CN106434595A - Phytase mutants YkAPPA-L327V, YeAPPA-L327V and coding genes and application thereof - Google Patents
Phytase mutants YkAPPA-L327V, YeAPPA-L327V and coding genes and application thereof Download PDFInfo
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Abstract
The invention relates to the field of genetic engineering, and in particular relates to phytase mutants YkAPPA-L327V, YeAPPA-L327V and coding genes and application thereof. The phytase mutants are obtained by mutating the 327th leucine of phytase of which the amino acid sequence is as shown in the SEQ ID NO.1 or 3 into valine. Compared with a wild type, the two phytase mutants YkAPPA-L327V and YeAPPA-L327V have apparently improved gastrointestinal adaptability and catalytic efficiency, reduced optimum pH values and benefit for development and application of feed enzymes.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to phytic acid enzyme mutant YkAPPA-L327V, YeAPPA-L327V
And its encoding gene and application.
Background technology
Phytic acid is phytic acid, is the storage vault of P elements in plant.Due to some albumen and mineral often with albumen-
Phytic acid-mineral element composite form is present, so as to reduce certain plants property feedstuff and vegetable protein separator mineral
The nutrition balance of these materials.Phytase can decompose phytic acid and discharge Phos, increase the nutrition balance of albumen and mineral, because
And become the important industrial enzyme of a class.As enzyme Preparations Used for Feeds volume of production and marketing grows at top speed, the ratio of enzyme preparation used in feedstuff
Constantly expand, start during using enzyme preparation to consider the potential nutritive value of enzyme.Selection-breeding is suitable for various animal alimentary canal environment bars
The enzyme of part and the good properties such as efficiently has become one of important development direction of feed enzyme preparation.
Phytase is a kind of exoenzyme, is widely present in animal, plant and microorganism.According to three dimensional structure and catalytic machine
Phytase can be divided into 4 classes by system：HAP (Histidine Acid Phosphatase) phytase, BPP (β-Propellar
Phytase) phytase, CP (Cysteine Phosphatase) phytase, and PAP (Purple Acid
Phosphatase) phytase.The evolution of different phytases be in order to meet the nutrition of its place host animals, plant, microorganism
Need, some constituents are that catalysis is necessary, and some away unnecessary parts can also be adapted to different substrates by changing
Catalysis.It is the effective means for carrying out zymologic property improvement by design and rational.Application site-directed mutagenesis technique transformation of the present invention is planted
Sour enzyme, obtains phytic acid enzyme mutant YkAPPA-L327V and YeAPPA-L327V, the intestinal such as its acid resistance and protease resistant respectively
Stomach adaptability is remarkably reinforced, and heat stability and catalytic efficiency are significantly improved, and optimum pH reduces, with very big application potential.
Content of the invention
The purpose of the present invention is by the method for rite-directed mutagenesises, phytase to be transformed, and makes improved phytic acid enzyme mutant
Body YkAPPA-L327V or YeAPPA-L327V property on the intestines and stomach adaptability and catalytic efficiency is more excellent.
The present invention another object is that and provide the above-mentioned phytic acid enzyme mutant YkAPPA-L327V's or YeAPPA-L327V of coding
Gene.
It is a further object of the present invention to provide including above-mentioned phytic acid enzyme mutant YkAPPA-L327V or YeAPPA-L327V
Encoding gene recombinant vector.
It is a further object of the present invention to provide including above-mentioned phytic acid enzyme mutant YkAPPA-L327V or YeAPPA-L327V
Encoding gene recombinant bacterial strain.
Present invention also offers a kind of host cell, its contain foregoing phytic acid enzyme mutant YkAPPA-L327V or
The gene of YeAPPA-L327V or foregoing recombinant vector.
The present invention is to Yerinia kristensenii (Yersinia kristensenii) source phytase YkAPPA or small intestinal knot
Film inflammation yersinia (Y.enterocolitica) source phytase YeAPPA gene carries out point mutation, the maturation of the phytase
Albumen has aminoacid sequence as shown in SEQ ID NO.1 or 3, the maturation protein be by such as SEQ ID NO.2 or 4, shown
Nucleotide sequence coded.
SEQ ID NO.1
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTP
RGAGLVTLMGGFYGDYFRSYGLLPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHT
VEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKEG
TKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
SEQ ID NO.2
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaagtgctgcacc
gcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgttccccgacga
aacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggctatttaacgccg
cgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatgggttgttaccggcggg
gtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccgggcaggcatttctgg
acggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacccattgttccatacc
gtcgaggcgggggtatgtaaattggacccagagaaaactcatcaggctgttgaaaaacgcttgggtgggccattaaa
tgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcggcctcaccttattgca
actcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcgaggtaaataaagaaggg
acaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcctattacaaaattcacaggc
catgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattgtcactgcataatgcacagt
tcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgcaacaaattgatacggcatta
gtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcgggggacatga
caccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcagcaacctgataataccccgccag
gcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatgtggcggtgaaaatgttctatcaa
acgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggcaagaattgttcccattgctattga
agggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaaaagaaagtcgcccaagtgatcgagc
cagcctgccatatttaa
SEQ ID NO.3
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLWPVKAGYLTP
RRAELVTLMGGFYGDYFRSQGLLSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKVDPLFHT
VEAGVCKLDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATFTANEIKVNEEG
TKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
SEQ ID NO.4
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcaggtgctgcccc
gattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgctccccgacaa
aacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggctatttaacaccg
cgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaagggttgttgtctgcggg
gtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccggacaggcattcttgg
atgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacccgctatttcatacc
gtcgaagcgggggtctgtaaactggactcagcgaaaactcatcaggctgttgaggagcgattgggcgggccattgag
tgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgcagcatcgccttattgca
agtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaattaaggtaaacgaagaaggt
actaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcctgttacaagactcacaagc
tatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattgtcgttgcgcaatgcgcaat
ttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgcaacaaattgatacggcatta
gtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaattgctgttcctcggcgggcatga
caccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacagcaacctgataataccccgcctg
gtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatgtcgccgtgaaaatgttctatcaa
acgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagctaaaattgttccaattaccattga
aggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaaaagaaagttgcccaagtgatcgagc
cagcctgccatatttaa
Using the method for rite-directed mutagenesises, 2 mutants for improving the intestines and stomach adaptability and catalytic efficiency are obtained, is named respectively
L-Valine is sported for the Leucine of the 327th of YkAPPA-L327V and YeAPPA-L327V, i.e. YkAPPA or YeAPPA.
Therefore according to the intestines and stomach adaptability of the present invention and the phytic acid enzyme mutant YkAPPA-L327V of catalytic efficiency raising, its
Aminoacid sequence is as shown in SEQ ID NO.5
SEQ ID NO.5
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQWPVKAGYLTP
RGAGLVTLMGGFYGDYFRSYGLLPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKIDPLFHT
VEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATFAANEIEVNKEG
TKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKVLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The aminoacid sequence of mutant YeAPPA-L327V is as shown in SEQ ID NO.6
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLWPVKAGYLTP
RRAELVTLMGGFYGDYFRSQGLLSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQADLKKVDPLFHT
VEAGVCKLDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATFTANEIKVNEEG
TKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHKGTPLLQQIDTAL
VLQRDAQGQTLPLSPQTKVLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQ
TMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
Present invention also offers the phytic acid enzyme mutant YkAPPA- that the above-mentioned the intestines and stomach adaptability of coding and catalytic efficiency are improved
The gene order of L327V and YeAPPA-L327V, its nucleotide sequence is as shown in SEQ ID NO.7 and 8.
SEQ ID NO.7
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaagtgctgcacc
gcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgttccccgacga
aacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggctatttaacgccg
cgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatgggttgttaccggcggg
gtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccgggcaggcatttctgg
acggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacccattgttccatacc
gtcgaggcgggggtatgtaaattggacccagagaaaactcatcaggctgttgaaaaacgcttgggtgggccattaaa
tgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcggcctcaccttattgca
actcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcgaggtaaataaagaa
gggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcctattacaaaattcaca
ggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattgtcactgcataatgcac
agttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgcaacaaattgatacggca
ttagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaaGtgctgttcctcgggggaca
tgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcagcaacctgataataccccgc
caggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatgtggcggtgaaaatgttctat
caaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggcaagaattgttcccattgctat
tgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaaaagaaagtcgcccaagtgatcg
agccagcctgccatatttaa
SEQ ID NO.8
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcaggtgctgcccc
gattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgctccccgacaa
aacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggctatttaacaccg
cgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaagggttgttgtctgcggg
gtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccggacaggcattcttgg
atgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacccgctatttcatacc
gtcgaagcgggggtctgtaaactggactcagcgaaaactcatcaggctgttgaggagcgattgggcgggccattgag
tgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgcagcatcgccttattgca
agtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaattaaggtaaacgaagaaggt
actaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcctgttacaagactcacaagc
tatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattgtcgttgcgcaatgcgcaat
ttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgcaacaaattgatacggcatta
gtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaaGtgctgttcctcggcgggcatga
caccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacagcaacctgataataccccgcctg
gtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatgtcgccgtgaaaatgttctatcaa
acgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagctaaaattgttccaattaccattga
aggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaaaagaaagttgcccaagtgatcgagc
cagcctgccatatttaa
The mutant enzyme YkAPPA-L327V that above-mentioned coding the intestines and stomach adaptability and catalytic efficiency are improved or YeAPPA-L327V
Reading frame be inserted between EcoRI the and NotI restriction enzyme site of the carrier so as to nucleotide sequence is exercisable
It is connected with expression regulation sequence.Currently preferred carrier be pET-22b (+), obtain the recombined pronucleus expression matter of mutant
Grain.Currently preferred Host Strains are BL21 (DE3).The nucleotide sequence of the phytic acid enzyme mutant is located under T7 promoter
Trip is simultaneously regulated and controled by which.Compared to wild type, two phytic acid enzyme mutant YkAPPA-L327V and YeAPPA-L327V of the present invention
The intestines and stomach adaptability and catalytic efficiency significantly improve, and optimum pH reduce, be conducive to feed enzyme development and application.
Description of the drawings
Fig. 1 is that the protease resistant of the phytase before and after transformation compares；
Fig. 2 is that the acid resistance of the phytase before and after transformation compares.
Specific embodiment
Experiment material
Bacterial strain and carrier：E. coli bl21 (DE3) cell is purchased from Tiangeng company；Prokaryotic expression carrier pET-22b (+) purchase
From Novagen company.Enzyme and other biochemical reagents：Pfu archaeal dna polymerase, restricted enzyme, T4DNA ligase are purchased from day
Root company.Phytate (sodium) and pepsin (p0685) are purchased from Sigma company, and other are all that domestic reagent (all can be from ordinary student
Change Reagent Company to be commercially available).
Embodiment 1：The acquisition of mutant gene
Mutant enzyme YkAPPA-L327V and YeAPPA-L327V is produced using Overlap PCR method.The method is to contain
The recombiant plasmid pEASY-T3-YkAPPA of wild phytase gene and pEASY-T3-YeAPPA is template, anti-by two-wheeled PCR
Mutation should be introduced.The upstream and downstream primer of amplification mutant gene complete encoding sequence is with EcoR I and Not I recognition sequence, difference
For YkAPPA Forward：5 '-cgcgaattcgcaccgcttgcagcacaatctac-3 ' and YkAPPA Reverse：5’-
gatgcggccgcttaaatatggcaggctggctcG-3’；YeAPPA Forward：5’-
Cgcgaattcgcaccgcttgcagcacaatctac-3 ' and YeAPPA Reverse：5’-
gatgcggccgcttaaatatggcaggctggctcg-3’.The upstream and downstream primer for mutation being introduced in ad-hoc location is L327V
Forward:5 '-tcaccgcaaaccaaagtgctgttcctc-3 ' and L327V Reverse:5’-
gaggaacagcactttggtttgcggtga-3’.Required mutant gene is connected on pEASY-T3 carrier and through sequencing card
Real.
Embodiment 2：Mutation phytase expression and purification in antibacterial with wild enzyme
After wild enzyme and mutant enzyme remove signal peptide sequence, be inserted into expression vector pET-22b (+) on, in escherichia coli
2mM IPTG (isopropyl-β-D- galactoside) abduction delivering is received in BL21 (DE3) cell.Crude enzyme liquid is through Ni-NTA (nickel-secondary nitrogen
Base triacetic acid) post and DEAE (diethylamino ethyl) post carry out purification, and use 10%SDS-PAGE detected through gel electrophoresis.Wild
Enzyme encodes 441 aminoacid with mutant enzyme, and N-terminal contains the signal peptide sequence of 23 aminoacid, and maturation protein theoretical molecular is
48.6kDa.Remove the wild enzyme after signal peptide sequence and all show as a treaty 46kDa's in SDS-PAGE electrophoresis with mutant enzyme
Specific band (data do not show).
Embodiment 3：Mutation phytase is compared with the protease resistant of wild enzyme
Wild enzyme and mutant enzyme are respectively with pepsin (pH2) and trypsin pH7) 2h, albumen is processed at 37 DEG C
The ratio of enzyme and phytase is between 1/1000 to 1/20.Sample after Protease Treatment is diluted with optimum pH buffer,
Impact of the protease to phytase activity is studied under 37 DEG C and optimum pH part.Phytic acid enzyme activity is determined using ferrous sulfate molybdenum blue method
Property.Enzyme liquid after 50 μ L are diluted is added to 950 μ L 1.5mmol/L sodium phytate substrates (with the pH's 4.5 of 0.25mol/L
1.5mmol/L sodium phytate buffer) in, 30min being reacted at 37 DEG C, with 1mL 10%TCA terminating reaction, then uses 2mL
Nitrite ion (1% Ammonium Molybdate Tetrahydrate, 3.2% concentrated sulphuric acid, 7.32% ferrous sulfate) is developed the color.Control be before enzyme-added liquid
Being initially charged TCA mixing makes enzyme denaturation, and other are identical.After colour developing, under 700nm light absorbs, OD value is determined, calculate enzyme activity.
The trypsin-resistant of mutant enzyme YkAPPA-L327V and YeAPPA-L327V respectively with wild enzyme YKAPPA and
YeAPPA is similar to, when trypsin treatment concentration rises to 1/20 from 1/1000, the enzyme activity base of mutant enzyme YKAPPA-L327V
This is constant, and wild enzyme YKAPPA can keep more than 60% enzymatic activity (Fig. 2).And mutant enzyme YkAPPA-L327V and YeAPPA-
The pepsin resistance of L327V is apparently higher than wild enzyme (Fig. 1).Under 1/1000 to 1/20 pepsin concentration, mutant enzyme
YeAPPA-L327V can keep more than 21.1% enzymatic activity, and wild enzyme YeAPPA in 1/1000 pepsin concn
Complete deactivation.When pepsin concentration rises to 1/20 from 1/1000, mutant enzyme YkAPPA-L327V can keep 46.1% enzyme activity
Property, and wild enzyme YkAPPA almost complete deactivation.Therefore the 327th Leucine of phytase YkAPPA and YeAPPA is in pepsin
Play an important role in cleavage.
Embodiment 4：Mutation phytase of the present invention is compared with the acid resistance of wild enzyme
The absolute acid stability of wild enzyme and mutant enzyme 37 DEG C of process 2h studying enzymes under pH 1-4.Mutant enzyme YkAPPA-L327V
With stability of the YeAPPA-L327V under pH1-3.5 apparently higher than wild enzyme (Fig. 2).2h, mutant enzyme is processed under pH1-3.5
YkAPPA-L327V can keep more than 81.2% enzymatic activity, and wild enzyme YkAPPA only keeps 58.4% enzymatic activity.In pH
During 1 and pH 1.5 times process 2h, YeAPPA-L327V keeps 7.2% and 31.6% enzymatic activity respectively, and YeAPPA is then complete
Lose activity.
Embodiment 5：Mutation phytase is compared with the kinetic constant of wild enzyme
With 0.0625mmol/L sodium phytate as substrate, when reacting different time (1 to 15min) at enzyme optimum pH and 37 DEG C
1mL 10%TCA terminating reaction is added, enzymatic activity is determined, determine the response time of detection Km and Vmax.With 0.0625 to
The sodium phytate of 1.5mmol/L is substrate, and at enzyme optimum pH and 37 DEG C, reaction certain time determines enzyme activity, double using Michaelis-Menten equation
Counting backward technique asks Km value and Vmax and kcat value.
When with sodium phytate as substrate, mutant enzyme YkAPPA-L327V is similar to wild enzyme to the Km value of YeAPPA-L327V,
Respectively 0.10mM and 0.18mM (table 1).Kinetic constant Vmax of mutant enzyme YkAPPA-L327V and YeAPPA-L327V and
Kcat is respectively 12771U mg-1And 9700S-1With 24.27U mg-1And 18.57S-1, 3.8 times (tables 1) of up to wild enzyme.Prominent
The Kcat/Km for becoming enzyme YKAPPA-L327V and YeAPPA-L327V is respectively 94444S-1mM-1And 103.48S-1mM-1, it is respectively
3.2 and 4.0 times (tables 1) of wild enzyme.These results show the 327th leucine residue of YkAPPA and YeAPPA to catalysis effect
Rate has important function.
The kinetics of the phytase before and after the transformation of table 1 compare
Embodiment 6：Mutation phytase is compared with the zymologic property of wild enzyme
Wild enzyme and mutant enzyme react 30min under different pH (1-12) and different temperatures (30-80 DEG C), determine optimum pH
And optimum temperature.Enzyme liquid 37 DEG C of process 1h, pH stability of studying enzyme under pH 1-9.Buffer used is：0.1mol/L
Glycine-HCI buffer, pH 1-3；0.1mol/L Acetate-acetate buffer solution, pH 3-6；0.1mol/L Tris- hydrochloric acid
Buffer, pH 6-8；0.1mol/L Glycine-NaOH buffer, pH 8-12.To mutant enzyme YkAPPA-L327V and
The optimum pH of YeAPPA-L327V is determined and compares (table 2) with wild type YkAPPA.Mutant YkAPPA-
The optimum pH of L327V and YeAPPA-L327V is respectively 4 and 4.5, respectively reduces 0.5 pH mono- compared with wild enzyme YeAPPA
Position.Mutant enzyme YkAPPA-L327V and YeAPPA-L327V are respectively with higher stability (table under pH2-10 and pH3-9
2).Under pH1, mutant enzyme YkAPPA-L327V has preferable acid resistance, can keep 91.3% enzymatic activity, and wild enzyme
YkAPPA loses 36.3% enzymatic activity.In 2 times mutant enzyme YeAPPA-L327V of pH, there is more preferable acid resistance, can keep
77.3% enzymatic activity, and the enzyme activity (table 2) of wild enzyme YeAPPA only remaining 18.4%.Therefore, phytase YkAPPA and
The 327th site Leucine of YeAPPA sports L-Valine and reduces optimum pH and improve acid resistance.
Enzyme liquid is processed at 60 DEG C 0 respectively, after 2,5,10,20,30,60min, is measured heat stability.Mutant enzyme
YkAPPA-L327V is similar to wild enzyme with the optimum temperature of YeAPPA-L327V, respectively 55 DEG C and 45 DEG C (table 2).At 60 DEG C
Lower process 30min, mutant enzyme YKAPPA-L327V and YeAPPA-L327V have more preferable heat stability, mutant enzyme YKAPPA-
L327V and YeAPPA-L327V keep 34.3% and 14.0% enzymatic activity respectively, and wild enzyme YkAPPA and YeAPPA is respectively
Remaining 16.1% and 0.6% enzyme activity (table 2).Therefore, in phytase YkAPPA and YeAPPA L327V mutation improve thermally-stabilised
Property.
The zymologic property of the phytase before and after the transformation of table 2 compares
Different metal ions and the chemical reagent of final concentration of 1mmol/L is added in enzymatic reaction system, in optimum pH
With 37 DEG C under the conditions of determine enzymatic activity, study the impact of various materials to enzyme activity.With not plus metal ion and chemical reagent
React for control.Each metal ion species and chemical reagent have not to the enzyme activity of mutant enzyme YkAPPA-L327V and YeAPPA-L327V
Same impact (table 3).The activity of mutant enzyme YeAPPA-L327V is activated by Ca2+ but is suppressed by Cu2+.Ca2+ and Cu2+ is to prominent
The activity for becoming enzyme YkAPPA-L327V has not significant impact.Pd2+、Fe3+、Zn2+、Ag+、Hg2+With SDS strong inhibition mutant enzyme
The enzyme activity of YkAPPA-L327V and YeAPPA-L327V.Other chemical substances are to mutant enzyme YkAPPA-L327V and YeAPPA-
The enzyme activity of L327V has little to no effect.Compared with wild enzyme, L327V mutation in phytase YkAPPA and YeAPPA reduces Hg2 +, Fe3+, Zn2+, with Ag+Inhibitory action to phytase activity.
Impact of 3 chemical reagent of table to the enzyme activity of the phytase before and after transformation
Claims (9)
1. phytase YkAPPA mutant YkAPPA-L327V, it is characterised in that by by aminoacid sequence such as SEQ ID NO.1
327th Leucine of shown phytase sports L-Valine and obtains.
2. phytase YeAPPA mutant YeAPPA-L327V, it is characterised in that by by aminoacid sequence such as SEQ ID NO.3
327th Leucine of shown phytase sports L-Valine and obtains.
3. phytic acid enzyme mutant gene, it is characterised in that its phytase YkAPPA mutant described in coding claim 1
Phytase YeAPPA mutant YeAPPA-L327V described in YkAPPA-L327V or claim 2.
4. phytic acid enzyme mutant gene according to claim 3, it is characterised in that the nucleotide sequence of the gene is such as
Shown in SEQ ID NO.7 or SEQ ID NO.8.
5. the recombinant vector comprising the phytic acid enzyme mutant gene described in claim 3.
6. the recombinant bacterial strain comprising the phytic acid enzyme mutant gene described in claim 3.
7. a kind of phytic acid enzyme mutant method for preparing improved stability, it is characterised in that the method comprising the steps of：
1) with the recombinant vector transformed host cell described in claim 5, recombinant bacterial strain is obtained；
2) recombinant bacterial strain is cultivated, induces the phytase gene expression of restructuring；And
3) reclaim and the phytic acid enzyme mutant YkAPPA-L327V expressed by purification or phytic acid enzyme mutant YeAPPA-L327V.
8. the application of the phytic acid enzyme mutant YkAPPA-L327V described in claim 1.
9. the application of the phytase YeAPPA mutant YeAPPA-L327V described in claim 2.
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