Patent Description:
Amino acids are amphoteric compounds having one carboxyl and one amino at least, and may be classified into two categories in accordance with existing ways of the amino acids, that is, natural amino acids and unnatural amino acids. The natural amino acids are amino acids that exist in nature, while the unnatural amino acids are synthetic amino acids. Generally, some groups are introduced into side chains of the natural amino acids so as to optimize their properties. Due to special structural properties, the amino acids and derivatives thereof have wide applications in aspects such as agriculture, industry, chemical engineering, food and medicine. Optically active unnatural amino acids are chiral synthesis units of some bioactive peptides, and are also important intermediates of many medicines and fine chemicals.

With in-depth scientific researches and the development of new drugs, D-amino acids become more and more important in medicinal development and preparation and food fields.

During enzymatic synthesis of the D-amino acids, used enzymes mainly include transaminase (<NPL>) and amino acid dehydrogenase (<NPL>). By taking prochiral ketonic acid as a substrate and utilizing free NH<NUM>+ as an amino donor, the amino acid dehydrogenase can be used for synthesizing chrial amino acids in the presence of a co-enzyme cycle system, and the synthesis method is green and economic.

However, D-amino acid dehydrogenase existing in nature is very limited in substrate spectrum, and has extremely low reaction activity on most of the substrates, particularly substrates having higher steric hindrance. The concentration of the substrate is low in the reaction; loads on the enzyme are quite high; and cost is higher. Generally speaking, wild enzymes should be modified by the mean of orthogenesis, and various properties of the enzymes are increased, so that the enzymes can be applied to production.

A purpose of the present disclosure is to provide an amino acid dehydrogenase mutant and use thereof, for solving a technical problem in the prior art that wild amino acid dehydrogenase is unsuitable for industrial production.

To achieve the above purpose, according to one aspect of the present disclosure, an amino acid dehydrogenase mutant is provided.

An amino acid dehydrogenase mutant having the sequence SEQ ID NO: <NUM> with a mutation, wherein the mutation is one of the following mutation sites: lysine at the 64th site is mutated into aspartic acid; cysteine at the 133rd site is mutated into threonine; phenylalanine at the 137th site is mutated into alanine; phenylalanine at the 148th site is mutated into valine; proline at the 191st site is mutated into glutamic acid; arginine at the 183rd site is mutated into cysteine and tyrosine at the 207th site is mutated into arginine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and tyrosine at the 207th site is mutated into arginine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into valine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and tyrosine at the 207th site is mutated into glutamic acid; arginine at the 183rd site is mutated into valine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine; threonine at the 173rd site is mutated into histidine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine.

According to another aspect of the present disclosure, a DNA molecule is provided. The DNA molecule encodes any one of the above amino acid dehydrogenase mutants.

According to another aspect of the present disclosure, a recombinant plasmid is provided. The recombinant plasmid includes any of the above DNA molecules.

Further, the recombinant plasmid is pET-22b(+),pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-<NUM>, pGEX-6p-<NUM>, pGEX-6p-<NUM>, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-<NUM>, pUC-<NUM> or pUC-<NUM>.

According to another aspect of the present disclosure, a host cell is provided. The host cell includes any of the recombinant plasmids above.

Further, the host cell contains a prokaryotic cell, a yeast or an eukaryotic cell; preferably, the prokaryotic cell is an Escherichia coli BL21 cell or an Escherichia coli DH5α competent cell.

According to another aspect of the present disclosure, a method for producing D-amino acids is provided. The method includes a step of carrying out a catalytic reductive amination reaction on a ketone compound by using amino acid dehydrogenase, wherein the amino acid dehydrogenase is any of the above amino acid dehydrogenase mutants.

Further, the ketone compound is
<CHM>
and a reductive amination reaction product is
<CHM>.

Further, an amino donor in the reductive amination reaction is ammonium chloride.

The above amino acid dehydrogenase mutant of the present disclosure is obtained by mutating the amino acid dehydrogenase as shown in SEQ ID NO:<NUM> through a method of site-directed mutation and through a method of directed screening, thereby changing the amino acid sequence thereof, realizing a change of protein structure and function. The amino acid dehydrogenase mutant of the present disclosure has an advantage of greatly improving enzymatic activity; the enzymatic activity is increased by over <NUM> times compared with that of the wild amino acid dehydrogenase; and the enzyme specificity is correspondingly increased, thereby the cost in industrial production of the D-amino acids is greatly reduced.

Activity and stability of the following amino acid dehydrogenase shown as SEQ ID NO:<NUM> are increased by the inventor of the present disclosure by an orthogenesis method: (MGEKIRVAIVGYGNIGRYALDAIKAAPDMELAGVVRRSSSLGDKPAELADVPVV GSIKELTGVKVALLCTPTRSVPEYAREILALGINTVDSYDIHGQLADLRLELDKVAKEH NAVAVISAGWDPGTDSMVRCMFEFMAPKGITYTNFGPGMSMGHSVAVKAVKGVKN ALSMTIP LGTGVH RRMVYVELEPGADFAQVE KAVKTDPYFVKDETHVIQVE DVDALI DMGHGVLMERKGVSGGTHNQLLSFSMRINNPALTAQIMVASARASVKQKPGAYTMI QIPIIDYMYGDPDEIIRQLV, a corresponding nucleotide sequence SEQ ID NO:<NUM>: ATGGGTGAAAAAATTCGCGTTGCAATCGTTGGTTACGGCAACATTGGCCGTTATG CCCTGGATGCAATCAAAGCCGCACCGGATATGGAACTGGCCGGCGTGGTGCGC CGTAGTAGCAGTCTGGGCGACAAGCCGGCCGAACTGGCAGATGTGCCTGTTGT GGGCAGCATCAAAGAGCTGACCGGTGTGAAAGTTGCACTGCTGTGCACCCCGA CCCGCAGTGTTCCGGAATATGCCCGTGAGATTCTGGCCCTGGGCATCAACACCG TGGATAGCTATGACATCCACGGTCAGCTGGCCGATCTGCGTCTGGAGCTGGATA AAGTGGCCAAAGAACACAACGCCGTGGCCGTGATTAGCGCAGGTTGGGACCCT GGCACCGATAGCATGGTTCGCTGCATGTTCGAGTTTATGGCCCCGAAGGGCATC ACCTATACCAATTTCGGTCCGGGCATGAGCATGGGTCACAGCGTGGCCGTTAAA GCCGTGAAGGGCGTGAAAAATGCCCTGAGCATGACCATTCCGCTGGGCACCGG TGTTCATCGTCGTATGGTGTATGTGGAGCTGGAACCTGGTGCCGATTTCGCCCA GGTGGAAAAGGCCGTGAAAACCGATCCGTACTTCGTGAAGGATGAGACCCACG TGATTCAGGTGGAAGACGTGGACGCCCTGATTGATATGGGCCATGGCGTTCTGA TGGAACGTAAGGGCGTTAGCGGTGGCACCCATAACCAGCTGCTGAGCTTCAGTA TGCGTATCAATAACCCGGCCCTGACCGCCCAGATTATGGTGGCCAGCGCCCGTG CCAGCGTGAAACAGAAACCGGGCGCATACACCATGATCCAGATTCCGATCATTG ATTACATGTACGGCGACCCGGATGAGATCATTCGTCAGCTGGTGTAA), thereby decreasing the dosage of enzymes. Mutation sites were introduced into the amino acid dehydrogenase shown as SEQ ID NO:<NUM> by a whole-plasmid PCR manner; mutants were subjected to activity and stability detection; and mutants with increased activity or stability were selected.

Wild amino acid dehydrogenase shown as SEQ ID NO:<NUM> was taken as a template; <NUM> pairs of site-specific mutation primers (i.e., K64D, D94A, D94G, D94V, D94S, C133A, C133T, F137A, F148V, F148A, N168D, T173S, T173F, T173H, T173W, T173L, R183F, R183K, R183L, R183C, R183V, R183A, P191E, Y207F, Y207R, Y207E, Y207V, H229V, H229A, H229N, H229G, H229S, H229T, S248E, N255A, N255Q, N255D, Q282E) were designed; and by utilizing the site-specific mutation means, by taking pET-22b (+) as an expression vector, mutation plasmids with target genes were obtained.

Herein, site-directed mutagenesis: refers to the introduction of desired changes (usually characterizing changes in favorable directions) to the target DNA fragments (either genomes or plasmids) by polymerase chain reaction (PCR), including addition, deletion, point mutation, etc. of bases. Site-directed mutagenesis can rapidly and efficiently improve the properties and characterization of target proteins expressed by DNA, and is a very useful means in gene research.

The method of introducing site-directed mutation by whole plasmid PCR is simple and effective, and is a widely used method at present. The principle is as follows, a pair of primers containing mutation sites (forward and reverse), and the template plasmid is annealed, then "cycled extended" by polymerase, the so-called cyclic extension means that the polymerase extends the primers according to the template, and then returns to the <NUM>' end of the primers after a circle, after cycles of repeated heating and annealing, this reaction is different from rolling circle amplification, will not form multiple tandem copies. The extension products of the forward primer and the reverse primer are annealed and paired to form a nicked open circular plasmid. Dpn I digests the extension product, since the original template plasmid is derived from conventional E. coli, subjected to dam methylation modification and sensitive to Dpn I, it is chopped, and the plasmid with the mutant sequence synthesized in vitro is not cut due to no methylation, so that the plasmid is successfully transformed in subsequent transformation, and clone of the mutant plasmid can be obtained.

The above mutant plasmid is transformed into an escherichia coli cell, and over-expressed in the escherichia coli. After that, a crude enzyme is obtained through a method of ultrasonic cell-break. An optimum condition of amino acid dehydrogenase induced expression is as follows: <NUM>, and inducing overnight in <NUM> of IPTG.

According to a typical embodiment of the present disclosure, an amino acid dehydrogenase mutant is provided. An amino acid dehydrogenase mutant having the sequence SEQ ID NO: <NUM> with a mutation, wherein the mutation is one of the following mutation sites: lysine at the 64th site is mutated into aspartic acid; cysteine at the 133rd site is mutated into threonine; phenylalanine at the 137th site is mutated into alanine; phenylalanine at the 148th site is mutated into valine; proline at the 191st site is mutated into glutamic acid; arginine at the 183rd site is mutated into cysteine and tyrosine at the 207th site is mutated into arginine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and tyrosine at the 207th site is mutated into arginine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into valine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and tyrosine at the 207th site is mutated into glutamic acid; arginine at the 183rd site is mutated into valine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine; threonine at the 173rd site is mutated into histidine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine.

The above amino acid dehydrogenase mutant of the present disclosure is obtained by mutating the amino acid dehydrogenase as shown in SEQ ID NO:<NUM> through a method of site-directed mutation and through a method of directed screening, thereby changing the amino acid sequence thereof, realizing a change of protein structure and function. The amino acid dehydrogenase mutant of the present disclosure has an advantage of greatly improving enzymatic activity; the enzymatic activity is increased by over <NUM> times compared with that of the wild amino acid dehydrogenase; and the enzyme specificity is correspondingly increased, thereby the cost in industrial production is greatly reduced.

According to a typical embodiment of the present disclosure, a DNA molecule is provided. The amino acid dehydrogenase coded by the DNA is capable of improving enzymatic activity and stability of the amino acid dehydrogenase, reducing an added enzyme amount in industrial production of D-amino acids.

The above DNA molecule of the disclosure may also exist in the form of an "expression cassette". The "expression cassette" refers to a linear or circular nucleic acid molecule that encompasses DNA and RNA sequences capable of guiding expression of a specific nucleotide sequence in an appropriate host cell. Generally, including a promoter which is effectively linked with a target nucleotide, it is optionally effectively linked with a termination signal and/or other control elements. The expression cassette may also include a sequence required for proper translation of the nucleotide sequence. A coding region usually encodes a target protein, but also encodes a target function RNA in a sense or antisense direction, for example an antisense RNA or an untranslated RNA. The expression cassette including a target polynucleotide sequence may be chimeric, which means that at least one of components thereof is heterologous to at least one of the other components thereof. The expression cassette may also be existent naturally, but obtained with effective recombinant formation for heterologous expression.

According to a typical implementation of the disclosure, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecule in the above recombinant plasmid is placed in a proper position of the recombinant plasmid, so that the above DNA molecule may be correctly and smoothly copied, transcribed or expressed.

Although a qualifier used in the disclosure while the above DNA molecule is defined is "contain", it does not mean that other sequences which are not related to a function thereof may be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombination operations, it is necessary to add suitable enzyme digestion sites of a restriction enzyme at two ends of the DNA sequence, or additionally increase a start codon, a termination codon and the like, therefore, if the closed expression is used for defining, these situations may not be covered truly.

The term "plasmid" used in the disclosure includes any plasmids, cosmids, bacteriophages or agrobacterium binary nucleic acid molecules in double-strand or single-strand linear or circular form, preferably a recombinant expression plasmid, which may be a prokaryotic expression plasmid or may be a eukaryotic expression plasmid, preferably the prokaryotic expression plasmid, in some implementation, the recombinant expression plasmid is selected from pET-22b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-<NUM>, pGEX-6p-<NUM>, pGEX-6p-<NUM>, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-<NUM>, pUC-<NUM> or pUC-<NUM>. More preferably, the recombinant plasmid is the pET-22b(+).

According to a typical implementation of the present disclosure, a host cell is provided. The host cell includes any one of the above recombinant plasmids. The host cell suitable for the disclosure includes, but not limited to, a prokaryotic cell, yeast or a eukaryotic cell. Preferably the prokaryotic cell is a eubacterium, for example a Gram-negative bacterium or a Gram-positive bacterium. More preferably the prokaryotic cell is an Escherichia coli BL21 cell or an Escherichia coli DH5α competent cell.

According to a typical implementation of the present disclosure, a method for producing D-amino acids is provided. The method includes a step of carrying out a catalytic transamination reaction on a ketone compound and an amino donor with amino acid dehydrogenase, wherein the amino acid dehydrogenase is any one of the above amino acid dehydrogenase mutants. Since the amino acid dehydrogenase mutant in the present disclosure has higher enzyme catalytic activity, the D-amino acids prepared by utilizing the amino acid dehydrogenase mutant in the present disclosure can decrease the production cost, and a value ee of the prepared D-amino acids is greater than <NUM>%.

According to a typical embodiment of the present disclosure, the ketone compound is
<CHM>
a reductive amination reaction product is
<CHM>
and a reaction formula is
<CHM>.

Beneficial effects of the present disclosure are further described below in combination with embodiments.

Those skilled in the art all know that, many modifications may be made to the present disclosure without departing from the present disclosure. These modifications are included in the scope of the present disclosure. Unless otherwise specified, experimental methods below are all conventional methods; and unless otherwise specified, used experimental materials may be easily purchased from commercial corporations.

<NUM> of the substrate <NUM> was added into <NUM> of a reaction system including <NUM> of ammonium chloride, <NUM> of glucose, <NUM> of glucose dehydrogenase, <NUM> of NAD+, <NUM> of amino acid dehydrogenase and <NUM> of Tris-HCl buffer; a reaction was carried out at <NUM> for <NUM>; <NUM>µL of HCl and MeOH (<NUM>. 1N HCl: MeOH=<NUM>:<NUM>) were added into <NUM>µL of the system; centrifugation was performed at <NUM> rpm for <NUM>; and the supernatant was detected. The value ee was detected as follows: <NUM>µL of the reaction system was taken; <NUM>µL of ACN, <NUM>µL of H<NUM>O and <NUM>µL of <NUM> NaHCOs were added and then centrifuged at <NUM> rpm for <NUM>; the supernatant was taken out; <NUM>µL of <NUM>/mL Na-(<NUM>,<NUM>-binitro-<NUM>-fluorophenyl)-L-alaninamide was added and reacted at <NUM> for <NUM>; <NUM>µL of the ACN was added for centrifugation; and the supernatant was taken for liquid chromatography.

Compared with increase multiples of a female parent, the activity + is increased by <NUM>-<NUM> times; the activity ++ is increased by <NUM>-<NUM> times; the activity +++ is increased by <NUM>-<NUM> times; and the activity ++++ is increased by over <NUM> times.

The value ee is up to * of <NUM>-<NUM>%; the value ee is up to ** of <NUM>-<NUM>%; and the value ee is greater than *** of <NUM>%.

Mutation is continuously performed, thereby increasing a substrate concentration and decreasing a reaction volume.

<NUM> of the substrate <NUM> was added into <NUM> of a reaction system, <NUM> of ammonium chloride, <NUM> of glucose, <NUM> of glucose dehydrogenase, <NUM> of NAD+, <NUM> of amino acid dehydrogenase and <NUM> of Tris-HCl buffer; a reaction was carried out at <NUM> for <NUM>; <NUM>µL of HCl and MeOH (<NUM>. 1N HCl: MeOH=<NUM>:<NUM>) were added into <NUM>µL of the system; centrifugation was performed at <NUM> rpm for <NUM>; and the supernatant was detected. The value ee was detected as follows: <NUM>µL of the reaction system was taken; <NUM>µL of ACN, <NUM>µL of H<NUM>O and <NUM>µL of <NUM> NaHCOs were added and then centrifuged at <NUM> rpm for <NUM>; the supernatant was taken out; <NUM>µL of <NUM>/mL Na-(<NUM>,<NUM>-binitro-<NUM>-fluorophenyl)-L-alaninamide was added and reacted at <NUM> for <NUM>; <NUM>µL of the ACN was added for centrifugation; and the supernatant was taken for liquid chromatography.

Beneficial mutation sites were further combined, thereby further increasing the substrate concentration and decreasing the reaction volume.

Claim 1:
An amino acid dehydrogenase mutant having the sequence SEQ ID NO: <NUM> with a mutation, wherein the mutation is one of the following mutation sites: lysine at the 64th site is mutated into aspartic acid; cysteine at the 133rd site is mutated into threonine; phenylalanine at the 137th site is mutated into alanine; phenylalanine at the 148th site is mutated into valine; proline at the 191st site is mutated into glutamic acid; arginine at the 183rd site is mutated into cysteine and tyrosine at the 207th site is mutated into arginine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and tyrosine at the 207th site is mutated into arginine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into valine; arginine at the 183rd site is mutated into cysteine, histidine at the 229th site is mutated into serine and tyrosine at the 207th site is mutated into glutamic acid; arginine at the 183rd site is mutated into valine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine; threonine at the 173rd site is mutated into histidine, histidine at the 229th site is mutated into serine and phenylalanine at the 148th site is mutated into alanine.