Method for detecting activity of beta-glucosidase of litter
阅读说明：本技术 一种检测凋落物β-葡萄糖苷酶活性的方法 (Method for detecting activity of beta-glucosidase of litter ) 是由 刘兵 刘怡 徐杰 耿莉 葛炎 孙辉 于 2020-01-19 设计创作，主要内容包括：本发明公开了一种检测凋落物β-葡萄糖苷酶活性的方法,属于生物化学技术领域。本发明提供的检测凋落物中β-葡萄糖苷酶活性的方法主要步骤为：称取剪碎过筛的凋落物到离心过滤管中,加入培养缓冲液进行孵育,高速离心后混合上清液并调节至一定体积作为酶活待测液；将酶活待测液和灭活的酶活待测液分别加入样品活性孔和阴性对照孔,再加入荧光底物工作液4-MUB-β-D-葡萄糖苷溶液,在避光条件下震荡孵育；再向酶标孔加入Tris终止液；采用多功能酶标仪进行荧光检测；计算β-葡萄糖苷酶活性。本发明的优点为：反应体系优化后,灵敏度高,检测消耗待测酶液的量少,缩短了操作时间,短时间内能大批量获得测定数据,操作简单,效率高。(The invention discloses a method for detecting the activity of beta-glucosidase of a litter, and belongs to the technical field of biochemistry. The method for detecting the activity of the beta-glucosidase in the litter mainly comprises the following steps: weighing the litters which are cut and sieved into pieces and put into a centrifugal filter tube, adding a culture buffer solution for incubation, centrifuging at a high speed, mixing a supernatant, and adjusting to a certain volume to serve as a solution to be tested for enzyme activity; adding the enzyme activity solution to be detected and the inactivated enzyme activity solution to be detected into the sample active hole and the negative control hole respectively, adding the fluorescent substrate working solution 4-MUB-beta-D-glucoside solution, and performing shake incubation under the condition of keeping out of the sun; adding Tris stop solution into the enzyme-labeled hole; performing fluorescence detection by using a multifunctional microplate reader; the beta-glucosidase activity was calculated. The invention has the advantages that: after the reaction system is optimized, the sensitivity is high, the amount of the enzyme solution to be detected is less in detection consumption, the operation time is shortened, the measurement data can be obtained in a large scale in a short time, the operation is simple, and the efficiency is high.)
1. A method for detecting the activity of beta-glucosidase of a litter is characterized by comprising the following steps:
1) adding the culture buffer solution into the sheared and sieved litter to be detected, centrifuging the litter in a centrifugal filter tube to obtain enzyme filtrate, and further diluting the enzyme filtrate to prepare enzyme activity detection solution;
2) heating and inactivating a part of the enzyme activity solution to be detected prepared in the step 1) to prepare an inactivated enzyme solution;
3) preparing MUB fluorescent standard substance solution with gradient concentration, and performing fluorescence detection by using a multifunctional microplate reader;
4) adding enzyme activity to-be-detected liquid and inactivated enzyme liquid into enzyme labeling holes of an enzyme labeling plate respectively, adding a fluorescent substrate working solution containing 4-MUB-beta-D-glucoside for reaction respectively, and adding a stop solution to stop the reaction;
5) performing fluorescence detection on the reaction result of the step 4) by using a multifunctional microplate reader;
6) drawing a fluorescence standard curve by using the gradient concentration of the MUB fluorescence standard substance solution and the corresponding measured light absorption value A to obtain a standard curve slope b;
7) calculating a dilution factor of the enzyme activity solution to be detected;
8) and (4) calculating the specific enzyme activity of the enzyme activity to-be-detected solution.
2. The method for detecting the activity of litter beta-glucosidase of claim 1, comprising the steps of:
1) shearing the litters to be detected, sieving the litters with a 2mm sieve, weighing 3 samples, adding 0.15g of the litters to be detected into 3 centrifugal filter tubes, adding 600 mu L of culture buffer solution with the pH value of 4.5, incubating the samples at 4 ℃ for 1h, centrifuging the samples at 16000 Xg for 30min at 4 ℃ to obtain enzyme filtrate, mixing the enzyme filtrate and the enzyme filtrate, and adjusting the volume of the enzyme filtrate to 4mL by using the culture buffer solution to obtain enzyme activity detection solution;
2) sucking 1mL of the liquid to be detected with the enzyme activity prepared in the step 1), putting the liquid into a 2mL centrifuge tube, heating the liquid at 85 ℃ for 2h under the condition of keeping out of the sun to inactivate the enzyme liquid fully, and cooling the sample to room temperature;
3) preparing MUB fluorescent standard substance solution with gradient concentration, and performing fluorescence detection by using a multifunctional microplate reader;
4) adding 33.3 mu L of enzyme activity solution to be detected into the sample enzyme-labeled hole on a black enzyme-labeled plate; adding 33.3 mu L of inactivated enzyme solution into a negative control enzyme labeling hole; adding 750 mu M of fluorogenic substrate working solution, namely 4-MUB-beta-D-glucoside solution into the sample enzyme-labeled hole and the negative control enzyme-labeled hole according to 66.7 mu L of each hole; shaking and incubating at 160rpm for 15min under the condition of keeping out of light and 22 ℃, and quickly adding 1M Tris stop solution of 100 mu LpH10-11 into all enzyme-labeled holes to stop reaction;
5) performing fluorescence detection on the reaction result of the step 4) by using a multifunctional microplate reader;
6) drawing a fluorescence standard curve by using the gradient concentration of the MUB fluorescence standard substance solution and the corresponding measured light absorption value A to obtain a standard curve slope b;
7) calculating the dilution factor D of the sample to be detected (200 muL 4mL)/(33.3 muL X), wherein 200 muL is the volume of 100 muL stop solution added into 100 muL reaction solution, 4mL is the volume of enzyme activity detection solution after the mixed enzyme filtrate is adjusted by culture buffer solution, 33.3 muL is the volume of enzyme activity detection solution added into 100 muL reaction solution, and X is the volume of mixed enzyme filtrate;
8) calculating the specific enzyme activity of the sample to be detected: (As-A)Yin (kidney)) D/(b t m), the unit of specific enzyme activity is pmol/min/g, As is the light absorption value of the sample enzyme activity reaction liquid, AYin (kidney)The absorbance value of the sample corresponding to the inactivated enzyme solution is shown, D is a dilution factor, b is the slope of a standard curve, t is the oscillation incubation time of 15min, and m is the dry weight of the litter of 3 centrifugal filter tubes, and the unit is g.
3. The method for detecting the activity of the litter beta-glucosidase in claim 1 or 2, wherein the culture buffer in step 1) is prepared by: preparing a universal buffer stock solution by using 3.025g of Tris, 3.9g of maleic acid, 3.5g of citric acid or 3.825g of citric acid monohydrate, 1.56g of boric acid and 122mL of 1M sodium hydroxide and performing volume fixing to 250mL by using deionized water; and then adjusting the pH of 100mL of the universal buffer solution stock solution to 4.5 by using hydrochloric acid, diluting the solution to 1000mL by using deionized water, and sterilizing the solution at 4 ℃ for later use.
4. The method for detecting the activity of the litter beta-glucosidase in claim 2, wherein the step 3) is specifically as follows: weighing 8.8mg of MUB, dissolving in 1mL of 2-methoxyethanol and 4mL of sterile deionized water, and uniformly mixing to prepare MUB mother liquor; adding 20 mu L of MUB mother liquor into 20mL of sterile deionized water for further dilution to obtain 10 mu M of MUB calibration solution, and storing for no more than one week at the temperature of 4-6 ℃ in a dark place for later use; adding 10 mu M MUB calibration solution into sterile deionized water to sequentially dilute the calibration solution into 1 mu M, 2 mu M, 3 mu M, 4 mu M and 5 mu M MUB fluorescent standard substance solution; on a black enzyme label plate, 100 muL of MUB fluorescent standard substance solution with the concentration of 0 muM, 1 muM, 2 muM, 3 muM, 4 muM and 5 muM is added in sequence, repeated for 4 times, and then 100 muL of 1M Tris stop solution with the pH value of 10-11 is added respectively, and then fluorescence detection is carried out by adopting a multifunctional enzyme label instrument.
5. The method for detecting the activity of the litter beta-glucosidase of claim 2, wherein the enzyme activity test solution in step 4) is added to the sample enzyme-labeled well for 3 times; and adding a negative control enzyme labeling hole into the inactivated enzyme solution, wherein the number of the added negative control enzyme labeling holes is 1 sample and 1 hole.
6. The method for detecting the activity of litter beta-glucosidase of claim 2 or 4, wherein the 1M Tris stop solution with pH of 10-11 is prepared by the following steps: 129 g of Tris is added into 750mL of deionized water, the volume is increased to 1000mL by using the deionized water, and the Tris is stored at room temperature for standby after autoclaving.
7. The method for detecting the activity of the litter beta-glucosidase in claim 1 or 2, wherein the fluorescence detection in step 5) is specifically: the excitation light wave wavelength is 364nm, the emission light wave wavelength is 450nm, the sliding width is 5nm, the collection time is 500ms, and the measured corresponding light absorption value is A.
8. The method for detecting the activity of the beta-glucosidase of the litter according to claim 1 or 2, wherein the litter to be detected is prepared by the following steps: collecting dead branches or fallen leaves forming forest stand characteristic tree species in a forest, putting the dead branches or fallen leaves into a nylon decomposition bag after water removing and drying, burying the nylon decomposition bag in the forest, covering the nylon decomposition bag with 0-10cm of soil on the original surface layer, slightly compacting the nylon decomposition bag, burying the nylon decomposition bag for a preset time, taking out the half-degraded dead branches or fallen leaves, and removing sediment impurities to obtain the litters to be detected.
9. The method for detecting the activity of litter beta-glucosidase of claim 8, wherein the litter to be detected is prepared by: collecting dead branches with the diameter of 5mm forming forest stand characteristic tree species in a forest, deactivating enzyme at 105 ℃ for 15min, continuously drying at 80 ℃ for 48h, weighing 7-10 g, putting into a 300-mesh 10cm x 8cm nylon decomposition bag, burying the nylon decomposition bag in the forest, namely covering with soil 0-10cm away from the tree trunk 1m and lightly compacting, burying for a preset time, taking out the semi-degraded dead branches, and removing silt impurities to obtain the litters to be detected.
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a method for detecting activity of litter beta-glucosidase.
Background
The decomposition of litters is the basis of forest soil substance transformation, is a main source of plant and microorganism nutrients, and plays an important role in maintaining the carbon and nutrient circulation of a forest ecological system. The microorganisms are main contributors to the decomposition of the forest litter, and in the process of litter degradation, the microorganisms colonized on the litter secrete various extracellular enzymes to change the composition structure of the litter, so that the degradation of the litter is promoted. The enzyme activity is related to various biochemical processes as the most important biological active substances participating in litter decomposition, is directly related to litter decomposition, reflects nutrient circulation conditions such as soil C, N, P to a great extent, and is different according to litter types and environmental conditions. The research on the activity dynamics of various enzyme activities in the litters decomposition process has important significance for understanding the micro ecological process of the forest litters.
Beta-glucosidase (EC3.2.1.21), cellobiase, is responsible for hydrolyzing cellobiose, cellotriose and water-soluble cellodextrins in litter and converting them to glucose. Although the beta-glucosidase has no direct effect on the cellulose, the beta-glucosidase can eliminate the feedback inhibition effect on the final products of the reaction of exoglucanase and endoglucanase and is a key rate-limiting enzyme in the cellulose degradation process. Due to the fact that cellulose in the litters is degraded and has a certain relevance with the formation of soil humus, the activity of the beta-glucosidase can reflect the biochemical strength and dynamic process in the soil to a certain extent, and the beta-glucosidase is a reliable index for evaluating the soil fertility. Meanwhile, the decomposition and release of soil microorganisms are the main source of the beta-glucosidase, the connection and the action of the beta-glucosidase activity and the beta-glucosidase activity can not be ignored in the study of soil microbial community characteristics, and the enzyme activity can be used as an index for more stable and sensitive microbial activity.
The determination of the beta-glucosidase activity of the litter in China is an extension on the basis of a soil enzymology method, but the research on the determination of the beta-glucosidase activity of the litter in the forest has the problems of single extensive method, derailment with an international advanced method, slow replacement of experimental equipment, replacement of the beta-glucosidase activity by the total enzyme activity of cellulase and the like. Wherein, the Barush and Swiain methods which take salicin as a substrate, 4-aminoantipyrine is used as a color developing agent for an enzymolysis product, and the colorimetric determination is carried out by a spectrophotometry, but the reaction is easy to be interfered, and the detection sensitivity is low; the colorimetric method for enzymolysis by taking the nitrophenyl-beta-glucoside as the substrate is generally applied, and although the method is simple to operate and good in reproducibility, the method is easy to interfere, too much enzyme liquid is consumed, and the whole time consumption is too long. And because the original litter composition in the forest is complex: there may be surface litters with no or very little colonization by degrading microorganisms, with little extracellular β -glucosidase secretion by microorganisms, and there may be litters already at the end of degradation. This complex situation is not favorable for comparative study of enzyme activity and variation difference among various samples, so the detection of the litter enzyme activity requires more manual intervention than the soil enzyme activity measurement in the early stage, so that the litter enzyme activity is standardized, and the study of the litter degradation process is facilitated.
In summary, there is a need to provide a new, standardized, rapid and convenient method for detecting the activity of beta-glucosidase in litter.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is a method for detecting the activity of beta-glucosidase of a litter.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for detecting the activity of beta-glucosidase of a litter comprises the following steps:
1) adding the culture buffer solution into the sheared and sieved litter to be detected, centrifuging the litter in a centrifugal filter tube to obtain enzyme filtrate, and further diluting the enzyme filtrate to prepare enzyme activity detection solution;
2) heating and inactivating a part of the enzyme activity solution to be detected prepared in the step 1) to prepare an inactivated enzyme solution;
3) preparing MUB fluorescent standard substance solution with gradient concentration, and performing fluorescence detection by using a multifunctional microplate reader;
4) adding enzyme activity to-be-detected liquid and inactivated enzyme liquid into enzyme labeling holes of an enzyme labeling plate respectively, adding a fluorescent substrate working solution containing 4-MUB-beta-D-glucoside for reaction respectively, and adding a stop solution to stop the reaction;
5) performing fluorescence detection on the reaction result of the step 4) by using a multifunctional microplate reader;
6) drawing a fluorescence standard curve by using the gradient concentration of the MUB fluorescence standard substance solution and the corresponding measured light absorption value A to obtain a standard curve slope b;
7) calculating a dilution factor of the enzyme activity solution to be detected;
8) and (4) calculating the specific enzyme activity of the enzyme activity to-be-detected solution.
Preferably, the method for detecting the activity of the litter beta-glucosidase comprises the following steps:
1) shearing the litters to be detected, sieving the litters with a 2mm sieve, weighing 3 samples, adding 0.15g of the litters to be detected into 3 centrifugal filter tubes, adding 600 mu L of culture buffer solution with the pH value of 4.5, incubating the samples at 4 ℃ for 1h, centrifuging the samples at 16000 Xg for 30min at 4 ℃ to obtain enzyme filtrate, mixing the enzyme filtrate and the enzyme filtrate, and adjusting the volume of the enzyme filtrate to 4mL by using the culture buffer solution to obtain enzyme activity detection solution;
2) sucking 1mL of the liquid to be detected with the enzyme activity prepared in the step 1), putting the liquid into a 2mL centrifuge tube, heating the liquid at 85 ℃ for 2h under the condition of keeping out of the sun to inactivate the enzyme liquid fully, and cooling the sample to room temperature;
3) preparing MUB fluorescent standard substance solution with gradient concentration, and performing fluorescence detection by using a multifunctional microplate reader;
4) adding 33.3 mu L of enzyme activity solution to be detected into the sample enzyme-labeled hole on a black enzyme-labeled plate; adding 33.3 mu L of inactivated enzyme solution into a negative control enzyme labeling hole; adding 750 mu M of fluorogenic substrate working solution, namely 4-MUB-beta-D-glucoside solution into the sample enzyme-labeled hole and the negative control enzyme-labeled hole according to 66.7 mu L of each hole; shaking and incubating at 160rpm for 15min under the condition of keeping out of light and 22 ℃, and quickly adding 100 mu L of 1M Tris stop solution with the pH value of 10-11 into all enzyme-labeled holes to terminate the reaction;
5) performing fluorescence detection on the reaction result of the step 4) by using a multifunctional microplate reader;
6) drawing a fluorescence standard curve by using the gradient concentration of the MUB fluorescence standard substance solution and the corresponding measured light absorption value A to obtain a standard curve slope b;
7) calculating the dilution factor D of the sample to be detected (200 muL 4mL)/(33.3 muL X), wherein 200 muL is the volume of 100 muL stop solution added into 100 muL reaction solution, 4mL is the volume of enzyme activity detection solution after the mixed enzyme filtrate is adjusted by culture buffer solution, 33.3 muL is the volume of enzyme activity detection solution added into 100 muL reaction solution, and X is the volume of mixed enzyme filtrate;
8) calculating the specific enzyme activity of the sample to be detected: (As-A)Yin (kidney)) D/(b t m), the unit of specific enzyme activity is pmol/min/g, As is the light absorption value of the sample enzyme activity reaction liquid, AYin (kidney)The absorbance value of the sample corresponding to the inactivated enzyme solution is shown, D is a dilution factor, b is the slope of a standard curve, t is the oscillation incubation time of 15min, and m is the dry weight of the litter of 3 centrifugal filter tubes, and the unit is g.
Preferably, the culture buffer solution in step 1) is prepared by the following steps: preparing a universal buffer stock solution by using 3.025g of Tris, 3.9g of maleic acid, 3.5g of citric acid or 3.825g of citric acid monohydrate, 1.56g of boric acid and 122mL of 1M sodium hydroxide and performing volume fixing to 250mL by using deionized water; and then adjusting the pH of 100mL of the universal buffer solution stock solution to 4.5 by using hydrochloric acid, diluting the solution to 1000mL by using deionized water, and sterilizing the solution at 4 ℃ for later use.
Preferably, the step 3) is specifically as follows: weighing 8.8mg of MUB, dissolving in 1mL of 2-methoxyethanol and 4mL of sterile deionized water, and uniformly mixing to prepare MUB mother liquor; adding 20 mu L of MUB mother liquor into 20mL of sterile deionized water for further dilution to obtain 10 mu M of MUB calibration solution, and storing for no more than one week at the temperature of 4-6 ℃ in a dark place for later use; adding 10 mu M MUB calibration solution into sterile deionized water to sequentially dilute the calibration solution into 1 mu M, 2 mu M, 3 mu M, 4 mu M and 5 mu M MUB fluorescent standard substance solution; and sequentially adding 100 mu L of MUB fluorescent standard substance solutions with the concentrations of 0 mu M, 1 mu M, 2 mu M, 3 mu M, 4 mu M and 5 mu M to a black enzyme label plate, repeating for 4 times, respectively adding 100 mu LpH10-11 of 1M Tris stop solution, and then carrying out fluorescence detection by adopting a multifunctional enzyme label instrument.
Preferably, the enzyme activity solution to be detected in the step 4) is added into the sample enzyme-labeled hole, and the process is repeated for 3 times; and adding a negative control enzyme labeling hole into the inactivated enzyme solution, wherein the number of the added negative control enzyme labeling holes is 1 sample and 1 hole.
Preferably, the 1M Tris stop solution with the pH value of 10-11 is prepared by the following steps: 129 g of Tris is added into 750mL of deionized water, the volume is increased to 1000mL by using the deionized water, and the Tris is stored at room temperature for standby after autoclaving.
Preferably, the fluorescence detection in step 5) is specifically: the excitation light wave wavelength is 364nm, the emission light wave wavelength is 450nm, the sliding width is 5nm, the collection time is 500ms, and the measured corresponding light absorption value is A.
Preferably, the preparation method of the litter to be detected comprises the following steps: collecting dead branches or fallen leaves forming forest stand characteristic tree species in a forest, putting the dead branches or fallen leaves into a nylon decomposition bag after water removing and drying, burying the nylon decomposition bag in the forest, covering the nylon decomposition bag with 0-10cm of soil on the original surface layer, slightly compacting the nylon decomposition bag, burying the nylon decomposition bag for a preset time, taking out the half-degraded dead branches or fallen leaves, and removing sediment impurities to obtain the litters to be detected.
Preferably, the preparation method of the litter to be detected comprises the following steps: collecting dead branches with the diameter of 5mm forming forest stand characteristic tree species in a forest, deactivating enzyme at 105 ℃ for 15min, continuously drying at 80 ℃ for 48h, weighing 7-10 g, putting into a 300-mesh 10cm x 8cm nylon decomposition bag, burying the nylon decomposition bag in the forest, namely covering with soil 0-10cm away from the tree trunk 1m and lightly compacting, burying for a preset time, taking out the semi-degraded dead branches, and removing silt impurities to obtain the litters to be detected.
Has the advantages that: compared with the prior art, the invention has the advantages that:
1) according to the powerful analysis means of the fluorescence analysis technology, the fluorescence conjugate is used as an enzymolysis substrate, 4-MUB-beta-D-glucoside is subjected to enzymolysis by beta-glucosidase to release a fluorescence probe 4-MUB, and the activity of the enzyme is reflected by collecting the change of the fluorescence intensity of the 4-MUB, so that the interference of other substances in the enzyme solution to be detected is greatly reduced;
2) the micro-aperture centrifugal filter tube is adopted, so that interfering substances in the enzyme solution are greatly reduced, a reaction system is optimized, the detection method has high sensitivity and low demand on the enzyme solution to be detected, and the residual enzyme solution can be used for other enzyme activity determination purposes;
3) the enzyme solution to be detected and the substrate are placed in the enzyme labeling hole of the enzyme labeling plate together, after culture, the enzyme solution and the substrate are directly added into the stop solution and then are put on the computer for detection, so that the operation time is shortened, the measurement data can be obtained in a large scale in a short time, the operation is simple, and the efficiency is high.
5) The preparation method of the litter to be detected is provided, so that main factors influencing the enzyme activity of the litter, including litter type, environmental conditions, time and the like, are controlled, and thus detection results of the beta-glucosidase activity of the litter of different samples have the possibility of mutual reference and comparison, and the method is favorable for researching the influence of the beta-glucosidase on the micro-ecological process of the forest litter decomposition.
Drawings
FIG. 1 is a schematic diagram of an ELISA plate structure for detecting the activity of litter beta-glucosidase and a sample adding; s1 is 33.3 muL of
Detailed Description
The invention is further described with reference to specific examples.
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