CN107384895B - A kind of beta-glucosidase and its preparation method and application - Google Patents

A kind of beta-glucosidase and its preparation method and application Download PDF

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CN107384895B
CN107384895B CN201710581264.4A CN201710581264A CN107384895B CN 107384895 B CN107384895 B CN 107384895B CN 201710581264 A CN201710581264 A CN 201710581264A CN 107384895 B CN107384895 B CN 107384895B
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ginsenoside
beta
glucosidase
monomer
glu
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CN107384895A (en
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毕云枫
刘景圣
郗昕
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Jilin Agricultural University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
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    • C12P33/00—Preparation of steroids
    • C12P33/20—Preparation of steroids containing heterocyclic rings
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)

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Abstract

The present invention provides a kind of β glucuroides and its preparation method and application, belongs to technique for gene engineering and biomedicine field.The β glucuroides are selected from following protein 1) or 2)：1) protein of amino acid composition of the sequence as shown in SEQ ID NO.1；2) 1) amino acid residue sequence of the protein shown in by the substitution of one or several amino acid residues and/or is lacked and ored add and protein with glycosidase activity.β glucuroides provided by the invention have good thermal stability and wider pH range of hydrolysis, available for production rare ginsenoside and its mixture, it is with stronger hydrolysis ability, ginsenoside will can be converted to the micro existing bioactivity higher in the plants such as ginseng, easily by the rare ginsenoside of human body efficient absorption, can apply in the multiple fields such as food and medical and health.

Description

A kind of beta-glucosidase and its preparation method and application
Technical field
The invention belongs to technique for gene engineering and biomedicine fields, and in particular to a kind of beta-glucosidase and its preparation Methods and applications.
Background technology
The main active of ginseng is ginsenoside.Ginsenoside is the glycoside chemical combination being connected by sugar with aglycon Object belongs to Triterpene saponins.What content was most in ginsenoside is protopanaxadiol-type's (Protopanaxdiol) saponin(e and original Panaxatriol type (Protopanaxtriol) saponin(e, protopanaxadiol-type's saponin(e include ginsenoside Rb1, Rb2, Rc, Rd, F2, Rg3, Rh2, ginsenoside compound K (CK) etc.；Protopanaxatriol type saponin(e includes ginsenoside Re, Rg1, Rf, Rg2, Rh1 Deng.Wherein ginsenoside Rb1, the content of Rb2, Rc, Re, Rg1, Rd are higher, and ginsenoside Rg2, Rg3, Rh1, Rh2, CK etc. In natural ginseng content there was only 100,000/it is several or be not present, be rare ginsenoside.Many pharmacological researches show rare people Ginseng saponin(e pharmacological effect is more preferable, is easier absorbed into serum, bioavilability higher.Therefore, a large amount of rare ginsenoside is obtained It is the hot spot of numerous scientific research personnel's researchs with important social value.Traditional concocting method can improve these rare people Join the content of saponin(e, but its content is still very low.It would therefore be highly desirable to carry out the research of new green environment protection production technology to improve The conversion ratio of these rare ginsenosides and the utilization ratio of Chinese material medicine resource reduce the production cost of rare ginsenoside.
The method of common hydrolyzing saponin has acid-hydrolysis method, alkali hydrolysis method and biotransformation method.But their hydrolysis item Part all very acutely, is difficult to control, and due to that can make sapogenin that dehydration occur in hydrolytic process, cyclization, double-bond shift, take For variations such as base displacement, configuration conversions, increase the by-product of hydrolysis, target product hardly results in.More importantly acid, buck Solution causes environment harm largely, so need to find new transformation technology and method, can more effectively by Low activity, high-content saponin be converted to the low monomer saponin of high activity, natural content, and can accomplish not cause to environment The transformation technology and method of burden.The regio- and stereo-selectivity of bioconversion especially enzyme is strong, reaction condition is mild, operation letter Just, cost is relatively low, public hazards are few, and can complete the reaction that some chemical syntheses are difficult to, and overcomes classical acid, Base hydrolysis method The defects of, and reduce the generation of pollutant, meet the novel modern production pattern of the control conversion from end treatment to source, because And it is increasingly taken seriously.
Invention content
The purpose of the present invention is to solve existing hydrolyzing saponin method by-product more than and environmentally harmful problem, And provide a kind of beta-glucosidase and its preparation method and application.
Present invention firstly provides a kind of beta-glucosidases, are following protein 1) or 2)：
1) protein of amino acid composition of the sequence as shown in SEQ ID NO.1；
2) by the amino acid residue sequence of the protein shown in 1) by the substitution of one or several amino acid residues and/or Lack and or add and have the protein of glycosidase activity.
The present invention also provides a kind of above-mentioned beta-glucosidase enzyme dnas of coding, and nucleotide sequence is as shown in SEQ ID NO.2.
The present invention also provides the recombinant vectors containing above-mentioned beta-glucosidase enzyme dna.
The present invention also provides the recombinant bacteriums obtained by above-mentioned recombinant vector conversion host cell.
The present invention also provides a kind of preparation method of beta-glucosidase, this method includes：
DNA fragmentation shown in SEQ ID NO.2 is inserted into expression vector and obtains recombinant vector, recombinant vector is converted into host Cell cultivates transformant, beta-glucosidase is obtained from culture.
Preferably, the method includes：
Step 1：To extract the genomic DNA of Thermotoga neapolitana DSM 4359 as template, with The sense primer of nucleotide sequence shown in SEQ ID NO.3 and the downstream with the nucleotide sequence shown in SEQ ID NO.4 Primer carries out PCR amplification, obtains the DNA molecular shown in SEQ ID NO.2；
Step 2：The DNA molecular that step 1 obtains is subjected to double digestion with Nhe I and Ecor I, digestion products with through phase Expression vector 16 DEG C of connections of T4 ligases with digestion are stayed overnight, and obtain recombinant vector；
Step 3：Recombinant vector conversion expression host's bacterium competence cell that step 2 is obtained, obtains recombinant bacterium；
Step 4：The recombinant bacterium and IPTG that step 3 is obtained are added in fresh culture, 30 DEG C of progress induction fermentations 16h, thalline were collected by centrifugation, is centrifuged after broken thalline, obtains beta-glucosidase.
The present invention also provides above-mentioned beta-glucosidase in ginsenoside Rg2 or mixture containing ginsenoside Rg2 is prepared Application.
The present invention also provides above-mentioned beta-glucosidases to prepare ginsenoside Rh 1, mixture containing ginsenoside Rh 1, original Application in the mixture of Panaxoside A aglycone Aj's monomer or the monomer containing proto-ginseng triol aglycone.
The present invention also provides above-mentioned beta-glucosidases to prepare ginseng sapoglycoside Rg 3, mixture containing ginseng sapoglycoside Rg 3, original Application in the mixture of panoxadiol aglycon monomer or the monomer containing protopanaxadiol aglycone.
Preferably, the beta-glucosidase in pH3-10, under the conditions of being 40 DEG C -100 DEG C react by temperature.
Beneficial effects of the present invention
(1) beta-glucosidase provided by the invention has good thermal stability and wider pH range of hydrolysis；
(2) beta-glucosidase provided by the invention have it is efficient, single-minded, by-product is few, yield is high, product is easy to pure The advantages of changing, be at low cost.
(3) beta-glucosidase provided by the invention has relatively strong for producing rare ginsenoside and its mixture Hydrolysis ability, ginsenoside will can be converted to the micro existing bioactivity higher in the plants such as ginseng, it is easier to By the rare ginsenoside of human body efficient absorption, the pharmacological effect of the rare ginsenoside has obtained certification, can apply The multiple fields such as medical and health.
Description of the drawings
Fig. 1 is the line chart of beta-glucosidase optimal reactive temperature of the present invention；
Fig. 2 is the line chart of beta-glucosidase optimal reaction pH of the present invention；
Fig. 3 is the thin layer figure of beta-glucosidase enzyme hydrolysis ginsenoside Re of the present invention；
Fig. 4 is the thin layer figure of beta-glucosidase enzyme hydrolysis ginsenoside Rg1 of the present invention；
Fig. 5 is the thin layer figure of beta-glucosidase enzyme hydrolysis Ginsenoside Rc of the present invention, Rd, Rb1, Rb2.
Specific embodiment
Present invention firstly provides a kind of beta-glucosidases, are following protein 1) or 2)：
1) protein of amino acid composition of the sequence as shown in SEQ ID NO.1；
2) by the amino acid residue sequence of the protein shown in 1) by the substitution of one or several amino acid residues and/or Lack and or add and have the protein of glycosidase activity.
The substitution of said one or several amino acid residues and/or the egg for lacking and oring add and having glycosidase activity White matter is the substitution no more than 40 amino acid residues and/or lacks and ors add.
The present invention also provides a kind of above-mentioned beta-glucosidase enzyme dnas of coding, and nucleotide sequence is as shown in SEQ ID NO.2.
The present invention also provides the recombinant vectors containing above-mentioned beta-glucosidase enzyme dna.
The present invention also provides the recombinant bacteriums obtained by above-mentioned recombinant vector conversion host cell.
The present invention also provides a kind of preparation method of beta-glucosidase, this method includes：
DNA fragmentation shown in SEQ ID NO.2 is inserted into expression vector and obtains recombinant vector, recombinant vector is converted into host Cell cultivates transformant, beta-glucosidase is obtained from culture.
According to the present invention, the preparation method step is as follows：
Step 1：To extract the genomic DNA of Thermotoga neapolitana DSM 4359 as template, with The sense primer of nucleotide sequence shown in SEQ ID NO.3 and the downstream with the nucleotide sequence shown in SEQ ID NO.4 Primer carries out PCR amplification, obtains the DNA molecular shown in SEQ ID NO.2；The amplification condition is preferably：First 95 DEG C of pre- changes Property 4min, then 95 DEG C of 50s, 55 DEG C of 45s, 72 DEG C of 1min 30s, totally 30 cycles, last 72 DEG C of extensions 10min, recycling are above-mentioned PCR reaction products are detected into row agarose gel electrophoresis；
Step 2：The DNA molecular that step 1 obtains is subjected to double digestion with Nhe I and Ecor I, digestion products with through phase Expression vector 16 DEG C of connections of T4 ligases with digestion are stayed overnight, and obtain recombinant vector；The expression vector is preferably PET28a carriers；
Step 3：Recombinant vector conversion expression host's bacterium competence cell that step 2 is obtained, obtains recombinant bacterium；It is described Conversion expression host bacterium be E. coli expression strains, preferably Escherichia coli Escherichia coli BL21 (DE3) bacterium Strain；
Step 4：The recombinant bacterium and IPTG that step 3 is obtained are added in fresh culture, 30 DEG C of progress induction fermentations 16h, thalline were collected by centrifugation, is centrifuged after broken thalline, obtains beta-glucosidase.
According to the present invention, recombinant bacterium single bacterium colony is preferably seeded to and contains before fresh culture is added in by the recombinant bacterium In the LB fluid nutrient mediums for having 50 μ g/mL kanamycins, 37 DEG C of culture 12h collect zymotic fluid, by zymotic fluid according to 1% (v/v) Inoculum concentration be forwarded in the fresh LB fluid nutrient mediums of 100mL, 37 DEG C of cultures to OD600 reach 0.6, then in the medium plus Enter sterile IPTG and carry out induction fermentation；The final concentrations of the IPTG in the medium are preferably 0.4mM.
According to the present invention, described thalline were collected by centrifugation, and the means centrifuged after broken thalline are this field conventional technique, It is not particularly limited, preferably：Zymotic fluid centrifugation 10min is collected into thalline, by obtained coli somatic with a concentration of The NaAc_HAc buffer solution piping and druming that 50mmol/L pH value is 5.0 suspends, and it is thin that ultrasonic wave is carried out under conditions of ice-water bath Born of the same parents crush (200W, work 3s pause 3s, work 100 times), centrifugation (15700g, 4 DEG C, 30min) removal cell fragment, in collection Clear liquid, as beta-glucosidase enzyme solution.
The present invention also provides above-mentioned beta-glucosidase in ginsenoside Rg2 or mixture containing ginsenoside Rg2 is prepared Application, the application process is specially：By beta-glucosidase of the present invention in pH3-10,40 DEG C -100 DEG C of temperature Under the conditions of hydrolysis ginsenoside monomer Re or the mixture containing ginsenoside Re Rg2 or the mixture containing Rg2 is prepared.
The present invention also provides above-mentioned beta-glucosidases to prepare ginsenoside Rh 1, mixture containing ginsenoside Rh 1, original Application in the mixture of Panaxoside A aglycone Aj's monomer or the monomer containing proto-ginseng triol aglycone.The application process is specially： By beta-glucosidase of the present invention under the conditions of pH3-10,40 DEG C -100 DEG C of temperature hydrolysis ginsenoside monomer Ginsenoside Rh 1, mixture containing ginsenoside Rh 1, protopanaxatriol is prepared in Rg1 or mixture containing ginsenoside Rg1 The mixture of aglycon monomer or the monomer containing proto-ginseng triol aglycone.
The present invention also provides above-mentioned beta-glucosidases to prepare ginseng sapoglycoside Rg 3, mixture containing ginseng sapoglycoside Rg 3, original Application in the mixture of panoxadiol aglycon monomer or the monomer containing protopanaxadiol aglycone.The application process is specially： By beta-glucosidase of the present invention under the conditions of pH3-10,40 DEG C -100 DEG C of temperature hydrolysis ginsenoside monomer Rc, Rd, Rb1, Rb2 monomer and ginseng is prepared containing Ginsenoside Rc, two kinds of Rd, Rb1, Rb2 and two or more mixtures Saponin(e Rg3, mixture containing ginseng sapoglycoside Rg 3, protopanaxadiol aglycone monomer or the monomer containing protopanaxadiol aglycone mixture.
Further detailed description is done to the present invention with reference to specific embodiment, the various reactions arrived involved in embodiment Raw material and reaction reagent can be commercially available by commercial channel.
The preparation of 1 beta-glucosidase of embodiment
Extracting Thermotoga neapolitana DSM 4359, (source is Germany Microbiological Culture Collection Center, number It is primer with P1 and P2 for genomic DNA 4359) and using it as template, primer gives birth to work biotechnology service by Shanghai Co., Ltd synthesizes, and primer sequence is as follows：
P1：
5’-CGCGGCAGCCATATGGCTAGCATGAAGATGGAAAAGGTGAATGA-3’
(forward), underscore represents Nhe I sites (P1 nucleotide sequences are as shown in SEQ ID NO.3)；
P2：
5’-TTGTCGACGGAGCTCGAATTCTCACGGTTTGAATCTTCTCTCCT3’
(reverse), underscore represents Ecor I sites (P2 nucleotide sequences are as shown in SEQ ID NO.4)；
The sense primer of nucleotide sequence and with the nucleotides sequence shown in SEQ ID NO.4 shown in SEQ ID NO.3 The downstream primer of row carries out PCR amplification, and PCR amplification condition is：First 95 DEG C of pre-degeneration 4min, then 95 DEG C of 50s, 55 DEG C of 45s, 72 DEG C 1min 30s, totally 30 cycles, last 72 DEG C of extensions 10min recycle above-mentioned PCR reaction products, carry out Ago-Gel electricity Swimming detection, as a result obtains the piece segment DNA of about 2170bp sizes, nucleotide sequence is as shown in SEQ ID NO.2；
By the PCR product of above-mentioned acquisition Nhe I and Ecor I double digestions, digestion products and the pET28a through identical digestion Carrier overnight, obtains recombinant vector with the 16 DEG C of connections of T4 ligases；
Above-mentioned recombinant vector is converted into Escherichia coli Escherichia coli BL21 (DE3) competent cell, is weighed Group bacterium；
The 4359 beta-glucosidase recombinant bacterium single bacterium colonies of Thermotoga neapolitana DSM of above-mentioned acquisition are connect Kind in the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, 37 DEG C of culture 12h collect zymotic fluid, by zymotic fluid according to The inoculum concentration of 1% (v/v) is forwarded in the fresh LB fluid nutrient mediums of 100mL, 37 DEG C of cultures to OD600Reach 0.6, then training It supports in base and adds in sterile IPTG, make the final concentration of 0.4mM of IPTG in the medium, induction fermentation 16h is carried out at 30 DEG C.It will Zymotic fluid 4000g centrifugations 10min collects thalline.It is 5.0 by a concentration of 50mmol/L pH value of obtained coli somatic NaAc_HAc buffer solution piping and druming suspends, and ultrasonic cell-break is carried out under conditions of ice-water bath, and (200W, work 3s are temporary Stop 3s, work 100 times), and centrifugation (15700g, 4 DEG C, 30min) removal cell fragment, collect supernatant, as beta-glucosidase Enzyme solution.
2 beta-glucosidase zymologic property of embodiment measures
Enzyme activity is defined as：Catalysis generates the enzyme amount needed for 1 μm of ol p-nitrophenol (pNP) in 1min.
1st, the measure of the optimal reactive temperature of beta-glucosidase
Measure pH 5.0, the enzyme activity of the dilution enzyme solution of 40-100 DEG C of temperature condition respectively, each temperature set three it is parallel, With enzyme activity, soprano is set to 100%, is mapped with enzyme activity to temperature.The results are shown in Figure 1.
It will be seen from figure 1 that the optimal reactive temperature of beta-glucosidase of the present invention is 85 DEG C.
2nd, the measure of the optimal reaction pH of beta-glucosidase
The buffer solution (pH range 3.0-10.0) of the 50mM of different pH is taken to be configured to the pNPG solution of 5mM respectively, adds in 25 μ L dilutes enzyme solution, and setting three is parallel, measures the enzyme activity under each pH value condition at 85 DEG C.With enzyme activity, soprano is defined as 100%, it is mapped with enzyme activity to pH.The results are shown in Figure 2.
Figure it is seen that the pH of beta-glucosidase optimal reaction of the present invention is 5.0.
Rg2 is prepared in 3 beta-glucosidase hydrolysis ginsenoside monomer Re of embodiment
200 μ L enzyme solutions obtained above are added in into 200 μ L ginsenoside Res, are reacted Re final concentrations 2mg/mL, instead It is carried out under conditions of should being 5.0 in pH value, 85 DEG C of reaction 15-60min.
It vacuum dried sample and is re-dissolved after reaction with methanol, carries out thin-layer chromatography detection.Fig. 3 is β-grape of the present invention Thin layer figure (the thin layer condition of glucosides enzyme hydrolysis ginsenoside Re：Solvent is chloroform：Methanol：Water=65:35:10, expansion Color developing agent is sprayed in 65 DEG C of drying afterwards, and color developing agent is 10% sulfuric acid-ethyl alcohol, and low temperature drying color developing agent is dried to colour developing after 110 DEG C, with Lower embodiment condition thus), it can be seen from the figure that ginsenoside Re is just almost completely converted into Rg2 in 40min, with The extension in reaction time, ginsenoside Re, which is hydrolyzed, is converted into ginsenoside Rg2, conversion ratio 99%.
Ginsenoside Rh 1 or protoplast is prepared in 4 beta-glucosidase hydrolysis ginsenoside monomer Rg1 of embodiment Join triol aglycon monomer
200 μ L enzyme solutions obtained above are added in into 200 μ L ginsenoside Rg1s, are reacted Rg1 final concentrations 2mg/mL, Reaction carries out under conditions of being 5.0 in pH value, 85 DEG C of reaction 25min-8h.
It vacuum dried sample and is re-dissolved after reaction with methanol, carries out thin-layer chromatography detection.Fig. 4 is β-grape of the present invention The thin layer figure of glucosides enzyme hydrolysis ginsenoside Rg1, it can be seen from the figure that ginsenoside Rg1 is just almost all turned in 25min Rh1 is turned to, while has a small amount of proto-ginseng triol aglycone (PPT) generation, with the extension in reaction time, during to 8h, Rh1 wholes again It is converted into PPT, conversion ratio 99%.
Ginseng is prepared in 5 beta-glucosidase hydrolysis ginsenoside monomer Rc, Rd, Rb1, Rb2 monomer of embodiment Saponin(e Rg3 or protopanaxadiol aglycone monomer
200 μ L enzyme solutions obtained above are added in into 200 μ L Ginsenoside Rcs, Rd, Rb1, Rb2, make Rc, Rd, Rb1, Rb2 whole Concentration 2mg/mL is reacted, and is reacted in pH value to carry out under conditions of 5.0,85 DEG C of reaction 5-10h.
It vacuum dried sample and is re-dissolved after reaction with methanol, carries out thin-layer chromatography detection.Fig. 5 is β-grape of the present invention Glucosides enzyme hydrolysis Ginsenoside Rc, the thin layer figure of Rd, Rb1, Rb2, it can be seen from the figure that ginsenoside turns under the action of enzyme Rg3 and protopanaxadiol aglycone (PPD) are turned to, and is increased with the extension product of time.
Ginsenoside Rg2, Rh1 and original is prepared in 6 beta-glucosidase hydrolysis panaxsaponin mixture of embodiment Panaxoside A aglycone Aj's mixture
1mL enzyme solutions obtained above are added in into 1mL triol groups ginsenoside (triol group ginsenoside or triol group three Seven saponin(es), saponin(e final concentration 5mg/mL is reacted, is reacted in pH value to carry out under conditions of 5.0,85 DEG C of reaction 6h.
Ginseng sapoglycoside Rg 3 and protoplast's ginseng is prepared in 7 beta-glucosidase hydrolysis panaxsaponin mixture of embodiment Glycol aglycon mixture
1mL enzyme solutions obtained above are added in into 1mL glycol groups ginsenoside (glycol group ginsenoside or glycol group three Seven saponin(es), saponin(e final concentration 5mg/mL is reacted, is reacted in pH value to carry out under conditions of 5.0,85 DEG C of reaction 6h.
SEQUENCE LISTING
<110>Jilin Agriculture University
<120>A kind of beta-glucosidase and its preparation method and application
<160> 4
<210> 1
<211> 723
<212> PRT
<213>Artificial sequence
<400> 1
Met Lys Met Glu Lys Val Asn Glu Ile Leu Ser Gln Leu Thr Leu Glu
1 5 10 15
Glu Lys Val Lys Leu Val Val Gly Val Gly Leu Pro Gly Leu Phe Gly
20 25 30
Asn Pro His Ser Arg Val Ala Gly Ala Ala Gly Glu Thr His Pro Val
35 40 45
Pro Arg Val Gly Leu Pro Ala Phe Val Leu Ala Asp Gly Pro Ala Gly
50 55 60
Leu Arg Ile Asn Pro Thr Arg Glu Asn Asp Glu Asn Thr Tyr Tyr Thr
65 70 75 80
Thr Ala Phe Pro Val Glu Ile Met Leu Ala Ser Thr Trp Asn Arg Glu
85 90 95
Leu Leu Glu Glu Val Gly Lys Ala Met Gly Glu Glu Val Arg Glu Tyr
100 105 110
Gly Val Asp Val Leu Leu Ala Pro Ala Met Asn Ile His Arg Asn Pro
115 120 125
Leu Cys Gly Arg Asn Phe Glu Tyr Tyr Ser Glu Asp Pro Val Leu Ser
130 135 140
Gly Glu Met Ala Ser Ser Phe Val Lys Gly Val Gln Ser Gln Gly Val
145 150 155 160
Gly Ala Cys Ile Lys His Phe Val Ala Asn Asn Gln Glu Thr Asn Arg
165 170 175
Met Val Val Asp Thr Ile Val Ser Glu Arg Ala Leu Arg Glu Ile Tyr
180 185 190
Leu Arg Gly Phe Glu Ile Ala Val Lys Lys Ser Lys Pro Trp Ser Val
195 200 205
Met Ser Ala Tyr Asn Lys Leu Asn Gly Lys Tyr Cys Ser Gln Asn Glu
210 215 220
Trp Leu Leu Lys Lys Val Leu Arg Glu Glu Trp Gly Phe Glu Gly Phe
225 230 235 240
Val Met Ser Asp Trp Tyr Ala Gly Asp Asn Pro Val Glu Gln Leu Lys
245 250 255
Ala Gly Asn Asp Leu Ile Met Pro Gly Lys Ala Tyr Gln Val Asn Thr
260 265 270
Glu Arg Arg Asp Glu Ile Glu Glu Ile Met Glu Ala Leu Lys Glu Gly
275 280 285
Lys Leu Ser Glu Glu Val Leu Asp Glu Cys Val Arg Asn Ile Leu Lys
290 295 300
Val Leu Val Asn Ala Pro Ser Phe Lys Asn Tyr Arg Tyr Ser Asn Lys
305 310 315 320
Pro Asp Leu Glu Lys His Ala Lys Val Ala Tyr Glu Ala Gly Ala Glu
325 330 335
Gly Val Val Leu Leu Arg Asn Glu Glu Ala Leu Pro Leu Ser Glu Asn
340 345 350
Ser Lys Ile Ala Leu Phe Gly Thr Gly Gln Ile Glu Thr Ile Lys Gly
355 360 365
Gly Thr Gly Ser Gly Asp Thr His Pro Arg Tyr Ala Ile Ser Ile Leu
370 375 380
Glu Gly Ile Lys Glu Arg Gly Leu Asn Phe Asp Glu Glu Leu Ala Lys
385 390 395 400
Thr Tyr Glu Asp Tyr Ile Lys Lys Met Arg Glu Thr Glu Glu Tyr Lys
405 410 415
Pro Arg Arg Asp Ser Trp Gly Thr Ile Ile Lys Pro Lys Leu Pro Glu
420 425 430
Asn Phe Leu Ser Glu Lys Glu Ile His Lys Leu Ala Lys Lys Asn Asp
435 440 445
Val Ala Val Ile Val Ile Ser Arg Ile Ser Gly Glu Gly Tyr Asp Arg
450 455 460
Lys Pro Val Lys Gly Asp Phe Tyr Leu Ser Asp Asp Glu Thr Asp Leu
465 470 475 480
Ile Lys Thr Val Ser Arg Glu Phe His Glu Gln Gly Lys Lys Val Ile
485 490 495
Val Leu Leu Asn Ile Gly Ser Pro Val Glu Val Val Ser Trp Arg Asp
500 505 510
Leu Val Asp Gly Ile Leu Leu Val Trp Gln Ala Gly Gln Glu Thr Gly
515 520 525
Arg Ile Val Ala Asp Val Leu Thr Gly Arg Ile Asn Pro Ser Gly Lys
530 535 540
Leu Pro Thr Thr Phe Pro Arg Asp Tyr Ser Asp Val Pro Ser Trp Thr
545 550 555 560
Phe Pro Gly Glu Pro Lys Asp Asn Pro Gln Lys Val Val Tyr Glu Glu
565 570 575
Asp Ile Tyr Val Gly Tyr Arg Tyr Tyr Asp Thr Phe Gly Val Glu Pro
580 585 590
Ala Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Tyr Ser
595 600 605
Asp Leu Asn Val Ser Phe Asp Gly Glu Thr Leu Arg Val Gln Tyr Arg
610 615 620
Ile Glu Asn Thr Gly Gly Arg Ala Gly Lys Glu Val Ser Gln Val Tyr
625 630 635 640
Ile Lys Ala Pro Lys Gly Lys Ile Asp Lys Pro Phe Gln Glu Leu Lys
645 650 655
Ala Phe His Lys Thr Arg Leu Leu Asn Pro Gly Glu Ser Glu Glu Val
660 665 670
Val Leu Glu Ile Pro Val Arg Asp Leu Ala Ser Phe Asn Gly Glu Glu
675 680 685
Trp Val Val Glu Ala Gly Glu Tyr Glu Val Arg Val Gly Ala Ser Ser
690 695 700
Arg Asn Ile Lys Leu Lys Gly Thr Phe Ser Val Gly Glu Glu Arg Arg
705 710 715 720
Phe Lys Pro
<210> 2
<211> 2172
<212> DNA
<213>Artificial sequence
<400> 2
atgaagatgg aaaaggtgaa tgaaatcctg tctcaactca ctctggaaga aaaagtgaaa 60
cttgtagtgg gggtaggact tccggggttg tttggaaatc cccattcccg ggtggcgggt 120
gccgctggag agacgcatcc tgtcccaaga gtgggtcttc ctgctttcgt tctggcggat 180
ggcccagcag gacttagaat aaatcctacg agagaaaacg atgagaacac ctattacacc 240
accgcttttc ctgttgagat catgcttgct tccacctgga acagagagct cctcgaagaa 300
gtgggaaagg caatgggtga agaggtgaga gagtacggtg tggatgtgct ccttgctcct 360
gcgatgaaca tacacagaaa tccactttgt ggaagaaact ttgaatacta ctcggaagat 420
cctgtcctct ccggtgagat ggcctcttcc tttgtgaagg gagtccagtc acagggagtt 480
ggtgcgtgta taaagcactt cgtggcgaac aaccaggaga cgaacagaat ggtcgtggac 540
acgatcgtgt ccgaacgtgc tctcagagag atatatctca ggggattcga gatcgctgtg 600
aagaaatcaa aaccgtggag cgtgatgagt gcttacaaca aactcaatgg gaagtactgc 660
tcgcagaacg agtggctcct gaagaaggtt ctcagggaag aatggggttt cgaaggtttc 720
gttatgagcg actggtacgc tggagacaat ccggtagaac agctcaaagc aggtaacgat 780
ctcatcatgc ctggaaaggc ctaccaggtg aacacagaac gaagagacga aatagaagag 840
atcatggagg ccctgaaaga aggaaaactc agcgaagaag ttctcgatga atgtgtaaga 900
aacatcttga aagtccttgt gaacgcacct tctttcaaaa actacagata ctccaacaaa 960
cccgatcttg agaagcacgc aaaggttgct tatgaagcag gagcagaagg tgttgtcctt 1020
ttgaggaacg aagaggctct tcctctttct gaaaactcga aaatagccct ctttggaacg 1080
ggccagatcg aaacgataaa aggtggaaca ggaagcggcg acacccatcc aaggtacgct 1140
atttccatcc ttgaggggat aaaagaaagg ggtctgaatt tcgacgaaga actcgcaaaa 1200
acctacgaag actacatcaa gaagatgaga gaaacagaag agtacaaacc aagaagggat 1260
tcctggggaa cgatcataaa accaaaactt ccagaaaact tcctttcgga gaaggaaata 1320
cacaaactgg cgaaaaagaa cgacgtggcg gtcatcgtga tcagcaggat ttccggagaa 1380
ggctatgaca gaaagccggt gaagggagac ttttaccttt ctgacgatga gactgatctc 1440
ataaagactg tctccagaga gttccatgaa cagggcaaga aagtgatcgt tcttctcaac 1500
ataggaagtc ctgttgaggt tgttagctgg agagatctgg tggatgggat tctccttgtg 1560
tggcaggcag ggcaggaaac cggcaggatc gttgccgatg ttctcactgg aaggatcaat 1620
ccatctggaa aacttccaac cacctttccg agagactact ctgatgtacc ctcctggacc 1680
tttcctggag agccaaagga caatccacag aaggtggtct acgaagagga catctacgtg 1740
ggatacaggt actacgacac cttcggtgtg gagccggcgt acgagttcgg atacggcctt 1800
tcttacacga cctttgagta cagtgacctg aacgtttcgt tcgacggtga aacactcaga 1860
gttcagtaca gaatagaaaa cacgggcggt cgtgcaggaa aggaagtctc gcaggtttac 1920
atcaaggcac cgaaaggaaa aatcgacaaa cccttccagg aactcaaggc gtttcacaaa 1980
accagacttt tgaatcctgg agagtctgaa gaagtggtgc ttgagatacc tgtcagagat 2040
cttgcaagtt tcaacggtga agaatgggtt gtcgaagcgg gtgaatacga agtaagggtt 2100
ggtgcgtctt cgaggaacat aaaacttaaa ggaacgtttt ccgtcggtga ggagagaaga 2160
ttcaaaccgt ga 2172
<210> 3
<211> 44
<212> DNA
<213>Artificial sequence
<400> 3
cgcggcagcc atatggctag catgaagatg gaaaaggtga atga
<210> 4
<211> 44
<212> DNA
<213>Artificial sequence
<400> 4
ttgtcgacgg agctcgaatt ctcacggttt gaatcttctc tcct

Claims (3)

1. beta-glucosidase of the amino acid sequence as shown in SEQ ID NO.1 is in preparation ginsenoside Rg2 or containing ginsenoside Application in Rg2 mixtures, which is characterized in that for the beta-glucosidase in pH5, temperature is hydrolysis people under the conditions of 85 DEG C Rg2 or the mixture containing Rg2 is prepared in ginseng saponin monomer Re or the mixture containing ginsenoside Re.
2. beta-glucosidase of the amino acid sequence as shown in SEQ ID NO.1 prepare ginsenoside Rh 1, containing ginsenoside Application in the mixture of Rh1 mixtures, proto-ginseng triol aglycone monomer or the monomer containing proto-ginseng triol aglycone, feature exist In, the beta-glucosidase in pH5, hydrolysis ginsenoside monomer Rg1 or containing ginsenoside under the conditions of temperature is 85 DEG C Ginsenoside Rh 1, mixture containing ginsenoside Rh 1, proto-ginseng triol aglycone monomer or containing protoplast is prepared in the mixture of Rg1 Join the mixture of triol aglycon monomer.
3. beta-glucosidase of the amino acid sequence as shown in SEQ ID NO.1 prepare ginseng sapoglycoside Rg 3, containing ginsenoside Application in the mixture of Rg3 mixtures, protopanaxadiol aglycone monomer or the monomer containing protopanaxadiol aglycone, feature exist In the beta-glucosidase is in pH5, and hydrolysis ginsenoside monomer Rc, Rd, Rb1, Rb2 are mono- under the conditions of temperature is 85 DEG C Body and ginseng sapoglycoside Rg 3, soap containing ginseng is prepared containing Ginsenoside Rc, two kinds of Rd, Rb1, Rb2 and two or more mixtures The mixture of glycosides Rg3 mixtures, protopanaxadiol aglycone monomer or the monomer containing protopanaxadiol aglycone.
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CN110791490B (en) * 2019-11-15 2021-03-30 东北师范大学 Recombinant beta-glucosidase CB-3B and application thereof in production of ginsenoside Rg3
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