CN106754599B

CN106754599B - The engineering bacteria and its construction method of expression glucose dehydrogenase and application

Info

Publication number: CN106754599B
Application number: CN201611153688.2A
Authority: CN
Inventors: 孙静文; 周卫; 程明芳; 李书田; 王玉军
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2016-12-14
Filing date: 2016-12-14
Publication date: 2018-10-26
Anticipated expiration: 2036-12-14
Also published as: CN109055419B; CN106754599A; CN109055419A

Abstract

The invention discloses the engineering bacteria of expression glucose dehydrogenase and its construction method and applications.The engineering bacteria of expression glucose dehydrogenase provided by the present invention is with the active recombinant microorganism of Soluble phosphorus.It should be prepared with the active recombinant microorganism of Soluble phosphorus by assigning recipient microorganism glucose dehydrogenase activity；The Soluble phosphorus activity with the active recombinant microorganism of Soluble phosphorus is higher than the recipient microorganism.The present invention has the active recombinant microorganism of Soluble phosphorus by the activity structure for assigning recipient microorganism (bacillus megaterium WH320) glucose dehydrogenase CrGDH3A, the recombinant microorganism is to 11.59 times that the phosphate solubilization of tricalcium phosphate is recipient microorganism, phosphate solubilization to aluminum phosphate is 16.23 times of its corresponding recipient microorganism, and the phosphate solubilization to ground phosphate rock is 14.00 times of its corresponding recipient microorganism.

Description

The engineering bacteria and its construction method of expression glucose dehydrogenase and application

Technical field

The present invention relates to the engineering bacteria that glucose dehydrogenase is expressed in biological field and its construction method and applications.

Background technology

In agricultural production, increasing the p application rate is the approach of a kind of " high investment, low output ".China consumes about 2100 every year Ten thousand~22,000,000 tons of phosphate fertilizer, but phosphate fertilizer this season crop utilization rate is only 5%-25%, after 90% or so phosphate fertilizer is manured into soil It is fixed by chemistry quickly, form the compounds such as dissolubility extremely low calcium phosphate, iron, aluminium.Phosphorus is non-renewable resources, with phosphorus ore The continuous consumption of deposit, China may face phosphorus ore shortage and seriously restrict grain-production.This in soil does not lack phosphorus in fact, still Its validity in the soil is very low, and mostly the Phos of slightly solubility, plant are difficult to be absorbed and utilized.Therefore, in activating soil Invalid phosphorus element is one of agricultural production urgent problem.

Glucose dehydrogenase (glucose dehydrogenase, GDH) belongs to a member of short chain alcohols dehydrogenase family, In the presence of coenzyme, D-Glucose can be catalyzed and be converted to maltonic acid-delta-lactone, and the maltonic acid-δ-generated is interior Ester further can spontaneously be hydrolyzed into gluconic acid.It researches and develops the glucose dehydrogenase with high activity and then builds glucose dehydrogenation Enzyme Soluble phosphorus engineering bacteria can significantly improve the ability that phosphorus bacteria fertilizer decomposes Inorganic Phosphorus Fractions in Soil, in the invalid phosphorus element of activating soil, raising Phosphate fertilizer utilization efficiency reduces phosphate fertilizer input side face with substantial worth.

Invention content

The technical problem to be solved by the present invention is to how build with the active microorganism of Soluble phosphorus.

In order to solve the above technical problems, the present invention provides with the active recombinant microorganism of Soluble phosphorus.

It is provided by the present invention that there is the active recombinant microorganism of Soluble phosphorus, it is by assigning recipient microorganism glucose dehydrogenation It is prepared by enzymatic activity；The Soluble phosphorus activity with the active recombinant microorganism of Soluble phosphorus is higher than the recipient microorganism.

In above-mentioned recombinant microorganism, the imparting recipient microorganism glucose dehydrogenase activity is by by glucose dehydrogenase Encoding gene import recipient microorganism in realize.

In order to solve the above technical problems, the present invention provides method of the structure with Soluble phosphorus reconstituted protein microorganism.

Method of the structure with Soluble phosphorus reconstituted protein microorganism provided by the present invention, includes by the volume of glucose dehydrogenase Code channel genes recipient microorganism, obtain Soluble phosphorus activity has Soluble phosphorus reconstituted protein microorganism higher than the recipient microorganism； The glucose dehydrogenase is protein a) or b) or c) or d)：

A) protein that amino acid sequence forms shown in SEQ ID No.2；

B) protein that amino acid sequence forms shown in SEQ ID No.6；

C) fusion protein that the c-terminus of the protein shown in a) or b) or/and aminoterminal fusion protein label obtain；

D) amino acid sequence shown in SEQ ID No.2 or SEQ ID No.6 is passed through into one or several amino acid residues Substitution and/or the protein with glucose dehydrogenase activity that lacks and ors add.

In the application, the Soluble phosphorus activity refers to converting inorganic phosphorus into the ability of titanium pigment.Wherein, titanium pigment refers to The phosphorus that can be dissolved into water or can be absorbed and used by plants after being dissolved into weak acid.Titanium pigment includes water-soluble phosphorus and/or can Exchangeability phosphorus.The Phos can be the phosphate (such as tricalcium phosphate or aluminum phosphate) or ground phosphate rock of indissoluble.

In the above method, a) shown in protein entitled CrGDH3A；SEQ ID No.2 are by 796 amino acid residues Composition.

In the above method, b) shown in protein entitled CrGDH3A-His, CrGDH3A shown in SEQ ID No.2 The obtained fusion proteins of N-terminal connection MGSSHHHHHHSSGLVPRGSHM, SEQ ID No.6 are by 817 amino acid residue groups At.

In the above method, protein tag refers to utilizing DNA extracorporeal recombinations, the one of amalgamation and expression together with destination protein Kind polypeptide or albumen, in order to the expression of destination protein, detection, tracer and/or purifying etc..

The encoding gene concretely it is following 1) or 2) or 3) shown in glucose dehydrogenase encoding gene：

1) coded sequence (CDS) is DNA molecular shown in SEQ ID No.1, entitled CrGDH3A genes；

2) coded sequence is DNA molecular shown in SEQ ID No.5, entitled CrGDH3A-His genes；

1) or 2) 3) with the DNA molecular that limits with 90% or more homogeneity and the coding glucose dehydrogenase DNA molecular.

In the encoding gene, " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " can use meat Eye or computer software are evaluated.Using computer software, the homogeneity between two or more sequences can use percentage (%) is indicated, can be used for evaluating the homogeneity between correlated series.

In above-mentioned recombinant microorganism or method, the recipient microorganism can be prokaryotic micro-organisms.

In above-mentioned recombinant microorganism or method, concretely gramnegative bacterium or gram are positive for the prokaryotic micro-organisms Property bacterium.

In above-mentioned recombinant microorganism or method, the gramnegative bacterium concretely Escherichia bacteria.It is described Gram-positive bacterium concretely bacillus.

In above-mentioned recombinant microorganism or method, the Escherichia bacteria concretely Escherichia coli.The gemma bar Campylobacter bacteria can be bacillus megaterium.

In above-mentioned recombinant microorganism or method, the encoding gene of above-mentioned glucose dehydrogenase can pass through recombinant expression carrier PET-CrGDH3A import the recipient microorganism；PET-the CrGDH3A are the CrGDH3A genes shown in SEQ ID No.1 Replace the recombinant expression carrier that the segment between NdeI the and BamHI recognition sites of pET-28a (+) obtains.PET-CrGDH3A contain CrGDH3A-His encoding genes shown in SEQ ID No.5, the protein C rGDH3A- of CrGDH3A-His encoding genes coding The amino acid sequence of His is as shown in SEQ ID No.6.CrGDH3A-His is that the N-terminal of CrGDH3A shown in SEQ ID No.2 connects Connect the fusion protein that MGSSHHHHHHSSGLVPRGSHM is obtained.

In above-mentioned recombinant microorganism or method, the encoding gene of above-mentioned glucose dehydrogenase can also pass through recombinant expression carrier PWH-CrGDH3A import the recipient microorganism；PWH-the CrGDH3A are the CrGDH3A genes shown in SEQ ID No.1 Forward direction replaces the recombinant expression carrier that the segment between 2 BamHI recognition sites of pWH1520 obtains.PWH-CrGDH3A are containing orderly CrGDH3A genes shown in SEQ ID No.1 in list, pWH-CrGDH3A expression is protein shown in SEQ ID No.2 CrGDH3A。

The application of above-mentioned recombinant microorganism or method in dissolved metals also belongs to protection scope of the present invention.

In above application, the Phos can be the phosphate (such as tricalcium phosphate or aluminum phosphate) or ground phosphate rock of indissoluble.

Protection scope of the present invention is also belonged to the relevant biomaterial of the glucose dehydrogenase, the biomaterial is B1) or B2):B1) contain the expression cassette of the encoding gene；B2) contain the recombinant vector of the encoding gene.

Above-mentioned B2) in recombinant vector concretely above-mentioned pWH-CrGDH3A or pET-CrGDH3A.

Application of the above-mentioned biomaterial in preparing glucose dehydrogenase also belongs to protection scope of the present invention.

Above-mentioned biomaterial also belongs to the protection model of the present invention preparing the application in having Soluble phosphorus reconstituted protein microorganism It encloses.

It is demonstrated experimentally that in pattern bacterium prokaryotic expression (Escherichia coli are recipient bacterium), glucose dehydrogenase CrGDH3A and CrGDH3A-His all has higher glucose dehydrogenase activity, and glucose dehydrogenase CrGDH3A-His is 25 DEG C of pH7.8's Under the conditions of its glucose dehydrogenase enzyme activity be 39.47 ± 1.03U/mg albumen；(the bacillus megaterium in Soluble phosphorus engineering bacteria WH320 is recipient bacterium), glucose dehydrogenase CrGDH3A is at 40 DEG C, the enzyme activity of the glucose dehydrogenase under conditions of pH7.4 For 36.53 ± 1.16U/mg.The present invention is by assigning recipient microorganism (bacillus megaterium WH320) glucose dehydrogenase CrGDH3A activity structure have the active recombinant microorganism of Soluble phosphorus, the recombinant microorganism to the phosphate solubilization of tricalcium phosphate be by 11.59 times of body microorganism, the phosphate solubilization to aluminum phosphate is 16.23 times of its corresponding recipient microorganism, to ground phosphate rock Phosphate solubilization is 14.00 times of its corresponding recipient microorganism.The present invention is to cultivate phosphorus efficiency farming using genetic engineering means The bioengineered strain of object new varieties and efficient activating soil phosphorus nutrients provides important gene resource, helps to push Soluble phosphorus work Journey bacterium strides forward from laboratory development phase to the Field information stage.

Description of the drawings

Fig. 1 is the physical map of pET-CrGDH3A and pET-CrGDH3B.Wherein, gdh3 be CrGDH3A genes or CrGDH3B genes.

Fig. 2 is the SDS-PAGE collection of illustrative plates of the induced expression glucose dehydrogenase in Escherichia coli.Wherein, 1：Albumen Marker；2：E. coli bl21 (DE3)；3：pET–CrGDH3A/BL21；4：pET-30a(+)/BL21；5：pET- CrGDH3B/BL21.Arrow shows purpose band.

Fig. 3 is the physical map of pWH-CrGDH3A.Wherein, gdh3gene is CrGDH3A genes.

Fig. 4 is the SDS-PAGE collection of illustrative plates that glucose dehydrogenase is expressed in glucose dehydrogenase engineering bacteria.Wherein, M：Albumen Marker；1：Intracellular precipitates；2：Intracellular supernatant；3：Extracellular supernatant.

Fig. 5, which is pH, influences the enzymatic activity for the glucose dehydrogenase expressed in glucose dehydrogenase engineering bacteria.

Fig. 6, which is temperature, influences the enzymatic activity for the glucose dehydrogenase expressed in glucose dehydrogenase engineering bacteria.

Specific implementation mode

The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The preparation and functional verification of embodiment 1, glucose dehydrogenase CrGDH3

One, the structure of recombinant expression carrier

In order to improve the activity of glucose dehydrogenase, by Genbank Accession Number WP_012904518 institutes Show from citric acid bacillus Citrobacter rodentium grape glucocorticoid dehydrogenases (hereinafter referred to as CrGDH3B, abbreviation 3B) into The replacement of row amino acid residue obtains glucose dehydrogenase CrGDH3A (abbreviation 3A).The amino acid sequence of CrGDH3A is SEQ ID The amino acid sequence of No.2, CrGDH3B are differing amino acid residues such as 1 He of table of SEQ ID No.4, CrGDH3A and CrGDH3B Table 2.

The differing amino acid residues of table 1, CrGDH3A and CrGDH3B

The differing amino acid residues of table 2, CrGDH3A and CrGDH3B

CrGDH3B genes shown in CrGDH3A genes and SEQ ID No.3 shown in SEQ ID No.1 are prepared respectively.

The CrGDH3A genes shown in SEQ ID No.1 replace pET-28a (+), and (EMD Biosciences, are purchased in north Capital company of fresh warp thread section, size 5369bp) NdeI and Bam HI recognition sites between segment, keep pET-28a (+) other sequences It arranges constant, obtains recombinant expression carrier, be named as pET-CrGDH3A (Fig. 1).PET-CrGDH3A contain SEQ ID No.5 Shown in His tag fusion protein CrGDH3A-His encoding genes, CrGDH3A-His encoding genes coding protein The amino acid sequence of CrGDH3A-His is as shown in SEQ ID No.6.CrGDH3A-His is shown in SEQ ID No.2 The fusion protein that the N-terminal connection MGSSHHHHHHSSGLVPRGSHM of CrGDH3A is obtained.

The CrGDH3B genes shown in SEQ ID No.3 replace pET-28a (+), and (EMD Biosciences, are purchased in north Capital company of fresh warp thread section, size 5369bp) NdeI and Bam HI recognition sites between segment, keep pET-28a (+) other sequences It arranges constant, obtains recombinant expression carrier, be named as pET-CrGDH3B (Fig. 1).PET-CrGDH3B contain SEQ ID No.7 Shown in His tag fusion protein CrGDH3B-His encoding genes, CrGDH3B-His encoding genes coding protein The amino acid sequence of CrGDH3B-His is as shown in SEQ ID No.8.CrGDH3B-His is shown in SEQ ID No.4 The fusion protein that the N-terminal connection MGSSHHHHHHSSGLVPRGSHM of CrGDH3B is obtained.

Two, the preparation of the recombination bacillus coli of expression glucose dehydrogenase

1, the expression of CrGDH3A-His

PET-the CrGDH3A of step 1 Calcium Chloride Methods are converted into e. coli bl21 (DE3) (Tiangeng company), utilize card That chloramphenicol resistance screening positive clone screening and culturing, picking monoclonal, with P1 (5 '-ATGGCTATTAACAATACAGGCTC-3 ') It is that primer carries out PCR identifications with P2 (5 '-TTATTTCACATCATCCGGCAGCG-3 '), PCR is identified to obtain 2391bp PCR The positive colony of product is named as pET-CrGDH3A/BL21 as genetic engineering bacterium.Picking pET-CrGDH3A/BL21 bacterial strains, (it is a concentration of to kanamycins that kanamycins is added in the LB culture mediums for being inoculated in the kanamycins containing 100ug/ml in LB culture mediums The culture medium that 100 μ g/ml are obtained) in, 37 DEG C of cultures to 0D₆₀₀Value is (using the LB culture mediums containing 100 μ g/ml kanamycins as blank Control) when reaching 0.6, IPTG to final concentration l mM is added, 28 DEG C of induction 6h under the rotating speed of 150r/min collect culture solution warp After 4000r/min centrifuges 20min, it is 10 that thalline, which is resuspended, to obtain thalline content with 50mM Tris-HCl (pH7.1)⁸Cfu/ml's Thallus suspension liquid, thallus suspension liquid are suspended in smudge cells through ultrasonication 30min (50% power, work 10s, interval 20s) Triton-X100 to final concentration of 1% is added in liquid, is extracted at 4 DEG C overnight, 12 000r/min centrifuge 10min, collect supernatant Liquid (mycetome gross protein), CrGDH3A-His crude enzyme liquids are named as by the supernatant.

2, the expression of CrGDH3B-His

PET-the CrGDH3B of step 2 Calcium Chloride Methods are converted into e. coli bl21 (DE3) (Tiangeng company), utilize card That chloramphenicol resistance screening positive clone screening and culturing, picking monoclonal, with P3 (5 '-ATGGCTGAAAACAATGCACG-3 ') and P4 (5 '-TTACTTCTCGTCGTCCGGCA-3 ') is that primer carries out PCR identifications, and PCR is identified to obtain 2391bp PCR products Positive colony is named as pET-CrGDH3B/BL21 as genetic engineering bacterium.Picking pET-CrGDH3B/BL21 bacterial strains, are inoculated in (kanamycins is added to a concentration of 100 μ g/ of kanamycins in LB culture mediums containing 100 μ g/ml kanamycins in LB culture mediums The culture medium that ml is obtained) in, 37 DEG C of cultures to 0D₆₀₀It is worth (using the LB culture mediums containing 100 μ g/ml kanamycins as blank control) When reaching 0.6, IPTG to final concentration l mM is added, 28 DEG C of induction 6h under the rotating speed of 150r/min collect culture solution warp After 4000r/min centrifuges 20min, it is 10 that thalline, which is resuspended, to obtain thalline content with 50mM Tris-HCl (pH7.1)⁸Cfu/ml's Thallus suspension liquid, thallus suspension liquid are suspended in smudge cells through ultrasonication 30min (50% power, work 10s, interval 20s) Triton-X100 to final concentration of 1% is added in liquid, is extracted at 4 DEG C overnight, 12 000r/min centrifuge 10min, collect supernatant Liquid (mycetome gross protein), CrGDH3B-His crude enzyme liquids are named as by the supernatant.

3, empty vector control bacterium

PET-28a (+) is transferred to e. coli bl21 (DE3) according to method identical with step 1, obtained recombination is big Entitled pET-28a (+)/BL21 of enterobacteria.Using pET-28a (+)/BL21 as empty vector control bacterium according to the side of above-mentioned steps 1 Method carries out induced expression and prepares bacterial protein.Picking pET-28a (+)/BL21 bacterial strains is inoculated in containing 100 μ g/ml kanamycins LB culture mediums (the obtained culture mediums of a concentration of 100 μ g/ml that kanamycins to kanamycins are added in LB culture mediums) in, 37 DEG C are cultivated to 0D₆₀₀When value reaches 0.6 (using the LB culture mediums containing 100 μ g/ml kanamycins as blank control), IPTG is added Culture solution is collected after 4000r/min centrifuges 20min, is used to final concentration l mM, 28 DEG C of induction 6h under the rotating speed of 150r/min It is 10 that 50mM Tris-HCl (pH7.1), which are resuspended thalline and obtain thalline content,⁸The thallus suspension liquid of cfu/ml, thallus suspension liquid warp Ultrasonication 30min (50% power, work 10s, interval 20s), is added Triton-X100 to end in smudge cells suspension A concentration of 1%, it is extracted at 4 DEG C overnight, 12 000r/min centrifuge 10min, supernatant (mycetome gross protein) are collected, by this Supernatant is named as empty vector control bacterium crude enzyme liquid.

4, blank control bacterium e. coli bl21 (DE3)

E. coli bl21 (DE3) is subjected to induced expression preparation as blank control bacterium according to the method for above-mentioned steps 1 Bacterial protein.Picking e. coli bl21 (DE3) bacterial strain, is inoculated in LB culture mediums, 37 DEG C of cultures to 0D₆₀₀Value (is trained with LB It is blank control to support base) when reaching 0.6, IPTG to final concentration l mM is added, 28 DEG C of induction 6h, are received under the rotating speed of 150r/min Collect culture solution after 4000r/min centrifuges 20min, obtaining thalline content with 50mM Tris-HCl (pH7.1) resuspension thalline is 10⁸The thallus suspension liquid of cfu/ml, thallus suspension liquid through ultrasonication 30min (50% power, work 10s, interval 20s), Triton-X100 to final concentration of 1% is added in smudge cells suspension, is extracted at 4 DEG C overnight, 12 000r/min centrifugations 10min collects supernatant (mycetome gross protein), which is named as blank control bacterium crude enzyme liquid.

30 μ L CrGDH3A-His crude enzyme liquids are taken (to come from 10⁸cfu/ml pET–CrGDH3A/BL21)、30μL CrGDH3B-His crude enzyme liquids (come from 10⁸Cfu/ml pET-CrGDH3B/BL21), 30 μ L empty vector control bacterium crude enzyme liquids (come from 10⁸Cfu/ml pET-28a (+)/BL21) and 30 μ L blank control bacterium crude enzyme liquids (come from 10⁸Cfu/ml e. coli bl21s (DE3)) SDS-PAGE analyses (resolving gel concentration 12%), the sample-adding pore volume on the glue and shape are carried out on same glue Consistent, sample-adding pore volume is 80 μ L.

The results are shown in Figure 2 by SDS-PAGE, although showing CrGDH3A-His crude enzyme liquids, CrGDH3B-His crude enzyme liquids, sky Have the band of 87kD in vehicle Control bacterium crude enzyme liquid and blank control bacterium crude enzyme liquid, but CrGDH3A-His crude enzyme liquids and The content of 87kD polypeptides is apparently higher than empty vector control bacterium crude enzyme liquid and blank control bacterium crude enzyme liquid in CrGDH3B-His crude enzyme liquids The content of middle 87kD polypeptides, and the content of the 87kD polypeptides in CrGDH3A-His crude enzyme liquids is higher than CrGDH3B-His crude enzyme liquids The content of middle 87kD polypeptides.Illustrate that CrGDH3A-His and CrGDH3B-His have obtained table in e. coli bl21 (DE3) It reaches, and expression quantity of the CrGDH3A-His in e. coli bl21 (DE3) is apparently higher than CrGDH3B-His in Escherichia coli Expression quantity in BL21 (DE3).

Three, the catalytic activity of the glucose dehydrogenase of CrGDH3A-His and CrGDH3B-His is measured

Take CrGDH3A-His crude enzyme liquids, CrGDH3B-His crude enzyme liquids, empty vector control bacterium crude enzyme liquid and the sky of step 2 White control bacterium crude enzyme liquid use respectively nickel column (the high-affinity Ni-NTA Rasin products for being purchased from AM General company) into Row purifying, nickel column is pre-processed, and crude enzyme liquid is added, and (the 50mM NaH containing imidazole elution are then added₂PO₄, 300mM NaCl, 250mM imidazole, pH8.0) 4 DEG C of effect 10min, 3000rpm centrifuges 1min and collects eluent, repeats to elute primary, receives Collect eluent, 1ml eluents is taken to carry out SDS-PAGE analyses.The sequencing results of CrGDH3A-His show the 15 of its N-terminal A amino acid is the 1-15 amino acids of sequence 2 in sequence table, and the sequencing results of CrGDH3B-His show its N-terminal 15 amino acid be sequence table in sequence 4 1-15 amino acids.

The eluent of above-mentioned collection is dialysed with distilled water, removes salt ion, obtain respectively pure CrGDH3A-His enzyme solutions, Pure CrGDH3B-His enzyme solutions, pure empty vector control bacterium enzyme solution and pure blank control bacterium enzyme solution, as enzyme solution to be measured.It waits for It surveys enzyme solution and measures protein content using BCA quantification of protein kit quantifications.

Glucose dehydrogenase activity measures, and using glucose as substrate, colorimetric analysis grape is carried out according to red is generated The activity of glucocorticoid dehydrogenase.To 50 μ L enzyme solutions to be measured (pure CrGDH3A-His enzyme solutions, pure CrGDH3B-His enzyme solutions, pure sky Vehicle Control bacterium enzyme solution or pure blank control bacterium enzyme solution) in be added 50 μ L pH7.8 buffer solution (to 100mmol/L MOPS PQQ (pyrroloquinoline quinone) and CaCl is added in buffer solution₂, it is 10 μm of ol/L and CaCl to make PQQ contents₂Content obtains for 2mol/L Liquid), 37 DEG C pretreatment 1 hour to stablize the structure of enzyme, obtain pretreatment enzyme solution.Then, it is added 1mL's into cuvette The Tris-HCl buffer solutions of the pH7.8 of 50mmol/L, the 20mmol/L phenazine methosulfates for being then separately added into 100 μ L again are molten Liquid, 2,6-sodium dichlorophenol indophenolate (DCIP) solution of 6.7mmol/L and 1mol/L glucose solutions, are added 50 μ L after mixing Pretreatment enzyme solution, be finally settled to 3mL, reaction temperature is 25 DEG C, measures the variation of light absorption value under 600nm per minute.Enzyme activity Unit of force (U) is defined as：Under conditions of 25 DEG C of pH7.8, glycoxidative (or the 1 μm of ol of the grape of 1 μm of ol can be made in 1min DCIP restore) enzyme amount.Glucose dehydrogenase Rate activity is calculated with the vigor of enzyme in per unit total protein, unit U/ mg。

Experiment is set to be repeated three times.The result shows that pure empty vector control bacterium enzyme solution and pure blank control bacterium enzyme solution do not have Portugal Grape glucocorticoid dehydrogenase activity, the enzyme activity by the glucose dehydrogenase of the CrGDH3A-His of pET-CrGDH3A/BL21 expression are 39.47 ± 1.03U/mg albumen, by the enzyme activity of the glucose dehydrogenase of the CrGDH3B-His of pET-CrGDH3B/BL21 expression For 7.39 ± 0.26U/mg albumen.The glucose dehydrogenase enzyme activity of CrGDH3A-His is CrGDH3B-His glucose dehydrogenases 5.34 times of enzyme activity.The glucose dehydrogenase yield of pET-CrGDH3A/BL21 is 25.65/10⁸cfu pET–CrGDH3A/ The glucose dehydrogenase yield of BL21, pET-CrGDH3B/BL21 are 5.09U/10⁸cfu pET–CrGDH3B/BL21。pET– The glucose dehydrogenase yield of CrGDH3A/BL21 is 5 times of pET-CrGDH3B/BL21.

Embodiment 2, the cultivation with Soluble phosphorus reconstituted protein microorganism-glucose dehydrogenase Soluble phosphorus engineering bacteria and its Identification

1. the structure of glucose dehydrogenase CrGDH3A gene shuttle expression carriers

In order to obtain the Soluble phosphorus engineering bacteria of high efficient expression glucose dehydrogenase CrGDH3A genes, it is necessary first to which structure can Across the shuttle expression carrier of host expresses.PWH1520 expression vectors (7929bp) are the efficient shuttling expressings of bacillus megaterium Carrier (German MoBiTec Products are purchased in Beijing Baeyer enlightening biotech company), xylA promoters downstream carry BamHI restriction enzyme sites have ammonia benzyl and tetracycline resistance gene, can stablize express express target protein.

CrGDH3A gene orders are analyzed using DNAMAN softwares, discovery does not have BamHI restriction enzyme sites, according to CrGDH3A bases Because of complete coding region primers, Bam HI restriction enzyme sites (GGATCC) are added in upstream and downstream primer.Upstream and downstream primer Respectively：P5：5′-ATGGATCCATGGCTATTAACAATACAGGCTC-3 ' and P6：5′- GCGGATCCTTATTTCACATCATCCGGCAGCG-3′).Using the pET-CrGDH3A of step 1 as template, using above-mentioned P5 and P6 is as primer, and using the method for PCR amplification, BamHI enzymes are introduced respectively at 5 ' ends of CrGDH3A genes complete coding region and 3 ' Recognition site obtains the CrGDH3A gene PCR products with enzyme recognition site；With BamHI digestion shuttle expression carriers PWH1520 and CrGDH3A gene PCR products with enzyme recognition site, the digestion products T of recycling₄Ligase connects, connection Screening positive clone after product conversion, sequencing.Due to being to be connected to expression vector after single endonuclease digestion, so also needing using PCR simultaneously The positive bacterial plaque of shuttle expression carrier pWH1520, extraction weight are inserted into conjunction with the method screening CrGDH3A gene forward directions that sequencing compares Group plasmid, the final shuttle expression carrier for obtaining CrGDH3A genes.Sequencing result is shown to be shown in SEQ ID No.1 CrGDH3A gene forward directions are replaced the recombinant expression carrier that the segment between 2 BamHI recognition sites of pWH1520 obtains and are named as PWH-CrGDH3A (Fig. 3).PWH-CrGDH3A contain CrGDH3A genes, pWH-shown in SEQ ID No.1 in ordered list CrGDH3A expression is protein C rGDH3A shown in SEQ ID No.2.

2. the zymetology feature of the CrGDH3A of acquisition and its expression of glucose dehydrogenase engineering bacteria

Using protoplast transformation, recombinant vector pWH-CrGDH3A is transferred to bacillus megaterium, and (WH320 is purchased Hai Beinuo biotechnologies company), obtain glucose dehydrogenase engineering bacteria.Glucose dehydrogenase engineering bacteria is accessed containing tetracycline In LB culture mediums (culture medium that a concentration of 100 μ g/ml of tetracycline to tetracycline are obtained is added in LB culture mediums), cultivated Night.It is transferred in the above-mentioned LB culture mediums containing tetracycline with 2% inoculum concentration and continues culture to exponential phase, xylose is added and arrives Final concentration of 0.5%, Fiber differentiation 6h, 4000r/min rotating speeds centrifugation 15min, collects supernatant as extracellular supernatant at room temperature；It receives Collection precipitation, adds 2 times of volume phosphate buffers (pH6.0), smudge cells to obtain smudge cells suspension.In smudge cells suspension Middle addition Triton-X100 to final concentration of 1% is extracted overnight at 4 DEG C, and 12000r/min centrifuges 10min, and supernatant is intracellular Supernatant is precipitated as intracellular precipitation.Respectively to intracellular supernatant, intracellular precipitation and extracellular supernatant carry out SDS-PAGE electrophoretic analysis and Enzyme activity determination.Enzyme solution to be measured measures protein content using BCA quantification of protein kit quantifications.

The activity of glucose dehydrogenase CrGDH3A is measured according to the method for embodiment 1, experiment is in triplicate.SDS-PAGE Electrophoresis result (Fig. 4) shows that CrGDH3A genes can be with normal expression, expression product in glucose dehydrogenase engineering bacteria For intracellular protein (intracellular supernatant swimming lane), expression product molecular weight is about 87kD.Glucose dehydrogenase engineering bacterium expression The glucose dehydrogenase enzyme activity of CrGDH3A is 33.85 ± 1.53U/mg.

3, influences of the pH to the catalytic activity of glucose dehydrogenase engineering bacteria

The glucose dehydrogenase engineering bacteria of step 2 is accessed the LB culture mediums containing ampicillin and tetracycline (to train in LB It is the culture that 100 μ g/ml are obtained to support the concentration of addition ampicillin and tetracycline to ampicillin and tetracycline in base Base) in, overnight incubation.It is transferred in the above-mentioned LB culture mediums containing tetracycline with 2% inoculum concentration and continues culture to logarithmic growth Xylose is added to final concentration of 0.5%, Fiber differentiation 6h in phase, and 4000r/min rotating speeds centrifugation 15min, collects precipitation at room temperature, 2 times of volume phosphate buffers (pH7.0), smudge cells are added to obtain smudge cells suspension.It is added in smudge cells suspension Triton-X100 to final concentration of 1% is extracted overnight at 4 DEG C, and 12000r/min centrifuges 10min, and supernatant is that glucose is de- Hydrogen enzyme crude enzyme liquid, as enzyme solution to be measured.

Using the different reaction system of 10 pH value：Citrate-phosphate buffer solution system (pH5.4 reaction systems, PH5.8 reaction systems, pH6.2 reaction systems, pH6.6 reaction systems and pH7.0 reaction systems)；Tris buffer solution systems (pH7.4 reaction systems, pH7.8 reaction systems, pH8.2 reaction systems, pH8.6 reaction systems and pH9.0 reaction systems) measures Glucose dehydrogenase engineering bacteria is catalyzed the ability of glucose dehydrogenation.

The different reaction system of above-mentioned 10 pH value is by enzyme solution to be measured, phenazine methosulfate, 2,6-sodium dichlorophenol indophenolate (DCIP), glucose and corresponding buffer solution composition.

The buffer solution that the corresponding pH value of 50 μ L is added into 50 μ L enzyme solutions to be measured (is added into 100mmol/L MOPS buffer solutions PQQ (pyrroloquinoline quinone) and CaCl₂, it is 10 μm of ol/L and CaCl to make PQQ contents₂Content is the liquid that 2mol/L is obtained), 37 DEG C Pretreatment obtains pretreatment enzyme solution in 1 hour to stablize the structure of enzyme.

It is separately added into the different pH buffer of the 50mmol/L of 1mL into cuvette, is then separately added into 20mmol/ again L phenazine methosulfates, 2,6-sodium dichlorophenol indophenolate (DCIP) of 6.7mmol/L and each 100 μ L of 1mol/L glucose are uniformly mixed The pretreatment enzyme solution of 50 μ L is added afterwards, is finally settled to 3mL, reaction temperature is 25 DEG C, measures light absorption value under 600nm per minute Variation.Enzyme activity unit (U) is defined as：Under the conditions of 25 DEG C, glycoxidative (or the 1 μm of ol of the grape of 1 μm of ol can be made in 1min DCIP restore) enzyme amount.Glucose dehydrogenase Rate activity is calculated with the vigor of enzyme in per unit total protein, unit U/ Mg converts relative activity using highest enzyme activity as 100%.Experiment is in triplicate.

The optimal pH of glucose dehydrogenase engineering bacteria catalytic activity is 7.4, is most in the enzymatic activity of pH 6.6-7.8 ranges The 80% of suitable pH enzymatic activitys, as pH5.4 and pH5.8, enzymatic activity is about the 20% of optimal pH enzymatic activity, as pH8.6 and pH 9 When, 15% (Fig. 5) of enzymatic activity less than optimal pH enzymatic activity.

4, influence of the temperature to the catalytic activity of glucose dehydrogenase engineering bacteria

The glucose dehydrogenase engineering bacteria of step 2 is accessed in the above-mentioned LB culture mediums containing ampicillin and tetracycline, Overnight incubation.It is transferred in the above-mentioned LB culture mediums containing tetracycline with 2% inoculum concentration and continues culture to exponential phase, be added Xylose centrifuges 15min to final concentration of 0.5%, Fiber differentiation 6h, at room temperature 4000r/min rotating speeds, collects precipitation, adds 2 times of bodies Product phosphate buffer (pH7.0), smudge cells obtain smudge cells suspension.Triton- is added in smudge cells suspension X100 to final concentration of 1% is extracted overnight at 4 DEG C, and 12000r/min centrifuges 10min, and supernatant is that glucose dehydrogenase is thick Enzyme solution, as enzyme solution to be measured.The buffer solution that 50 μ L pH7.4 are added into 50 μ L enzyme solutions to be measured (is buffered to 100mmol/L MOPS PQQ (pyrroloquinoline quinone) and CaCl is added in liquid₂, it is 10 μm of ol/L and CaCl to make PQQ contents₂Content is the liquid that 2mol/L is obtained Body), 37 DEG C pre-process 1 hour to stablize the structure of enzyme, obtain pretreatment enzyme solution.In Tris buffer solution systems (pH7.4), The ability of glucose dehydrogenase engineering bacteria catalysis glucose is measured in 20 DEG C of -70 DEG C of temperature ranges.Reaction system is by enzyme to be measured Liquid, phenazine methosulfate, 2,6-sodium dichlorophenol indophenolate (DCIP), glucose are each and Tris buffers molten (pH7.4) liquid system composition.To It is separately added into the Tris buffer solutions (pH7.4) of the 50mmol/L of 1mL in cuvette, is then separately added into 20mmol/L azophenlyene again Methylsulfate, 2,6-sodium dichlorophenol indophenolate (DCIP) of 6.7mmol/L and each 100 μ L of 1mol/L glucose, are added after mixing The pretreatment enzyme solution of 50 μ L, is finally settled to 3mL, measures the variation of light absorption value under 600nm per minute.Enzyme activity unit (U) is fixed Justice is：Under the conditions of relevant temperature pH7.4, the grape of 1 μm of ol can be made glycoxidative (or 1 μm of ol DCIP reduction) in 1min Enzyme amount.Glucose dehydrogenase Rate activity is calculated with the vigor of enzyme in per unit total protein, unit U/mg.

The optimal reactive temperature of the glucose dehydrogenase CrGDH3A of the glucose dehydrogenase engineering bacteria induced expression of step 2 It is 40 DEG C, the activity of enzyme is up to 36.53 ± 1.16U/mg, and the activity of enzyme maintains higher level in the range of 30 DEG C -45 DEG C, and 50 The activity of enzyme then shows rapid downward trend after DEG C, is difficult then to detect enzymatic activity (Fig. 6) more than 70 DEG C.

5, the effect of solubilizing phosphate of glucose dehydrogenase engineering bacteria

The glucose dehydrogenase engineering bacteria of step 2 and bacillus megaterium WH320 (recipient bacterium) are inoculated in phosphorus ore respectively In powder liquid culture medium, tricalcium phosphate fluid nutrient medium and aluminum phosphate fluid nutrient medium, make glucose dehydrogenase engineering bacteria and huge The content of Bacterium anthracoides WH320 is 10⁸Cfu/mL, at 37 DEG C, culture is to exponential phase, and xylose is added to final concentration of 0.5%, 37 DEG C of 160r/min shaking table cultures, the tricalcium phosphate culture medium and aluminum phosphate culture medium of inoculation took culture solution at the 7th day, And the ground phosphate rock culture medium being inoculated with took culture solution at the 14th day.10000r/min rotating speeds centrifuge 10min at 4 DEG C, collect supernatant, Using molybdenum antimony resistance colorimetric method, inoculation glucose dehydrogenase engineering bacteria is directly measured in wavelength 700nm with 722 type spectrophotometers And water-soluble phosphorus (also referred to as available phosphorus or rapid available phosphorus) content in bacillus megaterium WH320 (recipient bacterium) culture solution, if not connecing The corresponding control (CK) of bacterium, available phosphorus content given below are the value for deducting the corresponding control (CK) for not connecing bacterium, and experiment repeats 3 It is secondary.

Wherein, the pH of ground phosphate rock fluid nutrient medium is 7.0, and preparation method is as follows：It is water by solvent, solute and its concentration are such as Under culture solution sterilize at 115 DEG C 30min：Glucose 5g/L, xylose 5g/L, NaCl 0.2g/L, MgSO₄·7H₂O 0.1g/L, KCl 0.2g/L, (NH₄)₂SO₄0.5g/L, yeast extract 0.5g/L, 5 grams of ground phosphate rock (Chengjiang County of Yunnan Province Dong Tai phosphate fertilizer Co., Ltd, 30% P₂O₅Content, 13% phosphorus content), add distilled water to 1000ml.

The pH of tricalcium phosphate fluid nutrient medium is 7.0, and preparation method is as follows：It is water by solvent, solute and its concentration are as follows Culture solution sterilize at 115 DEG C 30min：Glucose 5g/L, xylose 5g/L, NaCl 0.2g/L, MgSO₄·7H₂O 0.1g/L, KCl 0.2g/L, (NH₄)₂SO₄0.5g/L, yeast extract 0.5g/L, tricalcium phosphate 5.0g/L add distilled water to 1000ml.

The pH of aluminum phosphate fluid nutrient medium is 7.0, and preparation method is as follows：It is water by solvent, solute and its concentration are following Culture solution sterilizes 30min at 115 DEG C：Glucose 5g/L, xylose 5g/L, NaCl 0.2g/L, MgSO₄·7H₂O 0.1g/L, KCl 0.2g/L, (NH₄)₂SO₄0.5g/L, yeast extract 0.5g/L, aluminum phosphate 5.0g/L add distilled water to 1000ml.

The result shows that the glucose dehydrogenase engineering bacteria of step 2 cultivates culture in 7 days in tricalcium phosphate fluid nutrient medium The content of the available phosphorus of liquid is 143.15 ± 7.16 μm of ol/L, and having for 7 days culture solutions is cultivated in aluminum phosphate fluid nutrient medium The content for imitating phosphorus is 78.90 ± 3.95 μm of ol/L, and the available phosphorus of 14 days culture solutions is cultivated in ground phosphate rock fluid nutrient medium Content is 34.57 ± 2.07 μm of ol/L；Bacillus megaterium WH320 as recipient bacterium is trained in tricalcium phosphate fluid nutrient medium The content for supporting the available phosphorus of 7 days culture solutions is 12.35 ± 0.62 μm of ol/L, is cultivated in aluminum phosphate fluid nutrient medium 7 days The content of the available phosphorus of culture solution is 4.86 ± 0.27 μm of ol/L, and 14 days culture solutions are cultivated in ground phosphate rock fluid nutrient medium The content of available phosphorus is 2.47 ± 0.12 μm of ol/L.As it can be seen that Soluble phosphorus of the glucose dehydrogenase engineering bacteria of step 2 to tricalcium phosphate Ability is 11.59 times of the bacillus megaterium WH320 as recipient bacterium, and the phosphate solubilization to aluminum phosphate is as recipient bacterium 16.23 times of bacillus megaterium WH320, the phosphate solubilization to ground phosphate rock are the bacillus megaterium WH320 as recipient bacterium 14.00 times.

<110>INST OF AGRICULTURAL RESOURCES

<120>The engineering bacteria and its construction method of expression glucose dehydrogenase and application

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 2391

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aaacgttccg cactctggct gtacgccgcc ctgctcctcg ccaccctgat ttggggcgtg 240

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ggcatctggc tgatcctgcc gtttgtctgg cgtcgcctgg tcattcctgc cagcggcgca 360

gttgccgcac tggtggtcgc gctgttgatt agcggtggta tcctcacctg ggcgggcttc 420

aacgacccgc aggagatcga cggcgcgctc agcgcggagt cgacgcctgc acaggccatc 480

tcaccagtgg ctgacggcga ctggccggcg tatggccgca atcaggaagg tcaacgcttt 540

tcaccactga agcaaattca cgccgataac gtccacaagc tgaaagaagc ctgggtgttc 600

cgtactggcg atgtgaagca gccgaacgat ccgggtgaaa tcaccaatga agtgacgcca 660

attaaagtgg gcgacacgct gtatctgtgc accgctcacc agcgtctgtt cgcgctggag 720

gcggcgacgg gtaaagaaaa atggcattac gatcctgagc tgaaaaccaa cgagtctttc 780

cagcatgtaa cctgccgtgg tgtctcttat catgaagcca aagcagaaac tgcttcgccg 840

gaagtgatgg cggattgccc gcgtcgtatc attctcccgg tcaatgatgg ccgcctgatt 900

gcgattaacg ctgaaaacgg caagctgtgc gaaaccttcg ctaataaagg cgtgctcaat 960

ctgcaaagca atatgccaga caccaaaccg ggtctgtatg agccgacttc gccgccgatt 1020

atcaccgata aaacgattgt gattgctggt tcagtaacgg ataacttctc cacccgcgaa 1080

acctcgggcg tgatccgtgg ttttgacgtc aataacggta aactgctgtg ggcgttcgat 1140

ccgggtgcga aagacccgaa tgcaatccct tccgatgagc actcttttac ctttaactcg 1200

ccgaactcct gggcgccagc ggcctatgac gcgaagctgg acctcgttta cctgccgatg 1260

ggggtctcga cgccggatat ctggggcgga caccggacgc cggagcagga gcgctacgcc 1320

agttccattc tggcgctgaa cgcgaccacc ggtaaactcg cctggagcta tcagacggtt 1380

caccacgatc tgtgggatat ggacatgccg tcccagccga cgctggcgga tattaccgtc 1440

aacggtgaga aagtcccggt tatctacgcg ccagcgaaaa ccggtaacat ctttgtcctc 1500

gaccgccgta acggcgagct ggtcgttcct gcaccggaaa aaccggttcc gcagggggcc 1560

gcgaaaggcg attacgttac ccctactcaa ccgttctctg agctgagctt ccgtccgaca 1620

aaagatctaa gcggtgcgga tatgtggggt gccaccatgt ttgaccaact ggtgtgccgc 1680

gtgatgttcc accagatgcg ctatgaaggc attttcaccc caccatctga acagggtacg 1740

ctggtcttcc cgggtaacct ggggatgttc gaatggggcg gtatttcggt cgatccgaac 1800

cgtcaggtgg cgattgccaa cccgatggcg ctgccgttcg tctctaagct tattccacgc 1860

ggtccgggca acccgatgga acagccgaaa gatgcaaaag gcacaggcac cgaatccggc 1920

atccagccgc agtacggtgt accgtatggc gtcacgctca atccgttcct ctcaccgttt 1980

ggtctgccat gtaaacagcc agcatggggt tatatttcgg cgctggatct gaaaaccaat 2040

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Met Ala Ile Asn Asn Thr Gly Ser Arg Arg Leu Leu Val Val Leu Thr

1 5 10 15

Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu Leu Ile Gly Gly Gly

20 25 30

Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr Pro Ile Ala Gly Leu

35 40 45

Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg Ser Lys Arg Ser Ala

50 55 60

Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr Leu Ile Trp Gly Val

65 70 75 80

Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr Pro Arg Ser Asp Ile

85 90 95

Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro Phe Val Trp Arg Arg

100 105 110

Leu Val Ile Pro Ala Ser Gly Ala Val Ala Ala Leu Val Val Ala Leu

115 120 125

Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly Phe Asn Asp Pro Gln

130 135 140

Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr Pro Ala Gln Ala Ile

145 150 155 160

Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr Gly Arg Asn Gln Glu

165 170 175

Gly Gln Arg Phe Ser Pro Leu Lys Gln Ile His Ala Asp Asn Val His

180 185 190

Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly Asp Val Lys Gln Pro

195 200 205

Asn Asp Pro Gly Glu Ile Thr Asn Glu Val Thr Pro Ile Lys Val Gly

210 215 220

Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg Leu Phe Ala Leu Glu

225 230 235 240

Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp Pro Glu Leu Lys Thr

245 250 255

Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly Val Ser Tyr His Glu

260 265 270

Ala Lys Ala Glu Thr Ala Ser Pro Glu Val Met Ala Asp Cys Pro Arg

275 280 285

Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu Ile Ala Ile Asn Ala

290 295 300

Glu Asn Gly Lys Leu Cys Glu Thr Phe Ala Asn Lys Gly Val Leu Asn

305 310 315 320

Leu Gln Ser Asn Met Pro Asp Thr Lys Pro Gly Leu Tyr Glu Pro Thr

325 330 335

Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val Ile Ala Gly Ser Val

340 345 350

Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly Val Ile Arg Gly Phe

355 360 365

Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe Asp Pro Gly Ala Lys

370 375 380

Asp Pro Asn Ala Ile Pro Ser Asp Glu His Ser Phe Thr Phe Asn Ser

385 390 395 400

Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala Lys Leu Asp Leu Val

405 410 415

Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile Trp Gly Gly His Arg

420 425 430

Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile Leu Ala Leu Asn Ala

435 440 445

Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr Val His His Asp Leu

450 455 460

Trp Asp Met Asp Met Pro Ser Gln Pro Thr Leu Ala Asp Ile Thr Val

465 470 475 480

Asn Gly Glu Lys Val Pro Val Ile Tyr Ala Pro Ala Lys Thr Gly Asn

485 490 495

Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu Val Val Pro Ala Pro

500 505 510

Glu Lys Pro Val Pro Gln Gly Ala Ala Lys Gly Asp Tyr Val Thr Pro

515 520 525

Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro Thr Lys Asp Leu Ser

530 535 540

Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp Gln Leu Val Cys Arg

545 550 555 560

Val Met Phe His Gln Met Arg Tyr Glu Gly Ile Phe Thr Pro Pro Ser

565 570 575

Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu Gly Met Phe Glu Trp

580 585 590

Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val Ala Ile Ala Asn Pro

595 600 605

Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro Arg Gly Pro Gly Asn

610 615 620

Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr Gly Thr Glu Ser Gly

625 630 635 640

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

645 650 655

Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro Ala Trp Gly Tyr Ile

660 665 670

Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val Trp Lys Lys Arg Ile

675 680 685

Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met Pro Val Pro Leu Pro

690 695 700

Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro Ile Ser Thr Ala Gly

705 710 715 720

Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn Tyr Leu Arg Ala Tyr

725 730 735

Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Gly Arg Leu Pro Ala Gly

740 745 750

Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn Gly Lys Gln Tyr Val

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Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly Thr Lys Met Gly Asp

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Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Val Lys

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agacgtacgg cgctatggct gtatgccgcc ctgctcctcg ccaccatgat ctggggcgta 240

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ggcatctggc tgattttgcc ttttgtctgg catcgcctga tggtgccttc ccgcggcgcg 360

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aacgacccgc aggagatcga cggcgcgctc agcgcggagt cgacgcctgc acaggccatc 480

tcaccagtgg ctgacggcga ctggccggcg tatggccgca atcaggaagg ccagcgctat 540

tcgccgctga agcaaattaa cgccgataac gttcacaagc tgaaagaagc atgggtgttc 600

cgtaccggcg atctgaagca gccggacgat ccgggcgaac tgaccaatga agtgacgcca 660

attaaagtgg gcgacacgct gtatctgtgc accgctcacc agcgtctgtt cgcgctggag 720

gcggcgacgg gtaaagaaaa atggcactac gacccggagc tgaaaaccaa cgagtccttc 780

cagcacgtta cctgccgcgg cgtttcatac catgaggcga ctgcgggtaa cgcttcgccg 840

gaagtgattg ccgactgccc gcgccgcatt attctgccgg taaacgacgg tcgtctgatt 900

gcgcttaacg ctgaaaccgg caagctgtgc gagactttcg gcaacaaagg cgtgctcaat 960

ctgcaaacca acatgccgga tcaaacgccg gggctgtatg agccaacctc gccgccgatc 1020

atcaccgata aaaccatcgt cattgccggt tcggtgaccg ataacttctc gacccgcgag 1080

acttccggcg tcattcgcgg cttcgatgtt aacaacggca agctgctgtg ggcgttcgat 1140

ccgggcgcga aagacccgaa tgcgatcccg tccgatgagc acacgtttac ctttaactcg 1200

ccgaactcct gggcgccagc ggcctatgac gcgaagctgg acctcgttta cctgccgatg 1260

ggggtctcga cgccggatat ctggggcgga caccggacgc cggagcagga gcgctacgcc 1320

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caccacgatc tgtgggatat ggacctgccc gctcagccga cgctggcgga cattaccgtc 1440

aacggccaga ccgttccggt catttacgcc ccggcgaaaa ccggcaatat ctttgtgctg 1500

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gcgaaaggcg attacgtcag caaaacgcag ccgttctctg aactgagctt ccgtccgaag 1620

aaagatctca gcggcgcgga tatgtggggc gccaccatgt tcgaccagct ggtatgccgc 1680

gtgatgttcc accagctgcg ctatgaaggc atcttcactc cgccatctga gcagggcacg 1740

ctggtgttcc cgggcaacct cgggatgttc gaatggggcg gtatttccgt cgatccgaac 1800

cgtcaggtag cgattgctaa cccgatggcg ctgccgttcg tctctaagct tattccacgc 1860

ggtccgggca acccgatgga gcagccgaag gatgcgaaag gcaccggcac cgaagccggt 1920

attcagccgc agtacggcgt accgtacggc gtgacgctga acccgttcct gtcgccgttt 1980

ggcctgccgt gtaagcaacc ggcctggggt tatatttccg cgctggatct gaaaaccaat 2040

gaagtggtgt ggaaaaaacg tatcggtacg ccgcaggaca gtatgccgtt cccgatgccg 2100

gttccgcttc ccttcaacat ggggatgccg atgctcggcg ggcccatctc gactgccggt 2160

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gaggtgaatg gcaagcagta cgttgttatt tccgcgggtg gtcacggttc gtttggtacg 2340

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<213>Citric acid bacillus Citrobacter rodentium

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Met Ala Glu Asn Asn Ala Arg Ser Pro Arg Leu Leu Val Thr Leu Thr

1 5 10 15

Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu Leu Ile Gly Gly Gly

20 25 30

Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr Pro Ile Ala Gly Leu

35 40 45

Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg Ser Arg Arg Thr Ala

50 55 60

Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr Met Ile Trp Gly Val

65 70 75 80

Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr Pro Arg Ser Asp Ile

85 90 95

Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro Phe Val Trp His Arg

100 105 110

Leu Met Val Pro Ser Arg Gly Ala Val Ala Ala Leu Val Ala Ala Leu

115 120 125

Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly Phe Asn Asp Pro Gln

130 135 140

Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr Pro Ala Gln Ala Ile

145 150 155 160

Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr Gly Arg Asn Gln Glu

165 170 175

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

180 185 190

Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly Asp Leu Lys Gln Pro

195 200 205

Asp Asp Pro Gly Glu Leu Thr Asn Glu Val Thr Pro Ile Lys Val Gly

210 215 220

Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg Leu Phe Ala Leu Glu

225 230 235 240

Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp Pro Glu Leu Lys Thr

245 250 255

Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly Val Ser Tyr His Glu

260 265 270

Ala Thr Ala Gly Asn Ala Ser Pro Glu Val Ile Ala Asp Cys Pro Arg

275 280 285

Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu Ile Ala Leu Asn Ala

290 295 300

Glu Thr Gly Lys Leu Cys Glu Thr Phe Gly Asn Lys Gly Val Leu Asn

305 310 315 320

Leu Gln Thr Asn Met Pro Asp Gln Thr Pro Gly Leu Tyr Glu Pro Thr

325 330 335

Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val Ile Ala Gly Ser Val

340 345 350

Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly Val Ile Arg Gly Phe

355 360 365

Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe Asp Pro Gly Ala Lys

370 375 380

Asp Pro Asn Ala Ile Pro Ser Asp Glu His Thr Phe Thr Phe Asn Ser

385 390 395 400

Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala Lys Leu Asp Leu Val

405 410 415

Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile Trp Gly Gly His Arg

420 425 430

Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile Leu Ala Leu Asn Ala

435 440 445

Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr Val His His Asp Leu

450 455 460

Trp Asp Met Asp Leu Pro Ala Gln Pro Thr Leu Ala Asp Ile Thr Val

465 470 475 480

Asn Gly Gln Thr Val Pro Val Ile Tyr Ala Pro Ala Lys Thr Gly Asn

485 490 495

Ile Phe Val Leu Asp Arg Arg Asn Gly Glu Leu Val Val Pro Ala Pro

500 505 510

Glu Thr Pro Val Pro Gln Gly Ala Ala Lys Gly Asp Tyr Val Ser Lys

515 520 525

Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro Lys Lys Asp Leu Ser

530 535 540

Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp Gln Leu Val Cys Arg

545 550 555 560

Val Met Phe His Gln Leu Arg Tyr Glu Gly Ile Phe Thr Pro Pro Ser

565 570 575

Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu Gly Met Phe Glu Trp

580 585 590

Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val Ala Ile Ala Asn Pro

595 600 605

Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro Arg Gly Pro Gly Asn

610 615 620

Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr Gly Thr Glu Ala Gly

625 630 635 640

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

645 650 655

Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro Ala Trp Gly Tyr Ile

660 665 670

Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val Trp Lys Lys Arg Ile

675 680 685

Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met Pro Val Pro Leu Pro

690 695 700

Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro Ile Ser Thr Ala Gly

705 710 715 720

Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn Tyr Leu Arg Ala Tyr

725 730 735

Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Ala Arg Leu Pro Ala Gly

740 745 750

Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn Gly Lys Gln Tyr Val

755 760 765

Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly Thr Lys Met Gly Asp

770 775 780

Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Glu Lys

785 790 795

<210> 5

<211> 2454

<212> DNA

<213>Artificial sequence

<220>

<221> CDS

<222> (1)..(2454)

<223>

<400> 5

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgatggcta ttaacaatac aggctcgcga cgactcctcg tggtgttaac ggccctcttt 120

gcagctcttt gcgggctgta tcttctcatt ggcggaggct ggttggtcgc cattggcggc 180

tcctggtact acccgatcgc cggtctggcg atgctgggcg tcgcctggct gctgtggcgc 240

agcaaacgtt ccgcactctg gctgtacgcc gccctgctcc tcgccaccct gatttggggc 300

gtgtgggaag ttggtttcga cttctgggcg ctgactccgc gcagcgacat tctggtcttc 360

ttcggcatct ggctgatcct gccgtttgtc tggcgtcgcc tggtcattcc tgccagcggc 420

gcagttgccg cactggtggt cgcgctgttg attagcggtg gtatcctcac ctgggcgggc 480

ttcaacgacc cgcaggagat cgacggcgcg ctcagcgcgg agtcgacgcc tgcacaggcc 540

atctcaccag tggctgacgg cgactggccg gcgtatggcc gcaatcagga aggtcaacgc 600

ttttcaccac tgaagcaaat tcacgccgat aacgtccaca agctgaaaga agcctgggtg 660

ttccgtactg gcgatgtgaa gcagccgaac gatccgggtg aaatcaccaa tgaagtgacg 720

ccaattaaag tgggcgacac gctgtatctg tgcaccgctc accagcgtct gttcgcgctg 780

gaggcggcga cgggtaaaga aaaatggcat tacgatcctg agctgaaaac caacgagtct 840

ttccagcatg taacctgccg tggtgtctct tatcatgaag ccaaagcaga aactgcttcg 900

ccggaagtga tggcggattg cccgcgtcgt atcattctcc cggtcaatga tggccgcctg 960

attgcgatta acgctgaaaa cggcaagctg tgcgaaacct tcgctaataa aggcgtgctc 1020

aatctgcaaa gcaatatgcc agacaccaaa ccgggtctgt atgagccgac ttcgccgccg 1080

attatcaccg ataaaacgat tgtgattgct ggttcagtaa cggataactt ctccacccgc 1140

gaaacctcgg gcgtgatccg tggttttgac gtcaataacg gtaaactgct gtgggcgttc 1200

gatccgggtg cgaaagaccc gaatgcaatc ccttccgatg agcactcttt tacctttaac 1260

tcgccgaact cctgggcgcc agcggcctat gacgcgaagc tggacctcgt ttacctgccg 1320

atgggggtct cgacgccgga tatctggggc ggacaccgga cgccggagca ggagcgctac 1380

gccagttcca ttctggcgct gaacgcgacc accggtaaac tcgcctggag ctatcagacg 1440

gttcaccacg atctgtggga tatggacatg ccgtcccagc cgacgctggc ggatattacc 1500

gtcaacggtg agaaagtccc ggttatctac gcgccagcga aaaccggtaa catctttgtc 1560

ctcgaccgcc gtaacggcga gctggtcgtt cctgcaccgg aaaaaccggt tccgcagggg 1620

gccgcgaaag gcgattacgt tacccctact caaccgttct ctgagctgag cttccgtccg 1680

acaaaagatc taagcggtgc ggatatgtgg ggtgccacca tgtttgacca actggtgtgc 1740

cgcgtgatgt tccaccagat gcgctatgaa ggcattttca ccccaccatc tgaacagggt 1800

acgctggtct tcccgggtaa cctggggatg ttcgaatggg gcggtatttc ggtcgatccg 1860

aaccgtcagg tggcgattgc caacccgatg gcgctgccgt tcgtctctaa gcttattcca 1920

cgcggtccgg gcaacccgat ggaacagccg aaagatgcaa aaggcacagg caccgaatcc 1980

ggcatccagc cgcagtacgg tgtaccgtat ggcgtcacgc tcaatccgtt cctctcaccg 2040

tttggtctgc catgtaaaca gccagcatgg ggttatattt cggcgctgga tctgaaaacc 2100

aatgaagtgg tgtggaagaa acgcattggt acgccgcagg acagcatgcc gttcccgatg 2160

ccggttccgc ttcccttcaa catggggatg ccgatgctcg gcgggcccat ctcgactgcc 2220

ggtaacgtgc tgtttatcgc cgctacggca gataactacc tgcgcgctta caacatgagc 2280

aacggtgaaa aactgtggca gggtcgtcta ccagcgggcg gtcaggcaac accgatgacc 2340

tatgaggtga acggcaagca gtatgtcgtg atttcagccg ggggccacgg ctcgtttggt 2400

acgaagatgg gcgattatat tgtcgcgtat gcgctgccgg atgatgtgaa ataa 2454

<210> 6

<211> 817

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 6

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Met Ala Ile Asn Asn Thr Gly Ser Arg Arg Leu

20 25 30

Leu Val Val Leu Thr Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu

35 40 45

Leu Ile Gly Gly Gly Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr

50 55 60

Pro Ile Ala Gly Leu Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg

65 70 75 80

Ser Lys Arg Ser Ala Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr

85 90 95

Leu Ile Trp Gly Val Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr

100 105 110

Pro Arg Ser Asp Ile Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro

115 120 125

Phe Val Trp Arg Arg Leu Val Ile Pro Ala Ser Gly Ala Val Ala Ala

130 135 140

Leu Val Val Ala Leu Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly

145 150 155 160

Phe Asn Asp Pro Gln Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr

165 170 175

Pro Ala Gln Ala Ile Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr

180 185 190

Gly Arg Asn Gln Glu Gly Gln Arg Phe Ser Pro Leu Lys Gln Ile His

195 200 205

Ala Asp Asn Val His Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly

210 215 220

Asp Val Lys Gln Pro Asn Asp Pro Gly Glu Ile Thr Asn Glu Val Thr

225 230 235 240

Pro Ile Lys Val Gly Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg

245 250 255

Leu Phe Ala Leu Glu Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp

260 265 270

Pro Glu Leu Lys Thr Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly

275 280 285

Val Ser Tyr His Glu Ala Lys Ala Glu Thr Ala Ser Pro Glu Val Met

290 295 300

Ala Asp Cys Pro Arg Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu

305 310 315 320

Ile Ala Ile Asn Ala Glu Asn Gly Lys Leu Cys Glu Thr Phe Ala Asn

325 330 335

Lys Gly Val Leu Asn Leu Gln Ser Asn Met Pro Asp Thr Lys Pro Gly

340 345 350

Leu Tyr Glu Pro Thr Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val

355 360 365

Ile Ala Gly Ser Val Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly

370 375 380

Val Ile Arg Gly Phe Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe

385 390 395 400

Asp Pro Gly Ala Lys Asp Pro Asn Ala Ile Pro Ser Asp Glu His Ser

405 410 415

Phe Thr Phe Asn Ser Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala

420 425 430

Lys Leu Asp Leu Val Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile

435 440 445

Trp Gly Gly His Arg Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile

450 455 460

Leu Ala Leu Asn Ala Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr

465 470 475 480

Val His His Asp Leu Trp Asp Met Asp Met Pro Ser Gln Pro Thr Leu

485 490 495

Ala Asp Ile Thr Val Asn Gly Glu Lys Val Pro Val Ile Tyr Ala Pro

500 505 510

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

515 520 525

Val Val Pro Ala Pro Glu Lys Pro Val Pro Gln Gly Ala Ala Lys Gly

530 535 540

Asp Tyr Val Thr Pro Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro

545 550 555 560

Thr Lys Asp Leu Ser Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp

565 570 575

Gln Leu Val Cys Arg Val Met Phe His Gln Met Arg Tyr Glu Gly Ile

580 585 590

Phe Thr Pro Pro Ser Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu

595 600 605

Gly Met Phe Glu Trp Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val

610 615 620

Ala Ile Ala Asn Pro Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro

625 630 635 640

Arg Gly Pro Gly Asn Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr

645 650 655

Gly Thr Glu Ser Gly Ile Gln Pro Gln Tyr Gly Val Pro Tyr Gly Val

660 665 670

Thr Leu Asn Pro Phe Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro

675 680 685

Ala Trp Gly Tyr Ile Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val

690 695 700

Trp Lys Lys Arg Ile Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met

705 710 715 720

Pro Val Pro Leu Pro Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro

725 730 735

Ile Ser Thr Ala Gly Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn

740 745 750

Tyr Leu Arg Ala Tyr Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Gly

755 760 765

Arg Leu Pro Ala Gly Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn

770 775 780

Gly Lys Gln Tyr Val Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly

785 790 795 800

Thr Lys Met Gly Asp Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Val

805 810 815

Lys

<210> 7

<211> 2454

<212> DNA

<213>Artificial sequence

<220>

<221> CDS

<222> (1)..(2454)

<223>

<400> 7

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgatggctg aaaacaatgc acgttcgcca cgacttctcg tgacgctgac ggccctcttt 120

gcagcgcttt gcgggctgta tcttctgatc ggcggtggct ggctggtcgc catcggcggc 180

tcctggtact acccgatcgc cggtctggcg atgctgggcg tcgcctggct gctgtggcgc 240

agcagacgta cggcgctatg gctgtatgcc gccctgctcc tcgccaccat gatctggggc 300

gtatgggaag tcggcttcga cttctgggcg ctgacgccgc gcagcgatat cctggtcttc 360

ttcggcatct ggctgatttt gccttttgtc tggcatcgcc tgatggtgcc ttcccgcggc 420

gcggtggccg cactggttgc cgccctgctg attagcggcg gcatcctgac ctgggcgggc 480

ttcaacgacc cgcaggagat cgacggcgcg ctcagcgcgg agtcgacgcc tgcacaggcc 540

atctcaccag tggctgacgg cgactggccg gcgtatggcc gcaatcagga aggccagcgc 600

tattcgccgc tgaagcaaat taacgccgat aacgttcaca agctgaaaga agcatgggtg 660

ttccgtaccg gcgatctgaa gcagccggac gatccgggcg aactgaccaa tgaagtgacg 720

ccaattaaag tgggcgacac gctgtatctg tgcaccgctc accagcgtct gttcgcgctg 780

gaggcggcga cgggtaaaga aaaatggcac tacgacccgg agctgaaaac caacgagtcc 840

ttccagcacg ttacctgccg cggcgtttca taccatgagg cgactgcggg taacgcttcg 900

ccggaagtga ttgccgactg cccgcgccgc attattctgc cggtaaacga cggtcgtctg 960

attgcgctta acgctgaaac cggcaagctg tgcgagactt tcggcaacaa aggcgtgctc 1020

aatctgcaaa ccaacatgcc ggatcaaacg ccggggctgt atgagccaac ctcgccgccg 1080

atcatcaccg ataaaaccat cgtcattgcc ggttcggtga ccgataactt ctcgacccgc 1140

gagacttccg gcgtcattcg cggcttcgat gttaacaacg gcaagctgct gtgggcgttc 1200

gatccgggcg cgaaagaccc gaatgcgatc ccgtccgatg agcacacgtt tacctttaac 1260

tcgccgaact cctgggcgcc agcggcctat gacgcgaagc tggacctcgt ttacctgccg 1320

atgggggtct cgacgccgga tatctggggc ggacaccgga cgccggagca ggagcgctac 1380

gccagttcca ttctggcgct gaacgcgacc accggtaaac tcgcctggag ctatcagacg 1440

gttcaccacg atctgtggga tatggacctg cccgctcagc cgacgctggc ggacattacc 1500

gtcaacggcc agaccgttcc ggtcatttac gccccggcga aaaccggcaa tatctttgtg 1560

ctggatcgcc gtaacggcga actggtggtg cctgcgccgg aaacgccggt gccgcagggc 1620

gccgcgaaag gcgattacgt cagcaaaacg cagccgttct ctgaactgag cttccgtccg 1680

aagaaagatc tcagcggcgc ggatatgtgg ggcgccacca tgttcgacca gctggtatgc 1740

cgcgtgatgt tccaccagct gcgctatgaa ggcatcttca ctccgccatc tgagcagggc 1800

acgctggtgt tcccgggcaa cctcgggatg ttcgaatggg gcggtatttc cgtcgatccg 1860

aaccgtcagg tagcgattgc taacccgatg gcgctgccgt tcgtctctaa gcttattcca 1920

cgcggtccgg gcaacccgat ggagcagccg aaggatgcga aaggcaccgg caccgaagcc 1980

ggtattcagc cgcagtacgg cgtaccgtac ggcgtgacgc tgaacccgtt cctgtcgccg 2040

tttggcctgc cgtgtaagca accggcctgg ggttatattt ccgcgctgga tctgaaaacc 2100

aatgaagtgg tgtggaaaaa acgtatcggt acgccgcagg acagtatgcc gttcccgatg 2160

ccggttccgc ttcccttcaa catggggatg ccgatgctcg gcgggcccat ctcgactgcc 2220

ggtaacgtgc tgtttatcgc cgcgaccgcc gataactacc tgcgcgctta caacatgagc 2280

aacggggaaa agctgtggca ggctcgcctg ccagcgggcg gacaggccac gccgatgacc 2340

tatgaggtga atggcaagca gtacgttgtt atttccgcgg gtggtcacgg ttcgtttggt 2400

acgaagatgg gcgattatat tgtcgcgtat gcgctgccgg acgacgagaa gtaa 2454

<210> 8

<211> 817

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 8

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Met Ala Glu Asn Asn Ala Arg Ser Pro Arg Leu

20 25 30

Leu Val Thr Leu Thr Ala Leu Phe Ala Ala Leu Cys Gly Leu Tyr Leu

35 40 45

Leu Ile Gly Gly Gly Trp Leu Val Ala Ile Gly Gly Ser Trp Tyr Tyr

50 55 60

Pro Ile Ala Gly Leu Ala Met Leu Gly Val Ala Trp Leu Leu Trp Arg

65 70 75 80

Ser Arg Arg Thr Ala Leu Trp Leu Tyr Ala Ala Leu Leu Leu Ala Thr

85 90 95

Met Ile Trp Gly Val Trp Glu Val Gly Phe Asp Phe Trp Ala Leu Thr

100 105 110

Pro Arg Ser Asp Ile Leu Val Phe Phe Gly Ile Trp Leu Ile Leu Pro

115 120 125

Phe Val Trp His Arg Leu Met Val Pro Ser Arg Gly Ala Val Ala Ala

130 135 140

Leu Val Ala Ala Leu Leu Ile Ser Gly Gly Ile Leu Thr Trp Ala Gly

145 150 155 160

Phe Asn Asp Pro Gln Glu Ile Asp Gly Ala Leu Ser Ala Glu Ser Thr

165 170 175

Pro Ala Gln Ala Ile Ser Pro Val Ala Asp Gly Asp Trp Pro Ala Tyr

180 185 190

Gly Arg Asn Gln Glu Gly Gln Arg Tyr Ser Pro Leu Lys Gln Ile Asn

195 200 205

Ala Asp Asn Val His Lys Leu Lys Glu Ala Trp Val Phe Arg Thr Gly

210 215 220

Asp Leu Lys Gln Pro Asp Asp Pro Gly Glu Leu Thr Asn Glu Val Thr

225 230 235 240

Pro Ile Lys Val Gly Asp Thr Leu Tyr Leu Cys Thr Ala His Gln Arg

245 250 255

Leu Phe Ala Leu Glu Ala Ala Thr Gly Lys Glu Lys Trp His Tyr Asp

260 265 270

Pro Glu Leu Lys Thr Asn Glu Ser Phe Gln His Val Thr Cys Arg Gly

275 280 285

Val Ser Tyr His Glu Ala Thr Ala Gly Asn Ala Ser Pro Glu Val Ile

290 295 300

Ala Asp Cys Pro Arg Arg Ile Ile Leu Pro Val Asn Asp Gly Arg Leu

305 310 315 320

Ile Ala Leu Asn Ala Glu Thr Gly Lys Leu Cys Glu Thr Phe Gly Asn

325 330 335

Lys Gly Val Leu Asn Leu Gln Thr Asn Met Pro Asp Gln Thr Pro Gly

340 345 350

Leu Tyr Glu Pro Thr Ser Pro Pro Ile Ile Thr Asp Lys Thr Ile Val

355 360 365

Ile Ala Gly Ser Val Thr Asp Asn Phe Ser Thr Arg Glu Thr Ser Gly

370 375 380

Val Ile Arg Gly Phe Asp Val Asn Asn Gly Lys Leu Leu Trp Ala Phe

385 390 395 400

Asp Pro Gly Ala Lys Asp Pro Asn Ala Ile Pro Ser Asp Glu His Thr

405 410 415

Phe Thr Phe Asn Ser Pro Asn Ser Trp Ala Pro Ala Ala Tyr Asp Ala

420 425 430

Lys Leu Asp Leu Val Tyr Leu Pro Met Gly Val Ser Thr Pro Asp Ile

435 440 445

Trp Gly Gly His Arg Thr Pro Glu Gln Glu Arg Tyr Ala Ser Ser Ile

450 455 460

Leu Ala Leu Asn Ala Thr Thr Gly Lys Leu Ala Trp Ser Tyr Gln Thr

465 470 475 480

Val His His Asp Leu Trp Asp Met Asp Leu Pro Ala Gln Pro Thr Leu

485 490 495

Ala Asp Ile Thr Val Asn Gly Gln Thr Val Pro Val Ile Tyr Ala Pro

500 505 510

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

515 520 525

Val Val Pro Ala Pro Glu Thr Pro Val Pro Gln Gly Ala Ala Lys Gly

530 535 540

Asp Tyr Val Ser Lys Thr Gln Pro Phe Ser Glu Leu Ser Phe Arg Pro

545 550 555 560

Lys Lys Asp Leu Ser Gly Ala Asp Met Trp Gly Ala Thr Met Phe Asp

565 570 575

Gln Leu Val Cys Arg Val Met Phe His Gln Leu Arg Tyr Glu Gly Ile

580 585 590

Phe Thr Pro Pro Ser Glu Gln Gly Thr Leu Val Phe Pro Gly Asn Leu

595 600 605

Gly Met Phe Glu Trp Gly Gly Ile Ser Val Asp Pro Asn Arg Gln Val

610 615 620

Ala Ile Ala Asn Pro Met Ala Leu Pro Phe Val Ser Lys Leu Ile Pro

625 630 635 640

Arg Gly Pro Gly Asn Pro Met Glu Gln Pro Lys Asp Ala Lys Gly Thr

645 650 655

Gly Thr Glu Ala Gly Ile Gln Pro Gln Tyr Gly Val Pro Tyr Gly Val

660 665 670

Thr Leu Asn Pro Phe Leu Ser Pro Phe Gly Leu Pro Cys Lys Gln Pro

675 680 685

Ala Trp Gly Tyr Ile Ser Ala Leu Asp Leu Lys Thr Asn Glu Val Val

690 695 700

Trp Lys Lys Arg Ile Gly Thr Pro Gln Asp Ser Met Pro Phe Pro Met

705 710 715 720

Pro Val Pro Leu Pro Phe Asn Met Gly Met Pro Met Leu Gly Gly Pro

725 730 735

Ile Ser Thr Ala Gly Asn Val Leu Phe Ile Ala Ala Thr Ala Asp Asn

740 745 750

Tyr Leu Arg Ala Tyr Asn Met Ser Asn Gly Glu Lys Leu Trp Gln Ala

755 760 765

Arg Leu Pro Ala Gly Gly Gln Ala Thr Pro Met Thr Tyr Glu Val Asn

770 775 780

Gly Lys Gln Tyr Val Val Ile Ser Ala Gly Gly His Gly Ser Phe Gly

785 790 795 800

Thr Lys Met Gly Asp Tyr Ile Val Ala Tyr Ala Leu Pro Asp Asp Glu

805 810 815

Lys

Claims

1. having the active recombinant microorganism of Soluble phosphorus, it is characterised in that：It is described that there is the active recombinant microorganism of Soluble phosphorus to pass through tax Recipient microorganism glucose dehydrogenase activity is given to prepare；The Soluble phosphorus activity with the active recombinant microorganism of Soluble phosphorus is higher than The recipient microorganism；The imparting recipient microorganism glucose dehydrogenase activity is by by the encoding gene of glucose dehydrogenase It imports in recipient microorganism and realizes；

The glucose dehydrogenase is protein a) or c)：

A) protein that amino acid sequence forms shown in SEQ ID No.2；

C) fusion protein that the c-terminus of the protein shown in a) or/and aminoterminal fusion protein label obtain.

2. recombinant microorganism according to claim 1, it is characterised in that：Protein c) is by SEQ ID No.6 Shown in amino acid sequence composition protein.

3. recombinant microorganism according to claim 1 or 2, it is characterised in that：1) or 2) encoding gene is following institute The gene shown：

1) coded sequence is DNA molecular shown in SEQ ID No.1；

2) coded sequence is DNA molecular shown in SEQ ID No.5.

4. recombinant microorganism according to claim 1 or 2, it is characterised in that：The recipient microorganism is prokaryotic micro-organisms.

5. recombinant microorganism according to claim 1 or 2, it is characterised in that：The recipient microorganism is Gram-negative Bacterium or gram-positive bacterium.

6. recombinant microorganism according to claim 1 or 2, it is characterised in that：The recipient microorganism is Escherichia Bacterium or bacillus.

7. application of the recombinant microorganism in claim 1-6 described in any claim in dissolved metals.