Transaminase mutant and coding gene and application thereof
阅读说明：本技术 一种转氨酶突变体及其编码基因与应用 (Transaminase mutant and coding gene and application thereof ) 是由 金利群 申屠俊康 刘汉林 柳志强 薛亚平 郑裕国 于 2021-05-21 设计创作，主要内容包括：本发明公开了一种转氨酶突变体以及其编码基因,以及其在微生物催化2-羰基-4-[羟基(甲基)膦酰基]丁酸为底物不对称合成L-草铵膦中的应用。所述转氨酶的氨基酸序列如SEQ ID:1所示,其突变体是将SEQ ID:1所示的氨基酸序列的第138和141位中的一个进行单位点突变或组合突变获得的。本发明实现了高转化率转氨酶活力突变体基因的高效表达,酶活最高可达3827U/g。本发明转氨酶突变体在温度40℃,以pH 8.0缓冲液为反应介质进行生物催化反应获得L-草铵膦,收率高达99％,产物e.e.值达到99％,具有较好应用前景。(The invention discloses a transaminase mutant, a coding gene thereof and application thereof in asymmetric synthesis of L-glufosinate-ammonium by using 2-carbonyl-4- [ hydroxyl (methyl) phosphonyl ] butyric acid as a substrate under catalysis of microorganisms. The amino acid sequence of the transaminase is shown in SEQ ID:1, and the mutant is obtained by performing single-site mutation or combined mutation on one of 138 th and 141 th positions of the amino acid sequence shown in SEQ ID: 1. The invention realizes the high-efficiency expression of the transaminase activity mutant gene with high conversion rate, and the enzyme activity can reach 3827U/g at most. The transaminase mutant of the invention performs a biocatalytic reaction at 40 ℃ with a buffer solution with pH8.0 as a reaction medium to obtain L-glufosinate-ammonium, the yield is up to 99%, the value of the product e.e. is up to 99%, and the transaminase mutant has a good application prospect.)
1. A transaminase mutant is prepared from the amino acids 138 and 141 of transaminase shown in SEQ ID No.1 by single-point mutation or double mutation.
2. The transaminase mutant of claim 1, characterized in that its amino acid sequence is shown in SEQ ID No. 3.
3. A gene encoding the transaminase mutant of claim 1.
4. The encoding gene of claim 3, wherein the nucleotide sequence of the encoding gene is shown as SEQ ID No. 4.
5. A recombinant bacterium comprising the coding gene of claim 3.
6. The use of the transaminase mutant of claim 1 in the microbial catalysis of 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid as substrate in the asymmetric synthesis of L-glufosinate-ammonium.
7. The use according to claim 6, characterized in that the use is: carrying out ultrasonic disruption on wet thalli obtained by fermentation culture on recombinant genetic engineering bacteria containing the transaminase mutant coding gene, purifying a nickel column to serve as an enzyme source, reacting under the conditions of pH 6.0-10.0 buffer solution, 20-75 ℃ and 400-600 r/min by taking a glufosinate-ammonium precursor ketone PPO as a substrate, pyridoxal phosphate as a coenzyme and natural amino acid as an amino donor, and after the reaction is finished, separating and purifying the reaction solution to obtain the L-glufosinate-ammonium.
8. The use according to claim 7, wherein the initial concentration of the substrate in the reaction system is 20 to 500mM, the amount of the wet cells is 10 to 100g/L, and the amount of the coenzyme is 0 to 1 mM.
(I) technical field
The invention relates to a transaminase mutant, a coding gene thereof and application thereof in asymmetric synthesis of L-glufosinate-ammonium by using 2-carbonyl-4- [ hydroxyl (methyl) phosphonyl ] butyric acid as a substrate under catalysis of microorganisms.
(II) background of the invention
Glufosinate (PPT) is used as an unnatural amino acid herbicide, and is named as three major herbicides in the world of paraquat and glyphosate. The compound is widely used due to high efficiency and low toxicity and nonselectivity, is discovered from fermentation liquor of streptomyces hygroscopicus by a Bayer research team in Germany at first, has good weeding effect, mainly plays a role in inhibiting Glutamine Synthetase (GS) in plants, enables nitrogen metabolism to be disordered, causes the plants to die due to ammonium poisoning, simultaneously influences photosynthesis of the plants, cannot synthesize chlorophyll, and finally gradually withers, yellows and dies.
Currently, glufosinate-ammonium sold on the market is a racemic mixture, racemic PPT has two configurations of an L type and a D type, but only the L configuration has a weeding effect, and the glufosinate-ammonium can be rapidly degraded under the action of soil microorganisms. It would be of great significance to improve atom economy, reduce use costs, and alleviate environmental stresses if glufosinate products could be produced and sold in the L-configuration.
The current reports that the production of the optically pure L-glufosinate-ammonium mainly comprises a chemical synthesis method, a chiral resolution method and a biological catalytic synthesis method.
The chemical synthesis method mainly comprises a low-temperature directional synthesis method, a chiral auxiliary base method, a natural amino acid synthesis method and an asymmetric catalytic synthesis method, and the asymmetric synthesis method also comprises an asymmetric catalytic hydrogenation synthesis method, an asymmetric Michael addition method and the like. The existing chemical synthesis method has the disadvantages of complicated reaction route, harsh reaction conditions, serious later-period pollution and high price of asymmetric synthetic reagents, so that the production cost is high, and the large-scale preparation of the L-glufosinate-ammonium is not facilitated.
The chiral resolution method is to perform chiral resolution on racemic D, L-glufosinate-ammonium or derivatives thereof, and separate D-type and L-type isomers, thereby obtaining optically pure L-glufosinate-ammonium. However, the method needs expensive chiral resolution reagent, the theoretical yield can only reach 50%, the single resolution ratio is low, and the process is complicated.
The biocatalytic synthesis method has the advantages of mild reaction conditions, high catalytic efficiency and stereoselectivity, easy separation and purification of products, environmental protection, zero pollution and the like, and gradually becomes a current research hotspot. The research on the synthesis of the L-glufosinate-ammonium by the biological enzyme method has important significance and economic value. The biosynthesis of L-PPT mainly comprises an enzymatic hydrolysis method, an enzyme selective resolution method and an enzyme asymmetric synthesis method, wherein the related enzymes mainly comprise: protease, amidase, deacetylase, transaminase.
In a plurality of enzymatic synthesis routes of glufosinate-ammonium, the asymmetric synthesis method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, easy separation and purification and the like, and is a route suitable for industrial development and production of L-glufosinate-ammonium, enzymes for catalyzing the reactions mainly comprise glutamate dehydrogenase and transaminase, and the substrate is glufosinate-ammonium precursor ketone (2-carbonyl-4- [ hydroxy (methyl) phosphoryl ] -butyric acid, PPO). When glutamate dehydrogenase is catalyzed, expensive NAD (P) H is used as a cofactor, so that the catalysis cost is too high.
Transaminase (Transaminase, TA, EC 2.6.1.X) as a Pyridoxal phosphate (PLP) dependent enzyme catalyzes the transfer of an amino group on an amine compound to a potentially chiral ketone compound to yield a chiral amine and a byproduct ketone or an α -keto acid. Transaminases are ubiquitous in microbial, animal and plant tissues and play an important role in transamination during intracellular nitrogen metabolism. Transaminases are mainly used for the synthesis of chiral amines, chiral amino acids, chiral amino alcohols, etc., and these chiral compounds are often used as active ingredients or main intermediates of pharmaceuticals, for example, the broad-spectrum contact herbicide glufosinate-ammonium, sitagliptin, an antibiotic penicillin, etc., which are medicines for treating diabetes, have important roles in pharmaceutical industry, agriculture and chemical industry.
Depending on the position of the carboxyl group of the substrate to which the amino group is transferred during the catalytic process, it can be classified into two major classes, α -transaminase and ω -transaminase. Alpha-aminotransferases require the presence of a carboxyl group in the alpha position on the carbonyl function and therefore can only allow the production of alpha-amino acids, i.e. alpha-aminotransferases can only use alpha-amino acids and alpha-keto acids as substrates simultaneously, for example, aspartate TA (EC 2.6.1.1), tyrosine TA (EC 2.6.1.5), branched-chain amino acid TA (EC 2.6.1.11), etc., the remainder being classified as omega-aminotransferases, which are broadly classified, for example, as 4-aminobutyric acid: pyruvate transaminase (EC 2.6.1.19), β -alanine: pyruvate transaminase (EC 2.6.1.18). In general, all transaminases can also be classified according to the amino acid substrates utilized in their reversible reactions, which are named for their catalytic substrate amino acid activity, e.g., the two enzymes that catalyze the substrates aspartate and phenylalanine, respectively.
At present, some TA prepared L-PPT has been reported. Bartsch et al screened aspartate aminotransferase from soil and applied to the preparation of L-PPT, with aspartate as the amino donor and a substrate concentration of 40mM giving up to 75% conversion and a substrate concentration of 100mM giving up to 59% (K. Bartsch, Process for the preparation of L-phosphinothricine by enzymatic conversion with enzyme, U.S. patent (2005) 6936444.). Chinese patent CN107119084A reports that the PPO of Yanglirong et al is taken as a substrate, alanine is taken as an amino donor, when the concentration of the PPO substrate is 100mM, 400mM alanine and 1mM PLP are added for reaction for 19h, and the PPO conversion rate can reach 100%. The existing transaminase process has the problems of few enzyme sources, low enzyme activity, poor substrate tolerance and low product conversion rate.
Under the background, the invention provides a method for screening a novel transaminase recombinase by a gene mining technology, carries out molecular modification by a protein engineering technology, uses a dominant mutant catalyst for asymmetric synthesis preparation of L-PPT, and has important significance for improving poor substrate tolerance and low enzyme activity of TA in the process of preparing L-PPT.
Disclosure of the invention
The invention aims to provide a novel transaminase mutant which can be applied to the preparation of L-glufosinate-ammonium with excellent catalytic activity and substrate tolerance and by using a cheap amino donor, and an application of the transaminase mutant in the asymmetric synthesis of L-glufosinate-ammonium.
The technical scheme adopted by the invention is as follows:
a transaminase mutant is prepared from the amino acids 138 and 141 of transaminase shown in SEQ ID No.1 by single-point mutation or double mutation.
SEQ ID NO: 1 the sequence is as follows:
MNTNNALMQRRHNAVPRGVGQIHPIFAERAENCRVWDVEGREYLDFAGGIAVLN TGHLHPGIVSAVEAQLKKLSHTCFQVLAYEPYLALCERMNQKVPGDFAKKTLLVTTGS EAVENAVKIARAATKRSGAIAFSGAYHGRTHYTLSLTGKVHPYSAGMGLMPGHVYRAL YPCPLHNISDDDAIASIERIFKNDAAPEDIAAIIIEPVQGEGGFYAASPAFMQRLRALCDQ HGIMLIADEVQSGAGRTGTLFAMEQMGVAADITTFAKSIAGGFPLAGVTGRADVMDAI APGGLGGTYAGNPIACAAALAVLDIFEQENLLQKANTLGNTLRDGLMEIAETHREIGD VRGLGAMIAIELFENGDPGKPNAALTADIVTRAREKGLILLSCGPYYNILRILVPLTIEAS QIRQGLEIIAQCFDEAKQA
the coding gene is (SEQ ID NO: 2):
atgaacaccaacaacgctctgatgcagcgtcgtcacaacgctgttccgcgtggtgttggtcagatccacccgatcttcgctgaacg tgctgaaaactgccgtgtttgggacgttgaaggtcgtgaatacctggacttcgctggtggtatcgctgttctgaacaccggtcacctgcacc cgggtatcgtttctgctgttgaagctcagctgaaaaaactgtctcacacctgcttccaggttctggcttacgaaccgtacctggctctgtgcg aacgtatgaaccagaaagttccgggtgacttcgctaaaaaaaccctgctggttaccaccggttctgaagctgttgaaaacgctgttaaaatc gctcgtgctgctaccaaacgttctggtgctatcgctttctctggtgcttaccacggtcgtacccactacaccctgtctctgaccggtaaagttc acccgtactctgctggtatgggtctgatgccgggtcacgtttaccgtgctctgtacccgtgcccgctgcacaacatctctgacgacgacgct atcgcttctatcgaacgtatcttcaaaaacgacgctgctccggaagacatcgctgctatcatcatcgaaccggttcagggtgaaggtggttt ctacgctgcttctccggctttcatgcagcgtctgcgtgctctgtgcgaccagcacggtatcatgctgatcgctgacgaagttcagtctggtg ctggtcgtaccggtaccctgttcgctatggaacagatgggtgttgctgctgacatcaccaccttcgctaaatctatcgcgggcggcttcccg ctggctggcgttaccggtcgtgctgacgttatggacgctatcgctccgggtggtctgggtggtacctacgctggtaacccgatcgcttgcg ctgctgctctggctgttctggacatcttcgaacaggaaaacctgctgcagaaagctaacaccctgggtaacaccctgcgtgacggtctgat ggaaatcgctgaaacccaccgtgaaatcggtgacgttcgtggtctgggtgctatgatcgctatcgaactgttcgaaaacggtgacccggg taaaccgaacgctgctctgaccgctgacatcgttacccgtgctcgtgaaaaaggtctgatcctgctgtcttgcggtccgtactacaacatcc tgcgtatcctggttccgctgaccatcgaagcttctcagatccgtcagggtctggaaatcatcgctcagtgcttcgacgaagctaaacaggct ctcgagcaccaccaccaccaccac
preferably, the amino acid sequence of the transaminase mutant is shown as SEQ ID NO.3 (the tyrosine at the 138 th position is substituted by phenylalanine (Y138F), and the nucleotide sequence of the coding gene of the transaminase mutant is shown as SEQ ID NO. 4.
SEQ ID NO:3 the sequence is as follows:
MNTNNALMQRRHNAVPRGVGQIHPIFAERAENCRVWDVEGREYLDFAGGIAVLN TGHLHPGIVSAVEAQLKKLSHTCFQVLAYEPYLALCERMNQKVPGDFAKKTLLVTTGS EAVENAVKIARAATKRSGAIAFSGAFHGRTHYTLSLTGKVHPYSAGMGLMPGHVYRAL YPCPLHNISDDDAIASIERIFKNDAAPEDIAAIIIEPVQGEGGFYAASPAFMQRLRALCDQ HGIMLIADEVQSGAGRTGTLFAMEQMGVAADITTFAKSIAGGFPLAGVTGRADVMDAI APGGLGGTYAGNPIACAAALAVLDIFEQENLLQKANTLGNTLRDGLMEIAETHREIGD VRGLGAMIAIELFENGDPGKPNAALTADIVTRAREKGLILLSCGPYYNILRILVPLTIEAS QIRQGLEIIAQCFDEAKQALEHHHHHH
the coding gene is (SEQ ID NO: 4):
atgaacaccaacaacgctctgatgcagcgtcgtcacaacgctgttccgcgtggtgttggtcagatccacccgatcttcgctgaacg tgctgaaaactgccgtgtttgggacgttgaaggtcgtgaatacctggacttcgctggtggtatcgctgttctgaacaccggtcacctgcacc cgggtatcgtttctgctgttgaagctcagctgaaaaaactgtctcacacctgcttccaggttctggcttacgaaccgtacctggctctgtgcg aacgtatgaaccagaaagttccgggtgacttcgctaaaaaaaccctgctggttaccaccggttctgaagctgttgaaaacgctgttaaaatc gctcgtgctgctaccaaacgttctggtgctatcgctttctctggtgctttccacggtcgtacccactacaccctgtctctgaccggtaaagttc acccgtactctgctggtatgggtctgatgccgggtcacgtttaccgtgctctgtacccgtgcccgctgcacaacatctctgacgacgacgct atcgcttctatcgaacgtatcttcaaaaacgacgctgctccggaagacatcgctgctatcatcatcgaaccggttcagggtgaaggtggttt ctacgctgcttctccggctttcatgcagcgtctgcgtgctctgtgcgaccagcacggtatcatgctgatcgctgacgaagttcagtctggtg ctggtcgtaccggtaccctgttcgctatggaacagatgggtgttgctgctgacatcaccaccttcgctaaatctatcgcgggcggcttcccg ctggctggcgttaccggtcgtgctgacgttatggacgctatcgctccgggtggtctgggtggtacctacgctggtaacccgatcgcttgcg ctgctgctctggctgttctggacatcttcgaacaggaaaacctgctgcagaaagctaacaccctgggtaacaccctgcgtgacggtctgat ggaaatcgctgaaacccaccgtgaaatcggtgacgttcgtggtctgggtgctatgatcgctatcgaactgttcgaaaacggtgacccggg taaaccgaacgctgctctgaccgctgacatcgttacccgtgctcgtgaaaaaggtctgatcctgctgtcttgcggtccgtactacaacatcc tgcgtatcctggttccgctgaccatcgaagcttctcagatccgtcagggtctggaaatcatcgctcagtgcttcgacgaagctaaacaggct ctcgagcaccaccaccaccaccac
due to the specificity of the amino acid sequence, any fragment of the polypeptide of the amino acid sequence shown in SEQ ID NO.3 or its variants, such as conservative variants, bioactive fragments or derivatives thereof, is included in the scope of the present invention as long as the homology between the fragment of the polypeptide or the polypeptide variant and the amino acid sequence is above 95%. The alteration may comprise a deletion, insertion or substitution of an amino acid in the amino acid sequence; for conservative changes in a variant, the substituted amino acid has similar structural or chemical properties as the original amino acid, e.g., replacement of isoleucine with leucine, or the variant may have non-conservative changes, e.g., replacement of glycine with tryptophan.
The invention also relates to a coding gene of the transaminase mutant.
Preferably, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 4.
Due to the specificity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO.4 is within the scope of the present invention as long as it has more than 90% homology with the polynucleotide. A mutant of the polynucleotide refers to a polynucleotide sequence having one or more nucleotide changes. Mutants of the polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants, including substitution, deletion and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded amino acid.
The invention also relates to a recombinant bacterium containing the coding gene.
The key point of the invention lies in the selection of novel high-activity transaminase and mutation sites thereof, on the premise of knowing the sequence of the novel high-activity transaminase and the mutation sites thereof, a person skilled in the art can design a mutation primer of site-specific mutation according to the TA gene (ABAT2) of SEQ ID NO.1, construct a mutant by site-specific mutation by taking a cloning vector carrying the transaminase as a template, convert a recombinant plasmid into E.coli BL21(DE3) cells or host cells capable of expressing the transaminase by taking a plasmid pET28b or a vector capable of expressing the transaminase as an expression vector, and culture a positive single clone after high-throughput screening verification to obtain the wet bacterial strain containing the mutant.
The application of the transaminase mutant in asymmetric synthesis of L-glufosinate-ammonium by catalyzing 2-carbonyl-4- [ hydroxyl (methyl) phosphonyl ] butyric acid as a substrate by microorganisms.
The application is as follows: carrying out ultrasonic disruption on wet thalli obtained by fermentation culture on recombinant genetic engineering bacteria containing the transaminase mutant coding gene, purifying a nickel column to serve as an enzyme source, reacting under the conditions of pH 6.0-10.0 buffer solution, 20-75 ℃ and 400-600 r/min by taking a glufosinate-ammonium precursor ketone PPO as a substrate, pyridoxal phosphate as a coenzyme and natural amino acid as an amino donor, and after the reaction is finished, separating and purifying the reaction solution to obtain the L-glufosinate-ammonium.
In the reaction system, the initial concentration of the substrate is 20 to 500mM, the amount of wet cells is 10 to 100g/L (preferably 20 g/L), and the amount of coenzyme is 0 to 1mM (preferably 0.1 mM).
Preferably, the reaction is carried out at 38 to 45 ℃ (more preferably at 40 ℃).
Specifically, the wet cells can be prepared as follows: constructing a recombinant vector containing the TA mutant gene with excellent catalytic activity and substrate tolerance, converting the recombinant vector into E.coli, performing induced expression on the obtained recombinant gene engineering bacteria, and separating culture solution to obtain wet bacterial cells. The method specifically comprises the following steps: the transaminase mutant gene-containing engineered bacteria were inoculated into LB liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/mL, cultured at 37 ℃ for 9 hours at 200rpm, inoculated into fresh LB liquid medium containing kanamycin resistance at a final concentration of 50. mu.g/mL at an inoculum size of 1% by volume, and inoculated at 37 ℃ at 150rpmCulturing until the thallus OD600Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, performing induction culture at 28 ℃ for 12h, centrifuging at 4 ℃ and 8000rpm for 10min, discarding supernatant, and collecting wet thallus. The LB medium composition: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of NaCl, water as a solvent and 7.0 of pH value.
The invention has the following beneficial effects: the invention provides a novel transaminase and a high-activity mutant thereof, the mutant has higher catalytic activity (the highest activity can reach 3827U/g) on glufosinate-ammonium precursor ketone PPO, the optimum reaction temperature is 40 ℃, L-glufosinate-ammonium is obtained by performing biocatalytic reaction at 40 ℃ by taking a buffer solution with the pH value of 8.0 as a reaction medium, the yield is up to 99%, the value of a product e.e. reaches 99%, and the mutant has better application prospect.
(IV) description of the drawings
FIG. 1 shows the gel electrophoresis of transaminase: lane M is the protein molecular weight Marker, lane 1 is the transaminase ABAT2, and lane 2 is the transaminase mutant ABAT 2-Y138F.
FIG. 2 is a SDS-PAGE pattern of purified transaminase: lane M shows protein molecular weight Marker, lane 1 shows transaminase ABAT2, lane 2 shows purified transaminase variant ABAT2, lane 3 shows transaminase variants ABAT2-Y138F, and lane 4 shows purified transaminase variants ABAT 2-Y138F.
FIG. 3 is a schematic diagram of the optimum temperature of transaminase mutants.
FIG. 4 is a schematic diagram showing the optimum pH of the transaminase mutant.
FIG. 5 is a schematic diagram showing the effect of a metal ion transaminase mutant.
FIG. 6 shows the synthesis equation of L-glufosinate-ammonium.
(V) detailed description of the preferred embodiments
For the purpose of enhancing understanding of the present invention, the present invention will be described in further detail with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention.
Example 1: amplification of the transaminase Gene ABAT2
Pseudomonas was isolated from soil and stored in China center for type culture Collection (preservation number CCTCC NO: M2012539, published in the patent application, application No. CN 201210593105.3, published (bulletin) No. CN 103131649A).
According to the sequencing information of the transaminase gene derived from Pseudomonas (WP-076423369.1) recorded by Genbank, Pseudomonas (total genomic DNA of the cells) was extracted by a nucleic acid rapid analyzer, and PCR amplification was carried out using the genomic DNA as a template under the action of primer 1 (5'-ATGAACACCAACAACGCTC-3') and primer 2 (5'-TTAAGCCTGTTTAGCTTC-3'). The PCR reaction system (total volume: 50. mu.L) contained 10 Xpfu DNA Polymerase Buffer 5. mu.L, 10mM dNTP mix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu.L, cloning primer 1 and primer 2 each at a concentration of 50. mu.M, genomic DNA 1. mu.L, Pfu DNA Polymerase 1. mu.L, and non-nucleic acid water 40. mu.L.
PCR conditions using a BioRad PCR instrument: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 65 deg.C for 45s, extension at 72 deg.C for 1min for 30 cycles, and final extension at 72 deg.C for 10 min.
The PCR reaction solution was subjected to 0.9% agarose gel electrophoresis, and the fragment was recovered and purified by cutting the gel, and then a base A was introduced into the 5' end of the fragment using Taq DNA polymerase. The fragment is connected with pMD18-T vector under the action of T4 DNA ligase to obtain the cloned recombinant plasmid pMD18-T-ABAT 2. The recombinant plasmid is transformed into Escherichia coli JM109, a basket white spot screening system is used for screening, white clone sequencing is randomly selected, a software analysis sequencing result shows that: the length of the nucleotide sequence amplified by the primer 1 and the primer 2 is 1275bp (the nucleotide sequence of the ABAT2 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2), and the sequence encodes a complete open reading frame.
Example 2: construction of recombinant Escherichia coli BL21(DE3)/pET28b-ABAT2
Primer 3(5 ' -CCGCATATGAACACCAACAAC-3) and primer 4 (5'-TTGCTCGAG TTAAGCCTGTTTAGC-3') were designed based on the ABAT2 gene sequence in example 1, and Nde I and Xho I restriction sites (underlined) were introduced into primer 3 and primer 4, respectively. Under the initiation of primer 3 and primer 4, amplification was performed using high fidelity Pfu DNA polymerase, using recombinant plasmid pMD18-T-ABAT2 as a template (obtained in example 1), the ATA3 gene sequence was obtained, the amplified fragment was treated with Nde I and Xho I restriction enzymes (TaKaRa) after sequencing, and the fragment was ligated with commercial vector pET28b (Invitrogen) treated with the same restriction enzymes using T4 DNA ligase (TaKaRa), to construct expression vector pET28b-ab 2. The constructed expression vector pET28b-ABAT2 is transformed into Escherichia coli BL21(DE3) (Invitrogen) (42 ℃, 90s), spread on LB plate containing 50 ug/ml kanamycin resistance, cultured for 8-12h at 37 ℃, randomly picked clone extracted plasmid for sequencing and identification, and screened to obtain recombinant Escherichia coli BL21(DE3)/pET28b-ABAT2 containing expression recombinant plasmid pET28b-ABAT 2.
Example 3: inducible expression of transaminase
Recombinant E.coli BL21(DE3)/pET28b-ABAT2 was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin resistance, cultured at 37 ℃ and 200rpm for 12 hours, further inoculated into fresh LB liquid medium containing 50. mu.g/mL kanamycin resistance in an inoculum size of 1% (v/v), and cultured at 37 ℃ and 150rpm until the microbial OD600Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, inducing and culturing at 25 ℃ for 12h, centrifuging at 8000rpm for 20min at 4 ℃, discarding supernatant, and collecting precipitate to obtain recombinant Escherichia coli E.coli BL21/pET28b-ABAT2 wet thallus containing expression recombinant plasmid. The thallus can be directly used as a biocatalyst or used for protein purification.
Example 4: construction and screening of transaminase mutants
Designing a mutation primer of site-directed mutation according to a sequence (an amino acid sequence is shown as SEQ ID NO.1 and a nucleotide sequence is shown as SEQ ID NO. 2) of transaminase (ABAT2 is from Salmonella enterica, Genbank access No. CP026235.1), introducing the site-directed mutation to the 138 th site by using a recombinant vector pET28b/ABAT2 as a template by using a rapid PCR technology, wherein the primer is as follows:
forward primer TTTCTCTGGTGCTNNKCACGGTCGT (base mutation underlined)
Reverse primer GGGTACGACCGTGMNNAGCACCAGA (base mutation underlined)
PCR reaction System (Overall)Volume 50 μ L): 10 XDNA Polymerase Buffer 5. mu.L, 10mM dNTP mix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu.L, forward primer and reverse primer each at a concentration of 50. mu.M, template DNA 1. mu.L, DNA Polymerase 1. mu.L, ddH2O 40μL。
PCR conditions using a BioRad PCR instrument: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 5min for 30 cycles, and final extension at 72 ℃ for 10 min.
Adding 3 mu L of PCR product into 100 mu L of ice bath competent cell suspension, standing on ice for 30min, thermally shocking the transformation product at 42 ℃ for 90s, rapidly placing on ice for cooling for 2min, adding 600 mu L of LB liquid culture medium into the tube, culturing at 37 ℃ and 150r/min for 60min, coating 100 mu L of the bacterial liquid on a plate, and performing inverted culture at 37 ℃ for 12h after the bacterial liquid is completely absorbed by the culture medium.
Example 5: screening gene mutant library to obtain mutant ABAT2-Y138F
8000 single colonies of the LB resistant plates containing 50. mu.g/mL kanamycin in example 4 were picked up and inoculated into LB medium containing 50. mu.g/mL kanamycin, respectively, to induce expression under the conditions as in example 1, and 8000 recombinant E.coli wet cells containing the mutant gene, i.e., mutant wet cells, were obtained.
After obtaining Escherichia coli containing the mutant protein, carrying out biocatalysis on 0.36g of glufosinate-ammonium precursor ketone PPO, wherein the final concentration composition and catalysis conditions of a catalytic system (10mL) are as follows: 0.075g of wet mutant cell, Tris-HCl buffer (pH 8.0), 0.1mM pyridoxal phosphate, 70mM L-alanine, 20mM 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid. Reaction conditions are as follows: the temperature is 40 ℃, the stirring speed is 150r/min, and the reaction time is 24 h. Under the same conditions, the reaction solution with added sterile cells was used as a blank control, and the mutant wet cells were replaced with the E.coli wet cells containing empty vector as a negative control. After the reaction, samples were taken for HPLC detection (50:50 acetonitrile: water, 10mM ammonium acetate, 0.8mL/min flow rate, 268nm detection wavelength), and from 8000 muteins, 6298 muteins were lower in activity than the original protein, 1661 muteins were equivalent in activity to the original protein, and only 41 muteins were significantly higher in activity (increased by 20%) than the original protein, wherein the one mutant with the highest substrate conversion rate, pET28b-ABAT2-Y138F, was 96% in conversion rate, and ee > 99%. The amino acid sequence and the nucleotide sequence of the mutant pET28b-ABAT2-Y138F are shown as SEQ ID No.3 and SEQ ID No.4 in the sequence table. The mutant is prepared by mixing the amino acid sequence shown in SEQ ID NO: 2 to phenylalanine at amino acid position 138.
Example 6: recombinant escherichia coli fermentation enzyme production
The recombinant bacterium E.coli BL21(DE3)/pET28b-ABAT2-Y138F of example 5 was inoculated into LB liquid medium containing kanamycin to a final concentration of 50. mu.g/mL, and OD was cultured at 37 ℃ and 150r/min600About 0.6 to about 0.8, obtaining a seed solution; the seed solution was inoculated at 1% (v/v) into a fresh LB liquid medium containing 50. mu.g/mL kanamycin to culture OD at 37 ℃ at 150r/min600And (3) when the concentration is 0.6-0.8, adding IPTG with the final concentration of 0.1M into the culture solution, carrying out induced expression for 12 hours at the temperature of 28 ℃, centrifuging for 10 minutes at the temperature of 4 ℃ and 8000r/min, discarding supernatant, washing wet thalli twice by using 0.85% physiological saline, and collecting the wet thalli for later use.
Example 7: determination of transaminase and its mutant enzyme activity
Collecting wet thallus prepared in example 6, ultrasonic crushing for 6min under 40W condition, centrifuging the crushed mixture for 10min at 4 deg.C and 8000r/min, discarding the precipitate, collecting the supernatant, purifying with nickel column, balancing the chromatographic column with balance buffer (20mM phosphate buffer, 300mM NaCl, 20mM imidazole, pH 8.0), eluting with eluent (50mM phosphate buffer, 300mM NaCl, 500mM imidazole, pH 8.0), and collecting the corresponding eluent according to the signal response of ultraviolet detector, i.e. transaminase mutant pure enzyme solution. Enzyme activity detection standard conditions: 20mM PPO, 70mM L-alaine, 0.1mM PLP, 40 ℃ pH8.0 (0.05M Tris-HCl buffer) was incubated for 5min, then an appropriate amount of enzyme solution was added, the reaction was stopped with 6M HCl at 40 ℃ for 5min at 600rpm, and then 6M NaOH was added to adjust the pH of the reaction system back. The samples were subjected to derivatization reaction after centrifugation (12,000rpm, 1min) and dilution with ultrapure water, and then subjected to HPLC analysis after filtration through a 0.22 μm membrane.
Definition of enzyme activity: under the condition of enzyme activity measurement, the enzyme quantity required by generating 1 mu mol L-PPT per minute is defined as an enzyme activity unit U.
The specific enzyme activity is defined as the enzyme activity unit number per milligram of protein and is recorded as U/mg.
Table 1: transaminase activity determination
Example 8: determination of the optimum temperature for catalyzing transaminase mutants
The temperature optimum of the enzyme was measured using the pure enzyme solution of example 7 as the enzyme for catalysis. The specific operation is as follows: to 50mM Tris-HCl buffer (pH 8.0) were added 20mM PPO, 70mM L-alanine, 0.1mM PLP and appropriate amount of purified enzyme solution. At different catalytic temperatures: TA activity was measured at 20-75 deg.C (same procedure as in example 7), and the results are shown in FIG. 3. The optimum temperature of the transaminase mutant pET28b-ABAT2-Y138F is 40 ℃.
Example 9: determination of the optimum pH for catalyzing transaminase mutants
The pH optimum of the enzyme was measured using the pure enzyme solution of example 7 as the enzyme for catalysis. The specific operation is as follows: to 50mM Tris-HCl buffer (pH 8.0) were added 20mM PPO, 70mM L-alanine, 0.1mM PLP and appropriate amount of purified enzyme solution. TA activity was assayed by measuring the enzyme activity in acetic acid-sodium Acetate (Acetate buffer, pH 5.0-6.0), dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (PB buffer, pH 6.0-8.0), Tris-HCl buffer (Tris-HCl buffer, pH 8.0-9.0) and three different pH (5.0-10.0) buffers (the same procedure as in example 7). The results are shown in FIG. 4. The optimum pH of the transaminase mutant pET28b-ABAT2-Y138F is 8.0.
Example 10: determination of the Effect of Metal ions on the enzymatic Activity of transaminase mutants
The pure enzyme solution of example 7 was used as a catalytic enzyme, and the influence of metal ions on the enzymatic activity of the transaminase mutant was measured. The specific operation is as follows: the purified enzyme was placed in a Tris-HCl buffer at pH8.0, to which different metal ions (K) were added, respectively+、Cu2+、Fe3+、Ca2+、Ba2+、Na+、Mn2+、Ni2+、Li+、Co2+And Mg2+) EDTA (final concentration 1mM), incubated at 0 ℃ for 30min, and TA activity was measured (same as in example 5), and the results are shown in FIG. 5. Co2+、Ba2+And Fe3+Three metal ions have small promotion effect on the enzyme activity of the mutant, and other metal ions have obvious inhibition effect on the enzyme activity, especially Ni2+And Cu2+The inhibition effect of (A) is strongest.
Example 11: reaction process for synthesizing L-glufosinate-ammonium by transaminase mutant
Catalytic system (10 mL): 20mM PPO, 70mM L-alanine, 0.1mM PLP, Tris-HCl buffer system, pH8.0, wet bacterial mass of 20g/L, reaction at 40 ℃ and 600rpm for different time (1-24 h), timing sampling analysis, conversion rate results are shown in Table 2.
Table 2: effect of different reaction times on conversion
Example 12: reaction system (10 mL): Tris-HCl buffer, pH8.0, 100mM PPO, 350mM L-alanine, adding 1mM PLP, wet bacterial weight of 20g/L, 40 ℃, 600rpm under reaction for 24h, the conversion rate is 99.24%.
Example 13: reaction system (10 mL): Tris-HCl buffer, pH8.0, 200mM PPO, 700mM L-alanine, 1mM PLP added, wet bacterial mass of 20g/L, 40 ℃, 600rpm reaction for 24h, conversion of 91.02%.
Example 14: reaction system (10 mL): Tris-HCl buffer, pH8.0, 300mM PPO, 1050mM L-alanine, 1mM PLP added, wet biomass of 20g/L, reaction at 40 ℃ and 600rpm for 24h, conversion of 88.03%.
Example 15: reaction system (10 mL): Tris-HCl buffer, pH8.0, 400mM PPO, 1400mM L-alanine, adding 1mM PLP, wet bacterial amount of 20g/L, 40 ℃, 600rpm under reaction for 24h, the conversion rate is 87.02%.
Example 16: reaction system (10 mL): Tris-HCl buffer, pH8.0, 500mM PPO, 1750mM L-alanine, adding 1mM PLP, wet bacterial weight of 20g/L, 40 ℃, 600rpm under reaction for 24h, conversion is 86.32%.
The experimental results show that the recombinant escherichia coli containing the transaminase gene has stronger transaminase capacity, can directly take enzyme-containing bacterial cells as an enzyme source for biocatalysis or catalytic reaction, and can utilize 2-carbonyl-4- [ hydroxyl (methyl) phosphonyl ] -butyric acid as a substrate for biocatalysis asymmetric synthesis of high-optical-purity pesticide L-glufosinate-ammonium.
Sequence listing
<110> Zhejiang industrial university
<120> transaminase mutant and coding gene and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 427
<212> PRT
<213> Unknown (Unknown)
<400> 1
Met Asn Thr Asn Asn Ala Leu Met Gln Arg Arg His Asn Ala Val Pro
1 5 10 15
Arg Gly Val Gly Gln Ile His Pro Ile Phe Ala Glu Arg Ala Glu Asn
20 25 30
Cys Arg Val Trp Asp Val Glu Gly Arg Glu Tyr Leu Asp Phe Ala Gly
35 40 45
Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Gly Ile Val Ser
50 55 60
Ala Val Glu Ala Gln Leu Lys Lys Leu Ser His Thr Cys Phe Gln Val
65 70 75 80
Leu Ala Tyr Glu Pro Tyr Leu Ala Leu Cys Glu Arg Met Asn Gln Lys
85 90 95
Val Pro Gly Asp Phe Ala Lys Lys Thr Leu Leu Val Thr Thr Gly Ser
100 105 110
Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Lys Arg
115 120 125
Ser Gly Ala Ile Ala Phe Ser Gly Ala Tyr His Gly Arg Thr His Tyr
130 135 140
Thr Leu Ser Leu Thr Gly Lys Val His Pro Tyr Ser Ala Gly Met Gly
145 150 155 160
Leu Met Pro Gly His Val Tyr Arg Ala Leu Tyr Pro Cys Pro Leu His
165 170 175
Asn Ile Ser Asp Asp Asp Ala Ile Ala Ser Ile Glu Arg Ile Phe Lys
180 185 190
Asn Asp Ala Ala Pro Glu Asp Ile Ala Ala Ile Ile Ile Glu Pro Val
195 200 205
Gln Gly Glu Gly Gly Phe Tyr Ala Ala Ser Pro Ala Phe Met Gln Arg
210 215 220
Leu Arg Ala Leu Cys Asp Gln His Gly Ile Met Leu Ile Ala Asp Glu
225 230 235 240
Val Gln Ser Gly Ala Gly Arg Thr Gly Thr Leu Phe Ala Met Glu Gln
245 250 255
Met Gly Val Ala Ala Asp Ile Thr Thr Phe Ala Lys Ser Ile Ala Gly
260 265 270
Gly Phe Pro Leu Ala Gly Val Thr Gly Arg Ala Asp Val Met Asp Ala
275 280 285
Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Ile Ala
290 295 300
Cys Ala Ala Ala Leu Ala Val Leu Asp Ile Phe Glu Gln Glu Asn Leu
305 310 315 320
Leu Gln Lys Ala Asn Thr Leu Gly Asn Thr Leu Arg Asp Gly Leu Met
325 330 335
Glu Ile Ala Glu Thr His Arg Glu Ile Gly Asp Val Arg Gly Leu Gly
340 345 350
Ala Met Ile Ala Ile Glu Leu Phe Glu Asn Gly Asp Pro Gly Lys Pro
355 360 365
Asn Ala Ala Leu Thr Ala Asp Ile Val Thr Arg Ala Arg Glu Lys Gly
370 375 380
Leu Ile Leu Leu Ser Cys Gly Pro Tyr Tyr Asn Ile Leu Arg Ile Leu
385 390 395 400
Val Pro Leu Thr Ile Glu Ala Ser Gln Ile Arg Gln Gly Leu Glu Ile
405 410 415
Ile Ala Gln Cys Phe Asp Glu Ala Lys Gln Ala
420 425
<210> 2
<211> 1305
<212> DNA
<213> Unknown (Unknown)
<400> 2
atgaacacca acaacgctct gatgcagcgt cgtcacaacg ctgttccgcg tggtgttggt 60
cagatccacc cgatcttcgc tgaacgtgct gaaaactgcc gtgtttggga cgttgaaggt 120
cgtgaatacc tggacttcgc tggtggtatc gctgttctga acaccggtca cctgcacccg 180
ggtatcgttt ctgctgttga agctcagctg aaaaaactgt ctcacacctg cttccaggtt 240
ctggcttacg aaccgtacct ggctctgtgc gaacgtatga accagaaagt tccgggtgac 300
ttcgctaaaa aaaccctgct ggttaccacc ggttctgaag ctgttgaaaa cgctgttaaa 360
atcgctcgtg ctgctaccaa acgttctggt gctatcgctt tctctggtgc ttaccacggt 420
cgtacccact acaccctgtc tctgaccggt aaagttcacc cgtactctgc tggtatgggt 480
ctgatgccgg gtcacgttta ccgtgctctg tacccgtgcc cgctgcacaa catctctgac 540
gacgacgcta tcgcttctat cgaacgtatc ttcaaaaacg acgctgctcc ggaagacatc 600
gctgctatca tcatcgaacc ggttcagggt gaaggtggtt tctacgctgc ttctccggct 660
ttcatgcagc gtctgcgtgc tctgtgcgac cagcacggta tcatgctgat cgctgacgaa 720
gttcagtctg gtgctggtcg taccggtacc ctgttcgcta tggaacagat gggtgttgct 780
gctgacatca ccaccttcgc taaatctatc gcgggcggct tcccgctggc tggcgttacc 840
ggtcgtgctg acgttatgga cgctatcgct ccgggtggtc tgggtggtac ctacgctggt 900
aacccgatcg cttgcgctgc tgctctggct gttctggaca tcttcgaaca ggaaaacctg 960
ctgcagaaag ctaacaccct gggtaacacc ctgcgtgacg gtctgatgga aatcgctgaa 1020
acccaccgtg aaatcggtga cgttcgtggt ctgggtgcta tgatcgctat cgaactgttc 1080
gaaaacggtg acccgggtaa accgaacgct gctctgaccg ctgacatcgt tacccgtgct 1140
cgtgaaaaag gtctgatcct gctgtcttgc ggtccgtact acaacatcct gcgtatcctg 1200
gttccgctga ccatcgaagc ttctcagatc cgtcagggtc tggaaatcat cgctcagtgc 1260
ttcgacgaag ctaaacaggc tctcgagcac caccaccacc accac 1305
<210> 3
<211> 435
<212> PRT
<213> Unknown (Unknown)
<400> 3
Met Asn Thr Asn Asn Ala Leu Met Gln Arg Arg His Asn Ala Val Pro
1 5 10 15
Arg Gly Val Gly Gln Ile His Pro Ile Phe Ala Glu Arg Ala Glu Asn
20 25 30
Cys Arg Val Trp Asp Val Glu Gly Arg Glu Tyr Leu Asp Phe Ala Gly
35 40 45
Gly Ile Ala Val Leu Asn Thr Gly His Leu His Pro Gly Ile Val Ser
50 55 60
Ala Val Glu Ala Gln Leu Lys Lys Leu Ser His Thr Cys Phe Gln Val
65 70 75 80
Leu Ala Tyr Glu Pro Tyr Leu Ala Leu Cys Glu Arg Met Asn Gln Lys
85 90 95
Val Pro Gly Asp Phe Ala Lys Lys Thr Leu Leu Val Thr Thr Gly Ser
100 105 110
Glu Ala Val Glu Asn Ala Val Lys Ile Ala Arg Ala Ala Thr Lys Arg
115 120 125
Ser Gly Ala Ile Ala Phe Ser Gly Ala Phe His Gly Arg Thr His Tyr
130 135 140
Thr Leu Ser Leu Thr Gly Lys Val His Pro Tyr Ser Ala Gly Met Gly
145 150 155 160
Leu Met Pro Gly His Val Tyr Arg Ala Leu Tyr Pro Cys Pro Leu His
165 170 175
Asn Ile Ser Asp Asp Asp Ala Ile Ala Ser Ile Glu Arg Ile Phe Lys
180 185 190
Asn Asp Ala Ala Pro Glu Asp Ile Ala Ala Ile Ile Ile Glu Pro Val
195 200 205
Gln Gly Glu Gly Gly Phe Tyr Ala Ala Ser Pro Ala Phe Met Gln Arg
210 215 220
Leu Arg Ala Leu Cys Asp Gln His Gly Ile Met Leu Ile Ala Asp Glu
225 230 235 240
Val Gln Ser Gly Ala Gly Arg Thr Gly Thr Leu Phe Ala Met Glu Gln
245 250 255
Met Gly Val Ala Ala Asp Ile Thr Thr Phe Ala Lys Ser Ile Ala Gly
260 265 270
Gly Phe Pro Leu Ala Gly Val Thr Gly Arg Ala Asp Val Met Asp Ala
275 280 285
Ile Ala Pro Gly Gly Leu Gly Gly Thr Tyr Ala Gly Asn Pro Ile Ala
290 295 300
Cys Ala Ala Ala Leu Ala Val Leu Asp Ile Phe Glu Gln Glu Asn Leu
305 310 315 320
Leu Gln Lys Ala Asn Thr Leu Gly Asn Thr Leu Arg Asp Gly Leu Met
325 330 335
Glu Ile Ala Glu Thr His Arg Glu Ile Gly Asp Val Arg Gly Leu Gly
340 345 350
Ala Met Ile Ala Ile Glu Leu Phe Glu Asn Gly Asp Pro Gly Lys Pro
355 360 365
Asn Ala Ala Leu Thr Ala Asp Ile Val Thr Arg Ala Arg Glu Lys Gly
370 375 380
Leu Ile Leu Leu Ser Cys Gly Pro Tyr Tyr Asn Ile Leu Arg Ile Leu
385 390 395 400
Val Pro Leu Thr Ile Glu Ala Ser Gln Ile Arg Gln Gly Leu Glu Ile
405 410 415
Ile Ala Gln Cys Phe Asp Glu Ala Lys Gln Ala Leu Glu His His His
420 425 430
His His His
435
<210> 4
<211> 1305
<212> DNA
<213> Unknown (Unknown)
<400> 4
atgaacacca acaacgctct gatgcagcgt cgtcacaacg ctgttccgcg tggtgttggt 60
cagatccacc cgatcttcgc tgaacgtgct gaaaactgcc gtgtttggga cgttgaaggt 120
cgtgaatacc tggacttcgc tggtggtatc gctgttctga acaccggtca cctgcacccg 180
ggtatcgttt ctgctgttga agctcagctg aaaaaactgt ctcacacctg cttccaggtt 240
ctggcttacg aaccgtacct ggctctgtgc gaacgtatga accagaaagt tccgggtgac 300
ttcgctaaaa aaaccctgct ggttaccacc ggttctgaag ctgttgaaaa cgctgttaaa 360
atcgctcgtg ctgctaccaa acgttctggt gctatcgctt tctctggtgc tttccacggt 420
cgtacccact acaccctgtc tctgaccggt aaagttcacc cgtactctgc tggtatgggt 480
ctgatgccgg gtcacgttta ccgtgctctg tacccgtgcc cgctgcacaa catctctgac 540
gacgacgcta tcgcttctat cgaacgtatc ttcaaaaacg acgctgctcc ggaagacatc 600
gctgctatca tcatcgaacc ggttcagggt gaaggtggtt tctacgctgc ttctccggct 660
ttcatgcagc gtctgcgtgc tctgtgcgac cagcacggta tcatgctgat cgctgacgaa 720
gttcagtctg gtgctggtcg taccggtacc ctgttcgcta tggaacagat gggtgttgct 780
gctgacatca ccaccttcgc taaatctatc gcgggcggct tcccgctggc tggcgttacc 840
ggtcgtgctg acgttatgga cgctatcgct ccgggtggtc tgggtggtac ctacgctggt 900
aacccgatcg cttgcgctgc tgctctggct gttctggaca tcttcgaaca ggaaaacctg 960
ctgcagaaag ctaacaccct gggtaacacc ctgcgtgacg gtctgatgga aatcgctgaa 1020
acccaccgtg aaatcggtga cgttcgtggt ctgggtgcta tgatcgctat cgaactgttc 1080
gaaaacggtg acccgggtaa accgaacgct gctctgaccg ctgacatcgt tacccgtgct 1140
cgtgaaaaag gtctgatcct gctgtcttgc ggtccgtact acaacatcct gcgtatcctg 1200
gttccgctga ccatcgaagc ttctcagatc cgtcagggtc tggaaatcat cgctcagtgc 1260
ttcgacgaag ctaaacaggc tctcgagcac caccaccacc accac 1305