WO1982000833A1 - Method for the determination of total cholesterol - Google Patents

Method for the determination of total cholesterol Download PDF

Info

Publication number
WO1982000833A1
WO1982000833A1 PCT/EP1981/000139 EP8100139W WO8200833A1 WO 1982000833 A1 WO1982000833 A1 WO 1982000833A1 EP 8100139 W EP8100139 W EP 8100139W WO 8200833 A1 WO8200833 A1 WO 8200833A1
Authority
WO
WIPO (PCT)
Prior art keywords
cholesterol
bound
determination
dehydrogenase
nad
Prior art date
Application number
PCT/EP1981/000139
Other languages
German (de)
French (fr)
Inventor
Inst Battelle
Original Assignee
Betz J
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Betz J filed Critical Betz J
Publication of WO1982000833A1 publication Critical patent/WO1982000833A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol

Definitions

  • the invention relates to a method for determining total cholesterol including the bound cholesterol by releasing the bound cholesterol with cholesterol esterase and reacting the free cholesterol with a NAD- or NADP-dependent cholesterol dehydrogenase and measuring the reduced cosubstrate.
  • cholesterol esterase To determine total cholesterol, the bound cholesterol is generally released with cholesterol esterase and the free cholesterol is reacted with an enzyme specific for cholesterol.
  • the cholesterol-converting enzyme is called cholesterol oxidase which, in the presence of atmospheric oxygen, converts cholesterol into the formation of H 2 O 2 in cholestenone.
  • the particular advantage of this method is that it enables a fully enzymatic determination of cholesterol in biological material, where cholesterol is usually present as an ester both in free and in bound form.
  • This fully enzymatic determination considerably simplifies the previously customary, at least two-stage method, in which the saponification of the ester has to be carried out in a separate stage.
  • the reaction products from the oxidation of cholesterol can be determined using various methods.
  • a known method for determining H 2 O 2 is based on the oxidative coupling with p-amino-phenazone and phenol, in Presence of peroxidase.
  • a chromogen is formed with an absorption maximum at 500 nm, which can easily be determined quantitatively with a photometer.
  • This method can be used to determine cholesterol with cholesterol oxidase.
  • Cholesterol is determined using simple means. In many cases, however, large deviations are found, which are due to the formation of turbidity. Furthermore, at least two enzymes are involved in this reaction, which can result in errors.
  • Nocardia erythropolis It is known that an enzyme preparation with cholesterol dehydrogenase activity can be obtained from Nocardia erythropolis (DE-OS 23 05 232).
  • Nocardia erythropolis is an aerobic microorganism, but has no NAD or NADP dependency. For this reason, the detection of the reaction products formed is difficult and prone to failure.
  • the present invention is based on the object of specifying a method with which the disadvantages of known methods can be avoided.
  • Streptomyces hydrogenans as an aerobic microorganism allows cultivation in open ferment, the conditions for the recovery of the cholesterol dehydrogenase are considerably facilitated.
  • Streptomyces hydrogenans (ATCC 19631) is kept on oatmeal agar in inclined agar tubes.
  • Composition of the nutrient medium according to Heinz and Ring 3 g oatmeal, 2 g agar, 250 mg NaCl, 90 ml water and 10 ml soil extract.
  • the mixture is heated in a boiling water bath for 30 min, poured off over gauze, adjusted to pH 7.2 and sterilized in an autoclave at 177 ° C. for 5 min.
  • Vaccinations are carried out at intervals of approx. 6 weeks and after 10 days at 30 ° C in the incubator, the fully grown stock culture can be stored at room temperature.
  • bacteria are cultivated in shaking cultures of 150 ml or 30 ml nutrient medium in 1 l or 250 ml Erlenmeyer flasks.
  • the medium is prepared as follows: 10 g glucose, 1 g yeast extract, 2.5 g NaCl, 4 g meat extract, 4 g casein peptone and 1 l water are heated until all components have dissolved. After filtration, the pH is adjusted to 7.5 and the mixture is heated at 117 ° C. in an autoclave at 20 min. The slanted agar is inoculated with a platinum loop and an overnight culture is grown at 30 ° C. with shaking at approx. 100 rpm and 7 cm amplitude.
  • This culture can either be used immediately, or you inoculate a new 150 ml culture with 20 ml overnight culture, which you can harvest after 5 to 6 hours of growth.
  • the cells in the quasi-logarithmic phase are aspirated in a porcelain filter and washed with cold Tris buffer (10 nM Tris, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM sodium azide, pH 7.4).
  • Tris buffer 10 nM Tris, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM sodium azide, pH 7.4
  • the cells are already very sporulating and the filter pores are clogged, they are centrifuged and washed three times in a Christ IIK centrifuge at 1500 xg for 10 min each.
  • the cells are then resuspended in a suitable buffer and homogenized.
  • the homogenized cells are centrifuged for 20 min at 0-4 ° C in a Sorvall Superspeed SR-2 at 35,000 x g. Cell wall debris and membrane parts are then in the precipitation. The supernatant can now be sedimented by centrifugation at 105,000 x g and 0 - 4 oC in a Spinco L50 for 3 hours.
  • the extraction can be carried out by fractional ammonium sulfate precipitation in a manner known per se.
  • Another possibility to purify the enzyme to a higher specific activity is the ion exchange chromatography.
  • the enzyme obtained can be reacted in solution with the free cholesterol and NAD or NADP as cosubstrate in a manner known per se and NADH or NADPH as the end product can be determined photometrically.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

For the determination of total cholesterol, including bound cholesterol, the bound cholesterol is released by means of cholesterol-esterase and free cholesterol is brought in contact with a cholesterol-hydrogenase obtained from streptomyces hydrogenans, depending on NAD or NADP, and then the reduced co-substrate is measured.

Description

Verfahren zur Bestimmung von Gesamtcholesterin Method for the determination of total cholesterol
Die Erfindung betrifft ein Verfahren zur Bestimmung von Gesamtcholesterin einschließlich des gebundenen Cholesterins durch Freisetzung des gebundenen Cholesterins mit Cholesterinesterase und Umsetzung des freien Cholesterins mit einer NAD- oder NADP-abhängigen Cholesterindehydrogenase und Messung des reduzierten Cosubstrats.The invention relates to a method for determining total cholesterol including the bound cholesterol by releasing the bound cholesterol with cholesterol esterase and reacting the free cholesterol with a NAD- or NADP-dependent cholesterol dehydrogenase and measuring the reduced cosubstrate.
Bei der üblichen Cholesterinbestimmung nach der Liebermann- Burchard-Reaktion bildet Cholesterin mit Essigsäureanhydrid und konzentrierter Schwefelsäure blau-grün gefärbte Verbindungen, deren Farbintensität gemessen wird. Wegen stark korrodierenden und viskosen Reagenzien eignet sich diese Reaktion nicht für die Automatisierung. Sie ist überdies nicht völlig spezifisch, sondern spricht auch auf andere Steroide, z. B. 3 % Dihydrocholesterol und 0,5 - 1,4 % Δ7-Cholestenol an, die ebenfalls in Körperflüssigkeiten vorliegen können.In the usual cholesterol determination after the Liebermann-Burchard reaction, cholesterol with acetic anhydride and concentrated sulfuric acid forms blue-green colored compounds, the color intensity of which is measured. Because of the highly corrosive and viscous reagents, this reaction is not suitable for automation. Moreover, it is not entirely specific, but also speaks other steroids, e.g. B. 3% dihydrocholesterol and 0.5 - 1.4% Δ7-cholestenol, which can also be present in body fluids.
Zur Bestimmung von Gesamtcholesterin wird im allgemeinen das gebundene Cholesterin mit Cholesterinesterase freigesetzt und das freie Cholesterin mit einem für Cholesterin spezifischen Enzym umgesetzt. Als Cholesterin umsetzendes Enzym wird Cholesterinoxidase genannt, welche in Anwesenheit von Luftsauerstoff Cholesterin tinter Bildung von H2O2 in Cholestenon überführt. (DE-AS 22 24 132; DE-OS 22 65 121; DE-OS 22 65 122).To determine total cholesterol, the bound cholesterol is generally released with cholesterol esterase and the free cholesterol is reacted with an enzyme specific for cholesterol. The cholesterol-converting enzyme is called cholesterol oxidase which, in the presence of atmospheric oxygen, converts cholesterol into the formation of H 2 O 2 in cholestenone. (DE-AS 22 24 132; DE-OS 22 65 121; DE-OS 22 65 122).
Der besondere Vorteil dieses Verfahrens ist darin zu sehen, daß eine vollenzymatische Bestimmung des Cholesterins in biologischem Material ermöglicht wird, wo Cholesterin gewöhnlich sowohl in freier als auch in gebundener Form als Ester vorliegt. Durch diese vollenzymatische Bestimmung wird die früher übliche, mindestens zweistufige Methode, bei der die Verseifung des Esters in einer gesonderten Stufe durchgeführt werden muß, wesentlich vereinfacht.The particular advantage of this method is that it enables a fully enzymatic determination of cholesterol in biological material, where cholesterol is usually present as an ester both in free and in bound form. This fully enzymatic determination considerably simplifies the previously customary, at least two-stage method, in which the saponification of the ester has to be carried out in a separate stage.
Die Reaktionsprodukte aus der Oxidation des Cholesterins lassen sich nach verschiedenen Methoden bestimmen. Ein bekanntes Verfahren zur Bestimmung von H2O2 basiert auf der oxidativen Kupplung mit p-Amino-phenazon und Phenol, in Gegenwart von Peroxidase. Bei dieser Reaktion wird ein Chromogen gebildet mit einem Absorptionsmaximum bei 500 nm, welches sich leicht mit einem Photometer quantitativ bestimmen läßt. Diese Methode kann zur Cholesterinbestimmung mit Cholesterinoxidase herangezogen werden. Dabei wird Cholesterin mit einfachen Mitteln bestimmt. In vielen Fällen werden jedoch starke Abweichungen festgestellt, die auf eine Trübungsbildung zurückzuführen sind. Ferner sind an dieser Reaktion mindestens zwei Enzyme beteiligt, woraus Fehlermöglichkeiten resultieren können.The reaction products from the oxidation of cholesterol can be determined using various methods. A known method for determining H 2 O 2 is based on the oxidative coupling with p-amino-phenazone and phenol, in Presence of peroxidase. In this reaction, a chromogen is formed with an absorption maximum at 500 nm, which can easily be determined quantitatively with a photometer. This method can be used to determine cholesterol with cholesterol oxidase. Cholesterol is determined using simple means. In many cases, however, large deviations are found, which are due to the formation of turbidity. Furthermore, at least two enzymes are involved in this reaction, which can result in errors.
Bei analytischen und klinischen Bestimmungen wird sehr häufig von Methoden Gebrauch gemacht, die in gekoppelten Reaktionen schließlich zur Reduktion von NAD oder NADP unter Bildung von NADH bzw. NADPH führen, da sich letztere Umsetzung be sonders einfach in üblichen und weit verbreiteten Photometern verfolgen läßt.In analytical and clinical determinations, methods are very often used which, in coupled reactions, ultimately lead to the reduction of NAD or NADP with the formation of NADH or NADPH, since the latter implementation can be followed particularly easily in customary and widely used photometers.
Die bekannten vollenzymatischen Cholesterinbestimmungen lasse sich jedoch nur auf recht komplizierte und umständliche Weise mit den nachgeschalteten enzymatischen Reaktionen koppeln, daß schließlich NADH bzw. NADPH gemessen werden kann. Es ist bekannt, daß diese Schwierigkeit durch ein Verfahren beseitig werden kann, bei dem das gebundene Cholesterin mit einer Cholesterinesterase freigesetzt und gleichzeitig oder anschließend das freigesetzte Cholesterin mit einer NAD- bzw. NADP-abhängigen Dehydrogenase aus einem anaeroben Mikroorga nismus oder aus Warmbluterleber bestimmt wird (DE-OS 26 49 249 Der wesentliche Nachteil dieser Methode liegt jedoch darin, daß zur Gewinnung der Cholesterindehydrogenase schwierige Kulturbedingungen eingehalten werden müssen.However, the known fully enzymatic cholesterol determinations can only be coupled with the downstream enzymatic reactions in a very complicated and complicated manner, so that finally NADH or NADPH can be measured. It is known that this difficulty can be eliminated by a method in which the bound cholesterol is released with a cholesterol esterase and at the same time or subsequently the released cholesterol with a NAD- or NADP-dependent dehydrogenase from an anaerobic microorganism nism or from warm-blood liver is determined (DE-OS 26 49 249) The main disadvantage of this method is, however, that difficult culture conditions must be observed in order to obtain the cholesterol dehydrogenase.
Es ist bekannt, daß aus Nocardia erythropolis ein Enzympräparat mit Cholesterindehydrogenase-Aktivität gewonnen werden kann (DE-OS 23 05 232). Nocardia erythropolis ist ein aerober Mikroorganismus, besitzt jedoch keine NAD- bzw. NADP-Abhängigkeit. Aus diesem Grunde ist der Nachweis der gebildeten Reaktionsprodukte schwierig und störanfällig.It is known that an enzyme preparation with cholesterol dehydrogenase activity can be obtained from Nocardia erythropolis (DE-OS 23 05 232). Nocardia erythropolis is an aerobic microorganism, but has no NAD or NADP dependency. For this reason, the detection of the reaction products formed is difficult and prone to failure.
Der vorliegenden Erfindung liegt nun die Aufgabe zugrunde, ein Verfahren anzugeben, mit dem die Nachteile bekannter Verfahren vermieden werden können.The present invention is based on the object of specifying a method with which the disadvantages of known methods can be avoided.
Es hat sich gezeigt, daß sich diese Aufgabe in technisch fortschrittlicher Weise lösen läßt, wenn die Cholesterindehydrogenase aus Streptomyces hydrogenans (ATCC 19631) gewonnen wird. Zur Freisetzung des gebundenen Cholesterins kann auch eine Cholesterinesterase aus dem gleichen Mikroorganismus verwendet werden.It has been shown that this object can be achieved in a technically progressive manner if the cholesterol dehydrogenase is obtained from Streptomyces hydrogenans (ATCC 19631). A cholesterol esterase from the same microorganism can also be used to release the bound cholesterol.
Da Streptomyces hydrogenans als ein aerober Mikroorganismus eine Kultivierung in offenen Fermenten zuläßt, werden die Be dingungen bei der Gewinnung der Cholesterindehydrogenasewesentlich erleichtert. Darüber hinaus ist die aus Strepto myces hydrogenans erhaltene Cholesterindehydrogenase NAD- bzw. NADP-abhängig, so daß die Nachteile bekannter Methoden bei der Durchführung des Nachweises vermieden werden können.Since Streptomyces hydrogenans as an aerobic microorganism allows cultivation in open ferment, the conditions for the recovery of the cholesterol dehydrogenase are considerably facilitated. In addition, the is from Strepto myces hydrogenans obtained cholesterol dehydrogenase depending on NAD or NADP, so that the disadvantages of known methods can be avoided when carrying out the detection.
Im folgenden wird die Herstellung der Cholesterindehydrogenase anhand eines lediglich einen Ausführungsweg darstellenden Beispiels näher erläutert.The production of the cholesterol dehydrogenase is explained in more detail below with the aid of an example which merely shows one embodiment.
Für die Kultivierung des Mikroorganismus wird Streptomyces hydrogenans (ATCC 19631) auf Haferflockenagar in Schrägagar röhrchen gehalten. Zusammensetzung des Nährbodens nach Heinz und Ring: 3 g Haferflocken, 2 g Agar, 250 mg NaCl, 90 ml Wasser und 10 ml Erdextrakt. Das Gemisch wird 30 min im siedenden Wasserbad erhitzt, über Gaze abgegossen, auf pH 7,2 eingestellt und 5 min bei 177 °C im Autoklaven sterilisiert. In Abständen von ca. 6 Wochen wird überimpft und jeweils nach 10 Tagen bei 30 °C im Brutschrank kann die ausgewachsene Stammkultur bei Zimmertemeratur aufbewahrt werden.For the cultivation of the microorganism, Streptomyces hydrogenans (ATCC 19631) is kept on oatmeal agar in inclined agar tubes. Composition of the nutrient medium according to Heinz and Ring: 3 g oatmeal, 2 g agar, 250 mg NaCl, 90 ml water and 10 ml soil extract. The mixture is heated in a boiling water bath for 30 min, poured off over gauze, adjusted to pH 7.2 and sterilized in an autoclave at 177 ° C. for 5 min. Vaccinations are carried out at intervals of approx. 6 weeks and after 10 days at 30 ° C in the incubator, the fully grown stock culture can be stored at room temperature.
Für die Versuche werden Bakterien in Schüttelkulturen von 150 ml bzw. 30 ml Nährmedium in 1 l bzw. 250 ml Erlenmeyerkolben kultiviert. Das Medium wird wie folgt hergestellt: 10 g Glucose, 1 g Hefeextrakt, 2,5 g NaCl, 4 g Fleischextrakt, 4 g Caseinpepton und 1 l Wasser werden erhitzt bis sich alle Bestandteile gelöst haben. Nach Filtration wird auf pH 7,5 eingestellt und bei 20 min 117 °C im Autoklaven erhitzt. Vom Schrägagar wird mit einer Platinöse angeimpft und bei 30 °C unter Schütteln mit ca. 100 Upm und 7 cm Amplitude eine Übernachtkultur gezüchtet. Diese Kultur kann entweder sofort verwendet werden, oder man impft mit je 20 ml Übernachtkultur eine neue 150-ml-Kultur an, die man nach 5 bis 6 Stunden Wachstum ernten kann. Die Zellen in. der quasilogarithmischen Phase werden in einer Porzellanfilternusche abgesaugt und mit kalten Trispuffer (10 nM Tris, 1,5 mM MgCl2, 10 mM KCl, 1 mM Natriumazid, pH 7,4) gewaschen. In Fällen, in denen die Zellen schon stark sporulieren und die Filterporen verstopfen, werden sie dreimal in einer Christ- IIK-Zentrifuge bei 1500 x g jeweils 10 min zentrifugiert und gewaschen. Die Zellen werden dann in einem geeigneten Puffer resuspendiert und homogenisiert.For the experiments, bacteria are cultivated in shaking cultures of 150 ml or 30 ml nutrient medium in 1 l or 250 ml Erlenmeyer flasks. The medium is prepared as follows: 10 g glucose, 1 g yeast extract, 2.5 g NaCl, 4 g meat extract, 4 g casein peptone and 1 l water are heated until all components have dissolved. After filtration, the pH is adjusted to 7.5 and the mixture is heated at 117 ° C. in an autoclave at 20 min. The slanted agar is inoculated with a platinum loop and an overnight culture is grown at 30 ° C. with shaking at approx. 100 rpm and 7 cm amplitude. This culture can either be used immediately, or you inoculate a new 150 ml culture with 20 ml overnight culture, which you can harvest after 5 to 6 hours of growth. The cells in the quasi-logarithmic phase are aspirated in a porcelain filter and washed with cold Tris buffer (10 nM Tris, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM sodium azide, pH 7.4). In cases where the cells are already very sporulating and the filter pores are clogged, they are centrifuged and washed three times in a Christ IIK centrifuge at 1500 xg for 10 min each. The cells are then resuspended in a suitable buffer and homogenized.
Zum Aufbrechen der Zellwände haben sich vor allem zwei Methode bewährt: Ultraschallaufschluß und Aufschluß mit der X-Press. Bei der ültrasσhallmethode werden die Zellen in 3 ml Tris- Pulver resuspendiert und im Branson-Sonifier S 75 bis 90 sec bei Stufe 8 und 4,5 A homogenisiert. Wenn ein Detergeπs verwendet werden soll, setzt man es kurz vor Ende der Ultraschallbehandlung zu. Während der Beschallung wird die Probe mit Eis gekühlt. Zur Aufarbeitung der gesammelten Zellen in der X-Press werden diese in wenig entsprechendem Puffer je nach Extraktionsmethode und gegebenenfalls unter Zusatz von Detergenzien suspendiert und die tiefgekühlte , mindestens -25 °C kalte X-Press eingefüllt und noch eine halbe Stunde bei -30 °C gekühlt. Die gefrorenen Zellen werden dann mittels einer hydraulischen Presse in vier Durchläufen bei 1000 - 2000 N/mm 2 homogenisiert.Two methods have proven particularly useful for breaking open the cell walls: ultrasonic digestion and digestion with the X-Press. With the ültrasσhall method, the cells are resuspended in 3 ml Tris powder and homogenized in the Branson-Sonifier S for 75 to 90 sec at level 8 and 4.5 A. If a detergent is to be used, it is added shortly before the end of the ultrasound treatment. The sample is cooled with ice during sonication. To work up the collected cells in the X-Press, they are suspended in a little appropriate buffer depending on the extraction method and, if necessary, with the addition of detergents, and the frozen X-Press, which is at least -25 ° C cold, is poured in and another half an hour at -30 ° C chilled. The frozen cells are then removed using a hydraulic press homogenized in four passes at 1000 - 2000 N / mm 2 .
Die homogenisierten Zellen werden 20 min 0 - 4 °C in einer Sorvall Superspeed SR-2 mit 35.000 x g zentrifugiert. Im Niederschlag befinden sich sodann Zellwandtrümmer und Membranteile. Aus dem überstand können nun durch 3-stündige Zentrifugation bei 105.000 x g und 0 - 4 ºC in einer Spinco L50 die Ribosomen sedimentiert werden.The homogenized cells are centrifuged for 20 min at 0-4 ° C in a Sorvall Superspeed SR-2 at 35,000 x g. Cell wall debris and membrane parts are then in the precipitation. The supernatant can now be sedimented by centrifugation at 105,000 x g and 0 - 4 ºC in a Spinco L50 for 3 hours.
Die Gewinnung kann durch fraktionierte Ammonsulfatfällung in an sich bekannter Weise erfolgen. Eine weitere Möglichkeit, das Enzym zu höherer spezifischer Aktivität aufzureinigen, besteht in der Ionenaustauscher-Chromatographie. Das gewonnene Enzym kann in Lösung mit dem freien Cholesterin und NAD bzw. NADP als Cosubstrat in an sich bekannter Weise umgesetzt und NADH bzw. NADPH als Endprodukt photometrisch bestimmt werden. The extraction can be carried out by fractional ammonium sulfate precipitation in a manner known per se. Another possibility to purify the enzyme to a higher specific activity is the ion exchange chromatography. The enzyme obtained can be reacted in solution with the free cholesterol and NAD or NADP as cosubstrate in a manner known per se and NADH or NADPH as the end product can be determined photometrically.

Claims

Patentansprüche Claims
1. Verfahren zur Bestimmung von Gesamtcholesterin einschließlich des gebundenen Cholesterins durch Freisetzung des gebundenen Cholesterins mit Cholesterinesterase und Umsetzung des freien Cholesterins mit einer NAD- oder NADP-abhängigen Cholesterindehydro genäse und Messung des reduzierten Cosubstrates, dadurch gekennzeichnet, daß die Cholesterindehydrogenase aus Streptomyces hydrogenans gewonnen wird. 1. A method for determining total cholesterol including the bound cholesterol by releasing the bound cholesterol with cholesterol esterase and reacting the free cholesterol with a NAD- or NADP-dependent cholesterol dehydrogenase and measuring the reduced cosubstrate, characterized in that the cholesterol dehydrogenase is obtained from Streptomyces hydrogenans .
2. Verfahren nach Anspruch, dadurch gekennzeichnet, daß die zur Freisetzung des gebundenen Cholesterin verwendete Cholesterinesterase aus Streptomyces hydrogenans gewonnen wird. 2. The method according to claim, characterized in that the cholesterol esterase used to release the bound cholesterol is obtained from Streptomyces hydrogenans.
PCT/EP1981/000139 1980-08-28 1981-08-26 Method for the determination of total cholesterol WO1982000833A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19803032377 DE3032377A1 (en) 1980-08-28 1980-08-28 METHOD FOR DETERMINING TOTAL CHOLESTEROL
DE3032377800828 1980-08-28

Publications (1)

Publication Number Publication Date
WO1982000833A1 true WO1982000833A1 (en) 1982-03-18

Family

ID=6110557

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1981/000139 WO1982000833A1 (en) 1980-08-28 1981-08-26 Method for the determination of total cholesterol

Country Status (2)

Country Link
DE (1) DE3032377A1 (en)
WO (1) WO1982000833A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344580A1 (en) * 1988-05-25 1989-12-06 IMMUNO Aktiengesellschaft Method to determine the relative quantities of all cholesterol-containing lipoproteins in body fluids
EP0709456A2 (en) * 1994-10-26 1996-05-01 Roche Diagnostics GmbH Microbial cholesterol-dehydrogenase, process for the preparation and use
WO1998003675A1 (en) * 1996-07-24 1998-01-29 Helena Laboratories Corporation Cholesterol separation and fluorescent analysis
US5856156A (en) * 1994-10-26 1999-01-05 Boehringer Mannheim Gmbh Microbial cholesterol dehydrogenase, process for its production and use

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2047147A5 (en) * 1969-05-06 1971-03-12 Kyowa Hakko Kogyo Kk
DE2305232A1 (en) * 1973-02-02 1974-08-22 Thomas Patterson Pro Whitehead Cholesterol-free enzyme prepn. with cholesteroldehydrogenase activity - with cholesterol dehydrogenase activity, prepd by culturing No cardia erythropolis in nutrient medium contg. cholesterol
DE2506712A1 (en) * 1974-03-01 1975-09-04 Boehringer Mannheim Gmbh Cholesterol determination procedure - in which bound cholesterol is released using microbial cholinesterase
FR2369564A2 (en) * 1976-10-29 1978-05-26 Boehringer Mannheim Gmbh METHOD AND REAGENT FOR DETERMINING THE CONTENT OF TOTAL CHOLESTEROL OR OF COMBINED CHOLESTEROL

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2047147A5 (en) * 1969-05-06 1971-03-12 Kyowa Hakko Kogyo Kk
DE2305232A1 (en) * 1973-02-02 1974-08-22 Thomas Patterson Pro Whitehead Cholesterol-free enzyme prepn. with cholesteroldehydrogenase activity - with cholesterol dehydrogenase activity, prepd by culturing No cardia erythropolis in nutrient medium contg. cholesterol
DE2506712A1 (en) * 1974-03-01 1975-09-04 Boehringer Mannheim Gmbh Cholesterol determination procedure - in which bound cholesterol is released using microbial cholinesterase
FR2369564A2 (en) * 1976-10-29 1978-05-26 Boehringer Mannheim Gmbh METHOD AND REAGENT FOR DETERMINING THE CONTENT OF TOTAL CHOLESTEROL OR OF COMBINED CHOLESTEROL

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Chemical Abstracts, vol. 79, No. 19, 12 November 1973, (Columbus Ohio, US) H.H. Flegg:"Determination of serum cholesterol by an enzymic method", page 139, abstract 112935w; & Ann. Clin. Biochem. 1973, 10 (Pr3). 79-84 *
Chemical Abstracts, vol. 91, No 19, 5 November 1979, (Columbus Ohio,US) H. Duchmann u.a.: "Purification and characterization of a 17-béta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans" page 250, abstract 153358b; & Z. Naturforsch. C: Biosci. 1979, 34 C(7-8), 533-40 *
Chemical Abstracts, vol.83, No. 7, 18 August 1975 (Columbus Ohio, US) B. Kohler u.a.: "delta5-3 bétahydroxysteroid dehydrogenase from streptomyces hydrogenans" page 200, abstract 55098 g; & Naturwissenschaften 1975, 62 (6), 299 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0344580A1 (en) * 1988-05-25 1989-12-06 IMMUNO Aktiengesellschaft Method to determine the relative quantities of all cholesterol-containing lipoproteins in body fluids
US5385828A (en) * 1988-05-25 1995-01-31 "Immuno" Aktiengesellschaft Fur Chemisch-Medizinische Produkte Method for determining the relative amounts of all cholesterol-containing lipoproteins in body fluids
EP0709456A2 (en) * 1994-10-26 1996-05-01 Roche Diagnostics GmbH Microbial cholesterol-dehydrogenase, process for the preparation and use
US5856156A (en) * 1994-10-26 1999-01-05 Boehringer Mannheim Gmbh Microbial cholesterol dehydrogenase, process for its production and use
EP0709456A3 (en) * 1994-10-26 1999-08-11 Roche Diagnostics GmbH Microbial cholesterol-dehydrogenase, process for the preparation and use
WO1998003675A1 (en) * 1996-07-24 1998-01-29 Helena Laboratories Corporation Cholesterol separation and fluorescent analysis

Also Published As

Publication number Publication date
DE3032377A1 (en) 1982-04-01

Similar Documents

Publication Publication Date Title
DE2224133B2 (en) Use of certain strains of microorganisms to obtain cholesterol oxidase, which oxidizes cholesterol with O low 2 to cholestenone + H low 2 O low 2
DE2343587A1 (en) PROCESS FOR THE PRODUCTION OF 2-KETOL-GULONIC ACID
DE1939341A1 (en) Cephalosporin derivatives and processes for their preparation
DE2649749C2 (en)
DE1768215C3 (en) Process for the production of 1,4-androstadiene-3,17-dlon and 4-androstene-3,17-dione by microbiological degradation
DD232310A5 (en) PROCESS FOR THE PREPARATION OF L-CARNITINE
DE3127979C2 (en) Use of a specific cholesterase to hydrolyze cholesterol fatty acid esters
EP0120440B1 (en) Nad(p)-independent glycerol dehydrogenase, process for its preparation and its use in the determination of glycerol and triglycerides
WO1982000833A1 (en) Method for the determination of total cholesterol
DE2527068A1 (en) PROCESS FOR THE PRODUCTION OF CHOLESTEROLESTERASE
DE69019396T2 (en) Process for the fermentative oxidation of reducing disaccharides.
DE2619311A1 (en) PROCESS FOR THE PRODUCTION OF L (+) TARTARIC ACID
AT396480B (en) PROCESS FOR THE PREPARATION OF 9ALPHA-HYDROXY-4-ANDROSTENE-3,17-DIONE
DE3741198C2 (en)
CH656642A5 (en) Method for strengthening of steroid compounds microbiological implementation.
DE3991428C2 (en) Bile acid sulphate sulphatase produced by pseudomonas testosterone
EP0139985B1 (en) Process and reagent for the determination of transaminases
DE3783671T2 (en) 3-ALPHA-HYDROXYSTEROID OXYDASE AND MASS ANALYSIS OF 3-ALPHA-HYDROXYSTEROID USING THE SAME.
DE2712004C2 (en) Method for the determination of formate or compounds which can be converted into formate and a suitable reagent therefor
EP0027829B1 (en) Process for the preparation of 9-alpha-hydroxy-4-androstene-3,17-dione
DE3903759C2 (en)
EP0089640B1 (en) L(+)-tartrate dehydrogenase, method for its prepartion, as well as a method and reagent for the determination of l(+)-tartrate and d(+)-malate
DE2506712C3 (en) Method for the determination of cholesterol
DD139859A5 (en) PROCESS FOR PREPARING 21-HYDROXY-20-METHYLPREGNANESE
EP0609254B1 (en) Method of preparing 2,5-dihydroxyphenylacetic acids

Legal Events

Date Code Title Description
AK Designated states

Designated state(s): JP US

AL Designated countries for regional patents

Designated state(s): AT CH FR GB LU NL SE