CN116144713A

CN116144713A - Method for producing secoisolariciresinol

Info

Publication number: CN116144713A
Application number: CN202310096565.3A
Authority: CN
Inventors: 杨东辉; 刘树林; 蔡少青
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2023-01-20
Filing date: 2023-01-20
Publication date: 2023-05-23
Also published as: CN117925734A

Abstract

The invention relates to the technical field of biochemistry, and provides a method for producing secoisolariciresinol, which comprises the following steps: preparing a substrate solution by taking flaxseed meal as a substrate, and then adding glycoside hydrolase to perform enzymolysis catalysis to obtain a reaction solution containing secoisolariciresinol; the glycoside hydrolase is derived from glycoside hydrolase family 3; according to the invention, the first research shows that the glycoside hydrolase from the glycoside hydrolase family 3 can be used for hydrolyzing the Secoisolariciresinol (SDG) -3-hydroxy-3-methyl-glutaric acid (HMGA) oligomer in the linseed meal to directly obtain the secoisolariciresinol, so that the glycoside hydrolase from the glycoside hydrolase family 3 is used for producing the secoisolariciresinol, the process is simple, the environment is friendly, and the raw material cost is low and the method is easy to obtain.

Description

Method for producing secoisolariciresinol

Technical Field

The invention relates to the technical field of biochemistry, in particular to a method for producing secoisolariciresinol.

Background

The secoisolariciresinol is lignan compound, and has bidirectional regulating effect on estrogen and antiestrogen, i.e. phytoestrogen-like bioactivity. The research result shows that the secoisolariciresinol can prevent and treat breast cancer, colon cancer and prostate cancer, can reduce blood fat and cholesterol, resist oxidation, resist radiation, reduce blood sugar, protect cardiac muscle and resist climacteric syndrome, and has good medicinal value for preventing and treating diseases such as breast cancer, colon cancer, cardiovascular diseases, osteoporosis and the like. The secoisolariciresinol exists in various vegetables, fruits, leguminous grains and other plants, but the content of the secoisolariciresinol is generally low from natural plant sources.

At present, the step of chemically synthesizing the ring-opened iso-larch resin phenol is complicated, the cost is high, and the method is not friendly to the environment.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a method for producing the secoisolariciresinol.

For this purpose, the invention provides the following technical scheme:

a method for producing secoisolariciresinol comprises the following steps: preparing a substrate solution by taking flaxseed meal as a substrate, and then adding glycoside hydrolase to perform enzymolysis catalysis to obtain a reaction solution containing secoisolariciresinol; the glycoside hydrolase is derived from glycoside hydrolase family 3.

Optionally, the substrate solution is obtained by diluting flaxseed meal with phosphate buffer.

Optionally, the flaxseed meal comprises residues after the flaxseed oil extraction, residues after the flaxseed protein and the flaxseed gum removal, and residues after the flaxseed gum removal of the flaxseed cake shell or the flaxseed cake shell.

Optionally, the phosphate buffer solution has a concentration of 15-30mM and a pH of 6.0-7.0; or the concentration of the substrate solution is 100-400mg/mL;

optionally, the concentration of the substrate solution is 200-250mg/mL.

Alternatively, the glycoside hydrolase includes glycoside hydrolase derived from Bacteroides simplex (Bacteroides uniformis), glycoside hydrolase of Propionibacterium spelt (Prevotella bergensis) _、 Glycoside hydrolases of Bacteroides ruminalis (Prevotella ruminicola), bacteroides flowerans (Bacteroides fluxus), flavobacterium cold domain (Flavobacterium frigoris), thermotoga new Apollo (Thermotoga neapolitana) and/or thermophilic eubacteria (Fervidobacterium nodosum).

Optionally, the preparation of the glycoside hydrolase comprises: synthesizing a coding gene of a target enzyme, and constructing recombinant bacteria containing the coding gene; and (3) culturing, fermenting and inducing the recombinant bacteria to obtain protein, and extracting to obtain a crude enzyme extract of the glycoside hydrolase.

Optionally, mixing enzyme solution and substrate solution of glycoside hydrolase according to the volume ratio of 1:10-1:30, and then reacting for 0.5-35 hours at 25-37 ℃; the concentration of the enzyme solution of the glycoside hydrolase is 2.0-5.0mg/mL;

optionally, the enzyme solution concentration of the glycoside hydrolase is 2.5-3.5mg/mL.

Optionally, centrifuging the reaction solution containing the secoisolariciresinol to obtain a precipitate and a supernatant;

and separating, purifying and crystallizing the supernatant to obtain a crystal sample of the secoisolariciresinol.

Optionally, mixing the supernatant with macroporous resin according to the volume ratio of 1:10-1:20, loading the mixture on a chromatographic column, eluting 2-4 times of the column volume by using an ethanol water solution with the volume percentage of 10-30%, eluting 2-4 times of the column volume by using an ethanol water solution or ethanol with the volume percentage of 50-100%, collecting the flow eluted by the ethanol water solution or ethanol with the volume percentage of 50-100%, concentrating, adding 2-5 times of an organic solvent for dissolving, standing for crystallization, and obtaining the ring-opened alternaria resin phenol crystal.

Optionally, eluting with 10-20vol% ethanol water solution for 2-3 times of column volume; and/or

Eluting with 60-80% ethanol water solution or ethanol by volume percentage for 2-3 times of column volume; and/or

The macroporous resin is D101 macroporous resin; and/or

The organic solvent is methanol, acetonitrile, acetone or ethanol.

The compound ring-opened cloisonne resin phenol has the following structure:

linseed meal (including residues after oil extraction of flaxseed, residues after removal of flax protein and flax seed gum after oil extraction of flaxseed, flaxseed cake hulls or residues after removal of flax seed gum from flaxseed cake hulls) is rich in Secoisolariciresinol Diglucoside (SDG) -3-hydroxy-3-methyl-glutaric acid (HMGA) oligomers (commonly known as secoisolariciresinol diglucoside). The invention utilizes the technology and means of combining bioinformatics, molecular biology and chemistry to establish a preparation process for producing secoisolariciresinol by utilizing various glycoside hydrolase to catalyze flaxseed meal in vitro.

The technical scheme of the invention has the following advantages:

1. the invention provides a method for producing ring-opened iso-larch resin phenol, which comprises the following steps: preparing a substrate solution by taking flaxseed meal as a substrate, then adding glycoside hydrolase, and performing enzyme catalytic hydrolysis reaction to obtain a reaction solution containing secoisolariciresinol; the glycoside hydrolase is derived from glycoside hydrolase family 3; the first research of the invention shows that the glycoside hydrolase from glycoside hydrolase family 3 can enzymatically hydrolyze the Secoisolariciresinol Diglucoside (SDG) -3-hydroxy-3-methyl-glutaric acid (HMGA) oligomer (the chemical structure of which is shown in figure 1) in the linseed meal to directly obtain the secoisolariciresinol. Therefore, the method for producing the secoisolariciresinol by using the glycoside hydrolase from the glycoside hydrolase family 3 and using the flaxseed meal as a substrate has the advantages of simple process, environmental friendliness, low raw material cost and easy obtainment.

2. The invention provides a method for producing secoisolariciresinol, which comprises the steps of mixing enzyme solution of glycoside hydrolase and substrate solution according to the volume ratio of 1:10-1:30, and then reacting for 0.5-35 hours at 25-37 ℃; the yield of the secoisolariciresinol in the solution is 95-97%; the optimal reaction condition is determined by selecting and optimizing parameters such as the mixing proportion of enzyme solution and substrate solution of glycoside hydrolase, reaction solution, pH value, reaction temperature, reaction time and the like, so that the content of the secoisolariciresinol in the reaction solution is improved to the greatest extent.

3. The invention provides a method for producing secoisolariciresinol, which comprises the steps of adding supernatant of enzyme reaction solution into a macroporous resin chromatographic column for eluting, wherein the volume ratio of the supernatant to the macroporous resin is 1:10-1:20; eluting with 10% -30% ethanol water solution as eluent, wherein the amount of eluent is 2-4 times of column volume; eluting with 50-100% ethanol water solution or ethanol with the eluent amount of 2-4 times of column volume, collecting eluent, concentrating, adding 2-5 times of methanol, acetonitrile or acetone for dissolving, filtering the solution, and standing to obtain crystals of secoisolariciresinol (purity not less than 95%); the invention adopts macroporous resin chromatography purification, and the impurities are eluted by firstly selecting an ethanol water solution with the concentration of 10% -30%, then selecting an ethanol water solution with the concentration of 50% -100% or ethanol to elute and collect target fractions, and the samples of the secoisolariciresinol (the purity is more than 95%) can be obtained by a recrystallization technology after the fractions are concentrated, so that the method can be directly used as raw materials of medical products and health-care products.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the following description will briefly explain the drawings needed in the embodiments or the prior art description, and it is obvious that the drawings in the following description are some embodiments of the invention and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a chemical structure of an oligomer of Secoisolariciresinol Diglucoside (SDG) -3-hydroxy-3-methyl-glutaric acid (HMGA) contained in flaxseed meal;

FIG. 2 is a graph showing the yield versus time of secoisolariciresinol produced by converting flaxseed meal with the glycoside hydrolase of example 1 of the present invention;

FIG. 3 is an HPLC chart of a phenol sample (purity 98%) of the purified Secoisolariciresinol in example 1 of the present invention.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

The method for measuring the content of the target glycoside hydrolase in the crude enzyme solution in the following examples is as follows:

and respectively taking 20 mu L of 0.6,0.8,1.0,1.2,1.4,1.6,1.8 and 2.0mg/mL BSA standard protein solution, mixing with 3mL coomassie brilliant blue G-250 dye solution, standing for 5min, measuring the absorbance at 595nm, taking the absorbance as an abscissa and the protein concentration as an ordinate, and drawing a standard curve. Diluting the crude enzyme solution by 40 times, then sucking 20 mu L of the crude enzyme solution, mixing with 3mL of coomassie brilliant blue G-250 dye solution, standing for 5min, measuring the absorbance at 595nm, and calculating the protein content to obtain the content of the target glycoside hydrolase in the crude enzyme solution.

The method for detecting the purity of the secoisolariciresinol in the following examples comprises the following specific steps:

1mg of a secoisolariciresinol sample is taken, 1mL of methanol is added for dissolution, and 10 mu L of solution is taken for HPLC detection and analysis. HPLC analysis and detection, the conditions were as follows: column ZORBAX C18 (4.6mm.times.150 mm,5 μm, agilent Co., U.S.A.), guard column C18 (4 mm.times.3.0 mm,5 μm, phillips Co., U.S.A.). Mobile phase: water (a) -acetonitrile (B); gradient elution procedure: 0-5 min (15 v/v% B-15v/v% B), 5-25 min (15 v/v% B-40v/v% B), 25-27 min (40 v/v% B-100v/v% B), 27-35 min (100 v/v% B-100v/v% B); flow rate: 0.8 mL/min; detection wavelength: 280nm; column temperature: 25 ℃.

The flaxseed meal in the examples below is the residue of the oil extraction from flaxseeds.

Example 1

The embodiment provides a method for producing secoisolariciresinol, which comprises the following steps:

(1) Preparation and purification of glycoside hydrolase:

the gene encoding the target glycoside hydrolase (amino acid sequence NCBI accession No. AGU13704.1, derived from Bacteroides simplex (Bacteroides uniformis)) was synthesized (delegated to Beijing Liuhua Dairy Gene technologies Co., ltd.) and amplified by PCR. The PCR reaction system (100. Mu.L) comprises: 2×TransTaq ^TM HiFi PCR SuperMix 50. Mu.l, 4. Mu.l of forward primer (10. Mu.M), 4. Mu.l of reverse primer (10. Mu.M), 4. Mu.l of target gene DNA template, and sterile double distilled water was added to 100. Mu.l. The forward primer and the reverse primer were designed by conventional primer design software, and in this example, the forward primer sequence was 5'-CCGGGTGTGCCGCAGCTTGGAAAGT-3' (shown as SEQ ID NO. 1) and the reverse primer sequence was 5'-CAGACAATCCTACTCTCATCTCTTC-3' (shown as SEQ ID NO. 2).

The PCR reaction procedure was as follows: the reaction was pre-denatured at 94℃for 5min, and the amplification reaction was 30 cycles, namely: denaturation at 94℃for 30 seconds, annealing at 58℃for 30 seconds, extension at 72℃for 2 minutes and incubation for another 10 minutes. The PCR product was detected by 1% agarose gel electrophoresis, and the target DNA fragment of the PCR product was recovered by using an agarose gel electrophoresis recovery kit.

The PCR product is directly connected with a pEASY expression vector to construct a glycoside hydrolase expression plasmid, and the specific steps are as follows: in a 1.5mL EP tube, PCR product or agarose gel recovery product (2. Mu.L) and pEASY-E1 Expression vector (1. Mu.L) were added sequentially, and finally made up to 5. Mu.L with sterilized deionized water. The above liquids were gently mixed and reacted at 25℃for 20 minutes using a PCR instrument to obtain a ligation product (-20℃for preservation).

Glycoside hydrolase expression plasmid was added to 50mL of Trans1-T1 competent cells (ice bath for 30 min; heat shock at 42℃for 30 sec; ice bath for 2 min), 500mL of LB medium was added, and incubated at 200rpm for 1 hour at 37 ℃. 200. Mu.L of the bacterial liquid was plated on LB agar plates (containing 100ng/mL ampicillin) and incubated overnight at 37 ℃.

Extracting recombinant plasmid containing target gene, and transforming the recombinant plasmid vector into colibacillus expression cell BL21 (DE 3) pLysS to obtain target gene recombinant bacterium. The method comprises the following specific steps: mu.l of plasmid was added to 50mL of BL21 (DE 3) pLysS competent cells, and ice-incubated for 30 min; heat shock at 42 ℃ for 45 seconds and ice bath for 2 minutes; 500. Mu.L LB was added thereto and incubated at 37℃for 1 hour with stirring; 200. Mu.l of the bacterial liquid was plated on LB agar plates (containing 100. Mu.g/mL ampicillin) and incubated overnight at 37 ℃.

The monoclonal is selected and inoculated in LB culture medium, and is cultured overnight at 37 ℃ in a shaking way for activation. The next day, 1% of the culture was transferred to 100mL of LB medium (containing 100. Mu.g/mL of ampicillin) and cultured at 37℃with shaking at 250rpm until OD ₆₀₀ About 0.6, isopropyl thiogalactoside (IPTG) was added at a final concentration of 0.5mM, and expression was induced at 20℃and 200rpm for 16 hours. After the induction of the expression, the bacterial liquid was centrifuged at 9000rpm for 5 minutes at 4℃and the supernatant was discarded. The bacterial pellet was stirred with sodium phosphate buffer (20 mM, pH 6.0) and sonicated for 30 min (2 seconds at 5 second intervals). The bacterial liquid after ultrasonic disruption is centrifuged for 10min at the temperature of 4 ℃ and 14000rpm, and the supernatant is the crude enzyme solution (3 mg/mL) of the target glycoside hydrolase.

(2) Enzymolysis:

a substrate solution was prepared using 20mM sodium phosphate buffer (pH 6.0) and flaxseed meal to give a flaxseed meal solution (250 mg/mL). Mixing the crude enzyme solution prepared in the step (1) with a substrate solution according to the volume ratio of 1:30, reacting for 3 hours at 30 ℃ with the final constant volume of 5L, sampling and detecting the content of Secoisolariciresinol (SECO) in the fermentation broth at time points of 0.25, 0.5, 0.75, 1.0, 1.25, 1.75, 2.0, 2.25, 2.5, 2.75 and 3.0 hours respectively, and calculating the SECO yield, wherein the SECO yield is 98% (fig. 2, SECO yield% = ratio of the actual SECO content measured to the total SDG content converted into SECO in the flax seed meal theoretically).

(3) Purifying:

centrifuging (5000 rpm,10 minutes) the reaction solution obtained in the step (2) at normal temperature to obtain a supernatant; mixing the supernatant with D101 macroporous resin at a volume ratio of 1:10, loading onto a chromatographic column, eluting with 10% ethanol water solution by volume percentage for twice the column volume to remove impurities, eluting with 60% ethanol water solution for 2 times the column volume, collecting the target fraction containing secoisolariciresinol, concentrating the obtained fraction into paste, adding 3 times volume amount of acetonitrile for dissolving, standing to precipitate secoisolariciresinol crystals, and filtering to obtain a secoisolariciresinol sample (purity 98% in figure 3).

Example 2

(1) Preparation and purification of glycoside hydrolase:

the coding gene of the target glycoside hydrolase (sequence NCBI accession number is ZP_06005092.1, which is derived from Proteus spelt (Prevotella bergensis)), is connected to a pEASY expression vector by a Gibson splicing technology to construct a pEASY-bubgl8 plasmid, and the plasmid is introduced into a E.coli Rosetta (DE 3) (the method is the same as that of example 1, except that primers which can be conventionally designed by using primer design software) are used to obtain a recombinant gene bacterium capable of expressing the target glycoside hydrolase (the method is the same as that of example 1). The recombinant strain was inoculated into LB medium, cultured overnight at 37℃with shaking, and activated. The next day, 1% of the culture was transferred to 100mL of LB medium (containing 100g/mL of ampicillin), and the culture was performed at 37℃and 200rpm with shaking until OD ₆₀₀ About 0.6, IPTG was added at a final concentration of 0.5mM and induction was carried out at 20℃and 200rpm for 18 hours. After induction of expression, the bacterial liquid was centrifuged at 9000rpm at 4℃for 5 minutes, and the supernatant was discarded. The bacterial cell precipitate was stirred with 30mM potassium phosphate buffer solution at pH 6.5, and the bacterial cell was subjected to ultrasonic lysis for 30 minutes (ultrasonic treatment for 2 seconds at intervals)5 seconds, alternate). Centrifuging the bacterial liquid after ultrasonic disruption at 14000rpm for 10min at 4 ℃, and taking supernatant, wherein the supernatant is a crude enzyme solution of the target glycoside hydrolase (the target protein content is 2.5 mg/mL).

(2) Enzymolysis:

a substrate solution was prepared using 30mM potassium phosphate buffer (pH 6.5) and flaxseed meal to give a flaxseed meal solution (200 mg/mL). Mixing the crude enzyme solution prepared in the step (1) with a substrate solution according to a concentration ratio of 1:20, wherein the final constant volume of the system is 5L, and reacting at 37 ℃ for 2 hours, wherein the SECO conversion rate is 96% (SECO conversion rate% = ratio of actually measured SECO content to theoretically total conversion of SDG in flaxseed meal) is achieved.

(3) Purifying:

centrifuging (5000 rpm,10 minutes) the reaction solution obtained in the step (2) at normal temperature to obtain a supernatant; mixing the supernatant with D101 macroporous resin according to the volume ratio of 1:15, loading the mixture on a chromatographic column, eluting twice the column volume by adopting an ethanol water solution with the volume percentage of 20% to remove impurities, eluting 3 times the column volume by adopting an ethanol water solution with the volume percentage of 80%, collecting the target fluid containing the secoisolariciresinol, concentrating the obtained fluid into paste, adding 3 times the volume of acetone to dissolve, and standing to separate out the secoisolariciresinol crystals. Filtering to obtain a sample of the secoisolariciresinol. The purity of the secoisolariciresinol in the sample solution is greater than 95%.

Example 3

(1) Preparation and purification of glycoside hydrolase:

the coding gene of the target glycoside hydrolase (sequence NCBI accession number is YP_003575940.1, derived from Bacteroides ruminalis (Prevotella ruminicola)), a pEASY-bubgl8 plasmid is constructed by connecting the sequence NCBI accession number to a pEASY expression vector by a Gibson splicing technique, and the plasmid is introduced into a E.coli Rosetta (DE 3) (the method is the same as that of example 1, except that primers which can be conventionally designed by using primer design software) are used to obtain a recombinant gene bacterium capable of expressing the target glycoside hydrolase (the method is the same as that of example 1). Gene weightThe group bacteria were inoculated in LB medium, cultured overnight at 37℃with shaking, and activated. The next day, 1% of the culture was transferred to 100mL of LB medium (containing 100g/mL of ampicillin), and the culture was performed at 37℃and 200rpm with shaking until OD ₆₀₀ About 0.6, IPTG was added at a final concentration of 0.5mM and induction was carried out at 20℃and 200rpm for 18 hours. After induction of expression, the bacterial liquid was centrifuged at 9000rpm at 4℃for 5 minutes, and the supernatant was discarded. The bacterial pellet was stirred with a pH 7.0 and 15mM sodium phosphate buffer solution, and the bacterial pellet was subjected to ultrasonic lysis for 30 minutes (ultrasonic lysis for 2 seconds, at 5 seconds intervals, alternately). Centrifuging the bacterial liquid after ultrasonic disruption at 14000rpm for 10min at 4 ℃, and taking supernatant, wherein the supernatant is a crude enzyme solution of the target glycoside hydrolase (the target protein content is 3.5 mg/mL).

(2) Enzymolysis:

a substrate solution was prepared using 15mM sodium phosphate buffer (pH 7.0) and flaxseed meal to give a flaxseed meal solution (300 mg/mL). Mixing the crude enzyme solution prepared in the step (1) with a substrate solution according to a concentration ratio of 1:10, wherein the final constant volume of the system is 5L, and reacting at 30 ℃ for 1 hour, wherein the SECO conversion rate is 95% (SECO conversion rate% = ratio of actually measured SECO content to theoretically total conversion of SDG in flaxseed meal) is achieved.

(3) Purifying:

centrifuging (5000 rpm,10 minutes) the reaction solution obtained in the step (2) at normal temperature to obtain a supernatant; mixing the supernatant with D101 macroporous resin according to the volume ratio of 1:20, loading the mixture on a chromatographic column, eluting the mixture by adopting an ethanol water solution with the concentration of 15% for 3 times of column volume to remove impurities, eluting the mixture by adopting 100% ethanol for 2 times of column volume to obtain a target flow containing the secoisolariciresinol, concentrating the obtained flow into paste, adding 5 times of volume of methanol for dissolving, and standing to separate out the secoisolariciresinol crystals. Filtering to obtain a sample of the secoisolariciresinol. The purity of the secoisolariciresinol in the sample solution is greater than 95%.

Example 4

This example differs from example 1 in that the gene encoding the glycoside hydrolase of interest (sequence NCBI accession number ZP_08300624.1, derived from Bacteroides flowstream (Bacteroides fluxus)) was synthesized in step (1); the concentration of the crude enzyme solution was 2.0mg/mL.

In the step (2), the concentration of the substrate solution is 100mg/mL; the reaction was carried out at 25℃for 35 hours. The SECO yield is more than 85%.

In the step (3), after the chromatographic column is loaded, eluting twice the column volume by adopting an ethanol water solution with the volume percentage of 10% to remove impurities, eluting 4 times the column volume by adopting an ethanol water solution with the volume percentage of 50%, and collecting and obtaining the target flow containing the secoisolariciresinol. The purity of the secoisolariciresinol in the sample solution is greater than 95%.

Example 5

This example differs from example 1 in that the gene encoding the glycoside hydrolase of interest (sequence NCBI accession number ZP_09895978.1, derived from Flavobacterium cold domain (Flavobacterium frigoris)) was synthesized in step (1); the concentration of the crude enzyme solution was 5mg/mL.

In the step (2), the concentration of the substrate solution is 400mg/mL; the reaction was carried out at 37℃for 35 hours. The SECO yield is more than 80%.

In the step (3), after the chromatographic column is loaded, eluting by adopting an ethanol water solution with the concentration of 30% for 4 times of column volume to remove impurities, then eluting by adopting an ethanol water solution with the concentration of 60% for 2 times of column volume, and collecting a target flow containing the secoisolariciresinol. The purity of the secoisolariciresinol in the sample solution is greater than 95%.

Example 6

This example differs from example 1 in that the gene encoding the target glycoside hydrolase (sequence NCBI accession number YP_002534212.1, derived from Thermotoga new Apollo (Thermotoga neapolitana)) was synthesized in step (1).

The SECO yield in the step (2) is more than 75 percent.

The purity of the secoisolariciresinol in the sample solution in the step (3) is more than 95%.

Example 7

This example differs from example 1 in that the gene encoding the glycoside hydrolase of interest (sequence NCBI accession number YP_001409810.1, derived from thermophilic eubacteria (Fervidobacterium nodosum)) was synthesized in step (1).

The SECO yield in the step (2) is more than 70 percent.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. The method for producing the secoisolariciresinol is characterized by comprising the following steps: preparing a substrate solution by taking flaxseed meal as a substrate, and then adding glycoside hydrolase to perform enzymolysis catalysis to obtain a reaction solution containing secoisolariciresinol; the glycoside hydrolase is derived from glycoside hydrolase family 3.

2. The method for producing secoisolariciresinol according to claim 1, wherein the substrate solution is obtained by diluting flaxseed meal with phosphate buffer; and/or

The flaxseed meal comprises residues after the flaxseed oil extraction, which are used for removing flaxseed protein and flaxseed gum, flaxseed cake shells, which are used for removing the flaxseed gum, or residues after the flaxseed cake shells are used for removing the flaxseed gum and dietary fibers.

3. The method for producing secoisolariciresinol according to claim 2, wherein the phosphate buffer solution has a concentration of 15-30mm and a ph of 6.0-7.0; or the concentration of the substrate solution is 100-400mg/mL;

optionally, the concentration of the substrate solution is 200-250mg/mL.

4. A process for producing secoisolariciresinol according to any one of claims 1 to 3, wherein,the glycoside hydrolase comprises glycoside hydrolase derived from Bacteroides simplex (Bacteroides uniformis), and glycoside hydrolase of Propionibacterium spelt (Prevotella bergensis) _、 Glycoside hydrolases of Bacteroides ruminalis (Prevotella ruminicola), bacteroides flowerans (Bacteroides fluxus), flavobacterium cold domain (Flavobacterium frigoris), thermotoga new Apollo (Thermotoga neapolitana) and/or thermophilic eubacteria (Fervidobacterium nodosum).

5. The process for producing secoisolariciresinol according to any one of claims 1 to 4, wherein,

the preparation of the glycoside hydrolase comprises the following steps: synthesizing a coding gene of a target enzyme, and constructing recombinant bacteria containing the coding gene; and (3) culturing, fermenting and inducing the recombinant bacteria to obtain protein, and extracting to obtain a crude enzyme extract of the glycoside hydrolase.

6. The process for producing secoisolariciresinol according to any one of claims 1 to 5, wherein,

mixing enzyme solution and substrate solution of glycoside hydrolase according to the volume ratio of 1:10-1:30, and then reacting for 0.5-35 hours at 25-37 ℃; the concentration of the enzyme solution of the glycoside hydrolase is 2.0-5.0mg/mL;

7. The process for producing secoisolariciresinol according to any one of claims 1 to 6, wherein the reaction solution containing secoisolariciresinol is obtained by centrifugation to obtain a precipitate and a supernatant;

8. The method for producing the secoisolariciresinol according to claim 7, wherein the supernatant and the macroporous resin are mixed according to the volume ratio of 1:10-1:20, then the mixture is loaded on a chromatographic column, the volume of the column is 2-4 times of the volume of the column is eluted by 10-30% of ethanol aqueous solution by volume percent, then the volume of the column is 2-4 times of the volume of the column is eluted by 50-100% of ethanol aqueous solution or ethanol by volume percent, the fraction eluted by 50-100% of ethanol aqueous solution or ethanol is collected, concentrated, and 2-5 times of organic solvent is added for dissolution, and standing crystallization is carried out, thus obtaining the secoisolariciresinol crystal.

9. The method for producing secoisolariciresinol according to claim 8, wherein the column volume is 2 to 3 times as large as that of the secoisolariciresinol by eluting with 10 to 20% by volume of aqueous ethanol solution; and/or

The macroporous resin is D101 macroporous resin; and/or

The organic solvent is methanol, acetonitrile, acetone or ethanol.