CN110066849A - A kind of screening technique producing cholesterol oxidase bacterial strain - Google Patents

A kind of screening technique producing cholesterol oxidase bacterial strain Download PDF

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Publication number
CN110066849A
CN110066849A CN201910271075.6A CN201910271075A CN110066849A CN 110066849 A CN110066849 A CN 110066849A CN 201910271075 A CN201910271075 A CN 201910271075A CN 110066849 A CN110066849 A CN 110066849A
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bacterial strain
cholesterol
screening
cholesterol oxidase
screening technique
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迟乃玉
希伦
张庆芳
刘洋
于爽
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Dalian University
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Dalian University
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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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)

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  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The present invention relates to a kind of technical field of bacteria screening, specifically a kind of screening technique for producing cholesterol oxidase bacterial strain.The present invention is through primary dcreening operation, after sample is diluted with sterile water, by enrichment, is coated in the screening flat board using cholesterol as sole carbon source, selects the bacterial strain that can generate bluish violet；Using secondary screening, by primary dcreening operation bacterial strain it is activated after be inoculated into fermentation medium, 20-30 DEG C of culture 48h measurement extracellular cholesterol oxidase activity.Screening technique efficiently and accurately of the present invention, can obtain the bacterial strain of highly yielding cholesterol oxidase；Enzyme activity determination method, rapid, high sensitivity easy to operate, while ensure that the safety in continuous mode.

Description

A kind of screening technique producing cholesterol oxidase bacterial strain
Technical field
The present invention relates to a kind of technical field of bacteria screening, specifically a kind of screening side for producing cholesterol oxidase bacterial strain Method.
Background technique
Cholesterol oxidase (3 β-hydroxyl sterol oxidizing ferment；ChOX, EC1.1.3.6) it is found in nineteen forty-three earliest, it is flavine One of member in protein oxidoreductase family.The enzyme mainly acts on 3 position of cholesterol carbon, and oxidative dehydrogenation occurs, Furthermore it has allomerase effect, and isomerization reaction occurs for the first step product cholesteric -5- alkene -3- ketone after being catalyzed cholesterol oxidation Generate final product gallbladder Gona-4-en-3-one 3- ketone.
In recent years, microorganism cholesterol oxidase because its be widely used in serum cholesterol measurement medical application due to by Concern.Since cholesterol oxidase has stronger oxidation activity to sterol, it is solid that it has been used for gallbladder in clinical and food samples The measurement of pure and mild phytosterol.Currently, cholesterol oxidase has extensive clinical application in the lab, such as in food and serum Middle quantitating cholesterol is horizontal, this is critically important to the diagnosis of atherosclerosis, cardiovascular disease and other lipid disorders.In addition, Cholesterol oxide enzyme is also used for the bioconversion of many nonsteroidal compounds, allyl alcohol and sterol.Present screening technique Without apparent color change in screening process, it is not easy to observe；Or it is cumbersome using thin-layer chromatography.
Summary of the invention
The purpose of the present invention is being directed to the insufficient link of current screening technique, provide for using cholesterol as sole carbon source and A kind of screening technique of a large amount of efficiently and accuratelies for generating cholesterol oxidase bacterial strain.
For achieving the above object, the invention adopts the following technical scheme:
(1) it primary dcreening operation: after sample is diluted with sterile water, by enrichment, is coated in using cholesterol as the sieve of sole carbon source It selects on plate, selects the bacterial strain that can generate bluish violet；
(2) secondary screening: by above-mentioned primary dcreening operation bacterial strain it is activated after be inoculated into fermentation medium, 20-30 DEG C of culture 48h measures born of the same parents Outer cholesterol oxidation enzymatic activity.
The screening solid culture based component of primary dcreening operation process described in step (1) is as follows: cholesterol 1.5466g/L, yeast extract 0.5-10g/L, sodium chloride 2-10g/L, potassium dihydrogen phosphate 1-10g/L, dipotassium hydrogen phosphate 0.5-10g/L, magnesium sulfate 0.5-10g/ L, starch 10-20g/L, potassium iodide 1-10g/L, agar 20-40g/L.
The fermentation medium components of secondary screening process described in step (2) are as follows: glucose 10g/L, tryptone 10g/L, Yeast extract 5g/L, cholesterol 2g/L, Tween-80 1ml/L, pH6.5.
The measurement system ingredient of the enzyme activity determination process is as follows: 2,2- joins nitrogen (3- ethyl-benzothiazole -6- sulfonic acid) two Ammonium salt (abbreviation ABTS) and HRP mixed liquor 3mL, 200 μ L of cholesterol working solution, 50 μ L of crude enzyme liquid.
The ABTS-HRP mixed liquor configuration is as follows: weighing 0.0183g ABTS and 0.0021g HRP (250U/mg), uses 0.05M kaliumphosphate buffer (ph7.2) dissolution, is settled to 100ml.
It is characterized in that, the cholesterol working solution is formulated as follows: weighing 0.0190g cholesterol powder and be dissolved in deoxidation gallbladder Sour sodium (5%w/v), is settled to 10ml.
Compared with prior art, the invention has the following advantages:
(1) screening technique efficiently and accurately can obtain the bacterial strain of highly yielding cholesterol oxidase；Existing method was being screened Without apparent color change in journey, it is not easy to observe, and the method for the present invention has bluish violet when generating cholesterol oxidase, So as to observing for quicklook, it can embody and produce to a certain extent further according to the size and shade of bluish violet covering Amount.
(2) enzyme activity determination method of the invention, rapid, high sensitivity easy to operate, while ensure that in continuous mode Safety.
Detailed description of the invention
Fig. 1 is the bacterium colony plate for having bluish violet transparent circle.
Specific embodiment
Below with reference to embodiment, the present invention is further described.Sample used is from Dalian Bohai Bay in embodiment Ooze seawater sample.
The primary dcreening operation of 1 cholesterol oxidase producing strain of embodiment, secondary screening method
A kind of screening technique producing cholesterol oxidase bacterial strain provided in this embodiment, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training Case is supported, temperature setting is cultivated 2-3 days under 20-30 degree, constant temperature, can be carried out in next step；Wherein enriched medium (%): gallbladder Sterol 0.1-0.5, NH4NO30.1-0.5, KH2PO40.025-0.1, MgSO40.025-0.1, FeSO4 0.0001-0.001, PH 7.0, distilled water 1.0-1.5L are saved after high-temperature sterilization, and temperature is controlled at 100-200 DEG C；
(2) it produces the separation of cholesterol oxidase bacterial strain: sample in step (1) being diluted with sterile water, is coated on after dilution In screening and culturing medium, temperature setting is constant temperature incubation 4-5 days under the conditions of 20-30 degree；
Screening and culturing medium are as follows: cholesterol 1.5466g/L, yeast extract 0.5-10g/L, sodium chloride 2-10g/L, potassium dihydrogen phosphate 1-10g/L, dipotassium hydrogen phosphate 0.5-10g/L, magnesium sulfate 0.5-10g/L, starch 10-20g/L, potassium iodide 1-10g/L, agar 20-40g/L。
(3) produce the primary dcreening operation of cholesterol oxidase bacterial strain: picking has the bacterial clump of bluish violet transparent circle, passes through three rides It is isolated and purified, temperature setting is cultivated 4-5 days under 20-30 degree constant temperature, obtains single bacterium colony, carries out microscopy；
(4) it produces the secondary screening of cholesterol oxidase bacterial strain: above-mentioned bacterial strains is trained with the strain inoculated of 1% inoculum concentration in LB liquid Support shaken cultivation 12h or so in base；Bacterium solution is connected in the 50ml triangular flask containing 10ml fermentation medium by 2% inoculum concentration, 25 DEG C of shaken cultivation 48h are to stationary phase；Above-mentioned fermentation liquid is centrifuged, takes supernatant to obtain crude enzyme liquid, both cell-free extract, as The source of ectoenzyme.
Fermentation medium components are as follows: glucose 10g/L, tryptone 10g/L, yeast extract 5g/L, cholesterol 2g/L, Tween-801ml/L, pH6.5.
2,2- connection nitrogen (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts are added in cuvette (light path 10mm) (referred to as ABTS it) with HRP (horseradish peroxidase) mixed liquor 3mL and 200 μ L cholesterol working solutions, mixes and is balanced at 37 DEG C 5min；Wherein, ABTS-HRP mixed liquor, which is configured that, weighs 0.0183g ABTS and 0.0021g HRP (250U/mg), uses 0.05M Kaliumphosphate buffer (pH7.2) dissolution, is settled to 100ml；Cholesterol working solution is prepared are as follows: weighs 0.0190g cholesterol powder It is dissolved in NaTDC (5%w/v), is settled to 10ml；Gained 50 μ L of crude enzyme liquid is added, remixes, in constant temperature spectrophotometric It counts in (37 DEG C) at record 415nm, 3min internal absorbance is worth variation, takes linear data A415nm/ min brings formula calculating into:
Wherein, 33 molar absorption coefficient (the ε 415nm=3.3*10 for representing oxygen condition ABTS4M-1cm-1)。
One enzyme-activity unit is defined as 37 DEG C and generates enzyme amount required for 1 μm of ol hydrogen peroxide per minute.
The new screening technique and application for one plant of production cholesterol oxidase bacterial strain that the present embodiment obtains, can be quick, quasi- The bacterium of cholesterol oxidase is produced in true screening, and has the higher advantage of low-temperature epitaxy producing enzyme enzyme activity, is conducive to latter Step carries out the research of application aspect.
The primary dcreening operation of 2 cholesterol oxidase producing strain of embodiment, secondary screening method
The new screening technique of one plant of production cholesterol oxidase bacterial strain provided in this embodiment, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training Case is supported, temperature setting is cultivated 2 days under constant temperature at 20 degree, can be carried out in next step；Wherein enriched medium (%): cholesterol 0.1, NH4NO30.1, KH2PO40.025, MgSO40.025, FeSO40.0001, pH 7.0, distilled water 1.0, high-temperature sterilization After save, temperature control at 100 DEG C；
(2) it produces the separation of cholesterol oxidase bacterial strain: sample in step (1) being diluted with sterile water, is coated on after dilution In screening and culturing medium, temperature setting is constant temperature incubation 4 days under the conditions of 20 degree；
Screening and culturing medium are as follows: cholesterol 1.5466g/L, yeast extract 0.5g/L, sodium chloride 2g/L, potassium dihydrogen phosphate 1g/L, Dipotassium hydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, starch 10g/L, potassium iodide 1g/L, agar 20g/L.
(3) produce the primary dcreening operation of cholesterol oxidase bacterial strain: picking has the bacterial clump of bluish violet transparent circle, passes through three rides It is isolated and purified, temperature setting is cultivated 4 days under 20 degree of constant temperature, obtains single bacterium colony, carries out microscopy；
(4) it produces the secondary screening of cholesterol oxidase bacterial strain: above-mentioned bacterial strains is trained with the strain inoculated of 1% inoculum concentration in LB liquid Support shaken cultivation 12h or so in base；Bacterium solution is connected in the 50ml triangular flask containing 10ml fermentation medium by 2% inoculum concentration, 25 DEG C of shaken cultivation 48h are to stationary phase；Above-mentioned fermentation liquid is centrifuged, supernatant is taken to obtain crude enzyme liquid, measuring enzyme activity is 0.52U/mL, Both cell-free extract, the source as ectoenzyme.Enzyme activity method is measured with embodiment 1.
The primary dcreening operation of 3 cholesterol oxidase producing strain of embodiment, secondary screening method
The new screening technique of one plant of production cholesterol oxidase bacterial strain provided in this embodiment, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training Case is supported, temperature setting is cultivated 2.5 days under constant temperature at 25 degree, can be carried out in next step；Wherein enriched medium (%): cholesterol 0.3, NH4NO3 0.3, KH2PO4 0.05, MgSO4 0.05, FeSO4 0.0001, pH 7.0, distilled water 1.0, high-temperature sterilization After save, temperature control at 150 DEG C；
(2) it produces the separation of cholesterol oxidase bacterial strain: sample in step (1) being diluted with sterile water, is coated on after dilution In screening and culturing medium, temperature setting is constant temperature incubation 4 days under the conditions of 25 degree；
Screening and culturing medium are as follows: cholesterol 1.5466g/L, yeast extract 4g/L, sodium chloride 5g/L, potassium dihydrogen phosphate 5g/L, phosphorus Sour hydrogen dipotassium 5g/L, magnesium sulfate 5g/L, starch 15g/L, potassium iodide 5g/L, agar 30g/L.
(3) produce the primary dcreening operation of cholesterol oxidase bacterial strain: picking has the bacterial clump of bluish violet transparent circle, passes through three rides It is isolated and purified, temperature setting is cultivated 4 days under 25 degree of constant temperature, obtains single bacterium colony, carries out microscopy；
(4) it produces the secondary screening of cholesterol oxidase bacterial strain: above-mentioned bacterial strains is trained with the strain inoculated of 1% inoculum concentration in LB liquid Support shaken cultivation 12h or so in base；Bacterium solution is connected in the 50ml triangular flask containing 10ml fermentation medium by 2% inoculum concentration, 25 DEG C of shaken cultivation 48h are to stationary phase；Above-mentioned fermentation liquid is centrifuged, supernatant is taken to obtain crude enzyme liquid, measuring enzyme activity is 0.57U/mL, Both cell-free extract, the source as ectoenzyme.
The primary dcreening operation of 4 cholesterol oxidase producing strain of embodiment, secondary screening method
The new screening technique of one plant of production cholesterol oxidase bacterial strain provided in this embodiment, specifically includes the following steps:
(1) it will be accessed in enriched medium for the deep-sea ooze sample of examination by 5% inoculum concentration, and be put into constant-temperature table training Case is supported, temperature setting is cultivated 3 days under constant temperature at 30 degree, can be carried out in next step；Wherein enriched medium (%): cholesterol 0.5, NH4NO30.5, KH2PO40.1, MgSO40.1, FeSO40.001, pH 7.0, distilled water 1L are saved after high-temperature sterilization, Temperature is controlled at 200 DEG C；
(2) it produces the separation of cholesterol oxidase bacterial strain: sample in step (1) being diluted with sterile water, is coated on after dilution In screening and culturing medium, temperature setting is constant temperature incubation 5 days under the conditions of 30 degree；
Screening and culturing medium are as follows: cholesterol 1.5466g/L, yeast extract 10g/L, sodium chloride 10g/L, potassium dihydrogen phosphate 10g/L, Dipotassium hydrogen phosphate 10g/L, magnesium sulfate 10g/L, starch 20g/L, potassium iodide 10g/L, agar 40g/L.
(3) produce the primary dcreening operation of cholesterol oxidase bacterial strain: picking has the bacterial clump of bluish violet transparent circle, passes through three rides It is isolated and purified, temperature setting is cultivated 5 days under 30 degree of constant temperature, obtains single bacterium colony, carries out microscopy；
(4) it produces the secondary screening of cholesterol oxidase bacterial strain: above-mentioned bacterial strains is trained with the strain inoculated of 1% inoculum concentration in LB liquid Support shaken cultivation 12h or so in base；Bacterium solution is connected in the 50ml triangular flask containing 10ml fermentation medium by 2% inoculum concentration, 25 DEG C of shaken cultivation 48h are to stationary phase；Above-mentioned fermentation liquid is centrifuged, supernatant is taken to obtain crude enzyme liquid, measuring enzyme activity is 0.68U/mL, Both cell-free extract, the source as ectoenzyme.

Claims (4)

1. a kind of screening technique for producing cholesterol oxidase bacterial strain, which is characterized in that the described method comprises the following steps:
(1) it primary dcreening operation: after deep-sea ooze sample is diluted with sterile water, by enrichment, is coated in using cholesterol as sole carbon source Screening flat board on, select the bacterial strain that can generate bluish violet；
(2) secondary screening: by above-mentioned primary dcreening operation bacterial strain it is activated after be inoculated into fermentation medium, 20-30 DEG C of culture 48h measures extracellular gallbladder Sterol oxidase active.
2. a kind of screening technique for producing cholesterol oxidase bacterial strain as described in claim 1, which is characterized in that the method step Suddenly the screening solid culture based component of primary dcreening operation process is as follows in (1): cholesterol 1.5466g/L, yeast extract 0.5-10g/L, chlorination Sodium 2-10g/L, potassium dihydrogen phosphate 1-10g/L, dipotassium hydrogen phosphate 0.5-10g/L, magnesium sulfate 0.5-10g/L, starch 10-20g/L, Potassium iodide 1-10g/L, agar 20-40g/L.
3. a kind of screening technique for producing cholesterol oxidase bacterial strain as described in claim 1, which is characterized in that the method step Suddenly the fermentation medium components of secondary screening process are as follows in (2): glucose 10g/L, tryptone 10g/L, yeast extract 5g/L, gallbladder are solid Alcohol 2g/L, Tween-80 1ml/L, pH6.5.
4. a kind of screening technique for producing cholesterol oxidase bacterial strain as described in claim 1, which is characterized in that the method step Suddenly the measurement system ingredient of enzyme activity determination process is as follows in (2): 2,2- joins nitrogen (3- ethyl-benzothiazole -6- sulfonic acid) di-ammonium salts With HRP mixed liquor 3mL, 200 μ L of cholesterol working solution, 50 μ L of crude enzyme liquid；Wherein, ABTS-HRP mixed liquor, which is configured that, weighs 0.0183g ABTS and 0.0021g HRP (250U/mg) are dissolved with 0.05M kaliumphosphate buffer (pH7.2), are settled to 100ml；Cholesterol working solution is prepared are as follows: is weighed 0.0190g cholesterol powder and is dissolved in NaTDC (5%w/v), is settled to 10ml。
CN201910271075.6A 2019-04-04 2019-04-04 A kind of screening technique producing cholesterol oxidase bacterial strain Pending CN110066849A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215531A (en) * 2007-12-29 2008-07-09 四川师范大学 Method for separating extracellular cholesterol oxidase producing strain
CN103361399A (en) * 2013-06-20 2013-10-23 江南大学 Screening method for cholesterol oxidase producing strain
CN109097435A (en) * 2018-09-01 2018-12-28 长沙理工大学 A method of screening malaga carbohydrate oxidase strain from air

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101215531A (en) * 2007-12-29 2008-07-09 四川师范大学 Method for separating extracellular cholesterol oxidase producing strain
CN103361399A (en) * 2013-06-20 2013-10-23 江南大学 Screening method for cholesterol oxidase producing strain
CN109097435A (en) * 2018-09-01 2018-12-28 长沙理工大学 A method of screening malaga carbohydrate oxidase strain from air

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
冯艳頔: "酶法测定胆固醇氧化酶方法的比较及优化", 《轻工科技》 *

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