Standardized Glu-C incision enzyme activity spectrum detection method

Abstract

The invention relates to the technical field of enzyme activity detection, in particular to a standardized Glu-C incision enzyme activity spectrum detection method. The method provided by the invention realizes rapid, accurate and low-cost enzyme activity detection by using a specific substrate Z-Phe-Leu-Gl-4-pNA and optimized reaction conditions, and specifically comprises the following steps: diluting a Glu-C endonuclease sample, mixing the diluted Glu-C endonuclease sample with a substrate solution, putting the mixture into a preheated Tris-HCl buffer solution, monitoring absorbance change at the wavelength of 405nm, and calculating the enzyme activity through a specific formula. A standardized Glu-C incision enzyme activity detection system is established, the detection time is short, the cost is low, the detection method has a wide linear range, a low detection limit and high precision, and a technical basis is provided for establishing an industrial unified standard.

Description

Technical Field

[0001] This invention relates to the field of enzyme activity detection technology, and in particular to a standardized spectral detection method for Glu-C endonuclease activity. Background Technology

[0002] Glu-C endonuclease (also known as V8 protease, EC: 3.4.21.19) is an enzyme with significant applications in biochemical research and biopharmaceuticals. Accurate detection of Glu-C endonuclease activity is crucial for product quality control, scientific research, and related application development. However, existing methods for detecting Glu-C enzyme activity have many shortcomings and urgently need improvement.

[0003] Currently, Glu-C enzyme activity detection mainly employs conventional spectrophotometry and LC-MS. Conventional spectrophotometry, using Z-Phe-Leu-Glu-4-pNA as the substrate, is a widely adopted method by many manufacturers. However, a key issue with this method is the lack of standardized operating procedures. Although manufacturers use the same detection principle and substrate, significant differences exist in specific operational details, reaction condition control, quality control standards, and data processing methods. This leads to a lack of comparability and reproducibility of enzyme activity detection results between different manufacturers, seriously affecting the establishment of industry standards and the unified evaluation of product quality.

[0004] Furthermore, conventional spectroscopic methods lack precise specifications regarding parameters such as temperature control accuracy, pH buffer system selection, substrate concentration, and reaction time, resulting in inconsistent precision control and reproducibility. Manufacturers like New England Biolabs utilize LC-MS analysis for functional testing, such as cytochrome C digestion and ACTH(1-17) peptide digestion. However, this method is primarily used to verify enzyme functionality and specificity, rather than for standard enzyme activity quantification. It also has a long detection time, exceeding 16 hours, is complex to operate, and is costly, making it unsuitable for routine quality control and large-scale sample testing. Additionally, this method demands highly skilled equipment and technical personnel, resulting in substantial equipment investment, high maintenance costs, and unsuitability for general laboratories and enterprise applications.

[0005] In summary, existing detection methods suffer from low standardization, high technical barriers, high costs, and low efficiency, severely hindering the industrial application and scientific research development of Glu-C enzymes. Therefore, developing a standardized, rapid, accurate, and low-cost method for detecting Glu-C enzyme activity has significant scientific value and practical application implications. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a standardized spectral detection method for Glu-C endonuclease activity, comprising:

[0007] S1. Take a Glu-C endonuclease sample, dilute it with deionized water to obtain the enzyme solution to be tested;

[0008] Under light-protected conditions, the Z-Phe-Leu-Glu-4-pNA (CAS No.: 104634-10-8) substrate was diluted with Tris-HCl buffer to obtain the substrate solution.

[0009] S2. Add preheated Tris-HCl buffer and preheated substrate solution sequentially to a quartz cuvette, place it in a spectrophotometer, zero the baseline, add the enzyme solution to be tested, mix well, and react. Continuously monitor the absorbance change at a wavelength of 405 nm and record ΔA. 405 The / min value is then used to calculate the Glu-C endonuclease activity according to the enzyme activity formula.

[0010] Where: enzyme activity = (ΔA) 405 / min×V 反应 ×1000) / (ε×L×V 酶液 );

[0011] ΔA 405 / min represents the change in absorbance per minute, with units of abs / min;

[0012] V 反应 The total volume of the reaction system;

[0013] ε is the molar extinction coefficient, 9.87 × 10⁻⁶. 3 L / mol / cm;

[0014] L is the optical path length, taken as 1 cm;

[0015] V 酶液 This represents the volume of the enzyme solution to be tested added.

[0016] 1000 is the unit conversion factor, converting mol to μmol.

[0017] This invention establishes a standardized spectroscopic detection system specific to Glu-C endonuclease. Compared to traditional protein substrate enzymatic digestion-analysis detection methods, this invention uses Z-Phe-Leu-Glu-4-pNA as the substrate, which contains a specific recognition sequence for Glu-C, ensuring detection specificity. Simultaneously, the optimized reaction conditions guarantee both optimal enzyme activity and an ideal linear response range. The standardized enzyme activity calculation formula and complete quality control system provided by this invention solve the problem of inconsistent internal standards among manufacturers, and the entire detection process takes only 1 minute, significantly improving detection efficiency.

[0018] As an feasible example, the mass concentration of the enzyme solution to be tested is 0.02-1 mg / mL; the enzyme solution should be stored at 2-8℃ and used within 24 hours; vigorous shaking should be avoided during sample preparation to prevent denaturation and inactivation of the enzyme protein. For lyophilized powdered Glu-C endonuclease samples, they should be equilibrated at room temperature (approximately 25℃) for 10 minutes before dissolution.

[0019] Furthermore, the mass concentration of the enzyme solution to be tested is 0.02-0.8 mg / mL.

[0020] As an example of implementation, the molar concentration of the substrate solution is 1.0-2.0 mM.

[0021] As an example of implementation, the Tris-HCl buffer solution has a molar concentration of 50-100 mM and a pH of 7.5-7.8.

[0022] In this invention, the substrate solution should be prepared and used immediately, and stored at 4°C in the dark for no more than 4 hours. Because the substrate Z-Phe-Leu-Glu-4-pNA is photosensitive, it should be handled in the dark as much as possible during preparation and use.

[0023] As an example of implementation, the preheating temperature of the Tris-HCl buffer solution is 25-28°C.

[0024] As an example of implementation, the preheating temperature of the substrate solution is 25-28°C.

[0025] As an example of implementation, the monitoring time for absorbance changes is 30-60 seconds.

[0026] As an example of implementation, the scanning interval for monitoring absorbance changes is 0.2-1 s.

[0027] As an example of feasible implementation, the reaction temperature is 25±2℃.

[0028] Beneficial effects

[0029] (I) This invention primarily addresses the long-standing technical dilemma of "high precision - high cost - low efficiency" in the field of Glu-C endonuclease activity detection. By employing a synthesized chromogenic substrate Z-Phe-Leu-Glu-4-pNA and optimized reaction conditions, this invention successfully transforms high-end enzyme activity detection technology into a standardized method suitable for widespread application. Simultaneously, the required detection equipment has been reduced from expensive precision instruments to ordinary UV-Vis spectrophotometers, lowering equipment investment from over 800,000 RMB to 30,000-80,000 RMB, a reduction of over 90%. The detection equipment used has a high penetration rate in laboratories at all levels, greatly expanding the potential user base.

[0030] (II) Compared to traditional spectroscopic detection methods that require 20-40 minutes to complete a single test, the testing method provided by this invention only requires 1 minute, increasing efficiency by 20-40 times. Therefore, previously impossible application scenarios become possible, such as real-time quality monitoring of production lines, large-scale sample screening, and teaching experiments. Especially in the biopharmaceutical industry, rapid and accurate enzyme activity detection is of great significance for ensuring product quality and production efficiency.

[0031] (III) This invention lays the technical foundation for establishing unified industry standards by establishing a complete standardized operating procedure, including standardization of the entire chain of sample preparation, reaction conditions, detection procedures, data processing, and quality control. At the same time, the standardized operating procedure ensures good comparability of results between different laboratories, promoting the application and industrialization of the technology.

[0032] (IV) This invention achieves a significant cost reduction while maintaining detection accuracy; the detection cost is reduced from 45-85 yuan / test to 2-4 yuan / test, a cost reduction of over 95%. More importantly, this invention lowers the technical application threshold; ordinary laboratory technicians can master it after simple training, eliminating reliance on professional technicians and enabling more research institutes, enterprises, and educational institutions to conduct Glu-C-related research and applications. In terms of detection performance, this invention maintains excellent detection quality while significantly reducing cost and complexity, with a wide linear range (0.02-0.80 mg / mL), low detection limit (0.01 U / mL), high precision (RSD less than 5%), and a correlation of 0.9993 with traditional LC-MS detection methods, fully meeting the requirements of scientific research and industrial applications.

[0033] (V) The testing method provided by this invention is suitable for various application scenarios and has broad market prospects. In the biopharmaceutical field, it can be used for quality control, batch inspection, and production process monitoring of Glu-C enzyme products, helping enterprises establish a sound quality management system and improve product consistency and reliability. In proteomics research, it provides research institutes with convenient and accurate enzyme activity detection methods, promoting the development of related basic and applied research. In the field of biotechnology education, it provides standardized experimental methods and teaching tools, which is beneficial to the training of professional talents and the construction of disciplines. By providing complete technical specifications and operating standards, this invention provides a technical foundation and reference for establishing a unified industry standard for Glu-C enzyme detection. The promotion of standardization will promote the standardized development of related industries, improve product quality, reduce industry costs, and enhance international competitiveness. Detailed Implementation

[0034] Example 1

[0035] This example provides a standardized spectral detection method for Glu-C endonuclease activity, specifically:

[0036] S1. Take a Glu-C endonuclease sample, dilute it with 0.3 mL of deionized water to obtain the enzyme solution to be tested, and use an equal volume of Tris-HCl buffer with pH=7.8 to replace the enzyme solution for the blank control group for parallel determination.

[0037] Under light-protected conditions, Z-Phe-Leu-Glu-4-pNA substrate was diluted with 0.3 mL of Tris-HCl buffer at pH 7.8 to obtain a 1.0 mM substrate solution.

[0038] S2. Add 2.4 mL of preheated Tris-HCl buffer (25°C) and 0.3 mL of preheated substrate solution (25°C) to a quartz cuvette. Place the cuvette in a spectrophotometer, zero the baseline, add 0.3 mL of the enzyme solution to be tested, mix well, and allow the reaction to proceed. Continuously monitor the absorbance change at 405 nm and record ΔA. 405 The enzyme activity of Glu-C endonuclease was calculated based on the enzyme activity formula. The first 30 seconds were recorded as the background value, and then the enzyme activity was calculated by continuously monitoring for 60 seconds and taking the linear part.

[0039] Where: enzyme activity = (ΔA) 405 / min×V 反应 ×1000) / (ε×L×V 酶液 );

[0040] ΔA 405 / min: Absorbance change per minute (excluding blank control), unit is abs / min;

[0041] V反应 Total volume of the reaction system;

[0042] ε: The molar extinction coefficient of p-nitroaniline at pH 7.8 is 9.87 × 10⁻⁶. 3 L / mol / cm;

[0043] L: Optical path length is 1cm;

[0044] V 酶液 The volume of the enzyme solution to be tested added was 0.3 mL;

[0045] 1000 is the unit conversion factor, converting mol to μmol.

[0046] During the monitoring of absorbance changes, the reaction temperature was 25±2℃;

[0047] The scan interval is 0.2s.

[0048] For Example 1, systematic methodological validation was performed using standard Glu-C endonuclease test solutions with different concentration gradients, including linear range testing, precision validation, stability assessment, and accuracy evaluation, and a comparative study was conducted with existing LC-MS methods.

[0049] 1. Linear range test

[0050] Set different concentrations of the enzyme solution to be tested and detect ΔA. 405 The enzyme specific activity and relative standard deviation (RSD) were calculated by taking the / min value. Each group was tested 6 times, and the experimental results were recorded in Table 1.

[0051] Table 1

[0052]

[0053] Based on the experimental data in the table, the linear regression equation is: y = 94.73x + 0.08(R²). 2 =0.9996);

[0054] Limit of detection (LOD): 0.01 U / mL;

[0055] Limit of quantitation (LOQ): 0.05 U / mL.

[0056] By testing the reaction rate at different enzyme concentrations, a linear relationship between enzyme concentration and enzyme activity was established. Experimental results showed that within the concentration range of 0.02-0.80 mg / mL, enzyme concentration exhibited an excellent linear relationship with ΔA405 / min. The linear regression equation was y = 94.73x + 0.08, and the correlation coefficient R0 was [value missing]. 2 =0.9996, indicating that the method has excellent linearity.

[0057] The limit of detection (LOD) was set at 0.01 U / mL, and the limit of quantitation (LOQ) at 0.05 U / mL, meeting the detection requirements of various application scenarios. The relative standard deviation (RSD) for all concentration points was less than 5.5%, with the highest precision at a medium concentration of 0.4 mg / mL, where the RSD was as low as 2.8%.

[0058] 2. Precision testing

[0059] Based on the linear range test, the precision of the test method was further determined, including intra-day precision and inter-day precision (5 days), precision between different operators, and precision tests between different batches of substrates and equipment. The experimental results are detailed in Table 2.

[0060] Table 2

[0061]

[0062] As can be seen from the experimental data in Table 2, the RSD of all precision tests is less than 5%, proving that the test method provided by this invention has good reproducibility. Intra-day precision showed the best performance, with an RSD of only 3.1%, indicating excellent short-term stability. Inter-day precision and RSD between different operators were both within 4.5%, indicating that the enzyme activity detection method is not significantly affected by time factors or differences in operator skill. Furthermore, the test results of different batches of substrate and different instruments also showed good consistency, providing strong support for the standardization and promotion of the test method.

[0063] III. Stability Testing

[0064] The enzyme activity retention rates of substrate solutions and test enzyme solutions under different storage conditions were tested to evaluate the stability of the test method. The test conditions and results are detailed in Table 3.

[0065] Table 3

[0066]

[0067]

[0068] Stability test results showed that the Z-Phe-Leu-Glu-4-pNA substrate solution exhibited good stability at 4°C in the dark, with enzyme activity retention exceeding 98% within 4 hours and a relative deviation of less than 2%. Stability began to decrease after 4 hours, but remained acceptable within 6 hours. Stability was poor at room temperature (25°C), and it is recommended to store the enzyme solution at 4°C in the dark. The enzyme solution maintained good stability at 4°C for 24 hours, with enzyme activity retention exceeding 96%, providing a feasible time window for sample processing and batch detection in practical applications.

[0069] IV. Method Durability Testing

[0070] The robustness of the testing method was evaluated by performing minor variations on key operating parameters. The experimental results are detailed in Table 4.

[0071] Table 4

[0072] variable Setting value Test range Enzyme activity results (U / mL) Relative deviation (%) temperature 25℃ 24℃ 18.92 -3.8 temperature 25℃ 26℃ 20.15 +2.3 pH 7.8 7.6 18.76 -4.6 pH 7.8 8.0 20.28 +3.1 Substrate mass concentration 1.0mM 0.8mM 18.51 -5.9 Substrate mass concentration 1.0mM 1.2mM 19.84 +0.9 reaction time 60s 50s 19.26 -2.1 reaction time 60s 70s 19.89 +1.2 Mixing time 10s 5s 19.15 -2.6 Mixing time 10s 15s 19.73 +0.4

[0073] The results show that the impact of changes in all test parameters is within 6%, indicating that the test method provided by this invention has good robustness. Specifically, the impact of temperature changes of ±1℃ on the results is less than 4%, the impact of pH changes of ±0.2% on the results is within 5%, and the maximum deviation does not exceed 6% when the substrate mass concentration changes by ±20%. These data show that even small operational deviations in practical applications will not significantly affect the accuracy of the detection results, providing a reliable guarantee for the widespread application of the method.

[0074] 5. Accuracy Verification: Comparison with LC-MS Testing Method

[0075] The consistency evaluation of the Glu-C endonuclease activity detection method based on ultraviolet-visible spectrophotometry provided by the present invention with the conventional LC-MS test method in the art was carried out.

[0076] Manufacturers like New England Biolabs use LC-MS analysis for functional testing, including: cytochrome C digestion assay (20 μL reaction system containing 2 μg cytochrome C protein and 0.1 μg Glu-C endonuclease, incubated at 37°C for 16 h, LC-MS analysis of expected digestion products); and ACTH (1-17) peptide digestion assay (analysis of ACTH peptide digestion products under similar conditions). However, this method is mainly used to verify enzyme functionality and specificity, rather than as a standard quantitative enzyme activity assay, which is time-consuming (16 h), complex to operate, and expensive.

[0077] The consistency evaluation test results are shown in Table 5.

[0078] Table 5

[0079]

[0080] As can be seen from the experimental results in Table 5, the enzyme activity determination results of the test method provided by this invention are consistent with those of the LC-MS test method.

[0081] 6. Standardization Improvement Effect: Compared with Existing Non-Standardized Spectroscopic Methods

[0082] Currently, UV spectrophotometry using Z-Phe-Leu-Glu-4-nitranilide as a substrate is the industry-recognized mainstream method, widely adopted by manufacturers such as MedChemExpress, Sigma-Aldrich, Xibao Biotechnology, and Sangon Biotech. However, a key issue exists: each manufacturer has its own internal operating procedures and has not publicly released a unified, detailed standard operating procedure. This leads to differences in specific operational details, quality control standards, and data processing methods across different manufacturers' products, even if the enzyme activity definitions are the same, affecting the comparability of results.

[0083] The detection direction provided by this invention has higher testing accuracy than existing non-standardized spectral methods, as shown in Table 6.

[0084] Table 6

[0085]

[0086] Standardization Improvement Analysis:

[0087] Average deviation: 2.3% (good consistency among methods)

[0088] The precision of the detection method of this invention: average RSD = 3.0%.

[0089] The precision of existing non-standardized spectroscopic methods is: average RSD = 8.2%.

[0090] Precision improvement: over 63%

[0091] A comprehensive comparison of the testing method provided by this invention, the LC-MS testing method, and existing non-standardized spectroscopic methods is shown in Table 7.

[0092] Table 7

[0093]

[0094]

[0095] A comprehensive comparison with existing detection methods shows that the testing method provided by this invention has significant advantages in several key indicators. In terms of detection efficiency, the spectroscopic method of this invention completes a single detection in just 1 minute, which is 25-40 times faster than the LC-MS method. Regarding cost control, the cost per detection is 2-4 yuan, saving more than 90% compared to the LC-MS method. Equipment investment comparison shows that the spectroscopic method requires 30,000-80,000 yuan in equipment investment, while the LC-MS system requires 800,000-1,500,000 yuan. Ease of operation is a prominent feature of this invention; ordinary technicians can operate it proficiently after 2 hours of training, while the LC-MS detection method requires specialized technicians.

Claims

1. A standardized method for the spectral detection of Glu-C endonuclease activity, characterized in that, include: S1. Take a Glu-C endonuclease sample, dilute it with deionized water to obtain the enzyme solution to be tested; Under light-protected conditions, Z-Phe-Leu-Glu-4-pNA substrate was diluted with Tris-HCl buffer to obtain a substrate solution; S2. Add preheated Tris-HCl buffer and preheated substrate solution sequentially to a quartz cuvette, place it in a spectrophotometer, zero the baseline, add the enzyme solution to be tested, mix well, and react. Continuously monitor the absorbance change at a wavelength of 405 nm and record ΔA. 405 The / min value is then used to calculate the Glu-C endonuclease activity according to the enzyme activity formula. Where: enzyme activity = (ΔA) 405 / min×V 反应 ×1000) / (ε×L×V 酶液 ); ΔA 405 / min represents the change in absorbance per minute, with units of abs / min; V 反应 The total volume of the reaction system; ε is the molar extinction coefficient, 9.87 × 10⁻⁶. 3 L / mol / cm; L is the optical path length, taken as 1 cm; V 酶液 This represents the volume of the enzyme solution to be tested added. 1000 is the unit conversion factor, converting mol to μmol.

2. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The mass concentration of the enzyme solution to be tested is 0.02-1 mg / mL.

3. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The molar concentration of the substrate solution is 1.0-2.0 mM.

4. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The molar concentration of the Tris-HCl buffer solution is 50-100 mM.

5. The standardized Glu-C endonuclease activity detection method according to claim 4, characterized in that, The pH of the Tris-HCl buffer solution is 7.5-7.

8.

6. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The preheating temperature of the Tris-HCl buffer solution is 25-28℃.

7. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The preheating temperature of the substrate solution is 25-28℃.

8. The standardized Glu-C endonuclease activity detection method according to any one of claims 1-7, characterized in that, The monitoring time for absorbance changes is 30-60 seconds.

9. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The scanning interval for monitoring absorbance changes is 0.2-1 s.

10. The standardized Glu-C endonuclease activity detection method according to claim 1, characterized in that, The reaction temperature is 25±2℃.

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