Constant-temperature auxiliary measuring device for screening tannase crystals

Abstract

The invention relates to a constant-temperature auxiliary measuring device for screening tannase crystals, and belongs to the technical field of analysis and measurement equipment. Comprising a crystallization instrument, and the crystallization instrument comprises a crystallization vessel and a control center; the auxiliary box is rotationally arranged outside the crystallizing dish; the second measuring device is installed on the auxiliary box, the second measuring device is in communication connection with the control center, the second measuring device periodically measures the sampling viscosity value of the enzyme liquid in the crystallizing dish, and when the viscosity value reaches a preset value, it is judged that the enzyme liquid is fully crystallized. The method is used for solving the technical problem that when whether crystallization is sufficient or not cannot be judged and determined in the prior art, crystallized crystals cannot be sufficiently separated.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical measurement equipment, and particularly relates to a determination device for thermostatic-assisted crystallization screening of tannase. Background Art

[0002] During the process of screening and measuring samples in the laboratory, it is necessary to measure through the physical and chemical properties of materials. Tannase is a protein hydrolase that can decompose tannic acid, which is widely used in the food and pharmaceutical industries, especially in the treatment of tea and wine. Crystallization of tannase is required in protein purification and structure research, so the crystallization of tannase is of great significance for analyzing and determining its structure and function. The crystallization of tannase is different from that of general hydrolases and proteins, and polyphenolic interferents in the crystallization environment need to be removed to improve the crystallization purity.

[0003] Publication No.: CN117907163B discloses a determination device for lanthanum chloride cooling crystallization screening. It includes a determination box, a cooling component is installed at the top of the determination box, a crystallization component is movably clamped at the top of the cooling component, a recovery component is installed on one inner wall of the determination box, and a rotating component is installed on the bottom inner wall; in the present invention, by starting the fourth electric push rod to drive the sieve tube to shake up and down, and then starting the fourth motor to drive the sieve tube to rotate rapidly, during its rotation, the lanthanum chloride crystals collide to separate the possibly adhered lanthanum chloride crystals, starting the second vibration motor to drive the filter screen to vibrate, driving the fine particles and powder crystals to fall onto the bottom inner wall of the screening box, and then measuring the regularly screened crystals and particle powder crystals in sequence.

[0004] During the use of the prior art, there are at least the following problems:

[0005] Because there are amorphous precipitates during the crystallization of proteins, they cannot be screened through a sieve. Therefore, when the prior art cannot judge and measure whether the crystallization is sufficient, the crystals of the crystallization cannot be fully separated. Summary of the Invention

[0006] The present invention provides a determination device for thermostatic-assisted crystallization screening of tannase, which is used to solve the technical problem that when the prior art cannot judge and measure whether the crystallization is sufficient, the crystals of the crystallization cannot be fully separated.

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] A determination device for the crystallization screening of thermostatic-assisted tannase, comprising: a crystallizer, the crystallizer including a crystallization dish and a control center; an auxiliary box rotatably arranged outside the crystallization dish; a second determination device installed on the auxiliary box, the second determination device being in communication connection with the control center, the second determination device periodically measuring the sampling viscosity value of the enzyme solution in the crystallization dish, and when the viscosity value reaches a preset value, it is determined that sufficient crystallization has occurred.

[0009] Furthermore, it further includes a first determination device, which is used to measure the parameters of the components in the crystallization dish before crystallization to obtain a first set of measurement parameters, and measure the parameters after crystallization to obtain a second set of measurement parameters.

[0010] Furthermore, the auxiliary box includes: a first temperature component arranged around the crystallization dish, the first temperature component being used to control the crystallization environment temperature, and the first temperature component being in communication connection with the control center; a second temperature component for releasing cold air into the crystallization dish, and the second temperature component being in communication connection with the control center.

[0011] Furthermore, it further includes: a detection box detachably arranged on the crystallization dish, having a detection space; an input port opened on the detection box for communicating the detection space and the crystallization dish; an output port opened on the detection box; a waste liquid tank communicated with the output port for collecting detection waste liquid; a micro pump arranged on the input port and the output port for controlling the flow between the detection space and the crystallization dish, and the micro pump is also arranged on the first determination device and the second determination device for respectively controlling the sampling of the enzyme solution before crystallization, and the micro pump is in communication connection with the control center.

[0012] Furthermore, the second determination device includes: a viscosity measurement component arranged in the crystallization dish for measuring the viscosity of the enzyme solution in the detection space.

[0013] Furthermore, the second determination device further includes: a temperature sensor arranged in the detection box and the crystallization dish for detecting the ambient temperature.

[0014] Furthermore, it further includes: a cleaning component communicated with the detection box and the crystallization dish for respectively cleaning the detection box and the crystallization dish; an electric push rod arranged on the auxiliary box, one end of the electric push rod is fixed on the auxiliary box, and the other end is arranged on the limit ring for driving the displacement of the limit ring.

[0015] Furthermore, it further includes: a limit ring slidably arranged on the auxiliary box for compressing the space inside the auxiliary box and driving the gas to gather for assisting crystallization.

[0016] The present invention provides a measuring device for the crystallization screening of thermostatic-assisted tannase, and the beneficial effects are as follows:

[0017] Through two measurements by the first measuring device, high-performance liquid chromatography combined with an ultraviolet detector is used to accurately measure the initial concentration, pH value, and activity unit, so as to record the parameters before and after crystallization, facilitate data comparison, optimize the crystallization conditions, provide a reliable basis for variable control, and reduce the risk of mother liquor residue and recrystallization;

[0018] Through the second measuring device's periodic multi-point sampling and temperature compensation algorithm, the effect of dynamically monitoring the crystallization stage is achieved, accurately judging the crystallization end point, and preventing structural rupture or functional damage caused by over-crystallization;

[0019] Through the auxiliary box cooperating with the limiting ring to accelerate the crystallization process and improve the crystal uniformity, further avoiding the activity loss caused by metastable crystal defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0021] Figure 1 It is a schematic structural diagram of a measuring device for the crystallization screening of thermostatic-assisted tannase provided by an embodiment of the present invention;

[0022] Figure 2 It is a top view of a measuring device for the crystallization screening of thermostatic-assisted tannase provided by an embodiment of the present invention;

[0023] Figure 3 It is a schematic structural diagram of opening the auxiliary box of a measuring device for the crystallization screening of thermostatic-assisted tannase provided by an embodiment of the present invention;

[0024] Figure 4 For Figure 3 The enlarged view at A1 in

[0025] Figure 5 It is a schematic diagram of the periodic determination logic flow of a measuring device for the crystallization screening of thermostatic-assisted tannase provided by an embodiment of the present invention.

[0026] In the figure: 101 - the first measuring device; 102 - the crystallization dish; 103 - the control center; 201 - the auxiliary box; 202 - the limiting ring; 203 - the viscosity measuring piece; 1011 - the detection box; 1012 - the micro pump; 1013 - the cleaning component. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0028] In the description of the present application, it should be understood that the orientation or positional relationships indicated by the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. are based on the orientation or positional relationships shown in the accompanying drawings. These are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present application.

[0029] The terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0030] In the description of the present application, it should be noted that unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense. For example, it can be welding, bolt connection, or riveting; it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood according to specific circumstances.

[0031] Embodiment:

[0032] As Figures 1 to 4 shown, the present invention provides a measuring device for constant-temperature assisted crystallization screening of tannase, including: a first measuring device 101 for performing a first measurement on reaction components; a crystallizer, which is connected to the first measuring device 101 and includes a crystallization dish 102 and a control center 103; an auxiliary box 201, which is arranged outside the crystallization dish 102, and a temperature adjustment part is arranged on the auxiliary box 201 for controlling the temperature of the crystallization dish 102, and the first measuring device 101 is used for performing a second measurement on the components in the crystallization dish 102; a limiting ring 202, which is slidably arranged on the auxiliary box 201 for compressing the space inside the auxiliary box 201 and driving the gas to gather for assisted crystallization; a second measuring device, which is installed on the auxiliary box 201 and periodically measures the crystallization degree.

[0033] One side of the auxiliary box 201 is rotatably hinged to the crystallizer and is provided with a sealing structure. The first measuring device 101 is used to perform the first sampling measurement on the enzyme solution for the crystallization measurement experiment, so as to record the relevant data before the experiment, improve the recording and control of the crystallization conditions of the enzyme solution. The auxiliary box 201 is used to control the temperature of the crystallization environment at a constant temperature. Then, the limiting ring 202 is used to gather the local air pressure and cold air in the crystallization space, improve the crystallization speed and the uniformity of crystallization. Then, the second measuring device is used to measure the parameters related to the crystallization degree, specifically viscosity, and the viscosity is measured periodically, so as to obtain the crystallization stage and crystallization state reflected by different viscosities in the corresponding temperature environment, so as to actively judge whether the crystallization is sufficient. Insufficient crystallization will lead to incomplete screening, residual uncrystallized protein in the mother liquor, affecting subsequent function verification, and metastable crystals are prone to recrystallization, resulting in loss of specific activity; excessive crystallization will lead to crystal structure defects, specifically the appearance of twins or polycrystalline agglomerates, pseudo-peaks in the diffraction pattern, internal stress accumulation leading to lattice distortion, functional activity damage, and crystal rupture. In the first measuring device 101, high performance liquid chromatography combined with an ultraviolet detector is used to perform measurements before and after crystallization, specifically for detecting the concentration of tannic acid and the proportion of impurity proteins.

[0034] Furthermore, this embodiment also includes some other implementation contents. The temperature adjustment part includes: a first temperature element, arranged around the crystallization dish 102, and the first temperature element is used to control the ambient temperature and is communicatively connected to the control center 103; a second temperature element, used to add cold air into the crystallization dish 102, and the second temperature element is communicatively connected to the control center 103.

[0035] The first temperature element is specifically a heating component with constant temperature control, used to control the temperature of the solution to be crystallized. The specific experiment can be salting-out crystallization method. By controlling the temperature of the solution at a constant temperature, the stability of the crystallization environment is ensured. Then, through the cold air generating device of the second temperature element and the displacement of the limiting ring, the cold air is gathered, so as to realize the controllable decrease of the temperature in the crystallization area, and thus realize the crystallization process and the uniformity of crystallization.

[0036] Furthermore, as Figures 1 to 4As shown in the figure, this embodiment also includes some other implementation contents, including: a detection box 1011, detachably arranged on the crystallization dish 102, having a detection space; an input port, opened on the detection box 1011, for communicating the detection space and the crystallization dish 102; an output port, opened on the detection box 1011; a waste liquid tank, communicated with the output port, for collecting detection waste liquid; a micropump 1012, arranged on the input port and the output port, for controlling the flow between the detection space and the crystallization dish 102. The micropump 1012 is also arranged on the first measurement device 101 and the second measurement device, for respectively controlling the sampling of the enzyme solution before crystallization. The micropump 1012 is communicatively connected to the control center 103; a viscosity measurement piece 203, arranged in the crystallization dish 102, for measuring the viscosity of the enzyme solution in the detection space.

[0037] By setting the detection box 1011 to perform independent sampling operations for the first measurement and the second measurement, and using a small peristaltic pump to sample the solutions before and after crystallization and uniformly circulate them among the crystallization dish 102, the detection box 1011, and the waste liquid tank through the micropump 1012. The corresponding viscosity measurement piece 203 can specifically be a vibrating viscometer, arranged in the crystallization dish 102, and multiple detection points are set to perform multi-point sampling of the viscosity to control the influence brought by data fluctuations.

[0038] Furthermore, this embodiment also includes some other implementation contents. The first measurement of the first measurement device 101 includes the concentration measurement and environmental measurement of the enzyme solution and the measurement of the enzyme activity unit value, and records the initial concentration P1, pH value A1, and initial activity unit value B1.

[0039] The second measurement of the first measurement device 101 includes recording the concentration value P2 and activity unit value B2 of the enzyme solution after crystallization.

[0040] By detecting and recording the initial concentration P1, pH value A1, and initial activity unit value B1, and recording the concentration value P2 and activity unit value B2 of the enzyme solution after crystallization, it is used to establish a before-and-after comparison and control the crystallization variables for auxiliary observation.

[0041] Furthermore, this embodiment also includes some other implementation contents, including: a cleaning component 1013, communicated with the detection box 1011 and the crystallization dish 102, for respectively cleaning the detection box 1011 and the crystallization dish 102; a temperature sensor, arranged in the detection box 1011 and the crystallization dish 102, for detecting the environmental temperature; an electric push rod, arranged on the auxiliary box 201, with one end fixed on the auxiliary box 201 and the other end arranged on the limit ring 202, for driving the displacement of the limit ring 202.

[0042] The cleaning component 1013 is provided with a flushing device and a drying device. The pipeline is flushed with deionized water, disinfected by injecting 75% ethanol, and finally purged with dry air to complete the environmental control of the test kit 1011. By arranging temperature sensors, specifically using infrared sensors to collect temperature, contamination of the crystal is prevented. Temperature sensing is also used for thermal feedback of the constant temperature system and for temperature compensation in cooperation with the viscosity measuring member 203 to obtain the crystallization degree represented by the viscosity value at the corresponding temperature by comparison; the electric push rod is specifically a micro push rod provided to precisely control the displacement of the limit ring 202, thereby controlling the aggregation speed of cold air to indirectly control the change rate of the temperature gradient, so as to assist the crystallization dish 102 to accelerate the crystallization process and crystallization uniformity, and specifically cooperate with the viscosity measuring members 203 arranged in a matrix for accurate measurement.

[0043] Further, as Figure 5 shown, this embodiment also includes some other implementation contents. The viscosity measuring member 203 periodically samples and measures the enzyme solution in the crystallization dish 102, and the viscosity value is recorded as T. When the T value reaches the preset value, it is judged that crystallization is sufficient.

[0044] Specifically, the sampled and measured viscosity value T is specifically the average value obtained by the control center 103 after the measurement by each specific T value of Tn (T1, T2, T3......) of the array viscosity measuring member 203. When abnormal values and some numerical fluctuations are too large, re-measurement is performed and the abnormal points are recorded. Multiple detection frequency periods are set. The frequency period corresponding to the nucleation period is N1, the frequency period corresponding to the growth period is N2, and the frequency period corresponding to the stable period is N3. The measurement frequency period in this application is adaptively selected according to the specific measurement object and is not publicly disclosed herein. The detection parameters are sent to the control center 103 for analysis of the detection parameters and for judging whether the preset viscosity value T is reached. The preset viscosity values include the viscosity value Ta representing the termination of crystallization, the viscosity value Tb in the nucleation period, the viscosity value Tc in the growth period, and the viscosity value Td in the stable period, so as to judge which detection period corresponding to the viscosity value to continue the measurement or terminate crystallization for the next operation.

[0045] In summary, by setting up the first measuring device 101 for double-stage sampling measurement and analysis before and after crystallization, using high-performance liquid chromatography combined with an ultraviolet detector to accurately record the initial concentration, pH value, and activity unit, and comparing with the data after crystallization, the crystallization conditions are optimized to provide a reliable basis for variable control, reducing the risk of mother liquor residue and recrystallization; through the synergistic effect of constant temperature control and cold air aggregation, the crystallization process is accelerated and the crystal uniformity is improved, avoiding activity loss caused by metastable crystal defects; through the periodic multi-point sampling of the viscosity measuring part 203 and the temperature compensation algorithm, the effect of dynamically monitoring the crystallization stage is achieved, accurately judging the crystallization end point, and preventing structural rupture or functional damage caused by overcrystallization; through the independent detachable design of the detection box 1011 and the closed-loop flow control of the micro pump 1012, combined with the cleaning component 1013, the effect of reducing cross-contamination and ensuring the cleanliness of the detection environment is achieved, while improving the detection efficiency and operation convenience; by finely adjusting the displacement of the limiting ring 202 with the electric push rod, the effect of accurately controlling the cold air aggregation rate and temperature gradient is achieved, assisting in realizing the directional regulation of the crystallization rate and crystal size, and adapting to different experimental requirements; through the outlier retest mechanism and multi-cycle determination logic, the fluctuation of the measured data is dynamically corrected to ensure the accuracy of the crystallization state evaluation.

[0046] The above is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope recorded in the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A constant temperature assisted tannase crystal screening measuring device, characterized in that: include: A crystallizer, the crystallizer comprising a crystallization dish (102) and a control center (103); An auxiliary box (201) rotatably disposed outside the crystallization dish (102); The second measuring device is installed on the auxiliary box (201). The second measuring device is connected to the control center (103) for communication. The second measuring device periodically samples the enzyme solution in the crystallization dish (102) to measure the viscosity value. When the viscosity value reaches a preset value, it is determined to be fully crystallized.

2. A constant temperature assisted tannase crystal screening measuring device according to claim 1, characterized in that: Also includes A first measuring device (101), the first measuring device (101) is used to measure parameters of components in the crystallization dish (102) before crystallization to obtain a first set of measurement parameters, and to measure parameters after crystallization to obtain a second set of measurement parameters.

3. A constant temperature assisted tannase crystal screening measuring device according to claim 2, characterized in that: The auxiliary box (201) comprises: A first temperature element, arranged around the crystallization dish (102), the first temperature element being used to control the crystallization environment temperature, the first temperature element being communicatively connected to the control center (103); The second temperature element is used to release cold air into the crystallization dish (102), and the second temperature element is communicatively connected to the control center (103).

4. A constant temperature assisted tannase crystal screening measuring device according to claim 3, characterized in that: Also includes: A detection box (1011) is detachably arranged on the crystallization dish (102) and has a detection space; An input port, provided on the detection box (1011), and used for connecting the detection space and the crystallization dish (102); An output port, provided on the detection box (1011); A waste liquid tank, connected to the output port, for collecting detection waste liquid; A micro pump (1012) is arranged on the input port and the output port, and is used to control the flow between the detection space and the crystallization dish (102). The micro pump (1012) is also arranged on the first measuring device (101) and the second measuring device, and is used to control the sampling of the enzyme solution before crystallization. The micro pump (1012) is communicatively connected to the control center (103).

5. A constant temperature assisted tannase crystal screening measuring device according to claim 4, characterized in that: The second measuring device comprises: The viscosity measuring element (203) is arranged in the crystallization dish (102) and is used to measure the viscosity of the enzyme solution in the detection space.

6. A constant temperature assisted tannase crystal screening measuring device according to claim 5, characterized in that: The second measuring device also includes: A temperature sensor is arranged in the detection box (1011) and the crystallization dish (102) and is used to detect the ambient temperature.

7. A constant temperature assisted tannase crystal screening measuring device according to claim 6, characterized in that: Also includes: A cleaning component (1013), which is in communication with the detection box (1011) and the crystallization dish (102), and is used to clean the detection box (1011) and the crystallization dish (102), respectively; An electric push rod is arranged on the auxiliary box (201), one end of the electric push rod is fixed on the auxiliary box (201), and the other end of the electric push rod is arranged on the limiting ring (202) for driving the limiting ring (202) to move.

8. A constant temperature assisted tannase crystal screening measuring device according to claim 7, characterized in that: Also includes: A limiting ring (202) is slidably disposed on the auxiliary box (201) and is used to compress the space in the auxiliary box (201) and drive gas to gather to assist crystallization.

Images