CN113373126A - Transaminase mutant and coding gene and application thereof - Google Patents
Transaminase mutant and coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to a transaminase mutant, the amino acid sequence of which is SEQ ID NO: 1, and the amino acid sequence of the mutant amino acid sequence is shown in the specification; wherein the mutated amino acid sequence has at least 1 of the following mutation sites: 45 th, 75 th, 108 th, 159 th, 234 th, 272 th bits; the 45 th P is mutated into V or G, the 75 th L is mutated into K, the 108 th G is mutated into E or D, the 159 th T is mutated into Y, H or P, the 234 th L is mutated into C, and the 272 th C is mutated into R or K; the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and has more than 90% homology with the mutated amino acid sequence. The amino acid sequence of the transaminase mutant is shown as SEQ ID NO: 3, the enzyme mutant has high thermal stability and high catalytic activity on the substrate shown in the formula (a).
Description
Technical Field
The invention relates to the field of biotransformation, and particularly relates to a transaminase mutant for catalyzing and preparing chiral amine compounds and a coding gene thereof.
Background
The chiral amine is connected with a chiral centerFunctional group-NH2The compounds are important pesticides and medical intermediates, and chiral amine is used as an intermediate in the synthesis of nerve drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and the like. However, the traditional chemical method for producing chiral amine compounds has complex synthetic route, high cost, harsh reaction conditions, low yield and optical purity of the product, and is also easy to cause serious pollution to the environment.
Transaminase (Aminotransferase, EC 2.6.1.X) is a pyridoxal-5-phosphate dependent enzyme which is widely found in nature and plays an important role in transamination during the nitrogen metabolism of cells. Which is capable of catalyzing the transfer of an amino group from an amino donor (amino acid or simple amine) to a prochiral acceptor ketone to yield a chiral amine and a byproduct ketone or alpha-keto acid. However, such transaminases have some difficulties in industrial applications, such as substrate product inhibition, narrow substrate application range, and the like.
For wild enzyme transaminase, related research reports are lacked at present, and the enzyme has low catalytic activity on a substrate of the type shown in a formula (a) and poor self-thermal stability. Therefore, the production of important chiral pharmaceutical intermediates using the substrate represented by the formula (b) as an intermediate is affected, and the problems of high cost, incapability of large-scale production and the like are caused.
Disclosure of Invention
Based on the problems of multiple reaction steps, harsh reaction conditions and low optical purity existing in the existing preparation of chiral amine compounds by a chemical synthesis method and the problems of low catalytic activity and poor thermal stability existing in the existing wild transaminase, the invention aims at carrying out genetic engineering modification on the wild transaminase and obtaining the transaminase mutant with good thermal stability and high catalytic activity on the substrate shown in the formula (a) by high-throughput enzyme activity screening.
The technical scheme adopted by the invention for solving the technical problems is as follows: a transaminase mutant, which has an amino acid sequence of SEQ ID NO: 1, and the amino acid sequence of the mutant amino acid sequence is shown in the specification; wherein the mutated amino acid sequence has at least 1 of the following mutation sites: 45 th, 75 th, 108 th, 159 th, 234 th, 272 th bits; the 45 th P is mutated into V or G, the 75 th L is mutated into K, the 108 th G is mutated into E, D, the 159 th T is mutated into Y, H or P, the 234 th L is mutated into C, and the 272 th C is mutated into R or K; the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and has more than 90% homology with the mutated amino acid sequence.
Wherein, SEQ ID NO: 1 is the amino acid sequence of wild transaminase, and the nucleotide sequence is shown in SEQ ID NO: 2, from Aspergillus oryzae (strain ATCC 42149/RIB40) (Yellowkoji mold). Specifically, "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, that can be isolated from a source in nature, and that has not been manipulated or intentionally modified. However, enzymes obtained after the expression of the genes have the defects of low catalytic activity and poor thermal stability for certain substrates.
Preferably, the amino acid sequence of the high-activity transaminase mutant obtained by screening is shown as SEQ ID NO: 3 is shown in the specification; the transaminase mutant gene sequence used for coding the amino acid sequence is shown as SEQ ID NO: 4, respectively.
In another aspect of the present invention, a recombinant expression vector is provided, which can be constructed by connecting the nucleotide sequence containing the transaminase mutant gene of the present invention to various prokaryotic or eukaryotic expression vectors by conventional methods in the art. Prokaryotic expression vectors such as pGEX, pMAL, pET series and the like, eukaryotic expression vectors, more preferably pET series, and pET-24a as the vector plasmid used in the present invention.
The invention also provides a genetic engineering bacterium for producing the transaminase mutant, wherein the genetic engineering bacterium comprises the transaminase mutant gene or the recombinant expression vector; the host cell of the above-mentioned genetically engineered bacterium is preferably Escherichia coli (Escherichia coli BL21(DE 3)).
In still another aspect, the present invention provides a method for preparing the above transaminase mutant, comprising the steps of fermenting and culturing the genetically engineered bacteria, and collecting and preparing the recombinant transaminase mutant.
The method comprises the step of industrially preparing the transaminase mutant under certain fermentation conditions of a production tank; the fermentation conditions of the production tank are preferably as follows: more than 30% of DO, and the air flow is 1: 1-2 vvm.
In yet another aspect, the amino acid sequence of the invention is as set forth in SEQ ID NO: 3, which can be used for converting the compound shown as the formula (a) into a chiral amine compound shown as the formula (b); the structural general formulas of the compounds shown in the formula (a) and the formula (b) are as follows:
wherein: r1is-CH2-、-CH2-CH2-；R2is-CH2-。
Compared with the prior art, the invention has the following advantages and effects:
1. the invention obtains a high-activity transaminase mutant by rational design (individual amino acid in protein molecules is changed by site-directed mutagenesis or other methods on the basis of a space structure of a protein similar to wild transaminase) and methods such as overlapping extension PCR, recombination PCR, large primer PCR, circular plasmid PCR and the like to mutate the protein, and by establishing a transaminase mutant gene library and combining a high-throughput enzyme activity screening mode, the nucleotide sequence of the mutant is SEQ ID NO: 4.
2. the transaminase mutant provided by the invention has higher catalytic activity on a substrate shown as a formula (a), when R in the formula (a)1is-CH2-CH2-、R2is-CH2When the enzyme activity of the transaminase mutant is 31-40U/mg, the enzyme activity of the wild enzyme on the substrate is 8U/mg. Furthermore, the half-life of this enzyme mutant was greater than 48 hours at 40 ℃ whereas the half-life of the wild-type enzyme was only 8 hours under the same conditions.
3. In conclusion, the transaminase mutant has excellent catalytic activity for the substrate shown in the formula (a), and the catalyzed transamination reaction is simple and mild, has no waste discharge, high reaction conversion rate and better application prospect.
Detailed Description
The present invention will be described in further detail with reference to examples, which are illustrative of the present invention and are not to be construed as being limited thereto.
Example 1: establishment of wild transaminase gene engineering bacteria
The transaminase gene engineering strain was constructed by artificially synthesizing a whole gene fragment after sequence optimization based on the Aspergillus oryzae (strain ATCC 42149/RIB40) (Yellow koji mold) transaminase wild-type gene sequence (GenBank: AP007152 Genomic DNA 42896-43876) included in NCBI, inserting the gene into pET-24a plasmid by NdeI and BamHI endonucleases via a gene synthesis company, and transferring the ligated vector into E.coli BL21(DE 3).
Example 2: acquisition of transaminase mutant genes
Due to the three-dimensional structure of the transaminase wild-type gene, it has not been revealed so far; the present application found 74.1% identity with branched chain amino acid transferase from Neosartorya Fumigata (strain ATCC MYA-4609/Af293/CBS 101355/FGSC A1100) (Aspergillus fumigatus) (GenBank: AAHF 01000009.1: 374544..375515, PDB: 4CHI/4UUG) by performing Blast alignment on wild type gene sequences. Therefore, the analysis was carried out with reference to the three-dimensional structure of the enzyme, and the substrate (R) shown in the formula (a) was carried out by Docking1is-CH2-CH2-、R2is-CH2-time) simulation of protein binding, and finally selecting amino acids likely to be involved in substrate binding, PLP binding, and proton transfer as mutant amino acids by Pymol analysis.
In addition to the above rational design, the present application also makes use of an error-prone PCR random mutation method to perform protein engineering on the transaminase. When the error-prone PCR is used for target gene amplification through DNA polymerase, the mutation frequency in the amplification process is changed by adjusting reaction conditions (including increasing magnesium ion concentration, adding manganese ions, changing the concentration of four dNTPs in a system or applying low-fidelity DNA polymerase and the like), so that mutation is randomly introduced into the target gene at a certain frequency to obtain a random mutant of a protein molecule.
This example uses lower fidelity Taq polymerase with Mn2+Substitute for natural cofactor Mg2+Increasing the error-prone probability, and designing the system as follows:
the 50 μ L PCR system was as follows:
wherein: a transaminase template gene constructed by amplifying the transaminase gene by PCR and inserting the gene into the pET-24a plasmid according to the method in example 1; primer design, based on the construction of recombinant plasmid in example 1 of the upstream and downstream sequence design of the target gene.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2.5 min; denaturation at 94 ℃ for 15s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was continued for 10min at 72 ℃ and cooled to 4 ℃.
The resulting PCR amplification product was ligated into pET-24a vector and transferred into E.coli BL21(DE3) to create a library of transaminase gene mutations.
Escherichia coli BL21(DE3) is used as a host, pET-24a plasmid is used as a vector, and the extended transaminase mutant is expressed. Wherein the amino acid sequence of the transaminase mutant is SEQ ID NO: 1, and the amino acid sequence of the mutant amino acid sequence is shown in the specification; wherein the mutated amino acid sequence has the amino acid sequence of SEQ ID NO: 1 at least 1 mutation site in the amino acid sequence set forth in seq id no: 45 th, 75 th, 108 th, 159 th, 234 th, 272 th bits; the 45 th P is mutated into V or G, the 75 th L is mutated into K, the 108 th G is mutated into E or D, the 159 th T is mutated into Y, H or P, the 234 th L is mutated into C, and the 272 th C is mutated into R or K; the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and has more than 90% homology with the mutated amino acid sequence.
Further, a high-activity mutant strain is obtained by high-throughput screening by the enzyme activity detection method described in example 5, and the amino acid sequence of the mutant strain is shown as SEQ ID NO: 3, identifying the mutated high-activity transaminase mutant gene, wherein the nucleotide sequence of the mutated high-activity transaminase mutant gene is shown as SEQ ID NO: 4, respectively.
Example 3: Small-Scale production of wild-type transaminase and transaminase mutants in Shake flasks
Escherichia coli (containing a transaminase mutant gene) containing the recombinant plasmid constructed in example 1 or 2 was inoculated into 100mL of LB medium (peptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH7.2) containing kanamycin (50. mu.g/mL). The cells were cultured on a shaker at 37 ℃ for 16 hours with shaking at 210 rpm. Then, the cells were transferred at a ratio of 1: 100, cultured under the same conditions in 100mL of LB medium containing kanamycin with shaking, and the absorbance (OD) at 600nm of the cells was measured at regular intervals600) To monitor the growth density of the cells. When OD of culture600When the concentration is 0.6-0.8, isopropyl beta-D-thiogalactoside (IPTG) with the final concentration of 0.8mM is added to induce the expression of the target transaminase gene, and the induction culture is carried out overnight (more than or equal to 16 hours). Centrifuging at 10000rpm and 4 deg.C for 10min, discarding supernatant, resuspending the cell pellet with precooled 50mM Tris-HCl buffer (pH8.5) at 200g/L, ultrasonicating, centrifuging at 13000rpm and 4 deg.C for 30min, collecting supernatant, i.e. crude enzyme solution, and storing at-20 deg.C.
Example 4: fermentative production of transaminases
The fermentation scheme is as follows: coli constructed in examples 1 and 2 and recombinant E.coli (containing mutant transaminase gene), single colony of microorganism was inoculated in 400mL LB medium (containing 50. mu.g/mL kanamycin), shake-cultured overnight (5 hours or more) at 37 ℃ and 210rpm, and then fermented in a 30L fermenter: inoculating the seed liquid into 15L fermentation medium according to the inoculation amount of 2%, adding ammonia water into the fermentation liquid to maintain the pH value to be 7.0-7.2, keeping the temperature of a tank at 37 ℃, stirring at the rotating speed of 300-900 rpm, controlling the dissolved oxygen to be about 30% and controlling the air flow to be 1: 1-2 vvm in the process. After 8 hours of culture, IPTG (final concentration of 0.8mmol/L) is added to induce the expression of transaminase, the temperature of the tank is adjusted to 22 ℃, and the fermentation is continued for 12 to 16 hours. During the fermentation, a supplement (glucose 200g/L, yeast extract 100g/L, pH7.2) is added to maintain the growth of the culture. After the fermentation is finished, the culture is directly homogenized and crushed by a high-pressure homogenizer. After the fermentation liquid was disrupted, polyethyleneimine having a final concentration of 2g/L and diatomaceous earth having a final concentration of 150g/L were added thereto, and the mixture was stirred for 30 minutes. After the flocculation and sedimentation are finished, filtering by using filter cloth paved with diatomite. Filtering the filtered enzyme solution with ultrafiltration membrane, concentrating, and storing at-20 deg.C.
Example 5: determination of transaminase enzyme Activity
The transaminase activity determination system is as follows:
50mM Tris-HCl buffer solution containing 50g/L of the substrate represented by the formula (c), 1M phenethylamine, 1mM pyridoxal 5-phosphate, and 10% DMSO, adjusting pH to 8.5, diluting to 270. mu.L, mixing well, and adding into a 96-well plate. Adding transaminase crude enzyme solution (including wild transaminase and transaminase mutant) or its diluted solution 30 μ L, mixing, placing into enzyme labeling instrument, reacting at 45 deg.C, and detecting light absorption at 245 nm. Wherein: the phenethylamine as a co-substrate of the amino donor generates acetophenone as the reaction proceeds, which absorbs light at 245 nm.
Definition of enzyme activity: under the above conditions, the amount of enzyme required for the catalytic production of 1. mu. mol of acetophenone per minute was defined as 1 enzyme activity unit.
Obtained by high throughput screening: the amino acid sequence is as follows: SEQ ID NO: the specific activity of the recombinant transaminase mutant shown in 3 on the substrate shown in the formula (c) is 31-40U/mg. Before mutation, the specific activity of the wild-type transaminase was 8U/mg.
Example 6: effect of temperature on transaminase stability
20mL of crude transaminase solutions of wild transaminase and mutant transaminase were taken, incubated at different temperatures (20-60 ℃ C., 10 ℃ C. intervals) for 4, 8, 12, 16, 24 and 48 hours, cooled in an ice bath, and the residual enzyme activity was measured according to the method in example 5. The time that the residual enzyme activity is reduced to about 50 percent of the original enzyme activity is the half-life period of the enzyme at the temperature, so as to determine the temperature stability of the transaminase.
Table 1 gives the half-lives of the wild transaminases and mutants in their enzymatic activity at different temperatures: wherein the wild transaminase has a half-life of about 8h at 40 ℃; whereas the half-life of the transaminase mutants is greater than 48h at 40 ℃.
TABLE 1 half-lives of wild transaminases and transaminase mutants at different temperatures
| Temperature of heat preservation | Wild transaminase | Transaminase mutants |
| 20℃ | ＞48h | ＞48h |
| 30℃ | 24h | ＞48h |
| 40℃ | 8h | ＞48h |
| 50℃ | ＜4h | 16h |
| 60℃ | ＜4h | 4h |
In addition, it should be noted that the specific embodiments described in the present specification do not limit the present invention. Various modifications, additions and substitutions for the specific embodiments described may be made by those skilled in the art without departing from the scope of the invention as defined in the accompanying claims.
Sequence listing
<110> Tianjin Fa Mo xi biomedical science and technology Co., Ltd
<120> transaminase mutant and coding gene and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 326
<212> PRT
<213> wild transaminase protein sequence SEQ ID NO: 1(Aspergillus oryzae)
<400> 1
Met Thr Ser Met Asn Lys Val Phe Ser Gly Tyr Tyr Glu Arg Lys Ala
1 5 10 15
Arg Leu Asp Asn Ser Asp Asn Arg Phe Ala Lys Gly Ile Ala Tyr Val
20 25 30
Gln Gly Ser Phe Val Pro Leu Ala Asp Ala Arg Val Pro Leu Leu Asp
35 40 45
Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser Val Trp
50 55 60
Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Ser Arg Leu Glu Asp
65 70 75 80
Ser Cys Glu Lys Met Arg Leu Lys Ile Pro Leu Ser Arg Asp Glu Val
85 90 95
Lys Gln Thr Leu Arg Glu Met Val Ala Lys Ser Gly Ile Glu Asp Ala
100 105 110
Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg Gly Asn
115 120 125
Lys Pro Glu Asp Leu Phe Asp Asn His Leu Tyr Leu Ile Val Met Pro
130 135 140
Tyr Val Trp Val Met Glu Pro Ala Ile Gln His Thr Gly Gly Thr Ala
145 150 155 160
Ile Ile Ala Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro
165 170 175
Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Arg Gly Leu Phe Glu
180 185 190
Ala Ala Asp Arg Gly Ala Asp Tyr Pro Phe Leu Ser Asp Gly Asp Thr
195 200 205
Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp Gly
210 215 220
Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Glu Gly Ile Thr Arg Lys
225 230 235 240
Ser Val Phe Asp Ile Ala Gln Val Lys Asn Ile Glu Val Arg Val Gln
245 250 255
Val Val Pro Leu Glu His Ala Tyr His Ala Asp Glu Ile Phe Met Cys
260 265 270
Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Lys Leu Asp Gly Lys Pro
275 280 285
Ile Arg Asn Gly Glu Val Gly Pro Leu Thr Thr Lys Ile Trp Asp Glu
290 295 300
Tyr Trp Ala Met His Tyr Asp Pro Lys Tyr Ser Ser Ala Ile Asp Tyr
305 310 315 320
Arg Gly His Glu Gly Asn
325
<210> 2
<211> 981
<212> DNA
<213> wild transaminase nucleotide sequence SEQ ID NO: 2(Aspergillus oryzae)
<400> 2
atgacatcta tgaacaaagt attttccggt tactacgagc gcaaggctcg tctagataac 60
agtgacaacc gctttgcgaa aggaattgcc tacgtccagg gatctttcgt cccactcgcc 120
gacgcacgag tcccactcct cgacgagggt ttcatgcata gcgacctcac gtacgatgtg 180
ccatcggtct gggatgggcg ctttttccgc cttgatgatc atctcagtcg attggaagat 240
agttgtgaaa agatgcgact gaagatccca ctgtccaggg acgaagtcaa gcaaacccta 300
agggagatgg ttgctaagag tggaatcgaa gatgcctttg tggagctgat cgtcactcgt 360
ggcctgaaag gggtccgtgg caataagcca gaggatcttt tcgacaatca tctctatctg 420
atcgtcatgc cgtatgtctg ggtgatggag cccgccatcc aacataccgg aggtactgcg 480
atcattgccc gtacagtacg gcgcactccc cccggtgctt tcgatcctac catcaagaat 540
ctccagtggg gggacttgac acggggtcta tttgaagcgg ctgaccgtgg cgcggattac 600
ccatttctct cagatggaga taccaatctc acagaaggat ccggtttcaa tatagtgttg 660
gttaaagatg gtattatcta cacgcccgac cgtggtgttc tggaaggcat tacacgtaag 720
agtgtttttg atattgccca ggtcaagaac atcgaggtcc gcgttcaggt ggtgccactc 780
gaacatgcct atcacgccga tgagatattc atgtgtacta ctgctggtgg cattatgcct 840
atcacgaaac tcgatgggaa accgatccgg aatggagaag tcggtcccct tactacaaag 900
atatgggatg agtactgggc gatgcactat gacccgaaat atagctctgc tatcgattac 960
aggggccatg agggtaactg a 981
<210> 3
<211> 326
<212> PRT
<213> transaminase mutant protein sequence SEQ ID NO 3(Aspergillus oryzae)
<400> 3
Met Thr Ser Met Asn Lys Val Phe Ser Gly Tyr Tyr Glu Arg Lys Ala
1 5 10 15
Arg Leu Asp Asn Ser Asp Asn Arg Phe Ala Lys Gly Ile Ala Tyr Val
20 25 30
Gln Gly Ser Phe Val Pro Leu Ala Asp Ala Arg Val Val Leu Leu Asp
35 40 45
Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser Val Trp
50 55 60
Asp Gly Arg Phe Phe Arg Leu Asp Asp His Lys Ser Arg Leu Glu Asp
65 70 75 80
Ser Cys Glu Lys Met Arg Leu Lys Ile Pro Leu Ser Arg Asp Glu Val
85 90 95
Lys Gln Thr Leu Arg Glu Met Val Ala Lys Ser Glu Ile Glu Asp Ala
100 105 110
Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg Gly Asn
115 120 125
Lys Pro Glu Asp Leu Phe Asp Asn His Leu Tyr Leu Ile Val Met Pro
130 135 140
Tyr Val Trp Val Met Glu Pro Ala Ile Gln His Thr Gly Gly Tyr Ala
145 150 155 160
Ile Ile Ala Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro
165 170 175
Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Arg Gly Leu Phe Glu
180 185 190
Ala Ala Asp Arg Gly Ala Asp Tyr Pro Phe Leu Ser Asp Gly Asp Thr
195 200 205
Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp Gly
210 215 220
Ile Ile Tyr Thr Pro Asp Arg Gly Val Cys Glu Gly Ile Thr Arg Lys
225 230 235 240
Ser Val Phe Asp Ile Ala Gln Val Lys Asn Ile Glu Val Arg Val Gln
245 250 255
Val Val Pro Leu Glu His Ala Tyr His Ala Asp Glu Ile Phe Met Arg
260 265 270
Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Lys Leu Asp Gly Lys Pro
275 280 285
Ile Arg Asn Gly Glu Val Gly Pro Leu Thr Thr Lys Ile Trp Asp Glu
290 295 300
Tyr Trp Ala Met His Tyr Asp Pro Lys Tyr Ser Ser Ala Ile Asp Tyr
305 310 315 320
Arg Gly His Glu Gly Asn
325
<210> 4
<211> 981
<212> DNA
<213> transaminase mutant nucleotide sequence SEQ ID NO 4(Aspergillus oryzae)
<400> 4
atgacatcta tgaacaaagt attttccggt tactacgagc gcaaggctcg tctagataac 60
agtgacaacc gctttgcgaa aggaattgcc tacgtccagg gatctttcgt cccactcgcc 120
gacgcacgag tcgtgctcct cgacgagggt ttcatgcata gcgacctcac gtacgatgtg 180
ccatcggtct gggatgggcg ctttttccgc cttgatgatc ataaaagtcg attggaagat 240
agttgtgaaa agatgcgact gaagatccca ctgtccaggg acgaagtcaa gcaaacccta 300
agggagatgg ttgctaagag tgaaatcgaa gatgcctttg tggagctgat cgtcactcgt 360
ggcctgaaag gggtccgtgg caataagcca gaggatcttt tcgacaatca tctctatctg 420
atcgtcatgc cgtatgtctg ggtgatggag cccgccatcc aacataccgg aggttatgcg 480
atcattgccc gtacagtacg gcgcactccc cccggtgctt tcgatcctac catcaagaat 540
ctccagtggg gggacttgac acggggtcta tttgaagcgg ctgaccgtgg cgcggattac 600
ccatttctct cagatggaga taccaatctc acagaaggat ccggtttcaa tatagtgttg 660
gttaaagatg gtattatcta cacgcccgac cgtggtgttt gcgaaggcat tacacgtaag 720
agtgtttttg atattgccca ggtcaagaac atcgaggtcc gcgttcaggt ggtgccactc 780
gaacatgcct atcacgccga tgagatattc atgcgtacta ctgctggtgg cattatgcct 840
atcacgaaac tcgatgggaa accgatccgg aatggagaag tcggtcccct tactacaaag 900
atatgggatg agtactgggc gatgcactat gacccgaaat atagctctgc tatcgattac 960
aggggccatg agggtaactg a 981
Claims (10)
1. A transaminase mutant, characterized in that the amino acid sequence of the transaminase mutant is SEQ ID NO: 1, and the amino acid sequence of the mutant amino acid sequence is shown in the specification; wherein the mutated amino acid sequence has at least 1 of the following mutation sites: 45 th, 75 th, 108 th, 159 th, 234 th, 272 th bits; the 45 th P is mutated into V or G, the 75 th L is mutated into K, the 108 th G is mutated into E or D, the 159 th T is mutated into Y, H or P, the 234 th L is mutated into C, and the 272 th C is mutated into R or K; the amino acid sequence of the transaminase mutant has a mutation site in the mutated amino acid sequence, and has more than 90% homology with the mutated amino acid sequence.
2. The transaminase mutant according to claim 1, characterized in that the amino acid sequence of the transaminase mutant is as set forth in SEQ ID NO: 3, respectively.
3. A transaminase mutant gene, which encodes the transaminase mutant of claim 2 and has the nucleotide sequence shown in SEQ ID NO: 4, respectively.
4. A recombinant expression vector comprising the transaminase mutant gene of claim 3.
5. The recombinant expression vector of claim 4, wherein the recombinant expression vector is a vector plasmid that is pET-24 a.
6. A genetically engineered bacterium for producing the transaminase mutant of claim 2, characterized in that the genetically engineered bacterium comprises the recombinant expression vector of claim 4, and the host cell of the genetically engineered bacterium is Escherichia coli.
7. Use of the transaminase mutant gene of claim 3, the recombinant expression vector of claim 4, the genetically engineered bacterium of claim 6 in the preparation of the transaminase mutant of claim 2.
8. A process for the preparation of a transaminase mutant as claimed in claim 2, which comprises the following steps: culturing the genetically engineered bacterium of claim 6 to obtain a transaminase mutant.
9. The process according to claim 8, comprising the step of producing the transaminase mutant by fermentation.
10. Use of a transaminase mutant according to claim 2 in an amine transfer reaction, wherein the substrate of the transamination reaction is of the formula (a),
wherein R is1is-CH2-or-CH2-CH2-；R2is-CH2-。
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| US20100285541A1 (en) * | 2009-02-26 | 2010-11-11 | Codexis, Inc. | Transaminase biocatalysts |
| CN106995808A (en) * | 2017-04-27 | 2017-08-01 | 宿迁阿尔法科技有限公司 | One kind restructuring transaminase and its application |
| AU2016249780A1 (en) * | 2015-04-16 | 2017-10-19 | F. Hoffmann-La Roche Ag | Mutant transaminases as well as methods and uses relating thereto |
| CN107828751A (en) * | 2017-11-06 | 2018-03-23 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
| CN111549011A (en) * | 2020-06-03 | 2020-08-18 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus and application thereof |
| CN112522229A (en) * | 2020-10-26 | 2021-03-19 | 浙江工业大学 | Transaminase mutant and application thereof in preparation of sitagliptin intermediate |
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- 2021-06-17 CN CN202110669602.6A patent/CN113373126B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100285541A1 (en) * | 2009-02-26 | 2010-11-11 | Codexis, Inc. | Transaminase biocatalysts |
| AU2016249780A1 (en) * | 2015-04-16 | 2017-10-19 | F. Hoffmann-La Roche Ag | Mutant transaminases as well as methods and uses relating thereto |
| CN106995808A (en) * | 2017-04-27 | 2017-08-01 | 宿迁阿尔法科技有限公司 | One kind restructuring transaminase and its application |
| CN107828751A (en) * | 2017-11-06 | 2018-03-23 | 凯莱英生命科学技术(天津)有限公司 | Transaminase mutant and its application |
| CN111549011A (en) * | 2020-06-03 | 2020-08-18 | 重庆迪维斯生物科技有限公司 | Transaminase mutant derived from aspergillus terreus and application thereof |
| CN112522229A (en) * | 2020-10-26 | 2021-03-19 | 浙江工业大学 | Transaminase mutant and application thereof in preparation of sitagliptin intermediate |
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