CN104480080A - Recombinant apple phlorizin glycosyl transferase and separation and purification method thereof - Google Patents

Abstract

The invention discloses a recombinant apple phlorizin glycosyl transferase and a separation and purification method thereof and belongs to the technical field of biological gene engineering. The glycosyl transferase has an amino acid sequence which is shown as SEQ.ID.NO.1. On the one hand, the biological engineering technology is used for carrying out heterologous expression of the apple phlorizin glycosyl transferase of escherichia coli BL21(DE3) and the apple phlorizin glycosyl transferase is used for catalyzing to generate phlorizin; on the other hand, the separation and purification methods such as saturated ammonium sulfate precipitation, DEAE-Sepharose anion-exchange chromatography and Sephadex-75 gel filtration chromatography are adopted. The recombinant apple phlorizin glycosyl transferase has the advantages of being simple, liable to implement, short in period, high in repeatability, high in zymoprotein activity and the like.

Description

A kind of restructuring apple phlorizin glycosyltransferase and separation purification method

Technical field

The invention belongs to technical field of biological genetic engineering, be specifically related to a kind of restructuring apple phlorizin glycosyltransferase and separation purification method thereof.

Background technology

Phlorizin (Phloridzin) belongs to dihydrochalcone-type, it is the glucoside of Phloretin (Phloretin), mainly be present in the root skin of apple tree, stem, tender leaf and Apple, be the representational a kind of polyphenol components of most in apple polyphenol, in the sweet tea of Pasania cuspidata, found the existence of phlorizin again in recent years.Phlorizin except have antibacterial, anti-oxidant, remove except the biological activity such as interior free yl and cardioprotection, the most outstanding effect can reduce blood sugar exactly.Phlorizin suppresses sodium ion association glucose transporter (Sodium-linked glucose transporters, the SGLTs) transport to glucose molecule exclusively, competitively, thus suppresses glucose in the absorption of small intestine and kidney.Therefore, phlorizin as a kind of potential ofhypoglycemic medicine, can have a extensive future.

Glycosyltransferase (glycosyltransferase, GT, EC2.4.x.y) be the enzyme of special catalysis glycosylation, active glycosyl is transferred to glycosyl acceptor from glycosyl donor by them, form glycosidic link, product comprises oligosaccharides, polysaccharide, various compounding sugar (glycoprotein, glycolipid) and glycoside compounds (as anthocyanogen, flavone glycoside, resveratrol glucoside etc.).In CAZy (Carbohydrate Active enzymes Database) database, 94 families are divided at present.Plant glycosylated effect is the solvability changing aglycon by increasing wetting ability, provides the approach entering film movement system.Glycosylation by regulating the level of important cells metabolite, the function of activity and location keeping playing very large effect in cell inner equilibrium.The balance adjustment of some glycosylation involved in plant hormone in vivo.Glycosylation can also reduce or remove endogenous and toxicity that is allogenic material in addition.Glycosyltransferase has substrate specificity, therefore containing multiple glycosyltransferase in plant, as ginsenoside Rh ₂glucanotransferase, puerarin glycosyltransferase, rhodioside glycosyltransferase, cinnamophenone glycosyltransferase, anthocyanidin glycosyltransferase, genistein glycosyltransferase, Quercetin glycosyltransferase, stevioside glycosides glycosyltransferase etc.Apple phlorizin glycosyltransferase is present in the root skin of apple tree, stem, tender leaf and Apple, and catalysis Phloretin generates phlorizin.

Summary of the invention

The object of the present invention is to provide a kind of restructuring apple phlorizin glycosyltransferase and separation purification method, the method utilizes biotechnology, heterogenous expression apple phlorizin glycosyltransferase in competent cell, through chromatographic separation and purification, obtained restructuring apple phlorizin glycosyltransferase, method is simple, the cycle is short, repeatability is high.

The present invention is achieved through the following technical solutions:

A kind of restructuring apple phlorizin glycosyltransferase, this glycosyltransferase has the aminoacid sequence as shown in SEQ.ID.NO.1.

A kind of restructuring apple phlorizin glycosyltransferase, the nucleotide sequence of described restructuring apple phlorizin glycosyltransferase of encoding is as shown in SEQ.ID.NO.2.

To recombinate the separation purification method of apple phlorizin glycosyltransferase, comprise the following steps:

1) adopt CTAB method to extract Folium Mali pumilae total serum IgE, the cDNA obtained with reverse transcription is template, carries out pcr amplification by a pair primers F 01 with restriction enzyme site and R01, obtains goal gene band afterwards after testing, reclaims PCR primer;

2) by step 1) PCR primer that obtains is connected on pMD18-T carrier, and after adopting heat shock method transformed competence colibacillus cell, screening recon, obtains recombinant plasmid;

3) to recombinant plasmid double digestion qualification positive colony, order-checking, the gene order of restructuring apple phlorizin glycosyltransferase is obtained;

4) isopropyl-β-D-thiogalactoside(IPTG) abduction delivering is carried out to the thalline containing recombinant plasmid, heterogenous expression product is by saturated ammonium sulphate, DEAE-Sepharose anion-exchange chromatography and Sephadex-75 gel filtration chromatography purifying, again through enteropeptidase excision carrier tag albumen, obtain the restructuring phlorizin glycosyltransferase after purifying.

Step 1) described in the nucleotide sequence of primers F 01 as shown in SEQ.ID.NO.3, the nucleotide sequence of primer R01 is as shown in SEQ.ID.NO.4; It is BamH I and Hind III that enzyme cuts enzyme used.

Described competent cell is e. coli bl21 (DE3) competent cell.

The concrete operations of described heat shock method transformed competence colibacillus cell are:

1. being transferred to by recombinant plasmid is equipped with in the centrifuge tube of competent cell, mixes gently;

2. after ice bath 30min, at 42 DEG C of heat shock 90s, ice bath 3min;

3. the LB liquid nutrient medium of antibiotic-free is added, shaking culture 1h ~ 3h at 37 DEG C;

4. the bacterium liquid after recovery is centrifugal, Aspirate supernatant, resuspended to the bacterium liquid liquid-transfering gun piping and druming of staying bottom centrifuge tube;

5. with liquid-transfering gun resuspended bacterium liquid to be moved into Amp microbiotic and on the LB solid medium flat boards of 37 DEG C of insulations, with the spreader coating of sterilizing evenly;

6. flat-plate inverted is placed in 37 DEG C of thermostat containers and cultivates 12 ~ 16h.

Describedly the concrete operations of isopropyl-β-D-thiogalactoside(IPTG) abduction delivering carried out to the thalline containing recombinant plasmid be:

Single colony inoculation that picking contains recombinant plasmid is in in the antibiotic LB liquid medium of Amp, at 37 DEG C, under 200prm, shaking culture is spent the night, be inoculated into in the antibiotic fresh LB of Amp by the volume ratio of 1% again, at 37 DEG C, under 180rpm, shaking culture reaches 0.6 ~ 1.0 to OD600; Add isopropyl-β-D-thiogalactoside(IPTG), at 20 DEG C, induce 4h; Through collected by centrifugation thalline, thalline brine, then add Cell Buffer, carry out ultrasonication, then at 4 DEG C, centrifugal 10min under 12000rpm, obtains crude enzyme liquid I.

Described saturated ammonium sulphate concrete operations are:

Under ice bath, in crude enzyme liquid I, add (NH ₄) ₂sO ₄powder is to (NH ₄) ₂sO ₄whole mass concentration is 30%, centrifugal after stirring, and gets supernatant liquor, continues to add (NH ₄) ₂sO ₄powder is to (NH ₄) ₂sO ₄whole mass concentration is 70%, after stirring, 4 DEG C of standing 1h, then centrifuging and taking precipitation; Carry out the concentrated of enzyme liquid and desalination by after precipitation buffer solution with 30kDa ultra-filtration centrifuge tube, obtain crude enzyme liquid II.

The concrete operations of described DEAE-Sepharose anion-exchange chromatography are:

Crude enzyme liquid II is crossed the filter membrane of 0.22 μm, then AKTA protein purification system is adopted, linear elution is carried out with 1mL/min with NaCl, the glycosyltransferase enzyme detecting each elution peak is lived, the part having enzyme to live is carried out merging collect, proceed to dialysis tubing, concentrate at 4 DEG C with PEG20000, obtain enzyme liquid III.

The concrete operations of described Sephadex-75 gel filtration chromatography purifying are:

Enzyme liquid III is expelled to AKTA protein purification system, use Sephadex-75 gel column to carry out purifying, carry out wash-out with damping fluid with 1mL/min flow velocity, the glycosyltransferase enzyme detecting each elution peak is lived, merge the part of collecting and having enzyme to live, proceed to dialysis tubing PEG20000 and concentrate at 4 DEG C.

Compared with prior art, the present invention has following useful technique effect:

The invention discloses apple phlorizin glycosyltransferase gene sequence 1452 base pairs of cloning and obtaining, this genes encoding 483 amino acid.

One aspect of the present invention utilizes biotechnology, heterogenous expression apple phlorizin glycosyltransferase in e. coli bl21 (DE3), generates phlorizin for catalysis; Adopt the separation purification method such as saturated ammonium sulphate, negatively charged ion DEAE-Sepharose displacement chromatography and Sephadex-75 gel filtration chromatography on the other hand, have simple, the cycle is short, and repeatability is high, zymoprotein vigor advantages of higher.

The active optimum pH of the restructuring apple phlorizin glycosyltransferase obtained through the inventive method separation and purification is 8.5, and active optimum temperuture is 45 DEG C, when metal ion is when the final concentration of reaction system is 5mM, and Ca ²⁺and Mg ²⁺promoter action is had, Na to this restructuring apple phlorizin glycosyltransferase ⁺and K ⁺act on not obvious, Al ³⁺, Cu ²⁺, Mn ²⁺and Zn ²⁺there is significant restraining effect, Cu ²⁺restraining effect the strongest.

Accompanying drawing explanation

Fig. 1 is for adopting DEAE-Sepharose FF purification of Recombinant glycosyl transferase activity peak color atlas;

Fig. 2 is for adopting Superdex ^tM75 gel chromatography purification of Recombinant glycosyltransferase color atlass;

Fig. 3 is the restructuring apple phlorizin glycosyltransferase SDS-PAGE electrophorogram after purifying;

Fig. 4 is enzyme reaction product color atlas;

Fig. 5 is phlorizin mark product mass spectrums;

Fig. 6 is enzyme reaction product mass spectrum;

To be pH live on restructuring glycosyltransferase enzyme Fig. 7 affects result figure;

To be temperature live on restructuring glycosyltransferase enzyme Fig. 8 affects result figure;

To be metal ion live on restructuring glycosyltransferase enzyme Fig. 9 affects result figure.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.

Apple phlorizin glycosyltransferase gene clone technology scheme provided by the present invention is: adopt CTAB method (CTAB, hexadecyltrimethylammonium bromide, cetyl trimethylammonium bromide, be a kind of cationic detergent, there is the characteristic of precipitate nucleic acids and acidic polysaccharide from LISS.) extract Folium Mali pumilae total serum IgE, the cDNA obtained with reverse transcription is template, pcr amplification is carried out by a pair primers F 01 with restriction enzyme site and R01, detect with 1.0% agarose gel electrophoresis, extract goal gene band under ultraviolet light, adopt sepharose DNA to reclaim test kit and carry out PCR primer recovery, glue is reclaimed product and be connected to pMD18-T carrier, the bacillus coli DH 5 alpha of heat shock method transformed competence colibacillus, with blue hickie method screening recon, recombinant plasmid extracts and adopts SanPrep pillar plasmid DNA extraction agent box in a small amount, recombinant plasmid double digestion qualification positive colony, order-checking.Clone apple phlorizin glycosyltransferase gene sequence 1452 base pairs obtained, this genes encoding 483 amino acid.

The constructing technology scheme of expression vector provided by the present invention is: with BamH I and Hind III, the apple phlorizin glycosyltransferase gene obtained is carried out double digestion, connects with pET-32a (+) plasmid of T4 ligase enzyme with same BamH I and Hind III double digestion.

The technical scheme of the recombinant bacterial strain containing apple phlorizin glycosyltransferase gene provided by the invention is: be transformed in competent e. coli bl21 (DE3) by positive recombinant plasmid pET-32a-p2 ' the GT heat shock method carried after being transformed into DH5 α.

Restructuring phlorizin glycosyltransferase technology of preparing scheme provided by the invention is: the thalline containing recombinant plasmid is carried out isopropyl-β-D-thiogalactoside(IPTG) (IPTG) abduction delivering, heterogenous expression product is by saturated ammonium sulphate, DEAE-Sepharose anion-exchange chromatography and Sephadex-75 gel filtration chromatography purifying, adopt enteropeptidase (Enterokinase) to excise carrier tag albumen, obtain restructuring phlorizin glycosyltransferase.

1, gene amplification

Primer:

F01 (5'-ACG gGATCCaTG GGA GAC GTC ATT GTA CTG-3') (dashed part is BamH I restriction enzyme site);

R01 (5'-CCC aAGCTTtTA TGT AAT GCT ACT AAC AAA GTT G-3') (dashed part is Hind III restriction enzyme site).

The cDNA obtained with reverse transcription is template, carries out pcr amplification with F01 and R01 two primers, Takara Premix Taq test kit, and PCR reaction system is as table 1:

Table 1 PCR reaction system

Amplification program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 30s, and 53.1 DEG C of annealing 40s, 72 DEG C extend 1.5min, and 35 circulations, last 72 DEG C extend 10min again.After reaction terminates, PCR primer is stored in-20 DEG C.PCR primer 1.0% agarose gel electrophoresis detects, and reclaims object band.

2, PCR primer and pET-32a (+) plasmid double digestion

By identical endonuclease reaction system, as table 2:

Table 2 endonuclease reaction system

37 DEG C of water-bath 6h.Get 5 μ L digestion products 1.0% agarose gel electrophoresis after double digestion to detect, reclaim digestion products.

3, the connection of object fragment and expression vector pET-32a (+)

Reaction system is as table 3:

Table 3 ligation system

16 DEG C of connections of spending the night.

4, transform

Use CaCl ₂legal system is for BL21 (DE3) competent cell:

1. the e. coli bl21 (DE3) of picking purifying lines LB solid medium, 37 DEG C, is inverted overnight incubation;

2. picking e. coli bl21 (DE3) single bacterium colony is in 50mL liquid nutrient medium, and 37 DEG C of 180rpm shaking culture are 0.6 to OD600;

3. on average proceeded to by bacterium liquid in 50mL precooling sterile centrifugation tube, ice bath 30min, 4 DEG C, the centrifugal 5min of 6000rpm, abandons supernatant, with the thorough suck dry moisture of filter paper, and the precipitation 0.1MCaC1 of l0mL precooling ₂(filtration sterilization) suspends, and mixes gently, ice bath 30min with rifle head;

4. 4 DEG C, the centrifugal 5min of 6000rpm, abandons supernatant, and with the thorough suck dry moisture of filter paper, precipitation adds the 0.1MCaC1 of 1mL again ₂, resuspended gently, namely can be used for transforming; Or often pipe adds sterile glycerol to final concentration 20-30%, mixing, liquid nitrogen cold shock is placed on-80 DEG C of Refrigerator stores.

Heat shock method transforms:

1. plasmid 5 μ L is transferred in the 1.5mL centrifuge tube that 50 μ L competent cells are housed, mixes gently;

2. after ice bath 30min, 42 DEG C of heat shock 90s, ice bath 3min;

3. the LB liquid nutrient medium of 900 μ L antibiotic-frees is added, more than shaking culture 1h under being less than 180rpm at 37 DEG C;

4. under the bacterium liquid 12000rpm after recovery, the centrifugal 5min of normal temperature, Aspirate supernatant 700 μ L stays about 100 μ L bacterium liquid bottom EP pipe, and blows and beats resuspended gently with liquid-transfering gun;

5. resuspended bacterium liquid moved on the LB solid medium flat boards with 37 DEG C of insulations of corresponding microbiotic (Amp 100 μ g/mL) with liquid-transfering gun, with the spreader coating of sterilizing evenly;

6. flat-plate inverted is placed in 37 DEG C of thermostat containers and cultivates 12-16h.

5, protein induced expression

Picking contains single colony inoculation of recombinant plasmid in 10mL LB liquid medium (containing 100 μ g/mLAmp), 37 DEG C, 200rpm shaking culture is spent the night, next day is inoculated in fresh 10mLLB substratum (containing 100 μ g/mL Amp) in the ratio of 1% (v/v), 37 DEG C, 180rpm shaking culture reaches 0.6 ~ 1.0 to OD600.Add IPGT, make its final concentration 0.05-1.0mM, 20 DEG C of induction 4h, get 1m L bacterium liquid after induction, 4 DEG C, 12000rpm, centrifugal 2min, collect thalline, add 1m L0.9% brine cell, 4 DEG C, 5000rpm, centrifugal 10min, repeat to wash once, add 1mL Cell Buffer (0.05M potassium primary phosphate, 0.05M Sodium phosphate dibasic, 0.1M Repone K, 0.1% mercaptoethanol, pH7.5), ultrasonication, 130w, each 2min, interval 2min, altogether 6min.Bacterium liquid bleach after fragmentation is limpid, 4 DEG C, 12000rpm, and centrifugal 10min obtains crude enzyme liquid I.

6. enzyme purification

1. ammonium sulfate precipitation 10mL crude enzyme liquid I slowly adds (NH on ice bath ₄) ₂sO ₄powder is 30% to final concentration, slowly stirs 20min, the centrifugal 20min of 12000 × g.Get supernatant liquor, continue to add (NH ₄) ₂sO ₄be 70% to final concentration, slowly stir 20min, the centrifugal 20min of 4 DEG C of standing 1h, 12000 × g.Throw out a small amount of damping fluid (0.1mM Tris-HCl, pH 8.1,5mM dithiothreitol (DTT) (DTT), 5mMEDTA) dissolves.Carry out the concentrated of enzyme liquid and desalination with 30kDa ultra-filtration centrifuge tube, obtain crude enzyme liquid II.

2. 5mL crude enzyme liquid II is crossed 0.22 μm of filter membrane by DEAE anionresin, is expelled to AKTA protein purification system (Amersham Biosciences), carries out linear elution with 0 – 0.5M NaCl with 1mL/min.As shown in Figure 1, when elution volume is 40 ~ 54mL place, there is Peak Activity in wash-out result, the elution volume of peak point is 47mL.The glycosyltransferase enzyme detecting each elution peak is lived, and the part having enzyme to live is carried out merging and collects, proceed to dialysis tubing (molecular weight cut-off MWCO 0.8-1.4kDa) and concentrate at 4 DEG C with PEG20000.Obtain enzyme liquid III.

3. 1mL enzyme liquid III is expelled to AKTA protein purification system by gel-filtration, uses gel column to carry out purifying.Carry out wash-out wash-out result as shown in Figure 2 with damping fluid (0.02M Tris-HCl buffer, pH8.5) with 1mL/min flow velocity, when elution volume is 16.0-22.0mL place, occur Peak Activity, the elution volume of peak point is 19.0mL.The glycosyltransferase enzyme detecting each elution peak is lived, and merges the part of collecting and having enzyme to live, proceeds to dialysis tubing (molecular weight cut-off MWCO 0.8-1.4kDa) and concentrate at 4 DEG C with PEG20000 ,-20 DEG C of preservations.Through SDS-PAGE electrophoretic analysis, electrophorogram only occurs a protein band, and this enzyme apparent molecular weight is about 50kDa, and protein electrophoresis result as shown in Figure 3.

After this enzyme purification, the purification of target protein is 42.7 times, and Rate activity is 514.2U/mg, and yield is 10.5%.

7, HPLC-MS method qualification enzyme reaction product

Recombinase separation and purification obtained joins enzyme reaction system, and enzyme reaction system is as shown in table 4:

Table 4 enzyme reaction system

30 DEG C of reaction 1h, by 60 μ L glacial acetic acid termination reactions.Reaction solution after 0.45 μm of membrane filtration, with HPLC-MS detection assay reaction product phlorizin.By enzyme reaction product color atlas, as shown in Figure 4, in figure, UDPG is uridine diphosphoglucose; Phz is phlorizin; Pht is Phloretin, can find out, after adding two reaction substrate Phloretins and uridine diphosphoglucose (UDPG) and enzyme liquid, catalysis generates a kind of material, and its retention time is 6.486min.Through mass spectrum inspection, Fig. 5 is phlorizin mark product mass spectrums, as shown in Figure 6, and the m/z 435 [M-1] at peak 1 ^-1, with the mark product mass spectrum comparison shown in Fig. 5, determine that this material is phlorizin.

8, enzymatic property

1. pH is on the impact of enzyme activity

Respectively at pH4.0, the 50mmol/L Acetic acid-sodium acetate damping fluid of 4.6,5.0,5.6,6.0.6.5.7.0,50mmol/L potassium primary phosphate-dipotassium hydrogen phosphate the damping fluid of 7.5,8.0,8.5,9.0, enzyme activity is measured, to determine the optimum pH of glycosyltransferase in the 50mmol/LTris-HCl damping fluid of 9.5,10.0.

2. temperature affects enzyme activity

Respectively 25,30,35,40,45, at 50,60 DEG C, measure its enzyme activity.

3. metal ion is on the impact of enzyme activity

Metal ion Al is added in the reaction solution of enzyme ³⁺, Ca ²⁺, Mg ²⁺, Cu ²⁺, Mn ²⁺, Zn ²⁺, Na ⁺and K ⁺; The ultimate density of metal ion is 5mmol/L, measures its enzyme activity, not add the enzyme activity of metal ion for 100%.

Than enzyme (U/mg) alive definition, pH 7.5,30 DEG C, the every min of the every mg albumen enzyme amount generated needed for 1 μ g phlorizin is 1 Mei Huo unit.

As shown in figs. 7-9, its enzymatic property is optimal pH is 8.5, and optimum temperuture is 45 DEG C, when metal ion is when the final concentration of reaction system is 5mM, and Ca ²⁺and Mg ²⁺promoter action is had, Na to glycosyltransferase ⁺and K ⁺act on not obvious, Al ³⁺, Cu ²⁺, Mn ²⁺and Zn ²⁺there is significant restraining effect, Cu ²⁺restraining effect the strongest.

Claims (10)

1. a restructuring apple phlorizin glycosyltransferase, it is characterized in that, this glycosyltransferase has the aminoacid sequence as shown in SEQ.ID.NO.1.

2. one restructuring apple phlorizin glycosyltransferase according to claim 1, it is characterized in that, the nucleotide sequence of described restructuring apple phlorizin glycosyltransferase of encoding is as shown in SEQ.ID.NO.2.

3. to recombinate the separation purification method of apple phlorizin glycosyltransferase, it is characterized in that, comprise the following steps:

1) adopt CTAB method to extract Folium Mali pumilae total serum IgE, the cDNA obtained with reverse transcription is template, carries out pcr amplification by a pair primers F 01 with restriction enzyme site and R01, obtains goal gene band afterwards after testing, reclaims PCR primer;

2) by step 1) PCR primer that obtains is connected on pMD18-T carrier, and after adopting heat shock method transformed competence colibacillus cell, screening recon, obtains recombinant plasmid;

3) to recombinant plasmid double digestion qualification positive colony, order-checking, the gene order of restructuring apple phlorizin glycosyltransferase is obtained;

4) isopropyl-β-D-thiogalactoside(IPTG) abduction delivering is carried out to the thalline containing recombinant plasmid, heterogenous expression product is by saturated ammonium sulphate, DEAE-Sepharose anion-exchange chromatography and Sephadex-75 gel filtration chromatography purifying, again through enteropeptidase excision carrier tag albumen, obtain the restructuring phlorizin glycosyltransferase after purifying.

4. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 3, it is characterized in that, step 1) described in the nucleotide sequence of primers F 01 as shown in SEQ.ID.NO.3, the nucleotide sequence of primer R01 is as shown in SEQ.ID.NO.4; It is BamH I and Hind III that enzyme cuts enzyme used.

5. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 3, is characterized in that, described competent cell is e. coli bl21 (DE3) competent cell.

6. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 3, is characterized in that, the concrete operations of described heat shock method transformed competence colibacillus cell are:

1. being transferred to by recombinant plasmid is equipped with in the centrifuge tube of competent cell, mixes gently;

2. after ice bath 30min, at 42 DEG C of heat shock 90s, ice bath 3min;

3. the LB liquid nutrient medium of antibiotic-free is added, shaking culture 1h ~ 3h at 37 DEG C;

4. the bacterium liquid after recovery is centrifugal, Aspirate supernatant, resuspended to the bacterium liquid liquid-transfering gun piping and druming of staying bottom centrifuge tube;

5. with liquid-transfering gun resuspended bacterium liquid to be moved into Amp microbiotic and on the LB solid medium flat boards of 37 DEG C of insulations, with the spreader coating of sterilizing evenly;

6. flat-plate inverted is placed in 37 DEG C of thermostat containers and cultivates 12 ~ 16h.

7. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 3, is characterized in that, describedly carries out the concrete operations of isopropyl-β-D-thiogalactoside(IPTG) abduction delivering to the thalline containing recombinant plasmid and is:

Single colony inoculation that picking contains recombinant plasmid is in in the antibiotic LB liquid medium of Amp, at 37 DEG C, under 200prm, shaking culture is spent the night, be inoculated into in the antibiotic fresh LB of Amp by the volume ratio of 1% again, at 37 DEG C, under 180rpm, shaking culture reaches 0.6 ~ 1.0 to OD600; Add isopropyl-β-D-thiogalactoside(IPTG), at 20 DEG C, induce 4h; Through collected by centrifugation thalline, thalline brine, then add Cell Buffer, carry out ultrasonication, then at 4 DEG C, centrifugal 10min under 12000rpm, obtains crude enzyme liquid I.

8. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 7, it is characterized in that, described saturated ammonium sulphate concrete operations are:

Under ice bath, in crude enzyme liquid I, add (NH ₄) ₂sO ₄powder is to (NH ₄) ₂sO ₄whole mass concentration is 30%, centrifugal after stirring, and gets supernatant liquor, continues to add (NH ₄) ₂sO ₄powder is to (NH ₄) ₂sO ₄whole mass concentration is 70%, after stirring, 4 DEG C of standing 1h, then centrifuging and taking precipitation; Carry out the concentrated of enzyme liquid and desalination by after precipitation buffer solution with 30kDa ultra-filtration centrifuge tube, obtain crude enzyme liquid II.

9. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 8, it is characterized in that, the concrete operations of described DEAE-Sepharose anion-exchange chromatography are:

Crude enzyme liquid II is crossed the filter membrane of 0.22 μm, then AKTA protein purification system is adopted, linear elution is carried out with 1mL/min with NaCl, the glycosyltransferase enzyme detecting each elution peak is lived, the part having enzyme to live is carried out merging collect, proceed to dialysis tubing, concentrate at 4 DEG C with PEG20000, obtain enzyme liquid III.

10. the separation purification method of a kind of apple phlorizin glycosyltransferase of recombinating according to claim 9, is characterized in that, the concrete operations of described Sephadex-75 gel filtration chromatography purifying are:

Enzyme liquid III is expelled to AKTA protein purification system, use Sephadex-75 gel column to carry out purifying, carry out wash-out with damping fluid with 1mL/min flow velocity, the glycosyltransferase enzyme detecting each elution peak is lived, merge the part of collecting and having enzyme to live, proceed to dialysis tubing PEG20000 and concentrate at 4 DEG C.