Biological preparation method for synthesizing dihydroberberine based on imine reductase

Abstract

The invention discloses a biological preparation method for synthesizing dihydroberberine based on imine reductase, and relates to the technical field of medicines.The biological preparation method comprises the steps that S1, in a liquid reaction system, berberine serves as a substrate, reduction reaction is conducted under catalysis of imine reductase, and dihydroberberine is formed; and S2, separating out dihydroberberine from the reaction system used in the reaction in the step S1. The biological preparation method for synthesizing dihydroberberine based on imine reductase is high in yield, safe, simple and convenient to operate, low in production cost and suitable for industrial production, and a technological product prepared by the method is good in fluidity, free of static electricity, more suitable for food industrial production application such as capsule filling and tabletting and suitable for industrial production of dihydroberberine. Meanwhile, the content and the character of the product are not changed after the product is placed for 15 days in an open manner, the state is more stable, and the problems of degradation and product purity reduction caused by contact with air cannot occur after the product is placed in the open manner.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a biological preparation method for synthesizing dihydroberberine based on imine reductase. Background Art

[0002] Dihydroberberine, also known as dihydroberberine, is a natural plant alkaloid widely used in the treatment of diabetes. Dihydroberberine is a natural glucagon-like peptide 1 agonist, and its bioavailability, absorption rate, safety and efficacy are all superior to berberine. Some studies have shown that the intestinal absorption rate of dihydroberberine is higher than that of berberine, mainly acting locally in the intestine to prevent the absorption of disaccharides by the intestine. Dihydroberberine can improve insulin sensitivity by inhibiting mitochondrial respiratory complex to activate adenosine-activated protein kinase.

[0003] Dihydroberberine is usually synthesized by reducing the quaternary ammonium of berberine. Takemoto and Ishii H obtained dihydroberberine by reducing berberine with lithium aluminum hydride in 1962 and 1991 respectively;

[0004] In 2009, Ying-Hong Li et al. prepared dihydroberberine from berberine by using sodium borohydride reduction method instead of lithium aluminum hydride;

[0005] In 2018, patent CN108997332A disclosed a preparation method of dihydroberberine: in the presence of a catalyst, berberine undergoes catalytic transfer hydrogenation reaction with a hydrogen donor in a certain solvent to obtain dihydroberberine. The hydrogen donors used include hydrogen, ammonium formate, triethylamine / formic acid, hydrazine hydrate, isopropanol, etc., and the catalysts used are palladium carbon, palladium black, Raney nickel or platinum black, etc.;

[0006] In 2024, patent CN118063454A used potassium borohydride to prepare dihydroberberine, and the yield was 61.7%;

[0007] All of the above methods are traditional chemical reduction methods. Both of the two double bonds in berberine may be hydrogenated and reduced, and the yield and purity of the product cannot be well guaranteed.

[0008] In 2018, Clifts et al. reported a method for constructing dihydroberberine by Pictet-Spengler reaction in Org. Lett.;

[0009] The yields of each step of this route are medium to good, but there are still many problems in industrialization: including that the aldehyde in the Pictet-Spengler reaction needs to be in excess and the yield is low, partial acetal hydrolysis occurs during the reductive amination process, and the hydrolysis impurities increase greatly after amplification, resulting in a significant reduction in the yield. The separation yield of the intermediate dihydroberberine is only 55%, and it is even lower during the amplification process.

[0010] Therefore, there is an urgent need in the art to provide a method for preparing berberrubine that uses inexpensive and easily available raw materials and auxiliary materials, is safe and simple to operate, and is suitable for industrial production. Summary of the Invention

[0011] The main object of the present invention is to provide a biological preparation method for synthesizing berberrubine based on imine reductase, which can effectively solve the problems in the background art.

[0012] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0013] A biological preparation method for synthesizing berberrubine based on imine reductase, which comprises the following preparation steps:

[0014] S1: In a liquid reaction system, using berberine as a substrate, a reduction reaction is carried out under the catalysis of imine reductase to form berberrubine;

[0015] S2: Separate berberrubine from the reaction system used in step S1.

[0016] Preferably, the imine reductase is selected from: Mutant 1: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A; Mutant 2: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site D129A; Mutant 3: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A / D129G; Mutant 4: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A / L173V, and the imine reductase is modified based on the NcIRED imine reductase protein structure with the accession number: WP_014349966.1 predicted by AlphaFold.

[0017] Preferably, the initial concentration of berberine includes 1 - 1000 g / L; 5 - 500 g / L; 10 - 500 g / L.

[0018] Preferably, in step S1, the reaction temperature catalyzed by the enzyme includes 10°C to 50°C; 20°C to 40°C; 25°C to 35°C.

[0019] Preferably, in step S1, the reaction time catalyzed by the enzyme includes 0.1 - 72 h; 0.5 - 36 h; 1 - 24 h.

[0020] Preferably, in step S1, the pH of the reaction system catalyzed by the enzyme includes 6.0 - 9.0; 6.5 - 8.5; 7.0 - 8.0.

[0021] Preferably, in the step S1, the liquid reaction system includes a reaction solution, the reaction solution is a phosphate buffer system, the liquid reaction system further includes a coenzyme, and the coenzyme includes but is not limited to NADH, NADPH, NAD+, NADP+, glucose dehydrogenase, and the coenzyme is NADP+ and glucose dehydrogenase.

[0022] Preferably, in the step S1, the liquid reaction system further includes a cosubstrate, and the cosubstrate includes but is not limited to isopropanol, glucose, ammonium formate, and sodium formate.

[0023] Preferably, the coding gene of the imine reductase is a nucleic acid and an expression vector, the nucleic acid is the coding gene encoding the aforementioned imine reductase, and the expression vector is loaded with the coding gene of the imine reductase of the nucleic acid.

[0024] Preferably, the expression vector is a pET22a(+) vector, and the recombination of the expression vector is constructed by ligating the nucleic acid to various expression vectors. The expression vector includes a recombinant expression transformant, and the recombinant expression transformant includes a host cell and a target gene transferred into the host cell. The target gene contains the coding gene of the imine reductase loaded with the nucleic acid. The preparation method of the recombinant expression transformant is: transforming the expression vector into a host microorganism, and the host microorganism is preferably Escherichia coli E.coli; Escherichia coli BL21(DE3); Escherichia coli DH5α.

[0025] Beneficial effects

[0026] Compared with the prior art, the present invention provides a biological preparation method for synthesizing dihydroberberine based on imine reductase, which has the following beneficial effects:

[0027] 1. The biological preparation method for synthesizing dihydroberberine based on imine reductase has a high yield, is safe and simple to operate, has a low production cost, is suitable for industrial production. The process product prepared by this method has good fluidity and no static electricity, is more suitable for food industrial production applications such as capsule filling and tabletting. At the same time, its content and properties do not change after being placed open for 15 days, its state is more stable, and there will be no problem of degradation and reduction of product purity due to contact with air when it is placed open. Brief description of the drawings

[0028] Figure 1 It is a reaction diagram of the present invention in a liquid reaction system, where berberine is used as a substrate and undergoes a reduction reaction under the catalysis of imine reductase to form dihydroberberine;

[0029] Figure 2 It is a sample comparison diagram of the present invention.

[0030] Figure 2 On the left in is the process product prepared by this method;Figure 2 On the right side in the middle is a conventional product. Detailed implementation manners

[0031] To make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific implementation manners.

[0032] As Figure 1 shown, a biological preparation method for synthesizing dihydroberberine based on imine reductase includes the following preparation steps:

[0033] S1: In a liquid reaction system, using berberine as a substrate, a reduction reaction is carried out under the catalysis of imine reductase to form dihydroberberine;

[0034] The imine reductase is selected from: Mutant 1: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A; Mutant 2: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site D129A; Mutant 3: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A / D129G; Mutant 4: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A / L173V. The initial concentration of berberine includes 1 - 1000 g / L; 5 - 500 g / L; 10 - 500 g / L. The reaction temperature of enzyme catalysis includes 10°C to 50°C; 20°C to 40°C; 25°C to 35°C. The reaction time of enzyme catalysis includes 0.1 to 72 h; 0.5 to 36 h; 1 to 24 h. The pH of the reaction system of enzyme catalysis includes 6.0 to 9.0; 6.5 to 8.5; 7.0 to 8.0. The liquid reaction system includes a reaction solution, the reaction solution is a phosphate buffer system, the liquid reaction system further includes a coenzyme, and the coenzyme includes but is not limited to NADH, NADPH, NAD+, NADP+, glucose dehydrogenase, and the coenzyme is NADP+ and glucose dehydrogenase. The liquid reaction system further includes a cosubstrate, and the cosubstrate includes but is not limited to isopropanol, glucose, ammonium formate, sodium formate;

[0035] The coding gene of the imine reductase is a nucleic acid and an expression vector. The nucleic acid is the coding gene encoding the aforementioned imine reductase. The expression vector is loaded with the coding gene of the imine reductase of the nucleic acid. The expression vector is a pET22a(+) vector. The recombination of the expression vector is constructed by ligating the nucleic acid to various expression vectors. The expression vector includes a recombinant expression transformant. The recombinant expression transformant includes a host cell and a target gene transferred into the host cell. The target gene contains the coding gene of the imine reductase loaded with the nucleic acid. The preparation method of the recombinant expression transformant is: obtained by transforming the expression vector into a host microorganism. The host microorganism is preferably Escherichia coli E.coli; Escherichia coli BL21(DE3); Escherichia coli DH5α.

[0036] This application is modified based on the protein structure of NcIRED imine reductase (accession number WP_014349966.1) predicted by AlphaFold.

[0037] The nucleotide sequence and amino acid sequence listing are incorporated into this specification by reference, specifically as shown in the attached sequence listing. Specific Example 1:

[0039] The imine reductase genes derived from Nocardiacyriacigeorgica (NcIRED), Nocardiasp. XZ_19_231 (NxIRED), Nocardiasalmonicida (NsIRED), Streptomycescanus (ScIRED), and Jiangellamuralis (JmIRED) were respectively cloned into the pET22a(+) vector, transferred into competent Escherichia coli DH5α cells, cultured on kanamycin-resistant plates, positive transformant single colonies were picked and plasmid extraction and sequencing were performed to confirm. After that, the recombinant plasmids were extracted and then introduced into the host Escherichia coli BL21(DE3), cultured on plates, single colonies were picked and cultured in LB to obtain recombinant genetically engineered bacteria of 5 kinds of imine reductases, as shown in Table 1;

[0040] Using the obtained cells of 5 kinds of imine reductases, berberine was used as the substrate to prepare dihydroberberine by biocatalysis for preliminary screening. The reaction conditions were: 0.02 g of substrate, 0.01 g of wet cells, 2 mL of phosphate buffer (100 mM, pH 7), adding 0.01 g of NADP + , 0.05 g of glucose, 0.01 g of glucose dehydrogenase, reacting at 30 °C for 24 h, and then the conversion rate was analyzed by HPLC.

[0041] Among them, the reaction activities of 5 kinds of imine reductases (characterized by the substrate conversion rate; the higher the conversion rate, the higher the reaction activity is considered) are shown in Table 1. The reaction conversion rate of NcIRED enzyme is the highest and the activity is the best.

[0042] Table 1:

[0043]

[0044]

[0045] As can be seen from Table 1, the reduction effects of imine reductases from different sources on the substrate in the present invention are significantly different. When dihydroberberine is prepared by reducing the substrate berberine, imine reductase NcIRED has a significant advantage in terms of conversion rate compared with imine reductases from other sources;

[0046] The literature source of the accession number WP_014349966.1 is as follows: Zumbrgel N, Machui P, Nonnhoff J, et al. Enantioselective Biocatalytic Reduction of 2H-1,4-Benzoxazines Using Imine Reductases. [J]. The Journal of organic chemistry, 2019, 843:, 1440-1447. DOI: 10.1021 / acs.joc.8b02867; Wetzl D, Berrera M, Sandon N, et al. Expanding the Imine Reductase Toolbox by Exploring the Bacterial Protein-Sequence Space [J]. Chembiochem, 2015, 16(12): 1823-1823. DOI: 10.1002 / cbic.201590035). The nucleotide sequence and amino acid sequence are shown in the sequence listing. Specific Example 2:

[0048] MEGA6 was used to construct an evolutionary tree of imine reductases reported in existing databases. Combining information such as amino acid sequences, protein structures, species sources, substrate spectra, and activities, multiple key amino acid mutation sites were selected. Degenerate codon NNK was used to design mutant primers, and site-directed mutagenesis was performed on the amino acid sequence of imine reductase from Nocardiacyriacigeorgica strain using pET22a-NcIRED as a template. The sites for library mutagenesis were 73, 129, 173, and 194 respectively. The obtained mutants were tested for enzyme activity according to the method of Specific Example 1. The enzyme activity effects of some mutants are shown in Table 2;

[0049] Table 2:

[0050]

[0051]

[0052] Example 3:

[0053] As Figure 1 shown, the synthesis of dihydroberberine I:

[0054] Add 10 g of M4 cells to a 500 mL three-necked flask, and add 0.02 g of NADP +, 5 g of berberine, 10 g of glucose, 5 g of glucose dehydrogenase, add to 100 mL of phosphate buffer solution and stir evenly by mechanical stirring. Heat in a water bath with an internal temperature of 30 °C. Keep the pH at 7 - 8 throughout the reaction process. Add 200 mL of ethyl acetate to extract the product, stir mechanically for 1 h, filter the bacteria, separate the liquid. Extract the aqueous phase twice with 200 mL of ethyl acetate, combine the organic phases, wash twice with water, separate the liquid. Dry and concentrate the organic phase, crystallize with ethanol-water to obtain 4.4 g of the product, with a yield of 88% and a purity of 98%. Specific Example Four:

[0056] As Figure 1 shown, the synthesis of dihydroberberine II:

[0057] Add 20 g of M4 bacteria to a 1 L three-necked flask, add 0.04 g of NADP + , 10 g of berberine, 20 g of glucose, 10 g of glucose dehydrogenase, add to 200 mL of phosphate buffer solution and stir evenly by mechanical stirring. Heat in a water bath with an internal temperature of 30 °C. Keep the pH at 7 - 8 throughout the reaction process. Add 400 mL of ethyl acetate to extract the product, stir mechanically for 1 h, filter the bacteria, separate the liquid. Extract the aqueous phase twice with 400 mL of ethyl acetate, combine the organic phases, wash twice with water, separate the liquid. Dry and concentrate the organic phase, crystallize with ethanol-water to obtain 9.3 g of the product, with a yield of 93% and a purity of 97%. Specific Example Five:

[0059] As Figure 1 shown, the synthesis of dihydroberberine III:

[0060] Add 20 g of M4 bacteria to a 1 L three-necked flask, add 0.04 g of NADP + , 15 g of berberine, 20 g of glucose, 10 g of glucose dehydrogenase, add to 200 mL of phosphate buffer solution and stir evenly by mechanical stirring. Heat in a water bath with an internal temperature of 30 °C. Keep the pH at 7.2 throughout the reaction process. Add 400 mL of ethyl acetate to extract the product, stir mechanically for 2 h, filter the bacteria, separate the liquid. Extract the aqueous phase twice with 400 mL of ethyl acetate, combine the organic phases, wash twice with water, separate the liquid. Dry and concentrate the organic phase, crystallize with ethanol-water to obtain 14.2 g of the product, with a yield of 95% and a purity of 98%.

[0061] In summary, the above-mentioned examples are only the preferred examples of the present invention, and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0062] The present invention relates to a biological preparation method for synthesizing berberrubine by imine reductase, which has high yield, safe and simple operation, low production cost, and is suitable for industrial production. The process product prepared by this method has good fluidity, no static electricity, and is more suitable for food industrial production applications such as capsule filling and tabletting. At the same time, when it is placed open for 15 days, its content and properties remain unchanged, its state is more stable, and no degradation occurs due to contact with air and no problem of product purity reduction occurs when it is placed open.

[0063] Product open stability test:

[0064] Experimental method: For 3 batches of products prepared by this method and 1 batch of conventionally synthesized products, under normal temperature conditions, after being exposed to air open for 15 days, samples were taken and the content of berberrubine was determined by HPLC, and the difference in content results before and after being open was compared. The following is the test table:

[0065]

[0066] As Figure 2 shown, after products of batches 1 - 3 were placed open for 15 days, there were no changes in product appearance, fluidity, properties and content. For the conventionally synthesized product, there was slight water absorption after being placed open for one day, and it caked severely, and became a hard block after being placed open for 15 days.

[0067] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A biological preparation method for synthesizing dihydroberberine based on imine reductase, characterized in that: The preparation steps include: S1: In a liquid reaction system, berberine is used as a substrate and undergoes a reduction reaction catalyzed by imine reductase to form dihydroberberine; S2: Separating dihydroberberine from the reaction system used in step S1.

2. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: The imine reductase is selected from: mutant 1: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A; mutant 2: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site D129A; mutant 3: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A / D129G; mutant 4: having an amino acid sequence corresponding to SEQ ID NO: 1 and having a mutation site P73A / L173V, and the imine reductase is modified based on the NcIRED imine reductase protein structure predicted by AlphaFold with accession number: WP_014349966.

1.

3. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: The initial concentration of berberine includes 1-1000 g / L; 5-500 g / L; 10-500 g / L.

4. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: In step S1, the enzyme-catalyzed reaction temperature includes 10°C to 50°C; 20°C to 40°C; 25°C to 35°C.

5. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: In the step S1, the enzyme-catalyzed reaction time includes 0.1 to 72 hours; 0.5 to 36 hours; 1 to 24 hours.

6. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: In the step S1, the pH of the enzyme-catalyzed reaction system includes 6.0-9.0; 6.5-8.5; 7.0-8.

0.

7. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: In step S1, the liquid reaction system includes a reaction solution, the reaction solution is a phosphate buffer system, and the liquid reaction system also includes coenzymes, the coenzymes include but are not limited to NADH, NADPH, NAD+, NADP+, glucose dehydrogenase, and the coenzymes are NADP+ and glucose dehydrogenase.

8. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: In the step S1, the liquid reaction system further comprises a co-substrate, which includes but is not limited to isopropanol, glucose, ammonium formate, and sodium formate.

9. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 1, characterized in that: The imine reductase encoding gene is a nucleic acid and an expression vector, wherein the nucleic acid is the encoding gene for the aforementioned imine reductase, and the expression vector is loaded with the imine reductase encoding gene of the nucleic acid.

10. The biological preparation method for synthesizing dihydroberberine based on imine reductase according to claim 9, characterized in that: The expression vector is a pET22a(+) vector. The recombinant expression vector is constructed by connecting the nucleic acid to various expression vectors. The expression vector includes a recombinant expression transformant. The recombinant expression transformant includes a host cell and a target gene transferred into the host cell. The target gene includes a gene encoding an imine reductase loaded with nucleic acid. The preparation method of the recombinant expression transformant is: transforming the expression vector into a host microorganism to obtain the obtained host microorganism. The host microorganism is preferably Escherichia coli E. coli; Escherichia coli BL21 (DE3); Escherichia coli DH5α.

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