Method for detecting activity of exoglucanase of litters
阅读说明：本技术 一种检测凋落物外切葡聚糖酶活性的方法 (Method for detecting activity of exoglucanase of litters ) 是由 刘兵 李世杰 葛炎 李小丽 刘怡 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外切葡聚糖酶活性的方法,属于酶活检测技术领域。本发明提供的检测凋落物外切葡聚糖酶活性的方法包括：称取过筛的凋落物碎屑到离心过滤管中,加入培养缓冲液低温孵育,高速离心后将滤液混合并调节至一定体积；将待测酶液和灭活酶液分别加入黑色酶标板孔内,再加入荧光共轭纤维素二糖苷工作液,在避光条件下震荡孵育后,向各酶标孔加入Tris终止液；采用多功能酶标仪进行荧光检测；计算外切葡聚糖酶活性。本发明的优点为：优化了反应体系,反应酶液需求少,减少了其它酶活和人为操作干扰,灵敏度高,结果可靠,将反应体系置于酶标板内,避光震荡培养后添加终止液即可上机操作,批量获得结果数据,效率高。(The invention discloses a method for detecting the activity of exoglucanase of a litter, and belongs to the technical field of enzyme activity detection. The method for detecting the exoglucanase activity of the litter comprises the following steps: weighing the sieved litter fragments into a centrifugal filter tube, adding a culture buffer solution for low-temperature incubation, and mixing and adjusting the filtrate to a certain volume after high-speed centrifugation; respectively adding enzyme solution to be detected and inactivated enzyme solution into black enzyme label plate holes, adding fluorescent conjugated cellulose diglycoside working solution, performing shake incubation under the condition of keeping out of the sun, and adding Tris stop solution into each enzyme label hole; performing fluorescence detection by using a multifunctional microplate reader; the exoglucanase activity was calculated. The invention has the advantages that: the reaction system is optimized, the requirement of the reaction enzyme solution is low, the interference of other enzyme activities and manual operation is reduced, the sensitivity is high, the result is reliable, the reaction system is placed in an enzyme label plate, the reaction system can be operated on a computer by adding the stop solution after being subjected to light-proof oscillation culture, the result data can be obtained in batches, and the efficiency is high.)
1. A method for detecting the exoglucanase activity 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 obtain 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-cellulose diglycoside 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 exoglucanase activity of a litter according to 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 for 60min at 4 ℃, centrifuging for 30min at 16000 Xg at 4 ℃ to obtain enzyme filtrate, mixing, and adjusting the volume to 4mL by using the culture buffer solution to obtain enzyme activity detection solution;
2) absorbing 1mL of the enzyme activity solution to be detected prepared in the step 1), heating for 2h at 85 ℃ under the condition of keeping out of the sun, and cooling to room temperature to obtain 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 33.3 mu L of enzyme activity solution to be detected into the sample hole and adding 33.3 mu L of inactivated enzyme solution into the negative control hole on a black enzyme label plate; adding 600 mu M of fluorogenic substrate working solution, namely 600 mu M of 4-MUB-beta-D-cellulose diglycoside solution into the sample wells and the negative control wells according to 66.7 mu L of each well; after shaking incubation reaction at 160rpm for 30min under the condition of keeping out of light at 22 ℃, 100 mu L of 1M Tris stop solution with pH10-11 is rapidly added into all sample wells and negative control wells 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: (A)Activity device-AYin (kidney)) D/(b t m), specific activity unit pmol/min/g, AActivity deviceIs 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 exoglucanase activity of the litter according to claim 1 or 2, wherein the culture buffer in step 1) is prepared by: 6.05g Tris, 7.8g maleic acid, 7.0g citric acid or 7.65g citric acid monohydrate, 3.12g boric acid and 244mL 1M sodium hydroxide are subjected to volume fixing by deionized water to 500mL to obtain a universal buffer solution stock solution, the pH of the universal buffer solution stock solution is adjusted to 4.5 by hydrochloric acid, the volume of the universal buffer solution stock solution is fixed to 1000mL by deionized water, and the universal buffer solution stock solution is stored at 4 ℃ after sterilization to be used as a culture buffer solution for later use.
4. The method for detecting the exoglucanase activity of the litter according to claim 2, wherein the step 3) is specifically as follows: weighing 8.8mg of MUB, dissolving the MUB into 1mL of ethylene glycol monomethyl ether, adding 4mL of sterile deionized water, and uniformly mixing to prepare MUB mother solution; diluting 20 μ L of the MUB mother liquor obtained in the previous step with sterile deionized water to 20mL as a calibration solution, wherein the concentration of the calibration solution is 10 μ M, and storing the calibration solution in the dark at 4 ℃ for a week for later use; sequentially diluting the MUB calibration solution into MUB fluorescent standard substance solutions of 1 mu M, 2 mu M, 3 mu M, 4 mu M and 5 mu M by using sterile deionized water; 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 exoglucanase activity of the litter according to claim 2, wherein the 600 μ M4-MUB- β -D-cellulose diglycoside solution in step 4) is prepared by: weighing 17.2mg of 4-MUB-beta-D-cellulose diglycoside, dissolving into 2mL of ethylene glycol monomethyl ether, adding 8mL of sterile deionized water, and uniformly mixing to prepare 5mM 4-MUB-beta-D-cellulose diglycoside mother liquor; and adding sterile deionized water into the 4-MUB-beta-D-cellulose diglycoside mother liquor according to the proportion of 3: 22 for further dilution to obtain 600 mu M4-MUB-beta-D-cellulose diglycoside working solution, and freezing and storing at-20 ℃ for half a year for later use.
6. The method for detecting the exoglucanase activity of the litter according to claim 1 or 2, wherein the multifunctional microplate reader in step 3) or step 5) is set to have an excitation light wavelength of 364nm, an emission light wavelength of 450nm, a slip width of 5nm, and a collection time of 500 ms.
7. A method of litter exoglucanase activity according to claim 1 or 2, wherein the litter to be tested is prepared by: 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.
8. The litter exoglucanase activity method of claim 7, wherein the litter to be tested 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 enzyme activity detection, and particularly relates to a method for detecting the activity of exoglucanase of a litter.
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.
Exoglucanase (EC3.2.1.91), namely C1 enzyme, microcrystalline cellulose and cellobiohydrolase, can adsorb and act on a crystalline region of a cellulose molecule, and the structure of the cellulose molecule is destroyed from the end of the cellulose linear molecule, thereby being beneficial to the degradation of the endoglucanase and the beta-glucosidase. Therefore, the activity of the exoglucanase has important significance for degrading forest litters and soil organic matters and recycling nutrients, maintaining biochemical circulation and energy flow of a forest ecological system and improving soil fertility. The activity of the exoglucanase is sensitive to the change of environmental factors such as the quantity and the composition of litters, the physical and chemical properties of soil and the like, is a direct medium for soil microorganisms to act on the circulation of soil substances, and can be used as one of indexes for reflecting the activity of soil microorganism communities. Therefore, the method for detecting the activity of the exoglucanase, which has the advantages of rapid operation, high sensitivity, batch data reading and accurate result, is of great significance.
The determination of the exoglucanase activity of the litters in China is an improvement on the basis of a soil enzymology activity determination method, but the research on the determination of the exoglucanase activity of the litters in the forest has the problems of slow technical update, complicated method, easily interfered determination results by other cellulases and the like. The colorimetric method takes microcrystalline cellulose as a substrate, and an enzymolysis product is subjected to colorimetric determination after being developed by a DNS reagent, but the reaction is a synergistic effect result of endoglucanase, exoglucanase and beta-glucosidase, the activity of the exoglucanase cannot be truly reflected, and the reliability of the result is further reduced by the preparation time of the DNS developing reagent, the reaction intensity in a boiling water bath and the like; the colorimetric method for carrying out enzymolysis by taking p-nitrophenol cellobioside as a substrate is simple to operate, but inhibitors of other enzymes need to be added, the operation time is too long, too much enzyme solution is needed, and the inconvenience in application is increased. And because the original litter composition in the forest is complex: there may be surface litters with no or little colonization by degrading microorganisms, with little extracellular exoglucanase secretion by microorganisms, and there may be litters already at the end of degradation. The complex condition is not beneficial to the comparative study of the enzyme activity and the variation difference among various samples, so that the detection of the litter exoglucanase activity also needs more manual intervention than the soil enzyme activity determination in the early stage, so that the litter exoglucanase 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 exoglucanase activity of litter.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for detecting the exoglucanase activity 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 exoglucanase activity of 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 a part of the liquid to be tested for enzyme activity prepared in the step 1) to inactivate the enzyme to obtain an inactivated enzyme liquid;
3) preparing 4-Methylumbelliferone (MUB) fluorescence 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-cellulose diglycoside 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 exoglucanase 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 for 60min at 4 ℃, centrifuging for 30min at 16000 Xg at 4 ℃ to obtain enzyme filtrate, mixing, and adjusting the volume to 4mL by using the culture buffer solution to obtain enzyme activity detection solution;
2) absorbing 1mL of the enzyme activity solution to be detected prepared in the step 1), heating for 2h at 85 ℃ under the condition of keeping out of the sun, and cooling to room temperature to obtain 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 33.3 mu L of enzyme activity solution to be detected into the sample hole on a black enzyme label plate, and then adding 33.3 mu L of inactivated enzyme solution into the negative control hole; adding a 600 mu M fluorogenic substrate working solution, namely a 600 mu M4-MUB-beta-D-cellulose diglycoside solution into the sample wells and the negative control wells according to 66.7 mu L per well; after shaking incubation reaction at 160rpm for 30min under the condition of keeping out of light at 22 ℃, 100 mu L of 1M Tris stop solution with pH10-11 is rapidly added into all sample wells and negative control wells 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: (A)Activity device-AYin (kidney)) D/(b t m), specific activity unit pmol/min/g, AActivity deviceIs 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: 6.05g Tris, 7.8g maleic acid, 7.0g citric acid or 7.65g citric acid monohydrate, 3.12g boric acid and 244mL 1M sodium hydroxide are subjected to volume fixing by deionized water to 500mL to obtain a universal buffer solution stock solution, the pH of the universal buffer solution stock solution is adjusted to 4.5 by hydrochloric acid, the volume of the universal buffer solution stock solution is fixed to 1000mL by deionized water, and the universal buffer solution stock solution is stored at 4 ℃ after sterilization to be used as a culture buffer solution for later use.
Preferably, step 3) is specifically: weighing 8.8mg of MUB, dissolving the MUB into 1mL of ethylene glycol monomethyl ether, adding 4mL of sterile deionized water, and uniformly mixing to prepare MUB mother solution; diluting 20 μ L of the MUB mother liquor obtained in the previous step with sterile deionized water to 20mL as a calibration solution, wherein the concentration of the calibration solution is 10 μ M, and storing the calibration solution in the dark at 4 ℃ for a week for later use; sequentially diluting a proper amount of MUB calibration solution into MUB fluorescent standard substance solutions of 1 mu M, 2 mu M, 3 mu M, 4 mu M and 5 mu M by using sterile deionized water; 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 600 μ M4-MUB- β -D-cellobioside solution in step 4) is prepared by: weighing 17.2mg of 4-MUB-beta-D-cellulose diglycoside, dissolving into 2mL of ethylene glycol monomethyl ether, adding 8mL of sterile deionized water, and uniformly mixing to prepare 5mM 4-MUB-beta-D-cellulose diglycoside mother liquor; and adding sterile deionized water into the 4-MUB-beta-D-cellulose diglycoside mother liquor according to the proportion of 3: 22 for further dilution to obtain 600 mu M4-MUB-beta-D-cellulose diglycoside working solution, and freezing and storing at-20 ℃ for half a year for later use.
Preferably, the multifunctional microplate reader in step 3) or step 5) is set to have an excitation light wave length of 364nm, an emission light wave length of 450nm, a sliding width of 5nm, and a collection time of 500 ms.
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.
Compared with the traditional analysis method, the method has the main advantages that:
1) the method comprises the steps of adopting 4-Methylumbelliferone (MUB) conjugate cellulose diglycoside (4-MUB-beta-D-cellulose diglycoside) as an enzymolysis substrate, carrying out enzymolysis through exoglucanase to release fluorescence, collecting change of MUB fluorescence intensity through a multifunctional enzyme-labeling instrument, and calculating enzyme activity;
2) compared with the traditional method, the method has the advantages of optimized reaction system, less reaction enzyme liquid, consistent reaction conditions, high sensitivity, simple operation and the like, and can be operated in batches, so that the enzyme activity testing efficiency is greatly improved;
3) the adoption of a centrifugal filter tube with a micro-aperture greatly reduces interfering substances in enzyme liquid, reduces other enzyme activity and manual operation interference, and has high sensitivity and reliable result;
4) placing the reaction system in a hole of an enzyme label plate, performing light-shielding oscillation culture, adding a stop solution, and performing machine operation, so that result data can be obtained in batches in a short time, 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, so that the detection results of the exoglucanase activity of the litter of different samples have the possibility of mutual reference and comparison, and the influence of the litter enzyme on the micro-ecological process of forest litter decomposition is favorably researched.
Drawings
FIG. 1 is a schematic diagram of an ELISA plate structure and sample application for detecting the activity of the litter exoglucanase; s1 is 33.3 mu L of
Detailed Description
The invention is further described with reference to specific examples.