CN109486782A

CN109486782A - A kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency

Info

Publication number: CN109486782A
Application number: CN201811415908.3A
Authority: CN
Inventors: 韩瑞枝; 姚栋; 倪晔; 肖静; 王克芬
Original assignee: Shandong Longkete Enzyme Preparation Co Ltd; Jiangnan University
Current assignee: Shandong Longkete Enzyme Preparation Co Ltd; Jiangnan University
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2019-03-19
Anticipated expiration: 2038-11-26
Also published as: CN109486782B

Abstract

The invention discloses a kind of molecular chaperones to co-express the method for improving sucrose phosphorylase expression efficiency, belongs to bioengineering and technical field of enzyme engineering.For the present invention by co-expressing recombinant plasmid pET-20b-SPase and pGro7, the molecular chaperone protein that pGro7 is expressed effectively reduces the formation of inclusion body, promotes the raising of SPase Soluble expression levels and vigor.By Optimal Expression condition and molecular chaperones coexpression system is used, the enzyme activity intracellular of SPase has reached 24.33U/mL, and specific enzyme activity has reached 6.58U/mg.

Description

A kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency

Technical field

The present invention relates to a kind of molecular chaperones to co-express the method for improving sucrose phosphorylase expression efficiency, belongs to biological work Journey and technical field of enzyme engineering.

Background technique

Sucrose phosphorylase (SPase, EC 2.4.1.7) belongs to GH13 family, is primarily present in Bifidobacterium In longum, Leuconostoc mesenteroides, Pseudomonas saccharophila.Because its wide in range substrate is special The opposite sex has a wide range of applications in the industries such as food, cosmetics and medicine, and main includes three aspects: (1) being to supply with sucrose Body, rhamnose, xylose, fructose and galactolipin are catalyzed as receptor to obtain corresponding oligosaccharide, which can a more Portugal Grape glycosyl group；(2) substance of certain unstable earnest can be catalyzed to synthesize its stable derivative, if sucrose phosphorylase is in sugarcane The 2-O- alpha-glucosyl of L-AA is catalyzed to synthesize 2-O- α-with high efficiency and fabulous site selectivity in sugar juice D-Glucose glycosides (AA-2G), the latter are the highly stable derivatives of vitamin C, are had in cosmetics, foods and drugs important Industrial application；(3) compound with phenolic hydroxyl group, carboxyl and alcoholic extract hydroxyl group is modified and is transformed, it is some with industrialization valence to synthesize The product of value, such as using sucrose as glycosyl donor, hydroquinone is glycosyl acceptor to catalyze and synthesize alpha-arbutin, and alpha-arbutin has Anti-oxidant, antimicrobial and anti-inflammatory activity is a kind of mild, safely effectively product, is widely used to medical treatment and makeup Conduct industry.

However, sucrose phosphorylase is in these bacterium due to there is complicated metabolic regulation mechanism in wild-type strain Content is not high.By natural bacterial strain fermenting and producing sucrose phosphorylase, yield and production efficiency are all relatively low, it is difficult to meet work The requirement of industry application.Recently, many researchers construct recombinant bacterial strain by using Principles of Gene Engineering to realize SPase Overexpression, but disadvantages such as that there are solubility expression effects is not good enough, and enzyme activity is low.2006, Jin-Ha Lee etc. People uses the SPase gene cloning derived from Leuconostoc mesenteroides NRRL B-742 at E.coli BL21 (DE3) On pLysS, producing enzyme reaches 1.8U/mL.2018, Wang, MM et al. were with the SPase derived from bifidobacterium adolescentis in withered grass gemma It is expressed in bacillus, in the case where signal peptide is not present, SPase is effectively secreted into extracellular medium.It is anti-in 3L biology It answers after cultivating recombinant bacterial strain in device, thick SPase yield and activity respectively reach 7.5g/L and 5.3U/mL, this is in current report SPase yield and active highest level.So far, do not occur molecular chaperones coexpression also and improve SPase expression efficiency Report.Therefore it provides a kind of method for improving sucrose phosphorylase expression efficiency, very heavy for the industrialized production of SPase It wants.

Summary of the invention

The first purpose of the invention is to provide a kind of methods for improving sucrose phosphorylase expression efficiency, are by sucrose phosphorus Phosphorylase and pGro7 are co-expressed in Escherichia coli.

In one embodiment of the invention, the gene order of encoding sucrose phosphorylase gene such as SEQ ID NO.2 It is shown.

In one embodiment of the invention, the carrier for expressing the sucrose phosphorylase includes pET-20b, described big Enterobacteria includes E. coli BL21.

In one embodiment of the invention, by the L- of final concentration of 0.5-1g/L of the Escherichia coli after co-expression The IPTG of arabinose and 0.05-0.1mM induce 8-20h.

A second object of the present invention is to provide a kind of genetic engineering bacteriums, have co-expressed shown in SEQ ID NO.1 Sucrose phosphorylase and molecular chaperones plasmid pGro7.

In one embodiment of the invention, the genetic engineering bacterium is with e. coli bl21 for host, with pET system Column plasmid is carrier, expresses saccharose phosphorylation enzyme gene shown in SEQ ID NO.2.

Third object of the present invention is to provide a kind of saccharose phosphorylation enzyme genes, which is characterized in that its nucleotide sequence As shown in SEQ ID NO.2.

Fourth object of the present invention is to provide above-mentioned method in the application of food, cosmetics or pharmaceutical field.

Fifth object of the present invention is to provide said gene engineering bacteria food, cosmetics or pharmaceutical field application.

Sixth object of the present invention is to provide said gene engineering bacterias in product of the preparation containing sucrose phosphorylase Application.

Beneficial effects of the present invention:

The present invention improves the table of sucrose phosphorylase (SPase, EC 2.4.1.7) using the means of molecular chaperones coexpression Up to efficiency, by co-expressing recombinant plasmid pET-20b-SPase and pGro7, the molecular chaperone protein for enabling pGro7 to express The formation of inclusion body is enough effectively reduced, the raising of SPase Soluble expression levels and vigor are promoted.By Optimal Expression condition and Using molecular chaperones coexpression system, the enzyme activity intracellular of SPase has reached 24.33U/mL, and compared to Wang, MM et al. is in withered grass SPase is expressed in bacillus, the recombinant sucrose phosphorylase enzyme activity that the present invention obtains improves 3.5 times.When inducer L- Ah The concentration for drawing uncle's sugar is 0.5g/L, and IPTG final concentration of 0.05mM, molecular chaperones pGro7 and pET-20b-SPase co-express 8h When, specific enzyme activity is up to 6.58U/mg.

Detailed description of the invention

Fig. 1: the drafting of glucose standard curve.

Fig. 2: it co-expresses pET-20b-SPase-pGro7 and only expresses the supernatant of bacteria solution and sink that pET-20b-SPase is obtained The SDS-PAGE in shallow lake is analyzed, M:Blue Plus II Protein Marker；1: after coexpression pET-20b-SPase-pGro7 Supernatant of bacteria solution part；2: bacterium solution sediment fraction after coexpression pET-20b-SPase-pGro7；3: only expressing pET-20b-SPase Supernatant of bacteria solution part afterwards；4: only expressing bacterium solution sediment fraction after pET-20b-SPase.

Specific embodiment

(1) protein purification steps:

A. required reagent solution:

In conjunction with liquid: 0.5mol/L NaCl, 20mmol/L imidazoles, 20mmol/L PBS, 1% glycerol, pH=7.4.

Eluent: 0.5mmol/L NaCl, 500mmol/L imidazoles, 20mmol/L PBS, 1% glycerol, pH=7.0.

Lysate: 0.5mmol/L NaCl, 20mmol/L PBS, 50mmol/L EDTA, pH=7.0.

NiSO₄: 100mmol/L NiSO₄。

B. it operates:

Regeneration: it selects 1mL Ni-NTA prepackage gravity column to be used to carry out protein purification, cleans column with 10mL combination liquid first Son is then rinsed with 10mL lysate, then is cleaned with 10mL lysate, is then washed with 10mL, clear with 10mL NiSO4 later Wash, after washed with 10mL, cleaned with 10mL combination liquid, finally cleaned with 20% ethyl alcohol again.

In conjunction with: it is first cleaned with 20% ethyl alcohol, then the cleaning of 20mL combination liquid, then by sample upper prop, is combined later with 10mL Liquid cleaning.

Elution: 10mL combination liquid cleans first, flows through liquid with the collection of EP pipe, then (respectively with the imidazoles of various concentration 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L, 300mmol/L) cleaning and with EP pipe collection liquid Body is to get the eluent for arriving various concentration imidazoles, and then 10mL eluent cleans, and 20% ethyl alcohol cleans later.

It saves: being stored in 20% ethyl alcohol.

Loading: taking 40 μ L to flow through liquid and eluent respectively, and 10 μ L SDS-PAGE albumen loading buffers are added thereto Liquid is heated after 100 DEG C of 5min and is centrifuged, and 5 μ L is then taken to carry out the analysis of SDS-PAGE protein electrophoresis.

Analysis: according to SDS-PAGE electrophoresis, the higher sample of protein concentration is subjected to concentrating and desalinating operation, is just obtained pure Enzyme solution.

SDS-PAGE electrophoretic analysis:

Each 80 μ L of sample is taken, 20 μ L mercaptoethanols are added, mixing is placed on 100 DEG C of heating 10min, 12000rmin^-1From 10 μ L loading of supernatant is taken after heart 5min；Loading deposition condition is respectively that 130V, 15min flatten band, and 200V, 45min separate egg It is white；After electrophoresis, takes out protein adhesive and be rinsed with water completely, be immersed in after coomassie brilliant blue staining liquid and be placed on Universal table Middle dyeing；Dyeing liquor is removed, be immersed in after protein adhesive is rinsed well in Coomassie brilliant blue destainer and is placed on Universal table Middle decoloration；The protein adhesive that decoloration is completed is put into gel imager, electrophoretogram protein band is observed.

(2) enzyme activity determination method:

DNS surveys reducing sugar method: containing 900 μ L PBS buffer solution (pH=6.5) in 1.5mL reaction system, 1.48M sucrose is molten 400 μ L, SPase solution of liquid 200 μ L, 55 DEG C of water-bath 10min.The reaction of boiling water bath heating termination, then measures its life with DNS At the amount of fructose.

The definition of enzyme activity: 55 DEG C, when pH 6.5, it is a vigor list that 1min resolvase sucrose hydrolysis, which generates 1 μm of ol fructose, Position (U).

The definition of specific enzyme activity: enzyme activity unit number possessed by Unit Weight (mg) protein.

The drafting of standard curve: if taking the graduated test tube of 7 braces, the concentration for being separately added into different volumes is 1mg/mL's Glucose standard, DNS reagent, distilled water.It is specific as shown in table 1.

The different test tube number component content tables of table 1

Each pipe is shaken up, 10min is heated in boiling water bath, is cooled to room temperature, distilled water is added to be settled to 10mL, top up and down of jumping a queue , it is control school zero point with No. 1 test tube, surveys absorbance in wavelength 540nm with spectrophotometer.With OD₅₄₀For ordinate, grape Sugared content is abscissa, draws standard curve, as shown in Figure 1.

The preparation of glucose standard: preparation weighs 70 DEG C of 100g drying to constant weight glucose, is placed in small beaker, first A small amount of water dissolution is added, then volumetric flask constant volume to 100mL, mixes.

The preparation of 3,5- dinitrosalicylic acids (DNS): 10.6g DNS and 19.8g NaOH are added to containing for certain volume In the sodium potassium tartrate tetrahydrate hydrothermal solutions of 306g, 7.6mL phenol and 8.3g sodium pyrosulfite are added, is placed in brown bottle and saves, It can be used after seven days.

Enzyme activity calculation formula: glucose content m is calculated according to glucose standard curve.Unit volume enzyme activity U/mL= m/180╳1000/10/V

Specific enzyme activity U/mg=unit volume enzyme activity/protein concentration

(3) culture medium

LB culture medium (g/L): peptone 10g/L, yeast powder 5g/L, NaCl 10g/L

TB culture medium (g/L): yeast powder 24g/L, peptone 12g/L, glycerol 4g/L, KH₂PO₄17mM, K₂H PO₄· 3H₂O72mM。

(4) thermal conversion process

The E.coli BL21 melted on ice is added in recombinant plasmid (30-100ng) or connection reaction product (10 μ L) (DE3) in competent cell, it is embedded in ice bath 25min on ice, is then immersed in 42 DEG C of water-baths and is immediately placed in ice bath after thermal shock 90s Cooling 15min, is added the LB liquid medium of nonreactive in gnotobasis, and 37 DEG C of constant temperature incubation 15min are placed on 37 DEG C of shaking tables Shake culture 1h.It is finished wait cultivate, takes appropriate culture solution to be coated on the LB solid plate with resistance, plate is inverted in 37 DEG C insulating box is incubated overnight 12-16h.

The construction method of 1 molecular chaperones coexpression system of embodiment

1. cloning saccharose phosphorylation enzyme gene

Using chemical complete synthesizing process synthesis of sucrose phosphorylase gene, nucleotide sequence is as shown in SEQ ID NO.2.

2. construction recombination plasmid pET-20b-SPase

First with NcoI and XhoI to pET-20b double digestion, after by the genetic fragment of synthesis be inserted into pET-20b NcoI and Between two restriction enzyme sites of XhoI.

3.CaCl₂Prepare competent escherichia coli cell and thermal conversion step

E.coli BL21 (DE3) glycerol stocks bacterial strain is crossed on the LB solid plate of nonreactive plate, plate is inverted 12h or so is cultivated, in 37 DEG C of insulating boxs to isolate single colonie.Picking neat in edge, the single bacterium being of moderate size fall within fresh In culture medium, cultivate in 37 DEG C, 180rpm shaking table to OD₆₀₀Stop culture when being 0.3, bacterium solution is placed in ice bath on ice 30min；The bacterium solution of pre-cooling is dispensed into 50mL centrifuge tube, 10min is centrifuged in 4 DEG C, 4000rpm, abandons supernatant；Into centrifuge tube About 10mL 0.1mol/L MgCl is added₂·CaCl₂(being pre-chilled on ice) sufficiently dissolves to thallus in soft rotation on ice, continues to mend Add 30mL MgCl₂·CaCl₂Solution, in 4 DEG C, 4000rpm is centrifuged 10min, abandons supernatant；It is primary to repeat step previous action； 0.5mL 0.1mol/L CaCl is added into the centrifuge tube being centrifuged₂(being pre-chilled on ice) and 30% glycerol of 0.5mL are (pre- on ice It is cold), after softly being mixed well with liquid-transfering gun, dispenses into 1.5mL EP pipe and (be pre-chilled on ice) with 100 μ L/ pipes, in -80 DEG C of refrigerators Middle preservation.

4. inducing expression purifying and the SDS-PAGE electrophoretic analysis of destination protein

Recombinant plasmid pET-20b-SPase (amicillin resistance) and molecular chaperones plasmid pGro7 (chlorampenicol resistant), It is added to thermal transition in E.coli BL21 (DE3) competent cell simultaneously by molar concentration 1:1.To grow list on plate When bacterium colony, bacterium colony PCR verifying is carried out using primer, only verification result is that the positive is only positive clone molecule.Positive gram of picking Grand single bacterium falls on 25mL and contains in the fresh LB of two kinds of resistances of Amp and Cm, 37 DEG C of shake culture 10h.By 1% (v/ V) inoculum concentration is transferred to containing in 40mL TB (40 μ L Amp/Cm) culture medium, is cultivated in 37 DEG C, 180rpm to OD₆₀₀For 0.8- 0.9, carry out optimum induction experiment.

(1) by recombinant bacterium culture to OD₆₀₀It is respectively for 0.8-0.9, the IPTG and concentration that final concentration 0.05mM is added The L-arabinose of 0.25g/L, 0.5g/L, 0.75g/L, 1.0g/L, after 18 DEG C of Fiber differentiation 20h, in 4 DEG C, 8000rpm from 15 minutes collection wet cells of the heart.Utilize 50mM K₂HPO₄-KH₂PO₄(pH=6.5) it is broken that ultrasonic wave is carried out after buffer suspension cell It is broken, and centrifugation and enzyme activity determination are carried out to broken liquid, as shown in table 2.The result shows that when arabinose concentrations are 0.5g/L, Inducing effect is preferable, and unit volume enzyme activity is up to 20.88U/mL.

The enzyme activity measured is expressed under the conditions of the different arabinose concentrations of table 2

(2) by recombinant bacterium culture to OD₆₀₀For 0.8-0.9, the L-arabinose and final concentration 0.05mM of 0.5g/L is added IPTG, 18 DEG C of Fiber differentiations, 4h, 8h, 12h, 16h, 20h are separately sampled, and it is wet thin to be centrifuged collection in 15 minutes in 4 DEG C, 8000rpm Born of the same parents.Utilize 50mM K₂HPO₄-KH₂PO₄(pH=6.5) carry out ultrasonic disruption after buffer suspension cell, and to broken liquid into Row centrifugation and enzyme activity determination, as shown in table 3.

Coexpression pET-20b-SPase-pGro7 and the supernatant of bacteria solution and precipitating that only expression pET-20b-SPase is obtained SDS-PAGE result is as shown in Fig. 2, co-express the band of the sediment fraction of pET-20b-SPase-pGro7 obviously than only expressing PET-20b-SPase's is brighter.

The enzyme activity measured is expressed under the conditions of the different induction times of table 3

Induction time (h)	Unit volume enzyme activity U/mL	Specific enzyme activity (U/mg)
			4	4.24	2.91
8	12.93	6.58
			12	12.31	5.96
16	24.33	5.56
			20	22.43	4.99

In conclusion MM et al. expresses SPase in bacillus subtilis compared to Wang, by pET-20b-SPase with Molecular chaperones pGro7, which is transferred in Escherichia coli, to be co-expressed, and the enzyme activity intracellular of SPase has reached 24.33U/mL, improves 3.5 times, Simultaneously when the concentration of inducer L-arabinose is 0.5g/L, when the final concentration of 0.05mM of IPTG, 8h is co-expressed, specific enzyme activity also reaches 6.58U/mg is arrived.If the enzyme activity intracellular of SPase is only 3.24U/mL and only in expression in escherichia coli pET-20b-SPase.

PET-20b-SPase is transferred in Escherichia coli with molecular chaperones PKJE7 or PG-TF2 and co-expresses by comparative example 1, Remaining condition is the same as embodiment 1

PET-20b-SPase and molecular chaperones PKJE7 or PG-TF2 are transferred in Escherichia coli after coexpression, by the weight of acquisition Group bacterium is cultivated to OD₆₀₀For 0.8-0.9, it is added the L-arabinose of 0.5g/L or the IPTG of tetracycline and final concentration 0.05mM, 18 DEG C Fiber differentiation samples after 16h, is centrifuged 15 minutes collection wet cells in 4 DEG C, 8000rpm.Utilize 50mM K₂HPO₄-KH₂PO₄ (pH=6.5) ultrasonic disruption is carried out after buffer suspension cell, and centrifugation and enzyme activity determination are carried out to broken liquid.Such as 4 institute of table Show, the enzyme activity intracellular of SPase is respectively 3.72U/mL, 2.36U/mL.

The recombinase enzyme activity that table 4 co-expresses pET-20b-SPase and molecular chaperones PKJE7 or PG-TF2

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

<110>Southern Yangtze University

Shandong Longke Enzyme Co., Ltd.

<120>a kind of method that molecular chaperones coexpression improves sucrose phosphorylase expression efficiency

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 488

<212> PRT

<213> Thermoanaerobacterium thermosaccharolyticum

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Met Ala Leu Lys Asn Lys Val Gln Leu Ile Thr Tyr Pro Asp Ser Leu

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Gly Gly Asn Leu Lys Thr Leu Asn Asp Val Leu Glu Lys Tyr Phe Ser

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Asp Val Phe Gly Gly Val His Ile Leu Pro Pro Phe Pro Ser Ser Gly

35 40 45

Asp Arg Gly Phe Ala Pro Ile Thr Tyr Ser Glu Ile Glu Pro Lys Phe

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Gly Thr Trp Tyr Asp Val Lys Lys Met Ala Asp Asn Phe Asp Ile Leu

65 70 75 80

Leu Asp Leu Met Val Asn His Val Ser Arg Arg Ser Ile Tyr Phe Gln

85 90 95

Asp Phe Leu Lys Lys Gly Arg Lys Ser Lys Tyr Ala Asp Met Phe Ile

100 105 110

Thr Leu Asp Lys Leu Trp Lys Asp Gly Lys Pro Val Lys Gly Asp Ile

115 120 125

Glu Lys Met Phe Leu Arg Arg Thr Leu Pro Tyr Ser Thr Phe Lys Ile

130 135 140

Glu Glu Thr Gly Glu Glu Glu Lys Val Trp Thr Thr Phe Gly Lys Thr

145 150 155 160

Asp Pro Ser Glu Gln Ile Asp Leu Asp Val Asn Ser His Leu Val Lys

165 170 175

Glu Phe Leu Leu Glu Val Phe Arg Thr Phe Ser Asn Phe Gly Val Asn

180 185 190

Ile Val Arg Leu Asp Ala Val Gly Tyr Val Ile Lys Lys Ile Gly Thr

195 200 205

Ser Cys Phe Phe Val Glu Pro Glu Ile Tyr Glu Phe Leu Asn Trp Ile

210 215 220

Lys Lys Gln Ala Val Ser Tyr Gly Ile Glu Leu Leu Leu Glu Val His

225 230 235 240

Ser Gln Phe Glu Ile Gln Tyr Lys Leu Ala Glu Arg Gly Phe Trp Ile

245 250 255

Tyr Asp Phe Ile Leu Pro Phe Thr Val Leu Tyr Thr Leu Ile Asn Lys

260 265 270

Ser Asn Glu Met Leu Tyr Asp Tyr Leu Lys Asn Arg Pro Met Asn Gln

275 280 285

Phe Thr Met Leu Asp Cys His Asp Gly Ile Pro Val Lys Pro Asp Leu

290 295 300

Asp Gly Leu Ile Asp Thr Lys Lys Ala Lys Glu Val Val Asp Ile Cys

305 310 315 320

Val Gln Arg Gly Ala Asn Leu Ser Leu Ile Tyr Gly Asp Lys Tyr Lys

325 330 335

Ser Glu Asp Gly Phe Asp Val His Gln Ile Gly Cys Thr Tyr Tyr Ser

340 345 350

Ala Leu Asn Cys Asp Asp Asp Ala Tyr Leu Ala Ala Arg Ala Ile Gln

355 360 365

Phe Phe Thr Pro Gly Ile Pro Gln Val Tyr Tyr Val Gly Leu Leu Ala

370 375 380

Gly Val Asn Asp Phe Glu Ala Val Lys Arg Thr Gly Glu Gly Arg Glu

385 390 395 400

Ile Asn Arg His Asn Tyr Gly Leu Lys Glu Ile Glu Glu Ser Val Gln

405 410 415

Lys Asn Val Val Gln Arg Leu Leu Lys Leu Ile Arg Phe Arg Asn Glu

420 425 430

Tyr Glu Ala Phe Asn Gly Glu Phe Met Val Gln Asp Cys Gln Lys Asp

435 440 445

Glu Ile Arg Leu Thr Trp Glu Lys Asp Asp Lys Arg Cys Ser Leu Phe

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Gly Glu Glu Val Lys Tyr Leu Val

485

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cagctgatca cctatccgga tagtctgggc ggtaatctga aaaccctgaa cgacgtcctg 180

gagaaatact tcagcgacgt tttcggcggc gttcatattt taccgccgtt tccgagttct 240

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gattttgaag cggttaaacg taccggcgaa ggtcgcgaaa ttaaccgtca caactacggc 1320

ctgaaagaga tcgaagagag cgtccagaaa aacgtcgtcc aacgtctgct gaaactgatt 1380

cgcttccgta acgagtacga ggcgtttaac ggcgaattca tggtccagga ctgccagaaa 1440

gacgaaatcc gtctgacctg ggagaaagac gataaacgct gcagcctgtt catcgacctg 1500

aaaacctaca aaaccaccat cgactacatc aacgagaacg gcgaagaggt caaatatctg 1560

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Claims

1. a kind of method for improving sucrose phosphorylase expression efficiency, which is characterized in that by sucrose phosphorylase and pGro7 big It is co-expressed in enterobacteria.

2. the method as described in claim 1, which is characterized in that encode the gene order such as SEQ ID of the sucrose phosphorylase Shown in NO.2.

3. method according to claim 1 or 2, which is characterized in that the carrier for expressing the sucrose phosphorylase includes pET- 20b, the Escherichia coli include E. coli BL21.

4. method a method according to any one of claims 1-3, which is characterized in that by the Escherichia coli after co-expression with final concentration of The L-arabinose of 0.5-1g/L and the IPTG of 0.05-0.1mM induce 8-20h.

5. a kind of genetic engineering bacterium, which is characterized in that express sucrose phosphorylase and molecular chaperones shown in SEQ ID NO.1 Plasmid pGro7.

6. genetic engineering bacterium according to claim 5, which is characterized in that be with e. coli bl21 for host, with pET system Column plasmid is carrier.

7. a kind of saccharose phosphorylation enzyme gene, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO.2.

8. any method of claim 1-4 is in the application of food, cosmetics or pharmaceutical field.

9. the genetic engineering bacterium of claim 5 or 6 is in the application of food, cosmetics or pharmaceutical field.

10. application of the genetic engineering bacterium of claim 5 or 6 in product of the preparation containing sucrose phosphorylase.