CN107488642B - Phytase mutant and application thereof - Google Patents
Phytase mutant and application thereof Download PDFInfo
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- CN107488642B CN107488642B CN201710913105.XA CN201710913105A CN107488642B CN 107488642 B CN107488642 B CN 107488642B CN 201710913105 A CN201710913105 A CN 201710913105A CN 107488642 B CN107488642 B CN 107488642B
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- 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)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03008—3-Phytase (3.1.3.8)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
Abstract
The invention belongs to the technical field of biology, and particularly relates to a phytase mutant and application thereof. The invention obtains the heat-resistant phytase mutant gene A-2 from bacillus subtilis mutant bacteria through PCR, constructs the A-2 on pPIC9 plasmid to obtain recombinant plasmid pPIC9-A-2, converts the recombinant plasmid into pichia GS115, screens to obtain the recombinant bacteria of high-expression heat-resistant phytase, and the expressed heat-resistant phytase is heat-insulated at 80 ℃ for 30min, the relative activity is still about 75 percent, and is processed at 90 ℃ for 30min, the enzyme activity is still more than 25 percent, the heat resistance is good, and the invention can be widely used in industries of food, feed and the like.
Description
The technical field is as follows:
the invention relates to the technical field of biology, and particularly relates to a phytase mutant and application thereof.
Background art:
phosphorus is an essential mineral element of an animal body, most of phosphorus in plant tissues exists in the form of phytic acid (phytic acid) or phytate, phytic acid molecules can chelate metal ions and exist in the form of chelate, the solubility of the phytic acid molecules is low, the phytic acid molecules are equivalent to anti-nutritional factors, the phytic acid molecules are difficult to be absorbed by monogastric animals, and the phosphorus which is not fully utilized enters a water body through animal excrement to finally cause water body eutrophication.
The phytase is a general term of enzymes which catalyze the hydrolysis of phytic acid and salts thereof into inositol and phosphate, and the phytase is added into animal feed, so that the utilization rate of phosphorus in the feed can be improved, and the pollution of the discharge of the phosphorus to the environment can be reduced. In 1996, the FDA confirmed that phytase was safe for use in food and could be used in animal feed, and has become the third largest feeding enzyme. The phytase can be synthesized in a large amount by microorganisms and added into feed for animals to use so as to solve the problem that monogastric animals lack the phytase capable of utilizing phytate, but the heat treatment in the feed processing process requires that the phytase has certain heat resistance. The major problems faced at present are high production cost of phytase, low phytase yield and insufficient thermostability.
The invention content is as follows:
the invention aims to provide a phytase mutant with improved heat resistance and a pichia pastoris engineering strain thereof. The phytase mutant is from a bacillus subtilis BS2 obtained by ultraviolet mutagenesis, and the phytase mutant coding gene A-2 is cloned from BS2 and constructed into a recombinant plasmid, and then is expressed in pichia pastoris GS115, so that a pichia pastoris engineering strain is obtained.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of Phytase mutants
"amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the mutant. E.g., Gln166Asn, indicating the substitution of the amino acid at position 166 from wild-type Gln to Asn, the numbering of the positions corresponding to the numbering of the amino acid sequence of the wild-type phytase of SEQ ID No. 2; "bases substituted by the original base position" is likewise used to indicate the mutated base in the mutant, the numbering of the position corresponding to the numbering of the nucleotide sequence of the wild-type phytase in SEQ ID No. 1.
In the present invention, A-1 represents a gene encoding a wild-type phytase, and A-2 represents a gene encoding a phytase mutant, the information being shown in the following table.
The amino acid sequence of the phytase mutant is shown in a sequence table SEQ ID No. 4;
the nucleotide sequence of the phytase mutation coding gene A-2 is shown in SEQ ID No. 3;
the expression vector for expressing the phytase is pPIC9, and the microbial host cell for transforming the expression vector is Pichia pastoris GS 115;
the pichia pastoris engineering bacteria are obtained by connecting phytase mutation coding gene A-2 with an expression vector pPIC9 and expressing in pichia pastoris GS 115;
the invention also provides application of the phytase mutant in the field of feed.
The experimental steps of the invention are as follows:
1. carrying out enzyme digestion on the coding gene A-2 of the phytase mutant, and connecting the phytase mutant to an expression vector pPIC9 to obtain a recombinant vector;
2. transforming the recombinant vector into pichia pastoris GS115 to obtain a production strain GS115/pPIC9-A-2 of the phytase mutant;
3. GS115/pPIC9-A-2 is used as a production strain for producing phytase by fermentation.
The enzymatic properties of the phytase mutant are as follows:
(1) pH 4-6, the enzyme activity is stable, and the optimum action pH is 5.0.
(2) Temperature: the enzyme activity is stable at 50-80 ℃, and the optimal action temperature is 70 ℃.
(3) Heat resistance: the enzyme still has over 75 percent of enzyme activity after being insulated for 30min at 80 ℃, and the enzyme activity still remains over 25 percent after being insulated for 30min at 90 ℃.
Has the advantages that:
1. the invention discloses a brand-new phytase mutant which has the characteristics of high enzyme activity and good thermal stability. The enzyme activity of the mutant fermentation liquor can reach 359U/mL, and is improved by 200 percent compared with the wild phytase;
2. the phytase obtained by the invention still has over 75 percent of enzyme activity after being preserved for 30min at 80 ℃, and the enzyme activity still remains over 25 percent after being preserved for 30min at 90 ℃.
Description of the drawings:
FIG. 1 shows a PCR identification electrophoretogram of a colony;
wherein M is marker, and lane 1 is gene A-2;
FIG. 2 Phytase optimum pH curve;
FIG. 3 phytase optimum temperature curves;
FIG. 4 temperature stability curve of phytase.
The specific implementation mode is as follows:
the present invention is described in more detail below with reference to specific examples, which are provided by way of illustration only and are not intended to limit the scope of the invention. Modifications may be made by those skilled in the art, which would be within the principles of the invention, and such modifications are to be considered within the scope of the invention. The molecular biological experiment method not specifically described in this example can be referred to "molecular cloning Experimental Manual".
EXAMPLE 1 obtaining Phytase Gene A-2
Separating bacillus subtilis BS capable of producing phytase from soil, screening by ultraviolet mutagenesis to obtain a strain BS2 with heat-resistant phytase activity, designing a PCR primer according to the sequence of a mutant gene A-2, adding a restriction enzyme site Xho I into the 5 'end and adding a restriction enzyme site Not I into the 3' end, and obtaining a mutant gene A-2 by PCR, wherein the nucleotide sequence of the mutant gene A-2 is SEQ ID No. 3. The primer sequences are as follows:
A-2-F 5'-CCGCTCGAGATGAAGGTTCCAAAAACAAT-3'
A-2-R 5'-TTGCGGCCGCCTAGCCGTCAGAACGGTCTT-3'
EXAMPLE 2 construction of recombinant vector pPIC9-A-2
Respectively carrying out Xho I and Not I enzyme digestion on mutant gene A-2 and plasmid pPIC9, recovering products, mixing the recovered A-2 and pPIC9 in proportion, connecting the mixture by T4 ligase at 16 ℃ overnight, transforming Escherichia coli DH5 alpha competent cells by the connecting products, coating the transformed products on an LB (ampicillin-containing) solid plate, carrying out inverted culture at 37 ℃ overnight, picking single colonies to an LB liquid culture medium, carrying out culture at 37 ℃, carrying out colony PCR on bacterial liquid, and carrying out electrophoresis identification result as shown in figure 1, wherein the sequencing result shows that the sequence is correct and the sequence size is 1.15 kb. And extracting the recombinant plasmid for later use.
EXAMPLE 3 transformation of Pichia pastoris with recombinant plasmids
1. Preparation of Pichia pastoris GS115 competent cell
1) Selecting a single colony of a pichia pastoris plate, inoculating the single colony in 5mL of YPD culture medium, and oscillating overnight at 30 ℃ at 220 r/min;
2) inoculating 0.5mL of overnight-cultured bacterial liquid into 50mL of freshly prepared YPD medium, and performing shaking culture at 30 ℃ and 220r/min to enable the OD600 value to reach 1.3-1.5;
3) centrifuging the culture solution at 4 deg.C and 3000r/min for 5 min;
4) discarding the supernatant, adding 50mL of ice-precooled sterile water, and oscillating to resuspend the thalli;
5) centrifuging at 4 ℃ and 3000r/min for 5min, removing supernatant, sucking residual liquid on the tube wall, adding 25mL of ice-precooled sterile water, and oscillating to resuspend thalli;
6) centrifuging at 4 ℃ and 3000r/min for 5min, removing supernatant, sucking residual liquid on the tube wall, adding 10mL of 1mol/L sterile sorbitol solution precooled on ice, and resuspending thalli;
7) centrifuging at 4 deg.C and 3000r/min for 5min, discarding supernatant, sucking off residual liquid on tube wall, adding 1mL of 1mol/L sterile sorbitol solution precooled on ice (adding glycerol to final concentration of 15%), shaking and mixing.
8) 100 μ L/tube into sterile EP jar, and freezing at-70 deg.C (fresh competent cells are more effective).
2. Transformation of linearized plasmids
The recombinant plasmid pPIC9-A-2 obtained by extraction is subjected to single enzyme digestion by SalI to obtain a linearized plasmid. Freshly prepared (or frozen at-70 ℃) competent cells were placed in an ice bath and allowed to thaw completely.
1) Removing 100 μ L of competent cells into a new sterile EP tube, adding 10 μ L of linearized plasmid, mixing by gentle blowing, sucking out and transferring into a 0.2 cm-type electroporation transfer cup;
2) the transformation cup was placed in an ice bath for 5-10 minutes, maintaining the low temperature.
3) Electroporation transformation shock conditions: 1500V, 200 omega, 25 muF, discharge time of about 5ms, one electric shock.
4) After electric shock, 1mL of 1mol/L sorbitol solution precooled at 4 ℃ is added into an electric shock conversion cup immediately, and the mixture is blown and beaten uniformly by a liquid transfer gun and placed in an ice bath;
5) MD Medium (1.34% YNB; 4 × 10) was aseptically applied on a clean bench-5% biotin; 2% glucose plate), 100-;
6) two recombinant bacteria are obtained by screening on an MD plate, a target sequence is obtained by colony PCR, and the sequence comparison shows that the target sequence is the nucleotide sequence of a target gene A-2, namely the obtained strains are recombinant bacteria containing pPIC9-A-2 and are respectively named as P-1 and P-2.
Example 4 inducible expression of Yeast containing recombinant plasmid pPIC9-A-2
BMGY medium formulation comprising 1% yeast extract, 2% peptone, 0.1mol/LpH6.0 phosphate buffer, 1.34% YNB, 4 × 10-5% biotin, 1% glycerol.
The BMMY culture medium comprises 1% of yeast extract, 2% of peptone, 0.1mol/LpH6.0 phosphate buffer, 1.34% of YNB, 4 × 10-5% biotin, 0.5% methanol.
Respectively inoculating recombinant bacteria P-1 and P-2 and a recombinant strain (control) constructed by the original gene A-1 by the same method into a triangular flask filled with 30mLBMGY culture medium, culturing at 30 ℃ and 220r/min until the OD600 is about 10, centrifugally collecting thalli, suspending the thalli by using 35mL BMMY culture medium, continuously culturing for 48 hours at 30 ℃ and 220r/min, and measuring the phytase activity in supernatant after the fermentation liquor is centrifuged, wherein the results are as follows:
EXAMPLE 5 enzymatic Properties of Phytase
The determination is carried out by a vanadium ammonium molybdate method, and the definition of enzyme activity is as follows: the enzyme amount required for releasing 1. mu. mol of inorganic phosphorus from a sodium phytate solution with a concentration of 5.0mmol/L per minute at a temperature of 37 ℃ and a pH of 5.50 is 1 enzyme activity unit (U).
(1) Optimum pH for action
Taking the supernatant of the P-2 fermentation liquor obtained in the example 5 as a sample, taking the measured highest enzyme activity of the phytase as a reference, respectively reacting in a buffer solution with the pH value of 3.0-8.0 at the temperature of 37 ℃, and measuring the enzyme activity under different pH conditions. As can be seen from FIG. 2, the phytase has stable enzyme activity in the pH range of 4-6, and the optimum action pH of 5.0.
(2) Optimum temperature of action
Taking the supernatant of the P-2 fermentation liquor obtained in the example 5 as a sample, taking the measured highest enzyme activity of the phytase as a reference, respectively measuring the enzyme activity in a buffer solution with the pH value of 5.0 at the temperature of 30-90 ℃, and calculating the relative enzyme activity, wherein the result is shown in figure 3, the phytase produced by the P-2 has stable enzyme activity at the temperature of 50-80 ℃, and the optimal action temperature is 70 ℃.
(3) Thermal stability
Taking supernatant of P-2 obtained in example 5 and fermentation liquor of a control as a sample, taking the enzyme activity of the phytase which is not processed as a reference of 100%, carrying out heat preservation treatment on the sample at 50-90 ℃ for 30min under the condition of pH5.0 buffer solution, measuring the enzyme activity, and calculating the residual enzyme activity, wherein as can be seen from figure 4, the relative activity of the phytase is about 75% remained (the control residual enzyme activity is 20%) at 80 ℃, and the enzyme activity is still more than 25% (the control residual enzyme activity is 0) at 90 ℃, so that compared with the phytase produced by the recombinant bacterium (control) constructed by the original gene A-1, the heat stability is greatly improved, and the phytase produced by the recombinant bacterium P-2 has good heat resistance and can be widely used in the food and feed industries.
SEQUENCE LISTING
<110> Shandong Kete enzyme preparation Co., Ltd
<120> phytase mutant and application thereof
<130>1
<160>4
<170>PatentIn version 3.5
<210>1
<211>1149
<212>DNA
<213> Bacillus subtilis BS
<400>1
atgaaggttc caaaaacaat gctgctaagc actgccgcgg gtttattgct tagcctgaca 60
gcaacctcgg tgtcggctca ttatgtgaat gaggaacatc atttcaaagt gactgcacac 120
acggagacag atccggtcgc atctggcgat gatgcagcag atgacccggc catttgggtt 180
catgaaaaac acccggaaaa aagcaagttg attacaacaa ataagaagtc agggctcgtt 240
gtgtatgatt tagacggaaa acagcttcat tcttatgagt ttggcaagct caataatgtc 300
gatctgcgct atgattttcc attgaacggc gaaaaaattg atattgctgc cgcatccaac 360
cggtccgaag gaaaaaatac aattgaagta tatgcaatag acggggataa aggaaaattg 420
aaaagcatta cagatccgaa ccatcctatt tccaccaata tttctgaggt ttatggattc 480
agcttgtatc acagccagaa aacaggagca ttttacgcat tagtgacagg caaacaaggg 540
gaatttgagc agtatgaaat tgttgatggt ggaaagggtt atgtaacagg gaaaaaggtg 600
cgtgaattta agttgaattc tcagaccgaa ggccttgttg cggatgatga gtacggaaac 660
ctatacatag cagaggaaga tgaggccatc tggaaattta acgctgagcc cggcggaggg 720
tcaaaggggc aggttgttga ccgtgcgaca ggagatcatt tgacagctga tattgaagga 780
ctgacaatct attatgcacc aaatggcaaa ggatatctca tggcttcaag tcaaggaaat 840
aacagctatg caatgtatga acggcagggg aaaaatcgct atgtagccaa ctttgagatt 900
acagatggcg agaagataga cggtactagt gacacggatg gtattgatgt tctcggtttc 960
ggacttggcc caaaatatcc gtacgggatt tttgtggcgc aggacggcga aaatattgat 1020
aacggacaag ccgtcaatca aaatttcaaa attgtatcgt gggaacaaat tgcacagcat 1080
ctcggcgaaa tgcctgatct tcataaacag gtaaatccga ggaagctgaa agaccgttct 1140
gacggctag 1149
<210>2
<211>382
<212>PRT
<213> Bacillus subtilis BS
<400>2
Met Lys Val Pro Lys Thr Met Leu Leu Ser Thr Ala Ala Gly Leu Leu
1 5 10 15
Leu Ser Leu Thr Ala Thr Ser Val Ser Ala His Tyr Val Asn Glu Glu
20 25 30
His His Phe Lys Val Thr Ala His Thr Glu Thr Asp Pro Val Ala Ser
35 40 45
Gly Asp Asp Ala Ala Asp Asp Pro Ala Ile Trp Val His Glu Lys His
50 55 60
Pro Glu Lys Ser Lys Leu Ile Thr Thr Asn Lys Lys Ser Gly Leu Val
65 70 75 80
Val Tyr Asp Leu Asp Gly Lys Gln Leu His Ser Tyr Glu Phe Gly Lys
85 90 95
Leu Asn Asn Val Asp Leu Arg Tyr Asp Phe Pro Leu Asn Gly Glu Lys
100 105 110
Ile Asp Ile Ala Ala Ala Ser Asn Arg Ser Glu Gly Lys Asn Thr Ile
115 120 125
Glu Val Tyr Ala Ile Asp Gly Asp Lys Gly Lys Leu Lys Ser Ile Thr
130 135 140
Asp Pro Asn His Pro Ile Ser Thr Asn Ile Ser Glu Val Tyr Gly Phe
145 150 155 160
Ser Leu Tyr His Ser Gln Lys Thr Gly Ala Phe Tyr Ala Leu Val Thr
165 170 175
Gly Lys Gln Gly Glu Phe Glu Gln Tyr Glu Ile Val Asp Gly Gly Lys
180 185 190
Gly Tyr Val Thr Gly Lys Lys Val Arg Glu Phe Lys Leu Asn Ser Gln
195 200 205
Thr Glu Gly Leu ValAla Asp Asp Glu Tyr Gly Asn Leu Tyr Ile Ala
210 215 220
Glu Glu Asp Glu Ala Ile Trp Lys Phe Asn Ala Glu Pro Gly Gly Gly
225 230 235 240
Ser Lys Gly Gln Val Val Asp Arg Ala Thr Gly Asp His Leu Thr Ala
245 250 255
Asp Ile Glu Gly Leu Thr Ile Tyr Tyr Ala Pro Asn Gly Lys Gly Tyr
260 265 270
Leu Met Ala Ser Ser Gln Gly Asn Asn Ser Tyr Ala Met Tyr Glu Arg
275 280 285
Gln Gly Lys Asn Arg Tyr Val Ala Asn Phe Glu Ile Thr Asp Gly Glu
290 295 300
Lys Ile Asp Gly Thr Ser Asp Thr Asp Gly Ile Asp Val Leu Gly Phe
305 310 315 320
Gly Leu Gly Pro Lys Tyr Pro Tyr Gly Ile Phe Val Ala Gln Asp Gly
325 330 335
Glu Asn Ile Asp Asn Gly Gln Ala Val Asn Gln Asn Phe Lys Ile Val
340 345 350
Ser Trp Glu Gln Ile Ala Gln His Leu Gly Glu Met Pro Asp Leu His
355 360 365
Lys Gln Val Asn Pro Arg LysLeu Lys Asp Arg Ser Asp Gly
370 375 380
<210>3
<211>1149
<212>DNA
<213> Artificial sequence
<400>3
atgaaggttc caaaaacaat gctgctaagc actgccgcgg gtttattgct tagcctgaca 60
gcaacctcgg tgtcggctca ttatgtgaat gaggaacatc atttcaaagt gactgcacac 120
acggagacag atccggtcgc atctggcgat gatgcagcag atgacccggc catttgggtt 180
catgaaaaac acccggaaaa aagcaagttg attacaacaa ataagaagtc agggctcgtt 240
gtgtatgatt tagacggaaa acagcttcat tcttatgagt ttggcaagct caataatgtc 300
gatctgcgct atgattttcc attgaacggc gaaaaaattg atattgctgc cgcatccaac 360
cggtccgaag gaaaaaatac aattgaagta tatgcaatag acggggataa aggaaaattg 420
aaaagcatta cagatccgaa ccatcctatt tccaccaata tttctgaggt ttatggattc 480
agcttgtatc acagcaacaa aacaggagca ttttacgcat tagtgacagg caaacaaggg 540
gaatttgagc agtatgaaat tgttgatggt ggaaagggtt atgtaccagg gaaaaaggtg 600
cgtgaattta agttgaattc tcagaccgaa ggccttgttg cggatgatga gtacggaaac 660
ctatacatag cagaggaaga tgaggccatc tggaaattta acgctgagcc cggcggaggg 720
tcaaaggggc aggttgttga ccgtgcgaca ggagatcatt tgacagctga tattgaagga 780
ctgacaatct attatgcacc aaatggcaaa ggatatctca tggcttcaag tcaaggaaat 840
aacagctatg caatgtatga acggcagggg aaaaatcgct atgtagccaa ctttgagatt 900
acagatggcg agaagataga cggtactagt gacacggatg gtattgatgt tctcggtttc 960
ggacttggcc caaaatatcc gtacgggatt tttgtggcgc aggacggcga aaatattgat 1020
aacggacaag ccgtcaatca aaatttcaaa attgtatcgt gggaacaaat tgcacagcat 1080
ctcggcgaaa tgcctgatct tcataaacag gtaaatccga ggaagctgaa agaccgttct 1140
gacggctag 1149
<210>4
<211>382
<212>PRT
<213> Artificial sequence
<400>4
Met Lys Val Pro Lys Thr Met Leu Leu Ser Thr Ala Ala Gly Leu Leu
1 5 10 15
Leu Ser Leu Thr Ala Thr Ser Val Ser Ala His Tyr Val Asn Glu Glu
20 25 30
His His Phe Lys Val Thr Ala His Thr Glu Thr Asp Pro Val Ala Ser
35 40 45
Gly Asp Asp Ala Ala Asp Asp Pro Ala Ile Trp Val His Glu Lys His
50 55 60
Pro Glu Lys Ser Lys Leu Ile Thr Thr Asn Lys Lys Ser Gly Leu Val
65 70 75 80
Val Tyr Asp Leu Asp Gly Lys Gln Leu His Ser Tyr Glu Phe Gly Lys
85 90 95
Leu Asn Asn Val Asp Leu Arg Tyr Asp Phe Pro Leu Asn Gly Glu Lys
100 105 110
Ile Asp Ile Ala Ala Ala Ser Asn Arg Ser Glu Gly Lys Asn Thr Ile
115 120 125
Glu Val Tyr Ala Ile Asp Gly Asp Lys Gly Lys Leu Lys Ser Ile Thr
130 135 140
Asp Pro Asn His Pro Ile Ser Thr Asn Ile Ser Glu Val Tyr Gly Phe
145 150 155 160
Ser Leu Tyr His Ser Asn Lys Thr Gly Ala Phe Tyr Ala Leu Val Thr
165 170 175
Gly Lys Gln Gly Glu Phe Glu Gln Tyr Glu Ile Val Asp Gly Gly Lys
180 185 190
Gly Tyr Val Pro Gly Lys Lys Val Arg Glu Phe Lys Leu Asn Ser Gln
195 200 205
Thr Glu Gly Leu Val Ala Asp Asp Glu Tyr Gly Asn Leu Tyr Ile Ala
210 215 220
Glu Glu Asp Glu Ala Ile Trp Lys Phe Asn Ala Glu Pro Gly Gly Gly
225 230 235 240
Ser Lys Gly Gln Val Val Asp Arg Ala Thr Gly Asp His Leu Thr Ala
245 250255
Asp Ile Glu Gly Leu Thr Ile Tyr Tyr Ala Pro Asn Gly Lys Gly Tyr
260 265 270
Leu Met Ala Ser Ser Gln Gly Asn Asn Ser Tyr Ala Met Tyr Glu Arg
275 280 285
Gln Gly Lys Asn Arg Tyr Val Ala Asn Phe Glu Ile Thr Asp Gly Glu
290 295 300
Lys Ile Asp Gly Thr Ser Asp Thr Asp Gly Ile Asp Val Leu Gly Phe
305 310 315 320
Gly Leu Gly Pro Lys Tyr Pro Tyr Gly Ile Phe Val Ala Gln Asp Gly
325 330 335
Glu Asn Ile Asp Asn Gly Gln Ala Val Asn Gln Asn Phe Lys Ile Val
340 345 350
Ser Trp Glu Gln Ile Ala Gln His Leu Gly Glu Met Pro Asp Leu His
355 360 365
Lys Gln Val Asn Pro Arg Lys Leu Lys Asp Arg Ser Asp Gly
370 375 380
Claims (9)
1. A phytase mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID No. 4.
2. A gene encoding the phytase mutant according to claim 1.
3. The coding gene of claim 2, which is represented by SEQ ID No.3 of the sequence Listing.
4. Use of a phytase mutant according to claim 1 or a gene according to claim 2 in the fields of food and feed.
5. A recombinant vector comprising the gene of claim 3.
6. A recombinant strain comprising the gene of claim 3.
7. The recombinant vector according to claim 5, wherein the gene represented by SEQ ID No.3 is inserted into the expression vector pPIC 9.
8. The recombinant strain of claim 6, wherein the gene represented by SEQ ID No.3 is expressed in host cell Pichia pastoris GS 115.
9. The method for producing the phytase mutant according to claim 1, which comprises the following steps:
using a strain containing a gene coding for the phytase mutant according to claim 3 as a production strain; the strain takes pPIC9 as an expression vector and pichia pastoris GS115 as a host cell;
inoculating the strain in BMGY culture medium, culturing at 30 deg.C and 220r/min until OD600 is 10, centrifuging, collecting thallus, re-suspending the thallus in BMMY culture medium, and continuously culturing at 30 deg.C and 220r/min for 48 hr to obtain phytase;
the BMGY culture medium comprises 1% of yeast extract, 2% of peptone, 0.1mol/L phosphate buffer solution with pH of 6.0, 1.34% of YNB, 4 × 10-5% biotin, 1% glycerol;
the BMMY culture medium comprises 1% of yeast extract, 2% of peptone, 0.1mol/L phosphate buffer solution with pH of 6.0, 1.34% of YNB, 4 × 10-5% biotin, 0.5% methanol.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710913105.XA CN107488642B (en) | 2017-09-30 | 2017-09-30 | Phytase mutant and application thereof |
| CN202010595893.4A CN111593063B (en) | 2017-09-30 | 2017-09-30 | Application of recombinant vector or recombinant strain containing phytase mutant |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710913105.XA CN107488642B (en) | 2017-09-30 | 2017-09-30 | Phytase mutant and application thereof |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010595893.4A Division CN111593063B (en) | 2017-09-30 | 2017-09-30 | Application of recombinant vector or recombinant strain containing phytase mutant |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107488642A CN107488642A (en) | 2017-12-19 |
| CN107488642B true CN107488642B (en) | 2020-08-18 |
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| CN110484521B (en) * | 2018-05-14 | 2022-09-23 | 广东溢多利生物科技股份有限公司 | High-thermostability phytase mutant KspHIPHY 9, and gene and application thereof |
| CN110724676B (en) * | 2019-10-21 | 2021-09-07 | 广东溢多利生物科技股份有限公司 | Phytase mutant and carrier and application thereof |
| CN113862237B (en) * | 2021-12-02 | 2022-03-25 | 中国农业科学院北京畜牧兽医研究所 | Method for improving heat stability of phytase, mutant, gene and application |
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| TWI326708B (en) * | 2001-11-21 | 2010-07-01 | Univ Hong Kong | Recombinant bacillus phytases and uses thereof |
| MY139056A (en) * | 2001-12-28 | 2009-08-28 | Ab Enzymes Gmbh | Microbially-expressed thermotolerant phytase for animal feed |
| CN101935617A (en) * | 2010-07-13 | 2011-01-05 | 湖北大学 | Heat-resisting phytase Pichia pastoris engineering bacterial strain and production method of heat-resisting phytase |
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