CN106754599A - Express engineering bacteria and its construction method and the application of GDH - Google Patents

Abstract

Engineering bacteria and its construction method and application the invention discloses expression GDH.The engineering bacteria of expression GDH provided by the present invention is the recombinant microorganism with Soluble phosphorus activity.The recombinant microorganism with Soluble phosphorus activity is prepared by assigning recipient microorganism glucose dehydrogenase activity；The Soluble phosphorus activity of the recombinant microorganism with Soluble phosphorus activity is higher than the recipient microorganism.The present invention builds the recombinant microorganism with Soluble phosphorus activity by assigning the activity of recipient microorganism (bacillus megaterium WH320) GDH CrGDH3A, the recombinant microorganism is 11.59 times of recipient microorganism to the phosphate solubilization of tricalcium phosphate, it is 16.23 times of its corresponding recipient microorganism to the phosphate solubilization of aluminum phosphate, is 14.00 times of its corresponding recipient microorganism to the phosphate solubilization of ground phosphate rock.

Description

Express engineering bacteria and its construction method and the application of GDH

Technical field

Engineering bacteria and its construction method and application the present invention relates to express GDH in biological field.

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 does not lack phosphorus in soil in fact, but Its validity in soil is very low, mostly the Phos of slightly solubility, and plant is difficult to absorb.Therefore, in activating soil Invalid phosphorus element, is one of agricultural production urgent problem.

GDH (glucose dehydrogenase, GDH) belongs to a member of short chain alcohols dehydrogenase family, In the presence of coenzyme, D-Glucose can be catalyzed and change into maltonic acid-delta-lactone, and the maltonic acid-δ for generating-interior Ester further can spontaneously be hydrolyzed into gluconic acid.GDH of the research and development with high activity and then structure 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, reduction phosphate fertilizer input side face have substantial worth.

The content of the invention

The technical problems to be solved by the invention are how to build the microorganism with Soluble phosphorus activity.

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

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

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

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

It is provided by the present invention to build the method with Soluble phosphorus reconstituted protein microorganism, including by the volume of GDH Code channel genes recipient microorganism, obtains Soluble phosphorus activity higher than the recipient microorganism with Soluble phosphorus reconstituted protein microorganism； The GDH is protein a) or b) or c) or d)：

A) protein that the amino acid sequence shown in SEQ ID No.2 is constituted；

B) protein that the amino acid sequence shown in SEQ ID No.6 is constituted；

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

D) by the amino acid sequence shown in SEQ ID No.2 or SEQ ID No.6 by one or several amino acid residues Substitution and/or missing and/or the protein with glucose dehydrogenase activity that obtains of addition.

In the application, the Soluble phosphorus activity refers to the ability for converting inorganic phosphorus into titanium pigment.Wherein, titanium pigment refers to The phosphorus that can be absorbed and used by plants after being dissolved into water or be 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, the CrGDH3A shown in SEQ ID No.2 The N-terminal connection fusion proteins that obtain of MGSSHHHHHHSSGLVPRGSHM, SEQ ID No.6 are by 817 amino acid residue groups Into.

In the above method, protein tag refers to using DNA extracorporeal recombinations, the one of amalgamation and expression together with destination protein Polypeptide or albumen are planted, in order to the expression of destination protein, detection, spike and/or purifying etc..

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

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

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

1) or 2) 3) there is more than 90% homogeneity with the DNA molecular for limiting and the GDH is encoded 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 (%) represents that it can be used to evaluate 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 GDH can be by recombinant expression carrier PET-CrGDH3A import the recipient microorganism；PET-the CrGDH3A are with the CrGDH3A genes shown in SEQ ID No.1 Replace the recombinant expression carrier that the fragment between NdeI the and BamHI recognition sites of pET-28a (+) is obtained.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 the 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 GDH can also be by recombinant expression carrier PWH-CrGDH3A import the recipient microorganism；PWH-the CrGDH3A are with the CrGDH3A genes shown in SEQ ID No.1 Forward direction replaces the recombinant expression carrier that the fragment between 2 BamHI recognition sites of pWH1520 is obtained.PWH-CrGDH3A are containing orderly CrGDH3A genes in list shown in SEQ ID No.1, pWH-CrGDH3A expression is the protein shown in SEQ ID No.2 CrGDH3A。

The application of above-mentioned recombinant microorganism or method in dissolved metals falls within protection scope of the present invention.

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

The biomaterial related to the GDH falls within protection scope of the present invention, and the biomaterial is B1) or B2):B1) the expression cassette containing the encoding gene；B2) the recombinant vector containing the encoding gene.

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

Application of the above-mentioned biomaterial in GDH is prepared falls within protection scope of the present invention.

Application of the above-mentioned biomaterial in preparing with Soluble phosphorus reconstituted protein microorganism falls within protection model of the invention Enclose.

It is demonstrated experimentally that in pattern bacterium prokaryotic expression (Escherichia coli are recipient bacterium), GDH CrGDH3A and CrGDH3A-His is respectively provided with glucose dehydrogenase activity higher, and GDH CrGDH3A-His is 25 DEG C of pH7.8's Under the conditions of its GDH enzyme activity be 39.47 ± 1.03U/mg albumen；(the bacillus megaterium in Soluble phosphorus engineering bacteria WH320 is recipient bacterium), GDH CrGDH3A is in 40 DEG C, the enzyme activity of the GDH under conditions of pH7.4 It is 36.53 ± 1.16U/mg.The present invention is by assigning recipient microorganism (bacillus megaterium WH320) GDH CrGDH3A activity builds the recombinant microorganism with Soluble phosphorus activity, and the recombinant microorganism is to receive to the phosphate solubilization of tricalcium phosphate 11.59 times of body microorganism, are 16.23 times of its corresponding recipient microorganism to the phosphate solubilization of aluminum phosphate, 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 thing new varieties and efficient activating soil phosphorus nutrients provides important gene resource, helps to promote Soluble phosphorus work Journey bacterium strides forward from laboratory development to the Field information stage.

Brief 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 GDH 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 GDH is expressed in GDH engineering bacteria.Wherein, M：Albumen Marker；1：Intracellular is precipitated；2：Intracellular supernatant；3：Extracellular supernatant.

Fig. 5 is the enzymatic activity influence of GDHs of the pH on being expressed in GDH engineering bacteria.

Fig. 6 is the enzymatic activity influence of GDH of the temperature on being expressed in GDH engineering bacteria.

Specific embodiment

The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining The bright present invention, rather than in order to limit the scope of the present invention.Experimental technique in following embodiments, unless otherwise specified, is Conventional method.Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.

The preparation and functional verification of embodiment 1, GDH CrGDH3

First, the structure of recombinant expression carrier

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

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

(EMD Biosciences, purchase in north to replace pET-28a (+) with the CrGDH3A genes shown in SEQ ID No.1 Capital company of fresh warp thread section, size is 5369bp) NdeI and Bam HI recognition sites between fragment, keep pET-28a (+) other sequences Row are constant, obtain recombinant expression carrier, are named as pET-CrGDH3A (Fig. 1).PET-CrGDH3A contain SEQ ID No.5 Shown His tag fusion protein CrGDH3A-His encoding genes, the protein of CrGDH3A-His encoding genes coding 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.

(EMD Biosciences, purchase in north to replace pET-28a (+) with the CrGDH3B genes shown in SEQ ID No.3 Capital company of fresh warp thread section, size is 5369bp) NdeI and Bam HI recognition sites between fragment, keep pET-28a (+) other sequences Row are constant, obtain recombinant expression carrier, are named as pET-CrGDH3B (Fig. 1).PET-CrGDH3B contain SEQ ID No.7 Shown His tag fusion protein CrGDH3B-His encoding genes, the protein of CrGDH3B-His encoding genes coding 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.

2nd, the preparation of the recombination bacillus coli of expression GDH

1st, the expression of CrGDH3A-His

PET-the CrGDH3A of step one are converted into e. coli bl21 (DE3) (Tiangeng company) with Calcium Chloride Method, using card That chloramphenicol resistance screening positive clone screening and culturing, picking monoclonal, with P1 (5 '-ATGGCTATTAACAATACAGGCTC-3 ') With P2 (5 '-TTATTTCACATCATCCGGCAGCG-3 ') for primer enters performing PCR identification, PCR identifications are obtained into 2391bp PCR The positive colony of product is named as pET-CrGDH3A/BL21 as genetic engineering bacterium.Picking pET-CrGDH3A/BL21 bacterial strains, (addition kanamycins to the concentration of kanamycins is in LB culture mediums to be inoculated in the LB culture mediums containing 100ug/ml kanamycins The culture medium that 100 μ g/ml are obtained) in, 37 DEG C of cultures to 0D₆₀₀Value (is blank with the LB culture mediums containing 100 μ g/ml kanamycins Control) when reaching 0.6, IPTG is added to final concentration l mM, 28 DEG C of induction 6h under the rotating speed of 150r/min collect nutrient solution warp After 4000r/min centrifugations 20min, it is 10 to obtain thalline content with 50mM Tris-HCl (pH7.1) resuspended thalline⁸Cfu/ml's Thallus suspension liquid, thallus suspension liquid is suspended in smudge cells through ultrasonication 30min (50% power, work 10s, interval 20s) Triton-X100 to final concentration of 1% is added in liquid, in 4 DEG C of extractions overnight, 12 000r/min centrifugation 10min collect supernatant Liquid (mycetome gross protein), CrGDH3A-His crude enzyme liquids are named as by the supernatant.

2nd, the expression of CrGDH3B-His

PET-the CrGDH3B of step 2 are converted into e. coli bl21 (DE3) (Tiangeng company) with Calcium Chloride Method, using card That chloramphenicol resistance screening positive clone screening and culturing, picking monoclonal, with P3 (5 '-ATGGCTGAAAACAATGCACG-3 ') and P4 (5 '-TTACTTCTCGTCGTCCGGCA-3 ') enters performing PCR identification for primer, and PCR identifications are obtained into 2391bp PCR primers Positive colony is named as pET-CrGDH3B/BL21 as genetic engineering bacterium.Picking pET-CrGDH3B/BL21 bacterial strains, are inoculated in (it is 100 μ g/ that kanamycins to the concentration of kanamycins is added in LB culture mediums to LB culture mediums containing 100 μ g/ml kanamycins The culture medium that ml is obtained) in, 37 DEG C of cultures to 0D₆₀₀Value (being blank with the LB culture mediums containing 100 μ g/ml kanamycins) When reaching 0.6, IPTG is added to final concentration l mM, 28 DEG C of induction 6h under the rotating speed of 150r/min collect nutrient solution warp After 4000r/min centrifugations 20min, it is 10 to obtain thalline content with 50mM Tris-HCl (pH7.1) resuspended thalline⁸Cfu/ml's Thallus suspension liquid, thallus suspension liquid is suspended in smudge cells through ultrasonication 30min (50% power, work 10s, interval 20s) Triton-X100 to final concentration of 1% is added in liquid, in 4 DEG C of extractions overnight, 12 000r/min centrifugation 10min collect supernatant Liquid (mycetome gross protein), CrGDH3B-His crude enzyme liquids are named as by the supernatant.

3rd, empty vector control bacterium

PET-28a (+) is transferred to e. coli bl21 (DE3) according to step 1 identical method, the restructuring that will be obtained is big Entitled pET-28a (+)/BL21 of enterobacteria.Using pET-28a (+)/BL21 as empty vector control bacterium according to above-mentioned steps 1 side 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 (it is the culture medium that 100 μ g/ml are obtained that kanamycins to the concentration of kanamycins is added in the LB culture mediums) in, 37 DEG C are cultivated to 0D₆₀₀When value (being blank with the LB culture mediums containing 100 μ g/ml kanamycins) reaches 0.6, IPTG is added To final concentration l mM, 28 DEG C of induction 6h under the rotating speed of 150r/min after collecting nutrient solution through 4000r/min centrifugations 20min, are used It is 10 that 50mM Tris-HCl (pH7.1) resuspended thalline obtains thalline content⁸The thallus suspension liquid of cfu/ml, thallus suspension liquid warp Ultrasonication 30min (50% power, work 10s, interval 20s), adds Triton-X100 to end in smudge cells suspension Concentration is 1%, and in 4 DEG C of extractions overnight, 12 000r/min centrifugation 10min collect supernatant (mycetome gross protein), by this Supernatant is named as empty vector control bacterium crude enzyme liquid.

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

Using e. coli bl21 (DE3) as blank bacterium induced expression preparation is carried out 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 to support base) when reaching 0.6, IPTG is added to final concentration l mM, 28 DEG C of induction 6h, receive under the rotating speed of 150r/min After collection nutrient solution is through 4000r/min centrifugations 20min, obtaining thalline content with 50mM Tris-HCl (pH7.1) resuspended 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, in 4 DEG C of extractions overnight, 12 000r/min centrifugations 10min, collects supernatant (mycetome gross protein), and the supernatant is named as into blank bacterium crude enzyme liquid.

30 μ L CrGDH3A-His crude enzyme liquids are taken (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 bacterium crude enzyme liquids (come from 10⁸Cfu/ml e. coli bl21s (DE3) SDS-PAGE analyses (resolving gel concentration is 12%)) are carried out on same glue, sample-adding pore volume and shape on the glue Consistent, sample-adding pore volume is 80 μ L.

Although SDS-PAGE results are as shown in Fig. 2 show 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 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 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) Reach, and expression quantity of the CrGDH3A-His in e. coli bl21 (DE3) apparently higher than CrGDH3B-His in Escherichia coli Expression quantity in BL21 (DE3).

3rd, the catalysis activity of the GDH of CrGDH3A-His and CrGDH3B-His is determined

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 is entered with nickel post (purchased from the high-affinity Ni-NTA Rasin products of AM General company) respectively Row purifying, by the pretreatment of nickel post, adds crude enzyme liquid, is subsequently adding containing imidazole elution (50mM NaH₂PO₄, 300mM NaCl, 250mM imidazole, pH8.0) 4 DEG C of effect 10min, 3000rpm centrifugation 1min collect eluent, repeat wash-out once, receive Collection eluent, taking 1ml eluents carries out SDS-PAGE analyses.The sequencing results of CrGDH3A-His show the 15 of its N-terminal Individual 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 the 1-15 amino acids of sequence 4 in sequence table.

By the eluent of above-mentioned collection with distilled water dialyse, remove salt ion, obtain respectively pure CrGDH3A-His enzyme liquids, Pure CrGDH3B-His enzyme liquids, pure empty vector control bacterium enzyme liquid and pure blank bacterium enzyme liquid, as enzyme liquid to be measured.Treat Survey enzyme liquid and determine protein content using BCA quantification of protein kit.

Glucose dehydrogenase activity is determined, and with glucose as substrate, colorimetric analysis grape is carried out according to red is produced The activity of glucocorticoid dehydrogenase.To 50 μ L enzyme liquids to be measured (pure CrGDH3A-His enzyme liquids, pure CrGDH3B-His enzyme liquids, pure sky Vehicle Control bacterium enzyme liquid or pure blank bacterium enzyme liquid) in add 50 μ L pH7.8 buffer solution (to 100mmol/L MOPS PQQ (PQQ) and CaCl is added in buffer solution₂, make PQQ contents be 10 μm of ol/L and CaCl₂Content is obtained for 2mol/L Liquid), 37 DEG C pretreatment 1 hour structure to stablize enzyme, obtain pre-process enzyme liquid.Then, to addition 1mL in cuvette The Tris-HCl buffer solutions of the pH7.8 of 50mmol/L, the 20mmol/L phenazine methosulfates that 100 μ L are then separately added into again are molten Liquid, 6.7mmol/L 2,6-sodium dichlorophenol indophenolate (DCIP) solution and 1mol/L glucose solutions, 50 μ L are added after being well mixed Pretreatment enzyme liquid, be finally settled to 3mL, reaction temperature is 25 DEG C, determines the change 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 that can make 1 μm of ol in 1min DCIP reduce) enzyme amount.GDH Rate activity is calculated with the vigor of enzyme in per unit total protein, and unit is U/ mg。

Experiment sets three repetitions.Result shows that pure empty vector control bacterium enzyme liquid and pure blank bacterium enzyme liquid do not have Portugal Grape glucocorticoid dehydrogenase is active, and the enzyme activity of the GDH of the CrGDH3A-His expressed by pET-CrGDH3A/BL21 is 39.47 ± 1.03U/mg albumen, the enzyme activity of the GDH of the CrGDH3B-His expressed by pET-CrGDH3B/BL21 It is 7.39 ± 0.26U/mg albumen.The GDH enzyme activity of CrGDH3A-His is CrGDH3B-His GDHs 5.34 times of enzyme activity.The GDH yield of pET-CrGDH3A/BL21 is 25.65/10⁸cfu pET–CrGDH3A/ The GDH yield of BL21, pET-CrGDH3B/BL21 is 5.09U/10⁸cfu pET–CrGDH3B/BL21。pET– The GDH yield of CrGDH3A/BL21 is 5 times of pET-CrGDH3B/BL21.

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

1. the structure of GDH CrGDH3A genes shuttle expression carrier

In order to obtain the Soluble phosphorus engineering bacteria of high efficient expression GDH 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 carries BamHI restriction enzyme sites, with ammonia benzyl and tetracycline resistance gene, can stablize express express target protein.

Using DNAMAN software analysis CrGDH3A gene orders, 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′).PET-CrGDH3A with step one are template, using above-mentioned P5 and P6 introduces BamHI enzymes respectively as primer, the method expanded using PCR 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 the CrGDH3A gene PCR products with enzyme recognition site, the digestion products T of recovery₄Ligase is connected, 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 the positive insertion shuttle expression carrier pWH1520 of method screening CrGDH3A genes compared with reference to sequencing, extracts weight Group plasmid, the final shuttle expression carrier for obtaining CrGDH3A genes.Sequencing result is shown to be with shown in SEQ ID No.1 The recombinant expression carrier that fragment between the positive 2 BamHI recognition sites for replacing pWH1520 of CrGDH3A genes is obtained is named as PWH-CrGDH3A (Fig. 3).PWH-CrGDH3A containing the CrGDH3A genes shown in SEQ ID No.1 in ordered list, pWH- CrGDH3A expression is the protein C rGDH3A shown in SEQ ID No.2.

2. GDH engineering bacteria acquisition and its expression CrGDH3A zymetology feature

Using protoplast transformation, recombinant vector pWH-CrGDH3A is transferred into bacillus megaterium, and (WH320 is purchased Hai Beinuo biotechnologies company), obtain GDH engineering bacteria.GDH engineering bacteria is accessed containing tetracycline In LB culture mediums (it is the culture medium that 100 μ g/ml are obtained that tetracycline to the concentration of tetracycline 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 to cultivate to exponential phase, adds xylose to arrive Final concentration of 0.5%, Fiber differentiation 6h, 4000r/min rotating speeds centrifugation 15min, collects supernatant as extracellular supernatant at room temperature；Receive Collection precipitation, plus 2 times of volume phosphate buffers (pH6.0), smudge cells obtain smudge cells suspension.In smudge cells suspension Middle addition Triton-X100 to final concentration of 1%, in 4 DEG C of extractions overnight, 12000r/min centrifugation 10min, supernatant is intracellular Supernatant, is precipitated as intracellular precipitation.Respectively to intracellular supernatant, intracellular precipitate and extracellular supernatant carry out SDS-PAGE electrophoretic analysis and Enzyme activity determination.Enzyme liquid to be measured determines protein content using BCA quantification of protein kit.

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

3rd, influences of the pH to the catalysis activity of GDH engineering bacteria

The GDH engineering bacteria of step 2 is accessed into the LB culture mediums containing ampicillin and tetracycline (in LB trainings Ampicillin and tetracycline to the concentration of ampicillin and tetracycline is added to be the culture that 100 μ g/ml are obtained in foster base Base) in, overnight incubation.It is transferred in the above-mentioned LB culture mediums containing tetracycline with 2% inoculum concentration and continues to cultivate to logarithmic growth Phase, xylose to final concentration of 0.5%, Fiber differentiation 6h is added, 4000r/min rotating speeds centrifugation 15min, collects precipitation at room temperature, Plus 2 times of volume phosphate buffers (pH7.0), smudge cells obtains smudge cells suspension.Added in smudge cells suspension Triton-X100 to final concentration of 1%, in 4 DEG C of extractions overnight, 12000r/min centrifugation 10min, supernatant is glucose and takes off Hydrogen enzyme crude enzyme liquid, as enzyme liquid 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) is determined GDH engineering bacteria is catalyzed the ability of glucose dehydrogenation.

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

(added in 100mmol/L MOPS buffer solutions to the buffer solution of the corresponding pH value of 50 μ L is added in 50 μ L enzyme liquids to be measured PQQ (PQQ) and CaCl₂, make PQQ contents be 10 μm of ol/L and CaCl₂The liquid that content is obtained for 2mol/L), 37 DEG C 1 hour structure to stablize enzyme of pretreatment, obtains pre-processing enzyme liquid.

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

The optimal pH of GDH engineering bacteria catalysis activity is 7.4, is most in the enzymatic activity of pH 6.6-7.8 scopes 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.

4th, influence of the temperature to the catalysis activity of GDH engineering bacteria

During the GDH engineering bacteria of step 2 accessed into 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 to cultivate to exponential phase, is added To final concentration of 0.5%, Fiber differentiation 6h, 4000r/min rotating speeds centrifugation 15min, collects precipitation, plus 2 times of bodies to xylose at room temperature Product phosphate buffer (pH7.0), smudge cells obtains smudge cells suspension.Triton- is added in smudge cells suspension X100 to final concentration of 1%, in 4 DEG C of extractions overnight, 12000r/min centrifugation 10min, it is thick that supernatant is GDH Enzyme liquid, as enzyme liquid to be measured.(buffered to 100mmol/L MOPS to the buffer solution of 50 μ L pH7.4 is added in 50 μ L enzyme liquids to be measured PQQ (PQQ) and CaCl is added in liquid₂, make PQQ contents be 10 μm of ol/L and CaCl₂The liquid that content is obtained for 2mol/L Body), 37 DEG C of pretreatments, 1 hour structure to stablize enzyme obtains pre-processing enzyme liquid.In Tris buffer solution systems (pH7.4), The ability that GDH engineering bacteria is catalyzed glucose is determined 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 The Tris cushioning liquid (pH7.4) of the 50mmol/L of 1mL is separately added into cuvette, 20mmol/L azophenlyene is then separately added into again Methylsulfate, 6.7mmol/L 2,6-sodium dichlorophenol indophenolate (DCIP) and each 100 μ L of 1mol/L glucose, add after being well mixed The pretreatment enzyme liquid of 50 μ L, is finally settled to 3mL, determines the change 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 is reduced) in 1min Enzyme amount.GDH Rate activity is calculated with the vigor of enzyme in per unit total protein, and unit is U/mg.

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

5th, the effect of solubilizing phosphate of GDH engineering bacteria

The GDH 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 GDH engineering bacteria and huge The content of Bacterium anthracoides WH320 is 10⁸Cfu/mL, in 37 DEG C of cultures to exponential phase, adds xylose 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 nutrient solution at the 7th day, And the ground phosphate rock culture medium being inoculated with took nutrient solution at the 14th day.10000r/min rotating speeds centrifugation 10min, collects supernatant at 4 DEG C, Using molybdenum antimony resistance colorimetric method, with 722 type spectrophotometers, in wavelength 700nm, inoculation GDH engineering bacteria is directly determined And water-soluble phosphorus (also referred to as available phosphorus or rapid available phosphorus) content in bacillus megaterium WH320 (recipient bacterium) nutrient solution, if not connecing The corresponding control (CK) of bacterium, available phosphorus content given below is 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 compound method is as follows：It is water by solvent, solute and its concentration are such as Under nutrient solution 115 DEG C sterilize 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), plus distilled water is to 1000ml.

The pH of tricalcium phosphate fluid nutrient medium is 7.0, and compound method is as follows：It is water by solvent, solute and its concentration are as follows Nutrient solution 115 DEG C sterilize 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, plus distilled water is to 1000ml.

The pH of aluminum phosphate fluid nutrient medium is 7.0, and compound method is as follows：It is water by solvent, solute and its concentration are following Nutrient solution is in 115 DEG C of sterilizing 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, aluminum phosphate 5.0g/L, plus distilled water is to 1000ml.

Result shows that the GDH engineering bacteria of step 2 cultivates the culture of 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 the nutrient solution of culture 7 days has 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 the nutrient solution of 14 days is cultivated in ground phosphate rock fluid nutrient medium Content is 34.57 ± 2.07 μm of ol/L；Trained in tricalcium phosphate fluid nutrient medium as the bacillus megaterium WH320 of recipient bacterium The content for supporting the available phosphorus of the nutrient solution of 7 days 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 nutrient solution is 4.86 ± 0.27 μm of ol/L, the nutrient solution of culture 14 days in ground phosphate rock fluid nutrient medium The content of available phosphorus is 2.47 ± 0.12 μm of ol/L.It can be seen that, the Soluble phosphorus of the GDH engineering bacteria of step 2 to tricalcium phosphate Ability is 11.59 times as the bacillus megaterium WH320 of 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 is as the bacillus megaterium WH320 of recipient bacterium 14.00 times.

<110>INST OF AGRICULTURAL RESOURCES

<120>Express engineering bacteria and its construction method and the application of GDH

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 2391

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<213>Artificial sequence

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<222> (1)..(2391)

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atggctatta acaatacagg ctcgcgacga ctcctcgtgg tgttaacggc cctctttgca 60

gctctttgcg ggctgtatct tctcattggc ggaggctggt tggtcgccat tggcggctcc 120

tggtactacc cgatcgccgg tctggcgatg ctgggcgtcg cctggctgct gtggcgcagc 180

aaacgttccg cactctggct gtacgccgcc ctgctcctcg ccaccctgat ttggggcgtg 240

tgggaagttg gtttcgactt ctgggcgctg actccgcgca gcgacattct ggtcttcttc 300

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

gaagtggtgt ggaagaaacg cattggtacg ccgcaggaca gcatgccgtt cccgatgccg 2100

gttccgcttc ccttcaacat ggggatgccg atgctcggcg ggcccatctc gactgccggt 2160

aacgtgctgt ttatcgccgc tacggcagat aactacctgc gcgcttacaa catgagcaac 2220

ggtgaaaaac tgtggcaggg tcgtctacca gcgggcggtc aggcaacacc gatgacctat 2280

gaggtgaacg gcaagcagta tgtcgtgatt tcagccgggg gccacggctc gtttggtacg 2340

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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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tggtactacc cgatcgccgg tctggcgatg ctgggcgtcg cctggctgct gtggcgcagc 180

agacgtacgg cgctatggct gtatgccgcc ctgctcctcg ccaccatgat ctggggcgta 240

tgggaagtcg gcttcgactt ctgggcgctg acgccgcgca gcgatatcct ggtcttcttc 300

ggcatctggc tgattttgcc ttttgtctgg catcgcctga tggtgccttc ccgcggcgcg 360

gtggccgcac tggttgccgc cctgctgatt agcggcggca tcctgacctg ggcgggcttc 420

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

agttccattc tggcgctgaa cgcgaccacc ggtaaactcg cctggagcta tcagacggtt 1380

caccacgatc tgtgggatat ggacctgccc gctcagccga cgctggcgga cattaccgtc 1440

aacggccaga ccgttccggt catttacgcc ccggcgaaaa ccggcaatat ctttgtgctg 1500

gatcgccgta acggcgaact ggtggtgcct gcgccggaaa cgccggtgcc gcagggcgcc 1560

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

aacgtgctgt ttatcgccgc gaccgccgat aactacctgc gcgcttacaa catgagcaac 2220

ggggaaaagc tgtggcaggc tcgcctgcca gcgggcggac aggccacgcc gatgacctat 2280

gaggtgaatg gcaagcagta cgttgttatt tccgcgggtg gtcacggttc gtttggtacg 2340

aagatgggcg attatattgt cgcgtatgcg ctgccggacg acgagaagta a 2391

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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 (10)

1. there is the recombinant microorganism of Soluble phosphorus activity, the recombinant microorganism with Soluble phosphorus activity is by assigning recipient microorganism It is prepared by glucose dehydrogenase activity；The Soluble phosphorus activity of the recombinant microorganism with Soluble phosphorus activity is higher than the micro- life of the acceptor Thing.

2. recombinant microorganism according to claim 1, it is characterised in that：The imparting recipient microorganism GDH Activity is realized by the way that the encoding gene of GDH is imported in recipient microorganism.

3. the method for building the recombinant microorganism with Soluble phosphorus activity described in claim 1 or 2, including by GDH Encoding gene import recipient microorganism, obtain Soluble phosphorus activity micro- higher than the restructuring of the recipient microorganism with Soluble phosphorus activity It is biological.

4. recombinant microorganism according to claim 1 and 2 or the method described in claim 3, it is characterised in that：The Portugal Grape glucocorticoid dehydrogenase is protein a) or b) or c) or d)：

A) protein that the amino acid sequence shown in SEQ ID No.2 is constituted；

B) protein that the amino acid sequence shown in SEQ ID No.6 is constituted；

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

D) by amino acid sequence the taking by one or several amino acid residues shown in SEQ ID No.2 or SEQ ID No.6 The protein with glucose dehydrogenase activity that generation and/or missing and/or addition obtain.

5. recombinant microorganism according to claim 4 or method, it is characterised in that：The encoding gene for it is following 1) or 2) Or 3) shown in gene：

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

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

1) or 2) 3) have with the DNA molecular for limiting more than 90% homogeneity and coding claim 1 described in protein DNA molecular.

6. the recombinant microorganism or method according to claim 3,4 or 5, it is characterised in that：The recipient microorganism be M1, M2 or M3：

M1, prokaryotic micro-organisms；

M2, gramnegative bacterium or gram-positive bacterium；

M3, Escherichia bacteria or bacillus.

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

8. the biomaterial related to GDH described in claim 4, the biomaterial is B1) or B2):

B1 the expression cassette of encoding gene described in claim 4 or 5) is contained；

B2 the recombinant vector of encoding gene described in claim 4 or 5) is contained.

9. application of the biomaterial described in claim 8 in GDH is prepared.

10. application of the biomaterial described in claim 8 in preparing with Soluble phosphorus reconstituted protein microorganism.