KR20160134487A - Microorganism including a gene encoding a protein having hydroxylase activity and method for reducing concentration of fluorinated methane in sample - Google Patents

Abstract

Provided are a microorganism comprising a gene for coding protein having hydroxylase activities, a composition used for reducing the concentration of CHnF4-n (n is an integer from 0 to 3) in a sample comprising the microorganism comprising a gene for coding protein having hydroxylase activities, and a method for reducing the concentration of CHnF4-n in the sample.

Description

FIELD OF THE INVENTION The present invention relates to a microorganism containing a gene coding for a protein having hydroxylase activity and a method for reducing the concentration of fluorinated methane in a sample using the same, methane in sample}

For use in reducing the concentration of fluorinated methane in a sample comprising a microorganism comprising a gene encoding a protein having a hydroxylase activity and a microorganism comprising a gene encoding a protein having a hydroxylase activity, Compositions and methods for reducing the concentration of fluorinated methane in a sample.

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 them, perfluorocarbons (PFCs), hydrofluorocarbons (HFCs), and fluorinated gases such as SF6 (F-gas) have lower emissions but have a longer half-life and a much higher 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.

Hydroxylase is an enzyme that catalyzes the introduction of a hydroxyl group (-OH) to the carbon of the carbon-containing compound (RH). Hydroxylases catalyze the conversion of the CH groups of carbon containing compounds to COH groups. The above-mentioned hydroxylase includes monooxygenase and dehalogenase. The monooxygenase catalyzes a reaction in which one carbon of oxygen is introduced into the carbon position of the carbon-containing compound while the other oxygen atom is reduced to water. The monooxygenase includes cytochrome P450 and methane monooxygenase (MMO). Cytochrome P450 belongs to the superfamily of proteins, including the heme cofactor, and is a hemoprotein. The dehalogenase is one type of enzyme that catalyzes the removal of a halogen atom from a substrate.

However, even in the above-mentioned prior art, there is no disclosure of a microorganism including a hydroxylase gene acting on fluorinated methane, and a composition and a method for reducing the concentration of fluorinated methane in a sample using the same.

One aspect is a recombinant microorganism comprising one or more foreign genes encoding a protein having a hydroxylase activity, wherein the microorganism has increased hydroxylase activity relative to the parent strain.

Another aspect is a recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has an increased hydroxylase activity relative to the parent strain, a lysate thereof, (N is an integer from 0 to 3) in a sample, comprising the aqueous fraction of the degradation product.

Another aspect is a recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has an increased hydroxylase activity as compared to the parent strain, a degradation product thereof or a degradation product thereof Contacting the aqueous fraction of CHnF4-n with a sample containing CHnF4-n (n is an integer of 0 to 3) to reduce the concentration of CHnF4-n (n is an integer of 0 to 3) in the sample. n < / RTI >

One aspect is a recombinant microorganism comprising one or more foreign genes encoding a protein having a hydroxylase activity, wherein the microorganism has increased hydroxylase activity relative to the parent strain.

In the microorganism, the protein having the hydroxylase activity may be an enzyme catalyzing the introduction of a hydroxyl group (-OH) to the carbon of the carbon-containing compound (RH). The hydroxylase can catalyze the conversion of the CH group of the carbon-containing compound to the COH group. The protein having the hydroxylase activity may be one which acts on the bond between carbon-fluorine or carbon-hydrogen of the fluoralkane compound.

The protein having the hydroxylase activity may be selected from the group consisting of desalase and monooxygenase.

The dehalogenase is one type of enzyme that catalyzes the removal of a halogen atom from a substrate. The denaturation agent may be selected from the group consisting of chloroform reductive dehalogenase CfrA, tetrachloroethene dehydrogenase, dichloromethane dehalogenase, haloalkane dehalogenase, alkylhalide, (S) -2-halo acid dehalogenase, (R) -2-halo acid dehalogenase, 2-halo acid configuration-inverting, haloacetate dehalogenase, Lt; / RTI >

The protein having the hydroxylase activity may have 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with SEQ ID NO: 1 or 2. The protein having the hydroxylase activity may have the amino acid sequence of SEQ ID NO: 1 or 2. The protein having the amino acid sequence of SEQ ID NO: 1 may be classified as a haloalkane dehalogenase. The protein having the amino acid sequence of SEQ ID NO: 1 may be an enzyme that catalyzes a reaction to produce primary alcohol and halide using 1-haloalkane and water as a substrate. The protein having the amino acid sequence of SEQ ID NO: 2 may be classified as (S) -2-halo acid desaturase. The protein having the amino acid sequence of SEQ ID NO: 2 may be an enzyme that catalyzes the reaction to produce (R) -hydroxy acid and halide using (S) -2-halo acid and water as a substrate. The at least one foreign gene encoding the protein having the hydroxylase activity may be one having the nucleotide sequence of SEQ ID NOs: 3 and 4. In addition, the gene may be codon-optimized for a recombinant microorganism that is a host cell. The codon optimization indicates that one or more of the endogenous codons encode the same amino acid but produce a gene substituted with a codon that is favorable for expression in the host. The nucleotide sequences of SEQ ID NOS: 3 and 4 are genes coding for haloalkane dehalogenase (dhlA) and (S) -2-halohydrate dehalogenase (dhlB) derived from zottoberterautotrophicus, respectively. Chloroform reductive dehalogenase CfrA has the amino acid sequence of SEQ ID NO: 57 and may be the one encoded by the nucleotide sequence of SEQ ID NO: 56. CfrA is known to dechlorinate chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA) but not 1,1-dichloroethane to dechlorination.

In the recombinant microorganism, the monooxygenase may be one which introduces one carbon of oxygen to the carbon position of the carbon-containing compound while the other oxygen atom catalyzes a monooxygenase reaction which is reduced to water. The monooxygenase includes methane monooxygenase (MMO) and cytochrome P450. Methane monooxygenase is an enzyme capable of oxidizing the C-H bond of methane. Cytochrome P450 (CYP) belongs to the superfamily of proteins, including hem auxiliaries, and is a hemoprotein.

The methane monooxygenase may be soluble methane monooxygenase (sMMO), particulate methane monooxygenase, ammonia monooxygenase, camphor 5-monooxygenase, or a combination thereof. 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 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: 5, 7, 9, 11, 13 and 15, respectively. The polynucleotides encoding MmoX, MmoY, MmoZ, MmoB, MmoC, and MmoD may be those having the nucleotide sequences of SEQ ID NOS: 6, 8, 10, 12, 14 and 16, respectively. In the recombinant microorganism, the gene comprises a polynucleotide having a nucleotide sequence of SEQ ID NO: 6, a polynucleotide having a nucleotide sequence of SEQ ID NO: 8, a polynucleotide having a nucleotide sequence of SEQ ID NO: 10, a polynucleotide having a nucleotide sequence of SEQ ID NO: A polynucleotide having the nucleotide sequence of SEQ ID NO: 14, and a polynucleotide having the nucleotide sequence of SEQ ID NO: 16. In the recombinant microorganism, the gene may be contained in a polynucleotide having a nucleotide sequence of SEQ ID NO: 35. That is, the recombinant microorganism may comprise a polynucleotide having the nucleotide sequence of SEQ ID NO: 35.

The recombinant microorganism may further comprise a foreign gene encoding MmoG. MmoG may have the amino acid sequence of SEQ ID NO: 17. The polynucleotide encoding MmoG may be the one having the nucleotide sequence of SEQ ID NO: 18.

The recombinant microorganism may be one belonging to the genus Escherichia or the genus Zotobacter. The Escherichia genus may contain E. coli. The genus Zanthorrhoea may include Zantho bactorautotrophicus.

In the recombinant microorganism, the cytochrome P450 may be any of those expressed in the microorganism and having monooxygenase activity. The cytochrome P450 may be bacterial P450. The cytochrome P450 may belong to EC 1.14.15.1 or EC 1.14.14.1. The cytochrome P450 protein may be P450Cam or P450BM3. P450Cam may be derived from Pseudomonas putida PpG786. P450BM3 may be derived from Bacillus megaterium (ATCC 14581). The cytochrome P450 protein may be a complex of CamA, CamB, and CamC. CamA, CamB, and CamC may be those having the amino acid sequences of SEQ ID NOS: 37, 39, and 41, respectively. The genes encoding CamA, CamB, and CamC may be those having the nucleotide sequences of SEQ ID NOS: 36, 38, and 40, respectively.

P450BM3 may be a protein of the amino acid sequence of SEQ ID NO: 43. The gene encoding P450BM3 may be the one having the nucleotide sequence of SEQ ID NO: 42.

The gene may comprise at least one of the nucleotide sequences of SEQ ID NOS: 36, 38, 40 and 42.

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.

In the recombinant microorganism, the microorganism may comprise at least one, eg, at least 2, at least 3, at least 3, at least 3, at least 4, at least 5, and at least 10 foreign genes encoding the protein having the hydroxylase activity Or more, or 50 or more. When a plurality of genes are contained in the microorganism, they may be different genes. The gene may be integrated into the genome of the microorganism, or may be independent thereof.

The recombinant microorganism can reduce the concentration of CHnF4-n (n is an integer of 0 to 3) (hereinafter also referred to as "fluorinated methane") in the sample. The reduction may be carried out by introducing a hydroxyl group into the carbon by the action of the protein on the CF or the CH bond of the fluorinated methane, or by accumulating the fluorinated methane in the cells of the microorganism. In addition, the decrease may be that the cleavage of the CF bond of CHnF4-n is cut, or CHnF4-n is converted to another substance, or CHnF4-n is accumulated in the cells. The sample may be liquid or gaseous. The sample may be factory waste water or waste. The sample includes any of the fluorinated methane. The fluorinated methane may be one comprising CF ₄ , CHF ₃ , CH ₂ F ₂ , CH ₃ F, or mixtures thereof.

The recombinant microorganism may be selected from the group consisting of a haloalkane dehalogenase (dhlA) derived from zotto bacteriototrophicus, (s) -2-halohydrate dehalogenase (dhlB) derived from zottoferterautotrophicus, P450CAM derived from water monasufutida, Bacillus megaterium-derived P450BM3, and soluble methane monooxygenase (sMMO) from Methylloccocusucapulatase. The present invention also encompasses a foreign gene encoding a protein having at least one hydroxylase activity selected from the group consisting of have.

In the recombinant microorganism, the gene may be introduced into the microorganism by a conventional method known in the art, for example, transformation, electroporation and the like.

Another aspect is a recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has an increased hydroxylase activity as compared to the parent strain, a degradation product thereof or a degradation product thereof (N is an integer from 0 to 3) in a sample comprising an aqueous fraction of CHnF4-n (where n is an integer from 0 to 3).

In the above composition, the recombinant microorganism, the sample and the fluorinated methane are as described above.

The decomposition product refers to a state in which the microorganism is destroyed and its contents are exposed to the outside of the cell. The degradation products can be obtained by destroying the cells by enzymes such as protease and lipase, heat, pressure, and the like. The "aqueous fraction" may be taken from a material dissolved in an aqueous solvent. The aqueous solvent may be water.

In the above composition, the term "reduction" is intended to reduce the concentration of fluorinated methane in the sample and includes complete removal. The sample may be a gas or a liquid. The sample may not contain the microorganism. The composition may further comprise a substance that increases the solubility of the fluorinated methane relative to the medium or culture.

The composition may be one which reduces the concentration of fluorinated methane in the sample by contacting it with the sample. The contact may be in liquid or solid form. The contacting may be carried out, for example, by bringing the sample into contact with a culture of a microorganism cultured in a medium. The cultivation may be carried out under the condition of growing microorganisms.

Another aspect is a recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has an increased hydroxylase activity as compared to the parent strain, a degradation product thereof or a degradation product thereof Contacting the aqueous fraction of CHnF4-n with a sample containing CHnF4-n (n is an integer of 0 to 3) to reduce the concentration of CHnF4-n (n is an integer of 0 to 3) in the sample. n < / RTI >

In the above method, a recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has increased hydroxylase activity as compared to the parent strain, The aqueous fraction of the decomposed product and the sample containing CHnF4-n (n is an integer of 0 to 3) are as described above.

In the above method, the contact may be in liquid or solid form. The contacting may be carried out, for example, by bringing the sample into contact with a culture of a microorganism cultured in a medium. The culture may be carried out under the condition that the microorganism grows. The contacting may be carried out in an airtight container. The contact may be made when the growth stage of the microorganism is an exponential phase or a stationary phase. The culture may be carried out under aerobic or anaerobic conditions. The contacting may be carried out under conditions in which the recombinant microorganism can survive in a closed container. The viable condition may be a condition allowing the recombinant microorganism to be in a proliferative state or a resting state.

In the above method, the sample may be in a liquid or gaseous state. The sample may be factory waste water or waste. The sample includes not only passive contact with the culture of the microorganism, but also active contact. The sample may be, for example, sparging into the culture medium of the microorganism. That is, the sample may be blown through a medium or a culture medium. The sparging may be blowing from the bottom of the culture medium or the culture medium to the top. The sparging may be injecting while making droplets of the sample.

In the above method, the contact may be performed batchwise or continuously. Said contacting is, for example, a recombinant microorganism comprising at least one foreign gene encoding a protein having fresh hydroxylase activity, wherein said microorganism is an increased hydride With the aqueous fraction of the microorganism, its degradation product or degradation product thereof, which has a rocky silage activity. The contact of the fresh microorganism, the degradation product or the degradation product with the aqueous fraction may be performed more than 2 times, for example, 2, 3, 5, or 10 times or more. The contacting may be continued or repeated for a period of time until a desired reduced concentration of fluorinated methane in the sample is achieved.

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

Compositions according to other aspects may be used to reduce the concentration of CHnF4-n in the sample.

The method of reducing the concentration of CHnF4-n in the sample according to another aspect can effectively reduce the concentration of CHnF4-n in the sample.

Fig. 1A shows the result of decomposition of fluoroform by recombinant E. coli.
Fig. 1 (b) shows the result of decomposition of fluoroform by E. coli into which haloalkane dehalogenase is introduced.
Fig. 2A shows the result of decomposition of perfluoromethane by E. coli into which haloalkane dehalogenase is introduced.
FIG. 2B is a graph showing normalized changes in the concentration of CF ₄ in a sample by E. coli BL21 star / pMALc2-CfrA with negative control values. FIG. In Fig. 2B, the DELTA peak area represents the CfrA area-negative control area and the degradation rate (%) represents the (DELTA peak area / negative control) x100 value.
Figure 3 shows a vector map of the pETDuet-camC-camAB vector.
Fig. 4 shows changes with time in CHF ₃ in head space when E. coli BL21 / pETDuet-camC-camAB was cultured in a medium in contact with CHF ₃ -containing gas.
FIG. 5A shows changes with time of CHCl _{3 in} the head space when E. coli BL21 / pETDuet-camC-camAB was cultured in a CHCl _3- containing medium.
FIG. 5B 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. 6 shows the results of the measurement of pET28a-P450 _BM3 Represents a vector map of a vector.
Figure 7 shows pACYCDuet-zwf Represents a vector map of a vector.
Figure 8 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 .
FIG. 9A shows the change with time of CHCl ₃ in headspace when E. coli BL21 / pET28a-P450 _BM3 was cultured in a CHCl ₃ -containing solution.
FIG. 9B shows changes with time in CF _{4 in} the headspace when Escherichia coli BL21 / pET28a-P450 _BM3 strain was cultured in a medium in contact with CF ₄ -containing gas for 7 days.
Figure 10a shows a vector map of the pET28a-mmoXYBZDC vector.
Figure 10B shows a vector map of the pETDuet-mmoXY-ZD vector.
Figure 10C shows a vector map of the pACYCDuet-mmoBC vector.
Fig. 10D shows a vector map of pACYCDuet-mmoG-BC vector.
11 shows the change in 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.
12A shows changes in the concentration of CHCl _{3 in} the head space when the recombinant E. coli was cultured in a CHCl ₃ -containing medium.
FIG. 12B shows the change with time of CF _{4 in} the headspace when the recombinant E. coli BL21 / pET28a-mmoXYBZDC was cultured in the medium in contact with the CF ₄ -containing gas for 7 days.
13A is a diagram showing that X. autotrophicus GJ10 degrades tetrafluoromethane.
13B is a diagram showing that X. autotrophicus GJ10 Xantho_dhlA degrades tetrafluoromethane.

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: Hedalogaina  By the introduced E. coli Fluoroform  decomposition

(One) Deralogenase  Introduction of genes into E. coli

(1.1) dhlA  And dhlB  Introduction of genes

(DhlA) and (S) -2-halohydrate dehalogenase (dhlB) of Jatobacteriototrophicus GJ10 were selected as enzymes having activity to hydrolyze fluoro-containing hydrocarbons. Xanthobacter autotrophicus GJ10 was purchased from the German Bio Resource Center (DSMZ).

(SEQ ID NO: 3) encoding the haloalkane dehalogenase (dhlA) (SEQ ID NO: 3) and the gene (SEQ ID NO: 4) encoding the (S) -2-halo acid dehalogenase (dhlB) NdeI and HindIII, respectively, to obtain pET28a_dhlA as a dhlA expression vector and pET28a_dhlB as a dhlB expression vector. These vectors were introduced into Escherichia coli, respectively, and sequenced to confirm whether or not they were introduced. The E. coli bacteria into which the haloalkane dehalogenase was introduced and the E. coli into which the (S) -2-halo acid dehalogenase was introduced were designated E. coli_dhlA and E. coli_dhlB, respectively.

(1.2) CfrA  Introduction of genes

Dehalobacter sp. The gene encoding the chloroform reductive dehalogenase CfrA (SEQ ID NO: 56) of CF was inserted into the EcoRI site of the pMALc2 vector (New England Biolabs Inc.) to obtain CrfA expression vector pMALc2-CfrA . This vector was introduced into Escherichia coli BL21 Star and sequenced to confirm whether it was introduced or not. E. coli BL21 star / pMALc2-CfrA, in which the CfrA gene was introduced, was named.

(2) Haloalkane Hedalogaina  By the introduced E. coli Fluoroform  decomposition

The E. coli_dhlA and E. coli_dhlB obtained in Section (1) were placed in a shaking incubator (Daihan Labtech) containing M9 medium at a concentration of 2 × 10 ⁹ cells / ml and the initial concentration of 200 ppm (see FIG. 1A) or 600 ppm only to .coli_dhlA performed: the amount of CHF ₃ and 48 hours of incubation with stirring at 30 to 230rpm while ℃, the headspace of the CHF ₃ see Fig. 1b) it was analyzed. Analysis was taken from the headspace using a syringe and 0.5 ml was injected into the GC (Agilent 7890, Palo Alto, CA, USA). The injected CHF ₃ was isolated via a CP-PoraBOND Q column (25 m length, 0.32 mm id, 5 μm film thickness, Agilent) and analyzed for changes in CHF ₃ concentration via MSD (Agilent 5973, Palo Alto, CA, USA) . The carrier gas was helium and flowed to the column at a rate of 1.5 ml / min. GC conditions were as follows: the inlet temperature was 250 ° C, the initial temperature was 40 ° C for 2 minutes, and the temperature was increased to 290 ° C at 20 ° C / min. MS conditions were 70eV ionization energy, interface temperature was 280 ℃, ion source temperature was 230 ℃, and quadrupole temperature was 150 ℃. Unless otherwise stated, the above methods were used for the analysis of gases such as CHF _3, CHCl _3, and CF ₄ . For the control group, 200 ppm of CHF ₃ without cells was incubated under the same conditions and then measured. M9 medium contained 0.015 g / l CaCl ₂ , 6 g / l Na ₂ HPO ₄ , ₃ g / l KH ₂ PO ₄ , 0.5 g / l NaCl, 1 g / l NH ₄ Cl, 0.5 g / l MgSO ₄ , / l. < / RTI >

Fig. 1A shows the result of decomposition of fluoroform by recombinant E. coli. As shown in Figure 1A, E. coli_dhlA and E. coli_dhlB reduced the amount of fluoroform by 6% and 7%, respectively. The results of FIG. 1A indicate that the haloalkane dehalogenase and (S) -2-halohydrate desaturase have decomposition activity of fluoroform.

Fig. 1 (b) shows the result of decomposition of fluoroform by E. coli into which haloalkane dehalogenase is introduced. As shown in Figure 1b, E. coli_dhlA reduced the amount of fluoroform by 12.4% compared to the control. The results of FIG. 1 b show that when the initial concentration of trifluoromethane is increased, the E. coli into which the haloalkane dehalogenase is introduced can decompose larger amounts of trifluoromethane per hour.

(3) Haloalkane Hedalogaina  The amount of perfluoromethane of  decomposition

It was confirmed whether or not Escherichia coli in which the Zotobacterium avtotrophicus GJ10 -derived haloalkane dehalogenase was introduced had decomposition activity against perfluoromethane (CF ₄ ).

The degree of reduction of the concentration of CF ₄ was analyzed in the same manner as described in (2), except that E. coli_dhlA was used and 600 ppm of CF ₄ was added in the headspace.

Fig. 2A shows the result of decomposition of perfluoromethane by E. coli into which haloalkane dehalogenase is introduced. As shown in Figure 2a, E. coli_dhlA reduced the amount of perfluoromethane by 7.6% compared to the control. From the results of FIG. 2A, it was confirmed that the E. coli into which the haloalkane dehalogenase was introduced had a decomposing activity against perfluoromethane.

The E. coli BL21 star / pMALc2-CfrA prepared in (1) was inoculated into the medium in a shaking incubator and incubated in the presence of 0.2 mM IPTG and 1 μM cobalamin cofactor at 20 ° C. for 20 hours to obtain CfrA Expression of the gene was induced. Cell pellets were obtained from the cultures and disrupted in PBS buffer (Sigma-Aldrich Inc.) as the disruption solution to obtain lysate, and a crude extract was obtained from the lysate. Next, 2 mM Ti (III) -NTA, 2 mM methylviologen and 5 ml crude extract were added to the serum bottle and 1,000 ppm CF ₄ was added to the headspace, followed by incubation at 30 ° C. for the designated time. Negative control (NC) was performed in the same way except that E. coli BL21 star was used. As a result, the final 12% CF ₄ was decomposed. The specific activity of the cells was 0.0044 μmol / cell. Analysis of CF ₄ are as described above.

FIG. 2B is a graph showing normalized changes in the concentration of CF ₄ in a sample by E. coli BL21 star / pMALc2-CfrA with negative control values. FIG. In Fig. 2B, the DELTA peak area represents the CfrA area-negative control area and the decomposition rate represents the DELTA peak area / negative control value.

Example  2: 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 nucleotides of SEQ ID NOS: 40, 36, and 38, respectively, and encode the amino acid sequences of SEQ ID NOS: 41, 37, and 39, 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: 46 and 47, using this CAM plasmid as a template; A primer set of the nucleotide sequences of SEQ ID NOS: 48 and 49; And primer sets of the nucleotide sequences of SEQ ID NOS: 50 and 51 as primers, 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: 46 and 47 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 prepared 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 (Clontech Laboratories, Inc.) to obtain pETDuet-camC -camAB vector.

Figure 3 shows a vector map of the pETDuet-camC-camAB vector.

Next, the prepared pETDuet-camC-camAB vector was introduced into Escherichia coli BL21 strain by heat shock method (Sambrook, J & Russell, DW, New York: Cold Spring Harbor Laboratory Press, 2001), and ampicillin , 100 / / mL), and strains showing resistance to ampicillin were selected. Finally, the selected strains were named as recombinant E. coli BL21 / pETDuet-camC-camAB.

(2) P450 _CAM  In a sample of recombinant E. coli expressing the gene CHF ₃  or CHCl3 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 a TB medium at 30 ° C with stirring at 230 rpm, 0.5 mM IPTG at an OD ₆₀₀ of about 0.5, and then cultured overnight at 25 ° C with stirring at 230 rpm. Recovering the cells and the cell concentration was suspended using M9 medium so that the 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.

Next, CHF ₃ in the gaseous phase was injected through the elastic material of the cap of the serum 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. The gas in the headspace was analyzed at a constant time.

Fig. 4 shows changes with time in CHF ₃ in head space when E. coli BL21 / pETDuet-camC-camAB was cultured in a medium in contact with CHF ₃ -containing gas.

FIG. 5A shows changes with time of CHCl _{3 in} the head space when E. coli BL21 / pETDuet-camC-camAB was cultured in a CHCl _3- containing medium. In FIGS. 4 and 5A and 5B, NC represents a negative control, and 'CAM' represents an experiment using E. coli BL21 / pETDuet-camC-camAB. As shown in Fig. 4, when the cells were cultured for 62 hours and 152 hours, the concentration of CHF _{3 in} the headspace decreased by 5.62% and 17.3%, respectively, as compared with the control. In addition, as shown in FIG. 5A, when cultured for 18 hours, the concentration of CHCl _{3 in} the headspace decreased by 14.8% as compared with the control.

(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. 5B.

FIG. 5B 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 5b, when the culture for 7 days, the concentration of CF ₄ in the headspace than in the control group decreased by 3.57%.

Example  3: 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: 43, respectively, having the nucleotide sequence of SEQ ID NO: 42. 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: 52 and 53 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 6 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: 44 and codes for the amino acid sequence of SEQ ID NO: 45. 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 sequence of SEQ ID NOS: 54 and 55 as a template and the zwf gene was amplified. The amplified zwf gene was ligated with pACYCDuet (Novagen, Cat. No. 71147-3) cut with restriction enzymes NcoI and SacI through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to prepare pACYCDuet-zwf vector. Figure 7 shows pACYCDuet-zwf Represents a vector map of a vector.

Next, the prepared pET28a-P450 _BM3 vector was introduced into Escherichia coli BL21 by thermal shock method and cultured in LB plate medium containing kanamycin (50 μg / mL) 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 heat shocked into Escherichia coli strain BL21, and LB plate medium containing kanamycin (50 / / mL) and chloramphenicol (35 / / mL) And strains showing resistance to kanamycin and chloramphenicol 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 the mixture was incubated overnight at 25 ° C with stirring at 230 rpm. These cells were harvested and suspended in M9 medium to a cell concentration OD ₆₀₀ of 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 2.

Next, CHF ₃ in the gaseous phase was injected through the elastic material of the cap of the serum 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. 8 shows the 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 .

FIG. 9A shows the change in CHCl _{3 in} the head space when E. coli BL21 / pET28a-P450 _BM3 was cultured in CHCl _3- containing medium for 15 hours. 8 and 9A and 9B, NC represents an experiment using negative control, 'BM3' represents an experiment using Escherichia coli BL21 / pET28a-P450 _BM3 + pACYCDuet-zwf for E. coli BL21 / pET28a-P450 _BM3 and 'BM3 + Zwf' . As shown in Fig. 8, when the cells were cultured for 70 hours and 142 hours, the concentration of CHF _{3 in} the headspace was higher than that of the control cells 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 FIG. 9A, when the cells were cultured for 15 hours, the concentration of CHCl _{3 in} the headspace decreased by 4.1% as compared with the control.

(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 result is as shown in Fig. 9B.

FIG. 9B shows changes with time in CF _{4 in} the headspace when Escherichia coli BL21 / pET28a-P450 _BM3 strain was cultured in a medium in contact with CF ₄ -containing gas for 7 days. As it is shown in Figure 9b, when the culture 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} %.

Example  4: sMMO  The recombinant E. coli expressing the gene and the sample using the recombinant E. coli Halomethane  remove

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) strains. 8, 10, 12, 14, 16 and 18 and the nucleotide sequences of SEQ ID NOS: 5, 7, 9, 11, 13, 15, and 17, respectively.

Specifically, PCR was performed 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: 19 and 20 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 10a 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). SEQ ID NOS: 21 and 24, using as primers a PCR amplified mmoX gene fragment using the primer set of the nucleotide sequences of SEQ ID Nos. 21 and 22 and a primer set of the nucleotide sequences of SEQ ID Nos. 23 and 24 as PCR- 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 . Further, the mmoZ gene fragment amplified by PCR using the primer set of the nucleotide sequences of SEQ ID Nos. 25 and 26 and the primer set of the nucleotide sequences of SEQ ID Nos. 27 and 28 were used as a template and PCR was carried out using SEQ ID NO: 25 And a primer set of the nucleotide sequence of 26 were used to amplify the region 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 10B shows a vector map of the pETDuet-mmoXY-ZD vector.

Nos. 29 and 32, using a PCR-amplified mmoB gene fragment using the primer set of the nucleotide sequences of SEQ ID Nos. 29 and 30 and a primer set of the nucleotide sequences of SEQ ID NOS: 31 and 32 as a template, 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 10C 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 primer sets of nucleotide sequences of SEQ ID NOs: 29 and 30 and a primer set of nucleotide sequences of SEQ ID NOs: 31 and 32 were used as a template and PCR was carried out using SEQ ID NO: 29 And a primer set of nucleotide sequence of 32 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. Fig. 10D shows a vector map of 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 introduced into Escherichia coli BL21 strain by heat shock, mL of ampicillin and 35 μg / mL of chloramphenicol or 50 μg / mL of kanamycin, and strains showing resistance to ampicillin and chloramphenicol or kanamycin 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 by culturing with stirring at rpm, OD ₆₀₀ 0.5 degree in IPTG 0.1 mM, and ferric citrate (ferric citrate) 0.1 mg / ml , ferrule's sulfate (ferrous sulfate) 0.1 mg / ml , and ferric ammonium citrate (ferric ammonium citrate) 0.1 mg / ml, and cysteine 1 mM were added, followed by overnight incubation 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.

Next, in the case of CHF _3, using a syringe a CHF ₃ on the base body so that the 1,000 ppm of the headspace was injected through the elastic material of the cap of the serum sickness. In the case of CHCl ₃ it was injected through the elastic material of the cap of the serum sickness using a syringe to be 0.02 mM from the CHCl ₃ in the liquid 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 time during the culture, CHF ₃ or CHCl ₃ in the headspace of the sera was analyzed in the same manner as described in (2) of Example 2.

11 shows the change in 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. 11, 1 is a control group, and 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. 11, 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.

12A shows changes in the concentration of CHCl _{3 in} the head space when the recombinant E. coli was cultured in a CHCl ₃ -containing medium. 12A, 1 to 4 are as described with reference to FIG. As shown in Figure 12a, when a culture of 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.

(3) sMMO  In a sample by recombinant E. coli expressing the gene CF ₄  Removal effect

In this section, we have confirmed the effect of the recombinant E. coli BL21 / pET28a-mmoXYBZDC with the sMMO gene prepared in (1) on the removal of CF ₄ in the sample. As a control, E. coli BL21 / pET28a into which an empty vector without sMMO gene was introduced was used.

Experiments (2) in the process performed with respect to the CHF ₃ in the section, using CF ₄ in place of CHF _3, and using the syringe to be 1000ppm the CF ₄ on the gas head space over the injection through the elastic material of the serum sickness cap And thereafter, the serum bottle was incubated for 7 days with stirring at 30 DEG C and 200 rpm. The result is as shown in Fig. 12B.

FIG. 12B shows the change with time of CF _{4 in} the headspace when the recombinant E. coli BL21 / pET28a-mmoXYBZDC was cultured in the medium in contact with the CF ₄ -containing gas for 7 days. In FIG. 12B, NC represents a negative control, and 'MMO' represents an experiment using E. coli BL21 / pET28a-mmoXYBZDC. As it is shown in Figure 12b, a case, and decreases the amount of CF ₄ in the headspace than in the control group 3.42% to incubate for 7 days.

Example  5: Hedalogaina  Introduced Xanthobacter autotrophicus On by Of tetrafluoromethane  decomposition

58 and 59 as primers and the genomic sequence of Xanthobacter autotrophicus GJ10 purchased from the German Center for Biological Resources (DSMZ) as a template and as a primer, the amplified dhlA gene No. 3) was introduced into a pTSa vector using an In-Fusion HD Cloning Kit (Clontech) to prepare a pTSa_DhlA vector (SEQ ID NO: 60) (ORF: 2982-3914).

The prepared vector was introduced into X. autotrophicus GJ10 strain by electrophoresis and Xantho_dhlA was designated as a strain which was confirmed to have introduced the dhlA gene. This strain was cultured at 30 DEG C in 250 mL plastic flask in 50 mL M9 medium with stirring at 230 rpm overnight.

The sera containing 10 ml of 2x10 ⁹ cells / ml Xantho_dhlA in M9 medium and containing 600 ppm or 1000 ppm CF ₄ in the headspace were incubated for 48 hours at 30 ° C with stirring in a shaking incubator (Daihan Labtech) at 230 rpm. Thereafter, CF ₄ in the head space was analyzed, and the control group was carried out in the same manner except that the control group contained 600 ppm or 1000 ppm CF ₄ in the head space without cells and X. autotrophicus GJ10 was used under the same conditions. The results are shown in Figs. 13A and 13B. 13A and 13B, Xantho represents X. autotrophicus GJ10, Xantho_dhlA represents X. autotrophicus GJ10 Xantho_dhlA, and the vertical axis represents the peak area, i.e., AP peak area.

13A is a diagram showing that X. autotrophicus GJ10 degrades tetrafluoromethane. As shown in FIG. 13A, when 600 ppm CF ₄ was contained in the head space, X. autotrophicus GJ10 reduced the amount of tetrafluoromethane by 12.94% as compared with the control group.

13B is a diagram showing that X. autotrophicus GJ10 Xantho_dhlA degrades tetrafluoromethane. As shown in Fig. 13B, when 1000 ppm CF ₄ was contained in the head space, the amount of tetrafluoromethane was reduced by 16.29% for X. autotrophicus GJ10 Xantho_dhlA and 12.04% for X. autotrophicus GJ10 compared to the control group. Thus, X. autotrophicus GJ10 Xantho_dhlA degraded CF ₄ significantly more efficiently than X. autotrophicus GJ10.

<110> SAMSUNG ELECTRONIS CO., LTD. <120> Microorganism including a gene encoding a protein having          hydroxylase activity and method for reducing concentration of          fluorinated methane in sample <130> PN112574KR <160> 60 <170> Kopatentin 2.0 <210> 1 <211> 311 <212> PRT <213> Xanthobacter autotrophicus <400> 1 Met Ile Asn Ala Ile Arg Thr Pro Asp Gln Arg Phe Ser Asn Leu Asp   1 5 10 15 Gln Tyr Pro Phe Ser Pro Asn Tyr Leu Asp Asp Leu Pro Gly Tyr Pro              20 25 30 Gly Leu Arg Ala His Tyr Leu Asp Glu Gly Asn Ser Asp Ala Glu Asp          35 40 45 Val Phe Leu Cys Leu His Gly Glu Pro Thr Trp Ser Tyr Leu Tyr Arg      50 55 60 Lys Met Ile Pro Val Phe Ala Glu Ser Gly Ala Arg Val Ile Ala Pro  65 70 75 80 Asp Phe Phe Gly Phe Gly Lys Ser Asp Lys Pro Val Asp Glu Glu Asp                  85 90 95 Tyr Thr Phe Glu Phe His Arg Asn Phe Leu Leu Ala Leu Ile Glu Arg             100 105 110 Leu Asp Leu Arg Asn Ile Thr Leu Val Val Gln Asp Trp Gly Gly Phe         115 120 125 Leu Gly Leu Thr Leu Pro Met Ala Asp Pro Ser Arg Phe Lys Arg Leu     130 135 140 Ile Ile Met Asn Ala Cys Leu Met Thr Asp Pro Val Thr Gln Pro Ala 145 150 155 160 Phe Ser Ala Phe Val Thr Gln Pro Ala Asp Gly Phe Thr Ala Trp Lys                 165 170 175 Tyr Asp Leu Val Thr Pro Ser Asp Leu Arg Leu Asp Gln Phe Met Lys             180 185 190 Arg Trp Ala Pro Thr Leu Thr Glu Ala Glu Ala Ser Ala Tyr Ala Ala         195 200 205 Pro Phe Pro Asp Thr Ser Tyr Gln Ala Gly Val Arg Lys Phe Pro Lys     210 215 220 Met Val Ala Gln Arg Asp Gln Ala Cys Ile Asp Ile Ser Thr Glu Ala 225 230 235 240 Ile Ser Phe Trp Gln Asn Asp Trp Asn Gly Gln Thr Phe Met Ala Ile                 245 250 255 Gly Met Lys Asp Lys Leu Leu Gly Pro Asp Val Met Tyr Pro Met Lys             260 265 270 Ala Leu Ile Asn Gly Cys Pro Glu Pro Leu Glu Ile Ala Asp Ala Gly         275 280 285 His Phe Val Gln Glu Phe Gly Glu Gln Val Ala Arg Glu Ala Leu Lys     290 295 300 His Phe Ala Glu Thr Glu Glx 305 310 <210> 2 <211> 254 <212> PRT <213> Xanthobacter autotrophicus <400> 2 Met Ile Lys Ala Val Val Phe Asp Ala Tyr Gly Thr Leu Phe Asp Val   1 5 10 15 Gln Ser Val Ala Asp Ala Thr Glu Arg Ala Tyr Pro Gly Arg Gly Glu              20 25 30 Tyr Ile Thr Gln Val Trp Arg Gln Lys Gln Leu Glu Tyr Ser Trp Leu          35 40 45 Arg Ala Leu Met Gly Arg Tyr Ala Asp Phe Trp Gly Val Thr Arg Glu      50 55 60 Ala Leu Ala Tyr Thr Leu Gly Thr Leu Gly Leu Glu Pro Asp Glu Ser  65 70 75 80 Phe Leu Ala Gly Met Ala Gln Ala Tyr Asn Arg Leu Thr Pro Tyr Pro                  85 90 95 Asp Ala Ala Gln Cys Leu Ala Glu Leu Ala Pro Leu Lys Arg Ala Ile             100 105 110 Leu Ser Asn Gly Ala Pro Asp Met Leu Gln Ala Leu Val Ala Asn Ala         115 120 125 Gly Leu Thr Asp Ser Phe Asp Ala Val Ile Ser Val Asp Ala Lys Arg     130 135 140 Val Phe Lys Pro His Pro Asp Ser Tyr Ala Leu Val Glu Glu Val Leu 145 150 155 160 Gly Val Thr Pro Ala Glu Val Leu Phe Val Ser Ser Asn Gly Phe Asp                 165 170 175 Val Gly Gly Ala Lys Asn Phe Gly Phe Ser Val Ala Arg Val Ala Arg             180 185 190 Leu Ser Gln Glu Ala Leu Ala Arg Glu Leu Val Ser Gly Thr Ile Ala         195 200 205 Pro Leu Thr Met Phe Lys Ala Leu Arg Met Arg Glu Glu Thr Tyr Ala     210 215 220 Glu Ala Pro Asp Phe Val Val Pro Ala Leu Gly Asp Leu Pro Arg Leu 225 230 235 240 Val Arg Gly Met Ala Gly Ala His Leu Ala Pro Ala Val Glx                 245 250 <210> 3 <211> 933 <212> DNA <213> Xanthobacter autotrophicus <400> 3 atgataaatg caattcgcac cccggaccaa cgcttcagca atctcgatca gtatccgttc 60 agccccaact acctggacga cctccccggc tacccgggat tgcgggcaca ctacctcgac 120 gagggcaatt ctgacgctga agacgttttt ctctgccttc atggcgagcc cacctggagt 180 tacctgtatc gcaagatgat cccggtattt gctgaatcag gcgcacgagt tattgcgcca 240 gacttttttg gattcggaaa atccgacaag ccagtagacg aagaagacta caccttcgaa 300 tttcaccgca acttcctgct tgcactaatc gaacggcttg acttgcgcaa cattacgctg 360 gtcgttcagg actggggcgg atttttgggg ctgaccttac cgatggccga cccttcccgc 420 ttcaagcgcc tgatcatcat gaacgcctgc ttgatgaccg acccggtcac ccagcctgcg 480 tttagcgcct ttgtcaccca gcctgcggat ggctttaccg cctggaaata cgatctggtt 540 acgccatcag acctgcgcct tgaccagttc atgaagcgtt gggcgcccac actgaccgaa 600 gctgaggcct ccgcgtatgc tgcgcctttc cctgacactt cctatcaggc tggtgtacgc 660 aagtttccca agatggtcgc gcaacgcgac caggcctgca tcgacatttc aaccgaagcg 720 atttcgttct ggcagaacga ctggaatggc cagaccttca tggccattgg catgaaagac 780 aaattgctgg gaccggacgt catgtatcct atgaaggcgc tcattaatgg ctgcccggaa 840 cccctcgaaa tagcggacgc tggccatttc gtacaggagt ttggcgagca agtggctcgc 900 gaggccctga aacactttgc cgagacagaa tag 933 <210> 4 <211> 762 <212> DNA <213> Xanthobacter autotrophicus <400> 4 atgatcaagg cagtcgtgtt cgacgcttac ggtacgctct tcgacgtcca gtcggtggcc 60 gcgccaccg agcgggcgta tccaggccgg ggcgagtaca tcacgcaggt ctggcggcag 120 aagcagctgg aatacagctg gctccgcgcg ctgatggggc gctatgccga cttttggggc 180 gtcacgcggg aagcgctggc ctataccctc ggaacgctgg ggctggagcc ggacgagtcc 240 ttcctcgccg ggatggcgca ggcctacaac cgcctcacgc cctatccgga cgccgcgcaa 300 tgcctcgcgg agctggcgcc cctcaagcgc gccatcctct ccaacggcgc gcccgacatg 360 ctgcaggcgc tcgtggccaa tgcgggcctg acggacagct tcgatgccgt catcagcgtc 420 gatgccaagc gcgtgttcaa gcctcatccc gactcctacg cgctggtgga ggaggtacta 480 ggcgtgacgc ccgcggaggt gctgttcgtg tcctccaacg gcttcgacgt cggcggcgcg 540 aagaatttcg gcttcagcgt cgcccgggtc gcgcgcctgt cgcaggaggc gctggcgcgc 600 gaactcgtct cgggtaccat cgcgcccctg accatgttca aggcgctgag gatgcgggaa 660 gaaacctatg cggaggcgcc tgatttcgtg gtgcccgccc ttggcgacct gccgcggctg 720 gttcgcggga tggccggcgc tcatctcgca ccagcggtgt ga 762 <210> 5 <211> 527 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 5 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> 6 <211> 1584 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 6 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> 7 <211> 389 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 7 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> 8 <211> 1170 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 8 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> 9 <211> 170 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 9 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> 10 <211> 513 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 10 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> 11 <211> 141 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 11 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> 12 <211> 426 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 12 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> 13 <211> 348 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 13 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> 14 <211> 1047 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 14 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> 15 <211> 103 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 15 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> 16 <211> 312 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 16 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> 17 <211> 559 <212> PRT <213> Methylococcus capsulatus (Bath) <400> 17 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> 18 <211> 1680 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 18 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> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aagaaggaga tataccatgg cacttagcac cgcaac 36 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gtggtggtgg tggtgctcga ttaggccgcc ccggacggca 40 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 aagaaggaga tataccatgg cacttagcac cgcaac 36 <210> 22 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaattctgtt tcctgtgtga ttaattgaat gccttcacc 39 <210> 23 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tcacacagga aacagaattc atgagcatgt taggagaaa 39 <210> 24 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 cattatgcgg ccgcaagctt tatttcaatc ctgccaga 38 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 aagaaggaga tatacatatg gcgaaactgg gtatac 36 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 gaattctgtt tcctgtgtga ttagtgcggc gactgcaga 39 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 tcacacagga aacagaattc atggtcgaat cggcatttc 39 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 gtttctttac cagactcgat taatgttgaa ctccgccggt 40 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 aagaaggaga tatacatatg agcgtaaaca gcaacg 36 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 atgtatatct ccttcttata ttaagcgtga tagtcttcg 39 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 tataagaagg agatatacat atgcagcgag ttcacacta 39 <210> 32 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 gatcgcgtgg ccggccgatt taggccgccc cggacggca 39 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 taataaggag atataccatg gcaaaggaag tggttt 36 <210> 34 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 cattatgcgg ccgcaagctt tagacccgcg gatcctcc 38 <210> 35 <211> 5324 <212> DNA <213> Methylococcus capsulatus (Bath) <400> 35 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 <210> 36 <211> 1269 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 36 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> 37 <211> 422 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 37 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> 38 <211> 324 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 38 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> 39 <211> 107 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 39 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> 40 <211> 1248 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 40 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> 41 <211> 415 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 41 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> 42 <211> 3150 <212> DNA <213> Bacillus megaterium (ATCC 14581) <400> 42 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> 43 <211> 1049 <212> PRT <213> Bacillus megaterium (ATCC 14581) <400> 43 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> 44 <211> 1476 <212> DNA <213> E. coli K12 (MG1655) <400> 44 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> 45 <211> 491 <212> PRT <213> E. coli K12 (MG1655) <400> 45 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> 46 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer: CAM_F <400> 46 taagaaggag atatacatat gaacgcaaac gacaacg 37 <210> 47 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer: camA_R <400> 47 catgaattct gtttcctgtg tgattaggca ctactcagtt ca 42 <210> 48 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer: camB_F <400> 48 taatcacaca ggaaacagaa ttcatgtcta aagtagtgta tg 42 <210> 49 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: camB_R <400> 49 ggtttcttta ccagactcga ttaccattgc ctatcgggaa 40 <210> 50 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer: camC_F <400> 50 aagaaggaga tataccatga cgactgaaac cataca 36 <210> 51 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: camC_R <400> 51 gcattatgcg gccgcaagct ttataccgct ttggtagtcg 40 <210> 52 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer: P450bm3_F <400> 52 aagaaggaga tataccatga caattaaaga aatgcct 37 <210> 53 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: P450bm3_R <400> 53 gtggtggtgg tggtgctcga ttacccagcc cacacgtctt 40 <210> 54 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer: Zwf_F <400> 54 ttaagaagga gatataccat ggcggtaacg caaacagc 38 <210> 55 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer: Zwf_R <400> 55 tcgacctgca ggcgcgccgt tactcaaact cattccagg 39 <210> 56 <211> 1371 <212> DNA <213> Dehalobacter sp. CF <400> 56 atggacaagg aaaaaagtaa caacgataag ccggcaacaa aaattaatcg cagacaattc 60 cttaaatttg gagctggagc ttcttcgggt attgcaattg ccactgcagc tactgcattg 120 ggagggaaat cacttatcga tcccaaacag gtatatgctg gaacggtcaa ggaactggat 180 gaacttccct ttaatatccc ggcagactac aaaccgttta ccaatcaaag gaatatatat 240 ggccaggctg tattgggagt acccgaacct ctagcacttg tagagcgttt tgatgaagta 300 agatggaatg gttggcagac agatggttcg cccggtctta ctgtacttga tggtgcggct 360 gctcgtgcaa gctttgccgt tgattattat tttaacgggg aaaatagcgc ctgcagggcc 420 aataaaggtt tttttgaatg gcatcccaaa gtggccgagc tgaactttaa gtggggcgat 480 ccggagagaa atattcattc ccccggtgta aaaagtgccg aagaaggaac gatggcagta 540 aaaaaaatag ctagattttt cggcgctgct aaagctggga tagcgccttt tgacaaacgt 600 tgggttttta ctgaaacgta tgcctttgtt aaaacgcctg agggtgaaag tctgaaattt 660 atccctccgg attttgggtt tgagcccaag catgtaatct cgatgattat cccacagtcg 720 ccagaaggag taaagtgtga cccgtccttt ttaggatcaa ctgaatatgg attaagttgt 780 gcccagattg gatatgctgc attcggttta tccatgttta ttaaagatct gggatatcat 840 gcggttccaa tcggatctga cagtgcatta gctataccta tagctattca ggcgggtctg 900 ggggaataca gcaggtcggg gctaatgatt acgcctgaat ttggttcaaa tgttagactc 960 tgtgaagtat ttactgacat gcctttaaat catgataaac ctatttcatt cggagtaact 1020 gaattttgca aaacctgcaa aaaatgcgct gaagcatgcg cccctcaagc tattagctat 1080 gaagatccta ccattgatgg acctcgtggg caaatgcaaa attcgggaat aaagagatgg 1140 tatgttgacc cggtgaagtg cttagaattc atgtcgcgtg ataacgtcgg aaactgctgc 1200 ggagcttgta tagctgcttg cccatttact aagccggaag cctggcacca taccttaatt 1260 aggagtctag taggagcacc tgttattact ccattcatga aagatatgga tgatattttt 1320 ggatacggaa agctgaatga tgaaaaagcg atagcagatt ggtggaaata a 1371 <210> 57 <211> 456 <212> PRT <213> Dehalobacter sp. CF <400> 57 Met Asp Lys Glu Lys Ser Asn Asn Asp Lys Pro Ala Thr Lys Ile Asn   1 5 10 15 Arg Arg Gln Phe Leu Lys Phe Gly Ala Gly Ala Ser Ser Gly Ile Ala              20 25 30 Ile Ala Thr Ala Ala Thr Ala Leu Ily Asp Pro          35 40 45 Lys Gln Val Tyr Ala Gly Thr Val Lys Glu Leu Asp Glu Leu Pro Phe      50 55 60 Asn Ile Pro Ala Asp Tyr Lys Pro Phe Thr Asn Gln Arg Asn Ile Tyr  65 70 75 80 Gly Gln Ala Val Leu Gly Val Pro Glu Pro Leu Ala Leu Val Glu Arg                  85 90 95 Phe Asp Glu Val Arg Trp Asn Gly Trp Gln Thr Asp Gly Ser Pro Gly             100 105 110 Leu Thr Val Leu Asp Gly Ala Ala Ala Arg Ala Ser Phe Ala Val Asp         115 120 125 Tyr Tyr Phe Asn Gly Glu Asn Ser Ala Cys Arg Ala Asn Lys Gly Phe     130 135 140 Phe Glu Trp His Pro Lys Val Ala Glu Leu Asn Phe Lys Trp Gly Asp 145 150 155 160 Pro Glu Arg Asn Ile His Ser Pro Gly Val Lys Ser Ala Glu Glu Gly                 165 170 175 Thr Met Ala Val Lys Lys Ile Ala Arg Phe Phe Gly Ala Ala Lys Ala             180 185 190 Gly Ile Ala Pro Phe Asp Lys Arg Trp Val Phe Thr Glu Thr Tyr Ala         195 200 205 Phe Val Lys Thr Pro Glu Gly Glu Ser Leu Lys Phe Ile Pro Pro Asp     210 215 220 Phe Gly Phe Glu Pro Lys His Val Ser Ser Ile Pro Gln Ser 225 230 235 240 Pro Glu Gly Val Lys Cys Asp Pro Ser Phe Leu Gly Ser Thr Glu Tyr                 245 250 255 Gly Leu Ser Cys Ala Gln Ile Gly Tyr Ala Ala Phe Gly Leu Ser Met             260 265 270 Phe Ile Lys Asp Leu Gly Tyr His Ala Val Pro Ile Gly Ser Asp Ser         275 280 285 Ala Leu Ala Ile Pro Ile Ala Ile Gln Ala Gly Leu Gly Glu Tyr Ser     290 295 300 Arg Ser Gly Leu Met Ile Thr Pro Glu Phe Gly Ser Asn Val Arg Leu 305 310 315 320 Cys Glu Val Phe Thr Asp Met Pro Leu Asn His Asp Lys Pro Ile Ser                 325 330 335 Phe Gly Val Thr Glu Phe Cys Lys Thr Cys Lys Lys Cys Ala Glu Ala             340 345 350 Cys Ala Pro Gln Ala Ile Ser Tyr Glu Asp Pro Thr Ile Asp Gly Pro         355 360 365 Arg Gly Gln Met Gln Asn Ser Gly Ile Lys Arg Trp Tyr Val Asp Pro     370 375 380 Val Lys Cys Leu Glu Phe Met Ser Arg Asp Asn Val Gly Asn Cys Cys 385 390 395 400 Gly Ala Cys Ile Ala Ala Cys Pro Phe Thr Lys Pro Glu Ala Trp His                 405 410 415 His Thr Leu Ile Arg Ser Leu Val Gly Ala Pro Val Ile Thr Pro Phe             420 425 430 Met Lys Asp Met Asp Asp Ile Phe Gly Tyr Gly Lys Leu Asn Asp Glu         435 440 445 Lys Ala Ile Ala Asp Trp Trp Lys     450 455 <210> 58 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 58 gacgcttacg gaggctctat gataaatgca attcgcac 38 <210> 59 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 59 tggcagttcc ctactctcct attctgtctc ggcaaa 36 <210> 60 <211> 3914 <212> DNA <213> Artificial Sequence <220> <223> pTSa_DhlA vector <400> 60 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg 60 aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg 120 cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag 180 gccatcctga cggatggcct ttttggaatt cagccagcaa gacagcgata gagggtagtt 240 atccacgtga aaccgctaat gccccgcaaa gccttgattc acggggcttt ccggcccgct 300 ccaaaaacta tccacgtgaa atcgctaatc agggtacgtg aaatcgctaa tcggagtacg 360 tgaaatcgct aataaggtca cgtgaaatcg ctaatcaaaa aggcacgtga gaacgctaat 420 agccctttca gatcaacagc ttgcaaacac ccctcgctcc ggcaagtagt tacagcaagt 480 agtatgttca attagctttt caattatgaa tatatatatc aattattggt cgcccttggc 540 ttgtggacaa tgcgctacgc gcaccggctc cgcccgtgga caaccgcaag cggttgccca 600 ccgtcgagcg ccagcgcctt tgcccacaac ccggcggccg gccgcaacag atcgttttat 660 aaattttttt ttttgaaaaa gaaaaagccc gaaaggcggc aacctctcgg gcttctggat 720 ttccgatcac ctgtaagtcg gacgaattcg gcgctcttcc gcttcctcgc tcactgactc 780 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 840 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 900 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 960 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 1020 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 1080 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 1140 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 1200 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 1260 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 1320 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac 1380 agcatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 1440 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 1500 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 1560 tcagtggaac gaaaactcac gttaattctc atgtttgaca gcttatcatc gataagcttt 1620 aatgcggtag tttatcacag ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta 1680 acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc ataggcttgg 1740 ttatgccggt actgccgggc ctcttgcggg atatcgtcca ttccgacagc atcgccagtc 1800 actatggcgt gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg 1860 gagcactgtc cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta cttggagcca 1920 ctatcgacta cgcgatcatg gcgaccacac ccgtcctgtg gatcctctac gccggacgca 1980 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca 2040 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta 2100 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc 2160 ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta atgcaggagt 2220 cgcataaggg agagcgtcga ccgatgccct tgagagcctt caacccagtc agctccttcc 2280 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac 2340 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga 2400 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag 2460 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca 2520 tggcggccga cgcgctggcc tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct 2580 tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg caggccatgc 2640 tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc gcggctctta 2700 ccagcctaac ttcgatcact ggaccgctga tcgtcacggc gatttatgcc gcctcggcga 2760 gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc tgcctccccg 2820 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct 2880 cgctaacgga ttcaccactc ttgacattgt aggtcaggcg acctactttg tcattgctag 2940 gtcacccgac ctaacttttg acagacgctt acggaggctc tatgataaat gcaattcgca 3000 ccccggacca acgcttcagc aatctcgatc agtatccgtt cagccccaac tacctggacg 3060 acctccccgg ctacccggga ttgcgggcac actacctcga cgagggcaat tctgacgctg 3120 aagacgtttt tctctgcctt catggcgagc ccacctggag ttacctgtat cgcaagatga 3180 tcccggtatt tgctgaatca ggcgcacgag ttattgcgcc agactttttt ggattcggaa 3240 aacccgacaa gccagtagac gaagaagact acaccttcga atttcaccgc aacttcctgc 3300 ttgcactaat cgaacggctt gacttgcgca acattacgct ggtcgttcag gactggggcg 3360 gatttttggg gctgacctta ccgatggccg acccttcccg cttcaagcgc ctgatcatca 3420 tgaacgcctg cttgatgacc gacccggtca cccagcctgc gtttagcgcc tttgtcaccc 3480 agcctgcgga tggctttacc gcctggaaat acgatctggt tacgccatca gacctgcgcc 3540 ttgaccagtt catgaagcgt tgggcgccca cactgaccga agctgaggcc tccgcgtatg 3600 ctgcgccttt ccctgacact tcctatcagg ctggtgtacg caagtttccc aagatggtcg 3660 cgcaacgcga ccaggcctgc atcgacattt caaccgaagc gatttcgttc tggcagaacg 3720 actggaatgg ccagaccttc atggccattg gcatgaaaga caaattgctg ggaccggacg 3780 tcatgtatcc tatgaaggcg ctcattaatg gctgcccgga acccctcgaa atagcggacg 3840 ctggccattt cgtacaggag tttggcgagc aagtggctcg cgaggccctg aaacactttg 3900 ccgagacaga atag 3914

Claims (20)

A recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has an increased hydroxylase activity as compared to the parent strain, a lysate thereof or a degradation product thereof (N is an integer from 0 to 3) in a sample, wherein the concentration of CHnF &lt; 4 &gt; The composition according to claim 1, wherein the protein having the hydroxylase activity acts on the bond between carbon-fluorine or carbon-hydrogen of the fluoralkane compound. The composition of claim 1, wherein the protein is selected from the group consisting of dehalogenase, and monooxygenase. [4] The method of claim 3, wherein the derogagenase is selected from the group consisting of tetrachloroethene dehydrogenase, dichloromethane dehalogenase, haloalkane dehalogenase, alkylhalide, (S) -2- (R) -2-halo acid dehalogenase, 2-halo acid configuration-inverting, haloacetate dehalogenase, or a combination thereof. 4. The composition of claim 3, wherein the monooxygenase is soluble methane monooxygenase (sMMO), ammonia monooxygenase, camphor 5-monooxygenase, cytochrome P450, or a combination thereof. 6. The composition of claim 5, wherein the cytochrome P450 is bacterial cytochrome P450. [2] The method according to claim 1, wherein the protein is selected from the group consisting of haloalkane dehalogenase (dhlA) derived from zottoferterautotrophicus, (s) -2-halohydrate dehalogenase (dhlB) derived from zottoferterautotrophicus, Wherein the composition is at least one selected from the group consisting of multi-derived P450CAM, P450BM3 derived from Bacillus megaterium, and soluble methane monooxygenase (sMMO) derived from methylrococcus capsulatus. The method according to claim 1, wherein the reduction is performed by cutting the CF bond of CHnF4-n (n is an integer of 0 to 3), converting CHnF4-n into another fragment, or accumulating CHnF4-n in the cell &Lt; / RTI &gt; The composition according to claim 1, wherein the sample is liquid or gaseous. The composition according to claim 1, wherein the microorganism belongs to the genus Escherichia or genus Xanthobacter. An Escherichia recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has increased hydroxylase activity relative to the parent strain, a microorganism whose lysate ) Or an aqueous fraction thereof is contacted with a sample containing CHnF4-n (n is an integer of 0 to 3) to reduce the concentration of CHnF4-n (n is an integer of 0 to 3) in the sample. A method for reducing the concentration of CHnF4-n in a sample. 12. The method of claim 11, wherein the protein is selected from the group consisting of dehalogenases, and monooxygenases. [Claim 13] The method of claim 12, wherein the derogagenase is selected from the group consisting of tetrachloroethene dehydrogenase, dichloromethane dehalogenase, haloalkane dehalogenase, alkylhalide, (S) -2- (R) -2-halo acid dehalogenase, 2-halo acid configuration-inverting, haloacetate dehalogenase, or a combination thereof. 13. The method of claim 12, wherein the monooxygenase is soluble methane monooxygenase (sMMO), ammonia monooxygenase, camphor 5-monooxygenase, cytochrome P450, or a combination thereof. 14. The method of claim 13, wherein the cytochrome P450 is bacterial cytochrome P450. 12. The method of claim 11, wherein the protein is selected from the group consisting of haloalkane dehalogenase (dhlA) derived from zotonburterautotrophicus, (s) -2-halohydrate dehalogenase (dhlB) derived from zotobacterautotrophicus, P450BM3 derived from Bacillus megaterium, and soluble methane monooxygenase (sMMO) derived from Methylorcoccus capsulatus. 12. The method of claim 11, wherein the contacting is performed in an airtight container. 12. The recombinant microorganism according to claim 11, wherein the contact is a recombinant microorganism comprising at least one foreign gene encoding a protein having a hydroxylase activity, wherein the microorganism has increased hydroxylase activity as compared to the parent strain, And culturing or incubating the lysate or an aqueous fraction of the lysate with contacting with a sample containing CHnF4-n (n is an integer of 0 to 3). 12. The method of claim 11, wherein said contacting comprises culturing under conditions in which the recombinant microorganism is grown in an enclosed vessel. 12. The method according to claim 11, wherein the microorganism belongs to the genus Escherichia or Xanthobacter.

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