High-throughput detection method of 2-hydroxy acid and application of high-throughput detection method

Abstract

The invention discloses a high-throughput detection method of 2-hydroxy acid and application of the high-throughput detection method. In particular to a high-throughput detection method of 2-hydroxy-4-methylthiobutyric acid and a high-throughput rapid screening method of protease for producing methionine. The detection method comprises the following steps: mixing a to-be-detected sample with oxidase, catalase and ABTS for a chromogenic reaction, and detecting the absorbance of a chromogenic system to obtain the content of 2-hydroxy acid in the to-be-detected sample. The detection method is efficient, convenient and low in cost, and compared with an HPLC detection method, the efficiency is higher.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and specifically to a high-throughput detection method for 2-hydroxy acid and its application, and further to a high-throughput detection method for 2-hydroxy-4-methylthiobutyric acid and a high-throughput rapid screening method for proteases producing methionine. Background Art

[0002] Methionine, also known as methionine and 2-amino-4-methylthio-butyric acid, is an essential amino acid that plays an important role in human growth, development and health. It is widely used in many industries such as medicine, food and cosmetics. At the same time, methionine is also an indispensable additive in animal feed, which can be used to increase the lean meat content of poultry and shorten the feeding cycle. Currently, the commercially available methionine used in feed is mainly divided into methionine and hydroxymethionine (also known as "2-hydroxy-4-methylthiobutyric acid" and "liquid methionine"), both of which have the same physiological function in organisms. In recent years, the global demand for methionine has increased year by year, and my country's methionine production cannot meet the needs of the rapid development of the feed industry, so it mainly relies on imports from overseas.

[0003] At present, the production methods of methionine are mainly chemical synthesis and fermentation. Due to the low yield of fermentation, chemical synthesis is generally used by methionine manufacturers around the world. Chemical synthesis can be divided into hydantoin method and cyanohydrin method. Hydantoin method can be used to synthesize methionine. The process technology is relatively mature, but there will be a large number of by-products such as sodium sulfate in the production process, and the technology and equipment are complex and energy consumption is high; the cyanohydrin method has a short process route, but there are also highly toxic substances such as hydrogen cyanide in the production process, which has high requirements for equipment safety and produces three wastes. The biological synthesis of methionine is still in its early stages and requires a large number of experiments for transformation, such as protein engineering to screen mutant libraries in large quantities, and 2-hydroxy-4-methylthiobutyric acid is the main product in the process of bioengineering transformation to high-yield methionine. At present, the HPLC method is mainly used to detect the content of 2-hydroxy-4-methylthiobutyric acid. The detection time for each sample is about 40 minutes, which greatly limits the research and development efficiency. Therefore, it is very urgent to form a set of low-cost high-throughput rapid detection methods for 2-hydroxymethylthiobutyric acid. Summary of the invention

[0004] Based on this, it is necessary to provide a high-throughput detection method for 2-hydroxy acid and its application, and further provide a high-throughput detection method for 2-hydroxy-4-methylthiobutyric acid (hereinafter referred to as 2-hydroxymethylthiobutyric acid) and a high-throughput rapid screening method for proteases that produce methionine.

[0005] The first aspect of the present application provides a high-throughput detection method for 2-hydroxy acid, comprising the following steps:

[0006] Mixing the sample to be tested with a color developing reagent to obtain a color developing system, performing a color developing reaction, and detecting the absorbance of the color developing system to obtain the content of 2-hydroxy acid in the sample to be tested;

[0007] The color developing reagent includes: oxidase, peroxidase and ABTS. Based on the feed amount, the usage ratio of the ABTS, the peroxidase and the oxidase is (38-42) mM: (15-20) U: (28-32) U.

[0008] In some embodiments, the oxidase is selected from one or more of AvLOX, LctO, AOX2 and SoGO.

[0009] In some embodiments, the AvLOX, LctO, AOX2 and SoGO have the amino acid sequences shown in SEQ ID NOs: 1-4, respectively.

[0010] In some embodiments, the volume ratio of the sample to be tested to the color developing reagent is 1:(0.95~1.05).

[0011] In some embodiments, relative to the volume of the sample to be tested, the content of the oxidase is 0.4 U to 1.2 U per 100 μL of the sample to be tested.

[0012] In some embodiments, the sample to be tested is a liquid sample containing 2-hydroxy acid, wherein the concentration of 2-hydroxy acid in the sample to be tested is 0.3 mM to 1.0 mM.

[0013] In some embodiments, the sample to be tested can be diluted with a buffer to obtain a desired 2-hydroxy acid concentration, wherein the buffer is an M9 buffer, and the M9 buffer includes Na2HPO4, KH2PO4, NaCl, NH4Cl, MgSO4 and CaCl2; optionally, in the M9 buffer, the concentration of Na2HPO4 is 6.7 g / L~6.9 g / L, the concentration of KH2PO4 is 2.9g / L~3.1 g / L, the concentration of NaCl is 0.4 g / L~0.6 g / L, the concentration of NH4Cl is 0.9 g / L~1.1 g / L, the concentration of MgSO4 is 1.9 mM~2.1 mM, and the concentration of CaCl2 is 2.4 mM~2.6 mM.

[0014] In some embodiments, the step of detecting the absorbance of the color development system to obtain the content of 2-hydroxy acid in the sample to be tested comprises:

[0015] The linear interval average value of the absorbance of the color development system is detected, and the content of 2-hydroxy acid in the sample to be tested is calculated using the linear interval average value of the absorbance and the standard curve of the 2-hydroxy acid concentration; wherein the R 2 Value greater than or equal to 0.99.

[0016] In some embodiments, the step of detecting the linear interval average value of the absorbance of the color development system comprises:

[0017] After the color development system is formed, the absorbance value at 420 nm of the color development system is detected every 30 seconds to 60 seconds for 10 minutes to 30 minutes; optionally, the detection temperature is 25°C to 40°C.

[0018] In some embodiments, the 2-hydroxy acid includes one or more of 2-hydroxy-4-methylthiobutyric acid, glycolic acid, lactic acid, malic acid, and 2-hydroxybutyric acid.

[0019] The second aspect of the present application provides a high-throughput screening method for a protease producing methionine, comprising the following steps:

[0020] Producing methionine under the action of multiple different proteases, using the high-throughput detection method for 2-hydroxy acid described in the first aspect of the present application, and detecting the content of 2-hydroxy acid in multiple intermediates at the same time, and evaluating the catalytic ability of the multiple different proteases for methionine production according to the content of 2-hydroxy acid, thereby high-throughput screening of proteases with strong catalytic ability;

[0021] Wherein, the 2-hydroxy acid is 2-hydroxy-4-methylthiobutyric acid,

[0022] The intermediate product comprises the supernatant of the fermentation broth for producing methionine after solid impurities are removed.

[0023] In some embodiments, the intermediate product further comprises one or more of the by-products methylthiopropionaldehyde, methylthiopropionic acid and methylthiopropanol.

[0024] In some embodiments, the step of simultaneously detecting the content of 2-hydroxy acid in multiple intermediates includes: placing the intermediates on an ELISA plate and detecting them using an ELISA reader. Preferably, the ELISA plate is a 96-well ELISA plate.

[0025] The above method can be used to achieve high-throughput detection of 2-hydroxy acids. The content of 2-hydroxy acids in the system can be obtained by the oxidation reaction of 2-hydroxy acids by oxidase and the detection of the generated hydrogen peroxide by the ABTS method. This detection method has high accuracy, short time, high efficiency and low cost.

[0026] Furthermore, the detection method can be used to screen out methionine-producing proteases with strong catalytic ability in high throughput, thereby increasing the production of methionine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0028] Figure 1 This is a flow chart of a detection method in one embodiment of the present application;

[0029] Figure 2 A standard curve of hydrogen peroxide in one embodiment of the present application;

[0030] Figure 3 The standard curve of 2-hydroxymethylthiobutyric acid in one embodiment of the present application;

[0031] Figure 4 The absorbance curve of 2-hydroxymethylthiobutyric acid at different temperatures in one embodiment of the present application;

[0032] Figure 5 This is a correlation analysis diagram in one embodiment of the present application. DETAILED DESCRIPTION

[0033] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0035] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0036] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0037] The terms "having", "containing", "including" and "comprising" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0038] In this application, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0039] In this application, if the unit of the data range is followed only by the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 1~2μg / mL means that the units of the left endpoint "1" and the right endpoint "2" are both μg / mL, which have the same meaning as 1μg / mL~2μg / mL. In addition, similar descriptions involving other parameters also apply to the above understanding.

[0040] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0041] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0042] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0043] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0044] 2-Hydroxy-4-methylthiobutyric acid is the main product in the process of bioengineering to produce high methionine. Currently, the HPLC method is mainly used to detect the content of 2-Hydroxy-4-methylthiobutyric acid. The detection time for each sample is about 40 minutes. The long detection time greatly limits the research and development efficiency.

[0045] Based on this, the embodiments of the present application at least provide a high-throughput detection method for 2-hydroxy acid and its application, and further provide a high-throughput detection method for 2-hydroxy-4-methylthiobutyric acid and a high-throughput rapid screening method for proteases that produce methionine.

[0046] In a first aspect of the present application, a high-throughput detection method for 2-hydroxy acid is provided, comprising the following steps:

[0047] S100: Mix the sample to be tested with the color developing reagent to form a color developing system and perform a color developing reaction;

[0048] S200: Detect the absorbance of the color development system to obtain the content of 2-hydroxy acid in the sample to be tested;

[0049] The color developing reagent includes: oxidase, peroxidase and ABTS. Based on the feed amount, the dosage ratio of ABTS, peroxidase and oxidase is (28-32) mM: (15-20) U: (28-32) U.

[0050] Further, in some embodiments, the usage ratio of ABTS, peroxidase and oxidase is 40 mM:18 U:30 U.

[0051] In some embodiments, the color development system is placed in a 96-well ELISA plate, and the 2-hydroxy acid in the sample to be tested is detected in conjunction with an ELISA reader.

[0052] In some embodiments, the oxidase is selected from one or more of AvLOX, LctO, AOX2, and SoGO.

[0053] In some embodiments, AvLOX, LctO, AOX2 and SoGO have the amino acid sequences shown in SEQ ID NOs: 1-4, respectively.

[0054] In some embodiments, the volume ratio of the sample to be tested to the color developing reagent may be 1:(0.95-1.05). In a non-limiting manner, the volume ratio of the sample to be tested to the color developing reagent may be, but is not limited to, 1:0.95, 1:1, 1:1.05, or a ratio or range between any two of the above ratios.

[0055] In some embodiments, the content of the oxidase is 0.4 U to 1.2 U per 100 μL of the sample to be tested, relative to the volume of the sample to be tested. In a non-limiting manner, the content of the oxidase may be, but is not limited to, 0.4 U, 0.5 U, 0.6 U, 0.7 U, 0.8 U, 0.9 U, 1 U, 1.1 U, 1.2 U, or a value or range between any two of the above values.

[0056] In the present application, unless otherwise specified, U is defined as the amount of enzyme that converts 1 micromole of substrate into product within 1 minute as 1 unit.

[0057] In some embodiments, the sample to be tested is a liquid sample containing 2-hydroxy acid, wherein the concentration of 2-hydroxy acid in the sample to be tested is 0.3 mM to 1.0 mM. In a non-limiting manner, the concentration of 2-hydroxy acid in the sample to be tested can be, but is not limited to, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM or a value or range between any two of the above values.

[0058] In some embodiments, the sample to be tested can be diluted with a buffer to obtain a desired 2-hydroxy acid concentration, wherein the buffer is an M9 buffer comprising Na2HPO4, KH2PO4, NaCl, NH4Cl, MgSO4 and CaCl2.

[0059] In some embodiments, in the M9 buffer, the concentration of Na2HPO4 is 6.7 g / L to 6.9 g / L, the concentration of KH2PO4 is 2.9 g / L to 3.1 g / L, the concentration of NaCl is 0.4 g / L to 0.6 g / L, the concentration of NH4Cl is 0.9 g / L to 1.1 g / L, the concentration of MgSO4 is 1.9 mM to 2.1 mM, and the concentration of CaCl2 is 2.4 mM to 2.6 mM. Further, the concentration of Na2HPO4 is 6.78 g / L, the concentration of KH2PO4 is 3 g / L, the concentration of NaCl is 0.5 g / L, the concentration of NH4Cl is 1 g / L, the concentration of MgSO4 is 2 mM, and the concentration of CaCl2 is 2.5 mM.

[0060] In some embodiments, in step S200, the step of detecting the absorbance of the color development system to obtain the content of 2-hydroxy acid in the sample to be tested includes:

[0061] The linear interval average value of the absorbance of the colorimetric system is detected, and the content of 2-hydroxy acid in the sample to be tested is calculated using the linear interval average value of the absorbance and the standard curve of 2-hydroxy acid concentration; wherein the R 2 Value greater than or equal to 0.99.

[0062] In some embodiments, the high-throughput detection flow chart of 2-hydroxy acid in the sample to be tested is as follows: Figure 1 shown.

[0063] In some embodiments, the step of detecting the linear interval average value of the absorbance of the color development system comprises: after the color development system is formed, the 420nm absorbance value of the color development system is detected every 30s to 60s, and the duration or range between 10min to 30min. In a non-limiting manner, after the color development system is formed, the 420nm absorbance value of the color development system is detected every 30s, 40s, 50s, 60s or any two of the above durations or ranges, and the duration or range between 10min, 20min, 30min or any two of the above durations or ranges.

[0064] In some embodiments, the detection temperature is 25°C to 40°C. Non-limitingly, the detection temperature can be but is not limited to 25°C, 30°C, 35°C, 40°C or a value or range between any two of the above values.

[0065] In some embodiments, the 2-hydroxy acid includes one or more of 2-hydroxy-4-methylthiobutyric acid, glycolic acid, lactic acid, malic acid, and 2-hydroxybutyric acid.

[0066] In a second aspect of the present application, a high-throughput screening method for a protease producing methionine is provided, comprising the following steps:

[0067] Producing methionine under the action of multiple different proteases, using the high-throughput detection method of the first aspect of the present application, simultaneously detecting the content of 2-hydroxy acid in multiple intermediates, and evaluating the catalytic ability of the multiple different proteases for methionine production according to the content of 2-hydroxy acid, thereby high-throughput screening out proteases with strong catalytic ability;

[0068] Wherein, the 2-hydroxy acid is 2-hydroxy-4-methylthiobutyric acid;

[0069] The intermediate product includes the supernatant of the fermentation broth for producing methionine after solid impurities are removed.

[0070] In some embodiments, the intermediate product may include one or more of the by-products methylthiopropionaldehyde, methylthiopropionic acid, and methylthiopropanol.

[0071] In some embodiments, the step of simultaneously detecting the content of 2-hydroxy acid in multiple intermediate products includes: placing the intermediate products on an ELISA plate and detecting them using an ELISA instrument.

[0072] In some embodiments, the ELISA plate is a 96-well ELISA plate.

[0073] Some examples are provided below.

[0074] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0075] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0076] The peroxidase (HRP), 2,2-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 2-hydroxy acid oxidase involved in the following examples were purchased from Beijing Puxitang Biotechnology Co., Ltd., and 2-hydroxymethylthiobutyric acid and other standard products were purchased from Merck Life Sciences Sigma-Aldrich®.

[0077] In the following examples, methionine was synthesized by bioengineering, wherein the construction host used was Escherichia coli DH5α, the expression host used was Escherichia coli BL21 (DE3), the plasmid vector used was pET-28a, and the resistance was kanamycin resistance.

[0078] The specific process of synthesizing methionine using bioengineering includes:

[0079] Construction of protein expression strain: Based on the target gene and plasmid sequence, specific primers were designed and homology arms were added to perform PCR amplification of the target gene and linearized pET28a plasmid, and then purified and recovered; the two fragments were subjected to one-step homologous recombination to form a recombinant vector; the plasmid was transformed into DH5α competent cells using heat shock, the plasmid was extracted, and then the plasmid was transformed into BL21 (DE3) competent cells using the heat shock method.

[0080] Protein purification process:

[0081] 1. Inducible expression

[0082] The verified recombinant plasmid was transformed into the expression host bacterium Escherichia coli BL21 (DE3), and a single colony was picked and inoculated into LB liquid medium containing kanamycin. The culture was shaken at 37°C and 200 rpm overnight to obtain seed liquid. Then, the seed liquid was inoculated into fresh LB liquid medium containing kanamycin at a certain ratio (such as 1:100), and the culture was continued until the OD 600 When the p-value reaches 0.6 - 0.8, IPTG (isopropyl-β-D-thiogalactoside) is added to induce expression. The final concentration of IPTG is 0.1 mM and the induction is carried out at 16°C for 16 hours.

[0083] 2. Cell collection and disruption

[0084] After induction, the bacterial solution was centrifuged at 4°C and 8000 rpm for 10 minutes to collect the cells and discard the supernatant. The cells were resuspended in PBS buffer and the cells were broken by ultrasonic disruption to release the protein. The ultrasonic disruption was set for 3 seconds, 7 seconds interval, and a total of 20 minutes. After disruption, the cells were centrifuged at 4°C and 8000 rpm for 30 minutes and the supernatant was collected.

[0085] 3. Nickel column affinity purification

[0086] Column pretreatment: Wash the column with ultrapure water and then equilibrate the column with binding buffer containing 20 mM imidazole, pH 7.5.

[0087] Loading: Slowly load the collected sample containing the target protein onto the equilibrated nickel column to allow the target protein to specifically bind to the nickel ions on the nickel column.

[0088] Washing: After loading, continue to wash the column with binding buffer containing 50 mM imidazole to wash away unbound proteins.

[0089] Elution: Use elution buffer containing 300 mM imidazole for elution, and collect the eluate, which is the purified protein solution.

[0090] The 96-well ELISA plate used in the high-throughput screening process in the following examples is a sterile 96-well ELISA plate, and the amount of the reaction system added to the 96-well ELISA plate is 200 μL.

[0091] The oxidases in the following examples are selected from oxidases AvLOX and LctO derived from Aerococcus viridans, oxidase AOX2 derived from Arabidopsis thaliana, and oxidase SoGO derived from Convolvulus sarvensis. The amino acid sequences of the oxidases are shown in Table 1 below.

[0092] Table 1

[0093]

[0094] The dilution buffer involved in the following examples is as follows:

[0095] M9: Na2HPO4 6.78 g / L, KH2PO4 3 g / L, NaCl 0.5 g / L, NH4Cl 1 g / L, MgSO4 2 mM, CaCl2 2.5 mM, Mops.

[0096] The detection methods involved in the following embodiments are as follows:

[0097] Hydrogen peroxide detection method: Place the ELISA plate with the reaction system in an ELISA reader, vibrate the plate for 30 seconds, and measure the absorbance at 420nm. The ELISA plate can be a 96-well plate or more, and multiple samples can be tested at one time.

[0098] HPLC product detection method:

[0099] The sample was centrifuged at 4000 rpm for 10 min, the supernatant was removed, and then the sample was filtered with a 0.22 μm filter membrane. Chromatographic conditions: Waters HPLC chromatograph, chromatographic column is Aminex HPX-87H Column, 300 x 7.8 mm CT-1250140-1, mobile phase is 10 mM sulfuric acid, column temperature is 30°C, flow rate is 0.5 mL / min.

[0100] Example 1: ABTS method for detecting H2O2 standard sample

[0101] Take 100 μL of diluted H2O2 standard samples of different concentrations, 20 μL of 40 U / mL HRP, and 50 μL of 32mM ABTS, make up to 200 μL with ddH2O to form a color development system, add it to the ELISA plate, and detect the absorbance at 420 nm.

[0102] The concentrations of H2O2 standard samples are: 1μM, 0.5μM, 0.25μM, 0.125μM, 0.06μM, and 0.03 μM.

[0103] The experiment found that the detection samples of H2O2 standards with different concentrations quickly changed from light green to dark green. The end point value 420nm absorbance value was linearly correlated with the H2O2 concentration. The lowest detectable H2O2 concentration was 0.03~0.06 μM. The standard curve is shown in Figure 2 As shown, R 2 The value is 0.996. This shows that the absorbance of the colorimetric system is linearly related to the H2O2 concentration. Therefore, in practice, the measurement of H2O2 concentration can be converted into the measurement of system absorbance without affecting the accuracy of H2O2 concentration.

[0104] Example 2: Detection of 2-Hydroxymethylthiobutyric Acid Standard

[0105] (1) Initial condition test:

[0106] Take 100 μL of diluted 2-hydroxymethylthiobutyric acid (desmeninol) standard sample of different concentrations (the dilution solution is M9 culture medium), 20 μL 40 U / mL HRP, 50 μL 32mM ABTS, 30 μL 40 U / mL oxidase (AvLOX), a total volume of 200 μL, add them to the ELISA plate, then put it into the ELISA plate (preheated to 30°C) and vibrate the plate for 30 seconds. After vibrating the plate, detect the absorbance every 50 seconds with the ELISA plate reader for 30 minutes.

[0107] The concentrations of 2-hydroxymethylthiobutyric acid standard samples are: 0 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1 mM, respectively.

[0108] The experiment found that after the mixed reagent was colored at 420nm, an obvious absorption peak would appear, and the linear interval MeanValue (average value) was linearly correlated with the concentration of 2-hydroxymethylthiobutyric acid. The standard curve is as follows: Figure 3 As shown, R 2 The value is 0.9966.

[0109] (2) Reaction temperature and sample concentration test:

[0110] Taking the system in the initial condition test (1) as the benchmark, change the reaction temperature to 37℃ and 30℃, and test the reaction under different conditions. Take the gradient concentration of 2-hydroxymethylthiobutyric acid standard sample for detection, set the temperature of the microplate reader to 37℃ and 30℃, and detect the absorbance at 420 nm within 30 minutes.

[0111] The absorbance value change curve obtained with time is as follows Figure 4 . As can be seen from the figure, at 37°C, the high concentration of 2-hydroxymethylthiobutyric acid curve drops significantly, and the available detection interval is short, making the result unstable. When the reaction temperature is 30°C, the curve has a better linear interval, indicating that 30°C is a more preferred reaction temperature. When the concentration of 2-hydroxymethylthiobutyric acid is 0.3mM~1.0mM, the absorbance value shows a good linear relationship with time, indicating that the concentration of 2-hydroxymethylthiobutyric acid is in the range of 0.3mM~1.0mM, and the detection result is accurate and reliable.

[0112] Example 3: Effects of different concentrations of substrate / byproduct on the detection results of 2-hydroxymethylthiobutyric acid

[0113] Based on the method of Example 2, the substrate methylthiopropionaldehyde for producing 2-hydroxymethylthiobutyric acid and the byproducts methylthiopropanol and methylthiopropionic acid produced in the production were added to the M9 solution to detect the effects of these three compounds on the high-throughput detection method of the target sample 2-hydroxymethylthiobutyric acid. 1 mM~10 mM methylthiopropionaldehyde (methional), methylthiopropionic acid (3-methylthio)propionic acid) and methylthiopropanol (3-methylthiopropaol) were added to the M9 solution, and the Mean V obtained by detection is shown in Table 2. The results show that under the conditions of adding 1 mM methylthiopropionaldehyde, methylthiopropionic acid and methylthiopropanol, there is no significant effect on the high-throughput detection method of 2-hydroxymethylthiobutyric acid, and it does not affect the accuracy of the concentration trend of 2-hydroxymethylthiobutyric acid.

[0114] Table 2

[0115]

[0116] Example 4: Actual sample detection (high-throughput detection verification)

[0117] 80 samples were taken for producing 2-hydroxymethylthiobutyric acid using different proteases for methylthiopropionaldehyde as substrate. The samples were centrifuged at 4000 rpm for 10 minutes at room temperature to precipitate solid impurities such as bacteria and collect the supernatant. The supernatant was then tested by HPLC and the high-throughput method of the present application (according to the method of Example 2). The corresponding relationship between the results of the two methods is shown in FIG. Figure 5 The two were significantly correlated ρ=0.67 (95% CI), indicating that the high-throughput method of the present application was consistent with the HPLC method in accuracy.

[0118] Example 5: Detection of different oxidase contents

[0119] Four samples that had been subjected to HPLC quantitative analysis were selected: C3, C11, C12 and D1, whose 2-hydroxymethylthiobutyric acid concentrations were 0.61 mM, 0.81 mM, 0.89 mM and 0.55 mM, respectively.

[0120] 0.4 U, 0.8 U and 1.2 U of oxidase LctO were added to the 200 μL system (diluted at different times), and the linear interval mean V obtained by microplate reader detection is shown in Table 3. At different oxidase concentrations, the relative values ​​of each sample were consistent with the HPLC detection values, that is, C12>C11>C3>D1, but the addition of 1.2 U had a more significant differentiation.

[0121] Table 3

[0122]

[0123] Example 6: Glycolic Acid Detection

[0124] The high-throughput detection method of the present application was used to detect glycolic acid samples. The concentration of the glycolic acid standard sample was selected to be 0.5~1 mM. According to the method of Example 2, the oxidase was replaced with SoGO, and the same concentration of 2-hydroxymethylthiobutyric acid was used as a control. Glycolic acid can also obtain detection values ​​under this detection method, as shown in Table 4, indicating that the detection method is applicable to a variety of 2-hydroxy acids.

[0125] Table 4

[0126]

[0127] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0128] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the scope of the claims.

Claims

1. A high-throughput detection method for 2-hydroxy acids, characterized in that: The following steps are involved: Mixing the sample to be tested with a color developing reagent to obtain a color developing system, performing a color developing reaction, and detecting the absorbance of the color developing system to obtain the content of 2-hydroxy acid in the sample to be tested; The color developing reagent includes: oxidase, peroxidase and ABTS. Based on the feed amount, the usage ratio of the ABTS, the peroxidase and the oxidase is (38-42) mM: (15-20) U: (28-32) U.

2. The detection method according to claim 1, characterized in that The oxidase is selected from one or more of AvLOX, LctO, AOX2 and SoGO.

3. The detection method according to claim 2, characterized in that The AvLOX, LctO, AOX2 and SoGO have the amino acid sequences shown in SEQ ID NOs: 1-4, respectively.

4. The detection method according to claim 1, characterized in that The volume ratio of the sample to be tested to the color developing reagent is 1:(0.95~1.05).

5. The detection method according to claim 1, characterized in that Relative to the volume of the sample to be tested, the content of the oxidase is 0.4 U to 1.2 U per 100 μL of the sample to be tested.

6. The detection method according to claim 1, characterized in that The sample to be tested is a liquid sample containing 2-hydroxy acid, wherein the concentration of 2-hydroxy acid in the sample to be tested is 0.3 mM to 1.0 mM.

7. The detection method according to claim 6, characterized in that The sample to be tested can be diluted with a buffer to obtain a desired 2-hydroxy acid concentration, wherein the buffer is an M9 buffer, and the M9 buffer includes Na2HPO4, KH2PO4, NaCl, NH4Cl, MgSO4 and CaCl2; Optionally, in the M9 buffer, the concentration of Na2HPO4 is 6.7 g / L~6.9 g / L, the concentration of KH2PO4 is 2.9 g / L~3.1 g / L, the concentration of NaCl is 0.4 g / L~0.6 g / L, the concentration of NH4Cl is 0.9 g / L~1.1 g / L, the concentration of MgSO4 is 1.9 mM~2.1 mM, and the concentration of CaCl2 is 2.4 mM~2.6 mM.

8. The detection method according to any one of claims 1 to 7, characterized in that The step of detecting the absorbance of the color development system to obtain the content of 2-hydroxy acid in the sample to be tested comprises: The linear interval average value of the absorbance of the color development system is detected, and the content of 2-hydroxy acid in the sample to be tested is calculated using the linear interval average value of the absorbance and the standard curve of the 2-hydroxy acid concentration; wherein the R 2 Value greater than or equal to 0.

99.

9. The detection method according to claim 8, characterized in that: The step of detecting the linear interval average value of the absorbance of the color development system comprises: After obtaining the color system, detect the absorbance value of the color system at 420 nm every 30s to 60s for 10min to 30min; Optionally, the detection temperature is 25°C~40°C.

10. The detection method according to any one of claims 1 to 9, characterized in that: The 2-hydroxy acid includes one or more of 2-hydroxy-4-methylthiobutyric acid, glycolic acid, lactic acid, malic acid and 2-hydroxybutyric acid.

11. A high-throughput screening method for a protease producing methionine, characterized in that: The steps include: Producing methionine under the action of multiple different proteases, using the high-throughput detection method for 2-hydroxy acid described in any one of claims 1 to 9, and detecting the content of 2-hydroxy acid in multiple intermediates at the same time, and evaluating the catalytic ability of the multiple different proteases for methionine production according to the content of 2-hydroxy acid, thereby high-throughput screening of proteases with strong catalytic ability; Wherein, the 2-hydroxy acid is 2-hydroxy-4-methylthiobutyric acid; The intermediate product comprises the supernatant of the fermentation broth for producing methionine after solid impurities are removed.

12. The high-throughput screening method for methionine-producing protease according to claim 11, characterized in that: The intermediate product also includes one or more of the by-products methylthiopropionaldehyde, methylthiopropionic acid and methylthiopropanol.

13. The high-throughput screening method for methionine-producing protease according to claim 12, characterized in that: The step of simultaneously detecting the content of 2-hydroxy acid in multiple intermediate products comprises: placing the intermediate products on an ELISA plate and detecting them using an ELISA reader. Preferably, the ELISA plate is a 96-well ELISA plate.

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