Method for simply and efficiently measuring beta-amylase activity
An amylase and activity technology, applied in the field of amylase to achieve accurate results
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0024] Example 1: Determination steps of DNS method and p-nitrophenol maltopentoside method
[0025] Enzyme activity is defined as: 1mL enzyme solution hydrolyzes 1% (w / v) soluble starch solution at 55°C and pH6.0 for 10 minutes to generate 1mg maltose, which is 1 enzyme activity unit, expressed in U / mL.
[0026] DNS method: drawing of standard curve: take 6 25mL colorimetric tubes with stoppers, add 0.0, 0.4, 0.8, 1.2, 1.6, 2.0ml maltose standard solution respectively, then add distilled water to each tube to 2mL, then add 3mL DNS Reagent, heated in boiling water bath for 10min, took out and cooled to room temperature, and diluted to 25mL with distilled water. After mixing, take the colorimetric tube without maltose standard solution as a contrast and measure the absorbance at 540nm, take the absorbance as the ordinate, and the maltose content as the abscissa, draw a standard curve, and draw its linear regression equation as y=2979+0.0226( R2 = 0.9935).
[0027] Determinati...
Embodiment 2
[0029] Example 2: Purification of β-amylase
[0030] A recombinant Escherichia coli strain capable of expressing the barley β-amylase gene was induced to express and the bacteria were collected, and the β-amylase was extracted by ultrasonic crushing method, centrifuged and concentrated, and then passed through a Ni-NTA affinity chromatography column. Proteins are purified. Purification result as figure 1 As shown, the purified β-amylase is a single band in the SDS-PAGE pattern, indicating that the β-amylase has reached electrophoretic purity and can be used for further analysis.
[0031] Embodiment 2: Determination of linear concentration range
[0032] For the same concentration of β-amylase, the absorbance value measured by the DNS method is generally high, while the absorbance value measured by the p-nitrophenol maltopentoside kit method is low, as shown in Table 1. When the enzyme When the liquid concentration gradient is high, the absorbance value measured by the DNS m...
Embodiment 3
[0036] Embodiment 3: repeatability experiment
[0037] Five copies of β-amylase solutions with different concentrations were precisely prepared, and the corresponding absorbance values were obtained by DNS method and p-nitrophenol maltopentoside method respectively. The results are shown in Table 2.
[0038] Table 2 Precision experiment (n=5)
[0039]
[0040]As can be seen from Table 2, the standard deviations (RSD) of the two methods are respectively 1.6-8.8% and 3.3-7.3%. The absorbance value measured by the phenol maltopentoside method was linearly fitted, and the results showed that the correlation between the two was good, and the R 2 The value is 0.9886, and the regression equation is y=0.0079x+0.0089. A linear fitting curve such as Figure 4 .
PUM
| Property | Measurement | Unit |
|---|---|---|
| concentration | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More