KR20120079676A - Novel oleate hydratase and method for the production of 10-hydroxystearic acid by recombinant cells comprising the same - Google Patents

Abstract

PURPOSE: A method for preparing 10-hydroxystearic acid is provided to obtain 10-hydroxystearic acid at high yield and to use the product as a starting material of lactone. CONSTITUTION: An oleate hydratase used for producing 10-hydroxystearic acid is selected from the group of sequence numbers 1-4. The enzyme is derived from Lysinibacillus fusiformis, Macrococcus caseolyticus, Propionibacterium acnes, and Stenotrophomonas maltophilia. A gene encoding the oleate hydratase is selected from sequence numbers 5-8. A method for preparing oleate hydratase comprises: a step of preparing the recombinant expression vector containing oleate hydratase gene; a step of culturing microorganism transformed with the recombinant expression vector; a step of inducing the expression of the gene; and a step of purifying and isolating the recombinant proteins.

Description

Novel oleate hydratase and method for the production of 10-hydroxystearic acid by recombinant cells comprising the same

The present invention relates to a method for preparing 10-hydroxystearic acid by using an oleate hydratase and a transformed microorganism including the enzyme, and more specifically, 10-hydroxystearic acid. The present invention relates to a recombinant expression vector comprising a hydroxystearic acid gene, a microorganism transformed therewith, and a production method for obtaining 10-hydroxystearic acid in high yield using the same.

Hydroxy fatty acids are naturally occurring substances found in triglycerols, waxes, cerebrosides and other lipids present in plants, insects and microorganisms. There are three types of hydroxy fatty acids in which microorganisms produce unsaturated fatty acids through biological conversion: monohydroxy, dihydroxy and trihydroxy fatty acids. Hydroxy fatty acids are important industrial materials as precursors to lactones that make up spices and are used as starting materials for resins, waxes, nylons, plastics, lubricants and coatings because they are more reactive than non-hydroxy fatty acids. It is also used as a cosmetic ingredient.

10-hydroxystearic acid is an important substance that produces sebacic acid, which is used as a lubricant and used to synthesize plasticizers and resins. Hydroxy fatty acids, such as 10-hydroxystearic acid, are converted to gamma-decalactone by yeast. Gamma-dekaralactone is characterized by a peach or apricot flavor and is essential for food processing and is increasingly used in the food industry. However, since these substances are present in a small amount in some fruits, they are synthesized by organic synthesis.However, due to the increase in national income and the demand of natural foods and fragrances, and the high quality of consumer goods, natural substances are stable and highly There is an absolute need for methods using biotransformation as the preferred trend in.

Microorganisms producing 10-hydroxystearic acid include Flavobacterium (sp. NRRL B-14859), Nocardia cholesterolicum , and runimal bacteria. bacteria) are, but when these microorganisms are not only produced the 10-hydroxy stearic acid in low yields are produced as a by-product, such as mountains and 10 KEDO stearic industrial uneconomical. In addition, there have been no reports of the use of recombinant microorganisms containing enzymes or related enzymes.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel oleic hydrase.

Another object of the present invention to provide a method for preparing the oleic acid hydratase.

Another object of the present invention is to provide a method for preparing 10-hydroxystearic acid.

In order to achieve the above object, the present invention provides an oleate hydratase used for the production of 10-hydroxystearic acid. The enzyme of the present invention preferably has an animonic sequence selected from the group consisting of SEQ ID NOs: 1 to 4, but at least one substitution, deletion, inversion and All mutants having the activity of the present invention by causing the same mutation are also included in the protection scope of the present invention.

In one embodiment of the invention, the enzyme is Lysinibacillus ( Lysinibacillus) fusiformis ), Macrococcus caseolyticus, Propionibacterium acnes ), Stenotrophomonas maltophilia ) is preferably derived from, but not limited to.

The present invention also provides a gene encoding the enzyme protein of the present invention.

In one embodiment of the present invention, the gene sequence is preferably selected from the group consisting of SEQ ID NOs: 5 to 8, respectively, but considering the gene code degeneracy and the gene encoding the mutant of the enzyme, the gene sequence and at least All gene sequences with 80% homology, preferably 85%, more preferably 90%, are also within the scope of the present invention.

In addition, the present invention Lysinibacillus Fushiformis ( Lysinibacillus fusiformis ), Macrococcus caseolyticus ), Propionibacterium acnes acnes), stems from the Pomona's malto pilriah (Stenotrophomonas maltophilia) strain by stacking notes, provides a recombinant expression vector pET 28 containing the oleic acid hydration enzyme gene of the present invention (+) a / oleate hydratase.

In the present invention, as long as the recombinant expression vector can be used for the production of 10-hydroxy stearic acid, any vector used in the gene recombination method including the expression vector pET-28a (+) can be used. E. coli ER 2566 is preferably used as the microorganism to be transformed. However, any strain may be used as long as it is transformed with a recombinant expression vector to overexpress the desired gene and produce an active enzyme protein.

In one embodiment of the invention, the expression vector preferably has a cleavage map of Figure 6, but is not limited thereto.

The present invention also provides a recombinant expression vector of the present invention comprising an oleic acid hydratase gene;

b) culturing the microorganism transformed with said recombinant expression vector;

c) induce expression of said oleic acid hydratase gene;

d) provides a method for preparing oleic acid hydratase comprising the step of isolating and purifying the expressed recombinant protein.

The present invention also provides a 10-hydroxy fatty acid production method comprising the step of treating the oleic acid hydratase of the present invention on a substrate.

In one embodiment of the present invention, the method is preferably, but not limited to using oleic acid as a substrate.

In another embodiment of the present invention, the hydroxy fatty acid is preferably 10-hydroxystearic acid, but is not limited thereto.

Hereinafter, the present invention will be described.

The present inventors investigated enzymes capable of converting oleic acid, which is abundant unsaturated fatty acids of plants into hydroxy fatty acids, and selected enzymes and recombinant microorganisms capable of producing 10-hydroxystearic acid in high yield without by-products.

Lysinibacillus fusiformis ), Macrococcus construct and use a recombinant expression vector comprising the oleic acid hydratase gene derived from caseolyticus ), Propionibacterium acnes , Stenotrophomonas maltophilia and microorganisms transformed therewith. A large amount of oleic acid hydrase was prepared and the optimal reaction conditions for obtaining 10-hydroxystearic acid in high yield without the production of by-products by using an enzyme and a transformed microorganism were completed and the present invention was completed.

In a preferred embodiment of the present invention, Lysinibacillus fusiformis , Macrococcus caseriticus ( Macrococcus) caseolyticus ), Propionibacterium acnes, Stenotrophomonas maltophilia) the genomic DNA (genomic DNA) of the strain as a template and SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, by carrying out PCR oleic hydrated gene with primers of SEQ ID NOS: 15 and 16 Amplifying a DNA fragment comprising (Fig. 1a, Fig. 1b, Fig. 1c, Fig. 1d); DNA fragments containing the amplified oleic acid hydrase genes were treated with restriction enzymes Nhe and Xho, Nhe and Xho, Nde and Hind, Nhe and Xho, respectively, and cloned into pEt 28 (+), an expression vector pET28 ( +) a / oleic acid hydratase was prepared; Transforming E. coli ER 2566 strain by conventional transformation method; Culturing the transformed Escherichia coli and then inducing overexpression to produce oleic acid hydrase; And the expressed oleic hydrase is isolated and purified.

In addition, the expressed oleic acid hydrase is obtained by crushing the cell solution of the culture medium of the transformed strain and then centrifuged to obtain a supernatant; The supernatant may be separated and purified through the process of separation by fast protein liquid chromatography.

In addition, the present invention provides a production method for obtaining a high yield of 10-hydroxystearic acid using the oleic acid hydrase prepared by the above method.

Specifically, the present invention provides a method for producing 10-hydroxystearic acid using the oleic acid hydrolase which can selectively synthesize 10-hydroxystearic acid by adding a redox cofactor as a coenzyme without generating by-products. do.

The oleic acid hydrase is prepared by inducing the expression of the recombinant enzyme gene by culturing E. coli transformed with the recombinant expression vector containing the oleic acid hydratase gene as described above, and then separating and purifying the expressed protein. It is preferable.

It is also preferable to use fatty acids as the substrate, more preferably oleate. In addition, the concentration of the substrate is preferably in the range of 5 to 30 g /, more preferably in the range of about 10 g / l to 20 g / l.

In addition, the enzyme and strain reaction is preferably made in the range of pH 5 to 7.0, more preferably in the range of pH 7.0.

In addition, the enzyme and the strain reaction is preferably made in the temperature range of 20 ℃ to 40 ℃, more preferably in the temperature range of about 30 ℃.

In addition, the time of the enzyme and strain reaction can be appropriately adjusted according to a conventional method.

The present invention provides a recombinant expression vector containing an oleic acid hydratase gene, a microorganism transformed therewith, a method for producing a large amount of oleic acid hydrase using them, and a high yield of 10-hydroxystearic acid using oleic acid hydratase. It is effective to provide a production method.

Oleic acid derived from Lysinibacillus fusiformis, Macrococcus caseolyticus, Propionibacterium acnes, Stenotrophomonas maltophilia of the present invention The hydratase can produce 10-hydroxystearic acid, a raw material of industrial products, in high yield in a high specificity and environmentally friendly way, and the 10-hydroxystearic acid thus produced can be usefully used as a starting material for synthesis of various lactones. .

Figure 1 shows the gene sequence of the oleic acid hydratase of the present invention, Figure 1a: Lysinibacillus Fushiformis, Figure 1b: Macrococcus kageritix, Figure 1c: Propionibacterium Acnes, Figure 1d: Stenott Ropomonas maltophilia strain derived sequence is shown.
Figure 2 shows a comparison of the conversion rate of 10-hydroxystearic acid from oleic acid using the recombinant oleic acid hydrase of the present invention.
Figure 3 shows a comparison of the conversion rate of 10-hydroxystearic acid from oleic acid using the recombinant strain of the present invention. (■: oleic acid, □: linoleic acid)
Figure 4 shows the hourly production of 10-hydroxystearic acid using the recombinant oleic acid hydrase of the present invention. (●: Propionibacterium Acnes Oleic acid, ○: Propionibacterium acnes 10-hydroxystearic acid, ■: stenotropomonas maltophilia oleic acid, □: stenotropomonas maltophilia 10-hydroxystearic acid, ▲: macrococcus casericicus oleic acid, △: macrococos casericitis 10-hydroxystearic acid, ◆: Ricinibacillus fusiformis oleic acid, ◇: Ricinibacillus fusiformis 10-hydroxystearic acid)
Figure 5 shows the 10-hydroxystearic acid hourly production using the recombinant strain of the present invention. (●: Propionibacterium Acnes Oleic acid, ○: Propionibacterium acnes 10-hydroxystearic acid, ■: stenotropomonas maltophilia oleic acid, □: stenotropomonas maltophilia 10-hydroxystearic acid, ▲: macrococcus casericicus oleic acid, △: macrococos casericitis 10-hydroxystearic acid, ◆: Ricinibacillus fusiformis oleic acid, ◇: Ricinibacillus fusiformis 10-hydroxystearic acid)

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art of fatty acids that the scope of the present invention is not to be construed as being limited by these examples.

Example  1. Preparation of recombinant expression vector and transformed microorganism comprising oleic acid hydratase gene

In order to prepare the oleic acid hydratase gene of the present invention, Lysinibacillus fusiformis, Macrococcus caseriticus ( Macrococcus) caseolyticus ), Propionibacterium acnes , Stenotrophomonas a gene derived from oleic acid, hydrated maltophilia) the strain was first separated.

Specifically, the gene sequence and re shinny Bacillus Fushimi in the amino acid sequence has already been specified formate miss (Lysinibacillus fusiformis) KCTC 3454, a mark Cocos helicase utility kusu (Macrococcus caseolyticus ) KCTC 3582 , Propionibacterium acnes ) KCTC 3314 , Stenotrophomonas maltophilia .) KCCM 40552 strains were selected and DNA sequences of oleic acid hydratase derived therefrom (Genebank Accession No.ZP_07049769; Fig. 1a), (Genebank Accession No. YP_002559479.1; Fig. 1b), (Genebank Accession No. The following primers were designed based on AE017283.1; FIG. 1c), (Genebank Accession No. AM743169; FIG. 1d).

  SEQ ID NO: 9 (forward primer):

5'- GCTAGC ATGTATTACAGTAATGGTAACTATGAA-3 '

  SEQ ID NO: 10 (reverse primer):

5'-GG CTCGAG CTATATTAGTTTACTTTCTTTCA-3 '

  SEQ ID NO: 11 (forward primer):

5'-GGG GCTAGC ATGTACTATAGTAATGGA-3

  SEQ ID NO: 12 (reverse primer):

5'-AGG CTCGAG TTATATCAAATTTGCTTC-3 '

  SEQ ID NO: 13 (Forward primer):

5'-GGG CATATG ACGAACTTCCAACAC-3 '

  SEQ ID NO: 14 (Reverse primer):

5'-TTT AAGCTT TCACGGCAGGATGTC-3 '

  SEQ ID NO: 15 (Forward primer):

5'-GGG GCTAGC ATGTACTACAGCAGTGGC-3 '

  SEQ ID NO: 16 (reverse primer):

5'-AAA CTCGAG TCAGTCCTCCTGCACCAG-3 '

The primers SEQ ID NOS: 9-12 and 15-16 were designed for Nhe and Xho , and SEQ ID NOs: 13-14 were designed for cleavage of Nde and Hind restriction enzymes. The sequence was amplified.

The oleic hydrase gene obtained in large quantities is a restriction enzyme. Nhe and Xho or Nde and Hind Was inserted into the plasmid vector pET 28 (+) (manufactured by Novagen) to prepare pET 28 (+) a / oleic acid hydrolase.

The recombinant expression vector obtained as described above was transformed into E. coli ER 2566 strain (New Englands Biolabs, Herfordshire, UK) by a conventional transformation method, the transformed microorganism was added by adding 20% glycerin solution 10 It was stored frozen before incubation for the production of hydroxystearic acid.

Example  2. Preparation of Oleic Acid Hydrolase

In order to mass-produce the oleic acid hydrase of the present invention, the frozen E. coli ER 2566 strain was inoculated into a test tube containing 3 ml of LB medium, and shaker at 37 ° C. at 600 nm until the absorbance was 2.0. The spawn culture was carried out. Then, the seed cultured culture was added to a 2,000 ml flask containing 500 ml of LB medium to carry out the main culture.

In addition, when the absorbance became 0.6 at 600 nm, 0.1 mM IPTG was added to induce mass expression of oleic acid hydratase. At this time, the stirring speed was maintained at 200rpm, the culture temperature was maintained at 37 ℃, after the addition of IPTG was adjusted to the stirring speed 150rpm, culture temperature 16 ℃ and incubated.

In addition, the oleic acid hydrase produced by overexpression as described above, the culture medium of the transformed strain was washed twice with 0.85% sodium chloride (NaCl) by centrifugation at 6,000xg for 30 minutes at 4 ℃, 50mM phosphate The cell solution was disrupted with a sonicator by adding sodium, 300 mM sodium chloride, 10 mM immidazole, 0.1 mM protease inhibitor (phenylmethylsulfonyl fluoride). The cell lysate was again centrifuged at 13,000 × g for 20 minutes at 4 ° C., cell pellets were removed, and only cell supernatant was obtained for fast protein liquid chromatography (Bio-Rad Laboratories, Hercules, CA, USA). It was equipped with a Hisrap HP adsorption column using a His-tag, and separated as an enzyme liquid used for the production of 10-hydroxystearic acid.

Example  3.10- of Recombinant Oleic Acid Hydrolase Hydroxy stearic acid  Acid conversion capacity comparison

Lysinibacillus of the present invention ( Lysinibacillus) fusiformis ), Macrococcus caseolyticus , Propionibacterium acnes ), Stenotrophomonas maltophilia) to the enzymes of the strains derived from oleic acid hydrate to determine the conversion capacity of 10-hydroxystearic acid was separated as described above, the 10-hydroxy oleic acid as a substrate in the optimal pH 6.5, the temperature (30 ℃) of enzyme hydroxy The ability to convert to stearic acid was compared. As a result, Lee shinny Bacillus backsight formate miss (Lysinibacillus fusiformis) derived from the conversion of oleic acid, hydrated enzyme was 17.2%, a mark Cocos helicase utility kusu (Macrococcus caseolyticus) conversion of the resulting oleic hydrated enzyme was 15.5%, propionic sludge tumefaciens arc Ness (Propionibacterium acnes ) derived from the conversion of oleic acid, hydrated enzyme was 3.6%, the conversion of malto Pomona's pilriah (Stenotrophomonas maltophilia) derived from oleic acid, hydrated enzyme Ste note was identified as 11.8%. (Fig. 2)

Example  4. 10- of recombinant strains Hydroxy Stearic Exsidic  Conversion ability comparison

Recombinant Lysinibacillus Fushiformis of the present invention ( Lysinibacillus) fusiformis ), Macrococcus caseolyticus ), Propionibacterium acnes acnes ), Stenotrophomonas In order to compare the conversion ability of 10-hydroxystearic acid using maltophilia ) strains, the conversion ability of 10-hydroxystearic acid was compared with oleic acid as a substrate at an optimum pH of 6.5 and temperature (25 ° C). The result is Lysinibacillus lysinibacillus fusiformis ) has a conversion rate of 10.6% and Macrococcus Conversion of caseolyticus) has a conversion rate of 0%, propionic sludge tumefaciens arc Ness (conversion rate of 4.6%, Pomona's malto pilriah (Stenotrophomonas maltophilia) in Ste note of Propionibacterium acnes) was identified as 11.8%. (Fig. 3)

Example  5. 10- Using Oleic Acid Hydase Hydroxy stearic acid  Production of acid

In order to confirm the productivity of 10-hydroxystearic acid using the oleic acid hydrase of the present invention, 10-hydroxystearic acid was prepared using 10 g / l oleic acid as a substrate at an optimum pH of 6.5 and a temperature of 25 ° C. Hourly yield was measured.

As a result, Lysinibacillus fusiformis ) produced 7 g / l of 10-hydroxystearic acid at 10 g / l of oleic acid after 5.5 hours of reaction, yielding 1.27 g / l of productivity and 70% conversion yield. Macrococcus Kaceritikkusu ( Macrococcus caseolyticus ) produced 6.6 g / l of 10-hydroxystearic acid, yielding 1.2 g / l of productivity and 66% conversion yield. Propionibacterium acnes ) produced 1.8 g / l of 10-hydroxystearic acid, yielding 0.33 g / l of productivity and 18% conversion yield. Stenotrophomonas maltophilia ) produced 8.2 g / l of 10-hydroxystearic acid, yielding 1.5 g / l of productivity and 82% conversion yield per hour (FIG. 4).

Example  6. 10- Using Recombinant Strains Hydroxy stearic acid  Production of acid

In order to confirm the productivity of 10-hydroxystearic acid using the recombinant strain of the present invention, the optimum time of 10-hydroxystearic acid based on 20 g / l oleic acid at the optimum pH 6.5, temperature (25 ℃) of the enzyme The yield was measured.

As a result, Lysinibacillus fusiformis ) produced 19 g / l of 10-hydroxystearic acid at 20 g / l of oleic acid after 3 hours of reaction, yielding 6.3 g / hour of productivity and 91% conversion yield. Macrococcus Kaceritikkusu ( Macrococcus caseolyticus ) produced 18.3 g / l of 10-hydroxystearic acid, yielding 6.1 g / l of productivity and 88% conversion yield. Propionibacterium acnes ) produced 8.5 g / l of 10-hydroxystearic acid, yielding 2.8 g / l of productivity and 18% conversion yield. Producing malto Pomona's pilriah (Stenotrophomonas maltophilia), is 18 g / ℓ 10- hydroxy stearic acid to stearyl note indicated by the hour 6 g / ℓ of productivity and conversion yield of 90%. (5)

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art of fatty acids, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp. <120> Novel oleate hydratase and Method for the production of          10-hydroxystearic acid by recombinant cells comprising the same <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 590 <212> PRT <213> Lysinibacillus fusiformis <400> 1 Met Tyr Tyr Ser Asn Gly Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys   1 5 10 15 Pro Glu Gly Val Asp Gly Lys Ser Ala Tyr Leu Ile Gly Ser Gly Leu              20 25 30 Ala Ser Leu Ser Ala Ala Cys Phe Leu Ile Arg Asp Gly Gln Met Lys          35 40 45 Gly Glu Asn Ile His Ile Leu Glu Glu Leu Asp Ile Ser Gly Gly Ser      50 55 60 Leu Asp Gly Ile Leu Asn Pro Thr Arg Gly Phe Ile Ile Arg Gly Gly  65 70 75 80 Arg Glu Met Glu Asp His Phe Glu Cys Leu Trp Asp Leu Phe Arg Ser                  85 90 95 Ile Pro Ser Leu Glu Val Asp Asn Ala Ser Val Leu Asp Glu Phe Tyr             100 105 110 Trp Leu Asn Lys Glu Asp Pro Asn Tyr Ser Lys Cys Arg Leu Ile Lys         115 120 125 Asp Arg Gly Gln Arg Leu Glu Asp Asp Gly Lys Phe Thr Leu Ser Asp     130 135 140 Gln Ser Ser Glu Glu Met Ile Lys Leu Phe Phe Thr Pro Glu Glu Lys 145 150 155 160 Leu Glu Asp Lys Lys Ile Thr Asp Val Phe Ser Glu Glu Phe Phe Glu                 165 170 175 Ser Asn Phe Trp Leu Tyr Trp Ser Thr Met Phe Ala Phe Glu Lys Trp             180 185 190 His Ser Ala Met Glu Met Arg Arg Tyr Ile Met Arg Phe Ile His His         195 200 205 Ile Gly Gly Leu Pro Asp Leu Ser Ala Leu Lys Phe Thr Lys Tyr Asn     210 215 220 Gln Tyr Glu Ser Leu Val Leu Pro Met Ile Lys Tyr Leu Glu Gly His 225 230 235 240 Asp Val Asp Phe Gln Tyr Asn Thr Val Val Glu Asn Val Leu Val Asp                 245 250 255 Lys Val Gly Asp Lys Lys Val Ala His Thr Leu Val Leu Arg Lys Asn             260 265 270 Gly Val Lys Glu Asn Ile Glu Leu Thr Glu Asn Glu Leu Val Phe Val         275 280 285 Thr Asn Gly Ser Ile Thr Glu Ser Thr Thr Tyr Gly Asp Asn Tyr Thr     290 295 300 Pro Ala Pro Val Asn Lys Glu Leu Gly Gly Ser Trp Ser Leu Trp Lys 305 310 315 320 Asn Ile Ala Ala Gln Asp Thr Asp Phe Gly Arg Pro Glu Lys Phe Cys                 325 330 335 Asp Asn Leu Pro Lys Glu Ser Trp Phe Val Ser Ala Thr Leu Thr Thr             340 345 350 Leu Asp Asp Arg Val Ala Pro Tyr Ile Glu Lys Ile Ser Lys Arg Asp         355 360 365 Pro Tyr Ala Gly Lys Val Val Thr Gly Gly Ile Val Thr Ala Thr Asp     370 375 380 Ser Asn Trp Met Leu Ser Tyr Thr Leu Asn Arg Gln Pro His Phe Lys 385 390 395 400 His Gln Pro Lys Asp Gln Leu Val Val Trp Ile Tyr Gly Leu Leu Ser                 405 410 415 Asn Lys Pro Gly Asp Phe Ile Lys Lys Ser Ile Thr Glu Cys Ser Gly             420 425 430 Ile Glu Ile Ala Gln Glu Trp Leu Tyr His Met Gly Val Pro Val Asp         435 440 445 Glu Ile Pro Asp Ile Ala Gln Asn Ser Cys Asn Thr Ile Pro Cys Tyr     450 455 460 Met Pro Tyr Ile Thr Ser Tyr Phe Met Pro Arg Ala Met Gly Asp Arg 465 470 475 480 Pro Leu Val Val Pro Glu Gly Ser Ala Asn Leu Ala Phe Ile Gly Asn                 485 490 495 Phe Ser Glu Thr Val Arg Asp Thr Val Phe Thr Thr Glu Tyr Ser Val             500 505 510 Arg Thr Ala Met Glu Ala Val Tyr Gln Leu Leu Asn Ile Asp Arg Gly         515 520 525 Val Pro Glu Val Phe Ala Ser Ser Phe Asp Val Arg Thr Leu Leu Ala     530 535 540 Ser Thr Ala Arg Leu Leu Asp Gly Lys Lys Leu Thr Asp Ile Asp Ala 545 550 555 560 Pro Phe Ile Leu Lys Gln Ile Gly Lys Leu Gly Ile His Lys Thr Lys                 565 570 575 Asp Thr Ile Ile Tyr Asp Leu Leu Lys Glu Ser Lys Leu Ile             580 585 590 <210> 2 <211> 589 <212> PRT <213> Macrococcus caseolyticus <400> 2 Met Tyr Tyr Ser Asn Gly Asn Tyr Glu Ala Phe Ala Arg Pro Lys Lys   1 5 10 15 Pro Glu Gly Val Asp Asn Lys Ser Ala Tyr Leu Val Gly Ser Gly Leu              20 25 30 Ala Ser Leu Ala Ala Ala Ser Phe Leu Ile Arg Asp Gly Lys Met Lys          35 40 45 Gly Glu Asn Ile His Ile Leu Glu Glu Leu Asp Leu Pro Gly Gly Ser      50 55 60 Leu Asp Gly Ile Leu Asn Pro Glu Arg Gly Tyr Ile Met Arg Gly Gly  65 70 75 80 Arg Glu Met Glu Asn His Phe Glu Cys Leu Trp Asp Leu Phe Arg Ser                  85 90 95 Val Pro Ser Leu Glu Val Glu Asp Ala Ser Val Leu Asp Glu Phe Tyr             100 105 110 Trp Leu Asn Lys Glu Asp Pro Asn Tyr Ser Lys Cys Arg Val Ile Glu         115 120 125 Asn Arg Gly Gln Arg Leu Glu Ser Asp Gly Lys Met Thr Leu Thr Lys     130 135 140 Lys Ala Asn Lys Glu Ile Ile Gln Leu Cys Leu Met Lys Glu Glu Gln 145 150 155 160 Leu Asn Asp Val Lys Ile Ser Asp Val Phe Ser Lys Asp Phe Leu Asp                 165 170 175 Ser Asn Phe Trp Ile Tyr Trp Lys Thr Met Phe Ala Phe Glu Pro Trp             180 185 190 His Ser Ala Met Glu Met Arg Arg Tyr Leu Met Arg Phe Ile His His         195 200 205 Ile Gly Gly Leu Ala Asp Phe Ser Ala Leu Lys Phe Thr Lys Phe Asn     210 215 220 Gln Phe Glu Ser Leu Val Met Pro Leu Ile Glu His Leu Lys Ala Lys 225 230 235 240 Asn Val Thr Phe Glu Tyr Gly Val Thr Val Lys Asn Ile Gln Val Glu                 245 250 255 Cys Ser Lys Glu Ser Lys Val Ala Lys Ala Ile Asp Ile Val Arg Arg             260 265 270 Gly Asn Glu Glu Ser Ile Pro Leu Thr Glu Asn Asp Leu Val Phe Val         275 280 285 Thr Asn Gly Ser Ile Thr Glu Ser Thr Thr Tyr Gly Asp Asn Asp Thr     290 295 300 Pro Ala Pro Pro Thr Thr Lys Pro Gly Gly Ala Trp Gln Leu Trp Glu 305 310 315 320 Asn Leu Ser Thr Gln Cys Glu Glu Phe Gly Asn Pro Ala Lys Phe Tyr                 325 330 335 Lys Asp Leu Pro Glu Lys Ser Trp Phe Val Ser Ala Thr Ala Thr Thr             340 345 350 Asn Asn Lys Glu Val Ile Asp Tyr Ile Gln Lys Ile Cys Lys Arg Asp         355 360 365 Pro Leu Ser Gly Arg Thr Val Thr Gly Gly Ile Val Thr Val Asp Asp     370 375 380 Ser Asn Trp Gln Leu Ser Phe Thr Leu Asn Arg Gln Gln Gln Phe Lys 385 390 395 400 Asn Gln Pro Asp Asp Gln Val Ser Val Trp Ile Tyr Ala Leu Tyr Ser                 405 410 415 Asp Glu Arg Gly Glu Arg Thr Asn Lys Thr Ile Val Glu Cys Ser Gly             420 425 430 Lys Glu Ile Cys Glu Glu Trp Leu Tyr His Met Gly Val Pro Glu Glu         435 440 445 Lys Ile Ser Ala Leu Ala Ala Glu Cys Asn Thr Ile Pro Ser Tyr Met     450 455 460 Pro Tyr Ile Thr Ala Tyr Phe Met Pro Arg Lys Glu Gly Asp Arg Pro 465 470 475 480 Leu Val Val Pro His Gly Ser Lys Asn Ile Ala Phe Ile Gly Asn Phe                 485 490 495 Ala Glu Thr Glu Arg Asp Thr Val Phe Thr Thr Glu Tyr Ser Val Arg             500 505 510 Thr Ala Met Glu Ala Val Tyr Lys Leu Leu Glu Val Asp Arg Gly Val         515 520 525 Pro Glu Val Phe Ala Ser Val Tyr Asp Val Arg Ile Leu Leu His Ala     530 535 540 Leu Ser Val Leu Asn Asp Gly Lys Lys Leu Asp Glu Ile Asp Met Pro 545 550 555 560 Ile Tyr Glu Arg Leu Val Glu Lys Arg Leu Leu Lys Lys Ala Ser Gly                 565 570 575 Thr Phe Ile Glu Glu Leu Leu Glu Glu Ala Asn Leu Ile             580 585 <210> 3 <211> 594 <212> PRT Propionibacterium acnes <400> 3 Met Thr Asn Phe Gln His Ile Tyr Gln Arg Ser Tyr Pro Val Ser Pro   1 5 10 15 Asp Gly Asn Pro Phe Val Gln Asn Asp Leu Gly Arg Tyr Thr Gln Asn              20 25 30 His Pro Val Pro Pro Asp Asp Val Ala Glu Arg Lys Ala Tyr Leu Val          35 40 45 Gly Ser Gly Ile Ala Ser Leu Leu Ser Ala Ala Tyr Leu Ile Arg Asp      50 55 60 Ala Gln Met Pro Gly Glu Asn Ile Thr Ile Leu Glu Glu Met Ser Glu  65 70 75 80 Pro Gly Gly Ala Phe Asp Gly Ala Gly Asp Thr Glu Lys Gly Phe Ile                  85 90 95 Ala Arg Gly Gly Arg Glu Met Gly Gln His Phe Glu Cys Phe Trp Asp             100 105 110 Ile Met Lys Asp Ile Pro Ala Leu Glu Met Pro Glu Pro Tyr Ser Val         115 120 125 Leu Asp Glu Phe Arg Ile Val Asn Glu Asn Asp Pro Asn Ile Asn Pro     130 135 140 Cys Arg Ile Ile Asn Asn Arg Gly His Lys Arg Asp Ala Ser Lys Met 145 150 155 160 Gly Leu Asn Lys Lys Gly Gln Leu Asp Ile Val Arg Leu Leu Leu Ala                 165 170 175 Lys Glu Ser Asp Thr Tyr Tyr Lys Ser Ile Glu Asp Trp Phe Asp Glu             180 185 190 Asp Phe Leu Gln Ser Thr Phe Tyr Leu Leu Trp Lys Thr Met Phe Ala         195 200 205 Phe Glu Gln Trp Gln Ser Leu Thr Glu Leu Lys Arg Tyr Met His Arg     210 215 220 Phe Leu Gln Tyr Leu Pro Gly Phe Ser Asn Leu Ser Cys Leu Arg Phe 225 230 235 240 Ser Arg Tyr Asn Gln His Asp Ser Phe Val Val Pro Leu Val Lys Trp                 245 250 255 Leu Thr Glu Lys Gly Val Asn Phe Gln Tyr Asp Thr Leu Val Tyr Asp             260 265 270 Val Asp Leu Glu Ile Thr Ala His Arg Lys Ile Ala Arg Gly Ile Leu         275 280 285 Trp Arg Asp Lys Glu Gly Gly Glu His Arg Ile Asp Met Ser Ala Lys     290 295 300 Asp Leu Val Phe Val Thr Asn Gly Ser Leu Thr Glu Cys Thr Gly Tyr 305 310 315 320 Gly Asp Met Asp Thr Pro Ala Pro Tyr His Lys Asp Met Gln Ala Gly                 325 330 335 Trp Glu Leu Trp Arg Asn Leu Val Arg Arg Ser Pro Ala Phe Gly Arg             340 345 350 Pro Asp Val Phe Cys Gly Asp Ala Asp Lys Thr Val Trp Gln Ser Ile         355 360 365 Ser Phe Asn Phe Ile Gly Arg Asp His Pro Phe Leu Glu Lys Ile Lys     370 375 380 Glu Leu Thr Gly Asn Asp Pro Leu Ser Gly Arg Thr Val Thr Gly Gly 385 390 395 400 Ile Ile Thr Ala Glu Asp Ser Ser Trp Cys Ile Ser Leu Thr Met Asn                 405 410 415 Arg Gln Pro Gln Phe His Gly Gln Pro Glu Asp Trp Gly Val Ala Trp             420 425 430 Ala Tyr Gly Leu Tyr Pro Phe Glu Lys Gly Asp Val Val Asn Lys Thr         435 440 445 Met Leu Glu Cys Thr Gly Glu Glu Leu Leu Lys Glu Tyr Cys Tyr His     450 455 460 Phe Gly Leu Leu Asp Gln Phe Glu Glu Val Lys Ala His Thr Lys Val 465 470 475 480 Arg Ile Ala Thr Met Pro Trp Ile Thr Ala Phe Phe Met Pro Arg Gly                 485 490 495 Lys Gly Asp Arg Pro Glu Val Ile Pro Asp Gly Cys Val Asn Leu Ala             500 505 510 Cys Leu Gly Gln Phe Val Glu Thr Pro Asp Asp Cys Val Phe Thr Thr         515 520 525 Glu Gly Ser Ala Arg Thr Ala Met Met Ala Val Tyr Gly Leu Leu Asp     530 535 540 Leu Asp Arg Asp Ile Pro Pro Ile Trp Pro Thr Gln Tyr Asp Ile Arg 545 550 555 560 Ser Leu Leu Ala Ser Ala Lys Thr Leu Asn Asn Gly Arg Leu Pro Gly                 565 570 575 Ser Trp Leu Leu Ser Lys Leu Leu Lys Asn Thr Tyr Tyr Glu Asp Ile             580 585 590 Leu pro         <210> 4 <211> 589 <212> PRT <213> Stenotrophomonas maltophilia <400> 4 Met Tyr Tyr Ser Ser Gly Asn Tyr Glu Ala Phe Ala Arg Pro Arg Lys   1 5 10 15 Pro Ala Gly Val Asp Gly Lys Arg Ala Trp Phe Val Gly Ser Gly Leu              20 25 30 Ala Ser Leu Ala Gly Ala Ala Phe Leu Val Arg Asp Gly His Met Ala          35 40 45 Gly Glu Cys Ile Thr Ile Leu Glu Gln Gln Gln Ile Pro Gly Gly Ala      50 55 60 Leu Asp Gly Leu Lys Val Pro Glu Lys Gly Phe Val Ile Arg Gly Gly  65 70 75 80 Arg Glu Met Glu Asp His Phe Glu Cys Leu Trp Asp Leu Phe Arg Ser                  85 90 95 Ile Pro Ser Leu Glu Ile Glu Asp Ala Ser Val Leu Asp Glu Phe Tyr             100 105 110 Trp Leu Asn Lys Asp Asp Pro Asn Tyr Ser Leu Gln Arg Ala Thr Ile         115 120 125 Asn Arg Gly Glu Asp Ala His Thr Asp Gly Leu Phe Thr Leu Thr Glu     130 135 140 Gln Ala Gln Arg Asp Ile Val Ala Leu Phe Leu Ala Thr Arg Gln Glu 145 150 155 160 Met Glu Asn Lys Arg Ile Asn Glu Val Leu Gly Arg Asp Phe Leu Asp                 165 170 175 Ser Asn Phe Trp Leu Tyr Trp Arg Thr Met Phe Ala Phe Glu Glu Trp             180 185 190 His Ser Ala Leu Glu Met Lys Leu Tyr Leu His Arg Phe Ile His His         195 200 205 Ile Gly Gly Leu Pro Asp Phe Ser Ala Leu Lys Phe Thr Lys Tyr Asn     210 215 220 Gln Tyr Glu Ser Leu Val Leu Pro Leu Val Lys Trp Leu Gln Asp Gln 225 230 235 240 Gly Val Val Phe Gln Tyr Gly Thr Glu Val Thr Asp Val Asp Phe Asp                 245 250 255 Leu Gln Pro Asp Arg Lys Gln Ala Thr Arg Ile His Trp Met His Asp             260 265 270 Gly Val Ala Gly Gly Val Glu Leu Gly Ala Asp Asp Leu Leu Phe Met         275 280 285 Thr Ile Gly Ser Leu Thr Glu Asn Ser Asp Asn Gly Asp His His Thr     290 295 300 Ala Ala Arg Leu Asn Glu Gly Pro Ala Pro Ala Trp Asp Leu Trp Arg 305 310 315 320 Arg Ile Ala Ala Lys Asp Asp Ala Phe Gly Arg Pro Asp Val Phe Gly                 325 330 335 Ala His Ile Pro Glu Thr Lys Trp Glu Ser Ala Thr Val Thr Thr Leu             340 345 350 Asp Ala Arg Ile Pro Ala Tyr Ile Gln Lys Ile Ala Lys Arg Asp Pro         355 360 365 Phe Ser Gly Lys Val Val Thr Gly Gly Ile Val Ser Val Arg Asp Ser     370 375 380 Arg Trp Leu Met Ser Trp Thr Val Asn Arg Gln Pro His Phe Lys Asn 385 390 395 400 Gln Pro Lys Asp Gln Ile Val Val Trp Val Tyr Ser Leu Phe Val Asp                 405 410 415 Thr Pro Gly Asp Tyr Val Lys Lys Pro Met Gln Asp Cys Thr Gly Glu             420 425 430 Glu Ile Thr Arg Glu Trp Leu Tyr His Leu Gly Val Pro Val Glu Glu         435 440 445 Ile Asp Glu Leu Ala Ala Thr Gly Ala Lys Thr Val Pro Val Met Met     450 455 460 Pro Tyr Ile Thr Ala Phe Phe Met Pro Arg Gln Ala Gly Asp Arg Pro 465 470 475 480 Asp Val Val Pro Glu Gly Ala Val Asn Phe Ala Phe Ile Gly Gln Phe                 485 490 495 Ala Glu Ser Lys Gln Arg Asp Cys Ile Phe Thr Thr Glu Tyr Ser Val             500 505 510 Arg Thr Pro Met Glu Ala Val Tyr Thr Leu Leu Gly Ile Glu Arg Gly         515 520 525 Val Pro Glu Val Phe Asn Ser Thr Tyr Asp Val Arg Ser Leu Leu Ala     530 535 540 Ala Thr Gly Arg Leu Arg Asp Gly Lys Glu Leu Asp Ile Pro Gly Pro 545 550 555 560 Ala Phe Leu Arg Asn Leu Leu Met Asn Lys Leu Asp Lys Thr Gln Ile                 565 570 575 Gly Gly Leu Leu Arg Glu Phe Lys Leu Val Gln Glu Asp             580 585 <210> 5 <211> 1770 <212> DNA <213> Lysinibacillus fusiformis <400> 5 atgtattaca gtaatggtaa ctatgaagct ttcgcacgtc ctaaaaaacc ggagggagta 60 gatggaaaat ctgcttatct cattggttcg ggcctagcct cgctgtcagc agcatgtttt 120 ttaattcgtg atggccaaat gaaaggtgag aacattcata ttctggaaga gctagatatt 180 tcaggaggca gtcttgatgg catacttaat ccgacgagag gatttattat tcgcggtggt 240 cgagaaatgg aagatcattt tgaatgctta tgggatttat tccgttccat tccttcctta 300 gaggtagaca atgcttctgt gttagatgaa ttttattggt taaacaaaga ggaccccaat 360 tattcaaaat gtcgtttaat aaaagataga ggtcaaagac ttgaagatga tggcaagttt 420 actttatcgg atcagtcatc tgaggaaatg attaaattat tctttacacc tgaggaaaag 480 ttagaggata aaaaaattac agatgttttc tcagaggaat tttttgaatc gaacttttgg 540 ctgtattggt ccactatgtt tgcgtttgaa aaatggcatt ctgcaatgga aatgcgccgt 600 tatattatgc gtttcattca ccatattggt ggattaccag atctatcagc attgaaattt 660 acaaaatata accaatatga atcgttagtg ctaccgatga taaaatattt agaaggtcat 720 gatgttgatt ttcaatataa tacggtagta gagaatgttt tggtcgataa agtgggcgac 780 aaaaaagtag ctcatacatt agttttaaga aaaaatggtg tgaaagagaa tattgagcta 840 acagaaaatg aattggtatt tgtgacaaat ggtagtatta cggagagtac aacatacggg 900 gataattata ccccagcccc tgtcaataaa gaattaggtg gaagctggtc actgtggaaa 960 aacatcgctg cccaagatac tgactttgga cgaccagaaa agttctgtga taatcttcca 1020 aaagaaagct ggtttgtttc cgccactcta acgactttag acgatcgtgt ggctccttat 1080 attgaaaaaa ttagtaaaag ggacccgtat gcaggcaaag tagtaacagg aggtattgtc 1140 acagctacag attctaattg gatgctaagc tatacgttaa accgacaacc tcattttaaa 1200 catcaaccaa aagaccaatt agtcgtttgg atttatggtt tactatcaaa taaaccaggt 1260 gattttatta aaaaaagcat taccgaatgt tcaggtattg agatagcaca agagtggttg 1320 taccatatgg gtgtaccggt tgatgaaatt ccagacatcg cacaaaattc ttgtaacacg 1380 attccatgct atatgcctta catcacctct tatttcatgc caagagccat gggggatcgt 1440 ccattagttg tgccagaagg gtctgctaat ttagccttca ttggcaactt tagtgaaact 1500 gtaagagata ccgtttttac tactgaatat tcagtaagaa ctgcgatgga ggcagtctat 1560 caattattaa atattgatcg tggagttcca gaagtattcg catcttcttt tgatgttcgc 1620 acattattag cttcaactgc tcgcttatta gatggcaaaa aattaacaga tattgatgcg 1680 ccatttattt taaaacaaat tggtaaatta ggaattcaca aaacaaaaga tacaattatt 1740 tatgacttac tgaaagaaag taaactaata 1770 <210> 6 <211> 1767 <212> DNA <213> Macrococcus caseolyticus <400> 6 atgtactata gtaatggaaa ctatgaggca tttgcaagac cgaagaagcc ggaaggggta 60 gataataagt ctgcatattt agttggttct ggtttagcgt cattagcagc ggcaagtttt 120 ttaatacgag atggtaaaat gaaaggtgaa aatattcata tattagaaga actcgatctc 180 cctggaggaa gcttggatgg aatattgaat cctgaacgtg gctatataat gcgtggcggt 240 cgtgagatgg agaatcattt tgaatgctta tgggatttat ttcgttcagt accatcattg 300 gaagtcgaag atgcttctgt tctggatgaa ttttactggt taaataaaga agatccaaac 360 tattcgaagt gccgcgtaat agaaaatcgt ggacaacgcc tagaatcaga tggaaaaatg 420 actctaacaa aaaaagcaaa taaagaaatt atccagctat gcttaatgaa agaagaacag 480 ctgaatgatg tgaagatctc tgatgtcttc agtaaagact tcttagactc aaacttctgg 540 atctactgga aaacgatgtt tgcatttgaa ccttggcatt ctgctatgga gatgcgtcga 600 tatttaatgc gtttcatcca tcatattggc ggacttgcag acttttcagc cctaaaattt 660 acgaagttca atcagttcga atcacttgtt atgcctctga ttgagcatct taaagcgaag 720 aacgttacat ttgaatatgg tgtaaccgtt aagaatatac aagttgaatg ttcaaaagag 780 tcaaaagttg caaaggcaat agacatcgtg cgcagaggta acgaggaatc aattccttta 840 actgaaaatg atttagtatt tgtaacaaat ggcagcatca ctgaaagtac tacttatgga 900 gataatgata cacctgcacc gcctacaaca aaacctggtg gcgcatggca actatgggaa 960 aacttaagta cgcaatgtga ggagtttggt aatccagcta aattctataa agatttacca 1020 gaaaaaagct ggttcgtgtc tgctacagca acaacaaata ataaagaagt tatagattat 1080 attcaaaaaa tttgtaaacg cgatccatta tcaggtcgta cagtaaccgg cggtatcgtt 1140 actgtagatg attcaaattg gcagttaagc tttacgctaa atcgacaaca gcagtttaaa 1200 aatcaacctg atgatcaagt gagtgtatgg atttacgcac tttattcaga tgaacgtgga 1260 gaacgtacaa ataaaacaat tgttgagtgt tctggtaaag aaatttgtga agaatggctt 1320 tatcatatgg gtgttcctga agagaagatt tcagcactag cagcagaatg taatacaatt 1380 ccaagctata tgccgtacat taccgcttac tttatgccgc gtaaagaagg agaccgtcct 1440 ttagtagtac cacatggttc aaagaatatt gcatttatag gtaactttgc agaaacagaa 1500 agagataccg tatttacaac agaatattca gtaagaactg ctatggaagc ggtgtataaa 1560 cttctagaag tagaccgtgg agtgcctgaa gtattcgctt cagtatacga tgtgagaatt 1620 ttattacatg cgttatctgt actgaatgat ggcaagaaac tagatgaaat tgatatgcca 1680 atctatgaaa gattggtaga aaaacgctta ttgaagaaag catctggtac attcattgaa 1740 gaactattag aagaagcaaa tttgata 1767 <210> 7 <211> 1782 <212> DNA Propionibacterium acnes <400> 7 atgacgaact tccaacacat ttatcaacgc agctaccctg tgagcccgga tggcaaccct 60 ttcgtgcaga acgacctcgg ccgctacacc cagaaccacc ctgtaccgcc cgatgacgtc 120 gctgagcgca aggcatacct cgtcggctcg ggcatcgcct cgcttctttc cgctgcctac 180 ttgattcgcg acgcccagat gcctggggag aacatcacca tcctcgagga aatgtcagaa 240 cccggcggcg cgttcgacgg agcaggggat acggagaagg gcttcatcgc tcgcggtggt 300 cgtgagatgg ggcaacactt cgagtgtttc tgggacatca tgaaagacat cccagccttg 360 gagatgcccg aaccctacag cgttctggat gaattccgga tcgtgaacga gaacgacccc 420 aacatcaatc cgtgtcggat tatcaataat cgcggtcaca agcgcgacgc ctcaaagatg 480 gggctcaaca agaaaggcca gctcgacatc gtgaggctct tgctggccaa ggagtcggac 540 acctactaca agtcgatcga ggactggttc gacgaggact tcctgcagtc caccttctac 600 ctgctgtgga agacgatgtt cgccttcgag cagtggcagt cccttaccga gctcaaacgg 660 tacatgcacc gcttcctgca gtatcttccc ggtttctcga atctgtcctg tctgcgattc 720 agtcgctaca accagcacga ctccttcgtc gtcccgttgg tgaagtggct caccgaaaag 780 ggcgtgaact tccagtacga caccctcgtc tacgacgtcg acctagagat caccgcccac 840 cgcaagatcg cccgcggcat cctgtggcgc gataaggagg gtggcgagca tcgcatcgac 900 atgtccgcca aggacctggt gttcgtgacc aacggttcac ttaccgagtg caccggatac 960 ggcgacatgg acacccctgc cccttaccac aaagacatgc aggccggctg ggagctgtgg 1020 cgcaacctgg ttcgccgctc ccccgcgttc ggacgtcccg atgtcttctg cggcgacgct 1080 gacaagacgg tgtggcaatc aatcagcttc aacttcatcg gtcgtgacca cccgttcctc 1140 gagaagatca aggagctgac cggcaacgat ccgttgtccg ggcgcaccgt caccggcggc 1200 atcatcaccg ccgaggactc gtcgtggtgc atctcgctga cgatgaatcg tcagccacag 1260 ttccatggcc agcctgaaga ctggggcgtg gcatgggcct acgggctgta cccgttcgaa 1320 aagggcgacg tcgtcaacaa gacaatgctg gaatgcaccg gcgaggaact gctcaaagag 1380 tactgctatc acttcggact gctcgaccag ttcgaggagg tcaaggccca caccaaggtg 1440 cgaatcgcga cgatgccgtg gatcacggcc ttcttcatgc cccgcggcaa gggcgatcgc 1500 ccggaggtca tcccggacgg ctgcgtcaac ctggcttgcc taggccagtt cgtcgaaacc 1560 cccgatgact gcgtcttcac caccgagggt tcggcacgca ccgcaatgat ggccgtctac 1620 ggcctgctcg accttgaccg tgacatcccg ccgatctggc caacccagta cgacatccgc 1680 tccctgctgg cctccgccaa aaccctcaac aatggtcgct tgcccggtag ctggctgttg 1740 tcgaagctat tgaagaacac ctactacgag gacatcctgc cg 1782 <210> 8 <211> 1767 <212> DNA <213> Stenotrophomonas maltophilia <400> 8 atgtactaca gcagtggcaa ttacgaagcc ttcgcgcgcc cgcgcaagcc cgccggtgtg 60 gatggcaagc gtgcatggtt cgtcggttcg ggcctggcct cgttggccgg tgccgcgttc 120 ctggtgcgcg acggccacat ggccggtgag tgcatcacca tccttgagca gcagcagatt 180 ccgggcggcg cgctggacgg cctgaaagtg ccggaaaagg gtttcgtgat ccgtggtggc 240 cgcgagatgg aagatcattt cgagtgcctg tgggacctgt tccgctcgat accgtcgctg 300 gagatcgaag atgccagcgt gctggacgag ttctactggt tgaacaagga cgacccgaac 360 tattcgctgc agcgcgccac catcaatcgc ggcgaggatg cacacaccga tggcctgttc 420 acgctgaccg agcaggcaca gagggacatc gtcgcgctgt tcctggccac ccgtcaggag 480 atggagaaca agcgcatcaa cgaagtactg ggccgtgatt tcctggacag caacttctgg 540 ctgtactggc gaaccatgtt cgcgttcgaa gagtggcatt cggcgctgga gatgaagctg 600 tacctgcatc gcttcatcca ccatatcggc ggcctgccgg atttctcggc gctgaagttc 660 acgaagtaca accagtacga atcgctggtg ctgccgctgg tgaagtggtt gcaggaccag 720 ggcgtggtgt tccagtacgg taccgaggtg accgacgtcg actttgatct gcagccggac 780 cgcaagcagg ccacgcgcat ccattggatg catgacggtg tagccggtgg cgtggaactc 840 ggcgcggatg atctgctgtt catgaccatc ggttcgctga ccgagaactc ggataatggc 900 gaccaccaca ctgcggcacg cctgaacgaa ggcccggcgc ctgcctggga cctgtggcgc 960 cgcattgccg cgaaggatga cgcgttcggg cgtccggatg tgttcggcgc gcacattcca 1020 gaaaccaagt gggaatcggc aacggtgacc acgctggatg cgcgcattcc ggcctacatc 1080 cagaagatcg ccaagcgcga tcccttcagc ggcaaagtgg tgaccggcgg catcgtcagc 1140 gtgcgcgact cgcgctggct gatgagctgg acggtgaatc gccagccaca tttcaagaac 1200 cagcccaagg accagatcgt ggtctgggtg tattcgttgt tcgtggatac gcccggcgac 1260 tacgtgaaga agccgatgca ggactgcacc ggcgaggaga tcacgcgcga atggctgtat 1320 cacctgggcg tgccggtgga agagatcgat gagctggccg cgaccggcgc gaagacagtg 1380 ccggtgatga tgccgtacat cacggcgttc ttcatgccac gccaggcagg tgaccgcccg 1440 gacgtggtgc cggaaggtgc ggtgaacttc gctttcatcg gccagttcgc cgaatcgaag 1500 cagcgcgact gcatcttcac caccgagtac tcggtgcgca cgccgatgga ggcggtgtac 1560 accttgctgg ggatcgagcg tggcgtgccg gaggtgttca attccacgta tgacgtgcgc 1620 tcgctgctgg ccgcgaccgg gcgcctgcgt gatggcaagg aactggacat tcccgggcca 1680 gcgttcctgc gcaacctgct gatgaacaag ctggacaaga cccagatcgg tggtctgctg 1740 cgcgagttca agctggtgca ggaggac 1767 <210> 9 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gctagcatgt attacagtaa tggtaactat gaa 33 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ggctcgagct atattagttt actttctttc a 31 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggggctagca tgtactatag taatgga 27 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aggctcgagt tatatcaaat ttgcttc 27 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gggcatatga cgaacttcca acac 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tttaagcttt cacggcagga tgtc 24 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ggggctagca tgtactacag cagtggc 27 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 aaactcgagt cagtcctcct gcaccag 27

Claims (10)

Oleate hydratase used for the production of 10-hydroxystearic acid selected from the group consisting of SEQ ID NOs: 1-4. The method of claim 1, wherein the enzyme is Lysinibacillus fusiformis , Macrococcus caseolyticus, Propionibacterium acnes, respectively acnes ), Stenotrophomonas maltophilia ) oleate hydratase, characterized in that it is derived from. A gene encoding the enzyme protein of claim 1. The gene of claim 3, wherein the gene sequence is selected from the group consisting of SEQ ID NOs: 5 to 8, respectively. Lysinibacillus fusiformis ), Macrococcus caseolyticus), propynyl sludge tumefaciens arc Ness (Propionibacterium acnes), Pomona's malto pilriah (Stenotrophomonas maltophilia) derived from the strain, and the recombinant expression containing 3 or claim 4 of the oleic acid hydrate gene by stacking notes vector pET 28a ( +). The recombinant expression vector pET 28 (+) a according to claim 5, wherein the expression vector has a cleavage map of FIG. a) preparing a recombinant expression vector of claim 5 or 6 comprising an oleic acid hydratase gene;
b) culturing the microorganism transformed with said recombinant expression vector;
c) induce expression of said oleic acid hydratase gene;
d) a method for preparing oleic acid hydratase comprising the step of isolating and purifying the expressed recombinant protein. 10-hydroxyfatty acid production method comprising the step of treating the oleic acid hydrase of claim 1 to the substrate. The method of claim 8, wherein the method uses oleic acid as a substrate. The method of claim 8,
The hydroxy fatty acid is 10-hydroxystearic acid production method, characterized in that 10-hydroxystearic acid.

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