CA1046437A - Enzyme preparations and method of preparing same - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Enzyme preparations which will convert cholesterol to .DELTA.4-cholestenone and hydrogen peroxide are obtained from certain Nocardia species belonging to the Mycobacterium rhodocrous group. The preparations have a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen and, when in liquid form, a potency of at least 10-2 units/ml. They are prepared by growing the organism and recovering the enzyme preparation, preferably by extracting the harvested cells with a surface active agent such as TRITON X-100*.
The enzyme preparations are used to assay for cholesterol by measuring the amount in which one of the products of the cholesterol oxidase reaction, preferably hydrogen peroxide, is formed or the quantity in which oxygen is used.

Description

~()4~;43'7 I This application is a divisional application o~ appli-cation serial number 163,288 filed on February 6, 1973.
This invention relates to enzyme preparations suitable for use in the assay of cholesterol in liquids and particularly biological fluids such as serum, and to the production of such enzyme preparations.
The assay of total cholesterol, in its role as an indi-cator of atherolsclerosis and incipient coronary heart disease, now constitutes about 3~ of the total number of tests performed in 1~ the average clinical chemistry laboratory. In Britain at the present time this represents about 1~ million cholesterol assays per year.
Cholesterol is currently assayed by the Liebermann-Burchard reaction which involves the use of highly corrosive and viscous reagents and presents many obstacles to automation.
It is an object of the present invention~to provide an assay for cholesterol which does not have the disadvantages of the Liebermann-Burchard reaction and which can be readily automated.
Many Nocardia species are capable of metabolising choles-terol and in particular Stadtman et al IJ. Biol. Chem. (1954) 206511 - 523) has disclosed that a soil Mycobacterlum is capable of oxidising cholesterol to ~4-cholestenone. The so-called "cholesterol dehydrogenase" responsible for this reaction can be obtained in a cell-free form of low activity. The production of this "cholesterol dehydrogenase" from the same soil Mycobacterium as a slightly purer soluble enzyme preparation and the determination of the activlty of the enzyme is described by Stadtman in Methods in Enzymology 11955) 1 678 - 681 but the soluble enzyme is still of low activity and no significant purification was achieved.

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- 10~37 1 1~ has now been found that two micro-organisms identified as ~ocardia species belonging to the so-called Mycobacterium rhodocrous group have cholesterol oxidase activity, i.e. they convert chole-sterol to ~4-cholestenone and hydrogen peroxide, and that this provides the basis for an enzymic assay for cholesterol which can be readily automate~ and which obviates the difficulties of the Liebermann-Burchard reaction. The present invention is not limited however, to the two micro-organisms identified as Nocardia species but is applicable to any micro-organism having cholesterol oxidase activityO
`- These two micro-organisms are re~erred to as "rough" and "smooth" strains and have been given the numbers NCIB 10554 and NCIB 10555 respectively by the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen. The micro-organisms have also been deposited with the Agricultural Research Service of the United States Department of Agriculture as the ARS Culture ! Collection Investigations Fermentation Laboratory, Peoria, Illinois, U.S.A. where they have been given the numbers NRRL 5635 and NRRL
~ 5636.
; 20 Full details of the organisms are as follows:
Rough Colony NCIB 10554 (NRRL 5635) orphology (Nutrient agar 30C) , , ' ' Gram-positive, coryneform organisms. No well-developed mycelium, but rudimentary branching present. Cocoid forms appear as the culture ages. Non-motile.
Colonial morphology (nutrient agar 5 days 30C) Circular, flat, entire dry, opaque, craamy-orange colonies.
1.5 - 3 mm in diameter.
~- Yeast Dextrose Agar 5 days 30C
, Circular, raised, entire, opaque, pale pink colonies. Dry crusted surface. 1 mm diameter.

-~. , 10~6437 1 i~ela-in Agar .
Irregular colony edge, raised, dull crusted surface; opaque off-white colour.
Egg Yolk Agar plates ; .
Irregular colony edge, raised, dull rough surface, opaque, buff-coloured.
Characteristics in liquid culture White surface pellicle, white floccular deposit which does not completely disperse on shaking, no turbidity.
. 10 PhysiologY
Strict aerobe Gelatin hydrolysis +
Casein hydrolysis Starch hydrolysis Kovacs oxidase Catalase +
Urease +
Indole - _ Vogue-Proskauer ~V-P) test - -Methyl Red Deamination of phenylalanine ` Hippurate hydrolysis Litmus milk alkaline Utilisation of compounds as sole carbon sources -; Citrate +
Lactate +
. Malate Succinate +
Carbohydrates (acid) No acid was detactable in peptone water sugars.
Ammonium based sugars ' ~04~4;37 1 ~urctose +
`~ Glucose Sucrose +
~laltose .
Glycerol -~
` Sorbitol +
Trehalose Raffinose Dulcitol +
. ~ .
10 Lactose Mannitol Starch Arabinose Smooth colony NCIB 10555 (NRRL 5636) ~ Morphology (Nutrient agar 30 C) - Gram-positive, coryneform organisms. No well-developed mycelium, but rudimetary branching present. Coccoid forms appear as the culture ages. Non-motile.
Colonial morphology (Nutrient a~_r 5 days 30C) :................................................................. : . . .:
~ 20 Circular, entire, convex, semi mucoid, opaque creamy off-white . . .
~ colonies, 0.5 - 2 mm diameter.
- Yeast Dextrose agar 5 days 30 C
;- Convex, entire, smooth shiny, semi mucoid, pale pink colonies.
Gelatin agar ..
Convex, entire, smooth shiny, off-white, opaque colonies.
Egg Yolk agar plates Convex, entire, opaque, smooth shiny colonies. Moist surface.
l - 3 mm diameter.
Characteristics ln liquid culture White surface pellicle, white floccular deposit which does not completely disperse on shaking.
-~?

~, '~; ' r -~LO~ 37 Physiology , ^:` S~rict aerobe Gelatin hydrolysis +
- Casein hydrolysis Starch hydrolysis Kovacs oxidase Catalase +

- 10 Indole Voges-Proskauer (V-P) test Methyl Red Deamination of phenylalanine Hippurate hydrolysis Litmus milk alkaline Utilisation of compounds as sole carbon sources Citrate +
Lactate Malate +
Succinate +
Carbohydrates ¦acid) Acid was not detactable in peptone water sugars.
Ammonium based sugars.
Fructose ~- Glucose :~ 30 Sucrose ~ Maltose +
,: Glycerol +

,~ .

3~
1 Sorbitol -~
Trehalose ~affinose +
~ulcitol +
Xylose +
Arabinose ~ +
Mannitol -~ Starch The present invention provides an enzyme preparation derived : 10 from a cholesterol oxidase producing micro-organism of the Myco-bacterium rhodocrous group (two examples of which are derived from Nocardia species NCIB 10554 or NCIB 10555) having a cholesterol oxidase specific activity of at least l unit per 5 mg of protein nitrogen.
The preparation may be in liquid form or in solid, e.g.
freeze-dried form. When the preparation is in solid form it must be reconstituted with buffer into a liquid form before it can be used in an assay.
Of ~le two micro-organisms ocardia species WCIB 10554 (the "rough" strain) is preferred because of the greater abllity of the cells of this strain to oxidise cholesterol to 4-cholestenone and hydrogen peroxide.
The minimum potency of the liquid cholesterol oxidase preparation depends on the assay method for which it is to be used.
Thus in the case of a preparation which is to be used for the assay of cholesterol by the fluorimetric determination of hydrogen peroxide produced the potency need only be relatively small, a potency of lO 2 units/ml of liquid preparation being sufficient.

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L3~7 1 The assay for chole~terol can also be carried out by using non-fluorimetric methods for the determination of hydrogen peroxide, e.g. colorimetric methods, or by measuring the amount in which ~ -cholestenone is formed or oxygen is consumed in the cholesterol oxidase reaction. In this case the potency of the cholesterol oxidase in the liquid preparation should be at least 10 1 units/ml.
Particularly for use in automated analysis it is pre-ferred that the enzyme preparation have a cholesterol oxidase specific activity of at least 1 unit per 50 ~g protein nitrogen and when made up in liquid form have a potency of at least 0.5 unit/ml.

When the assay depends on the determination of hydrogen peroxide the presence of catalase in the enzyme preparation reduces the sensltivity of the assay. Generally a cathode activity of less than 10% of the cholesterol oxidase acti~ity i.e. less than 10 1 units of catalase activity per unit of cholesterol oxidase activity, is tolerable and preferably for the preparation should have a catalase activity of less than 1%
of the cholesterol oxidase activity, i.eO less than 10 2 units of catalase activity per unit of cholesterol oxidase activity.

- The catalase activity of the enzyme preparation depends on the method by which is has been prepared and when the preferred method is used as described below the preparation contains only small amounts of catalase which do not reduce the sensitivity of the assay. However if other methods are used and the preparation does include catalase this can either be removed in the purification step or inhibited with a catalase inhibitor, such as an azide, e.g. sodium azide.

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1 As used herein one unit of cholesterol oxidase activity is defined as that activity which will oxidase 1 ~ mol - ~la 6 mol) of cholesterol to Q4-cho:Lestenone and hydroyen peroxide per minute at 30C and pH7. One unit of catalase activity is defined as that activity which will convert 1 ~u mol of hydrogen peroxide to water and oxygen per minute at 25C
and pH7.

.. In general the enzyme preparations according to the invention are prepared by growing Nocardia species NCIB 10554 and NCIB 10555 and recovering therefrom an enzyme.preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen.

; The production of the enzyme preparation can generally be divided into the following stages: ~

(1) Growth of the micro-organism and harvesting of the cells;
~ - .

(2) Extraction of the cholesterol oxidase activity from the cells; and

(3) Purification and concentration of the preparation . with cholesterol oxidase activity.

- The micro-organism can be grown on any suitable medium and the cells harvested. Preferably the organism is grown in ` a culture medium comprising glycerol as a carbon source. An example of a suitable medium is:

.~ 30 - 8 ~

~ ' . '.

.
. , '', ' :, ~ ''.

g %

(NH4)2SOa 0.2 12 2H20 0.001

4 7 2 0.001 K2HP0~ 0.2 MgS04.7H20 0.02 Glycerol 0.5 Yeast Extract 2.0 ~: .
To avoid precipitation of sparingly soluble salts, the above constituents are dissolved separately and then added to a conveniently large volume of distilled water in the order shown. The medium is then made up to the required volume.
The preferred incubation temperature is about 29C and the optimum pH is between pH 6.0 and pH 7.6. Preferably the pH is controlled at about pH 6.7.

Increased yields of cells can be obtained by aeration and agitation but the culture has a tendency to foam and high rates of air flow and agitation can not generaIly be used because of excessive foaming. An air flow rate of 0.2 v/v/min and an impeller~speed of 760 rpm. has given rapid growth without excessive foaming. An anti-foam such as polypropylene glycol is preferably also used to control foaming.

If the initial inoculation of micro-organism into the culture medium is small there may be a lag phase of up to 16 hours. The lag phase is a function of the size of the inoculum and no lag is obtained if an inoculum greater than 2~ (based on the final cell weight) is used.

: , ~9~ ` '',' ' ~ i. ,. ~ : . . ' . . !, 1 The total incubatlon time required is generally about 18 to 24 hours. Under optim~m conditions the doubling time of the culture is about 2 hours and a final cell harvest of at least 25 g (wet weight) per litre of cells (5 g dry weight) can be obtained. The culture can be carried out on a small scale in 5 litre or lO litre vessels but excellent results have also been obtained in larger batches in 100 litre and 1000 litre vessels, in the latter case using a 500 litre batch.

Cholesterol oxidase production is growth associated with a lag behind cell growth. The enzyme level continues to increase for at least 2 hours after the cells stop growing.
Although the enzyme cholesterol oxidase is produced in the absence of an inducer the yield of enzyme can be increased by inducing with cholesterol. Small quantities of cholesterol may be added to the culture as a saturated solution in acetone but for larger quantities the cholesterol is preferably made up as a slurry in water and a wetting agent (e.g. TWEEN 80*) and ;

the resulting slurry sonicated to break up the cholesterol particles into a fine suspension befoFe addit1on to the culture.
As examples of the effect of the inducer 0.2 g/l cholesterol added at a cell concentration of 5 g (wet weight)/l give an increase in enzyme production by the "rough" strain NCIB 10554 of the order of 4 fold and 1.5 g/l cholesterol added at the same cell concentration given an increase in enzyme production of the order of 30 fold.

, , *Trade Mark ~041~437 1 The cel~s may bo harvested by centrifugation. It can be demonstrated a. this stage that the cells themselves have cholesterol oxidase activity with a typical specific activity of the cells of the "rough" strain being about 0.5 ,u mols/mg/hour.
- Cholesterol oxidase activity can be extracted by disrupting the cells and subsequently removing cell debris.
: Electron microscopy has revealed that the forces encountered in grinding and sonication tend to produce fragmentation without secondary damage to the cell wall. In disruption pro-cesses giving more efficient release of cholesterol oxidase activity little fragmentation occurs but damage to the cell wall is extensive. The efficiency of a variety of techniques of cell rupture are illustrated below:

. _ . ~ .

otfhd % Total Activity Rupture Recovered in Recovered as Homogenate -Soluble Enzyme .
~ _ . - _ ', . ~
Grinding with cardice 95~ 8.0% ~

20 Ultrasonication 28% 7.0~ : :
Acetone Powder 52% 12.5%

Mickle apparatus 33% 22.0% ..

LKB X-press 95% 65.0% ~ :

. . _ ....... . ~_ The preferred method of cell rupture is thus the use `~
of an X-press. ~.
Although the cholesterol oxidase activity can be removed by disrupting the cells and recovering the enzyme in a cell-free supernatsnt it has bsen round that the enzyme can be -10~43~7 e~tra~ted without disruption of the cells. There are, in fact, indications that the cholesterol oxidase is situated on the surrace of the organism and it has been found that it can be re~oved with good recovery of activity using a surface active agent. Non-ionic surface active agents have proved particularly suitable and very good results have been obtained with Triton X-100* (iso-octylphenoxypolyethoxy-ethanol containing appro-ximately 10 moles of ethylene oxide).
To remove the cholesterol oxidase the cells are sus-pended in a solution of the surface active agent buffered at a suitable pH and the suspension stirred vigorously at, for example, room temperature. The cholesterol oxidase is recovered in the supernatant by removing the cells, for example by centrifugation. As the cells are not ruptured in this process protein release is low, for example about 100 yg/ml.
In experiments to demonstrate extraction with Triton X-100* 5 g cells (wet weight) of the "rough" strain were suspended in 45 ml. 0.5M Tris/HCl buffer at pH 8.0 containing concentrations of 1%, 3% and 5% Triton X-100*. The suspension was stirred vigorously at room temperature and 10 ml aliquots were removed at 15 minute lntervals. On removal each aliquot was immediately centrifuged at 3500 rpm. in a Mistral 6L
centrifuge. The extracts were assayed for cholesterol oxidase activity with the following results.

*Trade Mark :~ .
'; ' 1~4~f~43'7 1 TA~LE 1 : Extraction time ~ug cholesterol % recovery . mins oxidised/50 ~:L/15 mins in supernatant .~, 1% Triton X-100*
30.0 56.0%
-- 30 35.0 65.5%
35.0 65.5~
lO60 37 5 70 0%
' 120 36.5 68.3%
whole cells 53,5 ' ~
,::
3% Triton X-100*
36.0 68.5%
; 30 35.5 67.5%
38.5 73.5%
, -60 - 38.5 73.5%
`: 120 41.0 78.0 20 whole cells 52.5 ~' ' . . .
. . . .. _ _ _ .. .. .. . . _ _ _ _ _

- 5% Triton X-100*
34.0 62.5%
. :
.30 37.5 69.0%
38.5 71.0%
38.0 70.0%
120 41.0 75.0%
whole cells 54,5 -.: 30 ---- . .. .. _ , *Trade Mark ~ .

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10~37 1 Thus 70% o~ the ac.ivity of the whole cells can be reco~ered at 1% TRITON X-100* and up to 78% at 3~ TRITON X-100*.
T~e surface active agent will, however, remain in the cholesterol oxidase solution. In the assay method as described below it has been found ~hat the presence of TRITON X-100* may be ; advantageous but that the optimum concentration in the assay mixture is about 0.25%, higher concentrations being inhibitory.
The enzyme preparation should thus not have a level of surface ; active agent greater than that which produces an acceptable level - 10 in the assay mixture. Thus although the most rapid release of enzyme can be obtained at 3~ T~ITON X-100* it may be preferable to work at a lower concentration, e.g. 1~ TRITON
X-100*. It may, however, be necessary to remove the surface active agent, for example by dialysis or gel filtration, or alternatively by precipitating the protein tenzyme) out of the surface active agent solution.

Although the cholesterol oxidase preparation produced by extraction of the cells with a surface active agent may, ; in some cases, have a specific activity and a potency which is 2~
sufficient to conduct an assay it is almost always necessary to purify and concentrate the extract before it can be used - practically in a cholesterol assay. In the case of a fluorimetric determination of hydrogen peroxide the enzyme preparation should have a potency of at least 10 2 units/ml to provide a result in a short enough time for an assay to be useful in practice and particularly where the analysis is automated the preparation should have a potency of at least 10 and preferably 0.5 units/ml.

In the case of a non-fluorimetric assay the preparation should have a potency of at least 10 1 unitsjml and preferably *Trade Mark 1 ~or automated analysis at least 0.5 units/ml. The preparation may be prepared with a hi~her potency for example 5 units/ml or ~cre but at higher levels or potency will generally be diluted wi~h buffer be~ore use in a test.
In all cases the preparation must have a cholesterol oxidase specific activity of at least 1 unit per 5 mg protein nitrogen. At higher levels a protein nitrogen the amount of protein present may make the assay solution too viscous or ~-interfere in the assay. A specific activity of at least 1 unit 0 per 50 ~ug protein nitrogen is preferred. A particularly suitable enzyme preparation has been found to be an aqueous preparation with a specific activity of 1 unit of cholesterol oxidase activity per 28 lug protein nitrogen, a potency of 5 units/ml and con-taining 3~ v/v TRITON X-100*. This preparation may be diluted for use in an assay, for example with 0.01M phosphate buffer, so that for automated analysis it contains 0.5 units/ml or for manual analysis 0.1 units/ml.
The enzyme preparation having cholesterol oxidase activity need not be in aqueous form and it may be, for example, in the form of a freeze-dried powder. In addition to the preparation of a soluble lyophilised powder, the enzyme can be present as a concentrated, buffered solution or in suspension with ammonium sulphate ~with or without added buffer).
Although very little protein is extracted with the cholesterol oxidase some catalase activity is generally present in the suractant extract. The amount of catalase activity which can be tolerated in the final enzyme preparation depends upon the assay method to be used, the catalase activity of the preparation beiny important where the assay involves measuring the amount of H202 produced. As described in more *Trade Mark - 15 -: ' ' .

~4~i9L37 1 detail below some assay methods in which H2O2 is determined includethe use of peroxidase and in some cases it is possible to over-~ower limited amounts of catalase with peroxidase. In addition ~~ it is possible to inhibit any concentration of catalase likely to be found in the surfactant extract with an inhibitor for - example sodium azide.
- The effect of catalase on the sensitivity of an assay can be demonstrated as follows:
A catalase solution was prepared by dissolving 20 ~ul of a crystalline suspension of catalase in 50 ml 0.05 M
phosphate buffer pH 7Ø
0.5 ml of this catalase solution was found to break down 7.04 ~M H2O2 (in a final volume of 2.5 ml) in 2.0 minutes.
On this basis the catalase activity of the solution can be said to be 7.04 units per ml at pH 7.0 and 25C.
Varying amounts of this catalase solution were introduced into a cholesterol assay system containing 0.5 units ; of cholesterol oxidase in 2.0 ml and the reduction in sensitivity of the assay observed. ~ ;

Units catalase added % reduction in sensitivity '' :
0.704 95%
0.352 66.4%
0.074 34.3% -However, the inclusion of 0.1% sodium azide in the reaction mixture completely inhibited even the highest level of catalase, thus giving 100% sensitivity even in the presence of catalase.
Catalast should, however, generally be removed from the preparation or at least reduced in amount, for example by ,. .

.

~0~3~
1 chromatography on D~ ceLlulDse.
Suitable means ror purifying the enzyme preparation obtained by surface active agent extxaction and optionally catalase removal include ammonium sulphate precipitation and/or chromatographic methods and/or reduced pressure evaporation.
For example the preparation may be concentrated by ammonium sulphate precipitation or polyethylene glycol precipitation, desalted on a Sephadex* column and then subjected to ion exchange chromatography. It may be necessary in this step to concentrate the activity of the preparation by about 15 to 40 fold.
For example concentration and purification may be carried out by substrate affinity chromatography. Sephadex LH-20*, prepared by hydroxy propylation of G-25, has both hydrophilic and lipophilic properties and can be swollen in polar organic solvents, water, or mixtures thereof. LH-20 may be swollen in ethanol saturated with cholesterol (e.g. approximately 4.0 g~) and a column may be packed with LH-20 prepared in this fashion. Cholesterol may be uniformly distributed throughout the gel by washing the column with distilled water and finally the column may be equilibrated with 0.05M potassium phosphate buffer at pH 7.5. Substantial purification of the enzyme preparation may be achieved by the use of such a column.
Preferably the enzyme preparation is first subjected to ion-exchange chromatography or substrate affinity chroma-tography, preferably chromatography as DEAE cellulose, and then further concentrated by ultrafiltration or reduced pressure evaporation.
In the parent application serial number 163,288, there are disclosed methods for assaying cholesterol in a liquid, in particular, in a biological fluid sush as serum or plasma.

*Trade Mark ~04~37 ~he method generally comprisi,es incubating the liquid with an enzyIre preparation capable of oxidising the cholesterol into ~4-cholestenone and hydrogen peroxide and determing the amount of cholesterol present by measuring the amount in which hydrogen peroxide or ~4-cholestenone is formed or the amount in which oxygen is absorbed in the reaction.
The parent application also describes and claims a kit for use in assaying the amount of cholesterol in a liquid comprising, in combination;
(i) an enzyme preparation capable of oxidising cholesterol to ~4-cholestenone and hydrogen peroxide; and ~ ii) at least one reagent which is capable of being used in the determination of the amount in which hydrogen peroxide or -cholestenone is formed.
Preferably, the enzyme preparation is derived from a cholesterol oxidase producing micro-organism of the Mycobacteri~m rhodocrous group or, more preferably, Nocardia species NCIB
10554 ~NRRL 5635) or NCIB 10555 ~NRRL 5636).
The invention is illustrated by the following 20 example, although it is to be understood that they do not limit the invention in any way.

500 litres of sterile growth media were inoculated with 1 litre of a seed culture of Nocardia species NCIB 10554. The growth media for both seed and production cultures were:

; .

.

~0~6~3~
1 g/litre (NH4)2SO4 2.0 2. H2O 0.01 FeS04 . 7H20 0 . 01 ~ ,.
K2HPO4 2.0 MgSO4.7H2O 0.2 Glycerol 10 Yeast Extract 20 The pH is 6.7 The culture was allowed to grow at 30C for 24 hours.

The culture was agitated by an agitator fitted with 3 turbine impellers at 250 rpm and sterile air was supplied through a -sparge pipe at 150 litres/min. Foaming was controlled by intermittent addition of polypropylene glycol antifoam. After -about 14 hours when the culture had reached a concentration -of 1 - 2 g dry wt/litre, 600 g of cholesterol suspended in 2 litres of a mixture of Tween 80* and water ~0.03:1, v/v)was added to the culture. During the growth phase the maximum ....
doubling time for growth was about 2 hours. After 24 hours when the cell concentration was about 6-7 g dry wt/litre the microorganisms were harvested by passage through an intermittent-discharge disc bowl centrifuge at 400 litresjhour. tAlternatively a rotary vacuum precoat filter may be used. The fermentation time may be reduced by inoculating with a larger seed culture).
The harvested cells were suspended in 0.01 ~ potassium phosphate buffer pH 7.0 containing 0.5~ (v/v) TRITON X-100* at 10 C to give a final volume of 60 litres. A~ter gentle stirring for 2 hours ~shorter times may be used with only a slight reduction in the amount of enzyme extracted) the extracted cells were removed by passage through a tu~ular bowl centri~uge (model *Trade Mark .

.. . . . . . .. . . . . . . .

~O~L~9L37 1 6P, Sharples). The c~ar supernatant obtained was passed through a column (5 litre capacity) at 5 C containing DE-52 cellulose ~previously equilibrated with 0.01 ~ pH. 7.0 potassium phosphate bu~fer containing 0.5% TRITON X-100*). The cholesterol oxidase ; present and any catalase present was retained by the DE-52 cellulose. The cholesterol oxidase was released by step-wise elution with increasing molarities of potassium phosphate buffer pH 7.0 (coniaining 0.5~ TRITON X-100*) at 5 C. Any catalase present remained on the column.

The eluent fractions rich in cholesterol oxidase were further concentrated by ultrafiltration at 5C. using a PM-30 membrane. The retentate solution obtained t3 litres) had a cholesterol oxidase activity of 5.5 ~mole cholesterol oxidised/ml/min at 37C. The overall yield was about 20%.
This may be increased by washing of discarded solids at the extraction and by collecting a larger fraction at the ion-exchange step. The enzyme solution was stored in a liquid form at 5C with azide added as a preservative. The solution retained virtually total activity for at least 2 months.
The enzyme preparation had a cholesterol oxidase specific activity of about 1 unit per 28 ~ug protein nitrogen and a potency of about 5 units/ml. The preparation contained about 3% v/v, TRITON X-100*.

Although the disclosure describes and illustrates a preferred embodiment of the invention, it is to be understood that the invention is not restricted to this particular embodiment.

*Trade Mark , . . . ~ ~ .
. . . . ..
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Claims (25)

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

1. An enzyme preparation derived from a cholesterol oxidase producing micro-organism of the Mycobacterium rhodocrous group, said enzyme preparation having a cholesterol oxidase specific activity of at least one unit per 5 mg of protein nitrogen.

2. An enzyme preparation derived from Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), said enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen.

3. An enzyme preparation as claimed in claim 2, said enzyme preparation being in liquid form and having a potency of at least 10-2 units/ml.

4. An enzyme preparation as claimed in claim 3 having a potency of at least 10-1 units/ml.

5. An enzyme preparation derived from Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), said enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 50 µg of protein nitrogen.

6. An enzyme preparation as claimed in claim 5, said enzyme preparation being in liquid form and having a potency of at least 0.5 unit/ml.

7. An enzyme preparation as claimed in claim 5 in freeze-dried or other solid form.

8. An enzyme preparation derived from Nocardia species, NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636) said enzyme preparation having a cholesterol oxidase activity of at least 1

Claim 8 continued:

unit per 5 mg or protein nitrogen and a catalase activity of less than 10-1 units per unit of cholesterol oxidase activity.

9. An enzyme preparation as claimed in claim 8, said enzyme preparation having a catalase activity of less than 10-2 units per unit of cholesterol oxidase activity.

10. An enzyme preparation as claimed in claim 9, said enzyme containing a catalase inhibitor.

11. An enzyme preparation as claimed in claim 8, said enzyme preparation also containing peroxidase activity.

12. A method of preparing an enzyme preparation having cholesterol oxidase activity, said method comprising growing a cholesterol oxidase producing micro-organism of the Mycobacterium rhodocrous group and recovering therefrom an enzyme preparation having a cholesterol oxidase specific activity of at least one unit per 5 mg of protein nitrogen.

13. A method of preparing an enzyme preparation having cholesterol oxidase activity, said method comprising growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636), and recovering therefrom an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen.

14. A method as claimed in claim 13 in which said enzyme preparation is obtained by harvesting the cells and extracting said cells with a surface active agent.

15. A method as claimed in claim 14 in which said surface active agent is a non-ionic surface active agent.

16. A method as claimed in claim 14 in which said surface active agent is iso-octyl-phenoxypolyethoxyethanol containing approximately 10 moles of ethylene oxide.

17. A method as claimed in claim 13 in which said enzyme preparation is obtained by harvesting the cells, disrupting said cells and removing cell debris.

18. A method as claimed in claim 17 in which said cells are disrupted by means of an X-press.

19. A method of preparing an enzyme preparation having cholesterol oxidase activity, said method comprising aerobically growing Nocardia species NCIB 10554 (NRRL 5635) or NCIB 10555 (NRRL 5636) in a medium comprising glycerol as a carbon source, harvesting the cells, extracting said cells with a surface active agent and recovering an enzyme preparation having a cholesterol oxidase specific activity of at least 1 unit per 5 mg of protein nitrogen.

20. A method as claimed in claim 19 in which said medium has a pH of from 6 to 8.

21. A method as claimed in claim 19 in which growth is carried out in the presence of cholesterol.

22. A method as claimed in claim 19 in which after extraction of said cells with said surface active agent to produce an extract said extract is further purified by ion exchange chroma-tography or substrate affinity chromatography.

23. A method as claimed in claim 19 in which after extraction of said cells with said surface active agent to produce an extract, said extract is further purified by ion exchange chromatography on DEAE cellulose.

24. A method as claimed in claim 22 in which ssid extract purified by chromatography is further concentrated by ultra-filtration or reduced pressure evaporation.

25. A method as claimed in claim 19 in which said enzyme preparation is converted into freeze-dried or other solid form.