CN109666714A - A method of preparing 12 β-O-Glc-PPD and 12 β-O-Glc-PPT - Google Patents

A method of preparing 12 β-O-Glc-PPD and 12 β-O-Glc-PPT Download PDF

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CN109666714A
CN109666714A CN201710955862.3A CN201710955862A CN109666714A CN 109666714 A CN109666714 A CN 109666714A CN 201710955862 A CN201710955862 A CN 201710955862A CN 109666714 A CN109666714 A CN 109666714A
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glc
ppd
ppt
synthase
substrate
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杨金玲
朱平
梁会超
张婷婷
胡宗风
李燕
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Abstract

The present invention relates to glycosyl hydrolase LXYL-P1-2 to prepare the purposes in 12 β-O-Glc-PPD of non-natural ginsenoside and 12 β-O-Glc-PPT, the method for preparing 12 β-O-Glc-PPD and 12 β-O-Glc-PPT, and the recombinant cell of production 12 β-O-Glc-PPD and 12 β-O-Glc-PPT.Therefore, the present invention realizes the prepare with scale of 12 β-O-Glc-PPD and 12 β-O-Glc-PPT by biological method.

Description

A method of preparing 12 β-O-Glc-PPD and 12 β-O-Glc-PPT
Technical field
The present invention relates to glycosyl hydrolase LXYL-P1-2 to prepare non-natural ginsenoside 12 β-O-Glc-PPD and 12 β- Purposes in O-Glc-PPT, the preparation method of 12 β-O-Glc-PPD and 12 β-O-Glc-PPT, and 12 β-O-Glc- of production The recombinant cell of PPD and 12 β-O-Glc-PPT.
Background technique
Pharmaceutical research proves that the main active of ginseng is ginsenoside (ginsenoside).Ginsenoside category In triterpene compound, can be divided into two types according to aglycon skeleton difference: one kind is dammarane type four-ring triterpenoid saponins； Another kind of is Triterpenoids sapogenins saponins.Dammarane saponins account for the overwhelming majority in ginsenoside, are therein Main active.Dammarane saponins include panoxadiol type saponin(e and panaxatriol type saponin(e again.In glycosyl transferase Under effect, one or several glycosyls are introduced on the C3-OH and C20-OH of protopanoxadiol (protopanoxadiol, PPD) Form panoxadiol type saponin(e；One is introduced on the C6-OH and C20-OH of protopanaxatriol (protopanaxatriol, PPT) A or several glycosyls form panaxatriol type saponin(e.
PPD and PPT is in addition to C3-OH, C6-OH and C20-OH, and there are also C12-OH, but does not find C12-OH also in ginseng Glycosylated saponin(e.Atopkina etc. is using PPD as precursor, using acetyl acetylbromoglycose as glycosyl donor, in silver oxide and dichloro Under the catalysis of methane, pass through the glycosylated 12 β-O-D- glucopyra of non-natural ginsenoside of the semi-synthetic C12-OH of chemical method Glycosyl-Da Ma -24- alkene -3 β, 12 β, and 20S- triol (12 β-O- β-D-glucopyranosyl-dammar-24-ene-3 β, 12 β, 20S-triol, referred to as 12 β-O-Glc-PPD), and carried out pharmacology activity research.As a result, it has been found that-O-Glc-PPD couples of 12 β The cytotoxic activity of lung carcinoma cell is apparently higher than the natural ginsengs such as Rg3, Rh2 saponin(e (Atopkina LN, Malinovskaya GV, Elyakov GB, et al., Cytotoxicity of natural ginseng glycosides and Semisynthetic analogues, Planta Medica, 1999,65 (1): 30-34).Niu Yiming etc. is with C3 and C6 hydroxyl Base acetylation protection and the exposed PPT of C12 hydroxyls is precursor, using specific neighbour's alkynylbenzoate as glycosyl donor, PPh3AuNTf2 is to have synthesized Chikusetsusaponin L under catalysts conditions10(i.e. 12 β-O- β-D- Glucopyranosyl-dammar-24-ene-3 β, 6 α, 12 β, 20S-tetraol, referred to as 12 β-O-Glc-PPT) (ox one Ring, ginsenoside efficiently synthesize, Zhengzhou: Zhengzhou University, 2012 are integrally incorporated herein by reference).But change Learning synthetic method has that by-product is more, yield is low and at high cost, and a large amount of pollutants can be generated in synthesis process, right Environment causes damages.
In view of this, it would be highly desirable to excavate the new life of 12 β-O-Glc-PPD of non-natural ginsenoside and 12 β-O-Glc-PPT Production approach.
Summary of the invention
The technical problem to be solved by the present invention is to provide a kind of glycosyl hydrolase LXYL-P1-2 to prepare non-natural ginseng soap Purposes in 12 β-O-Glc-PPD of glycosides and/or 12 β-O-Glc-PPT.Further provide for a kind of production non-natural ginsenoside 12 The method of β-O-Glc-PPD and/or 12 β-O-Glc-PPT, and the recombinant cell of 12 β-O-Glc-PPD of production.
In order to solve the above-mentioned technical problem, through research, the inventor has found that, from fungi mushroom (Lentinula Edodes, M95.33) in the glycosyl hydrolase LXYL-P1-2 (Genbank number of registration: JN167171.1) that is cloned into can be with water Solve 3 β, 12 β-Di-O-Glc-PPD and 3 β, the position the C3 glucosyl group of 12 β-Di-O-Glc-PPT.In addition, changing LXYL-P1-2 It is catalyzed 3 β, 12 β-Di-O-Glc-PPD and 3 β, the reaction time of 12 β-Di-O-Glc-PPT and condition, discovery only has one kind Hydrolysate (12 β-O-Glc-PPD or 12 β-O-Glc-PPT) generates, without detecting what C12 glucosyl groups were hydrolyzed Product, this illustrates that LXYL-P1-2 can hydrolyze 3 β, 12 β-Di-O-Glc-PPD and 3 β with specificity, 12 β-Di-O-Glc-PPT's C3 glucosyl groups.
In addition, the present inventor is cloned into from bacillus subtilis (Bacillus subtilis) to convert PPD For 3 β of non-natural ginsenoside, the sugar of 12 β-Di-O-Glc-PPD (C3-OH and C12-OH while glycosylated product of PPD) Based transferase BsUGT1.On this basis, the present inventor utilizes dammarendiol-II synthase, protopanoxadiol synthase/protoplast Join triol synthase, nicotinamide adenine dinucleoside phosphate-cytochrome P450 reductase, glycosyl transferase BsUGT1 (Genbank number of registration: KY952161) and glycosyl hydrolase LXYL-P1-2, which have been constructed, can directly produce 12 β-O-Glc- The recombinant bacterium of PPD and 12 β-O-Glc-PPT.
Glycosyl transferase BsUGT1 is also disclosed in Chinese Patent Application No. 201710232360.8, herein with reference Mode is integrally incorporated herein.Glycosyl transferase LXYL-P1-2 is also disclosed in Chinese patent CN102296053B, herein It is integrally incorporated by reference herein.
In the first aspect, the present invention provides glycosyl hydrolase LXYL-P1-2 to prepare 12 β of non-natural ginsenoside- Purposes in O-Glc-PPD and/or 12 β-O-Glc-PPT.
Substrate is 3 β, 12 β-Di-O-Glc-PPD and/or 3 β, 12 β-Di-O-Glc-PPT.
In embodiments of the present invention, substrate can be at form below: 3 β of form of mixtures or unpack format, 3 β of 12 β-Di-O-Glc-PPD, form of mixtures or unpack format, 12 β-Di-O-Glc-PPT, production 3 β, 12 β-Di-O- The recombinant cell of Glc-PPD and/or 3 β, the recombinant cell or their combination of 12 β-Di-O-Glc-PPT.
In second aspect, the present invention provides production 12 β-O-Glc-PPD of non-natural ginsenoside and 12 β-O-Glc-PPT Method, the method includes use glycosyl hydrolase LXYL-P1-2 hydrolyze 3 β, 12 β-Di-O-Glc-PPD and/or 3 β, 12 The position the C3 glucosyl group of β-Di-O-Glc-PPT, to obtain 12 β-O-Glc-PPD and/or 12 β-O-Glc-PPT.
In a preferred embodiment, the method for the present invention includes the following steps:
(1) by 3 β of substrate, 12 β-Di-O-Glc-PPD and/or 3 β, 12 β-Di-O-Glc-PPT dissolution；
(2) it in the buffer that pH is 3.0~6.0, preferably 3.5~5.0, more preferable 4.0~4.5, is added step (1) Substrate solution and glycosyl hydrolase LXYL-P1-2, reaction temperature be 25~50 DEG C, preferably 35~50 DEG C, it is more preferable 35~ Under conditions of 45 DEG C, 3 β of substrate, 12 β-Di-O-Glc-PPD and/or 3 β, the position the C3 glucose of 12 β-Di-O-Glc-PPT are hydrolyzed Base, to obtain 12 β-O-Glc-PPD and/or 12 β-O-Glc-PPT.
3 β, 12 β-Di-O-Glc-PPD and/or 3 β are dissolved, the solvent of 12 β-Di-O-Glc-PPT can exist for above-mentioned substance It wherein can dissolve or sl. sol. any solvent be, it is preferable to use dimethyl sulfoxide is dissolved.
Buffer used is preferably Acetic acid-sodium acetate buffer.Wherein, the concentration of Acetic acid-sodium acetate buffer can be 10~200mM, preferably 50~100mM.
In the third aspect, the present invention provides the recombinant cell of 12 β-O-Glc-PPD of production, led in the recombinant cell Enter to have coding gene below: dammarendiol-II synthase, protopanoxadiol synthase, nicotinamide adenine dinucleoside phosphate- Cytochrome P450 reductase, glycosyl transferase BsUGT1 and glycosyl hydrolase LXYL-P1-2.
In fourth aspect, the present invention provides the recombinant cell of 12 β-O-Glc-PPT of production, led in the recombinant cell Enter to have coding gene below: dammarendiol-II synthase, protopanoxadiol synthase, protopanaxatriol synthase, nicotinoyl amine gland Purine dinucleotide phosphoric acid-cytochrome P450 reductase, glycosyl transferase BsUGT1 and glycosyl hydrolase LXYL-P1-2.
In some embodiments, the form that the gene of importing can dissociate and/or integrate exists.Preferably, importing Gene is present in recombinant cell in a free form.Preferably, the gene of importing is to be integrated into host cell gene group Form exists.
Preferably, recombinant cell can be Escherichia coli, saccharomyces cerevisiae and/or Pichia pastoris.Recombinant cell is most preferably Brewing yeast cell.
Saccharomyces cerevisiae of the invention can be any saccharomyces cerevisiae obtained by this field.For example, commercially available wine brewing Yeast INVSc1, saccharomyces cerevisiae BY4742, saccharomyces cerevisiae YPH499 or saccharomyces cerevisiae W303-1B etc..
Channel genes can be recombinated thin by the commercially available any carrier in this field or with the carrier of similar functions In born of the same parents.For example, episomal vector can be pESC serial carrier, including pESC-HIS, pESC-URA, pESC-TRP and pESC- TRP；pYES2；Or pAUR123.
In a preferred embodiment, by dammarendiol-II synthase and green fluorescent protein GFP amalgamation and expression.For Yield is further increased, by the lactosterol synthase ERG7 gene deregulation in recombinant cell.
Any method well known by persons skilled in the art can be used to make the expression of target gene (such as erg7 gene) Decline or ergosterol synthase activity is made to decline (including inactivating target gene), such method includes but is not limited to: clpp gene It removes, rite-directed mutagenesis or RNA interference (RNAi).
In some embodiments, the method for reducing ergosterol synthase activity level is RNAi.For example, in a reality It applies in mode, the method for reducing ergosterol synthase activity level is the antisense fragments for importing ergosterol synthase gene erg7 (erg7 of antisense expression vector expression).
It yet still another aspect, the present invention also provides recombinant cells described above to prepare 12 β-O- of non-natural ginsenoside Purposes in Glc-PPD and/or 12 β-O-Glc-PPT.
Beneficial effect
Provide the glycosyl hydrolase LXYL-P1-2 for capableing of specificity ginsenoside C3 glucosyl group of hydrolysis.So that logical Biological method acquisition 12 β-O-Glc-PPD and 12 β-O-Glc-PPT are crossed to be possibly realized.
Meanwhile first passage synthetic biology technology acquisition of the present invention can generate 12 β-O-Glc- of non-natural ginsenoside The recombinant cell of PPD is laid the foundation for 12 β-O-Glc-PPD of prepare with scale with carrying out antitumor innovation drug research.
Detailed description of the invention
Fig. 1 shows HPLC the and LC-MS testing result of recombination BsUGT1 catalysis PPD glycosylation product.A: Transetta-32a crude enzyme liquid is catalyzed PPD reaction product；B:Transetta-BsUGT1 crude enzyme liquid is catalyzed PPD reaction product； C: the MS map of product.
Fig. 2 shows HPLC the and LC-MS testing results of recombination BsUGT1 catalysis PPT glycosylation product.A: Transetta-32a crude enzyme liquid is catalyzed PPT reaction product；B:Transetta-BsUGT1 crude enzyme liquid is catalyzed PPT reaction product； C: the MS map of product.
Fig. 3 shows recombination LXYL-P1-2 and is catalyzed 3 β, the glycosyl hydrolase reaction product of 12 β-Di-O-Glc-PPD HPLC and LC-MS testing result.
Fig. 4 shows recombination LXYL-P1-2 catalysis 3 β, the HPLC of 12 β-Di-O-Glc-PPT glycosyl hydrolase reaction products With LC-MS testing result.
Fig. 5 shows the recombinant expression plasmid map constructed in the embodiment of the present invention 3.
Fig. 6 shows the HPLC testing result of the 12 β-Di-O-Glc-PPD generated in recombinant bacterium.A:12 β-Di-O-Glc-PPD Standard items；B: the extract of recombinant bacterium INVSc1-DS-GFP/CYP1/Re/BsUGT1/P1-2.
Fig. 7 shows the LC-MS result of the 12 β-Di-O-Glc-PPD generated in recombinant bacterium.
Specific embodiment
Exemplary embodiments of the present invention are described below, those skilled in the art are it will be obvious that following implementation Mode is not intended to limit only certain exemplary embodiments of this invention, it is thus understood that including in the spirit and scope of the present invention all variants, Equivalent or substitute.A variety of adjustment and other embodiment are expected in the limit of power of those of ordinary skill in the art It falls within the scope of the present invention.
Unless otherwise indicated, the conventional method that experimental method used herein below is well known to the skilled person, For example, can be implemented using standardization program described in following works: Sambrook etc., Molecular Cloning:A Laboratory Manual (the 3rd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001)；Davis etc., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995)；And Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino etc. writes, John Wiley and Sons, Inc.).
It is better understood the present invention by means of following embodiments, these embodiments are only used for illustrating the present invention, no It should be interpreted limitation of the present invention.
In the examples below, UDP-glucose, Escherichia coli Trans1-T1 competent cell and Transetta impression State cell is purchased from Beijing Quanshijin Biotechnology Co., Ltd, and saccharomyces cerevisiae INVSc1 is purchased from Invitrogen company.Clone carries Body pEASY-Blunt simple vector is purchased from Beijing Quanshijin Biotechnology Co., Ltd, and expression vector pESC series carries Body is purchased from Invitrogen company.Restriction enzyme, T4 DNA ligase and Q5DNA polymerase are purchased from New England Biolabs (NEB) company, bacterial genomes DNA extraction kit and bacteria plasmid DNA extracts kit are purchased From TIANGEN Biotech (Beijing) Co., Ltd..The primer and sequencing are by Beijing Bioisystech Co., Ltd, farsighted Boxing section It completes.
Standard items 12 β-O-Glc-PPD and 12 β-O-Glc-PPT according to Atopkina LN, Malinovskaya GV, Elyakov GB, et al., Cytotoxicity of natural ginseng glycosides and Semisynthetic analogues, Planta Medica, 1999,65 (1): 30-34 and Niu Yiming, ginsenoside It efficiently synthesizes, Zhengzhou: Zhengzhou University, the preparation of method described in 2012.
Embodiment 1. 3 β, 12 β-Di-O-Glc-PPD and 3 β, the preparation of 12 β-Di-O-Glc-PPT
The expression of 1.1 glycosyl transferase BsUGT1
According to Genbank number of registration: KY952161 synthesizes BsUGT1 full-length gene, and by itself and cloning vector pEASY- Blunt simple vector connection, the reaction condition of connection are as follows: (20 DEG C -37 DEG C) reaction 20min of room temperature.Connection is produced Object directly converts Trans1-T1 competent cell, and picking single colonie carries out PCR verifying, chooses the correct transformant of PCR result Sequencing confirmation is carried out, the plasmid vector for being named as pEASY-Blunt-BsUGT1 is obtained.
Using plasmid pEASY-Blunt-BsUGT1 as template, by BsUGT1 gene cloning to prokaryotic expression plasmid pET-32a In (+), while restriction enzyme site BamH I and Sal I is introduced, to obtain recombinant plasmid pET-32a (+)-BsUGT1.
Correct transformant is sequenced in picking, is incubated overnight, and extracts plasmid pET-32a (+)-BsUGT1.By control plasmid PET-32a (+) and recombinant plasmid pET-32a (+)-BsUGT1 convert Transetta competent cell respectively, and it is negative right to obtain According to conversion bacterial strain Transetta-32a and Transetta-BsUGT1.Picking transformant is inoculated in 30mL LB culture medium and (contains 100 μ g/mL of ampicillin), 37 DEG C, 220rpm is cultivated to OD600=0.6~1；It is transferred with 1% inoculum concentration in 100mL LB Culture medium (the 100 μ g/mL containing ampicillin) is inoculated with 30 bottles altogether.37 DEG C, 220rpm is cultivated to OD600=0.6~1, it is added IPTG to final concentration 1mM, 16 DEG C, 180rpm cultivates 16h, induction recombinant protein expression.It is examined using SDS-PAGE electrophoresis It surveys.The result shows that BsUGT1 is expressed in Escherichia coli with soluble form.
1.2 3 β, the preparation of 12 β-Di-O-Glc-PPD
Using Transetta-32a cell as negative control group, by Transetta-32a cell and Transetta- BsUGT1 cell carries out ultrasonication in 20mM Tris-HCl (pH 8.0).Using broken supernatant as crude enzyme liquid, with UDP- Glucose is glycosyl donor, and PPD is substrate, carries out external enzymatic reaction.
Reaction system:
88 μ L of crude enzyme liquid
2 μ L of 50mM substrate
50mM UDPG 10μL
Reaction system mixes, and 37 DEG C, stands reaction for 24 hours, and 200 μ L ice methanol are added and terminate reaction, mix, 12000rpm It is centrifuged 10min, supernatant is taken to cross 0.22 μm of filter membrane, utilizes the glycation product of HPLC detection BsUGT1 catalysis PPD.
HPLC testing conditions: Cosmosil C18 reversed-phase column, 4.6 × 150mm, flow velocity 1mL/min, ultraviolet detection wave A length of 203nm, 30 μ L of sample introduction.Mobile phase: 0min, 20% acetonitrile；20min, 85% acetonitrile；30min, 100% acetonitrile； 40min, 100% acetonitrile；41min, 20% acetonitrile；50min, 20% acetonitrile.
The results show that only detecting that Rt is the PPD substrate peak (Figure 1A) of 24.44min in control group；With the thick enzyme of BsUGT1 Liquid carries out in Catalysis experiments, is that 12.93min has apparent product peak (Figure 1B) in Rt, UV absorption is consistent with substrate.Benefit The product is detected with LC-MS, its molecular weight is 784.5 as the result is shown, compared to PPD molecular weight 460.5, is increased 324 (Fig. 1 C)；1H-NMR and13It is that sugar occurs two positions by PPD under BsUGT1 catalytic action that C-NMR, which confirms the product, Base metaplasia at product 3 β, 12 β-Di-O-Glc-PPD.
3 β, 12 β-Di-O-Glc-PPD:ESI-MS m/z 807.48 [M+Na]+；13C-NMR(125MHz, Methanol- d4)δ131.85,(C-25),126.32,(C-24),90.66,(C-3),71.67,(C-12),70.96, (C-20),57.54, (C-5),55.03,(C-14),53.19,(C-17),51.19,(C-9),49.63,(C-13), 47.32,(C-22),40.97, (C-4),40.36,(C-1),40.03,(C-10),38.16,(C-8),36.71,(C-7), 35.76,(C-11),31.87, (C-15),28.67,(C-2),28.39,(C-16),27.17,(C-21),26.19, (C-26),25.92,(C-28), 23.30,(C-23),19.21,(C-6),17.76,(C-27),17.39,(C-19), 16.78,(C-18and C-29), 16.19,(C-30),106.78,(C-1′),77.68,(C-2′),79.46,(C-3′), 75.14,(C-4′),78.28,(C- 5′),62.81,(C-6′),100.58,(C-1″),75.66,(C-2″),78.40, (C-3″),74.77,(C-4″),78.01, (C-5″),62.37,(C-6″)。
1.3 3 β, the preparation of 12 β-Di-O-Glc-PPT
Using PPT as substrate, reacted with the identical enzymatic reaction condition for preparing with 3 β, 12 β-Di-O-Glc-PPD.
HPLC testing conditions: Cosmosil C18 reversed-phase column, 4.6 × 150mm, flow velocity 1mL/min, ultraviolet detection wave A length of 203nm, 30 μ L of sample introduction.Mobile phase: 0min, 10% acetonitrile；20min, 55% acetonitrile；30min, 70% acetonitrile；31min, 100% acetonitrile；40min, 100% acetonitrile；41min, 10% acetonitrile；50min, 10% acetonitrile.
The results show that only detecting that Rt is the PPT substrate peak (Fig. 2A) of 23.72min in control group；In the thick enzyme of BsUGT1 Liquid carries out in Catalysis experiments, is that 15.12min has apparent product peak (Fig. 2 B) in Rt, UV absorption is consistent with substrate.Benefit The product is detected with LC-MS, its molecular weight is 800.5 as the result is shown, compared to substrate PPT molecular weight 476.5, is increased 324 (Fig. 2 C) are added；1H-NMR and13It is in the case where recombinating BsUGT1 catalytic action, by PPT two positions that C-NMR, which confirms the product, It sets and product 3 β, 12 β-Di-O-Glc-PPT that glycosylation generates occurs.
3 β, 12 β-Di-O-Glc-PPT:ESI-MS m/z 823.48 [M+Na]+；13C-NMR(125MHz, Methanol- d4)δ131.85,(C-25),126.29,(C-24),90.68,(C-3),74.76,(C-20),70.97, (C-12),68.79, (C-6),62.82,(C-5),55.05,(C-17),53.02,(C-14),50.54,(C-9), 47.10,(C-13),46.95, (C-7),41.93,(C-8),40.94,(C-4),40.00,(C-10),39.82,(C-1), 36.64,(C-22),31.82, (C-11),31.19,(C-28),28.60,(C-15),27.60,(C-2),26.94, (C-21),26.14,(C-16), 25.92,(C-26),23.29,(C-23),17.76,(C-18and C-27),17.63, (C-19),17.40,(C-30), 16.80,(C-29),107.00,(C-1′),78.28,(C-2′),79.31,(C-3′), 71.68,(C-4′),78.00,(C- 5′),62.35,(C-6′),100.55,(C-1″),75.14,(C-2″),75.71, (C-3″),77.69,(C-4″),78.43, (C-5″),62.38,(C-6″)。
2 glycosyl hydrolase LXYL-P1-2 of embodiment is catalyzed rare ginsenoside glycosyl hydrolase Activity determination
The pure enzyme of LXYL-P1-2 is prepared according to Chinese patent CN102296053B.
With 3 β, 12 β-Di-O-Glc-PPD and 3 β, 12 β-Di-O-Glc-PPT are substrate, be utilized respectively LXYL-P1-2 into Row catalysis.
Hydrolysis reaction system:
The pure enzyme 500ng of LXYL-P1-2
With the 2 μ L of 50mM substrate of dmso solution
100mM HAc-NaAc (pH 4.5) buffer complements to 100 μ L
After catalyst system mixes, 45 DEG C of shaking baths, reaction for 24 hours, is added 200 μ L ice methanol and terminates reaction, after mixing 12000rpm is centrifuged 10min, takes supernatant to cross 0.22 μm of filter membrane, detected using HPLC to hydrolysate.
HPLC condition:
Agilent1200 high performance liquid chromatography, agilent company；
Cosmosil C18 reversed-phase column, 4.6 × 150mm, flow velocity 1mL/min, ultraviolet detection wavelength are 203nm, sample introduction 30μL；
It is substrate: 0min, 20%ACN with 3 β, 12 β-Di-O-Glc-PPD；20min, 85%ACN；30min, 100% ACN；40min, 100%ACN；41min, 20%ACN；50min, 20%ACN；
It is substrate: 0min, 10%ACN with 3 β, 12 β-Di-O-Glc-PPT；20min, 55%ACN, 30min, 70% ACN；31min, 100%ACN；40min, 100%ACN；41min, 10%ACN；50min, 10%ACN.
It is substrate with 3 β, 12 β-Di-O-Glc-PPD, LXYL-P1-2 is added, catalysate is detected by HPLC.As a result Display is that 17.87min has apparent product peak, i.e. compound 1 (Fig. 3 A, B), UV absorption and bottom at retention time (Rt) Object is consistent.The product is detected using LC-MS.The results show that the molecular weight of the compound is 622.5, compared to 3 β, 12 β-Di-O-Glc-PPD molecular weight 784.5, reduce 162, i.e., compound 1 be by 3 β, 12 β-Di-O-Glc-PPD one of them The product (Fig. 3 C) that glucosyl group hydrolysis generates.
It is substrate with 3 β, 12 β-Di-O-Glc-PPT, LXYL-P1-2 is added, catalysate is detected by HPLC.As a result Display is that 17.06min has apparent product peak, i.e. compound 2 (Fig. 4 A, B) in Rt, and UV absorption is consistent with substrate.Benefit The product is detected with LC-MS.The results show that 2 molecular weight of compound is 638.5, relative to 3 β, 12 β-Di-O-Glc- PPT molecular weight 800.5 reduces 162, i.e. compound 2 is by 3 β, the one of glucosyl group hydrolysis of 12 β-Di-O-Glc-PPT The product (Fig. 4 C) of generation.
The preparation of hydrolysate and Structural Identification:
According to above-mentioned reaction condition, 3 β, 12 β-Di-O-Glc-PPD and 3 β, 12 β-Di-O-Glc- are catalyzed to LXYL-P1-2 The hydrolysis of PPT has carried out the amplification of 5mL catalyst system, has carried out the enzyme law catalysis synthesis of compound 1 and 2.Using the side HPLC Method prepares hydrolysate (Shimadzu SPD-20A preparative high-efficient liquid phase analysis work station, Japanese Shimadzu Corporation；Preparation condition: Aglient C18 reversed-phase column, 9.6 × 250mm, flow velocity 4mL/min, ultraviolet detection wavelength are 203nm；Mobile phase: compound 1 use 62% acetonitrile, compound 2 use 42% acetonitrile), prepare product be utilized respectively LC-MS,1H-NMR、13C-NMR and HMBC It is detected, determine glycosyl position and structure: compound 1 is 3 β, and the C3 position glucosyl group of 12 β-Di-O-Glc-PPD hydrolyzes 12 β-the O-Glc-PPD generated；Compound 2 is 3 β, 12 β-that the C3 position glucosyl group hydrolysis of 12 β-Di-O-Glc-PPT generates O-Glc-PPT, structural formula are as follows.
The LC-MS of hydrolysate,1H-NMR and13C-NMR data:
12 β-O-Glc-PPD:ESI-MS m/z 645.48 [M+Na]+；13C-NMR(125MHz,Methanol-d4) δ 131.85,126.32,100.59,79.51,79.46,78.38,77.99,75.15,74.77,70.96,62.37,57.24, 55.03,53.19,51.20,47.31,40.94,40.07,40.05,38.41,36.70,35.75,31.86,28.67, 28.61, 27.97,27.62,26.18,25.92,23.30,19.38,17.76,17.37,16.72,16.18,16.11.With Know that document NMR data is consistent.
12 β-O-Glc-PPT:ESI-MS m/z 661.48 [M+Na]+；13C-NMR(125MHz,Methanol-d4) δ 131.86,126.30,100.56,79.50,79.30,78.41,77.98,75.16,74.76,70.97,68.88,62.38, 62.10,55.06,53.02,50.54,47.06,46.93,41.96,40.53,40.36,39.88,36.64,31.82, 31.40, 28.59,27.74,27.60,26.14,25.92,23.28,17.75,17.68,17.62,17.38,16.11.With Know that document NMR data is consistent.
Embodiment 3. produces the building of 12 β-Di-O-Glc-PPD engineering bacterias
The building of 3.1 recombinant expression carriers
Recombinant expression carrier pESC-HIS-DS-GFP can be superfine according to beam, and ginseng dammarendiol-II synthase is being made wine Expression, positioning and functional study in yeast, Acta Pharmaceutica Sinica, 2016,51 (6): the building of method described in 998-1003.
Recombinant expression carrier pESC-URA-erg7-According to Wang Qinghua etc., inhibit saccharomyces cerevisiae sheep using Antisense RNA Technique The expression of hair sterol synthase gene, Acta Pharmaceutica Sinica, 2015,50 (1): the building of method described in 118-122.
According to GenBank number of registration: JN167171.1 synthesizes LXYL-P1-2 full length gene, and is connected to clone In carrier pEASY-Blunt simple vector, vector plasmid pEASY-Blunt-P1-2 is obtained, wherein in P1-2 gene Both ends introduce restrictive restriction endonuclease Apa I and Xho I.
Method according to the embodiment 1 constructs plasmid pEASY-BsUGT1, and introduces at the both ends of BsUGT1 gene Restriction enzyme Not I and Spe I.
The building of pEASY-Blunt-CYP1 and pEASY-Blunt-CYP-Re: the ginseng protoplast registered according to GenBank Join the cDNA sequence (No.JN604536.1) of glycol synthase gene cyp1, synthesizes overall length cyp1 gene, and be connected to gram In grand carrier pEASY-Blunt simple vector, vector plasmid pEASY-Blunt-CYP1 is obtained, wherein in cyp1 base Because both ends introduce restrictive restriction enzyme site BamH I and Sal I；
According to the cDNA sequence of the arabidopsis CYP450 reductase gene cyp-re of GenBank registration (No.X66017.1), overall length cyp-re gene is synthesized, and is connected to cloning vector pEASY-Blunt simple In vector, vector plasmid pEASY-Blunt-CYP-Re is obtained, wherein introduce restrictive enzyme at cyp-re gene both ends Enzyme site BamH I and Sal I.
The building of pESC-HIS-DS-GFP-P1-2: with restriction enzyme A pa I and Xho I to plasmid pESC-HIS- DS-GFP and pEASY-Blunt-P1-2 (LXYL-P1-2 GenBank number of registration: JN167171.1) carry out double digestion, with solidifying Plastic recovery kit recycles plasmid vector and target gene fragment after digestion, by T4DNA Ligase linked system, by base Because lxyl-p1-2 is connected to the MCS2 (Fig. 5 A) of pESC-HIS-DS-GFP.
pESC-URA-erg7-The building of-CYP1: with restriction enzyme BamH I and Sal I to plasmid pESC-URA- erg7-Double digestion is carried out with pEASY-Blunt-CYP1, recycles the plasmid vector and mesh after digestion using gel reclaims kit Genetic fragment gene cyp1 is connected to by pESC-URA-erg7 by T4DNA Ligase linked system-MCS2 (figure 5B)。
The building of pESC-TRP-CYP-Re: with restriction enzyme BamH I and Sal I to plasmid pESC-TRP and PEASY-Blunt-CYP-Re carries out double digestion, recycles the plasmid vector and purpose base after digestion using gel reclaims kit Because gene cyp-re is connected to the MCS2 (Fig. 5 C) of pESC-TRP by T4DNA Ligase linked system by segment.
The building of pESC-LEU-BsUGT1: with restriction enzyme Not I and Spe I to plasmid pESC-LEU and PEASY-Blunt-BsUGT1 carries out double digestion, recycles the plasmid vector and purpose base after digestion using gel reclaims kit Because of segment, by establishing eFusion linked system, gene BsUGT1 is connected to the MCS1 (Fig. 5 D) of pESC-LEU.
Above-mentioned linked system is converted into Trans1-T1 competent cell respectively, screens positive transformant, extracts plasmid simultaneously Sequence verification.Recombinant expression plasmid is respectively designated as pESC-HIS-DS-GFP-P1-2, pESC-URA-erg7--CYP1、 PESC-LEU-BsUGT1 and pESC-TRP-CYP-Re.
Expression casette in 1. recombinant expression plasmid of table
Wherein, double digestion system used and digestion condition are as follows:
Double digestion system:
Wherein, T4DNA ligase linked system used and condition of contact are as follows:
Linked system (20 μ L):
At room temperature, 30min is connected, converts Escherichia coli Trans1-T1 competent cell immediately, screens positive transformant, And extract its plasmid.
Wherein, eFusion linked system used and condition of contact are as follows:
At room temperature, 30min is connected, converts Escherichia coli Trans1-T1 competent cell immediately, screens positive transformant And extract its plasmid.
The building of 3.2 recombinant yeasts
Using LiAc/SS Carrier DNA/PEG conversion method, by recombinant expression plasmid pESC-HIS-DS-GFP-P1- 2、pESC-URA-erg7-- CYP1, pESC-LEU-BsUGT1 and pESC-TRP-CYP-Re while transformed saccharomyces cerevisiae INVSc1 screens recombinant bacterial strain on auxotroph culture medium SD/-HIS-URA-LEU-TRP.Picking transformant extracts and turns Beggar's plasmid.Using plasmid as template, PCR amplification is carried out with the special primer of dependent conversion gene, gene is verified by PCR It imports properly, to obtain recombinant bacterial strain, is named as INVSc1-DS-GFP/CYP1/Re/BsUGT1/P1-2.By upper Method is stated by empty carrier pESC-His, pESC-Ura, pESC-Leu and pESC-Trp while transformed saccharomyces cerevisiae INVSc1, is obtained The recombinant bacterium obtained is as negative control bacterium.
The separation and identification of 12 β-Di-O-Glc-PPD in 3.3 recombinant bacteriums
Under the conditions of 30 DEG C and 220rpm, with 2% on auxotroph culture medium SG/-HIS-URA-LEU-TRP 10 d of galactolipin Fiber differentiation recombinant bacterium INVSc1-DS-GFP/CYP1/Re/BsUGT1/P1-2, thalline were collected by centrifugation and fermentation Liquid, thallus is cold dry.Dry bacterium 1.0g is weighed, 70% ethyl alcohol of 150mL, 70 DEG C of reflux 2h is added, natural cooling filters and goes degerming Body residue, extracting solution evaporated under reduced pressure are dissolved with 100 mL distilled waters, use 100mL water-saturated n-butanol extraction 3 times respectively, every time Stand 30min.Combining extraction liquid is simultaneously evaporated n-butanol, with methanol lysate, 100 μ L of sample introduction after 0.22 μm of membrane filtration, Whether there is 12 β-Di-O-Glc-PPD generation in HPLC detection thallus.
Supernatant after taking 1L fermentation liquid to be centrifuged is concentrated under reduced pressure and arrives 100mL, respectively with 100mL water-saturated n-butanol extraction 3 It is secondary, 30min is stood every time.Combining extraction liquid is simultaneously evaporated n-butanol, with methanol lysate, sample introduction after 0.22 μm of membrane filtration Whether there is 12 β-Di-O-Glc-PPD generation in 100 μ L, HPLC detection fermentation liquids.
Product in recombinant bacterium INVSc1-DS-GFP/CYP1/Re/BsUGT1/P1-2 cell is extracted, HPLC, LC-MS are carried out It is detected with NMR.HPLC in recombinant bacterium the results show that occur consistent with 12 β-Di-O-Glc-PPD standard items UV absorptions and Rt Compound (Fig. 6), LC-MS is the results show that the fragment ion peak of this compound is consistent with 12 β-Di-O-Glc-PPD standard items (Fig. 7).As the result is shown, data are consistent with the 12 β-Di-O-Glc-PPD prepared by enzymatic reaction by NMR.Fermentation medium In be not detected and 12 β-Di-O-Glc-PPD standard items UV absorptions and the consistent compound of Rt.
HPLC testing conditions: Cosmosil C18 reversed-phase column, 4.6 × 150mm, flow velocity 1mL/min, ultraviolet detection wave A length of 203nm, 100 μ L of sample introduction.Mobile phase: 0min, 42% acetonitrile；30min, 42% acetonitrile.

Claims (12)

1. glycosyl hydrolase LXYL-P1-2 is preparing 12 β-O-Glc-PPD of non-natural ginsenoside and/or 12 β-O-Glc-PPT In purposes.
2. purposes according to claim 1, which is characterized in that substrate is 3 β, 12 β-Di-O-Glc-PPD and/or 3 β, 12 β-Di- O-Glc-PPT。
3. a kind of method for producing 12 β-O-Glc-PPD of non-natural ginsenoside and/or 12 β-O-Glc-PPT, which is characterized in that The method includes using glycosyl hydrolase LXYL-P1-2 to hydrolyze 3 β of substrate, 12 β-Di-O-Glc-PPD and/or 3 β, 12 β-Di- The position the C3 glycosyl of O-Glc-PPT generates 12 β-O-Glc-PPD and/or 12 β-O-Glc-PPT.
4. according to the method described in claim 3, it is characterized in that, which comprises
(1) by 3 β of substrate, 12 β-Di-O-Glc-PPD and/or 3 β, it is molten that substrate is made in 12 β-Di-O-Glc-PPT dissolution Liquid；
(2) in the buffer that pH is 3.0~6.0, the substrate solution and glycosyl hydrolase LXYL-P1-2 of step (1) is added, Under conditions of reaction temperature is 25~50 DEG C, reaction for 24 hours, hydrolyzes 3 β of substrate, 12 β-Di-O-Glc-PPD and/or 3 β, 12 β-Di- The position the C3 glycosyl of O-Glc-PPT, to obtain 12 β-O-Glc-PPD and/or 12 β-O-Glc-PPT.
5. according to the method described in claim 4, it is characterized in that, by the substrate dmso solution；In step (2) The buffer is Acetic acid-sodium acetate buffer, and the concentration of the Acetic acid-sodium acetate buffer is 10~200mM；Described PH is 3.5~5, and the reaction temperature is 35~50 DEG C.
6. method according to claim 5, which is characterized in that the concentration of the Acetic acid-sodium acetate buffer be 50~ 100mM；The pH is 4.0~4.5；Reaction temperature is 35~45 DEG C.
7. a kind of recombinant cell for producing 12 β-O-Glc-PPD, is imported with the gene for encoding following albumen in the recombinant cell: Dammarendiol-II synthase, protopanoxadiol synthase, nicotinamide adenine dinucleoside phosphate-cytochrome P450 reductase, sugar Based transferase BsUGT1 and glycosyl hydrolase LXYL-P1-2.
8. a kind of recombinant cell for producing 12 β-O-Glc-PPT, is imported with the gene for encoding following albumen in the recombinant cell: Dammarendiol-II synthase, protopanoxadiol synthase, protopanaxatriol synthase, nicotinamide adenine dinucleoside phosphate-cell color Plain P450 reductase, glycosyl transferase BsUGT1 and glycosyl hydrolase LXYL-P1-2.
9. according to the described in any item recombinant cells of claim 7 or 8, which is characterized in that the recombinant cell is selected from large intestine Bacillus, saccharomyces cerevisiae and/or Pichia pastoris.
10. according to the described in any item recombinant cells of claim 7 or 8, which is characterized in that described in the recombinant cell Dammarendiol-II synthase and green fluorescent protein GFP amalgamation and expression.
11. according to the described in any item recombinant cells of claim 7 or 8, which is characterized in that the recombinant cell is lanosterol The cell that synthase ERG7 gene expression is lowered.
12. the described in any item recombinant cells of claim 7-11 prepare 12 β-O-Glc-PPD of non-natural ginsenoside and/or Purposes in 12 β-O-Glc-PPT.
CN201710955862.3A 2017-10-15 2017-10-15 A method of preparing 12 β-O-Glc-PPD and 12 β-O-Glc-PPT Pending CN109666714A (en)

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