CN109679974A - Marine microorganism arthrobacterium YJ34 produces dextranase genes and its recombination engineering - Google Patents

Abstract

The present invention be a kind of marine microorganism arthrobacterium (ArthrobacterSp.) YJ34 produces dextranase genes, and enzyme gene sequence 1923bp, using ATG as initiation codon, using TAG as terminator codon, GC base contents are 59.23%.The invention also discloses aforementioned dextranase genes clone and in the obtained dextranase genes recombination engineering of high efficient expression of Escherichia coli.The invention belongs to zymetologys and technical field of enzyme engineering to construct recombination engineering, inducing expression by gene cloning and sequencing, and the purifying of recombinase obtains the recombinase of purifying.Greatly increase enzyme activity and enzyme stability.Measuring the enzyme optimal reactive temperature by zymologic property is 55 DEG C, and most suitable action pH is 7.5.

Description

Marine microorganism arthrobacterium YJ34 produces dextranase genes and its recombination engineering

Technical field

The present invention relates to a kind of ocean arthrobacterium (ArthrobacterSp.) the dextranase genes that YJ34 is produced, with And marine microorganism arthrobacterium YJ34 produces dextranase genes clone and the obtained recombination work of high efficient expression in large intestine strain Journey bacterium, belongs to zymetology and technical field of enzyme engineering.

Background technique

Ocean arthrobacterium (ArthrobacterSp.) can be in cheap raw material such as corn flour, syrup, molasses, dregs of beans, It is grown on the industrial and agricultural wastes such as wheat bran, large-scale culture easy to accomplish, and there is dextran enzymatic activity.But due to the enzyme It is induced enzyme, after growth logarithmic phase enzyme activity reaches peak value, enzyme activity decline is very fast, and stability is poor, and thallus cannot long-time, repetition It utilizes, causes production cost high.To improve enzyme activity and stability, need to divide enzyme using molecular biosciences means Son transformation, constructs engineering bacteria, is used for large-scale production.

Summary of the invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of ocean arthrobacterium (Arthrobacter Sp.) YJ34 dextranase genes.

Another object of the present invention is logical gene cloning and to obtain ocean micro- in the method for E. coli Biological arthrobacterium YJ34 produces the recombination engineering of dextranase genes.

Ocean arthrobacterium according to the present invention (ArthrobacterSp.) YJ34 is from Lianyungang inshore sample What separation screening obtained, in Chinese Academy of Sciences's Organism Depositary, deposit number is CGMCC N0.7385 for culture presevation.

The present invention is achieved through the following technical solutions.The present invention is a kind of marine microorganism arthrobacterium (ArthrobacterSp.) YJ34 produces dextranase genes, its main feature is that: the dextranase genes sequence such as SEQID Shown in NO.1:

atgcccggaa cagggctggg ccgattggcc aaacgcatga cagcggccgc cgccgtcttc 60

cttatcagca gtggcgccgt cctcccggcg caggcagcca cgacagcggg gcacaccccc 120

tctacggcgc ctgctgcgcc taccgacaaa cacgccatta ccactgcgga caacggcaac 180

cttcacacct ggtggcatga caacgccgtt ttcaacacca ccggcccaac tggcaacgac 240

gaagtgcggc ggtcctcctt ctacgacctg caggtggccc aggagaacca gccggacaag 300

gcttatgacg ccttcactta catgagcatc ccccgcagcg gcaaggacaa gatcggctac 360

accaaggaag acggcgccga attctcctcc caggccggcc ttaccatgag ctggtccagc 420

ttcgaatatg ccaaagacgt ctgggtggac gtaagcctcc gcaccggcca gaccatcacc 480

tcagcggacc aggtgcagat ccgccccagc agctacaact tcgaaaagca gctcgttgac 540

gcggacaccg tcaaaatcaa agtgccctac tccgatgccg gctaccggtt ctcagtggaa 600

ttcgagccac agttgtatac ggcctacaac gacatgtccg gggacagcgg caagctgacc 660

accgaagccg agggcaaccg cgccatccac accgaacccc gcaattcaat gatgatcttc 720

gcggaaccta aactccgcgg agagcaaaag gaacgattgg ttcccacaga ggagtcgggc 780

agcatccatt accctgccga aggtgaggtg accaacctca atgccgttac cgaggaaatc 840

atctacttca agcccggcac ctacagcatg ggatcggact accacgcggt cctgccgccc 900

aacgtcaagt gggtctacct ggcaccgggc gcttacgtaa agggagcctt ccggttcctc 960

catgacaacc agagccagta caaggtcacc ggctacggtg tcctctccgg cgagcagtac 1020

gtgtacgagg cagacaccaa taacaactac aaccacctca gcggagcgtc caactgccac 1080

tcgtcgtgcg tgaagatgct gcagttcgct tccgccgatg ccgagcagaa gctggacctt 1140

cagggcgtta ctgtcgccga gcccccgtac cactcctttg tggtctacgg aaacgagcag 1200

accttccaca tgaacgttga gaactacaag caggtgggca gctggtactg gcagaccgac 1260

ggcatcgaac tgtacaaggg cagcaccatg aagaacacct tcttcaacgc gaacgacgac 1320

gtgctgaaga tgtaccacag cgacgtcacc atcgataaca ccgtgatttg gaagaacgag 1380

aacggacccg tgatccagtg gggctggacg ccccggaaca tcgacaacgt gaacgtcacc 1440

aacaccacgg tcatccacaa ccggatgtac tggaaggacg tcaagtacaa cacctgcatc 1500

ctgaactcct cgtcgcactg ggaagacatg ggttccacca ccaaggcgga tcccaacacc 1560

accgtgaaga acatgcgctt cgagaacatc acggtggagg gcatgaccaa ctgtgccatc 1620

cgcgtctatg ccctctccga taccgagaac atccacgtga aaaacctcaa catcgacgct 1680

tggaacgggc tggactggac ctcacaggtc agccacctca agcgctacac caaccctgcc 1740

ggcgaaaaag tcacaatcgg aaacgagatt cctgacggca acggactagc cctggagaac 1800

tactccgtgg gcggcgaagt gatcgaaaaa accgccgaca actgggccga ccaccagctg 1860

ggccgcatcg gcttcgacgg cgaaaactgg aacagctgga acgcctggcg gaccaccccg 1920

tag 1923。

The present invention also provides a kind of dextranase genes recombination engineerings, its main feature is that, the recombination engineering by with The upper dextranase genes, which clone and construct recombination bacillus coli, to be obtained.The cloning vector and expression vector of building are distinguished For pMD-18T-Dextranase and pET-21a (+)-Dextranase.Clone strain and expression bacterial strain be respectively DH5 α and BL21(DE3).Inducing expression is carried out by IPTG using Escherichia coli as host strain, with SDS-PAGE for identifying expression.Using Portugal Glycan 70,000 carries out enzyme assay substrate.Purify recombinase with NI affinity chromatography.

Invention described above dextranase genes recombination engineering, its main feature is that: specific clone and recombinant expression Method is as follows:

(1) strain culturing and extracting genome DNA: strain culturing takes the 100 μ l of ocean arthrobacterium YJ34 of refrigerator culture to access In triangular flask equipped with culture medium, 30 DEG C of culture 18-20h extract genomic DNA using bacterial genomes extracts kit, use Bis- generation of DNA sequencing technologies are scanned sequencing to the genomic DNA, obtain the dextranase genes as shown in SEQID NO.1 Sequence；

(2) dextranase genes recombinant bacterium constructs:

Using primer:

Primer F:5 '-GGGAATTCCATATGAAGCATTACCTCCGTCTA -3 '

Primer R:5 '-CCCAAGCTTCCACGCGTTCCAGTTATCCCA -3 ',

PCR condition: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 30 are followed Ring；PCR product gel extraction is connect with pMD-18T carrier, is transferred to E.coli DH5 α, picking single colonie, the acquisition gram of upgrading grain Grand carrier pMD-18T-Dextranase；NdeI and HindIII is respectively to pMD-18T-Dextranase and empty carrier pET-21a (+) carries out double digestion and after glue recycling overnight in 16 DEG C of connections E.coli DH5 α is transferred to, in the LB containing amicillin resistance Plate chooses positive restructuring bacterium, and PCR verifies positive restructuring bacterium；

(3) prepared by recombinant bacterium crude enzyme liquid: the recombinant bacterium of activation being taken to be inoculated in the LB liquid medium of 1L according to the ratio of 1:100 In, the IPTG of final concentration of 0.5mM is added in 20 DEG C of inductions overnight in 37 DEG C of cultures when OD600nm to 0.6-0.8；It will induction Supernatant is removed in overnight culture centrifugation, and thallus is suspended with the PBS of 40ml 50mM pH6.5, obtained thallus suspension liquid ultrasound 12000rpm is centrifuged 10min after broken, supernatant is transferred in new 50mL centrifuge tube, the crude enzyme liquid as obtained；

(4) recombinate enzyme purification: by 100ml crude enzyme liquid with 30ml NTA-Ni in conjunction with 4h, ceaselessly stir therebetween, in conjunction with after 4h divide 0mM, 10 mM, 20 mM, 30 mM, 40 mM, the 10mM pH7.0 Tris- of 50mM, 500mM imidazole salts are not contained with 100ml It elutes respectively for HCl times, eluent is recycled and surveys activity, the protein sample 10mM that 500mM imidazoles salting liquid is eluted PH7.0 Tris-HCl buffer is dialysed 2-3 days, while carrying out SDS-PAGE detection purification result；

(5) vigor of measurement recombination dextranase: by the recombination dextranase of purifying respectively in 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, at a temperature of 60 DEG C and 65 DEG C, in the buffer system of pH 6.0, measurement recombination dextranase Vigor detects the optimal reactive temperature of enzyme；Respectively pH be 4.0,5.0,6.0,7.0,7.5,8.0,8.5,9.0,10.0, 11.0 and 12.0 pH condition measures enzymatic activity, measures the most suitable action pH of recombinase.

Compared with existing technology, the present invention provides provide ocean arthrobacterium dextranase (Dextranase) base Cause, and by clone's ocean arthrobacterium dextranase (Dextranase) gene, recombination bacillus coli is constructed, realizes dextrose The high efficient expression of acid anhydride enzyme.Using ammonium sulfate fractional precipitation, anion exchange resin, gel filtration refines recombinase, obtains pure enzyme. Greatly increase enzyme activity and stability.Measuring the enzyme optimal reactive temperature by zymologic property is 55 DEG C, most suitable action pH It is 7.5.

Detailed description of the invention

Fig. 1 be arthrobacterium YJ34 (ArthrobacterSp.) genome dna electrophoresis figure；

Fig. 2 is dextranase genes PCR amplification result figure；

Fig. 3 is pET-21a (+)-Dextranase double digestion result figure；

Fig. 4 is recombinant bacterium inducing expression SDS-PAGE result figure；

Fig. 5 is that recombination dextranase purifies electrophoretogram；

Fig. 6 is influence diagram of the temperature pH to dextran enzyme effect；

Fig. 7 is influence diagram of the pH to dextran enzyme effect.

Specific embodiment

Referring to the drawings, a specific embodiment of the invention is further described, so that those skilled in the art are further Ground understands the present invention, without constituting the limitation to right of the present invention.

Embodiment 1, marine microorganism arthrobacterium (ArthrobacterSp.) YJ34 dextranase genes cloning experimentation:

1 ocean arthrobacterium Genome DNA extraction

Bacteria Culture

Activated spawn is inoculated in 5.0mL fluid nutrient medium (yeast powder 1.0, peptone 5.0, MgSO₄0.4, NaCl 4.0, dextrorotation Sugared acid anhydride 20,000 10.0,1L, pH7.5,121 DEG C of high pressure steam sterilization 30min of distilled water.) in, 37 DEG C, 220rpm, shaking table culture 12h obtains inoculum.

(2) microorganism collection

Take above-mentioned inoculum into Eppendorf pipe, room temperature 12000g is centrifuged 10min, abandons supernatant, and precipitating suspends again In 1mL TE (pH 8.0).

(3) cellular lysate

Lysozyme 6 the μ L, 37 DEG C of effect 2h of 50mg/mL is first added.Again plus 50 μ L, 10%SDS (dodecyl sulphur of 2M NaCl Sour sodium) 110 μ L, 20mg/mL Proteinase K 3 μ L, 50 DEG C of effect 3h crack thallus sufficiently, obtain cellular lysate liquid.

(4) it extracts

Cellular lysate liquid is gone in new Eppendorf pipe, adds isometric phenol: chloroform: isoamyl alcohol (25: 24: 1), after mixing It is placed at room temperature for 10min, 12000g is centrifuged 10min.Extracting is twice.

(5) it precipitates

It takes supernatant in new Eppendorf pipe, adds the isopropanol of 0.6 times of volume, be placed at room temperature for 10min after mixing, 12000g is centrifuged 10min, abandons supernatant.

(6) it washs

Precipitating is air-dried at room temperature after abandoning supernatant with ethanol washing 2 times of 75%.

(7) it dissolves

Precipitating is dissolved in 50 μ L TE buffers and (in 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 7.4), takes 2-5 μ L electric Swimming, remaining sample are used as pcr template.

Its integrality and purity, such as Fig. 1 are observed by 1% agarose gel electrophoresis by said extracted Genomic DNA solution, 1 is the genomic DNA for eliminating RNA in figure, it can be seen that extracted genome is complete, with high purity.

2 genomic DNAs are scanned and are compared

Known arthrobacterium YJ34 (ArthrobacterSp. dextranase) can be secreted, it is thus determined that it has dextran Enzyme gene, so the Arthrobacter gene sequence group DNA that we extract carries out genome-wide screening sequencing.Sequencing result is shown after comparing Show and obtain a new dextranase genes, overall length 1923bp, sequence is as shown in SEQID NO.1.

3 PCR amplifications

Design a pair of specific primer primer F:5 '-GGGAATTCCATATGAAGCATTACCTCCGTCTA -3 '； PrimerR:5 '-CCCAAGCTTCCACGCGTTCCAGTTATCCCA -3 '.1 μ L DNA is added in the centrifuge tube of 0.25mL, Each 1 μ L of primerF and R, the 5 μ L μ L pfu archaeal dna polymerases of 10 × buffer, 5 μ L dNTP (2mM), 0.5 add deionized water To 50 μ L, 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 30 are recycled.

The detection of 4 agarose gel electrophoresis

2 μ L PCR products are taken, is prepared in 1 × TAE and carries out electrophoresis, electrophoresis on 1% Ago-Gel added with appropriate GoldView Voltage 100V, electrophoresis time 30min use gel imaging system observation analysis result.

Using primers F F and FR, with YJ34(ArthrobacterSp.) strain gene group DNA is template, carries out PCR expansion Increase, as a result see that Fig. 2, Fig. 2 are PCR result: it is about 2kb that PCR, which clones ocean dextranase genes full length fragment size,.

5, the glue recycling of PCR product

All products of PCR are added in Ago-Gel loading hole, after 100V electrophoresis 30min, cut target in the UV lamp Band, with DNA Ago-Gel QIAquick Gel Extraction Kit recovery purifying object tape, recovery purifying operating process is following (to be said referring to kit Bright book).

(1) purpose band is scaled off from the Ago-Gel after electrophoresis, is put into clean centrifuge tube after weighing quality In;

(2) sol solutions of 3 times of volumes are added in centrifuge tube, 50 DEG C of heat preservation 10min must ensure that blob of viscose is completely dissolved.

(3) above-mentioned sol solutions are transferred in adsorption column, room temperature 12000g is centrifuged 30s, abandons waste liquid.

(4) 700 μ L of rinsing liquid is added in adsorption column, room temperature 12000g is centrifuged 30s, abandons waste liquid.

(5) 500 μ L rinsing liquids are added in adsorption column, room temperature 12000g is centrifuged 30s, abandons waste liquid, adsorption column 13000g centrifugation 2min abandons waste liquid.

(6) adsorption column is put into a clean centrifuge tube, 40 μ L of eluent, room temperature is added dropwise to adsorption column centre is hanging 2min is placed, 12000g is centrifuged 2min, and the solution in centrifuge tube is PCR glue recovery product.

6, cloning vector pMD-18T-Dextranase is constructed

(1) 5 μ L PCR products, 1 μ 10 × Taq of L buffer, 1 μ L dATP (2mM), 1 μ are added into the centrifuge tube of 0.25mL LTaq enzyme (5U/ μ L) adds water to the imitative extracting of phenol after 10 μ L, 70 DEG C of reaction 30min, ethanol precipitation, aseptic operating platform wind 15min Completely to ethyl alcohol volatilization, it is resuspended with 10 μ L sterile waters.

(2) 4 μ L of previous step reaction solution is taken, 5 μ L connection buffer are added, 1 μ L pMD-18T carrier connects overnight in 16 DEG C It connects.

(3) 5 μ L of connection product is taken, is added into the E.coli DH5 α competence of 50 μ L, ice bath 15min, 42 DEG C of thermal shocks 90sec, 2min, is added the fresh LB of 100 μ L on ice, and 37 DEG C of culture 30-60min take 100 μ L coating to contain ammonia benzyl The LB plate of penicillin (50ug/mL), 37 DEG C of culture 10h, picking single colonie access ampicillin of the 5mL containing 50ug/mL LB culture medium in 37 DEG C of culture 10h, take 2mL bacterium solution, upgrading grain, the pMD-18T- Dextranase of PCR verifying building, sieve Positive colony bacterium is selected to be sequenced.

(4) column are sequenced and submit ncbi database, and carry out bioinformatic analysis, obtain entire ocean dextran Enzyme gene overall length.Complete ORF is by 1923 base compositions, and using ATG as initiation codon, TAG is terminator codon, coding 640 amino acid, it is consistent with genome sequencing result.

Embodiment 2, marine microorganism arthrobacterium (ArthrobacterSp.) YJ34 dextranase genes are in large intestine bar Expression in bacterium:

1, construction of expression vector pET-21a (+)-Dextranase

Nde1 and EcoR1 carries out double digestion to pMD-18T-Dextranase and empty carrier pET-28a (+) respectively, after glue recycling Overnight in 16 DEG C of connections, conversionE.coliJM109 chooses positive recombinant in the LB plate of resistance containing kanamycin, extracts weight Group plasmid simultaneously carries out PCR and double digestion verifying, and positive colony, that is, pET-28a (+)-Dextranase(is as shown in Figure 3).

2, inducing expression

1 μ L pET-21a (+)-Dextranase is taken, E.coli BL21(DE3 is transferred to), take 100 μ L coating to contain ammonia benzyl mould The LB plate of plain (100ug/mL), 37 DEG C of culture 10h, picking single bacterium drop down onto 37 DEG C of culture 10h in the LB culture medium of 5mL, take 1mL culture solution into the LB culture medium of 100mL 37 DEG C culture to OD600 be 0.6 when be added to the IPTG of final concentration of 0.4mM, Thalline were collected by centrifugation after 20 DEG C of inducing expression 12h, carries out SDS-PAGE detection and enzyme assay (as shown in Figure 4).

3, enzyme purification is recombinated

(1) by 100ml crude enzyme liquid in conjunction with 30ml NTA-Ni 4h, ceaselessly stir therebetween, in conjunction with 4h;

(2) contain 0mM, 10 mM, 20 mM, 30 mM, 40 mM, the 10mM of 50mM, 500mM imidazole salts with 100ml respectively It elutes respectively for pH7.0 Tris-HCl times, collects 500mM elution fraction;

(3) the protein component 10mM pH7.0 Tris-HCl buffer dialysis 2-3 that 500mM imidazoles salting liquid is eluted It, while carrying out SDS-PAGE detection purification result (as shown in Figure 5).

4, zymologic property measures

(1) recombinate dextranase optimum temperature: 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C and At a temperature of 65 DEG C, in the buffer system of pH 6.0, measurement recombination dextranase vigor, as the result is shown 55 DEG C when enzymatic activity Highest (as shown in Figure 6).

(2) recombinate dextranase Optimun pH: respectively pH be 4.0,5.0,6.0,7.0,7.5,8.0,8.5, 9.0,10.0,11.0 and 12.0 pH condition measures enzymatic activity, and 40 DEG C are reacted, enzymatic activity when measurement result shows pH7.5 Highest, pH enzymatic activity within the scope of 7-10 is higher, as shown in Figure 7 more than the 65%(of most highly active).

Embodiment 3, the experiment of dextranase genes recombination engineering:

One, material and detection method

Ocean arthrobacterium (ArthrobacterSp.) YJ34, voluntarily separation screening obtains in this laboratory.

Enzyme activity determination method: by 100 μ L enzyme solutions be added to 100 μ L 3% dextran 70,000 (50 mmol/L, PH6.5PBS buffer solution is prepared), after 37 DEG C of 30 min of water-bath, 200 μ LDNS are added in reaction solution and terminate reaction, 3ml distilled water is added after boiling water bath 5min to be diluted, reads the light absorption value at 520nm after contact plate in microplate reader.

Arthrobacterium YJ34 in ocean is accessed in the triangular flask of 250mL equipped with 50mL culture medium, 30 DEG C of culture 18-20h are extremely Near OD600=1.0, genomic DNA is extracted using Hangzhou Bao Sai biotech firm bacterial genomes extracts kit, to genome DNA carries out genome scanning sequencing.

Two, dextranase genes recombinant bacterium constructs, using primer

Primer F:5 '-GGGAATTCCATATGAAGCATTACCTCCGTCTA -3 '

Primer R:5 '-CCCAAGCTTCCACGCGTTCCAGTTATCCCA -3 ',

PCR condition: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 30 are followed Ring.PCR product gel extraction is connect with pMD-18T carrier, is transferred to E.coli DH5 α, picking single colonie, the acquisition gram of upgrading grain Grand carrier pMD-18T-Dextranase.NdeI and HindIII is respectively to pMD-18T-Dextranase and empty carrier pET-21a (+) carries out double digestion and after glue recycling overnight in 16 DEG C of connections E.coli DH5 α is transferred to, in the LB containing amicillin resistance Plate chooses positive restructuring bacterium, and PCR verifies positive restructuring bacterium.

Three, prepared by recombinant bacterium crude enzyme liquid, and the recombinant bacterium of activation is taken to be inoculated in the LB Liquid Culture of 1L according to the ratio of 1:100 In base, the IPTG of final concentration of 0.5mM is added in 20 DEG C of inductions overnight in 37 DEG C of cultures when OD600nm to 0.6-0.8；It will lure Overnight culture centrifugation is led, supernatant is removed, thallus is suspended with the PBS of 40ml 50mM pH6.5, and obtained thallus suspension liquid is super 12000rpm is centrifuged 10min after sound is broken, supernatant is transferred in new 50mL centrifuge tube, the crude enzyme liquid as obtained.

Four, recombinate enzyme purification, by 100ml crude enzyme liquid in conjunction with 30ml NTA-Ni 4h, ceaselessly stir therebetween, in conjunction with 4h Contain 0mM, 10 mM, 20 mM, 30 mM, 40 mM, the 10mM pH7.0 of 50mM, 500mM imidazole salts with 100ml respectively afterwards It elutes respectively for Tris-HCl times, eluent is recycled and surveys activity, the protein sample that 500mM imidazoles salting liquid is eluted is used 10mM pH7.0 Tris-HCl buffer is dialysed 2-3 days, while carrying out SDS-PAGE detection purification result.

Five, by the recombination dextranase of purifying respectively in 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C and 65 DEG C at a temperature of, in the buffer system of pH 6.0, measurement recombination dextranase vigor, detect the optimal reaction of enzyme Temperature；Enzyme is measured in the pH condition that pH is 4.0,5.0,6.0,7.0,7.5,8.0,8.5,9.0,10.0,11.0 and 12.0 respectively Activity measures the most suitable action pH of recombinase.

Sequence table

<110>Huaihai Institute of Technology

<120>marine microorganism arthrobacterium YJ34 produces dextranase genes and its recombination engineering

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1923

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgcccggaa cagggctggg ccgattggcc aaacgcatga cagcggccgc cgccgtcttc 60

cttatcagca gtggcgccgt cctcccggcg caggcagcca cgacagcggg gcacaccccc 120

tctacggcgc ctgctgcgcc taccgacaaa cacgccatta ccactgcgga caacggcaac 180

cttcacacct ggtggcatga caacgccgtt ttcaacacca ccggcccaac tggcaacgac 240

gaagtgcggc ggtcctcctt ctacgacctg caggtggccc aggagaacca gccggacaag 300

gcttatgacg ccttcactta catgagcatc ccccgcagcg gcaaggacaa gatcggctac 360

accaaggaag acggcgccga attctcctcc caggccggcc ttaccatgag ctggtccagc 420

ttcgaatatg ccaaagacgt ctgggtggac gtaagcctcc gcaccggcca gaccatcacc 480

tcagcggacc aggtgcagat ccgccccagc agctacaact tcgaaaagca gctcgttgac 540

gcggacaccg tcaaaatcaa agtgccctac tccgatgccg gctaccggtt ctcagtggaa 600

ttcgagccac agttgtatac ggcctacaac gacatgtccg gggacagcgg caagctgacc 660

accgaagccg agggcaaccg cgccatccac accgaacccc gcaattcaat gatgatcttc 720

gcggaaccta aactccgcgg agagcaaaag gaacgattgg ttcccacaga ggagtcgggc 780

agcatccatt accctgccga aggtgaggtg accaacctca atgccgttac cgaggaaatc 840

atctacttca agcccggcac ctacagcatg ggatcggact accacgcggt cctgccgccc 900

aacgtcaagt gggtctacct ggcaccgggc gcttacgtaa agggagcctt ccggttcctc 960

catgacaacc agagccagta caaggtcacc ggctacggtg tcctctccgg cgagcagtac 1020

gtgtacgagg cagacaccaa taacaactac aaccacctca gcggagcgtc caactgccac 1080

tcgtcgtgcg tgaagatgct gcagttcgct tccgccgatg ccgagcagaa gctggacctt 1140

cagggcgtta ctgtcgccga gcccccgtac cactcctttg tggtctacgg aaacgagcag 1200

accttccaca tgaacgttga gaactacaag caggtgggca gctggtactg gcagaccgac 1260

ggcatcgaac tgtacaaggg cagcaccatg aagaacacct tcttcaacgc gaacgacgac 1320

gtgctgaaga tgtaccacag cgacgtcacc atcgataaca ccgtgatttg gaagaacgag 1380

aacggacccg tgatccagtg gggctggacg ccccggaaca tcgacaacgt gaacgtcacc 1440

aacaccacgg tcatccacaa ccggatgtac tggaaggacg tcaagtacaa cacctgcatc 1500

ctgaactcct cgtcgcactg ggaagacatg ggttccacca ccaaggcgga tcccaacacc 1560

accgtgaaga acatgcgctt cgagaacatc acggtggagg gcatgaccaa ctgtgccatc 1620

cgcgtctatg ccctctccga taccgagaac atccacgtga aaaacctcaa catcgacgct 1680

tggaacgggc tggactggac ctcacaggtc agccacctca agcgctacac caaccctgcc 1740

ggcgaaaaag tcacaatcgg aaacgagatt cctgacggca acggactagc cctggagaac 1800

tactccgtgg gcggcgaagt gatcgaaaaa accgccgaca actgggccga ccaccagctg 1860

ggccgcatcg gcttcgacgg cgaaaactgg aacagctgga acgcctggcg gaccaccccg 1920

tag 1923

<210> 2

<211> 32

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

gggaattcca tatgaagcat tacctccgtc ta 32

<210> 3

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cccaagcttc cacgcgttcc agttatccca 30

Claims (8)

1. a kind of marine microorganism arthrobacterium (ArthrobacterSp.) YJ34 produces dextranase genes, it is characterised in that: The dextranase genes sequence is as shown in SEQID NO.1.

2. a kind of dextranase genes recombination engineering, which is characterized in that the recombination engineering is by the right side described in claim 1 The sugared acid anhydride enzyme gene of rotation, which clones and constructs recombination bacillus coli, to be obtained.

3. dextranase genes recombination engineering according to claim 2, it is characterised in that: constructing cloning vector is PMD-18T- Dextranase and pET-21a (+)-Dextranase.

4. dextranase genes recombination engineering according to claim 2, it is characterised in that: clone strain and expression bacterium Strain is respectively DH5 α and BL21 (DE3).

5. dextranase genes recombination engineering according to claim 2, it is characterised in that: with Escherichia coli be host Bacterium carries out inducing expression by IPTG, with SDS-PAGE for identifying expression.

6. dextranase genes recombination engineering according to claim 2, it is characterised in that: using glucan 70,000 into Row enzyme assay substrate.

7. dextranase genes recombination engineering according to claim 2, it is characterised in that: pure with NI affinity chromatography Change recombinase.

8. dextranase genes recombination engineering according to claim 2, it is characterised in that: specific clone and recombination table It is as follows up to method:

(1) strain culturing and extracting genome DNA: strain culturing takes the 100 μ l of ocean arthrobacterium YJ34 of refrigerator culture to access In triangular flask equipped with culture medium, 30 DEG C of culture 18-20h extract genomic DNA using bacterial genomes extracts kit, use Bis- generation of DNA sequencing technologies are scanned sequencing to the genomic DNA, obtain the dextranase genes as shown in SEQID NO.1 Sequence；

(2) dextranase genes recombinant bacterium constructs:

Using primer:

Primer F:5 '-GGGAATTCCATATGAAGCATTACCTCCGTCTA -3 '

Primer R:5 '-CCCAAGCTTCCACGCGTTCCAGTTATCCCA -3 ',

PCR condition: 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 30 are followed Ring；PCR product gel extraction is connect with pMD-18T carrier, is transferred to E.coli DH5 α, picking single colonie, the acquisition gram of upgrading grain Grand carrier pMD-18T-Dextranase；NdeI and HindIII is respectively to pMD-18T-Dextranase and empty carrier pET-21a (+) carries out double digestion and after glue recycling overnight in 16 DEG C of connections E.coli DH5 α is transferred to, in the LB containing amicillin resistance Plate chooses positive restructuring bacterium, and PCR verifies positive restructuring bacterium；

(3) prepared by recombinant bacterium crude enzyme liquid: the recombinant bacterium of activation being taken to be inoculated in the LB liquid medium of 1L according to the ratio of 1:100 In, the IPTG of final concentration of 0.5mM is added in 20 DEG C of inductions overnight in 37 DEG C of cultures when OD600nm to 0.6-0.8；It will induction Supernatant is removed in overnight culture centrifugation, and thallus is suspended with the PBS of 40ml 50mM pH6.5, obtained thallus suspension liquid ultrasound 12000rpm is centrifuged 10min after broken, supernatant is transferred in new 50mL centrifuge tube, the crude enzyme liquid as obtained；

(4) recombinate enzyme purification: by 100ml crude enzyme liquid with 30ml NTA-Ni in conjunction with 4h, ceaselessly stir therebetween, in conjunction with after 4h divide 0mM, 10 mM, 20 mM, 30 mM, 40 mM, the 10mM pH7.0 Tris- of 50mM, 500mM imidazole salts are not contained with 100ml It elutes respectively for HCl times, eluent is recycled and surveys activity, the protein sample 10mM that 500mM imidazoles salting liquid is eluted PH7.0 Tris-HCl buffer is dialysed 2-3 days, while carrying out SDS-PAGE detection purification result；

(5) vigor of measurement recombination dextranase: by the recombination dextranase of purifying respectively in 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, at a temperature of 60 DEG C and 65 DEG C, in the buffer system of pH 6.0, measurement recombination dextranase Vigor detects the optimal reactive temperature of enzyme；Respectively pH be 4.0,5.0,6.0,7.0,7.5,8.0,8.5,9.0,10.0, 11.0 and 12.0 pH condition measures enzymatic activity, measures the most suitable action pH of recombinase.