BACTERIAL CYTOCHROME P450 PROTEIN VARIANT AND METHOD OF REDUCING CONCENTRATION OF FLUORINATED METHANE IN SAMPLE USING THE SAME

Abstract

Provided is a recombinant microorganism including an exogenous gene encoding a bacterial cytochrome P450 protein or a variant thereof, a composition including the recombinant P450 protein or the variant thereof, which is used for removing CH.sub.nF.sub.4-n (n is an integer of 0 to 3) in a sample, and a method of reducing a concentration of CH.sub.nF.sub.4-n in the sample.

Claims

1. A recombinant microorganism comprising an exogenous gene encoding a bacterial cytochrome P450 protein or a variant thereof, wherein the variant is a P450Cam variant having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 and having an activity belonging to EC 1.14.15.1, or wherein the variant is a P450BM3 variant having an amino acid alteration at an amino acid residue corresponding to position N320 of SEQ ID NO: 8 and having an activity belonging to EC 1.14.14.1.

2. The recombinant microorganism of claim 1, wherein the recombinant microorganism comprises an exogenous gene encoding the P450Cam variant comprising a F351Y, F351T, F351N, F351Q, F351H, or F351D mutation.

3. The recombinant microorganism of claim 1, wherein the recombinant microorganism comprises an exogenous gene encoding the P450BM3 variant comprising a N320W, N320F, N320G, N320P, N320S, or N320E mutation.

4. The recombinant microorganism of claim 1, further comprising an exogenous gene encoding a protein belonging to EC 1.1.1.49.

5. A composition comprising a recombinant P450 protein variant, wherein the variant is a P450Cam variant having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 and having an activity belonging to EC 1.14.15.1, or a P450BM3 variant having an amino acid alteration at an amino acid residue corresponding to position N320 of SEQ ID NO: 8 and having an activity belonging to EC 1.14.14.1.

6. The composition of claim 5, wherein the composition comprises a P450Cam variant comprising a F351Y, F351T, F351N, F351Q, F351H, or F351D mutation.

7. The composition of claim 5, wherein the composition comprises a P450BM3 variant comprising a N320W, N320F, N320G, N320P, N320S, or N320E mutation.

8. A method of reducing a concentration of fluorinated methane in a sample, the method comprising contacting a recombinant P450 protein or a variant thereof with the sample comprising fluorinated methane represented by CH.sub.nF.sub.4-n (n is an integer of 0 to 3) to reduce the concentration of fluorinated methane in the sample, wherein the variant is a P450Cam variant having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 and having an activity belonging to EC 1.14.15.1, or a P450BM3 variant having an amino acid alteration at an amino acid residue corresponding to position N320 of SEQ ID NO: 8 and having an activity belonging to EC 1.14.14.1.

9. The method of claim 8, wherein the contacting is performed in a sealed container.

10. The method of claim 8, wherein the variant of the recombinant P450 protein is in a recombinant microorganism.

11. The method of claim 10, wherein the contacting is performed in a sealed container under conditions where the recombinant microorganism survives or is viable.

12. The method of claim 10, wherein the contacting comprises culturing the recombinant microorganism in the presence of the sample.

13. The method of claim 12, wherein the sample is industrial waste water or waste gas.

14. A variant bacterial P450Cam polypeptide having an amino acid alteration at an amino acid residue corresponding to position F351 of SEQ ID NO: 6 and belonging to EC 1.14.15.1, or a variant bacterial P450BM3 polypeptide having an amino acid alteration at an amino acid residue corresponding to position N320 of SEQ ID NO: 8 and belonging to EC 1.14.14.1.

15. A polynucleotide encoding the variant of bacterial P450Cam or the variant of bacterial P450BM3 of claim 17.

16. The recombinant microorganism of claim 1, wherein the recombinant microorganism comprises a P450Cam variant comprising SEQ ID NO: 6 with a substitution at position F351 of SEQ ID NO: 6, or a P450BM3 variant comprising SEQ ID NO: 8 with a substitution at position N320 of SEQ ID NO: 8.

17. The variant polypeptide of claim 14, wherein the variant polypeptide comprises a P450Cam variant comprising SEQ ID NO: 6 with a substitution at position F351 of SEQ ID NO: 6, or a P450BM3 variant comprising SEQ ID NO: 8 with a substitution at position N320 of SEQ ID NO: 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] 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 in which:

[0013] FIG. 1 shows a vector map of a pETDuet-camC-camAB vector;

[0014] FIG. 2 shows changes in a headspace concentration of CHF.sub.3 over time when E. coli BL21/pETDuet-camC-camAB was cultured in a medium contacted with CHF.sub.3-containing gas;

[0015] FIG. 3A shows changes in a headspace concentration of CHCl.sub.3 over time when E. coli BL21/pETDuet-camC-camAB was cultured in a CHCl.sub.3-containing medium;

[0016] FIG. 3B shows changes in a headspace concentration of CF.sub.4 over time when E. coli BL21/pETDuet-camC-camAB was cultured in a medium contacted with CF.sub.4-containing gas;

[0017] FIG. 3C shows changes of CF.sub.4 in a sample over time by E. coli BL21/pETDuet-camCmt-camAB introduced with a mutant camC gene;

[0018] FIG. 4 shows a vector map of a pET28a-P450.sub.BM3 vector;

[0019] FIG. 5 shows a vector map of a pACYCDuet-zwf vector;

[0020] FIG. 6 shows changes in a headspace concentration of CHF.sub.3 over time when recombinant E. coli BL21/pET28a-P450.sub.BM3 or recombinant E. coli BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf was cultured in a solution contacted with CHF.sub.3-containing gas;

[0021] FIG. 7 shows changes in a headspace concentration of CHCl.sub.3 over time when E. coli BL21/pET28a-P450.sub.BM3 was cultured in a CHCl.sub.3-containing solution;

[0022] FIG. 8 shows changes in a headspace concentration of CF.sub.4 over time when E. coli BL21/pET28a-P450.sub.BM3 was cultured for 7 days in a medium contacted with CF.sub.4-containing gas; and

[0023] FIG. 9 shows changes in a concentration of CF.sub.4 in a sample over time by E. coli BL21/pET28a-P450BM3mt introduced with a mutant P450BM3 gene.

DETAILED DESCRIPTION

[0024] An aspect provides a recombinant microorganism including an exogenous gene encoding a bacterial cytochrome P450 protein or a variant thereof.

[0025] Cytochromes P450 (CYPs) belong to the superfamily of proteins containing a heme cofactor, and therefore, are hemoproteins. Cytochromes P450 use a variety of small and large molecules as substrates in enzymatic reactions. They are, in general, terminal oxidase enzymes in electron transfer chains, broadly categorized as P450-containing systems.

[0026] Bacterial cytochromes P450 are often soluble enzymes and are involved in diverse metabolic processes. Some bacteria such E. coli have no cytochrome P450. Cytochrome P450 (CYP101) derived from P. putida is part of a camphor-hydroxylating catalytic cycle consisting of two electron transfer steps from putidaredoxin, which is a 2Fe-2S cluster-containing protein cofactor.

[0027] Cytochrome P450 BM3 (CYP102) derived from B. megaterium catalyzes the NADPH-dependent hydroxylation of several long-chain fatty acids at the -1 through -3 positions. Cytochrome P450 BM3 constitutes a natural fusion protein between the CYP domain and an electron donating cofactor.

[0028] With regard to the recombinant microorganism, the cytochrome P450 protein may belong to EC 1.14.15.1 or EC 1.14.14.1. The cytochrome P450 protein may be P450Cam or P450.sub.BM3.

[0029] P450Cam may be derived from Pseudomonas putida PpG786. P450.sub.BM3 may be derived from Bacillus megaterium (ATCC 14581). The cytochrome P450.sub.CAM protein may be a complex of CamA, CamB, and CamC, which constitutes the bacterial CYP101 system. CamA may be FAD-containing reductase. CamA may be NADH or NADPH-dependent. The CamA may belong to EC 1.18.1.5. CamB may be [2Fe2S]-type ferredoxin. CamC, also called P450Cam(CYP101), may include cytochrome P450 and may belong to EC 1.14.15.1. CamA, CamB, and CamC may have amino acid sequences of SEQ ID NOS: 2, 4, and 6, respectively. Genes encoding CamA, CamB, and CamC may have nucleotide sequences of SEQ ID NOS: 1, 3, and 5, respectively.

[0030] The P450Cam variant may have an amino acid alteration at an amino acid residue corresponding to position F351 of an amino acid sequence of SEQ ID NO: 6, and may have an activity belonging to EC 1.14.15.1. The amino acid alteration may be replacement of the amino acid residue corresponding to position F351 with a different amino acid, for example, any of the 19 natural amino acids. For instance, the variant may have replacement (substitution) of the amino acid residue corresponding to the position F351 of SEQ ID NO: 6 with Y, T, N, Q, H, or D (e.g., a F351Y, F351T, F351N, F351Q, F351H, or F351D variant). EC 1.14.15.1 may represent an enzyme that catalyzes the reaction of (+)-camphor+reduced putidaredoxin+O.sub.2(+)-exo-5-hydroxycamphor+oxidized putidaredoxin+H.sub.2O.

[0031] A gene encoding the P450CAM variant may be a gene encoding the F351Y, F351T, F351N, F351Q, F351H, or F351D variant in P450CAM having the amino acid sequence of SEQ ID NO: 6. The gene may have a nucleotide sequence of SEQ ID NO: 51, 52, 53, 54, 55, or 56, or corresponding sequence by virtue of the degeneracy of the genetic code (e.g., a codon-optimized sequence). The microorganism may further include a gene encoding CamA and a gene encoding CamB.

[0032] P450.sub.BM3 may be a polypeptide having an amino acid sequence of SEQ ID NO: 8. A gene encoding P450.sub.BM3 may have a nucleotide sequence of SEQ ID NO: 7. The variant may have an amino acid alteration at an amino acid residue corresponding to the position N320 of the amino acid sequence of SEQ ID NO: 8, and may have an activity belonging to EC 1.14.14.1. The amino acid alteration may be replacement (substitution) of the amino acid residue corresponding to the position N320 with a different amino acid, for example, any of the 19 natural amino acids. The variant may have replacement of the amino acid residue corresponding to the position N320 of SEQ ID NO: 8 with W, F, G, P, S, or E (e.g., a N320W, N320F, N320G, N320P, N320S, or N320E variant). A gene encoding the P450BM3 variant may be a gene encoding the variant having a N320W, N320F, N320G, N320P, N320S, or N320E substation in P450BM3 having the amino acid sequence of SEQ ID NO: 8. The gene may have a nucleotide sequence of SEQ ID NO: 45, 46, 47, 48, 49, or 50, or corresponding sequence by virtue of the degeneracy of the genetic code (e.g., a codon-optimized sequence).

[0033] EC 1.14.14.1 may catalyze the following reaction: RH+reduced NADPH - - - hemoprotein reductase+O.sub.2=ROH+oxidized NADPH - - - hemoprotein reductase+H.sub.2O.

[0034] As used herein, the term corresponding refers to the amino acid position of a protein of interest that aligns with the mentioned position (e.g., position F351 of SEQ ID NO: 6 or position N320 of SEQ ID NO: 8) of a reference protein when amino acid sequences of the protein of interest and the reference protein are aligned using an art-acceptable protein alignment program, including the NCBI BLAST pairwise alignment or the well known Lipman-Pearson Protein Alignment program, with the following parameters: Ktuple=2, Gap Penalty=4, and Gap length penalty=12. In this regard, the range included in the corresponding sequence may be a range of E-value 0.00001 and H-value 0.001.

[0035] Examples of proteins homologs of P450CAM with an amino acid substitution at a position corresponding to position F351 of SEQ ID NO: 6, obtained according to the above alignment conditions, are listed in the following Tables 1, 2, and 3.

[0036] Also, examples of homologs of P450BM3 with an amino acid substitution at a position corresponding to position N320 of SEQ ID NO: 8, obtained according to the above alignment conditions, are listed in Table 4.

[0037] Thus, in some embodiments, the P450 variant can comprise SEQ ID NO: 6 with the described substitution at F351 of SEQ ID NO: 6, or can comprise SEQ ID NO: 8 with the described substitution at N320 of SEQ ID NO: 8, or can comprise a different amino acid sequence with a substation at a corresponding amino acid residue, provided it catalyzes the same reaction as the p450 variant comprising SEQ ID NO: 6 or 8 with the indicated substitution. In some embodiments, the p450 variant comprises an amino acid sequence with at least 75, 80, 85, 90, 91, 93, 94, 95, 95, 97, 98, or 99% sequence identity to SEQ ID NO: 6 or SEQ ID NO: 8, including the amino acid alteration at F351 of SEQ ID NO: 6 or N320 of SEQ ID NO: 8. Also contemplated are fragments (e.g., N or C terminal truncations or internal deletions) that retain the recited activity.

TABLE-US-00001 TABLE 1 NO. NCBI ID 1 gi|163930960 2 gi|612182735 3 gi|497125935 4 gi|310942843 5 gi|657832383 6 gi|498088271 7 gi|544829275 8 gi|861974080 9 gi|738620841 10 gi|499763441 11 gi|746290673 12 gi|503614840 13 gi|861969570 14 gi|662139213 15 gi|544827262 16 gi|498088269 17 gi|861974085 18 gi|737512009 19 gi|817101596 20 gi|494439068 21 gi|496309894 22 gi|746289514 23 gi|753796069 24 gi|545454562 25 gi|648417306 26 gi|666681698 27 gi|567402060 28 gi|826044703 29 gi|826049125 30 gi|806905723 31 gi|551338874 32 gi|730289226 33 gi|654615031 34 gi|656116930 35 gi|826046029 36 gi|737567226 37 gi|98976439 38 gi|612108073 39 gi|515116019 40 gi|817101463 41 gi|398136480 42 gi|757698965 43 gi|126194726 44 gi|737644672 45 gi|496199226 46 gi|654614522 47 gi|759387698 48 gi|739341634 49 gi|783098869 50 gi|783093808 51 gi|783094059 52 gi|759427060 53 gi|861968800 54 gi|497509581 55 gi|497810537 56 gi|783098865 57 gi|739341585 58 gi|783097592 59 gi|783099978 60 gi|783097681 61 gi|728824802 62 gi|746238551 63 gi|494955160 64 gi|496104589 65 gi|567412687 66 gi|98976470 67 gi|358240604 68 gi|494898237 69 gi|504740033 70 gi|648547795 71 gi|808659667 72 gi|551292036 73 gi|551292470 74 gi|502616812 75 gi|545316934 76 gi|491842667 77 gi|750353906 78 gi|858007594 79 gi|665981585 80 gi|806833869 81 gi|504197805 82 gi|564970689 83 gi|499924508 84 gi|765348796 85 gi|497424876 86 gi|739367531 87 gi|517897868 88 gi|702839727 89 gi|739883834 90 gi|750419370

TABLE-US-00002 TABLE 2 NO. NCBI ID 91 gi|494019567 92 gi|498089540 93 gi|826041014 94 gi|566044904 95 gi|566044935 96 gi|501296495 97 gi|517247251 98 gi|516019877 99 gi|288913356 100 gi|512734904 101 gi|783100677 102 gi|764994003 103 gi|517247290 104 gi|652940833 105 gi|169821418 106 gi|835531786 107 gi|551362264 108 gi|764960072 109 gi|759387686 110 gi|737512189 111 gi|820802680 112 gi|646534628 113 gi|820802864 114 gi|334103741 115 gi|826041595 116 gi|746241621 117 gi|764993875 118 gi|748599849 119 gi|764997807 120 gi|662354752 121 gi|783096369 122 gi|530258733 123 gi|657922982 124 gi|654614517 125 gi|739616950 126 gi|783098022 127 gi|806908467 128 gi|21467173 129 gi|401808868 130 gi|514397583 131 gi|749201634 132 gi|528182079 133 gi|647795133 134 gi|651636595 135 gi|661269250 136 gi|695263348 137 gi|696511194 138 gi|493217416 139 gi|493377553 140 gi|563565380 141 gi|665834124 142 gi|665888884 143 gi|665827329 144 gi|751294725 145 gi|827106327 146 gi|639165413 147 gi|652899356 148 gi|652914977 149 gi|652694819 150 gi|496153669 151 gi|518973859 152 gi|808102361 153 gi|664078266 154 gi|663221595 155 gi|663326563 156 gi|493424288 157 gi|502993954 158 gi|518949456 159 gi|750417303 160 gi|750543392 161 gi|377530614 162 gi|377532462 163 gi|737965741 164 gi|739538229 165 gi|739543125 166 gi|750407524 167 gi|739645497 168 gi|493919745 169 gi|737792710 170 gi|750519055 171 gi|521297725 172 gi|482632482 173 gi|494797766 174 gi|493993588 175 gi|648280499 176 gi|750519223 177 gi|498814706 178 gi|519015945 179 gi|639007804 180 gi|518767974

TABLE-US-00003 TABLE 3 NO. NCBI ID 181 gi|739625293 182 gi|820802866 183 gi|739651753 184 gi|502742128 185 gi|515118033 186 gi|820802677 187 gi|391860290 188 gi|737643185 189 gi|544823238 190 gi|763095543 191 gi|739611016 192 gi|145322598 193 gi|825391797 194 gi|759685456 195 gi|836723496 196 gi|488703345 197 gi|763384158 198 gi|528059914 199 gi|783097229 200 gi|494017068 201 gi|739663478 202 gi|739620206 203 gi|746237691 204 gi|567412712 205 gi|550925359 206 gi|746344573 207 gi|530255704 208 gi|739669024 209 gi|654478200 210 gi|490753280 211 gi|497922631 212 gi|740896970 213 gi|652908779 214 gi|503298839 215 gi|740869740 216 gi|503612867 217 gi|646519758 218 gi|494981163 219 gi|490214493 220 gi|736859678 221 gi|739577671 222 gi|736886954 223 gi|654534319 224 gi|549129549 225 gi|653383901 226 gi|703388673 227 gi|653777500 228 gi|655968891 229 gi|655882347 230 gi|630947972 231 gi|495218410 232 gi|768967538 233 gi|746229913 234 gi|746230981 235 gi|746236533 236 gi|544823589 237 gi|746239269 238 gi|490319630 239 gi|494981649 240 gi|494957004 241 gi|763090173 242 gi|738613213 243 gi|746229737 244 gi|754958228 245 gi|499912932 246 gi|657825087 247 gi|655586613 248 gi|739190742 249 gi|518714103 250 gi|503189844 251 gi|739186131 252 gi|739186149 253 gi|516607102 254 gi|522116265 255 gi|522150263 256 gi|703225980 257 gi|703223632 258 gi|703223663 259 gi|494300956 260 gi|808659227 261 gi|489969104 262 gi|806822276 263 gi|556618018 264 gi|738609029 265 gi|403646243 266 gi|737785331 267 gi|703226655 268 gi|602519307 269 gi|739367513 270 gi|737980497 271 gi|737981631 272 gi|817101442 273 gi|497809551 274 gi|545453717 275 gi|497809089

TABLE-US-00004 TABLE 4 NO. NCBI ID 1 gi|515136080 2 gi|757757972 3 gi|822528663 4 gi|544838284 5 gi|491696887 6 gi|655149838 7 gi|512150124 8 gi|493729782 9 gi|738856821 10 gi|655112080 11 gi|648634781 12 gi|522106669 13 gi|504462655 14 gi|783152040 15 gi|759010788 16 gi|545381104 17 gi|548617766 18 gi|648623486 19 gi|738714376 20 gi|639453808 21 gi|497281073 22 gi|494207912 23 gi|843075790 24 gi|518517905 25 gi|655094715 26 gi|517805393 27 gi|518469404 28 gi|655084756 29 gi|764415731 30 gi|491699287 31 gi|518251998 32 gi|493730772 33 gi|817723893 34 gi|228697407 35 gi|228736549 36 gi|692165489 37 gi|489315595 38 gi|498015014 39 gi|749037577 40 gi|763303489 41 gi|830323790 42 gi|857573616 43 gi|654951198 44 gi|647569946 45 gi|738784028 46 gi|515717624 47 gi|517613324 48 gi|507035289 49 gi|661257874 50 gi|655116131 51 gi|736161405 52 gi|493687687 53 gi|806498422 54 gi|532550849 55 gi|757435944 56 gi|737448097 57 gi|542116840 58 gi|764608412 59 gi|518088806 60 gi|768926886 61 gi|498013687 62 gi|498020927 63 gi|498487619 64 gi|530665825 65 gi|753200845 66 gi|495633284 67 gi|748815403 68 gi|738932691 69 gi|738896417 70 gi|652405427 71 gi|764371274 72 gi|701527930 73 gi|751587021 74 gi|736758744 75 gi|657859536 76 gi|657039097 77 gi|852221735 78 gi|850337075 79 gi|550547409 80 gi|495772021 81 gi|504454491 82 gi|737572351 83 gi|654483633 84 gi|495911896 85 gi|737423431 86 gi|737423433

[0038] The recombinant microorganism may be bacteria or fungi. The bacteria may be Gram-positive or Gram-negative bacteria. The Gram-negative bacteria may belong to the family Enterobacteriaceae. The Gram-negative bacteria may belong to the genus Escherichia, the genus Salmonella, the genus Xanthomonas, or the genus Pseudomonas. The genus Escherichia microorganism may be E. coli. The genus Xanthomonas microorganism may include Xanthobacter autotrophicus. Gram-positive bacteria may belong to the genus Corynebacterium or the genus Bacillus.

[0039] The recombinant microorganism may have a genetic modification that increases the level (activity or protein level) of an enzyme that catalyzes a NADPH production reaction to increase an intracellular NADPH level by the reaction. The genetic modification can be amplification of an endogenous gene or introduction of an exogenous gene. The enzyme may be a protein belonging to EC 1.1.1.49. The enzyme may be glucose-6-phosphate dehydrogenase (G6PD or G6PDH). The recombinant microorganism may further include an exogenous gene encoding G6PDH.

[0040] Another aspect provides a composition including the recombinant P450 protein or the variant thereof, which is useful for removing a halogenated methane such as fluorinated methane represented by CH.sub.nF.sub.4-n (n is an integer of 0 to 3) in a sample. Unless otherwise specified, the recombinant P450 protein or the variant thereof is the same as described above.

[0041] With regard to the composition, fluorinated methane represented by CH.sub.nF.sub.4-n may be, CHF.sub.3, CH.sub.2F.sub.2, CH.sub.3F, or CF.sub.4. The term removing includes reducing of a concentration of fluorinated methane in the sample. The reducing includes complete removal.

[0042] With regard to the composition, the recombinant P450 protein or the variant thereof may be in a recombinant microorganism, or the composition can comprise a lysate thereof, or a water-soluble material fraction of the lysate. When in a recombinant microorganism, the bacterial cytochrome P450 or the variant thereof may be expressed from an exogenous gene.

[0043] The recombinant microorganism may be bacteria or fungi. The bacteria may be Gram-positive or Gram-negative bacteria. The Gram-negative bacteria may belong to the family Enterobacteriaceae. The Gram-negative bacteria may belong to the genus Escherichia, the genus Salmonella, the genus Xanthomonas, or the genus Pseudomonas. The genus Escherichia microorganism may be E. coli. The genus Xanthomonas microorganism may include Xanthobacter autotrophicus. Gram-positive bacteria may belong to the genus Corynebacterium or the genus Bacillus.

[0044] Removing fluorinated methane may include cleaving of CF bonds of fluorinated methane, converting of fluorinated methane into other materials, or reducing of the concentration of fluorinated methane in the sample by intracellular accumulation. The converting may be introducing of a hydrophilic group such as a hydroxyl group into fluorinated methane or introducing of a carbon-carbon double bond or a carbon-carbon triple bond thereto.

[0045] With regard to the composition, the sample may be in a liquid or gas state. The sample may be industrial waste water or waste gas.

[0046] Still another aspect provides a method of reducing a concentration of fluorinated methane in a sample; the method includes contacting the recombinant P450 protein or the variant thereof with the sample containing fluorinated methane represented by CH.sub.nF.sub.4-n (n is an integer of 0 to 3) or other halogenated methane to reduce the concentration of halogenated methane in the sample. Unless otherwise specified, the recombinant P450 protein or the variant thereof is the same as described above.

[0047] Contacting of the recombinant P450 protein or the variant thereof with the sample may be performed in a sealed container. The contacting may be gas-liquid contact of contacting a gas sample with a liquid containing the recombinant P450 protein or the variant thereof. Further, the contacting may be liquid-liquid contact of contacting a liquid sample with a liquid containing the recombinant P450 protein or the variant thereof. The liquid-liquid contact includes mixing thereof.

[0048] With regard to the method, the recombinant P450 protein or the variant thereof may be in a recombinant microorganism that expresses bacterial cytochrome P450 protein, or a lysate thereof or the water-soluble material fraction of the lysate, or the recombinant P450 protein itself (e.g., isolated protein).

[0049] The contacting may be performed in the sealed container under conditions where the recombinant microorganism may survive or be viable. The conditions where the recombinant microorganism may survive or be viable may be conditions where the recombinant microorganism may be allowed to proliferate or to be in a resting state. In this case, the contacting may be culturing of the microorganism in the presence of fluorinated methane. The culturing may be performed under aerobic or anaerobic conditions.

[0050] The recombinant microorganism may be bacteria or fungi. The bacteria may be Gram-positive or Gram-negative bacteria. The Gram-negative bacteria may belong to the family Enterobacteriaceae. The Gram-negative bacteria may belong to the genus Escherichia, the genus Salmonella, the genus Xanthomonas, or the genus Pseudomonas. The genus Escherichia microorganism may be E. coli. The genus Xanthomonas microorganism may include Xanthobacter autotrophicus. Gram-positive bacteria may belong to the genus Corynebacterium or the genus Bacillus.

[0051] With regard to the method, the sample may be in a liquid or gas state. The sample may be industrial waste water or waste gas.

[0052] Still another aspect provides the variant of bacterial cytochrome P450 protein and a polynucleotide encoding the same.

[0053] The variant may be as described above. For instance, the variant may have an amino acid alteration at an amino acid residue corresponding to position F351 of an amino acid sequence of SEQ ID NO: 6, and may have an activity belonging to EC 1.14.15.1. The variant may have replacement of the amino acid residue at position F351 with a different amino acids, for example, any of the 19 natural amino acids, in camC of P450CAM having the amino acid sequence of SEQ ID NO: 6. The variant may be a F351Y, F351T, F351N, F351Q, F351H, or F351D mutant in camC of P450CAM having the amino acid sequence of SEQ ID NO: 6. In another aspect, the variant may have an amino acid alteration at an amino acid residue corresponding to position N320 of an amino acid sequence of SEQ ID NO: 8, and may have an activity belonging to EC 1.14.14.1. The variant may have replacement of the amino acid residue at the position N320 with other amino acids, for example, any of the other 19 natural amino acids in P450BM3 having the amino acid sequence of SEQ ID NO: 8. The variant be N320W, N320F, N320G, N320P, N320S, or N320E in P450BM3 having the amino acid sequence of SEQ ID NO: 8.

[0054] The polynucleotide encoding the variant can be codon optimized for use in various organisms. The polynucleotide encoding the variant may be included in the vector. The vector may be any vector, as long as it is used to introduce the polynucleotide into microorganisms. The vector may be a plasmid or viral vector. The polynucleotide may be operably linked to suitable regulatory sequences.

[0055] The recombinant microorganism according to an aspect may be used for removing fluorinated methane represented by CH.sub.nF.sub.4-n (or other halogenated methane) in the sample.

[0056] The variant of the recombinant P450 protein according to an aspect may be used for removing fluorinated methane in the sample.

[0057] The composition including the recombinant P450 protein or the variant thereof according to another aspect may be used for removing fluorinated methane in the sample.

[0058] The method of reducing the concentration of fluorinated methane in the sample according to still another aspect may efficiently reduce the concentration of fluorinated methane in the sample. For example, a headspace concentration of fluorinated methane can be reduced, by at least 3, 4, 5, 10, 15, or 20% when measured according to the protocol of any of the Examples below. The activity of the p450 variant can be a multiple of 1.5, 2, 2.5, 3, 3.5, 4, or greater, of the wild-type enzyme (in vitro or in an otherwise genetically identical strain).

[0059] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects.

[0060] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited by these Examples.

Example 1: Recombinant E. coli Expressing P450.SUB.CAM .Gene and Removal of Halomethane in Sample by Using the Same

[0061] In this Example, a recombinant E. coli expressing a P450.sub.CAM gene was prepared, and an effect of removing halomethane, i.e., CHF.sub.3, CF.sub.4, or CHCl.sub.3 in a sample by using the same was examined.

[0062] (1) Preparation of Recombinant E. coli Expressing P450.sub.CAM Gene

[0063] As P450.sub.CAM genes, camC, camA, and camB genes were amplified from CAM plasmid of Pseudomonas putida PpG786 strain, respectively. camC, camA, and camB genes have nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 1, and SEQ ID NO: 3, respectively. These genes encode amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 2, and SEQ ID NO: 4, respectively. In detail, P. putida PpG786 strain DSM 7162 was cultured in an LB medium at 30 C. under stirring at 230 rpm overnight, and then CAM plasmid was isolated using a total DNA extraction kit (Invitrogen Biotechnology). PCR was performed using the CAM plasmid as a template and a set of primers having nucleotide sequences of SEQ ID NOS: 11 and 12; a set of primers having nucleotide sequences of SEQ ID NOS: 13 and 14; and a set of primers having nucleotide sequences of SEQ ID NOS: 15 and 16 to amplify and obtain camA, camB, and camC genes, respectively.

[0064] The camC gene which was amplified by PCR using a set of primers of nucleotide sequences of SEQ ID NOS: 11 and 12 was ligated with pETDuet (Novagen, Cat. No. 71146-3), which was digested with restriction enzymes, NcoI and HindII, using an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pETDuet-camC vector. Further, the prepared pETDuet-camC vector was digested with restriction enzymes, NdeI and XhoI, and ligated with the amplified camA and the amplified camB gene fragment using the InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pETDuet-camC-camAB vector.

[0065] FIG. 1 shows a vector map of the pETDuet-camC-camAB vector.

[0066] Next, E. coli BL21 strain was introduced with the prepared pETDuet-camC-camAB vector by a heat shock method, and then cultured on a LB plate containing 100 g/mL of ampicillin. A strain showing ampicillin resistance was selected. Finally, the strain thus selected was designated as a recombinant E. coli BL21/pETDuet-camC-camAB.

[0067] (2) Effect of Removing CHF.sub.3 or CHCl.sub.3 in Sample by Recombinant E. coli Expressing P450.sub.CAM Gene

[0068] In this section, it was examined whether the P450.sub.CAM gene-introduced, E. coli BL21/pETDuet-camC-camAB strain prepared in section (1) affects removal of CHF.sub.3 or CHCl.sub.3 in a sample. In detail, E. coli BL21/pETDuet-camC-camAB was cultured in a TB medium at 30 C. under stirring at 230 rpm. At OD.sub.600 of about 0.5, 0.5 mM of IPTG was added thereto, followed by culturing at 25 C. and 230 rpm overnight. The cells were harvested and suspended in an M9 medium supplemented with 4 g/L to a cell density of OD.sub.600 of 2.5. 10 ml of this cell suspension was added to a 60 ml-serum bottle, and then the bottle was sealed. The terrific broth (TB) medium included 12 g of tryptone, 24 g of yeast extract, 5 g of glycerol, and 89 mM phosphate buffer per 1 L of distilled water. Further, the M9 medium included 6 g of Na.sub.2HPO.sub.4, 3 g of KH.sub.2PO.sub.4, 0.5 g of NaCl, and 1 g of NH.sub.4Cl per 1 L of distilled water.

[0069] Next, gas-phase CHF.sub.3 was injected through a rubber stopper of a cap of the serum bottle using a syringe to its headspace concentration of 200 ppm. Further, liquid-phase CHCl.sub.3 was injected through the rubber stopper of the cap of the serum bottle using the syringe to its concentration of 0.02 mM in the medium. Thereafter, the serum bottle was incubated for 18 hrs to 152 hrs, while stirring at 30 C. and 200 rpm. Each experiment was performed in triplicate.

[0070] At a predetermined time interval during incubation, 0.5 ml of the headspace gas containing no medium in the serum bottle was collected using a 1.0 ml-headspace syringe and injected into GC (Agilent 7890, Palo Alto, Calif., USA). The injected CHF.sub.3 or CHCl.sub.3 was separated through a CP-PoraBOND Q column (25 m length, 0.32 mm i.d., 5 um film thickness, Agilent), and changes in the CHF.sub.3 or CHCl.sub.3 concentration were analyzed by mass spectrometry (Agilent 5973, Palo Alto, Calif., USA). As a carrier gas, helium was used, and applied to the column at a flow rate of 1.5 ml/min. GC conditions were as follows: An inlet temperature was 250 C., an initial temperature was maintained at 40 C. for 2 minutes, and temperature was raised to 290 C. at a rate of 20 C./min. MS conditions were as follows: Ionization energy was 70 eV, an interface temperature was 280 C., an ion source temperature was 230 C., and a quadrupole temperature was 150 C.

[0071] FIG. 2 shows changes in a headspace concentration of CHF.sub.3 over time when E. coli BL21/pETDuet-camC-camAB was cultured in the medium contacted with CHF.sub.3-containing gas.

[0072] FIG. 3A shows changes in a headspace concentration of CHCl.sub.3 over time when E. coli BL21/pETDuet-camC-camAB was cultured in a CHCl.sub.3-containing medium. In FIGS. 2, 3A and 3B, NC represents a negative control group, and CAM represents an experiment performed by using E. coli BL21/pETDuet-camC-camAB. As shown in FIG. 2, when the E. coli BL21/pETDuet-camC-camAB was cultured for 62 hours and 152 hours, the headspace concentration of CHF.sub.3 was decreased by about 5.6% and about 17.3%, respectively, compared to the control group. Further, as shown in FIG. 3A, when the E. coli BL21/pETDuet-camC-camAB was cultured for 18 hours, the headspace concentration of CHCl.sub.3 was decreased by about 14.8%, compared to the control group.

[0073] (3) Effect of Removing CF.sub.4 in Sample by Recombinant E. coli Expressing P450.sub.CAM Gene

[0074] In this section, it was examined whether the P450.sub.CAM gene-introduced, E. coli BL21/pETDuet-camC-camAB strain prepared in section (1) affects removal of CF.sub.4 in a sample.

[0075] The experiment was performed in the same manner as the procedure performed for CHF.sub.3 in Section (2), except that CF.sub.4 was used instead of CHF.sub.3 and gas-phase CF.sub.4 was injected through a rubber stopper of a cap of the serum bottle using a syringe to its headspace concentration of 1000 ppm, and then the serum bottle was incubated for 7 days, while stirring at 30 C. and 200 rpm. The results are as shown in FIG. 3B.

[0076] FIG. 3B shows changes in a headspace concentration of CF.sub.4 over time when E. coli BL21/pETDuet-camC-camAB was cultured in a medium contacted with CF.sub.4-containing gas. As shown in FIG. 3B, when the E. coli BL21/pETDuet-camC-camAB was cultured for 7 days, the headspace concentration of CF.sub.4 was decreased by about 3.57%, compared to the control group.

[0077] (4) Recombinant E. coli Expressing Mutant P450.sub.CAM Gene and Effect of Removing CF.sub.4 in Sample Thereby

[0078] In this section, mutants were prepared in order to improve the activity of removing fluorinated methane in a sample by P450.sub.CAM. Phenylalanine (hereinafter, referred to as F351) at position 351 of the amino acid sequence of SEQ ID NO: 6 was replaced by other 19 natural amino acids (hereinafter, referred to as F351X. Here, X represents 19 natural amino acids other than phenylalanine), and each of the genes encoding the mutants was introduced into E. coli, and their activity of removing CF.sub.4 in a sample was examined. camC corresponds to heme domain and F351 is one of conserved amino acids in the amino acid sequences of camC derived from many different species.

[0079] (4.1) Preparation of 19 Mutants

[0080] Preparation of the F351X mutants of SEQ ID NO: 6 was performed using a QuikChange II Site-Directed Mutagenesis Kit (Agilent Technology, USA). Site-directed mutagenesis using the kit was performed using PfuUltra high-fidelity (HF) DNA polymerase for mutagenic primer-directed replication of both plasmid strands with the highest fidelity. The basic procedure utilized a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two synthetic oligonucleotide primers, both containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, were extended during temperature cycling by PfuUltra HF DNA polymerase, without primer displacement. Extension of the oligonucleotide primers generated a mutated plasmid containing staggered nicks. Following temperature cycling, the product was treated with Dpn I. The Dpn I endonuclease (target sequence: 5-Gm.sup.6ATC-3) was specific for methylated and hemimethylated DNA and was used to digest the parental DNA template and to select for mutation-containing synthesized DNA. The nicked vector DNA incorporating the desired mutations was then transformed into XL1-Blue supercompetent cells. The sequence identifiers for the primer sets used to produce the mutations are given in the following Table 6.

TABLE-US-00005 TABLE 6 Mutation NO. type Primer sequence 1 F351Y SEQ ID NOS: 21 and 22 2 F351T SEQ ID NOS: 23 and 24 3 F351N SEQ ID NOS: 25 and 26 4 F351Q SEQ ID NOS: 27 and 28 5 F351H SEQ ID NOS: 29 and 30 6 F351D SEQ ID NOS: 31 and 32

[0081] In detail, PCR was performed using the pETDuet-camC-camAB vector prepared in (1) as a template and each of the primer sets described in Table 6 as a primer and PfuUlta HF DNA polymerase to obtain mutated vectors. These vector products were treated with DpnI to select mutation-containing synthesized DNAs. The vector DNA incorporating the desired mutations was then transformed into XL1-Blue supercompetent cells to clone a pETDuet-camCmt-camAB vector.

[0082] Lastly, the cloned pETDuet-camCmt-camAB vector and pETDuet-camCwt-camAB vector were introduced into E. coli BL21 strain in the same manner as in (1), and a finally selected strain was designated as recombinant E. coli BL21/pETDuet-camCmt-camAB.

[0083] (4.2) Effect of Removing CF.sub.4 in Sample by Recombinant E. coli BL21/pETDuet-camCmt-camAB

[0084] In this section, it was examined whether the mutant camC gene-introduced, E. coli BL21/pETDuet-camCmt-camAB prepared in section (4.1) affects removal of CF.sub.4 in a sample.

[0085] The experiment was performed in the same manner as the procedure performed for CHF.sub.3 in Section (2), except that CF.sub.4 was used instead of CHF.sub.3 and gas-phase CF.sub.4 was injected through a rubber stopper of a cap of the serum bottle using a syringe to a headspace concentration of 1000 ppm, and then the serum bottle was incubated for 6 days, while stirring at 30 C. and 230 rpm. The results are as shown in Table 7.

TABLE-US-00006 TABLE 7 Residual amount of CF.sub.4 Reduction rate of CF.sub.4 Mutation (Percentage relative to (Percentage relative to NO. type control group) control group) 1 F351Y 91.82 8.18 2 F351T 95.42 4.58 3 F351N 92.56 7.44 4 F351Q 94.12 5.88 5 F351H 89.85 10.15 6 F351D 94.31 5.69 7 F351* 96.43 3.57

[0086] In Table 7, the control group represents E. coli introduced with the pETDuet vector instead of the pETDuet-camCmt-camAB vector, and F351* represents wild-type camC.

[0087] Further, in this section, the experiment was performed in the same manner as the procedure performed for CHF.sub.3 in Section (2), except that 20 mL of mutant camC-introduced E. coli BL21/pETDuet-camCmt-camAB (OD.sub.600=3.0) prepared in Section (4.1) was injected to a 175-mL flask, CF.sub.4 was used instead of CHF.sub.3, and gas-phase CF.sub.4 was injected through a rubber stopper of a cap of the serum bottle using a syringe to a headspace concentration of 1000 ppm, and then the serum bottle was incubated for 6 days, while stirring at 30 C. and 230 rpm. A residual amount of CF.sub.4 over time, that is, a remaining percentage (%) of CF.sub.4 was examined. The results are shown in FIG. 3C.

[0088] FIG. 3C shows changes of CF.sub.4 in a sample over time by E. coli BL21/pETDuet-camCmt-camAB introduced with the mutant camC gene. As shown in FIG. 3C, when the recombinant E. coli P450CAM strain, namely, F351N or F351H mutant gene-containing strain was cultured for 6 days, the CF.sub.4 level was further decreased by about 7.02% or about 8.92%, compared to the control group. In contrast, the wild-type strain further decreased the CF.sub.4 level by about 3.14%, compared to the control group.

Example 2: Recombinant E. coli Expressing P450.SUB.BM3 .Gene and Removal of Halomethane in Sample by Using the Same

[0089] In this Example, a recombinant E. coli expressing a P450.sub.BM3 gene was prepared, and an effect of removing halomethane, i.e., CHF.sub.3, CF.sub.4, or CHCl.sub.3 in a sample by using the same was examined.

[0090] (1) Preparation of Recombinant E. coli Expressing P450BM3 Gene

[0091] P450.sub.BM3 gene of Bacillus megaterium (ATCC 14581) strain was amplified. P450.sub.BM3 gene has a nucleotide sequence of SEQ ID NO: 7, and encodes an amino acid sequence of SEQ ID NO: 8. In detail, B. megaterium (ATCC 14581) was cultured in an LB medium at 30 C. under stirring at 230 rpm overnight, and then a genomic DNA was isolated using the total DNA extraction kit (Invitrogen Biotechnology). PCR was performed using this genomic DNA as a template and a set of primers having nucleotide sequences of SEQ ID NOS: 17 and 18 to amplify and obtain the P450.sub.BM3 gene. The P450.sub.BM3 gene thus amplified was ligated with pET28a (Novagen, Cat. No. 69864-3), which was digested with restriction enzymes, NcoI and XhoI, using the InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pET28a-P450.sub.BM3 vector. FIG. 4 shows a vector map of the pET28a-P450.sub.BM3 vector.

[0092] Further, in order to increase an intracellular NADPH level, a zwf gene encoding glucose 6-phosphate dehydrogenase of E. coli K12 (MG1655) was amplified. The Zwf gene has a nucleotide sequence of SEQ ID NO: 9, and encodes an amino acid sequence of SEQ ID NO: 10. In detail, E. coli was cultured in an LB medium at 37 C. under stirring at 230 rpm overnight, and then a genomic DNA was isolated using the total DNA extraction kit (Invitrogen Biotechnology). PCR was performed using this genomic DNA as a template and a set of primers having nucleotide sequences of SEQ ID NOS: 19 and 20 to amplify and obtain the zwf gene. The zwf gene thus amplified was ligated with pACYCDuet (Novagen, Cat. No. 71147-3), which was digested with restriction enzymes, NcoI and SacI, using the InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pACYCDuet-zwf vector.

[0093] FIG. 5 shows a vector map of the pACYCDuet-zwf vector.

[0094] Next, E. coli BL21 strain was introduced with the prepared pET28a-P450.sub.BM3 vector by a heat shock method, and then cultured on a LB plate containing 50 g/mL of kanamycin. A strain showing kanamycin resistance was selected. Finally, the strain thus selected was designated as a recombinant E. coli BL21/pET28a-P450.sub.BM3.

[0095] Further, E. coli BL21 strain was introduced with the prepared pET28a-P450.sub.BM3 vector and pACYCDuet-zwf vector by a heat shock method, and then cultured on a LB plate containing 50 g/mL of kanamycin and 35 g/mL of chloramphenicol. A strain showing kanamycin resistance and chloramphenicol resistance was selected. Finally, the strain thus selected was designated as a recombinant E. coli BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf.

[0096] (2) Effect of Removing CHF.sub.3 or CHCl.sub.3 in Sample by Recombinant E. coli Expressing P450.sub.BM3 Gene

[0097] In this section, it was examined whether the P450.sub.BM3 gene-introduced, recombinant E. coli BL21/pET28a-P450BM3 strain or BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf strain prepared in section (1) affects removal of CHF.sub.3 or CHCl.sub.3 in a sample.

[0098] In detail, E. coli BL21/pET28a-P450.sub.BM3 or BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf strain was cultured in the TB medium at 30 C. under stirring at 230 rpm. At OD.sub.600 of about 0.5, 0.2 mM of IPTG was added thereto, followed by culturing at 25 C. and 230 rpm overnight. The cells were harvested and suspended in the M9 medium to a cell density of OD.sub.600 of 2.5. 10 ml of this cell suspension was added to a 60 ml-serum bottle, and then the bottle was sealed. The TB medium and the M9 medium are the same as those described in Example 1.

[0099] Next, gas-phase CHF.sub.3 was injected through a rubber stopper of a cap of the serum bottle using a syringe to a headspace concentration of 200 ppm. Further, liquid-phase CHCl.sub.3 was injected through the rubber stopper of the cap of the serum bottle using the syringe to its concentration of 0.02 mM in the medium. Thereafter, the serum bottle was incubated for 15 hrs to 142 hrs, while stirring at 30 C. and 230 rpm. Each experiment was performed in triplicate.

[0100] At a predetermined time interval during incubation, the headspace concentration of CHCl.sub.3 or CHCl.sub.3 in the serum bottle was analyzed under the same conditions as described in (2) of Example 2.

[0101] FIG. 6 shows changes in headspace concentration of CHF.sub.3 over time when E. coli BL21/pET28a-P450.sub.BM3 or BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf was cultured for 142 hours in a medium contacted with CHF.sub.3-containing gas.

[0102] FIG. 7 shows changes in headspace concentration of CHCl.sub.3 over time when E. coli BL21/pET28a-P450.sub.BM3 was cultured for 15 hours in a CHCl.sub.3-containing medium. In FIGS. 6, 7, and 8, NC represents a negative control group, BM3 represents an experiment performed by using E. coli BL21/pET28a-P450.sub.BM3, and BM3+Zwf represents an experiment performed by using E. coli BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf. As shown in FIG. 6, when the E. coli BL21/pET28a-P450.sub.BM3 and E. coli BL21/pET28a-P450.sub.BM3+pACYCDuet-zwf were cultured for 70 hours and 142 hours, the headspace concentration of CHF.sub.3 was decreased, compared to the control group, by about 3.93% and about 4.57% upon culturing for 70 hours and by about 4.15% and about 11.03% upon culturing for 142 hours, respectively. Further, as shown in FIG. 7, when they were cultured for 15 hours, the headspace concentration of CHCl.sub.3 was decreased by about 4.1%, compared to the control group.

[0103] (3) Effect of Removing CF.sub.4 in Sample by Recombinant E. coli Expressing P450.sub.BM3 Gene

[0104] In this section, it was examined whether the P450.sub.BM3 gene-introduced, E. coli BL21/pET28a-P450.sub.BM3 strain prepared in section (1) affects removal of CF.sub.4 in a sample.

[0105] The experiment was performed in the same manner as the procedure performed for CHF.sub.3 in Section (2), except that CF.sub.4 was used instead of CHF.sub.3 and gas-phase CF.sub.4 was injected through a rubber stopper of a cap of the serum bottle using a syringe to its headspace concentration of 1000 ppm, and then the serum bottle was incubated for 7 days, while stirring at 30 C. and 200 rpm. The results are as shown in FIG. 8.

[0106] FIG. 8 shows changes in a headspace concentration of CF.sub.4 over time when E. coli BL21/pET28a-P450.sub.BM3 was cultured for 7 days in a medium contacted with CF.sub.4-containing gas. As shown in FIG. 8, when the E. coli BL21/pET28a-P450.sub.BM3 was cultured for 7 days, the headspace concentration of CF.sub.4 was decreased by about 3.03%, compared to the control group.

[0107] (4) Recombinant E. coli Expressing Mutant P450.sub.BM3 Gene and Effect of Removing CF.sub.4 in Sample Thereby

[0108] In this section, mutants were prepared in order to improve the activity of removing fluorinated methane in a sample by P450.sub.BM3. Asparagine (hereinafter, referred to as N320) at position 320 of the amino acid sequence of SEQ ID NO: 8 was replaced by other 19 natural amino acids (hereinafter, referred to as N320X. Here, X represents 19 natural amino acids other than asparagine), and each of the genes encoding the mutants was introduced into E. coli, and their activity of removing CF.sub.4 in a sample was examined. N320 is included in the heme-containing P450 oxygenase domain, and N320 is one of conserved amino acids in the amino acid sequences of enzymes having the same function.

[0109] (4.1) Preparation of 19 Mutants

[0110] Preparation of the N320X mutants of SEQ ID NO: 8 was performed using a QuikChange II Site-Directed Mutagenesis Kit (Agilent Technology, USA). Mutagenesis using the kit was performed in the same manner as described above.

[0111] Of respective primer sets used to induce N320X mutation, primer sets regarding to the increased activity of removing fluorinated methane in a sample, compared to that of the wild-type E. coli, are given in the following Table 8.

TABLE-US-00007 TABLE 8 NO. Mutation type Primer sequence 1 N320W SEQ ID NOS: 33 and 34 2 N320F SEQ ID NOS: 35 and 36 3 N320G SEQ ID NOS: 37 and 38 4 N320P SEQ ID NOS: 39 and 40 5 N320S SEQ ID NOS: 41 and 42 6 N320E SEQ ID NOS: 43 and 44

[0112] In detail, PCR was performed using the pET28a-P450BM3 vector prepared in (1) as a template and each of the primer sets described in Table 8 as a primer and PfuUlta HF DNA polymerase to obtain mutated vectors. These vector products were treated with DpnI to select mutation-containing synthesized DNAs. The vector DNA incorporating the desired mutations was then transformed into XL1-Blue supercompetent cells to clone a pET28a-P450BM3mt vector.

[0113] Lastly, the cloned pET28a-P450.sub.BM3 vector and pET28a-P450BM3mt vector were introduced into E. coli BL21 strain in the same manner as in (1), and a finally selected strain was designated as recombinant E. coli BL21/pET28a-P450BM3mt.

[0114] (4.2) Effect of Removing CF.sub.4 in Sample by Recombinant E. coli BL21/pET28a-P450BM3mt

[0115] In this section, it was examined whether the mutant P450BM3mt-introduced, E. coli BL21/pET28a-P450BM3mt prepared in section (4.1) affects removal of CF.sub.4 in a sample.

[0116] The experiment was performed in the same manner as the procedure performed for CHF.sub.3 in Section (2), except that CF.sub.4 was used instead of CHF.sub.3 and gas-phase CF.sub.4 was injected through a rubber stopper of a cap of the serum bottle using a syringe to a headspace concentration of 1000 ppm, and then the serum bottle was incubated for 6 days, while stirring at 30 C. and 230 rpm. The results are as shown in Table 9.

TABLE-US-00008 TABLE 9 Residual amount of CF.sub.4 Reduction rate of CF.sub.4 Mutation (Percentage relative to (Percentage relative to NO. type control group) control group) 1 N320W 94.42 5.58 2 N320F 87.38 12.62 3 N320G 89.82 10.18 4 N320P 86.89 13.11 5 N320S 82.03 17.97 6 N320E 88.48 11.52 7 N320* 96.97 3.03

[0117] In Table 9, the control group represents E. coli introduced with the pET28a vector instead of the pET28a-P450BM3mt vector, and N320* represents wild-type P450BM3.

[0118] Further, in this section, the experiment was performed in the same manner as the procedure performed for CHF.sub.3 in Section (2), except that 100 mL of mutant P450BM3-introduced E. coli BL21/pET28a-P450BM3mt (OD.sub.600=3.0) prepared in Section (4.1) was injected to a 250-mL flask, CF.sub.4 was used instead of CHF.sub.3, and gas-phase CF.sub.4 was injected through a rubber stopper of a cap of the serum bottle using a syringe to its headspace concentration of 1000 ppm, and then the serum bottle was incubated for 48 hours, while stirring at 30 C. and 230 rpm. Culturing was performed in the same manner as for CHF.sub.3, and a residual amount of CF.sub.4 over time, that is, a remaining percentage (%) of CF.sub.4 was examined. The results are shown in FIG. 9.

[0119] FIG. 9 shows changes of CF.sub.4 in a sample over time by E. coli BL21/pET28a-P450BM3mt introduced with the mutant P450BM3 gene. As shown in FIG. 9, when the recombinant E. coli P450BM3 strain, namely, N320E mutant gene-containing strain was cultured for 48 hours, the CF.sub.4 level was further decreased by about 14.3%, compared to the control group. In contrast, the wild-type strain further decreased the CF.sub.4 level by about 5.5%, compared to the control group.

[0120] The use of the terms a and an and the and at least one and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term at least one followed by a list of one or more items (for example, at least one of A and B) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms comprising, having, including, and containing are to be construed as open-ended terms (i.e., meaning including, but not limited to,) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0121] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.