CN104390919A - Quantitative determination method of specific activity of crop lipoxidase isozyme LOX-3 - Google Patents
Quantitative determination method of specific activity of crop lipoxidase isozyme LOX-3 Download PDFInfo
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- CN104390919A CN104390919A CN201410668086.5A CN201410668086A CN104390919A CN 104390919 A CN104390919 A CN 104390919A CN 201410668086 A CN201410668086 A CN 201410668086A CN 104390919 A CN104390919 A CN 104390919A
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Abstract
The invention provides a quantitative determination method of specific activity of crop lipoxidase isozyme LOX-3. The method is characterized by comprising the steps of firstly crushing crops, adding the crushed crops into an extracting solution, grinding the mixture of the crops and the extracting solution into pulp, carrying out centrifugal separation under the assist of ultrasound to obtain an enzyme solution, placing the enzyme solution into a cuvette, respectively determining a light absorption value at 454nm before and after reaction, determining the protein concentration of the enzyme solution according to a formula, and finally calculating the enzyme specific activity according to an enzyme specific activity determination formula. According to the method, the specific activity of the lipoxidase isozyme LOX-3 can be rapidly, accurately and quantitatively determined, the process is simple, the cost is low, and the precision is obviously better than that of the existing relevant detection technologies.
Description
Technical field
The present invention relates to a kind of biochemical detection methods, be specifically related to a kind of method for quantitatively determining of crop fat oxidation enzyme isoenzyme LOX-3 Rate activity.
Background technology
Lipoxidase (LOX) is a kind of protein containing nonheme iron, and single-minded catalysis has the polybasic unsaturated fatty acid Oxygenation of suitable, cis-Isosorbide-5-Nitrae-pentadiene structure, and oxidation generates the hydroperoxide with conjugated double bond.Lipoxidase has three kinds of isodynamic enzymes Lox-1, Lox-2, Lox-3, and they are extensively present in higher plant body, relevant with photosynthesis, traumatic response and other Stress responses etc. with the growing of plant, the aging of plant, lipid peroxidation.Lox content in the soya seeds of maturation very high (accounting for 1% ~ 2% of seed protein content), can the Oxygenation of catalysis unsaturated fatty acid, form the volatile matter such as hydrogen peroxide derivant, can protein directly in food and amino acid be combined, produce beany flavor and bitter taste, reducing local flavor and the nutritive value of food, and destroy above-mentioned several essential fatty acid, is the ANFs in soybean.
The vigor of lipoxidase directly affects crop seed shelf-life and the processing qualities such as soybean, peanut, paddy rice, and vigor is less, and it is less on the impact of seed.Researcher has turned out the new crop varieties of lipoxidase Lox disappearance in research seed selection, therefore how quantitatively to detect that lipoxidase Lox is active rapidly and accurately, data can be detected more exactly for researcher provides, thus increase work efficiency, accelerate the research steps of new varieties, simultaneously also for crops storage, purchase provide accurately practical data.
Summary of the invention
The invention provides a kind of method for quantitatively determining of crop fat oxidation enzyme isoenzyme LOX-3 Rate activity.
The technical solution used in the present invention is as follows:
A method for quantitatively determining for crop fat oxidation enzyme isoenzyme LOX-3 Rate activity, is characterized in that, comprise the following steps:
(1) crop plant embryos is ground, add extract, grind to form homogenate, homogenate loaded in ultrasonic extraction still, ultrasound wave transmission frequency is set in 30-40KHz, temperature regulable control is at 20-25 DEG C, extraction time 5-8 minute, then goes to tool lid centrifuge tube, then divides 3-4 washing with extract, cleansing solution goes to tool lid centrifuge tube, the centrifugal 8-10min of decolorization swinging table vibration 8-10min, 8000rpm, leaves standstill 3-5min, get supernatant liquor, i.e. enzyme liquid;
(2) the enzyme liquid of extraction is got in cuvette, add appropriate reactant liquor, measure light absorption value at 454nm place, when reaction starts, record light absorption value, takes out reaction system, to centrifuge tube, 4 DEG C of lucifuge reactions, measure the light absorption value after 24h and record, represent LOX-3 vigor with the change of light absorption value, return to zero as reference with pure water, replace enzyme liquid in contrast with damping fluid;
(3) enzyme liquid protein concentration is measured:
Get appropriate enzyme liquid, first working sample 280nm OD value and 260nm OD value, then according to following formulae discovery enzyme liquid protein concentration: (assay method of enzyme liquid protein concentration can with reference to Li Jianwu etc., Biochemistry Experiment principle and method, 1994)
Enzyme liquid protein concentration (mg/mL)=280nm OD value * 1.45-0.74*260nm OD value;
(4) specific activity of enzyme is calculated:
Measure formulae discovery according to specific activity of enzyme and go out specific activity of enzyme,
Specific activity of enzyme=(the sample OD of 0 hour value-sample OD value of 24 hours)-(contrasting OD value-the contrast OD value of 24 hours of 0 hour)/enzyme liquid protein concentration.
The method for quantitatively determining of described crop fat oxidation enzyme isoenzyme LOX-3 Rate activity, is characterized in that: described crop is paddy rice or peanut.
The method for quantitatively determining of described crop fat oxidation enzyme isoenzyme LOX-3 Rate activity, it is characterized in that: the compound method of described extract is: get glycerine 10ml, Tween-201.5ml, 100ml is settled to the phosphate buffer of PH6.6, even through magnetic stirrer, obtain LOX3 extract;
The preparation of described pH6.6 phosphate buffer: Na
2hPO
471.64g/L, NaH
2pO
431.21g/L, gets 26.865g Na
2hPO
4, 19.506g NaH
2pO
4, be dissolved in water, be settled to 1000ml.
The method for quantitatively determining of described crop fat oxidation enzyme isoenzyme LOX-3 Rate activity, it is characterized in that: the preparation method of described reactant liquor is: by the phosphate buffer of PH6.6, pure water, saturated beta carotene-the acetone soln of linoleic acid sodium and dilution 1.5 times, mix in the ratio of 5:1:1:1 (v/v), obtain LOX-3 reactant liquor, the compound method of described linoleic acid sodium: quantitatively take 70mg linoleic acid, add equivalent Tween-20 again, add 4ml oxygen-free water again, abundant mixing, be about 0.55ml at the NaOH adding 0.5N and become clarification to solution, be settled to 25ml again, equivalent, divide and be filled to-20 DEG C of refrigerations, stand-by, the described preparation releasing the saturated beta carotene-acetone soln of 1.5 times: first join saturated beta carotene-acetone soln, then use acetone diluted 1.5 times, now with the current.
Principle of the present invention: utilize lipoxidase LOX-3 energy coupling to be oxidized beta carotene and produce metachromasia, this reaction has an absorption peak at 454nm place, and the rate of change measuring its light absorption value can reflect its catalytic rate.
Beneficial effect of the present invention: the inventive method can detect lipoxidase Lox-3 Rate activity fast, accurately, quantitatively, its process is simple, and cost is low, and its degree of accuracy is obviously better than existing correlation detection technology; Data can be detected more exactly for researcher provides, can increase work efficiency, accelerate the breeding of new variety paces of LOX-3 disappearance, simultaneously also for crops storage, purchase provide more accurately practical data.
Embodiment
1, reactant liquor, extract is prepared
Detection method
(1) sample
Rice paddy seed more than 30, shells, and cuts or tweezers take EMBRYO IN RICE with blade, amounts to about 100 embryos, is placed in-80 DEG C of refrigerators, stand-by.Get peanut single seed cotyledon, after blade chopping, mixing, is placed in-80 DEG C of refrigerators, stand-by.
(2) leaching process
For paddy rice, peanut seed.Accurately take EMBRYO IN RICE: 10mg; Peanut cotylcdon: 5mg.Add 1ml said extracted liquid, grind to form homogenate, homogenate is loaded in ultrasonic extraction still, ultrasound wave transmission frequency is set in 35KHz, temperature regulable control at 24 DEG C, extraction time 6 minutes, then go to 5ml tool lid centrifuge tube, then divide three washings with 3ml extract, go to 5ml tool lid centrifuge tube, decolorization swinging table vibration 10min, the centrifugal 10min of 8000rpm, leaves standstill 3min, gets supernatant liquor, i.e. enzyme liquid, 3 repetitions are established in test.
(3) testing process
Get the enzyme liquid of 1.5ml extraction in cuvette, add 2ml reactant liquor, measure light absorption value at 454nm place, when reaction starts, record light absorption value, takes out reaction system, to centrifuge tube, 4 DEG C of lucifuge reactions, measure the light absorption value after 24h, represent LOX-3 vigor with the change of light absorption value, return to zero as reference with pure water, replace enzyme liquid in contrast with damping fluid.
Measure enzyme liquid protein concentration:
Get 1.5ml enzyme liquid, first working sample 280nm OD value and 260nm OD value, then according to following formulae discovery: (with reference to Li Jianwu etc., Biochemistry Experiment principle and method, 1994)
Enzyme liquid protein concentration (mg/mL)=280nm OD value * 1.45-0.74*260nm OD value calculates specific activity of enzyme:
Measure formulae discovery according to specific activity of enzyme and go out specific activity of enzyme:
Specific activity of enzyme=(the sample OD of 0 hour value-sample OD value of 24 hours)-(contrasting OD value-the contrast OD value of 24 hours of 0 hour)/enzyme liquid protein concentration.
Claims (4)
1. a method for quantitatively determining for crop fat oxidation enzyme isoenzyme LOX-3 Rate activity, is characterized in that, comprise the following steps:
(1) crop plant embryos is ground, add extract, grind to form homogenate, homogenate loaded in ultrasonic extraction still, ultrasound wave transmission frequency is set in 30-40KHz, temperature regulable control is at 20-25 DEG C, extraction time 5-8 minute, then goes to tool lid centrifuge tube, then divides 3-4 washing with extract, cleansing solution goes to tool lid centrifuge tube, the centrifugal 8-10min of decolorization swinging table vibration 8-10min, 8000 rpm, leaves standstill 3-5 min, get supernatant liquor, i.e. enzyme liquid;
(2) the enzyme liquid of extraction is got in cuvette, add appropriate reactant liquor, measure light absorption value at 454nm place, when reaction starts, record light absorption value, takes out reaction system, to centrifuge tube, 4 DEG C of lucifuge reactions, measure the light absorption value after 24h and record, represent LOX-3 vigor with the change of light absorption value, return to zero as reference with pure water, replace enzyme liquid in contrast with damping fluid;
(3) enzyme liquid protein concentration is measured:
Get appropriate enzyme liquid, first working sample 280nm OD value and 260nm OD value, then according to following formulae discovery enzyme liquid protein concentration:
Enzyme liquid protein concentration (mg/mL)=280nm OD value * 1.45-0.74*260nm OD value;
(4) specific activity of enzyme is calculated:
Measure formulae discovery according to specific activity of enzyme and go out specific activity of enzyme,
Specific activity of enzyme=(the sample OD of 0 hour value-sample OD value of 24 hours)-(contrasting OD value-the contrast OD value of 24 hours of 0 hour)/enzyme liquid protein concentration.
2. the method for quantitatively determining of crop fat oxidation enzyme isoenzyme LOX-3 Rate activity according to claim 1, is characterized in that: described crop is paddy rice or peanut.
3. the method for quantitatively determining of crop fat oxidation enzyme isoenzyme LOX-3 Rate activity according to claim 1, it is characterized in that: the compound method of described extract is: get glycerine 10ml, Tween-20 1.5ml, 100ml is settled to the phosphate buffer of PH6.6, even through magnetic stirrer, obtain LOX3 extract;
The preparation of described pH6.6 phosphate buffer: Na
2hPO
471.64g/L, NaH
2pO
431.21g/L, gets 26.865g Na
2hPO
4, 19.506 g NaH
2pO
4, be dissolved in water, be settled to 1000ml.
4. the method for quantitatively determining of crop fat oxidation enzyme isoenzyme LOX-3 Rate activity according to claim 1, it is characterized in that: the preparation method of described reactant liquor is: by the phosphate buffer of PH6.6, pure water, linoleic acid sodium and dilution 1.5 times saturated-carrotene-acetone soln, in 5:1:1:1(v/v) ratio mixing, obtain LOX-3 reactant liquor, the compound method of described linoleic acid sodium: quantitatively take 70mg linoleic acid, add equivalent Tween-20 again, add 4ml oxygen-free water again, abundant mixing, be about 0.55ml at the NaOH adding 0.5N and become clarification to solution, be settled to 25ml again, equivalent, divide and be filled to-20 DEG C of refrigerations, stand-by, described release 1.5 times saturated-preparation of carrotene-acetone soln: first join saturated-carrotene-acetone soln, then use acetone diluted 1.5 times, now with the current.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112813185A (en) * | 2021-02-20 | 2021-05-18 | 嘉兴市农业科学研究院 | Primer composition, kit and method for identifying genotype of rice storage-resistant gene lox3 |
| CN113848202A (en) * | 2021-09-03 | 2021-12-28 | 东北农业大学 | Screening and identifying method for soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny |
Citations (5)
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| JP2000287700A (en) * | 1999-02-05 | 2000-10-17 | Shinotesuto:Kk | Measurement of lipase activity value, cholesterol concentration and neutral lipid concentration |
| CN101797038A (en) * | 2010-04-14 | 2010-08-11 | 山东省花生研究所 | Peanut dietary fiber ultrasonic wave or microwave auxiliary extraction and purification method |
| CN102279181A (en) * | 2011-04-18 | 2011-12-14 | 中国农业大学 | Method for testing activity of corn seed embryo lipoxidase |
| CN102323229A (en) * | 2011-08-09 | 2012-01-18 | 丹阳市江南面粉有限公司 | Method for quickly measuring activity of lipoxidase in grain processing byproduct |
| CN102807629A (en) * | 2011-06-01 | 2012-12-05 | 鹤岗市三江平原米业集团有限公司 | Method for extracting rice bran polysaccharides by using continuous countercurrent ultrasonic equipment |
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2014
- 2014-11-20 CN CN201410668086.5A patent/CN104390919A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000287700A (en) * | 1999-02-05 | 2000-10-17 | Shinotesuto:Kk | Measurement of lipase activity value, cholesterol concentration and neutral lipid concentration |
| CN101797038A (en) * | 2010-04-14 | 2010-08-11 | 山东省花生研究所 | Peanut dietary fiber ultrasonic wave or microwave auxiliary extraction and purification method |
| CN102279181A (en) * | 2011-04-18 | 2011-12-14 | 中国农业大学 | Method for testing activity of corn seed embryo lipoxidase |
| CN102807629A (en) * | 2011-06-01 | 2012-12-05 | 鹤岗市三江平原米业集团有限公司 | Method for extracting rice bran polysaccharides by using continuous countercurrent ultrasonic equipment |
| CN102323229A (en) * | 2011-08-09 | 2012-01-18 | 丹阳市江南面粉有限公司 | Method for quickly measuring activity of lipoxidase in grain processing byproduct |
Non-Patent Citations (1)
| Title |
|---|
| 高奇等: "《花生种子LOX3活力测定及其对储藏特性的影响》", 《中国油料作物学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112813185A (en) * | 2021-02-20 | 2021-05-18 | 嘉兴市农业科学研究院 | Primer composition, kit and method for identifying genotype of rice storage-resistant gene lox3 |
| CN113848202A (en) * | 2021-09-03 | 2021-12-28 | 东北农业大学 | Screening and identifying method for soybean lipoxygenase and 7S and 11S globulin subunit deletion hybrid progeny |
| CN113848202B (en) * | 2021-09-03 | 2024-03-01 | 东北农业大学 | Soybean lipoxygenase and screening and identifying method of 7S and 11S globulin subunit deletion hybrid offspring |
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