A method for preparing retinamide glutamic acid by biological enzyme

Abstract

The present application relates to the technical field of cosmetics, and discloses a method for preparing retinamide glutamic acid by using a biological enzyme, which comprises the following steps: performing an enzyme catalysis reaction on retinol and glutamic acid in the presence of a biological enzyme to prepare retinamide glutamic acid; wherein the biological enzyme is a lipase from Bacillus laevolacticus; and the enzyme catalysis reaction is performed at a pH of 7.0-9.0 and a temperature of 25-40 DEG C for 1-3 h. Bacillus velezensis The present application successfully synthesizes retinamide glutamic acid by using the lipase from Bacillus laevolacticus as a biological enzyme to catalyze the reaction of retinol and glutamic acid; and high-yield production and high conversion rate of the retinamide glutamic acid are realized by optimizing the reaction conditions. Bacillus velezensis The retinamide glutamic acid prepared by the present application has good anti-saccharification effect and can be widely applied to cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a method for preparing retinamide glutamic acid using a bio-enzymatic process. Background Technology

[0002] Retinol is a class of low-molecular-weight compounds with a unique chemical structure that can act on the skin's epidermis. After being converted into retinoic acid by enzymes on the skin surface, it can diminish signs of skin aging, promote skin cell proliferation, thicken the epidermis, and thus form a stronger skin barrier. Furthermore, the use of retinol can improve skin appearance and enhance skin elasticity, making it an undeniably advantageous ingredient in the anti-aging field. However, retinol and retinoic acid have drawbacks such as high irritation and poor chemical stability, limiting their application. To address these issues, retinol or retinoic acid can be synthesized into retinamide derivatives. These retinamide derivatives release retinoic acid and other amide-like active ingredients upon application, reducing irritation during application and providing a synergistic effect.

[0003] Currently, most retinamide compounds are synthesized using traditional chemical synthesis methods. These methods require high temperatures and strong acid / base catalysis, resulting in problems such as numerous byproducts, low product purity, and environmental pollution. Furthermore, they can easily lead to the isomerization and inactivation of retinoic acid. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing retinamide glutamic acid using a bio-enzymatic approach.

[0005] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing retinamide glutamic acid by a biological enzymatic method, comprising the following steps: In the presence of biological enzymes, retinol and glutamic acid are subjected to an enzyme-catalyzed reaction to obtain retinamide glutamic acid. The bio-enzyme is derived from Bacillus reizei. Bacillus velezensis Lipase; The enzyme-catalyzed reaction was carried out for 1 to 3 hours at a pH of 7.0–9.0 and a temperature of 25–40 °C.

[0006] As a preferred embodiment, the amino acid sequence of the bioenzyme is shown in SEQ ID NO.1.

[0007] As a preferred embodiment, the bio-enzyme is prepared by the following method: A1. The gene sequence of the synthesized biological enzyme is constructed on a vector, and the constructed plasmid is transformed into Escherichia coli to obtain recombinant bacteria; A2. After inducing expression of recombinant bacteria, the obtained bacterial cell precipitate is broken and centrifuged, and the resulting supernatant is the crude enzyme solution containing biological enzymes.

[0008] As a preferred embodiment, in step A1, the vector is pET-28a(+) and the Escherichia coli is BL21(ED3).

[0009] As a preferred embodiment, in step A2, the step of inducing expression of the recombinant bacteria is as follows: when the recombinant bacteria are cultured until the bacterial cell concentration OD600 reaches 0.7-0.8, an inducing agent is added for induction culture; The inducing agent was isopropyl-β-D-thiopyranoside, and the induction culture temperature was 24–26 °C, and the culture time was 10–15 h.

[0010] As a preferred embodiment, the final concentration of the added bio-enzyme is 0.05–0.4 g / L. More preferably, the final concentration of the added bio-enzyme is 0.15–0.3 g / L, and most preferably, the final concentration of the added bio-enzyme is 0.2 g / L.

[0011] As a preferred embodiment, the final concentration of retinol added is 20-50 mM, and the final concentration of glutamic acid added is 20-45 mM. More preferably, the final concentration of retinol added is 20-40 mM, and the final concentration of glutamic acid added is 20-40 mM. Further preferably, the final concentration of retinol added is 20 mM or 30-40 mM, and the final concentration of glutamic acid added is 20-40 mM. Even more preferably, the final concentration of retinol added is 30 mM, and the final concentration of glutamic acid is 20-40 mM, or the final concentration of retinol added is 35 mM, and the final concentration of glutamic acid is 30 mM.

[0012] As the optimal solution, the final concentration of retinol added is 30 mM and the final concentration of glutamic acid added is 25 mM.

[0013] As a preferred embodiment, the enzyme-catalyzed reaction is carried out under conditions of pH 7.5–8.5 and temperature 35–40 °C.

[0014] As the most preferred embodiment, the enzyme-catalyzed reaction is carried out at a pH of 8.5 and a temperature of 35 °C.

[0015] As a preferred embodiment, the pH is adjusted by adding at least one of a buffer solution, an acid, or a base.

[0016] As a preferred embodiment, the buffer solution is a phosphate buffer or a Tris-HCl buffer with a pH of 6.8 to 7.8; The acid is hydrochloric acid; The alkali is sodium hydroxide.

[0017] Secondly, the present invention provides a retinamide glutamic acid prepared according to the aforementioned method.

[0018] Thirdly, the present invention provides a retinamide glutamic acid prepared according to the aforementioned method or the application of the retinamide glutamic acid prepared according to the aforementioned method in the preparation of cosmetics.

[0019] As a preferred embodiment, the cosmetic product is one with anti-glycation and anti-aging effects.

[0020] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention utilizes Bacillus leucovora. Bacillus velezensis The lipase, as a biological enzyme, catalyzes the reaction of retinol and glutamic acid to successfully synthesize retinamide glutamic acid (the carboxyl terminus of retinoic acid is linked to the α-amino group of glutamic acid to form an amide bond, thus obtaining retinamide glutamic acid). The entire preparation process is characterized by good substrate stability, high enzyme conversion rate, and simple and convenient operation.

[0021] 2) The bio-enzyme used in this invention has high activity and good specificity; through the bio-enzymatic method and further optimization of reaction conditions, high yield and high conversion rate of retinamide glutamic acid were achieved, with a maximum yield of 9.6 g / L and a maximum conversion rate of 92%.

[0022] 3) Compared with chemical synthesis, the present invention has the advantages of short reaction time, simple process, few impurities, easy product purification and no environmental pollution, and has a very good prospect for industrial production.

[0023] 4) The retinamide glutamic acid prepared by this invention has a good anti-glycation effect and can be widely used in cosmetics. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0025] In a specific embodiment of the present invention, a method for preparing retinamide glutamic acid by a biological enzymatic method is provided, comprising the following steps: In the presence of biological enzymes, retinol and glutamic acid are subjected to an enzyme-catalyzed reaction to obtain retinamide glutamic acid. The bio-enzyme is derived from Bacillus reizei. Bacillus velezensis Lipase; The enzyme-catalyzed reaction was carried out for 1 to 3 hours at a pH of 7.0–9.0 and a temperature of 25–40 °C.

[0026] Retinamide glutamic acid can be prepared under the above reaction conditions.

[0027] All biological materials, raw materials, and reagents used in the experiments of this invention were provided by our company and / or purchased commercially. For example, the plasmid pET-28a(+) in the examples was purchased from Qingke Biotechnology Co., Ltd., the Escherichia coli competent cells BL21 (ED3) were purchased from Beyotime Biotechnology Co., Ltd., kanamycin (K+) was purchased from Sigma-Aldrich, isopropyl-β-D-thiogalactopyranoside was purchased from Sigma-Aldrich, retinol was purchased from Sigma-Aldrich, glutamate was purchased from Sigma-Aldrich, and sodium phosphate buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0028] The biological enzyme used in this invention is lipase from Bacillus velezensis, and its amino acid sequence is shown in SEQ ID NO.1.

[0029] SEQ ID NO.1: mpkgkeitvsdvtdkeyyrlsqaaynykmlalhmkyktpykisktsywyvekvkqdsdtgldafvfskgikskngkwiksknpenvvvafagtdigkdpindgvkadggnivfgn dpktevhyivkkdakdtsktlgtyhgtpsqdamlttgkyklitktsqidqadqlvrgvkkkyagtstiisttghslggaeaeysavnnnvyavafnspsvvklhdeetqkeinsgiydpy irsiinpddmvgagywneydrhngttiytknpslsqfsrnqrldgklseqigknlayffttvilrnpdthglneanfvfdrngtianpngdelvfdknlgallpagaigsgdaikvtptn akklaekvqamaedlrtmkkeaqnayqehdekiaelktefygqvghglfdqlqaqdvtnsiediaqsydkgpifydtqaeqafidslqaaitdleeiggflhkiadefqekdqmlanwlr Example 1 This embodiment provides a method for preparing a biological enzyme, including the following steps: 1. GenScript was commissioned to synthesize the lipase gene sequence of Bacillus velezensis using conventional methods and construct it in the pET-28a(+) vector. The gene sequence was optimized according to the codon preference of Escherichia coli. The restriction enzyme sites of the plasmid were NdeI and XhoI. The prepared plasmid was named pET-28a(+)-BVLIPs.

[0030] 2. The plasmid pET-28a(+)-BVLIPs was transformed into *E. coli* BL21(ED3) to obtain positive clones. These clones were plated onto kanamycin (K+) resistant plates and incubated overnight at 37 ℃ with the plates inverted. A single clone was picked and incubated overnight at 37 ℃ and 220 rpm in 5 mL of LB medium containing 50 mg / mL K+. 1 mL of this incubation was then transferred to 100 mL of LB medium containing 50 mg / mL K+ and incubated overnight at 37 ℃ and 220 rpm to prepare the seed culture. The LB medium formulation was as follows: Tryptone 10 g / L; Yeast extract 5 g / L; Sodium chloride (NaCl) 10 g / L; the pH of the medium was adjusted to 7.4 with NaOH. The above-mentioned resistance plates are solid LB medium plates. Add 1.5g of agar powder to 100ml of LB medium, autoclave, and add kanamycin (K+) when the medium temperature drops to 55℃. Seal the edges with sealing glue to obtain the plate.

[0031] 3. Take 10 mL of seed culture and put it into 1 L of LB medium containing 50 mg / mL K+, and incubate at 37℃ and 220 r / min.

[0032] 4. When the bacterial cell concentration OD600 reaches 0.7-0.8, add isopropyl-β-D-thiopyranogalactoside (final concentration 0.1 mM), continue culturing at 25 ℃ for 12 h, collect the bacterial solution, centrifuge at 4 ℃, 6793×g for 3 min to obtain the bacterial cell precipitate.

[0033] 5. At 4 ℃, the collected bacterial cell precipitate was resuspended in sodium phosphate buffer (100 mM, pH 8.0), sonicated, and then centrifuged at 10012×g for 30 min at 4 ℃ to remove cell debris and undisturbed cells. The resulting supernatant was the crude enzyme solution containing Bacillus velezensis lipase. The concentration of the bio-enzyme in the crude enzyme solution was detected using a BCA kit (Beyotime), and the content was approximately 4 g / L.

[0034] Example 2 This embodiment provides a method for preparing retinamide glutamic acid using a bio-enzymatic method, including the following steps: 1. Preparation of diluted crude enzyme solutions with different enzyme concentrations: The crude enzyme solution obtained in Example 1 was added to deionized water to prepare diluted crude enzyme solutions with enzyme contents of 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L and 3 g / L respectively. 2. Construction of the enzyme catalytic system: In a 1L enzyme bioreactor (total tank volume 2L), add 100 ml of the diluted crude enzyme solution prepared in step 1, 50 ml of 100 mM sodium phosphate buffer (pH 8.0), 20 mM retinol, and 20 mM glutamic acid, and then add water to bring the volume to 1L to construct an enzyme catalytic system with different enzyme contents (the final concentrations of the biological enzymes in the enzyme catalytic system are 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, and 0.3 g / L, respectively). Adjust the pH of the enzyme catalytic system to 8.5 using sodium hydroxide or hydrochloric acid. 3. Preparation of retinamide glutamic acid: The reaction vessels containing each enzyme-catalyzed system were placed at 35 °C for 1.5 h. Samples were taken every 30 min for HPLC analysis to determine the amount of retinamide glutamic acid produced. Once the maximum production of retinamide glutamic acid was reached, the reaction was terminated by boiling in water for 5 min. The retinol content in the system after the reaction was determined by HPLC, and the conversion rate of retinamide glutamic acid was calculated. The conversion rate was calculated as: 100% - (molar concentration of remaining retinol in the system after the reaction / initial molar concentration of retinol added before the reaction)%.

[0035] The results showed that at final enzyme concentrations of 0.05 g / L, 0.1 g / L, 0.15 g / L, 0.2 g / L, and 0.3 g / L, the highest yields of retinamide glutamate were 3.1 g / L, 3.4 g / L, 6.7 g / L, 9.1 g / L, and 8.9 g / L, respectively, with corresponding conversion rates of 32%, 39%, 67%, 86%, and 84%. Therefore, 0.15–0.3 g / L is the optimal concentration for the enzymatic reaction, with 0.2 g / L being the most suitable concentration.

[0036] Example 3 This embodiment provides a method for preparing retinamide glutamic acid using a bio-enzymatic method, including the following steps: 1. Constructing the enzyme catalytic system: In a 1L enzyme reactor (total tank volume 2L), add 100ml of diluted crude enzyme solution with a concentration of 2 g / L prepared in Example 2, 50ml of 100 mM sodium phosphate buffer with pH 8.0, add water to make up to 1L, and add retinol and glutamate with a final concentration of 20 mM to construct the enzyme catalytic system. Adjust the pH of the enzyme catalytic system to 8.5 with sodium hydroxide or hydrochloric acid. 2. Preparation of retinamide glutamic acid: The reaction vessels containing each enzyme-catalyzed system were placed at different temperatures (25 ℃, 30 ℃, 35 ℃, and 40 ℃) for 1.5 h. Samples were taken every 30 min for HPLC analysis to determine the amount of retinamide glutamic acid produced. Once the maximum production of retinamide glutamic acid was reached, the reaction was terminated by boiling in water for 5 min. The retinol content in the system after the reaction was determined by HPLC, and the conversion rate of retinamide glutamic acid was calculated.

[0037] The results showed that the highest production amounts of retinamide glutamic acid were 4.2 g / L, 8.6 g / L, 9.1 g / L, and 8.1 g / L at reaction temperatures of 25 ℃, 30 ℃, 35 ℃, and 40 ℃, respectively, with corresponding conversion rates of 78%, 79%, 86%, and 76%. The results indicate that the highest production amount and conversion rate of retinamide glutamic acid were achieved at a reaction temperature of 35 ℃.

[0038] Example 4 This embodiment provides a method for preparing retinamide glutamic acid using a bio-enzymatic method, including the following steps: 1. Constructing the enzyme catalytic system: In a 1L enzyme reactor (total tank volume 2L), add 100 ml of diluted crude enzyme solution with a concentration of 2 g / L prepared in Example 2, 50 ml of 100 mM sodium phosphate buffer with a pH of 8.0, 20 mM retinol, and 20 mM glutamic acid, and add water to make up to 1L. Adjust the pH of the enzyme catalytic system to 7.0, 7.5, 8.0, and 8.5 with sodium hydroxide or hydrochloric acid, respectively. 2. Preparation of retinamide glutamic acid: The reaction vessels of the enzyme catalytic systems at various pH values ​​were placed at 35 °C for 1.5 h. Samples were taken every 30 min, and the amount of retinamide glutamic acid produced was detected by HPLC. Once the maximum production of retinamide glutamic acid was reached, the reaction was terminated by boiling in water for 5 min. The content of retinol in the system after the reaction was detected by HPLC, and the conversion rate of retinamide glutamic acid was calculated.

[0039] The results showed that the highest production amounts of retinamide glutamate were 5 g / L, 8.2 g / L, 8.5 g / L, and 9.1 g / L at pH values ​​of 7.0, 7.5, 8.0, and 8.5, respectively, with corresponding conversion rates of 63%, 79%, 82%, and 86%. These results indicate that the highest production and conversion rate of retinamide glutamate were achieved at pH 8.5 of the enzyme-catalyzed system.

[0040] Example 5 This embodiment provides a method for preparing retinamide glutamic acid using a bio-enzymatic method, including the following steps: 1. Construction of enzyme catalytic system: In a 1 L enzyme reactor (total tank volume 2 L), 100 ml of diluted crude enzyme solution with an enzyme concentration of 2 g / L prepared in Example 2, 50 ml of 100 mM sodium phosphate buffer at pH 8.0, different final concentrations of retinol (20 mM, 25 mM, 30 mM, 35 mM and 40 mM), and different final concentrations of glutamate (20 mM, 25 mM, 30 mM, 35 mM and 40 mM) were added sequentially to construct enzyme catalytic system groups 1-25 with pH 8.5; as shown in Table 1.

[0041] 2. Preparation of retinamide glutamic acid: The reaction vessels of each enzyme catalytic system were placed at 35 °C for 1.5 h. Samples were taken every 30 min for HPLC analysis to detect the amount of retinamide glutamic acid produced. The reaction was terminated by boiling in water for 5 min after the maximum production of retinamide glutamic acid was reached. The content of retinol in the system after the reaction was detected by HPLC, and the conversion rate of retinamide glutamic acid was calculated. The results are shown in Table 1.

[0042] Table 1

[0043] The results showed that under different concentration gradients, the conversion rates were all above 85% when the final concentrations of retinol were 20 mM or 30–40 mM and glutamate were 20–40 mM (groups 1–5, 11–25). When the final concentrations of retinol were 30 mM and glutamate were 20–40 mM or 35 mM and glutamate were 30 mM, the conversion rates were all above 90% (groups 11–15, 18). The highest conversion rate of 92% and the highest amount of retinamide glutamate production (9.6 g / L) were achieved when the retinol concentration was 30 mM and the glutamate concentration was 25 mM. Therefore, the optimal final substrate concentrations were: 30 mM retinol and 25 mM glutamate.

[0044] Verification Example: Verification of the Anti-aging Efficacy of Retinamide Glutamate MGO is an intermediate product of glycation reactions, which can react with amino and thiol groups on protein side chains to generate AGEs, the final products of the Maillard reaction. During skin aging, on the one hand, AGEs directly act on RAGE receptors on the cell surface, activating the NF-κB signaling pathway and oxidative stress, leading to decreased cell function and inducing skin aging, wrinkles, and other problems; on the other hand, AGEs cross-link with proteins in the extracellular matrix, causing the skin to lose elasticity.

[0045] In this embodiment, MGO was used to induce glycation in cells, and then different concentrations of retinamide glutamate were applied. The anti-aging effect of retinamide glutamate was evaluated by detecting the content of AGEs.

[0046] The reaction product prepared under the conditions of 30 mM retinol and 25 mM glutamic acid in Example 5 was spray-dried after the reaction was terminated to obtain a powdered retinamide glutamic acid sample.

[0047] Test subjects: 10 mg / mL bovine collagen and porcine elastin (prepared with sterile water), with a final reaction concentration of 5 mg / mL.

[0048] Inducing agent: 1 M MGO, final reaction concentration 0.1 M.

[0049] Test samples: The powdered retinamide glutamic acid sample obtained in the above steps was added to water to prepare test samples with final reaction concentrations of 1.28%, 0.64%, 0.32%, 0.16%, 0.08%, 0.04%, 0.02%, and 0.01%.

[0050] Positive control samples: 0.016%, 0.008%, 0.004%, 0.002%, and 0.001% aminoguanidine aqueous solutions.

[0051] Competitive control samples: N-acetylhydroxyproline (prepared with water to concentrations of 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 1%, and 5%), Salvia miltiorrhiza extract (prepared with water to concentrations of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, and 25%), and egg yolk choline (prepared with water to concentrations of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, and 25%).

[0052] Test method: 1) Prepare 100 μL reaction systems: The model group includes 50 μL of 10 mg / mL bovine collagen or porcine elastin, 25 μL of 1 M GO, and 25 μL of PBS solution; the protein control group includes 50 μL of 10 mg / mL bovine collagen or porcine elastin and 50 μL of PBS solution; each well of the sample group contains 25 μL of test samples or different concentrations of competitor control samples or positive control samples at different concentrations (1.28%, 0.64%, 0.32%, 0.16%, 0.08%, 0.04%, 0.02%, 0.01%), as well as 25 μL of 1 M GO and 50 μL of 10 mg / mL bovine collagen or porcine elastin; each well of the sample control group contains 25 μL of 1 M GO and 50 μL of 10 mg / mL bovine collagen or porcine elastin. Test samples of different concentrations (1.28%, 0.64%, 0.32%, 0.16%, 0.08%, 0.04%, 0.02%, 0.01%) or control samples of different concentrations of competing products or positive control samples of different concentrations and 75 μL of PBS solution.

[0053] 2) Using black 96-well plates, add 100 μL of each reaction system to each well according to the group, with 3-4 replicates per group. Detect fluorescence values ​​after 3 days of reaction. AGEs have characteristic absorption peaks at excitation / emission at 360 / 446 nm; and the fluorescent cross-linked AGEs—pentoside—have characteristic absorption peaks at 335 / 385 nm.

[0054] Data calculation: The inhibition rate of each sample group was calculated using formula (1): Inhibition rate = 1 - (fluorescent value sample group - fluorescent value sample control group) / (fluorescent value model group - fluorescent value protein control group) (1) After using Graphpad Prism to plot the dose-response relationship, the IC50 of each group was obtained. The test results of each sample obtained by the experimental method are shown in Table 2 below (all results are the average values ​​of repeated experimental data).

[0055] Table 2

[0056] As can be seen from the results in Table 2, the retinamide glutamic acid prepared in this invention can inhibit protein glycation reaction, significantly reduce the fluorescence values ​​of AGEs and pentoglycosides, and inhibit the formation of AGEs, thus exhibiting a good anti-glycation effect.

[0057] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. A method for preparing retinamide glutamic acid via a bio-enzymatic process, characterized in that, Includes the following steps: In the presence of biological enzymes, retinol and glutamic acid are subjected to an enzyme-catalyzed reaction to obtain retinamide glutamic acid. The bio-enzyme is derived from Bacillus reesei. Bacillus velezensis Lipase; The enzyme-catalyzed reaction was carried out for 1 to 3 hours at a pH of 7.0–9.0 and a temperature of 25–40 °C.

2. The method for preparing retinamide glutamic acid by bio-enzymatic method according to claim 1, characterized in that, The amino acid sequence of the bioenzyme is shown in SEQ ID NO.

1.

3. The method for preparing retinamide glutamic acid by bio-enzymatic method according to claim 2, characterized in that, The bioenzyme is prepared by the following method: A1. The gene sequence of the synthesized biological enzyme is constructed on a vector, and the constructed plasmid is transformed into Escherichia coli to obtain recombinant bacteria; A2. After inducing expression of recombinant bacteria, the obtained bacterial cell precipitate is broken and centrifuged, and the resulting supernatant is the crude enzyme solution containing biological enzymes.

4. The method for preparing retinamide glutamic acid by bio-enzymatic method according to claim 1, characterized in that, The final concentration of the added bio-enzyme is 0.05–0.4 g / L.

5. The method for preparing retinamide glutamic acid by bio-enzymatic method according to claim 1, characterized in that, The final concentration of retinol added is 20–50 mM, and the final concentration of glutamic acid added is 20–45 mM.

6. The method for preparing retinamide glutamic acid by bioenzymatic method according to claim 1, 4, or 5, characterized in that, The final concentration of the added bio-enzyme is 0.15–0.3 g / L; The final concentration of retinol added is 20-40 mM, and the final concentration of glutamic acid added is 20-40 mM. The enzyme-catalyzed reaction was carried out at a pH of 7.5–8.5 and a temperature of 35–40 °C.

7. The method for preparing retinamide glutamic acid by bio-enzymatic method according to claim 1, characterized in that, The pH is adjusted by adding at least one of a buffer solution, an acid, or a base.

8. The method for preparing retinamide glutamic acid by bio-enzymatic method according to claim 7, characterized in that, The buffer solution is a phosphate buffer or Tris-HCl buffer with a pH of 6.8 to 7.8; The acid is hydrochloric acid; The alkali is sodium hydroxide.

9. A retinamide glutamic acid prepared by the method according to any one of claims 1-8.

10. The use of retinamide glutamic acid prepared according to any one of claims 1-8 or the retinamide glutamic acid according to claim 9 in the preparation of cosmetics.

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