starch and 200mM sodium phosphate buffer (pH 7.0) as substrate was found to be 1.253Kg, approximately 50% of the initial starch amount was bioconverted. there was. In addition, the addition of pullulanase to the reaction solution was observed to increase the initial G1P yield by more than 10 to 50%.

Example 3 Isolation and Purification of 1-Phosphate Glucose

The resulting glucose phosphate was subjected to separation and purification by modifying well-known methods such as ion exchange resins and crystallization (JACS 66: 560-563 (1944)) according to the situation of the present invention. The 1-phosphate glucose (G1P) reactant obtained in Example 2 contains other impurities such as starch, phosphate buffer solution, and protein. This was done by sequentially applying strong acid cation exchange resin and weakly basic anion exchange resin column. The strongly acidic cation exchange resin column was washed and pretreated with 4% HCl, eluted with ultrapure water, and the fractions containing G1P were collected. Subsequently, the G1P fraction was recovered by applying the weak base anion exchange resin column and eluting with 4% ammonia water. After crystallization, the yield of 1-phosphate glucose was at least 80% and the purity was at least 95%. Additional column manipulations, such as activated carbon, can be performed to increase the chromaticity of the final product.

Example 4 Preparation of 6-phosphate fructose from 1-phosphate glucose

Pure mono-phosphate glucose (G1P) prepared in Example 3 is used as a substrate for 6-phosphate fructose (F6P) production. The enzymes used in this example were a single molecular weight of 61 kDa recombinant phosphoglucomutase (EC 5.4.2.2.) Derived from the genus Thermus sp. And a 46 kDa phosphoglucose isomerase (EC 5.3.1.9). )to be. These enzymes are recombinant enzymes from the genus Dermouth, and the reaction conditions as well as the reaction temperature are the same, and can simultaneously perform two enzyme reactions in one step. 25 g / L 1-glucose phosphate (G1P), 2 mM MgCl ₂ , two enzymes: recombinant phosphoglucomutase and phosphoglucose isomerase from Thermus sp. Reactions included were left in the reactor at 70 ° C. for 72 hours or lightly shaken. The maximum 6-phosphate fructose (F6P) production after the reaction was yield 21% after 44 hours of culture, after which the amount of 6-phosphate glucose increased and eventually equilibrated as the amount of 6-phosphate fructose decreased.

4mM 1-glucose and 2mM MgCl ₂ , starch or phosphate buffers at various concentrations as inhibitors, recombinant transferase from Thermus sp. The reaction was added to the reaction mixture of phosphoglucomutase and phosphoglucose isomerase at 70 ° C. for 21 hours. Inhibition results of starch and phosphate buffer, which are the major inhibitors in producing 6-phosphate fructose, are shown in Table 3 and Table 4 below.

Water Soluble Starch (g / ℓ) % Conversion G1P G6P F6P 5 10.6 82.7 6.7 One 8.9 82.3 8.8 0.5 7.9 81.4 10.7 0.1 8.3 81.9 9.8

Phosphate Buffer (mM) % Conversion G1P G6P F6P 50 26 74 n.d. * 25 19.9 71.5 8.6 10 9 82 9 5 8.1 77.8 14.1

* n.d .: not determined

In Table 3 and Table 4 above, starch and phosphate buffer solution both show an inhibitory effect on the production of 6-phosphate fructose, the final product. Therefore, in order to efficiently produce 6-phosphate fructose from starch, it can be seen that there is an optimal concentration ratio between the two substances.

Example 5 6-Phosphate Fructose Production Optimization Using Statistical Techniques

In this example, statistical techniques were used to investigate more efficient reaction conditions in the production of 6-phosphate fructose. The reaction was performed using 10 mM 1-glucose glucose, 2 mM MgCl ₂ , 20 mM buffer, and the reaction condition variables were temperature, pH, enzyme ratio (activity ratio of introduced relocation enzyme and isomerase), and final product 6-phosphate fructose yield. Evaluation based on the results obtained as shown in Table 5.

Experiment number Enzyme Ratio (GM: GI) Temperature (℃) pH Actual F6P yield (g / l) Theoretical F6P Yield (g / l) One 1: 2 70 5 1.652144067 1.59684175 2 1: 2 70 9 0.138300833 0.08443214 3 1: 2 50 7 1.167886 1.48221169 4 1.5: 1.5 70 7 1.229571 1.21223371 5 1: 2 30 9 0.125368533 0.05371415 6 2: 1 30 9 0.154782833 0.17076195 7 1.5: 1.5 50 7 1.382317067 1.2509859 8 2: 1 70 9 0.314321133 0.40849824 9 2: 1 70 5 1.919574667 1.95190585 10 1.5: 1.5 50 7 1.434240333 1.2509859 11 2: 1 30 5 0.1071289 0.12167439 12 1.5: 1.5 50 5 0.9238225 1.06574844 13 2: 1 50 7 1.875300533 1.71826764 14 1.5: 1.5 30 7 0.1071289 0.28175898 15 1: 2 30 5 0.1071289 -0.0263714 16 1.5: 1.5 50 9 0.318720567 0.33408742

R = 0.964, R ² = 0.911

As shown in this example, the correlation between the experimental data and the theoretical data showed that the R value was 0.96 and the R ² value was 0.91. According to the response surface analysis, the ratio of enzyme was GM: GI = 1: 1.23, temperature 63.5 ° C, pH 6.85, and this condition was determined as the optimum reaction condition. The produced 6-phosphate fructose was obtained by using the separation method mentioned in Example 3 and the separation method using a silica column to obtain a pure 6-phosphate fructose of more than 50% yield, 95% purity.

Through the present invention, a method for preparing phosphate sugar using a heat resistant enzyme, and more specifically, a biochemical process for preparing 1-glucose phosphate, 6-glucose phosphate, and 6-phosphate fructose was established. In particular, it is an important effect that phosphate sugar can be used as a high value-added potential drug using starch, which is an industrial substrate.

Claims (6)

Reaction temperature 40 ~ 80 using heat resistant starch phosphorylase, phosphoglucomutase, and 6-glucose 6-phosphate isomerase from Thermus sp. A process for producing 6-phosphate fructose from water soluble or insoluble starch at < RTI ID = 0.0 > The method of claim 1, wherein the fructose derived from Thermus sp. Is added to produce 6-phosphate fructose from starch. The method of claim 1, wherein the glucose phosphate transferase: 6-phosphate isomerase is in a ratio of 1: 1 to 1: 1.5. A process for producing 6-phosphate fructose from starch according to claim 1, wherein the reaction temperature is 60 to 70 ° C. The method of claim 1, wherein the insoluble starch has a concentration of 0.01 g / l to 20 g / l. The method for producing 6-phosphate fructose from starch by using a heat-resistant enzyme according to claim 1, wherein after the 6-phosphate fructose production process, a separation and purification process are added.

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