CA1067844A - Method and medium for producing cholesterol oxidase - Google Patents

Abstract

Abstract of the Disclosure An improved fermentation method comprises growing a cholesterol oxidase-producing microorganism in a medium comprising a carbon source, such as glycerol, yeast extract, a nonionic surfactant, an auxiliary carbon source which is also an inducer of cholesterol oxidase, and trace salts. Yields of cholesterol oxidase up to about 900 U. (international units) per liter have been obtained using the improved method of the present invention.

Description

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~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, ~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.

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.

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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:

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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 ~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:

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(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.

~ 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 . ~ . ~

~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 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 ~.
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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.

~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

~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.

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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 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.

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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.

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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.

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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.

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.
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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 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.

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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.

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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.

Claims (12)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for producing cholesterol oxidase comprising:
growing a cholesterol oxidase-producing microorganism in a medium comprising a cholesterol oxidase inducer, from about 1.0 to about 5.0 g/liter of a nonionic surfactant which is non-toxic to the microorganism, and at least about 10 g./liter of yeast extract, thus producing cholesterol oxidase; and recovering the cholesterol oxidase.

2. A method for producing cholesterol oxidase com-prising:
growing a cholesterol oxidase-producing microorganism of the order Actinomycetales in a medium comprising a cholesterol oxidase inducer, from about 1.0 to about 5.0 g./liter of a non-ionic surfactant which is non-toxic to the microorganism, and at least about 10 g./liter of yeast extract, thus producing cholesterol oxidase; and recovering the cholesterol oxidase.

3. A method for producing cholesterol oxidase com-prising:
growing a strain of microorganism selected from the group consisting of the families Mycobacteriaceae, Nocardiaceae, Streptomycetaceae and Corynebacteriaceae in a medium comprising a cholesterol oxidase inducer, from about 1.0 to about 5.0 g./
liter of a nonionic surfactant which is non-toxic to the bacterium, and at least about 10 g./liter of yeast extract, thus producing cholesterol oxidase; and recovering the cholesterol oxidase.

4. The method for producing cholesterol oxidase as described in claim 3 wherein said inducer is cholesterol, a cholesterol ester or a 3-.beta.-hydroxysterol.

5. The method for producing cholesterol oxidase as described in claim 3 wherein said inducer is present in a concentration of from about 1.0 to about 10 g./liter and said yeast extract is present in a concentration of from about 10 to about 30 g./liter.

6. A method for producing cholesterol oxidase comprising:
growing a strain of microorganism selected from the group consisting of NRRL 5635, NRRL 5636, NRRL 5767, NRRL 5768, ATCC 4277, ATCC 14349, and ATCC 17895 in a medium comprising from about 1.0 to about 10 g./liter of an inducer selected from the group consisting of cholesterol, cholest-4-en-3-one, .beta.-sitosterol, 5-.alpha.-cholestan-3-.beta.-ol, cholesteryl linoleate, cholesteryl oleate, cholesteryl formate, cholesteryl propionate and cholesterol linolenate, from about 1.0 to about 5.0 g./liter of a nonionic surfactnat having a polyoxyethylene or polyglycidol hydrophilic moiety and a lipophilic moiety comprising at least 9 carbon atoms, and from about 10 to about 30 g./liter of yeast extract, thus producing cholesterol oxidase; and recovering the cholesteorl oxidase.

7. The method for producing cholesterol oxidase as described in claim 6 wherein said nonionic surfactant comprises a hydrophilic moiety having at least about 20 units of oxy-ethylene and a lipophilic moiety having a fatty acid chain containing at least 16 carbon atoms.

8. The method for producing cholesterol oxidase as described in claim 6 wherein said inducer is selected from the group consisting of cholesterol, .beta.-sitosterol, cholesteryl oleate, cholesteryl linoleate and cholesteryl propionate, and said nonionic surfactant comprises a hydrophilic moiety con-sisting of 1 unit of sorbitan and 20 units of oxyethylene and a lipophilic moiety consisting of 1 unit of palmitic acid.

9. A method for producing cholesterol oxidase comprising:
growing a strain of microorganism selected from the group consisting of NRRL 5635, NRRL 5636, NRRL 5767, NRRL 5768, ATCC 4277, ATCC 14349, and ATCC 17895 in a medium comprising from about 2.0 to about 5.0 g./liter of an inducer selected from the group consisting of cholesterol, .beta.-sitosterol, cholesteryl oleate, cholesteryl linoleate and cholesteryl propionate, from about 1.0 to about 5.0 g./liter of a nonionic surfactant com-prising a hydrophilic moiety consisting of 1 unit of sorbitan and 20 units of oxyethylene and a lipophilic moiety consisting of 1 unit of palmitic acid, and from about 10 to about 30 g./liter of yeast extract, thus producing cholesterol oxidase; and recovering the cholesterol oxidase.

10. The-method for producing cholesterol oxidase as described in claim 9 wherein said inducer is cholesterol or .beta.-sitosterol and said yeast extract is present in a con-centration of about 20 g./liter.

11. A method for the production of cholesterol oxidase which comprises:
growing a bacterium selected from Nocardia cholester-olicum species NRRL 5767 and NRRL 5768 in a medium comprising from about 1.0 to about 10 g/liter of a cholesterol oxidase in-ducer, from about 1.0 to about 5.0 g/liter of a nonionic sur-factant having a hydrophilic moiety comprising at least 20 units of oxyethylene and a lipophilic moiety comprising a fatty acid chain with at least 16 carbon atoms, and from about 10 to about 30 g/liter of yeast extract; and separating the cholesterol oxidase from the bacterium.

12. A method for the production of cholesterol esterase which comprises:
growing a bacterium selected from Nocardia cholester-olicum species NRRL 5767 and NRRL 5768 in a medium comprising a cholesterol esterase inducer and at least 10 g/liter of yeast extract and separating the cholesterol esterase from the bacterium.