Lysine incision enzyme activity detection method and application thereof

Abstract

The invention provides a lysine endonuclease enzyme activity detection method and application thereof, and the lysine endonuclease enzyme activity detection method comprises the following steps: mixing a substrate solution, a buffer solution and a to-be-detected enzyme solution, then adopting ultraviolet-visible spectrophotometry for detection, and calculating the enzyme activity of to-be-detected lysine endonuclease according to the detection result. The detection method provided by the invention can be realized only by a conventional ultraviolet-visible spectrophotometer, is low in equipment requirement, low in cost and high in detection efficiency, can complete detection only in 5-10 minutes, is simple and standardized in overall detection process, reduces professional requirements, and is suitable for popularization and application. The method has the advantages of strong specificity, high precision, good reproducibility, small relative standard deviation, wide linear range and low detection limit, and can meet the quality control requirements of scientific research and industrial application.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of enzyme activity detection, and particularly relates to a lysine endonuclease enzyme activity detection method and application thereof. BACKGROUND

[0002] Currently, the detection related to lysine protease is mainly applied to proteomics research, rather than enzyme activity detection itself. The technical characteristics of the existing enzyme activity detection methods are as follows:

[0003] Mass spectrometry: LC-MS / MS technology is used to detect enzyme products, the detection precision is high, multiple products can be analyzed at the same time, but the equipment investment is large (usually more than 1 million yuan), the operation is complex, the single detection time is 15-30 minutes, and the detection cost is 50-100 yuan / time.

[0004] High performance liquid chromatography: HPLC is used to separate and detect enzyme products, the technology is relatively mature, the equipment investment is 20-50 million yuan, the single detection time is 30-60 minutes, the detection cost is 20-30 yuan / time, but the detection flux is limited.

[0005] Existing colorimetric method: A few research reports the detection method based on spectrophotometry, but lacks standardized operation process, the comparability of results between different laboratories is poor, and the precision is general.

[0006] The above methods generally have high detection technical threshold, strict equipment requirements, high detection cost, and lack of clear standards. Therefore, how to provide a lysine protease enzyme activity detection method with low cost, high detection efficiency and high standardization has become a problem to be solved. SUMMARY

[0007] In view of the deficiencies of the prior art, the purpose of the present application is to provide a lysine endonuclease enzyme activity detection method and application thereof. The detection method provided by the present application can be realized by using a conventional ultraviolet-visible spectrophotometer, has low equipment requirements, low cost, high detection efficiency, and can complete the detection in 5-10 minutes, has a simple and standardized overall detection process, reduces the professional requirements, has strong method specificity, high precision, good reproducibility, small relative standard deviation, wide linear range and low detection limit, and can meet the quality control requirements of scientific research and industrial application.

[0008] In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0009] On the one hand, the present application provides a lysine endonuclease (Lys-C enzyme) enzyme activity detection method, which comprises the following steps:

[0010] The substrate solution, the buffer and the enzyme solution to be detected are mixed, and then the ultraviolet-visible spectrophotometry is used for detection, and the enzyme activity of the lysine endo-enzyme to be detected is calculated according to the detection result.

[0011] The above method only needs a conventional ultraviolet-visible spectrophotometer, has low equipment requirement and low cost, high detection efficiency, and can complete the detection in 5-10 minutes, and has simple and standardized overall detection process, without complex sample pretreatment and post-treatment steps, reduces professional requirements, and has strong specificity, high precision, good reproducibility, small relative standard deviation, wide linear range and low detection limit, and can meet the quality control requirements of scientific research and industrial application.

[0012] Preferably, the substrate comprises N-acetyl-L-lysine p-nitroaniline.

[0013] The above specific substrate can effectively improve the detection effect and improve the specificity and reproducibility of the detection.

[0014] Preferably, the concentration of the substrate solution is 0.8-1.2 mM, for example, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM or 1.2 mM, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0015] Preferably, the pH of the buffer is 8.5-9.5, for example, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4 or 9.5, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0016] Preferably, the concentration of the enzyme solution to be detected is 0.1-2 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL or 2 mg / mL, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0017] Preferably, the volume ratio of the buffer, the substrate solution and the enzyme solution to be detected is (7-10):1:1, for example, 7:1:1, 8:1:1, 9:1:1 or 10:1:1, etc., but is not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0018] Preferably, the temperature of the mixing is 36-38℃, such as 36℃, 36.5℃, 37℃, 37.5℃ or 38℃, etc., but not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0019] Preferably, the wavelength of the detection is 400-410nm, such as 400nm, 401nm, 402nm, 403nm, 404nm, 405nm, 406nm, 407nm, 408nm, 409nm or 410nm, etc., but not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0020] Preferably, the formula of the calculation is as follows:

[0021] Enzyme activity = (ΔA405 / min x V1 x 1 x 10 6 ) / (ε x L x V2);

[0022] In the formula, ΔA405 / min is the absorbance change value per minute (unit: abs / min), V1 is the total volume of the reaction system, ε is the molar extinction coefficient of p-nitroaniline (9.87 x 103L / mol / cm), L is the optical path length, and V2 is the volume of the enzyme to be detected.

[0023] The above method establishes a standardized enzyme activity calculation formula and quality control standard, and lays a foundation for industry standardization.

[0024] In another aspect, the present application also provides the use of the lysine endonuclease activity detection method as described above in lysine endonuclease quality control, batch consistency test, enzyme activity standardization in proteomics research, enzyme activity monitoring in biopharmaceutical production process, enzyme preparation stability evaluation or shelf life determination.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] The present application provides a lysine endonuclease activity detection method, which can be realized only with a conventional ultraviolet-visible spectrophotometer, has low equipment requirements and low cost, has high detection efficiency, can complete the detection in only 5-10 minutes, has a simple and standardized overall detection process, does not require complex sample pretreatment and post-treatment steps, reduces the professional requirements, and has strong method specificity, high precision, good reproducibility, small relative standard deviation, wide linear range and low detection limit, and can meet the quality control requirements of scientific research and industrial application. DETAILED DESCRIPTION

[0027] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0028] Example 1

[0029] The present embodiment provides a method for detecting the activity of a lysine endonuclease, and the specific steps are as follows:

[0030] (I) Sample preparation

[0031] An appropriate amount of Lys-C enzyme to be tested is dissolved with 0.001 mol / L hydrochloric acid solution and diluted to 1 mg / mL as a stock solution, which is further diluted to a working concentration of 0.5 mg / mL when used. The stock solution is stored at 2-8°C and used within 24 hours.

[0032] (II) Preparation of substrate solution

[0033] An appropriate amount of N-acetyl-L-lysine p-nitroaniline is accurately weighed, dissolved with 50 mM Tris-HCl buffer (pH 9.0), and prepared into a 1.0 mM substrate working solution. The substrate solution is prepared fresh and used, and stored at 4°C in the dark for no more than 2 hours.

[0034] (III) Setting of core detection conditions

[0035]

[0036]

[0037] (IV) Standardized detection process

[0038] The total volume of the reaction system is 3.0 mL, and the composition is as follows:

[0039]

[0040] The blank control group uses an equal volume of buffer instead of enzyme solution. Each component is preheated at 37°C for 3 minutes, and the buffer, substrate solution and enzyme solution are added in order, mixed quickly, and then the absorbance change is continuously monitored at 405 nm wavelength.

[0041] (V) Enzyme activity calculation formula

[0042] Enzyme activity (U / mL) = (ΔA405 / min x V1 x 1 x 10 6 ) / (ε x L x V2)

[0043] Wherein:

[0044] ΔA405 / min: absorbance change value per minute (blank control should be deducted), unit: abs / min;

[0045] V1: total volume of the reaction system;

[0046] ε: molar extinction coefficient of p-nitroaniline (9.87 x 103 L / mol / cm);

[0047] L: optical path length;

[0048] V2: enzyme liquid volume added;

[0049] 1000: unit conversion factor, converting mol to pmol.

[0050] The methodological study was carried out according to the above method, and the results are as follows:

[0051] Linear range test:

[0052] (1) Sample preparation: Take lysine endonuclease standard, and prepare working solutions of different concentrations (0.1, 0.3, 0.5, 0.8, 1.0, 1.5 mg / mL) with 0.001 mol / L hydrochloric acid solution;

[0053] (2) Detection conditions: According to the detection method of Example 1, each concentration was determined in parallel for 5 times;

[0054] (3) Data processing: Take enzyme concentration as abscissa and ΔA405 / min as ordinate, and use least squares method for linear regression analysis;

[0055]

[0056] (4) Acceptance criteria: Correlation coefficient R 2 ≥ 0.998, and RSD of each concentration point ≤ 5%.

[0057] The results are as follows:

[0058]

[0059] Linear regression equation: y = 31.95x + 0.21 (R 2 = 0.998)

[0060] Limit of detection (LOD): 0.05 U / mL

[0061] Limit of quantification (LOQ): 0.15 U / mL

[0062] Data analysis: Within the concentration range of 0.1-1.5 mg / mL, the enzyme concentration showed good linear relationship with the reaction rate, the correlation coefficient R 2 = 0.998, indicating that the method has wide linear range and high precision. The RSD of all concentration points is less than 5%, meeting the precision requirement of the analysis method.

[0063] Precision verification:

[0064] ​(1) Intra-day precision: select a medium concentration sample (0.5 mg / mL), measure 10 times in one day by the same operator;

[0065] (2) Inter-day precision: measure 5 times per day for 3 consecutive days, calculate the total RSD;

[0066] (3) Different operator precision: measure the same sample 4 times by 3 different operators;

[0067] (4) Acceptance criteria: all RSDs should be < 5%.

[0068] Results are as follows:

[0069]

[0070] Data analysis: all RSDs of precision tests are less than 5%, which indicates that the method has good reproducibility and stability, and is not significantly affected by operators and time differences.

[0071] Recovery verification:

[0072] Test design and sample preparation

[0073] Accuracy verification was performed by the method of spiked recovery test, which is the standard practice for evaluating the accuracy of an analytical method. Three lysine endo-enzyme samples with different activity levels were selected as matrix samples, and three concentration levels of spiked recovery tests were performed for each matrix sample. This design can comprehensively evaluate the accuracy performance of the method at different concentration levels.

[0074] Matrix sample and spiked design

[0075] Matrix type Enzyme activity (U / mL) Spiked level Low activity matrix 8.2 50％、100％、150％ Medium activity matrix 25.6 50％、100％、150％ High activity matrix 48.3 50％、100％、150％

[0076] The design of spiked concentration follows the standard requirements of analytical chemistry method verification: 50% spiking is used to evaluate the accuracy of the low concentration range, 100% spiking to evaluate the accuracy of the normal concentration level, and 150% spiking to evaluate the accuracy of the high concentration range.

[0077] Standard preparation

[0078] The standard spiking operation uses standard enzyme preparations with known activity, which are diluted to the required concentration with the buffer system of this method.

[0079] Spiked sample preparation process

[0080] Step 1: Preparation of standard stock solution. 10.0 mg of lysine endo-enzyme standard was precisely weighed into a 10 mL volumetric flask, dissolved with 0.001 mol / L hydrochloric acid solution and diluted to the mark to prepare a 1.0 mg / mL standard stock solution. After thorough mixing, it was stored at 2-8°C and used within 24 hours.

[0081] Step 2: Preparation of standard working solution series. An appropriate volume was removed from the standard stock solution, diluted with 50 mM Tris-HCl buffer (pH 9.0) to prepare standard working solutions of different concentrations. The low-concentration standard solution was prepared at 0.20 mg / mL (corresponding to about 6.4 U / mL), the medium-concentration standard solution was prepared at 0.80 mg / mL (corresponding to about 25.6 U / mL), and the high-concentration standard solution was prepared at 1.50 mg / mL (corresponding to about 48.0 U / mL).

[0082] Step 3: Matrix sample pretreatment. The background enzyme activity of the matrix samples to be tested was determined using the detection method of the present application, and the background values of each matrix sample were recorded. The background value of the low-activity matrix was 8.2±0.3 U / mL, the background value of the medium-activity matrix was 25.6±0.8 U / mL, and the background value of the high-activity matrix was 48.3±1.2 U / mL.

[0083] Step 4: Spiking operation. Each matrix sample was treated at three spiking levels, including 50%, 100% and 150% spiking levels. 1.0 mL of the matrix sample was taken and the corresponding concentration of the standard working solution was added to make the final spiking amount 50%, 100% and 150% of the matrix background value, respectively. The spiking volume was strictly controlled within 5% of the total volume to avoid dilution effect.

[0084] Step 5: Mixing and equilibration of spiked samples. The spiked samples were mixed on a vortex mixer for 30 seconds to ensure uniformity. Then they were equilibrated at room temperature for 30 minutes to allow the spiked enzyme to fully bind with the matrix, and during this period, they were gently inverted and mixed every 10 minutes.

[0085] Step 6: Preparation of parallel samples

[0086] Six independent parallel samples were prepared for each spiking concentration, and corresponding blank matrix control samples (without standard) were also prepared. All samples were treated under the same conditions to ensure consistency of experimental conditions.

[0087] Step 7: Preparation of quality control samples

[0088] Quality control samples were prepared by preparing QC samples at an intermediate concentration level using standard samples with known activity. The QC samples were treated simultaneously with the test samples to monitor the system performance of the entire spiking recovery experiment, and the recovery rate of the QC samples should be within the range of 95-105%.

[0089] Each spiked concentration was prepared in 6 replicates to ensure the reliability of statistical analysis. The corresponding blank matrix samples were prepared at the same time as controls to deduct the matrix interference and calculate the true recovery rate.

[0090] Detection and data processing

[0091] All samples were subjected to enzyme activity determination according to the detection method established in Example 1, and the consistency of the detection conditions was strictly controlled. The data processing was analyzed by using the standard statistical method.

[0092] Detection condition control:

[0093] Control item Standard requirement Acceptance standard Parallel determination 3 times for each sample RSD≤5% Detection of in-batch quality control Recovery rate of quality control sample 95-105％ System suitability Standard response Deviation from expected value ≤3%

[0094] Recovery rate calculation formula:

[0095] Recovery rate (%) = [(spiked sample measured value-blank matrix measured value) / theoretical spiked amount] x 100%

[0096] Recovery rate test results:

[0097] The recovery rate test results show that the detection method established by the present application has excellent accuracy. The recovery rates under all test conditions are within the acceptable range, and the systematic error and random error of the method meet the requirements.

[0098] Recovery rate test results

[0099]

[0100] Statistical analysis results

[0101] Statistical item Calculation result Evaluation standard Conclusion Average recovery rate 98.7±1.7％ 95-105％ Pass Recovery rate range 97.1-101.9％ Deviation ≤5% Pass Precision RSD <3.5％ ≤5％ Pass t-test P>0.05 No significant difference from 100% Pass System error <2％ ≤3％ Pass

[0102] Statistical analysis shows that the recovery rates at all spiked levels are within the range of 97.1%-101.9%, the average recovery rate is 98.7±1.7%, the t-test result P>0.05, indicating that there is no significant difference between the recovery rate and the theoretical value 100%.

[0103] Stability test:

[0104] The samples were stored under different conditions for a certain period of time, and then detected, and the results were as follows:

[0105]

[0106] Data analysis: The substrate solution remained good stability within 24 hours under the condition of 4℃ and light avoidance, with a deviation of less than 3%. The stability was relatively poor at room temperature, and it was recommended to be stored at low temperature and light avoidance.

[0107] Method robustness test:

[0108] The samples were treated under different processing conditions, and then detected, and the results were as follows:

[0109]

[0110] Data analysis: the detection result deviation is less than 5% within the condition change range of temperature ± 1 ℃, pH ± 0.2 and substrate concentration ± 20%, indicating that the method has good durability.

[0111] Comparison and verification with the traditional method:

[0112] Ten different samples were tested by the method of the application and the traditional HPLC method respectively, and the HPLC method was as follows:

[0113] HPLC test method:

[0114] Instrument and reagent

[0115] The HPLC control method is detected by a high performance liquid chromatography system equipped with an ultraviolet detector and a column temperature control module. The chromatographic separation uses a C18 reversed-phase column (4.6*250mm, 5μm particle size), which has good retention and separation effect on p-nitroaniline compounds. The mobile phase A is 0.1% TFA aqueous solution, and the mobile phase B is 0.1% TFA acetonitrile solution, and the addition of TFA helps to improve the peak shape and improve the separation effect.

[0116] Main reagent:

[0117] Standard: N-acetyl-L-lysine p-nitroaniline, p-nitroaniline (both are analytical pure grade)

[0118] Mobile phase A: 0.1% TFA aqueous solution

[0119] Mobile phase B: 0.1% TFA acetonitrile solution

[0120] Other reagents: methanol (HPLC grade)

[0121] Chromatographic conditions

[0122] Parameter Set value Detection wavelength 405 nm Flow rate 1.0 mL / min Column temperature 30℃ Injection volume 20 μL Total analysis time 35 min

[0123] Gradient elution program:

[0124] Time (min) Mobile phase B (%) 0-5 5 5-20 5→95 20-25 95 25-30 95→5 30-35 5

[0125] Sample processing

[0126] Sample processing was carried out according to the standardized procedure: first, enzyme reaction was carried out according to the detection method established in the present application to the specified time, and then an equal volume of methanol solution was added to terminate the enzyme reaction. The addition of methanol can rapidly inactivate the enzyme and precipitate the protein. After the reaction was terminated, the sample was centrifuged at 12,000 rpm for 10 min to separate the supernatant and precipitate. The supernatant was appropriately diluted with mobile phase A according to the expected concentration range to ensure that the concentration of the component to be detected was within the linear range of the standard curve. The diluted sample should be stored at 4°C, and the detection should be completed within 24 h.

[0127] Quantitative analysis method

[0128] The external standard method was used for quantitative analysis. Different volumes were accurately removed from the stock solution and diluted with mobile phase A (0.1% TFA aqueous solution) to prepare six concentration points of standard working solution, with concentrations of 1.0 μg / mL, 5.0 μg / mL, 20.0 μg / mL, 40.0 μg / mL, 70.0 μg / mL, and 100.0 μg / mL, respectively. Each concentration point was determined in triplicate.

[0129] Technical indicators of the standard curve:

[0130] Item Requirement Concentration range 1-100 μg / mL Specific concentration 1.0, 5.0, 20.0, 40.0, 70.0, 100.0 μg / mL Correlation coefficient r 2 >0.999]]> Parallel determination 3 times for each point

[0131] Enzyme activity calculation formula:

[0132] Enzyme activity (U / mL) = [product concentration (μg / mL) x reaction volume (mL)] / [reaction time (min) x enzyme protein amount (mg)]

[0133] The results are as follows:

[0134]

[0135] Statistical analysis:

[0136] Correlation coefficient r 2 = 0.996

[0137] Average deviation: 1.9%

[0138] Data analysis: The method of the present application has good consistency with the existing HPLC method, the correlation coefficient is close to 1, and the average deviation is less than 2%, proving the accuracy and reliability of the method.

[0139] Comparison of detection efficiency:

[0140]

[0141] Data analysis: The method of the present application is significantly superior to the traditional method in terms of detection time, cost, and equipment requirements, and has obvious economic advantages and practical value.

[0142] Comparison of different substrate specificity:

[0143] Purpose and materials of the experiment

[0144] This experiment aims to verify the advantage of N-acetyl-L-lysine p-nitroanilide as a specific substrate of lysine endo-enzyme compared with other potential substrates. The experiment adopts a systematic comparative research design to comprehensively evaluate the specificity and applicability of the substrates.

[0145] List of test substrates

[0146] Number Substrate name Abbreviation Classification 1 N-acetyl-L-lysine p-nitroaniline Ac-Lys-pNA Substrate selected by the application 2 N-benzoyl-L-arginine p-nitroaniline Bz-Arg-pNA Trypsin substrate control 3 N-butyloxycarbonyl-L-lysine p-nitroaniline Boc-Lys-pNA Substitute substrate 1 4 L-lysine p-nitroaniline Lys-pNA Substitute substrate 2 5 Z-lysine thioaniline Z-Lys-SBzl Literature reported substrate

[0147] Enzyme preparation

[0148] Enzyme type Source Lysine endonuclease Commercial standard Trypsin Commercial standard Chymotrypsin Commercial standard

[0149] Experimental methods and conditions

[0150] The substrate hydrolysis activity assay was performed using a standardized reaction system. The concentration of each substrate solution was uniformly prepared as 1.0 mM, the reaction buffer was 50 mM Tris-HCl buffer (pH 9.0), the enzyme concentration was set as 0.5 mg / mL, and the reaction temperature was controlled at 37°C.

[0151] Reaction system composition

[0152] Component Concentration / amount Substrate solution 1.0 mM Tris-HCl buffer 50 mM, pH 9.0 Enzyme solution 0.5 mg / mL Reaction temperature 37℃ Reaction time 1-30 min

[0153] In order to obtain accurate kinetic parameters, the substrate concentration range was set as 0.1-5.0 mM, by measuring the initial reaction rate under different substrate concentrations, the Michaelis-Menten kinetic curve was drawn, and then the Km and Vmax values of each substrate were calculated.

[0154] Kinetic parameter determination results

[0155] Through systematic substrate activity comparison experiments, N-acetyl-L-lysine p-nitroanilide showed the best enzymatic properties.

[0156] Substrate activity comparison data

[0157] Substrate name Km (mM) Vmax (μmol / min / mg) kcat / Km (M-1s-1) Specificity index Ac-Lys-pNA 0.85±0.05 12.3±0.8 2.41 x 10 5 ]] 1.00 Boc-Lys-pNA 1.23±0.08 9.7±0.6 1.64 x 10 5 ]]> 0.68 Lys-pNA 1.67±0.12 7.2±0.9 8.95 x 10 4 ]] 0.37 Z-Lys-SBzl 2.15±0.18 5.1±0.7 4.92 x 10 4 ]]> 0.20 Bz-Arg-pNA >10 <1.0 <2 x 10 3 ]]> <0.01

[0158] The Km value of N-acetyl-L-lysine p-nitroanilide was 0.85±0.05 mM, which was significantly lower than that of other test substrates, indicating that it had the highest affinity with lysine endo-enzyme. The maximum reaction rate reached 12.3±0.8 μmol / min / mg protein, and the catalytic efficiency kcat / Km value was 2.41×10 5 M-1s-1, which were the highest among all test substrates.

[0159] Enzyme specificity verification results

[0160] Enzyme specificity verification experiments further confirmed the superior performance of N-acetyl-L-lysine p-nitroanilide. Experiments used cross-verification design of different enzymes acting on various substrates.

[0161] Enzyme specificity verification data

[0162]

[0163]

[0164] The hydrolytic activity of lysine endopeptidase on Ac-Lys-pNA was set as 100% reference value, while the activity on trypsin substrate Bz-Arg-pNA was less than 5%, with a selectivity ratio of more than 20:1, showing extremely high substrate specificity. In reverse verification, the activity of trypsin on its specific substrate was 100%, and the activity on Ac-Lys-pNA was only 15%.

[0165] Substrate stability and spectral properties

[0166] In addition to catalytic performance, the physicochemical properties of the substrate are also important indicators for evaluating its practical value. The stability test and spectral property analysis results are as follows:

[0167] Substrate stability test

[0168] Substrate pH 8.5 stability pH 9.0 stability pH 9.5 stability 24 h retention rate Ac-Lys-pNA >98％ >99％ >97％ >95％ Boc-Lys-pNA >95％ >96％ >92％ >90％ Lys-pNA >93％ >94％ >89％ >85％ Z-Lys-SBzl >88％ >90％ >82％ >78％

[0169] Spectral detection properties

[0170] Substrate Maximum absorption wavelength Molar extinction coefficient Detection suitability Ac-Lys-pNA 405 nm 9.87 x 10 3 L / mol / cm Excellent Boc-Lys-pNA 405 nm 8.95 x 10 3 L / mol / cm Good Lys-pNA 405 nm 8.12 x 10 3 L / mol / cm Good Z-Lys-SBzl 324 nm 1.24 x 10 4 L / mol / cm General

[0171] N-acetyl-L-lysine p-nitroanilide showed excellent chemical stability in a wide range of pH 8.5-9.5, with a 24-hour retention rate of more than 95%. The substrate has good spectral properties at 405 nm wavelength, with a molar extinction coefficient of 9.87 x 10 3 L / mol / cm, which is conducive to spectrophotometric detection.

[0172] Comprehensive evaluation and advantage analysis

[0173] Through comprehensive comparative experiments, it can be concluded that N-acetyl-L-lysine p-nitroanilide has significant comprehensive advantages as a lysine endopeptidase detection substrate:

[0174] Technical advantage summary

[0175] Evaluation index Ac-Lys-pNA performance Relative advantage Substrate affinity Km = 0.85 mM Best Catalytic efficiency kcat / Km = 2.41 x 10 5 M-1s-1]] Highest Enzyme specificity Selectivity ratio > 20:1 Strongest Chemical stability Stable in the range of pH 8.5-9.5 Best Detection suitability λmax = 405 nm, high ε Most suitable

[0176] Comprehensive analysis shows that the substrate not only has the highest substrate affinity and catalytic efficiency, but also shows excellent enzyme specific recognition ability, good chemical stability and spectral characteristics suitable for spectrophotometric detection, which ensures that the detection method established by the application has high accuracy, high specificity and good practicability.

[0177] The applicant declares that the lysine endonuclease enzyme activity detection method and application thereof of the present application are illustrated by the above-mentioned embodiments, but the present application is not limited to the above-mentioned embodiments, that is, it does not mean that the present application must rely on the above-mentioned embodiments to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes and the like all fall within the protection scope and disclosure scope of the present application.

[0178] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above-mentioned embodiments, and within the technical concept scope of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0179] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined by any suitable method without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination methods.

Claims

1. A method for detecting endolysin enzyme activity, characterized by, The lysine endonuclease enzyme activity detection method comprises the following steps: The substrate solution, the buffer and the enzyme solution to be detected are mixed, and then ultraviolet-visible spectrophotometry is used for detection, and the lysine endonuclease enzyme activity to be detected is calculated according to the detection result.

2. The lysine endo-enzyme enzyme activity detection method according to claim 1, wherein, The substrate comprises N-acetyl-L-lysine para-nitroaniline.

3. The lysine endo-enzyme activity detection method according to claim 1 or 2, characterized by, The concentration of the substrate solution is 0.8-1.2 mM.

4. The lysine endo-enzyme activity detection method according to any one of claims 1 to 3, characterized in that, The pH of the buffer is 8.5-9.

5.

5. The lysine endo-enzyme activity detection method according to any one of claims 1 to 4, wherein, The concentration of the enzyme solution to be detected is 0.1-2 mg / mL.

6. The lysine endo-enzyme activity detection method according to any one of claims 1 to 5, wherein, The volume ratio of the buffer, the substrate solution and the enzyme solution to be detected is (7-10):1:

1.

7. The lysine endo-enzyme activity detection method according to any one of claims 1 to 6, wherein, The mixing temperature is 36-38 DEG C.

8. The lysine endoendonuclease enzyme activity assay method according to any one of claims 1 to 7, wherein, The detection wavelength is 400-410 nm.

9. The lysine endoendonuclease enzyme activity assay method according to any one of claims 1 to 8, wherein, The calculation formula is as follows: Enzyme activity = (ΔA405 / min x V1 x 1 x 10 6 ) / (ε x L x V2); In the formula, ΔA405 / min is the absorbance change value per minute, V1 is the total volume of the reaction system, ε is the molar extinction coefficient of para-nitroaniline, L is the optical path length, and V2 is the volume of the enzyme solution to be detected. 10.A lysine endonuclease enzyme activity detection method according to any one of claims 1-9 is applied in lysine endonuclease quality control, batch consistency test, enzyme activity standardization in proteomics research, enzyme activity monitoring in biopharmaceutical production process, enzyme preparation stability evaluation or shelf life determination.

Images