Note: Descriptions are shown in the official language in which they were submitted.
~6~6'71~4~
~ield of the Invention
The present invention relates to an improved method
for producing cholesterol oxidase by cultivating of a
cholesterol oxidase-producing microorganism.
Background of the In~ention
Microorganisms capable of metabolizing cholesterol
are potential sources of enzymes useful in an enzymatic
` 10 assay of cholesterol in complex mixtures such as blood
serum, etc. This is particularly so if the microorganisms
can use cholesterol as a sole carbon source, for in this
assay process cholesterol must be degraded by oxidative
enzymes.
Stadtman, T. C., Methods in Enzymology, Vol. l;
Colowick, S. P., and Kaplan, N. 0., Eds. Academic Press,
N. Y., 1955, p. 678; and Stadtman, T. C., Cherkes, A., and
- Anfinsen~ J., Biol. Chem., 206, 510 (1954), reported the
preliminary purification of an enzyme from Nocardia cho-
; 20 lesterolicum, an organism originally isolated by Schatz et
al (Schatz, A., Savard, K., and Pintner, I. J., J. Bac-
teriol., 58, 117-125 (1949). Stadtman's enzyme, "choles-
terol dehydrogenase", was purified sufficiently for use in a
cholesterol assay based on the measurement of the increase
in absorbance at 250 nm. owing to the formation of cholest-
4-en-3-one. Since, as we have now determined, the direct
acceptor Or cholesterol electrons in this oxidation is
oxygen, the enzyme should properly be called cholesterol
oxida.se according to current convention.
3 The bacterial strains described by Stadtman when
cultured as described ln the aforementioned references
produce very low enzyme levels which are not practical for
-2_ ~x
10~:;'7844
commercial operations. These levels are so low that commercial
production of purified enzyme is a very remote possibility.
Goodhue et al, in U.S. Patent 3,909,359 issued
September 30, 1975, describe an improved method for the production
of the Stadtman cholesterol oxidase, which method comprises the
steps of:
(a) growing the bacterium Nocardia cho'lesteroli'cum species
NRRL 5767 or NRRL 5768 in a medium in which cholesterol
or a suitable derivative thereof serves as an auxiliary
source of carbon and
(b) isolating from said medium a cell-free extract containing
the active enzyme.
The method described by Goodhue et al is greatly improved over the
original synthesis described by Stadtman and can be said to render
the process commercially practical.
German OLS 2,246,695 published March 29, 1973, de-
scribes a method for isolating a cholesterol oxidase enzyme pro-
duced by a culture of Nocar'dia microorganism identified as NRRL
5635 and NRRL 5636. According to the method described therein,
the harvested cells are treated with a nonionic surfactant and
stirred at room temperature to release a large proportion of the
enzyme from the cells into the supernatant, thereby eliminating
the need for mechanical disruption of cells. From the data '
reported,~we have calculated the extraction yields enzyme activity
on the order of about 40 to 160 U/liter.
Reese, E. T., and Maguire, A., in "Surfactants as
Stimulants of Enzyme Production by Microorganisms", Applied
Microbiolog~, February, 1969, pp. 242-245, describe the observa-
tion that the addition of sorbitan polyoxyethylene monoleate
(Tween 80 from Atlas Chemical Co., Wilmington,
--3--
~Q6~
Delaware) and other nonionic surfactants to fungal cultures
which normally produce extracellular enzymes results in a
marked increase in enzyme yield.
British Patent 1,~85~319 describes the production
of cholesterol oxidase from Nocardia species NRRL 5635 and
NRRL 5636. The fermentation is conducted in a growth medium
containing 20 g./liter of yeast extract. During the fer-
mentation, cholesterol is slowly added to the medium, pref-
erably in the form of a dispersion containing a nonionic
surfactant. Cholesterol is added in quantities totaling up
to 1.2 g./liter of medium during this addition. The total
concentration of nonionic surfactant added in this manner is
minute.
Republic of South Africa Patent 73/3259 describes
the production of cholesterol oxidase using Proactinomyces
erythropolis NBC 9158, ATC 17895, ATCC 4277 and Nocardia
; formica ATCC 14811. The fermentation is conducted in a
peptone-containing mineral salt medium and, when the loga-
rithmic growth phase is reached, cholesterol in the form of
an aqueous suspension is slowly added to the medium in
proportion to the growth of the microorganism such that the
cholesterol added totals 1 to 20 g./liter of medium. A
small amount of Gholesterol (0.05% in Example 2) may be
added to the medium initially. Increase in the yield of
cholesterol oxidase activity is obtained by adding yeast
extract to the cholesterol suspension as an emulsifying
agent in an amount of 0.02 to 1% by weight of the choles-
terol suspension. It can be seen that only a very small
amount of yeast extract is added to the medium in this
3 manner. No surfactant is used during the fermentation.
44
Summary of the Invention
The present invention provides an improved method
for the cultivation of a cholesterol oxidase-producing micro-
organism which substantially increases the production of choles-
terol oxidase over prior-art methods. The improved fermentation
method is accomplished by growing the cholesterol oxidase-pro-
ducing microorganism in a growth medium having an enzyme inducer
which may also be an auxiliary carbon source. The growth medium
also has from about 1.0 to about 5.0 g'./liter of a nonionic sur-
factant which is nontoxic to the'microorganism and at least about10 g./liter of yeast extract. The above ingredients in the
growth medium apparently have a synergistic effect on the pro-
duction of cholesterol oxidase, giving yields of up to about
900 U./liter. The cholesterol oxidase can then be recovered by
any conventional means.
Detalled Descri_t on'of the'Inv'e'n*ion
A microbial cell has the genetic information to
synthesize virtually thousands of enzymes. However, enzyme pro-
duction is tightly controlled to avoid the waste of energy
and the intermediates. Therefore, to develop a ermentation,
existing control mechanisms in the microbial cells need to
be modified so that the desired enzyme or metabolite is
overproduced. There are two ways to alter the regulation
of the biochemical pathways, namely, (1) environmental and
(2) genetic. The production of cholesterol oxidase is
Ç;78~4
greatly increased by the pxocedure of this invention by pro-
viding appropxiate environmental conditions.
According to the present invention, the production
of intracellular cholesterol oxidase is substantially increased
by growing a cholesterol oxidase-producing microorganism in a
medium comprising yeast extract, a nonionic surfactant and an
inducer for cholesterol oxidase, in addition to a carbon source
and trace metal salts. Proper selection of the inducer and other
environmental conditions can also result in the coproduction of
cholesterol esterase in usable quantities.
Any cholesterol oxidase producing microorganism can
be used in the improved method of this invention. Such useful
microorganisms ganerally belong to the order Actinomycetale~
or to related microorganisms of the coryneform group. Parti-
cularly useful microorganisms of th~ order Actinomycetales belong
to the families Mycobacteriaceae, Nocardiaceae and Streptomycet-
aceae. Other such useful microorganisms include, for example,
microorganisms belonging to the fam:ily Corynebacteriaceae of
the coryneform group. Examples o species o~ microorgani~ms
use~ul for the practice o this i~vention include, ~or instance,
NRRL 56-35, NRRL 5636, NRRL 5767, NR~L 5768, ATCC 4277, ATCC 14349,
ATCC 14811, and ATCC 17895. The ~RRL designation indicates
that cultures of the species so designated are on deposit with
the Agricultural Collection Investigations Fermentation ~abora-
tory, Peoria, Illinois~ The ATCC designation indicat~s such
deposit with the American Type Culture Collection. The above
species have been identified in the past by various genus names
including Mycobacterium, Nocardia, Proactinomycetes and
Streptomyces. Although the criteria for classifying these
~0~;7~
species of microorganisms into particular genera appears somewhat
uncertain and the classification of particular species has changed
periodically as far as placing them withîn certain genera of micro-
organisms, they are readily identifiable as members of the order
Actinomycetales.
Two preferred species for the practice of this
invention are characterized as the "rough" and "smooth" strains
of Nocardia cholesterolicum, identified as NRRL 5767 and ~RRL
5768 respectively.
1 0 _,,
According to the method for producing cholesterol
oxidase described by Goodhue et al in U.S. Patent 3~900,359
which produced intracellular cholesterol oxidase, the
use of a conventional primary carbon source such as glyc-
erol, in combination with a secondary or auxiliary carbon
source such as cholesterol,~cholest-4-en-3-one or choles-
teryl linoleate, all of which act as cholesterol oxidase
inducers, increases the yield of cholesterol oxidase enzyme
to levels about 100 times higher than those produced when a
cholesterol oxidase inducer is not used or when cholesterol
is used as the sole carbon source as described in the prior
art.
Thus, according to Goodhue et al, improved yields
were obtained when the bacterium , Nocardia cholesterolicum, was
grown in a conventional nutrient medium of the type well-known in
the art which generally comprises a nitrogen source such as ammonium
sulfate, a potassium and a phosphorus source such as potas-
sium phosphate, trace metal ions, and a mixture of a primary
carbon source such as glycerol and a cholesterol oxidase
inducer selected from the group consisting of cholesterol,
cholest-4-en-3-one, cholesteryl linoleate, and mixtures
~)6';J89L~
thereof. The pH value of the medium i5 maintained between about
5.0 and 8.0, preferably between about 6.5 and 7.5, at a tempera-
ture of from about 25 to about 35 C., preferably about 30C.,
for a period of from about 18 to about 40 hours, preferably from
about 20 to about 2~ hours.
The quantities of nitrogen, potassium phosphorus and
trace metal ions used in the culture are those conventionally
used in processes of this -type and are well-known to those skilled
in the art. Specifically, those described in the aforementioned
references provide useful levels of these constituents.
Among the primary carbon sources which were found
useful by Goodhue et al and which are similarly useful herein are
glycerol, glucose and acetic acid. Conventional concentrations
of primary carbon source are used. These generally range from
about 5 g./liter to about 50 g./liter. The concentration of the
cholesterol oxidase inducer utilized generally ranges from about
1.0 g./liter to about 10.0 g./liter. A preferred range of
inducer is from about 2 g./liter to about 5 g./liter.
According to the improved process described herein,
cholesterol oxidase is prepared substantially as described by
Goodhue et al except that other cholesterol oxidase-producing
microorganisms can optionally be employed, and the growth medium
further includes yeast extract and a nonionic surfactant.
Inclusion of such materials in the fermentation medium unex-
pectedly results in a substantial increase in the production of
intracellular cholesterol oxidase.
~o~
The inclusion of yeast extract is essential to
; obtain the high yield of cholesterol oxidase in the improved
fermentation process of this invention. The addition of
; increasing amounts of yeast extract improves the yield of
cholesterol oxidase until it reaches a maximum, after which
point the continued addition of yeast extract represses the
yield of cholesterol oxidase, as will be demonstrated by the
examples which follow. The yield of cholesterol oxidase has
been increased as much as 5000 percent over the prior art by
incorporating optimum quantities of yeast extract into the
growth medium. Generally, yeast extract is used in a con-
centration of at least about 10 g./liter; however, a con-
centration of from about 10 g./liter to about 30 g./liter is
; preferred. Optimum results have been achieved using yeast
extract in the particularly preferred amount of about 20
g./liter. It should be noted, however, that the quantity of
yeast extract which produces optimum results may vary some-
what depending upon the source of the yeast extract.
The production of cholesterol oxidase is strongly
affected by the addition of nonionic surfactant to the
growth medium. Up to threefold increases in the enzyme
yield were obtained by incorporating small amounts of non-
ionic surfactant in the growth medium. Nonionic surfactants
are well-known in the art and no further definition thereof
is required herein. Typical examples of such materiaIs
include polyethylene glycol, polyvinyl alcohol, polyethers,
polyesters and polyhalides.
Of critical importance to the successful practice
of the invention are the criteria that:
3 (1) neither the particular nonionic surfactant used
nor its decomposition product is toxic to the micro-
'71~
organism in the concentrations required to increasethe yield of enzyme and
(2) the amount of surfactant used does not inhibit
enzyme production.
The toxicity of the decomposition products of the
surfactant can be theorized as dessribed briefly herein-
after; however, the only positive test for such a criterion
is evaluatlon in the growth medium and observation of the
effects of by-products produced therein.
Nonionic surfactants include a broad range of
materials and any such material which meets the two criteria
described hereinabove are useful in the successful practice
of the invention.
According to a preferred embodiment wherein cho-
lesterol oxidase is prepared as described hereinabove, the
nonionic surfactant has a hydrophilic moiety comprisin~ 20
units of ~xyethylene and a lipophilic moiety comprising
a fatty acid chain having 16 carbon atoms. For the practice
o~ the present invention, nonionic surfactants equivalent to
the preferred surfactant described above include nonionic
- surfactants having a polyoxyethylene or polyglycidol hydro-
philic moiety and a lipophilic moiety comprising at least 9
carbon atoms. Particularly useful nonionic surfactants are
those in which the lipophilic moiety comprises a fatty acid
chain of at least 10 carbon atoms.
Optimum results are achieved when the fatty acid
chain contains at least 16 carbon atoms and the hydrophilic
moiety comprises about 20 oxyethylene units.
Examples of specific surfactants which are useful
in the practice of the instant invention and their structure
include:
-- 10 --
106'7~
U~
O
~;
, '
S
~ ~ .,~ ~ ~ S 5
~d t~ ~ -1-) ~ ~ ~1 ~ O O O O O
~1 ~ ~ tn u~ tq O O
U~
.
~10 ~ 0 ~0 ~ 0 ~0 ~ O ~ o ~D O
O ~\J ~ 1 N ~1 ~ ~1 ~ ~1 i
. .
; .
~ s s ~ s s s s s ~ s s ~ ~
O
~ ~ O ~ O ~ O ~1 0 rl O rl OX ~ O ~ O X X X
5: O O O O O O O O O O O O O O O O O O O O O
~ . ~
~d ~ D ~ CO O~ 1
~ .
~D C7~ ~ U~ O O ,1 0
m¦ ~O ~ u~ ~ ~ o Ln o ~ Lr~
-- 11 --
67l34~
The concentration of nonionic surfactant used in
any particular growth medium will vary considerably depend-
ing upon the composition of the medium, the sensitivity of
the medium to the particular surfactant and the particular
surfactant used. Generally, however, surfactant concen-
trations of up to about 5.0 g./liter of medium have been
found useful in at least certain fermentation media with
certain surfactant compositions. At levels above 5.O g./liter,
the surfactant generally tends to inhibit the production of
cholesterol oxidase. It is generally preferred, as dem-
onstrated by the exemplary results set forth below, to
utilize surfactant concentrations of from about 1.0 to about
3.0 g./liter of medium.
In the production of cholesterol oxidase using a
nutrient growth medium containing a nonionic surfactant in
accordance with the teachings of this invention, foaming may
be encountered. In order to control the foaming, especially
when producing large batches, the use of a foam-control
agent is advisable. One such foam-control agent found
useful in the practice of this invention is Polyglycol P-
2000, available from Dow Chemical, Midland, Michigan. Other
foam-control agents can also be used, the main criterion for
selection and use being the lack of inhibition of enzyme
synthesis at a concentration level which will control the
foam.
The growth medium includes a primary carbon source
such as, for example, glycerol, glucose, corn syrup, or the
like, and an inducer of cholesterol oxidase which may also
be a secondary carbon source. Besides the cholesterol,
3 cholest-4-en-3-one and cholesteryl linoleate which were
taught as inducers by Goodhue et al, supra, other sterols
and cholesterol esters are useful as inducers of cholesterol
- 12 -
~06~8~
oxidase. Other preferred inducers include~ for example, 3-~-
hydroxy sterols such as ~-sitosterol and 5-~-cholestan-3-~-ol,
and other cholesterol esters such as cholesteryl oleate, choles-
terol linolenate, cholesteryl formate and cholesteryl propionate.
The fermentation process described herein is desir-
; ably run at a temperature of from about 18C. to about 35C.
Temperatures below 30C. are preferred, with temperatures in the
range of from 20C. to about 30C. being most preferred.
We have further discovered, as noted hereinabove,
that Nocardia'chole's-tero'licum also produces a cholesterol esterase.
This enzyme is induced by cholesterol, cholesterol esters, and
certain sterols such as those described above and in the examples.
In general, the sterols are better inducers of the esterase.
There is a good correlation between the production of cholesterol
oxidase and cholesterol esterase from growing Nocardia choles-
terolicum. However, the ratio of esterase to oxidase varies
depending on the inducer.
In the following examples, which are presented to
demonstrate better the successful practice of the invention, the
following definitions apply:
(1) Cultu're
Noc'ardia chol`es'terol'i'c'um was obtained from Dr. Theresa
Stadtman (N.I.H., Bethesda, Md.). A rough colony variant (NRRL
5767) was used unless otherwise stated.
(2) Nutrient media
The compositions of media used in the examples are
as follows:
- 13 -
6789L~L
(A) Glycerol medium
per lite-r
ammonium sulfate 2.0 g.
potassium phosphate (dibasic 2.0 g.
anhydrous)
salt solution "C" 5.0 ml.
glycerol 5.0 g.
tryptone O.l g.
distilled water to 1 liter
salt solution "C": per liter of O.lN HCl
MgS0 ~iH 0 25.0 g.
CaCl2.2H2 0.1 g.
FeS04 7H 0 2.8 g.
MnS04 H~ 1.7 g.
ZnS04 7~ o.o6 g.
NaCl 0.6 g.
(B) Inoculum medium
per liter
glucose lO.0 g.
yeast extract~ 10.0 g.
potassium phosphate (dibasic l.0 g.
anhydrous)
salt solution A-l 2.0 ml.
salt solution A-2 2.0 ml.
agar 20.0 g.
adjust pH to 7.0 and make up to l liter with
; distilled water
salt solution A-l:
per liter
` MgS04 7H20 lO0.0 g.
3 FeS04 7H 0 lO.0 g.
MnSOJ, H ~ l.0 g.
NaMo~4 ~H20 0.5 g.
make up to l liter in O.lN HCl
salt solution A-2:
per liter
CaCl2 2H 0 lO.0 g.
distllle~ water to l liter
*The yeast extract ~sed is commercially
available as BactoWyeast extract from Difco
4 Laboratories, Detroit, Michigan.
- 14 -
~ 1:)67 !344
- (C) Modified glycerol medium
per liter
ammonium sulfate 2.0 g.
potassium phosphate (dibasic, 2.0 g.
anhydrous)
salt solution "C" 5.0 ml.
glycerol 5.0 g.
surfactant S-3 3.0 g.
yeast extract* 20.0 g.
cholesterol 1.0 g.
distilled waterto 1 liter
*The yeast extract used is commercially
available as Bacto yeast extract from Difco
Laboratories, Detroit, Michigan.
(3) Maintenance of the culture
The cultures were maintained on the slants of
glycerol medium containing cholesterol and were transferred
every second day. In addition, the cultures were also kept
frozen in liquid nitrogen.
(4) Preparation of inoculum (small-scale use)
An inoculum medium slant was inoculated with
Nocardia cholesterolicum (rough) from a 2-day-old glycerol
medium slant and was incubated at ~0 C. for 48 hr. The
culture from this slant was scraped off with a wire loop and
was resuspended in 25 ml. of sterile distilled water by
vigorous shaking. The turbidity of this suspension was
generally between 1.8-2.2 O.D. units at 660 nm. Sixty ml.
of this suspension were used as inoculum per liter of the
growth medium.
3 (5) Preparation of inoculum for large-scale fermenta-
tion
Nocardia cholesterolicum (rough) grown for 48 hr.
on inoculum medium slants was used to inoculate 7.5 liters
of sterilized modified glycerol medium in a 14-liter fer-
menter (Chemapec, M~nnedorf, Switzerland). Eight slants
; were used for this purpose. The medium was aerated at 0.5
vvm. and agitated with flat 3-blade turbine impellers at
- 15 -
. ~ . ~
~L06784~
1300 rpm. The temperature was maintained at 30 C. After
18 hr. of incubation, the contents of the fermenter were
aseptically transferred to a 150-liter fermenter.
(6) Fermentation
(A) Small-scale
The fermentations were carried out in 250-ml.
Erlenmeyer flasks. The volume of medium used in Erlenmeyer
flasks was 25 ml. The medium in the flasks was inoculated
as described above and was incubated at 30 C. The shaker
speed was adjusted to 200 rpm. (2-in. throw). The samples
were withdrawn aseptically every 24 hr. for the measurement
of the cholesterol oxidase activity.
(B) Large-scale
The 150-liter fermenter used for the large-scale
fermentation contained 75 liters of sterilized modified
glycerol medium. After inoculation as described in (5), the
medium was maintained at 30 C. and was aerated and agitated
at 0.5 vvm. (volume of air per volume of medium per minute) and
250 rpm., respectively. Every 2 5 hr., samples were withdrawn
aseptically with`an automatic sampler. The samples were assayed
for cholesterol oxidase activity as described in (8). The cells
were harvested when the level of cholesterol oxidase reached the
maximum. The time required for the fermentation was between 17-25 hr.
(7) Harvesting cells ~~~-~ ~~
(A) Small-scale
The cells were harvested (i.e., separated from the
fermentation broth) by 15 min. centrifugation in a refrig-
erated centrifuge (duPont Co., Instrument Products Div.,
Sorvall Operations, Newtown, Conn.) at 12,350 x g.
(B) Large-scale
The fermenter was cooled with cold water when the
production of the enzyme reached the maximum. The cells
- 16 -
67~
were separated from the br~h with a continuous centrifu~e
having a bowl with a capacity of 8 liters (Cepa Centrifuge,
West Germany). The cells were further processed for the
isolation and purification of cholesterol oxidase.
' (8) Determination of cholesterol _xidase activity
.~ (A) Preparation of cell fractions for the assay
of cholesterol oxidase
~holesterol oxldase can be present outside the
cell in ~he fermentation medium (or extracellularly) and inside
3 10 the cell (or intracellularly). Further, the intracellular enzyme
,
can be present as free or sQluble enzyme and as bound or insoluble
¦ enzyme. ~he extracellular enzyme can be assayed in the broth after
; the removal of the cells by centrifugation. To measure the
, intracellular enzyme, the cells are disrupted by sonication.
j The cell pellet obtained by centrifugation is
suspended in l ml. of distilled water and diluted to 20 ml.
with 50 mM potassium phosphate buffer (pH 7.0). It is
sonicated for 5 min. in an ice-water bath, in l-min. bursts
at 30-sec. intervals. The sonicated suspensions are cen-
tri~uged at 27,000 x g for 15 min. in the cold. The activity
¦ in the supernatant is called the intracellular, soluble
activity The pellet is resuspended in 2% sodium deoxy-
cholate and allowed to stand on ice ~or 10 min. It then is
r centrifuged at 27,000 x g for 15 min. in the cold. The
cholesterol oxidase activity in the supernatant is called
; the intracellular, insoluble activity. The sum of the
.:
'~ extracellular, the intracellular soluble and the intracel-
lular insoluble activities is called the total activlty. It
is also possible to measure the total activity witho~t
breaking the cells. For this purpose, the whole fermen-
tation broth containing cells is diluted to minimize the
e ~.
:,
~ - 17 -
... ~
~04~71~4~`
interference due to its turbidity and is used as an enzyme solu-
tion.
(9) Enzym _ ssay
Cholesterol oxidase activity is measured by the follow-
ing technique:
(A) Reagents:
1. 50 mM potassium phosphate buffer, pH 7.0 (KP buffer):
30.5 ml- 0-2 M K2HPO4 + 19.5 ml. 0.2 M KH2PO4 + water to 200 ml.
final volume.
2. 0.1~ dianisidine solution: 10 mg. 3,3'-dimethoxy-
benzidine dihydroc ~ ide per ml. water. No pH adjustment.
3. Reagent buffer: Add 0.5 ml. dianisidine solution
and 1.4 mg. peroxidase powder (Sigma Type II, horseradish per-
oxidase, RZ 1.0 - 1.5 No. P8250) to 40 ml. KP buffer, mix, dilute
to 50 ml. with KP bufer. The solution will turn turbid when
the dianisidine is added but clears w;hen mixed. This solution
should be kept cold until ready to use. We have stored reagent
buffer at 4 C. for 3 days without problems, but routinely this
reagent is prepared fresh daily.
4. Cholesterol solution: To 10 ml. Triton X 10 ~
(available from Rohm and Haas, Philadelphia, Pa.) heated on a hot
plate, add 500 mg. cholesterol powder and mix wi-~h a magnetic
stirrer until solution clears. Add 90 ml. water and stir. The
solution will be cloudy. Now continue mixing the flask by
swirling it under a stream of cold water; the solution will become
clear.
This solution is stable for 1 wk. when stored at room
temperature.
- 18 -
~067~4
(B) Reactions:
detergent
cholesterol ~ 2 ~ cholest-4-en-3-one
oxidase
~: H202
peroxidase
H2O2 ~ dianisidine -~2H2O ~ dye
(C) Assay:
Six ml. of reagent buffer plus 0.1 ml. substrate plus
0.9 ml. water are combined in a test tube, mixed, and placed in
a water bath set at 37 C. After 5 min., 1.0 ml. enzyme is
added to give 8 ml. final volume in the tube. An initial
reading at 430 nm. on a Spectronic 20 spectrophotometer (Bausch
and Lomb) is recorded. The tube is replaced in the water bath.
Tubes are read in the spectrophotometer every 5 min. for 25 min.
Rate of color development is determined from a plot of O.D.
change vs. time by averaging the O.D. change throughout the
linear portion of the curve. Activity is calculated using a
constant (molar extinction coefficient) previously determined
for the dye system from a standard curve. Enzyme preparations
are diluted so that 0.005 to 0.06 unit of cholesterol oxidase
are used per assay tube.
One unit of cholesterol oxidase activity is that
amount of enzyme ca-talyzing the production of l~mole H202/min.
at 37 C. and pH 7Ø
- 19 -
~Cl16'~44
(10) De~ermination_of cholesterol esterase activity
The esterase activity is assayed by enzymatically
measuring cholesterol released by hydrolysis of cholesterol
linoleate~ Cell fractions for the assay of cholesterol
esterase are prepared the same as in ~8)(A) above.
(A) Reagents:
1. Substrate emulsion: Six-tenths gram of cholesteryl
linoleate is dissolved in 10.0 g of hot Triton X-100 and made
up to 100 ml with deionized water. The solution is cooled under
running cold water to dissolve the detergent. This results in
milky white emulsion of cholesteryl linoleate.
2. Esterase assay mixture: The esterase assay mix-
ture contains 6.7 ml of buffer solution, 0.3 ml of cholesteryl
linoleate emulsion, 0.5 ml o cholesterol oxidase solution
~l U/ml) and 0.5 ml of appropriately diluted call suspension.
(B) Procedure:
Tubes containing the assa~ mixture without the cell
suspension are shaken in a water bath at 37C. for 15 minutes.
Then the cell suspension is added alld transmittance at 430 nm
is measured at 5-mlnute in~ervals. The amount of the enzyme
is computed from the rate of calor development. This assay
procedure is similar to ~hat used to measure cholesterol oxidase.
One unit of cholesterol esterase activity is that
amount catalyzing the hydrolysis of 1 ~mole cholesterol
linoleate/minute at 37~C and pH 7Ø
The invention can be il-Lu~r~ted by the following
examples. Unless otherwise indicated, concentrations given
in pPrcent are weight per~ent. The yields from small-scale
experiments conducted using flasks are generally lower than
those obtained in large-scale ~ermenters; however, reIative
effects of various fermentation parameters have been found
to be similar.
_ 20
:~L06'7~4~
Example 1: Effect of yeast extract
Yeast extract was essential for the production o~
cholesterol oxidase (Table 1). The yield of the enzyme
increased from 2.7 U per liter obtained in the absence of
yeast extract to 140.4 U per liter obtained in the medium
with 2.0 percent yeast extract. Further increase in the
concentration of yeast extract repressed the synthesis of
the enzyme. Yeast extract also affected the location of the
enzyme. In the presence of yeast extract, at most 5 percent
of the enzyme was extracellular, and the rest was intra-
cellular. The maximum enzyme titre was obtained in 24 hr.
Table_l
Effect of Yeast Extract on the
Production of Cholesterol Oxidase
Concentration Cholesterol Oxidase Enzyme
of Yeast U/Liter Released
Extract ~ _ Extracellular Total % of Total
0.0 2-7 2.7 100
0.1 0.50.5 100
0.5 0.320.5 2
1.0 2.556.2 5
2.0 2.7140.5 2
3.0 0.268.1 0
Glycerol medium used in this experiment contained 0.01
percent trypkone, 0.5 percent surfactant S-3 and was sup-
plemented with Bacto yeast extract as shown.
Surfactant S-3 is commercially available as Tween~
40 from Atlas Chemicals, Wilmington, Delaware.
Example 2:- Effect of cholesterol (small-scale in Erlenmeyer
3 flasks)
There was no enzyme synthe~is in the absence Or
cholesterol (Table 2). In the medium containing 0.5 percent
S-~, there was a dramatic increase in the amount of the
enzyme produced on supplementing the medium with cholesterol
(Table 2). The production of cholesterol oxidase reached a
maximum in the medium containing 0.1 percent cholesterol.
- 21 -
7~34~
There was no further increase in the enzyme yield on increas-
ing the concentration Or cholesterol.
Cholesterol had a less pronounced effect on the
cell growth. There was, at most, a 14 percent increase in
the dry cell weight upon the addition of cholesterol to the
` medium.
Essentially similar observations were made on the
effect Or cholesterol on the production of the enzyme in a
modified glycerol medium with 0.3 percent S-3. There was
some increase in the yield of cholesterol oxidase when the
concentration of cholesterol was increased abo~e 0.1 percent
in the medium containing 0.3 percent S-3.
Table 2
Effect of Cholesterol on the Production
of Cholesterol Oxidase in the Presence
of 0.5 Percent Surfactant S-3
Concentration Dry Cell
of Cholesterol Oxidase Weight
Cholesterol %U/Liter g./Liter
;, - .
o.o o.o - 8.3
0.02 76-7 8.9
o.o5 126.2 8.8
0.1 143.5 8.5
0.2 124.8 7.7
`0.5 142.2 9.5
The medium used to obtain the results in Table 2 contained
0.5 percent S-3; otherwise, it had the same composition as
modified glycerol medium. Cholesterol concentration of the
medium was adjusted as described.
3 Example 3: Effect Or Surfactant S-3
The production Or the enzyme was strongly afrected
by the surfactant S-3 (Table 3). A threefold increase in
the enzyme yield was obtained on supplementation of the
medium with 0. 2 percent surfactant (Table 3). The maximum
production of the enzyme was obtained with the concentration
of S-3 betweén 0. 2 percent to 0.3 percent (Table 3). Higher
- 22 -
concentrations Or S-3 (>0.3%) inhibited the production of
the enzyme. At the most, o.6 percent of the enzyme was
released into the medlum.
Table 3
E~fect o~ Surfactant S-3 on the
Production of Cholesterol Oxidase
Dry Cell
Concentration Weight Cholesterol Oxidase
of S-3 g./Liter U/Liter
.
- o.0 6.o 39.6
0.1 6.9 85.4
0.2 4.7 129.1
0.3 6.6 124.7
0.5 6.o 72.4
Modified glycerol medium with varying concentration of
Surfactant S-3 was used to obtain the results in Table 3.
Example 4: Effect of glycerol
Omission of glycerol from the medium reduced the
enzyme production by 28 percent (Table 4). The enzyme level
was restored almost to the control level upon addition of
0.25 percent glycerol. Further increase in the glycerol
concentrat~on did not change the enzyme level significantly.
The growth of the culture also was affected by
glycerol. Almost 50-percent increase in the dry cell weight
was obtained in the medium with glycerol over that obtained
in the medium without glycerol (Table 4).
Table 4
Requirement of Glycerol for the
Production of Cholesterol Oxidase
3 Dry Cell
Concentration Weight Cholesterol Oxidase
of Glycerol % ~/Liter - U/Llter % of Control
0.0 5.8 108.9 72
0.25 8.2 142.3 95
0.5 8-3 150.5 100
aModified glycerol medium which contained 0.5%
glycerol was considered the control.
- 23 -
,. ~
~067~
Example 5: Sterols and cholesterol esters as inducers
- for cholesterol oxidase
Example 2 demonstrates the effect of cholesterol
on the synthesis of cholesterol oxidase. Four sterols other
than cholesterol and twelve cholesterol esters were tested
for their ability to induce cholesterol oxidase. All the
inducers were studied at a concentration of 0.1 percent.
The most efficient inducer of cholesterol oxidase was ~-
sitosterol (Table 5). The total enzyme activity induced was
122 percent of that induced by cholesterol. Two other
steroids, 5-~-cholestan-3-~-ol and cholest-4-en-3-one, were
moderately effective in inducing the enzyme. Of the choles-
terol esters tested (Table 6), cholesteryl oleate and cho-
lesteryl l~inoleate induced approximately 90 percent of the
enzyme induced by cholesterol while cholesteryl propionate
and cholesteryl linolenate induced 75 percent and 65 per-
cent~ respectively.
:
Table 5
Effectiveness of Sterols as Inducers
Enzyme
Total Activity Activity %
Sterol (U/Liter) _ of Controla
cholesterol 163.6 100
~-sitosterol 199.5 122
5-~-cholestan-3-~-ol105.6 65
' cholest-4-en-3-one101.0 62
7-dehydrocholesterol 0 0
Modified glycerol medium was used in this
aeXperiment .
The control medium contained cholesterol as the
,binducer.
Concentration Or sterol tested was 0.1 percent.
- 24 -
~o~
Table 6
Effectiveness_of Cholesterol Esters as Inducers
Concen- Oxidase
b tration Activity %
Cholesterol Ester (mmoles) of Controla
cholesterol 2.6 100
cholesteryl formate 2.4 64
cholesteryl propionate 2.3 75
cholesteryl butyrate 2.2 54
cholesteryl hexanoate 2.1 52
cholesteryl benzoate 2.0 13
cholesteryl _-nitrobenzoate 1.9 0 -
cholesteryl decanoate 1.9 26
cholesteryl laurate 1.8 36
cholesteryl myristate 1.7 21
cholesteryl palmitate 1.6 14
cholesteryl oleate 1.5 91
cholesteryl linoleate 1.5 89
cholesteryl linolenate 1.6 65
Modified glycerol medium was used in this
experiment.
aThe control medium contained cholesterol as the
binducer .
Concentration of cholesterol esters tested was
0.1 percent.
Example 6: Substitutes for Bacto yeast extract
,
Example 1 illustrates the importance of yeast
extract to the present invention. In this example several
yeas~ hydrolysates were studied (Table 7). The effective-
3 ness of 2.0 percent Amber BYF 100 and 2.0 percent Amber BYF
~:
50X were comparable with 2.0 percent Bacto yeast extract.
The best yeast hydrolysate, Amberex 1003, at a concentration
of 1.0 percent stimulated enzyme production to a level 112
percent of the control. Further studies to determine the
optimum concentration of Amberex 1003 demonstrated maximum
activity, well over twice that of the control. Amber and
Amberex yeast hydrolysates are available commercially frorn
Amber Laboratories, Juneau, Wisconsin.
- 25 -
1~67~4~
, ~ .
Table 7
Effectiveness of Yeast Hydrolysates for
the Production of Cholesterol Oxidase
Concen- Cholesterol Enzyme
Yeast tration Oxidase Activity %
Hydrolysates % (U/Liter) of Controla
Difco (Bacto 2.0141.2 loO
Yeast Extract
Amber BYF 100 O. 5 51.7 37
l.o91.8 65
2.0152.4 108
5.044.2 34
Amber BYF 50X 0.5 0 0
1.0 0 '
~ 2.0127.3 go
; 5.0 o o
: Amber BYF 300 o.5 53.6 38
l.o65.7 47
2.095.0 67
Amberex 1003 o.5122.4 87
1.0157.8 112
2.0153.9 lo9
5.057.0 40
Modified glycerol medium was used in this
aeXperiment .
The control medium contained Difco Yeast
Extract.
Yeast extract obtained from sources other than
Difco does not always work as well. In fact, considerable
3 batch-to-b,atch variation from alternate sources has been
found, although no such difficulty has been experienced with
Difco yeast extract. Each supply of yeast extract should be
tested using 2 percent Difco yeast extract as a control as
in Table 7. Any yeast extract which yields 70 percent of
the control when using a concentration of up to 5.0 percent
,in the modified glycerol medium is considered to be equiva-
lent to Difco yeast extract for the purposes described
herein.
- 26 -
3L067~4
Example 7: Effect of cholesterol on production of cho-
lesterol oxidase in large-scale fermenter
The 150-liter fermenter used for the large-scale
fermentation was charged with 75 liters Or modified glycerol
medium containing 0.3 g /liter o~ Polyglycol P-2000 and
various concentrations of cholesterol as shown in Table 8
below. After sterilization, the medium was inoculated as
described hereinabove with inoculum prepared as described
above. The medium was aerated at O.~ vvm. and agitated with
flat 3-bladed turbine impellers at 250 rpm. Samples were
- withdrawn and assayed every 2.5 hr. until the production of
cholesterol oxidase reached the optimum value.
; . ~
Table 8
I
Effect of Cholesterol on the Production of
Cholesterol ~Oxidase in the Large-Scale Fermenter
Concentra~ion of Cholesterol Oxidase
` Cholesterol g./Liter U/Liter
1.0 177
~ 2.0 356
- 20 3.0 749
4.0 888 -
,. ~: ,.
-~ Each value in Table 8 above is an average of four
or more experiments.
Example 8: Comparison of modified glycerol medium to prior
art media
Samples of various strains known to produce cholesterol
oxidase were obtained from the NRRL and ATCC culture collections and
were used to compare the effect of the present modified glycerol
medium to that of two prior art media discussed hereinabove. The
NRDC medium was prepared on a small scale according to the ingredients,
proportions, and methods specified in British P~atent 1,385,319. The
B-M medium was prepared on a small scale according to the ingredients,
proportions, and methods specified in Example 2 of Republic of South
Africa Patent 73/3259 exept using ammonium acetate instead of casein
peptone. The various bacteria were then grown in each of the three
redia, and their cholesterol oxidase activity was measured. The
- 27 -
106~71344
results in Table 9 illustrate the improvement and utility of
the present medium in comparison to the prior art. The data for
each strain has been normalized against the control for easy com-
parison of results.
'
.
~ .
.
.
- 28 -
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3 ta a
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,~
o
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0 ~J I` O ~I Q ~ O C~-rl
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'~ ~ ~:~o
:: e
.C .
q~ ~3
,` ~ ~ o ~ U~ ~ S ~l
~ ~ ~ ~ ~ ldX~
.U~ 0 ~1 r4
.,~ .,. .a) ;~i o o o o o o o ~rl
Sl ~ . OOO OOO U~r-l~
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29 --
~16'i~
In one other experiment the strain NRRL 5767 apparently
produced about 93~ of control when grown in the NRDC medium but
we were no able to repeat this experiment~
E~ Esterase and oxidase production in
modified glycerol medium
(a) To test their effect, the sterols were added in
place of cholesterol a~ the concentration of 0.1%. Cholesterol
concentration in modified glycerol medium was 0.1~.
The highest level of the enzyme was induced by ~-
sitosterol and 5-~-cholestan-3 ~-ol (Table 10~. Intermediate
en~yme yields were obtained with cholesterol and cholest-4-
en-3-one. No enzyme was found in the medium with 7-dehydro-
cholesterol.
Tab
Effect of Sterol~ on the Production of
Cholesterol Esterase a~d Cholesterol
Oxidase in Modified Glycerol Medium _ _
Dry Cell
Weight E~terase Oxidase
Sterols ~ U/Liter U/Liter
.
cholesterol 6.3 37.9121.6
-sitosterol 7.7 48.5119.9
5-~-cholestan-3-~-ol 6.8 49.2 72.3
chole~t-4-en-3-one 7.9 21.5 44.0
7-dehydrocholesterol 7.6 0.0 0.0
(b) To te~t the effect of various esters, 0.1% of
the ester to be tested was added in place of cholesterol. Modi-
fied glycerol medium contained 0.1% cholesterol.
All six of the cholesterol esters ~ested in modified
glycerol medium induced the esterase (Table 11)~ The m~ximum
yield of the enzyme was obtained in the presence of cholesteryl
linoleate. The levels of the esterase induced by the remaining
five esters were about half that produced with the linoleate
ester.
- 30 -
~Ot;'7~
Table 11
Ef~ect of Cholesterol Esters on the
Production of Cholesterol Esterase and
Cholesterol Oxidase in Modified GlYcerol Medium
Dry Ce~l
Weight Esterase Oxidase
Cholesterol Estersg/Liter U~Liter U/Liter
cholesterol 6.3 37.9121.6
cholesterol propionate 7.6 14.9 45.3
cholesteryl butyrate 7.6 19.638.6
cholesteryl hexanoate 8.2 14.959.5
cholesteryl oleate 8.2 16.087.2
cholesteryl linoleate 6.8 49.795.9
cholesteryl linolenate 7.6 23.0 71.6
Cholesterol oxidase production in the presence of
cholesterol and ~-sitosterol was significantly higher ~40-60~)
~han the other three sterols tested (Table 10). Cholester~l
oleate and cholesteryl linoleate also induced high levels of
the oxidase (Table 11). Moderate le~els of the enzyme were
produced in the medium with the oth~r four cholesterol esters
(Table 11).
, Although the in~ention has been described in consider
able detail with particular reference to certain preferred em-
bodLments thereof, variations and modifications can be effected
within the spirit and scope of the invention.
- 31 -
