Note: Descriptions are shown in the official language in which they were submitted.
1 15B~7()
GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND PROCESS
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
-- 2
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
- 3 -
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
3 -
1 l5B~n
-- 4
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.
- 4
- 5
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)
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~()
-- 6
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 -
- 7
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.
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
- 8
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
1 1 5B570
- 10 -
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
- 10 -
11~8~0
- 11 -
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.
- 13 -
, .. .
115~5~)
- 14 -
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
- 14 -
115B57()
- 15 -
~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~)
- 16 -
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
- 16 -
n
- 17 -
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
- 17 -
1158570
- 18 -
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
- - 18 -
115~70
- 19 -
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
- 19
11~6$70
- 20 -
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
- 20 -
11565~()
- 21 -
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
- 21 -
1 156570
- 22 -
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 ()
- 23 -
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
23 -
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-
. ~ .
~ . ~,
