CN102943083B - Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof - Google Patents
Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof Download PDFInfo
- Publication number
- CN102943083B CN102943083B CN 201210489052 CN201210489052A CN102943083B CN 102943083 B CN102943083 B CN 102943083B CN 201210489052 CN201210489052 CN 201210489052 CN 201210489052 A CN201210489052 A CN 201210489052A CN 102943083 B CN102943083 B CN 102943083B
- Authority
- CN
- China
- Prior art keywords
- phytase
- high temperature
- temperature resistant
- gene
- resistant phytase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention provides site-specific mutagenesis high temperature resistant phytase gene TP and an expression vector and application thereof. Amino acid in a specific area in a phytase amino acid sequence is mutated into praline and arginine so as to enable the phytase to have a special stable protein structure. Simultaneously, a gene sequence is optimized to synthesize a phytase gene according to pichia pastoris codon preference and GC content, recombinant plasmids are built and transferred to pichia pastoris, and positive converters are obtained through screening to conduct induction expression to obtain the high temperature resistant phytase. Experiments prove that the phytase obtained through expression of the pichia pastoris can resist high temperature plasmids with the temperature higher than 85 DEG C, and survival rate reaches 85%. Popularization and application of the high temperature resistant phytase have substantial economical benefit and social benefit on development of feed animal husbandry of our country, and simultaneously great ecological benefit can be obtained.
Description
Technical field
The invention belongs to the molecular biology feed additive field, be specifically related to a kind of high temperature resistant phytase gene TP and expression vector and application of rite-directed mutagenesis.
Background technology
Phytic acid (phytinic acid) is the major storage form of phosphorus in plant, and phytic acid and its esters are the main storage forms of phosphorus in the crops such as cereal, beans and oil plant.Can not be hydrolyzed and utilize phytic acid owing to lacking corresponding enzyme in the stomach of monogastric animal, this has caused the waste in phosphorus source, and the phosphorus in the phytic acid that can not be utilized directly excreted, and has caused the phosphorus in soil and waters to pollute.In addition, phytic acid can also with mineral element and protein chelating, these nutritive elements can not be used effectively, caused the reduction of plant feed nutritive value, so phytic acid also is generally considered a kind of antinutritional factor in feed.
Phytase extensively is present in animal, plant and microorganism, and the phytase of practical application at present all derives from microorganism.In natural microbial, phytase content is too low, is difficult to a large amount of cheap phytase products that obtain, and can not meet the requirement of feed industrial development.Utilize at present genetic engineering bacterium to obtain the phytase of high expression level amount, to the widespread use of phytase and reduce production costs and brought into play vital role.There is at present the phytase gene in multiple-microorganism source to be separated, as aspergillus niger, genus bacillus, intestinal bacteria etc.According to investigation and comparison, the strongest phytase of decomposition phytic acid ability known to deriving from colibacillary phytase (appA phytase) and be so far.Within 1985, cloned first escherichia coli phytase encoding gene appA; Nineteen ninety has been carried out sequential analysis to this gene; Methods by rite-directed mutagenesis in 1992 have realized the overexpression of appA gene.At present, the thermostability that the key issue of phytase preparation in application is enzyme is not solved all the time well, granulation high temperature produces greatly and destroys phytase activity, although the investigator has carried out much fruitful research from different aspects to the thermotolerance of phytase, as the bacterial strain that produces heat-stable phytase by screening, use the methods such as protein engineering, improvement of production process to obtain heat-stable phytase, but the phytase heat-resisting effect obtained is still undesirable.
Summary of the invention
The invention provides a kind of high temperature resistant phytase gene TP and expression vector and application of rite-directed mutagenesis, the present invention is by the colibacillary phytase of rite-directed mutagenesis (appA phytase) aminoacid sequence, improve the thermostability of phytase, and transform its gene according to the pichia spp codon preference, to obtain the high efficient expression of phytase mutant.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The invention provides a kind of high temperature resistant phytase gene TP of rite-directed mutagenesis, its base sequence is as shown in SEQ ID No:4; The high temperature resistant phytase of described correspondence, its aminoacid sequence is as shown in SEQ ID No:5.
The present invention also provides the carrier that contains described high temperature resistant phytase gene TP, and concrete described carrier is pPIC9K-TP.
The invention provides described high temperature resistant phytase in the application as in fodder additives, the consumption of described high temperature resistant phytase is 100-200 gram/ton feed.
Compared with prior art, advantage of the present invention and positively effect are: for improving thermostability of phytase, the decomposition phytic acid ability the strongest phytase-escherichia coli phytase (appA phytase) of the present invention known to having selected so far, first the amino acid mutation of specific region in the phytase aminoacid sequence is become to proline(Pro) (P) and arginine (R), make phytase there is special stabilizing protein structure, after the phytase aminoacid sequence is translated to the acquisition gene order in the other direction, when rite-directed mutagenesis is carried out in the subregion to the phytase aminoacid sequence to its gene order by the Pichia yeast codon preference, GC content is optimized its gene order, simultaneously according to after pichia spp codon preference synthetic phytase gene, it is cloned in eukaryotic vector pPIC9K, built recombinant plasmid pPIC9K-TP.Recombinant plasmid pPIC9K-TP linearizing is forwarded in Pichia pastoris GS115 by the electric shock conversion method, screening is obtained to positive transformant and carry out the phytase that the abduction delivering acquisition is high temperature resistant and expression amount is high.By the enzymic activity of spectrophotometry phytase, and the enzyme that detects phytase under the differential responses temperature condition ratio of living.The experiment proved that, can tolerate the high temperature granulating more than 85 ℃ by the phytase obtained after Pichia anomala expression, Retention reaches 85%.
The phytase mutant that the present invention obtains by the transformation aminoacid sequence has good thermotolerance, obtains the high expression level amount in pichia spp.Applying of this phytase, not only produce considerable economic benefit and social benefit to the development of China's feed livestock industry, also can produce huge ecological benefits simultaneously.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, it is clearer that the other features and advantages of the invention will become.
The accompanying drawing explanation
Fig. 1 is that the PCR of recombinant plasmid pUC18-T-TP in the present invention identifies electrophoretogram, and M means DL2000 DNA marker; 1 means the PCR product of plasmid pUC18-T-TP.
Fig. 2 is that the PCR of recombinant expression plasmid pPIC9K-TP in the present invention identifies electrophoretogram, and M means DL2000 DNA marker; 1 means the PCR product of plasmid pPIC9K-TP.
Fig. 3 is the gel pattern of phytase mutant protein SDS-PAGE in the present invention, and in figure, M means albumen MW Marker; 1 means that recombinant bacterial strain GS115/pPIC9K-TP expresses supernatant; 2 mean that recombinant bacterial strain GS115/pPIC9K induces supernatant.
Fig. 4 is the rear phytase phytase activity figure under the differential responses temperature condition of transformation in the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
One, the acquisition of escherichia coli phytase (appA phytase) protein mutant gene
1, the phytase gene sequence in selection coli strain Escherichia coli str.K-12 is as shown in SEQ ID No:2, and appA phytase aminoacid sequence is as shown in SEQ ID No:1, and the avtive spot conserved sequence of phytase is RHGXRXP.
Phytase aminoacid sequence in NCBI (SEQ IDNo:1):
QSEPELKLESVVIVSRHGVRAPTKATQLMQDVTPDAWPTWPVKLGWLTPRGGELIAYLG
HYQRQRLVADGLLAKKGCPQSGQVAIIADVDERTRKTGEAFAAGLAPDCAITVHTQADTS
SPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGHRQTAFRELERVLNFPQSNLCLK
REKQDESCSLTQALPSELKVSADNVSLTGAVSLASMITEIFLLQQAQGMPEPGWGRITDSH
QWNTLLSLHNAQFYLLQRTPEVARSRATPLLDLIKTALTPHPPQKQAYGVTLPTSVLFIAGH
DTNLANLGGALELNWTLPGQPDNTPPGGELVFERWRRLSDNSQWIQVSLVFQTLQQMRD
KTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQIVNEARIPACSL
Phytase base sequence in NCBI (SEQ IDNo:2):
CAGAGTGAGCCGGAGCTGAAGCTGGAAAGTGTGGTGATTGTCAGT
CGTCATGGTGTGCGTGCTCCAACCAAG?
ACGCAACTGATGCAGGATGTCACCCCAGACGCATGGCCAACCTGGCCGGTA?
CTGGGTTGGCTGACACCGCGCGGTGGTGAGCTAATCGCCTATCTCGGACATTACCAACGCCAGCGTCTGGTAGCCGACGGATTGCTGGCGAAAAAGGGCTGCCCGCAGTCTGGTCAGGTCGCGATTATT?
GATGTCGACGAGCGTACCCGTAAAACAGGCGAAGCCTTCGCCGCCGGGCTGGCACCTGACTGTGCAATAACCGTACATACCCAGGCAGATACGTCCAGTCCCGATCCGTTATTTAATCCTCTAAAAACTGGCGTTTGCCAACTGGATAACGCGAACGTGACTGACGCGATCCTCAGCAGGGCAGGAGGGTCAATTGCTGACTTTACCGGGCATCGGCAAACGGCGTTTCGCGAACTGGAACGGGTGCTTAATTTTCCGCAATCAAACTTGTGCCTTAAACGTGAGAAACAGGACGAAAGCTGTTCATTAACGCAGGCATTACCATCGGAACTCAAGGTGAGCGCCGACAATGTCTCATTAACCGGTGCGGTAAGCCTCGCATCAATGCTGACGGAGATATTTCTCCTGCAACAAGCACAGGGAATGCCGGAGCCGGGGTGGGGAAGGATCACCGATTCACACCAGTGGAACACCTTGCTAAGTTTGCATAACGCGCAATTTTATTTGCTACAACGCACGCCAGAGGTTGCCCGCAGCCGCGCCACCCCGTTATTAGATTTGATCAAGACAGCGTTGACGCCCCATCCACCGCAAAAACAGGCGTATGGTGTGACATTACCCACTTCAGTGCTGTTTATCGCCGGACACGATACTAATCTGGCAAATCTCGGCGGCGCACTGGAGCTCAACTGGACGCTTCCCGGTCAGCCGGATAACACGCCGCCAGGTGGTGAACTGGTGTTTGAACGCTGGCGTCGGCTAAGCGATAACAGCCAGTGGATTCAGGTTTCGCTGGTCTTCCAGACTTTACAGCAGATGCGTGATAAAACGCCGCTGTCATTAAATACGCCGCCCGGAGAGGTGAAACTGACCCTGGCAGGATGTGAAGAGCGAAATGCGCAGGGCATGTGTTCGTTGGCAGGTTTTACGCAAATCGTGAATGAAGCACGCATACCGGCGTGCAGTTTGTAA
2, apppA phytase aminoacid sequence is carried out to rite-directed mutagenesis
Leucine in thermostable enzyme albumen (L), proline(Pro) (P), L-glutamic acid (E) and arginine (R) content are all higher than normal temperature bacterium institute phytase generating.Little and the foldable of proline(Pro) structure entropy wherein, and need very high energy just can untie after folding, thus improve protein stability.So by the orthomutation technology, the partial amino-acid in the contiguous active centre of phytase is sported to proline(Pro), can effectively improve the thermostability of phytase.Because proline(Pro) easily forms β-pleated sheet structure, thereby make conformation more stable firmly, aspect stabilizing protein structure and raising enzyme heat stability, there is very important effect.Arginine has larger side chain than the amino acid with same electric charge, and the hydrophobic interaction that side chain provides and interionic interact and can improve the stability of protein.Therefore codon mutation corresponding to the L-Ala (A) of 25th, 87th of the present invention in phytase maturation protein aminoacid sequence becomes the codon of proline(Pro) (P), the codon that the codon mutation that the 43rd amino acids Methionin (K) is corresponding is arginine (R), by the base sequence obtained according to the pichia spp codon preference, its base sequence is carried out to codon modify, by Pichia anomala expression, obtain phytase high temperature resistant, the high expression level amount.Phytase base sequence after sudden change as shown in SEQ ID No:3, suddenly change and optimize after the phytase base sequence as shown in SEQ ID No:4, the sudden change after the phytase aminoacid sequence as shown in SEQ ID No:5.
Phytase gene sequence after sudden change (SEQ ID No:3) as follows, underscore is partly the gene order that the avtive spot conserved sequence is corresponding.Respectively corresponding the 25th, 43 and 87 amino acids of square frame part base.
CAGAGTGAGCCGGAGCTGAAGCTGGAAAGTGTGGTGATTGTCAGT
CGTCATGGTGTGCGTGCTCCAACCAAG?
ACGCAACTGATGCAGGATGTCACCCCAGACGCATGGCCAACCTGGCCGGTA?
CTGGGTTGGCTGACACCGCGCGGTGGTGAGCTAATCGCCTATCTCGGACATTACCAACGCCAGCGTCTGGTAGCCGACGGATTGCTGGCGAAAAAGGGCTGCCCGCAGTCTGGTCAGGTCGCGATTATT?
GATGTCGACGAGCGTACCCGTAAAACAGGCGAAGCCTTCGCCGCCGGGCTGGCACCTGACTGTGCAATAACCGTACATACCCAGGCAGATACGTCCAGTCCCGATCCGTTATTTAATCCTCTAAAAACTGGCGTTTGCCAACTGGATAACGCGAACGTGACTGACGCGATCCTCAGCAGGGCAGGAGGGTCAATTGCTGACTTTACCGGGCATCGGCAAACGGCGTTTCGCGAACTGGAACGGGTGCTTAATTTTCCGCAATCAAACTTGTGCCTTAAACGTGAGAAACAGGACGAAAGCTGTTCATTAACGCAGGCATTACCATCGGAACTCAAGGTGAGCGCCGACAATGTCTCATTAACCGGTGCGGTAAGCCTCGCATCAATGCTGACGGAGATATTTCT?CCTGCAACAAGCACAGGGAATGCCGGAGCCGGGGTGGGGAAGGATCACCGATTCACACCAGTGGAACACCTTGCTAAGTTTGCATAACGCGCAATTTTATTTGCTACAACGCACGCCAGAGGTTGCCCGCAGCCGCGCCACCCCGTTATTAGATTTGATCAAGACAGCGTTGACGCCCCATCCACCGCAAAAACAGGCGTATGGTGTGACATTACCCACTTCAGTGCTGTTTATCGCCGGACACGATACTAATCTGGCAAATCTCGGCGGCGCACTGGAGCTCAACTGGACGCTTCCCGGTCAGCCGGATAACACGCCGCCAGGTGGTGAACTGGTGTTTGAACGCTGGCGTCGGCTAAGCGATAACAGCCAGTGGATTCAGGTTTCGCTGGTCTTCCAGACTTTACAGCAGATGCGTGATAAAACGCCGCTGTCATTAAATACGCCGCCCGGAGAGGTGAAACTGACCCTGGCAGGATGTGAAGAGCGAAATGCGCAGGGCATGTGTTCGTTGGCAGGTTTTACGCAAATCGTGAATGAAGCACGCATACCGGCGTGCAGTTTGTAA
Phytase gene sequence after optimization (SEQ ID No:4) as follows:CAATCTGAACCAGAATTGAAGTTGGAATCTGTTGTTATCGTTTCTAGACACGGTGTTAGAGCTCCAACTAAGCCAACTCAATTGATGCAAGACGTTACTCCAGACGCTTGGCCAACTTGGCCAGTTAGATTGGGTTGGTTGACTCCAAGAGGTGGTGAATTGATCGCTTACTTGGGTCACTACCAAAGACAAAGATTGGTTGCTGACGGTTTGTTGGCTAAGAAGGGTTGTCCACAATCTGGTCAAGTTGCTATCATCCCAGACGTTGACGAAAGAACTAGAAAGACTGGTGAAGCTTTCGCTGCTGGTTTGGCTCCAGACTGTGCTATCACTGTTCACACTCAAGCTGACACTTCTTCTCCAGACCCATTGTTCAACCCATTGAAGACTGGTGTTTGTCAATTGGACAACGCTAACGTTACTGACGCTATCTTGTCTAGAGCTGGTGGTTCTATCGCTGACTTCACTGGTCACAGACAAACTGCTTTCAGAGAATTGGAAAGAGTTTTGAACTTCCCACAATCTAACTTGTGTTTGAAGAGAGAAAAGCAAGACGAATCTTGTTCTTTGACTCAAGCTTTGCCATCTGAATTGAAGGTTTCTGCTGACAACGTTTCTTTGACTGGTGCTGTTTCTTTGGCTTCTATGTTGACTGAAATCTTCTTGTTGCAACAAGCTCAAGGTATGCCAGAACCAGGTTGGGGTAGAATCACTGACTCTCACCAATGGAACACTTTGTTGTCTTTGCACAACGCTCAATTCTACTTGTTGCAAAGAACTCCAGAAGTTGCTAGATCTAG AGCTACTCCATTGTTGGACTTGATCAAGACTGCTTTGACTCCACACCCACCACAAAAGCAAGCTTACGGTGTTACTTTGCCAACTTCTGTTTTGTTCATCGCTGGTCACGACACTAACTTGGCTAACTTGGGTGGTGCTTTGGAATTGAACTGGACTTTGCCAGGTCAACCAGACAACACTCCACCAGGTGGTGAATTGGTTTTCGAAAGATGGAGAAGATTGTCTGACAACTCTCAATGGATCCAAGTTTCTTT GGTTTTCCAAACTTT GCAACAAATGAGAGACAAGACTCCATTGTCTTTGAACACTCCACCAGGTGAAGTTA AGTTGACTTTGGCTGGTTGTGAAGAAAGAAACGCTCAAGGTATGTGTTCTTTGGCT GGTTTCACTCAAATCGTTAACGAAGCTAGAATCCCAGCTTGTTCTTTGTAA
Aminoacid sequence (SEQ ID No:5) as follows after sudden change:
QSEPELKLESVVIVSRHGVRAPTKPTQLMQDVTPDAWPTWPVRLGWLTPRGGELIAYLGHYQRQRLVADGLLAKKGCPQSGQVAIIPDVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILSRAGGSIADFTGHRQTAFRELERVLNFPQSNLCLKREKQDESCSLTQALPSELKVSADNVSLTGAVSLASMLTEIFLLQQAQGMPEPGWGRITDSHQWNTLLSLHNAQFYLLQRTPEVARSRATPLLDLIKTALTPHPPQKQAYGVTLPTSVLFIAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFERWRRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQIVNEARIPACSL
3, the phytase gene sequence of acquisition is sent biotech firm synthetic, synthetic phytase gene is cloned on the pUC18-T carrier.
Two, the structure of recombinant plasmid pPIC9K-TP
The Yeast expression carrier adopted is pPIC9K, and the host cell of carrier construction is bacillus coli DH 5 alpha.
1, design primer
According to new synthetic phytase gene sequences Design upstream and downstream primers F 1, R1, described primer is synthetic by Shanghai Sheng Gong company, and the sequence of described primer is as follows, and underscore part base represents corresponding restriction enzyme site:
Table 1:PCR amplification the primer
2, the clone of phytase gene TP
At first the recombinant plasmid pUC18-T-TP obtained of take is template, with F1, R1 primer, carries out pcr amplification.The PCR reaction system is as follows:
Reaction conditions: 95 ℃, 5min; 94 ℃, 30s, 66 ℃, 30s, 72 ℃, 1min20s, 35 circulations; Last 72 ℃ are extended 10min.
The PCR product of gained runs 0.8% agarose gel electrophoresis, and the 1233bp of being about band is arranged as seen, and electrophoretogram as shown in Figure 1.
3, the enzyme of phytase gene TP and Yeast expression carrier pPIC9K cuts back to close
Reclaim with two kinds of restriction enzymes double zyme cutting PCR of EcoR I, Not I purpose fragment TP and the pPIC9K carrier obtained, it is as follows that enzyme is cut system:
Reclaim enzyme and cut product.
4, the preparation of DH5 α competent cell (pressing molecular cloning guide method)
5, the conversion that is connected of phytase gene TP and pPIC9K carrier endonuclease bamhi
TP and pPIC9K endonuclease bamhi are mixed, then with the connection of spending the night of 16 ℃ of T4DNA ligase enzymes.Linked system is as follows:
Then will connect product and be transformed in DH5 α competent cell, step of converting as mentioned above, obtains recombinant expression plasmid.
6, the extraction of recombinant expression plasmid (with reference to the operation of molecular cloning experiment guide)
7, the PCR of recombinant expression plasmid identifies
Carry out pcr amplification with F1, R1 primer pair recombinant expression plasmid, the recombinant plasmid of the above-mentioned doubtful positive is carried out to the PCR evaluation.The PCR reaction system is as follows:
Reaction conditions: 95 ℃, 5min; 94 ℃, 30s, 66 ℃, 30s, 72 ℃, 1min20s, 35 circulations; Last 72 ℃ are extended 10min.
The PCR product of gained runs 0.8% agarose gel electrophoresis, and the 1233bp of being about band is arranged as seen, and electrophoretogram as shown in Figure 2.
8, the enzyme of recombinant expression plasmid is cut evaluation
Pcr amplification is accredited as to positive recombinant plasmid and carries out the double digestion evaluation with restriction enzyme EcoR I and Not I again.20 μ L endonuclease reaction systems are as follows:
After centrifugal mixing, in 37 ℃ of water-bath digestion 2h, enzyme is cut 0.8% sepharose for product (containing 0.5 μ g/mL EB) electrophoretic examinations.
9, the sequencing of recombinant plasmid
In order further to determine resulting recombinant plasmid, positive bacteria liquid is delivered to Shanghai Sheng Gong company and carry out sequencing, and by the exactness of DNAStar software analysis Insert Fragment.Correct recombinant plasmid called after pPIC9K-TP will finally be identified.
Three, the acquisition of recombinant bacterial strain GS115/pPIC9K-TP and abduction delivering phytase
1, linearizing recombinant plasmid pPIC9K-TP
With Sac I, by the pPIC9K-TP single endonuclease digestion, it is as follows that enzyme is cut system:
Sepharose DNA reclaims test kit recovery enzyme and cuts product.
2, the competent preparation of GS115 yeast and electricity transform (pressing Invitrogen company Pichia anomala expression service manual method)
3, the phenotypic evaluation of pichia spp transformant
Treat 28 ℃ of milky yeast conversion bacterium colonies of the dull and stereotyped appearance of hatching 4～6d to MD, after transforming, resulting positive transformant is connected on respectively on MD and MM flat board again, carry out the Mut+/Muts phenotypic evaluation, screening can be on MD and MM the bacterium colony of well-grown Mut+, transformant at the equal energy of two kinds of flat boards normal growth is the His+/Mut+ type, and can normal growth on the MD flat board, on the MM flat board, do not grow or poky be the His+/Muts type.
4, the screening of high resistance positive transformant
Single bacterium colony with the toothpick picking of sterilizing at the initial His+/Mut+ by growing in MD and MM culture medium flat plate, photolithography respectively dibbling be 1,2,3 in the G418 mass concentration of preparation, on the YPD flat board of 4mg/mL.Flat board is inverted, is placed in constant incubator, cultivate 2～3d for 30 ℃.The clone of high density resistance is picked out, and the YPD flat board that the G418 mass concentration of ruling respectively is 4mg/mL, with the purifying mono-clonal.
5, the PCR method is identified recombinant bacterial strain
Adopt PCR method to analyze the pichia spp transformant, the PCR reaction system:
After centrifugal mixing, carry out following response procedures on the PCR instrument: 95 ℃, 5min; 94 ℃, 40s, 53 ℃, 40s, 72 ℃, 1min, 35 circulations; Last 72 ℃ are extended 10min.Set up the negative control without template simultaneously.
6, the abduction delivering of positive transformant
(1) the mono-clonal recombination microzyme GS115/pPIC9K-TP transformed is inoculated in respectively containing in the 3mLYPD liquid nutrient medium, and 28 ℃, incubated overnight in the shaking table of 250rpm.
(2) get bacterium liquid 100 μ L in the ratio of 1: 100 and join in the Erlenmeyer flask (100mL) containing 25mL BMGY, 28 ℃, incubated overnight in the shaking table of 250rpm.
(3), when the OD600 value reaches 2.0-6.0, centrifugal collection thalline is transferred in the Erlenmeyer flask (250mL) that 50mL BMMY is housed, and 28 ℃, abduction delivering in the shaking table of 250rpm.
(4) every 24h adds the methyl alcohol that final concentration is 2%, to guarantee successive induction, expresses.
(5) get sample one time every 24h, to determine the Best Times of expression.Continuous induction is expressed 7d.After inducing end, the centrifugal 8min of nutrient solution 7000rpm room temperature collects supernatant, and-20 ℃ save backup.
7, the SDS-PAGE of expressing protein
Expressing protein is carried out to SDS-PAGE (with reference to the operation of molecular cloning experiment guide).Electrophoretogram as shown in Figure 3.
Four, the enzymic activity of phytase detects (spectrophotometer method)
1, phytase activity unit definition
Under the condition that sample is 5.0mmol/L, 37 ℃ of temperature, pH value 5.00 in sodium phytate concentration, per minute discharges 1 μ mol inorganic phosphorus from sodium phytate, is a phytase activity unit, with U, means.
2, Method And Principle
Phytase is under certain temperature and pH condition, and the hydrolysis substrate sodium phytate, generate ortho-phosphoric acid and inositol derivative, in acidic solution, with vanadium ammonium molybdate, processes and can generate yellow [(NH
4)
3pO
4nH
4vO
316MoO
3] mixture, carry out colorimetric estimation under wavelength 415nm.
3, determination step
3.1 typical curve
Accurately take 0.6804g at 105 ℃ of benchmark potassium primary phosphates that dry to constant weight in the 100mL volumetric flask, with acetate buffer, dissolve, and to be settled to 100mL concentration be 50.0mmol/L.Dilution proportion by table 1 becomes different concns, and reaction assay together with sample be take inorganic phosphorus concentration as X-coordinate, and light absorption value is ordinate zou, lists linear regression equation (y=ax+b).
Table 1
3.2 the preparation of sample solution
Take two parts, sample, be accurate to 0.0001g, be placed in the 100mL volumetric flask, add about 70mL acetate buffer, a magnetic bar, high-speed stirring 30min on magnetic stirring apparatus, be settled to scale (deducting the volume of magnetic bar) with acetate buffer.Shake up, on whizzer with the centrifugal 10min of 4000r/min.Divide acetate buffer dilution (generally getting the 2ml supernatant liquor in the 50ml volumetric flask) for the supernatant liquor of getting different volumes, make sample liquid concentration remain on 0.4U/mL.Left and right, question response.
3.3 reaction
Get the 10mL test tube and operated by following reaction sequence, in reaction process, from adding substrate, in every test tube, add the timed interval of reagent to want definitely consistent, 37 ℃ of hydrolysis 30min.
Reactions steps and reagent, solution usage are in Table 2.
Table 2
3.4 sample determination
Reacted sample is standing 10min at room temperature, as become turbid need be with the centrifugal 10min of 4000r/min on whizzer, and supernatant liquor is with the blank zeroing of typical curve, the light absorption value of and sample solution blank at spectrophotometer 415nm wavelength place working sample.For the actual measurement light absorption value.Calculate the activity of phytase with linear regression equation.
4, result is calculated and is meaned
4.1 calculation formula
The activity of phytase in sample, mean with the activity unit " U " in every gram (or ml) sample, and calculation formula is as follows:
In formula:
The activity of phytase in the U-sample, U/g;
The x value that C-is calculated by straight-line regression ten thousand journeys according to the light absorption value of actual sample liquid, U;
Total extension rate before the reaction of F-sample solution;
The m-sample mass, g or mL;
The 30-reaction times, min.
5, high temperature resistant analysis
Reaction pH5.0 and other conditions are constant, detect its enzyme according to above phytase activity detection method respectively under 37 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ conditions and live.The highest enzyme work of take is 100%, under its temperature condition, survey enzyme work account for percentage ratio that this enzyme lives and be this enzyme enzyme with this understanding ratio of living.Result is as shown in table 3 and Fig. 4.
Table 3: phytase activity under the differential responses temperature condition
Experimental result shows: the phytase optimal reactive temperature after sudden change is 70 ℃, and thus obtained phytase can tolerate the high temperature granulating more than 85 ℃, and Retention reaches 85%.
The L-Ala (A) of 25th, 87th of the present invention in phytase maturation protein aminoacid sequence is mutated into the little and proline(Pro) (P) of foldable of structure entropy, thereby makes conformation more stable firmly, improved the thermostability of phytase.Sport than the amino acid with same electric charge larger side chain arginine (R) is arranged at the 43rd amino acids Methionin (K), the hydrophobic interaction that its side chain provides and interionic interact and can improve the stability of protein.Evidence, thus obtained phytase albumen has resistant to elevated temperatures characteristic, and optimal reactive temperature is 70 ℃, and thus obtained phytase can tolerate the high temperature granulating more than 85 ℃, and Retention reaches 85%.This explanation is passed through some amino acid whose sudden change of phytase, stablized the conformation of phytase albumen, improve its thermostability, prevent sex change under the high temperature granulating condition, thereby effectively improve the utilization ratio of phosphorus in plant feed and the monogastric animal specific absorption to mineral element, and alleviate the pollution of phosphorus to environment in animal excrements.
Above embodiment is only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment, the present invention is had been described in detail, for the person of ordinary skill of the art, the technical scheme that still can put down in writing previous embodiment is modified, or part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
Claims (6)
1. the high temperature resistant phytase gene TP of a rite-directed mutagenesis, its base sequence is as shown in SEQ ID No:4.
2. a high temperature resistant phytase, its aminoacid sequence is as shown in SEQ ID No:5.
3. recombinant vectors, it contains high temperature resistant phytase gene TP claimed in claim 1.
4. recombinant vectors according to claim 3, it is characterized in that described carrier is pPIC9K-TP, with EcoR I, two kinds of restriction enzymes double zyme cutting phytase gene TP of Not I and pPIC9K carrier, with the T4DNA ligase enzyme, connect, connect product and be transformed in DH5 α competent cell, obtain recombinant vectors pPIC9K-TP.
5. high temperature resistant phytase according to claim 2 is in the application as in fodder additives.
6. high temperature resistant phytase according to claim 5 is in the application as in fodder additives, and the consumption that it is characterized in that described high temperature resistant phytase is 100-200 gram/ton feed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210489052 CN102943083B (en) | 2012-11-27 | 2012-11-27 | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210489052 CN102943083B (en) | 2012-11-27 | 2012-11-27 | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102943083A CN102943083A (en) | 2013-02-27 |
| CN102943083B true CN102943083B (en) | 2013-12-25 |
Family
ID=47726071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210489052 Active CN102943083B (en) | 2012-11-27 | 2012-11-27 | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102943083B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK2617814T3 (en) | 2006-09-21 | 2016-02-22 | Basf Enzymes Llc | Phytases, nucleic acids encoding them, and methods of producing and using them |
| GB201308828D0 (en) | 2013-03-12 | 2013-07-03 | Verenium Corp | Phytase |
| GB201308843D0 (en) | 2013-03-14 | 2013-07-03 | Verenium Corp | Phytase formulation |
| CN106399273B (en) * | 2013-07-24 | 2019-09-20 | 东莞泛亚太生物科技有限公司 | The phytase of enzymatic activity with promotion |
| KR20160033711A (en) | 2013-07-25 | 2016-03-28 | 바스프 엔자임스 엘엘씨 | Phytase |
| CN104312931B (en) * | 2014-03-24 | 2017-01-25 | 安徽科技学院 | Torulaspora delbrueckii and application thereof |
| ES2759083T3 (en) * | 2014-11-21 | 2020-05-07 | Qingdao Vland Biotech Group Co Ltd | Phytase mutants |
| CN104450643B (en) * | 2014-12-19 | 2020-07-28 | 青岛蔚蓝生物集团有限公司 | Phytase mutant and application thereof |
| CN107236717B (en) * | 2016-03-28 | 2021-03-30 | 青岛蔚蓝生物集团有限公司 | Phytase mutant |
| CN105969750B (en) * | 2016-06-24 | 2019-04-26 | 北京昕大洋科技发展有限公司 | A kind of phytic acid enzyme mutant and its application |
| CN107164344B (en) * | 2017-06-28 | 2020-03-17 | 青岛红樱桃生物技术有限公司 | Heat-resistant phytase mutant and encoding gene and application thereof |
| EA202092569A1 (en) * | 2018-05-30 | 2021-07-08 | Наньцзин Бестзайм Био-Энжиниринг Ко., Лтд. | MODIFIED FITASES |
| CN117535267A (en) * | 2020-04-29 | 2024-02-09 | 南京百斯杰生物工程有限公司 | Parent phytase variant |
| CN116555219B (en) * | 2023-03-14 | 2023-12-08 | 江西省科学院微生物研究所(江西省流域生态研究所) | Phytase mutant with improved thermal stability and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392002A (en) * | 2011-11-03 | 2012-03-28 | 青岛蔚蓝生物集团有限公司 | Improved escherichia coli phytase HTP6M and gene and application thereof |
-
2012
- 2012-11-27 CN CN 201210489052 patent/CN102943083B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102392002A (en) * | 2011-11-03 | 2012-03-28 | 青岛蔚蓝生物集团有限公司 | Improved escherichia coli phytase HTP6M and gene and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 《植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量》;陈惠 等;《中国生物化学与分子生物学报》;20050430;第21卷(第2期);全文 * |
| 《植酸酶最新研究进展》;康伟 等;《生命的化学》;20051231;第25卷(第2期);全文 * |
| 康伟 等.《植酸酶最新研究进展》.《生命的化学》.2005,第25卷(第2期),全文. |
| 陈惠 等.《植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量》.《中国生物化学与分子生物学报》.2005,第21卷(第2期),全文. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102943083A (en) | 2013-02-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102943083B (en) | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof | |
| KR100206453B1 (en) | A novel strain e coli jm83/pkp2 transformed with a novel plasmid and phytase produced from it | |
| CN104450643B (en) | Phytase mutant and application thereof | |
| CN105624131A (en) | Phytase mutant | |
| CN102002487A (en) | Optimized and improved high temperature resistance phytase PHYTH as well as gene and application thereof | |
| CN110029120B (en) | Phytase high-yield strain and application thereof | |
| CN102392002A (en) | Improved escherichia coli phytase HTP6M and gene and application thereof | |
| CN113528476A (en) | Glucose oxidase mutant and coding gene and efficient recombinant expression thereof | |
| Huang et al. | Novel low-temperature-active phytase from Erwinia carotovora var. carotovota ACCC 10276 | |
| CN103468660B (en) | High reactivity neutral phytase mutant and gene thereof and purposes | |
| CN103555688A (en) | Construction of engineered strain capable of efficiently expressing phytase | |
| CN101724611B (en) | Acid phytase APPA as well as mutant and preparation method thereof | |
| EP3569704A2 (en) | Phytase ykappa mutant having improved pepsin resistance and increased catalytic efficiency | |
| CN104480087A (en) | Novel high-temperature resistant alpha-amylase, preparing method of novel high-temperature resistant alpha-amylase and application of novel high-temperature resistant alpha-amylase | |
| CN112824527B (en) | Artificially designed lysyl endonuclease, coding sequence and fermentation method | |
| CN102559632B (en) | Optimized and improved escherichia coli phytase APPA-M with enhanced catalytic activity in acidic range, and gene and application of optimized and improved escherichia coli phytase APPA-M | |
| DK3215610T3 (en) | HOW TO UNKNOWN PHYTASE, PROCEDURE FOR OBTAINING THE SAME AND USE THEREOF | |
| CN103725707B (en) | Genetic engineering strain for recombinant expression of phytase | |
| CN101955957B (en) | Gene engineered Pichia pastoris highly secreting and expressing phytase | |
| CN108251439B (en) | Artificially-modified trypsin-resistant phytase as well as preparation method and application thereof | |
| CN103849607B (en) | High temperature resistant phytase and uses thereof | |
| CN101812433B (en) | Use of hevea brasiliensis invertase and coding gene thereof | |
| CN101812434B (en) | Invertase and application of encoding gene thereof | |
| CN104560917A (en) | Beta-glucosaccharase, beta-glucosaccharase mutant and application | |
| CN104404012B (en) | A kind of novel phytase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230707 Address after: No. 17, Seventh Avenue, Binhai Economic and Technological Development Zone, Tianjin, 300000 Patentee after: C.P. PREMIX (TIANJIN) CO.,LTD. Address before: Qingdao cross strait agricultural cooperation experimental zone, No.42, Youyi Avenue, Nancun Town, Pingdu City, Qingdao City, Shandong Province 266736 Patentee before: QINGDAO GENYUAN BIOLOGICAL TECHNOLOGY GROUP Co.,Ltd. |