CN108251439B - Artificially-modified trypsin-resistant phytase as well as preparation method and application thereof - Google Patents
Artificially-modified trypsin-resistant phytase as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an artificially modified trypsin-resistant phytase and a preparation method and application thereof, wherein a potential trypsin enzyme cutting site is found out by analyzing the sequence and structure of a reported phytase appA gene, the trypsin enzyme cutting site of the gene is replaced by utilizing a site-directed mutagenesis technology to obtain a mutant gene appA-M6, the mutant gene is constructed on a eukaryotic expression vector pPIC9 and is transformed into Pichia pastoris GS115, and the trypsin-resistant phytase appA-M6 is efficiently expressed. The optimum pH value of the appA-M6 is 4.5, the optimum reaction temperature is 60 ℃, and the appA-M is not obviously different from the wild appA. The improved phytase appA-M6 has improved tolerance to trypsin, after being treated by trypsin, the wild phytase appA is basically inactivated, the residual enzyme activity of appA-M6 can reach 30%, and the tolerance of trypsin is improved by 8.7 times. The improved phytase can be applied to feed additives.
Description
Technical Field
The invention relates to the field of bioengineering, and particularly belongs to gene design, site-directed mutagenesis modification and expression purification and identification of recombinant phytase of artificially modified trypsin-resistant phytase, and application of the gene design, site-directed mutagenesis modification and recombinant phytase in feed additives.
Background
Phytic acid (IP)6Myinonothiakis dihydrogen phosphate) widely exists in grains, oil and beans, has a chelating effect on inorganic ions, proteins and the like, is one of main anti-nutritional factors existing in plant feed, and influences the absorption of nutrients by animals. While plant feeds contain significant amounts of phosphorus, more than 50% of which is present in the form of phytate phosphorus, monogastric animals cannot utilize phytate phosphorus effectively due to the lack of enzymes necessary for the decomposition of phytic acid. The phytase isThe generic name of enzymes catalyzing the hydrolysis of phytic acid and its salts into inositol and phosphate belongs to the field of phosphoric monoester hydrolase. The phytase has a special spatial structure, can sequentially separate phosphorus in phytic acid molecules, degrades phytic acid (salt) into inositol and inorganic phosphorus, and releases other nutrients combined with the phytic acid (salt). The phytase is added into the ration of monogastric animals such as pigs, poultry and the like, so that the anti-nutritional effect of phytate can be degraded, the utilization rate of phosphorus in the feed is improved, the discharge amount of phosphorus in the excrement of the animals and the poultry is reduced, and the environmental pollution is reduced. Therefore, the phytase added into the feed has important economic and ecological significance for improving the production benefit of livestock and poultry industry and reducing the pollution of phytate phosphorus to the environment.
The E.coli phytase appA, belonging to the family of histidine acid phosphatases, is one of the phytases known to date to have the highest capacity to decompose phytic acid, having a pH range which is more suitable for functioning in the gastrointestinal tract of animals than the Aspergillus phytases currently produced commercially. However, the presence of a large number of proteases including trypsin in the digestive system of monogastric animals such as pigs and birds, and the sensitivity to trypsin has limited the widespread use of phytase in the industrial production of feed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide an improved trypsin-resistant phytase gene and a phytase coded by the same, wherein the improved phytase has better tolerance to trypsin, and the improved phytase can be applied to feed additives.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
an artificially modified trypsin-resistant phytase gene appA-M6 is obtained by site-directed mutagenesis of a wild-type gene, wherein bases at positions +220, +222, +223, +540, +541, +542, +547, +548 and +1089 are changed; the nucleotide sequence is shown as SEQ ID No. 1.
An artificially modified trypsin-resistant phytase appA-M6, wherein amino acids at positions +74, +75, +180, +181, +183, +363 are mutated from corresponding +74K, +75K, +180K, +181R, +183K, +363K to +74D, +75Q, +180N, +181N, +183S, + 363N; the phytase amino acid sequence is shown as SEQ ID No. 2.
A method for preparing artificially-modified trypsin-resistant phytase comprises the following steps:
the improved phytase gene is constructed on a eukaryotic expression vector pPIC9 and is transformed into Pichia pastoris GS115, and phytase expression and purification are carried out by screening and identifying strains which highly express phytase.
Compared with the prior art, the invention has the beneficial effects that:
the results of the enzymological property comparison of the purified phytase show that the tolerance of the invention to trypsin is obviously improved, after the treatment of the trypsin, the wild phytase appA is basically inactivated, the residual enzyme activity of appA-M6 can still reach 30%, the tolerance of the trypsin enzyme is improved by 8.7 times, and the improved phytase can be applied to feed additives and has good application prospect.
Drawings
FIG. 1: the result of the appA-M6 mutation (bases after mutation are underlined)
FIG. 2 is a drawing: eukaryotic expression vector pPIC9-appA-M6 enzyme digestion identification result
(Lane M: nucleic acid Marker Trans 2K Plus II; Lane 1: eukaryotic expression vector pPIC9-appA-M6 with EcoRI and NotI double cleavage products)
FIG. 3: transformation of eukaryotic expression vector pPIC9-appA-M6 and screening of positive expression strain
(Lane M: Low molecular weight Standard protein; Lane 1-7: Positive Yeast expression Strain No. 1-7)
FIG. 4 is a drawing: SDS-PAGE results of purified Phytase
(Lane M: Low molecular weight Standard protein; Lane 1: purified wild-type phytase appA; Lane 2: purified phytase appA-M6)
FIG. 5: the phytase appA and appA-M6 are relatively tolerant to trypsin
FIG. 6: comparison of enzymatic Properties of Phytase appA and appA-M6
(A: optimum pH; B: optimum temperature; C: heat resistance; D: heat stability)
Detailed Description
Example 1 obtaining of the Phytase Gene appA
According to the appA sequence (DQ513832) reported in GenBank, a biotechnology company is entrusted to synthesize the phytase gene appA, and the phytase gene appA is cloned to a T-Easy vector to obtain a recombinant plasmid T-Easy-appA.
Example 2 Trypsin cleavage site analysis and mutation
The trypsin belongs to a serine protease family, can specifically recognize arginine or lysine residues in a polypeptide chain, and arginine or lysine residues in a loop on the surface of the protein are easy to recognize. The invention selects six trypsin enzyme cutting sites 74K, 75K, 180K, 181R, 183K and 363K positioned on the phytase loop ring as candidate mutation sites. These six sites were mutated to 74D, 75Q, 180N, 181N, 183S and 363N, respectively, by homology modeling and hydrogen bond comparison analysis.
And (3) replacing the trypsin enzyme cutting site by utilizing the combination of the site-directed mutagenesis technology so as to improve the trypsin tolerance of the phytase. Site-directed mutagenesis primers were synthesized and underlined to mark the site of the mutation.
Primer K74D/K75Q:
FK74D/K75Q
5’-TGCCGACGGATTGTTGCCCGACCAGGGTTGTCCACAATCTG-3’(SEQ ID No.3)
RK74D/K75Q
5’-CAGATTGTGGACAACCCTGGTCGGGCAACAATCCGTCGGCA-3’(SEQ ID No.4)
K180N/R181N/K183S primer:
FK180N/R181N/K183S
5’-CCCACAATCCAACTTGTGCCTTAACAATGAGTCGCAAGACGAATCCTG-3’(SEQ ID No.5)
RK180N/R181N/K183S
5’-CAGGATTCGTCTTGCGACTCATTGTTAAGGCACAAGTTGGATTGTGGG-3’(SEQ ID No.6)
primer K363N:
F363N
5’-AGCAGATGAGAGACAACACTCCAC-3’(SEQ ID No.7)
R363N
5’-CAAAGACAGTGGAGTGTTGTCTCTC-3’(SEQ ID No.8)
three pairs of mutation primers (SEQ ID No.3-8) are used, the recombinant plasmid T-Easy-appA with the synthetic phytase gene appA is used as a template, and the mutant recombinant plasmid T-Easy-appA-M6 is finally obtained through PCR additive mutation. The nucleotide sequence of the mutated appA-M6 is shown in SEQ ID No.1, the amino acid sequence is shown in SEQ ID No.2, and the mutation result is shown in FIG. 1.
EXAMPLE 3 construction of eukaryotic expression vector pPIC9-appA-M6
The recombinant plasmids T-Easy-appA-M6 and pPIC9 are subjected to double enzyme digestion by EcoRI and NotI respectively, so that the target gene appA-M6 is directionally inserted between EcoRI sites and NotI sites of the eukaryotic expression vector pPIC9 in a correct reading frame, and the recombinant eukaryotic expression vector pPIC9-appA-M6 is obtained. The restriction enzyme of pPIC9-appA-M6 is identified as shown in figure 2, the restriction enzyme fragments of pPIC9-appA-M6 are 8000bp and 1233bp respectively, and the construction is proved to be correct by sequencing.
Example 4 transformation of Pichia pastoris
The recombinant eukaryotic expression vector pPIC9-appA-M6 is used for transforming Pichia pastoris GS115 after being extracted in a large amount and linearized by a sufficient amount of BglII. And (3) converting the pichia pastoris by a lithium chloride conversion method. The specific operation is as follows:
a competent cell preparation
Pichia pastoris was cultured in 50ml YPD to OD600 ═ 0.8-1.0. The cells were harvested, washed with 25m L sterile water, centrifuged at 4000rpm for 10min at room temperature, and then the yeast cells were resuspended in 1.5mL centrifuge tubes using 1mL100mM LiCl. Cells were pelleted at maximum speed for 15S, suspended in 400. mu.L of 100mM LiCl and aliquoted at 50. mu.L/tube for future use.
B transformation
Yeast competent cells were centrifuged at maximum speed for 15S to remove LiCl, and then 240. mu.L of 50% PEG, 36. mu.L of 1M LiCl, 25. mu.L of 2mg/mL single stranded DNA were added in order to linearize the plasmid DNA and vortexed vigorously until well mixed. The transformed yeast was incubated at 30 ℃ for 30min and then heat-shocked in a 42 ℃ water bath for 25 min. After centrifugation at 8000rpm, 100. mu.L of sterilized water was applied to the MD plate and the yeast cells were incubated at 30 ℃ for 2-4 days.
EXAMPLE 5 screening of Positive expression strains
Transformants were picked from the MD plates and numbered, shake-cultured in 2mL BMGY liquid medium, and enrichment-cultured at 30 ℃ and 220rpm for 48 hours. The culture solution is centrifuged at 8000rpm for 10 minutes, the supernatant is discarded, fresh 2mL BMMY culture medium is added for shake culture, and induction is carried out at 30 ℃ and 220rpm for 48 hours. And (3) screening out a positive high-expression strain as an expression strain by performing activity detection and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) protein gel detection on the supernatant obtained in the step (2). A total of 7 positive expression strains were selected, as shown in FIG. 3.
EXAMPLE 6 expression and purification of Phytase
Inducible expression of A Phytase
The recombinant yeast was inoculated into a 1L flask of 500mL BMGY, and cultured for 48 hours at 220rpm and 30 ℃. Centrifuging the culture solution at 8000rpm for 10min, discarding supernatant, replacing fresh 500mL BMMY induction culture medium, shake culturing, and supplementing methanol every 12h to ensure methanol supply.
Purification of recombinant Phytase B
The induction medium was centrifuged at 8000rpm at 4 ℃ for 15min, the supernatant was precipitated with 80% ammonium sulfate, and after centrifugation at 12000rpm at 4 ℃ for 15min, the precipitate was collected, dissolved in 30mL of sodium acetate buffer (40mM, pH 4.5) and dialyzed. The enzyme solution after dialysis was subjected to cation exchange chromatography (SP Sepharose)TMFast Flow) and desalting (Hiprep 26/10desalting), and the SDS-PAGE detection result of the phytase is shown in FIG. 4. Analysis of the SDS-PAGE results showed that: the relative molecular weight of wild-type phytase appA is about 53kDa, while appA-M6 is about 53-55 kDa. The phytase appA and appA-M6 expressed by pichia have glycosylation modification, and the difference of the expressed protein molecular weight is caused by the difference of glycosylation degree.
Example 7 Trypsin tolerance assay
The anti-trypsin ability of appA-M6 was enhanced relative to appA by treating phytase appA and appA-M6 with trypsin at different ratios, respectively (FIG. 5). When the mixing ratio of the trypsin/protein is 1.5, the wild phytase appA is basically inactivated, the residual enzyme activity of the appA-M6 can reach 30%, the tolerance of the trypsin is improved by 8.7 times, and when the mixing ratio of the trypsin/protein is 2.1, the residual enzyme activity of the appA-M6 can reach 20%.
Example 8 determination of enzymatic Properties
The purified phytase was enzymatically reacted under different pH and temperature conditions to determine the optimum pH and temperature, and the results are shown in FIGS. 6A-6B. The phytase is treated at different temperatures (40, 50, 60, 70, 80, 90 ℃) for 15min, the enzyme activity is determined, and the heat-resistant range of the enzyme is determined, and the result is shown in figure 6C. The phytase is treated in constant temperature water bath at 75 deg.C for 5-30min, the residual enzyme activity is determined under standard conditions, and the thermal stability of the enzyme is compared, the result is shown in FIG. 6D. The results of the enzyme property measurement show that the optimum pH value of the appA-M6 is 4.5, the optimum reaction temperature is 60 ℃, the method has no obvious difference from the wild appA, and the heat resistance and the heat stability are good.
Sequence listing
<110> university of Shanxi
<120> artificially-modified trypsin-resistant phytase, and preparation method and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1233
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 1
cagagtgagc ctgagttgaa actggaatcc gttgtcatcg tctctagaca tggtgttaga 60
gcaccaacca aggccaccca acttatgcaa gatgtcaccc cagacgcttg gccaacctgg 120
ccagtcaagc tgggttggtt gacacctaga ggtggtgagc tcattgctta cttgggtcac 180
taccaaagac agcgtcttgt tgccgacgga ttgttggccg accagggttg tccacaatct 240
ggtcaagtag ctattattgc tgacgtcgac gaaagaaccc gtaagacagg tgaagccttc 300
gccgccggtc ttgctcctga ctgtgccatt accgttcaca cccaagctga cacttcttct 360
ccagatccat tgttcaaccc tttgaagact ggtgtttgcc aattggacaa cgctaacgtt 420
actgacgcta tcttgtccag agctggagga tccattgctg acttcaccgg tcacagacag 480
actgccttca gagagttgga aagagttctt aacttcccac aatccaactt gtgccttaac 540
aatgagtcgc aagacgaatc ctgttccttg actcaagcat taccatctga gttgaaggtc 600
tccgccgaca acgtctcttt gaccggtgct gtcagcttgg cttccatgtt gactgaaatc 660
tttcttctgc aacaagctca aggtatgcct gagccaggtt ggggtagaat caccgactct 720
caccaatgga acaccttgtt gtccttgcac aacgctcaat tctacttgct gcagagaact 780
ccagaggttg ctagatccag agccacccca ttgttggact tgatcaagac tgctttgact 840
cctcacccac ctcaaaagca agcctacggt gttaccttgc ccacttctgt cttgttcatt 900
gccggtcacg atactaactt ggcaaatctc ggcggtgctt tggagttgaa ctggactctt 960
cctggtcaac ctgataacac tccaccaggt ggtgagctcg ttttcgaaag atggcgtaga 1020
ctatctgata actctcaatg gattcaggtt tcgttggtct tccaaacttt gcagcagatg 1080
agagacaaca ctccactgtc tttgaacacg cctccaggag aagtcaaatt gaccttggct 1140
ggatgtgaag agagaaatgc tcagggtatg tgttccttgg ctggtttcac tcaaatcgtt 1200
aacgaagcta gaatcccagc ttgttccttg tag 1233
<210> 2
<211> 410
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 2
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His Gly Val Arg Ala Pro Thr Lys Ala Thr Gln Leu Met Gln Asp Val
20 25 30
Thr Pro Asp Ala Trp Pro Thr Trp Pro Val Lys Leu Gly Trp Leu Thr
35 40 45
Pro Arg Gly Gly Glu Leu Ile Ala Tyr Leu Gly His Tyr Gln Arg Gln
50 55 60
Arg Leu Val Ala Asp Gly Leu Leu Ala Asp Gln Gly Cys Pro Gln Ser
65 70 75 80
Gly Gln Val Ala Ile Ile Ala Asp Val Asp Glu Arg Thr Arg Lys Thr
85 90 95
Gly Glu Ala Phe Ala Ala Gly Leu Ala Pro Asp Cys Ala Ile Thr Val
100 105 110
His Thr Gln Ala Asp Thr Ser Ser Pro Asp Pro Leu Phe Asn Pro Leu
115 120 125
Lys Thr Gly Val Cys Gln Leu Asp Asn Ala Asn Val Thr Asp Ala Ile
130 135 140
Leu Ser Arg Ala Gly Gly Ser Ile Ala Asp Phe Thr Gly His Arg Gln
145 150 155 160
Thr Ala Phe Arg Glu Leu Glu Arg Val Leu Asn Phe Pro Gln Ser Asn
165 170 175
Leu Cys Leu Asn Asn Glu Ser Gln Asp Glu Ser Cys Ser Leu Thr Gln
180 185 190
Ala Leu Pro Ser Glu Leu Lys Val Ser Ala Asp Asn Val Ser Leu Thr
195 200 205
Gly Ala Val Ser Leu Ala Ser Met Leu Thr Glu Ile Phe Leu Leu Gln
210 215 220
Gln Ala Gln Gly Met Pro Glu Pro Gly Trp Gly Arg Ile Thr Asp Ser
225 230 235 240
His Gln Trp Asn Thr Leu Leu Ser Leu His Asn Ala Gln Phe Tyr Leu
245 250 255
Leu Gln Arg Thr Pro Glu Val Ala Arg Ser Arg Ala Thr Pro Leu Leu
260 265 270
Asp Leu Ile Lys Thr Ala Leu Thr Pro His Pro Pro Gln Lys Gln Ala
275 280 285
Tyr Gly Val Thr Leu Pro Thr Ser Val Leu Phe Ile Ala Gly His Asp
290 295 300
Thr Asn Leu Ala Asn Leu Gly Gly Ala Leu Glu Leu Asn Trp Thr Leu
305 310 315 320
Pro Gly Gln Pro Asp Asn Thr Pro Pro Gly Gly Glu Leu Val Phe Glu
325 330 335
Arg Trp Arg Arg Leu Ser Asp Asn Ser Gln Trp Ile Gln Val Ser Leu
340 345 350
Val Phe Gln Thr Leu Gln Gln Met Arg Asp Asn Thr Pro Leu Ser Leu
355 360 365
Asn Thr Pro Pro Gly Glu Val Lys Leu Thr Leu Ala Gly Cys Glu Glu
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Arg Asn Ala Gln Gly Met Cys Ser Leu Ala Gly Phe Thr Gln Ile Val
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Asn Glu Ala Arg Ile Pro Ala Cys Ser Leu
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<210> 3
<211> 41
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 3
tgccgacgga ttgttgcccg accagggttg tccacaatct g 41
<210> 4
<211> 41
<212> DNA
<213> Escherichia coli (Escherichia coli)
<400> 4
cagattgtgg acaaccctgg tcgggcaaca atccgtcggc a 41
<210> 5
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<213> Escherichia coli (Escherichia coli)
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<210> 6
<211> 48
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<213> Escherichia coli (Escherichia coli)
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caggattcgt cttgcgactc attgttaagg cacaagttgg attgtggg 48
<210> 7
<211> 24
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<213> Escherichia coli (Escherichia coli)
<400> 7
agcagatgag agacaacact ccac 24
<210> 8
<211> 25
<212> DNA
<213> Escherichia coli (Escherichia coli)
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caaagacagt ggagtgttgt ctctc 25
Claims (3)
1. An artificially modified trypsin-resistant phytase gene, the nucleotide sequence of which is SEQ ID No. 1.
2. The gene coding phytase of claim 1, whose amino acid sequence is SEQ ID No. 2.
3. A preparation method of artificially modified trypsin-resistant phytase is characterized by comprising the following steps: the phytase gene of claim 1 is constructed on a eukaryotic expression vector pPIC9, and is transformed into Pichia pastoris GS115, and phytase expression and purification are carried out by screening and identifying strains which highly express phytase.
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| WO2020063268A1 (en) * | 2018-09-28 | 2020-04-02 | 青岛蔚蓝生物集团有限公司 | Phytase mutant |
| WO2020063267A1 (en) * | 2018-09-28 | 2020-04-02 | 青岛蔚蓝生物集团有限公司 | Phytase mutant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101260390A (en) * | 2007-03-06 | 2008-09-10 | 四川禾本生物工程有限公司 | High specific activity phytase gene and application thereof |
| 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 |
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| CA2231948C (en) * | 1997-03-25 | 2010-05-18 | F. Hoffmann-La Roche Ag | Modified phytases |
| US6514495B1 (en) * | 1998-03-23 | 2003-02-04 | Novozymes A/S | Phytase varinats |
| BR0311287A (en) * | 2002-05-30 | 2005-03-29 | Basf Ag | Polypeptide, polynucleotide, vector, host cell, process for producing a polypeptide, and, composition |
| CN101792749B (en) * | 2002-08-12 | 2012-09-05 | 金克克国际有限公司 | Mutant colibacillus appa phytase enzymes |
| EP1604008B1 (en) * | 2002-09-13 | 2010-12-22 | Cornell Research Foundation, Inc. | Using mutations to improve aspergillus phytases |
| DE102004050410A1 (en) * | 2004-10-15 | 2006-06-08 | Ab Enzymes Gmbh | Polypeptide with phytase activity and this coding nucleotide sequence |
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| WO2016078168A1 (en) * | 2014-11-21 | 2016-05-26 | 青岛蔚蓝生物集团有限公司 | Phytase mutants |
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| 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 |
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| CN106591256B (en) * | 2017-01-18 | 2022-12-30 | 中国农业科学院饲料研究所 | Phytase YeAPPA mutant with improved pepsin resistance and acid resistance as well as coding gene and application thereof |
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| CN107488642B (en) * | 2017-09-30 | 2020-08-18 | 山东隆科特酶制剂有限公司 | Phytase mutant and application thereof |
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