Process for producing halogenated compounds

Abstract

The present disclosure features methods of modulating the yield of small halogenated organic compounds with peroxidases, as well as related compositions and methods of use thereof.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. Application No. 63 / 335,010 filed on April 26, 2022 and U.S. Application No. 63 / 451,399 filed on March 10, 2023; the entire contents of each of the foregoing applications are incorporated herein by reference. Background Art

[0003] There is growing concern about greenhouse gas emissions from animal agriculture. A large portion of these emissions can be attributed to biogenic enteric methane (CH4) emissions from all domestic ruminants (3.2% of total U.S. emissions; EPA, 2019). It has long been known that small halogenated organic compounds, such as chloroform, bromochloromethane, and 2-bromoethane sulfonate, can act as inhibitors of enteric methane production (Hristov, AN et al. J Anim Sci (2013) 91 (11): 5045-69). These small organic halogenated compounds can competitively inhibit the activity of methyl-Coenzyme M reductase (mMCR), which catalyzes the final step of CH4 synthesis by methanogens found in the rumen (Wood, JM et al. Biochemistry (1968) 7 (5): 1707-1713; Ferry, JG Annu Rev Microbiol (2010) 64: 3117-3126). However, these small halogenated organic compounds cannot be used directly as livestock feed additives for animal and human safety and environmental considerations. To address this problem, researchers have found that the halogen-rich red algae of the Asparagopsis genus is a natural source of small halogenated organic compounds that act as CH4 inhibitors (Machado L. et al., J Appl Phycol (2016) 28 (5): 3117-3126). Although many seaweeds produce halogenated compounds, Asparagopsis is unusual because it has glandular cells that can accumulate very high concentrations of bromoform (CHBr3). A series of independent in vivo experiments showed that when Asparagopsis was added to the diets of sheep, dairy cows and beef cattle, CH4 production was significantly reduced by 50-80% (Li X., et al. Anim Prod Sci (2018) 58 (4): 681-688). Although research is devoted to large-scale production of Asparagopsis, it is unlikely that the scale of growth of this seaweed will meet the future demand for feed additives in the dairy and beef industries. Therefore, there is a need to develop scalable alternatives to reduce methane production. Summary of the invention

[0004] The present disclosure features peroxidases and related compositions, and methods for regulating the production of small halogenated organic compounds with the peroxidases. 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) under conditions sufficient to produce a small halogenated organic compound to form a reaction mixture; and / or (iii) evaluating the produced small halogenated organic compound. 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 each of (i) and (iii). In one embodiment, the method is characterized by each of (ii) and (iii). In one embodiment, the method is characterized by each of (i)-(iii). In one embodiment, regulation includes increasing the production of small halogenated organic compounds. In one embodiment, the method includes regulating the production of multiple small halogenated organic compounds. In one embodiment, the method includes providing multiple small organic compounds, such as for halogenation.

[0005] Peroxidase can be any peroxidase known in nature, including haloperoxidase. In one embodiment, haloperoxidase is vanadium haloperoxidase (VHPO). In one embodiment, VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO) or vanadium iodoperoxidase (VIPO). In one embodiment, VHPO is VBPO. Peroxidase can be algae haloperoxidase (e.g., derived from algae species) or fungal haloperoxidase (e.g., derived from fungal species). In one embodiment, peroxidase is fungal haloperoxidase (e.g., derived from fungal species). Peroxidase can be derived from an organism selected from Curvularia inaequalis, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium erythraeum, Aphanocapsa montana, Lyngbya confervoides, Synechococcus PCC7335 and Corallina officinalis. In one embodiment, peroxidase is derived from coral algae. In one embodiment, peroxidase is derived from mountain cryptococcus. In one embodiment, peroxidase is derived from Curvularia inaequalis. In one embodiment, peroxidase comprises the sequence of peroxidase as described herein, for example, the peroxidase sequence provided in Table 2.

[0006] Peroxidase can be produced in host cell microorganism, for example, crosses expression in host cell microorganism.In one embodiment, host cell microorganism is selected from Pichia pastoris (Pichia pastoris), Aspergillus niger (Aspergillus niger), Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Escherichia coli (Escherichiacoli).In one embodiment, the expression of the peroxidase produced in the host cell microorganism increases by about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 10 times, for example, relative to the peroxidase produced in its natural host.In one embodiment, the amino acid sequence of peroxidase is selected from the amino acid sequence 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) with 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) with a peroxidase sequence selected from SEQ ID NOs: 1-50. In one embodiment, the peroxidase is selected from the amino acid sequence of any one of SEQ ID NOs. 1-50.

[0007] Small organic compounds can be naturally occurring or non-naturally occurring compounds. For example, small organic compounds may include natural products, lipids, sterols, steroids, amino acids, sugars, brown algae tannins, tannins, lignin or lignin derivatives. In one embodiment, small organic compounds include functional groups, such as aldehydes, ketones, acetyl groups, acyl groups, hydroxyls, esters, ethers, amines, amides, aryls, heteroaryls, heterocyclic 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 α-β unsaturated ketones. In one embodiment, small organic compounds are acetone or acetylacetone. In one embodiment, small organic compounds include 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. In one embodiment, small organic compounds include ketones or aldehydes.

[0008] In one embodiment, the small halogenated organic compound comprises a natural product, a lipid, a sterol, a steroid, an amino acid, a sugar, a brown algae tannin, a tannin, a lignin, or a lignin derivative is chlorinated, brominated or iodinated. In one embodiment, the small halogenated organic compound is brominated. In one embodiment, the small halogenated organic compound comprises a functional group, such as an aldehyde, a ketone, an acetyl group, an acyl group, a hydroxyl group, an ester, an ether, an amine, an amide, an aryl group, a heteroaryl group, a heterocyclic group or a cycloalkyl group. In one embodiment, the small halogenated organic compound comprises an alkenyl group or an alkynyl group. In one embodiment, the small halogenated organic compound comprises an aldehyde or ketone group. In one embodiment, the small halogenated organic compound comprises an α-β unsaturated ketone. In one embodiment, the small halogenated organic compound comprises 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. In one embodiment, the small halogenated organic compound comprises 1, 2, 3 halogen atoms. In one embodiment, the small halogenated organic compound comprises 1, 2, 3 bromine atoms. In one embodiment, the small halogenated organic compound comprises an acetone moiety. In one embodiment, the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform or tribromoacetone. In one embodiment, the small halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone or 1,1,1-tribromoacetone. In one embodiment, the small halogenated compound comprises dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane or diiodobromomethane. In one embodiment, the small 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.

[0009] In one aspect, the small organic compound comprises a compound of formula (Y):

[0010]

[0011] or a salt, 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 5cEach 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 optionally substituted by one or more R 6 Replacement; R 6 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; R B and R C are each independently hydrogen, C1-C6 alkyl or C1-C6 heteroalkyl; m and n are each independently an integer between 0 and 24; and is a single bond or a double bond, where When it is a double bond, R 2b and R 3b Each does not exist independently.

[0012] In embodiments of formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b and R 5c Each of is 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), is a single bond. 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 Each of is independently hydrogen, each of m and n is independently selected from 0, 1, 2 or 3, and It is a single bond.

[0013] In embodiments of formula (Y), R 1a , R 1b , R 1c , R 5a , R 5b and R5c Each of is 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 Each of is independently hydrogen, n is 0, m is 1, and In the embodiment of formula (Y), R 1a C l Alkyl; R 1b , R 1c , R 5a , R 5b and R 5c are each independently hydrogen, and m and n are each 0. In an embodiment of formula (Y), R 1a is halogen (e.g., chlorine, bromine or iodine); R 1b , R 1c , R 5a , R 5b and R 5c are each independently hydrogen, and m and n are each 0.

[0014] In another aspect, the small halogenated organic compound comprises a compound of formula (Z):

[0015]

[0016] or a salt, 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 5c Each 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 optionally substituted by one or more R 6 Replace; and R 1a , R 1b , R 1c , R 2a , R 2b , R3a , 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 ; R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl or C2-C6 alkenyl; 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; and is a single bond or a double bond, where When it is a double bond, R 2b and R 3b Each does not exist independently.

[0017] In an embodiment of formula (Z), R 1a , R 1b and R 1c Each of R is independently halogen or hydrogen, wherein R 1a , R 1b and R 1c At least one of is halogen. In one embodiment, halogen is selected from chlorine, bromine or iodine. In the embodiment of formula (Z), R 1a , R 1b , R 1c Each of R is independently halogen or hydrogen, wherein R 1a , R 1b and R 1c At least two of are halogen. In one embodiment, the halogen is selected from two of chlorine, bromine or iodine. In the embodiment of formula (Z), R 1a , R 1b , R 1c Each of is independently halogen. In one embodiment, halogen is selected from chlorine, bromine or iodine. In an embodiment of formula (Z), R 5a , R 5b and R 5c Each of R is independently halogen or hydrogen, wherein R 5a , R 5b and R 5c At least one of is halogen. In the embodiment of formula (Z), R 5a , R 5b , R 5cEach of R is independently halogen or hydrogen, wherein R 5a , R 5b and R 5c At least two of are halogen. In the embodiment of formula (Z), R 5a , R 5b , R 5c Each of is independently halogen. In an embodiment of formula (Z), is a single bond. In an embodiment of formula (Z), each of m and n is independently selected from 0, 1, 2 or 3, and It is a single bond.

[0018] In one embodiment, conditions sufficient to produce small halogenated organic compounds include one or more of the following: (a) temperature between 10°C and 85°C; (b) pH between 4-10; and (c) ionic strength between 0.1mM and 4M. In one embodiment, conditions sufficient to produce small halogenated organic compounds include temperature between 10°C and 85°C. In one embodiment, conditions sufficient to produce small halogenated organic compounds include pH between 4-10. In one embodiment, conditions sufficient to produce small halogenated organic compounds include ionic strength between 0.1mM and 4M. In one embodiment, the evaluation includes analyzing the small halogenated organic compounds by analytical techniques. In one embodiment, analytical techniques include HPLC, GC-MS, NMR.

[0019] This little halogenated organic compound can be used for many agricultural, marine and / or industrial processes. In one embodiment, the little halogenated organic compound can reduce the methane production of microorganisms. In one embodiment, methane production reduces 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 reduces 10-75%. In one embodiment, microorganisms are present in the rumen community.

[0020] In another aspect, the disclosure features a method for reducing the production of small hydrocarbons (e.g., methane) in a rumen community, comprising: (i) providing a small organic compound (e.g., acetylacetone); (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) under conditions sufficient to produce a small halogenated organic compound to form a reaction mixture; (iii) separating the small halogenated organic compound from the reaction mixture; and / or (iv) providing the small halogenated organic compound to the rumen community under conditions sufficient to reduce the production of small hydrocarbons (e.g., methane). 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)-(iv).

[0021] In one embodiment, the method is characterized by (iii) isolating a small halogenated organic compound from the reaction mixture, and (iv-a) incorporating the isolated small halogenated organic compound into a matrix so as to deliver it to the rumen community under conditions sufficient to reduce the production of small hydrocarbons (e.g., methane). In one embodiment, the method is characterized by (iv-a) incorporating the isolated small halogenated organic compound into a matrix so as to deliver it to the rumen community under conditions sufficient to reduce the production of small hydrocarbons (e.g., methane). In one embodiment, the method is characterized by each of (i), (ii), (iii), and (iv-a). In one embodiment, the method is characterized by each of (ii) and (iv-a). In one embodiment, the method is characterized by each of (i), (ii), and (iv-a). In one embodiment, the method is characterized by each of (iii) and (iv-a). In one embodiment, the method is characterized by (i), (iii), and (iv-a). In one embodiment, the method is characterized by each of (ii), (iii), and (iv-a).

[0022] In one embodiment, the method also includes obtaining the value of small hydrocarbon (e.g., methane) level before (a) providing peroxidase or after (b) providing peroxidase. In one embodiment, the method also includes (a). In one embodiment, the method also includes (b). In one embodiment, the method also includes (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%.

[0023] Peroxidase can be any peroxidase known in nature, including haloperoxidase. In one embodiment, haloperoxidase is vanadium haloperoxidase (VHPO). In one embodiment, VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO) or vanadium iodoperoxidase (VIPO). In one embodiment, VHPO is VBPO. Peroxidase can be algae haloperoxidase (for example, derived from algae species) or fungal haloperoxidase (for example, derived from fungal species). In one embodiment, peroxidase is fungal haloperoxidase (for example, derived from fungal species). Peroxidase can be derived from an organism selected from the following: inequilateral Curvularia, Halomicronema hongdechloris, Moorea bouillonii, Red Sea Trichoderma, Mountain Cryptococcus, filamentous sheathed filamentous algae, Synechococcus PCC7335 and coralline algae. In one embodiment, peroxidase is derived from coralline algae. In one embodiment, peroxidase is derived from mountain cryptococcus. In one embodiment, the peroxidase is derived from Curvularia inaequalis.

[0024] Peroxidase can be produced in host cell microorganism, for example, overexpressed in host cell microorganism.In one embodiment, host cell microorganism is selected from pichia pastoris, aspergillus niger or escherichia coli.In one embodiment, the expression of the peroxidase produced in host cell microorganism increases by about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 10 times, for example, relative to the peroxidase produced in its natural host.In one embodiment, the amino acid sequence of peroxidase is selected from the amino acid sequence listed in Table 2.In one embodiment, peroxidase has at least 75% sequence identity (for example, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99% or 99.5% sequence identity) with the peroxidase sequence of the list selected from Table 2.In one embodiment, peroxidase is a sequence selected from any one of SEQ ID NO.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 at least 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.

[0025] In one embodiment, the method is characterized by: (i) providing a small organic compound comprising an α-β unsaturated ketone (e.g., acetylacetone); and (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) under conditions sufficient to produce a small halogenated organic compound to form a reaction mixture. In one embodiment, for step (ii), the method further includes 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, for step (ii), the method further includes providing a halogen source and a peroxide source in the presence of an amine, for example, to produce ammonium chloride, such as NH2Cl. In one embodiment, providing a halogen layer includes providing ammonium chloride (e.g., NH2Cl).

[0026] In one embodiment, for step (ii), also include, for example, providing a peroxide source and a halogen source (e.g., a bromine source, a chlorine source, or an iodine source) to peroxidase in the presence of an amine, to generate halogenated amines, such as NH2Cl. In one embodiment, for step (ii), the method also includes reacting halogenated amines with an additional halogen source to further halogenate small halogenated organic compounds. In one embodiment, the additional halogen source is a bromine anion, a chlorine anion, or an iodine anion. In one embodiment, the reaction of halogenated amines with an additional halogen source occurs in a container different from step (i) or (ii).

[0027] In one embodiment, the method is characterized by: (ii) providing a peroxide source and a chlorine source to a peroxidase (e.g., VCPO) in the presence of an amine to generate chlorinated amines, such as NH2Cl; and (iii) reacting the chlorinated amines with two equivalents of an iodide anion (I - ) reaction, to generate a mixture of hypoiodite anion and iodine (I2). In one embodiment, (ii) occurs in a reaction vessel different from (iii). In one embodiment, the method also includes (iv) contacting a mixture of hypoiodite anion and iodine from (iii) with a small halogenated compound such as produced in (i), to generate other small halogenated organic compounds (e.g., further halogenated small halogenated organic compounds, 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), (iii) and (iv). In all embodiments, (ii) can be replaced by (iib).

[0028] In one embodiment, the yield of step (i) is between about 50% and 99%. In one embodiment, the yield of step (i) is between about 75% and 99. In one embodiment, the yield of step (i) is between about 85% and 99%. In one embodiment, the yield of steps (i), (ii), (iii) and (iv) is between about 10% and 99%. In one embodiment, the yield of steps (i), (ii), (iii) and (iv) is between about 10% and 75%. In one embodiment, the yield of steps (i), (ii), (iii) and (iv) is between about 10% and 50%. In one embodiment, the yield of steps (i), (ii), (iii) and (iv) is between about 25% and 99%. In one embodiment, the method is carried out at a pH value of about 5.0 to 9.0. In one embodiment, step (ii) and / or (iii) is performed at a pH between 7.0 and 8.0, such as pH 7.2 to 7.8, pH 7.5 to 8.0. Without being bound by theory, maintaining the pH of step (ii) and / or (iii) between about 7.0 and 8.0 may help reduce the formation of I3 - or iodate anion IO3 - side effects.

[0029] In one embodiment, the method comprising an enzymatic reaction of an organic peroxide (e.g., peracetic acid (PAA)) and a peroxidase (e.g., VCPO) is characterized in that hypohalite anions and excess iodide anions are converted into diatomic iodine I2 and triiodide anions I3. - In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO at pH 7, the conversion of the subhalogenate anion and the excess iodide anion into diatomic iodine I2 and triiodide anion I3 at pH 7 is reduced by about 10% to 99%. - In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that the conversion rate of the subhalate anion and the bromide anion into diatomic bromine Br2 at pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO at pH 0-5.

[0030] In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that the conversion rate of hypohalite anion and excess chloride anion to diatomic chlorine Cl2 between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO between pH 0-5. In one embodiment, the method comprising the enzymatic reaction of an organic peroxide such as PAA with a peroxidase (e.g., VCPO) is characterized in that the conversion rate of peracetic acid and bromide anion to hypobromite anion or its conjugate acid between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 with VHPO between pH 0-5. In one embodiment, the method comprising the enzymatic reaction of an organic peroxide such as PAA and a peroxidase (e.g., VCPO) is characterized in that the conversion rate of peracetic acid and iodide anions to hypoiodite anions or their conjugate acids between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of an organic peroxide such as PAA and VHPO between pH 0-5.

[0031] In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) (e.g., step (ii)) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) and a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 and VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) and a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 and VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) and a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 and VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, kcat Increased by 90%-110%.

[0032] In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) (e.g., step (ii)) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) and a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 and VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) and a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 and VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k catIn one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of an organic peroxide (e.g., PAA) with a peroxidase (e.g., VCPO) is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat The increase was 90%-110%, among which dibromoiodomethane was produced.

[0033] Small organic compounds can be naturally occurring or non-naturally occurring compounds. In one embodiment, the small organic compound comprises 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. In one embodiment, the small organic compound comprises a ketone or an aldehyde. In one embodiment, the small halogenated organic compound is chlorinated, brominated or iodinated. In one embodiment, the small halogenated organic compound is brominated. In one embodiment, the small halogenated organic compound comprises 1, 2, 3 halogen atoms. In one embodiment, the small halogenated organic compound comprises 1, 2, 3 bromine atoms. In one embodiment, the small halogenated organic compound comprises an acetone moiety. In one embodiment, the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform or tribromoacetone. In one embodiment, the small halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone or 1,1,1-tribromoacetone.

[0034] In one embodiment, conditions sufficient to produce small halogenated organic compounds include one or more of the following: (a) temperature between 10°C and 85°C; (b) pH between 4-10; and (c) ionic strength between 0.1mM and 4M. In one embodiment, conditions sufficient to produce small halogenated organic compounds include temperature between 10°C and 85°C. In one embodiment, conditions sufficient to produce small halogenated organic compounds include pH between 4-10. In one embodiment, conditions sufficient to produce small halogenated organic compounds include ionic strength between 0.1mM and 4M. In one embodiment, the evaluation includes analyzing the small halogenated organic compounds by analytical techniques. In one embodiment, analytical techniques include HPLC, GC-MS, NMR.

[0035] This little halogenated organic compound can be used for many agricultural, marine and / or industrial processes. In one embodiment, the little halogenated organic compound can reduce the methane production of microorganisms. In one embodiment, methane production reduces 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 reduces 10-75%. In one embodiment, microorganisms are present in the rumen community.

[0036] In another aspect, the method features a method for increasing expression of a peroxidase in a host cell or host microorganism.

[0037] In another aspect, the method is also characterized by a reactor for continuous production of small halogenated organic compounds, which includes: (i) a reaction chamber; (ii) a temperature control module; (iii) a peristaltic pump; and / or (iv) a module containing a catalyst.

[0038] Additional embodiments of the disclosure are described in further detail herein in the figures, description, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is an example of a chemical reaction catalyzed by a vanadium haloperoxidase.

[0041] Figure 2A-2C This is the enzymatic reaction mechanism catalyzed by vanadium haloperoxidase. Figure 2A is the equation that describes the general ping-pong bi-bi reaction mechanism. Figure 2B is the Cleland equation for the ping-pong double reaction with substrate inhibition. Figure 2C is the equation describing the reaction parameters of vanadium haloperoxidase constructed by the King-Altman method.

[0042] Figures 3A-3C The effects of pH and ionic strength on the kinetics of an exemplary CoVBPO are shown. Figure 3A The K at high (2.0 M) and low (0.2 M) MgSO4 concentrations is shown. mBr- Tables 3 and 4 give more details of the kinetics. Figure 3B It shows that k cat and the relationship between the deuterium isotope fractions in each ionic strength solution; Figure 3C The solvent isotope effect (SIE) on k cat and K mBr- This information is used to explain the reaction mechanism and to convert K mBr Reduced by 12 times (K mBr =0.8mM.

[0043] Figures 4A-4C The CiVCPO-driven production of small halogenated organic compounds from the exemplary small organic compound acetylacetone is shown. Figure 4A Shown is the change in concentration of reaction products over time in an exemplary CiVCPO reaction. Figure 4B This is an HPLC source chromatogram of the reaction products of the CiVCPO reaction, illustrating how to fractionate 1,1,dibromoacetone for use in the methane suppression experiment. Figure 4C is a GC-MS chromatogram showing the components of the HPLC fractionated n-hexane extract of 1,1-dibromoacetone. The identity of the major product was determined by comparing the product MS spectrum with a 1,1-dibromoacetone standard.

[0044] Figure 5 An exemplary workflow for generating and testing small halogenated organic compounds for their methane inhibition potential is illustrated.

[0045] Figures 6A-6C is a graph showing the effect of small halogenated compounds on methane production by rumen-derived microbial cultures. The methane production rates for the addition of 1,1-dibromoacetone, bromoform, and dibromomethane are shown.

[0046] FIG. 7 is a sequence alignment of several exemplary VHPOs.

[0047] Figure 8 is a SDS-PAGE polyacrylamide gel after purification of cchVBPO from heterologous production in E. coli.

[0048] Fig. 9 is an SDS-PAGE polyacrylamide gel after purification of synVBPO from heterologous production in E. coli.

[0049] Fig. 10A-B shows the reaction kinetics of synVBPO and the fitting of the kinetic parameters to Michael-Menten and Hill type kinetics. Fig. 10A The surface plot of SynVBPO halogenation activity shows k cat As a function of H2O2 and KBr concentrations. Fig. 10B The fitting of the kinetic parameters for the halogenation reaction rate using Michaelis-Menten and Hill type kinetics is described. Akaike's Information Criterion Test (AIC), Bayesian Information Criterion Test (BIC) and related statistics are also shown to determine the best fit of the candidate models.

[0050] Fig.11A -D is the Salwin test, depicting the enzyme stability of synVBPO under various concentrations of KBr and H2O2. Fig.11A Salwin assay on syn VBPO in 160 mM KBr and 50 μM H2O2. Fig. 11B Salwin assay on synVBPO in 40 mM KBr and 50 μM H2O2. Fig. 11C Salwin assay of synVBPO in 1 mM KBr and 50 μM H2O2. Fig.11D The Salwin assay was performed on synVBPO in 1 mM KBr and 250 μM H2O2.

[0051] Fig. 12A -D shows the determination of the enzymatic activity of synVBPO as a function of ionic strength and the Michaelis-Menten kinetic parameters as a function of pH. Fig. 12A is a graph showing the fluorescence change per second as a function of ionic strength for the VBPO reaction rate in the presence of peroxide and halide substrates. Fig. 12B SynVBPO k cat Plot as a function of pH. Fig. 12C SynVBPO k cat / Km H2O2 Plot as a function of pH. Fig.12D SynVBPO k cat / Km KBr Plot as a function of pH.

[0052] Fig.13The reaction rate diagram of VCPO in the presence of peroxide source H2O2 and peracetic acid (PAA) and the calculation results of Michaelis-Menten kinetic parameters show that V max,PAA About V max,H2O2 twice as much.

[0053] Fig.14 The graph of the VCPO reaction rate with PAA and H2O2 as substrates and the Michaelis Menten curve fitting equation shows that PAA has a higher v than hydrogen peroxide. max and K m .

[0054] Fig.15 is a graph of DCIM concentration versus time produced in a two-step fed-batch system.

[0055] Fig.16 is a graphic representation of the dissolved oxygen cascade in the glycerol batch stage.

[0056] Fig.17 is an SDS-PAGE polyacrylamide gel showing the results of the purification protocol outlined in the Examples. DETAILED DESCRIPTION

[0057] The present disclosure features peroxidases (e.g., haloperoxidases) and related compositions thereof for producing small halogenated organic compounds or a plurality of small halogenated organic compounds. These small halogenated organic compounds can be used to reduce methane production in microbial or rumen communities. In addition, the present 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.

[0058] definition

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] The term "a" or "an" refers to one or more than one (ie, at least one) grammatical object of the article. For example, "an element" means one element or more than one element.

[0061] When "about" is used herein to modify a numerically defined parameter, it means that the parameter can vary by up to 15% above and below the stated value of the parameter. For example, a small organic compound defined as having a molecular weight of 100 Da can have a molecular weight between 85 Da and 115 Da. In some embodiments, the term "about" means that the parameter can vary by up to 10% above and below the stated value of the parameter.

[0062] As used herein, the term "acquire" or "acquiring" refers to the process of acquiring a value (e.g., a numerical value) or an image or physical entity (e.g., a sample) by "directly acquiring" or "indirectly acquiring" a value or physical entity. "Direct acquisition" means performing a process (e.g., performing an analytical method or protocol) to acquire a value or physical entity. "Indirect acquisition" refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquires a physical entity or value). Directly acquiring a value or physical entity includes performing a process that includes a physical change of a physical substance or the use of a machine or device. Examples of directly acquiring values ​​include obtaining a sample from a human subject. Directly acquiring values ​​includes performing a process that uses a machine or device (e.g., a mass spectrometer) to acquire molecular weight information.

[0063] The term "halogenating agent" as used herein refers to an active agent (e.g., a small molecule or protein) that can modify 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 a salt, such as potassium bromide. In another embodiment, the halogenating agent is a protein, such as a halogenase or a haloperoxidase (e.g., a vanadium haloperoxidase).

[0064] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds comprising 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 can constitute a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising 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, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof. "Multiple polypeptides" refers to two or more polypeptides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200 or 500 or more polypeptides.

[0065] As used herein, the term "peroxidase" refers to an enzyme that reduces a peroxide-containing substrate or a hydroperoxide-containing substrate. Many peroxidases contain a cofactor, such as a heme cofactor, orthovanadate, or a redox-active side chain, such as cysteine ​​or selenocysteine. Exemplary peroxidases include haloperoxidase, ascorbate peroxidase, lactoperoxidase, thyroid peroxidase, and the like.

[0066] The term "rumen" refers to a specialized intestinal compartment found in certain animals (eg, ruminants) that performs various digestive functions in the animal, such as fermentation processes.

[0067] The term "ruminant" refers to an animal that has specialized intestinal compartments that perform various digestive functions within the animal, such as fermentation processes.

[0068] The term "rumen community" or "rumen microbial community" refers to a community of microorganisms, including bacteria, archaea, and protozoa, that live in the digestive tract of large animals, such as ruminants. The rumen microbial community performs a variety of digestive functions in the animal, including assisting digestion to provide key nutrients to the host animal. Exemplary organisms that make up the microbial community include archaeal methanogens, such as Methanobrevibacter, Methanosarcina, and Methanocorpulusum. Exemplary fermentative bacterial species found in the rumen include Coriobacteraceae, Fibrobacter, Ruminococcus, Butyrivibrio, Streptococcus, Prevotella, Succinic acid mononas, Lunar mononas, Lachnospira, and Succinic acid vibrio.

[0069] Selected chemical definitions

[0070] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the periodic table CAS version, Handbook of Chemistry and Physics, 75th edition, inside cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry and specific functional moieties and reactivity are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th 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, 3rd edition, Cambridge University Press, Cambridge, 1987.

[0071] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valence known in the chemical arts.

[0072] When a range of values ​​is listed, it is intended to encompass each value and subrange within that range. For example, "C1-C6 alkyl" is intended to encompass 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.

[0073] As used herein, "alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C 12 alkyl”), 1 to 10 carbon atoms (“C1-C 12 In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), s-propyl (C4), s-propyl (C5), s-propyl (C6), s-propyl (C7), s-propyl (C8), s-propyl (C9), s-propyl (C10), s-propyl (C11), s-propyl (C12), s-propyl (C13), s-propyl (C14), s-propyl (C15), s-propyl (C16), s-propyl (C17), s-propyl (C18), s-propyl (C19), s-propyl (C21), s-propyl (C22), s-propyl (C23), s-propyl (C24), s-propyl (C25), s-propyl (C26), s-propyl (C27), s-propyl (C28), s-propyl (C29), s-propyl (C31), s-propyl (C32), s-propyl (C33), s-propyl (C34), s-propyl (C35), s-propyl (C36), s-propyl (C37), s-propyl (C38), s-propyl (C39). ), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), tert-pentyl (C5) and n-hexyl (C6). Additional examples of alkyl include n-heptyl (C7), n-octyl (C8), etc. Each example of alkyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents, such as 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0074] As used herein, "alkenyl" refers to 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 has 2 to 12 carbon atoms ("C2-C 12 alkenyl”), 2 to 10 carbon atoms (“C2-C 10The carbon-carbon double bond(s) may be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include vinyl (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 and pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each instance of alkenyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkenyl").

[0075] As used herein, the term "alkynyl" refers to a straight chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ("C2-C 24 In some embodiments, an alkynyl group has 2 to 12 carbon atoms ("C2-C 10 alkynyl”), 2 to 10 carbon atoms (“C2-C 10 The carbon-carbon triple bond(s) may be internal (as in 2-butynyl) or terminal (as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each example of an alkynyl group may independently be optionally substituted, i.e., unsubstituted (“unsubstituted alkynyl”), or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted alkynyl”).

[0076] As used herein, the term "heteroalkyl" refers to a non-cyclic stable straight or branched chain or a combination thereof including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen atom and the sulfur atom may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, P, S and Si may be placed at any position of the heteroalkyl. Exemplary heteroalkyls 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)2-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 may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. After reciting "heteroalkyl," a specific heteroalkyl group such as -CH2O, -NR C R D It will be understood that the term heteroalkyl and -CH2O or -NR C R D are not redundant or mutually exclusive. Instead, specific heteroalkyl groups are described to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups, such as -CH2O, -NR C R D Etc. Each instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted (“unsubstituted heteroalkyl”) or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted heteroalkyl”).

[0077] As used herein, "cycloalkyl" refers to a non-aromatic ring system having 3 to 10 ring carbon atoms ("C3-C 10 In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C8 cycloalkyl"). 10Cycloalkyl"). Cycloalkyl can be described, for example, as a C4-C7 membered cycloalkyl, where the term "membered" refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyls 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 cycloalkyls include The cycloalkyl radicals include, but are not limited to, the aforementioned C3-C6 cycloalkyl radicals and 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), etc. Exemplary C3-C6 cycloalkyl radicals include, but are not limited to, 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), etc. 10 Cycloalkyl includes, but is not limited to, the aforementioned C3-C8 cycloalkyl and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C9), 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. As illustrated by the foregoing examples, in certain embodiments, the cycloalkyl is monocyclic ("monocyclic cycloalkyl") or contains a fused, bridged or spiro ring system (such as a bicyclic ring system ("bicyclic cycloalkyl")), and may be saturated or may be partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the cycloalkyl ring, and in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each example of a cycloalkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl"), or substituted ("substituted cycloalkyl") with one or more substituents.

[0078] As used herein, "heterocyclyl" refers to a group of a 3-10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon ("3-10 membered heterocyclyl"). In heterocyclyls containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as long as valence permits. The heterocyclyl group may be a monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiro ring system, such as a bicyclic system ("bicyclic heterocyclyl"), and may be saturated or may be partially unsaturated. Heterocyclyl bicyclic ring systems may include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which the heterocyclyl ring as defined above is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl or heterocyclyl ring; or ring systems in which the heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Heterocyclyl can be described as, for example, 3-7 membered heterocyclyl, wherein the term "member" refers to the non-hydrogen ring atoms within the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted ("substituted heterocyclyl") by one or more substituents. In certain embodiments, heterocyclyl is an unsubstituted 3-10 membered heterocyclyl. In certain embodiments, heterocyclyl is a substituted 3-10 membered heterocyclyl.

[0079]

[0043] As used herein, "halo" or "halogen," by itself or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0080] As used herein, "hydroxy" refers to the group -OH.

[0081] As defined herein, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, and heterocyclyl 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). In general, the term "substituted", whether or not preceded by the term "optionally", means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permitted substituent, such as a substituent that, upon substitution, results in a stable compound, such as a compound that does not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when substitution occurs at more than one position in any given structure, the substituent at each position is the same or different. The term "substituted" is intended to include substitution with all permissible substituents of an organic compound, any substituent that results in the formation of a stable compound as described herein. The present disclosure contemplates any and all such combinations to achieve a stable compound. For purposes of the present disclosure, a heteroatom (such as nitrogen) may have a hydrogen substituent and / or any suitable substituent that satisfies the valence of the heteroatom and results in the formation of a stable moiety as described herein.

[0082] Haloperoxidase

[0083] Vanadium haloperoxidases (VHPOs) (EC 1.11.1.18) are members of a subfamily of oxidoreductases containing vanadate as a prosthetic group. VHPOs catalyze the two-electron oxidation of halide ions to the corresponding hypohalous acids under a substrate-inhibited double-ping-pong mechanism, usually without substrate specificity and 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 chloroperoxidases (VCPOs) oxidize chloride, bromide, and iodide; vanadium bromoperoxidases (VBPOs) oxidize bromide and iodide; and vanadium iodoperoxidases (VIPOs) oxidize iodide. VHPOs are able to halogenate a wide range of organic compounds of both commercial and pharmaceutical interest and exhibit stability under a wide range of conditions, including elevated temperatures, acidic and alkaline pH values, oxidizing conditions, and organic solvents. These properties make VHPOs attractive candidates for use in a variety of industrial transformations.

[0084] Among the most studied VHPOs, there is a class of VBPOs found in the marine algae of the genus Coralline. Coralline is a calcareous red alga commonly found in tidal pools and marine habitats. VBPO from Coralline (CoVBPO) has been successfully isolated from the marine algae and biochemical and structural studies have been performed. CoVBPO is characterized by having a large dodecamer (12x64kDa) structure composed of twelve identical subunits. CoVBPO has been shown to exhibit functional stability in the pH range of 5-10 and at temperatures up to 90°C; in addition, it is tolerant to organic solvents such as ethanol, methanol, and propan-1-ol, which makes it an attractive enzyme in commercial biocatalysis. However, it has been challenging to directly isolate appropriate amounts of CoVBPO from Coralline for industrial use.

[0085] Industrial enzymes are enzymes used commercially in a variety of industries such as pharmaceuticals, chemical production, biofuels, food and beverages, and consumer products. Due to advances in heterologous expression in hosts such as E. coli and various fungi, biocatalysis by isolated enzymes may be more economical than using whole cells. However, in many cases, heterologous expression of foreign proteins in E. coli will produce insoluble inclusion bodies due to protein misfolding. Specifically, since pure CoVBPO in E. coli easily forms large insoluble inclusion bodies, it is difficult to perform heterologous expression of large amounts of pure CoVBPO in E. coli. Therefore, we searched for CoVBPO analogs that may show improved properties from publicly available databases.

[0086] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises the 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., haloperoxidase) comprises the 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., haloperoxidase) comprises the amino acid sequence of formula (Ia): GPQTGPX1 or GPLTGPX1, wherein X1 is proline or threonine.

[0087] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (II): X3WKE, wherein 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., haloperoxidase) comprises an amino acid sequence of formula (II-a): GX4FX3WKE, wherein 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., haloperoxidase) comprises an amino acid sequence of formula (II-b): GIX5X3WX6, wherein 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, GIFAWK, GIFAWE, GIFAWQ, GILSWK, GILSWE, GILSWQ, GILAWK, GILAWE and GILAWQ.

[0088] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (III): X7KW or X7KY, wherein 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, wherein 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-a): X7KWX8FEFW or X7KYX8FEFW, wherein 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 QKYHF EFW.

[0089] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of formula (IV): YHX9, wherein 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-a): YH X9PFY, wherein 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 10 is 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 the amino acid sequence of formula (IV-c): FRX 11 YHX9PFY, where 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, FRM YHAPFY and FRIYHAPFY.

[0090] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (V): GVHX 12 , where 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (Va): GVHWX 13 F, where X 13 is arginine, histidine, valine or glutamic acid. In one embodiment, the amino acid sequence of formula (Va) is selected from GVHWRF, GVHWHF, GVHWVF and GVHWEF. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises the amino acid sequence of formula (Vb): WX 13 FDAX 14 , where X 13is arginine, histidine, valine or glutamic acid; and X 14 is 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.

[0091] In one embodiment, the peroxidase (eg, haloperoxidase) comprises an amino acid sequence of formula (VI): X 15 LI, where 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (VI-a): DGX 16 X 15 LI, where X 15 is asparagine or lysine, and 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (VI-b): WX 17 YDX 16 X 15 LI, where X 15 is asparagine or lysine, 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 the group consisting of WGYDGSNLI, WAYDGSNLI, WAYDGANLI, WGYDGANLI, WGYDGSKLI, WAYDGSKLI, WAYDGAKLI and WGYDGAKLI.

[0092] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (VII): GTPX 18 , where X 18 is proline or valine. In one embodiment, the amino acid sequence of formula (VII) is selected from GTPP and GTPV. In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (VII-a): X 19 LIGTPX 18 , where X 19 is proline or valine, and X 18is asparagine or lysine. 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) comprises the amino acid sequence of formula (VII-b): DGSX 19 LIGTPX 18 , where 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (VII-c): X 20 YDX 19 LIGTPX 18 , where 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 the group consisting of AYDGSNLIGTPP, GYDGSNLIGTPP, AYDGSKLIGTPP, GYDGSKLIGTPP, AYDGSNLIGTPV, GYDGSNLIGTPV, AYDGSKLIGTPV and GYDGSKLIGTPV.

[0093] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (VIII): AYLNACX 21 I, where X 21 is leucine or isoleucine. In one embodiment, the amino acid sequence of formula (VIII) is selected from AYLNAACLI and AYLNACII. In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (VIII-a): AYLNACX 21 IX 22 L, where 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 the group consisting of AYLNACLILL, AYLNACIILL, AYLNACLIML and AYLNACIIML.

[0094] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (IX): IPX23 or LPX 23 , where 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 the amino acid sequence of formula (IX-a): DX 25 X 24 IPX 23 or DX 25 X 24 LPX 23 , where X 23 is methionine or phenylalanine; X 24 is glycine, histidine or asparagine; X 25 is glutamine, lysine, proline or serine. In one embodiment, the amino acid sequence of formula (IX-a) is selected from 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 selected from 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.

[0095] In one embodiment, the peroxidase (eg, haloperoxidase) comprises an amino acid sequence of 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., haloperoxidase) comprises the 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; and X 27is 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.

[0096] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XI): EVATRALKX 28 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 the amino acid sequence of formula (XI-a): EVATRALKX 28 X 29 RX 30 QK, where 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) comprises the amino acid sequence of 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.

[0097] In one embodiment, the peroxidase (eg, haloperoxidase) comprises an amino acid sequence of formula (XII): EGSPX 32 HP, where 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 the amino acid sequence of formula (XII-a): EGSPX 32HPX 33 YG, where 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 EGSP FHPAYG. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises the amino acid sequence of formula (XII-b): EGSP X 32 HPX 33 YGX 34 GHA, where 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 EGSPMHPSYG AGHA, EGSPMHPAYGSGHA, EGSPMHPSYGAGHA, EGSPMHPA YGSGHA, EGSPFHPSYGAGHA, EGSPFHPAYGSGHA, EGSPFHP SYGAGHA and EGSPFHPAYGSGHA.

[0098] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (XIII-a): VAGACX 35 TLKAFFX 36 , where 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, VAGACT TLKAFFQ and VAGACTTLKAFFD.

[0099] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XIV): DGX 37 X 38 LD or DEX 37 X 38 LD, where X 37is serine, lysine, arginine, threonine or aspartic acid; and 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.

[0100] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XV): LTVX 39 , where 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) comprises the amino acid sequence of formula (XV-a): X 40 ELNK, where 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) comprises the amino acid sequence of formula (XV-b): LTVX 39 X 40 ELNK, where X 39 is alanine, aspartic acid or glutamic acid; and X 40 is 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.

[0101] In one embodiment, Formula (XVI) comprises NISIGR, NISVGR, or NVAIGR.

[0102] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XVII): AGVHYX 41 X 42 D, where X 41 is phenylalanine or tyrosine, and X42 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (XVII-a): AGVHYX 41 X 42 DX 43 X 44 ES, where 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. In one embodiment, the amino acid sequence of formula (XVII-a) is selected from AGVHYFTDYIES, AGVHYFSDYIES, AGVHYFTDQIES, AGVHYFSDQIES, AGVHYFTDYRES, AGVHYFS DYRES, AGVHYFTDQRES, AGVHYFSDQRES, AGVHYYTDYFES, AGVHYYSDYFES, AGVHYYTDQFES, AGVHYYSDQFES, AGVHYYTDYVES, AGVHYYSDYVES, AGVHYYTDQVES and AGV HYYSDQVES.

[0103] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (XVIII-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.

[0104] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XIX): GEX47 X 48 A, where X 47 is glutamine, lysine or glutamate; and X 48 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) comprises the amino acid sequence of formula (XIX-a): GEX 47 X 48 AX 49 G, where X 47 is glutamine, lysine or glutamate; 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 the group consisting of GEQIALG, GEQIAIG, GEQIALG, GEQIAIG, GEKIALG, GEKIAIG, GEKVALG, GEKVAIG, GEEVALG, GEEVAIG, GEEVALG and GEEVAIG.

[0105] In one embodiment, the peroxidase (eg, haloperoxidase) comprises an amino acid sequence of formula (XX): LX 50 EQ, where 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 (eg, haloperoxidase) comprises the amino acid sequence of formula (XX-a): LX 50 EQX 51 LT, where X 50 is glutamate, glutamine or lysine; and X 51 is methionine, lysine or serine. In one embodiment, the amino acid sequence of formula (XVIII) is selected from LEEQMLT, LEEQKLT, LEEQSLT, LQEQMLT, LQEQKLT, LQEQSLT, LKEQMLT, LKEQKLT and LKEQ SLT. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises the amino acid sequence of formula (XX-b): LX 50 EQX 51 LTFX 52 E or LX 50 EQX 51 LTYX 52 E, where X 50 is glutamate, glutamine or lysine; X 51is methionine, lysine or serine; and X 52 It is proline, alanine, glycine or serine. In one embodiment, the amino acid sequence of formula (XX-b) is selected from the group consisting of LEEQMLTPE, LEEQMLTAE, LEE QMLTGE, LEEQMLTSE, LEEQKLTPE, LEEQKLTAE, LEEQKLTGE, LEEQKLTSE, LEEQSLTPE, LEEQSLTAE, LEEQSLTGE, LE EQSLTSE, LQEQMLTPE, LQEQMLTAE, LQEQMLTGE, LQEQML LKEQKLTPE SLTAE, LKEQSLTGE, and LKEQSLTSE.

[0106] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XXI): EX 53 F, where 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) comprises the amino acid sequence of formula (XXI-a): EX 53 FX 54 M or EX 53 FX 54 F, where 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, ETFFM, EEFFM, EDFFM, EKFFM and ENFFM. In one embodiment, the amino acid sequence of formula (XXI-a) is selected from ETFTF, EEFMF, EDFTF, EKFTF, ENFTF, ETFSF, EEFSF, EDFSF, EKFSF, ENFSF, ETFFF, EEFFF, EDFFF, EKFFF and ENFFF.

[0107] In one embodiment, the peroxidase (eg, haloperoxidase) comprises the amino acid sequence of formula (XXII): PSGHAX 55 FX 56 , where 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, PSGH ATFG, PSGHATFS, PSGHATFG and PSGATFS. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises the amino acid sequence of formula (XXII-a): FP X 57 YPSGHAX 55 FX 56 , where X 55 is serine or threonine, X 56 is glycine or serine, and X 57 is proline or asparagine. In one embodiment, the amino acid sequence of formula (XXII) is selected from FPNYPSGHASFG, FPNYPSGHASFS, FPNYPSG HASFG, FPNYPSGHASFS, FPNYPSGHATFG, FPNYPSGHATFS, FPNYPSGHATFG, FPNYPSGATFS, FPPYPSGHASFG, FPPYPSGH ​​ASFS, FPPYPSGHASFG, FPPYPSGHASFS, FPPYPSGHATFG, FP PYPSGHATFS, FPPYPSGHATFG and FPPYPSGATFS.

[0108] The peroxidase (e.g., haloperoxidase) of the present disclosure can contain any amino acid sequence described herein. In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of one of the following: Formula (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), (XV I), (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) comprises an amino acid sequence of two of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of three of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of four of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of five of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of six of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of seven of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of eight of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any one of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (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) comprises an amino acid sequence of any of the following: Formula (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), (X VI), (XVII), (XVII-a), (XVIII), (XVIII-a), (XIX), (XIX-a), (XX), (XX-a), (XX-b), (XXI), (XXI-a), (XXII) or (XXII-a).

[0109] In one embodiment, the peroxidase (e.g., haloperoxidase) comprises an amino acid sequence of one 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 amino acid sequence of two 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 amino acid sequence of three 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 amino acid sequence of four 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 amino acid sequence of five 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 amino acid sequence of six 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 amino acid sequence of seven 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 amino acid sequence of eight 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 amino acid sequence of nine 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 amino acid sequence of 10 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 amino acid sequence of 11 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 amino acid sequence of 12 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 amino acid sequence of 13 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 amino acid sequence of 14 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 amino acid sequence of 15 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 amino acid sequence of 16 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 amino acid sequence of 17 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 amino acid sequence of 18 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 amino acid sequence of 19 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 amino acid sequence of 20 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 amino acid sequence of 21 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 amino acid sequence of 22 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).

[0110] Table 1. Exemplary motifs and associated subvariables X in haloperoxidases n Summary table

[0111]

[0112]

[0113]

[0114]

[0115] Sequences of exemplary haloperoxidases found in various organisms, including Curvularia ini, Coralline algae, Halomicronema hongdechloris, Moore bouillonii, Trichodesmium erythrorhizium, Cryptococcus montana, Synechococcus sp. PCC7335, Coralilin, and Saccharomyces cerevisiae, are provided below in Table 2. These sequences include naturally occurring and engineered VHPO sequences.

[0116] Table 2. Exemplary VHPO sequences

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Host cell microorganism

[0136] The present disclosure provides peroxidases and related methods for producing a variety of small halogenated organic compounds, which compounds can be used, for example, to reduce methane in the rumen of agricultural animals. In one embodiment, peroxidase (VHPO) is heterologously produced in a host cell microorganism. In one embodiment, producing peroxidase (VHPO) in a host cell microorganism causes, for example, an increase in the total amount of peroxidase (VHPO) compared to a reference standard. As used herein, producing peroxidase (VHPO) in a host cell microorganism includes expression, translation and / or secretion of peroxidase (e.g., VHPO).

[0137] Host cell microorganisms suitable for the present disclosure are capable of producing peroxidases (VHPO) as described herein. In one embodiment, the host cell microorganism naturally produces peroxidases (VHPO), such as expressing endogenous peroxidases (VHPO). In one embodiment, the host cell microorganism produces exogenous peroxidases (VHPO). In one embodiment, the host cell microorganism is genetically modified to produce peroxidases (VHPO), such as expressing heterologous peroxidases (VHPO). In such embodiments, nucleic acids encoding heterologous peroxidases (VHPO) are introduced into the host cell microorganism using standard methods known in the art, such as by electroporation, transfection or transduction. Heterologous peroxidases (VHPO) can be peroxidases (VHPO) naturally produced in different microorganisms, or can be modified peroxidases (VHPO) comprising amino acid sequences different from corresponding naturally occurring peroxidases (VHPO) or different functions and / or activities (e.g., increased or decreased activities).

[0138] The host cell microorganism can be a fungus (e.g., yeast), a bacterium, a protozoan, an archaeon, a synthetic organism, or a semisynthetic organism that produces one or more proteins, e.g., one or more enzymes, such as one or more peroxidases (e.g., VHPO). 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 archaeon. Exemplary host cell microorganisms include Pichia pastoris, Aspergillus niger, Saccharomyces cerevisiae, Escherichia coli, and Synechococcus PCC11901.

[0139] Pichia pastoris is a very successful heterologous expression system. Yeast Pichia pastoris has been used as an expression system for proteins and enzymes in biotechnology because it has been shown to obtain increased protein yields compared to many natural systems. Pichia can grow in specific culture media to reach high cell densities, resulting in wet cell densities of up to 250 g / L for the produced proteins (e.g., secreted proteins), and has excellent scalability (e.g., scalability of up to about 200,000 liters of cell culture). Proper folding and secretion are two major advantages of the Pichia pastoris system. The system is also commonly used to produce complex eukaryotic cell proteins, and has been successfully used to produce fungal enzymes, including β-glucosidases, lignocellulase, and oxidases from macroalgae. Although the Pichia pastoris expression system may be easy to use on an experimental and industrial scale, the production of recombinant proteins in Pichia pastoris usually requires an optimization process to achieve the maximum yield of the target protein. It is well known that to obtain the desired results, many conditions must be tested, including optimizing the culture medium composition, growth temperature, and protein and nucleic acid sequences. Pichia pastoris cells can also be converted into cell factories for large-scale production.

[0140] Aspergillus niger is a filamentous mold that was originally developed for the production of citric acid and is now the basis for the production of a wide variety of proteins, enzymes and secondary metabolites. Aspergillus niger is now increasingly used as an alternative biospecies. Products include proteins (including enzymes) but also pharmaceuticals for human and animal health.

[0141] Saccharomyces cerevisiae is a commonly used host cell microorganism for improving heterologous production of proteins (e.g., enzymes). In addition to the large-scale production potential of baker's yeast, there are also a large number of studies on methods for concentrating products in organelles (such as peroxisomes) within cells, which has significant advantages in biomanufacturing. The peroxisomes of Saccharomyces cerevisiae have been engineered for equivalent reactions, and large-scale production of the yeast has been fully established in the food industry (e.g., wine, beer, bread). Intracellular compartmentalization can provide optimal conditions for the desired enzymatic reactions by increasing metabolic flux and reducing metabolic crosstalk, making it a powerful strategy for separating toxic products from host cell sols, thereby limiting cytotoxicity, improving growth, and increasing product titers.

[0142] Cyanobacteria are prokaryotic oxyphotoautotrophs that can convert CO2 and inorganic sources of nitrogen, phosphorus, and trace elements into biomass. Synechoccus PCC11901 is a durable marine cyanobacterial strain that has been identified for high biomass production on an industrial scale. PCC11901 can be cultured in seawater that is unsuitable for agricultural use or direct human drinking; other promising features include: (i) it can grow at high light intensities and over a wide range of salinities, (ii) it can accumulate up to about 33 g dry cell weight / L, and (iii) the genome and genetic tools are already available. Interestingly, PCC 11901 lacks VHPO, but can heterologously express the CiVHPO enzyme. In addition, there is a large literature on the ecology, genetics, cell biology, and biotechnological applications of Synechococcus that can be directly applied to PCC11901. The target strain has been engineered for rapid growth, solvent tolerance, product biosynthesis and accumulation, and bioprotection.

[0143] In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence that 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 at least 99% identical to a peroxidase (e.g., VHPO) described herein or a functional fragment thereof, such as an amino acid sequence that is 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% identical to a peroxidase (e.g., VHPO) described herein or a functional fragment thereof.

[0144] 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) comprises an amino acid sequence that 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 at least 99% identical to an amino acid sequence listed in Table 2. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 2. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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: 2. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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) comprises an amino acid sequence that 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 at least 99% identical to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 4.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence of SEQ ID NO: 5. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises the amino acid sequence of SEQ ID NO: 6. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 8. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:9.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence of SEQ ID NO: 10. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence having 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 one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 13. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:14.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 15. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence that 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 at least 99% identical to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 18. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:19.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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: 19. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 20. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 one embodiment, the peroxidase (e.g., VHPO) comprises the amino acid sequence of SEQ ID NO: 21. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises the amino acid sequence of SEQ ID NO: 22. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 23. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:24.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 25. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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) comprises an amino acid sequence that 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 at least 99% identical 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., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence of SEQ ID NO: 28. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 one embodiment, the peroxidase (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:29.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 30. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence having 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., VHPO) comprises the amino acid sequence of SEQ ID NO: 32. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 33. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:34.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 35. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises the amino acid sequence of SEQ ID NO: 36. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence that 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 at least 99% identical 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) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 38. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:39.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 40. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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: 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) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence of SEQ ID NO: 43. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence having 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., VHPO) comprises an amino acid sequence of SEQ ID NO: 45. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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) comprises an amino acid sequence that 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 at least 99% identical 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) comprises an amino acid sequence having 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 an amino acid sequence of SEQ ID NO: 48. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 (eg, VHPO) comprises the amino acid sequence of SEQ ID NO:49.In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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 one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence of SEQ ID NO: 50. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 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.

[0145] In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to a peroxidase (e.g., VHPO) described herein or a functional fragment thereof. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to a peroxidase (e.g., VHPO) listed in Table 2. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 1. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 2. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 3. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 4. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 5. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 7. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO:8.In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 9. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 10. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 11. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 12. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 13. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 14. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 15. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 16. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 17. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO:18.In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 19. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 20. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 21. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 22. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 23. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 24. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 25. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 26. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 27. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO:28.In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 29. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 30. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 31. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 32. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 33. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 34. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 35. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 36. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 37. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO:38.In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 39. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 40. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 41. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 42. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 43. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 44. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 45. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 46. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 47. In one embodiment, the peroxidase (e.g., VHPO) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO:48.In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 49. In one embodiment, the peroxidase (e.g., VHP0) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more mutations relative to the amino acid sequence of SEQ ID NO: 50.

[0146] In the context of two or more amino acids or nucleic acid sequences, percentage identity refers to two or more identical, e.g., conservative sequences. If, when compared or aligned for maximum correspondence over a comparison window, in a specified region or over the entire sequence, or using one of the following sequence comparison algorithms or by manually comparing and visually inspecting the specified region measured, the two sequences have a specified percentage of identical amino acid residues or nucleotides (e.g., in a specified region or when not specified, 60% identity within the entire sequence, 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), then the two sequences are "substantially identical". Optionally, the identity exists over a region that is at least about 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 75 amino acids, 100 amino acids, or 150 amino acids in length. The identity can exist over a region that is about 10 amino acids to about 100 amino acids, or about 50 amino acids to about 250 amino acids, or about 200 amino acids to about 500 amino acids in length.

[0147] For sequence comparison, a sequence usually serves as a reference sequence to which one or more test sequences are compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters may be used or alternative parameters may be specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters. The method for comparing sequences for comparison is well known in the art. Optimal alignment of sequences for comparison can be performed by methods such as 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; a search for similarity to the method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444; computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software 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).

[0148] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (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 through the National Center for Biotechnology Information.

[0149] Functional variants may include one or more mutations such that the variant retains a certain level of activity, such as a peroxidase described herein (e.g., VHPO) produced by a microorganism from which the enzyme is derived. In one embodiment, the 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 a peroxidase (e.g., VHPO) as a corresponding naturally occurring peroxidase (e.g., VHPO). In embodiments, the functional variant has at least 200%, at least 300%, at least 400%, at least 500%, at least 1000% or more of a peroxidase (e.g., VHPO) as a 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 that measures consumption of bromide activity can be performed. In addition, functional assays can be combined with each other; for example, if the peroxidase is VBPO, VBPO activity can be combined with consumption of peroxide-containing compounds (e.g., H2O2 or peracetic acid (PAA)). Other exemplary assays include MCD and APF assays.

[0150] 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. Mutagenesis can also be achieved by using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas system. The CRISPR / Cas system is naturally present in bacteria and archaea, and has been modified for gene editing (silencing, enhancing or mutating specific genes) in eukaryotic organisms 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 into the cell.

[0151] Mutation can be a conservative amino acid substitution, wherein the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-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 within the peroxidase (e.g., VHPO) can be replaced by other amino acids from the same side chain family, and the resulting peroxidase (e.g., VHPO) activity is comparable to the wild-type peroxidase (e.g., VHPO) activity (e.g., at least 80%, 85%, 90%, 95%, or 99% of the peroxidase (e.g., VHPO) activity). Alternatively, the mutation can be an amino acid substitution, in which an amino acid residue is replaced by an amino acid residue having a different side chain.

[0152] Such mutations may change or affect various enzyme properties of peroxidases (e.g., VHPO). For example, such mutations may change or affect activity, such as peroxidase activity, thermostability, optimal pH of the reaction, enzyme kinetics, or enzymes, such as substrate recognition of peroxidases (e.g., VHPO). In some embodiments, the mutation causes the peroxidase (e.g., VHPO) activity of the variant to increase compared to the peroxidase (e.g., VHPO) activity of the wild-type peroxidase (e.g., VHPO). In some embodiments, the mutation causes the thermostability of the variant to increase or decrease compared to the wild-type peroxidase (e.g., VHPO). In one embodiment, the mutation changes the pH range in which the variant performs the peroxidase reaction optimally compared to the wild-type peroxidase (e.g., VHPO). In one embodiment, the mutation causes the kinetics (e.g., k of the peroxidase (e.g., VHPO) to increase compared to the wild-type peroxidase (e.g., VHPO). cat , K M、 k cat / K M or K D ) is increased or decreased. In one embodiment, the mutation results in an increase or decrease in the ability of the peroxidase (e.g., VHPO) to recognize or bind to a substrate 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 compared to a wild-type peroxidase (e.g., VHPO).

[0153] In addition, such mutations may change or affect the stability or expression level of peroxidase (e.g., VHPO). For example, such mutations can increase the stability of peroxidase (e.g., VHPO) under specific conditions (e.g., temperature or pH), or increase the expression level of peroxidase (e.g., VHPO) in a specific host cell microorganism. In one embodiment, compared to the wild-type sequence, the mutation causes the stability of peroxidase (e.g., VHPO) to increase. In one embodiment, compared to the wild-type sequence, the mutation causes the expression level of peroxidase (e.g., VHPO) to increase.

[0154] 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).

[0155] Cell culture and protein expression

[0156] Host cell microorganisms capable of producing proteins, such as peroxidases (e.g., VHPO), can be in cell culture. The cell culture comprises one or more cells in a cell culture medium. The cell culture medium can be an aqueous cell culture medium comprising components that support cell maintenance, cell activity, cell growth and / or cell proliferation. The cell culture medium typically comprises 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 culturing the host cell microorganisms described herein comprises ammonium salts, such as ammonium sulfate and / or ammonium hydroxide; potassium salts, such as potassium hydroxide; calcium salts, such as calcium chloride; magnesium salts, such as magnesium sulfate; manganese salts, such as manganese sulfate; iron salts, such as iron sulfate; zinc salts, such as zinc sulfate; cobalt salts, such as cobalt chloride, phthalic acid; lactose; antibiotics, such as and a carbon source, such as glycerol or carbon dioxide.

[0157] Host cell microorganism or cell culture is contacted with for example feed carbon source (for example sugar) to support the growth or propagation of host cell microorganism.In one embodiment, host cell microorganism or cell culture is contacted with glucose, for example, feed glucose.Along with the host cell microorganism breeding in culture, cell culture can be transferred to a larger container from a container (for example, cell culture container), to allow and promote the host cell microorganism to continue to breed.For example, the host cell microorganism contacts with sugar in the first container under suitable conditions, as described herein, so that the host cell microorganism breeds.Breeding can be monitored, and once reaching required growth level (for example specific growth stage) or required proliferation level (for example, measured by the turbidity or cell number of culture), the host cell microorganism can be transferred to a second container, wherein the second container is larger (for example, by volume) than the first container.Microorganism is transferred to a second larger container to allow and promote the microorganism to continue to breed.In embodiments, microorganism is transferred once, for example, transferred to a second larger container from the first container. In embodiments, the host cell-microorganism is transferred more than once, e.g., two, three, four, five, six, seven, eight, nine, or ten times, wherein for each transfer, the host cell-microorganism is transferred to a larger container than the container from which the host cell-microorganism was transferred.

[0158] Containers suitable for transferring and culturing host cell microorganisms described herein include any cell culture container known in the art. Examples of suitable containers include, but are not limited to, cell culture bottles, roller bottles, bioreactors or tanks. Other cell culture conditions suitable for maintaining cell activity or promoting cell proliferation are known in the art. Cell culture conditions to be considered include pH, temperature, oxygen level and movement. The pH of cell culture, such as culture medium, is generally physiological pH, such as between pH 4-8, or between pH 5-7, such as pH 5, pH 6 or pH 7. The growth temperature of host cell microorganisms producing peroxidase (such as VHPO) is generally between 20 and 40°C, such as 30°C. In some embodiments, specific strains of host cell microorganisms can show enhanced proliferation or enzyme production at elevated temperatures (such as 32 or 37°C) or at lower temperatures (such as 27°C), respectively. Cell culture can be static, or movement can be used to promote maintenance or proliferation. For example, cell culture can be rolled, shaken or agitated to enhance cell proliferation. The cell culture conditions disclosed herein are merely exemplary and should not be construed as limiting. Those conditions different from the cell culture conditions explicitly listed herein can be envisioned or determined experimentally, and may depend on the species or strain of the host cell microorganism used. Cell culture conditions sufficient to proliferate a host cell microorganism capable of producing a peroxidase (e.g., VHPO) result in an increase in the number of cells of the host cell microorganism culture. Cell culture conditions sufficient to produce a peroxidase (e.g., VHPO) result in one or more cells of the microorganism producing a peroxidase (e.g., VHPO).

[0159] Once the cell culture reaches the desired growth level, such as a specific growth stage or culture volume size, or when the cell culture (e.g., aqueous portion) is substantially free of a carbon source (e.g., sugar) for stimulating proliferation, the cell culture can be induced to produce a protein, such as a peroxidase (e.g., VHPO) as described herein. The composition comprising the small organic compound starting material as described herein is added (e.g., feed supplement) 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 comprising the small organic compound is directly added to the culture. In one embodiment, the composition comprising the small organic compound is added to an enzyme production medium, the medium comprising components that support and promote protein production, such as peroxidase (e.g., VHPO). The host cell microorganism is then transferred or cultured in an enzyme production medium. The enzyme production medium can include physiological salts, such as ammonium salts, and compositions including caramelized sugar products and / or a second active agent, and is adjusted to pH 4-7, such as pH 6. In one embodiment, the enzyme production medium comprises ammonium sulfate, rice bran, and a composition comprising small organic compounds.

[0160] In one embodiment, small organic compound and second activating agent are added to host cell microorganism or cell culture simultaneously. Small organic compound and second activating agent (for example, substrate containing peroxide, for example H2O2 or PAA) can be present in the same composition, or can be present in a separate composition. When small organic compound and second activating agent are present in the same composition, small organic compound and second activating agent can be components of enzyme production culture medium. In another embodiment, small organic compound and second activating agent are located in a separate composition, and are added to host cell microorganism or cell culture successively. For example, small organic compound can be added to host cell microorganism or cell culture before or after the second activating agent is added to host cell microorganism or cell culture. In this sequential induction process, the duration between adding small organic compound and adding second activating agent can be several hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours or longer, or several days, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more days.

[0161] The little organic compound can be introduced into the host cell microorganism, for example, by being directly added into the culture or by producing enzyme culture medium twice a day, once a day, every other day, every three days or once a week. The little organic compound can be added with a concentration range of 1-20g / L, 1-15g / L, 1-10g / L, 1-5g / L, 2-15g / L, 2-10g / L, 2-5g / L, 5-20g / L, 5-15g / L, 5-10g / L, 4-5g / L, 10-20g / L or 10-15g / L of the host cell microorganism cell culture. The little organic compound can be added with 0.5g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 15g / L or 20g / L or higher concentration of the host cell microorganism culture. In one embodiment, the small organic compound is added to the host cell microorganism at 4 g / L once a day or 5 g / L once a day.

[0162] A second active agent (e.g., a peroxide-containing substrate, e.g., H2O2 or PAA) can be introduced into the host cell microorganism, e.g., by adding it directly to the culture twice a day, once a day, every other day, every 3 days, or once a week, or by passing through an enzyme production medium. The second active agent can be added in 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 host cell microorganism cell culture. The second active agent can be added at a concentration 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 of the host microorganism culture. In one embodiment, the second active agent is added to the host cell microorganism once a day at 5 g / L.

[0163] In embodiments, the concentration of the small organic compound or second active agent used to induce the production of peroxidase (e.g., VHPO) is greater than or equal to 0.1% weight volume (w / v), 0.5% w / v, 1% w / v, 2% w / v, or 5% w / v, and less than or equal to 25% w / v, 20% w / v, 15% w / v, and 10% w / v. The host cell microorganism can be induced to produce peroxidase (e.g., VHPO) for one or more days, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 30 days or more. The duration of induction may depend on the size (e.g., volume or cell number) of the host cell microorganism culture, the microorganism used, or the amount of peroxidase (e.g., VHPO) required. In one embodiment, the host cell microorganism is induced to produce a peroxidase (e.g., VHPO) for 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, or 1-12 days.

[0164] By measuring the level of peroxidase (e.g., VHPO) present in the cell culture and / or the activity of the peroxidase (e.g., VHPO) produced by the cells, the output of peroxidase (e.g., VHPO) can be measured from the cell culture. For example, the aqueous portion of the culture can be separated, for example, by centrifuging the cell culture or aliquots or samples of the cell culture. Protein assays known in the art, such as Bradford assays or nanodrop protein quantification, can be used to measure the total protein level or titer, e.g., g / L, in the aqueous portion of the culture. The total protein titer indicates the amount of peroxidase (e.g., VHPO) produced by a microorganism or cell culture. Control samples can be used to standardize the amount of protein present in a cell culture that is not induced to produce peroxidase (e.g., VHPO).

[0165] Peroxidases (e.g., VHPO) produced by host cell microorganisms as described herein can be used in biological or industrial processes, for example, peroxidases themselves or products of peroxidase reactions can be used in rumen compositions as described herein. Methods for regulating rumen characteristics are further described herein, which include reducing methane production in the rumen. As described herein, host cell microorganisms or cultures thereof that have been induced to produce peroxidases (e.g., VHPO) can be added directly to the rumen. Alternatively, small organic compounds or multiple small organic compounds to be treated can be added directly to host cell microorganisms or cultures that have been induced to produce peroxidases (e.g., VHPO). On the other hand, halogenated organic compounds separated and produced as peroxidase catalysis products are added to the rumen as inhibitors of methane generation. On the other hand, halogenated organic compounds produced as products catalyzed by peroxidases are delivered to the rumen to inhibit methane generation by being encapsulated in a matrix.

[0166] Peroxidases (e.g., VHPO) produced by host cell microorganisms described herein can also be separated or purified before being used in culture or rumen samples. Peroxidases (e.g., VHPO) can be separated from one or more of the following components: microorganisms, such as cells of microorganisms; small organic compound starting materials; small organic halogenated compound products; second active agents; components of cell culture media, such as glucose, physiological salts; and one or more proteins present in the culture that do not have peroxidase activity. Peroxidases (e.g., VHPO) can be purified so that the peroxidases (e.g., VHPO) are substantially free of other proteins, cell debris, nucleic acids (e.g., from host cell microorganisms), small organic compounds, and / or active agents that do not have peroxidase activity. Methods for separating or purifying peroxidases (e.g., VHPO) are known in the art and may include centrifugation, filtration, protein fractionation, size exclusion chromatography, affinity chromatography, ion exchange chromatography, or any combination thereof.

[0167] Halogenation of compounds

[0168] The present disclosure features methods for producing small halogenated organic compounds and related compositions thereof using peroxidases. In one embodiment, the method comprises (i) providing a small organic compound or a plurality of small organic compounds; (ii) providing a peroxide source, such as H2O2 or peracetic acid (PAA), and a halogen source, such as a halide salt, such as KBr, NaCl or KI; (iii) contacting the small organic compound or a plurality of small organic compounds with a peroxidase (such as a vanadium haloperoxidase (i.e., VHPO)) or its reaction product (i.e., hypohalous acid) under conditions sufficient to produce the small halogenated organic compound or a plurality of small halogenated organic compounds to form a reaction mixture; and (iv) evaluating the produced small halogenated organic compound or a plurality of small halogenated organic compounds.

[0169] 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, brown algae tannins, tannins, lignins or lignin derivatives. In one embodiment, the small organic compound comprises a functional group, such as an aldehyde, a ketone, an acetyl group, an acyl group, a hydroxyl group, an ester, an ether, an amine, an amide, an aryl group, a heteroaryl group, a heterocyclic group or a cycloalkyl group. In one embodiment, the small organic compound comprises an alkenyl group or an alkynyl group. In one embodiment, the small organic compound comprises an aldehyde or a ketone group. In one embodiment, the small organic compound comprises an alpha-beta unsaturated ketone. In one embodiment, the small organic compound is acetone or acetylacetone.

[0170] Small organic compounds contain at least 1 carbon atom, for example at least 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 12 carbon atoms or more. In one embodiment, the small organic compound comprises 1 to 10 carbon atoms, 1 to 6 carbon atoms, 2 to 10 carbon atoms, 3 to 10 carbon atoms, 4 to 10 carbon atoms, 5 to 10 carbon atoms or 6 to 10 carbon atoms. In some embodiments, the small organic compound is saturated. In some embodiments, the small organic compound comprises unsaturated elements, for example 2, 3, 4, 5, 6, 7, 8 or more unsaturated elements. In one embodiment, the small organic compound also comprises oxygen atoms, nitrogen atoms, sulfur atoms or phosphorus atoms. In one embodiment, the small organic compound comprises 1, 2, 3, 4, 5 or 6 oxygen atoms. In one embodiment, the small organic compound comprises 1, 2, 3, 4, 5 or 6 nitrogen atoms. In one embodiment, the small organic compound comprises 1, 2, 3, 4, 5 or 6 sulfur atoms. In one embodiment, the small organic compound comprises 1, 2, 3, 4, 5 or 6 phosphorus atoms.

[0171] In one embodiment, the small organic compound has a molecular weight or molecular mass of about 15 Da to 1,500 Da, e.g., about 15 Da to 1,250 Da, about 15 Da to about 1,000 Da, about 15 Da to about 750 Da, about 15 Da to about 500 Da, about 15 Da to about 250 Da, about 15 Da to about 100 Da, about 25 Da to 500 Da, about 25 Da to 100 Da, about 50 Da to 500 Da, about 100 Da to 250 Da, about 100 Da to 500 Da, or about 100 Da to 1,000 Da.

[0172] In one embodiment, the small organic compound comprises an aldehyde, a ketone, an acetyl group, an acyl group, a hydroxyl group, an ester, an ether, an amine, an amide, an aryl group, a heteroaryl group, a heterocyclic group or a cycloalkyl group. In one embodiment, the small organic compound comprises an alkenyl group or an alkynyl group. In one embodiment, the small organic compound comprises an aldehyde or a ketone group. In one embodiment, the small organic compound comprises an α-β unsaturated ketone. In one embodiment, the small organic compound is acetone or acetylacetone. In one embodiment, the small organic compound is a natural product, a lipid, a sterol, a steroid, an amino acid, a sugar, a brown algae tannin, a tannin, a lignin or a lignin derivative.

[0173] In one aspect, the small organic compound comprises a compound of formula (Y):

[0174]

[0175] or a salt, 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 5c Each 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 optionally substituted by one or more R 6 Replacement; R 6 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; R B and RC are each independently hydrogen, C1-C6 alkyl or C1-C6 heteroalkyl; m and n are each independently an integer between 0 and 24; and is a single bond or a double bond, where When it is a double bond, R 2b and R 3b Each does not exist independently.

[0176] In embodiments of formula (Y), R 1a , R 1b , R 1c , R 2a , R 2b , R 3a , R 3b , R 5a , R 5b and R 5c Each of is 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), is a single bond. 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 Each of is independently hydrogen, each of m and n is independently selected from 0, 1, 2 or 3, and It is a single bond.

[0177] In embodiments of formula (Y), R 1a , R 1b , R 1c , R 5a , R 5b and R 5c Each of is 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 Each of is independently hydrogen, n is 0, m is 1, and In the embodiment of formula (Y), R 1aC l Alkyl; R 1b , R 1c , R 5a , R 5b and R 5c are each independently hydrogen, and m and n are each 0. In an embodiment of formula (Y), R 1a is halogen (e.g., chlorine, bromine or iodine); R 1b , R 1c , R 5a , R 5b and R 5c are each independently hydrogen, and m and n are each 0.

[0178] In one embodiment, the method is characterized by providing a peroxide source, such as PAA, to a VHPO (e.g., VCPO), such as a VHPO comprising a protein sequence motif provided in Table 1 or a VHPO sequence provided in Table 2. In one embodiment, the method is characterized by: (i) providing a peroxide source, such as PAA and a chloride anion to the VCPO to generate a hypochlorite anion or its conjugate acid. In one embodiment, the method is characterized by: (i) providing a peroxide source (e.g., PAA) and a chloride source to the VCPO to generate a hypochlorite anion or its conjugate acid; and (ii): providing a hypochlorite anion to an amine to generate ammonium chloride, such as NH2Cl. In one embodiment, the method is characterized by: (i) providing a peroxide source (e.g., PAA) and a chloride source to the VCPO to generate a hypochlorite anion or its conjugate acid; (ii): providing a hypochlorite anion to the amine to generate ammonium chloride, such as NH2Cl; and (iii) reacting the ammonium chloride with an iodide anion to generate a hypoiodite anion or its conjugate acid. In one embodiment, the method is characterized by: (i) providing a peroxide source (such as PAA) and a chlorine source to VCPO to generate a hypochlorite anion or its conjugate acid; and (ii-a) reacting the hypochlorite anion with an iodide anion to generate a hypoiodite anion or its conjugate acid. In one embodiment, the method is characterized by: (i) providing a peroxide source (such as PAA) and a chlorine source to VCPO to generate a hypochlorite anion or its conjugate acid; (ii) providing a hypochlorite anion to an amine to generate ammonium chloride, such as NH2Cl; (iii) reacting ammonium chloride with an iodide anion to generate a hypoiodite anion or its conjugate acid; and (iv) reacting the hypoiodite anion with a small organic compound, dichloroacetic acid, to generate a small halogenated organic 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 generate a hypochlorite anion or its conjugate acid; (ii-a): reacting the hypochlorite anion with an iodide anion to generate a hypoiodite anion or its conjugate acid; and (iv) reacting the hypoiodite anion with a small organic compound, dichloroacetic acid, to generate a small halogenated organic 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 (i), (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).

[0179] In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that the hypohalite anion and excess iodide anion are converted to diatomic iodine I2 and triiodide anion I3 between pH 0-5 compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 0-5. - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 and VHPO at pH 7, the hypohalite anion and the excess iodide anion are converted into diatomic iodine I2 and triiodide anion I3 at pH 7. - The conversion rate of chlorine is reduced by 10%-99%. In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO between pH 0-5 is characterized in that the conversion rate of hypohalite anion and excess bromine anion to diatomic bromine Br2 between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 and VHPO. In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO between pH 0-5 is characterized in that the conversion rate of hypohalite anion and excess chlorine anion to diatomic chlorine Cl2 between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 and VHPO. In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that the conversion rate of peracetic acid and bromine anion to hypobromite anion or its conjugate acid between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 0-5. In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that the conversion rate of peracetic acid and iodide anion to hypoiodite anion or its conjugate acid between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of PAA and VHPO between pH 0-5.

[0180] In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that the conversion of hypochlorite and hypoiodite anions to iodite anions IO2 at pH 7-14 is achieved compared to the method 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 and VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 and VHPO at pH 7-14, the conversion of hypobromite and hypoiodite to iodite anion IO2 at pH 7-14 is reduced by 10% to 99%. - and iodate anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that the conversion of hypoiodite and hypoiodite to iodite anion IO2 is reduced by 10% to 99% between pH 7 and 14, compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 7 and 14. - and iodate anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 and VHPO at pH 7-14, the conversion of hypohalite and hypoiodite to iodite anion IO2 at pH 7-14 is reduced by 10% to 99%. - and iodate anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 and VHPO between pH 7-14, the conversion of hypohalite (comprising HOCl, HOBr or HOI or a combination thereof) and hypoiodite is reduced by 10% to 99%. - and iodate anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that monochloramine and hypoiodite are converted to iodite anion IO2 at pH 7-14, compared to the method comprising the enzymatic reaction of H2O2 and VHPO at pH 7-14. - and iodate anion IO3 - In one embodiment, the method comprising the enzymatic reaction of PAA and VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 and VHPO between pH 7-14, the conversion of diatomic iodine I2 and hypoiodite to iodite anion IO2 between pH 7-14 is reduced by 10% to 99%. - and iodate anion IO3 - The conversion rate was reduced by 10%-99%.

[0181] In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat Increased by 90%-110%.

[0182] In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k catIn one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared to the method comprising the enzymatic reaction of H2O2 with VHPO, k cat In one embodiment, the method comprising the enzymatic reaction of PAA with VCPO is characterized in that, compared with the method comprising the enzymatic reaction of H2O2 with VHPO, k cat The increase was 90%-110%, among which dibromochloromethane was produced.

[0183] As described herein, peroxidase (e.g., VHPO) can be used, for example, in the presence of a halogen source to convert a small organic compound into a small halogenated organic compound. In one embodiment, the small halogenated organic compound includes natural products, lipids, sterols, steroids, amino acids, sugars, brown algae tannins, tannins, lignin, or lignin derivatives are chlorinated, brominated or iodinated. In one embodiment, the small halogenated organic compound is brominated. In one embodiment, the small halogenated organic compound comprises a functional group, such as an aldehyde, ketone, acetyl, acyl, hydroxyl, ester, ether, amine, amide, aryl, heteroaryl, heterocyclic or cycloalkyl. In one embodiment, the small halogenated organic compound comprises an alkenyl or alkynyl. In one embodiment, the small halogenated organic compound comprises an aldehyde or ketone group. In one embodiment, the small halogenated organic compound comprises an α-β unsaturated ketone. In one embodiment, the small halogenated organic compound comprises 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. In one embodiment, the small halogenated organic compound comprises 1, 2, 3 halogen atoms. In one embodiment, the small halogenated organic compound comprises 1, 2, or 3 bromine atoms. In one embodiment, the small halogenated organic compound comprises an acetone moiety. In one embodiment, the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone. In one embodiment, the small halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone, or 1,1,1-tribromoacetone. In one embodiment, the small halogenated compound comprises dichloroiodomethane, dichlorobromomethane, dibromoiodomethane, diiodochloromethane, or diiodobromomethane. In one embodiment, the small halogenated organic compound includes 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.

[0184] The small halogenated organic compound may include any halogen atom, such as a chlorine atom, a bromine atom or an iodine atom. In one embodiment, the small halogenated organic compound includes 1, 2, 3, 4, 5, 6 or more halogen atoms. In one embodiment, the small halogenated organic compound includes 1, 2, 3, 4, 5, 6 or more chlorine atoms. In one embodiment, the small halogenated organic compound includes 1, 2, 3, 4, 5, 6 or more bromine atoms. In one embodiment, the small halogenated organic compound includes 1, 2, 3, 4, 5, 6 or more iodine atoms. In one embodiment, the small halogenated organic compound includes bromoacetone, dibromoacetone, bromopentanedione, bromoform, tribromoacetone or their variants or analogs. In one embodiment, the small halogenated organic compound includes 1, 1-dibromoacetone, bromoacetone, 3-bromo-2, 4-pentanedione, bromoform, 1, 1, 3-tribromoacetone, 1, 1, 1-tribromoacetone or their variants or analogs. In one embodiment, the small halogenated organic compound includes a halogenated methane, such as a dihalogenated methane and a trihalogenated methane. In one embodiment, the small halogenated organic compound includes a halogenated acetone, such as a dihalogenated acetone and a trihalogenated acetone. In one embodiment, the small halogenated organic compound includes bromoacetone, 1,1-dibromoacetone, 1,1,3-tribromoacetone, 1,1,1-tribromoacetone, or a variant or analog thereof. In one embodiment, the small halogenated organic compound includes methyl bromide, dibromomethane, bromoform, or a variant or analog thereof.

[0185] In another aspect, the small halogenated organic compound comprises a compound of formula (Z):

[0186]

[0187] or a salt, 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 5c Each 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 optionally substituted by one or more R 6 Replace; and 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 ; R A is hydrogen, C1-C6 alkyl, C1-C6 heteroalkyl or C2-C6 alkenyl; 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; and is a single bond or a double bond, where When it is a double bond, R 2b and R 3b Each does not exist independently.

[0188] In an embodiment of formula (Z), R 1a , R 1b and R 1c Each of R is independently halogen or hydrogen, wherein R 1a , R 1b and R 1c At least one of is halogen. In one embodiment, halogen is selected from chlorine, bromine or iodine. In the embodiment of formula (Z), R 1a , R 1b , R 1c Each of R is independently halogen or hydrogen, wherein R 1a , R 1b and R 1c At least two of are halogen. In one embodiment, the halogen is selected from two of chlorine, bromine or iodine. In the embodiment of formula (Z), R 1a , R 1b , R 1c Each of is independently halogen. In one embodiment, halogen is selected from chlorine, bromine or iodine. In an embodiment of formula (Z), R 5a , R 5b and R 5c Each of R is independently halogen or hydrogen, wherein R 5a , R 5b and R 5c At least one of is halogen. In the embodiment of formula (Z), R 5a , R 5b , R 5cEach of R is independently halogen or hydrogen, wherein R 5a , R 5b and R 5c At least two of are halogen. In the embodiment of formula (Z), R 5a , R 5b , R 5c Each of is independently halogen. In an embodiment of formula (Z), is a single bond. In an embodiment of formula (Z), each of m and n is independently selected from 0, 1, 2 or 3, and It is a single bond.

[0189] The peroxidase described herein (e.g., VHPO) can adjust the yield of small halogenated organic compounds according to reaction conditions. For example, under a first set of reaction conditions, the peroxidase may produce a first small halogenated organic compound, and under a second set of reaction conditions, the peroxidase may produce a second halogenated organic compound. In addition, the peroxidase described herein (e.g., VHPO) may be able to adjust the ratio of certain small halogenated organic compounds to each other in a plurality of small halogenated organic compounds according to reaction conditions. For example, under a first set of reaction conditions, the peroxidase may produce a first ratio of one small halogenated organic compound to another small halogenated organic compound in a plurality of small halogenated organic compounds, and under a second set of reaction conditions, the peroxidase may produce a second ratio of one small halogenated organic compound to another small halogenated organic compound in a plurality of small halogenated organic compounds.

[0190] In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the output of small halogenated organic compounds is a peroxidase derived from algae species. In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the output of small halogenated organic compounds is a peroxidase derived from fungal species. In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the output of small halogenated organic compounds is a peroxidase derived from cyanobacteria species. In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the output of small halogenated organic compounds is a peroxidase derived from cyanobacteria species. In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the output of small halogenated organic compounds is from Curvularia inequality, coralline algae, fleshy Nostoc, H.hongdechloris, M.bouillonii, Red Sea Trichoderma, Mountain Cryptococcus, Synechococcus, Saccharomyces cerevisiae, filamentous sheath silk algae or sheath silk algae. In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the production of small halogenated organic compounds comprises an amino acid sequence having 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 with, for example, the amino acid sequences listed in Table 1 provided herein. In one embodiment, the peroxidase (e.g., VHPO) capable of regulating the production of small halogenated organic compounds is produced in a host cell microorganism described herein (e.g., Pichia pastoris, Aspergillus niger or Saccharomyces cerevisiae).

[0191] Halogenation of the small organic compound or compounds occurs under specific conditions, such as conditions sufficient for halogenation. In one embodiment, conditions sufficient to produce a small halogenated organic compound or compounds include a temperature between 0°C and 85°C, such as about 4°C to about 65°C, about 10°C to about 65°C, about 25°C to about 50°C. In one embodiment, conditions sufficient to produce a small halogenated organic compound or compounds include a pH between about 2 and about 10, such as about 2 to about 9, about 2 to about 8, about 4 to about 10, about 4 to about 8, about 5 to about 8.

[0192] List of implementation plans

[0193] 1. A method for regulating the production of small halogenated organic compounds using peroxidase, comprising:

[0194] (i) providing a small organic compound (e.g. acetylacetone);

[0195] (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) under conditions sufficient to produce the small halogenated organic compound to form a reaction mixture;

[0196] (iii) evaluating the produced small halogenated organic compound; thereby regulating the production of the small halogenated organic compound.

[0197] 2. The method of embodiment 1, wherein the peroxidase is a haloperoxidase.

[0198] 3. The method of embodiment 2, wherein the haloperoxidase is a vanadium haloperoxidase (VHPO).

[0199] 4. The method of embodiment 3, wherein the VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO) or vanadium iodoperoxidase (VIPO).

[0200] 5. The method of any one of embodiments 3-4, wherein the VHPO is VBPO.

[0201] 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).

[0202] 7. The method of any one of the embodiments, wherein the peroxidase is a fungal haloperoxidase (eg, derived from a fungal species).

[0203] 8. The method of any one of the preceding embodiments, wherein the peroxidase is derived from an organism selected from the group consisting of Curvularia inaequalis, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium rubrum, Cryptococcus montaneus, Synechococcus sp. PCC7335, and Coralline algae.

[0204] 9. The method of any one of the preceding embodiments, wherein the peroxidase is derived from coralline algae.

[0205] 10. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Cryptococcus montana.

[0206] 11. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Curvularia inaequalis.

[0207] 12. The method of any one of the preceding embodiments, wherein the peroxidase is produced in a host cell microorganism.

[0208] 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.

[0209] 14. method as described in any one of the preceding embodiments, wherein the expression of the peroxidase produced in the host cell microorganism increases by about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 10 times, for example with respect to the peroxidase produced in its natural host.

[0210] 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 provided in Table 1.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 19. The method of any 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 SEQ ID NOs: 1-50.

[0215] 20. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase has at least 1, 2, 3, 4, 5 or 6 amino acid substitutions relative to the amino acid sequence selected from any one of SEQ ID NOs. 1-50.

[0216] 21. The method of any one of the preceding embodiments, wherein the small organic compound comprises 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.

[0217] 22. The method of any one of the preceding embodiments, wherein the small organic compound comprises a ketone or an aldehyde.

[0218] 23. A method as described in any of the preceding embodiments, wherein the small halogenated organic compound is chlorinated; brominated; iodinated; chlorinated and iodinated; chlorinated and brominated; brominated and iodinated; or chlorinated, brominated and iodinated.

[0219] 24. The method of any one of the preceding embodiments, wherein the small halogenated organic compound is brominated.

[0220] 25. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1, 2, or 3 halogen atoms.

[0221] 26. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1, 2, or 3 bromine atoms.

[0222] 27. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises an acetone moiety.

[0223] 28. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone.

[0224] 29. The method of any one of the preceding embodiments, wherein the small 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.

[0225] 30. The method of any one of the preceding embodiments, further comprising providing a peroxide source, such as H2O2 or PAA, and a halogen source, such as a halide salt such as KBr, NaCl or KI.

[0226] 31. The method of any one of the preceding embodiments, wherein the conditions sufficient to produce small halogenated organic compounds include one or more of the following:

[0227] (a) Temperature between 10℃ and 85℃

[0228] (b) pH between 4 and 10; and

[0229] (c) The ionic strength is between 0.1 mM and 4 M.

[0230] 32. The method of embodiment 31, wherein the conditions sufficient to produce small halogenated organic compounds include a temperature between 10°C and 85°C.

[0231] 33. The method of any one of embodiments 31-32, wherein the conditions sufficient to produce small halogenated organic compounds include a pH between 4-10.

[0232] 34. The method of any one of embodiments 31-33, wherein conditions sufficient to produce small halogenated organic compounds include an ionic strength between 0.1 mM and 4M.

[0233] 35. The method of any one of the preceding embodiments, wherein evaluating comprises analyzing small halogenated organic compounds by analytical techniques.

[0234] 36. The method of embodiment 45, wherein the analytical techniques include HPLC, GC-MS, NMR.

[0235] 37. The method of any one of the preceding embodiments, wherein the small halogenated organic compound is capable of reducing methane production by the microorganism.

[0236] 38. A method as described in 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.

[0237] 39. The method of any one of embodiments 37-38, wherein methane production is reduced by 10-75%.

[0238] 40. The method of any one of embodiments 37-39, wherein the microorganism is present in a rumen community.

[0239] 41. A method for reducing methane production in a rumen community, comprising:

[0240] (i) providing a small organic compound (e.g. acetylacetone);

[0241] (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) under conditions sufficient to produce the small halogenated organic compound to form a reaction mixture;

[0242] (iii) separating the small halogenated organic compound from the reaction mixture;

[0243] (iv) providing small halogenated organic compounds to the rumen community under conditions sufficient to reduce methane production.

[0244] 42. The method of embodiment 41, further comprising obtaining a value of methane level before (a) providing the peroxidase or after (b) providing the peroxidase.

[0245] 43. A method as described in embodiment 42, comprising (a).

[0246] 44. The method of embodiment 42, comprising (b).

[0247] 45. The method of embodiment 42, comprising (a) and (b).

[0248] 46. ​​A method as described in any 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.

[0249] 47. The method of any one of embodiments 41-46, wherein methane production is reduced by 10-75%.

[0250] 48. The method of any one of embodiments 40-46, wherein the peroxidase is a haloperoxidase.

[0251] 49. The method of embodiment 47, wherein the haloperoxidase is a vanadium haloperoxidase (VHPO).

[0252] 50. The method of embodiment 49, wherein the VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO) or vanadium iodoperoxidase (VIPO).

[0253] 51. The method of any one of embodiments 49-50, wherein the VHPO is VBPO.

[0254] 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).

[0255] 53. The method of any one of the preceding embodiments, wherein the peroxidase is a fungal haloperoxidase (eg, derived from a fungal species).

[0256] 54. The method of any one of the preceding embodiments, wherein the peroxidase is derived from an organism selected from the group consisting of Curvularia inaequalis, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium rubrum, Cryptococcus montaneus, Sphaerotheca filamentosa, Synechococcus sp. PCC7335, Coralline algae, and Saccharomyces cerevisiae.

[0257] 55. The method of any one of the preceding embodiments, wherein the peroxidase is derived from coralline algae.

[0258] 56. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Cryptococcus montana.

[0259] 57. The method of any one of the preceding embodiments, wherein the peroxidase is derived from Curvularia inaequalis.

[0260] 58. The method of any one of the preceding claims, wherein the peroxidase is produced in a host cell microorganism.

[0261] 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.

[0262] 60. Method as described in any one of the preceding embodiments, wherein the expression of the peroxidase produced in the host cell microorganism increases by about 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times or 10 times, for example relative to the peroxidase produced in its natural host.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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 sequence selected from SEQ ID NOs: 1-50.

[0267] 65. The method of any one of the preceding embodiments, wherein the amino acid sequence of the peroxidase has at least 1, 2, 3, 4, 5 or 6 amino acid substitutions relative to the amino acid sequence selected from any one of SEQ ID NOs. 1-50.

[0268] 66. The method of any one of the preceding embodiments, wherein the small organic compound comprises 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.

[0269] 67. The method of any one of the preceding embodiments, wherein the small organic compound comprises a ketone or an aldehyde.

[0270] 68. The method of any one of the preceding embodiments, wherein the small halogenated organic compound is chlorinated, brominated or iodinated.

[0271] 69. The method of any one of the preceding embodiments, wherein the small halogenated organic compound is brominated.

[0272] 70. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1, 2, or 3 halogen atoms.

[0273] 71. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1, 2, or 3 bromine atoms.

[0274] 72. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises an acetone moiety.

[0275] 73. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone.

[0276] 74. The method of any one of the preceding embodiments, wherein the small halogenated organic compound comprises 1,1-dibromoacetone, bromoacetone, 3-bromo-2,4-pentanedione, bromoform, 1,1,3-tribromoacetone or 1,1,1-tribromoacetone.

[0277] 75. The method of any one of embodiments 40-73, wherein conditions sufficient to produce small halogenated organic compounds include a pH between 4-10.

[0278] 76. The method of any one of embodiments 40-74, wherein conditions sufficient to produce small halogenated organic compounds include an ionic strength between 0.1 mM and 4M.

[0279] 77. The method of any of the preceding embodiments, further comprising evaluating the small halogenated organic compounds produced, such as by analytical techniques.

[0280] 78. The method of embodiment 77, wherein the analytical techniques include HPLC, GC-MS, NMR.

[0281] 79. A method for increasing the expression of a peroxidase in a host cell or a host microorganism.

[0282] 80. A reactor for the continuous production of small halogenated organic compounds, comprising:

[0283] (i) a reaction chamber;

[0284] (ii) temperature control module;

[0285] (iii) a peristaltic pump; and

[0286] (iv) A module for accommodating a catalyst.

[0287] 81. A reactor for continuous production of a small halogenated compound as claimed in claim 79, further comprising:

[0288] (i) devices for regulating the interior atmosphere;

[0289] (ii) an impeller for agitating the reaction mixture;

[0290] (iii) Multiple sensors for monitoring the reaction.

[0291] 82. A method for regulating the production of small halogenated organic compounds as described in any of the preceding embodiments, further comprising providing a 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 chloride salt, such as KCl or NaCl) to a peroxidase (e.g., VCPO).

[0292] 84. The method for regulating the production of a small halogenated organic compound as described in any one of the preceding embodiments, further comprising:

[0293] (i) providing a peroxide source (e.g., PAA) and a chlorine source to the VCPO to generate a hypochlorite anion or its conjugate acid;

[0294] (ii): donating hypochlorite anions to amines to generate chlorinated amines, such as NH2Cl;

[0295] (iii) reacting ammonium chloride with an iodide anion to form a hypoiodite anion or a conjugate acid thereof; and

[0296] (iv) reacting the hypoiodite anion with a small organic compound, dichloroacetic acid, to produce a small halogenated organic compound product, dichloroiodomethane.

[0297] 85. A method for regulating the production of small halogenated organic compounds as described in embodiments 83-84, wherein compared with the method of claim 1 comprising an enzymatic reaction of H2O2 with VHPO between pH 0-5, hypohalite anions and excess iodide anions are converted to diatomic iodine I2 and triiodide anions I3 between pH 0-5. - The conversion rate was reduced by 10%-99%.

[0298] 86. A method for regulating the production of small halogenated organic compounds as described in embodiments 83-84, wherein the hypohalite anion and excess iodide anion are converted to diatomic iodine I2 and triiodide anion I3 at pH 7 compared to a method comprising an enzymatic reaction of H2O2 with VHPO at pH 7. -The conversion rate was reduced by 10%-99%.

[0299] 87. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein the conversion rate of hypohalite anions and bromide anions into diatomic iodine Br2 between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 0-5 as described in claim 1.

[0300] 88. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein the conversion rate of hypohalite anions and chloride anions into diatomic chlorine Cl2 between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 0-5 as described in claim 1.

[0301] 89. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein the conversion rate of peracetic acid and bromide anions to hypobromite anions or their conjugate acids between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 0-5 as described in claim 1.

[0302] 90. The method for regulating the production of small halogenated organic compounds as described in any one of embodiments 83-84, wherein the conversion rate of peracetic acid and iodide anion to hypoiodite anion or its conjugate acid between pH 0-5 is reduced by 10%-99% compared to the method comprising the enzymatic reaction of PAA and VHPO between pH 0-5 as described in claim 1.

[0303] 91. A method for regulating the production of small halogenated organic compounds as described in any one of embodiments 83-90, wherein:

[0304] (i) Compared with the method of claim 1 comprising the enzymatic reaction of H2O2 with VHPO at pH 0-5, the hypohalite anion and the excess iodide anion are converted into diatomic iodine I2 and triiodide anion I3 at pH 0-5. - The conversion rate decreased by 10%-99%;

[0305] (ii) Compared with the method of claim 1 comprising the enzymatic reaction of H2O2 with VHPO at pH 7, the hypohalite anion and excess iodide anion are converted into diatomic iodine I2 and triiodide anion I3 at pH 7. - The conversion rate decreased by 10%-99%;

[0306] (iii) the conversion of hypohalite anions and bromide anions to diatomic bromine Br2 at pH 0-5 is reduced by 10% to 99% compared to the method of claim 1 comprising the enzymatic reaction of H2O2 and VHPO at pH 0-5;

[0307] (iv) the conversion of hypohalite anions and chloride anions to diatomic chlorine Cl2 at pH 0-5 is reduced by 10% to 99% compared to the method of claim 1 comprising the enzymatic reaction of H2O2 and VHPO at pH 0-5;

[0308] (v) the conversion rate of peracetic acid and bromide anion to hypobromite anion or its conjugate acid at pH 0-5 is reduced by 10% to 99% compared to the method of claim 1 comprising the enzymatic reaction of H2O2 and VHPO at pH 0-5; or

[0309] (vi) the conversion rate of peracetic acid and iodide anion to hypoiodite anion or its conjugate acid at pH 0-5 is reduced by 10% to 99% compared to the method of claim 1 comprising the enzymatic reaction of PAA and VHPO at pH 0-5,

[0310] or a combination thereof.

[0311] 92. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein hypochlorite and hypoiodite are converted to iodite anion IO2 at pH 7-14 compared to the method of claim 1 comprising an enzymatic reaction of H2O2 and VHPO at pH 7-14. - and iodate anion IO3 - The conversion rate was reduced by 10%-99%.

[0312] 93. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein hypobromite and hypoiodite are converted to iodite anion IO2 between pH 7-14 compared to a method comprising an enzymatic reaction of H2O2 and VHPO between pH 7-14. - and iodate anion IO3 - The conversion rate was reduced by 10%-99%.

[0313] 94. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein the conversion of hypoiodite and hypoiodite anion IO2 to iodite anion IO2 between pH 7-14 is compared to the method comprising the enzymatic reaction of H2O2 and VHPO between pH 7-14 as described in claim 1. -and iodate anion IO3 - The conversion rate was reduced by 10%-99%.

[0314] 95. A method for regulating the production of small halogenated organic compounds as described in any of embodiments 83-84, wherein compared with the method of claim 1 comprising an enzymatic reaction of H2O2 and VHPO between pH 7-14, hypohalite and hypoiodite are converted to iodite anion IO2 between pH 7-14. - and iodate anion IO3 - The conversion rate was reduced by 10%-99%.

[0315] 96. The method for regulating the production of small halogenated organic compounds as described in any one of embodiments 83-84, wherein:

[0316] (i) Compared with the method of claim 1 comprising an enzymatic reaction of H2O2 and VHPO at a pH between 7 and 14, hypochlorite and hypoiodite are converted to iodite anion IO2 at a pH between 7 and 14. - and iodate anion IO3 - The conversion rate is reduced by 10%-99%; or,

[0317] (ii) Compared with the method involving the enzymatic reaction of H2O2 with VHPO at pH 7-14, hypobromite and hypoiodite are converted to iodite anion IO2 at pH 7-14. - and iodate anion IO3 - The conversion rate is reduced by 10%-99%; or,

[0318] (iii) Compared with the method comprising the enzymatic reaction of H2O2 with VHPO at pH 7-14, the conversion of hypoiodite and hypoiodite to iodite anion IO2 at pH 7-14 - and iodate anion IO3 - The conversion rate decreased by 10%-99%.

[0319] or a combination thereof.

[0320] 97. The method for regulating the production of small halogenated organic compounds as described in any of embodiments 82-96, wherein the kcat of the method comprising the enzymatic reaction of PAA and VCPO is 10%-500% higher than the kcat of the method comprising the enzymatic reaction of H2O2 and VHPO.

[0321] 98. The method for regulating the production of small halogenated organic compounds as described in any of embodiments 82-97, wherein the kcat of the method comprising the enzymatic reaction of PAA and VCPO is 25%-250% higher than the kcat of the method comprising the enzymatic reaction of H2O2 and VHPO.

[0322] 99. The method for regulating the yield of small halogenated organic compounds as described in any of embodiments 82-98, wherein the kcat of the method comprising the enzymatic reaction of PAA and VCPO is 50%-150% higher than the kcat of the method comprising the enzymatic reaction of H2O2 and VHPO.

[0323] 100. The method for regulating the production of small halogenated organic compounds as described in any of embodiments 82-99, wherein the kcat of the method comprising the enzymatic reaction of PAA and VCPO is 75%-125% higher than the kcat of the method comprising the enzymatic reaction of H2O2 and VHPO.

[0324] 101. The method for regulating the yield of small halogenated organic compounds as described in any of embodiments 82-100, wherein the kcat of the method comprising the enzymatic reaction of PAA and VCPO is 80%-120% higher than the kcat of the method comprising the enzymatic reaction of H2O2 and VHPO.

[0325] 102. The method for regulating the yield of small halogenated organic compounds as described in any of embodiments 82-101, wherein the kcat of the method comprising the enzymatic reaction of PAA and VCPO is 90%-110% higher than the kcat of the method comprising the enzymatic reaction of H2O2 and VHPO.

[0326] 103. A method for regulating the production of small halogenated compounds as described in any of embodiments 82-102, wherein the product is dichloroiodomethane.

[0327] 104. The method for regulating the production of small halogenated compounds as described in any of embodiments 82-102, wherein the product is dibromochloromethane.

[0328] 105. The method for regulating the production of small halogenated compounds as described in any of embodiments 82-102, wherein the products are dichloroiodomethane and dibromochloromethane.

[0329] Example

[0330] The present disclosure may be further described in detail with reference to the following experimental examples. Unless otherwise indicated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present disclosure should never be considered to be limited to the following examples, but should be understood to cover any and all variations that become apparent due to the teachings provided herein. Without further description, it is believed that those of ordinary skill in the art can use the aforementioned description and the following illustrative examples to prepare and utilize the compositions, methods and devices of the present disclosure and practice the claimed methods. The following working examples clearly point out various aspects of the present invention and should not be interpreted as limiting the remainder of the present disclosure in any way.

[0331] Example 1: Heterologous Expression and Purification of Exemplary Vanadium Haloperoxidases in E. coli

[0332] Exemplary vanadium haloperoxidase (VBPO) genes were overexpressed in E. coli according to the following steps. The complete nucleotide sequence of each VBPO was chemically synthesized using a commercial supplier (GeneScript) and cloned into the pET-28a(+) vector suitable for E. coli protein expression. The resulting protein was designed to contain an N-terminal HRV 3C protease recognition signal and a C-terminal 6x-His tag to enable affinity purification. After culturing E. coli cells using standard techniques, the recombinant VBPO was purified to homogeneity using a two-step protocol that included an affinity column-based protein purification step (HisTrap FF Crude) followed by a size exclusion step (Amicon TM Ultra centrifugal filter device 100 kDa) and then used without further purification.

[0333] Example 2: Purification and kinetic characterization of exemplary vanadium haloperoxidases from coralline algae

[0334] This example provides the purification of VBPO naturally produced in the seaweed Coralline algae (CoVBPO). After harvesting the seaweed, a crude extract was prepared by rapid freezing in liquid nitrogen and grinding into fine particles. Next, Tris-sulfate buffer solution 0.1 M, pH = 8.5 was added in a 1:5 ratio. After centrifugation, the supernatant constituted the crude extract. CoVBPO was purified using the 3-step protocol summarized in Table 3. SDS-PAGE analysis showed that the final enzyme preparation had a purity of over 90%.

[0335] Table 3: Summary of enzyme purification

[0336]

[0337]

[0338] CoVBPO was then studied and various kinetic parameters were determined based on 3D fitting of the Cleland equation using CoVBPO compositions in MOPS buffer pH = 7.0 and MES buffer pH = 6.5 at 0.2 and 2.0 M ionic strengths, respectively.

[0339] All kinetic measurements were performed based on an initial rate analysis 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 fluorimeter. The excitation wavelength was set to 490 nm and emission was recorded at 515 nm by a parallel photomultiplier detection system; lamp output was 74 W).

[0340] like Figure 2A-2C As shown in the ping-pong double reaction system, the first product P1 (H2O) is released after the first substrate (hydrogen peroxide) binds to the enzyme molecule and before the second substrate (bromide) binds. Figure 2B The nonlinear multiparameter equation shown was used to fit the experimental kinetic data. The nonlinear multiparameter equation was formulated using the King-Altman method, in which substrate inhibition leads to the formation of a dead-end complex (e.g., see Figure 2C ). All fitting procedures as well as goodness of fit were performed using OriginPro version 2021; unweighted least squares tests were used as convergence criteria in all cases. Tables 4 and 5 summarize the resulting parameters determined by this analysis at the two ionic strengths tested.

[0341] Table 4: Exemplary kinetic parameters of CoVBPO at 2M ionic strength

[0342] parameter value Standard error t-value <![CDATA[k cat ]]> 18.3 0.24 75 <![CDATA[Km Br ]]> 8.21E-4 4.5E-5 18 <![CDATA[Km H2O2 ]]> 1.28E-5 5.1E-7 25 <![CDATA[ki H2O2 ]]> 1.14E-4 1.0E-5 11 <![CDATA[k cat / Km Br ]]> 2.23E4 <![CDATA[k cat / Km H2O2 ]]> 1.43E6

[0343] Table 5: Exemplary kinetic parameters of CoVBPO at 0.2 M ionic strength

[0344] parameter value Standard error t-value <![CDATA[k cat ]]> 30.2 0.48 63 <![CDATA[Km Br ]]> 0.011 2.7E-4 40 <![CDATA[Km H2O2 ]]> 5.84E-5 1.7E-6 35 <![CDATA[ki H2O2 ]]> 5.57E-4 3.0E-5 19 <![CDATA[k cat / Km Br ]]> 2.83E3 <![CDATA[k cat / Km H2O2 ]]> 5.18E5

[0345] The results suggest that CoVBPO may proceed via a ping-pong mechanism involving dead-end inhibition of the hydrogen peroxide substrate. At low ionic strength (0.2 M), an increased turnover parameter (k cat ). In addition, the parameter Km H2O2 and Km BrThe estimated values ​​of and respectively indicate that at high ionic strength, VBPO has a 4.5-fold increase in affinity for hydrogen peroxide and more than an order of magnitude (12-fold) increase in affinity for bromide. H2O2 The pattern is similar.

[0346] Example 3: Effects of pH and Temperature on Exemplary VBPOs

[0347] The pH dependence of Michaelis-Menten kinetics was examined in high and low ionic strength media using the CoVBPO enzyme. The objectives were: i) to explain the pH dependence of the second substrate (bromide, Br- ) ii) reduce the corresponding KmBr- .

[0348] The presence of high ionic strength in solution disrupts the ionic interactions that occur in the solvent accessible amino acids of the enzyme. - The affinity of mBr )( Figure 3A ).

[0349] Example 4: Characterization of Halogenated Organic Compounds Produced by Exemplary VBPOs

[0350] This example describes how to preferentially produce certain halogenated compounds by carefully adjusting the VBPO reaction conditions. Acetylacetone was incubated with VBPO at a near constant concentration in a fed-batch bioreactor. The VHPO substrates H2O2 and KBr were added simultaneously to the fed-batch bioreactor in a controlled manner by a peristaltic pump. The concentration of the reaction products was monitored by taking aliquots of the reaction over time and analyzing the reaction curve using GC-MS. Figure 4A As shown, the major product produced was 1,1-dibromoacetone, which accumulated to 50 mM. Other halogenated compounds were produced in small amounts, 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 the concentration of halogenated analytes.

[0351] 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 identification of the n-hexane extracted product was determined by comparing the MS spectrum of the n-hexane extracted product with the MS spectrum of the standard in the NIST library ( Figure 4C ).

[0352] Example 5: Inhibition of Methane Production by Exemplary Small Halogenated Organic Compounds in Rumen-Derived Microbial Communities

[0353] Figure 5 The production and testing process of small organic halogenated compounds for inhibiting methane production is shown. In this example, small organic halogenated compounds produced by VBPO are incubated in a microbial culture system derived from rumen fluid obtained from dairy cows to determine their relative ability to inhibit methane production. Briefly, 1,1-dibromoacetone, byproduct bromoform, and derivative dibromomethane are added to the culture system separately. The rate of methane production is monitored in each reaction ( Figures 6A-6C ).

[0354] like Figures 6A-6C As shown, 1,1-dibromoacetone was the least effective in inhibiting methane production among the screened compounds. In the rumen-derived microbial community tested, 1,1-dibromoacetone provided 50% inhibition of methane production at 125uM, compared to bromoform showing 50% inhibition of methane production at 0.20uM, and dibromomethane showing 50% inhibition of methane production at 0.15uM.

[0355] Example 6: Heterologous production of cchVBPO in E. coli

[0356] This example describes the heterologous expression of Chondrus crispus VBPO (cchVBPO) in E. coli, namely the inoculation culture of the model microorganism in a bioreactor, the induction of cchVBPO production, protein extraction, purification and biochemical characterization.

[0357] Inoculation and induction

[0358] Briefly, 4x5 ml of LB medium containing kanamycin (4.0 μl, maintained at about 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 and the OD was measured after overnight (i.e., 15-18 hours). 600 The culture (4% inoculum / 20 ml) was then added to a 0.5-L (pre-warmed in 37°C medium) bioreactor containing 400 μl of kanamycin ([kanamycin] = 40 μg / ml; stock solution is 50 mg / ml) and shaken at 200 rpm at 37°C until the OD 600 Approximately 0.6–0.7. Monitor OD during growth by aseptically removing aliquots 600 When OD 600=0.6–0.7, the culture was cooled to 18°C ​​and 0.1 mM IPTG (0.5 ml from a stock solution of 100 mM IPTG) was added. A 3-ml sample was taken for t=0 h (uninduced). The culture was incubated overnight at 18°C ​​and stirred at 200 rpm under oxygen supply. It was then placed in a pre-weighed 500 ml centrifuge tube and centrifuged at 10,000 rpm for 10 min at 4°C (where the precipitate was drained by inversion and the excess medium was tapped onto a paper towel). The sample was then kept on ice.

[0359] Extraction and preparation

[0360] Resuspend the pellet from the previous step in Master (available from EMD Millipore), use 5 ml of 1x reagent per gram of wet cell paste. Gently mix 5 grams of cell paste with 22.5 ml of borate buffer 20 mM, pH=8.0, 30 mM imidazole [binding buffer] by gentle vortexing. Then add 2.5 ml of 10x and a protease inhibitor to the mixture. Master Mix is ​​compatible and therefore optional to include. If the target protein is to be treated with thrombin, Factor Xa, or recombinant enterokinase, the use of serine protease inhibitors should be avoided. If the target protein is to be treated with HRV 3C, a cysteine ​​protease inhibitor is required. Although purification can remove active inhibitors, dialysis or gel filtration is recommended before lysis. Incubate the cell suspension on a shaking platform or rotating mixer at room temperature for 20 minutes on a slow setting. The extract should not become viscous after incubation. The suspension is then centrifuged at 75,600g (JA-25.50, 8x30ml) for 15 minutes at 4°C to remove insoluble cell debris. The pellet is then optionally washed again with 5ml of binding buffer for 5 minutes and the centrifugation is repeated. Repeat the above steps for the pre-induced sample. Save 0.5ml of the induced and pre-induced samples and transfer the induced supernatant to a new tube.

[0361] purification

[0362] Assemble HisTrap FF Crude 5ml column and remove storage buffer with 5CV binding buffer (20mM boric acid, pH 8+30mM imidazole). Then balance the column with 5CV binding buffer (20mM boric acid, pH 8.0+30mM imidazole) and load the sample from the previous step. Next wash the sample with 6CV binding buffer, or until UV 280After reaching a stable value, the solution was finally eluted with elution buffer (20 mM boric acid, pH 8.0 + 300 mM imidazole) to obtain cchVBPO eluate.

[0363] Characterization

[0364] The protein concentration of the eluate produced in the previous step was determined by Bradford analysis. Briefly, Bradford reagent and 5 μl of protein sample were incubated in a 250-μl well plate for 5-10 min at room temperature, and the OD was measured. 600 The total protein concentration was determined based on a calibration curve with BSA standards (0.125 mg / ml - 2 mg / ml). The Bradford calibration curve with known BSA standards and the measured protein concentrations of the pre-induced and purified, induced samples are given in Tables 6 and 7 below.

[0365]

[0366] Table 6. Bradford calibration curve using BSA standards to measure total protein concentration of induced and pre-induced samples of the heterologous cchVBPO expression system in E. coli.

[0367]

[0368]

[0369] Table 7. Total protein concentrations of pre-induced and purified, induced samples of the heterologous cchVBPO expression system in E. coli.

[0370] SDS-PAGE electrophoresis was also used to verify whether the eluate contained cchVBPO protein, such as Figure 8 As shown. Uninduced, induced, unbound 4μg cchVBPO borate buffer containing 20mM imidazole buffer, 6μg cchVBPO borate buffer containing 20mM imidazole, and 8μg cchVBPO borate buffer containing 20mM imidazole were compared to form the ladder shown. Bromoperoxidase activity was determined by APF analysis (490nm / 515nm). In short, 2930ml buffer containing 30μl KBr (18mM) + 5μl APF (2μM) + 5μl VBPO (containing Na3VO4) + 30μl hydrogen peroxide (250μM) was mixed in a 3ml cuvette. After stirring at 25°C and adding VBPO, the reaction started. Bromoperoxidase activity was evaluated at 490nm / 515nm.

[0371] Example 7: Heterologous production of cchVBPO in E. coli

[0372] This example describes the heterologous expression of Synechococcus VBPO (synVBPO) in E. 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 performed according to the protocol described in Example 6 with minor modifications as described below. 17-18 h after inoculation, the optical density (OD) at 600 nm of a 5-ml sample was 600 The culture was transferred to a 0.5-L bioreactor and shaken at 200 rpm at 37°C. The OD 600 Until it reaches 0.6-0.7. OD 600 The results were 0.312, 0.535 and 0.638 at 4, 5 and 5.5 h, respectively, which was considered sufficient to start the induction process. After induction with 0.1 mM IPTG at 18 °C for 18 h, OD 600 The result was 0.920. After centrifugation, the weight of the precipitate was 4.162 grams. The protein was resuspended and purified using a HisTrap FF column as previously described in Example 6. The eluate was then subjected to total protein concentration, SDS-PAGE, and haloperoxidase activity assay. The Bradford calibration curves for known BSA standards and the measured protein concentrations of the pre-induced and purified, induced samples are given in Tables 8 and 9 below. Fig. 9 The SDS-PAGE results show ladder, uninduced, induced, unbound 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, 12 μg synVBPO in 20 mM borate buffer + 30 kDa cutoff.

[0373] Bradford Calibration Curve

[0374]

[0375] Table 8. Bradford calibration curve using BSA standards to measure total protein concentration of induced and pre-induced samples of the heterologous synVBPO expression system in E. coli.

[0376]

[0377] Table 9. Total protein concentrations of pre-induced and purified, induced samples of the heterologous synVBPO expression system in E. coli.

[0378] The synVBPO enzyme kinetic surface was plotted using k cat (s -1 ) as a function of hydrogen peroxide concentration [H2O2] (M) and potassium bromide concentration [KBr] (M), as shown in Figure 10. The kinetic parameters, standard errors, t values, and Prob>|t| of two kinetic models were determined: Cleland Michaelis-Menten (model 1) and Cleland Hill type (model 2). The Akaike information criterion test (AIC) and the Bayesian information criterion test (BIC) showed that the Cleland Hill type (model 2) had a lower AIC value and a lower BIC value, respectively; therefore, it was inferred that synVBPO kinetics followed the Cleland Hill type kinetic paradigm. OriginPro (2021 version) was used to calculate kinetic parameters and perform statistical tests according to Michaelis-Menten and Hill type kinetics.

[0379] In addition, as depicted in Figures 11-12, the stability of synVBPO enzyme activity at high concentrations of KBr and H2O2, its dependence on ionic strength, and the Michaelis-Menten parameter as a function of pH were determined. To explore the inactivation effect of KBr on synVBPO activity, a Salwin test was performed using 100 μM MCD, three VBPO concentrations (2.5 nM, 5 nM, and 7.5 nM) E=2.5|5|7.5 nM in 20 mM phosphate buffer (pH 7), 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 and 50 μM H2O2, respectively. Sequential testing at 40 mM and 160 mM KBr concentrations showed that at these high bromide concentrations, the enzyme activity was significantly inactivated, while at 1 mM KBr, the enzyme retained its activity at either peroxide concentration. The relationship between ionic strength and synVBPO activity is shown as the change in fluorescence per μ(ionic strength) and is shown in Figure 12. In addition, the kinetic parameter k was determined using a least squares fit of the experimental data. cat (s -1 ), k cat / Km H2O2 (M - 1 s -1 ) and k cat / Km KBr (M -1 s -1 ), as shown in the formula in Figure 12.

[0380] Example 8. Addition of PAAM to ciVCPO

[0381] This example describes a method for producing small halogenated organic compounds using peracetic acid as a substrate, using ciVCPO 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. Since peracetic acid is not an efficient two-electron reducing agent, and unlike hydrogen peroxide, peroxyacetic acid cannot generate oxygen in the presence of hypochlorous acid and catalytic amounts of chloride ions, hydrogen peroxide must be removed to avoid oxygen generation using the enzyme catalase derived from bovine liver.

[0382] Fig.14 The reaction rates of ciVCPO in the presence of chloride ions are shown using PAA and H2O2 as substrates. The reaction rates were fitted according to Michaelis-Menten kinetics to obtain v = f([PAA] or [H2O2]). As shown, for a constant concentration of 150 mM NaCl, PAA as a substrate gave a higher v than hydrogen peroxide. max and K m .

[0383] Process Design

[0384] In each of the following process designs for the production of small halogenated organic compounds in a bioreactor, dibromoacetone (DBA) or dichloroacetone (DCA) is produced enzymatically, as previously described.

[0385] Design 8.1

[0386] PAA+NaCl+DBA+ciVCPO→DBCA, where DBA is dibromoacetone and DBCA is dibromochloroacetone (pH=7-8 and T=20-55°C)

[0387] DBCA→DBCM (After the enzymatic reaction, set pH=8-9 in pH-STAT)

[0388] Design 8.2

[0389] Step 1:

[0390] PAA+Cl - +ciVCPO→HOCl

[0391] HOCl+NH3→NH2Cl (at pH=7-8 and T=20-55°C)

[0392] Step 2:

[0393] NH2Cl+I - →HOI

[0394] HOI+I - ←→I2

[0395] DCA+I2→DCIA (DCIA=dichloroiodoacetone)

[0396] DCIA→DCIM (pH=8, T=20-55℃)

[0397] (DCIM = dichloroiodomethane).

[0398] DCIM production in a two-batch system

[0399] The present embodiment describes the production of dichloroiodomethane (DCIM) in a two-step batch system according to the following conditions, as described in the following steps. In the first step, 0.1 M phosphate buffer (pH 7, containing 12 mM H2O2, 100 mM NaCl, 10 mM DCA, at T = 22°C) was added to the bioreactor, and H2O2 was added at a rate of 25 μl / min. 1 equivalent of H2O2 was added to 1.2 equivalents of NH3, i.e., C NH3 is 0.24 M, the molar rate of H2O2 entering the tank is equal to the molar rate of H2O2 leaving the tank, or C H2O2 The initial concentrations in the bioreactor were 100 mM NaCl, 100 μM H2O2, 480 μM NH4Cl, 264 μM Na3VO4 and 2.64 μM enzyme.

[0400] Every ten minutes, 500 μl of the bioreactor sample was combined with 500 μl of a DCA stock solution in phosphate buffer (solution (A)) and an appropriate amount of KI (solution (B)). Solution (A) contained 10 mM DCA diluted from a 9.93 M DCA stock solution in 0.5 M phosphate (pH 8.5). Solution (B) contained 1 mM KI diluted from a 400 mM stock solution. The vials were reacted overnight and 50 μl of the reaction sample was combined with 950 μl of the extractant and then analyzed by GC-MS for DCIM. Fig.15 is a plot of DCIM concentration versus reaction coordinate, showing that DCIM concentration increases linearly with time, consistent with Michaelis-Menten kinetics.

[0401] Example 9. Pichia pastoris glycerol batch fermentation and fed-batch fermentation

[0402] According to the following examples, the enzyme ciVCPO from Curvularia inaequalis was produced in Pichia pastoris and purified to homogeneity, with typical results being 100-500 mg / l of more than 75-95% pure ciVCPO enzyme.

[0403] Inoculum bottle preparation

[0404] A 250 mL baffled shake flask containing 25 mL of BMGY medium was inoculated with a single colony from an agar plate containing Pichia pastoris ciVCPO. The shake flask was incubated in an incubator shaker at 30°C and 250 RPM for 24 hours. A second 2x500 mL baffled shake flask containing 150 mL of BMGY medium was inoculated with 5 mL (OD 600 Approximately 0.2-0.4) from a 250 mL baffled shake flask culture and incubated in an incubator shaker at 30°C and 250 RPM for 24 hours.

[0405] Glycerol batch stage

[0406] First, calibrate the pH probe, internal and external peristaltic pumps in the EVA software. Autoclave the bioreactor containing 3 L of basal salt medium (FM22) plus 4% v / v glycerol and 0.6 ml of Defoamer C at 121°C for 30 minutes. After sterilization and cooling, aseptically add 4 ml / liter total volume (9 ml) of PTM1 salts and ampicillin. Take an initial 10 ml sample. Calibrate the O2 probe (1000 rpm, 1 vvm air until stable reading) and automatically zero the OD 600 (turbidity), and the tubes were filled with ammonium hydroxide (25%), phosphoric acid (10%), defoamer and feed. The temperature was set to 30°C, stirring and aeration minimum and setpoints → 800 rpm, 0.5 vvm air | 1.5 L / min, pO2 = 40% v / v, and the pH of the medium was adjusted to 5.0 with 25% ammonium hydroxide (for better salt solubility). Fig.16 The dissolved oxygen (DO) cascade is described in. The initial fermentation volume of about 5-10% of the culture generated in the inoculation shake flask is inoculated into the fermentor. The seed culture is spun down and redissolved in 50ml BMGY medium or water, and 60ml is added to a syringe with a pink nail. Batch cultures are grown until glycerol is completely consumed (14 to 20 hours). This is indicated by O2 peak (pO2>85% and time>14 hours). The cell growth (OD600, cell wet weight (CWW), cell dry weight), ethanol, glycerol and methanol concentrations and enzyme activity of the samples are analyzed. If not analyzed immediately, the cell pellet and supernatant of each sample are frozen at -80°C for subsequent analysis.

[0407] Sample preparation

[0408] Transfer 5 ml of sample to a pre-weighed 15 ml falcon tube with high accuracy. Remove 1 ml and pour into a 1.5 ml Eppendorf tube. Use 0.1 ml of this sample for OD 600, 0.1 < absorbance < 0.8. The residue was spun down (0.9 ml), and the supernatant was transferred to a new 1.5 ml Eppendorf tube for EtOH, MeOH and glycerol determination. If the sample has been induced, after the cells are broken with yeast cell lysis buffer, 0.9 ml of slurry is placed at -80 ° C for VCPO activity determination. The tube is spun down, washed with water, and then spun down again, and then the precipitate is decanted and weighed. The CWW and CWW g / L of 4 ml cell culture were calculated. For cell dry weight (CDW): the precipitate is washed again by resuspension and centrifugation, and the washed precipitate is finally resuspended to a final volume of 4 ml (initial). 3 ml of the suspension is transferred to a pre-weighed aluminum weighing dish, dried overnight in an oven at 80 ° C for 16 hours, and weighed again. The dry mass of each sample is calculated to obtain CDW.

[0409] Glycerol fed-batch phase

[0410] Once all the glycerol in the batch growth phase is consumed, glycerol feeding is started to increase the cell biomass under limiting (carbon) conditions. Use DO peak to ensure that glycerol is limited. After observing the DO peak, 50% w / v glycerol feed containing 12ml PTM1 salt per liter of glycerol feed is used to start glycerol fed batch. A constant feed rate of 20ml / h*L (60ml / h) initial fermentation volume is used. Glycerol feeding is carried out until the required cell density is reached. After about 22 hours or 6 hours of feeding, a cell yield of 230g / L wet cells should be achieved. At the end of this stage, no obvious recombinant enzyme was produced.

[0411] Methanol fed-batch phase of the MutS Pichia strain

[0412] Before starting the methanol feed, all glycerol needs to be consumed to fully induce the AOX1 promoter on methanol. The methanol is introduced slowly to acclimate the culture to growth in methanol. The methanol feed rate is adjusted to keep the methanol excess in the medium at no more than 0.3%, as determined by GC / MS.

[0413] Methanol induction period of MutS strain.

[0414] The temperature was lowered to 28°C, the glycerol feed rate was gradiently reduced to 0 g / L*h (26.1% (60 ml / h) → 0% in 2 hours), and the culture was induced. The 2 L induction feed contained 50% v / v methanol, 12 ml / L PTM1 salts (24 ml) and 0.0075% v / v (0.15 ml) antifoam 204, and was started with an initial fermentation volume of 2 ml / h*L in the first two hours. It was then gradiently increased from 2 ml / h*L to 6 ml / h*L over 12 hours and maintained throughout the fermentation. The vessel was then harvested after 4 days in methanol.

[0415] Enzyme recovery: cell disruption

[0416] The total cell pellet was resuspended in water at a ratio of 1:4. Then 200 ml of the suspension was added to a 500 ml Beckman centrifuge tube (about 50 ml of cell slurry) and centrifuged at 4000 g for 5 min at 4 ° C. The water was removed, leaving 50 ml of cell slurry 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 cell slurry. The mixture was treated for 10 minutes for homogenization. After this step, the broken yeast was black under a microscope. Next, the mixture was centrifuged, the supernatant was collected and placed on ice. The slurry was centrifuged with 40 ml of binding buffer and combined with the previous supernatant. The test tube containing the cell lysate was centrifuged at 75,600 g (JA-25.50, up to 8x30 ml, polycarbonate) for 15 min at 4 ° C to remove insoluble cell debris and collect the clarified lysate. The lysate was then filtered using a syringe and then purified.

[0417] Enzyme recovery: purification

[0418] Enzyme purification was performed using heat treatment of the crude extract (e.g. 55°C for 45 min) and classical affinity purification (His Tag). The binding buffer was 0.1 M Tris-HCl pH, and the elution buffer was the same as the binding buffer but with the addition of 500 mM imidazole.

[0419] Enzyme characterization: SDS-PAGE electrophoresis

[0420] Add 5 μg of pure protein or 10-20 μg of a mixture to each well. In an Eppendorf, mix 200 μl of sample buffer with 2 μl of fresh DTT 10 mM (stock solution is 1 M), immediately heat on a heating block at 90°C for 5 min, then place on ice or freeze. The running buffer for SDS-Page contains 10x Tris / Glycine / SDS (900 ml mili-Q water + 100 ml 10xTris / Glycine / SDS). The staining buffer used is One-Step Blue. Fig.17 It is SDS-PAGE of ladder, crude extract, crude extract after heat treatment and 60% pool HisTap crude peak fraction.

[0421] Buffer exchange and concentration

[0422] The enzyme peak was selected and a subsample was buffer exchanged with the desired buffer on a 20K MWCO dialyzer membrane. The samples were then frozen in liquid nitrogen and stored at -80°C.

[0423] Enzyme Characterization: Activity Assays

[0424] MCD measurement (292nm), ε=20mM -1 *cm -1

[0425] Add the following to a quartz cuvette in order: 2900 μl buffer (phosphate 50 mM pH 7), 50 μl 3M KBr, 20 μl 10 mM MCD, 4 μl 10 mM vanadium, and 8 μl 300 mM H2O. 2。 Then add 20uL to the UV-Vis and read at 292nm every 6 seconds for 1 min.

[0426] Equivalent solutions

[0427] The disclosure of each and every patent, patent application and citation cited herein is hereby incorporated by reference in its entirety. Although the present invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of the present invention may be designed by other persons skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be interpreted as including all such aspects and equivalent variations.

Claims

1. A method for regulating the production of small halogenated organic compounds using peroxidase, comprising: (i) providing a small organic compound (e.g. acetylacetone); (ii) contacting the small organic compound with a peroxidase (e.g., VHPO) under conditions sufficient to produce the small halogenated organic compound to form a reaction mixture; (iii) evaluating the small halogenated organic compound produced; thereby regulating the production of the small halogenated organic compound.

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

3. The method of claim 2, wherein the haloperoxidase is a vanadium haloperoxidase (VHPO).

4. The method of claim 3, wherein the VHPO is vanadium chloroperoxidase (VCPO), vanadium bromoperoxidase (VBPO) or vanadium iodoperoxidase (VIPO).

5. The method of any one of claims 3-4, wherein the VHPO is VBPO.

6. The method of claim 1, wherein the peroxidase can be 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).

7. The method of claim 1, wherein the peroxidase is a fungal haloperoxidase (eg, derived from a fungal species).

8. The method of claim 1, wherein the peroxidase is derived from an organism selected from the group consisting of Curvularia inequili, Halomicronema hongdechloris, Moorea bouillonii, Trichodesmium rubrum, Cryptococcus montaneus, Synechococcus sp. PCC7335, and Coralline algae.

9. The method of claim 1, wherein the peroxidase is produced in a host cell microorganism.

10. The method of claim 9, wherein the host cell microorganism is selected from Pichia pastoris, Aspergillus niger, Saccharomyces cerevisiae or Escherichia coli.

11. The method of any one of claims 9-10, wherein the expression of the peroxidase produced in the host cell microorganism is increased by about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 10-fold, for example relative to the peroxidase produced in its natural host.

12. The method of claim 1, wherein the amino acid sequence of the peroxidase is selected from the amino acid sequences listed in Table 1.

13. The method of claim 1, 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 1.

14. The method of claim 1, wherein the peroxidase is a sequence selected from any one of SEQ ID NOs. 1-50.

15. The method of claim 1, wherein the amino acid sequence of the peroxidase has at least 1, 2, 3, 4, 5 or 6 amino acid substitutions relative to the amino acid sequence selected from any one of SEQ ID NOs. 1-50.

16. The method of claim 1, wherein the small organic compound has a structure such as Formula (Y) as described herein.

17. The method of claim 1, wherein the small organic compound comprises 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

18. The method of claim 1, wherein the small organic compound comprises a ketone or an aldehyde.

19. The method of claim 1, wherein the small halogenated organic compound comprises a structure having formula (Y), such as described herein.

20. The method of claim 1, wherein the small halogenated organic compound is chlorinated; brominated; iodinated; chlorinated and iodinated; chlorinated and brominated; brominated and iodinated; or chlorinated, brominated and iodinated.

21. The method of claim 1, wherein the small halogenated organic compound is brominated.

22. The method of claim 1, wherein the small halogenated organic compound comprises 1, 2, or 3 halogen atoms.

23. The method of claim 1, wherein the small halogenated organic compound contains 1, 2, or 3 bromine atoms.

24. The method of claim 1, wherein the small halogenated organic compound comprises an acetone moiety.

25. The method of claim 1, wherein the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform or tribromoacetone.

26. The method of claim 1, wherein the small 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.

27. The method of claim 1, further comprising providing a peroxide source, such as H2O2 or PAA, and a halogen source, such as a halide salt such as KBr, NaCl or KI.

28. The method of claim 1, wherein the conditions sufficient to produce small halogenated organic compounds include one or more of the following: (a) Temperature between 10℃ and 85℃ (b) pH between 4 and 10; and (c) The ionic strength is between 0.1 mM and 4 M.

29. The method of claim 1, further comprising reducing methane production by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.

30. The method of claim 29, wherein the methane production is reduced by 10-75%.

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

32. The method of claim 31, further comprising obtaining a value for the methane level before (a) providing the peroxidase or after (b) providing the peroxidase.

33. The method of claim 32, comprising (a).

34. The method of claim 32, comprising (b).

35. The method of claim 32, comprising (a) and (b).

36. The method of claim 31, 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.

37. The method of claim 31, wherein the methane production is reduced by 10-75%.

38. The method of claim 31, wherein the peroxidase is a haloperoxidase.

39. The method of claim 38, wherein the haloperoxidase is a vanadium haloperoxidase (VHPO).

40. The method of claim 39, wherein the VHPO is a vanadium chloroperoxidase (VCPO), a vanadium bromoperoxidase (VBPO), or a vanadium iodoperoxidase (VIPO).

41. The method of claim 31, wherein the peroxidase is produced in a host cell microorganism such as selected from Pichia pastoris, Aspergillus niger or Escherichia coli.

42. The method of claim 41, wherein the expression of the peroxidase produced in the host cell microorganism is increased by about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold or 10-fold, e.g., relative to the peroxidase produced in its natural host.

43. The method of claim 31, wherein the amino acid sequence of the peroxidase is selected from the amino acid sequences listed in Table 2.

44. The method of claim 31, 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.

45. The method of claim 31, 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 SEQ ID NOs: 1-50.

46. ​​The method of claim 31, wherein the small organic compound has a structure such as Formula (Y) as described herein.

47. The method of claim 31, wherein the small organic compound comprises 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms.

48. The method of claim 31, wherein the small organic compound comprises a ketone or an aldehyde.

49. The method of claim 31, wherein the small halogenated organic compound comprises a structure having formula (Y), such as described herein.

50. The method of claim 31, wherein the small halogenated organic compound is chlorinated; brominated; iodinated; chlorinated and iodinated; chlorinated and brominated; brominated and iodinated; or chlorinated, brominated and iodinated.

51. The method of claim 31 , wherein the small halogenated organic compound is brominated.

52. The method of claim 31, wherein the small halogenated organic compound comprises 1, 2, or 3 halogen atoms.

53. The method of claim 31, wherein the small halogenated organic compound contains 1, 2, or 3 bromine atoms.

54. The method of claim 31, wherein the small halogenated organic compound comprises an acetone moiety.

55. The method of claim 31, wherein the small halogenated organic compound comprises dibromoacetone, bromoacetone, bromopentanedione, bromoform, or tribromoacetone.

56. The method of claim 31, wherein the small 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.

57. The method of claim 31 further comprising providing a peroxide source, such as H2O2 or PAA, and a halogen source, such as a halide salt such as KBr, NaCl or KI.

58. The method of claim 31, wherein the conditions sufficient to produce small halogenated organic compounds include one or more of the following: (a) Temperature between 10℃ and 85℃ (b) pH between 4 and 10; and (c) The ionic strength is between 0.1 mM and 4 M.

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