CN106591256A - Pepsin resistance and acid resistance improved phytase YeAPPA mutant, and coding gene and application thereof - Google Patents
Pepsin resistance and acid resistance improved phytase YeAPPA mutant, and coding gene and application thereof Download PDFInfo
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- CN106591256A CN106591256A CN201710039951.3A CN201710039951A CN106591256A CN 106591256 A CN106591256 A CN 106591256A CN 201710039951 A CN201710039951 A CN 201710039951A CN 106591256 A CN106591256 A CN 106591256A
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- C12N15/09—Recombinant DNA-technology
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Abstract
The invention relates to the field of gene engineering, and concretely relates to a pepsin resistance and acid resistance improved phytase YeAPPA mutant, and a coding gene and an application thereof. Leucine in the 99th position of phytase with the amino acid sequence represented by SEQ ID NO.1 mutates to alanine, leucine in the 162th position of the phytase mutates to glycine, and/or glutamic acid in the 230th position of the phytase mutates to glycine. Compared with wild phytase YeAPPA, the phytase YeAPPA mutant has the advantages of obviously improved pepsin resistance and acid resistance, and great application potential in animal production.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to the phytase YeAPPA that pepsin resistance and acid resistance are improved
Mutant and its encoding gene and application.
Background technology
Phytic acid is a kind of antinutritional factor, can often result in the nutrition of animal with reference to several mineral materials and albumen etc. in vivo
It is bad.Phytase is the catalyst of a class Hydrolysis of Phytic Acid, can progressively discharge phosphoric acid group and form rudimentary inositol monophosphate derivant.It is single
Stomach animal can not utilize phytate phosphorus due to a lack of endogenous phytase, thus need to add the anti-battalion that source of phytase eliminates phytic acid in feedstuff
Effect is supported, the utilization rate of feedstuff phosphorus is improved, is conducive to saving feed cost and environmental protection.
Carried out extensive investigations to the phytase in various sources at present, find phytase be prevalent in plant,
In funguses, antibacterial and yeast, wherein microbial phytase activity is higher, becomes the focus of research and development.According to structure and urging
Change mechanism, phytase is divided into histidine acid phosphatase phytase, β-propeller phytase, cysteine phosphoric acid phytase and purple
Four kinds of color acid acid phosphatase phytase.Feeding phytase major function site is the intestines and stomach of animal.The high protein enzyme of the intestines and stomach and acid
Property environment can usually limit giving full play to for some phytase functions.Therefore the phytic acid of enzymatic property improvement is obtained by rite-directed mutagenesises
Enzyme is the major issue that feed enzyme industry needs to solve.
The content of the invention
It is an object of the invention to provide the phytic acid enzyme mutant that a kind of pepsin resistance and acid resistance are improved.
Another object of the present invention is to provide the phytic acid enzyme mutant of the above-mentioned pepsin resistance of coding and acid resistance improvement
Gene.
It is a further object of the present invention to provide the phytase improved comprising above-mentioned coding pepsin resistance and acid resistance is dashed forward
The recombinant vector of the gene of variant.
It is a further object of the present invention to provide the phytase improved comprising above-mentioned coding pepsin resistance and acid resistance is dashed forward
The recombinant bacterial strain of the gene of variant.
It is a further object of the present invention to provide above-mentioned pepsin resistance and acid resistance improvement phytic acid enzyme mutant should
With.
The present invention is to yersinia enterocolitica (Yersinia enterocolitica) source phytase YeAPPA
Gene carries out rite-directed mutagenesises, and the maturation protein of phytase YeAPPA has the aminoacid sequence as shown in SEQ ID NO.1, should
Maturation protein is by nucleotide sequence coded as shown in SEQ ID NO.2.
SEQ ID NO.1
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLWPVKAGYLTP
RRAELVTLMGGFYGDYFRSQGLLSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKVDPLFHT
VEAGVCKLDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATFTANEIKVNEEG
TKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
SEQ ID NO.2
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcaggtgctgcccc
gattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgctccccgacaa
aacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggctatttaacaccg
cgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaagggttgttgtctgcggg
gtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccggacaggcattcttgg
atgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacccgctatttcatacc
gtcgaagcgggggtctgtaaactggactcagcgaaaactcatcaggctgttgaggagcgattgggcgggccattgag
tgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgcagcatcgccttattgca
agtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaattaaggtaaacgaagaaggt
actaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcctgttacaagactcacaagc
tatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattgtcgttgcgcaatgcgcaat
ttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgcaacaaattgatacggcatta
gtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcggcgggcatga
caccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacagcaacctgataataccccgcctg
gtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatgtcgccgtgaaaatgttctatcaa
acgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagctaaaattgttccaattaccattga
aggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaaaagaaagttgcccaagtgatcgagc
cagcctgccatatttaa
The mutant of the phytase YeAPPA that pepsin resistance of the invention and acid resistance are improved, aminoacid sequence
The leucine of the 99th of the phytase as shown in SEQ ID NO.1 sports alanine, further, the leucine of the 162nd
Glycine is sported, further, the glutamic acid mutation of the 230th is glycine.
Specific embodiment of the invention, using the method for rite-directed mutagenesises, obtains 3 pepsin resistances and resistance to
The mutant of the phytase YeAPPA that acidity is improved, is respectively designated as L99A, L99A/L162G and L99A/L162G/E230G,
I.e.：L99A sports L-Valine for the 99th leucine；L99A/L162G sports L-Valine and the 162nd for the 99th leucine
Position leucine sports glycine；L99A/L162G/E230G sports L-Valine and the 162nd bright ammonia for the 99th leucine
Acid mutation is glycine and the 230th glutamic acid mutation is glycine.
Therefore the mutant L99A of pepsin resistance of the invention and the phytase YeAPPA of acid resistance improvement, its
In the 99th leucin sport alanine, its aminoacid sequence is as shown in SEQ ID NO.3
SEQ ID NO.3
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLWPVKAGYLTP
RRAELVTLMGGFYGDYFRSQGALSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKVDPLFHT
VEAGVCKLDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATFTANEIKVNEEG
TKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The mutant of the phytase YeAPPA of pepsin resistance improvement of the invention, wherein the 99th leucin
Sport alanine and the 162nd leucine sports glycine, its aminoacid sequence is as shown in SEQ ID NO.4
SEQ ID NO.4
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLWPVKAGYLTP
RRAELVTLMGGFYGDYFRSQGALSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKVDPLFHT
VEAGVCKGDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATFTANEIKVNEEG
TKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The mutant L99A/L162G/E230G of the phytase YeAPPA of pepsin resistance improvement of the invention, its
In the 99th leucin sport alanine, and the 162nd leucine sports glycine, and the 230th glutamic acid mutation
For glycine, its aminoacid sequence is as shown in SEQ ID NO.5
SEQ ID NO.5
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLWPVKAGYLTP
RRAELVTLMGGFYGDYFRSQGALSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKVDPLFHT
VEAGVCKGDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATFTANEIKVNEGG
TKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
Present invention also offers encoding phytase YeAPPA mutant L99A, L99A/ of above-mentioned pepsin resistance improvement
The gene order of L162G and L99A/L162G/E230G, its nucleotide sequence is respectively as shown in SEQ ID NO.6,7 and 8.
SEQ ID NO.6
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcaggtgctgcccc
gattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgctccccgacaa
aacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggctatttaacaccg
cgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaaggggcgttgtctgcggg
gtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccggacaggcattcttgg
atgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacccgctatttcatacc
gtcgaagcgggggtctgtaaactggactcagcgaaaactcatcaggctgttgaggagcgattgggcgggccattgag
tgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgcagcatcgccttattgca
agtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaattaaggtaaacgaagaaggt
actaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcctgttacaagactcacaagc
tatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattgtcgttgcgcaatgcgcaat
ttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgcaacaaattgatacggcatta
gtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcggcgggcatga
caccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacagcaacctgataataccccgcctg
gtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatgtcgccgtgaaaatgttctatcaa
acgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagctaaaattgttccaattaccattga
aggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaaaagaaagttgcccaagtgatcgagc
cagcctgccatatttaa
SEQ ID NO.7
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcaggtgctgcccc
gattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgctccccgacaa
aacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggctatttaacaccg
cgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaaggggcgttgtctgcggg
gtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccggacaggcattcttgg
atgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacccgctatttcatacc
gtcgaagcgggggtctgtaaaggcgactcagcgaaaactcatcaggctgttgaggagcgattgggcgggccattgag
tgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgcagcatcgccttattgca
agtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaattaaggtaaacgaagaaggt
actaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcctgttacaagactcacaagc
tatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattgtcgttgcgcaatgcgcaat
ttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgcaacaaattgatacggcatta
gtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcggcgggcatga
caccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacagcaacctgataataccccgcctg
gtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatgtcgccgtgaaaatgttctatcaa
acgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagctaaaattgttccaattaccattga
aggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaaaagaaagttgcccaagtgatcgagc
cagcctgccatatttaa
SEQ ID NO.8
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcaggtgctgcccc
gattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgctccccgacaa
aacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggctatttaacaccg
cgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaaggggcgttgtctgcggg
gtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccggacaggcattcttgg
atgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacccgctatttcatacc
gtcgaagcgggggtctgtaaaggcgactcagcgaaaactcatcaggctgttgaggagcgattgggcgggccattgag
tgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgcagcatcgccttattgca
agtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaattaaggtaaacgaaggcggt
actaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcctgttacaagactcacaagc
tatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattgtcgttgcgcaatgcgcaat
ttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgcaacaaattgatacggcatta
gtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcggcgggcatga
caccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacagcaacctgataataccccgcctg
gtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatgtcgccgtgaaaatgttctatcaa
acgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagctaaaattgttccaattaccattga
aggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaaaagaaagttgcccaagtgatcgagc
cagcctgccatatttaa
The cDNA molecules of the mutant of the phytase YeAPPA that above-mentioned coding pepsin resistance is improved are suitably taking
To and correct reading frame be inserted between the restriction enzyme site of the carrier so as to nucleotide sequence is exercisable and table
It is connected up to regulating and controlling sequence.Currently preferred carrier is pET-22b (+), and the phytase gene for making transformation is inserted into plasmid
Between EcoRI and NotI restriction enzyme sites on pET-22b (+), the nucleotide sequence is set to be located under T7lac promoteres
Trip is simultaneously regulated and controled by it, obtains the recombinant bacteria expression plasmid of each mutant.Currently preferred Host Strains are BL21 (DE3).
Compared to wild type, mutant L99A, L99A/L162G and L99A/L162G/ of the phytase YeAPPA of the present invention
The pepsin resistance of E230G and acid resistance are significantly improved, and have very big application potential in animal productiong.
Description of the drawings
Fig. 1 is tolerability curves figure of the forward and backward phytase of transformation to variable concentrations pepsin；
Fig. 2 is enzyme activity curve chart of the forward and backward phytase of transformation under different pH；
Fig. 3 is enzyme activity curve chart after the forward and backward phytase of transformation processes different time at 60 DEG C.
Specific embodiment
Experiment material
Prokaryotic expression carrier pET-22b (+) is purchased from Novagen companies.Escherichia coli Trans1-T1 and BL21 (DE3) are thin
Born of the same parents are purchased from Tiangeng, respectively as plasmid amplification and prokaryotic expression Host Strains.DNA purification kits are purchased from TaKaRa.Pfu DNA gather
Synthase, restricted enzyme, T4DNA ligases are purchased from Tiangeng.Sodium phytate and pepsin (p0685) are purchased from Sigma.By Shanghai
Handsome Bioisystech Co., Ltd's synthesizing ribonucleotide primer.
Embodiment 1：The acquisition of mutant gene
The base of the phytase YeAPPA originated with yersinia enterocolitica (Yersinia enterocolitica)
Because sequence (SEQ IDNO.2) is transformed, mutation is introduced by way of Overlap PCR, and it is sequenced, obtained
Mutant gene.The method passes through with the pEASY-T3-YeAPPA recombiant plasmid containing wild phytase gene YeAPPA as template
Two-wheeled PCR reactions introduce mutation, and required mutant gene is connected on carrier pEASY-T3 carriers and Jing DNA sequencings confirm.
Mutation uses eight primers：Ye-F, Ye-R, L99A-F, L99A-R, L162G-F, L162G-R, E230G-F and
E230G-R。
Primer sequence is as follows：
Ye-F：5’-cgcgaattcgccccgattgctacaccgcc-3’
Ye-R：5’-gatgcggccgcttaaatatggcaggctggctcga-3’
L99A-F：5’-tccgcagccaaggggcgttgtctgcggggtg-3’
L99A-F：5’-caccccgcagacaacgccccttggctgcgga-3’
L162G-F：5’-cgggggtctgtaaaggcgactcagcgaaaac-3’
L162G-R：5’-gttttcgctgagtcgcctttacagacccccg-3’
E230G-F：5’-ttaaggtaaacgaaggcggtactaaagtttc-3’
E230G-R：5’-gaaactttagtaccgccttcgtttaccttaa-3’
Italic represents restriction enzyme site EcoR I and Not I in primer, and the nucleotides sequence of underscore labelling is classified as prominent
Become site.
Embodiment 2：Expression and purification of the phytase before and after transformation in escherichia coli
The coding region sequence insertion of mutant L99A, L99A/L162G and L99A/L162G/E230G of phytase YeAPPA
To between EcoRI the and NotI restriction enzyme sites of prokaryotic expression carrier pET-22b (+), receive in e. coli bl21 (DE3) cell
The regulating and expressing of T7 lac promoteres.Derivant is IPTG (isopropyl-β-D- galactoside).In 24 under 1mM IPTG inductions
DEG C and 220rpm shaking table in cultivate 5h carry out great expression.Crude enzyme liquid Jing nickel-nitrilotriacetic acid(NTA) (Ni-NTA) post and diethyl
Base amino-ethyl (DEAE) post carries out chromatography purification.Jing 10%SDS-PAGE electrophoresis detection, mutant enzyme divides with the surface of wild enzyme
Son amount is consistent, for about 46kDa.
Embodiment 3：The pepsin resistance of mutation phytase
The pepsin resistance of mutation phytase is according to remaining phytase activity after the h of variable concentrations pepsin 2
It is determined with phytase protein.It is 1/1000,1/500,1/200,1/100,1/ with the mass ratio of pepsin and phytase
40 and 1/20 pepsin concentration processes mutation phytase.
Remaining phytase activity after pepsin is determined using ferrous sulfate molybdenum blue method.By 50 μ L debita spissitudo enzymes
Liquid is added in 950 μ L 1.5mmol/L sodium phytate substrates (with the NaAc-HAc buffers of pH 4.5 and 0.25M), 37
30min is reacted in DEG C water-bath, the trichloroacetic acid terminating reactions of 1mL 10% are added, (1% 4 hydration of 2mL nitrite ions is eventually adding
Ammonium molybdate, 3.2% concentrated sulphuric acid, 7.32 ferrous sulfate) developed the color.Mensuration absorbance value weighs the nothing of release under 700 nanometers
The phosphatic amount of machine.One phytase activity unit is defined as under condition determination, 1 micromolar phosphate institute of release per minute
The enzyme amount for needing.As a result (Fig. 1) shows that when processing 2h with 1/1000 pepsin concn, wild enzyme YeAPPA loses completely work
Property, being mutated phytase L99A can keep 24% enzyme activity, mutation phytase L99A/L162G and L99A/L162G/E230G difference
Keep 72% and 99% enzyme activity；When pepsin concn increases to 1/20, wild enzyme YeAPPA and mutation phytase L99A
Lose activity, and being mutated phytase L99A/L162G and L99A/L162G/E230G can still keep 26% and 49% enzyme
It is living.Therefore the wilder enzymes of mutation phytase L99A, L99A/L162G and L99A/L162G/E230G show higher pepsin
Resistance.
The residual protein Jing PAGE of phytase are separated after variable concentrations pepsin 2h, and are commented with Image J softwares
The gray value of valency its protein band.The pepsin hydrolysiss constant of phytase is calculated according to the residual protein of mutation phytase.Such as
Shown in table 1, mutation phytase has stronger pepsin resistance, under pepsin, is mutated phytase YkAPPA-
The percent hydrolysiss of the wilder enzyme of L99A, YkAPPA-L99A/L162G and YkAPPA-L99A/L162G/E230G are less, and it partly declines
Phase is bigger.
Percent hydrolysiss and half-life of the phytase before and after the transformation of table 1 under pepsin process
Embodiment 4：The acid resistance of mutation phytase
Phytase before and after transformation carries out enzymatic reaction 30min at pH1-12 and 37 DEG C, determines optimum pH.Enzyme liquid is in pH
The lower 37 DEG C of process 2h of 1h or pH1-4 are processed at 1-9 and 37 DEG C, the active patterns of enzyme under difference pH are studied.Buffer used is：
0.1mol/L glycine-HCI buffer, pH1-3；0.1mol/L Acetate-acetate buffer solutions, pH3-6；0.1mol/L
Tris- hydrochloride buffers, pH6-8；0.1mol/L Glycine-NaOH buffer, pH8-12.The optimum pH of mutant enzyme is similar to
It is pH 5.0 in wild type.Mutant enzyme L99A, L99A/L162G and L99A/L162G/E230G process under pH 1-2.5 1h compared with
Wild type has more preferable absolute acid stability；1h, mutant enzyme L99A, L99A/L162G and L99A/L162G/ are processed under pH2.5
E230G keeps the enzyme activity of 50-76%, and wild enzyme only keeps 28% enzyme activity；When pH value is down to 1.0, mutant enzyme L99A,
L99A/L162G and L99A/L162G/E230G can keep respectively 11.3%, 21.8% and 38.8% enzyme activity, and wild type is then
Lose activity (Fig. 2).
Mutant enzyme processes 30min with wild enzyme at optimum pH and 30-80 DEG C, determines optimum temperature.Thermal stability determination
It is respectively to process enzyme liquid after different time at 60 DEG C to be measured.Mutation phytase L99A, L99A/L162G and L99A/
The optimum temperature of L162G/E230G is consistent with mutant enzyme, is 45 DEG C.Mutant enzyme L99A/L162G/E230G is processed at 60 DEG C
The wilder enzymes of 30min and mutation phytase L99A and L99A/L162G have more preferable heat stability, can keep 22% enzyme activity, and wild
Raw enzyme and mutation phytase L99A and L99A/L162G then lose activity (Fig. 3).Therefore, the 230th of phytase YeAPPA
Glutamic acid mutation improves the heat stability of phytase for glycine, but its 99th leucine sports alanine or
The leucine of 162 sports glycine does not affect its heat stability.
<110>Institute of Feeds,China Academy of Agriculture Sciences
<120>Phytase YeAPPA mutants and its encoding gene and application that pepsin resistance and acid resistance are improved
<160>8
<210> 1
<211> 441
<212> PRT
<213>Yersinia enterocolitica
<400> 1
MSVAKRNLHL SALTLIMGCF TAGAAPIATP PASYTLERVV ILSRHGVRSP TKQTQLMNDV 60
TPDKWPLWPV KAGYLTPRRA ELVTLMGGFY GDYFRSQGLL SAGCPVDGSV YAQADVDQRT 120
RLTGQAFLDG IAPDCGLKVH YQADLKKVDP LFHTVEAGVC KLDSAKTHQA VEERLGGPLS 180
DLSQRYAKPF AQMGEVLNFA ASPYCKSLQK NGKTCDFATF TANEIKVNEE GTKVSLSGPL 240
ALSSTLGEIF LLQDSQAMPD VAWHRLSGEE NWVSLLSLRN AQFDLMAKTP YIARHKGTPL 300
LQQIDTALVL QRDAQGQTLP LSPQTKLLFL GGHDTNIANI AGMLGANWQL PQQPDNTPPG 360
GGLVFELWQN PDNHQRYVAV KMFYQTMDQL RNAEKLDMKN NPAKIVPITI EGCENEGDNK 420
LCQLETFQKK VAQVIEPACH I 441
<210> 2
<211> 1320
<212> DNA
<213>Yersinia enterocolitica
<400> 1
atgtcagttg caaagagaaa tctgcactta tccgcactca ctttgataat gggctgtttt 60
accgcaggtg ctgccccgat tgctacaccg ccggccagct acacattaga gcgtgtggtt 120
attttgagtc gacatggtgt tcgctccccg acaaaacaaa cccagctaat gaatgatgtc 180
acacctgata aatggcccct gtggccagta aaagcgggct atttaacacc gcgaagggct 240
gagttagtga ctttgatggg gggattttat ggtgattatt tccgcagcca agggttgttg 300
tctgcggggt gtccggtaga tggctccgtt tatgcacagg cagatgttga ccaacgaacc 360
cgcttaaccg gacaggcatt cttggatggg atcgcaccgg attgtggtct gaaagtacat 420
tatcaggctg atttgaagaa agttgacccg ctatttcata ccgtcgaagc gggggtctgt 480
aaactggact cagcgaaaac tcatcaggct gttgaggagc gattgggcgg gccattgagt 540
gatcttagcc agcgctatgc caaacccttt gctcagatgg gcgaagtgct gaattttgca 600
gcatcgcctt attgcaagtc attgcaaaaa aatggaaaaa cctgtgattt tgcaactttt 660
acggcaaatg aaattaaggt aaacgaagaa ggtactaaag tttctctgag tgggccattg 720
gcactatcgt cgacattggg tgaaattttc ctgttacaag actcacaagc tatgccggat 780
gtggcctggc atcggctcag cggtgaagag aactgggttt cgctattgtc gttgcgcaat 840
gcgcaatttg atttgatggc caaaaccccg tatatcgctc gccataaagg gaccccgctg 900
ttgcaacaaa ttgatacggc attagtgctg caacgcgatg cccaagggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc ggcgggcatg acaccaatat tgctaatatc 1020
gctggtatgt taggggccaa ttggcaatta ccacagcaac ctgataatac cccgcctggt 1080
ggcggattag tctttgagct atggcagaac ccagataatc atcagcgcta tgtcgccgtg 1140
aaaatgttct atcaaacgat ggatcagctg cgaaatgccg agaaattaga tatgaaaaac 1200
aacccagcta aaattgttcc aattaccatt gaaggttgtg agaacgaggg tgataacaaa 1260
ctttgccaac ttgagacttt ccaaaagaaa gttgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
<210> 3
<211> 441
<212> PRT
<213>Artificial sequence
<400> 3
MSVAKRNLHL SALTLIMGCF TAGAAPIATP PASYTLERVV ILSRHGVRSP TKQTQLMNDV 60
TPDKWPLWPV KAGYLTPRRA ELVTLMGGFY GDYFRSQGAL SAGCPVDGSV YAQADVDQRT 120
RLTGQAFLDG IAPDCGLKVH YQADLKKVDP LFHTVEAGVC KLDSAKTHQA VEERLGGPLS 180
DLSQRYAKPF AQMGEVLNFA ASPYCKSLQK NGKTCDFATF TANEIKVNEE GTKVSLSGPL 240
ALSSTLGEIF LLQDSQAMPD VAWHRLSGEE NWVSLLSLRN AQFDLMAKTP YIARHKGTPL 300
LQQIDTALVL QRDAQGQTLP LSPQTKLLFL GGHDTNIANI AGMLGANWQL PQQPDNTPPG 360
GGLVFELWQN PDNHQRYVAV KMFYQTMDQL RNAEKLDMKN NPAKIVPITI EGCENEGDNK 420
LCQLETFQKK VAQVIEPACH I 441
<210> 4
<211> 441
<212> PRT
<213>Artificial sequence
<400> 4
MSVAKRNLHL SALTLIMGCF TAGAAPIATP PASYTLERVV ILSRHGVRSP TKQTQLMNDV 60
TPDKWPLWPV KAGYLTPRRA ELVTLMGGFY GDYFRSQGAL SAGCPVDGSV YAQADVDQRT 120
RLTGQAFLDG IAPDCGLKVH YQADLKKVDP LFHTVEAGVC KGDSAKTHQA VEERLGGPLS 180
DLSQRYAKPF AQMGEVLNFA ASPYCKSLQK NGKTCDFATF TANEIKVNEE GTKVSLSGPL 240
ALSSTLGEIF LLQDSQAMPD VAWHRLSGEE NWVSLLSLRN AQFDLMAKTP YIARHKGTPL 300
LQQIDTALVL QRDAQGQTLP LSPQTKLLFL GGHDTNIANI AGMLGANWQL PQQPDNTPPG 360
GGLVFELWQN PDNHQRYVAV KMFYQTMDQL RNAEKLDMKN NPAKIVPITI EGCENEGDNK 420
LCQLETFQKK VAQVIEPACH I 441
<210> 5
<211> 441
<212> PRT
<213>Artificial sequence
<400> 5
MSVAKRNLHL SALTLIMGCF TAGAAPIATP PASYTLERVV ILSRHGVRSP TKQTQLMNDV 60
TPDKWPLWPV KAGYLTPRRA ELVTLMGGFY GDYFRSQGAL SAGCPVDGSV YAQADVDQRT 120
RLTGQAFLDG IAPDCGLKVH YQADLKKVDP LFHTVEAGVC KGDSAKTHQA VEERLGGPLS 180
DLSQRYAKPF AQMGEVLNFA ASPYCKSLQK NGKTCDFATF TANEIKVNEG GTKVSLSGPL 240
ALSSTLGEIF LLQDSQAMPD VAWHRLSGEE NWVSLLSLRN AQFDLMAKTP YIARHKGTPL 300
LQQIDTALVL QRDAQGQTLP LSPQTKLLFL GGHDTNIANI AGMLGANWQL PQQPDNTPPG 360
GGLVFELWQN PDNHQRYVAV KMFYQTMDQL RNAEKLDMKN NPAKIVPITI EGCENEGDNK 420
LCQLETFQKK VAQVIEPACH I 441
<210> 6
<211> 1320
<212> DNA
<213>Artificial sequence
<400> 6
atgtcagttg caaagagaaa tctgcactta tccgcactca ctttgataat gggctgtttt 60
accgcaggtg ctgccccgat tgctacaccg ccggccagct acacattaga gcgtgtggtt 120
attttgagtc gacatggtgt tcgctccccg acaaaacaaa cccagctaat gaatgatgtc 180
acacctgata aatggcccct gtggccagta aaagcgggct atttaacacc gcgaagggct 240
gagttagtga ctttgatggg gggattttat ggtgattatt tccgcagcca aggggcgttg 300
tctgcggggt gtccggtaga tggctccgtt tatgcacagg cagatgttga ccaacgaacc 360
cgcttaaccg gacaggcatt cttggatggg atcgcaccgg attgtggtct gaaagtacat 420
tatcaggctg atttgaagaa agttgacccg ctatttcata ccgtcgaagc gggggtctgt 480
aaactggact cagcgaaaac tcatcaggct gttgaggagc gattgggcgg gccattgagt 540
gatcttagcc agcgctatgc caaacccttt gctcagatgg gcgaagtgct gaattttgca 600
gcatcgcctt attgcaagtc attgcaaaaa aatggaaaaa cctgtgattt tgcaactttt 660
acggcaaatg aaattaaggt aaacgaagaa ggtactaaag tttctctgag tgggccattg 720
gcactatcgt cgacattggg tgaaattttc ctgttacaag actcacaagc tatgccggat 780
gtggcctggc atcggctcag cggtgaagag aactgggttt cgctattgtc gttgcgcaat 840
gcgcaatttg atttgatggc caaaaccccg tatatcgctc gccataaagg gaccccgctg 900
ttgcaacaaa ttgatacggc attagtgctg caacgcgatg cccaagggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc ggcgggcatg acaccaatat tgctaatatc 1020
gctggtatgt taggggccaa ttggcaatta ccacagcaac ctgataatac cccgcctggt 1080
ggcggattag tctttgagct atggcagaac ccagataatc atcagcgcta tgtcgccgtg 1140
aaaatgttct atcaaacgat ggatcagctg cgaaatgccg agaaattaga tatgaaaaac 1200
aacccagcta aaattgttcc aattaccatt gaaggttgtg agaacgaggg tgataacaaa 1260
ctttgccaac ttgagacttt ccaaaagaaa gttgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
<210> 7
<211> 1320
<212> DNA
<213>Artificial sequence
<400> 7
atgtcagttg caaagagaaa tctgcactta tccgcactca ctttgataat gggctgtttt 60
accgcaggtg ctgccccgat tgctacaccg ccggccagct acacattaga gcgtgtggtt 120
attttgagtc gacatggtgt tcgctccccg acaaaacaaa cccagctaat gaatgatgtc 180
acacctgata aatggcccct gtggccagta aaagcgggct atttaacacc gcgaagggct 240
gagttagtga ctttgatggg gggattttat ggtgattatt tccgcagcca aggggcgttg 300
tctgcggggt gtccggtaga tggctccgtt tatgcacagg cagatgttga ccaacgaacc 360
cgcttaaccg gacaggcatt cttggatggg atcgcaccgg attgtggtct gaaagtacat 420
tatcaggctg atttgaagaa agttgacccg ctatttcata ccgtcgaagc gggggtctgt 480
aaaggcgact cagcgaaaac tcatcaggct gttgaggagc gattgggcgg gccattgagt 540
gatcttagcc agcgctatgc caaacccttt gctcagatgg gcgaagtgct gaattttgca 600
gcatcgcctt attgcaagtc attgcaaaaa aatggaaaaa cctgtgattt tgcaactttt 660
acggcaaatg aaattaaggt aaacgaagaa ggtactaaag tttctctgag tgggccattg 720
gcactatcgt cgacattggg tgaaattttc ctgttacaag actcacaagc tatgccggat 780
gtggcctggc atcggctcag cggtgaagag aactgggttt cgctattgtc gttgcgcaat 840
gcgcaatttg atttgatggc caaaaccccg tatatcgctc gccataaagg gaccccgctg 900
ttgcaacaaa ttgatacggc attagtgctg caacgcgatg cccaagggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc ggcgggcatg acaccaatat tgctaatatc 1020
gctggtatgt taggggccaa ttggcaatta ccacagcaac ctgataatac cccgcctggt 1080
ggcggattag tctttgagct atggcagaac ccagataatc atcagcgcta tgtcgccgtg 1140
aaaatgttct atcaaacgat ggatcagctg cgaaatgccg agaaattaga tatgaaaaac 1200
aacccagcta aaattgttcc aattaccatt gaaggttgtg agaacgaggg tgataacaaa 1260
ctttgccaac ttgagacttt ccaaaagaaa gttgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
<210> 8
<211> 1320
<212> DNA
<213>Artificial sequence
<400> 8
atgtcagttg caaagagaaa tctgcactta tccgcactca ctttgataat gggctgtttt 60
accgcaggtg ctgccccgat tgctacaccg ccggccagct acacattaga gcgtgtggtt 120
attttgagtc gacatggtgt tcgctccccg acaaaacaaa cccagctaat gaatgatgtc 180
acacctgata aatggcccct gtggccagta aaagcgggct atttaacacc gcgaagggct 240
gagttagtga ctttgatggg gggattttat ggtgattatt tccgcagcca aggggcgttg 300
tctgcggggt gtccggtaga tggctccgtt tatgcacagg cagatgttga ccaacgaacc 360
cgcttaaccg gacaggcatt cttggatggg atcgcaccgg attgtggtct gaaagtacat 420
tatcaggctg atttgaagaa agttgacccg ctatttcata ccgtcgaagc gggggtctgt 480
aaaggcgact cagcgaaaac tcatcaggct gttgaggagc gattgggcgg gccattgagt 540
gatcttagcc agcgctatgc caaacccttt gctcagatgg gcgaagtgct gaattttgca 600
gcatcgcctt attgcaagtc attgcaaaaa aatggaaaaa cctgtgattt tgcaactttt 660
acggcaaatg aaattaaggt aaacgaaggc ggtactaaag tttctctgag tgggccattg 720
gcactatcgt cgacattggg tgaaattttc ctgttacaag actcacaagc tatgccggat 780
gtggcctggc atcggctcag cggtgaagag aactgggttt cgctattgtc gttgcgcaat 840
gcgcaatttg atttgatggc caaaaccccg tatatcgctc gccataaagg gaccccgctg 900
ttgcaacaaa ttgatacggc attagtgctg caacgcgatg cccaagggca aacactgccg 960
ctgtcaccgc aaaccaaatt gctgttcctc ggcgggcatg acaccaatat tgctaatatc 1020
gctggtatgt taggggccaa ttggcaatta ccacagcaac ctgataatac cccgcctggt 1080
ggcggattag tctttgagct atggcagaac ccagataatc atcagcgcta tgtcgccgtg 1140
aaaatgttct atcaaacgat ggatcagctg cgaaatgccg agaaattaga tatgaaaaac 1200
aacccagcta aaattgttcc aattaccatt gaaggttgtg agaacgaggg tgataacaaa 1260
ctttgccaac ttgagacttt ccaaaagaaa gttgcccaag tgatcgagcc agcctgccat 1320
atttaa 1326
Claims (9)
1. phytase YeAPPA mutants, it is characterised in that the 99th of phytase of the aminoacid sequence as shown in SEQ ID NO.1
The leucine of position sports alanine.
2. phytase YeAPPA mutants, it is characterised in that the 99th of phytase of the aminoacid sequence as shown in SEQ ID NO.1
The leucine of position sports alanine, and the leucine of the 162nd sports glycine.
3. phytase YeAPPA mutants, it is characterised in that the 99th of phytase of the aminoacid sequence as shown in SEQ ID NO.1
The leucine of position sports alanine, and the leucine of the 162nd sports glycine, and the glutamic acid mutation of the 230th is sweet ammonia
Acid.
4. phytase YeAPPA mutant genes, it is characterised in that the phytase YeAPPA described in coding claim 1,2 or 3
Mutant.
5. phytase YeAPPA mutant genes according to claim 4, it is characterised in that the nucleotides sequence of the gene
Row are as shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
6. comprising the recombinant vector of the phytase YeAPPA mutant genes described in claim 4.
7. comprising the recombinant bacterial strain of the phytase YeAPPA mutant genes described in claim 4.
8. a kind of method of the phytic acid enzyme mutant for preparing improved stability, it is characterised in that comprise the following steps：
1) with the recombinant vector transformed host cell of claim 6, recombinant bacterial strain is obtained；
2) recombinant bacterial strain, abduction delivering phytase YeAPPA mutants are cultivated；And
3) phytase YeAPPA mutants simultaneously expressed by purification are reclaimed.
9. the application of the phytase YeAPPA mutants described in claim 1,2 or 3.
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| CN108251439A (en) * | 2018-01-11 | 2018-07-06 | 山西大学 | A kind of artificial reconstructed phytase of resistance to trypsase and its preparation method and application |
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| CN107418939A (en) * | 2017-08-19 | 2017-12-01 | 中国农业科学院饲料研究所 | The phytic acid enzyme mutant F89S/K226H and application that protease resistant and catalytic efficiency improve |
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