CA1156570A - Glucose-6-phosphate dehydrogenase and process for preparation thereof - Google Patents
Glucose-6-phosphate dehydrogenase and process for preparation thereofInfo
- Publication number
- CA1156570A CA1156570A CA000363960A CA363960A CA1156570A CA 1156570 A CA1156570 A CA 1156570A CA 000363960 A CA000363960 A CA 000363960A CA 363960 A CA363960 A CA 363960A CA 1156570 A CA1156570 A CA 1156570A
- Authority
- CA
- Canada
- Prior art keywords
- glucose
- phosphate dehydrogenase
- enzyme
- phosphate
- buffer solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
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- 235000018417 cysteine Nutrition 0.000 description 1
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- 206010012601 diabetes mellitus Diseases 0.000 description 1
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- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
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- 229910052742 iron Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
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- 239000011777 magnesium Substances 0.000 description 1
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- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
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- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
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- 239000004474 valine Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
Abstract
ABSTRACT OF THE DISCLOSURE
A heat-resistant glucose-6-phosphate dehydro-genase which when incubated for about 15 minutes in a buffer solution at about 50°C, retains at least about 80% of the initial activity, and a process for producing such de-hydrogenase by culturing a microorganism of the genus Bacillus and recovering the desired heat-resistant glucose-6-phosphate dehydrogenase from the culture,are disclosed.
The enzyme can be stored for an extended period of time and so is very effective for use in biochemical research, food industry and for applications in clinical tests.
A heat-resistant glucose-6-phosphate dehydro-genase which when incubated for about 15 minutes in a buffer solution at about 50°C, retains at least about 80% of the initial activity, and a process for producing such de-hydrogenase by culturing a microorganism of the genus Bacillus and recovering the desired heat-resistant glucose-6-phosphate dehydrogenase from the culture,are disclosed.
The enzyme can be stored for an extended period of time and so is very effective for use in biochemical research, food industry and for applications in clinical tests.
Description
1 15B~7() FOR PREPARATION THEREOF
BACKGROUND OF THE INVENTION
This invention relates to a novel and useful glucose-6-phosphate dehydrogenase and a process for pr~paration thereof More particularly, the invention relates to a glucose-6-phosphate dehydrogenase that can be used in the determination of glucose, glucose-6-phosphate, hexokinase and hexose-6-phosphate isomerase in clinical tests in the medical field, and the determination of fructose and glucose in the food industry, as well as a process for preparing such a dehydrogenase.
Recently, enzymes, because of their high specificity in type of reaction, substrate ~i.e., material acted upon), and optical characteristics, have begun to be used widely as cata-lysts in medical and food analyses. As is well known, the determination of glucose and hexokinase levels in body fluids constitutes an important parameter in clinical tests. The analysis of glucose and fructose levels in foods is also an impor-tant parameter in the manufacture of invert sugar. Currently, glucose-6-phosphate dehydrogenase is widely used in determining these parameters, by virtue of its very high specificity in reaction, substrate andoptical characteristics, and hence is one Of the more important enzymes in the state of art.
While microanalysis using enzymes has the above mentioned advantages, enzymes are very labile, and lose their catalytic activity in relatively short periods of from a few .
,.
1 lsss7n days to several weeks, even when maintained at lower than room temperature. Thus, the lability of enzymes constitutes a serious bar to microanalysis using enzymes. Glucose-6-phosphate dehydrogenase preparations known to date are also labile, and yeast-derived glucose-6-phosphate dehydrogenase, described in the Journal of_Biological Chemistry, Vol. 236, p. 1225, (1961) generally loses most of its activity within one to three weeks in aqueous solution at room temperature.
High cost is another reason that has prevented large-scale application-of glucose-6-phosphate dehydrogenase to microanalyses in the fields of medical and food analyses. The reaction of glucose-6-phosphate dehydrogenase always requires a coenzyme, and most of the conventional analyses using glucose-6-phosphate dehydrogenases require the use of nicotinamide adenine dinucleotide phosphate (hereunder referred to as NADP).
As is well known, NADP is very expensive. Many glucose-6-phosphate dehydrogenase preparations are known, as described, e.g., in Advances in Enzymology, ed. by Alton Meister, Vol. 48, pp. 97-191, and most of them require NADP as a coenzyme in an enzymatic reaction, with the exception of those which are derived from Leuconostoc mesenteroides and Pseudomonas W 6, which are compatible for use with nicotinamide adenine dinucleotide (here-inafter NAD) that is one tenth or less the cost of NADP.
However, the glucose-6-phosphate dehydrogenases produced from Leuconostoc mesenteroides and Pseudomonas W 6 are not heat-stable B~7n and do not keep long, i.e., the inactivation of the enzyme is observed when the enzyme is allowed to stand for 1 to 2 weeks at a room temperature. Therefore, to maximize the advantages of analyses using a glucose-6-phosphate dehydrogenase, a glucose-6-phosphate dehydrogenase which is stable and compatible with inexpensive coenzymes (i.e., other than the expensive NADP) has been the subject of much research.
SUMMARY OF THE INVENTION
An object of this invention is to provide a glucose-6-phosphate dehydrogenase which is heat-stable, does not lose its activity for an extended period of time, and which is com-patible with an inexpensive coenzyme ~i.e., other than the expensive NADP).
As a result of various efforts to achieve this object, the inventors of this invention have found that a microorganism of the genus Bacillus produces a glucose-6-phosphate dehydrogenase that has the above described properties.
Therefore, this lnvention provides a glucose-6-phosphate dehydrogenase which, when incubated for about 15 minutes in a buf-fer solution at about 50C, retains at least 80 % of its i,~tial activity, as well as a process for producing such glucose-6-phosphate dehydrogenase by culturing a microorganism of the genus Bacillus and recovering from the culture a glucose-6-phosphate dehydrogenase which, when incubated for about 15 minutes with a buffer solution at about 50C, retains at least 80 % of its initial activity.
The glucose-6-phosphate dehydrogenase of this invention is very stable against heat, so after isolation, it can be stored 1 l5B~n for a longer period than the conventional glucose-6-phosphate dehydrogenase. The enzyme is very effective for use }n biochem-ical research, the food industry, and for applications in clinical test. In addition, the enzyme is compatible with NAD, which is much less expensive than NADP, and so it can be used with advantage as an inexpensive reagent. -- BRIEF DESCRIPTION OF THE DRAWIN~
FIG. l is a graph showing the residual activities of the glucose-6-phosphate dehydrogenase of this invention (curve A) and yeast-derived glucose-6-phosphate dehydrogenase (curve B) after heating at various temperatures for 15 minutes; and FIG. 2 is a graph showing the residual activities of the glucose-6-phosphate dehydrogenase of this invention (curve C) and yeast-derived glucose-6-phosphate dehydrogenase (curve D) after storage at 30C.
DETAILED DESCRIPTION OF THE INVENTION
The glucose-6-phosphate dehydrogenase of this invention, when incubated for about 15 minutes in a buffer solution at about 50C, retains at least about 80 %, preferably at least about 90 %, and more preferably about 100 %, of its initial activity. In par-ticular, the dehydrogenase of this invention retains at least about 80 % of its initial activity when it is treated for about 15 minutes with a buffer solution at about 57C. The concentration and pH of the buffer solution are not limited to a specific value, but generally, the concentration is in the range of rrom 5 to 500 millimole/Q (hereinafter, referred to as "mM"), and the pH is from 7 to 10.5. For the purpose of this invention, 100 mM of tris-HCl buffer solution ~pH: about 9.0) containg_l00 mM of potassium chloride is used with advantage.
The physicochemical properties of the glucose-6-phosphate dehydrogenase of this invention are set forth below.
1. Function of enzyme The glucose-6-phosphate dehydrogenase of this invension catalyzes the following reactions:
Hexose-6-phosphate + coenzyme ~oxidized form) Hexose aldonic acid-6-phosphate + coenzyme (reduced form) "Hexose-6-phosphate" is a generic term for glucose-6-phosphate, mannose-6-phosphate and galactose-6-phosphate, and "hexose -aldonic acid-6-phosphate" is a generic-term for gluconic acid-6-phosphate,-mannonic acid-6-phosphate and galactonic acid-6-phosphate. The coenzyme may be either NADP OT NAD.
BACKGROUND OF THE INVENTION
This invention relates to a novel and useful glucose-6-phosphate dehydrogenase and a process for pr~paration thereof More particularly, the invention relates to a glucose-6-phosphate dehydrogenase that can be used in the determination of glucose, glucose-6-phosphate, hexokinase and hexose-6-phosphate isomerase in clinical tests in the medical field, and the determination of fructose and glucose in the food industry, as well as a process for preparing such a dehydrogenase.
Recently, enzymes, because of their high specificity in type of reaction, substrate ~i.e., material acted upon), and optical characteristics, have begun to be used widely as cata-lysts in medical and food analyses. As is well known, the determination of glucose and hexokinase levels in body fluids constitutes an important parameter in clinical tests. The analysis of glucose and fructose levels in foods is also an impor-tant parameter in the manufacture of invert sugar. Currently, glucose-6-phosphate dehydrogenase is widely used in determining these parameters, by virtue of its very high specificity in reaction, substrate andoptical characteristics, and hence is one Of the more important enzymes in the state of art.
While microanalysis using enzymes has the above mentioned advantages, enzymes are very labile, and lose their catalytic activity in relatively short periods of from a few .
,.
1 lsss7n days to several weeks, even when maintained at lower than room temperature. Thus, the lability of enzymes constitutes a serious bar to microanalysis using enzymes. Glucose-6-phosphate dehydrogenase preparations known to date are also labile, and yeast-derived glucose-6-phosphate dehydrogenase, described in the Journal of_Biological Chemistry, Vol. 236, p. 1225, (1961) generally loses most of its activity within one to three weeks in aqueous solution at room temperature.
High cost is another reason that has prevented large-scale application-of glucose-6-phosphate dehydrogenase to microanalyses in the fields of medical and food analyses. The reaction of glucose-6-phosphate dehydrogenase always requires a coenzyme, and most of the conventional analyses using glucose-6-phosphate dehydrogenases require the use of nicotinamide adenine dinucleotide phosphate (hereunder referred to as NADP).
As is well known, NADP is very expensive. Many glucose-6-phosphate dehydrogenase preparations are known, as described, e.g., in Advances in Enzymology, ed. by Alton Meister, Vol. 48, pp. 97-191, and most of them require NADP as a coenzyme in an enzymatic reaction, with the exception of those which are derived from Leuconostoc mesenteroides and Pseudomonas W 6, which are compatible for use with nicotinamide adenine dinucleotide (here-inafter NAD) that is one tenth or less the cost of NADP.
However, the glucose-6-phosphate dehydrogenases produced from Leuconostoc mesenteroides and Pseudomonas W 6 are not heat-stable B~7n and do not keep long, i.e., the inactivation of the enzyme is observed when the enzyme is allowed to stand for 1 to 2 weeks at a room temperature. Therefore, to maximize the advantages of analyses using a glucose-6-phosphate dehydrogenase, a glucose-6-phosphate dehydrogenase which is stable and compatible with inexpensive coenzymes (i.e., other than the expensive NADP) has been the subject of much research.
SUMMARY OF THE INVENTION
An object of this invention is to provide a glucose-6-phosphate dehydrogenase which is heat-stable, does not lose its activity for an extended period of time, and which is com-patible with an inexpensive coenzyme ~i.e., other than the expensive NADP).
As a result of various efforts to achieve this object, the inventors of this invention have found that a microorganism of the genus Bacillus produces a glucose-6-phosphate dehydrogenase that has the above described properties.
Therefore, this lnvention provides a glucose-6-phosphate dehydrogenase which, when incubated for about 15 minutes in a buf-fer solution at about 50C, retains at least 80 % of its i,~tial activity, as well as a process for producing such glucose-6-phosphate dehydrogenase by culturing a microorganism of the genus Bacillus and recovering from the culture a glucose-6-phosphate dehydrogenase which, when incubated for about 15 minutes with a buffer solution at about 50C, retains at least 80 % of its initial activity.
The glucose-6-phosphate dehydrogenase of this invention is very stable against heat, so after isolation, it can be stored 1 l5B~n for a longer period than the conventional glucose-6-phosphate dehydrogenase. The enzyme is very effective for use }n biochem-ical research, the food industry, and for applications in clinical test. In addition, the enzyme is compatible with NAD, which is much less expensive than NADP, and so it can be used with advantage as an inexpensive reagent. -- BRIEF DESCRIPTION OF THE DRAWIN~
FIG. l is a graph showing the residual activities of the glucose-6-phosphate dehydrogenase of this invention (curve A) and yeast-derived glucose-6-phosphate dehydrogenase (curve B) after heating at various temperatures for 15 minutes; and FIG. 2 is a graph showing the residual activities of the glucose-6-phosphate dehydrogenase of this invention (curve C) and yeast-derived glucose-6-phosphate dehydrogenase (curve D) after storage at 30C.
DETAILED DESCRIPTION OF THE INVENTION
The glucose-6-phosphate dehydrogenase of this invention, when incubated for about 15 minutes in a buffer solution at about 50C, retains at least about 80 %, preferably at least about 90 %, and more preferably about 100 %, of its initial activity. In par-ticular, the dehydrogenase of this invention retains at least about 80 % of its initial activity when it is treated for about 15 minutes with a buffer solution at about 57C. The concentration and pH of the buffer solution are not limited to a specific value, but generally, the concentration is in the range of rrom 5 to 500 millimole/Q (hereinafter, referred to as "mM"), and the pH is from 7 to 10.5. For the purpose of this invention, 100 mM of tris-HCl buffer solution ~pH: about 9.0) containg_l00 mM of potassium chloride is used with advantage.
The physicochemical properties of the glucose-6-phosphate dehydrogenase of this invention are set forth below.
1. Function of enzyme The glucose-6-phosphate dehydrogenase of this invension catalyzes the following reactions:
Hexose-6-phosphate + coenzyme ~oxidized form) Hexose aldonic acid-6-phosphate + coenzyme (reduced form) "Hexose-6-phosphate" is a generic term for glucose-6-phosphate, mannose-6-phosphate and galactose-6-phosphate, and "hexose -aldonic acid-6-phosphate" is a generic-term for gluconic acid-6-phosphate,-mannonic acid-6-phosphate and galactonic acid-6-phosphate. The coenzyme may be either NADP OT NAD.
2. Substrate specificity The Michaelis constant ~Km value) of the enzyme for glucose-6-phosphate is about 0.16 mM. The reaction rates for mannose-6-phosphate and galactose-6-phosphate at a given substrate concentration are about 40 ~ and 20 ~ of that for glucose-6-phosphate. The Km values for NADP and NAD are about 0.016 and 1.64 mM, respectively.
3. Optimum pH
About 9.0 (at 30C)
About 9.0 (at 30C)
4. Stable pH range Little deactivation of the enzyme OCCUTS if it is incubated at a pH of 7.0 to 10.5 and 4C for 24 hours.
- 1 15B5~()
- 1 15B5~()
5. Optimal Functioning t~mperature range The optimal functioning temperature ranges for reaction are from 25C to 75C. The activity incTeases at a pH of 8.9 as the temperature incre~ses from 25C to 75C.
6. Heat resistance The enzyme is stable against heating~at 57aC for at least 15 minutes.
7. ~lolecular weight Gel filtration chromatography on Sephacry~ S-200 (product of Pharmacia Fine Chemical) showed that the enzyme has- a molecular weight of about ~30,000. Yeast- and Leuconostoc mesenteroides-derived glucose-6-phosphate dehydrogenases were found to have a molecular weight o~ about 100,000 upon gel filtration chromatography on Sephacryl S-200.
8. DeteTmination and definition o~ activity A mixture of 2mM glucose-6-phosphate, 0.5 m~l NADP
and 5 mM magnesium chloride in 50 m~l tris-HCQ buffer having a pH of 8.9 was prepared. A suitable amount of glucose-6-phosphate dehydrogenase was added to the mixLure, and the increase in absorption of reduced form NADP (~DP Hj at 340 nm for a given period of time was measured. Assu~ing the enzymatic activity that increased the absorption of 1 micromol of NADP H at 340 nm per minute to be one unit, the purified enzyme had a potency of about 100 units/mg at 30C.
*Trade Mark - 6 -
and 5 mM magnesium chloride in 50 m~l tris-HCQ buffer having a pH of 8.9 was prepared. A suitable amount of glucose-6-phosphate dehydrogenase was added to the mixLure, and the increase in absorption of reduced form NADP (~DP Hj at 340 nm for a given period of time was measured. Assu~ing the enzymatic activity that increased the absorption of 1 micromol of NADP H at 340 nm per minute to be one unit, the purified enzyme had a potency of about 100 units/mg at 30C.
*Trade Mark - 6 -
9. Purity Upon 7.5 % acrylamide disc electrophoresis at a pH
of 9.4, a purified sample of the enzyme migrated to a positive electrode and gave a single band. In SDS electrophoresis, the sample also migrated to a positive electrode and gave a single band.
of 9.4, a purified sample of the enzyme migrated to a positive electrode and gave a single band. In SDS electrophoresis, the sample also migrated to a positive electrode and gave a single band.
10. Compositional analysis The proportions of amino acids in the enzyme are shown below in terms of mol%.
11.04 % aspartic acid, 5.42 % threonine, 4.56 % serine, 11.86 % glutamic acid, 3.66 % proline, 6.67 % glycine, 7.92 %
alanine, 0.62 % cystine (cysteine), 6.09 % valine, 1.97 %
methionine, 5.59 % isoleucine, 8.04 % leucine, 3.72 % tyrosine, 4.88 % phenylalanine, 5.28 % lysine, 3.40 % his~idine, 6.56 %
arginine, and 2.72 % tryptophan.
11. Crystalline structure The crystalline structure of the enzyme is yet to be determined because it has not yet been crystallized.
The glucose-6-phosphate dehydrogenase of this i.~ntion can be produced by recovering it from a culture of a microorganism of the genus Bacillus. The microorganism used in the production of the glucose-6-phosphate dehydrogenase of this invention is any of the microorganisms that belong to the genus Bacillus and which are capable of producing said dehydrogenase. Preferred Bacillus microorganisms is Bacillus stearothermophilus, examples of which are ATCC 7953, 7954, 8005, 10149, 12980 and NCA 1503.
. , ;, .
, 115~570 Various nutrients can be used in culturing a micro-organism of the genus Bacillus in this invention: carbon sources include saccharides such as glucose, sucrose, fructose, starch hydrolyzate, molasses and sulfite pulp waste liquor, organic acids such as acetic acid and lactic acid, alcohols that can be utilized by the microorganism used, such as ethyl alcohol and butyl alcohol, fats and oils, aliphatic acids and glycerin;
nitrogen sources include inorganic and organic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, amino acid, peptone, meat extract and yeast extract;
inorganic salts include potassium, sodium, phosphoric acid, zinc, iron, magnesium, manganese, copper, calcium, and cobalt salts; optional nutrients include trace metal salts, corn steep liquor, vitamins and nucleic acids. Generally known nutrient media for the cultivation of microorganisms can be used.
A microorganism of the genus Bacillus is cultivated aerobically on media as described above for from about 2 to 6 hours at a temperature from about 20 to 80C, preferably from about 40 and 70C, and more preferably at about 60C. For industrial operation, it is preferred to perform continuous cultivation with the dilution rate being controlled to be greater than 90 % of the maximum specific growth rate of a microorganism cultured under continuous cultivation conditions (~max), expressed in terms of the rate of dilution thereof in l/hr, of the microorganism used. Continuous cultivation at .
11~857/) g a dilution rate controlled to be greater than 90 % of the ~max of the microorganism produces more cells than can be produced by batchwise process, and the cells contains more glucose-6-phosphate dehydrogenase than the maximum achieved in batchwise process per unit number of cells. In particular, continuous cultivation at a dilution rate maintained close to ~max produces about 1.3 times as much glucose-6-phosphate dehydrogenase as in batchwise prosess. If continuous cultivation is performed at a dilution rate lower than 0.9 ~max, the glucose-6-phosphate dehydrogenase level in the cell tends to be lower than in a batchwise process.
The dilution rate (hereunder referred to as D) as used in this invention is represented by the following formula ~
lS -- D = V ~I) wherein D: dilution rate ~l/Hr) F: rate (QtHr) at which fermentation liquor is supplied to and withdrawn from the fermentor V: volume (Q) of fermentation liquor in the ~- fermentor The symbol "~max" as used in this invention is a maximum specific growth rate (l/Hr) of a microorganism cultivated under continuous cultivation conditions, and is a specificgrowth rate observed at "washout" time, the time when the microorganism being cultivated in a chemostat (see Herbert, Elsworth and Telling, Journal of General Microbiology, Vol. 14, No. 8, pp.
601-622, 1956) no longer maintains a stationary cell concen-tration as a result of an increase in D. The ~max of a thermo-in this philic microorganism used/invention can be determined as follaws:
1.5 to 20 liters of a nutrient medium in a 2-30 liter fermentor is inoculated with ~he microorganism for batchwise cultivation at from 40C to 75C and preferably from 48C to 61C, at a pH
between 4.5 and 9.0 and preferably between 6.0 and 8.0; when the growth of the microorganism reduces the content of carbon source in the broth to less than 0.01 wt~, a nutrient medium having the same composition as that charged into the fermentor initially is supplied to the fermentor to start continuous cultivation wherein the only growth inhibiting factor is a carbon source. In this way, a chemostat is established. After the continuous cultivation reaches a stationary phase, D is increased stepwise by measuring the cell concentra*ion in the fermentation liquor and the residual carbon source at given time intervals, and when D exceeds the specific growth rate of the microortanism, the stable cell concentration begins to decrease whereas the carbon source content begins to increase.
The phenomenon wherein an increasing D causes the continuous cultivation to come out of the stationary phase is referred to as "washout" and the specific growth rate at the washout time is ~max. The ~max varies for a given organism depending upon the type of nutrient m-edium and cultivation conditions, but for 11~8~0 a given combination of microoganism, nutrient medium and cultivation conditions, the ~max is constant, and hence provides a reliable value for operation over an extended period once it is measured.
According to this invention, D is limited to a pre-determined value in continuous cultivation that is perfoTmed after a desired cell concentration is achieved in a conventional pre-cultivation and batchwise cultivation. TransfeT to con-tinuous cultivation may be effected at any time of the batchwise cultivation, but prefeTably, the cultivation is transferTed for continuous cultivation in the last stage of the logarithmic growth phase of the batchwise cultivaition and D should be fixed at a predeteTmined level as soon as possible.
One embodiment of the process of this invention is hereundeT described by refeTence to the cultivation of Bacillus sterotheTmophilus NCA 1503 on a nutrient medium having glucose as a carbon source. When chemostatic continuous cultivation was performed in a 30-liter fermentor (charged with 20 liters of the medium) at an optimum temperatuTe of about 57C and an o~imum pH of about 6.8, the ~max of the miCTOOTganiSm was 1.1 (l/Hr). Therefore, to perform continuous cultivation at a D
equal to ~max, a fresh nutrient medium of the same formulation as used in the batchwise cultivation must be continously supplied to and withdrawn from the fermentor in an amount 1.1 times per hour as much as the volume initially charged in the fermentor, 115~57~
or at a rate of 22 liters per hour as calculated from the formula (I). Such supply and ~ithdrawal can be achieved by a meteTing pump.
The glucose-6-phosphate dehydrogenase of this invention is then recovered from the culture. The enzyme may be recovered' as separated live cells, treated live cells, a crude enzyme, or a purified enzyme. For purification, the conventional technique of enzyme purification can be employed; after centrifugation or other sui~able'means, the cells are homogenized with a Manton Gaulin* (Homogenizer), Dyno-mil~ ~Homogenizer), French press or supersonic waves, and by centrifugation, the cell membranes are' remo~ed to provide a cell extract which is then treated'with 'sulfate streptomycin or sulfate protamine, optionally followed by precipitation with ammonia sulfate, acetone or heat treatment.
If-further purification is necessary, these purification tech- ~-niques may be combined with ion exchange chromatography on a DEAE-cellulose column, adsorption chromatography on hydro-xyapatite or gel permeation chromatography on Sephadex*. This way, the glucose-6-phosphate dehydrogenase of this invention can be separated from the culture and purified.
~ The glucose-6-phosphate dehydrogenase of this invention is very stable against heat, so after isolation, it can be stored for a longer period than the existing glucose-6-phsphate dehydrogenase preparations. Therefore, the dehydrogenase o~ this invention is used with particular *Trade Marks - 12 -B
115~5~0- 13 -advantage for biochemical research, the food industry; and forapplications in clinical tests. For instance, the glucose level in f~ods or body fluid can be measured with high accuracy using a reaction liquor consisting of the glucose-6-phosphate dehydrogenase of this invention~ hexokinase, NAD, ATP and - magnesium chloride. The glucose level in foods is an important parameter for operation analysis in the manufacture of invert sugar, and the glucose level in the body fluid is used in diag-nosis of diabetes. In another application, the activity of phosphoglucose isomerase can be determined by measuring the glucose-6-phosphate level in the body fluid with a reaction liquor consisting of the glucose-6-phosphate dehydrogenase of this invention, NAD and magnesium chloride. That activity is used as a parameter for examination of cancers such as lS metastatic cancer of the breast.
The glucose-6-phosphate dehydrogenase of this invention acts as a co-enzyme with NAD,:which is a much less ~expensive coenzyme than NADP, so that the coenzymes can be used with advantage as an inexpensive reagent.
_ The invention is now described in greater detail by reference to the following examples, comparative example ~ .
and reference examples which are given here for illustrative purposes only, and are not intended to limit the scope of the invention.
, .. .
115~5~) Example l and Comparative Example 1 A medium ~250 liters) containing 0.5 g/dl (deci liter) polypeptone, 0.5 g/dl of yeast extract, 1.0 g/dl of saccharose, 0.13 g/dl of potassium sulfate, 0.644 g/dl of disodium phosphate, 0.027 g/dl of magnesium sulfate, 0.032 g/dl of citric acid, 0.0007 g/dl of ferrous sulfate and 0.015 g/dl of manganese sulfate was adjusted to a pH of 7.0 and sterilized with heat at 115C for 10 minutes. Thereafter, the medium was inoculated with a strain of Bacillus stearothermophilus NCA 1503 and aeration cultivation was performed at 60C for 3 hours at an internal pressure of 0.5 kg/m2G tgauge).
Immediately after the cultivation, 700 g of the cells were recovered with a De Laval centrifuge while cooling with water. The cells were frozen and 300 g of the frozen sample was suspended in 600 g of a 0.1 M phosphate buffer ~pH: 7.5) and after thorough homogenization with a French press, the cell membranes were removed by centrifugation to provide a crude extract containing glucose-6-phosphate dehydrogenase. To 600 mQ
of the crude extract, 300 mQ of 1 % aqueous sulfate protamine was added, and the mixture was thoroughly stirred. By removing the resulting precipitate with centrifugation, protamine super-natant was obtained. Solid ammonium sulfate was gradually added to the supernatant until it was 50 % saturated at 4C.
The resultant precipitate was collected with centrifugation and dissolved in 0.1 M phosphate buffer ~pH: 7.5). The solution 115B57() ~as desalted by dialysis against 20-fold 0.1 M phosphate buffer ~pH: 7.5).
The liquid crude enzyme thus obtained was passed through a DEAE-cellulose column equilibrated with 20 mM
phosphate buf-fer (p~: 7.5) containing 2 mM mercaptoethanol and 2 mM sodium ethylenediaminetetraacetate. Upon elution with a solution comprising potassium chloride in a phosphate buffer of ~he same formulation as above, the desired glucose-6-phosphate dehydrogenase was obtained at a KCl concentration close to 0.15 M. The active fractions were combined, concen-tTated, desalted and passed through a hydroxyapatite column equilibrated with 5 mM phosphate buffer ~pH: 7.5) and eluted -with a linear gradient star~ing from 5 m~l phosphate buffer and ending with 250 mM phosphate buffer. The sesired glucose-6-phosphate dehydrogenase was obtained at a buffer concentration close to 75 mM. The active fractions were combined, concentrated, desaited and sùbjected to Ultrogel ACA 34 chromatograph (product of LKB-Produkter A.B., Sweden) using an eluting agent comprised of 50 mM tris-HCl buffer containing 0.1 M potassium chloride.
~ resulting active fractions were passed through a DEAE-Sephadex~A-50 column equilibrated with 30 mM phosphate buffer (pH: 7.7) containing 2 mM mercaptoethanol and 2 mM sodium ethylenediaminetetraacetate, and eluted with a linear gradient from 0.1 to 0.4 M of potassium chloride in a buffer of the same formulation as used above. As a results, the glucose-6-phosphate dehydrogenase purified at a potassium chloride concentration close to 0.2 M was obtained.
*Trade ~5arks - 15 -. ~'j -1 156~7~) The glucose-6-phosphate dehydrogenase so obtained migrated to a positive electrode in a disc electrophoresis at pH of 9.4 using 7.5 % acrylamide and gave a single band.
It also gave a single peak for a molecular weight of about 230,000 in Sephadex G-200 chromatography.
The yield of *he enzyme was about 10 mg and it had a potency of about 100 units/mg. The degree of purification of the enzyme was about 1,500 in comparison with the crude extract which was assumed to be 1.
The glucose-6-phosphate dehydrogenase of this invention was compared for stability with a yeast-derived glucose-6-phosphate dehydrogenase prepared in Comparative Example 1. The results are shown in FIGS~ 1 and 2. FIG.
1 shows the residual activities of the two enzymes after heating for 15 minutes at various temperatures in 100 mM
Tris-buffer (pH: 9.0~ containing 100 mM of potassium chloride. In the figure, curve A represents the glucose-6-phosphate dehydrogenase of this invention, and curve B
represents the yeast-derived glucose-6-phosphate dehydro-genase. FIG. 2 shows a time-dependent change in the residual actlvities of the two enzymes when they were stored at 30bC in 100 mM Tris-buffer (pH: 9.0). In the figure, curve C represents the glucose-6-phosphate de-hydrogenase of this invention, and curve D represents n yeast-derived glucose-6-phosphate dehydrogenase. As is clear from the two figures, almost all activity of the yeast-derived glucose-6-phosphate dehydrogenase was lost irreversibly upon heat treatment at 50C for 15 minutes, whereas the glucose-6-phosphate de~ydrogenase of this invention did not lose its activity at all upon treatment at 50C. Upon treatment at 30C, the yeast-drived glucose-6-phosphate dehydrogenase substantially lost its activity -in 10 to 20 days, but the glucose-6-phosphate dehydro-genase of this invention experienced no decrease in activity even after 50-day storage. Therefore, the glucose-6-phosphate dehydrogenase of this invention~is surprisingly stable against heat, indicating its long keeping quality. This quality is entirely absent from the previous-ly known glucose-6-phosphate dehydrogenase preparations.
EXAMPLES 2 to 5 Microorganism used: Bacillus stearothermophilus NCA 1503 Formulation of nutrient medium: A medium of the following formulation that used glucose as a carbon source was prepared by dissolving in one liter of tap water.
glucose 1.3 g, yeast ext~act (product of Oriental Yeast Co., Ltd.) 1.0 g, peptone (product of Difico) 0.5 g, KH2PO4 0.5 g, Na2HPO4 12H2O 0-5 g, MgSO4 7H2O 0.1 g, ZnSO4-7H2O 0.01 g, MnSO4-7H2O 0.01 g, CuSO4 5H2O 0.01 g, CoCQ2 6H2O 0.01 g Germination stage: 20 mQ of the above nutrient medium was put in a 100-mQ conical flask, and 100 mQ of the same nutrient was put in a 500-mQ conical flask, and after stoppering each flask with a cotton plug, the media were sterilized with pressurized steam for 10 minutes at 121C
and l kg/cm2. After cooling, the medium in the 100-mQ
flask was aseptically inoculated with about 5 mg of a freeze-dried sample of Bacillus stearothermophilus NCA
1503 obtained from the American Type Culture Collection.
When the medium was subjected to rotary shake cultivation (160 rpm) for 24 hours at 55C with a rotary shaker ~product of Takasaki Seisakusho Co., Ltd.), the micro-organism grew and the degree of turbidity increased to such a level that the absorption at 660 nm (as measured with Model 101 spectrophotometer of Hitachi Ltd. and to be hereunder referred to as OD660 nm) was 0.8 to 1Ø
About 5 mQ of the germinated microorganism tinoculum) was transplanted on the medium in the 500-mQ flask, and the flask was subjected to rotary shake cultiration for a few hours under the same conditions as used above. l~hen the OD660 nm reached about 1.0, the cultivation was terminated Fermentation stage: A 30-liter fermentor was charged with 20 liters of a nutrient medium of the same formulation as used in the germination stage, and sterilization was performed at 121C and 1 kg/cm2 for 15 minutes. To the 115~70 medium, about one liter of the inoculum was transferred and it was subjected to batchwise cultivation at 55 + 1C, pH of 6.5 to 7.0 ~adjusted with 4 N NaOH) with air supplied at 20 liters/min at 900 rpm. Since the cultivation was accompanied by foaming, a small amount of defoaming agent (KM-70 of Shinetsu Chemical Industry Co., Ltd.) was added.
About 2.5 hours after the start of cultivation, the OD660 nm reached 1.2 (0.56 g of dry cell per liter) and the glucose level in the fermentation liquor became less than 0.01 wt%, so continuous fermentation was started immediately.
Since the ~max of Bacillus stearothermophilus NCA 1503 was found to have a ~max of 1.4 ~l/hr), a sterilized nutrient medium of the same formulation as used in the germination stage was supplied to the fermentor at a rate of 28.0 liters/hr and the fermentation liquor was discharged from the fermentor at the same rate. In this way the ~max was held at 1.00 (in Example 2) while continuous cultivation was performed using a nutrient medium five times the volume of the fermentation liquor in the fermentor Continuous cultivation was performed in the same q~anner as above except that D was changed to 0.9 ~max (medium supplied and fermentation liquor withdrawn at - 25.2 liters/hr) in Example 3 and to 0.75 ~max (supply and withdrawal rate: 21.0 liters/hr) in Example 4.
Thé level of glucose-6-phosphate dehydrogenase in the cells obtained in Examples 2, 3 and 4 was measured 11~6$70 and the results are shown in Table 1 below. The Table lalsoindicates glucose-6-phosphate dehydrogenase leve'l in cells produced by the batchwise processing (in Example 5) that was performed before transfer to the continuous cultivation.
Table Glucose-6-phosphate Yield of Yield of glucose-dehydrogenase level cell phosphate de-Run No. (U/g of dry cell) (g of dry hydrogenase(U/Q/hr) cell/Q/hr) Example 2 98 0.65 63.7 Example 3 71 0.59 41.9 Example 4 65 0.54 35.1 Example 5 69 0.23 15.9 - As is clear from the table, the cells produced ,by continuous cultivation with the D held at higher than 0.9 ~max gave a,glucose-6-phosphate dehydrogenase level higher than that obtained in batchwise processing.
,Example '6 A 30-liter fermentor was charged with 20 liters of a medium prepared by dissolving in one liter of tap water a mixture of 1.3 g of glucose, 1.0 g of ammonium sulfate, 0.5 g of yeast extract, 0.5 g of monopotassium phosphate, 0.5 g of dissodium phosphate and 0.1 g of magnesium sulfate, and the medium was sterilized with pressurized steam at 121C and 1 kg/cm2 for 15 minutes. Onelliter of a liquid ' inoculum of Bacillus stearothermophilus ATCC 12980 germinated 11565~() on a nutrient medium of the same formulation as defined above and the absorption of which at 660 nm reached about 1.0 was transferred onto the sterilized medium and cultured at 57C and a pH between 6.5 and 7.0 (adjusted with 4N
NaOH) and with air supplied at a rate of 20 liters/min at 900 rpm. When the absorption at 660 nm reached 1.0 by a batch cultivation for about 2.5 hours, a sterilized nutrient medium of the same formulation as indicated above was - supplied continuously with a metering pump at a rate of 24.0~liters/hr and the fermentation liquor was withdrawn from the fermentor at the same rate with the same machine.
A total of 100 liters of nutrient medium was used in the continuous cultivation. Immediately after the fermentation, a De Laval centrifuge was used to recover 400 g of the cell.
The cells were suspended in 1.5-fold 0.1 M phosphate ,(Homogeni~er) buffer and homogenized with a Dyno-mill' By removing the soluble matter with a centrifuge, a crude extract contain-ing~glucose-6-phosphate dehydrogenase was obtained. To , 400 mQ of the extract, 200 mQ of 10 ~ aqueous streptomycin 20 ~ sulfate and the resulting precipitate was removed with a centrifuge to give a streptomycin supernatant. The super--~ ~ natant was treated with ammonium sulfate and fractions for 25 % saturation (4C) thru 50 % saturation ~4C) were obtained. The fractions were dissolved in 50 mM tris-HCQ
. ~ z5 buffer (pH: 8.0) and the solution was passed through a DEAE- ephadex column equilibrated with a buffer of the same formulation as indicated above and was eluted with a bufer of the same formulation except that it contained sodium chloride. The desired glucose-6-phosphate dehydro-genase ~as obtained at a NaCQ concentration close to 0.2 M.
The active fraction was subjected to hydroxyapatite column chromatography under the same conditions as employed in Example 1. The active fraction obtained was passed through a Sepharcyl* S-200 column and elutèd with 30 m~l tris-HCQ bufer (pH: 8.0) containina 0.1 M sodium chloride.
A glucose-6-phosphate dehydrogenase sample resulted that gave a single band in a disc electrophoresis with acryl-amide as in Example 1. The enzyme gave a singe peak indlcat-- ing an average molecular weight of about 230,000 in Sephade~ G-200 chromatography as in Example 1.
The yield of the enzyme was about 20 mg and it had a potency of about 100 units/mg. The degree of purification of the enzyme was about 1100 in comparison . with the crude extract which was assumed to be 1.
Reference Examples 1, 2 and 3 The glucose-6-phosphate dehydrogenase prepared in Example 1 was used to determine the glucose level o standard sera which were already kno-~n to contain 76 mg/dl (Ref. Ex. 1), 155 mg/dl ~Ref. Ex. 2) and 43 mg/dl ~Ref.
Ex. 3), of glucose.
.
*Trade Marks - 22 -7 () Procedure A reaction liquor was prepared by dissolving 1 unit/mQ of glucose-6-phosphate dehydrogenase, 2 units/mQ
of hexokinase, 2 mM NAD, Z mM ATP, and 2 mM MgCQ2 in 1 mQ
of 100 mM phosphate buffer ~pH: 8.5). After standing at 30C for 5 minutes, the reaction liquor was mixed with 20 ~Q of each standard serum. Following a reaction at 30C for 5 minutes, the absorption at 340 nm was determined on a spectrophotometer. As a control, 20 ~Q of pure water was added to a reaction liquor of the same formulation as defined above, and following a reaction at 30C for 5 minutes, the absorption at 340 nm was determined The absorption of the control was subtracted from the adsorption of each standard serum to determine the increase in adsorption.
The glucose level (mg) in 1 dQ of each standard serum ; was calculated by the following equation:
; 145.8 x ~increase in absorption) = glucose level (mg) in 1 dQ of sample The results are shown in Table 2 below.
Table 2 Run No. Measurements Ref. Ex. 1 79 mg/dQ
Ref. Ex. 2 151 mg/dQ
Ref. Ex. 3 43 mg/dQ
115857~) 1 Fxom the results of Table 2, it can be seen that since the known glucose concentrations in sample are very consistent with the measured glucose concentrations, respectively, it is possible to measure the glucose concentration by the above described procedure.
The following table identifies enzymes referred to in this disclosure by name with a corresponding enzyme number in accordance with the numbering scheme of Florkin, M. ~ Stotz, E.H. "Comprehensive Biochemistry", Volume 13, 3rd edition, Elsevier Pub. Co. New York ~1973). The numberings of enzymes are as follows:
Enzyme Enzyme No.
Glucose 6- phosphate dehydrogenase E.C. 1.1.1.49 Hexokinase E.C. 2.7.1.1 Hexose-6-phosphate isomeraseE.C. 5.3.1.-Phosphoglucose isomeraseE.C. 5.3.1.9 While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the sprit and scope thereof.
-~4-. ~ .
~ . ~,
alanine, 0.62 % cystine (cysteine), 6.09 % valine, 1.97 %
methionine, 5.59 % isoleucine, 8.04 % leucine, 3.72 % tyrosine, 4.88 % phenylalanine, 5.28 % lysine, 3.40 % his~idine, 6.56 %
arginine, and 2.72 % tryptophan.
11. Crystalline structure The crystalline structure of the enzyme is yet to be determined because it has not yet been crystallized.
The glucose-6-phosphate dehydrogenase of this i.~ntion can be produced by recovering it from a culture of a microorganism of the genus Bacillus. The microorganism used in the production of the glucose-6-phosphate dehydrogenase of this invention is any of the microorganisms that belong to the genus Bacillus and which are capable of producing said dehydrogenase. Preferred Bacillus microorganisms is Bacillus stearothermophilus, examples of which are ATCC 7953, 7954, 8005, 10149, 12980 and NCA 1503.
. , ;, .
, 115~570 Various nutrients can be used in culturing a micro-organism of the genus Bacillus in this invention: carbon sources include saccharides such as glucose, sucrose, fructose, starch hydrolyzate, molasses and sulfite pulp waste liquor, organic acids such as acetic acid and lactic acid, alcohols that can be utilized by the microorganism used, such as ethyl alcohol and butyl alcohol, fats and oils, aliphatic acids and glycerin;
nitrogen sources include inorganic and organic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonia, amino acid, peptone, meat extract and yeast extract;
inorganic salts include potassium, sodium, phosphoric acid, zinc, iron, magnesium, manganese, copper, calcium, and cobalt salts; optional nutrients include trace metal salts, corn steep liquor, vitamins and nucleic acids. Generally known nutrient media for the cultivation of microorganisms can be used.
A microorganism of the genus Bacillus is cultivated aerobically on media as described above for from about 2 to 6 hours at a temperature from about 20 to 80C, preferably from about 40 and 70C, and more preferably at about 60C. For industrial operation, it is preferred to perform continuous cultivation with the dilution rate being controlled to be greater than 90 % of the maximum specific growth rate of a microorganism cultured under continuous cultivation conditions (~max), expressed in terms of the rate of dilution thereof in l/hr, of the microorganism used. Continuous cultivation at .
11~857/) g a dilution rate controlled to be greater than 90 % of the ~max of the microorganism produces more cells than can be produced by batchwise process, and the cells contains more glucose-6-phosphate dehydrogenase than the maximum achieved in batchwise process per unit number of cells. In particular, continuous cultivation at a dilution rate maintained close to ~max produces about 1.3 times as much glucose-6-phosphate dehydrogenase as in batchwise prosess. If continuous cultivation is performed at a dilution rate lower than 0.9 ~max, the glucose-6-phosphate dehydrogenase level in the cell tends to be lower than in a batchwise process.
The dilution rate (hereunder referred to as D) as used in this invention is represented by the following formula ~
lS -- D = V ~I) wherein D: dilution rate ~l/Hr) F: rate (QtHr) at which fermentation liquor is supplied to and withdrawn from the fermentor V: volume (Q) of fermentation liquor in the ~- fermentor The symbol "~max" as used in this invention is a maximum specific growth rate (l/Hr) of a microorganism cultivated under continuous cultivation conditions, and is a specificgrowth rate observed at "washout" time, the time when the microorganism being cultivated in a chemostat (see Herbert, Elsworth and Telling, Journal of General Microbiology, Vol. 14, No. 8, pp.
601-622, 1956) no longer maintains a stationary cell concen-tration as a result of an increase in D. The ~max of a thermo-in this philic microorganism used/invention can be determined as follaws:
1.5 to 20 liters of a nutrient medium in a 2-30 liter fermentor is inoculated with ~he microorganism for batchwise cultivation at from 40C to 75C and preferably from 48C to 61C, at a pH
between 4.5 and 9.0 and preferably between 6.0 and 8.0; when the growth of the microorganism reduces the content of carbon source in the broth to less than 0.01 wt~, a nutrient medium having the same composition as that charged into the fermentor initially is supplied to the fermentor to start continuous cultivation wherein the only growth inhibiting factor is a carbon source. In this way, a chemostat is established. After the continuous cultivation reaches a stationary phase, D is increased stepwise by measuring the cell concentra*ion in the fermentation liquor and the residual carbon source at given time intervals, and when D exceeds the specific growth rate of the microortanism, the stable cell concentration begins to decrease whereas the carbon source content begins to increase.
The phenomenon wherein an increasing D causes the continuous cultivation to come out of the stationary phase is referred to as "washout" and the specific growth rate at the washout time is ~max. The ~max varies for a given organism depending upon the type of nutrient m-edium and cultivation conditions, but for 11~8~0 a given combination of microoganism, nutrient medium and cultivation conditions, the ~max is constant, and hence provides a reliable value for operation over an extended period once it is measured.
According to this invention, D is limited to a pre-determined value in continuous cultivation that is perfoTmed after a desired cell concentration is achieved in a conventional pre-cultivation and batchwise cultivation. TransfeT to con-tinuous cultivation may be effected at any time of the batchwise cultivation, but prefeTably, the cultivation is transferTed for continuous cultivation in the last stage of the logarithmic growth phase of the batchwise cultivaition and D should be fixed at a predeteTmined level as soon as possible.
One embodiment of the process of this invention is hereundeT described by refeTence to the cultivation of Bacillus sterotheTmophilus NCA 1503 on a nutrient medium having glucose as a carbon source. When chemostatic continuous cultivation was performed in a 30-liter fermentor (charged with 20 liters of the medium) at an optimum temperatuTe of about 57C and an o~imum pH of about 6.8, the ~max of the miCTOOTganiSm was 1.1 (l/Hr). Therefore, to perform continuous cultivation at a D
equal to ~max, a fresh nutrient medium of the same formulation as used in the batchwise cultivation must be continously supplied to and withdrawn from the fermentor in an amount 1.1 times per hour as much as the volume initially charged in the fermentor, 115~57~
or at a rate of 22 liters per hour as calculated from the formula (I). Such supply and ~ithdrawal can be achieved by a meteTing pump.
The glucose-6-phosphate dehydrogenase of this invention is then recovered from the culture. The enzyme may be recovered' as separated live cells, treated live cells, a crude enzyme, or a purified enzyme. For purification, the conventional technique of enzyme purification can be employed; after centrifugation or other sui~able'means, the cells are homogenized with a Manton Gaulin* (Homogenizer), Dyno-mil~ ~Homogenizer), French press or supersonic waves, and by centrifugation, the cell membranes are' remo~ed to provide a cell extract which is then treated'with 'sulfate streptomycin or sulfate protamine, optionally followed by precipitation with ammonia sulfate, acetone or heat treatment.
If-further purification is necessary, these purification tech- ~-niques may be combined with ion exchange chromatography on a DEAE-cellulose column, adsorption chromatography on hydro-xyapatite or gel permeation chromatography on Sephadex*. This way, the glucose-6-phosphate dehydrogenase of this invention can be separated from the culture and purified.
~ The glucose-6-phosphate dehydrogenase of this invention is very stable against heat, so after isolation, it can be stored for a longer period than the existing glucose-6-phsphate dehydrogenase preparations. Therefore, the dehydrogenase o~ this invention is used with particular *Trade Marks - 12 -B
115~5~0- 13 -advantage for biochemical research, the food industry; and forapplications in clinical tests. For instance, the glucose level in f~ods or body fluid can be measured with high accuracy using a reaction liquor consisting of the glucose-6-phosphate dehydrogenase of this invention~ hexokinase, NAD, ATP and - magnesium chloride. The glucose level in foods is an important parameter for operation analysis in the manufacture of invert sugar, and the glucose level in the body fluid is used in diag-nosis of diabetes. In another application, the activity of phosphoglucose isomerase can be determined by measuring the glucose-6-phosphate level in the body fluid with a reaction liquor consisting of the glucose-6-phosphate dehydrogenase of this invention, NAD and magnesium chloride. That activity is used as a parameter for examination of cancers such as lS metastatic cancer of the breast.
The glucose-6-phosphate dehydrogenase of this invention acts as a co-enzyme with NAD,:which is a much less ~expensive coenzyme than NADP, so that the coenzymes can be used with advantage as an inexpensive reagent.
_ The invention is now described in greater detail by reference to the following examples, comparative example ~ .
and reference examples which are given here for illustrative purposes only, and are not intended to limit the scope of the invention.
, .. .
115~5~) Example l and Comparative Example 1 A medium ~250 liters) containing 0.5 g/dl (deci liter) polypeptone, 0.5 g/dl of yeast extract, 1.0 g/dl of saccharose, 0.13 g/dl of potassium sulfate, 0.644 g/dl of disodium phosphate, 0.027 g/dl of magnesium sulfate, 0.032 g/dl of citric acid, 0.0007 g/dl of ferrous sulfate and 0.015 g/dl of manganese sulfate was adjusted to a pH of 7.0 and sterilized with heat at 115C for 10 minutes. Thereafter, the medium was inoculated with a strain of Bacillus stearothermophilus NCA 1503 and aeration cultivation was performed at 60C for 3 hours at an internal pressure of 0.5 kg/m2G tgauge).
Immediately after the cultivation, 700 g of the cells were recovered with a De Laval centrifuge while cooling with water. The cells were frozen and 300 g of the frozen sample was suspended in 600 g of a 0.1 M phosphate buffer ~pH: 7.5) and after thorough homogenization with a French press, the cell membranes were removed by centrifugation to provide a crude extract containing glucose-6-phosphate dehydrogenase. To 600 mQ
of the crude extract, 300 mQ of 1 % aqueous sulfate protamine was added, and the mixture was thoroughly stirred. By removing the resulting precipitate with centrifugation, protamine super-natant was obtained. Solid ammonium sulfate was gradually added to the supernatant until it was 50 % saturated at 4C.
The resultant precipitate was collected with centrifugation and dissolved in 0.1 M phosphate buffer ~pH: 7.5). The solution 115B57() ~as desalted by dialysis against 20-fold 0.1 M phosphate buffer ~pH: 7.5).
The liquid crude enzyme thus obtained was passed through a DEAE-cellulose column equilibrated with 20 mM
phosphate buf-fer (p~: 7.5) containing 2 mM mercaptoethanol and 2 mM sodium ethylenediaminetetraacetate. Upon elution with a solution comprising potassium chloride in a phosphate buffer of ~he same formulation as above, the desired glucose-6-phosphate dehydrogenase was obtained at a KCl concentration close to 0.15 M. The active fractions were combined, concen-tTated, desalted and passed through a hydroxyapatite column equilibrated with 5 mM phosphate buffer ~pH: 7.5) and eluted -with a linear gradient star~ing from 5 m~l phosphate buffer and ending with 250 mM phosphate buffer. The sesired glucose-6-phosphate dehydrogenase was obtained at a buffer concentration close to 75 mM. The active fractions were combined, concentrated, desaited and sùbjected to Ultrogel ACA 34 chromatograph (product of LKB-Produkter A.B., Sweden) using an eluting agent comprised of 50 mM tris-HCl buffer containing 0.1 M potassium chloride.
~ resulting active fractions were passed through a DEAE-Sephadex~A-50 column equilibrated with 30 mM phosphate buffer (pH: 7.7) containing 2 mM mercaptoethanol and 2 mM sodium ethylenediaminetetraacetate, and eluted with a linear gradient from 0.1 to 0.4 M of potassium chloride in a buffer of the same formulation as used above. As a results, the glucose-6-phosphate dehydrogenase purified at a potassium chloride concentration close to 0.2 M was obtained.
*Trade ~5arks - 15 -. ~'j -1 156~7~) The glucose-6-phosphate dehydrogenase so obtained migrated to a positive electrode in a disc electrophoresis at pH of 9.4 using 7.5 % acrylamide and gave a single band.
It also gave a single peak for a molecular weight of about 230,000 in Sephadex G-200 chromatography.
The yield of *he enzyme was about 10 mg and it had a potency of about 100 units/mg. The degree of purification of the enzyme was about 1,500 in comparison with the crude extract which was assumed to be 1.
The glucose-6-phosphate dehydrogenase of this invention was compared for stability with a yeast-derived glucose-6-phosphate dehydrogenase prepared in Comparative Example 1. The results are shown in FIGS~ 1 and 2. FIG.
1 shows the residual activities of the two enzymes after heating for 15 minutes at various temperatures in 100 mM
Tris-buffer (pH: 9.0~ containing 100 mM of potassium chloride. In the figure, curve A represents the glucose-6-phosphate dehydrogenase of this invention, and curve B
represents the yeast-derived glucose-6-phosphate dehydro-genase. FIG. 2 shows a time-dependent change in the residual actlvities of the two enzymes when they were stored at 30bC in 100 mM Tris-buffer (pH: 9.0). In the figure, curve C represents the glucose-6-phosphate de-hydrogenase of this invention, and curve D represents n yeast-derived glucose-6-phosphate dehydrogenase. As is clear from the two figures, almost all activity of the yeast-derived glucose-6-phosphate dehydrogenase was lost irreversibly upon heat treatment at 50C for 15 minutes, whereas the glucose-6-phosphate de~ydrogenase of this invention did not lose its activity at all upon treatment at 50C. Upon treatment at 30C, the yeast-drived glucose-6-phosphate dehydrogenase substantially lost its activity -in 10 to 20 days, but the glucose-6-phosphate dehydro-genase of this invention experienced no decrease in activity even after 50-day storage. Therefore, the glucose-6-phosphate dehydrogenase of this invention~is surprisingly stable against heat, indicating its long keeping quality. This quality is entirely absent from the previous-ly known glucose-6-phosphate dehydrogenase preparations.
EXAMPLES 2 to 5 Microorganism used: Bacillus stearothermophilus NCA 1503 Formulation of nutrient medium: A medium of the following formulation that used glucose as a carbon source was prepared by dissolving in one liter of tap water.
glucose 1.3 g, yeast ext~act (product of Oriental Yeast Co., Ltd.) 1.0 g, peptone (product of Difico) 0.5 g, KH2PO4 0.5 g, Na2HPO4 12H2O 0-5 g, MgSO4 7H2O 0.1 g, ZnSO4-7H2O 0.01 g, MnSO4-7H2O 0.01 g, CuSO4 5H2O 0.01 g, CoCQ2 6H2O 0.01 g Germination stage: 20 mQ of the above nutrient medium was put in a 100-mQ conical flask, and 100 mQ of the same nutrient was put in a 500-mQ conical flask, and after stoppering each flask with a cotton plug, the media were sterilized with pressurized steam for 10 minutes at 121C
and l kg/cm2. After cooling, the medium in the 100-mQ
flask was aseptically inoculated with about 5 mg of a freeze-dried sample of Bacillus stearothermophilus NCA
1503 obtained from the American Type Culture Collection.
When the medium was subjected to rotary shake cultivation (160 rpm) for 24 hours at 55C with a rotary shaker ~product of Takasaki Seisakusho Co., Ltd.), the micro-organism grew and the degree of turbidity increased to such a level that the absorption at 660 nm (as measured with Model 101 spectrophotometer of Hitachi Ltd. and to be hereunder referred to as OD660 nm) was 0.8 to 1Ø
About 5 mQ of the germinated microorganism tinoculum) was transplanted on the medium in the 500-mQ flask, and the flask was subjected to rotary shake cultiration for a few hours under the same conditions as used above. l~hen the OD660 nm reached about 1.0, the cultivation was terminated Fermentation stage: A 30-liter fermentor was charged with 20 liters of a nutrient medium of the same formulation as used in the germination stage, and sterilization was performed at 121C and 1 kg/cm2 for 15 minutes. To the 115~70 medium, about one liter of the inoculum was transferred and it was subjected to batchwise cultivation at 55 + 1C, pH of 6.5 to 7.0 ~adjusted with 4 N NaOH) with air supplied at 20 liters/min at 900 rpm. Since the cultivation was accompanied by foaming, a small amount of defoaming agent (KM-70 of Shinetsu Chemical Industry Co., Ltd.) was added.
About 2.5 hours after the start of cultivation, the OD660 nm reached 1.2 (0.56 g of dry cell per liter) and the glucose level in the fermentation liquor became less than 0.01 wt%, so continuous fermentation was started immediately.
Since the ~max of Bacillus stearothermophilus NCA 1503 was found to have a ~max of 1.4 ~l/hr), a sterilized nutrient medium of the same formulation as used in the germination stage was supplied to the fermentor at a rate of 28.0 liters/hr and the fermentation liquor was discharged from the fermentor at the same rate. In this way the ~max was held at 1.00 (in Example 2) while continuous cultivation was performed using a nutrient medium five times the volume of the fermentation liquor in the fermentor Continuous cultivation was performed in the same q~anner as above except that D was changed to 0.9 ~max (medium supplied and fermentation liquor withdrawn at - 25.2 liters/hr) in Example 3 and to 0.75 ~max (supply and withdrawal rate: 21.0 liters/hr) in Example 4.
Thé level of glucose-6-phosphate dehydrogenase in the cells obtained in Examples 2, 3 and 4 was measured 11~6$70 and the results are shown in Table 1 below. The Table lalsoindicates glucose-6-phosphate dehydrogenase leve'l in cells produced by the batchwise processing (in Example 5) that was performed before transfer to the continuous cultivation.
Table Glucose-6-phosphate Yield of Yield of glucose-dehydrogenase level cell phosphate de-Run No. (U/g of dry cell) (g of dry hydrogenase(U/Q/hr) cell/Q/hr) Example 2 98 0.65 63.7 Example 3 71 0.59 41.9 Example 4 65 0.54 35.1 Example 5 69 0.23 15.9 - As is clear from the table, the cells produced ,by continuous cultivation with the D held at higher than 0.9 ~max gave a,glucose-6-phosphate dehydrogenase level higher than that obtained in batchwise processing.
,Example '6 A 30-liter fermentor was charged with 20 liters of a medium prepared by dissolving in one liter of tap water a mixture of 1.3 g of glucose, 1.0 g of ammonium sulfate, 0.5 g of yeast extract, 0.5 g of monopotassium phosphate, 0.5 g of dissodium phosphate and 0.1 g of magnesium sulfate, and the medium was sterilized with pressurized steam at 121C and 1 kg/cm2 for 15 minutes. Onelliter of a liquid ' inoculum of Bacillus stearothermophilus ATCC 12980 germinated 11565~() on a nutrient medium of the same formulation as defined above and the absorption of which at 660 nm reached about 1.0 was transferred onto the sterilized medium and cultured at 57C and a pH between 6.5 and 7.0 (adjusted with 4N
NaOH) and with air supplied at a rate of 20 liters/min at 900 rpm. When the absorption at 660 nm reached 1.0 by a batch cultivation for about 2.5 hours, a sterilized nutrient medium of the same formulation as indicated above was - supplied continuously with a metering pump at a rate of 24.0~liters/hr and the fermentation liquor was withdrawn from the fermentor at the same rate with the same machine.
A total of 100 liters of nutrient medium was used in the continuous cultivation. Immediately after the fermentation, a De Laval centrifuge was used to recover 400 g of the cell.
The cells were suspended in 1.5-fold 0.1 M phosphate ,(Homogeni~er) buffer and homogenized with a Dyno-mill' By removing the soluble matter with a centrifuge, a crude extract contain-ing~glucose-6-phosphate dehydrogenase was obtained. To , 400 mQ of the extract, 200 mQ of 10 ~ aqueous streptomycin 20 ~ sulfate and the resulting precipitate was removed with a centrifuge to give a streptomycin supernatant. The super--~ ~ natant was treated with ammonium sulfate and fractions for 25 % saturation (4C) thru 50 % saturation ~4C) were obtained. The fractions were dissolved in 50 mM tris-HCQ
. ~ z5 buffer (pH: 8.0) and the solution was passed through a DEAE- ephadex column equilibrated with a buffer of the same formulation as indicated above and was eluted with a bufer of the same formulation except that it contained sodium chloride. The desired glucose-6-phosphate dehydro-genase ~as obtained at a NaCQ concentration close to 0.2 M.
The active fraction was subjected to hydroxyapatite column chromatography under the same conditions as employed in Example 1. The active fraction obtained was passed through a Sepharcyl* S-200 column and elutèd with 30 m~l tris-HCQ bufer (pH: 8.0) containina 0.1 M sodium chloride.
A glucose-6-phosphate dehydrogenase sample resulted that gave a single band in a disc electrophoresis with acryl-amide as in Example 1. The enzyme gave a singe peak indlcat-- ing an average molecular weight of about 230,000 in Sephade~ G-200 chromatography as in Example 1.
The yield of the enzyme was about 20 mg and it had a potency of about 100 units/mg. The degree of purification of the enzyme was about 1100 in comparison . with the crude extract which was assumed to be 1.
Reference Examples 1, 2 and 3 The glucose-6-phosphate dehydrogenase prepared in Example 1 was used to determine the glucose level o standard sera which were already kno-~n to contain 76 mg/dl (Ref. Ex. 1), 155 mg/dl ~Ref. Ex. 2) and 43 mg/dl ~Ref.
Ex. 3), of glucose.
.
*Trade Marks - 22 -7 () Procedure A reaction liquor was prepared by dissolving 1 unit/mQ of glucose-6-phosphate dehydrogenase, 2 units/mQ
of hexokinase, 2 mM NAD, Z mM ATP, and 2 mM MgCQ2 in 1 mQ
of 100 mM phosphate buffer ~pH: 8.5). After standing at 30C for 5 minutes, the reaction liquor was mixed with 20 ~Q of each standard serum. Following a reaction at 30C for 5 minutes, the absorption at 340 nm was determined on a spectrophotometer. As a control, 20 ~Q of pure water was added to a reaction liquor of the same formulation as defined above, and following a reaction at 30C for 5 minutes, the absorption at 340 nm was determined The absorption of the control was subtracted from the adsorption of each standard serum to determine the increase in adsorption.
The glucose level (mg) in 1 dQ of each standard serum ; was calculated by the following equation:
; 145.8 x ~increase in absorption) = glucose level (mg) in 1 dQ of sample The results are shown in Table 2 below.
Table 2 Run No. Measurements Ref. Ex. 1 79 mg/dQ
Ref. Ex. 2 151 mg/dQ
Ref. Ex. 3 43 mg/dQ
115857~) 1 Fxom the results of Table 2, it can be seen that since the known glucose concentrations in sample are very consistent with the measured glucose concentrations, respectively, it is possible to measure the glucose concentration by the above described procedure.
The following table identifies enzymes referred to in this disclosure by name with a corresponding enzyme number in accordance with the numbering scheme of Florkin, M. ~ Stotz, E.H. "Comprehensive Biochemistry", Volume 13, 3rd edition, Elsevier Pub. Co. New York ~1973). The numberings of enzymes are as follows:
Enzyme Enzyme No.
Glucose 6- phosphate dehydrogenase E.C. 1.1.1.49 Hexokinase E.C. 2.7.1.1 Hexose-6-phosphate isomeraseE.C. 5.3.1.-Phosphoglucose isomeraseE.C. 5.3.1.9 While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the sprit and scope thereof.
-~4-. ~ .
~ . ~,
Claims (16)
1. A glucose-6-phosphate dehydrogenase enzyme obtained from a microorganism of the genuse Basillus which, when incubated for about 15 minutes in a buffer solution having a temperature of about 50°C, retains an activity value of at least about 80 %
of its initial activity.
of its initial activity.
2. An enzyme according to Claim 1 wherein said activity value is at least about 90 %.
3. An enzyme according to Claim 2 wherein said activity value is about 100 %.
4. An enzyme according to Claim 1 wherein the buffer solution has a temperature of about 57°C.
5. An enzyme according to Claim 1 or 4 wherein the glucose-6-phosphate dehydrogenase acts on nicotineamide adenine dinucleotide used as a coenzyme.
6. A process for preparing a glucose-6-phosphate dehydrogenase which comprises culturing a microorganism of the genus Bacillus and recovering from the culture a glucose-6-phosphate dehydrogenase which, when treated for about 15 minutes in a buffer solution having a temperature of about 50°C, retains at least about 80 % of the initial activity.
7. A process according to Claim 6 wherein continuous cultivation is performed under conditions such that the dilution rate D is at least 0.9 µmax and µmas is the maximum specific growth rate of a microoganism being cultured under continuous cultivation conditions.
8. A process according to Claim 6 wherein the micro-organism of the genus Bacillus is Bacillus stearothermophilus.
9. A co-enzyme composition consisting essentially of a glucose-6-phosphate dehydrogenase which, when incubated for about 15 minutes in a buffer solution having a temperature of about 50°C, retains an activity value of at least about 80 %
of its initial activity, and nicotine-amide adenine dinucleotide thosphate.
of its initial activity, and nicotine-amide adenine dinucleotide thosphate.
10. A co-enzyme composition as according to Claim 9 wherein said activity value is at least about 90 %.
11. A process according to Claim 8 wherein said Bacillus stearothermophilus is selected from ATCC 7953, 7954, 8005, 10149, 12980, and NCA 1503.
12. A process according to Claim 11 wherein the micro-organism is NCA 1503 and the buffer solution has a temperature of about 57°C and a pH of about 6.8.
13. An enzyme according to Claim 1 wherein the micro-organism of the genus Bacillus is Bacillus stearothermophilus.
14. An enzyme according to Claim 13 wherein the micro-organism is selected from a group consisting of ATCC 7953, 7954, 8005, 10149, 12980, and NCA 1503.
15. A glucose-6-phosphate dehydrogenase which, when incubated for about is minutes in a buffer solution having a temperature of about 50 degree C, retains an activity value of at least 80 percent of its initial activity.
16. A co-enzyme composition consisting essentially of a glucose-6-phosphate dehydrogenase obtained from a microorganism of the genus bacillus which, when incubated for about 15 minutes in a buffer solution having a temperature of about 50 degree C
retains an activity value of at least about 80 percent of its initial activity, and nicotine-amide adenine dinucleotide.
retains an activity value of at least about 80 percent of its initial activity, and nicotine-amide adenine dinucleotide.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP14485879A JPS5668391A (en) | 1979-11-07 | 1979-11-07 | Glucose-6-phosphate dehydrogenase and its preparation |
| JP144858/79 | 1979-11-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1156570A true CA1156570A (en) | 1983-11-08 |
Family
ID=15372034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000363960A Expired CA1156570A (en) | 1979-11-07 | 1980-11-04 | Glucose-6-phosphate dehydrogenase and process for preparation thereof |
Country Status (8)
| Country | Link |
|---|---|
| JP (1) | JPS5668391A (en) |
| CA (1) | CA1156570A (en) |
| DE (1) | DE3041744A1 (en) |
| DK (1) | DK471880A (en) |
| FR (1) | FR2472608B1 (en) |
| GB (1) | GB2066261B (en) |
| IT (1) | IT1146060B (en) |
| SE (1) | SE8007818L (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5971687A (en) * | 1982-10-12 | 1984-04-23 | Toyobo Co Ltd | Stable composition containing glucose-6-phosphate dehydrogenase |
| JPS6152282A (en) * | 1984-08-22 | 1986-03-14 | Toyobo Co Ltd | Thermostable glucose-6-phosphate dehydrogenase |
| JP2742281B2 (en) * | 1988-12-29 | 1998-04-22 | 旭化成工業株式会社 | Glucose-6-phosphate dehydrogenase gene |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1567606A (en) * | 1976-08-24 | 1980-05-21 | Secr Defence | Glycerokinases |
| GB1592413A (en) * | 1976-11-19 | 1981-07-08 | Kyowa Hakko Kogyo Kk | Preparation of choline oxidase |
-
1979
- 1979-11-07 JP JP14485879A patent/JPS5668391A/en active Granted
-
1980
- 1980-11-04 CA CA000363960A patent/CA1156570A/en not_active Expired
- 1980-11-05 IT IT50089/80A patent/IT1146060B/en active
- 1980-11-05 DE DE19803041744 patent/DE3041744A1/en active Granted
- 1980-11-06 SE SE8007818A patent/SE8007818L/en unknown
- 1980-11-06 GB GB8035669A patent/GB2066261B/en not_active Expired
- 1980-11-06 DK DK471880A patent/DK471880A/en unknown
- 1980-11-07 FR FR8023926A patent/FR2472608B1/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| DE3041744C2 (en) | 1990-07-12 |
| JPS5668391A (en) | 1981-06-09 |
| GB2066261B (en) | 1983-09-21 |
| IT8050089A0 (en) | 1980-11-05 |
| JPS6343079B2 (en) | 1988-08-26 |
| DE3041744A1 (en) | 1981-05-21 |
| DK471880A (en) | 1981-05-08 |
| FR2472608A1 (en) | 1981-07-03 |
| SE8007818L (en) | 1981-05-08 |
| IT1146060B (en) | 1986-11-12 |
| GB2066261A (en) | 1981-07-08 |
| FR2472608B1 (en) | 1985-12-06 |
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