US3869349A - Method for the enzymatic hydrolysis of cholesterol esters - Google Patents

Method for the enzymatic hydrolysis of cholesterol esters Download PDF

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Publication number
US3869349A
US3869349A US454659A US45465974A US3869349A US 3869349 A US3869349 A US 3869349A US 454659 A US454659 A US 454659A US 45465974 A US45465974 A US 45465974A US 3869349 A US3869349 A US 3869349A
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Prior art keywords
lipase
cholesterol
protease
improved method
serum
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US454659A
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Charles T Goodhue
Hugh A Risley
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Eastman Kodak Co
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Eastman Kodak Co
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Priority to US454659A priority Critical patent/US3869349A/en
Priority to CA220,426A priority patent/CA1046389A/en
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Publication of US3869349A publication Critical patent/US3869349A/en
Priority to DE19752512605 priority patent/DE2512605B2/de
Priority to GB12202/75A priority patent/GB1498461A/en
Priority to JP3504475A priority patent/JPS5615240B2/ja
Priority to FR7509199A priority patent/FR2265759B1/fr
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Expired - Lifetime legal-status Critical Current

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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus
    • Y10S435/839—Bacillus subtilis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces
    • Y10S435/897—Streptomyces griseus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/913—Aspergillus
    • Y10S435/918—Aspergillus oryzae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
    • Y10S435/921—Candida

Definitions

  • ABSTRACT A totally enzymatic method for the hydrolysis of cholesterol esters using a lipase having cholesterol esterase activity and a protease and a method for quantitative determination of total cholesterol in compositions containing both free and esterified cholesterol comprising enzymatically hydrolyzing the cholesterol esters with a lipase having cholesterol esterase activity and a protease and determining total cholesterol by gas-liquid chromatography or :some other suitable technique are described.
  • This invention relates to an enzymatic method for hydrolyzing cholesterol esters in complex aqueous solutions which may contain both free and esterified cholesterol, for example, blood serum and in particular to a process comprising enzymatically hydrolyzing cholesterol esters using a lipase having cholesterol esterase activity and a protease.
  • hydrolysis of cholesterol esters in complex aqueous solutions is achieved by treating the blood serum with a mixture comprising per ml of serum from about 20 to about 50 mg 'of a lipase having cholesterol ester ase activity and from about 5 to about mg of a protease at a temperature of from about 25 to about C and a pH of from about 6.5 to about 9.5 for about 5 to about 15 minutes, preferably with agitation and in an inert atmosphere.
  • the lipase should containat least about 30 international units per mg and the protease at least about 10 units per mg.
  • One unit of lipase is defined as the amount of the enzyme which will liberate 1 micro mole of fatty acid in a given time at a given pH and temperature using a substrate containing esterified fatty acid.
  • the conditions are 1 minute at pH 7 and 37C with olive oil as substrate.
  • protease hydrolyze casein to produce color equivalent to 1 micro mole (181 ug) tyrosine per minute at pH 7.5 and a temperature of 37C. (Color per Folin-Ciocalteu Reagent). It should, of course, be clear that as the level of enzyme activity per unit by weight of preparation increases or decreases, so also will the quantity of'enzyme preparation added vary. Most preferably, the ratio of lipase to protease on an activity basis should range from about 3 to about 10 and at least about I000 units of lipase should be used per ml of serum. Relative lipase activity to esteraseactivity is usually about 10 to about 50.
  • aqueous medium containing the cholesterol ester preferably blood serum, which contains both esterified and free cholesterol
  • a lipase which demonstrates cholesterol esterase activity as defined in Example 3 and a protease.
  • the lipase maybe of plant or animal origin, but we prefer and find best a microbial lipase such as the lipase from'Cana'ida cylindracca. Lipasesfrom Chronzobacterium viscosum, variant paralipolyticum, crude or purified, the lipase from Rhizopus delemar, purified, for example as noted in Fukumoto et al, J. Gen. Appli.
  • lipases having similar activity which are those described in the aforementioned Bucolo and Davis publication, do not demonstrate the required cholesterol esterase activity.
  • Specifically preferred commercial lipase preparations include wheat germ lipase supplied by Miles Laboratories of Elkhart, Ind.,
  • Proteases in general may be used in the successful practice of this invention. These include by way of example, chymotrypsin, Strepromyces griseus protease (commercially available under the registered trademark Pronase), Aspergillus oryzae protease, Bacillus subtilis protease, elastas, papain and bromelain. Mixtures of such enzymes, of course, may also be emtein containing solution the most notable and important of which is, of course, blood serum. In the case where a simple non protenaceous solution is being assayed, it is possible, as demonstrated in Example 3 below, to obtain ester hydrolysis using only the lipase. As also demonstrated by the Examples, however, in a protein containing solution such as serum, the presence of the protease is essential to useful results.
  • the free cholesterol liberated by'the action of the foregoing enzyme mixture may be assayed in a number of ways. According to a preferred embodiment of the instant invention, this assay is performed using gas-liquid chromatography.
  • an aliquot generally from about 0.5 to about 5 ml of the hydrolyzed aqueous composition to be tested, hereinafter blood serum, is mixed with from about 0.5 to about 2 ml of heptane or some other suitable organic solvent, for example, isooctane, containing from about 25 mg to about 50 mg weight percent octacosane or some other organic suitable for use as an internal standard.
  • heptane may, of course, be replaced with any other solvents suitable for gas-liquid chromatography, for example, isooctane.
  • the solvent mixture is then extracted with water according to conventional techniques, preferably using from about 3 to about ml of water per ml of solvent solution.
  • the water extracted solvent solution is then reacted with a silylating agent, for example, (N,O-bis(- trimethylsilyl) trifluoroacetanide with 1% trimethylchlorosilane or a mixture of equal volumes of trimethylchlorosilane and 1,1,1,3,3,3,-hexamethyldisilazane for a period of from about 2 to about minutes.
  • a silylating agent for example, (N,O-bis(- trimethylsilyl) trifluoroacetanide with 1% trimethylchlorosilane or a mixture of equal volumes of trimethylchlorosilane and 1,1,1,3,3,3,-hexamethyldisilazane for a period of from about 2 to about minutes.
  • the silylated solvent solution is then passed through a conventional gas-liquid chromatograph to determine the total cholesterol in the sample under examination. This method for determining cholesterol concentration is an adaptation of the technique described in detail in J. L. Driscoll, D. Au
  • Validate a reconstituted serum standard produced by the Warner-Lambert Company, was used in the examples below.
  • the total cholesterol content of Validate (lot 2560121) was checked by saponifying an aliquot according to the method of Driscoll et a1, and analyzing the heptane extract by both glc and the Liebermann-Burchard method. Values of 160 and 162 mg% respectively were obtained. These are well within the range of values quoted by the supplier 148-192 mg%).
  • the hydrolysis is performed in an atmosphere of nitrogen in order to minimize artifacts introduced by autooxidation of cholesterol and its esters; of course, proper correction for such autooxidation factors will permit hydrolysis to be performed in a normal atmosphere.
  • EXAMPLE 1 EXAMPLE 2 QUANTITATIVE ESTIMATION OF TOTAL CHOLESTEROL BY GAS-LIQUID CHROMATOGRAPHY (GLC)
  • serum or reconstituted serum standards containing up to 150 mg% cholesterol are mixed with 5 ml ethanol and shaken 3 minutes with 1 ml heptane containing 50 mg7c octacosane.
  • Five ml of water is added and the mixture is shaken again for 3 minutes.
  • the layers separate equal portions of the heptane layer and N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane are mixed.
  • reaction 1 ul samples are injected into a Hewlett Packard F and M 810 chromatograph with a single stainless steel column (l/8 inch X 4 feet) packed with 3% SE30 on 0v 1.
  • Gas flow rate 20 ml/min, oven temperature 250C, injection pop 260C, flame detector 265C, range 10 attenuation X l, chart speed k inch/min.
  • Octacosane retention time is about 1 min.
  • Cholesterol retention time is about 2% min. Runs are complete in about 4 min. Under these conditions the amount of cholesterol in the sample is proportional to the peak height ratios of cholesterol to octacosane. This method is adapted from Driscoll et a1 referred to above.
  • EXAMPLE 5 ESTERASE ACTIVITY OF LIPASE-PROTEASE COMBINATIONS ON SERUM CHOLESTEROL ESTERS Combinations of Lipase M and various proteases were tested in the same manner as in Example 4. Proteases, except for a-chymotrypsin, were added directly to serum at 40 mg per ml; a-chymotrypsin was added at 8 mg per ml. The amount of cholesterol (mg%) released by each combination is shown in Table III.
  • Lipase M to be optimally effective on serum cholesterol esters, a protease must also be present.
  • EXAMPLE 6 A series of commercially prepared proteases were tested for their ability to enhance esterase activity of Lipase M in serum. Protease was added directly to serum in the amounts shown in Table IV. Lipase M concentration was 40 mg per ml serum. Otherwise assay and conditions are the same as in Example 4. The results are contained in Table IV.
  • Lipase M 36 All of the proteases tested appear to enhance the activity ofLipase M somewhat more than a-chymotrypsin and papain. However, it is difficult: to compare the activities of these proteases on the basis of units given by suppliers since several different assays are used. In general, the enzymes judged less pure were used in the higher amounts.
  • EXAMPLE 7 Four different buffers were tested in a system containing three enzymes. Each sample consists of I ml Validate," 40 mg Lipase M. 40 mg papain, 8 mg a-chymotrypsin and 0.I M buffer to 3 ml total volume. The mixtures were incubated and tested as in Example 4. The results are depicted in FIG. I.
  • EXAMPLE 8 Studies with tris buffer were conducted on combinations of Lipase M with the series of proteases described in Example 6. The amount of each protease used was that which gave the best result in the experiment shown in Table IV. Three pH values between 7 and 9 were tested. The assay was the same as described in Example Most of these proteases show an optimum within the three values tested. The best results were obtained with 6 except that time was reduced to 5 minutes so that it bromelain at pH 8.1, Calbiochem bacterial protease at pH values from 8 to 9, A. oryzae protease at pH 8.1 and a-chymotrypsin at pH 7.2.
  • Each incubation mixture contained 0.5 ml Validate, 0.5 ml 0.2M tris buffer at pH shown, mg 1ipase M and the protease in the amounts shown. lncubation was for 5 minutes under N at C, 250 rpm. Samples were analyzed by glc as in Example 2. The results are indicated in Table V.
  • any ofthe well known conventional techniques for the analysis of total free cholesterol may be used. These include the Pearson, Stern and McGarack, Carr and Drecker, and Zak methods described at pages 355-361 of Fundamentals of Clinical Chemistry, Tl- ETZ, N. W., W. B. SAUNDERS CO. (1970) as well as the well known Lieberman-Burchard technique and the cholesterol oxidase method described in German Offenlegungschrifft No.
  • aqueous liquid is serum and said treating step is accomplished with a mixture comprising from about 600 to about 1500 units of said lipase having cholesterol ester- 10 ase activity and from about 50 to about 500 units of said protease per ml of serum.
  • said lipase is selected from the group consisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.
  • protease is selected from the group consisting of a-chymotrypsin, papain, bromelain, Bacillus subll'lis protease, Aspergillus oryzae protease, Streptomyces griseus protease and mixtures thereof.
  • said lipase is selected from the group consisting of wheat germ lipase, pancreatic lipases and the lipase from Candida cylindracca.

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US454659A 1974-03-25 1974-03-25 Method for the enzymatic hydrolysis of cholesterol esters Expired - Lifetime US3869349A (en)

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Application Number Priority Date Filing Date Title
US454659A US3869349A (en) 1974-03-25 1974-03-25 Method for the enzymatic hydrolysis of cholesterol esters
CA220,426A CA1046389A (en) 1974-03-25 1975-02-19 Method for the enzymatic hydrolysis of chlolesterol esters
DE19752512605 DE2512605B2 (de) 1974-03-25 1975-03-21 Verfahren zur bestimmung des gesamt- cholesteringehaltes waessriger fluessigkeiten
GB12202/75A GB1498461A (en) 1974-03-25 1975-03-24 Quantitative determination of cholesterol
JP3504475A JPS5615240B2 (online.php) 1974-03-25 1975-03-25
FR7509199A FR2265759B1 (online.php) 1974-03-25 1975-03-25

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FR (1) FR2265759B1 (online.php)
GB (1) GB1498461A (online.php)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4011045A (en) * 1975-02-14 1977-03-08 Bonderman Dean P Turbidity reduction in triglyceride standards
US4011138A (en) * 1974-06-17 1977-03-08 Kyowa Hakko Kogyo Co., Ltd. Process for the preparation of cholesterol esterase
EP0019253A1 (en) * 1979-05-21 1980-11-26 Miles Laboratories, Inc. Test device, composition and method for the determination of triglycerides
US4275151A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4275152A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4680259A (en) * 1984-09-26 1987-07-14 Eastman Kodak Company Analytical element and method for colorimetric determination of total cholesterol
US5116741A (en) * 1988-04-12 1992-05-26 Genex Corporation Biosynthetic uses of thermostable proteases
US5219733A (en) * 1985-03-06 1993-06-15 Yoshikawa Oil & Fat Co., Ltd. Process for preparing fatty acid esters

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112877219A (zh) * 2021-01-29 2021-06-01 江西科技师范大学 一种高浓度胆固醇培养基及其制备方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2224132A1 (online.php) * 1972-05-17 1973-11-22

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2224132A1 (online.php) * 1972-05-17 1973-11-22

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. Hyun et al., " The J. of Biol. Chem.," 244, No. 7, pp 1937-1945, 1969. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4011138A (en) * 1974-06-17 1977-03-08 Kyowa Hakko Kogyo Co., Ltd. Process for the preparation of cholesterol esterase
US4011045A (en) * 1975-02-14 1977-03-08 Bonderman Dean P Turbidity reduction in triglyceride standards
US4275151A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
US4275152A (en) * 1977-02-03 1981-06-23 Eastman Kodak Company Hydrolysis of protein-bound cholesterol esters
EP0019253A1 (en) * 1979-05-21 1980-11-26 Miles Laboratories, Inc. Test device, composition and method for the determination of triglycerides
US4680259A (en) * 1984-09-26 1987-07-14 Eastman Kodak Company Analytical element and method for colorimetric determination of total cholesterol
US5219733A (en) * 1985-03-06 1993-06-15 Yoshikawa Oil & Fat Co., Ltd. Process for preparing fatty acid esters
US5116741A (en) * 1988-04-12 1992-05-26 Genex Corporation Biosynthetic uses of thermostable proteases

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DE2512605B2 (de) 1976-11-18
GB1498461A (en) 1978-01-18
CA1046389A (en) 1979-01-16
JPS50131588A (online.php) 1975-10-17
FR2265759B1 (online.php) 1977-11-25
DE2512605A1 (de) 1975-10-02
JPS5615240B2 (online.php) 1981-04-09
FR2265759A1 (online.php) 1975-10-24

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