WO2015078258A1 - R型ω-转氨酶及其应用 - Google Patents

R型ω-转氨酶及其应用 Download PDF

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WO2015078258A1
WO2015078258A1 PCT/CN2014/089845 CN2014089845W WO2015078258A1 WO 2015078258 A1 WO2015078258 A1 WO 2015078258A1 CN 2014089845 W CN2014089845 W CN 2014089845W WO 2015078258 A1 WO2015078258 A1 WO 2015078258A1
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group
seq
transaminase
amino acid
acid sequence
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PCT/CN2014/089845
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English (en)
French (fr)
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洪浩
高峰
李艳君
张艳
李少贺
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凯莱英医药集团（天津）股份有限公司
凯莱英生命科学技术(天津)有限公司
天津凯莱英制药有限公司
凯莱英医药化学（阜新）技术有限公司
吉林凯莱英医药化学有限公司
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Application filed by 凯莱英医药集团（天津）股份有限公司, 凯莱英生命科学技术(天津)有限公司, 天津凯莱英制药有限公司, 凯莱英医药化学（阜新）技术有限公司, 吉林凯莱英医药化学有限公司 filed Critical 凯莱英医药集团（天津）股份有限公司
Priority to KR1020167016927A priority Critical patent/KR20160089492A/ko
Priority to EP14865169.8A priority patent/EP3075856A1/en
Priority to US15/039,736 priority patent/US20170002338A1/en
Priority to JP2016535175A priority patent/JP2016537991A/ja
Publication of WO2015078258A1 publication Critical patent/WO2015078258A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)

Definitions

  • the present invention relates to the field of synthesis of chiral compounds, and in particular to an R-type ⁇ -transaminase and its use.
  • Chiral amines are widely found in nature, are structural units of many important biologically active molecules, and are important intermediates for the synthesis of natural products and chiral drugs. Many chiral amines can also become important chiral auxiliaries and chiral separations. Reagents. Therefore, the preparation of chiral amine compounds has important economic significance.
  • the preparation of chiral amines is mainly carried out by chemical reduction, and optically active amines are prepared by using prochiral ketones.
  • the prochiral ketone reacts with formic acid and inorganic ammonia/organic primary amine to form a chiral amine.
  • Another researcher uses a ruthenium complex as a catalyst to pass the prochiral ketone asymmetry.
  • Amination to give a chiral amine (Renat Kadyrov et al. Highly Enantioselective Hydrogen-Transfer Reductive Amination: Catalytic Asymmetric Synthesis of Primary Amines. Angewandte Chemie International Edition.
  • Omega-transaminase is one of the transaminase enzymes, but it is slightly different. Omega-transaminase refers to a class of enzymes which can be said to be ⁇ -transaminase as long as the substrate or product of the reaction does not contain an ⁇ -amino acid in the catalyzed transamination reaction.
  • the ⁇ -transaminase can utilize a ketone compound as a raw material to efficiently produce a chiral amine by stereoselective transamination. Due to its relatively low substrate quality and high product purity, researchers are receiving more and more attention. People hope to fully explore its potential and promote it for the industrial production of chiral amines, but the research and application of the enzyme is still relatively small.
  • the present invention is directed to an R-type ⁇ -transaminase and its use to improve the deficiencies of the prior art ⁇ -transaminase which does not meet the industrial production requirements of chiral amines.
  • an R-type ⁇ -transaminase or a modification, a functional equivalent, a functional fragment or a variant thereof wherein the amino acid sequence of the R-type ⁇ -aminotransferase comprises a sequence selected from the group consisting of One of the sequences: a) an amino acid sequence as shown in SEQ ID NO.: 2; b) at least 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 and having a highly stereoselective-R configuration
  • An amino acid sequence of catalytically active ⁇ -transaminase activity wherein the amino acid sequence is not the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.: 4; c) the amino acid shown in SEQ ID NO.
  • amino acid sequence of the above R-type ⁇ -aminotransferase is an amino acid sequence in which the leucine at position 199 in the amino acid sequence shown in SEQ ID NO.: 2 is substituted with valine.
  • nucleotide encoding the above-described R-type ⁇ -transaminase or a modification, functional equivalent, functional fragment or variant thereof.
  • sequence of the nucleotide comprises a sequence selected from one of the following sequences: a) a nucleotide sequence as shown in SEQ ID NO.: 1; b) a nucleotide represented by SEQ ID NO.: A nucleotide sequence having at least 80% identity and encoding an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, wherein the nucleotide sequence is not a nucleotide as set forth in SEQ ID NO.: a sequence; c) a nucleotide sequence which hybridizes to the nucleotide sequence shown by SEQ ID NO.: 1 under high stringency conditions and which encodes an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, wherein the nucleus
  • the nucleotide sequence is not the nucleotide sequence as shown in SEQ ID NO.: 4; wherein, highly stereoselective means that one of the
  • a recombinant vector in which any one of the above nucleotides is operably linked.
  • the recombinant vector is pET22b-taC.
  • a host cell which is transformed or transfected with a recombinant vector of any of the above.
  • a method of synthesizing a chiral amine which comprises the steps of: a ketone compound, the R-type ⁇ -transaminase of claim 1, or a modification thereof, a functional equivalent, a functional fragment Alternatively, the pyridoxal phosphate and the amino donor are reacted in the reaction system, thereby obtaining a chiral amine.
  • R 1 and R 2 are each independently a C 1 -C 8 alkyl group, a C 5 -C 10 cycloalkyl group, a C 5 -C 10 aryl group or a C 5 -C 10 heteroaryl group, or R 1 and R 2 together with carbon on the carbonyl group to form a C 5 -C 10 heterocyclic group, a C 5 -C 10 carbocyclic group or a C 5 -C 10 heteroaryl group, a C 5 -C 10 heterocyclic group and a C 5 -C 10 hetero
  • the heteroatoms in the aryl group are each independently selected from at least one of nitrogen, oxygen and sulfur, an aryl group in a C 5 -C 10 aryl group, a heteroaryl group in a C 5 -C 10 heteroaryl group, C 5
  • the carbocyclic group in the -C 10 carbocyclic group or the heterocyclic group in the C 5 -C 10 heterocyclic group are
  • the above reaction system further contains a solubilizing agent, and the solubilizing agent is dimethyl sulfoxide or polyethylene glycol, and preferably polyethylene glycol is PEG-400.
  • the C1-C8 alkyl group is a C1-C8 linear alkyl group
  • the C5-C10 heteroaryl group is a pyridyl group
  • the alkoxy group is a C1-C6 alkoxy group
  • the heterocyclic group in the C5-C10 heterocyclic group is a heterocyclic ring.
  • the base is piperidine, an aryl group in a C5-C10 aryl group, a heteroaryl group in a C5-C10 heteroaryl group, a carbocyclic group in a C5-C10 carbocyclic group or a heterocyclic group in a C5-C10 heterocyclic group.
  • the substituents on each are independently a C1-C6 linear alkyl group, a C1-C6 alkoxy group, and the amino donor is isopropylamine or D-alanine.
  • Figure 1 is a flow chart showing the chemical reaction of the R type ⁇ -transaminase of the present invention in the synthesis of chiral amines;
  • Figure 2 shows the chemical reaction equation for the application of the R-type ⁇ -transaminase of the present invention in the synthesis of chiral amines
  • FIG. 3 shows the results of enzyme digestion identification in Example 1 of the present invention
  • Figure 4 is a view showing the sequencing result after PCR of the mutant gene in Example 1 of the present invention.
  • Figure 5 shows the protein expressed by the recombinant plasmid pET22b-taB in Example 1 of the present invention.
  • optionally substituted alkyl means “unsubstituted alkyl” (alkyl substituted without a substituent) or “substituted alkyl” (alkyl substituted with a substituent) .
  • C1 to Cn as used herein include C1 to C2, C1 to C3, ..., C1 to Cn.
  • the "C1-C4" group means having 1 to 4 carbon atoms in the moiety, that is, the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms.
  • alkyl refers to an optionally substituted straight or optionally substituted branched aliphatic hydrocarbon.
  • the "alkyl group” herein preferably has 1 to 20 carbon atoms, for example, 1 to 10 carbon atoms, 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms or 1 ⁇ 3 carbon atoms.
  • alkoxy refers to an alkyl ether group (O-alkyl), non-limiting examples of which include methoxy, ethoxy, n-propoxy, isopropyl Oxyl, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy groups.
  • halo or halogen-substituted as used herein, alone or in combination, means that one or more hydrogen atoms of an optionally substituted group (eg, alkyl, alkenyl, and alkynyl) are replaced with fluorine, chlorine. , bromine, iodine atoms or a combination thereof.
  • an optionally substituted group eg, alkyl, alkenyl, and alkynyl
  • aryl/aryl refers to an optionally substituted aromatic hydrocarbon group having from 6 to 20, such as from 6 to 12 or from 6 to 10 ring-forming carbon atoms. It may be a fused aromatic ring or a non-fused aromatic ring.
  • heteroaryl refers to an optionally substituted monovalent heteroaryl group containing from 5 to 20, such as from 5 to 12 or from 5 to 10 backbones, wherein one or more (eg, 1-4, 1-3, 1-2) ring atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, and A hetero atom in tin, but is not limited thereto.
  • the ring of the group does not contain two adjacent O or S atoms.
  • Heteroaryl groups include monocyclic heteroaryl or polycyclic heteroaryl (eg bicyclic heteroaryl, tricyclic heteroaryl, etc.).
  • heterocycle refers to a non-aromatic heterocycle, including heterocycloalkyl and heterocycloalkenyl.
  • One or more (eg, 1 to 4, 1 to 3, or 1 to 2) ring-forming atoms are heteroatoms such as oxygen, nitrogen or sulfur atoms.
  • the heterocyclic group may include a monocyclic heterocyclic group (heterocyclic group having one ring) or a polycyclic heterocyclic group (for example, a bicyclic heterocyclic group (heterocyclic group having two rings), a tricyclic heterocyclic group, or the like).
  • Carbocyclyl refers to a non-aromatic carbocyclic ring, including cycloalkyl and cycloalkenyl.
  • the cycloalkyl group may be a monocyclic cycloalkyl group or a polycyclic cycloalkyl group (for example, having 2, 3 or 4 rings; such as a bicyclic cycloalkyl group), which may be a spiro ring or a bridged ring.
  • the carbocyclic group may have 3 to 20 carbon atoms, for example, 3 to 15 ring-forming carbon atoms or 3 to 10 ring-forming carbon atoms or 3 to 6 ring-forming carbon atoms, and may have 0, 1, 2 or 3 Double keys and / or 0, 1 or 2 triple keys.
  • a cycloalkyl group having 3 to 8 or 3 to 6 ring-forming carbon atoms.
  • Halogen means fluoro, chloro, bromo, iodo, preferably fluoro, chloro and bromo. Cyano refers to "-CN”; hydroxy refers to “-OH”; thiol refers to "-SH”; amino refers to "-NH2”.
  • substituted means that one or more hydrogens are replaced by a specified group on a particular atom. If the normal valence of the specified atom is not exceeded in the existing case, the result of the substitution is one. A stable compound.
  • the present invention provides an R-type ⁇ -transaminase or a modification thereof, A functional equivalent, a functional fragment or variant, the amino acid sequence of the R-type ⁇ -transaminase comprising a sequence selected from the group consisting of: a) the amino acid sequence set forth in SEQ ID NO.: 2; b) and SEQ ID NO An amino acid sequence having an amino acid sequence of at least 80% identity and having a highly stereoselective-R configuration catalytic activity of ⁇ -transaminase activity, wherein the amino acid sequence is not as shown in SEQ ID NO.: The amino acid sequence encoded by the nucleotide sequence; c) the amino acid sequence shown in SEQ ID NO.: 2 is derived from SEQ ID NO.: 2 by substitution, deletion or addition of one or
  • a chiral amine having a higher chiral purity in the R configuration can be efficiently synthesized. Suitable for industrial production of chiral amines.
  • the above-mentioned R-type ⁇ -transaminase of the present invention means an ⁇ -transaminase having a high R configuration stereoselectivity, and in one embodiment, the transaminase of the present invention refers to a transaminase having a sequence of SEQ ID NO.: 2.
  • the above transaminase is a molecular biological technique in which a transaminase gene derived from Arthobacter sp. is mutated and molecularly engineered to obtain an R-type ⁇ -transaminase, wherein the amino acid sequence is derived without changing the amino acid sequence.
  • the nucleotide sequence of the transaminase gene taA after optimization of the nucleotide sequence of the transaminase gene of Arthrobacter is shown in SEQ ID NO.: 4, and the corresponding amino acid sequence is shown in SEQ ID NO.: 5.
  • amino acid sequence of the above-mentioned ⁇ -transaminase activity having at least 80% identity with the amino acid sequence of SEQ ID NO.: 2 and having a highly stereoselective-R configuration catalytic activity means that it is SEQ ID NO.: 2
  • the amino acid sequence shown has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% identity and is not the amino acid sequence set forth in SEQ ID NO.: 5. the sequence of.
  • the amino acid sequence which retains the key function of the catalytic activity of the transaminase in the amino acid sequence shown by SEQ ID NO.: 2 remains unchanged, those skilled in the art can change the amino acid sequence of the remaining inactive sites, so that The amino acid sequence of the transaminase to be obtained has at least 80% identity with the amino acid sequence of SEQ ID NO.: 2.
  • the transaminase was obtained to have the same transaminase activity as the aminotransferase of SEQ ID NO.: 2.
  • the amino acid sequence which plays a key role in the catalytic activity of the transaminase in the amino acid sequence shown by SEQ ID NO.: 2 it can be in the amino acid sequence shown in SEQ ID NO.: 2 above.
  • the amino acid is substituted, deleted or added with one or more amino acids, such that the protein derived from SEQ ID NO.: 2 maintains the high stereoselectivity of the transaminase shown by SEQ ID NO.: 2.
  • the substituted, deleted or added base may be one or more, such as 1, 2, 3, 4, 5, 10, 20, 30 or 50 amino acids, such as conservative amino acids.
  • amino acid sequence is not the amino acid sequence as shown in SEQ ID NO.: 5; "substitution of conservative amino acids” means, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr ; Lys, Arg; and a combination of Phe, Tyr.
  • stereoselectivity means that when one reaction produces two stereoisomers A and B, the yield of A is more than B.
  • Highly stereoselective means that one of the stereoisomers is at least about 1.1, such as at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 2 times, at least about the other. 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 70 times, at least about 90 times, at least about 100 times, or higher.
  • the above-mentioned modification of the R-type ⁇ -transaminase may be a chemical modification such as an acylation, alkylation, or PEGylation product as long as these modifications retain the above-mentioned highly stereoselective-R configuration catalytic activity of ⁇ - Transaminase activity can be.
  • the above functional equivalents refer to other polypeptide fragments capable of achieving R-type ⁇ -transaminase activity.
  • the above functional fragment refers to a protein fragment which retains the ⁇ -transaminase activity of the highly stereoselective-R configuration catalytic activity.
  • the above variant refers to a polypeptide derived from a parent protein by one or more amino acids at one or more (several) positions, ie, by substitution, insertion and/or deletion.
  • the amino acid sequence of the R-type ⁇ -transaminase is an amino acid sequence in which the leucine at position 199 in the amino acid sequence shown in SEQ ID NO.: 2 is substituted with valine.
  • the amino acid-substituted transaminase has the same activity and function as the transaminase shown by SEQ ID NO.: 2.
  • a nucleotide which encodes the above-described R-type ⁇ -transaminase or a modification, functional equivalent, functional fragment or variant thereof.
  • the coding rules for the nucleotides of the above-described R-type ⁇ -transaminase or a modification, functional equivalent, functional fragment or variant thereof of the present invention conform to the conventional codon usage table.
  • the sequence of the above nucleotide comprises a sequence selected from one of the following: a) a nucleotide sequence as set forth in SEQ ID NO.: 1; b) and SEQ ID
  • the nucleotide sequence shown by NO.: 1 has at least 80% identity and encodes a nucleotide sequence of an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, wherein the nucleotide sequence is not as SEQ ID Nucleotide sequence shown by NO.: 4; c) hybridizing to the nucleotide sequence shown by SEQ ID NO.: 1 under high stringency conditions and encoding ⁇ - having a highly stereoselective-R configuration catalytic activity a nucleotide sequence of a transaminase, wherein the nucleotide sequence is not a nucleotide sequence as shown in SEQ ID NO.: 4; wherein, highly stereoselective means
  • nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO.: 1 and encoding an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8% or 99.9% identity, wherein the nucleotide sequence is not as shown in SEQ ID NO.: Nucleotide sequence.
  • the person skilled in the art can nucleate the remaining inactive sites.
  • the nucleotide sequence is altered such that the nucleotide sequence of the resulting transaminase is at least 80% identical to the nucleotide sequence set forth in SEQ ID NO.: 1.
  • the transaminase was obtained to have the same transaminase activity as the transaminase having the nucleotide sequence of SEQ ID NO.: 1.
  • nucleotide sequence which hybridizes to the nucleotide sequence shown by SEQ ID NO.: 1 under high stringency conditions and which encodes an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, wherein the nucleotide sequence Is not a nucleotide sequence as shown in SEQ ID NO.: 4; similarly, based on the nucleotide sequence shown in SEQ ID NO.: 1, it is possible to hybridize with the plasmid under high stringency conditions and encode with a height
  • the nucleotide sequence of the ⁇ -transaminase catalytically active in the stereoselective-R configuration, the variant sequence of the nucleotide sequence shown in SEQ ID NO.: 1 thus obtained, having the nucleotide sequence of SEQ ID NO .:1 transaminase has the same transaminase activity.
  • stereoselectivity means that when one reaction produces two stereoisomers A and B, the yield of A is more than B.
  • Highly stereoselective means that one of the stereoisomers is at least about 1.1, such as at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 2 times, at least about the other. 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 70 times, at least about 90 times, at least about 100 times, or higher.
  • An exemplary high stringency condition can be a 6X SSC, 0.5% SDS solution, hybridized at 65 °C, and then washed once with 2X SSC, 0.1% SDS and 1X SSC, 0.1% SDS.
  • identity has the meaning commonly known in the art, that is, sequence similarity or homology, and those skilled in the art are also familiar with the rules and standards for determining the identity between different sequences.
  • sequences defined by the present invention with varying degrees of identity must also have the activity of a highly stereoselective-R configuration catalytically active ⁇ -transaminase.
  • Methods and means for screening variant sequences for the activity of the highly stereoselective-R configuration catalytically active ⁇ -transaminase are well known to those skilled in the art. Such variant sequences can be readily obtained by those skilled in the art in light of the teachings of the present disclosure.
  • one or more codons in the above nucleotide sequence can be equivalently replaced without changing the encoded amino acid, such as replacing the leucine Leu encoded by CTT with CTA, CTC. Or CTG.
  • the number of codons substituted can be one or several codons, such as 1, 2, 3, 4, 5, 6, 8, 9, 10, 15, 20, 30, 40, 50 codons. Codon usage tables are well known in the art.
  • a recombinant vector in which any one of the above nucleotides is operably linked.
  • Recombinant vectors of the invention include, but are not limited to, recombinant expression vectors, and may also include recombinant cloning vectors.
  • the recombinant vector may be a prokaryotic expression vector or a eukaryotic expression vector.
  • the recombinant vector is a recombinant prokaryotic expression vector capable of inducing expression, such as a pET series vector for inducing gene expression using IPTG, such as a pET22b vector. .
  • operably linked refers to a manner in which a polynucleotide is placed in position on a vector such that the polynucleotide is correctly, smoothly replicated, transcribed, and/or translated.
  • the above recombinant vector of the present invention is pET22b-taC comprising the nucleotide sequence shown in SEQ ID NO.: 1.
  • a host cell which is transformed or transfected with a recombinant vector of any of the above.
  • Host cells of the invention include prokaryotic host cells and eukaryotic host cells.
  • the host cell is a prokaryotic host cell, such as E. coli, more preferably E. coli DH5[alpha] (DE3).
  • a method of synthesizing a chiral amine comprising the steps of: a ketone compound, the R-type ⁇ -transaminase of claim 1, or a modification thereof, a functional equivalent, a function A fragment or variant, pyridoxal phosphate and an amino donor are reacted in a reaction system, thereby obtaining a chiral amine.
  • the method for synthesizing a chiral amine of the present invention is based on a conventional method for preparing a chiral compound by a bio-enzymatic catalytic reaction, and appropriately adjusting the composition, ratio, amount, pH, temperature, and various reaction raw materials of the reaction system. The reaction time and other parameters can be used.
  • the ketone compound is wherein R 1 and R 2 are each independently a C 1 -C 8 alkyl group, a C 5 -C 10 cycloalkyl group, a C 5 -C 10 aryl group or a C 5 -C 10 heteroaryl group, or R 1 and R 2 together with carbon on the carbonyl group to form a C 5 -C 10 heterocyclic group, a C 5 -C 10 carbocyclic group or a C 5 -C 10 heteroaryl group, a C 5 -C 10 heterocyclic group and a C 5 -C 10 hetero
  • the heteroatoms in the aryl group are each independently selected from at least one of nitrogen, oxygen and sulfur, an aryl group in a C 5 -C 10 aryl group, a heteroaryl group in a C 5 -C 10 heteroaryl group, C 5 The carbocyclic group in the -C 10 carbocyclic group or the heterocyclic group in the C 5 -C
  • the above reaction system further contains a solubilizing agent, and the solubilizing agent is dimethyl sulfoxide or polyethylene glycol, and preferably polyethylene glycol is PEG-400.
  • the solubilizing agent can dissolve the above raw materials well to facilitate the reaction, and the solvating effect of PEG-400 is better.
  • the C1-C8 alkyl group is a C1-C8 linear alkyl group
  • the C5-C10 heteroaryl group is a pyridyl group
  • the alkoxy group is a C1-C6 alkoxy group, C5.
  • the heterocyclic group in the -C10 heterocyclic group is piperidine, an aryl group in a C5-C10 aryl group, a heteroaryl group in a C5-C10 heteroaryl group, a carbocyclic group in a C5-C10 carbocyclic group or a C5 ⁇
  • the substituents on the heterocyclic group in the C10 heterocyclic group are each independently a C1 to C6 linear alkyl group, a C1 to C6 alkoxy group, and the amino donor is isopropylamine or D-alanine.
  • the above raw materials are commercial raw materials or easily prepared raw materials and are inexpensive, and can meet the needs of large-scale production.
  • the reaction system contains a buffer for maintaining the pH of the reaction system in the range of 7.0 to 9.5; and/or wherein the ratio of the ketone compound to the solvent is 1 g/1 mL. 15mL; and/or wherein the ratio of the ketone compound to the buffer is from 1g/15mL to 50mL; and/or the ratio of the ketone compound to the pyridoxal phosphate is from 1g/0.01g to 0.1g; and/or Wherein the ratio of the ketone compound to the amino donor is from 1 eq/1 eq to 5 eq; and/or the ratio of the ketone compound to the R-type ⁇ -transaminase is from 1 g/0.2 g to 10 g; and/or the temperature of the reaction system therein It is 20 to 45 ° C and reacts for 12 to 48 hours; and/or the buffer is a phosphate buffer or a triethanolamine buffer having a pH of 9.3 to 9.5
  • the method further comprises the steps of: adjusting the reaction system to pH ⁇ 10 with a base, and extracting the product chiral amine in the aqueous phase with an organic solvent, preferably, the base is sodium hydroxide or hydrogen.
  • the organic solvent is ethyl acetate, methyl tert-butyl ether or 2-methyltetrahydrofuran.
  • the aminotransferase gene taA (sequence) optimized by the biotransformation gene (TA) was optimized by changing the amino acid sequence of the transaminase gene derived from Arthrobacter sp. As shown in SEQ ID NO.: 4, the corresponding amino acid sequence is shown in SEQ ID NO.: 5), the synthetic taA gene was ligated to the vector pUC57 to obtain the recombinant plasmid pUC-taA, and then restriction endonuclease with NdeI and XhoI.
  • the recombinant plasmid pUC-taA and the vector pET-22b(+) were simultaneously digested, the digested product was ligated with T4 DNA ligase, and the ligated product was transformed into competent cells of Escherichia coli DH5 ⁇ strain by shaker. After resuscitation, it was applied to an LB culture dish containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured overnight in a 37 ° C incubator.
  • Fig. 3 is a diagram showing the identification of the pET22b-taA plasmid by NdeI enzyme and XhoI enzyme, wherein 1 indicates a pET22b empty vector; 2 indicates a DNA molecular size marker (10000 bp, 8000 bp, 6000 bp from top to bottom, respectively). 5000bp, 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp); 3 denotes pET22b-taA-DH5 ⁇ . As can be seen from Fig.
  • the relatively weak band with a fragment size of about 1000 bp after digestion is the target fragment (the plasmid strip after the excision fragment is relatively strong).
  • the insertion direction and size of the inserted sequence of the recombinant plasmid pET22b-taA were correct, and then the correct recombinant plasmid pET22b-taA was used as a template for the next mutation.
  • the whole plasmid PCR was carried out using the above recombinant plasmid pET22b-taA as a template, and the PCR product was digested with DpnI and transformed into E. coli BL21 (DE3), which was resuscitated by a shaker and then coated.
  • the LB culture dish containing ampicillin at a final concentration of 50 ⁇ g/ml was cultured overnight at 37 °C.
  • a single colony in the above culture dish was picked and inoculated into 5 ml of LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured at 37 ° C, 180 r / min overnight.
  • the bacterial solution was sent to Bioengineering (Shanghai) Co., Ltd. for sequencing, and the recombinant plasmid of the sequencing bacterial solution was named BL21/pET22b-taB, and the sequencing result was compared with the primer sequence (SEQ_ID_NO.3) and taA. 4.
  • the 198 bp sequence carried by the BL21/pET22b-taB plasmid from the ATG was completely replaced with the sequence designed above, which was completely identical to the expected, without the mutated base. .
  • the recombinant plasmid is the target plasmid sequence.
  • the above bacterial solution was transferred to 5 ml of LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured at 37 ° C, shaking at 180 r/min until the OD600 value was 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM.
  • the culture solution was transposed at 25 ° C for induction expression, and the culture medium without the IPTG inducer was set as a negative control. After induction for 16 h, the bacterial solution was taken out, and the cells were collected by centrifugation at 12000 r/min for 5 min. The cells were disrupted by ultrasonic sonicator.
  • the ultrasonic parameters were: probe diameter 6mm, power 200W, working 2s, intermittent 6s, total 10min, after ultrasonication, centrifuged at 12000r/min for 20min to obtain ultrasonic supernatant and sediment.
  • the supernatant was subjected to SDS-PAGE detection using a vertical electrophoresis apparatus.
  • Primer antisense strand 5-CCATTGGAAGTTCTTAACTTGCGGATCGATGC-3 (SEQ ID NO.: 7)
  • the PCR product was digested with DpnI and transformed into E. coli BL21 (DE3), and plated in LB culture containing ampicillin at a final concentration of 50 ⁇ g/ml.
  • the dish was cultured overnight at 37 ° C to obtain a large number of single colonies.
  • a single colony in the above culture dish was picked and inoculated into 5 ml of LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml to obtain a large number of mutants.
  • the mutant bacterial solution was transferred to 100 ml of LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured at 37 ° C, shaking at 180 r/min until the OD600 value was 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM.
  • the culture solution was transposed at 25 ° C for induction expression, and the culture medium without the IPTG inducer was set as a negative control. After induction for 16 h, the bacterial solution was taken out, and the cells were collected by centrifugation at 12000 r/min for 5 min. Weigh 0.5g of bacterial mud and resuspend with 2.5mL of triethanolamine buffer (pH 9.5).
  • the cells were disrupted by ultrasonic sonicator.
  • the ultrasonic parameters were: probe diameter 6mm, power 200W, working 2s, intermittent 6s, total 10min, ultrasound After completion, the ultrasonic supernatant and the precipitate were obtained by centrifugation at 12000 r/min for 20 min at 4 ° C, and the supernatant was used for the reaction to verify the enzyme activity.
  • the obtained mutant having the best enzyme activity has the sequence of SEQ ID NO.: 1, and the expressed R-type aminotransferase is named BL21/pET22b-taC.
  • the above strain BL21/pET22b-taC was inoculated into LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml, and shaken at 37 ° C, 180 r/min until the OD600 value was 0.6-0.8, and IPTG was added to the final concentration. 0.2 mM, and the culture solution was transposed to 25 ° C for induction expression. After induction for 16 h, the fermentation broth was taken out, 12000 r/min. The cells were collected by centrifugation for 10 min.
  • the nuclear magnetic data of the obtained product are as follows: 1H-NMR (300 MHz, CDCl3) ⁇ 4.00-3.78 (m, 2H), 3.80 (m, 2H), 3.60 (m, 1H), 1.90 (m, 1H), 1.70 (m) , 1H), 1.60-1.40 (m, 12H), 1.30 (m, 1H) ppm.
  • 0.1 g of the main raw material (2,4-dichloroacetophenone, CAS: 2234-16-4) and 1.5 mL of polyethylene glycol PEG-400 were added to the reaction flask. After the raw materials were dispersed, 23.5 ml of phosphate buffer was added. (pH 8.0), 0.031 g of isopropylamine, 0.0075 g of pyridoxal phosphate and 0.02 g of the above-mentioned mutant R-type ⁇ -transaminase having the amino acid sequence shown in SEQ ID NO.: 2, the pH of the system was 8.0, and the temperature was stirred at 45 ° C. 20h.
  • the enzyme activity test of the transaminase is carried out, and the detection steps are as follows:
  • the mutant bacterial solution was transferred to 100 ml of LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured at 37 ° C, shaking at 180 r/min until the OD600 value was 0.6-0.8, and IPTG was added to a final concentration of 0.2 mM.
  • the culture solution was transposed at 25 ° C for induction expression, and the culture medium without the IPTG inducer was set as a negative control. After induction for 16 h, the bacterial solution was taken out, and the cells were collected by centrifugation at 12000 r/min for 5 min. Weigh 0.5g of bacterial mud and resuspend with 2.5mL of triethanolamine buffer (pH 9.5).
  • the cells were disrupted by ultrasonic sonicator.
  • the ultrasonic parameters were: probe diameter 6mm, power 200W, working 2s, intermittent 6s, total 10min, ultrasound After completion, the ultrasonic supernatant and the precipitate were obtained by centrifugation at 12000 r/min for 20 min at 4 ° C, and the supernatant was used for the reaction to verify the enzyme activity.
  • 0.1 g of the main raw material (N-BOC-piperidone, CAS: 79099-07-3) and 0.1 mL of dimethyl sulfoxide were added to the reaction flask, and after the raw materials were dispersed, 7.5 mL of 0.2 mol/L was added in an ice bath condition.
  • the triethanolamine buffer having a pH of 9.3-9.5, 0.591 g of isopropylamine, 0.0025 g of pyridoxal phosphate and 2.5 mL of the above-mentioned mutant R-type ⁇ -transaminase were adjusted with concentrated hydrochloric acid, and the pH of the system was 9.5, and the mixture was stirred at 30 ° C for 12 hours.
  • the nuclear magnetic data of the obtained product are as follows: 1H-NMR (300 MHz, CDCl3) ⁇ 4.00-3.78 (m, 2H), 3.80 (m, 2H), 3.60 (m, 1H), 1.90 (m, 1H), 1.70 (m) , 1H), 1.60-1.40 (m, 12H), 1.30 (m, 1H) ppm.
  • the novel transaminase disclosed by the present invention catalyzes the transfer of an amino group in an amino donor to a prochiral ketone or an aldehyde, thereby producing a corresponding R.
  • the chiral amine of the configuration, using the novel transaminase of the invention for the preparation of the synthetic amine, can not only more substrates The conversion was carried out to obtain a high purity of the R-form chiral amine, which was stable at 98% or more.
  • the raw material used in the synthesis method is easy to obtain, the method is simple, the chemical reaction conditions are mild, the yield and the purity of the enantiomer are high, and the operation is simple in the whole production process, which is a feasible and less polluting synthetic process.
  • the preparation of chiral amines provides a new approach and method.

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Abstract

提供了一种R型ω-转氨酶。该R型ω-转氨酶含有SEQ ID NO: 2所示的氨基酸序列，或与SEQ ID NO: 2具有至少80%同一性，或经过取代、缺失或添加一个或多个氨基酸的，具有高度立体选择性-R构型催化活性的ω-转氨酶活性的蛋白质的氨基酸序列；且不是如SEQ ID NO: 4所示的核苷酸序列所编码的氨基酸序列。高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。提供了R型ω-转氨酶的应用，可用于高效合成手性纯度较高的R构型手性胺，适用于手性胺的工业化生产。

Description

R型ω-转氨酶及其应用 技术领域
本发明涉及手性化合物的合成领域，具体而言，涉及一种R型ω-转氨酶及其应用。
背景技术
手性胺广泛存在于自然界中，是很多重要生物活性分子的结构单元，是合成天然产物和手性药物的重要中间体，很多手性胺还可成为重要的手性助剂和手性拆分试剂。所以，手性胺化合物的制备有很重要的经济意义。
现阶段，手性胺的制备主要采用化学还原的方法，利用前手性酮制备得到光学活性的胺。在Pd/C及喹宁的催化作用下，前手性酮与甲酸以及无机氨/有机伯胺进行反应生成手性胺；另有研究者以钌配合物为催化剂，通过前手性酮不对称胺化还原得到手性胺(Renat Kadyrov et al.Highly Enantioselective Hydrogen-Transfer Reductive Amination:Catalytic Asymmetric Synthesis of Primary Amines.Angewandte Chemie International Edition.2003,42(44)，第5472-5474页)，此类反应中金属催化剂是非常关键的因素，且对金属催化剂要求苛刻，反应需要在高压条件下完成，操作设备要求高，同时金属催化剂价格昂贵，对环境污染也较大(Ohkuma T et al.Trans-RuH(eta1-BH4)(binap)(1，2-diamine):a catalyst for asymmetric hydrogenation of simple ketones under base-free conditions.Journal of the American Chemical Society.2002,124(23)，第6508-6509页)。
氨基转移酶，也称为转氨酶，可以催化一个氨基与羰基互换的过程。ω-转氨酶属于转氨酶之一，但有少许不同。ω-转氨酶是指一类酶，只要在其催化的转氨反应中，反应的底物或产物中不含有α-氨基酸，就可以称该酶为ω-转氨酶。ω-转氨酶可以利用酮类化合物为原料，通过立体选择性地转氨基作用，高效生产手性胺。因其底物相对廉价、产物纯度高的特点，受到研究人员越来越多的关注。人们希望能充分发掘其潜力，将其推广用于手性胺的工业生产，但目前对该酶的研究及应用仍然比较少。
本领域仍然存在对具有高度立体选择性R构型催化活性的ω-转氨酶的需求，以满足制备手性胺化合物的需求。
发明内容
本发明旨在提供一种R型ω-转氨酶及其应用，以改善现有技术中的ω-转氨酶不能满足手性胺的工业生产需求的缺陷。
为了实现上述目的，根据本发明的一个方面，提供了一种R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，该R型ω-转氨酶的氨基酸序列包含选自如下序列之一的序列：a)如SEQ ID NO.：2所示的氨基酸序列；b)与SEQ ID NO.：2所示的氨基酸序列具有至少80％同一性且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的氨基酸序列，其中该氨基酸序列不是如SEQ ID NO.：4所示的核苷酸序列所编码的氨基酸序列；c)在SEQ ID NO.：2中所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而由SEQ ID NO.：2衍生而来且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的蛋白质，其中该氨基酸序列不是如SEQ ID NO.：4所示的核苷酸序列所编码的氨基酸序列；其中，高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。
进一步地，上述R型ω-转氨酶的氨基酸序列是在SEQ ID NO.：2中所示的氨基酸序列中第199位的亮氨酸取代为缬氨酸的氨基酸序列。
根据本发明的另一方面，提供了一种核苷酸，该核苷酸编码上述R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体。
进一步地，核苷酸的序列包含选自如下序列之一的序列：a)如SEQ ID NO.：1所示的核苷酸序列；b)与SEQ ID NO.：1所示的核苷酸序列具有至少80％同一性且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；c)在高严谨条件下与SEQ ID NO.：1所示的核苷酸序列杂交且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；其中，高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。
根据本发明的又一方面，提供了一种重组载体，重组载体中有效连接有上述任一种核苷酸。
进一步地，重组载体为pET22b-taC。
根据本发明的再一方面，提供了一种宿主细胞，该宿主细胞转化或转染有上述任一种的重组载体。
根据本发明的另一方面，提供了一种合成手性胺的方法，该方法包括如下步骤：使酮类化合物、权利要求1的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，磷酸吡哆醛和氨基供体在反应体系中反应，由此获得手性胺。
进一步地，上述酮类化合物为
Figure PCTCN2014089845-appb-000001
其中，R1和R2各自独立地为C1～C8烷基、C5～C10环烷基、C5～C10芳基或C5～C10杂芳基，或者R1和R2与羰基上的碳共同形成C5～C10杂环基、C5～C10碳环基或C5～C10杂芳基，C5～C10杂环基和C5～C10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，C5～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，酮类化合物
Figure PCTCN2014089845-appb-000002
选自
Figure PCTCN2014089845-appb-000003
或
Figure PCTCN2014089845-appb-000004
进一步地，上述反应体系中还含有促溶剂，促溶剂为二甲基亚砜或聚乙二醇，优选聚乙二醇为PEG-400。
进一步地，上述C1～C8烷基为C1～C8直链烷基，C5～C10杂芳基为吡啶基团，烷氧基为C1～C6烷氧基，C5～C10杂环基中的杂环基为哌啶，C5～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基上的取代基各自独立为C1～C6直链烷基、C1～C6烷氧基，氨基供体为异丙胺或D-丙氨酸。
应用本发明的技术方案，通过利用具有高度立体选择性的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，可以高效地合成手性纯度较高的R构型的手性胺，适合用于手性胺的工业化生产。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明的R型ω-转氨酶在手性胺合成中的应用的化学反应流程图；
图2示出了本发明R型ω-转氨酶在手性胺合成中的应用的化学反应方程式；
图3示出了本发明的实施例1中的酶切鉴定结果；
图4示出了本发明实施例1中的突变基因PCR后的测序结果；以及
图5示出了本发明实施例1中的重组质粒pET22b-taB所表达的蛋白。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
术语解释
术语“任选/任意”或“任选地/任意地”是指随后描述的事件或情况可能发生或可能不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，根据下文的定义，“任选取代的烷基”是指“未取代的烷基”(未被取代基取代的烷基)或“取代的烷基”(被取代基取代的烷基)。
本文所用C1～Cn包括C1～C2、C1～C3、……C1～Cn。举例而言，所述“C1～C4”基团是指该部分中具有1～4个碳原子，即基团包含1个碳原子，2个碳原子、3个碳原子或4个碳原子。
本文单独或组合使用的术语“烷基”是指任选取代的直链或任选取代的支链的脂肪族烃类。本文的“烷基”优选可具有1～20个碳原子，例如具有1～10个碳原子，具有1～8个碳原子，或1～6个碳原子，或1～4个碳原子或1～3个碳原子。本文单独或组合使用的术语“烷氧基”是指烷基醚基(O-烷基)，烷氧基的非限定性实施例包括甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、仲丁氧基和叔丁氧基等。
本文单独或组合使用的术语“卤代”或“卤素取代”是指任选被取代的基团(如烷基、烯基和炔基)的其中一个或多个氢原子被替换成氟、氯、溴、碘原子或其组合。
本文单独或组合使用的术语“芳香基/芳基”是指任选取代的芳香烃基，其具有6至20个，如6～12个或6～10个成环碳原子。其可以是稠合芳环或非稠合芳环。
本文单独或组合使用的术语“杂芳基”是指任意取代的一价杂芳基，其包含5至20个，如5至12个或5至10个骨架成环原子，其中一个或多个(如1～4个、1～3个、1～2个)成环原子为杂原子，所述杂原子独立地选自氧、氮、硫、磷、硅、硒和 锡中的杂原子，但不限于此。所述基团的环不包含两个相邻的O或S原子。杂芳基包括单环杂芳基或多环杂芳基(例如双环杂芳基、三环杂芳基等)。
本文单独或组合使用的术语“杂环”或“杂环基”是指非芳香杂环，包括杂环烷基和杂环烯基。其中一个或者多个(如1～4个、1～3个、1～2个)成环的原子是杂原子，如氧、氮或硫原子。杂环基可以包括单环杂环基(杂环基具有一个环)或多环杂环基(例如，双环杂环基(杂环基具有两个环)、三环杂环基等)。
本文单独或组合使用的术语“碳环基”是指非芳香族的碳环，包括环烷基和环烯基。环烷基可以是单环环烷基或多环环烷基(例如，有2、3或4个环；如双环环烷基)，其可以是螺环或桥环。碳环基可以具有3至20碳原子，例如具有3～15个成环碳原子或3～10个成环碳原子或3～6个成环碳原子，并可以具有0、1、2或3个双键和/或0、1或2个三键。例如具有3～8个或3～6个成环碳原子的环烷基。
“卤素”是指氟、氯、溴、碘，优选氟、氯和溴。氰基是指“-CN”；羟基是指“-OH”；巯基是指“-SH”；氨基是指“-NH2”。
术语“被取代的”意思是在一个特定的原子上一个或更多的氢被指定的基团所替代，如果指定的原子的正常化合价在现有的情况下没有超出，那么取代后结果是一个稳定的化合物。
如背景技术所提到的，现有技术中的ω-转氨酶仍存在不能满足制备手性胺化合物需求的缺陷，为了改善上述状况，本发明提供了一种R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，该R型ω-转氨酶的氨基酸序列包含选自如下序列之一的序列：a)如SEQ ID NO.：2所示的氨基酸序列；b)与SEQ ID NO.：2所示的氨基酸序列具有至少80％同一性且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的氨基酸序列，其中该氨基酸序列不是如SEQ ID NO.：4所示的核苷酸序列所编码的氨基酸序列；c)在SEQ ID NO.：2中所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而由SEQ ID NO.：2衍生而来且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的蛋白质，其中该氨基酸序列不是如SEQ ID NO.：4所示的核苷酸序列所编码的氨基酸序列；其中，高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。
通过利用本发明的上述具有高度立体选择性的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，可以高效地合成手性纯度较高的R构型的手性胺，适合用于手性胺的工业化生产。
本发明的上述R型ω-转氨酶是指具有高度R构型立体选择性的ω-转氨酶，在一个实施方案中，本发明的转氨酶是指序列如SEQ ID NO.：2所示的转氨酶。上述转氨酶是本发明采用分子生物学技术，将来源于节杆菌属(Arthobacter sp)的转氨酶基因进行突变及分子改造获得R型ω-转氨酶，其中，在不改变氨基酸序列的前提下，对来源于节杆菌属的转氨酶基因的核苷酸序列进行优化改造后的转氨酶基因taA的核苷酸序列如SEQ ID NO.：4所示，对应的氨基酸序列如SEQ ID NO.：5所示。
上述与SEQ ID NO.：2所示的氨基酸序列具有至少80％同一性且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的氨基酸序列，是指与SEQ ID NO.：2所示的氨基酸序列至少具有例如85％、90％、95％、96％、97％、98％、99％、99.5％或99.7％的同一性且不为SEQ ID NO.：5所示的氨基酸序列的序列。在保留SEQ ID NO.：2所示的氨基酸序列中对转氨酶的催化活性起关键作用的氨基酸序列保持不变的情况下，本领域技术人员可以对其余非活性位点的氨基酸序列进行改变，使得到的转氨酶的氨基酸序列与SEQ ID NO.：2所示的氨基酸序列的同一性至少在80％以上。这样得到转氨酶具有与氨基酸序列为SEQ ID NO.：2的转氨酶相同的转氨酶活性。
同理，在保留SEQ ID NO.：2所示的氨基酸序列中对转氨酶的催化活性起关键作用的氨基酸序列保持不变的情况下，可以对上述SEQ ID NO.：2所示的氨基酸序列中的氨基酸进行取代、缺失或添加一个或多个氨基酸，这样，由SEQ ID NO.：2所衍生的蛋白质就能保持SEQ ID NO.：2所示的转氨酶的高度立体选择性。其中，取代、缺失或添加的碱基可以为一个或多个，例如1个、2个、3个、4个、5个、10个、20个、30个或50个氨基酸，例如保守氨基酸的取代，其中该氨基酸序列不是如SEQ ID NO.：5所示的氨基酸序列；“保守氨基酸的替换”是指如Gly、Ala；Val、Ile、Leu；Asp、Glu；Asn、Gln；Ser、Thr；Lys、Arg；及Phe、Tyr的组合。
其中立体选择性是指，当一个反应生成A、B两个立体异构体时，A的产量比B多。高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1，例如至少约1.2倍，至少约1.3倍，至少约1.4倍，至少约1.5倍，至少约2倍，至少约3倍，至少约4倍，至少约5倍，至少约10倍，至少约15倍，至少约20倍，至少约30倍，至少约40倍，至少约50倍，至少约70倍，至少约90倍，至少约100倍，或更高。
在发明中，上述R型ω-转氨酶的修饰物可以是化学修饰物，如酰基化、烷基化、PEG化产物，只要这些修饰物保留了上述高度立体选择性-R构型催化活性的ω-转氨酶活性即可。上述功能等同物是指能够实现R型ω-转氨酶活性的其他多肽片段。上述功能片段是指保留了高度立体选择性-R构型催化活性的ω-转氨酶活性的蛋白质片段。 上述变体是指通过在一个或多个(几个)位置的一个或多个氨基酸进行改变，即通过取代、插入和/或缺失而从亲本蛋白衍生出来的多肽。
在本发明一种优选的实施例中，上述R型ω-转氨酶的氨基酸序列是对SEQ ID NO.：2所示的氨基酸序列中第199位的亮氨酸取代为缬氨酸的氨基酸序列。该经过氨基酸替换的转氨酶与SEQ ID NO.：2所示的转氨酶具有相同的活性和功能。
在另一种典型的实施方式中，提供了一种核苷酸，该核苷酸编码上述R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体。本发明的上所述的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体的核苷酸的编码规则符合常规的密码子使用表。
在本发明一种更优选的实施例中，上述核苷酸的序列包含选自如下序列之一的序列：a)如SEQ ID NO.：1所示的核苷酸序列；b)与SEQ ID NO.：1所示的核苷酸序列具有至少80％同一性且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；c)在高严谨条件下与SEQ ID NO.：1所示的核苷酸序列杂交且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；其中，高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。
上述与SEQ ID NO.：1所示的核苷酸序列具有至少80％同一性且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，例如至少具有85％、90％、95％、96％、97％、98％、99％、99.5％、99.7％、99.8％或99.9％的同一性，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列。在SEQ ID NO.：1所示的核苷酸序列的基础上，对转氨酶催化活性起关键作用的核苷酸序列保持不变的情况下，本领域技术人员可以对其余非活性位点的核苷酸序列进行改变，使得到的转氨酶的核苷酸序列与SEQ ID NO.：1所示的核苷酸序列的同一性至少在80％以上。这样得到转氨酶具有与核苷酸序列为SEQ ID NO.：1的转氨酶相同的转氨酶活性。
上述在高严谨条件下与SEQ ID NO.：1所示的核苷酸序列杂交且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；同样，在SEQ ID NO.：1所示的核苷酸序列的基础上，通过在高严谨条件下筛选能够与其杂交并编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，这样得到的SEQ ID NO.：1所示的核苷酸序列的变体序列，具有与核苷酸序列为SEQ ID NO.：1的转氨酶相同的转氨酶活性。
其中立体选择性是指，当一个反应生成A、B两个立体异构体时，A的产量比B多。高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1，例如至少约1.2倍，至少约1.3倍，至少约1.4倍，至少约1.5倍，至少约2倍，至少约3倍，至少约4倍，至少约5倍，至少约10倍，至少约15倍，至少约20倍，至少约30倍，至少约40倍，至少约50倍，至少约70倍，至少约90倍，至少约100倍，或更高。
一个示例性的高严谨条件可为用6X SSC，0.5％SDS的溶液，在65℃下杂交，然后用2X SSC，0.1％SDS和1X SSC，0.1％SDS各洗膜一次。
本发明中所使用的术语“同一性”具有本领域通常已知的含义即序列相似性或同源性，本领域技术人员也熟知测定不同序列间同一性的规则、标准。本发明用不同程度同一性限定的序列还必须要同时具有高度立体选择性-R构型催化活性的ω-转氨酶的活性。本领域技术人员公知如何利用该高度立体选择性-R构型催化活性的ω-转氨酶的活性筛选变体序列的方法和手段。本领域技术人员可以在本申请公开内容的教导下容易地获得这样的变体序列。
本领域技术人员知晓，虽然本发明在限定所述氨基酸序列或多核苷酸时所用限定语为“包括”，但其并不意味着可以在上述氨基酸序列或核苷酸序列两端任意加入与其功能不相关的其他序列。本领域技术人员知晓，为了满足重组操作的要求，需要在所述多核苷酸的两端添加合适的限制性内切酶的酶切位点，或者额外增加启动密码子、终止密码子等，因此，如果用封闭式的表述来限定上述序列将不能真实地覆盖这些情形。
本领域技术人员公知，在不改变所编码的氨基酸的情况下，上述核苷酸序列中的一个或多个密码子可以进行等义替换，如将由CTT编码的亮氨酸Leu替换为CTA、CTC或CTG.。替换的密码子数目可以是一个或几个密码子，如1、2、3、4、5、6、8、9、10、15、20、30、40、50个密码子。密码子使用表是本领域公知的。
在发明的又一方面，提供了一种重组载体，该重组载体中有效连接有上述任一种核苷酸。本发明的重组载体包括但不限于重组表达载体，还可以包括重组克隆载体。重组载体可以是原核表达载体或真核表达载体，在本发明一个具有的实施例中，上述重组载体是可诱导表达的重组原核表达载体，如利用IPTG诱导基因表达的pET系列载体，如pET22b载体。其中，“有效连接”是指这样的连接方式，即将多核苷酸置于载体的适当位置，使得多核苷酸正确地、顺利地复制、转录和/或翻译。在本发明又一种优选的实施例中，本发明的上述重组载体为含有SEQ ID NO.：1所示的核苷酸序列的pET22b-taC。
在本发明的再一方面，提供了一种宿主细胞，该宿主细胞转化或转染有上述任一种的重组载体。本发明的宿主细胞包括原核宿主细胞和真核宿主细胞，在本发明的一个实施例中，上述宿主细胞是原核宿主细胞，如大肠杆菌，更优选大肠杆菌DH5α(DE3)。
在本发明的另一方面中，提供了一种合成手性胺的方法，该方法包括如下步骤：使酮类化合物、权利要求1的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，磷酸吡哆醛和氨基供体在反应体系中反应，由此获得手性胺。本发明的合成手性胺的方法在本领域常规的通过生物酶催化反应来制备手性化合物的方法基础上，适当调整反应体系的各种反应原料的成分、比例、用量、pH值、温度、反应时间等参数即可。
在本发明一种优选的实施例中，上述酮类化合物为
Figure PCTCN2014089845-appb-000005
其中，R1和R2各自独立地为C1～C8烷基、C5～C10环烷基、C5～C10芳基或C5～C10杂芳基，或者R1和R2与羰基上的碳共同形成C5～C10杂环基、C5～C10碳环基或C5～C10杂芳基，C5～C10杂环基和C5～C10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，C5～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，酮类化合物
Figure PCTCN2014089845-appb-000006
选自
Figure PCTCN2014089845-appb-000007
或
Figure PCTCN2014089845-appb-000008
上述酮类化合物为商业化的原料或者易制备的原料且价格低廉，可以满足规模化生产的需要。
在本发明另一种优选的实施例中，上述反应体系中还含有促溶剂，促溶剂为二甲基亚砜或聚乙二醇，优选聚乙二醇为PEG-400。促溶剂的作用能够很好地溶解上述原料，以方便反应的进行，PEG-400的促溶效果更好。
在本发明又一种优选的实施例中，上述C1～C8烷基为C1～C8直链烷基，C5～C10杂芳基为吡啶基团，烷氧基为C1～C6烷氧基，C5～C10杂环基中的杂环基为哌啶，C5～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基上的取代基各自独立为C1～C6直链烷基、C1～C6烷氧基，氨基供体为异丙胺或D-丙氨酸。上述原料为商业化的原料或者易制备的原料且价格低廉，可以满足规模化生产的需要。
在本发明的一个优选的实施例中，上述反应体系含有使反应体系的pH值维持在7.0～9.5范围内的缓冲液；和/或其中酮类化合物与促溶剂的用量比为1g/1mL～15mL；和/或其中，酮类化合物与缓冲液的用量比为1g/15mL～50mL；和/或其中酮类化合物与磷酸吡哆醛的用量比为1g/0.01g～0.1g；和/或其中酮类化合物与氨基供体的用量比为1eq/1eq～5eq；和/或其中酮类化合物与R型ω-转氨酶的用量比为1g/0.2g～10g；和/或其中反应体系的温度为20～45℃且反应12h～48h；和/或其中缓冲液为磷酸盐缓冲液或pH＝9.3～9.5的三乙醇胺缓冲液。
在本发明另一个优选的实施例中，上述方法还包括用碱调节反应体系至pH≥10，用有机溶剂萃取水相中的产品手性胺的步骤，优选地，碱为氢氧化钠或氢氧化钾，有机溶剂为乙酸乙酯、甲基叔丁基醚或2-甲基四氢呋喃。
下面将结合具体的实施例来说明本发明的有益效果。
实施例1：制备R型ω-转氨酶
本发明的R型ω-转氨酶的制备方法的具体步骤如下：
(1)模板的构建：
在不改变氨基酸序列的前提下，通过对节杆菌属(Arthrobacter sp.)来源的转氨酶基因TAS部分核苷酸进行优化，合成(生工生物工程(上海)有限公司)优化的转氨酶基因taA(序列如SEQ ID NO.:4所示，对应的氨基酸序列如SEQ ID NO.:5所示)，合成的taA基因连接到载体pUC57上获得重组质粒pUC-taA，然后利用NdeⅠ和XhoⅠ限制性内切酶将重组质粒pUC-taA和载体pET-22b(+)同时进行酶切，酶切产物用T4DNA连接酶进行连接反应，并将连接产物转化到大肠杆菌DH5α菌株的感受态细胞中，经摇床复苏后涂布于含氨苄青霉素终浓度为50μg/ml的LB培养皿中，37℃培养箱培养过夜。挑取上述培养皿上的单菌落接种于含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，37℃，180r/min振荡培养过夜，提取质粒，经PCR和酶切鉴定，酶切鉴定结果见图3。
图3显示的是pET22b-taA质粒经NdeⅠ酶和XhoⅠ酶双酶切后的鉴定图，其中，1表示pET22b空载体；2表示DNA分子大小标记(从上至下分别为10000bp、8000bp、6000bp、5000bp、4000bp、3500bp、3000bp、2500bp、2000bp、1500bp、1000bp、750bp、500bp、250bp)；3表示pET22b-taA-DH5α。从图3中可以看出，酶切后的片段大小为1000bp左右的比较弱的条带即为目的片段(切除目的片度后的质粒条带相对较强)， 从而可以确定，重组质粒pET22b-taA的插入序列的插入方向和大小正确，然后将正确的重组质粒pET22b-taA作为下一步突变的模板。
(2)突变基因的克隆与表达：
根据NCBI报道设计一段基因：5'-ATGGCGTTTACTGTAGAAAGCCCGGCGAGTATGGATAAGGTCTTCGCCGGCTATGCTGCCCGCCAGGCTATCCTCGAAAGTACGGAGACGACGAACCCATTCGCCAAAGGTATTGCATGGGTCGAAGGTGAGTTAGTTCCGCTGGCCGAAGCACGTATCCCGATTCTCGATCAGGGCTTTTACTCCAGCGACGCGACC-3'(SEQ ID NO.：3)
以上述基因的正反向序列为引物，以上述重组质粒pET22b-taA为模板，进行全质粒PCR，PCR产物经DpnⅠ消化后转化至大肠杆菌BL21(DE3)中，经摇床复苏后涂布于含氨苄青霉素终浓度为50μg/ml的LB培养皿中，37℃培养过夜。挑取上述培养皿中的单菌落接种于5ml含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，37℃，180r/min培养过夜。取菌液送至生工生物工程(上海)有限公司测序，并将测序菌液的重组质粒命名为BL21/pET22b-taB，其测序结果与引物序列(SEQ_ID_NO.3)及taA比对结果见图4。
从图4中可以看出，测序结果中BL21/pET22b-taB质粒所携带的已将上述taA基因从ATG开始的198bp的序列完全替换为上述所设计的序列，与预期完全一致，无突变碱基。经测序验证正确后，将该重组质粒即为目标质粒序列。
将上述菌液转接于5ml含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，37℃，180r/min振荡培养至OD600值为0.6～0.8时，加入IPTG至终浓度为0.2mM，并将培养液转置25℃进行诱导表达，同时设立不加IPTG诱导剂的培养液为阴性对照。诱导16h后，取出菌液，12000r/min离心5min收集菌体。菌体用超声破碎仪破碎细胞，超声参数为：探头直径6mm，功率200W，工作2s，间歇6s，共计10min，超声完毕后于4℃，12000r/min离心20min获得超声上清液和沉淀，上清液用垂直电泳仪进行SDS-PAGE检测。
检测结果见图5的重组质粒pET22b-taB蛋白表达图，其中，1表示pET22b-taB；2表示pET22b-taA；3表示pET22b；4表示蛋白MARKER(从上至下分别为：97.4KDa、66.2KDa、43KDa、31KDa)。从图5中可以看出，结果表明蛋白表达良好，与TaA蛋白表达情况无明显区别。测序结果也表明插入的核苷酸序列是正确的。
(3)BL21/pET22b-taC的获取：
以上述重组质粒pET22b-taB为模版，进行标准的连续易错PCR(易错PCR反应体系为：10×PCR缓冲液10ul，dNTP 10mM 2ul，dCTP 10mM 1ul，dTTP 10mM 1ul，引物各10uM 1ul，模版50ng/ul 3ul，Taq DNA聚合酶0.5ul，MgCl2 7mM 1.4ul，MnCl20.05mM 0.2ul，H2O 76.9ul。引物有义链：5-CGCAAGTTAAGAACTTCCAATGGGGCG-3(SEQ ID NO.：6)。引物反义链：5-CCATTGGAAGTTCTTAACTTGCGGATCGATGC-3(SEQ ID NO.：7)，PCR产物经DpnⅠ消化后转化到大肠杆菌BL21(DE3)中，涂布于含氨苄青霉素终浓度为50μg/ml的LB培养皿中，37℃培养过夜，获得大量单菌落。挑取上述培养皿中上的单菌落接种于5ml含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，获得大量的突变体。
然后对所获得的大量的突变体进行转氨酶酶活验证，验证步骤如下：
将突变体菌液转接于100ml含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，37℃，180r/min振荡培养至OD600值为0.6～0.8时，加入IPTG至终浓度为0.2mM，并将培养液转置25℃进行诱导表达，同时设立不加IPTG诱导剂的培养液为阴性对照。诱导16h后，取出菌液，12000r/min离心5min收集菌体。称取0.5g菌泥重悬与2.5mL三乙醇胺缓冲液(pH9.5)菌体用超声破碎仪破碎细胞，超声参数为：探头直径6mm，功率200W，工作2s，间歇6s，共计10min，超声完毕后于4℃，12000r/min离心20min获得超声上清液和沉淀，上清液用于反应验证酶活。
向反应瓶中加入0.1g主原料(N-BOC-哌啶酮，CAS：79099-07-3)和0.1mL二甲基亚砜，原料分散后，加入7.5mL 0.2mol/L在冰浴条件下用浓盐酸调节pH为9.3-9.5的三乙醇胺缓冲液、0.591g异丙胺、0.0025g磷酸吡哆醛和2.5mL上述大量的突变的R型ω-转氨酶，体系pH为9.5，30℃恒温搅拌12h。体系用2N NaOH调节pH至10以上，用乙酸乙酯萃取两次，有机相经干燥，过滤，浓缩得到粗品(中文名称：(R)-1-N-Boc-3-氨基哌啶，CAS：188111-79-7)。经气相色谱(GC)检测，筛选到反应最好的一株突变体，转化率92.7％，e.e值100％。
经测序，即得到的酶活性最好的突变体的序列SEQ ID NO.：1，其表达的R型转氨酶命名为BL21/pET22b-taC。
(4)R型ω-转氨酶的制备：
取上述菌株BL21/pET22b-taC菌液接种于含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，37℃，180r/min振荡培养至OD600值在0.6-0.8时，加入IPTG至终浓度为0.2mM，并将培养液转置25℃进行诱导表达，诱导16h后，取出发酵液，12000r/min 离心10min收集菌体。菌体经细胞破碎后，于4℃，12000r/min离心20min获得上清液，即为制备所得R型ω-转氨酶，其氨基酸序列如SEQ ID NO.：2所示。
实施例2：R型ω-转氨酶的活性实验1
向反应瓶中加入1g主原料(N-BOC-哌啶酮，CAS：79099-07-3)和1mL二甲基亚砜，原料分散后，加入50mL 0.2mol/L在冰浴条件下用浓盐酸调节pH为9.3-9.5的三乙醇胺缓冲液、0.765g异丙胺、0.01g磷酸吡哆醛和0.01g上述氨基酸序列如SEQ ID NO.：2所示的R型ω-转氨酶，体系pH为9.5，30℃恒温搅拌12h。体系用2N NaOH调节pH至10以上，用乙酸乙酯萃取两次，有机相经干燥，过滤，浓缩得到粗品(中文名称：(R)-1-N-Boc-3-氨基哌啶，CAS：188111-79-7)，气相色谱(GC)检测，转化率90.8％，e.e值100％。
所得产品的核磁数据如下：1H-NMR(300MHz，CDCl3)δ4.00-3.78(m，2H)，3.80(m，2H)，3.60(m，1H)，1.90(m，1H)，1.70(m，1H)，1.60-1.40(m，12H)，1.30(m，1H)ppm。
实施例3：R型ω-转氨酶的活性实验2
向反应瓶中加入0.1g主原料(2,4-二氯苯乙酮，CAS:2234-16-4)和1.5mL聚乙二醇PEG-400，原料分散后，加入23.5ml磷酸盐缓冲液(pH8.0)、0.031g异丙胺、0.0075g磷酸吡哆醛和0.02g上述氨基酸序列如SEQ ID NO.：2所示的突变的R型ω-转氨酶,体系pH为8.0，45℃恒温搅拌20h。体系用2N NaOH调节pH至10以上，用乙酸乙酯萃取两次，有机相经干燥，过滤，浓缩得到粗品((R)-2,4-二氯苯乙胺)，GC检测，转化率95％，e.e值100％。
所得产品的核磁数据如下：1H NMR(400MHz，DMSO D6)：δ＝7.67(d 1H),7.60(d，1H)，7.47(dd，1H)，7.34(dd，4H)，7.23-7.12(m，6H)，4.84(s，1H)，4.47(quartet，1H)，1.31(d，3H)。
实施例4：R型ω-转氨酶的活性实验3
向反应瓶中加入0.1g主原料(2-萘乙酮，CAS：93-08-3)和1mL聚乙二醇PEG-400，原料分散后，加入24ml磷酸盐缓冲液(pH为7.0)、0.17g异丙胺、0.01g磷酸吡哆醛和0.004g上述突变的R型ω-转氨酶，体系pH为7.0，20℃恒温搅拌48h。体系用2NNaOH调节pH至10以上，用乙酸乙酯萃取两次，有机相经干燥，过滤，浓缩得到粗 品(R)-(+)-1-(2-萘基)乙胺CAS：3906-16-9)，GC检测，转化率27％，e.e值99.5％。
所得产品的核磁数据如下：1H NMR(400MHz，CDCl3)δ7.86-7.76(m，4H)，7.52-7.41(m，3H)，4.29(q，J＝6.4Hz，1H)，1.74(br s，2H)，1.48(d，J＝6.4Hz，3H)。
实施例5
在氨基酸序列为SEQ ID NO.：2的转氨酶的基础上，对该转氨酶在199位的异亮氨酸进行定点突变，将异亮氨酸替换为缬氨酸，得到由氨基酸序列为SEQ ID NO.：8所编码的转氨酶。
对该转氨酶进行酶活性实验进行检测，检测步骤如下：
将突变体菌液转接于100ml含氨苄青霉素终浓度为50μg/ml的LB液体培养基中，37℃，180r/min振荡培养至OD600值为0.6～0.8时，加入IPTG至终浓度为0.2mM，并将培养液转置25℃进行诱导表达，同时设立不加IPTG诱导剂的培养液为阴性对照。诱导16h后，取出菌液，12000r/min离心5min收集菌体。称取0.5g菌泥重悬与2.5mL三乙醇胺缓冲液(pH9.5)菌体用超声破碎仪破碎细胞，超声参数为：探头直径6mm，功率200W，工作2s，间歇6s，共计10min，超声完毕后于4℃，12000r/min离心20min获得超声上清液和沉淀，上清液用于投反应验证酶活。
向反应瓶中加入0.1g主原料(N-BOC-哌啶酮，CAS：79099-07-3)和0.1mL二甲基亚砜，原料分散后，加入7.5mL 0.2mol/L在冰浴条件下用浓盐酸调节pH为9.3-9.5的三乙醇胺缓冲液、0.591g异丙胺、0.0025g磷酸吡哆醛和2.5mL上述突变的R型ω-转氨酶，体系pH为9.5，30℃恒温搅拌12h。体系用2N NaOH调节pH至10以上，用乙酸乙酯萃取两次，有机相经干燥，过滤，浓缩得到粗品(中文名称：(R)-1-N-Boc-3-氨基哌啶，CAS：188111-79-7)，气相色谱(GC)检测，转化率91.6％，e.e值100％。
所得产品的核磁数据如下：1H-NMR(300MHz，CDCl3)δ4.00-3.78(m，2H)，3.80(m，2H)，3.60(m，1H)，1.90(m，1H)，1.70(m，1H)，1.60-1.40(m，12H)，1.30(m，1H)ppm。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：本发明公开的新型转氨酶催化了氨基供体中的氨基转移给前手性酮或醛类，从而产生相应R构型的手性胺，利用本发明的新型转氨酶进行合成胺的制备，不仅可以对更多的底物 进行转化，而得到R构型手性胺的纯度高，稳定在98％以上。所述合成方法采用的原料易得，方法简单，化学反应条件温和，收率和对映体的纯度均很高，整个生产过程中，操作简单，是可行的、污染较低的合成工艺，为制备手性胺提供了一种新的途径和方法。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 一种R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，其特征在于，所述R型ω-转氨酶的氨基酸序列包含选自如下序列之一的序列：
    a)如SEQ ID NO.：2所示的氨基酸序列；
    b)与SEQ ID NO.：2所示的氨基酸序列具有至少80％同一性且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的氨基酸序列，其中该氨基酸序列不是如SEQ ID NO.：4所示的核苷酸序列所编码的氨基酸序列；
    c)在SEQ ID NO.：2中所示的氨基酸序列经过取代、缺失或添加一个或多个氨基酸而由SEQ ID NO.：2衍生而来且具有高度立体选择性-R构型催化活性的ω-转氨酶活性的蛋白质，其中该氨基酸序列不是如SEQ ID NO.：4所示的核苷酸序列所编码的氨基酸序列；
    其中，高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。
  2. 根据权利要求1所述的转氨酶，其特征在于，所述R型ω-转氨酶的氨基酸序列是对SEQ ID NO.：2所示的氨基酸序列中第199位的亮氨酸取代为缬氨酸的氨基酸序列。
  3. 一种核苷酸，其特征在于，所述核苷酸编码权利要求1或2所述的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体。
  4. 根据权利要求3所述的核苷酸，其特征在于，所述核苷酸的序列包含选自如下序列之一的序列：
    a)如SEQ ID NO.：1所示的核苷酸序列；
    b)与SEQ ID NO.：1所示的核苷酸序列具有至少80％同一性且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；
    c)在高严谨条件下与SEQ ID NO.：1所示的核苷酸序列杂交且编码具有高度立体选择性-R构型催化活性的ω-转氨酶的核苷酸序列，其中该核苷酸序列不是如SEQ ID NO.：4所示的核苷酸序列；
    其中，高度立体选择性是指其中一个立体异构体的含量是另一个的至少约1.1倍。
  5. 一种重组载体，其特征在于，所述重组载体中有效连接有权利要求3或4所述的核苷酸。
  6. 根据权利要求5所述的重组载体，其特征在于，所述重组载体为pET22b-taC。
  7. 一种宿主细胞，其特征在于，所述宿主细胞转化或转染有权利要求5或6所述的重组载体。
  8. 一种合成R型手性胺的方法，其特征在于，所述方法包括如下步骤：使酮类化合物、权利要求1所述的R型ω-转氨酶或其修饰物、功能等同物、功能片段或变体，磷酸吡哆醛和氨基供体在反应体系中反应，由此获得R型手性胺。
  9. 根据权利要求8所述的方法，其特征在于，所述酮类化合物为
    Figure PCTCN2014089845-appb-100001
    其中，R1和R2各自独立地为C1～C8烷基、C5～C10环烷基、C5～C10芳基或C5～C10杂芳基，或者R1和R2与羰基上的碳共同形成C5～C10杂环基、C5～C10碳环基或C5～C10杂芳基，所述C5～C10杂环基和C5～C10杂芳基中的杂原子各自独立地选自氮、氧和硫中的至少一种，所述C5～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基各自独立地未被取代或被卤素、烷氧基或烷基中的至少一个基团所取代，优选地，所述酮类化合物
    Figure PCTCN2014089845-appb-100002
    选自
    Figure PCTCN2014089845-appb-100003
  10. 根据权利要求8或9所述的方法，其特征在于，所述反应体系中还含有促溶剂，所述促溶剂为二甲基亚砜或聚乙二醇，优选所述聚乙二醇为PEG-400。
  11. 根据权利要求9所述的合成手性胺的方法，其特征在于，所述C1～C8烷基为C1～C8直链烷基，所述C5～C10杂芳基为吡啶基团，所述烷氧基为C1～C6烷氧基，所述C5～C10杂环基中的杂环基为哌啶，所述C5～C10芳基中的芳基、C5～C10杂芳基中的杂芳基、C5～C10碳环基中的碳环基或C5～C10杂环基中的杂环基上的取代基各自独立为C1～C6直链烷基、C1～C6烷氧基，所述氨基供体为异丙胺或D-丙氨酸。
PCT/CN2014/089845 2013-11-26 2014-10-29 R型ω-转氨酶及其应用 WO2015078258A1 (zh)

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CN109468297B (zh) * 2018-11-30 2020-11-03 江南大学 一种能够催化西他沙星五元环中间体的ω-转氨酶突变体
WO2021077425A1 (zh) * 2019-10-25 2021-04-29 凯莱英医药化学（阜新）技术有限公司 转氨酶突变体及其应用
EP3977863A1 (en) * 2020-09-30 2022-04-06 Erber Aktiengesellschaft Means and methods to detoxify mycotoxins
WO2022069610A1 (en) * 2020-09-30 2022-04-07 Erber Aktiengesellschaft Means and methods to detoxify mycotoxins
CN113789310A (zh) * 2021-01-25 2021-12-14 上海予君生物科技发展有限公司 一种转氨酶及其在制备莫西沙星或其中间体中的应用
CN113789310B (zh) * 2021-01-25 2023-11-10 上海予君生物科技发展有限公司 一种转氨酶及其在制备莫西沙星或其中间体中的应用

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