CN103397078A - Determination method for enzymatic activity of sucrose synthetase in cassava leaf - Google Patents
Determination method for enzymatic activity of sucrose synthetase in cassava leaf Download PDFInfo
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Abstract
The invention discloses a determination method for enzymatic activity of sucrose synthetase in a cassava leaf. The method comprises the following steps: determination of enzymatic activity of sucrose synthetase both in a synthesis direction and in a decomposition direction; and extracting enzyme liquid from a fresh cassava leaf, carrying out desugaring by using a dialysis bag and then determining enzymatic activity. Compared with the prior art, the invention has the following advantages: since dialysis desugaring treatment and enzymatic activity determination technology are combined together after extraction of the enzyme liquid, interference of high-background cane sugar content in the enzyme extract of the cassava leaf on determination of enzymatic activity of sucrose synthetase can be effectively eliminated, determination sensitivity is improved, determination process is easy to control, and determination results are reliable and have good comparability and reproducibility.
Description
Technical field
The present invention relates to a kind of method of measuring the sucrose synthase enzymic activity, specifically a kind of method of measuring sucrose synthase enzymic activity in the cassava blade.
Background technology
Cassava stores much starch with the form of piece root, is important starch source.Sucrose is important photosynthate, is the essential substance of transportation in body.Sucrose synthase in the cassava blade is one of key enzyme in the tapioca (flour) building-up process, sucrose synthase is can either catalysis sucrose synthetic again can the catalysis sucrose decomposition, be one of key of controlling Qiang Yu storehouse, storehouse activity, the substrate of its active high synthetic starch is just sufficient.In the cassava blade, the mensuration of sucrose synthase activity is subjected to various factors, because itself there is a large amount of photosynthates---sucrose in the cassava blade, far away higher than the amount of measuring the product sucrose that generates in the short period of time in the enzymic activity process, therefore the existence of original a large amount of sucrose in cassava blade zyme extract, disturbed detecting of a small amount of sucrose that in the mensuration process, reaction generates.Find a kind of operation relatively simple, measurement result is reliable, favorable reproducibility, and sucrose synthase activity determination method in the strong cassava blade of different sample room comparabilities is significant to research tapioca (flour) synthesis mechanism.
The material Extraction buffer A(100mmol/L of the 126th page of " mensuration of sucrose synthase, Sucrose Phosphate Synthase activity " (what the is newly-built) collection in Shanghai Inst. of Plant Physiology, Chinese Academy of Sciences, the chief editor's of Shanghai City plant physiology association " modern plants Physiology Experiment guide " (1999), the Tris-HCl of pH=7.0,10mmol/L MgCl
2, 2mmol/L EDTA-Na
2, 20mmol/L mercaptoethanol, 2% ethylene glycol) in ice bath, extract after, with 4 layers of filtered through gauze, abandon residue, the centrifugal 30min of 7000 * g, get supernatant liquor ammonium sulfate fractional precipitation, collect the ammonium sulfate precipitation part of 30%～40% saturation ratio, redissolve in buffer B (20mmol/L, the Tris-HCl of pH=7.0,10mmol/L MgCl
2, 2mmol/L EDTA-Na
2, 5mmol/L mercaptoethanol, 10% ethylene glycol) in dialysed overnight, dialysis buffer liquid is changed once in centre, through the centrifugal 10min of 27500 * g, the supernatant liquor constant volume namely obtains mensuration enzyme liquid to 5mL.The sucrose amount that activity determination method adopts Resorcinol color reaction method assaying reaction to generate.The pre-treatment of enzyme liquid comprises ammonium sulfate precipitation and dialysis, the ammonium sulfate precipitation complicated operation, and wayward, the operating time is longer.The present invention improves Extraction buffer, is mainly to add polyvinylpyrrolidone (PVP), significantly improves the extraction result of extraction of proteolytic enzyme; Dialysis buffer liquid is also improved, greatly reduced the consumption of ethylene glycol, effectively avoid the precipitation of enzyme liquid in dialysis procedure.
[during Litchi Ripening, sugar accumulation and carbohydrate metabolism related enzyme activity change Li Jianguo.Agricultural University Of South China's journal (natural science edition), 2003,24(2): 87-88 ］ measure in litchi fruits in the sucrose synthase activity extraction of crude enzyme liquid: get about 4.0g pulp+10mmol/L Tris-HCl (pH7.0) damping fluid and [include 5mmol/L MgCl
2, 1mmol/L DTT, φ (BSA)=0.1%, (under TritonX-l00)=0.05% ice bath, grind homogenate, under 9000r/min, centrifugal 10min, pour out supernatant liquor to φ, get 2mL and cross the desugar of Sephadex G-25 post, collect the part of rich in proteins in elutriant as crude enzyme liquid.The desugar of Sephadex G-25 post is adopted in the pre-treatment of enzyme liquid, wash-out, collection process complicated operation, and the operating time is long, and operating process need to be carried out in low temperature chromatography cabinet.
Summary of the invention
The present invention is the defect that overcomes prior art, and a kind of method of measuring sucrose synthase enzymic activity in the cassava blade is provided.After the method adopts the enzyme liquid Extraction buffer of improvement to extract crude enzyme liquid, the improvement dialysis buffer liquid in by dialysis, remove or significantly reduce crude enzyme liquid in glucide, the interference to follow-up sucrose synthase enzyme assay of high background sucrose in cassava organ crude enzyme liquid, glucose content can be effectively eliminated, sensitivity and the reliability of sucrose synthase enzyme assay can be improved.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
A kind of method of measuring sucrose synthase enzymic activity in the cassava blade, the method comprises sucrose synthase compound direction and the mensuration of decomposing the direction enzymic activity in the cassava blade, after with fresh cassava blade, extracting enzyme liquid, by the dialysis tubing desugar, measure enzymic activity, concrete operation step is as follows again:
Acquired for materials with preserve: gathers fresh cassava blade, puts into rapidly low-temperature (low temperature) vessel, if can not measure at once, with liquid nitrogen grinding, be placed in-80 ℃ of cryogenic refrigerators preservation stand-by.
2. enzyme liquid extracts: get the cassava blade of 1g left and right precooling, add a small amount of quartz sand and 5mL enzyme liquid Extraction buffer, it consists of: 100mmol/L, the Tris-HCl of pH=7.2,10mmol/L MgCl
2, 1mmol/L EDTA-Na
2, 10mmol/L beta-mercaptoethanol, volumetric concentration 2% ethylene glycol, weight concentration l% polyvinylpyrrolidone, in mortar, grind to form fast even pasty state, after cleaning mortar, pour in centrifuge tube, the centrifugal 15min of 12000r/min on the cryogenic freezing ultracentrifuge, get supernatant liquor, abandon precipitation, above leaching process all carries out under 4 ℃.
3. enzyme liquid dialysis desugar: get supernatant liquor 2.5mL and pack in dialysis tubing, dialysis tubing is placed in dialysis buffer liquid and spends the night, and dialysis buffer liquid consists of: the Tris-HCl of 20mmol/L, pH=7.2,2.5mmol/L MgCl
2, 1mmol/L EDTA-Na
2, the 5mmol/L beta-mercaptoethanol, volumetric concentration 1% ethylene glycol, during change dialysis buffer liquid 1 time, use 125mL at every turn.Above dialysis procedure is carried out under 4 ℃, after dialysis, enzyme liquid is for enzyme assay.
4. sucrose synthase compound direction enzyme assay: 4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, respectively add enzyme liquid after the above-mentioned dialysis desugar of 0.2mL, this moment, the blank pipe added the 0.2mL2mmol/L NaOH enzyme that goes out to live, then in above-mentioned 4 test tubes, add 0.4mL building-up reactions medium, it consists of: Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDPG, 10mmol/L D-Fructose, after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.2mL2mol/L NaOH and mix, then boiling water bath 10min, cooling, add 1mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric; From the sucrose typical curve, calculate enzymic activity, with micromole's sucrose/gram fresh weight hour, namely μ mol sucrose/gFWh represents.
5. the drafting of sucrose typical curve: accurately take 0.1000g sucrose and dissolve, be settled to 250ml with ultrapure water, be mixed with 400 μ g/mL sucrose solutions, get respectively 0.1,0.2,0.3,0.4,0.5, the above-mentioned sucrose solution of 0.6mL is in test tube, not enough 0.6mL mends to 0.6mL with ultrapure water, adds 0.2mL2mol/L NaOH and mixes, and adds 1mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric.With the result that the sucrose of different concns records, draw the sucrose typical curve.
6. sucrose synthase decomposes the direction enzyme assay: 4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, respectively add 0.2mL enzyme liquid, this moment, the blank pipe added the 0.2mL2mol/L NaOH enzyme that goes out to live, then at 4 test tubes, add 0.4mL building-up reactions medium, it consists of: Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDP, 50mmol/L sucrose, after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.1mL2mol/L NaOH and mix, and adds 0.5mL DNS reagent, then boiling water bath 5min, and is cooling, then adds 4mL distilled water, in 540nm place colorimetric; From the glucose typical curve, calculate enzymic activity, enzymic activity micromole's glucose/gram fresh weight hour, i.e. μ mol glucose/gFWh.
7. the drafting of glucose typical curve: accurately take 0.1000g analytical pure glucose, with ultrapure water, dissolve, be settled to 500mL, be mixed with 200 μ g/mL glucose solutions, get respectively 0.1,0.2,0.3,0.4,0.5, the above-mentioned glucose solution of 0.6mL is in test tube, not enough 0.6mL mends to 0.6mL with ultrapure water, adding 0.2mL2mol/L NaOH mixes, add 0.5mL DNS reagent, boiling water bath 5min, cooling, add again the 4mL ultrapure water, in 540nm place colorimetric, with the result that the glucose of different concns records, draw the glucose typical curve.
Above-mentioned steps 3) enzyme liquid dialysis desugar: described dialysis tubing refers to that molecular weight cut-off makes with semi-permeable membranes bag or the pipe that can pack into and treat dialysis liquid more than 3500～14000Dalton.
Enzyme assay that sucrose synthase measuring method in above-mentioned cassava blade, is characterized in that, mainly comprises the following steps: enzyme liquid extracts---enzyme liquid dialysis desugar---.
The present invention's advantage compared with the prior art is: after enzyme liquid dialysis desugar, eliminated the interference of high background sucrose content to the sucrose synthase enzyme assay in cassava blade zyme extract, improve and measure sensitivity, the mensuration process is easy to control, and measurement result is reliable, comparability is strong, favorable reproducibility.
Embodiment:
Below in conjunction with embodiment, the present invention is further described.
Embodiment 1
Measure the method for sucrose synthase enzymic activity in the cassava blade:
Acquired for materials with preserve: gathers fresh cassava blade, puts into rapidly low-temperature (low temperature) vessel, if can not measure at once, with liquid nitrogen grinding, be placed in-80 ℃ of cryogenic refrigerators preservation stand-by.
2. enzyme liquid extracts: get the cassava blade of 1g left and right precooling, add a small amount of quartz sand and 5mL enzyme liquid Extraction buffer (100mmol/L, the Tris-HCl of pH=7.2,10mmol/L MgCl
2, 1mmol/L EDTA-Na
2, 10mmol/L beta-mercaptoethanol, volumetric concentration 2% ethylene glycol, weight concentration l% polyvinylpyrrolidone (PVP) grind to form even pasty state fast in mortar, after cleaning mortar, pour in centrifuge tube, the centrifugal 15min of 12000r/min on the cryogenic freezing ultracentrifuge, get supernatant liquor, abandon precipitation, above leaching process all carries out under 4 ℃.
3. enzyme liquid dialysis desugar: supernatant liquor 2.5mL packs in dialysis tubing, and dialysis tubing is placed in dialysis buffer liquid (Tris-HCl of 20mmol/L, pH=7.2,2.5mmol/L MgCl
2, 1mmol/L EDTA-Na
2, 5mmol/L beta-mercaptoethanol, volumetric concentration 1% ethylene glycol) in spend the night, during change dialyzate 1 time, use 125mL at every turn.Above process dialysis procedure is carried out under 4 ℃.After dialysis, enzyme liquid is for enzyme assay.
4. sucrose synthase compound direction enzyme assay: 4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, respectively add enzyme liquid (this moment, the blank pipe added 0.2mL2mmol/L NaOH) after 0.2mL dialysis desugar, add 0.4mL building-up reactions medium (Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDPG, 10mmol/L D-Fructose), after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.2mL2mol/L NaOH and mix, then boiling water bath 10min, cooling, add 1mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric.By Units of Account, in the time, react the amount that generates sucrose and calculate the activity of enzyme, from typical curve, calculate the sucrose amount of sucrose synthase compound direction reaction generation and calculate enzymic activity, enzymic activity represents with mmole sucrose/(gram fresh weight hour), i.e. μ mol sucrose/(gFWh) expression.
5. the drafting of sucrose typical curve: preparation 400 μ g/mL sucrose solutions (accurately take 0.1000g sucrose and dissolve, be settled to 250ml with ultrapure water), get respectively 0.1,0.2,0.3,0.4,0.5, the 0.6mL sucrose solution puts into test tube, not enough 0.6mL mends to 0.6mL with ultrapure water, adding 0.2mL2mol/LNaOH mixes, add 1mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath 10min, cooling, 480nm place colorimetric.With the result that the sucrose of different concns records, draw the sucrose typical curve.
6. sucrose synthase decomposes the direction enzyme assay: 4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, respectively add 0.2mL enzyme liquid (this moment, the blank pipe added 0.2mL2mol/L NaOH), add 0.4mL building-up reactions medium (Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDP, 50mmol/L sucrose).After 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.1mL2mol/L NaOH and mix, and adds 0.5mL DNS reagent, then boiling water bath 5min, and is cooling, then adds 4mL distilled water, in 540nm place colorimetric.Enzymic activity represents with μ mol sucrose/(gram fresh weight hour), i.e. μ mol glucose/(gFWh) expression.
7. the drafting of glucose typical curve: preparation 200 μ g/mL glucose solutions (accurately take 0.1000g analytical pure glucose, with ultrapure water, dissolve, be settled to 500mL), get respectively 0.1,0.2,0.3,0.4,0.5, the 0.6mL glucose solution puts into test tube, not enough 0.6mL mends to 0.6mL with ultrapure water, adds 0.2mL2mol/L NaOH and mixes, and adds 0.5mL DNS reagent, boiling water bath 5min, cooling, then add the 4mL ultrapure water, in 540nm place colorimetric.With the result that the glucose of different concns records, draw the glucose typical curve.
Adopt the result of present method mensuration cassava blade sucrose synthase enzymic activity as follows:
Table 1 cassava blade sucrose synthase compound direction enzyme assay result
Annotate: " sucrose synthase " english abbreviation " SS "
Table 2 cassava blade sucrose synthase decomposes direction enzyme assay result
8) although the present invention describes with reference to embodiment, this description not meaning that is construed as limiting the present invention.With reference to the description of the embodiment of the present invention, other of disclosed embodiment change, and the variation that can expect for those skilled in the art should belong in affiliated claim limited range.
Claims (2)
1. method of measuring sucrose synthase enzymic activity in the cassava blade, it is characterized in that, the method comprises sucrose synthase compound direction and the mensuration of decomposing the direction enzymic activity in the cassava blade, after with fresh cassava blade, extracting enzyme liquid, by the dialysis tubing desugar, measure again enzymic activity, concrete operation step is as follows: 1) acquired for materials and preservation: gather fresh cassava blade, put into rapidly low-temperature (low temperature) vessel, if can not measure at once, with liquid nitrogen grinding be placed in-80 ℃ of cryogenic refrigerators preserve stand-by;
2) enzyme liquid extracts: get the cassava blade of 1g left and right precooling, add a small amount of quartz sand and 5mL enzyme liquid Extraction buffer, it consists of: 100mmol/L, the Tris-HCl of pH=7.2,10mmol/L MgCl
2, 1mmol/L EDTA-Na
2, 10mmol/L beta-mercaptoethanol, volumetric concentration 2% ethylene glycol, weight concentration l% polyvinylpyrrolidone, in mortar, grind to form fast even pasty state, after cleaning mortar, pour in centrifuge tube, the centrifugal 15min of 12000r/min on the cryogenic freezing ultracentrifuge, get supernatant liquor, abandon precipitation, above leaching process all carries out under 4 ℃;
3) enzyme liquid dialysis desugar: get supernatant liquor 2.5mL and pack in dialysis tubing, dialysis tubing is placed in dialysis buffer liquid and spends the night, and dialysis buffer liquid consists of: the Tris-HCl of 20mmol/L, pH=7.2,2.5mmol/L MgCl
2, 1mmol/L EDTA-Na
2, the 5mmol/L beta-mercaptoethanol, volumetric concentration 1% ethylene glycol, during change dialysis buffer liquid 1 time, more than using 125mL at every turn; Dialysis procedure is carried out under 4 ℃, after dialysis, enzyme liquid is for enzyme assay;
4) enzyme assay of sucrose synthase compound direction: 4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, respectively add enzyme liquid after the above-mentioned dialysis desugar of 0.2mL, this moment, the blank pipe added the 0.2mL2mmol/L NaOH enzyme that goes out to live, then in above-mentioned 4 test tubes, add 0.4mL building-up reactions medium, it consists of: Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDPG, 10mmol/L D-Fructose, after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.2mL2mol/L NaOH and mix, then boiling water bath 10min, cooling, add 1mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric; From the sucrose typical curve, calculate enzymic activity, with micromole's sucrose/gram fresh weight hour, namely μ mol sucrose/gFWh represents;
5) drafting of sucrose typical curve: accurately take 0.1000g sucrose and dissolve, be settled to 250ml with ultrapure water, be mixed with 400 μ g/mL sucrose solutions, get respectively 0.1,0.2,0.3,0.4,0.5, the above-mentioned sucrose solution of 0.6mL is in test tube, not enough 0.6mL mends to 0.6mL with ultrapure water, add 0.2mL2mol/L NaOH and mix, add 1mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric, draw the sucrose typical curve with the result that the sucrose of different concns records;
6) sucrose synthase decomposes the mensuration of direction enzymic activity: 4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, respectively add enzyme liquid after the above-mentioned dialysis desugar of 0.2mL, this moment, the blank pipe added the 0.2mL2mol/L NaOH enzyme that goes out to live, then at 4 test tubes, add 0.4mL building-up reactions medium, it consists of: Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDP, 50mmol/L sucrose, after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.1mL2mol/L NaOH and mix, add 0.5mL DNS reagent, then boiling water bath 5min, cooling, add again 4mL distilled water, in 540nm place colorimetric, from the glucose typical curve, calculate enzymic activity, enzymic activity micromole's glucose/gram fresh weight hour, i.e. μ mol glucose/gFWh;
7) drafting of glucose typical curve: accurately take 0.1000g analytical pure glucose, with ultrapure water, dissolve, be settled to 500mL, be mixed with 200 μ g/mL glucose solutions, get respectively 0.1,0.2,0.3,0.4,0.5, the above-mentioned glucose solution of 0.6mL is in test tube, not enough 0.6mL mends to 0.6mL with ultrapure water, adding 0.2mL2mol/L NaOH mixes, add 0.5mL DNS reagent, boiling water bath 5min, cooling, add again the 4mL ultrapure water, in 540nm place colorimetric, with the result that the glucose of different concns records, draw the glucose typical curve;
Above-mentioned steps 3) enzyme liquid dialysis desugar: described dialysis tubing refers to that molecular weight cut-off makes with semi-permeable membranes bag or the pipe that can pack into and treat dialysis liquid more than 3500～14000Dalton.
2. enzyme assay that sucrose synthase measuring method in the described cassava blade of claim 1, is characterized in that, mainly comprises the following steps: enzyme liquid extracts---enzyme liquid dialysis desugar---.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108195646A (en) * | 2017-12-29 | 2018-06-22 | 河海大学 | A kind of long-range sampling rice grain carbon metablism key enzyme activity detection method |
| CN112268870A (en) * | 2020-10-28 | 2021-01-26 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Method for evaluating storage-resistant characteristic of sweet potatoes |
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| CN102747055A (en) * | 2012-06-19 | 2012-10-24 | 南京农业大学 | Method for extracting sucrose synthase and sucrose phosphate synthase of pear fruit and activity determination method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108195646A (en) * | 2017-12-29 | 2018-06-22 | 河海大学 | A kind of long-range sampling rice grain carbon metablism key enzyme activity detection method |
| CN112268870A (en) * | 2020-10-28 | 2021-01-26 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Method for evaluating storage-resistant characteristic of sweet potatoes |
| CN112268870B (en) * | 2020-10-28 | 2023-09-29 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | Evaluation method for storage-resistant characteristics of sweet potatoes |
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