JPS6343079B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel and useful glucose-6-phosphate dehydrogenase and a method for producing the same. -Glucose-6-phosphate dehydrogenase used for measurement of phosphate, hexokinase, hexose-6-phosphate isomerase, fructose, glucose in the food industry, etc., and its production method. Recently, enzymes have attracted attention for their excellent reaction, substrate, and optical specificity, which are characteristics of enzymatic reactions, and are widely used as catalysts in medical analysis, food analysis, etc. Among them, glucose in body fluids,
It is well known that measuring the content of hexokinase and the like is important in clinical testing. Furthermore, analysis of the content of glucose and fructose in foods has become an important inspection item in the field of invert sugar industry. In recent years, methods using glucose-6-phosphate dehydrogenase have been widely used to analyze such components due to the advantages of the enzyme's excellent reaction, substrate, and optical specificities. It's being used. Glucose-6-phosphate dehydrogenase is an important enzyme in this regard. Although microanalytical methods using enzymes have the above-mentioned advantages, enzymes are generally very unstable and typically lose their catalytic activity within a few days to a few weeks at temperatures below room temperature. This instability is a major obstacle in trace component analysis using enzymes. The previously known glucose-6-phosphate dehydrogenase is similarly unstable; for example, Journal of Biological Chemistry (J.Biol.Chem.), vol. 236, p. 1225 (1961) Glucose-6 obtained from yeast, described in
- Phosphate dehydrogenase is 1 to 1 in aqueous solution (room temperature)
It was usual for most of the activity to be lost within 3 weeks. Furthermore, most glucose-6-phosphate dehydrogenases contain nicotinamide as a coenzyme.
Adenine dinucleotide phosphate (NADP)
That's what it means. ) only worked. This NADP
was, as is widely known, extremely expensive. To date, Advances in Enzymology (Ed. Altman Meister) Volume 48, pp. 97-191 (Advances in Enzymology, ed., by
As described by Alton Meister, many glucose-6-phosphate dehydrogenases are known, and most of them require NADP as a coenzyme for the reaction.
Nicotinamide is about 10 times cheaper than NADP.
Adenine dinucleotide (hereinafter referred to as NAD)
Leuconostoc mesenteroides (Leuconostoc mesenteroides), which can be used as a coenzyme in reactions.
Glucose obtained from Pseudomonas w6 and Pseudomonas mesenteroides
6-phosphate dehydrogenase. However, the enzymes obtained from Leuconostochus mesenteroides and Pseudomonas w6 have the major drawback of lacking the above-mentioned thermostability and long-term stability. Therefore, glucose-
Glucose-6-phosphate dehydrogenation has properties that are stable and allow the use of inexpensive coenzymes other than NADP in order to maximize the advantages of analytical methods using 6-phosphate dehydrogenase. There is a strong demand for the emergence of enzymes. Therefore, from this perspective, the present inventors developed glucose-6-phosphate, which is heat-stable, does not lose its activity for a long period of time, and has properties that allow the use of inexpensive coenzymes other than NADP. As a result of intensive research in search of dehydrogenase, glucose-6, which has the above properties, was found in microorganisms belonging to the genus Bacillus.
- Discovered the existence of phosphate dehydrogenase and completed the present invention. That is, the present invention provides a solution for about 15
The activity after treatment is approximately 80% of the activity before treatment.
Glucose-6-phosphate dehydrogenase and bacteria belonging to the genus Bacillus that have the above values and the following physical and chemical properties are cultured, and the culture is placed in a buffer solution at approximately 50°C. Collect glucose-6-phosphate dehydrogenase whose activity after treatment for approximately 15 minutes retains approximately 80% or more of the activity before treatment and which has the following physical and chemical properties. This is a method for producing glucose-6-phosphate dehydrogenase. (a) Action and substrate specificity Hexose-6-phosphate + coenzyme (oxidized type) ↑↓ Hexose-aldonic acid-6-phosphate + coenzyme (reduced type) Catalyzes the above reaction. In addition, hexose-6-phosphoric acid in the above formula is glucose-6-phosphoric acid,
It is a general term for mannose-6-phosphate, galactose-6-phosphate, and hexose aldonic acid-6.
- Phosphoric acid is a general term for gluconic acid-6-phosphoric acid, mannoic acid-6-phosphoric acid, and galactonic acid-6-phosphoric acid. In addition, as a coenzyme in the above formula,
Acts on NADP or NAD. (b) Optimum pH is approximately 9.0 (temperature 30°C). (c) Molecular weight The value measured by gel chromatography is
Approximately 230,000. By treating the glucose-6-phosphate dehydrogenase of the present invention in a buffer solution at about 50°C for about 15 minutes, the residual activity of glucose-6-phosphate dehydrogenase can be reduced to about 80% of the original activity. % or more, preferably about 90
% or more, optimally about 100%, and in particular, the activity after processing for about 15 minutes in a buffer at about 57°C is about 80% or more of the activity before treatment. It possesses excellent qualities. The concentration and pH of the buffer are not particularly limited, but generally the concentration is 5mM to 50mM and the pH is 7 to 10.5. In particular, in the present invention, 100mM
It is preferable to use a 100 mM Tris-HCl buffer (PH approximately 9.0) containing potassium chloride. Next, the physicochemical properties of the glucose-6-phosphate dehydrogenase of the present invention will be shown. 1 Action and substrate specificity As described above. Note that the Michaelis constant (km value) of glucose-6-phosphate is approximately 0.16 mM. The reaction rates of mannose-6-phosphate and galactose-6-phosphate at the same substrate concentration are approximately 40% and 20%, respectively, when glucose-6-phosphate is used as the substrate.
It is. The Km values for coenzymes NADP and NAD are approximately 0.016mM and approximately 1.64mM, respectively. 2 Optimum PH As mentioned above. 3. Stable PH range: Almost no deactivation occurs when treated at 4°C for 24 hours at PH7.0 to 10.5. 4. Suitable temperature range for action At pH 8.9, activity increases as temperature rises from 25°C to 75°C. Usually, the reaction is carried out at 30°C. 5 Heat resistance Stable when heated at 57℃ for 15 minutes. 6 Molecular weight As described above. Note that Sephacryl S-200 (manufactured by Pharmacia) was used for gel chromatography. Glucose-6-phosphate dehydrogenase from yeast and Leuconostoccus mesenteroides is approximately 10% under the same conditions.
It had a molecular weight of 1,000,000. 7. Method for measuring titer Prepare a mixed solution containing 2mM glucose-6-phosphate, 0.5mM NADP, and 5mM magnesium chloride in 50mM Tris-HCl buffer at pH 8.9, and add an appropriate amount of glucose-6- to the mixture. Phosphate dehydrogenase was added, and the titer was measured based on the increase in absorbance of NADP reduced form (NADPH) at 340 nm per unit time, and 1 micromole per minute was determined.
The titer of the purified enzyme is 30 units, where the enzyme activity that increases the absorbance of NADPH at 340 nm is taken as 1 unit.
Approximately 100 units/mg purified enzyme at °C. The glucose-6-phosphate dehydrogenase of the present invention has excellent heat resistance, so it can react even at temperatures at which conventional yeast enzymes are inactivated, that is, about 50°C to 60°C. It is possible. The titer at that temperature is even higher. 8. Unity The purified sample migrated to the anode side by 7.5% acrylamide disc electrophoresis at pH 9.4 and gave a single band. In addition, by SDS electrophoresis, it migrated to the anode side and gave a single band. 9 Elemental analysis Instead of elemental analysis values, amino acid composition is shown in mol%. Aspartic acid 11.04%, threonine 5.42%,
Serine 4.56%, glutamic acid 11.86%, proline
3.66%, glycine 6.67%, alanine 7.92%, cystine (cysteine) 0.62%, valine 6.09%, methionine 1.97%, isoleucine 5.59%, leucine
8.04%, Tyrosine 3.72%, Phenylalanine 4.88
%, lysine 5.28% histidine 3.40%, arginine
6.56%, tryptophan 2.72% 10 Crystal structure Currently, it has not been crystallized, so it has not been measured. In order to produce the glucose-6-phosphate dehydrogenase of the present invention, the following method can be adopted. That is, bacteria belonging to the genus Pacillus are cultured, and the glucose-6- of the present invention is obtained from the culture.
It can be obtained by collecting phosphate dehydrogenase. The bacteria used in the present invention are Pachylus bacteria that can produce the glucose-6-phosphate dehydrogenase of the present invention, and any such bacteria can be used. Preferred bacteria include
An example is Bacillus stearothermophilus.
Specific examples of stearothermophiles include ATCC7953, 7954, 8005, 10149, 12980,
Examples include NCA1503. As a carbon source in the nutrient medium used for culturing bacteria in the present invention, for example,
Glucose, sucrose, fructose, starch hydrolyzate, molasses, sugars from sulfite pulp waste liquid,
Organic acids such as acetic acid and lactic acid, as well as alcohols, oils and fats, fatty acids, glycerin, etc. that can be assimilated by the bacteria used can be used, and as nitrogen sources, for example, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, amino acids, peptone, Inorganic or organic substances such as meat extract and yeast extract can be used. Furthermore, as inorganic salts, for example, potassium, sodium, phosphoric acid, zinc, iron, magnesium, manganese, copper, calcium, cobalt salts, trace metal salts, corn stave liquor, vitamins, chopped An acid or the like may be used, and a general nutrient medium for bacteria can be used. Using these media, bacteria belonging to the genus Bacillus may be cultured aerobically at 20°C to 80°C, preferably 40°C to 70°C, optimally 60°C, for about 2 to 6 hours. In addition, industrially, for example, the maximum specific growth rate of a strain is determined using the dilution rate (the value obtained by dividing the rate of supplying the culture medium into the fermenter and the rate at which it is simultaneously withdrawn from the fermenter by the amount of culture solution in the fermenter). A continuous culture method can also be used, in which the concentration is controlled continuously in the range of 0.3 to 1.0, preferably 0.5 to 1.0, optimally 0.7 to 1.0. Next, the glucose-6-phosphate dehydrogenase of the present invention is collected from the obtained culture.
Purified enzymes can be collected at any stage. As a purification method, a normal enzyme purification method can be used. That is, after obtaining bacterial cells by centrifugation etc.
After disrupting the bacterial cells using Manton-Gaulin, Dynomill, French press, ultrasonication, etc., cell debris is removed by centrifugation to obtain a cell extract, which is treated with streptomycin sulfate or protamine sulfate, and then treated with sulfuric acid. ammonia precipitation,
Acetone precipitation, heat treatment, etc. are performed, and for further purification, ion exchange chromatography such as DEAE-cellulose column, adsorption chromatography such as hydroxyapatite column, gel filtration chromatography such as Sephadex chromatography is performed. It can be carried out in combination with other chromatography methods. In this manner, the glucose-6-phosphate dehydrogenase of the present invention can be isolated and purified. Since the glucose-6-phosphate dehydrogenase of the present invention is very stable against heat, it can be used for a long period of time after enzyme isolation compared to conventional glucose-6-phosphate dehydrogenase. Can be saved.
Therefore, it is very effective in biochemical research, food, and clinical testing fields. For example, the glucose-6-phosphate dehydrogenase of the present invention, hexokinase,
Using a reaction solution consisting of NAD, ATP, and magnesium chloride, glucose concentrations in foods and body fluids can be measured with high accuracy. Glucose concentration in foods is important for operational analysis in the invert sugar industry, and glucose concentration in body fluids is used for diagnosis of diabetes and the like. Furthermore, phosphoglucose isomerase activity is measured by measuring the concentration of glucose-6-phosphate in body fluids using a reaction solution consisting of the glucose-6-phosphate dehydrogenase of the present invention, NAD, and magnesium chloride. can do. This is used to screen for cancers such as metastatic breast cancer. In addition, the glucose-6-phosphate dehydrogenase of the present invention has the property of acting on NAD, which is much cheaper than NADP as a coenzyme, and therefore exhibits an economic advantage as an inexpensive test drug. can do. Next, the present invention will be specifically explained using examples. Example 1, Comparative Example 1 Polypeptone 0.5g/dl, vinegar mother extract 0.5g/
dl, satsucrose 1.0g/dl, potassium sulfate 0.13
g/dl, nitrium phosphate 0.644g/dl, magnesium sulfate 0.027g/dl, citric acid 0.032g/dl,
Ferrous sulfate 0.0007g/dl, manganese sulfate 0.015
g/dl, medium 250 adjusted to PH7.00 at 115℃ for 10
After heat sterilization for a minute, Patillus stearothermophile strain NCA1503 was inoculated and cultured with aeration at 60°C for 3 hours at an internal pressure of 0.5 kg/m ² G. After culturing, while cooling with water, the cells were immediately collected using a DeLaval centrifuge to obtain 700 g of cells. After storing the obtained bacterial cells in a frozen state,
300g of frozen bacterial cells was diluted with twice the amount of 0.1M phosphate buffer (PH
7.5), thoroughly disrupt the cells using a French press, and remove cell debris by centrifugation.
A crude extract containing glucose-6-phosphate dehydrogenase was obtained. After adding 300 ml of 1% protamine sulfate solution to 600 ml of this crude extract and stirring thoroughly,
The resulting precipitate was removed by centrifugation to obtain a protamine supernatant. Solid ammonium sulfate was gradually added to this supernatant to achieve 50% saturation (4°C). The generated precipitate was collected by centrifugation, dissolved again in 0.1M phosphate buffer (PH7.5), and then dialyzed against 20 times the amount of 0.1M phosphate buffer (PH7.5) and desalted. 2mM mercaptoethanol in advance,
Contains 2mM Sodium Ethylenediaminetetraacetate
Equilibrated with 20mM phosphate buffer (PH7.5)
When the above crude enzyme solution was passed through a DEAE-cellulose column and eluted with a solution in which potassium chloride was added to the above buffer, the target glucose-6-phosphate dehydrogenase was eluted at a potassium chloride concentration of approximately 0.15M. After collecting, concentrating and desalting this fraction,
The eluate was passed through a hydroxyapatite column equilibrated with 5mM phosphate buffer (PH7.5),
When a linear gradient elution from 5mM phosphate buffer to 250mM phosphate buffer was performed, the target glucose-6-phosphate dehydrogenase was eluted at a concentration near 75mM. After concentrating and desalting this active fraction, it was subjected to Ultrogel ACA34 chromatography using 50mM Tris/HCl buffer containing 0.1M potassium chloride as the eluent. The mixture was passed through a DEAE-Sephadex A-50 column equilibrated with 30mM phosphate buffer (PH7.7) containing sodium tetraacetate, and potassium chloride was added to the above buffer, starting at a potassium chloride concentration of 0.1M.
Elution was performed with a 0.4M linear gradient. As a result, purified glucose-6-phosphate dehydrogenase was eluted near a potassium chloride concentration of 0.2M. Glucose-6-phosphate dehydrogenase thus obtained migrated to the anode side in 7.5% acrylamide disc electrophoresis at pH 9.4 and gave a single band, and was further analyzed in Sephadex G-200 chromatography. also gave a single peak at a molecular weight of approximately 230,000. The yield was about 10 mg, the titer was about 100 units per mg of enzyme, and the degree of purification was about 1500 when the crude extract was 1. Next, for comparison with the glucose-6-phosphate dehydrogenase thus obtained, the stability of glucose-6-phosphate dehydrogenase obtained from yeast (Comparative Example 1) was investigated. The results are shown in FIGS. 1 and 2. In addition,
Figure 1 shows PH9.0 containing 100mM potassium chloride.
It shows the residual activity when heated for 15 minutes at each temperature in 100 mM Tris buffer, where curve A is Example 1 and curve B is Comparative Example 1. Figure 2 shows PH9.0
The figure shows the change in residual activity over time when the sample was stored in 100mM Tris buffer at 30°C, where curve C is for Example 1 and curve D is for Comparative Example 1. From these results, it is clear that glucose-6-phosphate dehydrogenase obtained from yeast irreversibly loses its activity almost completely when treated at 50°C for 15 minutes, whereas this The glucose-6-phosphate dehydrogenase of the invention did not lose any activity at 50°C. Furthermore, at 30°C, glucose-6-phosphate dehydrogenase obtained from yeast loses its activity within 10 to 20 days, whereas the glucose-6-phosphate dehydrogenase of the present invention loses its activity after 50 days. No decrease in activity was observed even at this point. As described above, the glucose-6-phosphate dehydrogenase of the present invention is surprisingly stable against heat and has the property of being able to be stored for a long period of time. This property is not present in conventional glucose-6-phosphate dehydrogenases. Example 2 A medium obtained by dissolving 1.3 g of glucose, 1.0 g of ammonium sulfate, 0.5 g of yeast extract, 0.5 g of potassium phosphate, 0.5 g of disodium phosphate, and 0.1 g of magnesium sulfate in 1 part of tap water in a 30-volume fermenter. of
20 times, and sterilized with pressure steam at 121° C. and 1 Kg/cm ² for 15 minutes. Culture conditions were 57℃, PH6.5-7.0 (4N-
Prepared with NaOH. ), ventilation rate 20/min (air),
After setting the stirring number to 900 rpm, add Bacillus to this.
Stearothermophilus ATCC12980 strain was obtained by pre-culture in the above medium. One inoculum of a solution whose absorbance at 660 nm reached approximately 1.0 was inoculated. first,
Batch culture was carried out for about 2.5 hours, and when the absorbance at 660 nm reached 1.0, a sterilized nutrient medium with the above composition was continuously supplied using a metering pump at a rate of 24.0/hr, and the same rate was removed from the fermenter. By extracting the culture solution, continuous culture was performed using 100 nutrient medium to obtain a culture solution. Immediately collect the bacterial cells using a DeLaval centrifuge while cooling the culture solution with water.
g of bacterial cells were obtained. The cells were suspended in 1.5 times the volume of 0.1M phosphate buffer, the cells were disrupted using a Dynomill, and insoluble matter was removed by centrifugation, resulting in a crude extract containing glucose-6-phosphate dehydrogenase. I got it. This crude extract
10% streptomycin sulfate solution per 400ml
After adding 200ml, remove the resulting precipitate by centrifugation,
Streptomycin supernatant was obtained. This supernatant was subjected to ammonium sulfate separation and saturated at 25% (4℃).
From this, fractions between 50% saturation (4°C) were obtained. Add this fraction to 50mM Tris-HCl buffer (PH8.0).
After dissolving the mixture, it is passed through a DEAE-Sephadex column equilibrated with the above buffer solution and eluted with a solution containing sodium chloride in the above buffer solution. -6-phosphate dehydrogenase was eluted. This eluted portion was subjected to hydroxyapatite column chromatography under the same conditions as in Example 1, and then passed through a Sephacryl S-200 column with 30mM Tris-HCl buffer (PH8.0) containing 0.1M sodium chloride. By elution, a sample of glucose-6-phosphate dehydrogenase was obtained which gave a single band in acrylamide disc electrophoresis in the same manner as in Example 1. Furthermore, as in Example 1, in Sephadex G-200 chromatography,
A single peak was given at a molecular weight of approximately 230,000. The yield was about 20 mg. The titer was approximately 100 units per mg of enzyme. The degree of purification was approximately 1100 when the crude extract was 1. Reference Examples 1, 2, 3 Using the glucose-6-phosphate dehydrogenase obtained in Example 1 and using standard serum with a known glucose concentration as a test solution, the glucose concentration was measured as follows. In addition, as a standard serum, the glucose concentration in the standard serum is 76 mg/dl (Reference Example 1), 155
mg/dl (Reference Example 2) and 43 mg/dl (Reference Example 3)
The one with the following was used. As a measurement method, first, per 100mM phosphate buffer pH8.51ml, glucose-6-phosphate dehydrogenase 1 unit/ml, hexokinase 2 units/ml,
A reaction solution was obtained by dissolving NAD at 2mM, ATP at 2mM, and _Mgcl2 at 2mM. This reaction solution was heated to 30℃ for 5 minutes.
After keeping the mixture for a minute, 20 μl of standard serum was added, mixed, and reacted at 30° C. for 5 minutes, and then the absorbance value at 340 nm was read using a spectrophotometer. At the same time, 20 μl of pure water was added to the reaction solution with the above composition, and after reacting at 30°C for 5 minutes, the absorbance at 340 nm was read, and this was used as a control, and from the absorbance at 340 nm of standard serum,
The increase in absorbance was determined by subtracting the absorbance of the control.
Next, the amount of glucose (mg) in 1 dl of standard serum was calculated using the following formula. 145.8×(increase in absorbance) = amount of glucose in 1 dl of subject (mg) The results are shown in Table 1. 【table】

[Brief explanation of drawings]

Figure 1 shows the residual activity of glucose-6-phosphate dehydrogenase of the present invention (curve A) and glucose-6-phosphate dehydrogenase obtained from yeast (curve B) after heating for 15 minutes at various temperatures. Figure 2 shows the glucose-6-phosphate dehydrogenase of the present invention (curve C) and the glucose-6-phosphate dehydrogenase obtained from yeast (curve D) left at 30°C. It is a figure showing the residual activity after.

Claims (1)

[Claims] 1. Has the property that the activity after being treated in a buffer solution at about 50°C for about 15 minutes maintains a value of about 80% or more of the activity before the treatment, and the following physical and chemical Glucose-6-phosphate dehydrogenase with properties. (a) Action and substrate specificity Hexose-6-phosphate + coenzyme (oxidized type) ↑↓ Hexose-aldonic acid-6-phosphate + coenzyme (reduced type) Catalyzes the above reaction. In addition, hexose-6-phosphoric acid in the above formula is glucose-6-phosphoric acid,
It is a general term for mannose-6-phosphate, galactose-6-phosphate, and hexose aldonic acid-6.
- Phosphoric acid is a general term for gluconic acid-6-phosphoric acid, mannoic acid-6-phosphoric acid, and galactonic acid-6-phosphoric acid. Furthermore, as a coenzyme in the above formula, it acts on nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide. (b) Optimum pH is approximately 9.0 (temperature 30°C). (c) Molecular weight The value measured by gel chromatography is
Approximately 230,000. 2. Bacteria belonging to the genus Bacillus are cultivated and the culture is treated in a buffer solution at approximately 50°C for approximately 15 minutes, and the activity thereof retains approximately 80% or more of the activity before treatment. A method for producing glucose-6-phosphate dehydrogenase, which comprises collecting glucose-6-phosphate dehydrogenase having the following physical and chemical properties. (a) Action and substrate specificity Hexose-6-phosphate + coenzyme (oxidized type) ↑↓ Hexose-aldonic acid-6-phosphate + coenzyme (reduced type) Catalyzes the above reaction. In addition, hexose-6-phosphoric acid in the above formula is glucose-6-phosphoric acid,
It is a general term for mannose-6-phosphate, galactose-6-phosphate, and hexose aldonic acid-6.
- Phosphoric acid is a general term for gluconic acid-6-phosphoric acid, mannoic acid-6-phosphoric acid, and galactonic acid-6-phosphoric acid. Furthermore, as a coenzyme in the above formula, it acts on nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide. (b) Optimum pH is approximately 9.0 (temperature 30°C). (c) Molecular weight The value measured by gel chromatography is
Approximately 230,000.