KR101816620B1 - Endo-1,4-beta-xylanase cel10-KG99 gene from rumen microorganism of black goat and uses thereof - Google Patents
Endo-1,4-beta-xylanase cel10-KG99 gene from rumen microorganism of black goat and uses thereof Download PDFInfo
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Abstract
The present invention relates to endo-1,4-beta-xylanase cel10-KG99 gene derived from a black goat rumen microorganism and its use, and more particularly to a novel endo-1,4-beta-xylanase selected from a black goat rumen microorganism Lanease cel10-KG99 gene and a protein product thereof, and a method for preparing a feed additive, a detergent composition and a biofuel using the same.
Description
The present invention relates to endo-1,4-beta-xylanase cel10-KG99 gene derived from a black goat rumen microorganism and its use, and more particularly to a novel endo-1,4-beta-xylanase selected from a black goat rumen microorganism Lanease cel10-KG99 gene and a protein product thereof, and a method for preparing a feed additive, a detergent composition and a biofuel using the same.
The main constituents of plant cell walls are celluloase, hemi-cellulose, and pectin, which are degraded and absorbed by microorganisms through complex and effective processes in herbivore rumen. Rumen microorganisms are extreme anaerobes, including fungi, protozoa and bacteria, and the breakdown of fibrin is mainly responsible for bacteria and fungi. However, most of these microorganisms are not well known because they are difficult to cultivate. Among the ruminal microorganisms known to date, bacteria that are mainly involved in digesting plant cell walls include Fibrobacter succinogenes), luminometer Caucus Playa Pacifica beret Enschede (Ruminococcus flavefaciens), luminometer Cocu live bus (Ruminococcusalbus), Libby brick Booty avoid debris Sol Bence (Butyrivibrio fibrisolvens), oil cake Te Solarium cellulite with a brush Bence (Eubacterium The bacteria belonging to the genus cellulosolvens), frame Beam telra (Prevotella), and some of Clostridium (Clostridium) have been identified. To date, most microbial DNA in the digestive organs of cows, rabbits, and kangaroos, or the metagenome (referred to as the total DNA of environmental microorganisms) has been studied to identify the most abundant enzymes in the rumen, A number of glucanases, beta-glucosidase and cellulosome complexes have been identified through a number of studies.
However, most of the above cases have evolved from soft fungal fibrinogen to food. In industry, degradation ability of fibrinolytic enzyme to harder fibrin source is required.
Black goats evolved to digest feeds of the less coarse fibrinogen, such as twigs, bark, and vegetation. This means that the ruminal microbial population of the black goat evolved differently from other herbivores. Techniques for searching for novel substances using fosmid, cosmid, and bacterial artificial chromosome (GAC) gene banks have been widely used for gene search for promiscuous microorganisms. In this way, a number of fibrinolytic enzyme genes have been found to date.
Under these technical backgrounds, the present inventors have made intensive efforts to accomplish the present invention.
Finally, it is an object of the present invention to provide endo-1,4-beta-xylanase cel10-KG99 gene derived from black goat rumen microorganism and its use.
According to one aspect of the present invention, endo-1,4-beta-xylanase cel10-KG99 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence shown in SEQ ID NO: 1 can be provided.
According to another aspect of the present invention, there is provided a GH10 domain derived from a black goat rumen microorganism and an endo-1,4-beta-xylanase protein encoded by the GH2C domain, which is encoded by the gene and is composed of the amino acid sequence represented by SEQ ID NO: .
According to another aspect of the present invention, a recombinant vector containing the gene may be provided.
According to another aspect of the present invention, a host cell transformed with the recombinant vector may be provided.
According to another aspect of the present invention, there can be provided a feed additive for promoting fibrin degradation comprising the endo-1,4-beta-xylanase protein.
According to another aspect of the present invention, a detergent composition comprising the endo-1,4-beta-xylanase protein can be provided.
According to another aspect of the present invention, there is provided a method for producing biofuel comprising the step of hydrolyzing the biomass material by adding the endo-1,4-beta-xylanase protein.
The present invention provides a novel chlorine-derived fibrinolytic enzyme capable of degrading a harder fibrin source. By utilizing the present invention, it is possible to develop a low-cost feed additive against the rise in the price of feed grains, develop biofuels using the most abundant resource on the planet, and apply it to various fields such as a fiber softener and a process for adding a cellulose degrading enzyme can do.
FIG. 1 shows the results of meta genome DNA fragment cleavage conditions and insertion DNA cleavage for the construction of the Fosmid gene bank.
Fig. 2 shows the results of confirming the average insertion size of the phosphide gene bank for the Ad fraction and the Lq fraction.
Fig. 3 shows clonal selection results of inoculation on Esculin-Agar plates and the change of the surrounding brown color according to the esculin degradation.
Figure 4 shows nucleotide blast results according to the present invention.
FIG. 5 shows the major domain identification results according to the protein blast according to the present invention.
Figure 6 shows the major analogous proteins according to the protein blast results of the present invention.
FIG. 7 shows the phylogenetic tree and homology of the highest homologous protein and other fibrinolytic enzymes among the protein blast results of the present invention.
Hereinafter, the present invention will be described in more detail.
The term " superfamily " used in the present invention means a group of proteins having similar amino acid sequences, protein functions, and three-dimensional structures of proteins.
The term " biomass " used in the present invention refers to a whole of a biological organism including a plant carrying out a carbon assimilation process for fixing carbon dioxide using sunlight and a living animal eating it. Various fuels such as alcohol, diesel, and hydrogen produced from the biomass are called biofuel, and biomass such as biomass such as cellulose is proposed as a substitute for chemical substances obtained from petroleum . Plant-based biomass is more advantageous in terms of energy balance than petroleum as raw material in producing various chemical products.
The reason for this is that, in the case of the plant-based biomass, various functional groups including oxygen are included, so that it is possible to convert to a lower activation energy when various chemical products with functional groups are produced.
The term 'biofuel' used in the present invention includes all the byproducts derived from metabolism activities such as feces and living organisms as well as fossil fuels. It is a renewable energy different from fossil fuels, and bioethanol and biodiesel And the like.
The present invention focuses on fibrin degrading ability of a black goat rumen microorganism, and constructs a fosmid gene bank against a black goat rumen microorganism metagenome, from which an exocellulase system, beta-glucosidase activity And the plasmid DNA of the clone was decoded to secure the genetic information. The gene encoding Escherichia coli was transformed into Escherichia coli. The plasmid DNA of the active phosmid clone was extracted using the Qiagen Midi Prep Kit, and the extracted DNA produced 100 times more shotgun sequencing data with an average length of 220 bp. Sequence information was obtained in fasta format. The major domain and similar genes were searched for NCBI blastp sequence of the obtained active fosmid plasmid DNA sequence to identify the fibrinolytic enzyme related gene.
The present invention is very different from the recently disclosed fibrinolytic enzyme gene sequence.
According to one aspect of the present invention, endo-1,4-beta-xylanase cel10-KG99 gene derived from a black goat rumen microorganism and consisting of the nucleotide sequence shown in SEQ ID NO: 1 can be provided.
The endo-l, 4-beta-xylanase cel10-KG99 gene according to the present invention may be DNA or RNA encoding endo-l, 4-beta-xylanase protein. DNA includes cDNA, genomic DNA, or artificial synthetic DNA. The DNA may be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.
Preferably, the endo-1,4-beta-xylanase cel10-KG99 gene according to the present invention may comprise the nucleotide sequence shown in SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. More specifically, the gene comprises a nucleotide sequence having a sequence homology of 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably 95% or more, from the nucleotide sequence of SEQ ID NO: 1 .
The 'percentage of sequence homology to polynucleotides' is identified by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (addition or deletion) (I. E., A gap) relative to the < / RTI >
According to an embodiment of the present invention, the endo-l, 4-beta-xylanase cel10-KG99 gene may preferably be derived from a black goat rumen microorganism. However, the present invention is not necessarily limited thereto. Examples of the present invention may include a homologous gene having high homology (for example, 70% or more, preferably 80% or more, , More preferably at least 90%, most preferably at least 95% sequence homology) and may include genes derived from rumen microbes of other organisms other than the black goat. Methods for sequencing and means for determining sequence homology (e.g., BLAST, etc.) are well known to those skilled in the art.
According to another aspect of the present invention, there is provided a GH10 domain derived from a black goat rumen microorganism and an endo-1,4-beta-xylanase protein encoded by the GH2C domain domain, which is encoded by the gene and is composed of the amino acid sequence represented by SEQ ID NO: Can be provided.
The range of endo-1, 4-beta-xylanase protein according to the present invention includes the GH1 and BGL domain-family proteins consisting of the amino acid sequence shown in SEQ ID NO: 2 isolated from the black goat rumen microorganism and functional equivalents of said proteins do.
The term "functional equivalent" means that at least 70% or more, preferably 80% or more, more preferably 90% or more, or more preferably 90% or more, of the amino acid sequence shown in SEQ ID NO: 2 as a result of addition, 2, and most preferably at least 95%, of the amino acid sequence shown in SEQ ID NO: 2, and has substantially the same physiological activity as the protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The term " substantially homogenous bioactivity " means the same endo-1,4-beta-xylanase activity.
The invention also includes fragments, derivatives and analogues of endo-1, 4-beta-xylanase proteins. The term "fragments", "derivatives" and "analogues" refer to polypeptides having substantially the same biological function or activity as the endo-1, 4-beta-xylalanase protein according to the present invention.
Fragments, derivatives and analogs of endo-1,4-beta-xylanase proteins according to the present invention are useful for the detection of (i) one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) (Ii) a polypeptide having a substituent (s) at one or more amino acid residues, or (iii) a polypeptide having a half-life of the polypeptide (s) (Iv) an additional amino acid sequence (e. G., A sequence derived from a parent sequence, a secretion sequence, a sequence derived from the sequence used to purify the polypeptide), or a polypeptide derived from a mature polypeptide conjugated to a compound , A proteinogen sequence (or a fusion protein), or a polypeptide derived from such a polypeptide. The fragments, derivatives and analogs defined in accordance with the present invention are well known to those skilled in the art.
The amino acid sequence represented by SEQ ID NO: 2, that is, the polynucleotide encoding the mature polypeptide, is a coding sequence that encodes only the mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences coding for noncoding sequences.
The term " polynucleotide encoding the polypeptide " refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.
The present invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequence as described above, or fragments, analogs and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The nucleotide mutant includes a substitution mutant, a deletion mutant, and an insertion mutant.
As is known to those of ordinary skill in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides, Lt; RTI ID = 0.0 > polypeptide < / RTI >
According to another aspect of the present invention, a recombinant vector containing the gene may be provided.
The vector according to the invention can typically be constructed as a vector for cloning or expression. In addition, the vector according to the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector according to the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter (for example, pL promoter, trp promoter, lac promoter, T7 promoter, tac promoter, etc.) , Ribosome binding sites for initiation of detoxification, and transcription / translation termination sequences. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway, and the left-handed promoter of the phage (pL promoter) can be used as a regulatory region.
Herein, the vectors that can be used in the present invention include plasmids (e.g., pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX, pQE80L series, pET series and pUC19, etc.) commonly used in the technical field to which the present invention belongs, (E.g., gt4B, -Charon, z1, and M13) or viruses (e.g., SV40, etc.).
In addition, when the vector according to the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a mammalian virus (e.g., Adeno Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.
The vectors according to the present invention may be used as selectable markers and include antibiotic resistance genes commonly used in the art to which the present invention belongs, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, , ≪ / RTI > neomycin, and tetracycline.
According to another aspect of the present invention, a host cell transformed with the recombinant vector may be provided.
The host cell for stably transforming the vector according to the present invention into a prokaryotic cell and then expressing the vector may be a host cell commonly used in the technical field of the present invention, for example, E. coli JM109 , E. coli BL21 , E. coli RR1 , E. coli LE392 , E. coli B , E. coli X 1776 , E. coli W3110, Bacillus subtilis, and Bacillus thuringiensis, or enterobacteria and strains such as Salmonella typhimurium, Serratia marcesensus and various Pseudomonas spp. Can be used.
When the vector according to the present invention is transformed into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (for example, Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells.
The method of delivering a vector according to the present invention into a host cell can be carried out by the CaCl 2 method, Hanahan, D., J. MoI. Biol., 166: 557-580 (1983) ) And electric drilling method. When the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, have.
According to still another aspect of the present invention, there can be provided a feed additive for promoting fibrin degradation comprising the endo-1,4-beta-xylanase active enzyme protein.
Feed additives according to the present invention include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid; Phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate) and the like; Natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like have.
The feed additive according to the present invention may include adjuvant components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes and other microbial agents in the form of live cells; Grain, for example, ground or crushed wheat, oats, barley, corn and rice; Vegetable protein feedstuffs, for example, based on rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A dry component consisting of sugar and dairy products, for example various powdered milk and whey powders; Lipids such as animal fat and vegetable fat optionally liquefied by heating; Nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents, and the like.
The feed additive according to the present invention may be in the form of a dry or liquid preparation, and may contain an excipient for feed addition. Examples of the excipient for adding the feed include zeolite, corn or rice bran, and the present invention is not limited to the above examples.
The feed additive according to the present invention may be used as an enzyme preparation, for example, a lipase such as lipase, a phytase which decomposes phytic acid to form phosphate and inositol phosphate, starch and glycogen Amylase, which is an enzyme that hydrolyzes the α-1,4-glycoside bond, phosphatase, which is an enzyme that hydrolyzes the organic phosphate ester, and maltose, And a conversion enzyme which hydrolyzes maltase and saccharose to produce a glucose-fructose mixture, and the like.
The feed additive according to the present invention may be administered to animals alone or in combination with other feed additives in edible carriers. The feed additives can also be administered as top dressing or they can be mixed directly with the livestock feed or separately from the feed, in separate oral formulations, or in combination with other ingredients.
A single daily intake or divided daily intake can be used as is commonly known in the art.
The animal in which the feed additive according to the present invention can be used can be, for example, an animal such as a cow, a cow, a calf, a pig, a pig, a sheep, a goat, a horse, a rabbit, a dog, , Poultry such as ducks, geese, turkeys, quails, small birds, and the like, and are not necessarily limited to the above examples.
The amount of endo-1, 4-beta-xylalanase according to the present invention contained in the feed additive according to the present invention is not particularly limited, but as is commonly known in the technical field of the present invention, Fibrous, and semi-fibrous materials.
According to another aspect of the present invention, a detergent composition comprising the endo-1,4-beta-xylalanase activator protein can be provided.
The detergent composition according to the present invention may be in the form of a part and a part of an aqueous detergent composition, a non-aqueous liquid detergent composition, a cast solid, a granular form, a granular form, a compressed tablet, a gel, a paste or a slurry. The detergent composition can be used to quickly remove food stains, food residue films, and other small amounts of food compositions.
The detergent compositions according to the present invention may be effective in removing dried stains through the catalytic-mediated hydrolysis of starch polysaccharides.
The detergent composition according to the present invention may be provided in the form of a detergent composition for cleaning hard surfaces, a detergent composition for cleaning fabrics, a detergent composition for washing dishes, a detergent composition for oral cleaning, a detergent for cleaning denture or a contact lens cleaning solution .
According to another aspect of the present invention, there is provided a method for producing biofuel comprising the step of hydrolyzing the biomass material by adding the endo-1,4-beta-xylanase protein.
The biomass material according to the present invention is a biomass material containing a high molecular weight carbohydrate. The method for preparing a biofuel according to the present invention comprises: pre-treating a biomass material containing a high molecular weight carbohydrate; Hydrolyzing the pretreated biomass with an enzyme; And a step of fermenting the hydrolyzed biomass material. The method of manufacturing the biofuel may be performed by a method of manufacturing a biofuel commonly known in the art.
In the method for producing a biofuel according to the present invention, the biomass may be selected from the group consisting of starchy (cereals, potatoes), cellulose (herb, wood, chaff) Organic material such as manure, carcass, microbial cells and paper and food waste derived therefrom), and the like, and the present invention is not limited to the above examples.
Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention and the contents of the present invention are not limited to the following examples.
Example
Isolation and DNA extraction of black goat rumen microorganisms
Two males and one female black goat were raised for only one month with rice straw and mineral supplements, and then slaughtered. The rumen was obtained from the four rumen, and the content was obtained. The obtained contents were first separated into a liquid phase (Lq) and an adherent phase (Ad) by using gauze. Feed scoop Ad was obtained by suspending the residue after filtering with gauze in 0.15% (volume / volume) Tween-80, PBS solution, and recovering the microorganisms attached to rice straw. A total of 5 g of the precipitated sample was prepared and extracted with CTAB (hexadecylmethylammonium bromide) and SDS (sodium dodecyl sulfate).
Black goat rumen microbe fosmid Construction of gene bank
For the DNA extracted from Ad and Lq, the amount of several HindIII restriction enzymes was adjusted for the appropriate truncation conditions to construct a fosmid gene bank. As a result, Ad was determined to use 5 units of HindIII and 2.5 units of Lq (Fig. 1). The partially cleaved DNA fragments were clipped from 10 kb to 50 kb using the Pulse-Field Gel electrophoresis method, from which the insert DNA fragments were again prepared. The prepared DNA was subjected to terminal smoothing reaction and ligated to the pCC1FOS vector by ligation reaction. After the ligation reaction, it was transformed into DH5alpha E. coli by electrical shock method to construct a gene bank. In order to confirm the average insertion size of the constructed fosmid gene bank, NotI enzyme treatment was performed and it was confirmed by electrophoresis that 30 kb for Ad and 31 kb for Lq (FIG. 2). For each of these, 150 plates of 384 well plates were inoculated on cryopreservation media and stored in a cryogenic freezer (-70 ° C).
Esculin ( Esculin ) Decomposing activity Fosmid ( fosmind ) Clone starter
A total of 115,200 clones of Ad and Lq phosphodiester clones were inoculated with 0.5 μl of LB chloramphenicol agar medium containing 1.65% esculin iron substrate in 384 clone units using a VIAFLO 384 instrument from INTEGRA, Time. Clones having the activity of degrading esculin in the cultured plate exhibited a brown color around them, and thus active clones were selected visually (Fig. 3).
Esculin ( Esculin ) Decomposition activity Fosmid Sequence decode for clones
The fosmid plasmid extraction was performed using the Qiagen plasmid midi prep kit for the identified phosmid clone Ad_006O06. The extracted plasmid DNA was amplified by a semi-conductor sequencing method, Ion Personal Genome Machine (PGM) We generated shotgun sequencing data of drainage. The resulting data was converted to fasta format and assembled using the Newbler 2.5 de novo assembler program. A total of three consensus sequences were constructed, and a total of 34,870 bp of metagenomic sequence information was obtained with a length of 22,495 bp, 9,103 bp, and 3,272 bp, respectively.
Sequence List
- Sequence Locations: National Livestock Research Institute, RDA
- Black goat derived from the sequence: Rumen obtained from slaughtering after feeding for 3 months (2 males, 1 female) born in the livestock genetics laboratory in the National Livestock Research Institute, RDA Microbial sample
- Fosmid gene bank vector: pCC1Fos
- Genomic Fosmid clone name: Ad_051B10
- DNA sequence of the gene (SEQ ID NO: 1) Length: 1,548 bp
- the amino acid sequence of the gene (SEQ ID NO: 2) length: 516 amino acids
- DNA and protein sequence of the gene: See the following table and sequence listing
Gene search and similarity survey
The inserted DNA sequence of fosmid clones was searched for the gene region using the MetaGeneMark program. Regarding the genetic information, the sequence similarity information of each gene was firstly obtained by protein blast search of NCBI. In this process, a novel cellulolytic enzyme gene was extracted by referring to the main domain search result and the similar protein. Cel10-KG99 is the nucleotide blast result (Fig. 4) as of October 2015, and Contig20b (KC246833) among the ruminal microorganism metagenomic decoy sequences registered by Wang et al in 2014 is 76% 72% homology, indicating that the cel10-KG99 gene is a novel gene sequence that is different from the KC246833 sequence. As a result of the protein blast search, it is confirmed that the carbohydrate binding module (CBM) 4_9 series having the function of allowing the enzyme to adhere to the cellulose contains the GH10 domain and the GH2C domain which are common in the xylanase family , Suggesting that the cel10-KG99 gene is a xylanase-based enzyme (Fig. 5). Similar protein searches revealed that Wang and his colleagues had 68% homology with the endo-1,4-beta-xylanase (AHF25759) in the entire 99% of the bovine rumen meta- genomic genes released in 2014 (Fig. 6). As a result of comparison with other types of the proteolytic enzyme-related protein sequence, the product of cel10-KG99 gene can be said to be an endo-1,4-beta-xylanase family of fibrinolytic enzymes (FIG. 7).
The product of the cel10-KG99 gene, which is a kind of the endo-1,4-beta-xylanase according to the present invention, can be used not only as a feed additive but also in the production of detergents and biofuels, Lt; / RTI >
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> RURAL DEVELOPMENT ADMINISTRATION <120> Endo-1,4-beta-xylanase cel10-KG99 gene from rumen microorganism of black goat and uses thereof <130> NPF-29355 <160> 2 <170> PatentIn version 3.2 <210> 1 <211> 1548 <212> DNA <213> unidentified bacterium <400> 1 atgcggaaat tgttgctgat cctgctggcg ctgctgatgg cgctgaatgt atcccccgtt 60 ctggccgagg gaacggtata cgcttccaac tttgcatccg gtcctgacgg ctggtatggc 120 cggggtgcgc aagtgtctgt tatcaatggc gtactgcgca cccagggccg ccaaagcgac 180 tggaactctc ccggccgtga ttttgatctg atccaggacg ggcattacct gctttccgtt 240 caggtgtatc agaatgaaaa agataccgcg gacttcatga tttccgtcgc ccactccaag 300 gacggcatgg aaagctatga aaacctggcc catggcaccg cgaaaaaggg tgaatggacc 360 accctgcagg gcgactatac agccggcgat tatgaaaaat tcgtgctgta cgtggaaacc 420 accggcgctt ctgagctggt tttcgacatc aaggatttca ccgtcacagc cccggacggc 480 ctgccccagg ccaaacctac cgagccgccc atggtaatcg cccaggtgga cgaagtaccg 540 tcactgaaag aaatctatgc cgataagttt gatttcggcg cggctgcccc ccagcaggcg 600 ttccgggata tcggcctgat gaagctgatg aagcagcagt tcagcatcct cacaccggaa 660 aacgaattga agccggatgc tgtgctggat acctctgcca gcaaaaagct ggtgcaggaa 720 accggtgatg agaccgccgt tgccgttcac tttgacagcg cgaagccatt gctgaatttt 780 gctcagaaga acggcatcaa ggtgcatggc catgtgctgg tttggcacag ccagaccccg 840 gaagtatttt tccgtgaagg ctacgacgcc aagaaaccgc tcctgaaaag agaaatgatg 900 ctgggccgcc ttgaaaacta catcaaaaac gtaatggctt acctggagga aaactatccc 960 ggggtggtgg tcagctggga cgtggtgaac gaagccatcg atgacggcac gggctggctg 1020 cgcaagggct ccgcctggta taaggtggtg ggcgaagatt tcgttgcccg ggcctttgag 1080 tacgcgcgga aatacgcccc cgaaggcacg ctgctgtatt acaacgatta caacaccgcc 1140 atgtccggca agcgcaaggg catcgtgcgc ctgctggaat ccctgatccc ggaaggcaat 1200 atcgacggtt acggcttcca gatgcaccac agcgtgggca gcccttccat gcagcagatc 1260 gcggattccg taaaggatat cgccgccctg ggcctgcgtc tccgcgtcag cgaactggat 1320 gtgggcacgg ataaccgtac cgaagccagc ttcaaaaagc aggcgcagaa gtattcctgg 1380 atcatgaagg tgctgcgccg ctacagcgat cagctggaag ccgtccaggt atggggcctc 1440 accgacaaca tgagctggcg cgccaaggat ttcccgcttc tgttcgattc ctcccgcaac 1500 cccaagcccg ctttttgggc ggtggccgat ccggattccg tccagtga 1548 <210> 2 <211> 515 <212> PRT <213> unidentified bacterium <400> 2 Met Arg Lys Leu Leu Leu Leu Leu Leu Leu Leu Met Ala Leu Asn 1 5 10 15 Val Ser Pro Val Leu Ala Glu Gly Thr Val Tyr Ala Ser Asn Phe Ala 20 25 30 Ser Gly Pro Asp Gly Trp Tyr Gly Arg Gly Ala Gln Val Ser Val Ile 35 40 45 Asn Gly Val Leu Arg Thr Gln Gly Arg Gln Ser Asp Trp Asn Ser Pro 50 55 60 Gly Arg Asp Phe Asp Leu Ile Gln Asp Gly His Tyr Leu Leu Ser Val 65 70 75 80 Gln Val Tyr Gln Asn Glu Lys Asp Thr Ala Asp Phe Met Ile Ser Val 85 90 95 Ala His Ser Lys Asp Gly Met Glu Ser Tyr Glu Asn Leu Ala His Gly 100 105 110 Thr Ala Lys Lys Gly Glu Trp Thr Thr Leu Gln Gly Asp Tyr Thr Ala 115 120 125 Gly Asp Tyr Glu Lys Phe Val Leu Tyr Val Glu Thr Thr Gly Ala Ser 130 135 140 Glu Leu Val Phe Asp Ile Lys Asp Phe Thr Val Thr Ala Pro Asp Gly 145 150 155 160 Leu Pro Gln Ala Lys Pro Thr Glu Pro Pro Met Val Ile Ala Gln Val 165 170 175 Asp Glu Val Pro Ser Leu Lys Glu Ile Tyr Ala Asp Lys Phe Asp Phe 180 185 190 Gly Ala Ala Ala Pro Gln Gln Ala Phe Arg Asp Ile Gly Leu Met Lys 195 200 205 Leu Met Lys Gln Gln Phe Ser Ile Leu Thr Pro Glu Asn Glu Leu Lys 210 215 220 Pro Asp Ala Val Leu Asp Thr Ser Ala Ser Lys Lys Leu Val Gln Glu 225 230 235 240 Thr Gly Asp Glu Thr Ala Val Ala Val His Phe Asp Ser Ala Lys Pro 245 250 255 Leu Leu Asn Phe Ala Gln Lys Asn Gly Ile Lys Val His Gly His Val 260 265 270 Leu Val Trp His Ser Gln Thr Pro Glu Val Phe Phe Arg Glu Gly Tyr 275 280 285 Asp Ala Lys Lys Pro Leu Leu Lys Arg Glu Met Met Leu Gly Arg Leu 290 295 300 Glu Asn Tyr Ile Lys Asn Val Met Ala Tyr Leu Glu Glu Asn Tyr Pro 305 310 315 320 Gly Val Val Val Ser Trp Asp Val Val Asn Glu Ala Ile Asp Asp Gly 325 330 335 Thr Gly Trp Leu Arg Lys Gly Ser Ala Trp Tyr Lys Val Val Gly Glu 340 345 350 Asp Phe Val Ala Arg Ala Phe Glu Tyr Ala Arg Lys Tyr Ala Pro Glu 355 360 365 Gly Thr Leu Leu Tyr Tyr Asn Asp Tyr Asn Thr Ala Met Ser Gly Lys 370 375 380 Arg Lys Gly Ile Val Arg Leu Leu Glu Ser Leu Ile Pro Glu Gly Asn 385 390 395 400 Ile Asp Gly Tyr Gly Phe Gln Met His His Ser Val Gly Ser Ser Ser 405 410 415 Met Gln Gln Ile Ala Asp Ser Val Lys Asp Ile Ala Ala Leu Gly Leu 420 425 430 Arg Leu Arg Val Ser Glu Leu Asp Val Gly Thr Asp Asn Arg Thr Glu 435 440 445 Ala Ser Phe Lys Lys Gln Ala Gln Lys Tyr Ser Trp Ile Met Lys Val 450 455 460 Leu Arg Arg Tyr Ser Asp Gln Leu Glu Ala Val Gln Val Trp Gly Leu 465 470 475 480 Thr Asp Asn Met Ser Trp Arg Ala Lys Asp Phe Pro Leu Leu Phe Asp 485 490 495 Ser Ser Arg Asn Pro Lys Pro Ala Phe Trp Ala Val Ala Asp Pro Asp 500 505 510 Ser Val Gln 515
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