CN106350565A - Production method of rare ginsenoside Rh2 - Google Patents
Production method of rare ginsenoside Rh2 Download PDFInfo
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Abstract
The invention discloses a production method of rare ginsenoside Rh2 and belongs to the field of enzymatic synthesis of ginsenoside. The production method includes that protopanoxadiol is used as a substrate, glycosyltransferase YjiC or YojK screened from bacillus subtilis is used as a catalyst, and the glycosylation of the C3 hydroxyl of the protopanoxadiol is efficiently catalyzed through in-vitro enzyme reaction to generate the ginsenoside Rh2. The production method is cheap in raw materials, high in ginsenoside Rh2 yield, low in production cost and capable of achieving large-scale production of the precious and rare ginsenoside Rh2.
Description
Technical field
A kind of a kind of the present invention relates to method that ginsenoside rh2 is produced by protopanoxadiol, more particularly, it relates to profit
With glycosyl transferase yjic or yojk to the c3 position hydroxyl glycosylation of protopanoxadiol the method to produce ginsenoside rh2.
Background technology
Ginsenoside rh2(ginsenoside rh2), it is commonly called as shield life element, prolong raw effect and gain the name because it has shield life.
Ginsenoside rh2 is one of topmost anti-tumor active substance in Radix Ginseng, has suppression growth of tumour cell, suppresses tumor thin
The effects such as activity of born of the same parents' telomerase, inducing apoptosis of tumour cell, antitumor cell transfer, abnormal differentiation of reversing tumor cell,
Thus having functions that good antitumor and preventing and treating tumor recurrence transfer.Clinically, ginsenoside rh2 and radiation and chemotherapy
Combine Deng therapeutic modality, effect in treatment of cancer for the radiation and chemotherapy can be strengthened.Additionally, ginsenoside rh2 also has
The a series of physiological functions such as antiallergic, raising immunity, resisting fatigue and antiinflammatory.
Ginsenoside rh2 is the extremely rare a kind of ginsenoside of content in Radix Ginseng, and its content in Radix Ginseng Rubra only has ten
Ten thousand/left and right, and there's almost no in Radix Ginseng.At present, the production of ginsenoside rh2 has three kinds of modes.(1) with Chinese medicine
The Radix Ginseng total saponinss extracting acquisition in material Radix Ginseng are raw material, using acid hydrolysis, basic hydrolysiss, enzymatic hydrolysises and fermentable water
The mode of solution is hydrolyzed to Radix Ginseng total saponinss production ginsenoside rh2, because Ginseng Growth cycle length, growth conditionss are harsh,
Total Ginsenosides Content is low, extraction process is complicated and the yield of ginsenoside rh2 is low, leads to the production of ginsenoside rh2 to become
This high it is difficult to meet the requirement of its large-scale industrial production.(2) two are joined with the precursor substance protoplast of ginsenoside rh2
Alcohol is substrate, carries out glycosylation modified production ginsenoside rh2 by chemical method, chemical method complex synthetic route, relatively costly,
Conversion ratio is low, stereo selectivity is poor and has the generation of a large amount of by-products, has been rarely employed in recent years.(3) using synthesis life
The biosynthesis pathway that thing technology builds ginsenoside rh2 in Microbial cell factories produces ginsenoside rh2(wang,
pingping, et al. "production of bioactive ginsenosides rh2 and rg3 by
Metabolically engineered yeasts. " metabolic engineering 29 (2015): 97-105.)；
But, ginsenoside rh2 has higher toxicity to microbial cell, leads to Microbial cell factories to be difficult to synthesize in a large number Radix Ginseng
Saponin rh2, equally can not meet the demand of industrialized production.
Protopanoxadiol is the precursor substance of ginsenoside rh2, and its price is relatively inexpensive, and by integrating protopanoxadiol
Biosynthesis related genes can produce protopanoxadiol (dai, zhubo, et al. in brewing yeast cell factory in a large number
"metabolic engineering of saccharomyces cerevisiae for production of
Ginsenosides. " metabolic engineering 20 (2013): 146-156.).Meanwhile, enzymatic reaction has and urges
Change efficiency high, the features such as specificity is strong, substrate tolerance is good, product purification is simple.Therefore, with price more cheap and supply fill
The protopanoxadiol of foot is substrate, excavates that expression is high, catalysis activity is high, region and the strong glycosyl transferase of stereo selectivity exist
External protopanoxadiol is carried out glycosylation modified can the extremely high ginsenoside rh2 of production prices in large quantities.
The glycosyl transferase obtaining with respect to screening in plant (such as: Radix Ginseng), screens the glycosyl obtaining from microorganism and turns
Move enzyme and generally there is broader substrate spectrum and higher enzyme activity, it is catalyzed glycosylated mode and also has more multiformity, or even can
Some non-natural glycation products can be obtained.For example, to 137 kinds different from the glycosyl transferase oled of antibiosis chain enzyme bacteria
Substrate all has higher activity, these substrates be all much natural product in plant (gantt r w, goff r d,
williams g j, et al. probing the aglycon promiscuity of an engineered
glycosyltransferase[j]. angewandte chemie international edition, 2008, 47
(46): 8889-8892).Therefore, carry out the excacation of glycosyl transferase from microorganism, it may be found that living to ginsenoside
Property high and even can be with the glycosyl transferase of synthesizing new ginsenoside.
Content of the invention
Technical problem
At present, the production method of ginsenoside rh2 includes the hydrolysis of Radix Ginseng total saponinss, the chemical method of protopanoxadiol is catalyzed and micro-
The biosynthesiss of biological cell factory.Radix Ginseng total saponinss hydrolysis produce ginsenoside rh2 be subject to raw material is rare, extraction process is complicated,
The low impact of efficiency of pcr product leads to the production cost of rh2 higher it is difficult to meet the demand of its large-scale industrial production.In addition,
Chemical method carries out glycosylation modified production ginsenoside rh2 and there is complex synthetic route, relatively costly, conversion to protopanoxadiol
Rate is low, stereo selectivity is poor and has the technical problems such as a large amount of by-products generations.Simultaneously as ginsenoside rh2 is to microorganism
Cell there is higher toxicity it is difficult to by the method for synthetic biology in Microbial cell factories to its a large amount of synthesis.
Therefore, it is an aspect of the present invention to provide a kind of production method of ginsenoside rh2, its with produce ginsenoside before
The typical method of rh2 is compared more economical and efficient and can be met industrial production demand.
Technical solution
Embodiments of the invention provide a kind of production method of ginsenoside rh2, comprising: with protopanoxadiol as substrate, with
Udp- glucose is glycosyl donor, excavates the glycosyl transferase that the expression obtaining is high, catalysis activity is strong to its c3 position using new
Hydroxyl glycosylation generates ginsenoside rh2；Glycation product described in purification；And the glycation product that crystallization is purified.
Beneficial effect
The present invention provides one kind using cheap and well-off protopanoxadiol as raw material, using the new height excavating and obtaining
The method that the glycosyl transferase of effect carries out glycosylation modified production ginsenoside rh2 in vitro to protopanoxadiol, thus avoid
The biosynthesiss of the hydrolysis of Radix Ginseng total saponinss, the chemical method catalysis of protopanoxadiol and Microbial cell factories produce rh2's
During have such problems as that production cost is high, extraction process is complicated, efficiency of pcr product is low, by-product is many, with more economical, more efficient
Mode produce ginsenoside rh2, thus promoting its large-scale production.
Brief description
Fig. 1 is the synthetic method of ginsenoside rh2 and f12.
Fig. 2 is the expression that sds-page analyzes glycosyl transferase yjic.
Fig. 3 is the expression that sds-page analyzes glycosyl transferase yojk.
Fig. 4 is the impact to glycosyl transferase yjic and yojk enzymatic activity for the ph.
Fig. 5 is the impact to glycosyl transferase yjic and yojk enzymatic activity for the temperature.
Fig. 6 is the impact to glycosyl transferase yjic and yojk enzymatic activity for the metal ion.
Fig. 7 is the process chart producing ginsenoside rh2 according to one embodiment of present invention.
Fig. 8 is the production technological process of the ginsenoside rh2 being described according to the present invention.
Fig. 9 is the protopanoxadiol or ginsenoside rh2 catalytic production ginsenoside through glycosyl transferase yjic or yojk
Rh2 and the liquid chromatogram of ginsenoside f12.
Figure 10 is the mass spectrum of ginsenoside's rh2 product.
Figure 11 is the mass spectrum of ginsenoside's f12 product.
Figure 12 is the nucleus magnetic hydrogen spectrum figure of ginsenoside's rh2 product.
Figure 13 is the nuclear-magnetism carbon spectrogram of ginsenoside's rh2 product.
Figure 14 is the nucleus magnetic hydrogen spectrum figure of ginsenoside's f12 product.
Figure 15 is the nuclear-magnetism carbon spectrogram of ginsenoside's f12 product.
Specific embodiment
One embodiment of the present of invention, with reference to Fig. 7, a kind of production method of ginsenoside rh2, comprising: (a) is joined with protoplast
Glycol is substrate, with udp- glucose as glycosyl donor, using the c3 position to protopanoxadiol for glycosyl transferase yjic or yojk
Hydroxyl glycosylation obtains ginsenoside rh2；B () purification is to obtain described glycation product；C () enters to the glycation product of purification
Row crystallization.
With reference to Fig. 7, an alternative embodiment of the invention, the source of protopanoxadiol include extracting from Radix Ginseng obtain, water
Solution ginsenoside obtains, combines acquisition using the acquisition of Microbial cell factories biosynthesiss or these modes.In addition, glycosylation
Glycosyl donor needed for reaction include udp- glucose, tdp- glucose, adp- glucose, gdp- glucose, cdp- glucose or
Derivant of other nucleoside diphosphate glucoses and combinations thereof.
Glycosyl transferase encoding gene yjic and yojk of the present invention derive from bacillus subtilises (bacillus subtilis168) or its mutant enzyme, the nucleotide coding sequence of described glycosyl transferase yjic and yojk is respectively seq
Id no 1 and 2, or with the homology 85%(of nucleotide sequence shown in seq id no 1 or 2 preferably 95%) and encode
The polypeptide product nucleotide sequence active to protopanoxadiol.The peptide sequence of described glycosyl transferase yjic and yojk is respectively
For seq id no 3 and 4, or with the homology 85%(of seq id no 3 or 4 preferably 95%) and protopanoxadiol had
Activated peptide sequence.
Wherein, described glycosyl transferase can be by conversion or the heterologous table of recombination bacillus coli incorporating nucleotide sequence 1 or 2
Reach acquisition.In addition, the bacterial strain for expressing glycosyl transferase also include saccharomyces cerevisiae (saccharomyces cerevisiae),
Pichia sp. (pichia pastoris), corynebacterium glutamicum (corynebacterium glutamicum), hay spore
Bacillus (bacillus subtilis) or aspergillus niger (aspergillus nigar).
Therefore, an alternative embodiment of the invention is recombination bacillus coli bl21 (de3)/pet28-yjic and escherichia coli
Bl21 (de3)/pet28-yojk, as shown in Figures 2 and 3, in escherichia coli bl21(de3) in, yjic and yojk is completely
Solubility expression, the glycosyl transferase of expression is in the supernatant of bacterial cell disruption liquid.
Produce ginsenoside rh2 using glycosyl transferase to carry out under conditions of 15 DEG C -50 DEG C and ph 4-ph 11,
Preferably carry out under conditions of 30 DEG C of -40 DEG C and ph 8-ph 9, under the conditions of this temperature and ph, glycosyl transferase is to former
Panoxadiol carries out glycosylation, has reaction rate the fastest and the yield of highest ginsenoside rh2.In other temperature
And under the conditions of ph, glycosyl transferase yjic and yojk can also produce ginsenoside rh2 with protopanoxadiol as substrate, but
Catalysis activity is poor, and the yield of ginsenoside rh2 is relatively low.
In addition, the impact to enzyme activity with reference to the ph described in Fig. 4.Within the specific limits, when ph value increases, enzymatic activity increases therewith
Plus, if however, the ph value of enzyme reaction system is less than 6 or more than 10, enzyme activity then drastically declines.The ph value of enzyme reaction system is permissible
Configured using different buffer, for example, the ph value of the buffer regulatable enzyme reaction of trihydroxy aminomethane (tris)
To 8.0-9.0.With reference to the impact to enzyme activity for the temperature described in Fig. 5, under the conditions of 30-40 DEG C, glycosyl transferase is to protopanoxadiol
Conversion ratio highest, less than 30 DEG C or higher than 40 DEG C when, enzyme activity all can decline.
In one embodiment, producing ginsenoside rh2 using glycosyl transferase yjic and yojk can be in metal ion
In the presence of carry out, metal ion can be used as the activator of glycosyl transferase, and specific metal ion includes mn2+、mg2+、ca2+And
The slaine of these metal ions can be produced.With reference to Fig. 6, enzymatic activity is activated by part metals ion, wherein, mn2+、mg2 +、ca2+To glycosyl transferase yjic and yojk, there is significant activation.In addition, the activation to glycosyl transferase for the metal ion
Effect has concentration dependent, and its concentration is the most notable to the activation of glycosyl transferase in the range of 5-10 mm.
In the present invention, it is possible to use in art, known various purification process are carried out to the ginsenoside rh2 producing
Purification.The example of purification process includes organic solvent extraction and preparing chromatograph in industry purification, but not limited to this.By organic solvent
Extract and separate protopanoxadiol and ginsenoside rh2 are to dissolve sex differernce in aqueous phase with organic faciess based on product to be separated to carry out
, in one embodiment, organic solvent can be ethyl acetate, but not limited to this.Separated by preparing chromatograph in industry method
Ginsenoside rh2 is to be carried out based on the difference of product to be separated and chromatographic column absorption, in one embodiment, industrially prepared
Chromatographic column can be c18 type reverse preparative hplc post, but not limited to this.Specifically, chromatography is using being configured with system
The high voltage chromatograph of standby type c18 reverse chromatograms post carries out to glycation product separating.
In one embodiment, before or after glycation product purification, need to carry out desalination, decolouring, deproteinization or same
These process of Shi Jinhang.Decolouring can add the activated carbon of 0.03%3% weight (mass/volume) to carry out by stirring, concrete bar
Part is stirring 1h-4h under the rotating speed of 50-200 rpm.
Desalination and deproteinization can be carried out in the way of being extracted using organic solvent.Organic solvent and the addition of glycation product
Ratio can be 1:0.5 to 5:1, specifically 1:1-2:1.Specific organic solvent includes ethyl acetate, n-butyl alcohol, oil
Ether etc., but not limited to this.When being extracted with organic solvent, can be owned with efficient recovery as far as possible by way of multiple extraction
Glycation product, specifically extracts 4-5 time.Meanwhile, after multiple extraction, its response rate is not less than glycation product
95%.
Further decolouring, desalination and Deproteinated glycation product are concentrated, be beneficial to subsequent purification process.Specifically
For, glycation product can be concentrated to 30 g/l or be more than 30 g/l, such as 40 g/l.
By way of slow cold cut, glycation product can be lowered the temperature, so that purified ginsenoside rh2 is crystallized,
Thus reaching detached purpose.Specifically, can with per hour 0.5 DEG C to 3 DEG C of speed by the glycation product of purification from 60
DEG C cold cut is crystallized to 10 DEG C.In addition, the further dehydrate of ginsenoside rh2 that also crystallization can be obtained, it is beneficial to
The storage of ginsenoside rh2 and transport that purification obtains.
With reference to Fig. 8, a kind of production method of ginsenoside rh2 comprises to carry out sugar using glycosyl transferase to protopanoxadiol
Base comprises the glycation product of ginsenoside rh2 to obtain；Purifying glycosylation product；The glycation product concentrating and purifying；Knot
The brilliant glycation product concentrating is to obtain ginsenoside's rh2 crystal；And it is brilliant ginsenoside rh2 to be further dried by dehydration
Body.
The purity of the ginsenoside rh2 of the production according to an embodiment can be 90% or be more than 90%, specifically
Be 95% or more than 95%, more particularly 98% or be more than 98%.The detection of the purity of ginsenoside rh2 can be in step
After (b) and before step (c).In another embodiment, the inspection of the detection of the purity of ginsenoside rh2 can be
After step (c).
According to another embodiment of the invention, the unreacted protopanoxadiol that purification reclaims after the step (b) can
To generate ginsenoside rh2 to carry out glycosylation again being added to circulation in step (a), or after step (c) with
The detached protopanoxadiol of ginsenoside's rh2 crystal can be added to recirculation in step (b) again, or step (a) and step
Suddenly (b) all can be carried out.
With reference to Fig. 8, after ginsenoside rh2 and the purified separation of protopanoxadiol in glycation product, separate obtain former
Panoxadiol can be used as the substrate of the glycosylation in step (a) again.In addition, with reference to Fig. 8, the ginsenoside of crystal form
Rh2 is partially separated in crystallisation step with the remaining charging removing ginsenoside's rh2 crystal, also deposits in the charging of remaining after crystallization
In a small amount of ginsenoside rh2, it can be re-used for purification.With reference to Fig. 8, the present invention describes another embodiment simultaneously,
Wherein reuse detached protopanoxadiol in step (b) can enter with reusing the charging removing crystal in step (c) simultaneously
OK.
According to one embodiment of present invention with protopanoxadiol as substrate, carry out glycosylation acquisition using glycosyl transferase
Product can be at least one glycation product selected in the component that ginsenoside rh2, ginsenoside f12 are formed.Glycosyl
Change product to extract with crude separation and concentration using organic solvent, then refined using process-scale chromatography and separated.
In general, protopanoxadiol is larger with its glycation product polarity spectrum, it is possible to use preparative c18 industry color
Spectrum post is efficiently separated by high voltage chromatograph, but not limited to this.
Hereinafter, will be described in further detail to the present invention in conjunction with specific embodiments.It should be understood that given is concrete
Embodiment only for illustrating the present invention, rather than in order to limit the range of the present invention.
The preparation of embodiment 1 glycosyl transferase
Using bacterial genomes dna extracts kit extract bacillus subtilises (bacillus subtilis168) gene
Group dna, then to extract Bacillus subtilis genes group dna obtaining as template, according to the primer sequence in table 1, utilizes
Primer pair yjic-f and yjic-r and primer pair yojk-f and yojk-r expand respectively acquisition glycosyltransferase gene yjic and
yojk.The amplification system of pcr is: q5 buffer(5 ×) 10 μ l, dntp (2.5 mm) 3 μ l, genome dna template: 1 μ
L, primer (10 μm) each 2.5 μ l, q5 exo+ polymerase 0.5 μ l, polishing distilled water to 50 μ l.Pcr reaction condition be
98 DEG C of predeformation 3 minutes (1 circulation), 98 DEG C deform 10 seconds, annealing 20 seconds (annealing temperature is 56 DEG C), 72 DEG C extend 1 point
Clock (31 circulations), 72 DEG C of extensions 2 minutes (1 circulation).
Table 1 primer sequence
After pcr reaction terminates, by dna QIAquick Gel Extraction Kit purification glycosyltransferase gene yjic and yojk.Then, using restriction
Property restriction endonuclease bamhi and sali carries out enzyme action process respectively to glycosyltransferase gene yjic and yojk and pet28 plasmid.Enzyme
After cutting into, dna QIAquick Gel Extraction Kit is used to reclaim digestion products, in the presence of t4 ligase, by glycosyl transferase base again
Because yjic and yojk is connected respectively on plasmid pet-28, thus building recombinant vector pet28-yjic and pet28- respectively
yojk.Pet28-yjic and pet28-yojk through sequence verification correct after, convert respectively and obtain in escherichia coli bl21 (de3)
Recombinant bacterial strain bl21 (de3)/pet28-yjic and bl21 (de3)/pet28-yojk.
In order to obtain glycosyl transferase, by recombination bacillus coli bl21 (de3)/pet28-yjic and bl21 (de3)/
Pet28-yojk is inoculated in the lb fluid medium containing 50 μ g/ml kanamycin respectively, at 37 DEG C and 200 rpm
Under the conditions of, cultivate 2.5 h-3 h.Using spectrophotometer in 600 nm wavelength (od600) under the conditions of detect cell density, when
od600When value reaches 0.6-0.8, add isopropyl ss-d-1- thio-pyrylium galactose (iptg) in lb culture medium to final concentration
For 0.2 mm, and under conditions of 16 DEG C and 200 rpm, continue culture 16 h-20 h to induce the table of glycosyl transferase
Reach, sds-page result shows (Fig. 2 and Fig. 3), yjic and yojk is complete solubility expression in escherichia coli, and express
Glycosyl transferase size be about 45 kda, consistent (theory of yjic and yojk is divided with the glycosyl transferase molecular size of prediction
Son amount size is respectively 44 kda and 45 kda).
In order to measure glycosyl transferase yjic and yojk of expression in escherichia coli to protopanoxadiol and ginsenoside
The activity of rh2, prepares glycosyl transferase crude enzyme liquid using following methods.(1) take the cultured cells of 1l, be centrifuged under 8000 rpm
10 min, collect coli somatic, then will collect disruption buffer (the 25 mm tris- of the cell obtaining 100 ml
Hcl, ph 8.0) suspend；(2) make cellular lysate using the method that ultrasonication or high-pressure homogenization crush, then at 4 DEG C and
Under conditions of 20000 rpm, the thalline of cracking is centrifuged, the broken liquid supernatant obtaining after collecting centrifugation is glycosyl and turns
Move enzyme crude enzyme liquid.
Embodiment 2 glycosyl transferase yjic is used for producing ginsenoside rh2 and f12
The system of glycosylation includes the tris-hcl(ph8.0 of 25 mm/l), the protopanoxadiol of 1 mm/l or ginsenoside
The udp- glucose of rh2,3 mm/l, the mgcl of 10 mm/l2.According to 5%(v/v) ratio add the thick enzyme of glycosyl transferase yjic
Liquid to reactant liquor, 35 DEG C, 120 rpm, react 4 h.
After glycosylation terminates, add ethyl acetate extraction glycation product, and repeat to extract 4 times.It is then combined with total
The glycation product of totally 5 extractions, is concentrated using Rotary Evaporators, is subsequently adding the sugar of chromatograph methanol dissolution extraction again
Base product, carries out detection and quantitation using liquid chromatograph mass spectrography to glycation product.Chromatograph of liquid is Agilent
1260 types, chromatographic column be the moon rising sun science and technology (Shanghai) limited company c18 chromatographic column (model: xdb-c18,250 × 4.6
Mm, 5 μm), condition of gradient elution is: 0-25min, 25%-85% acetonitrile；25-45min, 85% acetonitrile.30 DEG C of column oven, flow velocity
For 1 ml/min, ultraviolet detection wavelength 203 nm.Mass Spectrometry Conditions are: positive ion mode, ion source: esi ion source.As Fig. 9 institute
Show, with protopanoxadiol as substrate, yjic is 85% to the conversion ratio of protopanoxadiol, and protopanoxadiol can be catalyzed and generate 2 kinds
Glycation product (f12 and rh2).Wherein the ratio of product f12 and rh2 is 1.3:1.In addition, yjic also has to ginsenoside rh2
Activity, can be catalyzed ginsenoside rh2 and generate single ginsenoside's f12 product, and the conversion ratio to ginsenoside rh2 for the yjic
For 86%.Mass spectral results show (Figure 10 and Figure 11), and the molecular weight of f12 and rh12 is respectively 785.5033 [m+h]+With
623.4502 [m+h]+, so that it is determined that two products generate after being respectively protopanoxadiol glycosylation with two glucoses
Molecule and the glycation product of a glucose molecule.
Embodiment 3 glycosyl transferase yojk catalysis protopanoxadiol produces ginsenoside rh2
The system of glycosylation includes the tris-hcl(ph8.0 of 25 mm/l), the protopanoxadiol of 1 mm/l, 3 mm/l's
Udp- glucose, the mgcl of 10 mm/l2.According to 10%(v/v) ratio add glycosyl transferase yojk crude enzyme liquid to reactant liquor
In, 35 DEG C, 120 rpm, react 4 h.
After glycosylation terminates, add ethyl acetate extraction glycation product, and repeat to extract 4 times.Merge all extractions
After taking product, the product utilization Rotary Evaporators of extraction are dried, are subsequently adding the glycosylation of chromatograph methanol dissolution extraction
Product, carries out detection and quantitation using liquid chromatograph mass spectrography to glycation product.Chromatograph of liquid is Agilent 1260
Type, chromatographic column is the c18 chromatographic column (model: xdb-c18,250 × 4.6 mm, 5 μm) of upper Placuna placenta (L.) rising sun science and technology, gradient elution
Condition is: 0-25min, 25%-85% acetonitrile；25-45min, 85% acetonitrile.30 DEG C of column oven, flow velocity is 1 ml/min, and ultraviolet is examined
Survey wavelength 203 nm.Mass Spectrometry Conditions are: positive ion mode, ion source: esi ion source.As shown in figure 9, yojk catalysis protoplast's ginseng
Glycol can generate single a kind glycation product (rh2), and yjic is 72% to the conversion ratio of protopanoxadiol.Mass spectral results
Display (Figure 11), the molecular weight of this product (ginsenoside rh2) is 623.4502 [m+h]+, so that it is determined that this product is protoplast
Ginseng glycol connects the glycation product of a upper glucose molecule.
Embodiment 4 process-scale chromatography purifying glycosylation product
Configure the reactant liquor of 1000 ml according to the reaction system in embodiment 2 and 3.Under the conditions of 35 DEG C, respectively with yjic and yojk
For the catalyst of glycosylation, react 12 h.With isopyknic ethyl acetate (1000 ml), glycosylation is produced after the completion of reaction
Thing extracts 4 times, and combined ethyl acetate extraction product is simultaneously concentrated by Rotary Evaporators and is dried, then, using c18 industry
Chromatographic column carries out purification to glycation product, and high pressure liquid chromatography condition is: flow velocity 10 ml/min, mobile phase be distilled water and
Chromatograph methanol, condition of gradient elution is: 0min-50min, 50%-100% methanol.By chromatogram purification, protoplast's ginseng can be obtained simultaneously
The glycation product ginsenoside f12 of glycol and ginsenoside rh2, the purity of ginsenoside f12 and ginsenoside rh2 is respectively
95% and 92%.
The crystallization of embodiment 5 ginsenoside rh2 and f12 and drying
To isolating and purifying the ginsenoside rh2 obtaining from embodiment 4 by process-scale chromatography and ginsenoside's f12 solution is tied
Brilliant.First, the ginsenoside's rh2 solution being concentrated and purified by Rotary Evaporators heating and ginsenoside's f12 solution, make Radix Ginseng soap
The concentration of glycosides rh2 solution and ginsenoside f12 is 30 g/l, then, makes ginsenoside rh2 molten with per hour 0.5 DEG C of speed
Liquid and ginsenoside's f12 solution are slowly lowered to 10 DEG C from 55 DEG C and so that ginsenoside rh2 and ginsenoside f12 is crystallized respectively.
After obtaining life saponin rh2 and ginsenoside's f12 crystal, by crystal is collected by centrifugation.Then fluid bed is utilized to do
Dry device, processes 2 h under the conditions of 70 DEG C, and ginsenoside rh2 and ginsenoside f12 crystal are dried.Meanwhile, the detection to crystal is tied
Fruit shows, it is 91% and 94% that the response rate of ginsenoside rh2 and ginsenoside f12 is respectively, and ginsenoside rh2 and Radix Ginseng soap
The purity of glycosides f12 is more than 99.9%, thus obtaining high-purity ginsenoside rh2 and ginsenoside's f12 product.
The Structural Identification of embodiment 6 ginsenoside rh2 and f12
The deuterated dimethyl sulfoxide of 600 μ l and deuterated methanol is taken to dissolve ginsenoside rh2 and the people of the high and dry process of 30 mg respectively
Ginseng saponin f12 powder, under the conditions of 400mhz, using two kinds of glycation product (ginsenoside rh2 to purification for the nuclear magnetic resonance analyser
With ginsenoside f12) carry out Structural Identification, as shown in table 2, the result of carbon spectrum and hydrogen spectrum shows that this two products are Radix Ginseng respectively
The c3 position hydroxyl glycation product of saponin rh2(protopanoxadiol) and the c3 position of ginsenoside's f12(protopanoxadiol and c12 hydroxyl
Base glycation product simultaneously) (nuclear magnetic spectrum is as shown in Figure 12-Figure 15).Wherein, ginsenoside f12 is non-existent in nature
Novel ginsenoside.
The carbon spectrum of table 2 ginsenoside rh2 and ginsenoside f12 and hydrogen spectrum
The carbon spectrum of table 2 ginsenoside rh2 and ginsenoside f12 and hydrogen spectrum
sequence listing
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>a kind of production method of rare ginsenoside rh2
<130> 2016
<160> 4
<170> patentin version 3.5
<210> 1
<211> 1179
<212> dna
<213>bacillus subtilises (bacillus subtilis 168)
<400> 1
atgaaaaagt accatatttc gatgatcaat atcccggcgt acggacatgt caatcctacg 60
cttgctttag tagagaagct ttgtgagaaa gggcaccgtg tcacgtacgc gacgactgag 120
gagtttgcgc ccgctgttca gcaagccggt ggagaagcat tgatctatca tacatccttg 180
aatattgatc ctaagcaaat cagggagatg atggaaaaga atgacgcgcc cctcagcctt 240
ttgaaagaat cactcagcat tctgccgcag cttgaggagt tatataagga tgatcagcct 300
gatctgatca tctatgactt tgttgcgctg gctggtaaat tgtttgctga aaagcttaat 360
gttccggtca ttaagctctg ttcgtcatat gcccaaaatg aatcctttca gttaggaaat 420
gaagacatgc tgaaaaaaat aagagaagca gaggctgaat ttaaagccta cttggagcaa 480
gagaagttgc cggctgtttc atttgaacag ttagctgtgc cggaagcatt aaatattgtc 540
tttatgccga agtcttttca gattcagcat gagacgttcg atgaccgttt ctgttttgtc 600
ggcccctctc tcggagaacg gaaggaaaaa gaaagcctgt tgattgacaa ggatgatcgc 660
ccgcttatgc tgatttcttt gggtacggcg tttaacgcat ggccggaatt ttacaagatg 720
tgcatcaagg catttcggga ttcttcatgg caagtgatca tgtcggttgg gaaaacgatt 780
gatccagaaa gcttggagga tattcctgct aactttacca ttcgccaaag tgtgccgcag 840
cttgaggtgt tagagaaagc tgatttgttc atctctcatg gcgggatgaa cagtacgatg 900
gaagcgatga acgcaggtgt gccgcttgtc gtcattccgc aaatgtatga gcaggagctc 960
actgcaaatc gggttgatga attaggcctt ggcgtttatt tgccgaaaga ggaagtgact 1020
gtttccagcc tgcaggaagc ggttcaggct gtatccagtg atcaagagct gctcagccgc 1080
gtcaagaata tgcaaaagga tgtaaaagaa gctggcggag cggagcgtgc ggcagctgag 1140
attgaagcgt ttatgaaaaa atccgctgtc ccgcagtaa 1179
<210> 2
<211> 1218
<212> dna
<213>bacillus subtilises (bacillus subtilis 168)
<400> 2
atggctaatg tattaatgat cggtttcccc ggtgaagggc atattaatcc ctctatcggt 60
gtgatgaagg agctgaaatc ccggggagaa aacattacgt actacgcagt gaaggaatat 120
aaagaaaaaa tcacagctct tgatatagag tttcgtgagt atcatgattt cagaggagat 180
tacttcggga aaaacgcaac cggcgatgaa gaaagagatt tcacagaaat gctctgcgct 240
tttttgaaag cctgtaagga tatcgcgact catatttatg aggaagtcaa acatgaatcg 300
tatgattatg tcatatatga tcaccatctt ctcgcgggta aagtcattgc caacatgctg 360
aagctgccaa gattttcatt gtgtacaacc tttgcgatga atgaggaatt tgcgaaggaa 420
atgatgggag cgtacatgaa aggatcactt gaagattcgc ctcattatga atcataccag 480
cagcttgcag aaacgttaaa tgctgatttt caagcagaga tcaagaagcc atttgatgtt 540
tttttagctg atggtgactt gacaatcgtc tttacatcaa ggggatttca gccactggct 600
gagcaatttg gcgagcgata tgtatttgtc ggtccttcca ttacagaaag agccggaaac 660
aatgatttcc catttgatca gattgacaat gaaaacgtgc tgtttatttc aatgggaacc 720
atttttaata atcaaaagca gttttttaat caatgccttg aagtgtgtaa ggactttgac 780
ggtaaagttg tgctttccat cggcaagcat attaaaacaa gtgagttaaa cgacattccg 840
gagaatttca ttgtacgccc gtatgtccct cagcttgaga tcttgaaaag agccagctta 900
tttgtgaccc acggcggaat gaacagcaca agtgaaggtt tgtattttga aaccccgctc 960
gttgtcattc cgatgggagg cgaccaattt gttgtcgcag atcaggtaga aaaagtcggc 1020
gcaggaaaag taattaaaaa ggaagaattg tctgaaagcc tactgaaaga gacgatacaa 1080
gaagtaatga ataatcgttc gtatgctgaa aaggcaaaag aaattggaca atcactgaaa 1140
gcggcaggcg gctctaaaaa agcagccgac agcattcttg aagctgtaaa acaaaaaact 1200
caatcagcaa atgcatag 1218
<210> 3
<211> 392
<212> prt
<213>bacillus subtilises (bacillus subtilis 168)
<400> 3
met lys lys tyr his ile ser met ile asn ile pro ala tyr gly his
1 5 10 15
val asn pro thr leu ala leu val glu lys leu cys glu lys gly his
20 25 30
arg val thr tyr ala thr thr glu glu phe ala pro ala val gln gln
35 40 45
ala gly gly glu ala leu ile tyr his thr ser leu asn ile asp pro
50 55 60
lys gln ile arg glu met met glu lys asn asp ala pro leu ser leu
65 70 75 80
leu lys glu ser leu ser ile leu pro gln leu glu glu leu tyr lys
85 90 95
asp asp gln pro asp leu ile ile tyr asp phe val ala leu ala gly
100 105 110
lys leu phe ala glu lys leu asn val pro val ile lys leu cys ser
115 120 125
ser tyr ala gln asn glu ser phe gln leu gly asn glu asp met leu
130 135 140
lys lys ile arg glu ala glu ala glu phe lys ala tyr leu glu gln
145 150 155 160
glu lys leu pro ala val ser phe glu gln leu ala val pro glu ala
165 170 175
leu asn ile val phe met pro lys ser phe gln ile gln his glu thr
180 185 190
phe asp asp arg phe cys phe val gly pro ser leu gly glu arg lys
195 200 205
glu lys glu ser leu leu ile asp lys asp asp arg pro leu met leu
210 215 220
ile ser leu gly thr ala phe asn ala trp pro glu phe tyr lys met
225 230 235 240
cys ile lys ala phe arg asp ser ser trp gln val ile met ser val
245 250 255
gly lys thr ile asp pro glu ser leu glu asp ile pro ala asn phe
260 265 270
thr ile arg gln ser val pro gln leu glu val leu glu lys ala asp
275 280 285
leu phe ile ser his gly gly met asn ser thr met glu ala met asn
290 295 300
ala gly val pro leu val val ile pro gln met tyr glu gln glu leu
305 310 315 320
thr ala asn arg val asp glu leu gly leu gly val tyr leu pro lys
325 330 335
glu glu val thr val ser ser leu gln glu ala val gln ala val ser
340 345 350
ser asp gln glu leu leu ser arg val lys asn met gln lys asp val
355 360 365
lys glu ala gly gly ala glu arg ala ala ala glu ile glu ala phe
370 375 380
met lys lys ser ala val pro gln
385 390
<210> 4
<211> 405
<212> prt
<213>bacillus subtilises (bacillus subtilis 168)
<400> 4
met ala asn val leu met ile gly phe pro gly glu gly his ile asn
1 5 10 15
pro ser ile gly val met lys glu leu lys ser arg gly glu asn ile
20 25 30
thr tyr tyr ala val lys glu tyr lys glu lys ile thr ala leu asp
35 40 45
ile glu phe arg glu tyr his asp phe arg gly asp tyr phe gly lys
50 55 60
asn ala thr gly asp glu glu arg asp phe thr glu met leu cys ala
65 70 75 80
phe leu lys ala cys lys asp ile ala thr his ile tyr glu glu val
85 90 95
lys his glu ser tyr asp tyr val ile tyr asp his his leu leu ala
100 105 110
gly lys val ile ala asn met leu lys leu pro arg phe ser leu cys
115 120 125
thr thr phe ala met asn glu glu phe ala lys glu met met gly ala
130 135 140
tyr met lys gly ser leu glu asp ser pro his tyr glu ser tyr gln
145 150 155 160
gln leu ala glu thr leu asn ala asp phe gln ala glu ile lys lys
165 170 175
pro phe asp val phe leu ala asp gly asp leu thr ile val phe thr
180 185 190
ser arg gly phe gln pro leu ala glu gln phe gly glu arg tyr val
195 200 205
phe val gly pro ser ile thr glu arg ala gly asn asn asp phe pro
210 215 220
phe asp gln ile asp asn glu asn val leu phe ile ser met gly thr
225 230 235 240
ile phe asn asn gln lys gln phe phe asn gln cys leu glu val cys
245 250 255
lys asp phe asp gly lys val val leu ser ile gly lys his ile lys
260 265 270
thr ser glu leu asn asp ile pro glu asn phe ile val arg pro tyr
275 280 285
val pro gln leu glu ile leu lys arg ala ser leu phe val thr his
290 295 300
gly gly met asn ser thr ser glu gly leu tyr phe glu thr pro leu
305 310 315 320
val val ile pro met gly gly asp gln phe val val ala asp gln val
325 330 335
glu lys val gly ala gly lys val ile lys lys glu glu leu ser glu
340 345 350
ser leu leu lys glu thr ile gln glu val met asn asn arg ser tyr
355 360 365
ala glu lys ala lys glu ile gly gln ser leu lys ala ala gly gly
370 375 380
ser lys lys ala ala asp ser ile leu glu ala val lys gln lys thr
385 390 395 400
gln ser ala asn ala
405
Claims (9)
1. a kind of production method of ginsenoside rh2 is it is characterised in that comprise the following steps:
(a) with protopanoxadiol as substrate, udp- glucose be glycosyl donor, using screening obtain glycosyl transferase yjic or
Yojk is to protopanoxadiol glycosylation to obtain ginsenoside rh2；
B () purification is to obtain described glycation product；
C () crystallizes to the glycation product of purification.
2. the production method of ginsenoside rh2 according to claim 1, the volume of wherein said glycosyl transferase yjic and yojk
Code gene source in bacillus subtilises (bacillus subtilis168) or its mutant, described glycosyl transferase yjic
Be respectively seq id no 1 and 2 with the nucleotide coding sequence of yojk, or with nucleotide sequence shown in seq id no 1 or 2
Homology 85%(preferably 95%) and the polypeptide product that the encodes nucleotide sequence active to protopanoxadiol；Described sugar
The peptide sequence of based transferase yjic and yojk is respectively seq id no 3 and 4, or the homology with seq id no 3 or 4
85%(preferably 95%) and the peptide sequence active to protopanoxadiol.
3. the production method of ginsenoside rh2 according to claim 1, wherein said glycosyl transferase be by conversion or
Incorporate glycosyltransferase gene yjic or yojk escherichia coli (escherichia coli), saccharomyces cerevisiae
(saccharomyces cerevisiae), Pichia sp. (pichia pastoris), corynebacterium glutamicum
(corynebacterium glutamicum), bacillus subtilises (bacillus subtilis) or aspergillus niger
(aspergillus nigar) produce acquisition.
4. the production method of ginsenoside rh2 according to claim 3, wherein said glycosyl transferase yjic's and yojk
Produce bacterial strain and be respectively escherichia coli bl21 (de3)/pet28-yjic and escherichia coli bl21 (de3)/pet28-yojk.
5. the production method of ginsenoside rh2 according to claim 1, wherein said glycosylation step includes making described former
Panoxadiol and described glycosyl transferase carry out enzyme reaction, described glycosyl under the scope of 15 DEG C of -50 DEG C of temperature and ph4-ph11
Transferring enzyme is to carry out catalytic reaction in the presence of ionizable metal salt, and wherein, described metal ion includes mncl2、cacl2And
mgcl2In at least one.
6. the production method of ginsenoside rh2 according to claim 1, wherein said protopanoxadiol is to carry from Radix Ginseng
Take, ginsenoside hydrolyzes, Microbial cell factories biosynthesiss obtain or these modes combine acquisition；Described glycosyl donor
For at least one in udp- glucose, adp- glucose, tdp- glucose, cdp- glucose or gdp- glucose or a combination thereof
Thing.
7. the production method of ginsenoside rh2 according to claim 1, wherein removing protein, decolouring, de-salting operation are pure
Carry out before changing step, the glycation product that enzyme reaction obtains is to separate by process-scale chromatography to obtain, and separates the glycosylation obtaining
Product, again through concentrating, crystallizing, be dried, finally obtains ginsenoside's rh2 product.
8. the production method of the ginsenoside rh2 according to any one claim in claim 1 to claim 7,
Wherein, in claim 1, after the step (b), by isolating and purifying the protopanoxadiol that can obtain not reaction completely and inciting somebody to action
It is recycled into step (a) again acting as the raw material of glycosylation, or after step (c), makes to separate in crystal
The protopanoxadiol obtaining is recycled to the separation that step (a) is used for protopanoxadiol for enzyme reaction or step (b), or two
Step is all carried out.
9. present invention also offers a kind of synthetic method of Novel ginsenoside f12 is it is characterised in that with protopanoxadiol or people
Ginseng saponin rh2 is substrate, with udp- glucose as glycosyl donor, joins two using the glycosyl transferase yjic that screening obtains to protoplast
Alcohol or ginsenoside rh2 carry out glycosylation to obtain ginsenoside f12.
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| CN108728423A (en) * | 2017-04-13 | 2018-11-02 | 中国医学科学院药物研究所 | Bacillus subtilis glycosyl transferase and its application |
| CN109796516A (en) * | 2017-11-17 | 2019-05-24 | 中国科学院天津工业生物技术研究所 | One group of natural synthetic method with non-natural protopanaxatriol type ginsenoside |
| CN111763705A (en) * | 2019-04-01 | 2020-10-13 | 伽蓝（集团）股份有限公司 | Preparation method and application of ginsenoside composition |
| CN112457359A (en) * | 2020-04-20 | 2021-03-09 | 吉林省华惠生物科技有限公司 | Method for preparing converted ginsenoside from ginseng fruit and preparation thereof |
| CN113493795A (en) * | 2021-08-03 | 2021-10-12 | 昆明理工大学 | Preparation method of ginsenoside Rh2 |
| CN114164191A (en) * | 2021-11-17 | 2022-03-11 | 江南大学 | Method for efficiently biosynthesizing rebaudioside D by utilizing glycosyltransferase |
| CN115992108A (en) * | 2022-08-15 | 2023-04-21 | 烟台大学 | Recombinant glycosyltransferase of Salmonella tropicalis CNB-440 and application thereof in novel ginsenoside conversion |
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| CN108728423B (en) * | 2017-04-13 | 2021-12-07 | 中国医学科学院药物研究所 | Bacillus subtilis glycosyltransferase and application thereof |
| CN108728423A (en) * | 2017-04-13 | 2018-11-02 | 中国医学科学院药物研究所 | Bacillus subtilis glycosyl transferase and its application |
| CN107929296A (en) * | 2017-11-17 | 2018-04-20 | 中国科学院天津工业生物技术研究所 | A kind of preparation method and application of non-natural ginsenoside |
| CN109796516A (en) * | 2017-11-17 | 2019-05-24 | 中国科学院天津工业生物技术研究所 | One group of natural synthetic method with non-natural protopanaxatriol type ginsenoside |
| CN107868115A (en) * | 2017-11-17 | 2018-04-03 | 中国科学院天津工业生物技术研究所 | A kind of non-natural ginsenoside Rd 12 and its preparation method and application |
| CN107868115B (en) * | 2017-11-17 | 2021-02-26 | 中国科学院天津工业生物技术研究所 | Non-natural ginsenoside Rd12, and preparation method and application thereof |
| CN109796516B (en) * | 2017-11-17 | 2022-07-22 | 中国科学院天津工业生物技术研究所 | A method for synthesizing natural and unnatural protopanaxatriol type ginsenoside |
| CN111763705A (en) * | 2019-04-01 | 2020-10-13 | 伽蓝（集团）股份有限公司 | Preparation method and application of ginsenoside composition |
| CN111763705B (en) * | 2019-04-01 | 2024-04-19 | 上海自然堂集团有限公司 | Preparation method and application of ginsenoside composition |
| CN112457359A (en) * | 2020-04-20 | 2021-03-09 | 吉林省华惠生物科技有限公司 | Method for preparing converted ginsenoside from ginseng fruit and preparation thereof |
| CN113493795A (en) * | 2021-08-03 | 2021-10-12 | 昆明理工大学 | Preparation method of ginsenoside Rh2 |
| CN113493795B (en) * | 2021-08-03 | 2022-10-28 | 昆明理工大学 | Preparation method of ginsenoside Rh2 |
| CN114164191A (en) * | 2021-11-17 | 2022-03-11 | 江南大学 | Method for efficiently biosynthesizing rebaudioside D by utilizing glycosyltransferase |
| WO2023087518A1 (en) * | 2021-11-17 | 2023-05-25 | 江南大学 | Method for efficient biosynthesis of rebaudioside d using glycosyltransferase |
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