TWI507527B - Method of manufacturing 6-hydroxyapigenin by biotransformation - Google Patents

Description

Method for preparing 6-hydroxy apigenin by biotransformation

The present invention relates to a method for preparing 6-hydroxy apigenin by biotransformation, in particular to a recombinant plastid having a fusion gene of CYP57B3 and sCPR, which can be produced in a large amount after being cultured for a period of time in a medium containing apigenin. - Method of hydroxy apigenin; the method of the present invention reveals for the first time that 6-hydroxy apigenin can be prepared by biotransformation.

According to flavonoids, flavonoids are an important secondary metabolite in plants. In recent years, flavonoids have been found to have many effects on human physiology, including antioxidants, prevention of osteoporosis, reduction of cardiovascular diseases and slowing of menopausal symptoms. . According to chemical structural differences, flavonoids can be mainly divided into eight subtypes: chalcones, flavones, flavanones, isoflavones, flavonols (flavonols). ), flavandiols, anthocyanins, and tannins. Recently, although many studies have confirmed that flavonoids have many different biological activities, low productivity is still the main reason for hindering the application of flavonoids to health foods or pharmaceutical products.

It is currently known that certain flavonoids can be synthesized by chemical conversion. However, the synthesis process usually involves toxic chemicals and extreme reaction conditions, making the entire preparation process difficult to scale up. In order to avoid these problems, inventors in related fields began to adopt The flavonoids are prepared by biosynthesis, such as microbial hosts, Escherichia coli and yeast, which may be helpful for mass production.

Cytochrome P450 monooxygenases (CYP) are a large class of heme-containing enzymes that catalyze various chemical reactions in a regioselective and hemi-containing enzymes. In general, such enzymes act in a similar manner to monooxygenases, introducing an oxygen atom in one oxygen molecule into the chemical structure of a substrate molecule, and a hydrogen donor must be present in another oxygen atom ( For example, NADPH or NADH, etc., reduces another oxygen atom to a water molecule. In chemical synthesis, the hydroxylation of non-activated carbon atoms can only be achieved by a free radical reaction. Most of this reaction does not have sufficient selectivity to add a chiral hydroxyl group to the hope. The location. Therefore, CYP's extraordinary ability to introduce oxygen into non-activated carbon-hydrogen bonds has attracted the attention of chemists and biochemistry. In the catalysis process, CYP must be accompanied by an electron donor: cytochrome P450 reductase (CPR) to transfer electrons from NADPH or NADH to the heme domain of CYP.

Aspergillus oryzae (A. oryzae) is the best known, soy isoflavones may convert the biological of microorganism ortho hydroxy derivative (ortho -hydroxyl derivatives) of (Biosci.Biotechnol.Biochem. 2007, 71, 1330-1333 . ). In general, most bacterial CYPs with flavonoid hydroxylase activity usually only catalyze one of the subfamilies of flavonoids; however, recent studies have indicated that CYP (CYP57B3) and Saccharomyces cerevisiae ( S) .cerevisiae ) CPR (sCPR) can biotransform flavonoids (genistein) in isoflavone and naringenin in flavanone ( Appl.Environ.Microbiol. 2011) , 77 , 3147-3150.), it can be seen that CYP57B3 is very different from the specificity of the receptor and the conventional CYP.

However, it is still unclear whether CYP57B3 can also biotransform apigenin in flavonoids. Therefore, this subject has become the direction of the inventors in related fields.

Nowadays, the inventor is in view of the fact that the above-mentioned existing methods for preparing flavonoids still have multiple defects in practical use, and thus it is a tireless spirit, and is supported by its rich professional knowledge and years of practical experience. Improvements have been made, and the present invention has been developed based on this.

The main object of the present invention is to provide a method for preparing 6-hydroxy apigenin by biotransformation, which mainly comprises biosynthesis of synthetic apigenin by CYP57B3 and sCPR to obtain 6-hydroxy apigenin (6- Hydroxyapigenin).

In order to achieve the above-mentioned object, the present invention provides a method for preparing 6-hydroxy apigenin by biotransformation, which comprises the following steps: Step 1: Amplification of CYP57B3 gene from cDNA of rice bran bacteria by polymerase chain reaction, and by Saccharomyces cerevisiae The gDNA regenerates a cytochrome reductase gene, wherein the cytochrome reductase gene sCPR (cytochrome P450 reductase) gene; step 2: CYP57B3 gene and cytochrome reductase gene are cleaved by the first and second sets of restriction enzymes, respectively Engaging to form a fusion gene; Step 3: cleavage of a pGAPZA vector with a third set of restriction enzymes, and ligating the fusion gene to the corresponding restriction site of the pGAPZA vector to form a circular recombinant plastid; 4: placing the circular recombinant plastid in a suitable microbial expression system, and culturing the microbial expression system for a period of time using a medium containing apigenin to produce 6-hydroxy apigenin; wherein the microbial expression system is Pichia pastoris .

In one embodiment of the invention, the first group of restriction enzymes is EcoR I/Bgl II, Two sets of restriction enzymes are Bgl II/XhoI, and a third set of restriction enzymes are EcoR I/XhoI.

In one embodiment of the invention, the medium is a YPD (yeast extract peptone dextrose) medium.

In one embodiment of the invention, the yield of 6-hydroxy apigenin can be up to 0.22 mg/L after a period of more than 72 hours.

The invention also provides a 6-hydroxy apigenin (6-Hydroxyapigenin) obtained by the above method, which is obtained by biotransformation of apigenin.

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First Figure: Flow chart of the preparation method of the preferred embodiment of the present invention

Second Figure: (A) Schematic diagram of a specific embodiment of the present invention (B) Schematic diagram of a cyclic recombinant plastid of a specific embodiment of the present invention

Figure 3: (A) HPLC analysis of standard apigenin, 6-hydroxy apigenin, 3-hydroxy apigenin (B) HPLC analysis of the product after 72 hours of fermentation.

Figure 4: Schematic diagram of the biotransformation of apigenin of the present invention

Figure 5: Analysis of yield of 6-hydroxy apigenin in a preferred embodiment of the invention

The object of the present invention and its structural and functional advantages will be explained in conjunction with the specific embodiments according to the structure shown in the following drawings, so that the reviewing committee can have a more in-depth and specific understanding of the present invention.

First, please refer to the first figure, which is a flow chart of the steps of the preparation method according to the preferred embodiment of the present invention. The present invention provides a method for preparing 6-hydroxy apigenin by biotransformation, which comprises the following steps: Step 1 (S1): utilizing polymerization The enzyme chain-locking reaction amplifies the CYP57B3 gene from the cDNA of Aspergillus oryzae , and a cytochrome reductase gene is amplified from the gDNA of Saccharomyces cerevisiae , wherein the cytochrome reductase gene sCPR (cytochrome P450) Reductase gene; step 2 (S2): the CYP57B3 gene and the cytochrome reductase gene are cleaved by the first and second restriction enzymes, respectively, and then joined to form a fusion gene; wherein, the first group of restriction enzymes is EcoR I/Bgl II, the second group of restriction enzymes is Bgl II/XhoI, and the third group of restriction enzymes is EcoR I/XhoI; Step 3 (S3): a pGAPZA vector is cleaved with a third group of restriction enzymes, and the fusion gene is ligated to The corresponding position of the pGAPZA vector is restricted to the cleavage site to form a circular recombinant plastid; and the fourth step (S4): the circular recombinant plastid is placed in a suitable microbial expression system, which can be, for example, placed in the Pichia Parent (Pichia pastoris) in using the medium containing apigenin (YPD medium may be for example) culturing a microbial expression systems This action time, to produce 6-hydroxy-apigenin (6-Hydroxyapigenin, scutellarein), in the longer duration of action The more 6-hydroxy apigenin can be produced, for example, when the action time is 72 hours, the yield of 6-hydroxy apigenin is 0.22 mg/L.

The invention also discloses a 6-Hydroxyapigenin prepared by the above method, which is obtained by biotransformation of apigenin.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

Briefly, the present invention converts apigenin to 6-hydroxyapinalin by means of biotransformation, and establishes a condition for mass production of 6-hydroxy apigenin in a fermentation tank.

Experiment 1: Construction of a circular recombinant plastid containing the fusion gene of CYP57B3 and sCPR

<Microorganisms and Materials>

A. oryzae (BCRC32288) and S. cerevisiae (ATCC57896) strains were obtained from the Bioresources Collection and Research Center (BCRC) and strains were cultured according to the BCRC protocol. Comprising Pichia (P.pastoris X-33), vector (plasmid vector pGAPZA ^TM), and antibiotics (Zeocin) the Pichia expression kit (Eazy Select ^TM Pichia expression kit) were purchased from Invitrogen (Carlsbad, CA ). Apigenin, 6-hydroxy apigenin, and 3'-hydroxy apigenin were purchased from Indofine Chemical Company (Hillsborough, NJ). All other chemicals were purchased from Tokyo Chemical Industry (Tokyo) and were all analytical reagent grades.

<Constructing Recombinant Pichia pastoris>

The present invention intends to integrate the rice CYP57B3 gene with the sCPR gene of Saccharomyces cerevisiae to form a fusion gene to investigate the biotransformation of apigenin. First, please refer to the second figure-(A), which is a schematic flow chart of a specific embodiment of the present invention, using generative PCR by polymerase chain reaction: respectively, from the cDNA of rice bran bacteria and the yeast of Saccharomyces cerevisiae The gDNA amplifies the CYP57B3 gene and the sCPR (cytochrome P450 reductase) gene, and the sequence of the primer pair used is shown in Table 1.

Next, the amplified CYP57B3 and sCPR genes were sequenced and cloned respectively on the EcoR I/Bgl II cleavage of the pETDuet-1 ^TM vector and the Bgl II/XhoI cleavage to form a fusion gene pET-CYPsCPR. . The fusion gene was digested with the restriction enzymes EcoR I and XhoI, and cloned into the corresponding restriction sites of the pGAPZA vector to form a circular recombinant plastid pGAP-CYPsCPR (as shown in the second figure-(B) Show). The pGAP-CYPsCPR was cleaved by restriction enzyme SpaI, and pGAP-CYPsCPR was transformed into a microbial expression system using electroporation according to the instructions of the expression kit ( P. pastoris X- 33) Formation of recombinant P. pastoris (Recombinant P. pastoris ). The recombinant was screened using the antibiotic zeocin, and the fusion gene was confirmed to be inserted into the genomic DNA of the recombinant using the above PCR method.

Experiment 2: Fermentation and HPLC analysis

The Pichia pastoris recombinant was cultured in 20 mL YPD (1% yeast extract, 2% peptone, 2% dextrose) medium at 28 ° C, 200 rpm. The medium still contained 100 μg/ml antibiotic Zeocin and 250 μM δ-aminolevulinic. Acid (heme precursor) and 25 mg/L apigenin. The analysis was carried out using high performance liquid chromatography (HPLC) at predetermined time intervals.

For large-scale reactions, 30 mL of seed culture was inoculated into a 7 L fermentation tank containing 3 L of YPD medium supplemented with 100 μg/ml antibiotic Zeocin and 250 μM. Δ-aminolevulinic acid, 25mg/L of parsley The phytochemical, and 1% (v/v) defoamer (Sigma), was then reacted at 28 ° C with aeration (0.5, v/v/m) and stirring (280 rpm). 10 mL of each culture was collected at different time points, and the content of 6-hydroxy apigenin was determined by HPLC analysis.

The operating conditions for HPLC analysis by analytical C18 reversed-phase column (Spherisorb, 5 μm, 4.6 id × 250 mm, ODS 2, Phase Separation, Deeside Industrial Park, Clwyd, UK) include: 1.5% (v/v) acetic acid aqueous solution A and acetonitrile solution B gradient elution, and 15% solution B initial isocratic elution for 4 minutes; Linear gradient 15% to 35% solution B is rinsed for 20 minutes, and 35% solution B is used for the last 10 minutes of isocratic elution at a flow rate of 1 mL/min; all samples are The absorbance was detected at 269 nm. A standard curve obtained by HPLC analysis of the standard was calculated, and the resulting 6-hydroxyapigenin content was calculated.

Experimental result

Result one

P. pastoris has been widely used in industrial utilization and basic research, and it is known that Pichia cannot itself digest apigenin (data not shown). Thus, in this embodiment based Pichia expression kit (Eazy Select ^TM Pichia expression kit) Construction of a recombinant microorganism of the present specific embodiment. The ability of CPR to transfer electrons to the corresponding cytochrome P450 plays a crucial role in the catalytic efficiency of CYP. Some synthetic self-sufficient P450 fusions are non-self-sufficient. P450) has a high catalytic activity after CPR. Thereby, the inventors fused CYP57B3 and sCPR to study the biotransformation reaction of apigenin.

The construction of this fusion gene has two important features: (1) removing the stop codon of CYP57B3, and placing an Arg-Ser linker sequence containing the BglII restriction enzyme cleavage site into CYP57B3; (2) removing the sCPR membrane - the first 41 amino acids of the membrane-binding domain, and an Arg-Ser-Pro (RSP) ligation sequence is placed in the N-terminal region of sCPR (as shown in Figure 2 - (B)).

The results showed that apigenin was biotransformed from a recombinant P. pastoris having a circular recombinant plastid (pGAP-CYPsCPR) to produce other derivatives (hydroxylation products).

Result two

In the Pichia pastoris recombinant fermentation process, the standard was used as a reference by HPLC, and the biotransformed hydroxylated product was identified. Please refer to the third figure-(A) for the HPLC analysis of the standard apigenin, 6-hydroxy apigenin, 3-hydroxy apigenin, the vertical axis is the absorbance measured at 269 nm, and the horizontal axis is retained. Retention time; the residence time of the three standards is: 6-hydroxyapigenin-17.7 minutes, 3'-hydroxyapigenin-22.1 minutes, apigenin -27.1 minutes.

Please refer to the third figure-(B) for the HPLC analysis of the product for 72 hours of fermentation. The results showed that compared to the HPLC results measured without the addition of apigenin, two new peaks were produced after the addition of apigenin, wherein the residence time was 17.7 minutes compared to the standard of the third figure-(A). The new peak is 6-hydroxy apigenin, and the other new peak is the unknown product, which is not discussed here. This result confirms CYP57B3 apigenin may be converted into its biologically ortho hydroxy derivative (ortho -hydroxyl derivatives) a 6-hydroxy-apigenin (as shown in FIG. IV).

In order to further expand the biotransformation reaction using recombinant Pichia pastoris, the present invention The fermentation was carried out in a 7 L fermentation tank containing 3 L of YPD medium. Fermentation samples at different time points were collected and the yield of 6-hydroxy apigenin was calculated from a standard curve obtained from HPLC analytical standards. Please refer to the fifth figure for the analysis of the yield of 6-hydroxy apigenin according to a preferred embodiment of the present invention. The vertical axis represents the yield of 6-hydroxyapigenin (■) and biomass (biomass, □), respectively. The axis represents the time of fermentation (culture); the results show that after the initial concentration of 25 mg/L of apigenin is added for fermentation, the yield of 6-hydroxy apigenin increases with the increase of fermentation time (24 hours to 72 hours). At the 72 hour time point, the highest yield of 6-hydroxy apigenin was 0.22 mg/L.

In summary, the method of the present invention reveals for the first time that 6-hydroxy apigenin can be prepared by biotransformation: biotransformation of apigenin to 6-hydroxy apigenin by CYP57B3 synergistic with sCPR of rice bran bacteria; The yield of 6-hydroxy apigenin can be as high as 0.22 mg/L in a 7 L fermentation tank.

It can be seen from the above description that the present invention has the following advantages compared with the prior art:

1. The present invention is the first method for synthesizing 6-hydroxy apigenin by biotransformation, and its yield can be as high as 0.22 mg/L.

2. Prior art points out that the CYP57B3 gene of rice bran bacteria can biotransform genistein and daidzein in the isoflavone, and naringenin in flavanone. The present invention further confirms that the CYP57B3 gene can also biotransform flavonoid apigenin.

In summary, the method for preparing 6-hydroxy apigenin by biotransformation of the present invention can achieve the intended use efficiency by the above disclosed embodiments, and the present invention has not been disclosed before the application. Full compliance with the requirements and requirements of the Patent Law.提出Proposed according to law If you apply for a patent, you can ask for a review and grant a patent.

The illustrations and descriptions of the present invention are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention; those skilled in the art, which are characterized by the scope of the present invention, Equivalent variations or modifications are considered to be within the scope of the design of the invention.

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Claims (5)

A method for preparing 6-hydroxy apigenin by biotransformation, comprising the following steps: Step 1: Amplification of CYP57B3 gene from cDNA of Aspergillus oryzae by polymerase chain reaction, and Saccharomyces cerevisiae The gDNA regenerates a cytochrome reductase gene, wherein the cytochrome reductase gene sCPR (cytochrome P450 reductase) gene; step two: the CYP57B3 gene and the cytochrome reductase gene are respectively the first and second sets of restriction enzymes Cutting, recombining to form a fusion gene; Step 3: cleavage of a pGAPZA vector with a third set of restriction enzymes, and ligating the fusion gene to the corresponding restriction site of the pGAPZA vector to form a circular recombination Plastid; and step 4: placing the circular recombinant plastid in a suitable microbial expression system, and culturing the microbial expression system for a period of time using a medium containing apigenin to produce 6-hydroxy apigenin (6- Hydroxyapigenin). The method for preparing 6-hydroxy apigenin by biotransformation according to claim 1, wherein the first group of restriction enzymes is EcoR I/Bgl II, and the second group of restriction enzymes is Bgl II/XhoI. The third group of restriction enzymes is EcoR I/XhoI. A method for producing 6-hydroxy apigenin by biotransformation as described in claim 1, wherein the microorganism expression system is Pichia pastoris . A method for producing 6-hydroxy apigenin by biotransformation as described in claim 1, wherein the medium is YPD (yeast extract peptone dextrose) medium. The method for producing 6-hydroxy apigenin by biotransformation as described in claim 1, wherein the 6-hydroxy apigenin yield is 0.22 mg/L after the action time exceeds 72 hours.