CN110951705B - Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell and preparation method and application thereof - Google Patents
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Abstract
The invention relates to an amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell, a preparation method and an application thereof. The amine dehydrogenase mutant is obtained by mutating 104 th glutamic acid of wild amine dehydrogenase into alanine; or the wild amine dehydrogenase is obtained by mutating the 137 th asparagine of the wild amine dehydrogenase into proline; or the valine at the 174 th position of the wild amine dehydrogenase is mutated into the cysteine to obtain the amino acid; or mutation from valine at position 197 of wild-type amine dehydrogenase to aspartic acid. The catalytic efficiency of the amine dehydrogenase is high.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell, a preparation method and an application thereof.
Background
Chiral amines are widely used in organic synthesis, perfumes, medicines, etc., and are important intermediates. The difference of the stereo structures of different enantiomers of chiral amine makes the different chiral enantiomers have different activities and different physiological activities and toxic actions when acting on life bodies. Therefore, chiral amines are becoming important indicators for evaluating the safety and efficacy of drugs.
At present, the method for preparing chiral amine mainly comprises a chemical catalyst and a biological enzyme catalysis method, and compared with the chemical catalyst, the enzyme catalysis method has better catalysis effect, and particularly has obvious technical advantages in the aspect of identifying two enantiomers of the chiral amine when the biological enzyme preparation catalyzes the selective synthesis of chiral drugs. Among them, amine dehydrogenase (AmDH) can catalyze prochiral ketone to synthesize chiral amine, and is an effective biocatalyst for preparing chiral amine. However, the catalytic efficiency of the existing amine dehydrogenases is low, and the requirements of practical application cannot be met.
Disclosure of Invention
Accordingly, it is necessary to provide an amine dehydrogenase having high catalytic efficiency.
In addition, the application of the amine dehydrogenase mutant, an enzyme preparation, a recombinant vector, a recombinant cell and a preparation method thereof are also provided.
An amine dehydrogenase mutant is obtained by mutating 104 th glutamic acid of wild amine dehydrogenase into alanine;
or the wild amine dehydrogenase is obtained by mutating the 137 th asparagine of the wild amine dehydrogenase into proline;
or the valine at the 174 th position of the wild amine dehydrogenase is mutated into the cysteine to obtain the amino acid;
or mutation from valine at position 197 of wild-type amine dehydrogenase to aspartic acid.
As a result of extensive studies on amine dehydrogenase, it was found that glutamic acid at position 104 of wild-type amine dehydrogenase was mutated to alanine, or wild-type amine dehydrogenase was mutatedOr mutating valine at position 174 of the wild-type amine dehydrogenase into cysteine, or mutating valine at position 197 of the wild-type amine dehydrogenase into aspartic acid, so that the obtained amine dehydrogenase mutant has higher catalytic efficiency. Proved by experiments, the K of the amine dehydrogenase mutantcatValue of 468.34s-1～501.22s-1The results show that the amine dehydrogenase mutant has higher catalytic rate and higher catalytic efficiency.
In one embodiment, the amino acid sequence of the wild-type amine dehydrogenase is shown as SEQ ID No. 1.
In one embodiment, the nucleotide sequence of the wild-type amine dehydrogenase is shown as SEQ ID No. 2.
An enzyme preparation comprising the amine dehydrogenase mutant.
A recombinant vector comprising the coding sequence of the amine dehydrogenase mutant.
A recombinant cell comprising the coding sequence of the above amine dehydrogenase mutant.
The preparation method of the recombinant engineering bacteria cell comprises the following steps:
carrying out error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase, and connecting the coding sequence to an empty vector after enzyme digestion to obtain an amine dehydrogenase mutant library plasmid;
respectively transforming the amine dehydrogenase mutant library plasmids into host cells to obtain transformed cells; and
and (3) carrying out high-throughput screening on the transformed cells by adopting an IVC-FACS method to obtain the recombinant cells.
In one embodiment, in the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase, the error-prone PCR amplification is performed by using primer pairs with sequences shown as SEQ ID No. 3-SEQ ID No. 4.
In one embodiment, the step of performing error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase further comprises the following steps: and adopting a primer pair with the sequence shown as SEQ ID No. 5-SEQ ID No.6 to amplify the coding sequence of the wild amine dehydrogenase by PCR.
The use of the above-described amine dehydrogenase mutant, the above-described enzyme preparation, the above-described recombinant vector or the above-described recombinant cell for the preparation of chiral amines.
Drawings
FIG. 1 is a schematic structural diagram of a protein crystal model of wild-type amine dehydrogenase.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention. The nucleotide sequences in the sequence listing are all in the order from 5 'to 3' unless otherwise specified.
The amine dehydrogenase mutant has high catalytic efficiency, and can catalyze prochiral ketone and free ammonia to asymmetrically synthesize chiral amine under the action of coenzyme so as to be used for preparing the chiral amine. Specifically, the amine dehydrogenase mutant is obtained by mutating 104 th glutamic acid of wild-type amine dehydrogenase into alanine; or the wild amine dehydrogenase is obtained by mutating the 137 th asparagine of the wild amine dehydrogenase into proline; or the valine at the 174 th position of the wild amine dehydrogenase is mutated into the cysteine to obtain the amino acid; or mutation from valine at position 197 of wild-type amine dehydrogenase to aspartic acid.
The present inventors have conducted extensive studies on amine dehydrogenases and found that an amine dehydrogenase mutant obtained by mutating the 104 th glutamic acid of a wild-type amine dehydrogenase to alanine, or mutating the 137 th asparagine of a wild-type amine dehydrogenase to proline, or mutating the 174 th valine of a wild-type amine dehydrogenase to cysteine, or mutating the 197 th valine of a wild-type amine dehydrogenase to aspartic acid has high catalytic efficiency.
Wherein the amino acid sequence of the wild-type amine dehydrogenase is shown in SEQ ID No. 1. Specifically, the sequence shown as SEQ ID No.1 is: MEKIRVIIWGLGAMGGGMARMILQKKGMEIVGAIASRPEKSGKDLGEVLDLGLKTGVTISCDPETVLKQPADIVLLATSSFTREVYPQLQRIIASGKNVITIAEEMAYPAYREPELAAKIDKMAKDHGVTVLGTGINPGFVLDTLIIALSGVCMDIKKITARRINDLSPFGTTVMRTQGVGTTVDEFRKGLEEGTIVGHIGFPESISLISEALGLEIDEIREMREPIVSNVYRETPYARVEPGMVAGCKHTGIGYRKGEPVIVLEHPQQIRPELEDVETGDYIEIEGTPNIKLSIKPEIPGGIGTIAIAVNMIPKVISANTGLVTMKDLPVPAALMGDIRKLAKDGVNNA are provided.
Further, the nucleotide sequence of the wild-type amine dehydrogenase is shown in SEQ ID No. 2. Specifically, the sequence shown as SEQ ID No.2 is: ATGGAAAAAATCCGTGTTATCATCTGGGGTCTGGGTGCTATGGGTGGTGGTATGGCTCGTATGATCCTGCAGAAAAAAGGTATGGAAATCGTTGGTGCTATCGCTTCTCGTCCGGAAAAATCTGGTAAAGACCTGGGTGAAGTTCTGGACCTGGGTCTGAAAACCGGTGTTACCATCTCTTGCGACCCGGAAACCGTTCTGAAACAGCCGGCTGACATCGTTCTGCTGGCTACCTCTTCTTTCACCCGTGAAGTTTACCCGCAGCTGCAGCGTATCATCGCTTCTGGTAAAAACGTTATCACCATCGCTGAAGAAATGGCTTACCCGGCTTACCGTGAACCGGAACTGGCTGCTAAAATCGACAAAATGGCTAAAGACCACGGTGTTACCGTTCTGGGTACCGGTATCAACCCGGGTTTCGTTCTGGACACCCTGATCATCGCTCTGTCTGGTGTTTGCATGGACATCAAAAAAATCACCGCTCGTCGTATCAACGACCTGTCTCCGTTCGGTACCACCGTTATGCGTACCCAGGGTGTTGGTACCACCGTTGACGAATTCCGTAAAGGTCTGGAAGAAGGTACCATCGTTGGTCACATCGGTTTCCCGGAATCTATCTCTCTGATCTCTGAAGCTCTGGGTCTGGAAATCGACGAAATCCGTGAAATGCGTGAACCGATCGTTTCTAACGTTTACCGTGAAACCCCGTACGCTCGTGTTGAACCGGGTATGGTTGCTGGTTGCAAACACACCGGTATCGGTTACCGTAAAGGTGAACCGGTTATCGTTCTGGAACACCCGCAGCAGATCCGTCCGGAACTGGAAGACGTTGAAACCGGTGACTACATCGAAATCGAAGGTACCCCGAACATCAAACTGTCTATCAAACCGGAAATCCCGGGTGGTATCGGTACCATCGCTATCGCTGTTAACATGATCCCGAAAGTTATCTCTGCTAACACCGGTCTGGTTACCATGAAAGACCTGCCGGTTCCGGCTGCTCTGATGGGTGACATCCGTAAACTGGCTAAAGACGGTGTTAACAACGCT are provided.
It should be noted that, since the same amino acid can be determined by several different codons, the same amino acid can correspond to different coding sequences. Thus, in the present application, the amino acid sequence shown in SEQ ID No.1 can be encoded by a coding sequence having a codon-synonymous mutation obtained by substituting 1 or several nucleotides in the coding sequence shown in SEQ ID No.2, in addition to the coding sequence shown in SEQ ID No. 2. The nucleotide sequence of the wild-type ammonia amine dehydrogenase of the present application can be obtained by those skilled in the art by the method of cDNA cloning and site-directed mutagenesis or other suitable methods based on the amino acid sequence shown in SEQ ID No.1 disclosed in the present application, and thus, the coding sequence of the nucleotide sequence encoding the wild-type ammonia amine dehydrogenase is not limited to the coding sequence shown in SEQ ID No. 2. It is also within the scope of the present invention if the nucleotide sequence encoding the resulting protein does not differ significantly from the wild-type ammonia amine dehydrogenase of the present application.
In addition, due to polymorphism and variation of protein coding sequences, naturally occurring proteins may have genetic mutations, in which bases are deleted, substituted or added, or amino acids are deleted, inserted, substituted or otherwise varied in the coding sequences, resulting in deletion, substitution or addition of one or more amino acids in the amino acid sequence of the protein. Thus, there are some proteins that are substantially equivalent to the non-mutated proteins in terms of their physiological and biological activities. These polypeptides or proteins which differ structurally from the corresponding protein, but which do not differ significantly in function from the protein, are referred to as functionally equivalent variants.
Functionally equivalent variants are also suitable for polypeptides made by introducing such variations into the amino acid sequence of a protein by altering one or more codons by artificial means such as deletions, insertions, and mutations. Although this allows more variant variants to be obtained, the resulting variants are functionally equivalent variants provided that their physiological activity is substantially equivalent to that of the original non-variant protein.
Generally, functionally equivalent variant coding sequences are homologous, and thus a polypeptide or protein resulting from at least one alteration, such as a deletion, insertion or substitution of one or more bases in the coding sequence of the protein or a deletion, insertion or substitution of one or more amino acids in the amino acid sequence of the protein, generally has a functionally equivalent activity to the protein in question, and therefore a polypeptide resulting from a polypeptide encoded by a coding sequence as described above or a polypeptide consisting of an amino acid sequence as described above is also included within the scope of the present application if the amino acid sequences encoding the wild-type amino dehydrogenase of the present application do not differ significantly in function.
In one embodiment, the substrates for the action of the amine dehydrogenase mutant include prochiral ketones and free ammonia.
Wherein the prochiral ketone is 4-ketopentanoic prochiral ketone. The free ammonia is ammonia free ammonia. The coenzyme is reduced coenzyme I (NADH).
The amine dehydrogenase mutant has higher catalytic efficiency. Proved by experiments, the K of the amine dehydrogenase mutantcatValue of 468.34s-1～501.22s-1The results show that the amine dehydrogenase mutant has higher catalytic rate and higher catalytic efficiency.
The research shows that the wild-type amine dehydrogenase has high affinity to the substrate, but the wild-type amine dehydrogenase mainly plays a role in a milder environment in vivo, and the industrial application process requires that the enzyme plays a role in a harsher environment (such as high temperature, extreme pH value, organic solvent, non-natural substrate, product inhibition and the like), so the wild-type amine dehydrogenase often has the problems of low catalytic efficiency and the like in application. K of the above-mentioned amine dehydrogenase mutantMA value of 17.12 to 23.21. mu.M, and K of a wild-type amine dehydrogenaseMThe values are comparable, indicating that the above-described amine dehydrogenase mutants still retain a higher affinity for the substrate.
Some researches show that the activity of the wild amine dehydrogenase can be improved to a certain extent, but the stability is reduced along with the improvement of the activity of the enzyme. The amine dehydrogenase mutant has high activity and thermal stability equivalent to that of wild amine dehydrogenase. Experiments prove that the amine dehydrogenase mutant can be kept for 30min at 42 ℃ and the activity is kept unchanged.
In conclusion, the amine dehydrogenase mutant has high catalytic efficiency and high affinity to a substrate, can catalyze prochiral ketone and free ammonia to asymmetrically synthesize chiral amine under the action of coenzyme, has excellent thermal stability, can be used for preparing chiral amine, and can be applied to the fields of food, medicine and the like.
An enzyme preparation according to an embodiment includes the amine dehydrogenase mutant according to the above embodiment.
Wherein the enzyme preparation is soluble protein or immobilized enzyme.
In one embodiment, the enzyme preparation further comprises a coenzyme. Wherein the coenzyme is reduced coenzyme I (NADH) coenzyme.
The enzyme preparation comprises the amine dehydrogenase mutant of the embodiment, has high catalytic efficiency and high affinity to a substrate, can catalyze prochiral ketone and free ammonia to asymmetrically synthesize chiral amine under the action of coenzyme, can be used for preparing chiral amine, and can be applied to the fields of food, medicine and the like.
The recombinant vector of an embodiment includes a coding sequence of the dehydrogenase mutant of the above embodiment.
In one embodiment, the recombinant vector is a recombinant expression vector or a recombinant cloning vector.
In one embodiment, the recombinant vector contains a purification tag. And the purification label is arranged, so that the separation and purification of the active peptide are facilitated. Further, the purification tag is a His tag, a GST tag, or a SUMO tag. It should be noted that the purification tag is not limited to the above-mentioned purification tags, and other common purification tags can also be used as the purification tag of the recombinant vector.
In one embodiment, the recombinant vector comprises a genetically engineered vector. The nucleotide encoding the dehydrogenase mutant is inserted into a genetic engineering vector. Furthermore, the genetic engineering vector is a pET-32a vector, a pET28a vector, a pGEX-6P-1 vector, a pPIC-9K vector or a pPIC-Z alpha vector. It should be noted that the genetic engineering vector is not limited to the above-mentioned genetic engineering vector, and other common genetic engineering vectors may be used as the genetic engineering vector of the recombinant vector.
The recombinant vector can better preserve the nucleotide for coding the dehydrogenase mutant, is favorable for the expression of the dehydrogenase mutant, and can be applied to the preparation of chiral amine.
A recombinant cell comprising the coding sequence of the amine dehydrogenase mutant of the above embodiments.
In one embodiment, the recombinant cell is a cell that clones a coding sequence encoding the above-described amine dehydrogenase mutant.
In one embodiment, the recombinant cell is a cell that expresses a coding sequence encoding the above-described amine dehydrogenase mutant.
In one embodiment, the recombinant cell comprises a recipient cell. The coding sequence encoding the above-mentioned amine dehydrogenase mutant or the above-mentioned recombinant vector is located in the recipient cell.
In one embodiment, the recipient cell is escherichia coli, saccharomyces cerevisiae, pichia pastoris, an animal cell, or a plant cell. Further, the recipient cell is Escherichia coli 10G, Escherichia coli DH5 alpha, Escherichia coli Top10, Escherichia coli Orgami (DE3), Pichia pastoris GS115 or Pichia pastoris SMD 1168.
The recipient cell is not limited to the above-mentioned recipient cell, and other common recipient cells may also function as the recipient cell of the recombinant cell.
The recombinant cell can clone or express the amine dehydrogenase mutant, so that the amine dehydrogenase mutant can be prepared on a large scale, and the amine dehydrogenase mutant can be directionally expressed by the recombinant cell, so that the amine dehydrogenase mutant with high purity can be obtained, and the application of the amine dehydrogenase mutant is facilitated, therefore, the recombinant cell can be used for preparing chiral amine.
The method for producing a recombinant cell according to the above embodiment includes steps S110 to S130 of:
s110: the coding sequence of the wild-type amine dehydrogenase of the above embodiment is subjected to error-prone PCR amplification, and is ligated into an empty vector after enzyme cleavage, thereby obtaining an amine dehydrogenase mutant library plasmid.
Wherein, in the step of carrying out error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase of the embodiment, a primer pair with the sequence shown as SEQ ID No. 3-SEQ ID No.4 is adopted for carrying out error-prone PCR amplification. Specifically, the sequence shown as SEQ ID No.3 is: 5'-ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3', respectively; the sequence shown as SEQ ID No.4 is: 5'-TCAGCTCTCGAGTTAAGCGTTGTTAACACCG-3' are provided.
Wherein, the reaction system of the error-prone PCR is as follows: 0.05U/. mu.L of DreamTaqTM250 mu M dATP, 250 mu M dGTP, 1050 mu M dCTP, 1050 mu M dTTP, 0.4 mu M primer with sequence shown in SEQ ID No.3, 0.4 mu M primer with sequence shown in SEQ ID No.4, 0.2 ng/mu L empty carrier and 0.2 mM-0.8 mM manganese chloride.
Wherein, the reaction conditions of the error-prone PCR are as follows: 95 ℃, 3min, 1 cycle; 95 deg.C, 15s, 55 deg.C, 30s, 72 deg.C, 1min, 30 cycles; 72 ℃, 5min, 1 cycle.
Wherein the empty vector is pET-28a plasmid. The empty vector is not limited to the pET-28a plasmid, and may be any other empty vector commonly used in the art.
In one embodiment, the error-prone PCR amplification step of the coding sequence of the wild-type amine dehydrogenase of the above embodiment is preceded by the following steps: the coding sequence of wild amine dehydrogenase is amplified by PCR by using a primer pair with the sequence shown in SEQ ID No. 5-SEQ ID No. 6. This arrangement facilitates enrichment of the coding sequence of wild-type amine dehydrogenase to facilitate construction of amine dehydrogenase mutant library plasmids by error-prone PCR amplification.
Specifically, the sequence shown as SEQ ID No.5 is: 5' -ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3', wherein the underlined bases are recognition sites for restriction enzyme NdeI; the sequence shown as SEQ ID No.6 is: 5' -TCAGCTCTCGAGTTAAGCGTTGTTAACACCG-3', wherein the underlined bases are recognition sites for the restriction enzyme XhoI.
S120: and (3) respectively transforming the amine dehydrogenase mutant library plasmids into host cells to obtain transformed cells.
Wherein the host cell is Escherichia coli. The Escherichia coli expression system has the advantages of clear genetic background, high expression level of target genes, short culture period, strong pollution resistance and the like, and is an important tool in molecular biology research and the industrialized development process of biotechnology. Further, the host cell is Escherichia coli 10G. The host cell is not limited to the above-mentioned cells, and may be other host cells commonly used in the art.
Wherein the transformation mode is electric transformation. The transformation method is not limited to electrotransformation, and other transformation methods commonly used in the art may be used.
S130: and (3) carrying out high-throughput screening on the transformed cells by adopting an IVC-FACS method to obtain the recombinant cells.
Directed evolution is a powerful tool for molecular engineering of enzymes to improve their properties in various ways. The principle is to simulate the natural evolution principle, artificially construct a gene mutation library of target protein in a laboratory, and then select out mutants which accord with expected properties by utilizing a screening means. However, because the positive rate in the mutation library is low, the conventional screening method based on the micro-porous plate or the substrate plate is time-consuming and labor-consuming due to low flux, and cannot obtain good effect in the directed evolution screening. According to the research, an ultrahigh-flux screening method based on an in vitro compartmentalization-fluorescence activated cell sorting (IVC-FACS) technology is established, so that the enzyme mutants can be efficiently and quickly screened, the screening flux can reach one million per hour, and the method can be used for high-flux screening of an amine dehydrogenase random mutation library so as to obtain the positive mutants with improved activity.
The recombinant cell prepared by the preparation method of the recombinant cell can clone or express the amine dehydrogenase mutant, so that the amine dehydrogenase mutant can be prepared on a large scale, and the amine dehydrogenase mutant is directionally expressed by the recombinant cell, so that the amine dehydrogenase mutant with higher purity can be obtained, and the preparation method has better thermal stability, is beneficial to the application of the amine dehydrogenase mutant, and can be used for preparing chiral amine.
The following are specific examples.
Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, are usually carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer of the kits. The reagents used in the examples are all commercially available.
In the following examples, T4DNA ligase was purchased from NEB; pET28a plasmid was purchased from vast Ling plasmid; XhoI enzyme was purchased from NEB; NdeI enzyme was purchased from NEB.
Example 1
Cloning of the coding sequence of the wild amine dehydrogenase
The coding sequence of wild amine dehydrogenase (GenBank: CP002131.1) is codon optimized by taking Escherichia coli as a host, synthesized by Jinwei Zhi, Suzhou, primers with sequences shown as SEQ ID No. 5-SEQ ID No.6 are adopted to amplify the coding sequence of the wild amine dehydrogenase by PCR, KOD high-fidelity polymerase of Toyobo (Shanghai) Biotechnology Limited is used for PCR amplification, and the amplification conditions are as follows: at 95 ℃ for 2 min; then 56 ℃, 20sec, 72 ℃, 90sec, total 30 cycles; finally 72 ℃ for 10 min. Wherein the amino acid sequence of the wild-type amine dehydrogenase is shown as SEQ ID No. 1; the nucleotide sequence of the wild-type amine dehydrogenase is shown in SEQ ID No. 2.
After the reaction is finished, agarose gel electrophoresis with the mass percentage content of 1.5% is used for detecting the PCR product, a 1.0kb band is obtained, and the length accords with an expected result. The target fragment was recovered and purified according to the instructions of a nucleic acid recovery kit (purchased from Takara). The recovered fragment and pET28a plasmid were double-digested with restriction endonucleases XhoI and NdeI, ligated with T4DNA ligase, and the ligated product was transformed into E.coli BL21(DE3) competent cells, plated on LB plate containing kanamycin (50ug/mL) and cultured for 12 hours. The positive strains were picked, and the positive cloning plasmids were extracted using a plasmid extraction kit (purchased from Takara) and sequenced. The sequencing detection result shows that the cloned amine dehydrogenase ANDD-TDO gene has correct sequence and is correctly inoculated into pET28a plasmid, named recombinant plasmid pET28a-ANDD-TDO, and stored in glycerol.
Example 2
Expression, purification and activity determination of amine dehydrogenase
Mixing glycerol tube with pET28The engineering bacteria of the a-ANDD-TDO recombinant plasmid are inoculated into a 4mL LB culture medium test tube containing 100 mu g/mL kanamycin according to the volume ratio of 1 percent, and cultured for 12h at 37 ℃ and 220 rpm. Transferring all the cultured bacterial liquid into 1L LB culture medium shake flask containing 50. mu.g/mL kanamycin, culturing at 37 deg.C and 220rpm for about 2.5h to OD600Reaching about 0.9, adding 0.1mM IPTG inducer, and performing induced culture at 25 deg.C and 200rpm for 16 h. And ultrasonically crushing the escherichia coli suspension obtained after the induction is finished, and performing Ni-NTA affinity chromatography treatment to obtain the wild amine dehydrogenase with the purity of more than 95%. The activity of the wild-type amine dehydrogenase was determined, and the results were: k of wild-type amine dehydrogenaseMK of 19.22. mu.M, wild-type amine dehydrogenasecatValue of 128.34s-1. Among these, the reference Catal.Sci.Technol.2016:10.1039.C6CY01625A determines the activity of wild-type amine dehydrogenases.
Example 3
Construction of Large-Capacity random mutation library of amine dehydrogenase
A mutation library of amine dehydrogenase is constructed by an error-prone PCR (ep-PCR) method, wherein the high and low mutation rates are realized by adjusting the concentration of manganese ions in a PCR system. Specifically, error-prone PCR amplification was performed using the coding sequence for the wild-type amine dehydrogenase of example 1. The error-prone PCR reaction system is as follows: 0.05U/. mu.L of DreamTaqTM250 mu M dATP, 250 mu M dGTP, 1050 mu M dCTP, 1050 mu M dTTP, 0.4 mu M primer with the sequence shown in SEQ ID No.3, 0.4 mu M primer with the sequence shown in SEQ ID No.4, 0.2 ng/mu L empty carrier and 0.2 mM-0.8 mM manganese chloride; the total volume of each tube was 25. mu.L, and the empty vector was pET-28a plasmid. The error-prone PCR reaction conditions were: 95 ℃, 3min, 1 cycle; 95 deg.C, 15s, 55 deg.C, 30s, 72 deg.C, 1min, 30 cycles; 72 ℃, 5min, 1 cycle.
The amplification product obtained by error-prone PCR is subjected to agarose electrophoresis, gel cutting, purification and recovery, then NdeI and BamHI are used for double enzyme digestion, then T4 ligase is used for cloning onto pET28a plasmid, and the purified ligation system is electrically transformed into Escherichia coli 10G competent cells. The transformed cells were recovered, inoculated into 50mL of LB medium (containing 100. mu.g/mL of kanamycin), and cultured overnight at 37 ℃. The plasmid was extracted from the culture plasmid recovery kit (purchased from Takara) to obtain mutant library plasmids. Meanwhile, the culture broth was spread on an LB agarose plate containing 20. mu.g/mL of kanamycin and cultured at 37 ℃ for 12 hours, and the pool capacity of the mutant pool plasmids was calculated to be about 200 ten thousand. Several clones were subjected to pyrosequencing to determine the mutation rate, and it was found that the mutation rate was about 2 changed amino acid residues per gene on average at a manganese ion concentration of 0.6 mM. The plasmid of the mutant library was transformed into a host BL21(DE3), inoculated into a 4mL LB medium tube containing 100. mu.g/mL Kan, and cultured at 37 ℃ and 220rpm for 12 hours; transferring 4mL of the bacterial liquid into a shake flask of 1L LB culture medium containing 50 mu g/mL Kan, culturing for 2.5h at 37 ℃ and 220rpm to enable OD600 to reach about 0.9, adding 0.1mM IPTG inducer, performing induced culture for 16h at 25 ℃ and 200rpm, and then screening to obtain BL21(DE3) cells expressing the amine dehydrogenase mutant library.
Example 4
IVC-FACS high throughput screening of amine dehydrogenase random mutation pools
BL21(DE3) cells expressing the mutant library of amine dehydrogenase were encapsulated in w/o/w (water-in-oil-in-water) secondary microdroplets for enzymatic reaction.
Specifically, microdroplets were prepared using a micro membrane extruder (Avanti Polar Lipids, AL, USA), two syringes (Gastight 1001syring, 1mL, Hamilton, NV, USA) in a set and a Track-Etch polycarbonate membrane (Millipore, USA) with a pore size of 8 microns.
First, the film was set in a film extruder, and then 0.5mL of an oil phase (oil phase component: light paraffin oil containing 2.9% by volume of ABIL EM90 emulsifier) was rinsed twice with a syringe. When emulsifying, 100 μ L of internal water phase (i.e. Escherichia coli BL21(DE3) -Codonplus cell suspension with cell concentration OD 600: 0.5) and 400 μ L of oil phase (oil phase component: light paraffin oil containing ABIL EM90 emulsifier with volume percentage of 2.9%) are sucked into the same syringe, the mixed system is pushed into another syringe by a membrane extruder and then pushed back into the first syringe, and the process is called primary emulsification to generate w/o (water-in-oil type) primary micro-liquid. The generated w/o primary micro-droplets are observed in real time by a microscope (50i, Nikon, Japan, 40 Xobject), and the diameters of the micro-droplets are distributed within 3-5 μm by optimizing the emulsification times.
The prepared w/o primary micro-droplets were dispersed into a secondary aqueous phase (i.e., 1 XPBS containing 1% Triton X-102 by volume, pH7.4) through a membrane with an aperture of 8 μm to form w/o/w secondary micro-droplets. The method comprises the following specific steps: a new piece of the film was placed in the film extruder and rinsed twice with 0.5mL of aqueous phase. Respectively sucking 200 mu L of w/o first-level micro-droplets and 400 mu L of secondary aqueous phase into two syringes; specifically, firstly, injecting the w/o primary micro-droplets into the secondary water phase of the second injector through a membrane extruder, and then pushing the mixed system back into the original injector through the membrane extruder to complete primary emulsification. The morphological distribution of the generated w/o/w secondary micro-droplets is observed in real time through a microscope, and the final w/o/w secondary micro-droplets have the diameter of about 10 mu m and relatively uniform size by optimizing the emulsification times. To 0.2mL of an external aqueous phase containing w/o/w secondary micro-droplets (the external aqueous phase is water, and the content of the w/o/w secondary micro-droplets is 80%), 0.02mL of a fluorogenic substrate (i.e., dimethyl sulfoxide containing 10mM fluorescein dibutyrate, available from Sigma) was added, and the mixture was incubated with shaking on a metal bath at 37 ℃ and 1000rpm for 30min to perform an enzymatic reaction, wherein the final concentration of the fluorogenic substrate fluorescein dibutyrate was 0.5 mM.
Using sorting type flow cytometer (BD FACSAria)TMII) detecting a fluorescence signal of a reaction system, wherein the inner diameter of a nozzle is 100 mu m, droplets (accounting for about 0.1 percent of the droplets containing cells) with the highest fluorescence intensity at the detection speed of 10000 cells/sec of a sample are sorted into an empty 2mL eppendorf tube, and positive genes are subjected to PCR amplification by taking the droplets as templates, wherein the PCR reaction system is as follows: 0.05U/. mu.L of DreamTaqTM(purchased from Takara), 250. mu.M dATP, 250. mu.M dGTP, 250. mu.M dCTP, 250. mu.M dTTP, 0.4. mu.M primer having the sequence shown in SEQ ID No.3, 0.4. mu.M primer having the sequence shown in SEQ ID No. 4. Of these, 1000 cells were sorted to a volume of about 5. mu.L. The PCR reaction conditions are as follows: 95 ℃, 3min, 1 cycle; 95 deg.C, 15s, 55 deg.C, 30s, 72 deg.C, 1min, 30 cycles; 72 ℃, 5min, 1 cycle.
The PCR product was recloned into pET-28a (+) plasmid and after plate culture, the resulting monoclonal was picked into 96-well plates containing 200. mu.L LB medium at 37 ℃ and 400 rpm; when the cell OD600 reached 0.6-0.8, 1.0mM IPTG was added and induction was carried out at 25 ℃ for 20 hours. After induction, cells were collected by centrifugation at 3000rpm for 30min, and the supernatant was discarded. And (3) performing freeze thawing on the cells, adding 200 mu L of PBS into the lysate obtained after the lysis, uniformly mixing, centrifuging at 3000rpm for 30min, and taking the supernatant to obtain a crude enzyme solution. mu.L of the crude enzyme solution was reacted with 10. mu.L of acetonitrile solution of 4-nitrophenylbutyrate (4-nitrophenylbutyrate concentration: 10mM, available from Sigma) and 180. mu.L of PBS in a new 96-well plate at 37 ℃ for 5min, and after the reaction, the enzyme activities of the different clones were detected with a spectrophotometer at a wavelength of 405 nm. Amine dehydrogenase mutants with activity higher than that of wild amine dehydrogenase are selected and sequenced, and the positive mutants are subjected to mass culture expression and then purified by nickel column affinity chromatography to obtain four amine dehydrogenase mutants of E104A, N137P, V174C and V197D. Wherein "E104A" represents an amine dehydrogenase mutant obtained by mutating glutamic acid at position 104 of a wild-type amine dehydrogenase to alanine; "N137P" represents an amine dehydrogenase mutant obtained by mutating asparagine at position 137 of a wild-type amine dehydrogenase to proline; "V174C" represents an amine dehydrogenase mutant obtained by mutating valine at position 174 of a wild-type amine dehydrogenase to cysteine; "V197D" represents an amine dehydrogenase mutant obtained by mutating valine at position 197 of a wild-type amine dehydrogenase to aspartic acid.
A protein crystal model of wild-type amine dehydrogenase was constructed by the on-line software Swiss-model, as shown in FIG. 1. In FIG. 1, the box structure is the corresponding mutation site of the four amine dehydrogenase mutants on the wild-type amine dehydrogenase.
The kinetic parameters of the prochiral ketones of the four amine dehydrogenase mutants and the wild-type amine dehydrogenase mentioned above were determined, and the kinetic parameters of the enzymes were determined in the references Catal.Sci.Technol.2016:10.1039.C6CY 01625A. The results are shown in Table 1. Wherein prochiral ketone is XX prochiral ketone; in Table 1, "WT" represents a wild-type amine dehydrogenase, and "E104A" represents an amine dehydrogenase mutant obtained by mutating glutamic acid at position 104 of the wild-type amine dehydrogenase to alanine; "N137P" represents an amine dehydrogenase mutant obtained by mutating asparagine at position 137 of a wild-type amine dehydrogenase to proline; "V174C" represents an amine dehydrogenase mutant obtained by mutating valine at position 174 of a wild-type amine dehydrogenase to cysteine; "V197D" represents an amine dehydrogenase mutant obtained by mutating valine at position 197 of a wild-type amine dehydrogenase to aspartic acid.
TABLE 1 enzymological Properties of wild-type amine dehydrogenase and amine dehydrogenase mutants
Screening to obtain four amine dehydrogenase mutants of E104A, N137P, V174C and V197D, and K of the mutantsMK with wild-type amine dehydrogenaseMEquivalent values, K of four amine dehydrogenase mutantscatThe value is significantly higher than the K of the wild-type amine dehydrogenasecatThe values indicate that the amine dehydrogenase mutant of the above embodiment has high catalytic efficiency and high affinity for a substrate, can catalyze asymmetric synthesis of chiral amine from prochiral ketone and free ammonia under the action of a coenzyme, can be used for preparing chiral amine, and can be applied to the fields of food, medicine and the like.
In conclusion, the amine dehydrogenase mutant has high catalytic efficiency and high affinity to a substrate, can catalyze prochiral ketone and free ammonia to asymmetrically synthesize chiral amine under the action of coenzyme, has excellent thermal stability, can be used for preparing chiral amine, and can be applied to the fields of food, medicine and the like.
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 invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> institute of biomedical engineering technology of Suzhou, China academy of sciences
<120> amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell, preparation method and application thereof
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20 25 30
Ala Ile Ala Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
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Leu Asp Leu Gly Leu Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
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Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
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Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
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Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
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Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
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Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
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Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
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Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
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Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
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Ile Ser Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
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Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
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Thr Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
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ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
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Claims (9)
1. An amine dehydrogenase mutant, which is obtained by mutating 104 th glutamic acid of wild-type amine dehydrogenase into alanine;
or the wild amine dehydrogenase is obtained by mutating the 137 th asparagine of the wild amine dehydrogenase into proline;
or the valine at the 174 th position of the wild amine dehydrogenase is mutated into the cysteine to obtain the amino acid;
or obtained by mutating the 197 th valine of the wild type amine dehydrogenase into the aspartic acid;
the amino acid sequence of the wild-type amine dehydrogenase is shown in SEQ ID No. 1.
2. The amine dehydrogenase mutant according to claim 1, wherein the wild-type amine dehydrogenase has a nucleotide sequence shown in SEQ ID No. 2.
3. An enzyme preparation comprising the amine dehydrogenase mutant according to any one of claims 1 to 2.
4. A recombinant vector comprising the coding sequence of the amine dehydrogenase mutant according to any one of claims 1 to 2.
5. A recombinant cell comprising the coding sequence of the amine dehydrogenase mutant according to any one of claims 1 to 2.
6. The method for producing a recombinant cell according to claim 5, comprising the steps of:
carrying out error-prone PCR amplification on the coding sequence of the wild-type amine dehydrogenase, and connecting the coding sequence to an empty vector after enzyme digestion to obtain an amine dehydrogenase mutant library plasmid;
respectively transforming the amine dehydrogenase mutant library plasmids into host cells to obtain transformed cells;
and (3) carrying out high-throughput screening on the transformed cells by adopting an IVC-FACS method to obtain the recombinant cells.
7. The method for preparing recombinant cells according to claim 6, wherein in the step of error-prone PCR amplification of the coding sequence of the wild-type amine dehydrogenase, the error-prone PCR amplification is performed by using primer pairs with sequences shown as SEQ ID Nos. 3 to 4.
8. The method of claim 6, wherein the error-prone PCR amplification of the coding sequence of the wild-type amine dehydrogenase is preceded by the following steps: and adopting a primer pair with the sequence shown as SEQ ID No. 5-SEQ ID No.6 to amplify the coding sequence of the wild amine dehydrogenase by PCR.
9. Use of the amine dehydrogenase mutant of any one of claims 1 to 2, the enzyme preparation of claim 3, the recombinant vector of claim 4 or the recombinant cell of claim 5 for the preparation of chiral amines.
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| CN107002050A (en) * | 2014-11-25 | 2017-08-01 | 科德克希思公司 | It is engineered imine reduction enzyme and the method for ketone and the reduction amination of amines |
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