CN102250853A - Low-temperature neutral phytase PhyH with double structure domains as well as gene and application thereof - Google Patents
Low-temperature neutral phytase PhyH with double structure domains as well as gene and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, in particular to a low-temperature neutral phytase PhyH with double structure domains as well as a gene and application thereof. The invention provides the neutral phytase PhyH from Bacillus sp.HJB17, and the amino acid sequence of the neutral phytase PhyH is shown as SEQ ID NO.1; and the invention provides a gene PhyH for coding the neutral phytase. The phytase provided by the invention has good pH stability and still keeps higher enzyme activity in the alkaline range; the phytase has higher relative enzyme activity in a low-temperature range; the phytase has double structure domains, an N terminal is an incomplete BPP (Butyl Pivalate Peroxide) phytase structure domain and a C terminal is a complete phytase structure domain. In short, the PhyH obtained by the invention can be used as the low-temperature neutral phytase which can be applied to cultivation of fish; the PhyH-DI structure domain can increase the catalytic efficiency of other phytases and is good for improving the production and the application of the other phytases.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of low temperature neutral phytase PhyH and gene and application with geminus territory.
Background technology
Myo-phytinic acid (IP
6, myo-inositol hexakisphosphate) and be called phytic acid (phytic acid) again, be the modal form of occurring in nature inositol monophosphate.The molecular formula of phytic acid is C
6H
18O
24P
6, molecular weight is 660.09.
Phytic acid mainly extensively is present in the plant seed with the form of phytin sylvite, also is present in the animal erythroblast, all finds to have the existence of phytase in soil, plant and the microorganism secretion enzyme outside born of the same parents.Well imagine that phytase has important effect for the metabolic cycles of phosphorus in the physical environment.In addition, phytic acid all has extremely strong complex ability to most metal ions, and complexing power is similar to EDTA, but wider than the range of application of EDTA.Under usual conditions, the phosphoric acid ester bond in the phytic acid is very stable, for the degraded of phytic acid two kinds of methods is arranged generally, the one, and chemical process is extremely difficult but utilize the chemical process phytic acid of degrading; Another kind method is an enzymolysis process, at present the phytase that has only of our the known phytic acid of can effectively degrading.
Phytase has been widely used in feedstuff industry at present, particularly derives from Histidine acid phosphatase enzyme (HAP) phytase of fungi and bacterium, because of its optimal pH meta-acid with than characteristics such as height alive, can improve their production performances chicken and pig significantly.Be widely used in animal farming industry as intestinal bacteria and aspergillus source place phytase.But because temperature is lower in fish growth environment in culture fishery and the body, the pH partial neutral causes the application of HAP class phytase to be restricted, and more and more receives publicity so have the BPP class phytase of neutral pH feature.But the disadvantage of present known BPP class phytic acid is that catalytic efficiency is low, is difficult to satisfy the actual production demand.So obtain novel high-quality BPP phytase, improve the catalytic efficiency of BPP class phytase, all can expand its actual application space greatly, also present just urgent problem and focus.
Summary of the invention
The purpose of this invention is to provide a kind of low temperature neutral phytase with geminus territory.
Another object of the present invention provides the gene of the above-mentioned geminus of coding territory low temperature neutral phytase.
Another object of the present invention provides above-mentioned phytase N end and C end structure territory and encoding gene thereof, and wherein, the catalytic efficiency that improves other phytases can be worked in coordination with in N end structure territory.
Another object of the present invention provides and comprises above-mentioned recombinant vectors with the low temperature neutral phytase gene in geminus territory.
Another object of the present invention provides and comprises above-mentioned recombinant bacterial strain with the low temperature neutral phytase gene in geminus territory.
Another object of the present invention provides above-mentioned application with the low temperature neutral phytase in geminus territory.
(microbial preservation number is: CGMCC No.4868 from genus bacillus (Bacillus sp.HJB17) in the present invention; The preservation time is: 2011 05 month No. 18; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.100101) separate in and obtain a kind of new low temperature neutral phytase PhyH with geminus territory.
The invention provides a kind of low temperature neutral phytase PhyH with geminus territory, its aminoacid sequence is shown in SEQID NO.1.
SEQ ID NO.1：
MLVAKKLATKNMFLLGAVSLTLLAMLSGCQAGKFATPISAAVQPLSVNASHFHKVQLAGQDYQLFTTTTALQIRQGNRELAQQAGQFSRLVLQPLDTDALLLAAMDINSNTLFLWRFSTKQTPSLQLLQRRLISSRVVDDLCFYHSTENQQLSLFLLGGRGGADQLLLQQQQQWLAQPVVIRELNIPYDSTACVVDQTAGALYIAEADRAIWRYQAEPEADEGRSLVQVNKPFGQLQGEVKALQTLSDGSLLALEEAPARLLHINSDGQLRSAIAVPALAEASGLAVSMQGNTATAYISTEDAGAVQQLAVVPQAKPERQPKAPVVQLLPTLQTEPASQRGDVMDDPAVWHHPARPELSLILGTDKRAGLDVYNMQGTRVQQLSVGRLNNVDVRYGLNWQGSAHDIAVASLRDDNSLQLFAIDSSGTLHNAGKVATSMSEIYGLCMYHSAQ SNKHYVFVNDKAGLIQQYRIDTDGDHWQGSLVRALQVPSQPEGCVADDIRGLLFVGEEDAAIWRFAAEAEATTTGEAIIRVDGERLVDDIEGITLAEHNGSSYLLVSSQGNDSYLVFDAAPPYTERPHFRIGTNPLLGIDGASETDGVDVTTRSLGPGFEQGAFIVQDGRNRMPEQGQNLKLVPWADILQQLN
Wherein, this enzyme comprises 644 amino acid and a terminator codon, and preceding 40 amino acid are signal peptides, and therefore, the theoretical molecular of sophisticated phytase PhyH is 67kDa.
Phytase PhyH of the present invention has the geminus territory simultaneously, and low temperature active is arranged.
The neutral phytase PhyH that the present invention obtains from screen Bacillus sp.HJB17, its optimum pH is 7.0, pH 7.0-12.0 in alkaline range, activity remains unchanged substantially; Optimum temperuture is 35 ℃, and (20-35 ℃) still has the enzyme activity about 60% in low temperature range.Neutral phytase PhyH of the present invention comprises the complete typical BPP phytase structural domain PhyH-DII that has of the incomplete structural domain PhyH-DI of two phytase structural domain: N end and C end.Wherein PhyH-DII and PhyH all have phytase activity.
The invention provides the gene of the above-mentioned geminus of coding territory phytase PhyH.Particularly, the sequence of this gene is shown in SEQ ID NO.2:
SEQ ID NO.2
atgttagtagctaaaaagttagccacaaaaaatatgtttttactcggtgcagtcagtctgacgctgctggccatgctaagcggctgtcaggctggcaagtttgccacacctatatcagctgcagtccaacctttatcggtcaatgccagccacttccataaggtgcagctggccgggcaggattatcagctattcaccacaaccacggccttgcagatccgccagggcaatcgcgagctggcgcagcaagctggccagtttagccgcctggtgctgcagccactggataccgatgcgctgttattggcagcgatggatataaacagtaatactttgtttttatggcgtttttcaacaaagcaaacgcccagcctgcaattgctgcaacgccggttgatcagcagccgggtggtagacgatctgtgcttttaccacagtactgaaaaccagcagctcagcctgtttttactcggtggccgcggaggcgctgatcagctgctgttgcaacagcagcaacagtggctggcgcaaccggtggtaatacgcgagttaaatatcccttatgacagcacggcttgtgtagtggatcagaccgctggtgcgctgtatatcgccgaagctgatcgcgctatctggcgctatcaggccgaacccgaagccgatgagggccgcagtttagttcaggtaaataagccgtttggccagttgcagggcgaagtaaaagcgctgcaaacgttgtctgacggcagcctgctggcgctggaggaagcaccggccagattgttacatattaacagcgacggccagctgcgcagcgccatagctgtcccggcgttggccgaggccagcggtttagcggtcagtatgcagggcaacacggcaacagcctatatcagcactgaggatgccggtgcagtacagcagctagcggtggtgccgcaggcgaagccggagcgtcagcccaaagcgccggtagtccagctgttgccgaccttacagactgagcccgccagtcagcgtggcgatgtgatggatgaccccgcggtgtggcatcatccggcgcggcctgagcttagcctgatcctcggcactgacaaacgcgccggactggatgtgtacaatatgcagggtacgcgcgtgcagcagcttagcgtgggccggttaaataatgtcgatgtgcgctacggcctgaactggcagggtagtgcgcacgatattgccgtagccagtttgcgcgacgacaacagcctgcaactttttgctattgatagcagtggcacgctgcataatgccggtaaggtcgcaaccagcatgagcgagatttacggcctgtgtatgtatcacagtgcacaaagcaataagcattatgtgtttgttaatgataaagccggtttaattcagcagtatcgcatcgacacagacggcgaccactggcagggcagcttagtgcgcgctttgcaggtaccgtcgcaaccggaaggctgtgtggccgatgatatacgcggcctgttatttgtcggcgaagaagatgctgccatctggcgttttgccgctgaagccgaagcgacaaccacaggtgaagcgatcatccgcgttgatggtgagcggctggtcgacgatattgaaggcataacattggcagagcataacggcagcagttatctgttggtatcaagccagggtaatgacagttacctggtgtttgacgccgcgccaccttatacagagcggccgcattttcgtattggcactaacccattactgggcatagatggcgcctctgagactgatggcgttgatgttaccacccgttcgctggggcctggctttgagcagggcgcctttatcgtacaggacggccgtaaccgtatgccggaacagggccaaaaccttaaactggtaccctgggcagatatattgcagcaattaaac
The invention provides a kind of low temperature neutral phytase with geminus territory, its N holds the aminoacid sequence of imperfect structural domain (41-318aa) PhyH-DI shown in SEQ ID NO.3, and the theoretical molecular of this imperfect structural domain PhyH-DI is 31kDa.。
SEQ ID NO.3：
AVQPLSVNASHFHKVQLAGQDYQLFTTTTALQIRQGNRELAQQAGQFSRLVLQPLDTDALLLAAMDINSNTLFLWRFSTKQTPSLQLLQRRLISSRVVDDLCFYHSTENQQLSLFLLGGRGGADQLLLQQQQQWLAQPVVIRELNIPYDSTACVVDQTAGALYIAEADRAIWRYQAEPEADEGRSLVQVNKPFGQLQGEVKALQTLSDGSLLALEEAPARLLHINSDGQLRSAIAVPALAEASGLAVSMQGNTATAYISTEDAGAVQQLAVVPQAKPE
The incomplete structural domain PhyH-DI of the N of the neutral phytase PhyH of the present invention end phytic acid (IP that can not degrade
6).Further discover the PhyH-DI low-phosphorous substrate IP that can degrade
4, and can act synergistically with PhyH-DII and other phytases, improve the catalytic efficiency of phytase.
The invention provides the gene of the above-mentioned imperfect structure thresholding acid enzyme PhyH-DI of coding.Particularly, the sequence of this gene is shown in SEQ ID NQ.4:
SEQ ID NO.4
GCAGTCCAACCTTTATCGGTCAATGCCAGCCACTTCCATAAGGTGCAGCTGGCCGGGCAGGATTATCAGCTATTCACCACAACCACGGCCTTGCAGATCCGCCAGGGCAATCGCGAGCTGGCGCAGCAAGCTGGCCAGTTTAGCCGCCTGGTGCTGCAGCCACTGGATACCGATGCGCTGTTATTGGCAGCGATGGATATAAACAGTAATACTTTGTTTTTATGGCGTTTTTCAACAAAGCAAACGCCCAGCCTGCAATTGCTGCAACGCCGGTTGATCAGCAGCCGGGTGGTAGACGATCTGTGCTTTTACCACAGTACTGAAAACCAGCAGCTCAGCCTGTTTTTACTCGGTGGCCGCGGAGGCGCTGATCAGCTGCTGTTGCAACAGCAGCAACAGTGGCTGGCGCAACCGGTGGTAATACGCGAGTTAAATATCCCTTATGACAGCACGGCTTGTGTAGTGGATCAGACCGCTGGTGCGCTGTATATCGCCGAAGCTGATCGCGCTATCTGGCGCTATCAGGCCGAACCCGAAGCCGATGAGGGCCGCAGTTTAGTTCAGGTAAATAAGCCGTTTGGCCAGTTGCAGGGCGAAGTAAAAGCGCTGCAAACGTTGTCTGACGGCAGCCTGCTGGCGCTGGAGGAAGCACCGGCCAGATTGTTACATATTAACAGCGACGGCCAGCTGCGCAGCGCCATAGCTGTCCCGGCGTTGGCCGAGGCCAGCGGTTTAGCGGTCAGTATGCAGGGCAACACGGCAACAGCCTATATCAGCACTGAGGATGCCGGTGCAGTACAGCAGCTAGCGGTGGTGCCGCAGGCGAAGCCGGAG
The invention provides the application that above-mentioned phytase structural domain PhyH-DI is used to improve phytase activity.
The invention provides the aminoacid sequence of PhyH-DII, particularly, the sequence of this gene is shown in SEQ ID NO.5, and its theoretical molecular is 36kDa, the PhyH-DII phytic acid of can effectively degrading.
SEQ ID NO.5
RQPKAPVVQLLPTLQTEPASQRGDVMDDPAVWHHPARPELSLILGTDKRAGLDVYNMQGTRVQQLSVGRLNNVDVRYGLNWQGSAHDIAVASLRDDNSLQLFAIDSSGTLHNAGKVATSMSEIYGLCMYHSAQSNKHYVFVNDKAGLIQQYRIDTDGDHWQGSLVRALQVPSQPEGCVADDIRGLLFVGEEDAAIWRFAAEAEATTTGEAIIRVDGERLVDDIEGITLAEHNGSSYLLVSSQGNDSYLVFDAAPPYTERPHFRIGTNPLLGIDGASETDGVDVTTRSLGPGFEQGAFIVQDGRNRMPEQGQNLKLVPWADILQQLN
The invention provides the nucleotide sequence of PhyH-DII, particularly, the sequence of this gene is shown in SEQ ID NO.6:
SEQ ID NO.6
cgtcagcccaaagcgccggtagtccagctgttgccgaccttacagactgagcccgccagtcagcgtggcgatgtgatggatgaccccgcggtgtggcatcatccggcgcggcctgagcttagcctgatcctcggcactgacaaacgcgccggactggatgtgtacaatatgcagggtacgcgcgtgcagcagcttagcgtgggccggttaaataatgtcgatgtgcgctacggcctgaactggcagggtagtgcgcacgatattgccgtagccagtttgcgcgacgacaacagcctgcaactttttgctattgatagcagtggcacgctgcataatgccggtaaggtcgcaaccagcatgagcgagatttacggcctgtgtatgtatcacagtgcacaaagcaataagcattatgtgtttgttaatgataaagccggtttaattcagcagtatcgcatcgacacagacggcgaccactggcagggcagcttagtgcgcgctttgcaggtaccgtcgcaaccggaaggctgtgtggccgatgatatacgcggcctgttatttgtcggcgaagaagatgctgccatctggcgttttgccgctgaagccgaagcgacaaccacaggtgaagcgatcatccgcgttgatggtgagcggctggtcgacgatattgaaggcataacattggcagagcataacggcagcagttatctgttggtatcaagccagggtaatgacagttacctggtgtttgacgccgcgccaccttatacagagcggccgcattttcgtattggcactaacccattactgggcatagatggcgcctctgagactgatggcgttgatgttaccacccgttcgctggggcctggctttgagcagggcgcctttatcgtacaggacggccgtaaccgtatgccggaacagggccaaaaccttaaactggtaccctgggcagatatattgcagcaattaaac
The method separating clone of the present invention by PCR phytase gene phyH, the DNA complete sequence analysis is the result show, this full length gene 1224bp.The aminoacid sequence of phyH and the aminoacid sequence of deriving are carried out the BLAST comparison in GenBank, PhyH has the highest consistence (51%) with the supposition phytase that derives from Idiomarina loihiensis L2TR, PhyH-DI is 25% with the phytase consistence that derives from B.amyloliquefaciens TS-Phy, the consistence of PhyH-DII and TS-Phy is 49%, with the consistence that derives from B.subtilis PhyC be 40%, illustrate that phyH is a kind of new phytase.
The present invention also provides the recombinant vectors that comprises above-mentioned phytase gene phyH, called after pET22b (+)-phyH.Phytase gene of the present invention is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As the most preferred embodiment of the present invention, be preferably phytase gene of the present invention is inserted between the EcoR I and Xho I restriction enzyme site on the plasmid pET22b (+), make this nucleotide sequence be positioned at the downstream of T7 promotor and regulated and control by it, obtain expression of recombinant e. coli plasmid pET22b (+)-phyH.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned phytase gene PhyH, and preferred described bacterial strain is intestinal bacteria, yeast, genus bacillus or lactobacillus, is preferably e. coli bl21 (DE3).
The present invention also provides a kind of method for preparing neutral phytase PhyH, may further comprise the steps:
1) with above-mentioned carrier transformed host cell, obtains active bacterial strain;
2) cultivate recombinant bacterial strain, induce recombinant phytase to express;
3) reclaim the also expressed phytase PhyH of purifying.
The present invention also provides the application of above-mentioned phytase PhyH.
The present invention has found a kind of BPP phytase with geminus territory, and it is made up of the incomplete structural domain PhyH-DI of N end and the complete typical BPP phytase structural domain PhyH-DII that has of C end.Discover, on the one hand, the incomplete structural domain PhyH-DI phytase substrate IP that can not degrade
6, but the intermediate product IP in the hydrolysis of phytic acid process of can degrading
4Thereby, promote the degraded fully of phytic acid substrate; On the other hand, this structural domain can with other phytases synergies such as PhyH-DII, thereby the ratio that improves other single structure territory matter acid enzymes live and catalytic efficiency, for the catalytic efficiency that improves BPP class phytase provides a kind of thinking.Simultaneously, this enzyme has low temperature active and good alkalescence stability, has the potentiality of practical application.
The present invention's technical problem at first to be solved is at the low present situation of BPP class phytase catalytic efficiency, provide a kind of character good, be suitable in fish feed using new phytase.Phytase optimal pH of the present invention is 7.0, at pH 7.0～12.0 good stability all; 35 ℃ of optimum temperutures, 20-35 ℃ of enzyme work that keeps more than 60%, and 0 ℃ of enzyme work of 20% in addition.Its low temperature and pH neutral characteristic can make it use in fish feed.The present invention has found the incomplete structural domain PhyH-DI IP that can degrade
4, and can act synergistically with complete structural domain PhyH-DII, improve the catalytic efficiency of geminus territory phytic acid.By amalgamation and expression, find that this synergy is equally applicable to other phytase.
Description of drawings
The expression of Fig. 1 recombinant phytase and purifying, wherein, 1 PhyH is purifying 2 PhyH-DII purifying 3 PhyH-DI purifying 4 PhyH (67kDa) 5 PhyH-DII (36kDa) 6 PhyH-DI (31kDa) not not
The calcium ion concn influence of Fig. 2 recombinant phytase PhyH and PhyH-DII.
The optimal pH of Fig. 3 recombinant phytase PhyH and PhyH-DII.
The pH stability of Fig. 4 recombinant phytase PhyH and PhyH-DII.
The optimum temperuture of Fig. 5 recombinant phytase PhyH and PhyH-DII.
The thermostability of Fig. 6 recombinant phytase PhyH.
The thermostability of Fig. 7 recombinant phytase PhyH-DII.
Fig. 8 fusion expression vector design of graphics.
(microbial preservation number is genus bacillus (Bacillus sp.HJB17): CGMCC No.4868; The preservation time is: 2011 05 month No. 18; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; 100101)
Embodiment
Test materials and reagent
1, bacterial strain and carrier: (microbial preservation number is: CGMCC No.4868 from genus bacillus (Bacillus sp.HJB17) in the present invention; The preservation time is: 2011 05 month No. 18; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; 100101) separate obtaining a kind of new low temperature neutral phytase with geminus territory in, coli expression carrier pET22b (+) and bacterial strain BL21 (DE3) preserve for the laboratory.
2, enzyme and other biochemical reagents: restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Available from Sigma company, other all is domestic reagent (all can buy from common biochemical reagents company and obtain).Bacterial genomes is extracted test kit available from sky root company.
3, substratum:
Intestinal bacteria substratum LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH 7.0).
Illustrate: make the experimental methods of molecular biology specify in following examples, all carry out, perhaps carry out according to test kit and product description with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Sa nurse Brooker one book.
An Xinjiang dirt band sample in the sky is after the phytase screening culture medium is cultivated (low-phosphorous substratum LPM), and dilution plate respectively 4,20, is cultivated for 37 ℃ routinely.The transparent circle list bacterium colony that has that will grow is then drawn and dull and stereotyped is divided purely, and will have active bacterial strain to identify, this bacterial strain is a bacillus after 16S identifies, called after Bacillus sp.HJB17.
The prescription of low-phosphorous substratum LPM is as follows:
Casein peptone 1g; Soy peptone 0.45g; Glucose 4g; Glutaminate 0.5g; Sodium-chlor 5g; MgCl
21.7mM; MgSO
41.4mM; KCl, 0.47mM; CaCl
20.3mM; Be dissolved among the 900mL 50mMpH 7.5Tris-HCl, be settled to 1000mL, packing, 15min is stand-by in sterilization.
The optimum growing condition of Bacillus sp.HJB17 is 37 ℃, and pH 7.0.Detect this bacterium at 37 ℃ and have phytase activity (0.33 ± 0.05UmL
-1).
The clone of embodiment 2 bacillus sp.HJB17 phytase encoding gene phyH
Extract bacillus sp.HJB17 genomic dna, adopt bacterial genomes to extract test kit (day root company), specification sheets between concrete operations.Degenerated primer BPP-F and BPP-R:BPP-F:5 '-GACGCAGCCGAYGAYCCNGCNITNTGG-3 ' and BPP-R:5 '-CAGGSCGCANRTCIACRTTRTT-3 ' have been synthesized according to the conserved regions sequences Design of BPP phytase gene.With Bacillus sp.HJB 17 total DNA is that template is carried out pcr amplification.Obtain an about 180bp fragment, this fragment is reclaimed the back order-checking that links to each other with the pEASY-T3 carrier.
According to the nucleotide sequence that order-checking obtains, each three TAIL-PCR Auele Specific Primer of design upstream and downstream: design direction is for needing the zone of ignorance direction of amplification, and the Position Design of sp2 is in the inboard of sp1, and sp3 is positioned at the inboard of sp2.Distance between per two primers does not have strict regulation, the general 22～30nt of primer length, and annealing temperature is at 60～65 ℃.And with they difference called after usp1, usp2, usp3 (upstream Auele Specific Primer), dsp1, dsp2, dsp3 (downstream Auele Specific Primer) sees Table 1.
The TAIL-PCR Auele Specific Primer of table 1 phytase PhyH
| The primer title | Primer sequence (5 '---3 ') | Primer length (bp) |
| USP-1 | ACCGGCCCACGCTAAGCTGCTG | 22 |
| USP-2 | CTGCTGCACGCGCGTACCCTGC | 22 |
| USP-3 | TCAGGCTAAGCTCAGGCCGCGC | 22 |
| DSP-1 | TCATCCGGCGCGGCCTGAGCTTAG | 24 |
| DSP-2 | TAGCCTGATCCTCGGCACTGACAAACGC | 28 |
| DSP-3 | GCGTGCAGCAGCTTAGCGTGGG | 22 |
Obtain the flanking sequence of known sequence by TAIL-PCR, amplification obtains sending the order-checking of Bo Maide company after product reclaims.Splicing back phyH full length gene 1224bp, encode 664 amino acid and a terminator codon.The theoretical molecular of predicting the maturation protein of this coded by said gene is 67kDa.PhyH sequence and the aminoacid sequence derived are carried out the BLAST comparison in GenBank, PhyH has the highest consistence (51%) with the supposition phytase that derives from Idiomarina loihiensis L2TR, PhyH-DI is 25% with the phytase consistence that derives from B.amyloloquefaciens TS-Phy, the consistence of PhyH-DII and TS-Phy is 49%, with the consistence that derives from B.subtili PhyC be 40%, illustrate that phyH is a kind of new phytase.Through comparison and structural analysis, this phytase has the imperfect structural domain PhyH-DI of two phytase structural domain: N ends and the structural domain PhyH-DII that the C end has typical phytase structure.
The preparation of embodiment 3 recombinant phytases
The reorganization of PhyH and purifying:
With after removing the gene phyH of coding phytase of signal peptide and expression vector pET22b (+) and carrying out double digestion (EcoRI+XhoI) simultaneously, the connection of spending the night, be converted in the TransI competence, acquisition contains recombinant plasmid pET22 (+)-phyH of Bacillus sp.HJB 17BPP phytase gene phyH, and recombinant plasmid pET22 (+)-phyH through checking order correct is converted into BL21 (DE3) then.
Get BL21 (DE3) bacterial strain that contains plasmid, be inoculated in the 300mL LB nutrient solution, behind 37 ℃ of about 2h of 220rpm shaking culture, add 1mM IPTG and 1mM CaCl
2, place 30 ℃ of 220rpm to induce, measure in the born of the same parents behind about 8h and the outer phytase vigor of born of the same parents.In born of the same parents, all detect the activity of phytase outside the born of the same parents, and will be higher than outside the born of the same parents in the born of the same parents.Through ni-sepharose purification, SDS-PAGE result shows that recombinant phytase has obtained expressing and reaching electrophoresis pure (Fig. 1).
The reorganization of PhyH-DII and purifying:
Express primer according to phytase phyH-DII protein coding gene sequences Design, and introducing restriction enzyme site EcoRI and XhoI, with gene phyH-DII and pET22 (+) while double digestion, the connection of spending the night transforms BL21 (DE3) and obtains correct pET22 (+)-phyH-DII recombinant plasmid.The bacterial strain that will contain correct recombinant plasmid is induced, and condition is 37 ℃, and IPTG (1mM) after inducing 8 hours, all detects phytase activity in last cleer and peaceful born of the same parents.To go up clear enzyme solution and and intracellular enzyme liquid collect, by ni-sepharose purification, the albumen in the elutriant of NTA200 has reached electrophoresis pure (Fig. 1), collects enzyme liquid and carries out next step activation analysis.
The activation analysis of embodiment 4 recombinant phytases
With the Tris damping fluid dilution of the albumen behind the purifying with the pH7.0 that 0.1mol/L is arranged, get 50 μ L dilution enzyme liquid and add substrate 4mmol/L sodium phytate 950 μ L (using the acetate buffer solution preparation of the pH4.5 of 0.1mol/L) usefulness, 37 ℃ of reaction 30min, add 1mL 10%TCA termination reaction, add 2mL colour developing liquid (10g Ammonium Molybdate Tetrahydrate+32mL sulfuric acid+73.2g ferrous sulfate adds water and is settled to 1L).Add the TCA mixing earlier after contrasting then enzyme-added liquid, add substrate again.Colour developing back 700nm surveys its OD value down, calculates enzyme and lives.
The property testing of embodiment 5 recombinant phytase PhyH
1, calcium ion is to the influence of recombinant phytase PhyH and PhyH-DII
The CaCl that phytase PhyH behind the purifying and PhyH-DII is added different concns in the sodium phytate substrate of 1.5mmol/L pH 7.0
2(0-5.0mM), in 37 ℃, measure enzymic activity, research Ca
2+Concentration is to the influence of enzymic activity.The result shows that the suitableeest calcium ion concn of PhyH and PhyH-DII is 1mM, and the calcium ion concn of high density is little to the PhyH influence, but has suppressed the activity (Fig. 2) of PhyH-DII.
2, the measuring method of the optimal pH of recombinant phytase PhyH and pH stability is as follows:
The recombinant phytase of embodiment 3 purifying is carried out enzymatic reaction to measure its optimal pH under different pH.The substrate sodium phytate carries out the phytase vitality test under in the 0.2mol/L damping fluid of different pH 37 ℃.Result (Fig. 3) shows that the optimal pH of recombinase PhyH is 7.0, at pH6.0-8.0 relative activity more than 80% is arranged.Phytase is handled 120min at 37 ℃ in the damping fluid of above-mentioned various different pH, measure enzymic activity again under 70 ℃ in the pH7.0 buffer solution system, with the pH patience of research enzyme.Result (Fig. 4) shows phytase when 37 ℃ are handled, and PhyH is all very stable between the pH 7.0-12.0 in alkaline range, in this pH scope, handle 120min after the residual enzyme activity more than 90%.And under 60 ℃ of high-temperature conditions, near optimal pH 7.0, handle 120min, enzyme work has dependency to the calcium ion of high density.
3, the optimum temperuture of phytase PhyH and thermal stability determination method are as follows:
The optimum temperuture of PhyH and PhyH-DII is 35 ℃, keeps the enzyme more than 60% to live at 20-35 ℃, and also has 20% enzyme (Fig. 5) alive at 0 ℃.PhyH and PhyH-DII have certain dependency to calcium ion simultaneously, and 1mM is the optimum concn of calcium ion.Aspect thermostability; calcium ion has strengthened the stability of phytase significantly; complete structural domain PhyH is under the protection of 10mM calcium ion; handled 1 hour for 60 ℃; also keep 90% relative enzyme (Fig. 6) alive, the thermostability of PhyH-DII is poor slightly, under the condition of 10mM calcium ion; handled 1 hour for 60 ℃, can keep 70% relative enzyme (Fig. 7) alive.
4, the K of phytase
mValues determination method is as follows:
Sodium phytate with different concns is a substrate, in Tris hydrochloride buffer (pH7.0) buffer solution system, measures enzymic activity down, calculates its K under 35 ℃ for 35 ℃
mValue.When after measured, being substrate with the sodium phytate at 35 ℃ K
mValue is respectively 4.43 ± 0.55 and 1.82 ± 0.23mg/mL, maximum reaction velocity V
MaxBe respectively 24.82 and 15.93 μ mol/minmg.
5, the substrate specificity of PhyH-DI
PhyH-DI is acted on phytic acid and corresponding low-phosphorous inositol monophosphate simultaneously, is respectively D-Ins (2) P1 of 1mM, D-Ins (1,4) P2, D-Ins (1,3,4) P3, D-Ins (1,3,4,5) P4, Ins (1,3,4,5,6) P5 or InsP
6, in 100mM Tris hydrochloride buffer (pH7.0) buffer solution system, 37 ℃ are reacted 30min down, measure its phosphoric acid burst size.The result shows, the phytic acid IP though PhyH-DI can not degrade
6, but can degrade d-Ins (1,3,4,5) P4, other low-phosphorous inositol monophosphates there is not degradation capability yet, this is to find that first incomplete phytase structural domain has degraded IP
4Ability (table 2).This hydrolysis ability can ensure that itself and other phytase does the time spent altogether and strengthen thorough hydrolysis to the phytic acid substrate.
The substrate specificity of table 2 PhyH-DI
| The substrate title | Than (the Umg that lives -1) |
| D-Ins(2)P 1 | ND a |
| D-Ins(1，4)P 2 | ND |
| D-Ins(1，3，4)P 3 | ND |
| D-Ins(1，3，4，5)P 4 | 4.28±0.56 |
| Ins(1，3，4，5，6)P 5 | ND |
| InsP 6 | ND |
aND refers to not detect enzyme and lives.
6, the synergy of PhyH-DI and single structure territory phytase
With the experiment that acts synergistically of the phytase of PhyH-DI and BPP class, they are the PhyP that derive from Pedobacter nyackensis, derive from the colibacillary phytase APPa that derives from of the 168phy of Bacillus.subtilis 168 and HAP class.At first the phytase with the single structure territory acts on for some time (5min or 120min) separately for 37 ℃, places boiling water to boil 5min and makes first enzyme deactivation, adds 37 ℃ of reactions of PhyH-DI 120min then, stops with trichoroacetic acid(TCA) then and adding colour developing liquid, at OD
700Measure the burst size of inorganic phosphorus down.The result shows: PhyH-DI can produce collaborative effect (table 3) with the phytase in single structure territory really, by being that substrate is degraded with the intermediate product that produces after these phytase effects, thereby improves the catalytic capability of single structure territory phytase.
Simultaneously, we have contrasted the degradation capability of geminus territory PhyH and two structural domain PhyH-DI and PhyH-DII, be the mixed enzyme of the PhyH-DI of 25nmol PhyH and 25nmol and 25nmol PhyH-DII or 50nmol PhyH-DII 37 ℃ react 120min simultaneously after, measure its inorganic phosphorus burst size respectively.The result shows: the catalytic capability of the PhyH in geminus territory will be higher than independent structural domain accumulative total sum far away, and the PhyH-DII that is higher than the twice molecule, this has illustrated that geminus territory phytase may form certain special catalyst mechanism (as the superstructure territory), thereby more effectively phytic acid is degraded.
The synergy of the phytase in table 3, PhyH-DI and single structure territory
aTwo enzymes are made the inorganic phosphorus burst size sum of time spent separately.
bThe ratio of practical measurement value and theoretical value.
7, the synergy of the structure of fusion rotein and PhyH-DI structural domain
We carry out amalgamation and expression (Fig. 8) with the phytase in PhyH-DI and other single structure territory, and measure the kinetic parameter of fusion rotein.Found that PhyH-DI can improve the degradation efficiency (table 4) of single structure territory phytase equally, thereby checking PhyH-DI improves the ubiquity meaning of catalytic efficiency.
The proteic kinetic parameter of table 4, PhyH, PhyH-DII and amalgamation and expression
Claims (10)
1. the low temperature neutral phytase PhyH with geminus territory is characterized in that its aminoacid sequence is shown in SEQ ID NO.1.
2. the gene with the low temperature neutral phytase PhyH in geminus territory is characterized in that, the described low temperature neutral phytase PhyH with geminus territory of coding claim 1.
3. the gene of the low temperature neutral phytase PhyH in geminus as claimed in claim 2 territory is characterized in that its nucleotide sequence is shown in SEQ ID NO.2.
4. a phytase structural domain PhyH-DI is characterized in that, its aminoacid sequence is shown in SEQ ID NO.3.
5. the gene of the described structural domain PhyH-DI of claim 4 is characterized in that, its nucleotide sequence is shown in SEQ ID NO.4.
6. the described phytase structural domain of claim 4 PhyH-DI is used to improve the active application of phytase.
7. phytase structural domain PhyH-DII with phytase activity, its aminoacid sequence is shown in SEQ ID NO.5.
8. the gene of the described structural domain PhyH-DII of claim 7 is characterized in that, its nucleotide sequence is shown in SEQID NO.6.
9. the described application of claim 1 with the low temperature neutral phytase PhyH in geminus territory.
10. genus bacillus (Bacillus sp.HJB17), its preserving number is: CGMCC No.4868.
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| CN107090465B (en) * | 2017-04-13 | 2019-09-17 | 河北省农林科学院遗传生理研究所 | A kind of neutral phytase gene for being suitble to express in crop, expression cassette and application thereof |
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