CN109085230B - Zymogram method for identifying xylanase protein band and activity - Google Patents
Zymogram method for identifying xylanase protein band and activity Download PDFInfo
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Abstract
The invention discloses an zymogram method for identifying xylanase protein bands and activity, which comprises the following steps: preparing a xylanase liquid sample to be measured; preparing separation gel in a vertical electrophoresis tank, and preparing a concentrated gel layer with a sample tank on the upper layer of the separation gel; after sampling in a sample tank for concentrating gel, applying voltage to the vertical electrophoresis tank, and running gel; cutting the glue of the lane where the sample is located into strips by using a glue cutting plate, removing the concentrated glue, and reserving the separation glue to obtain strip separation glue containing the sample; putting the strip-shaped separation gel containing the sample into a sodium acetate solution, and incubating; and (5) closely sticking the incubated strip-shaped separation gel containing the sample on the surface of the substrate gel in the culture dish, placing the culture dish in a biochemical incubator at the temperature of 30-50 ℃, and culturing for 1-36 hours until clear strips and transparent circles appear. The method is simple to operate, and the xylanase protein band and activity can be identified simultaneously by using the same glue.
Description
Technical Field
The invention relates to the technical field of detection, in particular to an zymogram method for identifying xylanase protein bands and activity.
Background
Xylan is a plant cell wall polysaccharide, accounts for about 15-35% of plant cell dry matter, is the main component of plant hemicellulose, widely exists in agricultural byproducts such as straws, wheat bran, corncobs and the like, and is very rich in content. The xylanase can decompose xylan into xylo-oligosaccharide and xylose with different lengths, so that the resources are fully utilized, and the potential application value of the xylanase is exerted. The xylanase is widely applied to the fields of feed, food, paper making, energy and the like, and brings great economic benefit.
At present, xylanase production mainly comes from microbial fermentation, including microorganisms secreting xylanase in nature and engineering bacteria containing recombinant xylanase gene constructed by using genetic engineering technology (Chenhoniang, etc., research progress of xylanase, brewing in China, 2016 (11): 1-5). In the process of producing xylanase by using microorganisms, the characteristics, activity, position in non-denatured polyacrylamide gel electrophoresis gel (Native-Page), existence of isozyme and the like of xylanase are required to be identified so as to purify the xylanase, and zymogram technology is required. The zymogram technology is a method for analyzing enzyme and isoenzyme thereof by utilizing electrophoresis technology combined with specific enzymatic chemical reaction color development. The enzyme protein molecules are separated by electrophoresis according to their sizes and charged amounts, and the enzyme protein molecules are subjected to a different method of determination after their characteristic electrophoretic bands (enzyme spectrum) are developed by a different method using the catalytic activity of the enzyme, thereby obtaining a model (Kogaku, the French dictionary: chemical industry Press, 2009). For xylanase, zymography technology has two main purposes, namely, identifying whether active xylanase exists in a sample or not and where a protein band is; secondly, the presence or absence of an isozyme of xylanase in the sample. At present, a method combining Native-Page with Congo red staining (Ninave et al, Purification and simulation of extracellular xylanase from Streptomyces cyaneus SN32, Bioresource Technology, 2008, 99: 1252-.
Therefore, the search for a xylanase zymogram identification method which is simple and easy to operate is a problem to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of the above, the present invention provides an zymogram method for identifying xylanase protein bands and activities, which is simple to operate and color by using the zymogram method, and can identify the xylanase protein bands and activities by using the same glue.
In order to achieve the purpose, the invention adopts the following technical scheme: an zymogram method for identifying protein bands and activities of xylanase, which is characterized by comprising the following steps:
preparing a xylanase liquid electrophoresis sample to be measured;
preparing a separation glue layer, and preparing a concentrated glue layer with a sample groove on the upper layer of the separation glue layer;
putting the glue prepared in the step (2) in a vertical electrophoresis tank, adding a Tris-glycine buffer solution, carrying out sample application in a sample tank of a concentrated glue layer, applying voltage to the vertical electrophoresis tank, and running the glue until a bromophenol blue strip in a xylanase liquid sample runs to the bottom layer of the vertical electrophoresis tank;
cutting the glue of a lane where the xylanase liquid sample is located into strips by using a glue cutting plate, removing a concentrated glue layer, and reserving a separation glue layer to obtain strip separation glue containing the sample; putting the strip-shaped separation gel containing the sample into a sodium acetate solution, and incubating;
step (5) closely attaching the strip-shaped separation gel containing the incubated sample to the surface of the substrate gel of a culture dish, placing the culture dish in a biochemical incubator at 30-50 ℃ and culturing for 1-36 hours until clear strips and transparent circles appear; the substrate glue is prepared from remainders brilliant blue R cross-linked xylan, agar powder and a sodium acetate buffer solution with the pH value of 2.0-8.0.
The invention has the beneficial effects that: according to the identification method provided by the invention, the substrate glue prepared by adopting remainders brilliant blue R cross-linked xylan as a main raw material and the strip-shaped separation glue containing the sample are cultured in the incubator, so that the position of xylanase and activity in the same glue can be identified in the same time, and the steps are simple and easy to operate.
Preferably, the preparation step of the substrate glue in the step (5): preparing a substrate glue: weighing 10-100 mg of remainders brilliant blue R cross-linked xylan and 0.15-0.6 g of agar powder, putting the remainders brilliant blue R cross-linked xylan into a sodium acetate buffer solution with the pH value of 2.0-8.0, heating and dissolving the mixture on an electric furnace, cooling the mixture to 30-50 ℃, pouring the mixture into a culture dish, putting the culture dish on a table top, and cooling and solidifying the mixture to obtain the substrate glue. The thickness of the substrate glue is preferably 2-8 mm.
Preferably, the xylanase liquid electrophoresis sample in step (1) comprises xylanase liquid and loading buffer. The loading buffer solution comprises 0.5M Tris-HCl buffer solution, glycerol, 0.5% bromophenol blue and distilled water.
Preferably, the preparation of the loading buffer: 1.25 ml of 0.5M Tris-HCl buffer solution, 3 ml of glycerol, 2 ml of 0.5% bromophenol blue and 5.5 ml of distilled water are mixed uniformly to obtain the loading buffer solution.
Preferably, the concentration of the xylanase liquid is 30-150 micrograms/ml, and the volume ratio of the xylanase liquid to the loading buffer solution is 1: 1-2: 1 and mixing.
Preferably, the separation gel in step (2) is made of distilled water, 30% acrylamide, 1.5% MTris-HCl buffer, 10% ammonium persulfate and tetraethyl ethylenediamine; the volume ratio of the distilled water, 30% of acrylamide, 1.5% of MTris-Hcl buffer solution, 10% of ammonium persulfate and tetraethyl ethylenediamine stock solution is (1.60-1.70): (1.8-2.1): (1.2-1.4): (0.02-0.07): (0.003-0.005).
Preferably, the concentrated gel in step (2) is made of distilled water, 30% acrylamide, 0.5% MTris-HCl buffer, 10% ammonium persulfate and tetraethyl ethylenediamine; the volume ratio of the distilled water, 30% of acrylamide, 0.5% of MTris-Hcl buffer solution, 10% of ammonium persulfate and tetraethyl ethylenediamine stock solution is (2.0-2.2): (0.4-0.7): (0.35-0.4): (0.02-0.04): (0.002-0.004).
Preferably, the glue running step in the step (3) is: adding the xylanase liquid sample prepared in the step (1) into a sample groove, adjusting the voltage to 80v, starting glue running, and adjusting the voltage to 120v twenty minutes later.
Preferably, the incubation time in step (4) is 20-50 minutes.
Preferably, the temperature of the culture box in the step (5) is 30-50 ℃, and the culture time is 1-36 h.
According to the technical scheme, compared with the prior art, the xylanase protein band and activity can be identified simultaneously on the same glue by adopting the substrate glue and the separation glue.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a drawing showing the identification of xylanase protein bands and activity in example 3 of the present invention, cultured in a biochemical incubator for 12 h;
FIG. 2 is a drawing showing the identification of xylanase protein bands and activity in example 3 of the present invention cultured in a biochemical incubator for 24 h;
FIG. 3 is a drawing showing the identification of xylanase protein bands and activity after culturing for 36h in a biochemical incubator in example 3 of the present invention;
FIG. 4 is a drawing showing the identification of xylanase protein bands and activity in the biochemical incubator of example 3 of the present invention after 48h incubation;
FIG. 5 is a figure showing the identification of xylanase protein bands and activities in comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The raw materials used in the following examples:
bromophenol blue, glycerol, Tris (hydroxymethyl) aminomethane (Tris), hydrochloric acid, acrylamide, methylene bisacrylamide, tetraethylethylenediamine, ammonium persulfate, glycine, Coomassie Brilliant blue R-250, glacial acetic acid, and ethanol were purchased from general reagents; common Xylan, ramalin Brilliant Blue R cross-linked Xylan (Remazol Brilliant Blue R-D-Xylan) was purchased from Sigma-Aldrich.
Preparation of the relevant solutions used in the examples of the invention:
(1)0.5M Tris-HCl buffer: and 5.98 g of Tris is dissolved by adding distilled water, the pH value is adjusted to 6.7 by concentrated hydrochloric acid, and the volume is adjusted to 100 ml.
(2)1.5M Tris-HCl buffer: 18.17 g of Tris is dissolved by adding distilled water, the pH value is adjusted to 8.9 by concentrated hydrochloric acid, and the volume is adjusted to 100 ml.
(3) Loading buffer solution: 1.25 ml of 0.5M Tris-HCl buffer, 3 ml of glycerol, 2 ml of 0.5% bromophenol blue and 5.5 ml of distilled water are mixed uniformly.
(4) 30% acrylamide stock solution: in a fume hood, 29 g of acrylamide and 1 g of methylene bisacrylamide are weighed, and are dissolved by adding distilled water to a constant volume of 100 ml.
(5) Tris-glycine buffer: weighing 3.02 g of Tris and 18.8 g of glycine, dissolving in distilled water, and fixing the volume to 1000 ml.
(6) 10% ammonium persulfate: 0.1 g of ammonium persulfate was added to 1 ml of distilled water and used as it was.
(7)0.1M sodium acetate buffer: 0.6 ml of glacial acetic acid is absorbed, and distilled water is added to dissolve the mixture until the volume is 100 ml, so as to prepare 0.1M acetic acid solution. 0.82 g of anhydrous sodium acetate is weighed, dissolved by adding distilled water, and prepared into 0.1M sodium acetate solution after the volume is up to 100 ml. A0.1M acetic acid solution and a 0.1M sodium acetate solution are mixed to form a solution having a pH of 2.0 to 8.0.
Example 1
Step (1), mixing xylanase liquid and a sample buffer solution according to the ratio of 1: 1, uniformly mixing to obtain a xylanase liquid sample to be measured;
step (2) 1.65 ml of distilled water, 2.0 ml of 30% acrylamide, 1.3 ml of 1.5M Tris-HCl buffer, 50. mu.l of 10% ammonium persulfate and 4. mu.l of tetraethylethylenediamine stock solution were mixed uniformly.
4 ml of the separation gel was pipetted into a gel plate for vertical electrophoresis, and the liquid surface was sealed with distilled water.
2.13 ml of distilled water, 0.5 ml of 30% acrylamide, 0.38 ml of 0.5M Tris-HCl buffer, 30. mu.l of 10% ammonium persulfate, and 3. mu.l of tetraethylethylenediamine stock solution were mixed uniformly.
And after the separation gel is solidified for 30 minutes, pouring out the liquid seal water, adding the concentrated gel on the separation gel to the position 1mm away from the top layer of the short glass plate of the glue-making plate, inserting a comb, and solidifying for 40 minutes.
After the glue is solidified in the step (3), pulling out a comb of the sample groove, putting the prepared glue into a vertical electrophoresis groove, applying voltage, and filling Tris-glycine buffer solution;
adding 10 microliter xylanase liquid sample into the sample groove, adjusting the voltage to 80v, starting to run glue, and adjusting the voltage to 120v twenty minutes later until the bromophenol blue band in the xylanase liquid electrophoresis sample runs to the bottom layer of the glass plate.
Cutting the glue of the lane where the sample is located into strips by using a glue cutting plate, removing the concentrated glue, and reserving the separation glue to obtain strip separation glue containing the sample; putting the strip-shaped separation gel into 0.1M sodium acetate solution, and incubating for 30 minutes;
weighing 50 mg of remainders brilliant blue R cross-linked xylan and 0.3 g of agar powder, putting the remainders brilliant blue R cross-linked xylan and the agar powder into 30 ml of sodium acetate buffer solution with the pH value of 4.5, heating and dissolving the mixture on an electric furnace, cooling the mixture to 37 ℃, pouring the mixture into a culture dish to obtain a substrate glue with the thickness of about 5 mm, and placing the culture dish on a table until the mixture is cooled and solidified to obtain the substrate glue. The incubated strip-shaped separation gel containing the sample was placed on the surface of the substrate gel in a petri dish and incubated in a biochemical incubator at 40 ℃ for 12, 24, 36, and 48 hours until clear bands and transparent circles appeared, as shown in FIG. 3.
Comparative example 1
0.06-0.45 g of ordinary xylan was used in place of the remainders brilliant blue R cross-linked xylan of example 1 and a substrate gum was prepared in the same manner as in example 1.
Performance testing
The separation gel of example 1 was placed in the substrate gel containing general xylan of comparative example 1, and incubated in a biochemical incubator for 36 hours until a transparent circle appeared (FIG. 5). The results of example 1 were compared with those of comparative example 1.
The result shows that the transparent circle and the xylanase protein band can be clearly seen at the arrow in FIG. 3, and the transparent circle is arranged around the band, which indicates that the band is the active xylanase in the sample of the invention; wherein the strips and transparent circles are most clear at 36 hours of incubation, and the effect is not achieved at other time points. While only the obscured transparent ring is visible in the arrow in figure 5 without any visible bands. Therefore, the method can simultaneously and effectively identify the activity of the xylanase and the position of the band in the Native-Page gel, and provides powerful support for subsequent research.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. An zymogram method for identifying protein bands and activities of xylanase, which is characterized by comprising the following steps:
preparing a xylanase liquid sample to be measured;
preparing a separation glue layer, and preparing a concentrated glue layer with a sample groove on the upper layer of the separation glue layer;
putting the glue prepared in the step (2) in a vertical electrophoresis tank, adding a Tris-glycine buffer solution, carrying out sample application in a sample tank of a concentrated glue layer, applying voltage to the vertical electrophoresis tank, and running the glue until a bromophenol blue strip in a xylanase liquid sample runs to the bottom layer of the vertical electrophoresis tank;
cutting the glue of a lane where the xylanase liquid sample is located into strips by using a glue cutting plate, removing a concentrated glue layer, and reserving a separation glue layer to obtain strip separation glue containing the sample; putting the strip-shaped separation gel containing the sample into a sodium acetate solution, and incubating for 20-50 minutes;
step (5) tightly pasting the strip-shaped separation gel containing the sample incubated in the step (4) on the surface of the substrate gel of a culture dish, placing the culture dish in a biochemical incubator at 30-50 ℃ and culturing for 1-36 hours until clear strips and transparent circles appear; the substrate glue is prepared from remainders brilliant blue R cross-linked xylan, agar powder and sodium acetate buffer solution with the pH value of 2.0-8.0;
the xylanase liquid sample in the step (1) comprises xylanase and a loading buffer solution, the concentration of the xylanase liquid is 30-150 micrograms/ml, and the volume ratio of the xylanase liquid to the loading buffer solution is 1: 1-2: 1, mixing; the loading buffer solution comprises 0.5M Tris-HCl buffer solution, glycerol, 0.5% bromophenol blue and distilled water;
preparation of the loading buffer: 1.25 ml of 0.5M Tris-HCl buffer solution, 3 ml of glycerol, 2 ml of 0.5% bromophenol blue and 5.5 ml of distilled water are mixed uniformly to obtain the loading buffer solution.
2. The zymogram method for identifying xylanase protein bands and activity according to claim 1, characterized in that, the preparation step of the substrate glue in step (5): weighing 10-100 mg of remainders brilliant blue R cross-linked xylan and 0.15-0.6 g of agar powder, putting the remainders brilliant blue R cross-linked xylan and the agar powder into 30-50 ml of sodium acetate buffer solution with the pH value of 2.0-8.0, heating and dissolving the mixture on an electric furnace, cooling the mixture to 30-50 ℃, pouring the mixture into a culture dish, and placing the culture dish on a table until the mixture is cooled and solidified to obtain the substrate glue.
3. The zymogram method for identifying xylanase protein band and activity according to claim 1, characterized in that the separation gel in step (2) is made of distilled water, 30% acrylamide, 1.5M Tris-HCl buffer, 10% ammonium persulfate, tetraethyl ethylenediamine stock solution; the volume ratio of the distilled water, 30% of acrylamide, 1.5% of MTris-Hcl buffer solution, 10% of ammonium persulfate and tetraethyl ethylenediamine stock solution is (1.60-1.70): (1.8-2.1): (1.2-1.4): (0.02-0.07): (0.003-0.005).
4. The zymogram method for identifying xylanase protein band and activity according to claim 1, characterized in that the concentrated gel in step (2) is made of distilled water, 30% acrylamide, 0.5M Tris-HCl buffer, 10% ammonium persulfate, tetraethyl ethylenediamine stock solution; the volume ratio of the distilled water, 30% of acrylamide, 0.5% of MTris-Hcl buffer solution, 10% of ammonium persulfate and tetraethyl ethylenediamine stock solution is (2.0-2.2): (0.4-0.7): (0.35-0.4): (0.02-0.04): (0.002-0.004).
5. The zymogram method for identifying xylanase protein bands and activity according to claim 1, wherein the gel-running step in step (3) is: adding the xylanase liquid sample prepared in the step (1) into a sample groove, adjusting the voltage to 80v, starting glue running, and adjusting the voltage to 120v twenty minutes later.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000070063A1 (en) * | 1999-05-13 | 2000-11-23 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | CONIOTHYRIUM MINITANS XYLANASE GENE $i(CXY1) |
| CN103105427A (en) * | 2013-01-18 | 2013-05-15 | 中南大学 | Zymogram active electrophoresis detection method |
| CN106967674A (en) * | 2017-05-22 | 2017-07-21 | 江西农业大学 | A kind of isolated culture method of sheep rumen epithelial cell |
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- 2018-07-30 CN CN201810853978.0A patent/CN109085230B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000070063A1 (en) * | 1999-05-13 | 2000-11-23 | Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food | CONIOTHYRIUM MINITANS XYLANASE GENE $i(CXY1) |
| CN103105427A (en) * | 2013-01-18 | 2013-05-15 | 中南大学 | Zymogram active electrophoresis detection method |
| CN106967674A (en) * | 2017-05-22 | 2017-07-21 | 江西农业大学 | A kind of isolated culture method of sheep rumen epithelial cell |
Non-Patent Citations (3)
| Title |
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| Purification and characterization of extracellular xylanase from Streptomyces cyaneus SN32;Suchita Ninawe等;《Bioresource Technology》;20070403;第99卷;第1252-1258页 * |
| Simple, Sensitive Zymogram Technique for Detection of Xylanase Activity in Polyacrylamide Gels;JOHN C. ROYER等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19900630;第56卷(第6期);第1516-1517页 * |
| 交联木聚糖制备工艺的优化和活性检测研究;尹李锋 等;《湖北大学学报（自然科学版）》;20180330;第40卷(第2期);第179-182页 * |
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