Process for preparing L-tertiary leucine by enzyme method

Abstract

The invention provides a process for preparing L-tertiary leucine by an enzyme method. The preparation method comprises the following steps: firstly carrying out carboxylation treatment on graphene oxide, promoting the graphene oxide to be combined with a phenolic prepolymer by increasing surface active sites to form a graphene oxide phenolic copolymer, and loading gold nanoparticles through a photodeposition method to prepare the modified phenolic aldehyde photocatalyst. In the catalyst, a quinone type-benzene type unit formed by condensation polymerization of a phenolic aldehyde structure forms an electron D-A conjugate pair, and the electron D-A conjugate pair is combined with the plasma resonance effect of gold nanoparticles, so that the light absorption range can be effectively widened, photo-induced electrons are enriched, carrier separation is accelerated, and the NADH regeneration efficiency is remarkably improved; meanwhile, due to the photocatalytic activity of the graphene oxide and the two-dimensional lamella and surface functional groups of the graphene oxide, the electron transmission path in the catalyst can be optimized, and the graphene oxide cooperates with other components to enhance the electron transmission efficiency of the system. According to the modification strategy, an efficient coenzyme regeneration system is constructed through the synergistic effect of multiple components, and high substrate conversion rate and high light purity preparation of the L-tertiary leucine are achieved.

Description

Technical Field

[0001] The present invention relates to the field of L-tert-leucine synthesis, and in particular to a process for preparing L-tert-leucine by an enzymatic method. Background Art

[0002] L-tert-leucine, an important chiral amino acid, is widely used in pharmaceuticals, food additives, and cosmetics. Currently, the main methods for preparing L-tert-leucine include chemical synthesis and biological methods. Chemical synthesis often faces challenges such as cumbersome steps, low atom utilization, severe environmental pollution, and difficulty achieving high stereoselectivity. Enzymatic biological methods, however, have become a research hotspot for L-tert-leucine preparation due to their advantages such as mild reaction conditions, high catalytic efficiency, and strong stereoselectivity.

[0003] The enzymatic preparation of L-tert-leucine usually uses trimethylpyruvate as a substrate and uses leucine dehydrogenase to catalyze its reductive amination reaction with ammonia. This process requires the coenzyme NAD + However, coenzymes are expensive and easily inactivated, so it is often necessary to build a coenzyme regeneration system. At the same time, there are also high requirements for the stability of the catalyst and the compatibility of the reaction system.

[0004] However, in the existing enzymatic preparation process, the coenzyme NAD + The regeneration efficiency is low, and the catalyst is easily affected by external factors and deactivated during the reaction, making it difficult to further improve the reaction substrate conversion rate and enantiomeric purity. In addition, the light absorption capacity and electron transfer efficiency of traditional catalysts are limited, further restricting the coenzyme NAD + The regeneration of L-tert-leucine is restricted, which limits the conversion of the substrate and the industrial application of enzymatic preparation of L-tert-leucine.

[0005] In summary, a new technical solution is urgently needed to solve the problems existing in the existing technology. Summary of the Invention

[0006] Based on this, the present invention provides a process for preparing L-tert-leucine by an enzymatic method. The process first carboxylates graphene oxide to increase its surface active sites, enabling it to better bind to a phenolic prepolymer to form a graphene oxide-phenolic copolymer. Gold nanoparticles are then loaded onto the copolymer via a photodeposition method to form a modified phenolic photocatalyst. On the one hand, the quinone-benzene units formed by the phenolic structure of the catalyst form electronic DA conjugated pairs. Combined with the plasmon resonance effect of the gold nanoparticles, this effectively broadens the light absorption range and enriches photogenerated electrons, achieving rapid carrier separation and significantly improving NADH regeneration efficiency. On the other hand, graphene oxide itself possesses certain photocatalytic activity. Its two-dimensional sheet structure and surface functional groups can optimize the electron transport pathway within the catalyst. It also synergizes with the phenolic resin and gold nanoparticles to further enhance the system's electron transfer efficiency, thereby synergistically improving NADH regeneration efficiency. This results in high substrate conversion and high light purity for the preparation of L-tert-leucine, providing an efficient and environmentally friendly solution for industrial production.

[0007] An object of the present invention is to provide a process for preparing L-tert-leucine by an enzymatic method, wherein the process for preparing L-tert-leucine by an enzymatic method comprises the following steps:

[0008] S1, mixing graphene oxide, sodium hydroxide and bromoacetic acid, and heating them to react to obtain carboxylated graphene oxide;

[0009] S2, mixing resorcinol, formaldehyde and sodium hydroxide, and heating them to react to obtain a phenolic prepolymer;

[0010] S3, blending the carboxylated graphene oxide and the phenolic prepolymer, adjusting the pH, and heating the mixture to react to obtain a graphene oxide phenolic copolymer;

[0011] S4, mixing the graphene oxide phenolic copolymer with chloroauric acid, and reacting them under light conditions to obtain a modified phenolic photocatalyst;

[0012] S5, dispersing the modified phenolic photocatalyst in a buffer system containing ammonia and triethanolamine, adding an electron mediator and trimethylpyruvic acid, and then adding leucine dehydrogenase coenzyme NAD + , react under light conditions to obtain L-tert-leucine.

[0013] Furthermore, the electron mediator is a rhodium-based ligand.

[0014] Furthermore, in step S1, the mass ratio of the graphene oxide, sodium hydroxide and bromoacetic acid is 1:(5-15):(15-30).

[0015] Furthermore, in step S1, the heating temperature is 30-50°C.

[0016] Furthermore, in step S2, the molar ratio of resorcinol, formaldehyde and sodium hydroxide is 1:(0.1-0.9):(0.5-1.5).

[0017] Furthermore, in step S2, the heating temperature is 30-50°C.

[0018] Furthermore, in step S3, the mass ratio of the carboxylated graphene oxide to the phenolic prepolymer is 1:(20-40).

[0019] Furthermore, in step S3, the heating temperature is 80-100°C.

[0020] Furthermore, in step S4, the mass ratio of the graphene oxide phenolic copolymer to chloroauric acid is 250:(1-5).

[0021] Furthermore, in step S5, the illumination does not include ultraviolet light.

[0022] The present invention has the following beneficial effects:

[0023] The invention first performs carboxylation treatment on graphene oxide to increase its surface active sites so that it can better combine with phenolic prepolymer to form graphene oxide phenolic copolymer. Then, gold nanoparticles are loaded on the copolymer by photodeposition to form a modified phenolic photocatalyst. On the one hand, the quinone-benzene units formed by the condensation of the phenolic structure of the catalyst constitute an electronic DA conjugated pair. Combined with the plasmon resonance effect of gold nanoparticles, it can effectively broaden the light absorption range and enrich photogenerated electrons, realize rapid separation of carriers, and significantly improve the NADH regeneration efficiency; on the other hand, the combination of graphene oxide and phenolic prepolymer effectively improves the dispersibility of graphene oxide while providing abundant interface adsorption sites, effectively increasing the loading capacity of gold nanoparticles, thereby improving the NADH regeneration efficiency; in addition, graphene oxide itself has certain photocatalytic activity, and its two-dimensional layer structure and surface functional groups can optimize the electron transfer path inside the catalyst, synergistically with phenolic resin and gold nanoparticles, further enhancing the electron transfer efficiency of the system, thereby synergistically improving the NADH regeneration efficiency, and then achieving high substrate conversion rate and high light purity preparation of L-tert-leucine, providing an efficient and environmentally friendly solution for industrial production. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the technical solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0025] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0026] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.

[0027] The present invention uses the following raw materials:

[0028] Leucine dehydrogenase: brand 120-04391, purchased from Beijing Biode Biotechnology Co., Ltd.

[0029] Coenzyme NAD + β-NAD, brand N111609, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0030] The preparation method of rhodium-based ligand [Cp*Rh(bpy)Cl] solution comprises the following steps:

[0031] First, weigh 0.4 mmol of rhodium trichloride into a glass container containing 20 mL of methanol. Then, add 0.4 mmol of pentamethylcyclopentadiene and reflux at room temperature for 12 hours to produce a red precipitate. Next, mix this solution with 0.8 mmol of 2,2'-bipyridine and stir for 1 hour to produce an orange solution. Allow to evaporate naturally in a fume hood to a volume of approximately 3 mL. Then, add 10 mL of ether and air-dry at room temperature for several hours to produce an orange solid of the rhodium-based ligand. This solid was weighed and collected to prepare a 20 mM rhodium-based ligand solution, which was stored in a refrigerator at 4°C until ready for use.

[0032] The water in the present invention is all deionized water.

[0033] The "parts" in the present invention refer to parts by mass.

[0034] Example 1

[0035] A process for preparing L-tert-leucine by an enzymatic method, comprising the following steps:

[0036] S1. Using water as solvent, graphene oxide, sodium hydroxide and bromoacetic acid were mixed (graphene oxide: sodium hydroxide: bromoacetic acid = 1:8:17, m / m / m), ultrasonically dispersed uniformly, reacted at 30° C. for 7 h, filtered, washed, and dried to obtain carboxylated graphene oxide;

[0037] S2. Blending resorcinol and formaldehyde, adding 50 wt % sodium hydroxide aqueous solution (resorcinol: formaldehyde: sodium hydroxide = 1:0.9:1, n / n / n), and reacting at 30° C. for 45 h to obtain a phenolic prepolymer;

[0038] S3, using water as a solvent, blending the carboxylated graphene oxide and the phenolic prepolymer (carboxylated graphene oxide: phenolic prepolymer = 1:30, m / m), adjusting the pH to 9, reacting at 90° C. for 2 h, filtering, washing, drying, and grinding to obtain a graphene oxide phenolic copolymer;

[0039] S4. Using water as a solvent, the graphene oxide phenolic copolymer and chloroauric acid were mixed (graphene oxide phenolic copolymer: chloroauric acid = 250:3.5, m / m), and the mixture was placed under a 300 W xenon lamp for 2 h of irradiation under stirring. The mixture was then centrifuged, washed, dried, and ground to obtain a modified phenolic photocatalyst.

[0040] S5, 0.5 mg of the modified phenolic photocatalyst was dispersed in 3 mL of phosphate buffer (0.1 M NH4Cl, 15 wt% triethanolamine) system, rhodium ligand solution (the final concentration of rhodium ligand was 0.375 mM) and substrate trimethylpyruvate (the final concentration was 10 mM), and then coenzyme NAD was added. + (final concentration of 1 mM) and leucine dehydrogenase (7.5 μL); react under irradiation of a 300 W xenon lamp equipped with a 420 nm filter for 8 h. After the reaction is completed, the mixture is purified using a cationic resin and dried to obtain L-tert-leucine.

[0041] Example 2

[0042] A process for preparing L-tert-leucine by an enzymatic method. The difference between this embodiment and embodiment 1 is that in step S5, the amount of the modified phenolic photocatalyst used is 0.4 mg, the amount of the leucine dehydrogenase used is 6 μL, and the remaining steps and amounts are the same as those in embodiment 1.

[0043] Example 3

[0044] A process for preparing L-tert-leucine by an enzymatic method. The difference between this embodiment and Example 1 is that in step S5, the amount of the modified phenolic photocatalyst used is 0.7 mg, the amount of the leucine dehydrogenase used is 15 μL, and the remaining steps and amounts are the same as in Example 1.

[0045] Comparative Example 1

[0046] A process for preparing L-tert-leucine by an enzymatic method. The difference between this comparative example and Example 1 is that step S4 is not performed. In step S5, the mass of the modified phenolic photocatalyst is replaced by graphene oxide phenolic copolymer. The remaining steps and amounts are the same as those in Example 1.

[0047] Comparative Example 2

[0048] A process for preparing L-tert-leucine by an enzymatic method. The difference between this comparative example and Example 1 is that steps S1 and S3 are not performed, and in step S4, the mass of the graphene oxide phenolic copolymer is replaced by a phenolic prepolymer. The remaining steps and amounts are the same as those in Example 1.

[0049] Test Case

[0050] The performance of the processes for preparing L-tert-leucine by the enzymatic method of Examples 1-3 and Comparative Examples 1-2 was tested.

[0051] Test method:

[0052] (1) During the reaction of step S5, samples were taken at 2, 4, 6, and 8 hours to test the substrate conversion rate.

[0053] Detection method: HPLC was used with a mobile phase consisting of 0.25 wt% NH₄H₂PO₄ and 100% methanol (v:v = 100:5), a detection wavelength of 205 nm, a Kromasil 700-5C₁₈ column (5 μm, 4.6 mm × 250 mm), a flow rate of 0.8 mL / min, and a column temperature of 30°C.

[0054] (2) The prepared product was dissolved in water and filtered through a 0.22 μm filter membrane. The enantiomeric excess was then determined by high performance liquid chromatography (HPLC). The chiral column Chirex 3126 (D-penicillamine, Phenomenex) was used for detection and analysis. The detection conditions were: a mobile phase of water / isopropanol (volume ratio) = 95:5 (containing 2 mM copper sulfate), a flow rate of 1 mL / min, a detection wavelength of 254 nm, and a column temperature of 35°C.

[0055] The enantiomeric excess percentage is calculated as follows:

[0056]

[0057] Here, A1 represents the content of L-tert-leucine, and A2 represents the content of D-tert-leucine.

[0058] The test results are shown in Table 1.

[0059] Table 1 Test results of substrate conversion and enantiomeric excess percentage

[0060]

[0061] The above test results show that the method for preparing L-tert-leucine by the present invention can achieve a substrate conversion rate of more than 42% in 8 hours and an ee% of more than 99%, which has high substrate conversion rate and optical purity and good application prospects.

[0062] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0063] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A process for preparing L-tert-leucine by enzymatic method, characterized in that: The process for preparing L-tert-leucine by enzymatic method comprises the following steps: S1, mixing graphene oxide, sodium hydroxide and bromoacetic acid, and heating them to react to obtain carboxylated graphene oxide; S2, mixing resorcinol, formaldehyde and sodium hydroxide, and heating them to react to obtain a phenolic prepolymer; S3, blending the carboxylated graphene oxide and the phenolic prepolymer, adjusting the pH, and heating the mixture to react to obtain a graphene oxide phenolic copolymer; S4, mixing the graphene oxide phenolic copolymer with chloroauric acid, and reacting them under light conditions to obtain a modified phenolic photocatalyst; S5, dispersing the modified phenolic photocatalyst in a buffer system containing ammonia and triethanolamine, adding an electron mediator and trimethylpyruvic acid, and then adding leucine dehydrogenase coenzyme NAD + , react under light conditions to obtain L-tert-leucine.

2. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: The electron mediator is a rhodium-based ligand.

3. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S1, the mass ratio of graphene oxide, sodium hydroxide and bromoacetic acid is 1:(5-15):(15-30).

4. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S1, the heating temperature is 30-50°C.

5. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S2, the molar ratio of resorcinol, formaldehyde and sodium hydroxide is 1:(0.1-0.9):(0.5-1.5).

6. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S2, the heating temperature is 30-50°C.

7. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S3, the mass ratio of the carboxylated graphene oxide to the phenolic prepolymer is 1:(20-40).

8. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S3, the heating temperature is 80-100°C.

9. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S4, the mass ratio of the graphene oxide phenolic copolymer to chloroauric acid is 250:(1-5).

10. The process for preparing L-tert-leucine by enzymatic method according to claim 1, characterized in that: In step S5, the illumination does not include ultraviolet light.

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