CN109402084A

CN109402084A - Construct the method and its nucleic acid molecule used therefor with Soluble phosphorus reconstituted protein microorganism

Info

Publication number: CN109402084A
Application number: CN201811351550.2A
Authority: CN
Inventors: 孙静文; 周卫; 程明芳; 李书田; 王玉军
Original assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Current assignee: Institute of Agricultural Resources and Regional Planning of CAAS
Priority date: 2016-01-26
Filing date: 2016-01-26
Publication date: 2019-03-01
Anticipated expiration: 2036-01-26
Also published as: CN109234251B; CN105779408A; CN105779408B; CN109234251A; CN109402084B

Abstract

The invention discloses methods and its nucleic acid molecule used therefor that building has Soluble phosphorus reconstituted protein microorganism.This method includes that the encoding gene of following protein is imported recipient microorganism, and obtaining Soluble phosphorus activity higher than the recipient microorganism has Soluble phosphorus reconstituted protein microorganism: a) protein that the amino acid sequence shown in SEQ ID No.2 1-203 forms；B) protein that the amino acid sequence shown in SEQ ID No.2 26-203 forms；C) fusion protein that the c-terminus of the protein shown in a) or b) or/and aminoterminal fusion protein label obtain；D) protein with activity of acid phosphatase for obtaining amino acid sequence shown in SEQ ID No.2 or SEQ ID No.6 by the substitution and/or deletion and/or addition of one or several amino acid residues.The present invention can be used for cultivating the bioengineered strain of efficient activating soil phosphorus nutrients.

Description

Construct the method and its nucleic acid molecule used therefor with Soluble phosphorus reconstituted protein microorganism

It is on 01 26th, 2016 that the application, which is application No. is the 201610051877.2, applying date, invention and created name is The divisional application of " application of acid phosphatase and its relevant biological material in building Soluble phosphorus engineering bacteria ".

Technical field

The present invention relates to methods and its nucleic acid molecule used therefor that building in biological field has Soluble phosphorus reconstituted protein microorganism.

Background technique

Phosphorus is one of big indispensable element of plant growth and development three, is played an important role in life process.The phosphorus that plant utilizes Element is mainly derived from soil.Currently, China is since available phosphorus contains in soil the main reason for having 2/3 arable land to lack phosphorus, lack phosphorus Amount is insufficient, and about 95% phosphorus is the invalid phosphorus of slightly solubility, and plant is difficult to be absorbed and utilized.China consumes about 1050- every year 12000000 tons of phosphate fertilizer (China Chemical Industry Information Network in 2010), but phosphate fertilizer this season plant utilization rate is only 5%-25%, 90% is left Right phosphate fertilizer is fixed by chemistry quickly after being manured into soil.Therefore, phosphate fertilizer utilization efficiency is improved, the invalid phosphorus element of activating soil is agricultural Produce one of the problem in science urgently solved.

Acid phosphatase, phytase play crucial work in the decomposition and release of Organic phosphate in Soluble phosphorus microorganism With (Yamamura et al., 2004；Zhao little Rong etc., 2001；Chen Zhe etc., 2009).Acid phosphatase (Acid Phosphatase, abbreviation ACPase, E.C.3.1.3.2), it is the enzyme of catalytic phosphatase monoesters hydrolysis in acid condition.This enzyme removes The metabolism of phosphate is participated in, also the important vital movement such as participation Metabolism regulation, energy conversion and signal transduction.Acid phosphatase Enzyme has very important function, first, acid phosphatase has the activity of phosphohydrolase, by the phosphorus one for decomposing organic matter Ester bond and one acid anhydride key of phosphorus discharge phosphorus, so that invalid phosphorus in activating soil, is making full use of soil phosphor resource, reducing on phosphate fertilizer With significant application value；Second, acid phosphatase also has phosphate transferase activity, it under suitable conditions, can be by low energy Phosphate group is transferred on the hydroxyl of nucleosides, has biggish application value in the synthesis of nucleotide biochemistry.Nucleotide is usually made For food additives and medicine intermediate, wherein inosinicacid (hypoxanthine -5 '-nucleotide) is because fresh with more obviously helping Effect is widely used in food processing field.There are mainly two types of methods to produce nucleotide at present, and one is chemical synthesis, benefits It is fermented with thallus and generates inosine, then use POCl₃Phosphorylation, this method by-product is more, purification difficult；Another method is to utilize Escherichia coli inosine kinase phosphorylating inosine, this process needs the participation of ATP, and ATP is needed by producing the regeneration of ammonia bacillus fermentation, Limit the application of enzymatic clarification.

Summary of the invention

A technical problem to be solved by this invention is to provide a kind of higher acid phosphatase of phosphohydrolase enzymatic activity Enzyme.

In order to solve the above technical problems, the present invention provides protein a) or b) or c) or d) in as phosphohydrolase Application:

A) protein that the amino acid sequence shown in SEQ ID No.2 1-203 forms；

B) protein that the amino acid sequence shown in SEQ ID No.2 26-203 forms；

C) c-terminus (C-terminal) of the protein shown in a) or b) or/and aminoterminal (N-terminal) fusion protein label obtain The fusion protein arrived；

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 activity of acid phosphatase of deletion and/or addition.

In above-mentioned application, a) shown in protein be from bacillus megaterium (Bacillus megaterium) Complete acid phosphatase, entitled BmacpA；SEQ ID No.2 is made of 203 amino acid residues, and 1-25 are signal Peptide.

In above-mentioned application, b) shown in protein be remove that the signal peptide of BmacpA obtains remove signal peptide acid phosphatase Enzyme, entitled NSBmacpA.

In above-mentioned application, 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..C) protein shown in specifically may be used To merge the fused protein that histidine tag obtains in the c-terminus of NSBmacpA or/and aminoterminal, such as SEQ ID No.6 institute The protein shown, entitled NSBmacpA-His.SEQ ID No.6 is made of 192 amino acid residues.

In order to solve the above technical problems, the present invention provides nucleic acid molecules and is preparing the application in phosphohydrolase；The core Acid molecule encodes above-mentioned protein a) or b) or c) or d).

In above-mentioned application, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid point Son is also possible to RNA, such as mRNA or hnRNA.

Above-mentioned nucleic acid molecules concretely it is following 1) or 2) or 3) or 4) shown in gene:

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

2) coded sequence is DNA molecular shown in the 76th to 612 of SEQ ID No.1, entitled NSBmacpA base Cause；

3) coded sequence is DNA molecular shown in SEQ ID No.5；Its entitled NSBmacpA-His gene；

1) or 2) or 3) 4) identity and the above-mentioned acid phosphatase of coding with the DNA molecular that limits with 90% or more DNA molecular.

In above-mentioned application, " identity " refers to the sequence similarity with native sequence nucleic acid." identity " can with the naked eye or Computer software is evaluated.Using computer software, the identity between two or more sequences can use percentage (%) It indicates, can be used to evaluate the identity between correlated series.

Another technical problem to be solved by this invention how is constructed with the active microorganism of Soluble phosphorus.

In order to solve the above technical problems, the present invention provides above-mentioned nucleic acid molecules to have Soluble phosphorus active microorganism in building In application.

In order to solve the above technical problems, the present invention provides a kind of specific buildings to have Soluble phosphorus reconstituted protein microorganism Method.

The method that building provided by the present invention has Soluble phosphorus reconstituted protein microorganism, including by it is above-mentioned a) or b) or c) or D) encoding gene of protein imports recipient microorganism, and obtain Soluble phosphorus activity has Soluble phosphorus living higher than the recipient microorganism Property recombinant microorganism.

In the above method, the Soluble phosphorus activity is presented as phosphoric acid hydrolysis enzymatic activity.

In the above method, the encoding gene be it is following 1) or 2) or 3) or 4) shown in DNA molecular:

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

2) coded sequence is DNA molecular shown in the 76th to 612 of SEQ ID No.1；

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

1) or 2) or 3) 4) with the DNA molecular that limits with 90% or more identity and the DNA of code for said proteins Molecule.

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

In the above method, the prokaryotic micro-organisms concretely gramnegative bacterium.

In the above method, concretely Escherichia bacteria or Citrobacter are thin for the gramnegative bacterium Bacterium.

In the above method, the Escherichia bacteria concretely Escherichia coli, such as e. coli bl21 (DE3).Institute Stating Citrobacter bacterium can be citric acid bacillus ACCC02187.

In order to solve the above technical problems, the present invention provides the biomaterials for containing the nucleic acid molecules.

Biomaterial provided by the present invention containing the nucleic acid molecules, the biomaterial be B1), B2), B3) or B4):

B1) contain the expression cassette of the nucleic acid molecules；

B2) contain the recombinant vector of the nucleic acid molecules；

B3) contain the recombinant vector of the expression cassette；

B4) any of the above-described kind of the described of method building has Soluble phosphorus reconstituted protein microorganism.

In above-mentioned biomaterial, the expression cassette containing the nucleic acid molecules, be refer to express in host cell it is above-mentioned A) DNA of protein or b) or c) or d), the DNA not only may include the promoter for starting above-mentioned protein gene transcription, also It may include the terminator for terminating above-mentioned protein gene transcription.The recombinant vector can be pET-NSBmacpA or pHT- BmacpA.PET-the NSBmacpA imports the recipient microorganism；PET-the NSBmacpA is with sequence 5 in sequence table the Segment between NdeI the and HindIII recognition site of the replacement of DNA molecular shown in 4 to 537 nucleotide pET-30b (+) obtains Recombinant expression carrier；PHT-the BmacpA be in sequence table DNA molecular shown in sequence 1 replacement pHT43 BamHI and The recombinant expression carrier that segment between XbaI recognition site obtains.

It is demonstrated experimentally that BmacpA and NSBmacpA all have phosphoric acid hydrolysis enzymatic activity, BmacpA and NSBmacpA 37 DEG C, Phosphoric acid hydrolysis enzyme activity in the 50mmol/L NaAc_HAc buffer solution of pH5.0 be respectively 33.96 ± 1.32U/ml albumen and 37.35 ± 1.55U/ml albumen；Acid phosphatase enzyme engineering bacteria that BmacpA channel genes citric acid bacillus is obtained with as by The citric acid bacillus of body bacterium is compared, (containing salmon sperm dna), phosphatide fluid nutrient medium (phosphorus containing L-A- in nucleic acid fluid nutrient medium Acyl inositol) and phosphoinositide fluid nutrient medium (containing Isosorbide-5-Nitrae, 5- InsP3), the content of available phosphorus has increased separately 14.59 μm ol/L, 16.63 μm of ol/L and 18.55 μm of ol/L (Fig. 7).The present invention can be used for cultivating the life of efficient activating soil phosphorus nutrients Object engineering bacteria.

Detailed description of the invention

Fig. 1 is the SDS-PAGE map of the inducing expression acid phosphatase in Escherichia coli.

In Fig. 1, M: albumen Marker；1: blank control bacterium crude enzyme liquid；2: empty vector control bacterium crude enzyme liquid；3: NSBmacpB-His crude enzyme liquid；4:NSBmacpA-His crude enzyme liquid.

Fig. 2 is the influence of different pH and reaction time to the phosphate transferase activity of NSBmacpA-His.

Fig. 3 is the influence of different pH and reaction time to the phosphate transferase activity of NSBmacpB-His.

Fig. 4 is different divalent ions on the active influence of NSBmacpA-His and NSBmacpB-His phosphohydrolase.

In Fig. 4, BmacpA NSBmacpA-His；BmacpB is NSBmacpB-His.

Fig. 5 is the SDS-PAGE expression map of acid phosphatase in recombination engineering.

In Fig. 5, M: albumen Marker；1: supernatant intracellular；2: extracellular supernatant.

Fig. 6 is influence of the pH to the acid phosphatase enzymatic activity of acid phosphatase enzyme engineering bacteria.

Fig. 7 is the effect of solubilizing phosphate of the acid phosphatase enzyme engineering bacteria in addition organic phosphorus sources culture medium.

In Fig. 7, citric acid bacillus is citric acid bacillus ACCC02187, is transferred to the citric acid bacillus of acid phosphatase as acid Acid phosphatase 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.

Bacillus megaterium (Bacillus megaterium) ACCC10010 (Granted publication as used in the following examples The Chinese invention patent that day is on 2 16th, 2005, Authorization Notice No. is CN1189086C) it was collected before the applying date of the application In China Committee for Culture Collection of Microorganisms agricultural microorganism center (abbreviation ACCC, address: Zhongguangcun, Haidian District, Beijing City South Street 12, INST OF AGRICULTURAL RESOURCES, postcode 100081), it is public from collecting Crowd can obtain the bacterial strain from China Committee for Culture Collection of Microorganisms agricultural microorganism center.Referred to as huge bud in the present invention Spore bacillus ACCC10010.

Bacillus megaterium (Bacillus megaterium) ACCC02970 as used in the following examples is in the application The applying date before be concealed in China Committee for Culture Collection of Microorganisms agricultural microorganism center (abbreviation ACCC, address: Beijing City, Zhong Guan-cun South Street, Haidian District 12, INST OF AGRICULTURAL RESOURCES, postcode 100081), It collects day as on December 20th, 2007, and from collecting, the public can be from China Committee for Culture Collection of Microorganisms's agriculture Industry microorganism center obtains the bacterial strain.Abbreviation bacillus megaterium ACCC02970 in the present invention.

Citric acid bacillus (Citrobacter sp.) ACCC02187 was concealed in the micro- life of China before the applying date of the application Object culture presevation administration committee agricultural microorganism center (abbreviation ACCC, address: ZhongGuanCun south Street, Haidian District, BeiJing City 12 Number, INST OF AGRICULTURAL RESOURCES, postcode 100081), collection day is November 1 in 2005 Day, from collecting, the public can obtain the bacterial strain from China Committee for Culture Collection of Microorganisms agricultural microorganism center. Abbreviation citric acid bacillus ACCC02187 in the present invention.

The preparation and functional verification of embodiment 1, acid phosphatase BmacpA

One, the clone of BmacpA gene and BmacpB gene

The genomic DNA of bacillus megaterium ACCC10010 and bacillus megaterium ACCC02970 are extracted, respectively with huge The genomic DNA of Bacterium anthracoides ACCC10010 is template, using P1 and P2 as primer, PCR amplification BmacpA gene；With huge The genomic DNA of bacillus ACCC02970 is template, using P3 and P4 as primer, PCR amplification BmacpB gene.Wherein, P1, P2, P3 and P4 sequence are as follows: P1:5 '-ATGTATGTGAAACGATATCG-3 ', P2:5 '-CTACTTTTGTCGAACACATA- 3 ', P3:5 '-ATGGTAAATCGCACTACAAA-3 ', P4:5 '-CTATTTTTGGTTATATAAGC-3 '.

Obtained BmacpA gene PCR product and BmacpB gene PCR product are subjected to electrophoresis respectively, the results showed that The band that BmacpA gene PCR product and BmacpB gene PCR product are about 600bp, is separately recovered BmacpA gene PCR product It with BmacpB gene PCR product, is individually connected on cloning vector, screening and identification positive colony, and carries out sequencing； Sequencing result shows the DNA sequence dna of BmacpA gene PCR product as shown in sequence 1 in sequence table, the sequence 1 in sequence table by 612 nucleotide form, protein shown in sequence 2 in polynucleotide, and sequence 2 is by 203 amino acid residues in sequence table Protein shown in sequence 2 in sequence table is named as BmacpA by composition, and 1-25 of sequence 2 are signal peptide sequence, sequence DNA molecular shown in sequence 1 in table is BmacpA gene；Sequence in the DNA sequence dna of BmacpB gene PCR product such as sequence table Shown in 3, sequence 3 is made of 627 nucleotide in sequence table, protein shown in sequence 4 in polynucleotide, in sequence table Sequence 4 is made of 208 amino acid residues, protein shown in sequence 4 in sequence table is named as BmacpB, in sequence table 1-25 of sequence 4 are signal peptide sequence, and DNA molecular shown in sequence 3 is BmacpB gene in sequence table.

Two, the building of recombinant expression carrier

1, the building of pET-NSBmacpA

The BmacpA gene expression primer of design removal signal peptide sequence.According to bacillus megaterium ACCC10010

Signal peptide primer is removed in the design of BmacpA gene function structural domain, and NdeI enzyme is added in the 5 ' end primers for removing signal peptide HindIII restriction enzyme site (AAGCTT) is added in 3 ' end primers in enzyme site (CATATG).Upstream and downstream primer is respectively as follows: P5 primer, 5 '-AT-CATATG-TTTAATACACCTTGGGTGAA-3 ' and P6 primer, 5 '-GC-AAGCTT-CTTTTGT CGAACACATAA-3′.Using the method for PCR amplification, in the gene coding region BmacpA, removal signal peptide coding DNA is (in sequence table The 1-75 nucleotide of sequence 1), and NdeI and HindIII enzyme recognition site is introduced respectively at 5 ' ends and 3 ' ends, it is not taken a message Number peptide BmacpA gene PCR product will be NSBmacpA gene without signal peptide BmacpA unnamed gene, will be without signal peptide BmacpA gene PCR product is named as NSBmacpA gene PCR product.NSBmacpA gene PCR product is containing sequence in ordered list The 76th to 609 nucleotide of column 1 (the 4th to 537 nucleotide of sequence 5 in sequence table).

Target fragment is recycled in NSBmacpA gene PCR product NdeI and the HindIII digestion that above-mentioned steps one are obtained (NSBmacpA gene)；With NdeI and HindIII digestion carrier pET-30b (+), (EMD Biosciences, is purchased in north simultaneously Capital company, fresh warp thread section), recycle carrier large fragment；The target fragment of recycling and 16 DEG C of the carrier large fragment of recycling are connected, obtained Purpose plasmid.By purpose plasmid CaCl₂Method converts bacillus coli DH 5 alpha competent cell.It is spread evenly across the blueness of benzyl containing ammonia On the LB plate of mycin, 37 DEG C are cultivated 16 hours.Single colonie shaken cultivation is stayed overnight, and is extracted plasmid and is carried out with NdeI and HindIII Double digestion identification, the correct plasmid of digestion verification is sequenced, sequencing result is shown to be with the sequence 5 the 4th in sequence table The recombination table obtained to the segment between NdeI the and HindIII recognition site of the replacement of DNA molecular shown in 537 pET-30b (+) PET-NSBmacpA is named as up to carrier.PET-NSBmacpA contains His tag fusion protein NSBmacpA-His encoding gene, The nucleotide sequence of NSBmacpA-His encoding gene is the sequence 5 in sequence table, and NSBmacpA-His is the sequence in sequence table Protein shown in column 6.

2, the building of pET-NSBmacpB

The BmacpB gene expression primer of design removal signal peptide sequence.According to bacillus megaterium ACCC02970's NdeI restriction enzyme site (CATATG) is added in the 5 ' end primers for removing signal peptide in BmacpB coding sequence design primer, HindIII restriction enzyme site (AAGCTT) is added in 3 ' end primers.Upstream and downstream primer is respectively as follows: P7 primer, 5 '-AT-CATATG– TTTAA TACACCTTGG GTGAA-3 ' and P8 primer, 5 '-GC-AAGCTT-TTTTTGG TTATATAAGCG-3′.Utilize PCR The method of amplification removes signal peptide in the gene coding region BmacpB and introduces NdeI and HindIII identification respectively at 5 ' ends and 3 ' Site is obtained without signal peptide BmacpB gene PCR product, will be NSBmacpB base without signal peptide BmacpB unnamed gene Cause will be named as NSBmacpB gene PCR product without signal peptide BmacpB gene PCR product.NSBmacpB gene PCR product Containing the 76th to 624 nucleotide of sequence 3 in ordered list (the 4th to 552 nucleotide of sequence 7 in sequence table).

Purpose piece is recycled in NSBmacpB gene PCR product NdeI and the HindIII digestion that above-mentioned steps one are obtained Section；With NdeI and HindIII digestion carrier pET-30b (+), (EMD Biosciences is purchased in Beijing fresh warp thread section public affairs simultaneously Department), recycle carrier large fragment；The target fragment of recycling and 16 DEG C of the carrier large fragment of recycling are connected, purpose plasmid is obtained.It will Purpose plasmid CaCl₂Method converts bacillus coli DH 5 alpha competent cell.LB with ampicillin is spread evenly across to put down On plate, 37 DEG C are cultivated 16 hours.Single colonie shaken cultivation is stayed overnight, and is extracted plasmid with NdeI and HindIII and is carried out double digestion identification, The correct plasmid of digestion verification is sequenced, sequencing result is shown to be with the 4th to 552 nucleotide of sequence 7 in sequence table Shown in DNA molecular replacement pET-30b (+) NdeI and HindIII recognition site between the obtained recombinant expression carrier of segment It is named as pET-NSBmacpB.PET-NSBmacpB contains His tag fusion protein NSBmacpB-His encoding gene, The nucleotide sequence of NSBmacpB-His encoding gene is the sequence 7 in sequence table, and NSBmacpB-His is the sequence in sequence table Protein shown in column 8.

Three, the preparation of the recombination bacillus coli of acid phosphatase is expressed

1, the expression of NSBmacpA-His

PET-the NSBmacpA of step 2 Calcium Chloride Method is converted into e. coli bl21 (DE3) (Tiangeng company), is utilized Kanamycin resistance screening positive colony screening and culturing, picking monoclonal carry out PCR identification by primer of above-mentioned P5 and P6, will PCR identifies to obtain the positive colony of 539bp or so PCR product as genetic engineering bacterium, is named as pET-NSBmacpA/BL21. Picking pET-NSBmacpA/BL21 bacterial strain, the LB culture medium for being inoculated in the kanamycins containing 100ug/ml (are added in LB culture medium The concentration of kanamycins to kanamycins is the culture medium that 100ug/ml is obtained) in, 37 DEG C of cultures to 0D₆₀₀Value is (to contain 100ug/ The LB culture medium of ml kanamycins is blank control) when reaching 0.6, IPTG to final concentration l mM is added, in turning for 150r/min The lower 28 DEG C of inductions 6h of speed, collects culture solution after 4000r/min is centrifuged 20min, and bacterium is resuspended with 50mM Tris-HCl (pH7.1) It is 10 that body, which obtains thallus content,⁸The thallus suspension liquid of cfu/ml, thallus suspension liquid is through ultrasonication, 12 000r/min centrifugation 10min collects supernatant (mycetome gross protein), which is named as NSBmacpA-His crude enzyme liquid.

2, the expression of NSBmacpB-His

PET-the NSBmacpB of step 2 Calcium Chloride Method is converted into e. coli bl21 (DE3) (Tiangeng company), is utilized Kanamycin resistance screening positive colony screening and culturing, picking monoclonal carry out PCR identification by primer of above-mentioned P7 and P8, will PCR identifies to obtain the positive colony of 552bp or so PCR product as genetic engineering bacterium, is named as pET-NSBmacpB/BL21. Picking pET-NSBmacpB/BL21 bacterial strain, the LB culture medium for being inoculated in the kanamycins containing 100ug/ml (are added in LB culture medium The concentration of kanamycins to kanamycins is the culture medium that 100ug/ml is obtained) in, 37 DEG C of cultures to 0D₆₀₀Value is (to contain 100ug/ The LB culture medium of ml kanamycins is blank control) when reaching 0.6, IPTG to final concentration l mM is added, in turning for 150r/min The lower 28 DEG C of inductions 6h of speed, collects culture solution after 4000r/min is centrifuged 20min, and bacterium is resuspended with 50mM Tris-HCl (pH7.1) It is 10 that body, which obtains thallus content,⁸The thallus suspension liquid of cfu/ml, thallus suspension liquid is through ultrasonication, 12 000r/min centrifugation 10min collects supernatant (mycetome gross protein), which is named as NSBmacpB-His crude enzyme liquid.

3, empty vector control bacterium

PET-30b (+) is transferred to e. coli bl21 (DE3) according to method identical with step 1, obtained recombination is big Entitled pET-30b (+)/BL21 of enterobacteria.Using pET-30b (+)/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-30b (+)/BL21 bacterial strain, is inoculated in kanamycins containing 100ug/ml LB culture medium (concentration that kanamycins is added to kanamycins in LB culture medium is the obtained culture medium of 100ug/ml) in, 37 DEG C are cultivated to 0D₆₀₀When value (using the LB culture medium of the kanamycins containing 100ug/ml 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, 12 000r/min are centrifuged 10min, collect supernatant (mycetome gross protein), which is named as zero load Body compares 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 the LB culture medium of the kanamycins containing 100ug/ml (in LB The concentration that kanamycins is added to kanamycins in culture medium is the culture medium that 100ug/ml is obtained) in, 37 DEG C of cultures to 0D₆₀₀ When value (using the LB culture medium of the kanamycins containing 100ug/ml as blank control) reaches 0.6, IPTG to final concentration l mM is added, The lower 28 DEG C of inductions 6h of the revolving speed of 150r/min collects culture solution after 4000r/min is centrifuged 20min, with 50mM Tris-HCl (pH7.1) it is 10 that thallus, which is resuspended, and obtains thallus content⁸The thallus suspension liquid of cfu/ml, thallus suspension liquid is through ultrasonication, and 12 000r/min is centrifuged 10min, collects supernatant (mycetome gross protein), which is named as the thick enzyme of blank control bacterium Liquid.

Take 30 μ L NSBmacpA-His crude enzyme liquids (from 10⁸cfu pET–NSBmacpA/BL21)、30μL NSBmacpB-His crude enzyme liquid (comes from 10⁸Cfu pET-NSBmacpB/BL21), 30 μ L empty vector control bacterium crude enzyme liquids (come from 10⁸Cfu pET-30b (+)/BL21) and 30 μ L blank control bacterium crude enzyme liquids (come from 10⁸Cfu e. coli bl21 (DE3)) same SDS-PAGE analysis (resolving gel concentration 12%) is carried out on one piece of glue, the sample-adding pore volume on the glue is consistent with shape, adds Sample pore volume is 80 μ L.

Although SDS-PAGE result as shown in Figure 1, show NSBmacpA-His crude enzyme liquid, NSBmacpB-His crude enzyme liquid, Have the band of 27kD in empty vector control bacterium crude enzyme liquid and blank control bacterium crude enzyme liquid, but NSBmacpA-His crude enzyme liquid and The content of 27kD polypeptide is apparently higher than empty vector control bacterium crude enzyme liquid and the thick enzyme of blank control bacterium in NSBmacpB-His crude enzyme liquid The content of 27kD polypeptide in liquid, and the content of the 27kD polypeptide in NSBmacpA-His crude enzyme liquid is thick higher than NSBmacpB-His The content of 27kD polypeptide in enzyme solution.Illustrate that NSBmacpA-His and NSBmacpB-His are obtained in e. coli bl21 (DE3) Expression is arrived, and expression quantity of the NSBmacpA-His in e. coli bl21 (DE3) is higher than NSBmacpB-His in large intestine Expression quantity in bacillus BL21 (DE3).

Four, the phosphoric acid hydrolysis enzymatic activity of NSBmacpA-His and NSBmacpB-His is measured

Take the NSBmacpA-His crude enzyme liquid of step 3, NSBmacpB-His crude enzyme liquid, empty vector control bacterium crude enzyme liquid and Blank control bacterium crude enzyme liquid uses nickel column (the high-affinity Ni-NTA Rasin product purchased from AM General company) respectively It is purified, nickel column is pre-processed, crude enzyme liquid is added, (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 NSBmacpA-His show that 15 amino acid of its N-terminal are the of sequence 6 in sequence table 1-15 amino acids, the sequencing results of NSBmacpB-His show that 15 amino acid of its N-terminal are sequence 8 in sequence table 1-15 amino acids.

The eluent of above-mentioned collection is dialysed with distilled water, removes salt ion, obtain respectively pure NSBmacpA-His enzyme solution, Pure NSBmacpB-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.

Using the phosphorus of common p-nitrophenol sodium phosphate (pNPP) method measurement NSBmacpA-His and NSBmacpB-His Sour hydrolytic enzyme activities.Used reaction system is by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L acetic acid- Sodium acetate buffer and MnCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, MnCl₂Concentration For 1mmol/L.10min is reacted at 37 DEG C, the Na0H that 0.1ml 5mmol/L is added after reaction immediately, which is terminated, to be reacted and measure A_405nm.Use blank reaction system as blank control.The blank reaction system inactivates enzyme solution to be measured by isometric heat, to nitro Phenol sodium phosphate (pNPP), 50mmol/L NaAc_HAc buffer solution and MnCl₂Composition, the pH of the blank reaction system are 5.0, pNPP concentration is 1mmol/L, MnCl₂Concentration be 1mmol/L.Enzyme activity unit (U) is defined as: 37 DEG C, pH5.0 item Under part, the amount that catalysis per minute generates 1 μm of ol phosphoric acid hydrolysis product pNP (p-nitrophenol) is 1 enzyme activity unit.

Experiment is set to be repeated three times.The result shows that not having in pure empty vector control bacterium enzyme solution and pure blank control bacterium enzyme solution Phosphoric acid hydrolysis enzymatic activity, by pET-NSBmacpA/BL21 expression NSBmacpA-His phosphoric acid hydrolysis enzyme activity be 37.35 ± 1.55U/mg albumen, by pET-NSBmacpB/BL21 expression NSBmacpB-His phosphoric acid hydrolysis enzyme activity be 12.49 ± 1.26U/mg albumen.The phosphoric acid hydrolysis enzyme activity of NSBmacpA-His is 2.99 times of NSBmacpB-His.

Five, the influence of differential responses time and pH to the phosphate transferase activity of NSBmacpA-His and NSBmacpB-His

Converting inosine rate (by converting inosine for the ability of inosinicacid) is used to embody NSBmacpA-His and NSBmacpB- The phosphate transferase activity of His.The specific method is as follows:

The pure NSBmacpA-His enzyme solution and pure NSBmacpB-His enzyme solution that step 4 is obtained, as enzyme to be measured Liquid.Enzyme solution to be measured measures protein content using BCA quantification of protein kit quantification.

Using the different reaction system of 5 pH value (pH4.0 reaction system, pH5.0 reaction system, pH6.0 reaction system, PH7.0 reaction system and pH8.0 reaction system) measurement NSBmacpA-His and NSBmacpB-His by converting inosine be inosinicacid Ability.

The different reaction system of above-mentioned 5 pH value is gentle by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP), inosine Rush solution composition.The concentration of the pNPP of the different reaction system of above-mentioned 5 pH value is 5mmol/L, and the concentration of inosine is 1mmol/L.The pH of pH4.0 reaction system is 4.0, and buffer solution is 0.2mmol/L NaAc_HAc buffer solution.PH5.0 is anti- Answering the pH of system is 5.0, and buffer solution is 0.2mmol/L NaAc_HAc buffer solution.The pH of pH6.0 reaction system is 6.0, buffer solution is 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.The pH of pH7.0 reaction system is 7.0, buffer solution For 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.The pH of pH8.0 reaction system is 8.0, and buffer solution is 0.2mmol/L phosphorus Acid dihydride sodium-disodium hydrogen phosphate.

Above-mentioned each reaction system respectively at 37 DEG C react 15min, 30min, 45min, 60min, 75min, 90min, 120min, the Na0H that 0.1ml 5mmol/L is added after reaction immediately terminates reaction, with containing for HPLC assay inosinicacid Amount, according to converting inosine rate (converting inosine rate=inosine acid content/Inosine Content × 100%) embody NSBmacpA-His and The phosphate transferase activity of NSBmacpB-His.Use corresponding blank reaction system as blank control.The blank of each pH value Reaction system is that the enzyme solution to be measured in corresponding pH value reaction system is replaced with isometric heat to inactivate enzyme solution to be measured, other Ingredient is identical as corresponding pH value reaction system.HPLC condition are as follows: chromatographic column: 5 μm of Hypersil SAX (4.6mm × 250mm), mobile phase: 60mmol/L pH3.0 phosphoric acid-ammonium dihydrogen phosphate buffer, flow velocity: 1mL/min, Detection wavelength 254nm, Column temperature: 25 DEG C.

Experiment is set to be repeated three times.As a result as shown in Figures 2 and 3, show that the phosphate transferase activity of NSBmacpA-His exists (pH4.0-6.0) is in a higher level under acid condition, and converting inosine rate can reach 31%-38% within 45min, It is 5, reacts the converting inosine rate highest of 30min in pH value, is 38% (Fig. 2)；The phosphate transferase activity of NSBmacpB-His (pH4.0-6.0) is lower than NSBmacpA-His in acid condition, and NSBmacpB-His is 5, reacts the inosine of 30min in pH value Conversion ratio is up to 20% (Fig. 3).The converting inosine rate of NSBmacpA-His and NSBmacpB-His is 5, reacts in pH value Time is highest under conditions of 30min, and the converting inosine rate of NSBmacpA-His is higher than NSBmacpB-His, NSBmacpA-His Converting inosine rate be 1.90 times of NSBmacpB-His.

Illustrate that NSBmacpA-His and NSBmacpB-His all have phosphoric acid hydrolysis enzymatic activity and phosphoric acid in acid condition Transferase active is acid phosphatase.

Six, metal ion is on the active influence of NSBmacpA-His and NSBmacpB-His phosphohydrolase

Using 9 different reaction system (Mn²⁺Reaction system, Cu²⁺Reaction system, Fe²⁺Reaction system, Zn²⁺Reactant System, Co²⁺Reaction system, Ca²⁺Reaction system, control reaction system and EDTA reaction system) measurement NSBmacpA-His and The phosphoric acid hydrolysis enzymatic activity of NSBmacpB-His.

Control reaction system is by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Buffer solution composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L.

Mn²⁺Reaction system is buffered by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and MnCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, MnCl₂Concentration be 1mmol/L。

Cu²⁺Reaction system is buffered by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and CuCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, CuCl₂Concentration be 1mmol/L。

Fe²⁺Reaction system is buffered by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and FeCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, FeCl₂Concentration be 1mmol/L。

Zn²⁺Reaction system is buffered by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and ZnCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, ZnCl₂Concentration be 1mmol/L。

Co²⁺Reaction system is buffered by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and CoCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, CoCl₂Concentration be 1mmol/L。

Ca²⁺Reaction system is buffered by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and CaCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, CaCl₂Concentration be 1mmol/L。

EDTA reaction system is slow by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L Acetic acid-sodium acetate Solution composition and EDETATE SODIUM composition are rushed, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/L, and the concentration of EDTA is 5mmol/L。

10min is reacted at 37 DEG C of above-mentioned each reaction system, the Na0H that 0.1ml 5mmol/L is added after reaction immediately is terminated It reacts and measures A_405nm.Use corresponding blank reaction system as blank control.The blank reactant of above-mentioned each reaction system Enzyme solution to be measured in corresponding reaction system is only replaced with isometric heat and inactivates enzyme solution to be measured by system, other ingredients with phase The reaction system answered is identical.Enzyme activity unit (U) is defined as: 37 DEG C, under the conditions of pH5.0, catalysis generates 1 μm of ol phosphoric acid per minute The amount of hydrolysate pNP (p-nitrophenol) is 1 enzyme activity unit.

As a result as shown in figure 4, in addition to Ca²⁺+ with outside EDTA, the addition of divalent metal can improve NSBmacpA- The phosphoric acid hydrolysis enzymatic activity of His and NSBmacpB-His, wherein Mn²⁺、Zn²⁺It is best to improve activity of acid phosphatase effect, NSBmacpA-His adds Mn in external source²⁺、Zn²⁺Later, enzymatic activity be respectively 37.35 ± 1.55U/mg albumen and 32.54 ± 1.36U/mg albumen, and NSBmacpB-His adds Mn in external source²⁺、Zn²⁺Later, enzymatic activity is respectively 12.49 ± 1.26U/mg The two is compared and shows that the phosphoric acid hydrolysis enzymatic activity of NSBmacpA-His is by albumen and 10.12 ± 1.17U/mg albumen Active 2.99-3.22 times of the phosphohydrolase of NSBmacpB-His.

The cultivation and its Function Identification of embodiment 2, acid phosphatase Soluble phosphorus engineering bacteria

1. the building of Bacillus megatherium acid phosphatase BmacpA gene shuttle expression carrier

In order to obtain the Soluble phosphorus engineering bacteria of high efficient expression Bacillus megatherium acid phosphatase BmacpA gene, need first Building can be across the shuttle expression carrier of host expresses.PHT43 (purchase in the vast spirit biology in Wuhan by German MoBiTec Products Science and Technology Ltd.) be common shuttle expression carrier, carry BamHI and XbaI enzyme recognition site, have ampicillin and Chloramphenicol resistance gene is induced by IPTG, being capable of high-level secretory expression destination protein.It is analyzed using DNAMAN software huge Bacillus acid phosphatase BmacpA gene order, finds no BamHI and XbaI enzyme cutting site, can use shuttling expressing Carrier pHT43, the recombinant plasmid that building can be expressed in citric acid bacillus.

According to Bacillus megatherium BmacpA coding sequence design primer, BamHI digestion is added in 5 ' end primers XbaI enzyme cutting site (TCTAGA) is added in 3 ' end primers in site (GGATCC).Underscore is restriction enzyme site.Upstream and downstream primer point Not are as follows: P9:5 '-AT-GGATCC- ATGTATGTGA AACGATATCG-3 ' and P10:5 '-GC-TCTAGA-CTACTTTTGT CGAACACATA-3′).Using the method for PCR amplification, introduced respectively at 5 ' ends of BmacpA gene complete coding region and 3 ' BamHI and XbaI enzyme recognition site obtains BmacpA gene PCR product；With BamHI and XbaI while digestion shuttle expression carrier PHT43 and BmacpA gene PCR product with enzyme recognition site, the digestion products T of recycling₄Ligase connection, connection produce Sequencing result is shown to be the replacement of the DNA molecular shown in sequence 1 in sequence table by screening positive clone after object conversion, sequencing The recombinant expression carrier that segment between BamHI the and XbaI recognition site of pHT43 obtains is named as pHT-BmacpA.pHT– For BmacpA containing BmacpA gene shown in sequence 1 in ordered list, pHT-BmacpA expression is shown in the sequence 2 in sequence table Protein.

2. the phosphoric acid hydrolysis enzymatic activity of the BmacpA of acquisition and its expression of acid phosphatase enzyme engineering bacteria

Using electrotransformation, recombinant vector pHT-BmacpA is transferred to citric acid bacillus ACCC02187, obtains recombinant bacterium, The recombinant bacterium is acid phosphatase enzyme engineering bacteria.The access of acid phosphatase enzyme engineering bacteria is trained containing the LB of ampicillin and chloramphenicol It supports in base, overnight incubation.It is transferred in the LB culture medium containing ampicillin and chloramphenicol with 2% inoculum concentration and is continued at 35 DEG C To logarithmic growth phase IPTG to final concentration of 0.5mM, Fiber differentiation 6h is added, 4000r/min revolving speed is centrifuged at room temperature in culture 15min, collects supernatant and thallus respectively, which is extracellular supernatant.After bacterial cell disruption, 4 DEG C of 4000r/min revolving speeds from Heart 15min, collects supernatant, which is supernatant intracellular.Then SDS- is carried out to the extracellular supernatant of supernatant intracellular respectively PAGE electrophoretic analysis and enzyme activity determination.Using the extracellular supernatant of supernatant intracellular as enzyme solution to be measured.Enzyme solution to be measured utilizes BCA quantification of protein kit quantification measures protein content.It is measured using common p-nitrophenol sodium phosphate (pNPP) method The phosphoric acid hydrolysis enzymatic activity of enzyme solution to be measured.Used reaction system by enzyme solution to be measured, p-nitrophenol sodium phosphate (pNPP) and 50mmol/L NaAc_HAc buffer solution and MnCl₂Composition, the concentration that the pH of the reaction system is 5.0, pNPP is 1mmol/ L, MnCl₂Concentration be 1mmol/L.10min is reacted at 37 DEG C, the Na0H that 0.1ml 5mmol/L is added after reaction immediately is terminated It reacts and measures A_405nm.Use blank reaction system as blank control.The blank reaction system is to be measured by isometric heat inactivation Enzyme solution, p-nitrophenol sodium phosphate (pNPP), 50mmol/L NaAc_HAc buffer solution and MnCl₂Composition, blank reaction The concentration that the pH of system is 5.0, pNPP is 1mmol/L, MnCl₂Concentration be 1mmol/L.Enzyme activity unit (U) is defined as: 37 DEG C, under the conditions of pH5.0, the amount that catalysis per minute generates 1 μm of ol phosphoric acid hydrolysis product pNP (p-nitrophenol) is 1 enzyme activity Unit.Experiment is in triplicate.

SDS-PAGE electrophoresis result (Fig. 5) display, BmacpA gene can be normal in acid phosphatase enzyme engineering bacteria Expression, expression product is secretory protein, and expression product molecular weight is about 24kD.The phosphoric acid hydrolysis of acid phosphatase engineering bacterium expression The enzyme activity of enzyme is 33.96 ± 1.32U/mg.

3, the influence of reaction time and pH to the phosphate transferase activity of acid phosphatase enzyme engineering bacteria

The phosphoric acid for using converting inosine rate (by converting inosine for the ability of inosinicacid) to embody acid phosphatase enzyme engineering bacteria turns Move enzymatic activity.The specific method is as follows:

By in LB culture medium of the acid phosphatase enzyme engineering bacteria access containing ampicillin and chloramphenicol of step 2, cultivated Night.It is transferred in the LB culture medium containing ampicillin and chloramphenicol with 2% inoculum concentration and continues culture at 35 DEG C to logarithm life For a long time, IPTG to final concentration of 0.5mM is added, in 28 DEG C of Fiber differentiation 6h, 4000r/min revolving speed is centrifuged 15min at room temperature, receives Collect thallus, using the different reaction system of 5 pH value (pH4.0 reaction system, pH5.0 reaction system, pH6.0 reaction system, PH7.0 reaction system and pH8.0 reaction system) measure ability of the acid phosphatase enzyme engineering bacteria by converting inosine for inosinicacid.

The different reaction system of above-mentioned 5 pH value is molten by thallus, p-nitrophenol sodium phosphate (pNPP), inosine and buffering Liquid composition.The concentration of the pNPP of the different reaction system of above-mentioned 5 pH value is 5mmol/L, and the concentration of inosine is 1mmol/ L, the content of thallus are 10⁸cfu/mL.The pH of pH4.0 reaction system is 4.0, and buffer solution is 0.2mmol/L Acetic acid-sodium acetate Buffer solution.The pH of pH5.0 reaction system is 5.0, and buffer solution is 0.2mmol/L NaAc_HAc buffer solution.pH6.0 The pH of reaction system is 6.0, and buffer solution is 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.The pH of pH7.0 reaction system It is 7.0, buffer solution is 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.The pH of pH8.0 reaction system is 8.0, is buffered molten Liquid is 0.2mmol/L sodium dihydrogen phosphate-disodium hydrogen phosphate.

Above-mentioned each reaction system respectively at 37 DEG C react 15min, 30min, 45min, 60min, 75min, 90min, 120min, the Na0H that 0.1ml 5mmol/L is added after reaction immediately terminates reaction, with containing for HPLC assay inosinicacid Amount embodies acid phosphatase enzyme engineering bacteria according to converting inosine rate (converting inosine rate=inosine acid content/Inosine Content × 100%) Phosphate transferase activity.Use corresponding blank reaction system as blank control.The blank reaction system of each pH value is Enzyme solution to be measured in corresponding pH value reaction system is replaced with into isometric heat and inactivates enzyme solution to be measured, other ingredients are and accordingly PH value reaction system it is identical.HPLC condition are as follows: chromatographic column: 5 μm of Hypersil SAX (4.6mm × 250mm), mobile phase: 60mmol/L pH3.0 phosphoric acid-ammonium dihydrogen phosphate buffer, flow velocity: 1mL/min, Detection wavelength 254nm, column temperature: 25 DEG C.

The optimal pH that converting inosine is inosinicacid is 5.0 by acid phosphatase enzyme engineering bacteria, in acid condition (pH4.0- 6.0) converting inosine rate is in a higher level, is attained by 25% or more.Wherein, under conditions of 37 DEG C, pH value are 5 The converting inosine rate highest of reaction 45 minutes is 36% (Fig. 6).

4, the effect of solubilizing phosphate of acid phosphatase enzyme engineering bacteria

The acid phosphatase enzyme engineering bacteria and citric acid bacillus (recipient bacterium) of step 2 are inoculated in the training of phosphoinositide liquid respectively It supports in base, phosphatide fluid nutrient medium and nucleic acid fluid nutrient medium, keeps the content of acid phosphatase enzyme engineering bacteria and citric acid bacillus equal It is 10⁸IPTG to final concentration of 0.5mM is added in 35 DEG C of cultures to logarithmic growth phase in cfu/mL, in 28 DEG C of Fiber differentiation 6h, 4000r/min revolving speed is centrifuged 15min at room temperature, collects supernatant, using the anti-colorimetric method of key antimony, with 722 type spectrophotometers, Wavelength 700nm, the content of available phosphorus directly in measurement inoculation acid phosphatase recombinant liquid.

Wherein, the pH of phosphoinositide fluid nutrient medium is 6.0, and preparation method is as follows: being water, solute and its concentration by solvent Following culture solution is in 115 DEG C of sterilizing 30min:0.3g/L MgSO₄·7H₂O, 0.2g/L (NH₄)₂SO₄, 0.03g/L CaCl₂, 0.9g/L NaCl, 0.5mL trace element solution/L, 20g/L glucose.Wherein, the solvent of trace element solution is water, solute And its concentration is as follows: 1.23g/L MnSO₄, 0.356g/L ZnSO₄, 0.256g/L FeSO₄, 0.31g/L CuSO₄·5H₂O.It will Isosorbide-5-Nitrae, 5- InsP3 pass through biofilter degerming, are added in the culture solution of above-mentioned sterilizing, obtain the training of phosphoinositide liquid Base, Isosorbide-5-Nitrae in phosphoinositide fluid nutrient medium are supported, the concentration of 5- InsP3 is 50 μm of ol/L.

The pH of phosphatide fluid nutrient medium is 6.0, and preparation method is as follows: it is water by solvent, solute and its following training of concentration Nutrient solution is in 115 DEG C of sterilizing 30min:0.3g/L MgSO₄·7H₂O, 0.2g/L (NH₄)₂SO₄, 0.03g/L CaCl₂, 0.9g/L NaCl, 0.5mL trace element solution/L, 20g/L glucose.Wherein, the solvent of trace element solution is water, solute and its concentration It is as follows: 1.23g/L MnSO₄, 0.356g/L ZnSO₄, 0.256g/L FeSO₄, 0.31g/L CuSO₄·5H₂O.By L-A- phosphatide Acyl inositol passes through biofilter degerming, is added in the culture solution of above-mentioned sterilizing, obtains phosphatide fluid nutrient medium, the training of phosphatide liquid The concentration for supporting L-A- phosphatidylinositols in base is 50 μm of ol/L.

The pH of nucleic acid fluid nutrient medium is 6.0, and preparation method is as follows: it is water by solvent, solute and its following training of concentration Nutrient solution is in 115 DEG C of sterilizing 30min:0.3g/L MgSO₄·7H₂O, 0.2g/L (NH₄)₂SO₄, 0.03g/L CaCl₂, 0.9g/L NaCl, 0.5mL trace element solution/L, 20g/L glucose.Wherein, the solvent of trace element solution is water, solute and its concentration It is as follows: 1.23g/L MnSO₄, 0.356g/L ZnSO₄, 0.256g/L FeSO₄, 0.31g/L CuSO₄·5H₂O.By salmon essence DNA passes through biofilter degerming, is added in the culture solution of above-mentioned sterilizing, obtains nucleic acid fluid nutrient medium, nucleic acid Liquid Culture The concentration of salmon sperm dna is 50 μm of ol/L in base.The result shows that compared with the citric acid bacillus as recipient bacterium, in nucleic acid solution (containing salmon sperm dna), phosphatide fluid nutrient medium (phosphatidylinositols containing L-A-) and phosphoinositide fluid nutrient medium in body culture medium (containing Isosorbide-5-Nitrae, 5- InsP3), the content of available phosphorus has increased separately 14.59 μm of ol/L, 16.63 μm of ol/L and 18.55 μ Mol/L (Fig. 7).

<110>INST OF AGRICULTURAL RESOURCES

<120>building has the method and its nucleic acid molecule used therefor of Soluble phosphorus reconstituted protein microorganism

<130> GNCFH181963

<160> 8

<170> PatentIn version 3.5

<210> 1

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115 120 125

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ctcgctttat atatcacgct cgttttttgg ggagttcgcg ctttaaattg gggattgaag 240

gagatttttg caagaccaag acctgattgg agtcaagtcg ttcccgcctc tcactatagt 300

tttccgagtg gacatgccat gaactcaatg gcgttttaca gcggaatact tttgttaata 360

tggatgtata caagaagcag ggctgttaaa acggcagctg catgcgtaat agctattgtt 420

attttattaa ttggatttag ccgtttgtat ttaggggtac actttttaac ggatatacta 480

gcaggatatt gtttaggact tgtttggtcc ttaggagtct atctcctttc taagcgctta 540

tataaccaaa aaaagcttgc ggccgcactc gagcaccacc accaccacca ctga 594

<210> 8

<211> 197

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Met Phe Asn Thr Pro Trp Val Lys Gln Leu Asp Phe Asn Val Leu His

1 5 10 15

Thr Ile Glu Gly Trp Arg Thr Asp Thr Leu Thr Pro Ile Ile Ile Phe

20 25 30

Ile Thr Thr Val Gly Ser Trp Tyr Val Thr Ala Pro Ile Trp Phe Ala

35 40 45

Ile Ile Val Phe Leu Leu Tyr Lys Arg Lys Gly Leu Leu Ala Leu Tyr

50 55 60

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

65 70 75 80

Glu Ile Phe Ala Arg Pro Arg Pro Asp Trp Ser Gln Val Val Pro Ala

85 90 95

Ser His Tyr Ser Phe Pro Ser Gly His Ala Met Asn Ser Met Ala Phe

100 105 110

Tyr Ser Gly Ile Leu Leu Leu Ile Trp Met Tyr Thr Arg Ser Arg Ala

115 120 125

Val Lys Thr Ala Ala Ala Cys Val Ile Ala Ile Val Ile Leu Leu Ile

130 135 140

Gly Phe Ser Arg Leu Tyr Leu Gly Val His Phe Leu Thr Asp Ile Leu

145 150 155 160

Ala Gly Tyr Cys Leu Gly Leu Val Trp Ser Leu Gly Val Tyr Leu Leu

165 170 175

Ser Lys Arg Leu Tyr Asn Gln Lys Lys Leu Ala Ala Ala Leu Glu His

180 185 190

His His His His His

195

Claims

1. nucleic acid molecules have the application in Soluble phosphorus active microorganism in building；The nucleic acid molecule encoding a) or b) or c) or d) Protein:

A) protein that the amino acid sequence shown in SEQ ID No.2 1-203 forms；

B) protein that the amino acid sequence shown in SEQ ID No.2 26-203 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 activity of acid phosphatase that generation and/or deletion and/or addition obtain.

2. the method with Soluble phosphorus reconstituted protein microorganism is constructed, including by the albumen in claim 1 a) or b) or c) or d) The encoding gene of matter imports recipient microorganism, and obtain Soluble phosphorus activity has Soluble phosphorus reconstituted protein micro- higher than the recipient microorganism Biology.

3. according to the method described in claim 2, it is characterized by: the encoding gene is following 1) or 2) or 3) or 4) shown DNA molecular:

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

2) coded sequence is DNA molecular shown in the 76th to 612 of SEQ ID No.1；

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

4) with 1) or 2) or 3) albumen described in the identity and coding claim 1 of the DNA molecular with 90% or more that limit The DNA molecular of matter.

4. according to the method in claim 2 or 3, it is characterised in that: the recipient microorganism is prokaryotic micro-organisms.

5. according to the method described in claim 4, it is characterized by: the prokaryotic micro-organisms is gramnegative bacterium.

6. according to the method described in claim 5, it is characterized by: the gramnegative bacterium be Escherichia bacteria or Citrobacter bacterium.

7. there is Soluble phosphorus reconstituted protein microorganism as described in the method any in claim 2-6 building.