Method for immobilizing N-BOC-piperidone reductase

Abstract

The invention discloses an N-BOC-piperidone reductase immobilization method, which comprises the following steps: soaking epoxy resin in a PBS buffer solution to ensure that the epoxy resin is immersed, standing and soaking overnight, filtering to enable liquid to flow out, and taking out a soaked filter cake to obtain pretreated epoxy resin for later use; through precise proportioning of experimental materials, the temperature is precisely set to be 25 DEG C, the stirring rotating speed of 200 rpm is matched, the activity temperature interval of enzyme is adapted, the maximum catalytic activity is stimulated, enzyme structure damage caused by too high rotating speed is prevented, 24-hour stable reaction is maintained, meanwhile, the enzyme is immobilized on epoxy resin in a covalent immobilization mode, and the reaction time is shortened. The conversion rate of T28-108 under the conditions of 20 DEG C, pH 7 and 5ml of crude enzyme liquid is optimal through screening, the conversion rate and ee value of the finally prepared immobilized enzyme are stably kept at 99% when the immobilized enzyme is repeatedly used for 1-10 times, the whole process adopts an immobilization technology, the operation is simple, the condition is mild, the enzyme activity is obviously improved, the whole stability is good, and the production is easy.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme immobilization, and particularly to a method for immobilizing N-BOC-piperidone reductase. Background Art

[0002] N-BOC-piperidone reductase is a biological enzyme with specific catalytic functions. It can act on N-BOC-piperidone molecules, promote reduction reactions, precisely modify and transform substrates with specific structures, namely N-BOC-piperidone, into target products, and help construct complex and bioactive molecular structures.

[0003] For example, the publication number CN104059952 discloses a whole-cell immobilization method. However, the immobilized enzyme obtained by this method has poor mechanical properties, is fragile, and is difficult to reuse. At the same time, the fragments cause filtration difficulties and reduce product quality, making it difficult to apply industrially. For another example, the publication number CN114410619 discloses an immobilized enzyme method, which mainly involves carrier preparation and protein purification, undoubtedly increasing the overall cost and being unfavorable for production.

[0004] However, the above enzyme immobilization methods are generally cumbersome, resulting in an increase rather than a decrease in production costs. At the same time, they are easily interfered by various physical or chemical factors, causing the spatial structure of the enzyme to be damaged, and ultimately the obtained immobilized enzyme has low activity. Summary of the Invention

[0005] The purpose of this part is to outline some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Simplifications or omissions may be made in this part, as well as in the abstract and title of the present application, to avoid obscuring the purpose of this part, the abstract, and the title. However, such simplifications or omissions shall not be used to limit the scope of the present invention.

[0006] In view of the problems existing in the above-mentioned existing method for immobilizing N-BOC-piperidone reductase, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to provide a method for immobilizing N-BOC-piperidone reductase, which solves the problem of "the above enzyme immobilization methods are generally cumbersome, resulting in an increase rather than a decrease in production costs. At the same time, they are easily interfered by various physical or chemical factors, causing the spatial structure of the enzyme to be damaged, and ultimately the obtained immobilized enzyme has low activity".

[0008] To solve the above technical problems, the present invention provides the following technical solutions: including the following steps:

[0009] Step 1: Immerse the epoxy resin in PBS buffer to ensure complete immersion, then let it stand overnight and filter to allow the liquid to flow out, thus obtaining the soaked filter cake and getting the pretreated epoxy resin for standby.

[0010] Step 2: Suspend the cultured bacteria in the buffer, break the bacteria to release the substances inside the cells, and then through centrifugation, make the denser cell debris precipitate, take out the supernatant to obtain the crude enzyme solution for standby.

[0011] Step 3: Then put the pretreated epoxy resin into the crude enzyme solution to allow interaction between the epoxy resin and the crude enzyme solution.

[0012] Step 4: Set the stirring temperature at 15 - 35°C and stir the interacting epoxy resin and crude enzyme solution for 24 hours.

[0013] Step 5: After stirring, perform suction filtration to remove some insoluble solid impurities to separate the liquid component from the solid component. Meanwhile, wash the filter cake with buffer and then take it out to obtain the immobilized enzyme.

[0014] Step 6: Add N - BOC - 3 - piperidone / 0.5 g, isopropanol / 1 g, PBS buffer / 4 ml, and NADP / 0.1 mg into the immobilized enzyme respectively, set the temperature at 30°C and react for 1 hour, and take samples at 5 hours to measure the conversion rate.

[0015] As a preferred scheme of the method for immobilizing N - BOC - piperidone reductase according to the present invention, wherein: the epoxy resin is 0.5 g, the PBS buffer is 5 ml, and the ratio of the epoxy resin to the PBS buffer is 0.5 g / 5 ml.

[0016] As a preferred scheme of the method for immobilizing N - BOC - piperidone reductase according to the present invention, wherein: the bacteria are 0.5 g, the PBS buffer is 5 ml, and the ratio of the bacteria to the PBS buffer is 0.5 g / 5 ml.

[0017] As a preferred scheme of the method for immobilizing N - BOC - piperidone reductase according to the present invention, wherein: the pretreated epoxy resin is 0.5 g, the crude enzyme solution is 5 ml, and the ratio of the pretreated epoxy resin to the crude enzyme solution is 0.5 g / 5 ml.

[0018] As a preferred scheme of the method for immobilizing N - BOC - piperidone reductase according to the present invention, wherein: the stirring temperature is specifically set at 25°C and the stirring speed is 200 rpm.

[0019] As a preferred embodiment of the method for immobilizing N-BOC-piperidone reductase according to the present invention, the resin model is T28-108, the conversion time is set to 1 hour, the immobilization temperature is set to 20 °C, the conversion rate at this time with a pH value of 7 is 99%, the amount of crude enzyme solution is 5 ml, and the number of uses is investigated. The number of uses is set to 1-10, and the conversion rate and ee value are both 99%.

[0020] The beneficial effects of the present invention: Through the precise ratio of experimental materials, the temperature is precisely set to 25 °C, combined with a stirring speed of 200 rpm, which not only adapts to the active temperature range of the enzyme, stimulates the maximum catalytic activity, but also prevents damage to the enzyme structure due to too high a rotation speed, maintaining a stable reaction for 24 hours. At the same time, through the covalent immobilization method, the enzyme is immobilized on the epoxy resin, and through screening, it is found that T28-108 has the best conversion rate under the conditions of 20 °C, pH 7, and 5 ml of crude enzyme solution. Finally, the prepared immobilized enzyme, when reused 1-10 times, the conversion rate and ee value are both stably maintained at 99%. The overall process adopts the immobilization technology, which is simple to operate, mild in conditions, significantly improves the enzyme activity, has good overall stability, and is easy to produce. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. 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. Among them:

[0022] Figure 1 It is a schematic diagram of the steps of a method for immobilizing N-BOC-piperidone reductase proposed by the present invention;

[0023] Figure 2 It is a schematic diagram of resin screening proposed by the present invention;

[0024] Figure 3 It is a schematic diagram of immobilization temperature screening proposed by the present invention;

[0025] Figure 4 It is a schematic diagram of immobilization pH screening proposed by the present invention;

[0026] Figure 5 It is a schematic diagram of immobilized enzyme amount screening proposed by the present invention;

[0027] Figure 6 It is a schematic diagram of the number of uses of the immobilized enzyme proposed by the present invention;

[0028] Figure 7 It is a schematic diagram of the peak conversion rate of T28-108 resin proposed by the present invention;

[0029] Figure 8 Schematic diagram of the peak ee value of T28-108 resin proposed by the present invention. Specific embodiments

[0030] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings of the specification.

[0031] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar generalizations without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0032] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure or characteristic that may be included in at least one implementation manner of the present invention. The appearances of "in one embodiment" in different places in this specification do not all refer to the same embodiment, nor are they separate or alternative embodiments that exclude each other from other embodiments.

[0033] Referring to Figures 1-6 , the present invention provides a method for immobilizing N-BOC-piperidone reductase, comprising the following steps:

[0034] Step 1: Soak 0.5 g of epoxy resin in 10 ml of PBS buffer to ensure that the epoxy resin is completely immersed, then let it stand overnight and filter to allow the liquid to flow out, thereby taking out the soaked filter cake to obtain pretreated epoxy resin for standby;

[0035] Step 2: Suspend 0.5 g of cultured bacteria in 5 ml of buffer, ultrasonically disrupt the bacteria to release the substances inside the cells, and then centrifuge at 1300 rpm to precipitate the denser cell debris, and take out the supernatant to obtain crude enzyme solution for standby;

[0036] Step 3: Then put the pretreated epoxy resin into the crude enzyme solution to allow interaction between the epoxy resin and the crude enzyme solution;

[0037] Step 4: Set the stirring temperature at 15-35 °C and stir the interacting epoxy resin and crude enzyme solution for 24 hours;

[0038] Step 5: After stirring, filter at room temperature to remove some insoluble solid impurities to separate the liquid component from the solid component. At the same time, wash the filter cake with buffer and then take it out to obtain immobilized enzyme;

[0039] Step 6: Add 0.5 g of N-BOC-3-piperidone, 1 g of isopropanol, 4 ml of PBS buffer, and 0.1 mg of NADP into the immobilized enzyme. Set the temperature at 30°C and react for 1 hour. Take samples at 5 hours to measure the conversion rate. See the attached drawings for details.

[0040] Among them, the epoxy resin is 0.5 g, the PBS buffer is 5 ml, and the ratio of epoxy resin to PBS buffer is 0.5 g / 5 ml. In the preparation stage of experimental materials, take 0.5 g of epoxy resin and pour it into 10 ml of PBS buffer to ensure that the epoxy resin can fully contact the buffer environment during the subsequent soaking pretreatment process, laying a foundation for the subsequent property optimization.

[0041] Furthermore, the bacterial cells are 0.5 g, the PBS buffer is 5 ml, and the ratio of bacterial cells to PBS buffer is 0.5 g / 5 ml. In the bacterial cell treatment step, accurately weigh 0.5 g of bacterial cells and add 5 ml of PBS buffer that matches them. This precise ratio can not only meet the requirement of bacterial cell suspension but also ensure the efficient and stable release of intracellular substances during the subsequent bacterial cell disruption and centrifugation operations, providing strong support for the high-quality preparation of crude enzyme solution.

[0042] Furthermore, the pretreated epoxy resin is 0.5 g, the crude enzyme solution is 5 ml, and the ratio of pretreated epoxy resin to crude enzyme solution is 0.5 g / 5 ml. When entering the crucial stage of the interaction between epoxy resin and crude enzyme solution, take 0.5 g of pretreated epoxy resin and put it into 5 ml of crude enzyme solution, so that the active sites on the surface of the epoxy resin can fully contact the enzyme molecules in the crude enzyme solution, thereby carrying out efficient immobilization.

[0043] Furthermore, the temperature is specifically set at 25°C and the stirring speed is 200 rpm. During the dynamic process of the interaction between epoxy resin and crude enzyme solution, temperature and stirring speed are two crucial control parameters. Precisely set the temperature at 25°C, which is close to the suitable activity temperature range of many enzymes and can maximize the catalytic activity of the enzyme. At the same time, with a stirring speed of 200 rpm, this speed can not only ensure the full and continuous mixing of epoxy resin and crude enzyme solution but also prevent the destruction of the enzyme molecular structure due to too high a speed, thus ensuring that the entire reaction system operates in a stable and efficient state for 24 hours.

[0044] Furthermore, the resin models are T28-103B, T28-108, T28-109, and T28-110 respectively, and the conversion rate duration is set to 1 hour. The optimal conversion rate among the above resin models is the T28-108 resin model. Under unified experimental conditions, the conversion rates of each resin model after enzyme immobilization were accurately measured. For details, see the attached drawings. The results show that the conversion rate corresponding to the T28-103B resin model is 80%, the conversion rate of the T28-108 resin model is as high as 99%, the conversion rate of the T28-109 resin model is 90%, and the conversion rate of the T28-110 resin model is only 56%. Through comparative analysis, it is obvious that the T28-108 resin model performs the best in promoting enzyme immobilization and improving the conversion rate, so it is determined as the optimal choice.

[0045] Furthermore, take the T28-108 resin. The optimal immobilization temperature is 20°C. Experimental data show that when the immobilization temperature is 15°C, the conversion rate reaches 90%. When the temperature rises to 20°C, the conversion rate increases to 99%. For details, see the attached drawings, reaching the peak. When the temperature continues to rise to 25°C, the conversion rate drops to 93%. When the temperature reaches 30°C, the conversion rate is only 80%. Therefore, 20°C is the most suitable immobilization temperature for the T28-108 resin, and the high-efficiency immobilization of enzymes and the maximization of catalytic activity can be achieved at this temperature.

[0046] Furthermore, take the T28-108 resin. The immobilization temperature is set to 20°C, and the optimal pH is 7. The conversion rates at various pH values were carefully measured. The measured data show that when the pH value is 6, the conversion rate is 81%. When the pH value is adjusted to 7, the conversion rate rises to 99%. For details, see the attached drawings, presenting the best state. When the pH value rises to 8, the conversion rate drops back to 90%. When the pH value is 9, the conversion rate drops to 70%. It can be seen that when the pH value is 7, it is the optimal pH value choice for the T28-108 resin at the immobilization temperature of 20°C, which can create the most ideal acid-base environment for the enzymatic reaction.

[0047] Furthermore, take the T28-108 resin. The immobilization temperature is set to 20°C, and the pH is set to 7. The optimal amount of crude enzyme solution is 5 ml, and the resin is 0.5 g each. The experimental results show that when the amount of crude enzyme solution is 2 ml, the conversion rate is 75%. When the amount of crude enzyme solution increases to 5 ml, the conversion rate reaches 99%. For details, see the attached drawings, and the effect is the best at this time. When the amount of crude enzyme solution is further increased to 7 ml, the conversion rate is 93%. When the amount of crude enzyme solution reaches 10 ml, the conversion rate is 92%. Therefore, the state of 5 ml of crude enzyme solution is the optimal choice, which can not only ensure the full contact between the enzyme and the resin but also avoid resource waste and potential side reaction interference caused by excessive enzyme solution.

[0048] Furthermore, the immobilized enzyme was prepared under the above optimal conditions, and the number of uses was investigated. The number of uses was set from 1 to 10, and the conversion rate and ee value were both 99%. The above series of optimal conditions were selected, that is, T28-108 resin was used, the immobilization temperature was 20°C, the pH value was 7, and the amount of crude enzyme solution was 5 ml, so as to prepare a high-quality immobilized enzyme. On this basis, the number of uses of the immobilized enzyme was systematically investigated. The number of uses was gradually increased from 1 to 10 times, and the conversion rate and ee value were accurately measured after each use. During these 10 uses, the conversion rate was always stably maintained at 99%, and the ee value was also stably maintained at 99%. For details, see the attached drawings, which fully clarify that the immobilized enzyme prepared under the optimal conditions has excellent stability and high reusability.

[0049] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for immobilizing N-BOC-piperidone reductase, characterized in that: The following steps are involved: Step 1: Soak the epoxy resin in PBS buffer to ensure that the epoxy resin is immersed, then let it stand and soak overnight and filter to allow the liquid to flow out, thereby taking out the filter cake after soaking to obtain the pretreated epoxy resin for use; Step 2: suspend the cultured bacteria in a buffer solution, break the bacteria to release the substances in the cells, and then centrifuge to precipitate the cell fragments with higher density, take out the supernatant, and obtain the crude enzyme solution for use; Step 3: placing the pretreated epoxy resin into the crude enzyme solution to allow the epoxy resin and the crude enzyme solution to interact with each other; Step 4: Set the stirring temperature at 15-35°C to stir the interacting epoxy resin and crude enzyme solution for 24 hours; Step 5: After the stirring is completed, some insoluble solid impurities are removed by suction filtration to separate the liquid component from the solid component. At the same time, the filter cake is washed with a buffer solution and then taken out to obtain the immobilized enzyme; Step 6: Add N-BOC-3-piperidone / 0.5g, isopropanol / 1g, PBS buffer / 4ml, and NADP / 0.1mg to the immobilized enzyme respectively, set the temperature to 30°C and react for 1 hour, and take samples after 5 hours to measure the conversion rate.

2. The method for immobilizing N-BOC-piperidone reductase according to claim 1, characterized in that: The epoxy resin is 0.5 g, the PBS buffer is 5 ml, and the ratio of epoxy resin to PBS buffer is 0.5 g / 5 ml.

3. The method for immobilizing N-BOC-piperidone reductase according to claim 1, characterized in that: The bacterial cells were 0.5 g, the PBS buffer was 5 ml, and the ratio of bacterial cells to PBS buffer was 0.5 g / 5 ml.

4. The method for immobilizing N-BOC-piperidone reductase according to claim 1, characterized in that: The pretreated epoxy resin was 0.5 g, the crude enzyme solution was 5 ml, and the ratio of the pretreated epoxy resin to the crude enzyme solution was 0.5 g / 5 ml.

5. The method for immobilizing N-BOC-piperidone reductase according to claim 1, characterized in that: The stirring temperature was specifically set to 25°C and the stirring speed was 200 rpm.

6. The method for immobilizing N-BOC-piperidone reductase according to claim 1, characterized in that: The resin model is T28-108, the conversion time is set to 1 hour, the immobilization temperature is set to 20°C, the conversion rate at pH 7 is 99%, the crude enzyme liquid volume is 5 ml, and the number of uses is investigated, the number of uses is set to 1-10, and the conversion rate and ee value are both 99%.

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