CN105695552A - Method using enzymatic method to prepare rare ginsenoside Rh1 - Google Patents

Abstract

The invention relates to a method using an enzymatic method to prepare rare ginsenoside Rh1. The method is characterized in that beta-glucosidase from Thermotoga petrophila and alpha-rhamnosidase from Aspergillus niger are utilized to degrade ginsenoside Re so as to produce the Rh1. The method has the advantages that the enzymatic method is used to catalyze the ginsenoside Re to produce the rare ginsenoside Rh1, the produced rare ginsenoside Rh1 is high in purity, no byproducts are produced, the enzymatic method is high in conversion efficiency, and mole yield reaches 95%.

Description

A kind of enzyme process prepares the method for rare ginsenoside Rh1

Technical field

The invention belongs to biomedicine field, the preparation method relating to rare ginsenoside Rh1, particularly relate to utilize and recombinant expressed derive from α-rhamnosidase and beta-glucosidase converts ginsenoside Re and generates Rh1。

Background technology

Radix Ginseng (Panaxginseng) is the rare Chinese medicine that Asia is traditional, and ginsenoside is the bioactive substance that it is main, has the features such as biological activity is notable, pharmacological action is unique。Raising along with analytical technology level, the chemical composition of ginsenoside obtains further clearly, now isolation identification goes out more than 180 kind ginsenoside monomers, wherein 5 kinds of main saponin (ginsenoside Rb1, Rb2, Rc, Re and Rg1) account for more than the 80% of total saponins, and ginsenoside (Rg3, Rh2, CompoundK, Rg2, F1 and Rh1) content is very low, it is called rare saponin (AppliedMicrobiologyandBiotechnology, 2012,94:673-682), but its pharmacologically active is better than the ginsenoside of above-mentioned high-load。

Rare saponin Rh1 has startup and strengthens immunne response, promotes the physiological actions such as hepatocyte growth, killing tumor cell, and aging resistance, can be used for regulating body's immunity, treats and prevents hepatitis etc.。But rare saponin Rh1 content in plant is very low, extracting directly complex process, and cost is too high。And ginsenoside Re's content in Radix Ginseng total saponins is higher, to rare ginsenoside Rh1, there is similar chemical constitution, therefore, it can obtain Rh1 (Fig. 1) by ginsenoside Re carries out conversion, but need the rhamanopyranosyl on specific degraded C20 position and the glucosyl group on C6 position。

Utilize chemical method and biotransformation method ginsenoside Re can be converted into Rh1。But chemical transformation is not used because of shortcomings such as its reaction are violent, poor selectivity, by-product are many。The features such as bioconversion has reaction condition gentleness, and specificity is good and favored。The biotransformation method of current ginsenoside mainly has microbe transformation method and enzyme transforming process。Microbe transformation method is complicated because of its transformation system, it is easy to produce by-product, the purity of product is had certain impact。As, Dong Aling etc. finds that aspergillus niger and Absidia coerulea can convert ginsenoside Rg1 and produce ginsenoside Rh1, but it is longer to there is the transformation period, and has the shortcomings such as other saponin by-product (China's medicine, 2001,10 (3): 115-118)。Enzymatic conversion method is avoided that the defect of microbe transformation method, but various enzymes are different to the specificity of substrate, the glycosidic bond type of degraded is also different, therefore, finds and can become research key by glycoside hydrolase efficient, that selective degradation ginsenoside Re produces ginsenoside Rh1。At present, but without the report utilizing enzymatic conversion ginsenoside Re to produce ginsenoside Rh1。

Summary of the invention

Solving the technical problem that: the invention provides a kind of method that enzyme process prepares rare ginsenoside Rh1, the recombinase that the method uses is respectively derived from the beta-glucosidase of Thermotogapetrophila and the α-rhamnosidase of aspergillus niger (Aspergillusniger)。

Technical scheme: a kind of enzyme process prepares the method for rare ginsenoside Rh1, utilizing and derive from the beta-glucosidase of Thermotogapetrophila and the α-rhamnosidase of aspergillus niger (Aspergillusniger), degraded ginsenoside Re produces Rh1。

The nucleotide sequence of described beta-glucosidase is such as shown in SEQIDNO:1, and aminoacid sequence is such as shown in SEQIDNO:2。

The nucleotide sequence of described α-rhamnosidase is such as shown in SEQIDNO:3, and aminoacid sequence is such as shown in SEQIDNO:4。

Described beta-glucosidase is by recombinant expressed preparation, and step is:

(1) with the Thermotogapetrophila genomic DNA of extraction for template, being classified as forward primer with having the nucleotides sequence shown in SEQIDNO:5 and have the nucleotides sequence shown in SEQIDNO:6 and be classified as downstream primer, pcr amplification obtains the DNA molecular of beta-glucosidase；

(2) DNA molecular of above-mentioned beta-glucosidase and pET-20b are carried out double digestion with NdeI and XhoI respectively, connect and convert the recombiant plasmid obtaining the DNA molecular containing beta-glucosidase；

(3) by recombinant plasmid transformed expressive host E.coliBL21 (DE3) of above-mentioned acquisition, IPTG induces beta-glucosidase expression of enzymes, collects thalline, smudge cells, 80 DEG C of heat treatments 40 minutes, it is thus achieved that the beta-glucosidase of purification。

Described α-rhamnosidase is by recombinant expressed preparation, and step is:

(1) with aspergillus niger (Aspergillusniger) NL-1cDNA of extraction for template, being classified as forward primer with having the nucleotides sequence shown in SEQIDNO:7 and have the nucleotides sequence shown in SEQIDNO:8 and be classified as downstream primer, pcr amplification obtains the DNA molecular of α-rhamnosidase；

(2) DNA molecular of above-mentioned beta-glucosidase and pET-20b are carried out double digestion with NdeI and XhoI respectively, connect and convert the recombiant plasmid obtaining the DNA molecular containing α-rhamnosidase；

(3) by recombinant plasmid transformed expressive host E.coliBL21 (DE3) of above-mentioned acquisition, induce α-rhamnoside expression of enzymes, collect thalline, smudge cells, be centrifuged and obtain α-rhamnosidase。

Described enzyme process prepares the degradation condition in the method for rare ginsenoside Rh1:

(1) 1g/L ginsenoside Re, under beta-glucosidase 0.2U, pH5.0,85 DEG C condition, degrade 1h；

(2) above-mentioned reactant liquor being added α-rhamnosidase 6.6U, adjusting pH is under 6.5,35 DEG C of conditions, and degrade 1h, and ginsenoside Re may finally be converted into ginsenoside Rh1, and molar yield is 95%。

Beneficial effect: 1. produce rare ginsenoside Rh1 by enzyme law catalysis ginsenoside Re, the rare ginsenoside Rh1 purity of generation is high, it does not have by-product。2. the efficiency of enzymatic conversion method is high, and molar yield reaches 95%。

Accompanying drawing explanation

Fig. 1 is the structural representation of ginsenoside Re and Rh1；

Fig. 2 is that activity of beta-glucosidase is affected figure by the temperature that the embodiment of the present invention provides；

Fig. 3 is that activity of beta-glucosidase is affected figure by the pH that the embodiment of the present invention provides；

Fig. 4 is the Re that the embodiment of the present invention the provides time plot being converted into Rg2；

Fig. 5 is that α-rhamnosidase activity is affected figure by the temperature that the embodiment of the present invention provides；

Fig. 6 is that α-rhamnosidase activity is affected figure by the pH that the embodiment of the present invention provides；

Fig. 7 is the Rg2 that the embodiment of the present invention the provides time plot being converted into Rh1。

Detailed description of the invention

Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments。Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention。

In order to be further appreciated by the present invention, below in conjunction with embodiment, the present invention will be described in detail, and wherein, if no special instructions, the various reaction reagents related in embodiment all can be commercially available by commercial channel；If no special instructions, the concrete operations related in embodiment are referring to " the Molecular Cloning: A Laboratory guide third edition "。

Embodiment 1: the structure of recombiant plasmid pET-20b-bgl

The cultivation of 1.1Thermotogapetrophila

Thermotogapetrophila is purchased from DSMZ DSMZ (www.dsmz.de) and is numbered DSM13995。Its culture medium prescription is: 10g/L soluble starch, 3g/L yeast powder, 5g/L tryptone, 5g/L meat extract, 10g/L2-morpholino b acid, 10mg/L seven ferric sulfate hydrate, 1mg/L "diazoresorcinol", adjusting pH is 7.2, boils inflated with nitrogen, after removing oxygen, culture medium loads anaerobism bottle sterilizing under anaerobic。Inoculate according to 0.5wt.% inoculum concentration with syringe, 85 DEG C of static gas wave refrigerator 24h, collect cell。

The extraction of 1.2 genomic DNAs

(1) quiescent culture Thermotogapetrophila24h, takes the centrifugal 10min of 30mL bacterium solution 4,000g and collects cell。

(2) with the resuspended thalline of 9.5mLTE buffer, 0.5mL10wt.% sodium lauryl sulphate (SDS) and 50 μ L E.C. 3.4.21.64 (20mg/mL), mix homogeneously, 37 DEG C of insulation 1h are added。

(3) 1.8mL5mol/LNaCl, 1.5mL cetyltriethylammonium bromide (CTAB)/NaCl is added, mixing, 65 DEG C of incubation 20min。

(4) equal-volume chloroform/isoamyl alcohol (volume ratio 24:1) is added, mixing, 6,000g centrifugal 10min。

(5) for preventing shearing force from causing genomic DNA to rupture, with thick mouth suction pipe, supernatant is proceeded in another centrifuge tube, add equal-volume phenol/chloroform/isoamyl alcohol mixing (volume ratio 25:24:1), 6,000g centrifugal 10min。

(6) in another centrifuge tube, add 0.6 times of volume isopropanol, rock to white thread DNA precipitation high-visible gently。

(7) with suction pipe, DNA is wound on it, clean in 70vt.% ethanol。

(8) with sterile toothpick, DNA is scraped from suction pipe, proceed in 1.5mL centrifuge tube。

(9) air-dry under room temperature, add 500 μ LTE buffer solution。

(10) 50 μ L nucleic acid-protein detector detection DNA concentration are taken。

The structure of 1.3 recombiant plasmid pET-20b-bgl

Primer is designed: (SEQIDNO:5) underscore represents NdeI restriction enzyme site according to the extremely heat-resisting beta-glucosidase gene (YP_001244492.1) of known Thermotogapetrophila；(SEQIDNO:6) underscore represents XhoI restriction enzyme site, and removes termination codon；With the genomic DNA of the Thermotogapetrophila of extraction for template, carrying out pcr amplification with the primer of synthesis, the condition of amplification is 94 DEG C, 3min；30 circulations (94 DEG C, 30s；58 DEG C, 30s；72 DEG C, 2min10s)；72 DEG C, 10min；Reaction stops, 4 DEG C of insulations。Reclaim test kit by gel pcr amplification product is purified。Obtain the extremely heat-resisting beta-glucosidase gene of T.petrophila。

Obtain the extremely heat-resisting beta-glucosidase gene of Thermotogapetrophila and pET-20b carries out double digestion with NdeI and XhoI respectively, and recovery of tapping rubber respectively, concentrate latter 16 DEG C to connect overnight, product will be connected and convert escherichia coli Top10F ' competent cell, converted product is applied to 37 DEG C of incubated overnight on LB (add ampicillin to final concentration 100mg/L) solid medium, inoculate after several single bacterium colony cultivates 8-10 hour in LB (add ampicillin to final concentration 100mg/L) fluid medium, collect thalline and extract plasmid, digestion verification removes empty plasmid, recombiant plasmid is carried out determining nucleic acid sequence, obtain correct recombinant expression carrier pET20b-bgl。

2. recombinate the expression of extremely heat-resisting beta-glucosidase and purification

Recombiant plasmid pET-20b-bgl is converted e. coli bl21 (DE3) Host Strains (Novagen), in LB flat board (the LB culture medium: tryptone 10g/L containing Amp (100 μ g/mL), yeast extract 5g/L, NaCl5g/L, agar 15g/L) on through 37 DEG C of overnight incubation, choosing transformant (100 μ g/mLAmp) 37 DEG C in the LB culture medium of 200mL, 200rpm shaken cultivation is to OD₆₀₀When being 0.6, add final concentration of 0.5mM isopropyl ss-D-Thiogalactopyranoside (IPTG) derivant, 30 DEG C of inducing culture 8h, with High speed refrigerated centrifuge by culture fluid at 4 DEG C, with 13,000rpm centrifugal 15min, collect thalline, supernatant is gone to add sterilized water, ultrasonic disruption cell, subsequently 70 DEG C of heat treatment 30min, with High speed refrigerated centrifuge by culture fluid at 4 DEG C, with 13,000rpm centrifugal 15min, supernatant is the pure enzyme of extremely heat-resisting beta-glucosidase of recombinating。

3. the extremely heat-resisting beta-glucosidase enzymatic conversion Re that recombinates is the condition of Rg2

3.1 enzyme activity determinations

Reaction system 200 μ L, 10 μ L20mmol/LPNPG add 100 μ L100mmol/L citrate-phosphate disodium hydrogen buffer (pH5.0) and appropriate water, first hatch 2min at 90 DEG C, add 5 μ L enzyme liquid (being diluted to suitable multiple) and react 10min。Reaction is added immediately the Na of 600 μ L1M after terminating₂CO₃, under 405nm, measure its light absorption value with spectrophotometer。Enzyme activity unit (U) is defined as: under condition determination, and the enzyme amount used by 1 μm of ol ginsenoside Rg2 of generation per minute is 1 enzyme activity unit。

The mensuration of 3.2 optimal reactive temperatures

Within the scope of 70-90 DEG C, every 5 DEG C, measure enzyme respectively and live。Buffering is 100mmol/L citrate-phosphate disodium hydrogen buffer, and pH5.0, it has been found that the optimal reactive temperature of extremely heat-resisting glycosidase is 85 DEG C (Fig. 2), higher reaction temperature can make substrate saponin Re dissolubility in water increase。

The mensuration of 3.3 optimal reaction pH

Under different pH (4.0-8.0,100mmol/L citrate-phosphate disodium hydrogen buffer) conditions, 85 DEG C measure enzyme respectively and live, it has been found that the optimal reaction pH of extremely heat-resisting glycosidase is 5.0 (Fig. 3)。

4. ginsenoside Re converts production ginsenoside Rg2

The concentration of ginsenoside Re is 1g/L, and conversion condition is 85 DEG C, pH5.0100mmol/L citrate-phosphate disodium hydrogen buffer, and different time points (0,5,10,20,30,40,50,60min) is separately sampled, is detected by HPLC。Found that ginsenoside Re can be converted into ginsenoside Rg2 by extremely heat-resisting glycosidase (0.2U) in 60min, molar yield is 100% (Fig. 4)。

5. the structure of recombiant plasmid pET-20b-Rha

The cultivation of 5.1 aspergillus nigers (Aspergillusniger) NL-1

AspergillusnigerNL-1 (" aspergillus niger is secreted the impact of beta-glucosidase by carbon source and nitrogenous source ", " Nanjing Forestry University's journal: natural science edition ", 2008,32 (6): 1-4), its culture medium prescription is: rhamnose 10g/L, MgSO₄·7H₂O0.5g/L, KH₂PO₄1.5g/L, (NH₄)₂SO₄4g/L, ZnSO₄·7H₂O0.09g/L, CaCl₂0.1g/L, yeast extract 1g/L, bean cake 2.0g/L, peptone 2.0g/L。At 30 DEG C, 180rpm cultivates 3-4 days。

The synthesis of 5.2cDNA

The mycelium phosphate buffer solution getting the aspergillus niger of collection cleans after once, it is used in pulverized under liquid nitrogen mycelium to Powdered, collect in 2mL centrifuge tube, acutely shake after adding 1mLTrizol, at 4 DEG C, after the centrifugal 10min of 12000g, transfer supernatant to 2mL centrifuge tube adds at 200 μ L chloroforms concussion 15s mix latter 30 DEG C and hatches 2-3min, at 4 DEG C, the centrifugal 10min of 12000g takes supernatant, add the centrifugal 10min of 12000g at 0.8 times of volume isopropanol mixes latter 4 DEG C, remove after supernatant that (DEPC processed with 75wt.% ethanol water, eliminate mRNA enzyme) clean 2 times after at 4 DEG C the centrifugal 5min of 7500g be precipitated, after making its natural drying, add appropriate DEPC water dissolution RNA precipitate, as template ribonucleic acid, preserve at-20 DEG C。

With aspergillus niger AspergillusnigerNL-1 bacterial strain RNA for template, utilize " PrimeScriptTM1stStrandcDNASynthesisKit " test kit (being purchased from Takara company) reverse transcription synthesis cDNA Article 1 chain (concrete reactant liquor proportioning reference reagent box)。

The structure of 5.3 recombiant plasmid pET-20b-Rha

Primer is designed: (SEQIDNO:7) underscore represents NdeI restriction enzyme site according to known AspergillusnigerNL-1 α-rhamnose glycosidase genes (accession number: XM_001402229.1)；(SEQIDNO:8) underscore represents Hind III site, and removes termination codon；With the genome cDNA of the AspergillusnigerNL-1 of extraction for template, carrying out pcr amplification with the primer of synthesis, the condition of amplification is 94 DEG C, 3min；30 circulations (94 DEG C, 30s；52 DEG C, 30s；72 DEG C, 2min50 ')；72 DEG C, 10min；Reaction stops, 4 DEG C of insulations。Reclaim test kit by gel pcr amplification product is purified。Obtain A.nigerNL-1 α-rhamnose glycosidase genes。

AspergillusnigerNL-1 α-rhamnose the glycosidase genes obtained and pET-20b carry out double digestion with NdeI and Hind III respectively, and recovery of tapping rubber respectively, concentrate latter 16 DEG C to connect overnight, product will be connected and convert escherichia coli Top10F ' competent cell, converted product is applied to 37 DEG C of incubated overnight on LB (add ampicillin to final concentration 100mg/L) solid medium, inoculate after several single bacterium colony cultivates 8-10 hour in LB (add ampicillin to final concentration 100mg/L) fluid medium, collect thalline and extract plasmid, digestion verification removes empty plasmid, recombiant plasmid is carried out determining nucleic acid sequence, obtain correct recombinant expression carrier pET20b-Rha。

6. the expression of recombinant alpha-rhamnosidase and purification

Recombiant plasmid pET-20b-Rha is converted e. coli bl21 (DE3) Host Strains (Novagen), in LB flat board (the LB culture medium: tryptone 10g/L containing Amp (100 μ g/mL), yeast extract 5g/L, NaCl5g/L, agar 15g/L) on through 37 DEG C of overnight incubation, choose transformant (100 μ g/mLAmp) 37 DEG C of inducing culture 12h in the LB culture medium of 200mL, with High speed refrigerated centrifuge by culture fluid at 4 DEG C, with 13, 000rpm is centrifuged 15min, collect thalline, supernatant is gone to add sterilized water, ultrasonic disruption cell, with High speed refrigerated centrifuge by culture fluid at 4 DEG C, with 13, 000rpm is centrifuged 15min, take supernatant, it is purified with Ni-NTA affinity column。The recombinase obtained is the pure enzyme of recombinant alpha-rhamnosidase。

The specific operation process of purification:

A. the process of sample

(1) by washed thalline, resuspended with 1 × BindingBuffer8mL, supersonic wave wall breaking。

(2) after breaking cellular wall, 13,000g centrifugal 30min, take supernatant and be sample。

B. pillar is processed

(1) 1mL filler dress post is taken。

(2) with the aseptic washing pillar of 3mL。

(3) pillar is washed with the 1 × ChargeBuffer of 5mL。

(4) pillar is washed with the 1 × BindingBuffer of 3mL。

C. loading

(1) sample is added pillar, 6 about per minute of coutroi velocity。

(2) wash pillar with 3mL1 × BindingBuffer, remove unconjugated protein。

(3) wash pillar with the 4mL eluent containing 20mM imidazoles, remove foreign protein。

(4) wash pillar with the eluent of 100mmol/L imidazoles, destination protein is eluted。

(5) pillar is washed with 4mL1 × StripBuffer。

Recombinant alpha-the rhamnosidase of purification is obtained by this process。

7. recombinant alpha-rhamnoside enzymatic conversion Rg2 is the condition of Rh1

7.1 enzyme activity determinations

Reaction system 200 μ L, adds 100 μ L100mmol/L citrate-phosphate disodium hydrogen buffer (pH6.5) and 85 μ L water in 40 μ L5mmol/LpNPR, first hatch 2min at 35 DEG C, add 10 μ L enzyme liquid and (make OD₄₀₅Value between 0.2～1.0) reaction 10min。Reaction is added immediately the Na of 600 μ L1M after terminating₂CO₃, under 405nm, measure its light absorption value with spectrophotometer。Enzyme activity unit (U) is defined as: under condition determination, and the enzyme amount used by 1 μm of olPNP of generation per minute is 1 enzyme activity unit。

The mensuration of 7.2 optimal reactive temperatures

Within the scope of 25-45 DEG C, every 5 DEG C, measure enzyme respectively and live。Buffering is 100mmol/L citrate-phosphate disodium hydrogen buffer, pH6.5, it has been found that the optimal reactive temperature of α-rhamnosidase is 35 DEG C (Fig. 5)。

The mensuration of 7.3 optimal reaction pH

Under different pH (5.0-7.5,100mmol/L citrate-phosphate disodium hydrogen buffer) conditions, 35 DEG C measure enzyme respectively and live, it has been found that the optimal reaction pH of α-rhamnosidase is 6.5 (Fig. 6)。

8. ginsenoside Rg2 converts and produces ginsenoside Rh1

Ginsenoside Rg2 is the reactant liquor in above-mentioned 4.th part, and conversion condition is 35 DEG C, pH6.550mmol/L citrate-phosphate disodium hydrogen buffer, different time points (0,5,10,20,30,40,50,60min) separately sampled, detected by HPLC。Found that ginsenoside Rg2 can be converted into ginsenoside Rh1 by α-rhamnosidase (6.6U) in 60min, molar yield is 95% (Fig. 7)。

SEQUENCELISTING

<110>Nanjing Forestry University

<120>a kind of method that enzyme process prepares rare ginsenoside Rh1

<130>

<160>8

<170>PatentInversion3.3

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ProHisSerArgValAlaGlyAlaAlaGlyGluThrHisProValPro

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ValValAspThrIleValSerGluArgAlaLeuArgGluIleTyrLeu

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GlyAsnAspMetIleMetProGlyLysAlaTyrGlnValAsnThrGlu

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ArgArgAspGluIleGluGluIleMetGluAlaLeuLysGluGlyArg

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LeuSerGluGluValLeuAsnGluCysValArgAsnIleLeuLysVal

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LeuValAsnAlaProSerPheLysGlyTyrArgTyrSerAsnLysPro

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AspLeuGluSerHisAlaLysValAlaTyrGluAlaGlyValGluGly

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ArgThrAspSerTrpGlyThrValIleLysProLysLeuProGluAsn

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PheLeuSerGluLysGluIleLysLysAlaAlaLysLysAsnAspAla

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ProValLysGlyAspPheTyrLeuSerAspAspGluLeuGluLeuIle

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LysThrValSerArgGluPheHisGluGlnGlyLysLysValValVal

485490495

LeuLeuAsnIleGlySerProIleGluValAlaSerTrpArgAspLeu

500505510

ValAspGlyIleLeuLeuValTrpGlnAlaGlyGlnGluMetGlyArg

515520525

IleValAlaAspValLeuValGlyArgValAsnProSerGlyLysLeu

530535540

ProThrThrPheProLysAspTyrSerAspValProSerTrpThrPhe

545550555560

ProGlyGluProLysAspAsnProGlnArgValValTyrGluGluAsp

565570575

IleTyrValGlyTyrArgTyrTyrAspThrPheGlyValGluProAla

580585590

TyrGluPheGlyTyrGlyLeuSerTyrThrLysPheGluTyrLysAsp

595600605

LeuLysIleAlaIleAspGlyAspIleLeuArgValSerTyrThrIle

610615620

ThrAsnThrGlyAspArgAlaGlyLysGluValSerGlnValTyrVal

625630635640

LysAlaProLysGlyLysIleAspLysProPheGlnGluLeuLysAla

645650655

PheHisLysThrLysLeuLeuAsnProGlyGluSerGluLysIlePhe

660665670

LeuGluIleProLeuArgAspLeuAlaSerPheAspGlyLysGluTrp

675680685

ValValGluSerGlyGluTyrGluValArgValGlyAlaSerSerArg

690695700

AspIleArgLeuArgAspIlePheLeuValGluGlyGluLysArgPhe

705710715720

LysPro

<210>3

<211>2766

<212>DNA

<213>Aspergillusniger

<400>3

atggccagccaaatcttcattgaaacccctacagttgagcaacactcaaccgggtttggc60

attggaactgcgactccgcgtctgtcatggcgattcctcaccaccgacagtagtcctcgc120

gactgggagcagacggcctacgaagtggaagttgttcgttccggatctcgagaggaaacc180

taccatgtgaacagcagtgcatcggtgctggtcccttggcccagtggcccactacaatcc240

cgggaaatcgcacaggtccgtgttcgagcatacgggtgcagcgctggcaaggagcagcag300

agtgactgtgccaccgcttggtctccctggcggactattgagtgtggactactcgaccgc360

gctgactgggtagcccgacctattgccagtcctgaggaaccacagccggatcatccatta420

cgaccggtgcgattccgtaaagagttccaactccctgcggcaggtaccatagaaaaagct480

cgcctttatatcacgagctttggagtataccgcgccttcatcaacgggcatcgagtaggg540

gatcagtgtctcgcgcccggatggaccagctaccgccatcgcttgaactatcaagtgttc600

gacatcgcgtccctcctgaatgctgaaggtcctaatgtccttgcggtagaggttgcagag660

ggctggtacgccactcgcctcggcttccttggcgggcgaagacagctgtatggggatcga720

ctggccgtgttggcccagctggagatccaacttggatctaatggagaccgattctatatg780

tctaccgatagcacctggacgtgtactcccagtgccatcatccggagtgagctctatgac840

ggcgaggtttacgatacccgtgaagaagattcgacctggaactgcttgcgtcttgaccaa900

acctcccgctgggtggccgttcaggaactcgagttcccaaccgcagccttggtggcaccg960

aacgctcccccagtccgtatcaccgaggagatatctccagtctccgtccagaagacccca1020

tccggtgccaccgtcatcgacttcggccagaacctggtcggtcgactttgcgtgcgctcg1080

ttgaacaagccctcgggctctcgcgtttccttcattcacgccgaagtgcttgagaatggc1140

gagctcggggttcgacccctgcgacacgccaagtgcaccgacgaagtcattctaagtgat1200

acagagctggtagattggtcgcctcagtatacattccacgggtttcggttcgtccaggtc1260

aatggttgggatgaagaaagcgatggatcactgctcctcaacatcaacgcgcttgtgatg1320

cacactgatatgactcgcagcggatggttttcctgctcgcaccctatggtcaatcaactc1380

cacaccaatgcctggtggagtatgcgtgggaatttcctttccatccctaccgattgcccc1440

cagagggatgaacgcctcggctggaccggggatattcagatattctgcccatcggccaac1500

ttcctctacaatactgctggcatgctgagtgactggttgcaagatgtcgcggccgagcag1560

ctgagggagaaagatggttgcgtgcctccatttacagtccccaatatcattagtgagacc1620

ctgtggccgcatacgccgcaggctgtatgggatgatgtggtgatcctaacgccctgggcc1680

ctctaccggtcgtatggggacagtgaaattctccgccgtcaatatgaaagcatgctcgcc1740

tggatcgatcgcggcatccgtcgcggctcagatggactctgggaccctgagctttggcaa1800

ctgggagactggttagacccaacagcgccgcccgaggaacctggggatgcacgcaccagc1860

ggaaccctcgttgctgatgcctatctcgtgcacatcacttcggtgatgtccgaaatcagc1920

caggtcctcggccagagccaggatgccgcacggtttaagacggactataatcgactcaag1980

gcgaggttccaggccaagtatataacggcgactgggttactagtcggcgatacccaaacg2040

gcgcttagccttgccatcgtgtacgaccttcactccacccctgaagcagcgcaagcagca2100

gcatcacgtctcgtccacctcgtccggctcgccaagtttcgcgtcgccaccgggttcgcg2160

ggcactccaatcatcacgcatgctctcaccaagagcggaaatccccagatcgcctaccgg2220

atgttgctagagaaatctcgtccgtcgtggatgtatcccatcaccatgggggcgacgacc2280

atgtgggagaggtgggacagcatgctccccgacgggtccatcaaccctggcgaaatgacc2340

agcttcaaccactacgccctgggttcgatcatcaactggctgcactccgttgtagccggc2400

gtcagccctctggccccaggctggaaacacatacaggtcgccccgacacctgggcctaca2460

attcactctgctgaagccatgtatgacactccttatgggcgactggaatgccggtggtcg2520

atcgagactgatgcagaccgcttccacatggacctgttgataccccctaactctcgcgcg2580

cgtgtaatcttgccaactcgagagaagctatcacagcccgttggttctcgtgaagacgga2640

ggcttttgggtgggttcggggcgtcacaagttctcagcgaccttcgagtggaaggactat2700

tctagggactggccaccaaagcctctgaatccgattatgcgtgaacctgaaccggaggat2760

attgcc2766

<210>4

<211>922

<212>PRT

<213>Aspergillusniger

<400>4

MetAlaSerGlnIlePheIleGluThrProThrValGluGlnHisSer

151015

ThrGlyPheGlyIleGlyThrAlaThrProArgLeuSerTrpArgPhe

202530

LeuThrThrAspSerSerProArgAspTrpGluGlnThrAlaTyrGlu

354045

ValGluValValArgSerGlySerArgGluGluThrTyrHisValAsn

505560

SerSerAlaSerValLeuValProTrpProSerGlyProLeuGlnSer

65707580

ArgGluIleAlaGlnValArgValArgAlaTyrGlyCysSerAlaGly

859095

LysGluGlnGlnSerAspCysAlaThrAlaTrpSerProTrpArgThr

100105110

IleGluCysGlyLeuLeuAspArgAlaAspTrpValAlaArgProIle

115120125

AlaSerProGluGluProGlnProAspHisProLeuArgProValArg

130135140

PheArgLysGluPheGlnLeuProAlaAlaGlyThrIleGluLysAla

145150155160

ArgLeuTyrIleThrSerPheGlyValTyrArgAlaPheIleAsnGly

165170175

HisArgValGlyAspGlnCysLeuAlaProGlyTrpThrSerTyrArg

180185190

HisArgLeuAsnTyrGlnValPheAspIleAlaSerLeuLeuAsnAla

195200205

GluGlyProAsnValLeuAlaValGluValAlaGluGlyTrpTyrAla

210215220

ThrArgLeuGlyPheLeuGlyGlyArgArgGlnLeuTyrGlyAspArg

225230235240

LeuAlaValLeuAlaGlnLeuGluIleGlnLeuGlySerAsnGlyAsp

245250255

ArgPheTyrMetSerThrAspSerThrTrpThrCysThrProSerAla

260265270

IleIleArgSerGluLeuTyrAspGlyGluValTyrAspThrArgGlu

275280285

GluAspSerThrTrpAsnCysLeuArgLeuAspGlnThrSerArgTrp

290295300

ValAlaValGlnGluLeuGluPheProThrAlaAlaLeuValAlaPro

305310315320

AsnAlaProProValArgIleThrGluGluIleSerProValSerVal

325330335

GlnLysThrProSerGlyAlaThrValIleAspPheGlyGlnAsnLeu

340345350

ValGlyArgLeuCysValArgSerLeuAsnLysProSerGlySerArg

355360365

ValSerPheIleHisAlaGluValLeuGluAsnGlyGluLeuGlyVal

370375380

ArgProLeuArgHisAlaLysCysThrAspGluValIleLeuSerAsp

385390395400

ThrGluLeuValAspTrpSerProGlnTyrThrPheHisGlyPheArg

405410415

PheValGlnValAsnGlyTrpAspGluGluSerAspGlySerLeuLeu

420425430

LeuAsnIleAsnAlaLeuValMetHisThrAspMetThrArgSerGly

435440445

TrpPheSerCysSerHisProMetValAsnGlnLeuHisThrAsnAla

450455460

TrpTrpSerMetArgGlyAsnPheLeuSerIleProThrAspCysPro

465470475480

GlnArgAspGluArgLeuGlyTrpThrGlyAspIleGlnIlePheCys

485490495

ProSerAlaAsnPheLeuTyrAsnThrAlaGlyMetLeuSerAspTrp

500505510

LeuGlnAspValAlaAlaGluGlnLeuArgGluLysAspGlyCysVal

515520525

ProProPheThrValProAsnIleIleSerGluThrLeuTrpProHis

530535540

ThrProGlnAlaValTrpAspAspValValIleLeuThrProTrpAla

545550555560

LeuTyrArgSerTyrGlyAspSerGluIleLeuArgArgGlnTyrGlu

565570575

SerMetLeuAlaTrpIleAspArgGlyIleArgArgGlySerAspGly

580585590

LeuTrpAspProGluLeuTrpGlnLeuGlyAspTrpLeuAspProThr

595600605

AlaProProGluGluProGlyAspAlaArgThrSerGlyThrLeuVal

610615620

AlaAspAlaTyrLeuValHisIleThrSerValMetSerGluIleSer

625630635640

GlnValLeuGlyGlnSerGlnAspAlaAlaArgPheLysThrAspTyr

645650655

AsnArgLeuLysAlaArgPheGlnAlaLysTyrIleThrAlaThrGly

660665670

LeuLeuValGlyAspThrGlnThrAlaLeuSerLeuAlaIleValTyr

675680685

AspLeuHisSerThrProGluAlaAlaGlnAlaAlaAlaSerArgLeu

690695700

ValHisLeuValArgLeuAlaLysPheArgValAlaThrGlyPheAla

705710715720

GlyThrProIleIleThrHisAlaLeuThrLysSerGlyAsnProGln

725730735

IleAlaTyrArgMetLeuLeuGluLysSerArgProSerTrpMetTyr

740745750

ProIleThrMetGlyAlaThrThrMetTrpGluArgTrpAspSerMet

755760765

LeuProAspGlySerIleAsnProGlyGluMetThrSerPheAsnHis

770775780

TyrAlaLeuGlySerIleIleAsnTrpLeuHisSerValValAlaGly

785790795800

ValSerProLeuAlaProGlyTrpLysHisIleGlnValAlaProThr

805810815

ProGlyProThrIleHisSerAlaGluAlaMetTyrAspThrProTyr

820825830

GlyArgLeuGluCysArgTrpSerIleGluThrAspAlaAspArgPhe

835840845

HisMetAspLeuLeuIleProProAsnSerArgAlaArgValIleLeu

850855860

ProThrArgGluLysLeuSerGlnProValGlySerArgGluAspGly

865870875880

GlyPheTrpValGlySerGlyArgHisLysPheSerAlaThrPheGlu

885890895

TrpLysAspTyrSerArgAspTrpProProLysProLeuAsnProIle

900905910

MetArgGluProGluProGluAspIleAla

915920

<210>5

<211>35

<212>DNA

<213>artificial sequence

<400>5

ggaattccatatggccagccaaatcttcattgaaa35

<210>6

<211>34

<212>DNA

<213>artificial sequence

<400>6

cccaagcttggcaatatcctccggttcaggttca34

<210>7

<211>35

<212>DNA

<213>artificial sequence

<400>7

ggaattccatatggccagccaaatcttcattgaaa35

<210>8

<211>34

<212>DNA

<213>artificial sequence

<400>8

cccaagcttggcaatatcctccggttcaggttca34

Claims (6)

1. the method that an enzyme process prepares rare ginsenoside Rh1, it is characterized in that the α-rhamnosidase utilizing the beta-glucosidase deriving from Thermotogapetrophila and aspergillus niger (Aspergillusniger), degraded ginsenoside Re produces Rh1。

2. the method that enzyme process described in claim 1 prepares rare ginsenoside Rh1, it is characterised in that the nucleotide sequence of beta-glucosidase is such as shown in SEQIDNO:1, and aminoacid sequence is such as shown in SEQIDNO:2。

3. the method that enzyme process described in claim 1 prepares rare ginsenoside Rh1, it is characterised in that the nucleotide sequence of α-rhamnosidase is such as shown in SEQIDNO:3, and aminoacid sequence is such as shown in SEQIDNO:4。

4. the method that enzyme process described in claim 1 prepares rare ginsenoside Rh1, it is characterised in that described beta-glucosidase is by recombinant expressed preparation, and step is:

(1) with the Thermotogapetrophila genomic DNA of extraction for template, being classified as forward primer with having the nucleotides sequence shown in SEQIDNO:5 and have the nucleotides sequence shown in SEQIDNO:6 and be classified as downstream primer, pcr amplification obtains the DNA molecular of beta-glucosidase；

(2) DNA molecular of above-mentioned beta-glucosidase and pET-20b are carried out double digestion with NdeI and XhoI respectively, connect and convert the recombiant plasmid obtaining the DNA molecular containing beta-glucosidase；

(3) by recombinant plasmid transformed expressive host E.coliBL21 (DE3) of above-mentioned acquisition, IPTG induces beta-glucosidase expression of enzymes, collects thalline, smudge cells, 80 DEG C of heat treatments 40 minutes, it is thus achieved that the beta-glucosidase of purification。

5. the method that enzyme process described in claim 1 prepares rare ginsenoside Rh1, it is characterised in that described α-rhamnosidase is by recombinant expressed preparation, and step is:

(1) with aspergillus niger (Aspergillusniger) NL-1cDNA of extraction for template, being classified as forward primer with having the nucleotides sequence shown in SEQIDNO:7 and have the nucleotides sequence shown in SEQIDNO:8 and be classified as downstream primer, pcr amplification obtains the DNA molecular of α-rhamnosidase；

(2) DNA molecular of above-mentioned beta-glucosidase and pET-20b are carried out double digestion with NdeI and XhoI respectively, connect and convert the recombiant plasmid obtaining the DNA molecular containing α-rhamnosidase；

(3) by recombinant plasmid transformed expressive host E.coliBL21 (DE3) of above-mentioned acquisition, induce α-rhamnoside expression of enzymes, collect thalline, smudge cells, be centrifuged and obtain α-rhamnosidase。

6. the method that enzyme process prepares rare ginsenoside Rh1 according to claim 1, it is characterised in that degradation condition is:

(1) 1g/L ginsenoside Re, under beta-glucosidase 0.2U, pH5.0,85 DEG C condition, degrade 1h；

(2) above-mentioned reactant liquor being added α-rhamnosidase 6.6U, adjusting pH is under 6.5,35 DEG C of conditions, and degrade 1h, and ginsenoside Re may finally be converted into ginsenoside Rh1, and molar yield is 95%。