High-efficiency heat-resistant luciferase reaction system

Abstract

This invention relates to the field of luciferase reaction system technology, specifically to a high-efficiency, heat-resistant luciferase reaction system. The high-efficiency, heat-resistant luciferase reaction system comprises the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 17.1-68.4 g / L trehalose, 0.4-0.8 g / L reducing agent, 0.2-0.5 g / L L-proline, 20 mg / L pyruvate kinase, and 25 mM Tris-acetic acid buffer as the solvent. This invention's high-efficiency, heat-resistant luciferase reaction system improves the thermal stability of luciferase by adding a protective agent and using recombinant, heat-resistant luciferase, and by adding other enzymes to the reaction system to reduce fluorescence decay rate, thereby improving the stability of the luciferase reaction system and ultimately enhancing the quality of related products.

Description

Technical Field

[0001] This invention relates to the field of luciferase reaction system technology, and more specifically to a high-efficiency, heat-resistant luciferase reaction system. Background Technology

[0002] In the cleaning process of medical devices, microbial content is a crucial indicator of cleanliness. Many methods exist for detecting microbial content, including plate count, nucleic acid detection, and ATP fluorescence detection. Among these, ATP fluorescence detection, with its advantages of simple operation, short detection time, and high sensitivity, has been widely used in related industries. ATP fluorescence detection can simply and efficiently measure microbial content; its detection principle is as follows:

[0003] ATP fluorescence detection utilizes a luciferase-luciferase system for the rapid detection of adenosine triphosphate (ATP). Since all living cells contain a constant amount of ATP, the ATP content clearly indicates the amount of microorganisms and other biological residues in a sample, thus assessing its cleanliness. The chemical formula for the ATP fluorescence reaction under the action of magnesium ions and luciferase is as follows:

[0004] ATP + luciferin + O2 → oxidized luciferin + CO2 + AMP + PPI + Light.

[0005] Since there is a certain functional relationship between the number of bacteria in the sample and the amount of ATP, as well as between the amount of ATP and the relative light units (RLU), the bacterial content of the sample can be obtained by detecting the RLU.

[0006] In the ATP fluorescence reaction system, luciferase is the core component, and its stability largely determines the reliability of the entire reaction system. Luciferase in the reagent (luciferase-luciferin system) is susceptible to heat damage and loses its enzyme activity, posing a drawback for ATP content detection. Due to the instability of luciferase, most current ATP fluorescence detection reagents contain protective agents to prevent its oxidation.

[0007] Patent CN200810154596.5 discloses a luciferase stabilizer for insects, comprising 80g / L-120g / L glycerol, 0.08-0.12mol / L trehalose, and the balance being double-distilled water. This stabilizer protects the activity of luciferase, making it less sensitive to temperature and freeze-thaw cycles. When used, it can be aliquoted and stored at 4℃, further enhancing the stability of the luciferase, extending its shelf life, and making it more convenient and quick to use.

[0008] Patent CN201510427377.X also discloses a luciferase stabilizer, comprising 100-250 g / L glycerol, 0.08-0.12 mol / L glycine, and double-distilled water as the solvent. This patented luciferase stabilizer protects the activity of luciferase, reduces its sensitivity to temperature and freeze-thaw cycles, and allows the luciferase to be stored in a 4°C refrigerator after being dispensed, effectively improving its stability, extending its shelf life, and making it more convenient and faster to use.

[0009] However, both patents mentioned above protect luciferase activity from a formulation perspective, specifically by adding protective agents such as glycerol, glycine, and trehalose to the reaction system. Trehalose, for example, is a non-reducing disaccharide widely found in nature. It can protect biological cells and bioactive substances from damage under adverse environmental conditions, such as high temperature, freezing, dehydration, high osmotic pressure, and toxic reagents. While adding glycerol, glycine, and trehalose to the reagent can effectively protect luciferase activity after repeated freeze-thaw cycles, the luciferase reaction system is often prepared at room temperature. Therefore, the protective efficacy of glycerol, glycine, and trehalose on luciferase needs further verification. Furthermore, luciferase inactivation is affected not only by temperature but also by environmental factors. Therefore, reducing agents should be added to the reaction system for protection against oxidation, such as reduced glutathione and dithiothreitol. Luciferase is a sulfhydryl enzyme, easily oxidized and inactivated in air. Adding reducing agents can protect the active sulfhydryl group at the enzyme's active site, preventing oxidation. Dithiothreitol and reduced glutathione can reduce the oxidized sulfhydryl groups in the enzyme molecule, allowing it to regain high activity. Simultaneously, it can reduce the inactivation caused by excessive binding sites on luciferase. The two patented formulations mentioned above do not provide perfect protection for luciferase, lacking protection for the easily oxidized groups.

[0010] Patent CN201510290281.3 relates to a method for preparing magnetically immobilized luciferase, comprising the following steps: ① preparing magnetite magnetic microspheres; ② immobilizing luciferase. The obtained magnetically immobilized enzyme exhibits good magnetic responsiveness and can maintain high enzyme activity even after repeated use. This invention uses an immobilization method to embed luciferase, which can enhance the stability of luciferase catalysis to a certain extent. However, its preparation process is complex, and the prepared reaction system is not suitable for detecting the cleaning effect of medical devices. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a high-efficiency, heat-resistant luciferase reaction system. By adding a protective agent to the reaction system and using recombinant heat-resistant luciferase, the thermal stability of luciferase is improved. In addition, other enzymes are added to the reaction system to reduce the fluorescence decay rate and improve the stability of the luciferase reaction system, thereby improving the quality of related products.

[0012] The high-efficiency, heat-resistant luciferase reaction system of the present invention comprises the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 17.1-68.4 g / L trehalose, 0.4-0.8 g / L reducing agent, 0.2-0.5 g / L D-proline, 20 mg / L pyruvate kinase, and 25 mM Tris-acetic acid buffer as the solvent.

[0013] More preferably, the high-efficiency, heat-resistant luciferase reaction system comprises the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 34.2 g / L trehalose, 0.5 g / L reducing agent, 0.4 g / L D-proline, 20 mg / L pyruvate kinase, and 25 mM Tris-acetic acid buffer as the solvent.

[0014] The recombinant luciferase was a recombinant thermostable luciferase from *E. coli*. It was purchased from Merck Biotechnology, model L9420.

[0015] In the luciferase reaction system, the most crucial component is luciferase. Since traditional luciferase is not heat-resistant, this reaction system uses a recombinant thermostable luciferase from *E. coli*, which exhibits higher enzyme activity (3 × 10⁻⁶). 11 (units / mgsolid), which is beneficial for the reaction system to quickly capture the maximum luminescence value.

[0016] The reducing agent is dithiothreitol or reduced glutathione.

[0017] The pH of 25mM Tris-acetic acid buffer is 7.8.

[0018] The purpose of this invention is to improve the stability of the luciferase reaction system. Stability includes three parts: thermal stability, antioxidant properties, and luminescence stability.

[0019] Thermal stability primarily addresses the challenge of heat-induced inactivation of luciferase, specifically preventing a decrease in the detection sensitivity of the reaction system after thermal inactivation. Currently, many types of luciferases are available on the market, some extracted directly from bioluminescent animals or bacteria. These directly extracted and purified enzymes are prone to inactivation and are expensive, suitable for research but unsuitable for product manufacturing. Therefore, recombinant luciferases are the obvious choice. The genes of these enzymes are redesigned and expressed in an E. coli vector. The enzymes are moderately priced, and their catalytic activity and stability are significantly improved, allowing for widespread application in the research and development and production of luciferase-related products.

[0020] Regarding the antioxidant properties of luciferase, it is not only easily inactivated by high temperatures, but also susceptible to damage from harmful environmental factors, such as oxidizing agents. Therefore, specific reducing agents need to be added to the luciferase reaction system to neutralize oxidizing agents and thus protect the active groups of luciferase. Currently, we have selected dithiothreitol and reduced glutathione as reducing agents.

[0021] The interpretation of ATP fluorescence detection results is characterized by the amount of fluorescence emitted. There are two types of fluorescence emission: flash and glow. ATP fluorescence detection emits flash-type light, which has a short duration and fades easily. In ATP fluorescence detection, it is easy to miss the optimal detection time, leading to inaccurate results. Therefore, delaying fluorescence fading is crucial. In the ATP fluorescence detection reaction, ATP is degraded into ADP. Allowing ADP to regenerate ATP will delay ATP fading, thus maintaining a high ATP concentration for a certain period, which is beneficial for detection accuracy. Adding pyruvate kinase (a key enzyme in the glycolysis pathway that catalyzes the conversion of phosphoenolpyruvate to enolpyruvate and produces ATP) to the reaction system can slow down the ATP degradation process, i.e., reduce the fluorescence fading rate, making the ATP fluorescence detection results more accurate.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) This invention starts from luciferase itself and improves the thermal stability of the reaction system from the source by selecting recombinant thermostable luciferase from Escherichia coli. Moreover, the recombinant thermostable luciferase from Escherichia coli has higher enzyme activity (3×10¹¹ units / mg solid), which is conducive to the reaction system to quickly capture the maximum luminescence value.

[0024] (2) The present invention adds reducing agents and other protective components to the reaction system, which can protect luciferase from oxidation while ensuring its heat resistance, thereby improving the stability of luciferase or its reaction system in many ways.

[0025] (3) In this invention, pyruvate kinase is added to the reaction system, which can catalyze the conversion of phosphoenolpyruvate into enolpyruvate and generate ATP, which can delay the degradation of ATP, i.e. reduce the fluorescence decay rate and make the ATP fluorescence detection results more accurate. Attached Figure Description

[0026] Figure 1 This is a comparison diagram of luciferase activity between Example 6 and Comparative Example 1 of the present invention;

[0027] Figure 2 This is a comparison diagram of the fluorescence fading of the reaction systems in Example 6 and Comparative Example 2 of the present invention;

[0028] Figure 3 This is a comparison chart of the relative luminescence percentages of the reaction systems of Comparative Example 3, Example 6, and Example 11 of the present invention. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. This should not be construed as a limitation on the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above content.

[0030] Examples 1-10

[0031] The study investigated the protective effect of reducing agents on the luciferase reaction system, which consisted of the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 20 mg / L pyruvate kinase, 34.2 g / L trehalose, 0.2 g / L D-proline, and 0.1-0.9 g / L reduced glutathione, in 25 mM Tris-acetic acid buffer.

[0032] The content of reduced glutathione and the relative luminescence of the luciferase reaction system are shown in Table 1.

[0033] Table 1. Content of reduced glutathione and relative luminescence of the luciferase reaction system

[0034]

[0035] As can be seen from Table 1, adding reducing agents before luciferase during the preparation of the reaction system can significantly protect the luciferase reaction system from oxidation.

[0036] Examples 11-16

[0037] The study investigated the protective effect of D-proline on the luciferase reaction system, which consisted of the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 20 mg / L pyruvate kinase, 34.2 g / L trehalose, 0.5 g / L reduced glutathione, and 0.1-0.5 g / L D-proline, in 25 mM Tris-acetic acid buffer.

[0038] The content of D-proline and the relative luminescence of the luciferase reaction system are shown in Table 2.

[0039] Table 2. D-proline content and relative luminescence of the luciferase reaction system

[0040] Example 11 0 (blank) 4403 Example 12 0.1 4519 Example 13 0.2 5788 Example 14 0.3 5920 Example 15 0.4 6254 Example 16 0.5 5877

[0041] Examples 17-21

[0042] The protective effect of trehalose on the luciferase reaction system was studied. The luciferase reaction system consisted of the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 20 mg / L pyruvate kinase, 0.2 g / L D-proline, 0.5 g / L reduced glutathione, 17.1-68.4 g / L trehalose, and 25 mM Tris-acetic acid buffer.

[0043] The trehalose content and the relative luminescence of the luciferase reaction system are shown in Table 3.

[0044] Table 3. Trehalose content and relative luminescence of the luciferase reaction system

[0045]

[0046] Comparative Example 1

[0047] The only difference between this comparative example and Example 5 is that the recombinant luciferase is replaced with an equal concentration of wild-type luciferase (unrecombined).

[0048] The luciferase activity of recombinant luciferase and wild-type luciferase, for example... Figure 1 As shown in the figure, the changes in luciferase activity were observed when two luciferases were added to the same reaction system, and the system was sealed and left at room temperature for 7 days. After seven days, the wild-type luciferase activity decreased by 77.6%, while the recombinant luciferase activity decreased by 34.3%. Therefore, the selected recombinant luciferase exhibits high thermostability.

[0049] Comparative Example 2

[0050] The only difference between this comparative example and Example 6 is that pyruvate kinase is not added to the reaction system.

[0051] The fluorescence fading of reaction systems with and without pyruvate kinase is as follows: Figure 2 As shown in the figure, when 20 mg / L pyruvate kinase was added to the reaction system, the fluorescence decay rate was 15.3% after 3 minutes of reaction, while the fluorescence decay rate without pyruvate kinase was 20.6%. After 5 minutes of reaction, the fluorescence decay rate was 38.8% for the system with pyruvate kinase and 51.5% for the system without pyruvate kinase.

[0052] Comparative Example 3

[0053] This comparative study investigated the protective effects of trehalose and D-proline on the luciferase reaction system. The only difference between this comparative study and Example 6 is that trehalose and D-proline were not added to the reaction system.

[0054] The relative luminescence percentages of the reaction systems in Comparative Examples 3, 6, and 11 are as follows: Figure 3 As shown in the figure, the addition of trehalose alone or trehalose and D-proline to the luciferase reaction system during the 38°C aging experiment promoted the thermal stability of the reaction system.

Claims

1. A highly efficient, heat-resistant luciferase reaction system, characterized in that: Includes the following components: 20 mg / L recombinant luciferase, 50 mg / L luciferin, 34.2 g / L trehalose, 0.5 g / L reducing agent, 0.4 g / L L-proline, 20 mg / L pyruvate kinase, in 25 mM Tris-acetic acid buffer; The recombinant luciferase is a recombinant thermostable luciferase from Escherichia coli; The reducing substance is reduced glutathione.

2. The high-efficiency, heat-resistant luciferase reaction system according to claim 1, characterized in that: The pH of the 25 mM Tris-acetic acid buffer is 7.8.

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