A multi-enzymatic method for the synthesis of hydroxytyrosol

Abstract

The present application relates to a kind of synthetic hydroxytyrosol multi-enzyme catalytic method.The synthetic hydroxytyrosol multi-enzyme catalytic method described in the present application includes first module reaction, second module reaction, third module reaction in turn;The first module reaction is that glycerol is synthesized pyruvic acid by catalysis, wherein the concentration of the glycerol is 1-5mM;The second module reaction is that pyruvic acid is condensed with catechol and ammonium chloride to be L-levodopa by catalysis;Wherein, the concentration of the catechol is 1-5mM, and the concentration of the catechol is greater than the concentration of the glycerol in the first module reaction;The third module reaction is that L-levodopa is synthesized hydroxytyrosol by catalysis.The synthetic hydroxytyrosol multi-enzyme catalytic method described in the present application, reaction 4 hours hydroxytyrosol molar conversion rate can reach 71.5%, compared with one-pot method has obvious advantage, in the same reaction time molar conversion rate is compared with one-pot method and increased nearly 6 times.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, and in particular to a multi-enzyme catalytic method for synthesizing hydroxytyrosol. Background Technology

[0002] Hydroxytyrosol is a bisphenol compound mainly found in olives. It has excellent antioxidant activity and has been proven to be beneficial to human health. It has applications in many areas, including anti-cancer, promoting bone growth and development, preventing and treating diseases such as hypertension, arteriosclerosis, and heart disease, as well as pest and disease control in agriculture.

[0003] Currently, the main methods for synthesizing hydroxytyrosol include extraction, chemical synthesis, microbial fermentation, and whole-cell catalysis. Extraction involves separating and extracting hydroxytyrosol from olive oil, but this process is complex and causes severe environmental pollution. Chemical synthesis faces problems such as expensive substrates and catalysts, and significant pollution. Microbial fermentation uses host bacteria such as *E. coli* to convert glucose into hydroxytyrosol, but due to the long metabolic pathway and high carbon loss, the conversion rate is low. Whole-cell catalysis mainly uses L-tyrosine as a substrate, converting it into hydroxytyrosol through host bacteria such as *E. coli*. Although the conversion rate is improved, the substrate L-tyrosine remains too expensive.

[0004] Currently, the one-pot method is the most common catalytic strategy in in vitro multi-enzyme catalysis, which involves adding all substrates and all enzymes to the reaction system at the same time to carry out multi-enzyme catalysis. However, the one-pot method has many problems. For example, in a multi-enzyme catalytic pathway, the substrate of the enzyme that catalyzes the reaction earlier may inhibit the enzymatic reaction that follows, and the substrate of the enzyme that catalyzes the reaction later may also inhibit the enzymatic reaction that follows earlier. Therefore, in a one-pot reaction system, it is not easy for all enzymes to exert their optimal catalytic effect, which will affect the yield of the target product.

[0005] Another catalytic strategy is to add all enzymes sequentially according to the order of the catalytic reaction. This avoids to some extent the complex interaction between small molecules and enzymes in a one-pot system. However, the time cost of this strategy is much greater than that of the one-pot method. In addition, in multi-enzyme catalytic reactions, some enzyme-catalyzed reactions are reversible. In the sequential addition strategy, the conversion rate of these reversible reactions will be thermodynamically limited and difficult to reach a high level because the products of the preceding enzyme-catalyzed reactions are not consumed immediately.

[0006] Therefore, it is necessary to design appropriate catalytic strategies to achieve optimal synergy among different enzymatic reactions, thereby maximizing the molar conversion of hydroxytyrosol. Summary of the Invention

[0007] Based on this, the purpose of this invention is to provide a multi-enzyme catalytic method for synthesizing hydroxytyrosol, which has many advantages such as low substrate cost, mild reaction conditions, low environmental pollution, fast reaction rate, and high conversion rate.

[0008] A multi-enzyme catalytic method for synthesizing hydroxytyrosol includes a first module reaction, a second module reaction, and a third module reaction performed sequentially.

[0009] The first module reaction involves the catalytic synthesis of pyruvate from glycerol, wherein the concentration of the glycerol is 1-5 mM;

[0010] The second module reaction involves the catalytic condensation of pyruvate with catechol and ammonium chloride to form L-levodopa; wherein the concentration of catechol is 1-5 mM, and the concentration of catechol is greater than the concentration of glycerol in the first module reaction;

[0011] The third module reaction involves the catalytic synthesis of hydroxytyrosol from L-levodopa.

[0012] This invention proposes a modular reaction strategy for the synthesis of hydroxytyrosol from glycerol. Modularization typically uses key metabolites or metabolic branches as nodes. Research in this invention revealed that in the second module reaction, pyruvate undergoes catalytic condensation with catechol and ammonium chloride to form L-L-DOPA. The added catechol affects the catalytic activity of the catalysts in the first and third module reactions of the glycerol-to-hydroxytyrosol pathway. Therefore, this invention separates the reaction catalyzing the synthesis of L-L-DOPA from pyruvate into a separate module. Based on this, the other two modules are the module for the synthesis of pyruvate from glycerol and the module for the synthesis of hydroxytyrosol from L-L-DOPA. In the multi-enzyme catalytic reaction for the synthesis of hydroxytyrosol, the first, second, and third module reactions are executed sequentially, using modules as units. This modular reaction strategy minimizes the influence of catechol on the catalytic activity of the catalysts in the first and third module reactions of the glycerol-to-hydroxytyrosol pathway, ensuring optimal synergy among the enzymatic reactions of different modules and thus improving the conversion rate of hydroxytyrosol.

[0013] The design of the glycerol concentration in the first module reaction being 1-5 mM and the catechol concentration in the second module reaction being 1-5 mM, with the catechol concentration being greater than the glycerol concentration in the first module reaction, is based on the consideration that the second module reaction is a reversible reaction, requiring a higher catechol concentration than the glycerol concentration to promote the reaction. Furthermore, considering the inhibitory effect of catechol on the third module reaction, the catechol concentration should not be too high. Therefore, this invention relates to a catechol concentration of 1-5 mM, with the catechol concentration being greater than the glycerol concentration in the first module reaction.

[0014] The multi-enzyme catalytic method for synthesizing hydroxytyrosol described in this invention differs from previous synthetic methods, particularly in that it employs a novel synthetic route. Furthermore, compared to other methods, the multi-enzyme catalytic method for synthesizing hydroxytyrosol described in this invention offers numerous advantages, including inexpensive substrates, mild reaction conditions, minimal environmental pollution, rapid reaction rate, and high conversion rate.

[0015] Furthermore, the concentration of glycerol in the first module reaction is 2 mM, and the concentration of catechol in the second module reaction is 3 mM. As a preferred embodiment, the concentration of glycerol in the first module reaction is 2 mM, and the concentration of catechol in the second module reaction is 3 mM. This allows catechol to promote the reaction while avoiding the inhibitory effect of the substrate catechol on the third module reaction.

[0016] Furthermore, in the first module reaction, glycerol is first converted to D-glyceric acid by sugar alcohol oxidase, and then the D-glyceric acid is converted to pyruvate by dehydrating enzyme. Glycerol is converted to D-glyceric acid by sugar alcohol oxidase, and D-glyceric acid is converted to pyruvate by dehydrating enzyme.

[0017] Further, in the second module reaction, pyruvate is condensed with catechol and ammonium chloride to L-L-DOPA via tyrosine phenol lyase catalysis. Pyruvate is condensed with catechol and ammonium chloride to L-L-DOPA via tyrosine phenol lyase.

[0018] Furthermore, in the third module reaction, L-L-DOPA is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase, and then the 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase. L-L-DOPA is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase, and 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase.

[0019] Furthermore, during the conversion of 3,4-dihydroxyphenylacetaldehyde to hydroxytyrosol via phenylacetaldehyde reductase catalysis, 5 μM glucose dehydrogenase and 1-5 mM glucose are added to the reaction system for coenzyme regeneration. Coenzyme regeneration maintains the reaction stability of the system.

[0020] Further, the first module reaction involves adding 1-5 mM glycerol, 1-10 mM MgCl2, 1 μL-10 μL of H2O2 enzyme at a concentration of 26.6 mg / mL, 7.5 μM sugar alcohol oxidase, and 5 μM dehydrating enzyme to the reaction system, controlling the total volume to 10 μL, and reacting for 1 hour; wherein, glycerol is converted to D-glyceric acid by sugar alcohol oxidase catalysis, and then D-glyceric acid is converted to pyruvate by dehydrating enzyme catalysis;

[0021] The second module reaction: After the first module reaction was completed, 3 mM catechol, 80 mM ammonium chloride as substrate, 0.2 mM pyridoxal phosphate as cofactor, 2 mM ascorbic acid, and 20 μM tyrosine phenol lyase were added, and the total volume was controlled at 15 μL. The reaction was continued for 1 hour. During this process, pyruvate was converted to L-L-DOPA by tyrosine phenol lyase.

[0022] The third module reaction: After the second module reaction is completed, 0.2-2 mM NADH, 1 μL-10 μL of H2O2 enzyme at a concentration of 26.6 mg / mL, 1 μM 3,4-dihydroxyphenylacetaldehyde synthase, and 5 μM phenylacetaldehyde reductase are added to the reaction system. 5 μM glucose dehydrogenase and 1-5 mM glucose are added to the reaction system for coenzyme regeneration. The total volume is controlled at 100 μL, the reaction temperature is 30℃-40℃, the pH is 6-9, and the reaction continues for 4 hours to obtain the product hydroxytyrosol. Specifically, L-L-DOPA is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase, and then 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase.

[0023] The concentration of substances added in the first module reaction, the second module reaction, and the third module reaction is based on a system volume of 100 μL. The concentration of substances added based on a system volume of 100 μL means adding substances that can make a 100 μL volume reach a specified concentration.

[0024] In the first module reaction, glycerol is converted to D-glyceric acid by sugar alcohol oxidase, and then D-glyceric acid is catalyzed to pyruvate by dehydrating enzyme. In the second module reaction, by adding catechol and other substances after the first module reaction to generate L-L-DOPA, catechol can be rapidly consumed, minimizing its potential oxidative loss and minimizing its inhibitory effect on the third module reaction. In the third module, L-L-DOPA is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase, and then 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase.

[0025] Further, in the first module reaction, the concentration of glycerol is 2 mM, the concentration of MgCl2 is 5 mM, and the amount of commercial H2O2 enzyme added is 2.5 μL; in the second module reaction, the concentration of catechol is 3 mM, the concentration of ammonium chloride is 80 mM, the concentration of pyridoxal phosphate is 0.2 mM, and the concentration of ascorbic acid is 2 mM; in the third module reaction, the concentration of NADH is 0.2 mM, the amount of commercial H2O2 enzyme added is 2.5 μL, the concentration of glucose is 2.5 mM, the reaction temperature is 35℃, the total reaction time is 6 h, the reaction pH is 8.0, and the pH buffer is 250 mM HEPES-NaOH buffer.

[0026] Furthermore, the sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase, and glucose dehydrogenase were all obtained through genetic engineering. By screening for high-activity and high-expression sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase, and glucose dehydrogenase through genetic engineering, the enzymes in the glycerol-to-hydroxytyrosol pathway can achieve the highest catalytic efficiency, ensuring optimal synergy among different enzymatic reactions and thus improving the conversion rate of hydroxytyrosol.

[0027] Furthermore, the method for obtaining the sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase, and glucose dehydrogenase through genetic engineering includes the following steps:

[0028] (1) Construction of gene expression vectors: The genes of sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase and glucose dehydrogenase were subcloned into E. coli expression vectors, resulting in six gene expression vectors respectively.

[0029] (2) Construction of genetically engineered bacteria: The constructed gene expression vectors were transformed into Escherichia coli expression hosts and positive transformants were identified, resulting in six types of genetically engineered bacteria;

[0030] (3) Culture of genetically engineered bacteria: Select positive single colonies of the six constructed genetically engineered bacteria for culture;

[0031] (4) Disruption of genetically engineered bacteria and purification of enzymes: Six kinds of cultured genetically engineered bacteria were separated by centrifugation and ultrasonically disrupted. Crude enzyme solutions were obtained by centrifugation and the target enzymes were purified by nickel ion chromatography to obtain genetically engineered sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase and glucose dehydrogenase.

[0032] To better understand and implement this invention, the following detailed description is provided in conjunction with the accompanying drawings. Attached Figure Description

[0033] Figure 1 The reaction circuit for the synthesis of hydroxytyrosol in Example 2 is shown below;

[0034] Figure 2 The conversion rate-time curve of hydroxytyrosol (molar) in the multi-enzyme catalytic reaction for the synthesis of hydroxytyrosol in the third module reaction of Example 2 is shown.

[0035] Figure 3 Example 2 compares the effects of the modular reaction strategy with the one-pot method. Detailed Implementation

[0036] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the scope of the invention.

[0037] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0038] Preparation of solutions required for the experiment:

[0039] LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, prepared with deionized water, autoclaved, and kept at room temperature for later use.

[0040] TB medium: 12 g / L peptone, 24 g / L yeast extract, 4 mL / L glycerol, containing potassium phosphate buffer.

[0041] Isopropyl β-D-Thiogalactoside solution (IPTG) (1M): Weigh 2.383g of IPTG, dissolve it in deionized water, and after complete dissolution, bring the volume to 10mL. Aliquot the solution into 2mL seed tubes and store at -20℃ for later use.

[0042] Kanamycin solution (50mg / mL): Weigh 0.5g of kanamycin sulfate, dissolve it in deionized water, and after complete dissolution, bring the volume to 10mL. Aliquot the solution into 2mL preservation tubes and store at -20℃ for later use.

[0043] In the following examples, all enzymes used were expressed in *Escherichia coli* BL21(DE3): alditol oxidase (EC: 1.1.3.41, ALDO), dehydratase (DHT), tyrosine phenol lyase (EC: 4.1.99.2, TPL), 3,4-dihydroxyphenylacetaldehyde synthase (4.1.1.107, DHPAAS), phenylacetaldehyde reductase (PAR), and glucose dehydrogenase (1.1.1.47, GDH).

[0044] Example 1

[0045] Construction of gene expression vectors and expression and purification of enzymes

[0046] (1) Constructing gene expression vectors:

[0047] Optimization and synthesis of gene sequences for sugar alcohol oxidase (ALDO), dehydratase (DHT), tyrosine phenol lyase (TPL), 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS), phenylacetaldehyde reductase (PAR), and glucose dehydrogenase (GDH)

[0048] The sugar alcohol oxidase (ALDO) gene (Gene ID: 1101588) from *Streptomyces coelicolor* A3, containing mutations at four sites: V125M, A244T, V133M, and G399R, was obtained from the NCBI database. The dehydratase (DHT) gene from *Paralcaligenes ureilyticus* was obtained from literature. The tyrosine phenol lyase (TPL) gene from *Citrobacterfreundii* (GenBank: SYX30154.1) was obtained from the NCBI database. The 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) gene from *B. mori* (GenBank: XM_004930959.4) was obtained from the NCBI database. The phenylacetaldehyde reductase (PAR) gene from *S. lycopersicum* (NCBI) was obtained from the NCBI database. Reference Sequence: NP_001234823.1); The glucose dehydrogenase (GDH) gene from *S. solfataricus* was obtained from the NCBI database (GenBank: AAK43106.1). All six genes were sent to Suzhou Genewise Biotechnology Co., Ltd. for whole-gene synthesis and subcloning into *E. coli* gene expression vectors. The gene sequence of 3,4-dihydroxyphenylacetaldehyde synthase was subcloned into plasmid pET28a-SUMO to obtain the recombinant plasmid pET28a-SUMO-BmDHPAAS. The gene sequences of sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, phenylacetaldehyde reductase, and glucose dehydrogenase were subcloned into plasmid pET28a to obtain the recombinant plasmids pET28A-ScALDO, pET28A-PuDHT, pET28a-CfTPL, pET28a-SlPAR, and pET28a-SsGDH, respectively.

[0049] (2) Constructing BL21(DE3) genetically engineered bacteria for expressing the enzymes related to step (1).

[0050] (21) Transformation of E. coli competent cells with recombinant plasmid

[0051] The six recombinant plasmids constructed in step (1), namely pET28a-SUMO-BmDHPAAS, pET28A-ScALDO, pET28A-PuDHT, pET28a-CfTPL, pET28a-SlPAR, and pET28a-SsGDH, were transformed into the Escherichia coli expression host BL21(DE3) using the calcium chloride method.

[0052] In a clean bench, BL21(DE3) competent cells were placed on ice for 2 minutes to thaw. The six recombinant plasmids pET28a-SUMO-BmDHPAAS, pET28A-ScALDO, pET28A-PuDHT, pET28a-CfTPL, pET28a-SlPAR, and pET28a-SsGDH were added to the BL21(DE3) competent cells, and gently pipetted to mix them. The cells were then placed on ice for 5 minutes and then spread on LB agar plates containing 50 mg / L kanamycin. The plates were incubated overnight at 37°C.

[0053] (22) Colony PCR identification

[0054] Positive single colonies were picked from the plates in step (21) and preliminarily identified by colony PCR using 2×Utaq PCR MasterMix (Beijing Zhuangmeng International Biotechnology Co., Ltd., catalog number: ZT201A-1). The colony PCR reaction system is shown in Table 1, and the colony PCR amplification reaction procedure is shown in Table 2. After colony PCR amplification, the colony PCR products were spotted into the wells of a 1% agarose gel electrophoresis gel (another well was spotted with a DS 5000 DNA Marker as a control). The gel was separated in the electrophoresis tank (110V) for 30 min. After separation, the agarose gel was immersed in Gelred staining solution for 15 min. The approximate size of the colony PCR product bands was determined by the DS 5000 DNA Marker in the gel electrophoresis apparatus and compared with the expected band size to determine whether the expression strain construction was complete. In this embodiment, positive transformants were identified by colony PCR, and the expression strains BL21(DE3) / pET28a-SUMO-BmDHPAAS, BL21(DE3) / pET28a-ScALDO, BL21(DE3) / pET28a-PuDHT, BL21(DE3) / pET28a-CfTPL, BL21(DE3) / pET28a-SlPAR, and BL21(DE3) / pET28a-SsGDH were obtained.

[0055] Table 1 Colony PCR Reaction System

[0056]

[0057]

[0058] Table 2 Colony PCR Amplification Reaction Procedure

[0059]

[0060] (3) Cultivation of BL21(DE3) genetically engineered bacteria in step (2)

[0061] Select the corresponding positive single colonies, namely BL21(DE3) / pET28a-SUMO-BmDHPAAS, BL21(DE3) / pET28a-ScALDO, BL21(DE3) / pET28a-PuDHT, BL21(DE3) / pET28a-CfTPL, BL21(DE3) / pET28a-SlPAR, and BL21(DE3) / pET28a-SsGDH, and incubate them overnight at 37°C and 250 rpm to obtain seed culture. Inoculate the seed culture into 100 mL of TB medium containing 50 mg / L kanamycin, and control the initial OD of the fermentation medium. 600 The concentration was 0.1, and the mixture was incubated at 37°C and 220 rpm. When the OD value was... 600 When the concentration reaches above 0.6, add 50 μL of 1M IPTG for induction, so that the final concentration of IPTG in the fermentation medium is 0.5 mM. The induction temperature is 16℃, the rotation speed is 180 rpm, and the induction time is 16 h.

[0062] (4) Disruption of genetically engineered bacteria and purification of enzymes

[0063] In step (3), after induction, the bacterial culture is transferred to a 100mL centrifuge tube and centrifuged at 6000r / min at room temperature for 5min. The supernatant is discarded, and the culture is resuspended in sterile water and centrifuged at 6000r / min at room temperature for 5min. The supernatant is then discarded.

[0064] Add 10 mL of buffer A to each of the centrifuged cells and treat them in a Toshiba ultrasonic homogenizer. Sonicate at 35% power for 3 seconds, with a 3-second interval, for a total of 40 minutes. Centrifuge at 10000 r / min at 4℃ for 30 minutes to obtain crude enzyme solution.

[0065] The target enzyme was purified using an AKTA purifier and a nickel ion affinity chromatography column with an imidazole concentration of 0.3M-0.5M. The purified enzyme was then desalted using a desalting column to remove imidazole.

[0066] By disrupting and purifying the genetically engineered bacteria BL21(DE3) / pET28a-SUMO-BmDHPAAS, BL21(DE3) / pET28a-ScALDO, BL21(DE3) / pET28a-PuDHT, BL21(DE3) / pET28a-CfTPL, BL21(DE3) / pET28a-SlPAR, and BL21(DE3) / pET28a-SsGDH, genetically engineered 3,4-dihydroxyphenylacetaldehyde synthase (BmDHPAAS), sugar alcohol oxidase (ScALDO), dehydratase (PuDHT), tyrosine phenol lyase (CfTPL), phenylacetaldehyde reductase (SlPAR), and glucose dehydrogenase (SsGDH) were obtained.

[0067] Example 2 Hydroxytyrosol synthesized by multi-enzyme catalysis using glycerol as a substrate

[0068] Using the genetically engineered sugar alcohol oxidase (ScALDO), dehydratase (PuDHT), tyrosine phenol lyase (CfTPL), phenylacetaldehyde reductase (SlPAR), 3,4-dihydroxyphenylacetaldehyde synthase (BmDHPAAS), and glucose dehydrogenase (SsGDH) obtained in Example 1, hydroxytyrosol was synthesized using glycerol as a substrate through multi-enzyme catalysis.

[0069] Please see Figure 1 Using glycerol as a substrate, glycerol is first converted to D-glyceric acid by sugar alcohol oxidase (ScALDO); then D-glyceric acid is converted to pyruvate by dehydratase (PuDHT); then pyruvate is condensed with catechol and ammonium chloride by tyrosine phenol lyase (CfTPL) to form L-L-DOPA; then L-L-DOPA is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase (BmDHPAAS); then 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase (SlPAR), and glucose and glucose dehydrogenase (SsGDH) are added to the reaction system as coenzyme cycle modules to achieve NADH regeneration.

[0070] We carried out the reaction of glycerol to hydroxytyrosol according to a modular strategy. The concentration of all added substances was based on a system volume of 100 μL, that is, the added substances could achieve the specified concentration in a volume of 100 μL.

[0071] Specifically, the multi-enzyme catalytic method for synthesizing hydroxytyrosol in this embodiment includes the following steps:

[0072] Module 1 reaction: First, add the substrate and enzymes for this module. Specifically, add 2 mM glycerol, 5 mM MgCl2, 2.5 μL of commercially available H2O2 enzyme at a concentration of 26.6 mg / mL, 7.5 μM sugar alcohol oxidase (ScALDO), and 5 μM dehydrating enzyme (PuDHT), maintaining a total volume of 10 μL. React for 1 hour. In this module reaction, glycerol is converted to D-glyceric acid by sugar alcohol oxidase (ScALDO), and D-glyceric acid is converted to pyruvate by dehydrating enzyme (PuDHT); glycerol is completely converted to pyruvate.

[0073] Second module reaction: After the first module reaction, the substrate and enzyme for this module are added. Specifically, 3 mM catechol, 80 mM ammonium chloride as substrate, 0.2 mM pyridoxal phosphate as cofactor, 2 mM ascorbic acid, and 20 μM tyrosine phenol lyase (CfTPL) are added, maintaining a total volume of 15 μL, and the reaction continues for 1 hour. In this module, considering that the reaction is reversible, the catechol concentration needs to be slightly higher than the glycerol concentration to drive the reaction. Also considering the inhibitory effect of the catechol substrate on the subsequent third module reaction, the catechol concentration should not be too high. Therefore, a catechol concentration of 3 mM is chosen for this module reaction.

[0074] The third module reaction: After the second module reaction was completed, the substrate and enzymes for this module were added. Specifically, 0.2 mM NADH, 2.5 μL of commercially available H2O2 enzyme at a concentration of 26.6 mg / mL, 1 μM 3,4-dihydroxyphenylacetaldehyde synthase (BmDHPAAS), and 5 μM phenylacetaldehyde reductase (SlPAR) were added to catalyze the synthesis of hydroxytyrosol. In addition, 5 μM glucose dehydrogenase (SsGDH) and 2.5 mM glucose were added to the reaction system for coenzyme regeneration. The total volume was controlled at 100 μL, 35℃, pH 8.0, and the reaction was continued for 4 h to obtain the product hydroxytyrosol.

[0075] Comparative Example 1

[0076] This comparative example uses a one-pot method, in which all the substrates and enzymes of Example 2 are added to the reaction system at the same time to carry out multi-enzyme catalytic reactions.

[0077] The initial volume of this comparative example is 100 μL, and it is ensured that the amount of substance added is sufficient to achieve the specified concentration in the first, second, and third module reactions of Example 2.

[0078] Specifically, in this comparative example, all substrates and enzymes from the first, second, and third module reactions of Example 2 were introduced into the reaction system simultaneously to carry out multi-enzyme catalytic reactions. All other conditions were the same as in Example 2.

[0079] Please see Figure 2 Within the total 4-hour multi-enzyme catalytic reaction time for the synthesis of hydroxytyrosol from glycerol, specifically within the second hour of the third module reaction, the molar conversion of hydroxytyrosol reached 71.5%, and the substrate glycerol was completely consumed. Please refer to [link to relevant documentation]. Figure 3 In a multi-enzyme catalytic reaction for the synthesis of hydroxytyrosol from glycerol, lasting a total time of 6 hours, the molar conversion rate of hydroxytyrosol achieved by the multi-enzyme catalytic method (modular reaction strategy) of the present invention was 69.1%. Compared to a total time of 4 hours (i.e., the second hour of the third module reaction), the slight decrease in conversion rate (2.4%) may be due to the reverse of the reversible reaction or measurement errors. The molar conversion rate of hydroxytyrosol achieved by the one-pot method was only 12.1%. These results clearly demonstrate that the multi-enzyme catalytic method (modular reaction strategy) for the synthesis of hydroxytyrosol described in this invention is significantly superior to the traditional one-pot reaction strategy, increasing the molar conversion rate by nearly 6 times within the same reaction time.

[0080] The reasons for the significant improvement in conversion rate are mainly as follows: First, by performing the first module reaction before the second module reaction, the influence of catechol in the second module reaction on the first module reaction is avoided. After adding the substrate and enzyme for the second module reaction, tyrosine phenol lyase (CfTPL) condenses catechol, pyruvate, and ammonium chloride into L-L-DOPA. Catechol is rapidly consumed in this module reaction, which largely avoids the inhibitory effect of catechol on the third module reaction in the subsequent execution. Second, the applicant's research found that if catechol is added in the first module reaction, it is easily oxidized in the system of glycerol to pyruvate synthesis in the first module reaction. Even with the addition of ascorbic acid as an antioxidant, this side reaction seems difficult to avoid. Therefore, in the one-pot system, the added catechol does not have time to react with pyruvate and is spontaneously lost to a certain extent, which also reduces the conversion rate of the system to some extent. The multi-enzyme catalytic method for synthesizing hydroxytyrosol described in this invention, namely a modular reaction strategy, converts glycerol to pyruvate in the first module reaction before executing the second module reaction. This rapidly consumes catechol and minimizes its potential oxidative loss. Furthermore, in a one-pot reaction, all substrates and enzymes are added at once, resulting in a system complexity far greater than that of a modular sequential reaction. The complex mutual interference within this system may, to some extent, limit the reaction rate of the one-pot method.

[0081] This invention designs a multi-enzyme catalytic pathway for the synthesis of hydroxytyrosol from glycerol and proposes a modular reaction strategy. This strategy executes the reactions of the three modules sequentially as a unit, and its conversion rate and reaction rate are much higher than the one-pot reaction strategy that adds all enzymes and substrates at the same time.

[0082] Compared with existing technologies, the multi-enzyme catalytic method for synthesizing hydroxytyrosol described in this invention has many advantages, such as low substrate cost, mild reaction conditions, low environmental pollution, fast reaction rate, and high conversion rate.

[0083] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments and features of the embodiments of the present invention can be combined with each other unless otherwise specified. The above-described embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and the present invention also intends to include these modifications and variations.

Claims

1. A multi-enzyme catalytic method for synthesizing hydroxytyrosol, characterized in that: This includes the first module reaction, the second module reaction, and the third module reaction, which are carried out sequentially. The first module reaction involves the catalytic synthesis of pyruvate from glycerol, wherein the concentration of glycerol is 2 mM; in the first module reaction, glycerol is first converted to D-glyceric acid by sugar alcohol oxidase, and then D-glyceric acid is converted to pyruvate by dehydrating enzyme. The second module reaction involves the condensation of pyruvate with catechol and ammonium chloride to L-L-DOPA via catalysis; wherein the concentration of catechol is 3 mM; in the second module reaction, pyruvate is condensed with catechol and ammonium chloride to L-L-DOPA via tyrosine phenol lyase catalysis. The third module reaction involves the catalytic synthesis of hydroxytyrosol from L-levodopa. In this reaction, L-levodopa is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase, and then the 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase. During the conversion of 3,4-dihydroxyphenylacetaldehyde to hydroxytyrosol by phenylacetaldehyde reductase, 5 μM glucose dehydrogenase and 1-5 mM glucose are added to the reaction system for coenzyme regeneration.

2. The multi-enzyme catalytic method for synthesizing hydroxytyrosol according to claim 1, characterized in that: The first module reaction: 2 mM glycerol, 1-10 mM MgCl2, 1 μL-10 μL of H2O2 enzyme at a concentration of 26.6 mg / mL, 7.5 μM sugar alcohol oxidase, and 5 μM dehydrating enzyme were added to the reaction system, and the total volume was controlled at 10 μL. The reaction was carried out for 1 hour. Glycerol was converted to D-glyceric acid by sugar alcohol oxidase, and then D-glyceric acid was converted to pyruvate by dehydrating enzyme. The second module reaction: After the first module reaction was completed, 3 mM catechol, 80 mM ammonium chloride as substrate, 0.2 mM pyridoxal phosphate as cofactor, 2 mM ascorbic acid, and 20 μM tyrosine phenol lyase were added, and the total volume was controlled at 15 μL. The reaction was continued for 1 hour. During this process, pyruvate was converted to L-L-DOPA by tyrosine phenol lyase. The third module reaction: After the second module reaction is completed, 0.2-2 mM NADH, 1 μL-10 μL of H2O2 enzyme at a concentration of 26.6 mg / mL, 1 μM 3,4-dihydroxyphenylacetaldehyde synthase, and 5 μM phenylacetaldehyde reductase are added to the reaction system. 5 μM glucose dehydrogenase and 1-5 mM glucose are added to the reaction system for coenzyme regeneration. The total volume is controlled at 100 μL, the reaction temperature is 30℃-40℃, the pH is 6-9, and the reaction continues for 4 hours to obtain the product hydroxytyrosol. Specifically, L-L-DOPA is converted to 3,4-dihydroxyphenylacetaldehyde by 3,4-dihydroxyphenylacetaldehyde synthase, and then 3,4-dihydroxyphenylacetaldehyde is converted to hydroxytyrosol by phenylacetaldehyde reductase. The concentration of substances added in the first module reaction, the second module reaction, and the third module reaction is based on a system volume of 100 μL. The concentration of substances added based on a system volume of 100 μL means adding substances that can make a 100 μL volume reach a specified concentration.

3. The multi-enzyme catalytic method for synthesizing hydroxytyrosol according to claim 2, characterized in that: In the first module reaction, the concentration of glycerol was 2 mM, the concentration of MgCl2 was 5 mM, and the amount of H2O2 enzyme added was 2.5 μL. In the second module reaction, the concentration of catechol was 3 mM, the concentration of ammonium chloride was 80 mM, the concentration of pyridoxal phosphate was 0.2 mM, and the concentration of ascorbic acid was 2 mM. In the third module reaction, the concentration of NADH was 0.2 mM, the amount of H2O2 enzyme added was 2.5 μL, the concentration of glucose was 2.5 mM, the reaction temperature was 35℃, the total reaction time was 6 h, the reaction pH was 8.0, and the pH buffer was 250 mM HEPES-NaOH buffer.

4. The multi-enzyme catalytic method for synthesizing hydroxytyrosol according to claim 3, characterized in that: The sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase, and glucose dehydrogenase were all obtained through genetic engineering.

5. The multi-enzyme catalytic method for synthesizing hydroxytyrosol according to claim 4, characterized in that: The method for obtaining the sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase, and glucose dehydrogenase through genetic engineering includes the following steps: (1) Constructing gene expression vectors: The genes of sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase and glucose dehydrogenase were subcloned into E. coli expression vectors, resulting in six gene expression vectors respectively. (2) Construction of genetically engineered bacteria: The constructed gene expression vectors were transformed into Escherichia coli expression hosts and positive transformants were identified, resulting in six genetically engineered bacteria; (3) Cultivating genetically engineered bacteria: Select positive single colonies of the six constructed genetically engineered bacteria for cultivation; (4) Disruption of genetically engineered bacteria and purification of enzymes: Six kinds of cultured genetically engineered bacteria were separated by centrifugation and ultrasonically disrupted. Crude enzyme solutions were obtained by centrifugation and the target enzymes were purified by nickel ion chromatography to obtain genetically engineered sugar alcohol oxidase, dehydratase, tyrosine phenol lyase, 3,4-dihydroxyphenylacetaldehyde synthase, phenylacetaldehyde reductase and glucose dehydrogenase.

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