CN110499273A - Spore surface shows engineered strain and the application of glucose oxidase and catalase altogether - Google Patents

Abstract

The present invention relates to engineered strain and applications that spore surface shows glucose oxidase and catalase altogether, the bacillus subtilis engineering bacteria, the fusion segment cotX-god-SpyTag of preparation is converted into bacillus subtilis WB800n, obtain recombined bacillus subtilis WB800n-cotX-god-SpyTag, again by fusion segment P43-cat-SpyCatcher electrotransformation recombined bacillus subtilis WB800n-cotX-god-SpyTag competent cell to get；Experiment discovery is independent of each other to the glucose oxidase and catalase of SpyTag/SpyCatcher connection by the polypeptide that interacts, and the glucose oxidase of expression and the zymologic property of catalase have and substantially change, temperature, pH wide adaptation range and Heat-stable performance improve.

Description

Spore surface show altogether glucose oxidase and catalase engineered strain and Using

Technical field

The present invention relates to engineered strain and applications that spore surface shows glucose oxidase and catalase altogether, belong to Technical field of biotechnology.

Background technique

Glucose oxidase (GOD) is a kind of oxidoreducing enzyme, can be catalyzed β-D- glucose in specific manner under aerobic conditions Generate gluconic acid and hydrogen peroxide.GOD is distributed widely in animals and plants and microbial body.Due to microbial growth is fast, Source is wide, is the main source for producing GOD, and the main bacterial strain that produces is aspergillus niger and mould.The application field of GOD is constantly opened up at present Exhibition, domestic and international market demand sharply increase.Low output, enzyme activity are low, detection method it is complicated be GOD industrialization it is restricted because Element.

Catalase (CAT) is the enzyme that catalyzing hydrogen peroxide resolves into oxygen and water, is present in the peroxidase precursor of cell It is interior.Catalase is the marker enzyme of peroxisome, accounts for about the 40% of peroxisome enzyme total amount.Catalase is deposited It is in each tissue of all known animals, especially exists in liver with high concentration.Catalase is in food industry In be used to remove hydrogen peroxide in the milk for manufacturing cheese.Catalase is also used for food packaging, prevents from eating Object is oxidized.

Spore surface displaying is the technology that can express and show biologically active foreign protein in spore surface, It merges the gene of foreign protein with gemma capsid protein gene, so that foreign protein is anchored on spore surface. (Isticatoet al.2001)) for the first time realize foreign protein and bacillus subtilis spore capsid protein amalgamation and expression, It is widely paid close attention to from this spore surface display technique, bacillus subtilis spore surface display technologies apply most common original Because being because having following several advantages:

(1) bacillus subtilis is aerobic microbiological, and is typical gram-positive microorganism, belongs to GRAS points Class, nutritional requirement needed for growing are low；

(2) gemma is the self-assembling formation in mother cell；

(3) molecular chaperones in cytoplasm can suitably promote the correct folding of heterologous protein.

SpyTag and SpyCatche can form covalent bond by spontaneous reaction, generate stable molecular self-assembling body.Enzyme point Sub- self-assembly applies valence with important because of the catalysis characteristics with effective, in synthetic biology and field of nanometer technology Value.

Chinese patent literature CN105132450A (application number 201510571328.3) discloses a kind of bacillus subtilis The method of gemma capsid protein Cot surface display trehalose synthase.The invention is using Cot series gemma capsid protein as gemma The molecular vehicle of surface display foreign protein designs the serial primer of Cot molecular vehicle, can both have been replaced by double digestion point Subcarrier can change external source destination protein again, and trehalose synthase is showed in bacillus subtilis bud finally by gene technology Spore surface filters out the gemma capsid protein that can efficiently show trehalose synthase.

GOD and CAT is shown to total in withered grass by gemma capsid protein and SpyTag and SpyCatche interacting peptide The temperature of bacillus spore surface, GOD and CAT, pH wide adaptation range and Heat-stable performance are good, illustrate tempting industry Application prospect and commercial value.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides spore surfaces to show glucose oxidase and catalase altogether Engineered strain and application.The present invention is using SpyTag/SpyCatcher interaction protein to bacillus subtilis spore clothing Glutelin is as molecular vehicle, and building can be in gemma on the basis of integrative plasmid pDG1730 and episomal plasmids pHT01 Recombinant vector is transformed into withered by the carrier of surface display glucose oxidase and catalase by the method for electrotransformation twice In careless bacillus, to obtain a kind of to show the withered grass bud of glucose oxidase and catalase altogether in spore surface Spore bacillus, is found through experiments that, the zymologic property of the glucose oxidase and catalase that obtain by this way has bright It is aobvious to change.

Technical solution of the present invention is as follows:

Spore surface shows the bacillus subtilis engineering bacteria of glucose oxidase and catalase altogether, pass through to The cotX-god- of gemma capsid protein gene, glucose oxidase gene and SpyTag gene is inserted into pDG1730 carrier SpyTag genetic fragment constructs recombinant vector pDG1730-cotX-god-SpyTag；It is opened by being inserted into P43 into pHT01 plasmid The P43-cat-SpyCatcher genetic fragment of mover gene, catalase gene and SpyCatcher gene, building recombination Carrier pHT01-P43-cat-SpyCatcher, then conversion bacillus subtilis is made twice；

The cotX-god-SpyTag nucleotide sequencing is as shown in SEQ ID NO.3, the cat- SpyCatcher nucleotide sequencing is as shown in SEQ ID NO.4.

Preferred according to the present invention, the carrier is integrating vector pDG1730 and episomal vector pHT01.

It is preferred according to the present invention, the nucleotide sequence such as SEQ of the recombinant vector pDG1730-cotX-god-SpyTag Shown in ID No.1, the nucleotide sequence of the recombinant vector pHT01-P43-cat-SpyCatcher such as SEQ ID No.2 institute Show.

Above-mentioned bacillus subtilis engineering bacteria is preparing the application in gluconate.

Above-mentioned application, steps are as follows:

(i) will the activated culture of above-mentioned bacillus subtilis engineering bacteria, expand culture after, in 35~38 DEG C of fermentation 96h, from The heart takes gemma, and the glucose oxidase and catalase shown altogether in spore surface is made；

(ii) glucose oxidase and catalase that spore surface made from step (i) is shown altogether are added to grape In sugar juice, reaction solution is made；

(iii) by reaction solution made from step (ii) under the conditions of 30~70 DEG C, pH4.0~8.0, it is stirred to react 12~ For 24 hours, mixed solution is made；

(iv) metal aqueous slkali will be added in mixed solution made from step (iii) to pH is 10, and metal base mixing is made Liquid；

(v) immobilised enzymes in metal base mixed liquor made from step (iv) being separated, washing recycling rotates reaction solution, Crystallization, is dried to obtain gluconate.

Preferred according to the present invention, in the step (i), activation culture condition is 35~38 DEG C, 180~220rpm is cultivated 12~16h, activation medium are LB liquid medium, and component is as follows:

10g/L peptone, 5g/L yeast extract, 10g/L NaCl, pH 7.0.

It is preferred according to the present invention, in the step (i), expands condition of culture and cultivated for 35~38 DEG C, 180~220rpm 84~108h, expansion culture medium are TB culture medium, and component is as follows:

Glycerine 15mL/L, tryptone 12g/L, yeast extract 24g/L, MgCl₂2.5g/L, 17mM KH₂PO₄, 72mM K₂HPO₄。

It is preferred according to the present invention, in the step (i), centrifugal condition are as follows: 4 DEG C, be centrifuged under conditions of 8000rpm 10min。

Preferred according to the present invention, in the step (ii), the concentration of glucose solution is 20~200mM；Preferably 100mM。

Preferred according to the present invention, in the step (iii), reaction temperature is 40~50 DEG C, and reaction pH is 6.0~7.0, Stirring rate is 150~400rpm, and the reaction time is 4~8h；

It is further preferred that reaction temperature is 40 DEG C, reaction pH is 6.0, and stirring rate is 200~240rpm, when reaction Between be 6h.

Preferred according to the present invention, in the step (iv), the concentration of metal aqueous slkali is 50~500mM；It is further excellent Choosing, the concentration of the metal aqueous slkali is 200~400mM；

Preferably, in the step (iv), separation is using centrifugation.

Beneficial effect

1, the present invention is for the first time simultaneously using gemma capsid protein as molecular vehicle and interaction polypeptide to SpyTag/ Glucose oxidase and catalase are shown on bacillus subtilis spore surface by SpyCatcher altogether, and obtaining one kind can To show the genetic engineering bacterium of glucose oxidase and catalase altogether in spore surface, experiment discovery is more by interacting Peptide is independent of each other to the glucose oxidase and catalase of SpyTag/SpyCatcher connection, and the grape glycosyloxy expressed The zymologic property of change enzyme and catalase, which has, to be substantially change, and temperature, pH wide adaptation range and Heat-stable performance improve；

2, bacillus subtilis engineering bacterium expression system of the present invention has the characteristics that safety, endotoxin-free, is eating Product and medicinal industry field are by approval；

3, the spore surface that the present invention constructs shows the bacillus subtilis work of glucose oxidase and catalase altogether Journey bacterium, neither influence bacterial strain itself form gemma, while also improving the stability of glucose oxidase and catalase.

Detailed description of the invention

Fig. 1 is pDG1730-cotX-god-SpyTag vector construction schematic diagram；

Fig. 2 is pHT01-P43-cat-SpyCatcher vector construction schematic diagram；

Fig. 3 a is the agarose gel electrophoresis figure of gemma capsid protein cotX gene and glucose oxidase god gene；

Fig. 3 b is the agarose gel electrophoresis of P43 promoter, catalase cat gene and SpyCatcher protein gene Figure；

Fig. 4 is the agarose gel electrophoresis of fusion segment cotX-god-SpyTag and P43-cat-SpyCatcher Figure；

Fig. 5 is the optimum temperature curve graph for the glucose oxidase that spore surface is shown；

Fig. 6 is the glucose oxidase thermal stability curve graph that spore surface is shown；

Fig. 7 is the optimal pH curve graph for the glucose oxidase that spore surface is shown；

Fig. 8 is the glucose oxidase pH stability curve figure that spore surface is shown；

Fig. 9 is the optimum temperature curve graph for the catalase that spore surface is shown；

Figure 10 is the hydrogen peroxide enzyme heat stability curve graph that spore surface is shown；

Figure 11 is the optimal pH curve graph for the catalase that spore surface is shown；

Figure 12 is the catalase pH stability curve figure that spore surface is shown.

Specific embodiment

Technical solution of the present invention is further elaborated below with reference to embodiment, but institute's protection scope of the present invention is not limited to This.

Biological material source:

Bacillus subtilis WB800n matches biological Co., Ltd purchased from Hangzhou treasured；

Integrative plasmid pDG1730 and episomal plasmids pHT01 is purchased from You Bao Bioisystech Co., Ltd；

Aspergillus niger (Aspergillus niger ATCC 6275) is purchased from BioVector plasmid vector bacterium cell gene Collection；

Escherichia coli laboratory saves.

Embodiment 1: construction recombination plasmid

(1) clone obtains gemma capsid protein gene segment

Using bacillus subtilis WB800n genome as template, design primer carries out PCR amplification cotX gemma capsid protein Genetic fragment and P43 promoter genetic fragment.

The PCR primer of the CotX protein gene sequence is as follows:

CotX-F:5 '-aaaactggtctgatcggatccGATCAGACAAAGACGGAAAAACG-3 '

CotX-R:5 '-gtctgcatGAGGACAAGAGTGATAACTAGGATGG-3 '

The PCR reaction system is as follows:

Under 2 × Phanta Max Master Mix 25 μ L, 10 μm of ol/L upstream primer cotX-F 2.5 μ L, 10 μm of ol/L 2.5 μ L of primer cotC-X is swum, 2.5 μ L of template uses ddH₂O complements to 50 μ L；

Above-mentioned PCR reaction carries out according to the procedure below:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 15s, 30 recycle；72 DEG C of extensions 5min；4 DEG C of preservations.

The PCR primer of the P43 promoter gene sequence is as follows:

P43-F:5 '-caattaaaggaggaaggatccAGCTTCGTGCATGCAGGC-3 '

P43-R:5 '-gacgtgctcatAAGCTTCTGTTATTAATTCTTGTCTGTTC-3 '

The PCR reaction system is as follows:

Under 2 × Phanta Max Master Mix 25 μ L, 10 μm of ol/L upstream primer P43-F 2.5 μ L, 10 μm of ol/L 2.5 μ L of primer P43-R is swum, 2.5 μ L of template uses ddH₂O complements to 50 μ L；

Above-mentioned PCR reaction carries out according to the procedure below:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 15s, 30 recycle；72 DEG C of extensions 5min；4 DEG C of preservations.

(2) clone obtains glucose oxidase gene segment

Using aspergillus niger Aspergillus niger as the gene order of stencil design PCR amplification glucose oxidase god Primer carries out PCR amplification:

The PCR primer of the glucose oxidase god-SpyTag gene order is as follows:

god-SpyTag-F:5’-actcttgtcctcATGCAGACGCTTCTGGTTAGCT-3’

god-SpyTag-R:5’-ctgcaggaattcgataagcttTTACTTTGTTGGCTTGTATGCATCTACCATT ACT ATGTGTGCATGGTGGTGATGATGGTGC-3’

PCR reaction system is as follows:

2 × Phanta Max Master Mix 25 μ L, 10 μm of ol/L upstream primer god-SpyTag-F 2.5 μ L, 10 μ 2.5 μ L of mol/L downstream primer god-SpyTag-R, 2.5 μ L of template, uses ddH₂O complements to 50 μ L；

Above-mentioned PCR reaction carries out according to the procedure below:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 70 DEG C of annealing 15s, 72 DEG C of extension 70s, 30 recycle；72 DEG C of extensions 5min；4 DEG C of preservations.

It is short by 1% agarose gel electrophoresis analytic plate segment length after PCR, purpose item is cut according to clip size Band cuts glue using the raw work plastic recovery kit recycling in Shanghai.

(3) clone obtains catalase gene segment

Using E. coli as the primer of the gene order of stencil design PCR amplification catalase, PCR expansion is carried out Increase:

The PCR primer of the catalase cat gene order gene order is as follows:

cat-F:5’-cagaagcttATGAGCACGTCAGACGATATCCA-3’

cat-R:5’-gtgtgtcaaccatggccatTTTATCATCATCATCTTTATAATCCAGCAGGTCGAAACGG TCG-3’

PCR reaction system is as follows:

Under 2 × Phanta Max Master Mix 25 μ L, 10 μm of ol/L upstream primer cat-F 2.5 μ L, 10 μm of ol/L 2.5 μ L of primer cat-R is swum, 2.5 μ L of template uses ddH₂O complements to 50 μ L；

Above-mentioned PCR reaction carries out according to the procedure below:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 62 DEG C of annealing 15s, 72 DEG C of extension 80s, 30 recycle；72 DEG C of extensions 5min；4 DEG C of preservations.

It is short by 1% agarose gel electrophoresis analytic plate segment length after PCR, purpose item is cut according to clip size Band cuts glue using the raw work plastic recovery kit recycling in Shanghai.

(4) amplimer of the SpyCatcher protein gene sequence is as follows:

SpyCatcher-F:5’-aATGGCCATGGTTGACACACTTA-3’

SpyCatcher-R:5’-cccggggacgtcgactctagaTTAATCGATATGGGCATCTCCTT-3’

PCR reaction system is as follows:

2 × Phanta Max Master Mix 25 μ L, 10 μm of ol/L upstream primer SpyCatcher-F 2.5 μ L, 10 μ 2.5 μ L of mol/L downstream primer SpyCatcher-R, 2.5 μ L of template, uses ddH₂O complements to 50 μ L；

Above-mentioned PCR reaction carries out according to the procedure below:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 60 DEG C of annealing 15s, 72 DEG C of extension 15s, 30 recycle；72 DEG C of extensions 5min；4 DEG C of preservations.

It is short by 1% agarose gel electrophoresis analytic plate segment length after PCR, purpose item is cut according to clip size Band cuts glue using the raw work plastic recovery kit recycling in Shanghai.

(5) by glucose oxidase gene segment made from cotX segment made from step (1) and step (2), step (1) Catalase gene segment made from P43 promoter gene segment obtained, step (3) and step (4) are obtained SpyCatcher protein gene segment carries out over-lap PCR, and cotX-god-SpyTag and P43-cat-SpyCatcher is made；

The preparation of cotX-god-SpyTag fusion segment:

The first amplification system of over-lap PCR is 25 μ L；

4 μ L of glucose oxidase gene sequence god；4 μ L of cotC protein gene sequence；2×Phanta Max Master Mix12.5μL；ddH₂O 4.5μL；

The first amplification program of the over-lap PCR is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 70 DEG C of annealing 15s, 72 DEG C of extension 90s, 5 recycle；72 DEG C of extensions 5min；

The supplement system of the over-lap PCR is 25 μ L:

2 μ L of upstream primer (cotX-F), 2 μ L, 2 × Phanta Max Master of downstream primer (god-SpyTag-R) Mix12.5 μ L, ddH₂O 8.5μL；

The over-lap PCR supplement amplification program is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 70 DEG C of annealing 15s, 72 DEG C of extension 90s, 30 recycle；72 DEG C of extensions 5min, 4 DEG C of preservations.

The preparation of P43-cat-SpyCatcher fusion segment:

The first amplification system of first time over-lap PCR is 25 μ L；

4 μ L of P43 promoter gene sequence, 4 μ L, 2 × Phanta Max Master of catalase cat gene order Mix12.5 μ L, ddH₂O 4.5μL；

The first amplification program of the first time over-lap PCR is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 62 DEG C of annealing 15s, 72 DEG C of extension 75s, 5 recycle；72 DEG C of extensions 5min；

The supplement system of the first time over-lap PCR is 25 μ L:

12.5 μ of 2 μ L of upstream primer (P43-F), 2 μ L, 2 × Phanta Max Master Mix of downstream primer (cat-R) L, ddH₂O 8.5μL；

The over-lap PCR supplement amplification program is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 62 DEG C of annealing 15s, 72 DEG C of extension 75s, 30 recycle；72 DEG C of extensions 5min, 4 DEG C of preservations.

The first amplification system of second of over-lap PCR is 25 μ L:

4 μ L, SpyCatcher protein gene of P43-cat fusion, 4 μ L, 2 × Phanta Max Master Mix 12.5 μ L, ddH₂O 4.5μL；

The first amplification program of second of over-lap PCR is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 62 DEG C of annealing 15s, 72 DEG C of extension 90s, 5 recycle；72 DEG C of extensions 5min；

The supplement system of second of over-lap PCR is 25 μ L:

2 μ L of upstream primer (P43-F), 2 μ L, 2 × Phanta Max Master of downstream primer (SpyCatcher-R) Mix12.5 μ L, ddH₂O 8.5μL；

Second of over-lap PCR supplement amplification program is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 62 DEG C of annealing 15s, 72 DEG C of extension 90s, 30 recycle；72 DEG C of extensions 5min, 4 DEG C of preservations.

(6) by integrative plasmid pDG1730 through cotX- obtained in BamHI and HindIII double digestion and step (5) God-SpyTag is merged segment and is connected using seamless clone, is transferred in bacillus coli DH 5 alpha；It, will after identifying successfully and being sequenced correctly Recombinant vector obtained is named as pDG1730-cotX-god-SpyTag.Through detecting, contain gemma capsid protein cotX and grape The vector nucleotide sequence of carbohydrate oxidase fusion is as shown in SEQ ID No.1.

The pDG1730 plasmid enzyme restriction system are as follows:

16 μ L of pDG1730 plasmid；BamHI 1μL；HindIII 1μL；10×buffer 2.5μL；ddH₂O 4.5μL；

Reaction condition: 37 DEG C of reaction 2h of metal bath.

Product after plasmid double digestion is detected through 1% agarose gel electrophoresis, and is returned using DNA plastic recovery kit It receives.

Seamless clone's linked system are as follows:

PDG1730 plasmid 152ng；cotX-god-SpyTag 100ng；Exnase 2μL；5×CE buffer 4μL； ddH₂O complements to 20 μ L.

Reaction condition: 37 DEG C of reaction 30min of metal bath.

Seamless clone's connection product is converted and is imported in bacillus coli DH 5 alpha competent cell.

(7) by episomal plasmids pHT01 through P43-cat- obtained in BamHI and XabI double digestion and step (5) SpyCatcher is merged segment and is connected using seamless clone, is transferred in bacillus coli DH 5 alpha；It, will after identifying successfully and being sequenced correctly Recombinant vector obtained is named as pHT01-P43-cat-SpyCatcher.Through detecting, contain P43 promoter gene, glucose The vector nucleotide sequence of oxidase gene and the fusion of SpyCatcher protein gene is as shown in SEQ ID No.2.

The pHT01 plasmid enzyme restriction system are as follows:

16 μ L of pHT01 plasmid；BamHI 1μL；XabI 1μL；10×buffer 2.5μL；ddH₂O 4.5μL；

Reaction condition: 37 DEG C of reaction 2h of metal bath.

Product after plasmid double digestion is detected through 1% agarose gel electrophoresis, and is returned using DNA plastic recovery kit It receives.

Seamless clone's linked system are as follows:

PHT01 plasmid 160ng；P43-cat-SpyCatcher 120ng；Exnase 2μL；5×CE buffer 4μL； ddH₂O complements to 20 μ L.

Reaction condition: 37 DEG C of reaction 30min of metal bath.

Seamless clone's connection product is converted and is imported in bacillus coli DH 5 alpha competent cell.

Embodiment 2: bacillus subtilis WB800n electricity turns the preparation of competent cell

The bacillus subtilis WB800n single bacterium of the fresh LB solid culture primary surface of picking is fallen in 5mL LB culture medium, mistake Night culture.It takes in 1mL overnight culture access 50mL GM culture medium (LB+0.5M sorbierite), 37 DEG C of shaken cultivations to OD₆₀₀For 1.0.By 10min, 5000rpm, 4 DEG C of centrifugation 8min of bacterium solution ice-water bath, thallus is collected.ETM culture medium (the 0.5M being pre-chilled with 20mL + 10% glycerol of sorbierite+0.5M mannitol) thallus is resuspended, 5000rpm, 4 DEG C of centrifugation 8min remove supernatant, so washing 3 times.It will Thallus after washing is resuspended in 500 μ L ETM culture mediums, and with EP pipe, every pipe dispenses 60 μ L for packing.

Embodiment 3: recombinant plasmid pDG1730-cotX-god-SpyTag is transferred in bacillus subtilis WB800n

5 μ L recombinant plasmid pDG1730-cotX-god-SpyTag are added in 50 μ L competent cell WB800n, on ice It is incubated for 5min, is added in the electric revolving cup (2mm) of pre-cooling, electrotransformation is carried out under the conditions of 2500V, 5ms.After electric shock, immediately The RM culture medium (LB+0.5M sorbierite+0.38M mannitol) of 1mL37 DEG C of preheating, 37 DEG C of oscillation recovery trainings are added in electric revolving cup 3h is supported, is coated on the LB plate containing spectinomycin.37 DEG C of inversion cultures, screen the bacterial strain of anti-spectinomycin.

Embodiment 4: the Medium on Identification of positive restructuring bacterium WB800n

It is raw using Shanghai by overnight incubation in above-mentioned positive restructuring colony inoculation to LB liquid medium (containing spectinomycin) The kit that object Engineering Co., Ltd provides extracts genomic DNA, with the genomic DNA position template of acquisition, respectively with cotX-F It is that primer carries out PCR amplification with god-SpyTag-R.

The colony PCR amplification system is 20 μ L:

1 μ L of upstream primer；1 μ L of downstream primer；1 μ L of template；2×Phanta Max Master Mix 10μL；ddH₂O 7 μL；

The colony PCR amplification program is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 70 DEG C of annealing 15s, 72 DEG C of extension 80min, 30 recycle；72 DEG C are prolonged Stretch 5min；4 DEG C of preservations；

Agarose gel electrophoresis proves that exogenous sequences cotX-god-SpyTag has been transferred in bacillus subtilis WB800n, Recombinant bacterium is named as WB800n-cotX-god-SpyTag.

Embodiment 5: recombined bacillus subtilis WB800n-cotX-god-SpyTag electricity turns the preparation of competent cell

The bacillus subtilis WB800n-cotX-god- on the fresh LB solid medium of picking (containing spectinomycin) surface SpyTag single bacterium is fallen in 5mL LB culture medium (containing spectinomycin), is incubated overnight.1mL overnight culture is taken to access 50mL GM In culture medium (LB+0.5M sorbierite), 37 DEG C of shaken cultivations to OD₆₀₀It is 1.0.By bacterium solution ice-water bath 10min, 5000rpm, 4 DEG C It is centrifuged 8min, collects thallus.Bacterium is resuspended with the ETM culture medium (+10% glycerol of 0.5M sorbierite+0.5M mannitol) of 20mL pre-cooling Body, 5000rpm, 4 DEG C of centrifugation 8min remove supernatant, so washing 3 times.Thallus after washing is resuspended in 500 μ L ETM culture mediums In, with EP pipe, every pipe dispenses 60 μ L for packing.

Embodiment 6: recombinant plasmid pHT01-P43-cat-SpyCatcher is transferred to recombined bacillus subtilis In pDG1730-cotX-god-SpyTag

5 μ L recombinant plasmid pHT01-P43-cat-SpyCatcher are added to 50 μ L and are transferred to recombined bacillus subtilis In pDG1730-cotX-god-SpyTag competent cell, it is incubated for 5min on ice, is added in the electric revolving cup (2mm) of pre-cooling, In Electrotransformation is carried out under the conditions of 2500V, 5ms.After electric shock, the RM culture medium of 37 DEG C of 1mL preheatings is added in electric revolving cup immediately (LB+0.5M sorbierite+0.38M mannitol), 37 DEG C of oscillation recovery culture 3h, it is flat to be coated on the LB containing spectinomycin and chloramphenicol On plate.37 DEG C of inversion cultures, screen the bacterial strain of anti-spectinomycin and chloramphenicol.

Embodiment 7: the Medium on Identification of positive restructuring bacterium pDG1730-cotX-god-SpyTag

By overnight incubation, benefit in above-mentioned positive restructuring colony inoculation to LB liquid medium (containing spectinomycin and chloramphenicol) Genomic DNA is extracted with the kit that Shanghai bioengineering Co., Ltd provides, using the genomic DNA of acquisition as template, respectively PCR amplification is carried out by primer of P43-F and SpyCatcher-R.

The colony PCR amplification system is 20 μ L:

1 μ L of upstream primer；1 μ L of downstream primer；1 μ L of template；2×Phanta Max Master Mix 10μL；ddH₂O 7 μL；

The colony PCR amplification program is as follows:

95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 62 DEG C of annealing 15s, 72 DEG C of extension 90min, 30 recycle；72 DEG C are prolonged Stretch 5min；4 DEG C of preservations；

Agarose gel electrophoresis proves that exogenous sequences cotX-god-SpyTag has been transferred to recombined bacillus subtilis In pDG1730-cotX-god-SpyTag, recombinant bacterium is named as WB800n-cotX-god-cat.

Embodiment 8: the fermentation of positive restructuring bacterium WB800n-cotX-god-cat

The recombinant bacterium WB800n-cotX-god-cat that embodiment 7 is built is inoculated in LB liquid medium (is containing concentration The spectinomycin and concentration of 100 μ g/mL be 25 μ g/mL chloramphenicol), 37 DEG C are incubated overnight, next day by 1% inoculum concentration switching with In TB culture medium (containing the spectinomycin that concentration is 100 μ g/mL and chloramphenicol that concentration is 25 μ g/mL), 37 DEG C of culture 48h, 4 DEG C, 7500rpm is centrifuged 10min, and with being centrifuged after 37 DEG C of processing 2h of lysozyme of final concentration 2mg/mL, precipitating is recombinant spore.

Above-mentioned LB liquid medium formula are as follows:

Peptone 10g, yeast extract 5g, NaCl 10g, pH 7.0.

Above-mentioned every liter of component of TB culture medium is as follows:

20g glucose, 30g soy peptone, 2.5g MgCl₂, 17mmol KH₂PO₄, 72mmol K₂HPO₄, pH7.0.

Embodiment 9: glucose oxidase Enzyme activity assay and characterization analysis

The definition of glucose oxidase enzyme activity unit: 1 enzyme activity unit is at 30 DEG C, phosphoric acid buffer body that pH is 5.6 In system, it can be catalyzed 1 μm of oL glucose in 1min and be converted into gluconic acid.

It takes the dextrose buffer solution 25mL of 20g/L in the conical flask of 250mL, is preheated in 30 DEG C of waters bath with thermostatic control 3min, it is accurate that enzyme solution 1mL is added, clock reaction immediately is mixed, the Na of 0.1moL/L is added after accurate response 60min immediately (OH)₂Standard solution 20mL is uniformly mixed and terminates reaction, 1 drop phenolphthalein is added, with the HCl standard solution of 0.1moL/L, record disappears The volume V of consumption.

Blank control: replacing enzyme solution with 1mL water, remaining step is identical, and consumption HCl standard solution volume is V₀。

It calculates: GOD (U/g)=(V₀-V)×C×N×1000/(60×m)

In formula, C is the accurate response concentration of HCl standard solution；N is the extension rate of enzyme solution；1000 be conversion coefficient；60 For reaction time, unit min；M is dry cell weight.

The result shows that the specific enzyme activity of glucose oxidase is 315.2U/g in recombinant spore.

(1) thermal stability of influence and enzyme of the temperature to enzymatic activity

By recombinant spore obtained in embodiment 8 in the reaction system that pH value is 6.0, respectively at 0 DEG C, 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 Enzyme activity is surveyed at a temperature of DEG C, observes influence of the different temperatures to enzymatic reaction；Enzyme solution be placed in it is above it is different at a temperature of keep the temperature 60min measures remaining enzyme activity, the stability of observation GOD at different temperatures.Analysis experimental result (Fig. 5) shows in pH6.0 Under conditions of, the optimal reactive temperature of the enzyme is 40 DEG C.Still there is 60% or more enzyme activity between 0-70 DEG C.Thermal stability Experimental result (Fig. 6) shows that glucose oxidase provided by the invention stands 1 hour in 30-60 DEG C of water-bath, and enzyme activity compares Stablize, have almost no change, the enzyme activity that reservation is more than 80% is remained to after standing 1 hour in 60-70 DEG C of water-bath, in 80 DEG C of water-baths Still it can retain the vigor more than 50% after standing 1 hour, heat-resisting effect is significant.

(2) the pH stability of influence and enzyme of the pH to enzymatic activity

By recombinant spore obtained in embodiment 8 at 30 DEG C of temperature, pH value is respectively 2.0,3.0,4.0,5.0,6.0, 7.0, enzyme activity is measured in 8.0,9.0,10.0,11.0,12.0 reaction system；Enzyme is placed in the buffer of different pH value in 40 60min is handled at DEG C, measures enzymatic activity under optimal pH to carry out the research of enzyme pH stability.Experimental result (Fig. 7) display: The Optimun pH of glucose oxidase provided by the invention is 6.0, and the enzyme is basicly stable between pH5.0-9.0, can Maintain 60% or more enzyme activity；Analysis result (Fig. 8) shows that the enzyme is basicly stable between pH5.0-pH9.0, is able to maintain that 80% or more enzyme activity, enzyme activity only remains 20% after pH4.0 and pH10.0 processing.

Embodiment 10: catalase Enzyme activity assay and characterization analysis

By the hydrogen peroxide enzyme detection kit purchased from the green skies Bioisystech Co., Ltd in Shanghai to recombination withered grass bud The CAT of spore surface display carries out Enzyme activity assay.Measuring method is as follows:

Standard curve determination

It takes the prepared 5mM hydrogenperoxide steam generator of 0,12.5,25,50 and 75 μ L into 1.5mL centrifuge tube, was separately added into Hydrogen oxide enzyme detects buffer to last 100 μ L of volume, mixes.4 μ L are respectively taken, are added in a hole total in 96 orifice plates, are added 200 μ L colour developing working solution.A520 is measured after 25 DEG C of at least incubation 15min, but incubation time is no more than 45min.

Sample measurement

System is added in 1.5mL centrifuge tube by upper table, 450 μ L catalase reactions are added in 25 DEG C of reaction 1-5min Terminate liquid is mixed by inversion to terminate reaction, and the detection of 40 μ L catalases is taken to be buffered in centrifuge tube, and 10 μ L are added and have terminated simultaneously The above-mentioned reaction system mixed mixes.It takes 10 μ L of mixing liquid to be added in a hole in 96 orifice plates, 200 μ L colour developing work is added Liquid measures A520 after 25 DEG C of incubation at least 15min, but incubation time is no more than 45min.

The calculating of catalase enzyme activity in sample

Calculate standard curve: A520=k [hydrogen peroxide micromole number]+b is calculated the value of k and b by standard curve；

Calculate hydrogen peroxide micromole's number remaining in sample: residual hydrogen peroxide micromole number=(A520-b)/k；

The definition of catalase enzyme activity unit: 1 enzyme activity unit is at 25 DEG C, under conditions of pH7.0, in 1min 1 μ L hydrogen peroxide can be catalytically decomposed；

Sample catalase enzyme activity=[consumption hydrogen peroxide micromole number] × [extension rate]/([reaction minute Number] × [sample volume])

Sample catalase enzyme activity unit is U/mg；

Consume hydrogen peroxide micromole number=[blank control residual hydrogen peroxide micromole number]-[sample remnants peroxidating Hydrogen micromole number]；

Extension rate=250；

Reacting the number of minutes is actual reaction the number of minutes

The result shows that the specific enzyme activity of the catalase in recombinant spore is 1803.8U/g.

(1) thermal stability of influence and enzyme of the temperature to enzymatic activity

By recombinant spore obtained in embodiment 8 in the reaction system that pH value is 6.0, respectively at 0 DEG C, 5 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 Enzyme activity is surveyed at a temperature of DEG C, observes influence of the different temperatures to enzymatic reaction；Enzyme solution be placed in it is above it is different at a temperature of keep the temperature 60min measures remaining enzyme activity, the stability of observation CAT at different temperatures.Analysis experimental result (Fig. 9) shows in pH6.0 Under conditions of, the optimal reactive temperature of the enzyme is 50 DEG C.Still there is 60% or more enzyme activity between 20-75 DEG C.Thermostabilization Property experimental result (Figure 10) shows that catalase provided by the invention stands 1 hour in 20-70 DEG C of water-bath, enzyme activity ratio It is more stable, it has almost no change, the enzyme activity that reservation is more than 70%, 85 DEG C of water-baths is remained to after standing 1 hour in 70-80 DEG C of water-bath Middle standing still can retain 30% vigor after 1 hour, heat-resisting effect is significant.

(2) the pH stability of influence and enzyme of the pH to enzymatic activity

By recombinant spore obtained in embodiment 8 at 30 DEG C of temperature, pH value is respectively 2.0,3.0,4.0,5.0,6.0, 7.0, enzyme activity is measured in 8.0,9.0,10.0,11.0,12.0 reaction system；Enzyme is placed in the buffer of different pH value in 40 60min is handled at DEG C, measures enzymatic activity under optimal pH to carry out the research of enzyme pH stability.Experimental result (Figure 11) is aobvious Show: the Optimun pH of catalase provided by the invention is 5.0, and the enzyme is basicly stable between pH4.0-9.0, can Maintain 60% or more enzyme activity；Analysis result (Figure 12) shows that the enzyme is basicly stable between pH4.0-pH9.0, Neng Gouwei Hold 80% or more enzyme activity.

The result shows that engineering bacteria constructed by the present invention can be realized the spore surface in bacillus subtilis and show exhibition altogether Show glucose oxidase and catalase, the glucose oxidase and catalase that spore surface is shown all have temperature, The characteristics of pH wide adaptation range and Heat-stable, and the most suitable enzyme activity temperature of glucose oxidase and catalase and most suitable PH is close, is conducive to the concerted reaction of the two, to be conducive to the application in gluconate production.

Comparative example 1

It uses flexible peptide linker (GGGGS)₃Glucose oxidase gene is connected with catalase gene, with withered grass bud Spore bacillus gemma capsid protein CotX will using integrative plasmid pDG1730 and episomal plasmids pHT01 as molecular vehicle Glucose oxidase and catalase are shown to spore surface altogether.The results show that the ratio enzyme of its glucose oxidase shown Living is 272.3U/g, and for thermal stability at 30-40 DEG C, most suitable enzyme activity temperature is 30 DEG C, and pH stability is most suitable in pH5.5-pH7.0 PH is 7.0；The specific enzyme activity of catalase is 1608.3U/g, and thermal stability is at 25-45 DEG C, and most suitable enzyme activity temperature is 45 DEG C, pH Stability is in pH5.0-pH8.0, optimal pH 5.0.

Comparative example 2

Use rigid connection peptide (EAAAK)₃Glucose oxidase gene is connected with catalase gene, with withered grass bud Spore bacillus gemma capsid protein CotX will using integrative plasmid pDG1730 and episomal plasmids pHT01 as molecular vehicle Glucose oxidase and catalase are shown to spore surface altogether.The results show that the ratio enzyme of its glucose oxidase shown Living is 298.5U/g, and thermal stability is at 30-40 DEG C, and 30 DEG C of enzyme activity temperature most suitable, pH stability is in pH5.0-pH7.0, optimal pH It is 6.0；The specific enzyme activity of catalase is 1754.7U/g, and thermal stability is at 30-45 DEG C, most suitable enzyme activity temperature 45 C, and pH stablizes Property is in pH5.0-pH8.0, optimal pH 8.0.

Comparative example 3

Directly glucose oxidase gene and catalase gene are merged using overlapping pcr, with withered grass gemma Bacillus gemma capsid protein CotX is as molecular vehicle, using integrative plasmid pDG1730 and episomal plasmids pHT01, by Portugal Grape carbohydrate oxidase and catalase are shown to spore surface altogether.The results show that the specific enzyme activity of its glucose oxidase shown For 283.4U/g, thermal stability is 30 DEG C of enzyme activity temperature most suitable at 30-35 DEG C, and pH stability is most suitable in pH5.0-pH7.0 pH5.0；The specific enzyme activity of catalase is 1527.8U/g, and thermal stability is at 30-50 DEG C, most suitable enzyme activity temperature 50 C, and pH stablizes Property is in pH5.0-pH7.0, optimal pH 7.0.

Interpretation of result

Can be seen that target gene by the experimental data of comparative example and comparative example 1-3 can be in recombinant bacillus bud It obtains stablizing heredity in spore bacillus WB800n-cotX-god-SpyTag.Spore surface shows glucose oxidase and peroxidating altogether In the recombinant bacterial strain of hydrogen enzyme, the specific enzyme activity of glucose oxidase is 315.2U/g, and the specific enzyme activity of catalase is 1803.8U/ G, thermal stability and pH stability are more extensive, and the most suitable enzyme activity temperature and optimal pH of glucose oxidase and catalase are more It is close, be conducive to concerted reaction of the two in gluconate production.It compares using other link peptides and directly merges Method is shown altogether for glucose oxidase and catalase, shows glucose oxidase and mistake altogether using method of the invention Hydrogen oxide enzyme seems more advantageous.

Sequence table

<110>Qilu University of Technology

<120>spore surface shows engineered strain and the application of glucose oxidase and catalase altogether

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 10122

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 1

aacaaaattc tccagtcttc acatcggttt gaaaggagga agcggaagaa tgaagtaaga 60

gggatttttg actccgaagt aagtcttcaa aaaatcaaat aaggagtgtc aagaatgttt 120

gcaaaacgat tcaaaacctc tttactgccg ttattcgctg gatttttatt gctgtttcat 180

ttggttctgg caggaccggc ggctgcgagt gctgaaacgg cgaacaaatc gaatgagctt 240

acagcaccgt cgatcaaaag cggaaccatt cttcatgcat ggaattggtc gttcaatacg 300

ttaaaacaca atatgaagga tattcatgat gcaggatata cagccattca gacatctccg 360

attaaccaag taaaggaagg gaatcaagga gataaaagca tgtcgaactg gtactggctg 420

tatcagccga catcgtatca aattggcaac cgttacttag gtactgaaca agaatttaaa 480

gaaatgtgtg cagccgctga agaatatggc ataaaggtca ttgttgacgc ggtcatcaat 540

cataccacca gtgattatgc cgcgatttcc aatgaggtta agagtattcc aaactggaca 600

catggaaaca cacaaattaa aaactggtct gatcggatcc gatcagacaa agacggaaaa 660

acgataacaa ttagccaatc ataaaaaata gggttcttca tcaggatata tgactcagtc 720

aaaataagag gctcgctcat ttaataacag taaaagaaaa ggaggaatag atatggaaag 780

cagaccatat tcttgggttg cacttgatcc tgactgtgac catccgttag atgacaaaga 840

gaaagataaa gaaaaacacg aaagaaaatg tcattgcgac gtttgctgta atggcaatgg 900

tttttttggc aacgacaacg ccttcatcga ccaagatcta gctcaagcaa atctcaacaa 960

acaagtttca gatgaaacga ttattattag agattcttgt gacatcaatg ttacatctac 1020

agacgttcaa gccgtaacat cagttgtaac agcacttaat gccgctgtcg taacggcaac 1080

tctgacatca attgcagacg gcgtaattgc cgaattagtc gcacaagatt tgttacagct 1140

tacagctaac aaacaagtaa accgccaaaa acttctcatc gaatgttccc gcggcgtaaa 1200

cgtcacaaca gtagatgccg atatcgcaac ccttatttct acagcaacaa atacactcgt 1260

agccatccta gttatcactc ttgtcctcat gcagacgctt ctggttagct ctcttgtcgt 1320

ttcccttgcc gccgctcttc cgcattacat ccgcagcaat ggcatcgagg ccagccttct 1380

gacggatcca aaggatgtca cgggccgcac ggtcgactac attattgctg gcggaggact 1440

tacgggactt acgacggctg ctagacttac ggaaaacccg aacatcacgg ttctggtcat 1500

tgagagcggc agctatgaga gcgatcgcgg cccgattatc gaagatctta atgcctacgg 1560

cgacatcttc ggatcctccg tcgaccatgc ctacgaaacg gtcgagctgg ccacgaacaa 1620

tcaaacggct cttatcagat ccggaaacgg acttggaggc agcacgcttg ttaatggagg 1680

aacgtggaca cgcccgcata aagcccaagt cgatagctgg gaaacggtct tcggaaacga 1740

gggctggaat tgggactccg tcgccgctta ttctcttcaa gccgagcgcg ctcgcgcccc 1800

gaatgctaag cagatcgccg ctggccatta ctttaacgcc agctgccacg gacttaatgg 1860

cacagttcat gctggcccga gagacacggg cgacgactat agcccgattg tcaaggctct 1920

tatgagcgtt gttgaggatc gcggagttcc gacaaagaaa gatcttggct gtggcgatcc 1980

gcatggagtt agcatgttcc cgaatacgct gcatgaggat caagttcgct ccgatgctgc 2040

tcgcgaatgg ctgcttccga attatcagcg cccaaacctt caagttctta cgggccagta 2100

cgttggcaaa gttcttctga gccaaaacgc tacaacacca cgcgccgttg gagtcgaatt 2160

tggcacgcat aagggcaaca cgcacaacgt ctacgccaaa catgaggttc tgcttgctgc 2220

cggaagcgcc gttagcccga caattcttga gtatagcggc atcggcatga agagcattct 2280

tgagccactt ggaatcgaca cagtcgttga tctgccggtc ggacttaacc ttcaagacca 2340

gacgacgtcc acggtccgca gccgcattac aagcgctgga gctggacaag gccaagccgc 2400

ttggttcgct acattcaacg agacgttcgg cgactacaca gagaaggccc acgaactgct 2460

gaacacgaaa ctggagcaat gggccgaaga agctgtcgcc agaggcggct tccacaatac 2520

gacggccctt cttatccagt acgaaaacta tcgcgactgg atcgtcaaag acaatgtcgc 2580

ctactccgaa ctgttccttg atacggctgg cgtcgcctcc tttgatgtct gggaccttct 2640

gccgttcaca cgcggctacg tccacattct tgacaaagac ccgtatcttc gccatttcgc 2700

ctacgatccg cagtactttc ttaatgagct ggatcttctt ggacaagccg ccgctacaca 2760

actggcccgc aatatttcca attccggcgc catgcagacg tactttgctg gagaaacgat 2820

cccgggagat aaccttgcct atgatgccga tcttagcgct tgggttgagt atatcccgta 2880

caacttccgc ccaaattatc acggagttgg cacatgcagc atgatgccga aggaaatggg 2940

cggagtcgtt gataatgctg cccgcgtcta tggcgtccaa ggccttagag ttatcgacgg 3000

ctccatcccg ccaacacaga tgagcagcca cgtcatgacg gtcttctacg ctatggctct 3060

taaaatcgct gacgccgtcc ttgctgatta cgcttccatg cagcaccatc atcaccacca 3120

ttaagcacac atagtaatgg tagatgcata caagccaaca aagaagctta tcgaattcct 3180

gcagccctgg cgaatggcga ttttcgttcg tgaatacatg ttataataac tataactaat 3240

aacgtaacgt gactggcaag agatattttt aaaacaatga ataggtttac acttacttta 3300

gttttatgga aatgaaagat catatcatat ataatctaga ataaaattaa ctaaaataat 3360

tattatctag ataaaaaatt tagaagccaa tgaaatctat aaataaacta aattaagttt 3420

atttaattaa caactatgga tataaaatag gtactaatca aaatagtgag gaggatatat 3480

ttgaatacat acgaacaaat taataaagtg aaaaaaatac ttcggaaaca tttaaaaaat 3540

aaccttattg gtacttacat gtttggatca ggagttgaga gtggactaaa accaaatagt 3600

gatcttgact ttttagtcgt cgtatctgaa ccattgacag atcaaagtaa agaaatactt 3660

atacaaaaaa ttagacctat ttcaaaaaaa ataggagata aaagcaactt acgatatatt 3720

gaattaacaa ttattattca gcaagaaatg gtaccgtgga atcatcctcc caaacaagaa 3780

tttatttatg gagaatggtt acaagagctt tatgaacaag gatacattcc tcagaaggaa 3840

ttaaattcag atttaaccat aatgctttac caagcaaaac gaaaaaataa aagaatatac 3900

ggaaattatg acttagagga attactacct gatattccat tttctgatgt gagaagagcc 3960

attatggatt cgtcagagga attaatagat aattatcagg atgatgaaac caactctata 4020

ttaactttat gccgtatgat tttaactatg gacacgggta aaatcatacc aaaagatatt 4080

gcgggaaatg cagtggctga atcttctcca ttagaacata gggagagaat tttgttagca 4140

gttcgtagtt atcttggaga gaatattgaa tggactaatg aaaatgtaaa tttaactata 4200

aactatttaa ataacagatt aaaaaaatta taaaaaaatt gaaaaaatgg tggaaacact 4260

tttttcaatt tttttgtttt attatttaat atttgggaaa tattcattct aattggtaat 4320

cagattttag aaaacaataa acccttgcat agggggatct cgacatggat gagcgatgat 4380

gatatccgtt taggctgggc ggtgatagct tctcgttcag gcagtacgcc tcttttcttt 4440

tccagacctg agggaggcgg aaatggtgtg aggttcccgg ggaaaagcca aataggcgat 4500

cgcgggagtg ctttatttga agatcaggct atcactgcgg tcaatagatt tcacaatgtg 4560

atggctggac agcctgagga actctcgaac ccgaatggaa acaaccagat atttatgaat 4620

cagcgcggct cacatggcgt tgtgctggca aatgcaggtt catcctctgt ctctatcaat 4680

acggcaacaa aattgcctga tggcaggtat gacaataaag ctggagcggg ttcatttcaa 4740

gtgaacgatg gtaaactgac aggcacgatc aatgccaggt ctgtagctgt gctttatcct 4800

gatgatattg caaaagcgcc tcatgttttc cttgagaatt acaaaacagg tgtaacacat 4860

tctttcaatg atcaactgac gattaccttg cgtgcagatg cgaatacaac aaaagccgtt 4920

tatcaaatca ataatggacc agacgacagg cgtttaagga tggagatcaa ttcacaatcg 4980

gaaaaggaga tccaatttgg caaaacatac accatcatgt taaaaggaac gaacagtgat 5040

ggtgtaacga ggaccgagaa atacagtttt gttaaaagag atccagcgtc ggccaaaacc 5100

atcggctatc aaaatccgaa tcattggagc caggtaaatg cttatatcta taaacatgat 5160

gggagccgag taattgaatt gaccggatct tggcctggaa aaccaatgac taaaaatgca 5220

gacggaattt acacgctgac gctgcctgcg gacacggata caaccaacgc aaaagtgatt 5280

tttaataatg gcagcgccca agtgcccggt cagaatcagc ctggctttga ttacgtgcta 5340

aatggtttat ataatgactc gggcttaagc ggttctcttc cccattgagg gcaaggctag 5400

acgggactta ccgaaagaaa ccatcaatga tggtttcttt tttgttcata aatcagacaa 5460

aacttttctc ttgcaaaagt ttgtgaagtg ttgcacaata taaatgtgaa atacttcaca 5520

aacaaaaaga catcaaagag aaacataccc tgcaaggatg ctgatattgt ctgcatttgc 5580

gccggagcaa accaaaaacc tggtgagaca cgccttgaat tagtagaaaa gaacttgaag 5640

attttcaaag gcatcgttag tgaagtcatg gcgagcggat ttgacggcat tttcttagtc 5700

gcgacgcgag gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg 5760

atgcccgcgt tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt 5820

caaggatcgc tcgcggctct taccagccta acttcgatca ctggaccgct gatcgtcacg 5880

gcgatttatg ccgcctcggc gagcacatgg aacgggttgg catggattgt aggcgccgcc 5940

ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctactga 6000

agtggatttc tttaagagct cctttaactt cctcaccagt agttgtatcg gtaccataag 6060

tagaagcagc aacccaagta gctttaccag catccggttc aaccagcata gtaagaatct 6120

tactggacat cggcagttct tcgaacagtg cgccaactac cagctctttc tgcagttcat 6180

tcagggcacc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca 6240

tcctgtctct tgatccatgg attacgcgtt aacccgggcc cgcggatgca tatgatcaga 6300

tcctttaact ctggcaaccc tcaaaattga atgagacatg ctacacctcc ggataataaa 6360

tatatataaa cgtatataga tttcataaag tctaacacac tagacttatt tacttcgtaa 6420

ttaagtcgtt aaaccgtgtg ctctacgacc aaaactataa aacctttaag aactttcttt 6480

ttttacaaga aaaaagaaat tagataaatc tctcatatct tttattcaat aatcgcatcc 6540

gattgcagta taaatttaac gatcactcat catgttcata tttatcagag ctcgtgctat 6600

aattatacta attttataag gaggaaaaaa tatgggcatt tttagtattt ttgtaatcag 6660

cacagttcat tatcaaccaa acaaaaaata agtggttata atgaatcgtt aataagcaaa 6720

attcatataa ccaaattaaa gagggttata atgaacgaga aaaatataaa acacagtcaa 6780

aactttatta cttcaaaaca taatatagat aaaataatga caaatataag attaaatgaa 6840

catgataata tctttgaaat cggctcagga aaaggccatt ttacccttga attagtaaag 6900

aggtgtaatt tcgtaactgc cattgaaata gaccataaat tatgcaaaac tacagaaaat 6960

aaacttgttg atcacgataa tttccaagtt ttaaacaagg atatattgca gtttaaattt 7020

cctaaaaacc aatcctataa aatatatggt aatatacctt ataacataag tacggatata 7080

atacgcaaaa ttgtttttga tagtatagct aatgagattt atttaatcgt ggaatacggg 7140

tttgctaaaa gattattaaa tacaaaacgc tcattggcat tacttttaat ggcagaagtt 7200

gatatttcta tattaagtat ggttccaaga gaatattttc atcctaaacc taaagtgaat 7260

agctcactta tcagattaag tagaaaaaaa tcaagaatat cacacaaaga taaacaaaag 7320

tataattatt tcgttatgaa atgggttaac aaagaataca agaaaatatt tacaaaaaat 7380

caatttaaca attccttaaa acatgcagga attgacgatt taaacaatat tagctttgaa 7440

caattcttat ctcttttcaa tagctataaa ttatttaata agtaagttaa gggatgcata 7500

aactgcatcc cttaacttgt ttttcgtgtg cctatttttt gtgaatcgat tatgtctttt 7560

gcgcagtcgg cttaaaccag ttttcgctgg tgcgaaaaaa gagtgtcttg tgacacctaa 7620

attcaaaatc tatcggtcag atttataccg atttgatttt atatattctt gaataacata 7680

cgccgagtta tcacataaaa gcgggaacca atcatcaaat ttaaacttca ttgcataatc 7740

cattaaactc ttaaattcta cgattccttg ttcatcaata aactcaatca tttctttaat 7800

taatttatat ctatctgttg ttgttttctt taataattca tcaacatcta caccgccata 7860

aactatcata tcttcttttt gatatttaaa tttattagga tcttaaggcc taggtctaga 7920

gtctttgttt tgacgccatt agcgtacgta acaatcctcg ttaaaggaca aggacctgag 7980

cggaagtgta tcgtacagta gacggagtat actagtatag tctatagtcc gtggaattat 8040

tatatttatc tccgacgata ttctcatcag tgaaatccag ctggagttct ttagcaaatt 8100

tttttattag ctgaacttag tattagtggc catactcctc caatccaaag ctatttagaa 8160

agattactat atcctcaaac aggcggtaac cggcctcttc atcgggaatg cgcgcgacct 8220

tcagcatcgc cggcatgtcc ccctggcgga cgggaagtat ccagctcgag gtcgggccgc 8280

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 8340

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 8400

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 8460

cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 8520

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 8580

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 8640

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 8700

gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 8760

gaagccagtt accttcggaa aaagagttga tagctcttga tccggcaaac aaaccaccgc 8820

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 8880

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 8940

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 9000

atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 9060

cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 9120

actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 9180

aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 9240

cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 9300

ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 9360

cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 9420

ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 9480

cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 9540

ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 9600

tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 9660

ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 9720

aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 9780

gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 9840

gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 9900

ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 9960

catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 10020

atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 10080

taaaaatagg cgtatcacga ggccctttcg tcttcaagaa tt 10122

<210> 2

<211> 10988

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 2

ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa 60

tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa 120

gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat 180

aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt 240

tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat 300

ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag 360

gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt 420

ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt 480

tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc 540

cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga 600

aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa 660

tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct 720

cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa 780

tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc 840

ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat 900

ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa 960

aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc 1020

ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac 1080

tatggcgtgc tgctagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc 1140

ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 1200

aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 1260

cgagctcagg ccttaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 1320

aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 1380

tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 1440

tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 1500

gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 1560

cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 1620

ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 1680

tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 1740

ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 1800

ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 1860

gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 1920

tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 1980

tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 2040

gattgtgcac cgccgtttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 2100

cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 2160

gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 2220

gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 2280

ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 2340

cggcatactc tgcgacatcg tataacgtta ctggtttcat caaaatcgtc tccctccgtt 2400

tgaatatttg attgatcgta accagatgaa gcactctttc cactatccct acagtgttat 2460

ggcttgaaca atcacgaaac aataattggt acgtacgatc tttcagccga ctcaaacatc 2520

aaatcttaca aatgtagtct ttgaaagtat tacatatgta agatttaaat gcaaccgttt 2580

tttcggaagg aaatgatgac ctcgtttcca ccggaattag cttggtacca gctattgtaa 2640

cataatcggt acgggggtga aaaagctaac ggaaaaggga gcggaaaaga atgatgtaag 2700

cgtgaaaaat tttttatctt atcacttgaa attggaaggg agattcttta ttataagaat 2760

tgtggaattg tgagcggata acaattccca attaaaggag gaaggatcca gcttcgtgca 2820

tgcaggccgg ggcatatggg aaacagcgcg gacgcagcgg aatttccaat ttcatgccgc 2880

agccgcctgc gctgttctca tttgcggctt ccttgtagag ctcagcatta ttgagtggat 2940

gattatattc cttttgatag gtggtatgtt ttcgcttgaa cttttaaata cagccattga 3000

acatacggtt gatttaataa ctgacaaaca tcaccctctt gctaaagcgg ccaaggacgc 3060

tgccgccggg gctgtttgcg tttttgccgt gatttcgtgt atcattggtt tacttatttt 3120

tttgccaaag ctgtaatggc tgaaaattct tacatttatt ttacattttt agaaatgggc 3180

gtgaaaaaaa gcgcgcgatt atgtaaaata taaagtgata gcggtaccat tataggtaag 3240

agaggaatgt acacatgaac agacaagaat taataacaga agcttatgag cacgtcagac 3300

gatatccata acaccacagc cactggcaaa tgcccgttcc atcagggcgg tcacgaccag 3360

agtgcggggg cgggcacaac cactcgcgac tggtggccaa atcaacttcg tgttgacctg 3420

ttaaaccaac attctaatcg ttctaaccca ctgggtgagg actttgacta ccgcaaagaa 3480

ttcagcaaat tagattacta cggcctgaaa aaagatctga aagccctgtt gacagaatct 3540

caaccgtggt ggccagccga ctggggcagt tacgccggtc tgtttattcg tatggcctgg 3600

cacggcgcgg ggacttaccg ttcaatcgat ggacgcggtg gcgcgggtcg tggtcagcaa 3660

cgttttgcac cgctgaactc ctggccggat aacgtaagcc tcgataaagc gcgtcgcctg 3720

ttgtggccaa tcaaacagaa atatggtcag aaaatctcct gggccgacct gtttatcctc 3780

gcgggtaacg tggcgctaga aaactccggc ttccgtacct tcggttttgg tgccggtcgt 3840

gaagacgtct gggaaccgga tctggatgtt aactggggtg atgaaaaagc ctggctgact 3900

caccgtcatc cggaagcgct ggcgaaagca ccgctgggtg caaccgagat gggtctgatt 3960

tacgttaacc cggaaggccc ggatcacagc ggcgaaccgc tttctgcggc agcagctatc 4020

cgcgcgacct tcggcaacat gggcatgaac gacgaagaaa ccgtggcgct gattgcgggt 4080

ggtcatacgc tgggtaaaac ccacggtgcc ggtccgacat caaatgtagg tcctgatcca 4140

gaagctgcac cgattgaaga acaaggttta ggttgggcga gcacttacgg cagcggcgtt 4200

ggcgcagatg ccattacctc tggtctggaa gtagtctgga cccagacgcc gacccagtgg 4260

agcaactatt tcttcgagaa cctgttcaag tatgagtggg tacagacccg cagcccggct 4320

ggcgcaatcc agttcgaagc ggtagacgca ccggaaatta tcccggatcc gtttgatccg 4380

tcgaagaaac gtaaaccgac aatgctggtg accgacctga cgctgcgttt tgatcctgag 4440

ttcgagaaga tctctcgtcg tttcctcaac gatccgcagg cgttcaacga agcctttgcc 4500

cgtgcctggt tcaaactgac gcacagggat atggggccga aatctcgcta catcgggccg 4560

gaagtgccga aagaagatct gatctggcaa gatccgctgc cgcagccgat ctacaacccg 4620

accgagcagg acattatcga tctgaaattc gcgattgcgg attctggtct gtctgttagt 4680

gagctggtat cggtggcctg ggcatctgct tctaccttcc gtggtggcga caaacgcggt 4740

ggtgccaacg gtgcgcgtct ggcattaatg ccgcagcgcg actgggatgt gaacgccgca 4800

gccgttcgtg ctctgcctgt tctggagaaa atccagaaag agtctggtaa agcctcgctg 4860

gcggatatca tagtgctggc tggtgtggtt ggtgttgaga aagccgcaag cgccgcaggt 4920

ttgagcattc atgtaccgtt tgcgccgggt cgcgttgatg cgcgtcagga tcagactgac 4980

attgagatgt ttgagctgct ggagccaatt gctgacggtt tccgtaacta tcgcgctcgt 5040

ctggacgttt ccaccaccga gtcactgctg atcgacaaag cacagcaact gacgctgacc 5100

gcgccggaaa tgactgcgct ggtgggcggc atgcgtgtac tgggtgccaa cttcgatggc 5160

agcaaaaacg gcgtcttcac tgaccgcgtt ggcgtattga gcaatgactt cttcgtgaac 5220

ttgctggata tgcgttacga gtggaaagcg accgacgaat cgaaagagct gttcgaaggc 5280

cgtgaccgtg aaaccggcga agtgaaattt acggccagcc gtgcggatct ggtgtttggt 5340

tctaactccg tcctgcgtgc ggtggcggaa gtttacgcca gtagcgatgc ccacgagaag 5400

tttgttaaag acttcgtggc ggcatgggtg aaagtgatga acctcgaccg tttcgacctg 5460

ctggattata aagatgatga tgataaaatg gccatggttg acacacttag cggccttagc 5520

tccgaacaag gccagagcgg cgatatgacg atcgaagaag actccgccac gcacatcaag 5580

ttcagcaaac gcgacgagga tggcaaggaa ctggccggcg ccacaatgga acttcgcgac 5640

agcagcggca aaacgatcag cacatggatc agcgatggcc aagttaagga cttctatctt 5700

tatccgggca agtacacgtt cgtcgaaaca gccgccccag atggctatga ggttgccaca 5760

gccatcacgt ttacggtcaa cgaacaaggc caagttacgg ttaatggcaa ggccacgaaa 5820

ggagatgccc atatcgatta atctagagtc gacgtccccg gggcagcccg cctaatgagc 5880

gggctttttt cacgtcacgc gtccatggag atctttgtct gcaactgaaa agtttatacc 5940

ttacctggaa caaatggttg aaacatacga ggctaatatc ggcttattag gaatagtccc 6000

tgtactaata aaatcaggtg gatcagttga tcagtatatt ttggacgaag ctcggaaaga 6060

atttggagat gacttgctta attccacaat taaattaagg gaaagaataa agcgatttga 6120

tgttcaagga atcacggaag aagatactca tgataaagaa gctctaaaac tattcaataa 6180

ccttacaatg gaattgatcg aaagggtgga aggttaatgg tacgaaaatt aggggatcta 6240

cctagaaagc cacaaggcga taggtcaagc ttaaagaacc cttacatgga tcttacagat 6300

tctgaaagta aagaaacaac agaggttaaa caaacagaac caaaaagaaa aaaagcattg 6360

ttgaaaacaa tgaaagttga tgtttcaatc cataataaga ttaaatcgct gcacgaaatt 6420

ctggcagcat ccgaagggaa ttcatattac ttagaggata ctattgagag agctattgat 6480

aagatggttg agacattacc tgagagccaa aaaacttttt atgaatatga attaaaaaaa 6540

agaaccaaca aaggctgaga cagactccaa acgagtctgt ttttttaaaa aaaatattag 6600

gagcattgaa tatatattag agaattaaga aagacatggg aataaaaata ttttaaatcc 6660

agtaaaaata tgataagatt atttcagaat atgaagaact ctgtttgttt ttgatgaaaa 6720

aacaaacaaa aaaaatccac ctaacggaat ctcaatttaa ctaacagcgg ccaaactgag 6780

aagttaaatt tgagaagggg aaaaggcgga tttatacttg tatttaacta tctccatttt 6840

aacattttat taaaccccat acaagtgaaa atcctctttt acactgttcc tttaggtgat 6900

cgcggaggga cattatgagt gaagtaaacc taaaaggaaa tacagatgaa ttagtgtatt 6960

atcgacagca aaccactgga aataaaatcg ccaggaagag aatcaaaaaa gggaaagaag 7020

aagtttatta tgttgctgaa acggaagaga agatatggac agaagagcaa ataaaaaact 7080

tttctttaga caaatttggt acgcatatac cttacataga aggtcattat acaatcttaa 7140

ataattactt ctttgatttt tggggctatt ttttaggtgc tgaaggaatt gcgctctatg 7200

ctcacctaac tcgttatgca tacggcagca aagacttttg ctttcctagt ctacaaacaa 7260

tcgctaaaaa aatggacaag actcctgtta cagttagagg ctacttgaaa ctgcttgaaa 7320

ggtacggttt tatttggaag gtaaacgtcc gtaataaaac caaggataac acagaggaat 7380

ccccgatttt taagattaga cgtaaggttc ctttgctttc agaagaactt ttaaatggaa 7440

accctaatat tgaaattcca gatgacgagg aagcacatgt aaagaaggct ttaaaaaagg 7500

aaaaagaggg tcttccaaag gttttgaaaa aagagcacga tgaatttgtt aaaaaaatga 7560

tggatgagtc agaaacaatt aatattccag aggccttaca atatgacaca atgtatgaag 7620

atatactcag taaaggagaa attcgaaaag aaatcaaaaa acaaatacct aatcctacaa 7680

catcttttga gagtatatca atgacaactg aagaggaaaa agtcgacagt actttaaaaa 7740

gcgaaatgca aaatcgtgtc tctaagcctt cttttgatac ctggtttaaa aacactaaga 7800

tcaaaattga aaataaaaat tgtttattac ttgtaccgag tgaatttgca tttgaatgga 7860

ttaagaaaag atatttagaa acaattaaaa cagtccttga agaagctgga tatgttttcg 7920

aaaaaatcga actaagaaaa gtgcaataaa ctgctgaagt atttcagcag ttttttttat 7980

ttagaaatag tgaaaaaaat ataatcaggg aggtatcaat atttaatgag tactgattta 8040

aatttattta gactggaatt aataattaac acgtagacta attaaaattt aatgagggat 8100

aaagaggata caaaaatatt aatttcaatc cctattaaat tttaacaagg gggggattaa 8160

aatttaatta gaggtttatc cacaagaaaa gaccctaata aaatttttac tagggttata 8220

acactgatta atttcttaat gggggaggga ttaaaattta atgacaaaga aaacaatctt 8280

ttaagaaaag cttttaaaag ataataataa aaagagcttt gcgattaagc aaaactcttt 8340

actttttcat tgacattatc aaattcatcg atttcaaatt gttgttgtat cataaagtta 8400

attctgtttt gcacaacctt ttcaggaata taaaacacat ctgaggcttg ttttataaac 8460

tcagggtcgc taaagtcaat gtaacgtagc atatgatatg gtatagcttc cacccaagtt 8520

agcctttctg cttcttctga atgtttttca tatacttcca tgggtatctc taaatgattt 8580

tcctcatgta gcaaggtatg agcaaaaagt ttatggaatt gatagttcct ctctttttct 8640

tcaacttttt tatctaaaac aaacacttta acatctgagt caatgtaagc ataagatgtt 8700

tttccagtca taatttcaat cccaaatctt ttagacagaa attctggacg taaatctttt 8760

ggtgaaagaa tttttttatg tagcaatata tccgatacag caccttctaa aagcgttggt 8820

gaatagggca ttttacctat ctcctctcat tttgtggaat aaaaatagtc atattcgtcc 8880

atctacctat cctattatcg aacagttgaa ctttttaatc aaggatcagt cctttttttc 8940

attattctta aactgtgctc ttaactttaa caactcgatt tgtttttcca gatctcgagg 9000

gtaactagcc tcgccgatcc cgcaagaggc ccggcagtca ggtggcactt ttcggggaaa 9060

tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 9120

gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 9180

acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 9240

cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 9300

catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 9360

tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 9420

cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 9480

accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 9540

cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 9600

ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 9660

accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 9720

ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 9780

attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 9840

ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 9900

tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 9960

tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 10020

gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 10080

tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 10140

ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 10200

ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 10260

agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 10320

cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 10380

caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 10440

tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 10500

ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 10560

ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 10620

gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 10680

gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 10740

tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 10800

cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 10860

gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 10920

ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 10980

acgcatgc 10988

<210> 3

<211> 2523

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 3

gatcagacaa agacggaaaa acgataacaa ttagccaatc ataaaaaata gggttcttca 60

tcaggatata tgactcagtc aaaataagag gctcgctcat ttaataacag taaaagaaaa 120

ggaggaatag atatggaaag cagaccatat tcttgggttg cacttgatcc tgactgtgac 180

catccgttag atgacaaaga gaaagataaa gaaaaacacg aaagaaaatg tcattgcgac 240

gtttgctgta atggcaatgg tttttttggc aacgacaacg ccttcatcga ccaagatcta 300

gctcaagcaa atctcaacaa acaagtttca gatgaaacga ttattattag agattcttgt 360

gacatcaatg ttacatctac agacgttcaa gccgtaacat cagttgtaac agcacttaat 420

gccgctgtcg taacggcaac tctgacatca attgcagacg gcgtaattgc cgaattagtc 480

gcacaagatt tgttacagct tacagctaac aaacaagtaa accgccaaaa acttctcatc 540

gaatgttccc gcggcgtaaa cgtcacaaca gtagatgccg atatcgcaac ccttatttct 600

acagcaacaa atacactcgt agccatccta gttatcactc ttgtcctcat gcagacgctt 660

ctggttagct ctcttgtcgt ttcccttgcc gccgctcttc cgcattacat ccgcagcaat 720

ggcatcgagg ccagccttct gacggatcca aaggatgtca cgggccgcac ggtcgactac 780

attattgctg gcggaggact tacgggactt acgacggctg ctagacttac ggaaaacccg 840

aacatcacgg ttctggtcat tgagagcggc agctatgaga gcgatcgcgg cccgattatc 900

gaagatctta atgcctacgg cgacatcttc ggatcctccg tcgaccatgc ctacgaaacg 960

gtcgagctgg ccacgaacaa tcaaacggct cttatcagat ccggaaacgg acttggaggc 1020

agcacgcttg ttaatggagg aacgtggaca cgcccgcata aagcccaagt cgatagctgg 1080

gaaacggtct tcggaaacga gggctggaat tgggactccg tcgccgctta ttctcttcaa 1140

gccgagcgcg ctcgcgcccc gaatgctaag cagatcgccg ctggccatta ctttaacgcc 1200

agctgccacg gacttaatgg cacagttcat gctggcccga gagacacggg cgacgactat 1260

agcccgattg tcaaggctct tatgagcgtt gttgaggatc gcggagttcc gacaaagaaa 1320

gatcttggct gtggcgatcc gcatggagtt agcatgttcc cgaatacgct gcatgaggat 1380

caagttcgct ccgatgctgc tcgcgaatgg ctgcttccga attatcagcg cccaaacctt 1440

caagttctta cgggccagta cgttggcaaa gttcttctga gccaaaacgc tacaacacca 1500

cgcgccgttg gagtcgaatt tggcacgcat aagggcaaca cgcacaacgt ctacgccaaa 1560

catgaggttc tgcttgctgc cggaagcgcc gttagcccga caattcttga gtatagcggc 1620

atcggcatga agagcattct tgagccactt ggaatcgaca cagtcgttga tctgccggtc 1680

ggacttaacc ttcaagacca gacgacgtcc acggtccgca gccgcattac aagcgctgga 1740

gctggacaag gccaagccgc ttggttcgct acattcaacg agacgttcgg cgactacaca 1800

gagaaggccc acgaactgct gaacacgaaa ctggagcaat gggccgaaga agctgtcgcc 1860

agaggcggct tccacaatac gacggccctt cttatccagt acgaaaacta tcgcgactgg 1920

atcgtcaaag acaatgtcgc ctactccgaa ctgttccttg atacggctgg cgtcgcctcc 1980

tttgatgtct gggaccttct gccgttcaca cgcggctacg tccacattct tgacaaagac 2040

ccgtatcttc gccatttcgc ctacgatccg cagtactttc ttaatgagct ggatcttctt 2100

ggacaagccg ccgctacaca actggcccgc aatatttcca attccggcgc catgcagacg 2160

tactttgctg gagaaacgat cccgggagat aaccttgcct atgatgccga tcttagcgct 2220

tgggttgagt atatcccgta caacttccgc ccaaattatc acggagttgg cacatgcagc 2280

atgatgccga aggaaatggg cggagtcgtt gataatgctg cccgcgtcta tggcgtccaa 2340

ggccttagag ttatcgacgg ctccatcccg ccaacacaga tgagcagcca cgtcatgacg 2400

gtcttctacg ctatggctct taaaatcgct gacgccgtcc ttgctgatta cgcttccatg 2460

cagcaccatc atcaccacca tgcacacata gtaatggtag atgcatacaa gccaacaaag 2520

taa 2523

<210> 4

<211> 3032

<212> DNA

<213>artificial sequence (artificial sequence)

<400> 4

agcttcgtgc atgcaggccg gggcatatgg gaaacagcgc ggacgcagcg gaatttccaa 60

tttcatgccg cagccgcctg cgctgttctc atttgcggct tccttgtaga gctcagcatt 120

attgagtgga tgattatatt ccttttgata ggtggtatgt tttcgcttga acttttaaat 180

acagccattg aacatacggt tgatttaata actgacaaac atcaccctct tgctaaagcg 240

gccaaggacg ctgccgccgg ggctgtttgc gtttttgccg tgatttcgtg tatcattggt 300

ttacttattt ttttgccaaa gctgtaatgg ctgaaaattc ttacatttat tttacatttt 360

tagaaatggg cgtgaaaaaa agcgcgcgat tatgtaaaat ataaagtgat agcggtacca 420

ttataggtaa gagaggaatg tacacatgaa cagacaagaa ttaataacag aagcttatga 480

gcacgtcaga cgatatccat aacaccacag ccactggcaa atgcccgttc catcagggcg 540

gtcacgacca gagtgcgggg gcgggcacaa ccactcgcga ctggtggcca aatcaacttc 600

gtgttgacct gttaaaccaa cattctaatc gttctaaccc actgggtgag gactttgact 660

accgcaaaga attcagcaaa ttagattact acggcctgaa aaaagatctg aaagccctgt 720

tgacagaatc tcaaccgtgg tggccagccg actggggcag ttacgccggt ctgtttattc 780

gtatggcctg gcacggcgcg gggacttacc gttcaatcga tggacgcggt ggcgcgggtc 840

gtggtcagca acgttttgca ccgctgaact cctggccgga taacgtaagc ctcgataaag 900

cgcgtcgcct gttgtggcca atcaaacaga aatatggtca gaaaatctcc tgggccgacc 960

tgtttatcct cgcgggtaac gtggcgctag aaaactccgg cttccgtacc ttcggttttg 1020

gtgccggtcg tgaagacgtc tgggaaccgg atctggatgt taactggggt gatgaaaaag 1080

cctggctgac tcaccgtcat ccggaagcgc tggcgaaagc accgctgggt gcaaccgaga 1140

tgggtctgat ttacgttaac ccggaaggcc cggatcacag cggcgaaccg ctttctgcgg 1200

cagcagctat ccgcgcgacc ttcggcaaca tgggcatgaa cgacgaagaa accgtggcgc 1260

tgattgcggg tggtcatacg ctgggtaaaa cccacggtgc cggtccgaca tcaaatgtag 1320

gtcctgatcc agaagctgca ccgattgaag aacaaggttt aggttgggcg agcacttacg 1380

gcagcggcgt tggcgcagat gccattacct ctggtctgga agtagtctgg acccagacgc 1440

cgacccagtg gagcaactat ttcttcgaga acctgttcaa gtatgagtgg gtacagaccc 1500

gcagcccggc tggcgcaatc cagttcgaag cggtagacgc accggaaatt atcccggatc 1560

cgtttgatcc gtcgaagaaa cgtaaaccga caatgctggt gaccgacctg acgctgcgtt 1620

ttgatcctga gttcgagaag atctctcgtc gtttcctcaa cgatccgcag gcgttcaacg 1680

aagcctttgc ccgtgcctgg ttcaaactga cgcacaggga tatggggccg aaatctcgct 1740

acatcgggcc ggaagtgccg aaagaagatc tgatctggca agatccgctg ccgcagccga 1800

tctacaaccc gaccgagcag gacattatcg atctgaaatt cgcgattgcg gattctggtc 1860

tgtctgttag tgagctggta tcggtggcct gggcatctgc ttctaccttc cgtggtggcg 1920

acaaacgcgg tggtgccaac ggtgcgcgtc tggcattaat gccgcagcgc gactgggatg 1980

tgaacgccgc agccgttcgt gctctgcctg ttctggagaa aatccagaaa gagtctggta 2040

aagcctcgct ggcggatatc atagtgctgg ctggtgtggt tggtgttgag aaagccgcaa 2100

gcgccgcagg tttgagcatt catgtaccgt ttgcgccggg tcgcgttgat gcgcgtcagg 2160

atcagactga cattgagatg tttgagctgc tggagccaat tgctgacggt ttccgtaact 2220

atcgcgctcg tctggacgtt tccaccaccg agtcactgct gatcgacaaa gcacagcaac 2280

tgacgctgac cgcgccggaa atgactgcgc tggtgggcgg catgcgtgta ctgggtgcca 2340

acttcgatgg cagcaaaaac ggcgtcttca ctgaccgcgt tggcgtattg agcaatgact 2400

tcttcgtgaa cttgctggat atgcgttacg agtggaaagc gaccgacgaa tcgaaagagc 2460

tgttcgaagg ccgtgaccgt gaaaccggcg aagtgaaatt tacggccagc cgtgcggatc 2520

tggtgtttgg ttctaactcc gtcctgcgtg cggtggcgga agtttacgcc agtagcgatg 2580

cccacgagaa gtttgttaaa gacttcgtgg cggcatgggt gaaagtgatg aacctcgacc 2640

gtttcgacct gctggattat aaagatgatg atgataaaat ggccatggtt gacacactta 2700

gcggccttag ctccgaacaa ggccagagcg gcgatatgac gatcgaagaa gactccgcca 2760

cgcacatcaa gttcagcaaa cgcgacgagg atggcaagga actggccggc gccacaatgg 2820

aacttcgcga cagcagcggc aaaacgatca gcacatggat cagcgatggc caagttaagg 2880

acttctatct ttatccgggc aagtacacgt tcgtcgaaac agccgcccca gatggctatg 2940

aggttgccac agccatcacg tttacggtca acgaacaagg ccaagttacg gttaatggca 3000

aggccacgaa aggagatgcc catatcgatt aa 3032

Claims (10)

1. spore surface shows the bacillus subtilis engineering bacteria of glucose oxidase and catalase altogether, integration vector is utilized PDG1730 merge with gemma capsid protein cotX and glucose oxidase gene god-SpyTag the fusion piece of acquisition Section cotX-god-SpyTag connection, then converts bacillus subtilis WB800n, obtains recombined bacillus subtilis WB800n- CotX-god-SpyTag, recycle free plasmid pHT01 and withered grass strong promoter P43 gene, catalase cat gene and SpyCatcher gene carries out the fusion segment P43-cat-SpyCatcher connection of fusion acquisition, and then electrotransformation recombinates Bacillus subtilis WB800n-cotX-god-SpyTag competent cell to get.

2. bacillus subtilis engineering bacteria as described in claim 1, which is characterized in that the fusion segment cotX- God-SpyTag nucleotide sequence is as shown in SEQ ID No.3.

3. bacillus subtilis engineering bacteria as described in claim 1, which is characterized in that the fusion segment P43-cat- SpyCatcher nucleotide sequence is as shown in SEQ ID No.4.

4. bacillus subtilis engineering bacteria described in claim 1 is preparing the application in gluconate.

5. application as claimed in claim 4, which is characterized in that steps are as follows:

(i) will the activated culture of above-mentioned bacillus subtilis engineering bacteria, expand culture after, in 35~38 DEG C of fermentation 96h, be centrifuged, Gemma is taken, the glucose oxidase and catalase shown altogether in spore surface is made；

(ii) that glucose oxidase and catalase that spore surface made from step (i) is shown altogether are added to glucose is molten In liquid, reaction solution is made；

(iii) by reaction solution made from step (ii) under the conditions of 30~70 DEG C, pH4.0~8.0, it is stirred to react 12~for 24 hours, Mixed solution is made；

(iv) metal aqueous slkali will be added in mixed solution made from step (iii) to pH is 10, and metal base mixed liquor is made；

(v) immobilised enzymes in metal base mixed liquor made from step (iv) being separated, reaction solution is rotated, is crystallized by washing recycling, It is dried to obtain gluconate.

6. application as claimed in claim 5, which is characterized in that in the step (i), activation culture condition is 35~38 DEG C, 180~220rpm cultivates 12~16h, and activation medium is LB liquid medium, and component is as follows:

10g/L peptone, 5g/L yeast extract, 10g/L NaCl, pH7.0.

7. application as claimed in claim 5, which is characterized in that in the step (i), expand condition of culture be 35~38 DEG C, 180~220rpm cultivates 84~108h, and expansion culture medium is TB culture medium, and component is as follows:

Glycerine 15mL/L, tryptone 12g/L, yeast extract 24g/L, MgCl₂2.5g/L, 17mM KH₂PO₄, 72mM K₂HPO₄；

Preferably, in the step (i), centrifugal condition are as follows: 4 DEG C, be centrifuged 10min under conditions of 8000rpm.

8. application as claimed in claim 5, which is characterized in that in the step (ii), the concentration of glucose solution is 20~ 200mM；Preferably 100mM.

9. application as claimed in claim 5, which is characterized in that in the step (iii), reaction temperature is 40~50 DEG C, instead Answering pH is 6.0~7.0, and stirring rate is 150~400rpm, and the reaction time is 4~8h；

It is further preferred that reaction temperature is 40 DEG C, reaction pH is 6.0, and stirring rate is 200~240rpm, and the reaction time is 6h。

10. application as claimed in claim 5, which is characterized in that in the step (iv), the concentration of metal aqueous slkali is 50~ 500mM；

It is further preferred that the concentration of the metal aqueous slkali is 200~400mM；

Preferably, in the step (iv), separation is using centrifugation.