D-amino acid oxidase, its use and method for enzymatic production of l-norvaline

Abstract

The application discloses a D-amino acid oxidase and application thereof and a method for preparing L-norvaline by using an enzyme method, and the amino acid sequence of the D-amino acid oxidase is shown as SEQ ID NO. 1. In order to prepare L-norvaline by using the enzyme method, the application provides an optimized D-amino acid oxidase sequence, the enzyme has high activity and substrate tolerance. By optimizing experimental conditions, the best conversion conditions are determined, the conversion rate can be improved by the method, and the production efficiency is improved. The method has small pollution and is more friendly to the environment. Meanwhile, raw materials are easy to obtain, the production cost can be reduced, and the method is beneficial to large-scale application.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to D-amino acid oxidases, their applications, and methods for the enzymatic preparation of L-valine. Background Technology

[0002] L-norvaline is a non-protein branched-chain amino acid that has a wide range of applications in the pharmaceutical industry as a precursor for the synthesis of many drugs. Perindopril, an effective drug for treating hypertension and heart failure, is synthesized using L-norvaline as an important intermediate.

[0003] Existing methods for synthesizing L-valine include chemical and enzymatic methods. Chemical methods, such as those described in patent CN202111031517.3, involve organic synthesis of DL-valine followed by tartaric acid resolution, resulting in a yield of only 36%. Another method directly uses DL-valine as a raw material and tartaric acid for resolution, achieving a yield of 83%. However, chemical methods generally suffer from significant pollution and high costs. Existing enzymatic methods, including CN202110276664.0, use valproic acid hydrochloride as a substrate, catalyzing the process with hydrolases and racemic enzymes to prepare L-valine. However, the raw materials are not readily available, making them unsuitable for industrial production. The literature "Multi-enzyme Catalytic Resolution of DL-valine to Produce L-valine" uses multi-enzyme catalysis, achieving a conversion rate of up to 96%, but the highest substrate concentration is only around 65 g / L, resulting in low production efficiency.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide D-amino acid oxidase, its application, and a method for the enzymatic preparation of L-valine, in order to improve the above-mentioned problems.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a D-amino acid oxidase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Secondly, the present invention also provides a nucleic acid molecule for encoding the above-mentioned D-amino acid oxidase, optionally, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0009] Thirdly, the present invention also provides the application of the above-mentioned D-amino acid oxidase in the enzymatic preparation of L-valine.

[0010] Fourthly, the present invention also provides a method for the enzymatic preparation of L-valine, comprising: adding the above-mentioned D-amino acid oxidase and catalase to a substrate to carry out an oxidation reaction; after the oxidation reaction, adding isopropanol dehydrogenase, leucine dehydrogenase and isopropanol to the reaction system, adjusting the pH with ammonia, and adding zinc acetate and NAD. + It undergoes a dehydrogenation reaction;

[0011] The substrate mentioned above is DL-valine.

[0012] The present invention has the following beneficial effects:

[0013] This invention provides an optimized D-amino acid oxidase sequence for the enzymatic preparation of L-valine, exhibiting high activity and substrate tolerance. Optimal conversion conditions were determined through experimental optimization, resulting in improved conversion rate and thus increased production efficiency. Furthermore, this method is less polluting and more environmentally friendly; the readily available raw materials not only reduce production costs but also facilitate large-scale application. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a flowchart of the enzymatic preparation of L-valine according to the present invention;

[0016] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of the target product in Example 1. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0018] The present invention provides a D-amino acid oxidase, the amino acid sequence of which is shown in SEQ ID NO.1.

[0019] The present invention also provides a nucleic acid molecule for encoding the aforementioned D-amino acid oxidase, the nucleotide sequence of which is shown in SEQ ID NO.2. The aforementioned D-amino acid oxidase can be obtained by encoding this nucleic acid molecule using existing genetic engineering methods.

[0020] This invention also provides the application of the above-mentioned D-amino acid oxidase in the enzymatic preparation of L-valine.

[0021] The method for preparing L-valine using the above-mentioned D-amino acid oxidase includes: using DL-valine as a substrate, adding the above-mentioned D-amino acid oxidase and catalase to carry out an oxidation reaction; after the oxidation reaction, adding isopropanol dehydrogenase, leucine dehydrogenase and isopropanol to the reaction system, adjusting the pH with ammonia, and adding zinc acetate and NAD. + The dehydrogenation reaction is carried out; after the dehydrogenation reaction is completed, the obtained reaction solution is filtered, concentrated twice, and dried to obtain the L-valine. The specific preparation process is as follows: Figure 1 As shown.

[0022] The synthetic route of the above preparation method is as follows:

[0023]

[0024] To achieve higher conversion rates, the inventors optimized the raw materials, enzymes and their ratios, and reaction conditions in the preparation process. By selecting specific raw materials, enzymes and their ratios, as well as reaction temperatures and pH within a certain range, the reaction can be made more complete, thereby obtaining higher conversion rates and purer target products.

[0025] In some embodiments, the substrate concentration in the reaction system can convert DL-n-valine to L-n-valine at a concentration of 50-300 g / L. Specifically, the substrate concentration in the reaction system can be 50 g / L, 100 g / L, 150 g / L, 200 g / L, 250 g / L, or 300 g / L, or any value between 50-300 g / L. Preferably, the substrate concentration in the reaction system is 100-250 g / L; more preferably, the substrate concentration in the reaction system is 125-250 g / L.

[0026] In some embodiments, a substrate-to-D-amino acid oxidase mass ratio of 1:0.2-0.4 exhibits good conversion rates. Specifically, the substrate-to-D-amino acid oxidase mass ratio can be 1:0.2, 1:0.25, 1:0.3, 1:0.35, or 1:0.4, or any value between 1:0.2 and 0.4. Preferably, the substrate-to-D-amino acid oxidase mass ratio is 1:0.2-0.3; more preferably, the substrate-to-D-amino acid oxidase mass ratio is 1:0.25-0.3.

[0027] In some embodiments, a better conversion rate is achieved when the amount of catalase added is 0.5-9 vt% of the reaction system. Specifically, the amount of catalase added can be 0.5 vt%, 1 vt%, 2 vt%, 2.5 vt%, 3.5 vt%, 4 vt%, 5.5 vt%, 6 vt%, 8 vt%, or 9 vt, or any value between 0.5-9 vt%. Preferably, the amount of catalase added is 1-8 vt% of the reaction system; more preferably, the amount of catalase added is 1-6 vt% of the reaction system.

[0028] In some embodiments, the substrate is reacted with isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD. + A good conversion rate was observed when the mass ratio of the substrate to isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD+ was 1:10%-30%:5%-25%:0.2%-0.6%:0.03%-0.05%. Specifically, the substrate reacted well with isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD+. + The mass ratio can be 1:10:5:0.2:0.03, 1:15:10:0.3:0.035, 1:20:15:0.4:0.04, 1:25:20:0.5:0.045, or 1:30:25:0.6:0.05, or any value between 1:10%-30%:5%-25%:0.2%-0.6%:0.03%-0.05%. Preferably, the substrate and isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD are... + The mass ratio is 1:15%-30%:10%-25%:0.3%-0.6%:0.035%-0.05%; more preferably, the substrate contains isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD. + The mass ratio is 1:20%-30%:15%-25%:0.4%-0.6%:0.04%-0.05%.

[0029] In some embodiments, the pH of the reaction system in the oxidation reaction is 7-7.5, and the reaction temperature is 28-32°C. Specifically, the pH of the reaction system in the oxidation reaction can be 7, 7.1, 7.2, 7.3, 7.4, or 7.5, or any value between 7 and 7.5. The reaction temperature can be 28°C, 29°C, 30°C, 31°C, or 30°C, or any value between 28 and 32°C.

[0030] In some embodiments, the pH of the reaction system in the dehydrogenation reaction is 8-8.5, and the reaction temperature is 36-38°C. Specifically, the pH of the reaction system in the dehydrogenation reaction can be 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any value between 8 and 8.5. The reaction temperature can be 36°C, 37°C, or 38°C, or any value between 36 and 38°C.

[0031] In some embodiments, the pH of the resulting reaction solution needs to be adjusted to 1-2 before filtration after the dehydrogenation reaction is completed. Specifically, the pH of the reaction solution can be adjusted to 1, 1.1, 1.3, 1.5, 1.8 or 2, or any value between 1 and 2.

[0032] In some embodiments, a better filtration effect is achieved when the filter membrane has a pore size of 45-55 nm and a molecular weight cutoff of 800-1200 D. Specifically, the filter membrane pore size can be 45 nm, 50 nm, or 55 nm. The molecular weight cutoff can be 800 D, 1000 D, or 1200 D.

[0033] In some embodiments, the two concentration steps include concentrating the filtered filtrate to 0.2-0.4 of the filtrate volume, filtering to obtain a first crystal, rinsing twice with methanol, collecting the crystal, further concentrating to 0.1-0.2 of the filtered filtrate volume, filtering to obtain a second crystal, rinsing twice with methanol, and collecting the crystal.

[0034] In some embodiments, the temperature for drying the crystals collected during the above process is 55-60°C. Specifically, the drying temperature can be 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C, or any value between 55-60°C.

[0035] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0036] The amino acid sequence of the D-amino acid oxidase used in the following examples is shown in SEQ ID NO.1.

[0037] Example 1

[0038] This embodiment provides a method for the enzymatic preparation of L-valine, the steps of which are as follows:

[0039] (1) Oxidation reaction

[0040] Weigh 200g of DL-valine and add it to a 2L reactor. First, add 400ml of water and stir until a suspension is formed. Then, add water to bring the volume to 700ml. Adjust the pH to 7.5 with ammonia solution (approximately 1mL of ammonia added dropwise). Heat to 30℃ and introduce air. Weigh 40g of D-amino acid oxidase, suspend it in 200ml of water, and add it to the reaction system. Add 40ml of catalase. Adjust the pH of the reaction system to 7.5 with ammonia solution. Add 1mL of defoamer and maintain the temperature at 30℃, keeping the pH around 7.5 throughout the process. During the reaction, add D-amino acid oxidase as needed based on chiral detection results. After 7-8 hours of reaction, when D-valine is completely reacted, add a total of 20g of D-amino acid oxidase. Determine the reaction endpoint, heat to 80℃, hold for 30 minutes, and then cool to 37℃.

[0041] (2) Dehydrogenation reaction

[0042] Weigh 40g of isopropanol dehydrogenase and 20g of leucine dehydrogenase, suspend them in 80ml of tap water, and add them to the reaction system; add 80ml of isopropanol to the reaction system, adjust the pH to approximately 8.5 with ammonia water, and weigh 0.05g of zinc acetate and NAD+. + Add 0.05g to the reactor, and carry out the reaction at 37℃ with ventilation. During the reaction, stop the reaction once the dehydrogenation reaction is complete, based on the liquid phase detection results.

[0043] (3) Product extraction

[0044] The reaction solution was adjusted to pH 1-2 and filtered sequentially through a 50 nm ceramic membrane and a 1 KD ultrafiltration membrane. After filtration, the filtrate was concentrated at 60 °C to a volume of approximately 300 mL. The first crystal was obtained by filtration, and 20 mL of methanol was added to wash twice. The crystals were collected. The solution was further concentrated to approximately 150 mL, and the second crystal was obtained by filtration. 10 mL of methanol was added to wash twice. The crystals were collected and dried at 60 °C to obtain 186.4 g of product, with a yield of 93.2% and a chiral purity of 99.9%.

[0045] like Figure 2 The detection results of the product in this embodiment were obtained by high-performance liquid chromatography (HPLC). The HPLC detection conditions were as follows:

[0046] Column: CROWNOAK CR-I(+) (3.0*150mm, 5um);

[0047] Flow rate: 0.2 mL / min; Wavelength: 230 nm; Injection volume: 20 μL; Column temperature: 25 °C;

[0048] Mobile phase: 1000 mL of water, pH adjusted to 1.50 ± 0.1 with perchloric acid, filtered and ultrasonically degassed.

[0049] Example 2

[0050] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 50 g / L, the D-amino acid oxidase accounts for 20% of the substrate mass, and the catalase accounts for 0.5% of the reaction system.

[0051] Example 3

[0052] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 100 g / L, the D-amino acid oxidase accounts for 20% of the substrate mass, and the catalase accounts for 1% of the reaction system.

[0053] Example 4

[0054] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 125 g / L, the D-amino acid oxidase accounts for 25% of the substrate mass, and the catalase accounts for 2% of the reaction system.

[0055] Example 5

[0056] The preparation method in this embodiment is the same as in Example 1, except that the concentration of DL-valine is 150 g / L, the D-amino acid oxidase accounts for 25% of the substrate mass, and the catalase accounts for 2.5% of the reaction system.

[0057] Example 6

[0058] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 175 g / L, the D-amino acid oxidase accounts for 30% of the substrate mass, and the catalase accounts for 3.5% of the reaction system.

[0059] Example 7

[0060] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 200 g / L, the D-amino acid oxidase accounts for 30% of the substrate mass, and the catalase accounts for 4% of the reaction system.

[0061] Example 8

[0062] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 225 g / L, the D-amino acid oxidase accounts for 35% of the substrate mass, and the catalase accounts for 5.5% of the reaction system.

[0063] Example 9

[0064] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 250 g / L, the D-amino acid oxidase accounts for 35% of the substrate mass, and the catalase accounts for 6% of the reaction system.

[0065] Example 10

[0066] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 275 g / L, the D-amino acid oxidase accounts for 40% of the substrate mass, and the catalase accounts for 8% of the reaction system.

[0067] Example 11

[0068] The preparation method of this embodiment is the same as that of Example 1, except that the concentration of DL-valine is 300 g / L, the D-amino acid oxidase accounts for 40% of the substrate mass, and the catalase accounts for 9% of the reaction system.

[0069] Example 12

[0070] The preparation method in this embodiment is the same as in Example 1, except that isopropanol dehydrogenase accounts for 10% of the substrate mass, leucine dehydrogenase accounts for 5% of the substrate mass, isopropanol accounts for 0.2% of the substrate mass, and NAD... + It accounts for 0.03% of the substrate mass.

[0071] Example 13

[0072] The preparation method in this embodiment is the same as in Example 1, except that isopropanol dehydrogenase accounts for 15% of the substrate mass, leucine dehydrogenase accounts for 10% of the substrate mass, isopropanol accounts for 0.3% of the substrate mass, and NAD... + It accounts for 0.035% of the substrate mass.

[0073] Example 14

[0074] The preparation method in this embodiment is the same as in Example 1, except that isopropanol dehydrogenase accounts for 20% of the substrate mass, leucine dehydrogenase accounts for 15% of the substrate mass, isopropanol accounts for 0.4% of the substrate mass, and NAD... + It accounts for 0.04% of the substrate mass.

[0075] Example 15

[0076] The preparation method in this embodiment is the same as in Example 1, except that isopropanol dehydrogenase accounts for 25% of the substrate mass, leucine dehydrogenase accounts for 20% of the substrate mass, isopropanol accounts for 0.5% of the substrate mass, and NAD... + It accounts for 0.045% of the substrate mass.

[0077] Example 16

[0078] The preparation method in this embodiment is the same as in Example 1, except that isopropanol dehydrogenase accounts for 30% of the substrate mass, leucine dehydrogenase accounts for 25% of the substrate mass, isopropanol accounts for 0.6% of the substrate mass, and NAD... +It accounts for 0.05% of the substrate mass.

[0079] Comparative Example 1

[0080] The difference from Example 1 is that the comparative example used D-amino acid oxidase before sequence optimization, whose nucleotide sequence is shown in SEQ ID NO.3. The concentration of DL-valine was 25 g / L, the D-amino acid oxidase accounted for 20% of the substrate mass, and the catalase accounted for 0.25% of the reaction system.

[0081] Comparative Example 2

[0082] The difference from Example 1 is that this comparative example uses D-amino acid oxidase before sequence optimization, whose nucleotide sequence is shown in SEQ ID NO.3. The concentration of DL-valine is 50 g / L, the D-amino acid oxidase accounts for 20% of the substrate mass, and the catalase accounts for 0.5% of the reaction system.

[0083] Comparative Example 3

[0084] The difference from Example 1 is that the comparative example used D-amino acid oxidase before sequence optimization, whose nucleotide sequence is shown in SEQ ID NO.3. The concentration of DL-valine was 75 g / L, the D-amino acid oxidase accounted for 20% of the substrate mass, and the catalase accounted for 0.75% of the reaction system.

[0085] Comparative Example 4

[0086] The difference from Example 1 is that this comparative example uses D-amino acid oxidase before sequence optimization, whose nucleotide sequence is shown in SEQ ID NO.3. The concentration of DL-valine is 100 g / L, the D-amino acid oxidase accounts for 20% of the substrate mass, and catalase accounts for 1% of the reaction system.

[0087] Experimental Example 1

[0088] The remaining amount of D-amino acid in Examples 2-11 was detected by central control, and the results are shown in Table 1:

[0089] Table 1. Effects of different substrate concentrations and enzyme dosages on the results of the oxidation reaction.

[0090]

[0091] As can be seen from the results in Table 1, high conversion rates can be obtained by using substrate concentrations and enzyme amounts within the scope of this invention. The optimal conversion rate is achieved when the substrate concentration is 125-250 g / L, the D-amino acid oxidase accounts for 0.25-0.35% of the substrate mass, and the catalase accounts for 1-6% of the reaction system volume.

[0092] Experimental Example 2

[0093] The degree of reaction of 2-oxopentanoic acid in Example 2-11 was detected by central control, and the results are shown in Table 2:

[0094] Table 2. Effects of different reaction conditions on the dehydrogenation reaction results.

[0095]

[0096] As can be seen from the results in Table 2, the use of isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD within the scope of this invention is effective. + High conversion rates can be achieved with appropriate dosages of isopropanol dehydrogenase (20-30% substrate mass), leucine dehydrogenase (15-25% substrate mass), isopropanol (0.4-0.6% substrate mass), and NAD+. + The conversion rate is optimal when the substrate content is 0.04-0.05% by mass.

[0097] Experimental Example 3

[0098] The residual amount of D-nova amino acid in Comparative Examples 1-4 was detected by central control, and the results are shown in Table 3:

[0099] Table 3 shows the remaining amounts of D-nova amino acids in Comparative Examples 1-4.

[0100]

[0101] As shown in Table 3, when using the D-amino acid oxidase before sequence optimization to prepare L-valine, under the same reaction conditions, the conversion efficiency decreased with the increase of DL-valine substrate concentration. Moreover, compared with the example, the residual amount of D-valine in the comparative example was greater at the same DL-valine substrate concentration. This proves that the conversion rate of L-valine prepared by using the D-amino acid oxidase after sequence optimization is significantly improved.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A D-amino acid oxidase, characterized in that, The amino acid sequence of the D-amino acid oxidase is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, It is used to encode the D-amino acid oxidase as described in claim 1.

3. The application of the D-amino acid oxidase as described in claim 1 in the enzymatic preparation of L-valine.

4. A method for preparing L-valine enzymatically, characterized in that, The method includes: adding the D-amino acid oxidase of claim 1 and catalase to the substrate to carry out an oxidation reaction; After the oxidation reaction, isopropanol dehydrogenase, leucine dehydrogenase, and isopropanol are added to the reaction system. The pH is adjusted with ammonia, and zinc acetate and NAD are added. + It undergoes a dehydrogenation reaction; The substrate is DL-valine.

5. The method for preparing L-valine by enzymatic method according to claim 4, characterized in that, The concentration of the substrate in the reaction system is 50-300 g / L.

6. The method for preparing L-valine by enzymatic method according to claim 5, characterized in that, The concentration of the substrate in the reaction system is 100-250 g / L.

7. The method for preparing L-valine by enzymatic method according to claim 6, characterized in that, The concentration of the substrate in the reaction system is 125-250 g / L.

8. The method for preparing L-valine by enzymatic method according to claim 4, characterized in that, The mass ratio of the substrate to D-amino acid oxidase is 1:0.2-0.

4.

9. The method for preparing L-valine by enzymatic method according to claim 8, characterized in that, The mass ratio of the substrate to D-amino acid oxidase is 1:0.2-0.

3.

10. The method for preparing L-valine by enzymatic method according to claim 9, characterized in that, The mass ratio of the substrate to D-amino acid oxidase is 1:0.25-0.

3.

11. The method for preparing L-valine by enzymatic method according to claim 4, characterized in that, The amount of catalase added is 0.5-9 VT of the reaction system.

12. The method for preparing L-valine by enzymatic method according to claim 11, characterized in that, The amount of catalase added is 1-8 vt of the reaction system.

13. The method for preparing L-valine by enzymatic method according to claim 12, characterized in that, The amount of catalase added is 1-6 vt of the reaction system.

14. The method for preparing L-valine by enzymatic method according to claim 4, characterized in that, The substrate contains isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD. + The mass ratio is 1:10%-30%:5%-25%:0.2%-0.6%:0.03%-0.05%.

15. The method for preparing L-valine by enzymatic method according to claim 14, characterized in that, The substrate contains isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD. + The mass ratio is 1:15%-30%:10%-25%:0.3%-0.6%:0.035%-0.05%.

16. The method for preparing L-valine by enzymatic method according to claim 15, characterized in that, The substrate contains isopropanol dehydrogenase, leucine dehydrogenase, isopropanol, and NAD. + The mass ratio is 1:20%-30%:15%-25%:0.4%-0.6%:0.04%-0.05%.

17. The method for preparing L-valine by enzymatic method according to claim 4, characterized in that, The pH of the reaction system in the oxidation reaction is 7-7.5, and the reaction temperature is 28-32℃.

18. The method for preparing L-valine by enzymatic method according to claim 17, characterized in that, The dehydrogenation reaction system has a pH of 8-8.5 and a reaction temperature of 36-38℃.

19. The method for preparing L-valine by enzymatic method according to any one of claims 4-18, characterized in that, The method further includes filtering, concentrating twice and drying the obtained reaction solution after the dehydrogenation reaction is completed to obtain the L-valine.

20. The method for preparing L-valine by enzymatic method according to claim 19, characterized in that, The pH of the reaction solution was adjusted to 1-2 before filtration.

21. The method for preparing L-valine by enzymatic method according to claim 19, characterized in that, The filter membrane has a pore size of 45-55 nm and a molecular weight cutoff of 800-1200 D.

22. The method for preparing L-valine by enzymatic method according to claim 19, characterized in that, The two concentrations involve concentrating the filtered filtrate to 0.2-0.4 of its volume, filtering to obtain the first crystal, rinsing twice with rinsing solution, collecting the crystals, further concentrating to 0.1-0.2 of the filtered filtrate volume, filtering to obtain the second crystal, rinsing twice with rinsing solution, and collecting the crystals.

23. The method for preparing L-valine by enzymatic method according to claim 22, characterized in that, The rinsing solution is methanol.

24. The method for preparing L-valine by enzymatic method according to claim 19, characterized in that, The drying temperature is 55-60℃.

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