Method for producing halogenated compounds

Abstract

The present disclosure features methods for modulating the production of small halogenated organic compounds using peroxidase enzymes, as well as related compositions and methods of use. The present disclosure features peroxidase enzymes and related compositions, as well as methods for modulating the production of small halogenated organic compounds using peroxidase enzymes. In one embodiment, the methods described herein include (i) providing a small organic compound (e.g., acetone or acetylacetone), (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) to form a reaction mixture under conditions sufficient to produce the small halogenated organic compound, and / or (iii) evaluating the amount of halogenated organic compound produced.

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Application No. 63 / 335,010, filed April 26, 2022, and U.S. Application No. 63 / 451,399, filed March 10, 2023, the entire contents of each of the foregoing applications being incorporated herein by reference. [Background technology]

[0002] There is growing concern about greenhouse gas emissions from agricultural animals. A large portion of these emissions can be attributed to biogenic enteric methane (CH4) emissions from all livestock ruminants (3.2% of total U.S. emissions, EPA, 2019). Small halogenated organic compounds such as chloroform, bromochloromethane, and 2-bromoethanesulfonate have long been known to act as inhibitors of enteric methane production (Hristov, ANet et al. J Anim Sci (2013) 91(11):5045-69). These small-molecule organic halogenated compounds can competitively inhibit the activity of methyl-coenzyme M reductase (mMCR), an enzyme found in the rumen that catalyzes the final step in CH synthesis by methanogenesis (Wood, J. Met al. Biochemistry (1968) 7(5):1707-1713; Ferry, J. G. Annu Rev Microbiol (2010) 64:3117-3126). However, due to animal and human safety and environmental concerns, the direct application of these small-molecule organic halogenated compounds as livestock feed additives is prohibited. To circumvent this problem, researchers have discovered that the halogen-rich red sea sponge Asparagopsis is a natural source of small-molecule halogenated organic compounds that are CH inhibitors (Machado L. et al. J Appl Phycol (2016) 28(5):3117-3126). While many seaweeds produce halogenated compounds, Asparagopsis is rare in that it possesses glandular cells that accumulate extremely high concentrations of methane, primarily bromoform (CHBr). A series of independent in vivo studies showed that including Asparagopsis in the diets of sheep, dairy, and beef cattle significantly reduced CH4 production by 50-80% (Li X., et al. Anim Prod Sci (2018) 58(4):681-688). Despite research efforts to mass-produce Asparagopsis, growth of this marine sponge is unlikely to match the scale of future demand for feed additives in the dairy and beef industries. Therefore, scalable alternatives to reduce methane production must be developed. [Prior art documents]

Non-Patent Literature

[0003]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Non-Patent Literature 5

Summary of the Invention

[0004] The present disclosure features peroxidase enzymes and related compositions, as well as methods for modulating the production of small halogenated organic compounds using peroxidase enzymes. In one embodiment, a method described herein includes (i) providing a small organic compound (e.g., acetone or acetylacetone); (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) to form a reaction mixture under conditions sufficient to produce the small halogenated organic compound; and / or (iii) evaluating the small amount of halogenated organic compound produced. In one embodiment, the method is characterized by (i). In one embodiment, the method is characterized by (ii). In one embodiment, the method is characterized by (iii). In one embodiment, the method is characterized by each of (i) and (ii). In one embodiment, the method is characterized by (i) and (iii). In one embodiment, the method is characterized by (ii) and (iii). In one embodiment, the method is characterized by (i) and (iii). In one embodiment, the method is characterized by (i) and (iii). In one embodiment, the method is characterized by (i) and (iii). In one embodiment, the modulating includes increasing the production of the small halogenated organic compound. In one embodiment, the method includes modulating the production of a plurality of small halogenated organic compounds. In one embodiment, the method includes, for example, providing a plurality of small molecule organic compounds for halogenation. The peroxidase can be any peroxidase known in nature, including haloperoxidases. In one embodiment, the haloperoxidase is a vanadium haloperoxidase (VHPO). In one embodiment, the VHPO is a vanadium chloroperoxidase (VCPO), a vanadium bromoperoxidase (VBPO), or a vanadium iodoperoxidase (VIPO). In one embodiment, the VHPO is a VBPO. The peroxidase can be an algal haloperoxidase (e.g., derived from an algal species) or a fungal haloperoxidase (e.g., derived from a fungal species). In one embodiment, the peroxidase is a fungal haloperoxidase (e.g., derived from a fungal species). The peroxidase may be derived from an organism selected from Curvularia inaequalis, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium erythraeum, Aphanocapsa montana, Lyngbya confervoides, Synechococcus sp. PCC7335, and Corallina officinalis. In one embodiment, the peroxidase is derived from Corallina officinalis. In one embodiment, the peroxidase is derived from Aphanocapsa montana. In one embodiment, the peroxidase is derived from Curvularia inaequalis. In one embodiment, the peroxidase comprises a peroxidase sequence described herein, e.g., a peroxidase sequence set forth in Table 2.

[0005] The peroxidase can be produced in a host cell microorganism, for example, overexpressed in the host cell microorganism. In one embodiment, the host cell microorganism is selected from Pichia pastoris, Aspergillus niger, Saccharomyces cerevisiae, or Escherichia coli. In one embodiment, the expression of the peroxidase produced in the host cell microorganism is, for example, about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 10-fold higher than the peroxidase produced in the native host. In one embodiment, the amino acid sequence of the peroxidase is selected from the amino acid sequences listed in Table 2. In one embodiment, the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from the list in Table 2. In one embodiment, the peroxidase is a sequence selected from any one of SEQ ID NOs: 1-50. In one embodiment, the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from SEQ ID NOs: 1-50. In one embodiment, the peroxidase is an amino acid sequence selected from any one of SEQ ID NOs: 1-50.

[0006] Small organic compounds can be naturally occurring or non-naturally occurring compounds. For example, small organic compounds can include natural products, lipids, sterols, steroids, amino acids, sugars, phlorotannins, tannins, lignin, or lignin derivatives. In one embodiment, small organic compounds contain functional groups, such as aldehyde, ketone, acetyl, acyl, hydroxyl, ester, ether, amine, amide, aryl, heteroaryl, heterocyclyl, or cycloalkyl groups. In one embodiment, small organic compounds contain alkenyl or alkynyl groups. In one embodiment, small organic compounds contain aldehyde or ketone groups. In one embodiment, small organic compounds contain alpha-beta unsaturated ketones. In one embodiment, small organic compounds are acetone or acetylacetone. In one embodiment, small organic compounds contain 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one embodiment, small organic compounds contain ketone or aldehyde groups.

[0007] In one embodiment, the small molecule halogenated organic compound comprises a natural product, lipid, sterol, steroid, amino acid, sugar, phlorotannin, tannin, lignin, or lignin derivative, and is chlorinated, brominated, or iodinated. In one embodiment, the small molecule halogenated organic compound is brominated. In one embodiment, the small molecule halogenated organic compound comprises a functional group, such as an aldehyde group, a ketone group, an acetyl group, an acyl group, a hydroxyl group, an ester group, an ether group, an amine group, an amide group, an aryl group, a heteroaryl group, a heterocyclyl group, or a cycloalkyl group. In one embodiment, the small molecule halogenated organic compound comprises an alkenyl group or an alkynyl group. In one embodiment, the small molecule halogenated organic compound comprises an aldehyde or ketone group. In one embodiment, the small molecule halogenated organic compound comprises an alpha-beta unsaturated ketone. In one embodiment, the small molecule halogenated organic compound comprises 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one embodiment, the small molecule halogenated organic compound contains one, two, or three halogen atoms. In one embodiment, the small molecule halogenated organic compound contains one, two, or three bromine atoms. In one embodiment, the small molecule halogenated organic compound contains an acetone moiety. In one embodiment, the small molecule halogenated organic compound includes dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone. In one embodiment, the small molecule halogenated organic compound includes 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone. In one embodiment, the small molecule halogenated compound includes dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane. In one embodiment, the small molecule halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone, dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.

[0008] In one embodiment, the small molecule organic compound is a compound of formula (Y): [ka] or a salt thereof, a tautomer thereof, or an isomer thereof, wherein R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , and R 5c is independently hydrogen, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl is selected from one or more R 6 and optionally replaced with ;R 6 is halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR A , or -NR B R C and R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C2-C6 alkenyl, and R B and R C are each independently hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl, and m and n are each independently an integer of 0 to 24; [ka] is a single or double bond, [ka] is a double bond, R 2b and R 3b Each of these does not exist independently.

[0009] In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b , and R 5c are each independently hydrogen. In one embodiment of Formula (Y), m is selected from 0, 1, 2, or 3. In one embodiment of Formula (Y), n is selected from 0, 1, 2, or 3. In one embodiment of Formula (Y), [ka] is a single bond. In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R5b , and R 5c are each independently hydrogen; m and n are each independently selected from 0, 1, 2, or 3; [ka] is a single bond.

[0010] In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 5a , R 5b , and R 5c is each independently hydrogen, and each of m and n is 0. In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b , and R 5c are each independently hydrogen, n is 0, and m is 1; [ka] is a single bond. In one embodiment of formula (Y), R 1a is C l alkyl, and R 1b , R 1c , R 5a , R 5b , and R 5c are each independently hydrogen, and each of m and n is 0. In one embodiment of Formula (Y), R 1a is a halogen (e.g., chlorine, bromine, or iodine), R 1b , R 1c , R 5a , R 5b , and R 5c are each independently hydrogen, and each of m and n is 0.

[0011] In another embodiment, the small molecule halogenated organic compound is a compound of formula (Z): [ka] or a salt thereof, a tautomer, or an isomer thereof, wherein R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , and R 5c is independently hydrogen, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl is selected from one or more R 6 and optionally replaced with ;R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R3b , R 4a , R 4b , R 5a , R 5b , and R 5c At least one of R is independently halogen; 6 is halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR A , or -NR B R C and R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C2-C6 alkenyl, and R B and R C are each independently hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl; m and n are each independently selected from 0, 1, 2, or 3; [ka] is a single or double bond, [ka] is a double bond, R 2b and R 3b Each of these does not exist independently.

[0012] In one embodiment of Formula (Z), R 1a , R 1b , and R 1c are independently halogen or hydrogen, and R 1a , R 1b , and R 1c At least one of R is a halogen. In one embodiment, the halogen is selected from chlorine, bromine, or iodine. In one embodiment of Formula (Z), R 1a , R 1b , and R 1c are independently halogen or hydrogen, and R 1a , R 1b , and R 1cAt least two of are halogen. In one embodiment, the halogens are selected from two of chlorine, bromine, or iodine. In one embodiment of Formula (Z), R 1a , R 1b , R 1c is independently a halogen. In one embodiment, the halogen is selected from chlorine, bromine, or iodine. In one embodiment of Formula (Z), R 5a , R 5b , and R 5c are independently halogen or hydrogen, and R 5a , R 5b , and R 5c At least one of R is halogen. 5a , R 5b , and R 5c are independently halogen or hydrogen, and R 5a , R 5b , and R 5c At least two of R are halogen. 5a , R 5b , R 5c Each of is independently a halogen. In some embodiments of Formula (Z), [ka] In some embodiments of Formula (Z), each of m and n is independently selected from 0, 1, 2, or 3; [ka] is a single bond.

[0013] In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound include one or more of: (a) a temperature of 10°C to 85°C, (b) a pH of 4 to 10, and (c) an ionic strength of 0.1 mM to 4 M. In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound include a temperature of 10°C to 85°C. In one embodiment, the small molecule halogenated organic compound includes a pH of 4 to 10. In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound include an ionic strength of 0.1 mM to 4 M. In one embodiment, evaluating includes analyzing the small molecule halogenated organic compound by an analytical technique. In one embodiment, the analytical technique includes HPLC, GC-MS, or NMR.

[0014] Small molecule halogenated organic compounds may be useful in several agricultural, marine, and / or industrial processes. In one embodiment, the small molecule halogenated organic compounds can reduce methane production by microorganisms. In one embodiment, methane production is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In one embodiment, methane production is reduced by 10-75%. In one embodiment, the microorganism is present within the rumen community.

[0015] In another aspect, the disclosure provides a method for reducing light hydrocarbon (e.g., methane) production in a rumen community, comprising: (i) providing a small molecule organic compound (e.g., acetylacetone); (ii) contacting the small molecule organic compound with a peroxidase (e.g., VHPO) to form a reaction mixture under conditions sufficient to produce a small molecule halogenated organic compound; (iii) separating the small molecule halogenated organic compound from the reaction mixture; and / or (iv) providing the small molecule halogenated organic compound to the rumen community under conditions sufficient to reduce light hydrocarbon (e.g., methane) production. In one embodiment, the method is characterized by (i). In one embodiment, the method is characterized by (ii). In one embodiment, the method is characterized by (iii). In one embodiment, the method is characterized by (iv). In one embodiment, the method is characterized by (i) and (ii). In one embodiment, the method is characterized by (i) and (iii). In one embodiment, the method is characterized by (i) and (iv). In one embodiment, the method is characterized by (ii) and (iii). In one embodiment, the method is characterized by (ii) and (iv). In one embodiment, the method is characterized by (iii) and (iv). In one embodiment, the method is characterized by each of (i) through (iv).

[0016] In one embodiment, the method comprises (iii) separating the small molecule halogenated organic compounds from the reaction mixture, and (iv-a) incorporating the separated small molecule halogenated organic compounds into a matrix for delivery to the rumen community under conditions sufficient to reduce light hydrocarbon production (e.g., methane). In one embodiment, the method comprises (iv-a) incorporating the separated small molecule halogenated organic compounds into a matrix for delivery to the rumen community under conditions sufficient to reduce light hydrocarbon production (e.g., methane). In one embodiment, the method comprises (i), (ii), (iii), and (iv-a). In one embodiment, the method comprises (ii) and (iv-a). In one embodiment, the method comprises each of (i), (ii), and (iv-a). In one embodiment, the method comprises (iii) and (iv-a). In one embodiment, the method comprises (i), (iii), and (iv-a). In one embodiment, the method comprises (ii), (iii), and (iv-a). In one embodiment, the method comprises (ii), (iii), and (iv-a).

[0017] In one embodiment, the method further comprises obtaining a value for the level of light hydrocarbons (e.g., methane) (a) before providing the peroxidase or (b) after providing the peroxidase. In one embodiment, the method further comprises (a). In one embodiment, the method further comprises (b). In one embodiment, the method further comprises (a) and (b). In one embodiment, methane production is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In one embodiment, methane production is reduced by 10-75%.

[0018] The peroxidase can be any peroxidase known in nature, including haloperoxidases. In one embodiment, the haloperoxidase is a vanadium haloperoxidase (VHPO). In one embodiment, the VHPO is a vanadium chloroperoxidase (VCPO), a vanadium bromoperoxidase (VBPO), or a vanadium iodoperoxidase (VIPO). In one embodiment, the VHPO is a VBPO. The peroxidase can be an algal haloperoxidase (e.g., derived from an algal species) or a fungal haloperoxidase (e.g., derived from a fungal species). In one embodiment, the peroxidase is a fungal haloperoxidase (e.g., derived from a fungal species). The peroxidase may be derived from an organism selected from Curvularia inaequalis, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium erythraeum, Aphanocapsa montana, Lyngbya confervoides, Synechococcus sp. PCC7335, and Corallina officinalis. In one embodiment, the peroxidase is derived from Corallina officinalis. In one embodiment, the peroxidase is derived from Aphanocapsa montana. In one embodiment, the peroxidase is derived from Curvularia inaequalis.

[0019] The peroxidase can be produced in a host cell microorganism, e.g., overexpressed in the host cell microorganism. In one embodiment, the host cell microorganism is selected from Pichia pastoris, Aspergillus niger, or Escherichia coli. In one embodiment, expression of the peroxidase produced in the host cell microorganism is, for example, about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 10-fold higher than that of the peroxidase produced in the native host. In one embodiment, the amino acid sequence of the peroxidase is selected from the amino acid sequences listed in Table 2. In one embodiment, the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from the list in Table 2. In one embodiment, the peroxidase is a sequence selected from any one of SEQ ID NOs: 1-50. In one embodiment, the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from SEQ ID NOs: 1-50. In one embodiment, the amino acid sequence of the peroxidase has 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to an amino acid sequence selected from any one of SEQ ID NOs: 1-50.

[0020] In one embodiment, the method includes (i) providing a small organic compound comprising an alpha-beta unsaturated ketone (e.g., acetylacetone); and (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) to form a reaction mixture under conditions sufficient to produce a small halogenated organic compound. In one embodiment, the method further includes, for step (ii), providing a halogen source and a peroxide source. In one embodiment, the halogen source provides a halogen anion (e.g., F, Cl, Br, and / or I). In one embodiment, the peroxide source is hydrogen peroxide. In one embodiment, the method further includes, for step (ii), providing a halogen source and a peroxide source in the presence of an amine, for example, to produce a chlorinated amine, such as NH2Cl. In one embodiment, providing the halogen source includes providing a chlorinated amine (e.g., NH2Cl).

[0021] In one embodiment, for step (ii), the method further comprises providing a peroxide source and a halogen source (e.g., a bromide source, a chloride source, or an iodine source) to the peroxidase, e.g., in the presence of an amine, to produce a halogenated amine, e.g., NH2Cl. In one embodiment, for step (ii), the method further comprises reacting the halogenated amine with an additional halogen source to further halogenate the small molecule halogenated organic compound. In one embodiment, the additional halogen source is bromide anion, perchlorate anion, or iodate anion. In one embodiment, reacting the halogenated amine with the additional halogen source is carried out in a separate vessel from step (i) or (ii).

[0022] In one embodiment, the method includes (ii) providing a peroxide source and a chloride source to a peroxidase (e.g., VCPO) in the presence of an amine to produce a chlorinated amine, e.g., NH2Cl, and (iii) reacting the chlorinated amine with two equivalents of iodate anion (I -) to produce a mixture of hypoiodite anion and iodine (I2). In one embodiment, (ii) occurs in a separate reaction vessel from (iii). In one embodiment, the method further includes (iv) contacting the mixture of hypoiodite anion and iodine from (iii) with, for example, a small molecule halogenated compound produced in (i) to produce an additional small molecule halogenated organic compound (e.g., a further halogenated small molecule halogenated organic compound, such as dichloroiodomethane or dibromoiodomethane). In one embodiment, the method is characterized by (i). In one embodiment, the method is characterized by (ii) and (iii). In one embodiment, the method is characterized by (i), (ii), (iii), and (iv). In one embodiment, the method is characterized by (ii) and (iv). In one embodiment, the method is characterized by (ii), (iii), and (iv). In all embodiments, (ii) can be replaced with (iib).

[0023] In one embodiment, the yield from step (i) is about 50% to 99%. In one embodiment, the yield from step (i) is about 75% to 99%. In one embodiment, the yield from step (i) is about 85% to 99%. In one embodiment, the yield from steps (i), (ii), (iii), and (iv) is about 10% to 99%. In one embodiment, the yield from steps (i), (ii), (iii), and (iv) is about 10% to 75%. In one embodiment, the yield from steps (i), (ii), (iii), and (iv) is about 10% to 50%. In one embodiment, the yield from steps (i), (ii), (iii), and (iv) is about 25% to 99%. In one embodiment, the method is performed at a pH of about 5.0 to 9.0. In one embodiment, steps (ii) and / or (iii) are carried out at a pH of 7.0-8.0, e.g., pH 7.2-7.8, pH 7.5-8.0. Without wishing to be bound by theory, maintaining the pH of steps (ii) and / or (iii) at about 7.0-8.0 may be beneficial in preventing the formation of I3 - or iodate anion IO3 - This may help reduce side reactions that produce

[0024] In one embodiment, the method involves the enzymatic reaction of an organic peroxide, such as peracetic acid (PAA), with a peroxidase (e.g., VCPO) to produce diatomic iodine, I2, and triiodate, I3, from hypochlorite anions and excess iodide anions. - In one embodiment, a method comprising the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) converts hypochlorite anions and excess iodate anions into diatomic iodine, I2, and triiodate anions, I3. - The method is characterized by a 10% to 99% reduction in the conversion of hypochlorite anions and bromide anions to diatomic bromine, Br, compared to a method comprising the enzymatic reaction of HO and VHPO at pH 7. In one embodiment, the method is characterized by a 10% to 99% reduction in the conversion of hypochlorite anions and bromide anions to diatomic bromine, Br, compared to a method comprising the enzymatic reaction of HO and VHPO at pH 0 to 5.

[0025] In one embodiment, a method comprising an enzymatic reaction of an organic peroxide (e.g., PAA) and a peroxidase (e.g., VCPO) is characterized by a 10% to 99% reduction in the conversion of hypochlorite anions and chloride anions to diatomic chlorine, Cl, compared to a method comprising an enzymatic reaction of HO with VHPO at a pH of 0 to 5. In one embodiment, a method comprising an enzymatic reaction of an organic peroxide, e.g., PAA, and a peroxidase (e.g., VCPO) is characterized by a 10% to 99% reduction in the conversion of peracetic acid and bromide anions to hypobromite anions or their conjugate acids compared to a method comprising an enzymatic reaction of HO with VHPO at a pH of 0 to 5. In one embodiment, for example, a method comprising the enzymatic reaction of PAA and peroxidase (e.g., VCPO) is characterized by a 10% to 99% reduction in the conversion rate of peracetic acid and iodate anion to hypoiodite anion or its conjugate acid at pH 0 to 5 compared to a method comprising the enzymatic reaction of a peroxide, e.g., PAA, and VCPO at pH 0 to 5.

[0026] In one embodiment, a method comprising a peroxide, e.g., PAA, and a peroxidase (e.g., VCPO) (e.g., step (ii)) may provide a reduction in the production of hydroxybenzoates compared to a method comprising the enzymatic reaction of HO and VCPO. cat In one embodiment, a method comprising the enzymatic reaction of a peroxide, e.g., PAA, with a peroxidase (e.g., VCPO) is characterized by a 10% to 500% increase in the conversion of peracetic acid and bromide anions to hypobromite anions or their conjugate acids compared to a method comprising the enzymatic reaction of HO with VCPO. cat It is characterized by an increase of 25% to 250%.

[0027] In one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) exhibits a kJ / kcal reduction compared to a method involving the enzymatic reaction of H2O2 with VCPO. cat In one embodiment, the process involving the enzymatic reaction of organic peroxide, e.g., PAA, with VCPO has a 50% to 150% increase in k compared to the process involving the enzymatic reaction of H2O2 with VCPO. cat In one embodiment, the method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) exhibits a 75% to 125% increase in kJ / kcal compared to the method involving the enzymatic reaction of H2O2 with VCPO. cat In one embodiment, the process involving the enzymatic reaction of an organic peroxide, such as PAA, with VCPO has a k cat It is characterized by an increase of 90% to 110%.

[0028] In one embodiment, a method comprising an enzymatic reaction of an organic peroxide, e.g., PAA, with a peroxidase (e.g., VCPO) (e.g., step (ii)) has a reduced k cat In one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) produces a 10% to 500% increase in k compared to a method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. catIn one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) produces a 25% to 250% increase in k compared to a method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) produces a 50% to 150% increase in k compared to a method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, the process involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) results in a 75% to 125% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, the process involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) results in an 80% to 120% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) results in a 90% to 110% increase in k compared to a method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, the method involving the enzymatic reaction of organic peroxide, such as PAA, with peroxidase (e.g., VCPO) reduces k by 10% to 99% compared to the method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) produces a 25% to 250% increase in k compared to a method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. catIn one embodiment, a method involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) produces a 50% to 150% increase in k compared to a method involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, the process involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) results in a 75% to 125% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat In one embodiment, the process involving the enzymatic reaction of an organic peroxide, such as PAA, with a peroxidase (e.g., VCPO) results in an 80% to 120% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO to produce dichloroiodomethane. cat It is characterized by an increase of 90% to 110%.

[0029] The small molecule organic compound can be a naturally occurring or non-naturally occurring compound. In one embodiment, the small molecule organic compound contains 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one embodiment, the small molecule organic compound contains a ketone group or an aldehyde. In one embodiment, the small molecule halogenated organic compound is a chloride, bromide, or iodide. In one embodiment, the small molecule halogenated organic compound is brominated. In one embodiment, the small molecule halogenated organic compound contains 1, 2, or 3 halogen atoms. In one embodiment, the small molecule halogenated organic compound contains 1, 2, or 3 bromine atoms. In one embodiment, the small molecule halogenated organic compound contains an acetone moiety. In one embodiment, the small molecule halogenated organic compound includes dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone. In one embodiment, the small molecule halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone.

[0030] In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound include one or more of: (a) a temperature of 10°C to 85°C, (b) a pH of 4 to 10, and (c) an ionic strength of 0.1 mM to 4 M. In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound include a temperature of 10°C to 85°C. In one embodiment, the small molecule halogenated organic compound includes a pH of 4 to 10. In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound include an ionic strength of 0.1 mM to 4 M. In one embodiment, evaluating includes analyzing the small molecule halogenated organic compound by an analytical technique. In one embodiment, the analytical technique includes HPLC, GC-MS, or NMR.

[0031] Small molecule halogenated organic compounds may be useful in several agricultural, marine, and / or industrial processes. In one embodiment, the small molecule halogenated organic compounds can reduce methane production by microorganisms. In one embodiment, methane production is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In one embodiment, methane production is reduced by 10-75%. In one embodiment, the microorganism is present within the rumen community.

[0032] In another aspect, a method features a method for increasing expression of a peroxidase in a host cell or host microorganism.

[0033] In another aspect, a method features a reactor for continuously producing small molecule halogenated organic compounds, the reactor including: (i) a reaction chamber; (ii) a module for temperature control; (iii) a peristaltic pump; and / or (iv) a module for containing a catalyst.

[0034] Descriptions of further embodiments of the present disclosure are provided herein in the drawings, detailed description, examples, and claims. [Brief explanation of the drawings]

[0035] [Figure 1] Examples of chemical reactions catalyzed by vanadium haloperoxidases. [Figure 2] Figures A to C show the enzyme reaction mechanisms catalyzed by vanadium haloperoxidase. Equation A describes the general ping-pong-Bibi reaction mechanism. Equation B is the Cleland equation for the ping-pong-Bibi reaction with substrate inhibition. Equation C describes the reaction parameters of vanadium haloperoxidase formulated via the King-Altman method. [Figure 3A] Figure 1 shows the effect of pH and ionic strength on exemplary CoVBPO kinetics. Figure 2 shows the pH dependence of KmBr at high (2.0 M) and low (0.2 M) concentrations of MgSO. Further details on reaction rates are provided in Tables 3 and 4. [Figure 3B] Figure 1 shows the effect of pH and ionic strength on exemplary CoVBPO kinetics. For each ionic strength solution, the relationship between kcat and deuterium isotope fraction is shown. [Figure 3C] The effect of pH and ionic strength on exemplary CoVBPO kinetics is shown. Solvent isotope effects (SIE) for kcat and KmBr are shown. This information was used to interpret the reaction mechanism, resulting in a 12-fold decrease in KmBr (KmBr = 0.8 mM). [Figure 3D] Not specified. [Figure 3E] Not specified. [Figure 4A] 1 shows the CiVCPO-driven production of small molecule halogenated organic compounds from the exemplary small molecule organic compound acetylacetone. 2 shows the concentration of reaction products over time in an exemplary CiVCPO reaction. [Figure 4B] Figure 1 shows the CiVCPO-driven production of small halogenated organic compounds from the exemplary small organic compound acetylacetone. Figure 2 shows a chromatogram derived from an HPLC of the reaction products from the CiVCPO reaction, illustrating how 1,1,dibromoacetone was fractionated for use in methane suppression experiments. [Figure 4C]This figure shows the CiVCPO-driven production of small halogenated organic compounds from the exemplary small organic compound acetylacetone. This figure shows a GC-MS chromatogram showing the components of an n-hexane extract of HPLC fraction 1,1-dibromoacetone. The identity of the major product was confirmed by comparing the MS spectrum of the product with a 1,1-dibromoacetone standard. [Figure 5] FIG. 1 is an illustration of an exemplary workflow used to generate and test the methane suppression potential of small molecule halogenated organic compounds. [Figure 6A] 1 is a graph showing the effect of small halogenated compounds on methane production by rumen-derived microbial cultures, showing the rate of methane production when 1,1-dibromoacetone, bromoform, and dibromomethane were added. [Figure 6B] 1 is a graph showing the effect of small halogenated compounds on methane production by rumen-derived microbial cultures, showing the rate of methane production when 1,1-dibromoacetone, bromoform, and dibromomethane were added. [Figure 6C] 1 is a graph showing the effect of small halogenated compounds on methane production by rumen-derived microbial cultures, showing the rate of methane production when 1,1-dibromoacetone, bromoform, and dibromomethane were added. [Figure 7-1] 1 is a sequence alignment of several exemplary VHPOs. [Figure 7-2] 1 is a sequence alignment of several exemplary VHPOs. [Figure 7-3] 1 is a sequence alignment of several exemplary VHPOs. [Figure 8] 10 is an SDS-PAGE polyacrylamide gel after purification of cchVBPO from heterologous production in Escherichia coli. [Figure 9] 10 is an SDS-PAGE polyacrylamide gel after purification of synVBPO from heterologous production in Escherichia coli. [Figure 10A]Figure 1 shows the reaction rate of synVBPO and fitting of the kinetic parameters to Michaelis-Menten and Hill-type kinetics. Figure 2 shows a surface plot of the halogenation activity of synVBPO showing kcat as a function of H2O2 and KBr concentrations. [Figure 10B] The reaction rate of synVBPO and fitting of kinetic parameters to Michaelis-Menten and Hill-type kinetics are shown. Fitting of kinetic parameters to the halogenation reaction rate using Michaelis-Menten and Hill-type kinetics is also illustrated. Akaike Information Criterion Test (AIC), Bayesian Information Criterion Test (BIC), and related statistics are also shown to determine the best fit of the candidate model. [Figure 11A] 1 is a Salwin test showing the enzyme stability of synVBPO at various KBr and H2O2 concentrations. FIG. 2 is a Salwin test of synVBPO at 160 mM KBr and 50 μM H2O2. [Figure 11B] 1 is a Salwin test showing the enzyme stability of synVBPO at various KBr and H2O2 concentrations. FIG. 2 is a Salwin test of synVBPO at 40 mM KBr and 50 μM H2O2. [Figure 11C] 1 is a Salwin test showing the enzyme stability of synVBPO at various KBr and H2O2 concentrations. FIG. 2 is a Salwin test of synVBPO at 1 mM KBr and 50 μM H2O2. [Figure 11D] 1 is a Salwin test showing the enzyme stability of synVBPO at various KBr and H2O2 concentrations. FIG. 2 is a Salwin test of synVBPO at 1 mM KBr and 250 μM H2O2. [Figure 12A] Figure 1 shows the enzymatic activity of synVBPO as a function of ionic strength, and the determination of Michaelis-Menten kinetic parameters as a function of pH. Figure 2 shows a plot of the change in fluorescence per second as a function of ionic strength for the reaction rate of VBPO in the presence of peroxide and halide substrates. [Figure 12B]Determination of enzymatic activity of synVBPO as a function of ionic strength, and Michaelis-Menten kinetic parameters as a function of pH. Plot of synVBPOkcat as a function of pH. [Figure 12C] Determination of enzymatic activity of synVBPO as a function of ionic strength and Michaelis-Menten kinetic parameters as a function of pH. Plot of synVBPOkcat / KmH2O2 as a function of pH. [Figure 12D] Determination of enzymatic activity of synVBPO as a function of ionic strength and Michaelis-Menten kinetic parameters as a function of pH. Plot of synVBPOkcat / KmKBr as a function of pH. [Figure 13] Plot of the VCPO reaction rate in the presence of the peroxide sources H2O2 and peracetic acid (PAA), and calculation of the Michaelis-Menten kinetic parameters demonstrating that Vmax, PAA, is approximately twice that of Vmax, H2O2. [Figure 14] Figure 1 shows the reaction rate of VCPO using a Michaelis-Menten curve fit equation with PAA and H2O2 as substrates, demonstrating that PAA has both a higher vmax and Km than hydrogen peroxide. [Figure 15] 1 is a graph of DCIM concentration produced versus time in a two-step fed-batch system. [Figure 16] FIG. 1 is an illustration of the dissolved oxygen cascade in the glycerol batch phase. [Figure 17] 1 is an SDS-PAGE polyacrylamide gel showing the results of the purification protocol outlined in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0036] This disclosure features peroxidases (e.g., haloperoxidases) and related compositions for use in producing a small molecule halogenated organic compound or compounds. These small molecule halogenated organic compounds can be useful for reducing methane production in microbial or ruminal communities. Additionally, this disclosure features methods for producing peroxidases in a robust and efficient manner, as well as reactors and other devices for containing and monitoring peroxidase activity.

[0037] definition 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 to which this invention belongs.

[0038] The articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0039] "About," when used herein to modify a numerically defined parameter, means that the parameter may vary 15% above or below the stated numerical value for that parameter. For example, a small organic compound defined as having a molecular weight of 100 Da may have a molecular weight of 85 Da to 115 Da. In some embodiments, the term "about" may mean that the parameter may vary by as much as 10% above or below the stated numerical value for that parameter.

[0040] "Obtain" or "obtaining," as used herein, refers to obtaining a property of a value, e.g., a numerical value, or an image, or a physical entity (e.g., a sample), by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. "Indirectly obtaining" refers to receiving a value or physical entity from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process or using a machine or device that involves a physical change of a physical substance. An example of directly obtaining a value includes obtaining a sample from a human subject. Directly obtaining a value includes performing a process that obtains molecular weight information using a machine or device, e.g., a mass spectrometer.

[0041] The term "halogenating agent," as used herein, refers to an agent (e.g., a small molecule or protein) capable of modifying an entity with a halogen, e.g., a fluorine, chlorine, bromine, or iodine atom. In one embodiment, the halogenating agent is a small molecule or salt, such as potassium bromide. In another embodiment, the halogenating agent is a protein, such as a halogenase or haloperoxidase (e.g., vanadium haloperoxidase).

[0042] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof. "Multiple polypeptides" refers to two or more polypeptides, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, or 500 or more polypeptides.

[0043] The term "peroxidase" as used herein refers to an enzyme that reduces peroxide-containing or hydroperoxidase-containing substrates.Many peroxidases contain cofactors, such as heme cofactors, orthovanadate, or redox-active side chains, such as cysteine ​​or selenocysteine.Exemplary peroxidases include haloperoxidase, ascorbate peroxidase, lactoperoxidase, thyroid peroxidase, etc.

[0044] The term "rumen" refers to a special intestinal compartment found within certain animals, such as ruminants, within which several digestive functions take place, such as the fermentation process.

[0045] The term "ruminant" refers to an animal that has a specialized intestinal compartment in which several digestive functions, such as fermentation processes, take place.

[0046] The terms "rumen community" or "rumen microbial community" refer to the population of microorganisms, including bacteria, archaea, and protozoa, that colonize the digestive tract of large animals, e.g., ruminants. The rumen microbial community performs several digestive functions within the animal, including assisting digestion to provide important nutrients to the host animal. Exemplary organisms that make up the microbial community include methanogenic archaea, such as Methanobrevibacter, Methanosarcina, and Methanocopalus. Exemplary fermenting bacterial genera found in the rumen include Colibacteraceae, Fibrobacter, Ruminococcus, Butylvibrio, Streptococcus, Prevotella, Succinimonas, Selenomonas, Lachanospira, and Succinivibrio.

[0047] Selected Chemical Definitions Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are defined in the Handbook of Chemistry and Physics, 75 th The general principles of organic chemistry, as well as specific functional sites and reactivities, are defined in accordance with Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0048] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0049] When a range of values ​​is listed, it is intended to encompass each value and sub-range within the range. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0050] As used herein, "alkyl" refers to the radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group is a group having 1 to 12 carbon atoms ("C-C 12 alkyl), 1 to 10 carbon atoms ("C1-C 12In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C-C alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C-C alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Each example of an alkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents; for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0051] As used herein, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C2-C 24 In some embodiments, an alkenyl group refers to an alkyl group having 2 to 12 carbon atoms ("C-C"). 12 alkenyl), 2 to 10 carbon atoms ("C2-C 10C2-C4 alkenyl groups have 2 to 8 carbon atoms ("C2-C8 alkenyl"), 2 to 6 carbon atoms ("C2-C6 alkenyl"), 2 to 5 carbon atoms ("C2-C5 alkenyl"), 2 to 4 carbon atoms ("C2-C4 alkenyl"), 2 to 3 carbon atoms ("C2-C3 alkenyl"), or 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each example of an alkenyl group may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkenyl").

[0052] As used herein, the term "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C2-C 24 In some embodiments, an alkynyl group refers to an alkyl group having 2 to 12 carbon atoms ("C-C"). 10 alkynyl), 2 to 10 carbon atoms ("C2-C 10C-C alkynyl groups have 2 to 8 carbon atoms ("C-C alkynyl"), 2 to 6 carbon atoms ("C-C alkynyl"), 2 to 5 carbon atoms ("C-C alkynyl"), 2 to 4 carbon atoms ("C-C alkynyl"), or 2 to 3 carbon atoms ("C-C alkynyl"), or 2 carbon atoms ("C alkynyl"). The one or more carbon-carbon triple bonds can be internal (e.g., as in 2-butynyl) or terminal (e.g., as in 1-butynyl). Examples of C-C alkynyl groups include ethynyl (C), 1-propynyl (C), 2-propynyl (C), 1-butynyl (C), 2-butynyl (C), and the like. Each instance of an alkynyl group independently may be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents; for example, with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkynyl").

[0053] As used herein, the term "heteroalkyl" refers to an acyclic, stable, straight or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. The heteroatom(s) O, N, P, S, and Si can be located at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. A "heteroalkyl" is recited followed by a particular heteroalkyl group, e.g., -CHO, -NR C RD When the term heteroalkyl is used, the term -CH2O or -NR C R D It is understood that the terms "heteroalkyl" and "heteroalkyl-" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" refers to specific heteroalkyl groups, e.g., -CHO, -NR C R D Nothing herein should be construed as excluding, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. Each example of a heteroalkyl group independently may be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents; for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted heteroalkyl").

[0054] As used herein, "cycloalkyl" refers to a group having 3 to 10 ring carbon atoms in a non-aromatic ring system ("C3-C 10 "cycloalkyl" refers to a radical of a non-aromatic ring system that has no heteroatoms. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C-C cycloalkyl"), 3 to 6 ring carbon atoms ("C-C cycloalkyl"), or 5 to 10 ring carbon atoms ("C-C 10Cycloalkyl groups may have the following meanings: "cycloalkyl" (e.g., C4-C7 membered cycloalkyl), where the term "membered" refers to a non-hydrogen ring atom within the moiety. Exemplary C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, but are not limited to, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. Exemplary C3-C 10 Cycloalkyl groups include, but are not limited to, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), and the like. As the foregoing examples illustrate, in certain embodiments, a cycloalkyl group is monocyclic ("monocyclic cycloalkyl") or contains fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic cycloalkyl"), and can be saturated or partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring, as defined above, is fused to one or more aryl groups, and the point of attachment is on the cycloalkyl ring; in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group independently can be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl").

[0055] "Heterocyclyl," as used herein, is the radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic heterocyclyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring, as defined above, is fused to one or more cycloalkyl groups, with the point of attachment being on either the cycloalkyl or heterocyclyl ring, or in which a heterocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, with the point of attachment being on the heterocyclyl ring; in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. Heterocyclyl groups may be described, for example, as 3- to 7-membered heterocyclyls, where the term "members" refers to the non-hydrogen ring atoms in the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0056] As used herein, "halo" or "halogen," independently or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0057] As used herein, "hydroxy" refers to the radical --OH.

[0058] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl groups, as defined herein, are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" cycloalkyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl groups). Typically, the term "substituted," whether preceded by the term "optionally," means that at least one hydrogen present on the group (e.g., a carbon or nitrogen atom) is replaced with an acceptable substituent, e.g., a substituent that results in a stable compound (e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reaction).

[0059] Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group; when multiple positions in any given structure are substituted, the substituents are either the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, including any of the substituents described herein, that result in the formation of stable compounds. The present disclosure contemplates all such combinations to arrive at stable compounds. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0060] Haloperoxidase Vanadium haloperoxidases (VHPOs) (EC 1.11.1.18) are members of the oxidoreductase enzyme subfamily, which contain vanadate as a prosthetic group. VHPOs catalyze the two-electron oxidation of halide ions to the corresponding hypohalous acids in a substrate-inhibited bi-biping-pong mechanism, often without substrate specificity or regioselectivity. VHPOs are commonly found in brown, red, and green marine algae and are classified according to the most electronegative halide they oxidize. For example, vanadium chloroperoxidase (VCPO) oxidizes chloride, bromide, and iodide; vanadium bromoperoxidase (VBPO) oxidizes bromide and iodide; and vanadium iodoperoxidase (VIPO) oxidizes iodide. VHPO is capable of halogenating a wide range of organic compounds of both commercial and pharmaceutical interest and exhibits stability over a wide range of conditions, including high temperatures, acidic and basic pH, oxidizing conditions, and organic solvents. These characteristics make VHPO an attractive candidate for use in a variety of industrial transformations.

[0061] Among the most studied VHPOs is the VBPO class found in the Corallina genus of marine sponges. Corallina officinalis is a calcareous red sponge commonly found in tidepools and marine habitats. VBPO from Corallina officinalis (CoVBPO) has been successfully isolated from the sponge and studied both biochemically and structurally. CoVBPO is characterized by a large dodecamer (12 × 64 kDa) structure containing 12 identical subunits. CoVBPO has been shown to exhibit functional stability over a pH range of 5–10 and at temperatures up to 90°C. Furthermore, it is tolerant to organic solvents such as ethanol, methanol, and propan-1-ol, making it an attractive enzyme for commercial biocatalysis. However, it has been difficult to directly isolate adequate quantities of CoVBPO from Corallina officinalis for industrial applications.

[0062] Industrial enzymes are enzymes used commercially in various industries, including pharmaceuticals, chemical production, biofuels, food and beverages, and consumer products. Advances in heterologous expression in hosts such as Escherichia coli and various fungi have made biocatalysis with isolated enzymes more economical than using whole cells. However, heterologous expression of foreign proteins in Escherichia coli often produces insoluble inclusion bodies due to protein misfolding. In particular, heterologous expression of large amounts of pure CoVBPO from Escherichia coli has proven difficult due to its tendency to form large, insoluble inclusion bodies. Therefore, we searched public databases for analogs of CoVBPO that may exhibit improved properties.

[0063] In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (I): TGPX1, wherein X1 is proline or threonine. In one embodiment, the amino acid sequence of formula (I) is selected from TGPP and TGPT. In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (Ia): GPX2TGPX1, wherein X1 is proline or threonine and X2 is glutamine or leucine. In one embodiment, the amino acid sequence of formula (Ia) is selected from GPPTGPQ, GPTTGPQ, GPPTGPL, and GPTTGPL. In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (Ia): GPQTGPX1 or GPLTGPX1, wherein X1 is proline or threonine.

[0064] In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (II): X3WKE, where X3 is serine or alanine. In one embodiment, the amino acid sequence of formula (II) is selected from SWKE and AWKE. In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (II-a): GX4FX3WKE, where X3 is serine or alanine and X4 is isoleucine or valine. In one embodiment, the amino acid sequence of formula (II-a) is selected from GIFSWKE, GIFAWKE, GVFSWKE, and GVFAWKE. In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (II-b): GIX5X3WX6, where X3 is serine or alanine, X5 is phenylalanine or leucine, and X6 is lysine, glutamic acid, or glutamine. In one embodiment, the amino acid sequence of formula (II-b) is selected from GIFSWK, GIFSWE, GIFSWQ, GFAWK, GIFAWE, GFAWQ, GILSWK, GILSWE, GILSWQ, GILAWK, GILAWE, and GILAWQ.

[0065] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (III): X7KW or X7KY, where X7 is glutamic acid or glutamine. In one embodiment, the amino acid sequence of formula (III) is selected from QKW and EKW. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (III-a): X7KWX8FE, where X7 is glutamic acid or glutamine and X8 is glutamic acid, arginine, or histidine. In one embodiment, the amino acid sequence of formula (III-a) is selected from EKWEFE, EKWRFE, EKWHFE, QKWEFE, QKWRFE, and QKWHFE. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (III-b): X7KWX8FEFW or X7KYX8FEFW, where X7 is glutamic acid or glutamine, and X8 is glutamic acid, arginine, or histidine. In one embodiment, the amino acid sequence of formula (III-b) is selected from EKWEFEFW, EKWRFEFW, EKWHFEFW, QKWEFEFW, QKWRFEFW, QKWHFEFW, EKYEFEFW, EKYRFEFW, EKYHFEFW, QKYEFEFW, QKYRFEFW, and QKYHFEFW.

[0066] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (IV): YHX9, where X9 is glycine or alanine. In one embodiment, the amino acid sequence of formula (IV) is selected from YHG and YHA. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (IV): YHX9PFY, where X9 is glycine or alanine. In one embodiment, the amino acid sequence of formula (Iv-a) is selected from YHGPFY and YHAPFY. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (IV-b): X 10 YHGPFY or X 10 YHAPFY, (where X 10is glutamine, glycine, methionine, isoleucine). In one embodiment, the amino acid sequence of formula (IV-b) is selected from QYHGPFY, GYHGPFY, MYHGPFY, IYHGPFY, QYHAPFY, GYHAPFY, MYHAPFY, and IYHAPFY. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (IV-c): FRX 11 YHX9PFY, wherein X 11 is glutamine, glycine, methionine, or isoleucine, and X9 is glycine or alanine. In one embodiment, the amino acid sequence of formula (IV-c) is selected from FRQYHGPFY, FRGYHGPFY, FRMYHGPFY, FRIYHGPFY, FRQYHAPFY, FRGYHAPFY, FRMYHAPFY, and FRIYHAPFY.

[0067] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (V): GVHX 12 (In the formula, X 12 is tryptophan or tyrosine). In one embodiment, the amino acid sequence of formula (V) is selected from GVHW and GVHF. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (Va): GVHWX 13 F, wherein X 13 is arginine, histidine, valine, or glutamic acid. In one embodiment, the amino acid sequence of formula (Va) is selected from GVHWRFGVHWHF, GVHWVF, and GVHWEF. In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (Vb): WX 13 FDAX 14 (In the formula, X 13 is arginine, histidine, valine, or glutamic acid, X14is alanine or phenylalanine). In one embodiment, the amino acid sequence of formula (Vb) is selected from WRFDAA, WHFDAA, WVFDAA, WEFDAA, WRFDAF, WHFDAF, WVFDAF, and WEFDAF.

[0068] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (VI):X 15 LI (wherein X 15 is asparagine or lysine). In one embodiment, the amino acid sequence of formula (VI) is selected from NLI and KLI. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (VI-a): DGX 16 X 15 LI, wherein X 15 is asparagine or leucine, X 16 is serine or alanine. In one embodiment, the amino acid sequence of formula (VI-a) is selected from DGSNLI, DGANLI, DGSKLI, and DGAKLI. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (VI-b): WX 17 YDGX 16 X 15 LI, wherein X 15 is asparagine or leucine, X 16 is serine or alanine, and X 17 is alanine or glycine. In one embodiment, the amino acid sequence of formula (VI-b) is selected from WGYDGSNLI, WAYDGSNLI, WAYDGANLI, WGYDGANLI, WGYDGSKLI, WAYDGSKLI, WAYDGAKLI, and WGYDGAKLI.

[0069] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (VII): GTPX 18 wherein X 18is proline or valine. In one embodiment, the amino acid sequence of formula (VII) is selected from GTPP and GTPV. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (VII-a): X 19 LIGTPX 18 wherein X 19 is proline or valine, and X 18 is asparagine or leucine. In one embodiment, the amino acid sequence of formula (VII-a) is selected from NLIGTPP, KLIGTPP, NLIGTPV, and KLIGTPV. In one embodiment, the peroxidase (e.g., haloperoxidase) has formula (VII-b): DGSX 19 LIGTPX 18 wherein X 18 is proline or valine, and X 19 is asparagine or lysine. In one embodiment, the amino acid sequence of formula (VII-b) is selected from DGSNLIGTPP, DGSKLIGTPP, DGSNLIGTPV, and DGSKLIGTPV. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (VII-c): X 20 YDGSX 19 LIGTPX 18 wherein X 18 is proline or valine, X 19 is asparagine or lysine, and X 20 is alanine or glycine. In one embodiment, the amino acid sequence of formula (VII-c) is selected from AYDGSNLIGTPP, GYDGSNLIGTPP, AYDGSKLIGTPP, GYDGSKLIGTPP, AYDGSNLIGTPV, GYDGSNLIGTPV, AYDGSKLIGTPV and GYDGSKLIGTPV.

[0070] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (VIII): AYLNACX 21 I (where X 21is leucine or isoleucine). In one embodiment, the amino acid sequence of formula (VIII) is selected from AYLNACLI and AYLNACII. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (VIII-a): AYLNACX 21 IX 22 L, wherein X 21 is leucine or isoleucine, and X 22 is leucine or methionine. In one embodiment, the amino acid sequence of formula (VIII-a) is selected from AYLNACLILL, AYLNACIILL, AYLNACLIML, and AYLNACIIML.

[0071] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (IX): IPX 23 or LPX 23 (In the formula, X 23 is methionine or phenylalanine). In one embodiment, the amino acid sequence of formula (IX) is selected from IPM, IPF, LPM, and LPF. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (IX-a):DX 25 X 24 IPX 23 or DX 25 X 24 LPX 23 wherein X 23 is methionine or phenylalanine, and X 24 is glycine, histidine, or asparagine, and X 25is glutamine, lysine, proline, or serine. In one embodiment, the amino acid sequence of formula (IX-a) is DQGIPM, DQHIPF, DQNIPM, DQGIPF, DQHIPM, DQNIPF, DQGIPM, DKHIPF, DKNIPM, DKGIPF, DKHIPM, DKNIPF, DKGIPM, DPHIPF, DPNIPM, DPGIPF, DPHIPM, DPNIPF, DPGIPM, DSHIPF, DSNIPM, DSGIPF, DSHIPM, and DSNIPF. In one embodiment, the amino acid sequence of formula (IX-a) is DQGLPM, DQHLPF, DQNLPM, DQGLPF, DQHLPM, DQNLPF, DQGLPM, DKHLPF, DKNLPM, DKGLPF, DKHLPM, DKNLPF, DKGLPM, DPHLPF, DPNLPM, DPGLPF, DPHLPM, DPNLPF, DPGLPM, DSHLPF, DSNLPM, DSGLPF, DSHLPM, and DSNLPF.

[0072] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (X):DX 26 Q, (where X 26 is histidine, lysine, or asparagine. In one embodiment, the amino acid sequence of formula (X) is selected from DHQ, DKQ, and DNQ. In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises an amino acid sequence of formula (Xa):DX 26 QX 27 GF or DX 26 QX 27 VF (where X 26 is histidine, lysine, or asparagine); 27 is arginine, glutamine, or aspartic acid. In one embodiment, the amino acid sequence of formula (Xa) is selected from DHQRGF, DHQQGF, DHQDGF, DKQRGF, DKQQGF, DKQDGF, DNQRGF, DNQQGF, and DNQDGF.

[0073] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XI): EVATRALKX28 X 29 R (where X 28 is alanine or glycine, and X 29 is phenylalanine or tyrosine). In one embodiment, the amino acid sequence of formula (XI) is selected from EVATRALKAFR, EVATRALKAYR, EVATRALKGFR, and EVATRALKGYR. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (VI-a): EVATRALKX 28 X 29 RX 30 QK, wherein X 28 is alanine or glycine, X 29 is phenylalanine or tyrosine, and X 30 is phenylalanine or tyrosine. In one embodiment, the amino acid sequence of formula (XI-a) is selected from EVATRALKAFRYQK, EVATRALKAYRFQK, EVATRALKAFRFQK, EVATRALKAYRYQK, EVATRALKGFRYQK, EVATRALKGYRFQK, EVATRALKGFRFQK, and EVATRALKGYRYQK. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (XI-b): HRRLRPEAX 31 G (where X 31 is valine, isoleucine, or threonine. In one embodiment, the amino acid sequence of formula (XI-b) is selected from HRRLRPEAVG, HRRLRPEAIG, and HRRLRPEATG.

[0074] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XII): EGSPX 32 (In the formula, X 32 is methionine or phenylalanine). In one embodiment, the amino acid sequence of formula (XII) is selected from EGSPMHP and EGSPFHP. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (XII-a):EGSPX 32 HPX 33YG (in the formula, X 32 is methionine or phenylalanine, and X 33 is serine or alanine). In one embodiment, the amino acid sequence of formula (XII-a) is selected from EGSPMHPSYG, EGSPMHPAYG, EGSPFHPSYG, and EGSPFHPAYG. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (XII-b): EGSPX 32 HPX 33 YGX 34 GHA (in the formula, X 32 is methionine or phenylalanine, X 33 is serine or alanine, and X 34 is serine or alanine). In one embodiment, the amino acid sequence of formula (XII-b) is selected from EGSPMHPSYGAGHA, EGSPMHPAYGSGHA, EGSPMHPSYGAGHA, EGSPMHPAYGSGHA, EGSPFHPSYGAGHA, EGSPFHPAYGSGHA, EGSPFHPSYGAGHA, and EGSPFHPAYGSGHA.

[0075] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XIII): VAGACX 35 T (where X 35 is valine or threonine). In one embodiment, the amino acid sequence of formula (XIII) is selected from VAGACVT and VAGACTT. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (XIII-a): VAGACX 35 TLKAFFX 36 (In the formula, X 35 is valine or threonine, and X 36 is glutamine or aspartic acid). In one embodiment, the amino acid sequence of formula (XIII-a) is selected from VAGACVTLKAFFQ, VAGACVTLKAFFD, VAGACTTLKAFFQ, and VAGACTTLKAFFD.

[0076] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XIV): DGX 37 X 38 LD or DEX 37 X 38 LD, wherein X 37 is serine, lysine, arginine, threonine, or aspartic acid; X 38 is arginine, lysine, or glycine. In one embodiment, the amino acid sequence of formula (XIV) is selected from DGSRLD, DGSKLD, DGSGLD, DGKRLD, DGKKLD, DGKGLD, DGRRLD, DGRKLD, DGRGLD, DGTRLD, DGTKLD, DGTGLD, DGDRLD, DGDKLD, and DGDGLD. In one embodiment, the amino acid sequence of formula (XIV) is selected from DESRLD, DESKLD, DESGLD, DEKRLD, DEKKLD, DEKGLD, DERRLD, DERKLD, DERGLD, DETRLD, DETKLD, DETGLD, DEDRLD, DEDKLD, and DEDGLD.

[0077] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XV): LTVX 39 wherein X 39 is alanine, aspartic acid, or glutamic acid. In one embodiment, the amino acid sequence of formula (XV) is selected from LTVA, LTVD, and LTVE. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (XV-a):X 40 ELNK, wherein X 40 is glycine or aspartic acid. In one embodiment, the amino acid sequence of formula (XV-a) is selected from GELNK and DELNK. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (XV-b): LTVX 39 X 40 ELNK, wherein X 39 is alanine, aspartic acid, or glutamic acid, and X 40is glycine or aspartic acid. In one embodiment, the amino acid sequence of formula (XV-b) is selected from LTVAGELNK, LTVDGELNK, LTVEGELNK, LTVADELNK, LTVDDELNK, and LTVEDELNK.

[0078] In one embodiment, formula (XVI) comprises NISIGR, NISVGR, or NVAIGR.

[0079] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XVII):AGVHYX 41 X 42 D (where X 41 is phenylalanine or tyrosine, and X 42 is threonine or serine). In one embodiment, the amino acid sequence of formula (XVII) is selected from AGVHYFSD, AGVHYFTD, AGVHYYSD, and AGVHYYTD. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (XVII-a):AGVHYX 41 X 42 DX 43 X 44 ES, wherein X 41 is phenylalanine or tyrosine; X 42 is threonine or serine, X 43 is tyrosine or glutamine, and X 44 is isoleucine, arginine, phenylalanine, or valine.

[0080] In one embodiment, the amino acid sequence of formula (XVII-a) is selected from AGVHYFTDYIES, AGVHYFSDYIES, AGVHYFTDQIES, AGVHYFSDQIES, AGVHYFTDYRES, AGVHYFSDYRES, AGVHYFTDQRES, AGVHYFSDQRES, AGVHYYTDYFES, AGVHYYSDYFES, AGVHYYTDQFES, AGVHYYSDQFES, AGVHYYTDYVES, AGVHYYSDYVES, AGVHYYTDQVES, and AGVHYYSDQVES.

[0081] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XVIII): EQX 45 LT (where X 45 is methionine, lysine, or serine). In one embodiment, the amino acid sequence of formula (XVIII) is selected from EQMLT, EQKLT, and EQSLT. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (XVII-a):LX 46 EQX 45 LT, (where X 45 is methionine, lysine, or serine, and X 46 is glutamic acid, glutamine, or lysine. In one embodiment, the amino acid sequence of formula (XVIII-a) is selected from LEEQMLT, LQEQMLT, LKEQMLT, LEEQKLT, LQEQKLT, LKEQKLT, LEEQSLT, LQEQSLT, and LKEQSLT.

[0082] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XIX): GEX 47 X 48 A, wherein X 47 is glutamine, lysine, or glutamic acid, and X 48is isoleucine or valine. In one embodiment, the amino acid sequence of formula (XIX) is selected from GEQIA, GEQVA, GEKIA, GEKVA, GEEIA, and GEEVA. In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XIX-a): GEX 47 X 48 AX 49 G, wherein X 47 is glutamine, lysine, or glutamic acid; X 48 is isoleucine or valine, and X 49 is leucine or isoleucine. In one embodiment, the amino acid sequence of formula (XIX-a) is selected from GEQIALG, GEQIAIG, GEQIALG, GEQIAIG, GEKIALG, GEKIAIG, GEKVALG, GEKVAIG, GEEVALG, GEEVAIG, GEEVALG, and GEEVAIG.

[0083] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XX):LX 50 EQ, wherein X 50 is glutamic acid, glutamine, or lysine. In one embodiment, the amino acid sequence of formula (XX) is selected from LEEQ, LQEQ, and LKEQ. In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XX-a): LX 50 EQX 51 LT, wherein X 50 is glutamic acid, glutamine, or lysine, and X 51 is methionine, lysine, or serine. In one embodiment, the amino acid sequence of formula (XX-a) is selected from LEEQMLT, LEEQKLT, LEEQSLT, LQEQMLT, LQEQKLT, LQEQSLT, LKEQMLT, LKEQKLT, and LKEQSLT. In one embodiment, the peroxidase (e.g., haloperoxidase) has the amino acid sequence of formula (XX-b): LX 50 EQX 51 LTFX 52 E or LX 50 EQX51 LTYX 52 E, wherein X 50 is glutamic acid, glutamine, or lysine, and X 51 is methionine, lysine, or serine, and X 52 is proline, alanine, glycine, or serine. In one embodiment, the amino acid sequence of formula (XX-b) is Selected from EQKLTAE, LQEQKLTGE, LQEQKLTSE, LQEQSLTPE, LQEQSLTAE, LQEQSLTGE, LQEQSLTSE, LKEQMLTPE, LKEQMLTAE, LKEQMLTG, LKEQMLTSE, LKEQKLTPE, LKEQKLTAE, LKEQKLTGE, LKEQKLTSE, LKEQSLTPE, LKEQSLTAE, LKEQSLTGE, and LKEQSLTSE.

[0084] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XXI):EX 53 F, wherein X 53 is threonine, glutamic acid, aspartic acid, lysine, or asparagine. In one embodiment, the amino acid sequence of formula (XXI) is selected from ETF, EEF, EDF, EKF, or ENF. In one embodiment, the peroxidase (e.g., haloperoxidase) is represented by formula (XXI-a):EX 53 FX 54 M or EX 53 FX 54 F, wherein X 53 is threonine, glutamic acid, aspartic acid, lysine, or asparagine, and X 54is threonine, serine, or phenylalanine. In one embodiment, the amino acid sequence of formula (XXI-a) is selected from ETFTM, EEFMT, EDFTM, EKFTM, ENFTM, ETFSM, EEFSM, EDFSM, EKFSM, ENFSM, EFFFM, EFFFM, EKFFM, and ENFFM. In one embodiment, the amino acid sequence of formula (XXI-a) is selected from ETFTF, EEFMF, EDFTF, EKFTF, ENFTF, EFSF, EDFSF, EKFSF, ENFSF, ETFFF, EFFFF, EKFFF, and ENFFF.

[0085] In one embodiment, the peroxidase (e.g., haloperoxidase) has the formula (XXII): PSGHAX 55 FX 56 (In the formula, X 55 is serine or threonine, and X 56 is glycine or serine). In one embodiment, the amino acid sequence of formula (XXII) is selected from PSGHASFG, PSGHASFS, PSGHASFG, PSGHASFS, PSGHATFG, PSGHATFS, PSGHATFG, and PSGATFS. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (XXII-a): FPX 57 YPSGHAX 55 FX 56 wherein X 55 is serine or threonine, X 56 is glycine or serine, and X 57is proline or asparagine. In one embodiment, the amino acid sequence of formula (XXII) is selected from FPNYPSGHASFG, FPNYPSGHASFS, FPNYPSGHASFG, FPNYPSGHASFS, FPNYPSGHATFG, FPNYPSGHATFS, FPNYPSGHATFG, FPNYPSGATFS, FPPYPSGHASFG, FPPYPSGHASFS, FPPYPSGHASFG, FPPYPSGHASFS, FPPYPSGHATFG, FPPYPSGHATFS, FPPYPSGHATFG, and FPPYPSGATFS.

[0086] The peroxidases (e.g., haloperoxidases) of the present disclosure can comprise any of the amino acid sequences described herein. In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X ... The amino acid sequence of any one of Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (Xa ), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), or two of the amino acid sequences (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X ... The amino acid sequence comprises three of the following: Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X ... The amino acid sequence includes four of the following: Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (Xa ), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), or (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (Xa ), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), or (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X ... The amino acid sequence includes seven of the following: Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X ... The amino acid sequence comprises eight of the following: Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).

[0087] In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X ... The amino acid sequence includes nine of the following: Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises 20 amino acid sequences selected from the group consisting of Formula (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (II), (III-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises 20 amino acid sequences selected from the group consisting of Formula (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (II). Ib), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b ), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (Xa), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII -b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a). In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).In one embodiment, the peroxidase (e.g., haloperoxidase) is selected from the group consisting of: (I), (Ia), (II), (II-a), (II-b), (III), (III-a), (III-b), (IV), (IV-a), (IV-b), (IV-c), (V), (Va), (Vb), (VI), (VI-a), (VI-b), (VII), (VII-a), (VII-b), (VII-c), (VIII), (VIII-a), (IX), (IX-a), (X), (X a), (XI), (XI-a), (XI-b), (XII), (XII-a), (XII-b), (XIII), (XIII-a), (XIV), (XV), (XV-a), (XV-b), (XVI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII), or (XXII-a).

[0088] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of one of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises two amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises three amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises four amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises five amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII).In one embodiment, the peroxidase (e.g., haloperoxidase) comprises six amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises seven amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., a haloperoxidase) comprises eight amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises nine amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 10 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII).In one embodiment, the peroxidase (e.g., haloperoxidase) comprises eleven amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 12 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 13 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 14 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 15 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII).In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 16 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises a 17 amino acid sequence of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an 18 amino acid sequence of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises a 19 amino acid sequence of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 20 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII).In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 21 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII). In one embodiment, the peroxidase (e.g., haloperoxidase) comprises 22 amino acid sequences of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), or (XXII).

[0089] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0090] Exemplary haloperoxidase sequences found in a variety of organisms, including Curvularia inaeqalis, Corallina officinalis, Halomicronema hongdechloris, Moore bouillonii, Trichodesmium erythraeum, Aphanocapsa montana, Synechococcus sp. PCC7335, Coralilin, and Saccharomyces cerevisiae, are shown below in Table 2. These sequences include both naturally occurring and engineered VHPO sequences.

[0091] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25

[0092] Host cell microorganisms The present disclosure provides peroxidases and related methods for producing multiple small halogenated organic compounds useful, for example, for methane reduction in the rumen of agricultural animals. In one embodiment, the peroxidase (VHPO) is heterologously produced in a host cell microorganism. In one embodiment, production of the peroxidase (VHPO) in the host cell microorganism results in an increase in the total amount of peroxidase (VHPO), e.g., compared to a reference standard. As used herein, producing a peroxidase (VHPO) in a host cell microorganism includes expression, translation, and / or secretion of a peroxidase (e.g., a VHPO).

[0093] Host cell microorganisms suitable for use in the present disclosure are capable of producing the peroxidases (VHPOs) described herein. In one embodiment, the host cell microorganism naturally produces the peroxidase (VHPO), e.g., expresses an endogenous peroxidase (VHPO). In one embodiment, the host cell microorganism produces an exogenous peroxidase (VHPO). In one embodiment, the host cell microorganism is genetically engineered to produce the peroxidase (VHPO), e.g., to express a heterologous peroxidase (VHPO). In such embodiments, a nucleic acid encoding the heterologous peroxidase (VHPO) is introduced into the host cell microorganism using standard methods known in the art, e.g., by electroporation, transfection, or transduction. A heterologous peroxidase (VHPO) may be a peroxidase (VHPO) that is naturally produced in a different microorganism, or may be a modified peroxidase (VHPO) that comprises a different amino acid sequence or a different function and / or activity, e.g., increased or decreased activity, from that of the corresponding naturally occurring peroxidase (VHPO).

[0094] The host cell microorganism can be a fungus (e.g., yeast), bacterium, protozoan, archaea, synthetic organism, or semi-synthetic organism that produces one or more proteins, such as one or more enzymes, one or more peroxidases, etc. (e.g., VHPOs). In one embodiment, the host cell microorganism is a fungus. In one embodiment, the host cell microorganism is a bacterium. In one embodiment, the host cell microorganism is a protozoan. In one embodiment, the host cell microorganism is an archaea. Exemplary host cell microorganisms include Pichia pastoris, Aspergillus niger, Saccharomyces cerevisiae, Escherichia coli, and Synechococcus sp. PCC11901.

[0095] Pichia pastoris is a highly successful heterologous expression system. The yeast Pichia pastoris has been used in biotechnology as an expression system for protein and enzyme production, as it has been shown to result in increased protein production compared to many native systems. Pichia can grow in defined media and reach high cell densities, resulting in wet cell densities of produced protein (e.g., secreted protein) of 250 g / L or more, and has excellent scalability (e.g., scalability up to approximately 200,000 liters of cell culture). Proper folding and secretion are two of the main advantages of the P. pastoris system. This system is also often used for the production of complex eukaryotic proteins and has been successfully used to produce fungal enzymes, including beta-glucosidases, lignocellulosic enzymes, and oxidases from macroalgae. While the P. pastoris expression system can be easily used on both experimental and industrial scales, recombinant protein production in P. pastoris often requires an optimization process to achieve maximum production of the protein of interest. It is widely known that many conditions need to be tested to achieve the desired results, including optimization of medium components, growth temperature, and protein and nucleic acid sequences. P. pastoris cells can also be converted into cell factories for large-scale production.

[0096] Aspergillus niger is a filamentous fungus that was first developed to produce citric acid and is now the basis for the production of a wide variety of proteins, enzymes, and secondary metabolites. A. niger is now increasingly used as an alternative biotechnological system. This includes proteins, including enzymes, as well as pharmaceuticals used in human and animal health.

[0097] Saccharomyces cerevisiae is a commonly utilized host cell microorganism for improving heterologous production of proteins, such as enzymes. In addition to the potential for large-scale production of baker's yeast, extensive research has explored approaches to concentrate products in intracellular organelles, such as peroxisomes, which offer significant advantages in biomanufacturing. S. cerevisiae peroxisomes have been engineered to produce comparable reactions, and large-scale production of this yeast is well established in the food industry (e.g., wine, beer, and bread). Subcellular compartmentalization can provide optimal conditions for desired enzymatic reactions by increasing metabolic flux and reducing metabolic crosstalk, making it a powerful strategy for isolating toxic products from the host cytosol, thereby limiting cytotoxicity, improving growth, and increasing product titer.

[0098] Cyanobacteria are prokaryotic oxyphotoautotrophs capable of converting CO2 and inorganic nitrogen sources, phosphorus, and trace elements into biomass. Synechococcus PCC11901 is a robust sponge cyanobacterial strain established for industrial-scale high biomass production. PCC11901 can be cultivated in seawater unsuitable for agricultural use or direct human consumption. Other promising features include (i) growth at high light intensities and a wide range of salinities, (ii) accumulation up to ≈33 g dry cell weight / L, and (iii) the availability of genomic and genetic tools. Interestingly, PCC 11901 lacks a VHPO, but the CiVHPO enzyme can be heterologously expressed. Furthermore, there is a large literature on the ecology, genetics, cell biology, and biotechnological applications of Synechococcus sp. that can be directly applied to PCC11901. The strains of interest are engineered for fast growth, solvent tolerance, product biosynthesis and accumulation, and biocontainment.

[0099] In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a peroxidase (e.g., VHPO) described herein, or a functional fragment thereof, e.g., comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a peroxidase (e.g., VHPO) described herein, or a functional fragment thereof.

[0100] In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence listed in Table 2. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence listed in Table 2. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO:1. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 3.In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 4. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 5. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 6. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 6.In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 7. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 8. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 8. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 9. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 9. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 10.In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 11. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 11. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 12. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 12. In another embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 14. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 14.In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 15. In one embodiment, the peroxidase (e.g., VHPO) comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 16. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 18. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 18.In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, the peroxidase (e.g., VHPO) is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or less, to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 20. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 21. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 21. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 22. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 22. In some embodiments, the peroxidase (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:23.In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 23. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 24. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 25. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 26. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 27.In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 27. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 28. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 28. In some embodiments, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 29. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 29. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 30. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 30. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 31.In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 31. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 32. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 32. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 33. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 33. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 34. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 34. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 35.In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 35. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 36. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 36. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 37. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 37. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 38. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 38. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 39.In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 39. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 40. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identity to the amino acid sequence of SEQ ID NO: 40. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 41. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 41. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 42. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 42. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 43. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 43. In one embodiment, the peroxidase (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:44.In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 44. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 45. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 45. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 46. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 46. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 47. In one embodiment, the peroxidase (e.g., VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 47. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 48.In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 48. In one embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 49. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 49. In another embodiment, the peroxidase (e.g., a VHPO) comprises the amino acid sequence of SEQ ID NO: 50. In one embodiment, the peroxidase (e.g., a VHPO) has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 50.

[0101] In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to a peroxidase (e.g., a VHPO) described herein or a functional fragment thereof. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to a peroxidase (e.g., a VHPO) listed in Table 2. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 2. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 4. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 5. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO:8.In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 9. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 11. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 12. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 14. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO:18.In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 20. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 21. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 22. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 23. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 27. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO:28.In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 29. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 30. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 31. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 32. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 33. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 34. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 35. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 36. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 37. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO:38.In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 39. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 40. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 41. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 42. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 43. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 44. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 45. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 46. In one embodiment, the peroxidase (e.g., a VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 47. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO:48.In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 49. In one embodiment, the peroxidase (e.g., VHPO) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mutations compared to the amino acid sequence of SEQ ID NO: 50.

[0102] Percent identity in two or more amino acid or nucleic acid sequences refers to two or more sequences that are the same, e.g., conserved. Two sequences are "substantially identical" if they have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60%, optionally 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity over a specified region, or, if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, or 150 amino acids in length. The identity can exist over a region that is about 10 to about 100 amino acids, or about 50 to about 250 amino acids, or about 200 to about 500 amino acids in length.

[0103] For sequence comparison, typically, one sequence serves as a reference sequence and is compared with one or more test sequences. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, and the similarity search method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of the algorithms described (GAP, BESTFIT, FASTA, and TFASTA Wisconsin Genetics Software solvent package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

[0104] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms (described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively). Software for performing BLAST analyses is publicly available via the website of the National Center for Biotechnology Information.

[0105] A functional variant can contain one or more mutations such that the variant retains some activity, e.g., of a peroxidase (e.g., VHPO) described herein produced by the microorganism from which the enzyme originates. In one embodiment, a functional variant has at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) of the peroxidase (e.g., VHPO) of its corresponding naturally occurring peroxidase (e.g., VHPO). In one embodiment, the functional variant has at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000% or more peroxidase (e.g., VHPO) activity as the corresponding naturally occurring peroxidase (e.g., VHPO). Peroxidase activity can be tested using functional assays known in the art. For example, if the peroxidase (e.g., VHPO) is VBPO, a functional assay measuring the consumption of bromide activity can be performed. Furthermore, functional assays may be coupled to one another. For example, if the peroxidase is VBPO, VBPO activity can be coupled to the consumption of peroxide-containing compounds, such as HO or peracetic acid (PAA). Other exemplary assays include the MCD and APF assays.

[0106] Mutations present in functional variants include amino acid substitutions, additions, and deletions. Mutations can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Mutations can also be achieved by using the CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system. The CRISPR / Cas system is naturally found in bacteria and archaea and has been modified for use in gene editing (silencing, enhancing, or mutating specific genes) in eukaryotes such as mice or primates (Wiedenheft et al. (2012) Nature 482:331-8). This is achieved by introducing a plasmid containing a specially designed CRISPR and one or more appropriate Cas sequences into cells.

[0107] A "mutation" may be a "conservative amino acid substitution," in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a peroxidase (e.g., VHPO) can be replaced with other amino acids from the same side chain family, and the resulting peroxidase (e.g., VHPO) activity can be equivalent to that of a wild-type peroxidase (e.g., VHPO) (e.g., at least 80%, 85%, 90%, 95%, or 99% of the peroxidase (e.g., VHPO) activity). Alternatively, a "mutation" can be an "amino acid substitution," in which an amino acid residue is replaced with an amino acid residue having a different side chain.

[0108] Such mutations may alter or affect various enzymatic properties of the peroxidase (e.g., VHPO). For example, such mutations may alter or affect the activity, e.g., peroxidase activity, thermostability, optimal pH for the reaction, enzyme kinetics, or substrate recognition, of the peroxidase (e.g., VHPO). In some embodiments, the mutations result in increased peroxidase (e.g., VHPO) activity of the variant compared to the peroxidase (e.g., VHPO) activity of the wild-type peroxidase (e.g., VHPO). In some embodiments, the mutations result in increased or decreased thermostability of the variant compared to the wild-type peroxidase (e.g., VHPO). In one embodiment, the mutations alter the pH range at which the variant optimally performs a peroxidase reaction compared to the wild-type peroxidase (e.g., VHPO). In one embodiment, the mutations alter the pH range at which the variant optimally performs a peroxidase reaction compared to the wild-type peroxidase (e.g., VHPO). In one embodiment, the mutations alter the pH range at which the variant optimally performs a peroxidase reaction compared to the wild-type peroxidase (e.g., VHPO). cat , K. M、 k cat / K M , or K D In one embodiment, the mutation results in an increase or decrease in the kinetics of the peroxidase (e.g., VHPO) to recognize or bind to a substrate, as compared to a wild-type peroxidase (e.g., VHPO). In one embodiment, the mutation results in an increase or decrease in the selectivity of the peroxidase (e.g., VHPO) for small organic compounds, as compared to a wild-type peroxidase (e.g., VHPO).

[0109] Furthermore, such mutations may alter or affect the stability or expression level of the peroxidase (e.g., VHPO). For example, such mutations may increase the stability of the peroxidase (e.g., VHPO) under certain conditions (e.g., temperature or pH) in a particular host cell microorganism, or may increase the expression level of the peroxidase (e.g., VHPO). In one embodiment, the mutations result in increased stability of the peroxidase (e.g., VHPO) compared to the wild-type sequence. In one embodiment, the mutations result in increased expression levels of the peroxidase (e.g., VHPO) compared to the wild-type sequence.

[0110] In one embodiment, the peroxidase sequence contains an N-terminal signal sequence. In one embodiment, the peroxidase sequence contains an N-terminal protein purification tag (e.g., a cleavable or non-cleavable protein purification tag). In one embodiment, the peroxidase sequence contains a C-terminal protein purification tag (e.g., a cleavable or non-cleavable protein purification tag).

[0111] Cell culture and protein expression A host cell microorganism capable of producing a protein, such as a peroxidase (e.g., VHPO), can be present in a cell culture. The cell culture includes one or more cells in a cell culture medium. The cell culture medium can be an aqueous cell culture medium containing components that support cell maintenance, cell viability, cell growth, and / or cell proliferation. Cell culture media typically include physiological salts, such as ammonium salts, phosphate salts, potassium salts, magnesium salts, calcium salts, iron salts, manganese salts, zinc salts, or cobalt salts; amino acids; water; and, optionally, a carbon source. In one embodiment, a cell culture medium suitable for growing a host cell microorganism described herein comprises an ammonium salt, e.g., ammonium sulfate and / or ammonium hydroxide, a potassium salt (e.g., potassium hydroxide), a calcium salt (e.g., calcium chloride), a magnesium salt (e.g., magnesium sulfate), a manganese salt (e.g., manganese sulfate), an iron salt (e.g., iron sulfate), a zinc salt (e.g., zinc sulfate), a cobalt salt (e.g., cobalt chloride), phthalic acid; lactose; an antibiotic, e.g., ACETOBAN®; and a carbon source, (e.g., glycerol or carbon dioxide).

[0112] The host cell microorganism or cell culture is contacted with a supplied carbon source, such as, for example, sugar, to support the growth or proliferation of the host cell microorganism. In one embodiment, the host cell microorganism or cell culture is contacted with, for example, supplied glucose. As the host cell microorganism grows in culture, the cell culture can be transferred from one container, such as a cell culture container, to a larger container to allow and promote continued growth of the host cell microorganism. For example, the host cell microorganism is contacted with sugar in a first container under appropriate conditions, such as those described herein, so that the host cell microorganism grows. Growth can be monitored, and upon reaching a desired growth level, for example, a specific growth phase, or a desired growth level (e.g., as measured by the turbidity of the culture or by cell count), the host cell microorganism can be transferred to a second container, for example, a container with a larger volume than the first container. Transferring the microorganism to the larger second container allows and promotes continued growth of the microorganism. In an embodiment, the microorganism is transferred, for example, from the first container to the larger second container once. In embodiments, the host cell microorganisms are transferred multiple times, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, and for each transfer, the host cell microorganisms are larger than the vessel into which they were transferred.

[0113] Suitable vessels for transporting and culturing the host cell microorganisms described herein include any cell culture vessel known in the art. Examples of suitable vessels include, but are not limited to, cell culture flasks, roller bottles, bioreactors, or tanks. Other cell culture conditions suitable for maintaining cell viability or promoting cell growth are known in the art. Cell culture conditions to consider include pH, temperature, oxygen level, and movement. The pH of the cell culture, e.g., the medium, is generally at a physiological pH, e.g., pH 4-8, or pH 5-7, e.g., pH 5, pH 6, or pH 7. The temperature for growth of host cell microorganisms that produce peroxidase (e.g., VHPO) is generally 20-40°C, e.g., 30°C. In some embodiments, certain strains of host cell microorganisms may exhibit enhanced growth or enzyme production at higher temperatures, e.g., 32°C or 37°C, or at lower temperatures (e.g., 27°C), respectively. Cell cultures may be static, or movement may be used to promote maintenance or growth. For example, cell cultures may be rolled, shaken, or agitated to enhance cell growth. The cell culture conditions disclosed herein are merely exemplary and should not be construed as limiting. Cell culture conditions that differ from those explicitly listed herein may be envisioned or may be determined experimentally and may depend on the species or strain of the host cell microorganism used. Cell culture conditions sufficient for the growth of a host cell microorganism capable of producing a peroxidase (e.g., VHPO) result in an increase in the cell number of the culture of the host cell microorganism. Cell culture conditions sufficient for the production of a peroxidase (e.g., VHPO) result in one or more cells of the microorganism producing the peroxidase (e.g., VHPO).

[0114] After the cell culture reaches a desired growth level, e.g., a specific growth phase or culture volume size, or when the cell culture, e.g., the aqueous portion, is substantially free of the carbon source, e.g., sugar, utilized to stimulate growth, the cell culture can be induced to produce a protein, e.g., a peroxidase (e.g., VHPO) described herein. A composition described herein containing a small organic compound starting material is added, e.g., fed, to a host cell microorganism or cell culture capable of producing a peroxidase (e.g., VHPO), thereby inducing the host cell microorganism to produce a peroxidase (e.g., VHPO). In one embodiment, the composition containing the small organic compound is added directly to the culture. In one embodiment, the composition containing the small organic compound is added to an enzyme production medium containing components that support and promote the production of a protein, e.g., a peroxidase (e.g., VHPO). The host cell microorganism is then transferred or cultivated in the enzyme production medium. The enzyme production medium may comprise a composition comprising a physiological salt, e.g., an ammonium salt, and a caramelized sugar product and / or a second agent, and is adjusted to a pH of 4 to 7, e.g., pH 6. In one embodiment, the enzyme production medium comprises a composition comprising ammonium sulfate, rice bran, and a low molecular weight compound.

[0115] In one embodiment, the small molecule organic compound and the second agent are added simultaneously to the host cell microorganism or cell culture. The small molecule organic compound and the second agent (e.g., a peroxide-containing substrate, such as HO or PAA) may be present in the same composition or in separate compositions. When the small molecule organic compound and the second agent are present in the same composition, they may be components of an enzyme production medium. In another embodiment, the small molecule organic compound and the second agent are present in separate compositions and added sequentially to the host cell microorganism or cell culture. For example, the small molecule organic compound may be added to the host cell microorganism or cell culture before or after the second agent is added to the host cell microorganism or cell culture. In such a continuous induction process, the period between the addition of the small molecule organic compound and the addition of the second agent may be several hours, e.g., 1, 2, 3, 4, 5, 6, 12, 18 hours or more, or may include, e.g., 1, 2, 3, 4, 5, 6, 7 days or more.

[0116] The small molecule organic compound can be introduced into the host cell microorganism, for example, by direct addition to the culture or via an enzyme production medium, twice a day, once a day, every other day, every three days, or once a week. The small molecule organic compound can be added to the host cell microorganism cell culture at a concentration range of 1-20 g / L, 1-15 g / L, 1-10 g / L, 1-5 g / L, 2-15 g / L, 2-10 g / L, 2-5 g / L, 5-20 g / L, 5-15 g / L, 5-10 g / L, 4-5 g / L, 10-20 g / L, or 10-15 g / L. Small molecule organic compounds can be added to a host cell microbial culture at a concentration range of 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, or 20 g / L or more. In one embodiment, small molecule organic compounds are added to a host cell microbial culture at 4 g / L once daily or 5 g / L once daily.

[0117] The second agent (e.g., a peroxide-containing substrate, such as HO or PAA) can be introduced to the host cell microorganism, for example, by direct addition to the culture or via the enzyme production medium, twice a day, once a day, every other day, every three days, or once a week. The host small molecule organic compound can be added to the host cell microorganism at a concentration range of 1-20 g / L, 1-15 g / L, 1-10 g / L, 1-5 g / L, 2-15 g / L, 2-10 g / L, 2-5 g / L, 5-20 g / L, 5-15 g / L, 5-10 g / L, 10-20 g / L, or 10-15 g / L of the cell culture. The second agent can be added at a concentration range of 0.5 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / L, or 20 g / L or more to the host microbial culture. In one embodiment, the second agent is added to the host microbial culture at 5 g / L once daily.

[0118] In embodiments, the concentration of the small molecule compound or second agent used to induce peroxidase (e.g., VHPO) production is 0.1 weight per volume (w / v), 0.5% w / v or more, 1%, 2% w / v, or 5% w / v, and 25%, 20%, 15%, and 10% w / v or less. The host cell microorganism can be induced to produce peroxidase (e.g., VHPO) for 1 day or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 30 days or more. The duration of induction can depend on the size of the host cell microorganism culture, e.g., volume or cell number, the microorganism used, or the amount of peroxidase (e.g., VHPO) required. In one embodiment, the host cell microorganism is induced to produce peroxidase (e.g., VHPO) for 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, or 1 to 12 days.

[0119] Peroxidase (e.g., VHPO) production can be measured from a cell culture by measuring the level of peroxidase (e.g., VHPO) present in the cell culture and / or the activity of peroxidase (e.g., VHPO) produced by the cells. For example, the aqueous portion of the culture can be isolated, for example, by centrifuging the cell culture or an aliquot or sample of the cell culture. Protein assays known in the art, such as the Bradford assay or Nanodrop protein quantification, can be used to determine the total level or titer (e.g., g / L) of protein in the aqueous portion of the culture. The total protein titer indicates the amount of peroxidase (e.g., VHPO) produced by the microorganism or cell culture. A control sample can be used to normalize for the amount of protein present in a cell culture that has not been induced to produce peroxidase (e.g., VHPO).

[0120] Peroxidases (e.g., VHPOs) produced by host cell microorganisms as described herein can be useful in biological or industrial processes; for example, the peroxidase enzyme itself or the product of the peroxidase reaction can be used in ruminal compositions as described herein. Further described herein are methods for modulating ruminal characteristics, including reducing methane production within the rumen. Host cell microorganisms or cultures thereof induced to produce peroxidases (e.g., VHPOs) described herein can be added directly to the rumen. Alternatively, a processed small molecule compound or multiple small molecule organic compounds can be added directly to host cell microorganisms or cultures induced to produce peroxidases (e.g., VHPOs). In another embodiment, isolated halogenated organic compounds produced as products of peroxidase catalysis are added to the rumen as inhibitors of methane production. In another embodiment, halogenated organic compounds produced as products of peroxidase catalysis are delivered to the rumen via encapsulation in a matrix for methane production inhibition.

[0121] As described herein, peroxidases (e.g., VHPOs) produced by host cell microorganisms can also be separated or purified prior to use in cultures or ruminal samples. The peroxidases (e.g., VHPOs) can be separated from one or more of the following components: the microorganisms (e.g., microbial cells), the starting small organic compounds, the small organic halogenated compound products, the second agent, components of the cell culture medium (e.g., glucose, physiological salts), and one or more proteins present in the culture that do not have peroxidase activity. The peroxidases (e.g., VHPOs) can be purified so that they are substantially free of other proteins, cellular debris, nucleic acids, and the like that do not have peroxidase activity, for example, from the host cell microorganisms, the small organic compounds, and / or the agent. Methods for separating or purifying peroxidases (e.g., VHPOs) are known in the art and can include centrifugation, filtration, protein fractionation, size exclusion chromatography, affinity chromatography, ion exchange chromatography, or any combination thereof.

[0122] Halogenation of compounds The present disclosure features a method for producing small molecule halogenated organic compounds and related compositions using peroxidase. In one embodiment, the method includes (i) providing a small molecule organic compound or compounds, (ii) providing a peroxide source, e.g., HO or peracetic acid (PAA), and a halogen source, e.g., a halide salt such as KBr, NaCl, or KI, (iii) contacting the small molecule organic compound or compounds with a peroxidase, e.g., vanadium haloperoxidase (i.e., VHPO), or its reaction product, i.e., hypohalous acid, to form a reaction mixture under conditions sufficient to produce the small molecule halogenated organic compound or compounds, and (iv) evaluating the produced small molecule halogenated organic compound or compounds.

[0123] Small organic compounds can be naturally occurring or non-naturally occurring compounds. For example, small organic compounds can include natural products, lipids, sterols, steroids, amino acids, sugars, phlorotannins, tannins, lignin, or lignin derivatives. In one embodiment, small organic compounds include functional groups such as aldehyde groups, ketone groups, acetyl groups, acyl groups, hydroxyl groups, ester groups, ether groups, amine groups, amide groups, aryl groups, heteroaryl groups, heterocyclyl groups, or cycloalkyl groups. In one embodiment, small organic compounds include alkenyl or alkynyl groups. In one embodiment, small organic compounds include aldehyde or ketone groups. In one embodiment, small organic compounds include alpha-beta unsaturated ketones. In one embodiment, small organic compounds are acetone or acetylacetone.

[0124] Small organic compounds contain at least one carbon atom, e.g., at least two, three, four, five, six, seven, eight, nine, ten, twelve, or more carbon atoms. In certain embodiments, small organic compounds contain 1-10 carbon atoms, 1-6 carbon atoms, 2-10 carbon atoms, 3-10 carbon atoms, 4-10 carbon atoms, 5-10 carbon atoms, or 6-10 carbon atoms. In some embodiments, small organic compounds are saturated. In some embodiments, small organic compounds contain unsaturated elements, e.g., 2, 3, 4, 5, 6, 7, 8, or more unsaturated elements. In certain embodiments, small organic compounds further contain oxygen, nitrogen, sulfur, or phosphorus atoms. In certain embodiments, small organic compounds contain 1, 2, 3, 4, 5, or 6 oxygen atoms. In certain embodiments, small organic compounds contain 1, 2, 3, 4, 5, or 6 nitrogen atoms. In one embodiment, the small molecule organic compound contains 1, 2, 3, 4, 5, or 6 sulfur atoms. In one embodiment, the small molecule organic compound contains 1, 2, 3, 4, 5, or 6 phosphorus atoms.

[0125] In one embodiment, the small molecule organic compound has a molecular weight, or molecular mass, between about 15 Da and 1,500 Da, e.g., between about 15 Da and 1,250 Da, between about 15 Da and about 1,000 Da, between about 15 Da and about 750 Da, between about 15 Da and about 500 Da, between about 15 Da and about 250 Da, between about 15 Da and about 100 Da, between about 25 Da and 500 Da, between about 25 Da and 100 Da, between about 50 Da and 500 Da, between about 100 Da and 250 Da, between about 100 Da and 500 Da, or between about 100 Da and 1,000 Da.

[0126] In one embodiment, the small molecule organic compound comprises an aldehyde group, a ketone group, an acetyl group, an acyl group, a hydroxyl group, an ester group, an ether group, an amine group, an amide group, an aryl group, a heteroaryl group, a heterocyclyl group, or a cycloalkyl group. In one embodiment, the small molecule organic compound comprises an alkenyl group or an alkynyl group. In one embodiment, the small molecule organic compound comprises an aldehyde or a ketone group. In one embodiment, the small molecule organic compound comprises an alpha-beta unsaturated ketone. In one embodiment, the small molecule organic compound is acetone or acetylacetone. In one embodiment, the small molecule organic compound may comprise a natural product, a lipid, a sterol, a steroid, an amino acid, a sugar, a phlorotannin, a tannin, a lignin, or a lignin derivative.

[0127] In one embodiment, the small molecule organic compound is a compound of formula (Y): [ka] or a salt thereof, tautomer, or isomer thereof, wherein R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , and R 5care each independently hydrogen, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl is selected from the group consisting of one or more R 6 and optionally replaced with ;R 6 is halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR A , or -NR B R C and R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C2-C6 alkenyl, and R B and R C are each independently hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl, and m and n are each independently an integer of 0 to 24; [ka] is a single or double bond, [ka] is a double bond, R 2b and R 3b Each of these does not exist independently.

[0128] In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b , and R 5c are each independently hydrogen. In an embodiment of Formula (Y), m is selected from 0, 1, 2, or 3. In an embodiment of Formula (Y), n is selected from 0, 1, 2, or 3. In an embodiment of Formula (Y), [ka] is a single bond.

[0129] In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b , and R 5c are each independently hydrogen; m and n are each independently selected from 0, 1, 2, or 3; [ka] is a single bond. In one embodiment of Formula (Y), R 1a , R 1b , R 1c , R 5a , R 5b , and R 5c are each independently hydrogen, and each of m and n is 0. In an embodiment of Formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b , and R 5c are each independently hydrogen, n is 0, and m is 1; [ka] is a single bond. In one embodiment of formula (Y), R 1a is C l alkyl, and R 1b , R 1c , R 5a , R 5b , and R 5c are each independently hydrogen, and each of m and n is 0. In one embodiment of Formula (Y), R 1a is a halogen (e.g., chlorine, bromine). 1b , R1c , R 5a , R 5b , and R 5c are each independently hydrogen, and each of m and n is 0.

[0130] In one embodiment, a method comprises providing a peroxide source, e.g., PAA, to a VHPO (e.g., VCPO), e.g., a VHPO comprising a protein sequence motif listed in Table 1 or a VHPO sequence listed in Table 2. In one embodiment, the method comprises (i) providing a peroxide source, e.g., PAA, and a chloride anion to the VCPO to produce a hypochlorite anion or its conjugate acid. In one embodiment, the method comprises (i) providing a peroxide source, e.g., PAA, and a chlorine source to the VCPO to produce a hypochlorite anion or its conjugate acid, and (ii): providing the hypochlorite anion to an amine to produce a chlorinated amine, e.g., NH2Cl. In one embodiment, the method comprises: (i) providing a peroxide source, e.g., PAA, and a chlorine source to VCPO to produce hypochlorite anions or their conjugate acids; (ii) providing the hypochlorite anions to an amine to produce a chlorinated amine (e.g., NHCl); and (iii) reacting the chlorinated amine with iodate anions to produce hypoiodite anions or their conjugate acids. In one embodiment, the method comprises: (i) providing a peroxide source, e.g., PAA, and a chlorine source to VCPO to produce hypochlorite anions or their conjugate acids; and (ii-a) reacting the hypochlorite anions with iodate anions to produce hypoiodite anions or their conjugate acids. In one embodiment, the method comprises: (i) providing a peroxide source, e.g., PAA, and a chlorine source to VCPO to produce hypochlorite anions or their conjugate acids; (ii) providing the hypochlorite anions to an amine to produce a chlorinated amine (e.g., NHCl); (iii) reacting the chlorinated amine with iodate anions to produce hypoiodite anions or their conjugate acids; and (iv) reacting the hypoiodite anions with a small organic compound, dichloroacetic acid, to produce a small organic halogenated compound product, dichloroiodomethane.In one embodiment, the method is characterized by (i) providing a peroxide source, e.g., PAA, and a chlorine source to VCPO to produce hypochlorite anions or their conjugate acids; (ii-a): reacting the hypochlorite anions with iodate anions to produce a chlorinated amine (e.g., NHCl); (iii) reacting the chlorinated amine with iodate anions to produce hypoiodate anions or their conjugate acids; and (iv) reacting the small iodate anions with a small organic compound to produce the small organic halogenated compound product, dichloroiodomethane. In one embodiment, the method is characterized by (ii). In one embodiment, the method is characterized by (ii) and (iii). In one embodiment, the method is characterized by (ii), (iii), and (iv). In one embodiment, the method is characterized by (ii) and (iv). In one embodiment, the method is characterized by (i) and (iii). In one embodiment, the method is characterized by (ii), (iii), and (iv). In one embodiment, the method is characterized by (iii) and (iv). In one embodiment, the method is characterized by (ii-a). In one embodiment, the method is characterized by (ii-a) and (iv). In one embodiment, the method is characterized by (iii). In one embodiment, the method is characterized by (iv).

[0131] In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO produces diatomic iodine, I2, and triiodide anion, I3, from hypochlorite anion and excess iodate anion at a pH of 0-5. -The method is characterized in that the conversion rate of hypohalite anions and excess iodate anions to diatomic iodine I2 and triiodate anions I3 is reduced by 10% to 99% at pH 7 compared to a method involving the enzymatic reaction of H2O2 with VHPO at pH 0 to 5. In one embodiment, the method involves the enzymatic reaction of PAA with VCPO is characterized in that the conversion rate of hypohalite anions and excess iodate anions to diatomic bromine Br2 is reduced by 10% to 99% at pH 7 compared to a method involving the enzymatic reaction of H2O2 with VHPO at pH 0 to 5. In one embodiment, the method involves the enzymatic reaction of PAA and VCPO is characterized in that the conversion rate of hypohalite anions and bromide anions to diatomic bromine Br2 is reduced by 10% to 99% at pH 0 to 5 compared to a method involving the enzymatic reaction of H2O2 with VHPO at pH 0 to 5. In one embodiment, a method comprising an enzymatic reaction of PAA and VCPO is characterized by a 10% to 99% reduction in the conversion rate of hypochlorite anions and chloride anions to diatomic chlorine, Cl, at pH 0 to 5, compared to a method comprising an enzymatic reaction of H0 and VHPO at pH 0 to 5. In one embodiment, a method comprising an enzymatic reaction of PAA and VCPO is characterized by a 10% to 99% reduction in the conversion rate of peracetic acid and bromide anions to hypobromite anions or their conjugate acids at pH 0 to 5, compared to a method comprising an enzymatic reaction of H0 and VHPO at pH 0 to 5. In one embodiment, a method comprising an enzymatic reaction of PAA and VCPO is characterized by a 10% to 99% reduction in the conversion rate of peracetic acid and iodate anions to hypoiodite anions or their conjugate acids at pH 0 to 5, compared to a method comprising an enzymatic reaction of PAA and VHPO at pH 0 to 5.

[0132] In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO provides a faster conversion of iodate anion IO from hypochlorite and hypoiodite at pH 7-14 than the process comprising the enzymatic reaction of H2O2 with VHPO at pH 7-14. - and iodate anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized by a 10% to 99% reduction in the conversion of iodate anion IO2 from hypobromite and hypoiodite at pH 7 to 14. - and iodate anion IO3 -In one embodiment, the process involving the enzymatic reaction of PAA and VCPO is characterized by a 10% to 99% reduction in the conversion of iodide anions IO2 from hypoiodite and hypoiodite at pH 7 to 14. - and iodide anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO at pH 7 to 14 is characterized by a 10% to 99% reduction in the conversion of iodate anion IO2 from hypochlorite and hypoiodite to iodate anion IO2. - and iodate anion IO3 - In one embodiment, the enzymatic reaction of PAA with VCPO is characterized by a 10% to 99% reduction in the conversion of PAA to iodate anion IO2 by a method comprising the enzymatic reaction of HO2O2 with VCPO at a pH of 7 to 14. In another ... - and iodate anion IO3 - The method is characterized by a 10% to 99% reduction in the conversion of iodate anion IO2 from monochloramine and hypoiodite at pH 7 to 14 compared to a method involving the enzymatic reaction of PAA with VCPO. In one embodiment, the method involving the enzymatic reaction of PAA with VCPO at pH 7 to 14 is characterized by a 10% to 99% reduction in the conversion of iodate anion IO2 from monochloramine and hypoiodite at pH 7 to 14. - and iodate anion IO3 - In one embodiment, the process involving the enzymatic reaction of PAA and VCPO is characterized by a 10% to 99% reduction in the conversion of diatomic iodine, I, and hypoiodite to iodate anion, IO, at pH 7 to 14. - and iodate anion IO3 - The method is characterized by a 10% to 99% reduction in the conversion rate to VHPO compared to a method involving the enzymatic reaction of H2O2 with VHPO at pH 7 to 14.

[0133] In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO has a k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO has a k cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO has a k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO has a k cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 75% to 125% increase in k compared to the process comprising the enzymatic reaction of H2O2 with VCPO. cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO has an 80% to 120% increase in k compared to the method comprising the enzymatic reaction of H2O2 with VCPO. cat It is characterized by an increase of 90% to 110%.

[0134] In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO has a k cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 10% to 500% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dichloroiodomethane. cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 25% to 250% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dichloroiodomethane. cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 50% to 150% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dichloroiodomethane. catIn one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 75% to 125% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dichloroiodomethane. cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by an 80% to 120% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dichloroiodomethane. cat In one embodiment, the process involving the enzymatic reaction of PAA with VCPO is characterized by a 90% to 110% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO, which produces dibromochloromethane. cat In one embodiment, the process involving the enzymatic reaction of PAA with VCPO is characterized by a 10% to 500% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO, which produces dibromochloromethane. cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 25% to 250% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dibromochloromethane. cat In one embodiment, the process involving the enzymatic reaction of PAA with VCPO is characterized by a 50% to 150% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO, which produces dibromochloromethane. cat In one embodiment, the process comprising the enzymatic reaction of PAA with VCPO is characterized by a 75% to 125% increase in k compared to a process comprising the enzymatic reaction of H2O2 with VCPO, which produces dibromochloromethane. cat In one embodiment, the process involving the enzymatic reaction of PAA with VCPO is characterized by an 80% to 120% increase in k compared to a process involving the enzymatic reaction of H2O2 with VCPO, which produces dibromochloromethane. cat It is characterized by an increase of 90% to 110%.

[0135] As described herein, peroxidases (e.g., VHPO) can be used to convert small organic compounds to small halogenated organic compounds, for example, in the presence of a halogen source. In one embodiment, the small halogenated organic compounds include natural products, lipids, sterols, steroids, amino acids, sugars, phlorotannins, tannins, lignin, or lignin derivatives, and are chlorinated, brominated, or iodinated. In one embodiment, the small halogenated organic compounds are brominated. In one embodiment, the small halogenated organic compounds include functional groups, such as aldehyde groups, ketone groups, acetyl groups, acyl groups, hydroxyl groups, ester groups, ether groups, amine groups, amide groups, aryl groups, heteroaryl groups, heterocyclyl groups, or cycloalkyl groups. In one embodiment, the small halogenated organic compounds include alkenyl or alkynyl groups. In one embodiment, the small halogenated organic compounds include aldehyde or ketone groups. In one embodiment, the small halogenated organic compounds include alpha-beta unsaturated ketones. In one embodiment, the small molecule halogenated organic compound contains 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one embodiment, the small molecule halogenated organic compound contains 1, 2, or 3 halogen atoms. In one embodiment, the small molecule halogenated organic compound contains 1, 2, or 3 bromine atoms. In one embodiment, the small molecule halogenated organic compound contains an acetone moiety. In one embodiment, the small molecule halogenated organic compound includes dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone. In one embodiment, the small molecule halogenated organic compound includes 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone. In one embodiment, the small molecule halogenated compound includes dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.In one embodiment, the small molecule halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone, dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane.

[0136] The small molecule halogenated organic compound can contain any halogen atom, such as chlorine, bromine, or iodine. In one embodiment, the small molecule halogenated organic compound contains 1, 2, 3, 4, 5, 6, or more halogen atoms. In one embodiment, the small molecule halogenated organic compound contains 1, 2, 3, 4, 5, 6, or more chlorine atoms. In one embodiment, the small molecule halogenated organic compound contains 1, 2, 3, 4, 5, 6, or more bromine atoms. In one embodiment, the small molecule halogenated organic compound contains 1, 2, 3, 4, 5, 6, or more iodine atoms. In one embodiment, the small molecule halogenated organic compound includes bromoacetone, dibromoacetone, bromopentanedione, bromoform, tribromoacetone, or a variant or analog thereof. In one embodiment, the small molecule halogenated organic compound includes 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone, or a variant or analog thereof. In one embodiment, the small molecule halogenated organic compound includes a halomethane, such as a dihalomethane or trihalomethane. In one embodiment, the small molecule halogenated organic compound includes a haloacetone, such as a dihaloacetone or trihaloacetone. In one embodiment, the small molecule halogenated organic compound includes 1,1-dibromoacetone, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone, or a variant or analog thereof. In one embodiment, the small molecule halogenated organic compound includes bromomethane, dibromethane, bromoform, or a variant or analog thereof.

[0137] In another embodiment, the small molecule halogenated organic compound is a compound of formula (Z): [ka] or a salt thereof, a tautomer, or an isomer thereof, wherein R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , and R 5c is independently hydrogen, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, or heterocyclyl, wherein each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl is selected from one or more R 6 and optionally replaced with ;R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5a , R 5b , and R 5c At least one of R is independently halogen; 6 is halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR A , or -NR B R C and R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C2-C6 alkenyl, and R B and R C are each independently hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl; m and n are each independently selected from 0, 1, 2, or 3; [ka] is a single or double bond, [ka] is a double bond, R 2b and R 3b Each of these does not exist independently.

[0138] In one embodiment of Formula (Z), R 1a , R 1b , and R 1c are independently halogen or hydrogen, and R 1a , R 1b , and R 1c At least one of R is a halogen. In one embodiment, the halogen is selected from chlorine, bromine, or iodine. In one embodiment of Formula (Z), R 1a , R 1b , and R 1c are independently halogen or hydrogen, and R 1a , R 1b , and R 1c At least two of are halogen. In one embodiment, the halogen is selected from chlorine, bromine, or iodine. In one embodiment of Formula (Z), R 1a , R 1b , R 1c is independently a halogen. In one embodiment, the halogen is selected from chlorine, bromine, or iodine. In one embodiment of Formula (Z), R 5a , R 5b , and R 5c are independently halogen or hydrogen, and R 5a , R 5b , and R 5c At least one of R is halogen. 5a , R 5b , and R 5c are independently halogen or hydrogen, and R 5a , R 5b , and R 5c At least two of R are halogen. 5a , R 5b , R5c Each of is independently a halogen. In some embodiments of Formula (Z), [ka] In some embodiments of Formula (Z), each of m and n is independently selected from 0, 1, 2, or 3; [ka] is a single bond.

[0139] The peroxidases described herein (e.g., VHPOs) can regulate the production of small halogenated organic compounds based on the reaction conditions. For example, under a first set of reaction conditions, the peroxidase can produce a first small halogenated organic compound, and under a second set of reaction conditions, the peroxidase can produce a second halogenated organic compound. Furthermore, the peroxidases described herein (e.g., VHPOs) can regulate the ratio of a particular small halogenated organic compound to another compound within the plurality of small halogenated organic compounds, depending on the reaction conditions. For example, under a first set of reaction conditions, the peroxidase can produce a first ratio of small halogenated organic compounds to another small halogenated organic compound within the plurality of compounds, and under a second set of reaction conditions, the peroxidase can produce a second ratio of small halogenated organic compounds to another small halogenated organic compound within the plurality of compounds.

[0140] In one embodiment, the peroxidase (e.g., VHPO) capable of modulating the production of small halogenated organic compounds is a peroxidase derived from an algae species. In one embodiment, the peroxidase (e.g., VHPO) capable of modulating the production of small halogenated organic compounds is a peroxidase derived from a fungal species. In one embodiment, the peroxidase (e.g., VHPO) capable of modulating the production of small halogenated organic compounds is a peroxidase derived from a cyanobacterial species. In one embodiment, the peroxidase (e.g., VHPO) capable of modulating the production of small halogenated organic compounds is a peroxidase derived from C. inaequalis, C. officinalis, N. carneum, H. hongdechloris, M. bouillonii, T. erythraeum, A. montana, Synechococcus, S. cerevisiae, L. confervoides, or Lyngbya. In one embodiment, the peroxidase (e.g., VHPO) capable of modulating the production of small halogenated organic compounds has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, for example, an amino acid sequence listed in Table 1 provided herein. In one embodiment, the peroxidase (e.g., VHPO) capable of modulating the production of small halogenated organic compounds is produced in a host cell microorganism described herein, e.g., P. pastoris, A. niger, or S. cerevisiae.

[0141] The halogenation of the small molecule organic compound or compounds occurs under certain conditions, e.g., conditions sufficient for halogenation. In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound or compounds include a temperature of 0°C to 85°C, e.g., about 4°C to about 65°C, about 10°C to about 65°C, and about 25°C to about 50°C. In one embodiment, the conditions sufficient to produce the small molecule halogenated organic compound or compounds include a pH of about 2 to about 10, e.g., about 2 to about 9, about 2 to about 8, about 4 to about 10, about 4 to about 8, or about 5 to about 8.

[0142] Enumerated Embodiments 1. A method for modulating the production of small molecule halogenated organic compounds using a peroxidase enzyme, comprising: (i) providing a low molecular weight organic compound (e.g., acetylacetone); (ii) contacting the small molecule organic compound with a peroxidase (e.g., VHPO) to form a reaction mixture under conditions sufficient to produce a small molecule halogenated organic compound; (iii) evaluating the small molecule halogenated organic compound produced, and thereby regulating the production of the small molecule halogenated organic compound.

[0143] 2. The method of embodiment 1, wherein the peroxidase is a haloperoxidase.

[0144] 3. The method of embodiment 2, wherein the haloperoxidase is a vanadium haloperoxidase (VHPO).

[0145] 4. The method of embodiment 3, wherein said VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO), or vanadium iodoperoxidase (VIPO).

[0146] 5. The method of any one of embodiments 3-4, wherein the VHPO is VBPO.

[0147] 6. The method of any one of the preceding embodiments, wherein the peroxidase is an algal haloperoxidase (e.g., derived from an algal species), a fungal haloperoxidase (e.g., derived from a fungal species), or a cyanobacterial haloperoxidase (e.g., derived from a cyanobacterium).

[0148] 7. The method of any one of embodiments, wherein the peroxidase is a fungal haloperoxidase (e.g., derived from a fungal species).

[0149] 8. The method of any one of the preceding embodiments, wherein the peroxidase is derived from an organism selected from Curvularia inaequalis, Halomicronema hongdecroris, Moorea bouillonii, Trichodesmium erythraeum, Aphanocapsa montana, Lyngbya confervoides, Synechococcus sp. PCC7335, and Corallina officinalis.

[0150] 9. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Corallina officinalis.

[0151] 10. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Aphanocapsa montana.

[0152] 11. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Curvularia inaequalis.

[0153] 12. The method of any one of the preceding embodiments, wherein the peroxidase is produced in a host cell microorganism.

[0154] 13. The method of any one of the preceding embodiments, wherein the host cell microorganism is selected from Pichia pastoris, Aspergillus niger, Saccharomyces cerevisiae, or Escherichia coli.

[0155] 14. The method of any one of the preceding embodiments, wherein expression of the peroxidase produced in the host cell microorganism is about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or, for example, 10-fold higher than the peroxidase produced in its native host.

[0156] 15. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase comprises a motif selected from the protein sequence motifs presented in Table 1.

[0157] 16. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase is selected from the amino acid sequences listed in Table 2.

[0158] 17. The method of any one of the preceding embodiments, wherein the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from the list in Table 2.

[0159] 18. The method of any one of the preceding embodiments, wherein the peroxidase is a sequence selected from any one of SEQ ID NOs: 1-50.

[0160] 19. The method of any one of the preceding embodiments, wherein the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase selected from SEQ ID NOs: 1-50.

[0161] 20. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase has 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to an amino acid sequence selected from any one of SEQ ID NOs: 1-50.

[0162] 21. The method of any one of the preceding embodiments, wherein the small molecule organic compound comprises 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0163] 22. The method of any one of the preceding embodiments, wherein the small molecule organic compound comprises a ketone or an aldehyde.

[0164] 23. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound is chlorinated, brominated, iodinated, chlorinated and iodinated, chlorinated and brominated, brominated and iodinated, or chlorinated, brominated, and iodinated.

[0165] 24. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound is brominated.

[0166] 25. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises one, two, or three halogen atoms.

[0167] 26. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound contains 1, 2, or 3 bromine atoms.

[0168] 27. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises an acetone moiety.

[0169] 28. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone.

[0170] 29. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises dichloroiodomethane, dibromochloromethane, 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone.

[0171] 30. The method of any one of the preceding embodiments, further comprising providing a peroxide source, e.g., H2O2 or PAA, and a halogen source, e.g., a halide salt such as KBr, NaCl, or KI.

[0172] 31. The conditions sufficient to produce the small molecule halogenated organic compound include: (a) Temperatures between 10℃ and 85℃ (b) pH 4 to 10, and (c) an ionic strength of 0.1 mM to 4 M.

[0173] 32. The method of embodiment 31, wherein the conditions sufficient to produce the small molecule halogenated organic compound comprise a temperature of 10°C to 85°C.

[0174] 33. The method of any one of embodiments 31-32, wherein the conditions sufficient to produce the small molecule halogenated organic compound comprise a pH between 4 and 10.

[0175] 34. The method of any one of embodiments 31-33, wherein the conditions sufficient to produce the small molecule halogenated organic compound comprise an ionic strength of 0.1 mM to 4 M.

[0176] 35. The method of any one of the preceding embodiments, wherein said assessing comprises analyzing said small molecule halogenated organic compounds by analytical techniques.

[0177] 36. The method of embodiment 45, wherein the analytical techniques include HPLC, GC-MS, and NMR.

[0178] 37. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound is capable of reducing methane production by microorganisms.

[0179] 38. The method of embodiment 36, wherein methane production is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.

[0180] 39. The method of any one of embodiments 37-38, wherein the methane production is reduced by 10-75%.

[0181] 40. The method of any one of embodiments 37-39, wherein the microorganism is present in the rumen community.

[0182] 41. A method for reducing methane production in a rumen community, comprising: (i) providing a low molecular weight organic compound (e.g., acetylacetone); (ii) contacting a small molecule organic compound with a peroxidase (e.g., VHPO) to form a reaction mixture under conditions sufficient to produce a small molecule halogenated organic compound; (iii) separating the small molecule halogenated organic compound from the reaction mixture; and (iv) providing said small molecule halogenated organic compound to a rumen community under conditions sufficient to reduce said production of methane.

[0183] 42. The method of embodiment 41, further comprising obtaining a value for the level of methane (a) before providing the peroxidase or (b) after providing the peroxidase.

[0184] 43. The method of embodiment 42, comprising (a).

[0185] 44. The method of embodiment 42, comprising (b).

[0186] 45. The method of embodiment 42, comprising (a) and (b).

[0187] 46. ​​The method of any one of embodiments 41-45, wherein methane production is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.

[0188] 47. The method of any one of embodiments 41 to 46, wherein the methane production is reduced by 10 to 75%.

[0189] 48. The method of any one of embodiments 40 to 46, wherein the peroxidase is a haloperoxidase.

[0190] 49. The method of embodiment 47, wherein the haloperoxidase is a vanadium haloperoxidase (VHPO).

[0191] 50. The method of embodiment 49, wherein said VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO), or vanadium iodoperoxidase (VIPO).

[0192] 51. The method of any one of embodiments 49-50, wherein the VHPO is VBPO.

[0193] 52. The method of any one of the preceding embodiments, wherein the peroxidase is an algal haloperoxidase (e.g., derived from an algal species) or a fungal haloperoxidase (e.g., derived from a fungal species).

[0194] 53. The method of any one of the preceding embodiments, wherein the peroxidase is a fungal haloperoxidase (e.g., derived from a fungal species).

[0195] 54. The method of any one of the preceding embodiments, wherein the peroxidase is derived from an organism selected from Curvularia inaequalis, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium erythraeum, Aphanocapsa montana, Lyngbya confervoides, Synechococcus sp. PCC 7335, Corallina officinalis, and Saccharomyces cerevisiae.

[0196] 55. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Corallina officinalis.

[0197] 56. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Aphanocapsa montana.

[0198] 57. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Curvularia inaequalis.

[0199] 58. The method of any one of the preceding claims, wherein the peroxidase is produced in a host cell microorganism.

[0200] 59. The method of any one of the preceding embodiments, wherein the host cell microorganism is selected from Pichia pastoris, Aspergillus niger, or Escherichia coli.

[0201] 60. The method of any one of the preceding embodiments, wherein expression of the peroxidase produced in the host cell microorganism is about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or, for example, 10-fold higher than the peroxidase produced in its native host.

[0202] 61. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase is selected from the amino acid sequences listed in Table 2.

[0203] 62. The method of any one of the preceding embodiments, wherein the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase sequence selected from the list in Table 2.

[0204] 63. The method of any one of the preceding embodiments, wherein the peroxidase is a sequence selected from any one of SEQ ID NOs: 1-50.

[0205] 64. The method of any one of the preceding embodiments, wherein the peroxidase has at least 75% sequence identity (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99%, or 99.5% sequence identity) to a peroxidase selected from SEQ ID NOs: 1-50.

[0206] 65. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase has 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to an amino acid sequence selected from any one of SEQ ID NOs: 1-50.

[0207] 66. The method of any one of the preceding embodiments, wherein the small molecule organic compound contains 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

[0208] 67. The method of any one of the preceding embodiments, wherein the small molecule organic compound comprises a ketone or an aldehyde.

[0209] 68. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound is chlorinated, brominated, or iodinated.

[0210] 69. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound is brominated.

[0211] 70. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises one, two, or three halogen atoms.

[0212] 71. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound contains 1, 2, or 3 bromine atoms.

[0213] 72. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises an acetone moiety.

[0214] 73. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone.

[0215] 74. The method of any one of the preceding embodiments, wherein the small molecule halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone.

[0216] 75. The method of any one of embodiments 40-73, wherein the conditions sufficient to produce the small molecule halogenated organic compound comprise a pH between 4 and 10.

[0217] 76. The method of any one of embodiments 40-74, wherein the conditions sufficient to produce the small molecule halogenated organic compound comprise an ionic strength of 0.1 mM to 4 M.

[0218] 77. The method of any one of the preceding embodiments, further comprising evaluating the produced small molecule halogenated organic compounds, e.g., by analytical techniques.

[0219] 78. The method of embodiment 77, wherein the analytical techniques include HPLC, GC-MS, and NMR.

[0220] 79. A method for increasing the expression of peroxidase in a host cell or host microorganism.

[0221] 80. A reactor for the continuous production of low molecular weight halogenated organic compounds, comprising: (i) a reaction chamber; (ii) a module for temperature control; (iii) a peristaltic pump; and (iv) a module for containing a catalyst.

[0222] 81. A reactor for the continuous production of low molecular weight halogenated organic compounds according to claim 79, comprising: (i) a device for adjusting the internal atmosphere; (ii) an impeller for agitating the reaction mixture; (iii) a plurality of sensors for monitoring the reaction.

[0223] 82. The method of any of the preceding embodiments for modulating the production of small molecule halogenated organic compounds, further comprising providing the peroxide source (e.g., peracetic acid) and a halogen source (e.g., a bromine source, a chlorine source, or an iodine source, e.g., a chlorinated salt such as KCl or NaCl) to the peroxidase (e.g., VCPO).

[0224] 84. The method of any of the preceding embodiments for modulating the production of small molecule halogenated organic compounds, comprising: (i) providing a peroxide source, e.g., PAA, and a chlorine source to VCPO to produce hypochlorite anion or its conjugate acid; (ii) donating hypochlorite anions to an amine to produce a chlorinated amine (e.g., NH2Cl); (iii) reacting the chlorinated amine with iodate anion to produce hypoiodite anion or its conjugate acid; and (iv) reacting the hypoiodite anion with the small molecule organic compound dichloroacetic acid to produce the small molecule halogenated organic compound product dichloroiodomethane.

[0225] 85. A method for controlling the production of low molecular weight halogenated organic compounds, comprising the steps of: hypochlorite anion and excess iodate anion, diatomic iodine I2 and triiodide anion I3 at pH 0-5. - 85. The method of any one of embodiments 83 to 84, wherein the rate of conversion to is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO as described in claim 1 at a pH of 0 to 5.

[0226] 86. A method for preparing low molecular weight halogenated organic compounds, comprising the steps of: diatomic iodine I2 and triiodide I3 with hypochlorite anion and excess iodide anion at pH 7. - 85. The method of any one of embodiments 88 to 84, wherein the conversion to is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO at pH 7.

[0227] 87. The method of any of embodiments 83-84 for modulating the production of small molecule halogenated organic compounds, wherein the conversion of hypochlorite anions and bromide anions to diatomic bromine, Br2, at pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO4 of claim 1 at pH 0-5.

[0228] 88. The method of any of embodiments 83-84 for modulating the production of low-halogenated organic compounds, wherein the conversion rate of hypochlorite anions and perchlorate anions to diatomic chlorine, Cl2, at a pH of 0-5 is reduced by 10% to 99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO of claim 1 at a pH of 0-5.

[0229] 89. The method of any of embodiments 83-84 for modulating the production of small molecule halogenated organic compounds, wherein the conversion rate of peracetic acid and bromide anions to hypobromite anions or their conjugate acids at pH 0-5 is reduced by 10%-99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO4 of claim 1 at pH 0-5.

[0230] 90. The method of any of embodiments 83-84 for modulating the production of small molecule halogenated organic compounds, wherein the conversion rate of peracetic acid and iodide anions to hypoiodite anions or their conjugate acids at pH 0-5 is reduced by 10%-99% compared to a method comprising the enzymatic reaction of PAA with VHPO according to claim 1 at pH 0-5.

[0231] 91. The method according to any one of embodiments 83-90 for modulating the production of small molecule halogenated organic compounds, comprising: (i) Hypochlorite anion and excess iodate anion to diatomic iodine I2 and triiodide anion I3 at pH 0-5 - the conversion rate to is reduced by 10% to 99% compared to the method comprising the enzymatic reaction with H2O2 and VHPO as described in claim 1 at a pH of 0 to 5; (ii) Hypochlorite anion and excess iodate anion to diatomic iodine I2 and triiodide anion I3 at pH 7 - the conversion rate to is reduced by 10% to 99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO as described in claim 1 at pH 7; (iii) the conversion rate of hypochlorite anion and bromide anion to diatomic bromine BrI2 at pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 as set forth in claim 1 at pH 0-5; (iv) the rate of conversion of hypochlorite anions and chloride anions to diatomic chlorine, Cl, at pH 0-5 is reduced by 10% to 99% compared to the method comprising the enzymatic reaction with H0 and VHPO of claim 1 at pH 0-5; (v) the rate of conversion of peracetic acid and bromide anions to hypobromite anions or their conjugate acids at pH 0-5 is reduced by 10% to 99% compared to the method comprising the enzymatic reaction of HO with VHPO as set forth in claim 1 at pH 0-5; or (i) The method, wherein the conversion rate of peracetic acid and iodide anions to hypoiodite anions or their conjugate acids at pH 0 to 5 is the enzymatic reaction of PAA with VHPO according to claim 1 at pH 0 to 5, or a combination thereof.

[0232] 92. A method for controlling the production of low molecular weight halogenated organic compounds, comprising the steps of: hypochlorite and hypoiodite at pH 7-14; and hypochlorite anion IO2. - and iodate anion IO3 - 85. The method according to any one of embodiments 83-84, wherein the conversion rate to is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO as claimed in claim 1 at a pH of 7 to 14.

[0233] 93. A method for controlling the production of low molecular weight halogenated organic compounds, comprising the steps of: hypobromite and hypoiodite; hypoiodite anion (IO2) at pH 7-14; - and iodate anion IO3 - 85. The method of any of embodiments 83-84, wherein the conversion of Br2 to diatomic bromine is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO at pH 7-14.

[0234] 94. A method for controlling the production of low molecular weight halogenated organic compounds, comprising the step of converting hypoiodite and hypoiodite to hypoiodite anion IO2 at pH 7-14. - and iodate anion IO3 - 85. The method according to any one of embodiments 83-84, wherein the conversion rate to is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO as described in claim 1 at a pH of 7 to 14.

[0235] 95. A method for controlling the production of low molecular weight halogenated organic compounds, comprising the steps of: (a) reacting hypochlorite anion and hypoiodite anion with iodate anion IO2 at pH 7-14; - and iodate anion IO3 - 85. The method according to any one of embodiments 83-84, wherein the conversion to is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 with VHPO as claimed in claim 1 at a pH of 7 to 14.

[0236] 96. The method according to any one of embodiments 83-84 for modulating the production of small molecule halogenated organic compounds, comprising: (i) Iodate anion IO2 for hypochlorite and hypoiodous acid at pH 7-14 - and iodate anion IO3 - the conversion rate to VHPO is reduced by 10% to 99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO as described in claim 1 at a pH of 7 to 14; (ii) Iodate anion IO2 from hypobromite and hypoiodite at pH 7-14 - and iodate anion IO3 - the conversion rate to VHPO is reduced by 10% to 99% compared to the method involving the enzymatic reaction of H2O2 with VHPO at pH 7 to 14; (iii) Hypoiodite and hypoiodite to hypoiodite anion IO2 at pH 7-14 - and iodate anion IO3 - the conversion rate to is reduced by 10% to 99% compared to a method comprising the enzymatic reaction of H2O2 and VHPO, or a combination thereof, at a pH of 7 to 14; The method.

[0237] 97. The method comprising the enzymatic reaction of PAA and VCPO. cat The k of the method includes an enzymatic reaction of H2O2 with VHPO cat 97. The method according to any one of embodiments 82 to 96, for modulating the production of small molecule halogenated organic compounds by 10% to 500% higher compared to the method according to any one of embodiments 82 to 96.

[0238] 98. The method comprising the enzymatic reaction of PAA and VCPO. cat k of the method comprising an enzymatic reaction of H2O2 with VHPO cat 98. The method according to any one of embodiments 82 to 97, for modulating the production of small molecule halogenated organic compounds by 25% to 250% compared to

[0239] 99. The method comprising the enzymatic reaction of PAA and VCPO. cat The k of the method includes an enzymatic reaction of H2O2 with VHPO cat 99. The method according to any one of embodiments 82 to 98, for modulating the production of small molecule halogenated organic compounds by 50% to 150% compared to

[0240] 100. The method of any of embodiments 82-99 for modulating the production of small molecule halogenated organic compounds, wherein the kcat of the method comprising the enzymatic reaction of PAA with VCPO is 75% to 125% higher than the kcat of the method comprising the enzymatic reaction of H2O2 with VCPO.

[0241] 101. The method of any of embodiments 82-100 for modulating the production of small molecule halogenated organic compounds, wherein the kcat of the method comprising the enzymatic reaction of PAA with VCPO is 80% to 120% higher than the kcat of the method comprising the enzymatic reaction of H2O2 with VCPO.

[0242] 102. The method according to any of embodiments 82-101 for modulating the production of small molecule halogenated organic compounds, wherein the kcat of the method comprising the enzymatic reaction of PAA with VCPO is 90% to 110% higher than the kcat of the method comprising the enzymatic reaction of H2O2 with VCPO.

[0243] 103. The method of any of embodiments 82-102 for modulating the production of the small molecule halogenated compound, wherein the product is dichloroiodomethane.

[0244] 104. The method of any of embodiments 82-102 for modulating the production of the small halogenated compound, wherein the product is dibromochloromethane.

[0245] 105. The method of any of embodiments 82-102 for modulating the production of small halogenated compounds, wherein the products are dichloroiodomethane and dibromochloromethane. [Example]

[0246] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present disclosure should in no way be construed as limited to the following examples, but rather to encompass any and all variations that become apparent as a result of the teachings provided herein. Without further description, it is believed that one of ordinary skill in the art will be able, using the foregoing description and the following illustrative examples, to make and utilize the compositions, methods, and devices of the present disclosure, and to practice the claimed methods. The following examples illustrate various aspects of the present disclosure, but are not to be construed as limiting in any way the remainder of the disclosure.

[0247] Example 1: Heterologous expression and purification of an exemplary vanadium haloperoxidase from Escherichia coli Exemplary vanadium haloperoxidase (VBPO) genes were overexpressed in Escherichia coli according to the following procedure. The complete nucleotide sequences of various VBPOs were chemically synthesized using a commercial vendor (GeneScript) and cloned into the pET-28a(+) vector, suitable for E. coli protein expression. The resulting proteins were designed to contain HRV 3C protease recognition signals at the N- and C-termini and a 6x-His tag at the C-terminus to enable affinity purification. After culturing E. coli cells using standard techniques, recombinant VBPOs were purified to homogeneity using a two-step protocol involving an affinity column-based protein purification step (HisTrap FF Crude) and a second size-exclusion step (Amicon™ ultracentrifugal filter units 100 kDa).

[0248] Example 2: Purification and kinetic characterization of an exemplary vanadium haloperoxidase from Corallina officinalis This example provides the purification of VBPO naturally produced in the seaweed Corallina officinalis (CoVBPO). After harvesting, the seaweed was quickly frozen in liquid nitrogen and ground into fine particles to prepare a crude extract. Next, a 1:5 Tris sulfate 0.1 M, pH 8.5 buffer solution was added. After centrifugation, the supernatant constituted the crude extract. CoVBPO was purified using a three-step protocol summarized in Table 3. Analysis by SDS-PAGE showed that the purity of the final enzyme preparation was greater than 90%.

[0249] [Table 3]

[0250] Next, CoVBPO was investigated to determine various kinetic parameters based on 3D fitting using the Cleland equation, with compositions of CoVBPO in MOPS buffer pH = 7.0 and MES buffer pH = 6.5 with ionic strengths of 0.2 and 2.0 M, respectively.

[0251] All kinetic measurements were performed based on the analysis of the initial rates of an exemplary reaction, namely the dearylation of aminophenylfluorescein (APF) to fluorescein (Archer et al. 2019). Fluorescence was measured using a Photon Technology International (PTI) QuantaMaster fluorometer. The excitation wavelength was set at 490 nm, and emission was recorded at 515 nm with a parallel photomultiplier tube detection system. Lamp power was 74 W.

[0252] As shown in Figures 2A-2C, in the ping-pong reaction system, the first product P1 (HO) is released after the first substrate (hydrogen peroxide) binds to the enzyme molecule and before the second substrate (bromide) binds. Surface fitting of the experimental kinetic data was performed using the nonlinear multiparametric equation shown in Figure 2B. A nonlinear multiparametric equation, in which substrate inhibition leads to the formation of one final complex, was formulated using the King-Altman method (see, for example, Figure 2C). All fitting procedures and goodness of fit were performed using OriginPro 2021. In all cases, an unweighted least-squares test was used as the convergence criterion. Tables 4 and 5 summarize the resulting parameters determined by this analysis at both ionic strengths tested.

[0253] [Table 4]

[0254] [Table 5]

[0255] These results indicate that CoVBPO may proceed via a ping-pong mechanism, which involves lethal inhibition of the hydrogen peroxide substrate. At low ionic strength (0.2 M), the turnover parameter (k cat ) was observed. Furthermore, the parameter Km H2O2 and km Br Estimates of Km indicated a 4.5-fold increase in affinity of VBPO for hydrogen peroxide and an over one order of magnitude (12-fold) increase in affinity for bromide at high ionic strength, respectively. The inhibition constant for hydrogen peroxide was Km H2O2 follows a similar pattern.

[0256] Example 3: Effect of pH and Temperature on Exemplary VBPOs Using the CoVBPO enzyme, the pH dependence of the Michaelis-Menten reaction rate was investigated in high and low ionic strength media. The objectives were to: i) increase the pH of the second substrate (bromide, Br), a reaction aspect that is poorly understood and prevents the utilization of VHPO; - ) binding mechanism, and ii) the corresponding K mBr- The goal was to reduce

[0257] The presence of high ionic strength in solution disrupts the ionic interactions that occur among the solvent-accessible amino acids of the enzyme. - The affinity for β decreases at higher pH values ​​(although K mBr (increase in ) (Figure 3A).

[0258] Example 4: Characterization of Halogenated Organic Compounds Produced by Exemplary VBPOs This example demonstrates how careful adjustment of VBPO reaction conditions can favor the production of certain halogenated compounds over others. Acetylacetone was incubated with VBPO at a nearly constant concentration in a fed-batch bioreactor. The VBPO substrates HO and KBr were simultaneously added to the bioreactor in a controlled manner via a peristaltic pump. Reaction aliquots were removed over time, and the reaction profile was analyzed by GC-MS to monitor the reaction product concentrations. As shown in Figure 4A, the primary product produced was 1,1-dibromoacetone, which accumulated to 50 mM. Small amounts of other halogenated compounds were produced, including bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, and 1,1,1-tribromoacetone. Naphthalene was used as an internal standard to determine halogenated analyte concentrations.

[0259] The resulting product was purified in batch mode using HPLC. An exemplary HPLC chromatogram is shown in Figure 4B. After HPLC fractionation, 1,1-dibromoacetone was extracted with n-hexane and further analyzed by GC-MS. The identity of the n-hexane-extracted product was confirmed by comparing its MS spectrum with that of a standard from the NIST library (Figure 4C).

[0260] Example 5: Inhibition of methane production by exemplary small molecule halogenated organic compounds in rumen-derived microbial communities Figure 5 shows the production and testing process for small organic halide compounds for the inhibition of methane production. In this example, small organic halide compounds produced by VBPO were incubated in a microbial culture system derived from ruminal fluid obtained from dairy cows to measure their relative ability to inhibit methane production. Briefly, 1,1-dibromoacetone, the co-product bromoform, and the derivative dibromomethane were added separately to the culture system. The rate of methane production was monitored in each reaction (Figures 6A-6C).

[0261] As shown in Figures 6A-6C, 1,1-dibromoacetone was the least effective compound screened for inhibiting methane production. 1,1-dibromoacetone produced a 50% inhibition of methane production at 125 μM in the rumen-derived microbial community tested, compared to bromoform, which showed a 50% inhibition of methane production at 0.20 μM, and dibromomethane, which showed a 50% inhibition of methane production at 0.15 μM.

[0262] Example 6: Heterologous production of cchVBPO in Escherichia coli This example describes the heterologous expression of Chrondora crispus VBPO (cchVBPO) in Escherichia coli: inoculation and cultivation of the model microorganism in a bioreactor, induction of cchVBPO production, protein extraction, purification, and biochemical characterization.

[0263] Seeding and induction Briefly, 4 x 5 ml of LB medium containing kanamycin (4.0 μl, maintained at approximately 0°C) was inoculated into a culture tube (15 ml) with a single colony from a kanamycin + agar plate (or 5-10 μl from a glycerol stock). The inoculum was then incubated at 37°C with shaking at 250 rpm overnight, i.e., after 15-18 hours. OD600 The β-glucan concentration (βg) was measured to be approximately 0.4-0.6 (200 μl in a multiwell plate). The culture (4% inoculum / 20 ml) was then added to a 0.5 L bioreactor (preheated in 37°C medium) containing 400 μl of kanamycin ([kanamycin] = 40 μg / ml, stock solution 50 mg / ml). OD600 The mixture was shaken at 200 rpm at 37°C until the OD reached approximately 0.6-0.7. 600 The OD was monitored during growth by aseptically removing aliquots. 600If the RI was between 0.6 and 0.7, the culture was cooled to 18°C ​​and 0.1 mM IPTG (0.5 ml of 100 mM IPTG from stock) was added. A 3 ml sample was taken and used as t = 0 time (no induction). The culture was incubated overnight at 18°C ​​and stirred at 200 rpm under oxygenated conditions. It was then centrifuged at 10,000 rpm for 10 minutes at 4°C in a pre-weighed 500 ml centrifuge tube (whereupon the pellet was expelled by inversion and excess medium was tapped on a paper towel). The sample was then kept on ice.

[0264] Extraction and preparation The pellet from the previous step was resuspended in BugBuster® Master (commercially available from EMD Millipore) using 5 ml of 1x reagent per gram of wet cell paste. Five grams of cell paste was gently mixed with 22.5 ml of 20 mM borate buffer, pH 8.0, 30 mM imidazole (binding buffer) by light vortexing. 2.5 ml of 10x BugBuster® and one round of protease inhibitor were then added to the mixture. Protease inhibitors were optionally included due to their compatibility with the BugBuster® Master Mix. Serine protease inhibitors were avoided when treating the target protein with thrombin, factor Xa, or recombinant enterokinase. Cysteine ​​protease inhibitors were used when treating the target protein with HRV 3C. While purification removes active inhibitors, dialysis or gel filtration is recommended prior to cleavage. The cell suspension was incubated at room temperature for 20 minutes at a low speed on a shaking platform or rotary mixer. After incubation, the extract should not be viscous. The suspension was then centrifuged at 75,600 g for 15 minutes at 4°C (JA-25.50, 8 x 30 ml) to remove insoluble cell debris. Optionally, the pellet was then washed again with 5 ml of binding buffer for 5 minutes, and the centrifugation was repeated. The above steps were repeated for the pre-induction sample. 0.5 ml of the induced and pre-induction samples were saved, and the induced supernatant was transferred to a new tube.

[0265] purification: A HisTrap FF Crude 5 ml column was assembled and the storage buffer was removed using 5 CV of binding buffer (20 mM boric acid, pH 8 + 30 mM imidazole). The column was then equilibrated with 5 CV of binding buffer (20 mM boric acid, pH 8.0 + 30 mM imidazole) and the sample from the previous step was loaded. The sample was then washed with 6 CV of binding buffer or UV280 The column was washed until the pH reached a stable value, and finally, the column was eluted with elution buffer (20 mM boric acid, pH 8.0 + 300 mM imidazole) to obtain the cchVBPO eluate.

[0266] Characterization The protein concentration of the eluate from the last step was determined by Bradford assay. Briefly, 5 μl of protein sample was incubated with Bradford reagent in a 250 μl well plate at room temperature for 5–10 min, and the OD 600 Total protein concentrations were determined against a calibration curve using BSA standards (0.125 mg / ml to 2 mg / ml). Tables 6 and 7 below show the Bradford calibration curves of known BSA standards, as well as the measured protein concentrations of pre-induced and purified induced samples.

[0267] [Table 6]

[0268] [Table 7]

[0269] SDS-PAGE electrophoresis was also performed to verify that the eluate contained cchVBPO protein, as shown in Figure 3. 8. Uninduced, induced, unbound, 4 μg of cchVBPO in borate buffer with 20 mM imidazole, 6 μg of cchVBPO in borate buffer with 20 mM imidazole, and 8 μg of cchVBPO in borate buffer with 20 mM imidazole were compared to the ladder as shown. Bromoperoxidase activity was determined by APF assay (490 nm / 515 nm). Briefly, 2930 ml of buffer containing 30 μl of KBr (18 mM) + 5 μl of APF (2 μM) + 5 μl of VBPO (containing Na3VO4) + 30 μl of hydrogen peroxide (250 μM) was mixed with 3 ml of curette. The reaction was initiated by adding VBPO at 25°C with stirring. Bromoperoxidase activity was assessed at 490 nm / 515 nm.

[0270] Example 7: Heterologous production of synVBPO in Escherichia coli This example describes the heterologous expression of Synechococcus sp. VBPO (synVBPO) in Escherichia coli, i.e., inoculation and cultivation of the model microorganism in a bioreactor, induction of synVBPO production, protein extraction, purification, and biochemical characterization. Inoculation, induction, protein extraction, protein purification, and characterization (i.e., Bradford assay, SDS-PAGE, APF assay) were completed according to the protocol detailed in Example 6 with minor modifications as described below. The optical density at 600 nm (OD ) of a 5 ml sample was measured. 600 The OD was 0.55 at 17–18 h after inoculation. After transferring the culture to a 0.5 L bioreactor with shaking at 200 rpm at 37 °C, the OD was increased until it reached 0.6–0.7. 600 The OD was monitored. 600 The OD was measured at 4, 5, and 5.5 hours as 0.312, 0.535, and 0.638, respectively, at which time it was deemed sufficient to start the induction process. After 18 hours of induction with 0.1 mM IPTG at 18 °C, the OD 600The pH was determined to be 0.920. After centrifugation, the pellet weighed 4.162 grams. The protein was resuspended and purified using a HisTrap FF column as previously described in Example 6. The eluate was then characterized for total protein concentration, SDS-PAGE, and haloperoxidase activity. Tables 8 and 9 below show the Bradford calibration curve of known BSA standards, as well as the measured protein concentrations of the pre-induced and purified induced samples. Figure 9 shows SDS-PAGE results showing uninduced, induced, and unbounded 4 μg synVBPO in 20 mM borate buffer, 6 μg synVBPO in 20 mM borate buffer, 8 μg synVBPO in 20 mM borate buffer, ladder, 12 μg synVBPO in 20 mM borate buffer, and 12 μg synVBPO in 20 mM borate buffer + 30 kDa cutoff.

[0271] [Table 8]

[0272] [Table 9]

[0273] The kinetic surface of the synVBPO enzyme was plotted as a function of hydrogen peroxide concentration [H2O2] (M) and potassium bromide concentration [KBr] (M), as shown in Figure 10. cat (s -1) were used to plot the data. The kinetic parameters, standard errors, t-values, and Prob > |t| were determined for two kinetic models: Cleland Michaelis-Menten (Model 1) and Cleland Hill (Model 2). The Akaike information criterion test (AIC) and Bayesian information criterion test (BIC) showed that the Cleland Hill model (Model 2) had lower AIC and BIC values, respectively. Therefore, the kinetics of synVBPO were inferred to follow the Cleland Hill kinetic paradigm. OriginPro (2021 version) was used to calculate kinetic parameters according to Michaelis-Menten and Hill kinetics, and statistical tests were performed.

[0274] Furthermore, the stability of synVBPO enzyme activity at high concentrations of KBr and HO, its dependence on ionic strength, and Michaelis-Menten parameters as a function of pH were determined, as shown in Figures 11-12. To investigate the deactivating effect of KBr on synVBPO activity, a Salwin test was performed using 100 μM MCD (E = 2.5|5|7.5 nM) in 20 mM phosphate buffer containing 1 mM KBr and 250 μM H2O2, 1 mM KBr and 50 μM H2O2, 40 mM KBr and 50 μM H2O2, and 160 mM KBr and 50 μM H2O2, respectively, at pH 7. Consecutive tests at 40 mM and 160 mM KBr concentrations showed a clear deactivation of the enzyme activity at these high bromide concentrations, whereas at 1 mM KBr, the enzyme retained its activity at both peroxide concentrations. The relationship between ionic strength and synVBPO activity is shown as the change in fluorescence per μ (ionic strength) as shown in Figure 12. Furthermore, the kinetic parameter k cat (s -1 ), k cat / Km H2O2 (M -1 s -1 ), and k cat / Km KBr (M -1 s -1) was determined using at least a -squared algorithm of the experimental data (shown in the equation in Figure 12).

[0275] Example 8. Addition of PAAM to ciVCPO This example demonstrates the use of ciVCPO to utilize peracetic acid as a substrate to generate small halogenated organic compounds instead of hydrogen peroxide. PAAM is a readily available source of peracetic acid, consisting of 32 w / v% peracetic acid and 6 w / v% hydrogen peroxide. Because peracetic acid is not an efficient two-electron reducing agent and, unlike hydrogen peroxide, is unable to generate oxygen in the presence of hypochlorous acid and catalytic amounts of chloride ions, the enzyme catalase derived from bovine liver must be used to remove hydrogen peroxide to avoid oxygen generation.

[0276] Figure 14 shows the reaction rate of ciVCPO in the presence of chloride ions using PAA and H2O2 as substrates. The reaction rate was fitted according to Michaelis-Menten kinetics to obtain v = f([PAA] or [H2O2]). As shown by the graph, using PAA as the substrate resulted in a higher v than hydrogen peroxide for a constant concentration of 150 mM NaCl. max and K. m is obtained.

[0277] Process Design In each of the following process designs for the production of small molecule halogenated organic compounds in bioreactors, dibromoacetone (DBA) or dichloroacetone (DCA) is enzymatically produced as described above.

[0278] Design 8.1 PAA + NaCl + DBA + ciVCPO → DBCA, where DBA is dibromoacetone and DBCA is dibromochloroacetone (at pH = 7-8 and T = 20-55°C) DBCA → DBCM (After the enzyme reaction is complete, set the pH to 8-9 using pH-STAT)

[0279] Design 8.2 Step 1: PAA+Cl - +ciVCPO→HOCl HOCl + NH3 → NH2Cl (at pH = 7-8 and T = 20-55°C) Step 2: NH2Cl+I - →HOI HOI+I - ←→I2 DCA + I2 → DIA (DIA = dichloroiodoacetone) DIA → DCIM (pH = 8, T = 20-55 °C) (DCIM = dichloroiodomethane).

[0280] DCIM production in a two-batch system One embodiment of the present invention describes the production of dichloroiodomethane (DCIM) in a two-step batch system depicted in the following steps, according to the following conditions: In the first step, at T=22°C, 1 equivalent of 0.1 M phosphate buffer, pH 7, containing 12 mM HO, 100 mM NaCl, 10 mM DCA was added to the bioreactor along with HO at 25 μl / min. HO was added to the bioreactor with 1.2 equivalents of aqueous NH, i.e., C NH3 is 0.24M, and the molar rate of H2O2 entering the tank is equal to the molar rate coming out, or C H2O2 The concentration of NaCl was 0.2 mol / L. The reaction time was set to 120 min. The initial concentrations in the bioreactor were 100 mM NaCl, 100 μM H2O2, 480 μM NH4Cl, 264 μM Na3VO4, and 2.64 μM enzyme.

[0281] Every 10 minutes, 500 μl of bioreactor sample was mixed with 500 μl of DCA stock in phosphate buffer (Solution (A)) and the appropriate amount of KI (Solution (B)). Solution (A) contained 10 mM DCA, diluted from a 9.93 M DCA stock solution into 0.5 M phosphate, pH 8.5. Solution (B) contained 1 mM KI, diluted from a 400 mM stock solution. These vials were reacted overnight, and 50 μl of the reaction sample was mixed with 950 μl of the extract and then analyzed by GC-MS to detect DCIM. Figure 15 shows a graph of DCIM concentration versus reaction coordinate, demonstrating a linear increase in DCIM concentration with time, consistent with Michaelis-Menten kinetics.

[0282] Example 9. Pichia pastoris glycerol batch and fed-batch fermentation According to the examples below, the enzyme ciVCPO from Curvularia inaequalis was produced in Pischia pastoris and purified to homogeneity, with typical results being 100-500 mg / L of over 75-95% pure ciVCPO enzyme.

[0283] Preparation of seeded seed flasks One 250 mL baffled shake flask containing 25 mL of BMGY medium was inoculated with a single colony from the agar plate with Pichia pastoris ciVCPO. The shake flask was incubated in an incubator shaker at 30°C and 250 RPM for 24 hours. 2 x 500 mL baffled shake flasks containing 150 mL of BMGY medium were inoculated with 5 mL (OD ) of the 250 mL baffled shake flask. 600 The culture was inoculated with approximately 0.2-0.4 ml of the medium and incubated in an incubator shaker at 30°C and 250 RPM for 24 hours.

[0284] Glycerol batch phase The pH probe, internal and external peristaltic pumps were first calibrated with the EVA software. The bioreactor was autoclaved at 121°C for 30 minutes with 3 L of basal salts medium (FM22) containing 4% (v / v) glycerol and 0.6 ml of antifoam C. After sterilization and cooling, a total volume (9 ml) of 4 ml / liter PTM1 salts and ampicillin were aseptically added. An initial sample of 10 ml was taken. The O2 probe (1000 rpm, 1 vvm air until a stable reading) was calibrated and the OD 600 The turbidity was autozeroed, and the tube was filled with ammonium hydroxide (25%), phosphoric acid (10%), antifoam, and feed. The temperature was set to 30°C, and stirring and aeration were enabled. Minimum and setpoints were 800 rpm, 0.5 v / v, air 1.5 L / min, and pO2 = 40% v / v. The medium pH was adjusted to 5.0 with 25% ammonium hydroxide (for better salt dissolution). The dissolved oxygen (DO) cascade is depicted in Figure 16. The fermenter was ionized with approximately 5-10% of the initial fermentation volume from a culture generated in an inoculated shake flask. This seed culture was spun down and redissolved in 50 ml of BMGY medium or water, and 60 ml was added to a syringe with a pink prong. The batch culture was grown until glycerol was completely consumed (14-20 h), indicated by an O2 spike (pO2 > 85% and time > 14 h). Samples were analyzed for cell growth (OD600, cell wet weight (CWW), dry cell weight), ethanol, glycerol, and methanol concentrations, and enzyme activity. If not analyzed immediately, the cell pellet and supernatant of each sample were frozen at -80°C for later analysis.

[0285] Sample preparation: A 5 ml sample was precisely transferred into a pre-weighed 15 ml Falcon tube. 1 ml was removed and poured into a 1.5 ml Eppendorf tube. 0.1 ml of this sample was measured at OD 600It was used for 0.1 < Abs < 0.8. The rest was centrifuged down (0.9 ml), and the supernatant was transferred to a new 1.5 ml Eppendorf tube for assays of EtOH, MeOH, and glycerol. If the sample was after induction, the cells were disrupted with yeast insect buster cell lysis buffer, and then 0.9 ml of the paste was placed at -80 °C for VCPO activity assay. The tube was centrifuged, washed with water, centrifuged again, and then the pellet was decanted and weighed. CWW was calculated for 4 ml of cell culture and for CWW g / L. For cell dry weight (CDW): The pellet was washed again by resuspension and centrifugation, and the washed pellet was finally resuspended to a final volume of 4 mL (initial). 3 mL of this suspension was transferred to a pre-weighed aluminum weighing dish and dried in an oven at 80 °C overnight for 16 hours and weighed again. The dry mass of each sample was calculated to derive CDW.

[0286] Glycerol fed-batch phase When all the glycerol was consumed from the batch growth phase, glycerol feeding was started to increase cell biomass under limited (carbon) conditions. A DO spike was used to ensure glycerol limitation. After starting glycerol fed-batch and observing a DO spike, a 50% w / v glycerol feed containing 12 ml of PTM1 salts per liter of glycerol feed was used. An initial fermentation rate [20 ml / h*L (60 ml / h) of a constant feed rate was used. The supply of glycerol was carried out until the desired cell density was reached. A cell yield of 230 g / L of wet cells should be achieved in about 22 hours or 6 hours of supply. At the end of this stage, no appreciable recombinant enzyme was produced.

[0287] Methanol fed-batch phase of MutS Pichia strain To fully induce the AOX1 promoter with methanol, all glycerol must be consumed before the methanol feed is initiated. Methanol was slowly introduced to adapt the culture to growth on methanol. The methanol feed rate was adjusted to maintain an excess of no more than 0.3% methanol in the medium, as determined by GC / MS.

[0288] Methanol induction phase of MutS strains. The temperature was reduced to 28 °C, and the glycerol feed rate was ramped down to 0 g / L·h (26.1% (60 ml / h) → 0% at 2 h) to induce the culture. A 2 L induction feed containing 50% v / v methanol, 12 ml / L PTM1 salt (24 ml), and 0.0075% v / v (0.15 ml) antifoam 204 was started at 2 ml / h*L for the first 2 h at the initial fermentation volume. It was then ramped up from 2 ml / h*L to 6 ml / h*L over 12 h and maintained for the duration of the fermentation. The vessel was then harvested after 4 days in methanol.

[0289] Enzyme recovery: cell disruption The whole cell pellet was resuspended in water at a 1:4 ratio. Then, 200 ml of this suspension was added to a 500 ml Beckman centrifuge tube (resulting in approximately 50 ml of cell paste) and centrifuged at 4000 g for 5 minutes at 4°C. The water was removed, leaving 50 ml of cell paste in each Beckman centrifuge tube. 50 ml of beads, 200 ml of binding buffer (50 mM Tris-HCl, pH = 8.0), and a protease inhibitor cocktail were added to the 50 ml of cell paste. The mixture was homogenized for 10 minutes. After this step, the disrupted yeast appeared dark under a microscope. The mixture was then centrifuged, and the supernatant was collected and placed on ice. 40 ml of binding buffer was added to the paste, centrifuged, and combined with the previous supernatant. The tubes containing the cell lysates were centrifuged at 75,600 g (JA-25.50, 8 × 30 ml max, polycarbonate) for 15 min at 4 °C to remove insoluble cell debris and collect the clear lysates, which were then syringe filtered prior to purification.

[0290] Enzyme recovery: purification Heat treatment of crude extracts (e.g., 55°C for 45 min) and classical affinity purification (His Tag) were used for enzyme purification. The binding buffer was 0.1 M Tris-HCl pH, and the elution buffer was the same as the binding buffer, supplemented with 500 mM imidazole.

[0291] Enzyme characterization: SDS-PAGE electrophoresis Five micrograms of pure protein or 10–20 micrograms of the mixture was added to each well. In an Eppendorf tube, 200 microliters of sample buffer was mixed with 2 microliters of fresh 10 mM DTT (1 M stock), immediately heated to 90°C on a thermoblock for 5 minutes, and then placed on ice or frozen. The SDS-Page running buffer consisted of 10x Tris / glycine / SDS (900 ml Milli-Q water + 100 ml 10x Tris / glycine / SDS). The staining buffer used was One-Step Blue. Figure 17 shows an SDS-PAGE of the ladder, crude extract, heat-treated crude extract, and 60% pooled HisTap crude peak fractions.

[0292] Buffer exchange and concentration The enzyme peak was selected and a subsample was buffer exchanged against a 20K MWCO dialyzer membrane with the desired buffer, after which the sample was frozen in liquid nitrogen and stored at -80°C. Enzyme characterization: activity assays MCD assay (292 nm), ε = 20 mM -1 *cm -1

[0293] To a quartz cuvette, 2900 μl of buffer (50 mM phosphate pH 7), 50 μl of 3 M KBr, 20 μl of 10 mM MCD, 4 μl of 10 mM vanadium, and 8 μl of 300 mM HO were added in that order. 20 μL was then added to a UV-Vis read at 292 nm every 6 seconds for 1 minute.

[0294] equivalent The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. While the present invention has been disclosed with reference to particular embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A method for regulating the production of low molecular weight halogenated organic compounds using peroxidase enzymes, (i) To supply low molecular weight organic compounds, (ii) Contacting the low molecular weight organic compound with the peroxidase to form a reaction mixture under conditions sufficient to produce a low molecular weight halogenated organic compound, (iii) The method comprising evaluating the generated low molecular weight halogenated organic compound and thereby regulating the generation of the low molecular weight halogenated organic compound.

2. The method according to claim 1, wherein the peroxidase is a haloperoxidase.

3. The method according to claim 2, wherein the peroxidase is vanadium chloroperoxidase (VCCO), vanadium bromoperoxidase (VBPO), or vanadium iodoperoxidase (VIPO).

4. The method according to claim 1, wherein the peroxidase is an algal haloperoxidase, a fungal haloperoxidase, or a cyanobacterial haloperoxidase.

5. The method according to claim 1, wherein the peroxidase is produced in a host cell microorganism selected from Pichia pastoris, Aspergillus niger, Saccharomyces cerevisiae, or Escherichia coli.

6. The method according to claim 1, wherein the amino acid sequence of the peroxidase includes the amino acid sequences listed in Table 1.

7. The method according to claim 1, wherein the peroxidase is a sequence selected from any one of sequence numbers 1 to 50.

8. The method according to claim 1, wherein the amino acid sequence of the peroxidase has 1, 2, 3, 4, 5, or 6 amino acid substitutions in an amino acid sequence selected from any one of SEQ ID NOs: 1 to 50.

9. The aforementioned low molecular weight organic compound has the structure of formula (Y): 【Chemistry 12】 or having a salt, tautomer, or isomer thereof, in the formula, Each of R1a, R1b, R1c, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5c is independently hydrogen, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, or heterocyclyl, and each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl is optionally substituted with one or more R6s; R6 is halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR A, or -NR B R C R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C2-C6 alkenyl, and R B and R C are each independently hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. Each of m and n is an integer between 0 and 24, independently. 【Chemistry 13】 It is either a single bond or a double bond. 【Chemistry 14】 The method according to claim 1, wherein, if the bond is a double bond, each of R2b and R3b does not exist independently.

10. The method according to claim 1, wherein the low molecular weight organic compound comprises 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

11. The method according to claim 1, wherein the low molecular weight organic compound comprises a ketone or an aldehyde.

12. The aforementioned low molecular weight halogenated organic compound has the structure of formula (Z): [Chemistry 18] or having a salt, tautomer, or isomer thereof, in the formula, Each of R1a, R1b, R1c, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5c is independently hydrogen, halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, cycloalkyl, or heterocyclyl, and each alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, or heterocyclyl is optionally substituted with one or more R6s, and R1a, R1b, R1c, R2a, R2b, R3a, R3b, R4a, R4b, R5a, R5b, and R5c At least one of them is independently a halogen, R6 is a halogen, C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 alkynyl, -OR A, or -NR B R C, R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl, or C2-C6 alkenyl, and R B and R C are each independently hydrogen, C1-C6 alkyl, or C1-C6 heteroalkyl. Each of m and n is independently selected from 0, 1, 2, or 3. 【Chemistry 19】 It is either a single bond or a double bond. 【Chemistry 20】 The method according to claim 1, wherein, if the bond is a double bond, each of R2b and R3b does not exist independently.

13. The method according to claim 1, wherein the low molecular weight halogenated organic compound is chlorinated, brominated, iodized, chlorinated and iodized, chlorinated and brominated, brominated and iodized, or chlorinated, brominated and iodized.

14. The method according to claim 1, wherein the low molecular weight halogenated organic compound contains one, two, or three halogen atoms.

15. The method according to claim 1, wherein the low molecular weight halogenated organic compound includes an acetone portion.

16. The method according to claim 1, wherein the low molecular weight halogenated organic compound includes dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone.

17. The method according to claim 1, wherein the low molecular weight halogenated organic compound comprises dichloroiodomethane, dibromochloromethane, 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone.

18. H ２ O ２ The method according to claim 1, further comprising providing a peroxide source containing PAA, and a halogen source containing KBr, NaCl, or KI.

19. A method for reducing methane production in a lumen community, (i) To supply low molecular weight organic compounds, (ii) Contacting the low molecular weight organic compound with peroxidase to form a reaction mixture under conditions sufficient to produce a low molecular weight halogenated organic compound, (iii) Separating the low molecular weight halogenated organic compound from the reaction mixture, (iv) The method comprising providing the low molecular weight halogenated organic compound to the lumen community under conditions sufficient to reduce the production of methane.

20. A reactor for the continuous production of low molecular weight halogenated organic compounds, (i) reaction chamber and (ii) A module for temperature control, (iii) Peristaltic pump and, (iv) The reactor comprising a module for housing a catalyst.

Images