KR20170075630A - Soluble methane monooxygenase protein and method for reducing concentration of fluorinated methane in sample - Google Patents

Abstract

A recombinant microorganism comprising a foreign gene encoding a soluble methane monooxygenase protein, a soluble methane monooxygenase for use in removing CH _n F _{4 -n} (n is an integer from 0 to 3) A method for reducing the concentration of CH _n F _{4 -n in} a composition and a sample.

Description

[0001] The present invention relates to a soluble methane monooxygenase protein and a method for reducing the concentration of fluorinated methane in a sample using the same,

A recombinant microorganism comprising a foreign gene encoding a soluble methane monooxygenase protein, a soluble methane monooxygenase protein for use in removing CH _n F _{4 -n} (n is an integer from 0 to 3) in the sample And a method for reducing the concentration of CH _n F _{4 -n} in a sample using the protein.

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. Among these, fluorinated gas (F-gas) such as perfluorocarbons (PFCs), hydrofluorocarbons (HFCs), and sulfur hexafluoride (SF ₆ ) Has a low half-life and a high global warming potential. 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.

Soluble methane monooxygenase (sMMO) is an oxidoreductase enzyme, which contains three components: hydroxylase, reductase, and regulatory elements. The hydroxyl group element is a dimer of a hydroxyl group element monomer composed of MmoX, MmoY, and MmoZ. The reductase element, MmoC, contains a prosthetic group and oxidizes NADH to NAD +. The regulatory element, MmoB, is involved in transferring electrons from the reductase element to the hydroxylase element. MmoD is not known about the elements of sMMO or its exact function.

To date, it is not known whether soluble methane monooxygenase catalyzes removal of fluorinated methane in a sample.

One aspect provides a recombinant microorganism comprising a foreign gene encoding a soluble methane monooxygenase protein.

Another aspect provides a composition comprising a soluble methane monooxygenase protein for use in removing CH _n F _{4 -n} (where n is an integer from 0 to 3) in a sample.

Another aspect relates to a method of reducing the concentration of CH _n F _{4 -n} in a sample by contacting a soluble methane monooxygenase protein with a sample containing CH _n F _{4 -n} (n is an integer from 0 to 3) Thereby reducing the concentration of CH _n F _{4 -n in} the sample.

One aspect provides a recombinant microorganism comprising a foreign gene encoding soluble methane monooxygenase (sMMO) protein.

In the recombinant microorganism, the sMMO protein may belong to EC 1.14.13.25. The sMMO protein may be derived from Methylococcus capsulatus (Bath). The sMMO protein may be one comprising a complex of MmoX, MmoY and MmoZ, MmoB, MmoC, and MmoD. MmoD can also be named OrfY. MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD may have the amino acid sequences of SEQ ID NOS: 1, 3, 5, 7, 9 and 11, respectively. The polynucleotides encoding MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD may have the nucleotide sequences of SEQ ID NOS: 2, 4, 6, 8, 10 and 12, respectively. Wherein the gene comprises a polynucleotide having the nucleotide sequence of SEQ ID NO: 2, a polynucleotide having the nucleotide sequence of SEQ ID NO: 4, a polynucleotide having the nucleotide sequence of SEQ ID NO: 6, a polynucleotide having the nucleotide sequence of SEQ ID NO: A polynucleotide having the nucleotide sequence of SEQ ID NO: 10, and a polynucleotide having the nucleotide sequence of SEQ ID NO: 12. In the recombinant microorganism, the gene may be contained in a polynucleotide having a nucleotide sequence of SEQ ID NO: 31. That is, the recombinant microorganism may comprise a polynucleotide having the nucleotide sequence of SEQ ID NO: 31.

The recombinant microorganism may further comprise a foreign gene encoding MmoG. MmoG may have the amino acid sequence of SEQ ID NO: 13. The polynucleotide encoding MmoG may be the one having the nucleotide sequence of SEQ ID NO: 14.

The recombinant microorganism may belong to the genus Escherichia. The Escherichia microorganism may be E. coli. The recombinant microorganism may be one in which a polynucleotide having the nucleotide sequence of SEQ ID NO: 31 has been introduced.

Another aspect provides a composition comprising a soluble methane monooxygenase (sMMO) protein for use in removing CH _n F _{4 -n} (n is an integer from 0 to 3) in a sample.

In this composition, CH _n F _{4 -n} can be, for example, CHF ₃ , CH ₂ F ₂ , CH ₃ F, or CF ₄ . The term "removal" includes reducing the concentration of CH _n F _{4 -n in} the sample. The reduction includes complete removal.

In the above composition, the sMMO protein may belong to EC 1.14.13.25. The sMMO protein may be derived from Methylococcus capsulatus (Bath). The sMMO protein may be one comprising a complex of MmoX, MmoY and MmoZ, MmoB, MmoC, and MmoD. MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD may have the amino acid sequences of SEQ ID NOS: 1, 3, 5, 7, 9 and 11, respectively. The polynucleotides encoding MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD are as described above.

In such a composition, the sMMO protein may be expressed from a recombinant microorganism comprising a foreign gene encoding the protein. The composition may comprise the recombinant microorganism, a lysate thereof, or a water soluble fraction of the lysate. The recombinant microorganism may be Escherichia genus. The microorganism Escherichia may be E. coli. The foreign gene contained in the recombinant microorganism is a polynucleotide having a nucleotide sequence of SEQ ID NO: 2, a polynucleotide having a nucleotide sequence of SEQ ID NO: 4, a polynucleotide having a nucleotide sequence of SEQ ID NO: 6, a polynucleotide having a nucleotide sequence of SEQ ID NO: A polynucleotide having the nucleotide sequence of SEQ ID NO: 10, and a polynucleotide having the nucleotide sequence of SEQ ID NO: 12. The foreign gene contained in the recombinant microorganism may be contained in a polynucleotide having the nucleotide sequence of SEQ ID NO: 31. The recombinant microorganism may be one in which a polynucleotide having the nucleotide sequence of SEQ ID NO: 31 has been introduced.

In the composition, the ratio of the CH _n F _{4 -n} Removal may be accomplished by cleaving the bond of CF _n F _{4 -n} (where n is an integer from 0 to 3), or converting CH _n F _{4 -n} to another substance, or accumulating it in the cell to increase the concentration of CHnF _{4 -n} ≪ / RTI > The conversion may be to introduce a hydrophilic group such as a hydroxyl group into CH _n F _{4 -n} , or to introduce 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.

In addition, the composition can be used to remove ethyl acetate.

The concentration of the further aspect is soluble methane mono oxide dioxygenase claim (sMMO) _{4 -n} CHnF protein (n is an integer of 0 to 3) is contacted with a sample containing the sample CHnF _{4 -n} (n is an integer of 0 to 3) decreasing; containing, provides a method for reducing the concentration of the sample of CHnF _{4 -n.}

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 the sMMO protein containing liquid. The contact may also be a liquid-liquid contact contacting the liquid sample with the sMMO protein containing liquid. The liquid-liquid contact includes mixing.

In this method, the sMMO protein may be expressed from a recombinant microorganism comprising a foreign gene encoding the protein. The composition may comprise the recombinant microorganism, a lysate thereof, or a water soluble fraction of the lysate. The recombinant microorganism may be Escherichia genus. The microorganism Escherichichia can be E. coli. The foreign gene contained in the recombinant microorganism is as described above.

In the above method, the contacting may be carried out under conditions in which the recombinant microorganism can survive in the closed container. Said viable condition may be a condition allowing the recombinant microorganism to be in a reproducible condition or in a resting state. In this case, the contacting may be to cultivate the microorganism in the presence of CH _n F _{4 -n} . The culture may be carried out under aerobic or anaerobic conditions.

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

In addition, the process can be used to remove ethyl acetate.

Recombinant microorganisms according to one aspect can be used to remove CH _n F _{4 -n} in the sample.

A composition comprising a sMMO protein according to another aspect can be used to remove CH _n F _{4 -n} in the sample.

A method of reducing the concentration of CH _n F _{4 -n} in a sample according to another aspect can effectively reduce the concentration of CH _n F _{4-n in} the sample.

Figure 1a shows a vector map of the pET28a-mmoXYBZDC vector.
Figure 1B shows a vector map of the pETDuet-mmoXY-ZD vector.
Figure 1c shows a vector map of the pACYCDuet-mmoBC vector.
Figure 1d shows a vector map of the pACYCDuet-mmoG-BC vector.
2 shows variation in the concentration of CHF ₃ in the case of cultivation of recombinant E. coli in a culture medium in contact with the gas containing CHF _3, the headspace.
Figure 3 when a culture of recombinant E. coli in CHCl ₃ containing medium, shows a change in concentration of the CHCl ₃ in the head space.
4 is CF _4, if containing the recombinant E. coli BL21 / pET28a-mmoXYBZDC out of the medium in contact with the substrate were incubated for seven days, shows a change with time of the CF ₄ in the head space.

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: sMMO  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 sMMO gene was prepared, and the removal effect of halomethane, i.e., CHF ₃ or CHCl ₃ , in the sample was confirmed using the recombinant E. coli.

(One) sMMO  Production of recombinant Escherichia coli expressing the gene

MmOX, mmoY, mmoZ, mmoB, mmoC, mmoD, and mmoG genes were amplified from Methylococcus capsulatus (Bath) strain. 2, 4, 6, 8, 10, 12, and 14, respectively, which have the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13, respectively. Specifically, PCR was carried out using the chromosomal DNA (ATCC 33009D-5) of the strain of Methylococcus capsulatus (Bath) as a template and the primer set of the nucleotide sequences of SEQ ID NOs: 15 and 16 to obtain the following primers: mmoX, mmoY, mmoZ , mmoB, mmoC, and mmoD genes were amplified. The amplified gene fragment was ligated with pET28a (Novagen, Cat. No. 69864-3) digested with restriction enzymes NcoI and XhoI through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare pET28a-mmoXYBZDC vector . Figure 1a shows a vector map of the pET28a-mmoXYBZDC vector.

MmoX, mmoY, mmoZ, mmoB, mmoC, and mmoD were amplified and inserted into the expression vector to express the sMMO gene using a ribosome binding site (RBS). Nos. 17 and 20 were prepared using the PCR-amplified mmoX gene fragment using the primer set of the nucleotide sequences of SEQ ID Nos. 17 and 18 and the primer set of the nucleotide sequences of SEQ ID Nos. 19 and 20 as a template, Was used to amplify the region containing the mmoX and mmoY genes. The amplified gene fragment was ligated with pETDuet (Novagen, Cat. No. 71146-3) cut with restriction enzymes NcoI and HindIII through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pETDuet-mmoXY vector . The mmoZ gene fragment PCR-amplified using the primer set of the nucleotide sequences of SEQ ID NOS: 21 and 22 and the primer set of the nucleotide sequences of SEQ ID NOS: 23 and 24 were used as a template, And a primer set of 24 nucleotide sequences were used to amplify sites containing the mmoZ and mmoD genes. The amplified gene fragment was ligated with pETDuet-mmoXY digested with restriction enzymes NdeI and XhoI through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare pETDuet-mmoXY-ZD vector. Figure 1B shows a vector map of the pETDuet-mmoXY-ZD vector.

Nos. 25 and 28, using a primer set of the nucleotide sequences of SEQ ID Nos. 25 and 26 as a template, and a primer set of the nucleotide sequences of SEQ ID Nos. 27 and 28 as primers of the mmoB gene fragment PCR amplified using the primer set of the nucleotide sequence of SEQ ID Nos. Were used to amplify the region containing the mmoB and mmoC genes. The amplified gene fragment was ligated with pACYCDuet (Novagen, Cat. No. 71147-3) cut with restriction enzymes NdeI and EcoRV through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pACYCDuet-mmoBC vector . Figure 1c shows a vector map of the pACYCDuet-mmoBC vector.

PCR-amplified mmoG gene fragments were amplified by PCR using pACYCDuet (Novagen, Cat. No. 71147-3) and InFusion Cloning Kit (Clontech Laboratories, Inc.) cut with restriction enzymes NcoI and HindIII using primer sets of the nucleotide sequences of SEQ ID NOs: , Inc.) to prepare pACYCDuet-mmoG vector. Further, a mmoB gene fragment PCR-amplified using the primer set of the nucleotide sequences of SEQ ID Nos. 25 and 26 and a primer set of the nucleotide sequences of SEQ ID Nos. 27 and 28 were used as templates, And primer sets of 28 nucleotide sequences were used to amplify sites containing the mmoB and mmoC genes. The amplified gene fragment was ligated with pACYCDuet-mmoG digested with restriction enzymes NdeI and EcoRV through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare a pACYCDuet-mmoG-BC vector. Figure 1d shows a vector map of the pACYCDuet-mmoG-BC vector.

Next, the prepared pETDuet-mmoXY-ZD vector and pACYCDuet-mmoBC vector, pETDuet-mmoXY-ZD and pACYCDuet-mmoG-BC vector, and pET28a-mmoXYBZDC vector were respectively introduced into Escherichia coli BL21 strain by heat shock method And cultured in an LB plate medium containing 100 / / mL of ampicillin and 35 / / mL of chloramphenicol or 50 / / mL of kanamycin to obtain ampicillin and chloramphenicol resistance or kanamycin resistance Were selected. The three finally selected strains were named as recombinant Escherichia coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoBC, BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoG-BC and BL21 / pET28a-mmoXYBZDC, respectively.

(2) sMMO  In a sample by recombinant E. coli expressing the gene CHF ₃  or CHCl3 ₃  Removal effect

In this section, recombinant Escherichia coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoBC, BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoG-BC and BL21 / pET28a-mmoXYBZDC The effect on the removal of CHF ₃ or CHCl ₃ in the sample was confirmed. As a control, Escherichia coli BL21 / pETDuet + pACYCDuet or BL21 / pET28a in which an empty vector containing no sMMO gene was introduced was used.

Specifically, recombinant E. coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoBC, BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoG- BC, and BL21 / pET28a-mmoXYBZDC were cultured in Terrific Broth rpm. After adding 0.1 mM IPTG, 0.1 mg / ml of ferric citrate, 0.1 mg / ml of ferul sulfate, 0.1 mg / ml of ferric ammonium sulfate and 1 mM of cysteine at an OD _{600 of} about 0.5 , And cultured overnight at 25 ° C with stirring at 230 rpm. For each recombinant E. coli, the cells were collected and suspended in M9 medium containing 4 g / L of glucose to a cell concentration of 2.5 at OD ₆₀₀ . 10 ml of this cell solution was added to a 60 ml serum bottle and sealed. The TB 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. The M9 medium also contained 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, in the case of CHF _3, using a syringe so that the CHF ₃ to 1000 ppm of the head space of the gas phase was injected through the rubber cap of the serum sickness. In the case of CHCl ₃ , the liquid phase of CHCl ₃ was injected through the rubber material of the cap of the serum bottle using a syringe so as to be 0.02 mM in the medium. Then, the serum bottle was incubated for 94 hours in the case of CHF ₃ and for 25 hours in the case of CHCl ₃ with stirring at 200 rpm at 30 ° C. The experiment was repeated in triplicate.

After a certain period of incubation, 0.5 ml of the gas in the headspace containing no serum medium was collected using a 1.0 ml headspace syringe and injected into GC (Agilent 7890, Palo Alto, CA, USA). The injected CHF ₃ or CHCl ₃ is PoraBOND CP-Q column were separated through (25m length, 0.32mm id, 5um film thickness, Agilent), MSD CHF 3 or CHCl through (Agilent 5973, Palo Alto, CA , USA) ₃ The concentration changes were analyzed. The carrier gas was helium and flowed to the column at a rate of 1.5 ml / min. The GC conditions were maintained at an inlet temperature of 250 캜 and an initial temperature of 40 캜 for 2 minutes and a temperature of 20 캜 / min up to 290 캜. MS conditions were 70eV ionization energy, interface temperature 280 ℃, ion source temperature 230 ℃, and quadrupole temperature 150 ℃.

2 shows variation in the concentration of CHF ₃ in the case of cultivation of recombinant E. coli in a culture medium in contact with the gas containing CHF _3, the headspace. In FIG. 2, 1 is a control group, 2 to 4 are experiments using recombinant E. coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoBC, BL21 / pETDuet-mmoXY-ZD + pACYCDuet- mmoG- BC, and BL21 / pET28a-mmoXYBZDC . As shown in FIG. 2, when the recombinant E. coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoBC was cultured for 94 hours, the concentration of CHF _{3 in} the headspace decreased by about 10% as compared with the control. In addition, when the recombinant E. coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoG-BC and BL21 / pET28a-mmoXYBZDC were cultured for 94 hours, the concentration of CHF _{3 in} the headspace was reduced by about 15% as compared with the control.

Figure 3 when a culture of recombinant E. coli in CHCl ₃ containing medium, shows a change in concentration of the CHCl ₃ in the head space. In Fig. 3, 1 to 4 are as described in Fig. 3, the case of culturing the recombinant E. coli BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoBC and BL21 / pETDuet-mmoXY-ZD + pACYCDuet-mmoG-BC for 25 hours, CHCl in the headspace than in the control group ₃ Was reduced by about 20%, and the concentration of CHCl ₃ decreased to similar levels when BL21 / pET28a-mmoXYBZDC was cultured for 25 hours.

Example  2: sMMO  In a sample by recombinant E. coli expressing the gene CF ₄  Removal effect

In this section, the effect of recombinant Escherichia coli BL21 / pET28a-mmoXYBZDC containing the sMMO gene prepared in Example 1 (1) on the removal of CF ₄ in the sample was confirmed. As a control, Escherichia coli BL21 / pET28a into which a vector free of sMMO gene was introduced was used.

The experiment in Example 1 (2) in the process performed with respect to the CHF _3, CHF ₃ instead of the rubber cap of the serum sickness using a syringe using a CF _4, and such that the head space than 1,000ppm the CF ₄ of the gas phase in the , And then the serum bottle was incubated for 7 days with stirring at 30 DEG C at 200 rpm. The results are shown in Fig.

4 is CF _4, if containing the recombinant E. coli BL21 / pET28a-mmoXYBZDC out of the medium in contact with the substrate were incubated for seven days, shows a change with time of the CF ₄ in the head space. In FIG. 4, NC represents a negative control, and 'MMO' represents an experiment using E. coli BL21 / pET28a-mmoXYBZDC. 4, in one case, and decreased the concentration of CF ₄ in the headspace than in the control group cultured for 7 days 3.42% as shown in FIG.

<110> SAMSUNG ELECTRONICS CO., LTD. <120> Soluble methane monooxygenase protein and method for reducing          concentration of fluorinated methane in sample <130> PN114343 <160> 31 <170> Kopatentin 2.0 <210> 1 <211> 527 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 1 Met Ala Leu Ser Thr Ala Thr Lys Ala Ala Thr Asp Ala Leu Ala Ala   1 5 10 15 Asn Arg Ala Pro Thr Ser Val Asn Ala Gln Glu Val His Arg Trp Leu              20 25 30 Gln Ser Phe Asn Trp Asp Phe Lys Asn Asn Arg Thr Lys Tyr Ala Thr          35 40 45 Lys Tyr Lys Met Ala Asn Glu Thr Lys Glu Gln Phe Lys Leu Ile Ala      50 55 60 Lys Glu Tyr Ala Arg Met Glu Ala Val Lys Asp Glu Arg Gln Phe Gly  65 70 75 80 Ser Leu Gln Asp Ala Leu Thr Arg Leu Asn Ala Gly Val Arg Val His                  85 90 95 Pro Lys Trp Asn Glu Thr Met Lys Val Val Ser Asn Phe Leu Glu Val             100 105 110 Gly Glu Tyr Asn Ale Ala Ala Thr Gly Met Leu Trp Asp Ser Ala         115 120 125 Gln Ala Ala Glu Gln Lys Asn Gly Tyr Leu Ala Gln Val Leu Asp Glu     130 135 140 Ile Arg His Thr His Gln Cys Ala Tyr Val Asn Tyr Tyr Phe Ala Lys 145 150 155 160 Asn Gly Gln Asp Pro Ala Gly His Asn Asp Ala Arg Arg Thr Arg Thr                 165 170 175 Ile Gly Pro Leu Trp Lys Gly Met Lys Arg Val Phe Ser Asp Gly Phe             180 185 190 Ile Ser Gly Asp Ala Val Glu Cys Ser Leu Asn Leu Gln Leu Val Gly         195 200 205 Glu Ala Cys Phe Thr Asn Pro Leu Ile Val Ala Val Thr Glu Trp Ala     210 215 220 Ala Ala Asn Gly Asp Glu Ile Thr Pro Thr Val Phe Leu Ser Ile Glu 225 230 235 240 Thr Asp Glu Leu Arg His Met Ala Asn Gly Tyr Gln Thr Val Val Ser                 245 250 255 Ile Ala Asn Asp Pro Ala Ser Ala Lys Tyr Leu Asn Thr Asp Leu Asn             260 265 270 Asn Ala Phe Trp Thr Gln Gln Lys Tyr Phe Thr Pro Val Leu Gly Met         275 280 285 Leu Phe Glu Tyr Gly Ser Lys Phe Lys Val Glu Pro Trp Val Lys Thr     290 295 300 Trp Asn Arg Trp Val Tyr Glu Asp Trp Gly Gly Ile Trp Ile Gly Arg 305 310 315 320 Leu Gly Lys Tyr Gly Val Glu Ser Pro Arg Ser Leu Lys Asp Ala Lys                 325 330 335 Gln Asp Ala Tyr Trp Ala His His Asp Leu Tyr Leu Leu Ala Tyr Ala             340 345 350 Leu Trp Pro Thr Gly Phe Phe Arg Leu Ala Leu Pro Asp Gln Glu Glu         355 360 365 Met Glu Trp Phe Glu Ala Asn Tyr Pro Gly Trp Tyr Asp His Tyr Gly     370 375 380 Lys Ile Tyr Glu Glu Trp Arg Ala Arg Gly Cys Glu Asp Pro Ser Ser 385 390 395 400 Gly Phe Ile Pro Leu Met Trp Phe Ile Glu Asn Asn His Pro Ile Tyr                 405 410 415 Ile Asp Arg Val Ser Gln Val Pro Phe Cys Pro Ser Leu Ala Lys Gly             420 425 430 Ala Ser Thr Leu Arg Val His Glu Tyr Asn Gly Gln Met His Thr Phe         435 440 445 Ser Asp Gln Trp Gly Glu Arg Met Trp Leu Ala Glu Pro Glu Arg Tyr     450 455 460 Glu Cys Gln Asn Ile Phe Glu Gln Tyr Glu Gly Arg Glu Leu Ser Glu 465 470 475 480 Val Ile Ala Glu Leu His Gly Leu Arg Ser Asp Gly Lys Thr Leu Ile                 485 490 495 Ala Gln Pro His Val Arg Gly Asp Lys Leu Trp Thr Leu Asp Asp Ile             500 505 510 Lys Arg Leu Asn Cys Val Phe Lys Asn Pro Val Lys Ala Phe Asn         515 520 525 <210> 2 <211> 1584 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 2 atggcactta gcaccgcaac caaggccgcg acggacgcgc tggctgccaa tcgggcaccc 60 accagcgtga atgcacagga agtgcaccgt tggctccaga gcttcaactg ggatttcaag 120 aacaaccgga ccaagtacgc caccaagtac aagatggcga acgagaccaa ggaacagttc 180 aagctgatcg ccaaggaata tgcgcgcatg gaggcagtca aggacgaaag gcagttcggt 240 agcctgcagg atgcgctgac ccgcctcaac gccggtgttc gcgttcatcc gaagtggaac 300 gagaccatga aagtggtttc gaacttcctg gaagtgggcg aatacaacgc catcgccgct 360 accgggatgc tgtgggattc cgcccaggcg gcggaacaga agaacggcta tctggcccag 420 gtgttggatg aaatccgcca cacccaccag tgtgcctacg tcaactacta cttcgcgaag 480 aacggccagg acccggccgg tcacaacgat gctcgccgca cccgtaccat cggtccgctg 540 tggaagggca tgaagcgcgt gttttccgac ggcttcattt ccggcgacgc cgtggaatgc 600 tccctcaacc tgcagctggt gggtgaggcc tgcttcacca atccgctgat cgtcgcagtg 660 accgaatggg ctgccgccaa cggcgatgaa atcaccccga cggtgttcct gtcgatcgag 720 accgacgaac tgcgccacat ggccaacggt taccagaccg tcgtttccat cgccaacgat 780 ccggcttccg ccaagtatct caacacggac ctgaacaacg ccttctggac ccagcagaag 840 tacttcacgc cggtgttggg catgctgttc gagtatggct ccaagttcaa ggtcgagccg 900 tgggtcaaga cgtggaaccg ctgggtgtac gaggactggg gcggcatctg gatcggccgt 960 ctgggcaagt acggggtgga gtcgccgcgc agcctcaagg acgccaagca ggacgcttac 1020 tgggctcacc acgacctgta tctgctggct tatgcgctgt ggccgaccgg cttcttccgt 1080 ctggcgctgc cggatcagga agaaatggag tggttcgagg ccaactaccc cggctggtac 1140 gaccactacg gcaagatcta cgaggaatgg cgcgcccgcg gttgcgagga tccgtcctcg 1200 ggcttcatcc cgctgatgtg gttcatcgaa aacaaccatc ccatctacat cgatcgcgtg 1260 tcgcaagtgc cgttctgccc gagcttggcc aagggcgcca gcaccctgcg cgtgcacgag 1320 tacaacggcc agatgcacac cttcagcgac cagtggggcg agcgcatgtg gctggccgag 1380 ccggagcgct acgagtgcca gaacatcttc gaacagtacg aaggacgcga actgtcggaa 1440 gtgatcgccg aactgcacgg gctgcgcagt gatggcaaga ccctgatcgc ccagccgcat 1500 gtccgtggcg acaagctgtg gacgttggac gatatcaaac gcctgaactg cgtcttcaag 1560 aacccggtga aggcattcaa ttga 1584 <210> 3 <211> 389 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 3 Met Ser Met Leu Gly Glu Arg Arg Gly Leu Thr Asp Pro Glu Met   1 5 10 15 Ala Ala Val Ile Leu Lys Ala Leu Pro Glu Ala Pro Leu Asp Gly Asn              20 25 30 Asn Lys Met Gly Tyr Phe Val Thr Pro Arg Trp Lys Arg Leu Thr Glu          35 40 45 Tyr Glu Ala Leu Thr Val Tyr Ala Gln Pro Asn Ala Asp Trp Ile Ala      50 55 60 Gly Gly Leu Asp Trp Gly Asp Trp Thr Gln Lys Phe His Gly Gly Arg  65 70 75 80 Pro Ser Trp Gly Asn Glu Thr Thr Glu Leu Arg Thr Val Asp Trp Phe                  85 90 95 Lys His Arg Asp Pro Leu Arg Arg Trp His Ala Pro Tyr Val Lys Asp             100 105 110 Lys Ala Glu Glu Trp Arg Tyr Thr Asp Arg Phe Leu Gln Gly Tyr Ser         115 120 125 Ala Asp Gly Gln Ile Arg Ala Met Asn Pro Thr Trp Arg Asp Glu Phe     130 135 140 Ile Asn Arg Tyr Trp Gly Ala Phe Leu Phe Asn Glu Tyr Gly Leu Phe 145 150 155 160 Asn Ala His Ser Gln Gly Ala Arg Glu Ala Leu Ser Asp Val Thr Arg                 165 170 175 Val Ser Leu Ala Phe Trp Gly Phe Asp Lys Ile Asp Ile Ala Gln Met             180 185 190 Ile Gln Leu Glu Arg Gly Phe Leu Ala Lys Ile Val Pro Gly Phe Asp         195 200 205 Glu Ser Thr Ala Val Pro Lys Ala Glu Trp Thr Asn Gly Glu Val Tyr     210 215 220 Lys Ser Ala Arg Leu Ala Val Glu Gly Leu Trp Gln Glu Val Phe Asp 225 230 235 240 Trp Asn Glu Ser Ala Phe Ser Val His Ala Val Tyr Asp Ala Leu Phe                 245 250 255 Gly Gln Phe Val Arg Arg Glu Phe Phe Gln Arg Leu Ala Pro Arg Phe             260 265 270 Gly Asp Asn Leu Thr Pro Phe Phe Ile Asn Gln Ala Gln Thr Tyr Phe         275 280 285 Gln Ile Ala Lys Gln Gly Val Gln Asp Leu Tyr Tyr Asn Cys Leu Gly     290 295 300 Asp Asp Pro Glu Phe Ser Asp Tyr Asn Arg Thr Val Met Arg Asn Trp 305 310 315 320 Thr Gly Lys Trp Leu Glu Pro Thr Ile Ala Ala Leu Arg Asp Phe Met                 325 330 335 Gly Leu Phe Ala Lys Leu Pro Ala Gly Thr Thr Asp Lys Glu Glu Ile             340 345 350 Thr Ala Ser Leu Tyr Arg Val Val Asp Asp Trp Ile Glu Asp Tyr Ala         355 360 365 Ser Arg Ile Asp Phe Lys Ala Asp Arg Asp Gln Ile Val Lys Ala Val     370 375 380 Leu Ala Gly Leu Lys 385 <210> 4 <211> 1170 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 4 atgagcatgt taggagaaag acgccgcggt ctgaccgatc cggaaatggc ggccgtcatt 60 ttgaaggcgc ttcctgaagc tccgctggac ggcaacaaca agatgggtta tttcgtcacc 120 ccccgctgga aacgcttgac ggaatatgaa gccctgaccg tttatgcgca gcccaacgcc 180 gactggatcg ccggcggcct ggactggggc gactggaccc agaaattcca cggcggccgc 240 ccttcctggg gcaacgagac cacggagctg cgcaccgtcg actggttcaa gcaccgtgac 300 ccgctccgcc gttggcatgc gccgtacgtc aaggacaagg ccgaggaatg gcgctacacc 360 gccgcttcc tgcagggtta ctccgccgac ggtcagatcc gggcgatgaa cccgacctgg 420 cgggacgagt tcatcaaccg gtattggggc gccttcctgt tcaacgaata cggattgttc 480 aacgctcatt cgcagggcgc ccgggaggcg ctgtcggacg taacccgcgt cagcctggct 540 ttctggggct tcgacaagat cgacatcgcc cagatgatcc aactcgaacg gggtttcctc 600 gccaagatcg tacccggttt cgacgagtcc acagcggtgc cgaaggccga atggacgaac 660 ggggaggtct acaagagcgc ccgtctggcc gtggaagggc tgtggcagga ggtgttcgac 720 tggaacgaga gcgctttctc ggtgcacgcc gtctatgacg cgctgttcgg tcagttcgtc 780 cgccgcgagt tctttcagcg gctggctccc cgcttcggcg acaatctgac gccattcttc 840 atcaaccagg cccagacata cttccagatc gccaagcagg gcgtacagga tctgtattac 900 aactgtctgg gtgacgatcc ggagttcagc gattacaacc gtaccgtgat gcgcaactgg 960 accggcaagt ggctggagcc cacgatcgcc gctctgcgcg acttcatggg gctgtttgcg 1020 aagctgccgg cgggcaccac tgacaaggaa gaaatcaccg cgtccctgta ccgggtggtc 1080 gacgactgga tcgaggacta cgccagcagg atcgacttca aggcggaccg cgatcagatc 1140 gttaaagcgg ttctggcagg attgaaataa 1170 <210> 5 <211> 170 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 5 Met Ala Lys Leu Gly Ile His Ser Asn Asp Thr Arg Asp Ala Trp Val   1 5 10 15 Asn Lys Ile Ala Gln Leu Asn Thr Leu Glu Lys Ala Ala Glu Met Leu              20 25 30 Lys Gln Phe Arg Met Asp His Thr Thr Pro Phe Arg Asn Ser Tyr Glu          35 40 45 Leu Asp Asn Asp Tyr Leu Trp Ile Glu Ala Lys Leu Glu Glu Lys Val      50 55 60 Ala Val Leu Lys Ala Arg Ala Phe Asn Glu Val Asp Phe Arg His Lys  65 70 75 80 Thr Ala Phe Gly Glu Asp Ala Lys Ser Val Leu Asp Gly Thr Val Ala                  85 90 95 Lys Met Asn Ala Ala Lys Asp Lys Trp Glu Ala Glu Lys Ile His Ile             100 105 110 Gly Phe Arg Gln Ala Tyr Lys Pro Pro Ile Met Pro Val Asn Tyr Phe         115 120 125 Leu Asp Gly Glu Arg Gln Leu Gly Thr Arg Leu Met Glu Leu Arg Asn     130 135 140 Leu Asn Tyr Tyr Asp Thr Pro Leu Glu Glu Leu Arg Lys Gln Arg Gly 145 150 155 160 Val Arg Val Val His Leu Gln Ser Pro His                 165 170 <210> 6 <211> 513 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 6 atggcgaaac tgggtataca cagcaacgac acccgcgacg cctgggtgaa caagatcgcg 60 cagctcaaca ccctggaaaa agcggccgag atgctgaagc agttccggat ggaccacacc 120 acgccgttcc gcaacagcta cgaactggac aacgactacc tctggatcga ggccaagctc 180 gaagagaagg tcgccgtcct caaggcacgc gccttcaacg aggtggactt ccgtcataag 240 accgctttcg gcgaggatgc caagtccgtt ctggacggca ccgtcgcgaa gatgaacgcg 300 gccaaggaca agtgggaggc ggagaagatc catatcggtt tccgccaggc ctacaagccg 360 ccgatcatgc cggtgaacta tttcctggac ggcgagcgtc agttggggac ccggctgatg 420 gaactgcgca acctcaacta ctacgacacg ccgctggaag aactgcgcaa acagcgcggt 480 gtgcgggtgg tgcatctgca gtcgccgcac tga 513 <210> 7 <211> 141 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 7 Met Ser Val Asn Ser Asn Ala Tyr Asp Ala Gly Ile Met Gly Leu Lys   1 5 10 15 Gly Lys Asp Phe Ala Asp Gln Phe Phe Ala Asp Glu Asn Gln Val Val              20 25 30 His Glu Ser Asp Thr Val Val Leu Val Leu Lys Lys Ser Asp Glu Ile          35 40 45 Asn Thr Phe Ile Glu Glu Ile Leu Leu Thr Asp Tyr Lys Lys Asn Val      50 55 60 Asn Pro Thr Val Asn Val Glu Asp Arg Ala Gly Tyr Trp Trp Ile Lys  65 70 75 80 Ala Asn Gly Lys Ile Glu Val Asp Cys Asp Glu Ile Ser Glu Leu Leu                  85 90 95 Gly Arg Gln Phe Asn Val Tyr Asp Phe Leu Val Asp Val Ser Ser Thr             100 105 110 Ile Gly Arg Ala Tyr Thr Leu Gly Asn Lys Phe Thr Ile Thr Ser Glu         115 120 125 Leu Met Gly Leu Asp Arg Lys Leu Glu Asp Tyr His Ala     130 135 140 <210> 8 <211> 426 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 8 atgagcgtaa acagcaacgc atacgacgcc ggcatcatgg gcctgaaagg caaggacttc 60 gccgatcagt tctttgccga cgaaaaccaa gtggtccatg aaagcgacac ggtcgttctg 120 gtcctcaaga agtcggacga gatcaatacc tttatcgagg agatccttct gacggactac 180 aagaagaacg tcaatccgac ggtaaacgtg gaagaccgcg cgggttactg gtggatcaag 240 gccaacggca agatcgaggt cgattgcgac gagatttccg agctgttggg gcggcagttc 300 aacgtctacg acttcctcgt cgacgtttcc tccaccatcg gccgggccta taccctgggc 360 aacaagttca ccattaccag tgagctgatg ggcctggacc gcaagctcga agactatcac 420 gcttaa 426 <210> 9 <211> 348 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 9 Met Gln Arg Val His Thr Ile Thr Ala Val Thr Glu Asp Gly Glu Ser   1 5 10 15 Leu Arg Phe Glu Cys Arg Ser Asp Glu Asp Val Ile Thr Ala Ala Leu              20 25 30 Arg Gln Asn Ile Phe Leu Met Ser Ser Cys Arg Glu Gly Gly Cys Ala          35 40 45 Thr Cys Lys Ala Leu Cys Ser Glu Gly Asp Tyr Asp Leu Lys Gly Cys      50 55 60 Ser Val Gln Ala Leu Pro Pro Glu Glu Glu Glu Glu Leu Val Leu  65 70 75 80 Leu Cys Arg Thr Tyr Pro Lys Thr Asp Leu Glu Ile Glu Leu Pro Tyr                  85 90 95 Thr His Cys Arg Ile Ser Phe Gly Glu Val Gly Ser Phe Glu Ala Glu             100 105 110 Val Val Gly Leu Asn Trp Val Ser Ser Asn Thr Val Gln Phe Leu Leu         115 120 125 Gln Lys Arg Pro Asp Glu Cys Gly Asn Arg Gly Val Lys Phe Glu Pro     130 135 140 Gly Gln Phe Met Asp Leu Thr Ile Pro Gly Thr Asp Val Ser Ser Ser 145 150 155 160 Tyr Ser Pro Ala Asn Leu Pro Asn Pro Glu Gly Arg Leu Glu Phe Leu                 165 170 175 Ile Arg Val Leu Pro Glu Gly Arg Phe Ser Asp Tyr Leu Arg Asn Asp             180 185 190 Ala Arg Val Gly Gln Val Leu Ser Val Lys Gly Pro Leu Gly Val Phe         195 200 205 Gly Leu Lys Glu Arg Gly Met Ala Pro Arg Tyr Phe Val Ala Gly Gly     210 215 220 Thr Gly Leu Ala Pro Val Val Ser Met Val Arg Gln Met Gln Glu Trp 225 230 235 240 Thr Ala Pro Asn Glu Thr Arg Ile Tyr Phe Gly Val Asn Thr Glu Pro                 245 250 255 Glu Leu Phe Tyr Ile Asp Glu Leu Lys Ser Leu Glu Arg Ser Met Arg             260 265 270 Asn Leu Thr Val Lys Ala Cys Val Trp His Pro Ser Gly Asp Trp Glu         275 280 285 Gly Glu Gln Gly Ser Pro Ile Asp Ala Leu Arg Glu Asp Leu Glu Ser     290 295 300 Ser Asp Ala Asn Pro Asp Ile Tyr Leu Cys Gly Pro Pro Gly Met Ile 305 310 315 320 Asp Ala Cys Glu Leu Val Arg Ser Ser Gly Ile Pro Gly Glu Gln                 325 330 335 Val Phe Phe Glu Lys Phe Leu Pro Ser Gly Ala Ala             340 345 <210> 10 <211> 1047 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 10 atgcagcgag ttcacactat cacggcggtg acggaggatg gcgaatcgct ccgcttcgaa 60 tgccgttcgg acgaggacgt catcaccgcc gccctgcgcc agaacatctt tctgatgtcg 120 tcctgccggg agggcggctg tgcgacctgc aaggccttgt gcagcgaagg ggactacgac 180 ctcaagggct gcagcgttca ggcgctgccg ccggaagagg aggaggaagg gttggtgttg 240 ttgtgccgga cctacccgaa gaccgacctg gaaatcgaac tgccctatac ccattgccgc 300 atcagttttg gtgaggtcgg cagtttcgag gcggaggtcg tcggcctcaa ctgggtttcg 360 agcaacaccg tccagtttct tttgcagaag cggcccgacg agtgcggcaa ccgtggcgtg 420 aaattcgaac ccggtcagtt catggacctg accatccccg gcaccgatgt ctcccgctcc 480 tactcgccgg cgaaccttcc taatcccgaa ggccgcctgg agttcctgat ccgcgtgtta 540 ccggagggac ggttttcgga ctacctgcgc aatgacgcgc gtgtcggaca ggtcctctcg 600 gtcaaagggc cactgggcgt gttcggtctc aaggagcggg gcatggcgcc gcgctatttc 660 gtggccggcg gcaccgggtt ggcgccggtg gtctcgatgg tgcggcagat gcaggagtgg 720 accgcgccga acgagacccg catctatttc ggtgtgaaca ccgagccgga attgttctac 780 atcgacgagc tcaaatccct ggaacgatcg atgcgcaatc tcaccgtgaa ggcctgtgtc 840 tggcacccga gcggggactg ggaaggcgag cagggctcgc ccatcgatgc gttgcgggaa 900 gacctggagt cctccgacgc caacccggac atttatttgt gcggtccgcc gggcatgatc 960 gatgccgcct gcgagctggt acgcagccgc ggtatccccg gcgaacaggt cttcttcgaa 1020 aaattcctgc cgtccggggc ggcctaa 1047 <210> 11 <211> 103 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 11 Met Val Glu Ser Ala Phe Gln Pro Phe Ser Gly Asp Ala Asp Glu Trp   1 5 10 15 Phe Glu Glu Pro Arg Pro Gln Ala Gly Phe Phe Pro Ser Ala Asp Trp              20 25 30 His Leu Leu Lys Arg Asp Glu Thr Tyr Ala Ala Tyr Ala Lys Asp Leu          35 40 45 Asp Phe Met Trp Arg Trp Val Ile Val Arg Glu Glu Arg Ile Val Gln      50 55 60 Glu Gly Cys Ser Ile Ser Leu Glu Ser Ser Ile Arg Ala Val Thr His  65 70 75 80 Val Leu Asn Tyr Phe Gly Met Thr Glu Gln Arg Ala Pro Ala Glu Asp                  85 90 95 Arg Thr Gly Gly Val Gln His             100 <210> 12 <211> 312 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 12 atggtcgaat cggcatttca gccattttcg ggcgacgcag acgaatggtt cgaggaacca 60 cggccccagg ccggtttctt cccttccgcg gactggcatc tgctcaaacg ggacgagacc 120 tacgcagcct atgccaagga tctcgatttc atgtggcggt gggtcatcgt ccgggaagaa 180 aggatcgtcc aggagggttg ctcgatcagc ctggagtcgt cgatccgcgc cgtgacgcac 240 gtactgaatt attttggtat gaccgaacaa cgcgccccgg cagaggaccg gaccggcgga 300 gttcaacatt ga 312 <210> 13 <211> 559 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 13 Met Ala Lys Glu Val Val Tyr Arg Gly Ser Ala Arg Gln Arg Met Met   1 5 10 15 Gln Gly Ile Glu Ile Leu Ala Arg Ala Ala Ile Pro Thr Leu Gly Ala              20 25 30 Thr Gly Pro Ser Val Met Ile Gln His Arg Ala Asp Gly Leu Pro Pro          35 40 45 Ile Ser Thr Arg Asp Gly Val Thr Val Ala Asn Ser Ile Val Leu Lys      50 55 60 Asp Arg Val Ala Asn Leu Gly Ala Arg Leu Leu Arg Asp Val Ala Gly  65 70 75 80 Thr Met Ser Arg Glu Ala Gly Asp Gly Thr Thr Thr Ala Ile Val Leu                  85 90 95 Ala Arg His Ile Ala Arg Glu Met Phe Lys Ser Leu Ala Val Gly Ala             100 105 110 Asp Pro Ile Ala Leu Lys Arg Gly Ile Asp Arg Ala Val Ala Arg Val         115 120 125 Ser Glu Asp Ile Gly Ala Arg Ala Trp Arg Gly Asp Lys Glu Ser Val     130 135 140 Ile Leu Gly Val Ala Ala Val Ala Thr Lys Gly Glu Pro Gly Val Gly 145 150 155 160 Arg Leu Leu Leu Glu Ala Leu Asp Ala Val Gly Val His Gly Ala Val                 165 170 175 Ser Ile Glu Leu Gly Gln Arg Arg Glu Asp Leu Leu Asp Val Val Asp             180 185 190 Gly Tyr Arg Trp Glu Lys Gly Tyr Leu Ser Pro Tyr Phe Val Thr Asp         195 200 205 Arg Ala Arg Glu Leu Ala Glu Leu Glu Asp Val Tyr Leu Leu Met Thr     210 215 220 Asp Arg Glu Val Val Asp Phe Ile Asp Leu Val Pro Leu Leu Glu Ala 225 230 235 240 Val Thr Glu Ala Gly Gly Ser Leu Leu Ile Ala Ala Asp Arg Val His                 245 250 255 Glu Lys Ala Leu Ala Gly Leu Leu Leu Asn His Val Arg Gly Val Phe             260 265 270 Lys Ala Val Ala Val Thr Ala Pro Gly Phe Gly Asp Lys Arg Pro Asn         275 280 285 Arg Leu Leu Asp Leu Ala Ala Leu Thr Gly Gly Arg Ala Val Leu Glu     290 295 300 Ala Gln Gly Asp Arg Leu Asp Arg Val Thr Leu Ala Asp Leu Gly Arg 305 310 315 320 Val Arg Arg Ala Val Val Ser Ala Asp Asp Thr Ala Leu Leu Gly Ile                 325 330 335 Pro Gly Thr Glu Ala Ser Arg Ala Arg Leu Glu Gly Leu Arg Leu Glu             340 345 350 Ala Glu Gln Tyr Arg Ala Leu Lys Pro Gly Gln Gly Ser Ala Thr Gly         355 360 365 Arg Leu His Glu Leu Glu Glu Ile Glu Ala Arg Ile Val Gly Leu Ser     370 375 380 Gly Lys Ser Ala Val Tyr Arg Val Gly Gly Val Thr Asp Val Glu Met 385 390 395 400 Lys Glu Arg Met Val Arg Ile Glu Asn Ala Tyr Arg Ser Val Val Ser                 405 410 415 Ala Leu Glu Gly Gly Val Leu Pro Gly Gly Gly Val Gly Phe Leu Gly             420 425 430 Ser Met Pro Val Leu Ala Glu Leu Glu Ala Arg Asp Ala Asp Glu Ala         435 440 445 Arg Gly Ile Gly Ile Val Arg Ser Ala Leu Thr Glu Pro Leu Arg Ile     450 455 460 Ile Gly Glu Asn Ser Gly Leu Ser Gly Glu Ala Val Val Ala Lys Val 465 470 475 480 Met Asp His Ala Asn Pro Gly Trp Gly Tyr Asp Gln Glu Ser Gly Ser                 485 490 495 Phe Cys Asp Leu His Ala Arg Gly Ile Trp Asp Ala Ala Lys Val Leu             500 505 510 Arg Leu Ala Leu Glu Lys Ala Ala Ser Val Ala Gly Thr Phe Leu Thr         515 520 525 Thr Glu Ala Val Val Leu Glu Ile Pro Asp Thr Asp Ala Phe Ala Gly     530 535 540 Phe Ser Ala Glu Trp Ala Ala Ala Thr Arg Glu Asp Pro Arg Val 545 550 555 <210> 14 <211> 1680 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 14 atggcaaagg aagtggttta cagggggagt gcgcggcagc gcatgatgca aggcatcgag 60 atactcgcgc gggcggcgat accgacgctg ggagccaccg gccccagcgt catgatccag 120 caccgcgccg atggcctgcc ccccatttcg acgcgggacg gcgtcacggt ggctaactcc 180 atcgtactca aggaccgtgt cgcgaatctc ggtgcccggc tgctgcggga cgtcgccggc 240 accatgtccc gcgaagcagg ggatggcacc accaccgcca tcgtgctggc ccgccatatc 300 gcccgggaga tgttcaagag cctcgccgtc ggtgccgatc ccatcgctct caagcgtggt 360 atcgaccgtg ccgtcgcccg cgtgagcgag gacatcgggg ctcgggcctg gcgcggcgac 420 aaggaatcgg tcatcctggg ggtggccgcg gtggcgacca agggcgagcc gggcgtgggc 480 cggctgctgc tggaggcgct ggacgcggtc ggcgtccatg gcgccgtgtc gatcgaactg 540 gggcagcggc gcgaggacct gctcgacgtg gtcgacgggt atcgttggga aaaaggttat 600 ctgtcgccct attttgtgac cgatcgggct cgcgagctgg ccgaactcga agacgtctac 660 ctcttgatga ccgatcggga ggtggtcgat ttcatcgatt tggtacccct gctggaggcg 720 gtgaccgagg ctggtggcag cctcctgatc gccgccgacc gtgtccacga gaaggcactg 780 gccggccttt tgctcaatca cgttcgcggc gtcttcaagg ccgtcgcggt caccgcgccc 840 gggttcggcg acaagcggcc gaaccgcctt ttggatctgg cggcgttgac cggtgggcgg 900 gcggtcctgg aagcccaggg cgaccgattg gaccgggtca cgctggccga cctggggcgg 960 gtgcggcggg cggtcgtcag cgctgacgac accgcgctgc tcggcatacc gggcaccgaa 1020 gcctcccggg cccgcttgga gggtttgcgc ctggaagcgg agcagtaccg ggcgctcaag 1080 cccggtcagg gatcggcgac ggggcgcttg cacgagctcg aggaaatcga ggcccggatc 1140 gtcggtctga gcggcaagtc cgcggtctac cgcgtgggcg gcgtgaccga cgtggagatg 1200 aaggagcgga tggtacggat cgaaaatgcc taccgctcgg tggtgtctgc actggaggag 1260 ggggtgttgc ccggcggcgg tgtcgggttt ctgggcagca tgcccgtttt ggccgagctg 1320 gaagcgcgcg atgccgacga agcacgcggc atcggcatcg tccgttccgc gctgacggag 1380 cccctccgga tcatcggaga aaattcggga ctgtcagggg aggccgtcgt cgccaaggtc 1440 atggatcacg ccaatcccgg ttggggttac gatcaggaaa gcggaagttt ctgcgacctc 1500 cacgccaggg gcatttggga tgccgccaag gtgctcaggc tggccctgga aaaagccgcg 1560 tcggtggccg gcacgtttct caccaccgaa gccgtggtac tggagattcc ggacactgac 1620 gctttcgccg gtttcagtgc ggagtgggcc gccgcgaccc gggaggatcc gcgggtctaa 1680                                                                         1680 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 aagaaggaga tataccatgg cacttagcac cgcaac 36 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gtggtggtgg tggtgctcga ttaggccgcc ccggacggca 40 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 aagaaggaga tataccatgg cacttagcac cgcaac 36 <210> 18 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gaattctgtt tcctgtgtga ttaattgaat gccttcacc 39 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tcacacagga aacagaattc atgagcatgt taggagaaa 39 <210> 20 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cattatgcgg ccgcaagctt tatttcaatc ctgccaga 38 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aagaaggaga tatacatatg gcgaaactgg gtatac 36 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaattctgtt tcctgtgtga ttagtgcggc gactgcaga 39 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tcacacagga aacagaattc atggtcgaat cggcatttc 39 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gtttctttac cagactcgat taatgttgaa ctccgccggt 40 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 aagaaggaga tatacatatg agcgtaaaca gcaacg 36 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 atgtatatct ccttcttata ttaagcgtga tagtcttcg 39 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tataagaagg agatatacat atgcagcgag ttcacacta 39 <210> 28 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gatcgcgtgg ccggccgatt taggccgccc cggacggca 39 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 taataaggag atataccatg gcaaaggaag tggttt 36 <210> 30 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 cattatgcgg ccgcaagctt tagacccgcg gatcctcc 38 <210> 31 <211> 5324 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 31 atggcactta gcaccgcaac caaggccgcg acggacgcgc tggctgccaa tcgggcaccc 60 accagcgtga atgcacagga agtgcaccgt tggctccaga gcttcaactg ggatttcaag 120 aacaaccgga ccaagtacgc caccaagtac aagatggcga acgagaccaa ggaacagttc 180 aagctgatcg ccaaggaata tgcgcgcatg gaggcagtca aggacgaaag gcagttcggt 240 agcctgcagg atgcgctgac ccgcctcaac gccggtgttc gcgttcatcc gaagtggaac 300 gagaccatga aagtggtttc gaacttcctg gaagtgggcg aatacaacgc catcgccgct 360 accgggatgc tgtgggattc cgcccaggcg gcggaacaga agaacggcta tctggcccag 420 gtgttggatg aaatccgcca cacccaccag tgtgcctacg tcaactacta cttcgcgaag 480 aacggccagg acccggccgg tcacaacgat gctcgccgca cccgtaccat cggtccgctg 540 tggaagggca tgaagcgcgt gttttccgac ggcttcattt ccggcgacgc cgtggaatgc 600 tccctcaacc tgcagctggt gggtgaggcc tgcttcacca atccgctgat cgtcgcagtg 660 accgaatggg ctgccgccaa cggcgatgaa atcaccccga cggtgttcct gtcgatcgag 720 accgacgaac tgcgccacat ggccaacggt taccagaccg tcgtttccat cgccaacgat 780 ccggcttccg ccaagtatct caacacggac ctgaacaacg ccttctggac ccagcagaag 840 tacttcacgc cggtgttggg catgctgttc gagtatggct ccaagttcaa ggtcgagccg 900 tgggtcaaga cgtggaaccg ctgggtgtac gaggactggg gcggcatctg gatcggccgt 960 ctgggcaagt acggggtgga gtcgccgcgc agcctcaagg acgccaagca ggacgcttac 1020 tgggctcacc acgacctgta tctgctggct tatgcgctgt ggccgaccgg cttcttccgt 1080 ctggcgctgc cggatcagga agaaatggag tggttcgagg ccaactaccc cggctggtac 1140 gaccactacg gcaagatcta cgaggaatgg cgcgcccgcg gttgcgagga tccgtcctcg 1200 ggcttcatcc cgctgatgtg gttcatcgaa aacaaccatc ccatctacat cgatcgcgtg 1260 tcgcaagtgc cgttctgccc gagcttggcc aagggcgcca gcaccctgcg cgtgcacgag 1320 tacaacggcc agatgcacac cttcagcgac cagtggggcg agcgcatgtg gctggccgag 1380 ccggagcgct acgagtgcca gaacatcttc gaacagtacg aaggacgcga actgtcggaa 1440 gtgatcgccg aactgcacgg gctgcgcagt gatggcaaga ccctgatcgc ccagccgcat 1500 gtccgtggcg acaagctgtg gacgttggac gatatcaaac gcctgaactg cgtcttcaag 1560 aacccggtga aggcattcaa ttgaaacggg tgtcgggctc cgtcacaggg cggggcccga 1620 cgcacgatcg ttcgatcaac ctcaaaccaa aaaggaacat cgatatgagc atgttaggag 1680 aaagacgccg cggtctgacc gatccggaaa tggcggccgt cattttgaag gcgcttcctg 1740 aagctccgct ggacggcaac aacaagatgg gttatttcgt caccccccgc tggaaacgct 1800 tgacggaata tgaagccctg accgtttatg cgcagcccaa cgccgactgg atcgccggcg 1860 gcctggactg gggcgactgg acccagaaat tccacggcgg ccgcccttcc tggggcaacg 1920 agaccacgga gctgcgcacc gtcgactggt tcaagcaccg tgacccgctc cgccgttggc 1980 atgcgccgta cgtcaaggac aaggccgagg aatggcgcta caccgaccgc ttcctgcagg 2040 gttactccgc cgacggtcag atccgggcga tgaacccgac ctggcgggac gagttcatca 2100 accggtattg gggcgccttc ctgttcaacg aatacggatt gttcaacgct cattcgcagg 2160 gcgcccggga ggcgctgtcg gacgtaaccc gcgtcagcct ggctttctgg ggcttcgaca 2220 agatcgacat cgcccagatg atccaactcg aacggggttt cctcgccaag atcgtacccg 2280 gtttcgacga gtccacagcg gtgccgaagg ccgaatggac gaacggggag gtctacaaga 2340 gcgcccgtct ggccgtggaa gggctgtggc aggaggtgtt cgactggaac gagagcgctt 2400 tctcggtgca cgccgtctat gacgcgctgt tcggtcagtt cgtccgccgc gagttctttc 2460 agcggctggc tccccgcttc ggcgacaatc tgacgccatt cttcatcaac caggcccaga 2520 catacttcca gatcgccaag cagggcgtac aggatctgta ttacaactgt ctgggtgacg 2580 atccggagtt cagcgattac aaccgtaccg tgatgcgcaa ctggaccggc aagtggctgg 2640 agcccacgat cgccgctctg cgcgacttca tggggctgtt tgcgaagctg ccggcgggca 2700 ccactgacaa ggaagaaatc accgcgtccc tgtaccgggt ggtcgacgac tggatcgagg 2760 actacgccag caggatcgac ttcaaggcgg accgcgatca gatcgttaaa gcggttctgg 2820 caggattgaa ataatagagg aactattacg atgagcgtaa acagcaacgc atacgacgcc 2880 ggcatcatgg gcctgaaagg caaggacttc gccgatcagt tctttgccga cgaaaaccaa 2940 gtggtccatg aaagcgacac ggtcgttctg gtcctcaaga agtcggacga gatcaatacc 3000 tttatcgagg agatccttct gacggactac aagaagaacg tcaatccgac ggtaaacgtg 3060 gaagaccgcg cgggttactg gtggatcaag gccaacggca agatcgaggt cgattgcgac 3120 gagatttccg agctgttggg gcggcagttc aacgtctacg acttcctcgt cgacgtttcc 3180 tccaccatcg gccgggccta taccctgggc aacaagttca ccattaccag tgagctgatg 3240 ggcctggacc gcaagctcga agactatcac gcttaaggag aatgacatgg cgaaactggg 3300 tatacacagc aacgacaccc gcgacgcctg ggtgaacaag atcgcgcagc tcaacaccct 3360 ggaaaaagcg gccgagatgc tgaagcagtt ccggatggac cacaccacgc cgttccgcaa 3420 cagctacgaa ctggacaacg actacctctg gatcgaggcc aagctcgaag agaaggtcgc 3480 cgtcctcaag gcacgcgcct tcaacgaggt ggacttccgt cataagaccg ctttcggcga 3540 ggatgccaag tccgttctgg acggcaccgt cgcgaagatg aacgcggcca aggacaagtg 3600 ggaggcggag aagatccata tcggtttccg ccaggcctac aagccgccga tcatgccggt 3660 gaactatttc ctggacggcg agcgtcagtt ggggacccgg ctgatggaac tgcgcaacct 3720 caactactac gacacgccgc tggaagaact gcgcaaacag cgcggtgtgc gggtggtgca 3780 tctgcagtcg ccgcactgaa gggaggaagt ctcgccctgg acgcgacggc atcgccgtga 3840 agtccagggg gcagggatgc cgttccgggc cggcaggctg gcccggaatc tctggttttc 3900 agggggcgtg ccggtccacg gctcccccct ccatctttcg taaggaaatc accatggtcg 3960 aatcggcatt tcagccattt tcgggcgacg cagacgaatg gttcgaggaa ccacggcccc 4020 aggccggttt cttcccttcc gcggactggc atctgctcaa acgggacgag acctacgcag 4080 cctatgccaa ggatctcgat ttcatgtggc ggtgggtcat cgtccgggaa gaaaggatcg 4140 tccaggaggg ttgctcgatc agcctggagt cgtcgatccg cgccgtgacg cacgtactga 4200 attattttgg tatgaccgaa caacgcgccc cggcagagga ccggaccggc ggagttcaac 4260 attgaacagg taagtttatg cagcgagttc acactatcac ggcggtgacg gaggatggcg 4320 aatcgctccg cttcgaatgc cgttcggacg aggacgtcat caccgccgcc ctgcgccaga 4380 acatctttct gatgtcgtcc tgccgggagg gcggctgtgc gacctgcaag gccttgtgca 4440 gcgaagggga ctacgacctc aagggctgca gcgttcaggc gctgccgccg gaagaggagg 4500 aggaagggtt ggtgttgttg tgccggacct acccgaagac cgacctggaa atcgaactgc 4560 cctataccca ttgccgcatc agttttggtg aggtcggcag tttcgaggcg gaggtcgtcg 4620 gcctcaactg ggtttcgagc aacaccgtcc agtttctttt gcagaagcgg cccgacgagt 4680 gcggcaaccg tggcgtgaaa ttcgaacccg gtcagttcat ggacctgacc atccccggca 4740 ccgatgtctc ccgctcctac tcgccggcga accttcctaa tcccgaaggc cgcctggagt 4800 tcctgatccg cgtgttaccg gagggacggt tttcggacta cctgcgcaat gacgcgcgtg 4860 tcggacaggt cctctcggtc aaagggccac tgggcgtgtt cggtctcaag gagcggggca 4920 tggcgccgcg ctatttcgtg gccggcggca ccgggttggc gccggtggtc tcgatggtgc 4980 ggcagatgca ggagtggacc gcgccgaacg agacccgcat ctatttcggt gtgaacaccg 5040 agccggaatt gttctacatc gacgagctca aatccctgga acgatcgatg cgcaatctca 5100 ccgtgaaggc ctgtgtctgg cacccgagcg gggactggga aggcgagcag ggctcgccca 5160 tcgatgcgtt gcgggaagac ctggagtcct ccgacgccaa cccggacatt tatttgtgcg 5220 gtccgccggg catgatcgat gccgcctgcg agctggtacg cagccgcggt atccccggcg 5280 aacaggtctt cttcgaaaaa ttcctgccgt ccggggcggc ctaa 5324

Claims (18)

A recombinant microorganism comprising a foreign gene encoding a soluble methane monooxygenase (sMMO) protein, wherein the microorganism is Escherichia coli. The microorganism according to claim 1, wherein the sMMO protein belongs to EC 1.14.13.25. The method of claim 1, wherein the sMMO protein is selected from the group consisting of Methylococcus capsulatus (Bath). The method of claim 1, wherein the sMMO protein comprises a complex of MmoX, MmoY and MmoZ, MmoB, MmoC, and MmoD, and MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD include SEQ ID NOS: 1, , 9, and 11, respectively. 3. The polynucleotide of claim 1, wherein the gene comprises a polynucleotide having the nucleotide sequence of SEQ ID NO: 2, a polynucleotide having the nucleotide sequence of SEQ ID NO: 4, a polynucleotide having the nucleotide sequence of SEQ ID NO: 6, A polynucleotide having a nucleotide sequence of SEQ ID NO: 10, and a polynucleotide having a nucleotide sequence of SEQ ID NO: 12. The microorganism according to claim 1, wherein the gene is contained in a polynucleotide having a nucleotide sequence of SEQ ID NO: 31. The microorganism according to claim 1, wherein the microorganism further comprises a foreign gene coding for MmoG, and MmoG has an amino acid sequence of SEQ ID NO: 13. A composition comprising a soluble methane monooxygenase (sMMO) protein for use in removing CH _n F _{4 -n} (n is an integer from 0 to 3) in a sample. The method of claim 8, wherein the sMMO protein comprises a complex of MmoX, MmoY and MmoZ, MmoB, MmoC, and MmoD, and MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD include SEQ ID NOS: 1, , 9, and 11, respectively. 9. The method of claim 8, wherein the sMMO protein is expressed from a recombinant microorganism comprising a foreign gene encoding the protein, wherein the composition comprises the microorganism, a lysate thereof, or a water soluble fraction of the lysate &Lt; / RTI &gt; 11. The composition of claim 10, wherein the microorganism is an Escherichia genus. 11. The polynucleotide of claim 10, wherein the gene comprises a polynucleotide having a nucleotide sequence of SEQ ID NO: 2, a polynucleotide having a nucleotide sequence of SEQ ID NO: 4, a polynucleotide having a nucleotide sequence of SEQ ID NO: 6, A polynucleotide having the nucleotide sequence of SEQ ID NO: 10, and a polynucleotide having the nucleotide sequence of SEQ ID NO: 12. The method according to claim 8, wherein the removal of CH _n F _{-n 4} is CH _n F _{-n 4} cutting the binding of the CF (n is an integer of 0 to 3), or switch the CH _n F _{-n 4} with other material, or , And accumulating in the cell to reduce the concentration of CH _n F _4-n . Soluble methane mono-oxide concentration of the dioxygenase (sMMO) protein to CH _n F _{4 -n (n} is an integer of 0 to 3) containing the sample is contacted with a sample of CH _n F _{4 -n (n} is an integer of 0 to 3) _Wherein the concentration of CH _n F _{4 -n in} the sample is reduced. The method of claim 14, wherein the sMMO protein comprises a complex of MmoX, MmoY and MmoZ, MmoB, MmoC, and MmoD, and MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD include SEQ ID NOS: 1, , 9, and 11, respectively. 15. The method of claim 14, wherein the sMMO protein is expressed from a recombinant microorganism comprising a foreign gene encoding the protein, wherein the contacting comprises contacting the microorganism, its lysate, or a water soluble fraction of the lysate with the sample / RTI &gt; 17. The method of claim 16, wherein the contacting is to culture the microorganism in the presence of CH _n F _{4 -n} . 17. The method of claim 16, wherein the contacting is performed in a closed vessel in which the microorganism is viable.

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