WO2015078267A1 - 转氨酶及其应用 - Google Patents

转氨酶及其应用 Download PDF

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WO2015078267A1
WO2015078267A1 PCT/CN2014/090080 CN2014090080W WO2015078267A1 WO 2015078267 A1 WO2015078267 A1 WO 2015078267A1 CN 2014090080 W CN2014090080 W CN 2014090080W WO 2015078267 A1 WO2015078267 A1 WO 2015078267A1
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group
transaminase
seq
amino acid
acid sequence
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PCT/CN2014/090080
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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 US15/039,804 priority Critical patent/US10131926B2/en
Priority to ES14865656T priority patent/ES2734579T3/es
Priority to KR1020167017037A priority patent/KR101910259B1/ko
Priority to EP14865656.4A priority patent/EP3075847B1/en
Priority to JP2016535181A priority patent/JP6367944B2/ja
Publication of WO2015078267A1 publication Critical patent/WO2015078267A1/zh

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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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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    • 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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    • 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
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    • 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
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    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
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    • 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)
    • C12Y206/01018—Beta-alanine-pyruvate transaminase (2.6.1.18)

Definitions

  • the present invention relates to the field of synthesis of chiral compounds, in particular, a 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 aims to provide a novel transaminase and its use to meet the industrial production requirements of chiral amines.
  • a transaminase or a modification thereof, a functional equivalent, a functional fragment or a variant thereof wherein the amino acid sequence of the transaminase comprises a sequence selected from the group consisting of: a) SEQ ID NO.: amino acid sequence shown in 2 or 4; b) ⁇ -transaminase having at least 80% identity with the amino acid sequence shown in SEQ ID NO.: 2 or 4 and having a highly stereoselective-R configuration catalytic activity
  • An amino acid sequence which is active wherein the amino acid sequence is not the amino acid sequence encoded by the nucleotide sequence as shown in SEQ ID NO.: 5 or 6; c) the amino acid sequence shown in SEQ ID NO.: 2 or 4 a protein which is substituted, deleted or added with one or more amino acids and which is derived from SEQ ID NO.: 2 and has a highly stereoselective-R configuration catalytic activity of ⁇ -transa
  • amino acid sequence of the transaminase is an amino acid sequence in which the leucine at the 38th position of the amino acid sequence shown by SEQ ID NO.: 2 is replaced with the isoleucine.
  • nucleotide encoding a transaminase as described above or a modification, functional equivalent, functional fragment or variant thereof.
  • sequence of the above nucleotide comprises a sequence selected from the group consisting of a) a nucleotide sequence as shown in SEQ ID NO.: 1 or 3; b) and SEQ ID NO.: 1 or 3 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 as set forth in SEQ ID NO.: 5 or 6.
  • nucleotide sequence as shown; c) a nucleus of an ⁇ -transaminase which hybridizes to the nucleotide sequence shown in SEQ ID NO.: 1 or 3 under high stringency conditions and which encodes a catalytic activity having a highly stereoselective-R configuration a nucleotide sequence, wherein the nucleotide sequence is not a nucleotide sequence as shown in SEQ ID NO.: 5 or 6; wherein, highly stereoselective means that at least one of the stereoisomers is at least about the other 1.1 times.
  • a recombinant vector in which the above nucleotide is operably linked.
  • the recombinant vector is pET22b-CM32 or pET22b-CM33.
  • a host cell which is transformed or transfected with the above recombinant vector.
  • a method of a chiral amine comprising the steps of: a ketone compound, the above transaminase or a modification thereof, a functional equivalent, a functional fragment or a variant, pyridoxal phosphate and The amino donor is 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
  • 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 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 are each independently a C1-C6 linear alkyl group, a C1-C6 alkoxy group, and the amino donor is isopropylamine or D-alanine.
  • FIG. 1 is a flow chart showing the chemical reaction of a transaminase derived from Aspergillus terreus and S. cerevisiae in the synthesis of chiral amines according to an embodiment of the present invention
  • FIG. 3 shows the results of enzyme digestion identification in Example 1 of the present invention
  • Figure 4 is a view showing the sequencing results of the mutated gene after PCR in Example 1 of the present invention.
  • Figure 5 shows the results of the enzyme digestion identification in Example 4 of the present invention.
  • Fig. 6 shows the results of sequencing after PCR of the mutated gene in Example 4 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 may preferably have from 1 to about 20 carbon atoms, for example from 1 to about 10 carbon atoms, from 1 to about 8 carbon atoms, or from 1 to about 6 carbon atoms, or from 1 to about 4 carbon atoms.
  • alkoxy refers to alkyl ether groups (O to 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 radical having from 6 to about 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 about 5 to about 20, such as from 5 to 12 or from 5 to 10 backbones, wherein one or A plurality of (eg, 1 to 4, 1 to 3, 1 to 2) ring-forming atoms are heteroatoms independently selected from the group consisting of oxygen, nitrogen, sulfur, phosphorus, silicon, selenium, and tin. Atom, but not limited to this.
  • 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 about 15 ring-forming carbon atoms or 3 to about 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. The preferred are fluorine, chlorine and bromine.
  • the cyano group means "-CN”; the hydroxy group means “-OH”; the thiol group means “-SH”; and the amino group means "-NH 2 ".
  • 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) an amino acid sequence as set forth in SEQ ID NO.: 2 or 4; b) and SEQ ID NO ..
  • an amino acid sequence having an amino acid sequence of 2 or 4 having at least 80% identity and having a highly stereoselective-R configuration catalytic activity of ⁇ -transaminase activity wherein the amino acid sequence is not SEQ ID NO.: 5 or The amino acid sequence encoded by the nucleotide sequence shown in 6; c) the amino acid sequence shown in SEQ ID NO.: 2 or 4 is substituted, deleted or added with one or more amino acids and has a high stereoselectivity-R A protein derived from SEQ ID NO.: 2 or 4 that modulates catalytically active ⁇ -transaminase activity, wherein highly stereoselective means that one of the stereoisomers is at least about 1.1 times more than the other.
  • 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 or 4. .
  • the aminotransferase is a novel transaminase obtained by mutating and molecularly modifying the transaminase genes taAT and taHN derived from Aspergillus terreus and Hyphomonas neptunium.
  • the amino acid sequence of the above-mentioned ⁇ -transaminase activity having at least 80% identity with the amino acid sequence of SEQ ID NO.: 2 or 4 and having a highly stereoselective-R configuration catalytic activity means 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 set forth in SEQ ID NO.: The sequence of the sequence.
  • the amino acid sequence of the remaining inactive sites can be changed by those skilled in the art in the case where the amino acid sequence which retains the key function of the catalytic activity of the transaminase remains unchanged in the amino acid sequence shown by SEQ ID NO.: 2 or 4.
  • the amino acid sequence of the obtained transaminase is at least 80% identical to the amino acid sequence shown by SEQ ID NO.: 2.
  • the transaminase has the same transaminase activity as the aminotransferase shown in SEQ ID NO.: 2 or 4.
  • amino acid sequence which retains the catalytic activity of the transaminase in the amino acid sequence shown in SEQ ID NO.: 2 or 4 remains unchanged, it can be shown in SEQ ID NO.: 2 or 4 above.
  • the amino acid in the amino acid sequence is substituted, deleted or added with one or more amino acids, such that the protein derived from SEQ ID NO.: 2 or 4 maintains the height of the transaminase shown in SEQ ID NO.: 2 or 4.
  • Stereoselective 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 aminotransferase is an amino acid sequence in which the leucine at position 38 of the amino acid sequence shown by SEQ ID NO.: 2 is replaced with isoleucine.
  • the substitution between amino acids of similar nature is such that the transaminase having such an amino acid sequence after replacement maintains the activity and highly stereoselectivity of the transaminase having the amino acid sequence shown by SEQ ID NO.: 2.
  • the above-mentioned transaminase obtained by the present invention has a highly stereoselective-R configuration catalytic activity of ⁇ -transaminase, and can efficiently synthesize a chiral amine having a higher chiral purity in the R configuration, and is suitable for industrialization of chiral amine synthesis.
  • the invention optimizes the selection of splicing objects and splicing sites, so that the new transaminase variants obtained by the transformation not affect the folding of the protein, but also retain the activity of the transaminase, have higher transaminase activity, and have high stereoselection. Sex.
  • 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 the group consisting of: a) a nucleotide sequence as set forth in SEQ ID NO.: 1 or 3; b) and SEQ ID Nucleotide sequence of NO.: 1 or 3 having at least 80% identity and encoding a ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, wherein the nucleotide sequence is not SEQ ID NO.: the nucleotide sequence shown in 5 or 6; c) hybridizes under high stringency conditions to the nucleotide sequence set forth in SEQ ID NO.: 1 or 3 and encodes a highly stereoselective-R construct a nucleotide sequence of a catalytically active ⁇ -transaminase, wherein the nucleotide sequence is not a nucleotide sequence as shown in SEQ ID NO.: 5 or 6; highly stereoselective refer
  • nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO.: 1 or 3 and encoding an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, for example, having 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 SEQ ID NO.: 5 or The nucleotide sequence shown in 6.
  • the nucleotide sequence which plays a key role in the catalytic activity of transaminase remains unchanged on the basis of the nucleotide sequence shown in SEQ ID NO.: 1 or 3, those skilled in the art can leave 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 or 3.
  • the transaminase was obtained to have the same transaminase activity as the transaminase having the nucleotide sequence of SEQ ID NO.: 1 or 3.
  • nucleotide sequence which hybridizes to the nucleotide sequence shown by SEQ ID NO.: 1 or 3 under high stringency conditions and which encodes an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity, wherein the nucleoside
  • the acid sequence is not the nucleotide sequence shown in SEQ ID NO.: 5 or 6; likewise, the nucleotide shown in SEQ ID NO.: 1 or 3.
  • SEQ ID NO.: 1 or 3 thus obtained is obtained by screening a nucleotide sequence capable of hybridizing thereto and encoding an ⁇ -transaminase having a highly stereoselective-R configuration catalytic activity under high stringency conditions.
  • 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, 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 which induces gene expression by IPTG.
  • Vector such as the pET22b vector.
  • the recombinant vectors having the nucleotide sequences shown in SEQ ID NO.: 1 and SEQ ID NO.: 3 are pET22b-CM32 and pET22b-CM33.
  • "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.
  • 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: bringing a ketone compound, an R-type ⁇ -transaminase of any of the above, or a modification thereof, a functional equivalent, A functional 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 reaction, using the transaminase of the present invention, and appropriately adjusting the composition, ratio and amount of various reaction raw materials of the reaction system. , pH, temperature, reaction time and other parameters can be.
  • 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 a piperidine.
  • a pyridine 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 groups are each independently a C1-C6 linear alkyl group, a C1-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.
  • an R-type chiral amine which is synthesized by any of the above methods.
  • the R-type chiral amine prepared by using the above transaminase of the present invention has high chiral purity and can be as high as 98% or more.
  • Example 1 Preparation of transaminase AH-TACM33 derived from Aspergillus terreus and S.
  • the gene sequence, the amino acid sequence is shown in SEQ ID NO.: 23) and the taHN (S. faecalis) (the nucleotide sequence thereof is the gene sequence shown in SEQ ID NO.: 6 in the sequence listing, and the amino acid sequence is SEQ.
  • the synthetic taAT gene and the taHN gene were ligated to the pUC57 vector, respectively, to obtain recombinant plasmids pUC57-taAT and pUC57-taHN.
  • the recombinant plasmid pUC57-taAT and then the Nde I and Xho I restriction enzymes were used.
  • pUC57-taHN was simultaneously digested, and the purified recovered fragments taAT and taHN were obtained by gel recovery, and used as a template for the next PCR.
  • taAT A 5'-CCGCTCGAGGTTACGCTCGTTGTAGTCAATTTC-3' (SEQ ID NO.: 7)
  • taAT S 5'-GGAATTCCATATGGCGTCTATGGACAAAG-3' (SEQ ID NO.: 8)
  • taHN A 5'-CCGCTCGAGCGGTGCATAGGTTACCGGTTC-3' (SEQ ID NO.: 9)
  • taHN S 5'-GGAATTCCATATGCTGACCTTCCAAAAAGTACTGAC-3' (SEQ ID NO.: 10)
  • CM31A 5'-GAACTTCAGACCGCGGGTGACAATCAG-3' (SEQ ID NO.: 11)
  • CM31S 5'-CACCCGCGGTCTGAAGTTCCTGC-3' (SEQ ID NO.: 12)
  • CM32A 5'-CGGCGGAACACGACGAACGGTACG-3' (SEQ ID NO.: 13)
  • CM32S 5'-TTCGTCGTACTCCGCCGGGCGCAC-3' (SEQ ID NO.: 14)
  • CM33A 5'-TAGCCTGCGCCCTCGGTCAGGTGAG-3' (SEQ ID NO.: 15)
  • CM33S 5'-GACCGAGGGCGCAGGCTACAATATC-3' (SEQ ID NO.: 16)
  • CM34A 5'-CCCTTCAGACCACGCGTAACGATGATC-3' (SEQ ID NO.: 17)
  • CM34S 5'-TTACGCGTGGTCTGAAGGGTGTGCGTG-3' (SEQ ID NO.: 18)
  • CM35A 5'-CCAGGCGGAGTACGACGTACAGTACGAG-3' (SEQ ID NO.: 19)
  • CM35S 5'-TACGTCGTGTTCCGCCTGGCGCAATC-3' (SEQ ID NO.: 20)
  • CM36A 5'-GCCGCTGCCTTCCGTCGCGTTACC-3' (SEQ ID NO.: 21)
  • CM36S 5'-GACGGAAGGCAGCGGCTTCAACATC-3' (SEQ ID NO.: 22)
  • the specific primer taAT S (forward primer) designed on the transaminase gene derived from Aspergillus terreus is combined with any of the three reverse primers (CM31A, CM32A, CM33A) of the above six pairs of primers, a fragment for amplifying a transaminase gene derived from Aspergillus terreus; or using taHN A (reverse primer) in combination with any of the three forward primers (CM36S, CM35S, and CM34S) of the above six pairs of primers, Amplification of the source of S. cerevisiae A fragment of the transaminase gene. The two fragments from different sources obtained by the above amplification are then integrated to obtain a modified transaminase gene.
  • taAT A reverse primer
  • the two fragments from different sources obtained by the above amplification are then integrated to obtain a modified transaminase gene.
  • the specific transformation step is a transaminase obtained by integrating the taAT S forward primer and the CM33A reverse primer amplified fragment with the taHN A reverse primer and the CM33S forward primer, and the taAT S forward primer and the CM32A reverse primer.
  • the transaminase obtained by integrating the amplified fragment with the taHN A reverse primer and the CM32S forward primer amplified fragment is described in detail.
  • Fragment A The above-mentioned recovered fragment taAT was used as a PCR template, and taAT S and CM33A were used as primers to carry out PCR amplification, and the product was purified by gel recovery and was fragment A.
  • Fragment B was obtained: using the above-mentioned recovered fragment taHN as a PCR template, and using taHN A and CM33S as primers, PCR amplification was carried out, and the product was purified by gel recovery and was fragment B.
  • Fragment CM33 The fragment A and the fragment B obtained above were used as templates and primers. After 5 cycles of PCR amplification, primers taAT S and taHN A were directly added to the PCR system, and PCR amplification was performed. The product was recovered by gel. After purification, it was fragment CM33.
  • PCR system fragment A 1 ⁇ L, fragment B 1 ⁇ L, PCR MIX 5 ⁇ L, ddH 2 O 4.5 ⁇ l;
  • the primers taAT S and taHN A were each added to the system at 0.2 ⁇ L.
  • CM33 and pET-22b(+) were simultaneously digested with Nde I and Xho I, T4 DNA ligase was ligated, and the ligation product was transformed into the competent state of Escherichia coli DH5 ⁇ strain.
  • the cells were resuscended on a shaker and plated in an LB culture dish containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured overnight in a 37 ° C incubator.
  • Figure 3 is a diagram showing the identification of pET22b-CM33 plasmid by Nde I enzyme and Xho I enzyme digestion, wherein 1 indicates pET22b empty vector; 2 indicates DNA molecular size marker (10000 bp, 8000 bp from top to bottom, respectively) 6000bp, 5000bp, 4000bp, 3500bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp); 3 denotes pET22b-CM33-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), and thus the recombinant plasmid pET22b- can be determined.
  • the insertion direction and size of the insertion sequence of CM33 are correct.
  • 5BL21/pET22b-CM33 The recombinant plasmid pET22b-CM33 obtained above was directly transformed into E. coli BL21 (DE3), and was resuscitated by shaker and applied to an LB culture dish containing ampicillin at a final concentration of 50 ⁇ g/ml. Incubate 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 bacteria solution was sent to the Bioengineering (Shanghai) Co., Ltd. for sequencing, and after being verified by gene sequencing, it was named BL21/pET22b-CM33.
  • the supernatant is obtained by centrifugation at 12000 r/min for 20 min at 4 ° C, which is the prepared transaminase AH-TACM33, the amino acid sequence of which is shown in SEQ ID NO.: 4, and the corresponding nucleotide sequence is as follows. SEQ ID NO.: 3 is shown.
  • 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, 0.02 g of the above AH-TACM33 transaminase, the system pH was 8.0, and the mixture was stirred at 45 ° C for 20 h. The system was adjusted to pH 10 with 2N NaOH, extracted twice with ethyl acetate. The organic phase was dried, filtered and concentrated to give crude ((R)-2,4-dichlorophenethylamine). %, ee value is 100%.
  • Example 4 Preparation of transaminase AH-TACM32 derived from Aspergillus terreus and S.
  • Recombinant plasmids pUC57-taAT and pUC57-taHN were obtained as described in Example 1.
  • the recombinant plasmids pUC57-taAT and pUC57-taHN were simultaneously digested with Nde I and Xho I restriction enzymes, and the recovered fragments taAT and taHN were obtained by gel recovery and used as templates for the next PCR.
  • Fragment E was obtained: using the above-mentioned recovered fragment taAT as a PCR template, and using taAT S and CM32A as primers, PCR amplification was carried out, and the product was purified by gel recovery and was fragment E.
  • Fragment F was obtained: using the above-mentioned recovered fragment taHN as a PCR template, and using taHN A and CM32S as primers, PCR amplification was carried out, and the product was purified by gel recovery and was fragment F.
  • Fragment CM32 was obtained: the fragment E and the fragment F obtained above were used as templates and primers, and after 5 cycles of PCR amplification, primers taAT S and taHN A were directly added to the PCR system, and PCR amplification was carried out, and the product was recovered by gel. After purification, it was the fragment CM32.
  • Recombinant plasmid pET22b-CM32 was obtained: Nd I and Xho I were used to simultaneously digest the above fragments CM32 and pET-22b(+), T4 DNA ligase was ligated, and the ligation product was transformed into Escherichia coli DH5 ⁇ strain. The cells were incubated in a LB culture dish containing ampicillin at a final concentration of 50 ⁇ g/ml after shaking through a shaker, and cultured overnight in a 37 ° C incubator.
  • a single colony on the above culture dish was inoculated into LB liquid medium containing ampicillin at a final concentration of 50 ⁇ g/ml, and cultured overnight at 37 ° C, shaking at 180 r/min, and the plasmid was extracted, identified by PCR and enzyme digestion, and identified by enzyme digestion. The results are shown in Figure 5.
  • Figure 5 is a diagram showing the identification of the pET22b-CM32 plasmid by Nde I enzyme and Xho I enzyme digestion, wherein 1 indicates the DNA molecular size marker (10000 bp, 8000 bp, 6000 bp, 5000 bp, 4000 bp from top to bottom, respectively). 3500 bp, 3000 bp, 2500 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp); 2 indicates pET22b-CM32-DH5 ⁇ , and 3 indicates pET22b empty vector.
  • 1 indicates the DNA molecular size marker (10000 bp, 8000 bp, 6000 bp, 5000 bp, 4000 bp from top to bottom, respectively). 3500 bp, 3000 bp, 2500 bp, 2000 bp, 1500 bp, 1000 bp, 750 bp, 500 bp, 250 bp
  • 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), and thus the recombinant plasmid pET22b- can be determined.
  • the insertion direction and size of the insertion sequence of CM32 were correct, and the recombinant plasmid pET22b-CM32 was obtained.
  • 5BL21/pET22b-CM32 The recombinant plasmid pET22b-CM32 obtained above was directly transformed into E. coli BL21 (DE3), and was resuscitated by shaker and applied to an LB culture dish containing ampicillin at a final concentration of 50 ⁇ g/ml. Incubate 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 bacteria solution was sent to the Bioengineering (Shanghai) Co., Ltd. for sequencing, and after being verified by gene sequencing, it was named BL21/pET22b-CM32.
  • the supernatant is obtained by centrifugation at 12000 r/min for 20 min at 4 ° C, which is the prepared transaminase AH-TACM32, the amino acid sequence of which is shown in SEQ ID NO.: 2, and the corresponding nucleotide sequence is as follows. SEQ ID NO.: 1 is shown.
  • 0.1 g of the main raw material (2-naphthylethyl ketone, CAS: 93-08-3) and 1 mL of polyethylene glycol PEG-400 were added to the reaction flask, and after the raw materials were dispersed, 24 ml of phosphate buffer (pH 7.0) was added. 0.17 g of isopropylamine, 0.01 g of pyridoxal phosphate and 0.004 g of the above AH-TACM32 transaminase, the system pH was 7.0, and the mixture was stirred at a constant temperature of 20 ° C for 48 h. The system was adjusted to pH 10 or more with 2N NaOH and extracted twice with ethyl acetate. The organic phase was dried, filtered and concentrated to give a crude product, which was obtained by GC, with a conversion of 20% and e.e.
  • the present invention also verified the transaminase activity of the transaminase AH-TACM33 using the main raw material (2-naphthylethyl ketone, CAS: 93-08-3), using the main raw material (N-BOC-piperidone, CAS: 79099-07-3 And the main starting material (2,4-dichloroacetophenone, CAS: 2234-16-4) verified the transaminase activity of the transaminase AH-TACM32, and the specific method steps were the same as the above examples.
  • transaminase On the basis of the aminotransferase SEQ ID NO.: 2 transaminase, AH-TACM32, the transaminase was subjected to site-directed mutagenesis at position 38, and leucine was replaced with isoleucine to obtain the sequence of SEQ ID. NO.: 25 transaminase.
  • 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 sludge and resuspend the cells with 2.5mL of 0.1M phosphate buffer (pH 8.0).
  • the ultrasonic parameters are: probe diameter 6mm, power 200W, work 2s, intermittent 6s, total 10min
  • 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 (acetophenone, CAS: 98-86-2) was added to the reaction flask, and the raw material was dispersed with 13.5 mL of 0.1 M phosphate buffer (pH 8.0), 0.356 g of D-alanine, and 0.002 g of ⁇ . -NAD+, 0.0192 g lactate dehydrogenase 0.006 g glucose dehydrogenase, 0.432 glucose, 0.004 g pyridoxal phosphate and 2.5 mL sequence R-type ⁇ -transaminase encoded by SEQ ID NO.: 25, system pH 7.0, 30 Stir at °C for 16 h.
  • the system adjusts the pH with 2N NaOH To 10 or more, it is extracted twice with ethyl acetate, and the organic phase is dried, filtered and concentrated to give a crude product (Chinese name: (R)-1-phenylethylamine, CAS: 3886-69-9), gas chromatography (GC) Detection, conversion rate of 83%, ee value of 99.5%.
  • GC gas chromatography
  • 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 can obtain a high-purity target product by the synthesis method of the novel transaminase of the present invention, and the optical purity of the obtained product is stabilized 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

提供了一种转氨酶及其应用。所述转氨酶具有SEQ ID NO:2或4所示的氨基酸序列，或与SEQ ID NO:2或4所示的氨基酸序列具有至少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，其中所述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。所述转氨酶能够合成手性纯度较高的R构型手性胺，适合手性胺合成的工业化应用。

Description

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，一种转氨酶及其应用。
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新的转氨酶及其应用，以适应手性胺的工业化生产需求。
为了实现上述目的，根据本发明的一个方面，提供了一种转氨酶或其修饰物、功能等同物、功能片段或变体，该转氨酶的氨基酸序列包含选自如下序列的序列：a)如SEQ ID NO.：2或4所示的氨基酸序列；b)与SEQ ID NO.：2或4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.：5或6所示的核苷酸序列所编码的氨基酸序列；c)在SEQ ID NO.：2或4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.：5或6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，转氨酶的氨基酸序列是将SEQ ID NO.：2所示的氨基酸序列的第38位上的亮氨酸替换为异亮氨酸的氨基酸序列。
根据本发明的另一个方面，提供了一种核苷酸，该核苷酸编码上述的转氨酶或其修饰物、功能等同物、功能片段或变体。
进一步地，上述核苷酸的序列包含选自如下序列的序列：a)如SEQ ID NO.：1或3所示的核苷酸序列；b)与SEQ ID NO.：1或3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.：5或6所示的核苷酸序列；c)在高严谨条件下与SEQ ID NO.：1或3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.：5或6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。
根据本发明的再一个方面，提供了一种重组载体，该重组载体中有效连接有上述核苷酸。
进一步地，重组载体为pET22b-CM32或pET22b-CM33。
根据本发明的又一个方面，提供了一种宿主细胞，该宿主细胞转化或转染有上述重组载体。
根据本发明的一个方面，提供了一种手性胺的方法，该方法包括如下步骤：使酮类化合物、上述转氨酶或其修饰物、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 PCTCN2014090080-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 PCTCN2014090080-appb-000002
选自
Figure PCTCN2014090080-appb-000003
或
Figure PCTCN2014090080-appb-000004
进一步地，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，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为本发明一个实施方式所述的来源于土曲霉和海王生丝单胞菌的转氨酶在手性胺合成中的应用的化学反应流程图；
图2为本发明一个实施方式所述的来源于土曲霉和海王生丝单胞菌的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中将突变基因PCR后的测序结果；
图5示出了本发明的实施例4中的酶切鉴定结果；以及
图6示出了本发明实施例4中的突变基因PCR后的测序结果。
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。
定义
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～约20个碳原子，例如具有1～约10个碳原子，具有1～约8个碳原子，或1～约6个碳原子，或1～约4个碳原子或1～约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～约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，其包含约5至约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～约15个成环碳原子或3～约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。首选是氟，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的序列：a)如SEQ ID NO.：2或4所示的氨基酸序列；b)与SEQ ID NO.：2或4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.：5或6所示的核苷酸序列所编码的氨基酸序列；c)在SEQ ID NO.：2或4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-转氨酶活性的由SEQ ID NO.：2或4所衍生的蛋白质，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。
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或4所示的转氨酶。上述 转氨酶是本发明采用分子生物学技术，将来源于土曲霉(Aspergillus terreus)和海王生丝单胞菌(Hyphomonas neptunium)的转氨酶基因taAT和taHN进行突变及分子改造获得新的转氨酶。
上述与SEQ ID NO.：2或4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或4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或4所示的转氨酶相同的转氨酶活性。
同理，在保留SEQ ID NO.：2或4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或4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或4所衍生的蛋白质就能保持SEQ ID NO.：2或4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，该转氨酶的氨基酸序列是将SEQ ID NO.：2所示的氨基酸序列的第38位上的亮氨酸替换为异亮氨酸的氨基酸序列，这种性质相似的氨基酸之间的替换使得具有这种替换后的氨基酸序列的转氨酶保持了具有SEQ ID NO.：2所示的氨基酸序列的转氨酶的活性和高度立体选择性。
本发明所获得的上述转氨酶拥有高度立体选择性-R构型催化活性的ω-转氨酶，可以高效地合成手性纯度较高的R构型的手性胺，适合用于手性胺合成的工业化。本发明通过对拼接对象及拼接位点的优化选择，使得改造所得的新的转氨酶变体在既不影响蛋白的折叠，又能保留转氨酶的活性，拥有较高的转氨酶活性，且有高度立体选择性。
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的序列：a)如SEQ ID NO.：1或3所示的核苷酸序列；b)与SEQ ID NO.：1或3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.：5或6所示的核苷酸序列；c)在高严谨条件下与SEQ ID NO.：1或3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.：5或6所示的核苷酸序列；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。
上述与SEQ ID NO.：1或3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.：5或6所示的核苷酸序列。在SEQ ID NO.：1或3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或3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或3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或3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.：5或6所示的核苷酸序列；同样，在SEQ ID NO.：1或3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或3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或3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，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。在本发明中，更优选带有SEQ ID NO.：1和SEQ ID NO.：3所示核苷酸序列的重组载体为pET22b-CM32和pET22b-CM33。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、上述任一种的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，利用本发明转氨酶，并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 PCTCN2014090080-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 PCTCN2014090080-appb-000006
选自
Figure PCTCN2014090080-appb-000007
或
Figure PCTCN2014090080-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。
在本发明另一种典型的实施方式中，提供了一种R型手性胺，该R型手性胺采用上述任一种方法合成。利用本发明的上述转氨酶所制备的R型手性胺，手性纯度高，可高达98％以上。
下面将结合具体的实施例来说明本发明的有益效果。
下述实施例中如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例1：制备来源于土曲霉和海王生丝单胞菌的转氨酶AH-TACM33
本发明的转氨酶AH-TACM33的制备方法的具体步骤如下：
(1)模板的构建：
委托生工生物工程(上海)有限公司全基因合成来源于土曲霉和海王生丝单胞菌的转氨酶基因taAT(土曲霉)(其核苷酸序列为序列表中SEQ ID NO.：5所示的基因序列，氨基酸序列如SEQ ID NO.：23所示)和taHN(海王生丝单胞菌)(其核苷酸序列为序列表中SEQ ID NO.：6所示的基因序列，氨基酸序列如SEQ ID NO.：24所示)，合成的taAT基因和taHN基因分别连接到pUC57载体上，获得重组质粒pUC57-taAT和pUC57-taHN。然后利用Nde Ⅰ和Xho Ⅰ限制性内切酶将重组质粒pUC57-taAT和 pUC57-taHN同时进行酶切，经胶回收获得纯化的回收片段taAT和taHN，并将其作为下一步PCR的模板。
(2)引物设计：
根据土曲霉来源的转氨酶基因设计的特异性引物如下：
taAT A:5’-CCGCTCGAGGTTACGCTCGTTGTAGTCAATTTC-3’(SEQ ID NO.：7)
taAT S:5’-GGAATTCCATATGGCGTCTATGGACAAAG-3’(SEQ ID NO.：8)
根据海王生丝单胞菌来源的转氨酶基因设计的特异性引物如下：
taHN A:5’-CCGCTCGAGCGGTGCATAGGTTACCGGTTC-3’(SEQ ID NO.：9)
taHN S:5’-GGAATTCCATATGCTGACCTTCCAAAAAGTACTGAC-3’(SEQ ID NO.：10)
同时根据不同位点设计了6对引物，分别为：
CM31A：5’-GAACTTCAGACCGCGGGTGACAATCAG-3’(SEQ ID NO.：11)
CM31S：5’-CACCCGCGGTCTGAAGTTCCTGC-3’(SEQ ID NO.：12)
CM32A：5’-CGGCGGAACACGACGAACGGTACG-3’(SEQ ID NO.：13)
CM32S：5’-TTCGTCGTACTCCGCCGGGCGCAC-3’(SEQ ID NO.：14)
CM33A：5’-TAGCCTGCGCCCTCGGTCAGGTGAG-3’(SEQ ID NO.：15)
CM33S：5’-GACCGAGGGCGCAGGCTACAATATC-3’(SEQ ID NO.：16)
CM34A：5’-CCCTTCAGACCACGCGTAACGATGATC-3’(SEQ ID NO.：17)
CM34S：5’-TTACGCGTGGTCTGAAGGGTGTGCGTG-3’(SEQ ID NO.：18)
CM35A：5’-CCAGGCGGAGTACGACGTACAGTACGAG-3’(SEQ ID NO.：19)
CM35S：5’-TACGTCGTGTTCCGCCTGGCGCAATC-3’(SEQ ID NO.：20)
CM36A：5’-GCCGCTGCCTTCCGTCGCGTTACC-3’(SEQ ID NO.：21)
CM36S：5’-GACGGAAGGCAGCGGCTTCAACATC-3’(SEQ ID NO.：22)
(3)新转氨酶的获得：
按照在土曲霉来源的转氨酶基因上设计的特异性引物taAT S(正向引物)与上述6对引物中的3条反向引物(CM31A、CM32A、CM33A)中的任一条反向引物进行组合，用来扩增土曲霉来源的转氨酶基因的片段；或者利用taHN A(反向引物)与上述6对引物中的3条正向引物(CM36S、CM35S以及CM34S)中的任一条进行组合，用来扩增海王生丝单胞菌来源 的转氨酶基因的片段。然后将上述扩增得到的来源不同的两个片段进行整合，从而得到改造的转氨酶基因。
同样，按照在土曲霉来源的转氨酶基因上设计的特异性引物taAT A(反向引物)与上述6对引物中的3条正向引物(CM33S、CM32S、CM31S)中的任一条正向引物进行组合，用来扩增土曲霉来源的转氨酶基因的片段；或者利用taHN S(正向引物)与上述6对引物中的3条反向引物(CM34A、CM35A以及CM36A)中的任一条进行组合，用来扩增海王生丝单胞菌来源的转氨酶基因的片段。然后将上述扩增得到的来源不同的两个片段进行整合，从而得到改造的转氨酶基因。
具体改造步骤以taAT S正向引物和CM33A反向引物扩增的片段与taHN A反向引物和CM33S正向引物扩增得到的片段整合得到的转氨酶以及taAT S正向引物和CM32A反向引物扩增的片段与taHN A反向引物和CM32S正向引物扩增的片段整合得到的转氨酶为例进行详细说明。
以下是获得转氨酶AH-TACM33的步骤：
①片段A的获得：以上述回收片段taAT为PCR模板，以taAT S和CM33A为引物，进行PCR扩增，产物经胶回收纯化后即为片段A。
②片段B的获得：以上述回收片段taHN为PCR模板，以taHN A和CM33S为引物，进行PCR扩增，产物经胶回收纯化后即为片段B。
③片段CM33的获得：以上述获得的片段A和片段B互为模板和引物，PCR扩增5个循环后直接向PCR体系中添加引物taAT S和taHN A，重叠PCR扩增，产物经胶回收纯化后即为片段CM33。
PCR体系：片段A 1μL，片段B 1μL，PCR MIX 5μL，ddH2O 4.5μl；
PCR程序：95℃3min；(95℃30s，57℃30s，72℃90s，5个循环)；72℃1min；
体系中加入引物taAT S和taHN A各0.2μL。
PCR程序：95℃3min；(95℃30s，57℃30s，72℃90s，30个循环)；72℃10min。
④重组质粒pET22b-CM33的获得：利用Nde Ⅰ和Xho Ⅰ将上述片段CM33和pET-22b(+)同时酶切，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-CM33质粒经Nde Ⅰ酶和Xho Ⅰ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-CM33-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-CM33的插入序列的插入方向和大小正确。
⑤BL21/pET22b-CM33的获得：将上述获得的重组质粒pET22b-CM33直接转化至大肠杆菌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，经基因测序验证正确后，命名为BL21/pET22b-CM33。
其中，测序结果见图4。从图4中可以看出，测序结果中BL21/pET22b-CM33质粒所携带的基因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。
⑥AH-TACM33转氨酶的制备：取上述BL21/pET22b-CM33菌液转接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＝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，取出菌液，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，即为制备所得的转氨酶AH-TACM33，其氨基酸序列如SEQ ID NO.:4所示，对应的核苷酸序列如SEQ ID NO.:3所示。
实施例2：AH-TACM33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AH-TACM33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)，气相色谱法(GC)检测，转化率90％，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：AH-TACM33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，0.02g上述AH-TACM33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82％，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：制备来源于土曲霉和海王生丝单胞菌的转氨酶AH-TACM32
本发明的转氨酶AH-TACM32的制备方法的具体步骤如下：
(1)模板的构建：
按实施例1所述的方法获得重组质粒pUC57-taAT和pUC57-taHN。利用Nde Ⅰ和Xho Ⅰ限制性内切酶将重组质粒pUC57-taAT和pUC57-taHN同时进行酶切，经胶回收纯化后获得回收片段taAT和taHN，并将其作为下一步PCR的模板。
(2)引物设计：
同实施例1。
(3)新转氨酶的获得：
①片段E的获得：以上述回收片段taAT为PCR模板，以taAT S和CM32A为引物，进行PCR扩增，产物经胶回收纯化后即为片段E。
②片段F的获得：以上述回收片段taHN为PCR模板，以taHN A和CM32S为引物，进行PCR扩增，产物经胶回收纯化后即为片段F。
③片段CM32的获得：以上述获得的片段E和片段F互为模板和引物，PCR扩增5个循环后直接向PCR体系中添加引物taAT S和taHN A，重叠PCR扩增，产物经胶回收纯化后即为片段CM32。
④重组质粒pET22b-CM32的获得：利用Nde Ⅰ和Xho Ⅰ将上述片段CM32和pET-22b(+)同时酶切，T4 DNA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5。
图5显示的是pET22b-CM32质粒经Nde Ⅰ酶和Xho Ⅰ酶双酶切后的鉴定图，其中，1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)；2表示pET22b-CM32-DH5α，3表示pET22b空载体。
从图5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-CM32的插入序列的插入方向和大小正确，获得重组质粒pET22b-CM32。
⑤BL21/pET22b-CM32的获得：将上述获得的重组质粒pET22b-CM32直接转化至大肠杆菌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，经基因测序验证正确后，命名为BL21/pET22b-CM32。
其中，测序结果见图6。从图6中可以看出，测序结果中BL21/pET22b-CM32质粒所携带的基因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。
⑥AH-TACM32转氨酶的制备：取上述BL21/pET22b-CM32菌液转接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＝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，取出菌液，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，即为制备所得的转氨酶AH-TACM32，其氨基酸序列如SEQ ID NO.:2所示，对应的核苷酸序列如SEQ ID NO.:1所示。
实施例5：AH-TACM32转氨酶的活性实验
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AH-TACM32转氨酶，体系pH为7.0，20℃恒温搅拌48h。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，GC检测，转化率20％，e.e值100％。
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)
本发明还用主原料(2-萘乙酮，CAS：93-08-3)验证了转氨酶AH-TACM33的转氨酶活性，用主原料(N-BOC-哌啶酮，CAS：79099-07-3)和主原料(2，4-二氯苯乙酮，CAS:2234-16-4)验证了转氨酶AH-TACM32的转氨酶活性，具体方法步骤同上述实施例。
实施例6
在氨基酸序列为SEQ ID NO.：2的转氨酶即AH-TACM32的基础上，对该转氨酶在38位的亮氨酸进行定点突变，将亮氨酸替换为异亮氨酸，得到序列为SEQ ID NO.：25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2.5mL0.1M磷酸盐缓冲液(pH8.0)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(苯乙酮，CAS：98-86-2)，原料分散与13.5mL0.1M磷酸盐缓冲液(pH8.0)、0.356g D-丙氨酸、0.002gβ-NAD+，0.0192g乳酸脱氢酶0.006g葡萄糖脱氢酶，0.432葡萄糖，0.004g磷酸吡哆醛和2.5mL序列为SEQ ID NO.：25编码的R型Ω-转氨酶，体系pH为7.0，30℃恒温搅拌16h。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-苯乙胺，CAS：3886-69-9)，气相色谱(GC)检测，转化率83％，e.e值99.5％。
所得产品的核磁数据如下：1H NMR(CDCl3，400MHz，300K)δ(ppm)：7.36-7.29(m，4H)，7.26-7.19(m，1H)，4.11(q，J＝6.6Hz，1H)，1.53(bs，2H)，1.38(d，J＝6.6Hz，3H)。
结果表明，本发明的上述实施例中的转氨酶均可得到类似的收率和对映体纯度，获得相应的R构型手性胺。
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的合成方法可以得到高纯度的目标产物，所得产品光学纯度稳定在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思路和方法。
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或4所示的氨基酸序列；
    b)与SEQ ID NO.：2或4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.：5或6所示的核苷酸序列所编码的氨基酸序列；
    c)在SEQ ID NO.：2或4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或4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.：5或6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，所述转氨酶的氨基酸序列是将SEQ ID NO.：2所示的氨基酸序列的第38位上的亮氨酸替换为异亮氨酸的氨基酸序列。
  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或3所示的核苷酸序列；
    b)与SEQ ID NO.：1或3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.：5或6所示的核苷酸序列；
    c)在高严谨条件下与SEQ ID NO.：1或3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.：5或6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-CM32或pET22b-CM33。
  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。
  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 PCTCN2014090080-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 PCTCN2014090080-appb-100002
    选自
    Figure PCTCN2014090080-appb-100003
    或
    Figure PCTCN2014090080-appb-100004
  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。
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