CN118421581A - Imine reductase and application thereof in synthesis of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline - Google Patents
Imine reductase and application thereof in synthesis of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline Download PDFInfo
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- 150000002466 imines Chemical class 0.000 title claims abstract description 80
- QPILYVQSKNWRDD-QMMMGPOBSA-N (1s)-1-methyl-1,2,3,4-tetrahydroisoquinoline Chemical compound C1=CC=C2[C@H](C)NCCC2=C1 QPILYVQSKNWRDD-QMMMGPOBSA-N 0.000 title claims abstract description 26
- 230000015572 biosynthetic process Effects 0.000 title claims description 4
- 238000003786 synthesis reaction Methods 0.000 title claims description 4
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 51
- 230000035772 mutation Effects 0.000 claims abstract description 32
- JZZLDIIDMFCOGF-UHFFFAOYSA-N 1-methyl-3,4-dihydroisoquinoline Chemical compound C1=CC=C2C(C)=NCCC2=C1 JZZLDIIDMFCOGF-UHFFFAOYSA-N 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 108090000854 Oxidoreductases Proteins 0.000 claims description 74
- 239000002773 nucleotide Substances 0.000 claims description 58
- 125000003729 nucleotide group Chemical group 0.000 claims description 58
- 239000013604 expression vector Substances 0.000 claims description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 16
- 102000008300 Mutant Proteins Human genes 0.000 claims description 16
- 108010021466 Mutant Proteins Proteins 0.000 claims description 16
- 241000894006 Bacteria Species 0.000 claims description 15
- 239000004280 Sodium formate Substances 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 9
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims description 9
- 235000019254 sodium formate Nutrition 0.000 claims description 9
- 108090000698 Formate Dehydrogenases Proteins 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000010276 construction Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims 2
- 108010048581 Lysine decarboxylase Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 29
- 108090000790 Enzymes Proteins 0.000 abstract description 29
- 238000006243 chemical reaction Methods 0.000 abstract description 26
- 230000000694 effects Effects 0.000 abstract description 24
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- 102000004169 proteins and genes Human genes 0.000 description 25
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- 238000002474 experimental method Methods 0.000 description 9
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 150000001412 amines Chemical class 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
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- 238000003259 recombinant expression Methods 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- -1 cyclic imine Chemical class 0.000 description 5
- QPILYVQSKNWRDD-UHFFFAOYSA-N 1-methyl-1,2,3,4-tetrahydroisoquinoline Chemical compound C1=CC=C2C(C)NCCC2=C1 QPILYVQSKNWRDD-UHFFFAOYSA-N 0.000 description 4
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 4
- 108091007187 Reductases Proteins 0.000 description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 150000002576 ketones Chemical class 0.000 description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000012460 protein solution Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
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- 238000001514 detection method Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000006268 reductive amination reaction Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102100040438 Epithelial cell-transforming sequence 2 oncogene-like Human genes 0.000 description 2
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 2
- 101000817241 Homo sapiens Epithelial cell-transforming sequence 2 oncogene-like Proteins 0.000 description 2
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000011914 asymmetric synthesis Methods 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 238000009010 Bradford assay Methods 0.000 description 1
- GSNUFIFRDBKVIE-UHFFFAOYSA-N DMF Natural products CC1=CC=C(C)O1 GSNUFIFRDBKVIE-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- 241001495113 Thermostaphylospora chromogena Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000001195 anabolic effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000007036 catalytic synthesis reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000033116 oxidation-reduction process Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 150000003141 primary amines Chemical class 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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)
- C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
- C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C12Y—ENZYMES
- C12Y105/00—Oxidoreductases acting on the CH-NH group of donors (1.5)
- C12Y105/01—Oxidoreductases acting on the CH-NH group of donors (1.5) with NAD+ or NADP+ as acceptor (1.5.1)
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Abstract
The invention provides imine reductase, the amino acid sequence of which is obtained by mutating the sequence shown in a sequence table SEQ ID No.1, wherein the mutation is at least one of the following (A) - (D): (a) mutation of amino acid residue 127 from E to P; (B) mutating the amino acid residue at position 177 from L to M; (C) mutation of amino acid residue 221 from E to a; (D) mutation of amino acid residue 236 from S to A. The invention also provides application of the iminoreductase in synthesizing (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline. The imine reductase provided by the invention has higher specific enzyme activity, higher thermal stability and larger pH optimum range, and has higher conversion rate and enantiomeric excess percentage when being used for catalyzing 1-methyl-3, 4-dihydro-isoquinoline to prepare (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline, thus having very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of protein modification, and particularly relates to imine reductase and application thereof in synthesis of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
Background
Imine reductase (ImineReductase, IRED, EC1.5.1.48) is a kind of oxidation-reduction enzyme which depends on coenzyme NADPH, can catalyze prochiral imine to be asymmetrically reduced into corresponding chiral amine, and has the unique advantages of high stereoselectivity and mild reaction conditions, wherein the substrate recognition range of S-type imine reductase is wider than that of R-type imine reductase. IRED can catalyze the reduction hydrogenation of cyclic imine to generate corresponding chiral amine, and can also use ketone and ammonia as substrates to synthesize acyclic imine and perform asymmetric reductive amination reaction.
Imine refers to an organic compound containing a carbon-nitrogen double bond (c=n bond); in vivo imines are often present in the metabolic process of nitrogen containing compounds, which are reduced to amine compounds for anabolic products by the action of a reductase. Although the imine reduction process is well known, the low substrate versatility of the reductases limits the expansion of the use of these enzymes for other imine substrates. Meanwhile, the imine is extremely unstable in water, and a great challenge is brought to IRED screening work.
The first report in 2011 that imine reductase is capable of asymmetrically reducing cyclic imines to form chiral amines; imine reductases were first reported in 2014 for asymmetric reductive amination of ketones with amines. In recent years, a plurality of subject groups begin to research and utilize imine reductase to catalyze asymmetric reductive amination of ketone and amine, the route starts from simple and easily available raw material ketone and cheap amine donor, and a plurality of chiral primary amine, secondary amine and even tertiary amine compounds with high added value can be synthesized by only one-step reaction, so that the method has important application prospect in the field of asymmetric synthesis of chiral amine.
Although a plurality of imine reductases have been developed, there are many problems that require intensive studies, such as a low variety of high-activity ketoaminated reductases, an insufficient source, a further expansion of the application range, etc.
Disclosure of Invention
In order to solve the problems existing in the prior art, the invention provides imine reductase for catalyzing 1-methyl-3, 4-dihydroisoquinoline to prepare (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline, and improving the conversion rate and the enantiomeric excess percentage.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the first object of the present invention is to provide an imine reductase having an amino acid sequence obtained by mutating a sequence shown in SEQ ID No.1 of the sequence Listing, wherein the mutation is at least one of the following (A) - (D):
(A) Mutating the 127 th amino acid residue from E to P;
(B) Mutation of amino acid residue 177 from L to M;
(C) Mutating the 221 th amino acid residue from E to A;
(D) The 236 th amino acid residue is mutated from S to A.
Preferably, the mutation is any one of the following (1) to (8):
(1) Mutating 127 th amino acid residue from E to P, 221 th amino acid residue from E to A, and 236 th amino acid residue from S to A;
(2) Mutating the 127 th amino acid residue from E to P;
(3) Mutation of amino acid residue 177 from L to M;
(4) Mutating the 221 th amino acid residue from E to A;
(5) Mutation of amino acid residue 236 from S to A;
(6) Mutating 127 th amino acid residue from E to P, and mutating 221 th amino acid residue from E to A;
(7) Mutating 127 th amino acid residue from E to P, 177 th amino acid residue from L to M, and 221 th amino acid residue from E to A;
(8) The 127 th amino acid residue is mutated from E to P, the 177 th amino acid residue is mutated from L to M, the 221 th amino acid residue is mutated from E to A, and the 236 th amino acid residue is mutated from S to A;
preferably, the mutation is: the 127 th amino acid residue is mutated from E to P, the 221 th amino acid residue is mutated from E to A, and the 236 th amino acid residue is mutated from S to A.
A second object of the present invention is to provide a nucleotide sequence encoding the above imine reductase.
Preferably, the nucleotide sequence is obtained by carrying out the following mutation on the sequence shown in the sequence table SEQ ID No.2, wherein the mutation is any one of the following (1) - (8):
(1) The 379-381 nucleotide is mutated from gaa to ccg, the 662-663 nucleotide is mutated from aa to cg, and the 706-708 nucleotide is mutated from agc to gcg;
(2) The 379-381 nucleotide is changed from gaa to ccg;
(3) Mutation of nucleotide 529 from c to a;
(4) The 662-663 nucleotide is changed from aa to cg;
(5) The 706-708 nucleotide is mutated from agc to gcg;
(6) Nucleotides 379-381 are mutated from gaa to ccg, and nucleotides 662-663 are mutated from aa to cg;
(7) Nucleotides 379-381 are mutated from gaa to ccg, nucleotide 529 is mutated from c to a, and nucleotides 662-663 are mutated from aa to cg;
(8) The 379-381 nucleotide is mutated from gaa to ccg, the 529 nucleotide is mutated from c to a, the 662-663 nucleotide is mutated from aa to cg, and the 706-708 nucleotide is mutated from agc to gcg;
Preferably, the mutation is: nucleotides 379-381 are mutated from gaa to ccg, nucleotides 662-663 are mutated from aa to cg, and nucleotides 706-708 are mutated from agc to gcg.
It is a third object of the present invention to provide an expression vector comprising the above nucleotide sequence;
preferably, the expression vector is pET28a.
A fourth object of the present invention is to provide a cell comprising the above expression vector;
Preferably, the cells include bacteria and fungi;
further preferably, the bacterium is Escherichia coli.
The fifth object of the present invention is to provide a preparation method of imine reductase mutant protein, constructing the above nucleotide sequence on an expression vector, introducing the expression vector into cells, culturing the cells, crushing, centrifuging, and collecting supernatant to obtain imine reductase mutant protein;
preferably, the restriction sites selected for construction on the expression vector are NdeI and EcoRI.
A sixth object of the present invention is to provide the use of the above-mentioned imine reductase, nucleotide sequence, expression vector or cell for synthesizing (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
A seventh object of the present invention is to provide a method for producing (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline by adding 1 to 3% by volume of dimethyl sulfoxide (DMSO), 20 to 100mM 1-methyl-3, 4-dihydroisoquinoline, 400 to 500U/L of imine reductase mutant protein, 640 to 1000U/L of Formate Dehydrogenase (FDH), 0.5 to 0.7mMNADP + and 0.6 to 0.8mol/L of sodium formate at a temperature of 30 to 40℃and a pH of 7.0 to 8.0, and reacting for 20 to 28 hours to obtain (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline; the imine reductase mutant protein is prepared by the method of claim 7;
Preferably, the nucleotide sequence of the imine reductase mutant protein is: the following mutations were performed on the sequence shown in SEQ ID No.2 of the sequence Listing:
Nucleotides 379-381 are mutated from gaa to ccg, nucleotides 662-663 are mutated from aa to cg, and nucleotides 706-708 are mutated from agc to gcg.
Preferably, 2% DMSO, 100mM 1-methyl-3, 4-dihydroisoquinoline, 400U/L imine reductase mutant protein, 640U/LFDH, 0.5mMNADP + and 0.6mol/L sodium formate are added in volume percent at 35℃and pH 7.5, and reacted for 24 hours to obtain (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
The imine reductase provided by the invention has higher specific enzyme activity, higher thermal stability and larger optimal pH range. When the catalyst is used for catalyzing 1-methyl-3, 4-dihydro isoquinoline to prepare (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline, the conversion rate and the enantiomeric excess percentage are higher, and the catalyst has very broad application prospect.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a graph showing the standard curve of the protein for IRED test in example 3 of the present invention.
FIG. 2 shows the results of the reaction temperature optimization experiment of the imine reductase mutant 7 protein.
FIG. 3 shows the results of pH optimization experiments of the imine reductase mutant 7 protein.
FIG. 4 is a graph showing the progress of the imine reductase of the present invention in catalyzing the preparation of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline by 1-methyl-3, 4-dihydroisoquinoline (before optimization of various parameters).
FIG. 5 shows the results of the reaction temperature optimization experiment of the imine reductase mutant 7 protein.
FIG. 6 shows the results of pH optimization experiments of the imine reductase mutant 7 protein.
FIG. 7 shows the results of an organic solvent type optimization experiment for the imine reductase mutant 7 protein.
FIG. 8 shows the results of an organic solvent concentration optimization experiment for the imine reductase mutant 7 protein.
FIG. 9 shows the results of an experiment for optimizing the substrate concentration of the imine reductase mutant 7 protein.
FIG. 10 shows the results of the experiment for optimizing the enzyme activity of the imine reductase mutant 7 protein.
FIG. 11 shows the results of an experiment for optimizing the enzyme activity addition ratio of the imine reductase mutant 7 protein.
FIG. 12 shows the results of an NADP + concentration-optimized experiment for the imine reductase mutant 7 protein.
FIG. 13 shows the results of sodium formate concentration optimization experiments of the imine reductase mutant 7 protein.
FIG. 14 is a graph showing the progress of the imine reductase of the present invention in catalyzing the preparation of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline by 1-methyl-3, 4-dihydroisoquinoline (after optimization of various parameters).
FIG. 15 is a High Performance Liquid Chromatography (HPLC) diagram. Wherein, the A diagram is the HPLC diagram of rac-1-methyl-1, 2,3, 4-tetrahydroisoquinoline, and the B diagram is the HPLC diagram of the reaction product of the invention, namely (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
The invention will be described in detail below with reference to the drawings in connection with embodiments.
The imine reductase is obtained by mutating imine reductase shown as SEQ ID NO.1, wherein the mutation comprises at least one mutation of 127 th, 177 th, 221 th and 236 th amino acids of SEQ ID NO. 1.
In order to prepare the imine reductase, the preparation raw materials selected in the invention are as follows:
pET28a vector: product catalog number from Novagen: 69864-3.
Coli BL21 (DE 3): from Beijing full gold biotechnology Co., ltd., product number: CD601-02.
LB medium: 10g tryptone, 5g yeast extract, 5g NaCl per liter, and deionized water to a volume of 1L. Steam sterilization under high pressure for 20min.
TB medium: each liter of 12g tryptone, 24g yeast extract, 4mL of 87% glycerol, 100mL of phosphate buffer solution (1 mol/L, pH 6.5), and deionized water was used to determine the volume to 1L. Steam sterilization under high pressure for 20min.
Example 1 selection determination of mutation sites
The sequence of natural imine reductase protein (shown as SEQ ID NO.1, and the coding gene (IRED gene) is shown as SEQ ID NO. 2) from Thermostaphylosporachromogena is analyzed, and the sequence conservation is primarily analyzed by comparing the amino acid sequences of imine reductase from different sources. Amino acid residues in the active domain are obtained by superposition and analysis of the protein structure. The amino acid sequences of imine reductase reported in the literature are collected, the mutation of active center amino acid is analyzed through site alignment, the distribution of each amino acid site in sequence homology is known, and proper substituent groups are selected for mutation sites. Through analysis of the structure, 4 important amino acid sites were screened out. The 4 amino acid loci are subjected to different forms of mutation to obtain mutant proteins. The 4 amino acid positions and their mutated forms are shown in Table 1.
TABLE 18 mutant forms of imine reductase
The amino acid sequence of the natural imine reductase is as follows (sequence table SEQ ID NO. 1):
the coding gene of the natural imine reductase is as follows (sequence table SEQ ID NO. 2):
EXAMPLE 2 preparation of recombinant bacteria
1. Construction of wild-type recombinant expression vector
The nucleotide sequence shown in SEQ ID NO.1 is inserted between NdeI and EcoRI restriction sites of the pET28a vector to obtain a recombinant expression vector pET28a-IRED, and sequencing is verified to be correct.
2. Construction of mutant recombinant expression vectors
The nucleotide sequences of the coding mutants a1-a8 are respectively inserted between NdeI and EcoRI restriction sites of the pET28a vector to obtain a recombinant expression vector pET28a-1-8, and sequencing is verified to be correct.
3. Preparation of recombinant bacteria
1. And (3) converting the recombinant expression vector pET28a-IRED obtained in the step one into escherichia coli BL21 (DE 3) to obtain wild-type recombinant bacteria.
2. And (3) respectively converting the recombinant expression vectors pET28a-1-8 prepared in the step two into escherichia coli BL21 (DE 3) to obtain mutant recombinant bacteria, wherein the mutant recombinant bacteria are sequentially numbered as mutant recombinant bacteria 1-8.
Example 3 determination of enzyme Activity of imine reductase mutant protein
The wild-type recombinant bacteria and mutant recombinant bacteria 1-8 obtained in example 2 were cultured, proteins were extracted, and the enzyme activity was detected.
1. 10. Mu.L of the cells were inoculated into a liquid LB medium containing 50. Mu.g/mL kanamycin and 10mL, and subjected to activation culture at 37℃for 12-16 hours in a shaker at 180rpm to obtain a seed solution. Inoculating 1% seed solution into 50mL TB liquid culture medium containing 50 μg/mL kanamycin, fermenting at 37deg.C in 180rpm shaker for 2.5-3 hr, adding inducer IPTG with final concentration of 0.1mM when the bacterial solution concentration OD 600 is 0.6-0.8, inducing expression at 20deg.C in 180rpm shaker for 20-24 hr, and stopping fermentation. Centrifuging the fermented bacterial liquid at 8000rpm for 10min, discarding supernatant, and collecting precipitate.
The recombinant bacterial cells were suspended in 50mLPBS buffer (100 mM, pH 7.0) and crushed with a high-pressure crusher at 700-900bar for 15s and cycled 3 times. And (3) centrifuging again to obtain intracellular supernatant and precipitate, and collecting the supernatant to obtain crude enzyme liquid.
2. The protein solution obtained by the above steps of the wild-type recombinant bacterium is named as a wild-type protein solution. The protein solutions obtained by the steps of the mutant recombinant bacteria 1-8 are sequentially named as mutant 1-mutant 8 protein solutions. Protein concentration was measured by Bradford method.
The specific enzyme activity calculation formula:
Wherein, U: enzyme activity; g: protein content.
3. The enzyme activity unit U is defined as: the amount of IRED required to catalyze the oxidation of 1 μ MNADPH to NADP + per minute at 30℃and pH8.0 is defined as 1U. This example shows the decrease in absorbance at 340nm for 1min in a 1mL reaction system containing 10mM of substrate 1-methyl-3, 4-dihydroisoquinoline, 0.15mM of NADPH,100mMPBS buffer (pH 7.0) and an appropriate amount of enzyme at 30 ℃. The results are shown in Table 2.
FIG. 1 is a graph showing the standard curve of the protein for IRED test in example 3 of the present invention.
TABLE 2 enzyme activity statistics
| Specific enzyme activity U/g | Specific activity (%) | |
| Wild type | 39.48±0.94 | 13.15 |
| Mutant 1 | 202.64±3.29 | 67.48 |
| Mutant 2 | 100.86±2.68 | 33.59 |
| Mutant 3 | 145.17±2.51 | 48.34 |
| Mutant 4 | 109.51±2.87 | 36.47 |
| Mutant 5 | 234.61±4.34 | 78.13 |
| Mutant 6 | 260.67±2.90 | 86.81 |
| Mutant 7 | 300.28±3.35 | 100 |
| Mutant 8 | 61.59±3.25 | 20.51 |
As can be seen from Table 2, the specific enzyme activity of mutant 7 was maximized, so that the following test was performed with mutant 7.
Example 4 optimal temperature testing of enzymatic Properties of imine reductase mutant proteins
In a 1mL reaction system containing 10mM of substrate 1-methyl-3, 4-dihydroisoquinoline, 0.15mM of NADPH,100mM of PBS buffer (pH 7.0) and an appropriate amount of enzyme solution, respectively incubating for 5min at a temperature ranging from 10 ℃ to 70 ℃, measuring enzyme activities at different temperatures, and calculating relative enzyme activities by taking the enzyme activities at the optimal temperature as a control (100%).
The test results are shown in FIG. 2.
Example 5 optimal pH determination of enzymatic Properties of imine reductase mutant proteins
The relative enzyme activities were calculated by incubating 10mM substrate 1-methyl-3, 4-dihydroisoquinoline, 0.15mM NADPH, and an appropriate amount of enzyme solution in 100mM PBS buffer solutions of different pH (4.0-12.0) at 40℃for 5min in 1mL reaction system, and measuring the enzyme activities in the different pH buffer solutions, with the enzyme activities under the optimal pH condition as a control (100%).
The test results are shown in FIG. 3.
Example 6 asymmetric Synthesis of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline
The imine reductase mutant recombinant 7 enzyme solution and FDH enzyme solution of example 2 are added into a 15mL reaction system in equal proportion, wherein 20mM 1-methyl-3, 4-dihydro isoquinoline, 0.5mMNADP + and 40mM sodium formate are contained, a solvent is 100mM PBS buffer solution (pH 7.5), the reaction is carried out for more than 12 hours in a shaking table at 30 ℃ and 400rpm, after the reaction is finished, the product is extracted three times by methyl tertiary butyl ether, and concentrated by vacuum rotary evaporation. The conversion and enantiomeric excess of the reaction were measured by HPLC at different times to give the reaction progress curves shown in FIG. 4, the reaction formula shown below:
FIG. 4 is a graph showing the progress of the imine reductase of the present invention in catalyzing the preparation of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline by 1-methyl-3, 4-dihydroisoquinoline (before optimization).
The enantiomeric excess and chemical purity of the (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline obtained by the conversion is determined by: the sample was eluted with CHIRALPAKAD-H column at 35℃at a flow rate of 1mL/min, with a mobile phase of n-hexane, ethanol, diethylamine (volume ratio 97.95:2:0.05) and a detection wavelength of 220nm.
The reaction conditions were optimized from the reaction temperature (20 to 60 ℃), pH of PBS buffer solution (6.0 to 11.0), organic solvent type (DMF, DMSO, ethanol, acetone, methanol, acetonitrile, isopropyl alcohol and n-hexane), organic solvent (DMSO) concentration (volume percentage 0 to 6%), substrate (1-methyl-3, 4-dihydroisoquinoline, abbreviated as "1 to MeDIQ") concentration (20 to 200 mmol/L), enzyme activity addition amount and ratio (imine reductase mutant 7 addition amount is 100 to 500U/L, FDH: imine reductase mutant 7 enzyme activity addition ratio gradient is 0.8 to 2.0), NADP + concentration (0.1 to 0.7 mmol/L) and sodium formate concentration (200 to 800 mmol/L).
FIG. 5 shows the results of the reaction temperature optimization experiment of the imine reductase mutant 7 protein.
FIG. 6 shows the results of pH optimization experiments of the imine reductase mutant 7 protein.
FIG. 7 shows the results of an organic solvent type optimization experiment for the imine reductase mutant 7 protein.
FIG. 8 shows the results of an organic solvent concentration optimization experiment for the imine reductase mutant 7 protein.
FIG. 9 shows the results of an experiment for optimizing the substrate concentration of the imine reductase mutant 7 protein.
FIG. 10 shows the results of the experiment for optimizing the enzyme activity of the imine reductase mutant 7 protein.
FIG. 11 shows the results of an experiment for optimizing the enzyme activity addition ratio of the imine reductase mutant 7 protein.
FIG. 12 shows the results of an NADP + concentration-optimized experiment for the imine reductase mutant 7 protein.
FIG. 13 shows the results of sodium formate concentration optimization experiments of the imine reductase mutant 7 protein.
The optimal reaction conditions for the final catalytic synthesis of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline are:
At 35 ℃ and PBS buffer solution pH7.5, adding 2% DMSO, 100mM 1-methyl-3, 4-dihydroisoquinoline, 400U/L imine reductase recombinant bacteria 7, 640U/LFDH, 0.5mMNADP + and 0.6mol/L sodium formate, reacting for 24 hours, wherein the conversion rate is >99%, and the ee value is >98%. The graph of the performance under optimal reaction conditions is shown in FIG. 14.
FIG. 14 is a graph showing the progress of the imine reductase of the present invention in catalyzing the preparation of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline by 1-methyl-3, 4-dihydroisoquinoline (after optimization of various parameters).
As is clear from FIG. 14, the imine reductase enzyme solution of the present invention was reacted with 1-methyl-3, 4-dihydroisoquinoline at 35℃for 24 hours or more, and the final conversion rate of the reaction was 99% and the enantiomeric excess was 98%.
The reaction solution is extracted three times by methyl tertiary butyl ether, concentrated by vacuum rotary evaporation, and then subjected to high performance liquid chromatography analysis, and meanwhile, rac-1-methyl-1, 2,3, 4-tetrahydroisoquinoline is used as a reference. The high performance liquid chromatography analysis conditions are as follows: CHIRALPAKAD-H column (250X 4.6mmid (inner diameter)), mobile phase is n-hexane, ethanol, diethylamine (volume ratio 97.95:2:0.05), detection wavelength 220nm, detection temperature 35 ℃, flow rate: 1mL/min. The HPLC diagram of rac-1-methyl-1, 2,3, 4-tetrahydroisoquinoline is shown as A in FIG. 15, and the High Performance Liquid Chromatography (HPLC) diagram of the reaction product is shown as B in FIG. 15, namely (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
FIG. 15 is a High Performance Liquid Chromatography (HPLC) diagram. Wherein, the A diagram is the HPLC diagram of rac-1-methyl-1, 2,3, 4-tetrahydroisoquinoline, and the B diagram is the HPLC diagram of the reaction product of the invention, namely (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An imine reductase, characterized in that: the amino acid sequence is obtained by mutating the sequence shown in a sequence table SEQ ID No.1, and the mutation is at least one of the following (A) - (D):
(A) Mutating the 127 th amino acid residue from E to P;
(B) Mutation of amino acid residue 177 from L to M;
(C) Mutating the 221 th amino acid residue from E to A;
(D) The 236 th amino acid residue is mutated from S to A.
2. The imine reductase according to claim 1, characterized in that: the mutation is any one of the following (1) - (8):
(1) Mutating 127 th amino acid residue from E to P, 221 th amino acid residue from E to A, and 236 th amino acid residue from S to A;
(2) Mutating the 127 th amino acid residue from E to P;
(3) Mutation of amino acid residue 177 from L to M;
(4) Mutating the 221 th amino acid residue from E to A;
(5) Mutation of amino acid residue 236 from S to A;
(6) Mutating 127 th amino acid residue from E to P, and mutating 221 th amino acid residue from E to A;
(7) Mutating 127 th amino acid residue from E to P, 177 th amino acid residue from L to M, and 221 th amino acid residue from E to A;
(8) The 127 th amino acid residue is mutated from E to P, the 177 th amino acid residue is mutated from L to M, the 221 th amino acid residue is mutated from E to A, and the 236 th amino acid residue is mutated from S to A;
preferably, the mutation is: the 127 th amino acid residue is mutated from E to P, the 221 th amino acid residue is mutated from E to A, and the 236 th amino acid residue is mutated from S to A.
3. A nucleotide sequence encoding the imine reductase according to claim 1 or 2.
4. A nucleotide sequence encoding a lysine decarboxylase according to claim 3, wherein: the nucleotide sequence is obtained by carrying out the following mutation on the sequence shown in a sequence table SEQ ID No.2, and the mutation is any one of the following (1) - (8):
(1) The 379-381 nucleotide is mutated from gaa to ccg, the 662-663 nucleotide is mutated from aa to cg, and the 706-708 nucleotide is mutated from agc to gcg;
(2) The 379-381 nucleotide is changed from gaa to ccg;
(3) Mutation of nucleotide 529 from c to a;
(4) The 662-663 nucleotide is changed from aa to cg;
(5) The 706-708 nucleotide is mutated from agc to gcg;
(6) Nucleotides 379-381 are mutated from gaa to ccg, and nucleotides 662-663 are mutated from aa to cg;
(7) Nucleotides 379-381 are mutated from gaa to ccg, nucleotide 529 is mutated from c to a, and nucleotides 662-663 are mutated from aa to cg;
(8) The 379-381 nucleotide is mutated from gaa to ccg, the 529 nucleotide is mutated from c to a, the 662-663 nucleotide is mutated from aa to cg, and the 706-708 nucleotide is mutated from agc to gcg;
Preferably, the mutation is: nucleotides 379-381 are mutated from gaa to ccg, nucleotides 662-663 are mutated from aa to cg, and nucleotides 706-708 are mutated from agc to gcg.
5. An expression vector comprising the nucleotide sequence of claim 3 or 4;
preferably, the expression vector is pET28a.
6. A cell comprising the expression vector of claim 5;
Preferably, the cells include bacteria and fungi;
further preferably, the bacterium is Escherichia coli.
7. A preparation method of an imine reductase mutant protein is characterized by comprising the following steps of: constructing the nucleotide sequence of claim 3 or 4 on an expression vector, introducing the expression vector into cells, culturing the cells, crushing, centrifuging, and collecting supernatant to obtain an imine reductase mutant protein;
preferably, the restriction sites selected for construction on the expression vector are NdeI and EcoRI.
8. Use of the imine reductase according to claim 1 or 2, the nucleotide sequence according to claim 3 or 4, the expression vector according to claim 5 or the cell according to claim 6 for the synthesis of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
9. A process for the production of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline characterized by: adding 1-3% dimethyl sulfoxide, 20-100mM 1-methyl-3, 4-dihydroisoquinoline, 400-500U/L imine reductase mutant protein, 640-1000U/L formate dehydrogenase, 0.5-0.7mM NADP + and 0.6-0.8mol/L sodium formate at 30-40 ℃ and pH 7.0-8.0, reacting for 20-28h to obtain (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline; the imine reductase mutant protein is prepared by the method of claim 7;
Preferably, the nucleotide sequence of the imine reductase mutant protein is: the following mutations were performed on the sequence shown in SEQ ID No.2 of the sequence Listing:
Nucleotides 379-381 are mutated from gaa to ccg, nucleotides 662-663 are mutated from aa to cg, and nucleotides 706-708 are mutated from agc to gcg.
10. A process for the production of (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline according to claim 9 characterized in that: at 35 ℃, pH 7.5, adding 2% dimethyl sulfoxide, 100mM 1-methyl-3, 4-dihydroisoquinoline, 400U/L imine reductase mutant protein, 640U/L formate dehydrogenase, 0.5mM NADP + and 0.6mol/L sodium formate by volume percentage, and reacting for 24 hours to obtain (S) -1-methyl-1, 2,3, 4-tetrahydroisoquinoline.
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