CN109321542A

CN109321542A - A kind of recombinant type dextransucrase and the preparation method and application thereof

Info

Publication number: CN109321542A
Application number: CN201811287674.9A
Authority: CN
Inventors: 鄢明辉; 刘振民; 游春苹; 罗力文
Original assignee: Bright Dairy and Food Co Ltd
Current assignee: Bright Dairy and Food Co Ltd
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2019-02-12

Abstract

The present invention provides a kind of recombinant type dextransucrase and the preparation method and application thereof, the recombinant type dextransucrase is synthesized by dextransucrase recombinant strains, and dextransucrase has the amino acid sequence as shown in SEQ ID No.1.The present invention provides a kind of completely new recombinant type dextransucrases, realize its expression by expanding glucansucrase enzyme coding gene from Leuconostoc mesenteroides CGMCC10064, and by constructing recombinant bacterium.A large amount of alpha-glucans can be obtained using gained recombination dextransucrase, which there is oxygen pressure to exclude effect, can promote growth of the oxygen sensitivity probiotics under aerobic conditions, there is important application value in probiotics and related industry.

Description

A kind of recombinant type dextransucrase and the preparation method and application thereof

Technical field

The invention belongs to microbial engineering fields, and in particular to a kind of recombinant type dextransucrase and its preparation side Method and application.

Background technique

Dextransucrase (glucansucrase, dextransucrase) is a kind of dextransucrase of GH70 family, De novo formation alpha-glucans can be catalyzed, free fructose is discharged using sucrose as substrate.Alpha-glucans have in food health care industry Wide application value.For example, dextran is used as dextran clinically because of its good characteristic.Because its product is being eaten Good application prospect in product and medical industry, dextransucrase and its encoding gene are constantly subjected to extensive concern.At present There are multiple glucansucrase enzyme coding genes to be cloned identification, the structure of the polysaccharide product catalyzed and synthesized is also parsed. Resulting polysaccharide product has a wide range of applications in food health care industry.

Dextransucrase and its encoding gene are primarily present in lactic acid bacteria, such as common streptococcus (Streptococcus), leukonid (Leuconostoc) etc..Leuconostoc mesenteroides subsp mesenteroides (Leuconostoc Mesenteroides subsp.mesenteroides) it is that edible strain catalogue (Ministry of Public Health's bulletin is included in by the Ministry of Public Health of China 2012 No. 8) strain, be also acknowledged as by the polysaccharide product in its source safe.Therefore, Leuconostoc mesenteroides, especially Be goldbeater's skin subspecies source dextransucrase field of food have major application prospect.

Summary of the invention

In order to solve the above-mentioned technical problems, the present invention provides a kind of recombinant type dextransucrase and preparation method thereof with Using present invention gained recombinant type glucansucrase enzyme activity is high, and gained alpha-glucans are easily isolated purifying, raw in probiotics There is important application value in production and relevant industries.

Specifically, on the one hand, the present invention provides a kind of recombinant type dextransucrase, the recombinant type glucan sugarcane Carbohydrase is synthesized by dextransucrase recombinant strains, and dextransucrase has the amino acid as shown in SEQ ID No.1 Sequence.

Specific preferred, the encoding gene of the dextransucrase has the nucleotides sequence as shown in SEQ ID No.2 Column.

Specific preferred, the encoding gene of the dextransucrase derives from Leuconostoc mesenteroides CGMCC10064.

Second aspect, the present invention also provides the preparation method of the recombinant type dextransucrase, this method includes Following steps:

(1) using Leuconostoc mesenteroides CGMCC10064 genomic DNA as template, SEQ ID NO.3, SEQ ID are used Primer shown in NO.4 carries out PCR amplification；

(2) amplified production, expression vector are subjected to digestion, digestion products are connected using ligase；

(3) recombinant vector of connection is transformed into host strain, obtains recombinant strains；

(4) cultivate recombinant strains, inducible protein expression, after purification to obtain the final product.

Wherein, in step (2), double digestion is carried out using BamHI restriction endonuclease and XhoI restriction endonuclease.Expression vector is plasmid pET-28a。

In step (3), host strain is Escherichia coli Rosetta bacterial strain.

In step (4), expressed using isopropyl-β-D-thiogalactoside induction recombinant protein.

The third aspect, the present invention also provides the recombinant type dextransucrases in production there is oxygen pressure to exclude effect The application in alpha-glucans answered.

And the recombinant type dextransucrase is preparing the application in growth of probiotics promotor

The invention has the benefit that

The present invention provides a kind of completely new recombinant type dextransucrase, by from Leuconostoc mesenteroides CGMCC10064 Middle amplification glucansucrase enzyme coding gene, and its expression is realized by constructing recombinant bacterium.Glucan sugarcane is recombinated using gained Carbohydrase can obtain a large amount of alpha-glucans, which there is oxygen pressure to exclude effect, and oxygen sensitivity probiotics can be promoted to exist Growth under aerobic conditions has important application value in probiotics and related industry.

Biomaterial preservation information:

Leuconostoc mesenteroides (Leuconostoc mesenteroides) BD3749 provided by the invention, in 2014 November 26 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation address: Beijing The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101, classification naming are as follows: goldbeater's skin is bright Beading bacterium (Leuconostoc mesenteroides), the deposit number of the bacterial strain are as follows: CGMCC No.10064.

Detailed description of the invention

Fig. 1 is the coomassie brilliant blue staining result figure after dextransucrase SDS-PAGE electrophoresis of the present invention；

Fig. 2 is the sugared synthetic reaction result figure in situ after dextransucrase SDS-PAGE electrophoresis of the present invention；

Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of present invention gained alpha-glucans；

Fig. 4 is that the dissolved oxygen of present invention gained alpha-glucans excludes effect result；

Fig. 5 is that present invention gained alpha-glucans promote the growth of lactobacillus plantarum ST-III and Bifidobacterium longum NCC2705 Into result.

Specific embodiment

Technical solution of the present invention is illustrated to be clearer, below in conjunction with specific embodiment to skill of the invention Art scheme is further elaborated:

In a specific embodiment, the present invention provides a kind of recombinant type dextransucrase, the recombinant types Dextransucrase is synthesized by dextransucrase recombinant strains, and dextransucrase has shown in SEQ ID NO.1 Amino acid sequence.The encoding gene of the dextransucrase has nucleotide sequence shown in SEQ ID NO.2.

The present invention passes through design specific primer: upstream primer shown in SEQ ID NO.3, shown in SEQ ID NO.4 Downstream primer, 1312 sequence of PCR amplification glucansucrase gene from Leuconostoc mesenteroides CGMCC10064 genomic DNA； Digestion amplified production, expression vector, then connect amplified production into expression vector；It is thin that the recombinant vector of connection is transformed into host The prokaryotic expression of glucansucrase gene can be realized by inducing by born of the same parents.

In another particular embodiment of the invention, the preparation method of recombinant type dextransucrase is additionally provided, it is specific to wrap Include following steps:

(1) using Leuconostoc mesenteroides CGMCC10064 genomic DNA as template, using nucleotide sequence respectively such as SEQ ID Primer sets shown in NO.3 (upstream primer), SEQ ID NO.4 (downstream primer) prepare PCR reaction system, to expand the Portugal Glycan sucrose enzyme coding gene；Reaction system is as follows:

(2) PCR amplification is carried out to the PCR reaction system that step (1) obtains, setting thermal circulation parameters are as follows:

(3) amplified production that step (2) is obtained, with gel extraction after 1% Ago-Gel progress electrophoresis.

(4) DNA fragmentation obtained in step (3) and pET-28a carrier are carried out using restriction endonuclease BamHI and XhoI is carried out Double digestion.Use Invitrogen restriction enzyme, 37 DEG C of water-bath 2h.Reaction system is as follows:

(5) to gel extraction after the digestion products progress 1% Ago-Gel progress electrophoresis in step (4), glue recycles institute Product is obtained after concentration mensuration for connecting reaction.

(6) the resulting segment of recycling in step (5) and carrier DNA are attached using T4DNA ligase, are connected at room temperature Take over night.Reaction system is as follows:

(7) 5 μ L of connection product is taken to be transformed into escherichia coli DH5a competent cell.Resistance sieve is carried out using kanamycins Choosing, gained transformant extract plasmid and carry out double digestion identification.

(8) the transformant sample presentation that the positive is accredited as in (7) is sequenced；2 μ L are taken to be transformed into Escherichia coli Rosetta sense simultaneously By state cell.

(9) it is screened with kanamycins, positive transformant carries out culture presevation and spreads cultivation.

(10) it spreads cultivation to positive transformant, it is to be grown to addition 0.1mM isopropyl-beta D-thio after logarithmic growth phase The expression of galactoside (IPTG) inducible protein.

(11) 16 DEG C of Fiber differentiations are centrifuged after overnight and collect thallus, carry out ultrasonication after being resuspended with lysate.

(12) sample after ultrasound is centrifuged, takes supernatant, using nickel column (Ni-NTA) affinitive layer purification, with The elution of 150mM imidazole buffer, gained are the glucansucrase transferase purified.

Detailed explanation is carried out to the present invention below with reference to embodiment and attached drawing.

Embodiment 1

A kind of preparation method of recombinant type dextransucrase, specifically includes the following steps:

(1) using Leuconostoc mesenteroides CGMCC10064 genomic DNA as template, using nucleotide sequence respectively such as SEQ ID Primer sets shown in NO.3 (upstream primer), SEQ ID NO.4 (downstream primer) prepare PCR reaction system, described with amplification Glucansucrase enzyme coding gene；Reaction system is as follows:

(8) the transformant sample presentation that the positive is accredited as in (7) is sequenced.

(9) it is sequenced using the universal primer on carrier, universal primer sequence such as SEQ ID NO.5, SEQ ID NO.6 It is shown.Sequencing result can be obtained the sequence of glucansucrase gene by splicing (shown in SEQ ID NO.2)；In glucan On the basis of saccharase gene sequence, amino acid sequence (the SEQ ID of dextransucrase can be obtained by codon translation Shown in NO.1), the taa of dextransucrase base gene order end is terminator codon.

Embodiment 2The recombinant expression of dextransucrase and the enzyme activity determination of recombinant type dextransucrase

Step (1)-(6) are the same as step (1)-(6) in embodiment 1；

(7) the recombinant plasmid pET-28a-1312 built is transformed into Escherichia coli Rosetta bacterial strain, gained transformant It spreads cultivation in LB culture medium containing kanamycin after identified.The formula of LB culture medium are as follows: tryptone 10g/L, ferment Female powder 5g/L, sodium chloride 10g/L.

(8) it is to be grown to logarithmic growth phase when, the isopropyl-β-D-thiogalactoside induction of final concentration 0.1mM is added Recombinant protein expression.16 DEG C of Fiber differentiations are centrifuged after overnight and collect thallus, and ultrasonication, gained sample are carried out after lysate is resuspended Product are added 5X albumen sample-loading buffer after high speed centrifugation removes cell fragment and mix, and SDS-PAGE points are carried out after 37 DEG C of incubation 2h Analysis.Using 8%SDS-PAGE electrophoresis, same sample does two parts in parallel.A copy of it coomassie brilliant blue staining, coloration result As shown in Figure 1, another does sugared synthetic reaction in situ, reaction result is as shown in Figure 2.

As shown in Figure 1, inducing expression goes out the protein band of 170kDa in Escherichia coli Rosetta；The sugar in situ of Fig. 2 closes Show that the band has the enzyme activity characteristic that polysaccharide is synthesized using sucrose as substrate at reaction.

Embodiment 3Purifying, vitality test and the enzyme activity reaction of recombinant type dextransucrase

The Escherichia coli Rosetta bacterial strain recombinated to embodiment 2 expands culture, the inducing expression in the system of 1L. Gained thallus is centrifuged after ultrasonication, and gained protein lysate is purified by Ni-NTA affinity chromatography.Finally with 2mL's 150mM imidazole buffer is eluted.Albumen is quantified through Bradford method, the concentration of gained recombinase is 2.8mg/ mL.Meanwhile enzyme activity determination is carried out to the recombinase of purifying by the burst size of fructose in enzymatic polysaccharide synthesis process.By enzyme Unit of activity U is defined as discharging enzyme amount corresponding to 1 μM of fructose per minute, then the opposite enzyme activity of gained recombinase is 270mU/ mg。

The external synthesis of alpha-glucans is carried out using the recombinase of purifying.Reaction system are as follows: sodium acetate 20mM, calcium chloride 0.05g/L, sucrose 200g/L, sodium azide 1g/L (are adjusted to pH 5.4)；The recombinase (5.0U/L) of purifying, 30 DEG C of items are added After being reacted under part 1 week be added Sevag reagent (n-butanol:chloroform=1:4, vol/vol) to remove isolating protein, 15000g is centrifuged 10min, takes supernatant, and the dehydrated alcohol of 3 times of volumes pre-cooling is added, and (4 DEG C) are stood overnight under refrigerated condition, 15000g is centrifuged 10min, collects sediment and after being dissolved in water, vacuum freeze drying up to alpha-glucans, gained alpha-glucans Amount is 53.2g/L.

The measurement of 4 alpha-glucans glycosidic bond ratio of embodiment

Alpha-glucans prepared by above-described embodiment 3 carry out the detection (as shown in Figure 3) of 1H-NMR, by comparing chromatogram On α-(1,6), α-(1,3), integral area at α-(Isosorbide-5-Nitrae) absorption peak determines that the molar ratio of glycosidic bond in alpha-glucans is closed System, and according to the relative molecular mass of molecular exclusion chromatography measurement alpha-glucans.Glycosidic bond α-in gained alpha-glucans (1, 6), (1,3) α-, the molar ratio of α-(Isosorbide-5-Nitrae) are 1:0.48:0.41, and the relative molecular mass of alpha-glucans is 1.0x10⁵~ 5.0x10⁶Da。

Embodiment 5Exclusion effect of the alpha-glucans to dissolved oxygen in aqueous solution

The resulting alpha-glucans of above-described embodiment 3 are dissolved in sterile distilled water by final concentration 0,0.5,1,2,4,8g/L In, stirring to the alpha-glucans aqueous solution for obtaining series of concentrations gradient after completely dissolution.After standing 2h at room temperature, dissolution is used Oxygen electrode is measured the dissolved oxygen content in alpha-glucans aqueous solution, obtains the dissolution in various concentration alpha-glucans solution Oxygen content draws the relation curve between dissolved oxygen content (mg/L) and alpha-glucans concentration according to measurement result, as a result as attached Shown in Fig. 4.From fig. 4, it can be seen that the dissolved oxygen content in solution is gradually reduced with the increase of alpha-glucans concentration, It can thus be seen that there is alpha-glucans good dissolved oxygen to exclude effect.

Embodiment 6Growth promoting function of the gained alpha-glucans to oxygen sensitivity probiotics

With lactobacillus plantarum (Lb.plantarum) ST-III and bifidobacterium longum (B.longum subsp.longum) NCC2705 is tested bacterium, and test promotees the growth of oxygen sensitivity probiotics using alpha-glucans obtained by recombination dextransucrase Into effect.

(1) actication of culture is carried out first, and the Lb.plantarum ST-III strain of freezen protective is inoculated in MRS liquid In culture medium, 18-22h, 2-3 strain activated of such secondary culture are cultivated at 37 DEG C；The MRS Liquid Culture Base composition is as follows: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g Trisodium citrate, 1g Tween 80,200mg magnesium sulfate, 54mg manganese sulfate and 1000mL distilled water adjust pH to 6.5, and 121 DEG C go out Bacterium 15min.

The B.longum NCC2705 strain of freezen protective is inoculated in TPY fluid nutrient medium, cultivates 18- at 37 DEG C 22h, 2-3 strain activated of such secondary culture；The TPY Liquid Culture based formulas: caseinhydrolysate 10g, plant Peptone 5g, yeast powder 2g, glucose 5g, dipotassium hydrogen phosphate (K₂HPO₄·7H₂O) 2g, magnesium chloride (MgCl₂·6H₂O) 0.5g, sulfuric acid Zinc (ZnSO₄·7H₂O) 0.25g, calcium chloride (CaCl₂) 0.15g, iron chloride (FeCl₃) 0.1mg, cysteine-hydrochloric acid 0.5g be molten Solution adjusts pH to 6.5,121 DEG C of sterilizing 15min after 1mL Tween-80 is added in 1000mL distilled water.

(2) culture obtained to step (1) is dispensed after distilled water cleaning three times by the way that thalline were collected by centrifugation, Dispense the freeze-dried rear preservation of later bacterial sediment.A small amount of sample is taken to carry out cytometer by the method that plate pours into before centrifugation Number (inoculation quantity and ratio that count results are used to adjust two kinds of fermenting microbes).

(3) alpha-glucans through isolating and purifying are tested in TYC synthetic media and skimmed milk respectively for probiotics Growth-promoting effect.

TYC synthetic media (CDM) (tryptone 1.5%, yeast extract 0.5%, sucrose 5%, Na₂HPO₄· 12H₂O 0.2%, sodium acetate 2%, sodium chloride 0.1%, sodium bicarbonate 0.2%, sodium sulphate 0.01%, l-cysteine 0.02%, The percentage is the preparation of mass percent (Wade et al., 1986): by tryptone 15g, yeast extract 5g, sugarcane Sugared 50g, Na₂HPO₄·12H₂O2g, sodium acetate 20g, sodium chloride 1g, sodium bicarbonate 2g, sodium sulphate 0.1g, l-cysteine 0.2g with 1L distilled water mixes, and after completely dissolution, sterilizes up to required TYC synthetic media.

Skimmed milk (SKM): pressing whole-fat milk powder or skimmed milk powder 10%, sucrose 5%, is transferred to 100ml with distilled water and prepares again Former cream, or final concentration of 5% sucrose is added in fresh cow milk and is cooled to 40- after completely dissolution in 95 DEG C of sterilizing 5-10min 45 DEG C stand-by.

(4) the alpha-glucans product of acquisition is separately added into after 0.22 μm of membrane filtration degerming with the final concentration of 5g/L Into above-mentioned TYC culture medium and skimmed milk (while the control group that alpha-glucans EPS is not added is set).

(5) take the seed liquor prepared in step (1) respectively by the inoculum concentration of 5% (v/v) be inoculated into above-mentioned TYC culture medium and In skimmed milk (group containing alpha-glucans and control group), 30 DEG C of culture 48h, the 180rpm shaken cultivation in TYC synthetic media, It samples after stationary culture in skimmed milk, is counted after gradient dilution with colony counting method.As a result as shown in Fig. 5.

From fig. 5, it can be seen that alpha-glucans can significantly reduce the dissolved oxygen content in solution, deoxygenation pressure is effectively solved, To promote growth of the oxygen sensitivity probiotics under aerobic conditions.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modification, equivalent replacement and simple modifications etc., should all be included in the protection scope of the present invention in content.

Sequence table

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acagataata acggtagtaa aactgatgcc aatggtgtag gaaatcctac tgatatcagc 300

aatattccta ctgattcagt taaaattaat gctaatggtc agacaacaac agtcattaac 360

ccaacaacag ctactaatag cggtagtacg gatgataaaa caacagctgt tgatcaaact 420

acaaatgaca ccaaaaatag tacaacaatt ataattacaa caccagaggc gaaagcggaa 480

acatccaagt taaaactagt aggtggagta gatggttact atgatagtga taaaaatggt 540

aattatcaat ttaagttgta taaaaataat gaccatatca aaagcaatga cgacaagcca 600

gttactggtc ttcaaaccat tgattatttt gtacagtatt ttgatactga tggtactcag 660

gttaaaggcg catatcgaac tgtcgatggg gacaaatatt attttgcctc agggtcagga 720

aacgcgttga aacaagcaac tgttgttcct aacgaaaatg gaacgggtac taaattagta 780

gggtttgaca gcactggtaa ggttgttaag aatggtttct cttcggataa tgaaggaaat 840

acatactatt ttgatgagaa tggtaatttt gtaactgggt ggcgtacagt tgatgggcaa 900

cagtactatt ttgaagataa tggtgcttta gttaaaagtg gacagaaaac tgttggcgat 960

aatgtttatt attttgatag ttcagatgga catgcgattg ttggaaataa gaatcaatat 1020

acagaaggac taactagtca aaatgatgac tttacagaac acaatgcaat tataaataat 1080

gatagcaaat caattgataa tgttaatggt tacataacag ctgcctcgtg gtaccgacca 1140

aaggatatat tagaaaacgg tcaaacgtgg gtttcttcaa cagaaacaga tcagcgccca 1200

ttgttgatga catggtggcc agataaaaat accgaagctg attatgtaaa ttttatggct 1260

aaaagaattg ctacggtaaa tagtgacggt aaaatatatt ctgctctaga ttcacaagaa 1320

gtgcttaata aacaagcgga agctattcaa atagaaattg aaaaatatat cagtcaaaac 1380

gcaacaaatt atactttaga attaaaaaaa ttgtttggtg actttattgc aacacaaccg 1440

aattggaata ttactagtga agacgttagc actgatcact ttcaaggtgg ggccttggtt 1500

tatgctaata gtacaatgac gccatgggct aattctgatt atcgcttttt gaatcgcaca 1560

ccgacaaatc aaaaagggga tgtgtatgga gataagggcg gttatgaact attattagcc 1620

aatgatgtag ataattctaa tccagttgta caagcagaac agttaaactg gctctattac 1680

ttgacacatt ttggcgaaat taccgcaaat gattctgatg ctaactttga tagtatccga 1740

attgatgctg ttgataacgt tgatgccgac ttgctacaaa ttgctgcaga ttacatgaag 1800

agtgcctatg gtgttggaga taacgatgct ataacaaata aacatttatc tattttagaa 1860

gattggtctg ataatgattt taagtatgtc agtgaaaatg gtaataatca attaactcta 1920

gataaaaaag tacaagataa tctattaaac gtattgacca agtttcctac taatcgtcaa 1980

aatatggaaa ctattattaa caatagaaat gtcgatcgta agaatgacga tggtagccaa 2040

actgtgacac caaactattc ttttgttcgc gcacacgata gtgaagtaca aaccgttatt 2100

gcccgaatca ttcaagataa gtttccggat tcaggtagtg gattaattca aacaacagat 2160

gagataaata aagcatttga aatttataat gcggatcaat tattagcaga caaacattat 2220

actcattaca atattccatc tgcttatgca ctattactta cgaataaaga tactatacca 2280

cgtgtttatt atggtgattt atttaccgat aatggtgatt atatggccaa tactacccct 2340

tactacaagg ctattgatgc gctgctaaaa ttacgtgtta agtatgtttc tggttcacaa 2400

acatcagagg ttgataaaaa tcatacggac gtgttaacta gtgttcgaat gggcaacaat 2460

acaactgaag gtgttggtat cattgtaagc aataatccgg atttgaattt aggtaatgat 2520

gaaattactc tgaatatggg tcaagcgcat gctaatcaaa catatcgtgc agcattgttg 2580

actacagata ccggcataac tgtatataat tcagatgaag gtgctcccat agcaacaact 2640

aattccaaag gacaattaat ttttagtgcc aaagaaatca acaaccaaag agacactaat 2700

attagaggag ttggtaaaga caataatcaa gtatcaggtt atctagcagt ttgggtaccg 2760

gtaggtgctg atactaatca caatgctaca acccaggcat tgactgaaac cacgactgat 2820

ggaaaaacat tgcacagtaa tgctgcctta gattctaacc ttatctacga aggattttct 2880

aattatcaga caataccgga tggttcagat ccagaaaagt atactaatgc tattattgca 2940

aagaatgtac aaacttttaa ggattggggt gttacaagct ttcagcttgc accacaatat 3000

cgctccagtg aaggaacatc atttttagat gaaacactga aaaatggata tgcttttact 3060

gatcgctatg atttaggatt tgataataat ccaacaaagt atggtacagc tgaacaatta 3120

agagcagcta ttaaggcatt gcattctgta ggaatacaag ccatggccga ctttgttcct 3180

gaccaaatat acacattacc aggagaagaa gcagttacag caactcgaac aaacaatcag 3240

ggtgtatata aacaagattc agattttaat aatcttgttt atattgctaa aacaaaaagt 3300

agcggacagg attatcaatc taagtatggt ggacagtatt tggacttatt aaaagataag 3360

tatagtaatt tgtttgagaa taaacaattg tctacaggtg aatctattga tcctagtgaa 3420

aaaatcacac aatggtcggc taaatacttc aatggaacaa atattcaagg tcgtggaaaa 3480

tattttgctt tgaaagactt tgcaagcaat cagtatttga cgattagtga tactagtacg 3540

tccgttttgc caaaacagtt aacaaatcaa aagacgcaaa cagggtttta taaagatgat 3600

aagggcattg gctattattc acttagtgga tatcaagcta aaaactcatt catccaagat 3660

gatcaaggga attggtatta tttcaataat gatggttaca tggtaacagg atatcaaaat 3720

attgataata aagattatta tttcttacaa aatggtgtca agttaaatgt caatggacta 3780

gttagtgaca atgatcaaac ttattatttc aaaaataatc agctacaaaa aggcagccaa 3840

acagttcagg ggataactta cttttttgat tctcaaacgg ggctgatgaa gaaagattat 3900

tttgacttta cagctgataa taaggtttat tattacggta ccgatggtgt tcgctacacg 3960

aataggtttt atagtaactg gggtaagatg tattactttg gtgaagacgg tgcgcgttat 4020

accaaccgtt tctacagtaa ttggggcaat atgtattact ttggtgaaga tggtgcgcgt 4080

tatactaatc gcttctatag taactggggc aatatgtatt actttggtga agatggtgcg 4140

cgttatacta accgcttcta tagtaattgg ggtaagttat attactttgg caatgatggt 4200

gcgcgctata caaatcaaag ctatagtaac tggggtaaaa cctattactt cggtaatgac 4260

ggtgcccgat taacaaacca atttagaagt gatagtcaag gtaatctgta ttattatggc 4320

aatgatggtg cgcgttatac taatcaattc tatagtaact ggggtaatac ctactacttt 4380

ggtgaaaatg gtgcacgtta cacgaataag ttctatagta actggggaca taaatattac 4440

tttgatgcca gtggtgtttt agtaaaaaat agagaaatca aagttgatgg tattgcgtat 4500

atagcaaatg ctgagggcgt tttgagtgag aaaaaacgct aa 4542

<210> 3

<211> 32

<212> DNA

<213>artificial sequence (unknown)

<400> 3

cgggatccaa gttgtataaa aataatgacc at 32

<210> 4

<211> 26

<212> DNA

<213>artificial sequence (unknown)

<400> 4

cgctcgaggc caatgccctt atcatc 26

<210> 5

<211> 17

<212> DNA

<213>artificial sequence (unknown)

<400> 5

taatacgact cactata 17

<210> 6

<211> 19

<212> DNA

<213>artificial sequence (unknown)

<400> 6

gctagttatt gctcagcgg 19

Claims

1. a kind of recombinant type dextransucrase, which is characterized in that the recombinant type dextransucrase is by dextransucrase Recombinant strains synthesis, dextransucrase have the amino acid sequence as shown in SEQ ID No.1.

2. recombinant type dextransucrase according to claim 1, which is characterized in that the coding of the dextransucrase Gene has the nucleotide sequence as shown in SEQ ID No.2.

3. recombinant type dextransucrase according to claim 2, which is characterized in that the coding of the dextransucrase Gene source is in Leuconostoc mesenteroides CGMCC10064.

4. the preparation method of recombinant type dextransucrase described in claim 1, which is characterized in that this method includes following step It is rapid:

(1) using Leuconostoc mesenteroides CGMCC10064 genomic DNA as template, SEQ ID NO.3, SEQ ID NO.4 institute are used The primer shown carries out PCR amplification；

5. the preparation method of recombinant type dextransucrase according to claim 4, which is characterized in that in step (2), make Double digestion is carried out with BamHI restriction endonuclease and XhoI restriction endonuclease.

6. the preparation method of recombinant type dextransucrase according to claim 4, which is characterized in that in step (2), table It is plasmid pET-28a up to carrier.

7. the preparation method of recombinant type dextransucrase according to claim 4, which is characterized in that in step (3), place Main bacterial strain is Escherichia coli Rosetta bacterial strain.

8. the preparation method of recombinant type dextransucrase according to claim 4, which is characterized in that in step (4), make With isopropyl-β-D-thiogalactoside induction recombinant protein expression.

9. recombinant type dextransucrase described in claim 1 is in the alpha-glucans that there is oxygen pressure to exclude effect for production Using.

10. recombinant type dextransucrase described in claim 1 is preparing the application in growth of probiotics promotor.