CN102660633A - Quick determination reagent kit for beta-glucosidase activity and beta-glucosidase activity determination method - Google Patents

Abstract

The invention discloses a quick quantitative determination reagent kit for beta-glucosidase activity and a beta-glucosidase activity determination method. The quick quantitative determination reagent kit for beta-glucosidase activity contains a reagent R1 and a reagent R2. The reagent R1 comprises 90-110mmol/L of buffer solution with the pH ranging from 6.8 to 7.0, 1.0-3.0U/L of glucose oxidase, 1.0-2.5U/L of peroxidase and 0.01-0.1% of preservative. The reagent R2 comprises 90-110mmol/L buffer solution with the pH ranging from 6.8 to 7.0, 100-115mmol/L of chromogenic agent, 70-100mmol/L of maltose, 0.2-1.0mmol/L of 4-aminoantipyrene and 0.01-0.1% of preservative. The invention further discloses an application of the quick quantitative determination reagent kit for beta-glucosidase activity in the process of quick determination of beta-glucosidase activity. Only two liquid reagents are utilized, and thus reagent type and usage are reduced greatly. Reaction time is shortened greatly by means of spectrophotometry or enzyme-labelled immunosorbent assay. Reaction steps are simplified, the reagents are saved, automatic determination in scale is achieved, and determination efficiency is increased greatly while accuracy and reliability of determination results are improved.

Description

A kind of activity of beta-glucosidase rapid determination test kit and detection method thereof

Technical field

The present invention relates to a kind of test kit and detection method thereof, relate in particular to a kind of activity of beta-glucosidase rapid determination test kit and detection method thereof.

Technical background

(β-glucosidase) is one type of lytic enzyme (EC 3.2.1.21) to beta-glucosidase, extensively is present in the various organisms, in the physiology of organism, metabolic process, plays an important role.In recent years, people have carried out deep research to aspects such as its catalytic mechanism, molecular structure, gene orders.Beta-glucosidase is of many uses, has been applied to having obtained huge economic benefit in many industrial production such as food, chemical industry, biology.

At present; β-D-glucosidase activity detection method has following several kinds: the one, and Barush and Swiain method; With the saligenin is enzyme reaction substrate, and enzymolysis product is made developer with the 4-aminoantipyrene, makes the saligenol colour developing that discharges; Use the spectrophotometer colorimetric estimation again, perhaps measure the vigor that the reducing sugar amount that generates is calculated enzyme with DNS reagent; The 2nd, fluorescent method utilizes Umbelliferone (umbelliferone) and 4-methyl umbelliferone to have the characteristics of intense fluorescence, can calculate enzyme activity; The 3rd, colourimetry is that substrate carries out enzymolysis with right-nitrophenyl β-D-glucoside (pNPG), and the right-nitrophenols that discharges behind the substrate hydrolysis can be used the spectrophotometer colorimetric estimation.

At present, the glycosidase activity analyzing and testing is that the colourimetry of enzyme substrates is used the most extensively with general with p-NP-β-D-glucoside in mikrobe and the plant, and this method is simple to operate, and is highly sensitive fast, favorable reproducibility.

How above-mentioned measuring principle is passed through absorbing wavelength, buffer type, pH value, temperature of reaction and time etc. is optimized, and producing a kind of mechanism beta-glucoside enzyme activity determination test kit simple, easy to use is the problem that the technician will solve.

Summary of the invention

The technical issues that need to address of the present invention have provided a kind of activity of beta-glucosidase rapid determination test kit and detection method thereof, are intended to address the above problem.

A kind of activity of beta-glucosidase detection by quantitative test kit comprises R1, R2, R3 and R4 reagent, wherein R1 reagent: 100-300mmol/L sodium-acetate buffer pH5-6; R2 reagent: 10-20mmol/L p-nitrophenyl-β-D-glucoside (pNPG) aqueous solution; R3 reagent: 200-400mmol/L sodium carbonate solution; R4 reagent: 10-30mmol/L p-NP storing solution.Wherein said chromogenic agent is a p-NP; Described damping fluid is an acetate buffer solution.

The application of described activity of beta-glucosidase detection by quantitative test kit in detecting beta-glucosidase fermentation broth enzyme activity.

Wherein, utilize activity of beta-glucosidase detection by quantitative test kit to detect the activity of beta-glucosidase, the step that comprises is following:

1) specimen preparation:

Through the centrifugal 10～20min of 3000～5000rpm, the supernatant that is obtained is maybe will make with extra care the solid enzyme to pulverize constant volume with fermented liquid.

2) drafting of typical curve:

Pipette a certain amount of p-NP standard stock solution successively, be settled to 100-200mL, be made into the p-NP standard operation liquid of respective concentration with sodium carbonate solution; Absorption 1-3mL in test tube, adds the 1-3mL sodium-acetate buffer to the p-nitrophenol standardized solution; Add the 1-3mL sodium-acetate buffer in the blank test tube of contrast; In all test tubes, add the 5-10mL sodium carbonate solution, absorbancy OD value is measured in vibration at the 405nm place; The drawing standard graphic representation.

3) detection of sample

Add 20-40 μ L R1 damping fluid mixing and add 20-30 μ L to the fermented liquid for preparing or refining solid zymin sample solution and go into zero(ppm) water; Under 40-60 ℃, hatch; Add 20-40 μ L R2 reagent immediately; Under 40-60C, carry out enzymatic reaction, add the reagent 100-200 μ L R3 termination reaction of precooling at last, and reaction product is detected on ELIASA or spectrophotometer;

The U of unit of enzyme activity definition: under above-mentioned reaction conditions, the required enzyme amount of p-nitrophenol that PM discharges 1 μ mol is defined as a unit of enzyme activity;

Activity of beta-glucosidase in the sample (U/L)

In the formula:

Ax---the absorbancy OD value of sample enzyme liquid;

The blank absorbency OD value of Ao---corresponding enzyme liquid;

The slope of the typical curve of K---p-nitrophenol;

The intercept that the standard of Co---p-nitrophenol is bent;

Df---extension rate;

V---the long-pending L of enzyme liquid that reaction is added;

T---reaction times min;

X---the vigor of sample beta-glucosidase, U/L;

Said R1 reagent is acetate buffer solution, is used to the reaction conditions that reaction provides matters;

Said R2 reagent is p-nitrophenyl-β-D-glucoside (pNPG) aqueous solution, and the content of pNPG is 5-10mmol/L, as the substrate of beta-glucoside enzyme reaction; When being lower than the lower limit 5mmol/L of substrate, can cause the linearity range of test kit to reduce, absorbancy changes not obvious; When being higher than 10mmol/L; The cost of test kit is increased, do not meet the economic benefit of reagent react, in the 5-10mmol/L scope; Concentration of substrate is high more, and the speed that the PM absorbancy changes is fast more.

Said R3 reagent is sodium carbonate solution, is used to stop the enzymatic color producing reaction.

Said R4 reagent is the p-NP storing solution, is used for the drafting of typical curve.

Compared with prior art, outstanding technique effect is:

This test kit adopts spectrophotometry to detect the activity of beta-glucosidase in the solution, only adopts 4 kinds of liquid reagents, and it is subsequent use to can be used for 4 ℃ of preservations; Compare with the traditional hand method, it is accurate, reliable, simple to operate, easy to use to measure; The fast quantification that adopts rate method to be more suitable for β-D-glucuroide detects, and the kind of agents useful for same and consumption all obviously reduce, and significantly shorten detection time; Solved the problem of reagent stability; Reduce testing staff's labour intensity, improved detection efficiency, guaranteed the repeatability of experimental data.

The practical implementation method:

According to following embodiment, can better understand the present invention.Yet, those skilled in the art will readily understand that the concrete material proportion, processing condition and the result thereof that describe among the embodiment only explain the present invention, do not say the present invention who describes in detail and should can not limit in the claim genus yet.

Embodiment 1

A kind of activity of beta-glucosidase rapid quantitative detection reagent box in the present embodiment is three kinds of reagent, comprises R1 reagent, R2 reagent, and R3 reagent and R4 reagent, respectively by following composition and consumption configuration:

1) R1 reagent: pH5.5 sodium-acetate buffer 200mmol/L;

2) R2 reagent: p-nitrophenyl-β-D-glucoside (pNPG) aqueous solution 10mmol/L;

3) R3 reagent: sodium carbonate solution 250mmol/L;

4) R4 reagent: p-NP storing solution 10mmol/L;

After mentioned reagent is dissolved fully, divide the bottle of packing into, process liquid four reagent, directly use.

Embodiment 2

Utilize the reagent among the embodiment 1 to draw p-NP (pNP) typical curve, the concrete operations step is following:

1) pipettes R4 reagent p-NP standard stock solution 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 7 μ L, 9 μ L among the embodiment 1 successively; Be dissolved to 100 μ L with sodium carbonate solution, being made into concentration is the p-NP standard operation liquid of 0.1mmol/L, 0.2mmol/L, 0.3mmol/L, 0.4mmol/L, 0.5mmol/L, 0.7mmol/L, 0.9mmol/L.

2) absorption 20 μ L in the EP test tube, add 20 μ L sodium-acetate buffers to the p-nitrophenol standardized solution; Add 40 μ L sodium-acetate buffers in the blank pipe of contrast; In all EP pipes, add 160 μ L sodium carbonate solutions, mixing is measured absorbancy OD value at the 405nm place.

3) be that Y axle, absorbancy OD value are the X axle with p-NP concentration, the drawing standard curve.

Embodiment 3

Utilize the activity methods of beta-glucosidase in the test kit detection fermented liquid among the embodiment 1, the concrete operations step is following:

1) specimen preparation: get the beta-glucosidase fermented liquid through the centrifugal 15min of 3500rpm, the supernatant that is obtained promptly is;

2) getting the above-mentioned sample solution 25 μ L of 25 μ L adds in the aseptic 1.5ml EP pipe (blank replaces with zero(ppm) water);

3) add R1 reagent 25 μ L among the 25 μ L embodiment 1, the vibration mixing;

4) add 25 μ L zero(ppm) water, vibration mixing, and 50 ℃ of preincubate 5min;

5) the R2 reagent among the adding 25 μ L embodiment 1 in the enzyme liquid to be measured of hatching, the vibration mixing;

6) hatch 10min for 50 ℃;

7) the R3 reagent among the embodiment 1 of adding 100 μ L precoolings in the reaction solution of hatching, vibration mixing, termination reaction;

8) the 405nm place measures absorbancy OD value;

9) calculate the relative reactivity of enzyme according to gained p-NP typical curve among the embodiment 3 and according to formula

.

The U of unit of enzyme activity definition: under above-mentioned reaction conditions, the required enzyme amount of p-nitrophenol that often minute discharges 1 μ mol is defined as a unit of enzyme activity.

Embodiment 4

Utilize test kit among the embodiment 1 to detect the activity methods of pure enzyme solution beta-glucosidase, the concrete operations step is following:

1) specimen preparation: solid, purified enzyme sample should be pulverized or fully pulverize; Cross 60 mesh sieves (aperture is 0.25mm) then; Take by weighing about solid sample 0.05g; Fully dissolve with 80mL zero(ppm) water, be settled to 100mL (as estimating that enzymic activity is higher than 200U and can dilutes 10 times of detections), with Whatman No.1 filter paper filtering.Liquid sample can directly dilute and constant volume;

2) getting the above-mentioned sample solution 25 μ L of 25 μ L adds in the aseptic 1.5ml EP pipe (blank replaces with zero(ppm) water);

3) add R1 reagent 25 μ L among the 25 μ L embodiment 1, the vibration mixing;

4) add 25 μ L zero(ppm) water, vibration mixing, and 50 ℃ of preincubate 5min

5) the R2 reagent among the adding 25 μ L embodiment 1 in the enzyme liquid to be measured of hatching, the vibration mixing;

6) hatch 10min for 50 ℃

7) the R3 reagent among the embodiment 1 of adding 100 μ L precoolings in the reaction solution of hatching, vibration mixing, termination reaction;

8) the 405nm place measures absorbancy OD value;

9) calculate the relative reactivity of enzyme according to gained p-NP typical curve among the embodiment 3 and according to formula

.

The U of unit of enzyme activity definition: under above-mentioned reaction conditions, the required enzyme amount of p-nitrophenol that often minute discharges 1 μ mol is defined as a unit of enzyme activity.

Claims (3)

1. an activity of beta-glucosidase rapid quantitative detection reagent box is characterized in that, comprises R1, R2, R3, R4 reagent, and wherein R1 reagent is 100-300mmol/L sodium-acetate buffer pH5-6; R2 reagent is 10-20mmol/L p-nitrophenyl-β-D-glucoside (pNPG) aqueous solution; R3 reagent is the 200-400mmol/L sodium carbonate solution; R4 reagent is 10-30mmol/L p-NP storing solution.Wherein said chromogenic agent is a p-NP, and described damping fluid is an acetate buffer solution.

2. the application of the described activity of beta-glucosidase detection by quantitative of claim 1 test kit in detecting beta-glucosidase fermentation broth enzyme activity.

3. application according to claim 2 is characterized in that comprising the steps:

1) specimen preparation:

Through the centrifugal 10～20min of 3000～5000rpm, the supernatant that is obtained is maybe will make with extra care the solid enzyme to pulverize constant volume with fermented liquid.

2) drafting of typical curve:

Pipette a certain amount of p-NP standard stock solution successively, be settled to 100-200mL, be made into the p-NP standard operation liquid of respective concentration with sodium carbonate solution; Absorption 1-3mL in test tube, adds the 1-3mL sodium-acetate buffer to the p-nitrophenol standardized solution; Add the 1-3mL sodium-acetate buffer in the blank test tube of contrast; In all test tubes, add the 5-10mL sodium carbonate solution, absorbancy OD value is measured in vibration at the 405nm place; The drawing standard graphic representation.

3) detection of sample

Add 20-40 μ L R1 damping fluid mixing and add 20-30 μ L to the fermented liquid for preparing or refining solid zymin sample solution and go into zero(ppm) water; Under 40-60 ℃, hatch; Add 20-40 μ L R2 reagent immediately; Under 40-60 ℃, carry out enzymatic reaction, add the reagent 100-200 μ L R3 termination reaction of precooling at last, and reaction product is detected on ELIASA or spectrophotometer;

The U of unit of enzyme activity definition: under above-mentioned reaction conditions, the required enzyme amount of p-nitrophenol that PM discharges 1 μ mol is defined as a unit of enzyme activity;

Activity of beta-glucosidase in the sample (U/L)

In the formula:

Ax---the absorbancy OD value of sample enzyme liquid;

The blank absorbency OD value of Ao---corresponding enzyme liquid;

The slope of the typical curve of K---p-nitrophenol;

The intercept that the standard of Co---p-nitrophenol is bent;

Df---extension rate;

V---the long-pending L of enzyme liquid that reaction is added;

T---reaction times min;

X---the vigor of sample beta-glucosidase, U/L.