KR20180023734A - Bacterial cytochrome 450 protein variant and method for reducing concentration of fluorinated methane in sample - Google Patents

Abstract

The present invention provides a recombinant microorganism comprising a foreign gene encoding bacterial cytochrome P450 protein; a composition comprising recombinant P450 protein for removing CHnF4-n (n is an integer from 0 to 3) in a sample; and a method for reducing the concentration of CHnF4-n inside a sample.

Description

Bacterial cytochrome P450 protein variants and methods for reducing the concentration of fluorinated methane in a sample using the same,

A recombinant microorganism comprising a foreign gene encoding a bacterial cytochrome P450 protein, a composition comprising a recombinant P450 protein for use in removing fluoromethane represented by CHnF4-n (n is an integer from 0 to 3) in a sample, Lt; RTI ID = 0.0 > CHnF4-n. ≪ / RTI >

Emissions of greenhouse gases that accelerate global warming are one of the most serious environmental problems, and regulations on greenhouse gas emissions are being tightened to regulate and prevent them. Of these, fluorinated gas (F-gas) such as perfluorocarbons (PFCs), hydrofluorocarbons (HFCs), and sulfohexafluoride (SF6) has low absolute emissions but a long half- And it is reported to have a more serious effect. In the semiconductor and electronics industries, which are major sources of F-gas, the amount of F-gas generated is increasing more than the GHG emission quota, and thus the burden of purchasing greenhouse gas decomposition and emission rights is increasing every year.

However, existing F-gas decomposition uses pyrolysis or catalytic thermal oxidation process, but there are limitations such as limited decomposition rate, secondary harmful substance discharge, and high cost. In order to solve this problem, biological F-gas decomposition process using microbial biocatalyst is expected to overcome the limitation of conventional chemical decomposition process and to be able to treat more economical and environmentally friendly F-gas.

Cytochrome P450 (CYP) belongs to the superfamily of proteins, including the heme cofactor, and is a hemoprotein. Cytochrome P450 uses a variety of small and large molecules as substrates in the enzyme reaction. Cytochrome P450 is generally the terminal oxidase enzyme of the electron transfer chain, broadly categorized as a system containing P450.

Bacterial cytochrome P450 is often a soluble enzyme and is involved in a variety of metabolic processes. Some bacteria, such as E. coli, do not contain cytochrome P450. Cytochrome P450 (CYP101) from P. putida is a camphor-hydroxylation catalytic cycle that constitutes two electron transfer steps from a 2Fe-2S cluster-containing protein cofactor, putidaredoxin. hydroxylating catalytic cycle.

Cytochrome P450 BM3 (CYP102) from B. megaterium catalyzes NADPH-dependent hydroxylation of several long chain fatty acids at the omega-1 to omega-3 positions. Cytochrome P450 BM3 is composed of a natural fusion protein between the CYP domain and an electron donating cofactor.

However, it is not known whether the bacterial cytochrome P450 catalyzes the removal of fluorinated methane in the sample.

One aspect provides a recombinant microorganism comprising a foreign gene encoding a bacterial cytochrome P450 protein or a variant thereof.

Another aspect provides a composition comprising a recombinant P450 protein or variant thereof for use in removing fluoromethane represented by CHnF4-n (n is an integer from 0 to 3) in a sample.

Another aspect includes contacting the recombinant P450 protein or variant thereof with a fluoromethane-containing sample represented by CHnF4-n, where n is an integer from 0 to 3, to reduce the concentration of fluoromethane in the sample. Lt; RTI ID = 0.0 > CHnF4-n. ≪ / RTI >

Another aspect provides variants of the bacterial cytochrome P450 protein and polynucleotides encoding the same.

One aspect provides a recombinant microorganism comprising a foreign gene encoding a bacterial cytochrome P450 protein or a variant thereof.

In 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 _BM3 . P450Cam may be derived from Pseudomonas putida PpG786. P450 _BM3 may be derived from Bacillus megaterium (ATCC 14581). The cytochrome P450 _CAM protein can constitute a bacterial CYP101 system in the form of a complex of CamA, CamB, and CamC. CamA can be a FAD-containing reductase. CamA may be NADH or NADPH dependent. The CamA may belong to EC 1.18.1.5. CamB may be ferredoxin of the [2Fe2S] type. CamC may also be referred to as P450Cam (CYP101) or P450Cam, including cytochrome 450 and belonging to EC 1.14.15.1. CamA, CamB, and CamC may be those having the amino acid sequences of SEQ ID NOS: 2, 4, and 6, respectively. The genes encoding CamA, CamB, and CamC may be those having the nucleotide sequences of SEQ ID NOS: 1, 3, and 5, respectively. The mutant may have an amino acid alteration at the amino acid residue corresponding to the F351 position of the amino acid sequence of SEQ ID NO: 6 and have an activity belonging to EC 1.14.15.1. The amino acid modification may be such that the amino acid residue corresponding to the F351 position is replaced with another amino acid, for example, 19 natural amino acids. The mutant may be one obtained by substituting Y, T, N, Q, H, or D for the amino acid residue corresponding to the F351 position in SEQ ID NO: EC 1.14.15.1 represents an enzyme that catalyzes the reaction of (+) - camphor + reduced putidaradexine + O2 ⇔ (+) - exo-5-hydroxycamphor + oxidized putidaradoxine + H ₂ O . The mutant may be F351 substituted with another amino acid, for example, 19 natural amino acids in P450CAM having the amino acid sequence of SEQ ID NO: 6. The mutant may be one having an amino acid sequence of SEQ ID NO: 6 with a substitution of F351Y, F351T, F351N, F351Q, F351H, or F351D. The gene encoding the P450CAM mutant may be a gene encoding a mutant having F351Y, F351T, F351N, F351Q, F351H, or F351D substitution in P450CAM having the amino acid sequence of SEQ ID NO: 6. This gene may be one having the nucleotide sequence of SEQ ID NO: 51, 52, 53, 54, 55, or 56. The microorganism may further comprise a gene encoding CamA and a gene encoding CamB.

P450 _BM3 may be a polypeptide having the amino acid sequence of SEQ ID NO: 8. The gene encoding P450 _BM3 may be the one having the nucleotide sequence of SEQ ID NO: 7. The mutant may have an amino acid alteration at the amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: 8 and have an activity belonging to EC 1.14.14.1. The amino acid modification may be such that the amino acid residue corresponding to the N320 position is replaced with another amino acid, for example, 19 natural amino acids. The mutant may be one obtained by substituting the amino acid residue corresponding to the N320 position of SEQ ID NO: 8 with W, F, G, P, S, or E. The mutant may be one in which N320 is replaced with another amino acid, for example, 19 natural amino acids in P450BM3 having the amino acid sequence of SEQ ID NO: 8. The mutant may be one having N320W, N320F, N320G, N320P, N320S, or N320E substitution in P450BM3 with the amino acid sequence of SEQ ID NO: The gene encoding the P450BM3 mutant may be a gene encoding a mutant having N320W, N320F, N320G, N320P, N320S, or N320E substitution in P450BM3 having the amino acid sequence of SEQ ID NO: This gene may be one having the nucleotide sequence of SEQ ID NO: 45, 46, 47, 48, 49, or 50. "EC 1.14.14.1" may be one which catalyzes the following reaction.

RH + Reduced NADPH --- hemoprotein reductase + O ₂ = ROH + oxidized NADPH --- hemoprotein reductase + H ₂ O

The term "corresponding ", as used herein, refers to an art-acceptable protein, such as a BLAST pairwise alignment, or a well-known Lipman-Pearson Protein Alignment program, When a protein alignment program is used with the following alignment parameters to align the amino acid sequence (e.g., SEQ ID NO: 6 or SEQ ID NO: 8) of a standard protein with a protein of interest, The F351 position of SEQ ID NO: 6 or the N320 position of SEQ ID NO: 8). The database (DB) in which the standard sequence is stored may be the RefSeq non-redundant proteins of NCBI. The parameters used for alignment of the sequences may be as follows: Ktuple = 2, Gap Penalty = 4, and Gap length penalty = 12. In this case, the range included in the "corresponding" sequence may be within the range of E-value 0.00001 and H-value 0.001.

Examples of proteins having an amino acid residue corresponding to the F351 position of the amino acid sequence of SEQ ID NO: 6 (hereinafter referred to as "homolog of P450CAM ") obtained according to the above alignment conditions are shown in Tables 1, 2 and 3 Respectively.

Examples of proteins having an amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: 8 (hereinafter referred to as "homologue of P450BM3 ") obtained according to the above alignment conditions are shown in Tables 4 and 5 .

number NCBI ID number NCBI ID number NCBI ID One gi | 163930960 31 gi | 551338874 61 gi | 728824802 2 gi | 612182735 32 gi | 730289226 62 gi | 746238551 3 gi | 497125935 33 gi | 654615031 63 gi | 494955160 4 gi | 310942843 34 gi | 656116930 64 gi | 496104589 5 gi | 657832383 35 gi | 826046029 65 gi | 567412687 6 gi | 498088271 36 gi | 737567226 66 gi | 98976470 7 gi | 544829275 37 gi | 98976439 67 gi | 358240604 8 gi | 861974080 38 gi | 612108073 68 gi | 494898237 9 gi | 738620841 39 gi | 515116019 69 gi | 504740033 10 gi | 499763441 40 gi | 817101463 70 gi | 648547795 11 gi | 746290673 41 gi | 398136480 71 gi | 808659667 12 gi | 503614840 42 gi | 757698965 72 gi | 551292036 13 gi | 861969570 43 gi | 126194726 73 gi | 551292470 14 gi | 662139213 44 gi | 737644672 74 gi | 502616812 15 gi | 544827262 45 gi | 496199226 75 gi | 545316934 16 gi | 498088269 46 gi | 654614522 76 gi | 491842667 17 gi | 861974085 47 gi | 759387698 77 gi | 750353906 18 gi | 737512009 48 gi | 739341634 78 gi | 858007594 19 gi | 817101596 49 gi | 783098869 79 gi | 665981585 20 gi | 494439068 50 gi | 783093808 80 gi | 806833869 21 gi | 496309894 51 gi | 783094059 81 gi | 504197805 22 gi | 746289514 52 gi | 759427060 82 gi | 564970689 23 gi | 753796069 53 gi | 861968800 83 gi | 499924508 24 gi | 545454562 54 gi | 497509581 84 gi | 765348796 25 gi | 648417306 55 gi | 497810537 85 gi | 497424876 26 gi | 666681698 56 gi | 783098865 86 gi | 739367531 27 gi | 567402060 57 gi | 739341585 87 gi | 517897868 28 gi | 826044703 58 gi | 783097592 88 gi | 702839727 29 gi | 826049125 59 gi | 783099978 89 gi | 739883834 30 gi | 806905723 60 gi | 783097681 90 gi | 750419370

number NCBI ID number NCBI ID number NCBI ID 91 gi | 494019567 121 gi | 783096369 151 gi | 518973859 92 gi | 498089540 122 gi | 530258733 152 gi | 808102361 93 gi | 826041014 123 gi | 657922982 153 gi | 664078266 94 gi | 566044904 124 gi | 654614517 154 gi | 663221595 95 gi | 566044935 125 gi | 739616950 155 gi | 663326563 96 gi | 501296495 126 gi | 783098022 156 gi | 493424288 97 gi | 517247251 127 gi | 806908467 157 gi | 502993954 98 gi | 516019877 128 gi | 21467173 158 gi | 518949456 99 gi | 288913356 129 gi | 401808868 159 gi | 750417303 100 gi | 512734904 130 gi | 514397583 160 gi | 750543392 101 gi | 783100677 131 gi | 749201634 161 gi | 377530614 102 gi | 764994003 132 gi | 528182079 162 gi | 377532462 103 gi | 517247290 133 gi | 647795133 163 gi | 737965741 104 gi | 652940833 134 gi | 651636595 164 gi | 739538229 105 gi | 169821418 135 gi | 661269250 165 gi | 739543125 106 gi | 835531786 136 gi | 695263348 166 gi | 750407524 107 gi | 551362264 137 gi | 696511194 167 gi | 739645497 108 gi | 764960072 138 gi | 493217416 168 gi | 493919745 109 gi | 759387686 139 gi | 493377553 169 gi | 737792710 110 gi | 737512189 140 gi | 563565380 170 gi | 750519055 111 gi | 820802680 141 gi | 665834124 171 gi | 521297725 112 gi | 646534628 142 gi | 665888884 172 gi | 482632482 113 gi | 820802864 143 gi | 665827329 173 gi | 494797766 114 gi | 334103741 144 gi | 751294725 174 gi | 493993588 115 gi | 826041595 145 gi | 827106327 175 gi | 648280499 116 gi | 746241621 146 gi | 639165413 176 gi | 750519223 117 gi | 764993875 147 gi | 652899356 177 gi | 498814706 118 gi | 748599849 148 gi | 652914977 178 gi | 519015945 119 gi | 764997807 149 gi | 652694819 179 gi | 639007804 120 gi | 662354752 150 gi | 496153669 180 gi | 518767974

number NCBI ID number NCBI ID number NCBI ID 181 gi | 739625293 213 gi | 652908779 245 gi | 499912932 182 gi | 820802866 214 gi | 503298839 246 gi | 657825087 183 gi | 739651753 215 gi | 740869740 247 gi | 655586613 184 gi | 502742128 216 gi | 503612867 248 gi | 739190742 185 gi | 515118033 217 gi | 646519758 249 gi | 518714103 186 gi | 820802677 218 gi | 494981163 250 gi | 503189844 187 gi | 391860290 219 gi | 490214493 251 gi | 739186131 188 gi | 737643185 220 gi | 736859678 252 gi | 739186149 189 gi | 544823238 221 gi | 739577671 253 gi | 516607102 190 gi | 763095543 222 gi | 736886954 254 gi | 522116265 191 gi | 739611016 223 gi | 654534319 255 gi | 522150263 192 gi | 145322598 224 gi | 549129549 256 gi | 703225980 193 gi | 825391797 225 gi | 653383901 257 gi | 703223632 194 gi | 759685456 226 gi | 703388673 258 gi | 703223663 195 gi | 836723496 227 gi | 653777500 259 gi | 494300956 196 gi | 488703345 228 gi | 655968891 260 gi | 808659227 197 gi | 763384158 229 gi | 655882347 261 gi | 489969104 198 gi | 528059914 230 gi | 630947972 262 gi | 806822276 199 gi | 783097229 231 gi | 495218410 263 gi | 556618018 200 gi | 494017068 232 gi | 768967538 264 gi | 738609029 201 gi | 739663478 233 gi | 746229913 265 gi | 403646243 202 gi | 739620206 234 gi | 746230981 266 gi | 737785331 203 gi | 746237691 235 gi | 746236533 267 gi | 703226655 204 gi | 567412712 236 gi | 544823589 268 gi | 602519307 205 gi | 550925359 237 gi | 746239269 269 gi | 739367513 206 gi | 746344573 238 gi | 490319630 270 gi | 737980497 207 gi | 530255704 239 gi | 494981649 271 gi | 737981631 208 gi | 739669024 240 gi | 494957004 272 gi | 817101442 209 gi | 654478200 241 gi | 763090173 273 gi | 497809551 210 gi | 490753280 242 gi | 738613213 274 gi | 545453717 211 gi | 497922631 243 gi | 746229737 275 gi | 497809089 212 gi | 740896970 244 gi | 754958228

number NCBI ID number NCBI ID number NCBI ID One gi | 515136080 30 gi | 542116840 59 gi | 518469404 2 gi | 757757972 31 gi | 764608412 60 gi | 655084756 3 gi | 822528663 32 gi | 518088806 61 gi | 764415731 4 gi | 544838284 33 gi | 768926886 62 gi | 491699287 5 gi | 491696887 34 gi | 498013687 63 gi | 518251998 6 gi | 655149838 35 gi | 498020927 64 gi | 493730772 7 gi | 512150124 36 gi | 498487619 65 gi | 817723893 8 gi | 493729782 37 gi | 530665825 66 gi | 228697407 9 gi | 738856821 38 gi | 753200845 67 gi | 228736549 10 gi | 655112080 39 gi | 495633284 68 gi | 692165489 11 gi | 648634781 40 gi | 748815403 69 gi | 489315595 12 gi | 522106669 41 gi | 738932691 70 gi | 498015014 13 gi | 504462655 42 gi | 738896417 71 gi | 749037577 14 gi | 783152040 43 gi | 652405427 72 gi | 763303489 15 gi | 759010788 44 gi | 764371274 73 gi | 830323790 16 gi | 545381104 45 gi | 701527930 74 gi | 857573616 17 gi | 548617766 46 gi | 751587021 75 gi | 654951198 18 gi | 648623486 47 gi | 736758744 76 gi | 647569946 19 gi | 738714376 48 gi | 657859536 77 gi | 738784028 20 gi | 639453808 49 gi | 657039097 78 gi | 515717624 21 gi | 497281073 50 gi | 852221735 79 gi | 517613324 22 gi | 494207912 51 gi | 850337075 80 gi | 507035289 23 gi | 843075790 52 gi | 550547409 81 gi | 661257874 24 gi | 518517905 53 gi | 495772021 82 gi | 655116131 25 gi | 655094715 54 gi | 504454491 83 gi | 736161405 26 gi | 517805393 55 gi | 737572351 84 gi | 493687687 27 gi | 518469404 56 gi | 654483633 85 gi | 806498422 28 gi | 655084756 57 gi | 495911896 86 gi | 532550849 29 gi | 764415731 58 gi | 737423431 87 gi | 757435944

number NCBI ID number NCBI ID number NCBI ID 88 gi | 491699287 117 gi | 737423433 145 gi | 737448097 89 gi | 518251998 118 gi | 515136080 146 gi | 542116840 90 gi | 493730772 119 gi | 757757972 147 gi | 764608412 91 gi | 817723893 120 gi | 822528663 148 gi | 518088806 92 gi | 228697407 121 gi | 544838284 149 gi | 768926886 93 gi | 228736549 122 gi | 491696887 150 gi | 498013687 94 gi | 692165489 123 gi | 655149838 151 gi | 498020927 95 gi | 489315595 124 gi | 512150124 152 gi | 498487619 96 gi | 498015014 125 gi | 493729782 153 gi | 530665825 97 gi | 749037577 126 gi | 738856821 154 gi | 753200845 98 gi | 763303489 127 gi | 655112080 155 gi | 495633284 99 gi | 830323790 128 gi | 648634781 156 gi | 748815403 100 gi | 857573616 129 gi | 522106669 157 gi | 738932691 101 gi | 654951198 130 gi | 504462655 158 gi | 738896417 102 gi | 647569946 131 gi | 783152040 159 gi | 652405427 103 gi | 738784028 132 gi | 759010788 160 gi | 764371274 104 gi | 515717624 133 gi | 545381104 161 gi | 701527930 105 gi | 517613324 134 gi | 548617766 162 gi | 751587021 106 gi | 507035289 135 gi | 648623486 163 gi | 736758744 107 gi | 661257874 136 gi | 738714376 164 gi | 657859536 108 gi | 655116131 137 gi | 639453808 165 gi | 657039097 109 gi | 736161405 138 gi | 497281073 166 gi | 852221735 110 gi | 493687687 139 gi | 494207912 167 gi | 850337075 111 gi | 806498422 140 gi | 843075790 168 gi | 550547409 112 gi | 532550849 141 gi | 518517905 169 gi | 495772021 113 gi | 757435944 142 gi | 655094715 170 gi | 504454491 114 gi | 737448097 143 gi | 517805393 171 gi | 737572351 115 gi | 654483633 144 gi | 495911896 172 gi | 737423431 116 gi | 737423433

The recombinant microorganism may be bacteria or fungi. The bacteria may be gram-positive or gram-negative bacteria. The gram negative bacterium may belong to the Enterobacteriaceae family. The gram negative bacterium may be one belonging to the genus Escherichia, the genus Samonella, the genus Xanthomonas or the genus Pseudomonas. The Escherichia genus microorganism may be E. coli. The microorganism in Zanthomobacter may be Zantho bacteriototrophicus. Gram-positive bacteria may be of the genus Corynebacterium, or of the genus Bacillus.

The recombinant microorganism may be an enzyme that catalyzes the reaction to produce NADPH, and has a genetic modification that increases the level of the enzyme that increases the level of NADPH in the cell by the reaction. The genetic modification includes amplification of an endogenous gene and introduction of a foreign gene. The enzyme may be a protein belonging to EC 1.1.1.49. The enzyme may be a glucose-6-phosphate dehydrogenase (G6PD or G6PDH). The recombinant microorganism may further comprise a foreign gene encoding G6PDH.

Another aspect provides a composition comprising a recombinant P450 protein or variant thereof for use in removing fluoromethane represented by CHnF4-n (n is an integer from 0 to 3) in a sample. Unless otherwise stated, the recombinant P450 protein or its variants are as described above.

In the above compositions, fluoro-n represented by CHnF4 methane, for example, CHF _3, CH ₂ F _2, may be CH ₃ F, or CF _4. The term "removal" includes reducing the concentration of fluoromethane in the sample. The reduction includes removing completely.

In such a composition, the recombinant P450 protein or variant thereof may be in the form of a recombinant microorganism, a lysate thereof, a water-soluble substance fraction of the lysate thereof, or a recombinant P450 protein, which contains the expressed bacterial cytochrome P450 protein. The bacterial cytochrome P450 or variant thereof may be one expressed from a foreign gene.

The recombinant microorganism may be bacteria or fungi. The bacteria may be gram-positive or gram-negative bacteria. The gram negative bacterium may belong to the Enterobacteriaceae family. The gram negative bacterium may be one belonging to the genus Escherichia, the genus Samonella, the genus Xanthomonas or the genus Pseudomonas. The Escherichia genus microorganism may be E. coli. The microorganism in Zanthomobacter may be Zantho bacteriototrophicus. Gram-positive bacteria may be of the genus Corynebacterium, or of the genus Bacillus.

In such a composition, the removal may include cutting the binding of C-F of fluoromethane, converting fluoromethane to another material, or accumulating it in the cell to reduce the concentration of fluoromethane. The conversion may be by introducing a hydrophilic group such as a hydroxyl group into the fluorinated methane, or introducing a carbon-carbon double bond or a carbon-carbon triple bond.

In the composition, the sample may be in a liquid or gaseous state. The sample may be factory waste water or waste.

Another aspect includes contacting the recombinant P450 protein or variant thereof with a fluoromethane-containing sample represented by CHnF4-n, where n is an integer from 0 to 3, to reduce the concentration of fluoromethane in the sample. Lt; RTI ID = 0.0 > fluoromethane. ≪ / RTI > Unless otherwise stated, the recombinant P450 protein or its variants are as described above.

In the above method, the contact may be performed in an airtight container. The contacting may be a gas-liquid contact contacting the gaseous sample with a liquid containing the recombinant P450 protein or variant thereof. In addition, the contacting may be a liquid-liquid contact contacting the liquid sample with a liquid containing the recombinant P450 protein or its variants. The liquid-liquid contact includes mixing.

In this method, the recombinant P450 protein or a variant thereof may be in the form of a recombinant microorganism, a lysate thereof, a water-soluble substance fraction of the lysate thereof, or a recombinant P450 protein, which contains the expressed bacterial cytochrome P450 protein.

In the above method, the contacting may be carried out under conditions in which the recombinant microorganism can survive in the closed container. The viable condition may be a condition allowing the recombinant microorganism to be in a proliferative state or a resting state. In this case, the contacting may be to culture the microorganism in the presence of fluoromethane. The culture may be carried out under aerobic or anaerobic conditions.

The recombinant microorganism may be bacteria or fungi. The bacteria may be gram-positive or gram-negative bacteria. The gram negative bacterium may belong to the Enterobacteriaceae family. The gram negative bacterium may be one belonging to the genus Escherichia, the genus Samonella, the genus Xanthomonas or the genus Pseudomonas. The Escherichia genus microorganism may be E. coli. The microorganism in Zanthomobacter may be Zantho bacteriototrophicus. Gram-positive bacteria may be of the genus Corynebacterium, or of the genus Bacillus.

In the above method, the sample may be in a liquid or gaseous state. The sample may be factory waste water or waste.

Another aspect provides variants of the bacterial cytochrome P450 protein and polynucleotides encoding the same.

The mutant may have an amino acid alteration at the amino acid residue corresponding to the F351 position of the amino acid sequence of SEQ ID NO: 6 and have an activity belonging to EC 1.14.15.1. The mutant may be F351 substituted with another amino acid, for example, 19 natural amino acids, in the camC of P450CAM having the amino acid sequence of SEQ ID NO: 6. The mutant may be one having a F351Y, F351T, F351N, F351Q, F351H, or F351D substitution in the camC of P450CAM having the amino acid sequence of SEQ ID NO: 6. The variant may have an alteration in the amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: 8 and belong to EC 1.14.14.1. The mutant may be one in which N320 is replaced with another amino acid, for example, 19 natural amino acids in P450BM3 having the amino acid sequence of SEQ ID NO: 8. The mutant may be one having N320W, N320F, N320G, N320P, N320S, or N320E substitution in P450BM3 with the amino acid sequence of SEQ ID NO:

The polynucleotide encoding the mutant may be contained in a vector. Such vectors include any that can be used to introduce polynucleotides into microorganisms. The vector may be a plasmid or a viral vector.

Recombinant microorganisms according to one aspect can be used to remove fluoromethane represented by CHnF4-n in the sample.

Variants of the recombinant P450 protein according to one embodiment can be used to remove fluoromethane in the sample.

A composition comprising a recombinant P450 protein or variant thereof according to another aspect can be used to remove fluoromethane in the sample.

A method of reducing the concentration of fluoromethane in a sample according to another aspect may effectively reduce the concentration of fluoromethane in the sample.

Figure 1 shows a vector map of the pETDuet-camC-camAB vector.
Fig. 2 shows changes with time in CHF _{3 in} the head space when Escherichia coli BL21 / pETDuet-camC-camAB was cultured in a medium in contact with CHF ₃ -containing gas.
Figure 3a is when a culture of E. coli BL21 / pETDuet-camC-camAB from ₃ CHCl containing medium, shows a change with time of the CHCl ₃ in the head space.
FIG. 3B shows the change with time of CF _{4 in} the head space when Escherichia coli BL21 / pETDuet-camC-camAB was cultured in a medium in contact with CF ₄ -containing gas.
FIG. 3C shows the degree of CF ₄ in the sample with time of the E. coli BL21 / pETDuet-camCmt-camAB into which the mutant camC gene was introduced.
FIG. 4 is a graph showing the effect of the pET28a-P450 _BM3 Represents a vector map of a vector.
Figure 5 shows the pACYCDuet-zwf Represents a vector map of a vector.
6 shows a change with time of the CHF ₃ containing case of culturing the solution from recombinant E. coli BL21 / pET28a-P450 _BM3 or recombinant E. coli BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf contact with the gas, the headspace of the CHF ₃ .
7 is a case of culturing Escherichia coli BL21 / pET28a-P450 _BM3 in CHCl ₃ containing solution, it shows the variation with time of the CHCl ₃ in the head space.
8 is CF _4, if contained in the medium in contact with a base in the E. coli BL21 / pET28a-P450 _BM3 strains were cultured for 7 days, it shows a change with time of the CF ₄ in the head space.
FIG. 9 shows the degree of CF ₄ in the sample with time of the E. coli BL21 / pET28a-P450BM3mt into which the mutant P450BM3 gene was introduced.

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 limited to these examples.

Example  One: P450 _CAM  Removal of halomethane in a recombinant E. coli expressing a gene and a sample using the recombinant E. coli

In this Example, a recombinant Escherichia coli expressing P450 _CAM gene was prepared, and the removal effect of halomethane, i.e., CHF ₃ , CF ₄ or CHCl ₃ , in the sample was confirmed using the recombinant E. coli.

(One) P450 _CAM  Production of recombinant Escherichia coli expressing the gene

The P450 _CAM gene amplified the camC, camA, and camB genes from the CAM plasmid of Pseudomonas putida PpG786. The camC, camA, and camB genes have the nucleotides of SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 3, respectively, and these genes encode the amino acid sequences SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 4, respectively. Specifically, the P. putida PpG786 strain DSM 7162 was cultured in LB medium at 30 ° C with stirring at 230 rpm overnight, and the CAM plasmid was isolated by a total DNA extraction kit (Invitrogen Biotechnology) , A primer set of the nucleotide sequences of SEQ ID NOS: 11 and 12, using this CAM plasmid as a template; A primer set of the nucleotide sequences of SEQ ID NOS: 13 and 14; And a primer set of the nucleotide sequences of SEQ ID NOS: 15 and 16, respectively, to obtain amplified camA, camB, and camC genes, respectively.

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

Figure 1 shows a vector map of the pETDuet-camC-camAB vector.

Next, the prepared pETDuet-camC-camAB vector was introduced into a strain of Escherichia coli BL21 by heat shock method and cultured in an LB plate medium containing 100 μg / mL of ampicillin to select an ampicillin resistant strain . Finally, the selected strains were named as recombinant E. coli BL21 / pETDuet-camC-camAB.

(2) P450 _CAM  In a sample by recombinant E. coli expressing the gene CHF ₃  Or CHCl ₃  Removal effect

In this section, the effect of the E. coli BL21 / pETDuet-camC-camAB strain introduced with the P450 _CAM gene prepared in (1) on the removal of CHF ₃ or CHCl ₃ in the sample was confirmed. Specifically, Escherichia coli BL21 / pETDuet-camC-camAB was cultured in TB medium at 30 ° C with stirring at 230 rpm, 0.5 mM IPTG was added at OD ₆₀₀ = 0.5, and the mixture was incubated overnight at 25 ° C with stirring at 230 rpm. The cells were harvested and suspended in M9 medium supplemented with 4 g / L glucose to a cell concentration of OD ₆₀₀ = 2.5. 10 ml of this cell solution was added to a 60 ml serum bottle and sealed. The TB (Terrific broth) medium contained 12 g of tryptone per liter of distilled water, 24 g of yeast extract, 5 g of glycerol, and 89 mM of phosphate buffer. In addition, the M9 medium contained components of 6 g of Na ₂ HPO ₄ , ₃ g of KH ₂ PO ₄ , 0.5 g of NaCl, and 1 g of NH ₄ Cl per liter of distilled water.

Next, CHF ₃ in the gaseous phase was injected through a rubber stopper of the cap of the sera bottle using a syringe so as to be 200 ppm relative to the head space. In addition, CHCl ₃ in the liquid phase was injected through the elastic material of the cap of the serum bottle using a syringe to 0.02 mM in the medium. The serum bottle was then incubated for 18 to 152 hours at 30 DEG C with stirring at 200 rpm. The experiment was conducted in triplicate.

0.5 ml of the gas in the headspace containing no medium in the sera was collected in a 1.0 ml headspace syringe and injected into GC (Agilent 7890, Palo Alto, CA, USA) at regular intervals during the incubation. The injected CHF ₃ or CHCl ₃ was isolated via a CP-PoraBOND Q column (25 m length, 0.32 mm id, 5 μm film thickness, Agilent) and mass spectrometry (Agilent 5973, Palo Alto, over CHF _3, or CHCl ₃ The concentration changes were analyzed. The carrier gas was helium and flowed to the column at a rate of 1.5 ml / min. Gas chromatography (GC) conditions were maintained at an inlet temperature of 250 ° C and an initial temperature of 40 ° C for 2 minutes, and the temperature was increased to 290 ° C at 20 ° C / min. Mass spectrometry conditions were 70eV ionization energy, interface temperature 280 ℃, ion source temperature 230 ℃, and quadrupole temperature 150 ℃.

Fig. 2 shows changes with time in CHF _{3 in} the head space when Escherichia coli BL21 / pETDuet-camC-camAB was cultured in a medium in contact with CHF ₃ -containing gas.

Figure 3a is when a culture of E. coli BL21 / pETDuet-camC-camAB from ₃ CHCl containing medium, shows a change with time of the CHCl ₃ in the head space. In FIGS. 2, 3A and 3B, NC represents a negative control, and 'CAM' represents an experiment using E. coli BL21 / pETDuet-camC-camAB. As shown in FIG. 2, when cultured for 62 hours and 152 hours, the concentration of CHF _{3 in} the head space was decreased by 5.6% and 17.3%, respectively, as compared with the control. In addition, as shown in FIG. 3A, when cultured for 18 hours, the concentration of CHCl _{3 in} the head space was 14.8% lower than that of the control group.

(3) P450 _CAM  In a sample of recombinant E. coli expressing the gene CF ₄  Removal effect

In this section (1) section is the P450 _CAM Gene E. coli BL21 / pETDuet-camC-camAB strain produced in a confirmed the effect of the removal of CF ₄ in the sample.

The experiment was carried out in the same manner as the procedure performed for CHF ₃ in the section (2), except that CF ₄ was used instead of CHF ₃ , and the elasticity of cap of the serum bottle was measured using a syringe so that CF ₄ on the gas phase was 1000 ppm relative to the head space And injected through the material. Thereafter, the sera were incubated for 7 days while stirring at 30 DEG C at 200 rpm. The result is as shown in Fig. 3B.

FIG. 3B shows the change with time of CF _{4 in} the head space when Escherichia coli BL21 / pETDuet-camC-camAB was cultured in a medium in contact with CF ₄ -containing gas. As it is shown in Figure 3b, if the culture for 7 days the concentration of CF ₄ in the headspace than in the control group decreased by 3.57%.

(4) P450 _CAM  The recombinant E. coli expressing the mutant gene and its sample CF ₄  Removal effect

In this section, mutants were prepared to improve the fluoromethane removal activity of P450 _CAM samples. (Hereinafter also referred to as " F351X ") in which the phenylalanine (hereinafter also referred to as "F351") at position 351 in the amino acid sequence of SEQ ID NO: 6 is replaced with another 19 natural amino acid It represents.), which was confirmed CF ₄ removal activity of the samples of the E. coli obtained by introducing a gene encoding a mutant obtained in E. coli. camC corresponds to the heme domain and F351 is one of the conserved amino acids of the amino acid sequence of camC from various species.

(4.1) Preparation of 19 mutants

The F354X mutant of SEQ ID NO: 6 was prepared using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technology, USA). The position-directed mutagenesis method using the kit described above can be used for PfuUlta high-fidelity PCR for mutagenic primer-directed replication of two plasmid strands with the highest fidelity. HF) DNA polymerase. The basic procedure utilizes two synthetic oligonucleotide primers, including a supercoiled double stranded DNA (dsDNA) vector with the desired insert and both of the desired mutations. The oligonucleotide primers, each complementary to the opposite strand of the vector, are extended during temperature cycling by the PfuUlta HF DNA polymerase, without primer displacement. Due to the extension of the oligonucleotide primers, a mutated plasmid containing a staggered nick is generated. After the temperature cycling, the product is treated with DpnI . DpnI endonuclease (target sequence: 5'-Gm ⁶ ATC-3 ') is specific for methylated and hemimethylated DNA and is used to digest parental DNA templates to select mutant-containing synthesized DNA. The nicked vector DNA containing the desired mutation is then transduced into XL1-Blue supercompetent cells. The increase in the activity of removing fluoromethane compared to wild-type E. coli among the primer sets used for F351X mutagenesis is shown in Table 6 below.

number Mutant form Primer sequence One 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

Specifically, a mutated vector was obtained by PCR using the pETDuet-camC-camAB vector prepared in (1) as a template and PCR using PfuUlta HF DNA polymerase with each of the primer sets described in Table 6 as a primer. This vector product was treated with DpnI to select mutant-containing synthesized DNA. Then vector DNA containing the desired mutation obtained was transformed into XL1-Blue supercompetent cells to clone the pETDuet-camCmt-camAB vector.

Finally, the cloned pETDuet-camCmt-camAB vector and the pETDuet-camCwt-camAB vector were introduced into E. coli BL21 strain through the same procedure as described in (1), and the finally selected strain was introduced into recombinant E. coli BL21 / pETDuet-camCmt-camAB Respectively.

(4.2) Recombinant E. coli BL21 / pETDuet - camCmt - CAMAB Of the sample CF ₄  Removal effect

In this section (4.1) the mutated camC introduced E. coli BL21 / pETDuet-camCmt-camAB gene produced in the section was confirmed the effect of the removal of CF ₄ in the sample.

The experiment was carried out in the same manner as the procedure performed for CHF ₃ in the section (2), except that CF ₄ was used instead of CHF ₃ , and the elasticity of cap of the serum bottle was measured using a syringe so that CF ₄ on the gas phase was 1000 ppm relative to the head space And injected through the material. Thereafter, the sera were incubated for 6 days at 30 DEG C with stirring at 230 rpm. The results are shown in Table 7.

number Mutant form CF4 residual ratio (percentage of control) CF4 reduction rate (percentage relative to control) One 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

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

In this section, 20 mL (OD ₆₀₀ = 3.0) of Escherichia coli BL21 / pETDuet-camCmt-camAB with the mutant camC gene prepared in Section 4.1 was injected into a 175 mL flask, CF ₄ was used instead of CHF ₃ , and the experiment was the same culture and the process performed for the CHF ₃ (2) section, except that the incubation on the CF ₄ for injection so that 1000ppm headspace compared and, while stirring the serum sickness in 30 ℃ to 230rpm 6 il The CF4 residual rate, CF4 remaining percentage, was determined by time. The results are shown in Fig. 3c.

FIG. 3C shows the degree of CF ₄ in the sample with time of the E. coli BL21 / pETDuet-camCmt-camAB into which the mutant camC gene was introduced. As shown in Figure 3c, when cultured 6 days, sikyeoteuna recombinant E. coli P450CAM strain i.e., F351N and the strain containing F351H mutant gene further reduce the level of the control group compared to CF ₄ 7.02% and 8.92% wild-type strain is CF control group compared to ₄ level by 3.14%.

Example  2: P450 _BM3  Removal of halomethane in a recombinant E. coli expressing a gene and a sample using the recombinant E. coli

In this Example, a recombinant Escherichia coli expressing P450 _BM3 gene was prepared, and the removal effect of halomethane, i.e., CHF ₃ , CF ₄ or CHCl ₃ , in the sample was confirmed using the recombinant E. coli.

(One) P450 _BM3  Production of recombinant Escherichia coli expressing the gene

P450 _BM3 gene was amplified P450 _BM3 gene of Bacillus megaterium (ATCC 14581) strain. The P450 _BM3 gene encodes the amino acid sequence of SEQ ID NO: 8, respectively, having the nucleotide sequence of SEQ ID NO: 7. Specifically, B. megaterium (ATCC 14581) was cultured in LB medium at 30 ° C with stirring at 230 rpm overnight. Then, genomic DNA was isolated using a total DNA extraction kit (Invitrogen Biotechnology) PCR was performed using the genomic DNA as a template and the nucleotide sequences of SEQ ID NOS: 17 and 18 as a primer set to obtain P450 _BM3 gene. The amplified P450 _BM3 gene was ligated with pET28a (Novagen, Cat. No. 69864-3) cleaved with restriction enzymes NcoI and XhoI through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to produce pET28a-P450 _BM3 vector Respectively. Figure 4 shows a vector map of pET28a-P450 _BM3 vector.

In addition, zwf gene encoding glucose 6-phosphate dehydrogenase of E. coli K12 (MG1655) was amplified to increase intracellular NADPH content. The Zwf gene has the nucleotide sequence of SEQ ID NO: 9 and encodes the amino acid sequence of SEQ ID NO: 10. Specifically, E. coli was cultured in LB medium at 37 ° C with stirring at 230 rpm overnight. Then, genomic DNA was isolated using a total DNA extraction kit (Invitrogen Biotechnology), and the genomic DNA PCR was carried out using the nucleotide sequences of SEQ ID NOs: 19 and 20 as a template and the zwf gene was amplified. The amplified zwf gene was ligated to the pACYCDuet (Novagen, Cat. No. 71147-3) cut with restriction enzymes NcoI and SacI through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to produce the pACYCDuet-zwf vector.

Figure 5 shows the pACYCDuet-zwf Represents a vector map of a vector.

Next, the produced pET28a-P450 _BM3 vector was introduced into Escherichia coli BL21 by a heat shock method and cultured in an LB plate medium containing 50 μg / mL kanamycin, to select strains showing kanamycin resistance . The finally selected strains were named recombinant E. coli BL21 / pET28a-P450 _BM3 .

The prepared pET28a-P450 _BM3 vector and pACYCDuet-zwf vector were introduced into Escherichia coli strain BL21 by heat shock method and cultured in LB plate medium containing 50 μg / ml kanamycin and 35 μg / ml chloramphenicol to obtain kanamycin And chloramphenicol resistant strains were selected. The finally selected strains were named recombinant E. coli BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf

(2) P450 _BM3  In a sample of recombinant E. coli expressing the gene CHF ₃  or CHCl3 ₃  Removal effect

In this section, the effect of recombinant Escherichia coli BL21 / pET28a-P450 _BM3 strain or BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf strain introduced with P450 _BM3 gene prepared in section (1) on the removal of CHF ₃ or CHCl ₃ Respectively.

Specifically, Escherichia coli BL21 / pET28a-P450 _BM3 Or BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf strain was cultured in TB medium at 30 ° C with stirring at 230 rpm, 0.2mM IPTG was added at OD ₆₀₀ = 0.5, and then cultured overnight at 25 ° C with stirring at 230 rpm. The cells were harvested and suspended in M9 medium to a cell concentration OD ₆₀₀ = 2.5. 10 ml of this cell solution was added to a 60 ml serum bottle and sealed. The TB medium and the M9 medium are as described in Example 1. [

Next, CHF ₃ in the gaseous phase was injected through a rubber stopper of the cap of the sera bottle using a syringe so as to be 200 ppm relative to the head space. In addition, CHCl ₃ in the liquid phase was injected through the elastic material of the cap of the serum bottle using a syringe to 0.02 mM in the medium. The serum bottle was then incubated for 15 to 142 hours at 30 DEG C with stirring at 230 rpm. The experiment was conducted in triplicate.

CHF ₃ or CHCl ₃ in the headspace of the sera was analyzed at the same time intervals during the culture in the same manner as described in Example 2 (2).

FIG. 6 shows a change with time in CHF _{3 in} the headspace when E. coli BL21 / pET28a-P450 _BM3 or BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf strains were cultured for 142 hours in medium contacted with CHF ₃ -containing gas .

7 is a case of 15 hour incubation of E. coli BL21 / pET28a-P450 _BM3 from ₃ CHCl containing medium, shows the change in CHCl ₃ in the head space. 6, 7 and 8, NC represents an experiment using negative control, 'BM3' represents an experiment using Escherichia coli BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf for Escherichia coli BL21 / pET28a-P450 _BM3 and 'BM3 + Zwf'. As shown in FIG. 6, when cultured for 70 hours and 142 hours, the concentration of CHF _{3 in} the headspace was higher than that of the control group for Escherichia coli BL21 / pET28a-P450 _BM3 and Escherichia coli BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf , 3.93% and 4.57% for 70 hour culture, and 4.15% and 11.03% for 142 hour culture, respectively. In addition, as shown in Figure 7, when cultured for 15 hours, the concentration of the CHCl ₃ in the headspace than in the control group was decreased by 4.1%.

(3) P450 _BM3  In a sample of recombinant E. coli expressing the gene CF ₄  Removal effect

In this section, the effect of recombinant E. coli BL21 / pET28a-P450 _BM3 strain introduced with P450 _BM3 gene prepared in (1) on the removal of CF ₄ in the sample was confirmed.

The experiment was carried out in the same manner as the procedure performed for CHF ₃ in the section (2), except that CF ₄ was used instead of CHF ₃ , and the elasticity of cap of the serum bottle was measured using a syringe so that CF ₄ on the gas phase was 1000 ppm relative to the head space , And then the serum cell was cultured for 7 days with stirring at 30 DEG C and 200 rpm. The results are shown in Fig.

8 is CF _4, if contained in the medium in contact with a base in the E. coli BL21 / pET28a-P450 _BM3 strains were cultured for 7 days, it shows a change with time of the CF ₄ in the head space. 8, when cultured for 7 days, the concentration of CF ₄ in the headspace than in the control group decreased relative to the E. coli _{BL21 / pET28a-P450 BM3, 3.03} %.

(4) P450 _BM3  The recombinant E. coli expressing the mutant gene and its sample CF ₄  Removal effect

In this section, mutants were prepared to improve the fluoromethane removal activity of P450 _BM3 samples. Asparagine (hereinafter also referred to as " N320 ") at position 320 in the amino acid sequence of SEQ ID NO: 8 is replaced with another 19 natural amino acids (hereinafter also referred to as" N320X ", where X is at least 19 natural amino acids other than asparagine) It represents.), which was confirmed CF ₄ removal activity of the samples of the E. coli obtained by introducing a gene encoding a mutant obtained in E. coli. N320 is contained in the heme domain P450 oxidase domain and N320 is one of the conserved amino acids of the amino acid sequence of the enzyme having the same function.

(4.1) Preparation of 19 mutants

The N320X mutant of SEQ ID NO: 8 was prepared using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technology, USA). Mutagenesis using the above Kit is as described above.

The increase in the activity of removing fluoromethane compared to the wild-type E. coli in the sample among the respective primer sets used for N320X mutagenesis is shown in Table 8 below.

number Mutant form Primer sequence One 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

Specifically, the mutated vector was obtained by PCR using the pET28a-P450BM3 vector prepared in (1) as a template and each primer set described in Table 8 as a primer and using PfuUlta HF DNA polymerase. This vector product was treated with DpnI to select mutant-containing synthesized DNA. Then vector DNA containing the desired mutation obtained was transformed into XL1-Blue supercompetent cells to clone the pET28a-P450BM3mt vector.

Finally, the cloned pET28a-P450BM3 vector and the pET28a-P450BM3mt vector were introduced into E. coli BL21 strain through the same procedure as described in (1), and the finally selected strain was named recombinant E. coli BL21 / pET28a-P450BM3mt.

(4.2) Recombinant E. coli BL21 / pET28a - P450BM3mt Of the sample CF ₄  Removal effect

In this section, the effect of E. coli BL21 / pET28a-P450BM3mt with the mutant P450BM3mt gene prepared in (4.1) on the removal of CF ₄ in the sample was confirmed.

The experiment was carried out in the same manner as the procedure performed for CHF ₃ in the section (2), except that CF ₄ was used instead of CHF ₃ , and the elasticity of cap of the serum bottle was measured using a syringe so that CF ₄ on the gas phase was 1000 ppm relative to the head space And injected through the material. Thereafter, the sera were incubated for 6 days at 30 DEG C with stirring at 230 rpm. The results are shown in Table 9.

number Mutant form CF4 residual ratio (percentage of control) CF4 reduction rate (percentage relative to control) One 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

In Table 9, the control group is E. coli in which the pET28a vector is introduced in place of the pET28a-P450BM3mt vector, and N320 * represents wild-type P450BM3.

In this section, 100 mL (OD ₆₀₀ = 15.0) of Escherichia coli BL21 / pET28a-P450BM3mt with the mutant P450BM3 gene prepared in Section 4.1 was injected into a 250 mL flask, CF ₄ was used instead of CHF ₃ , ₄ was injected at a concentration of 1000 ppm relative to the head space and the serum was incubated for 48 hours with stirring at 230 rpm at 30 ° C. The experiment was carried out in the same manner as in the procedure performed for CHF ₃ in (2) (CF4 remaining,%) of the remaining CF4 was determined. The results are shown in Fig.

FIG. 9 shows the degree of CF ₄ in the sample with time of the E. coli BL21 / pET28a-P450BM3mt into which the mutant P450BM3 gene was introduced. 9, the case of 48 hour incubation, recombinant E. coli P450BM3 strain that is, the strain containing the N320E mutation gene sikyeoteuna further reduce the level of the control group compared to CF ₄ 14.3% wild-type strain is the level of the control group compared to CF ₄ 5.5 %.

<110> Samsung Electronics Co., Ltd. <120> Bacterial cytochrome 450 protein and method for reducing          concentration of fluorinated methane in sample <130> PN114290KR <160> 56 <170> Kopatentin 2.0 <210> 1 <211> 1269 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 1 atgaacgcaa acgacaacgt ggtcatcgtc ggtaccggac tggctggcgt tgaggtcgcc 60 ttcggcctgc gcgccagcgg ctgggaaggc aatatccggt tggtggggga tgcgacggta 120 attccccatc acctaccacc gctatccaaa gcttacttgg ccggcaaagc cacagcggaa 180 agcctgtacc tgagaacccc agatgcctat gcagcgcaga acatccaact actcggaggc 240 acacaggtaa cggctatcaa ccgcgaccga cagcaagtaa tcctatcgga tggccgggca 300 ctggattacg accggctggt attggctacc ggagggcgtc caagacccct accggtggcc 360 agtggcgcag ttggaaaggc gaacaacttt cgatacctgc gcacactcga ggacgccgag 420 tgcattcgcc ggcagctgat tgcggataac cgtctggtgg tgattggtgg cggctacatt 480 ggccttgaag tggctgccac cgccatcaag gcgaacatgc acgtcaccct gcttgatacg 540 gcagcccggg ttctggagcg ggttaccgcc ccgccggtat cggcctttta cgagcaccta 600 caccgcgaag ccggcgttga catacgaacc ggcacgcagg tgtgcgggtt cgagatgtcg 660 accgaccaac agaaggttac tgccgtcctc tgcgaggacg gcacaaggct gccagcggat 720 ctggtaatcg ccgggattgg cctgatacca aactgcgagt tggccagtgc ggccggcctg 780 caggttgata acggcatcgt gatcaacgaa cacatgcaga cctctgatcc cttgatcatg 840 gccgtcggcg actgtgcccg atttcacagt cagctctatg accgctgggt gcgtatcgaa 900 tcggtgccca atgccttgga gcaggcacga aagatcgccg ccatcctctg tggcaaggtg 960 ccacgcgatg aggcggcgcc ctggttctgg tccgatcagt atgagatcgg attgaagatg 1020 gtcggactgt ccgaagggta cgaccggatc attgtccgcg gctctttggc gcaacccgac 1080 ttcagcgttt tctacctgca gggagaccgg gtattggcgg tcgatacagt gaaccgtcca 1140 gtggagttca accagtcaaa acaaataatc acggatcgtt tgccggttga accaaaccta 1200 ctcggtgacg aaagcgtgcc gttaaaggaa atcatcgccg ccgccaaagc tgaactgagt 1260 agtgcctaa 1269 <210> 2 <211> 422 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 2 Met Asn Ala Asn Asp Asn Val Val Ile Val Gly Thr Gly Leu Ala Gly   1 5 10 15 Val Glu Val Ala Phe Gly Leu Arg Ala Ser Gly Trp Glu Gly Asn Ile              20 25 30 Arg Leu Val Gly Asp Ala Thr Val Ile Pro His His Leu Pro Pro Leu          35 40 45 Ser Lys Ala Tyr Leu Ala Gly Lys Ala Thr Ala Glu Ser Leu Tyr Leu      50 55 60 Arg Thr Pro Asp Ala Tyr Ala Ala Gln Asn Ile Gln Leu Leu Gly Gly  65 70 75 80 Thr Gln Val Thr Ala Ile Asn Arg Asp Arg Gln Gln Val Ile Leu Ser                  85 90 95 Asp Gly Arg Ala Leu Asp Tyr Asp Arg Leu Val Leu Ala Thr Gly Gly             100 105 110 Arg Pro Pro Leu Pro Val Ala Ser Gly Ala Val Gly Lys Ala Asn         115 120 125 Asn Phe Arg Tyr Leu Arg Thr Leu Glu Asp Ala Glu Cys Ile Arg Arg     130 135 140 Gln Leu Ile Ala Asp Asn Arg Leu Val Val Ile Gly Gly Gly Tyr Ile 145 150 155 160 Gly Leu Glu Val Ala Ala Thr Ala Ile Lys Ala Asn Met His Val Thr                 165 170 175 Leu Leu Asp Thr Ala Ala Arg Val Leu Glu Arg Val Thr Ala Pro Pro             180 185 190 Val Ser Ala Phe Tyr Glu His Leu His Arg Glu Ala Gly Val Asp Ile         195 200 205 Arg Thr Gly Thr Gln Val Cys Gly Phe Glu Met Ser Thr Asp Gln Gln     210 215 220 Lys Val Thr Ala Val Leu Cys Glu Asp Gly Thr Arg Leu Pro Ala Asp 225 230 235 240 Leu Val Ile Ala Gly Ile Gly Leu Ile Pro Asn Cys Glu Leu Ala Ser                 245 250 255 Ala Ala Gly Leu Glu Val Asp Asn Gly Ile Val Ile Asn Glu His Met             260 265 270 Gln Thr Ser Asp Pro Leu Ile Met Ala Val Gly Asp Cys Ala Arg Phe         275 280 285 His Ser Gln Leu Tyr Asp Arg Trp Val Arg Ile Glu Ser Val Pro Asn     290 295 300 Ala Leu Glu Gln Ala Arg Lys Ile Ala Ala Ile Leu Cys Gly Lys Val 305 310 315 320 Pro Arg Asp Glu Ala Ala Pro Trp Phe Trp Ser Asp Gln Tyr Glu Ile                 325 330 335 Gly Leu Lys Met Val Gly Leu Ser Glu Gly Tyr Asp Arg Ile Ile Val             340 345 350 Arg Gly Ser Leu Ala Gln Pro Asp Phe Ser Val Phe Tyr Leu Gln Gly         355 360 365 Asp Arg Val Leu Ala Val Asp Thr Val Asn Arg Pro Val Glu Phe Asn     370 375 380 Gln Ser Lys Gln Ile Ile Thr Asp Arg Leu Pro Val Glu Pro Asn Leu 385 390 395 400 Leu Gly Asp Glu Ser Val Pro Leu Lys Glu Ile Ile Ala Ala Ala Lys                 405 410 415 Ala Glu Leu Ser Ser Ala             420 <210> 3 <211> 324 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 3 atgtctaaag tagtgtatgt gtcacatgat ggaacgcgtc gcgaactgga tgtggcggat 60 ggcgtcagcc tgatgcaggc tgcagtctcc aatggtatct acgatattgt cggtgattgt 120 ggcggcagcg ccagctgtgc cacctgccat gtctatgtga acgaagcgtt cacggacaag 180 gtgcccgccg ccaacgagcg ggaaatcggc atgctggagt gcgtcacggc cgaactgaag 240 ccgaacagca ggctctgctg ccagatcatc atgacgcccg agctggatgg catcgtggtc 300 gatgttcccg ataggcaatg gtaa 324 <210> 4 <211> 107 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 4 Met Ser Lys Val Val Tyr Val Ser His Asp Gly Thr Arg Arg Glu Leu   1 5 10 15 Asp Val Ala Asp Gly Val Ser Leu Met Gln Ala Ala Val Ser Asn Gly              20 25 30 Ile Tyr Asp Ile Val Gly Asp Cys Gly Gly Ser Ala Ser Cys Ala Thr          35 40 45 Cys His Val Tyr Val Asn Glu Ala Phe Thr Asp Lys Val Pro Ala Ala      50 55 60 Asn Glu Arg Glu Ile Gly Met Leu Glu Cys Val Thr Ala Glu Leu Lys  65 70 75 80 Pro Asn Ser Arg Leu Cys Cys Gln Ile Ile Met Thr Pro Glu Leu Asp                  85 90 95 Gly Ile Val Val Asp Val Pro Asp Arg Gln Trp             100 105 <210> 5 <211> 1248 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 5 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc tttggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 6 <211> 415 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 6 Met Thr Thr Glu Thr Ile Gln Ser Asn Ala Asn Leu Ala Pro Leu Pro   1 5 10 15 Pro His Val Pro Glu His Leu Val Phe Asp Phe Asp Met Tyr Asn Pro              20 25 30 Ser Asn Leu Ser Ala Gly Val Glu Glu Ala Trp Ala Val Leu Gln Glu          35 40 45 Ser Asn Val Pro Asp Leu Val Trp Thr Arg Cys Asn Gly Gly His Trp      50 55 60 Ile Ala Thr Arg Gly Gln Leu Ile Arg Glu Ala Tyr Glu Asp Tyr Arg  65 70 75 80 His Phe Ser Ser Glu Cys Pro Phe Ile Pro Arg Glu Ala Gly Glu Ala                  85 90 95 Tyr Asp Phe Ile Pro Thr Ser Met Asp Pro Pro Glu Gln Arg Gln Phe             100 105 110 Arg Ala Leu Ala Asn Gln Val Val Gly Met Pro Val Val Asp Lys Leu         115 120 125 Glu Asn Arg Ile Gln Glu Leu Ala Cys Ser Leu Ile Glu Ser Leu Arg     130 135 140 Pro Gln Gly Gln Cys Asn Phe Thr Glu Asp Tyr Ala Glu Pro Phe Pro 145 150 155 160 Ile Arg Ile Phe Met Leu Leu Ala Gly Leu Pro Glu Glu Asp Ile Pro                 165 170 175 His Leu Lys Tyr Leu Thr Asp Gln Met Thr Arg Pro Asp Gly Ser Met             180 185 190 Thr Phe Ala Glu Ala Lys Glu Ala Leu Tyr Asp Tyr Leu Ile Pro Ile         195 200 205 Ile Glu Gln Arg Arg Gln Lys Pro Gly Thr Asp Ile Ser Ile Val     210 215 220 Ala Asn Gly Gln Val Asn Gly Arg Pro Ile Thr Ser Asp Glu Ala Lys 225 230 235 240 Arg Met Cys Gly Leu Leu Leu Val Gly Gly Leu Asp Thr Val Val Asn                 245 250 255 Phe Leu Ser Phe Ser Met Glu Phe Leu Ala Lys Ser Pro Glu His Arg             260 265 270 Gln Glu Leu Ile Glu Arg Pro Glu Arg Ile Pro Ala Ala Cys Glu Glu         275 280 285 Leu Leu Arg Arg Phe Ser Leu Val Ala Asp Gly Arg Ile Leu Thr Ser     290 295 300 Asp Tyr Glu Phe His Gly Val Gln Leu Lys Lys Gly Asp Gln Ile Leu 305 310 315 320 Leu Pro Gln Met Leu Ser Gly Leu Asp Glu Arg Glu Asn Ala Cys Pro                 325 330 335 Met His Val Asp Phe Ser Arg Gln Lys Val Ser His Thr Thr Phe Gly             340 345 350 His Gly Ser His Leu Cys Leu Gly Gln His Leu Ala Arg Arg Glu Ile         355 360 365 Ile Val Thr Leu Lys Glu Trp Leu Thr Arg Ile Pro Asp Phe Ser Ile     370 375 380 Ala Pro Gly Ala Gln Ile Gln His Lys Ser Gly Ile Val Ser Gly Val 385 390 395 400 Gln Ala Leu Pro Leu Val Trp Asp Pro Ala Thr Thr Lys Ala Val                 405 410 415 <210> 7 <211> 3150 <212> DNA <213> Bacillus megaterium (ATCC 14581) <400> 7 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaaac 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 8 <211> 1049 <212> PRT <213> Bacillus megaterium (ATCC 14581) <400> 8 Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys   1 5 10 15 Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys              20 25 30 Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg          35 40 45 Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp      50 55 60 Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg  65 70 75 80 Asp Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn                  85 90 95 Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala             100 105 110 Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val         115 120 125 Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu     130 135 140 Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn 145 150 155 160 Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr                 165 170 175 Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala             180 185 190 Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu         195 200 205 Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg     210 215 220 Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn 225 230 235 240 Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg                 245 250 255 Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly             260 265 270 Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu         275 280 285 Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro     290 295 300 Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn 305 310 315 320 Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala                 325 330 335 Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp             340 345 350 Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp         355 360 365 Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser     370 375 380 Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala 385 390 395 400 Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly                 405 410 415 Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu             420 425 430 Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys         435 440 445 Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Ser Ser Thr     450 455 460 Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn 465 470 475 480 Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly                 485 490 495 Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro             500 505 510 Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly         515 520 525 Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn     530 535 540 Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val 545 550 555 560 Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala                 565 570 575 Thr Thr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala             580 585 590 Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp         595 600 605 Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp     610 615 620 Val Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys 625 630 635 640 Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu                 645 650 655 Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Ala Ser Lys Glu             660 665 670 Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu         675 680 685 Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile     690 695 700 Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly 705 710 715 720 Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu                 725 730 735 Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln             740 745 750 Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met         755 760 765 Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu     770 775 780 Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr 785 790 795 800 Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser                 805 810 815 Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile             820 825 830 Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser         835 840 845 Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile     850 855 860 Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys 865 870 875 880 Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu                 885 890 895 Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg             900 905 910 Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu         915 920 925 Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr     930 935 940 Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr 945 950 955 960 Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val                 965 970 975 Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp             980 985 990 Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro         995 1000 1005 Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val    1010 1015 1020 Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly 1025 1030 1035 1040 Arg Tyr Ala Lys Asp Val Trp Ala Gly                1045 <210> 9 <211> 1476 <212> DNA <213> E. coli K12 (MG1655) <400> 9 atggcggtaa cgcaaacagc ccaggcctgt gacctggtca ttttcggcgc gaaaggcgac 60 cttgcgcgtc gtaaattgct gccttccctg tatcaactgg aaaaagccgg tcagctcaac 120 ccggacaccc ggattatcgg cgtagggcgt gctgactggg ataaagcggc atataccaaa 180 gttgtccgcg aggcgctcga aactttcatg aaagaaacca ttgatgaagg tttatgggac 240 accctgagtg cacgtctgga tttttgtaat ctcgatgtca atgacactgc tgcattcagc 300 cgtctcggcg cgatgctgga tcaaaaaaat cgtatcacca ttaactactt tgccatgccg 360 cccagcactt ttggcgcaat ttgcaaaggg cttggcgagg caaaactgaa tgctaaaccg 420 gcacgcgtag tcatggagaa accgctgggg acgtcgctgg cgacctcgca ggaaatcaat 480 gatcaggttg gcgaatactt cgaggagtgc caggtttacc gtatcgacca ctatcttggt 540 aaagaaacgg tgctgaacct gttggcgctg cgttttgcta actccctgtt tgtgaataac 600 tgggacaatc gcaccattga tcatgttgag attaccgtgg cagaagaagt ggggatcgaa 660 gggcgctggg gctattttga taaagccggt cagatgcgcg acatgatcca gaaccacctg 720 ctgcaaattc tttgcatgat tgcgatgtct ccgccgtctg acctgagcgc agacagcatc 780 cgcgatgaaa aagtgaaagt actgaagtct ctgcgccgca tcgaccgctc caacgtacgc 840 gaaaaaaccg tacgcgggca atatactgcg ggcttcgccc agggcaaaaa agtgccggga 900 tatctggaag aagagggcgc gaacaagagc agcaatacag aaactttcgt ggcgatccgc 960 gtcgacattg ataactggcg ctgggccggt gtgccattct acctgcgtac tggtaaacgt 1020 ctgccgacca aatgttctga agtcgtggtc tatttcaaaa cacctgaact gaatctgttt 1080 aaagaatcgt ggcaggatct gccgcagaat aaactgacta tccgtctgca acctgatgaa 1140 ggcgtggata tccaggtact gaataaagtt cctggccttg accacaaaca taacctgcaa 1200 atcaccaagc tggatctgag ctattcagaa acctttaatc agacgcatct ggcggatgcc 1260 tatgaacgtt tgctgctgga aaccatgcgt ggtattcagg cactgtttgt acgtcgcgac 1320 gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 1380 gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 1440 acccgtgatg gtcgttcctg gaatgagttt gagtaa 1476 <210> 10 <211> 491 <212> PRT <213> E. coli K12 (MG1655) <400> 10 Met Ala Val Thr Gln Thr Ala Gln Ala Cys Asp Leu Val Ile Phe Gly   1 5 10 15 Ala Lys Gly Asp Leu Ala Arg Arg Lys Leu Leu Pro Ser Leu Tyr Gln              20 25 30 Leu Glu Lys Ala Gly Gln Leu Asn Pro Asp Thr Arg Ile Ile Gly Val          35 40 45 Gly Arg Ala Asp Trp Asp Lys Ala Ala Tyr Thr Lys Val Val Arg Glu      50 55 60 Ala Leu Glu Thr Phe Met Lys Glu Thr Ile Asp Glu Gly Leu Trp Asp  65 70 75 80 Thr Leu Ser Ala Arg Leu Asp Phe Cys Asn Leu Asp Val Asn Asp Thr                  85 90 95 Ala Ala Phe Ser Arg Leu Gly Ala Met Leu Asp Gln Lys Asn Arg Ile             100 105 110 Thr Ile Asn Tyr Phe Ala Met Pro Pro Ser Thr Phe Gly Ala Ile Cys         115 120 125 Lys Gly Leu Gly Aly Lys Leu Asn Aly Lys Pro Ala Arg Val Val     130 135 140 Met Glu Lys Pro Leu Gly Thr Ser Leu Ala Thr Ser Gln Glu Ile Asn 145 150 155 160 Asp Gln Val Gly Glu Tyr Phe Glu Glu Cys Gln Val Tyr Arg Ile Asp                 165 170 175 His Tyr Leu Gly Lys Glu Thr Val Leu Asn Leu Leu Ala Leu Arg Phe             180 185 190 Ala Asn Ser Leu Phe Val Asn Asn Trp Asp Asn Arg Thr Ile Asp His         195 200 205 Val Glu Ile Thr Val Ala Glu Glu Val Gly Ile Glu Gly Arg Trp Gly     210 215 220 Tyr Phe Asp Lys Ala Gly Gln Met Arg Asp Met Ile Gln Asn His Leu 225 230 235 240 Leu Gln Ile Leu Cys Met Ile Ala Met Ser Pro Pro Ser Asp Leu Ser                 245 250 255 Ala Asp Ser Ile Arg Asp Glu Lys Val Lys Val Leu Lys Ser Leu Arg             260 265 270 Arg Ile Asp Arg Ser Asn Val Arg Glu Lys Thr Val Arg Gly Gln Tyr         275 280 285 Thr Ala Gly Phe Ala Gln Gly Lys Lys Val Pro Gly Tyr Leu Glu Glu     290 295 300 Glu Gly Ala Asn Lys Ser Ser Asn Thr Glu Thr Phe Val Ala Ile Arg 305 310 315 320 Val Asp Ile Asp Asn Trp Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg                 325 330 335 Thr Gly Lys Arg Leu Pro Thr Lys Cys Ser Glu Val Val Val Tyr Phe             340 345 350 Lys Thr Pro Glu Leu Asn Leu Phe Lys Glu Ser Trp Gln Asp Leu Pro         355 360 365 Gln Asn Lys Leu Thr Ile Arg Leu Gln Pro Asp Glu Gly Val Asp Ile     370 375 380 Gln Val Leu Asn Lys Val Pro Gly Leu Asp His Lys His Asn Leu Gln 385 390 395 400 Ile Thr Lys Leu Asp Leu Ser Tyr Ser Glu Thr Phe Asn Gln Thr His                 405 410 415 Leu Ala Asp Ala Tyr Glu Arg Leu Leu Leu Glu Thr Met Arg Gly Ile             420 425 430 Gln Ala Leu Phe Val Arg Arg Asp Glu Val Glu Glu Ala Trp Lys Trp         435 440 445 Val Asp Ser Ile Thr Glu Ala Trp Ala Met Asp Asn Asp Ala Pro Lys     450 455 460 Pro Tyr Gln Ala Gly Thr Trp Gly Pro Val Ala Ser Val Ala Met Ile 465 470 475 480 Thr Arg Asp Gly Arg Ser Trp Asn Glu Phe Glu                 485 490 <210> 11 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer: CAM_F <400> 11 taagaaggag atatacatat gaacgcaaac gacaacg 37 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer: camA_R <400> 12 catgaattct gtttcctgtg tgattaggca ctactcagtt ca 42 <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer: camB_F <400> 13 taatcacaca ggaaacagaa ttcatgtcta aagtagtgta tg 42 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: camB_R <400> 14 ggtttcttta ccagactcga ttaccattgc ctatcgggaa 40 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer: camC_F <400> 15 aagaaggaga tataccatga cgactgaaac cataca 36 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: camC_R <400> 16 gcattatgcg gccgcaagct ttataccgct ttggtagtcg 40 <210> 17 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer: P450bm3_F <400> 17 aagaaggaga tataccatga caattaaaga aatgcct 37 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: P450bm3_R <400> 18 gtggtggtgg tggtgctcga ttacccagcc cacacgtctt 40 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer: Zwf_F <400> 19 ttaagaagga gatataccat ggcggtaacg caaacagc 38 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer: Zwf_R <400> 20 tcgacctgca ggcgcgccgt tactcaaact cattccagg 39 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for F351Y <400> 21 aaggtttcac acaccaccta tggccacggc agccatctg 39 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for F351Y <400> 22 cagatggctg ccgtggccat aggtggtgtg tgaaacctt 39 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for F351T <400> 23 aaggtttcac acaccaccac cggccacggc agccatctg 39 <210> 24 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for F351T <400> 24 cagatggctg ccgtggccgg tggtggtgtg tgaaacctt 39 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for F351N <400> 25 aaggtttcac acaccaccaa cggccacggc agccatctg 39 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for F351N <400> 26 cagatggctg ccgtggccgt tggtggtgtg tgaaacctt 39 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for F351Q <400> 27 aaggtttcac acaccaccca gggccacggc agccatctg 39 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for F351Q <400> 28 cagatggctg ccgtggccct gggtggtgtg tgaaacctt 39 <210> 29 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for F351H <400> 29 aaggtttcac acaccaccca tggccacggc agccatctg 39 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for F351H <400> 30 cagatggctg ccgtggccat gggtggtgtg tgaaacctt 39 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for F351D <400> 31 aaggtttcac acaccaccga tggccacggc agccatctg 39 <210> 32 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for F351D <400> 32 cagatggctg ccgtggccat cggtggtgtg tgaaacctt 39 <210> 33 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N320W <400> 33 tatgtcggca tggtcttatg ggaagcgctg cgcttatgg 39 <210> 34 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N320W <400> 34 ccataagcgc agcgcttccc ataagaccat gccgacata 39 <210> 35 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N320F <400> 35 tatgtcggca tggtcttatt tgaagcgctg cgcttatgg 39 <210> 36 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N320F <400> 36 ccataagcgc agcgcttcaa ataagaccat gccgacata 39 <210> 37 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N320G <400> 37 tatgtcggca tggtcttagg cgaagcgctg cgcttatgg 39 <210> 38 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N320G <400> 38 ccataagcgc agcgcttcgc ctaagaccat gccgacata 39 <210> 39 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N320P <400> 39 tatgtcggca tggtcttacc ggaagcgctg cgcttatgg 39 <210> 40 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N320P <400> 40 ccataagcgc agcgcttccg gtaagaccat gccgacata 39 <210> 41 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N320S <400> 41 tatgtcggca tggtcttaag cgaagcgctg cgcttatgg 39 <210> 42 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N320S <400> 42 ccataagcgc agcgcttcgc ttaagaccat gccgacata 39 <210> 43 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for N320E <400> 43 tatgtcggca tggtcttaga agaagcgctg cgcttatgg 39 <210> 44 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for N320E <400> 44 ccataagcgc agcgcttctt ctaagaccat gccgacata 39 <210> 45 <211> 3150 <212> DNA <213> Artificial Sequence <220> <223> P450BM3 N320W gene <400> 45 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttatgg 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 46 <211> 3150 <212> DNA <213> Artificial Sequence <220> <223> P450BM3 N320F gene <400> 46 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttattt 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 47 <211> 3150 <212> DNA <213> Artificial Sequence <220> <223> P450BM3 N320G <400> 47 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaggc 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 48 <211> 3150 <212> DNA <213> Artificial Sequence <220> <223> P450BM3 N320P <400> 48 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaccg 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 49 <211> 3150 <212> DNA <213> Artificial Sequence <220> <223> P450BM3 N320S <400> 49 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaagc 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 50 <211> 3150 <212> DNA <213> Artificial Sequence <220> <223> P450BM3 N320E <400> 50 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttagaa 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 51 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> CamC F351Y <400> 51 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc tatggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 52 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> CamC F351T <400> 52 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc accggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 53 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> CamC F351N <400> 53 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc aacggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 54 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> CamC F351Q <400> 54 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc cagggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 55 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> CamC F351H <400> 55 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc catggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 56 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> CamC F351D <400> 56 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc gatggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248

Claims (19)

A recombinant microorganism comprising a foreign gene encoding a mutant of the bacterial cytochrome P450 protein, said mutant having an amino acid alteration in the amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: 8 and having an activity belonging to EC 1.14.14.1 Lt; / RTI &gt; The microorganism according to claim 1, wherein the amino acid residue is substituted with W, F, G, P, S, or E in the amino acid residue corresponding to the N320 position. The microorganism according to claim 1, wherein the amino acid modification is N320W, N320F, N320G, N320P, N320S, or N320E substitution. The microorganism according to claim 1, further comprising a foreign gene encoding a protein belonging to EC 1.1.1.49. A composition comprising a variant of a recombinant P450 protein for use in removing fluoromethane represented by CHnF4-n (wherein n is an integer from 0 to 3) in a sample, wherein said variant is at the N320 position of the amino acid sequence of SEQ ID NO: 8 Having an amino acid alteration at the corresponding amino acid residue and having an activity belonging to EC 1.14.14.1. The composition of claim 5, wherein the variant of the recombinant P450 protein is in the form of a recombinant microorganism comprising a variant of the bacterial cytochrome P450 protein expressed from a foreign gene. 6. The composition of claim 5, wherein the bacterial cytochrome P450 is expressed from an exogenous gene. The microorganism according to claim 5, wherein the amino acid residue is substituted with W, F, G, P, S, or E in the amino acid residue corresponding to the N320 position. The microorganism according to claim 5, wherein the amino acid modification is N320W, N320F, N320G, N320P, N320S, or N320E substitution. 6. The composition of claim 5, wherein the sample is in a liquid or gaseous state. The composition of claim 6, wherein the microorganism is an Escherichia genus. Contacting a variant of the recombinant P450 protein with a fluoromethane-containing sample represented by CHnF4-n, wherein n is an integer from 0 to 3, to reduce the concentration of fluoromethane in the sample, Wherein the variant has an amino acid alteration in the amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: 8 and has an activity belonging to EC 1.14.14.1. 13. The method of claim 12, wherein the contacting is performed in an airtight container. 13. The method of claim 12, wherein the variant of the recombinant P450 protein is in the form of a recombinant microorganism comprising a variant of the expressed bacterial cytochrome P450 protein. 15. The method according to claim 14, wherein said contacting is carried out under conditions in which the recombinant microorganism can survive in an airtight container. 15. The method according to claim 14, wherein the contacting is to culture the microorganism in the presence of the fluoromethane. 15. The method of claim 14, wherein the sample is plant wastewater or waste. A variant of the bacterial P450BM3 belonging to EC 1.14.14.1 with an amino acid alteration at the amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: A polynucleotide encoding a variant of the bacterial P450BM3 having an amino acid alteration at the amino acid residue corresponding to the N320 position of the amino acid sequence of SEQ ID NO: 8 and belonging to EC 1.14.14.1.

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