WO2023185184A1 - 一种单胺氧化酶及其应用 - Google Patents

一种单胺氧化酶及其应用 Download PDF

Info

Publication number
WO2023185184A1
WO2023185184A1 PCT/CN2023/070432 CN2023070432W WO2023185184A1 WO 2023185184 A1 WO2023185184 A1 WO 2023185184A1 CN 2023070432 W CN2023070432 W CN 2023070432W WO 2023185184 A1 WO2023185184 A1 WO 2023185184A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
mutated
monoamine oxidase
serine
seq
Prior art date
Application number
PCT/CN2023/070432
Other languages
English (en)
French (fr)
Inventor
连磊
李小汝
刘桂智
华荣保
莫苏东
Original Assignee
青岛清原化合物有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 青岛清原化合物有限公司 filed Critical 青岛清原化合物有限公司
Publication of WO2023185184A1 publication Critical patent/WO2023185184A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C12Y104/03004—Monoamine oxidase (1.4.3.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present invention relates to a monoamine oxidase and its application in biocatalytic methods.
  • Patent CN102131813A discloses the use of monoamine oxidase to resolve and deracemize racemic chiral amines via stereospecific oxidation of one enantiomer to the corresponding imine with oxygen. It would nevertheless be desirable to provide new monoamine oxidases that can be used in the biocatalytic process.
  • the present invention also provides a monoamine oxidase, which comprises an amino acid sequence having the following mutations compared with the monoamine oxidase amino acid sequence shown in SEQ ID NO: 1: in the amino acid sequence corresponding to the monoamine oxidase shown in SEQ ID NO: 1
  • the 63rd amino acid was mutated from phenylalanine to leucine
  • the 65th amino acid was mutated from threonine to valine
  • the 100th amino acid was mutated from serine to proline
  • the 141st amino acid was mutated from threonine to Serine and/or the 234th amino acid is mutated from serine to cysteine.
  • the monoamine oxidase comprises an amino acid sequence having the following mutations compared to the monoamine oxidase amino acid sequence shown in SEQ ID NO: 1:
  • the 63rd amino acid is mutated from phenylalanine to leucine and the 65th amino acid is mutated from threonine to valine;
  • the 100th amino acid is mutated from serine to proline and the 234th amino acid is mutated from serine to cysteine;
  • the 141st amino acid is mutated from threonine to serine and the 234th amino acid is mutated from serine to cysteine;
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 100th amino acid is mutated from Serine mutates to proline
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 141st amino acid is mutated from Threonine mutates to serine
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 234th amino acid is mutated from Serine mutates to cysteine
  • the 100th amino acid is mutated from serine to proline
  • the 141st amino acid is mutated from threonine to serine
  • the 234th amino acid is mutated from serine to cysteine Acid
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 100th amino acid is mutated from Serine was mutated to proline and amino acid 141 was mutated from threonine to serine;
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 100th amino acid is mutated from Serine was mutated to proline and amino acid 234 was mutated from serine to cysteine;
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 141st amino acid is mutated from Threonine is mutated to serine and amino acid 234 is mutated from serine to cysteine; or,
  • the 63rd amino acid is mutated from phenylalanine to leucine
  • the 65th amino acid is mutated from threonine to valine
  • the 100th amino acid is mutated from Serine was mutated to proline
  • amino acid 141 was mutated from threonine to serine
  • amino acid 234 was mutated from serine to cysteine.
  • the amino acid sequence further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99% sequence identity.
  • the present invention provides a monoamine oxidase comprising at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any amino acid sequence selected from the group consisting of: Sexual amino acid sequence: SEQ ID NO: 2-17.
  • amino acid sequence of the monoamine oxidase is as shown in any one of SEQ ID NO: 2-17.
  • the present invention also provides a polynucleotide encoding the monoamine oxidase and a host cell containing the polynucleotide.
  • the present invention also provides a method for preparing substantially stereoisomerically pure compounds such as II
  • the method of the compound shown or its salt/hydrate, which includes making such as I The compounds shown are contacted with oxygen in the presence of the monoamine oxidase and cofactors.
  • the present invention also provides a method for preparing substantially enantiomerically pure compounds such as III
  • the compounds shown are contacted with oxygen in the presence of the monoamine oxidase, cofactors and bisulfite.
  • the present invention also provides a method for preparing substantially enantiomerically pure compounds such as IV
  • the compounds shown are contacted with oxygen in the presence of the monoamine oxidase, cofactors and bisulfite, and the resulting sulfamate compound is contacted with cyanide.
  • the cofactor is non-covalently associated with monoamine oxidase.
  • the cofactor is selected from the group consisting of: FAD, FMN, NAD, and NADP.
  • the method further comprises a component that catalyzes the disproportionation of hydrogen peroxide into molecular oxygen and water, preferably said component is selected from the group consisting of: Pd, Fe, and catalase.
  • the present invention also provides that the monoamine oxidase can catalyze such as I
  • the compounds shown are oxidized to substantially stereoisomerically pure compounds such as II Use of the indicated compounds or their salts/hydrates.
  • the present invention also provides that the monoamine oxidase can catalyze such as I
  • the compounds shown are prepared substantially enantiomerically pure as III
  • the sulfamate compound shown such as IV Application of the aminonitrile compound or its salt/hydrate as shown.
  • the present invention also provides that the monoamine oxidase can catalyze such as I Applications to desymmetrization of the compounds shown.
  • the practice of the techniques described herein may employ conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, bioemulsion generation, and sequencing techniques, which Within the skill of those skilled in the art.
  • Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and hybridization detection using labels.
  • Specific illustrations of suitable techniques can be obtained by reference to the examples herein. Of course, however, other equivalent conventional procedures may also be used.
  • Such general techniques and descriptions can be found in standard laboratory manuals such as Green et al., eds. (1999), Genome Analysis: A Laboratory Manual Series (Volume I-IV); Weiner, Gabriel, Stephens, eds.
  • oligonucleotide refers to one or more oligonucleotides
  • reference to an “automated system” includes reference to equivalents for use with systems known to those skilled in the art. Steps and methods, etc. Additionally, it should be understood that terms such as “left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width” may be used herein. ”, “upper”, “lower”, “interior”, “exterior”, “inner”, “outer”, etc.
  • the monoamine oxidase of the present disclosure capable of oxidizing an amine compound of structural formula (1) to the corresponding imine compound of structural formula (2) has one or more amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 1.
  • Such amino acid substitutions provide the monoamine oxidase with one or more improved properties, including increased enzyme activity, increased stereospecificity, increased thermal stability, increased solvent stability, decreased product inhibition, decreased substrate inhibition, or reaction byproducts. sensitivity is reduced.
  • Such amino acid substitutions may also improve the expression level, solubility and/or stability of the monoamine oxidase in host cells, such as as a recombinantly expressed protein in a heterologous host cell such as, but not limited to, an E. coli host cell.
  • the present disclosure also provides polynucleotides encoding such monoamine oxidases and methods of using the polypeptides in the disclosed biocatalytic methods.
  • “Monoamine oxidase” refers to a polypeptide that has the enzymatic ability to oxidize the compound of the above-mentioned structural formula I to the corresponding product of the above-mentioned structural formula II.
  • the polypeptide typically utilizes oxidized cofactors such as, but not limited to, flavin adenine dinucleotide (FAD), flavin adenine mononucleotide (FMN), nicotinamide adenine dinucleotide (NAD), or nicotinamide adenine dinucleotide (NAD).
  • FAD flavin adenine dinucleotide
  • FMN flavin adenine mononucleotide
  • NAD nicotinamide adenine dinucleotide
  • NADP nicotinamide adenine dinucleotide
  • the oxidized cofactor is FAD.
  • Monoamine oxidase includes naturally occurring
  • Coding sequence refers to that portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.
  • Naturally occurring or wild type refers to the form found in nature.
  • a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism that can be isolated from a natural source and that has not been intentionally modified by human manipulation.
  • Recombinant when used in connection with, for example, cells, nucleic acids or polypeptides means that has been modified in a manner that would not otherwise occur naturally, or is identical to the natural or inherent form of the material but is produced or derived from synthetic materials and/or by Materials produced or derived from operations using recombinant techniques, or materials that correspond to the natural or inherent form of the material.
  • Non-limiting examples include, but are not limited to, recombinant cells that express genes that are not present in the native (non-recombinant) form of the cell or that express native genes that are otherwise expressed at different levels.
  • Percent sequence identity and “percent homology” are used interchangeably herein and refer to a comparison between polynucleotides and polypeptides and are determined by comparing two optimally aligned sequences in a comparison window It is determined that the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence in which the two sequences are optimally aligned (which does not contain additions or deletions).
  • the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to give Percent sequence identity.
  • the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences or the number of positions where the nucleic acid base or amino acid residue aligns with the gap to derive the number of matching positions, using Divide the number of matching positions by the total number of positions in the comparison window and multiply the result by 100 to give percent sequence identity.
  • Those skilled in the art understand that there are many established algorithms for aligning two sequences.
  • Optimal alignment of the compared sequences can be performed by, for example, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482; by Needleman and Wunsch, 1970, J. Mol. Biol. .48:443 homology alignment algorithms; by similarity search methods of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; by computerized execution of these algorithms (in the GCG Wisconsin software package GAP, BESTFIT, FASTA, and TFASTA) or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., Editors, Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.
  • the word match string is then extended along each sequence in both directions as long as the cumulative alignment score can be increased.
  • the cumulative score is calculated using the parameters M (rewardscore for matched pairs of residues; always greater than 0) and N (penalty score for mismatched residues, always less than 0).
  • M return score for matched pairs of residues; always greater than 0
  • N penalty score for mismatched residues, always less than 0.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the comparison.
  • W wordlength
  • E expectation
  • BLASTP program uses the following default parameters: word length (W) of 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915).
  • Exemplary sequence alignments and determination of % sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI), using the default parameters provided.
  • Reference sequence refers to an established sequence used as a basis for sequence comparison.
  • a reference sequence may be a subsequence of a larger sequence, such as a segment of a full-length gene or polypeptide sequence.
  • a reference sequence is a nucleic acid or polypeptide that is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or full-length.
  • two polynucleotides or polypeptides can each (1) contain a sequence that is similar between the two sequences (i.e., a portion of the complete sequence) and (2) can also contain a sequence that is different between the two sequences, two ( Sequence comparisons between polynucleotides or polypeptides are typically performed by comparing the sequences of two polynucleotides within a "comparison window" to identify and compare local regions of sequence similarity.
  • Comparison window refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues in which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and in which the comparison The portion of the sequence in the window may contain 20% or less additions or deletions (ie, gaps) compared to a reference sequence (which contains no additions or deletions) to which the two sequences best align.
  • the comparison window can be longer than 20 contiguous residues and optionally includes 30 contiguous residues, 40 contiguous residues, 50 contiguous residues, 100 contiguous residues, or longer windows.
  • Substantial identity means at least 80% sequence identity, at least 85% identity compared to a reference sequence within a comparison window of at least 20 residue positions, usually within a window of at least 30-50 residues Identity and 89% to 95% sequence identity, and more typically at least 99% sequence identity, where the percent sequence identity is determined by comparing the reference sequence to a total of 20% or more including the reference sequence within the comparison window Fewer missing or added sequences than calculated.
  • the term "substantial identity” means that two polypeptide sequences have at least 80% when optimally aligned using default gap weights (gap weihgt), such as by the program GAP or BESTFIT Sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or higher (eg, 99% sequence identity).
  • residue positions that are not identical differ due to conservative amino acid substitutions.
  • Stereoselectivity refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which case one stereoisomer is favored over the other, or complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity refers to the enantioselectivity, that is, the fraction of one enantiomer in the sum of the two enantiomers (usually reported as a percentage).
  • stereoselectivity refers to diastereoselectivity, that is, the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), Often optionally reported as diastereomeric excess (d.e.).
  • Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.
  • High stereoselectivity refers to a monoamine oxidase polypeptide capable of converting a substrate into the corresponding product having a stereoisomer excess of at least about 99%.
  • Stereospecificity refers to the preferential transformation of one stereoisomer over another in a chemical or enzymatic reaction. Stereospecificity can be partial, in which case one stereoisomer is converted favorably over the other, or stereospecificity can be complete, in which case only one stereoisomer is converted.
  • Cromoselectivity refers to the preferential formation of one product over another in a chemical or enzymatic reaction.
  • “Improved enzymatic properties” refers to a monoamine oxidase polypeptide that exhibits any improvement in enzymatic properties compared to a reference monoamine oxidase.
  • comparisons to wild-type monoamine oxidase are generally performed, although in some embodiments, the reference monoamine oxidase may be another modified monoamine oxidase.
  • Enzyme properties that are desirable for improvement include, but are not limited to: enzyme activity (which can be expressed in terms of percent substrate conversion), thermal stability, pH activity profile, cofactor requirements, refractory to inhibitors (e.g., product inhibition), steric Specificity, stereoselectivity (including enantioselectivity), solubility and stability, and expression levels in host cells.
  • “Increased enzyme activity” refers to an improved property of an engineered monoamine oxidase polypeptide, which may be measured by an increase in specific activity (e.g., products produced/time/weight of protein) or percent substrate converted to product compared to a reference monoamine oxidase. An increase in conversion (eg, percent conversion of a starting amount of substrate to product over a specified period of time using a specified amount of monoamine oxidase) is represented. Exemplary methods for determining enzyme activity are provided in the Examples. Any property regarding enzyme activity can be affected, including the classic enzyme properties K m , V max or k cat , and their changes can lead to an increase in enzyme activity.
  • the improvement in enzymatic activity may be from about 1.5 times the enzymatic activity of the corresponding wild-type monoamine oxidase to as much as 2 times, 5 times, 10 times, 20 times the enzymatic activity of the naturally occurring monoamine oxidase or another engineered monoamine oxidase derived from the monoamine oxidase polypeptide. 25 times, 50 times, 75 times, 100 times or more.
  • the skilled artisan will understand that the activity of any enzyme is diffusion limited such that the rate of catalytic turnover does not exceed the diffusion rate of the substrate (including any required cofactors).
  • the theoretical maximum value of the diffusion limit or k cat /K m is typically about 10 8 to 10 9 (M ⁇ 1 s ⁇ 1 ).
  • any improvement in the enzymatic activity of a monoamine oxidase will have an upper limit related to the diffusion rate of the substrate on which the monoamine oxidase acts.
  • Monoamine oxidase activity can be measured using published methods for measuring monoamine oxidase or modifications thereof, such as, but not limited to, Zhou et al. ("A One-Step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity"Method)," 1997 Anal. Biochem. 253: 169-74) and Szutowicz et al.
  • Comparisons of enzymatic activities are performed using defined enzyme preparations, defined assays under set conditions and one or more defined substrates, as described in further detail herein or using, for example, the method of Zhou and Szutowicz.
  • comparing lysates determine the number of cells assayed and the amount of protein assayed and use the same expression system and the same host cells to minimize the difference in the amount of enzyme produced by the host cells and the amount of enzyme present in the lysate. change.
  • Conversion refers to the enzymatic oxidation of a substrate to the corresponding product.
  • Percent conversion refers to the percentage of substrate that is oxidized to product over a period of time under specified conditions.
  • the "enzymatic activity” or “activity” of a monoamine oxidase polypeptide may be expressed as the “percent conversion” of substrate to product.
  • Thermostable means that the monoamine oxidase polypeptide retains similar activity (e.g., greater than 60% to 80%).
  • solvent stable refers to a monoamine oxidase polypeptide that reacts well when exposed to varying concentrations (e.g., 5%-99%) of solvents (isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) maintain similar activity (greater than, for example, 60% to 80%) compared to the untreated enzyme after a period of time (eg, 0.5-24 hours).
  • solvents isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.
  • pH stable means that the monoamine oxidase polypeptide retains similar activity (greater than For example, 60% to 80%).
  • Heat-stable and solvent-stable refers to a monoamine oxidase polypeptide that is heat-stable and solvent-stable.
  • Hydrophilic amino acid or residue refers to an amino acid or residue having a side chain that exhibits hydrophobicity less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179:125-142 Residues.
  • Genetically encoded hydrophilic amino acids include L-Thr(T), L-Ser(S), L-His(H), L-Glu(E), L-Asn(N), L-Gln(Q), L -Asp(D), L-Lys(K) and L-Arg(R).
  • Acidic amino acid or residue refers to a hydrophilic amino acid or residue having a side chain that exhibits a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids often have negatively charged side chains due to the loss of hydrogen ions at physiological pH. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).
  • Basic amino acid or residue refers to a hydrophilic amino acid or residue having a side chain that exhibits a pK value greater than about 6 when the amino acid is included in a peptide or polypeptide.
  • Basic amino acids typically have positively charged side chains due to association with hydronium ions at physiological pH.
  • Genetically encoded basic amino acids include L-Arg(R) and L-Lys(K).
  • a "polar amino acid or residue” refers to a hydrophilic amino acid or residue having side chains that are uncharged at physiological pH but have an electron pair common to two atoms in which the electron pair is transferred between the two atoms. One is held more closely by at least one bond.
  • Genetically encoded polar amino acids include L-Asn(N), L-Gln(Q), L-Ser(S), and L-Thr(T).
  • Hydrophobic amino acid or residue refers to an amino acid or residue having a side chain that exhibits hydrophobicity greater than zero according to the standardized consensus hydrophobicity scale according to Eisenberg et al., 1984, J. Mol. Biol. 179:125-142 base.
  • Genetically encoded hydrophobic amino acids include L-Pro(P), L-Ile(I), L-Phe(F), L-Val(V), L-Leu(L), L-Trp(W), L- Met(M), L-Ala(A) and L-Tyr(Y).
  • Aromatic amino acid or residue refers to a hydrophilic or hydrophobic amino acid or residue having a side chain containing at least one aromatic or heteroaromatic ring.
  • Genetically encoded aromatic amino acids include L-Phe(F), L-Tyr(Y), and L-Trp(W).
  • L-His(H) is sometimes classified as a basic residue due to the pKa of its heterocyclic nitrogen atom or as an aromatic residue due to the inclusion of a heteroaromatic ring in its side chain, in this article histidine Acids are classified as hydrophilic residues or "constrained residues" (see below).
  • Restricted amino acid or residue refers to an amino acid or residue that has restricted geometric properties.
  • limiting residues include L-pro(P) and L-his(H). Histidine has restricted geometric properties due to its relatively small imidazole ring. Proline has restricted geometric properties since it also has a five-membered ring.
  • Nonpolar amino acid or residue means a hydrophobic amino acid or residue having side chains that are uncharged at physiological pH and have an electron pair common to two atoms in which the electron pair is generally held by each of the two atoms. A bond that is held to an equal degree (i.e. the side chain is not polar).
  • Genetically encoded non-polar amino acids include L-Gly(G), L-Leu(L), L-Val(V), L-Ile(I), L-Met(M), and L-Ala(A).
  • Aliphatic amino acid or residue refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala(A), L-Val(V), L-Leu(L), and L-Ile(I).
  • Cysteine The amino acid L-Cys(C) is unusual in that it can form disulfide bonds with other L-Cys(C) amino acids or other amino acids containing sulfoalkyl or sulfhydryl groups. "Cysteine-like residues” include cysteine and other amino acids containing sulfhydryl moieties that can be used to form disulfide bonds. The ability of L-Cys(C) (and other amino acids with -SH-containing side chains) to exist in peptides as reduced free -SH or oxidized disulfide forms affects whether L-Cys(C) renders the peptide net hydrophobic The characteristic is also hydrophilic.
  • L-Cys(C) exhibits a hydrophobicity of 0.29 according to the standardized consistency scale of Eisenberg (Eisenberg et al., 1984, supra), it should be understood that for the purposes of this disclosure, L-Cys(C) Classified into its own unique group.
  • Small amino acid or residue refers to an amino acid or residue having a side chain consisting of a total of three or fewer carbons and/or heteroatoms (excluding alpha-carbons and hydrogens). Small amino acids or residues may be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues according to the above definitions. Genetically encoded small amino acids include L-Ala(A), L-Val(V), L-Cys(C), L-Asn(N), L-Ser(S), L-Thr(T), and L- Asp(D).
  • Hydroxy-containing amino acid or residue refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser(S), L-Thr(T), and L-Tyr(Y).
  • Constant amino acid substitutions or mutations refer to the interchangeability of residues with similar side chains, and thus generally involve the substitution of amino acids in a polypeptide with amino acids in the same or similar defined classes of amino acids. However, as used herein, if a conservative mutation may instead be aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or For substitutions of restriction residues to restriction residues, conservative mutations do not include substitutions from hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residues to small residues. Furthermore, as used herein, A, V, L or I can be conservatively mutated to another aliphatic residue or to another non-polar residue. Table 1 below shows exemplary conservative substitutions. .
  • Non-conservative substitution refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between rather than within the defined groups listed above. In one embodiment, the non-conservative mutation affects (a) the structure of the peptide backbone in the region of substitution (eg, proline instead of glycine), (b) charge or hydrophobicity, or (c) side chain volume.
  • “Deletion” refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide.
  • Deletions may include removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids making up the reference enzyme, or up to 20% of the total number of amino acids making up the reference enzyme, while retaining enzymatic activity and/or retaining the improved properties of the engineered monoamine oxidase.
  • Deletions can be to the interior and/or ends of the polypeptide.
  • a deletion may comprise a contiguous segment or may be discontinuous.
  • “Insertion” refers to a modification of a polypeptide by the addition of one or more amino acids from a reference polypeptide.
  • improved engineered monoamine oxidases include the insertion of one or more amino acids into naturally occurring monoamine oxidases and the insertion of one or more amino acids into other modified monoamine oxidase polypeptides.
  • the insertion can be internal to the polypeptide, or at the carboxyl or amino terminus. Inserts as used herein include fusion proteins as known in the art. The insertion may be a continuous stretch of amino acids or separated by one or more amino acids in the naturally occurring polypeptide.
  • “Different from” or “different from” with respect to a given reference sequence refers to the difference in a given amino acid or polynucleotide sequence when aligned with the reference sequence. Generally, differences can be determined when two sequences are optimally aligned. Differences include insertions, deletions, or substitutions of amino acid residues compared to the reference sequence.
  • Fragment refers to a polypeptide having an amino-terminal and/or carboxyl-terminal deletion, but in which the remaining amino acid sequence is identical to the corresponding position in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer and up to 70%, 80%, 90%, 95%, 98% and 99% of the full length monoamine oxidase polypeptide.
  • isolated polypeptide refers to a polypeptide that is substantially separated from other contaminants with which it is naturally associated, such as proteins, lipids, and polynucleotides.
  • the term encompasses polypeptides removed or purified from their naturally occurring environment or expression system (eg, host cell or in vitro synthesis).
  • the modified monoamine oxidase can be present in the cells, in the cell culture medium, or prepared in various forms, such as lysates or isolated preparations.
  • the improved monoamine oxidase can be an isolated polypeptide.
  • substantially pure polypeptide refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant on a molar or weight basis than any other individual macromolecular species in the composition) and the composition
  • a substantially purified composition is generally one in which the subject matter constitutes at least about 50%, on a mole or weight percent basis, of the macromolecular species present.
  • a substantially pure monoamine oxidase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more.
  • the subject matter is purified to be substantially homogeneous (i.e., contaminating species cannot be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species. Solvent substances, small molecules ( ⁇ 500 Daltons) and elemental iron substances are not considered macromolecular substances.
  • the isolated improved monoamine oxidase polypeptide is a substantially pure polypeptide composition.
  • “Stringent hybridization” as used herein refers to conditions in which a nucleic acid hybrid is stable. As is known to those skilled in the art, the stability of a hybrid is reflected by the melting temperature ( Tm ) of the hybrid. Generally, the stability of hybrids depends on ionic strength, temperature, G/C content, and the presence of chaotropes.
  • Tm melting temperature
  • the Tm value of a polynucleotide can be calculated using known methods for predicting melting temperatures (see, e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al., 1962, Proc. Natl. Acad. Sci. USA 48: 1390; Bresslauer et al., 1986, Proc.
  • a polynucleotide encodes a polypeptide disclosed herein and hybridizes to the complement of a sequence encoding an engineered monoamine oxidase of the disclosure under defined conditions, such as moderately stringent or highly stringent condition.
  • Hybridization stringency refers to such wash conditions for nucleic acids. Typically, hybridization reactions are performed under lower stringency conditions, followed by different but higher stringency washes.
  • the term “moderately stringent hybridization” refers to a target DNA binding that is about 60% identical to the target DNA, preferably about 75% identical, about 85% identical; complementary to the target polynucleotide that is greater than about 90% identical. Nucleic acid conditions. Exemplary moderately stringent conditions are equivalent to the following: hybridization in 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS at 42°C, followed by 0.2x SSPE, Wash in 0.2% SDS.
  • High stringency hybridization generally refers to conditions that are about 10°C or less below the thermal melting temperature Tm determined under solution conditions defining the polynucleotide sequence.
  • high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C. (i.e., as expected here, if the hybrid is unstable in 0.018 M NaCl at 65°C, it will be unstable under highly stringent conditions).
  • High stringency conditions are provided by, for example, hybridization in conditions equivalent to 50% formamide, 5 ⁇ Denhart's solution, 5 ⁇ SSPE, 0.2% SDS at 42°C, followed by 0.1 ⁇ SSPE and 0.1 ⁇ SSPE at 65°C. Wash in 0.1% SDS.
  • Other highly stringent hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.
  • Heterologous polynucleotides refer to polynucleotides that are introduced into a host cell by laboratory techniques, and include polynucleotides that are removed from the host cell, subjected to laboratory manipulation, and then reintroduced into the host cell.
  • Codon optimized means that the codons of a polynucleotide encoding a protein are changed to those codons that are preferentially used in a particular organism so that the encoded protein is efficiently expressed in the organism of interest.
  • the genetic code is degenerate because most amino acids are represented by several codons (called “synonymous” or “synonymous” codons), it is well known that the codon usage of a given organism is non-random and a preference for specific codon triplets. This codon usage bias may be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins relative to low copy number proteins, and aggregated protein coding regions of the organism's genome.
  • a polynucleotide encoding a monoamine oxidase can be codon-optimized for optimal production from a host organism selected for expression.
  • Preferred, optimal, high codon usage bias codon refers interchangeably to a codon that is used with a higher frequency in a protein-coding region than a codon encoding the same amino acid.
  • Preferred codons can be determined with respect to codon usage in a single gene, codon usage in a group of genes with a common function or origin, codon usage in highly expressed genes, or in proteins aggregated throughout the organism.
  • codon frequency e.g., codon usage, relative synonymous codon usage
  • codon preference in a particular organism, including multivariate analysis, e.g., using cluster analysis or correspondence analysis and The effective number of codons used for a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J.O., 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29).
  • Codon usage tables are available for an increasing number of organisms (see, e.g., Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt, et al., eds., ASM Press, Washington D.C., pp. 2047-2066.
  • Obtaining codon usage data The source can rely on any available nucleotide sequence capable of encoding a protein.
  • nucleic acid sequences actually known to encode expressed proteins e.g., complete protein coding sequences-CDS
  • expressed sequence tags e.g., expressed sequence tags (ESTS)
  • Predicted coding regions of genomic sequences see, e.g., Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher , E.C., 1996, Methods Enzymol. 266: 259-281; Tiwari et al., 1997, Comput. Appl. Bioscl. 13: 263-270).
  • Control sequences are defined herein to include all components necessary or advantageous for expression of the polypeptides of the present disclosure.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide.
  • Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators.
  • control sequences include promoter, transcription, and translation termination signals.
  • the control sequences may have linker sequences for the purpose of introducing specific restriction sites that facilitate ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.
  • operably linked is defined herein as a construct in which a control sequence is appropriately positioned relative to the coding sequence of the DNA sequence such that the control sequence directs the expression of a polynucleotide and/or polypeptide.
  • a "promoter sequence” is a nucleic acid sequence recognized by a host cell for expression of a coding region. Control sequences may contain appropriate promoter sequences. The promoter sequence contains transcriptional control sequences that mediate expression of the polypeptide. The promoter can be any nucleic acid sequence that displays transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular genes that are homologous or heterologous to the host cell. Genetic acquisition of polypeptides.
  • stereoisomer As used herein, the terms "stereoisomer,” “stereoisomeric form” and similar terms are general terms used for all isomers of a single molecule, which differ only in the orientation of their atoms in space. It includes enantiomers and isomers ("diastereomers") of compounds having more than one chiral center that are not mirror images of each other.
  • chiral center refers to the carbon atoms to which four different groups are attached.
  • enantiomer or “enantiomeric” refers to molecules that are non-superimposable in their mirror images and are therefore optically active, where the enantiomer causes the plane of polarized light to rotate in one direction and its mirror image causes the plane of polarized light to rotate in the opposite direction direction of rotation.
  • racemic refers to a mixture of equal parts of optically inactive enantiomers.
  • resolution refers to the separation or concentration or elimination of one of the two enantiomeric forms of a molecule.
  • substantially enantiomerically pure as used herein means that a specified enantiomer of a compound is present to a greater extent than the other enantiomer of the same compound.
  • a substantially enantiomerically pure compound is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% enantiomeric excess is present.
  • substantially stereomerically pure as used herein means that a specified enantiomer or diastereomer of a compound is present to a greater extent than another enantiomer or diastereomer of the same compound.
  • enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.
  • a substantially stereoisomerically pure compound is present in a compound that is 80%, 85%, 90%, 91%, 92%, 93%, 94% less than another enantiomer or diastereomer of the same compound. %, 95%, 96%, 97%, 98% or 99% stereoisomer excess is present.
  • polypeptides described herein are not limited to genetically encoded amino acids.
  • the polypeptides described herein may comprise, in whole or in part, naturally occurring amino acids and/or synthetic non-encoded amino acids.
  • Monoamine oxidases described herein may contain certain commonly encountered non-encoded amino acids including, but not limited to: D-stereoisomers of genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); ⁇ -aminoisomers Butyric acid (Aib); ⁇ -aminocaproic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline ( Cit); tert-butylalanine (Bua); tert-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); n.
  • the monoamine oxidases disclosed herein may also comprise amino acids or residues having side chain protecting groups.
  • Such protected amino acids in this case belong to the aromatic category, non-limiting examples of which include (protecting groups are listed in brackets) but are not limited to: Arg (tos), Cys (methylbenzyl), Cys (nitropyridylsulfonyl), Glu ( ⁇ -benzyl ester), Gln (xanthyl), Asn (N- ⁇ -xanthyl), His (bom), His (benzyl), His (tos ), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).
  • Monoamine oxidases described herein may comprise conformationally restricted non-coding amino acids which may include, but are not limited to, N-methylamino acids (L-configuration); 1-aminocyclopent-(2 or 3)-en-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.
  • N-methylamino acids L-configuration
  • 1-aminocyclopent-(2 or 3)-en-4-carboxylic acid may include, but are not limited to, N-methylamino acids (L-configuration); 1-aminocyclopent-(2 or 3)-en-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.
  • the present disclosure provides polynucleotides encoding engineered monoamine oxidases disclosed herein.
  • the polynucleotide can be operably linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing the polypeptide.
  • Expression constructs containing heterologous polynucleotides encoding engineered monoamine oxidases can be introduced into appropriate host cells to express the corresponding monoamine oxidase polypeptides.
  • the polynucleotide comprises a nucleotide sequence encoding a monoamine oxidase having an amino acid sequence that has at least about 80% or more sequence identity as compared to any of the reference engineered monoamine oxidases described herein. , about 85% or higher sequence identity, about 90% or higher sequence identity, about 95% or higher sequence identity, about 96% or higher sequence identity, about 97% or higher High sequence identity, about 98% or higher sequence identity, or 99% or higher sequence identity.
  • codons that are appropriate for the host cell in which the protein is expressed.
  • a gene is expressed in a bacterium using preferred codons used in bacteria, expressed in yeast using preferred codons used in yeast; and expressed in a mammalian cell using preferred codons used in mammals. Express.
  • a codon-optimized polynucleotide encoding a monoamine oxidase may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of the codon positions of the full-length coding region.
  • the polynucleotides comprise polynucleotides that encode a monoamine oxidase described herein but have about 80% or greater sequence identity at the nucleotide level with a reference polynucleotide encoding an engineered monoamine oxidase. identity, about 85% or greater sequence identity, about 90% or greater sequence identity, about 95% or greater sequence identity, about 98% or greater sequence identity, or 99% or Higher sequence identity.
  • An isolated polynucleotide encoding a modified monoamine oxidase can be manipulated in a variety of ways to provide for expression of the polypeptide. Depending on the expression vector, manipulation of the isolated polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidelines are provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. Editor, Greene Pub. Associates, 1998, updated to 2006.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA ), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis ⁇ -amylase gene (amyL), Bacillus stearothermophilus maltose amylase gene (amyM) , Bacillus amyloliquefaciens ⁇ -amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic ⁇ -lactamase gene (Villa-Kamaroff et al., 1978, Proc.
  • promoters obtained from the E. coli lac operon Streptomyces coelicolor agarase gene (dagA ), Bacillus subtilis
  • suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include promoters obtained from the genes for the following enzymes: Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartate Acid protease, Aspergillus niger neutral ⁇ -amylase, Aspergillus niger acid-stable ⁇ -amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miha lipase, Aspergillus oryzae alkaline protease , Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase and Fusarium oxysporum trypsin-like protease (WO 96/00787); and the NA2-tpi promoter (from Aspergillus niger neutral ⁇ -
  • useful promoters may be derived from genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde- 3-phosphate dehydrogenase (ADH2/GAP) and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde- 3-phosphate dehydrogenase
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells are described by Romanos et al., 1992,
  • the control sequence may also be a suitable transcription terminator sequence, which is a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3'-end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the methods disclosed herein.
  • exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucoside enzyme and Fusarium oxysporum trypsin-like protease.
  • Exemplary terminators for use in yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.
  • Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
  • the control sequence may also be a suitable leader sequence, which is the untranslated region of the mRNA that is important for host cell translation.
  • the leader sequence is operably linked to the 5'-end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used.
  • Exemplary leader sequences for filamentous fungal host cells are obtained from the genes Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase.
  • Suitable leader sequences for use in yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde -3-phosphate dehydrogenase (ADH2/GAP).
  • ENO-1 Saccharomyces cerevisiae enolase
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
  • Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde -3-phosphate dehydrogenase ADH2/GAP
  • the control sequence may also be a polyadenylation sequence that is operably linked to the 3'-end of the nucleic acid sequence and that when transcribed is recognized by the host cell as adding polyadenylation to the transcribed mRNA. Sequence of signal for glycoside residues. Any polyadenylation sequence that is functional in the host cell of choice can be used in the methods disclosed herein.
  • Exemplary polyadenylation sequences for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum pancreatic Protease-like protein and Aspergillus niger alpha-glucosidase.
  • Polyadenylation sequences useful in yeast host cells are described by Guo and Sherman, 1995, Mol Cell Bio 15:5983-5990.
  • the control sequence may also be a signal peptide coding region encoding an amino acid sequence linked to the amino terminus of the polypeptide and directing the encoded polypeptide into the secretory pathway of the cell.
  • the 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame to the segment of the coding region encoding the secreted polypeptide.
  • the 5' end of the coding sequence may contain a signal peptide coding region foreign to the coding sequence. Foreign signal peptide coding regions may be required when the coding sequence does not naturally contain a signal peptide coding region.
  • the foreign signal peptide coding region can simply replace the native signal peptide coding region to enhance secretion of the polypeptide.
  • any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the host cell of choice can be used in the methods disclosed herein.
  • the effective signal peptide coding region of bacterial host cells is the signal peptide coding region obtained from the following genes: Bacillus NClB 11837 maltose amylase, Bacillus stearothermophilus ⁇ -amylase, Bacillus licheniformis subtilisin, Lichen Bacillus beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Other signal peptides are described by Simonen and Palva, 1993, Microbiol Rev 57:109-137.
  • Effective signal peptide coding regions for filamentous fungal host cells may be signal peptide coding regions obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor michelia Partic acid protease, Humicola insolens cellulase and Humicola lanuginosa lipase.
  • Signal peptides useful for yeast host cells can be derived from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra.
  • the control sequence may also be a propeptide coding region encoding an amino acid sequence located at the amino terminus of the polypeptide.
  • the resulting polypeptide is called a proenzyme or prepolypeptide (or in some cases, proenzyme).
  • Propolypeptides are generally inactive and can be converted to mature active polypeptides by catalytic cleavage from the propolypeptide or autocatalytic cleavage of the propeptide.
  • the propeptide coding region can be obtained from the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha factor, Rhizomucor michana aspartic protease, and thermomylolytic protease. Myceliophthorathermophila lactase (WO 95/33836).
  • the propeptide region is located adjacent the amino terminus of the polypeptide and the signal peptide region is located adjacent the amino terminus of the propeptide region.
  • regulatory sequences that permit modulation of polypeptide expression relative to host cell growth.
  • regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds.
  • suitable regulatory sequences include the lac, tac and trp operon systems.
  • suitable regulatory systems include, for example, the ADH2 system or the GAL1 system.
  • suitable regulatory sequences include the TAKA alpha amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.
  • regulatory sequences are those that allow gene amplification.
  • these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate and the metallothionein gene that is amplified in the presence of heavy metals.
  • the nucleic acid sequence encoding the monoamine oxidase of the present disclosure will be operably linked to the regulatory sequence.
  • the present disclosure also relates to a recombinant expression vector comprising a polynucleotide encoding an engineered monoamine oxidase or a variant thereof and one or more expression regulatory regions, such as a promoter and terminator subtypes, origins of replication, etc., depending on the type of host into which they are introduced.
  • the various nucleic acids and control sequences described above can be ligated together to produce a recombinant expression vector, which can include one or more convenient restriction sites to permit insertion or substitution of the nucleic acid sequence encoding the polypeptide at these sites.
  • nucleic acid sequences of the present disclosure may be expressed by inserting the nucleic acid sequences of the present disclosure or a nucleic acid construct comprising the sequences into a suitable expression vector.
  • the coding sequence is located in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.
  • the recombinant expression vector can be any vector (eg, plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that allows expression of a polynucleotide sequence.
  • the choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • Vectors can be linear plasmids or closed circular plasmids.
  • the expression vector may be an autonomously replicating vector, ie, a vector that exists as an extrachromosomal entity and replicates independently of chromosomal replication, for example, a plasmid, extrachromosomal element, minichromosome or artificial chromosome.
  • the vector may contain any means for ensuring self-replication.
  • the vector may be one that, when introduced into the host cell, integrates into the genome and is replicated together with the chromosome into which it is integrated.
  • a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or transposons, can be used.
  • Expression vectors of the present disclosure preferably contain one or more selectable markers that permit easy selection of transformed cells.
  • Selectable markers are genes whose products provide biocide or viral resistance, heavy metal resistance, auxotrophic prototrophy, and similar properties.
  • Examples of bacterial selectable markers are the dal gene from Bacillus subtilis or Bacillus licheniformis or markers conferring resistance to antibiotics such as ampicillin resistance, kanamycin resistance, chloramphenicol resistance or tetracycline resistance sex.
  • Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.
  • Selectable markers for filamentous fungal host cells include, but are not limited to: amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (serphosporin acetyltransferase), hph ( Hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotic acid nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate transferase) and trpC (anthranilic acid synthase) and their equivalents.
  • Embodiments for Aspergillus cells include the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.
  • Expression vectors of the present disclosure preferably contain elements that permit integration of the vector into the host cell genome or allow autonomous replication of the vector in said cell independent of the genome.
  • the vector may rely on a nucleic acid sequence encoding a polypeptide or any other element of the vector to integrate the vector into the genome by homologous or non-homologous recombination.
  • the expression vector may contain other nucleic acid sequences for directing integration into the host cell genome by homologous recombination.
  • the other nucleic acid sequences enable integration of the vector into the host cell genome at a precise location in the chromosome.
  • the integrating element should preferably contain a sufficient number of nucleic acids highly homologous to the corresponding target sequence to enhance the likelihood of homologous recombination, for example, 100 to 10,000 base pairs, 400 to 10,000 base pairs are preferred, and 800 to 10,000 base pairs are most preferred.
  • the integration element can be any sequence homologous to the target sequence in the genome of the host cell.
  • integration elements may be non-coding or coding nucleic acid sequences.
  • the vector can be integrated into the host cell genome by nonhomologous recombination.
  • the vector may also contain an origin of replication that enables the vector to replicate autonomously in the host cell under study.
  • bacterial origins of replication are the P15A ori or the origin of replication of the plasmids pBR322, pUC19, pACYC177 which has the P15A ori, or pACYC184 which allows replication in E. coli and pUB110, pE194, pTA1060 or pAM ⁇ 1 which allows replication in Bacillus .
  • origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
  • the origin of replication may be one with mutations that render its function temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proc Natl Acad Sci. USA 75:1433).
  • More than 1 copy of the nucleic acid sequence of the present disclosure can be inserted into a host cell to increase production of the gene product.
  • An increase in the copy number of a nucleic acid sequence may be obtained by integrating at least one additional copy of the sequence into the host cell genome; or by including an amplifiable selectable marker gene with the nucleic acid sequence when the cell contains an amplified copy of the selectable marker gene.
  • the marker gene is selected, and thus additional copies of the nucleic acid sequence can be selected by growing the cells in the presence of a suitable selection agent.
  • Suitable commercial expression vectors include: p3xFLAGTM expression vector from Sigma-Aldrich Chemicals, St. Louis MO., which includes the CMV promoter and hGH polyadenylation site for expression in mammalian host cells and uses pBR322 origin of replication and ampicillin resistance marker amplified in E. coli.
  • Suitable expression vectors are pBluescriptII SK(-) and pBK-CMV, commercially available from Stratagene, LaJolla CA, plasmids derived from pBR322 (GibcoBRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly ( Lathe et al., 1987, Gene 57:193-201).
  • the disclosure provides a host cell comprising a polynucleotide encoding an improved monoamine oxidase of the disclosure operably linked to one or more control sequences for expression of the monoamine oxidase in the host cell .
  • Host cells for expression of monoamine oxidase polypeptides encoded by expression vectors of the present disclosure are well known in the art and include, but are not limited to: bacterial cells, such as E.
  • yeast cells e.g., Saccharomyces cerevisiae or Pichia pastoris
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells
  • plant cells Appropriate culture media and growth conditions for the host cells described above are well known in the art.
  • Polynucleotides expressing monoamine oxidase can be introduced into cells by a variety of methods known in the art. Techniques include, but are not limited to: electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods of introducing polynucleotides into cells will be apparent to the skilled artisan.
  • An exemplary host cell is Escherichia coli W3110.
  • the expression vector is made by operably linking a polynucleotide encoding a modified monoamine oxidase into plasmid pCK110900, which is operably linked to the lac promoter under control of a lacI repressor.
  • the expression vector also contains the P15a origin of replication and the chloramphenicol resistance gene. Cells containing the subject polynucleotide in E. coli W3110 are isolated by subjecting the cells to chloramphenicol selection.
  • Engineered monoamine oxidases can be obtained by subjecting polynucleotides encoding naturally occurring monoamine oxidases to mutagenesis and/or directed evolution methods.
  • Exemplary directed evolution techniques are mutagenesis and/or DNA shuffling as described in: Stemmer, 1994, ProcNatl Acad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and US Patent 6,537,746.
  • StEP staggered extension method
  • in vitro recombination Zhao et al., 1998, Nat. Biotechnol. 16:258-261
  • mutagenesis PCR Caldwell et al., 1994, PCR Methods Appl. 3: S136-S140
  • cassette mutagenesis Black et al., 1996, Proc Natl Acad Sci USA 93: 3525-3529.
  • the clones obtained after the mutagenesis treatment are screened for engineered monoamine oxidases with desired improved enzyme properties.
  • Measurement of enzymatic activity from expression libraries can be performed using standard biochemical techniques, such as, but not limited to, published methods for measuring monoamine oxidase or modifications thereof, such as, but not limited to, Zhou et al. ("A One-Step" by Zhou et al. Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity (One-step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity," 1997 Anal. Biochem. 253: 169-74) and Szutowicz et al.
  • the number of cells and the amount of protein measured are determined and the same expression system and the same host cells are used to minimize differences in the amount of enzyme produced by the host cells and the amount of enzyme present in the lysate.
  • the enzyme activity can be measured by subjecting the enzyme preparation to a defined temperature and measuring the amount of enzyme activity remaining after heat treatment. A clone containing a polynucleotide encoding a monoamine oxidase is then isolated, sequenced to identify nucleotide sequence changes, if any, and used to express the enzyme in a host cell.
  • the polynucleotide encoding the enzyme can be prepared by standard solid phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases may be synthesized separately and then ligated (eg, by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence.
  • the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using methods such as the classic phosphoramidite method described by Beaucage et al., 1981, Tet Lett 22:1859-69; or The method described in Matthes et al., 1984, EMBO J.
  • 3:801-05 is, for example, as commonly practiced in automated synthesis methods. Oligonucleotides are synthesized, for example in an automated DNA synthesizer, according to the phosphoramidite method, purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources, such as The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc., Chicago, IL, Operon Technologies Inc., Alameda , CA and many other commercial sources.
  • the engineered monoamine oxidase expressed in the host cell may be recovered from the cells and or culture medium using any one or more of the well-known techniques for protein purification, including, but not limited to: lysozyme treatment, Sonication, filtration, salting out, ultracentrifugation, and chromatography.
  • Suitable solutions for lysis and efficient extraction of proteins from bacteria such as E. coli are commercially available from Sigma-Aldrich, St. Louis MO, under the trade designation CelLytic B TM .
  • Chromatographic techniques used to isolate monoamine oxidase include, but are not limited to: reversed phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis and affinity chromatography.
  • the conditions used to purify a particular enzyme will depend in part on factors such as electrostatic charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art.
  • affinity technology can be used to isolate improved monoamine oxidases.
  • any antibody that specifically binds to monoamine oxidase can be used.
  • antibody preparation a variety of host animals can be immunized by injection with the compound, including but not limited to: rabbits, mice, rats, etc.
  • the compound can be linked to a suitable support such as BSA by means of pendant functional groups or linkers attached to pendant functional groups.
  • a variety of adjuvants can be used to increase the immune response depending on the host species, including but not limited to: Freund's (complete and incomplete), mineral gums such as aluminum hydroxide, surface active substances such as lysolecithin, complex polypeptides, etc.
  • Alcohols, polyanions, peptides, oil emulsions, keyhole hemocyanin, dinitrophenol and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guérin) and Corynebacterium parvum.
  • oxidation reactions catalyzed by monoamine oxidases generally require cofactors.
  • the oxidation reactions catalyzed by monoamine oxidases described herein also generally require the cofactor flavin adenine nucleotide (FAD).
  • FAD cofactor flavin adenine nucleotide
  • the term "cofactor” refers to a non-protein compound that acts in combination with monoamine oxidase.
  • an oxidized form of the cofactor which may be linked non-covalently or covalently to the monoamine oxidase, is added to the reaction mixture.
  • the oxidized FAD form can be regenerated from the reduced form FAD- H2 by molecular oxygen.
  • the oxidized FAD form can be regenerated from NAD(P) to provide FAD and NAD(P)H.
  • NAD(P) can then be regenerated by reducing ketones to alcohols using NAD(P)H-dependent alcohol dehydrogenase/ketoreductase.
  • Suitable solvents include water, organic solvents (eg, ethyl acetate, butyl acetate, octacnol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, and similar organic solvents) and ionic solvents Liquids (e.g., 1-ethyl 4-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazole hexafluorophosphate, and similar to ionic liquids).
  • aqueous solvents are used, including water and aqueous cosolvent systems.
  • An exemplary aqueous co-solvent system has water and one or more organic solvents.
  • the organic solvent component of the aqueous cosolvent system is selected so that the organic solvent component does not completely inactivate the monoamine oxidase enzyme.
  • Suitable cosolvent systems can be readily identified by measuring the enzymatic activity of a given engineered monoamine oxidase with a defined substrate of interest in a candidate solvent system using enzyme activity assays such as those described herein.
  • the organic solvent component of the aqueous cosolvent system may be miscible with the aqueous component, resulting in a single liquid phase, or may be partially miscible or immiscible with the aqueous component, resulting in two liquid phases.
  • aqueous cosolvent systems are chosen to be biphasic, with water dispersed in the organic solvent or vice versa.
  • the ratio of water to organic solvent in the co-solvent system is usually in the range of about 90:10 to about 10:90 (v/v) organic solvent to water and 80:20 and 20:80 organic solvent to water. within the range between (v/v).
  • the co-solvent system can be preformed before being added to the reaction mixture, or it can be formed in situ in the reaction vessel.
  • the aqueous solvent may be pH buffered or unbuffered.
  • oxidation can be performed at a pH of about 10 or less, typically in the range of about 5 to about 10.
  • oxidation is performed at a pH of about 9 or less, typically in the range of about 5 to about 9.
  • the oxidation is performed at a pH of about 8 or less, typically in the range of about 5 to about 8, and typically at a pH in the range of about 6 to about 8.
  • Oxidation can also be performed at a pH of about 7.8 or below, or 7.5 or below.
  • the oxidation can be performed at neutral pH, i.e. about 7.
  • the pH of the reaction mixture can change. Typical amines of Formula I are protonated at and about neutral pH, whereas imine products of Formula II are generally not protonated at and about neutral pH. Thus, in typical embodiments where the reaction is conducted at or about neutral pH, oxidation of the protonated amine to the non-protonated imine releases protons into the aqueous solution.
  • the pH of the reaction mixture can be maintained at a desired pH or within a desired pH range by adding a base during the reaction. Alternatively, the pH can be controlled using an aqueous solvent containing a buffer. Suitable buffers to maintain the desired pH range are known in the art and include, for example, phosphate buffer, triethanolamine buffer, and similar buffers. Combinations of buffer or base additions can also be used.
  • Suitable bases for neutralizing acids are: organic bases, such as amines, alkoxides and similar organic bases; and inorganic bases, such as hydroxide salts (e.g. NaOH), carbonates (e.g. NaHCO3 ), bicarbonate Salts (eg, K 2 CO 3 ), alkaline phosphates (eg, K 2 HPO 4 , Na 3 PO 4 ), and similar inorganic bases.
  • the preferred base for neutralizing the protons released by the oxidation of the amine to the imine during the reaction is the amine substrate itself.
  • the simultaneous addition of base during the conversion can be done manually while monitoring the pH of the reaction mixture, or more conveniently, by using an automatic titrator as a pH fixator (pH stat). It is also possible to use a combination of partial buffering capacity and base addition for process control.
  • the base added to the unbuffered or partially buffered reaction mixture during the oxidation is added as an aqueous solution.
  • the engineered monoamine oxidase can be added to the reaction mixture as purified enzyme, whole cells transformed with a gene encoding monoamine oxidase, and/or cell extracts of such cells substances and/or lysates.
  • Whole cells transformed with genes encoding engineered monoamine oxidases or cell extracts and/or lysates thereof may be taken in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semi-solid (e.g., , coarse paste).
  • Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethylenimine, heat treatment, or similar methods) followed by a desalting procedure (eg, ultrafiltration, dialysis, and similar methods) before lyophilization.
  • Either cell preparation can be stabilized by cross-linking using known cross-linking agents such as glutaraldehyde or by immobilization on a solid phase (e.g., Eupergit C and the like).
  • Solid reactants e.g., enzymes, salts, etc.
  • the reactants can be provided to the reaction in a variety of different forms, including: powders (e.g., lyophilized, spray-dried, and the like), solutions, emulsions, suspensions, and the like. .
  • the reactants can be readily lyophilized or spray dried using methods and equipment known to those of ordinary skill in the art.
  • the protein solution can be frozen in aliquots at -80°C and then added to a pre-frozen lyophilization chamber, followed by application of vacuum. After removing water from the sample, the temperature is typically increased to 4°C for 2 hours before the vacuum is released and the lyophilized sample is retrieved.
  • the amounts of reactants used in the oxidation reaction will generally vary depending on the amount of product desired and the amount of monoamine oxidase substrate simultaneously employed. Generally, a concentration of substrate from about 5 g/liter to about 50 g/liter may be used when using about 50 mg/liter to about 5 g/liter of monoamine oxidase.
  • a concentration of substrate from about 5 g/liter to about 50 g/liter may be used when using about 50 mg/liter to about 5 g/liter of monoamine oxidase.
  • Appropriate amounts of optional reagents such as catalase, antifoaming agents, and sodium bisulfite or sodium metabisulfite can be readily determined by routine experimentation.
  • the order in which the reactants are added is not critical.
  • the reactants can be added to the solvent together at the same time (e.g., single-phase solvents, biphasic aqueous cosolvent systems, and similar solvents), or alternatively, some reactants can be added separately and some at different time points.
  • the reactants are added together.
  • one or more components of the reaction can be continuously added ("feeded") to the reaction at a level that minimizes or eliminates substrate and/or product inhibition of the monoamine oxidase.
  • the monoamine oxidase can be added intermittently during the reaction, for example, about every 1 hour, about every 2 hours, about every 3 hours, or about every 4 hours.
  • Suitable conditions for carrying out the monoamine oxidase-catalyzed oxidation reactions described herein include a number of conditions that can be readily optimized by routine experimentation, including but not limited to contacting the engineered monoamine oxidase and substrate at experimental pH and temperature and by e.g. The products were detected by the methods described in the examples provided herein.
  • Monoamine oxidase-catalyzed oxidation is typically performed at temperatures in the range of about 5°C to about 75°C.
  • the reaction is performed at a temperature in the range of about 20°C to about 55°C.
  • the reaction is performed at a temperature in the range of about 20°C to about 45°C, in the range of about 30°C to about 45°C, or in the range of about 40°C to about 45°C.
  • the reaction can also be carried out at ambient temperature (approximately 21°C).
  • the oxidation reaction is generally allowed to proceed until substantially complete or nearly complete oxidation of the substrate is achieved. Oxidation of substrate to product can be monitored using known methods for detecting substrate and/or product. Suitable methods include gas chromatography, HPLC and similar methods.
  • the conversion yield is generally greater than about 50%, may be greater than about 60%, may be greater than about 70%, may be greater than about 80%, may be greater than about 90%, and is usually greater than about 97%.
  • Figure 1 represents the standard curve for detecting H 2 O 2 concentration by Ampliflu Red fluorescence method.
  • Figure 2 represents the enzyme activity test results of MAON mutants.
  • the monoamine oxidase MAON protein expression gene derived from Aspergillus niger and the MAON mutant gene (SEQ ID NO: 1-17) designed based on stability and catalytic activity modification were connected to the E. coli expression vector pET15b and inserted Site NdeI+BamHI, retain N-ter 6 ⁇ His tag. After sequencing was correct, the recombinant vector was transformed into BL21(DE3) for protein expression.
  • the constructed expression vector was transferred into E. coli BL21 (DE3), induced by IPTG for expression, and purified using Ni-NTA column after harvest and lysis.
  • the specific method is as follows: Transform the MAON enzyme recombinant expression vector into the BL21 (DE3) strain. , pick a single clone into 10ml LB medium, ampicillin sodium resistance (100mg/L), culture overnight at 37°C, 200RPM, transfer to a 2L shake flask containing 1L LB medium, culture at 37°C, 200RPM When the OD600 reaches 0.6-0.8, cool down to 25°C, induce expression with 0.5mM IPTG overnight, and centrifuge at 5000xg to harvest the bacteria.
  • buffer A 50mM Tris pH 8.0, 500mM NaCl, 20mM imidazole, add a final concentration of 1mM PMSF, 250ul Cocktail inhibitor, and mix.
  • buffer A 50mM Tris pH 8.0, 500mM NaCl, 20mM imidazole, add a final concentration of 1mM PMSF, 250ul Cocktail inhibitor, and mix.
  • Ni-NTA column purification combine the lysate supernatant with resin for 20 minutes, wash the impurities with buffer A containing 50mM imidazole, and finally elute with an elution buffer containing 400mM imidazole.
  • SDS-PAGE detects protein purification effect.
  • Seed activation Transform the MAON enzyme recombinant expression vector into the BL21 (DE3) strain, pick a single clone into 10ml LB medium, ampicillin sodium resistance (100mg/L), culture at 37°C, 200RPM overnight, transfer to containing In a 1L shake flask of 500mL LB medium, culture at 37°C, 200RPM until OD600 reaches 0.8-1.0.
  • Fermentation culture Preheat the 10L fermentor culture medium containing 6L TB culture medium to 37°C, add ampicillin sodium with a final concentration of 100mg/L, ventilate and stir after inoculation to maintain 30% dissolved oxygen, when OD600 increases to 10, Start feeding.
  • Feed 1 is an aqueous solution containing 60g/L tryptone, 120g/L yeast extract, and 4% glycerol.
  • Feed 2 is 50% glycerol.
  • Ammonia and phosphoric acid are used to adjust the pH to stabilize at pH 7.0.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • Harvest cells by centrifugation at 8000xg. Harvested cells were used directly in subsequent purification processes or stored at -80°C until use as such.
  • Crude enzyme purification Resuspend the collected bacteria in 100mM Tris pH 8.0, 150mM NaCl, and resuspend at 200g wet bacteria/L and mix well. After crushing with an 800Bar high-pressure homogenizer, centrifuge at 18,400xg and 4°C for 20 minutes. Add ammonium sulfate powder (200g/L) with a final concentration of 36% saturation to the supernatant, and centrifuge to collect the protein precipitate. After freeze-drying, the precipitate was stored at 4°C until use.
  • the enzyme activity of the SEQ ID NO:7 mutant is 2 times that of the control SEQ ID NO:1
  • the enzyme activity of the SEQ ID NO:6 mutant is 4 times that of the control SEQ ID NO:1
  • the SEQ ID NO:12 mutation The body enzyme activity was 5 times that of the control SEQ ID NO:1.
  • Example 5 Monoamine oxidase catalyzes the oxidation of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane to produce (1R,5S)-6,6-dimethyl-3-azabicyclo[ 3.1.0]Hex-2-ene activity test
  • the reaction solution in the SEQ ID NO:6 sample reaction system was filtered through a pad of diatomaceous earth, and the MTBE phase and the water phase were separated into layers. After the water phase was extracted with MTBE three times, the MTBE phases were combined, dried with sodium sulfate, and spin-dried to obtain 950 mg of product. , the yield is 77%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

提供一种单胺氧化酶及其在生物催化方法中的应用。

Description

一种单胺氧化酶及其应用 技术领域
本发明涉及一种单胺氧化酶及其在生物催化方法中的应用。
背景技术
专利CN102131813A披露了使用单胺氧化酶经由用氧将一种对映体立体特异性地氧化为相应的亚胺而拆分和去外消旋化外消旋手性胺。然而仍期望提供可用于所述生物催化方法的新的单胺氧化酶。
发明简述
提供该概述是为了以简化的形式介绍概念的选择，所述概念在下文的详述中进一步描述。该概述既不意图确定所要求保护的主题的关键或基本特征，也不意图用于限制所要求保护的主题的范围。所要求保护的主题的其他特征、细节、效用和优势将是从下面撰写的详述，包括附图中阐明的和所附的权利要求中限定的那些方面明显的。
本发明还提供一种单胺氧化酶，其包含与如SEQ ID NO:1所示的单胺氧化酶氨基酸序列相比具有下述突变的氨基酸序列：在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸、第141位氨基酸由苏氨酸突变为丝氨酸和/或第234位氨基酸由丝氨酸突变为半胱氨酸。
在一个具体实施方式中，所述单胺氧化酶包含与如SEQ ID NO:1所示的单胺氧化酶氨基酸序列相比具有下述突变的氨基酸序列：
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸且第65位氨基酸由苏氨酸突变为缬氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第100位氨基酸由丝氨酸突变为脯氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸且第100位氨基酸由丝氨酸突变为脯氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸且第141位氨基酸由苏氨酸突变为丝氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第100位氨基酸由丝氨酸突变为脯氨酸、第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突 变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸且第141位氨基酸由苏氨酸突变为丝氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；或者，
在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸、第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸。
在另一个具体实施方式中，所述氨基酸序列进一步与SEQ ID NO:1所示的单胺氧化酶氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
本发明提供一种单胺氧化酶，其包含与选自由以下组成的组的任一氨基酸序列具有至少92％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列：SEQ ID NO:2-17。
在一个具体实施方式中，所述的单胺氧化酶的氨基酸序列如SEQ ID NO:2-17任一所示。
本发明还提供一种编码所述的单胺氧化酶的多核苷酸及包含所述多核苷酸的宿主细胞。
本发明还提供一种制备基本上立体异构纯的如Ⅱ
Figure PCTCN2023070432-appb-000001
所示的化合物或其盐/水合物的方法，其包括使如Ⅰ
Figure PCTCN2023070432-appb-000002
所示的化合物在所述单胺氧化酶和辅因子的存在下与氧接触。
本发明还提供一种制备基本上对映体纯的如Ⅲ
Figure PCTCN2023070432-appb-000003
所示的氨基磺酸盐化合物或其盐/水合物的方法，其包括使如Ⅰ
Figure PCTCN2023070432-appb-000004
所示的化合物在所述单胺氧化酶、辅因子和亚硫酸氢盐的存在下与氧接触。
本发明还提供一种制备基本上对映体纯的如Ⅳ
Figure PCTCN2023070432-appb-000005
所示的氨基腈化合物或其盐/水合物的方法，其包括使如Ⅰ
Figure PCTCN2023070432-appb-000006
所示的化合物在所述单胺氧化酶、辅因子和亚硫酸氢盐的存在下与氧接触，以及将所得的氨基磺酸盐化合物与氰化物接触。
在一个具体实施方式中，所述辅因子与单胺氧化酶非共价缔合。
在另一个具体实施方式中，所述辅因子选自由下列组成的组：FAD、FMN、NAD和NADP。
在另一个具体实施方式中，所述方法还包含催化过氧化氢歧化为分子氧和水的组分，优选地，所述组分选自由下列组成的组：Pd、Fe和过氧化氢酶。
本发明还提供所述的单胺氧化酶在催化如Ⅰ
Figure PCTCN2023070432-appb-000007
所示的化合物氧化为基本上立体异构纯的如Ⅱ
Figure PCTCN2023070432-appb-000008
所示的化合物或其盐/水合物上的应用。
本发明还提供所述的单胺氧化酶在催化如Ⅰ
Figure PCTCN2023070432-appb-000009
所示的化合物制备基本上对映体纯的如Ⅲ
Figure PCTCN2023070432-appb-000010
所示的氨基磺酸盐化合物、如Ⅳ
Figure PCTCN2023070432-appb-000011
所示的氨基腈化合物或其盐/水合物上的应用。
本发明还提供所述的单胺氧化酶在催化如Ⅰ
Figure PCTCN2023070432-appb-000012
所示的化合物去对称化上的应用。
发明详述
下文结合所附的附图阐述的描述意图描述所公开主题的多种说明性实施方案。结合每种 说明性实施方案描述了特定特征和功能；然而，对本领域技术人员将明显的是，所公开的实施方案可以在没有这些特定特征和功能的每一种的情况下实践。此外，除非明确声明或者特征或功能与另外的实施方案不兼容，否则结合一种实施方案描述的所有功能意图适用于本文描述的另外的实施方案。例如，除非特征或功能与替代实施方案不兼容，否则在结合一种实施方案明确描述给定特征或功能但没有结合替代实施方案明确提及的情况下，应当理解，该特征或功能可以结合替代实施方案来部署、利用或实现。
除非另外指出，否则本文描述的技术的实践可以采用有机化学、聚合物技术、分子生物学(包括重组技术)、细胞生物学、生物化学、生物乳液产生和测序技术的常规技术和描述，这些都在本领域从业的人员的技术内。这样的常规的技术包括聚合物阵列合成、多核苷酸的杂交和连接以及使用标记物的杂交检测。合适的技术的具体的说明可以通过参考本文的实例获得。然而，当然，也可以使用其他等同的常规程序。这样的常规技术和描述可以见于标准实验室手册，诸如Green等人编著(1999),Genome Analysis:A Laboratory ManualSeries(卷I-IV)；Weiner,Gabriel,Stephens,编著(2007),Genetic Variation:ALaboratory Manual；Dieffenbach,Dveksler,编著(2003),PCR Primer:A LaboratoryManual；Bowtell和Sambrook(2003),DNA Microarrays:A Molecular Cloning Manual；Mount(2004),Bioinformatics:Sequence and Genome Analysis；Sambrook和Russell(2006),Condensed Protocols from Molecular Cloning:A Laboratory Manual；以及Sambrook和Russell(2002),Molecular Cloning:A Laboratory Manual(全部来自ColdSpring Harbor Laboratory Press)；Stryer,L.(1995)Biochemistry(第4版)W.H.Freeman,New York N.Y.；Gait,“Oligonucleotide Synthesis:A PracticalApproach”1984,IRL Press,London；Nelson和Cox(2000),Lehninger,Principles ofBiochemistry第3版,W.H.Freeman Pub.,New York,N.Y.；Berg等人(2002)Biochemistry,第5版,W.H.Freeman Pub.,New York,N.Y.；Cell and Tissue Culture:LaboratoryProcedures in Biotechnology(Doyle&Griffiths,编著,John Wiley&Sons 1998)；Mammalian Chromosome Engineering–Methods and Protocols(G.Hadlaczky,编著,Humana Press2011)；Essential Stem Cell Methods,(Lanza和Klimanskaya,编著,Academic Press 2011)，所有文献出于所有目的通过引用以其整体并入本文。核酸酶特异性技术可以见于，例如，Genome Editing and Engineering From TALENs and CRISPRs toMolecular Surgery,Appasani和Church,2018；以及CRISPR:Methods and Protocols,Lindgren和Charpentier,2015；这两篇文献出于所有目的通过引用以其整体并入本文。酶工程化的基本方法可以见于，Enzyme Engineering Methods and Protocols,Samuelson,编著,2013；Protein Engineering,Kaumaya,编著,(2012)；以及Kaur和Sharma,“DirectedEvolution:An Approach to Engineer Enzymes”,Crit.Rev.Biotechnology,26:165-69(2006)。
注意，除非上下文另外清楚指出，否则如本文和所附的权利要求书中使用的，单数形式“一(a)”、“一(an)”和“该(the)”包括复数指代物。因此，例如，提及“寡核苷酸”是指一种或更多种寡核苷酸，并且提及“自动化系统”包括提及用于与本领域技术人员已知的系统一起使用的等同步骤和方法，等等。另外地，应当理解，本文可以使用的术语诸如“左”、“右”、“顶”、“底”、“前”、“后”、“侧”、“高度”、“长度”、“宽度”、“上”、“下”、“内部(interior)”、“外部(exterior)”、“内(inner)”、“外(outer)”等仅描述参考点，并且不必然将本公开内容的实施方案限制为任何特 定的方向或配置。此外，术语诸如“第一”、“第二”、“第三”等，仅标识如本文公开的许多部分、组件、步骤、操作、功能和/或参考点中的一个，并且同样不必然将本公开内容的实施方案限制为任何特定的配置或方向。
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域内的普通技术人员通常理解的相同含义。本文提及的所有的出版物为了描述和公开可以与本文描述的发明结合使用的设备、方法和细胞群体的目的，以引用方式并入。
在提供值的范围情况下，应理解，在该范围的上限值和下限值之间的每一个中间值和该规定的范围内的任何其他规定的值或中间值被涵盖在本发明内。这些较小的范围的上限值和下限值可以独立地被包括在较小的范围内，并且也被涵盖在本发明内，受限于规定的范围内的任何特定地排除的限值。在规定的范围包括限值中的一个或两个的情况下，将那些所包括的限值中的任一个或两个排除的范围也被包括在本发明中。
在以下的描述中，阐述了许多具体细节，以提供对本发明的更充分理解。然而，对本领域普通技术人员将明显的是，可以在没有一个或更多个这些具体细节的情况下，实践本发明。在其他情况下，为了避免使本发明含混不清，尚未描述本领域技术人员熟知的特征和熟知的程序。
能够将结构式(1)的胺化合物氧化为相应的结构式(2)的亚胺化合物的本公开内容的单胺氧化酶与SEQ ID NO:1的氨基酸序列相比具有一个或多个氨基酸取代。这种氨基酸取代为单胺氧化酶提供了一种或多种改善的特性，包括酶活性增加、立体特异性增加、热稳定性增加、溶剂稳定性增加、产物抑制降低、底物抑制降低或对反应副产物的敏感性降低。这种氨基酸取代还可以改善单胺氧化酶在宿主细胞中的表达水平、溶解性和/或稳定性，例如作为在异源宿主细胞中重组表达的蛋白，所述异源宿主细胞例如但不限于大肠杆菌宿主细胞。
本公开内容还提供编码这种单胺氧化酶的多核昔酸和在所公开的生物催化方法中使用多肤的方法。
如本文所用的下列术语旨在具有下列含义：
“单胺氧化酶”是指具有将上述结构式I的化合物氧化为相应的上述结构式II的产物的酶能力的多肽。该多肽通常利用氧化的辅因子，例如但不限于黄素腺嘌呤二核苷酸(FAD)、黄素腺嘌呤单核苷酸(FMN)、烟酰胺腺嘌呤二核苷酸(NAD)或烟酰胺腺嘌呤二核苷酸磷酸(NADP)。在具体的实施方案中，氧化的辅因子是FAD。如本文所用的单胺氧化酶包括天然存在的(野生型)单胺氧化酶以及通过人操作产生的非天然存在的工程化多肽。
“编码序列”是指编码蛋白的氨基酸序列的那部分核酸(例如，基因)。
“天然存在的”或“野生型”是指天然发现的形式。例如，天然存在的或野生型多肽或多核苷酸序列是在生物体中存在的可以从天然来源分离的序列并且该序列没有通过人操作被故意地修饰。
当结合例如细胞、核酸或多肽使用时，“重组”是指已经以否则不会天然存在的方式被修饰、或者与材料的天然形式或固有形式相同但是产自或源于合成材料和/或通过使用重组技术的操作产生或衍生的材料，或相应于该材料的天然形式或固有形式的材料。非限制性实例包 括但不限于：表达在固有(非重组)形式的细胞中不存在的基因或者表达否则以不同的水平表达的固有基因的重组细胞。
“序列同一性百分比”和“百分比同源性”在本文中可互换使用，是指多核苷酸和多肽之间的比较，并且是通过在比较窗口中比较两条最佳比对的序列来确定的，其中多核苷酸或多肽序列在比较窗口中的部分与两条序列最佳比对的参考序列(其不包含添加或缺失)相比可以包含添加或缺失(即，缺口)。百分比可以如下计算：确定在两条序列中出现相同核酸碱基或氨基酸残基的位置数从而产生匹配位置数，用匹配位置数除以比较窗口中位置的总数并将结果乘以100，得出序列同一性的百分比。可选择地，百分比可以如下计算：确定在两条序列中出现相同核酸碱基或氨基酸残基的位置数或者与缺口对齐的核酸碱基或氨基酸残基的位置数从而得出匹配位置数，用匹配位置数除以比较窗口中位置的总数并将结果乘以100，得出序列同一性的百分比。本领域的技术人员了解存在许多已确立的算法可用于比对两条序列。可以通过下列算法进行比较序列的最佳比对：例如，通过Smith和Waterman，1981，Adv.Appl.Math.2：482的局部同源性算法；通过Needleman和Wunsch，1970，J.Mol.Biol.48：443的同源比对算法；通过Pearson和Lipman，1988，Proc.Natl.Acad.Sci.USA 85：2444的相似性检索方法；通过这些算法的计算机化执行(在GCG Wisconsin软件包中的GAP、BESTFIT、FASTA和TFASTA)或通过视觉检查(大体参见，CurrentProtocols in Molecular Biology(分子生物学最新实验方案)，F.M.Ausubel等人，编辑，Current Protocols，Greene Publishing Associates，Inc.与JohnWiley&Sons，Inc.合资，(1995补充材料)(Ausubel))。适于确定百分比序列同一性和序列相似性的算法的实例是BLAST和BLAST 2.0算法，它们分别描述于：Altschul等人，1990，J.Mol.Biol.215：403-410和Altschul等人，1977，Nucleic Acids Res.3389-3402中。进行BLAST分析的软件通过美国国家生物技术信息中心网站可公开获得。这种算法包括首先通过鉴定在查询序列中长度为W的短字(word)来鉴定高评分序列对(HSP)，所述短字在与数据库序列中相同长度的字比对时符合或满足一些正值阈值评分T。T是指相邻字评分阈值(Altschul等人，如上)。这些最初的相邻字匹配字串(word hit)充当启动寻找含有它们的更长HSP的种子。然后字匹配字串沿着各序列在两个方向延伸，只要累积比对评分可以增加即可。对于核苷酸序列，累积评分使用参数M(匹配残基对的奖励评分(rewardscore)；总是大于0)和N(错配残基的惩罚评分，总是小于0)计算。对于氨基酸序列，使用评分矩阵来计算累积评分。在下列情况下停止各方向的字匹配字串延伸：累积比对评分由其达到的最大值降低了量X；累积评分由于一个或多个负评分残基比对的累积而变为零或低于零；或到达任一序列的末端。BLAST算法参数W、T和X决定比对的灵敏度和速度。BLASTN程序(对于核苷酸序列)使用下列作为缺省参数：字长(wordlength，W)为11，期望值(E)为10，M＝5，N＝-4，以及两条链的比较。对于氨基酸序列，BLASTP程序使用下列缺省参数：字长(W)为3，期望值(E)为10以及BLOSUM62评分矩阵(参见Henikoff和Henikoff，1989，Proc Natl Acad Sci USA 89：10915)。示例性的序列比对和％序列同一性的确定可以采用GCG Wisconsin软件包(Accelrys，Madison WI)中的BESTFIT或GAP程序，使用所提供的缺省参数。
“参考序列”是指用作序列比较的基础的确定序列。参考序列可以是较大序列的子序列，例如全长基因或多肽序列的区段。一般地，参考序列是至少20个核苷酸或氨基酸残基长、至 少25个残基长、至少50个残基长或全长的核酸或多肽。由于两条多核苷酸或多肽可以各自(1)包含两个序列之间相似的序列(即，完整序列的一部分)并且(2)还可以包含两个序列之间差异的序列，所以两条(或更多条)多核苷酸或多肽之间的序列比较通常通过在“比较窗口”内比较两条多核苷酸的序列而鉴定和比较局部区域的序列相似性来进行。
“比较窗口”是指其中可以将序列与至少20个连续核苷酸或氨基酸的参考序列相比较的至少约20个连续核苷酸位置或氨基酸残基的概念上的区段，并且其中在比较窗口中的序列部分与两条序列最佳比对的参考序列(其不包含添加或缺失)相比可以包含20％或更少的添加或缺失(即，缺口)。比较窗口可以长于20个连续残基并且任选包含30个连续残基、40个连续残基、50个连续残基、100个连续残基或更长的窗口。
“基本的同一性”是指在至少20个残基位置的比较窗口内、通常在至少30-50个残基的窗口内，与参考序列相比具有至少80％序列同一性、至少85％同一性和89％至95％序列同一性，更通常至少99％序列同一性的多核苷酸或多肽序列，其中序列同一性百分比是通过在比较窗口内将参考序列与包含参考序列的总计20％或更少的缺失或添加的序列相比计算而来的。在应用于多肽的特定实施方案中，术语“基本的同一性”意指在通过例如程序GAP或BESTFIT使用缺省空位权重(gap weihgt)进行最佳比对时，两条多肽序列具有至少80％序列同一性，优选至少89％序列同一性，至少95％序列同一性或更高的序列同一性(例如，99％序列同一性)。优选地，不相同的残基位置是由于保守的氨基酸取代而不同。
当用于给定氨基酸或多核苷酸序列编号的下文中时，“对应于”、“关于”或“相对于”是指在给定的氨基酸或多核苷酸序列与指定的参考序列相比较时，所述参考序列的残基编号。换言之，给定的聚合物的残基编号或残基位置是关于参考序列指定的而非通过给定氨基酸或多核苷酸序列中的残基的实际数字位置指定。例如，可以通过引入缺口以优化两条序列之间的残基匹配，将诸如工程化单胺氧化酶的氨基酸序列的给定氨基酸序列与参考序列比对。在这些情况下，尽管存在缺口，但给定氨基酸或多核苷酸序列中残基的编号是关于其所比对的参考序列而指定的。
“立体选择性”是指在化学反应或酶促反应中，一种立体异构体相对于另一立体异构体优先形成。立体选择性可以是部分的，这时一种立体异构体的形成比另一种有利，或者立体选择性可以是完全的，这时只形成一种立体异构体。当立体异构体是对映体时，立体选择性是指对映体选择性，即一种对映体在两种对映体总和中的分数(通常报道为百分比)。其(通常为百分比)在本领域中通常可选地报道为根据如下公式由其计算的对映体过量(e.e.)：[主要对映体-次要对映体]/[主要对映体+次要对映体]。在立体异构体是非对映异构体时，立体选择性是指非对映体选择性，即一种非对映体在两种非对映体混合物中的分数(通常报道为百分比)，通常可选地报道为非对映体过量(d.e.)。对映体过量和非对映体过量是立体异构体过量的类型。
“高立体选择性”：是指能够将底物转化为具有至少约99％立体异构体过量的相应产物的单胺氧化酶多肽。
“立体特异性”是指在化学反应或酶促反应中，一种立体异构体相对于另一立体异构体优先转化。立体特异性可以是部分的，这时一种立体异构体的转化比另一种有利，或者立体特异性可以是完全的，这时只转化一种立体异构体。
“化学选择性”是指在化学反应或酶促反应中，一种产物相对于另一产物优先形成。
“改良的酶特性”是指与参考单胺氧化酶相比表现出任何酶特性改良的单胺氧化酶多肽。对于本文所述的工程化单胺氧化酶多肽，一般进行与野生型单胺氧化酶的比较，尽管在一些实施方案中，参考单胺氧化酶可以是另一种改良的单胺氧化酶。期望改良的酶特性包括但不限于：酶活性(其可以根据百分比底物转化表示)、热稳定性、pH活性谱、辅因子需求、对抑制剂(例如，产物抑制)的不应性、立体特异性、立体选择性(包括对映体选择性)、溶解性和稳定性以及在宿主细胞中的表达水平。
“增加的酶活性”是指工程化单胺氧化酶多肽的改良的特性，它可以通过与参考单胺氧化酶相比的比活性(例如，产生的产物/时间/重量蛋白)的增加或底物转化为产物的百分比转化(例如，使用指定量的单胺氧化酶时在指定的时间段中起始量的底物转化为产物的转化百分比)的增加代表。确定酶活性的示例性方法在实施例中提供。可以影响关于酶活性的任何特性，包括经典的酶特性K m、V max或k cat，它们的改变可以导致酶活性增加。酶活性的改善可以为相应的野生型单胺氧化酶的酶活性的约1.5倍至多达天然存在的单胺氧化酶或单胺氧化酶多肽来源的另一工程化单胺氧化酶的酶活性的2倍、5倍、10倍、20倍、25倍、50倍、75倍、100倍或更多倍。技术人员应理解任何酶的活性是扩散限制的，使得催化转换速率不会超过底物(包括任何所需的辅因子)的扩散速率。扩散限制或k cat/K m的理论最大值一般是约10 8至10 9(M -1s -1)。因此，单胺氧化酶的酶活性的任何改善将具有与该单胺氧化酶所作用的底物的扩散速率有关的上限。单胺氧化酶活性可以使用已公布的测量单胺氧化酶的方法或其改造方法测量，例如但不限于Zhou等人(Zhou等人“A One-StepFluorometric Method for the Continuous Measurement of Monoamine OxidaseActivity(单胺氧化酶活性连续测量的一步式荧光法)，”1997 Anal.Biochem.253：169-74)和Szutowicz等人(Szutowicz等人，“Colorimetric Assay forMonoamine Oxidase in Tissues Using Peroxidase and2，2′-Azino(3-ethtylbenzthaizoline-6-sulfonic Acid)as Chromogen(使用过氧化物酶和2，2′-连氮基(3-乙基苯并噻唑啉-6-磺酸)作为色原进行的组织中单胺氧化酶的比色测定)，”1984，Anal.Biochem.138：86-94)所公开的那些方法。酶活性的比较是使用限定的酶制剂、在设定条件下的限定的测定和一种或多种限定的底物进行的，如本文进一步详细描述的或者使用例如Zhou和Szutowicz的方法。一般地，当比较裂解物时，确定测定的细胞数和测定的蛋白的量并且使用相同的表达系统和相同的宿主细胞来将宿主细胞产生的酶和裂解物中存在的酶的量的差异最小化。
“转化”：是指底物至相应产物的酶促氧化。“百分比转化”是指在指定条件下一段时间内被氧化为产物的底物的百分比。因此，单胺氧化酶多肽的“酶活性”或“活性”可以表示为底物至产物的“百分比转化”。
“热稳定的”是指单胺氧化酶多肽在暴露于升高的温度(例如40-80℃)一段时间(例如，0.5-24小时)后与未处理的酶相比保持相似的活性(例如大于60％至80％)。
“溶剂稳定的”是指是指单胺氧化酶多肽在暴露于不同浓度(例如，5％-99％)的溶剂(异丙醇、四氢呋喃、2-甲基四氢呋喃、丙酮、甲苯、乙酸丁酯、甲基叔丁醚等)一段时间(例如，0.5-24小时)后与未处理的酶相比保持相似的活性(大于例如60％至80％)。
“pH稳定的”是指单胺氧化酶多肽在暴露于高或低pH(例如4.5-6或8至12)一段时间(例如，0.5-24小时)后与未处理的酶相比保持相似的活性(大于例如60％至80％)。
“热稳定和溶剂稳定的”是指是热稳定和溶剂稳定的单胺氧化酶多肽。
“亲水氨基酸或残基”是指具有表现出根据Eisenberg等人，1984，J.Mol.Biol.179：125-142的标准化的一致性疏水量表小于零的疏水性的侧链的氨基酸或残基。遗传编码的亲水氨基酸包括L-Thr(T)、L-Ser(S)、L-His(H)、L-Glu(E)、L-Asn(N)、L-Gln(Q)、L-Asp(D)、L-Lys(K)和L-Arg(R)。
“酸性氨基酸或残基”是指当氨基酸包含在肽或多肽中时，具有表现出小于约6的pK值的侧链的亲水氨基酸或残基。酸性氨基酸在生理pH下由于氢离子的丢失而通常具有带负电荷的侧链。遗传编码的酸性氨基酸包括L-Glu(E)和L-Asp(D)。
“碱性氨基酸或残基”是指当氨基酸包含在肽或多肽中时，具有表现出大于约6的pK值的侧链的亲水氨基酸或残基。碱性氨基酸在生理pH下由于与水合氢离子的缔合而通常具有带正电荷的侧链。遗传编码的碱性氨基酸包括L-Arg(R)和L-Lys(K)。
“极性氨基酸或残基”是指具有如下的侧链的亲水氨基酸或残基：在生理pH下不带电荷，但是其具有其中两个原子所共同拥有的电子对被这两个原子之一更紧密地持有的至少一个键。遗传编码的极性氨基酸包括L-Asn(N)、L-Gln(Q)、L-Ser(S)和L-Thr(T)。
“疏水氨基酸或残基”是指具有表现出根据Eisenberg等人，1984，J.Mol.Biol.179：125-142的标准化的一致性疏水量表大于零的疏水性的侧链的氨基酸或残基。遗传编码的疏水氨基酸包括L-Pro(P)、L-Ile(I)、L-Phe(F)、L-Val(V)、L-Leu(L)、L-Trp(W)、L-Met(M)、L-Ala(A)和L-Tyr(Y)。
“芳族氨基酸或残基”是指具有包含至少一个芳环或杂芳环的侧链的亲水性或疏水性氨基酸或残基。遗传编码的芳族氨基酸包括L-Phe(F)、L-Tyr(Y)和L-Trp(W)。尽管L-His(H)由于其杂环氮原子的pKa而有时被归类为碱性残基或者由于其侧链包含杂芳环而被归类为芳族残基，但在本文中组氨酸被归类为亲水残基或“限制残基(constrained residue)”(参见下文)。
“限制氨基酸或残基”是指具有限制的几何性质的氨基酸或残基。本文中，限制残基包括L-pro(P)和L-his(H)。组氨酸由于其具有相对小的咪唑环而具有限制的几何性质。脯氨酸由于其还具有五元环而具有限制的几何性质。
“非极性氨基酸或残基”是指具有如下的侧链的疏水氨基酸或残基：在生理pH下不带电荷并且具有其中两个原子所共同拥有的电子对一般被这两个原子的每一个同等程度的持有的键(即侧链不是极性的)。遗传编码的非极性氨基酸包括L-Gly(G)、L-Leu(L)、L-Val(V)、L-Ile(I)、L-Met(M)和L-Ala(A)。
“脂肪族氨基酸或残基”是指具有脂肪族烃侧链的疏水氨基酸或残基。遗传编码的脂肪族氨基酸包括L-Ala(A)、L-Val(V)、L-Leu(L)和L-Ile(I)。
“半胱氨酸。”氨基酸L-Cys(C)的不寻常之处在于其可以与其他L-Cys(C)氨基酸或其他含硫烷基或巯基的氨基酸形成二硫键。“半胱氨酸样残基”包括半胱氨酸和含有可用于形成二硫键的巯基部分的其他氨基酸。L-Cys(C)(和具有含-SH侧链的其他氨基酸)以还原的游离-SH或氧化的二硫键形式存在于肽中的能力影响了L-Cys(C)是赋予肽净疏水特征还是亲水特征。虽 然L-Cys(C)表现出根据Eisenberg(Eisenberg等人，1984，如上)的标准化的一致性量表0.29的疏水性，但应了解，为了本公开内容的目的，将L-Cys(C)归类为其自身的独特组中。
“小氨基酸或残基”是指具有由共三个或更少的碳和/或杂原子(不包括α-碳和氢)构成的侧链的氨基酸或残基。小氨基酸或残基可以根据上述定义被进一步归类为脂肪族的、非极性的、极性的或酸性的小氨基酸或残基。遗传编码的小氨基酸包括L-Ala(A)、L-Val(V)、L-Cys(C)、L-Asn(N)、L-Ser(S)、L-Thr(T)和L-Asp(D)。
“含羟基氨基酸或残基”是指含有羟基(-OH)部分的氨基酸。遗传编码的含羟基氨基酸包括L-Ser(S)、L-Thr(T)和L-Tyr(Y)。
“保守的”氨基酸取代或突变是指具有相似侧链的残基的可互换性，并且因此通常包括用相同或相似的氨基酸定义类别中的氨基酸取代多肽中的氨基酸。然而，如本文所用，如果保守的突变可以代替地为脂肪族至脂肪族、非极性至非极性、极性至极性、酸性至酸性、碱性至碱性、芳族至芳族、或限制残基至限制残基的取代，则保守的突变不包括亲水至亲水、疏水至疏水、含羟基至含羟基或小残基至小残基的取代。此外，如本文所用，A、V、L或I可以保守地突变为另一脂肪族残基或另一非极性残基。下表1显示了示例性的保守取代。.
表1：保守取代
Figure PCTCN2023070432-appb-000013
“非保守取代”是指用具有显著不同的侧链特性的氨基酸进行的多肽中氨基酸的取代或突变。非保守取代可以使用上面所列的定义组之间而不是之内的氨基酸。在一个实施方案中，非保守突变影响(a)取代区域中肽主链的结构(例如，脯氨酸取代甘氨酸)、(b)电荷或疏水性、或(c)侧链体积。
“缺失”是指通过从参考多肽移除一个或多个氨基酸而对多肽进行的修饰。缺失可以包括移除1个或多个氨基酸、2个或更多个氨基酸、5个或更多个氨基酸、10个或更多个氨基酸、15个或更多个氨基酸、或20个或更多个氨基酸、多达构成参考酶的氨基酸总数的10％或多达构成参考酶的氨基酸总数的20％，同时保留酶活性和/或保留工程化单胺氧化酶的改良特 性。缺失可以针对多肽的内部和/或端部。在多个实施方案中，缺失可以包含连续的区段或者可以是不连续的。
“插入”是指通过从参考多肽添加一个或多个氨基酸而对多肽进行的修饰。在一些实施方案中，改良的工程化单胺氧化酶包括将一个或多个氨基酸插入天然存在的单胺氧化酶中以及将一个或多个氨基酸插入其他改良的单胺氧化酶多肽中。插入可以是在多肽的内部，或羧基端或氨基端。如本文所用的插入包括如本领域中已知的融合蛋白。插入可以是连续氨基酸区段或者被天然存在的多肽中的一个或多个氨基酸分隔开。
关于指定的参考序列的“与......不同”或“不同于”是指给定氨基酸或多核苷酸序列在与参考序列比对时的差异。一般地，可以在两条序列最佳比对时确定差异。差异包括与参考序列相比的氨基酸残基的插入、缺失或取代。
如本文所用的“片段”是指具有氨基端和/或羧基端缺失，但是其中剩余的氨基酸序列与序列的相应位置相同的多肽。片段可以是至少14个氨基酸长、至少20个氨基酸长、至少50个氨基酸长或更长以及多达全长单胺氧化酶多肽的70％、80％、90％、95％、98％和99％。
“分离的多肽”是指与其天然伴随的其他污染物如蛋白、脂和多核苷酸基本上分离的多肽。该术语涵盖由它们的天然存在的环境或表达系统(例如，宿主细胞或体外合成)中移出或纯化的多肽。改良的单胺氧化酶可以存在于细胞中、存在于细胞培养基中，或以各种形式制备，例如裂解物或分离的制剂。如此，在一些实施方案中，改良的单胺氧化酶可以是分离的多肽。
“基本上纯的多肽”是指其中多肽物质是存在的主要物质(即，以摩尔或重量计，其比组合物中的任何其他单独的大分子物质更丰富)的组合物，并且该组合物在主题物质以摩尔或％重量计构成至少约50％的存在的大分子物质时大体上是基本上纯化的组合物。一般地，基本上纯的单胺氧化酶组合物以摩尔数或％重量计占组合物中存在的全部大分子物质的约60％或更多、约70％或更多、约80％或更多、约90％或更多、约95％或更多和约98％或更多。在一些实施方案中，将主题物质纯化为基本上同质的(即，通过常规检测方法在组合物中不能检测到污染物质)，其中组合物基本上由单一的大分子物质组成。溶剂物质、小分子(＜500道尔顿)和元素铁物质不被认为是大分子物质。在一些实施方案中，分离的改良单胺氧化酶多肽是基本上纯的多肽组合物。
如本文所用的“严格杂交”是指其中核酸杂交物(hybrid)稳定的条件。如本领域的技术人员已知的，杂交物的稳定性是以杂交物的熔解温度(T m)反映的。一般地，杂交物的稳定性取决于离子强度、温度、G/C含量、以及离液剂的存在。多核苷酸的T m值可以使用预测熔解温度的已知方法计算(参见，例如Baldino等人，Methods Enzymology 168：761-777；Bolton等人，1962，Proc.Natl.Acad.Sci.USA 48：1390；Bresslauer等人，1986，Proc.Natl.Acad.Sci USA 83：8893-8897；Freier等人，1986，Proc.Natl.Acad.SciUSA 83：9373-9377；Kierzek等人，Biochemistry 25：7840-7846；Rychlik等人，1990，Nucleic Acids Res 18：6409-6412(勘误，1991，Nucleic Acids Res19：698)；Sambrook等人，如上)；Suggs等人，1981，在Developmental BiologyUsing Purified Genes(使用纯化基因的发育生物学)(Brown等人，编辑)，683-693页中，Academic Press；以及Wetmur，1991，Crit Rev Biochem MolBiol 26：227-259。所有出版物均通过引用并入本文)。在一些实施方案中，多核苷酸编码本文所公开的多肽并且在限定的条件下与编码本公开 内容的工程化单胺氧化酶的序列的互补序列杂交，所述限定的条件如中度严格或高度严格的条件。
“杂交严格性”指核酸的这种洗涤条件。一般地，杂交反应在较低严格性的条件下进行，随后进行不同的但是较高严格性的洗涤。术语“中度严格杂交”是指容许靶DNA结合与靶DNA具有约60％同一性，优选约75％同一性、约85％同一性；与靶多核苷酸具有大于约90％同一性的互补核酸的条件。示例性的中度严格条件是等同于下列的条件：在42℃下在50％甲酰胺、5×Denhart′s溶液、5×SSPE、0.2％SDS中杂交，随后42℃下在0.2×SSPE、0.2％SDS中洗涤。“高度严格性杂交”一般是指比在限定多核苷酸序列的溶液条件下确定的热熔解温度T m低约10℃或更少的条件。在一些实施方案中，高度严格性条件是指仅容许在65℃下0.018M NaCl中形成稳定的杂交物的那些核酸序列杂交的条件。(即，如本文所预期的，如果杂交物在65℃下0.018M NaCl中不稳定，则其在高度严格性条件下将是不稳定的)。高度严格性条件通过下列提供：例如在等同于42℃下在50％甲酰胺、5×Denhart′s溶液、5×SSPE、0.2％SDS中的条件中杂交，随后65℃下在0.1×SSPE和0.1％SDS中洗涤。其他高度严紧格杂交条件以及中度严格条件在上文所引用的参考文献中描述。
“异源的”多核苷酸是指通过实验室技术被引入宿主细胞的多核苷酸，并且包括从宿主细胞中移除，经受实验室操作，然后被再次引入宿主细胞的多核苷酸。
“密码子优化的”是指将编码蛋白的多核苷酸密码子改变为在具体生物体中优先使用以便使所编码的蛋白在感兴趣的生物体中有效地表达的那些密码子。尽管由于大部分氨基酸由几种密码子(称为“同义物”或“同义”密码子)代表而使遗传密码是简并的，但熟知的是特定生物体的密码子使用是非随机的并且偏爱特定的密码子三联体。这种密码子使用偏倚关于给定基因、共同功能或祖先起源(ancestra origin)的基因、相对于低拷贝数蛋白的高表达蛋白和生物体基因组的聚集蛋白编码区可能是更高的。在一些实施方案中，可以将编码单胺氧化酶的多核苷酸密码子优化以用于从被选择用于表达的宿主生物体中最佳地制备。
“优选的、最佳的、高密码子使用偏倚密码子”可互换地是指如下的密码子：其与编码相同氨基酸的密码子相比在蛋白编码区以更高的频率使用。优选的密码子可以就下列方面而确定：在单个基因中的密码子使用、一组共同功能或起源的基因的密码子使用、高表达的基因的密码子使用、在整个生物体中聚集的蛋白编码区的密码子频率、相关生物体的聚集的蛋白编码区的密码子频率或它们的组合。频率随基因表达水平增加的密码子通常是用于表达的最佳密码子。已知用于确定密码子频率(例如，密码子使用、相对的同义密码子使用)和特定生物体中密码子偏好的多种方法，包括多变量分析，例如使用聚类分析或相应分析和用于基因的有效密码子数(参见GCG CodonPreference，Genetics Computer Group WisconsinPackage；CodonW，John Peden，University of Nottingham；McInerney，J.O，1998，Bioinformatics 14：372-73；Stenico等人，1994，Nucleic Acids Res.222437-46；Wright，F.，1990，Gene 87：23-29)。越来越多的生物体的密码子使用表是可用的(参见，例如Wada等人，1992，Nucleic Acids Res.20：2111-2118；Nakamura等人，2000，Nucl.Acids Res.28：292；Duret，等人，如上；Henaut和Danchin，“Escherichia coli and Salmonella(大肠杆菌和沙门氏菌)，”1996，Neidhardt，等人编辑，ASM Press，Washington D.C.，第2047-2066页。获得密码子使用的数据来源可以 依赖于能够编码蛋白的任何可用的核苷酸序列。这些数据集包括实际上已知的编码表达的蛋白的核酸序列(例如，完整蛋白编码序列-CDS)、表达序列标签(ESTS)或基因组序列的预测编码区(参见，例如，Mount，D.，Bioinformatics：Sequence andGenome Analysis(生物信息学：序列和基因组分析)，第8章，Cold SpringHarbor Laboratory Press，Cold Spring Harbor，N.Y.，2001；Uberbacher，E.C.，1996，Methods Enzymol.266：259-281；Tiwari等人，1997，Comput.Appl.Bioscl.13：263-270)。
“控制序列”在本文中定义为包括对于表达本公开内容的多肽是必需的或有利的所有组分。每种控制序列对于编码多肽的核酸序列来说可以是固有的或外来的。这种控制序列包括但不限于：前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号肽序列和转录终止子。最低程度上，控制序列包含启动子、转录和翻译终止信号。控制序列可以为了引入促进控制序列与编码多肽的核酸序列的编码区连接的特定限制位点的目的而具有连接序列。
“可操作地连接”在本文中被定义为一种构造，其中控制序列被适当地放置在相对于DNA序列的编码序列的位置处以便使控制序列指导多核苷酸和/或多肽的表达。
“启动子序列”是宿主细胞所识别的用于表达编码区的核酸序列。控制序列可以包含适当的启动子序列。启动子序列含有介导多肽表达的转录控制序列。启动子可以是在选择的宿主细胞中显示转录活性的任何核酸序列，包括突变的、截短的和杂合的启动子，并且可以从编码对宿主细胞同源或异源的胞外或胞内多肽的基因获得。
如本文所用术语“立体异构体”、“立体异构形式”以及类似术语是用于单个分子的所有异构体的一般术语，它们仅在它们的原子在空间中的方向上不同。它包括对映体和具有彼此不为镜像的多于一个的手性中心的化合物的异构体(“非对映体”)。
术语“手性中心”是指四个不同的基团所连接的碳原子。
术语“对映体”或“对映体的”是指在其镜像上不可重叠并且因此是光学活性的分子，其中对映体使偏振光平面以一个方向旋转并且其镜像使偏振光平面以相反的方向旋转。
术语“外消旋的”是指光学上无活性的对映体的等份混合物。
术语“拆分”是指分子的两种对映体形式之一的分离或浓缩或排除。
如本文所用的“基本上对映体纯”意指化合物的指定对映体以比相同化合物的另一对映体更高的程度或度存在。因此，在具体实施方案中，基本上对映体纯的化合物以比相同化合物的另一对映体80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％对映体过量存在。
如本文所用的“基本上立体异构纯”意指化合物的指定对映体或非对映体以比相同化合物的另一对映体或非对映体更高的程度或度存在。如上文关于“立体选择性”所提到的，对映体过量和非对映体过量是立体异构体过量的类型。因此，在具体实施方案中，基本上立体异构纯的化合物以比相同化合物的另一对映体或非对映体80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％立体异构体过量存在。
如熟悉的技术人员将了解的，本文所述的多肽并不限于遗传编码的氨基酸。除遗传编码的氨基酸外，本文所述的多肽可以整体地或部分地包含天然存在的氨基酸和/或合成的非编码氨基酸。本文所描述的单胺氧化酶可以包含的某些常遇到的非编码氨基酸包括但不限于：遗 传编码的氨基酸的D-立体异构体；2，3-二氨基丙酸(Dpr)；α-氨基异丁酸(Aib)；ε-氨基己酸(Aha)；δ-氨基戊酸(Ava)；N-甲基甘氨酸或肌氨酸(MeGly或Sar)；鸟氨酸(Orn)；瓜氨酸(Cit)；叔丁基丙氨酸(Bua)；叔丁基甘氨酸(Bug)；N-甲基异亮氨酸(MeIle)；苯基甘氨酸(Phg)；环己基丙氨酸(Cha)；正亮氨酸(Nle)；萘基丙氨酸(Nal)；2-氯苯丙氨酸(Ocf)；3-氯苯丙氨酸(Mcf)；4-氯苯丙氨酸(Pcf)；2-氟苯丙氨酸(Off)；3-氟苯丙氨酸(Mff)；4-氟苯丙氨酸(Pff)；2-溴苯丙氨酸(Obf)；3-溴苯丙氨酸(Mbf)；4-溴苯丙氨酸(Pbf)；2-甲基苯丙氨酸(Omf)；3-甲基苯丙氨酸(Mmf)；4-甲基苯丙氨酸(Pmf)；2-硝基苯丙氨酸(Onf)；3-硝基苯丙氨酸(Mnf)；4-硝基苯丙氨酸(Pnf)；2-氰基苯丙氨酸(Ocf)；3-氰基苯丙氨酸(Mcf)；4-氰基苯丙氨酸(Pcf)；2-三氟甲基苯丙氨酸(Otf)；3-三氟甲基苯丙氨酸(Mtf)；4-三氟甲基苯丙氨酸(Ptf)；4-氨基苯丙氨酸(Paf)；4-碘苯丙氨酸(Pif)；4-氨基甲基苯丙氨酸(Pamf)；2，4-二氯苯丙氨酸(Opef)；3，4-二氯苯丙氨酸(Mpcf)；2，4-二氟苯丙氨酸(Opff)；3，4-二氟苯丙氨酸(Mpff)；吡啶-2-基丙氨酸(2pAla)；吡啶-3-基丙氨酸(3pAla)；吡啶-4-基丙氨酸(4pAla)；萘-1-基丙氨酸(1nAla)；萘-2-基丙氨酸(2nAla)；噻唑基丙氨酸(taAla)；苯并噻吩基丙氨酸(bAla)；噻吩基丙氨酸(tAla)；呋喃基丙氨酸(fAla)；高苯丙氨酸(hPhe)；高酪氨酸(hTyr)；高色氨酸(hTrp)；五氟苯丙氨酸(5ff)；苯乙烯丙氨酸(styrylkalanine，sAla)；蒽基丙氨酸(authrylalanine，aAla)；3，3-二苯基丙氨酸(Dfa)；3-氨基-5-苯基戊酸(phenypentanoic acid，Afp)；青霉胺(Pen)；1，2，3，4-四氢异喹啉-3-羧酸(Tic)；β-2-噻吩基丙氨酸(Thi)；蛋氨酸亚砜(Mso)；N(w)-硝基精氨酸(nArg)；高赖氨酸(hLys)；磷酸甲基苯丙氨酸(pmPhe)；磷酸丝氨酸(pSer)；磷酸苏氨酸(pThr)；高天冬氨酸(hAsp)；高谷氨酸(hGlu)；1-氨基环戊-(2或3)-烯-4羧酸；哌可酸(PA)，氮杂环丁烷-3-羧酸(ACA)；1-氨基环戊烷-3-羧酸；烯丙基甘氨酸(aOly)；炔丙基甘氨酸(pgGly)；高丙氨酸(hAla)；正缬氨酸(nVal)；高亮氨酸(hLeu)、高缬氨酸(hVal)；高异亮氨酸(hIle)；高精氨酸(hArg)；N-乙酰基赖氨酸(AcLys)；2，4-二氨基丁酸(Dbu)；2，3-二氨基丁酸(Dab)；N-甲基缬氨酸(MeVal)；高半胱氨酸(hCys)；高丝氨酸(hSer)；羟脯氨酸(Hyp)和高脯氨酸(hPro)。本文所述的单胺氧化酶可以包含的其他的非编码氨基酸对于本领域的技术人员来说将是明显的(参见，例如，在Fasman，1989，CRC Practical Handbook of Biochemistry and Molecular Biology(CRC生物化学和分子生物学实践手册)，CRC Press，Boca Raton，FL，在第3-70页和其中所引用的参考文献中所提供的多种氨基酸，该文献及其参考文献全部都通过引用并入)。这些氨基酸可以为L-构型或D-构型。
本领域的技术人员将认识到本文所公开的单胺氧化酶还可以包含具有侧链保护基团的氨基酸或残基。这种被保护的氨基酸在这种情况下属于芳族类别，它们的非限制性实例包括(保护基团在括号中列出)但不限于：Arg(tos)、Cys(甲基苄基)、Cys(硝基吡啶硫酰基)、Glu(δ-苄基酯)、Gln(呫吨基)、Asn(N-δ-呫吨基)、His(bom)、His(苄基)、His(tos)、Lys(fmoc)、Lys(tos)、Ser(O-苄基)、Thr(O-苄基)和Tyr(O-苄基)。
本文所述的单胺氧化酶可以包含的构象上受限的非编码氨基酸可以包括但不限于N-甲基氨基酸(L-构型)；1-氨基环戊-(2或3)-烯-4-羧酸；哌可酸；氮杂环丁烷-3-羧酸；高脯氨酸(hPro)；和1-氨基环戊烷-3-羧酸。
如上所述，可以将引入天然存在的多肽中来产生工程化单胺氧化酶的各种修饰靶向于特定的酶特性。
在另一方面，本公开内容提供编码本文所公开的工程化单胺氧化酶的多核苷酸。所述多核苷酸可与控制基因表达的一个或多个异源调节序列可操作地连接以产生能够表达多肽的重组多核苷酸。可以将含有编码工程化单胺氧化酶的异源多核苷酸的表达构建体引入适当的宿主细胞中以表达相应的单胺氧化酶多肽。
由于知道对应于各种氨基酸的密码子，所以蛋白序列的可用性提供了能够编码主题物的所有多核苷酸的说明。其中相同的氨基酸被可选的密码子或同义密码子编码的遗传密码的简并性容许制备极大量的核酸，它们全部编码本文所公开的改良的单胺氧化酶。因此，在鉴定了具体的氨基酸序列的情况下，本领域的技术人员能够通过以不改变蛋白氨基酸序列的方式简单地修饰一个或多个密码子序列来制备任何数目的不同核酸。
在一些实施方案中，多核苷酸包含编码具有如下的氨基酸序列的单胺氧化酶的核苷酸序列：与本文所述的参考工程化单胺氧化酶的任一个相比具有至少约80％或更多的序列同一性、约85％或更高的序列同一性、约90％或更高的序列同一性、约95％或更高的序列同一性、约96％或更高的序列同一性、约97％或更高的序列同一性、约98％或更高的序列同一性、或99％或更高的序列同一性。
在多个实施方案中，优选选择适合蛋白在其中表达的宿主细胞的密码子。例如，使用在细菌中使用的优选密码子来在细菌中表达基因，使用在酵母中使用的优选密码子来在酵母中表达；并且使用在哺乳动物中使用的优选密码子来在哺乳动物细胞中表达。
在某些实施方案中，不需要替代所有的密码子来优化单胺氧化酶的密码子使用，因为天然序列将包含优选的密码子并且因为优选密码子的使用可能不是所有氨基酸残基都需要的。因此，编码单胺氧化酶的密码子优化的多核苷酸可以在全长编码区的约40％、50％、60％、70％、80％或大于90％的密码子位置含有优选的密码子。
在其他实施方案中，多核苷酸包含如下的多核苷酸：编码本文所述的单胺氧化酶，但是在核苷酸水平上与编码工程化单胺氧化酶的参考多核苷酸具有约80％或更高的序列同一性、约85％或更高的序列同一性、约90％或更高的序列同一性、约95％或更高的序列同一性、约98％或更高的序列同一性、或99％或更高的序列同一性。
可以多种方式操作编码改良的单胺氧化酶的分离的多核苷酸以提供多肽的表达。依据表达载体，分离的多核苷酸在插入载体之前的操作可能是期望的或必需的。利用重组DNA方法修饰多核苷酸和核酸序列的技术是本领域中熟知的。指南提供在Sambrook等人，2001，Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)，第3版，Cold Spring HarborLaboratory Press；和Current Protocols in Molecular Biology(分子生物学最新实验方案)，Ausubel.F.编辑，Greene Pub.Associates，1998，更新至2006。
对于细菌宿主细胞，用于指导本公开内容的核酸构建体转录的合适启动子包括从下列基因中获得的启动子：大肠杆菌lac操纵子、天蓝色链霉菌(Streptomyces coelicolor)琼脂糖酶基因(dagA)、枯草芽孢杆菌(Bacillussubtilis)果聚糖蔗糖酶基因(sacB)、地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶基因(amyL)、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)麦芽糖淀粉 酶基因(amyM)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)α-淀粉酶基因(amyQ)、地衣芽孢杆菌青霉素酶基因(penP)、枯草芽孢杆菌xylA和xylB基因以及原核β-内酰胺酶基因(Villa-Kamaroff等人，1978，Proc.Natl Acad.Sci.USA 75：3727-3731)；以及tac启动子(DeBoer等人，1983，Proc.Natl Acad.Sci.USA 80：21-25)。其他启动子描述于Scientific American，1980，242：74-94中的“Useful proteins from recombinant bacteria(来自重组细菌的有用蛋白)”；和Sambrook等人，如上。
对于丝状真菌宿主细胞，用于指导本公开内容的核酸构建体转录的合适启动子包括从下列酶的基因获得的启动子：米曲霉TAKA淀粉酶、米赫根毛霉(Rhizomucor miehei)天冬氨酸蛋白酶、黑曲霉中性α-淀粉酶、黑曲霉酸稳定的α-淀粉酶、黑曲霉或泡盛曲霉(Aspergillus awamori)葡萄糖淀粉酶(glaA)、米赫根毛霉脂肪酶、米曲霉碱性蛋白酶、米曲霉磷酸丙糖异构酶、构巢曲霉乙酰胺酶和尖孢镰刀菌(Fusarium oxysporum)胰蛋白酶样蛋白酶(WO 96/00787)；以及NA2-tpi启动子(来自黑曲霉中性α-淀粉酶基因和米曲霉磷酸丙糖异构酶基因的启动子的杂合体)以及它们的突变启动子、截短启动子和杂合启动子。
在酵母宿主中，有用的启动子可以来自下列酶的基因：酿酒酵母(Saccharomyces cerevisiae)烯醇化酶(ENO-1)、酿酒酵母半乳糖激酶(GAL1)、酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)和酿酒酵母3-磷酸甘油酸激酶。用于酵母宿主细胞的其他有用启动子由Romanos等人，1992，Yeast 8：423-488描述。
控制序列还可以是合适的转录终止子序列，转录终止子序列是被宿主细胞识别以终止转录的序列。终止子序列与编码多肽的核酸序列的3’-端可操作地连接。在选择的宿主细胞中有功能的任何终止子可用于本文所公开的方法中。
例如，用于丝状真菌宿主细胞的示例性转录终止子可以从下列的基因获得：米曲霉TAKA淀粉酶、黑曲霉葡萄糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、黑曲霉α-葡萄糖苷酶和尖孢镰刀菌胰蛋白酶样蛋白酶。
用于酵母宿主细胞的示例性终止子可以从下列的基因获得：酿酒酵母烯醇化酶、酿酒酵母细胞色素C(CYC1)和酿酒酵母甘油醛-3-磷酸脱氢酶。用于酵母宿主细胞的其他有用终止子由Romanos等人，1992，如上描述。
控制序列还可以是合适的前导序列，前导序列是对于宿主细胞翻译重要的mRNA的非翻译区。前导序列与编码多肽的核酸序列的5’-端可操作地连接。可以使用在选择的宿主细胞中有功能的任何前导序列。用于丝状真菌宿主细胞的示例性前导序列从下列的基因获得：米曲霉TAKA淀粉酶和构巢曲霉磷酸丙糖异构酶。用于酵母宿主细胞的合适前导序列从下列的基因获得：酿酒酵母烯醇化酶(ENO-1)、酿酒酵母3-磷酸甘油酸激酶、酿酒酵母α-因子和酿酒酵母醇脱氢酶/甘油醛-3-磷酸脱氢酶(ADH2/GAP)。
控制序列还可以是多聚腺苷酸化序列，多聚腺苷酸化序列是与核酸序列的3’-端可操作地连接并且在被转录时被宿主细胞识别为向被转录的mRNA添加多聚腺苷残基的信号的序列。在选择的宿主细胞中有功能的任何多聚腺苷酸化序列可用于本文所公开的方法中。用于丝状真菌宿主细胞的示例性多聚腺苷酸化序列可以从下列的基因获得：米曲霉TAKA淀粉酶、黑曲霉葡萄糖淀粉酶、构巢曲霉邻氨基苯甲酸合酶、尖孢镰刀菌胰蛋白酶样蛋白和黑曲霉α-葡 萄糖苷酶。可用于酵母宿主细胞的多聚腺苷酸化序列由Guo和Sherman，1995，Mol Cell Bio15：5983-5990描述。
控制序列还可以是编码与多肽的氨基端连接并指导编码的多肽进入细胞分泌途径的氨基酸序列的信号肽编码区。核酸序列编码序列的5’端可以固有地含有在翻译阅读框中与编码分泌多肽的编码区区段天然连接的信号肽编码区。可选择地，编码序列的5′端可以含有对编码序列为外来的信号肽编码区。在编码序列不是天然含有信号肽编码区时可能需要外来信号肽编码区。
可选择地，外来信号肽编码区可以简单替代天然信号肽编码区以增强多肽的分泌。然而，指导表达的多肽进入选择的宿主细胞的分泌途径的任何信号肽编码区可用于本文所公开的方法中。
细菌宿主细胞的有效信号肽编码区是从下列的基因获得的信号肽编码区：芽孢杆菌(Bacillus)NClB 11837麦芽糖淀粉酶、嗜热脂肪芽孢杆菌α-淀粉酶、地衣芽孢杆菌枯草菌素、地衣芽孢杆菌β-内酰胺酶、嗜热脂肪芽孢杆菌中性蛋白酶(nprT、nprS、nprM)和枯草芽孢杆菌prsA。其他的信号肽由Simonen和Palva，1993，Microbiol Rev 57：109-137描述。
用于丝状真菌宿主细胞的有效信号肽编码区可以是从下列的基因获得的信号肽编码区：米曲霉TAKA淀粉酶、黑曲霉中性淀粉酶、黑曲霉葡萄糖淀粉酶、米赫根毛霉天冬氨酸蛋白酶、特异腐质霉(Humicola insolens)纤维素酶和绵毛状腐质霉(Humicola lanuginosa)脂肪酶。
可用于酵母宿主细胞的信号肽可以来自酿酒酵母α因子和酿酒酵母转化酶的基因。其他有用的信号肽编码区由Romanos等人，1992，如上描述。
控制序列还可以是编码位于多肽氨基端的氨基酸序列的前肽编码区。所得的多肽被称为前酶(proenzyme)或前多肽(或在一些情况下，酶原)。前多肽一般是无活性的并且其可以通过自前多肽催化裂解或自催化裂解前肽而转化为成熟的活性多肽。前肽编码区可以从下列的基因获得：枯草芽孢杆菌碱性蛋白酶(aprE)、枯草芽孢杆菌中性蛋白酶(nprT)、酿酒酵母α因子、米赫根毛霉天冬氨酸蛋白酶和嗜热毁丝霉(Myceliophthorathermophila)乳糖酶(WO 95/33836)。
在信号肽和前肽区均存在于多肽的氨基端时，前肽区位于多肽的氨基端相邻的位置并且信号肽区位于前肽区的氨基端相邻的位置。
还可能期望的是添加调节序列，该调节序列容许相对于宿主细胞生长调节多肽表达。调节系统的实例是响应于化学或物理刺激物而引起基因表达开启或关闭的那些调节系统，所述化学或物理刺激物包括调节化合物的存在。在原核宿主细胞中，合适的调节序列包括lac、tac和trp操纵子系统。在酵母宿主细胞中，合适的调节系统包括：例如，ADH2系统或GAL1系统。在丝状真菌中，合适的调节序列包括TAKAα淀粉酶启动子、黑曲霉葡萄糖淀粉酶启动子和米曲霉葡萄糖淀粉酶启动子。
调节序列的其他实例是容许基因扩增的那些调节序列。在真核系统中，这些调节序列包括在氨甲喋呤的存在下扩增的二氢叶酸还原酶基因和具有重金属时扩增的金属硫蛋白基因。在这些情况下，编码本公开内容的单胺氧化酶的核酸序列将与调节序列可操作地连接。
因此，在另一实施方案中，本公开内容还涉及包含编码工程化单胺氧化酶或其变体的多核苷酸和一个或多个表达调节区的重组表达载体，所述表达调节区如启动子和终止子、复制 起点等，这取决于它们要引入的宿主的类型。可以将上述各种核酸和控制序列连接在一起以产生重组表达载体，该重组表达载体可包括一个或多个方便的限制性位点以容许编码多肽的核酸序列在这些位点插入或取代。可选择地，可以通过将本公开内容的核酸序列或包含该序列的核酸构建体插入到合适的表达载体中来表达本公开内容的核酸序列。在制备表达载体中，编码序列位于载体中使得编码序列与用于表达的适当控制序列可操作地连接。
重组表达载体可以是可以方便地进行重组DNA程序并且可以使得多核苷酸序列表达的任何载体(例如，质粒或病毒)。载体的选择通常取决于载体与该载体要引入其中的宿主细胞的相容性。载体可以是线型质粒或闭合的环状质粒。
表达载体可以是自主复制的载体，即，作为染色体外实体存在的不依赖于染色体复制而复制的载体，例如，质粒、染色体外元件、微型染色体或人工染色体。载体可以含有用于确保自我复制的任何部件(means)。可选择地，载体可以是在被引入宿主细胞中时，整合到基因组中并与其所整合进的染色体一起被复制的载体。此外，可以使用单个载体或质粒、或一起含有要引入宿主细胞基因组的总DNA的两个或更多个载体或质粒、或者转座子。
本公开内容的表达载体优选含有一个或多个可选择标记物，该标记物容许容易地选择转化的细胞。可选择标记物是产物能提供杀生物剂或病毒抗性、重金属抗性、营养缺陷型的原养型以及类似特性的基因。细菌可选择标记物的实例是来自枯草芽孢杆菌或地衣芽孢杆菌的dal基因或赋予如下抗生素抗性的标记物：例如氨苄西林抗性、卡那霉素抗性、氯霉素抗性或四环素抗性。用于酵母宿主细胞的合适的标记物是ADE2、HIS3、LEU2、LYS2、MET3、TRP1和URA3。
用于丝状真菌宿主细胞的可选择标记物包括但不限于：amdS(乙酰胺酶)、argB(鸟氨酸氨甲酰转移酶)、bar(丝膦菌素乙酰基转移酶)、hph(潮霉素磷酸转移酶)、niaD(硝酸还原酶)、pyrG(乳清酸核苷-5′-磷酸脱羧酶)、sC(硫酸腺苷转移酶)和trpC(邻氨基苯甲酸合酶)以及它们的等同物。用于曲霉细胞的实施方案包括构巢曲霉或米曲霉的amdS和pyrG基因以及吸水链霉菌(Streptomyces hygroscopicus)的bar基因。
本公开内容的表达载体优选含有容许载体整合到宿主细胞基因组中或者容许载体在所述细胞中不依赖于基因组而自主复制的元件。对于向宿主细胞基因组中整合，载体可能依赖于编码多肽的核酸序列或载体的任何其他元件以通过同源重组或非同源重组将载体整合到基因组中。
可选择地，表达载体可以含有用于指导通过同源重组向宿主细胞基因组中整合的其他核酸序列。所述其他核酸序列能够在染色体中的精确位置将载体整合到宿主细胞基因组中。为了增加在精确位置整合的可能性，整合元件应优选含有足够数目的与相应的靶序列高度同源的核酸以增强同源重组的可能性，所述足够数目例如100至10,000个碱基对，优选400至10,000个碱基对，并且最优选800至10,000个碱基对。整合元件可以是与宿主细胞基因组中的靶序列同源的任何序列。此外，整合元件可以是非编码的或编码的核酸序列。另一方面，可以通过非同源重组将载体整合到宿主细胞基因组中。
对于自主复制，载体可以还包含复制起点，复制起点使得载体能够在所研究的宿主细胞中自主复制。细菌复制起点的实例是P15A ori或质粒pBR322、pUC19、pACYC177(该质粒 具有P15A ori)的复制起点、或容许在大肠杆菌中复制的pACYC184以及容许在芽胞杆菌中复制的pUB110、pE194、pTA1060或pAMβ1。用于酵母宿主细胞中的复制起点的实例是2微米复制起点、ARS1、ARS4、ARS1和CEN3的组合、以及ARS4和CEN6的组合。复制起点可以是具有使其机能在宿主细胞中温度敏感的突变的复制起点(参见，例如Ehrlich，1978，Proc Natl Acad Sci.USA 75：1433)。
可以将多于1拷贝的本公开内容的核酸序列插入到宿主细胞中以增加基因产物的产生。核酸序列拷贝数的增加可以如下来获得：通过将至少一个额外拷贝的序列整合到宿主细胞基因组中；或通过在细胞含有扩增拷贝的可选择标记物基因时随核酸序列包括可扩增的可选择标记物基因，并且从而可以通过在合适的选择剂存在下培育细胞来选择额外拷贝的核酸序列。
用于本文所公开的方法的许多表达载体是可商购获得的。合适的商业表达载体包括：来自Sigma-Aldrich Chemicals，St.Louis MO.的p3xFLAGTM TM表达载体，它包括用于在哺乳动物宿主细胞中表达的CMV启动子和hGH多聚腺苷酸化位点以及用于在大肠杆菌中扩增的pBR322复制起点和氨苄霉素抗性标记物。其他合适的表达载体是可从Stratagene，LaJolla CA商购获得的pBluescriptII SK(-)和pBK-CMV、自pBR322(GibcoBRL)、pUC(Gibco BRL)、pREP4、pCEP4(Invitrogen)或pPoly衍生的质粒(Lathe等人，1987，Gene 57：193-201)。
在另一方面，本公开内容提供包含编码本公开内容的改良单胺氧化酶的多核苷酸的宿主细胞，所述多核苷酸与用于在宿主细胞中表达单胺氧化酶的一个或多个控制序列可操作地连接。用于表达由本公开内容的表达载体编码的单胺氧化酶多肽的宿主细胞是本领域中熟知的并且包括但不限于：细菌细胞，如大肠杆菌、克菲尔乳杆菌(Lactobacillus kefir)、短乳杆菌(Lactobacillus brevis)、小乳杆菌(Lactobacillus minor)、链霉菌和鼠伤寒沙门氏菌(Salmonella typhimurium)细胞；真菌细胞，如酵母细胞(例如，酿酒酵母或巴斯德毕赤酵母(Pichia pastoris)(ATCC登录号201178))；昆虫细胞，如果蝇(Drosophila)S2和灰翅夜蛾(Spodoptera)Sf9细胞；动物细胞，如CHO、COS、BHK、293和Bowes黑素瘤细胞；以及植物细胞。用于上述宿主细胞的适当培养基和生长条件是本领域中熟知的。
可以通过本领域中已知的多种方法将表达单胺氧化酶的多核苷酸引入细胞中。技术包括但不限于：电穿孔、生物射弹粒子轰击、脂质体介导的转染、氯化钙转染和原生质体融合。将多核苷酸引入细胞中的多种方法对于熟悉的技术人员来说是明显的。
示例性的宿主细胞是大肠杆菌(Escherichia coli)W3110。表达载体通过将编码改良单胺氧化酶的多核苷酸可操作地连接到质粒pCK110900中来制造，所述质粒pCK110900与受lacI阻遏物控制的lac启动子可操作地连接。表达载体还含有P15a复制起点和氯霉素抗性基因。通过使细胞进行氯霉素选择来分离在大肠杆菌W3110中含有主题多核苷酸的细胞。
工程化单胺氧化酶可以通过使编码天然存在的单胺氧化酶的多核苷酸进行诱变和/或定向进化方法而获得。示例性的定向进化技术是如下列文献中所描述的诱变和/或DNA改组：Stemmer，1994，ProcNatl Acad Sci USA 91：10747-10751；WO 95/22625；WO 97/0078；WO97/35966；WO 98/27230；WO 00/42651；WO 01/75767和美国专利6,537,746。可以使用的其他定向进化程序包括但不限于：交错延伸方法(StEP)、体外重组(Zhao等人，1998， Nat.Biotechnol.16：258-261)、诱变PCR(Caldwell等人，1994，PCR Methods Appl.3：S136-S140)和盒式诱变(Black等人，1996，Proc Natl Acad Sci USA 93：3525-3529)。
筛选诱变处理之后获得的克隆中具有期望的改良酶特性的工程化单胺氧化酶。由表达文库测量酶活性可以使用标准生物化学技术进行，例如但不限于可以使用公布的用于测量单胺氧化酶的方法或其改造方法进行，例如但不限于Zhou等人(Zhou等人“A One-Step Fluorometric Methodfor the Continuous Measurement of Monoamine Oxidase Activity(单胺氧化酶活性连续测量的一步式荧光法)，”1997Anal.Biochem.253：169-74)和Szutowicz等人(Szutowicz等人，“Colorimetric Assay for MonoamineOxidase in Tissues Using Peroxidase and2，2′-Azino(3-ethtylbenzthaizoline-6-sulfonic Acid)as Chromogen(使用过氧化物酶和2，2′-连氮基(3-乙基苯并噻唑啉-6-磺酸)作为色原进行的组织中单胺氧化酶的比色测定)，”1984，Anal.Biochem.138：86-94)所公开的那些方法。酶活性的比较是使用限定的酶制剂、在设定条件下的限定的测定和一种或多种限定的底物进行的，如本文进一步详细描述的；或者使用例如Zhou和Szutowicz的方法进行。一般地，当比较裂解物时，测定细胞数和测定的蛋白的量并且使用相同的表达系统和相同的宿主细胞来将宿主细胞产生的酶和裂解物中存在的酶的量的差异最小化。在期望改良的酶特性是热稳定性时，可以在使酶制剂经受限定的温度并测量热处理后剩余的酶活性的量来测量酶活性。然后分离含有编码单胺氧化酶的多核苷酸的克隆，将该克隆测序以鉴定核苷酸序列改变(若存在)，并将其用于在宿主细胞中表达酶。
当已知工程化多肽的序列时，编码酶的多核苷酸可以根据已知的合成方法通过标准的固相方法来制备。在一些实施方案中，可以分别合成最多约100个碱基的片段，然后将它们连接(例如，通过酶连接或化学连接方法或聚合酶介导的方法)以形成任何期望的连续序列。例如，可以通过化学合成使用如下的方法来制备本文所公开的多核苷酸和寡核苷酸：例如Beaucage等人，1981，Tet Lett 22：1859-69所描述的经典的亚磷酰胺方法；或Matthes等人，1984，EMBO J.3：801-05所描述的方法，例如，如同该方法在自动化合成方法中所通常实践的一样。根据亚磷酰胺方法，在例如自动化DNA合成仪中合成寡核苷酸，将其纯化、退火、连接并克隆在适当的载体中。此外，基本上任何核酸可以从多种商业来源的任一种获得，例如The Midland Certified Reagent Company，Midland，TX、The GreatAmerican Gene Company，Ramona，CA、ExpressGen Inc.Chicago，IL、OperonTechnologies Inc.，Alameda，CA以及许多其他商业来源。
可以使用用于蛋白纯化的熟知技术的任一种或多种将在宿主细胞中表达的工程化单胺氧化酶从所述细胞和或培养基中回收，所述熟知技术包括但不限于：溶菌酶处理、超声处理、过滤、盐析、超速离心以及色谱。用于裂解和从诸如大肠杆菌的细菌中高效提取蛋白的合适的溶液可以商品名CelLytic B TM从St.Louis MO的Sigma-Aldrich商购获得。
用于分离单胺氧化酶的色谱技术包括但不限于：反相色谱高效液相色谱、离子交换色谱、凝胶电泳和亲和色谱。用于纯化特定酶的条件将部分取决于诸如静电荷、疏水性、亲水性、分子量、分子形状等因素，并且对于本领域技术人员来说将是明显的。
在一些实施方案中，可以使用亲和技术来分离改良的单胺氧化酶。对于亲和色谱纯化，可以使用与单胺氧化酶特异性结合的任何抗体。对于抗体制备，可以通过用化合物注射来免 疫多种宿主动物，包括但不限于：家兔、小鼠、大鼠等。可以借助于侧链官能团或与侧链官能团连接的连接物来将化合物连接到合适的载体如BSA上。可以依据宿主物种使用多种佐剂来增加免疫反应，所述佐剂包括但不限于：Freund’s(完整的和不完整的)、矿物胶如氢氧化铝、表面活性物质如溶血卵磷脂、复合多元醇、聚阴离子、肽、油乳液、匙孔血蓝蛋白、二硝基苯酚和可能有用的人佐剂如BCG(卡介苗)和短小棒状杆菌(Corynebacterium parvum)。
如本领域的技术人员已知的，单胺氧化酶催化的氧化反应通常需要辅因子。本文所述的单胺氧化酶催化的氧化反应通常也需要辅因子黄素腺嘌呤核苷酸(FAD)。如本文所用，术语“辅因子”是指与单胺氧化酶组合起作用的非蛋白化合物。一般地，将可以与单胺氧化酶非共价或共价连接的氧化形式的辅因子添加至反应混合物中。氧化的FAD形式可以通过分子氧而由还原形式FAD-H 2再生。在另一实施方案中，氧化的FAD形式可以由NAD(P)再生以提供FAD和NAD(P)H。NAD(P)继而可以通过使用NAD(P)H-依赖性醇脱氢酶/酮还原酶将酮还原为醇而再生。
本文所述的单胺氧化酶催化的氧化反应一般在溶剂中进行。合适的溶剂包括水、有机溶剂(例如，乙酸乙酯、乙酸丁酯、1-辛醇(octacnol)、庚烷、辛烷、甲基叔丁醚(MTBE)、甲苯以及类似有机溶剂)和离子液体(例如，1-乙基4-甲基咪唑四氟硼酸盐、1-丁基-3-甲基咪唑四氟硼酸盐、1-丁基-3-甲基咪唑六氟磷酸盐以及类似离子液体)。在一些实施方案中，使用含水溶剂，包括水和含水助溶剂系统。
示例性的含水助溶剂系统具有水和一种或多种有机溶剂。一般地，选择含水助溶剂系统的有机溶剂组分使得该有机溶剂组分不会完全灭活单胺氧化酶。可以通过利用如本文所述的那些酶活性测定，在候选溶剂系统中用感兴趣的限定底物测量指定的工程化单胺氧化酶的酶活性来容易地鉴定适当的助溶剂系统。
含水助溶剂系统的有机溶剂组分可以与含水组分混溶，得到单一的液相；或者可以与含水组分部分混溶或不混溶，得到两个液相。一般地，在采用含水助溶剂系统时，将其选择为双相性的，其中水分散在有机溶剂中或者反之亦然。一般地，在采用含水助溶剂系统时，期望的是选择可以容易地与水相分离的有机溶剂。一般地，助溶剂系统中水与有机溶剂的比通常在有机溶剂比水为约90∶10至约10∶90(v/v)的范围内和有机溶剂比水为80∶20与20∶80(v/v)之间的范围内。助溶剂系统可以在添加到反应混合物之前预形成，或者其可以在反应容器中原位形成。
含水溶剂(水或含水助溶剂系统)可以是pH缓冲的或未缓冲的。一般地，氧化可以在约10或以下，通常在约5至约10的范围内的pH下进行。在一些实施方案中，氧化在约9或以下，通常在约5至约9的范围内的pH下进行。在一些实施方案中，氧化在约8或以下，通常在约5至约8的范围内，并且通常在约6至约8的范围内的pH下进行。氧化还可以在约7.8或以下、或7.5或以下的pH下进行。可选择地，氧化可以在中性pH，即约7下进行。
在氧化反应过程中，反应混合物的pH可以改变。典型的结构式I的胺在中性pH和约中性pH下是质子化的，而结构式II的亚胺产物在中性pH和约中性pH下则通常不是质子化的。因此，在其中反应在中性pH或约中性pH下进行的典型实施方案中，质子化的胺向非质子化的亚胺的氧化将质子释放到水溶液中。可以通过在反应过程中添加碱来将反应混合物的pH 维持在期望pH下或期望pH范围内。可选择地，可以使用包含缓冲液的含水溶剂控制pH。维持期望的pH范围的合适的缓冲液是本领域中已知的并且包括：例如，磷酸盐缓冲液、三乙醇胺缓冲液以及类似缓冲液。还可以使用缓冲或碱添加的组合。
用于中和酸的合适碱是：有机碱，例如胺、醇盐以及类似有机碱；和无机碱，例如氢氧化物盐(例如，NaOH)、碳酸盐(例如，NaHCO 3)、碳酸氢盐(例如，K 2CO 3)、碱性磷酸盐(例如，K 2HPO 4、Na 3PO 4)以及类似无机碱。用于中和在反应过程中由胺氧化为亚胺而释放的质子的优选的碱是胺底物本身。在转化过程同时添加碱可以手动进行，同时监测反应混合物pH，或者更方便地，通过使用自动滴定器作为pH固定器(pH stat)。还可以使用部分缓冲能力和碱添加的组合进行过程控制。通常，以水溶液添加在氧化过程中添加到未缓冲的或部分缓冲的反应混合物中的碱。
在进行本文所述的立体选择性氧化反应中，可以将工程化单胺氧化酶以下列形式添加到反应混合物中：纯化的酶、用编码单胺氧化酶的基因转化的全细胞、和/或这种细胞的细胞提取物和/或裂解物。用编码工程化单胺氧化酶的基因转化的全细胞或其细胞提取物和/或裂解物可以多种不同的形式采用，包括固体(例如，冻干的、喷雾干燥的以及类似形式)或半固体(例如，粗糊料)。
可以通过沉淀(硫酸铵、聚乙烯亚胺、热处理或类似方法)部分纯化细胞提取物或细胞裂解物，随后在冻干之前是脱盐程序(例如，超滤、透析和类似方法)。可以通过使用诸如戊二醛的已知交联剂的交联或固定于固相(例如，Eupergit C和类似物)来稳定任一细胞制剂。
可以将固体反应物(例如，酶、盐等)以多种不同形式提供到反应中，包括：粉末(例如，冻干的、喷雾干燥的和类似形式)、溶液、乳液、悬浮液以及类似形式。可以使用本领域普通技术人员已知的方法和设备容易地将反应物冻干或喷雾干燥。例如，可以将蛋白溶液在-80℃以小份冷冻，然后将其添加到预冷冻的冻干室中，随后施加真空。从样品中移除水之后，在释放真空并取回冻干样品之前，通常将温度升至4℃持续2小时。
氧化反应中所用的反应物的量一般将依据所需的产物的量以及同时采用的单胺氧化酶底物的量而变化。一般地，使用约50mg/升至约5g/升的单胺氧化酶时可以使用约5克/升至50克/升的浓度的底物。本领域普通技术人员将容易地了解如何改变这些量来使它们适合期望的产力水平和产物规模。可以通过常规实验容易地确定任选试剂如过氧化氢酶、消泡剂和亚硫酸氢钠或偏亚硫酸氢钠的适当量。
反应物添加的顺序不是严格的。可以将反应物在同一时间一起添加到溶剂(例如，单相溶剂、双相含水助溶剂系统以及类似溶剂)中，或者可选择地，在不同的时间点，一些反应物可以分别添加，并且一些反应物一起添加。在某些实施方案中，可以将反应的一种或多种组分以将单胺氧化酶的底物和/或产物抑制最小化或消除的水平连续添加(“进料”)至反应中。在某些实施方案中，可以在反应过程中间断地添加单胺氧化酶，例如约每隔1小时、约每隔2小时、约每隔3小时或约每隔4小时添加。
进行本文所述的单胺氧化酶催化的氧化反应的合适条件包括可以通过常规实验容易地优化的大量条件，所述常规实验包括但不限于使工程化单胺氧化酶和底物在实验pH和温度下接触并通过例如本文所提供的实施例中描述的方法检测产物。
单胺氧化酶催化的氧化通常在约5℃至约75℃的范围内的温度下进行。对于一些实施方案，反应在约20℃至约55℃的范围内的温度下进行。在其他的实施方案中，反应在约20℃至约45℃的范围内、约30℃至约45℃的范围内或约40℃至约45℃的范围内的温度下进行。反应还可以在环境温度下(约21℃)进行。
一般容许氧化反应进行到直至获得基本上完全的或接近完全的底物氧化。可以使用检测底物和/或产物的已知方法来监测底物至产物的氧化。合适的方法包括气相色谱、HPLC以及类似方法。转化产率一般大于约50％、还可以大于约60％、还可以大于约70％、还可以大于约80％、还可以大于约90％并且通常大于约97％。
本说明书中提及的所有出版物和专利申请均通过引用并入本文，其程度如同每个单独的出版物或专利申请被具体地和单独地指出通过引用并入一样。
附图说明
图1代表Ampliflu Red荧光法检测H 2O 2浓度标准曲线。
图2代表MAON突变体酶活测试结果。
序号 名称
SEQ ID NO:1 单胺氧化酶MAON
SEQ ID NO:2 MAON变体F63L
SEQ ID NO:3 MAON变体T65V
SEQ ID NO:4 MAON变体S100P
SEQ ID NO:5 MAON变体T141S
SEQ ID NO:6 MAON变体S234C
SEQ ID NO:7 MAON变体F63L/T65V
SEQ ID NO:8 MAON变体S234C/S100P
SEQ ID NO:9 MAON变体S234C/T141S
SEQ ID NO:10 MAON变体F63L/T65V/S100P
SEQ ID NO:11 MAON变体F63L/T65V/T141S
SEQ ID NO:12 MAON变体F63L/T65V/S234C
SEQ ID NO:13 MAON变体S234C/T141S/S100P
SEQ ID NO:14 MAON变体F63L/T65V/S100P/T141S
SEQ ID NO:15 MAON变体F63L/T65V/S100P/S234C
SEQ ID NO:16 MAON变体F63L/T65V/T141S/S234C
SEQ ID NO:17 MAON变体F63L/T65V/S100P/T141S/S234C
具体实施方式
本公开内容的多种特征和实施方案在下列代表性实施例中示例，这些实施例旨在示例性的并且不旨在限制。
实施例1.单胺氧化酶突变体载体构建
将来源于黑曲霉(Aspergillus niger)的单胺氧化酶MAON蛋白表达基因，以及基于稳定性和催化活性改造设计的MAON突变体基因(SEQ ID NO:1-17)，连接到大肠杆菌表达载体pET15b上，插入位点NdeI+BamHI，保留N-ter 6×His tag。测序正确后，重组载体转化BL21(DE3)用于蛋白表达。
实施例2.单胺氧化酶表达与纯化：摇瓶生产
将构建好的表达载体转入大肠杆菌BL21(DE3)中，经IPTG诱导表达，收菌裂解后利用Ni-NTA柱进行纯化，具体方法如下：将MAON酶重组表达载体转化到BL21(DE3)菌株中，挑取单克隆到10ml LB培养基中，氨苄青霉素钠抗性(100mg/L)，37℃，200RPM培养过夜，转入含有1L LB培养基的2L摇瓶中，37℃，200RPM，培养到OD600达到0.6-0.8时，降温到25℃，0.5mM IPTG诱导表达过夜，5000xg离心收菌。将收集好的菌体利用buffer A：50mM Tris pH 8.0,500mM NaCl,20mM咪唑重悬，加入终浓度为1mM PMSF，250ul Cocktail inhibitor，混匀。高压匀浆破碎仪破碎后，43000xg，4℃离心30min，取上清过Ni柱。Ni-NTA柱纯化，将裂解液上清与resin结合20min后，用含有50mM咪唑的buffer A洗杂，最后用含400mM咪唑的洗脱缓冲液洗脱。SDS-PAGE检测蛋白纯化效果。透析换buffer至50mM Tris pH7.5,500mM NaCl,1mM DTT。最终样品SDS-PAGE检测蛋白纯化效果，超滤浓缩后，冻存于-80℃备用。
实施例3.单胺氧化酶表达与纯化：发酵生产
种子活化：将MAON酶重组表达载体转化到BL21(DE3)菌株中，挑取单克隆到10ml LB培养基中，氨苄青霉素钠抗性(100mg/L)，37℃，200RPM培养过夜，转入含有500mL LB培养基的1L摇瓶中，37℃，200RPM，培养到OD600达到0.8-1.0。发酵培养：将含有6L TB培养基的10L发酵罐培养基预热到37℃，加入终浓度为100mg/L的氨苄青霉素钠，接种后通气搅拌维持30％溶氧，当OD600增长到10时，开始补料，补料1为含有60g/L胰蛋白胨，120g/L酵母提取物，4％甘油的水溶液，补料2为50％甘油，利用氨水和磷酸调节酸碱度稳定在pH 7.0。当OD600值增长到20时，发酵液降温到25℃，加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度为1mM诱导单胺氧化酶表达，使培养物再生长20小时，直至收获。8000xg离心收获细胞。将收获的细胞直接用于随后的纯化过程或将其储存-80℃下直到如此使用。粗酶纯化：将收集好的菌体利用100mM Tris pH 8.0,150mM NaCl重悬，按照200g湿菌体/L重悬混匀。800Bar高压匀浆破碎仪破碎后，18400xg，4℃离心20min，取上清加入最终浓度为36％饱和度的硫酸铵粉末(200g/L)，离心收集蛋白沉淀。沉淀经过冷冻干燥后，存于4℃备用。
实施例4.单胺氧化酶活性测定
由于单胺氧化酶催化产生H 2O 2，通过测定过氧化氢的产生量，可以间接测定酶活参数，标准曲线绘制：取5ml的100mM K 2HPO 4·HCl pH 7.4缓冲液，加入终浓度为100uM的Ampliflu Red染料和1U/ml的辣根过氧化物酶配成工作液，利用工作液分别配制含有0、1.25、2.5、5、10、20、40浓度H 2O 2的反应液，利用酶标仪测定荧光数值，λex＝535nm/λem＝590nm，绘制标准曲线，如图1所示。MAON酶活参数测定利用100mM K 2HPO 4·HCl pH 7.4缓冲液，采用实施例2所述方法，纯化所得MAON酶，通过BCA法测定蛋白浓度，稀释成50nM工作液，并利用该缓冲液将底物配制成终浓度为0、47、94、188、375、750、1500、3000uM的反应液，取95ul反应液，加入5ul酶工作液，酶工作终浓度为2.5nM。利用酶标仪测定荧光数值变化，λex＝535nm/λem＝590nm，部分测试结果如图2所示。由图可知，SEQ ID NO:7突变体酶活性是对照SEQ ID NO:1的2倍，SEQ ID NO:6突变体酶活性是对照SEQ ID NO: 1的4倍，SEQ ID NO:12突变体酶活性是对照SEQ ID NO:1的5倍。
实施例5.单胺氧化酶催化6,6-二甲基-3-氮杂二环[3.1.0]己烷氧化生成(1R，5S)-6，6-二甲基-3-氮杂二环[3.1.0]己-2-烯活性测试
将由SEQ ID NO:1-17经实施例2表达和纯化的单胺氧化酶缓冲液40ml(2mg/ml，储存在pH 7.4的磷酸钾盐酸缓冲溶液中，黄色液体)加入到三口烧瓶中，向其中加入70mg过氧化氢酶，2mg消泡剂204，25℃条件下搅拌，氧气环境下。另外称取280mg 6,6-二甲基-3-氮杂二环[3.1.0]己烷用5ml pH 7.4的磷酸钾盐酸缓冲溶液溶解后，通过注射泵5小时内滴加到上述反应体系中，反应期间用3M氢氧化钠溶液调节pH维持在7.4，反应18小时后液质中控，转化率>95％，反应结束后加入甲基叔丁基醚萃取，甲基叔丁基醚相旋干得产品，液相测得ee>99％。
1H NMR(300MHz,Chloroform-d)δ7.39-7.35(m,1H),3.92-3.81(m,1H),3.62-3.52(m,1H),2.18–1.99(m,1H),1.72-1.63(m,1H),1.10(s,3H),0.76(s,3H).
实施例6.在亚硫酸氢盐存在下单胺氧化酶催化6,6-二甲基-3-氮杂二环[3.1.0]己烷去对称化
将40ml pH 7.4磷酸钾盐酸缓冲溶液加入到三口烧瓶中，加入500mg将由SEQ ID NO:1-17经实施例2表达和纯化的单胺氧化酶，后加入175mg过氧化氢酶，2.5mg消泡剂204，25℃条件下搅拌，氧气环境下搅拌。另外称取1.25g亚硫酸氢钠用8ml水溶解后加入1g 6,6-二甲基-3-氮杂二环[3.1.0]己烷，底物通过注射泵5小时内滴加到生物酶反应体系中，反应期间用3M氢氧化钠溶液调节pH维持在7.4，24小时后中控，液相质谱分析，原料转化完成，体系中为(1R,5S)-6,6-二甲基-3-氮杂二环[3.1.0]己-2-烯和(1R,2S,5S)-6,6-二甲基-3-氮杂二环[3.1.0]己烷-2-磺酸钠的混合物。其中，在反应进行1h、3h、6h、9h、11h时，分别取样检测，显示SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:12样品反应体系中，底物剩余量相比SEQ ID NO:1样品更少，说明活性更高。
实施例7.(1R,2S,5S)-6,6-二甲基-3-氮杂二环[3.1.0]己烷-2-腈的制备
将40ml pH 7.4磷酸钾盐酸缓冲溶液加入到三口烧瓶中，加入将由SEQ ID NO:1-17经实施例2表达和纯化的单胺氧化酶，后加入175mg过氧化氢酶，2.5mg消泡剂204，25℃条件下搅拌，氧气环境下搅拌。另外称取1.25g亚硫酸氢钠用8ml水溶解后加入1g 6,6-二甲基-3-氮杂二环[3.1.0]己烷，底物通过注射泵5小时内滴加到生物酶反应体系中，反应期间用3M氢氧化钠溶液调节pH维持在7.4，24小时后中控，液相质谱分析，原料转化完成，体系中为(1R,5S)-6,6-二甲基-3-氮杂二环[3.1.0]己-2-烯和(1R,2S,5S)-6,6-二甲基-3-氮杂二环[3.1.0]己烷-2-磺酸钠的混合物，将反应体系降至10℃后加入40毫升MTBE，30分钟内滴加1g TMSCN，反应30分钟液相质谱分析有产品生成。其中，SEQ ID NO:6样品反应体系中反应液垫硅藻土过滤后，MTBE相和水相分层，水相用MTBE萃取三次后，MTBE相合并，用硫酸钠干燥后旋干得950mg产品，收率77％。
虽然本发明通过许多不同形式的实施方案来满足，但是如结合本发明的优选的实施方案详细描述的，应理解本公开内容应被认为是对本发明的原理的示例而不意在将本发明局限于本文说明和描述的具体实施方案。本领域的技术人员可以作出许多变化而不脱离本发明的精神。本发明的范围将通过附加的权利要求和它们的等同物判断。摘要和标题不应被解释为限 制本发明的范围，因为它们的目的是使适当的机构以及一般公众能够迅速确定本发明的一般性质。

Claims (13)

  1. 一种单胺氧化酶，其包含与如SEQ ID NO:1所示的单胺氧化酶氨基酸序列相比具有下述突变的氨基酸序列：在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸、第141位氨基酸由苏氨酸突变为丝氨酸和/或第234位氨基酸由丝氨酸突变为半胱氨酸。
  2. 根据权利要求1所述的单胺氧化酶，其特征在于，所述单胺氧化酶包含与如SEQ ID NO:1所示的单胺氧化酶氨基酸序列相比具有下述突变的氨基酸序列：
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸且第65位氨基酸由苏氨酸突变为缬氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第100位氨基酸由丝氨酸突变为脯氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸且第100位氨基酸由丝氨酸突变为脯氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸且第141位氨基酸由苏氨酸突变为丝氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第100位氨基酸由丝氨酸突变为脯氨酸、第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸且第141位氨基酸由苏氨酸突变为丝氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸；或者，
    在对应于SEQ ID NO:1所示的单胺氧化酶氨基酸序列中的第63位氨基酸由苯丙氨酸突变为亮氨酸、第65位氨基酸由苏氨酸突变为缬氨酸、第100位氨基酸由丝氨酸突变为脯氨酸、第141位氨基酸由苏氨酸突变为丝氨酸且第234位氨基酸由丝氨酸突变为半胱氨酸。
  3. 根据权利要求1或2所述的单胺氧化酶，所述氨基酸序列进一步与SEQ ID NO:1所示的单胺氧化酶氨基酸序列具有至少80％、至少85％、至少90％、至少95％、至少96％、 至少97％、至少98％、至少99％序列同一性。
  4. 根据权利要求1-3任意一项所述的单胺氧化酶，其包含与选自由以下组成的组的任一氨基酸序列具有至少92％、至少95％、至少96％、至少97％、至少98％、至少99％或100％序列同一性的氨基酸序列：SEQ ID NO:2-17。
  5. 根据权利要求4所述的单胺氧化酶，其氨基酸序列如SEQ ID NO:2-17任一所示。
  6. 一种编码权利要求1-5任意一项所述的单胺氧化酶的多核苷酸及包含所述多核苷酸的宿主细胞。
  7. 一种制备基本上立体异构纯的如Ⅱ
    Figure PCTCN2023070432-appb-100001
    所示的化合物或其盐/水合物的方法，其包括使如Ⅰ
    Figure PCTCN2023070432-appb-100002
    所示的化合物在如权利要求1-5任意一项所示的单胺氧化酶和辅因子的存在下与氧接触。
  8. 一种制备基本上对映体纯的如Ⅲ
    Figure PCTCN2023070432-appb-100003
    所示的氨基磺酸盐化合物或其盐/水合物的方法，其包括使如Ⅰ
    Figure PCTCN2023070432-appb-100004
    所示的化合物在如权利要求1-5任意一项所示的单胺氧化酶、辅因子和亚硫酸氢盐的存在下与氧接触。
  9. 一种制备基本上对映体纯的如Ⅳ
    Figure PCTCN2023070432-appb-100005
    所示的氨基腈化合物或其盐/水合物的方法，其包括使如Ⅰ
    Figure PCTCN2023070432-appb-100006
    所示的化合物在如权利要求1-5任意一项所示的单胺氧化酶、辅因子和亚硫酸氢盐的存在下与氧接触，以及将所得的氨基磺酸盐化合物与氰化物接触。
  10. 根据权利要求7-9任意一项所述的方法，其特征在于，所述辅因子与单胺氧化酶非共价缔合；
    优选地，所述辅因子选自由下列组成的组：FAD、FMN、NAD和NADP；
    优选地，所述方法还包含催化过氧化氢歧化为分子氧和水的组分，更优选地，所述组分选自由下列组成的组：Pd、Fe和过氧化氢酶。
  11. 如权利要求1-5任意一项所述的单胺氧化酶在催化如Ⅰ
    Figure PCTCN2023070432-appb-100007
    所示的化合物氧化为基本上立体异构纯的如Ⅱ
    Figure PCTCN2023070432-appb-100008
    所示的化合物或其盐/水合物上的应用。
  12. 如权利要求1-5任意一项所述的单胺氧化酶在催化如Ⅰ
    Figure PCTCN2023070432-appb-100009
    所示的化合物制备基本上对映体纯的如Ⅲ
    Figure PCTCN2023070432-appb-100010
    所示的氨基磺酸盐化合物、如Ⅳ
    Figure PCTCN2023070432-appb-100011
    所示的氨基腈化合物或其盐/水合物上的应用。
  13. 如权利要求1-5任意一项所述的单胺氧化酶在催化如Ⅰ
    Figure PCTCN2023070432-appb-100012
    所示的化合物去对称化上的应用。
PCT/CN2023/070432 2022-03-31 2023-01-04 一种单胺氧化酶及其应用 WO2023185184A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210332720.2 2022-03-31
CN202210332720 2022-03-31

Publications (1)

Publication Number Publication Date
WO2023185184A1 true WO2023185184A1 (zh) 2023-10-05

Family

ID=88199010

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/070432 WO2023185184A1 (zh) 2022-03-31 2023-01-04 一种单胺氧化酶及其应用

Country Status (2)

Country Link
CN (1) CN116891839A (zh)
WO (1) WO2023185184A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102131813A (zh) * 2008-06-24 2011-07-20 科德克希思公司 用于制备基本上立体异构纯的稠合二环脯氨酸化合物的生物催化方法
CN105441401A (zh) * 2014-09-01 2016-03-30 南京博优康远生物医药科技有限公司 一种单胺氧化酶及其在合成手性氮杂双环化合物中的应用
CN105624128A (zh) * 2014-11-26 2016-06-01 南京博优康远生物医药科技有限公司 一种固定化单胺氧化酶及其在合成手性氮杂双环化合物中的应用
CN109897836A (zh) * 2017-12-11 2019-06-18 成都渊源生物科技有限公司 一种来源于米曲霉的单胺氧化酶用于手性胺中间体的制备
CN109971730A (zh) * 2017-12-28 2019-07-05 成都渊源生物科技有限公司 一种来源于黑曲霉的单胺氧化酶用于手性胺中间体的制备

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102131813A (zh) * 2008-06-24 2011-07-20 科德克希思公司 用于制备基本上立体异构纯的稠合二环脯氨酸化合物的生物催化方法
CN105441401A (zh) * 2014-09-01 2016-03-30 南京博优康远生物医药科技有限公司 一种单胺氧化酶及其在合成手性氮杂双环化合物中的应用
CN105624128A (zh) * 2014-11-26 2016-06-01 南京博优康远生物医药科技有限公司 一种固定化单胺氧化酶及其在合成手性氮杂双环化合物中的应用
CN109897836A (zh) * 2017-12-11 2019-06-18 成都渊源生物科技有限公司 一种来源于米曲霉的单胺氧化酶用于手性胺中间体的制备
CN109971730A (zh) * 2017-12-28 2019-07-05 成都渊源生物科技有限公司 一种来源于黑曲霉的单胺氧化酶用于手性胺中间体的制备

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ATKIN, K. E. ET AL.: "The Structure of Monoamine Oxidase from Aspergillus niger Provides a Molecular Context for Improvements in Activity Obtained by Directed Evolution", JOURNAL OF MOLECULAR BIOLOGY, vol. 384, 14 October 2008 (2008-10-14), pages 1218 - 1231, XP025744825, DOI: 10.1016/j.jmb.2008.09.090 *
CHEN, XUEJUN ET AL.: "Increasing Activity of a Monoamine Oxidase by Random Mutation", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 30, no. 1, 25 January 2014 (2014-01-25), pages 109 - 118, XP009549433 *
DUAN JIAQI, LI BEIBEI, QIN YOUCAI, DONG YIJIE, REN JIE, LI GUANGYUE: "Recent progress in directed evolution of stereoselective monoamine oxidases", BIORESOURCES AND BIOPROCESSING, vol. 6, no. 1, 1 December 2019 (2019-12-01), XP093097846, DOI: 10.1186/s40643-019-0272-6 *
LIU, RUI;YU, LIU; QIAOFENG, LI; XUDONG, FENG; CHUN, LI: "Strategies for Engineering the Thermo-Stability of Glycosidase", CHINESE JOURNAL OF BIOTECHNOLOGY, ZHONGGUO KEXUEYUAN WEISHENGWU YANJIUSUO, CHINESE ACADEMY OF SCIENCES, INSTITUTE OF MICROBIOLOGY, CN, vol. 37, no. 6, 25 June 2021 (2021-06-25), CN , pages 1919 - 1930, XP009549434, ISSN: 1000-3061, DOI: 10.13345/j.cjb.210050 *

Also Published As

Publication number Publication date
CN116891839A (zh) 2023-10-17

Similar Documents

Publication Publication Date Title
US11479756B2 (en) Ketoreductase polypeptides for the reduction of acetophenones
US11078505B2 (en) Transaminase biocatalysts
US20210324347A1 (en) Ketoreductase polypeptides for the production of azetidinone
US11345898B2 (en) Ketoreductase polypeptides for the preparation of phenylephrine
US9296992B2 (en) Ketoreductase-mediated stereoselective route to alpha chloroalcohols
US8617853B2 (en) Ketoreductase polypeptides for the production of (S,E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-hydroxypropyl)benzoate
WO2023185184A1 (zh) 一种单胺氧化酶及其应用
WO2023169071A1 (zh) 单胺氧化酶及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23777563

Country of ref document: EP

Kind code of ref document: A1