CN108195782A - It is a kind of to be simple and efficient the method for measuring beta amylase vigor - Google Patents
It is a kind of to be simple and efficient the method for measuring beta amylase vigor Download PDFInfo
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- CN108195782A CN108195782A CN201810073151.8A CN201810073151A CN108195782A CN 108195782 A CN108195782 A CN 108195782A CN 201810073151 A CN201810073151 A CN 201810073151A CN 108195782 A CN108195782 A CN 108195782A
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- beta amylase
- nitrophenol
- maltopentoside
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
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Abstract
The invention discloses a kind of methods for being simple and efficient and measuring beta amylase vigor, belong to amylase field.The assay method of beta amylase vigor has DNS reagent color developing methods and p-nitrophenol maltopentoside method.Time-consuming for DNS methods, sensitivity is low, and the beta amylase energy value measured can be influenced by alpha amylase, and p-nitrophenol maltopentoside method is easy to be quick, high sensitivity, but can only represent the opposite enzyme activity of beta amylase.Therefore, the present invention by DNS reagent color developing methods and p-nitrophenol maltopentoside method by carrying out linear fit, with reference to the advantage of the two, a kind of beta amylase vigour-testing method easy to operate, high sensitivity for measuring the absolute enzyme activity value of beta amylase is established.The absolute enzyme activity value for either purifying beta amylase in beta amylase or beta amylase enzyme preparation mixture can be quickly and easily determined using this method, Preliminary Applications result shows that the optimization method result is also more accurate, can be with further genralrlization.
Description
Technical field
The present invention relates to a kind of methods for being simple and efficient and measuring beta amylase vigor, belong to amylase field.
Background technology
Beta amylase, also known as maltoside enzyme are a kind of circumscribed-type carbohydrase.It can be opened from the non reducing end of starch
Beginning cuts the α-Isosorbide-5-Nitrae glycosidic bond at interval successively, and hydrolysate is mainly maltose and β-limit dextrin.Beta amylase extensive use
It is a kind of important industrial enzymes in industries such as brewing, food processings.The assay method of beta amylase enzyme activity has at present
DNS reagent color developing methods and p-nitrophenol maltopentoside RNA isolation kit.
DNS methods mainly determine the activity of beta amylase by measuring the content of reducing sugar in enzymolysis product, are to use
Most commonly used beta amylase enzyme activity determination method.However, what the crude enzyme liquid and certain micro-organisms due to being extracted in plant were expressed
Other Starch Hydrolysis enzymes (particularly alpha-amylase) may be mixed in beta amylase, in the case by content of reducing sugar table
The enzyme activity value shown is inaccurate, therefore there are certain difficulties for the absolute enzyme activity of beta amylase in Accurate Determining crude enzyme liquid.And DNS methods
Sensitivity is low, time-consuming, reagent has certain toxicity, therefore is relatively specific for the measure of beta amylase enzyme activity after purification.
P-nitrophenol maltopentoside RNA isolation kit is with the specificity substrate p-nitrophenol malt pentose of beta amylase
For glycosides as substrate, beta amylase reacts with it one molecule maltose of release and p-nitrophenol maltotriose so that it is determined that enzyme activity
Power, specificity is higher, and method is quick, sensitive, easy to operate, but its enzyme-activity unit is discharged in the unit interval to nitre
The amount of base phenol maltopentoside usually represents there is certain limitation come what is measured with the opposite enzyme activity of beta amylase.
Invention content
The technical problem to be solved by the present invention is to not only easy to operate, but also can Accurate Determining amylase crude enzyme liquid beta amylase
Absolute enzyme activity, to solve the above-mentioned problems, this experiment carry out DNS measuring methods and p-nitrophenol maltopentoside RNA isolation kit
With reference to, it is intended to establish a kind of accurate, easy method for measuring the absolute enzyme activity value of beta amylase in crude enzyme liquid.
The object of the present invention is to provide a kind of method for measuring beta amylase vigor, the method is joint using to nitro
Phenol maltopentoside method and DNS methods measure beta amylase vigor in sample to be tested.
In one embodiment of the invention, in the method in sample to be tested beta amylase a concentration of 12-60 μ g/
mL。
In one embodiment of the invention, linear matched curve regression equation is y=0.0079x+ in the method
0.0089, R2Be worth is 0.9886.
In one embodiment of the invention, the method is specifically：
(1) using purifying beta amylase be diluted to various concentration, be respectively adopted p-nitrophenol maltopentoside method and
DNS methods measure the light absorption value after reaction；
(2) light absorption value that p-nitrophenol maltopentoside method and DNS methods measure is subjected to correlation analysis, determined linear
Concentration range；
(3) beta amylase of purifying is diluted in the range of the linear concentration of step (2) and be detected, establish linear fit
Regression Equations y=0.0079x+0.0089, R2Be worth is 0.9886；
(4) sample to be tested using p-nitrophenol maltopentoside method and DNS methods is measured, utilizes regression equation meter
Calculate beta amylase vigor.
In one embodiment of the invention, the p-nitrophenol maltopentoside method is specially：Take 10~30 μ L
It is placed at 50~60 DEG C after keeping the temperature 2~4min and mixes together with enzyme solution and specificity substrate p-nitrophenol malt pentose solution, 50
10~15min is reacted at~60 DEG C, adds in 200~400 μ L stop buffers, extracts reaction solution the light absorption value measured at 400nm.
In one embodiment of the invention, the DNS methods are specially：1~2mL enzyme solutions and starch solution are put respectively
In 50~60 DEG C of 10~15min of water bath with thermostatic control, then 1~2mL, 1~2% starch solutions, 50~60 DEG C of constant temperature are added in into enzyme solution
10~15min in water-bath is placed in 2~4min of ice-water bath and terminates reaction, then adds in 2~4mL DNS reagents, mixing, boiling water bath
In boil 10~15min, take out cooling, 25mL be settled to water, the measure light absorption value 540nm at.
Second object of the present invention is to provide application of the method in brewing, food processing.
Beneficial effects of the present invention：
The linear fit curve R that the present invention is established2It is 0.9886 to be worth, and degree of correlation is higher, is applied to commercialization
Beta amylase product, measurement result are：Relative error is less than 10%, and can determine that in the sample and be substantially free of alphalise starch
Enzyme.Compared to the assay method of other two kinds of beta amylase enzyme activity, which combines DNS methods and p-nitrophenol maltopentoside
The respective advantage of method overcomes the shortcomings that respective simultaneously.Either purifying β-starch can quickly and easily be determined using this method
The absolute enzyme activity value of beta amylase, Preliminary Applications result show the optimization method knot in enzyme or beta amylase enzyme preparation mixture
Fruit is also more accurate, can be with further genralrlization.
Description of the drawings
Fig. 1 purifies SDS-PAGE results for Escherichia coli induced expression beta amylase；M：Protein standard marker；1：Greatly
Coli cell broken wall supernatant concentration；
Fig. 2 is DNS method measurement result linear relationships under high concentration gradient；
Fig. 3 is linear concentration range；
(A) the p-nitrophenol maltopentoside method range of linearity；(B) the DNS methods range of linearity；(C) the DNS methods range of linearity
(12-60μg/mL)；
Fig. 4 is linear fit curve.
Specific embodiment
Embodiment 1：DNS methods and p-nitrophenol maltopentoside method determination step
Enzyme activity is defined as：1mL enzyme solutions are under the conditions of 55 DEG C, pH6.0, hydrolysis 1% (w/v) soluble starch liquid life in 10 minutes
Into 1mg maltose, as 1 enzyme activity unit, represented with U/mL.
DNS methods：The drafting of standard curve：Take 6 25mL color-comparison tubes, be separately added into 0.0,0.4,0.8,1.2,1.6,
Then 2.0ml maltose standard solution adds in distilled water to 2mL into each pipe, adds 3mL DNS reagents, boiling water bath heating
10min, taking-up are cooled to room temperature, and distilled water is added to be diluted to 25mL.After mixing using not plus maltose standard solution colorimetric cylinder as
Absorbance is measured under 540nm to impinging upon, using absorbance as ordinate, maltose content is abscissa, draws standard curve, obtains
Its equation of linear regression is y=2979+0.0226 (R2=0.9935).
The measure of sample：Several 25mL color-comparison tubes is taken to number, being separately added into 1mL enzyme solutions, (20mmol/L phosphate delays
Fliud flushing dilutes), each colorimetric cylinder and starch solution are placed in 55 DEG C of water bath with thermostatic control 10min, then 1mL 1% is added in into each colorimetric cylinder
Starch solution, 10min in 55 DEG C of waters bath with thermostatic control are placed in ice-water bath 2min and terminate reaction, then addition 3mLDNS reagents, mixing,
10min is boiled in boiling water bath, cooling is taken out, 25mL is settled to distilled water, enzyme solution is replaced as blank using distilled water, in 540nm
Place measures absorbance, calculates enzyme activity.
P-nitrophenol maltopentoside RNA isolation kit：It is tried using Irish Megazyme companies Betamyl-3Method
Agent box measures enzyme activity, and experimental method reference reagent box specification is centainly changed.20 μ L enzyme solutions is taken to be managed in 2mL EP, and with
Specificity substrate p-nitrophenol malt pentose solution, which is placed in together at 55 DEG C, keeps the temperature 2min, and 10min is reacted at 55 DEG C, adds in
300 μ L stop buffers take 200 μ L reaction solutions, using distilled water as the absorbance at blank determination 400nm.
Embodiment 2：The purifying of beta amylase
The recombination bacillus coli that can express barley beta-amylase gene by one plant, after induced expression, microorganism collection,
It is pure to protein progress by Ni-NTA affinity columns after centrifugal concentrating by ultrasonic fragmentation extraction beta amylase
Change.Purification result illustrates beta amylase as shown in Figure 1, beta amylase is single band in SDS-PAGE collection of illustrative plates after purification
It is pure through reaching electrophoresis, it can be used for further analyzing.
Embodiment 2：Linear concentration range determines
For the beta amylase of same concentrations, since the absorbance value measured by DNS methods is generally higher, and p-nitrophenol
Absorbance value measured by maltopentoside RNA isolation kit is relatively low, as shown in table 1, when enzyme solution concentration gradient is higher, uses DNS
Absorbance value result measured by method can be more than its confidence interval (0.2-0.8), and correlation is poor that (correlation results are shown in Fig. 2
It is shown).And when enzyme solution concentration gradient is relatively low, the result measured using p-nitrophenol maltopentoside RNA isolation kit can be low
In its confidence interval, while the application range of standard curve is relatively narrow, so adjusting suitable beta amylase concentration gradient so that uses
When two methods are measured, the result of absorbance value is fallen in confidence interval, while it is to establish to have wider application range
The key of this method.
DNS methods and p-nitrophenol maltopentoside method measured value during the beta amylase of 1 various concentration of table
Measure after purification beta amylase protein concentration be 240 μ g/mL, by its according to 4,8,12,16,20,24% concentration
Gradient dilution measures the light absorption value after enzyme-to-substrate solution reaction using DNS methods and p-nitrophenol maltopentoside method respectively,
Data are subjected to correlation analysis, both as a result showing during a concentration of 12-60 μ g/mL of beta amylase has higher correlation.
(A), (B) and (C) of linear concentration range such as Fig. 3.
Embodiment 3：Repeated experiment
5 parts of the beta amylase solution of accurate formulation various concentration, and DNS methods and p-nitrophenol malt pentose is respectively adopted
Glycosides method measures to obtain corresponding absorbance value, and the results are shown in Table 2.
2 Precision Experiment of table (n=5)
As can be seen from Table 2, the standard deviation (RSD) of two methods is respectively 1.6-8.8% and 3.3-7.3%, herein
On the basis of, the absorbance value that beta amylase enzyme activity value and p-nitrophenol maltopentoside method that DNS methods measure measure is carried out
As a result linear fit shows that the two correlation is good, R2It is 0.9886 to be worth, regression equation y=0.0079x+0.0089.Line
Property matched curve such as Fig. 4.
Embodiment 4：Practical application
In order to verify the validity of linear fitting, the beta amylase product purchased from Beijing Sheng Dong scientific ＆ technical corporation is taken as sample
Product are further measured, first that beta amylase enzyme solution is dilute to concentration in table, and DNS methods and p-nitrophenol malt is respectively adopted
Pentoside method measures its enzyme activity, and p-nitrophenol maltopentoside method is measured absorbance value passes through Linear Quasi in embodiment 2
It closes curve and is scaled enzyme activity force value, and measure enzyme activity force value with DNS methods and be compared.The results are shown in Table 3, and DNS methods measure knot
Fruit and p-nitrophenol maltopentoside method measure absorbance value scaled value relatively, and relative error is respectively less than 10%, together
When being measured using this method, it may be determined that whether contain the amylases in addition to beta amylase in amylase samples.
The application of 3 linear fitting of table
Compared to the assay method of other two kinds of beta amylase enzyme activity, which combines DNS methods and p-nitrophenol malt
The respective advantage of pentoside method overcomes the shortcomings that respective simultaneously.It can quickly and easily be determined using this method and either purified
The absolute enzyme activity value of beta amylase, Preliminary Applications result show the optimization in beta amylase or beta amylase enzyme preparation mixture
Methods and results are also more accurate, can be with further genralrlization.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Claims (7)
- A kind of 1. method for measuring beta amylase vigor, which is characterized in that the method is joint using p-nitrophenol malt Pentoside method and DNS methods measure beta amylase vigor in sample to be tested.
- 2. according to the method described in claim 1, it is characterized in that, in the method in sample to be tested beta amylase it is a concentration of 12-60μg/mL。
- 3. according to the method described in claim 1, it is characterized in that, linear matched curve regression equation is y=in the method 0.0079x+0.0089, R2Be worth is 0.9886.
- 4. according to the method described in claim 1, it is characterized in that, the method is specifically：(1) various concentration is diluted to using the beta amylase of purifying, p-nitrophenol maltopentoside method and DNS methods is respectively adopted Measure the light absorption value after reaction；(2) light absorption value that p-nitrophenol maltopentoside method and DNS methods measure is subjected to correlation analysis, determines linear concentration Range；(3) beta amylase of purifying is diluted in the range of the linear concentration of step (2) and be detected, establish linear fit curve Regression equation y=0.0079x+0.0089, R2Be worth is 0.9886；(4) sample to be tested is measured using p-nitrophenol maltopentoside method and DNS methods, using regression equation calculation β- Amylase activity.
- 5. according to the method described in claim 4, it is characterized in that, the p-nitrophenol maltopentoside method is specially：It takes It is placed in together with 10~30 μ L enzyme solutions and specificity substrate p-nitrophenol malt pentose solution at 50~60 DEG C after keeping the temperature 2~4min It mixes, 10~15min is reacted at 50~60 DEG C, add in 200~400 μ L stop buffers, extract reaction solution the suction measured at 400nm Light value.
- 6. according to the method described in claim 4, it is characterized in that, the DNS methods are specially：Respectively by 1~2mL enzyme solutions and shallow lake Powder solution is placed in 50~60 DEG C of 10~15min of water bath with thermostatic control, then 1~2mL, 1~2% starch solutions are added in into enzyme solution, 50~ 10~15min in 60 DEG C of waters bath with thermostatic control is placed in 2~4min of ice-water bath and terminates reaction, then add in 2~4mL DNS reagents, mixes It is even, 10~15min is boiled in boiling water bath, cooling is taken out, 25mL is settled to water, light absorption value is measured at 540nm.
- 7. application of the method described in claim 1 in brewing, food processing.
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