A method for synthesizing optically active (R)-2-hydroxy-4-phenylbutyric acid using multi-enzyme catalysis in a microfluidic field

Abstract

The present invention discloses a method for synthesizing optically active ( R )-2-hydroxy-4-phenylbutyric acid, and established a three-step enzymatic reaction process based on continuous flow for the first time. The present invention uses cheap benzaldehyde and pyruvate as raw materials, and efficiently synthesizes ( R )-HPB. By applying the immobilized enzymes to three independent flow modules, the present invention overcomes the common problems of chemical imbalance and incompatible reaction conditions in multi-enzyme cascades. The present invention is for industrial production of ( R )‑HPB provides an efficient and economical technical solution and expands the application field of enzyme-catalyzed synthesis of high value-added compounds.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and in particular relates to a method for synthesizing optically active (R)-2-hydroxy-4-phenylbutyric acid by using multi-enzyme catalysis in a microfluidic field. Background Art

[0002] Angiotensin converting enzyme (ACE), also known as kininase II or peptidyl dipeptidase A (EC 3.4.15.1), plays a key role in cardiovascular regulation by promoting the production of vasoconstrictor angiotensin II and inactivating vasodilator bradykinin. ACE inhibitors lower blood pressure by blocking the production of angiotensin II. These inhibitors are commonly referred to as "pril" drugs, including benazepril, enalapril, quinapril and ramipril, which are widely used in the treatment of hypertension and congestive heart failure. Among them, ACE inhibitors account for about 3% of the total sales of antihypertensive drugs. Therefore, it is necessary to develop an economical and environmentally friendly synthetic ( R )-HPBE, a chiral precursor in the production of a variety of "pril" drugs, has attracted widespread attention in the research community.

[0003] In recent years, in order to produce ( R )-HPBE, a variety of organic synthesis methods have been developed, including multi-step chemical synthesis and chemical asymmetric reduction. However, some common problems still exist, including cumbersome procedures for substrate synthesis, low overall yield, poor enantioselectivity and high chemical waste. In contrast, enzymes, as green and mild catalysts, provide high chemical selectivity, regioselectivity and stereoselectivity, providing another option for numerous asymmetric chemical syntheses. They have been widely used in the synthesis of optically pure drugs or drug building blocks. At present, the synthesis of ( R The main enzymatic methods for the kinetic resolution of racemic 2-hydroxy-4-phenylbutyrate (HPB) catalyzed by lipases and the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBA) catalyzed by dehydrogenases / ketoreductases. In these methods, the asymmetric reduction of OPBA to ( R )-HPB has received the most attention. However, the limiting factor of this method is the high price of the compound OPBA, which makes it unsuitable for industrial production. Therefore, it is necessary to develop an efficient, economical and sustainable method to obtain ( R )-HPB is still very urgent. Summary of the invention

[0004] Purpose of the invention: In view of the shortcomings of the prior art, the present invention has established a method for synthesizing optically active (R)-2-hydroxy-4-phenylbutyric acid using multi-enzyme catalysis in a microfluidic field. The method is a three-step enzymatic reaction process based on continuous flow. The process uses cheap benzaldehyde and pyruvate as raw materials, and through the three-stage immobilized enzyme catalysis of aldolase, ene reductase and ketoreductase, it can efficiently synthesize optically pure (R)-2-hydroxy-4-phenylbutyric acid in a continuous flow. R )-2-hydroxy-4-phenylbutyric acid (( R )-HPB).

[0005] Specifically, the present application provides a method for synthesizing optically active (R)-2-hydroxy-4-phenylbutyric acid using multi-enzyme catalysis in a microfluidic field, comprising the following steps:

[0006] Step S1: using resin to fix aldolase, ene reductase and ketoreductase in the pipelines of the first reactor, the second reactor and the third reactor respectively, and optimizing the conditions for each reactor separately according to the substrate concentration of benzaldehyde to be reacted to obtain a suitable reaction flow rate so that the corresponding substrate in each reactor can react completely;

[0007] Specifically, in the first reactor, benzaldehyde is used as a substrate, and aldolase catalyzes benzaldehyde (BzH) and pyruvate through an Aldol reaction to form 2-oxo-4-phenylbut-3-enoic acid (OPEBA). In the second reactor, OPEBA is used as a substrate, and ene reductase catalyzes the asymmetric reduction of the C=C double bond in OPEBA to convert it into 2-oxo-4-phenylbutyric acid (OPBA), with NADH as a necessary cofactor. In the third reactor, OPBA is used as a substrate and is converted into ( R )-2-Hydroxy-4-phenylbutyric acid (( R )-HPB), NADPH as a cofactor; after the device and the concentration of the substrate to be reacted are determined, the optimal flow rate is optimized so that for each reactor, the substrate is completely reacted;

[0008] In some embodiments, in the first reactor, the dosage of benzaldehyde (BzH) and pyruvic acid is 10-50 mM and 20-100 mM, respectively; in the second reactor, the dosage of 2-oxo-4-phenylbut-3-enoic acid (OPEBA) and NAD + The dosages of 2-oxo-4-phenylbutyric acid (OPBA), NADP + , D-glucose dosages were 10~50 mM, 0.5~2 mM, and 20~100 mM, respectively;

[0009] Step S2: Add benzaldehyde, pyruvic acid, NADP + 、NAD + The mixed solution of D-glucose is introduced into the first reactor by a syringe pump to achieve a continuous flow reaction;

[0010] Wherein, the aldolase is derived from Pseudomonas putida NahE, the ene reductase is derived from Escherichia coli str. EcQOR of K-12; the ketoreductase is derived from Candida glabrata Specifically, the amino acid sequence of the aldolase is shown in SEQ ID NO.1, the amino acid sequence of the ene reductase is shown in SEQ ID NO.2, and the amino acid sequence of the ketoreductase is shown in SEQ ID NO.3.

[0011] The aldolase is prepared by the following method: constructing the aldolase NahE gene with a gene sequence as shown in SEQ ID NO.4 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding aldolase NahE; the aldolase is fixed in the pipeline of the first reactor by the following method: dispersing the cultured and purified NahE in a phosphate buffer soaked with a functionalized epoxy resin, slowly shaking at 16-20°C for 4-8 hours, washing with phosphate buffer for several times, draining with a suction funnel to obtain an immobilized resin, and filling the drained immobilized resin into the pipeline of the first microreactor.

[0012] The ene reductase is prepared by the following method: constructing an ene reductase EcQOR gene with a gene sequence as shown in SEQ ID NO.5 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding ene reductase EcQOR; the ene reductase is immobilized in a pipe of a second reactor by the following method: dispersing the ene reductase EcQOR obtained by culturing and purification in a phosphate buffer soaked with a functionalized epoxy resin, slowly shaking at 16-20° C. for 4-8 h, washing with phosphate buffer for several times, draining with a suction funnel to obtain an immobilized resin, and filling the drained immobilized resin into the pipe of the second reactor.

[0013] The ketoreductase is prepared by the following method: constructing a ketoreductase CgKR2 gene with a gene sequence as shown in SEQ ID NO.6 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding ketoreductase CgKR2; the ketoreductase is immobilized in a pipeline of a third reactor by the following method: dispersing the cultured and purified CgKR2 in a phosphate buffer soaked with a functionalized epoxy resin, slowly shaking at 16-20° C. for 4-8 h, washing with phosphate buffer for several times, draining with a suction funnel to obtain an immobilized resin, and filling the drained immobilized resin into a microchannel pipeline of the third reactor.

[0014] In step S1, the resin is any one of the hydrophobic epoxy resins ES-1, ES-103B, ES-108, LX-1000EP, NKA and ESD-1. Preferably, the aldolase is fixed by ES-103B epoxy resin, the ene reductase is fixed by ES-1 resin, and the ketoreductase is fixed by LX-1000EP resin.

[0015] In step S1, the ratio of the amount of resin to any one of aldolase, ene reductase and ketoreductase is 400-1400 mg: 20-70 mg; during the immobilization of the enzyme, the buffer ion concentration is 10-100 mM, the pH is 6-7.5; the immobilization time is 5-10 hours. Preferably, 2-4 mg of enzyme corresponds to each 100 mg of resin. Preferably, NahE achieves the best immobilization effect at an ion concentration of 100 mM, EcQOR performs best at an ion concentration of 10 mM, and CgKR2 performs excellently at an ion concentration of 20 mM; preferably, the pH during the immobilization of the three enzymes is 7; preferably, the immobilization time of NahE enzyme is 6 hours, and the immobilization time of EcQOR and CgKR2 is 8 hours.

[0016] The resin needs to be activated before it is put into use. The activation steps are as follows: First, 1~2 g of epoxy resin is suspended in 10~20 mL of modification solution (containing 0.1 M sodium borate and 2 M iminodiacetic acid). The mixture is then placed in a constant temperature shaker and gently incubated at 180 r / min for 2~6 h at 20~30°C. Subsequently, the mixture is filtered using a suction funnel to separate the epoxy resin and the modification solution. The resin is then thoroughly rinsed with deionized water, repeated three times, to ensure that the residual modification solution is removed. In the next step, the filtered epoxy resin is incubated with 10~20 mL of 0.5 M sulfuric acid solution at the same temperature and stirring conditions for 2~6 h. This step is intended to eliminate residual epoxy groups, thereby preventing unexpected reactions in future steps. After the incubation is completed, the resin is collected by filtration and rinsed three times with deionized water to remove residual acidic substances. After the acid treatment, the epoxy resin was further treated with 10-20 mL of 0.1 M sodium periodate solution at 20-30°C and 180 r / min for 2-6 h. This step introduces the key functional group aldehyde by promoting the oxidation of the epoxy resin. After incubation, the resin was filtered and rinsed three times with deionized water to remove the unreacted sodium periodate. Subsequently, the treated epoxy resin was mixed with 10-20 mL of metal modification solution and gently incubated for 2-6 h at 20-30°C and 180 r / min. This key step aims to introduce metal ions into the resin structure and enhance its affinity for binding to proteins. After the incubation, the resin was filtered and rinsed three times with deionized water to remove unbound metal ions. In the final step, the epoxy resin was incubated with 10-20 mL of desorption solution at 20-30°C and 180 r / min for 2-6 h to remove loosely bound metal ions and ensure that only tightly attached metal ions remain. The resin was then filtered, rinsed three times with deionized water, and dried using a pump to remove any residual moisture. The treated epoxy resin was then transferred to an appropriate container and stored at 4°C until further use.

[0017] Specifically, in step S2, the reaction solvent of the mixed solution is a mixture of PBS buffer and an organic solvent, and the organic solvent is any one or more of n-heptane, isooctane, methanol, ethanol, isopropanol, acetonitrile, and dimethyl sulfoxide; wherein the volume of the organic solvent is less than or equal to 10% of the total volume of the reaction solvent.

[0018] The reaction temperature of the first reactor, the second reactor and the third reactor is 20-30°C, the flow rate is 0.05-0.2 mL / min, and the reaction temperature of the first reactor, the second reactor and the third reactor is 20-30°C. + 、NAD +The molar ratio of D-glucose is: 10~50: 20~100: 0.5~2: 0.5~2: 20~100.

[0019] In some embodiments, after optimization, in step S2, benzaldehyde, pyruvic acid, NADP + 、NAD + and D-glucose were used in amounts of 10~50 mM, 20~100 mM, 0.5~2 mM, 0.5~2 mM, and 20~100 mM, respectively.

[0020] Among them, the first reactor is a packed bed reactor with a diameter of 1~3 cm and a length of 2~5 cm; the second reactor is a packed bed reactor with a diameter of 1~3 cm and a length of 2~5 cm; the third reactor is a packed bed reactor with an inner diameter of 1~3 cm and a length of 2~5 cm.

[0021] The present invention utilizes three consecutive enzyme-catalyzed steps to synthesize ( R )-HPB method, first, aldolase catalyzes the aldol reaction of benzaldehyde (BzH) and pyruvate to form 2-oxo-4-phenylbut-3-enoic acid (OPEBA), then ene reductase catalyzes the asymmetric reduction of the C=C double bond in OPEBA to convert it into 2-oxo-4-phenylbutyric acid (OPBA), with NADH as an essential cofactor, and then OPBA is converted into ( R )-2-Hydroxy-4-phenylbutyric acid (( R )-HPB), and NADPH as a cofactor. The immobilized enzyme modules in the three steps were connected together through a double pass and operated continuously.

[0022] This innovative synthesis strategy not only significantly reduces the cost of raw materials, but also improves the greenness of the synthesis process and the purity of the product, demonstrating its great potential in the pharmaceutical industry.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0024] (1) The present invention innovatively designs a multi-enzyme cascade reaction system, using cheap and readily available benzaldehyde as the starting material, and synthesizes the target product ( R )-HPB, this innovative synthesis strategy not only significantly reduces the cost of raw materials, but also improves the greenness of the synthesis process and the purity of the product, demonstrating its great potential in the pharmaceutical industry;

[0025] (2) By immobilizing the enzyme on a suitable carrier and optimizing the immobilization process, this study not only significantly improved the stability and reusability of the enzyme, but also greatly simplified the post-processing steps by integrating the reaction and biocatalyst separation steps into a single operation unit, paving the way for industrial production ( R )-HPB provides an efficient and economical technical solution, while expanding the application field of enzyme-catalyzed synthesis of high value-added compounds;

[0026] (3) In the present invention, the immobilized enzymes NahE@ES-103B, EcQOR-GDH@ES-1, and CgKR2-GDH@ES-103B were applied to a continuous flow reaction system for the first time, and each module was optimized to achieve ( R By combining the two technologies of immobilized enzyme and flow chemistry, we solved the stability and compatibility problems in the multi-enzyme reaction system designed in this study and realized the continuous flow synthesis of the whole process. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the reaction scheme of the present invention;

[0028] Figure 2 It is a schematic flow diagram of the reaction device of the present invention. DETAILED DESCRIPTION

[0029] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0030] The present invention establishes a three-step enzymatic reaction process based on continuous flow for the first time, using cheap benzaldehyde and pyruvate as raw materials, through the three-stage immobilized enzyme catalytic reaction of aldolase, ene reductase and ketoreductase, to efficiently synthesize optically active ( R )-2-hydroxy-4-phenylbutyric acid (( R )-HPB), synthesized using three consecutive enzymatic steps ( R )-HPB method, the reaction flow chart is as follows Figure 1 shown.

[0031] like Figure 2 As shown, the microchannel reaction device described in the following embodiment includes a 50 mL syringe connected by a pipeline, three microchannel reactors connected in series by a two-way connector, and a product collector. The pipelines in the three microchannel reaction devices are all polytetrafluoroethylene tubes with a length of 2-5 cm and a diameter of 1-3 cm.

[0032] The steps for constructing NahE described in the following examples are as follows: (1) Pseudomonas putidaThe NahE gene (the gene sequence is shown in SEQ ID NO.1) was constructed into the pET-28a (+) vector (the restriction endonuclease site is Ndel and HindIII ), and transferred into Escherichia coli BL 21 (DE 3); (2) Take 50 μL of the NahE glycerol strain constructed in step (1) and inoculate it into 5 mL of LB medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for 12 h to obtain a pre-cultured bacterial liquid. Transfer 5 mL of the pre-cultured bacterial liquid to 500 mL of TB (containing 100 mL of PBS) medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for about 4 h until the OD reaches 0. 600 When the pH reaches 0.6-0.8 and the culture medium temperature is cooled to 4°C, IPTG is added to a final concentration of 50 mM to induce expression, and the culture is continued at 16°C with shaking for 22 h. The culture solution is concentrated by centrifugation, the bacteria are collected, and the bacteria are suspended in the buffer solution with PBS buffer on a mixer to prepare a 0.2 g / mL whole cell solution. (3) The whole cell solution is broken with an ultrasonic cell crusher. The whole cell lysate is obtained at a power of 200 W, ultrasound on for 2 s, ultrasound off for 2 s, and ice water bath for 25 min. (4) After the cell lysate is fully centrifuged and filtered, the supernatant is taken and pumped into the pre-equilibrated Ni-NTA resin column by a peristaltic pump. After the supernatant is loaded, the resin is gradient eluted with 60 mL of lysis buffer with imidazole concentrations of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. After the impurities are eluted by ultraviolet detection analysis, the Ni-NTA is rinsed with lysis buffer containing 250 mM imidazole to collect the pure enzyme. The collected pure enzyme contains a high concentration of imidazole. The imidazole is eluted by ultrafiltration centrifugation using an ultrafiltration tube until the imidazole concentration is less than 10 mM, and then the pure enzyme is collected.

[0033] The steps for constructing the EcQOR of the olefin reductase described in the following example are as follows: (1) Escherichia coli str. The EcQOR gene of K-12 (the gene sequence is shown in SEQ ID NO. 2) was constructed into the pET-28a (+) vector (the restriction endonuclease site is Ndel and HindIII), and transferred into Escherichia coli BL 21 (DE 3); (2) Take 50 μL of the EcQOR glycerol strain constructed in step (1) and inoculate it into 5 mL of LB medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for 12 h to obtain a pre-cultured bacterial liquid. Transfer 5 mL of the pre-cultured bacterial liquid to 500 mL of TB (containing 100 mL PBS) medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for about 4 h until the OD 600 When the pH reaches 0.6-0.8 and the culture medium temperature is cooled to 4°C, IPTG is added to a final concentration of 50 mM to induce expression, and the culture is continued at 16°C with shaking for 22 h. The culture solution is concentrated by centrifugation, the bacteria are collected, and the bacteria are suspended in the buffer solution with PBS buffer on a mixer to prepare a 0.2 g / mL whole cell solution. (3) The whole cell solution is broken with an ultrasonic cell crusher. The whole cell lysate is obtained at a power of 200 W, ultrasound on for 2 s, ultrasound off for 2 s, and ice water bath for 25 min. (4) After the cell lysate is fully centrifuged and filtered, the supernatant is taken and pumped into the pre-equilibrated Ni-NTA resin column by a peristaltic pump. After the supernatant is loaded, the resin is gradient eluted with 60 mL of lysis buffer with imidazole concentrations of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. After the impurities are eluted by ultraviolet detection analysis, the Ni-NTA is rinsed with lysis buffer containing 250 mM imidazole to collect the pure enzyme. The collected pure enzyme contains a high concentration of imidazole. The imidazole is eluted by ultrafiltration centrifugation using an ultrafiltration tube until the imidazole concentration is less than 10 mM, and then the pure enzyme is collected.

[0034] The steps for constructing the ketoreductase CgKR2 described in the following example are as follows: (1) Candida glabrata The CgKR2 gene (the gene sequence is shown in SEQ ID NO.2) was constructed into the pET-28a (+) vector (the restriction endonuclease site is Ndel and HindIII ), and transferred into Escherichia coli BL 21 (DE 3); (2) Take 50 μL of the EcQOR glycerol strain constructed in step (1) and inoculate it into 5 mL of LB medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for 12 h to obtain a pre-cultured bacterial liquid. Transfer 5 mL of the pre-cultured bacterial liquid to 500 mL of TB (containing 100 mL PBS) medium, and add kanamycin with a final concentration of 100 μg / mL, and shake culture at 37°C and 200 rpm for about 4 h until the OD 600When the pH reaches 0.6-0.8 and the culture medium temperature is cooled to 4°C, IPTG is added to a final concentration of 50 mM to induce expression, and the culture is continued at 16°C with shaking for 22 h. The culture solution is concentrated by centrifugation, the bacteria are collected, and the bacteria are suspended in the buffer solution with PBS buffer on a mixer to prepare a 0.2 g / mL whole cell solution. (3) The whole cell solution is broken with an ultrasonic cell crusher. The whole cell lysate is obtained at a power of 200 W, ultrasound on for 2 s, ultrasound off for 2 s, and ice water bath for 25 min. (4) After the cell lysate is fully centrifuged and filtered, the supernatant is taken and pumped into the pre-equilibrated Ni-NTA resin column by a peristaltic pump. After the supernatant is loaded, the resin is gradient eluted with 60 mL of lysis buffer with imidazole concentrations of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM, respectively. After the impurities are eluted by ultraviolet detection analysis, the Ni-NTA is rinsed with lysis buffer containing 250 mM imidazole to collect the pure enzyme. The collected pure enzyme contains a high concentration of imidazole. The imidazole is eluted by ultrafiltration centrifugation using an ultrafiltration tube until the imidazole concentration is less than 10 mM, and then the pure enzyme is collected.

[0035] In the following examples, when the enzyme is immobilized, the resin needs to be activated before it is put into use. The activation steps are as follows: First, 1 g of epoxy resin is suspended in 10 mL of a modification solution (containing 0.1 M sodium borate and 2 M iminodiacetic acid). The mixture is then placed in a constant temperature shaker and gently incubated at 180 r / min for 4 h at 20°C. Subsequently, the mixture is filtered using a suction funnel to separate the epoxy resin and the modification solution. The resin is then rinsed thoroughly with deionized water, repeated three times, to ensure that the residual modification solution is removed. In the next step, the filtered epoxy resin is incubated with 10 mL of 0.5 M sulfuric acid solution for 4 h at the same temperature and stirring conditions. This step is intended to eliminate residual epoxy groups, thereby preventing unexpected reactions in future steps. After the incubation is completed, the resin is collected by filtration and rinsed three times with deionized water to remove residual acidic substances. After the acid treatment, the epoxy resin was further treated with 10 mL of 0.1 M sodium periodate solution at 20°C and 180 r / min for 2-6 h. This step introduces the key functional group aldehyde by promoting the oxidation of the epoxy resin. After incubation, the resin was filtered and rinsed three times with deionized water to remove the unreacted sodium periodate. Subsequently, the treated epoxy resin was mixed with 10-20 mL of metal modification solution and gently incubated for 4 h at 20°C and 180 r / min. This key step aims to introduce metal ions into the resin structure and enhance its affinity for binding to proteins. After the incubation, the resin was filtered and rinsed three times with deionized water to remove unbound metal ions. In the final step, the epoxy resin was incubated with 10 mL of desorption solution at 20°C and 180 r / min for 4 h to remove loosely bound metal ions and ensure that only tightly attached metal ions remain. The resin was then filtered, rinsed three times with deionized water, and dried using a pump to remove any residual moisture. The treated epoxy resin was then transferred to an appropriate container and stored at 4°C until further use.

[0036] The sources of the enzymes used in the following examples are as follows:

[0037] 1. Aldolase

[0038] 1) NahE, derived from Pseudomonas putida(JA LeVieux et al., Structural characterization of the hydratase-aldolases, NahE and PhdJ: Implications for the specificity, Biochem. 57(25) (2018) 3524-3536);

[0039] 2) MvPhdJ, from Mycobacterium vanbaalenii (JA LeVieux et al., Structural characterization of the hydratase-aldolases, NahE and PhdJ:Implications for the specificity, Biochem. 57(25) (2018) 3524-3536);

[0040] 3) SaKDG, derived from Stygiolobus azoricus (S. Wolterink-van Loo et al., Biochemical and structural exploration of the catalytic capacity of sulfolobus KDG aldolases, Biochem. J. 403(3) (2007) 421-430);

[0041] 4) BxBph1, derived from Burkholderia xenovorans LB400 (P. Baker et al., Rational Design of Stereoselectivity in the Class II pyruvate aldolase BphI, J. Am. Chem. Soc. 134(1) (2011) 507-513);

[0042] 5) Yfau-An, derived from Escherichia coli K-12 (K. Hernández et al., Nucleophilepromiscuity of engineered class II pyruvate aldolase YfaU from E. Coli , Angew.Chem. 130(14) (2018) 3645-3649);

[0043] (II) Oleoreductase

[0044] 1) EcQOR, derived from Escherichia coli str. K-12 (J.M. Thorn et al., Crystal structure of escherichia coli QOR quinone oxidoreductase complexed with NADPH, J. Mol. Biol. 249(4) (1995) 785-799);

[0045] 2) EcCurA, derived from Escherichia coli str. K-12 (P.A. Hubbard et al., The crystal structure and reaction mechanism of Escherichia coli 2,4-Dienoyl-CoA reductase, J. Biol. Chem. 278(39) (2003) 37553-37560);

[0046] 3) OYE1, derived from Saccharomyces pastorianus (N. Kress et al., Enantioselective reduction of citral isomers in NCR ene reductase: analysis of an active‐Site mutant library, ChemBioChem 18(8) (2017) 717-720);

[0047] 4) NCR, derived from Zymomonas mobilis (N. Kress et al., Enantioselective reduction of citral isomers in NCR ene reductase: analysis of an active‐Site mutant library, ChemBioChem 18(8) (2017) 717-720);

[0048] 5) TsER, derived from Thermus scotoductusSA-01 (N. Nett et al., A robust and stereocomplementary panel of ene-reductase variants for gram-scale asymmetric hydrogenation, Mol. Catal. 502 (2021));

[0049] (III) Keto reductase

[0050] 1) CgKR2, derived from Candida glabrata (N.D. Shen et al., Efficient synthesis of a chiral precursor for angiotensin-converting enzyme (ACE) inhibitors in high space-time yield by a new reductase without external cofactors, Org. Lett. 14(8) (2012) 1982-5);

[0051] 2) RasADH, derived from Ralstonia sp (H. Man et al., structures of alcohol dehydrogenases from ralstonia and sphingobium spp. reveal the molecular basis for their recognition of ‘Bulky–Bulky’ ketones, Top. Catal. 57(5) (2013) 356-365);

[0052] 3) LsADH, derived from Leifsonia sp. strain S749. (K. Inoue et al., Purification and characterization of a novel alcohol dehydrogenase from Leifsonia sp. Strain S749: A promising biocatalyst for an asymmetric hydrogen transfer bioreduction, Appl. Environ. Microbiol. 71(7) (2005) 3633-3641)

[0053] 4) CgkR1, derived from Candida glabrata. (G.-W. Zheng et al., Preparation ofstructurally diverse chiral alcohols by engineering ketoreductase CgKR1,ACSCatal. 7(10) (2017) 7174-7181)

[0054] 5) LkADH, derived from Lactobacillus kefir (C.V. Voss et al.,Deracemization ofsecondary alcohols through a concurrent tandem biocatalytic oxidation andreduction,Angew. Chem. Int. Ed. 47(4) (2008) 741-5);

[0055] 6) LbADH, derived from Lactobacillus brevis (P. Both et al.,Whole-cellbiocatalysts for stereoselective C-H amination reactions,Angew. Chem. Int.Ed. 55(4) (2016) 1511-3);

[0056] 7) TbsADH, derived from Thermoanaerobacter brockii (Z. Sun et al., Exploringthe substrate scope of mutants derived from the robust alcohol dehydrogenaseTbSADH,Tetrahedron Lett. 57(32) (2016) 3648-3651);

[0057] 8) HLADH, derived from Horse liver (L. Huang et al.,Horse liver alcoholdehydrogenase-catalyzed oxidative lactamization of amino alcohols,ACS Catal.8(9) (2018) 8680-8684);

[0058] 9) KtCR, derived from Kluyveromyces thermotolerans(G.-C. Xu et al.,Access tooptically active aryl halohydrins using a substrate-tolerant carbonylreductase discovered from Kluyveromyces thermotolerans , ACS Catal. 2(12)(2012) 2566-2571).

[0059] Example 1 Screening of the most suitable enzyme for the first step reaction.

[0060] In the first step, aldolase was selected to catalyze the aldehyde condensation reaction between benzaldehyde and pyruvate. Aldolase was overexpressed in Escherichia coli using the pET28a(+) vector and purified to obtain pure enzyme. Each 1 mL reaction system contained 50 mM sodium phosphate buffer, 50 mM benzaldehyde, 100 mM pyruvate, and 5 μM purified enzyme, and the pH was adjusted to 7.5. These systems were incubated at 30°C and shaken at 1500 rpm for durations ranging from 2 h to 24 h. It is worth noting that Yfau-An and BxBph1 belong to class II aldolases, which require metal ions as cofactors for their activity. Therefore, 1 mM Ni was added to the corresponding reaction systems. 2+ and Mn 2+ , to promote the activity of the enzyme. The results in Table 1 show that Pseudomonas putida The NahE enzyme and Stygiolobus azoricus All SaKDG enzymes can catalyze benzaldehyde and pyruvate to completely convert them into OPBEA, and the catalytic reaction time of NahE enzyme is significantly shorter than that of SaKDG enzyme. Therefore, NahE is selected as the best catalyst for CC bond formation in the cascade reaction.

[0061] Table 1 Summary of different aldolases for the synthesis of 2-oxo-4-phenylbut-3-enoic acid

[0062]

[0063] Example 2 Screening of the most suitable enzyme for the second step reaction.

[0064] In this experiment, in order to avoid the interference of olefin reductase contained in E. coli itself, olefin reductase was overexpressed in E. coli through pET28a(+) vector and purified to obtain pure enzyme. These enzymes were used to catalyze the conversion of OPEBA to OPBA. The 1 mL screening system of the reaction included 50 mM sodium phosphate buffer, 50 mM OPEBA, 0.5 mM NAD +, 1 U / mL of GDH (glucose dehydrogenase), and 100 mM of D-glucose. In addition, 5 μM of each purified enzyme was added to the mixture. The reaction was carried out at pH 7.5, shaking at 1500 rpm at 30°C, and the duration ranged from 2 h to 24 h. After analyzing the catalytic results of these enzyme candidates, EcQOR performed well, achieving complete reduction of OPBEA (Table 2, entry 1) and significantly exceeding other enzymes in efficiency. Therefore, EcQOR was selected as the best catalyst for the key step of C=C bond reduction in the cascade reaction.

[0065] Table 2 Summary of different ene reductases in the synthesis of 2-oxo-4-phenylbutyrate

[0066]

[0067] Example 3 Screening of the most suitable enzyme for the third step reaction.

[0068] In this study, 10 ketoreductases / alcohol dehydrogenases stored in the laboratory were overexpressed in E. coli using the pET28a(+) vector and purified to obtain pure enzymes. The total volume of the screening system was 1 mL, containing 50 mM sodium phosphate buffer, 50 mM OPBA, 0.5 mM NADP + , 1 U / mL of GDH, and 100 mM of D-glucose. In addition, 5 μM of each purified enzyme was added to the mixture. The reaction conditions were set at pH 7.5 and shaken at 1500 rpm at 30°C for durations ranging from 2 h to 24 h. The results showed that the Candida albicans Candida glabrata The obtained CgkR2 exhibited excellent conversion (≥99%) and outstanding enantioselectivity (99.9%) (Table 3, entry 1). Therefore, CgkR2 was confirmed to be an ideal candidate for the construction of chiral alcohols in the cascade reaction.

[0069] Table 3 Different ketoreductases / alcohol dehydrogenases in the synthesis of ( R Summary of )-2-Hydroxy-4-phenylbutyric acid

[0070]

[0071] Example 4

[0072] In this application, seven different functionalized epoxy resins were carefully evaluated to determine the best support material for the immobilization of three enzymes: NahE, EcQOR, and CgKR2.

[0073] First, for NahE, we mixed 15 mg of purified NahE with 20 mM sodium phosphate buffer, pH 8.0, in a 5 mL centrifuge tube to a final volume of 4 mL, and then vortexed thoroughly to ensure that the enzyme and buffer were mixed evenly. A 50 μL sample was taken from the mixture and the initial concentration before immobilization was determined using the BCA protein quantification method. Subsequently, the enzyme-buffer mixture was mixed with 300 mg of pre-weighed modified epoxy resin support and vortexed again to ensure uniform mixing. The mixture was stirred at 180 r / min at 20°C for 8 h to promote adequate immobilization. After immobilization, the mixture was allowed to stand for 10 min, and a 50 μL sample of the supernatant was taken to determine the concentration after immobilization using the BCA method. Next, the resin was filtered using a Buchner funnel and rinsed three times with 10 mM sodium phosphate buffer to remove any unbound proteins, and finally a vacuum pump was used to remove residual water to clean and dry the resin. The fixed epoxy resin was transferred to a 5 mL PE tube and stored at 4 °C until further use. The protein adsorption rate and enzyme activity recovery rate were determined according to equations (1) and (2), respectively.

[0074] (1)

[0075] (2)

[0076] Secondly, for EcQOR, we mixed 8 mg of purified EcQOR with 20 mM sodium phosphate buffer at pH 8.0 in a 5 mL centrifuge tube to ensure that the final volume of the mixture was 4 mL, and then vortexed it thoroughly to ensure that the enzyme and the buffer were mixed evenly. The subsequent immobilization procedure was the same as that of NahE. The protein adsorption rate and enzyme activity recovery rate were determined according to equations (1) and (2), respectively.

[0077] Finally, for CgKR2, we mixed 15 mg of purified CgKR2 with 4 mL of 20 mM pH 8.0 sodium phosphate buffer in a 5 mL centrifuge tube to ensure that the final volume of the mixture was 4 mL, and then vortexed it thoroughly to ensure that the enzyme and the buffer were mixed evenly. The subsequent immobilization procedure was the same as that of NahE and EcQOR. The protein adsorption rate and enzyme activity recovery rate were determined according to equations (1) and (2), respectively.

[0078] Table 4 Screening of the best resin for immobilizing NahE

[0079]

[0080] Table 5 Screening of the best resin for immobilizing EcQOR

[0081]

[0082] Table 6 Screening of the best resin for immobilizing CgKR2

[0083]

[0084] As shown in the results in Tables 4-6, based on the two key indicators of protein adsorption rate and enzyme activity recovery rate, we found that ES-103B epoxy resin performed best in terms of enzyme activity recovery rate and protein adsorption rate, and was identified as the best carrier for NahE immobilization; the screening results showed that ES-108 and HP-2MG exhibited the highest protein adsorption rate. However, the enzyme activity recovery efficiency of these two resins was not ideal. Considering that the recovery activity of the immobilized enzyme is a key parameter, ES-1 resin was selected as the immobilization carrier of EcQOR due to its excellent recovery activity; by adding purified CgKR2 to the modified epoxy resin carrier and comprehensively evaluating the protein adsorption rate and enzyme activity recovery rate, LX-1000EP resin was identified as the best choice for CgKR2 immobilization.

[0085] Example 5 Effect of enzyme amount on immobilization.

[0086] Before the experiment, 20 mM sodium phosphate buffer was prepared and the pH was adjusted to 8.0. First, 100 mg of epoxy resin (ES-103B, ES-1, LX-1000EP) was weighed. Next, solutions containing different concentrations of enzymes were prepared by dissolving 1, 2, 3, 4, 5, and 6 mg of enzymes (NahE, EcQOR, CgKR2) in 20 mM sodium phosphate buffer at pH 8.0, respectively, ensuring that the final mixed solution volume was 4 mL. Then, epoxy resin was added to each 5 mL centrifuge tube containing the corresponding enzyme solution. These tubes were placed in a shaking incubator and shaken at 20°C and 180 r / min for 8 h to promote the binding of enzymes to the resin. After the immobilization process was completed, the immobilized resin and the solution were separated by centrifugation. The immobilized resin was then washed three times with 10 mM sodium phosphate buffer to remove unbound enzymes. Finally, the immobilized resin was stored at 4°C for future use. The protein adsorption rate and enzyme activity recovery rate were determined according to equations (1) and (2), respectively.

[0087] Table 7 Screening for the optimal enzyme amount for NahE fixation

[0088]

[0089] Table 8 Screening for the optimal enzyme amount for immobilization of EcQOR

[0090]

[0091] Table 9 Screening the optimal enzyme amount for fixing CgKR2

[0092]

[0093] As shown in Tables 7 to 9, when 4 mg NahE was fixed per 100 mg ES-103B carrier, the enzyme activity recovery rate and protein adsorption rate reached the optimal value, and when 2 mg EcQOR was fixed per 100 mg ES-1 carrier, the enzyme activity recovery rate and protein adsorption rate reached the maximum value, which were 93.5% and 98.3%, respectively; when 4 mg CgKR2 was fixed per 100 mg LX-1000EP, the enzyme activity recovery rate and protein adsorption rate reached the optimal value.

[0094] Example 6 Effect of buffer ion concentration on enzyme immobilization.

[0095] In this experiment, we aimed to immobilize three enzymes, NahE (4 mg), EcQOR (2 mg), and CgKR2 (4 mg), onto 100 mg of epoxy-functionalized resin. Different concentrations of sodium phosphate buffer (pH 7.5) were used, including 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, and 250 mM sodium phosphate buffer. Before the experiment, each of the above enzymes was dissolved in the corresponding concentration of sodium phosphate buffer to ensure that the final mixed volume was 4 mL. Then, these enzyme solutions were transferred to their respective 5 mL centrifuge tubes. Next, epoxy resin was added to each 5 mL centrifuge tube containing enzyme solution. The centrifuge tubes containing solution and resin were placed in a shaking incubator and shaken at 20°C and 180 r / min for 8 h for immobilization. After immobilization, the immobilized resin and solution were separated using a centrifuge. Afterwards, the immobilized resin was washed 3 times with the corresponding concentration of sodium phosphate buffer to remove unbound enzyme molecules. Finally, the immobilized resin samples after washing were stored at 4°C for future use. In this study, we evaluated the immobilization effects of three enzymes, NahE, EcQOR, and CgKR2, at different ion concentrations by a single factor experimental method, specifically investigating the protein adsorption rate and enzyme activity recovery rate. The experimental results showed that NahE achieved the best immobilization effect at an ion concentration of 100 mM, EcQOR performed best at an ion concentration of 10 mM, and CgKR2 performed well at an ion concentration of 20 mM.

[0096] Table 10 Effect of ion concentration on NahE immobilization

[0097]

[0098] Table 11 Effect of ion concentration on EcQOR immobilization

[0099]

[0100] Table 12 Effect of ion concentration on CgKR2 immobilization

[0101]

[0102] Example 7 Effect of buffer pH on enzyme immobilization.

[0103] In this experiment, we explored the immobilization of three enzymes, NahE (4 mg), EcQOR (2 mg), and CgKR2 (4 mg), onto 100 mg of epoxy-functionalized resin. We specifically focused on the effect of different pH values ​​(from 5.0 to 9.0) on the immobilization process. At the beginning of the experiment, first, the required sodium phosphate solutions of different pH were prepared according to Table 13, where X is 0.2 M sodium dihydrogen phosphate aqueous solution and Y is 0.2 M sodium dihydrogen phosphate aqueous solution. Secondly, the specified amount of each enzyme was dissolved in 20 mM sodium phosphate buffer of different pH, ensuring that the final mixed solution volume was 4 mL. Next, 100 mg of epoxy-functionalized resin was added to each 5 mL centrifuge tube containing enzyme solution and ensured that it was thoroughly mixed. The centrifuge tubes containing solution and resin were placed in a shaking incubator and shaken at 20°C and 180 r / min for 8 h for immobilization. After incubation, we used a centrifuge to separate the immobilized resin and solution. The immobilized resin was then washed three times with 20 mM sodium phosphate buffer to remove any unbound enzyme molecules. Finally, the washed immobilized resin samples were stored at 4 °C for future use. In this study, we evaluated the immobilization effects of three enzymes, NahE, EcQOR, and CgKR2, at different buffer pHs using a single factor experimental method, specifically investigating the protein adsorption rate and enzyme activity recovery rate. The results showed that the pH value of the buffer had a significant effect on the activity of the immobilized enzymes. When the buffer pH was set to 7.0, the protein adsorption rate and enzyme activity recovery rate of the three enzymes reached the highest, indicating that immobilization at pH 7.0 can retain more enzyme activity. Specifically, as shown in Tables 14-16, the protein adsorption rate of NahE increased to 85.1%, and the enzyme activity recovery rate reached 86.7%; the protein adsorption rate and enzyme activity recovery rate of EcQOR were 93.5% and 92.5%, respectively; and the protein adsorption rate and enzyme activity recovery rate of CgKR2 were 99.1% and 95.6%, respectively.

[0104] Table 13 Preparation of phosphate buffer (PB) buffer with different pH values

[0105]

[0106] Table 14 Effect of immobilization pH on NahE immobilization

[0107]

[0108] Table 15 Effect of immobilization pH on EcQOR immobilization

[0109]

[0110] Table 16 Effect of immobilization pH on immobilization of CgKR2

[0111]

[0112] Example 8 Effect of immobilization time on enzyme immobilization.

[0113] In this experiment, we aimed to immobilize three enzymes, NahE (4 mg), EcQOR (2 mg), and CgKR2 (4 mg), onto 100 mg of epoxy functionalized resin using 20 mM sodium phosphate buffer (pH 7.4) and investigate the effect of different immobilization times (4 h, 6 h, 8 h, 10 h, 12 h, 20 h, and 24 h) on the immobilization efficiency. First, 4 mg of NahE, 2 mg of EcQOR, and 4 mg of CgKR2 were dissolved in 20 mM sodium phosphate buffer, pH 7.0, respectively, ensuring that the final volume of the mixture was 4 mL. Subsequently, 100 mg of epoxy resin was added to each centrifuge tube containing the enzyme solution and ensured that it was mixed thoroughly. The tubes were then placed in a shaking incubator at 20°C and 180 r / min. During the incubation process, samples were taken from each time point (4 h, 6 h, 8 h, 10 h, 12 h, 20 h, and 24 h) for analysis. After the incubation, the immobilized resin and the solution were separated using a centrifuge. The immobilized resin was then washed with fresh 20 mM sodium phosphate buffer to remove any unbound enzyme. Finally, the washed immobilized resin samples were stored at 4 °C for future use. Throughout the experiment, time and operation were strictly controlled to ensure accurate evaluation of the effects of different immobilization times on enzyme immobilization efficiency. As shown in Tables 17-19, the maximum enzyme activity of NahE was observed at 6 h, while the maximum enzyme activity of EcQOR and CgKR2 was observed at the 8 h immobilization time point, at which time the enzyme immobilization efficiency and activity remained at the optimal level.

[0114] (3)

[0115] Among them, the enzyme activity under standard conditions was set as 100%.

[0116] Table 17 Effect of immobilization time on NahE immobilization

[0117]

[0118] Table 18 Effect of immobilization time on EcQOR immobilization

[0119]

[0120] Table 19 Effect of immobilization time on CgKR2 immobilization

[0121]

[0122] Example 9 Preparation of immobilized enzymes NahE@ES-103B, EcQOR-GDH@ES-1, and CgKR2-GDH@ES-103B.

[0123] The immobilized enzyme obtained by fixing NahE on ES-103B resin was recorded as NahE@ES-103B, the immobilized enzyme obtained by fixing EcQOR-GDH on ES-1 was recorded as EcQOR-GDH@ES-1, and the immobilized enzyme obtained by fixing CgKR2-GDH on LX-1000EP was recorded as CgKR2-GDH@LX-1000EP.

[0124] For the immobilization of NahE@ES-103B, 90 mg of NahE was dissolved in 8 mL of 100 mM PBS and mixed with 2 g of bifunctional resin support ES-103B. The mixture was incubated in a shaking incubator at 20 °C and 180 rpm for 6 h. After incubation, the immobilized resin sample was washed with 10 mM PBS to remove unbound enzyme molecules. The washed resin sample was then stored at 4 °C for future use.

[0125] For the immobilization of EcQOR-GDH@ES-1, the immobilization process involved a sequential dual enzyme strategy. Initially, 100 UGDH was dissolved in 8 mL of 100 mM carbonate buffer (composed of sodium carbonate and sodium bicarbonate, adjusted to pH 10.0). This solution was incubated with 2 g of the bifunctional resin support ES-1 at 20 °C and 180 rpm for 16-18 h. After the completion of the first immobilization step, the GDH-loaded resin was washed three times with 10 mM PBS to remove any unbound enzyme and residues. Subsequently, 50 mg of EcQOR was dissolved in 100 mM PBS and added to the pretreated resin. The secondary immobilization was performed under the same conditions for an additional 8 h. The final product was washed with 10 mM PBS and stored at 4 °C for future use.

[0126] For the immobilization of CgKR2-GDH@LX-1000EP, the immobilization protocol of CgKR2 followed the same dual enzyme method as that of EcQOR and GDH. After the initial immobilization of GDH as described above, 90 mg of CgKR2 was dissolved in 100 mM PBS and mixed with the GDH-loaded resin. This mixture was incubated at 20 °C and 180 rpm for 8 h. After this stage, the immobilized resin was thoroughly washed with 10 mM PBS to remove any excess or unbound CgKR2. Subsequently, the resin was stored at 4 °C for further use.

[0127] Example 10

[0128] Weigh 1 g of each of the immobilized enzymes NahE@ES-103B, EcQOR-GDH@ES-1, and CgKR2-GDH@ES-103B and fill them in a 1 cm diameter polytetrafluoroethylene tube to make a continuous flow reactor for each module. a PBR, b PBR and c Finally, the filled immobilized enzyme reactor was placed in a 25°C water bath for insulation, and the reaction solution was injected into the reactor at a certain flow rate through a syringe pump for reaction.

[0129] a PBR refers to the NahE catalytic reactor setup: benzaldehyde was used as substrate at a concentration of 50 mM and pyruvate was 100 mM in 100 mM PBS buffer containing 10% DMSO. The reaction was carried out at room temperature 25°C. The packed bed reactor (PBR) used had an inner diameter of 1 cm, a length of 2.3 cm, and a column void volume of 550 µL. The reaction column was filled with 1 g of the immobilized enzyme NahE@ES-103B.

[0130] b PBR refers to the EcQOR catalytic reactor setup: This reaction involves the substrates OPBEA at a concentration of 50 mM, NAD + The column was filled with 1 g of immobilized enzyme EcQOR-GDH@ES-1.

[0131] c PBR refers to the CgKR2 catalytic reactor setup: the substrate used was OPBA at a concentration of 50 mM, supplemented with NADP +2.0 mM, GDH 80 U, D-glucose 200 mM, in 100 mM PBS buffer (pH 8.0) containing 10% DMSO. The reaction was also carried out at a constant 25°C. The reactor was a PBR with a diameter of 1 cm, a length of 2.3 cm, and a column void volume of 530 µL. The column was filled with 1 g of immobilized enzyme CgKR2-GDH@ES-103B.

[0132] Effect of flow rate in single-step continuous flow system on reactor catalytic efficiency:

[0133] After the successful development of immobilized catalysts, we further explored the use of continuous flow systems for efficient synthesis ( R In the aldolase-catalyzed CC bond formation module, the reaction solution is transported to a PBR. This setup facilitated the diffusion of substrates from the aqueous phase to the pores of the catalyst support and then to the active site of the enzyme, thus achieving NahE-mediated aldehyde condensation. When the flow rate of the syringe pump was set to 0.05 mL / min, the substrate was completely converted in only 11 min (Table 20, entry 1). When the flow rate was increased to 0.15 mL / min, the conversion rate decreased slightly but remained above 94% (Table 20, entry 3). However, when the flow rate was increased to 0.20 mL / min, the conversion rate decreased significantly due to the decrease in the reaction residence time (Table 20, entry 4).

[0134] For the olefin reductase reduction C=C double bond module, the reaction solution is delivered to the b PBR. At a flow rate of 0.05 mL / min, complete conversion of the substrate was achieved in only 16 min (Table 20, entry 5). Increasing the flow rate to 0.10 mL / min slightly decreased the conversion, but still remained above 80% (Table 20, entry 6). However, further increasing the flow rate to 0.15 mL / min resulted in a significant decrease in conversion due to a decrease in the reaction residence time (Table 20, entry 7).

[0135] In the chiral alcohol building block catalyzed by ketoreductase, the reaction solution is delivered to c In the PBR. At a flow rate of 0.05 mL / min, the substrate was completely converted in only 11.2 minutes (Table 20, entry 9), which is much higher than the traditional batch method. When the flow rate was increased to 0.10 mL / min, the conversion rate decreased, but still remained above 75% (Table 20, entry 10). As the flow rate was further increased, the conversion rate dropped to below 50% (Table 20, entries 11, 12), which was mainly attributed to the reduced residence time of the reaction in the reactor.

[0136] Table 20 Operational performance of immobilized enzyme in PBR at different flow rates

[0137]

[0138] According to the single-step continuous flow system established in the above embodiment, a separate flow rate test was performed on each immobilized module to determine that complete conversion could be achieved at a flow rate of 50 mL / min at a substrate concentration of 50 mM. The reactor was connected to the reactor through a two-way connector. a PBR, b PBR and c PBR connected together to establish a three-step continuous flow system (such as Figure 2 ). Therefore, the final reaction conditions were: a syringe pump was used to deliver a solution containing benzaldehyde (50 mM), pyruvate (100 mM), NADP + (2.0mM)、NAD + The system was operated continuously in PBS buffer (100 mM, pH 7.5, containing 10% DMSO) with 2-(2.0 mM) and D-glucose (200 mM) at 25 °C, with a substrate conversion rate of >99% and an ee value of >99.9%.

[0139] The present invention establishes a multi-enzyme continuous flow system for the first time for efficient production ( R )-2-hydroxy-4-phenylbutyric acid (( R )-HPB). By designing enzyme immobilization and continuous flow mode, we successfully integrated three key steps: 1) aldol reaction between commercially available benzaldehyde and pyruvate catalyzed by aldolase; 2) reduction of activated C=C bond by enolate reductase; 3) construction of chiral alcohol by ketoreductase. This route effectively solved the problems of chemical imbalance and incompatible reaction conditions in the multi-enzyme cascade reaction and achieved the production of ( R )-2-hydroxy-4-phenylbutyric acid (( R This innovative approach not only demonstrates the efficiency and controllability of continuous flow chemistry in complex synthesis, but also provides a new route for the synthesis of other high-value building blocks using inexpensive starting materials.

[0140] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for synthesizing optically active ( R )-2-hydroxy-4-phenylbutyric acid, characterized in that The steps include: Step S1: using resin to fix aldolase, ene reductase and ketoreductase in the pipelines of the first reactor, the second reactor and the third reactor respectively, and optimizing the conditions for each reactor separately according to the substrate concentration of benzaldehyde to be reacted to obtain a suitable reaction flow rate so that the corresponding substrate in each reactor can react completely; Step S2: Connect the first reactor, the second reactor and the third reactor in step S1 in sequence, and add benzaldehyde, pyruvic acid and NADP to be reacted. + 、NAD + The mixed solution of D-glucose is introduced into the reactor by a syringe pump to achieve a three-step continuous flow reaction; The amino acid sequence of the aldolase is shown in SEQ ID NO.1, the amino acid sequence of the ene reductase is shown in SEQ ID NO.2, and the amino acid sequence of the ketoreductase is shown in SEQ ID NO.3; The aldolase was fixed in the first reactor using epoxy resin ES-103B, the ene reductase was fixed in the second reactor using epoxy resin ES-1, and the ketoreductase was fixed in the third reactor using epoxy resin LX-1000EP. The reaction solvent of the mixed solution is a mixture of PBS buffer and an organic solvent, and the organic solvent is any one or more of n-heptane, isooctane, methanol, ethanol, isopropanol, acetonitrile, and dimethyl sulfoxide; wherein the volume of the organic solvent is less than or equal to 10% of the total volume of the reaction solvent.

2. The method according to claim 1, characterized in that The aldolase is prepared by the following method: constructing an aldolase NahE gene with a gene sequence as shown in SEQ ID NO.4 into a pET-28a (+) vector, transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding aldolase NahE; the aldolase is fixed in the pipeline of the first reactor by the following method: dispersing the cultured and purified NahE in a phosphate buffer soaked with a functionalized epoxy resin, slowly shaking at 16-20°C for 4-8 hours, washing with phosphate buffer for several times, draining with a suction funnel to obtain an immobilized resin, and filling the drained immobilized resin into the pipeline of the first microreactor.

3. The method according to claim 1, characterized in that: The ene reductase is prepared by the following method: constructing an ene reductase EcQOR gene with a gene sequence as shown in SEQ ID NO.5 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding ene reductase EcQOR; the ene reductase is immobilized in a pipe of a second reactor by the following method: dispersing the ene reductase EcQOR obtained by culturing and purification in a phosphate buffer soaked with a functionalized epoxy resin, slowly shaking at 16-20° C. for 4-8 h, washing with phosphate buffer for several times, draining with a suction funnel to obtain an immobilized resin, and filling the drained immobilized resin into the pipe of the second reactor.

4. The method according to claim 1, characterized in that: The ketoreductase is prepared by the following method: constructing a ketoreductase CgKR2 gene with a gene sequence as shown in SEQ ID NO.6 into a pET-28a (+) vector, and transferring it into Escherichia coli BL 21 (DE 3), culturing and purifying to obtain the corresponding ketoreductase CgKR2; the ketoreductase is immobilized in a pipeline of a third reactor by the following method: dispersing the cultured and purified CgKR2 in a phosphate buffer soaked with a functionalized epoxy resin, slowly shaking at 16-20° C. for 4-8 h, washing with phosphate buffer for several times, draining with a suction funnel to obtain an immobilized resin, and filling the drained immobilized resin into a microchannel pipeline of the third reactor.

5. The method according to claim 1, characterized in that In step S1, the usage ratio of the resin to any one of the aldolase, ene reductase and ketoreductase is 400-1400 mg: 20-70 mg; during the enzyme immobilization process, the buffer ion concentration is 10-100 mM, the pH is 6-7.5; and the immobilization time is 5-10 hours.

6. The method according to claim 1, characterized in that The reaction temperature of the first reactor, the second reactor and the third reactor was 20-30°C, and the flow rate was 0.05-0.2 mL / min; benzaldehyde, pyruvic acid, NADP + 、NAD + The molar ratio of D-glucose is: 10~50: 20~100: 0.5~2: 0.5~2: 20~100.

7. The method according to claim 1, characterized in that The first reactor is a packed bed reactor with a diameter of 1-3 cm and a length of 2-5 cm; the second reactor is a packed bed reactor with a diameter of 1-3 cm and a length of 2-5 cm; the third reactor is a packed bed reactor with a diameter of 1-3 cm and a length of 2-5 cm.

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