Method for detecting lytic polysaccharide monooxygenase enzyme activity and application thereof

A monooxygenase enzyme activity, polysaccharide monooxygenase technology, applied in the field of biology, can solve the problems of unstable ascorbate, unsuitable for non-purified samples, etc., and achieve short detection time, high sensitivity and low background. Effect

Inactive Publication Date: 2019-06-14
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires electron donors such as ascorbate, which is extremely unstable, and this method is not suitable for non-purified samples

Method used

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  • Method for detecting lytic polysaccharide monooxygenase enzyme activity and application thereof
  • Method for detecting lytic polysaccharide monooxygenase enzyme activity and application thereof
  • Method for detecting lytic polysaccharide monooxygenase enzyme activity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Experimental parameters for the determination of the activity of soluble polysaccharide monooxygenase:

[0025] (1) In a 200 μL reaction system, prepare the following enzyme activity reaction system:

[0026] 100mMPIPES buffer 50μL

[0027] Active substrate (10-acetyl-3,7-dihydroxyphenoxazine) 10μM

[0028] Coenzyme (horseradish catalase) 30U / ml

[0029] Carrier Disintegration Solution / Crude Enzyme Solution 100μL

[0030] Deionized water to 200μL

[0031] The carrier breaking solution / crude enzyme solution is derived from recombinant Escherichia coli BL21Gold(DE3)-pET28a capable of cloning and expressing soluble polysaccharide monooxygenase.

[0032] (2) Detection conditions: excitation wavelength 560nm, emission wavelength 590nm, detection temperature 30°C.

[0033] From figure 1 It can be seen from the curve that when no enzyme is added, the emission peak does not change, indicating that there is no intermolecular interaction between the systems. When the empty...

Embodiment 2

[0035] Construction of the Standard Curve of Enzyme Concentration and Fluorescence Intensification Rate of Soluble Polysaccharide Monooxygenase

[0036] Add different volumes of soluble polysaccharide monooxygenase into 2mL centrifuge tubes, and dilute to 1000μL with ultrapure water. The final concentration of enzyme protein is: 0μg / mL, 1.25μg / mL, 2.5μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, take 100ul into 96-well ELISA plate, add substrate 10-acetyl-3,7-dihydroxyphenoxazine (final concentration 10uM) and coenzyme horseradish catalase (final concentration 30U / ml), measure its fluorescence intensity at 30°C; take the concentration of soluble polysaccharide monooxygenase as the abscissa, and the rate of fluorescence enhancement as the ordinate to plot the activity of soluble polysaccharide monooxygenase standard curve (such as figure 2 shown).

[0037] figure 2 It shows that with the increase of the soluble polysaccharide monooxygenase enzyme concentration, the fluorescence enhanc...

Embodiment 3

[0039] Inhibitory effect of EDTA on soluble polysaccharide monooxygenase

[0040] Take 2 parts of 100ul soluble polysaccharide monooxygenase and add coenzyme horseradish catalase (final concentration 30U / ml) and substrate 10-acetyl-3,7-dihydroxyphenoxazine (final concentration 10uM) In the reaction system of , marked as reaction 1 and reaction 2, the rate of fluorescence enhancement was measured. After 10 min, 20ulPBS (20mM) buffer was added to reaction 1, and 20ulEDTA (20mM) was added to reaction 2, and the fluorescence enhancement rate was measured.

[0041] From image 3 The change of fluorescence rate shows that PBS buffer has no effect on the reaction, and EDTA can inhibit the activity of soluble polysaccharide monooxygenase.

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Abstract

The invention relates to a method for detecting a lytic polysaccharide monooxygenase and an application thereof. The detection principle is that: 10-acetyl-3,7-dihydroxyphenoxazine is used as a substrate, and horseradish peroxidase is used as a coenzyme; and when the lytic polysaccharide monooxygenase reacts with oxygen to form hydrogen peroxide, reaction with the substrate is performed to form aresorufin with fluorescence under the action of the coenzyme. The detection method does not require additional addition of an electron donor, which improves the stability of the reaction, can be usedfor identification of unpurified products, has high sensitivity, and has a short detection time. The detection of the lytic polysaccharide monooxygenase enzyme activity by means of the fluorescence detection method also has the characteristics of high detection flux, high accuracy, easy to realize automation, low cost and the like, which benefits performing scale application in screening work; andcombined with a flow cytometry, excellent strains with high enzyme activity can be efficiently and quickly selected.

Description

technical field [0001] The invention belongs to the technical field of biology, and in particular relates to a method for detecting enzyme activity of soluble polysaccharide monooxygenase and its application. Background technique [0002] Lytic polysaccharide monooxygenases (LPMO) are a class of oxidative enzymes that act on crystalline polysaccharides, which can break the glycosidic bonds of chitin (or cellulose) through oxidation to generate oxidized sugar chain ends And new non-reducing sugar chain ends, so that the structure of the crystalline substrate tends to be loose, providing a basis for the further action of glycoside hydrolase. The currently known LPMOs are mainly distributed in two protein families, AA9 and AA10, among which the LPMOs of the AA9 family all act on cellulose substrates, while some of the LPMOs of the AA10 family act on cellulose substrates, and some act on cellulose substrates. Chitin substrate. The discovery of soluble polysaccharide monooxygen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 马江锋成超姜岷
Owner NANJING UNIV OF TECH
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