Method for intelligently controlling RNA (Ribonucleic Acid) enzyme reaction by applying Raman

Abstract

The invention discloses a method for intelligently controlling RNA (Ribonucleic Acid) enzyme reaction by applying Raman, and particularly relates to the fields of molecular biology and biological reaction engineering. Comprising the following specific preparation steps: S1, establishing a reaction model; S2, finding a characteristic peak which is most suitable for tracking; S3, tracking the change of the characteristic peak along with time; S4, comparing a result with a model which is independently calculated by chemometrics; according to the present invention, the enzyme reaction conversion rate can be monitored by using Raman, the reaction process can be subjected to in-situ, real-time, online and non-destructive composition analysis monitoring, and the previous HPLC off-line test is not required; the content of four nucleotides and the reaction conversion rate can be quickly reflected in real time; meanwhile, the reaction can be controlled to a certain degree, the device is communicated with an ultrafiltration device, the enzyme utilization rate is increased, the total RNA reaction amount is increased, the conversion rate is increased, the production cost is saved, and efficient intelligent control is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and bioreaction engineering, and more specifically, to a new process combining Raman intelligent control of RNA enzyme chemical reaction and ultrafiltration technology. Background Art

[0002] The application of phosphodiesterase or nuclease P1 can hydrolyze RNA into four nucleotides, including 5'-adenylic acid (5'-AMP), 5'-cytidylic acid (5'-CMP), 5'-guanylic acid (5'-GMP), and 5'-uridylic acid (5'-UMP). The four single nucleotides obtained after separation, purification, and crystallization can be used to manufacture various nutritional, functional, and pharmaceutical products, or they can be mixed in a certain proportion as nutritional additives for infant formula. The above RNA enzyme method for manufacturing four nucleotides has been industrially applied and has good prospects for application and promotion.

[0003] In the above process, the degree of enzyme hydrolysis, i.e., the conversion rate of RNA into four kinds of nucleotides, is a very important parameter for reaction control. The quality of the reaction, i.e., the conversion rate, has a great impact on production costs and separation and purification processes. At present, the conversion rate must be tested offline in production, even if HPLC technology is used, it takes 30-40 minutes, and the enzyme reaction speed is very fast, about 1.5 hours, nearly half or even more RNA reactions have been completed. If the reaction time is extended too much, it is possible to further decompose into corresponding nucleosides and bases. The entire enzyme reaction process ends in about 2-4 hours. Therefore, it is quite necessary to establish a real-time online rapid reaction process control technology. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, an embodiment of the present invention provides a method for intelligently controlling RNA enzyme reactions using Raman. The technical problem to be solved by the present invention is: how to establish a real-time online rapid reaction process control technology for nucleotides.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for intelligently controlling RNA enzyme reaction using Raman, comprising the following specific preparation steps:

[0006] S1. Establish the reaction model: prepare 8L of RNA solution with a concentration of 32g / L, add 1g ZnSO4, stir and heat to 72℃, and add 2% phosphodiesterase;

[0007] S2. Find the most suitable characteristic peak for tracking: perform offline Raman spectrum comparison on the solution in step S1 to check the GMP characteristic peak, UMP characteristic peak, AMP characteristic peak and CMP characteristic peak;

[0008] S3, tracking the change of characteristic peaks over time: generating a two-dimensional trend graph of the rising and falling trends of the GMP and UMP characteristic peaks detected undisturbed in step S2 over time;

[0009] S4: Compare the results with the model calculated independently from the chemometrics;

[0010] S5: Determine the trend line that best characterizes the response;

[0011] S6: List the best options for application.

[0012] In a preferred embodiment, the enzyme activity of the phosphodiesterase in step S1 is 6000u / g.

[0013] In a preferred embodiment, in step S2, the model established in step S1 is used to process data with the aid of a data model function of a software, wherein the software uses Find Trends to analyze One Click reactions.

[0014] In a preferred embodiment, during the modeling process of step S1, quantitative samples are selected as modeling items and prediction items, and the above-established model is verified on the predicted set samples by offline Raman spectroscopy data.

[0015] In a preferred embodiment, the operation in step S1 is carried out in a 10L glass reactor with matching jacket heating, the stirring speed is 100 rpm, the temperature in the reactor is maintained at 72°C by a super thermostat, and a Raman probe is inserted into the reactor. When the temperature in the reactor reaches 72°C, the enzyme timing is added, and the total nucleotide concentration is determined by the Raman curve and modeling software.

[0016] In a preferred embodiment, the step S4 is performed by comparing the characteristic peaks with the independent model chemometric calculation results; the calculation formula includes: Lambert-Beer law ,in, is the extinction coefficient; is the optical path (cm); is the solute molar concentration (mol / L).

[0017] In a preferred embodiment, the reaction is carried out for 1.5 hours, and when the enzyme reaction conversion rate reaches 85%, an ultrafiltration device is connected, and the molecular weight of the ultrafiltration membrane is controlled to be 6000 or 10000, and ultrafiltration is carried out. The ultrafiltration filtrate is collected and transferred to the nucleotide separation and purification system; the macromolecular substances that have not passed the ultrafiltration solution, namely RNA and enzyme, are returned to the enzyme reactor, and RNA solution is added to continue to keep the reactor solution consistent, and ultrafiltration is continued while reacting until the additional 16L RNA solution (containing 2g of zinc sulfate) is used up; the reaction is completed after about 8-9 hours, and ultrafiltration is continued until the remaining volume of the ultrafiltrate that has not passed is 0.5-1L; 2L of pure water is added three times to continue ultrafiltration until only 0.5L of the solution that has not passed the ultrafiltration remains, and the total reaction solution that has passed the ultrafiltration is 25L.

[0018] Technical effects and advantages of the present invention:

[0019] The present invention obtains good results for RNA enzymatic reaction process control by applying online Raman technology; there are reports on the application of Raman to control fermentation, control juice brewing, cell culture, etc., but there are no reports on the control of the enzyme reaction process; the present invention finds that Raman can be used to monitor the enzyme reaction conversion rate, and to achieve in-situ, real-time, online and non-destructive composition analysis monitoring of the reaction process, and no previous HPLC offline test is required; the four nucleotide contents and reaction conversion rate can be quickly reflected in real time; at the same time, the reaction can be controlled to a certain extent, connected with an ultrafiltration device, to improve enzyme utilization, increase the total RNA reaction amount, improve conversion rate, save production costs, etc., and realize efficient and intelligent control. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the offline Raman spectrum comparison of the present invention.

[0021] Figure 2 This is the offline three-dimensional atlas of the present invention.

[0022] Figure 3 It is the GMP characteristic peak spectrum diagram of the present invention.

[0023] Figure 4 It is the UMP characteristic peak spectrum diagram of the present invention.

[0024] Figure 5 It is the linear correlation diagram of AMP of the present invention.

[0025] Figure 6 It is the CMP linear correlation diagram of the present invention.

[0026] Figure 7 It is the GMP linear correlation diagram of the present invention.

[0027] Figure 8It is the UMP linear correlation diagram of the present invention.

[0028] Fig. 9 It is the total concentration linear correlation diagram of the present invention.

[0029] Fig.10 It is a two-dimensional trend diagram of the characteristic peaks of GMP and UMP of the present invention.

[0030] Fig.11 It is the reaction flow chart of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The present invention provides a method for intelligently controlling RNA enzyme reaction using Raman, and the specific details of the invention are as follows:

[0033] The Raman measurement principle is based on the change in polarizability during molecular vibrations; when incident photons strike a molecule, the polarizability (bond properties) changes; the energy released (scattering), usually at a lower energy state, generates a spectrum that is unique to that molecule; the intensity of the Raman scattering is proportional to the number of molecules with that chemical bond and provides both qualitative and quantitative measurements;

[0034] The 785nm wavelength laser excitation is used, which is more suitable for organic chemical molecules and maximizes the scope of application; relying on the Mettler Raman instrument platform, the following steps are followed: first, the reaction model is built; the characteristic peak that is most suitable for tracking is found; the characteristic peak is tracked over time; the results are compared with the model independently calculated by chemometrics; the trend line that is most suitable for characterizing the reaction is determined; the optimal options are listed for application; in this way, the data model function of the OneClick reaction is analyzed with the help of FindTrends software to process the data, and the characteristic peaks are compared with the chemometric calculation results of the independent model; the calculation formula includes: Lambert-Beer law ,in, is the extinction coefficient; is the optical path (cm); is the solute molar concentration (mol / L), and the data reliability is guaranteed;

[0035] In RNA enzymatic hydrolysis, due to the characteristics of the enzyme reaction itself, the efficiency is high and the time is short; the reaction time is generally completed in 2-4 hours; if the reaction time is short, the enzyme reaction is incomplete; if the time is long, side reactions will occur, in which the nucleotides will be further degraded to generate corresponding nucleosides or bases; for example; 5'-AMP will be further degraded to adenosine or adenine; and 5'-CMP will be deaminated to generate 5'-UMP, so too high 5'-UMP generation also means that other nucleotides are degraded, and the overall conversion rate will be reduced; but the enzyme reaction conversion rate in current production is analyzed offline by HPLC after sampling, and the results are generally obtained after 30-40 minutes; this often delays the end of the reaction, resulting in not only a decrease in the reaction conversion rate, but also an increase in impurities, which increases the difficulty of the following separation process;

[0036] The application of Raman technology can realize real-time online detection of enzyme reactions; the concentration and conversion rate of various nucleotides can be accurately controlled, and the end point of the reaction can be accurately determined according to the amount of various nucleotides; according to the test results, the conversion rate can be increased by about 3-5%, and if unexpected situations occur during the reaction, they can also be handled in time;

[0037] In the experiment Figure 5 The fastest point of the enzyme reaction speed is 1-1.5h, and the end point is 2-2.5h, which is 0.5-1h earlier than the actual production operation, and it is found that the enzyme is still active at this time; the price of enzymes is relatively expensive, which is an important production cost. How to make full use of these enzymes; with the support of the Raman technology system, further exploration was carried out and the following measures were taken: 1. First, consider adding RNA substrate 1-1.5 hours after the enzyme reaction; 2. Considering that as the enzyme reaction proceeds, the pH drops from 6.0 to about 4.5, so buffer is added to prevent the pH from dropping rapidly; in short, various measures did not have the expected effect; but it was found that the addition of phosphate buffer decreased the total amount of nucleotides, that is, the conversion rate decreased; after repeated experiments, it was finally discovered that the reason was the inhibition of the substrate (nucleotide), which is consistent with the fact that the enzyme reaction speed is very fast in the early stage, but very slow after 1.5-2 hours; in addition, adjusting the initial pH of the reaction to 4.5, 5.0, 5.5, 6.0, etc. has no effect on the enzyme reaction;

[0038] Further use 6000 or 10,000 molecular weight, area 0.4M 2The ultrafiltration device is connected to a 10L reactor; ultrafiltration is performed after 1.5 hours of enzyme reaction, and 3.2% RNA is added to the enzyme reactor at the same time, with a total feed volume of 16L, and 2g ZnSO4 is contained therein, and the speed of adding RNA solution is consistent with the ultrafiltration speed to keep the volume balance of 8L feed liquid in the reactor; the reaction is carried out for a total of 9 hours, and 16L of ultrafiltration filtrate is collected; the feed liquid in the reactor continues to react for 1 hour until the enzyme reaction conversion rate reaches 90%±2% and ends; then the reaction liquid is also ultrafiltered, and finally 2L of pure water is added for washing 3 times until the circulating feed liquid contains almost no nucleotides, or the concentration is lower than 0.1g / L, and the ultrafiltration ends; A total of 25L of reaction liquid was obtained; during the ultrafiltration process, the enzyme and RNA macromolecules did not pass through the ultrafiltration membrane back to the reactor, while the molecular weight of nucleotides was 300-400, so they passed through the ultrafiltration membrane and were collected outside the reactor; in this way, the substrate left the reaction system and no longer inhibited the reaction, while the phosphodiesterase and RNA reacted quickly in the reactor, and good results were obtained with the support of Raman technology; with the same enzyme amount as 8L of reaction liquid, the reaction volume increased by 3 times, and the total conversion rate increased by 3%-5%, that is, reaching 93%-95%. Due to the increase in conversion rate, the enzyme amount was reduced by 2 / 3, and the total production cost decreased by 15%-20%;

[0039] The following examples further illustrate the present invention; (AMP, CMP, UMP, GMP are omitted 5'-)

[0040] Example 1: Modeling;

[0041] Prepare 8L of RNA solution with a concentration of 32g / L, add 1g of ZnSO4, stir and heat to 72℃, add 2% phosphodiesterase (enzyme activity 6000u / g), and compare the Raman spectra of the offline standard samples. Figure 1 shown by Figure 1 It can be seen that the characteristic peak of GMP is 1487cm -1 The characteristic peak position of UMP is 1677cm -1 and 1230cm -1 The characteristic peak position of CMP is 1293cm -1 The characteristic peak of AMP is at 728 cm -1 ; However, RNA is interfered by the fluorescence effect, its characteristic peak is not obvious, and it affects the characteristic peaks of other small molecules during the shearing process; the experimental results show that the characteristic peaks of UMP and CMP can still be tracked; the three-dimensional map is as follows Figure 2 As shown; the characteristic peak position of GMP is located at 1487cm by offline spectrum. -1 ; There will be a gradual upward trend in the reaction, and the peak will rise faster in the early stage and then slowly become stable, such as Figure 3As shown; the characteristic peak position of UMP is located at 1230cm by offline spectrum. -1 ; The same reaction will have a gradual upward trend, and the peak will rise well in the early stage, and then slowly become stable, such as Figure 4 As shown; the changes of its GMP and UMP characteristic peaks over time generate a two-dimensional trend graph, as shown Fig.10 As shown; it reaches its peak after about one and a half hours of reaction, after which the data fluctuates less and decreases slightly;

[0042] Example 2: Measurement;

[0043] During the modeling process, 10 samples from the first and second batches of reactions were used for modeling, and the remaining 4 samples were used for prediction, such as Figure 5-9 shown; Figure 5 is the linear correlation of AMP, Figure 6 is the CMP linear correlation, Figure 7 is the linear correlation of GMP, Figure 8 is the UMP linear correlation, Fig. 9 is the linear correlation of total concentration; the model established by the above data is verified on the predicted set samples; the prediction deviation of the model is shown in Table 1, where the average absolute deviation of 5-AMP is 0.11, CMP is 0.27, GMP is 0.37, UMP is 0.44, and the total concentration is 1.086. The overall average absolute deviation is within the acceptable range; the reaction conversion rate (W / W) can be calculated from the total concentration, that is: ;

[0044] Table 1 Prediction bias

[0045] Example 3: Raman control of RNase reaction;

[0046] According to the formula and operation of Example 1, the reaction was carried out in a 10L glass reactor with matching jacket heating, the stirring speed was 100rpm, the temperature in the reactor was kept at 72°C by a super thermostat, and the Raman probe was inserted into the reactor. When the temperature in the reactor reached 72°C, the enzyme was added and the reaction was carried out for 2.5 hours. The total nucleotide concentration was 28.8g / L as determined by Raman, that is, the conversion rate was equal to 90%; at this time, the temperature was quickly raised to 85°C and maintained for 10 minutes to stop the enzyme reaction; then the reaction was cooled to below 30°C, filtered, and microfiltered before entering the next separation stage; if the sample was taken at 2.5 hours, the HPLC analysis was delayed to 3 hours before the reaction was stopped. At this time, due to the increase of side reactions, the conversion rate dropped to 86%;

[0047] Example 4: A new process combining Raman control of RNA enzyme reaction with ultrafiltration;

[0048] Same formulation and operation as in Example 3; in addition, 0.4 M 2 , a 10,000 molecular weight ultrafiltration membrane device, and according to the formulation of Example 3, 16 liters of reaction solution are additionally configured; after reacting for 1 hour according to Example 3, the enzyme reactor is connected to the ultrafiltration device, and 16 liters of pre-prepared feed solution are added to the enzyme reactor; the feed rate is the same as the ultrafiltration filtrate rate, about 2 liters per hour; that is, the ultrafiltrate outflow rate is 2 liters / h, and 2 liters of feed solution are added to the enzyme reactor every hour; and the feed solution that has not passed the ultrafiltration, mainly enzymes and RNA, is still returned to the reactor, and its reaction loop is as follows Fig.11 As shown; the ultrafiltrate passing through the ultrafiltration device is mostly nucleotides and a small amount of nucleosides and bases of side reactions; this cycle is repeated, and the ultrafiltration and feeding are stopped after the enzyme reaction and ultrafiltration device have been running for 8 hours, while the enzyme reactor continues to react for another hour, and the reaction is stopped after the conversion rate reaches more than 88%; the reaction solution is continued to be ultrafiltered, and 2L of pure water is added 3 times to wash the final remaining liquid when it is almost finished, until the total nucleotide concentration is lower than 0.1g / L or it almost contains no nucleotides; the ultrafiltrate is combined to obtain a total of 25L reaction solution, which is detected by HPLC, and the total conversion rate reaches 93%, of which the concentration of AMP is 7.31g / L, the concentration of CMP is 5.64g / L, the concentration of GMP is 9.63g / L, and the concentration of UMP is 7.12g / L.

[0049] In summary, the application of online Raman to monitor the entire process of RNA enzymatic hydrolysis reaction can obtain the concentration and conversion rate of the real-time products 5'-AMP, 5'-CMP, 5'-GMP, and 5'-UMP in the reaction; and connect the ultrafiltration device at an appropriate time after the reaction to filter out the above products, and return the enzyme and RNA that have not passed the ultrafiltration to the enzyme reactor to continue the reaction; the RNA feed solution is supplemented during ultrafiltration to keep the total reaction volume consistent; the Raman real-time conversion rate can accurately control the enzyme reaction time and the timing of connecting the ultrafiltration device; as a result, the total amount of converted RNA increased by 3 times, the total conversion rate increased by 3%-5%, the total amount of enzyme used was saved by 2 / 3, and the production cost was saved by 15%-20%; because the product passed the ultrafiltration, the macromolecular enzyme, unreacted RNA and other impurities were removed, which is conducive to the subsequent process of further separation and purification.

[0050] Finally, a few points should be explained: First, in the description of this application, it should be noted that, unless otherwise specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, which may refer to mechanical connection or electrical connection, or internal communication between two components, or direct connection. "upper", "lower", "left", "right", etc. are only used to indicate relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may change;

[0051] Secondly: In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other;

[0052] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for intelligently controlling RNA enzyme reactions using Raman, characterized in that: The specific preparation steps are as follows: S1. Establish the reaction model: prepare 8L of RNA solution with a concentration of 32g / L, add 1g ZnSO4, stir and heat to 72℃, and add 2% phosphodiesterase; S2, tracking characteristic peaks: performing offline Raman spectrum comparison on the solution in step S1 to check the GMP characteristic peak, UMP characteristic peak, AMP characteristic peak and CMP characteristic peak; S3, selecting characteristic peaks that are not disturbed to track their changes over time: generating a two-dimensional trend graph of the rising and falling trends of the characteristic peaks of GMP and UMP detected in step S2 over time; S4: Compare the results with the model calculated independently from the chemometrics; S5: Determine the trend line that best characterizes the response; S6: List the best options for application.

2. The method for controlling RNA enzyme reaction by Raman intelligent method according to claim 1, characterized in that: The enzyme activity of the phosphodiesterase in step S1 is 6000u / g.

3. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 1, characterized in that: In step S2, the model established in step S1 is used to process data with the help of the data model function of the software, wherein the software uses FindTrends to analyze the One Click reaction.

4. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 1, characterized in that: In the modeling process of step S1, quantitative samples are selected as modeling items and prediction items, and the above-established model is verified on the prediction set samples by offline Raman spectroscopy data.

5. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 1, characterized in that: The operation in step S1 is carried out in a 10L glass reactor with matching jacket heating, with a stirring speed of 100 rpm, and a super thermostat is used to heat and maintain the temperature in the reactor at 72°C, and a Raman probe is inserted into the reactor. When the temperature in the reactor reaches 72°C, the enzyme timing is started, and the parameters of the entire reaction process of the enzyme reaction conversion rate can be monitored in situ, in real time, and online according to the Raman curve and modeling software, and the total nucleotide concentration is determined by Raman.

6. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 1, characterized in that: In step S4, the characteristic peaks are compared with the independent model chemometric calculation results; the calculation formula includes: Lambert-Beer law ,in, is the extinction coefficient; is the optical path length - cm; is the solute molar concentration - mol / L.

7. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 1, characterized in that: The reaction was carried out for 1.5 hours. When the enzyme reaction conversion rate reached 85%, the ultrafiltration device was connected, and the molecular weight of the ultrafiltration membrane was controlled at 6000 or 10000 for ultrafiltration. The ultrafiltration filtrate was collected and transferred to the nucleotide separation and purification system.

8. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 7, characterized in that: The macromolecular substances that have not passed the ultrafiltration solution, namely RNA and enzyme, are returned to the enzyme reactor, and RNA solution is added to keep the reactor solution consistent. Ultrafiltration and reaction are continued until the additional 16L RNA solution is used up.

9. The method for controlling RNA enzyme reaction by using Raman intelligent method according to claim 8, characterized in that: The reaction was completed after 8-9 hours, and ultrafiltration was continued until the volume of the remaining solution that did not pass through the ultrafiltration was 0.5-1 L; 2 L of pure water was added three times to continue ultrafiltration until only 0.5 L of the solution that did not pass through the ultrafiltration remained, and the total reaction solution that passed through the ultrafiltration was 25 L.

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