CN106399273A - Phytase with enzymatic activity elevating function - Google Patents
Phytase with enzymatic activity elevating function Download PDFInfo
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- CN106399273A CN106399273A CN201610921038.1A CN201610921038A CN106399273A CN 106399273 A CN106399273 A CN 106399273A CN 201610921038 A CN201610921038 A CN 201610921038A CN 106399273 A CN106399273 A CN 106399273A
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- phytase
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- 108010011619 6-Phytase Proteins 0.000 title claims abstract description 79
- 229940085127 phytase Drugs 0.000 title claims abstract description 72
- 230000002255 enzymatic effect Effects 0.000 title abstract description 11
- 230000003028 elevating effect Effects 0.000 title abstract 2
- 150000001413 amino acids Chemical group 0.000 claims abstract description 50
- 108090000623 proteins and genes Proteins 0.000 claims description 45
- 230000000694 effects Effects 0.000 claims description 43
- 235000001014 amino acid Nutrition 0.000 claims description 21
- 229940024606 amino acid Drugs 0.000 claims description 20
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 16
- 235000004279 alanine Nutrition 0.000 claims description 16
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 13
- 230000035772 mutation Effects 0.000 claims description 13
- 241000588724 Escherichia coli Species 0.000 claims description 11
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 11
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- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- 108020004705 Codon Proteins 0.000 claims description 3
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- 125000003147 glycosyl group Chemical group 0.000 claims 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 abstract description 8
- 239000004473 Threonine Substances 0.000 abstract description 8
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 abstract description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004474 valine Substances 0.000 abstract description 4
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- 102000004169 proteins and genes Human genes 0.000 description 19
- IMQLKJBTEOYOSI-GPIVLXJGSA-N Inositol-hexakisphosphate Chemical compound OP(O)(=O)O[C@H]1[C@H](OP(O)(O)=O)[C@@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@H](OP(O)(O)=O)[C@@H]1OP(O)(O)=O IMQLKJBTEOYOSI-GPIVLXJGSA-N 0.000 description 18
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- ZPEZUAAEBBHXBT-RZVRUWJTSA-N l-valine l-valine Chemical compound CC(C)[C@H](N)C(O)=O.CC(C)[C@H](N)C(O)=O ZPEZUAAEBBHXBT-RZVRUWJTSA-N 0.000 description 2
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- 238000005070 sampling Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
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- 229940083982 sodium phytate Drugs 0.000 description 2
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- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- BLGYKMPTFOIOCW-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;propanedioic acid Chemical compound OC(=O)CC(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O BLGYKMPTFOIOCW-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
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- 241001494522 Citrobacter amalonaticus Species 0.000 description 1
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- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241001646716 Escherichia coli K-12 Species 0.000 description 1
- 235000014683 Hansenula anomala Nutrition 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
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- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
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- 238000012365 batch cultivation Methods 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
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- 239000013604 expression vector Substances 0.000 description 1
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- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to phytase with an enzymatic activity elevating function. Specifically, the amino acid sequence of the phytase is characterized in that valine of the 90th position of SEQ ID No.2 is mutated into the amino acid sequence of threonine.
Description
The application is Application No. 201310315250.X, and the applying date is on July 24th, 2013, and invention entitled " have
The divisional application of the Chinese patent application of the phytase of the enzymatic activity of lifting ".
Technical field
The application is related to a kind of phytase, the phytase of espespecially a kind of enzymatic activity with lifting.
Prior art
Phytic acid (phytic acid) is also called phytic acid (myo-inositol (1,2,3,4,5,6)
Hexakisphosphate), it is the principal mode storing phosphorus in plant, in seed, content is especially abundant, and seed such as frumentum
And beans is the primary raw material of animal feed.Although the phytic acid in seed can become the important sources of phosphorus needed for letting animals feed,
Only ruminant ability energy metabolism phytic acid is with using phosphorus therein；For non-ruminant animal it is impossible to the phytic acid of digested metabolism is anti-
And it is considered anti-nutrient substance.And phytic acid to be phytic acid the reason being considered anti-nutrient substance carry abundant negative electricity, easily with carry
The ion of positive electricity, such as calcium ion, magnesium ion, zinc ion, manganese ion, copper ion, iron ion chelating, further with protein and
Starch formation complex, this complex not only hinders digesting and assimilating of metal ion, and also impact digests enzyme effect and hinders nutrition
Material absorbing.
Cannot metabolism phytic acid non-ruminant animal need to add in the diet Phos or add phytase (phytase).With
Add the mode of Phos, not only high cost it is impossible to eliminate anti-nutrition reaction, also can by the phytic acid not being metabolized in animal excrements
Cause water pollution, destroy ecological balance.And the interpolation in feedstuff via phytase, then can reduce the anti-nutrition phenomenon of phosphoric acid
Improve alimentation, increase animal and the utilizability of phosphorus in feedstuff is reached outside 60% moreover it is possible to the discharge reducing phosphorus reaches 50%.
Phytase be can from phytic acid hydrolysis phosphate radical enzyme general term, all can find in animal, plant, microorganism
The presence of phytase gene.Six phosphate radicals on phytic acid can sequentially be hydrolyzed out by phytase, the hydrolysis of different phytases
Final degree differs, and hydrolysis mechanism is also different, if according to this as classification, phytase can be divided into histidine acid phosphatase
(histidine acid phophatase), purple acid phosphatase (purple acid phosphatase), β propeller are planted
Sour enzyme (β-propeller phytase), and cysteine phosphatase (cysteine phytase), wherein histidine are acid
Phosphatase adds because its optimum pH and action condition are more suitable for feedstuff, is therefore widely studied.Histidine is acid
Phosphatase catalytic mechanism is by acid-base reaction, will connect the mono phosphoric acid ester ester bond effect of being hydrolyzed of inositol ring and phosphate radical, releases
Release phosphate radical, its hydrolysing step is divided into two stages, and histidine acid phosphatase first can be attacked using the histidine of active region
Hit one of phytic acid mono phosphoric acid ester ester bond, form phosphoric acid-histidine intermediate, then a water is utilized by the aspartic acid of active region
Molecule provides proton to the oxonium ion of phosphate radical in phosphoric acid-histidine intermediate, discharges a phosphate radical.
Although add phytase in feedstuff to have shown in animal feeding and can increase the productivity, and mono- using 500-1000
The phytase of position activity can replace 1 gram of Phos to add and reduce the discharge of 30-50% phosphorus, but industrially how to make to occupy
The phytase of feed market 60% can have economic benefit, is the lasting target of enzyme transformation and demand.Phytase is in feed industry
Processing procedure and application upper it is necessary to meet following characteristic：The most suitable pH-value is pH 2-6.5, acid-resisting and antipepsin and anti-pancreas
The thermostability when characteristic of proteases for decomposing, product are pelletized, product have high activity；Meet conditions above and could effectively reduce life
Produce cost, be applied to industrial.
The acquirement being suitable for industrial enzyme can by the biological screening of nature, but need to take considerable time and people
Power, the enzyme often obtaining does not meet commercial Application condition yet, needs to be transformed again, and is strengthened changing with existing enzyme again
Make as more economical mode.The strategy of enzyme transformation can be divided into two kinds, and one is to utilize orthogenesiss, and one is setting using theoretical property
Meter.Orthogenesiss include two steps, and one is to manufacture the mutant gene bank with rich and varied property, and then sieves from gene bank
Choosing has the mutant of certain optimisation characteristic.What the manufacture method of mutant gene bank was commonly used has error-prone PCR
(error-prone PCR) and DNA recombining reaction (DNA shuffling), but huge mutant gene bank needs a large amount of sieves
Choosing method, otherwise for screening on be suitable labor intensive and time.In recent years, with available protein structure and
Sequence increases, the also development of computer software for calculation technology, may recognize that in protein with Binding Capacity, active region, steady
The aminoacid of fixed structure, the enzyme that the limited mutation gene bank therefore set up with this theoretical basis can more effectively filter out optimization is special
Property.
Therefore, the application is intended to the sequence alignment by phytase and structural analyses, carries out base to some specific aminoacid
Because of mutation, and then the industrial value that effective lifting phytase is industrially applied.
Content of the invention
The purpose of the application is to transform existing phytase, using sequence alignment, structural analyses and site-directed mutagenesis technique, has
The action activity of effect lifting phytase, so as to increasing the industrial application value of phytase.
For reaching above-mentioned purpose, a better embodiment of the application provides a kind of phytase, its aminoacid sequence be by
The valine mutation of the 90th position of SEQ ID NO.2 becomes the aminoacid sequence of threonine.Encode the gene of this SEQ ID NO.2
It is the EcAppA gene being screened from escherichia coli (Escherichia coli), the protease that it is showed is histidine
Acid phosphatase.The aminoacid sequence of this phytase is the aminoacid sequence of SEQ ID NO.4.
For reaching above-mentioned purpose, a better embodiment of the application is to provide a kind of phytase, its aminoacid sequence be by
The asparagine mutation of the 204th position of SEQ ID NO.6 becomes alanine or the mutant serine of the 206th position is become the third ammonia
The aminoacid sequence of acid, to remove the glycosylation site in phytase activity area whereby.Encode the gene line of this SEQ ID NO.6 from
The EcAppA gene that escherichia coli (Escherichia coli) are screened uses sequence optimisation further across codon
The gene of gained, the protease that it is showed is histidine acid phosphatase.The aminoacid sequence of this phytase is SEQ ID
The aminoacid sequence of NO.8 or SEQ ID NO.10.
Brief description
Fig. 1 shows phytase EcAppA protein steric structure and the position of transformation point.
Fig. 2 shows gene and the aminoacid sequence of phytase EcAppA.
Fig. 3 shows the primer sequence that rite-directed mutagenesises are adopted.
Fig. 4 shows the gene of Ec-V90T mutant and the aminoacid sequence of phytase EcAppA.
Fig. 5 shows gene and the aminoacid sequence of phytase r-AppA.
Fig. 6 shows the gene of r-N204A mutant and the aminoacid sequence of phytase r-AppA.
Fig. 7 shows the gene of r-S206A mutant and the aminoacid sequence of phytase r-AppA.
Fig. 8 shows the phytase activity analysis test of wild type Ec-AppA and mutant Ec-V90T.
Fig. 9 shows the phytase activity analysis test of wild type r-AppA and mutant r-N204A and r-S206A.
Specific embodiment
Embody the application feature to describe in detail in the explanation of back segment with some exemplary embodiments of advantage.It should be understood that
It is that the application can have various changes on different embodiments, so it is all without departing from scope of the present application, and wherein
Explanation and the use that is inherently illustrated as of accompanying drawing, and be not used to limit the application.
Phytase gene (EcAppA) in the application is to be screened from escherichia coli (Escherichia coli)
, the protease that it is showed is histidine acid phosphatase.Because its high activity and high substrate specificity and the most suitable work
Use pH scope, all meet the application of feedstuff, therefore there is high industry using value, in addition, this gene can be successfully in industry
Produce expression in bacterial strain Pichia sp. (Pichia pastoris), and this enzyme has solved protein structure.Therefore, this phytase
It is the goodish target as the improvement of further enzyme, therefore the application is intended to the phytase EcAppA at a relatively high to this activity and enters
Row transformation, to lift its activity further.
For highly active EcAppA, will find in transformation increases the mutation of activity, and the low enzyme of probability specific activity comes
Low, therefore using the success rate of a large amount of screening of random point mutation relatively come low；Furthermore, even if activity increases also difficulty and is multiplied by again
The increase of rate, is difficult to distinguish with wild type phytase in screening, therefore increases the degree of difficulty in screening, so the application makes
To lift the activity of EcAppA with reason design (rational design), the available structure in maintenance data storehouse and sequence are in addition
Analysis, reduces the target zone of screening, effectively finds the mutant of activity increase.
First, by EcAppA with equally there is the CaAppA of high phytase activity (from no malonic acid citric acid bacillus
The phytase of Citrobacter amalonaticus) and CbAppA (from Bu Shi citric acid bacillus Citrobacter
The phytase of brakii) carry out sequence alignment, comparison result finds, CaAppA and CbAppA has the one of 60% respectively with EcAPPA
Cause degree and 70% similarity, and have mutually 70% consistent degree and 80% similarity.Had highly similar using these three
Property sequence and all have highly active phytase, can by range shorter to be mutated, reduce need screening activity optimization dash forward
Become.
Then, choose having concordance in CaAppA and CbAppA in the different amino acid sites of EcAppA, recycle
Known EcAppA protein steric structure analyzes the position in these sites, is retained in the amino acid sites near active region, because
It is that site causes the probability of activity change larger near active region, finding out activity with this has the mutant of increase.According to aforementioned
Analysis, on aminoacid sequence, the L-Valine (Valine) of the 90th position is selected as carrying out the position of rite-directed mutagenesises.
Additionally, agedoite may be produced when analyzing Pichia anomala expression using known EcAppA protein steric structure
(Asparagine) glycosylated site, that is, serine (Serine) in second aminoacid after agedoite in sequence
Or tyrosine (Tyrosine), and when the aminoacid after agedoite is not proline (Proline).From sequence,
EcAppA has three to be likely to form glycosylated position, using known EcAppA protein steric structure analysis and observation to wherein
One glycosylated position, therefore can be by the agedoite (Original amino of this glycosylation position around phytase activity area
The 205th position of sequence) or agedoite after second amino acid position serine (original amino acid the 207th
Position) carry out point mutation, to manufacture the mutant removing glycosylation site.
Refer to Fig. 1, the position of its display EcAppA protein steric structure and transformation point.EcAppA belongs to histidine acid
Phytase in acid phosphatase family, this histidine acid phosphatase family structure all has similar structure, is roughly divided into up and down
Two parts, the first half is made up of alpha-helix, has polytropy, and lower half is made up of alpha-helix and β-pleated sheet, and structure is more fixing.
IHS is the synthetic that the phosphorus in phytic acid is replaced with sulfur, is used to refer to phytic acid when doing phytase protein stereochemical structure in enzyme
The position of middle combination.An object of the application transformation point V90, N205, S207 are also shown in the structure of Fig. 1.
Hereinafter will be described the method that the application transforms phytase EcAppA, survey including rite-directed mutagenesises, albumen performance and activity
Method for testing, and its obtained improvement phytase protein.
First, phytase EcAppA gene order used in this application is to remove signal sequence (signal peptide)
Escherichia coli K12 AppA sequence (GenBank NC_000913.2), that is, from the 67th base start express,
But there are several natural mutation sites not affecting activity, the 266th base including original gene becomes C by T and (make aminoacid by figured silk fabrics
Propylhomoserin becomes alanine), the 302nd base becomes T (making aminoacid become leucine by L-Glutamine) by A, and the 835th base by
C becomes T (not changing aminoacid), as shown in Fig. 2 the total length of EcAppA gene is 1236 bases after sequence adds promoter ATG
(DNA sequence is indicated with SEQ ID NO.1) and 411 aminoacid (aminoacid sequence is indicated with SEQ ID NO.2).Will
EcAppA gene limits enzyme action position using EcoRI and XhoI and constructs in pET22b expression vector.Rite-directed mutagenesises (site-
Directed mutagenesis) method is to carry out polymerase chain reaction (polymerase chain using mutant primer
reaction；PCR), central mutant primer used is as shown in figure 3, the mutant primer of wherein Ec-V90T mutant is designed as
The 90th aminoacid of phytase EcAppA can be become threonine (Threonine) by valine mutation.The sequence row of this mutant
In Fig. 4, wherein the DNA sequence of mutant Ec-V90T is indicated with SEQ ID NO.3, and aminoacid sequence is then with SEQ ID NO.4
Indicate.
Then add DpnI to act at 37 DEG C in PCR product, primary template is removed.Again containing mutation base
The plasmid of cause is sent in escherichia coli XL1B competent cell, and bacterium solution is applied to the LB containing 100 μ g/ml ampicillin antibiotic
Culture carries out cultivating one day based at 37 DEG C.Choosing colony again, confirms successfully mutant gene sequence by sequencing steps.To be mutated
Successfully AppA gene transformation, in e. coli bl21 (DE3), carries out protein expression and purification.
Protein expression method is the e. coli bl21 (DE3) being converted into work(using containing 100 μ g/ml ampicillins
LB culture fluid cultivated, after wherein bacterial strain is first inoculated in 5ml LB culture 6 hours, then amplifies volume and train to 200ml LB
Support, be finally amplified to the LB culture of 2L.When OD value reaches 0.6-0.8, the protein being proceeded to using the IPTG induction of 1mM is a large amount of
Expression.Afterwards, bacterium solution is collected with the centrifugation of 6000g rotating speed for 20 minutes.Using ultrasound cell disruptor (sonicator)
Broken bacterium, then be centrifuged 30 minutes with 15000rpm, and collect supernatant, with fast protein liquid chromatography instrument (fast protein
liquid chromatography；FPLC) purification, using DEAE anion exchange column, isolate purity of protein reach 95% with
On phytase protein.
The active testing mode of phytase protein is by the 7.5mM sodium phytate of 4ml and 0.2ml pheron (enzyme egg
White buffer is 0.05%Triton X-100,0.05%BSA and 0.25M sodium acetate, and buffer is pH5.5) and 1.8ml
Reaction buffer 0.25M sodium acetate, pH be 5.5, at 37 DEG C act on 30 minutes.It is subsequently added into the color development stopping liquid of 4ml
(water:Salpeter solution:10% amine molybdate:0.2M metavanadic acid amine=4:2:1:1), measure light absorption value in OD415 wavelength, then be converted into
Unit of enzyme activity (unit).The standard curve of wherein enzymatic activity is to be made by between potassium dihydrogen phosphate standard liquid 0-25 μm ol/ml
Fixed, and the definition of 1 unit (unit) is to discharge 1 μm of ole Phos the sodium phytate solution from concentration 5mM per minute.
And in pichia yeast expression system, first by the sequent synthesis of AppA sequence reference GenBank DQ513832.1
It is optimized for the sequence (r-AppA) of Pichia sp. easily expression, this sequence codon uses (codon usage) sequence optimisation,
To be suitable for expression in Pichia sp., this sequence via secreting, expressing, increases signal sequence in sequence N-terminal in Pichia sp.
(signal peptide), and the methionine (methionine) positioned at the 1st position of original amino acid is moved to letter
In number sequence, because signal sequence can be removed in protein expression process, oozy protein therefore in Pichia sp.
Aminoacid sequence first methionine fewer than original sequence.As shown in figure 5, the total length of r-AppA gene is 1233 alkali
Base (DNA sequence is indicated with SEQ ID NO.5) and 410 aminoacid (aminoacid sequence is indicated with SEQ ID NO.6), and profit
Limit enzyme action position with EcoRI and NotI to construct in pPICZ α A carrier, wherein there occurs when constructing plasmid and do not affect activity
Natural mutation, makes the 116th aminoacid of r-AppA be changed into L-Valine from alanine.Directed mutagenesis method is recycled to draw to be mutated
Thing carries out polymerase chain reaction, and central mutant primer used is as shown in figure 3, the mutant primer of wherein r-N204A mutant is
It is designed as the 204th aminoacid of phytase r-AppA being become alanine (Alanine) by asparagine mutation, this mutant
Sequence is listed in Fig. 6, and wherein the DNA sequence of mutant r-N204A is indicated with SEQ ID NO.7, and aminoacid sequence is then with SEQ
ID NO.8 indicates.And the mutant primer of r-S206A mutant be then designed as can by the 206th aminoacid of phytase r-AppA by
Mutant serine becomes alanine, and the sequence of this mutant is listed in Fig. 7, and wherein the DNA sequence of mutant r-S206A is with SEQ ID
NO.9 indicates, and aminoacid sequence is then indicated with SEQ ID NO.10.
Then, after DNA plasmid being carried out linearisation using PmeI, by electric step of converting, DNA is sent into Pichia sp.
Interior.Then bacterium solution is applied to the YPD culture dish containing 100 μ g/mlZeocin antibiotic to carry out cultivating two days at 30 DEG C.Choose again
Select bacterium colony and be inoculated into 5ml YPD and cultivate at 30 DEG C, be inoculated into again in 50ml BMGY and cultivate to every other day at 30 DEG C.Connect
, culture medium is changed into the 20ml BMMY containing 0.5% methanol and is expressed with induced protein, be equally incubated at 30 DEG C.Every 24
Hour samples and supplements 0.5% methanol.Additionally, being centrifuged the bacterium solution of sampling and being collected supernatant, and then measure phytase
Activity, its method is for example above-mentioned.
In order to further amplify phytase volume of production, we first cultivate inoculation to 5mlYPD to every other day, then
It is amplified to after 2L cultivates at 30 DEG C, be amplified to again in the 50L fermentation tank containing 19L fermentation medium (FBSM).Fermentation
During, can complete monitoring temperature at 30 DEG C and by ammonia control ph 5.0 about, and the regulation and control of dissolved oxygen are then by air inlet
Amount and rotating speed maintain more than 40%.After batch cultivation, stream adds 50% glycerol by yeast growth to certain bacterium amount.Again
Methanol by gradient current plus in the way of add in fermentation tank, and then induce enzyme protein expression.Timing sampling simultaneously collects supernatant, enters
And detect expression and the activity of phytase.
Fig. 8 shows the phytase activity analysis test of wild type Ec-AppA and mutant Ec-V90T, and in figure enzymatic activity is
It is compared as under 100% benchmark with the enzymatic activity of wild type EcAppA, statistical analysiss are the double tails using T-test
Analysis, in P<It is judged to significant difference (*) when 0.05.Learnt by result, by the figured silk fabrics ammonia of the 90th position on aminoacid sequence
Sour (Valine) is mutated into the activity about 20% that threonine (Threonine) can lift phytase effectively.
Fig. 9 shows the phytase activity analysis test of wild type r-AppA and mutant r-N204A and r-S206A, in figure
Enzymatic activity is to be compared as under 100% benchmark with the enzymatic activity of wild type r-AppA, and statistical analysiss are to utilize T-
Double tail analyses of test, in P<It is judged to significant difference (*) when 0.05.Learnt by result, removed using rite-directed mutagenesises manufacture
Mutant r-N204A and r-S206A of active region glycosylation site, i.e. the agedoite (Asparagine) of the 204th position
It is mutated into alanine (Alanine) and the serine (Serine) of the 206th position is mutated into alanine (Alanine), equal energy
Phytase activity is made to rise about 10%.
In sum, in order to increase the industrial application value of phytase, the application labor and compare EcAppA phytic acid
The enzyme gene phytic acid enzyme sequence high with other activity, after adding the structural analyses arround active region, for central several aminoacid
Technology revulsion using rite-directed mutagenesises becomes target amino acid, in order to castering action activity.Wherein, by the 90th on aminoacid sequence
Ec-V90T mutant obtained by the L-Valine (Valine) of position is mutated into threonine (Threonine) can be lifted effectively
The activity of phytase about 20%.And consider the impact to protein for caused glycosylation when expressing using Pichia sp.,
The application also removes mutant r-N204A and r-S206A of active region glycosylation site using rite-directed mutagenesises manufacture, and that is, the 204th
The agedoite (Asparagine) of individual position is mutated into alanine (Alanine) and the serine of the 206th position
(Serine) it is mutated into alanine (Alanine), the mutant that result removes active region glycosylation site all can make phytic acid enzyme activity
Property rise about 10%.
This genus of catastrophe point looking for increase activity on the just at a relatively high enzyme of activity originally again is difficult, the width that activity can increase
Degree is also little, and the amplitude that the catastrophe point that the application finds increases activity is 10～20%, if the increase of this amplitude is screened with a large amount of
Method be also easily missed under expression variation, and the application combines enzyme known array and structure, using design and rational,
In the case of only screening a little mutant, it could be purified one by one, determine its activity, filter out effective mutant.
Because phytase occupies the 60% of 5,500,000,000 feed enzyme markets, the mutant proposing by the application lifts phytase activity further
Afterwards, more can effectively save production cost and increase productive value, therefore the phytase pole of the enzymatic activity with lifting of the application
Tool industrial value, then files an application in accordance with the law.
The application can make the spirit without departing from the present invention for the various changes, such change by those skilled in the art
Also each fall within protection scope of the present invention claim.
Claims (5)
1. a kind of phytase, its aminoacid sequence is that the asparagine mutation of the 204th position of SEQ ID NO.6 is become alanine
Or the mutant serine of the 206th position is become the aminoacid sequence of alanine, to remove the glycosyl in phytase activity area whereby
Change site.
2. phytase as claimed in claim 1, the gene wherein encoding this SEQ ID NO.6 is from escherichia coli
The EcAppA gene that Escherichia coli is screened uses the gene of sequence optimisation gained further across codon.
3. phytase as claimed in claim 1, wherein this phytase are histidine acid phosphatases.
4. phytase as claimed in claim 1, the aminoacid sequence of wherein this phytase is the aminoacid sequence of SEQ ID NO.8
Row.
5. phytase as claimed in claim 1, the aminoacid sequence of wherein this phytase is the aminoacid of SEQ ID NO.10
Sequence.
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| CN106119223B (en) * | 2016-07-05 | 2019-08-27 | 中国农业科学院饲料研究所 | Phytic acid enzyme mutant YkAPPA-L162V/L327V and YeAPPA-L162V/L327V and its encoding gene and application |
| JP6893000B2 (en) | 2018-08-17 | 2021-06-23 | ボンタック バイオエンジニアリング（シェンゼン） カンパニー リミテッド | Method for preparing acid phosphatase mutant and nicotinamide riboside |
| CN114317488B (en) * | 2021-12-17 | 2024-07-26 | 贵州六盘水厚全生态农业有限公司 | Phytase mutant with improved specific activity |
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| CN1309699A (en) * | 1998-03-23 | 2001-08-22 | 诺维信公司 | Phytase variants |
| 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 |
| CN102559632A (en) * | 2010-12-22 | 2012-07-11 | 武汉新华扬生物股份有限公司 | 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 |
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| US6720014B1 (en) * | 1997-08-13 | 2004-04-13 | Diversa Corporation | Phytase-containing foodstuffs and methods of making and using them |
| CN101792749B (en) * | 2002-08-12 | 2012-09-05 | 金克克国际有限公司 | Mutant colibacillus appa phytase enzymes |
| CN102002487B (en) * | 2010-11-03 | 2014-04-30 | 广东溢多利生物科技股份有限公司 | Optimized and improved high temperature resistance phytase PHYTH as well as gene and application thereof |
| CN102943083B (en) * | 2012-11-27 | 2013-12-25 | 青岛根源生物技术集团有限公司 | Site-specific mutagenesis high temperature resistant phytase gene TP and expression vector and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1309699A (en) * | 1998-03-23 | 2001-08-22 | 诺维信公司 | Phytase variants |
| CN102559632A (en) * | 2010-12-22 | 2012-07-11 | 武汉新华扬生物股份有限公司 | 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 |
| 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 |
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