Method for splitting flavonoid C-C glucosidic bond by using enzyme method

Abstract

The invention discloses a method for splitting a flavonoid C-C glucosidic bond by using an enzyme method. The invention provides application of a protein combination A or a protein combination B, namely application in splitting C-C glucosidic bonds. Application in preparation of aglycones; the invention also relates to an application in converting glucoside into aglycone. The invention also relates to an application in converting isoorientin and isovitexin into luteolin and apigenin. The invention also relates to an application in converting isoorientin into luteolin. The invention also relates to an application in converting isovitexin into apigenin. The protein combination A is an LD1 protein, an LD2 protein and an LD3 protein. And the protein combination B is an LD4 protein, an LD2 protein and an LD3 protein. The method has great application and popularization values in the production field of aglycones or aglycone derivatives and downstream products thereof.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for enzymatically cleaving the C-glycosidic bonds of flavonoid C-glycosides. Background Technology

[0002] Luteolin: A natural flavonoid compound found in various plants, possessing diverse pharmacological activities such as anti-inflammatory, anti-allergic, uric acid-lowering, anti-tumor, antibacterial, and antiviral effects. Clinically, luteolin is mainly used for cough relief, expectoration, anti-inflammation, uric acid lowering, treatment of cardiovascular diseases, treatment of amyotrophic lateral sclerosis (ALS), treatment of SARS, and treatment of hepatitis.

[0003] Apigenin, also known as apigenin flavonoid, is a flavonoid compound widely distributed in nature. It is mainly found in plants of the Thymelaeaceae, Verbenaceae, and Selaginellaceae families, and is widely distributed in warm and tropical vegetables and fruits, especially in celery. Apigenin has anti-tumor, cardiovascular protective, anti-neurodegenerative disease, and anti-type 2 diabetes functions.

[0004] Because the glycosyl group is directly linked to the aglycone via a C-C bond, the C-glycoside structure is relatively stable and not easily hydrolyzed. Compared with chemical methods, biological methods offer advantages such as good chemoselectivity, regioselectivity, and stereoselectivity; fast reaction rates; high product conversion rates; mild reaction conditions; environmental friendliness; simple conversion process; short cycle time; and low cost. The use of biocatalysis holds promise for the efficient cleavage of C-C glycosidic bonds, laying the foundation for the application of C-deglycosylation of C-glycosides. Summary of the Invention

[0005] The purpose of this invention is to provide a method for enzymatically cleaving the C-glycosidic bonds of flavonoid C-glycosides.

[0006] This invention provides applications of protein combination A or protein combination B, as follows (a1) or (a2) or (a3) ​​or (a4) or (a5) or (a6):

[0007] (a1) Application in cleaving C-glycosidic bonds;

[0008] (a2) Application in the preparation of aglycones;

[0009] (a3) Applications in converting glycosides into aglycones;

[0010] (a4) Application in the conversion of isoharmone and isovitexin into luteolin and apigenin;

[0011] (a5) Application in the conversion of isopropionol to luteolin;

[0012] (a6) Application in the conversion of isovitexin to apigenin.

[0013] The present invention also provides applications of protein combination A or protein combination B, as follows (b1) or (b2) or (b3) or (b4) or (b5) or (b6):

[0014] (b1) Application in the preparation of luteolin and apigenin;

[0015] (b2) Application in the preparation of luteolin and apigenin from isoherbine and isovitexin;

[0016] (b3) Application in the preparation of luteolin;

[0017] (b4) Application in the preparation of luteolin from isochorin;

[0018] (b5) Application in the preparation of apigenin;

[0019] (b6) Application in the preparation of apigenin from isovitexin.

[0020] This invention also provides the application of biomaterial A or biomaterial B in the preparation of products;

[0021] The uses of the product are as follows (c1) or (c2) or (c3) or (c4) or (c5) or (c6):

[0022] (c1) Cleavage of C-glycosidic bonds;

[0023] (c2) Preparation of aglycones;

[0024] (c3) Converting glycosides into aglycones;

[0025] (c4) Convert isoharmonin and isovitexin into luteolin and apigenin;

[0026] (c5) Convert isorhizonine to luteolin;

[0027] (c6) Convert isovitexin into apigenin.

[0028] This invention also provides the application of biomaterial A or biomaterial B in the preparation of products;

[0029] The uses of the product are as follows (d1) or (d2) or (d3) or (d4) or (d5) or (d6):

[0030] (d1) Preparation of luteolin and apigenin;

[0031] (d2) Luteolin and apigenin were prepared using isoharmone and isovitexin as raw materials;

[0032] (d3) Preparation of luteolin;

[0033] (d4) Luteolin was prepared using isochorin as a raw material;

[0034] (d5) Preparation of apigenin;

[0035] (d6) Apigenin was prepared using isovitexin as a raw material.

[0036] The present invention also provides a method for preparing luteolin and apigenin, comprising the following steps: using isoherbine and isovitexin as raw materials, and converting them using protein combination A or protein combination B to obtain luteolin and apigenin.

[0037] The present invention also provides a method for preparing luteolin and apigenin, comprising the following steps: using isoharonium and isovitelline as raw materials, and converting them using biological material A or biological material B to obtain luteolin and apigenin.

[0038] The present invention also provides a method for preparing luteolin, comprising the following steps: using isorhizonine as raw material, and converting it using protein combination A or protein combination B to obtain luteolin.

[0039] The present invention also provides a method for preparing luteolin, comprising the following steps: using isoherbine as a raw material, and converting it using biological material A or biological material B to obtain luteolin.

[0040] The present invention also provides a method for preparing apigenin, comprising the following steps: using isovitexin as raw material, and converting it using protein combination A or protein combination B to obtain apigenin.

[0041] The present invention also provides a method for preparing apigenin, comprising the following steps: using isovitexin as a raw material, and converting it using biological material A or biological material B to obtain apigenin.

[0042] The protein combination A described above consists of LD1 protein, LD2 protein, and LD3 protein.

[0043] The protein combination B described above consists of LD4 protein, LD2 protein, and LD3 protein.

[0044] The above-mentioned biomaterial A is either an LD1 protein-related biomaterial or an LD2 / LD3 protein-related biomaterial.

[0045] The biomaterial B mentioned above is either an LD4 protein-related biomaterial or an LD2 / LD3 protein-related biomaterial.

[0046] The LD1 protein is either (e1) or (e2) or (e3):

[0047] (e1) A protein with the amino acid sequence shown in SEQ ID NO: 1;

[0048] (e2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (e1);

[0049] (e3) is a protein that has more than 80% identity with (e1) and / or has the same function.

[0050] Preferably, the protein described in (e3) is derived from a strain of the genus Hungatella.

[0051] The LD2 protein is either (e4) or (e5) or (e6):

[0052] (e4) Proteins with amino acid sequences as shown in SEQ ID NO: 3;

[0053] (e5) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (e4);

[0054] (e6) and (e4) are proteins with more than 80% identity or / and the same function.

[0055] Preferably, the protein described in (e6) is derived from a strain of the genus Hungatella.

[0056] The LD3 protein is either (e7) or (e8) or (e9):

[0057] (e7) Proteins with amino acid sequences as shown in SEQ ID NO: 5;

[0058] (e8) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (e7);

[0059] (e9) and (e7) are proteins with more than 80% identity or / and the same function.

[0060] Preferably, the protein described in (e9) is derived from a strain of the genus Hungatella.

[0061] The LD4 protein is as follows (e10) or (e11) or (e12):

[0062] (e10) Protein with the amino acid sequence shown in SEQ ID NO: 7;

[0063] (e11) The fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of (e10);

[0064] (e12) is a protein that has more than 80% identity with (e10) and / or has the same function.

[0065] Preferably, the protein described in (e12) is derived from a strain of the genus Hungatella.

[0066] The LD1 protein-related biomaterials are nucleic acid molecules encoding the LD1 protein, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, or recombinant microorganisms containing the nucleic acid molecules or metabolites of the recombinant microorganisms.

[0067] The LD2 / LD3 protein-related biomaterials are nucleic acid molecules encoding LD2 and LD3 proteins, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, or recombinant microorganisms containing the nucleic acid molecules or metabolites of the recombinant microorganisms.

[0068] The LD4 protein-related biomaterials are nucleic acid molecules encoding the LD4 protein, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, or recombinant microorganisms containing the nucleic acid molecules or metabolites of the recombinant microorganisms.

[0069] Specifically, the nucleic acid molecule encoding the LD1 protein is any one of the following (f1) to (f3):

[0070] (f1) A DNA molecule with a coding sequence as shown in SEQ ID NO: 2;

[0071] (f2) and (f1) are DNA molecules that have more than 80% identity and encode the protein;

[0072] (f3) A DNA molecule that hybridizes to the specified nucleotide sequence of (f1) under stringent conditions and encodes the protein.

[0073] Preferably, the DNA molecule described in (f2) is derived from a strain of the genus Hungatella.

[0074] Specifically, nucleic acid molecules encoding LD2 and LD3 proteins contain segments encoding LD2 and LD3 proteins, respectively.

[0075] Specifically, the segment encoding the LD2 protein is any one of the following (f4) to (f6):

[0076] (f4) A DNA molecule with a coding sequence as shown in SEQ ID NO: 4;

[0077] (f5) and (f4) are DNA molecules that have more than 80% identity and encode the protein;

[0078] (f6) A DNA molecule that hybridizes to the specified nucleotide sequence of (f4) under stringent conditions and encodes the protein.

[0079] Preferably, the DNA molecule described in (f5) is derived from a strain of the genus Hungatella.

[0080] Specifically, the segment encoding the LD3 protein is any one of the following (f7) to (f9):

[0081] (f7) A DNA molecule with a coding sequence as shown in SEQ ID NO: 6;

[0082] (f8) and (f7) to a DNA molecule that has more than 80% identity and encodes the protein;

[0083] (f9) A DNA molecule that hybridizes to the specified nucleotide sequence of (f7) under stringent conditions and encodes the protein.

[0084] Preferably, the DNA molecule described in (f8) is derived from a strain of the genus Hungatella.

[0085] Specifically, the nucleic acid molecule encoding the LD4 protein is any one of the following (f10) to (f12):

[0086] (f10) A DNA molecule with a coding sequence as shown in SEQ ID NO: 8;

[0087] (f11) and (f10) to a DNA molecule that has more than 80% identity and encodes the protein;

[0088] (f12) A DNA molecule that hybridizes to the specified nucleotide sequence of (f10) under stringent conditions and encodes the protein.

[0089] Preferably, the DNA molecule described in (f11) is derived from a strain of the genus Hungatella.

[0090] The aforementioned 80% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0091] The 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or higher level of identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0092] The stringent conditions described above are hybridization in a 2×SSC, 0.1% SDS solution at 68°C with two washes of 5 min each, followed by hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C with two washes of 15 min each.

[0093] For example, the recombinant vector containing the nucleic acid molecule may be the recombinant expression plasmid pSmartI-LD1, the recombinant expression plasmid pSmartIII-LD2 / LD3, or the recombinant expression plasmid pSmartI-LD4.

[0094] The small fragment between the BamHI and HindIII restriction sites in the pSmartI plasmid was replaced with the LD1 gene shown in SEQ ID NO: 2 (while keeping the other sequences of the pSmartI plasmid unchanged) to obtain the recombinant expression plasmid pSmartI-LD1.

[0095] Using pSmartIII plasmid as the starting plasmid, the small fragment between the BamHI and HindIII restriction sites was replaced with the LD2 gene shown in SEQ ID NO:4, and the small fragment between the MfeI and KpnI restriction sites was replaced with the LD3 gene shown in SEQ ID NO:6. The other sequences of pSmartIII plasmid remained unchanged, resulting in the recombinant expression plasmid pSmartIII-LD2 / LD3.

[0096] The small fragment between the BamHI and HindIII restriction sites in the pSmartI plasmid was replaced with the LD4 gene shown in SEQ ID NO: 8 (while keeping the other sequences of the pSmartI plasmid unchanged) to obtain the recombinant expression plasmid pSmartI-LD4.

[0097] For example, the recombinant microorganism containing the nucleic acid molecule is engineered bacteria BL21 / pSmartI-LD1, BL21 / pSmartIII-LD2 / LD3, or BL21 / pSmartI-LD4.

[0098] Plasmid pSmartI-LD1 was introduced into Escherichia coli BL21(DE3) to obtain engineered strain BL21 / pSmartI-LD1.

[0099] Plasmid pSmartIII-LD2 / LD3 was introduced into Escherichia coli BL21(DE3) to obtain engineered strain BL21 / pSmartIII-LD2 / LD3.

[0100] Plasmid pSmartI-LD4 was introduced into Escherichia coli BL21(DE3) to obtain engineered strain BL21 / pSmartI-LD4.

[0101] Specifically, the metabolites of the recombinant microorganism are the total cell protein of the recombinant microorganism after being induced to express by IPTG.

[0102] Specifically, the method for preparing the metabolites of the recombinant microorganism is as follows: the recombinant microorganism is induced to express by IPTG, then the bacterial cells are collected and broken, and then the supernatant is obtained by centrifugation.

[0103] For example, the method for preparing the metabolites of the recombinant microorganism includes the following steps:

[0104] The recombinant microorganism was induced and cultured with IPTG; then, the bacterial cells were collected, and the cells were suspended in Tris-HCl buffer to obtain a bacterial solution (5 mg bacterial cells / ml, with the bacterial cells measured by wet weight of the precipitate); then, the bacterial solution was taken, the cells were lysed, and the supernatant was collected by centrifugation, which is the metabolite.

[0105] For example, the method for preparing the metabolites of the recombinant microorganism includes the following steps:

[0106] ① The recombinant microorganisms were inoculated into liquid LB medium and cultured at 37°C and 200 rpm until OD reached. 600nm The value is 0.8;

[0107] ②After completing step ①, add IPTG to the system and make its concentration in the system 0.5mM, and then incubate at 15℃ and 165rpm for 20 hours;

[0108] ③ After completing step ②, centrifuge at 4000g for 15 minutes and collect the bacterial precipitate;

[0109] ④ Take the bacterial precipitate obtained in step ③, add Tris-HCl buffer (pH 8.0, 50mM) to suspend it, and obtain the bacterial solution (5mg bacterial cells / ml, bacterial cells are based on the wet weight of the bacterial precipitate);

[0110] ⑤ Take the bacterial culture obtained in step ④, pressure break it (using a Constant high-pressure cell disruptor at a pressure of 20 klbs per square inch), then centrifuge at 4°C and 4000g for 25 min, and collect the supernatant, which is the metabolic product.

[0111] Specifically, the recombinant microorganism is a recombinant microorganism that has undergone IPTG-induced expression.

[0112] Specifically, the method for preparing recombinant microorganisms after IPTG-induced expression includes the following steps: inducing IPTG culture in the recombinant microorganisms; then, collecting the bacterial cells, adding Tris-HCl buffer to suspend them, and obtaining bacterial solution (5 mg bacterial cells / ml, bacterial cells are measured by wet weight of bacterial cell precipitate).

[0113] Specifically, the preparation method of recombinant microorganisms after IPTG-induced expression includes the following steps:

[0114] ① The recombinant microorganisms were inoculated into liquid LB medium and cultured at 37°C and 200 rpm until OD reached. 600nm The value is 0.8;

[0115] ②After completing step ①, add IPTG to the system and make its concentration in the system 0.5mM, and then incubate at 15℃ and 165rpm for 20 hours;

[0116] ③ After completing step ②, centrifuge at 4000g for 15 minutes and collect the bacterial precipitate;

[0117] ④ Take the bacterial precipitate obtained in step ③, add Tris-HCl buffer (pH 8.0, 50mM) to suspend it, and obtain the bacterial solution (5mg bacterial cells / ml, bacterial cells are based on the wet weight of the bacterial precipitate).

[0118] For example, the ratio of LD1 protein-related biomaterials to LD2 / LD3 protein-related biomaterials is equal in volume.

[0119] For example, the ratio of LD4 protein-related biomaterials to LD2 / LD3 protein-related biomaterials is equal in volume.

[0120] For example, the ratio of LD1 protein-related biomaterials to LD2 / LD3 protein-related biomaterials is equal in mass.

[0121] For example, the ratio of LD4 protein-related biomaterials to LD2 / LD3 protein-related biomaterials is equal in mass.

[0122] For example, the conversion temperature described above is 34-40°C.

[0123] For example, the conversion temperature described above is 37°C.

[0124] For example, the conversion time for any of the above-described conversions can be 9-36 hours.

[0125] For example, the conversion time for any of the above-described conversions could be 24 hours.

[0126] Specifically, the C-glycosidic bond is a flavonoid C-glycosidic bond.

[0127] For example, any of the above-mentioned C-glycosidic bonds are C-glycosidic bonds of flavonoid C-glycosides.

[0128] For example, any of the glycosides mentioned above are glycoside compounds.

[0129] For example, any of the glycosides mentioned above are flavonoid C-glycosides.

[0130] For example, any of the glycosides mentioned above are flavonoid C-glycosides.

[0131] For example, any of the aglycones mentioned above are flavonoid C-glycosides.

[0132] For example, any of the glycosides mentioned above may be isochorin or isovitexin.

[0133] For example, any of the aglycones mentioned above may be luteolin or apigenin.

[0134] This invention has significant application and promotion value in the production of aglycones or aglycone derivatives and their downstream products. Attached Figure Description

[0135] Figure 1 The HPLC chromatograms are of the standards isoharbitis and isovitexin.

[0136] Figure 2 The HPLC chromatograms are for the standards luteolin and apigenin.

[0137] Figure 3 The HPLC chromatogram of the product solution obtained by biotransformation using LD1-LD2 / LD3 is shown.

[0138] Figure 4 This is an HPLC chromatogram of the product solution obtained by biotransformation using LD4-LD2 / LD3. Detailed Implementation

[0139] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. The described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0140] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.

[0141] Isopropercin, molecular formula C 21 H 20 O 11 The CAS number is 4261-42-1, and the structural formula is shown in formula (Ⅰ).

[0142]

[0143] Luteolin, molecular formula C 15 H 10 O6, CAS number 491-70-3, has the structural formula shown in formula (Ⅱ).

[0144]

[0145] Isovitilione, with the molecular formula C 21 H 20 O 10 The CAS number is 29702-25-8, and the structural formula is shown in formula (Ⅲ).

[0146]

[0147] Apigenin, with the molecular formula C 15 H 10 O5, CAS number 520-36-5, has the structural formula shown in formula (Ⅳ).

[0148]

[0149] Example 1: Discovery of protein genes

[0150] Four proteins and their encoding genes were discovered in Hungatella sp.

[0151] LD1 protein, as shown in SEQ ID NO: 1. LD1 gene, as shown in SEQ ID NO: 2.

[0152] LD2 protein, as shown in SEQ ID NO: 3. LD2 gene, as shown in SEQ ID NO: 4.

[0153] LD3 protein, as shown in SEQ ID NO: 5. LD3 gene, as shown in SEQ ID NO: 6.

[0154] LD4 protein, as shown in SEQ ID NO: 7. LD4 gene, as shown in SEQ ID NO: 8.

[0155] Example 2: Preparation of engineered bacteria and preparation of enzymes using engineered bacteria

[0156] I. Construction of recombinant expression plasmids

[0157] The small fragment between the BamHI and HindIII restriction sites in the pSmartI plasmid was replaced with the LD1 gene shown in SEQ ID NO: 2 (while keeping the other sequences of the pSmartI plasmid unchanged) to obtain the recombinant expression plasmid pSmartI-LD1.

[0158] The small fragment between the BamHI and HindIII restriction sites in the pSmartI plasmid was replaced with the LD4 gene shown in SEQ ID NO: 8 (while keeping the other sequences of the pSmartI plasmid unchanged) to obtain the recombinant expression plasmid pSmartI-LD4.

[0159] Using pSmartIII plasmid as the starting plasmid, the small fragment between the BamHI and HindIII restriction sites was replaced with the LD2 gene shown in SEQ ID NO:4, and the small fragment between the MfeI and KpnI restriction sites was replaced with the LD3 gene shown in SEQ ID NO:6. The other sequences of pSmartIII plasmid remained unchanged, resulting in the recombinant expression plasmid pSmartIII-LD2 / LD3.

[0160] pSmartI plasmid, a circular plasmid, is shown in SEQ ID NO: 9.

[0161] pSmartIII plasmid, a circular plasmid, is shown in SEQ ID NO: 10.

[0162] II. Preparation of enzyme solution from engineered bacteria

[0163] 1. Plasmid pSmartI-LD1 was introduced into *E. coli* BL21(DE3) to obtain engineered bacteria BL21 / pSmartI-LD1. Plasmid pSmartIII-LD2 / LD3 was introduced into *E. coli* BL21(DE3) to obtain engineered bacteria BL21 / pSmartIII-LD2 / LD3. Plasmid pSmartI-LD4 was introduced into *E. coli* BL21(DE3) to obtain engineered bacteria BL21 / pSmartI-LD4.

[0164] 2. Inoculate the recombinant bacteria into liquid LB medium and incubate at 37°C and 200 rpm until OD reaches zero. 600nm The value is 0.8.

[0165] 3. After completing step 2, add IPTG to the system and make its concentration 0.5mM, then incubate at 15℃ and 165rpm for 20 hours.

[0166] 4. After completing step 3, centrifuge at 4000g for 15 minutes and collect the bacterial precipitate.

[0167] 5. Take the bacterial precipitate obtained in step 4, add Tris-HCl buffer (pH 8.0, 50mM) to suspend it, and obtain the bacterial solution (5mg bacterial cells / ml, bacterial cells are based on the wet weight of the bacterial precipitate).

[0168] 6. Take the bacterial culture obtained in step 5, pressure break it (using a Constant high-pressure cell disruptor at a pressure of 20 klbs per square inch), then centrifuge at 4°C and 4000g for 25 minutes, and collect the supernatant, which is the enzyme solution.

[0169] When the recombinant bacteria is the engineered strain BL21 / pSmartI-LD1, the enzyme solution obtained from steps 2 to 6 above is named LD1 enzyme solution. When the recombinant bacteria is the engineered strain BL21 / pSmartIII-LD2 / LD3, the enzyme solution obtained from steps 2 to 6 above is named LD2 / LD3 enzyme solution. When the recombinant bacteria is the engineered strain BL21 / pSmartI-LD4, the enzyme solution obtained from steps 2 to 6 above is named LD4 enzyme solution.

[0170] Example 3: Recombinase-mediated biotransformation

[0171] I. Application of LD1-LD2 / LD3 in biotransformation

[0172] 1. Prepare the reaction system, and then convert it at 37°C for 24 hours.

[0173] Composition of the reaction system (1 ml): containing 0.45 ml of LD1 enzyme solution prepared in Example 2, 0.45 ml of LD2 / LD3 enzyme solution prepared in Example 2, DTT, Mn 2+(Provided by manganese dioxide), NAD + (Provided by β-nicotinamide adenine dinucleotide hydrate), isothiazolinone and isovitexin, with the balance being Tris-HCl buffer (pH 8.0, 50 mM). In the reaction system, the concentration of DTT was 10 mM, and Mn... 2+ At a concentration of 1 mM, NAD + The concentration was 1 mM, the concentration of isoharbitis was 0.5 mM, and the concentration of isovitexin was 0.5 mM.

[0174] 2. After completing step 1, take the entire reaction system, add 3 times the volume of n-butanol, vortex for 10 min, then centrifuge at 4000g for 15 min, and then collect the organic phase.

[0175] 3. Take the organic phase obtained in step 2, concentrate it under reduced pressure at 38°C to remove the solvent, and redissolve the residue in methanol to obtain the catalytic product solution.

[0176] 4. Take the catalytic product solution and perform HPLC analysis.

[0177] Column: Agilent XDB-C18 (4.6×150mm, 5μm);

[0178] Sample introduction method: direct injection via autosampler; detection wavelength: 258 nm; column temperature: 35 °C;

[0179] Mobile phase: Phase A, Phase B, or a mixture of Phase A and Phase B; Phase B is a 0.2% (v / v) aqueous solution of acetic acid; Phase A is acetonitrile; Mobile phase flow rate: 1 mL / min;

[0180] Elution process: 0-5 min, phase A accounts for 10% of the mobile phase volume fraction, and the corresponding phase B accounts for 90% of the mobile phase volume fraction; 5-20 min, the volume fraction of phase A in the mobile phase linearly increases from 10% to 30%, and the corresponding volume fraction of phase B in the mobile phase linearly decreases from 90% to 70%; 20-30 min, the volume fraction of phase A in the mobile phase linearly increases from 30% to 100%, and the corresponding volume fraction of phase B in the mobile phase linearly decreases from 70% to 0%.

[0181] The standards for isoharmone (Chengdu Phytostandardized Pure Biotechnology Co., Ltd., PCS0497), isovitexin (Chengdu Phytostandardized Pure Biotechnology Co., Ltd., PCS0647), luteolin (Chengdu Phytostandardized Pure Biotechnology Co., Ltd., PCS0625), and apigenin (Chengdu Phytostandardized Pure Biotechnology Co., Ltd., PCS0751) were analyzed by HPLC according to the above parameters. The HPLC chromatograms of the standards isoharmone and isovitexin are shown below. Figure 1The retention times corresponding to the peak values ​​were 12.351 min and 14.355 min, respectively. The HPLC chromatograms of the standards luteolin and apigenin are shown below. Figure 2 The retention times corresponding to the peak values ​​were 21.929 min and 24.018 min, respectively.

[0182] The HPLC chromatogram of the catalytic product solution is shown in [reference needed]. Figure 3 The thick blue arrows indicate the substrates—isosalicylin and isovitexin, respectively. The thin red arrows indicate the products—luteolin and apigenin, respectively.

[0183] II. Application of LD4-LD2 / LD3 in biotransformation

[0184] Use the LD4 enzyme solution prepared in Example 2 instead of the LD1 enzyme solution, and follow the same procedure as in step one.

[0185] The HPLC chromatogram of the catalytic product solution is shown in [reference needed]. Figure 4 The thick blue arrows indicate the substrates—isosalicylin and isovitexin, respectively. The thin red arrows indicate the products—luteolin and apigenin, respectively.

[0186] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of protein combination A or protein combination B is as follows (a1) or (a2) or (a3) ​​or (a4) or (a5) or (a6): (a1) Application in cleaving C-glycosidic bonds; (a2) Application in the preparation of aglycones; (a3) Applications in converting glycosides into aglycones; (a4) Application in the conversion of isoharmone and isovitexin into luteolin and apigenin; (a5) Application in the conversion of isopropionol to luteolin; (a6) Application in the conversion of isovitexin to apigenin; The protein combination A consists of LD1 protein, LD2 protein, and LD3 protein; The protein combination B consists of LD4 protein, LD2 protein, and LD3 protein; The LD1 protein is either (e1) or (e2) or (e3): (e1) A protein with the amino acid sequence shown in SEQ ID NO: 1; (e2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (e1); (e3) is a protein that has more than 80% identity with (e1) and / or has the same function; The LD2 protein is either (e4) or (e5) or (e6): (e4) Proteins with amino acid sequences as shown in SEQ ID NO: 3; (e5) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (e4); (e6) and (e4) are proteins with more than 80% identity or / and the same function; The LD3 protein is either (e7) or (e8) or (e9): (e7) Proteins with amino acid sequences as shown in SEQ ID NO: 5; (e8) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of (e7); (e9) and (e7) are proteins with more than 80% identity or / and the same function; The LD4 protein is as follows (e10) or (e11) or (e12): (e10) Protein with the amino acid sequence shown in SEQ ID NO: 7; (e11) The fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of (e10); (e12) is a protein that has more than 80% identity with (e10) and / or has the same function.

2. The application of protein combination A or protein combination B is as follows: (b1) or (b2) or (b3) or (b4) or (b5) or (b6): (b1) Application in the preparation of luteolin and apigenin; (b2) Application in the preparation of luteolin and apigenin from isoherbine and isovitexin; (b3) Application in the preparation of luteolin; (b4) Application in the preparation of luteolin from isochorin; (b5) Application in the preparation of apigenin; (b6) Application in the preparation of apigenin from isovitexin; Protein combination A is as described in claim 1; protein combination B is as described in claim 1.

3. Application of biomaterial A or biomaterial B in product preparation; The uses of the product are as follows (c1) or (c2) or (c3) or (c4) or (c5) or (c6): (c1) Cleavage of C-glycosidic bonds; (c2) Preparation of aglycones; (c3) Converting glycosides into aglycones; (c4) Convert isoharmonin and isovitexin into luteolin and apigenin; (c5) Convert isorhizonine to luteolin; (c6) Converting isovitexin into apigenin; The biomaterial A is an LD1 protein-related biomaterial and an LD2 / LD3 protein-related biomaterial; The biomaterial B is an LD4 protein-related biomaterial and an LD2 / LD3 protein-related biomaterial; The LD1 protein-related biomaterials are nucleic acid molecules encoding LD1 protein, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, or recombinant microorganisms containing the nucleic acid molecules or metabolites of the recombinant microorganisms. The LD2 / LD3 protein-related biomaterials are nucleic acid molecules encoding LD2 and LD3 proteins, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, or recombinant microorganisms containing the nucleic acid molecules or metabolites of the recombinant microorganisms. The LD4 protein-related biomaterials are nucleic acid molecules encoding LD4 protein, expression cassettes containing the nucleic acid molecules, recombinant vectors containing the nucleic acid molecules, or recombinant microorganisms containing the nucleic acid molecules or metabolites of the recombinant microorganisms. The LD1 protein is as described in claim 1; the LD2 protein is as described in claim 1; the LD3 protein is as described in claim 1; and the LD4 protein is as described in claim 1.

4. Application of biomaterial A or biomaterial B in product preparation; The uses of the product are as follows (d1) or (d2) or (d3) or (d4) or (d5) or (d6): (d1) Preparation of luteolin and apigenin; (d2) Luteolin and apigenin were prepared using isoharmone and isovitexin as raw materials; (d3) Preparation of luteolin; (d4) Luteolin was prepared using isochorin as a raw material; (d5) Preparation of apigenin; (d6) Apigenin was prepared from isovitexin; The biomaterial A is as described in claim 3; the biomaterial B is as described in claim 3.

5. A method for preparing luteolin and apigenin, comprising the following steps: using isoherbine and isovitexin as raw materials, and converting them using protein combination A or protein combination B to obtain luteolin and apigenin; wherein protein combination A is as described in claim 1; and protein combination B is as described in claim 1.

6. A method for preparing luteolin and apigenin, comprising the following steps: using isoharonium and isovitelline as raw materials, and converting them using biological material A or biological material B to obtain luteolin and apigenin; wherein biological material A is as described in claim 3; and biological material B is as described in claim 3.

7. A method for preparing luteolin, comprising the following steps: using isoherbine as a raw material, and converting it using protein combination A or protein combination B to obtain luteolin; wherein protein combination A is as described in claim 1; and protein combination B is as described in claim 1.

8. A method for preparing luteolin, comprising the following steps: using isoherbine as a raw material, and converting it using biological material A or biological material B to obtain luteolin; wherein biological material A is as described in claim 3; and biological material B is as described in claim 3.

9. A method for preparing apigenin, comprising the following steps: using isovitexin as a raw material, and converting it using protein combination A or protein combination B to obtain apigenin; wherein protein combination A is as described in claim 1; and protein combination B is as described in claim 1.

10. A method for preparing apigenin, comprising the following steps: using isovitexin as a raw material, and converting it using biological material A or biological material B to obtain apigenin; wherein biological material A is as described in claim 3; and biological material B is as described in claim 3.

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