A method for preparing norepinephrine based on L-threonine aldolase R318L / H128N

Abstract

The present invention discloses a method for synthesizing norepinephrine by dual enzyme coupling, which utilizes the mutant R318L / H128N of L-threonine aldolase derived from the black bear intestinal microbial metagenomic database and the tyrosine decarboxylase derived from Enterococcus faecium to couple, and uses glycine and 3,4-dihydroxybenzaldehyde as substrates to synthesize norepinephrine, with 97% enantioselectivity and a yield of 76%. The amino acid sequence of the L-threonine aldolase mutant R318L / H128N is shown in SEQ ID NO:3, which is obtained by changing the 318th amino acid of the L-threonine aldolase of SEQ ID NO:1 from Arg to Leu and the 128th amino acid from His to Asn; the amino acid sequence of the tyrosine decarboxylase is shown in SEQ ID NO:5. The dual enzyme coupling method for synthesizing norepinephrine has the advantages of high efficiency, greenness, mild conditions, etc., and has great application potential in the synthesis of adrenaline drugs.

Description

Technical Field

[0001] The invention relates to a method for synthesizing norepinephrine by double-enzyme coupling of an L-threonine aldolase mutant R318L / H128N and an L-tyrosine decarboxylase. Background Art

[0002] Norepinephrine (NE), scientific name 1-(3,4-dihydroxyphenyl)-2-aminoethanol, is a substance formed by removing the N-methyl group of adrenaline. It belongs to catecholamine in chemical structure. NE is mainly synthesized and secreted by the adrenal medulla. It is an α receptor agonist and has very little effect on β2 receptors. It can cause strong contraction of small arteries and small veins in various systems of the body, thereby increasing blood pressure. It is mainly used for the rescue of acute hypotension caused by various reasons. The clinical medication form is the L-isomer, and its bitartrate, i.e. norepinephrine bitartrate, is often used.

[0003] The chemical synthesis of norepinephrine usually involves the co-heating of catechol, chloroacetic acid and phosphorus oxychloride to generate 3,4-dihydroxychloroacetophenone, which is then ammonolyzed with an alcoholic ammonia solution to generate the corresponding amine, and finally the carbonyl group is hydrogenated under palladium-carbon catalysis to obtain racemic norepinephrine. This method has no stereoselectivity, and the obtained racemic product requires chiral separation to obtain an optically pure product; and the hydrogenation step of the reaction needs to be carried out under pressure, which is very dangerous and requires high production equipment. These problems have led to high production costs and low efficiency, which do not conform to the principles of green and economy. Therefore, it is necessary to seek a more gentle, green and efficient synthesis route for norepinephrine. Summary of the invention

[0004] The invention provides a method for synthesizing norepinephrine by double enzyme coupling. The method is easy to control, has mild reaction conditions, and avoids the problems of many by-products and high separation cost in chemical synthesis.

[0005] The present invention relates to a method for synthesizing norepinephrine by dual-enzyme coupling of an L-threonine aldolase mutant R318L / H128N and an L-tyrosine decarboxylase, comprising the following steps:

[0006] Step 1, constructing a genetically engineered bacterium BL21(DE3) / pGEX-6p-2-L-TA expressing the L-threonine aldolase mutant R318L / H128N

[0007] The L-threonine aldolase mutant R318L / H128N was fully synthesized and subcloned into the vector pGEX-6p-2 to obtain the recombinant plasmid GEX-6p-2-L-TA. The constructed recombinant plasmid was transformed into the Escherichia coli expression vector BL21 to obtain the L-threonine aldolase mutant R318L / H128N strain BL21(DE3) / pGEX-6p-2-L-TA.

[0008] Step 2: Construction of genetically engineered bacteria BL21(DE3) / pGEX-6p-2-L-TyrDC expressing L-TyrDC

[0009] After codon optimization of L-TyrDC from Enterococcus faecium obtained from the NCBI database, the optimized sequence was synthesized by whole gene synthesis and subcloned into the vector pGEX-6p-2 to obtain the recombinant plasmid pGEX-6p-2-L-TyrDC, and the constructed recombinant plasmid was transformed into the Escherichia coli expression host BL21 (DE3) to obtain the strain BL21 (DE3) / pGEX-6p-2-L-TyrDC expressing L-tyrosine decarboxylase;

[0010] Step 3, Expression and purification of L-threonine aldolase mutant R318L / H128N

[0011] The L-threonine aldolase mutant R318L / H128N expression strain was inoculated into sterile LB medium, with a final concentration of ampicillin of 100 μg / mL, and cultured at 37°C and 180 rpm. When OD 600≈0.8-1, the inducer IPTG (50 μg / mL) was added, and the culture was induced overnight (12 h) at 16°C and 110 rpm. The bacteria were collected by centrifugation at 8000 rpm for 5 min. The bacteria were resuspended in a ratio of 1L culture system plus 30 mL lysis buffer (pH 7.4, 10 mM PBS), and the cell wall was broken with an ultra-high pressure nano homogenizer at 4°C. The broken bacteria were centrifuged (4°C, 12000 rpm for 20 min), and when the centrifugation was completed, the supernatant was transferred to a 50 mL sterile centrifuge tube pre-cooled at 4°C with a precision pipette. Take Glutathione Sepharose 4B filler (GE Healthcare) and load it into a chromatography column (GE Healthcare). The filler is used in a ratio of 5 mL filler per liter of culture system. Rinse 3 column volumes with 4°C precooled pH 7.4, 10mM sterile PBS to remove anhydrous ethanol. Combine the obtained supernatant with Glutathione Sepharose 4B and suspend vertically at 4°C for 3 hours. After the combination is complete, centrifuge at 500rpm for 5 minutes to precipitate the filler. Rinse the filler with 4°C precooled pH 7.4, 10mM sterile PBS for 10 column volumes to remove impurities. Add 4°C precooled enzyme cleavage buffer (pH 7.4, 10mM PBS), add PreScission Protease enzyme (GenScript, Z02799-100), and digest at 4°C overnight. After the digestion is complete, release the supernatant from the chromatography column to obtain L-TA R318L / H128N enzyme solution;

[0012] Step 4, Expression and purification of L-tyrosine decarboxylase

[0013] The L-tyrosine decarboxylase expression strain was inoculated into sterile LB medium, with a final concentration of ampicillin of 100 μg / mL, and cultured at 37°C and 180 rpm. When OD 600≈0.8-1, the inducer IPTG (50 μg / mL) was added, and the culture was induced overnight (12 h) at 16°C and 110 rpm. The bacteria were collected by centrifugation at 8000 rpm for 5 min. The bacteria were resuspended in a ratio of 30 mL lysis buffer (pH 7.4, 10 mM PBS) per 1 L culture system, and the cell wall was broken with an ultra-high pressure nano homogenizer at 4°C. The broken bacteria were centrifuged (4°C, 12000 rpm for 20 min), and when the centrifugation was completed, the supernatant was transferred to a 50 mL sterile centrifuge tube pre-cooled at 4°C with a precision pipette. Glutathione Sepharose 4B filler (GE Healthcare) was loaded into a chromatography column (GE Healthcare), and the filler was used in a ratio of 5 mL filler per liter of culture system. Rinse 3 column volumes with 4°C precooled pH 7.4, 10mM sterile PBS to remove anhydrous ethanol. Combine the obtained supernatant with Glutathione Sepharose4B and suspend vertically at 4°C for 3h. After the combination is completed, centrifuge at 500rpm for 5min to precipitate the filler. Rinse the filler with 4°C precooled pH 7.4, 10mM sterile PBS for 10 column volumes to remove impurities. Add 4°C precooled enzyme cleavage buffer (pH 7.4, 10mM PBS), add PreScission Protease enzyme (GenScript, Z02799-100), and digest at 4°C overnight. After the digestion is completed, release the supernatant from the chromatography column to obtain L-tyrosine decarboxylase enzyme solution;

[0014] Step 5, select 3,4-dihydroxybenzaldehyde as a substrate, glycine as a co-substrate, 5-pyridoxal phosphate as a cofactor, add L-threonine aldolase mutant R318L / H128N and L-tyrosine decarboxylase enzyme solution, add PBS buffer to adjust the pH of the reaction system to 6-11, and react at 20-60°C for 0.5-24h. After the reaction, use high performance liquid chromatography, XSelect HSS T3 column (4.6×250mm, 5μm), mobile phase is 23.3g sodium chloride (Shanghai Mairui Chemical Technology Co., Ltd.), 1ml glacial acetic acid, diluted with water to 1L, mixed with 2% pure acetonitrile (analytical grade) solution, column temperature 30°C, UV detection wavelength 280nm, flow rate 5mL / min.

[0015] The amino acid sequence of the L-threonine aldolase mutant R318L / H128N described in the present invention is shown in SEQ ID NO: 3, which is obtained by changing the 318th amino acid of the L-threonine aldolase with the amino acid sequence of SEQ ID NO: 1 from Arg to Leu and the 128th amino acid from His to Asn; the amino acid sequence of the tyrosine decarboxylase is shown in SEQ ID NO: 5.

[0016] In step 5 of the present invention, the concentration of 3,4-dihydroxybenzaldehyde is 5-200 mM, the concentration of glycine is 0.25-2.5 M, the concentration of pyridoxal 5-phosphate is 50 μM, the concentration of L-threonine aldolase is 7-9 U / mL, and the concentration of L-tyrosine decarboxylase is 0.3-1 U / mL.

[0017] A further improvement is that in step 5, the final concentration of 3,4-dihydroxybenzaldehyde is 50 mM and the final concentration of glycine is 2.5 M.

[0018] Compared with the prior art, the present invention has the following advantages: utilizing the L-threonine aldolase mutant R318L / H128N with excellent catalytic performance to participate in the dual-enzyme coupling synthesis of norepinephrine, with excellent product selectivity, high yield, low production cost, mild reaction conditions, greenness, high efficiency, and good industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure is a flow chart of the double-enzyme coupling synthesis of norepinephrine in the present invention. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific examples. It should be noted that the following examples are only for explanation and illustration and do not limit the scope of the present invention in any way.

[0021] Main reagents and consumables:

[0022] Prime STAR Max Premix (2×) (Cat. No. R045A), BamH I restriction endonuclease (Cat. No. 1010S), Xho I restriction endonuclease (Cat. No. 1094S), T4 DNA Ligase (Cat. No. D2011A), and 10× T4 Ligase buffer were all purchased from Bao Biotechnology Co., Ltd. (Dalian);

[0023] The pGEX-6p-2 plasmid is a known E. coli expression vector, also known as pGEX6P2, pGEX 6P 2, with a vector size of 4985 bp, Tac promoter, vector tag N-GST, and vector resistance Ampicillin (ampicillin), purchased from Shanghai Shenggong Biotechnology Co., Ltd., and also preserved in this laboratory;

[0024] Trans 5α competent cells (Cat. No. CD201-01) and E. coli BL21 (DE3) competent cells (Cat. No. CD601) were purchased from Quanshijin Biotechnology Co., Ltd.;

[0025] Glutathione Sepharose 4B, purchased from GE Healthcare, catalog number 10223836;

[0026] PreScission Protease, purchased from GenScript, catalog number Z02799-100;

[0027] Bradford protein concentration assay kit, purchased from Beyotime, catalog number P0006;

[0028] L-threo-DOPS standard (CAS: 23651-95-8, product number: D4235, molecular formula: C 9 H 11 NO 5 ), L-DOPS (L-threo-DOPS and L-erythro-DOPS racemate) standard (CAS: 23651-95-8, product number: D9446), 1-heptanesulfonic acid sodium salt (CAS: 22767-50-6, molecular formula: C 7 H 15 NaO 3 S), 1,4-dioxane (CAS: 123-91-1, molecular formula: C 4 H 8 O 2 ) were purchased from J&K Technology Co., Ltd.;

[0029] Glycine (CAS: 56-40-6, molecular formula: C 2 H 5 NO 2 , Product No. A502065), 3,4-dihydroxybenzaldehyde (CAS: 139-85-5, linear molecular formula: (HO) 2 C 6 H 3CHO, product number A601406), 5-pyridoxal phosphate (PLP, CAS: 41468-25-1, molecular formula C 8 H 12 NO 7 P, catalog number A610455), were purchased from Shanghai Biotechnology Co., Ltd.

[0030] LB medium

[0031] Each 100 mL of LB medium contains: 1 g tryptone, 0.5 g yeast extract, 1 g sodium chloride, pH 7.4.

[0032] Preparation method: in 950mL ddH 2 Dissolve 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride in 1% HO, then adjust the pH to 7.4 with NaOH and wash with ddHO. 2 If solid medium is to be prepared, add 15 g agar per liter. Sterilize with high pressure steam at 121°C for 20 min.

[0033] PBS buffer

[0034] Preparation of pH 7.4, 10 mM PBS: weigh 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 and 0.24 g KH 2 PO 4 , dissolve in 800 mL of distilled water, adjust the pH value of the solution to 7.4 with HCl, and finally add distilled water to make up to 1 L. Sterilize with high pressure steam at 121°C for at least 20 minutes and store at room temperature or in a refrigerator at 4°C for later use.

[0035] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and the experimental conditions used are all conventional experimental conditions in the art, and reference can be made to the relevant experimental manuals or manufacturer's instructions.

[0036] Example 1

[0037] Step 1, constructing a genetically engineered bacterium BL21(DE3) / pGEX-6p-2-L-TA expressing the L-threonine aldolase mutant R318L / H128N

[0038] The L-threonine aldolase mutant R318L / H128N was fully synthesized and subcloned into the vector pGEX-6p-2 to obtain the recombinant plasmid GEX-6p-2-L-TA. The constructed recombinant plasmid was transformed into the Escherichia coli expression vector BL21 to obtain the L-threonine aldolase mutant R318L / H128N strain BL21(DE3) / pGEX-6p-2-L-TA.

[0039] Step 2: Construction of genetically engineered bacteria BL21(DE3) / pGEX-6p-2-L-TyrDC expressing L-TyrDC

[0040] After codon optimization of L-TyrDC from Enterococcus faecium obtained from the NCBI database, the optimized sequence was synthesized by whole gene synthesis and subcloned into the vector pGEX-6p-2 to obtain the recombinant plasmid pGEX-6p-2-L-TyrDC, and the constructed recombinant plasmid was transformed into the Escherichia coli expression host BL21 (DE3) to obtain the strain BL21 (DE3) / pGEX-6p-2-L-TyrDC expressing L-tyrosine decarboxylase;

[0041] Step 3, Expression and purification of L-threonine aldolase mutant R318L / H128N

[0042] The L-threonine aldolase mutant R318L / H128N expression strain was inoculated into sterile LB medium, with a final concentration of ampicillin of 50 μg / mL, and cultured at 37°C and 180rpm. When OD 600≈0.8, IPTG was added at a final concentration of 0.2mM, and induced culture was carried out overnight (12h) at 16°C and 110rpm. The bacteria were collected by centrifugation at 8000rpm for 5min. The bacteria were resuspended in a ratio of 1L culture system plus 30mL lysis buffer (pH 7.4, 10mM PBS), and the cell wall was broken with an ultra-high pressure nano homogenizer at 4°C. The broken bacterial solution was evenly divided into 50mL sterile centrifuge tubes precooled at 4°C, and centrifuged at 4°C and 12000rpm for 20min. After the centrifugation was completed, the supernatant was transferred to a 50mL sterile centrifuge tube precooled at 4°C with a precision pipette. Take Glutathione Sepharose4B filler (GE Healthcare) and load it into a chromatography column (GE Healthcare). The filler is used in a ratio of 5 mL filler per liter of culture system. Rinse 3 column volumes with 4°C precooled pH 7.4, 10mM sterile PBS to remove anhydrous ethanol. Combine the obtained supernatant with Glutathione Sepharose 4B and combine at 4°C for 2h. Gently invert vertically to suspend. After the combination is completed, centrifuge at 500rpm for 5min to precipitate the filler. Rinse the filler with 4°C precooled pH 7.4, 10mM sterile PBS for 3-5 column volumes to remove impurities. Add 4°C precooled enzyme digestion buffer (pH7.4, 10mM PBS), add PreScission Protease (GenScript, Z02799-100), and digest overnight at 4°C. After the digestion is completed, release the supernatant from the chromatography column to obtain L-TAR318L / H128N enzyme solution. The L-TA R318L / H128N enzyme solution was subjected to SDS-PAGE detection to identify its molecular weight.

[0043] Step 4, Expression and purification of L-tyrosine decarboxylase

[0044] The L-tyrosine decarboxylase expression strain was inoculated into sterile LB medium, with a final concentration of ampicillin of 50 μg / mL, and cultured at 37°C and 110rpm. When OD 600≈0.8, IPTG was added at a final concentration of 0.2mM, and the culture was induced overnight (12h) at 16°C and 180rpm. The bacteria were collected by centrifugation at 8000rpm for 5min. The bacteria were resuspended in a ratio of 30mL lysis buffer (pH 7.4, 10mMPBS) per 1L culture system, and the cell wall was broken with an ultra-high pressure nano homogenizer at 4°C. The broken bacterial solution was evenly divided into 50mL sterile centrifuge tubes precooled at 4°C, and centrifuged at 4°C and 12000rpm for 20min. After the centrifugation was completed, the supernatant was transferred to a 50mL sterile centrifuge tube precooled at 4°C with a precision pipette. Take Glutathione Sepharose 4B filler (GE Healthcare) and load it into a chromatography column (GE Healthcare). The filler is used in a ratio of 5 mL filler per liter of culture system. Rinse 3 column volumes with 4°C precooled pH 7.4, 10mM sterile PBS to remove anhydrous ethanol. Combine the obtained supernatant with Glutathione Sepharose 4B and combine at 4°C for 2h. Gently invert vertically to suspend. After the combination is completed, centrifuge at 500rpm for 5min to precipitate the filler. Rinse the filler with 4°C precooled pH 7.4, 10mM sterile PBS for 3-5 column volumes to remove impurities. Add 4°C precooled enzyme digestion buffer (pH 7.4, 10mM PBS), add PreScission Protease enzyme (GenScript, Z02799-100), and digest at 4°C overnight. After the digestion is completed, release the supernatant from the chromatography column to obtain L-tyrosine decarboxylase enzyme solution. The L-tyrosine decarboxylase enzyme solution was subjected to SDS-PAGE detection to identify its molecular weight. The concentration of the L-tyrosine decarboxylase enzyme solution was determined using a Bradford protein concentration determination kit (Beyotime, P0006) according to the kit instructions.

[0045] Example 2

[0046] Catalytic synthesis of norepinephrine: the reaction system is 0.25-2.5M glycine, 25-200mM 3,4-dihydroxybenzaldehyde, 50μM pyridoxal 5-phosphate, then add 7-9U / mL L-threonine aldolase, 0.3-1U / mL L-tyrosine decarboxylase, adjust the system to 1mL with PBS buffer, pH 6.0, and react at 30℃ for 2-12h.

[0047] After the reaction, high performance liquid chromatography was used, XSelect HSS T3 column (4.6×250 mm, 5 μm), the mobile phase was 23.3 g sodium chloride (Shanghai Myrel Chemical Technology Co., Ltd.), 1 ml glacial acetic acid, diluted to 1 L with water, mixed with 2% pure acetonitrile (analytical grade) solution, column temperature 30° C., UV detection wavelength 280 nm, flow rate 5 ml / min.

[0048] After detection by high performance liquid chromatography, it was calculated that the enantioselectivity of the product was 97% and the yield was 76%.

Claims

1. A method for synthesizing norepinephrine by dual enzyme coupling, characterized in that: The mutant R318L / H128N of L-threonine aldolase from black bear intestinal microorganisms was coupled with L-tyrosine decarboxylase from Enterococcus faecium, using glycine and 3,4-dihydroxybenzaldehyde as substrates and pyridoxal 5-phosphate as a cofactor to synthesize norepinephrine. The amino acid sequence of the mutant R318L / H128N of L-threonine aldolase derived from black bear intestinal microorganisms is shown in SEQ ID NO: 3; The amino acid sequence of the L-tyrosine decarboxylase from Enterococcus faecium is shown in SEQ ID NO:

5.

2. The method for synthesizing norepinephrine by dual enzyme coupling according to claim 1, characterized in that: The final concentration of 3,4-dihydroxybenzaldehyde is 5-200 mM, the final concentration of glycine is 0.25-2.5 M, the final concentration of pyridoxal 5-phosphate is 50 μM, the concentration of the mutant R318L / H128N of L-threonine aldolase is 7-9 U / mL, and the concentration of L-tyrosine decarboxylase is 0.3-1 U / mL.

3. The method for synthesizing norepinephrine by dual enzyme coupling according to claim 1, characterized in that: The final concentration of 3,4-dihydroxybenzaldehyde was 50 mM, and the final concentration of glycine was 2.5 M.

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