CN109321550A - The extracting method of amylase enzyme activity in a kind of raising leaf tobacco extract - Google Patents
The extracting method of amylase enzyme activity in a kind of raising leaf tobacco extract Download PDFInfo
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- CN109321550A CN109321550A CN201811346040.6A CN201811346040A CN109321550A CN 109321550 A CN109321550 A CN 109321550A CN 201811346040 A CN201811346040 A CN 201811346040A CN 109321550 A CN109321550 A CN 109321550A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
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Abstract
The application belongs to tobacco metabolin technical field, and in particular to amylase (AMS) enzyme activity extracting method patent application matters in a kind of raising leaf tobacco extract.This method is specifically included for the extraction of amylase in fresh tobacco leaves: preparing extracting solution (using lysate RIPA, buffer NEB or PBS), tobacco leaf pretreatment, solwution method are extracted.Preliminary Determination the result shows that, after associated extraction process parameter optimizing, amylase (AMS) enzyme activity has obtained preferable raising in extracting solution, up to 9.7 × 10‑4U/ μ g holoprotein has preferably achieved the purpose that reduce impurity protein interference, has promoted AMS enzyme content.Simultaneously because associated extraction method is simple and quick, time-consuming shorter (only 1.5 h or so), so that the application has preferable scientific research value, technical foundation can be established for AMS activity analysis and the analysis of other GAP-associated protein GAPs and research.
Description
Technical field
The application belongs to tobacco metabolin technical field, and in particular to amylase (AMS) in a kind of raising leaf tobacco extract
The extracting method patent application matters of enzyme activity.
Background technique
For tobacco as a kind of more special industrial crops, yield and quality is directly related to the strong of Chinese national economy
Kang Fazhan.In order to study tobacco gene function, new breeding material is formulated, tobacco metabolin is furtherd investigate and is analyzed is aobvious
So have a very important significance.
Starch is the main sugar hydrate that tobacco leaf accumulates in growth and development process, and content is up to 30- in fresh tobacco leaf
40%.Cured tobacco leaf is a kind of main production raw material of China's cigarette product, is process by fresh tobacco leaves baking.Add in baking
During work, most of starch can degrade in tobacco leaf, by amylorrhexis at carbohydrate.And if starch drops in cured tobacco leaf
It solves insufficient, a series of miscellaneous gas substances will be generated, influence the quality of tobacco leaf and jealous.On the other hand, starch decomposition products sugar
The height of content directly affects the organoleptic attribute of tobacco leaf, while reduced sugar can generate a series of tobacco leaves by Maillard reaction again
Flavor component.Therefore content of starch is one of the conventional index of quality of tobacco evaluation.Due to the importance of content of starch index, with
And primary biological enzyme of the amylase as starch degradation, Starch in Tobacco enzyme (AMS) is furtherd investigate and is analyzed, it is raw for plant
Reason increment study and new variety of plant, new material screening and identification are all of great significance.
In the prior art, there are mainly two types of the extracting methods of vegetable protein (including biological enzyme): solwution method and the precipitation method.Its
The middle precipitation method will lead to part protease structure and change and lose activity, therefore is heavy because using denaturant in its extraction process
Shallow lake method is not appropriate for the extraction of tobacco leaf holoprotein.And when solution application method extracts, due to different albumen (biology
Enzyme) characteristic is different, therefore specifically extracts selected extracting solution and extraction process also tends to lack referentiability, often it is both needed to weight
New design.And it is mentioned in the prior art there is not yet preferably extracting 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR)
Method is taken to report.
Summary of the invention
The application is designed to provide amylase (AMS) enzyme activity extracting method in a kind of raising leaf tobacco extract, to be
Certain methodology basis is established in tobacco growing metabolism research.
Details are as follows for the technical solution that the application is taken.
The extracting method of amylase (AMS) enzyme activity in a kind of raising leaf tobacco extract, this method in fresh tobacco leaves for forming sediment
The extraction of powder enzyme (AMS), specifically comprises the following steps::
(1) extracting solution is prepared, lysate RIPA, NEB or PBS specifically can be used；Formula composition are as follows:
Lysate RIPA: Tris-Cl(pH 8.0), 50 mM, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%,
PMSF,1 mM；
Buffer NEB:Hepes(pH 7.5), 20mM, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
The PBS:NaCl of pH 8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1
mM；
The PBS:NaCl of pH7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH 6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1
mM；
(2) tobacco leaf pre-processes
Tobacco K326(or NC82 will be cultivated) fresh tobacco leaves of 8-12 leaf phase, after liquid nitrogen flash freezer, pulverize.
(3) solwution method is extracted
It pulverizes the extracting solution (lysate) for being added in tobacco sample and being prepared in step (1) in step (2), after concussion mixes,
It stands on ice and is no less than 1h；
In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution (lysate) dosage is 500 μ l-1mL；
After the completion of standing, 5000 ~ 15000rpm centrifugation be no less than 20min(centrifugal speed specifically be, for example, 5000 rpm,
9000rpm or 13000rpm etc.), retain supernatant, as contains the extracting solution of higher starch enzyme (AMS) enzyme activity；
Further, protein concentration in supernatant can be detected using BCA method, using ELISA method to AMS in supernatant
Activity detected.
In the prior art, when carrying out analysis measurement for amylase (AMS) enzyme activity in extracted holoprotein, by being extracted
Protein impurities are more, thus cause larger impact for accurate enzyme activity determination.In the application, done for holoprotein extracting mode
Part optimizes, to improving AMS enzyme activity.
Preliminary Determination the result shows that, after associated extraction process parameter optimizing, amylase (AMS) enzyme activity is obtained in extracting solution
Preferable raising is arrived, up to 9.7 × 10-4U/ μ g holoprotein has preferably reached the interference of reduction impurity protein, has promoted AMS enzyme content
Purpose.Simultaneously because associated extraction method is simple and quick, time-consuming shorter (only 1.5 h or so), so that the application has
Preferable scientific research value can establish technical foundation for AMS activity analysis and the analysis of other GAP-associated protein GAPs and research.
Detailed description of the invention
The AMS activity that Fig. 1 difference extracting solution obtains；
The AMS activity that the PBS of Fig. 2 difference pH is obtained；
Fig. 3 difference extracts the AMS activity of liquid proportional；
The AMS activity that PBS is obtained in Fig. 4 difference tobacco bred；
In each figure: K326, NEB, RIPA respectively indicate corresponding lysate, and P6, P7, P8 respectively indicate pH=6.0,7.2,8.0
PBS；It is 5000,13000 that number 5,13, which respectively indicates centrifugal speed,；1:5,1:10 respectively indicate extraction liquid proportional.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment, before introducing specific embodiment, with regard to following realities
It applies the backgrounds such as part Experiment material, experimental method involved in example and briefly introduces and be described as follows.
Experimental material:
Tobacco K326 and NC82 are cultivated, laboratory culture takes fresh tobacco leaves to the 8-12 leaf phase.
Experimental method:
1, when BCA method detection protein concentration, with specific reference to following operating procedure:
(1) supernatant is taken, dilutes 10 times；According to sample size, add 1 volume BCA reagent B(50:1 by 50 volume BCA reagent As) match
BCA working solution processed, mixes well；
(2) blank well, standard sample wells and sample well, each sample is arranged to repeat three times, 20 μ l protein extracts of every empty addition,
200 μ L BCA working solutions；
(3) ELISA Plate is put and vibrates 30sec on the oscillator, 37 DEG C are placed 30 minutes, and then microplate reader detects at 562nm,
With protein content (μ g) for abscissa, light absorption value is ordinate, draws standard curve；
According to the light absorption value of institute's sample, establishing criteria curve obtains corresponding protein content (μ g), total divided by sample diluting liquid
Volume (20 μ L) is sample actual concentrations (unit: μ g/ μ L) multiplied by sample extension rate；
2, when ELISA method detection enzymatic activity, with reference to following steps:
(1) equilibrium at room temperature 20min Enzyme assay reagent；
(2) standard sample wells is set and sample aperture, standard sample wells respectively add the 50 μ L of standard items of various concentration；It is added in sample aperture to be measured
50 μ L of sample, blank well are not added；
In addition to blank well, the detection antibody 100 of horseradish peroxidase (HRP) label is added in every hole in standard sample wells and sample aperture
μ L seals reacting hole with sealing plate film, and 37 DEG C of water-baths or insulating box incubate 60min；
(3) liquid is discarded, is patted dry on blotting paper, every hole is filled it up with cleaning solution (350 μ L), is stood 1min, is got rid of cleaning solution, blotting paper
On pat dry, so repeat board-washing 5 times (can also use board-washing machine-wash plate)；
Substrate A in kit, each 50 μ L of B is added in every hole, and 37 DEG C are protected from light incubation 15min；
Every hole is added in terminate liquid 50 μ L, 15min, and the OD value in each hole is measured at 450nm wavelength；
Using the OD value of surveyed standard items as abscissa, the concentration value of standard items is ordinate, draws standard curve, and obtain straight line
The OD value of sample is substituted into equation, calculates the concentration of sample by regression equation.
Embodiment
The extracting method of raising Starch in Tobacco enzyme (AMS) enzyme activity provided herein, for starch in fresh tobacco leaves
The extraction of enzyme (AMS), specific steps are briefly discussed below.
(1) extracting solution is prepared, lysate RIPA, NEB or PBS specifically can be used；Formula composition are as follows:
Lysate RIPA: Tris-Cl(pH 8.0), 50 mM, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%,
PMSF,1 mM；
Buffer NEB:Hepes(pH 7.5), 20mM, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
The PBS:NaCl of pH 8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1
mM；
The PBS:NaCl of pH7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH 6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1
mM；
(2) tobacco leaf pre-processes
Tobacco K326 and NC82 are cultivated, laboratory culture to 8-12 leaf phase acquires fresh tobacco leaves.After liquid nitrogen flash freezer, it is ground into
Powder.
(3) solwution method is extracted
It pulverizes the extracting solution (lysate) for being added in tobacco sample and being prepared in step (1) in step (2), after concussion mixes,
It stands on ice and is no less than 1h；
In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution (lysate) dosage is that 500 μ l-1mL(are specially
500 μ l or 1mL)；
After the completion of standing, 5000 ~ 15000rpm centrifugation be no less than 20min(centrifugal speed specifically be, for example, 5000 rpm,
9000rpm or 13000rpm etc.), retain supernatant, as contains the extracting solution of higher starch enzyme (AMS) enzyme activity；
Further, protein concentration in supernatant is detected using BCA method, the work using ELISA method to AMS in supernatant
Property is detected.
To in supernatant after the PBS of different extracting solutions, difference pH, different centrifugal speeds, different extraction liquid proportional processing
AMS activity is detected, as a result as shown in Figure 1, Figure 2, Figure 3, Figure 4 respectively.Analysis is it can be seen that using PBS(pH 7.2)
When RIPA and NEB is handled, low-speed centrifugal result is relatively preferable, according to the analysis, this is mainly the interaction between impurity protein
There is certain counteracting, reduces the influence for AMS enzyme activity determination, and the extraction effect of PBS is more preferable；At the PBS of different pH
When reason, AMS activity is higher when the PBS of pH 8.0 is extracted；In the processing of different extraction liquid proportionals, when 1:10 ratio, AMS activity is more
It is high.And it is also higher for the activity of the obtained AMS of different cultivar NC82, same processing method.
In general, with regard to AMS enzyme activity determination or for extracting, using PBS (pH 8.0), liquid proportional 1:10, centrifugation are extracted
Rate 5000rpm, processing result is optimal, main reason is that, after cracking processing, the interaction between impurity protein has centainly
It offsets, reduces for AMS enzyme activity determination, and suitable ph also makes the activity of AMS more preferable.
Claims (4)
1. a kind of extracting method for improving amylase enzyme activity in leaf tobacco extract, which is characterized in that this method is directed to fresh tobacco leaves
The extraction of middle amylase, specifically comprises the following steps:
(1) extracting solution is prepared, lysate RIPA, NEB or PBS are specifically used；Formula composition are as follows:
Tris-Cl, 50 mM of lysate RIPA: pH 8.0, EDTA, 1mM, SDS, 0.1%, NaCl, 150 mM, NP-40,1%,
PMSF,1 mM；
Hepes, 20mM of buffer NEB:pH 7.5, KCl, 40 mM, EDTA, 1 mM, PMSF, 1 mM；
(2) tobacco leaf pre-processes
By the fresh tobacco leaves of tobacco 8-12 leaf phase, pulverize spare；
(3) solwution method is extracted
It is stood on ice after concussion mixes in step (2) extracting solution for being added in tobacco sample and being prepared in step (1) of pulverizing
No less than 1h；In terms of specific dosage, pulverize tobacco sample 100mg, and extracting solution dosage is 500 μ l-1mL；
After the completion of standing, 5000 ~ 15000rpm centrifugation is no less than 20min, retains supernatant, as contains higher starch enzyme enzyme activity
Extracting solution.
2. improving the extracting method of amylase enzyme activity in leaf tobacco extract as described in claim 1, which is characterized in that step (2)
In, the tobacco is K326 NC82 kind.
3. improving the extracting method of amylase enzyme activity in leaf tobacco extract as claimed in claim 2, which is characterized in that step (1)
PH=8.0 of middle PBS, 7.2 or 6.0, concrete composition are as follows:
The PBS:NaCl of pH=8.0,137 mM, KCl, 2.7 mM, Na2HPO49.5 mM, NaH2PO4, 0.5 mM, PMSF, 1 mM；
The PBS:NaCl of pH=7.2,137 mM, KCl, 2.7 mM, Na2HPO47.2 mM, NaH2PO4, 2.8 mM, PMSF, 1 mM；
The PBS:NaCl of pH=6.0,137 mM, KCl, 2.7 mM, Na2HPO41.2 mM, NaH2PO4, 8.8 mM, PMSF, 1 mM.
4. improving the extracting method of amylase enzyme activity in leaf tobacco extract as claimed in claim 3, which is characterized in that step (1)
When middle extraction, using the PBS of ph=8.0；In step (3), extracting solution dosage is 1mL, and 5000 rpm are centrifuged 20min.
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| KR20150054249A (en) * | 2013-11-11 | 2015-05-20 | 경희대학교 산학협력단 | Method for preparing amylose bead by enzymatic synthesis |
| CN104928317A (en) * | 2015-06-09 | 2015-09-23 | 昆明理工大学 | Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector |
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2018
- 2018-11-13 CN CN201811346040.6A patent/CN109321550A/en active Pending
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|---|---|---|---|---|
| CN1745169A (en) * | 2003-02-26 | 2006-03-08 | 金克克国际有限公司 | Amylase producing an altered immunogenic response and methods of making and using the same |
| CN101870998A (en) * | 2010-07-13 | 2010-10-27 | 四川农业大学 | Method for detecting activity of plant superoxide dismutase, catalase and peroxidase |
| CN103145817A (en) * | 2013-02-28 | 2013-06-12 | 华南农业大学 | Sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and coding gene and application thereof |
| KR20150054249A (en) * | 2013-11-11 | 2015-05-20 | 경희대학교 산학협력단 | Method for preparing amylose bead by enzymatic synthesis |
| CN104928317A (en) * | 2015-06-09 | 2015-09-23 | 昆明理工大学 | Plant expression vector with nicotiana tabacum gibberellins synthesis transcription regulation factor gene, and application of plant expression vector |
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