CN116731991A

CN116731991A - Monoamine oxidase and application thereof

Info

Publication number: CN116731991A
Application number: CN202310006537.8A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Qingdao Kingagroot Chemical Compound Co Ltd
Current assignee: Qingdao Kingagroot Chemical Compound Co Ltd
Priority date: 2022-03-10
Filing date: 2023-01-04
Publication date: 2023-09-12
Also published as: WO2023169071A1

Abstract

The present disclosure provides a monoamine oxidase and its use in biocatalytic methods.

Description

Monoamine oxidase and application thereof

Technical Field

The invention relates to monoamine oxidase and application thereof in a biocatalytic method.

Background

Patent CN102131813a discloses resolution and deracemization of racemic chiral amines using monoamine oxidase via stereospecific oxidation of one enantiomer to the corresponding imine with oxygen. It is nevertheless still desirable to provide new monoamine oxidase enzymes useful in said biocatalytic processes.

Brief description of the invention

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written detailed description, including those aspects set forth in the accompanying drawings and defined in the appended claims.

The present invention also provides a monoamine oxidase comprising an amino acid sequence having the following mutations compared to the monoamine oxidase amino acid sequence as shown in SEQ ID NO: 1: the 382 th amino acid in the monoamine oxidase amino acid sequence shown in the corresponding SEQ ID NO. 1 is mutated from phenylalanine to leucine.

In a specific embodiment, the amino acid sequence further has one or more mutations selected from the group consisting of:

mutation of amino acid 63 from phenylalanine to any other amino acid, preferably leucine;

mutation of amino acid 65 from threonine to any other amino acid, preferably valine;

mutation of amino acid 100 from serine to any other amino acid, preferably proline;

mutation of amino acid 201 from leucine to any other amino acid, preferably proline;

mutation of amino acid 107 from threonine to any other amino acid, preferably serine;

mutation of amino acid 138 from threonine to any other amino acid, preferably serine;

mutation of amino acid 141 from threonine to any other amino acid, preferably serine;

mutation of amino acid 190 from glutamic acid to any other amino acid, preferably aspartic acid;

the amino acid at position 234 is mutated from serine to any other amino acid, preferably cysteine.

In another specific embodiment, 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 to the monoamine oxidase amino acid sequence shown in SEQ ID NO. 1.

The present invention provides a monoamine oxidase comprising an amino acid sequence having at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9 and 10.

The invention also provides a polynucleotide for encoding the monoamine oxidase and a host cell containing the polynucleotide.

The present invention also provides a process for preparing a stereoisomerically substantially pure compound of formula II ^Ⅱ The compounds or salts/hydrates thereof

The method comprises the steps of reacting a compound as shown in I ^Ⅰ The compounds shown are contacted with oxygen in the presence of the monoamine oxidase and cofactor.

The invention also provides a process for preparing a substantially enantiomerically pure compound of formula III ^Ⅲ Sulfamate compounds shown or

A salt/hydrate process comprising reacting a compound as defined in I ^Ⅰ The compounds shown are contacted with oxygen in the presence of the monoamine oxidase, cofactor and bisulphite.

The invention also provides a process for preparing a substantially enantiomerically pure compound such as IV ^Ⅳ The aminonitrile compound or its salt/water

A process for preparing a compound comprising reacting a compound as defined in I ^Ⅰ The compounds shown are contacted with oxygen in the presence of the monoamine oxidase, cofactor and bisulphite and the resulting sulfamate compound is contacted with cyanide.

In a specific embodiment, the cofactor is non-covalently associated with a monoamine oxidase.

In another specific embodiment, the cofactor is selected from the group consisting of: FAD, FMN, NAD and NADP.

In another embodiment, the method further comprises catalyzing the hydrogen peroxide to be bifidus to a component of molecular oxygen and water, preferably the component is selected from the group consisting of: pd, fe and catalase.

The invention also provides the use of said monoamine oxidase in catalyzing the oxidation of a compound as shown in II to substantially stereoisomerism

The application of the pure compound shown as II or the salt/hydrate thereof in preparation of medicines.

The invention also provides the use of said monoamine oxidase in the preparation of substantially enantiomerically pure compounds as shown in II

The sulfamate compound shown in III, the aminonitrile compound shown in IV or the salt/hydrate thereof.

The invention also provides application of the monoamine oxidase in catalyzing the desymmetrization of a compound shown as II.

Detailed Description

The description set forth below in connection with the appended drawings is intended to describe various illustrative embodiments of the disclosed subject matter. The specific features and functions are described in connection with each of the illustrative embodiments; however, it will be apparent to those skilled in the art that the disclosed embodiments may be practiced without each of these specific features and functions. Furthermore, unless explicitly stated or a feature or function is not compatible with another embodiment, all functions described in connection with one embodiment are intended to be applicable to the other embodiments described herein. For example, unless a feature or function is not compatible with an alternative embodiment, where a given feature or function is explicitly described in connection with one embodiment, but not explicitly recited in connection with an alternative embodiment, it is to be understood that the feature or function may be deployed, utilized, or implemented in connection with an alternative embodiment.

Practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, biological emulsion generation, and sequencing technology, which are within the skill of the art of practitioners. 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. However, of course, other equivalent conventional procedures may be used. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as those written by Green et al (1999), genome Analysis: A Laboratory ManualSeries (volumes I-IV); weiner, gabriel, stephens, et al (2007), genetic Variation: ALaboratory Manual; dieffnbach, dveksler, editions (2003), PCR Primer: A LaboratoryManual; bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual; mount (2004), bioinformatics: sequence and Genome Analysis; sambrook and Russell (2006), condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), molecular Cloning: A Laboratory Manual (all from ColdSpring Harbor Laboratory Press); strer, l. (1995) Biochemistry (4 th edition) w.h. freeman, new York n.y.; gait, "Oligonucleotide Synthesis: A PracticalApproach"1984,IRL Press,London; nelson and Cox (2000), lehninger, principles ofBiochemistry, 3 rd edition, W.H. Freeman Pub., new York, N.Y.; berg et al (2002) Biochemistry, 5 th edition, w.h. freeman pub., new York, n.y.; cell and Tissue Culture: laboratoryProcedures in Biotechnology (Doyle & Griffiths, eds., john Wiley & Sons 1998); mammalian Chromosome Engineering-Methods and Protocols (G.Hadlaczky, incorporated by reference, humana Press 2011); essential Stem Cell Methods, (Lanza and Klimanskaya, editions, academic Press 2011), all of which are incorporated herein by reference in their entirety for all purposes. Nuclease-specific techniques can be found, for example, in Genome Editing and Engineering From TALENs and CRISPRs toMolecular Surgery, appanani and Church,2018; and CRISPR Methods and Protocols, lindgren and Charpentier,2015; both of these documents are incorporated herein by reference in their entirety for all purposes. Basic methods of enzyme engineering can be found in Enzyme Engineering Methods and Protocols, samuelson, et al, 2013; protein Engineering, kaumaya, editions, (2012); and Kaur and Sharma, "directeEvolution: an Approach to Engineer Enzymes", crit. Rev. Biotechnology,26:165-69 (2006).

Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an oligonucleotide" refers to one or more oligonucleotides, and reference to "an automated system" includes reference to equivalent steps and methods for use with systems known to those of skill in the art, and so forth. Additionally, it should be understood that terms such as "left", "right", "top", "bottom", "front", "back", "side", "height", "length", "width", "upper", "lower", "inner", "outer", etc. as may be used herein describe only points of reference and do not necessarily limit embodiments of the disclosure to any particular orientation or configuration. Moreover, terms such as "first," "second," "third," and the like, identify only one of many parts, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the devices, methods and cell populations that might be used in connection with the invention described herein.

Where a range of values is provided, it is understood that each intervening value, to the extent any other stated or intervening value in that stated range, between the upper and lower limit of that range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and well-known procedures have not been described in order to avoid obscuring the present invention.

Monoamine oxidase of the present disclosure capable of oxidizing an amine compound of structural formula (1) to a 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 monoamine oxidase with one or more improved properties including increased enzymatic activity, increased stereospecificity, increased thermostability, increased solvent stability, decreased product inhibition, decreased substrate inhibition, or decreased sensitivity to reaction byproducts. Such amino acid substitutions may also improve the expression level, solubility and/or stability of monoamine oxidase in host cells, for example as a protein recombinantly expressed in heterologous host cells such as, but not limited to, e.coli host cells.

The present disclosure also provides polynucleic acids encoding such monoamine oxidase and methods of using the multifilments in the disclosed biocatalytic methods.

The following terms as used herein are intended to have the following meanings:

"monoamine oxidase" refers to a polypeptide having the enzymatic ability to oxidize a compound of formula I above to the corresponding product of formula II above. The polypeptide typically utilizes an oxidized cofactor such as, but not limited to, flavin Adenine Dinucleotide (FAD), flavin adenine mononucleotide (FMN), nicotinamide Adenine Dinucleotide (NAD), or Nicotinamide Adenine Dinucleotide Phosphate (NADP). In a specific embodiment, the oxidized cofactor is FAD. Monoamine oxidase as used herein includes naturally occurring (wild-type) monoamine oxidase as well as non-naturally occurring engineered polypeptides produced by human manipulation.

"coding sequence" refers to that portion of a nucleic acid (e.g., a gene) that encodes the amino acid sequence of a protein.

"naturally occurring" or "wild type" refers to a naturally occurring form. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that can be isolated from a natural source that is present in an organism and that has not been intentionally modified by human manipulation.

When used in connection with, for example, a cell, nucleic acid, or polypeptide, "recombinant" refers to a material that has been modified in a manner that would not otherwise occur in nature, or that is identical to the natural or intrinsic form of the material, but that is produced or derived from a synthetic material and/or by manipulation using recombinant techniques, or that corresponds to the natural or intrinsic form of the material. Non-limiting examples include, but are not limited to: recombinant cells expressing genes that are not present in the native (non-recombinant) form of the cell or expressing native genes that are otherwise expressed at different levels.

"percent sequence identity" and "percent homology" are used interchangeably herein to refer to a comparison between a polynucleotide and a polypeptide, and are determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentages can be calculated as follows: determining the number of positions at which the same nucleobase or amino acid residue occurs in both sequences to produce a number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentages may be calculated as follows: determining the number of positions at which the same nucleobase or amino acid residue occurs in both sequences or the number of positions at which the nucleobase or amino acid residue aligns with the gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to yield the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. The optimal alignment of the comparison sequences can be performed by the following algorithm: for example, by Smith and Waterman,1981, adv. Appl. Math.2:482, a local homology algorithm; by Needleman and Wunsch,1970, j.mol.biol.48:443 homology comparison algorithm; through Pearson and Lipman,1988,Proc.Natl.Acad.Sci.USA 85:2444 similarity retrieval method; computerized execution of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the GCG Wisconsin software package) or visual inspection (see generally CurrentProtocols in Molecular Biology (latest experimental protocols in molecular biology), f.m. Ausubel et al, editions, current Protocols, greene Publishing Associates, inc. Together with john wiley & Sons, inc. Together, (1995 supplementary material) (Ausubel)). Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in: altschul et al, 1990, J.mol. Biol.215:403-410 and Altschul et al, 1977,Nucleic Acids Res.3389-3402. Software for performing BLAST analysis is publicly available through the national center for biotechnology information website. Such algorithms include first identifying high scoring sequence pairs (HSPs) by identifying short words (words) of length W in the query sequence that meet or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T refers to the neighborhood word score threshold (Altschul et al, supra). These initial neighboring word matching strings (word hits) act as seeds for initiating searches for longer HSPs containing them. The word matching string then extends in both directions along each sequence, so long as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residue pairs; penalty score for mismatch residues always greater than 0) and N (penalty score for mismatch residues always less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The word match string extension in each direction is stopped when: the maximum value reached by the cumulative alignment score is reduced by an amount X; the cumulative score becomes zero or below due to the accumulation of one or more negative scoring residue alignments; or to the end of either sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as default parameters: word length (W) is 11, expected value (E) is 10, m= 5,N = -4, and the two chains are compared. For amino acid sequences, the BLASTP program uses the following default parameters: the word length (W) is 3, the expected value (E) is 10 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff,1989,Proc Natl Acad Sci USA 89:10915). Exemplary sequence alignments and% sequence identity determinations may employ the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, madison Wis.) using the default parameters provided.

"reference sequence" refers to a defined sequence that serves as the basis for sequence comparison. The reference sequence may be a subsequence of a larger sequence, e.g., a segment of the full-length gene or polypeptide sequence. Typically, the 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. Since two polynucleotides or polypeptides may each (1) comprise a sequence that is similar between the two sequences (i.e., a portion of the complete sequence) and (2) may also comprise a sequence that is different between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptides are typically made by comparing the sequences of the two polynucleotides within a "comparison window" to identify and compare sequence similarity of local regions.

"comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues in which a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can contain 20% or less additions or deletions (i.e., gaps) compared to the reference sequence in which the two sequences are optimally aligned (which does not contain additions or deletions). The comparison window may be longer than 20 consecutive residues and optionally comprises a window of 30 consecutive residues, 40 consecutive residues, 50 consecutive residues, 100 consecutive residues or longer.

"substantial identity" refers to a polynucleotide or polypeptide sequence that has at least 80% sequence identity, at least 85% identity, and 89% to 95% sequence identity, more typically at least 99% sequence identity over a comparison window of at least 20 residue positions, typically over a window of at least 30-50 residues, as compared to a reference sequence, wherein the percent sequence identity is calculated by comparing the reference sequence to sequences comprising a total of 20% or less deletions or additions of the reference sequence over the comparison window. In particular embodiments applied to polypeptides, the term "substantial identity" means that the two polypeptide sequences have at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or greater (e.g., 99% sequence identity) when optimally aligned using default GAP weights (GAP weihgt) by, for example, the programs GAP or BESTFIT. Preferably, the different residue positions differ due to conservative amino acid substitutions.

When used hereinafter with respect to a given amino acid or polynucleotide sequence number, "corresponding to," "about," or "relative to" refers to the residue number of the given amino acid or polynucleotide sequence when compared to a specified reference sequence. In other words, the residue number or residue position of a given polymer is specified with respect to a reference sequence rather than by the actual numerical position of the residue in a given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as an amino acid sequence of an engineered monoamine oxidase, can be aligned with a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, the numbering of residues in a given amino acid or polynucleotide sequence is specified with respect to the reference sequence to which it is aligned, despite gaps.

"stereoselectivity" means that one stereoisomer preferentially forms over the other stereoisomer in a chemical or enzymatic reaction. The stereoselectivity may be partial, where formation of one stereoisomer is favored over another, or the stereoselectivity may be complete, where only one stereoisomer is formed. When a stereoisomer is an enantiomer, stereoselectivity refers to enantioselectivity, i.e., the fraction of one enantiomer in the sum of the two enantiomers (usually reported as a percentage). It (typically as a percentage) is generally reported in the art alternatively as an enantiomeric excess (e.e.) calculated therefrom according to the following formula: [ major enantiomer-minor enantiomer ]/[ major enantiomer + minor enantiomer ]. When stereoisomers are diastereomers, stereoselectivity refers to diastereoselectivity, i.e. the fraction of one diastereomer in a mixture of two diastereomers (usually reported as a percentage), usually optionally reported as diastereomeric excess (d.e.). Enantiomeric excess and diastereomeric excess are types of stereoisomer excess.

"high stereoselectivity": refers to monoamine oxidase polypeptides capable of converting a substrate to the corresponding product with a stereoisomer excess of at least about 99%.

"stereospecific" means that one stereoisomer is preferentially converted over the other stereoisomer in a chemical or enzymatic reaction. The stereospecificity may be partial, where conversion of one stereoisomer is advantageous over the other, or the stereospecificity may be complete, where only one stereoisomer is converted.

"chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

By "improved enzymatic properties" is meant monoamine oxidase polypeptides that exhibit any improvement in enzymatic properties as compared to a reference monoamine oxidase. For the engineered monoamine oxidase polypeptides described herein, a comparison is generally made with the wild-type monoamine oxidase, although in some embodiments, the reference monoamine oxidase may be another modified monoamine oxidase. Desirable improved enzyme properties include, but are not limited to: enzyme activity (which may be expressed in terms of percent substrate conversion), thermostability, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g., product inhibition), stereospecificity, stereoselectivity (including enantioselectivity), solubility and stability, and expression levels in host cells.

"increased enzymatic activity" refers to an improved property of an engineered monoamine oxidase polypeptide, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of an initial amount of substrate to product in a specified time period when a specified amount of monoamine oxidase is used) as compared to a reference monoamine oxidase. Exemplary methods of determining enzyme activity are provided in the examples. Any property related to the enzymatic activity can be affected, including classical enzyme property K _m 、V _max Or k _cat Their alteration may lead to enzymatic activityThe sex increases. The improvement in enzymatic activity may be up to about 1.5 times the enzymatic activity of the corresponding wild-type monoamine oxidase up to 2, 5, 10, 20, 25, 50, 75, 100 or more times the enzymatic activity of the naturally occurring monoamine oxidase or another engineered monoamine oxidase from which the monoamine oxidase polypeptide is derived. The skilled artisan will appreciate that the activity of any enzyme is diffusion limited such that the catalytic conversion rate does not exceed the diffusion rate of the substrate (including any desired cofactors). Diffusion limiting or k _cat /K _m Is generally about 10 ⁸ To 10 ⁹ (M ^-1 s ^-1 ). Thus, any improvement in the enzymatic activity of a monoamine oxidase will have an upper limit related to the diffusion rate of the substrate to which the monoamine oxidase acts. Monoamine oxidase activity can be measured using published methods of measuring monoamine oxidase or modifications thereof, such as, but not limited to, those disclosed by Zhou et al (Zhou et al, "A One-StepFluorometric Method for the Continuous Measurement of Monoamine OxidaseActivity," One-step fluorescence method of continuous measurement of monoamine oxidase activity ")," 1997Anal. Biochem. 253:169-74), and Szutowicz et al (Szutowicz et al, "Colorimetric Assay forMonoamine Oxidase in Tissues Using Peroxidase and2,2'-Azino (3-ethylbenzotriazoline-6-sulfonic Acid) as Chromogen (colorimetric determination of monoamine oxidase in tissue using peroxidase and2,2' -Azino (3-ethylbenzothiazoline-6-sulfonic Acid) as chromogens)," 1984, anal. Biochem. 138:86-94). Comparison of enzyme activities is performed using defined enzyme preparations, defined assays under defined conditions, and one or more defined substrates, as described in further detail herein or using methods such as Zhou and Szutowicz. Generally, when comparing lysates, the number of cells assayed and the amount of protein assayed are determined and the same expression system and the same host cell are used to minimize the difference in the amount of enzyme produced by the host cell and the enzyme present in the lysate.

"transformation": refers to the enzymatic oxidation of a substrate to the corresponding product. "percent conversion" refers to the percentage of substrate oxidized to product over a period of time under the indicated conditions. Thus, the "enzymatic activity" or "activity" of a monoamine oxidase polypeptide may be expressed as a "percent conversion" of a substrate to a product.

By "thermostable" is meant that the monoamine oxidase polypeptide retains similar activity (e.g., greater than 60% to 80%) as compared to the untreated enzyme after exposure to an elevated temperature (e.g., 40-80 ℃) for a period of time (e.g., 0.5-24 hours).

By "solvent stable" is meant that the monoamine oxidase polypeptide retains similar activity (greater than, for example, 60% to 80%) as compared to untreated enzyme after exposure to different concentrations (for example, 5% -99%) of solvent (isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl t-butyl ether, etc.) for a period of time (for example, 0.5-24 hours).

By "pH stable" is meant that the monoamine oxidase polypeptide retains similar activity (greater than, for example, 60% to 80%) as compared to the untreated enzyme after exposure to a high or low pH (for example, 4.5-6 or 8 to 12) for a period of time (for example, 0.5-24 hours).

"thermostable and solvent stable" refers to thermostable and solvent stable monoamine oxidase polypeptides.

"hydrophilic amino acid or residue" means having a sequence which exhibits a sequence according to Eisenberg et al, 1984, J.mol.biol.179:125-142 are amino acids or residues of the hydrophobic side chains with a normalized consistent hydrophobicity scale of less than zero. 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 typically have negatively charged side chains at physiological pH due to loss of hydrogen ions. 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 of greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ions. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

"polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain as follows: is uncharged at physiological pH but has at least one bond in which an electron pair that is common to both atoms is more tightly held by one of the two atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).

"hydrophobic amino acid or residue" means having a sequence which exhibits a sequence according to Eisenberg et al, 1984, J.mol.biol.179:125-142 are amino acids or residues of the hydrophobic side chains with a normalized consistent hydrophobicity scale greater than zero. 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 comprising at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although 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 its side chain containing a heteroaromatic ring, histidine is herein classified as a hydrophilic residue or "limiting residue (constrained residue)" (see below).

"constrained amino acid or residue" refers to an amino acid or residue having constrained geometry. Here, the restriction residues include L-pro (P) and L-his (H). Histidine has limited geometry due to its relatively small imidazole ring. Proline has limited geometry because it also has a five-membered ring.

"nonpolar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain as follows: an electron pair that is uncharged at physiological pH and has two atoms in common has a bond that is generally held to the same extent by each of the two atoms (i.e., the side chains are not polar). Genetically encoded nonpolar 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 sulfanyl or sulfhydryl containing amino acids. "cysteine-like residues" include cysteine and other amino acids that contain a sulfhydryl moiety that may be used to form disulfide bonds. The ability of L-Cys (C) (and other amino acids having-SH-containing side chains) to exist in the peptide as reduced free-SH or oxidized disulfide bonds affects whether L-Cys (C) imparts net hydrophobic or hydrophilic characteristics to the peptide. While L-Cys (C) exhibits a hydrophobicity according to the standardized uniformity scale of 0.29 by Eisenberg (Eisenberg et al, 1984, supra), it is to be appreciated that L-Cys (C) is categorized as its own unique group for purposes of this disclosure.

"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 hydrogen). Small amino acids or residues may be further classified as aliphatic, nonpolar, polar or acidic according to the definition above. 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).

"hydroxyl-containing amino acid or residue" refers to an amino acid that contains a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T) and L-Tyr (Y).

"conservative" amino acid substitutions or mutations refer to the interchangeability of residues having similar side chains, and thus generally include the substitution of amino acids in polypeptides with amino acids in the same or similar amino acid definition categories. However, as used herein, a conservative mutation does not include a substitution of a hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue if the conservative mutation may instead be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or residue-limiting to residue-limiting substitution. Furthermore, as used herein A, V, L or I may conservatively mutate to another aliphatic residue or to another non-polar residue. Exemplary conservative substitutions are shown in table 1 below. .

Table 1: conservative substitutions

"non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with amino acids having significantly different side chain characteristics. Non-conservative substitutions may be made 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 substitution region (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain volume.

"deletion" refers to 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 comprising the reference enzyme, or up to 20% of the total number of amino acids comprising the reference enzyme, while retaining enzyme activity and/or retaining improved properties of the engineered monoamine oxidase. Deletions may be directed against the interior and/or ends of the polypeptide. In various embodiments, the deletions may comprise continuous segments or may be discontinuous.

"insertion" refers to modification of a polypeptide by adding one or more amino acids from a reference polypeptide. In some embodiments, the improved engineered monoamine oxidase comprises inserting one or more amino acids into a naturally occurring monoamine oxidase and inserting one or more amino acids into other improved monoamine oxidase polypeptides. The insertion may be internal to the polypeptide, or at the carboxy-or amino-terminus. Insertions as used herein include fusion proteins as known in the art. The insertions may be contiguous amino acid segments or separated by one or more amino acids in the naturally occurring polypeptide.

"different from" or "different from" with respect to a specified reference sequence refers to the difference of a given amino acid or polynucleotide sequence when aligned with the reference sequence. In general, the difference can be determined when two sequences are optimally aligned. Differences include insertions, deletions or substitutions of amino acid residues compared to a reference sequence.

"fragment" as used herein refers to a polypeptide having amino-and/or carboxy-terminal deletions, but wherein the remaining amino acid sequence is identical to the corresponding position of the sequence. Fragments may 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.

An "isolated polypeptide" refers to a polypeptide that is substantially isolated from other contaminants with which it is naturally associated, such as proteins, lipids, and polynucleotides. The term encompasses polypeptides that are removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). The modified monoamine oxidase may be present in the cells, in the cell culture medium, or prepared in various forms, such as lysates or isolated preparations. Thus, in some embodiments, the modified monoamine oxidase may be an isolated polypeptide.

"substantially pure polypeptide" refers to a composition in which the polypeptide material is the predominant material present (i.e., it is more abundant by mole or weight than any other macromolecular material alone in the composition), and which is substantially purified when the subject material comprises at least about 50% by mole or% by weight of the macromolecular material present. Generally, the substantially pure monoamine oxidase composition comprises 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 by mole or% by weight of all macromolecular species present in the composition. In some embodiments, the subject material is purified to be substantially homogeneous (i.e., no contaminating material can be detected in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular material. Solvent species, small molecules (< 500 daltons) and elemental iron species are not considered macromolecular species. In some embodiments, the isolated modified monoamine oxidase polypeptide is a substantially pure polypeptide composition.

"stringent hybridization" as used herein refers to conditions in which nucleic acid hybrids (hybrid) are stable. As known to those skilled in the art, the stability of a hybrid is determined by the melting temperature (T _m ) Reflected by the reflection. Generally, the stability of a hybrid depends on ionic strength, temperature, G/C content, and the presence of chaotropic agents. T of Polynucleotide _m The values may 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.Natl.Acad.Sci USA 83:8893-8897; freier et al, 1986,Proc.Natl.Acad.SciUSA 83:9373-9377; kierzek et al, biochemistry25:7840-7846; rychlik et al, 1990,Nucleic Acids Res 18:6409-6412 (Protect, 1991,Nucleic Acids Res19:698); sambrook et al, supra); suggs et al, 1981, academic Press at Developmental BiologyUsing Purified Genes (using the developmental biology of purified genes) (Brown et al, eds.) pages 683-693; and wetdur, 1991,Crit Rev Biochem MolBiol 26:227-259. All publications are incorporated herein by reference). In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to a complement of a sequence encoding an engineered monoamine oxidase of the disclosure.

"hybridization stringency" refers to the washing conditions of nucleic acids. Generally, the hybridization reaction is carried out under conditions of lower stringency, followed by a different but higher stringency wash. The term "moderately stringent hybridization" refers to allowing target DNA binding to have about 60% identity, preferably about 75% identity, about 85% identity, to the target DNA; conditions for a complementary nucleic acid having greater than about 90% identity to a target polynucleotide. Exemplary moderately stringent conditions are equalThe following conditions apply: hybridization was performed in 50% formamide, 5 XDenhart's solution, 5 XSSPE, 0.2% SDS at 42℃followed by washing in 0.2 XSSPE, 0.2% SDS at 42 ℃. "highly stringent hybridization" generally refers to a temperature T of fusion that is greater than the temperature T of fusion determined under solution conditions that define the polynucleotide sequence _m Conditions of about 10 ℃ or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids in 0.018MNaCl at 65 ℃. (i.e., if the hybrid is unstable in 0.018M NaCl at 65℃as contemplated herein, it will be unstable under high stringency conditions). The high stringency conditions are provided by: for example, hybridization in 50% formamide, 5 XDenhart's solution, 5 XSSPE, 0.2% SDS at 42℃followed by washing in 0.1 XSSPE and 0.1% SDS at 65 ℃. Other highly Yan Jinge hybridization conditions and moderately stringent conditions are described in the references cited above.

"heterologous" polynucleotide refers to a polynucleotide that is introduced into a host cell by laboratory techniques, and includes a polynucleotide that is removed from the host cell, subjected to laboratory procedures, and then reintroduced into the host cell.

"codon optimized" refers to altering the codons of a polynucleotide encoding a protein to those codons that are preferentially used in a particular organism in order to allow efficient expression of the encoded protein in the organism of interest. Although the genetic code is degenerate because most amino acids are represented by several codons (referred to as "synonyms" or "synonymous" codons), it is well known that codon usage for a particular organism is non-random and favors a particular codon triplet. Such codon usage bias may be higher for a given gene, a gene of common function or ancestral origin (accessra origin), a high expressed protein relative to a low copy number protein, and an collectin coding region of the organism's genome. In some embodiments, the polynucleotide codons encoding monoamine oxidase may be optimized for optimal production from the host organism selected for expression.

"preferred, optimal, high codon usage biased codons" interchangeably refer to codons as follows: which is used at a higher frequency in the protein coding region than the codons encoding the same amino acid. Preferred codons may be determined in terms of: codon usage in a single gene, codon usage of a group of genes of common function or origin, codon usage of highly expressed genes, codon frequency of aggregated protein coding regions in the whole organism, codon frequency of aggregated protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. Various methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism, including multivariate analysis, e.g., using cluster analysis or corresponding analysis and effective codon numbers for genes (see GCG CodonPreference, genetics Computer Group WisconsinPackage; codonW, john Peden, university of Nottingham; mclnerney, j.o,1998,Bioinformatics 14:372-73; stenico et al, 1994,Nucleic Acids Res.222437-46; wright, f.,1990, gene 87:23-29). More and more tables of codon usage for organisms are available (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; henout and Danchin, "Escherichia coli and Salmonella (E.coli and Salmonella)," 1996, neidhardt et al, editions, ASM Press, washington D.C., pages 2047-2066. The data sources from which codon usage can depend on any available nucleotide sequence capable of encoding a protein. These data sets include nucleic acid sequences encoding expressed proteins known in practice (e.g., complete protein coding sequences-CDS), expressed Sequence Tags (ESTS), or predictive coding regions of genomic sequences (see, e.g., mount, D., bioinformatics: sequence andGenome Analysis (Bioinformatics: sequence and genome analysis), chapter 8, cold SpringHarbor Laboratory Press, cold Spring Harbor, N.Y.,2001;Uberbacher,E.C, 1996,Methods Enzymol.266:259-281, tiwari et al, 1997, comput. Biol. 13:263 sc-270).

"control sequences" are defined herein to include all components necessary or advantageous for expression of a polypeptide 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, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence and transcription terminator. At a minimum, the control sequences comprise promoters, transcriptional and translational stop signals. The control sequences may have a linking sequence for the purpose of introducing specific restriction sites that facilitate linking the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

"operably linked" is defined herein as a configuration in which a control sequence is placed at a position relative to the coding sequence of a 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. The control sequence may comprise an appropriate promoter sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

The terms "stereoisomers," "stereoisomeric forms," and similar terms as used herein are generic terms for all isomers of a single molecule that differ only in the direction of their atoms in space. It includes enantiomers and isomers of compounds having more than one chiral center that are not mirror images of each other ("diastereomers").

The term "chiral center" refers to a carbon atom to which four different groups are attached.

The term "enantiomer" or "enantiomeric" refers to a molecule that is non-superimposable on its mirror image and is therefore optically active, wherein an enantiomer rotates a plane of polarized light in one direction and its mirror image rotates a plane of polarized light in the opposite direction.

The term "racemic" refers to an aliquot of a mixture of optically inactive enantiomers.

The term "resolution" refers to the separation or concentration or exclusion of one of the two enantiomeric forms of a molecule.

As used herein, "substantially enantiomerically pure" means that a given enantiomer of a compound is present to a greater degree or degree than another enantiomer of the same compound. Thus, in particular embodiments, a substantially enantiomerically pure compound is present in 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% enantiomeric excess of the other enantiomer of the same compound.

"substantially stereoisomerically pure" as used herein means that a given enantiomer or diastereomer of a compound is present to a greater extent or degree than another enantiomer or diastereomer of the same compound. As mentioned above with respect to "stereoselectivity", enantiomeric excess and diastereomeric excess are types of stereoisomer excess. Thus, in particular embodiments, a substantially stereoisomerically pure compound is present in 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% stereoisomer over the other enantiomer or diastereomer of the same compound.

As will be appreciated by those skilled in the art, the polypeptides described herein are not limited to genetically encoded amino acids. In addition 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. Some commonly encountered non-coding amino acids that monoamine oxidases described herein may comprise include, but are not limited to: a D-stereoisomer of a genetically encoded amino acid; 2, 3-diaminopropionic acid (Dpr); alpha-aminoisobutyric acid (Aib); epsilon-aminocaproic acid (Aha); delta-aminopentanoic acid (Ava); n-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); n-methyl isoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf); 3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf); 2-methyl phenylalanine (Omf); 3-methyl phenylalanine (Mmf); 4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf); 4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2, 4-dichlorophenylalanine (Opef); 3, 4-dichlorophenylalanine (Mpcf); 2, 4-difluorophenylalanine (Opff); 3, 4-difluorophenylalanine (Mpff); pyridin-2-ylalanine (2 pAla); pyridin-3-ylalanine (3 pAla); pyridin-4-ylalanine (4 pAla); naphthalen-1-ylalanine (1 nAla); naphthalen-2-ylalanine (2 nAla); thiazolylalanine (taAla); benzothiophenylalanine (btala); thienyl alanine (ttala); furyl alanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); high tryptophan (hTrp); pentafluorophenylalanine (5 ff); phenylalanine (sla); anthracenyl alanine (aAla); 3, 3-diphenylalanine (Dfa); 3-amino-5-phenylpentanoic acid (phenypentanoic acid, afp); penicillamine (Pen); 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid (Tic); beta-2-thienyl alanine (Thi); methionine sulfoxide (Mso); n (w) -nitroarginine (nArg); high lysine (hLys); methyl phenylalanine phosphate (pmPhe); phosphoserine (pSer); threonine phosphate (pThr); high aspartic acid (hAsp); homoglutamic acid (hGlu); 1-aminocyclopent- (2 or 3) -ene-4 carboxylic acid; pipecolic Acid (PA), azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid; allyl glycine (aOly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hlle); homoarginine (hArg); n-acetyl lysine (AcLys); 2, 4-diaminobutyric acid (Dbu); 2, 3-diaminobutyric acid (Dab); n-methylvaline (MeVal); homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Other non-coding amino acids that the monoamine oxidase described herein may comprise will be apparent to those skilled in the art (see, e.g., fasman,1989,CRC Practical Handbook of Biochemistry and Molecular Biology (handbook of CRC Biochemical and molecular biology practice), CRC Press, boca Raton, FL, pages 3-70 and the references cited therein, all of which are incorporated by reference). These amino acids may be in the L-or D-configuration.

Those skilled in the art will recognize that the monoamine oxidase disclosed herein may also comprise amino acids or residues having side chain protecting groups. Such protected amino acids fall within the aromatic category in this case, non-limiting examples of which include (protecting groups are listed in brackets) but are not limited to: arg (tos), cys (methylbenzyl), cys (nitropyridylsulfanyl), glu (delta-benzyl ester), gln (xanthenyl), asn (N-delta-xanthenyl), his (bom), his (benzyl), his (tos), lys (fmoc), lys (tos), ser (O-benzyl), thr (O-benzyl) and Tyr (O-benzyl).

Conformationally constrained non-coding amino acids that monoamine oxidase described herein may comprise may include, but are not limited to, N-methyl amino acids (L-configuration); 1-aminocyclopent- (2 or 3) -ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; high proline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

As described above, various modifications introduced into naturally occurring polypeptides to produce engineered monoamine oxidases can be targeted to specific enzyme properties.

In another aspect, the present disclosure provides polynucleotides encoding the engineered monoamine oxidase disclosed herein. The polynucleotide may 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 comprising heterologous polynucleotides encoding engineered monoamine oxidase may be introduced into suitable host cells to express the corresponding monoamine oxidase polypeptides.

The availability of protein sequences provides an indication of all polynucleotides capable of encoding the subject, as the codons corresponding to the various amino acids are known. The degeneracy of the genetic code, in which identical amino acids are encoded by alternative codons or synonymous codons, allows for the preparation of very large numbers of nucleic acids, all of which encode the improved monoamine oxidase disclosed herein. Thus, where a particular amino acid sequence is identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying one or more codon sequences in a manner that does not alter the amino acid sequence of the protein.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a monoamine oxidase having the amino acid sequence: has at least about 80% or more sequence identity, about 85% or more sequence identity, about 90% or more sequence identity, about 95% or more sequence identity, about 96% or more sequence identity, about 97% or more sequence identity, about 98% or more sequence identity, or 99% or more sequence identity as compared to any of the reference engineered monoamine oxidases described herein.

In various embodiments, it is preferred to select codons appropriate for the host cell in which the protein is expressed. For example, a preferred codon for use in bacteria is used to express a gene in bacteria, and a preferred codon for use in yeast is used to express in yeast; and the preferred codons used in the mammal are used for expression in mammalian cells.

In certain embodiments, it is not necessary to replace all codons to optimize codon usage of monoamine oxidase, as the native sequence will contain preferred codons and as the use of preferred codons may not be required for all amino acid residues. Thus, a codon-optimized polynucleotide encoding a monoamine oxidase may contain preferred codons at about 40%, 50%, 60%, 70%, 80% or more than 90% of the codon positions of the full-length coding region.

In other embodiments, the polynucleotide comprises a polynucleotide as follows: encodes a monoamine oxidase as described herein, but has about 80% or more sequence identity, about 85% or more sequence identity, about 90% or more sequence identity, about 95% or more sequence identity, about 98% or more sequence identity, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered monoamine oxidase.

Isolated polynucleotides encoding improved monoamine oxidase can be manipulated in a variety of ways to provide for expression of polypeptides. Depending on the expression vector, manipulation of the isolated polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides 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 (molecular cloning: laboratory Manual), 3 rd edition, cold Spring HarborLaboratory Press; and Current Protocols in Molecular Biology (latest experimental protocol in molecular biology), ausubel.f. edit, greene pub.associates,1998, update to 2006.

For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include those obtained from the following genes: coli lac operon, streptomyces coelicolor (Streptomyces coelicolor) agarase gene (dagA), bacillus subtilis levansucrase gene (sacB), bacillus licheniformis (Bacillus licheniformis) alpha-amylase gene (amyL), bacillus stearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene (amyM), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene (amyQ), bacillus licheniformis penicillinase gene (penP), bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al, 1978,Proc.Natl Acad.Sci.USA 75:3727-3731); and the tac promoter (DeBoer et al, 1983,Proc.Natl Acad.Sci.USA 80:21-25). Other promoters are described in Scientific American,1980, 242: "Useful proteins from recombinant bacteria (useful protein from recombinant bacteria)" in 74-94; and Sambrook et al, supra.

For filamentous fungal host cells, suitable promoters for directing the transcription of the nucleic acid constructs of the present disclosure include those obtained from the genes for: aspergillus oryzae TAKA amylase, rhizomucor miehei (Rhizomucor miehei) aspartic proteinase, aspergillus niger neutral alpha-amylase, aspergillus niger acid stable alpha-amylase, aspergillus niger or Aspergillus awamori (Aspergillus awamori) glucoamylase (glaA), rhizomucor miehei lipase, aspergillus oryzae alkaline proteinase, aspergillus oryzae triose phosphate isomerase, aspergillus nidulans acetamidase, and Fusarium oxysporum (Fusarium oxysporum) trypsin-like proteinase (WO 96/00787); and NA2-tpi promoters (hybrids from the promoters of the Aspergillus niger neutral alpha-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutant, truncated, and hybrid promoters thereof.

In yeast hosts, useful promoters may be derived from genes for the following enzymes: saccharomyces cerevisiae (Saccharomyces cerevisiae) enolase (ENO-1), saccharomyces cerevisiae galactokinase (GAL 1), saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH 2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al, 1992, yeast 8: 423-488.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' -terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the methods disclosed herein.

For example, exemplary transcription terminators for filamentous fungal host cells may be obtained from the following genes: aspergillus oryzae TAKA amylase, aspergillus niger glucoamylase, aspergillus nidulans anthranilate synthase, aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from the following genes: saccharomyces cerevisiae enolase, saccharomyces cerevisiae cytochrome C (CYC 1), 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 an untranslated region of an mRNA that is important for host cell translation. The leader sequence is operably linked to the 5' -terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leader sequences for filamentous fungal host cells are obtained from the following genes: aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for 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 (ADH 2/GAP).

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' -end of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the methods disclosed herein. Exemplary polyadenylation sequences for filamentous fungal host cells may be obtained from the genes: aspergillus oryzae TAKA amylase, aspergillus niger glucoamylase, aspergillus nidulans anthranilate synthase, fusarium oxysporum trypsin-like protein, and Aspergillus niger alpha-glucosidase. Polyadenylation sequences useful in yeast host cells are described by Guo and Sherman,1995,Mol Cell Bio15: 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 cell's secretory pathway. The 5' end of the nucleic acid sequence coding sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Foreign signal peptide coding regions may be required where the coding sequence does not naturally contain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the methods disclosed herein.

The effective signal peptide coding region of a bacterial host cell is a signal peptide coding region obtained from the following genes: bacillus NClB 11837 maltogenic amylase, bacillus stearothermophilus alpha-amylase, bacillus licheniformis subtilisin, bacillus licheniformis beta-lactamase, bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Other signal peptides are represented by Simonen and Palva,1993,Microbiol Rev 57: 109-137.

The effective signal peptide coding region for a filamentous fungal host cell may be a signal peptide coding region obtained from: aspergillus oryzae TAKA amylase, aspergillus niger neutral amylase, aspergillus niger glucoamylase, rhizomucor miehei aspartic proteinase, humicola insolens (Humicola insolens) cellulase, and Humicola lanuginosa (Humicola lanuginosa) lipase.

Signal peptides useful in yeast host cells may be derived from 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 that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme (proenzyme) or a pre-polypeptide (or in some cases a zymogen). The propeptide is generally inactive and may be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propeptide. The propeptide coding region may be obtained from the following genes: bacillus subtilis alkaline protease (aprE), bacillus subtilis neutral protease (nprT), saccharomyces cerevisiae alpha-factor, rhizomucor miehei aspartic proteinase, and myceliophthora thermophila (Myceliophothermophila) lactase (WO 95/33836).

Where both the signal peptide and the propeptide region are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow for the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause the turning on or off of gene expression in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include the lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include: for example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA alpha amylase promoter, aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene amplified in the presence of methotrexate and the metallothionein genes amplified with heavy metals. In these cases, the nucleic acid sequences encoding monoamine oxidase of the present disclosure will be operably linked to regulatory sequences.

Thus, in another embodiment, the present disclosure also relates to recombinant expression vectors comprising a polynucleotide encoding an engineered monoamine oxidase or variant thereof and one or more expression regulatory regions, such as promoters and terminators, origins of replication, etc., depending on the type of host into which they are to be introduced. The various nucleic acids and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the polypeptide at these sites. Alternatively, the 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. In preparing the 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 may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can allow expression of the polynucleotide sequence. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear plasmid or a closed circular plasmid.

The expression vector may be an autonomously replicating vector, i.e., a vector which replicates independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome, as an extrachromosomal entity. The vector may contain any means (means) for ensuring self-replication. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. In addition, a single vector or plasmid, or two or more vectors or plasmids, or transposons, which together contain the total DNA to be introduced into the host cell genome, may be used.

The expression vectors of the present disclosure preferably contain one or more selectable markers that allow for easy selection of transformed cells. Selectable markers are genes whose products provide biocidal or viral resistance, heavy metal resistance, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers are the dal genes from bacillus subtilis or bacillus licheniformis or markers conferring the following antibiotic resistance: such as ampicillin resistance, kanamycin resistance, chloramphenicol resistance or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.

Selectable markers for use in a filamentous fungal host cell include, but are not limited to: amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5' -phosphate decarboxylase), sC (sulfate adenyltransferase) and trpC (anthranilate 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 (Streptomyces hygroscopicus).

The expression vectors of the present disclosure preferably contain elements that allow the vector to integrate into the host cell genome or to autonomously replicate the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector to integrate the vector into the genome by homologous or non-homologous recombination.

Alternatively, the expression vector may contain other nucleic acid sequences for directing integration into the host cell genome by homologous recombination. The additional nucleic acid sequences are capable of integrating the vector into the host cell genome at precise locations in the chromosome. To increase the likelihood of integration at a precise location, the integration element should preferably contain a sufficient number of nucleic acids that are highly homologous to the corresponding target sequence, for example 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, to enhance the likelihood of homologous recombination. The integration element may be any sequence homologous to a target sequence in the host cell genome. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. Alternatively, the vector may be integrated into the host cell genome by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to autonomously replicate in the host cell in question. Examples of bacterial origins of replication are the P15A ori or the replication origins of plasmids pBR322, pUC19, pACYC177 (which have a P15A ori), or pACYC184 which allow replication in E.coli and pUB110, pE194, pTA1060 or pAM beta 1 which allow replication in Bacillus. Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN 6. The origin of replication may be one having 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 sequences of the present disclosure may be inserted into a host cell to increase production of a gene product. The increase in copy number of a nucleic acid sequence can be obtained as follows: 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 amplified copies of the selectable marker gene, and thus additional copies of the nucleic acid sequence may be selected by incubating the cell in the presence of a suitable selection agent.

Many expression vectors for use in the methods disclosed herein are commercially available. Suitable commercial expression vectors include: p3xFLAGTM from Sigma-Aldrich Chemicals, st. Louis MO. ^TM An expression vector comprising a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, a pBR322 replication origin and an ampicillin resistance marker for amplification in e. Other suitable expression vectors are pBluescriptII SK (-) and pBK-CMV, plasmids derived from pBR322 (GibcoBRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Latheta et al, 1987, gene 57:193-201) commercially available from Stratagene, laJolla CA.

In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an improved monoamine oxidase of the present disclosure operably linked to one or more control sequences for expressing the monoamine oxidase in the host cell. Host cells for expressing 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.coli, lactobacillus kefir (Lactobacillus kefir), lactobacillus brevis (Lactobacillus brevis), lactobacillus minor (Lactobacillus minor), streptomyces and Salmonella typhimurium (Salmonella typhimurium) cells; fungal cells, such as yeast cells (e.g., saccharomyces cerevisiae or Pichia pastoris (ATCC accession No. 201178)); insect cells, if fly (Drosophila) S2 and Spodoptera litura (Spodoptera) Sf9 cells; animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells; and a plant cell. Suitable media and growth conditions for such host cells are well known in the art.

Polynucleotides expressing monoamine oxidase may 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 those skilled in the art.

An exemplary host cell is E.coli (Escherichia coli) W3110. Expression vectors are made by operably linking a polynucleotide encoding an improved monoamine oxidase to plasmid pCK110900, which plasmid pCK110900 is operably linked to a lac promoter under the control of a lacI repressor. The expression vector also contains a P15a origin of replication and a chloramphenicol resistance gene. Cells containing the subject polynucleotide in E.coli W3110 were isolated by subjecting the cells to chloramphenicol selection.

Engineered monoamine oxidase can be obtained by subjecting polynucleotides encoding naturally occurring monoamine oxidase to mutagenesis and/or directed evolution methods. Exemplary directed evolution techniques are mutagenesis and/or DNA shuffling as described in the following documents: 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 U.S. Pat. No. 6,537,746. Other directed evolution programs that may be used include, but are not limited to: the staggered extension method (StEP), in vitro recombination (Zhao et al, 1998, nat. Biotechnol. 16:258-261), mutagenesis PCR (Caldwell et al, 1994,PCR Methods Appl.3:S136-S140) and cassette mutagenesis (Black et al, 1996,Proc Natl Acad Sci USA 93:3525-3529).

The clones obtained after mutagenesis treatment were screened for engineered monoamine oxidase with the desired improved enzymatic properties. Measurement of enzyme activity from the expression library may 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, the colorimetric determination of monoamine oxidase in tissue using peroxidase and2,2'-Azino (3-ethylbenzothiazoline-6-sulfonic Acid) as Chromogen, methods disclosed in "1997Anal. Biochem.253:169-74) and Szutowicz et al (Szutowicz et al," Colorimetric Assay for MonoamineOxidase in Tissues Using Peroxidase and2,2' -Azino (3-ethtylbenzoline-6-sulfonic Acid) as Chromogen, "1984, anal. Biochem.138:86-94". Comparison of enzyme activities is performed using defined enzyme preparations, defined assays under defined conditions, and one or more defined substrates, as described in further detail herein; or using methods such as Zhou and Szutowicz. In general, when comparing lysates, the number of cells and the amount of protein determined are determined and the same expression system and the same host cell are used to minimize the difference in the amount of enzyme produced by the host cell and the enzyme present in the lysate. Where the improved enzyme property is desired to be heat stability, the enzyme activity may be measured by subjecting the enzyme preparation to a defined temperature and measuring the amount of enzyme activity remaining after heat treatment. Clones containing the polynucleotide encoding the monoamine oxidase are then isolated, sequenced to identify nucleotide sequence changes (if present), and used to express the enzyme in a host cell.

When the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme may 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 (e.g., by enzymatic or chemical ligation methods or polymerase mediated methods) to form any desired contiguous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the following methods: for example Beaucage et al, 1981, tet Lett 22: 1859-69; or Matthes et al, 1984, EMBO J.3:801-05, for example, as commonly practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized in, for example, an automated DNA synthesizer, 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 GreatAmerican Gene Company, ramona, CA, expressGen inc.

The engineered monoamine oxidase to be expressed in the host cells 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 lysing and efficient extraction of proteins from bacteria such as E.coli are available under the trade name CelLytic B ^TM Commercially available from Sigma-Aldrich of st.

Chromatographic techniques for isolating 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, and the like, and will be apparent to those skilled in the art.

In some embodiments, affinity techniques may be used to isolate the modified monoamine oxidase. For affinity chromatography purification, any antibody that specifically binds to monoamine oxidase may be used. For antibody production, a variety of host animals may be immunized by injection with a compound, including but not limited to: rabbits, mice, rats, and the like. The compounds may be attached to a suitable carrier such as BSA by means of side chain functionalities or linkers attached to the side chain functionalities. A variety of adjuvants may be used to increase the immune response depending on the host species, including but not limited to: freund's (intact and incomplete), mineral gums such as aluminium hydroxide, surface active substances such as lysolecithin, complex polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and possibly useful human adjuvants such as BCG (bacillus calmette-guerin) and Corynebacterium parvum (Corynebacterium parvum).

Monoamine oxidase-catalyzed oxidation reactions, as known to those skilled in the artCofactors are often required. The monoamine oxidase-catalyzed oxidation reactions described herein also typically require the cofactor flavin adenine nucleotide (FAD). As used herein, the term "cofactor" refers to a non-protein compound that functions in combination with a monoamine oxidase. In general, cofactors in oxidized form, which may be non-covalently or covalently linked to monoamine oxidase, are added to the reaction mixture. Oxidized FAD forms can be converted from reduced forms of FAD-H by molecular oxygen ₂ And (5) regenerating. In another embodiment, the oxidized FAD form may be regenerated by NAD (P) to provide FAD and NAD (P) H. NAD (P) can then be regenerated by reducing the ketone to an alcohol using an NAD (P) H-dependent alcohol dehydrogenase/ketone reductase.

The monoamine oxidase-catalyzed oxidation reactions described herein are typically carried out in a solvent. Suitable solvents include water, organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol (octenol), heptane, octane, methyl Tertiary Butyl Ether (MTBE), toluene, and similar organic solvents), and ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and similar ionic liquids). In some embodiments, aqueous solvents are used, including water and aqueous co-solvent systems.

An exemplary aqueous co-solvent system has water and one or more organic solvents. Generally, the organic solvent component of the aqueous co-solvent system is selected such that the organic solvent component does not completely inactivate the monoamine oxidase. Suitable co-solvent 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 co-solvent system is miscible with the aqueous component to provide a single liquid phase; or may be partially miscible or immiscible with the aqueous component to provide two liquid phases. Generally, when an aqueous co-solvent system is employed, it is selected to be biphasic, with water dispersed in an organic solvent or vice versa. In general, where an aqueous co-solvent system is employed, it is desirable to select an organic solvent that can be readily separated from the aqueous phase. Generally, the ratio of water to organic solvent in the co-solvent system is typically in the range of about 90:10 to about 10:90 (v/v) organic solvent to water and between 80:20 and 20:80 (v/v) organic solvent to water. The co-solvent system may be preformed prior to addition to the reaction mixture, or it may be formed in situ in the reaction vessel.

The aqueous solvent (water or aqueous co-solvent system) may be pH buffered or unbuffered. Generally, oxidation can be carried out at a pH of about 10 or less, typically in the range of about 5 to about 10. In some embodiments, the oxidation is performed at a pH of about 9 or less, typically in the range of about 5 to about 9. In some embodiments, oxidation is at or below about 8, 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 may also be performed at a pH of about 7.8 or less, or 7.5 or less. Alternatively, the oxidation may be carried out at a neutral pH, i.e., about 7.

During the oxidation reaction, the pH of the reaction mixture may change. Typical amines of formula I are protonated at neutral pH and about neutral pH, whereas imine products of formula II are generally not protonated at neutral pH and about neutral pH. Thus, in typical embodiments in which 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 may be maintained at or within a desired pH range by adding a base during the reaction. Alternatively, an aqueous solvent comprising a buffer may be used to control the pH. Suitable buffers to maintain the desired pH range are known in the art and include: such as phosphate buffer, triethanolamine buffer, and the like. Combinations of buffering or base addition may also be used.

Suitable bases for neutralizing the acid are: organic bases such as amines, alkoxides, and the like; and inorganic bases, such as hydroxide salts (e.g., naOH), carbonates (e.g., naHCO) ₃ ) Bicarbonate (e.g. K) ₂ CO ₃ ) Alkaline phosphates (e.g., 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 imine during the reaction is the amine substrate itself. The simultaneous addition of base during the conversion can be done manually while monitoring the reaction mixture pH or, more conveniently, by using an auto-titrator as a pH holder (pH stat). A combination of partial buffering capacity and base addition may also be used for process control. Typically, the base added to the unbuffered or partially buffered reaction mixture during oxidation is added as an aqueous solution.

In carrying out the stereoselective oxidation reactions described herein, the engineered monoamine oxidase may be added to the reaction mixture in the following form: purified enzyme, whole cells transformed with a gene encoding monoamine oxidase, and/or cell extracts and/or lysates of such cells. Whole cells or cell extracts and/or lysates thereof transformed with a gene encoding an engineered monoamine oxidase may be employed in a variety of different forms, including solid (e.g., lyophilized, spray-dried, and the like) or semi-solid (e.g., coarse paste).

The cell extract or cell lysate may be partially purified by precipitation (ammonium sulfate, polyethylenimine, heat treatment, or the like), followed by a desalting procedure (e.g., ultrafiltration, dialysis, and the like) prior to lyophilization. Either cell preparation can be stabilized by crosslinking using a known crosslinking agent such as glutaraldehyde or immobilized on a solid phase (e.g., eupergit C and the like).

Solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a number 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. For example, the protein solution may be frozen in small portions at-80 ℃ and then added to a pre-frozen lyophilization chamber followed by application of vacuum. After removal of water from the sample, the temperature is typically raised to 4 ℃ for 2 hours before releasing the vacuum and retrieving the lyophilized sample.

The amount of reactant used in the oxidation reaction will generally vary depending on the amount of product desired and the amount of monoamine oxidase substrate employed simultaneously. In general, a substrate concentration of about 5 g/liter to 50 g/liter may be used when about 50 mg/liter to about 5 g/liter of monoamine oxidase is used. One of ordinary skill in the art will readily understand how to vary these amounts to tailor them to the desired level of productivity and product size. The appropriate amount of optional agents such as catalase, defoamer, and sodium bisulfite or sodium metabisulfite can be readily determined by routine experimentation.

The order of addition of the reactants is not critical. The reactants may be added together to the solvent (e.g., single phase solvent, dual phase aqueous co-solvent system, and the like) at the same time, or alternatively, at different points in time, some of the reactants may be added separately and some of the reactants added together. In certain embodiments, one or more components of the reaction may be added ("fed") continuously to the reaction at a level that minimizes or eliminates substrate and/or product inhibition by monoamine oxidase. In certain embodiments, monoamine oxidase may 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 performing the monoamine oxidase-catalyzed oxidation reactions described herein include a wide variety of conditions that can be readily optimized by routine experimentation, including, but not limited to, contacting an engineered monoamine oxidase and a substrate at experimental pH and temperature and detecting the product by methods such as those described in the examples provided herein.

Monoamine oxidase catalyzed oxidation is typically carried out at a temperature in the range of about 5 ℃ to about 75 ℃. For some embodiments, the reaction is conducted at a temperature in the range of about 20 ℃ to about 55 ℃. In other embodiments, the reaction is conducted at a temperature in the range of about 20 ℃ to about 45 ℃, in the range of about 30 ℃ to about 45 ℃, or in the range of about 40 ℃ to about 45 ℃. The reaction may also be carried out at ambient temperature (about 21 ℃).

The oxidation reaction is generally allowed to proceed until a substantially complete or near complete oxidation of the substrate is obtained. The oxidation of the substrate to the product may be monitored using known methods of detecting the substrate and/or the product. Suitable methods include gas chromatography, HPLC, and the like. Conversion yields are generally greater than about 50%, and may be greater than about 60%, and may be greater than about 70%, and may be greater than about 80%, and may be greater than about 90%, and typically greater than about 97%.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

FIG. 1 represents the detection of H by Ampliflu Red fluorescence ₂ O ₂ Concentration standard curve.

Sequence number	Name of the name
		SEQ ID NO:1	Monoamine oxidase MAON-1
SEQ ID NO:2	MAON-1 variant F382L
		SEQ ID NO:3	MAON-1 variant F382L/F63L/T65V
SEQ ID NO:4	MAON-1 variant F382L/S100P
		SEQ ID NO:5	MAON-1 variant F382L/L201P
SEQ ID NO:6	MAON-1 variant F382L/T107S
		SEQ ID NO:7	MAON-1 variant F382L/T138S
SEQ ID NO:8	MAON-1 variant F382L/T141S
		SEQ ID NO:9	MAON-1 variant F382L/E190D
SEQ ID NO:10	MAON-1 variant F382L/S234C

Detailed Description

Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be exemplary and not limiting.

EXAMPLE 1 monoamine oxidase mutant vector construction

The MAON protein expression gene derived from Aspergillus niger (Aspergillus niger) monoamine oxidase, and MAON mutant gene (SEQ ID NO: 1-10) engineered based on stability and catalytic activity were ligated into E.coli expression vector pET15b, inserted at the NdeI+BamHI site, retaining N-ter 6 XHis tag. After sequencing was correct, the recombinant vector transformed BL21 (DE 3) for protein expression.

Example 2 monoamine oxidase expression and purification: shaking bottle production

Transferring the constructed expression vector into escherichia coli BL21 (DE 3), performing IPTG induction expression, and purifying by using a Ni-NTA column after bacterial recovery and cracking, wherein the specific method is as follows: MAON enzyme recombinant expression vector is transformed into BL21 (DE 3) strain, monoclonal is selected and put into 10ml LB culture medium, ampicillin sodium resistance (100 mg/L), 37 ℃ and 200RPM are cultivated overnight, and transferred into 2L shake flask containing 1L LB culture medium, 37 ℃ and 200RPM are cultivated until OD600 reaches 0.6-0.8, then the temperature is reduced to 25 ℃,0.5mM IPTG is induced to express for overnight, and 5000Xg is centrifugated for bacterial collection. Utilizing buffer A to collect the thalli: 50mM Tris pH 8.0,500mM NaCl,20mM imidazole was resuspended and added to a final concentration of 1mM PMSF,250ul Cocktail inhibitor and mixed well. After crushing by a high-pressure homogenizing breaker, 43000Xg is centrifuged for 30min at 4 ℃, and the supernatant is taken out and passed through a Ni column. Ni-NTA column purification, after the lysate supernatant and resin combined for 20min, with 50mM imidazole buffer A washing, finally with 400mM imidazole elution buffer elution. SDS-PAGE detects the effect of protein purification. Buffer to 50mM Tris pH7.5,500mM NaCl,1mM DTT were dialyzed. And finally detecting the protein purification effect by SDS-PAGE of the sample, and freezing at-80 ℃ for later use after ultrafiltration and concentration.

Example 3 monoamine oxidase expression and purification: fermentation production

Seed activation: MAON enzyme recombinant expression vector is transformed into BL21 (DE 3) strain, monoclonal is selected and put into 10mL LB culture medium, ampicillin sodium resistance (100 mg/L), 37 ℃ and 200RPM are cultivated overnight, and then transferred into 1L shake flask containing 500mL LB culture medium, 37 ℃ and 200RPM are cultivated until OD600 reaches 0.8-1.0. Fermentation culture: 10L fermenter medium containing 6L TB medium was preheated to 37℃and ampicillin sodium at a final concentration of 100mg/L was added, aeration and agitation were carried out after inoculation to maintain 30% dissolved oxygen, and when OD600 increased to 10, feed 1 was an aqueous solution containing 60g/L tryptone, 120g/L yeast extract, 4% glycerol, feed 2 was 50% glycerol, and pH was regulated to pH 7.0 using ammonia and phosphoric acid. When the OD600 increased to 20, the broth was cooled to 25℃and monoamine oxidase expression was induced by adding isopropyl-. Beta. -D-thiogalactoside (IPTG) to a final concentration of 1mM, allowing the culture to grow for an additional 20 hours until harvest. Cells were harvested by centrifugation at 8000 Xg. The harvested cells were used directly in the subsequent purification process or stored at-80℃until used as such. Crude enzyme purification: the collected cells were resuspended by 100mM Tris pH 8.0,150mM NaCl, and the cells were resuspended at 200g wet cells/L. After crushing by an 800Bar high-pressure homogenizing breaker, centrifuging at 1840 xg and 4 ℃ for 20min, taking supernatant, adding ammonium sulfate powder (200 g/L) with the final concentration of 36% saturation, and centrifuging to collect protein precipitate. The precipitate was lyophilized and stored at 4℃until use.

EXAMPLE 4 monoamine oxidase Activity assay

Catalytic production of H due to monoamine oxidase ₂ O ₂ By measuring the amount of hydrogen peroxide produced, the enzyme activity parameter can be indirectly measured, and a standard curve is drawn: 5ml of 100mM K was taken ₂ HPO ₄ Buffer solution with HCl pH of 7.4, ampliflu Red dye with final concentration of 100uM and horseradish peroxidase with concentration of 1U/ml are added to prepare working solution, and H with concentration of 0, 1.25, 2.5, 5, 10, 20 and 40 is prepared by the working solution ₂ O ₂ The fluorescence value was measured by an enzyme-labeled instrument, λex=535 nm/λem=590 nm, and a standard curve was drawn. MAON enzyme Activity parameter determination Using 100mM K ₂ HPO ₄ HCl pH7.4 buffer, MAON enzyme was diluted to 100nM working solution, and the substrate was prepared into reaction solutions with final concentrations of 0, 11.7, 23.4, 47, 94, 188, 375, 750, 1500, 3000uM using the buffer, 95ul of the reaction solution was taken, and 5ul of enzyme working solution was added thereto, and the final enzyme concentration was 5nM. The change in fluorescence values was measured using a microplate reader, λex=535 nm/λem=590 nm, and the enzyme activity parameters were calculated according to the standard enzyme activity catalytic parameter measurement method and standard curve, and the results of some of the tests are shown in table 1.

TABLE 1 results of MAON mutant enzyme Activity test

EXAMPLE 5 monoamine oxidase catalyzes the oxidation of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane to give (1R, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hex-2-ene Activity test

40ml of monoamine oxidase buffer expressed and purified by SEQ ID NO:1-10 via example 2 (2 mg/ml, stored in potassium phosphate hydrochloric acid buffer solution at pH 7.4, yellow liquid) was added to a three-necked flask, to which 70mg of catalase, 2mg of antifoaming agent 204 was added, and stirred at 25℃under an oxygen atmosphere. In addition, 280mg of 6, 6-dimethyl-3-aza-bicyclo [3.1.0] hexane is weighed and dissolved by 5ml of potassium phosphate hydrochloric acid buffer solution with pH of 7.4, then is added into the reaction system dropwise by a syringe pump within 5 hours, the pH is regulated to be 7.4 by using 3M sodium hydroxide solution during the reaction, the liquid quality is controlled after the reaction is carried out for 18 hours, the conversion rate is more than 95%, methyl tertiary butyl ether is added for extraction after the reaction is finished, the methyl tertiary butyl ether is dried by spinning, and the ee is more than 98% by liquid phase measurement.

¹ H 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).

EXAMPLE 6 monoamine oxidase catalyzed 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane desymmetrization-1 in the Presence of bisulfite

40ml of a pH 7.4 potassium phosphate hydrochloric acid buffer solution was added to a three-necked flask, 500mg of monoamine oxidase to be expressed and purified by SEQ ID NO:1-10 via example 2 was added, followed by 175mg of catalase, 2.5mg of antifoaming agent 204, stirring at 25℃and stirring under an oxygen atmosphere. 1.25g of sodium bisulphite is weighed, dissolved in 8ml of water, 1g of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane is added, a substrate is dripped into a biological enzyme reaction system through a syringe pump within 5 hours, the pH is regulated to be 7.4 by using a 3M sodium hydroxide solution during the reaction, the reaction is controlled in a middle after 24 hours, liquid phase mass spectrometry analysis is carried out, and the raw material conversion is completed, wherein the system is a mixture of (1R, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hex-2-ene and (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-sodium sulfonate.

EXAMPLE 7 monoamine oxidase catalyzed 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane desymmetrization-2 in the Presence of bisulfite

40ml of a pH 7.4 potassium phosphate hydrochloric acid buffer solution was added to a three-necked flask, 1g of monoamine oxidase expressed and purified by SEQ ID NO:1-10 via example 2 was added, followed by 175mg of catalase, 2.5mg of antifoaming agent 204, and stirred at 25℃under an oxygen atmosphere. 1.25g of sodium bisulfite is weighed, dissolved in 8ml of water, 1g of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane is added, the mixed substrate is directly added into a biological enzyme reaction system, the pH is regulated by a 3M sodium hydroxide solution during the reaction and is maintained at 7.4, after 24 hours, the medium is controlled, the liquid phase mass spectrum analysis is carried out, and the raw material conversion is completed, wherein the system is a mixture of (1R, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hex-2-ene and (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-sodium sulfonate.

EXAMPLE 8 monoamine oxidase catalyzes the desymmetrization of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane in the presence of bisulfite, the effect of enzyme amount on the reaction

1g,500mg,250mg and 100mg of monoamine oxidase expressed and purified by SEQ ID NO:1-10 via example 2 were respectively added to three-necked flasks, 175mg of catalase, 2.5mg of antifoaming agent 204 were respectively added thereto, and stirred at 25℃and stirred under an oxygen atmosphere. 1.25g of sodium bisulphite is weighed and dissolved by 8ml of water, 1g of reactant 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane is added, the mixture is dropwise added into the reaction system within 5 hours through a syringe pump, the pH value is regulated to be 7.4 by using 3M sodium hydroxide solution during the reaction, and the comparison of reaction center control results shows that the higher the enzyme content is, the faster the reaction rate is.

EXAMPLE 9 preparation of (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-carbonitrile

40ml of a pH 7.4 potassium phosphate hydrochloric acid buffer solution was added to a three-necked flask, monoamine oxidase expressed and purified by SEQ ID NO:1-10 via example 2 was added, followed by 175mg of catalase, 2.5mg of antifoaming agent 204, stirring at 25℃and stirring at oxygen. 1.25g of sodium bisulfite is weighed, dissolved in 8ml of water, 1g of 6, 6-dimethyl-3-azabicyclo [3.1.0] hexane is added, the substrate is dripped into a biological enzyme reaction system through a syringe pump within 5 hours, the pH is regulated to be 7.4 by using a 3M sodium hydroxide solution during the reaction, the pH is controlled after 24 hours, liquid phase mass spectrometry analysis is carried out, the conversion of raw materials is completed, the system is a mixture of (1R, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hex-2-ene and (1R, 2S, 5S) -6, 6-dimethyl-3-azabicyclo [3.1.0] hexane-2-sodium sulfonate, 40ml of TMBE is added after the reaction system is reduced to 10 ℃, 1g of TMSCN is dripped within 30 minutes, and the product is generated after the reaction is carried out for 30 minutes.

While this invention is satisfied by embodiments in many different forms, as described in detail in connection with the preferred embodiments of the invention, it is to be understood that the present disclosure is to be considered exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Many variations may be made by those skilled in the art without departing from the spirit of the invention. The scope of the invention is to be determined by the appended claims and their equivalents. The abstract and the title should not be construed as limiting the scope of the invention, as they are intended to enable the appropriate mechanism and the general public to quickly ascertain the general nature of the invention.

Claims

1. A monoamine oxidase comprising an amino acid sequence having the following mutation compared to the monoamine oxidase amino acid sequence as set forth in SEQ ID NO: 1: the 382 th amino acid in the monoamine oxidase amino acid sequence shown in the corresponding SEQ ID NO. 1 is mutated from phenylalanine to leucine.

2. The monoamine oxidase of claim 1, said amino acid sequence further having one or more mutations selected from the group consisting of:

3. The monoamine oxidase of claim 1 or 2, said amino acid sequence further having 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 to the monoamine oxidase amino acid sequence shown in SEQ ID No. 1.

4. A monoamine oxidase according to any one of claims 1-3 comprising an amino acid sequence having at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of seq id no: SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9 and 10.

5. A polynucleotide encoding the monoamine oxidase of any one of claims 1 to 4 and a host cell comprising said polynucleotide.

6. Preparation of substantially stereoisomerically pure compounds of formula IIA method for producing the compound or a salt/hydrate thereof, which comprises reacting a compound as shown in I +.>The compound shown is contacted with oxygen in the presence of a monoamine oxidase as shown in any one of claims 1-4 and a cofactor.

7. Preparation of substantially enantiomerically pure compounds of formula III A process for the preparation of the sulfamate compound or salt/hydrate thereof which comprises reacting, for exampleⅠ/>The compound shown is contacted with oxygen in the presence of monoamine oxidase, cofactor and bisulphite as shown in any one of claims 1-4.

8. Preparation of substantially enantiomerically pure compounds such as IVA process for producing an aminonitrile compound or a salt/hydrate thereof which comprises reacting an aminonitrile compound as shown in I +.>The compound shown is contacted with oxygen in the presence of monoamine oxidase, cofactor and bisulphite as shown in any one of claims 1-4 and the resulting sulfamate compound is contacted with cyanide.

9. The method of any one of claims 6-8, wherein the cofactor is non-covalently associated with a monoamine oxidase;

preferably, the cofactor is selected from the group consisting of: FAD, FMN, NAD and NADP;

preferably, the method further comprises catalyzing the hydrogen peroxide to be bifidus to a component of molecular oxygen and water, more preferably, the component is selected from the group consisting of: pd, fe and catalase.

10. The monoamine oxidase of any one of claims 1-4 catalyzing a reaction as IThe compounds shown oxidize to substantially stereoisomerically pure compounds as II >The compounds orThe application of the salt/hydrate thereof.

11. The monoamine oxidase of any one of claims 1-4 catalyzing a reaction as IThe compounds shown are prepared to be substantially enantiomerically pure as III->Sulfamate compounds shown, e.g. IV->The use of the aminonitrile compounds shown or of their salts/hydrates.

12. The monoamine oxidase of any one of claims 1-4 catalyzing a reaction as IThe compounds shown are useful for the de-symmetrization.