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Method for detecting feed xylanase activity and application thereof
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A technology of xylanase and xylan, which is applied in the direction of measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problems that cannot be directly used to detect enzyme activity, etc.
Inactive Publication Date: 2014-06-04
广州市博仕奥生化技术研究有限公司
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Problems solved by technology
ELISA is a routine detection method in medicine and scientific research. It is often used for trace detection of analytes, but it cannot be directly used for detection of enzyme activity.
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Embodiment 1
[0053] (1) Material
[0054] 1. main instrument and equipment used in the present invention
[0055] Precision balance, PB203-N type, METTLER TOLEDO; electric heating constant temperature incubator, DPX-9082B2 type, Shanghai Fuma Experimental Equipment Co., Ltd.; digital precision pH meter, PHS-3C type, Lei Magnetic; magnetic heating stirrer, 78-1 type, Jintan Fuhua Instrument Co., Ltd.; full-temperature multi-amplitude orbital shaker, ZHW-200B, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.; low-temperature centrifuge, type 5415R, Eppendorf; microplate reader, type 680, BIO-RAD; pipettes (100-1000 μL, 20-200 μL, and 2-20 μL), Dragon; color multi-tip pipettes (50-300 μL), Thermo SCIENTIFIC.
[0056] ② The main reagents involved in the present invention
[0078] The operating process of the method for ELISA detection xylanase activity is as follows figure 1 Shown:
[0079] (1) Add xylanase (2500U / g) with known enzyme activity to the basal diet of feed wheat in the amount of 2%, 0.2% and 0.02% by mass percentage; take 1g sample and use 100mL pH4.7 , 25mmol / L acetate buffer at 40°C and 250rpm on a shaker for 30min, take 1mL of the extract and centrifuge at 5000rpm for 5min, the supernatant is the sample solution to be tested, and the theoretical enzyme activities of the sample solution to be tested are respectively 0.5 , 0.05 and 0.005U / mL;
[0080] (2) Dilute biotin-labeled xylan 40,000 times by volume with pH 9.6, 0.05 mol / L carbonate buffer, add 100 μL per well to the microtiter plate, and coat at room temperature for 2 hours or overnight at 4 °C; Wash 3 times with PBST solution; add the sample solution to be tested, set 3 parallels for each sample, seal with plastic wrap and incubate at 40°C for 30 minutes, wash 3 times wit...
Embodiment 3
[0087] Comparison of Xylanase Activity Detection by Enzyme-Linked Sorbent Assay (ELSA) and DNS
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Abstract
The invention discloses a method for detecting feed xylanase activity and application thereof. The method disclosed by the invention comprises the following steps: detecting the absorbance value of residual biotin labeled xylan in the sample to be detected after the xylanase hydrolysis by using ELSA (enzyme linked immunosorbent assay); and calculating the activity of xylanase in the sample to be detected according to an enzyme activity gradient curve, wherein the enzyme activity gradient curve is a curve of the corresponding relationship between the absorbance value of residual biotin labeled xylan after enzymolysis detected by ELSA and the known different xylanase activities. As the ELSA conditions are optimized in the invention, the method disclosed by the invention can perform trace detection on the xylanase activity in feed, and can realize sensitivity of 1*10<5>U / g. The method disclosed by the invention not only can detect activity of xylanase in feed, but also enables the enzymolysis temperature adopted for detection to be consistent with the temperature of intestinal canal of livestock / poultry due to high sensitivity; and thus, by adopting the method to obtain the enzyme activity, better activity evaluation can be performed on the xylanase in feed.
Description
technical field [0001] The invention relates to a method for detecting enzyme activity, in particular to a method for detecting feed xylanase activity and its application. Background technique [0002] From an international point of view, about 65% to 75% of grain feeds have been added with non-starch polysaccharases mainly xylanase. Adding xylanase to feed can improve animal performance and reduce feed The meat ratio has an obvious effect. The analysis and detection of xylanase activity in compound feed plays an important role in the feed industry: ① It is an important means for enzyme preparation manufacturers to inspect and monitor product quality; After the mixing process, it will inevitably cause a certain loss of enzyme activity. Therefore, the determination of the activity of the enzyme preparation in the mixed finished feed (that is, the survival rate of the enzyme in the feed) can directly reflect the effect and quality of the enzyme preparation in the finished fee...
Claims
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