CN109055419A - Construction method and application with the active recombinant microorganism of Soluble phosphorus - Google Patents

Construction method and application with the active recombinant microorganism of Soluble phosphorus Download PDF

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CN109055419A
CN109055419A CN201810965684.7A CN201810965684A CN109055419A CN 109055419 A CN109055419 A CN 109055419A CN 201810965684 A CN201810965684 A CN 201810965684A CN 109055419 A CN109055419 A CN 109055419A
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孙静文
周卫
程明芳
李书田
王玉军
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Institute of Agricultural Resources and Regional Planning of CAAS
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Abstract

The invention discloses with the active recombinant microorganism of Soluble phosphorus construction method and application.It includes that the encoding gene of glucose dehydrogenase is imported recipient microorganism that building provided by the present invention, which has the method for the active recombinant microorganism of Soluble phosphorus, and obtaining Soluble phosphorus activity higher than the recipient microorganism has the active recombinant microorganism of Soluble phosphorus.The present invention has the active recombinant microorganism of Soluble phosphorus by the activity building 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

Construction method and application with the active recombinant microorganism of Soluble phosphorus
It is that December 14, invention and created name in 2016 are that the application, which is application No. is the 201611153688.2, applying date, The divisional application of " engineering bacteria and its construction method of expression glucose dehydrogenase and application ".
Technical field
The present invention relates to the active recombinant microorganism of Soluble phosphorus construction method and application.
Background technique
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 constructs 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.
Summary of the invention
The technical problem to be solved by the present invention is to how construct 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 the dehydrogenation of recipient microorganism glucose 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 the methods that building has Soluble phosphorus reconstituted protein microorganism.
The method that building provided by the present invention has Soluble phosphorus reconstituted protein microorganism, including by the volume of glucose dehydrogenase Code channel genes recipient microorganism, obtaining Soluble phosphorus activity higher than the recipient microorganism has Soluble phosphorus reconstituted protein microorganism； The glucose dehydrogenase is protein a) or b) or c) or d):
A) protein that the amino acid sequence shown in SEQ ID No.2 forms；
B) protein that the amino acid sequence shown in SEQ ID No.6 forms；
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 obtained protein with glucose dehydrogenase activity of deletion and/or 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 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 is 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 protein of N-terminal connection MGSSHHHHHHSSGLVPRGSHM, SEQ ID No.6 is by 817 amino acid residue groups At.
In the above method, protein tag refers to using DNA extracorporeal recombination, 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 gene；
2) coded sequence is DNA molecular shown in SEQ ID No.5, entitled CrGDH3A-His gene；
1) or 2) 3) identity and the coding glucose dehydrogenase with the DNA molecular that limits with 90% or more DNA molecular.
In the encoding gene, " identity " refers to the sequence similarity with native sequence nucleic acid." identity " can use meat Eye or computer software are evaluated.Using computer software, the identity between two or more sequences can use percentage (%) is indicated, can be used to evaluate the identity 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 imports the recipient microorganism；PET-the CrGDH3A is the CrGDH3A gene shown in SEQ ID No.1 Replace the recombinant expression carrier that the segment between NdeI the and BamHI recognition site of pET-28a (+) obtains.PET-CrGDH3A contains CrGDH3A-His encoding gene shown in SEQ ID No.5, the protein C rGDH3A- of CrGDH3A-His encoding gene 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 imports the recipient microorganism；PWH-the CrGDH3A is the CrGDH3A gene shown in SEQ ID No.1 The recombinant expression carrier that segment between 2 BamHI recognition sites of forward direction replacement pWH1520 obtains.PWH-CrGDH3A is containing orderly CrGDH3A gene 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-mentioned application, the Phos can be the phosphate (such as tricalcium phosphate or aluminum phosphate) or ground phosphate rock of indissoluble.
Biomaterial relevant to the glucose dehydrogenase also belongs to protection scope of the present invention, and 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 preparation glucose dehydrogenase also belongs to protection scope of the present invention.
Above-mentioned biomaterial in preparation there is the application in Soluble phosphorus reconstituted protein microorganism to also belong to protection model of the invention It encloses.
It is demonstrated experimentally that in mode 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 building 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.
Detailed description of the invention
Fig. 1 is the physical map of pET-CrGDH3A and pET-CrGDH3B.Wherein, gdh3 be CrGDH3A gene or CrGDH3B gene.
Fig. 2 is the SDS-PAGE map of the inducing 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 gene.
Fig. 4 is the SDS-PAGE map that glucose dehydrogenase is expressed in glucose dehydrogenase engineering bacteria.Wherein, M: albumen Marker；1: precipitating intracellular；2: supernatant intracellular；3: extracellular supernatant.
Fig. 5 is that pH influences the enzymatic activity for the glucose dehydrogenase expressed in glucose dehydrogenase engineering bacteria.
Fig. 6 is that temperature influences the enzymatic activity for the glucose dehydrogenase expressed in glucose dehydrogenase engineering bacteria.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and 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 building of recombinant expression carrier
In order to improve the activity of glucose dehydrogenase, by Genbank Accession Number WP_012904518 institute Show from citric acid bacillus Citrobacter rodentium grape glucocorticoid dehydrogenase (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 gene shown in CrGDH3A gene shown in SEQ ID No.1 and SEQ ID No.3 is prepared respectively.
(EMD Biosciences, is purchased in north the CrGDH3A gene replacement pET-28a (+) shown in SEQ ID No.1 Capital company of fresh warp thread section, size 5369bp) NdeI and Bam HI recognition site between segment, keep pET-28a (+) other sequences It arranges constant, obtains recombinant expression carrier, be named as pET-CrGDH3A (Fig. 1).PET-CrGDH3A contains SEQ ID No.5 Shown in His tag fusion protein CrGDH3A-His encoding gene, CrGDH3A-His encoding gene 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.
(EMD Biosciences, is purchased in north the CrGDH3B gene replacement pET-28a (+) shown in SEQ ID No.3 Capital company of fresh warp thread section, size 5369bp) NdeI and Bam HI recognition site between segment, keep pET-28a (+) other sequences It arranges constant, obtains recombinant expression carrier, be named as pET-CrGDH3B (Fig. 1).PET-CrGDH3B contains SEQ ID No.7 Shown in His tag fusion protein CrGDH3B-His encoding gene, CrGDH3B-His encoding gene 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 glucose dehydrogenase is expressed
1, the expression of CrGDH3A-His
PET-the CrGDH3A of step 1 Calcium Chloride Method is converted into e. coli bl21 (DE3) (Tiangeng company), utilizes card That chloramphenicol resistance screening positive clone screening and culturing, picking monoclonal, with P1 (5 '-ATGGCTATTAACAATACAGGCTC-3 ') It is that primer carries out PCR identification 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 strain, Be inoculated in the kanamycins containing 100ug/ml LB culture medium (in LB culture medium be added kanamycins to kanamycins concentration be The culture medium that 100 μ g/ml are obtained) in, 37 DEG C of cultures to 0D600Value is (using the LB culture medium containing 100 μ g/ml kanamycins as blank Control) when reaching 0.6, IPTG to final concentration l mM, 28 DEG C of induction 6h under the revolving speed of 150r/min is added, it collects culture solution and passes through After 4000r/min is centrifuged 20min, it is 10 that thallus, which is resuspended, to obtain thallus 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 stayed overnight in 4 DEG C of extractions, 12 000r/min are centrifuged 10min, collect supernatant Liquid (mycetome gross protein), is named as CrGDH3A-His crude enzyme liquid for the supernatant.
2, the expression of CrGDH3B-His
PET-the CrGDH3B of step 2 Calcium Chloride Method is converted into e. coli bl21 (DE3) (Tiangeng company), utilizes 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 identification, and PCR is identified to obtain 2391bp PCR product Positive colony is named as pET-CrGDH3B/BL21 as genetic engineering bacterium.Picking pET-CrGDH3B/BL21 bacterial strain, is inoculated in (concentration that kanamycins is added to kanamycins in LB culture medium is 100 μ g/ to LB culture medium containing 100 μ g/ml kanamycins The culture medium that ml is obtained) in, 37 DEG C of cultures to 0D600Value (using the LB culture medium containing 100 μ g/ml kanamycins as blank control) When reaching 0.6, IPTG to final concentration l mM, 28 DEG C of induction 6h under the revolving speed of 150r/min is added, collects culture solution warp After 4000r/min is centrifuged 20min, it is 10 that thallus, which is resuspended, to obtain thallus 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 stayed overnight in 4 DEG C of extractions, 12 000r/min are centrifuged 10min, collect supernatant Liquid (mycetome gross protein), is named as CrGDH3B-His crude enzyme liquid for 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 inducing expression and prepares bacterial protein.Picking pET-28a (+)/BL21 bacterial strain is inoculated in containing 100 μ g/ml kanamycins LB culture medium (concentration that kanamycins is added to kanamycins in LB culture medium is the obtained culture medium of 100 μ g/ml) in, 37 DEG C are cultivated to 0D600When value (using the LB culture medium containing 100 μ g/ml kanamycins as blank control) reaches 0.6, IPTG is added To final concentration l mM, 28 DEG C of induction 6h under the revolving speed of 150r/min collect culture solution after 4000r/min is centrifuged 20min, use It is 10 that 50mM Tris-HCl (pH7.1), which is resuspended thallus and obtains thallus 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 Concentration is 1%, is stayed overnight in 4 DEG C of extractions, 12 000r/min are centrifuged 10min, collect supernatant (mycetome gross protein), 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 inducing expression preparation according to the method for above-mentioned steps 1 as blank control bacterium Bacterial protein.Picking e. coli bl21 (DE3) bacterial strain, is inoculated in LB culture medium, 37 DEG C of cultures to 0D600Value (is trained with LB Supporting base is blank control) when reaching 0.6, IPTG to final concentration l mM, 28 DEG C of induction 6h under the revolving speed of 150r/min, receipts are added Collect culture solution after 4000r/min is centrifuged 20min, obtaining thallus content with 50mM Tris-HCl (pH7.1) resuspension thallus 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 stayed overnight in 4 DEG C of extractions, 12 000r/min centrifugation 10min collects supernatant (mycetome gross protein), which is named as blank control bacterium crude enzyme liquid.
Take 30 μ L CrGDH3A-His crude enzyme liquids (from 108cfu/ml pET–CrGDH3A/BL21)、30μL CrGDH3B-His crude enzyme liquid (comes 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 bl21 (DE3)) SDS-PAGE analysis (resolving gel concentration 12%) is 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 result is as shown in Fig. 2, show CrGDH3A-His crude enzyme liquid, CrGDH3B-His crude enzyme liquid, 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 liquid and The content of 87kD polypeptide is apparently higher than empty vector control bacterium crude enzyme liquid and blank control bacterium crude enzyme liquid in CrGDH3B-His crude enzyme liquid The content of middle 87kD polypeptide, and the content of the 87kD polypeptide in CrGDH3A-His crude enzyme liquid is higher than CrGDH3B-His crude enzyme liquid The content of middle 87kD polypeptide.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 liquid, CrGDH3B-His crude enzyme liquid, 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 product 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 is then added2PO4, 300mM NaCl, 250mM imidazole, pH8.0) 4 DEG C of effect 10min, 3000rpm is centrifuged 1min and collects eluent, repeats to elute primary, receives Collect eluent, 1ml eluent is taken to carry out SDS-PAGE analysis.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 solution, Pure CrGDH3B-His enzyme solution, pure empty vector control bacterium enzyme solution and pure blank control bacterium enzyme solution, as enzyme solution to be measured.To It surveys enzyme solution and measures protein content using BCA quantification of protein kit quantification.
Glucose dehydrogenase activity measurement carries out colorimetric analysis grape according to red is generated using glucose as substrate The activity of glucocorticoid dehydrogenase.To 50 μ L enzyme solutions to be measured (pure CrGDH3A-His enzyme solution, pure CrGDH3B-His enzyme solution, pure sky Vehicle Control bacterium enzyme solution or pure blank control bacterium enzyme solution) in be added 50 μ L pH7.8 buffer (to 100mmol/L MOPS PQQ (pyrroloquinoline quinone) and CaCl are added in buffer2, make 10 μm of ol/L and CaCl of PQQ content2Content 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 of the pH7.8 of 50mmol/L, the 20mmol/L phenazine methosulfate 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 solution, 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 reduction) 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 dehydrogenase 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, have the function of the cultivation of Soluble phosphorus reconstituted protein microorganism-glucose dehydrogenase Soluble phosphorus engineering bacteria and its Identification
1. the building of glucose dehydrogenase CrGDH3A gene shuttle expression carrier
In order to obtain the Soluble phosphorus engineering bacteria of high efficient expression glucose dehydrogenase CrGDH3A gene, it is necessary first to which building can Shuttle expression carrier across host expresses.PWH1520 expression vector (7929bp) is the efficient shuttling expressing of bacillus megaterium Carrier (German MoBiTec Products are purchased in Beijing Baeyer enlightening biotech company), xylA promoter downstream carry BamHI restriction enzyme site has ammonia benzyl and tetracycline resistance gene, can stablize express express target protein.
CrGDH3A gene order is analyzed using DNAMAN software, discovery does not have BamHI restriction enzyme site, according to CrGDH3A base Because of complete coding region primers, Bam HI restriction enzyme site (GGATCC) is added in upstream and downstream primer.Upstream and downstream primer It is respectively as follows: 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, using the method for PCR amplification, introduces BamHI enzyme respectively at 5 ' ends of CrGDH3A gene complete coding region and 3 ' Recognition site obtains the CrGDH3A gene PCR product with enzyme recognition site；With BamHI digestion shuttle expression carrier PWH1520 and CrGDH3A gene PCR product with enzyme recognition site, the digestion products T of recycling4Ligase connection, 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 In conjunction with the positive bacterial plaque for the method screening CrGDH3A gene forward direction insertion shuttle expression carrier pWH1520 that sequencing compares, weight is extracted Group plasmid, the final shuttle expression carrier for obtaining CrGDH3A gene.Sequencing result is shown to be shown in SEQ ID No.1 The recombinant expression carrier that segment between 2 BamHI recognition sites of CrGDH3A gene forward direction replacement pWH1520 obtains is named as PWH-CrGDH3A (Fig. 3).PWH-CrGDH3A is containing CrGDH3A gene shown in SEQ ID No.1 in ordered list, pWH- 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 biotechnology company), obtain glucose dehydrogenase engineering bacteria.Glucose dehydrogenase engineering bacteria is accessed containing tetracycline In LB culture medium (concentration that tetracycline is added to tetracycline in LB culture medium is the culture medium that 100 μ g/ml are obtained), cultivated Night.It is transferred in the above-mentioned LB culture medium containing tetracycline with 2% inoculum concentration and continues culture to logarithmic growth phase, xylose is added and arrives Final concentration of 0.5%, Fiber differentiation 6h, 4000r/min revolving speed is centrifuged 15min at room temperature, collects supernatant as extracellular supernatant；It receives Collection precipitating, adds 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% is stayed overnight in 4 DEG C of extractions, and 12000r/min is centrifuged 10min, and supernatant is intracellular Supernatant is precipitated as precipitating intracellular.Respectively to supernatant intracellular, it is intracellular precipitating 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 quantification.
The activity of glucose dehydrogenase CrGDH3A is measured according to the method for embodiment 1, and experiment is in triplicate.SDS-PAGE Electrophoresis result (Fig. 4) display, CrGDH3A gene can be with normal expression, expression product in glucose dehydrogenase engineering bacteria For intracellular protein (supernatant swimming lane intracellular), 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, influence 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 medium containing ampicillin and tetracycline (to train in LB Supporting the concentration of addition ampicillin and tetracycline to ampicillin and tetracycline in base is the culture that 100 μ g/ml are obtained Base) in, overnight incubation.It is transferred in the above-mentioned LB culture medium 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 revolving speed is centrifuged 15min at room temperature, collects precipitating, Add 2 times of volume phosphate buffers (pH7.0), smudge cells obtain smudge cells suspension.It is added in smudge cells suspension Triton-X100 to final concentration of 1% is stayed overnight in 4 DEG C of extractions, and 12000r/min is centrifuged 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 system, PH5.8 reaction system, pH6.2 reaction system, pH6.6 reaction system and pH7.0 reaction system)；Tris buffer solution system (pH7.4 reaction system, pH7.8 reaction system, pH8.2 reaction system, pH8.6 reaction system and pH9.0 reaction system) measurement The ability of glucose dehydrogenase engineering bacteria catalysis 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 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 PQQ (pyrroloquinoline quinone) and CaCl2, make 10 μm of ol/L and CaCl of PQQ content2Content is the liquid that 2mol/L is obtained), 37 DEG C 1 hour structure to stablize enzyme is pre-processed, pretreatment enzyme solution is obtained.
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 methosulfate, 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 reduction) 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 range The 80% of suitable pH enzymatic activity, 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 medium containing ampicillin and tetracycline, Overnight incubation.It is transferred in the above-mentioned LB culture medium containing tetracycline with 2% inoculum concentration and continues culture to logarithmic growth phase, be added Xylose is to final concentration of 0.5%, Fiber differentiation 6h, and 4000r/min revolving speed is centrifuged 15min at room temperature, collects precipitating, 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 stayed overnight in 4 DEG C of extractions, and 12000r/min is centrifuged 10min, and supernatant is that glucose dehydrogenase is thick Enzyme solution, as enzyme solution to be measured.The buffer 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 are added in liquid2, make 10 μm of ol/L and CaCl of PQQ content2Content is the liquid that 2mol/L is obtained Body), 37 DEG C pre-process 1 hour structure to stablize enzyme, obtain pretreatment enzyme solution.In Tris buffer solution system (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 solution (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 are as follows: under the conditions of relevant temperature pH7.4, can make the grape of 1 μm of ol 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 inducing 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 then difficult 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 108Xylose is added to final concentration of in 37 DEG C of cultures to logarithmic growth phase in cfu/mL 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 revolving speed is centrifuged 10min at 4 DEG C, collects supernatant, Using molybdenum antimony resistance colorimetric method, with 722 type spectrophotometers, in wavelength 700nm, directly measurement is inoculated with glucose dehydrogenase engineering bacteria 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 test repeats 3 It is secondary.
Wherein, the pH of ground phosphate rock fluid nutrient medium is 7.0, and preparation method is as follows: being water by solvent, solute and its concentration are such as Under culture solution in 115 DEG C of sterilizings 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: being water by solvent, solute and its concentration are as follows Culture solution in 115 DEG C of sterilizings 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, preparation method is as follows: being water by solvent, solute and its concentration are following Culture solution is in 115 DEG C of sterilizings 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 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>construction method with the active recombinant microorganism of Soluble phosphorus and application
<130> GNCFH181846
<160> 8
<170> PatentIn version 3.5
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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
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 Val Lys
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atggctgaaa acaatgcacg ttcgccacga cttctcgtga cgctgacggc cctctttgca 60
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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
<210> 4
<211> 796
<212> PRT
<213>citric acid bacillus (Citrobacter rodentium)
<400> 4
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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (Artificial Sequence)
<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 (8)

1. constructing the method with the active recombinant microorganism of Soluble phosphorus, it is characterised in that: the method includes by glucose dehydrogenation The encoding gene of enzyme imports recipient microorganism, and obtaining Soluble phosphorus activity higher than the recipient microorganism has the active recombination of Soluble phosphorus Microorganism.
2. according to the method described in claim 1, it is characterized by: the glucose dehydrogenase is egg a) or b) or c) or d) White matter:
A) protein that the amino acid sequence shown in SEQ ID No.2 forms；
B) protein that the amino acid sequence shown in SEQ ID No.6 forms；
C) fusion protein that the c-terminus of the protein shown in a) or b) or/and aminoterminal fusion protein label obtain；
D) the taking by one or several amino acid residues by amino acid sequence shown in SEQ ID No.2 or SEQ ID No.6 The protein with glucose dehydrogenase activity that generation and/or deletion and/or addition obtain.
3. method according to claim 1 or 2, it is characterised in that: the encoding gene is following 1) or 2) or 3) shown Gene:
1) coded sequence is DNA molecular shown in SEQ ID No.1；
2) coded sequence is DNA molecular shown in SEQ ID No.5；
3) with 1) or 2) DNA molecular that limits with 90% or more identity and encode protein described in claim 1 DNA molecular.
4. method according to claim 1 or 2, it is characterised in that: the recipient microorganism is M1, M2 or M3:
M1, prokaryotic micro-organisms；
M2, gramnegative bacterium or gram-positive bacterium；
M3, Escherichia bacteria or bacillus.
5. application of the method described in any claim in dissolved metals in claim 1-4.
6. biomaterial relevant to glucose dehydrogenase described in claim 2, the biomaterial is B1) or B2):
B1 the expression cassette) containing encoding gene described in Claims 2 or 3；
B2 the recombinant vector) containing encoding gene described in Claims 2 or 3.
7. application of the biomaterial as claimed in claim 6 in preparation glucose dehydrogenase.
8. biomaterial as claimed in claim 7 has the application in Soluble phosphorus reconstituted protein microorganism in preparation.
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* Cited by examiner, † Cited by third party
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