Note: Descriptions are shown in the official language in which they were submitted.
w o 9l/09117 2 0 6 9 6 2 9 PCT/US90/06764
PROCESS FOR ISOLATING GALACTOSE OXIDASE
FIELD OF THE INYENTION
A method of isolating and purifying galactose oxidase from afungus media is disclosed. The supernatant liquid from a
fermentation broth is subjected to ultrafiltration and then
o separated on a carboxy sulfon cation exchange column using high
pressure liquid chromatography. Copper ions must be present to
insure enzyme activity.
BACKGROUND OF THE INV~N~ION
Galactose oxidase (E.C. 1.1.3.9, GOase) is a
copper-containing extracellular enzyme which oxidizes the primary
hydroxyl groups of many alcohols and of galactose. Although the
enzyme is produced by a number of fungus species, fermentation of
DactYlium Dendroides is currently the best practical source. A
simple method for the growth of this fungus and purification of
~0 the excreted enzyme from the growth medium is described by Tressel
and Kosman, AnalYtical Biochemistrv, I05, 150-153 (1930).
In this process the enzyme is grown in a fungal culture
aerobically in the dark for several days at 20- C. This fungus is
then transferred to a glucose-based liquid medium and grown
2; aerobically for about 2 days at 20-C. A completely artificial
medium is used; it is a mixture of sorbose, glucose, traces of
metal ions as micronutrients, and thiamine as the necessary
vitamin. Growth of the culture takes usually 5 to 7 days.
~'
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Isolation of the galactos~e ~xidase involves a number of steps
which begins with pre~ipitation in the presence of
microcrystalline cellulose. The purification is completed by
chromatography on a phosphocellulose column.
The enzyme contains 1 atom of copper per mole of protein and
the presence of the cupric (Cu+2) ions is necessary for enzymic
activity. When the galactose oxidase is grown under conditions of
copper deprivation, ~actYlium Dendroides synthesizes and excretes
a catalytically inactive protein, aplagalactose oxidase.
0 Catalytic activity of this protein appears when cupric ion is
added to the copper depleted solution (see, Shatzman and Kosman,
Biochim. BioPhvs. Acata, 1978, 544, 163-169 and Markus et al. , G.
Avigad. ADD1. Microbiol., 13 (5), 686-693 (1965)). This result
implies that although copper is necessary for the catalytic
activity of galactose oxidase, cupric ion neither induces
synthesis nor is necessary for the complete assembly and
secretion of proteins by DactYlium Dendroides. On the other hand,
~apanese workers (Aisaka and Terada, ~qric, ~lo. Chems., 45,
2311-2316 (1981)) concluded that the synthesis of galactose
oxidase protein by Gibberella Fuiikuroi is a phenomenon regulated
by copper.
Copper ion also preserves enzyme activity in unpurified
fermentation medium by preventing complexation of galactose
oxidase with an inhibitor (Avigad and Markus, Israel J. Chem., 3,
193 (1966)). It is known that DactYlium Dendroides produces at
least one galactose oxidase inhibitor identified as heptapeptide.
This protein forms a stable inactive complex with galactose
oxidase in the absence of copper. Presence of 1 to 10 mM cupric
ion not only prevents this inhibition but also causes slow
activation of the inhibited enzyme. The inhibiting components are
usually removed from galactose oxidase preparations by
chromatography.
The present process produces about a 45% yield of enzyme
(measured as recovered activity) over a three day isolation
period. It has now been found that this isolation and
2069629
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purification can be simplified and conducted on large
quantities of enzyme preparation mixtures by the use of
ultrafiltration and high pressure liquid chromatography
purification t~-hniques. Gram quantities of the
galactose oxidase can be prepared in a day.
Accordingly, it is an object of this invention to
prepare large quantities of galactose oxidase in yields
of above 70% (measured as recovered total activity) in
an efficient manner.
All percentages are by weight unless otherwise
indicated.
SUMMARY OF l~v~NllON
In one embodiment of the invention a process for
preparing and isolating pure galactose oxidase is
disclosed. The process comprises the steps of
(1) aerobically fermenting Dactylium Dendroides in
a medium comprising a nitrogen source, a carbon source
and trace metals and thiamine at a temperature of
between 20C to about 25C;
(2) filtering the supernatant liquid from the
fermentation broth using a 0.15 to 0.25 ~m filter;
(3) concentrating the enzyme by ultrafiltration
with a 10,000 molecular weight membrane;
(4) equilibrating the retentate from (3) with a
buffer containing copper ions and histidine;
(5) optionally, treating the retentate with DEAE
cellulose to remove contaminating proteins;
(6) purifying the enzyme on a carboxy-sulfon
column using high pressure liquid chromatography
methods.
Another aspect of this invention is as follows:
In a process for preparing galactose oxidase enzyme
solutions comprising the steps of:
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(1) aerobically fermenting a fungal source of
galactose oxidase in a medium comprising a nitrogen
source, a carbon source, trace metals, thiamine and from
10 to about 60 micromolar in copper ion at a temperature
of between 20C to about 25C;
(2) filtering the supernatant liquid from the
fermentation broth using a 0.15 to 0.25 ~m filter;
(3) concentrating the enzyme by ultrafiltration
with a 10,000 molecular weight membrane;
(4) equilibrating the retentate from (3) with a
buffer containing copper ions, histidine and hydroxide
ion;
the improvement comprising:
purifying the enzyme on a carboxy-sulfon column
using high pressure liquid chromatography methods and
elution with a buffer comprising histidiene, sodium
acetate and cupric ions.
DET~TT~n DESCRIPTION OF THE INVENTION
A. FERMENTATION
A starter broth of DactYlium Dendroides or other
fungal source of galactose oxidase is prepared. The
procedure of Kosman, ibid involves preparation of
stationary dextrose agar slants which
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are inoculated with DactYlium Dendroides. The inoculated slants
are grown aerobically in the dark for approximately two days at
about 20-C to form a uniformly white, fluffy coating of fungus.
Lengthy exposure to sunlight or an excessively warm incubation
temperature results in ~abnormal" cultures which are discolored
and finely matted.
A small portion of this starter fung~s is mixed with a liquid
medium to grow the galactose oxidase. This completely artificial
medium contains sorbose or glucose as the carbon sources, traces
o of metal ions as micronutrients and thiamine as the necessary
vitamin. Growth in the culture takes S to 7 days. The pH of the
growth media is neutral.
Copper sulfate (from 10 ~m to about 60 ~m) increases the
enzyme activity in the fermentation medium and improves
reproducibility of the fermentation runs as compared to batches
fermented by the standard method where the copper sulfate
concentration was ll~M. On the other hand, when the (Cu 2+)
cupric concentration was increased to 1 mM, growth of mycelia was
clearly retarded and only traces of galactose oxidase activity
were detected.
The usual batch size for growing the DactYlium Dendroides is
in 20 (L)liter containers. This process can easily be scaled up
to grow the fungus in kiloliter containers. Table 1 provides a
flow chart for the preparation of the enzyme broth in 250L
fermentor.
w o 91/09117 2 0 6 9 6 2 9 PCT/US90/06764
Table I
Using Dactylium Dendroides.
DactYlium dendroides NRRL 2903
grown on agar slants, 3-5 days,
25C, dark, stored,
wrapped in aluminum foil, 4C
I liter starter flasks
containing 500 ml starter medium
incubated at room temperature,
o 3 days, on gyratory shaker
(300 rpm, I" radius of gyration)
I.2 v/v inoculum
250 liter fermentor
containing 200 liters culture medium
21C; initiat aeration rate - I vvm,
to be adjusted to maintain
D0>50% saturation;
agitation rate - I00 rpm
Trace metals which can be used in the fermentation broth
include magnesium, manganese, zinc, calcium and iron. These
metals are added as water soluble salts. Anions such as sulfate,
chloride, nitrate and carbonate can be used. The level of copper
is very important and should be in the range of from about I0 to
about 60 micromoles. Preferably, the amount of copper is from
about 45 ~m to about 58 ~m. The level of trace metals is
generally equal to or less than the cupric level.
In addition to the trace metals, a source of nitrogen is
required. Ammonium nitrate is the preferred nitrogen source, but
other inorganic salts can be used, for example, sodium or
30 potassium nitrate, ammonium sulfate, ammonium hydrogen phosphate,
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ammonium phosphate, etc. Any alkali metal or alkaline earth metal
nitrate can be used. Urea can also be used as a nitrogen source.
The carbon source is preferably glucose or sorbose, but other
low molecular weight carbohydrates can be used. These include
fructose, sucrose and mannose. The level of carbohydrate is from
about 0.2% to about lOX in the fermentation broth.
Thiamine is added at about 1 to about 10 micromoles.
The pH of the medium is held at about 6.0 to about 7.8. The
pH is preferably controlled through the use of phosphate buffers,
but other buffers can be used.
The temperature of the fermentation is maintained at ambient
temperature and preferably between 20C and 25C. If necessary,
the level of water is maintained by replacing water lost to
evaporation.
During the fermentation reaction, the solution is stirred and
aerated with a flow of air. Compressed air which has been
filtered to remove any oil or other contaminants is used. For
example, the air can be passed through an oil filter and a glass
wool plug to remove most of the contaminants. The air flow is
adjusted to a rate which maintains the desired degree of oxygen
saturation in the broth. Preferably, atmospheric oxygen at above
50% saturation is desirable. Most preferably, the broth is about
75% saturated with oxygen.
The usual length of time that the fermentation is carried out
2s is from about 100 to about 200 hours, preferably from about 120 to
about 145 hours.
All of the metal ion solutions, sugar solutions and nitrogen
solutions used for preparing the fermentation broth, and any water
added during fermentation should be sterilized. This minimizes
the formation of interfering enzymes and other fungi, bacteria or
viruses.
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Table 2
Isolation of Galactose Oxidase
from ~hole Broth (-200 liters)
Filtration through 0.15 to 0.25 ~m filter
(using Millipore Pellicon)
Addition of CuS04 & histidine to filtrate
Filtration through 10,000 MWCO
membrane (using Millipore Pellicon)
v
retentate (-3.5 liters)
oDialysis against 2mM CuS04/5mM histidine/
SmM NaOH buffer (using Millipore Pellicon/
10,000 MWCO membrane)
retentate (-3.5 liters)
Addition of -450 gm. DEAE cellulose;
mixing for 30 minutes
Filtration (using Millipore Pellicon
microporous membrane)
Concentration using (Millipore Pellicon
10,000 MWCO membrane)
retentate (-100 mls)
i
Dialysis against 2mM CuS04/4mM histidine
/20 mM MES buffer using Amicon Cell/
10,000 MWCO membrane
for HPLC purification
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B. Isolation of the Galact~ose Oxidase
Table 2 illustrate~ the`isolation process.
The contents of the fermentation batches are cooled to about
3 to about 10C and filtered through a O.lS to 0.25 ~m filter.
Conventional filters such as millipore or other filters are
acceptable. Use of a millipore filter is a preferable filtration
technique because it also removes gel-like polymers which can foul
the membrane of the ultrafiltration apparatus in the next step.
However, other means of filtration such as nylon mesh sheet
0 CMN-210 (Small Points, Florida) can also be used.
Copper sulfate and histidine are added to the filtrate in the
amount necessary to make a 8mM to about a 12 mM solution of copper
ion (cupric) and about 15 mM to about 25 mM of histidine.
The filtrate is concentrated to about 10% to about 25% by
ultrafiltration through a 10,000 molecular weight cut off
membrane: Millipore Pellicon Cassette System can be used,as can
other ultrafiltration systems.
The retentate is optionally equilibrated with a freshly
prepared buffer (pH about 7.0) containing copper sulfate (3-8 mM),
histidine (3-8mM) and sodium hydroxide (3-8mM). Again the
solution is concentrated to about equal volumes as before.
The retentate is optionally treated with DEAE cellulose which
has been equilibrated with a phosphate buffer (pH about 7.0)
containing copper sulfate and histidine. The cellulose adsorbs
about 50% of the contaminating proteins from the retentate, i.e.
the galactose oxidase containing solution. Removal of these
proteins facilitates the final purification of the enzymes. The
cellulose is filtered and, if cloudy, centrifuged to remove any
materials. A 5 minute to about 30 minute cellulose treatment is
usually sufficient to remove these proteins.
This solution is again subjected to ultrafiltration to
further concentrate it to about 10% to about 25% of the starting
solution. The enzyme is equilibrated during this concentration
W O 91/09117 PC~r/US90/06764
206962q
_
with a solution of copper sulfate (2mM to about 20mM), histidine
(4mM to 40mM) and M~S (2-(N-morpholino) ethanesulfonic acid) (lOmM
to 30mM) at a pH of about 5.6 using a 10,000 molecular weight cut
off (MWCO) dialysis bag.
Any cloudiness or fine particles left in the solution can be
removed by filtration or centrifugation.
C. ChromatographY
The enzyme solution is finally purified using high
performance liquid chromatography (HPLC). A carboxy-sulfon 40
o micron column from J. T. Baker is used (Bakerbond Carboxy-
sulfon~). The carboxy-sulfon columns can contain particles
ranging from 5 microns to 50 microns in size. The carboxy-sulfon
substrate is described in U.S. 4,721,573.
After injection of a crude enzyme solution and elution of a
void volume with a buffer composed of MES, histidine and cupric
sulfate, a linear gradient is applied of a buffer containing
sodium acetate, histidine and cupric sulfate. Usually this
elution is done over about 1 to 2 hours with a flow rate of about
0.5 ml/min. to about 2 ml/min. A step gradient of about 10% to
about 15% is generally used.
The exact concentration gradients and times will depend on
the solution and the column length as well as the method of
loading the column. One skilled in the art will be able to
determine the exact method with minimal experimentation. The
enzyme position is determined using a ultraviolet light at 280 nm.
D. Assay Methods
An assay solution is prepared by boiling a phosphate buffer
(O.lM, pH 7.0) and then cooling it to a room temperature. To this
buffer is added 500 mg of D-galactose (available from Sigma as a
"substantially glucose free" material), 5 mg horseradish
peroxidase, (available from Sigma, type III mixture of basic
isozymes) and 5 mg o-dianisidine (3, 3'-dimethoxybenzidine),
dissolved in O.S ml of methanol. These solutions are added to the
phosphate buffer and the buffer is diluted to 100 ml in a
volumetric flask. O-Dianisidine (3-3'-dimethoxybenzidine) should
be added quickly otherwise a cloudy suspension will result. The
assay solution must be stored away from the light and be
refrigerated. The solution should be discarded when the
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absorbance at 460 nm becomes greater than 0.1 than the buffer
solution alone.
To assay the activity of the glactose oxidase, one ml of the
assay solution is added at room temperature to a microcuvette for
an ultraviolet spectrometer. A sample of the en~yme (5 to 50
microliters) is injected into the assay solution and stired for
about a second. ~he linear absorbance increas~ is followed for
one minute and the absorbance/per minute is calculated. ~he
amount of enzyme added should be adjusted until this value is
o between 0.2 and 0.6.
When using solid enzyme samples, the solid sample is
dissolved in 0.1 M phosphate buffer (pH 7.0) to make the amount of
enzyme equal to approximately 0.4 units in the assay mixture.
Protein Assay
Bio-Rad protein assay dye concentrate, catalogue number
500-006 is used for an assay standard. This protein assay dye
should be diluted to a concentration of about one (1) volume of
concentrate to four (4) volumes of distilled water. A solution of
bovine serum albumin in O.lS M sodium chloride is prepared (1 mg
per ml). ~en samples containing from 10 to 100 micrograms of the
protein are added to test tubes (10 microliter, 20 microliter,
etc.) The volume in each test tube is adjusted to 0.1 milliliter.
Five ml of the assay solution is measured into each test tube and
mixed for 30 seconds. After 2 minutes, the absorbance at 595 nm
against a blank solution prepared from 0.1 ml of 0.15 M sodium
chloride in 5 ml of assay solution is measured. ~he weight of the
protein is plotted against the absorbance.
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ExamPle 1
Fermentation of Galactose Oxidase in 20L Bottles
DactYlium Dendroides strain NRRL 2903 was obtained from
Dr. J. J. Ellis of the USDA of the Northern Regional Research
Laboratory (Pioria, IL.) and was maintained on agar slants.
A 20 liter autoclaveable plastic bottle was equipped with
four metal aerators and a three stage turbine propeller for
mixing. Compressed air was filtered through an oil removal
filter (DPS-19 from Grainview Products, California) followed
o by sterile glass wool filters. After passing through the
filters, the air is distributed through the four aerators in
the bottle. During the fermentation, the total air flow in
each bottle (fermentation flask) is maintained at 19
liters/minute as measured at the outlet of the air.
Agar Slants:
Starter Medium + 1.5X agar
Starter Medium:
5.88 parts Solution A + 1 part Solution B + 1 part
Solution C + 0.0006 parts Solution D
Culture Medium:
8 parts Solution A + 1 part Solution C + 0.0008 parts
Solution D
Solution A: (salts, nitrogen)
10.74 g/l Na2HP04
10.41 g/l KH2PO4
1.27 g/l (NH4)2N3
2.50 g/l (NH4)2SO4
0.93 g/l NaOH
1.0~ g/l KOH
w o 91/09117 2 0 6 9 6 2 9 PCT/USgo/06764
Solution B: (trace metals)
2.05 9/1 MgS04
19.48 mg/l MnS04 H2Q
30.00 mg/l ZnS047 ~2
17.75 mg/l CaC122 H20
28.44 mg/l FeS047 H20
135 mg/l CuS045 H20
Solution C: (carbon source)
for agar slants and starter flasks:
o 79.4 9/1 glucose
for cutture flask/fermentor
100.- 9/l sorbose
Solution D: (thiamine)
33.7 9/l thiamine, filter-sterilized
The fermentation flasks are maintained at 21 C in water
baths. Temperature control is provided by circulating water in
the tubing of the cooling or heating medium by a thermostat.
Solution A is placed in the fermentation flask. Stirrers and
aerators are then inserted into the flask and a slow flow of air
is turned on. Stirring rate is maintained at 490 RPM (gentle
stirring). Solutions B and C are added to each flask followed by
the addition of a thiamine stock solution (800 microliters). The
fermentators are then innoculated with 120 ml of the mycelia
suspension from the starter flask. The air flow is adjusted from
19 liters/minute and the fermentation was allowed to continue from
136 to 140 hours.
After 48 hours of fermentation, two liters of sterile water
is added to the flask to replace the water which is evaporated.
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The broth from the fermentation is filtered through 210
micrometer nylon mesh sheets and the mycelia is squeezed dry. The
volume of the filtrate is measured, and copper sulfate and
histamine are added to the filtrate in amounts to make a 10
millimolar solution of copper sulfate and a 20 millimoler solution
of histamine. The solution is filtered through a GF/D Whatman
glass fiber filter and a 0.22 micrometer millipore filter
(millidisc hydrophobic cartridge). The solution is fed into the
cartridge from a pressurized container. The fine filtration is
necessary to remoYe a gel-like polymer. Two cartridges are used,
one for each 15 liters of the solution. The filtered solution is
concentration to about 500 ml by ultrafiltration using a millipore
Pellicon cassette system equipped with a 10,000 MWC0 membrane
cassette. This concentration takes from 1-2 hours for the 30
liter solution. The retentate is equilibrated on the same
millipore Pellicon cassette system with a freshly prepared buffer
containing copper sulfate, (5 millimoles), histidine (5
millimoles) and sodium hydroxide (5 millimoles), a pH of about 5.6
The retentate is treated with DfAf cellulose (60 grams)
equilibrated with the same buffer as was used in the
ultrafiltration system and stirred for 15 minutes. The cellulose
is removed by suction filtration. The filtrate is centrifuged at
9,000 9 for 30 minutes to remove any cloudiness or precipitation.
The clear enzyme solution is concentrated to about 15 ml
using an Amicon stirred ultrafiltration cell equipped with 10,000
MWC0 membrane (PM-10 diameter 76 mm) at 40 psi. This
ultrafiltration takes 3-6 hours. The concentrated enzyme solution
is equilibrated with a solution of copper sulfate (2 millimoles),
histidine (4 millimoles), and MES (20 millimoles) at a pH of 5.6
using a dialysis bag fitted with a 10,000 MWC0 membrane size. The
retentate is centrifuged at 9,000 9 for 30 minutes to remove any
cloudiness. The concentrate is filtered through a plug of G/f
w o 91/09117 2 0 6 9 6 2 9 pCT/US90/06764
~hatman glass fiber filter to remove fine particles from this
solution.
A high pressure liquid chromatography column (40 microns) is
filled with carboxy sulfone (J. T. ~aker, Bakerbond carboxy-
sulfon ~) (40.6 X 250 mm). The ~ncentrated enzyme solution (1.75ml) is loaded onto the column-using a 2 ml loop. The void volume
peak is eluted and collected. Four to five portions of the
concentrated crude enzyme are injected onto the column and the
void volumes eluted. After the last void volume is eluted, a
0 linear gradient from 100X A to 100% B over a one hour period is
applied. ~he flow rate is 1 ml per minute. Buffer A is a mixture
of 20 mM 2-N morpholino ethane sulfonic acid (MES), 2 millimolar
copper sulfate, 4 millimolar histidine at a pH of 5.6 adjusted
with sodium hydroxide. Buffer B is a 1 molar solution of sodium
acetate, 2 millimolar copper sulfate, 4 millimolar histidine at a
pH of 5.8 adjusted with acetic acid or sodium hydroxide. The
galactose oxidase enzyme elutes in about 16 minutes. An
ultraviolet detector set at 280 nm can be used to detect the
enzyme.
Void volumes are analyzed for the presence of galactose
oxidase by standard activity analysis. If enzyme is present, the
void solution can be rechromatographed after concentration by
ultrafiltration) in the presence of copper ions and histidine.
A yield of 70% galactose oxidase is obtained.
