Monoamine oxidase and its applications

Abstract

The present invention provides monoamine oxidase and its application in biocatalysis.

Description

[Technical field]

[0001] The present invention relates to monoamine oxidase and its application in biocatalysis. [Background technology]

[0002] Patent CN102131813A discloses the use of monoamine oxidase to resolve and deracemize racemic chiral amines by stereospecifically oxidizing one enantiomer to the corresponding imine with oxygen. Summary of the Invention [Problem to be solved by the invention]

[0003] However, it remains desirable to provide novel monoamine oxidases useful in the above biocatalytic processes. [Means for solving the problem]

[0004] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description of the Invention. This Summary is not intended to identify key or essential features of the claims, nor is it intended to be used to limit the scope of the claims. Other features, details, utilities, and advantages of the claims will become apparent from the following Detailed Description of the Invention, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0005] The present invention provides a monoamine oxidase comprising an amino acid sequence having, compared to the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine to leucine at amino acid 63, a mutation from threonine to valine at amino acid 65, a mutation from serine to proline at amino acid 100, a mutation from threonine to serine at amino acid 141, and / or a mutation from serine to cysteine ​​at amino acid 234 in the amino acid sequence represented by SEQ ID NO:1.

[0006] In one specific embodiment, the monoamine oxidase has a structure similar to that of SEQ ID NO:1, as compared to the amino acid sequence represented by SEQ ID NO:1. In the amino acid sequence represented by SEQ ID NO: 1, a mutation from phenylalanine at the 63rd amino acid to leucine and a mutation from threonine at the 65th amino acid to valine; a mutation from serine to proline at the 100th amino acid and a mutation from serine to cysteine ​​at the 234th amino acid in the amino acid sequence represented by SEQ ID NO:1; a mutation from threonine to serine at amino acid 141 and from serine to cysteine ​​at amino acid 234 in the amino acid sequence represented by SEQ ID NO:1; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, and a mutation from serine to proline at the 100th amino acid are In the amino acid sequence represented by SEQ ID NO: 1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, and a mutation from threonine at the 141st amino acid to serine; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, and a mutation from serine to cysteine ​​at the 234th amino acid are a mutation from serine to proline at amino acid 100, a mutation from threonine to serine at amino acid 141, and a mutation from serine to cysteine ​​at amino acid 234 in the amino acid sequence represented by SEQ ID NO:1; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, a mutation from serine at the 100th amino acid to proline, and a mutation from threonine at the 141st amino acid to serine; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, a mutation from serine at the 100th amino acid to proline, and a mutation from serine at the 234th amino acid to cysteine; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at amino acid 63 to leucine, a mutation from threonine at amino acid 65 to valine, a mutation from threonine at amino acid 141 to serine, and a mutation from serine to cysteine ​​at amino acid 234, or This includes an amino acid sequence represented by SEQ ID NO:1, which has a mutation from phenylalanine to leucine at amino acid 63, a mutation from threonine to valine at amino acid 65, a mutation from serine to proline at amino acid 100, a mutation from threonine to serine at amino acid 141, and a mutation from serine to cysteine ​​at amino acid 234.

[0007] 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%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

[0008] 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 to any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 17.

[0009] In one specific embodiment, the amino acid sequence of the monoamine oxidase is represented by any one of SEQ ID NOs: 2 to 17.

[0010] The present invention also provides a polynucleotide encoding the monoamine oxidase and a host cell comprising the polynucleotide.

[0011] The present invention also provides a method for producing a substantially stereoisomerically pure compound represented by formula II below, or a salt / hydrate thereof, which comprises contacting a compound represented by formula I below with oxygen in the presence of the monoamine oxidase and a cofactor.

[0012] [ka]

[0013] The present invention also provides a method for producing a substantially enantiomerically pure aminosulfonate compound represented by the following formula III, or a salt / hydrate thereof, which comprises contacting a compound represented by the following formula I with oxygen in the presence of the monoamine oxidase, a cofactor and a bisulfite salt.

[0014] [ka]

[0015] The present invention also provides a method for producing a substantially enantiomerically pure aminonitrile compound represented by the following formula IV or a salt / hydrate thereof, which comprises contacting a compound represented by the following formula I with oxygen in the presence of the monoamine oxidase, a cofactor and a bisulfite to obtain an aminosulfonate compound, and contacting the resulting aminosulfonate compound with cyanide.

[0016] [ka]

[0017] In a specific embodiment, the cofactor is non-covalently bound to the monoamine oxidase. In another specific embodiment, said cofactor is selected from the group consisting of FAD, FMN, NAD and NADP. In another specific embodiment, the method further comprises a component that catalyzes the disproportionation of hydrogen peroxide to molecular oxygen and an aqueous component. Preferably, the component is selected from the group consisting of Pd, Fe, and catalase.

[0018] The present invention also provides a monoamine oxidase for use in catalysing the oxidation of a compound of formula I below to a substantially stereoisomerically pure compound of formula II below or a salt / hydrate thereof.

[0019] [ka]

[0020] The present invention also provides a monoamine oxidase for use in catalyzing the production of a substantially enantiomerically pure aminosulfonate compound represented by the following formula III, or an aminonitrile compound represented by the following formula IV, or a salt / hydrate thereof, from a compound represented by the following formula I.

[0021] [ka]

[0022] The present invention also provides a monoamine oxidase for use in catalysing the desymmetrisation of a compound of formula I below.

[0023] [ka] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The following description taken in conjunction with the accompanying drawings is intended to describe various exemplary embodiments of the disclosed invention. Specific features and functions are described in conjunction with each exemplary embodiment. However, it will be apparent to one skilled in the art that the disclosed embodiments may be practiced without any of these specific features and functions. Furthermore, all features described in conjunction with one embodiment are intended to be applicable to other embodiments described herein, unless the feature or function is expressly stated to be incompatible with other embodiments. For example, if a given feature or function is explicitly described in conjunction with one embodiment but not explicitly mentioned in conjunction with an alternative embodiment, it will be understood that the feature or function may be developed, utilized, or practiced in conjunction with the alternative embodiment, unless the feature or function is incompatible with the alternative embodiment.

[0025] The implementation of the techniques described herein may use conventional methods and explanations, such as organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, biological emulsion production, and sequencing technology, that are within the skill of the art, unless otherwise indicated. Such conventional methods include polymer array synthesis, polynucleotide hybridization and ligation, and hybridization detection using markers. Specific explanations of suitable techniques may be obtained by reference to the examples herein. Of course, other equivalent conventional procedures may also be used. Such conventional methods and explanations may be found in standard laboratory manuals. For example, Green et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: AL Laboratory Manual; Dieffenbach, Dveksler, Eds. (2003), PCR Primer: A Laboratory Manual; 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; Press);Stryer, L. (1995) Biochemistry (4th edition) WHFreeman, New York NYSee Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd ed., WH Freeman Pub., New York, NY; Berg et al., (2002) Biochemistry, 5th ed., WH Freeman Pub., New York, NY; Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, Eds., John Wiley & Sons 1998); Mammalian Chromosome Engineering - Methods and Protocols (G. Hadlaczky, Eds., Humana Press 2011); Essential Stem Cell Methods, (Lanza and Klimanskaya, Eds., Academic Press 2011). All references are incorporated herein by reference in their entirety for any purpose. Techniques for nuclease specificity are described, for example, in Genome Editing and Engineering From TALENs and CRISPRs to Molecular Surgery, Appasani and Church (2018); and CRISPR: Methods and Protocols, Lindgren and Charpentier (2015), both of which are incorporated by reference in their entirety for any purpose. Basic methods for enzyme engineering are described in Enzyme Engineering Methods and Protocols, Samuelson, Eds., (2013); Protein Engineering, Kaumaya, Eds., (2012); and Kaur and Sharma, “Directed Evolution: An Approach to Engineer Enzymes”, Crit. Rev.Biotechnology, 26:165-69(2006).

[0026] It should be noted that the singular forms "a" and "the" as used herein and in the claims include plural referents unless otherwise indicated. For example, reference to an "oligonucleotide" is intended to include one or more oligonucleotides, and reference to an "automated system" is intended to include equivalent steps and methods for use with systems known to those of skill in the art. Furthermore, it will be understood that terms such as "left," "right," "top," "bottom," "front," "back," "side," "height," "length," "width," "up," "down," "interior," "exterior," "inner," "outer," and the like, as used herein, are merely intended to describe a point of reference and do not necessarily limit embodiments of the present disclosure to a particular orientation or configuration. Furthermore, terms such as "first," "second," "third," and the like, are merely intended to identify one of the many parts, components, steps, operations, functions, and / or reference points disclosed herein and similarly do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the devices, methods and cell populations that can be used in accordance with the invention described herein.

[0028] For any numerical ranges described herein, it is understood that each intermediate value between the upper and lower limits of the range, and every other specified or intermediate value within the range, is encompassed within the invention. The upper and lower limits of any smaller range may independently be included in the smaller range, and may also be encompassed within the invention as if limited to any specifically excluded limit in a given range. Where a given range includes one or both limits, ranges excluding any one or both of those included limits are also included in the invention.

[0029] In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and steps well known to those of ordinary skill in the art have not been described in order to avoid obscuring the present invention.

[0030] The monoamine oxidase of the present invention, capable of oxidizing an amine compound of formula I to the corresponding imine compound of formula II, has one or more amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 1. Such amino acid substitutions may provide one or more improvements to the monoamine oxidase, including increased enzymatic activity, increased stereospecificity, increased thermal stability, increased solvent stability, reduced product inhibition, reduced substrate inhibition, or reduced sensitivity to reaction by-products. Furthermore, such amino acid substitutions may improve the expression level, solubility, and / or stability of the monoamine oxidase in a host cell, for example, but not limited to, a recombinantly expressed protein in a heterologous host cell, such as an E. coli host cell.

[0031] The invention also provides polynucleotides encoding such monoamine oxidases, and methods of using the polypeptides in the disclosed biocatalytic methods.

[0032] As used herein, the terms have the following meanings: A "monoamine oxidase" is a polypeptide having the enzymatic ability to oxidize a compound of formula I above to the corresponding product of formula II above. The polypeptide uses 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. As used herein, monoamine oxidase includes naturally occurring (wild-type) monoamine oxidases and non-naturally occurring artificial polypeptides produced by human manipulation.

[0033] A "coding sequence" is a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0034] "Naturally-occurring," "native," or "wild-type" refers to the form expressed in nature. For example, a naturally-occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that is isolated from a natural source and has not been intentionally modified by human manipulation.

[0035] "Recombinant," as used to refer, for example, to a cell, nucleic acid, or polypeptide, refers to material that has been altered to be different from that occurring in nature, the natural or native state of the material, or the same as the natural or native state of the material but produced or derived from synthetic material and / or produced or derived from synthetic material by manipulation using recombinant techniques. Non-limiting examples include, but are not limited to, recombinant cells that express genes that are not present in the native (non-recombinant) state of the cell, or that express native genes that are expressed at different levels.

[0036] "Percentage of sequence identity" and "percentage of homology" are used interchangeably herein and refer to the comparison between polynucleotides and polypeptides, which is determined by comparing the sequences of two optimally aligned sequences in a comparison window. However, the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence of two optimally aligned sequences (which does not contain additions or deletions). The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences, or the number of positions of nucleic acid base or amino acid residue that align with gaps to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms for aligning two sequences.Comparison of optimal alignment of sequences can be performed, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2:482; the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection (see generally Current Protocols in Molecular Biology, FMA Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 supplement) (Ausubel)). Examples of suitable algorithms 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, respectively. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring segment pairs (HSPs) by identifying short words of length W in the query sequence that match or meet a predefined positive threshold score T when aligned with words of the same length in the database sequence. T is the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing these word hits.The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, the cumulative score is calculated using a score matrix. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by an amount X from the maximum achieved, when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of any one sequence is reached. The sensitivity and speed of the alignment are determined by the BLAST algorithm parameters W, T, and X. The BLASTN program (for nucleotide sequences) uses as default arguments a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and double-stranded comparisons. For amino acid sequences, the BLASTP program uses as default arguments a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). By way of example, for sequence alignment and determination of percent sequence identity, the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison Wis.) can be used with the default arguments provided.

[0037] "Reference sequence" refers to a determined sequence used as the basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, or the full-length nucleic acid or polypeptide. Since each of the two polynucleotides or polypeptides (1) contains a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) may further contain a sequence that differs between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is generally performed by comparing the sequences of the two polynucleotides in a "comparison window" to identify and compare local regions of sequence similarity.

[0038] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, in which a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and further, the portion of the sequence in the comparison window can contain no more than 20% additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and can optionally be 30 contiguous residues, 40 contiguous residues, 50 contiguous residues, 100 contiguous residues, or longer windows.

[0039] "Substantially identical" refers to a polynucleotide or polypeptide sequence having at least 80% sequence identity, at least 85% identity, and 89%-95% sequence identity, typically at least 99% sequence identity, compared to a reference sequence in a comparison window of at least 20 residue positions (typically a window of at least 30-50 residues). Here, the percent sequence identity is calculated by comparing the reference sequence to a sequence that contains a total of 20% or less deletions or additions relative to the reference sequence in the comparison window. In certain embodiments applied to polypeptides, the term "substantially identical" means that two polypeptide sequences have at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity) when optimally aligned by the programs GAP or BESTFIT using default gap weights. Preferably, the differing residue positions differ by conservative amino acid substitutions.

[0040] "Corresponding," "relating to," or "against" as used with respect to a given amino acid or polynucleotide sequence number refers to the residue number of the given amino acid or polynucleotide sequence when compared to the specified reference sequence. In other words, the residue numbers or residue positions of a given polymer are not specified with respect to the actual numerical position of the residue in the given amino acid or polynucleotide sequence, but are specified with respect to the reference sequence. For example, a given 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, regardless of the presence of gaps, the residue numbers in the given amino acid or polynucleotide sequence are determined with respect to the reference sequence used for alignment.

[0041] "Stereoselectivity" refers to the preferential formation of one stereoisomer over the other in a chemical or enzymatic reaction. Stereoselectivity may be partial, with one stereoisomer being preferentially formed over the other, or complete, with only one stereoisomer being formed. When the stereoisomer is an enantiomer, stereoselectivity refers to the selectivity for the enantiomer, i.e., the proportion of one enantiomer in the sum of both stereoisomers (usually reported as a percentage), typically reported in the art as enantiomeric excess (ee), calculated according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomer is a diastereomer, stereoselectivity refers to the selectivity for the diastereomer, i.e., the proportion of one diastereomer in a mixture of both diastereomers (usually reported as a percentage), typically reported as diastereomeric excess (de). Types of stereoisomeric excess include enantiomeric excess and diastereomeric excess.

[0042] "Highly stereoselective" refers to a monoamine oxidase polypeptide capable of converting a substrate to the corresponding product in at least about 99% stereoisomeric excess.

[0043] "Stereospecificity" refers to the preferential conversion of one stereoisomer over another in a chemical or enzymatic reaction. Stereospecificity can be partial, where one stereoisomer is converted in preference to the other, or complete, where only one stereoisomer is converted.

[0044] "Chemoselectivity" refers to the preferential formation of one product over another in a chemical or enzymatic reaction.

[0045] "Improved enzymatic properties" refers to monoamine oxidase polypeptides that exhibit an improvement in any enzymatic property as compared to a reference monoamine oxidase. The engineered monoamine oxidase polypeptides described herein are generally compared to a wild-type monoamine oxidase, although in some embodiments the reference monoamine oxidase may be another improved monoamine oxidase. Improved enzymatic properties include, but are not limited to, enzymatic activity (as indicated by percent conversion of substrate), thermostability, pH activity profile, cofactor requirement, recalcitrance to inhibitors (e.g., product inhibition), stereospecificity, stereoselectivity (including enantioselectivity), solubility, and stability, as well as expression levels in a host cell.

[0046] "Increased enzymatic activity" refers to an improved property of an engineered monoamine oxidase polypeptide and can be expressed as 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 a starting amount of substrate to product in a specified period of time using a specified amount of monoamine oxidase). Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max or k cat The term "enzyme activity" refers to the ability to affect any property associated with enzyme activity, including the general enzyme properties of k and k = ... cat / K m The theoretical maximum value of is generally about 10 8 ~10 9 (M-1 s- 1 ) Therefore, any improvement in the enzymatic activity of monoamine oxidase has an upper limit related to the diffusion rate of the substrate on which monoamine oxidase acts. The activity of monoamine oxidase can be measured using previously published methods for measuring monoamine oxidase or modifications thereof. Examples include, but are not limited to, Zhou et al. ("A One-Step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity," 1997 Anal. Biochem. 253:169-74) and Szutowicz et al. ("Colorimetric Assay for Monoamine Oxidase in Tissues Using Peroxidase and 2,2'-Azino(3-ethylbenzthaizoline-6-sulfonic Acid) as Chromogen," 1984, Anal. Biochem. 138:86-94). Comparisons of enzyme activities are performed with one or more defined substrates under set conditions using defined enzyme preparations, as described in more detail herein or, for example, by the method of Zhou and Szutowicz. Generally, when comparing lysates, the cell numbers measured and the amounts of protein measured are determined using the same expression system and the same host cells to minimize variation in the amount of enzyme produced in the host cells and present in the lysates.

[0047] "Conversion" refers to the enzymatic oxidation of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is oxidized to a product under specified conditions within a given period of time. Thus, the "enzyme activity" or "activity" of a monoamine oxidase polypeptide can be expressed as the "percent conversion" of substrate to product.

[0048] "Thermostable" refers to the ability of a monoamine oxidase polypeptide to maintain similar activity (e.g., greater than 60%-80%) after exposure to a higher temperature (e.g., 40-80°C) for a period of time (e.g., 0.5-24 hours) compared to the untreated enzyme.

[0049] "Solvent stability" refers to the ability of a monoamine oxidase polypeptide to maintain similar activity (e.g., greater than 60% to less than 80%) after exposure to various concentrations (e.g., 5-99%) of solvents (e.g., isopropanol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 hours) compared to the untreated enzyme.

[0050] "pH stable" refers to the ability of a monoamine oxidase polypeptide to maintain similar activity (e.g., greater than 60% to 80%) after exposure to a higher or lower pH (e.g., 4.5-6 or 8-12) for a period of time (e.g., 0.5-24 hours) compared to the untreated enzyme.

[0051] "Heat and solvent stable" refers to a monoamine oxidase polypeptide that has both heat and solvent stability.

[0052] "Hydrophilic amino acid or residue" refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than 0 according to the standardized consensus hydrophobicity scale set forth in Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. 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).

[0053] "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 generally have a side chain that is negatively charged at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0054] "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 generally have a positively charged side chain due to association with a hydrated hydrogen ion at physiological pH. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0055] "Polar amino acid or residue" refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH but has at least one bond in which the electron pair shared by the two atoms is held closer to one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L-Thr (T).

[0056] "Hydrophobic amino acid or residue" refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of 0 or greater according to the standardized consensus hydrophobicity scale described in Eisenberg et al., 1984, J. Mol. Biol. 179:125-142. 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).

[0057] "Aromatic amino acid or residue" refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that contains at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), and L-Trp (W). Histidine is classified herein as a hydrophilic residue or "constrained residue" (see below), although L-His (H) may be classified as a basic residue because of the pKa of its heteroaromatic nitrogen atom, or as an aromatic residue because its side chain contains a heteroaromatic ring.

[0058] "Constrained amino acid or residue" refers to an amino acid or residue that has constrained geometric properties. As used herein, constrained residues include L-pro (P) and L-his (H). Histidine has constrained geometric properties because it has a relatively small imidazole ring. Proline also has constrained geometric properties because it also has a five-membered ring.

[0059] "Nonpolar amino acid or residue" refers to a hydrophobic amino acid or residue having a side chain that is uncharged at physiological pH and has a bond in which the electron pair shared by the two atoms is generally equally shared by either of the two atoms (i.e., the side chain is 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).

[0060] "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).

[0061] The amino acid L-Cys(C), referred to as "cysteine", 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 available for disulfide bond formation. The ability of L-Cys(C) (and other amino acids with -SH-containing side chains) to exist in peptides in the reduced, free -SH or oxidized disulfide bond form influences whether L-Cys(C) confers hydrophobic or hydrophilic properties to the peptide. Although L-Cys(C) exhibits a hydrophobicity of 0.29 according to the standardized consensus hydrophobicity scale described in Eisenberg (Eisenberg et.al., 1984, supra), it is understood that L-Cys(C) is classified in its own unique group for purposes of this disclosure.

[0062] "Small amino acid or residue" refers to an amino acid or residue having a side chain consisting of a total of three or less carbon and / or heteroatoms (excluding alpha carbon and hydrogen). Small amino acids or residues can be further classified as aliphatic, non-polar, polar or acidic small amino acids or residues according to the above definitions. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).

[0063] "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).

[0064] "Conservative" amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and generally includes the substitution of an amino acid in a polypeptide with an amino acid belonging to the same or similar defined amino acid group. However, as used herein, conservative mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue, although it may be aliphatic to aliphatic, non-polar to non-polar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or constrained residue to constrained residue. Furthermore, as used herein, A, V, L, or I can be conservatively mutated to another aliphatic or non-polar residue. Examples of conservative substitutions are shown in Table 1 below.

[0065] [Table 1]

[0066] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions cannot use amino acids within the defined set listed above, but can use amino acids in between. In one embodiment, non-conservative mutations affect (a) the structure of the peptide backbone in the region of substitution (e.g., proline to glycine), (b) the charge or hydrophobicity, or (c) the volume of the side chain.

[0067] "Deletion" refers to a modification to a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids that make up the reference enzyme, or up to 20% of the total number of amino acids that make up the reference enzyme, while retaining enzymatic activity and / or retaining the improved properties of the engineered monoamine oxidase. Deletions are made internally and / or at the ends of the polypeptide. In various embodiments, deletions can include continuous segments or can be discontinuous.

[0068] "Insertion" refers to a modification to a polypeptide by the addition of one or more amino acids to a reference polypeptide. In some embodiments, improved engineered monoamine oxidases include insertions of one or more amino acids into naturally occurring monoamine oxidases and insertions of one or more amino acids into other improved monoamine oxidase polypeptides. Insertions can be made internally in the polypeptide or at the carboxyl or amino terminus. As used herein, insertions include fusion proteins known in the art. Insertions can be contiguous amino acid segments or can be separated by one or more amino acids in naturally occurring polypeptides.

[0069] "Differing" or "dissimilar" to a designated reference sequence refers to the difference in a given amino acid or polynucleotide sequence when compared to and aligned with the reference sequence. In general, the difference can be determined when the two sequences are optimally aligned. The difference includes insertion, deletion, or substitution of amino acid residues compared to the reference sequence.

[0070] As used herein, a "fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion but which is identical to the remaining amino acid sequence at corresponding positions in the sequence. Fragments can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, up to 70%, 80%, 90%, 95%, 98% and 99% of the full-length monoamine oxidase polypeptide.

[0071] An "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants that naturally accompany it, such as proteins, lipids, and polynucleotides. The term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). The improved monoamine oxidase may be present intracellularly, in cell culture medium, or prepared in a variety of forms, such as a lysate or isolated preparation. Thus, in some embodiments, the improved monoamine oxidase may be an isolated polypeptide.

[0072] A "substantially pure polypeptide" refers to a composition in which the polypeptide material is present as the major substance (i.e., its molar or weight is more abundant than any other single macromolecular substance in the composition), and generally, the subject material is a substantially purified composition when it comprises at least about 50% (molar or weight %) of the macromolecular substance. Generally, a substantially pure monoamine oxidase composition will exhibit 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 (molar or weight %) of all the macromolecular substances in the composition. In some embodiments, the subject material is purified to a substantially homogenous composition consisting essentially of a single macromolecular substance (i.e., to the extent that contaminants cannot be detected in the composition by conventional detection methods). Solvent materials, small molecules (<500 Daltons), and elemental iron materials are not considered to be macromolecular substances. In some embodiments, the isolated improved monoamine oxidase polypeptide is a substantially pure polypeptide composition.

[0073] As used herein, "stringent hybridization" refers to conditions under which a nucleic acid hybrid is stable. As known to those skilled in the art, hybrid stability is determined by the melting temperature (T m ) In general, hybrid stability depends on ionic strength, temperature, G / C content, and the presence of chaotropic agents. The Tm value of a polynucleotide can be calculated using known methods for predicting melting temperatures (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. Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychlik et al., 1990, Nucleic Acids Res 18:6409-6412 (Errata, 1991, Nucleic Acids Res Res 19:698); Sambrook et al., supra); Suggs et al., 1981, Developmental Biology Using Purified Genes (Brown et al., Eds.), pp. 683-693; Academic Press; and Wetmur, 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 or highly stringent conditions, to the complementary sequence of an engineered monoamine oxidase-encoding sequence of the present disclosure.

[0074] "Stringency of hybridization" refers to the washing conditions of nucleic acids. Generally, hybridization reactions are performed under less stringent conditions, followed by different but more stringent washing. The term "moderately stringent hybridization" refers to conditions that allow the target DNA to bind to complementary nucleic acids that have about 60% identity with the target DNA, preferably about 75% identity, about 85% identity, and more than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are those that correspond to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "Highly stringent hybridization" generally refers to the thermal melting temperature T determined under solution conditions of a defined polynucleotide sequence. m Highly stringent conditions refer to conditions that are approximately 10°C or less lower than the temperature at which the nucleic acid sequence is hybridized. In some embodiments, highly stringent conditions refer to conditions that allow hybridization of only those nucleic acid sequences that can form stable hybrids in 0.018M NaCl at 65°C (i.e., as contemplated herein, if a hybrid is not stable in 0.018M NaCl at 65°C, it is not stable under highly stringent conditions). Highly stringent conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing with 0.1x SSPE and 0.1% SDS at 65°C. Other highly stringent hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0075] A "heterologous" polynucleotide refers to a polynucleotide that has been introduced into a host cell by laboratory techniques, and includes polynucleotides that have been removed from a host cell, manipulated in the laboratory, and then reintroduced into the host cell.

[0076] "Codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein can be efficiently expressed in the organism of interest. Although the genetic code is degenerate, with most amino acids represented by a few codons (called "synonyms" or "synonymous" codons), it is well known that the codon usage of a particular organism is not random, but is biased toward certain codon triplets. Such bias in codon usage is more likely in certain genes, genes of common function or ancestra origin, highly expressed proteins versus low copy number proteins, and aggregin-encoding regions of the organism's genome. In some embodiments, polynucleotides encoding monoamine oxidase are codon optimized for optimal production in the host organism selected for expression.

[0077] "Preferred codons, optimal codons, high codon usage bias codons" are used interchangeably with codons that are used more frequently in protein coding regions than other codons that code for the same amino acid. Preferred codons can be determined by codon usage in a single gene, codon usage in a set of genes of common function or origin, codon usage in highly expressed genes, codon frequency in aggregin coding regions across organisms, codon frequency in aggregin coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are generally optimal codons for expression. Various methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon bias in a particular organism, such as cluster analysis or correspondence analysis, and multivariate analysis using the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, JO, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables for an increasing number of organisms are becoming 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. ibid.; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt et al., Eds., ASM Press, Washington DC, pp. 2047-2066). Data sources for obtaining codon usage may rely on any available nucleotide sequence capable of encoding a protein.These datasets include nucleic acid sequences that actually code for known expressed proteins (e.g., complete protein-coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Uberbacher, EC, 1996, Methods Enzymol. 266:259-281; ​​Tiwari et al., 1997, Comput. Appl. Bioscl. 13:263-270).

[0078] "Control sequences" are defined herein to include all components necessary or advantageous for the 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 sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. Control sequences include at least promoters, transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation with the coding region of the nucleic acid sequence encoding the polypeptide.

[0079] "Operably linked" is defined herein as a structure in which a control sequence is suitably positioned relative to the coding sequence of a DNA sequence such that the control sequence can direct expression of a polynucleotide and / or polypeptide.

[0080] A "promoter sequence" is a nucleic acid sequence recognized by a host cell for expression of a coding region. The control sequence may include a suitable promoter sequence. The promoter sequence includes a transcriptional control sequence that mediates the expression of a polypeptide. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0081] As used herein, "stereoisomer," "stereoisomeric form," and similar terms are general terms used for all isomers of a single molecule. Isomers differ only in the orientation of their atoms in space and include enantiomers and isomers of compounds with one or more chiral centers that are not mirror images of one another ("diastereomers").

[0082] The term "chiral center" refers to a carbon atom to which four different groups are attached.

[0083] The term "enantiomer" or "enantiomeric" refers to a molecule that has optical activity because it cannot be superimposed on its mirror image. An enantiomer rotates the plane of polarized light in one direction, while its mirror image rotates the plane of polarized light in the opposite direction.

[0084] The term "racemate" refers to a mixture of equal parts of enantiomers that is optically inactive.

[0085] The term "resolution" refers to the separation, concentration, or removal of one of the two enantiomeric forms of a molecule.

[0086] As used herein, "substantially enantiomerically pure" means that a given enantiomer of a compound is present to a greater extent or amount than another enantiomer of the same compound. Thus, in certain embodiments, a substantially enantiomerically pure compound is present in an enantiomeric excess of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% over another enantiomer of the same compound.

[0087] As used herein, "substantially stereoisomerically pure" means that a given enantiomer or diastereomer of a compound is present to a greater extent or amount than another enantiomer or diastereomer of the same compound. As described above with respect to "stereoselectivity," types of stereoisomeric excess include enantiomeric excess and diastereomeric excess. Thus, in certain embodiments, a substantially stereoisomerically pure compound is present in a stereoisomeric excess of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% over another enantiomer or diastereomer of the same compound.

[0088] 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 contain, in whole or in part, naturally occurring and / or synthetic non-encoded amino acids. Common non-encoded amino acids that the monoamine oxidases described herein may contain include D-stereoisomers of genetically encoded amino acids, 2,3-diaminopropionic acid (Dpr), α-aminoisobutyric acid (Aib), ε-aminohexanoic acid (Aha), δ-aminovaleric acid (Ava), N-methylglycine or sarcosine (MeGly or Sar), ornithine (Orn), citrulline (Cit), t-butylalanine (Bua), t-butylglycine (Bug). , N-methylisoleucine (MeIle), phenylglycine (Phg), cyclohexylalanine (Cha), norleucine (Nle), naphthylalanine (NaI), 2-chlorophenylalanine (Ocf), 3-chlorophenylalanine (Mcf), 4-chlorophenylalanine (Pcf), 2-fluorophenylalanine (Off), 3-fluorophenylalanine (Mff), 4-fluorophenylalanine (Pff), 2-bromophenylalanine (Ob f), 3-bromophenylalanine (Mbf), 4-bromophenylalanine (Pbf), 2-methylphenylalanine (Omf), 3-methylphenylalanine (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 (2pAla), pyrid-3-ylalanine (3pAla), pyrid-4-ylalanine (4pAla), naphth-1-ylalanine (1nAla), naphth-2-ylalanine (2nAla), thiazolylalanine (taAla), benzothienylalanine (bAla), thienylalanine (tAla), furylalanine (fAla), homophenylalanine (hPhe), homotyrosine (hTyr), homotryptophan (hTrp), pentafluorophenylalanine (5ff), styrylalanine (Ala), anthrylalanine (aAla), 3,3-diphenylalanine (Dfa), 3-amino-5-phenylpentanoic acid (phenypentanoic acid,Afp), penicillamine (Pen), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), β-2-thienylalanine (Thi), methionine sulfoxide (Mso), N(w)-nitroarginine (nArg), homolysine (hLys), phosphonomethylphenylalanine (pmPhe), phosphoserine (pSer), phosphothreonine (pThr), homoaspartic acid (hAsp), homoglutamic acid (hGlu), 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid, pipecolic acid (PA), azetidine-3-carboxylic acid (ACA), 1- Examples of non-encoded amino acids include, but are not limited to, aminocyclopentane-3-carboxylic acid, allylglycine (aOly), propargylglycine (pgGly), homoalanine (hAla), norvaline (nVal), homoleucine (hLeu), homovaline (hVal), homoisoleucine (hIle), 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). Non-encoded amino acids that are included in the monoamine oxidases described herein will be apparent to those of skill in the art (see, for example, Fasman, 1989,See the various amino acids described in CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70 and references cited therein, all of which are incorporated by reference herein. These amino acids may be in the L- or D-configuration.

[0089] One of skill in the art will recognize that the monoamine oxidases disclosed herein may further comprise amino acids or residues bearing side chain protecting groups, where non-limiting examples of such protected amino acids in the aromatic category (with the protecting group listed in parentheses) include, but are not limited to, Arg(tos), Cys(methylbenzyl), Cys(nitropyridine sulfenyl), Glu(δ-benzyl ester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0090] Conformationally constrained non-coded amino acids included in the monoamine oxidase enzymes described herein 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, homoproline (hPro), and 1-aminocyclopentane-3-carboxylic acid.

[0091] As noted above, various modifications introduced into the native polypeptide to generate an engineered monoamine oxidase can be targeted to specific enzyme properties.

[0092] In another aspect, the disclosure provides a polynucleotide encoding an engineered monoamine oxidase as described herein. The polynucleotide may be operably linked to one or more heterologous regulatory sequences that control gene expression to generate a recombinant polynucleotide capable of expressing a polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered monoamine oxidase may be introduced into a suitable host cell to express the corresponding monoamine oxidase polypeptide.

[0093] The availability of a protein sequence provides a description of all polynucleotides that can code for the subject matter, since the codons corresponding to various amino acids are known. The degeneracy of the genetic code, in which identical amino acids are coded for by alternative or synonymous codons, allows for the creation of a large number of nucleic acids that code for the improved monoamine oxidases disclosed herein. Thus, after identifying a particular amino acid sequence, one skilled in the art can create a number of different nucleic acids by modifying the sequence of one or more codons without changing the amino acid sequence of the protein.

[0094] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a monoamine oxidase having an amino acid sequence having 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 to any one of the reference engineered monoamine oxidases described herein.

[0095] In various embodiments, the codons are preferably selected to be compatible with the host cell in which the protein is expressed.For example, the codons that are preferably used in bacteria are used to express genes in bacteria.The codons that are preferably used in yeast are used for expression in yeast.The codons that are preferably used in mammals are used for expression in mammalian cells.

[0096] In some embodiments, it is not necessary to replace all codons to optimize the codon usage of a monoamine oxidase, since the native sequence contains preferred codons, and it is not necessary to use preferred codons at every amino acid residue. Thus, a codon-optimized polynucleotide encoding a monoamine oxidase can contain preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions of the full-length coding region.

[0097] In other embodiments, the polynucleotides include those that encode a monoamine oxidase described herein and have about 80% or greater sequence identity, about 85% or greater sequence identity, about 90% or greater sequence identity, about 95% or greater sequence identity, about 98% or greater sequence identity, or 99% or greater sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered monoamine oxidase.

[0098] The isolated polynucleotide encoding the improved monoamine oxidase can be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the isolated polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance can be found in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F. Eds., Greene Pub. Associates, 1998, updated in 2006.

[0099] For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include the E. coli lac operon, the blue pigment actinorhodin (Streptomyces coelicolor) agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus licheniformis α-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyQ), the Bacillus amyloliquefaciens α-amylase gene (amyQ), the Bacillus Examples of such promoters include those derived from the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, the prokaryotic β-lactamase gene (VIIIa-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 "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94, and in Sambrook et al., supra.

[0100] For filamentous fungal host cells, suitable promoters for directing transcription of the nucleic acid constructs of the disclosure include those encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum f. Examples of such promoters include the promoter obtained from the gene for C. oxysporum trypsin-like protease (WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters derived from the genes for A. niger neutral α-amylase and A. oryzae triose phosphate isomerase), as well as mutant, truncated, and hybrid promoters thereof.

[0101] In yeast hosts, useful promoters can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for use in yeast host cells are described in Romanos et al., 1992, Yeast 8:423-488.

[0102] The control sequence may be a suitable transcription terminator sequence, which is a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that functions in the selected host cell can be used in the method disclosed herein.

[0103] For example, exemplary transcription terminators for use in filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0104] Exemplary terminators for use in yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for use in yeast host cells are described in Romanos et al., 1992, supra.

[0105] The control sequence may be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that functions in the host cell of choice may be used. Exemplary leader sequences for use in filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for use in yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0106] The control sequence may be a polyadenylation sequence, which is a sequence operably linked to the 3' end of a nucleic acid sequence and recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA during transcription. Any polyadenylation sequence that functions in a selected host cell can be used in the methods disclosed herein. Exemplary polyadenylation sequences for use in filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Polyadenylation sequences for use in yeast host cells are described in Guo and Sherman, 1995, Mol Cell Bio 15:5983-5990.

[0107] The control sequence may be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that 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. A foreign signal peptide coding region may be required when the coding sequence does not naturally contain a signal peptide coding region.

[0108] Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide, However, any signal peptide coding region which is capable of directing the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the methods disclosed herein.

[0109] Effective signal peptide coding regions for bacterial host cells include those obtained from the genes for Bacillus subtilis NC1B 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are described in Simonen and Palva, 1993, Microbiol Rev 57:109-137.

[0110] Useful signal peptide coding regions for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic protease, Humicola insolens cellulase, and Humicola lanuginosa lipase.

[0111] Signal peptides for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described in Romanos et al., 1992, supra.

[0112] The control sequence may be a propeptide coding region that codes for an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (also called a zymogen). A propolypeptide is generally inactive and may be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding regions can be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (WO 95 / 33836).

[0113] When both a signal peptide and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is adjacent to the amino terminus of the polypeptide and the signal peptide region is adjacent to the amino terminus of the propeptide region.

[0114] It may also be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that turn the expression of a gene on or off 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, by way of example, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include the TAKA α-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0115] Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the monoamine oxidase of the present disclosure is operably linked to the regulatory sequence.

[0116] Thus, in another embodiment, the present invention also provides a recombinant expression vector comprising a polynucleotide encoding an engineered monoamine oxidase or variant thereof and one or more expression control regions (e.g., promoter and terminator, origin of replication, etc.) depending on the type of host into which it will be introduced. The various nucleic acids and control sequences described above can be combined to produce a recombinant expression vector that contains one or more appropriate restriction sites that allow for the insertion or substitution of a nucleic acid sequence encoding a polypeptide. Alternatively, the nucleic acid sequence of the present invention can be expressed by inserting the nucleic acid sequence of the present invention or a nucleic acid construct comprising the sequence into a vector suitable for expression. To create an expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0117] The recombinant expression vector may be any vector (e.g., a plasmid or a virus) that can be conveniently subjected to recombinant DNA procedures and allows the expression of a polynucleotide sequence. The vector is generally selected according to the compatibility of the vector with the host cell into which it is to be introduced. The vector may be a linear plasmid or a closed circular plasmid.

[0118] The expression vector is a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means that ensures self-replication. Alternatively, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome in which it is integrated. Furthermore, a single vector or a single plasmid, or two or more vectors or plasmids that together comprise the total DNA that is introduced into the genome of the host cell, or a transposon, may be used.

[0119] The expression vectors of the present invention preferably contain one or more selectable markers to facilitate the selection of transformed cells. A selectable marker is a gene whose product can provide biocide or viral resistance, heavy metal resistance, prototrophy for auxotrophy, and the like. Examples of bacterial selectable markers include the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance (resistance to ampicillin, kanamycin, chloramphenicol, or tetracycline). Suitable markers for use in yeast host cells include ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0120] Selectable markers for use in filamentous fungal host cells 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 equivalents thereof. Embodiments for use in Aspergillus cells include the amdS and pyrG genes from Aspergillus nidulans or Aspergillus oryzae, and the bar gene from Streptomyces hygroscopicus.

[0121] The expression vectors of the invention preferably contain elements that allow the vector to integrate into the genome of the host cell or to autonomously replicate in the cell independent of the genome. Upon integration into the host cell genome, the vector may rely on any other elements encoding a polypeptide or vector to integrate the vector into the genome by homologous or non-homologous recombination.

[0122] Alternatively, the expression vector may contain other nucleic acid sequences to direct integration into the genome of the host cell by homologous recombination. Such other nucleic acid sequences allow the vector to integrate into the host cell genome at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integration elements preferably contain a sufficient number of nucleic acids that are highly homologous to the corresponding target sequence such that the likelihood of homologous recombination is increased, for example 100-10,000 base pairs, preferably 400-10,000 base pairs, most preferably 800-10,000 base pairs of nucleic acids. The integration elements may be any sequence that is homologous to the target sequence in the genome of the host cell. Furthermore, the integration elements may be non-coding or coding nucleic acid sequences. Alternatively, the vector may integrate into the genome of the host cell by non-homologous recombination.

[0123] In the case of self-replication, the vector may further comprise a replication origin that allows it to replicate in the target host cell. Examples of bacterial replication origins include P15A ori or plasmid pBR322, pUC19, pACYC177 (the plasmid has P15A ori), or pACYC184 that allows replication in E. coli, and pUB110, pE194, pTA1060 or pAMβ1 that allows replication in Bacillus subtilis. Examples of replication origins for yeast host cells are the 2 micron replication origin, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The replication origin may be a replication origin that has a mutation that makes its function temperature sensitive in the host cell (see, for example, Ehrlich, 1978, Proc Natl Acad Sci. USA 75:1433).

[0124] One or more copies of the nucleic acid sequences of the invention can be inserted into a host cell to increase production of the gene product. Increasing the copy number of the nucleic acid sequence can be achieved by integrating at least one additional copy of the sequence into the host cell genome, or, if the cell contains an amplified copy of a selectable marker gene, by including an amplifiable selectable marker gene in the nucleic acid sequence and selecting for the additional copies of the nucleic acid sequence by culturing the cells in the presence of an appropriate selection agent.

[0125] Many of the expression vectors for use in the methods disclosed herein are commercially available. Suitable commercially available expression vectors include p3xFLAG from Sigma-Aldrich Chemicals, St. Louis MO. TMExpression vectors include those containing a CMV promoter and hGH polyadenylation site for expression in mammalian host cells, and a pBR322 origin of replication and an ampicillin resistance marker for amplification in E. coli. Other suitable expression vectors include pBluescriptII SK(-) and pBK-CMV, available from Stratagene, LaJolla Calif., as well as plasmids derived from pBR322 (GibcoBRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

[0126] In another aspect, the invention provides a host cell comprising a polynucleotide encoding an improved monoamine oxidase of the invention, the polynucleotide being operably linked to one or more control sequences for expression of the monoamine oxidase in the host cell. Host cells for use in expressing the monoamine oxidase polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Lactobacillus kefir, Lactobacillus brevis, Lactobacillus minor, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris, ATCC Accession No. 201178); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culture media and growth conditions for the above host cells are well known in the art.

[0127] Polynucleotides for expressing monoamine oxidase can be introduced into cells by various methods well known in the art. Techniques include, but are not limited to, electroporation, gene gun particle bombardment, liposome-mediated transfection, calcium chloride transfection and protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0128] An exemplary host cell is Escherichia coli W3110. An expression vector is made by operably linking a polynucleotide encoding the improved monoamine oxidase to plasmid pCK110900, which is operably linked to a lac promoter controlled by a LadI repressor. The expression vector further comprises a P15a origin of replication and a chloramphenicol resistance gene. Cells containing the polynucleotide of interest in E. coli W3110 are isolated by chloramphenicol selection.

[0129] Engineered monoamine oxidases can be obtained by mutagenesis and / or directed evolution of polynucleotides encoding native monoamine oxidases. Exemplary directed evolution methods include the mutagenesis and / or DNA shuffling methods described in Stemmer, 1994, ProcNatl Acad Sci USA 91:10747-10751, WO 95 / 22625, WO 97 / 0078, WO 97 / 35966, WO 98 / 27230, WO 00 / 42651, WO 01 / 75767, and U.S. Patent No. 6,537,746. Other directed evolution procedures that can be used include, but are not limited to, the sessile extension process (StEP), in vitro recombination (Zhao et al., 1998, Nat. Biotechnol. 16:258-261), mutagenic 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).

[0130] The clones obtained after the mutagenesis treatment are screened to obtain engineered monoamine oxidases with the desired improved enzymatic properties. The activity of the enzyme from the expression library can be measured using standard biochemical techniques, such as, but not limited to, the methods for measuring monoamine oxidase disclosed by Zhou et al. ("A One-Step Fluorometric Method for the Continuous Measurement of Monoamine Oxidase Activity" 1997 Anal. Biochem. 253:169-74) and Szutowicz et al. ("Colorimetric Assay for Monoamine Oxidase in Tissues Using Peroxidase and 2,2'-Azino(3-ethylbenzthaizoline-6-sulfonic Acid) as Chromogen" 1984, Anal. Biochem. 138:86-94) or modified methods thereof. The comparison of enzyme activity is performed using a defined enzyme preparation, a defined assay under set conditions, one or more defined substrates as further described herein, or a method such as Zhou and Szutowicz. Generally, when comparing lysates, the same expression system and the same host cells are used to determine the measured cell number and protein amount, and to minimize the difference between the amount of enzyme produced in the host cells and the amount of enzyme present in the lysate. If the desired improved enzyme property is thermostability, the enzyme activity can be measured after exposing the enzyme preparation to a defined temperature and measuring the amount of enzyme activity remaining after heat treatment. The clone containing the polynucleotide encoding the monoamine oxidase is then isolated and sequenced to identify nucleotide sequence changes (if any) and used to express the enzyme in the host cell.

[0131] When the sequence of the engineered polypeptide is known, a polynucleotide encoding the enzyme can be prepared by standard solid-phase methods based on known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then joined (e.g., by enzymatic or chemical ligation methods, or by polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using, for example, the classical phosphoramidite method described in Beaucage et al., 1981, Tetra. Lett., 22:1859-69, or the method described in Matthes et al., 1984, EMBO J., 3:801-05, which is generally performed by automated synthesis. For the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, annealed, ligated into an appropriate vector, and cloned. Additionally, essentially any nucleic acid can be obtained from commercial sources such as The Midland Certified Reagent Company, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc. Chicago, IL, Operon Technologies Inc., Alameda, CA, and other commercial sources.

[0132] The engineered monoamine oxidase expressed in the host cells can be recovered from the cells and / or medium using 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. A suitable solution for lysis of bacteria such as E. coli and highly efficient protein extraction is available from Sigma-Aldrich, St. Louis MO, CelLytic B. TM It is commercially available under the trade name.

[0133] Chromatographic techniques for isolation of monoamine oxidase include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purification of a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to one of skill in the art.

[0134] In some embodiments, affinity techniques can be used to isolate the improved monoamine oxidase. Any antibody that specifically binds to monoamine oxidase can be used for affinity chromatography purification. For antibody generation, a host animal (e.g., but not limited to, rabbits, mice, rats, etc.) can be immunized by injection of the compound. The compound can be attached to a suitable carrier, such as BSA, by a side chain functional group or a linker attached to a side chain functional group. To increase the immune response, various adjuvants can be used depending on the host species. Adjuvants include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, complex polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0135] As known to those skilled in the art, monoamine oxidase-catalyzed oxidase reactions usually require a cofactor. The oxidation reactions catalyzed by monoamine oxidases described herein also usually require the cofactor flavin adenine dinucleotide (FAD). As used herein, the term "cofactor" refers to a non-protein compound that acts in combination with monoamine oxidase. Generally, an oxidized form of the cofactor, which may be non-covalently or covalently bound to monoamine oxidase, is added to the reaction mixture. The oxidized form of FAD is converted by molecular oxygen to the reduced form FAD-H. 2 In another embodiment, the oxidized form of FAD can be regenerated from NAD(P) to provide FAD and NAD(P)H. NAD(P) can in turn be regenerated by reducing ketones to alcohols using NAD(P)H-dependent alcohol dehydrogenase / ketoreductase.

[0136] The monoamine oxidase-catalyzed oxidation reactions described herein are generally carried out in a solvent. Suitable solvents include water, organic solvents (e.g., ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl t-butyl ether (MTBE), toluene, and similar organic solvents), and ionic liquids (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and similar organic solvents). In some embodiments, aqueous solvents are used, including water and aqueous co-solvent systems.

[0137] Exemplary aqueous co-solvent systems include water and one or more organic solvents.Generally, the organic solvent component of aqueous co-solvent systems is selected not to completely inactivate monoamine oxidase.Suitable co-solvent systems can be easily identified by using the enzyme activity assay described herein to measure the enzyme activity of a given engineered monoamine oxidase with a specific substrate of interest in a candidate solvent system.

[0138] The organic solvent component of the aqueous co-solvent system may be provided as a single liquid phase with the aqueous component, or may be provided as two liquid phases with partial or non-partial co-solvent with the aqueous component. When using an aqueous co-solvent system, it is common to select a two-phase system in which water is dispersed in the organic solvent or the organic solvent is dispersed in water. In general, when using an aqueous co-solvent system, it is desirable to select an organic solvent that can be easily separated from the aqueous phase. In general, the ratio of water to organic solvent in the co-solvent system is usually in the range of about 90:10 to about 10:90 (v / v) organic solvent to water, and may be in the range of 80:20 to 20:80 (v / v) organic solvent to water. The co-solvent system may be preformed before addition to the reaction mixture, or may be formed in situ in the reaction vessel.

[0139] The aqueous solvent (water or aqueous co-solvent system) may be pH buffered or unbuffered. Generally, the oxidation is carried out at a pH of about 10 or less, usually in the pH range of about 5 to about 10. In some embodiments, the oxidation is carried out at a pH of about 9 or less, usually in the pH range of about 5 to about 9. In some embodiments, the oxidation is carried out at a pH of about 8 or less, usually in the pH range of about 5 to about 8, and usually in the pH range of about 6 to about 8. The oxidation may be carried out 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.

[0140] During the oxidation reaction, the pH of the reaction mixture may change. The amine of general structural formula I is protonated at and near neutral pH, while the imine product of structural formula II is generally unprotonated at and near neutral pH. Thus, in the general embodiment where the reaction is carried out at or near neutral pH, the oxidation of the protonated amine to the unprotonated imine releases protons into the aqueous solution. The pH of the reaction mixture can be maintained at a desired pH or within a desired pH range by adding a base during the reaction. Alternatively, the pH can be controlled by using an aqueous solvent containing a buffer. Suitable buffers for maintaining a desired pH range are known in the art and include, for example, phosphate buffers, triethanolamine buffers, and similar buffers. A combination of buffers or base additions can also be used.

[0141] Suitable bases for neutralizing the acid include, for example, organic bases such as amines, alkoxides and similar organic bases, as well as hydroxide salts (e.g., NaOH), carbonate salts (e.g., NaHCO 3 ), bicarbonate (e.g., K 2 CO 3 ), basic phosphates (e.g., K 2 HPO 4 , Na 3 PO 4 ) and similar inorganic bases. A preferred base for neutralizing the protons released from the oxidation of the amine to the imine during the course of the reaction is the amine substrate itself. Addition of the base during the course of the conversion can be done manually while monitoring the pH of the reaction mixture, or more conveniently, an automatic titrator can be used as a pH stat. A combination of partially buffered and base addition can also be used to control the process. The base added to the unbuffered or partially buffered reaction mixture during the course of the oxidation is generally added as an aqueous solution.

[0142] In carrying out the stereoselective oxidation reactions described herein, the engineered monoamine oxidase can be added to the reaction mixture in the form of purified enzyme, whole cells transformed with a gene encoding the monoamine oxidase, and / or cell extracts and / or lysates of such cells. Whole cells transformed with a gene encoding the engineered monoamine oxidase, or cell extracts and / or lysates thereof, can be used in a variety of different forms, including solid (e.g., freeze-dried, spray-dried and similar forms) or semi-solid (e.g., crude paste).

[0143] Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment or similar methods) followed by a desalting step (e.g., ultrafiltration, dialysis and similar methods) before lyophilization. Cell preparations can be stabilized by cross-linking using known cross-linking agents such as glutaraldehyde, or fixation to a solid phase (e.g., Eupergit C and similar).

[0144] Solid reactants (e.g., enzymes, salts, etc.) can be subjected to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray-dried, and similar forms), solutions, emulsions, suspensions, and similar forms. Reactants can be readily lyophilized or spray-dried using methods and equipment known to those skilled in the art. For example, a protein solution can be frozen in small portions at -80°C and then added to a pre-cooled lyophilization chamber, which is then placed in a vacuum environment. After removing water from the sample, the temperature is typically raised to 4°C and held for 2 hours before releasing the vacuum and recovering the lyophilized sample.

[0145] The amount of reactants used in the oxidation reaction can generally vary depending on the amount of the desired product and the amount of monoamine oxidase substrate used in combination. In general, when about 50 mg / L to about 5 g / L of monoamine oxidase is used, the substrate can be used at a concentration of about 5 g / L to 50 g / L. Those skilled in the art will readily understand how to vary these amounts to adjust to the desired productivity level and production scale. The amounts of optional agents such as catalase, antifoaming agent, and sodium bisulfite or sodium metabisulfite can be appropriately determined by routine experimentation.

[0146] The order of addition of the reactants is not strictly limited. The reactants can be added together at the same time to a solvent (e.g., single-phase solvent, two-phase aqueous co-solvent system, and similar solvents), or some of the reactants can be added separately while others are added together at different times. In some embodiments, one or more reaction components can be added continuously ("fed") to the reaction to an extent that minimizes or eliminates inhibition of the monoamine oxidase substrate and / or product. In some embodiments, the monoamine oxidase can be added intermittently over the course of the reaction, for example, about every hour, about every two hours, about every three hours, or about every four hours.

[0147] Suitable conditions for carrying out the monoamine oxidase-catalyzed oxidation reactions described herein include those conditions that can be readily optimized by routine experimentation, including, for example, but not limited to, contacting an engineered monoamine oxidase with a substrate at an experimental pH and temperature, and detecting a product, such as using methods described in the Examples provided herein.

[0148] Monoamine oxidase-catalyzed oxidation is typically carried out at a temperature ranging from about 5° C. to about 75° C. In some embodiments, the reaction is carried out at a temperature ranging from about 20° C. to about 55° C. In other embodiments, the reaction is carried out at a temperature ranging from about 20° C. to about 45° C., from about 30° C. to about 45° C., or from about 40° C. to about 45° C. The reaction can also be carried out at room temperature (about 21° C.).

[0149] The oxidation reaction is generally allowed to proceed to such an extent that the substrate is essentially completely or nearly completely oxidized. The oxidation of the substrate to the product can be monitored using known methods for substrate and / or product detection. Suitable methods include gas chromatography, HPLC, and similar methods. The conversion yield is generally greater than about 50%, may be greater than about 60%, may be greater than about 70%, may be greater than about 80%, may be greater than 90%, and is usually greater than about 97%.

[0150] All publications and patent applications mentioned in this specification are herein incorporated by reference, that is, each publication or patent application is specifically and individually indicated to be incorporated by reference. [Brief description of the drawings]

[0151] [Figure 1] FIG. 1 shows the standard curve of H2O2 concentration detected by Ampliflu™ Red fluorescence method. [Diagram 2] FIG. 2 shows the results of an enzyme activity assay of MAON mutants. EXAMPLES

[0152] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting.

[0153] Example 1 Construction of a Vector for Monoamine Oxidase Mutants Monoamine oxidase MAON protein expression gene from Aspergillus niger and MAON mutant genes (SEQ ID NO: 1-17) modified and designed based on stability and catalytic activity were ligated into E. coli expression vector pET15b, NdeI+BamHI sites were inserted, and N-ter 6xHis tag was retained. After sequencing to confirm the accuracy, the recombinant vector was transformed into BL21(DE3) for protein expression.

[0154] [Table 2]

[0155] Example 2 Expression and purification of monoamine oxidase: Production in shake flasks The constructed expression vector was transformed into E. coli BL21(DE3) and induced with IPTG for expression. The bacteria were collected, lysed, and then purified with a Ni-NTA column. Specifically, the MAON enzyme recombinant expression vector was transformed into the BL21(DE3) strain. The monoclonal was collected in 10 mL of sodium ampicillin resistant LB medium (100 mg / L) and cultured overnight at 37°C and 200 rpm. It was transferred to a 2 L flask containing 1 L of LB medium and cultured at 37°C and 200 rpm until the OD600 reached 0.6-0.8, then cooled to 25°C, induced with 0.5 mM IPTG for expression overnight, and centrifuged at 5000×g to collect the cells. The collected cells were resuspended in buffer A containing 50 mM Tris, 500 mM NaCl, and 20 mM imidazole at pH 8.0, and 250 μL of PMSF and cocktail inhibitor (cocktail inhibitor) at a final concentration of 1 mM were added and mixed uniformly. After disruption with a high-pressure homogenizer, the cells were centrifuged at 4 ° C and 43,000 × g for 30 minutes, and the supernatant was collected and passed through a Ni column. Purification was performed using a Ni-NTA column, and the lysate supernatant was bound to the resin for 20 minutes, washed with buffer A containing 50 mM imidazole to remove impurities, and finally eluted with an elution buffer containing 400 mM imidazole. The purification effect of the protein was detected by SDS-PAGE. The buffer was changed to 50 mM Tris, 500 mM NaCl, and 1 mM DTT at pH 7.5, and dialysis was performed. The final sample was subjected to SDS-PAGE to detect the purified protein, and then concentrated by ultrafiltration and stored frozen at -80°C.

[0156] Example 3 Expression and purification of monoamine oxidase: fermentation production Seed activation: MAON enzyme recombinant expression vector was transformed into BL21 (DE3) strain, and monoclonal was taken up in 10mL of LB medium with ampicillin sodium resistance (100mg / L) and cultured overnight at 37℃ and 200rpm. This culture was transferred to 1L flask containing 500mL of LB medium and cultured at 37℃ and 200rpm until OD600 reached 0.8-1.0. Fermentation culture: The medium in a 10L fermenter containing 6L of TB medium was preheated to 37℃, and ampicillin sodium was added to a final concentration of 100mg / L. After inoculation, the medium was stirred while aerating to maintain dissolved oxygen at 30%. When OD600 reached 10, feeding was started. Feed 1 was an aqueous solution containing 60g / L tryptone, 120g / L yeast extract, and 4% glycerin. Feed 2 was 50% glycerin. The pH was adjusted to maintain pH 7.0 using ammonia water and phosphoric acid. When the OD600 reached 20, the culture was cooled to 25°C, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM to induce expression of monoamine oxidase, and the culture was grown for 20 hours and harvested. Cells were harvested by centrifugation at 8000g. The harvested cells were either used directly in the subsequent purification step or stored at -80°C until use. Crude enzyme purification: The harvested cells were resuspended at 200g / L wet cells in 100mM Tris, pH 8.0 and 150mM NaCl and mixed homogenously. The cells were disrupted using a high-pressure homogenizer at 800bar, and then centrifuged at 18,400g for 20 minutes at 4°C. The supernatant was collected and ammonium sulfate powder (200g / L) was added to a final concentration of 36% saturation, and the protein precipitate was collected by centrifugation. The precipitate was freeze-dried and stored at 4°C.

[0157] Example 4 Monoamine oxidase activity assay Monoamine oxidase produces hydrogen peroxide (H 2 O 2 ), so the enzyme activity parameter can be indirectly measured by measuring the amount of hydrogen peroxide produced. 2 HPO 4 100 μM Ampliflu in 5 mL of HCl buffer TMPrepare working solutions by adding Red dye and 1 U / mL horseradish peroxidase, and use them at concentrations of 0, 1.25, 2.5, 5, 10, 20, and 40. 2 O 2 The fluorescence value was measured using a microplate reader. ex =535nm / λ em The standard curve is shown in Figure 1. The MAON enzyme activity parameters were measured using a K 2 HPO 4 The MAON enzyme purified by the method described in Example 2 was used with 100 mM HCl buffer to measure its protein concentration by the BCA method, and diluted to a working solution of 50 nM. This buffer was used to prepare reaction solutions with the substrate at final concentrations of 0, 47, 94, 188, 375, 750, 1500, and 3000 μM. 95 μL of the reaction solution was taken and 5 μL of the enzyme working solution was added to make the final enzyme working concentration 2.5 nM. The change in fluorescence value was measured with a microplate reader, and λ ex =535nm / λ em = 590 nm, and the partial test results are shown in Figure 2. As can be seen from the figure, the enzyme activity of the mutant of SEQ ID NO: 7 was twice that of the control SEQ ID NO: 1, the enzyme activity of the mutant of SEQ ID NO: 6 was four times that of the control SEQ ID NO: 1, and the enzyme activity of the mutant of SEQ ID NO: 12 was five times that of the control SEQ ID NO: 1.

[0158] Example 5 Activity assay for monoamine oxidase-catalyzed oxidation of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane to (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene 40 mL of buffer solution of monoamine oxidase of SEQ ID NO: 1 to 17 expressed and purified in Example 2 (2 mg / mL, stored in potassium phosphate-hydrochloric acid buffer solution of pH 7.4, yellow liquid) was placed in a three-neck flask, 70 mg of catalase and 2 mg of antifoaming agent 204 were added, and the mixture was stirred at 25°C under oxygen environment. Furthermore, 280 mg of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane was weighed and dissolved in 5 mL of potassium phosphate-hydrochloric acid buffer solution of pH 7.4, and then dropped into the above reaction system using a syringe pump over 5 hours. During the reaction, the pH was adjusted to be maintained at 7.4 using a 3M sodium hydroxide solution. After 18 hours of reaction, in-process control was performed using LC-MS, and the conversion rate was >95%. After the reaction was completed, the mixture was extracted with methyl tert-butyl ether, and the methyl tert-butyl ether phase was spin-dried to obtain the product. The ee value of the liquid phase was >99%. 1 H NMR (300 MHz, chloroform-d) δ 7.39-7.35 (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).

[0159] Example 6: Experimental study demonstrating monoamine oxidase catalyzes the desymmetrization of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane in the presence of bisulfite 40 mL of potassium phosphate-hydrochloric acid buffer solution at pH 7.4 was placed in a three-neck flask, and 500 mg of monoamine oxidase of SEQ ID NO: 1 to 17 expressed and purified in Example 2 was added, followed by 175 mg of catalase and 2.5 mg of antifoaming agent 204, and the mixture was stirred at 25°C in an oxygen environment. Furthermore, 1.25 g of sodium hydrogen sulfite was dissolved in 8 mL of water, and 1 g of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane was added thereto. The substrate was dropped into the enzyme reaction system using a syringe pump over a period of 5 hours. During the reaction, the pH was adjusted to maintain 7.4 using a 3M sodium hydroxide solution. After 24 hours, in-process control was performed, and it was confirmed by LC-MS analysis that the conversion of the raw material was complete. The reaction system contained a mixture of (1R,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hex-2-ene and sodium (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-sulfonate. Samples were taken and measured at 1, 3, 6, 9 and 11 hours of reaction. The reaction systems of the samples of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:12 showed less residual substrate and higher activity than the reaction system of the sample of SEQ ID NO:1.

[0160] Example 7 Preparation of (1R,2S,5S)-6,6-dimethyl-3-azabicyclo[3.1.0]hexane-2-carbonitrile 40 mL of potassium phosphate-hydrochloric acid buffer solution of pH 7.4 was placed in a three-neck flask, and the monoamine oxidase of SEQ ID NO: 1 to 17 expressed and purified in Example 2 was added, followed by 175 mg of catalase and 2.5 mg of antifoaming agent 204, and the mixture was stirred at 25°C in an oxygen environment. Furthermore, 1.25 g of sodium bisulfite was weighed and dissolved in 8 mL of water, and then 1 g of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane was added. The mixture was added dropwise to the enzyme reaction system using a syringe pump over a period of 5 hours. During the reaction, the pH was adjusted to maintain 7.4 using a 3M sodium hydroxide solution. After 24 hours, in-process control was performed, and it was confirmed by LC-MS analysis that the conversion of the raw material was complete. The reaction system contained 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. After cooling the reaction system to 10°C, 40mL of MTBE was added, 1g of TMSCN was added dropwise over 30 minutes, and the reaction was allowed to proceed for 30 minutes. LC-MS analysis confirmed that the product was produced. The reaction solution of the reaction system of the sample of sequence number 6 was filtered through diatomaceous earth and then separated into an MTBE phase and an aqueous phase. The aqueous phase was extracted three times with MTBE, and then the MTBE phases were combined, dried with sodium sulfate, and spin-dried to obtain 950mg of the product with a yield of 77%.

[0161] Although the present invention has been described in many different forms of embodiments, as described in detail in connection with the preferred embodiments of the present invention, it is understood that the present disclosure should be considered as an exemplification of the principles of the present invention and is not intended to limit the present invention to the specific embodiments described and illustrated herein. Numerous modifications may be made by those skilled in the art without departing from the spirit of the present invention. The scope of the present invention is determined by the appended claims and their equivalents. The abstract and title are intended to enable the appropriate authorities and the general public to quickly determine the general nature of the invention and should not be construed as limiting the scope of the invention.

Claims

1. A monoamine oxidase comprising an amino acid sequence which, compared to the amino acid sequence represented by SEQ ID NO:1, has a mutation from phenylalanine to leucine at the 63rd amino acid, a mutation from threonine to valine at the 65th amino acid, a mutation from serine to proline at the 100th amino acid, a mutation from threonine to serine at the 141st amino acid, and / or a mutation from serine to cysteine ​​at the 234th amino acid.

2. Compared with the amino acid sequence represented by SEQ ID NO: 1, In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine to leucine at the 63rd amino acid and a mutation from threonine to valine at the 65th amino acid; A mutation from serine to proline at amino acid 100 and a mutation from serine to cysteine ​​at amino acid 234 in the amino acid sequence represented by SEQ ID NO:1; a mutation from threonine to serine at amino acid 141 and from serine to cysteine ​​at amino acid 234 in the amino acid sequence represented by SEQ ID NO:1; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, and a mutation from serine to proline at the 100th amino acid are In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, and a mutation from threonine at the 141st amino acid to serine; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, and a mutation from serine at the 234th amino acid to cysteine; In the amino acid sequence represented by SEQ ID NO:1, a serine to proline mutation at the 100th amino acid, a threonine to serine mutation at the 141st amino acid, and a serine to cysteine ​​mutation at the 234th amino acid are present; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, a mutation from serine at the 100th amino acid to proline, and a mutation from threonine at the 141st amino acid to serine; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at the 63rd amino acid to leucine, a mutation from threonine at the 65th amino acid to valine, a mutation from serine at the 100th amino acid to proline, and a mutation from serine at the 234th amino acid to cysteine; In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine at amino acid 63 to leucine, a mutation from threonine at amino acid 65 to valine, a mutation from threonine at amino acid 141 to serine, and a mutation from serine at amino acid 234 to cysteine, or In the amino acid sequence represented by SEQ ID NO:1, a mutation from phenylalanine to leucine at amino acid 63, a mutation from threonine to valine at amino acid 65, a mutation from serine to proline at amino acid 100, a mutation from threonine to serine at amino acid 141, and a mutation from serine to cysteine ​​at amino acid 234; The monoamine oxidase of claim 1, comprising an amino acid sequence having the formula:

3. The monoamine oxidase according to claim 1 or 2, wherein the amino acid sequence of the monoamine oxidase further has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence represented by SEQ ID NO:

1.

4. The monoamine oxidase according to any one of claims 1 to 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 any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 17.

5. The monoamine oxidase according to claim 4, wherein the amino acid sequence of the monoamine oxidase is represented by any one of SEQ ID NOs: 2 to 17.

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

7. 6. A method for producing a substantially stereoisomerically pure compound of formula II or a salt / hydrate thereof, comprising contacting a compound of formula I with oxygen in the presence of a monoamine oxidase enzyme and a cofactor according to any one of claims 1 to 5. 【Chemistry 1】

8. 6. A method for producing a substantially enantiomerically pure aminosulfonate compound of formula III or a salt / hydrate thereof, comprising contacting a compound of formula I with oxygen in the presence of a monoamine oxidase enzyme according to any one of claims 1 to 5, a cofactor and a bisulfite salt. 【Chemistry 2】

9. A method for producing a substantially enantiomerically pure aminonitrile compound represented by the following formula IV or a salt / hydrate thereof, comprising contacting a compound represented by the following formula I with oxygen in the presence of the monoamine oxidase according to any one of claims 1 to 5, a cofactor and a bisulfite, to obtain an aminosulfonate compound, and contacting the resulting aminosulfonate compound with cyanide. 【Chemistry 3】

10. the cofactor is non-covalently bound to monoamine oxidase; Preferably, the cofactor is selected from the group consisting of FAD, FMN, NAD and NADP; Preferably, the method further comprises a component that catalyzes the disproportionation of hydrogen peroxide to molecular oxygen and an aqueous component; More preferably, the method according to any one of claims 7 to 9, characterized in that said component is selected from the group consisting of Pd, Fe and catalase.

11. 6. The monoamine oxidase according to any one of claims 1 to 5, for use in catalysing the oxidation of a compound of formula I below to a substantially stereoisomerically pure compound of formula II below or a salt / hydrate thereof. 【Chemistry 4】

12. The monoamine oxidase according to any one of claims 1 to 5, which is used to catalyze the production of a substantially enantiomerically pure aminosulfonate compound represented by formula III below, an aminonitrile compound represented by formula IV below, or a salt / hydrate thereof, from a compound represented by formula I below. 【Chemistry 5】

13. The monoamine oxidase according to any one of claims 1 to 5, which is used to catalyse the desymmetrisation of a compound represented by formula I below. 【Chemistry 6】

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