Transaminase selection process for the production of sitagliptin phosphate

Abstract

This invention relates to a method for selecting transaminases for the production of sitagliptin phosphate, belonging to the field of transaminase screening technology. The method includes: site-specific protein modification of each pre-selected sitagliptin phosphate transaminase to obtain each sitagliptin phosphate transaminase, and calculating topological constraint matching conditions; injecting each enzyme solution into different channels of a micro-immobilized enzyme screening reactor, polymerizing each enzyme solution based on polymerization conditions to obtain the final polymerized product of each enzyme solution; inputting a sitagliptin phosphate precursor substrate solution into the micro-immobilized enzyme screening reactor, and performing an enzymatic catalytic reaction with the final polymerized product, calculating the product conversion rate for each channel; screening the sitagliptin phosphate transaminase with the highest product conversion rate as the optimal transaminase, and applying it to the production of sitagliptin phosphate raw materials. This invention improves the efficiency of screening optimal transaminases by combining site-specific protein modification and controlled polymerization, and provides highly efficient transaminases for industrial production.

Description

Technical Field

[0001] This invention relates to the field of transaminase screening technology, and more particularly to a method for selecting transaminases for the production of sitagliptin phosphate. Background Technology

[0002] Sitagliptin phosphate is the core active ingredient in dipeptidyl peptidase-4 inhibitors, a class of antidiabetic drugs, and is one of the top-selling drugs for treating type 2 diabetes. However, the chemical synthesis of sitagliptin phosphate has long faced significant challenges. Its molecular structure contains a chiral amine group, and traditional production processes require the use of a chiral rhodium catalyst under high-pressure hydrogenation conditions to synthesize this group. This process is not only costly but also results in low purity of the sitagliptin phosphate product. This bottleneck has prompted researchers to seek more efficient and greener alternative synthetic routes.

[0003] Current processes utilize transaminases in the production of sitagliptin phosphate, catalyzing the amino transfer reaction of sitagliptin phosphate precursor substrates to efficiently synthesize chiral amine compounds (compounds with chiral amine groups). However, traditional methods directly utilize natural amino acids, and natural transaminases exhibit low catalytic efficiency for sitagliptin phosphate precursor substrates, thus severely impacting the production efficiency of sitagliptin phosphate. Summary of the Invention

[0004] To solve the above-mentioned technical problems, this invention provides a method for selecting transaminases for the production of sitagliptin phosphate. The technical solution of this invention is as follows: Transaminase selection methods used in the production of sitagliptin phosphate include: S1, introduce non-natural amino acids into the specified position of each preselected sitagliptin phosphate transaminase to complete the site-specific protein modification of each preselected sitagliptin phosphate transaminase, obtain each sitagliptin phosphate transaminase, and calculate the topological constraint matching conditions of each sitagliptin phosphate transaminase. S2, generate the enzyme solution for each sitagliptin phosphate transaminase, inject each enzyme solution into different channels of the micro-immobilized enzyme screening reactor, adjust the initial polymerization conditions of the corresponding enzyme solution according to the topological constraint matching conditions of each sitagliptin phosphate transaminase to obtain the polymerization conditions, polymerize each enzyme solution based on the polymerization conditions to obtain the final polymer product of each enzyme solution. S3, Sitagliptin phosphate precursor substrate solution is fed into a micro-immobilized enzyme screening reactor and subjected to enzyme catalysis reaction with the final polymerization product of each enzyme solution to obtain the product after catalysis in each channel, and the product conversion rate of each channel is calculated. S4. The transaminase with the highest product conversion rate was selected as the optimal transaminase, and the optimal transaminase was applied to the production of sitagliptin phosphate raw material.

[0005] Preferably, S1 includes: S11, obtain the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase, and extract the modification sites in the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase as the designated locations of each preselected sitagliptin phosphate transaminase. S12, obtain the spatial coordinates of the specified location of each preselected sitagliptin phosphate transaminase in a high-resolution three-dimensional structure; S13, introduce non-natural amino acids into the specified positions of each preselected sitagliptin phosphate transaminase according to the spatial coordinates of the high-resolution three-dimensional structure at the specified positions, and complete the site-specific protein modification of each preselected sitagliptin phosphate transaminase to obtain each sitagliptin phosphate transaminase. S14, calculate the topological distance between the specified site of each preselected sitagliptin phosphate transaminase and its substrate binding site, and calculate the local density coefficient of the specified site of each preselected sitagliptin phosphate transaminase. The topological constraint matching condition of each preselected sitagliptin phosphate transaminase is constituted by the topological distance between the specified site of each preselected sitagliptin phosphate transaminase and its substrate binding site and its local density coefficient.

[0006] Preferably, S2 includes: S21, generate the enzyme solution for each sitagliptin phosphate transaminase, and inject each enzyme solution into a different channel of the micro-immobilized enzyme screening reactor. S22, obtain the initial polymerization conditions of the enzyme solution of each sitagliptin phosphate transaminase, construct an initial polymerization vector with each polymerization parameter in the initial polymerization conditions of the enzyme solution of each sitagliptin phosphate transaminase, generate the adjustment coefficient of each polymerization parameter of each sitagliptin phosphate transaminase according to the topological constraint matching conditions and the initial polymerization vector, and adjust the initial polymerization conditions of the corresponding enzyme solution according to the adjustment coefficient of each polymerization parameter of each sitagliptin phosphate transaminase to obtain the polymerization conditions; S23 controls the polymerization conditions of each enzyme solution in the micro-immobilized enzyme screening reactor and monitors the polymerization status parameter data of each channel in real time. S24, adjust the polymerization conditions of each channel in real time according to the polymerization state parameter data of each channel until each channel completes polymerization and obtains the final polymerization product of each enzyme solution.

[0007] Preferably, S22 includes: S221, obtain the initial polymerization conditions of the enzyme solution of the target sitagliptin phosphate transaminase according to the preset standard polymerization parameter group, and construct an initial polymerization vector with the initial value of each polymerization parameter in the initial polymerization conditions of the target sitagliptin phosphate transaminase, wherein the target sitagliptin phosphate transaminase is any sitagliptin phosphate transaminase. S222, Calculate the matching value between the target sitagliptin phosphate transaminase and each preset topological constraint based on the topological constraint matching conditions of the target sitagliptin phosphate transaminase; S223, Based on the matching value between the target sitagliptin phosphate transaminase and each preset topological constraint and the preset weighting coefficient between each preset topological constraint and each polymerization parameter, calculate the adjustment coefficient of each polymerization parameter of the target sitagliptin phosphate transaminase; S224, adjust the initial value of each polymerization parameter of the target sitagliptin phosphate transaminase enzyme solution according to the adjustment coefficient of each polymerization parameter to obtain the actual value of each polymerization parameter. The actual values ​​of all polymerization parameters constitute the polymerization conditions of the target sitagliptin phosphate transaminase enzyme solution.

[0008] Preferably, S24 includes: S241, construct the aggregated state vector of the target channel based on the aggregated state parameter data of the target channel at the current moment, wherein the target channel is any channel; S242, construct a control vector based on all aggregation parameters in the aggregation conditions of the target channel; S243, obtain the aggregation change characteristics of the target channel at the current moment, and construct the state transition equation between the control vector and the aggregation state vector based on the aggregation change characteristics; S244: Predict the future aggregation state vector of the target channel at the next moment based on the pre-trained Kalman filter, and input the future aggregation state vector into the state transition equation to obtain the execution control vector at the current moment. S245, adjust the aggregation conditions of the target channel according to the execution control vector at the current moment to obtain new aggregation conditions, and control the target channel to execute the new aggregation conditions; S246, continue steps S241 to S245 until the target channel completes polymerization, and obtain the final polymerized product of the enzyme solution corresponding to the target channel.

[0009] Preferably, S243 includes: S2431, obtain the polymerization product of the target channel at the current time, determine the peak area of ​​the polymerization product at the current time by CE method, and calculate the polymerization product generation rate of the target channel at the current time based on the peak area of ​​the polymerization product at the current time, and use the polymerization product generation rate of the target channel at the current time as the polymerization change characteristic of the target channel at the current time. S2432, obtain the initial product concentration equation of sitagliptin phosphate transaminase, correct the preset state transition matrix in the initial product concentration equation by the polymerization change characteristics, and obtain the corrected state transition matrix. Based on the corrected state transition matrix, the control vector and the polymerization state vector, construct the state transition equation between the control vector and the polymerization state vector.

[0010] Preferably, S3 includes: S31, Sitagliptin phosphate precursor substrate solution is fed into a micro-immobilized enzyme screening reactor, and the initial pH, initial conductivity and initial temperature of the sitagliptin phosphate precursor substrate solution in each channel of the micro-immobilized enzyme screening reactor are measured. S32, after the sitagliptin phosphate precursor substrate solution flows through each channel of the micro-immobilized enzyme screening reactor, it undergoes an enzyme-catalyzed reaction with the final polymerization product of each enzyme solution to obtain the catalyzed product of each channel. The pH value, conductivity and temperature of the catalyzed product of each channel are measured. The pH conversion rate of each channel is calculated based on the initial pH value of each channel and the pH value of the catalyzed product. S33, based on the initial conductivity, initial temperature, conductivity and temperature of each channel, the pH conversion rate of each channel is corrected to obtain the product conversion rate of each channel.

[0011] Preferably, S33 includes: S331, calculate the conductivity variation coefficient based on the initial conductivity and conductivity of each channel; S332, calculate the initial pH value and the difference between the pH values ​​for each channel to obtain the pH change value, and determine the conductivity correction factor for each channel based on the conductivity change coefficient and the pH change value; S333, adjust the initial pH value of each channel according to the initial temperature of each channel to obtain the corrected initial pH value, adjust the pH value of each channel according to the temperature of each channel to obtain the corrected pH value, and calculate the corrected pH conversion factor of each channel according to the corrected initial pH value, the corrected pH value and the pH conversion rate. S334, the pH conversion rate of each channel is jointly adjusted according to the conductivity correction factor and pH conversion correction factor of each channel to obtain the product conversion rate of each channel.

[0012] Preferably, the method further includes: The optimal transaminase was controlled to remain at multiple preset simulated production temperatures for preset time intervals, and its relative residual activity at each preset simulated production temperature was measured. Thermostability curves were constructed based on the relative residual activity of the optimal transaminase at each preset simulated production temperature, and the half-life of the optimal transaminase was determined by the thermostability curves. If the half-life of the optimal transaminase is less than the preset half-life threshold, the activity of the optimal transaminase is determined to be non-compliant, and a preselected sitagliptin phosphate transaminase is regenerated and a new optimal transaminase is screened.

[0013] All of the above-mentioned optional technical solutions can be combined arbitrarily, and the present invention will not provide a detailed description of the structure after each combination.

[0014] By means of the above solution, the beneficial effects of the present invention are as follows: This invention first involves site-directed protein modification of multiple pre-selected sitagliptin phosphate transaminases to obtain the corresponding sitagliptin phosphate transaminases, and then obtaining the polymerization conditions of the enzyme solution for each sitagliptin phosphate transaminases. Subsequently, multiple channels of a micro-immobilized enzyme screening reactor are used to simultaneously and controllably polymerize the multiple pre-selected sitagliptin phosphate transaminases. After simultaneous catalysis with sitagliptin phosphate precursor substrate solutions, the product conversion rate of each channel is calculated, and the optimal transaminase with the highest product conversion rate is screened. This provides a scientific method for screening transaminases for the production of sitagliptin phosphate. This method, by combining site-directed protein modification and controllable polymerization, can improve the efficiency of screening the optimal transaminase and can provide industrial enzymes that can improve product conversion rates for industrial production, thus promoting the development of green manufacturing industries to a certain extent.

[0015] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0016] Figure 1 This is a schematic flowchart of the transaminase selection method for producing sitagliptin phosphate provided by the present invention. Detailed Implementation

[0017] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0018] like Figure 1 As shown, the transaminase selection method for producing sitagliptin phosphate provided in this embodiment of the invention includes: S1, introduce non-natural amino acids into the specified positions of each preselected sitagliptin phosphate transaminase to complete the site-specific protein modification of each preselected sitagliptin phosphate transaminase, obtain each sitagliptin phosphate transaminase, and calculate the topological constraint matching conditions of each sitagliptin phosphate transaminase.

[0019] Specifically, each preselected sitagliptin phosphate transaminase is pre-selected. By site-specifically modifying non-natural amino acid proteins into designated locations of each preselected sitagliptin phosphate transaminase, the function of each resulting sitagliptin phosphate transaminase is guaranteed to be defined, facilitating subsequent catalytic studies. In this embodiment of the invention, the topological constraint matching condition refers to the topological distance and local density coefficient between the designated location of the preselected sitagliptin phosphate transaminase and its substrate binding site.

[0020] In one specific embodiment, S1 includes: S11, obtain the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase, and extract the modification sites in the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase as the designated locations for each preselected sitagliptin phosphate transaminase.

[0021] Specifically, the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase is determined in advance by X-ray crystallography. The modification site of a particular preselected sitagliptin phosphate transaminase is a pre-determined site for protein-specific modification of that preselected sitagliptin phosphate transaminase.

[0022] S12, obtain the spatial coordinates of the specified location of each preselected sitagliptin phosphate transaminase in a high-resolution three-dimensional structure.

[0023] S13, based on the specified location coordinates of the high-resolution three-dimensional structure, introduce non-natural amino acids to the specified position of each preselected sitagliptin phosphate transaminase, thereby completing the site-specific protein modification of each preselected sitagliptin phosphate transaminase and obtaining each sitagliptin phosphate transaminase.

[0024] Specifically, by adding fluorescent dye to a specified location of each preselected sitagliptin phosphate transaminase according to the spatial coordinates of the high-resolution three-dimensional structure, the protein modification of each preselected sitagliptin phosphate transaminase by non-natural amino acids can be precisely achieved.

[0025] S14, calculate the topological distance between the specified site of each preselected sitagliptin phosphate transaminase and its substrate binding site, and calculate the local density coefficient of the specified site of each preselected sitagliptin phosphate transaminase. The topological constraint matching condition of each preselected sitagliptin phosphate transaminase is constituted by the topological distance between the specified site of each preselected sitagliptin phosphate transaminase and its substrate binding site and its local density coefficient.

[0026] Specifically, the substrate binding site is a pre-determined location where the substrate of each preselected sitagliptin phosphate transaminase binds to the protein. The topological distance between the designated site of a preselected sitagliptin phosphate transaminase and its substrate binding site is calculated using the three-dimensional Euclidean distance. When calculating the local density coefficient of the designated site of the preselected sitagliptin phosphate transaminase, the number of neighboring atoms within the van der Waals radius of the designated site is first obtained. Then, the volume of the sphere of the neighborhood is calculated based on the van der Waals radius. Finally, the local density coefficient of the designated site of the preselected sitagliptin phosphate transaminase is obtained by dividing the number of neighboring atoms by the volume of the sphere.

[0027] S2, generate the enzyme solution for each sitagliptin phosphate transaminase, inject each enzyme solution into different channels of the micro-immobilized enzyme screening reactor, adjust the initial polymerization conditions of the corresponding enzyme solution according to the topological constraint matching conditions of each sitagliptin phosphate transaminase to obtain the polymerization conditions, polymerize each enzyme solution based on the polymerization conditions to obtain the final polymer product of each enzyme solution.

[0028] Specifically, this embodiment of the invention employs a micro-immobilized enzyme screening reactor, which ensures that multiple sitagliptin phosphate transaminase solutions react simultaneously, meeting the requirement for subsequent simultaneous observation of the polymerization parameters of multiple sitagliptin phosphate transaminase solutions and improving screening efficiency. The initial polymerization conditions of the enzyme solutions include polymerization parameters such as enzyme concentration, temperature, pH, and polymerization time. The initial polymerization conditions for each sitagliptin phosphate transaminase solution are preset.

[0029] In one specific embodiment, S2 includes: S21, generate the enzyme solution for each sitagliptin phosphate transaminase, and inject each enzyme solution into a different channel of the micro-immobilized enzyme screening reactor.

[0030] Specifically, in the process of generating a certain sitagliptin phosphate transaminase enzyme solution, the bacterial cells of sitagliptin phosphate transaminase are subjected to high-pressure homogenization to obtain an enzyme solution containing a large amount of sitagliptin phosphate transaminase.

[0031] S22, obtain the initial polymerization conditions of the enzyme solution for each sitagliptin phosphate transaminase, construct an initial polymerization vector with each polymerization parameter in the initial polymerization conditions of the enzyme solution for each sitagliptin phosphate transaminase, generate adjustment coefficients for each polymerization parameter of each sitagliptin phosphate transaminase according to the topological constraint matching conditions and the initial polymerization vectors, and adjust the initial polymerization conditions of the corresponding enzyme solution according to the adjustment coefficients of each polymerization parameter of each sitagliptin phosphate transaminase to obtain the polymerization conditions.

[0032] Specifically, the initial polymerization conditions of a certain sitagliptin phosphate transaminase solution and the initial values ​​of each polymerization parameter are: enzyme concentration 20%, temperature 30℃, pH value 7 and polymerization time 2 hours. Then its initial polymerization vector can be represented as [0.2,30,7,2].

[0033] In one specific embodiment, S22 includes: S221, obtain the initial polymerization conditions of the enzyme solution of the target sitagliptin phosphate transaminase according to the preset standard polymerization parameter group, and construct an initial polymerization vector with the initial value of each polymerization parameter in the initial polymerization conditions of the target sitagliptin phosphate transaminase, wherein the target sitagliptin phosphate transaminase is any sitagliptin phosphate transaminase.

[0034] Specifically, the preset standard polymerization parameter set consists of pre-determined initial values ​​for multiple polymerization parameters of the target sitagliptin phosphate transaminase enzyme solution. These initial values ​​constitute the initial polymerization conditions of the enzyme solution.

[0035] S222, Calculate the matching value between the target sitagliptin phosphate transaminase and each preset topological constraint based on the topological constraint matching conditions of the target sitagliptin phosphate transaminase.

[0036] Specifically, the preset topological constraints are pre-defined topological constraint evaluation criteria, including structural stability, spatial obstruction, and compatibility. When calculating the matching value between the target sitagliptin phosphate transaminase and structural stability, the local density coefficient is used as the matching value. When calculating the matching value between the target sitagliptin phosphate transaminase and spatial obstruction, the ratio of the topological distance between the specified location of the target sitagliptin phosphate transaminase and its substrate binding location to the standard distance is used as the matching value. In this embodiment, the standard distance is 0.4 nanometers. When calculating the matching value P between the target sitagliptin phosphate transaminase and compatibility, the formula is: P = a1 × L + a2 × R; where a1 represents the weighting coefficient of the topological distance, a2 represents the weighting coefficient of the local density coefficient, L represents the topological distance between the specified location of the target sitagliptin phosphate transaminase and its substrate binding location, and R represents the local density coefficient.

[0037] S223, based on the matching value between the target sitagliptin phosphate transaminase and each preset topological constraint and the preset weighting coefficient between each preset topological constraint and each polymerization parameter, calculate the adjustment coefficient of each polymerization parameter of the target sitagliptin phosphate transaminase.

[0038] Specifically, when calculating the adjustment coefficient D of a certain polymerization parameter of the target sitagliptin phosphate transaminase, the formula is: D=b1×A+b2×B+b3×C; where b1, b2 and b3 represent the preset weighting coefficients of the polymerization parameter with structural stability, steric hindrance and compatibility, respectively, and A, B and C represent the matching values ​​of the target sitagliptin phosphate transaminase with structural stability, steric hindrance and compatibility, respectively.

[0039] S224, adjust the initial value of each polymerization parameter of the target sitagliptin phosphate transaminase enzyme solution according to the adjustment coefficient of each polymerization parameter to obtain the actual value of each polymerization parameter. The actual values ​​of all polymerization parameters constitute the polymerization conditions of the target sitagliptin phosphate transaminase enzyme solution.

[0040] Specifically, the initial value of each aggregation parameter is multiplied by its adjustment factor to obtain the actual value of each aggregation parameter.

[0041] S23 controls the polymerization conditions of each enzyme solution in the micro-immobilized enzyme screening reactor and monitors the polymerization status parameter data of each channel in real time.

[0042] Specifically, polymerization state parameters include temperature, pH value, and electrical conductivity.

[0043] S24, adjust the polymerization conditions of each channel in real time according to the polymerization state parameter data of each channel until each channel completes polymerization and obtains the final polymerization product of each enzyme solution.

[0044] Specifically, in this embodiment of the invention, the completion of aggregation for each channel refers to the aggregation time in the aggregation completion conditions.

[0045] In one specific embodiment, S24 includes: S241, construct the aggregated state vector of the target channel based on the aggregated state parameter data of the target channel at the current moment, wherein the target channel is any channel.

[0046] Specifically, the polymerization state parameters of the target channel at the current moment are: temperature 30℃, pH value 5.1 and conductivity 5, so the polymerization state vector of the target channel at the current moment is [30, 5.1, 5].

[0047] S242, construct a control vector based on all aggregation parameters in the aggregation conditions of the target channel.

[0048] Specifically, the polymerization parameters include enzyme concentration, temperature, pH value, and polymerization time, and the corresponding control parameters in the control vector are temperature control value, pH control value, enzyme concentration control value, and polymerization time control value.

[0049] S243, obtain the aggregation change characteristics of the target channel at the current moment, and construct the state transition equation between the control vector and the aggregation state vector based on the aggregation change characteristics.

[0050] In one specific embodiment, S243 includes: S2431, obtain the polymerization product of the target channel at the current time, determine the peak area of ​​the polymerization product at the current time by CE method, and calculate the polymerization product generation rate of the target channel at the current time based on the peak area of ​​the polymerization product at the current time. Use the polymerization product generation rate of the target channel at the current time as the polymerization change characteristic of the target channel at the current time.

[0051] Specifically, in this embodiment of the invention, the time interval between any two adjacent moments is 1 second. The liquid containing the polymer product of the target channel at the current moment is input into the CE measuring instrument, allowing direct acquisition of the peak area of ​​the polymer product at the current moment. Similarly, the peak area of ​​the polymer product of the target channel at the previous moment is obtained. This peak area is then input into a preset concentration-peak area standard curve to obtain the concentration of the target channel at the previous moment. Similarly, the concentration of the target channel at the current moment is obtained. The difference between the concentration of the target channel at the previous moment and the concentration at the current moment is calculated as the concentration difference. Dividing the concentration difference by the time interval between any two adjacent moments yields the polymer product generation rate of the target channel at the current moment.

[0052] S2432, obtain the initial product concentration equation of sitagliptin phosphate transaminase, correct the preset state transition matrix in the initial product concentration equation by the polymerization change characteristics, and obtain the corrected state transition matrix. Based on the corrected state transition matrix, the control vector and the polymerization state vector, construct the state transition equation between the control vector and the polymerization state vector.

[0053] Specifically, the initial product concentration equation for sitagliptin phosphate transaminase was obtained by training on a large amount of historical product data from various sitagliptin phosphate transaminases. The initial product concentration equation represents the functional relationship between the polymerization product concentration and the polymerization state vector. The training objective of the initial product concentration equation for sitagliptin phosphate transaminase is to obtain a preset state transition matrix.

[0054] When correcting the preset state transition matrix in the initial product concentration equation by means of polymerization change characteristics, the polymerization state vector of the target channel at the current time is first input into the initial product concentration equation to obtain the predicted product concentration at the current time. Similarly, the predicted product concentration at the previous time is obtained. The predicted product generation rate at the current time is calculated based on the predicted product concentration at the current time and the predicted product concentration at the previous time. The absolute value of the difference between the predicted product generation rate at the current time and the polymerization product generation rate is calculated as the prediction error. The preset state transition matrix is ​​adjusted by an adaptive filtering algorithm and the prediction error to obtain the corrected state transition matrix.

[0055] The modified state transition matrix represents how, under ideal conditions—that is, without external control—the target channel naturally aggregates according to the aggregated state vector to obtain the aggregated product. Therefore, after applying an external control vector, the state transition equation of the target channel can be expressed as: X(k+1)=Z×x(k)+U(k); where x(k) represents the aggregated state vector of the target channel at the current moment, X(k+1) represents the future aggregated state vector of the target channel at the next moment after the application of the control vector, Z represents the modified state transition matrix, and U(k) represents the control vector at the current moment.

[0056] S244: Predict the future aggregated state vector of the target channel at the next moment based on the pre-trained Kalman filter, and input the future aggregated state vector into the state transition equation to obtain the execution control vector at the current moment.

[0057] Specifically, the Kalman filter is trained using a large number of historical aggregation state vectors. The pre-trained Kalman filter represents the change function of the aggregation state vectors between two adjacent time steps. The aggregation state vector at the current time step is input into the Kalman filter to obtain the future aggregation state vector at the next time step. The future aggregation state vector is then input into the state transition equation to obtain the execution control vector at the current time step. The execution control vector includes the specific values ​​of temperature control, pH control, enzyme concentration control, and polymerization time control.

[0058] S245, adjust the aggregation conditions of the target channel according to the execution control vector at the current moment to obtain new aggregation conditions, and control the target channel to execute the new aggregation conditions.

[0059] Specifically, assuming the temperature control value in the current execution control vector is 2, and the temperature in the target channel's polymerization conditions is 50℃, then the temperature in the new polymerization conditions for the target channel is 50 + 2 = 52℃. Assuming the pH control value in the current execution control vector is -0.2, and the pH value in the target channel's polymerization conditions is 5, then the pH value in the new polymerization conditions for the target channel is 5 - 0.2 = 4.8. Assuming the enzyme concentration control value in the current execution control vector is 10, and the enzyme concentration in the target channel's polymerization conditions is 30, then the enzyme concentration in the new polymerization conditions for the target channel is 10 + 30 = 40 (in percentage). Assuming the polymerization time control value in the current execution control vector is 1, and the polymerization time in the target channel's polymerization conditions is 3, then the enzyme concentration in the new polymerization conditions for the target channel is 1 + 3 = 4 hours.

[0060] S246, continue steps S241 to S245 until the target channel completes polymerization, and obtain the final polymerized product of the enzyme solution corresponding to the target channel.

[0061] S3, Sitagliptin phosphate precursor substrate solution is fed into a micro-immobilized enzyme screening reactor and subjected to enzyme catalysis reaction with the final polymerization product of each enzyme solution to obtain the product after catalysis in each channel, and the product conversion rate of each channel is calculated.

[0062] Specifically, sitagliptin phosphate precursor substrate solution is a liquid prepared from sitagliptin phosphate precursor substrate.

[0063] In one specific embodiment, S3 includes: S31, Sitagliptin phosphate precursor substrate solution is fed into a micro-immobilized enzyme screening reactor, and the initial pH, initial conductivity and initial temperature of the sitagliptin phosphate precursor substrate solution in each channel of the micro-immobilized enzyme screening reactor are measured.

[0064] Specifically, the initial pH value of the sitagliptin phosphate precursor substrate solution in each channel was measured using a pH sensor; the initial conductivity of the sitagliptin phosphate precursor substrate solution in each channel was measured using a conductivity sensor; and the initial temperature of the sitagliptin phosphate precursor substrate solution in each channel was measured using a thermistor.

[0065] S32 controls the flow of sitagliptin phosphate precursor substrate solution through each channel of the micro-immobilized enzyme screening reactor to carry out an enzyme catalytic reaction with the final polymerization product of each enzyme solution, thereby obtaining the catalytic product of each channel. The pH value, conductivity and temperature of the catalytic product of each channel are measured, and the pH conversion rate of each channel is calculated based on the initial pH value of each channel and the pH value of the catalytic product.

[0066] Specifically, pH conversion rate refers to the product conversion rate indirectly calculated using pH value. Since product conversion rate cannot be directly measured in actual enzyme-catalyzed reactions, this embodiment of the invention indirectly calculates the product conversion rate in enzyme-catalyzed reactions using pH value.

[0067] When calculating the pH conversion rate J of a channel based on its initial pH value and the pH value of the catalytic product, the formula is: ;in, This represents the pH conversion function (a function that converts pre-trained pH values ​​into conversion rates). Indicates the initial pH value. This indicates the pH value of the catalytic product.

[0068] S33, based on the initial conductivity, initial temperature, conductivity and temperature of each channel, the pH conversion rate of each channel is corrected to obtain the product conversion rate of each channel.

[0069] In one specific embodiment, S33 includes: S331, calculate the conductivity variation coefficient based on the initial conductivity and conductivity of each channel.

[0070] Specifically, the difference between the conductivity of each channel and the initial conductivity is calculated as the conductivity change value, and the ratio of the conductivity change value to the initial conductivity is calculated as the conductivity change coefficient of each channel.

[0071] S332, calculate the initial pH value and the difference between the pH values ​​for each channel to obtain the pH change value, and determine the conductivity correction factor for each channel based on the conductivity change coefficient and the pH change value.

[0072] Specifically, the formula for calculating the conductance correction factor k for a certain channel is as follows: ;in, This represents the coefficient of change in the conductivity of the channel. This indicates the pH change value of the channel.

[0073] S333, adjusts the initial pH value of each channel according to the initial temperature of each channel to obtain the corrected initial pH value, adjusts the pH value of each channel according to the temperature of each channel to obtain the corrected pH value, and calculates the corrected pH conversion factor of each channel based on the corrected initial pH value, the corrected pH value and the pH conversion rate.

[0074] Specifically, in calculating the corrected initial pH value for a certain channel. When, the formula is: ;in, This represents the initial temperature of the channel, and F represents the preset buffer system temperature coefficient. Similarly, the corrected pH value of the channel is calculated based on its temperature and pH value. The formula for calculating the corrected pH conversion factor g for this channel is: .

[0075] S334, the pH conversion rate of each channel is jointly adjusted according to the conductivity correction factor and pH conversion correction factor of each channel to obtain the product conversion rate of each channel.

[0076] Specifically, the combined factor is obtained by calculating the sum of the conductivity correction factor k and the pH conversion factor g of the channel, and then multiplying the combined factor by the pH conversion rate of the channel to obtain the product conversion rate of the channel.

[0077] S4. The transaminase with the highest product conversion rate was selected as the optimal transaminase, and the optimal transaminase was applied to the production of sitagliptin phosphate raw material.

[0078] Furthermore, to verify whether the optimal transaminase obtained through the above methods meets the standards, the method provided in this embodiment of the invention further includes: The optimal transaminase was controlled to remain at multiple preset simulated production temperatures for preset time intervals, and its relative residual activity at each preset simulated production temperature was measured. Thermostability curves were constructed based on the relative residual activity of the optimal transaminase at each preset simulated production temperature, and the half-life of the optimal transaminase was determined by the thermostability curves. If the half-life of the optimal transaminase is less than the preset half-life threshold, the activity of the optimal transaminase is determined to be non-compliant, and a preselected sitagliptin phosphate transaminase is regenerated and a new optimal transaminase is screened.

[0079] Specifically, the preset simulated production temperatures in this embodiment of the invention are 10℃, 20℃, 30℃, 40℃, 50℃, and 60℃. The preset time interval in this embodiment of the invention is 24 hours. The relative residual activity of the optimal transaminase at a certain preset simulated production temperature is determined by NADH-coupled colorimetric method.

[0080] The half-life of the optimal transaminase refers to the time required for its relative residual activity to decrease to half, which is equivalent to the relative residual activity decreasing to half of its original value. In the formula calculation, this is represented by -ln(2). Curve fitting (using the least squares method in this embodiment) is performed on the relative residual activity of the optimal transaminase at each preset simulated production temperature to obtain the thermal stability curve. The relative residual activity corresponding to the standard temperature in the thermal stability curve is obtained, and the standard temperature is calculated based on the relative residual activity DF corresponding to the standard temperature. The deactivation rate constant under the following conditions The formula is: Then, the optimal half-life of the transaminase is obtained by dividing -ln(2) by the inactivation rate constant.

[0081] The preset half-life threshold is a pre-set time threshold for half-life. Assuming the half-life of the optimal transaminase is 0.1 hours and the preset half-life threshold is 0.15 hours, it means that the half-life of the optimal transaminase is less than the preset half-life threshold, and the activity of the optimal transaminase is determined to be non-compliant.

[0082] When regenerating the preselected sitagliptin phosphate transaminase and screening for new optimal transaminases, the preselected sitagliptin phosphate transaminase is reselected, and this is achieved through S1 to S4 as described above.

[0083] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for selecting transaminases for the production of sitagliptin phosphate, characterized in that, include: S1, introduce non-natural amino acids into the specified position of each preselected sitagliptin phosphate transaminase to complete the site-specific protein modification of each preselected sitagliptin phosphate transaminase, obtain each sitagliptin phosphate transaminase, and calculate the topological constraint matching conditions of each sitagliptin phosphate transaminase. S2, generate the enzyme solution for each sitagliptin phosphate transaminase, inject each enzyme solution into different channels of the micro-immobilized enzyme screening reactor, adjust the initial polymerization conditions of the corresponding enzyme solution according to the topological constraint matching conditions of each sitagliptin phosphate transaminase to obtain the polymerization conditions, polymerize each enzyme solution based on the polymerization conditions to obtain the final polymer product of each enzyme solution. S3, Sitagliptin phosphate precursor substrate solution is fed into a micro-immobilized enzyme screening reactor and subjected to enzyme catalysis reaction with the final polymerization product of each enzyme solution to obtain the product after catalysis in each channel, and the product conversion rate of each channel is calculated. S4. The transaminase with the highest product conversion rate was selected as the optimal transaminase, and the optimal transaminase was applied to the production of sitagliptin phosphate raw material.

2. The method for selecting transaminases for producing sitagliptin phosphate according to claim 1, characterized in that, S1 includes: S11, obtain the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase, and extract the modification sites in the high-resolution three-dimensional structure of each preselected sitagliptin phosphate transaminase as the designated locations of each preselected sitagliptin phosphate transaminase. S12, obtain the spatial coordinates of the specified location of each preselected sitagliptin phosphate transaminase in a high-resolution three-dimensional structure; S13, introduce non-natural amino acids into the specified positions of each preselected sitagliptin phosphate transaminase according to the spatial coordinates of the high-resolution three-dimensional structure at the specified positions, and complete the site-specific protein modification of each preselected sitagliptin phosphate transaminase to obtain each sitagliptin phosphate transaminase. S14, calculate the topological distance between the specified site of each preselected sitagliptin phosphate transaminase and its substrate binding site, and calculate the local density coefficient of the specified site of each preselected sitagliptin phosphate transaminase. The topological constraint matching condition of each preselected sitagliptin phosphate transaminase is constituted by the topological distance between the specified site of each preselected sitagliptin phosphate transaminase and its substrate binding site and its local density coefficient.

3. The method for selecting transaminases for producing sitagliptin phosphate according to claim 1, characterized in that, S2 includes: S21, generate the enzyme solution for each sitagliptin phosphate transaminase, and inject each enzyme solution into a different channel of the micro-immobilized enzyme screening reactor. S22, obtain the initial polymerization conditions of the enzyme solution of each sitagliptin phosphate transaminase, construct an initial polymerization vector with each polymerization parameter in the initial polymerization conditions of the enzyme solution of each sitagliptin phosphate transaminase, generate the adjustment coefficient of each polymerization parameter of each sitagliptin phosphate transaminase according to the topological constraint matching conditions and the initial polymerization vector, and adjust the initial polymerization conditions of the corresponding enzyme solution according to the adjustment coefficient of each polymerization parameter of each sitagliptin phosphate transaminase to obtain the polymerization conditions; S23 controls the polymerization conditions of each enzyme solution in the micro-immobilized enzyme screening reactor and monitors the polymerization status parameter data of each channel in real time. S24, adjust the polymerization conditions of each channel in real time according to the polymerization state parameter data of each channel until each channel completes polymerization and obtains the final polymerization product of each enzyme solution.

4. The method for selecting transaminases for producing sitagliptin phosphate according to claim 3, characterized in that, S22 includes: S221, obtain the initial polymerization conditions of the enzyme solution of the target sitagliptin phosphate transaminase according to the preset standard polymerization parameter group, and construct an initial polymerization vector with the initial value of each polymerization parameter in the initial polymerization conditions of the target sitagliptin phosphate transaminase, wherein the target sitagliptin phosphate transaminase is any sitagliptin phosphate transaminase. S222, Calculate the matching value between the target sitagliptin phosphate transaminase and each preset topological constraint based on the topological constraint matching conditions of the target sitagliptin phosphate transaminase; S223, Based on the matching value between the target sitagliptin phosphate transaminase and each preset topological constraint and the preset weighting coefficient between each preset topological constraint and each polymerization parameter, calculate the adjustment coefficient of each polymerization parameter of the target sitagliptin phosphate transaminase; S224, adjust the initial value of each polymerization parameter of the target sitagliptin phosphate transaminase enzyme solution according to the adjustment coefficient of each polymerization parameter to obtain the actual value of each polymerization parameter. The actual values ​​of all polymerization parameters constitute the polymerization conditions of the target sitagliptin phosphate transaminase enzyme solution.

5. The method for selecting transaminases for producing sitagliptin phosphate according to claim 3, characterized in that, S24 includes: S241, construct the aggregated state vector of the target channel based on the aggregated state parameter data of the target channel at the current moment, wherein the target channel is any channel; S242, construct a control vector based on all aggregation parameters in the aggregation conditions of the target channel; S243, obtain the aggregation change characteristics of the target channel at the current moment, and construct the state transition equation between the control vector and the aggregation state vector based on the aggregation change characteristics; S244: Predict the future aggregation state vector of the target channel at the next moment based on the pre-trained Kalman filter, and input the future aggregation state vector into the state transition equation to obtain the execution control vector at the current moment. S245, adjust the aggregation conditions of the target channel according to the execution control vector at the current moment to obtain new aggregation conditions, and control the target channel to execute the new aggregation conditions; S246, continue steps S241 to S245 until the target channel completes polymerization, and obtain the final polymerized product of the enzyme solution corresponding to the target channel.

6. The method for selecting transaminases for producing sitagliptin phosphate according to claim 5, characterized in that, S243 includes: S2431, obtain the polymerization product of the target channel at the current time, determine the peak area of ​​the polymerization product at the current time by CE method, and calculate the polymerization product generation rate of the target channel at the current time based on the peak area of ​​the polymerization product at the current time, and use the polymerization product generation rate of the target channel at the current time as the polymerization change characteristic of the target channel at the current time. S2432, obtain the initial product concentration equation of sitagliptin phosphate transaminase, correct the preset state transition matrix in the initial product concentration equation by the polymerization change characteristics, and obtain the corrected state transition matrix. Based on the corrected state transition matrix, the control vector and the polymerization state vector, construct the state transition equation between the control vector and the polymerization state vector.

7. The method for selecting transaminases for producing sitagliptin phosphate according to claim 1, characterized in that, S3 includes: S31, Sitagliptin phosphate precursor substrate solution is fed into a micro-immobilized enzyme screening reactor, and the initial pH, initial conductivity and initial temperature of the sitagliptin phosphate precursor substrate solution in each channel of the micro-immobilized enzyme screening reactor are measured. S32, after the sitagliptin phosphate precursor substrate solution flows through each channel of the micro-immobilized enzyme screening reactor, it undergoes an enzyme-catalyzed reaction with the final polymerization product of each enzyme solution to obtain the catalyzed product of each channel. The pH value, conductivity and temperature of the catalyzed product of each channel are measured. The pH conversion rate of each channel is calculated based on the initial pH value of each channel and the pH value of the catalyzed product. S33, based on the initial conductivity, initial temperature, conductivity and temperature of each channel, the pH conversion rate of each channel is corrected to obtain the product conversion rate of each channel.

8. The method for selecting transaminases for producing sitagliptin phosphate according to claim 7, characterized in that, S33 includes: S331, calculate the conductivity variation coefficient based on the initial conductivity and conductivity of each channel; S332, calculate the initial pH value and the difference between the pH values ​​for each channel to obtain the pH change value, and determine the conductivity correction factor for each channel based on the conductivity change coefficient and the pH change value; S333, adjust the initial pH value of each channel according to the initial temperature of each channel to obtain the corrected initial pH value, adjust the pH value of each channel according to the temperature of each channel to obtain the corrected pH value, and calculate the corrected pH conversion factor of each channel according to the corrected initial pH value, the corrected pH value and the pH conversion rate. S334, the pH conversion rate of each channel is jointly adjusted according to the conductivity correction factor and pH conversion correction factor of each channel to obtain the product conversion rate of each channel.

9. The method for selecting transaminases for producing sitagliptin phosphate according to claim 1, characterized in that, The method further includes: The optimal transaminase was controlled to remain at multiple preset simulated production temperatures for preset time intervals, and its relative residual activity at each preset simulated production temperature was measured. Thermostability curves were constructed based on the relative residual activity of the optimal transaminase at each preset simulated production temperature, and the half-life of the optimal transaminase was determined by the thermostability curves. If the half-life of the optimal transaminase is less than the preset half-life threshold, the activity of the optimal transaminase is determined to be non-compliant, and a preselected sitagliptin phosphate transaminase is regenerated and a new optimal transaminase is screened.

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