CN112852894B - Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound - Google Patents

Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound Download PDF

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CN112852894B
CN112852894B CN202010499781.9A CN202010499781A CN112852894B CN 112852894 B CN112852894 B CN 112852894B CN 202010499781 A CN202010499781 A CN 202010499781A CN 112852894 B CN112852894 B CN 112852894B
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spamdh
gene
hydroxy
mutated
amino
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CN112852894A (en
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孙周通
王红月
曲戈
蒋迎迎
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Tianjin Institute of Industrial Biotechnology of CAS
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    • C—CHEMISTRY; METALLURGY
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/99—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with other acceptors (1.4.99)
    • C12Y104/99003—Amine dehydrogenase (1.4.99.3)

Abstract

The invention discloses an amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compounds. The amine dehydrogenase mutant disclosed by the invention is a protein obtained by mutating the 40 th, 61 th, 68 th, 69 th, 111 th, 113 th, 114 th, 116 th, 134 th, 150 th, 187 th, 239 th, 288 th, 291 th, 292 th, 295 th, 297 th and/or 294 th positions of an amine dehydrogenase (SpAmDH) derived from Sporosarcina psychrophila. The amine dehydrogenase mutant can catalyze hydroxy ketone substrates to synthesize chiral amino alcohol compounds represented by 1-hydroxy-2-butanone and 4-hydroxy-2-butanone, is convenient to operate, has the advantages of high optical purity of products and the like, can achieve the stereoselectivity of 99 percent, and has better industrial application prospect in preparing the chiral amino alcohol compounds through biocatalysis.

Description

Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound
Technical Field
The invention relates to an amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compounds in the field of biotechnology.
Background
Chiral amino alcohol is a structural unit of a plurality of bioactive molecules, is an important intermediate in medicine and fine chemical industry, wherein (S) -2-amino-1-butanol is mainly used for synthesizing Ethambutol hydrochloride (Ethambutol) and is used as a first-line drug for resisting mycobacterium tuberculosis for a long time; (R) -3-amino-1-butanol is an important raw material for synthesizing anti-AIDS integrase inhibitor Dolutegravir.
The (S) -2-amino-1-butanol is mainly synthesized by a chemical method and a biological enzyme method, wherein the current chemical synthesis methods comprise the following steps: using butyraldehyde, dibenzyl azodicarboxylate and D-proline as raw materials, adding NaBH4、H2Synthesizing (S) -2-amino-1-butanol under the catalytic action of nickel and the like; reducing 2-aminobutyric acid by using lithium aluminum hydride to obtain a corresponding chiral amino alcohol product; by means of H2And nickel at high pressureReducing L-2-aminobutyric acid under the condition to obtain (S) -2-amino-1-butanol; using NaHB4At H2SO4The corresponding alpha-amino acid is reduced under the THF condition to obtain; the dibenzoyl tartaric acid is used for preparing by a resolution method, and the L-tartaric acid is used for preparing (S) -2-amino-1-butanol and the like by a resolution method. The biological enzyme method comprises the following steps: under the condition of amino group protection, ester bonds formed by hydroxyl groups are selectively hydrolyzed by lipase to obtain chiral monomers; the immobilized penicillin G acylase selectively hydrolyzes (S) -configuration in the racemic mixture of N-phenylacetyl derivative 2-aminobutanol to obtain (S) -2-amino-1-butanol; recently, Weber et al reported that a new pathway for synthesizing (S) -2-amino-1-butanol from threonine by a four-step enzymatic method was constructed using yeast, but the yield of (S) -2-amino-1-butanol was only 1.1 mg/L.
The (R) -3-amino-1-butanol is mainly synthesized by a chemical method, and the yield is 60-70%. In 1998, Tatsuya et al reported a method for synthesizing (R) -3-amino-1-butanol, but in the method, the raw materials are expensive and the reaction conditions are severe; in 2005, Achmatowicz et al avoided expensive chemical raw materials and synthesized (R) -3-amino-1-butanol using inexpensive and readily available D-alanine as a starting material, but no specific yield and ee value were reported. In 2011, Breuer et al reported a method for obtaining (R) -3-amino-1-butanol by using mandelic acid to resolve racemic 3-aminobutanol. In 2018, (R) -3-amino-1-butanol was synthesized in one step from 4-hydroxy-2-butanone as a starting material by Khatik et al using a highly selective transaminase.
The synthesis method of the amino butanol requires high temperature, high pressure and a metal catalyst, and has harsh reaction conditions, great pollution and low safety coefficient. The biological method has mild reaction conditions but low conversion rate, so that a green and efficient method for synthesizing the chiral amino alcohol compound is urgently needed.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare chiral amino alcohol compounds, in particular to (S) -2-amino-1-butanol and (R) -3-amino-1-butanol.
In order to solve the above technical problems, the present invention provides, in a first aspect, a method for preparing a chiral aminoalcohol compound, the method comprising: taking hydroxyl ketone as a substrate, and carrying out catalytic reaction by using amine dehydrogenase or mutant protein thereof to obtain a chiral amino alcohol compound;
the amine dehydrogenase is derived from Sporosarcina psychrophilia (SpamoldH), the name of the amine dehydrogenase is SpAmDH, and the amino acid sequence of the amine dehydrogenase is SEQ ID No.2 in a sequence table; the SpAmDH mutant protein is obtained by mutating the 40 th, 61 th, 68 th, 69 th, 111 th, 113 th, 114 th, 116 th, 134 th, 150 th, 187 th, 239 th, 288 th, 291 th, 292 th, 295 th, 297 th and/or 294 th positions of SpAmDH.
In the above method, the chiral amino alcohol compound may be 2-amino-1-butanol, 3-amino-1-butanol, alaninol, phenethyl amine alcohol or 2-amino-2- (4-fluorophenyl) -1-ethanol;
the hydroxy ketone can be 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, acetol, 2-hydroxyacetophenone, and 1- (4-fluorophenyl) -2-hydroxy-1-ethanone.
Specifically, the chiral amine alcohol compound may be (S) -2-amino-1-butanol, (R) -3-amino-1-butanol, (S) -alaninol, (S) -phenethylamine alcohol or (S) -2-amino-2- (4-fluorophenyl) -1-ethanol.
In the above method, the spimdh mutein may be any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen, any seventeen, or all of the following modifications of spimdh:
x1, the leucine residue at the position 40 of SpAmDH is mutated into valine residue, cysteine residue or phenylalanine residue;
x2, mutation of leucine residue at position 61 of SpAmDH to alanine residue, cysteine residue or valine residue;
x3, the 68 th serine residue of SpAmDH is mutated into cysteine residue;
x4, the asparagine residue at the 69 th position of SpAmDH is mutated into serine residue or cysteine residue;
x5, the isoleucine residue at the 111 th position of SpAmDH is mutated into phenylalanine residue or tryptophan residue;
x6, mutation of alanine residue at position 113 of SpAmDH into cysteine residue;
x7, mutation of glutamic acid residue at position 114 of SpAmDH into valine residue or glutamine residue;
x8, mutation of valine residue at position 116 of SpAmDH to isoleucine, methionine, serine or tyrosine residue;
x9, mutation of the threonine residue at position 134 of SpAmDH to an alanine, phenylalanine, glycine or cysteine residue;
x10, mutation of the threonine residue at position 150 of SpAmDH to a cysteine residue;
x11, the alanine residue at the 187 th position of SpAmDH is mutated into lysine residue, asparagine residue, proline residue or threonine residue;
x12, the leucine residue at the 239 th site of SpAmDH is mutated into phenylalanine residue or methionine residue;
x13, the 288 th serine residue of SpAmDH is mutated into methionine residue or cysteine residue;
x14, mutation of valine residue at position 291 of SpAmDH into cysteine residue or leucine residue;
x15, the isoleucine residue at position 292 of SpAmDH is mutated into methionine residue or threonine residue;
x16, mutation of valine residue at position 294 of SpAmDH into cysteine residue or phenylalanine residue;
x17, mutation of alanine residue at position 295 of SpAmDH to an asparagine residue, a threonine residue, or a tryptophan residue;
x18, mutation of glutamic acid residue to asparagine residue at position 297 of SpAmDH.
In one embodiment of the invention, the SpAmDH mutant protein is SpAmDH-L40V, SpAmDH-L61A, SpAmDH-L61V, SpAmDH-L61C, SpAmDH-S68C, SpAmDH-N69S, SpAmDH-I111F, SpAmDH-I111W, SpAmDH-A113C, SpAmDH-E114V, SpAmDH-E114Q, SpAmDH-V116I, SpAmDH-V116M, SpAmDH-V116S, SpAmDH-V116Y, SpAmDH-T134A, SpAmDH-T150A, SpDH-36187-A A, SpAmDH-36187-A, SpAmDH-T A, SpAmDH-36239-A, SpAmDH-363672, SpAmDH-A, SpAmDH-36239-363672, SpAmDH-A, SpAmDH-36239-A, SpAmDH-36297, SpAmDH-A, SpAmDH-363672, SpAmDH-A, SpAmDH-36239-A, SpAmDH-36297, SpAmDH-3636363636363672, SpAmDH-A, SpAmDH-36295, SpAmDH-36297, SpAmDH-A, SpAmDH-36297, SpAmDH-36239-A, SpAmDH-36295, SpAmDH-36297, SpAmDH-A, SpAmDH-36239-3695, SpAmDH-A, SpAmDH-3695-A, SpAmDH-, SpAmDH-E114V/V291C, SpAmDH-L40C/I111C/T134C, SpAmDH-S68C/N69C/V291C, SpAmDH-I111C/A113C/T134C, SpAmDH-L40C/I111C/T134C/V294C, SpAmDH-S68C/N69C/E114C/V291C, SpAmDH-S68/N69C/I111C/V294C, SpAmDH-I111C/E114C/V294, SpAmDH-L40C/S68/N C/T C/V291C/V294, SpAmDH-S72/N C/N134/V C/V291/V294, SpAmDH-S68/S C/N C/T C/V36294, SpAmDH-S72/S C/N C/V36294, SpAmDH-S C/N C/V36294 and SpAmDH-S C/N C/V36294.
The SpAmDH-L40V is a protein obtained by mutating the position 40 of SpAmDH into a valine residue.
The SpAmDH-L61A is a protein obtained by mutating the 61 st position of SpAmDH to alanine residue.
The SpAmDH-L61C is a protein obtained by mutating the 61 st position of SpAmDH into a cysteine residue.
The SpAmDH-L61V is a protein obtained by mutating the 61 st position of SpAmDH into a valine residue.
The SpAmDH-S68C is a protein obtained by mutating the 68 th site of SpAmDH into a cysteine residue.
The SpAmDH-N69S is a protein obtained by mutating the 69 th site of SpAmDH into a serine residue.
The SpAmDH-I111F is a protein obtained by mutating the 111 th site of SpAmDH into a phenylalanine residue.
The SpAmDH-I111W is a protein obtained by mutating the 111 th site of SpAmDH into a tryptophan residue.
The SpAmDH-A113C is a protein obtained by mutating the 113 th site of SpAmDH into a cysteine residue.
The SpAmDH-E114V is a protein obtained by mutating the 114 th position of SpAmDH into a valine residue.
The SpAmDH-E114Q is a protein obtained by mutating the 114 th site of SpAmDH into a glutamine residue.
The SpAmDH-V116I is a protein obtained by mutating the 116 th site of SpAmDH into isoleucine residue.
The SpAmDH-V116M is a protein obtained by mutating the 116 th site of SpAmDH into a methionine residue.
The SpAmDH-V116S is a protein obtained by mutating the 116 th site of SpAmDH to a serine residue.
The SpAmDH-V116Y is a protein obtained by mutating the 116 th site of SpAmDH into a tyrosine residue.
The SpAmDH-T134A is a protein obtained by mutating the 134 th site of SpAmDH into an alanine residue.
The SpAmDH-T134F is a protein obtained by mutating the 134 th site of SpAmDH into a phenylalanine residue.
The SpAmDH-T134G is a protein obtained by mutating the 134 th site of SpAmDH into a glycine residue.
The SpAmDH-T150C is a protein obtained by mutating the 150 th site of SpAmDH into a cysteine residue.
The SpAmDH-A187K is a protein obtained by mutating the 187 th site of SpAmDH into a lysine residue.
The SpAmDH-A187N is a protein obtained by mutating the 187 th site of SpAmDH into an asparagine residue.
The SpAmDH-A187P is a protein obtained by mutating the 187 th site of SpAmDH into a proline residue.
The SpAmDH-A187T is a protein obtained by mutating the 187 th site of SpAmDH into a threonine residue.
The SpAmDH-L239F is a protein obtained by mutating the 239 th site of SpAmDH into a phenylalanine residue.
The SpAmDH-L239M is a protein obtained by mutating the 239 th site of SpAmDH into a methionine residue.
The SpAmDH-V291C is a protein obtained by mutating the 291 th site of SpAmDH into a cysteine residue.
The SpAmDH-V291L is a protein obtained by mutating the 291 th site of SpAmDH to a leucine residue.
The SpAmDH-I292M is a protein obtained by mutating the 292 th site of SpAmDH into a methionine residue.
The SpAmDH-I292T is a protein obtained by mutating the 292 th site of SpAmDH into a threonine residue.
The SpAmDH-A295N is a protein obtained by mutating the 295 th site of SpAmDH into an aspartic acid residue.
The SpAmDH-A295T is a protein obtained by mutating the 295 th position of SpAmDH into a threonine residue.
The SpAmDH-A295W is a protein obtained by mutating the 295 th position of SpAmDH into a tryptophan residue.
The SpAmDH-S288M is a protein obtained by mutating the 288 th position of SpAmDH into a methionine residue.
The SpAmDH-E297N is a protein obtained by mutating the 297 th site of SpAmDH into an asparagine residue.
The SpAmDH-N69C/V291C is a protein obtained by mutating the 69 th and 291 th positions of SpAmDH into cysteine residues.
The SpAmDH-S68C/V291C is a protein obtained by mutating the positions 68 and 291 of SpAmDH into cysteine residues.
The SpAmDH-L40C/I111F is a protein obtained by mutating positions 40 and 111 of SpAmDH into cysteine and phenylalanine residues respectively.
The SpAmDH-I111F/V294C is a protein obtained by mutating 111 th and 294 th sites of SpAmDH into phenylalanine residues and cysteine residues respectively.
The SpAmDH-E114V/V291C is a protein obtained by mutating positions 114 and 291 of SpAmDH into valine residues and cysteine residues respectively.
The SpAmDH-L40C/I111F/T134F is a protein obtained by mutating positions 40, 111 and 134 of SpAmDH into cysteine, phenylalanine and phenylalanine residues respectively.
The SpAmDH-S68C/N69C/V291C is a protein obtained by mutating positions 68, 69 and 291 of SpAmDH into cysteine residues.
The SpAmDH-I111F/A113C/T134C is a protein obtained by mutating 111 th, 113 th and 134 th positions of SpAmDH into phenylalanine residue, cysteine residue and cysteine residue respectively.
The SpAmDH-L40C/I111F/T134F/V294C is a protein obtained by mutating positions 40, 111, 134 and 294 of SpAmDH into cysteine, phenylalanine and cysteine residues respectively.
The SpAmDH-S68C/N69C/E114V/V291C is a protein obtained by mutating the positions 68, 69, 114 and 291 of SpAmDH into cysteine, valine and cysteine residues respectively.
The SpAmDH-S68C/N69C/I111F/V291C is a protein obtained by mutating the positions 68, 69, 111 and 291 of SpAmDH into cysteine, phenylalanine and cysteine residues respectively.
The SpAmDH-I111F/E114V/V294C is a protein obtained by mutating 111 th, 114 th and 294 th positions of SpAmDH into phenylalanine, valine and cysteine residues respectively.
The SpAmDH-L40F/S68C/N69C/T134C/V291C/V294C is a protein obtained by mutating positions 40, 68, 69, 134, 291 and 294 of SpAmDH into phenylalanine, cysteine and cysteine residues respectively.
The SpAmDH-S68C/N69C/T134C/V291C is a protein obtained by mutating positions 68, 69, 134 and 291 of SpAmDH into cysteine residues.
The SpAmDH-L40F/S68C/N69C/A113C/T134C/V291C/V294F is a protein obtained by mutating positions 40, 68, 69, 113, 134, 291 and 294 of SpAmDH into phenylalanine, cysteine and phenylalanine residues respectively.
The SpAmDH-S68C/I111F/V294C is a protein obtained by mutating the positions 68, 111 and 294 of the SpAmDH into cysteine, phenylalanine and cysteine residues respectively.
The SpAmDH-I111F/S288C/V294C is a protein obtained by mutating 111 th, 288 th and 294 th positions of SpAmDH into phenylalanine, cysteine and cysteine residues respectively.
The present invention also provides another method for preparing a chiral aminoalcohol compound, the method comprising: and (2) carrying out catalytic reaction by using hydroxyl ketone as a substrate and utilizing recombinant cells expressing SpAmDH or SpAmDH mutant protein or lysates of the recombinant cells to obtain the chiral amino alcohol compound.
In the above method, the recombinant cell may be obtained by introducing a recombinant vector capable of expressing spomdh or the spomdh mutein into a biological cell.
The biological cell may be a microorganism. The microorganism can be Escherichia coli, and can also be other bacteria. In one embodiment of the invention, the microorganism is escherichia coli BL21(DE 3).
The recombinant vector can be a recombinant plasmid obtained by replacing a small DNA fragment between NdeI and XhoI recognition sequences in a pET24a (+) vector with SpAmDH gene or a coding gene of SpAmDH mutant protein.
The SpAmDH gene can be a nucleic acid molecule shown as SEQ ID No.1 in a sequence table.
The encoding gene of the SpAmDH mutant protein can be SpAmDH-L40V gene, SpAmDH-L61A gene, SpAmDH-L61C gene, SpAmDH-L61V gene, SpAmDH-S68C gene, SpAmDH-N69S gene, SpAmDH-I111F gene, SpAmDH-A113F gene, SpAmDH-E114F gene, SpAmDH-V116F gene, SpAmDH-T134F gene, SpAmDH-T36150 gene, SpAmDH-F gene, SpAmDH-V187-L187-E187 gene, SpAmDH-S68 gene, SpAmDH-E F gene, SpAmDH-V239 gene, SpAmDH-V F gene, SpAmDH-V239 gene, SpAmDH-F gene, SpAmDH-36239 gene, SpAmDH-F gene, SpAmDH-36239 gene, SpAmDH-F gene, SpAmDH-36239-F, SpAmDH-A295 gene, SpAmDH-S288 gene, SpAmDH-E297 gene, SpAmDH-N69/V291 gene, SpAmDH-S68/V291 gene, SpAmDH-L40/I111 gene, SpAmDH-I111/V294 gene, SpAmDH-E114/V291 gene, SpAmDH-L40/I111/T134 gene, SpAmDH-S68/N69/V291 gene, SpAmDH-I111/A113/T134 gene, SpAmDH-L40/I111/T134/V294 gene, SpAmDH-S68/N69/E114/V291 gene, SpAmDH-S68/N69/I111/V291 gene, SpAmDH-I111/E114/V294 gene, SpAmDH-L40/S68/N69/T134/294/V291 gene, SpAmDH-S68/N69/V291 gene, SpAmDH-S68/N69/V291 gene, SpAmDH-I111/V294 gene, SpAmDH-L40/V294/S68/N69/N294 gene, SpAmDH-L113/V294 gene, SpAmDH-S68C/I111F/V294C gene or SpAmDH-I111F/S288C/V294C gene.
The gene SpAmDH-L40V is a double-stranded DNA molecule obtained by mutating the 118 th position of SpAmDH gene by C.
The gene SpAmDH-L61A was a double-stranded DNA molecule obtained by mutating SpAmDH gene from C at position 181 to G and from T at position 182 to C.
The gene SpAmDH-L61C is a double-stranded DNA molecule obtained by mutating the position 181 with C to T, the position 182 with T to G, and the position 183 with G to T of the SpAmDH gene.
The gene SpAmDH-L61V is a double-stranded DNA molecule obtained by mutating the 181 th position of the SpAmDH gene from C to G.
The gene SpAmDH-S68C is a double-stranded DNA molecule obtained by mutating the 202 nd position of the SpAmDH gene from A to T.
The gene SpAmDH-N69S is a double-stranded DNA molecule obtained by mutating the 206 th site of the SpAmDH gene from A to G.
The gene SpAmDH-I111F is a double-stranded DNA molecule obtained by mutating the 331 st position of the SpAmDH gene from A to T.
The gene SpAmDH-I111W was a double-stranded DNA molecule obtained by mutating the SpAmDH gene from A to T at position 331, from T to G at position 332 and from T to G at position 333.
The gene SpAmDH-A113C was a double-stranded DNA molecule obtained by mutating the gene SpAmDH from G at position 337 to T, from C at position 338 to G and from A at position 339 to T.
The gene SpAmDH-E114V was a double-stranded DNA molecule obtained by mutating the gene SpAmDH from A at position 341 to T and from A at position 342 to G.
The gene SpAmDH-E114Q is a double-stranded DNA molecule obtained by mutating the position 340 of the SpAmDH gene from G to C and from A to G at the position 342.
The gene SpAmDH-V116I is a double-stranded DNA molecule obtained by mutating G at the 346 th position of the SpAmDH gene into A.
The gene SpAmDH-V116M was a double-stranded DNA molecule obtained by mutating the position 346 of the SpAmDH gene from G to A and from T to G at the position 348.
The gene SpAmDH-V116S is a double-stranded DNA molecule obtained by mutating the 346 th site of the SpAmDH gene from G to A and the 347 th site from T to G.
The gene SpAmDH-V116Y was a double-stranded DNA molecule obtained by mutating the gene SpAmDH from G at position 346 to T and from T at position 347 to A.
The gene SpAmDH-T134A is a double-stranded DNA molecule obtained by mutating the 400 th position of SpAmDH gene from A to G and the 402 th position from C to G.
The gene SpAmDH-T134F is a double-stranded DNA molecule obtained by mutating the SpAmDH gene from A to T at position 400, from C to T at position 401 and from C to T at position 402.
The gene SpAmDH-T134G is a double-stranded DNA molecule obtained by mutating the SpAmDH gene from A to G at position 400, from C to G at position 401 and from C to T at position 402.
The gene SpAmDH-T150C is a double-stranded DNA molecule obtained by mutating the 448 th site of the SpAmDH gene from A to T, the 449th site from C to G and the 450 th site from C to T.
The gene SpAmDH-A187K is a double-stranded DNA molecule obtained by mutating the 559 th position of SpAmDH gene from G to A, mutating the 560 th position from C to A, and mutating the 561 th position from C to A.
The gene SpAmDH-A187N is a double-stranded DNA molecule obtained by mutating the 559 th position of SpAmDH gene from G to A, the 560 th position from C to A and the 561 th position from C to T.
The gene SpAmDH-A187P is a double-stranded DNA molecule obtained by mutating the 559 th position of SpAmDH gene from G to C and mutating the 561 th position from C to G.
The gene SpAmDH-A187T is a double-stranded DNA molecule obtained by mutating the 559 th position of SpAmDH gene from G to A and the 561 th position from C to G.
The gene SpAmDH-L239F is a double-stranded DNA molecule obtained by mutating the 715 th site of the SpAmDH gene from C to T and from G to T.
The gene SpAmDH-L239M is a double-stranded DNA molecule obtained by mutating the 715 th site of the SpAmDH gene from C to A.
The gene SpAmDH-V291C is a double-stranded DNA molecule obtained by mutating the 871 position of the SpAmDH gene from G to T, the 872 position from T to G and the 873 position from G to T.
The gene SpAmDH-V291L is a double-stranded DNA molecule obtained by mutating the 871 site of the SpAmDH gene with G to obtain C.
The gene SpAmDH-I292M is a double-stranded DNA molecule obtained by mutating the 876 th position of the SpAmDH gene with T to G.
The gene SpAmDH-I292T is a double-stranded DNA molecule obtained by mutating the 875 th site of the SpAmDH gene with T to C and the 876 th site with T to G.
The gene SpAmDH-A295N was a double-stranded DNA molecule obtained by mutating the gene SpAmDH from G at position 883 to A, from C at position 884 to A and from C at position 885 to T.
The gene SpAmDH-A295T is a double-stranded DNA molecule obtained by mutating the 883 position of the SpAmDH gene from G to A and the 885 position from C to G.
The gene SpAmDH-A295W was a double-stranded DNA molecule obtained by mutating the gene SpAmDH from G at position 883 to T, from C at position 884 to G and from C at position 885 to G.
The gene SpAmDH-S288M was a double-stranded DNA molecule obtained by mutating the gene SpAmDH from G at position 863 to T and from T at position 864 to G.
The gene SpAmDH-E297N is a double-stranded DNA molecule obtained by mutating the position 889 of the SpAmDH gene from G to A and the position 891 from A to T.
The SpAmDH-N69C/V291C gene is a double-stranded DNA molecule obtained by mutating the 205 th site of the SpAmDH gene from A to T, the 206 th site from A to G, the 207 th site from T to C, the 871 site from G to T, the 872 th site from T to G and the 873 rd site from G to T.
The SpAmDH-S68C/V291C gene is a double-stranded DNA molecule obtained by mutating the position 202 of the SpAmDH gene from A to T, the position 204 from T to C, the position 871 from G to T, the position 872 from T to G and the position 873 from G to T.
The SpAmDH-L40C/I111F gene is a double-stranded DNA molecule obtained by mutating the 118 th site of the SpAmDH gene from C to T, the 119 th site from T to G, the 120 th site from G to C and the 331 st site from A to T.
The gene SpAmDH-I111F/V294C is a double-stranded DNA molecule obtained by mutating the No. 331 position of SpAmDH gene from A to T, the No. 880 position from G to T, the No. 881 position from T to G and the No. 882 position from T to C.
The SpAmDH-E114V/V291C gene is a double-stranded DNA molecule obtained by mutating the position 341 of the SpAmDH gene from A to T, the position 342 from A to G, the position 871 from G to T, the position 872 from T to G and the position 873 from G to C.
The SpAmDH-L40C/I111F/T134F gene is a double-stranded DNA molecule obtained by mutating the 118 th position of the SpAmDH gene from C to T, the 119 th position from T to G, the 120 th position from G to C, the 331 st position from A to T, the 400 th position from A to T, the 401 th position from C to T and the 402 th position from C to T.
The SpAmDH-S68C/N69C/V291C gene is a double-stranded DNA molecule obtained by mutating the position 202 of the SpAmDH gene from A to T, the position 204 from T to C, the position 205 from A to T, the position 206 from A to G, the position 207 from T to C, the position 871 from G to T, the position 872 from T to G and the position 873 from G to T.
The SpAmDH-I111F/A113C/T134C gene is a double-stranded DNA molecule obtained by mutating the No. 331 position of SpAmDH gene from A to T, the No. 337 position from G to T, the No. 338 position from C to G, the No. 339 position from A to C, the No. 400 position from A to T and the No. 401 position from C to G.
The SpAmDH-L40C/I111F/T134F/V294C gene is a double-stranded DNA molecule obtained by mutating the 118 th position of SpAmDH gene from C to T, the 119 th position from T to G, the 120 th position from G to C, the 331 st position from A to T, the 400 th position from A to T, the 401 th position from C to T, the 402 th position from C to T, the 880 th position from G to T, the 881 th position from T to G and the 882 th position from T to C.
The SpAmDH-S68C/N69C/E114V/V291C gene is a double-stranded DNA molecule obtained by mutating the position 202 of the SpAmDH gene from A to T, the position 204 from T to C, the position 205 from A to T, the position 206 from A to G, the position 207 from T to C, the position 341 from A to T, the position 342 from A to G, the position 871 from G to T, the position 872 from T to G and the position 873 from G to T.
The SpAmDH-S68C/N69C/I111F/V291C gene is a double-stranded DNA molecule obtained by mutating the position 202 of the SpAmDH gene from A to T, the position 204 from T to C, the position 205 from A to T, the position 206 from A to G, the position 207 from T to C, the position 331 from A to T, the position 871 from G to T, the position 872 from T to G and the position 873 from G to T.
The SpAmDH-I111F/E114V/V294C gene is a double-stranded DNA molecule obtained by mutating the No. 331 position of SpAmDH gene from A to T, the No. 341 position from A to T, the No. 342 position from A to G, the No. 880 position from G to T, the No. 881 position from T to G and the No. 882 position from T to C.
The SpAmDH-L40F/S68C/N69C/T134C/V291C/V294C gene is a double-stranded DNA molecule obtained by mutating C at the 118 th position of SpAmDH gene to T, G at the 120 th position of SpAmDH gene to T, A at the 202 nd position of SpAmDH gene to T, T at the 204 th position of SpAmDH gene to C, A at the 205 th position of SpAmDH gene to T, A at the 206 th position of SpAmDH gene to G, T at the 207 th position of SpAmDH gene to C, A at the 400 th position of SpaDH gene to T, C at the 401 th position of SpaMth gene to G, T at the 872 th position of SpaMth position of SpaMdH, G at the 873 th position of SpaMth position of SpaMdH, G at the 400 th position of SpaMth position of SpaMdDH gene to T, G at the P position of the SpaMth position of SpaMth position, G at the No. of the No. P position of the No. P, G at the No. P, G, B at the No. P, G, B at the No. P, B at the No. P, B.
The SpAmDH-S68C/N69C/T134C/V291C gene is a double-stranded DNA molecule obtained by mutating the position 202 of the SpAmDH gene from A to T, the position 204 from T to C, the position 205 from A to T, the position 206 from A to G, the position 207 from T to C, the position 400 from A to T, the position 401 from C to G, the position 871 from G to T, the position 872 from T to G and the position 873 from G to T.
The SpAmDH-L40F/S68C/N69C/A113C/T134C/V291C/V294F gene is a double-stranded DNA molecule obtained by mutating the 118 th position of the SpAmDH gene from C to T, mutating the 120 th position from G to T, mutating the 202 nd position from A to T, mutating the 204 nd position from T to C, mutating the 205 th position from A to T, mutating the 206 th position from A to G, mutating the 207 th position from T to C, mutating the 337 th position from G to T, mutating the 338 th position from C to G, mutating the 339 rd position from A to C, mutating the 400 th position from A to T, mutating the 401 th position from C to G, mutating the 871 to T, mutating the 872 th position from T to G, mutating the 873 rd position from G, mutating the 880 rd position from G to T.
The SpAmDH-S68C/I111F/V294C gene is a double-stranded DNA molecule obtained by mutating the position 202 of the SpAmDH gene from A to T, the position 204 from T to C, the position 331 from A to T, the position 880 from G to T, the position 881 from T to G and the position 882 from T to C.
The SpAmDH-I111F/S288C/V294C gene is a double-stranded DNA molecule obtained by mutating the 331 st position of the SpAmDH gene from A to T, the 862 nd position from A to T, the 864 nd position from T to C, the 880 nd position from G to T, the 881 nd position from T to G and the 882 nd position from T to C.
In the above, in the reaction of catalyzing the substrate hydroxyketone to produce the chiral amino alcohol compound with the SpAmDH or the mutant thereof as the bio-enzyme, the reaction system may further contain the coenzyme NAD of the SpAmDH or the mutant thereof in addition to the substrate and the SpAmDH or the mutant thereof+And/or NH4 +。
The concentration of the substrate in the reaction system may be 1 to 100mmol/L (e.g., 40 mmol/L). The concentration of the recombinant cells in the reaction system may be 50-500g/L (e.g., 100 g/L). The concentration of the lysate in the reaction system may be10-50g/L (e.g., 20 g/L). The concentration of the pure amine dehydrogenase in the reaction system may be 0.1 to 2g/L (e.g., 0.5 g/L). The NAD+The concentration in the reaction system may be 0.1 to 2.0mmol/L (e.g., 1.0 mmol/L). The NH4 +The concentration in the reaction system can be 100 mmol/L-4 mol/L.
The reaction system of the catalytic reaction can be obtained by adding the following substances into 100 mmol/L-4 mol/L (such as 1mol/L) ammonium chloride/ammonia water buffer solution (obtained by mixing ammonium chloride and ammonia water in an equal molar ratio): the hydroxyketone, the recombinant cell or a lysate of the recombinant cell, NAD+(in the form of an aqueous solution of oxidized coenzyme I), glucose dehydrogenase, glucose, lysozyme, DNase I (deoxyribonuclease). The addition amount of each substance can be as follows: 1-100mmol/L (e.g., 20mmol/L) of the hydroxyketone, 50-500g/L (e.g., 100g/L) of the recombinant cell or 10-50g/L (e.g., 20g/L) of a lysate of the recombinant cell, NAD+0.1-2.0mmol/L (such as 1.0mmol/L) (in the form of oxidized coenzyme I aqueous solution), 2g/L of glucose dehydrogenase powder, 100mmol/L of glucose, 1g/L of lysozyme, and 6U/mL of DNase I (deoxyribonuclease).
The pH of the ammonium chloride/ammonia buffer solution can be 7-11.
The glucose dehydrogenase powder is prepared by the method comprising the following steps: replacing DNA fragments between pET24a (+) vector NdeI and XhoI recognition sequences with a glucose dehydrogenase gene (the nucleotide sequence of the coding gene is SEQ ID No.3) to obtain a recombinant vector which is marked as pET24 a-GDH; pET24a-GDH was introduced into E.coli BL21(DE3) to give a recombinant strain designated as BL21(DE3)/pET24 a-GDH. BL21(DE3)/pET24a-GDH was cultured, the obtained cells were disrupted and centrifuged, and the supernatant was put into a freeze-drying machine to obtain the glucose dehydrogenase powder.
The reaction temperature can be 20-35 ℃, and specifically can be 30 ℃; the reaction time is based on the completion of the reaction, and may be generally 0.5 to 24 hours, specifically 12 hours. The lysate of the recombinant cells may be obtained by lysing the recombinant cells.
The SpAmDH mutant protein also belongs to the protection scope of the invention.
The invention also provides a biological material related to SpAmDH or the SpAmDH mutant protein, wherein the biological material is any one of the following B1) to B4):
B1) a nucleic acid molecule encoding SpAmDH or the SpAmDH mutein;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);
B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.
Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.
B1) The nucleic acid molecule may be the SpAmDH gene, the SpAmDH-L40V gene, the SpAmDH-L61A gene, the SpAmDH-L61C gene, the SpAmDH-L61V gene, the SpAmDH-S68C gene, the SpAmDH-N69S gene, the SpAmDH-I111F gene, the SpAmDH-I111W gene, the SpAmDH-A113C gene, the SpAmDH-E114V gene, the SpAmDH-E114Q gene, the SpAmDH-V116I gene, the SpAmAmDH-V116M gene, the SpAmDH-V116S gene, the SpAmDH-V59116Y gene, the SpDH AmDH-T A gene, the SpAmDH-T F gene, the SpDH-T134 gene, the SpAmDH-T G gene, the SpAmDH-150 gene, the SpAmDH-V187-V Y gene, the SpAmDH-E84239 gene, the SpAmDH-E8653 gene, the SpAmDH-E Q gene, the SpAmDH-V116 gene, the SpAmDH-V57323 gene, the SpAmDH-V116 gene, the SpAmDH-V-III gene, the SpAmDH-III gene, The SpAmDH-V291 gene, the SpAmDH-I292 gene, the SpAmDH-A295 gene, the SpAmDH-S288 gene, the SpAmDH-E297 gene, the SpAmDH-N69/V291 gene, the SpAmDH-S68/V291 gene, the SpAmDH-L40/I111 gene, the SpAmDH-I111/V294 gene, the SpAmDH-E114/V291 gene, the SpAmDH-L40/I111/T134 gene, the SpAmDH-S68/N69/V291 gene, the SpAmDH-I111/A113/T134 gene, the SpAmDH-L40/I111/T134/294 gene, the SpAmDH-S68/N69/V291 gene, the SpAmDH-I111/A113/V114/V294 gene, the SpAmDH-L40/I111/T134 gene, the SpAmDH-S291 gene, the SpAmDH-S68/N291 gene, the SpAmDH-L114/V114/V291 gene, The SpAmDH-S C/N69C/I111F/V291C gene, the SpAmDH-I111F/E114V/V294C gene, the SpAmDH-L40F/S68/N69C/T134C/V291C/V294C gene, the SpAmDH-S68/N69C/T134C/V291C gene, the SpAmDH-L40C/S68/N69C/A113C/T134C/V291C/V294C gene, the SpAmDH-S68C/I111C/V294 gene or the SpAmDH-I111C/S288C/V294C gene.
The expression cassette of the nucleic acid molecule refers to a DNA capable of expressing SpAmDH or the SpAmDH mutant protein in a host cell, and the DNA not only can comprise a promoter for starting the transcription of the SpAmDH or the gene coding for the SpAmDH mutant protein, but also can comprise a terminator for the transcription of the SpAmDH or the SpAmDH mutant protein. Still further, the expression cassette may further comprise an enhancer sequence.
The recombinant vector of the nucleic acid molecule can be bacterial plasmid (such as expression vector based on T7 promoter expressed in bacteria, specifically pET-24a and the like), phage, yeast plasmid (such as YEp series vector and the like) or retrovirus packaging plasmid which carries SpAmDH or the SpAmDH mutant protein coding gene.
The recombinant vector is obtained by inserting the nucleic acid molecule into an expression vector.
The recombinant vector can be pET 24-SpAmDH-L40, pET 24-SpAmDH-L61, pET 24-SpAmDH-S68, pET 24-SpAmDH-N69, pET 24-SpAmDH-I111, pET 24-SpAmDH-A113, pET 24-SpAmDH-E114, pET 24-SpAmDH-V116, pET 24-SpAmDH-T134, pET 24-SpDH-pET 134, pET 24-SpAmDH-T134, pET 24-SpAmDH-V150, SpDH-187-SpA 187-AmDH-L239, SpDH-24, Sp-SpDH-239, SppET 24-SppET 187-SpDH-E, Sp 24-E, Sp-E, pET 24-Sp-E, pET 24-E-I, pET-I-, pET 24-SpAmDH-V291, pET 24-SpAmDH-I292, pET 24-SpAmDH-A295, pET 24-SpAmDH-S288, pET 24-SpAmDH-E297, pET 24-SpAmDH-N69/V291, pET 24-SpAmDH-S68/V291, pET 24-SpAmDH-L40/I111, pET 24-SpAmDH-I111/V294, pET 24-SpAmDH-E114/V291, pET 24-SpAmDH-L40/I111/T134, pET 24-SpAmDH-S68/N69/V291, pET 24-SpAmDH-111/I113/V113, pET 24-SpAmDH-L68/V294/E111/V294, pET 24-SpAmDH-L111/V294, pET 24/E114/V294, SpAmDH-L68/V294, and SpAMDH-L111/V294, pET 24-SpAmDH-S68/N69/I111/V291, pET 24-SpAmDH-I111/E114/V294, pET 24-SpAmDH-L40/S68/N69/T134/V291/V294, pET 24-SpAmDH-S68/N69/T134/V291, pET 24-SpAmDH-L40/S68/N69/A113/T134/V291/294, pET 24-SpAmDH-S68/I111/V294 or pET 24-SpAmDH-I111/S288/V294. The recombinant microorganism containing the nucleic acid molecule encoding the SpAmDH or the SpAmDH mutant protein can be yeast, bacteria, algae or fungi carrying the SpAmDH or the gene encoding the SpAmDH mutant protein, such as Escherichia coli and the like.
The recombinant microorganism may be BL (DE)/pET 24-SpAmDH-L40, BL (DE)/pET 24-SpAmDH-L61, BL (DE)/pET 24-SpAmDH-S68, BL (DE)/pET 24-SpAmDH-N69, BL (DE)/pET 24-SpAmDH-I111, BL (DE)/pET 24-SpAmDH-A113, BL (DE)/pET 24-SpAmDH-E114, BL (DE)/pET 24-SpAmDH-V116, BL (DE)/SpAmDH-V-116, SpAmDH-E116, Sp 24-E116, SpAmDH-E116, BL (DE)/SpAmDH-V-116, BL (DE)/pET 24-SpAmDH-T134, BL (DE)/pET 24-SpAmDH-T150, BL (DE)/pET 24-SpAmDH-A187, BL (DE)/pET 24-SpAmDH-L239, BL (DE)/pET 24-SpAmDH-V291, BL (DE)/pET 24-SpAmDH-I, pEBL (DE)/SpAmDH-I, BL (DE)/SpAmDH-T292, BL (DE)/SpAmDH-T24-T295-SpAmDH-V295, BL (DE)/SpAMDH-L295-SpAMDH-L295, SpAMDH-L295-L, BL (DE)/pET 24-SpAmDH-A295, BL (DE)/pET 24-SpAmDH-S288, BL (DE)/pET 24-SpAmDH-E297, BL (DE)/pET 24-SpAmDH-N69/V291, BL (DE)/pET 24-SpAmDH-S68/V291, BL (DE)/pET 24-SpAmDH-L40/I111, BL (DE)/pET 24-SpAmDH-I111/V294, BL (DE)/pET 24-SpAmDH-E114/V291, BL (DE)/pET 24-SpAmDH-L40/I111/T134, BL (DE)/pET 24-SpAmDH-S68/N69/V291, BL (DE)/pET 24-SpDH-I111/A113/pET 134, BL (DE)/SpAmDH-L24/SpAmDH-L40/I111/V294, BL (DE)/pET 24-SpAmDH-S68/N69/E114/V291, BL (DE)/pET 24-SpAmDH-S68/N69/I111/V291, BL (DE)/pET 24-SpAmDH-I111/E114/V294, BL (DE)/pET 24-SpAmDH-L40/S68/N69/T134/V291/294, BL (DE)/pET 24-SpAmDH-S68/N69/T134/V291, BL (DE)/pET 24-SpAmDH-L40/S68/N69/113/T134/V291/294, BL (DE)/pET 24-SpAmDH-S68/I111/V294 or BL (DE)/pET 24-SpAmDH-I111/S288/294.
The invention also provides any one of the following products:
m1, a kit consisting of 1-hydroxy-2-butanone and SpAmDH;
m2, a kit consisting of 1-hydroxy-2-butanone and the SpAmDH mutein;
m3, a kit consisting of 1-hydroxy-2-butanone and the biomaterial of claim 7;
m4, a kit consisting of 4-hydroxy-2-butanone and SpAmDH;
m5, a kit consisting of 4-hydroxy-2-butanone and the SpAmDH mutein;
m6, a kit consisting of 4-hydroxy-2-butanone and the biomaterial of claim 7;
m7, a kit consisting of acetol and SpAmDH;
m8, a kit consisting of acetol and the spomdh mutein;
m9, a kit consisting of acetol and the biomaterial of claim 7;
m10, a kit consisting of 2-hydroxyacetophenone and SpAmDH;
m11, a kit consisting of 2-hydroxyacetophenone and the spomdh mutein;
m12, a kit consisting of 2-hydroxyacetophenone and the biomaterial of claim 7;
m13, a kit consisting of 1- (4-fluorophenyl) -2-hydroxy-1-ethanone and SpAmDH;
m14, a kit consisting of 1- (4-fluorophenyl) -2-hydroxy-1-ethanone and the spomdh mutein;
m15, a kit consisting of 1- (4-fluorophenyl) -2-hydroxy-1-ethanone and a biomaterial as claimed in claim 7.
The kit of M1-M3 can be used for the preparation of (S) -2-amino-1-butanol.
The kit of M4-M6 can be used for the preparation of (R) -3-amino-1-butanol.
The kit of parts described by M7-M9 can be used for the preparation of (S) -alaninol.
The kit of parts described by M10-M12 can be used for the preparation of (S) -phenicol.
The kit described by M13-M15 was used for the preparation of (S) -2-amino-2- (4-fluorophenyl) -1-ethanol.
The invention also provides any one of the following applications of SpAmDH, SpAmDH mutein, biological material or product:
z1, in the preparation of chiral amino alcohol compounds;
z2, in the preparation of chiral amino alcohol compound products;
z3, in catalyzing hydroxy ketone to generate chiral amino alcohol compound;
z4, and the application in preparing products for catalyzing hydroxy ketone to generate chiral amino alcohol compounds.
In the present invention, the chiral amino alcohol compound may be 2-amino-1-butanol, 3-amino-1-butanol, alaninol, phenethyl amine alcohol or 2-amino-2- (4-fluorophenyl) -1-ethanol;
the hydroxy ketone can be 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, acetol, 2-hydroxyacetophenone, and 1- (4-fluorophenyl) -2-hydroxy-1-ethanone.
Specifically, the chiral amine alcohol compound may be (S) -2-amino-1-butanol, (R) -3-amino-1-butanol, (S) -alaninol, (S) -phenethylamine alcohol or (S) -2-amino-2- (4-fluorophenyl) -1-ethanol.
The invention applies directed evolution technology and method to carry out enzyme modification on amine dehydrogenase SpAmDH from Sporosarcina psychrophila to obtain a mutant thereof, the obtained mutant can catalyze hydroxyketone substrates to synthesize chiral amino alcohol compounds represented by 1-hydroxy-2-butanone and 4-hydroxy-2-butanone, and has the characteristics of high yield and high ee value. The method has the advantages of convenient operation, high optical purity of the product and the like, and has better industrial application prospect in preparing the chiral amino alcohol compound by biocatalysis.
Drawings
FIG. 1 is a schematic diagram of primer design for the construction of an amine dehydrogenase L40/A113/T134/V294 mutant library A and an M65/S68/N69/S288 mutant library A. F18 and R18, F19 and R19, F20 and R20 are SpAmDH-F18 and SpAmDH-R18, SpAmDH-F19 and SpAmDH-R19, SpAmDH-F20 and SpAmDH-R20 in Table 1, respectively.
FIG. 2 is a schematic diagram of amine dehydrogenase SpAmDH or a mutant thereof catalyzing reduction of 1-hydroxy-2-butanone to (S) -2-amino-1-butanol and production of 4-hydroxy-2-butanone to (R) -3-amino-1-butanol.
FIG. 3 is a HPLC detection result map of a reaction in which amine dehydrogenase SpAmDH or a mutant thereof catalyzes and reduces 1-hydroxy-2-butanone to produce (S) -2-amino-1-butanol and 4-hydroxy-2-butanone to produce (R) -3-amino-1-butanol. A: liquid chromatography results of a racemic-2-amino-1-butanol standard substance; b: the liquid chromatography result of the sample (S) -2-amino-1-butanol; c: liquid chromatography results of a racemic 3-amino-1-butanol standard substance; d: the sample (R) -3-amino-1-butanol was subjected to liquid chromatography.
FIG. 4 shows the results of the detection of hydroxyketone substrates catalyzed by amine dehydrogenase SpAmDH or its mutants, and "c" after percentage indicates the conversion rate.
FIG. 5 shows the results of detecting 1-hydroxy-2-butanone catalyzed by amine dehydrogenase SpAmDH and its mutants.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.
2 × High-fidelity Master Mix: department of biotechnology limited, cat #: TP 001;
1-hydroxy-2-butanone: shanghai Bide pharmaceutical science and technology Limited, Cat number: BD 59017;
4-hydroxy-2-butanone: shanghai Aladdin Biotechnology GmbH, cat #: h106330;
acetone alcohol: shanghai Aladdin Biotechnology GmbH, cat #: h111149;
2-hydroxyacetophenone: shanghai ai Expo chemical science and technology Limited, goods number: a42334;
1- (4-fluorophenyl) -2-hydroxy-1-ethanone: beijing Bailingwei science and technology Co., Ltd, cat #: 042861.
example 1 preparation of engineered bacteria of amine dehydrogenase SpAmDH Gene or its mutant
Preparation of amine dehydrogenase SpAmDH gene engineering strain
The encoding gene of amine dehydrogenase (SpAmDH) derived from Sporosarcina psychrophila is optimized by using Escherichia coli as host cells to obtain SpAmDH gene shown in SEQ ID No.1 in the sequence table. The amino acid sequence of SpAmDH is shown as SEQ ID No.2 in the sequence table.
The SpAmDH gene shown as SEQ ID No.1 in the sequence table is synthesized by the whole gene, a small DNA fragment between NdeI and XhoI recognition sequences in a pET24a (+) vector (Novagen) is replaced by the SpAmDH gene shown as SEQ ID No.1 to obtain a recombinant vector, the recombinant vector is marked as pET24a-SpAmDH, pET24a-SpAmDH can express the SpAmDH shown as SEQ ID No.2 in the sequence table, and the expression of the SpAmDH gene is driven by a T7 promoter.
The recombinant vector pET24a-SpAmDH is introduced into escherichia coli BL21(DE3) to obtain a recombinant strain, and the recombinant strain is marked as BL21(DE3)/pET24a-SpAmDH, namely the amine dehydrogenase SpAmDH gene engineering strain. pET24a (+) was introduced into E.coli BL21(DE3) to obtain a recombinant strain BL21(DE3)/pET24a as a control strain.
Preparation of amine dehydrogenase SpAmDH gene mutant engineering strain
And respectively constructing the amine dehydrogenase SpAmDH gene mutant engineering strains of the amine dehydrogenase SpAmDH mutants. The mutant of the amine dehydrogenase SpAmDH is obtained by designing a degenerate primer to construct a mutant library and screening, or by designing a mutant primer. Degenerate and mutant primer sequences were designed as shown in tables 1 and 2.
TABLE 1 mutant library construction primer sequences used
Figure BDA0002524285170000131
Figure BDA0002524285170000141
Figure BDA0002524285170000151
Note: in Table 1, there are primers for a plurality of single-stranded DNAs, including primers SpAmDH-F1, SpAmDH-F2, SpAmDH-F3, SpAmDH-F4, SpAmDH-F5, SpAmDH-F6, SpAmDH-F7, SpAmDH-F8, SpAmDH-F9, SpAmDH-F10, SpAmDH-R11, SpAmDH-R12, SpAmDH-R13, SpAmDH-R14, SpAmDH-R15, SpAmDH-R16, SpAmDH-R17, and SpAmDH-R18, in the order from the top to the bottom in terms of molar ratio: 12:6:1:1, and mixing primers SpAmDH-F18, SpAmDH-R18, SpAmDH-F19, SpAmDH-R19, SpAmDH-F21, SpAmDH-R21, SpAmDH-F22 and SpAmDH-R22 according to the molar ratio from top to bottom in the sequence shown in the table: 2:1, and mixing the primers SpAmDH-F20 and SpAmDH-F23 according to the molar ratio of the single-stranded DNA from top to bottom in the table: 4:2:2: 1; r represents A or G, Y represents C or T, M represents A or C, K represents G or T, S represents G or C, W represents A or T, H represents A or T or C, B represents G or T or C, V represents G or A or C, D represents G or A or T, and N represents A or T or G or C.
TABLE 2 mutants and primer sequences used
Figure BDA0002524285170000161
The primer design constructed by the L40/A113/T134/V294 mutant library A and the M65/S68/N69/S288 mutant library A is shown in a figure 1.
The specific construction method comprises the following steps:
1. two rounds of Mega-PCR reaction
Constructing an L40 mutant library, an L61 mutant library, an S68 mutant library, an N69 mutant library, an I111 mutant library, an A113 mutant library, an E114 mutant library, a V116 mutant library, a T134 mutant library, a T150 mutant library, an A187 mutant library, an L239 mutant library, an S288 mutant library, a V291 mutant library, an I292 mutant library, an A295 mutant library and an E297 mutant library by taking the recombinant vector pET24a-SpAmDH obtained in the first step as a template, constructing an M65/S68/N69/S288 mutant library A by taking the recombinant vector pET24a-SpAmDH-V291C below as a template, constructing an M65/S68/N69/S288 mutant library B by taking the recombinant vector pET24 a-SpDH Am I111F/V C below as a template, constructing an SpAmDH-E114/E5966/N291 mutant library B by taking the recombinant vector pET 24-SpDH 27-SpDH-a-Sp template, construction of SpAmDH-I111F/E114V/V294C mutant Using the recombinant vector pET24a-SpAmDH-I111F/V294C below as a template, SpAmDH-S68C/N69C/E114V/V291C and SpAmDH-S68C/N69C/I111F/V291C mutants were constructed using the recombinant vector pET24a-SpAmDH-S68C/N69C/V291C below as a template, and two rounds of Mega-PCR were performed using the primer pairs corresponding to the respective mutants. When the same mutant library or mutant is constructed, the template of the first round PCR is the same as that of the second round PCR.
The PCR reaction system and procedure were as follows:
the first round of PCR was 50 μ L: template (150 ng/. mu.L) 1. mu.L; 2 × High-fidelity Master Mix 25 μ L; ddH2O22 mu L; 1 μ L of the pre-primer (10 μmol/L); the rear primer (10. mu. mol/L) was 1. mu.L. The specific sequences of the primers used are shown in tables 1 and 2, and the front and rear primers in the same primer pair are labeled "F" and "R", respectively.
First round PCR procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds or 50 seconds, and circulating for 30 times; final extension at 72 ℃ for 5 min.
The second round of PCR was 50 μ L: template (150 ng/. mu.L) 1. mu.L; 2 × High-fidelity Master Mix 25 μ L; ddH2O22 mu L; first round PCR product 2. mu.L.
Second round PCR procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 2 minutes, and circulating for 30 times; final extension at 72 ℃ for 5 min.
2. Overlap PCR reaction
Construction of L40/A113/T134/V294 mutant library A Using the recombinant vector pET24a-SpAmDH-I111F below as a template (i.e., the templates of the first and third PCR rounds are both pET24a-SpAmDH-I111F), construction of L40/A113/T134/V294 mutant library B Using the recombinant vector pET24a-SpAmDH-S68C/N69C/V291C below as a template (i.e., the templates of the first and third PCR rounds are both pET24 a-SpAmDH-S68C/N69C/V291C). Taking the construction of L40/A113/T134/V294 mutant library A as an example, the design scheme of the mutant library construction primers is shown in FIG. 1, and an Overlap PCR reaction is carried out.
The PCR reaction system and procedure were as follows:
F18R18 and F19R19 products are obtained by the first round of PCR reaction amplification. The PCR system was 50. mu.L: template (150 ng/. mu.L) 1. mu.L; 2 × High-fidelity Master Mix 25 μ L; ddH2O22 mu L; 1 μ L of the pre-primer (10 μmol/L); the rear primer (10. mu. mol/L) was 1. mu.L. The specific sequences of the primers used are shown in Table 1, and the front and rear primers in the same primer pair are labeled "F" and "R", respectively.
First round PCR procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 55 ℃ for 15 seconds, extension at 72 ℃ for 30 seconds, and cycle times of 30 times; final extension at 72 ℃ for 5 min.
The PCR product obtained in the first round was recovered, and the product obtained from SpAmDH-F18 and SpAmDH-R18 was designated as F18R18, and the product obtained from SpAmDH-F19 and SpAmDH-R19 was designated as F19R 19.
The F18R19 product is obtained by the second PCR reaction. The PCR system was 50. mu.L: template (F18R 18 obtained in the first round: F19R19 molar mixed solution) 1. mu.L; 2 × High-fidelity Master Mix 25 μ L; ddH2O22 mu L; 1 μ L of the primer SpAmDH-F18(10 μmol/L); the rear primer SpAmDH-R19 (10. mu. mol/L) was 1. mu.L.
Second round PCR procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds; annealing at 55 ℃ for 15 seconds; stretching at 72 ℃ for 1 minute and circulating for 30 times; final extension at 72 ℃ for 5 min.
The product obtained from SpAmDH-F18 and SpAmDH-R19 was designated F18R 19.
And amplifying the third PCR reaction to obtain the recombinant plasmid. The PCR system was 50. mu.L: template (150 ng/. mu.L) 1. mu.L; 2 × High-fidelity Master Mix 25 μ L; ddH2O22 mu L; the second round of PCR F18R19 produced 2. mu.L.
Third round of PCR procedure: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 2 minutes, and circulating for 30 times; final extension at 72 ℃ for 5 min.
Construction of L40/A113/T134/V294 mutant library B was carried out as described above, replacing SpAmDH-F18, SpAmDH-R18, SpAmDH-F19 and SpAmDH-R19 with SpAmDH-F21, SpAmDH-R21, SpAmDH-F22 and SpAmDH-R22, respectively, to obtain PCR products.
3. Obtaining of amine dehydrogenase SpAmDH gene mutant engineering strain
After the last round of PCR is finished, adding 2 mu L of DpnI enzyme into each reaction system, digesting for 2 hours at 37 ℃, taking 1 mu L of DpnI enzyme, electrically transferring into E.coli BL21(DE3) competent cells, putting the E.coli BL21 competent cells into a 37 ℃ culture box, inversely culturing for 12-16 hours, picking transformants, extracting plasmids from the transformants, and sequencing to obtain transformants with the target nucleotides subjected to non-directional mutation or directional mutation, namely the amine dehydrogenase SpAmDH gene mutant engineering strain, wherein the recombinant plasmids with the target nucleotides subjected to non-directional mutation or directional mutation are the expression vector for expressing the amine dehydrogenase SpAmDH gene mutant.
Example 2 expression of amine dehydrogenase SpAmDH or its mutant and preparation of Whole cell, crude enzyme powder and pure enzyme solution
The recombinant strain BL21(DE3)/pET24a-SpAmDH, BL21(DE3)/pET24a and each amine dehydrogenase SpAmDH gene mutant engineering strain prepared in the example 1 are induced to express to obtain the whole cells, crude enzyme powder and pure enzyme solution of each strain, and the operation steps of each strain are as follows:
picking up the strain into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, shaking at 37 deg.C and 220rpm overnight for 12h, inoculating into TB liquid culture medium containing 50 ug/mL kanamycin according to the inoculum size of 1% volume percentage, and culturing at 37 deg.C to OD600When the concentration is 0.7, adding IPTG with the final concentration of 0.1mmol/L, inducing expression at 20 ℃ and 220rpm for 12h, centrifuging at 4 ℃ and 4,000 rpm for 10min, collecting precipitated thalli (namely whole cells), suspending the collected thalli by using phosphate buffer solution (50mmol/L and pH 7.4) to obtain thalli suspension, and then ultrasonically crushing the thalli cells under the ice bath condition to obtain a sample (namely crude enzyme liquid) after ultrasonic crushing. Centrifuging the crude enzyme solution at 8,000rpm at 4 deg.C for 30min, collecting supernatant, freezing the supernatant at-80 deg.C, and freeze-drying with vacuum drier to obtain crude enzyme powder. The crude enzyme solution was centrifuged at 12,000rpm at 4 ℃ for 60min to collect the supernatant, and the resulting supernatant was filtered through a 0.45 μm aqueous membrane and purified by an ATKA protein purifier using HisTrap HP 5mL prepacked column (GE), cat # 17524802. Loading and equilibrating buffer A (50mmol/L phosphate buffer, 0.5mol/L NaCl,20mmol/L imidazole, pH 8.0); elution buffer B (50mmol/L phosphate buffer, 0.5mol/L NaCl,500mmol/L imidazole, pH 8.0), elution flow rate 2 mL/min; collecting the eluent (namely pure enzyme solution).
Example 3 catalysis of 1-hydroxy-2-butanone to (S) -2-amino-1-butanol by amine dehydrogenase SpAmDH or mutants thereof
The crude enzyme powder or whole cells of the amine dehydrogenase SpAmDH or its mutant prepared in example 2 were used to catalyze the production of (S) -2-amino-1-butanol from 1-hydroxy-2-butanone to give (S) -2-amino-1-butanol, as shown in FIG. 2, and BL21(DE3)/pET24a was used as a control.
The reaction system for catalyzing the reaction of SpAmDH or the mutant thereof is obtained by adding the following substances to 1mol/L ammonium chloride/ammonia water buffer solution (obtained by mixing ammonium chloride and ammonia water in an equal molar ratio, and having a pH value of 8.5): substrate 1-hydroxy-2-butanone 20mmol/L or 40mmol/L, amine dehydrogenase SpAmDH or its mutant crude enzyme powder 20g/L or whole cell 100g/L, NAD+1mmol/L (in the form of oxidized coenzyme I aqueous solution), 2g/L GDH crude enzyme powder, 100mmol/L glucose, 1g/L lysozyme (CAS: 12650-88-3, specific activity of 20000U/mg, Beijing Solebao technology Co., Ltd.), DNase I (deoxyribonuclease, CAS:9003-98-9, specific activity of 2000U/mg, China Clay Biotech, Ltd.).
The GDH crude enzyme powder is glucose dehydrogenase powder capable of catalyzing substrates glucose and NAD+The enzyme is added to achieve the aim of regenerating coenzyme NADH, and the enzyme powder is prepared according to the following method:
replacing DNA fragments between pET24a (+) vector NdeI and XhoI recognition sequences with a glucose dehydrogenase gene (the nucleotide sequence of the coding gene is SEQ ID No.3) to obtain a recombinant vector which is marked as pET24 a-GDH; pET24a-GDH was introduced into E.coli BL21(DE3) to give a recombinant strain designated as BL21(DE3)/pET24 a-GDH. BL21(DE3)/pET24a-GDH was cultured, the obtained cells were disrupted and centrifuged, and the supernatant was put into a freeze-drying machine to obtain a crude GDH enzyme powder.
The GDH crude enzyme powder is detected, the specific enzyme activity is 2.04U/mg, and the enzyme activity determination method comprises the following steps:
the enzyme activity was calculated by monitoring the decrease in absorbance of NADH at 340nm, defined as: at pH 8.5, temperature 30 ℃ and substrate 1mmol/L NAD+The amount of enzyme required to consume 1. mu. mol/L NADH per minute under the condition of 10mmol/L glucose. The enzyme activity calculation formula is as follows: enzyme activity (U) ═ EW × V × 103(6220X 0.3) wherein EW represents a change in absorbance at 340nm per minute, V represents a volume of the reaction solution in mL, 6220 is a molar extinction coefficient of coenzyme, L/(mol X cm), and 0.3 is an optical path length in cm).
The reaction system for the catalytic reaction of SpAmDH or a mutant thereof was reacted at 30 ℃ for 12 hours. After the reaction is finished, calculating the conversion rate and carrying out stereoselectivity analysis, wherein the specific method comprises the following steps:
the reaction solution obtained by the above reaction was boiled for 5 minutes, centrifuged at 12,000rpm for 10 minutes, the precipitate was removed, the supernatant was retained, derivatized with a derivatizing agent (derivatizing agent preparation method: 0.343g of o-phthalaldehyde +5mL of absolute ethanol +0.147g N-acetyl-L cysteine, diluted to 25mL with 0.4mol/L of boric acid buffer (pH 9.5) and kept in the dark for use; derivatizing method: 300 μ L of 0.4mol/L of boric acid buffer (pH 9.5) +150 μ L of ultrapure water +200 μ L of derivatizing agent +100 μ L of reaction solution, mixed and left to stand for 2 minutes, centrifuged at 12,000rpm for 10 minutes, the supernatant was collected, filtered with a filter and then subjected to the reaction for HPLC detection. HPLC detection conditions: agilent SB-Aq C18 column (4.6 x 250mm,5 μm), detection wavelength 334nm, column temperature: 35 ℃, flow rate: 1mL/min, loading amount: 10 μ L. The gradient elution procedure is shown in table 3.
TABLE 3 gradient elution procedure for HPLC
Figure BDA0002524285170000181
Figure BDA0002524285170000191
Note: the% in table 3 represents the volume percentage.
The HPLC test results are shown in Table 4.
Conversion rate ═ A1/A2×100％；A1: the peak area value of the (S) -2-amino-1-butanol obtained by liquid chromatography analysis; a. the2: and (3) analyzing the peak area value of the obtained standard (S) -2-amino-1-butanol by liquid chromatography.
No (S) -2-amino-1-butanol was produced in the reaction product of BL21(DE3)/pET24a, and the HPLC test result of the reaction of catalytic reduction of 1-hydroxy-2-butanone to (S) -2-amino-1-butanol with amine dehydrogenase SpAmDH or its mutant is shown in FIG. 3, where A: liquid chromatography results of a racemic-2-amino-1-butanol standard substance; b: sample (S) -2-amino-1-butanol liquid chromatography results. The result shows that the amine dehydrogenase SpAmDH or the mutant thereof can asymmetrically reduce and catalyze 1-hydroxy-2-butanone to generate (S) -2-amino-1-butanol, the conversion rate is 47-99%, and the stereoselectivity is more than 99% (S).
Table 4 shows the results of the detection of the amine dehydrogenase SpAmDH and its mutant catalyzing 1-hydroxy-2-butanone
Figure BDA0002524285170000192
Figure BDA0002524285170000201
Note:a20mmol/L of substrate 1-hydroxy-2-butanone and 20g/L of crude enzyme powder of amine dehydrogenase SpAmDH or mutants thereof in a reaction system;bin the reaction system, 40mmol/L of substrate 1-hydroxy-2-butanone and 100g/L of whole cells of amine dehydrogenase SpAmDH or mutants thereof are added; -means no assay; ee (stereoselectivity) ═ aS-AR)/(AS+AR)×100％；AS: peak area values of the obtained (S) -2-amino-1-butanol by liquid chromatography; a. theR: the peak area value of the obtained (R) -2-amino-1-butanol was analyzed by liquid chromatography.
In column 1 of Table 4, the numbers indicate the sites of mutation of the amine dehydrogenase SpAmDH, and the letters before and after the numbers indicate the amino acids before and after the mutation, respectively. The DNA sequencing result of the corresponding strain shows that the strain expressing SpAmDH-L40V and the recombinant vector are respectively marked as BL21(DE3)/pET24a-SpAmDH-L40V and pET24a-SpAmDH-L40V, the 118 th position of SpAmDH gene in the strain and the vector is changed from C to G, and the mutated SpAmDH gene is marked as SpAmDH-L40V gene;
respectively marking a strain expressing SpAmDH-L61A and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-L61A and pET24a-SpAmDH-L61A, wherein the 181 th site of SpAmDH gene in the strain and the vector is mutated by C to G, the 182 th site is mutated by T to C, and the mutated SpAmDH gene is marked as SpAmDH-L61A gene;
respectively marking a strain expressing SpAmDH-L61C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-L61C and pET24a-SpAmDH-L61C, respectively marking the SpAmDH gene after mutation as SpAmDH-L61C gene, wherein the 181 th position of the SpAmDH gene in the strain and the vector is mutated by C to T, the 182 th position is mutated by T to G, and the 183 th position is mutated by G to T;
respectively recording a strain expressing SpAmDH-L61V and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-L61V and pET24a-SpAmDH-L61V, wherein the 181 th site of SpAmDH gene in the strain and the vector is mutated from C to G, and recording the mutated SpAmDH gene as SpAmDH-L61V gene;
respectively marking a strain expressing SpAmDH-S68C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-S68C and pET24a-SpAmDH-S68C, wherein the 202 nd position of SpAmDH gene in the strain and the vector is mutated from A to T, and the mutated SpAmDH gene is marked as SpAmDH-S68C gene;
respectively marking a strain expressing SpAmDH-N69S and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-N69S and pET24a-SpAmDH-N69S, wherein the 206 th site of SpAmDH gene in the strain and the vector is mutated from A to G, and marking the mutated SpAmDH gene as SpAmDH-N69S gene;
respectively marking a strain expressing SpAmDH-I111F and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I111F and pET24a-SpAmDH-I111F, wherein the No. 331 of SpAmDH gene in the strain and the vector is mutated from A to T, and the mutated SpAmDH gene is marked as SpAmDH-I111F gene;
respectively marking a strain expressing SpAmDH-I111W and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I111W and pET24a-SpAmDH-I111W, wherein the No. 331 of SpAmDH gene in the strain and the vector is mutated from A to T, No. 332 of SpAmDH gene is mutated from T to G, No. 333 of SpAmDH gene is mutated from T to G, and the mutated SpAmDH gene is marked as SpAmDH-I111W gene;
respectively marking a strain expressing SpAmDH-A113C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A113C and pET24a-SpAmDH-A113C, respectively marking the mutant SpAmDH gene as SpAmDH-A113C gene, wherein the 337 th position of the SpAmDH gene is mutated by G to be T, the 338 th position is mutated by C to be G, and the 339 th position is mutated by A to be T;
respectively marking a strain expressing SpAmDH-E114V and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-E114V and pET24a-SpAmDH-E114V, respectively marking the 341 th site of SpAmDH gene in the strain and the vector as T by A mutation, and the 342 th site of SpAmDH gene as G by A mutation, and marking the mutated SpAmDH gene as SpAmDH-E114V gene;
respectively marking a strain expressing SpAmDH-E114Q and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-E114Q and pET24a-SpAmDH-E114Q, respectively marking the 340 th site of SpAmDH gene in the strain and the vector as G mutation for C, the 342 th site of SpAmDH gene as A mutation for G, and marking the mutated SpAmDH gene as SpAmDH-E114Q gene;
respectively marking a strain expressing SpAmDH-V116I and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-V116I and pET24a-SpAmDH-V116I, wherein the 346 th site of SpAmDH gene in the strain and the vector is mutated by G to be A, and marking the mutated SpAmDH gene as SpAmDH-V116I gene;
respectively marking a strain expressing SpAmDH-V116M and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-V116M and pET24a-SpAmDH-V116M, respectively marking the 346 th site of SpAmDH gene in the strain and the vector as A by G mutation, and the 348 th site of SpAmDH gene as G by T mutation, and marking the mutated SpAmDH gene as SpAmDH-V116M gene;
respectively marking a strain expressing SpAmDH-V116S and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-V116S and pET24a-SpAmDH-V116S, wherein the 346 th site of SpAmDH gene in the strain and the vector is mutated by G so as to obtain A, the 347 th site is mutated by T so as to obtain G, and the mutated SpAmDH gene is marked as SpAmDH-V116S gene;
respectively marking a strain expressing SpAmDH-V116Y and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-V116Y and pET24a-SpAmDH-V116Y, wherein the 346 th site of SpAmDH gene in the strain and the vector is mutated by G to T, the 347 th site is mutated by T to A, and the mutated SpAmDH gene is marked as SpAmDH-V116Y gene;
respectively marking a strain expressing SpAmDH-T134A and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-T134A and pET24a-SpAmDH-T134A, wherein the 400 th site of SpAmDH gene in the strain and the vector is mutated from A to G, the 402 th site is mutated from C to G, and the mutated SpAmDH gene is marked as SpAmDH-T134A gene;
respectively marking a strain expressing SpAmDH-T134F and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-T134F and pET24a-SpAmDH-T134F, wherein the 400 th site of SpAmDH gene in the strain and the vector is mutated from A to T, the 401 th site is mutated from C to T, the 402 th site is mutated from C to T, and the mutated SpAmDH gene is marked as SpAmDH-T134F gene;
respectively marking a strain expressing SpAmDH-T134G and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-T134G and pET24a-SpAmDH-T134G, wherein the 400 th site of SpAmDH gene in the strain and the vector is mutated from A to G, the 401 th site is mutated from C to G, the 402 th site is mutated from C to T, and the mutated SpAmDH gene is marked as SpAmDH-T134G gene;
respectively marking a strain expressing SpAmDH-T150C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-T150C and pET24a-SpAmDH-T150C, marking the mutant SpAmDH gene as SpAmDH-T150C gene, wherein the 448 th position of the SpAmDH gene in the strain and the vector is mutated by A to T, the 449th position is mutated by C to G, and the 450 th position is mutated by C to T;
respectively marking a strain expressing SpAmDH-A187K and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A187K and pET24a-SpAmDH-A187K, wherein the 559 th site of SpAmDH gene in the strain and the vector is mutated by G to A, the 560 th site is mutated by C to A, the 561 th site is mutated by C to A, and the mutated SpAmDH gene is marked as SpAmDH-A187K gene;
respectively marking a strain expressing SpAmDH-A187N and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A187N and pET24a-SpAmDH-A187N, respectively marking the SpAmDH gene after mutation as SpAmDH-A187N gene, wherein the 559 th position of the SpAmDH gene in the strain and the vector is mutated by G to A, the 560 th position is mutated by C to A, and the 561 th position is mutated by C to T;
respectively marking a strain expressing SpAmDH-A187P and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A187P and pET24a-SpAmDH-A187P, wherein the 559 th site of SpAmDH gene in the strain and the vector is mutated by G to C, the 561 th site is mutated by C to G, and the mutated SpAmDH gene is marked as SpAmDH-A187P gene;
respectively marking a strain expressing SpAmDH-A187T and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A187T and pET24a-SpAmDH-A187T, respectively marking the 559 th site of SpAmDH gene in the strain and the vector as G mutation, the 561 th site of SpAmDH gene as C mutation, and marking the mutated SpAmDH gene as SpAmDH-A187T gene;
respectively marking a strain expressing SpAmDH-L239F and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-L239F and pET24a-SpAmDH-L239F, respectively marking the 715 th site of SpAmDH gene in the strain and the vector as T by C mutation and the 717 th site as G mutation, and marking the mutated SpAmDH gene as SpAmDH-L239F gene;
respectively recording a strain expressing SpAmDH-L239M and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-L239M and pET24a-SpAmDH-L239M, wherein the 715 th site of SpAmDH gene in the strain and the vector is mutated from C to A, and recording the mutated SpAmDH gene as SpAmDH-L239M gene;
respectively marking a strain expressing SpAmDH-V291C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-V291C and pET24a-SpAmDH-V291C, respectively marking the mutant SpAmDH gene as SpAmDH-V291C gene, wherein the No. 871 of the SpAmDH gene is mutated by G to be T, the No. 872 of the SpAmDH gene is mutated by T to be G, the No. 873 of the SpAmDH gene is mutated by G to be T;
respectively marking a strain expressing SpAmDH-V291L and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-V291L and pET24a-SpAmDH-V291L, wherein the No. 871 of SpAmDH gene in the strain and the vector is mutated by G to be C, and marking the mutated SpAmDH gene as SpAmDH-V291L gene;
respectively marking a strain expressing SpAmDH-I292M and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I292M and pET24a-SpAmDH-I292M, wherein the 876 th site of SpAmDH gene in the strain and the vector is changed from T mutation to G, and marking the mutated SpAmDH gene as SpAmDH-I292M gene;
respectively marking a strain expressing SpAmDH-I292T and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I292T and pET24a-SpAmDH-I292T, respectively marking the site 875 of the SpAmDH gene in the strain and the vector as a T mutation to a C site, the site 876 as a T mutation to a G site, and marking the mutated SpAmDH gene as a SpAmDH-I292T gene;
respectively marking a strain expressing SpAmDH-A295N and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A295N and pET24a-SpAmDH-A295N, respectively marking the SpAmDH gene subjected to mutation at the 883 th position of the strain and the vector as G, the B, the C and the T at the 884 th positions of the SpAmDH gene as A and the C and the T at the 885 th positions of the SpAmDH gene as SpAmDH-A295N genes;
respectively marking a strain expressing SpAmDH-A295T and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A295T and pET24a-SpAmDH-A295T, wherein the 883 th site of SpAmDH gene in the strain and the vector is mutated by G so that A is mutated, the 885 th site of the SpAmDH gene is mutated by C so that G is mutated, and the mutated SpAmDH gene is marked as SpAmDH-A295T gene;
respectively marking a strain expressing SpAmDH-A295W and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-A295W and pET24a-SpAmDH-A295W, wherein the 883 th site of the SpAmDH gene in the strain and the vector is mutated from G to T, the 884 th site is mutated from C to G, the 885 th site is mutated from C to G, and the mutated SpAmDH gene is marked as SpAmDH-A295W gene;
respectively marking a strain expressing SpAmDH-S288M and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-S288M and pET24a-SpAmDH-S288M, respectively marking the 863 th site of the SpAmDH gene in the strain and the vector as a G mutation to a T, and the 864 th site as a T mutation to a G, and marking the mutated SpAmDH gene as a SpAmDH-S288M gene;
respectively marking a strain expressing SpAmDH-E297N and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-E297N and pET24a-SpAmDH-E297N, wherein the 889 th site of SpAmDH gene in the strain and the vector is mutated by G to A, the 891 th site is mutated by A to T, and the mutated SpAmDH gene is marked as SpAmDH-E297N gene;
respectively marking a strain expressing SpAmDH-N69C/V291C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-N69C/V291C and pET24a-SpAmDH-N69C/V291C, wherein the 205 th site of the SpAmDH gene in the strain and the vector is mutated by A to T, the 206 th site is mutated by A to G, the 207 th site is mutated by T to C, the 871 site is mutated by G to T, the 872 th site is mutated by T to G, and the 873 th site is mutated by G to T, and marking the mutated SpAmDH gene as SpAmDH-N69C/V291C;
respectively marking a strain expressing SpAmDH-S68C/V291C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-S68C/V291C and pET24a-SpAmDH-S68C/V291C, wherein in the strain and the vector, the position 202 of the SpAmDH gene is mutated by A to T, the position 204 of the SpAmDH gene is mutated by T to C, the position 871 of the SpAmDH gene is mutated by G to T, the position 872 of the SpAmDH gene is mutated by T to G, and the position 873 of the SpAmDH gene is mutated by G to T, and the mutated SpAmDH gene is marked as SpAmDH-S68C/V291C gene;
respectively marking a strain expressing SpAmDH-L40C/I111F and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-L40C/I111F and pET24a-SpAmDH-L40C/I111F, wherein the 118 th site of the SpAmDH gene in the strain and the vector is mutated by C to T, the 119 th site is mutated by T to G, the 120 th site is mutated by G to C, the 331 st site is mutated by A to T, and the mutated SpAmDH gene is marked as SpAmDH-L40C/I111F gene;
respectively marking a strain expressing SpAmDH-I111F/V294C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I111F/V294C and pET24a-SpAmDH-I111F/V294C, wherein the No. 331 of the SpAmDH gene in the strain and the vector is mutated from A to T, No. 880 is mutated from G to T, No. 881 is mutated from T to G, No. 882 is mutated from T to C, and the mutated SpAmDH gene is marked as SpAmDH-I111F/V294C gene;
respectively marking a strain expressing SpAmDH-E114V/V291C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-E114V/V291C and pET24a-SpAmDH-E114V/V291C, wherein the 341 th site of SpAmDH gene in the strain and the vector is mutated from A to T, the 342 th site is mutated from A to G, the 871 site is mutated from G to T, the 872 th site is mutated from T to G, the 873 th site is mutated from G to C, and the mutated SpAmDH gene is marked as SpAmDH-E114V/V291C gene;
respectively recording a strain and a recombinant vector for expressing SpAmDH-L40C/I111F/T134F as BL21(DE3)/pET24a-SpAmDH-L40C/I111F/T134F and pET24a-SpAmDH-L40C/I111F/T134F, wherein the 118 th position of the SpAmDH gene is mutated by C to obtain T, the 119 th position is mutated by T to obtain G, the 120 th position is mutated by G to obtain C, the 331 th position is mutated by A to obtain T, the 400 th position is mutated by A to obtain T, the 401 th position is mutated by C to obtain T, and the 402 th position is mutated by C to obtain T, and recording the mutated SpAmDH gene as SpAmDH-L40C/I111F/T134F;
the strain and the recombinant vector for expressing SpAmDH-S C/N69C/V291C are respectively marked as BL21(DE3)/pET24a-SpAmDH-S68C/N69C/V291C and pET24a-SpAmDH-S68C/N69C/V291C, the 202 nd position of the SpAmDH gene is mutated by A to T, the 204 th position is mutated by T to C, the 205 th position is mutated by A to T, the 206 th position is mutated by A to G, the 207 th position is mutated by T to C, the 871 position is mutated by G to T, the 872 th position is mutated by T to G, the 873 rd position is mutated by G to T, and the mutated SpAmDH gene is marked as SpAmDH-S68/N8569/V291C gene;
respectively marking a strain expressing SpAmDH-I111F/A113C/T134C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I111F/A113C/T134C and pET24a-SpAmDH-I111F/A113C/T134C, wherein the No. 331 of the SpAmDH gene in the strain and the vector is mutated by A to T, No. 337 is mutated by G, No. 338 is mutated by C to G, No. 339 is mutated by A to C, No. 400 is mutated by A to T, No. 401 is mutated by C to G, and the mutated SpAmDH gene is marked as SpAmDH-I111F/A113C/T134C gene;
the strain and the recombinant vector for expressing SpAmDH-L40C/I111F/T134F/V294C are respectively marked as BL21(DE3)/pET24a-SpAmDH-L40C/I111F/T134F/V294C and pET24 a-SpAmDH-L40C/I111/T134F/V C, the 118 th position of the SpAmDH gene in the strain and the vector is mutated by C to T, the 119 th position is mutated by T to G, the 120 th position is mutated by G to C, the 331 th position is mutated by A to T, the 400 th position is mutated by A to T, the 401 th position is mutated by C to T, the 402 th position is mutated by C to T, the 880 th position is mutated by G, the 881 th position is mutated by T, the 882 position is mutated by T to C, and the mutant SpAmDH gene is marked as SpAmDH-L40/3543/F/C4;
the strain and the recombinant vector for expressing SpAmDH-S68C/N69C/E114V/V291C are respectively marked as BL21(DE3)/pET24 a-SpAmDH-S68/N69C/E114/V291C and pET24 a-SpAmDH-S68/N69C/E114V/V291C, wherein the 202 nd position of SpAmDH gene in the strain and the vector is mutated by A to T, the 204 nd position is mutated by T to C, the 205 th position is mutated by A to T, the 206 nd position is mutated by A to G, the 207 th position is mutated by T to C, the 341 th position is mutated by A to T, the 342 nd position is mutated by A to G, the 871 th position is mutated by G, the 873 rd position is mutated by G to T, the 872 th position is mutated by T, and the mutated SpAmDH gene is marked as SpAmDH 68/S46/V;
the strain and the recombinant vector for expressing SpAmDH-S68C/N69C/I111F/V291C are respectively marked as BL21(DE3)/pET24 a-SpAmDH-S68/N69C/I111F/V291C and pET24 a-SpAmDH-S68/N69C/I111F/V291C, the 202 nd position of SpAmDH gene in the strain and the vector is mutated by A to T, the 204 nd position is mutated by T to C, the 205 th position is mutated by A to T, the 206 nd position is mutated by A to G, the 207 th position is mutated by T to C, the 331 st position is mutated by A to T, the 871 th position is mutated by G to T, the 872 th position is mutated by T, the 873 rd position is mutated by G, and the mutated SpAmDH gene is marked as SpAmDH 68-S6/AmN 3569/C/V73727/F;
respectively marking a strain expressing SpAmDH-I111F/E114V/V294C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-I111F/E114V/V294C and pET24a-SpAmDH-I111F/E114V/V294C, wherein the No. 331 of the SpAmDH gene in the strain and the vector is subjected to A mutation to obtain T, the No. 341 is subjected to A mutation to obtain T, the No. 342 is subjected to A mutation to obtain G, the No. 880 is subjected to G mutation to obtain T, the No. 881 is subjected to T mutation to obtain G, the No. 882 is subjected to T mutation to obtain C, and marking the mutated SpAmDH-I111F/E114V/V294C gene;
the strain and the recombinant vector for expressing SpAmDH-L40F/S68C/N69C/T134C/V291C/V294C are respectively marked as BL21(DE3)/pET24a-SpAmDH-L40F/S68C/N69C/T134C/V291C/V294C and pET24a-SpAmDH-L40F/S68C/N69C/T134C/V291C/V294C, the 118 th position of SpAmDH gene in the strain and the vector is marked by C mutation for T, the 120 th position is marked by G mutation for T, the 202 th position is marked by A mutation for T, the 204 th position is marked by T mutation for C, the 205 th position is marked by A mutation for T, the 206 th position is marked by T mutation for C, the 400 th position is marked by A mutation for T872, the 204 th position is marked by T mutation for C, the 401 th position is marked by A mutation for T, the No. G for T87880, the No. G for T3, No.3 for T, Marking the mutant SpAmDH gene as SpAmDH-L40F/S68C/N69C/T134C/V291C/V294C gene with T mutation at position 881 for G and T mutation at position 882 for C;
the strain and the recombinant vector for expressing SpAmDH-S68C/N69C/T134C/V291C are respectively marked as BL21(DE3)/pET24 a-SpAmDH-S68/N69C/T134C/V291C and pET24 a-SpAmDH-S68/N69C/T134C/V291C, wherein the 202 nd position of SpAmDH gene is mutated by A to T, the 204 nd position is mutated by T to C, the 205 th position is mutated by A to T, the 206 nd position is mutated by A to G, the 207 th position is mutated by T to C, the 400 th position is mutated by A to T, the 401 th position is mutated by C to G, the fourth position is mutated by G to T, the 872 to G, the 873 rd position is mutated by G to T, and the mutated SpAmDH gene and the recombinant vector are respectively marked as BL21(DE3)/pET 24-a-SpAmDH-S68/N69/V C/V C, the 206;
the strains and recombinant vectors expressing SpAmDH-L40F/S68C/N69C/A113C/T134C/V291C/V294F are respectively marked as BL21(DE3)/pET24a-SpAmDH-L40F/S68C/N69C/A113C/T134C/V291C/V F and pET24a-SpAmDH-L40F/S68C/N69C/A113C/T134C/V291C/V294F, in which the 118 th position of SpAmDH gene is mutated by C for T, the 120 th position is mutated by G for T, the 202 nd position is mutated by A for T, the 204 nd position is mutated by T, the 206 nd position is mutated by A for G, the 207 th position is mutated by T, the 338 th position is mutated by C for T, the 401 th position is mutated by C for T, the 206 th position by A for G, the B is mutated by C for T, the 401 th position is mutated by C, and the third position is mutated by C, The mutant SpAmDH gene was designated as SpAmDH-L40F/S68C/N69C/A113C/T134C/V291C/V294F gene, wherein the G at position 871 is mutated to T, the T at position 872 is mutated to G, the G at position 873 is mutated to T, and the G at position 880 is mutated to T;
respectively marking a strain expressing SpAmDH-S68C/I111F/V294C and a recombinant vector as BL21(DE3)/pET24a-SpAmDH-S68C/I111F/V294C and pET24a-SpAmDH-S68C/I111F/V294C, wherein the 202 th site of the SpAmDH gene is subjected to mutation of A to obtain T, the 204 th site is subjected to mutation of T, the 331 th site is subjected to mutation of A to obtain T, the 880 th site is subjected to mutation of G to obtain T, the 881 th site is subjected to mutation of T to obtain G, and the 882 th site is subjected to mutation of T to obtain C, and marking the mutated SpAmDH-S68C/I111F/V294C gene;
the strain and the recombinant vector for expressing SpAmDH-I111F/S288C/V294C are respectively marked as BL21(DE3)/pET24a-SpAmDH-I111F/S288C/V294C and pET24a-SpAmDH-I111F/S288C/V294C, the No. 331 of the SpAmDH gene in the strain and the vector is subjected to A mutation to obtain T, the No. 862 of the SpAmDH gene is subjected to A mutation to obtain T, the No. 864 of the SpAmDH gene is subjected to T mutation to obtain T, the No. 880 of the SpAmDH gene is subjected to T mutation to G, the No. 882 of the T mutation to obtain C, and the mutated SpAmDH-I111F/S288C/V294C gene.
Example 4 catalysis of 4-hydroxy-2-butanone to (R) -3-amino-1-butanol by amine dehydrogenase SpAmDH or mutants thereof
Whole-cell catalysis of 4-hydroxy-2-butanone to produce (R) -3-amino-1-butanol using the amine dehydrogenase SpAmDH prepared in example 2 or a mutant thereof was performed using BL21(DE3)/pET24a as a control.
The reaction system for catalyzing the reaction of SpAmDH or the mutant thereof is obtained by adding the following substances to 1mol/L ammonium chloride/ammonia buffer solution (pH 8.5): substrate 4-hydroxy-2-butanone 20mmol/L, amine dehydrogenase SpAmDH or its mutant whole cell 100g/L, NAD+(in the form of an aqueous oxidized coenzyme I solution) 1mmol/L, crude GDH powder of example 32 g/L, glucose 100mmol/L, lysozyme 1g/L, DNase I (deoxyribonuclease) 6U/mL.
The reaction system for the catalytic reaction of SpAmDH or a mutant thereof was reacted at 30 ℃ for 12 hours. After the reaction was completed, the reaction solution was boiled for 5 minutes, centrifuged at 12,000rpm for 10 minutes, and the precipitate was removed, and the supernatant was retained, derivatized with o-phthalaldehyde (same as in example 3), followed by HPLC detection, and the procedure of HPLC gradient elution is the same as in Table 3.
The HPLC test results are shown in Table 5. Conversion rate ═ A1/A2×100％；A1: (R) -3-amino-1-butanol peak area value obtained by liquid chromatography; a. the2: and (3) analyzing the peak area value of the obtained standard product (R) -3-amino-1-butanol by liquid chromatography.
No (R) -3-amino-1-butanol was produced in the reaction product of BL21(DE3)/pET24a, and the HPLC test result of the reaction of catalyzing and reducing 4-hydroxy-2-butanone to produce (R) -3-amino-1-butanol with amine dehydrogenase SpAmDH or its mutant is shown in FIG. 3, C: liquid chromatography results of a racemic 3-amino-1-butanol standard substance; d: the sample (R) -3-amino-1-butanol was subjected to liquid chromatography. The result shows that the amine dehydrogenase SpAmDH or the mutant thereof can asymmetrically reduce and catalyze 4-hydroxy-2-butanone to generate (R) -3-amino-1-butanol, the conversion rate is 14-29%, and the stereoselectivity is more than 99% (R).
Table 5 shows the results of detecting the amine dehydrogenase SpAmDH and its mutant catalyzing 4-hydroxy-2-butanone
Amine dehydrogenase SpAmDH and mutant thereof Conversion (%) ee(％)
SpAmDH 23 >99(R)
SpAmDH-I111F 14 >99(R)
SpAmDH-V291C 17 >99(R)
SpAmDH-I111F/V294C 18 >99(R)
SpAmDH-E114V/V291C 20 >99(R)
SpAmDH-I111F/E114V/V294C 29 >99(R)
Example 5 catalysis of 1-hydroxy-2-butanone to (S) -2-amino-1-butanol by amine dehydrogenase SpAmDH or mutants thereof
The enzyme activity is detected according to the absorption value of coenzyme NADH at 340nm, and the molar absorption coefficient epsilon 340 is 6.22x 103(M- 1cm-1). The enzyme activity unit is defined as: conversion in one minuteThe amount of enzyme required to produce or consume 1. mu. mol/L NADH. The enzyme activity calculation formula is as follows: enzyme activity (U) ═ EW × V × 103(6220X 0.3) wherein EW represents a change in absorbance at 340nm per minute, V represents a volume of the reaction solution in mL, 6220 is a molar extinction coefficient of coenzyme, L/(mol X cm), and 0.3 is an optical path length in cm.
The reaction system used was obtained by adding the following amounts of substances to 1mol/L ammonium chloride/aqueous ammonia buffer (ammonium chloride and aqueous ammonia in equimolar ratio, pH 8.5): 0.2mmol/L NADH, 1-30 mmol/L1-hydroxy-2-butanone, the right amount of the pure enzyme solution of example 2. Reacting at 30 ℃, measuring the change of the light absorption value of NADH within 5min, measuring the initial reaction rate under different substrate concentrations, and then carrying out nonlinear fitting on the Mie equation by using Origin.
The results are shown in Table 6. The results show that the amine dehydrogenase SpAmDH or the mutant thereof catalyzes K of 1-hydroxy-2-butanonemThe value is 9.45-21.5mM, kcatThe value is 0.83-6.07s-1，kcat/KmThe value is 0.088-0.346s-1mM-1。
TABLE 6 Michaelis constants of amine dehydrogenase SpAmDH and its mutants
Amine dehydrogenase SpAmDH and mutant thereof Km(mM) kcat(s-1) kcat/Km(s-1mM-1)
SpAmDH 9.45±1.44 0.83±0.05 0.088
SpAmDH-I111F 18.52±1.42 1.74±0.07 0.094
SpAmDH-V291C 10.01±0.06 1.22±0.03 0.122
SpAmDH-I111F/V294C 21.5±3.37 3.14±0.27 0.146
SpAmDH-E114V/V291C 10.74±1.04 1.60±0.11 0.149
SpAmDH-I111F/E114V/V294C 17.53±1.11 6.07±0.19 0.346
Example 6 catalysis of Hydroxyketone substrates by amine dehydrogenase SpAmDH or mutants thereof
The amine dehydrogenase SpAmDH or its mutant prepared in example 2 (SpAmDH-E114V/V291C, SpAmDH-I111F/E114V/V294C) was used to catalyze hydroxy ketone substrates 1-hydroxy-2-butanone (1a), 4-hydroxy-2-butanone (1b), acetol (1c), 2-hydroxyacetophenone (1d), 1- (4-fluorophenyl) -2-hydroxy-1-ethanone (1E), the corresponding products are (S) -2-amino-1-butanol (2a), (R) -3-amino-1-butanol (2b), (S) -alaninol (2c), (S) -phenylglycinol (2d), and (S) -2-amino-2- (4-fluorophenyl) -1-ethanol (2e), respectively.
The reaction system for the above catalytic reaction was obtained by adding the following amounts of substances to 1mol/L ammonium chloride/aqueous ammonia buffer (pH 8.5): 100g/L, NAD of whole cells of substrate 20mmol/L, amine dehydrogenase SpAmDH or mutant thereof (SpAmDH-E114V/V291C, SpAmDH-I111F/E114V/V294C)+(in the form of an aqueous oxidized coenzyme I solution) 1mmol/L, 2g/L crude GDH powder of example 3, 100mmol/L glucose, 1g/L lysozyme, 6U/mL DNase I.
The reaction was carried out under the conditions of 30 ℃ for 12 hours.
Detecting the products generated by the catalytic substrates 1-hydroxy-2-butanone (1a), 4-hydroxy-2-butanone (1b) and acetol (1c) and deriving the products by using o-phthalaldehyde to perform HPLC detection reaction, detecting the products generated by the catalytic substrates 2-hydroxyacetophenone (1d) and 1- (4-fluorophenyl) -2-hydroxy-1-ethanone (1e) and deriving the products by using a Marfey's reagent (deriving method, taking 50 mu L of the reaction solution, 50 mu L of the ammonium chloride/ammonia buffer solution (pH 8.5) +500 mu L of acetonitrile, then filtering, taking 20 mu L of the filtered mixture, 20 mu L of the Marfey's reagent (14mmol/L, dissolved in acetonitrile) +36 mu L of NaHCO3(1mol/L) + 100. mu.L DMSO was mixed well, and after reaction at 40 ℃ and 1,000rpm for 2 hours, 40. mu.L HCl (1mol/L) was added thereto to terminate the reaction, and HPLC detection conditions were as shown in Table 7.
Table 7 HPLC detection methods
Figure BDA0002524285170000271
Note: in the context of Table 7, the following examples are,aHPLC detection conditions: agilent SB-Aq C18 column (4.6 x 250mm,5 μm), detection wavelength 334nm, column temperature: 35 ℃, flow rate: 1mL/min, loading amount: 10 mu L of the solution; mobile phase A: methanol, mobile phase B: 0.05mol/L sodium acetate;
bHPLC detection conditions: zorbax SB-C18 column (4.6 x 150mm, 5 μm), detection wavelength 340nm, column temperature: 25 ℃, flow rate: 0.45mL/min, loading: 10 mu L; mobile phase A: methanol containing 0.1% (v/v) trifluoroacetic acid, mobile phase B: double distilled water containing 0.1% (v/v) trifluoroacetic acid;
Athe standard substance is mixed rotary 2-amino-1-butanol, Maya reagent, the cargo number: MAYA-CR-3330;
Bthe standard substance is racemic 3-amino-1-butanol, Shanghai Shaoshao Yuan reagent, the product number: SY 030187;
Cthe standard substance is dl-alaninol, Shanghai Bide pharmaceutical science and technology Limited, Cat number: BD 70946;
Dthe standard substance is a DL-benzyl glycinol Shanghai Shao Yuan reagent with the following product number: SY 002083;
Ethe standard substance is racemic 2-amino-2- (4-fluorophenyl) -1-ethanol, Shanghai Bide pharmaceutical science and technology Limited, Cat number: BD 177526.
The detection results are shown in Table 8 and FIG. 4, and it can be seen that the amine dehydrogenase SpAmDH mutant (SpAmDH-I111F/E114V/V294C) has good catalytic activity and enantioselectivity for 1-hydroxy-2-butanone as a substrate, and also has good catalytic activity for other hydroxyketone substrates. WT: SpAmDH; m1: mutant SpAmDH-E114V/V291C; m2: mutant SpAmDH-I111F/E114V/V294C.
Table 8 shows the results of the detection of the substrate for hydroxyketone catalyzed by the amine dehydrogenase SpAmDH and its mutants
Figure BDA0002524285170000281
Note: the conversion and ee in Table 8 are, from top to bottom, the conversion and stereoselectivity of SpAmDH, SpAmDH-E114V/V291C or SpAmDH-I111F/E114V/V294C catalytic hydroxy ketone substrates 1-hydroxy-2-butanone (1a), 4-hydroxy-2-butanone (1b), acetol (1c), 2-hydroxyacetophenone (1d), 1- (4-fluorophenyl) -2-hydroxy-1-ethanone (1E), respectively.
Example 7 amine dehydrogenase SpAmDH or mutant thereof catalyzing amplification reaction of 1-hydroxy-2-butanone to (S) -2-amino-1-butanol
The amine dehydrogenase SpAmDH or its mutant (SpAmDH-E114V/V291C, SpAmDH-I111F/E114V/V294C) prepared in example 2 is used for catalyzing the amplification reaction of 1-hydroxy-2-butanone to generate (S) -2-amino-1-butanol, and the concentration of the substrate 1-hydroxy-2-butanone is increased to 200 mmol/L.
The reaction system was obtained by adding the following amounts of substances to 1mol/L ammonium chloride/aqueous ammonia buffer (pH 8.5): substrate 1-hydroxy-2-butanone 200mmol/L, whole cell 100g/L, NAD+(in the form of an aqueous oxidized coenzyme I solution) 1mmol/L, crude GDH powder of example 32 g/L, glucose 100mmol/L, lysozyme 1g/L, DNase I (deoxyribonuclease) 6U/mL.
The asymmetric reduction reaction conditions are 30 ℃ for 0.5h, 1h, 2h, 4h, 8h, 12h, 18h and 24 h.
After the reaction, the reaction solution was boiled for 5 minutes, centrifuged at 12,000rpm for 10 minutes, and the precipitate was removed, the supernatant was retained, and the reaction was detected by HPLC after derivatization with o-phthalaldehyde.
As shown in FIG. 5, it can be seen that the conversion rate of the substrate 1-hydroxy-2-butanone (200mmol/L) catalyzed by the amine dehydrogenase SpAmDH mutant (SpAmDH-I111F/E114V/V294C) is up to 91%, and the stereoselectivity is greater than 99% (S).
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound
<160> 3
<170> PatentIn version 3.5
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Ile Cys Gln Asp Lys Ala Ser Gly Leu Lys Ala Ile Ile Ala Ile His
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Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr
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Ala Ser Glu Glu Glu Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
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Met Thr Tyr Ser Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
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Thr Val Ile Ile Gly Asn Pro Lys Thr Asp Lys Asn Asp Glu Met Phe
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Arg Ala Phe Gly Arg Tyr Ile Glu Gly Leu Asn Gly Arg Tyr Ile Thr
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Ala Glu Asp Val Gly Thr Thr Glu Ala Asp Met Asp Leu Ile Asn Leu
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Glu Thr Asp Tyr Val Thr Gly Thr Ser Ala Gly Ala Gly Ser Ser Gly
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Asn Pro Ser Pro Val Thr Ala Tyr Gly Ile Tyr Tyr Gly Met Lys Ala
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Ala Ala Lys Glu Ala Phe Gly Asp Asp Ser Leu Ala Gly Lys Thr Val
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Ala Val Gln Gly Val Gly Asn Val Ala Tyr Ala Leu Cys Glu Tyr Leu
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His Glu Glu Gly Ala Lys Leu Ile Ile Thr Asp Ile Asn Glu Glu Ala
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Val Gln Arg Ala Val Asp Ala Phe Gly Ala Thr Ala Val Gly Ile Asn
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Glu Ile Tyr Ser Gln Glu Ala Asp Ile Phe Ala Pro Cys Ala Leu Gly
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Ala Ile Ile Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile
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Ala Gly Ser Ala Leu Asn Gln Leu Lys Glu Thr Arg His Gly Asp Leu
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Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ser
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Gly Gly Val Ile Asn Val Ala Asp Glu Leu Asp Gly Tyr Asn Arg Glu
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aaggcgatgg ccattcgctt cggcaaggag caggcaaaag tggttatcaa ctattatagt 120
aataaacaag atccgaacga ggtaaaagaa gaggtcatca aggcgggcgg tgaagctgtt 180
gtcgtccaag gagatgtcac gaaagaggaa gatgtaaaaa atatcgtgca aacggcaatt 240
aaggagttcg gcacactcga tattatgatt aataatgccg gtcttgaaaa tcctgtgcca 300
tctcacgaaa tgccgctcaa ggattgggat aaagtcatcg gcacgaactt aacgggtgcc 360
tttttaggaa gccgtgaagc gattaaatat ttcgtagaaa acgatatcaa gggaaatgtc 420
attaacatgt ccagtgtgca cgaagtgatt ccttggccat tatttgtcca ctatgcggca 480
agtaaaggcg ggatgaagct gatgacagaa acattagcgt tggaatacgc gccgaagggc 540
attcgcgtca ataatattgg gccaggtgcg atcaacacga cgatcaataa ggagaaattt 600
gctgaccctg aacagagagc tgatgtagaa agcatgattc caatgggata tatcggcgaa 660
ccggaggaga tcgccgcagt agcagcctgg cttgcttcga aggaagccag ctacgtcaca 720
ggcatcacgt tattcgcgga cggcggtatg acacaatatc cttcattcca ggcaggccgc 780
ggttaa 786

Claims (9)

1. A method of preparing a chiral aminoalcohol compound, comprising: carrying out catalytic reaction by using hydroxyketone as a substrate and SpAmDH or mutant protein thereof to obtain a chiral amino alcohol compound; the hydroxy ketone is 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, acetol, 2-hydroxyacetophenone or 1- (4-fluorophenyl) -2-hydroxy-1-ethanone;
the amino acid sequence of the SpAmDH is SEQ ID No.2 in the sequence table; the SpAmDH mutant protein is prepared by carrying out L40V, L61A, L61V, L61C, S68C, N69S, I111F, I111W, A113C, E114V, E114Q, V116I, V116M, V116S, V116Y, T134A, T134F, T134G, T150C, A187K, A187N, A187P, A187T, L239F and L239M on SpAmDH, V291C, V291L, I292M, I292T, A295N, A295T, A295W, S288M, E297N, N69C/V291C, S68C/V291C, L40C/I111F, I111F/V294C, E114V/V291C, L40C/I111F/T134F, S68C/N69C/V291C, I111F/A113C/T134C, L40C/I111F/T134F/V294C, S68C/N69C/E114C/V291C, S68C/N69/I C/V36291, I111/C/V C, I111/E72/V C/V36294, S68/N72/N C/V36294, S72/S C/N C/V36294 and S72/V C/V36294.
2. The method of claim 1, wherein: the chiral amine alcohol compound is 2-amino-1-butanol, 3-amino-1-butanol, alaninol, phenethylamine alcohol or 2-amino-2- (4-fluorophenyl) -1-ethanol.
3. The method according to claim 1 or 2, characterized in that: the chiral amine alcohol compound is (S) -2-amino-1-butanol, (R) -3-amino-1-butanol, (S) -alaninol, (S) -phenglycinol or (S) -2-amino-2- (4-fluorophenyl) -1-ethanol.
4. A method of preparing a chiral aminoalcohol compound, comprising: carrying out a catalytic reaction by using a recombinant cell expressing SpAmDH or SpAmDH mutant protein in claim 1 or a lysate of the recombinant cell by using hydroxyketone as a substrate to obtain a chiral amino alcohol compound; the hydroxy ketone is 1-hydroxy-2-butanone, 4-hydroxy-2-butanone, acetol, 2-hydroxyacetophenone or 1- (4-fluorophenyl) -2-hydroxy-1-ethanone.
5. The method of claim 4, wherein: the recombinant cell is obtained by introducing a recombinant vector capable of expressing SpAmDH or a mutant protein of SpAmDH according to claim 1 into a biological cell.
6. The SpAmDH mutein of any of claim 1.
7. A biological material related to spomdh or a spomdh mutein of claim 1, being any one of the following B1) to B4):
B1) a nucleic acid molecule encoding a spomdh or a spomdh mutein of claim 1;
B2) an expression cassette comprising the nucleic acid molecule of B1);
B3) a recombinant vector comprising the nucleic acid molecule of B1);
B4) a recombinant microorganism comprising the nucleic acid molecule of B1).
8. Any of the following products:
m1, a kit consisting of 1-hydroxy-2-butanone and SpAmDH;
m2, a kit consisting of 1-hydroxy-2-butanone and the SpAmDH mutein of claim 1;
m3, a kit consisting of 1-hydroxy-2-butanone and the biomaterial of claim 7;
m4, a kit consisting of 4-hydroxy-2-butanone and SpAmDH;
m5, a kit consisting of 4-hydroxy-2-butanone and the SpAmDH mutein of claim 1;
m6, a kit consisting of 4-hydroxy-2-butanone and the biomaterial of claim 7;
m7, a kit consisting of acetol and SpAmDH;
m8, a kit consisting of acetol and the SpAmDH mutein of claim 1;
m9, a kit consisting of acetol and the biomaterial of claim 7;
m10, a kit consisting of 2-hydroxyacetophenone and SpAmDH;
m11, a kit consisting of 2-hydroxyacetophenone and the spomdh mutein of claim 1;
m12, a kit consisting of 2-hydroxyacetophenone and the biomaterial of claim 7;
m13, a kit consisting of 1- (4-fluorophenyl) -2-hydroxy-1-ethanone and SpAmDH;
m14, a kit consisting of 1- (4-fluorophenyl) -2-hydroxy-1-ethanone and the spomdh mutein of claim 1;
m15, a kit consisting of 1- (4-fluorophenyl) -2-hydroxy-1-ethanone and a biomaterial as claimed in claim 7.
Use of any of SpAmDH, a SpAmDH mutein of claim 1, a biomaterial of claim 7 or a product of claim 8 in any of the following applications:
z1, in the preparation of chiral amino alcohol compounds;
z2, in the preparation of chiral amino alcohol compound products;
z3, in catalyzing hydroxy ketone to generate chiral amino alcohol compound;
z4, and the application in preparing products for catalyzing hydroxy ketone to generate chiral amino alcohol compounds.
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