Method for detecting activity of lysozyme

Abstract

The invention discloses a method for detecting the activity of lysozyme, which evaluates the activity of the lysozyme by detecting the release amount of nuclease in bacterial content, and realizes high-sensitivity quantification of the lysozyme by designing a special nuclease fluorescent probe substrate. Compared with the traditional nephelometry, the method has the advantages of remarkable sensitivity and robustness. According to the method, natural bacteria are used as a lysozyme substrate, so that the false negative problem that a fluorescence modified bacterial cell wall substrate is difficult to recognize and hydrolyze by part of lysozyme in the existing commodity kit is solved. Compared with an existing fluorescence method commercialized kit, the method has the advantages of being wide in application range and accurate in result.

Description

Technical Field

[0001] This invention patent relates to a method for detecting lysozyme activity, belonging to the field of biotechnology. Background Technology

[0002] Lysozyme is a class of enzymes that hydrolyze bacterial cell wall polysaccharides and is an important raw material enzyme in the food and pharmaceutical industries. Quantitative evaluation of lysozyme activity is crucial for the discovery of new lysozymes, quality control of lysozyme raw materials, and performance evaluation of lysozyme products. Lysozyme is also a component of the normal immune defense mechanism, present in neutrophils, monocytes, and macrophages, as well as in mucosal secretions, and is an important immune indicator enzyme. Lysozyme is absent in the urine of normal individuals. Significant differences in lysozyme activity levels in the serum or body fluids of patients with certain diseases have led to increasing clinical emphasis on lysozyme activity measurement.

[0003] Methods for detecting lysozyme activity include the agar plate method, turbidimetric method, and bacterial cell wall fluorescent labeling method.

[0004] 1) Turbidimetry is currently the most widely used method and is recommended by regulations such as the Chinese Pharmacopoeia. The national standard GB / T 30990-2014 provides detailed operational requirements for this method. Micrococci that lyse walls generally exist in irregular clusters, tetrads, or cubic clusters of bacterial microclusters. This method utilizes the property of lysozyme hydrolyzing the cell wall of micrococci, leading to the dissociation of bacterial microclusters and a decrease in the turbidity of the bacterial suspension. Enzyme activity is determined by detecting changes in absorbance at 450 nm. The sensitivity of this method is approximately 100 U / mL. Turbidimetry does not directly measure lysolytic activity but indirectly evaluates the lysolytic activity of lysozyme by measuring the dissociation of bacterial microclusters. Traditional turbidimetry suffers from poor sensitivity, poor stability, and poor reproducibility. It cannot be used for some high-turbidity solutions, such as tissue grinding solutions.

[0005] 2) The agar method utilizes the property of lysozyme to hydrolyze bacterial cell walls, causing bacterial death. Lysozyme samples are added to agar plates containing bacteria, and the activity of lysozyme is evaluated by observing the dissolution of bacteria near the sample addition point. The agar method directly detects the lysozyme activity by detecting the bacterial death caused by lysozyme. However, the operation is cumbersome, the sensitivity is extremely poor, and the reproducibility is poor. It is generally only used as an alternative to the turbidimetric method for the determination of lysozyme activity in high-turbidity solutions.

[0006] 3) The bacterial cell wall fluorescent labeling method utilizes bacterial cell walls labeled with fluorescent groups as lysozyme substrates. Lysozyme activity is evaluated by detecting changes in fluorescence signal caused by lysozyme hydrolysis of the substrate. This method has been developed into commercially available kits (such as the EnzChek™ Lysozyme Assay Kit). This method overcomes the poor sensitivity and stability of traditional turbidimetric methods. Because the fluorescence signal is not affected by turbidity, it can also be used for the direct quantitative detection of lysozyme in high-turbidity solutions, offering advantages of high sensitivity and high stability. However, because the fluorescent group is labeled onto the cell wall, located at or near the lysozyme recognition site, the fluorescent group of the substrate can affect the recognition of some lysozymes by the bacterial cell wall, leading to undetectable lysozyme activity and false negative results. For complex substrates like bacterial cell walls, the poor process stability can easily lead to unstable test results.

[0007] 4) Bacterial contents can serve as an indicator of bacterial disruption, and lysozyme activity can be evaluated by detecting the amount of bacterial contents released. Patent CN118599958A provides a lysozyme activity assay kit for detecting ATP content in cell contents; this method is based on the reaction of luciferase catalyzing ATP, oxygen, and D-luciferin to generate a luminescent product, which is then quantified using a special chemiluminescence detection device. This reaction is a flash-type luminescence detection, requiring immediate signal measurement after adding the working solution to the sample, and the luminescence signal decays rapidly within approximately 10 minutes of the reaction. As the data provided in this patent indicates, the activity stability interval of this reaction is less than 5 minutes (5-10 minutes). Figure 3 Therefore, only a small number of samples can be tested immediately, resulting in poor reliability and stability of the method. Furthermore, this reaction is a redox reaction and is easily affected by the oxygen concentration in the air; the presence of interfering substances such as ADP in the cell lysate also affects the reaction stability. Summary of the Invention

[0008] The purpose of this invention is to provide a rapid, stable, and sensitive evaluation method for detecting lysozyme activity.

[0009] The technical solution adopted in this invention is as follows: A method for detecting lysozyme activity, comprising the following steps: 1) Perform serial dilutions of the lysozyme standard, add the serially diluted lysozyme standard to the bacterial substrate, and incubate to obtain bacterial lysate; 2) Add a fluorescent-quenched oligonucleotide probe as an activity indicator to the bacterial lysate, detect the fluorescence value of the hydrolysis product of the activity indicator, and establish a standard curve between the enzyme activity of lysozyme and the fluorescence value of the hydrolysis product. 3) Incubate the lysozyme sample to be tested with the bacterial substrate under the same conditions as in step 2) to obtain the bacterial lysate to be tested. Add a fluorescent-quenched oligonucleotide probe with the same concentration as in step 1) to the bacterial lysate to be tested as an activity indicator. Detect the fluorescence value of the hydrolysis product of the activity indicator. Read the enzyme activity of the lysozyme to be tested according to the standard curve obtained in step 2). The fluorescent-quenched oligonucleotide probe described herein can be recognized and cleaved by non-specific nucleases in bacterial cells. Before cleavage, the fluorescent-quenched oligonucleotide probe does not release fluorescence, while after cleavage, it releases fluorescence.

[0010] Preferably, the fluorescent-quenched oligonucleotide probe is a nucleic acid molecule labeled with a fluorescent group and a quenching group, wherein the nucleic acid portion is a single-stranded oligonucleotide and its nucleotide sequence is shown in any one of SEQ ID No. 1-3; or the nucleic acid portion is a single-stranded oligonucleotide with a hairpin structure and its nucleotide sequence is shown in any one of SEQ ID No. 5-11.

[0011] Preferably, the fluorescent group of the fluorescence-quenched oligonucleotide is the FAM group, and the quenching group is the BHQ1 group.

[0012] Preferably, the incubation conditions in step 1) are: incubation at 16-37℃ for 0.5-2 hours.

[0013] Preferably, after adding the fluorescent-quenched oligonucleotide probe in step 2), the mixture is incubated at 16-37°C for 0.5-2 hours, and then EDTA solution is added to terminate the reaction. The fluorescence value of the hydrolysis product is then detected.

[0014] This invention also discloses the application of fluorescent-quenched oligonucleotide probes in the detection of lysozyme activity, wherein the fluorescent-quenched oligonucleotide probe is a nucleic acid molecule labeled with a fluorescent group and a quenching group, wherein the nucleic acid part is a single-stranded oligonucleotide with a nucleotide sequence as shown in any one of SEQ ID No. 1-3; or the nucleic acid part is a single-stranded oligonucleotide with a hairpin structure with a nucleotide sequence as shown in any one of SEQ ID No. 5-11, and the fluorescent-quenched oligonucleotide probe can be recognized and cleaved by non-specific nucleases in bacterial cells, the fluorescent-quenched oligonucleotide probe does not release fluorescence before cleavage, and releases fluorescence after cleavage.

[0015] Preferably, the fluorescent group of the fluorescence-quenched oligonucleotide is the FAM group, and the quenching group is the BHQ1 group.

[0016] This invention also discloses another method for detecting lysozyme activity, which evaluates lysozyme activity by assessing the degree of bacterial lysis caused by lysozyme treatment, and the steps include: 1) Add a certain concentration of fluorescent-quenched oligonucleotide probe as an activity indicator to a mixed turbid solution of bacterial lysate and bacteria, detect the fluorescence value of the hydrolysis product of the activity indicator, and establish a functional relationship between the degree of bacterial lysis and the fluorescence value; the bacterial lysate is a solution in which bacteria have been completely lysed by physical, chemical or biological methods. 2) Incubate the lysozyme sample to be tested with bacteria to form a partially or completely lysed bacterial lysate-bacterial mixed turbidity. Add a fluorescent-quenched oligonucleotide probe with the same concentration as in step 1) to the bacterial lysate-bacterial mixed turbidity as an activity indicator. Detect the fluorescence value of the hydrolysis product of the activity indicator. Based on the functional relationship obtained in step 1), read the degree of bacterial lysis of the lysozyme to be tested. The fluorescent-quenched oligonucleotide probe described herein can be recognized and cleaved by non-specific nucleases in bacterial cells. Before cleavage, the fluorescent-quenched oligonucleotide probe does not release fluorescence, while after cleavage, it releases fluorescence.

[0017] The physical methods include, but are not limited to, ultrasonic disruption, high-pressure homogenization, freeze-thaw cycles, immersion in hypotonic solutions, immersion in hypertonic solutions, or other methods that disrupt bacteria by oscillation, grinding, or changes in osmotic pressure, or a combination of at least one of the above methods; the chemical methods include, but are not limited to, methods that use surfactants, organic salts, acids, alkalis, or other chemical reagents to treat cells and cause them to lyse, or methods that combine at least one of the above preparations to treat cells and cause them to lyse; the biological methods refer to methods that use lysozyme, protease, lipase, or other biological enzymes to treat cells and cause them to lyse, or a combination of at least one of the above methods; and bacterial disruption methods that combine at least one of the physical, chemical, and biological methods.

[0018] Preferably, the fluorescent-quenched oligonucleotide probe can be recognized and cleaved by non-specific nucleic acids, and is a nucleic acid molecule labeled with a fluorescent group and a quenching group, wherein the nucleic acid portion is a single-stranded oligonucleotide, and its nucleotide sequence has 100% or greater than 90%, 80%, 70%, 60%, 50%, or 40% identity with the nucleotide sequence shown in any one of SEQ ID Nos. 1-3; or the nucleic acid portion is a single-stranded oligonucleotide with a hairpin structure, wherein the hairpin structure refers to the oligonucleotide portion being able to complementaryly pair to form a double-stranded region, and at least one base forming a single-stranded region in a single-stranded form, the single-stranded region being located on one side, both sides, or in the middle of the double-stranded region; or the double-stranded region is 8-20 bp in length, and the single-stranded region is 1-20 nt in length; or the double-stranded region is 10-15 bp in length, and the single-stranded region is 1-5 nt in length; or the nucleotide sequence of the nucleic acid portion is as shown in any one of SEQ ID Nos. 5-11; or the nucleotide sequence of the nucleic acid portion is identical to that of SEQ ID No. Any one of the comparisons in 5-11 is greater than 90%, 80%, 70%, 60%, 50%, or 40% and can form a hairpin structure.

[0019] Preferably, the steps specifically include: (1) Prepare a bacterial lysis buffer standard by mixing the bacterial lysate with the undiluted bacterial turbidity; mix the bacterial lysis buffer standard, the fluorescent-quenched oligonucleotide probe, and the nuclease activity detection buffer, incubate at 16-37℃ for 0.5-2 hours, add EDTA solution to terminate the reaction, detect the amount of fluorescent hydrolysis product generated, and plot a standard curve of lysis-fluorescent product amount. The method for determining the amount of fluorescent product generated is to use the probe fluorescent group or a compound with similar spectral properties as a standard, plot the product content-fluorescence standard curve, and substitute the sample fluorescence value into the product content-fluorescence standard curve to calculate the product content; (2) Add lysozyme sample to bacterial turbidity and incubate at 16-37℃ for 0.5-2 hours to obtain bacterial enzymatic hydrolysate; (3) React the bacterial enzymatic hydrolysate under the same conditions as (1) and the bacterial lysate standard, and detect the amount of hydrolysis products generated in the sample; (4) Substitute the amount of sample hydrolysis products into the standard curve of lysate-fluorescence product amount to calculate the degree of bacterial lysis. Use the amount of lysed bacteria per unit time as the standard to evaluate lysozyme activity.

[0020] Preferably, the fluorescence-quenched oligonucleotide probe has FAM and BHQ1 groups, and the corresponding hydrolysis product is FAM.

[0021] When bacterial lysate standard samples and test samples are on the same detection plate and their fluorescence values ​​are measured simultaneously, a simplified method of the present invention is to use the detected fluorescence value as an indicator of the amount of fluorescent hydrolysis products generated.

[0022] The fluorescent products described above are fluorescent groups of fluorescent-quenched oligonucleotide probes or compounds with similar fluorescence spectra.

[0023] This invention also discloses a fluorescent-quenched oligonucleotide probe with a hairpin structure for quantifying nonspecific nuclease activity. The fluorescent-quenched oligonucleotide can be specifically recognized and cleaved by intracellular bacterial nonspecific nucleases. Before cleavage, the fluorescent-quenched oligonucleotide probe does not release fluorescence, while after cleavage, it releases fluorescence. The hairpin structure refers to a region where the oligonucleotide portion can complementaryly pair to form a double-stranded region, and at least one base exists in single-stranded form to constitute a single-stranded region. The single-stranded region is located on one side, both sides, or in the middle of the double-stranded region. The double-stranded region is 8-20 bp in length, and the single-stranded region is 1-20 nt; or the double-stranded region is 10-15 bp in length, and the single-stranded region is 1-5 nt in length; or the nucleotide sequence of the nucleic acid portion is as shown in any one of SEQ ID No. 5-11, or the nucleotide sequence identity of the nucleic acid portion is greater than 90%, 80%, 70%, 60%, 50%, or 40% compared to any one of SEQ ID No. 5-11 and can form a hairpin structure.

[0024] The present invention also discloses the application of the above-mentioned fluorescent-quenched oligonucleotide probe in the detection of lysozyme activity.

[0025] The present invention also discloses a lysozyme activity assay kit, comprising the above-mentioned fluorescent-quenched oligonucleotide.

[0026] The present invention also discloses a non-specific nuclease activity detection kit or a nuclease residue analysis kit, comprising the above-mentioned fluorescent-quenched oligonucleotide probe.

[0027] The instructions for using the kit are as follows: 1) Incubate the nuclease with the above-mentioned fluorescent-quenched oligonucleotide probe at 16-60℃ for 15-120 minutes; 2) Terminate the reaction using EDTA or other metal chelating agents, proteases, inorganic bases, organic bases, organic acids, or inorganic acids; 3) Plot a concentration-fluorescence standard curve using the fluorescent group or a modification of the fluorescent group, or a compound having the same or similar fluorescence as the fluorescent group, from the fluorescence-quenched oligonucleotide probe in 1). 4) Calculate the amount of fluorescent product generated corresponding to the fluorescence of the nuclease sample using the standard curve described in 3).

[0028] The present invention also discloses a method or kit for analyzing the degree of cell lysis, comprising the above-mentioned fluorescent-quenched oligonucleotides.

[0029] During lysozyme lysis of cells or other cell lysis processes, bacterial contents are released. All cells contain nucleases, which can be used as typical cellular contents to assess the degree of cell lysis or fragmentation. This invention uses nucleases as markers of cell lysis, assessing the degree of bacterial fragmentation caused by lysozyme by detecting the amount of nuclease released, i.e., lysozyme activity. Through the design and screening of fluorescent nuclease probe substrates, accurate quantification and high linear range detection of bacterial nucleases are achieved, improving the sensitivity and stability of lysozyme activity assessment.

[0030] This invention presents a method for detecting lysozyme activity using natural bacteria as a substrate. It addresses the false-negative problem in existing commercially available fluorescent lysozyme activity detection kits, where the bacterial cell wall cannot be recognized by some lysozymes when fluorescent modification groups are present on the cell wall. The method of this invention has broader applicability.

[0031] The fluorescent substrate hydrolysis signal used in this invention has the characteristics of high sensitivity and no interference from material turbidity. Compared with the traditional turbidimetric method for detecting lysozyme activity, the method of this invention has advantages in sensitivity and reliability.

[0032] This invention uses nucleases as bacterial content markers and, through a specially designed probe, achieves high sensitivity and stability in the detection reaction. It also ensures batch-to-batch consistency through traceable fluorescent product standards, overcoming the low reliability and inability to compare batch-to-batch data issues of existing patents that use luciferase as a content marker and employ unstable redox reactions to detect luciferase activity. This method offers a simple, stable, and highly reproducible enzyme activity detection process.

[0033] This invention utilizes fluorescent product standards to trace the fluorescence intensity of the reaction product back to the amount of fluorescent product generated, directly calculating the released nuclease activity (the amount of product generated per unit time). This solves the problem of poor batch-to-batch consistency caused by the susceptibility of relative fluorescence intensity values ​​directly measured by fluorescence detection equipment to excitation light intensity and equipment parameters. Because this method uses traceable fluorescent product standards, it offers advantages such as traceable results, high batch-to-batch consistency, and high reproducibility.

[0034] One derivative of this invention is to directly use the fluorescent probes of the invention to detect the activity of sensitive nucleases for quantitative or residual detection of nuclease activity.

[0035] Another derivative of this invention is to use the fluorescent probes of this invention to detect nuclease activity in cell lysates, thereby evaluating the cell lysis capabilities of other cell lysis enzymes (such as lysozyme or chitinase), compounds (such as surfactants), or cell lysis processes (such as ultrasound or high-pressure homogenization). Attached Figure Description

[0036] Figure 1 The results are for the lysozyme activity assay in Example 5.

[0037] Figure 2 This is a standard curve for the lysozyme activity detection in Example 5.

[0038] Figure 3 The figure shows the comparison results of two methods for detecting different lysozyme activities in Example 6. The inset shows the fluorescence distribution of E. coli expressing fluorescent protein after treatment with lysozyme 38180 and control solutions. Detailed Implementation

[0039] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0041] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0042] Example 1: Preparation of bacterial substrate Escherichia coli BL21 was cultured overnight at 37°C in LB medium. The bacteria were washed three times by centrifugation with a bacterial wash buffer of 20 mM Tris-HCl, 150 mM NaCl, and 5 mM MgCl2. The bacterial cells were collected by centrifugation and then diluted to OD using bacterial wash buffer. 600 =0.1~5.0, which is the bacterial substrate.

[0043] Example 2: Nuclease substrate design Bacteria express numerous nonspecific nucleases during their life cycle for DNA molecule processing and degradation. These nucleases are highly active and generally do not exhibit sequence bias, thus they are selected as indicator enzymes for bacterial lysis. *Serratia marcescens* is a common Gram-negative bacterium. *Serratia marcescens* nonspecific nucleases (Sinochem Biotech, catalog number SSNP01) are widely used for the efficient removal of residual host nucleic acids and are among the most active nucleases currently available. Therefore, they have been selected as model molecules for bacterial nucleases in the development of lysozyme activity detection probes.

[0044] The DNA molecule's molecular chain structure (single-stranded, double-stranded, or other complex structures) and chain length both affect the recognition and further hydrolysis efficiency of nucleases. For example, excessively short nucleic acids may prevent nucleases from recognizing them, while excessively long nucleic acids may trigger multiple cleavages, leading to decreased method stability. We investigated the sensitivity of fluorescent probes with different structures and lengths. As is easily understood, non-specific nucleases generally do not possess significant sequence specificity, so we designed only one probe for each type.

[0045] To obtain highly sensitive fluorescent probes, single-stranded probes 1-3 (SEQ ID No. 1-3) with a length of 6-20 nt and probes 4-11 (SEQ ID No. 4-11) with a hairpin structure were designed. The 5' end of the probes was modified with the fluorescent group FAM, and the 3' end was modified with the quencher group BHQ1. To ensure that the probes could form an ideal structure, the single-stranded probes were selected to consist entirely of A and C bases to avoid forming a complementary double-stranded structure. The double-stranded portion of the hairpin probes used irregular A / C or T / G spacing to form two complementary bridging arms, with the middle single-stranded region dominated by A / T to facilitate the formation of a double hairpin structure probe.

[0046] After incubating the 10 μM probe with bacterial nonspecific nuclease for 30-60 minutes, immediately terminate the reaction with 0.2-0.5 M EDTA, detect the characteristic fluorescence of the FAM fluorescent group, plot a standard curve using FAM standards, calculate the amount of FAM fluorescent product generated, and divide by the reaction time to obtain the probe hydrolysis rate.

[0047] Bacterial nonspecific nucleases were serially diluted 5-fold, and the linear relationship between nuclease content and fluorescent product amount was detected according to the above method. The coefficient of determination R for linear fitting was used. 2 As a metric, R 2 The closer it is to 1, the better the linear relationship.

[0048] As shown in Table 1, hairpin probes exhibit higher sensitivity and are more easily hydrolyzed by bacterial non-specific nucleases than single-stranded probes, resulting in higher probe activity. For hairpin probes, linearity decreases significantly when the double-stranded region is less than 10 bp, and sensitivity decreases significantly when the double-stranded region is less than 8 bp. Probes with double-stranded regions of 10-15 bp show the highest activity, while single-stranded regions of 1-5 bp also exhibit high activity. Considering both probe activity and linearity, probes 6, 7, and 10 are preferred. It can be seen that probes with double-stranded regions of 10-15 bp and single-stranded regions of 3-5 bp have the highest activity and linearity. Probe 11 exhibited multiple cleavage issues in subsequent experiments; although the initial hydrolysis rate was fast, it ceased to produce fluorescence signals after a certain period, therefore it was not considered a preferred probe.

[0049] Table 1 Results of nuclease hydrolysis probe 1-11

[0050] Single-chain probe Probe 1: FAM-AGAGAG-BHQ1 Probe 2: FAM-ACACACAC-BHQ1 Probe 3: FAM- ACACACACACACACACACAC-BHQ1 Hairpin structure probe (double-chain regions are represented by uppercase letters, and single-chain regions are represented by lowercase letters). Probe 4: FAM-ACCACAatataTGTGGT-BHQ1 Probe 5: FAM-ACCACACAatataTGTGTGGT-BHQ1 Probe 6: FAM-AAGGAGGAGAataTCTCCTCCTT-BHQ1 Probe 7: FAM-ACCAACACCACAatataTGTGGTGTTGGT-BHQ1 Probe 8: FAM-AGAAGGAAGAGGAGAatataTCTCCTCTTCCTTCT-BHQ1 Probe 9: FAM-AACCACCACAtatTGTGGTGGTT-BHQ1 Probe 10: FAM-AGGAAGAGGAGAtatTCTCCTCTTCCT-BHQ1 Probe 11: FAM-ACAACCAACACCACAaTGGTGGTGTTGGTTGT-BHQ1.

[0051] Example 3: Escherichia coli lysate Lysozyme hydrolyzes bacteria, causing bacterial lysis and releasing intracellular nucleases. We mixed completely lysed bacteria with bacteria to form partially lysed bacterial lysates, which were used to simulate partially or completely lysed E. coli solutions. We then determined the relationship between the bacterial lysis ratio and the release of nucleases.

[0052] Completely break down OD using a high-pressure homogenizer 600 = 1.0 Escherichia coli BL21 bacterial fragments were obtained. The bacterial fragments and bacteria were added to a buffer containing 10 μM probe 7 (100 μL system, 20 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 5 mM MgCl2) according to the amounts specified in Table 2. The mixture was incubated at room temperature for 20 minutes, and the reaction was terminated by adding 100 μL of 0.5 mM EDTA-Na2 buffer. The mixture was centrifuged at 12,000 rpm for 5 minutes, and 100 μL of the supernatant was added to the same 96-well plate for fluorescence detection.

[0053] The results showed a linear positive correlation between the bacterial disruption ratio and nuclease activity. The nuclease activity assay method of this invention can be used to quantitatively assess the degree of bacterial disruption.

[0054] Table 2 Relationship between bacterial fragment volume and fluorescence value of active indicator product

[0055] Example 4 Lysozyme Activity Detection Commercial food-grade egg white lysozyme was dissolved to a concentration of 10 mg / mL using a lysozyme diluent consisting of 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5 mM MgCl2 to obtain the lysozyme stock solution.

[0056] Treatment Group 1: The lysozyme stock solution was diluted to 10, 30, 60, and 100 μg / mL using lysozyme diluent, respectively. After incubation with bacterial substrate for 60 minutes, 10 μL of the incubation product was added to a nuclease activity assay buffer containing 10 μM probe 7 (100 μL system, 20 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 5 mM MgCl2), and incubated for 40 minutes. The reaction was terminated by adding 50 μL of 0.5 mM EDTA-Na2 buffer, and the FAM fluorescence was detected. The amount of FAM generated was calculated by comparing the fluorescence with that of the FAM standard.

[0057] Treatment Group 2: The lysozyme stock solution was diluted to 10, 30, 60, and 100 μg / mL using lysozyme diluent, respectively. After incubation with bacterial substrate for 60 minutes, 20 μL of the incubation product was added to a nuclease activity assay buffer containing 10 μM probe 7 (100 μL system, 20 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 5 mM MgCl2). The mixture was incubated for 20 or 40 minutes, and the reaction was terminated by adding 50 μL of 0.5 mM EDTA-Na2 buffer. The FAM fluorescence was detected, and the amount of FAM generated was calculated by comparing the fluorescence with that of the FAM standard.

[0058] As shown in Table 3, the probe hydrolysis products are positively correlated with the amount of lysozyme added, the amount of incubation products added, and the probe incubation time.

[0059] It is easy to understand that, given a fixed detection limit, the detection sensitivity can be improved by extending the probe incubation time and increasing the amount of incubation product added.

[0060] Table 3 Relationship between the amount of incubation product added, probe incubation time, and amount of hydrolysis product

[0061] Example 5 Sensitivity and Linearity of Lysozyme Activity Detection Commercially available food-grade egg white lysozyme (200,000 U / mL) was dissolved in a lysozyme diluent containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 5 mM MgCl2. This solution was then serially diluted twofold to prepare lysozyme diluents with activity concentrations ranging from 6.1 to 100,000 U / mL. Each diluent was incubated with the bacterial substrate for 40 minutes. 10 μL of the incubation product was then added to a 100 μL nuclease activity assay buffer containing 10 μM probe 7 (100 μL system: 20 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 5 mM MgCl2) and incubated for 60 minutes. The reaction was terminated by adding 50 μL of 0.5 mM EDTA-Na2 buffer, and FAM fluorescence was detected. The results are as follows: Figure 1 As shown.

[0062] The range of 6.1–390.6 U / mL was linear, and the fitted standard curve is shown below. Figure 2 As shown, R 2 The linearity is 0.993, the linear range is wide, and the limit of quantitation is less than 6.1 U / mL, which is superior to other existing methods.

[0063] Sensitivity is primarily affected by the lower limit of detection of the equipment. It is easy to understand that the sensitivity of this method can be further improved by increasing the reaction time or the amount of incubated product.

[0064] Example 6: Detection of the activity of the new lysozyme Homologous protein sequences of egg white lysozyme and bacteriophage lysozyme were searched in the database. These lysozymes were recombinantly expressed in a cell-free system using fermentation supernatant. The methods of this invention (using probe 7 as an indicator probe) and the commercially available EnzChek were used respectively. TM A lysozyme assay kit (fluorescence method) is used to test the activity of various lysozymes. Results are as follows: Figure 3 As shown.

[0065] Enzyme-RFU standard curves were plotted using the method described in Example 5. Lysozyme activity was calculated based on the fluorescence data of each lysozyme sample.

[0066] According to EnzChek TM The instructions for the Lysozyme Assay Kit (Fluorescence Method) are as follows: Determine the activity of each lysozyme sample.

[0067] The method of this invention was found to be similar to that of EnzChek. TM The trend of lysozyme activity detected by the kit was generally consistent with the sensitivity, but EnzChek TM The activity of the two lysozymes in samples 38180 and 38169 could not be detected.

[0068] To confirm that 38180 indeed possesses lytic activity, *E. coli* expressing fluorescent protein were incubated with 38180 and a control negative buffer for a period of time, followed by centrifugation to settle the bacterial cells. Figure 3 As shown in the small image in the upper right corner, the fluorescence in the control negative buffer sample is concentrated in the precipitate, while fluorescence can be observed in both the precipitate and supernatant of the bacteria treated with lysozyme sample 38180. This indicates that 38180 has lysozyme activity.

[0069] Therefore, the method of the present invention overcomes the limitations of commercially available high-sensitivity reagent kits such as EnzChek. TM The problem of false negatives.

[0070] Example 7 Comparison of different cell lysis processes The lysis ability of different cell lysis processes on the same bacterial sample was tested using the method of this invention.

[0071] Process 1: Homogenize 5 times using a high-pressure homogenizer; Treatment 2: Repeated freeze-thaw cycles 5 times; Treatment 3: Add 1 mg / mL egg white lysozyme and incubate for 30 minutes; Treatment 4: Add 1 mg / mL lysozyme 38180 and incubate for 30 minutes; Treatment 5: Incubate with 1 mg / mL chitinase (fungal cell lysin) for 30 minutes.

[0072] Take 10 μL of the supernatant from each treatment and add it to a 96-well plate containing 90 μL of reaction solution (10 μM probe 7, 20 mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, 5 mM MgCl2). Incubate at room temperature for 20 minutes, then add 100 μL of 0.5 mM EDTA-Na2 buffer to terminate the reaction and detect FAM fluorescence.

[0073] As shown in Table 4, high-pressure homogenization five times resulted in the best cell lysis effect. Lysozyme 38180 was more effective than egg white lysozyme treatment and repeated freeze-thaw treatment in lysing E. coli. Chitinase could hardly lyse E. coli.

[0074] Table 4 Comparison of the effects of different cell lysis processes

[0075] Example 8: Method Stability Study This invention directly calibrates lysozyme activity by evaluating the content of nucleases released after bacterial lysis. Lysozyme standards can be used as a reference; a standard curve is plotted by diluting the lysozyme standards, and regression calculations are performed on the activity of the test sample. This invention also provides an improved method that directly quantifies the content of released nucleases using FAM standards, and then evaluates the bacterial lysis amount, i.e., lysozyme activity, by the amount of nuclease released per unit time. This embodiment aims to evaluate the method stability of the two approaches using the commercially available EnzChek kit. TM The results from the lysozyme assay kit (fluorescence method) are for reference only.

[0076] Lysozyme standards were aliquoted into different PCR tubes and frozen at -80°C to serve as lysozyme samples. One tube was taken out for each test.

[0077] Lysozyme and FAM standards were placed in a constant temperature incubator in the dark and subjected to accelerated spoilage at 37°C for 1 and 3 days, respectively. Lysozyme and FAM standards without accelerated spoilage were used as controls. Lysozyme activity was detected using the method of this invention and fluorescence method, respectively, with both lysozyme and FAM standards. The results are shown in Table 5. Because the lysozyme standard was unstable at 37°C for an extended period, the results in the treated groups were too high, and in some cases, undetectable. In contrast, the FAM standard was stable and exhibited high temperature tolerance.

[0078] Table 5 Results of the method stability study

[0079] The present invention has been illustrated with the above embodiments to explain the detailed method of the present invention. However, the present invention is not limited to the detailed method described above, that is, it does not mean that the present invention must rely on the detailed method described above to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for detecting lysozyme activity, characterized in that, The steps include: 1) Perform serial dilutions of the lysozyme standard, add the serially diluted lysozyme standard to the bacterial substrate, and incubate to obtain bacterial lysate; 2) Add fluorescent-quenched oligonucleotide probes as activity indicators to bacterial lysate, detect the fluorescence value of the hydrolysis products of the activity indicator, and establish a standard curve between the enzyme activity of lysozyme and the fluorescence value of the hydrolysis products. 3) Incubate the lysozyme sample to be tested with the bacterial substrate under the same conditions as in step 1) to obtain the bacterial lysate to be tested. Add a fluorescent-quenched oligonucleotide probe with the same concentration as in step 2) to the bacterial lysate to be tested as an activity indicator. Detect the fluorescence value of the hydrolysis product of the activity indicator. Read the enzyme activity of the lysozyme to be tested according to the standard curve obtained in step 2). The fluorescent-quenched oligonucleotide probe described herein can be recognized and cleaved by non-specific nucleases in bacterial cells. Before cleavage, the fluorescent-quenched oligonucleotide probe does not release fluorescence, while after cleavage, it releases fluorescence.

2. The method according to claim 1, characterized in that, The fluorescent-quenched oligonucleotide probe is a nucleic acid molecule labeled with a fluorescent group and a quenching group, wherein the nucleic acid part is a single-stranded oligonucleotide with a nucleotide sequence as shown in any one of SEQ ID No. 1-3; or the nucleic acid part is a single-stranded oligonucleotide with a hairpin structure with a nucleotide sequence as shown in any one of SEQ ID No. 5-11.

3. The method according to claim 2, characterized in that, The fluorescent group of the fluorescent-quenched oligonucleotide is the FAM group, and the quenching group is the BHQ1 group.

4. The method according to any one of claims 1-3, characterized in that, The incubation conditions in step 1) are: 16-37℃ for 0.5-2 hours.

5. The method according to claim 4, characterized in that, In step 2), after adding the fluorescent-quenched oligonucleotide probe, incubate at 16-37℃ for 0.5-2 hours, then add EDTA solution to terminate the reaction, and detect the fluorescence value of the hydrolysis product.

6. Application of fluorescent-quenched oligonucleotide probes in the detection of lysozyme activity, wherein the fluorescent-quenched oligonucleotide probe is a nucleic acid molecule labeled with a fluorescent group and a quenching group, wherein the nucleic acid part is a single-stranded oligonucleotide with a hairpin structure, and its nucleotide sequence is shown in any one of SEQ ID No. 5-11, and the fluorescent-quenched oligonucleotide probe can be recognized and cleaved by non-specific nucleases in bacterial cells, the fluorescent-quenched oligonucleotide probe does not release fluorescence before cleavage, and the fluorescent-quenched oligonucleotide probe releases fluorescence after cleavage.

7. The application according to claim 6, characterized in that, The fluorescent group of the fluorescent-quenched oligonucleotide is the FAM group, and the quenching group is the BHQ1 group.

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