CN109943546A - A kind of glutamine transaminage mutant and its preparation method and application - Google Patents
A kind of glutamine transaminage mutant and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of glutamine transaminage and its preparation method and application.The present invention is by extracting 23857 genomic DNA of Bacillus subtillis ATCC, PCR amplification obtains the glutamine transaminage btg gene order that wild type has proenzyme area, the wild type btg gene that amplification is obtained carries out random mutation by fallibility PCR, obtains a mutant gene btgm.Mutant gene is constructed into recombinant vector and the successful expression in bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, the recombinant bacterial strain of producing enzyme vigor raising is obtained, novel glutamine transaminage is further obtained by fermentation technology optimization.
Description
Technical field:
The invention belongs to technical field of bioengineering, and in particular to a kind of glutamine transaminage and preparation method thereof and answer
With.
Technical background:
Glutamine transaminage (Transglutaminase, abbreviation TG) is that one kind can be with catalyzing acyl transfer reaction
Enzyme, it can make the γ on epsilon-amino and glutamine residue in protein molecule on lysine residue-hydroxyl amide groups phase interaction
With formation ε-(γ-glutamyl) lysine key, so that covalent cross-linking occurs between protein (or polypeptide).So as to improve egg
The structure and function of white matter influences the property of protein, such as foaminess, emulsibility, emulsion stability, thermal stability, water-retaining property
With gelling ability etc., and then improve flavor, mouthfeel, quality and the appearance etc. of food, has been widely used in food at present, has spun
It knits, the fields such as pharmacy.This enzyme is promoted and applied in the food industry, such as meat products, dairy products, bean product.Pass through TG
Cross-linked proteins, thus it is possible to vary the structure of protein and the functional character for improving it, and do not destroy, it might even be possible to improve food
Nutrition.Such as TG can significantly improve the elasticity and hardness of soybean protein isolate, protein molecule can be made more closely to be incorporated in
Together, therefore Soybean Protein Isolate Gel intensity can be improved, so as to improve its mouthfeel, quality and appearance etc..
However currently, only cyclopentadienyl source streptomycete TG (abbreviation MTG) is realized and is commercially produced and be widely used in food and add
In work.But as food processing industry range constantly expands, food processing bottom material kind is growing day by day, and MTG is as currently the only life
Produce and application TG enzyme, substrate specificity, in terms of cannot meet various food processings well
The demand of substrate and process.Compared with MTG, the property of Bacillus subtillis TG (abbreviation BTG) has larger difference, can be food
Product processing provides a kind of new selection.BTG has gene source difference, substrate specificity compared with the MTG studied extensively at present
Not same advantages, but BTG Rate activity is low, which also limits commercially producing for BTG, therefore we pass through the side of molecular modification
The Rate activity of formula raising BTG.
Enzyme molecule lactam enzyme by directional anagenesis in vitro belongs to the nonideal explosives of protein, is the new strategy of protein engineering.Using point
Sub- biological means create the diversity of molecule in molecular level, in conjunction with sensitive screening technique, are preferably dashed forward rapidly
Variant.It is not required to understand the factors such as space structure, active site, the catalyst mechanism of protein in advance, but artificially creates special
Enzyme gene is transformed in different evolution conditions evolutionary mechanism in vitro, obtains the structure enzyme with certain expected features.Wherein, fallibility
PCR refers to while amplifying target genes do not have 3 ' → 5 ' proofreading functions using Taq enzyme, change simultaneously in reaction system
Mn2+、Mg2+With the concentration of various dNTP, base mispairing is randomly incorporated into target gene with certain frequency, leads to target gene
Random mutation occurs.However, the gene through being once mutated generally be difficult to obtain it is satisfied as a result, thus developing continuous error-prone again
PCR (Sequential Error-prone PCR) strategy.Will the obtained product of a PCR amplification expand as PCR next time
The template of increasing continuously repeatedly carries out fallibility, accumulates the micromutation obtained each time constantly and generates important beneficial
Mutation.Therefore, there is exclusive advantage.
Bacillus expression system has the advantage that 1, can efficiently secrete various protein；2, many brood cell's bars
Use of the bacterium in fermentation industry has quite long history, and no pathogenicity does not generate any endotoxin；3, Bacillus is micro-
Biogenetics background research it is fully aware of, and grow rapidly, the advantages that nutriment without particular/special requirement；4, codon
Preferences are unobvious；5, fermentation process is simple, and bacillus belongs to aerobic bacteria, is not necessarily to anaerobic fermentation equipment, after fermentation, letter
Single separation and fermentation liquid and microorganism can enter separation, the purification and recovery stage of destination protein；6, there is resistance, Ke Yisheng
Produce a variety of heat resistance enzyme preparations.
Therefore, in the present invention, the expression platform based on TG in Escherichia coli, using fallibility round pcr, to from withered
The TG gene btg of careless bacillus carries out molecular modification, and in Bacillus subtillis, bacillus amyloliquefaciens, lichens gemma bar
It is expressed in thallus system, is desirably to obtain the glutamine transaminage of enzyme activity raising.
Summary of the invention:
It is an object of that present invention to provide a kind of novel glutamine transaminages and its preparation method and application.
The present invention realizes that the technical route of above-mentioned purpose is as follows:
23857 genomic DNA of Bacillus subtillis ATCC is extracted, wild type is obtained with proenzyme area by PCR amplification
Glutamine transaminage btg gene (shown in SEQ ID NO.3) sequence (amino acid sequence is as shown in SEQ ID NO.4), will expand
Increase obtained wild type btg gene and random mutation is carried out by fallibility PCR, obtains a mutant gene btgm.It will mutation
The gene constructed recombinant vector of body and the successful expression in bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, obtain
The recombinant bacterial strain that producing enzyme vigor improves, further obtains novel glutamine transaminage by fermentation technology optimization.
It uses and such as gives a definition in the present invention:
1, the nomenclature of amino acid and DNA nucleic acid sequence
Using the generally acknowledged IUPAC nomenclature of amino acid residue, using three-letter codes form.DNA nucleic acid sequence is using public
Recognize IUPAC nomenclature.
2, the mark of glutamine transaminage mutant
The ammonia being mutated in glutamine transaminage mutant is indicated using " amino acid of Original amino acid position replacement "
Base acid.Such as Arg95Gly, indicate that the amino acid of position 95 is substituted for Gly, Arg95 by the Arg of wild type glutamine transaminage
The amino acid for indicating the 95th is Arg, and the number of position corresponds to the ammonia of wild type glutamine transaminage in SEQ ID NO.4
Base acid sequence number；Nucleotide representation method is similar with amino acid representation method, such as G95, indicates that the 95th base is G, position
The number set corresponds to the nucleotides sequence column number of wild type glutamine transaminage in SEQ ID NO.3.
In the present invention, btg represents wild type glutamine transaminage encoding gene, and BTG represents wild type glutamine and turns
Adnosine deaminase；Btgm is glutamine transaminage mutant gene；BTGM is glutamine transaminage mutant.It is mutated the base of front and back
And amino acid control is as follows:
| Glutamine transaminage | Base | Amino acid |
| BTG | A283、G284、A285 | Arg95 |
| BTGM | G283、G284、A285 | Arg95Gly |
The amino acid sequence of the glutamine transaminage mutant BTGM is as shown in sequence table SEQ ID NO.6；
The nucleotide sequence of the btgm gene is as shown in sequence table SEQ ID NO.5；
The host cell for expressing the mutant gene is bacillus subtilis, bacillus amyloliquefaciens or lichens brood cell's bar
Bacterium, expression vector pBSA43；
Preferably, the bacillus subtilis is WB600；
Preferably, the bacillus amyloliquefaciens are CGMCC No.11218；
Preferably, the bacillus licheniformis is TCCC11965.
The present invention also provides above-mentioned glutamine transaminages to be mutated preparation, includes the following steps:
(1) by PCR or gene chemical synthesis, glutamine transaminage mutant gene shown in SEQ ID NO.5 is obtained；
(2) by the glutamine transaminage mutant gene digestion, it is connected to expression vector, obtains carrying glutamy
The recombinant vector of amine transaminase mutant code gene；
(3) recombinant vector is converted to host cell, obtains recombinant bacterial strain；
(4) the expression recombinant bacterial strain obtains the mutation of the glutamine transaminage as shown in SEQ ID NO.6 after purification
Body BTGM；
After the expression of identical expression system, the specific enzyme activity of mutant BTGM is 2.3 times of wild type BTG.
The utility model has the advantages that
1, glutamine transaminase mutant gene of the present invention is expressed in expression system in bacillus, is obtained
To glutamine transaminage mutant recombinant bacterial strain, after recombinant bacterial strain fermentation, being handled accordingly can be obtained glutamine and turns ammonia
Enzyme mutant.
2, the present invention uses fallibility round pcr, carries out random mutation to wild type glutamine transaminage, obtains glutamy
Amine transaminase mutant BTGM, specific enzyme activity are 2.3 times of wild type glutamine transaminage specific enzyme activity.
Detailed description of the invention:
Fig. 1 is the PCR amplification electropherogram of wild type aminotransierase gene of glutamine；
Wherein: M DNAMarker, 1 is glutamine transaminage prochymosin gene btg；2 are mutated for glutamine transaminage
Body gene btgm；
Fig. 2 is JM109/pBAS43-btg and JM109/pBAS43-btgm digestion verification figure；
Wherein: M DNAMarker, 1 is pBSA43-btg through BamH I and Hind Ш double digestion；2 are
PBSA43-btgm is through BamH I and Hind Ш double digestion；
Fig. 3 is recombinant plasmid pBSA43-btg digestion verification figure of the present invention；
Wherein: M DNAMarker 1,2,3 is bacillus subtilis, bacillus amyloliquefaciens, weight in bacillus licheniformis
Plasmid pBSA43-btg is through BamH I and Hind Ш double digestion figure for group；
Fig. 4 is recombinant plasmid pBSA43-btgm digestion verification figure of the present invention
Wherein: M DNAMarker 1,2,3 is bacillus subtilis, bacillus amyloliquefaciens, weight in bacillus licheniformis
Plasmid pBSA43-btgm is through BamH I and Hind Ш double digestion figure for group；
Specific embodiment:
In order to which the objects, technical solutions and advantages of this patent are more clearly understood, below in conjunction with specific embodiment, to this
Patent is further elaborated.It should be appreciated that specific embodiment described herein is only to explain this patent, and do not have to
It is of the invention in limiting.
Bacillus licheniformis used in the present invention is TCCC11965, is disclosed in: Development and
application of a CRISPR/Cas9system for Bacillus licheniformis genome editing
[J] .International Journal of Biological Macromolecules, 2019,122:329-337, at present
It is stored in Microbiological Culture Collection administrative center, University Of Science and Technology Of Tianjin, the public can obtain strain from the center.
The acquisition of 1 wild type aminotransierase gene of glutamine btg of embodiment
1. bacillus subtilis (Bacillus subtilis) thallus ATCC 23857 is ground in liquid nitrogen, according to base
Because group extracts kit specification extracts Bacillus subtilis genes group.
2. being logged in using the genome of the bacillus subtilis of extraction as template according to Genbank sequence number CP032315.1
Glutamine transaminage sequence, ORF frame upstream and downstream design pair of primers, respectively introduce restriction enzyme site BamH I,
The amplimer of Hind III, aminotransierase gene of glutamine of the invention are as follows:
Upstream primer P1 (SEQ ID NO.1):
5’-CGCGGATCCGATGATTATTGTATCAGGACAATTGC-3’
Downstream primer P2 (SEQ ID NO.2):
5’-CCCAAGCTTTTAGCGGACGATGCGGA-3’
Using P1 and P2 as upstream and downstream primer, carried out by template of bacillus subtilis aminotransierase gene of glutamine group
Amplification；
Its reaction condition expanded are as follows:
| Upstream primer P1 | 1.5μL |
| Downstream primer P2 | 1.5μL |
| DNA profiling | 2.0μL |
| Pyrobest enzyme | 0.5μL |
| ddH2O | 34.5μL |
Amplification condition are as follows: 95 DEG C of initial denaturation 10min；94 DEG C of denaturation 30s, 54 DEG C of annealing 45s, 72 DEG C of extension 1min react 30
A circulation；72 DEG C of extension 10min.Pcr amplification product obtains the band (Fig. 1) of 735bp through 0.8% agarose gel electrophoresis, uses
Miniprep dna QIAquick Gel Extraction Kit recycles PCR product, obtains the proenzyme area gene btg of glutamine transaminage of the invention, will expand
The btg of acquisition is connect with carrier pBSA43 carrier, recombinant plasmid pBSA43-btg is obtained, and change and be transferred in JM109, through double digestion
It verifies (Fig. 2), such as SEQ ID NO.3 after sequencing.
The acquisition of 2 glutamine transaminage mutant gene of embodiment
1, random mutation is carried out based on fallibility round pcr, constructs novel glutamine transaminage, design primer is as follows:
Upstream primer P1 (SEQ ID NO.1):
5’-CGCGGATCCGATGATTATTGTATCAGGACAATTGC-3’
Downstream primer P2 (SEQ ID NO.2):
5’-CCCAAGCTTTTAGCGGACGATGCGGA-3’
In fallibility PCR reaction system, using P1 and P2 as upstream and downstream primer, with wild type aminotransierase gene of glutamine
Btg is template, carries out fallibility PCR.
Its reaction condition expanded are as follows:
| 10 × PCR buffer (no Mg2+) | 10μL |
| dATP | 0.2μL |
| dGTP | 0.2μL |
| dCTP | 1.0μL |
| dTTP | 1.0μL |
| Upstream primer P1 | 1.5μL |
| Downstream primer P2 | 1.5μL |
| Wild type aminotransierase gene of glutamine | 1.0μL |
| Taq archaeal dna polymerase | 1.0μL |
| Mg2+(7mM) | 28μL |
| Mn2+(0.15mM) | 2μL |
| ddH2O | 52.6μL |
Amplification condition are as follows: 95 DEG C of initial denaturation 10min；94 DEG C of denaturation 30s, 54 DEG C of annealing 45s, 72 DEG C of extension 1min react 30
A circulation；72 DEG C of extension 10min.
2, the glutamine transaminage mutant gene of acquisition is cloned into respectively in expression vector pBSA43, is obtained several
Recombinant plasmid pBSA43-btgmx, and change and be transferred in JM109, recombinant plasmid pBSA43-btg and pBSA43-btgmx are converted withered
In careless bacillus, recombinant bacterium is inoculated in the LB liquid medium (containing kanamycins, 50 μ g/mL) of 5mL, 37 DEG C, 220r/
Min overnight incubation continues according to the switching of 2% inoculum concentration in 50mL fresh LB with 37 DEG C, and 220r/min cultivates 48h,
Glutamine transaminage crude enzyme liquid can be prepared, glutamine transaminage is then precipitated using salt fractionation method, collects egg
White matter precipitating, after dissolution, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, be freeze-dried obtained glutamine and turn ammonia
The pure enzyme enzyme powder of enzyme, measures enzyme activity after dissolved in purified water.
3, glutamine transaminage enzyme activity determination
Reaction solution: with the reaction solution of Tris-HCl (pH=7.5) the configuration 300 μ L of total volume of 50mM: N, N- dimethyl junket
Albumen 0.2%, red 12.5 μ Μ, DTT 4.5mM of sulphur cadaverine (MDC).
Enzyme activity determination method is as follows:
Take the reaction solution (pH 7.5) of 200 μ L in 96 fluorescent plates, 37 DEG C of heat preservation 1min, the dilution that 100 μ L are added is fitted
When the enzyme solution of multiple, it is control so that the Tris-HCl buffer without enzyme is added, after reacting 20min, records excitation wavelength 350nm
With fluorescence intensity under launch wavelength 500nm.The MDC of catalysis 1nmol/L is inserted into N, mono- casein institute of N per minute
The enzyme amount needed is defined as a unit of activity.
Enzyme activity calculates:
Wherein:
I represents the fluorescence intensity of product after reaction.
I0Represent the fluorescence intensity of not enzyme control.
[MDC] is the concentration of MDC in reaction system.
Specific enzyme activity (U/g)=enzyme activity/protein concentration.
Compared with wild type glutamine transaminage enzyme activity, one plant of producing enzyme vigor will be filtered out after all mutant survey enzyme activity
It is 2.3 times of bacterial strain of wild type.
4, sequencing
Above-mentioned bacterial strains are extracted into aminotransierase gene of glutamine sequence, pcr amplification product is through 0.8% Ago-Gel electricity
Swimming, obtains the band (Fig. 1) of 735bp, and (Beijing Hua Da bio-engineering corporation) is sequenced, the results showed that, amplification obtains
Glutamine transaminage mutant gene nucleotide sequence is named as btgm as shown in SEQ ID NO.5, by the encoding gene.
The building of 3 bacillus mutant glutamine transaminage recombinant bacterium of embodiment
1, the building of expression vector pBSA43
PBSA43 is to be cloned into a strong brood cell using Escherichia coli-bacillus shuttle cloning vector pBE2 as skeleton
Bacillus constitutive promoter P43, and recombinant protein can be made directly to be secreted into levansucrase signal sequence in culture medium
SacB and obtain.It has AmprGene, can be in Escherichia coli using amicillin resistance as selection markers；Simultaneously
Also there is KmrGene, can be in Bacillus subtillis, bacillus licheniformis using kalamycin resistance as selection markers.
Referring to Chinese invention patent, " bacillus amyloliquefaciens add precursor fermentation method and produce antiviral drugs for the building of pBSA43 plasmid
The production technology of Ribavirin " CN 103146785B.
2, the building of glutamine transaminage expression vector pBSA43-btgm
It will be through PCR amplification and the glutamine transaminage mutant gene that is recycled after BamH I and Hind III double digestion
Btgm and same double digestion expression vector pBSA43 is attached with ligase, and connection product is converted e. coli jm109 sense
By in state cell, through Amp resistance screening, positive transformant is selected, extracts transformant plasmid, and carry out double digestion verifying (Fig. 2)
And sequencing, determine that building obtains correct recombinant bacterial strain JM109/pBSA43-btgm.
3, recombinant expression carrier pBSA43-btgm and pBSA43-btg converts Bacillus subtillis, bacillus licheniformis, solution
Bacillus amylobacter.
PBSA43-btgm the and pBSA43-btg recombinant plasmid of 1 μ L (50ng/ μ L) is added separately to the withered grass bud of 50 μ L
Born of the same parents bacillus WB600, bacillus amyloliquefaciens CGMCC No.11218, in bacillus licheniformis TCCC11965 competent cell simultaneously
It mixes, is transferred in the electric revolving cup (1mm) of pre-cooling later, after ice bath 1-1.5min, shock by electricity primary (25 μ F, 200 Ω, 4.5-
5.0ms).After electric shock finishes, 1mL recovery medium (LB+0.5mol/L sorbierite+0.38mol/L mannitol) is added immediately.
37 DEG C of shaking tables after shake culture 3h, recovery object are coated on LB plate containing kanamycin, 37 DEG C of culture 12-24h,
Picking positive transformant, and double digestion verifying (Fig. 3) (Fig. 4) is carried out, it determines and obtains expression mutant gene btgm and wild type
The Bacillus subtillis recombinant bacterial strain of gene btg, bacillus amyloliquefaciens recombinant bacterial strain, the recombination of bacillus licheniformis bacillus
Bacterial strain is respectively designated as WB600/pBSA43-probtgm, WB600/pBSA43-probtg, CGMCC No.11218/
pBSA43-probtgm、CGMCC No.11218/pBSA43-probtg、TCCC11965/pBSA43-probtgm、
TCCC11965/pBSA43-probtg。
Expression and preparation of the 4 glutamine transaminage mutant of embodiment in Bacillus subtillis recombinant bacterial strain.
Respectively by Bacillus subtillis mutant recombinant bacterial strain WB600/pBSA43-probtgm and wild type recombinant bacterium
WB600/pBSA43-probtg is inoculated in the LB liquid medium (containing kanamycins, 50 μ g/mL) of 5mL, and 37 DEG C, 220r/
Min overnight incubation continues according to the switching of 2% inoculum concentration in 50mL fresh LB with 37 DEG C, and 220r/min cultivates 48h,
Glutamine transaminage crude enzyme liquid can be prepared, glutamine transaminage is then precipitated using salt fractionation method, collects egg
White matter precipitating, after dissolution, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, be freeze-dried obtained glutamine and turn ammonia
The pure enzyme enzyme powder of enzyme measures enzyme activity using the method for embodiment 2 after dissolved in purified water, the results show that wild type BTG specific enzyme activity is
223U/g, mutant BTGM specific enzyme activity are 529U/g, improve 2.37 times compared with wild type BTG enzyme activity.
Expression and preparation of the 5 glutamine transaminage mutant of embodiment in bacillus amyloliquefaciens recombinant bacterial strain
Respectively by bacillus amyloliquefaciens mutant recombinant bacterial strain CGMCC No.11218/pBSA43-probtgm and wild
Type recombinant bacterium CGMCC No.11218/pBSA43-probtg is inoculated in the LB liquid medium of 5mL (containing kanamycins, 50 μ g/
ML in), 37 DEG C, 220r/min overnight incubation continues according to the switching of 2% inoculum concentration in 50mL fresh LB with 37 DEG C,
220r/min cultivates 48h, can prepare glutamine transaminage crude enzyme liquid, then precipitates glutamy using salt fractionation method
Amine transaminase collects protein precipitation, after dissolution, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, freeze-drying
The pure enzyme enzyme powder of glutamine transaminage is made, enzyme activity is measured using the method for embodiment 2 after dissolved in purified water, the results show that
Wild type glutamine transaminage specific enzyme activity is 251U/g, and mutant BTGM specific enzyme activity is 586U/g, and mutant enzyme vigor is wild
2.33 times of raw type enzyme activity.
Expression and preparation of the 6 glutamine transaminage mutant of embodiment in bacillus licheniformis recombinant bacterial strain
Respectively by wild type recombinant bacterium TCCC11965/pBSA43-probtg and bacillus licheniformis mutant recombinant bacterial strain
TCCC11965/pBSA43-probtgm is inoculated in the LB liquid medium (containing kanamycins, 50 μ g/mL) of 5mL, and 37 DEG C,
220r/min overnight incubation continues according to the switching of 2% inoculum concentration in 50mL fresh LB with 37 DEG C, 220r/min is trained
48h is supported, glutamine transaminage crude enzyme liquid can be prepared, glutamine transaminage is then precipitated using salt fractionation method,
Protein precipitation is collected, after dissolution, desalination of dialysing, then after ion-exchange chromatography, gel chromatography, be freeze-dried and glutamy is made
The pure enzyme enzyme powder of amine transaminase measures enzyme activity using the method for embodiment 2 after dissolved in purified water, the results show that wild type paddy ammonia
Amide transaminase specific enzyme activity power is 268U/g, and mutant BTGM specific enzyme activity is 643U/g, and mutant enzyme vigor is wild type enzyme activity
2.40 times of power.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
The limitation to the scope of the patents therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Under the premise of not departing from this patent design, the respective embodiments described above can also make several deformations, combination and improve, these all belong to
In the protection scope of this patent.Therefore, the protection scope of this patent should be subject to the claims.
Sequence table
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<120>a kind of glutamine transaminage mutant and its preparation method and application
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<212> PRT
<213>artificial sequence ()
<400> 6
Met Ile Ile Val Ser Gly Gln Leu Leu Arg Pro Gln Asp Ile Glu Asn
1 5 10 15
Trp Gln Ile Asp Gln Asp Leu Asn Pro Leu Leu Lys Glu Met Ile Glu
20 25 30
Thr Pro Val Gln Phe Asp Tyr His Ser Ile Ala Glu Leu Met Phe Glu
35 40 45
Leu Lys Leu Arg Met Asn Ile Val Ala Ala Ala Lys Thr Leu His Lys
50 55 60
Ser Gly Ala Lys Phe Ala Thr Phe Leu Lys Thr Tyr Gly Asn Thr Thr
65 70 75 80
Tyr Trp Arg Val Ser Pro Glu Gly Ala Leu Glu Leu Lys Tyr Gly Met
85 90 95
Pro Pro Ser Lys Ala Ile Arg Asp Ile Ala Glu Asn Gly Pro Phe Tyr
100 105 110
Ala Phe Glu Cys Ala Thr Ala Ile Val Ile Ile Tyr Tyr Leu Ala Leu
115 120 125
Ile Asp Thr Ile Gly Glu Asp Lys Phe Asn Ala Ser Phe Asp Arg Ile
130 135 140
Ile Leu Tyr Asp Trp His Tyr Glu Lys Leu Pro Ile Tyr Thr Glu Thr
145 150 155 160
Gly His His Phe Phe Leu Gly Asp Cys Leu Tyr Phe Lys Asn Pro Glu
165 170 175
Phe Asp Pro Gln Lys Ala Gln Trp Arg Gly Glu Asn Val Ile Leu Leu
180 185 190
Gly Glu Asp Lys Tyr Phe Ala His Gly Leu Gly Ile Leu Asn Gly Lys
195 200 205
Gln Ile Ile Asp Lys Leu Asn Ser Phe Arg Lys Lys Gly Ala Leu Gln
210 215 220
Ser Ala Tyr Leu Leu Ser Gln Ala Thr Arg Leu Asp Val Pro Ser Leu
225 230 235 240
Phe Arg Ile Val Arg
245
Claims (7)
1. a kind of glutamine transaminage mutant, which is characterized in that the amino acid sequence of the mutant such as sequence table SEQ
Shown in ID No.6.
2. the encoding gene of glutamine transaminage mutant described in claim 1.
3. the encoding gene of glutamine transaminage mutant described in claim 2, which is characterized in that such as sequence table SEQ ID
Shown in No.5.
4. a kind of recombinant vector or recombinant bacterial strain comprising gene described in claim 2.
5. the recombinant vector or recombinant bacterial strain stated such as claim 4, which is characterized in that expression vector pBSA43；Host cell
For bacillus subtilis WB600 or bacillus amyloliquefaciens CGMCC No.11218 or bacillus licheniformis
TCCC11965。
6. application of the recombinant vector or recombinant bacterial strain described in claim 4 in production glutamine transaminage.
7. the application of glutamine transaminage mutant described in claim 1.
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| CN111944778A (en) * | 2020-08-14 | 2020-11-17 | 安徽医学高等专科学校 | Glutamine transaminase mutant and encoding gene and application thereof |
| CN112391364A (en) * | 2020-11-02 | 2021-02-23 | 天津科技大学 | High-activity glutamine transaminase mutant and preparation method thereof |
| CN115161303A (en) * | 2019-11-21 | 2022-10-11 | 天津科技大学 | Phospholipase mutant and method for synthesizing glycerophospholipid by using same |
| CN115850086A (en) * | 2022-11-09 | 2023-03-28 | 重庆普佑生物医药有限公司 | Preparation method of ticagrelor intermediate and key intermediate compound |
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| CN112391364A (en) * | 2020-11-02 | 2021-02-23 | 天津科技大学 | High-activity glutamine transaminase mutant and preparation method thereof |
| CN115850086A (en) * | 2022-11-09 | 2023-03-28 | 重庆普佑生物医药有限公司 | Preparation method of ticagrelor intermediate and key intermediate compound |
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