CN106434595B - Phytic acid enzyme mutant YkAPPA-L327V, YeAPPA-L327V and its encoding gene and application - Google Patents
Phytic acid enzyme mutant YkAPPA-L327V, YeAPPA-L327V and its encoding gene and application Download PDFInfo
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Abstract
The present invention relates to genetic engineering fields, and in particular to phytic acid enzyme mutant YkAPPA-L327V, YeAPPA-L327V and its encoding gene and application.It is obtained and the 327th leucine of amino acid sequence phytase as shown in SEQ ID NO.1 or 3 is sported valine.Compared to wild type, the stomach adaptability and catalytic efficiency of two phytic acid enzyme mutants YkAPPA-L327V and YeAPPA-L327V of the invention are significantly improved, and optimal pH reduces, and is conducive to feed enzyme development and application.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to phytic acid enzyme mutant YkAPPA-L327V, YeAPPA-L327V
And its encoding gene and application.
Background technique
Phytic acid, that is, phytic acid is the repository of P elements in plant.Since some albumen and minerals are often with albumen-
Phytic acid-mineral element composite form exists, to reduce certain plants property feed and vegetable protein isolate mineral
The nutrition balance of these substances.Phytase can decompose phytic acid and discharge Phos, increase the nutrition balance of albumen and minerals, because
And become a kind of important industrial enzyme.As enzyme Preparations Used for Feeds volume of production and marketing grows at top speed, the ratio of enzyme preparation is used in feed
Constantly expand, starts the potential nutritive value for considering enzyme when using enzyme preparation.Breeding is suitable for various animal alimentary canal environment items
The enzyme of part and efficient equal good properties has become one of important development direction of feed enzyme preparation.
Phytase is a kind of ectoenzyme, is widely present in animal, plant and microorganism.According to three-dimensional structure and catalytic machine
Phytase can be divided into 4 classes: HAP (Histidine Acid Phosphatase) phytase, BPP (β-Propellar by system
Phytase) phytase, CP (Cysteine Phosphatase) phytase and PAP (Purple Acid
Phosphatase) phytase.The evolution of different phytases is the nutrition in order to meet its place host animals, plant, microorganism
It needs, some constituents are necessary to catalysis, and some away unnecessary parts can also be adapted to by changing to different substrates
Catalysis.It is to carry out the effective means of zymologic property improvement by design and rational.The transformation of present invention application site-directed mutagenesis technique is planted
Sour enzyme obtains phytic acid enzyme mutant YkAPPA-L327V and YeAPPA-L327V, the intestines such as acid resistance and protease resistant respectively
Stomach adaptability is remarkably reinforced, and thermal stability and catalytic efficiency significantly improve, and optimal pH reduces, and has very big application potential.
Summary of the invention
The purpose of the present invention is being transformed by the method for rite-directed mutagenesis to phytase, make improved phytic acid enzyme mutant
Body YkAPPA-L327V or YeAPPA-L327V property in stomach adaptability and catalytic efficiency is more excellent.
Another object of the present invention is to provide coding above-mentioned phytic acid enzyme mutant YkAPPA-L327V's or YeAPPA-L327V
Gene.
It is a further object of the present invention to provide include 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 include above-mentioned phytic acid enzyme mutant YkAPPA-L327V or YeAPPA-L327V
Encoding gene recombinant bacterial strain.
The present invention also provides a kind of host cell, containing 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 intestine knot
The source film inflammation Yersinia ruckeri (Y.enterocolitica) phytase YeAPPA gene carries out point mutation, the maturation of the phytase
Albumen have the amino acid sequence as shown in SEQ ID NO.1 or 3, the maturation protein be by such as SEQ ID NO.2 or 4, it is shown
It is nucleotide sequence coded.
SEQ ID NO.1
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQW
PVKAGYLTPRGAGLVTLMGGFYGDYFRSYGLLPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQAD
LKKIDPLFHTVEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATF
AANEIEVNKEGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHK
GTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPD
NHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
SEQ ID NO.2
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaag
tgctgcaccgcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgt
tccccgacgaaacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggct
atttaacgccgcgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatgggtt
gttaccggcggggtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccggg
caggcatttctggacggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacc
cattgttccataccgtcgaggcgggggtatgtaaattggacccagagaaaactcatcaggctgttgaaaaacgctt
gggtgggccattaaatgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcg
gcctcaccttattgcaactcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcg
aggtaaataaagaagggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcct
attacaaaattcacaggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattattg
tcactgcataatgcacagttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttgc
aacaaattgatacggcattagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaatt
gctgttcctcgggggacatgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgcag
caacctgataataccccgccaggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctatg
tggcggtgaaaatgttctatcaaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccggc
aagaattgttcccattgctattgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttccaa
aagaaagtcgcccaagtgatcgagccagcctgccatatttaa
SEQ ID NO.3
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLW
PVKAGYLTPRRAELVTLMGGFYGDYFRSQGLLSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQAD
LKKVDPLFHTVEAGVCKLDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATF
TANEIKVNEEGTKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHK
GTPLLQQIDTALVLQRDAQGQTLPLSPQTKLLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPD
NHQRYVAVKMFYQTMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
SEQ ID NO.4
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcagg
tgctgccccgattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgc
tccccgacaaaacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggct
atttaacaccgcgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaagggtt
gttgtctgcggggtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccgga
caggcattcttggatgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacc
cgctatttcataccgtcgaagcgggggtctgtaaactggactcagcgaaaactcatcaggctgttgaggagcgatt
gggcgggccattgagtgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgca
gcatcgccttattgcaagtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaatta
aggtaaacgaagaaggtactaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcct
gttacaagactcacaagctatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattg
tcgttgcgcaatgcgcaatttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgc
aacaaattgatacggcattagtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaatt
gctgttcctcggcgggcatgacaccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacag
caacctgataataccccgcctggtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatg
tcgccgtgaaaatgttctatcaaacgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagc
taaaattgttccaattaccattgaaggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaa
aagaaagttgcccaagtgatcgagccagcctgccatatttaa
Using the method for rite-directed mutagenesis, 2 mutant for improving stomach adaptability and catalytic efficiency are obtained, are named respectively
For YkAPPA-L327V and YeAPPA-L327V, i.e. the 327th leucine of YkAPPA or YeAPPA sport valine.
Therefore the phytic acid enzyme mutant YkAPPA-L327V that stomach adaptability according to the present invention and catalytic efficiency improve,
Amino acid sequence is as shown in SEQ ID NO.5
SEQ ID NO.5
MTIAKEYLRLSILTLVLSSFTLSAAPLAAQSTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPQW
PVKAGYLTPRGAGLVTLMGGFYGDYFRSYGLLPAGCPADESIYVQADVDQRTRLTGQAFLDGIAPDCGLKVHYQAD
LKKIDPLFHTVEAGVCKLDPEKTHQAVEKRLGGPLNELSQRYAKPFALMGEVLNFSASPYCNSLQQKGKTCDFATF
AANEIEVNKEGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWNRLSGEENWISLLSLHNAQFDLMAKTPYIARHK
GTPLLQQIDTALVLQRDAQGQTLPLSPQTKVLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPD
NHQRYVAVKMFYQTMEQLRNADKLDLKNNPARIVPIAIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The amino acid sequence of mutant YeAPPA-L327V is as shown in SEQ ID NO.6
MSVAKRNLHLSALTLIMGCFTAGAAPIATPPASYTLERVVILSRHGVRSPTKQTQLMNDVTPDKWPLW
PVKAGYLTPRRAELVTLMGGFYGDYFRSQGLLSAGCPVDGSVYAQADVDQRTRLTGQAFLDGIAPDCGLKVHYQAD
LKKVDPLFHTVEAGVCKLDSAKTHQAVEERLGGPLSDLSQRYAKPFAQMGEVLNFAASPYCKSLQKNGKTCDFATF
TANEIKVNEEGTKVSLSGPLALSSTLGEIFLLQDSQAMPDVAWHRLSGEENWVSLLSLRNAQFDLMAKTPYIARHK
GTPLLQQIDTALVLQRDAQGQTLPLSPQTKVLFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPD
NHQRYVAVKMFYQTMDQLRNAEKLDMKNNPAKIVPITIEGCENEGDNKLCQLETFQKKVAQVIEPACHI.
The present invention also provides the phytic acid enzyme mutant YkAPPA- for encoding above-mentioned stomach adaptability and catalytic efficiency raising
The gene order of L327V and YeAPPA-L327V, nucleotide sequence is as shown in SEQ ID NO.7 and 8.
SEQ ID NO.7
Atgacaatagcaaaagaatatctgcggttatccatactcactttggtgctcagtagttttacgctaag
tgctgcaccgcttgcagcacaatctaccggttacactttggagcgcgtggtgattttgagccgccacggtgttcgt
tccccgacgaaacaaacacagttaatgaatgatgttacaccggacaaatggccacaatggccagtaaaagcgggct
atttaacgccgcgaggggcaggattagtcactttaatgggcgggttctatggtgattatttccgcagctatgggtt
gttaccggcggggtgcccggcagacgaatccatctatgtgcaagctgatgttgaccaacgtacccgcttaaccggg
caggcatttctggacggtatagccccggattgcggcctgaaagtacattatcaagctgatttgaaaaaaattgacc
cattgttccataccgtcgaggcgggggtatgtaaattggacccagagaaaactcatcaggctgttgaaaaacgctt
gggtgggccattaaatgaactgagtcaacgctatgccaagccctttgccctgatgggcgaggtgctgaatttttcg
gcctcaccttattgcaactcactgcaacagaaaggaaaaacctgtgattttgcgacttttgcagcaaatgaaatcg
aggtaaataaagaa gggacaaaagtctcactgagtgggccattggcgctatcatcgacattaggtgaaattttcc
tattacaaaattcacaggccatgccagatgtcgcctggaaccgtctcagcggtgaagaaaattggatttcattatt
gtcactgcataatgcacagttcgatttgatggccaaaaccccttatatcgcccggcataaaggaactccgttgttg
caacaaattgatacggcattagtgttgcaacgtgatgctcaggggcaaacactgccgctgtcaccgcaaaccaaaG
tgctgttcctcgggggacatgacaccaatattgccaatattgcgggtatgttaggggccaattggcaattaccgca
gcaacctgataataccccgccaggcggagggctagtctttgagctatggcagaatccggataaccatcaacgctat
gtggcggtgaaaatgttctatcaaacgatggagcagttgcgcaatgcagataagttagatttgaaaaacaacccgg
caagaattgttcccattgctattgaagggtgtgaaaacgagggtgataacaaactttgtcagcttgaaacgttcca
aaagaaagtcgcccaagtgatcgagccagcctgccatatttaa
SEQ ID NO.8
Atgtcagttgcaaagagaaatctgcacttatccgcactcactttgataatgggctgttttaccgcagg
tgctgccccgattgctacaccgccggccagctacacattagagcgtgtggttattttgagtcgacatggtgttcgc
tccccgacaaaacaaacccagctaatgaatgatgtcacacctgataaatggcccctgtggccagtaaaagcgggct
atttaacaccgcgaagggctgagttagtgactttgatggggggattttatggtgattatttccgcagccaagggtt
gttgtctgcggggtgtccggtagatggctccgtttatgcacaggcagatgttgaccaacgaacccgcttaaccgga
caggcattcttggatgggatcgcaccggattgtggtctgaaagtacattatcaggctgatttgaagaaagttgacc
cgctatttcataccgtcgaagcgggggtctgtaaactggactcagcgaaaactcatcaggctgttgaggagcgatt
gggcgggccattgagtgatcttagccagcgctatgccaaaccctttgctcagatgggcgaagtgctgaattttgca
gcatcgccttattgcaagtcattgcaaaaaaatggaaaaacctgtgattttgcaacttttacggcaaatgaaatta
aggtaaacgaagaaggtactaaagtttctctgagtgggccattggcactatcgtcgacattgggtgaaattttcct
gttacaagactcacaagctatgccggatgtggcctggcatcggctcagcggtgaagagaactgggtttcgctattg
tcgttgcgcaatgcgcaatttgatttgatggccaaaaccccgtatatcgctcgccataaagggaccccgctgttgc
aacaaattgatacggcattagtgctgcaacgcgatgcccaagggcaaacactgccgctgtcaccgcaaaccaaaGt
gctgttcctcggcgggcatgacaccaatattgctaatatcgctggtatgttaggggccaattggcaattaccacag
caacctgataataccccgcctggtggcggattagtctttgagctatggcagaacccagataatcatcagcgctatg
tcgccgtgaaaatgttctatcaaacgatggatcagctgcgaaatgccgagaaattagatatgaaaaacaacccagc
taaaattgttccaattaccattgaaggttgtgagaacgagggtgataacaaactttgccaacttgagactttccaa
aagaaagttgcccaagtgatcgagccagcctgccatatttaa
The mutant enzyme YkAPPA-L327V or YeAPPA-L327V that above-mentioned coding stomach adaptability and catalytic efficiency are improved
Reading frame be inserted between EcoRI the and NotI restriction enzyme site of the carrier, keep its nucleotide sequence operable
It is linked to the expression control sequence.Currently preferred carrier is pET-22b (+), obtains the recombined pronucleus expression matter of mutant
Grain.Currently preferred host strain is BL21 (DE3).The nucleotide sequence of the phytic acid enzyme mutant is located under T7 promoter
It swims and is regulated and controled by it.Compared to wild type, two phytic acid enzyme mutants YkAPPA-L327V and YeAPPA-L327V of the invention
Stomach adaptability and catalytic efficiency significantly improve, and optimal pH reduce, be conducive to feed enzyme development and application.
Detailed description of the invention
Fig. 1 is that the protease resistant of the phytase of transformation front and back compares；
Fig. 2 is that the acid resistance of the phytase of transformation front and back compares.
Specific embodiment
Experimental 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, restriction enzyme, T4DNA ligase are purchased from day
Root company.Phytate (sodium) and pepsin (p0685) are purchased from Sigma company, and other is all that domestic reagent (can be from ordinary student
Change Reagent Company to be commercially available).
Embodiment 1: the acquisition of mutated gene
Mutant enzyme YkAPPA-L327V and YeAPPA-L327V are generated using Overlap PCR method.This method is to contain
The recombinant plasmid pEASY-T3-YkAPPA and pEASY-T3-YeAPPA of wild phytase gene are template, anti-by two-wheeled PCR
Mutation should be introduced.The upstream and downstream primer for expanding mutated gene complete encoding sequence identifies sequence with EcoR I and Not I, respectively
For YkAPPA Forward:5 '-cgcgaattcgcaccgcttgcagcacaatctac-3 ' and YkAPPA Reverse:5 '-g
atgcggccgcttaaatatggcaggctggctcG-3'；YeAPPA Forward:5 '-cgcgaattcgcaccgcttgcag
Cacaatctac-3 ' and YeAPPA Reverse:5 '-gatgcggccgcttaaatatggcaggctggctcg-3 '.Specific
Position introduce mutation upstream and downstream primer be L327V Forward:5 '-tcaccgcaaaccaaagtgctgttcctc-3 ' and
L327V Reverse:5'-gaggaacagcactttggtttgcggtga-3'.Required mutated gene is connected to pEASY-T3
It is confirmed on carrier and through sequencing.
Embodiment 2: mutation phytase and wild enzyme expression and purification in bacterium
After wild enzyme and mutant enzyme remove signal peptide sequence, it is inserted on expression vector pET-22b (+), in Escherichia coli
By 2mM IPTG (isopropyl-β-D- galactoside) inducing expression in BL21 (DE3) cell.Crude enzyme liquid is through Ni-NTA (the secondary nitrogen of nickel-
Base triacetic acid) column and DEAE (diethylamino ethyl) column purified, and with 10%SDS-PAGE detected through gel electrophoresis.It is wild
Enzyme and mutant enzyme encode 441 amino acid, and N-terminal contains the signal peptide sequence of 23 amino acid, and maturation protein theoretical molecular weight is
48.6kDa.Wild enzyme and mutant enzyme after removal signal peptide sequence show as a treaty 46kDa's in SDS-PAGE electrophoresis
Specific band (data are not shown).
Embodiment 3: mutation phytase is compared with the protease resistant of wild enzyme
Wild enzyme and mutant enzyme handle 2h, albumen with pepsin (pH2) and trypsase (pH7) respectively at 37 DEG C
The ratio of enzyme and phytase is between 1/1000 to 1/20.Sample after Protease Treatment optimal pH buffer is diluted,
Influence of the research protease to phytase activity under 37 DEG C and optimal pH part.Phytic acid enzyme activity is measured using ferrous sulfate molybdenum blue method
Property.Enzyme solution after 50 μ L dilution 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 is reacted at 37 DEG C, is terminated and is reacted with 1mL 10%TCA, then use 2mL
Developing solution (1% Ammonium Molybdate Tetrahydrate, 3.2% concentrated sulfuric acid, 7.32% ferrous sulfate) develops the color.Control is before adding enzyme solution
TCA mixing, which is first added, makes enzyme denaturation, other identical.After colour developing, OD value is measured under 700nm light absorption, calculates enzyme activity.
The trypsin-resistant of mutant enzyme YkAPPA-L327V and YeAPPA-L327V respectively with wild enzyme YKAPPA and
YeAPPA is similar, 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 60% or more 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 stomach cardia concentration, mutant enzyme
YeAPPA-L327V can keep 21.1% or more enzymatic activity, and wild enzyme YeAPPA in 1/1000 pepsin concn
Complete deactivation.When stomach cardia concentration rises to 1/20 from 1/1000, mutant enzyme YkAPPA-L327V can keep 46.1% enzyme activity
Property, and wild enzyme YkAPPA is almost inactivated.Therefore the 327th leucine of phytase YkAPPA and YeAPPA are in stomach cardia
Digestion plays an important role in cutting.
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 processing 2h studying enzymes at pH 1-4.Mutant enzyme YkAPPA-L327V
Wild enzyme (Fig. 2) is apparently higher than with stability of the YeAPPA-L327V at pH1-3.5.2h, mutant enzyme are handled at pH1-3.5
YkAPPA-L327V can keep 81.2% or more enzymatic activity, and wild enzyme YkAPPA only keeps 58.4% enzymatic activity.In pH
Under 1 and pH 1.5 when processing 2h, YeAPPA-L327V keeps 7.2% and 31.6% enzymatic activity respectively, and YeAPPA is then complete
It loses activity.
Embodiment 5: mutation phytase is compared with the kinetic constant of wild enzyme
Using 0.0625mmol/L sodium phytate as substrate, when reacting different time (1 to 15min) at enzyme optimal pH and 37 DEG C
1mL 10%TCA is added and terminates reaction, measures enzymatic activity, determines the reaction time of detection Km and Vmax.With 0.0625 to
The sodium phytate of 1.5mmol/L is substrate, and certain time is reacted at enzyme optimal pH and 37 DEG C and measures enzyme activity, double using Michaelis-Menten equation
Counting backward technique asks Km value and Vmax and kcat value.
Mutant enzyme YkAPPA-L327V and the Km value of YeAPPA-L327V are similar to wild enzyme when using sodium phytate as substrate,
Respectively 0.10mM and 0.18mM (table 1).The 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.It is 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 are the result shows that the 327th leucine residue of YkAPPA and YeAPPA imitates catalysis
Rate plays a significant role.
The dynamics of the phytase of the transformation of table 1 front and back compares
Embodiment 6: mutation phytase is compared with the zymologic property of wild enzyme
Wild enzyme and mutant enzyme react 30min at different pH (1-12) and different temperatures (30-80 DEG C), determine optimal pH
And optimum temperature.Enzyme solution 37 DEG C of processing 1h, pH stability of studying enzyme at pH 1-9.Buffer used are as follows: 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 optimal pH of YeAPPA-L327V is determined and compares (table 2) with wild type YkAPPA.Mutant YkAPPA-
The optimal pH of L327V and YeAPPA-L327V is respectively 4 and 4.5, and it is mono- that 0.5 pH is respectively reduced compared with wild enzyme YeAPPA
Position.Mutant enzyme YkAPPA-L327V and the YeAPPA-L327V stability (table with higher at pH2-10 and pH3-9 respectively
2).Mutant enzyme YkAPPA-L327V has preferable acid resistance at pH1, can keep 91.3% enzymatic activity, and wild enzyme
YkAPPA loses 36.3% enzymatic activity.Mutant enzyme YeAPPA-L327V has better acid resistance at pH 2, can keep
77.3% enzymatic activity, and wild enzyme YeAPPA only residue 18.4% enzyme activity (table 2).Therefore, phytase YkAPPA and
The 327th site leucine of YeAPPA, which sports valine, to be reduced optimal pH and improves acid resistance.
Thermal stability is measured after enzyme solution is handled 0,2,5,10,20,30,60min respectively at 60 DEG C.Mutant enzyme
The optimum temperature of YkAPPA-L327V and YeAPPA-L327V is similar to wild enzyme, and respectively 55 DEG C and 45 DEG C (table 2).At 60 DEG C
Lower processing 30min, mutant enzyme YKAPPA-L327V and YeAPPA-L327V have better thermal stability, mutant enzyme YKAPPA-
L327V and YeAPPA-L327V keeps 34.3% and 14.0% enzymatic activity respectively, and wild enzyme YkAPPA and YeAPPA distinguishes
The enzyme activity (table 2) of residue 16.1% and 0.6%.Therefore, L327V mutation improves thermostabilization in phytase YkAPPA and YeAPPA
Property.
The zymologic property of the phytase of the transformation of table 2 front and back compares
The different metal ions and chemical reagent of final concentration of 1mmol/L are added in enzymatic reaction system, in optimal pH
Enzymatic activity is measured under the conditions of with 37 DEG C, studies influence of the various substances to enzyme activity.With not plus metal ion and chemical reagent
Reaction is control.Each metal ion species and chemical reagent have not the enzyme activity of mutant enzyme YkAPPA-L327V and YeAPPA-L327V
Same influence (table 3).The activity of mutant enzyme YeAPPA-L327V is activated by Ca2+ but is inhibited 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 effect.Compared with wild enzyme, L327V mutation reduces Hg in phytase YkAPPA and YeAPPA2 +, Fe3+, Zn2+, with Ag+To the inhibiting effect of phytase activity.
Influence of 3 chemical reagent of table to the enzyme activity of the phytase of transformation front and back
Claims (9)
1. phytase YkAPPA mutant YkAPPA-L327V, which is characterized in that by by amino acid sequence such as SEQ ID NO.1
Shown in the 327th leucine of phytase sport valine and obtain.
2. phytase YeAPPA mutant YeAPPA-L327V, which is characterized in that by by amino acid sequence such as SEQ ID NO.3
Shown in the 327th leucine of phytase sport valine and obtain.
3. phytic acid enzyme mutant gene, which is characterized in that it encodes phytase YkAPPA mutant described in claim 1
YkAPPA-L327V or phytase YeAPPA mutant YeAPPA-L327V as claimed in claim 2.
4. phytic acid enzyme mutant gene according to claim 3, which is characterized in that the nucleotide sequence of the gene is such as
Shown in SEQ ID NO.7 or SEQ ID NO.8.
5. including the recombinant vector of phytic acid enzyme mutant gene as claimed in claim 3.
6. including the recombinant bacterial strain of phytic acid enzyme mutant gene as claimed in claim 3.
7. a kind of method for the phytic acid enzyme mutant for preparing improved stability, which is characterized in that the described method comprises the following steps:
1) recombinant vector described in claim 5 converts host cell, obtains recombinant bacterial strain；
2) recombinant bacterial strain is cultivated, the phytase gene expression of recombination is induced；And
3) it recycles and purifies expressed phytic acid enzyme mutant YkAPPA-L327V or phytic acid enzyme mutant YeAPPA-L327V.
8. the application that phytic acid enzyme mutant YkAPPA-L327V described in claim 1 is used for Hydrolysis of Phytic Acid.
9. the application that phytase YeAPPA mutant YeAPPA-L327V as claimed in claim 2 is used for Hydrolysis of Phytic Acid.
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| CN107475217A (en) * | 2017-08-19 | 2017-12-15 | 中国农业科学院饲料研究所 | Protease resistant improves and the phytic acid enzyme mutant E226H of heat resistance improvement and its application |
| CN107858364B (en) * | 2017-12-04 | 2023-01-06 | 上海市农业科学院 | High-temperature-resistant high-specific-activity bacterial phytase gene suitable for methanol yeast expression |
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