CN106191010A - A kind of rhamnosidase and hydrolysis Dioscorea zingiberensis dioscin prepare the application in diosgenin - Google Patents

A kind of rhamnosidase and hydrolysis Dioscorea zingiberensis dioscin prepare the application in diosgenin Download PDF

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Publication number
CN106191010A
CN106191010A CN201610854080.6A CN201610854080A CN106191010A CN 106191010 A CN106191010 A CN 106191010A CN 201610854080 A CN201610854080 A CN 201610854080A CN 106191010 A CN106191010 A CN 106191010A
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China
Prior art keywords
rhamnosidase
dioscin
diosgenin
hydrolysis
dioscorea zingiberensis
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Inventor
杨雪鹏
钟桂芳
马科
叶建斌
闫记
崔君竹
聂晶晶
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Zhengzhou University of Light Industry
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Zhengzhou University of Light Industry
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/20—Preparation of steroids containing heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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)
    • C12Y302/0104—Alpha-L-rhamnosidase (3.2.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Abstract

The invention belongs to bioengineering field, relate to a kind of rhamnosidase and the application preparing in diosgenin at hydrolysis Dioscorea zingiberensis dioscin thereof.Described rhamnosidase is α L Fructus rhamni (Rhamnus davurica Pall.) carbohydrase, and gene order is as shown in SEQ ID NO:1, and preparation process is as follows: build the recombinant vector with α L rhamnosidase gene；The conversion of recombinant vector and the expression of α L rhamnosidase；The purification of α L rhamnosidase.Rhamnosidase is as glycoside hydrolase, with Dioscorea zingiberensis dioscin as substrate, prepares glucosyl group dioscin, then with β glucosidase hydrolyzation of glucose base dioscin, prepares diosgenin.The present invention is developed to the enzyme of specific for hydrolysis dioscin glycosyl rhamnoside, prepare diosgenin, overcome existing for prior art is low to dioscin percent hydrolysis, the shortcomings such as destructiveness is big, seriously polluted, purposiveness is poor, have advantage pollution-free, that purposiveness is strong, the response time is disconnected, conversion ratio is high.

Description

A kind of rhamnosidase and hydrolysis Dioscorea zingiberensis dioscin prepare in diosgenin Application
Technical field
The invention belongs to bioengineering field, relate to a kind of rhamnosidase and prepare potato at hydrolysis Dioscorea zingiberensis dioscin Application in Chinese yam Saponin.
Background technology
Diosgenin is active substance important in Dioscorea zingiberensis, has the effect of estrogen and cholesterol reducing, can directly be used as medicine. Research shows, it has antibacterial, antiinflammatory, cough-relieving, eliminates the phlegm, haemolysis, blood fat reducing, desensitizes, repairs pathological tissues, stimulates heparinocyte raw Length and the effect of promotion bile secretion.Owing to the framing structure of diosgenin is very much like with steroid hormone structure, structural formula As in figure 2 it is shown, can be as synthesizing steroid hormonal medicaments and the main intermediate of contraceptive steroid.Therefore, a large amount of preparation Rhizoma Dioscoreae soaps Element has great importance.
Containing more diosgenin in Dioscorea zingiberensis, but it is all presented in glucosides.The glycosyl part of dioscin by Rhamnose and glucose composition, sugar chain structure be rhamnose respectively with α-Isosorbide-5-Nitrae and α-1,2-glycosidic bond is connected with glucose, Fructus Vitis viniferae Sugar links with aglycon, as shown in Figure 1 with β-Isosorbide-5-Nitrae glycosidic bond.Therefore, want to prepare diosgenin, it is important to develop α-Fructus rhamni (Rhamnus davurica Pall.) Exoglycosidase hydrolysis dioscin.
Prepare diosgenin, acid-hydrolysis method, microbe transformation method, enzymatic isolation method or the conbined enzymolysis etc. being disclosed.Pass The method of system is decomposed not thorough, and conversion ratio is the highest, yields poorly, and by-product contamination is serious.
Summary of the invention
The present invention solves that the glycosyl part of dioscin is difficult to the technical barrier hydrolyzed, it is provided that a kind of rhamnosidase And prepare the application in diosgenin at hydrolysis Dioscorea zingiberensis dioscin.
For solve above-mentioned technical barrier, the present invention by the following technical solutions:
A kind of rhamnosidase, described rhamnosidase is α-L-Fructus rhamni (Rhamnus davurica Pall.) carbohydrase, gene order as shown in SEQ ID NO:1, ammonia Base acid sequence is as shown in SEQ ID NO:2.
The preparation process of described α-L-Fructus rhamni (Rhamnus davurica Pall.) carbohydrase is as follows:
(1) recombinant vector with alpha-L-Rhamnosidase gene is built；
(2) conversion of recombinant vector and the expression of alpha-L-Rhamnosidase；
(3) purification of alpha-L-Rhamnosidase.
The recombinant vector built in described step (1), the primer of employing is to such as SEQ ID NO:3, SEQ ID NO:4 institute Show.
In described step (3), the purification of alpha-L-Rhamnosidase uses ni-sepharose purification.
A kind of rhamnosidase prepares the application in diosgenin, described alpha-L-rhamnoside at hydrolysis Dioscorea zingiberensis dioscin Enzyme is as glycoside hydrolysis enzyme catalyst I, with Dioscorea zingiberensis dioscin as substrate, prepares glucosyl group dioscin, then uses catalyst II hydrolyzation of glucose base dioscin, prepares diosgenin.
The addition of described catalyst I is 0.5-10 U, and the amount of substrate is 50-200mM, and catalyst II is beta-glucosidase Enzyme, the addition of beta-glucosidase is 0.5-10 U, and reaction temperature is at 20-60 DEG C, and pH is between 4-8.
The beneficial effects of the present invention is:
(1) present invention hydrolyzes the rhamnose of dioscin glycosyl end under the effect of rhamnosidase, reacts 1 hour, hydrolysis Rate reaches 99%, hydrolysed residual glycosyl part under beta-glucosidase effect, generates diosgenin, and conversion ratio reaches 98%；
(2) present invention is developed to the enzyme of specific for hydrolysis dioscin glycosyl rhamnoside, has prepared diosgenin, gram Taken existing for prior art is low to dioscin percent hydrolysis, destructive big, seriously polluted, the shortcoming such as purposiveness is poor, has Advantage pollution-free, that purposiveness is strong, the response time is disconnected, conversion ratio is high.
Accompanying drawing explanation
Fig. 1 is the structural formula of Dioscorea zingiberensis dioscin.
Fig. 2 is the structural formula of diosgenin.
Fig. 3 is that TLC detects hydrolysis effect figure.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that reality Execute concrete material proportion, process conditions and result thereof described by example and be merely to illustrate the present invention, and should also will not limit The present invention described in detail in claims processed.
One, the structure of recombination bacillus coli
1, the acquisition of alpha-L-Rhamnosidase gene
Activation Streptomyces griseus CICC11002 strain, collects cell, and cell turns after 1 minute through liquid nitrogen freezing again Move on to the middle temperature of boiling water bathe 10 minutes, using the cell pyrolysis liquid that processed as the template of PCR.
Primer used by construction of expression vector and restriction enzyme site thereof, primer sequence is as follows:
Forward primer is as shown in SEQ ID NO:3, and wherein ATGG is Nco I restriction enzyme site；Downstream primer such as SEQ ID NO:4 institute Showing, wherein GGATCC is BamHI restriction enzyme site.
All primers all have Shanghai Sheng Gong company to synthesize.
The PCR condition of gene: 94 DEG C of degeneration 5min, by 30 times: 94 DEG C of degeneration 1min of following parameter cyclic, 60 DEG C are moved back Fire 50s, 72 DEG C extend 2min.Last 72 DEG C extend 10min.
2, the structure of recombiant plasmid and conversion
WithNcoI andBamH I enzyme action pET-28b respectively (+) (pET-28b (+) be purchased from Novagen (Merck China)) and Being expanded the genes of interest containing two restriction enzyme sites, glue reclaims purpose fragment and the expression vector of double digestion respectively, by The expression vector pET-28b of double digestion and genes of interest T4 ligase are attached overnight, by the connection product of 10 μ L PET-28b-rha adds in Rosetta (DE3) competent cell of 100 μ L, places 30min, 42 DEG C of heat shocks on ice 90sec.Place 2min on ice.Add the 0.45mL culture medium of preheating.220rpm37℃ 1h.200 μ L bacterium solution are added Contain on the kanamycin of 100 μ g/mL and the LB flat board of chloromycetin respectively, 37 DEG C of incubated overnight 12-16h.
Two, the expression of recombinase and character research thereof
1, the expression of alpha-L-Rhamnosidase
Picking recombinant bacteriumE.coli/PET-28b-rha in LB fluid medium containing antibiotic, 37 DEG C of shaken cultivation mistakes Night.Then it is inoculated into respectively in fresh medium by 2% inoculum concentration, when 37 DEG C of cultivations are to OD 600 about 0.6, adds IPTG to final concentration 0.5mmolL-1, 20 DEG C, 220rpm, after abduction delivering 12h, centrifugal (4 DEG C, 10000rpm, 10min)。
2, the purification of alpha-L-Rhamnosidase
By resuspended with the phosphoric acid buffer (50mM, pH7.5) containing 300mM NaCl and 30% glycerol for the bacterium mud of collection, ultrasonic Smudge cells (power 300W, ultrasonic 5s, intermittently 5s, altogether 5min), centrifugal (4 DEG C, 12000rpm, 15min) take supernatant.
The upper clear enzyme solution collected is added in Ni-NTA post, on ice insulation 30 minutes.Discard after penetrating liquid, use Containing 60mM imidazoles, the phosphoric acid buffer (50mM, pH7.5) of 300mM NaCl and 30% glycerol washes away foreign protein.Again with containing 500mM imidazoles, the phosphoric acid buffer (50mM, pH7.5) of 300mM NaCl and 30% glycerol is by under target protein Rha eluting Come.SDS-PAGE with 12% detects the purity of enzyme, obtains electrophoretically pure Rha.Rha Tris-HCl after purification is delayed Punching (pH7.5) dialysis (dialyzer of M.W.3000 is dialysed 24 hours).Protein concentration uses Brandford method to survey Fixed.
Result shows, gained enzyme liquid concentration is 0.2 mg/L, cumulative volume 10 mL.
3, alpha-L-Rhamnosidase enzyme activity determination
Enzyme liquid 100 μ L joins the 0.2mol/LNa of pH6.62HPO4And NaH2PO4In buffer 840 μ L, pre-in 40 DEG C after mixing Hot 5min, adds warmed-up 20mmol/LoNPR solution 60 μ L, adds 4mL 0.4mol/L immediately after 40 DEG C of reaction 10min Na2CO3Solution terminates reaction, and room temperature places 5min, surveys extinction value OD at 420nm.With the enzyme liquid that is heated and inactivated according to same method Blank is made in process.
Enzyme is lived and is defined: under the above-described reaction conditions, producing the enzyme amount needed for the ONP of 1 μm ol per hour is 1 enzyme activity list Position U.
Result shows, the vigor of enzyme liquid is 1.6 mU/mL, is 7.8 U/mg than work.
Three, hydrolysis dioscin
The alpha-L-Rhamnosidase of 2U is added in 100mM dioscin solution, and pH value of solution is 6.5,40 DEG C, reacts 2 hours, Adding the beta-glucosidase of 2U again, react 1 hour, by thin plate chromatography detection response situation, result is as it is shown on figure 3, difference is tied The dioscin of structure is hydrolyzed by two kinds of glycosidase, and diosgenin occurs in a large number, and total conversion ratio reaches 98%.

Claims (6)

1. a rhamnosidase, it is characterised in that: described rhamnosidase is α-L-Fructus rhamni (Rhamnus davurica Pall.) carbohydrase, gene order such as SEQ ID Shown in NO:1, aminoacid sequence is as shown in SEQ ID NO:2.
2. rhamnosidase as claimed in claim 1, it is characterised in that the preparation process of described α-L-Fructus rhamni (Rhamnus davurica Pall.) carbohydrase is as follows:
(1) recombinant vector with alpha-L-Rhamnosidase gene is built；
(2) conversion of recombinant vector and the expression of alpha-L-Rhamnosidase；
(3) purification of alpha-L-Rhamnosidase.
3. rhamnosidase as claimed in claim 2, it is characterised in that: the recombinant vector built in described step (1), use Primer to as shown in SEQ ID NO:3, SEQ ID NO:4.
4. rhamnosidase as claimed in claim 2, it is characterised in that: in described step (3), alpha-L-Rhamnosidase is pure Change and use ni-sepharose purification.
5. a rhamnosidase prepares the application in diosgenin at hydrolysis Dioscorea zingiberensis dioscin, it is characterised in that: described α- L-rhamnosidase is as glycoside hydrolysis enzyme catalyst I, with Dioscorea zingiberensis dioscin as substrate, prepares glucosyl group dioscin, Again with catalyst II hydrolyzation of glucose base dioscin, prepare diosgenin.
6. rhamnosidase as claimed in claim 5 prepares the application in diosgenin at hydrolysis Dioscorea zingiberensis dioscin, and it is special Levying and be: the addition of described catalyst I is 0.5-10 U, the amount of substrate is 50-200mM, and catalyst II is beta-glucosidase Enzyme, the addition of beta-glucosidase is 0.5-10 U, and reaction temperature is at 20-60 DEG C, and pH is between 4-8.
CN201610854080.6A 2016-09-27 2016-09-27 A kind of rhamnosidase and hydrolysis Dioscorea zingiberensis dioscin prepare the application in diosgenin Pending CN106191010A (en)

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Cited By (6)

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CN110938613A (en) * 2018-09-25 2020-03-31 中国医学科学院医药生物技术研究所 Glycosyl hydrolase and related biological material and application thereof
CN110938612A (en) * 2018-09-25 2020-03-31 中国医学科学院医药生物技术研究所 Method for preparing diosgenin by using biological enzyme and biological material used by same
CN112575050A (en) * 2020-09-07 2021-03-30 北京化工大学 Method for preparing diosgenin by biological conversion
CN112813116A (en) * 2020-12-31 2021-05-18 涟源康麓生物科技有限公司 Method for preparing apigenin by biological enzymolysis
CN113584000A (en) * 2021-01-07 2021-11-02 北京化工大学 alpha-L-rhamnosidase as well as preparation method and application thereof
CN113584001A (en) * 2021-01-07 2021-11-02 北京化工大学 alpha-L-rhamnosidase, preparation method and application thereof

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110938613A (en) * 2018-09-25 2020-03-31 中国医学科学院医药生物技术研究所 Glycosyl hydrolase and related biological material and application thereof
CN110938612A (en) * 2018-09-25 2020-03-31 中国医学科学院医药生物技术研究所 Method for preparing diosgenin by using biological enzyme and biological material used by same
CN110938612B (en) * 2018-09-25 2021-06-15 中国医学科学院医药生物技术研究所 Method for preparing diosgenin by using biological enzyme and biological material used by same
CN112575050A (en) * 2020-09-07 2021-03-30 北京化工大学 Method for preparing diosgenin by biological conversion
CN112813116A (en) * 2020-12-31 2021-05-18 涟源康麓生物科技有限公司 Method for preparing apigenin by biological enzymolysis
CN113584000A (en) * 2021-01-07 2021-11-02 北京化工大学 alpha-L-rhamnosidase as well as preparation method and application thereof
CN113584001A (en) * 2021-01-07 2021-11-02 北京化工大学 alpha-L-rhamnosidase, preparation method and application thereof

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Application publication date: 20161207