CN107586764B - Glutamine transaminase mutant, gene, engineering bacteria and preparation method thereof - Google Patents
Glutamine transaminase mutant, gene, engineering bacteria and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to a glutamine transaminase mutant, a gene, an engineering bacterium and a preparation method thereof. The mutant is obtained by mutating and screening a target gene by an error-prone PCR method, and the specific enzyme activity of the mutant after expression in a hay expression system is respectively improved by 19% and 27% compared with that of wild glutamine transaminase, so that the mutant has a wide application prospect.
Description
The technical field is as follows:
the invention belongs to the technical field of bioengineering, and particularly relates to a glutamine transaminase mutant, a gene, an engineering bacterium and a preparation method thereof.
Technical background:
transglutaminase (TGase), an enzyme that catalyzes acyl transfer reactions, interacts with epsilon-amino groups on lysine residues and gamma-hydroxyamide groups on glutamine residues in protein molecules to form epsilon- (gamma-glutamyl) lysine bonds, resulting in covalent cross-linking between proteins (or polypeptides). Glutamine transaminase can change the structure and functional properties of protein, thereby improving the nutritional value of protein. Therefore, the glutamine transaminase has wide application prospect in the fields of food, textile, biological pharmacy and the like.
In the food industry, the appearance, flavor and texture of meat products can be improved by glutamine transaminase to improve the added value of the products. For example, in the process of forming gel by fish, the gel strength of the fish product can be improved by adding glutamine transaminase, the cooking loss is reduced, and the product quality is improved. The addition of glutamine transaminase to flour during the processing of baked food and flour products can increase the stability of the dough, improve the elasticity and extensibility of the dough, and thus increase the quality of the bread.
In the textile industry, the addition of glutamine transaminase to wool fabrics can improve the fiber strength of wool fabrics and reduce the damage to wool fabric fibers during hydrolysis treatment.
However, some defects of the glutamine transaminase itself, such as specific enzyme activity, thermal stability and the like, limit the application range of the glutamine transaminase, for example, the biological membrane forming process catalyzed by the glutamine transaminase, and the quality of membrane products is reduced due to the low activity of the glutamine transaminase under the high temperature condition.
The directed evolution of enzyme molecules in vitro belongs to the irrational design of proteins, and is a new strategy of protein engineering. The molecular diversity is created at the molecular level by means of molecular biology, and an ideal mutant is rapidly obtained by combining a sensitive screening technology. It does not need to know the space structure, active site, catalytic mechanism and other factors of protein in advance, but artificially creates a special evolutionary condition evolutionary mechanism, and modifies enzyme gene in vitro to obtain structural enzyme with some expected characteristics. Wherein, error-prone PCR means that when a target gene is amplified, the Taq enzyme does not have 3 '→ 5' proofreading function, and Mn in a reaction system is changed2+、Mg2+And the concentration of various dNTPs, and randomly introducing base mismatch to the target gene at a certain frequency to cause random mutation of the target gene. However, satisfactory results are generally difficult to obtain with a single mutated gene, and thus a Sequential Error-prone PCR (Sequential Error-prone PCR) strategy has been developed. That is, the product obtained by one PCR amplification is used as the template for the next PCR amplification, and the error is easily caused by continuous and repeated operation, so that the small mutation obtained in each time is continuously accumulated to generate important beneficial mutation. Therefore, the method has unique advantages.
Bacillus subtilis belongs to gram-positive bacteria. The bacillus subtilis expression system has the following advantages: 1. can secrete various proteins with high efficiency; 2. many Bacillus subtilis have a long history of use in the fermentation industry, are nonpathogenic, and do not produce any endotoxin; 3. the research on the microbial genetics background of the bacillus genus is very clear, the bacillus genus grows rapidly, and no special requirements on nutrient substances exist; 4. codon preference is not obvious; 5. the fermentation process is simple, the bacillus subtilis belongs to aerobic bacteria, anaerobic fermentation equipment is not needed, and after the fermentation is finished, fermentation liquor and bacterial thalli are simply separated, so that the separation, purification and recovery stages of target protein can be carried out; 6. has stress resistance, and can be used for producing various thermostable enzyme preparations.
Therefore, in the invention, based on an expression platform of the glutamine transaminase in escherichia coli, the error-prone PCR technology is utilized to carry out molecular modification on the glutamine transaminase gene promtg derived from the streptomyces mobaraensis, and the molecular modification is carried out in a bacillus subtilis system, so as to obtain the glutamine transaminase with improved enzyme activity.
The invention content is as follows:
the invention aims to provide a novel glutamine transaminase, a gene, an engineering bacterium and a preparation method thereof.
The technical circuit for realizing the purpose of the invention is as follows:
extracting a streptomyces mobaraensis genome, obtaining a wild type glutamine transaminase promtg gene (shown in SEQ ID NO. 3) sequence (shown in SEQ ID NO. 4) with an zymogen region through PCR amplification, and randomly mutating the wild type promtg gene obtained through amplification through error-prone PCR to obtain two mutant genes, namely promtgm1 and promtgm 2. Constructing a recombinant vector by the mutant gene and successfully expressing in the bacillus subtilis WB600 to obtain a recombinant strain with improved enzyme production activity, and further optimizing by a fermentation process to obtain the novel glutamine transaminase.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in the form of a three letter code. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of Glutamine transaminase mutants
"amino acid substituted at the original amino acid position" is used to indicate a mutated amino acid in a mutant transglutaminase. Such as Glu209Leu, indicating the substitution of the amino acid at position 209 from Glu of the wild-type transglutaminase to Leu, the numbering of the positions corresponding to the numbering of the amino acid sequence of the wild-type transglutaminase in SEQ ID NO. 4; if G625 is used, the base at position 625 is G, and the numbering of the positions corresponds to the numbering of the nucleotide sequence of the wild-type transglutaminase of SEQ ID NO. 3.
In the present invention, promtg represents a wild-type glutamine transaminase coding gene, and promtgm1 and promtgm2 are coding genes of glutamine transaminase mutants MTG1 and MTG2, respectively; base and amino acid controls before and after mutation are as follows:
| glutamine transaminase | Base | Amino acids |
| Wild type | G217、A218、G219、G625、A626 | Glu73、Glu209 |
| MTG1 | G217、A218、G219、C625、T626 | Glu209Leu |
| MTG2 | T217、T218、C219、C625、T626 | Glu73Phe、Glu209Leu |
The amino acid sequence of the glutamine transaminase mutant MTG1 is shown in a sequence table SEQ ID NO. 6;
the amino acid sequence of the glutamine transaminase mutant MTG2 is shown in a sequence table SEQ ID NO. 8;
the nucleotide sequence of the promtgm1 gene is shown in a sequence table SEQ ID NO. 5;
the nucleotide sequence of the promtgm2 gene is shown in a sequence table SEQ ID NO. 7;
the host cell for expressing the mutant gene is bacillus subtilis WB600, and the expression vector is pBSA 43.
The invention also provides a preparation method of the glutamine transaminase mutant, which comprises the following steps:
(1) randomly mutating a transglutaminase gene of Streptomyces mobaraensis by error-prone PCR to obtain a transglutaminase mutant gene shown in SEQ ID NO.5 and SEQ ID NO. 7;
(2) carrying out enzyme digestion on the glutamine transaminase mutant gene, and connecting the enzyme digestion product to an expression vector to obtain a recombinant vector carrying a glutamine transaminase mutant coding gene;
(3) transforming the recombinant vector into a host cell to obtain a recombinant strain;
(4) expressing the recombinant strain, and purifying to obtain glutamine transaminase mutants MTG1 and MTG2 shown as SEQ ID NO.6 and SEQ ID NO. 8;
the specific enzyme activity of the mutant MTG1 is improved by 19 percent compared with that of the wild MTG, and the specific enzyme activity of the mutant MTG2 is improved by 27 percent compared with that of the wild MTG.
Has the advantages that:
1. the glutamine transaminase mutant gene is expressed in a hay expression system to obtain a glutamine transaminase mutant recombinant strain, and the glutamine transaminase mutant can be obtained by corresponding treatment after the recombinant strain is fermented.
2. The invention uses error-prone PCR technology to carry out random mutation on wild type glutamine transaminase to respectively obtain a glutamine transaminase mutant MTG1 and a glutamine transaminase mutant MTG2, and the specific enzyme activity is respectively improved by 19 percent and 27 percent compared with that of the wild type glutamine transaminase;
description of the drawings:
FIG. 1 is a PCR amplification electrophoretogram of wild-type transglutaminase gene promtg;
wherein: m is DNA Marker, 1 is glutamine transaminase zymogen gene promtg;
FIG. 2 shows the restriction enzyme digestion verification map of recombinant plasmid pET22 b-promtg;
wherein M is DNA Marker, 1 is pET22b-promtg, and the double enzyme digestion is carried out by BamH I and Hind Ш;
FIG. 3 shows the restriction enzyme digestion verification of recombinant plasmids pET22b-promtgm1 and pET22b-promtgm2, wherein M is DNA Marker, 1 is pET22b-promtgm1, which is subjected to double restriction by BamH I and Hind Ш;
2, pET22b-promtgm2 is subjected to double enzyme digestion by BamH I and Hind Ш;
FIG. 4 shows the restriction enzyme digestion verification of recombinant plasmids pBSA43-promtgm1 and pBSA43-promtgm2, wherein M is DNA Marker, 1 is pBSA43-promtgm1, and BamH I and Hind Ш are used for double restriction enzyme digestion;
2 pBSA43-promtgm2 is subjected to double enzyme digestion by BamH I and Hind Ш;
FIG. 5 shows the principle of enzyme activity and color development in Grossowicz colorimetric method.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
EXAMPLE 1 acquisition of wild-type Glutamine transaminase Gene promtg
1. Streptomyces mobaraensis (Streptomyces mobaraensis) CICC 11018 thalli are ground in liquid nitrogen, and a Streptomyces mobaraensis genome is extracted according to the genome extraction kit specification.
2. Taking the extracted genome of streptomyces mobaraensis as a template, designing a pair of primers at the upstream and downstream of an ORF frame according to a glutamine transaminase sequence registered by a Genbank sequence number AY241675.1, and respectively introducing restriction enzyme sites BamH I and Hind III, wherein the amplification primers of the glutamine transaminase gene are as follows:
upstream primer P1(SEQ ID NO. 1):
5’-CGCGGATCCGGGCAGCGGCACCGGGGAAG-3’
downstream primer P2(SEQ ID NO. 2):
5’-CCCAAGCTTTCACGGCCAGCCCTGTGTCACCT-3’
taking P1 and P2 as upstream and downstream primers, and taking Streptomyces mobaraensis glutamine transaminase genome as a template for amplification;
the reaction conditions for amplification are as follows:
| upstream primer P1 | 1.5uL |
| Downstream primer P2 | 1.5uL |
| DNA template | 2.0uL |
| Pyrobest enzyme | 0.5uL |
| ddH2O | 34.5uL |
The amplification conditions comprise pre-denaturation at 95 ℃ for 10min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 45s, extension at 72 ℃ for 1min and reaction at 20s for 30 cycles, extension at 72 ℃ for 10min, electrophoresis of PCR amplification products through 0.8% agarose gel to obtain a 1147bp band (figure 1), recovery of PCR products by using a small amount of DNA recovery kit to obtain the proenzyme region gene promtg of the glutamine transaminase, connection of the promtg obtained by amplification with a pET22b vector to obtain a recombinant plasmid pET22b-promtg, and transformation into DH5 α, and sequencing to obtain SEQ ID NO. 3.
EXAMPLE 2 acquisition of Glutamine transaminase mutant Gene
1. Random mutation is carried out based on an error-prone PCR technology to construct a novel glutamine transaminase, and primers are designed as follows:
upstream primer P1(SEQ ID NO. 1):
5’-CGCGGATCCGGGCAGCGGCACCGGGGAAG-3’
downstream primer P2(SEQ ID NO. 2):
5’-CCCAAGCTTTCACGGCCAGCCCTGTGTCACCT-3’
in the error-prone PCR reaction system, P1 and P2 are used as upstream and downstream primers, and wild-type glutamine transaminase mature peptide is used as a template to perform error-prone PCR.
The reaction conditions for amplification are as follows:
the amplification conditions were: pre-denaturation at 95 ℃ for 10 min; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 45s, and extension at 72 ℃ for 1min for 30s reactions for 30 cycles; extension at 72 ℃ for 10 min.
2. Cloning glutamine transaminase mutant gene into expression vector pET22b, transforming Escherichia coli BL21(DE3), inoculating in 96-well cell culture plate containing 200 μ LLB liquid culture medium (containing 50 μ g/mL Amp) per well, shaking culturing at 37 deg.C for 200r/min, adding IPTG (final concentration of 1mmol/L) to each well when OD600 reaches 0.6, inducing at 16 deg.C for 16h, centrifuging at 4000r/min at 4 deg.C for 15min, collecting thallus, suspending in 15mL precooled PBS buffer solution with pH of 7.4, crushing cells with low-temperature ultrahigh-pressure continuous flow cell crusher, after crushing, centrifuging at 12000r/min at 4 deg.C for 45min, collecting supernatant to obtain crude enzyme solution of transglutaminase, incubating at 37 deg.C with 200 μ g/mL neutral protease solution for 20min to activate proenzyme of transglutaminase, and detecting enzyme activity.
3. Enzymatic activity assay of glutamine transaminases
The principle of determining enzyme activity by using a Grossowicz colorimetric method is as follows:
the enzyme activity is measured by Grossowicz colorimetric method, α -N-CBZ-GLN-GLY is taken as an action substrate, L-glutamic acid-gamma-monohydroxyamino acid is taken as a standard curve, and the chromogenic reaction is shown in figure 5.
The reagents required for enzyme activity determination are as follows:
reagent A1 g of α -N-CBZ-GLN-GLY was dissolved in 2mL of 0.2mol/L NaOH, and 4mL of 0.2mol/L Tris-HCI buffer solution (pH 6.0), 2mL of 0.l mol/L hydroxylamine, and 2mL of 0.01mol/L glutathione were added to adjust the pH to 6.0.
And (3) reagent B: 3mol/L hydrochloric acid, 12% TCA and 5% FeCl3According to the following steps: 1: 1, and mixing.
The enzyme activity determination method comprises the following steps:
taking 200 mu L of citric acid-disodium hydrogen phosphate buffer solution (pH 6.0) to a 96-well plate, preserving the temperature for 1min at 37 ℃, adding 10 mu L of enzyme solution diluted by a proper time, adding 10 mu L of substrate reagent A, adding 100 mu L of termination reagent B after reacting for 10min, and recording the absorbance value at the wavelength of 525 nm.
1 unit of transglutaminase enzyme activity is defined as: the amount of enzyme (U/mL) catalyzing the formation of 1. mu. mol L-glutamic acid-. gamma. -monohydroxyamino acid per minute at 37 ℃.
After the enzyme activity of all mutants is measured, comparing the enzyme activity with that of wild glutamine transaminase, two strains with enzyme production activity improved by 19 percent and 27 percent compared with that of the wild strains are screened out.
4. Sequence determination
The two glutamine transaminase mutant gene sequences are sequenced (Beijing Hua great bioengineering company), and the results show that the glutamine transaminase mutant gene promtgm1 and the glutamine transaminase mutant gene promtgm2 are obtained by amplification, and the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO. 7.
Example 3 construction of novel recombinant bacterium of Glutamine transaminase derived from Bacillus subtilis
1. Construction of expression vector pBSA43
pBSA43 is obtained by using an escherichia coli-bacillus subtilis shuttle cloning vector pBE2 as a framework, cloning a strong bacillus constitutive promoter P43 and directly secreting recombinant protein into a levansucrase signal sequence sacB in a culture medium. It carries AmprGenes allowing the use of ampicillin in E.coliThe resistance of the hormone is used as a screening marker; also has KmrThe gene can be used as a screening marker in bacillus subtilis and bacillus licheniformis by utilizing kanamycin resistance. The construction of pBSA43 plasmid is described in Chinese patent CN 103146785B of the invention 'production process of antiviral drug ribavirin by fermentation of Bacillus amyloliquefaciens with precursor added'.
2. Construction of Glutamine transaminase expression vectors pBSA43-promtgm1 and pBSA43-promtgm2
The glutamine transaminase mutant gene which is amplified by PCR and recovered by double enzyme digestion of BamH I and Hind III is connected with a Bacillus subtilis expression vector pBSA43 which is subjected to the same double enzyme digestion by ligase, the connection product is transformed into escherichia coli JM109 competent cells, positive transformants are selected by Amp resistance screening, transformant plasmids are extracted, single and double enzyme digestion verification and sequencing are carried out, and the correct recombinant JM strain 109/pBSA43-promtgm1 and JM109/pBSA43-promtgm2 are confirmed to be obtained.
3. Recombinant expression vectors pBSA43-promtgm1 and pBSA43-promtgm2 transform Bacillus subtilis WB600
mu.L (50 ng/. mu.L) of pBSA43-promtgm1 and pBSA43-promtgm2 recombinant plasmids were added to 50. mu.L of Bacillus subtilis WB600 competent cells and mixed well, after which they were transferred to a pre-cooled electric rotor (1mm) and shocked once (25uF, 200. omega., 4.5-5.0ms) after ice bath for 1-1.5 min. After the shock was completed, 1mL of recovery medium (LB +0.5mol/L sorbitol +0.38mol/L mannitol) was added immediately. And after shaking culture for 3h at 37 ℃ by a shaker, coating the resuscitate on an LB plate containing kanamycin, culturing for 12-24h at 37 ℃, picking positive transformants, and performing single-enzyme and double-enzyme digestion verification to obtain the bacillus subtilis recombinant strains WB600/pBSA43-promtgm1 and WB600/pBSA43-promtgm 2.
Example 4 expression and preparation of Glutamine transaminase mutants in Bacillus subtilis recombinant strains
The wild-type transglutaminase recombinant bacteria WB600/pBSA43-promtg constructed in the same manner as in example 3 are used as a control, the Bacillus subtilis recombinant strains WB600/pBSA43-promtgm1, WB600/pBSA43-promtgm2 and WB600/pBSA43-promtg are respectively inoculated into 5mL of LB liquid medium (containing kanamycin and 50 mu g/mL), cultured overnight at 37 ℃ at 220r/min, inoculated into 50mL of fresh LB medium according to the inoculation amount of 2%, and cultured at 37 ℃ at 220r/min for 48h to prepare a high-stability transglutaminase crude enzyme solution, and the enzyme activity of the crude enzyme solution is measured. The enzyme activity of the wild glutamine transaminase is 20.1U/mL, the enzyme activity of the mutant MTG1 is 23.9U/mL, the enzyme activity is improved by 19 percent compared with the wild MTG, the enzyme activity of the mutant MTG2 is 25.5U/mL, and the enzyme activity is improved by 27 percent compared with the wild MTG. Then, a fractional salting-out method is adopted to precipitate the novel glutamine transaminase, protein precipitate is collected and dissolved, dialysis is carried out to remove salt, and freeze drying is carried out after ion exchange chromatography and gel chromatography to prepare the novel pure glutamine transaminase powder.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
SEQUENCE LISTING
<110> Tianjin science and technology university
<120> glutamine transaminase mutant, gene, engineering bacteria and preparation method thereof
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Leu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser
210 215 220
Phe Lys Asp Arg Asn Gly Gly Asn His Asp Pro Ser Lys Met Lys Ala
225 230 235 240
Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Gly Ser
245 250 255
Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala Phe Arg Pro Asp Arg
260 265 270
Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser
275 280 285
Pro Thr Ser Pro Gly Glu Ser Phe Val Asn Phe Asp Tyr Gly Trp Phe
290 295 300
Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val TrpThr His Gly
305 310 315 320
Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr
325 330 335
Glu Ser Lys Phe Arg Asn Trp Ser Asp Gly Tyr Ser Asp Phe Asp Arg
340 345 350
Gly Ala Tyr Val Val Thr Phe Val Pro Lys Ser Trp Asn Thr Ala Pro
355 360 365
Asp Lys Val Thr Gln Gly Trp Pro
370 375
<210>7
<211>1128
<212>DNA
<213> Artificial sequence
<400>7
ggcagcggca ccggggaaga gaagaggtcc tacgccgaaa cgcaccgcct gacggcggat 60
gacgtcgacg acatcaacgc gctgaacgaa agcgctccgg ccgcttcgag cgccggtccg 120
tccttccggg cccccgactc cgacgagcgg gtgactcctc ccgccgagcc gctcgaccgg 180
atgcccgacc cgtaccggcc ctcgtacggc agggccttca cgatcgtcaa caactacata 240
cgcaagtggc agcaggtcta cagccaccgc gacggcagga aacagcagat gaccgaggaa 300
cagcgggagt ggctgtccta cggttgcgtc ggtgtcacct gggtcaactc gggccagtat 360
ccgacgaaca ggctggcttt cgcgttcttc gacgaggaca agtacaagaa cgagctgaag 420
aacggcaggc cccggtccgg cgaaacgcgg gcggagttcg aggggcgcgt cgccaaggac 480
agcttcgacg aggcgaaggg gttccagcgg gcgcgtgacg tggcgtccgt catgaacaag 540
gccctggaga acgcccacga cgagggggcg tacctcgaca acctcaagaa ggagctggcg 600
aacggcaacg acgccctgcg gaacctggat gcccgctcgc ccttctactc ggcgctgcgg 660
aacacgccgt ccttcaagga ccgcaacggc ggcaatcacg acccgtccaa gatgaaggcc 720
gtcatctact cgaagcactt ctggagcggc caggaccggt cgggctcctc cgacaagagg 780
aagtacggcg acccggaggc cttccgcccc gaccgcggca ccggcctggt cgacatgtcg 840
agggacagga acattccgcg cagccccacc agccccggcg agagtttcgt caatttcgac 900
tacggctggt tcggagcgca gacggaagcg gacgccgaca agaccgtatg gacccacggc 960
aaccactacc acgcgcccaa tggcagcctg ggtgccatgc acgtgtacga gagcaagttc 1020
cgcaactggt ccgacggtta ctcggacttc gaccgcggag cctacgtggt cacgttcgtc 1080
cccaagagct ggaacaccgc ccccgacaag gtgacacagg gctggccg 1128
<210>8
<211>376
<212>PRT
<213> Artificial sequence
<400>8
Gly Ser Gly Thr Gly Glu Glu Lys Arg Ser Tyr Ala Glu Thr His Arg
1 5 10 15
Leu Thr Ala Asp Asp Val Asp Asp Ile Asn Ala Leu Asn Glu Ser Ala
20 25 30
Pro Ala Ala Ser Ser Ala Gly Pro Ser Phe Arg Ala Pro Asp Ser Asp
35 40 45
Glu Arg Val Thr Pro Pro Ala Glu Pro Leu Asp Arg Met Pro Asp Pro
50 55 60
Tyr Arg Pro Ser Tyr Gly Arg Ala Phe Thr Ile Val Asn Asn Tyr Ile
65 70 75 80
Arg Lys Trp Gln Gln Val Tyr Ser His Arg Asp Gly Arg Lys Gln Gln
85 90 95
Met Thr Glu Glu Gln Arg Glu Trp Leu Ser Tyr Gly Cys Val Gly Val
100 105 110
Thr Trp Val Asn Ser Gly Gln Tyr Pro Thr Asn Arg Leu Ala Phe Ala
115 120 125
Phe Phe Asp Glu Asp Lys Tyr Lys Asn Glu Leu Lys Asn Gly Arg Pro
130 135 140
Arg Ser Gly Glu Thr Arg Ala Glu Phe Glu Gly Arg Val Ala Lys Asp
145 150 155 160
Ser Phe Asp Glu Ala Lys Gly Phe Gln Arg Ala Arg Asp Val Ala Ser
165 170 175
Val Met Asn Lys Ala Leu Glu Asn Ala His Asp Glu Gly Ala Tyr Leu
180 185 190
Asp Asn Leu Lys Lys Glu Leu Ala Asn Gly Asn Asp Ala Leu Arg Asn
195 200 205
Leu Asp Ala Arg Ser Pro Phe Tyr Ser Ala Leu Arg Asn Thr Pro Ser
210 215 220
Phe Lys Asp Arg Asn Gly Gly Asn His Asp Pro Ser Lys Met Lys Ala
225 230 235 240
Val Ile Tyr Ser Lys His Phe Trp Ser Gly Gln Asp Arg Ser Gly Ser
245 250 255
Ser Asp Lys Arg Lys Tyr Gly Asp Pro Glu Ala Phe Arg Pro Asp Arg
260 265 270
Gly Thr Gly Leu Val Asp Met Ser Arg Asp Arg Asn Ile Pro Arg Ser
275 280 285
Pro Thr Ser Pro Gly Glu Ser Phe Val Asn Phe Asp Tyr Gly Trp Phe
290 295 300
Gly Ala Gln Thr Glu Ala Asp Ala Asp Lys Thr Val Trp Thr His Gly
305 310 315 320
Asn His Tyr His Ala Pro Asn Gly Ser Leu Gly Ala Met His Val Tyr
325 330 335
Glu Ser Lys Phe Arg Asn Trp Ser Asp Gly Tyr Ser Asp Phe Asp Arg
340 345 350
Gly Ala Tyr Val Val Thr Phe Val Pro Lys Ser Trp Asn Thr Ala Pro
355 360 365
Asp Lys Val Thr Gln Gly Trp Pro
370 375
Claims (9)
1. A mutant glutamine transaminase which is characterized in that it has a mutation from Glu to Leu at position 209 based on the amino acid sequence of the wild-type glutamine transaminase as shown in SEQ ID No. 4.
2. The glutamine transaminase mutant according to claim 1, characterized in that the amino acid sequence of the mutant is as shown in SEQ ID No. 6.
3. The mutant glutamine transaminase enzyme according to claim 1, wherein the amino acid sequence of the mutant further comprises a mutation at position 73 from Glu to Phe, and the amino acid sequence is shown in SEQ ID No. 8.
4. The gene encoding a mutant glutamine transaminase of claim 1.
5. The gene encoding a mutant glutamine transaminase according to claim 4, which is represented by SEQ ID No.5 of the sequence Listing.
6. The gene encoding a mutant glutamine transaminase according to claim 3, which is represented by SEQ ID No.7 of the sequence Listing.
7. Use of a mutant transglutaminase described in claim 1 or 3 or a gene described in claim 5 or 6 for transacylation.
8. An expression vector and/or host cell comprising the gene of claim 5 or 6.
9. The expression vector and/or host cell of claim 8, wherein the expression vector is pBSA43 and the host cell is Bacillus subtilis WB 600.
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| CN108841851B (en) * | 2018-07-18 | 2021-12-24 | 中国科学院微生物研究所 | Method for expressing glutamine transaminase by using food-source-safe host |
| CN109943546B (en) * | 2019-04-12 | 2021-08-03 | 天津科技大学 | Glutamine transaminase mutant and preparation method and application thereof |
| CN110616208A (en) * | 2019-11-04 | 2019-12-27 | 泰兴市东圣生物科技有限公司 | Optimized fermentation method of glutamine transaminase based on hydrodynamics |
| CN112553176B (en) * | 2020-12-29 | 2022-04-29 | 江南大学 | Glutamine transaminase with improved thermal stability |
| 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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| CN102719411A (en) * | 2012-06-18 | 2012-10-10 | 华东师范大学 | Microbial transglutaminase and application thereof |
| CN103820411A (en) * | 2013-12-02 | 2014-05-28 | 华东师范大学 | Microbial transglutaminase and daily bleeding stopping products prepared thereby |
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