Process for the enzymatic preparation of l-tert-leucine

Abstract

The application provides a process for preparing L-tert-leucine by an enzymatic method. The application first performs carboxyl group treatment on graphene oxide, promotes the combination of the graphene oxide with a phenolic aldehyde prepolymer by increasing surface active sites, forms graphene oxide-phenolic aldehyde copolymer, and then loads gold nanoparticles by a light deposition method to prepare a modified phenolic aldehyde photocatalyst. In the catalyst, the quinone-type-benzene type units formed by the polycondensation of the phenolic aldehyde structure constitute an electronic D-A conjugate pair, the plasmonic resonance effect of the gold nanoparticles can effectively broaden the light absorption range, enrich the photo-generated electrons and accelerate the carrier separation, and the NADH regeneration efficiency is significantly improved; meanwhile, the photocatalytic activity of the graphene oxide and the two-dimensional sheet layer and the surface functional groups thereof can optimize the internal electron transport path of the catalyst, and synergistically enhance the electron transfer efficiency of the system with other components. The above modification strategy builds an efficient coenzyme regeneration system through the synergistic effect of multiple components, and realizes the high substrate conversion rate and high light purity preparation of L-tert-leucine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of L-tert-leucine synthesis, in particular to a process for preparing L-tert-leucine by enzyme method. BACKGROUND

[0002] L-tert-leucine, as an important chiral amino acid, has a wide range of applications in the fields of medicine, food additives and cosmetics, etc. At present, the preparation methods of L-tert-leucine mainly include chemical synthesis method and biological method. The chemical synthesis method often faces problems such as complicated steps, low atom utilization rate, serious environmental pollution, etc., and it is difficult to achieve high stereoselectivity; the enzyme method in the biological method has the advantages of mild reaction conditions, high catalytic efficiency and strong stereoselectivity, and has become a research hotspot for the preparation of L-tert-leucine.

[0003] The enzyme method for preparing L-tert-leucine usually takes trimethylpyruvic acid as the substrate, and utilizes leucine dehydrogenase to catalyze the reductive amination reaction with ammonia water. This process needs coenzyme NAD + , and the cost of coenzyme is high and easy to be inactivated, so it is often necessary to construct a coenzyme regeneration system, and the stability of the catalyst and the compatibility of the reaction system also have higher requirements.

[0004] However, in the existing enzyme preparation process, the regeneration efficiency of coenzyme NAD + is low, and the catalyst is easy to be inactivated by external factors during the reaction, which leads to the difficulty in further improving the conversion rate of the reaction substrate and the enantiomeric purity. In addition, the light absorption capacity and electron transfer efficiency of the traditional catalyst are limited, which further restricts the regeneration of coenzyme NAD + , thereby limiting the conversion of the substrate and limiting the industrial application of the enzyme preparation of L-tert-leucine.

[0005] In summary, a new technical solution is needed to solve the problems existing in the prior art. SUMMARY

[0006] Based on this, the application provides an enzyme method for preparing L-tert-leucine.

[0007] An object of the present application is to provide an enzyme method for preparing L-tert-leucine, which comprises the following steps:

[0008] S1, blending graphene oxide, sodium hydroxide and bromoacetic acid, heating and reacting to obtain carboxylated graphene oxide;

[0009] S2, blending resorcinol, formaldehyde and sodium hydroxide, heating and reacting to obtain a phenolic pre-polymer;

[0010] S3, blending the carboxylated graphene oxide and the phenolic pre-polymer, adjusting pH, heating and reacting to obtain a graphene oxide-phenolic copolymer;

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

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

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

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

[0015] Further, in step S1, the heating temperature is 30-50℃.

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

[0017] Further, in step S2, the heating temperature is 30-50℃.

[0018] Further, in step S3, the mass ratio of the carboxylated graphene oxide and the phenolic pre-polymer is 1:(20-40).

[0019] Further, in step S3, the heating temperature is 80-100℃.

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

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

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

[0023] The present application first carboxylates the graphene oxide, increases its surface active sites, enables it to better combine with the phenolic pre-polymer, forms the graphene oxide-phenolic copolymer, then loads gold nanoparticles on the copolymer through the photo-deposition method, forms the modified phenolic photocatalyst. On the one hand, the quinone-type-benzene-type units formed by the condensation of the phenolic structure of the catalyst constitute the electronic D-A conjugate pair, combined with the plasmonic resonance effect of the gold nanoparticles, can effectively broaden the light absorption range and enrich the photo-generated electrons, realize the rapid separation of the carriers, significantly improve the NADH regeneration efficiency; on the other hand, the combination of the graphene oxide and the phenolic pre-polymer effectively improves the dispersibility of the graphene oxide, provides abundant interface adsorption sites, effectively improves the loading amount of the gold nanoparticles, thereby improves the NADH regeneration efficiency; in addition, the graphene oxide itself has a certain photocatalytic activity, at the same time, its two-dimensional sheet structure and surface functional groups can optimize the electron transport path inside the catalyst, synergistically acts with the phenolic resin and the gold nanoparticles, further enhances the electron transfer efficiency of the system, thereby synergistically improves the NADH regeneration efficiency, and then realizes the high substrate conversion rate and high light purity preparation of L-tert-leucine, provides an efficient and environmentally friendly solution for industrial production. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the technical solutions of the present application, the following examples are listed. The raw materials, reactions and post-treatment means appearing in the examples are all common raw materials on the market, and technical means familiar to those skilled in the art, unless otherwise stated.

[0025] The words ''preferred'', ''preferably'', ''more preferred'', and the like, as used herein, mean that in certain situations, embodiments of the application can provide certain benefits, however, other embodiments can also provide the same or similar benefits in the same or other situations, and nothing in these terms should be taken to imply that other embodiments are not useful, unless otherwise indicated.

[0026] It should be understood that, except in any operating examples, or otherwise indicated herein and that the use of amounts or all numbers, as in the specification and claims, are understood to be prefaced by the term ''about'', i.e., amounts are understood to be approximate. Numerical parameters are thus specified with the understanding that some flexibility is inherent in the actual device, and further understanding that individual variations are a factor of manufacture and use.

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

[0028] Leucine dehydrogenase: brand 120-04391, purchased from Beijing Biodry Bio-technology Co., Ltd.

[0029] Coenzyme NAD + : beta-NAD, brand N111609, purchased from Shanghai Aldrin 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, reflux at room temperature for 12 h to obtain a red precipitate. Secondly, mix the solution with 0.8 mmol of 2,2'-bipyridine, stir for 1 h to obtain an orange solution, and evaporate naturally in a fume hood to about 3 mL. Then add 10 mL of ether, and after being dried at room temperature for several hours, an orange rhodium-based ligand solid is obtained, which is weighed and collected to prepare a 20 mM rhodium-based ligand solution, which is stored in a refrigerator at 4 DEG C for standby use.

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

[0033] The ''parts'' in the present application all refer to mass parts.

[0034] Example 1

[0035] A process for preparing L-tert-leucine by enzyme method, the process for preparing L-tert-leucine by enzyme method comprises the following steps:

[0036] S1, carboxylated graphene oxide was prepared by mixing graphene oxide, sodium hydroxide and bromoacetic acid (graphene oxide: sodium hydroxide: bromoacetic acid = 1:8:17, m / m / m) in water, uniformly ultrasonic dispersion, reaction at 30℃ for 7h, filtration, washing, drying to obtain carboxylated graphene oxide;

[0037] S2, phenol-formaldehyde prepolymer was prepared by mixing resorcinol and formaldehyde, adding 50wt% sodium hydroxide aqueous solution (resorcinol: formaldehyde: sodium hydroxide = 1:0.9:1, n / n / n), reaction at 30℃ for 45h to obtain phenol-formaldehyde prepolymer;

[0038] S3, graphene oxide-phenol-formaldehyde copolymer was prepared by mixing the carboxylated graphene oxide and phenol-formaldehyde prepolymer (carboxylated graphene oxide: phenol-formaldehyde prepolymer = 1:30, m / m) in water, adjusting pH to 9, reaction at 90℃ for 2h, filtration, washing, drying, grinding to obtain graphene oxide-phenol-formaldehyde copolymer;

[0039] S4, modified phenol-formaldehyde photocatalyst was prepared by mixing the graphene oxide-phenol-formaldehyde copolymer and chloroauric acid (graphene oxide-phenol-formaldehyde copolymer: chloroauric acid = 250:3.5, m / m) in water, irradiation under 300W xenon lamp for 2h under stirring, centrifugation, washing, drying, grinding to obtain modified phenol-formaldehyde photocatalyst;

[0040] S5, 0.5mg of the modified phenol-formaldehyde photocatalyst was dispersed in 3mL phosphate buffer (0.1M NH4Cl, 15wt% triethanolamine) system, rhodium-based ligand solution (final concentration of rhodium-based ligand was 0.375mM) and substrate trimethylpyruvic acid (final concentration was 10mM) were added, then coenzyme NAD + (final concentration was 1mM) and leucine dehydrogenase (7.5μL) were added; irradiation under 300W xenon lamp with 420nm filter for 8h, after the reaction was completed, cationic resin was used for purification, drying to obtain L-tert-leucine.

[0041] Example 2

[0042] A process for preparing L-tert-leucine by enzyme method, the difference between this embodiment and example 1 is that in step S5, the amount of the modified phenol-formaldehyde photocatalyst is 0.4mg, the amount of leucine dehydrogenase is 6μL, and the rest of the steps and amounts are the same as example 1.

[0043] Example 3

[0044] A process for preparing L-tert-leucine by enzyme method, the difference between this embodiment and example 1 is that in step S5, the amount of the modified phenol-formaldehyde photocatalyst is 0.7mg, the amount of leucine dehydrogenase is 15μL, and the rest of the steps and amounts are the same as example 1.

[0045] Comparative Example 1

[0046] A process for preparing L-tert-leucine by enzymatic method, the difference between the present example and example 1 is that step S4 is not carried out, and in step S5, the modified phenolic aldehyde photocatalyst is replaced with graphene oxide phenolic copolymer with the same mass, and the rest of the steps and the amount are the same as example 1.

[0047] Comparative example 2

[0048] A process for preparing L-tert-leucine by enzymatic method, the difference between the present example and example 1 is that step S1 and S3 are not carried out, and in step S4, the graphene oxide phenolic copolymer is replaced with phenolic prepolymer with the same mass, and the rest of the steps and the amount are the same as example 1.

[0049] Test example

[0050] The performance of the processes for preparing L-tert-leucine by enzymatic method of examples 1-3 and comparative examples 1-2 is tested.

[0051] Test method:

[0052] (1) During the reaction process of step S5, samples were taken at 2, 4, 6, and 8 h, respectively, and the substrate conversion rate was tested.

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

[0054] (2) After the product was prepared, it was dissolved in water, filtered with a 0.22μm filter membrane, and then the percentage of enantiomeric excess was determined by high performance liquid chromatography, and a chiral chromatographic column Chirex3126 (D-penicillamine, Phenomenex) was used for detection analysis. The detection conditions are as follows: water / isopropyl alcohol volume ratio=95:5 (containing 2mM copper sulfate) as the mobile phase, the flow rate is 1mL / min during detection, the detection wavelength is 254nm, and the column temperature is 35℃.

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

[0056]

[0057] Wherein, 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 Substrate conversion and percent enantiomeric excess test results

[0060]

[0061] From the above test results, it can be seen that the substrate conversion rate of L-tert-leucine prepared by the present application is more than 42% at 8h, and the e.e.% is more than 99%, which has higher substrate conversion rate and optical purity, and good application prospect.

[0062] It is apparent to those skilled in the art that the present application is not limited to the details of the foregoing exemplary embodiments, and that the present application can be carried out in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, it is to be understood that the embodiments are to be considered in all respects as illustrative and not restrictive, and the scope of the present application is to be determined not by the foregoing description but by the appended claims, and all changes which come within the meaning and range of equivalents of the claims are to be embraced therein.

[0063] Furthermore, it should be understood that although the specification is described in terms of exemplary embodiments, not every implementation embodies only one independent technical solution, and the specification is described in this way only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other implementations that those skilled in the art can understand.

Claims

1. A process for the enzymatic preparation of L-tert-leucine, characterized in that, The method for preparing L-tert-leucine by the enzyme method comprises the following steps: S1, blending graphene oxide, sodium hydroxide and bromoacetic acid, heating and reacting to obtain carboxylated graphene oxide; S2, blending resorcinol, formaldehyde and sodium hydroxide, heating and reacting to obtain a phenolic formaldehyde prepolymer; S3, blending the carboxylated graphene oxide and the phenolic formaldehyde prepolymer, adjusting pH, and heating and reacting to obtain a graphene oxide-phenolic formaldehyde copolymer; S4, blending the graphene oxide-phenolic formaldehyde copolymer with chloroauric acid, and reacting under light conditions to obtain a modified phenolic formaldehyde photocatalyst; S5, dispersing the modified phenolic photocatalyst in a buffer system containing ammonia water and triethanolamine, adding an electron mediator and trimethylpyruvic acid, and then adding leucine dehydrogenase and coenzyme NAD + under light irradiation to obtain L-tert-leucine; The electronic mediator is a rhodium-based ligand. In step S1, the heating temperature is 30-50 DEG C. In step S2, the heating temperature is 30-50 DEG C. In step S3, the heating temperature is 80-100 DEG C.

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

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

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

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

6. The process for the enzymatic preparation of L-tert-leucine according to claim 1, characterized in that, In step S5, the light does not include ultraviolet light.

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