阅读说明：本技术 卤酸脱卤酶超家族蛋白的变体及使用其降低样品中含氟化合物浓度的方法 (Variants of a halase superfamily protein and methods of using the same to reduce the concentration of a fluorine-containing compound in a sample ) 是由 郑有景 金兑勇 朴珍雨 朴焌盛 李陈肃 姜昌德 卞钟元 宋承勋 于 2019-04-11 设计创作，主要内容包括：提供了卤代酸脱卤素酶超家族蛋白的变体和利用所述变体降低样品中的含氟化合物浓度的方法。(Variants of haloacid dehalogenase superfamily proteins and methods of using the variants to reduce the concentration of a fluorine-containing compound in a sample are provided.)

1. A variant protein of the HAD superfamily (HAD) of haloacid dehalogenases, the variant comprising:

a substitution of the amino acid residue corresponding to position S184 of SEQ ID NO. 2 and a substitution of at least one amino acid residue corresponding to positions N206, T208 and V210 of SEQ ID NO. 2; alternatively, the first and second electrodes may be,

a substitution of at least one amino acid residue corresponding to positions N206, T208 and V210 of SEQ ID NO 2,

wherein the substitution of the amino acid residue corresponding to position S184 of SEQ ID NO 2 is S184H, S184K or S184R;

the substitution of the amino acid residue corresponding to position N206 of SEQ ID No. 2 is N206M, N206G, N206A, N206V, N206L, N206I, N206F, N206W or N206P;

the substitution of the amino acid residue corresponding to position T208 of SEQ ID NO 2 is T208Q, T208S, T208C, T208Y or T208N;

and the substitution of the amino acid residue corresponding to position V210 of SEQ ID NO 2 is V210D or V210E.

2. The variant of claim 1, wherein the variant protein comprises:

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184 and N206;

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184, N206 and T208; or

Corresponding to SEQ ID NO:2, amino acid residues at positions S184, N206 and V210.

3. The variant of claim 2, wherein said variant protein is as set forth in SEQ ID NO:2 comprises the following substitutions:

S184H and N206M;

S184H, N206M and T208D; or

S184H, N206M and V210D.

4. A polynucleotide encoding a halide acid dehalogenase superfamily (HAD) variant protein of claim 1.

5. The polynucleotide of claim 4, wherein the variant protein comprises:

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184 and N206;

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184, N206 and T208; or

Corresponding to SEQ ID NO:2, amino acid residues at positions S184, N206 and V210.

6. The polynucleotide of claim 5, wherein the variant protein comprises the following amino acid substitutions:

S184H and N206M;

S184H, N206M and T208Q; or

S184H, N206M and V210D.

7. A recombinant vector comprising the polynucleotide of claim 4.

8. The recombinant vector of claim 7, wherein the variant protein comprises:

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184 and N206;

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184, N206 and T208; or

Corresponding to SEQ ID NO:2, amino acid residues at positions S184, N206 and V210.

9. The recombinant vector of claim 8, wherein the variant protein comprises the following amino acid substitutions:

S184H and N206M;

S184H, N206M and T208Q; or

S184H, N206M and V210D.

10. A recombinant microorganism comprising the haloacid dehalogenase superfamily (HAD) variant protein of claim 1 or a polynucleotide encoding the same.

11. The recombinant microorganism of claim 10, wherein said variant protein comprises:

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184 and N206;

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184, N206 and T208; or

Corresponding to SEQ ID NO:2, amino acid residues at positions S184, N206 and V210.

12. The recombinant microorganism of claim 11, wherein said variant protein comprises the following amino acid substitutions:

S184H and N206M;

S184H, N206M and T208Q; or

S184H, N206M and V210D.

13. The recombinant microorganism of claim 10, wherein the recombinant microorganism belongs to the genus escherichia, pseudomonas, or bacillus.

14. The recombinant microorganism of claim 10, wherein the recombinant microorganism is escherichia coli, p.

15. A method of reducing the concentration of a fluorochemical in a sample, the method comprising:

contacting a sample comprising a fluorochemical represented by formula 1 or 2 with a variant protein of the halate dehalogenase superfamily (HAD) of claim 1 to reduce the concentration of the fluorochemical in the sample:

< formula 1>

C(R₁)(R₂)(R₃)(R₄)

< formula 2>

(R₅)(R₆)(R₇)C-[C(R₁₁)(R₁₂)]n-C(R₈)(R₉)(R₁₀)，

Wherein, in the formulae 1 and 2,

n is an integer from 0 to 10,

R₁，R₂，R₃and R₄Each independently is fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or hydrogen (H), wherein R is selected from₁、R₂、R₃And R₄At least one of is F, and

R₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁and R₁₂Each independently is F, Cl, Br, I or H, wherein R is selected from₅、R₆、R₇、R₈、R₉、R₁₀、R₁₁And R₁₂At least one of (a) is F.

16. The method of claim 15, wherein the variant protein is in a microorganism comprising a polynucleotide encoding the variant protein, and the sample is contacted with the variant protein by contacting the sample with the microorganism.

17. The method of claim 16, wherein the variant protein comprises:

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184 and N206;

corresponding to SEQ ID NO:2, substitution of amino acid residues at positions S184, N206 and T208; or

Corresponding to SEQ ID NO:2, amino acid residues at positions S184, N206 and V210.

18. The method of claim 17, wherein the variant protein comprises the following amino acid substitutions:

S184H and N206M;

S184H, N206M and T208Q;

or S184H, N206M, and V210D.

19. The method of claim 16, wherein the recombinant microorganism belongs to the genus escherichia, pseudomonas, or bacillus.

20. The method of claim 16, wherein the contacting comprises culturing the recombinant microorganism with a sample comprising the fluorine-containing compound.

Brief Description of Drawings

These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a vector map of pET-SF0757 vector;

FIG. 2 is a vector map of pTrc-BANF-SF0757 vector;

FIG. 3 is a schematic diagram of a Dimroth reflux condenser; and

figure 4 shows a flow chart of the microbubble process.

Detailed description of the invention

Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the like elements throughout. In this regard, the embodiments of the invention may take different forms and should not be construed as limited to the descriptions set forth herein. Accordingly, the embodiments are described below to explain aspects by referring to the figures only. Expressions such as "at least one of … …" when preceded by a list of elements modify the entire list of elements rather than modifying individual elements listed.

Additional aspects will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the illustrated embodiments.

The term "gene" or "polynucleotide" as used herein may refer to a fragment of a nucleic acid that expresses a particular protein. A gene may include regulatory sequences of a coding region or non-coding regions including 5 '-non-coding and 3' -non-coding sequences. Regulatory sequences may include promoters, enhancers, operators, ribosome binding sites, polyA binding sites, and terminator regions, among others.

As used herein, the term "sequence identity" of a nucleic acid or polypeptide refers to the degree of identity between the bases or amino acid residues of the sequences in certain regions of comparison, aligned for best match. Sequence identity is a value measured by comparing two sequences in a comparison region by optimal alignment of the two sequences, where portions of the sequences in the comparison region may be added or deleted compared to a reference sequence. For example, the percentage of sequence identity can be calculated as follows: comparing the two optimally aligned sequences over the entire comparison area; the number of positions at which the same amino acid or nucleic acid occurs in both sequences is determined as the number of matching positions; the number of matching locations divided by the total number of locations in the comparison area (i.e., the size of the range); and the result of the division is multiplied by 100 to obtain the percentage of sequence identity. The percentage of sequence identity can be determined using known sequence comparison programs, such as BLASTN or BLASTP (NCBI), CLC Main Workbench (CLC bio) or MegAlign (DNASTAR Inc). Unless otherwise stated in the specification, the selection of parameters for executing a program may be as follows: the E value is 0.00001 and the H value is 0.001.

Different levels of sequence identity can be used to identify peptides or polynucleotides having the same or similar function or activity of different species. For example, the sequence identity can be 50% or greater, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%.

One aspect of the present disclosure provides a variant of a haloacid dehalogenase superfamily (HAD) protein, the variant comprising an amino acid change in the amino acid residue corresponding to position S184 of SEQ ID No. 2 and at least one amino acid residue corresponding to positions N206, T208 and V210 of SEQ ID No. 2; or an amino acid change in at least one amino acid residue corresponding to positions N206, T208 and V210 of SEQ ID NO 2.

According to certain embodiments, the amino acid change in position S184 may comprise a substitution S184H, S184K, or S184R. The amino acid change at position N206 may comprise a substitution of N206M, N206G, N206A, N206V, N206L, N206I, N206F, N206W, or N206P. The amino acid change at position T208 may include a substitution T208Q, T208S, T208C, T208Y, or T208N. The amino acid change at position V210 may comprise a substitution V210D or V210E.

According to certain embodiments, the variant may have amino acid changes in the amino acid residues corresponding to positions S184 and N206 of SEQ ID No. 2; amino acid residue changes corresponding to positions S184, N206 and T208 of SEQ ID NO 2; or amino acid residue changes corresponding to positions S184, N206 and V210 of SEQ ID NO 2.

With respect to variants, the amino acid changes may include substitutions of S184H and N206M in the amino acid sequence of SEQ ID NO. 2; 2, substitutions of S184H, N206M and T208D in the amino acid sequence of SEQ ID NO; or substitutions of S184H, N206M and V210D in the amino acid sequence of SEQ ID NO. 2.

With respect to variants, the HAD superfamily protein can be a phosphatase, a phosphonate esterase, a P-type atpase, β -phosphoglucomutase, a phosphomannomutase, or a dehalogenase the HAD superfamily protein can include a HAD domain the HAD superfamily protein can be a phospholipid-translocating atpase belonging to EC 3.6.3.1, a 3-deoxy-D-mannose-caprylate (KDO) 8-phosphate phosphatase belonging to EC 3.1.3.45, a mannosyl-3-phosphoglycerate phosphatase belonging to EC 3.1.3.70, a phosphoglycolate phosphatase belonging to EC 3.1.3.18, or a HAD belonging to EC 3.8.1.2.

The ATPase may be a putative lipid flippase involved in cold tolerance in Arabidopsis (Arabidopsis). KDO 8-phosphate phosphatase may catalyze the final step in KDO biosynthesis, a component of lipopolysaccharide in gram-negative bacteria. The mannosyl-3-phosphoglycerate phosphatase may hydrolyze mannosyl-3-phosphoglycerate to form the permeate mannosyl glycerate. Phosphoglycerate phosphatase may catalyze the dephosphorylation of 2-phosphoglycerate.

The HAD superfamily protein or variant thereof may have 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with respect to the amino acid sequence of SEQ ID No. 2, 7, 9, 11 or 13.

In embodiments, the variant may have the activity of an enzyme belonging to the HAD superfamily protein, and for example, may have the activity of a halate dehalogenase belonging to EC 3.8.1.2.

Alterations may include substitutions with post-translationally modified amino acids at the indicated positions. The alteration can include a substitution at the indicated position with one of the 19 amino acids of the 20 natural amino acids that is different from the corresponding amino acid. Amino acids and their abbreviations used herein are shown in table 1.

TABLE 1

With respect to variants, each of the amino acids substituted at the respective positions 184, 206, 208 and 210 of SEQ ID NO. 2 may be in a "conservative substitution" relationship with each other. The term "conservative" or "conservative substitution" as used herein refers to the replacement of an amino acid with a similar amino acid in terms of amino acid characteristics. The first amino acid can include H184, K184, or R184; m206, G206, a206, V206, L206, I206, F206, W206 or P206; q208, S208, C208, Y208, or N208; SEQ ID NO:2, D210 or E210E in the amino acid sequence of seq id No. 2. For example, when a non-aliphatic amino acid residue (e.g., Ser) at a particular position is replaced with an aliphatic amino acid residue (e.g., Leu), a substitution at the same position with a different aliphatic amino acid (e.g., ILe or Val) is referred to as a conservative mutation. In addition, amino acid characteristics include the size, hydrophobicity, polarity, charge, pK value, and other amino acid characteristics known in the art of residues. Thus, conservative mutations may include substitutions, such as basic to basic, acidic to acidic, polar to polar, and the like. For example, conservative substitutions may be made according to table 2 below, which describes a generally accepted grouping of amino acid features.

TABLE 2

Group of	Amino acids
		Non-polar	G A V L I M F W P
Polarity	S T C Y N Q
		Acidity	D E
Basic property	K R H

The term "corresponding" as used herein means that the amino acid position of a protein of interest is identical to the position of a reference protein (e.g., position S184 of SEQ ID NO:2) when the amino acid sequence of the protein of interest and the reference protein (e.g., SEQ ID NO:2) are aligned using a protein alignment program that is acceptable in the art, such as a BLAST pairwise alignment or the Lipman-Pearson protein alignment program well known in the art. The protein of interest may be HAD, which belongs to e.g. EC3.8.1.2. The Database (DB) storing the reference sequences may be the NCBI reference sequence (RefSeq) non-redundant protein database. The parameters for sequence alignment may be as follows: e value 0.00001 and H value 0.001.

The protein obtained according to the above alignment conditions and having the amino acid residue corresponding to position S184 of the amino acid sequence of SEQ ID NO:2 may be a homologue of the amino acid sequence of SF0757(SEQ ID NO: 2). Homologs may have 85% or more sequence identity with the amino acid sequence of SEQ ID NO. 2.

In one embodiment, the variant HAD protein comprises the amino acid sequence of SEQ ID NO: 2:

(a) N206M, N206G, N206A, N206V, N206L, N206I, N206F, N206W or N206P;

(b) T208Q, T208S, T208C, T208Y or T208N; and/or

(c) V210D or V210E;

and optionally further comprising a substitution selected from S184H, S184K, or S184R.

Another aspect of the present disclosure provides polynucleotides encoding HAD superfamily variant proteins.

The polynucleotide may be linked to a polynucleotide encoding an anchoring motif that causes the variant polypeptide to be expressed on the cell surface. Fusion can be accomplished by a linkage between the C-terminus of the anchor motif and the N-terminus of the variant polypeptide.

The anchoring motif may include a transmembrane portion and a linker portion arranged from the transmembrane portion in a direction away from the cell surface. The anchor motif may be selected from the group consisting of membrane proteins, lipoproteins, and self-transporter proteins. The anchoring motif can be BclA of Bacillus, OmpA, Lpp-OmpA, OmpC, OmpS, LamB, OmpC, Lpp-OmpC, PhoE and FadL of E.coli, OmpC of Salmonella, OprF of Pseudomonas and AIDA-I of pathogenic E.coli, or a fragment thereof.

The anchor motif can be BclA having the amino acid sequence of SEQ ID NO 27, SEQ ID NO 28, or SEQ ID NO 29.

Another aspect of the present disclosure provides a vector comprising a polynucleotide encoding a variant. For use as a vector, any vector known in the art that can be used to introduce a polynucleotide into a microorganism can be used. The vector may be, for example, a plasmid or a viral vector.

Another aspect of the disclosure provides a recombinant microorganism comprising a HAD variant protein or a polynucleotide encoding a variant described herein.

The polynucleotide may optionally be part of a vector. The polynucleotide may be present in the microorganism chromosomally or extrachromosomally (e.g., the polynucleotide may be integrated into the host cell chromosome or may remain in the host cell without being integrated into the host cell chromosome). The microorganism can express the polynucleotide to produce a variant protein. The variant protein may be present intracellularly, expressed on the cell, or released extracellularly.

The recombinant microorganism may be a bacterium or a fungus, and the bacterium may be a gram-positive bacterium or a gram-negative bacterium. The gram-negative bacteria may belong to the family enterobacteriaceae. The gram-negative bacteria may belong to the genera Escherichia, Salmonella, Flavobacterium or Pseudomonas. The microorganism belonging to the genus Escherichia may be Escherichia coli. The microorganism belonging to the genus pseudomonas may be p.saitens SF 1. The microorganism belonging to the genus flavobacterium may be autotrophic flavobacterium (x. autotrophicus). The gram-positive bacterium may belong to the genus Corynebacterium or Bacillus. The microorganism belonging to the genus Bacillus may be Bacillus sternocleinii (B.bombysepticus) SF 3.

Another aspect of the present disclosure provides a composition for reducing a fluorine-containing compound in a sample, the composition comprising a recombinant microorganism comprising a variant of a HAD superfamily protein or a polynucleotide encoding the variant. The fluorine-containing compound mentioned herein may be an alkane compound having 1 to 12 carbon atoms substituted with at least one fluorine. The fluorine-containing compound may be represented by formula 1 or formula 2:

< formula 1>

C(R₁)(R₂)(R₃)(R₄)

< formula 2>

(R₅)(R₆)(R₇)C-[C(R₁₁)(R₁₂)]n-C(R₈)(R₉)(R₁₀).

Formula 1, R₁，R₂，R₃And R₄May each independently be F, Cl, Br, I or H,

wherein, is selected from R₁、R₂、R₃And R₄At least one of (a) is F,

in formula 2, n may be an integer of 0 to 10, and when n is equal to or greater than 2, R₁₁Are the same as or different from each other, and R₁₂Each of which may be the same or different from each other. In addition, R₅，R₆，R₇，R₈，R₉，R₁₀，R₁₁And R₁₂Can each independently be F, Cl, Br, I or H, wherein at least one is selected from R₅，R₆，R₇，R₈，R₉，R₁₀，R₁₁And R₁₂Is F. In some embodiments, the fluorine-containing compound may be, for example, CHF3, CH2F2, CH3F, or CF 4. The term "removal" as used herein refers to any reduction in the concentration of the fluorine-containing compound. Reduction includes partial or complete reduction, i.e., a concentration of zero or near zero.

The composition may comprise a variant of the HAD superfamily protein, which has an activity belonging to EC 3.8.1.2. The composition may include a recombinant microorganism, a lysate thereof, or a water-soluble material portion of the lysate.

Removal of the fluorochemicals can include any reduction in the concentration of the fluorochemicals in the sample, such as cleavage of the CF bonds of the fluorochemicals, conversion of the fluorochemicals to a different material, or accumulation of the fluorochemicals in the cells. The conversion of the fluorochemical may include introducing a hydrophilic group, such as a hydroxyl group, into the fluorochemical, or introducing a carbon-carbon double bond or a carbon-carbon triple bond into the fluorochemical.

The sample may be a liquid sample, a gas sample, or a combination of both. For example, the sample may be industrial sewage or waste gas, or both.

Another aspect of the present disclosure provides a method of reducing the concentration of a fluorine-containing compound in a sample, the method comprising:

contacting a HAD variant protein as described herein, or a recombinant microorganism comprising the HAD variant protein or a polynucleotide encoding the HAD variant protein, with a sample comprising a fluorochemical represented by formula 1 or formula 2 to reduce the concentration of the fluorochemical in the sample:

< formula 1>

C(R₁)(R₂)(R₃)(R₄)

< formula 2>

(R₅)(R₆)(R₇)C-[C(R₁₁)(R₁₂)]n-C(R₈)(R₉)(R₁₀)

In the formula 1, R₁，R₂，R₃And R₄May each independently be F, Cl, Br, I or H,

wherein is selected from R₁、R₂、R₃And R₄At least one of (a) is F,

in formula 2, n may be an integer of 0 to 10, and when n is equal to or greater than 2, R₁₁Are the same as or different from each other, and R₁₂Each of which may be the same or different from each other. In addition, R₅，R₆，R₇，R₈，R₉，R₁₀，R₁₁And R₁₂Can each independently be F, Cl, Br, I or H, wherein at least one is selected from R₅，R₆，R₇，R₈，R₉，R₁₀，R₁₁And R₁₂Is F.

The recombinant microorganism can further include a second polynucleotide encoding an unlinked dehalogenase. In some embodiments, the recombinant microorganism contains a first dehalogenase that is a variant HAD protein as described herein linked to (or contains a polynucleotide encoding) an anchor motif, and contains a second dehalogenase or a polynucleotide encoding the same that is not linked to an anchor. A motif (or polynucleotide encoding it), which may be the same or different from the first dehalogenase. Or vice versa. The first dehalogenase that is a variant HAD protein may be free of an anchor moiety and the second dehalogenase may comprise an anchor motif. Thus, the dehalogenase may be expressed both on the cell surface and within the cell. The second dehalogenase (or a polynucleotide encoding it) may be endogenous or exogenous, and the recombinant microorganism may allow the dehalogenase to be expressed intracellularly or recombinantly.

The recombinant microorganism may have activity in reducing the concentration of "fluorochemicals" in a sample. The fluorine-containing compound may be CH3F, CH2F2, CHF3, CF4, CH2FCOOH, or mixtures thereof.

Removal of the fluorochemicals can include any reduction in the concentration of the fluorochemicals in the sample, such as cleavage of the CF bonds of the fluorochemicals, conversion of the fluorochemicals to a different material, or accumulation of the fluorochemicals in the cells.

The sample may be a liquid sample or a gas sample, or a combination of both. For example, the sample may be industrial sewage or waste gas. For example, the sample may be sludge. The term "sludge" as used herein refers to a semi-solid slurry and may be obtained as sewage sludge from a wastewater treatment process or as a settled suspension obtained from conventional drinking water treatment or many other industrial processes.

The contacting of the recombinant microorganism with the sample can be carried out in a suitable manner, for example in the liquid or gas phase. The contacting can include culturing the recombinant microorganism in the presence of a fluorine-containing compound. The contacting may be performed in a closed vessel, e.g., an airtight vessel. The contacting may be performed when the growth state of the recombinant microorganism is in an exponential phase or a stationary phase. The cultivation may be carried out under aerobic or anaerobic conditions. The contacting can be performed under conditions under which the recombinant microorganism can survive in a closed container (e.g., a gas-tight container). Such conditions under which the recombinant microorganism survives may include conditions under which the recombinant microorganism may proliferate or may be allowed to be in a quiescent state.

The contact may include a passive contact and an active contact. The term "passive contact" refers to contact in the absence of an external driving force, and the term "active contact" refers to contact under an external driving force. The active contact may be achieved by injecting the fluorine-containing compound in the form of bubbles into the solution containing the recombinant microorganism and/or spraying the recombinant microorganism. For example, the contacting may be achieved by blowing the sample into the culture medium or broth. To inject the sample, the sample may be blown from the bottom of the culture medium or broth to the top thereof. Injection of the sample may be achieved by preparing a droplet of the sample. The contacting may be carried out batchwise or in a continuous manner. The contacting may be repeated, for example, two or more times, for example, three, five, or ten or more times. The contacting may be continued or repeated until the fluorine-containing compound is reduced to a desired concentration.

The recombinant microorganism may be in the form of a thin film layer. The film layer of such recombinant microorganisms may be a liquid film layer. The fluorine-containing compound may be in the form of a gaseous thin film layer. The liquid film layer of recombinant microorganisms and the gaseous film layer of fluorine-containing compounds may be in contact with each other.

The recombinant microorganism is subjected to a cyclic process, and in this regard, the contact area or contact time of the recombinant microorganism with the fluorine-containing compound can be increased. This cyclic process can increase the mass transfer coefficient (KLa) value, as well as the amount (or rate) of decomposition of the fluorine-containing compound.

With respect to the method, the contacting may further comprise: in an exhaust gas decomposition device comprising one or more reactors, each reactor comprising one or more first inlets and one or more first outlets, wherein a sample is injected into such an exhaust gas decomposition device, and

wherein the recombinant microorganism is injected into the exhaust gas decomposition device through the one or more first inlets such that the recombinant microorganism can contact the sample, and the resulting mixture can be discharged through the one or more outlets.

In this regard, the exhaust gas decomposition device may include a second inlet and a second outlet, and the sample may be injected through the second inlet and discharged through the second outlet. In such a configuration, the recombinant microorganism may move in a direction opposite to the direction in which the sample moves. A fluid film comprising recombinant microorganisms may be formed on the inner wall of one or more of the reactors.

With respect to the method, the exhaust gas decomposition device may further comprise a first recycle line for resupplying at least a portion of the fluid to the one or more first inlets, wherein the fluid contains recombinant microorganisms. Samples containing the fluorine-containing compound may be retained in one or more reactors. In addition, the exhaust gas decomposition device may further include a second inlet through which the sample may be supplied into the one or more reactors, and a second outlet through which the sample may be discharged to the outside of the one or more reactors. And moving in a second direction within the one or more reactors. The second direction may be different from the direction of fluid movement of, for example, a recombinant microorganism. In addition, in at least one of the fluid collection zone of the inner bottom of the one or more reactors and the fluid reaction zone of the inner top of the one or more reactors of the exhaust gas decomposition device, the fluid comprising the recombinant microorganism may contact each other to decompose the fluorine-containing compound. In the exhaust gas decomposition device, the fluid film comprising the fluid containing the recombinant microorganism may contact the fluid comprising the sample.

With respect to the method, the exhaust gas decomposition device may further comprise one or more structures internal to the reactor, wherein the structures may be configured to increase a contact area between the fluid comprising the recombinant microorganism and the sample comprising fluorine. For example, the structure may include at least one selected from a packing material and a return tube, but the embodiment of the present disclosure is not limited thereto. Any structure configured to increase the contact area between the fluid comprising the recombinant microorganism and the sample comprising the fluorine-containing compound may be included. The "packaging material" may be an inert solid material. The packaging material can have various shapes. The packaging material may be the same material used in the packaging of the packed bed column. The packaging material may be made of plastic, magnetic material, steel or aluminium. The packaging material may have a very thin thickness. The packaging material may have a ring shape, such as a rash ring, a pall ring and a berl saddle, a saddle shape and a protrusion shape. The packing material may be irregularly packed in the packed bed reactor. The packaging material may be effective to increase contact between the fluorochemical and microorganisms present in the liquid. By forming a microbial film on the surface of the packing material and on the inner surface of the reactor, the time or opportunity for contact between the fluorine-containing compound and the microbes can be maximized. In addition, the one or more first inlets may be connected to a fluid reaction zone at the interior top of the one or more reactors in the exhaust gas decomposition device, thereby supplying the fluid comprising the recombinant microorganisms through the one or more first inlets.

With respect to the method, the fluid comprising the recombinant microorganisms can be collected in a fluid collection zone at the interior bottom of one or more reactors in the exhaust gas decomposition device. The sample comprising the fluorine-containing compound fed into the one or more reactors through the second inlet may pass through the collected fluid comprising the recombinant microorganism in the form of bubbles, and then be transferred to the fluid reaction zone at the inner top of the one or more reactors, and then may be discharged to the outside of the one or more reactors through the second outlet.

With respect to the process, the aspect ratio (H/D) of the height H to the diameter D of one or more reactors in the exhaust gas decomposition device may be 2 or greater, 5 or greater, 10 or greater, 15 or greater, 20 or greater, or 50 or greater.

With regard to the method, the exhaust gas decomposition device may be arranged in such a way that the side wall of the reactor or reactors or some other inner surface thereof may be inclined or slanted at an angle of less than 90 ° or more than 90 °. For example, the sidewalls or other interior surfaces thereof may be inclined in a range of about 30 ° to about 150 ° (e.g., about 30 ° to less than 90 ° or greater than 90 ° to about 150 °), about 70 ° to about 110 ° (e.g., about 70 ° to less than 90 ° or greater than 90 ° to about 110 °), about 80 ° to about 100 ° (e.g., about 80 ° to less than 90 ° or greater than 90 ° to about 100 °), or about 50 ° to about 90 ° (e.g., about 50 ° to less than 90 °) relative to the earth's surface.

With respect to the method, one or more reactors in the exhaust gas decomposition device may be rotated. The fluid containing the recombinant microorganism may be a liquid and the sample containing the fluorine containing compound may be a gas.

Variants and polynucleotides encoding variants according to any of the above embodiments may be used to remove fluorochemicals from a sample or to generate variants.

The recombinant microorganism according to any of the above embodiments may be used to remove a fluorine-containing compound from a sample.

A composition comprising a HAD superfamily protein according to any of the above embodiments may be used to remove fluorochemicals from a sample.

The method of determining the concentration of a fluorine-containing compound in a sample according to any of the above embodiments may be effective to remove the fluorine-containing compound from the sample.

Example 1: preparation of recombinant microorganism comprising SF0757 Gene

Gene SF0757(SEQ ID NO:1), encoding dehalogenase, amplified from B.pleuroperineus SF3, deposited on 24 months 2.2017 in the Korean culture center for type cultures (KCTC), an international depository according to the Budapest treaty.

Mainly, Bacillus sternocleistris SF3 was cultured in LB medium overnight while stirring at a temperature of 30 ℃ and a speed of 230rpm, and its genomic DNA was isolated using a total DNA extraction kit (Invitrogen Biotechnology). PCR was performed using genomic DNA as a template and a set of primers having the nucleotide sequences of SEQ ID NO. 3 and SEQ ID NO. 4 to amplify and obtain the SF0757 gene. The genes thus amplified were each independently ligated with pET28a vector (Novagen, Cat. No. 69864-3) which was cleaved by restriction enzymes such as NcoI and XhoI using the InFusion Cloning Kit (Clontech laboratories, Inc.) to prepare pET-SF0757 vector. FIG. 1 is a vector map of pET-SF0757 vector. Here, the SF0757 protein has the amino acid sequence of SEQ ID NO 2.

Then, the prepared pET-SF0757 vector was introduced into E.coli BL21 by the heat shock method, and then cultured in LB plate agar containing 50. mu.g/mL kanamycin. A strain showing kanamycin resistance was selected from the plates, and the strain finally selected therefrom was named recombinant Escherichia coli BL21/pET-SF 0757.