CN105087514A - Method for extracting sucrose phosphate synthetase from muskmelon leaves and determining activity of sucrose phosphate synthetase - Google Patents

Method for extracting sucrose phosphate synthetase from muskmelon leaves and determining activity of sucrose phosphate synthetase Download PDF

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Publication number
CN105087514A
CN105087514A CN201510570664.6A CN201510570664A CN105087514A CN 105087514 A CN105087514 A CN 105087514A CN 201510570664 A CN201510570664 A CN 201510570664A CN 105087514 A CN105087514 A CN 105087514A
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sucrose phosphate
enzyme
enzyme liquid
muskmelon
extraction
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田红梅
方凌
严从生
王朋成
王艳
张其安
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Institute of Gardening of Anhui Academy Agricultural Sciences
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Institute of Gardening of Anhui Academy Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1066—Sucrose phosphate synthase (2.4.1.14)
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01014—Sucrose-phosphate synthase (2.4.1.14)
    • 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
    • G01N2333/91—Transferases (2.)
    • G01N2333/91091—Glycosyltransferases (2.4)
    • G01N2333/91097—Hexosyltransferases (general) (2.4.1)
    • G01N2333/91102—Hexosyltransferases (general) (2.4.1) with definite EC number (2.4.1.-)
    • G01N2333/91125—Sucrose phosphate synthases (2.4.1.14)

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

Abstract

The invention discloses a method for extracting sucrose phosphate synthetase from muskmelon leaves and determining activity of sucrose phosphate synthetase. The method comprises the following steps: (1), enzyme liquid extraction: taking fresh muskmelon leaves and putting the fresh muskmelon leaves into a mortar, grinding by an extraction buffer solution subjected to ice bathing, carrying out refrigerated centrifugation at a high speed after fully homogenizing, taking supernatant and desalting the supernatant by a centrifugal desalting column, wherein extracted enzyme liquid can be directly used for enzyme activity determination; and (2), enzyme activity determination: taking enzyme liquid, adding the enzyme liquid into a reaction substrate, wherein different reaction temperature and reaction time are determined and optimal reaction temperature and reaction time of sucrose phosphate synthetase in muskmelon leaves are determined, so that activity of enzyme can be reflected more accurately. According to the invention, salt, sugar, ions and other small molecular substances in enzyme liquid extracted primarily can be effectively removed, and reaction conditions of enzyme are optimized, so that the activity of sucrose phosphate synthetase in leaves can be determined more accurately.

Description

The extraction of muskmelon blade Sucrose Phosphate Synthase and activity determination method
Technical field
The present invention relates to a kind of from muskmelon blade extraction Sucrose Phosphate Synthase and the method measuring its activity.
Background technology
Sucrose Phosphate Synthase is a kind of sucrose metabolizing enzyme of key, is also a kind of important photosynthetic enzyme, and its activity is proportionate with Net Photosynthetic Rate, therefore studies this enzymic activity significant for improving muskmelon photosynthesis.What current Sucrose Phosphate Synthase extracted major part employing is G-25Sephadex post and dialysis tubing, and G-25Sephadex post is expensive, and extraction cost is higher, and dialysis tubing price is lower, but trivial operations, expend time in, and extraction efficiency is low; The centrifugal post price that desalts is slightly high, but has recyclability, and extraction efficiency is better than dialysis tubing and G-25Sephadex post.In enzyme assay, current Problems existing is enzyme-to-substrate reaction times and the less concern of temperature studies person, and its optimum reaction condition of different materials differs, and present invention optimizes muskmelon blade Sucrose Phosphate Synthase determination of activity condition.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of extraction and activity determination method of muskmelon blade Sucrose Phosphate Synthase.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is: the extraction of muskmelon blade Sucrose Phosphate Synthase and activity determination method, comprise the following steps:
(1) enzyme liquid extracts: take the fresh blade of 2g muskmelon, put into the mortar of precooling, add 10mL Extraction buffer; Add quartz sand, grind under ice bath, after abundant homogenate, under 0 ~ 4 DEG C of condition, the centrifugal 20min of 12000rpm, gets supernatant liquor, and remove the salinity just carried in enzyme liquid, the enzyme liquid after purifying can be directly used in enzyme assay;
(2) enzyme assay: with 0.2mL enzyme liquid boiling water bath 10min for contrast; Get 0.2mL enzyme liquid and add reaction substrate; 3 ~ 30min is reacted under 15 ~ 40 DEG C of conditions; Boiling water bath 1min, is settled to 1mL with distilled water, adds 0.1mL2molL -1naOH, boiling water bath 10min, running water cools; In reaction solution, add 3.5mL30%HCl, 1mL0.1% Resorcinol, after shaking up in 80 DEG C of water-baths water-bath 10min, flowing water cooling after, 480nm colorimetric.
As preferably, in step (1), Extraction buffer is composed of the following components: 50mmolL -1hEPES [N-(2-hydroxyethyl) piperazine-N '-2 sulfonic acid], 1mmolL -1eDTA.Na 2, 10mmolL -1mgCl 2, 2.5mmolL -1dTT, 10mmolL -1xitix; And the pH value of HEPES Extraction buffer is 7.5.
As preferably, the centrifugal post that desalts in step (1), is adopted to remove the salinity just carried in enzyme liquid.
As preferably, in step (2), reaction substrate comprises 0.1mL50mmolL- 1fructose-6-phosphate, 0.1mL30mmolL -1uDPG, 0.1mL100mmolL -1tris, 0.05mL10mmolL -1mgCl 2.
As preferably, in step (2), muskmelon blade Sucrose Phosphate Synthase and reaction substrate react 5min in 30 DEG C of water-baths can reach most enzymatic activity high.
The invention has the beneficial effects as follows:
The small-molecule substance such as salinity, sugar, the ion removing that can effectively will just extract in enzyme liquid, thus more accurately can measure the activity of Sucrose Phosphate Synthase in blade.
Embodiment
(1) experimental technique
1, for examination material: the melon variety " early sweet No. " of the autonomous seed selection of Institute of Gardening, Anhui Academy of Agricultural Sciences, the muskmelon mutation collected " goat's horn honey ", " S1 ".
2, experimental design
Muskmelon grow to three leaves wholeheartedly time get same position blade, blade is shredded mixing, and every part takes 2g, is placed in rapidly liquid nitrogen quick-frozen, is then kept in-80 DEG C of refrigerators for subsequent use.This test adopts the centrifugal post that desalts, G-25Sephadex post and dialysis tubing to extract Sucrose Phosphate Synthase respectively.Each process repeats for 3 times.
3, the extraction of Sucrose Phosphate Synthase
Testing sample is put into the mortar of precooling, add 10mL Extraction buffer (50mmolL -1hEPES, 1mmolL -1eDTA.Na 2, 10mmolL -1mgCl 2, 2.5mmolL -1dTT, 10mmolL -1xitix, pH7.5); Add quartz sand, grind under ice bath, after abundant homogenate, under 0 ~ 4 DEG C of condition, the centrifugal 20min of 12000rpm, gets supernatant liquor, gets 0.5mL supernatant liquor and slowly instills the centrifugal post (Spin-OUT that desalts tMgT-1200), the enzyme liquid collected can be directly used in enzyme assay.
4, the mensuration of Sucrose Phosphate Synthase activity
(0.1mL50mmolL in 0.35mL reaction solution -1fructose-6-phosphate, 0.1mL30mmolL -1uDPG, 0.1mL100mmolL -1tris, 0.05mL10mmolL -1mgCl 2) adding 0.2mL enzyme liquid, after reacting 5min under 30 DEG C of conditions, boiling water bath 1min, is settled to 1mL with distilled water, adds 0.1mL2molL -1naOH, boiling water bath 10min, running water cools; In reaction solution, add 3.5mL30%HCl, 1mL0.1% Resorcinol, after shaking up in 80 DEG C of water-baths water-bath 10min, flowing water cooling after, 480nm colorimetric.
(2) interpretation of result
1, different treatment method extraction muskmelon blade Sucrose Phosphate Synthase specific activity colour compares
Table 1
As shown in Table 1, muskmelon blade adopts diverse ways to extract Sucrose Phosphate Synthase, and its extraction efficiency differs greatly, and its colourimetric number of enzyme liquid that the centrifugal post that desalts extracts is far away higher than G-25Sephadex post and dialysis tubing, and dialysis tubing extraction efficiency is minimum.
2, standard curve making method
(1) analytical pure sucrose is dried to constant weight at 80 DEG C, accurately takes sucrose 1g, be dissolved in water, constant volume to 50mL, i.e. reference liquid (the 200 μ gmL of sucrose -1).
(2) 7 test tube numberings are got, according to the amount accurate formulation solution shown in table 2
The preparation of table 2 typical curve is compared
After each pipe solution mixing, boiling water bath 10min, running water cools; In reaction solution, add 3.5mL30%HCl, 1mL0.1% Resorcinol, after shaking up in 80 DEG C of water-baths water-bath 10min, flowing water cooling after, 480nm colorimetric.
Being X-coordinate with sugar degree, take absorbancy as ordinate zou, drawing standard curve, and obtains typical curve equation (y=433.5 × x+7.999, R 2=0.998).
3, enzymic activity calculates
Enzymic activity (μm olh -1g -1fW)=[calculating sucrose content (mg) according to typical curve by colourimetric number]/342.3 (sucrose molar mass) × zyme extract cumulative volume (mL)/[enzyme liquid added during sample fresh weight (g) × reaction amasss (mL) × reaction times (h).
Corresponding enzymic activity is gone out according to above formulae discovery, as shown in table 3.
Table 3
The Extraction buffer of the present embodiment does not have the poisonous or aggressive chemicalss such as mercaptoethanol, provides condition for ensureing that scientific research personnel is healthy.
The small-molecule substance such as salinity, sugar, the ion removing that the present embodiment adopts the centrifugal post that desalts effectively will just extract in enzyme liquid, thus more accurately can measure the activity of Sucrose Phosphate Synthase in blade, and the centrifugal post that desalts has recyclability, greatly can reduce scientific research cost.
The present embodiment optimizes muskmelon blade Sucrose Phosphate Synthase and substrate reactions condition, can actual response enzyme activity level, and shortens the time of enzyme assay, improves scientific research efficiency.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within claims of the present invention.

Claims (5)

1. the extraction of muskmelon blade Sucrose Phosphate Synthase and activity determination method, comprises the following steps:
(1) enzyme liquid extracts: take the fresh blade of 2g muskmelon, put into the mortar of precooling, add 10mL Extraction buffer; Grind under ice bath, after abundant homogenate, under 0 ~ 4 DEG C of condition, the centrifugal 20min of 12000rpm, gets supernatant liquor, and remove the salinity just carried in enzyme liquid, the enzyme liquid after purifying can be directly used in enzyme assay;
(2) enzyme assay: with 0.2mL enzyme liquid boiling water bath 10min for contrast; Get 0.2mL enzyme liquid and add reaction substrate, under 15 ~ 40 DEG C of conditions, react 3 ~ 30min; Boiling water bath 1min, is settled to 1mL with distilled water; Add 0.1mL2molL -1naOH, boiling water bath 10min, running water cools; In reaction solution, add 3.5mL30%HCl, 1mL0.1% Resorcinol, after shaking up in 80 DEG C of water-baths water-bath 10min, flowing water cooling after, 480nm colorimetric.
2. the extraction of muskmelon blade Sucrose Phosphate Synthase according to claim 1 and activity determination method, is characterized in that: in step (1), described Extraction buffer is composed of the following components: 50mmolL -1hEPES [N-(2-hydroxyethyl) piperazine-N '-2 sulfonic acid], 1mmolL -1eDTA.Na 2, 10mmolL -1mgCl 2, 2.5mmolL -1dTT, 10mmolL -1xitix; And the pH value of HEPES Extraction buffer is 7.5.
3. the extraction of muskmelon blade Sucrose Phosphate Synthase according to claim 1 and activity determination method, is characterized in that: adopt the centrifugal post that desalts to remove the salinity just carried in enzyme liquid in step (1).
4. the extraction of muskmelon blade Sucrose Phosphate Synthase according to claim 1 and activity determination method, is characterized in that: in step (2), described reaction substrate comprises 0.1mL50mmolL- 1fructose-6-phosphate, 0.1mL30mmolL -1uDPG, 0.1mL100mmolL -1tris, 0.05mL10mmolL -1mgCl 2.
5. the extraction of muskmelon blade Sucrose Phosphate Synthase according to claim 1 and activity determination method, it is characterized in that: in step (2), muskmelon blade Sucrose Phosphate Synthase and reaction substrate react 5min in 30 DEG C of water-baths can reach most enzymatic activity high.
CN201510570664.6A 2015-09-09 2015-09-09 Method for extracting sucrose phosphate synthetase from muskmelon leaves and determining activity of sucrose phosphate synthetase Pending CN105087514A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
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
CN109355353A (en) * 2018-10-29 2019-02-19 北京农学院 A kind of method of quick measurement non-plant sucrose synthase activity

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090068635A1 (en) * 2007-09-06 2009-03-12 Muerhoff Anthony S Indirectly labelled assay conjugates and methods of preparing and using same
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090068635A1 (en) * 2007-09-06 2009-03-12 Muerhoff Anthony S Indirectly labelled assay conjugates and methods of preparing and using same
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)

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Title
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蔡庆生: "《植物生理学实验》", 31 January 2013 *

Cited By (2)

* Cited by examiner, † Cited by third party
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
CN109355353A (en) * 2018-10-29 2019-02-19 北京农学院 A kind of method of quick measurement non-plant sucrose synthase activity

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