CN108195782B - Method for measuring activity of beta-amylase - Google Patents
Method for measuring activity of beta-amylase Download PDFInfo
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- CN108195782B CN108195782B CN201810073151.8A CN201810073151A CN108195782B CN 108195782 B CN108195782 B CN 108195782B CN 201810073151 A CN201810073151 A CN 201810073151A CN 108195782 B CN108195782 B CN 108195782B
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Abstract
The invention discloses a method for simply and efficiently measuring beta-amylase activity, and belongs to the field of amylase. The method for measuring the activity of the beta-amylase comprises a DNS reagent color development method and a p-nitrophenol maltoside method. The DNS method is long in time consumption and low in sensitivity, the activity value of the beta-amylase measured by the DNS method can be influenced by alpha-amylase, and the p-nitrophenol maltopenoside method is simple, convenient and quick and high in sensitivity, but can only represent the relative activity of the beta-amylase. Therefore, the invention establishes the beta-amylase activity measuring method which is simple to operate and high in sensitivity and can measure the absolute enzyme activity value of the beta-amylase by performing linear fitting on the DNS reagent color development method and the p-nitrophenol maltoside method and combining the advantages of the DNS reagent color development method and the p-nitrophenol maltoside method. The method can be used for simply and quickly measuring the absolute enzyme activity value of the beta-amylase in both purified beta-amylase and beta-amylase preparation mixtures, and the initial application result shows that the optimization method is relatively accurate in result and can be further popularized.
Description
Technical Field
The invention relates to a method for simply and efficiently determining activity of beta-amylase, belonging to the field of amylase.
Background
Beta-amylase, also known as maltosidase, is an exo-saccharifying enzyme. It can cut the alternate alpha-1, 4 glycosidic bond from the non-reducing end of starch, and the hydrolysis products are mainly maltose and beta-limit dextrin. The beta-amylase is widely applied to the industries of beer brewing, food processing and the like, and is an important industrial enzyme. The existing method for measuring the enzyme activity of the beta-amylase comprises a DNS reagent color development method and a p-nitrophenol maltopentoside kit method.
The DNS method is mainly used for determining the activity of beta-amylase by measuring the content of reducing sugar in an enzymolysis product, and is the most widely used method for determining the enzyme activity of the beta-amylase. However, since other starch hydrolyzing enzymes (particularly α -amylase) may be mixed in the crude enzyme solution extracted from plants and β -amylase expressed by some microorganisms, the enzyme activity value represented by the reducing sugar content is not accurate, and thus there is a difficulty in accurately determining the absolute enzyme activity of β -amylase in the crude enzyme solution. And the DNS method has low sensitivity, consumes time, and has certain toxicity of reagents, so the method is more suitable for measuring the enzyme activity of the purified beta-amylase.
The p-nitrophenol maltopenoside kit method takes a specific substrate p-nitrophenol maltopenoside of beta-amylase as a substrate, the beta-amylase reacts with the beta-amylase to release a molecule of maltose and p-nitrophenol maltotriose so as to determine the enzyme activity, the specificity is higher, the method is quick, sensitive and simple and convenient to operate, but the enzyme activity unit is measured by the amount of the p-nitrophenol maltopenoside released in unit time, usually expressed by the relative enzyme activity of the beta-amylase, and has certain limitation.
Disclosure of Invention
The invention aims to solve the technical problems that the operation is simple, and the absolute enzyme activity of the beta-amylase in the crude amylase liquid can be accurately measured.
The invention aims to provide a method for measuring beta-amylase activity, which measures the beta-amylase activity in a sample to be measured by jointly adopting a p-nitrophenol maltopenoside method and a DNS method.
In one embodiment of the invention, the concentration of beta-amylase in the sample to be tested in the method is 12-60. mu.g/mL.
In one embodiment of the present invention, in the method, the linear fitting curve regression equation is that y is 0.0079x +0.0089, and R is2The value is 0.9886.
In an embodiment of the present invention, the method specifically includes:
(1) diluting the solution to different concentrations by adopting purified beta-amylase, and respectively determining the light absorption values after reaction by adopting a p-nitrophenol maltoside method and a DNS method;
(2) carrying out correlation analysis on the light absorption values measured by a p-nitrophenol maltoside method and a DNS method, and determining a linear concentration range;
(3) diluting the purified beta-amylase to the linear concentration range of the step (2) for detection, and establishing a linear fitting curve regression equation y of 0.0079x +0.0089, wherein R is2A value of 0.9886;
(4) and (3) determining a sample to be detected by adopting a p-nitrophenol maltoside method and a DNS method, and calculating the activity of the beta-amylase by utilizing a regression equation.
In one embodiment of the present invention, the p-nitrophenol maltoside method specifically comprises: and (3) taking 10-30 mu L of enzyme solution and a specific substrate p-nitrophenol maltopentose solution, placing the enzyme solution and the specific substrate p-nitrophenol maltopentose solution together at 50-60 ℃, preserving the heat for 2-4 min, mixing, reacting at 50-60 ℃ for 10-15 min, adding 200-400 mu L of stop buffer solution, and taking the reaction solution to measure the light absorption value at 400 nm.
In an embodiment of the present invention, the DNS method specifically includes: respectively placing 1-2 mL of enzyme solution and starch solution in a 50-60 ℃ constant-temperature water bath for 10-15 min, then adding 1-2 mL of 1-2% starch solution into the enzyme solution, placing in a 50-60 ℃ constant-temperature water bath for 10-15 min, placing in an ice water bath for 2-4 min to terminate the reaction, then adding 2-4 mL of DNS reagent, uniformly mixing, boiling in a boiling water bath for 10-15 min, taking out and cooling, diluting to 25mL with water to a constant volume, and measuring the light absorption value at 540 nm.
The second purpose of the invention is to provide the application of the method in beer brewing and food processing.
The invention has the beneficial effects that:
linear fitting curve R established by the invention2The value is 0.9886, the correlation degree is higher, and the determination result is that when the product is applied to a commercial beta-amylase: the relative error is less than 10%, and the sample can be determined to be substantially free of alpha-amylase. Compared with other two methods for measuring the enzyme activity of the beta-amylase, the method combines the advantages of the DNS method and the p-nitrophenol maltopenoside method, and overcomes the disadvantages of the DNS method and the p-nitrophenol maltopenoside method. The method can be used for simply and quickly measuring the absolute enzyme activity value of the beta-amylase in both purified beta-amylase and beta-amylase preparation mixtures, and the initial application result shows that the optimization method is relatively accurate in result and can be further popularized.
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FIG. 1 shows the SDS-PAGE result of the beta-amylase purification induced by Escherichia coli; m: protein standard molecular weight; 1: concentrating the supernatant of the cell wall breaking of the Escherichia coli;
FIG. 2 is a linear relationship of DNS method determination results under a high concentration gradient;
FIG. 3 is a linear concentration range;
(A) normal linear range of p-nitrophenol maltopenoside; (B) a DNS normal linearity range; (C) DNS normal linearity range (12-60 μ g/mL);
fig. 4 is a linear fit curve.
Detailed Description
Example 1: DNS method and p-nitrophenol maltoside method determination steps
The enzyme activity is defined as: 1mL of enzyme solution hydrolyzes 1% (w/v) of soluble starch solution at 55 ℃ and pH6.0 for 10 minutes to generate 1mg of maltose, namely 1 unit of enzyme activity, which is expressed by U/mL.
The DNS method comprises the following steps: drawing a standard curve: taking 6 25mL colorimetric tubes with plugs, adding 0.0, 0.4, 0.8, 1.2, 1.6 and 2.0mL of maltose standard solution respectively, then adding distilled water into each tube to 2mL, adding 3mL of DNS reagent, heating in a boiling water bath for 10min, taking out, cooling to room temperature, and adding distilled water to dilute to 25 mL. After mixing, absorbance was measured at 540nm using a colorimetric cylinder without a maltose standard solution as a control, and a standard curve was drawn with absorbance as the ordinate and maltose content as the abscissa to find that the linear regression equation was 2979+0.0226 (R2-0.9935).
And (3) determination of a sample: numbering a plurality of 25mL colorimetric tubes with plugs, respectively adding 1mL of enzyme solution (20mmol/L of phosphate buffer solution for dilution), placing each colorimetric tube and the starch solution in a 55 ℃ constant-temperature water bath for 10min, then adding 1mL of 1% starch solution into each colorimetric tube, placing in a 55 ℃ constant-temperature water bath for 10min, placing in an ice water bath for 2min to terminate the reaction, then adding 3mL of a LDNS reagent, uniformly mixing, boiling in a boiling water bath for 10min, taking out and cooling, fixing the volume to 25mL by using distilled water, replacing the enzyme solution with the distilled water as a blank, measuring absorbance at 540nm, and calculating the enzyme activity.
The kit method of the p-nitrophenol maltopenoside comprises the following steps: the enzyme activity was measured using the Betamyl-3Method kit from Megazyme, Ireland, with some modifications to the protocol of the kit. 20 mu L of enzyme solution is taken in a 2mL EP tube, and is placed at 55 ℃ for 2min together with a specific substrate p-nitrophenol maltopentose solution, the temperature is kept for 10min at 55 ℃, 300 mu L of stop buffer solution is added, 200 mu L of reaction solution is taken, and the absorbance at 400nm is measured by taking distilled water as a contrast.
Example 2: purification of beta-amylase
After induced expression and thallus collection, a recombinant escherichia coli capable of expressing barley beta-amylase gene is subjected to wall breaking by an ultrasonic disruption method to extract beta-amylase, and after centrifugal concentration, protein is purified by a Ni-NTA affinity chromatography column. The purification result is shown in FIG. 1, the purified beta-amylase is a single band in SDS-PAGE pattern, which shows that the beta-amylase has reached electrophoretic purity and can be used for further analysis.
Example 2: determination of the Linear concentration Range
For the same concentration of beta-amylase, the absorbance values measured by the DNS method are generally higher, and the absorbance values measured by the p-nitrophenol maltoside kit method are lower, as shown in Table 1, when the concentration gradient of the enzyme solution is higher, the absorbance value measured by the DNS method exceeds the confidence interval (0.2-0.8), and the correlation is poor (the correlation result is shown in FIG. 2). When the concentration gradient of the enzyme solution is lower, the result measured by using the p-nitrophenol maltopenoside kit method is lower than the confidence interval, and the application range of the standard curve is narrower, so that the proper concentration gradient of the beta-amylase is adjusted, the result of the absorbance value falls within the confidence interval when the two methods are used for measuring, and the wider application range is the key for establishing the method.
TABLE 1 measurement of different concentrations of beta-amylase by DNS method and p-nitrophenol maltoside method
And (3) determining the concentration of the purified beta-amylase protein to be 240 mu g/mL, diluting the purified beta-amylase protein according to concentration gradients of 4%, 8%, 12%, 16%, 20 and 24%, respectively determining the light absorption value of the enzyme after the enzyme reacts with the substrate solution by adopting a DNS method and a p-nitrophenol maltopentoside method, and performing correlation analysis on the data to show that the beta-amylase protein and the substrate solution have higher correlation when the concentration of the beta-amylase is 12-60 mu g/mL. The linear concentration ranges are shown in FIG. 3 (A), (B) and (C).
Example 3: repeatability test
Accurately preparing 5 parts of beta-amylase solution with different concentrations, and respectively determining by a DNS method and a p-nitrophenol maltoside method to obtain corresponding absorbance values, wherein the results are shown in Table 2.
TABLE 2 precision experiment (n ═ 5)
As can be seen from Table 2, the standard deviation (RSD) of the two methods is 1.6-8.8% and 3.3-7.3%, respectively, on the basis, the enzyme activity value of the beta-amylase measured by the DNS method and the absorbance value measured by the p-nitrophenol maltoside method are subjected to linear fitting, and the result shows that the correlation between the two methods is good, and the standard deviation (RSD) is goodR2The value is 0.9886 and the regression equation is 0.0079x + 0.0089. The curve is linearly fit as in fig. 4.
Example 4: practical application
In order to verify the effectiveness of the linear fitting method, a beta-amylase product purchased from Beijing Shengdong science and technology company is taken as a sample to be further measured, firstly, the beta-amylase liquid is diluted to the concentration in the table, the enzyme activity is measured by respectively adopting a DNS method and a p-nitrophenol maltopenoside method, the absorbance value measured by the p-nitrophenol maltopenoside method is converted into an enzyme activity value through the linear fitting curve in the embodiment 2, and the enzyme activity value is compared with the enzyme activity value measured by the DNS method. As shown in Table 3, the results of the DNS method and the absorbance value conversion value of the p-nitrophenol maltoside method are relatively close, the relative errors are less than 10%, and when the method is used for measurement, whether the amylase sample contains amylase except beta-amylase or not can be determined.
TABLE 3 application of the Linear fitting method
Compared with other two methods for measuring the enzyme activity of the beta-amylase, the method combines the advantages of the DNS method and the p-nitrophenol maltopenoside method, and overcomes the disadvantages of the DNS method and the p-nitrophenol maltopenoside method. The method can be used for simply and quickly measuring the absolute enzyme activity value of the beta-amylase in both purified beta-amylase and beta-amylase preparation mixtures, and the initial application result shows that the optimization method is relatively accurate in result and can be further popularized.
Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and it is intended that the scope of the invention be defined by the appended claims.
Claims (2)
1. A method for determining beta-amylase activity is characterized in that a p-nitrophenol maltoside method and a DNS method are jointly used for determining the beta-amylase activity in a sample to be determined, the concentration of the beta-amylase in the sample to be determined in the method is 12-60 mu g/mL, and the method specifically comprises the following steps:
(1) diluting the solution to different concentrations by adopting purified beta-amylase, and respectively determining the light absorption values after reaction by adopting a p-nitrophenol maltoside method and a DNS method;
(2) carrying out correlation analysis on the light absorption values measured by a p-nitrophenol maltoside method and a DNS method, and determining a linear concentration range;
(3) diluting the purified beta-amylase to the linear concentration range in the step (2) for detection, and establishing a linear fitting curve regression equation y of 0.0079x +0.0089, wherein x is the enzyme activity of the beta-amylase measured by a DNS method, and y is OD measured by a p-nitrophenol maltopentoside method400Value of the OD400The absorbance value, R, of beta-amylase at 400nm2A value of 0.9886;
(4) measuring a sample to be measured by adopting a p-nitrophenol maltopenoside method, and calculating the activity of beta-amylase by utilizing a regression equation;
the method for preparing the p-nitrophenol maltoside specifically comprises the following steps: taking 10-30 mu L of enzyme solution and a specific substrate p-nitrophenol maltopentose solution, placing the enzyme solution and the specific substrate p-nitrophenol maltopentose solution together at 50-60 ℃, preserving heat for 2-4 min, mixing, reacting at 50-60 ℃ for 10-15 min, adding 200-400 mu L of termination buffer solution, and taking the reaction solution to measure the light absorption value at 400 nm;
the DNS method specifically comprises the following steps: respectively placing 1-2 mL of enzyme solution and starch solution in a 50-60 ℃ constant-temperature water bath for 10-15 min, then adding 1-2 mL of 1-2% starch solution into the enzyme solution, placing in a 50-60 ℃ constant-temperature water bath for 10-15 min, placing in an ice water bath for 2-4 min to terminate the reaction, then adding 2-4 mL of DNS reagent, uniformly mixing, boiling in a boiling water bath for 10-15 min, taking out and cooling, diluting to 25mL with water to a constant volume, and measuring the light absorption value at 540 nm.
2. Use of the method of claim 1 in beer brewing, food processing.
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