CN116606832A - Dibenzylbutane lignan oxymethyl transferase and application thereof - Google Patents

Abstract

The present application relates to a polypeptide molecule having an amino acid sequence selected from the group consisting of: (A) SEQ ID NO.2; (B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncate of (A) or (B); wherein the polypeptide molecule has an activity of catalyzing methylation modification of lignans. The lignan oxymethyl transferase of the present application can specifically catalyze dibenzylbutane lignan. The diversified methylation modification of the dibenzylbutane lignans provides more substrate choices for the generation of other cyclized lignans, and research results provide candidate genes for the research of the synthesis biology of the pharmaceutically active lignans, so that the method has important practical application significance for elucidating the formation mechanism of the structural diversity of the compounds, the development of active molecules and the like.

Description

Dibenzylbutane lignan oxymethyl transferase and application thereof

Technical Field

The application relates to the technical fields of genetic engineering and enzyme engineering, in particular to an oxymethyl transferase participating in biosynthesis of schisandra dibenzylbutane lignans and application thereof.

Background

The dibenzylbutane lignans are a class of acyclic lignans formed by coupling two C6-C3 units via the 8 and 8' positions. Is obtained by separating from fructus Schisandrae, euphorbiae radix, lauraceae, etc. Modern pharmacological research shows that the natural products have antioxidant activity, and various derivatives thereof have anti-HIV activity. Based on the chain structural characteristics of the compounds, lignans of other structural types can be formed through different cyclization modes, so that the lignans are positioned at the front position on the biosynthesis path of dimerized lignans.

The schisandra chinensis (Schisandra chinensis) is separated to obtain rich dibenzylbutane lignans and cyclized diphenyl cyclooctene lignans, and the structure of the lignans contains various oxymethyl substitutions, so that functional oxymethyl transferase (O-methyl transferases) is necessarily contained in the schisandra chinensis, and no related report exists at present.

Disclosure of Invention

Aiming at the technical problems in the prior art, the application provides a polypeptide molecule, the amino acid sequence of which is selected from the group consisting of: (A) SEQ ID NO.2; (B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncate of (A) or (B); wherein the polypeptide molecule has an activity of catalyzing methylation modification of lignans, and further, the polypeptide molecule has an activity of catalyzing methylation of phenolic hydroxyl groups of lignans.

A polypeptide molecule as described above, wherein said lignan is a dibenzylbutane-type lignan.

A polypeptide molecule as described above, wherein said lignan is regoramin (Pregomisin).

A polynucleotide molecule encoding a polypeptide molecule according to any one of the preceding claims, said polynucleotide molecule being selected from the group consisting of: (a) SEQ ID NO.1; (b) A nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and (c) a truncate of (a) or (b).

A recombinant vector comprising: a polynucleotide molecule as described above; an expression vector.

A fused cell comprising: a recombinant vector as described above; and expressing the cells.

A method of preparing a protein for catalyzing methylation modification of dibenzylbutane lignans, comprising: transforming the nucleic acid molecule of 4 above into an expression cell; a protein expressing the polypeptide molecule in an expression cell; and purifying the protein of the polypeptide molecule.

The use of a polypeptide molecule as defined in any one of the preceding claims for catalyzing the methylation modification of dibenzylbutane lignans.

A method of preparing a schisandra lignan compound D (Schisandrathera D), the method comprising: pre-gomisin (Pregomisin) is catalyzed with one or more of the following groups: a polypeptide molecule as claimed in any one of the preceding claims; a polypeptide molecule encoded by a nucleic acid molecule as described above; a polypeptide molecule expressed by a recombinant vector as described above; a polypeptide molecule obtained by fusing cells as described above; and polypeptide molecules prepared by the methods described above.

The schisandra lignan compound D (Schisandrathera D) prepared by the above method.

The dibenzylbutane type lignanoid methylation modification enzyme can methylate the dibenzylbutane type lignanoid, can be used for exploring lignanoid with novel functions, lays a foundation for the research of the synthesis biology of lignanoid diversification in shizandra berry, and has great application value. The diversified methylation modification of the dibenzylbutane lignans provides more substrate choices for the generation of other cyclized lignans, and research results provide candidate genes for the research of the synthesis biology of the pharmaceutically active lignans, so that the method has important practical application significance for elucidating the formation mechanism of the structural diversity of the compounds, the development of active molecules and the like. The compound obtained by using the dibenzylbutane type lignan oxymethyl transferase or the compound obtained by using the dibenzylbutane type lignan oxymethyl transferase as an intermediate can be used as an active ingredient of medicines, functional food materials and the like, so that the lignan oxymethyl transferase has positive application prospects in the medicine industry and the food industry.

Drawings

Preferred embodiments of the present application will be described in further detail below with reference to the attached drawing figures, wherein:

FIG. 1 is an SDS-PAGE electrophoresis of SchOMT7 protein according to an embodiment of the application, wherein: m: protein molecular mass standard; lane 1: supernatant of SchOMT 7; lane 2: schOMT7 precipitated cells; lane 3: purified protein of SchOMT 7;

FIG. 2 is an HPLC plot of an enzyme-catalyzed reaction of SchOMT7 pre-gomisin as a substrate in accordance with one embodiment of the present application; wherein, the enzyme activity catalytic reaction takes the catalytic reaction of the empty carrier as a control; and

FIG. 3 is a LC-MS spectrum and reaction scheme of an enzyme-catalyzed reaction of SchOMT7 pre-gomisin as substrate according to one embodiment of the application; FIG. 3A is a LC-MS spectrum of an enzyme-catalyzed reaction of SchOMT7 pre-gomisin as a substrate according to one embodiment of the present application; FIG. 3B is an enzyme catalyzed reaction of SchOMT7 with pre-gomisin as a substrate according to one embodiment of the application.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration specific embodiments of the application. In the drawings, like reference numerals describe substantially similar components throughout the different views. Various specific embodiments of the application are described in sufficient detail below to enable those skilled in the art to practice the teachings of the application. It is to be understood that other embodiments may be utilized or structural, logical, or electrical changes may be made to embodiments of the present application.

The terms described herein have the following meanings:

the term "lignans" as used herein refers to a class of natural organic compounds polymerized in different forms from 2 or 3 molecule phenylpropanoid derivatives, which are distributed throughout angiosperms and gymnosperms. Two main categories can be distinguished: a compound polymerized from two molecules of phenylpropyl derivative through its side chain beta-position is called lignan; compounds in which one molecule of phenylpropyl side chains is linked to the benzene ring of another molecule, or two moieties are linked by an oxygen atom, are called neolignans. More than 200 lignans and more than 100 neolignans have been isolated from the plant kingdom. Chinese medicinal materials of schisandra fruit, acanthopanax root, cuisine, arctium fruit, polygala root, capsule of weeping forsythia, carthamus flower and asarum herb, etc. all contain lignans. Many lignan components may have stereoisomers due to saturated cyclic moieties. It has been found that lignans present in some traditional Chinese medicines are unstable in nature, are affected by acid or alkali, are easily isomerized, and are converted into stereoisomers thereof. Various lignans have been found to have activities of anticancer, antifungal, insecticidal, leukocyte-increasing, glutamic pyruvic transaminase-reducing, liver-protecting, cough-relieving, purgation, etc., but are not so much used as pharmaceutical agents. The derivative of podophyllotoxin is used as anticancer agent, and lignanoid in fructus Schisandrae chinensis has effect in reducing glutamic pyruvic transaminase, and can be used for treating hepatitis.

As used herein, "Schisandra chinensis" refers to Schisandra chinensis, a woody plant of the genus Schisandra of the family Schisandraceae. Researches show that the schisandra chinensis has wide medicinal value, including: the composition has the effects of resisting liver injury, widely inhibiting central nervous system, strengthening heart, enhancing body defensive ability against nonspecific stimulation, inhibiting bacillus anthracis, staphylococcus aureus, staphylococcus albus, typhoid bacillus, vibrio cholerae and the like. Wherein the lignans are main effective components of fructus Schisandrae chinensis, the total lignans content in fructus Schisandrae chinensis fruit is 18.1%, and the total lignans content in stem is 10.25%. The lignan component has obvious antioxidation effect, and can protect heart from lipid peroxidation injury.

As used herein, "methylation" refers to the process of catalytically transferring methyl groups from an active methyl compound to other compounds, which may form various methyl compounds, or which may chemically modify certain proteins or nucleic acids, etc., to form a methylated product. In biological systems, methylation is enzymatically catalyzed, and involves regulation of gene expression, regulation of protein function, and ribonucleic acid processing.

As used herein, "OMT7" belongs to the OMTs family and is an oxygen methyl transferase (O-methyl transferases). The oxymethyl transferase (OMT) is an important enzyme which depends on S-adenosylmethionine to catalyze the generation of various secondary metabolites such as flavonoid, alkaloid, plant protection agent and the like, and plays an important role in various stages of plant growth and development, resisting invasion of external bacteria and the like. OMT is usually present in the animal and plant in the form of a gene family.

The protein of the present application, for example, the amino acid sequence shown in SEQ ID No.2, comprises (A) a protein having the amino acid sequence shown in SEQ ID No.2, or (B) a protein having an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the above amino acid sequence and having a function of catalyzing phenolic hydroxyl methylation of regoramin, or (C) a truncated form of the above two, such as truncated N-terminal or C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids, or extended N-terminal or C-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 30 amino acids, and having a function of catalyzing phenolic hydroxyl methylation of regoramin.

As used herein, "substitution, deletion, or addition of one or more bases" or "substitution, deletion, or addition of one or more amino acids" refers to the use of well-known mutagenesis methods such as site-directed mutagenesis [ Hashimoto-Gotoh, gene 152, 271-275 (1995) others ] to mutate a nucleotide sequence or amino acid sequence to a sequence having at least 80% homology to the original sequence. For example, the mutated sequence has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% or 80% homology to the original sequence. In some embodiments, the term "95% or more homology" in the above proteins and genes may be at least 96%, 97%, 98% identical. The term "homology above 90% may be at least 91%, 92%, 93%, 94% identical. The term "above 85% homology" may be at least 86%, 87%, 88%, 89% identical. The term "80% homology or greater" may be at least 81%, 82%, 83%, 84% identical.

The terms "coding gene", "nucleic acid molecule" refer to a Ribonucleotide (RNA) or Deoxyribonucleotide (DNA) sequence that can encode a peptide chain of a particular amino acid. In some embodiments, the encoding gene or nucleic acid molecule may be obtained by total gene synthesis, PCR amplification, chemical synthesis, or the like. The application is not limited to the manner in which the encoding genes and nucleic acid molecules are obtained.

The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells. The vector may include a vector mainly used for inserting DNA or RNA into a cell, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a plurality of functions as described above. The vector may be a polynucleotide capable of transcription and translation into a polypeptide when introduced into a suitable host cell. Typically, the vector will produce the desired expression product by culturing a suitable host cell comprising the vector.

In some embodiments of the application, the term "recombinant vector" is specifically a recombinant vector obtained by cloning a gene encoding a lignan oxymethyl transferase between expression vectors, such as pET28a vectors, which contain cleavage sites, such as bamhi and Xho i. In other embodiments, the expression vector is selected from one or more of the following vectors: other vectors such as pPIC9, pPIC9K, pPICZ a B, pPICZ a B vector, pET series vector, pGEX series vector, pMAL series vector, pQE series vector, pBADmycHis series vector, pTrcHis series vector, pTXB series vector, T series vector, and the like, and modified vectors of the above vectors. In some embodiments, the recombinant expression vector may be selected from prokaryotic expression vectors such as pET series, pGEX series, pCold series, etc., yeast expression vectors such as pPIC9, pHIL-D2, pPIC3.5, etc., and plant expression vectors such as PBI series, pCAMBIA series, etc. The application is not limited to the manner in which the recombinant vector is obtained, and recombinant vectors of the application obtained by other means are also within the scope of the application.

The terms "recombinant expression cell", "recombinant cell", "expression cell" refer to a cell having an exogenous gene integrated within its genome, or a host cell comprising an expression vector in vivo. In some embodiments, the "recombinant expression cell", "recombinant cell", "expression cell" may be a bacterial, fungal, higher plant cell, or the like. In some embodiments, the gene encoding a lignan oxymethyl transferase is obtained by introducing the gene into the E.coli, mammalian cells, yeast cells or Pichia pastoris genetically engineered with glycosylation modification pathways.

The term "comprising" as used herein generally refers to the meaning of containing, summarizing, comprising, or including. In some cases, the meaning of "as", "consisting of … …" is also indicated.

The oxygen methyl transferase (O-methyl transferases) is taken as an important structure modification enzyme and is a key enzyme for biosynthesis of Chinese medicine active compounds in schisandra chinensis. Aiming at the research on the biosynthesis path of the schisandra chinensis dibenzocyclooctene lignans, the path is deduced to be that the dibenzo-benzyl butane lignans are connected through benzene rings to form the dibenzocyclooctene lignans structure. Methylation modification may occur before or after coupling and is an important ring in the synthetic pathway of compounds containing oxymethyl groups.

Therefore, the application focuses on screening and identifying structural modification genes OMTs of schisandra lignans. The research result has important theoretical research significance and practical application significance for clarifying lignan synthesis pathway of schisandra chinensis, formation mechanism of compound structural diversity and the like.

The application aims to provide an oxymethyl transferase gene and a protein coded by the same, which can participate in methylation modification of schisandra dibenzylbutane lignans. The research shows that the methylation modified enzyme SchOMT7 from schisandra chinensis can catalyze the hydroxy methylation of the dibenzylbutane lignan pregoamine.

The nucleotide sequence of the SchOMT7 gene provided by the application is shown as SEQ ID No.1 or a mutant sequence thereof.

The amino acid sequence of the protein coded by the SchOMT7 gene is shown as SEQ ID No.2 or a mutant sequence thereof.

The application can be realized by the following technical scheme:

the technical scheme is as follows: the genome-based schisandra dibenzylbutane lignan methylation modification key enzyme gene screening comprises the following steps:

1) UPLC detects the content difference of the oxybenzyl-containing dibenzylbutane lignans in the fruits, mature stems, old leaves and roots of the schisandra chinensis.

2) Identifying members of the gene family of the schisandra chinensis oxymethyl transferase OMTs based on the schisandra chinensis genome, and primarily screening key enzyme protein sequences of the oxymethyl transferase OMTs in the schisandra chinensis by utilizing an HMM model of the OMT gene family.

3) Based on transcriptome data of different tissue parts of the schisandra chinensis, differential gene expression is analyzed, and key enzyme genes for modifying hydroxy methylation in the biosynthesis pathway of the schisandra chinensis dibenzylbutane lignan are further screened.

The second technical scheme is as follows: functional verification of the key enzyme gene SchOMT 7. The function of the oxymethyl transferase SchOMT7 was identified in vitro using a prokaryotic expression system with pre-gomisin as substrate.

The present application relates to a polypeptide molecule having an amino acid sequence selected from the group consisting of: (A) SEQ ID NO.2; (B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and (C) a truncate of (A) or (B); wherein the polypeptide molecule has an activity of catalyzing methylation modification of lignans, and further, the polypeptide molecule has an activity of catalyzing methylation of phenolic hydroxyl groups of lignans. Further, the above polypeptide molecule is encoded by a coding gene selected from the group consisting of: (a) SEQ ID NO.1; (b) A nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and (c) a truncate of (a) or (b).

In some embodiments, the lignan is a dibenzylbutane lignan. Further, in some embodiments, the dibenzylbutane lignan is regoramin (Pregomisin).

In some embodiments, the application relates to a recombinant vector comprising: the nucleic acid molecule as described above, which can express the polypeptide molecule as described above. In some embodiments, the polypeptide molecule has activity in catalyzing methylation modification of lignans; an expression vector. In some embodiments, the polypeptide molecule has activity in catalyzing the methylation of lignans phenolic hydroxyl groups. In some embodiments, the expression vector may be selected from prokaryotic expression vectors such as pET series, pGEX series, pCold series, etc., yeast expression vectors such as pPIC9, pHIL-D2, pPIC3.5, etc., and plant expression vectors such as PBI series, pCAMBIA series, etc.

In some embodiments, the application relates to a fusion cell comprising the aforementioned recombinant vector; and expressing the cells. In some embodiments, the expression cell may be a bacterium, a fungus, a higher plant cell, or the like. In some embodiments, the gene encoding a lignan oxymethyl transferase is obtained by introducing the gene into the E.coli, mammalian cells, yeast cells or Pichia pastoris genetically engineered with glycosylation modification pathways.

In some embodiments, the foregoing polypeptide molecules, or polynucleotide molecules encoding the foregoing polypeptide molecules, may be used to catalyze lignan methylation modifications. In some embodiments, the lignan is a dibenzylbutane lignan. Further, the dibenzylbutane lignan is regoramin (Pregomisin). In some embodiments, pregomisin (Pregomisin) may obtain schisandra lignan compound D (Schisandrathera D) under the oxymethyl catalytic activity of the aforementioned polypeptide molecules;

in some embodiments, the schisandra lignan compound D (Schisandrathera D) has the following structural formula:

the application discloses a screening and functional identification method of an encoding gene SchOMT7 of an oxymethyl transferase for catalyzing hydroxy methylation in a lignan biosynthesis pathway in shizandra berry, wherein SchOMT7 can catalyze hydroxy methylation of progamicin to generate a lignan compound D (Schisandrathera D) in shizandra berry, lays a foundation for the research of synthesis biology of various dibenzylbutane lignans in shizandra berry, and has great application value.

The technical scheme of the present application will be explained herein by the following examples.

EXAMPLE 1 cloning of expressed Gene SchOMT7

1.1 extraction of Schisandra chinensis total RNA by CTAB-PVP method

(1) Fresh schisandra plant material (leaf or fruit) is taken and rapidly ground into powder in liquid nitrogen.

(2) Estimating 50-100mg of powder by using a centrifuge tube, adding 600-800 mu L of CTAB-PVP extracting solution preheated at 65 ℃ into a 2mL inlet centrifuge tube precooled in advance, and placing the mixture in a vortex oscillator to oscillate for 30s to enable the mixture to be fully cracked.

The preparation method of the CTAB-PVP extraction buffer solution comprises the following steps:

100mM Tris-HCl (pH 8.0), 2% CTAB (w/v), 2% PVP (w/v), 25mM EDTA,2M NaCl, mercaptoethanol added to 0.2% after autoclaving; solution configuration double distilled water treated with DEPC (ddH ₂ O), autoclaving and then spare.

(3) The mixture was mixed by inverting it every 10 minutes in a water bath at 65℃for 30 minutes.

(4) After cooling to room temperature, 600-800. Mu.L of chloroform was added, and after mixing was reversed, centrifugation was carried out at 13,000rpm for 10min at 4 ℃.

(5) Transferring the supernatant to a new centrifuge tube with 2mL inlet, adding 600-800 μl of chloroform, shaking, mixing well, and centrifuging at 4deg.C at 13,000rpm for 10min.

(6) The above procedure was repeated (i.e., three extractions with chloroform).

(7) The supernatant was carefully pipetted into a fresh imported 1.5mL centrifuge tube, 1/3 of the 8M LiCl was added and allowed to stand at-20℃overnight.

(8) Centrifuge at 13,000rpm for 10min at 4℃and discard the supernatant.

(9) The precipitate was washed 2-3 times with 700. Mu.L of 75% ethanol (DEPC water formulation). Centrifuging, discarding the supernatant, and volatilizing residual ethanol.

(10) The total RNA was prepared by dissolving RNA in 30. Mu.L of sterilized water treated with protease K (Proteinase K). The concentration and quality of the extracted RNA were measured using a nucleic acid protein meter model BioPhotometer plus.

1.2SchOMT7 Gene full Length amplification

1.2.1 primer design

The gene was amplified by designing full-length primers SchOMT7-F/R on both sides of the Open Reading Frame (ORF) using the software SnapGene.

1.2.2cDNA Synthesis

The cDNA template strand is obtained by PCR technology with the total RNA of the extracted schisandra chinensis as a template and a PrimerScript RT Master Mix reverse transcription system.

The reverse transcription system and reverse transcription procedure were as follows:

reverse transcription PCR system:

reverse transcription procedure: 37 ℃ for 15min;

85℃,15s。

the reverse transcription product was stored at-20℃and diluted before use.

1.2.3 amplification of the Gene of interest

The diluted reverse transcribed Schisandra chinensis cDNA is used as a template and SchOMT7-F/R is used as a primer for amplification.

The amplification system and the amplification procedure were as follows:

the components are added into a 200 mu L PCR tube to be uniformly mixed, and the mixture is put into a PCR instrument for amplification after low-speed centrifugation according to the following procedures:

95℃,3min；

95 ℃ for 15s;52 ℃,15s;72 ℃,45s,30 cycles;

72℃,5min。

and (3) detecting the PCR reaction product by agarose gel electrophoresis, wherein the electrophoresis result is shown in figure 1, and cutting the target size strip into gel and recovering.

Example 2 construction of expression vector for SchOMT7 Gene

2.1 amplification of SchOMT7 fragment with homology arms

The SchOMT7 fragment was amplified with the homology arm-carrying primer SchOMT7-pET28 a-F/R. The amplification system and procedure are shown in example 1.

2.2 construction of expression vectors by homologous recombination

2.2.1 vector double enzyme digestion

Vector pET28a was digested with BamH I and Xho I in the following manner:

the enzyme digestion is carried out in a water bath at 37 ℃ for 30min. And adding 10 Xsample buffer (Loading buffer) into the enzyme digestion product to stop the reaction, then carrying out agarose gel electrophoresis, and selecting a proper strip for gel recovery, wherein the gel recovery method is the same as that described above and is not repeated.

2.2.2 homologous recombination, transformation and Positive validation

The target fragment with the homology arm is connected with the vector pET28a after enzyme digestion by a homologous recombination kit, and the system is as follows:

the components are fully and evenly mixed, reacted for 30min at 37 ℃, and immediately placed on ice for cooling. The ligation product was competent to transform E.coli DH 5. Alpha. Taking out competent cells of Escherichia coli DH5 alpha stored at-80 ℃, thawing on ice, adding all the connection products, gently blowing and mixing, and standing on ice for 30min; after 45s of heat shock in a water bath at 42 ℃, the mixture is rapidly placed on ice for 2min, 500 mu L of antibiotic-free LB culture medium is added, then the mixture is subjected to shaking culture for 1h in a culture box at 37 ℃, 200 mu L of transformation liquid is coated on LB solid culture medium (containing 100 mu g/mL kanamycin resistance), and the mixture is subjected to static culture at 37 ℃ for 12h to 16h.

And selecting the monoclonal to perform colony PCR (polymerase chain reaction) to verify positive, amplifying a bright monoclonal with single target size band as positive, sequencing the positive clone, taking the correctly sequenced monoclonal to store bacteria, and extracting the SchOMT7-pET32a plasmid. The constructed prokaryotic expression vector plasmid is transformed into competent cells of escherichia coli BL21 (DE 3) by a thermal shock method, and the transformation, screening and identification methods are the same as those described above and are not repeated.

Example 3 Gene protein expression and enzymatic Activity function analysis

3.1 prokaryotic expression of SchOMT7 recombinant protein

(1) The strain SchOMT7-pET28a-BL21 positive clone was picked and inoculated into 4mL of LB medium containing kanamycin (Kana) resistance, and shake cultured overnight at 37℃and 110 rpm.

(2) Inoculating the cultured bacterial liquid into 200mL of Kana-resistant culture medium according to the ratio of 1:100, and culturing under the same condition until OD ₆₀₀ And approximately 0.5. Adding 0.3mM IPTG into the bacterial liquid, and culturing in a shaking table at 16 ℃ and 110rpm for 16-18 hours to induce the expression of the target protein.

(3) And (3) bacterial collection: the bacterial solution was centrifuged at 5000rpm and the supernatant was discarded after 5 minutes.

(4) Washing: adding a proper amount of Binding buffer into a centrifuge tube according to the bacterial amount, re-suspending the bacterial body, centrifuging at 5,000rpm for 5min, collecting bacterial body, washing twice, and adding 15-20 mL of Binding buffer to re-suspend the bacterial body.

(5) Cracking: placing the bacterial liquid into an ice-water mixture, performing ultrasonic bacterial lysis on the bacterial liquid, centrifuging at 12,000rpm at 4 ℃ for 20min, and collecting the supernatant to obtain crude enzyme.

3.2 functional verification of SchOMT7 crude enzyme

The SchOMT7 performs in vitro enzyme activity function identification, and takes a reaction system added with pET28a protein as a control group. The substrate is pre-gomisin, gomisin J and gomisin K1. The enzyme activity reaction system is as follows:

the above components were mixed and allowed to react overnight at 30℃and then quenched by adding 50. Mu.L of methanol, and the supernatant was centrifuged at 12,000rpm for 30 minutes and analyzed by HPLC for enzymatic activity.

The results are shown in figure 2, where SchOMT7 catalyzes the production of the corresponding product of bisbenzylbutane type lignan pregoamide compared to the blank.

Purification and functional verification of 3.3pchomt 7 recombinant protein

Expression of recombinant proteins As described in 3.1, the supernatant of the disrupted bacterial solution was subjected to column purification, and a portion of the supernatant was prepared for SDS-PAGE to observe the protein expression.

(1) Separating: the collected supernatant was fed to an equilibrated Ni-NTA column, and after the supernatant was drained, a column volume of eluent (containing 20mM imidazole) was added to wash out the impurity proteins, followed by collection of the recombinant protein of interest with 5mL of Elution buffer (Elution buffer) (containing 250mM imidazole).

(2) Ultrafiltration: placing the eluted protein solution into a ultrafiltration tube with a protein molecule of 30,000Da, centrifuging for 10min at 4,000rcf, adding Binding buffer (Binding buffer) for 2-3 times, and concentrating target protein.

(3) The concentrate was aspirated into a 2mL collection tube, the protein concentration was determined, and the sample was left.

(4) After adding 10% glycerol into the protein, quick freezing the protein with liquid nitrogen and storing the protein in a refrigerator at the temperature of minus 80 ℃ for standby.

Binding buffer (Binding buffer): 2.42g Tris-HCl and 29.22g NaCl are respectively weighed, dissolved in water, pH is regulated to 8.0, volume is fixed to 1000mL, 70 mu L beta-mercaptoethanol is added after sterilization, and the mixture is preserved at 4 ℃.

Elution buffer (Elution buffer): 2.42g Tris-HCl, 29.22g NaCl and 34gimidazole are respectively weighed, dissolved in water, pH is regulated to 8.0, the volume is fixed to 1000mL, 70 mu L beta-mercaptoethanol is added after sterilization, and the mixture is preserved at 4 ℃.

Activity verification of purified proteins enzyme activity assay was performed using LC-MS as described in 3.2. As a result, as shown in FIG. 3, schOMT7 was purified to catalyze regrind efficiently and the molecular weight of the resulting product was that of methyl group.

The above embodiments are provided for illustrating the present application and not for limiting the present application, and various changes and modifications may be made by one skilled in the relevant art without departing from the scope of the present application, therefore, all equivalent technical solutions shall fall within the scope of the present disclosure.

Claims (10)

1. A polypeptide molecule having an amino acid sequence selected from the group consisting of:

(A)SEQ ID NO.2；

(B) An amino acid sequence which is generated by substituting, deleting and/or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.2; and

(C) A truncate of (a) or (B);

wherein the polypeptide molecule has an activity of catalyzing the methylation of the phenolic hydroxyl group of lignans.

2. The polypeptide molecule of claim 1, wherein the lignan is a dibenzylbutane-type lignan.

3. The polypeptide molecule of claim 2, wherein the lignan is regoramin (Pregomisin).

4. A polynucleotide molecule encoding the polypeptide molecule of any one of claims 1-3, said polynucleotide molecule selected from the group consisting of:

(a)SEQ ID NO.1；

(b) A nucleic acid sequence having more than 80% homology with SEQ ID NO.1; and

(c) The truncate of (a) or (b).

5. A recombinant vector comprising:

the polynucleotide molecule of claim 4; and

an expression vector.

6. A fused cell comprising:

the recombinant vector of claim 5; and

and expressing the cells.

7. A method of preparing a protein for catalyzing methylation modification of dibenzylbutane lignans, comprising:

transforming the nucleic acid molecule of claim 4 into an expression cell;

a protein expressing the polypeptide molecule in an expression cell; and

purifying the protein of the polypeptide molecule.

8. Use of a polypeptide molecule according to any one of claims 1-3 for catalyzing the methylation modification of dibenzylbutane lignans.

9. A method of preparing a schisandra lignan compound D (Schisandrathera D), the method comprising: pre-gomisin (Pregomisin) is catalyzed with one or more of the following groups:

a polypeptide molecule according to any one of claims 1 to 3;

a polypeptide molecule encoded by the nucleic acid molecule of claim 4;

a polypeptide molecule expressed by the recombinant vector of claim 5;

a polypeptide molecule obtained from the fusion cell of claim 6; and

a polypeptide molecule prepared by the method of claim 7.

10. The schisandra lignan compound D (Schisandrathera D) prepared by the method of claim 9.