KR20170067125A - Bacterial cytochrome 450 protein and method for reducing concentration of fluorinated methane in sample - Google Patents

Abstract

A recombinant microorganism comprising a foreign gene encoding a bacterial cytochrome P450 protein, a composition comprising a recombinant P450 protein for use in removing CHnF4-n (n is an integer from 0 to 3) in the sample, and a concentration of CHnF4-n in the sample / RTI >

Description

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

A recombinant microorganism comprising a foreign gene encoding a bacterial cytochrome P450 protein, a composition comprising a recombinant P450 protein for use in removing CHnF4-n (n is an integer from 0 to 3) in the sample, and a concentration of CHnF4-n in the sample / RTI >

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

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

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

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

Cytochrome P450 BM3 (CYP102A1) from B. megaterium catalyzes NADPH-dependent hydroxylation of several long chain fatty acids at the omega-1 to omega-3 positions. Cytochrome P450 BM3 consists of a natural fusion protein between the CYP domain and an electron donating cofactor.

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

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

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

Another aspect involves contacting a recombinant P450 protein with a sample containing CHnF4-n (n is an integer from 0 to 3) to reduce the concentration of CHnF4-n (n is an integer from 0 to 3) in the sample. Lt; RTI ID = 0.0 > CHnF4-n. ≪ / RTI >

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

In the recombinant microorganism, the cytochrome P450 protein may belong to EC 1.14.15.1 or EC 1.14.14.1. The cytochrome P450 protein may be P450Cam or P450 _BM3 . P450Cam may be derived from Pseudomonas putida PpG786. P450 _BM3 may be derived from Bacillus megaterium (ATCC 14581). The cytochrome P450 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: 2, 4, and 6, respectively. The genes encoding CamA, CamB, and CamC may be those having the nucleotide sequences of SEQ ID NOS: 1, 3, and 5, respectively.

P450 _BM3 may be a complex of the amino acid sequence of SEQ ID NO: 8. The gene encoding P450 _BM3 may be the one having the nucleotide sequence of SEQ ID NO: 7.

The recombinant microorganism may belong to the genus Escherichia. The Escherichia bacteria may be E. coli. The gene may comprise at least one of the nucleotide sequences of SEQ ID NOS: 1, 3, 5 and 7.

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

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

In this composition, CHnF4-n may be, for example, CHF3, CH2F2, CH3F, CF4, CHCl3, CH2Cl2, CH3Cl, or CCl4. The term "removal" includes reducing the concentration of CHnF4-n in the sample. The reduction includes complete removal.

In this composition, the recombinant P450 protein may be in the form of a recombinant microorganism, a lysate thereof, a water soluble fraction of the lysate thereof, or a recombinant P450 protein, which contains the expressed bacterial cytochrome P450 protein. Bacterial cytochrome P450 may be one expressed from a foreign gene. The microorganism may be an Escherichia genus. The microorganism Escherichia can be E. coli.

In such a composition, the removal may be accomplished by cleaving the bond of CF in CHnF4-n (where n is an integer from 0 to 3), or by converting CHnF4-n to another substance or accumulating it in the cell to reduce the concentration of CHnF4- ≪ / RTI > The conversion may be by introducing a hydrophilic group such as a hydroxyl group into CHnF4-n, or by introducing a carbon-carbon double bond or a carbon-carbon triple bond.

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

Another aspect involves contacting a recombinant P450 protein with a sample containing CHnF4-n (n is an integer from 0 to 3) to reduce the concentration of CHnF4-n (n is an integer from 0 to 3) in the sample. Lt; RTI ID = 0.0 > CHnF4-n. ≪ / RTI >

In the above method, the contact may be performed in an airtight container. The contact may be a gas-liquid contact contacting the gas sample with a recombinant P450 protein containing liquid. The contact may also be a liquid-liquid contact contacting the liquid sample with a liquid containing the recombinant P450 protein. The liquid-liquid contact includes mixing.

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

In the above method, the contacting may be carried out under conditions in which the recombinant microorganism can survive in the closed container. The viable condition may be a condition allowing the recombinant microorganism to be in a proliferative state or a resting state. In this case, the contacting may be to culture the microorganism in the presence of CHnF4-n. The culture may be carried out under aerobic or anaerobic conditions. The microorganism may be an Escherichia genus. The microorganism Escherichia can be E. coli.

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

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

A composition comprising a recombinant P450 protein according to another aspect can be used to remove 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.

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

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

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

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

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

The P450 _CAM gene amplified the camC, camA, and camB genes from the CAM plasmid of Pseudomonas putida PpG786. The camC gene has the nucleotide sequence of SEQ ID NO: 5 and encodes the amino acid sequence of SEQ ID NO: The camA gene has the nucleotide sequence of SEQ ID NO: 1 and encodes the amino acid sequence of SEQ ID NO: 2. The camB gene has the nucleotide sequence of SEQ ID NO: 3 and encodes the amino acid sequence of SEQ ID NO: 4. Specifically, P. putida PpG786 strain DSM 7162 was cultivated in LB medium at 30 ° C with stirring at 230 rpm overnight, and then cultured in a total DNA extraction kit (Invitrogen Biotechnology) The plasmid was separated, and using this CAM plasmid as a template,

A primer set of the nucleotide sequences of SEQ ID NOS: 11 and 12; A primer set of the nucleotide sequences of SEQ ID NOS: 13 and 14; And a primer set of the nucleotide sequences of SEQ ID NOS: 15 and 16, respectively, to obtain amplified camA, camB, and camC genes, respectively.

The camC gene amplified by PCR using the primer set of the nucleotide sequences of SEQ ID NOS: 11 and 12 was amplified by PCR using pETDuet (Novagen, Cat. No. 71146-3) and InFusion Cloning Kit (Clontech Laboratories , Inc.) to prepare a pETDuet-camC vector. The 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 1 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 by recombinant E. coli expressing the gene CHF ₃  Or CHCl ₃  Removal effect

In this section, the effect of the E. coli BL21 / pETDuet-camC-camAB strain introduced with the P450 _CAM gene prepared in (1) on the removal of CHF ₃ or CHCl ₃ in the sample was confirmed. Specifically, Escherichia coli BL21 / pETDuet-camC-camAB was cultured in a TB medium at 30 ° C with stirring at 230 rpm. After adding 0.5 mM IPTG at an OD ₆₀₀ of about 0.5, the cells were incubated overnight at 25 ° C with stirring at 230 rpm. Lt; / RTI > The cells recovered (harvest), and the cell concentration was suspended (suspension) with an 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. In addition, the M9 medium contained components of 6 g of Na ₂ HPO ₄ , ₃ g of KH ₂ PO ₄ , 0.5 g of NaCl, and 1 g of NH ₄ Cl per liter of distilled water.

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

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

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

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

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

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

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

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

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

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

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

P450 _BM3 gene was amplified P450 _BM3 gene of Bacillus megaterium (ATCC 14581) strain. The P450 _BM3 gene encodes the amino acid sequence of SEQ ID NO: 8, respectively, having the nucleotide sequence of SEQ ID NO: 7. Specifically, B. megaterium (ATCC 14581) was cultured in LB medium at 30 ° C with stirring at 230 rpm overnight,

Genomic DNA was isolated by a method using a total DNA extraction kit (Invitrogen Biotechnology), PCR was performed using the genomic DNA as a template and the nucleotide sequences of SEQ ID NOS: 17 and 18 as a primer set, P450 _BM3 gene was amplified. The amplified P450 _BM3 gene was ligated with pET28a (Novagen, Cat. No. 69864-3) cleaved with restriction enzymes NcoI and XhoI through an InFusion Cloning Kit (Clontech Laboratories, Inc.) to produce pET28a-P450 _BM3 vector Respectively. Figure 4 shows a vector map of pET28a-P450 _BM3 vector.

In addition, zwf gene encoding glucose 6-phosphate dehydrogenase of E. coli K12 (MG1655) was amplified to increase intracellular NADPH content. The Zwf gene has the nucleotide sequence of SEQ ID NO: 9 and encodes the amino acid sequence of SEQ ID NO: 10. Specifically, E. coli was cultured overnight in LB medium at 37 DEG C with stirring at 230 rpm,

Genomic DNA was isolated by a method using a total DNA extraction kit (Invitrogen Biotechnology), PCR was performed using this genomic DNA as a template and the nucleotide sequences of SEQ ID NOS: 19 and 20 as a primer set, zwf gene. 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 5 shows the pACYCDuet-zwf Represents a vector map of a vector.

Next, the prepared pET28a-P450 _BM3 vector was introduced into Escherichia coli BL21 by heat shock and cultured in an LB plate medium containing kanamycin (50 μg / mL) to produce kanamycin resistant The strains were selected. The finally selected strains were named recombinant E. coli BL21 / pET28a-P450 _BM3 .

The resulting pET28a-P450 _BM3 vector and pACYCDuet-zwf vector were introduced into Escherichia coli strain BL21 by heat shock method and LB (50 μg / mL) containing kanamycin (50 μg / mL) and chloramphenicol And cultured on a plate medium to select strains showing kanamycin and chloramphenicol resistance. 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 of IPTG was added at OD _{600 of} 0.5 and then incubated overnight at 25 ° C with stirring at 230 rpm Respectively. The 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 1. [

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

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

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

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

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

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

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

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

<110> Samsung Electronics Co., Ltd. <120> Bacterial cytochrome 450 protein and method for reducing          concentration of fluorinated methane in sample <130> PN111296 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 1269 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 1 atgaacgcaa acgacaacgt ggtcatcgtc ggtaccggac tggctggcgt tgaggtcgcc 60 ttcggcctgc gcgccagcgg ctgggaaggc aatatccggt tggtggggga tgcgacggta 120 attccccatc acctaccacc gctatccaaa gcttacttgg ccggcaaagc cacagcggaa 180 agcctgtacc tgagaacccc agatgcctat gcagcgcaga acatccaact actcggaggc 240 acacaggtaa cggctatcaa ccgcgaccga cagcaagtaa tcctatcgga tggccgggca 300 ctggattacg accggctggt attggctacc ggagggcgtc caagacccct accggtggcc 360 agtggcgcag ttggaaaggc gaacaacttt cgatacctgc gcacactcga ggacgccgag 420 tgcattcgcc ggcagctgat tgcggataac cgtctggtgg tgattggtgg cggctacatt 480 ggccttgaag tggctgccac cgccatcaag gcgaacatgc acgtcaccct gcttgatacg 540 gcagcccggg ttctggagcg ggttaccgcc ccgccggtat cggcctttta cgagcaccta 600 caccgcgaag ccggcgttga catacgaacc ggcacgcagg tgtgcgggtt cgagatgtcg 660 accgaccaac agaaggttac tgccgtcctc tgcgaggacg gcacaaggct gccagcggat 720 ctggtaatcg ccgggattgg cctgatacca aactgcgagt tggccagtgc ggccggcctg 780 caggttgata acggcatcgt gatcaacgaa cacatgcaga cctctgatcc cttgatcatg 840 gccgtcggcg actgtgcccg atttcacagt cagctctatg accgctgggt gcgtatcgaa 900 tcggtgccca atgccttgga gcaggcacga aagatcgccg ccatcctctg tggcaaggtg 960 ccacgcgatg aggcggcgcc ctggttctgg tccgatcagt atgagatcgg attgaagatg 1020 gtcggactgt ccgaagggta cgaccggatc attgtccgcg gctctttggc gcaacccgac 1080 ttcagcgttt tctacctgca gggagaccgg gtattggcgg tcgatacagt gaaccgtcca 1140 gtggagttca accagtcaaa acaaataatc acggatcgtt tgccggttga accaaaccta 1200 ctcggtgacg aaagcgtgcc gttaaaggaa atcatcgccg ccgccaaagc tgaactgagt 1260 agtgcctaa 1269 <210> 2 <211> 422 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 2 Met Asn Ala Asn Asp Asn Val Val Ile Val Gly Thr Gly Leu Ala Gly   1 5 10 15 Val Glu Val Ala Phe Gly Leu Arg Ala Ser Gly Trp Glu Gly Asn Ile              20 25 30 Arg Leu Val Gly Asp Ala Thr Val Ile Pro His His Leu Pro Pro Leu          35 40 45 Ser Lys Ala Tyr Leu Ala Gly Lys Ala Thr Ala Glu Ser Leu Tyr Leu      50 55 60 Arg Thr Pro Asp Ala Tyr Ala Ala Gln Asn Ile Gln Leu Leu Gly Gly  65 70 75 80 Thr Gln Val Thr Ala Ile Asn Arg Asp Arg Gln Gln Val Ile Leu Ser                  85 90 95 Asp Gly Arg Ala Leu Asp Tyr Asp Arg Leu Val Leu Ala Thr Gly Gly             100 105 110 Arg Pro Pro Leu Pro Val Ala Ser Gly Ala Val Gly Lys Ala Asn         115 120 125 Asn Phe Arg Tyr Leu Arg Thr Leu Glu Asp Ala Glu Cys Ile Arg Arg     130 135 140 Gln Leu Ile Ala Asp Asn Arg Leu Val Val Ile Gly Gly Gly Tyr Ile 145 150 155 160 Gly Leu Glu Val Ala Ala Thr Ala Ile Lys Ala Asn Met His Val Thr                 165 170 175 Leu Leu Asp Thr Ala Ala Arg Val Leu Glu Arg Val Thr Ala Pro Pro             180 185 190 Val Ser Ala Phe Tyr Glu His Leu His Arg Glu Ala Gly Val Asp Ile         195 200 205 Arg Thr Gly Thr Gln Val Cys Gly Phe Glu Met Ser Thr Asp Gln Gln     210 215 220 Lys Val Thr Ala Val Leu Cys Glu Asp Gly Thr Arg Leu Pro Ala Asp 225 230 235 240 Leu Val Ile Ala Gly Ile Gly Leu Ile Pro Asn Cys Glu Leu Ala Ser                 245 250 255 Ala Ala Gly Leu Glu Val Asp Asn Gly Ile Val Ile Asn Glu His Met             260 265 270 Gln Thr Ser Asp Pro Leu Ile Met Ala Val Gly Asp Cys Ala Arg Phe         275 280 285 His Ser Gln Leu Tyr Asp Arg Trp Val Arg Ile Glu Ser Val Pro Asn     290 295 300 Ala Leu Glu Gln Ala Arg Lys Ile Ala Ala Ile Leu Cys Gly Lys Val 305 310 315 320 Pro Arg Asp Glu Ala Ala Pro Trp Phe Trp Ser Asp Gln Tyr Glu Ile                 325 330 335 Gly Leu Lys Met Val Gly Leu Ser Glu Gly Tyr Asp Arg Ile Ile Val             340 345 350 Arg Gly Ser Leu Ala Gln Pro Asp Phe Ser Val Phe Tyr Leu Gln Gly         355 360 365 Asp Arg Val Leu Ala Val Asp Thr Val Asn Arg Pro Val Glu Phe Asn     370 375 380 Gln Ser Lys Gln Ile Ile Thr Asp Arg Leu Pro Val Glu Pro Asn Leu 385 390 395 400 Leu Gly Asp Glu Ser Val Pro Leu Lys Glu Ile Ile Ala Ala Ala Lys                 405 410 415 Ala Glu Leu Ser Ser Ala             420 <210> 3 <211> 324 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 3 atgtctaaag tagtgtatgt gtcacatgat ggaacgcgtc gcgaactgga tgtggcggat 60 ggcgtcagcc tgatgcaggc tgcagtctcc aatggtatct acgatattgt cggtgattgt 120 ggcggcagcg ccagctgtgc cacctgccat gtctatgtga acgaagcgtt cacggacaag 180 gtgcccgccg ccaacgagcg ggaaatcggc atgctggagt gcgtcacggc cgaactgaag 240 ccgaacagca ggctctgctg ccagatcatc atgacgcccg agctggatgg catcgtggtc 300 gatgttcccg ataggcaatg gtaa 324 <210> 4 <211> 107 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 4 Met Ser Lys Val Val Tyr Val Ser His Asp Gly Thr Arg Arg Glu Leu   1 5 10 15 Asp Val Ala Asp Gly Val Ser Leu Met Gln Ala Ala Val Ser Asn Gly              20 25 30 Ile Tyr Asp Ile Val Gly Asp Cys Gly Gly Ser Ala Ser Cys Ala Thr          35 40 45 Cys His Val Tyr Val Asn Glu Ala Phe Thr Asp Lys Val Pro Ala Ala      50 55 60 Asn Glu Arg Glu Ile Gly Met Leu Glu Cys Val Thr Ala Glu Leu Lys  65 70 75 80 Pro Asn Ser Arg Leu Cys Cys Gln Ile Ile Met Thr Pro Glu Leu Asp                  85 90 95 Gly Ile Val Val Asp Val Pro Asp Arg Gln Trp             100 105 <210> 5 <211> 1248 <212> DNA <213> P. putida PpG786 strain DSM 7162 <400> 5 atgacgactg aaaccataca aagcaacgcc aatcttgccc ctctgccacc ccatgtgcca 60 gagcacctgg tattcgactt cgacatgtac aatccgtcga atctgtctgc cggcgtgcag 120 gaggcctggg cagttctgca agaatcaaac gtaccggatc tggtgtggac tcgctgcaac 180 ggcggacact ggatcgccac tcgcggccaa ctgatccgtg aggcctatga agattaccgc 240 cacttttcca gcgagtgccc gttcatccct cgtgaagccg gcgaagccta cgacttcatt 300 cccacctcga tggatccgcc cgagcagcgc cagtttcgtg cgctggccaa ccaagtggtt 360 ggcatgccgg tggtggataa gctggagaac cggatccagg agctggcctg ctcgctgatc 420 gagagcctgc gcccgcaagg acagtgcaac ttcaccgagg actacgccga acccttcccg 480 atacgcatct tcatgctgct cgcaggtcta ccggaagaag atatcccgca cttgaaatac 540 ctaacggatc agatgacccg tccggatggc agcatgacct tcgcagaggc caaggaggcg 600 ctctacgact atctgatacc gatcatcgag caacgcaggc agaagccggg aaccgacgct 660 atcagcatcg ttgccaacgg ccaggtcaat gggcgaccga tcaccagtga cgaagccaag 720 aggatgtgtg gcctgttact ggtcggcggc ctggatacgg tggtcaattt cctcagcttc 780 agcatggagt tcctggccaa aagcccggag catcgccagg agctgatcga gcgtcccgag 840 cgtattccag ccgcttgcga ggaactactc cggcgcttct cgctggttgc cgatggccgc 900 atcctcacct ccgattacga gtttcatggc gtgcaactga agaaaggtga ccagatcctg 960 ctaccgcaga tgctgtctgg cctggatgag cgcgaaaacg cctgcccgat gcacgtcgac 1020 ttcagtcgcc aaaaggtttc acacaccacc tttggccacg gcagccatct gtgccttggc 1080 cagcacctgg cccgccggga aatcatcgtc accctcaagg aatggctgac caggattcct 1140 gacttctcca ttgccccggg tgcccagatt cagcacaaga gcggcatcgt cagcggcgtg 1200 caggcactcc ctctggtctg ggatccggcg actaccaaag cggtataa 1248 <210> 6 <211> 415 <212> PRT <213> P. putida PpG786 strain DSM 7162 <400> 6 Met Thr Thr Glu Thr Ile Gln Ser Asn Ala Asn Leu Ala Pro Leu Pro   1 5 10 15 Pro His Val Pro Glu His Leu Val Phe Asp Phe Asp Met Tyr Asn Pro              20 25 30 Ser Asn Leu Ser Ala Gly Val Glu Glu Ala Trp Ala Val Leu Gln Glu          35 40 45 Ser Asn Val Pro Asp Leu Val Trp Thr Arg Cys Asn Gly Gly His Trp      50 55 60 Ile Ala Thr Arg Gly Gln Leu Ile Arg Glu Ala Tyr Glu Asp Tyr Arg  65 70 75 80 His Phe Ser Ser Glu Cys Pro Phe Ile Pro Arg Glu Ala Gly Glu Ala                  85 90 95 Tyr Asp Phe Ile Pro Thr Ser Met Asp Pro Pro Glu Gln Arg Gln Phe             100 105 110 Arg Ala Leu Ala Asn Gln Val Val Gly Met Pro Val Val Asp Lys Leu         115 120 125 Glu Asn Arg Ile Gln Glu Leu Ala Cys Ser Leu Ile Glu Ser Leu Arg     130 135 140 Pro Gln Gly Gln Cys Asn Phe Thr Glu Asp Tyr Ala Glu Pro Phe Pro 145 150 155 160 Ile Arg Ile Phe Met Leu Leu Ala Gly Leu Pro Glu Glu Asp Ile Pro                 165 170 175 His Leu Lys Tyr Leu Thr Asp Gln Met Thr Arg Pro Asp Gly Ser Met             180 185 190 Thr Phe Ala Glu Ala Lys Glu Ala Leu Tyr Asp Tyr Leu Ile Pro Ile         195 200 205 Ile Glu Gln Arg Arg Gln Lys Pro Gly Thr Asp Ile Ser Ile Val     210 215 220 Ala Asn Gly Gln Val Asn Gly Arg Pro Ile Thr Ser Asp Glu Ala Lys 225 230 235 240 Arg Met Cys Gly Leu Leu Leu Val Gly Gly Leu Asp Thr Val Val Asn                 245 250 255 Phe Leu Ser Phe Ser Met Glu Phe Leu Ala Lys Ser Pro Glu His Arg             260 265 270 Gln Glu Leu Ile Glu Arg Pro Glu Arg Ile Pro Ala Ala Cys Glu Glu         275 280 285 Leu Leu Arg Arg Phe Ser Leu Val Ala Asp Gly Arg Ile Leu Thr Ser     290 295 300 Asp Tyr Glu Phe His Gly Val Gln Leu Lys Lys Gly Asp Gln Ile Leu 305 310 315 320 Leu Pro Gln Met Leu Ser Gly Leu Asp Glu Arg Glu Asn Ala Cys Pro                 325 330 335 Met His Val Asp Phe Ser Arg Gln Lys Val Ser His Thr Thr Phe Gly             340 345 350 His Gly Ser His Leu Cys Leu Gly Gln His Leu Ala Arg Arg Glu Ile         355 360 365 Ile Val Thr Leu Lys Glu Trp Leu Thr Arg Ile Pro Asp Phe Ser Ile     370 375 380 Ala Pro Gly Ala Gln Ile Gln His Lys Ser Gly Ile Val Ser Gly Val 385 390 395 400 Gln Ala Leu Pro Leu Val Trp Asp Pro Ala Thr Thr Lys Ala Val                 405 410 415 <210> 7 <211> 3150 <212> DNA <213> Bacillus megaterium (ATCC 14581) <400> 7 atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60 ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120 tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180 gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240 gattttgcag gagacgggtt atttacaagc tggacgcatg aaaaaaattg gaaaaaagcg 300 cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360 gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420 gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480 tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540 gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600 gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660 attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgca tatgctaaac 720 ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780 acattcttaa ttgcgggaca cgaaacaaca agtggtcttt tatcatttgc gctgtatttc 840 ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900 gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaaac 960 gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020 gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080 cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140 gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg tcagcgtgcg 1200 tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260 cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320 aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380 tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440 acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500 ttagcagata ttgcaatgag caaaggattt gcaccgcagg tcgcaacgct tgattcacac 1560 gccggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggtcat 1620 ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680 aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740 aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800 cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860 atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920 tctactcttt cacttcaatt tgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980 ggtgcgtttt caacgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040 agcacgcgac atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100 ttaggtgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160 ctagatgcat cacagcaaat ccgtctggaa gcagaagaag aaaaattagc tcatttgcca 2220 ctcgctaaaa cagtatccgt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280 acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340 cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400 atgcttgaac tgcttgaaaa atacccggcg tgtgaaatga aattcagcga atttatcgcc 2460 cttctgccaa gcatacgccc gcgctattac tcgatttctt catcacctcg tgtcgatgaa 2520 aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580 tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640 tttatttcca caccgcagtc agaatttacg ctgccaaaag accctgaaac gccgcttatc 2700 atggtcggac cgggaacagg cgtcgcgccg tttagaggct ttgtgcaggc gcgcaaacag 2760 ctaaaagaac aaggacagtc acttggagaa gcacatttat acttcggctg ccgttcacct 2820 catgaagact atctgtatca agaagagctt gaaaacgccc aaagcgaagg catcattacg 2880 cttcataccg ctttttctcg catgccaaat cagccgaaaa catacgttca gcacgtaatg 2940 gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000 ggagacggaa gccaaatggc acctgccgtt gaagcaacgc ttatgaaaag ctatgctgac 3060 gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120 cgatacgcaa aagacgtgtg ggctgggtaa 3150 <210> 8 <211> 1049 <212> PRT <213> Bacillus megaterium (ATCC 14581) <400> 8 Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu Leu Lys   1 5 10 15 Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu Met Lys              20 25 30 Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro Gly Arg          35 40 45 Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala Cys Asp      50 55 60 Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Ala Leu Lys Phe Val Arg  65 70 75 80 Asp Phe Ala Gly Asp Gly Leu Phe Thr Ser Trp Thr His Glu Lys Asn                  85 90 95 Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln Gln Ala             100 105 110 Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln Leu Val         115 120 125 Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val Pro Glu     130 135 140 Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly Phe Asn 145 150 155 160 Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe Ile Thr                 165 170 175 Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Leu Gln Arg Ala             180 185 190 Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe Gln Glu         195 200 205 Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala Asp Arg     210 215 220 Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met Leu Asn 225 230 235 240 Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn Ile Arg                 245 250 255 Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr Ser Gly             260 265 270 Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His Val Leu         275 280 285 Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro Val Pro     290 295 300 Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val Leu Asn 305 310 315 320 Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu Tyr Ala                 325 330 335 Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys Gly Asp             340 345 350 Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr Ile Trp         355 360 365 Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn Pro Ser     370 375 380 Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln Arg Ala 385 390 395 400 Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val Leu Gly                 405 410 415 Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr Glu Leu             420 425 430 Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val Val Lys         435 440 445 Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Ser Ser Thr     450 455 460 Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala His Asn 465 470 475 480 Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala Glu Gly                 485 490 495 Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe Ala Pro             500 505 510 Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg Glu Gly         515 520 525 Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro Asp Asn     530 535 540 Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp Glu Val 545 550 555 560 Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn Trp Ala                 565 570 575 Thr Thr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu Ala Ala             580 585 590 Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala Ser Asp         595 600 605 Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp Ser Asp     610 615 620 Val Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp Asn Lys 625 630 635 640 Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met Pro Leu                 645 650 655 Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Ala Ser Lys Glu             660 665 670 Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu Ile Glu         675 680 685 Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly Val Ile     690 695 700 Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg Phe Gly 705 710 715 720 Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu Lys Leu                 725 730 735 Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu Leu Gln             740 745 750 Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg Ala Met         755 760 765 Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu Ala Leu     770 775 780 Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg Leu Thr 785 790 795 800 Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys Phe Ser                 805 810 815 Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr Ser Ile             820 825 830 Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr Val Ser         835 840 845 Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys Gly Ile     850 855 860 Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile Thr Cys 865 870 875 880 Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp Pro Glu                 885 890 895 Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg             900 905 910 Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln Ser Leu         915 920 925 Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu Asp Tyr     930 935 940 Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile Ile Thr 945 950 955 960 Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr Tyr Val                 965 970 975 Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu Leu Asp             980 985 990 Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met Ala Pro         995 1000 1005 Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val His Gln Val    1010 1015 1020 Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu Glu Glu Lys Gly 1025 1030 1035 1040 Arg Tyr Ala Lys Asp Val Trp Ala Gly                1045 <210> 9 <211> 1476 <212> DNA <213> E. coli K12 (MG1655) <400> 9 atggcggtaa cgcaaacagc ccaggcctgt gacctggtca ttttcggcgc gaaaggcgac 60 cttgcgcgtc gtaaattgct gccttccctg tatcaactgg aaaaagccgg tcagctcaac 120 ccggacaccc ggattatcgg cgtagggcgt gctgactggg ataaagcggc atataccaaa 180 gttgtccgcg aggcgctcga aactttcatg aaagaaacca ttgatgaagg tttatgggac 240 accctgagtg cacgtctgga tttttgtaat ctcgatgtca atgacactgc tgcattcagc 300 cgtctcggcg cgatgctgga tcaaaaaaat cgtatcacca ttaactactt tgccatgccg 360 cccagcactt ttggcgcaat ttgcaaaggg cttggcgagg caaaactgaa tgctaaaccg 420 gcacgcgtag tcatggagaa accgctgggg acgtcgctgg cgacctcgca ggaaatcaat 480 gatcaggttg gcgaatactt cgaggagtgc caggtttacc gtatcgacca ctatcttggt 540 aaagaaacgg tgctgaacct gttggcgctg cgttttgcta actccctgtt tgtgaataac 600 tgggacaatc gcaccattga tcatgttgag attaccgtgg cagaagaagt ggggatcgaa 660 gggcgctggg gctattttga taaagccggt cagatgcgcg acatgatcca gaaccacctg 720 ctgcaaattc tttgcatgat tgcgatgtct ccgccgtctg acctgagcgc agacagcatc 780 cgcgatgaaa aagtgaaagt actgaagtct ctgcgccgca tcgaccgctc caacgtacgc 840 gaaaaaaccg tacgcgggca atatactgcg ggcttcgccc agggcaaaaa agtgccggga 900 tatctggaag aagagggcgc gaacaagagc agcaatacag aaactttcgt ggcgatccgc 960 gtcgacattg ataactggcg ctgggccggt gtgccattct acctgcgtac tggtaaacgt 1020 ctgccgacca aatgttctga agtcgtggtc tatttcaaaa cacctgaact gaatctgttt 1080 aaagaatcgt ggcaggatct gccgcagaat aaactgacta tccgtctgca acctgatgaa 1140 ggcgtggata tccaggtact gaataaagtt cctggccttg accacaaaca taacctgcaa 1200 atcaccaagc tggatctgag ctattcagaa acctttaatc agacgcatct ggcggatgcc 1260 tatgaacgtt tgctgctgga aaccatgcgt ggtattcagg cactgtttgt acgtcgcgac 1320 gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 1380 gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 1440 acccgtgatg gtcgttcctg gaatgagttt gagtaa 1476 <210> 10 <211> 491 <212> PRT <213> E. coli K12 (MG1655) <400> 10 Met Ala Val Thr Gln Thr Ala Gln Ala Cys Asp Leu Val Ile Phe Gly   1 5 10 15 Ala Lys Gly Asp Leu Ala Arg Arg Lys Leu Leu Pro Ser Leu Tyr Gln              20 25 30 Leu Glu Lys Ala Gly Gln Leu Asn Pro Asp Thr Arg Ile Ile Gly Val          35 40 45 Gly Arg Ala Asp Trp Asp Lys Ala Ala Tyr Thr Lys Val Val Arg Glu      50 55 60 Ala Leu Glu Thr Phe Met Lys Glu Thr Ile Asp Glu Gly Leu Trp Asp  65 70 75 80 Thr Leu Ser Ala Arg Leu Asp Phe Cys Asn Leu Asp Val Asn Asp Thr                  85 90 95 Ala Ala Phe Ser Arg Leu Gly Ala Met Leu Asp Gln Lys Asn Arg Ile             100 105 110 Thr Ile Asn Tyr Phe Ala Met Pro Pro Ser Thr Phe Gly Ala Ile Cys         115 120 125 Lys Gly Leu Gly Aly Lys Leu Asn Aly Lys Pro Ala Arg Val Val     130 135 140 Met Glu Lys Pro Leu Gly Thr Ser Leu Ala Thr Ser Gln Glu Ile Asn 145 150 155 160 Asp Gln Val Gly Glu Tyr Phe Glu Glu Cys Gln Val Tyr Arg Ile Asp                 165 170 175 His Tyr Leu Gly Lys Glu Thr Val Leu Asn Leu Leu Ala Leu Arg Phe             180 185 190 Ala Asn Ser Leu Phe Val Asn Asn Trp Asp Asn Arg Thr Ile Asp His         195 200 205 Val Glu Ile Thr Val Ala Glu Glu Val Gly Ile Glu Gly Arg Trp Gly     210 215 220 Tyr Phe Asp Lys Ala Gly Gln Met Arg Asp Met Ile Gln Asn His Leu 225 230 235 240 Leu Gln Ile Leu Cys Met Ile Ala Met Ser Pro Pro Ser Asp Leu Ser                 245 250 255 Ala Asp Ser Ile Arg Asp Glu Lys Val Lys Val Leu Lys Ser Leu Arg             260 265 270 Arg Ile Asp Arg Ser Asn Val Arg Glu Lys Thr Val Arg Gly Gln Tyr         275 280 285 Thr Ala Gly Phe Ala Gln Gly Lys Lys Val Pro Gly Tyr Leu Glu Glu     290 295 300 Glu Gly Ala Asn Lys Ser Ser Asn Thr Glu Thr Phe Val Ala Ile Arg 305 310 315 320 Val Asp Ile Asp Asn Trp Arg Trp Ala Gly Val Pro Phe Tyr Leu Arg                 325 330 335 Thr Gly Lys Arg Leu Pro Thr Lys Cys Ser Glu Val Val Val Tyr Phe             340 345 350 Lys Thr Pro Glu Leu Asn Leu Phe Lys Glu Ser Trp Gln Asp Leu Pro         355 360 365 Gln Asn Lys Leu Thr Ile Arg Leu Gln Pro Asp Glu Gly Val Asp Ile     370 375 380 Gln Val Leu Asn Lys Val Pro Gly Leu Asp His Lys His Asn Leu Gln 385 390 395 400 Ile Thr Lys Leu Asp Leu Ser Tyr Ser Glu Thr Phe Asn Gln Thr His                 405 410 415 Leu Ala Asp Ala Tyr Glu Arg Leu Leu Leu Glu Thr Met Arg Gly Ile             420 425 430 Gln Ala Leu Phe Val Arg Arg Asp Glu Val Glu Glu Ala Trp Lys Trp         435 440 445 Val Asp Ser Ile Thr Glu Ala Trp Ala Met Asp Asn Asp Ala Pro Lys     450 455 460 Pro Tyr Gln Ala Gly Thr Trp Gly Pro Val Ala Ser Val Ala Met Ile 465 470 475 480 Thr Arg Asp Gly Arg Ser Trp Asn Glu Phe Glu                 485 490 <210> 11 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer: CAM_F <400> 11 taagaaggag atatacatat gaacgcaaac gacaacg 37 <210> 12 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer: camA_R <400> 12 catgaattct gtttcctgtg tgattaggca ctactcagtt ca 42 <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer: camB_F <400> 13 taatcacaca ggaaacagaa ttcatgtcta aagtagtgta tg 42 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: camB_R <400> 14 ggtttcttta ccagactcga ttaccattgc ctatcgggaa 40 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer: camC_F <400> 15 aagaaggaga tataccatga cgactgaaac cataca 36 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: camC_R <400> 16 gcattatgcg gccgcaagct ttataccgct ttggtagtcg 40 <210> 17 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Primer: P450bm3_F <400> 17 aagaaggaga tataccatga caattaaaga aatgcct 37 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer: P450bm3_R <400> 18 gtggtggtgg tggtgctcga ttacccagcc cacacgtctt 40 <210> 19 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Primer: Zwf_F <400> 19 ttaagaagga gatataccat ggcggtaacg caaacagc 38 <210> 20 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer: Zwf_R <400> 20 tcgacctgca ggcgcgccgt tactcaaact cattccagg 39

Claims (20)

A recombinant microorganism comprising a foreign gene encoding a bacterial cytochrome P450 protein, wherein said microorganism is Escherichia coli. The microorganism according to claim 1, wherein the cytochrome P450 protein belongs to EC 1.14.15.1 or EC 1.14.14.1. The microorganism according to claim 1, wherein the cytochrome P450 protein is P450Cam or P450BM3. The microorganism according to claim 2, wherein the cytochrome P450 protein is a complex of CamA, CamB, and CamC, and each of CamA, CamB, and CamC has an amino acid sequence of SEQ ID NOs: 2, 4, The microorganism according to claim 1, further comprising a foreign gene encoding a protein belonging to EC 1.1.1.49. 4. The microorganism according to claim 3, wherein P450BM3 has the amino acid sequence of SEQ ID NO: 8. The microorganism according to claim 1, wherein the gene comprises at least one of SEQ ID NOS: 1, 3, 5, and 7. A composition comprising a recombinant P450 protein for use in removing CHnF4-n (n is an integer from 0 to 3) in a sample. 9. The composition of claim 8, wherein the recombinant P450 protein is a recombinant microorganism comprising the expressed bacterial cytochrome P450 protein, a lysate thereof, a water soluble fraction of the lysate thereof, or a recombinant P450 protein. 10. The composition of claim 9, wherein the bacterial cytochrome P450 is expressed from a foreign gene. The method according to claim 9, wherein the removal is performed by cutting off the binding of CF of CHnF4-n (n is an integer from 0 to 3), or converting CHnF4-n to another substance, or accumulating in the cell to reduce the concentration of CHnF4- &Lt; / RTI &gt; 10. The composition of claim 9, wherein the sample is in a liquid or gaseous state. The composition according to claim 9, wherein the sample is plant wastewater or waste. The composition according to claim 9, wherein the microorganism is an Escherichia genus. Contacting the recombinant P450 protein with a sample containing CHnF4-n (n is an integer from 0 to 3) to reduce the concentration of CHnF4-n (n is an integer from 0 to 3) in the sample, n. &lt; / RTI &gt; 16. The method of claim 15, wherein the contacting is performed in an airtight container. 16. The method of claim 15, wherein the recombinant P450 protein is in the form of a recombinant microorganism comprising the expressed bacterial cytochrome P450 protein, a lysate thereof, a water soluble fraction of the lysate, or a recombinant P450 protein. 18. The method of claim 17, wherein the contacting is performed under conditions in which the recombinant microorganism is viable in an enclosed vessel. 18. The method according to claim 17, wherein said contacting cultures the microorganism in the presence of CHnF4-n. 18. The method of claim 17, wherein the sample is factory waste water or waste.

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