CN103397079A - Determination method for enzymatic activity of sucrose phosphate synthase in cassava leaf - Google Patents
Determination method for enzymatic activity of sucrose phosphate synthase in cassava leaf Download PDFInfo
- Publication number
- CN103397079A CN103397079A CN2013103436075A CN201310343607A CN103397079A CN 103397079 A CN103397079 A CN 103397079A CN 2013103436075 A CN2013103436075 A CN 2013103436075A CN 201310343607 A CN201310343607 A CN 201310343607A CN 103397079 A CN103397079 A CN 103397079A
- Authority
- CN
- China
- Prior art keywords
- dialysis
- sucrose
- enzyme
- enzyme liquid
- phosphate synthase
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a determination method for enzymatic activity of sucrose phosphate synthase in a cassava leaf. The method comprises the following steps: rapidly placing a fresh cassava leaf in an ice box; extracting an enzyme liquid in an ice bath by using an extracting buffer solution; carrying out high-speed refrigerated centrifugation; taking out a supernatant for dialysis desugaring in a dialysis bag at a low temperature (4 DEG C), wherein a dialysis buffer solution is composed of Tris-HCI with a concentration of 20 mmol/L and a pH value of 7.2, MgCl2 with a concentration of 2.5 mmol/L, EDTA-Na2 with a concentration of 1 mmol/L, beta-mercaptoethanol with a concentration of 5 mmol/L and glycol with a volume concentration of 1%; and subjecting the enzyme liquid to dialysis and then determining activity of sucrose phosphate synthase. 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 phosphate synthase 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 Phosphate Synthase enzymic activity in the cassava blade.
Background technology
Cassava stores much starch with the form of piece root, is important starch source.Cassava blade photosynthate mainly exists and outwards output with the form of sucrose, controlling the synthetic enzyme of sucrose in blade is Sucrose Phosphate Synthase (SPS), and this enzyme is the sugared transferring enzyme take uridine diphosphoglucose (UDPG) as donor, take fructose-1, 6-diphosphate (F-6-P) as acceptor.It is generally acknowledged, the sucrose building-up process of SPS/Suc-6-Pase system catalysis is the synthetic main path of blade sucrose, the photosynthate that is transported in the piece root exists with the form of sucrose at first, and degraded thereafter just can be used to synthetic starch after generating uridine diphosphoglucose (UDPG) and fructose.In the cassava organ, the mensuration of Sucrose Phosphate Synthase activity is subjected to various factors, because itself there is a large amount of photosynthates---sucrose in the cassava organ, far away higher than the amount of measuring a small amount of 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 organ zyme extract, disturbed in the enzyme assay process and generated detecting of sucrose.Find a kind of operation relatively simple, measurement result is reliable, favorable reproducibility, and Sucrose Phosphate Synthase enzyme activity method in the strong cassava organ 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) extract in ice bath after, abandon residue with 4 layers of filtered through gauze, 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.
Summary of the invention
The present invention is for overcoming now methodical defect, a kind of method of measuring Sucrose Phosphate Synthase in the cassava organ is provided, after the method adopts the enzyme liquid Extraction buffer of improvement to extract crude enzyme liquid, glucide remove crude enzyme liquid by dialysis in the dialysis buffer liquid of improvement in, can effectively eliminate cassava organ crude enzyme liquid in high background sucrose content to the interference of follow-up Sucrose Phosphate Synthase enzyme assay, can improve sensitivity and the reliability of Sucrose Phosphate Synthase enzyme assay.
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 Phosphate Synthase enzymic activity in the cassava blade, after extracting enzyme liquid with fresh cassava blade, by the dialysis tubing desugar, then measure 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, adding a small amount of quartz sand and 5mL extracts medium grind to form fast even pasty state 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 ℃; Described extraction medium composition is: 100mmol/L, the Tris-HCl of pH=7.2,10mmol/L MgCl
2, l mmol/L EDTA-Na
2, 10mmol/L beta-mercaptoethanol, volumetric concentration 2% ethylene glycol, weight concentration 1% polyvinylpyrrolidone.
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, l mmol/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 process dialysis procedure is carried out under 4 ℃.After dialysis, enzyme liquid is used for enzyme assay.
4. the determination of activity of Sucrose Phosphate Synthase:
4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, 4 test tubes 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 add 0.4mL building-up reactions medium in above-mentioned 4 test tubes, it consists of: Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDPG, 10mmol/L fructose-6-phosphate, mix rear after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.2mL2mol/L NaOH and mix, then boiling water bath 10min, cooling, add l mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, in 480nm place colorimetric.Calculate enzymic activity from the sucrose typical curve, 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.Draw the sucrose typical curve with the result that the sucrose of different concns records.
Above-mentioned steps 3 enzyme liquid dialysis desugars: 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 3500～14000Da.
Enzyme assay that in above-mentioned cassava organ, the Sucrose Phosphate Synthase measuring method mainly comprises the following steps: enzyme liquid extracts---enzyme liquid dialysis desugar---.
The present invention's advantage compared with the prior art is: eliminated the interference of high background sucrose content to the Sucrose Phosphate Synthase enzyme assay in cassava blade zyme extract after enzyme liquid dialysis desugar, 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 Phosphate Synthase in the cassava organ:
1) acquired for materials with preserve: gathers fresh cassava blade, puts into rapidly low-temperature (low temperature) vessel,, if can not measure at once, namely 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 and extract medium (100mmol/L, the Tris-HCl of pH=7.2,10mmol/L MgCl
2, lmmol/L EDTA-Na
2, 10mmol/L beta-mercaptoethanol, 2% ethylene glycol, 1% 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, lmmol/L EDTA-Na
2, 5mmol/L beta-mercaptoethanol, 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 used for enzyme assay.
4) Sucrose Phosphate Synthase determination of activity: 4 test tubes are got in each processing, 1 blank wherein, 3 repetitions, respectively add enzyme liquid after the above-mentioned dialysis desugar of 0.2mL, this moment, the blank pipe added 0.2mL2mmol/L NaOH, added 0.4mL building-up reactions medium (Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDPG, 10mmol/L fructose-6-phosphate), after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.2mL2mol/L NaOH and mix, then boiling water bath 10min, cooling, add lmL0.1% Resorcinol and 3.5mL30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric.Calculate the activity of enzyme by the amount of Units of Account time internal reaction generation sucrose, calculate the sucrose amount of sucrose synthase compound direction reaction generation from typical curve and calculate enzymic activity, enzymic activity represents with mmole sucrose/(gram fresh weight hour), i.e. μ mol sucrose/(gFWh) expression.
5) drafting of sucrose typical curve: preparation 400 μ g/mL sucrose solutions (accurately take the 0.1000g sucrose dissolved, with ultrapure water, be settled to 250ml), get respectively 0.1,0.2,0.3,0.4,0.5,0.6mL sucrose reference liquid not enough 0.6mL, supply with ultrapure water, add 0.2mL2mol/L NaOH and mix, boiling water bath 10min, cooling, add 1mL0.1% Resorcinol and 3.5mL30%HCl, shake up, 80 ℃ of water-bath 10min, cooling, 480nm place colorimetric.Draw the sucrose typical curve with the result that the sucrose of different concns records.
Adopt the result of present method mensuration cassava blade Sucrose Phosphate Synthase enzymic activity as follows:
Table 1 cassava blade Sucrose Phosphate Synthase determination of activity result
Annotate: " Sucrose Phosphate Synthase " english abbreviation " SPS "
6) although the present invention describes with reference to embodiment, this description and 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. a method of measuring Sucrose Phosphate Synthase enzymic activity in the cassava blade, is characterized in that, after extracting enzyme liquid with fresh cassava blade, by the dialysis tubing desugar, then measures enzymic activity, and 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 leaves of 1g left and right precooling, adding a small amount of quartz sand and 5mL extracts medium grind to form fast even pasty state 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 ℃; Described extraction medium composition is: 100mmol/L, the Tris-HCl of pH=7.2,10mmol/L MgCl
2, lmmol/L EDTA-Na
2, 10mmol/L beta-mercaptoethanol, volumetric concentration 2% ethylene glycol, weight concentration 1% polyvinylpyrrolidone;
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, lmmol/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 process dialysis procedure is carried out under 4 ℃.Enzyme liquid after dialysis is used for enzyme assay;
4) determination of activity of Sucrose Phosphate Synthase:
4 test tubes are got in each processing, wherein 1 as blank, 3 are repeated as measuring, 4 test tubes respectively add enzyme liquid after the above-mentioned dialysis desugar of 0.2mL, this moment, the blank pipe added the 0.2mL2mmol/LNaOH enzyme that goes out to live, then add 0.4mL building-up reactions medium in above-mentioned 4 test tubes, it consists of: Tris-HCl of 100mmol/L pH=7.2,10mmol/L MgCl
2, 5mmol/L UDPG, 10mmol/L fructose-6-phosphate, mix rear after 30 ℃ of water-bath 30min, boiling water bath 5min, add 0.2mL2mol/L NaOH and mix, then boiling water bath 10min, cooling, add l mL weight concentration 0.1% Resorcinol and 3.5mL weight concentration 30%HCl, shake up, 80 ℃ of water-bath l0min, cooling, 480nm place colorimetric; Calculate enzymic activity from the sucrose typical curve, 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, 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.Draw the sucrose typical curve with the result that the sucrose of different concns records;
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 3500～14000Da.
2. enzyme assay that Sucrose Phosphate Synthase measuring method in the described cassava organ of claim 1, is characterized in that, mainly comprises the following steps: enzyme liquid extracts---enzyme liquid dialysis desugar---.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013103436075A CN103397079A (en) | 2013-08-08 | 2013-08-08 | Determination method for enzymatic activity of sucrose phosphate synthase in cassava leaf |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013103436075A CN103397079A (en) | 2013-08-08 | 2013-08-08 | Determination method for enzymatic activity of sucrose phosphate synthase in cassava leaf |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103397079A true CN103397079A (en) | 2013-11-20 |
Family
ID=49560778
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013103436075A Pending CN103397079A (en) | 2013-08-08 | 2013-08-08 | Determination method for enzymatic activity of sucrose phosphate synthase in cassava leaf |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103397079A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506061A (en) * | 2016-01-21 | 2016-04-20 | 苏州科铭生物技术有限公司 | Kit and method for detecting activity of sucrose phosphate synthase |
| CN107589081A (en) * | 2017-07-20 | 2018-01-16 | 中国农业科学院茶叶研究所 | A kind of anti-interference quick determination method of tealeaves China and foreign countries source doping sucrose |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2013
- 2013-08-08 CN CN2013103436075A patent/CN103397079A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| 唐秀梅等: "间作花生对木薯碳氮代谢产物及关键酶活性的影响", 《中国农学通报》 * |
| 许让伟: "砂梨果实和叶片中糖积累及代谢相关酶活性变化研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 高俊凤主编: "《植物生理学实验指导》", 31 May 2006, 高等教育出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506061A (en) * | 2016-01-21 | 2016-04-20 | 苏州科铭生物技术有限公司 | Kit and method for detecting activity of sucrose phosphate synthase |
| CN107589081A (en) * | 2017-07-20 | 2018-01-16 | 中国农业科学院茶叶研究所 | A kind of anti-interference quick determination method of tealeaves China and foreign countries source doping sucrose |
| CN107589081B (en) * | 2017-07-20 | 2020-05-08 | 中国农业科学院茶叶研究所 | Anti-interference rapid detection method for exogenous sucrose-doped tea |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| McCarthy et al. | Major biochemical composition of dissolved high molecular weight organic matter in seawater | |
| Weeks et al. | Extraction of collagenase from the involuting rat uterus | |
| CN102061332A (en) | Quantitative fructose assay kit and application thereof as well as quantitative seminal plasma fructose assay method | |
| CN103756974A (en) | Porcine epidemic diarrhea virus, and culture method and application thereof | |
| NZ586737A (en) | Compositions and methods of detecting tiabs | |
| CN103146835A (en) | Method and kit for detecting pathogenic bacterium of food source by test strip based on NASBA (Nucleic Acid Sequence Based Amplification) | |
| CN104387485A (en) | Method for extracting polysaccharides in flammulina velutipes by synergism of complex enzymes and high-pressure hot water extraction process | |
| WO2006086377A3 (en) | Method of detection of classical swine fever | |
| CN103397079A (en) | Determination method for enzymatic activity of sucrose phosphate synthase in cassava leaf | |
| CN102934680A (en) | Method for inhibiting lepidium meyenii activity | |
| CN103952473A (en) | UDP-glucuronic acid decarboxylase gene function detection | |
| CN101182344A (en) | Method for extracting and purifying DNA of plants like cactus | |
| CN103397078A (en) | Determination method for enzymatic activity of sucrose synthetase in cassava leaf | |
| Ren et al. | The use of amino sugars for assessing seasonal dynamics of particulate organic matter in the Yangtze River estuary | |
| CN103966362A (en) | Method for synchronously detecting four apple viruses | |
| CN102830183B (en) | Sample pre-treatment method for determining endogenesis hormone content of towel gourd seeds by adopting HPLC (High Performance Liquid Chromatography) | |
| WO2006091949A3 (en) | Phosphorylated p68 rna helicase as a marker for cancer and cancer metastasis | |
| CN106290661A (en) | The method of CYP2C9 enzyme activity in a kind of high performance liquid chromatography tandem mass spectrum method detection Lumbricus body | |
| CN103509821B (en) | The application of a kind of Plant P Nutrition quick diagnosis and Visual Dynamic monitoring method and recombinant expression vector thereof | |
| CN105486568A (en) | Universal extraction agent for soil and soil nutrient extraction method | |
| Liu et al. | The paleoclimatic events and cause in the Okinawa Trough during 50 kaBP | |
| CN102154522B (en) | Primer and system for detecting bombyx mori cytoplasmic polyhedrosis virus (BmCPV) | |
| Cheng et al. | Constituent analysis and proteomic evaluation of ovular secretions in Ginkgo biloba: not just a pollination medium | |
| CN101831484A (en) | New method for measuring nitric acid reductase activity | |
| CN202903670U (en) | Zeranol assay kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20131120 |