CN109234251A - Protein and the nucleic acid molecules for encoding the protein are preparing the application in phosphohydrolase - Google Patents

Protein and the nucleic acid molecules for encoding the protein are preparing the application in phosphohydrolase Download PDF

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CN109234251A
CN109234251A CN201811351441.0A CN201811351441A CN109234251A CN 109234251 A CN109234251 A CN 109234251A CN 201811351441 A CN201811351441 A CN 201811351441A CN 109234251 A CN109234251 A CN 109234251A
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protein
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nucleic acid
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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 protein and the nucleic acid molecules for encoding the protein to prepare the application in phosphohydrolase.The application is protein c) or d) as the application in phosphohydrolase: the c) fusion protein that the c-terminus of the protein shown in a) or b) or/and aminoterminal fusion protein label obtain;Protein a) formed for the amino acid sequence shown in SEQ ID No.2 1-203;Protein b) formed for the amino acid sequence shown in SEQ ID No.2 26-203;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

Protein and the nucleic acid molecules of the protein are encoded in preparing phosphohydrolase Using
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 protein in biological field and the nucleic acid molecules for encoding the protein in preparing phosphohydrolase Application.
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 (Acidphosphatase, abbreviation ACPase, E.C.3.1.3.2) is the enzyme of catalytic phosphatase monoesters hydrolysis in acid condition. Metabolism of this enzyme in addition to participating in phosphate, also participation Metabolism regulation, energy are converted and the important vital movements such as signal transduction.Acid Acid phosphatase has very important function, first, acid phosphatase has the activity of phosphohydrolase, by decomposing organic matter One ester bond of phosphorus and one acid anhydride key of phosphorus discharge phosphorus, thus invalid phosphorus in activating soil, making full use of soil phosphor resource, reducing phosphate fertilizer There is significant application value in application;Second, acid phosphatase also has phosphate transferase activity, under suitable conditions, energy Low energy phosphate group is transferred on the hydroxyl of nucleosides, there is biggish application value in the synthesis of nucleotide biochemistry.Nucleotide Usually as food additives and medicine intermediate, wherein inosinicacid (hypoxanthine -5 '-nucleotide) becomes apparent from because having Help fresh effect, be widely used in food processing field.There are mainly two types of methods to produce nucleotide at present, and one is chemical syntheses Method is fermented using thallus and generates inosine, then uses POCl3Phosphorylation, this method by-product is more, purification difficult;Another method It is using Escherichia coli inosine kinase phosphorylating inosine, this process needs the participation of ATP, and ATP is needed by producing ammonia bacillus hair Ferment regeneration, limits 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 Haidian District Zhong Guan-cun South Street 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 restriction enzyme site is added in the 5 ' end primers for removing signal peptide (CATATG), HindIII restriction enzyme site (AAGCTT) is added in 3 ' end primers.Upstream and downstream primer is respectively as follows: P5 primer, 5 '-AT-CATATG- TTTAATACACCTTGGGTGAA-3 ' and P6 primer, 5 '-GC-AAGCTT-CTTTTGT CGAACACATAA-3′.Benefit With the method for PCR amplification, signal peptide coding DNA (1-75 nucleosides of sequence 1 in sequence table are removed in the gene coding region BmacpA Acid), and NdeI and HindIII enzyme recognition site is introduced respectively at 5 ' ends and 3 ' ends, it obtains without signal peptide BmacpA gene PCR Product will be NSBmacpA gene without signal peptide BmacpA unnamed gene, will order without signal peptide BmacpA gene PCR product Entitled NSBmacpA gene PCR product.NSBmacpA gene PCR product is containing the 76th to 609 nucleotide of sequence 1 in ordered list (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 (+), (EMDBiosciences is purchased in north simultaneously Capital company of 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, NSBmacpB-His coding The nucleotide sequence of gene is the sequence 7 in sequence table, and NSBmacpB-His is protein shown in sequence 8 in sequence table.
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 lmM, 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 lmM 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 its N-terminal 15 amino acid are the 1-15 amino acids of sequence 6 in sequence table, and the sequencing results of NSBmacpB-His show the end its N 15 amino acid at end are the 1-15 amino acids of sequence 8 in sequence table.
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 A405nm.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
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 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 A405nm.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 A405nm.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>it protein and the nucleic acid molecules of the protein is encoded is preparing the application in phosphohydrolase
<130> GNCFH181962
<160> 8
<170> PatentIn version 3.5
<210> 1
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<213>bacillus megaterium (Bacillus megaterium)
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1 5 10 15
Leu Ile Cys Phe Leu Ile Leu Phe Thr Phe Asn Thr Pro Trp Val Lys
20 25 30
Gln Leu Asp Phe Asn Val Leu His Pro Val Ile Ala Tyr Val Gln Gly
35 40 45
Phe Ile Ser Asp Asn Met Thr Ser Ala Met Leu Val Ile Gly Ser Lys
50 55 60
Arg Ile Tyr Phe Pro Leu Leu Ile Ile Leu Val Met Tyr Phe Leu Val
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Phe Ser Lys Lys Leu Glu Glu Arg Gly Leu Leu Lys Phe Ser Lys Lys
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Glu Ser Asp Lys Arg Glu Arg Pro Ala Phe His Pro Leu Val His Glu
100 105 110
Thr Ser Tyr Ser Phe Pro Ser Gly Ser Gly His Ala Met Asn Ser Thr
115 120 125
Ala Phe Leu Leu Phe Leu Leu Tyr Leu Leu Ile His Ile Ala Tyr Val
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Ile Ala Ile Val Ile Leu Leu Ile Gly Phe Ser Arg Leu Tyr Leu Gly
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Val His Phe Leu Tyr Pro Ser Asp Ile Leu Ala Gly Trp Ala Ala Gly
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accattgaag gatggaggac ggacacgtta acacctatca tcatatttat aacaaccgta 180
ggatcttggt atgtcactgc gccaatatgg tttgcaatta tagtatttct tctttataaa 240
cgaaaaggat tgctcgcttt atatatcacg ctcgtttttt ggggagttcg cgctttaaat 300
tggggattga aggagatttt tgcaagacca agacctgatt ggagtcaagt cgttcccgcc 360
tctcactata gttttccgag tggacatgcc atgaactcaa tggcgtttta cagcggaata 420
cttttgttaa tatggatgta tacaagaagc agggctgtta aaacggcagc tgcatgcgta 480
atagctattg ttattttatt aattggattt agccgtttgt atttaggggt acacttttta 540
acggatatac tagcaggata ttgtttagga cttgtttggt ccttaggagt ctatctcctt 600
tctaagcgct tatataacca aaaatag 627
<210> 4
<211> 208
<212> PRT
<213>bacillus megaterium (Bacillus megaterium)
<400> 4
Met Val Asn Arg Thr Thr Lys Tyr Ser Leu Phe Ile Val Ser Leu Leu
1 5 10 15
Ala Phe Phe Leu Leu Ile Leu Phe Thr Phe Asn Thr Pro Trp Val Lys
20 25 30
Gln Leu Asp Phe Asn Val Leu His Thr Ile Glu Gly Trp Arg Thr Asp
35 40 45
Thr Leu Thr Pro Ile Ile Ile Phe Ile Thr Thr Val Gly Ser Trp Tyr
50 55 60
Val Thr Ala Pro Ile Trp Phe Ala Ile Ile Val Phe Leu Leu Tyr Lys
65 70 75 80
Arg Lys Gly Leu Leu Ala Leu Tyr Ile Thr Leu Val Phe Trp Gly Val
85 90 95
Arg Ala Leu Asn Trp Gly Leu Lys Glu Ile Phe Ala Arg Pro Arg Pro
100 105 110
Asp Trp Ser Gln Val Val Pro Ala Ser His Tyr Ser Phe Pro Ser Gly
115 120 125
His Ala Met Asn Ser Met Ala Phe Tyr Ser Gly Ile Leu Leu Leu Ile
130 135 140
Trp Met Tyr Thr Arg Ser Arg Ala Val Lys Thr Ala Ala Ala Cys Val
145 150 155 160
Ile Ala Ile Val Ile Leu Leu Ile Gly Phe Ser Arg Leu Tyr Leu Gly
165 170 175
Val His Phe Leu Thr Asp Ile Leu Ala Gly Tyr Cys Leu Gly Leu Val
180 185 190
Trp Ser Leu Gly Val Tyr Leu Leu Ser Lys Arg Leu Tyr Asn Gln Lys
195 200 205
<210> 5
<211> 579
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atgtttaata caccttgggt gaaacagctt gactttaatg tacttcatcc ggtaattgct 60
tatgtacaag gattcatctc agataacatg acaagtgcca tgcttgtgat cggttccaaa 120
agaatatatt ttccattact catcattctt gtaatgtatt ttctcgtttt ttcaaagaaa 180
cttgaagaac gcggccttct caaattttca aagaaagaaa gcgacaaaag agagcggccg 240
gcatttcacc cgctcgtcca tgaaacgtca tacagctttc cgagcggcag cgggcatgct 300
atgaattcaa ctgccttcct ccttttcttg ctttatttgc ttatacatat tgcatacgtc 360
acaattactg aagagcgtat ttcattgcat aaaaaactgt tgattattat agctattgtt 420
attttattaa ttggatttag ccgtttgtat ttaggggtac actttttata tccatctgac 480
atattagcag gatgggcagc gggcggtagc tggctcgttt tatgtgttcg acaaaagaag 540
cttgcggccg cactcgagca ccaccaccac caccactga 579
<210> 6
<211> 192
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Met Phe Asn Thr Pro Trp Val Lys Gln Leu Asp Phe Asn Val Leu His
1 5 10 15
Pro Val Ile Ala Tyr Val Gln Gly Phe Ile Ser Asp Asn Met Thr Ser
20 25 30
Ala Met Leu Val Ile Gly Ser Lys Arg Ile Tyr Phe Pro Leu Leu Ile
35 40 45
Ile Leu Val Met Tyr Phe Leu Val Phe Ser Lys Lys Leu Glu Glu Arg
50 55 60
Gly Leu Leu Lys Phe Ser Lys Lys Glu Ser Asp Lys Arg Glu Arg Pro
65 70 75 80
Ala Phe His Pro Leu Val His Glu Thr Ser Tyr Ser Phe Pro Ser Gly
85 90 95
Ser Gly His Ala Met Asn Ser Thr Ala Phe Leu Leu Phe Leu Leu Tyr
100 105 110
Leu Leu Ile His Ile Ala Tyr Val Thr Ile Thr Glu Glu Arg Ile Ser
115 120 125
Leu His Lys Lys Leu Leu Ile Ile Ile Ala Ile Val Ile Leu Leu Ile
130 135 140
Gly Phe Ser Arg Leu Tyr Leu Gly Val His Phe Leu Tyr Pro Ser Asp
145 150 155 160
Ile Leu Ala Gly Trp Ala Ala Gly Gly Ser Trp Leu Val Leu Cys Val
165 170 175
Arg Gln Lys Lys Leu Ala Ala Ala Leu Glu His His His His His His
180 185 190
<210> 7
<211> 594
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
atgtttaata caccttgggt gaaacagctt gactttaatg tacttcatac cattgaagga 60
tggaggacgg acacgttaac acctatcatc atatttataa caaccgtagg atcttggtat 120
gtcactgcgc caatatggtt tgcaattata gtatttcttc tttataaacg aaaaggattg 180
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 (6)

  1. Protein 1.c) or d) is as the application in phosphohydrolase:
    C) fusion protein that the c-terminus of the protein shown in a) or b) or/and aminoterminal fusion protein label obtain;It is described A) protein formed for the amino acid sequence shown in SEQ ID No.2 1-203;It is described b) for by SEQ ID No.2 The protein of the composition of amino acid sequence shown in 26-203;
    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. 2. application according to claim 1, it is characterised in that: protein c) is the carboxylic of the protein shown in b) The fused protein that cardinal extremity or/and aminoterminal fusion histidine tag obtain.
  3. 3. application according to claim 1 or 2, it is characterised in that: protein c) is shown in SEQ ID No.6 Protein, entitled NSBmacpA-His.
  4. 4. nucleic acid molecules are preparing the application in phosphohydrolase;Egg in the nucleic acid molecule encoding claim 1 c) or d) White matter.
  5. 5. application according to claim 4, it is characterised in that: the nucleic acid molecules be it is following 3) or 4) shown in gene:
    3) coded sequence is DNA molecular shown in SEQ ID No.5;
    4) there is 90% or more identity with the DNA molecular 3) limited and encode DNA points of protein described in claim 1 Son.
  6. 6. the biomaterial containing nucleic acid molecules described in claim 4 or 5, the biomaterial is B1), B2) or B3):
    B1 the expression cassette) containing nucleic acid molecules described in claim 4 or 5;
    B2 the recombinant vector) containing nucleic acid molecules described in claim 4 or 5;
    B3) contain B1) recombinant vector of the expression cassette.
CN201811351441.0A 2016-01-26 2016-01-26 Protein and application of nucleic acid molecule for coding protein in preparation of phosphohydrolase Active CN109234251B (en)

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