阅读说明：本技术 一种高通量同时测定混培微生物粗酶中多种酶活性的方法 (Method for simultaneously measuring multiple enzyme activities in mixed culture microbial crude enzyme in high flux ) 是由 牛启桂 薛含含 于 2020-12-21 设计创作，主要内容包括：本发明公开了一种高通量同时测定混培微生物粗酶中多种酶活性的方法,多种酶水解底物所得的产物均为还原糖,测定方法包括如下步骤：在96孔板上规定每一行酶标条测定酶的种类,其中一行酶标条用于测葡萄糖标准曲线；布底物：在每一行酶标条的各个孔中添加待测酶对应的底物,用于测葡萄糖标准曲线的一行酶标条添加不同浓度的葡萄糖标准溶液；添加样品：在96孔板上第1列各个孔中添加蒸馏水做空白对照,在第2-12列各个孔中依次添加混培微生物粗酶样品S1-S11,其中,添加葡萄糖标准溶液的一行酶标条不添加任何物质；酶促反应；终止反应；显色反应；计算。本发明所公开的方法成本低,测量快速全面,控制简单,适用于实验推广。(The invention discloses a method for simultaneously measuring the activities of various enzymes in mixed culture microbial crude enzyme in a high-throughput manner, wherein products obtained by hydrolyzing substrates by various enzymes are reducing sugars, and the measuring method comprises the following steps: specifying the type of enzyme in each row of enzyme standard bars on a 96-well plate, wherein one row of enzyme standard bars is used for measuring a glucose standard curve; cloth substrate: adding a substrate corresponding to the enzyme to be detected in each hole of each row of enzyme label strips, and adding glucose standard solutions with different concentrations into one row of enzyme label strips for detecting a glucose standard curve; adding a sample: adding distilled water into each hole in the 1 st column of a 96-well plate as a blank control, and sequentially adding mixed culture microorganism crude enzyme samples S1-S11 into each hole in the 2 nd to 12 th columns of the 96-well plate, wherein one line of enzyme standard strips added with glucose standard solution is not added with any substance; enzymatic reaction; terminating the reaction; carrying out color development reaction; and (4) calculating. The method disclosed by the invention has the advantages of low cost, quick and comprehensive measurement, simple control and suitability for experimental popularization.)

1. A method for simultaneously measuring the activities of a plurality of enzymes in crude enzymes of mixed culture microorganisms in a high-throughput manner is characterized in that products obtained by hydrolyzing substrates by a plurality of enzymes are reducing sugars, and the measuring method comprises the following steps:

(1) specifying the type of enzyme in each row of enzyme standard bars on a 96-well plate, wherein one row of enzyme standard bars is used for measuring a glucose standard curve;

(2) cloth substrate: adding a substrate corresponding to the enzyme to be detected in each hole of each row of enzyme label strips, and adding glucose standard solutions with different concentrations into one row of enzyme label strips for detecting a glucose standard curve;

(3) adding a sample: adding distilled water into each hole in the 1 st column of a 96-well plate as a blank control, and sequentially adding mixed culture microorganism crude enzyme samples S1-S11 into each hole in the 2 nd to 12 th columns of the 96-well plate, wherein one line of enzyme standard strips added with glucose standard solution is not added with any substance;

(4) enzymatic reaction: placing the 96-well plate in a water bath kettle at 38-40 deg.C for 30 min;

(5) and (3) terminating the reaction: boiling in water bath for 10-15 min;

(6) and (3) color development reaction: adding DNS reagent into all the holes of a 96-hole plate, developing in boiling water bath for 15min, and measuring the absorbance value of the solution in each hole at 540nm by using a rainbow microplate reader after the reaction is finished;

(7) and (3) calculating: drawing a glucose standard curve by using the absorbance value measured by the glucose standard solution, and calculating the corresponding reducing sugar content according to the glucose standard curve and the measured absorbance value of each pore solution;

and measuring the total protein content in each crude enzyme sample of the mixed culture microorganisms by using a BCA method, and then calculating the activity of each enzyme according to the reducing sugar content and the total protein content.

2. The method for high-throughput simultaneous determination of multiple enzyme activities in crude enzymes of mixed culture microorganisms according to claim 1, wherein the multiple enzymes comprise α -amylase, β -amylase, cellulase, xylanase, chitosanase, pectinase and chitinase.

3. The method for simultaneously measuring the activities of a plurality of enzymes in the crude enzyme of the mixed culture microorganisms at high throughput as claimed in claim 2, wherein the substrates added in the step (2) are soluble starch solution I, soluble starch solution II, sodium carboxymethyl cellulose solution, xylan solution, colloidal chitosan solution, pectin solution and colloidal chitin solution.

4. The method for simultaneously measuring the activities of a plurality of enzymes in the crude enzyme of the mixed culture microorganisms at high throughput as claimed in claim 3, wherein the enzyme label strip added with the soluble starch solution is taken out separately when the substrate is distributed, and is placed back to a 96-well plate after being kept at 70 ℃ for 15min, and then the sample is added.

5. According to the claimsThe method for simultaneously measuring the activities of a plurality of enzymes in the crude enzymes of the mixed culture microorganisms in a high throughput manner in claim 1 is characterized in that the method for measuring the total protein content in each crude enzyme sample of the mixed culture microorganisms by using the BCA method in the step (7) is as follows: taking two new enzyme label strips, adding 10uL of distilled water into the 1 st hole, adding 10uL of standard protein solution with the concentration of 563ug/mL into the 2 nd hole, sequentially adding 10uL of mixed culture microorganism crude enzyme samples S1-S11 into the 3 rd to 24 th holes, adding the same samples into the two adjacent holes, and taking an average value during calculation; then 100uL of BCA/CuSO with the volume ratio of 50:1 is added into each hole of the enzyme label strip₄Incubating the mixed solution for 30min at 37 ℃, and measuring the absorbance value at 562nm by using an enzyme-labeling instrument; finally, the total protein content of each sample is calculated according to the following formula:

wherein, OD_SFor the absorbance values, OD, measured for each sample in wells 3-24₀The absorbance value, OD, of the measured blank solution in well 1₁For the absorbance value of the standard solution in the 2 nd well, N is the dilution factor before sample test, and V is the sample volume 10 uL.

6. The method for simultaneously measuring the activities of a plurality of enzymes in the crude enzyme of the mixed culture microorganisms in high throughput according to claim 1, wherein the formula for calculating the activities of the enzymes in the step (7) is as follows:

Technical Field

The invention relates to the technical field of enzyme activity determination, in particular to a method for simultaneously determining multiple enzyme activities in mixed culture microbial crude enzyme in a high-throughput manner.

Background

Alpha-amylase, also called starch-1, 4-dextrin glycosidase, liquefying enzyme, is an incision enzyme, the products of starch hydrolysis are maltose and dextrin, the hydrolysis limit reaches 30% -90%, the main producing bacteria of the alpha-amylase are bacillus subtilis, aspergillus niger, aspergillus oryzae, etc., the alpha-amylase is not acid-resistant and is passivated quickly below pH3.6.

Beta-amylase, also known as starch-1, 4-maltosidase, is an exo-saccharifying enzyme. It can cut off the alpha-1, 4 glycosidic bond in turn from the non-reducing end of starch, the hydrolysis products are mainly maltose and beta-limit dextrin, and the main producing bacteria of beta-amylase are bacteria, etc. The beta-amylase is widely applied to the industries of beer brewing, food processing and the like, and is an important industrial enzyme. Beta-amylase is thermolabile and inactivated at 70 ℃ for 15 min.

Cellulase is a complex enzyme system consisting of a plurality of hydrolytic enzymes, and many fungi in nature can secrete the cellulase. Traditionally, cellulases are divided into three classes: c1 enzyme, Cx enzyme, and beta glucosidase. The C1 enzyme is the enzyme that acts initially on cellulose, disrupting the crystalline structure of the cellulose chain. Cx enzyme is a cellulase which acts on cellulose activated by C1 enzyme and decomposes beta-1, 4-glycosidic bonds. Beta-glucosidase can break down cellobiose, cellotriose and other low molecular cellodextrins to glucose. Cellulases are widely present in organisms in nature. Cellulase is produced in bacteria, fungi, animals, etc. Cellulases generally used for production are derived from fungi, typically Trichoderma, Aspergillus and Penicillium.

Xylanase is also called endo-1, 4-beta-xylanase, and is a key enzyme in the degradation process of xylan, and can degrade xylan into oligosaccharides and xylose which can be digested and absorbed by animals. Xylanases are widely distributed in nature and can be obtained from animals, plants and microorganisms. At present, xylanase is mainly produced by fermenting microorganisms such as fungi, bacteria and the like.

The chitosanase is a specific hydrolytic chitosanglycosyl hydrolase, and catalyzes and hydrolyzes beta- (1, 4) -glucosamine glycosidic bond in partially acetylated chitosan in an endo mode to generate chitosan oligosaccharide with different biological activities, and the final products are mainly chitobiose and chitotriose.

Pectinase is a general term for pectic enzyme decomposition, is polygalacturonic acid hydrolase essentially, comprises four major classes of protopectinase, pectinesterase, polygalacturonase and pectin lyase, is used for hydrolyzing pectin to generate beta-galacturonic acid, is widely present in plant fruits and microorganisms, and is mainly used in the industries of food, wine brewing, environmental protection, medicines, textiles and daily chemical products.

Chitinase is a glycoside hydrolase that acts on chitin to produce N-acetylglucosamine and chitooligosaccharides.

The products obtained by hydrolyzing substrates by alpha-amylase, beta-amylase, cellulase, xylanase, chitosanase, pectinase and chitinase are reducing sugars, oligosaccharide with a reducing end and monosaccharide with a reducing group are heated together with 3, 5-dinitrosalicylic acid reagent (DNS reagent) to generate brownish red amino compounds, the color shade of the generated brownish red substances is in direct proportion to the content of the reducing sugars generated by enzymolysis in a certain range, and the generation amount of the reducing sugars is in direct proportion to the activity of various enzymes in reaction liquid, so that the absorbance value is measured by an enzyme labeling instrument, and the activity of various enzymes can be calculated according to a glucose standard curve and a specific activity formula.

The mixed culture microorganism contains various crude enzymes, the workload of measuring the enzyme activity by using a single enzyme-related kit is large, the operation is complicated, the price of the kit is relatively high, and the use times are limited. Therefore, the method for simultaneously measuring the enzyme activities of various enzymes conveniently, efficiently and at low cost has important research significance for realizing the enzyme activity experiment of the crude enzyme of the optimized mixed culture microorganism.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for simultaneously measuring the activities of various enzymes in the crude enzyme of the mixed culture microorganism in a high-throughput manner, so as to achieve the purposes of low cost, rapid and comprehensive measurement, simple control and suitability for experimental popularization.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a high-throughput method for simultaneously measuring the activities of multiple enzymes in crude enzymes of mixed culture microorganisms, wherein products obtained by hydrolyzing substrates by multiple enzymes are reducing sugars, and the measuring method comprises the following steps:

(1) specifying the type of enzyme in each row of enzyme standard bars on a 96-well plate, wherein one row of enzyme standard bars is used for measuring a glucose standard curve;

(2) cloth substrate: adding a substrate corresponding to the enzyme to be detected in each hole of each row of enzyme label strips, and adding glucose standard solutions with different concentrations into one row of enzyme label strips for detecting a glucose standard curve;

(3) adding a sample: adding distilled water into each hole in the 1 st column of a 96-well plate as a blank control, and sequentially adding mixed culture microorganism crude enzyme samples S1-S11 into each hole in the 2 nd to 12 th columns of the 96-well plate, wherein one line of enzyme standard strips added with glucose standard solution is not added with any substance;

(4) enzymatic reaction: placing the 96-well plate in a water bath kettle at 38-40 deg.C for 30 min;

(5) and (3) terminating the reaction: boiling in water bath for 10-15 min;

(6) and (3) color development reaction: adding DNS reagent into all the holes of a 96-hole plate, developing in boiling water bath for 15min, and measuring the absorbance value of the solution in each hole at 540nm by using a rainbow microplate reader after the reaction is finished;

(7) and (3) calculating: drawing a glucose standard curve by using the absorbance value measured by the glucose standard solution, and calculating the corresponding reducing sugar content according to the glucose standard curve and the measured absorbance value of each pore solution;

and measuring the total protein content in each crude enzyme sample of the mixed culture microorganisms by using a BCA method, and then calculating the activity of each enzyme according to the reducing sugar content and the total protein content.

In the above scheme, the plurality of enzymes include alpha-amylase, beta-amylase, cellulase, xylanase, chitosanase, pectinase and chitinase.

In a further technical scheme, the substrates added in the step (2) are respectively a soluble starch solution I, a soluble starch solution II, a sodium carboxymethyl cellulose solution, a xylan solution, a colloidal chitosan solution, a pectin solution and a colloidal chitin solution.

In a further technical scheme, when substrates are distributed, the enzyme label strips added with the soluble starch solution I are independently taken out, and are placed back to a 96-well plate after being kept at 70 ℃ for 15min, and then samples are added.

In a further technical scheme, the method for determining the total protein content in each crude enzyme sample of the mixed culture microorganisms by using the BCA method in the step (7) comprises the following steps:

taking two new enzyme label strips, adding 10uL of distilled water into the 1 st hole, adding 10uL of standard protein solution with the concentration of 563ug/mL into the 2 nd hole, sequentially adding 10uL of mixed culture microorganism crude enzyme samples S1-S11 into the 3 rd to 24 th holes, adding the same samples into the two adjacent holes, and taking an average value during calculation; then 100uL of BCA/CuSO with the volume ratio of 50:1 is added into each hole of the enzyme label strip₄Incubating the mixed solution for 30min at 37 ℃, and measuring the absorbance value at 562nm by using an enzyme-labeling instrument; finally, the total protein content of each sample is calculated according to the following formula:

wherein, OD_SFor the absorbance values, OD, measured for each sample in wells 3-24₀The absorbance value, OD, of the measured blank solution in well 1₁For the absorbance value of the standard solution in the 2 nd well, N is the dilution factor before sample test, and V is the sample volume 10 uL.

In the scheme, the formula for calculating the enzyme activity in the step (7) is as follows:

through the technical scheme, the method for simultaneously measuring the activities of a plurality of enzymes in the crude enzyme of the mixed culture microorganism in a high-throughput manner has the following beneficial effects:

after alpha-amylase, beta-amylase, cellulase, xylanase, chitosanase, pectinase and chitinase react with various substrates, the generated reactants all belong to reducing sugar, the reducing sugar and DNS reagent perform color reaction, the enzyme activities are in direct proportion to the amount of the generated reducing sugar, the content of the corresponding reducing sugar can be calculated through a glucose standard curve, and then the corresponding reducing sugar content is substituted into a formula to calculate U/mg so as to express the enzyme activities of the enzymes.

When the activities of the alpha-amylase and the beta-amylase are measured, the beta-amylase is not heat-resistant, so that the enzyme label strip added with the soluble starch solution I is taken out independently, the temperature is accurately kept at 70 ℃ for 15min, the enzyme activity of the alpha-amylase is measured after the beta-amylase is passivated, the enzyme label strip added with the soluble starch solution II is not pretreated, the measured result is the sum of the enzyme activities of the alpha-amylase and the beta-amylase, and the difference value of the enzyme activities of the two enzyme label strips B is the enzyme activity of the beta-amylase.

The method disclosed by the invention is based on the high flux of a 96-well plate, utilizes a3, 5-dinitrosalicylic acid (DNS) colorimetric method, simultaneously determines and mixedly cultivates multiple enzyme activities in the microbial crude enzyme in one step, and is low in cost, rapid and comprehensive in measurement, simple to control and suitable for experimental popularization.

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.

FIG. 1 is a schematic diagram of a 96-well plate used in an embodiment of the present invention with corresponding sample additions;

FIG. 2 is a schematic diagram of two enzyme-labeled strip-added samples used in determining total protein content in the embodiment of the present invention.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

The invention provides a method for simultaneously measuring multiple enzyme activities in mixed culture microorganism crude enzyme in a high throughput manner, which comprises the following specific embodiments:

(1) as shown in fig. 1, the following are specified on a 96-well plate:

the enzyme label strip A is used for measuring the enzyme activity of alpha-amylase, the enzyme label strip B is used for measuring the enzyme activity of beta-amylase, the enzyme label strip C is used for measuring the enzyme activity of cellulase, the enzyme label strip D is used for measuring the enzyme activity of xylanase, the enzyme label strip E is used for measuring the enzyme activity of chitosanase, the enzyme label strip F is used for measuring the enzyme activity of pectinase, the enzyme label strip G is used for measuring the enzyme activity of chitinase, and the enzyme label strip H is used for measuring a glucose standard curve.

(2) Cloth substrate: adding 50uL of substrate corresponding to the enzyme to be detected into each hole of each row of enzyme label strips, which is as follows:

enzyme label strip A-1% soluble starch solution, enzyme label strip B-1% soluble starch solution, enzyme label strip C-1% sodium carboxymethylcellulose solution, enzyme label strip D-1% xylan solution, enzyme label strip E-1% colloidal chitosan solution, enzyme label strip F-1% pectin solution, and enzyme label strip G-1% colloidal chitin solution.

The mass concentration of the substrate is 1%, and the volume of each substrate solution is determined by 0.2mol/L phosphate buffer (disodium hydrogen phosphate-citric acid) with pH of 6.0. For example: the 1% soluble starch solution was prepared by dissolving 1g of soluble starch in 99g of phosphate buffer.

The enzyme label strip H was supplemented with glucose standard solutions of different concentrations, as shown in table 1:

TABLE 1 glucose Standard solution concentration

(3) Pretreatment: the enzyme label A is taken out separately and is accurately kept warm for 15min at 70 ℃, the beta-amylase is inactivated, and then the enzyme label A is put back to a 96-well plate.

(4) Adding a sample: adding distilled water into the 1 st row A-G hole on a 96-well plate as a blank control, sequentially adding mixed culture microorganism crude enzyme samples S1-S11 into the 2 nd row A-G hole to the 12 th row A-G hole, and adding no substance into each hole of an enzyme label strip H;

(5) enzymatic reaction: placing the 96-well plate in a water bath kettle at 40 ℃ for 30 min;

(6) and (3) terminating the reaction: boiling water bath for 15 min;

(7) and (3) color development reaction: adding 100uL DNS reagent (3, 5-dinitrosalicylic acid) into all the wells of a 96-well plate respectively, developing in boiling water bath for 15min, and measuring the absorbance value of the solution in each well at 540nm by using a rainbow microplate reader after the reaction is finished;

the reaction mechanism of this step is as follows:

(8) and (3) calculating: and (3) making a standard curve by using the absorbance value at 540nm measured by the glucose standard solution through Excel software, wherein the abscissa is the content of glucose, and the ordinate is the absorbance value of glucose with different concentrations. And calculating the corresponding reducing sugar content in each hole according to the glucose standard curve and the measured absorbance value of each hole solution.

The BCA method is used for determining the total protein content in each mixed culture microorganism crude enzyme sample, and the method comprises the following steps:

taking two new enzyme label strips, as shown in figure 2, adding 10uL of distilled water into the 1 st hole, adding 10uL of a 563ug/mL standard protein solution into the 2 nd hole, sequentially adding 10uL of mixed culture microorganism crude enzyme samples S1-S11 into the 3 rd to 24 th holes, adding the same samples into the two adjacent holes, and averaging during calculation; then 100uL of BCA/CuSO with the volume ratio of 50:1 is added into each hole of the enzyme label strip₄Incubating the mixed solution for 30min at 37 ℃, and measuring the absorbance value at 562nm by using an enzyme-labeling instrument; finally, the total protein content of each sample is calculated according to the following formula:

wherein, OD_SFor the absorbance values, OD, measured for each sample in wells 3-24₀The absorbance value, OD, of the measured blank solution in well 1₁For the absorbance value of the standard solution in the 2 nd well, N is the dilution factor before sample test, and V is the sample volume 10 uL.

The formula for calculating the enzyme activity is as follows:

when the mixed culture microorganism crude enzyme sample S1 is analyzed, the reducing sugar content of the holes A2 and C2-G2 and the total protein content of the mixed culture microorganism crude enzyme sample S1 are used for calculation, and the enzyme activities of alpha-amylase, cellulase, xylanase, chitosanase, pectinase and chitinase can be obtained.

When the activities of the alpha-amylase and the beta-amylase are measured, the beta-amylase is not heat-resistant, so that the enzyme label strip of the enzyme label strip A is taken out independently at first, the temperature is accurately kept for 15min at 70 ℃, the enzyme activity of the alpha-amylase is measured after the beta-amylase is passivated, the enzyme label strip B is not pretreated, the measured result is the sum of the enzyme activities of the alpha-amylase and the beta-amylase, and the difference value of the enzyme activities of the two enzyme label strips is the enzyme activity of the beta-amylase. Namely, the enzyme activity obtained by subtracting the enzyme activity obtained by the hole A2 from the enzyme activity obtained by the hole B2 is the enzyme activity of the beta-amylase in the mixed culture microorganism crude enzyme sample S1.

By analogy, when the mixed culture microorganism crude enzyme sample S2 is analyzed, the reducing sugar content of the holes A3-G3 and the total protein content of the mixed culture microorganism crude enzyme sample S2 are used for calculation.

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.