CN109355353A - A kind of method of quick measurement non-plant sucrose synthase activity - Google Patents
A kind of method of quick measurement non-plant sucrose synthase activity Download PDFInfo
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Abstract
The present invention provides a kind of method of quickly measurement non-plant sucrose synthase activity, comprising: 1) enzyme is extracted from plants, obtains crude enzyme liquid；2) salt plug is gone to purify crude enzyme liquid using centrifugal；3) it gained enzyme solution will be added in substrate UDPG and fructose after purification, it carries out enzymatic reaction and generates sucrose, after reaction, with the Brix angle value of saccharometer measurement reaction product, the concentration of sucrose is obtained according to sucrose Brix degree and the percent concentration table of comparisons, the enzyme activity of sucrose synthase is calculated according to the densimeter of sucrose.The present invention can substitute the measuring method that traditional bag filter purifying combines spectrophotometer, can not only save dialysis time, realize the unified standard of batch measurement using the measuring method of the centrifugal column purification combination saccharometer that desalts；The scavenging period of a large amount of cuvettes when spectrophotometric determination can also be saved simultaneously.
Description
Technical field
The present invention relates to the measuring methods of enzymatic activity, specifically, being related to a kind of quickly measurement non-plant sucrose synthetase activity
The method of property.
Background technique
Sucrose is photosynthesis of plant product from blade to the main matter form of " library " organ transportation.Sucrose synthase master
To directly affect that plant library is strong and the input and accumulation of sucrose by controlling the synthesis and decomposition of sucrose, it is each to be that sucrose participates in
A kind of a essential key enzyme of metabolic activity approach.For corn, the soluble sugar of accumulation has sucrose, grape
Sugar, fructose are several, and wherein sucrose accounts for the overwhelming majority, is the 60%~77% of total reducing sugar.And many foods such as corn, waxy corn
It is largely determined by sucrose with the mouthfeel of corn, quality.In addition, sucrose synthase is in terms of the synthesis of regulation starch
It plays an important role, sucrose synthase provides precursor substance UDPG as primary metabolic enzyme for the synthesis of starch.And it forms sediment in seed
The content of powder is to measure the important indicator of many Crops production and qualities.Neutral detergent fiber is the weight for measuring silage corn quality
Want evaluation index.Generally believe that sucrose synthase plays a key effect in neutral fibre in regulation silage corn at present.
For sucrose synthase using free fructose as receptor, reaction generates sucrose, mainly sucrose decomposition and nucleotide glucose
The system of synthesis.Therefore, Sucrose synthesis enzyme activity synthesizes closely related with sucrose accumulation and glucose.Studies have shown that Sucrose synthesis
The content of starch of enzyme and corn is closely bound up, and will affect the taste quality of corn, and in addition for silage corn, sucrose is closed
It is positively correlated at enzymatic activity and its stalk neutral detergent fiber (DNF).Measure raising of the sucrose synthase activity to crop yield
There is important directive significance with quality-improving.
In plant there are mainly two types of the existence forms of sucrose synthase, most of sucrose synthase is in plant cell
Exist in matter with soluble state, and small part is attached on cell membrane with insoluble state.Sucrose synthase was both
The synthesis of sucrose can be catalyzed or be catalyzed the decomposition of sucrose, and the enzyme is higher in the Km value for decomposing direction, works as sucrose concentration
Be conducive to reaction when high and carried out to direction is decomposed.Measuring the enzymatic activity of sucrose synthase both can also be with by its synthetic reaction
It is measured on decomposing direction, most of sucrose synthase activity measurement is to be synthesized by reaction at present.The measurement of use
The crude enzyme liquid of extraction is mainly directly generated sucrose with substrate reactions by method, and sucrose can divide in hydrochloric acid and resorcin reaction
Solution develops the color at fructose and glucose, then the method with spectrophotometric determination light absorption value to measure sucrose synthase activity carries out
Measurement.Because crude enzyme liquid is not purified, impurity is more, can produce bigger effect to measurement result.In addition, being carried out with spectrophotometer
When measurement, if sample size is larger, the cleaning of cuvette is also than relatively time-consuming when replacing sample.Therefore, it is necessary to develop a kind of quick
Convenient and fast measuring method, to achieve the purpose that optimize sucrose synthase activity measuring method.
Summary of the invention
The object of the present invention is to provide a kind of methods of quickly measurement non-plant sucrose synthase activity.
In order to achieve the object of the present invention, the present invention provides a kind of method of quickly measurement non-plant sucrose synthase activity, packet
Include following steps:
1) extraction of enzyme: enzyme is extracted from plants, obtains crude enzyme liquid；
2) purifying of enzyme；Salt plug is gone to purify crude enzyme liquid using centrifugal；
3) enzymatic reaction: gained enzyme solution will be added in substrate UDPG and fructose after purification, and carry out enzymatic reaction and generate sucrose,
After reaction, it with the Brix angle value of saccharometer measurement reaction product, is obtained according to sucrose Brix degree and the percent concentration table of comparisons
To the concentration of sucrose, the enzyme activity of sucrose synthase is calculated according to the densimeter of sucrose.
Method above-mentioned, the specific method is as follows for the extraction of step 1) enzyme: taking plant tissue 1-2g (preferably 2g), shreds and be put into
In mortar, liquid nitrogen grinding is added to crushing, takes 8-12mL (preferably 12mL) Hepes-NaOH Extraction buffer and a little quartz sand
It is added in mortar, is fully ground stirring, is transferred in centrifuge tube, be centrifuged 30min in 4 DEG C, 12000rpm, take supernatant to get thick
Enzyme solution.
Wherein, in the Hepes-NaOH Extraction buffer containing 50mmol/L Hepes, 1mmol/L EDTA,
10mmol/L MgCl2, 2.5mmol/L DTT, 0.05%Triton-X100 and 0.5mg/mL BSA, pH7.5.
Method above-mentioned, the specific method is as follows for step 2) enzyme purification: going salt plug to be put into 2mL centrifuge tube for centrifugal, so
Afterwards by Hepes-NaOH Extraction buffer be added it is centrifugal go in salt plug, in 4 DEG C, 1000g be centrifuged 2min, repeat 3-5 time with moisten
It washes and centrifugal removes salt plug；
After rinse, salt plug is gone to change new 2mL centrifuge tube by centrifugal, it is centrifugal after taking 1mL crude enzyme liquid that rinse is added
It goes in salt plug, is centrifuged 4min in 4 DEG C, 1000g, collects enzyme solution, reacted for subsequent enzymatic.
It is centrifugal to remove the preferred Spin-OUT of salt plug in the present inventionTM GT-1200。
Method above-mentioned, step 3) carries out enzymatic reaction, and the specific method is as follows: being added into Hepes-NaOH reaction solution
200 μ L enzyme solutions, 30 DEG C of heat preservation 30min；Then 100 DEG C of placement 4min；Then 200 μ L of 2mol/L NaOH, 100 DEG C of placements are added
10min。
Wherein, the formula of the Hepes-NaOH reaction solution are as follows: 100 μ L of 50mmol/L fructose, 50mmol/L UDPG100 μ
L、10mmol/L MgCl250 μ L, 100 μ L of elution buffer.
The formula of the elution buffer are as follows: 50mmol/L Hepes, 1mmol/L EDTA, 10mmol/L MgCl2、
2.5mmol/L DTT、0.5mg/mL BSA。
In the present invention, the plant includes but is not limited to corn.
The invention is particularly suited to quickly measure the work of sucrose synthase in maize leaf (especially Leaves of Maize Seedlings)
Property.
The present invention directly measures the cane sugar content of generation using saccharometer.The principle of saccharometer are as follows: a kind of medium enters
Refraction effect can be generated when another medium, and it is definite value that the ratio between incidence angle sine is permanent, this ratio is index of refraction.In a certain temperature
Under degree and environment, the concentration for generating sucrose solution has corresponding relationship to the refractive index of the light wave of certain wavelength with it, and use is this
Refractometer Method And Principle measures the index of refraction of the sample solution to be tested, directly reading soluble solid content, and measurement result is with sucrose matter
Measuring percentage concentration indicates, if containing non-sucrose matter in product, measurement result is approximation.The result that pol measures is used
Brix (Brix degree) is indicated, refers to the content of the soluble solid in product, the sucrose solubility in 100g sucrose solution.So
Afterwards according to sucrose Brix degree and the percent concentration table of comparisons, the concentration of you can get it sucrose.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
The present invention is using different purification process and activity determination method respectively to the Sucrose synthesis of different corn materials
Enzymatic activity is measured, filter out it is most easy, economical, efficiently measure scheme.Using Hepes-NaOH as Extraction buffer, from jade
Sucrose synthase is extracted in the blade of meter You Miao and obtains crude enzyme liquid, goes salt plug or bag filter to purify with centrifugal, then use pol
Meter or spectrophotometric determination sucrose synthase activity.It is pure that the centrifugal purification process for removing salt plug can replace traditional bag filter
Change method is to save dialysis time；Enzyme solution after purification, saccharometer consistent with the result of spectrophotometric determination with saccharometer
Resolution ratio it is higher.The present invention can be substituted traditional saturating using the measuring method of the centrifugal column purification combination saccharometer that desalts
The measuring method that bag purifying combines spectrophotometer is analysed, dialysis time can be not only saved, realize the unified standard of batch measurement；
The scavenging period of a large amount of cuvettes when spectrophotometric determination can also be saved simultaneously.
Detailed description of the invention
Fig. 1 is the standard curve drawn in the embodiment of the present invention 1 according to various concentration sucrose solution.
Fig. 2 is the thick enzymatic activity of sucrose synthase that different measuring methods measure in the embodiment of the present invention 1.
Fig. 3 is the sucrose synthase activity that the bag filter that different measuring methods measure in the embodiment of the present invention 1 purified.
Fig. 4 is the sucrose synthase that the centrifugal column purification that desalts that different measuring methods measure in the embodiment of the present invention 1 is crossed
Activity.
Fig. 5 is the sucrose synthase enzymatic activity of the different purification process measured in the embodiment of the present invention 1 with saccharometer.
Fig. 6 is the sucrose synthase enzyme activity of the different purification process measured in the embodiment of the present invention 1 with spectrophotometer
Property.
Fig. 7 be the embodiment of the present invention 1 in five corn variety sucrose synthase activity ratios compared with.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
It is centrifugal to go salt plug for Spin-OUT in following embodimentTM GT-1200。
The method that embodiment 1 quickly measures Leaves of Maize Seedlings sucrose synthase activity
1, experimental material
Corn inbred line: 2193, ZYQ061, ZYQ268, ZYQ335.
Corn hybrid seed: northern agriculture ensiling 368.
2, experimental method
The preparation of 2.1 thick enzymes
The fresh Leaves of Maize Seedlings of 2g is weighed respectively, shreds and is put into mortar, and liquid nitrogen grinding is added to crushing.Take 12mL
Hepes-NaOH (pH7.5) Extraction buffer (Hepes containing 50mmol/L, 1mmol/L EDTA, 10mmol/L MgCl2、
2.5mmol/L DTT, 0.05%Triton-X100,0.5mg/mL BSA) and quartz sand is added in mortar a little, sufficiently grinds
Mill stirring, is transferred in 15mL centrifuge tube.(4 DEG C, 12000rpm) centrifugation 30min of Extraction buffer low temperature, take supernatant, as slightly
Enzyme solution.
The purifying of 2.2 thick enzymes
The centrifugal Column methods that desalt: will be compacted the centrifugal of resin sheet and salt plug gone to be put into 2mL centrifuge tube, will be above-mentioned
Hepes-NaOH Extraction buffer be slowly added to it is centrifugal remove salt plug, low temperature (4 DEG C, 1000g) is centrifuged 2min, repeat 3-5 times with
Salt plug is removed in rinse.To going salt plug to change new 2mL centrifuge tube, take what 1mL crude enzyme liquid was slowly added to rinse to go to salt plug center, it is low
Warm (4 DEG C, 1000g) are centrifuged 4min, collect enzyme solution.
Bag filter purification process: crude enzyme liquid is added in bag filter, bag filter is then put into 250mLHepes-NaOH
(pH7.5) dialyzate (Hepes containing 50mmol/L, 1mmol/L EDTA, 10mmol/L MgCl2、2.5mmol/L DTT、
In 0.5mg/mLBSA), soaked overnight.
2.3 enzymatic reaction
To Hepes-NaOH reaction solution (100 μ L of 50mmol/L fructose, 100 μ L of 50mmol/L UDPG, 10mmol/
LMgCl250 μ L, elution buffer (50mmol/L Hepes, 1mmol/L EDTA, 10mmol/L MgCl2、2.5mmol/L
DTT, 0.5mg/mL BSA) 200 μ L enzyme solutions are added in 100 μ L, 30min is kept the temperature at 30 DEG C.
Then, 4min is saved under 100 DEG C of boiling water baths.The NaOH of 200 μ L 2mol/L is added.It is saved under 100 DEG C of boiling water baths
10min.Use UDPG in water surrogate response liquid as control.
2.4 enzyme activity determination
2.4.1 saccharometer measures
By saccharometer control zero, drawing reaction solution with liquid-transfering gun makes it cover the mirror surface of saccharometer, drives bubble away,
That is readable, reading are the Brix angle value of solution.
2.4.2 spectrophotometric determination
30% hydrochloric acid of 2mL is added into reaction solution to mix, 80 DEG C of heat preservation 10min.Add 1mL0.1% resorcinol, 80 DEG C
Keep the temperature 10min, after flowing water is cooling at 480nm colorimetric.
Enzyme activity unit is defined as: under the above conditions, total enzyme amount contained by every gram of sample dry mass, within a hour
Can be catalyzed and generate the amount of sucrose is a unit of activity (mmol/h/g FW).
The production of 2.5 standard curves
The formulation of standard curve: drying to constant weight at 80 DEG C for sucrose, weighs 1g, is dissolved in water, and is settled to 250mL, sugarcane
Standard for Sugars liquid is 4mg/mL.6 test tubes are taken accurately to configure by table 1.After mixing, boiling water bath 10min is cooling that 1.5mL 30% is added
Hydrochloric acid, 0.1% resorcinol of 0.5mL mix, and the water-bath 10min in 80 DEG C of water-baths is settled to 12mL after cooling, in 480nm
Lower colorimetric.Using sugar content as abscissa, using light absorption value as ordinate, standard curve is drawn, calibration curve equation is found out.Table 1 is
Cane sugar content Specification Curve of Increasing table.
1 cane sugar content Specification Curve of Increasing table of table
3. result and analysis
3.1 standard curve
According to above-mentioned standard curve plotting method, make the sucrose solution of a certain concentration gradient and with hydrochloric acid and isophthalic two
It develops the color under conditions of phenol heating, reads light absorption value (table 2) under spectrophotometer, and be depicted as standard curve and see Fig. 1.
2 standard curve light absorption value of table
The variation of 3.2 different measuring methods sucrose synthase activities
It can be seen that the maize leaf of five materials is surveyed in saccharometer and two kinds of spectrophotometer according to the experimental data of table 3
For the sucrose synthase enzymatic activity measured under amount method there are extremely significant difference, the enzymatic activity that saccharometer measures is apparently higher than light splitting light
Spend the enzymatic activity measured.
The sucrose synthase enzymatic activity (unit: μm ol/h/g FW) that 3 different measuring methods of table measure
Fig. 2 is the thick enzymatic activity of sucrose synthase that different measuring methods measure, and Fig. 3 is the dialysis that different measuring methods measure
The sucrose synthase activity that bag purified, Fig. 4 is the Sucrose synthesis that the centrifugal column purification that desalts that different measuring methods measure is crossed
Enzymatic activity.
According to fig. 2, the line chart of Fig. 3, Fig. 4 can be seen that Fig. 2,3 with saccharometer and spectrophotometer respectively to different jade
The sucrose synthase activity of not purified and bag filter purifying, which is measured, in rice material compares, and finds without any rule.And Fig. 4
The middle enzyme solution after the centrifugal column purification that desalts, is measured with spectrophotometer and hand-held digital display saccharometer, as a result respectively
Which kind of measuring method no matter display use, and the measurement result trend in different corn materials is almost the same.
3.3 different purification process sucrose synthase activity variations
The sucrose synthase enzymatic activity measured with different purification process is shown in Table 4.The sugarcane measured with different purification process
Sugared synzyme enzymatic activity is shown in Table 5.
The sucrose synthase enzymatic activity (saccharometer method, unit: μm ol/h/g FW) that table 4 is measured with different purification process
Sucrose synthase enzymatic activity (spectrophotometer method, the unit: μm ol/h/g that table 5 is measured with different purification process
FW)
Either spectrophotometer and sugar it can be seen from the line chart trend of table 4,5 experimental data of table and Fig. 5, Fig. 6
Degree counts any measuring method, and sucrose synthase activity variation tendency is almost the same.And use the centrifugal column purification that desalts
Enzymatic activity afterwards is generally higher than the enzymatic activity of bag filter after purification, it is seen that the centrifugal purification effect for removing salt plug is even more ideal.
From fig. 6, it can be seen that the enzyme of the enzyme activity of the not purified enzyme solution of spectrophotometric determination and enzyme solution after purification
Work is compared, and trend is opposite.Purify in enzyme solution, enzyme activity in corn hybrid seed 368 than in self-mating system (2193, ZYQ268,
ZYQ335, ZYQ061) activity want high, and in crude enzyme liquid, the difference of cenospecies and self-mating system is unobvious in addition cenospecies in
Enzyme activity will be lower than self-mating system.This may be because spectrophotometer is higher to the purity requirement of enzyme solution.
The sucrose synthase activity of 3.4 5 kinds of corn materials compares
According to the sucrose synthase activity (Fig. 7) after the centrifugal column purification that desalts for using saccharometer method to measure to corn from
Friendship is the Sucrose synthesis enzyme activity of 2193, ZYQ061, ZYQ268, ZYQ335 and this five kinds of corn materials of cenospecies 368 in Seedling Stage
Property is compared.It can be seen that in this five corn materials by Fig. 7 experimental data in addition to 368 sucrose synthase enzyme of Hybrid
Other than activity is relatively high, the enzymatic activity of remaining four strains is not much different.Not purified crude enzyme liquid is measured using saccharometer
Enzymatic activity and it is irregular follow, analyze its reason may is that hand-held digital display saccharometer be by measure solution index of refraction ask
The concentration (number of sugar content) of solution out.Because soluble solid content and index of refraction in solution are under certain condition
(same temperature, pressure) is directly proportional.And not purified crude enzyme liquid, because impure more, the influence to measurement result is very big.And
Enzyme solution after purification is measured with spectrophotometer and hand-held digital display saccharometer respectively, the results show that no matter being surveyed using which kind of
Determine method, the measurement result trend in different corn materials is almost the same.Moreover, the enzyme solution after the centrifugal column purification that desalts accords with
Conjunction degree is very high, therefore for material of the invention, and the centrifugal resolution ratio for removing salt plug is higher.After the centrifugal column purification that desalts
Enzyme solution, no matter use which kind of measuring method, show as, the enzyme activity of the sucrose synthase in corn hybrid seed 368 compares self-mating system
In the activity of (2193, ZYQ268, ZYQ335, ZYQ061) want high, illustrate that use desalts the purification process combination pol of formula centrifugal column
The measuring method of meter can substitute the measuring method of traditional bag filter purification process combination spectrophotometer.Not only may be used in this way
To save dialysis time, the unified standard of batch measurement is realized；A large amount of cuvettes when spectrophotometric determination can also be saved simultaneously
Scavenging period.
The present invention by corn inbred line 2193, ZYQ061, ZYQ268, ZYQ335 and Hybrid ensiling 368 this five
A corn material takes bag filter and centrifugal removes salt plug both different purification process and saccharometer, spectrophotometer
Two different measuring methods measure sucrose synthase activity respectively, are as a result compared, the centrifugal purification process for removing salt plug
Add the measuring method of saccharometer relatively the quickest, convenient, safe.Sucrose synthase is responsible for important in the decomposition direction of sucrose
Effect, in addition to this, also the synthesis for starch and cellulose provides precursor substance UDPG, so as primverose metabolic pathway
An important enzyme, the research of sucrose synthase has great meaning for the promotion of the quality and yield of corn.The present invention
More comprehensive research has been carried out to the measuring method of sucrose synthase activity, has passed through the test method of science and the ratio of system
Compared with having filtered out the most accurate, economic measuring method, laid a good foundation for the research of corn sucrose synthetase from now on.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Bibliography
[1]Komastu A,Takanokura Y,Moriguchi T,Differential expression of
three sucrose-phosphate synthase isoforms during accumulation in citrus fruit
(Citrus unshiu Marc) [J] .Plant Sci, 1999,140:169~178
[2] Lu Hequan, Shen Fafu, Liu Lingxiao, the side Sun Wei non-plant sucrose synzyme function and Progress on Molecular Biology
[J] China's agronomy notification, 2007,21 (7): 34~37
[3] Cui Yanhong, Zhou Hai, the nutritional quality of Li's uncle's boat corn seed and influence factor [J] Agricultural University Of Hebei
Journal, 1996,19 (4): 99~104
[4]WHITTINGHAM C P,KEYS AJ,BIRD I F.The enzymology of sucrose
synthesis in leaves[A].Gibbs M,Latzko E.Encyclopedia of plant physiology:New
Series Vol 6 [C] .Berlin:Springer-Verlag, 1979.313~326
[5] Zhu Jin, Hao Yulan, Zhang Qiuzhi, southern Zhang Jie, Li Yingying, Li Sukun, Pan Jin leopard Silage Corn Straw neutrality are washed
Wash relationship [J] China's agronomy notification between fiber and sucrose synthase activity, 2010,26 (12): 63~66
[6]Brown R M Jr,Saxena I M,Kudlicka K.Cellulose biosynthesis inhigher
Plants [J] .Trends in Plant Science, 1996 (5): 149~155
[7]Ruan Y L,Chourey P S,Delmer D P,et al.The differential expression
of sucrose synthase in relation to diverse patterns of carbon partitioning in
1997,115:375~385 developing cotton seed [J] .Plant Physiology
[8] Zhang Zhiliang, Qu Wei cyanines plant physiology experiment guidance [M] the .3 editions Beijing Higher Education Publishing House, 2004:
128~129.
[9] height faces south, Xu Fengcai, Zhao Yahua, and Mei Zhi expands conventional corn and super-sweet corn seedling stage sucrose synthase and phosphoric acid
Vigour [J] the Agricultural University Of South China journal of sucrose synthase, 2001,22 (2): 46~48
[10] Shanghai Xue Yinglong, Xia Zhen Australia plant physiology experiment handbook [M]: Shanghai Scientific & Technical Publishers, 1985.146
~150
[11] a kind of high-precision digital saccharometer [J] optical instrument of Yan Changxiang, Xu Xiangdong, 1998,20 (4): 34~
36
Claims (6)
1. a kind of method of quickly measurement non-plant sucrose synthase activity, which comprises the following steps:
1) extraction of enzyme: enzyme is extracted from plants, obtains crude enzyme liquid；
2) purifying of enzyme；Salt plug is gone to purify crude enzyme liquid using centrifugal；
3) enzymatic reaction: it gained enzyme solution will be added in substrate UDPG and fructose after purification, and carry out enzymatic reaction and generate sucrose, reaction
After, with the Brix angle value of saccharometer measurement reaction product, sugarcane is obtained according to sucrose Brix degree and the percent concentration table of comparisons
The concentration of sugar calculates the enzyme activity of sucrose synthase according to the densimeter of sucrose.
2. the method according to claim 1, wherein step 1) enzyme extracts, the specific method is as follows: taking plant group
1-2g is knitted, shreds and is put into mortar, liquid nitrogen grinding is added to crushing, takes 8-12mL Hepes-NaOH Extraction buffer and a little
Quartz sand is added in mortar, is fully ground stirring, is transferred in centrifuge tube, is centrifuged 30min in 4 DEG C, 12000rpm, takes supernatant,
Up to crude enzyme liquid；
Wherein, 50mmol/L Hepes, 1mmol/L EDTA, 10mmol/L are contained in the Hepes-NaOH Extraction buffer
MgCl2, 2.5mmol/L DTT, 0.05%Triton-X100 and 0.5mg/mL BSA, pH7.5.
3. the method according to claim 1, wherein the specific method is as follows for step 2) enzyme purification: will be centrifugal
Go salt plug to be put into 2mL centrifuge tube, then by Hepes-NaOH Extraction buffer be added it is centrifugal go in salt plug, in 4 DEG C, 1000g
It is centrifuged 2min, repeats to remove salt plug so that rinse is centrifugal 3-5 times；
After rinse, salt plug is gone to change new 2mL centrifuge tube by centrifugal, centrifugal desalting after taking 1mL crude enzyme liquid that rinse is added
In column, it is centrifuged 4min in 4 DEG C, 1000g, enzyme solution is collected, is reacted for subsequent enzymatic.
4. according to the method described in claim 3, it is characterized in that, step 3) carries out enzymatic reaction, the specific method is as follows: to
200 μ L enzyme solutions, 30 DEG C of heat preservation 30min are added in Hepes-NaOH reaction solution；Then 100 DEG C of placement 4min；Then 2mol/ is added
L NaOH 200 μ L, 100 DEG C of placement 10min；
Wherein, the formula of the Hepes-NaOH reaction solution are as follows: 100 μ L of 50mmol/L fructose, 50mmol/L UDPG100 μ L,
10mmol/L MgCl250 μ L, 100 μ L of elution buffer；
The formula of the elution buffer are as follows: 50mmol/L Hepes, 1mmol/L EDTA, 10mmol/L MgCl2、2.5mmol/
L DTT、0.5mg/mL BSA。
5. method according to claim 1-4, which is characterized in that the plant is corn.
6. according to the method described in claim 5, it is characterized in that, step 1) is to extract enzyme from maize leaf.
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| CN105087514A (en) * | 2015-09-09 | 2015-11-25 | 安徽省农业科学院园艺研究所 | Method for extracting sucrose phosphate synthetase from muskmelon leaves and determining activity of sucrose phosphate synthetase |
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| CN105087514A (en) * | 2015-09-09 | 2015-11-25 | 安徽省农业科学院园艺研究所 | Method for extracting sucrose phosphate synthetase from muskmelon leaves and determining activity of sucrose phosphate synthetase |
Non-Patent Citations (2)
| Title |
|---|
| YOUSHEN上传: "蔗糖合成酶的测定方法", 《原创力文档》 * |
| 郭璇，程高强: "《有机化工产品检验》", 31 December 2014 * |
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