KR101245304B1 - Metagenome Libraries with Alginate Lyase Activity - Google Patents

Abstract

The present invention relates to a metagenome library having alginate lyase activity. The present invention is the 1280-3652th nucleotide sequence, 5025-6128 nucleotide sequence, 6365-7492 nucleotide sequence, 8687-9202 nucleotide sequence, 9206-9823 nucleotide sequence, 9823-11250th nucleotide sequence of SEQ ID NO: 1 , 11377-12189 nucleotide sequence, 12290-13306 nucleotide sequence, 13611-14642 nucleotide sequence, 14646-15212 nucleotide sequence, 15417-16388 nucleotide sequence, 17370-19223 nucleotide sequence, 19452-21017 nucleotide sequence, 21253-22143 nucleotide sequence, 22309-23121 nucleotide sequence, 23333-24208 nucleotide sequence, 24294-24989 nucleotide sequence, 25446-26477 nucleotide sequence, 26549-28063 nucleotide sequence, 28362-29579 nucleotide Sequences, relates to meth genome library with a second nucleotide sequence 30103-30930 and 31010-31696 second nucleotide sequence alginate rayiah activity containing 22 open reading frames, including. The present invention provides novel microorganisms and proteins by analyzing unknown metagenomes present in natural microbial populations.

Description

Metagenome Libraries with Alginate Lyase Activity

The present invention relates to a metagenome library having alginate lyase activity.

Seaweeds are abundant habitats in the offshore waters of Korea and are a potential resource. Most seaweeds are used for food through simple processing, but they are expected to be used as bioenergy sources or as functional foods and pharmaceutical raw materials.

Polysaccharides make up the majority of seaweed, but the salt content is high and the surface layer is surrounded by mucopolysaccharide. Therefore, in order to utilize algae as a resource, decomposition of polysaccharides must be accompanied. Polysaccharides such as fibrin and agar are β-1,4-glycosides that can be degraded by various microorganisms. However, microorganisms using polysaccharides of land plants as substrates and microorganisms using polysaccharides of algae as substrates differ in terms of habitat environment and salinity of substrates. For example, if several passages of bacteria having excellent cell wall resolution of maesangi can be confirmed that the cell wall resolution is significantly reduced. In order to solve such a problem, it is necessary to isolate various microorganisms having excellent polysaccharide degrading ability and establish conditions under which these microorganisms can stably decompose algae.

In order to reduce the degree of polymerization of seaweed polysaccharides such as alginic acid, low molecular weight alginic acid is prepared by lowering the degree of polymerization by acid hydrolysis with hydrochloric acid and organic acid. Low molecular weight alginic acid production process by acid is required to neutralize and discharge the acid-resistant device and discarded strong acid due to excessive acid treatment in the process, the process cost is high because a large amount of alkali is required. However, by using abalone-derived microbial enzyme, it is possible to produce low molecular weight alginic acid under mild reaction conditions without strong acid and strong alkali. There is a need to establish technologies that reduce the amount of waste and increase the utilization of resources.

Cultivation of microorganisms in the natural environment is very complicated and difficult. Less than 1% of microorganisms can be isolated and cultured from the actual environmental ecosystem, and cannot be cultured using conventional culture techniques. In order to overcome these difficulties, molecular biological methods have been applied as a method for grasping the diversity, structure, and function of bacterial communities without separating and culturing microorganisms. Among them, the most commonly used part is systematically analyzed by sequencing analysis through nucleotide sequencing of 16S rRNA gene, which is a molecular biological method using nucleic acid, and researches using the same have been actively conducted. The 16S rRNA gene contains information that can differentiate bacteria at the species level, and the change in the nucleotide sequence of this gene is very useful for identifying the evolutionary flexibility between microorganisms. However, even if the information on the microorganisms in the ecosystem is confirmed by molecular biological methods, it is very important to isolate the microorganisms and to identify the characteristics of the microorganisms.

The demand for new functional foods and fermentable biomass increases the demand for new microbial enzymes. Lyase-producing microorganisms isolated from the intestinal tracts of marine organisms are algae-degradable mixtures of agarase, laminase, cellulase and alginate lyase. To produce enzymes. ^In addition, alginate lyase can be used to treat microorganism's brown algae by α-L-guluronic acid (G), β-D-mannuronic acid (M), alternating MG (GM) and heteropolymeric MG ( There are a number of reports of degradation to GM). ¹¹ abalone and Psuedoalteromonas sp. IAM 14594 (AF082561), Sphingomonas sp. A1 (AB120939), Klebsiella pneumonia subsp. aerogenes (L19657), Vibrio sp. QY101 (AY221030) and Psuedomonas sp. This enzyme is isolated from the intestines of many marine bacteria, including OS-ALG-9 (AB003330). ^2,4,13,15 So far, alginate ^lyase reported in the Genebank database is on the order of 23 ORF. ²⁴ Previous studies have shown that alginate lyases of many different microorganisms are G block-specific polyguluronate lyases (EC 4.2.2.11) and M block-specific polymannuronate lyases (EC 4.2). Can be classified according to the two substrate specificities. ¹⁹ Recently, promoting the root growth of higher plants, Bifidobacterium sp. Alginates have been found to be degraded by enzymes with certain biological activities such as acceleration of growth rate, induction of cytotoxic cytokine production in human mononuclear cells, inhibition of IgE, and antihypertensive effects. ⁵ , alginate lyases and alginate oligosaccharides have attracted attention from researchers in the food and pharmaceutical industry.

Independent studies using cultured clone library of the 16s rRNA gene Alpha -, Gamma -, Epsilonproteobacteria and mollicutes major microbial soldiers overturned Haliotis discus It shows the diversity of the whole bacteria in the viscera of hannai . ²¹ However, the information of the 16s rRNA gene does not provide effective information for knowing the biological function of the microorganism. ^17,23 In contrast, the metagenome library using a fosmid cloning system can identify new genes in organisms that are not easy to culture. ^1, the screening system kitty using the insert (insert) size of about 40 kb isolated directly from samples of the phosphine imide vector, and a variety of environmental xylanase (chitinase), dihydro-agent and the (dehydrogenase), oxy-dori virtue hydratase (oxidoreductase) It has been used to identify a wide range of genes such as amylase, esterase, endoglucanase and cyclodextrinase. ^{1,20 The} inventors identified a highly active alginate lyase gene in abalone intestinal microflora using metagenome library.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

We sought to build a metagenome library with alginate lyase activity. As a result, a microgenome library having alginate lyase activity was identified by separating microorganisms derived from abalone intestine, and the present invention was completed by analyzing the properties of alginate lyase.

It is an object of the present invention to provide a metagenome library having alginate lyase activity.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to an aspect of the present invention, the present invention provides the 1280-3652th nucleotide sequence, 5025-6128th nucleotide sequence, 6365-7492th nucleotide sequence, 8687-9202th nucleotide sequence, 9206-9823th nucleotide sequence of SEQ ID NO: 1 , 9823-11250 nucleotide sequence, 11377-12189 nucleotide sequence, 12290-13306 nucleotide sequence, 13611-14642 nucleotide sequence, 14646-15212 nucleotide sequence, 15417-16388 nucleotide sequence, 17370-19223 nucleotide sequence, 19452-21017 nucleotide sequence, 21253-22143 nucleotide sequence, 22309-23121 nucleotide sequence, 23333-24208 nucleotide sequence, 24294-24989 nucleotide sequence, 25446-26477 nucleotide sequence, 26549-28063 nucleotide sequence, 2836 A metagenome library having alginate lyase activity comprising 22 ORFs comprising a 2-29579 nucleotide sequence, 30103-30930 nucleotide sequence, and 31010-31696 nucleotide sequence is provided.

The term used herein, "nucleotide" includes analogs of the single-stranded or a deoxyribonucleotide or ribonucleotides present in double stranded form, the nature of the nucleotides that are not specifically stated otherwise (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990).

As used herein, the term “alginate lyase” refers to an enzyme that degrades and lowers alginate composed of G block-specific polyguluronate and M block-specific polymannuronate.

Preferably the metagenome library has a nucleotide sequence of SEQ ID NO: 1, more preferably the metagenome library is constructed using genome DNA obtained from the microflora of the abalone viscera.

As used herein, the term “metagenome” refers to the total microbial collection existing in nature, and the DNA of microorganisms collected from various natural environments such as soil, seawater, tidal flats, rivers, air, and the large intestine of animals are separated according to their species. The metagenome is not mixed but mixed together. It is also a technology that analyzes the function or diversity of microorganisms in the form of genes. Metagenome clones metagenome, which is the genome of total microorganisms existing in various environments in nature, maintains and expresses it in host cells to make a metagenome library to classify a line of egg cultured microorganisms and to search for useful enzymes and secondary metabolites. It is for research.

The metagenome library of the present invention can be carried out through various methods known in the art. Preferably, Bruce A. Voyles (2002) The biology of viruses 2nd ed.ISBN 0-07-237031-9 and Barry G. Hall (July 2004). "Predicting the evolution of antibiotic resistance genes. ". Nature Reviews Microbiology 2 (5). doi: 10.1038 / nrmicro888. PMID 15100696) to build a metagenome library.

The metagenome library of the present invention has utility as a research tool for searching for useful enzymes (eg, alginate lyases) and secondary metabolites.

The features and advantages of the present invention are summarized as follows:

(a) The present invention can provide new microorganisms and proteins by analyzing unknown metagenomes present in a natural microbial group.

(b) The present invention can provide an enzyme that can stably decompose algae by separating microorganisms having excellent polysaccharide degrading ability.

(c) The present invention can provide a technique for reducing the amount of waste derived from the seaweed processing process and increasing the utilization of resources by utilizing the technology of biotechnological mass production of functional polysaccharides.

1 is a result of the evaluation of alginate hydrolysis using the cetylpyridinium chloride (cetylpyridinium chloride) method. 1 shows plate screening confirming activity. Flavobacterium sp. Positive control group (A) derived from, negative control group (B) derived from E. coli , and the alginolatic metagenome fragment (AlyDW, C) are shown.
FIG. 2 is a schematic diagram of an alginolatic metagenome fragment (AlyDW). FIG. Differences in contrast shown in the figure indicate functional classification of putative genes. Diagonal stripes are ORFs associated with cell processes, blank arrows are metabolic ORFs, and light gray are ORFs associated with information storage. Dark gray color indicates unclear genes.
Figure 3 shows the results of reducing sugar analysis confirming the effect of pH (A) and temperature (B) on the alginate lyase activity of metazyme fragment (AlyDW).
Figure 4 shows the results of depolymerization of alginate by metagenome fragments (AlyDW) utilizing thin layer chromatography. The left panel (a) shows the hydrolysis profiles of poly (β-D-mannuronate, lane 1) and poly (α-L-guluronate, lane 2) after incubation at 40 ° C. for 3 hours. Right panel (b) shows the digested alginate product of the HPLC fractions obtained after incubation of alginate and alginolatic metagenome fragments (AlyDW). Flavobacterium sp. Positive control (lane 4), negative control (alginate only, lane 5), negative control (only enzyme, lane 6). M is G1 (glucose), G2 (cellobiose), G3 (cellotriose), G4 (cellotetraose), G5 (cellopentaose), G6 (cellohexaoz), G7 (celloheptaose) and G8 (Cellooctaose).
5-6 show the alignment results of putative alginate lyase and metagenome fragment (ORF-11) proteins from metazyme fragments. Amino acid sequences were aligned using the ClustalX program. Compared organisms are Klebsiella pneumoniae subsp. aerogenes , Saccharophagus degradans 2-40, Microbulbifer sp. 6532A, Vibrio harveyi aDA3, Cellulophaga lytica DSM 7489, Vibrio sp. A9m, Vibrio alginolyticus 40B, Pseudoalteromonas sp . CY2, Vibrio splendidus 12B01, Polaribacter sp. MED152 and Agarivorans sp. JAM-A1m and the gene bank entry numbers of the organisms are Q59478, ABD81738, BAJ62034, ZP_0617515, YP_004263738, BAH79133, ZP_06182095, ACM89454, ZP_00990010, ZP_01052859 and BAG70358, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

Metanomic Library Construction

In February 2009, an abalone sample was obtained near Yeosu, South Korea. DNA was prepared by direct DNA extraction and purification. ⁷ Metagenome libraries were constructed from samples using the appropriate protocol described previously. _28. The purified DNA was partial digested with Sau3AI restriction enzyme and the appropriate amount of DNA eluted only 40-50 kb piece by electrophoresis on 1% agarose gel was dephosphorylated by the DNA (Calf intestinal alkaline phosphatase) CIAP. Metagenome DNA was digested with a plasmid (pCC1FOS ready vector, Epicentre, USA) with BamHI restriction enzyme and then ligated at 16 ° C. for 16 hours with DNA digested with Sau3AI. Packaging was performed in vitro with the MAX lambda packaging extraction kit (Epicentre, USA) and only recombinant colonies were plated on LB solid medium containing kanamycin (50 μg / ml). Subsequently, Escherichia coli was transfected to construct a megnome library.

Library Screening and Shotgun Sequencing

In order to evaluate the resolution of the active alginate lyase, cetylpyridinium chloride (CPC) analysis was performed. Metagenome DNA was ligated to a phosmid vector (pCC1FOS ready vector, Epicentre, USA) and packed into lambda phage and infected with Escherichia coli . The resulting infected cells were incubated for one day in Luria-Bertani agar containing 0.1% alginate. 10% CPC solution was added to the cells and the clear zone formed by waiting at 37 ° C. for 1 hour (FIG. 1). Shotgun DNA libraries prepared from purified phosmid DNA and PHRED / PHRAP / CONSED packages were used to combine the shotgun sequencing reads to determine the total sequence of positive clones. DNA sequences were determined using ABI 9700 Applied Biosystems and ABI PRISM ^™ BigDye ^™ Terminator Cycle Sequencing kit (Applied Biosystems, version 3.1) according to the manufacturer's knowledge. The blast program of the National Center for Biotechnology Information (NCBI) was used for database searches and sequence comparisons. The complete phosphid insert was annotated using the Artemis program and the function of the protein coding portion was confirmed using the BLASTP and PSIBLAST programs. Prospective proteins were classified according to the cluster category of orthologous groups (Cor). The complete nucleotide sequence of the phosphide clone open reading frames (ORF) obtained using the ^12,22-23 CLUSTAL_X program was aligned with homologous genes obtained from the NCBI nucleotide database. ₆

Determination of Enzyme Activity

Endoclucanase activity of the enzyme was determined by the Nelson-Somogyi assay, which measures the increased color intensity by enzymatic hydrolysis of glycosidic bonds of carbonate by treating large amounts of reducing sugars. ^Three standard curves were prepared using a spectrophotometer (Mecassis, Korea) for glucose in the concentration range of 31.5 μg / ml-100 μg / ml based on the 525 nm absorbance measurement. Temperature using 0.1 M citrate-phosphate buffer (pH 4.0-7.0), 10 mM phosphate buffer (pH 8.0) and 50 mM glycine-NaOH buffer (pH 9.0-10.0) Optimal enzyme activity was measured for 2 hours at (15 ° C.-50 ° C.) and pH (4.0-10.0). Inhibition and enhancement of enzyme activity was determined in the presence of metal ions and nicotinamide adinine dinucleotide (NAD ⁺ ) at optimal conditions (pH 8.0 and 40 ° C.).

Alginate Lyase ( alginate lyase ) Active measurement

Clones were incubated in LB (Luria-Bertani) medium at 40 ° C. for 16 hours for the production of alginate lyase. Culture medium was concentrated using a protein concentration kit (Rapid-Con ^™ protein concentration kit, Elpis, Seoul, Korea). AlyDW was analyzed by treatment with a 2-mercaptoethanol-free sample buffer according to the method described by Laemmli and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% acrylamide gel. ^{After 3} SDS-PAGE, the protein in the gel was washed three times in a regeneration buffer (50 mM Tris-HCl buffer, pH 7.0, 10 mg / ml casein, 2 mM EDTA, 0.01% NaN ₃ ) with 25% methanol. And regenerated for 30 minutes. 1.5% agarose consisting of 1 mg / ml sodium alginate, 10 mg / ml NaCl and 100 mM phosphate buffer 1 (pH 8) after washing the polyacrylamide gel with 100 mM phosphate buffer 1 (pH 8) Covered with gel. The gel was incubated at 37 ° C. for 20 hours and then immersed in 10% CPC solution for 1 hour and then washed in distilled water.

Thin layer chromatography  Used Reaction products  analysis

Cellooligosaccharides Standard mixtures (cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, celloheptaose ) And cellooctaose) were purchased from Sigma-Aldrich. To determine the alginate lyase activity of the test clone, 0.1% alginate was reacted with 50 μl of AlyDW (2.19 mg / ml) in 100 mM phosphate buffer 1 (pH 8) at 37 ° C. HPLC using an Asahipak GS-310 column (21.5 mm ID × 500 mm, Showa Denko Kogyo Co. Ltd., Tokyo, Japan) to fraction of alginate oligomer by gel filtration chromatography (GFC). (Shimaju system, LC-6AD pump, RID-10A detector, SPD-M20A detector: Shimazu, Tokyo, Japan) was performed. The sample was separated by flowing water at 5 ml / min. Aliquot (2 mL) of digested alginate solution was injected to give a fraction. The reaction product was reacted in 1-propanol, nitromethane and water mixtures (5: 3: 2, v / v / v) for 2 hours before silica gel plate (Merck KGaA, The hydrolysis product was isolated from thin layer chromatography using 64271 Darmstadt, Germany). After separation, in sugar 1 ml of sulfuric acid and 10 ml stock solution (1 g diphenylamine, 1 ml aniline, 100 ml acetone) The mixture was visualized by spraying. ²⁶

Results and Discussion

Metagenome Of the fragment  Screening and ORF  analysis

One bacterial metagenome fragment (AlyDW) expressing alginate lyase activity was selected from 3,840 clones selected from a total of 90,000 clones composed of the intestinal microflora of alon (FIG. 1). Analysis of shot-gun sequencing data indicated that the positive metagnome fragments were 31.7 kb in length with an average G + C content of 43.3% and 22 expected ORFs (Figure 2 and Table 1). A in Table 2 was prepared based on TMHMM survey, and b indicates that it does not correspond to COG group.

ORF
No.
Homologous to BLASTP
Organism / Phylum or Domain
location
Blast
e-value
Number of TMHs ^a

COG
No.
COG
category One (NiFe) hydrogenase
maturation protein hypF Ferrimonas balearica
Of Gammaproteobacteria 1280..3652 0.00E + 00 0 O COG0068 2 Hydrogenase accessory
protein HypB Ferrimonas balearica
Of Gammaproteobacteria 5025..6128 5.00E-
105 0 OK COG0378 3 Hydrogenase expression
/ formation protein HypD Ferrimonas balearica
Of Gammaproteobacteria 6365..7492 2.00E-
137 0 O COG0409 4 Hypothetical protein Vibrio sp
./ Gammaproteobacteria 8687..9202 1.00E-47 One - NA ^b 5 Hypothetical protein Vibrio sp
./ Gammaproteobacteria 9206..9823 3.00E-
258 One - NA 6 Hypothetical protein Vibrio coralliilyticu
s / Gammaproteobacteria 9823..11250 1.00E-73 One - NA 7 Transcriptional regulator Vibrio parahaemolyticus
Of Gammaproteobacteria 11377..12189 2.00E-79 0 K COG0583 8 Hypothetical protein Photobacterium sp .
Of Gammaproteobacteria 12290..13306 2.00E-61 One - NA 9 Multidrug resistance efflux
pump Vibrio vulnificus
Of Gammaproteobacteria 13611..14642 2.00E-
166 2 Q COG1566 10 Hypothetical protein Vibrio vulnificus
Of Gammaproteobacteria 14646..15212 3.00E-55 3 - NA 11 Alginate lyase (= Poly β -Dmannuronate
lyase) Klebsiella pneumoniae subsp .
aerogenes Of Gammaproteobacteria 15417..16388 6.00E-
117 0 - NA 12 Lyase, putative Vibrio splendidus
Of Gammaproteobacteria 17370..19223 0.00E + 00 - - NA 13 Alginate lyase Vibrio sp .
Of Gammaproteobacteria 19452..21017 0.00E + 00 0 - NA 14 Transcriptional regulator,
LysR family Vibrio splendidus
Of Gammaproteobacter 21253..22143 9.00E-
122 0 K COG0583 15 Probable oxidoreductase Vibrio sp .
Of Gammaproteobacteria 22309..23121 1.00E-95 0 QR COG1028 16 Oxygen-independent
coproporphyrinogen III
oxidase, putative Vibrio splendidus
Of Gammaproteobacter 23333..24208 6.00E-
138 0 C COG1032 17 Flavodoxin reductase
family 1 protein Vibrio parahaemolyticus
Of Gammaproteobacteria 24294..24989 4.00E-72 0 R COG3217 18 Dihydroorotase Vibrio coralliilyticus
Of Gammaproteobacteria 25446..26477 2.00E-
171 0 F COG0418 19 Glycerol kinase Vibrio cholera
Of Gammaproteobacteria 26549..28063 0.00E + 00 0 C COG0554 20 Glucose-1-phosphate
adenylyltransferase Vibrio shilonii
Of Gammaproteobacteria 28362..29579 0.00E + 00 0 0 COG0448 21 ABC transporter,
periplasmic substratebinding
protein Vibrionales bacterium
/ Gammaproteobacteria 30103..30930 2.00E-88 0 P COG0226 22 NodN-related protein Vibrio orientalis
Of Gammaproteobacteria 31010..31696 6.00E-80 0 I COG32030

17 of the ORFs show significant similarity with genes of functions known to the NCBI nucleotide database, and 5 ORFs correspond to hypotheses proteins in the genome database of NCBI bacteria. Six ORFs are involved in cell processing and information storage, and 10 ORFs are involved in metabolism. 3 ORFs (11, 12 and 13) are Klebsiella pneumonia subspecies. aerogenes, Vibrio splendidus and Vibrio The species showed similarity with the protein sequence of the alginate lyase gene of Gammaproteobacteria .

AlyDW Features of

In the reducing sugar assay, the alginolytic activity showed more than 80% of activity at 30-45 ° C and peaked at 40 ° C. Even at low temperatures (15-25 ° C.), 60 ± 5% of the activity shown at 40 ° C. was maintained, demonstrating the characteristics of low temperature active enzymes (FIG. 3). It showed maximum alginolytic activity at pH 8 and about 90% residual activity at pH 4 to pH 9. However, activity dropped sharply above pH 9 (FIG. 3). The effects of metal ions and cofactors such as NAD ⁺ on the activity of alginate lyase are summarized in Table 2. Table 2 shows the relative value of the negative control group as '1' and the standard deviation value was derived by three independent experiments.

chemical substance Fold active single Combination (Cation + NAD ⁺ ) None 1.00 ± 0.05 - NAD + (0.1mM) 0.94 + - 0.01 - EDTA (1 mM) 0.80 ± 0.01 - CaCl2 (1 mM) 0.94 ± 0.02 1.51 ± 0.20 MgCl2 (1 mM) 0.93 ± 0.01 1.29 ± 0.10 KCl (1 mM) 0.90 ± 0.01 1.40 ± 0.36 MgSO4 (1 mM) 0.90 ± 0.15 1.68 ± 0.05 FeSO4 (1 mM) 0.86 ± 0.03 1.11 ± 0.04 CoCl2 (1 mM) 1.13 ± 0.02 1.36 ± 0.24 MnCl2 (1 mM) 0.81 ± 0.01 1.11 ± 0.14 CuCl2 (1 mM) 0.98 ± 0.01 1.17 ± 0.10 NaCl (1 mM) 0.94 ± 0.02 1.16 ± 0.05 AgNO3 (1 mM) 1.61 ± 0.02 2.08 ± 0.36

The results indicate that the combination of NAD ⁺ , Mg ^{2 +} and Ag ⁺ has at least 1.6-2.0 fold stimulatory effect on alginate lyase activity as compared to the control without metal cation and NAD ⁺ . NAD ⁺ causes an increase in alginate lyase activity while EDTA inhibits about 20% of alginate lyase activity. This may be due to the chelation of cations essential for enzymatic activity. Absolute enzyme activity is 0.15 μmoles / min / mg protein at optimal conditions. Haliotis spp . , Littorin spp . And alginate lyases obtained from marine mollusks including Turbo cornutus are endo-poly (M) and exo-poly (G) lyases having optimal values at 25-50 ° C. and pH 4.0-9.6. Enzyme activity is the enzyme activity was increased when the second of Ca ^{2 +} or Mg ^{2 +} cations have. ^9,16,26-27 It has been reported that NAD ⁺ and metal ions are required for the hydrolysis of α-1,4 or β-1,4-glucosidic linkages in a group of glycosidase such as GH4. . ²⁵

Another profile of alginate oligomers was confirmed by alginate degradation of alginate lyase (AlyDW). Alginate hydrolysis final product of AlyDW was analyzed by thin-layer chromatography after 3 hours (Fig. 4). 4, AlyDW has endolytic activity and preferentially degraded poly (β-D-mannuronate) over poly (α-L-guluronate). Alginate lyases isolated from marine molluscs are most preferred endo-poly (M) lyases. ²⁷

High performance liquid chromatography (HPLC) performed three of the 20 fractions Flavobacterium. sp . Unlike the commercial enzymes of origin, it consists of alginate products decomposed to 180 kDa-342 kDa and hexamers of 1,153-1,315 Da, which makes AlyDW a useful enzyme to make products of lower molecular weight than commercial enzymes.

Arginolatic ORF -11 Features

AlyDW has 3 ORFs (ORF11, 12 and 13) theoretically having molecular masses of 35, 67 and 57 kDa. After concentrating the metagenome bacterial cell suspension with an Amicon concentrator, the multibands appeared on 10% SDS-PAGE of unrelated protein. After SDS-PAGE, the protein in the gel was analyzed by SDS-PAGE under regeneration conditions and activated stained.

Guess amino acid sequence of ORF11 (AlyDW) and Sde_2478 ( Saccharophagus degradans 2-40) and alyA ( Klebsiella pneumoniae subsp. aerogenes) homologous relationship between the gene was 63% and 65%, respectively. Whereas algMsp ( Microbulbifer sp. 6532A), VME_15370 ( Vibrio harveyi 1DA3), Celly_3050 (Cellulophaga lytica DSM 7489), alg (Vibrio sp. A9m), VMC_35250 (Vibrio alginolyticus 40B ), alyPI (Pseudoalteromonas sp. CY2), V12B01_24259 (Vibrio splendidus 12B01), MED152_06195 ( Polaribactor sp. MED152) and AlgL ( Agarivorans sp. JAM-A1) gene was 54% -60% (Figs. 5-6). Polysaccharide lyase (PL) family 7 alginate lyases are known to act as substrate binding and catalytic sites for (R / E) (S / T / N) EL, Q (I / V) H and It has three kinds of highly conserved amino acid sequences of YFKAG (V / I) YNQ. ^The ORF11 of the ²⁴ AlyDW metagenome fragments is characterized by G and M specific lyases (AlyA) from K. pneumonia, M-specific lyases (AlxM) from marine bacteria ATCC 433367, and G-specific lyases from Corynebacterium sp. ) Also have RSEL, QIH and YFKAGVYNQ. The amino acid sequence is believed to be an essential part of maintaining the stable three-dimensional structure and function of alginate lyase. ^13-14

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Metagenome Libraries with Alginate Lyase Activity <130> PN110243 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 31713 <212> DNA <213> Unknown <220> <223> metagenomic library derived abalon tract <400> 1 agcttgcgct aaacaaggtg ttttcgcgtt tgccttacaa tgtggtgatc aggctgcgca 60 attgcttaaa tttggccaac aagtcttatt acatgccaat gcacctgagc cacagccact 120 gattagtcac cctgctgtgc aatcctcaat caccgccttg ggtctctctg ccagttggga 180 aaaagggaaa ctgttcatca aacgcgatca caattccttg cccctacctc gcccttggct 240 tagcgcagct tatgcactag aaaacatccg tcaacgtgac cgattcagta aaaagtgtgc 300 ggtgctgttt ttagacaatc aacagccgtc agggttatta caacgtgcca acgatacctc 360 gtctaatcac tatcaatggt tatttgaagt gcctcgccct ccgcaagggt tgagcttgac 420 tcaactgctc aatgacatca ccggcaatga tccaagcagt cagttattgg tggacgactt 480 tatctcgtct aaccctgagc taataaaaaa cgcgccagtt accaatgcta gcttgagcga 540 tatgcttgga ttgctggcaa tgatactggg cctaactgac agccaatcta ccttgcaaca 600 agggtgcaat cgcgccattg ctcacgtaat gagccaccgc tttgccacct tccctcgcat 660 tcaattcgag ttagcttccg aaggtgatgc gctgagtata gattggtcaa aagcgttcca 720 atctgtgctc agttatcgcg ctgctatgag tgacgataac accgtaatca tcgcaggatt 780 tttagacagc cttgccgatt acctttgtca atggattgaa aaactcgacc aacacattgg 840 ctttgaaaaa gtcaccctag cgggcaatga atttgataac cctattttta ctgagatgct 900 gcgcttacgt ctgagtaaaa acgtggcgtt agtgcacgag aacgactcag acttgaaaga 960 gatgaacctt gtatttggcg caatgtatgt cgccgaaaat gcgcaattga ttagagatta 1020 gtaaggggga tttatgtgct tttctattcc gtcaaaaatc accactttgg atgaaaccaa 1080 tcaacgagcc gtggtggata cgttaggtac tgccagagaa gtggataccc acttgatcag 1140 cgaaccgatt gccgttggcg attatgtgtt gattcaatcc ggtcgcgctg tacaaaccat 1200 gcccgaacaa gctgcattag aaagcttgga gctgtatcaa atcattttcg ataagatgca 1260 gagcggagag atataaccca tgtccggtgc tggaattcaa ttaacgatta caggccaagt 1320 gcaaggggtc ggctttcggc cgttcatttg gcaattggca aacgaatttc agctgtgtgg 1380 tgaagtgtat aataatgcgc aaggtgtcat aattaaggtg cataaaaccc accaaatgag 1440 cgcatttatt gaggccattc aagaacgtgc tccgcaactg gcgcatatct cagatatcag 1500 tcaaaaaacc attaccaccg cgaattatca cacatttaca atttcccatt cctgcttagg 1560 ggaaatgcac acacaaattc cggccgatgt ggcaacctgc caagactgtt taactgagtt 1620 aaatacgcca acggattccc gttatcatta tgcattgaac aactgtattc actgcgggcc 1680 acgctactca attattgagc gtatgcccta tgacagatcc gcgaccacaa tggccgactt 1740 tgctatgtgc gagccatgcg ccacacaata ccgtgatccg aaaaatagac gctttcacgc 1800 gcaacctatc gcttgtaata agtgtggtcc gacgctctct ctattaaata attccggaga 1860 aatcatcact caagatgcac agacttgcat tgatgaatgc atcacgcagc taaaacaggg 1920 aaagattgtc gcgataaaag gcgtcggtgg atttcatctt gccgtggacg ccacaaacac 1980 tgaagcagtg aatcgattgc gattaaaaaa acatcgacca cacaagcctt tcgcgattat 2040 ggcgcgtgac ataaacgcaa tacaacacat cgcgtttgct gatgaaacag agcgagcaca 2100 actgcaaagc cccgcggctc ccattgttct attgcgccaa aacaaaaatc aatccaacgc 2160 cgaacttgct cataacattg cccataacat tgcccataac ataacctttg acatgccatt 2220 tatcggcgta atgttggcca atacgcccat gcagcacttg attgccagtc gtatccactc 2280 gcctttagtg atgacatcgg ccaatttaag tggctgttcg cctgttattg acaacgtgca 2340 ggctattgaa caattgggtt ctatttgtga ttgtatctta actcaccacc gctcaatttt 2400 acgtcgttta gatgacagct tagtgcaggt cattgacggt aaacccagca ttatgcgccg 2460 cgcccgaggc tatgtgccac atatgcttga tttgcccaaa ggctttgaac tttgtgacag 2520 tgtcactgca ataggcgcag atattaaaaa tgcgccgtgt tttatcaagc aaggtaaagc 2580 tttgcttctg cctcaccttg gcgatgtatc taaccctgct attttggatg aactgtcacg 2640 tagccttact gacaatcaac agctgtttgc acacaagaat cagctaattg ttgccgataa 2700 acatgcaggc tatgtgactc atcaacaagc taagatactg gcaaatagcg aaaacttgca 2760 gttagtgaat catcatcatg cgcatatcgc agcttgttta gccgacaatg gcattgctag 2820 agaacacccg ccagtactgg ctttagtgct tgatggttta ggttggcacg atggtagtca 2880 ctctcaggcg acgcattcat tgtttggcgg tgagctattt attgccgatt atcagcactg 2940 ccaacacctt cgcggtttgc ctgctacccc gctaattggt ggtgataaag cggccactga 3000 gccgtggcga aatctatttg cacagttaga gtgtcatgtg gctgattggc aggctcaggc 3060 tcaagaaagt cgccaaggca aaccatgtgc gctatcgcag ttgcaacagc agcctatagc 3120 gttattaaaa agcgcaatac aaaacaacct caacactccc cttgccagta gcgcaggccg 3180 cctttttgat gcggtttcag cgctcgttag cgactgtttt gccgaacaaa gttttgaggg 3240 gcaagcggca atgaaactgg aagccttagc atggcacgac ttcaccgaaa atagccttga 3300 tctgagtgat tttaaacgga gttacccgag ccccaatatt aagcagatgt ggcaacaaat 3360 catgtcactt tatgcgcacc agacgccctc tgcaattgca ttgtttttcc atcgctattt 3420 agcgcaccta ctgttgtttt ggctgcgtca agctcaggtt caagtgcgca gtaacaaagt 3480 ggtattaagt ggcggcgtga tgcaaaacgc attattaacg cgcttgcttg tacatgggtt 3540 acagcaacaa cagcaagtgc cattgtgcca tcacaaagtt ccatgtaacg atggcggtat 3600 tagcttaggt caagccttag tggcatggac gcagcttcag gagcgcacat aatgccaagc 3660 gcaacactaa aattacaaca cgtaaatggc atcattaccc actgcgagcc taagataaac 3720 taccccaata ttgacgctat tttgcgccaa caaagcgcaa acatggtgca aaagcggcta 3780 aaccttatct atcagcaatg tggacaagca cacaatagcg ctttctttca tgcgcaaggc 3840 taccgtgcat ccatcatgca gcagcaggcg caaaatcaag ctttggcgtt ggaaaaaatc 3900 caagagttaa cttggcaatt gtggcgtatc catcaacatt tttttggcct acaacttgtt 3960 gaccacaagt ctgtgatgat aagcgcaacg caagcgcaag ataatgcact tttaagccaa 4020 tgccgcacac aactcatgag ctttaaatct caaatggcaa ccgattgttg gcacgataac 4080 agccaagcca agcctatcgc gattgattgg cctattgagc aattaacgtt atgggacaaa 4140 cacatttatc agccattacg caatgcgtta acgacgcaag catgggcaag tttgttatgt 4200 acaccacagt tgattctaac gcagtttgaa agtggcccta gcgtgcgcca agggcagcaa 4260 aaccccaacc ttattgaccg aattgatgcg ttatggcttg aattacaaca ggcccgctta 4320 gagctcaata cacccaccgc atatgccccc actcaacgca aagacggcac ggtagatgca 4380 gtgcgcggcg ccttacgtca tcacataact tggcaacaag ataaagtgga acaataccaa 4440 atcgaagtgc ctacagttgg cattatccat agttttagtc gcagtgttat tgggcttaat 4500 ttatcggcta tcggtaagga tttatccatt ttgtccttgt gggtcctgag ccatttccct 4560 tgcatggagg tgagtttgga attgtcagag gcccagtcta actgcactac gaaggcgcac 4620 acacccttaa acaaagtggc tcattcatct agccactgta acaagaaatt tagcggtaaa 4680 taattccatg catgaaatgt cgattgcgca aaatattgtc cagatagcac ttgagcacac 4740 ccaagcgggt gagcgtatag aacaagtggt gatcagcgtc ggtgagttat ctggcatcga 4800 aattgacgca ttacaatttg gctttagcgc agtgaccaac aacaccgccg ctcaaggcgc 4860 aatattaact atagaaaccg tgcccggcag catcttttgc atgaactgtc tggcgcaatt 4920 tgattaccat tcacgcacag atccctgtcc aaaatgccga cagcatcaat acgtggtcat 4980 tgaaggtaat gaaatgaaag taaaacatat cgaggtaaac taacatgtgt acagtctgcg 5040 gttgcggtac agaacaaatt gagggtcatc aacatgacca ccattcacat tcgcacgatc 5100 ataaccatga tcatgcgcat ccacacgatc ataatcatgg ccacagccac aataacgaca 5160 gtggcgcaca acctgtctct gtagttcacc accactacca caatagtggc gatgtgcact 5220 tacatattta tcatgcgcca aatactgtgg cttcagagca aataacgcca aactcaaatg 5280 cgagtcaacc tactcacagt catgcacatg agcaccacag tgctcctgtt catcattcac 5340 acgagcatca ttcacaccaa aacgccgaac atcaccatga acatgagcat gaacatcacc 5400 atgagcacac cccacatcat acaggtcata ccttagatta tggtgttggc gatgcatatg 5460 tgcatgtacc cggcttgtct cagcaacgaa tcatcgagat cgagcaagat atcttaggca 5520 aaaacaacca cattgcggcg cataaccgca gccactttac tgctgacaac ctaacggtat 5580 taaacctagt gtctagccca ggttcaggta aaacaacgtt actcactgaa accattaaac 5640 agatgcaaca cgcacacaat attgcggtga ttgaaggtga ccaacaaacg gccaatgatg 5700 ctgagcgtat ccgcgcgaca ggtgtgcaag ccattcaagt gaatacgggc aaaggctgcc 5760 atcttgatgc cagcatggtc gataaagctt atcaccaact acaactggcg aacgactcta 5820 tcttgtttat tgaaaatgtt ggcaatttgg tttgccctgc cgattttgat ttgggtgaac 5880 aagccaaagt tgccatttta tcggtgacag agggtgaaga aaaaccgctc aaatacccaa 5940 atatgtttgc ggcggccaaa gttatgatca tcaataaaac cgatttatta ccttatgtga 6000 attttgactt agctcagtgc atcgaaaacg cacgtaaagt aaatccagag attgaagtga 6060 tctgcctgtc agccacaact ggcgagggaa tggaacgctg gatcaactgg atcctagggg 6120 ataaataaca tgtgtttagg cgtacctgca caaatcattg ccattgctga tgcagagcaa 6180 caactggtga cagtttcctt aaacggcctt aaacgtgacg tcaatgcggc ctgtgtgtgg 6240 actgaaactc ctgaaacctt actcgagcaa tgggtgctag tgcatgtcgg ttttgctcta 6300 gcaattgtca gtgagcaaga agctaatgaa acccttgaac tgatacaaca aatgggatgc 6360 aacgatgaaa ttcattagtg aatatcgcca acctgaaaaa gtcatggcgc tagtgagcac 6420 tatcaatgac aaactcgata agctcgctcc tactcgacca ctgcaaatca tggaagtgtg 6480 tggcggtcat acgcatacta ttttccgttt tggtttagat aagttaatca ataaaggcat 6540 tgagtttgtg catggacccg gctgccctgt atgtgtactg cctcgtcaac gcatcaatga 6600 agcgattgaa attgccgagc aagaagatgt gattttttgt agctatggcg atgctattcg 6660 cgtacccgga gaaaacggta acttattggc ggctaaagcg cgcggcgccg atattcgcat 6720 tgtgtactca ccgctcgatg cacttgagtt ggccaaacag aacccacaaa aaaacatagt 6780 gttttttgcg atcggctttg aaacgaccac tcccccaact gcactgactt tacaacaagc 6840 caaacgccaa aacatcagca atttttcggt gctttgccat catattttgc ttgagccagc 6900 actgcgttct gtattagatg acgaaaatgt tcgtctagat ggtcttgttg cgccgggaca 6960 cgtttctatt gtgactgggc aacagcatta tcagttttgt aatgatgagt atcagttgcc 7020 agtggcaacg gccggatttg aaccgctgga ctttttaatg gcgttggaat ctatcgttga 7080 ccaagtgagt aatcatcaag ttaaggtggt taattgctac caacgcgccg tttccgaaac 7140 gggcaataaa atcgctcagc aagcgattaa tgatgtgttc actctatcag aatcaagcga 7200 ttggcgcgga ttgggtaata tagaacactc agggctgagt ctaaaaccgg cttatcagag 7260 ctttgatgca tcactgcgat accaattaaa tccaaaccgc attgcagagc ctgaaggatg 7320 tcgctgtgcc aacgtcatta aaggcttagc caaaccgacc gattgcccgc tatttgccaa 7380 aggttgtacg ccggaaaatc ctgtgggtgc gctgatggtg tctagcgaag gtgcttgctc 7440 agcttactat caatacaacg ttgaagctaa accaatcaca ctaagtaatt gatcagaact 7500 agcgcaggaa aaatactcga gaacaaggca gaatttttcg ataagcagtt attctataat 7560 caaaaattct aacgccgtta tctagcattt aacaagctag aatggctagt tatttagtac 7620 gattgctatt accgcataac cacaggcaca cgctacgatt aaaccgtatt gataacccta 7680 gagcggggtt atcaatactt aacatcatta tcaatgctta aaatcactac agcaaaaaca 7740 caccatagac cgctaccgca ataaaggctg attttgccgc atgttgcgcc cacacttttg 7800 tggttggaat acagccaatc cctaccccaa cgatatgaat aatgattgaa gcgagtaaaa 7860 aaccttgctg aaataaccaa ggattattca gaccgccaat ctcactgccg tgagcaaaac 7920 catgcgccaa cgcaaaaagc gtaataggta aggcaatgta ccccagacgc aaaggtttgt 7980 ttggtaccaa aagtaaacca aagaacacca aagataagcc aatcaaagat tcaaaataag 8040 gaaacggcag ttgcgctctg ccaagcataa aaccgaccat catcaataca ataaaataca 8100 gaggtactcg ccaaagagcc ttacccccta accgagaact ggtaataccg acacataaca 8160 tggcaaataa atgatcaagc ccaaacagcg gatgcaaaaa gcctgcctta aagccactac 8220 tggcgaccaa atgcccagta tgagcgtatg caggtacact caaacacaat agaaacaaaa 8280 cgaatgcgcg catatatcgc taacctatac acaaatgaaa aatgagggtc attgaagaag 8340 caattatctt tatcaatgaa acgcagacat gttatagcag attttatcgc cctagcacga 8400 cagggataag tcgttgtatc tgttatgccg tggcgactgc tcaataacaa ggcaattcca 8460 caccactgat tgcacaaact ctcgccagat acagtgaaaa ggcttagccc catatcgcat 8520 ttgccaccac ttagtgagcc ttatcacaca ttcagcaaat gaatagaaac tcactagtaa 8580 tacaaaatac aatttgttac cgtttttgca ttaaacctta catatattaa caaatccaca 8640 aatttaaccc tttaattatt aagggtttgc agtattttgt cccaacatgg aagtttggta 8700 cattttgaaa ttatcaacag tttcaccctg cataataaag atacgccata agcaacgtgg 8760 gatctccctt gtcgaatttt ctatcgtctt tagcttattt ttcctactgc tatttactat 8820 tgtcgatttt gctctgtttg gttacgtcaa actcaccatg caacatgcgg ttcgtgaagg 8880 agctcgttat gccattacgg gtcgaataga ttccgatcct gaccaagaaa atcctttgcg 8940 agaaaacgca gtccttactg tcatagaaca ctcatccatt ggcatattag aaaaagtgat 9000 ggatatcaaa gatattcgcg ttgaagacgt taatgggcaa agtgtttctg gttttggctc 9060 tccgggacag ttggtgtcta tccatctcga ctgtgagtgg cacagcctta gccctctcat 9120 tatccctatt ctgactgacg gtaaatacaa gtttacggtc agtgccacaa tgaaaaacga 9180 agaatttaac gagcaagtat aaaacatgca gatacgattc tttaaaaagc agtccggttt 9240 agccgccgtt gaaatgacct tagtcactcc tgtgttgttg ttactattga tggcaatttt 9300 tgagttcaca ctcattttgc aagctaagaa tattatcctt aacttgagtc gagagggggc 9360 aagccttatc tcacgcgcca gcagttacac cgaagaaacc gtcatggaca ttattgccaa 9420 cagcgccacc ccactgaata tgccccaaga tggcaccatt attatcaatc aagtatctag 9480 acgtgataac aatgacggcg aatttatcta tatatcgaga caagtaaaat ggatggattc 9540 ggccatttcg gccaccagcg cagtctgggg caattgtccc tcgtggagtg acgtcaatat 9600 tgatgacgat aacgaaaatg atgtgcaaga gtgcaatctt cccgatagtg acgctcagat 9660 caaaattgat gactttccta ccgtcaccat tgataacgca caaacctcag tcatccaaga 9720 tggcgagtcc acctatattg tcgaggtgta ttatcgctat acccctattt ctagtttttt 9780 actgagtaac gatttcatca tcagtgagcg tacatactta taatgcaagg aagcaaaatg 9840 cataagcgtt ggataaagac cctaactcat caaaaaggcg ccatctcatt atttatggtt 9900 atctcgcttc cttttatgat aattttggcc gctttcacca tagacactgg tcgtgcttat 9960 attgcgaaaa cgaaactgtt tgctgcagtt gacgcggctg gaattgccgc cgccagagta 10020 tcctcgttag gtgaagccac cgcaaaaaac gccgctcagc gctattttca ggtcaatttc 10080 cccagtgatt atctagatac ttccgctttt attaaaacat ggagttttag tgttgatgaa 10140 tggggaaaag tcaccgttga ccttaccggt caagccaata tgcccactat attagccaaa 10200 cttgtgggca ttgatacttg gacgctcacc gctgaatcac aaaccactcg tcgcactgtt 10260 gatatctcct tggttgtgga taattccagt tcattaagtg gcgtatttga tacagtgaaa 10320 acccgctcta aagagttcct gtcgcacttt aacccaacct atgacagagt tgcagtcacc 10380 aaatatggtt atggtgcaca aaccgttgtc gcatttgatc ctgacgtgcg ccaatacgat 10440 gctgacgcgg tagaaaaagc catcgatgac atgagctata acaacaacta caccaataca 10500 gccgaaggtt tttatcgagg ttatgagcag tttagcgtca ctggcacggc gttacccgcc 10560 aatttaaaag tgattgtgct gttcaccgat ggtgccccca atacctttag cgcgaatttt 10620 gatatagatg gcgtagataa ttttgcttct atttccacaa caggctcagc ggcgaaaggc 10680 ttatggagtg ccagcgaaat ggccagtcgt attagctata acatttggaa cgattaccaa 10740 aataaatggg taacaaagag cgcaagtaac aactctgaag atatttatcg ctatgtgaac 10800 atttcatctg atgacagtta tgcctctttt cctctattag gtggccctag agcaggcaac 10860 cagccttacg atgaaaatac acaaacaagc aactctcaca aaagcaagtt tcagcaatta 10920 attcgcggta tctctcggga tttacccgaa aaaatggcgt atgccgcgcg agctgatgac 10980 atctatattt ttacattggg actgggcagc tcactcacca gttcaatgga catcaatggc 11040 agcaacgttg gcacaggtga agatatgctc tacagaatgg caaatgatgc ccgaatggga 11100 ggcgctaatg gctatgatgc acaactttat ccgcaactgc attttcaagc caatcaaacc 11160 caagggctgt attgttatgc agaagatgaa cgcgatcttg ggccatgttt tgacaagata 11220 ttggacttta ttactcgcct gactttgtaa tctgagagca cagcaacaat taggataaat 11280 tagacaagtt acacgttata aacttattga ttctattgct tttttgcctt tgtttttttg 11340 ctattcttaa tcctaaaagg aattgatggc gacaggatga gaattaccga tctaaattca 11400 ctgaatgtgt ttttaacttt gatgcaaaca cactcaacgc aacgcgcagc caagaaattg 11460 gggcgttctc aatcgtatat ttcaaaggtg ctggcacaac ttcgtgacga actcgatgat 11520 cctttgttta cgcgcagtgc cgaagggctt tctcccacct cttacgcatt aagtattcaa 11580 cccaagttgc atcgcgccat tgaacaattg tcgcaatcac tagagccaga aaccttttgc 11640 cccacccatg ttgatcgcat tgtaattcac ggcctagagc caatgttaat tcaatttggc 11700 aaatctatca ttgaagccat tcgtcaagaa actgacgcaa tcattgaaat ccgtaattgg 11760 acaaagttat ctgaagggat gatcttagat gaagaagttg atttaggatt gcacgtttta 11820 accgataagc ctcaaagttt atatcagaaa aagctctaca ccagttcagg catagtcact 11880 ggcaatgaag acggcgaata catcaaatat attgctccgg gtgtcaacga atatgaaaac 11940 cgttttcaga gagtcttgcc tgacgtggaa gccaccattt ttgttgacaa ccatatgtta 12000 atgacaagtt taatggagat tgcctacacc atgcgtcttg acagttacag cgaagattct 12060 gaccttcctg aacttggcat cgacattgcc ctcatccaaa aaacctctaa aagaaaatca 12120 cctaaaaatc tatggctgga aaaactcatc acacgctgtg ttcgagaaca gaataagcat 12180 agtaactaat aataaagcta aataattcag cttaactttt cgattggcct gaaaaaatga 12240 aagcctgatg agtctcctca tcaggctttt tcgttctact cgctaaaaat tataaagcgg 12300 caggcttatg gatagtgaag gtaaagaact ggcttgtacc attgtcatta tcgctcacaa 12360 cataacccga tgcttgctcg ctagtttgct cagcaaccgc aatagattcc gctttcgtga 12420 tcaccggttt gccagcattt tgtatcaact ggctataagg cgtaagatca agactctctt 12 480 tattgttaag tgcatcgata ggtagcaccc caacaaaact gccttggatc tcaccatcat 12540 tcacgctact tgaattatta ccttcaaccg aagctgtata aatcaagctg ttactacttt 12600 gccaaaaatc agcaccagat aagcacgctt gtacaccgtt gatgctaggc aatgtaattt 12660 ctacaacatc caacgaagaa atatgatcga tcttaccttg cagataattc aacatatcgg 12720 cagttttcat tgtgaagatt gcattcttac cttcattacc acggttgaaa atgtaagttt 12780 catctgcaga attcgcaatg ccttcaatgt ttattttttc ttcgtctgtg aaacccgcag 12840 cgctgtgtaa atcagcatag aattgagaca acgataactt gatgttatcc ccgccatcag 12900 ctgggattag atacgcattt tcacgcaact cttttttgct gcccgaacca accacaaagt 12960 aataaggttg attctggtat ttcagctgcg tcatggcctc aaaatcaggt tttgttgcat 13020 gggctacgcg attgtcttta ttaactggga agttatcgat aagcgttttg ttatcaatgg 13080 aaaagtcatt agccacatca aataaccatg gtgagtcatc acctgaaaca ataattttac 13140 tattgtttac caccattgca gaaccagaag agagatttgg gtttatggtg gaattgccga 13200 tcacaaagtc aaatgactcc gtttgcgctg gagtttgaga gcagccacta acggcaatag 13260 ctgcagcagt tagaacactt gctacaacat agtttcttgt tttcatttta atctttacat 13320 ccttgatatt taaggatttt cactatatac cccgcaagca gttcttatag gaatggaaac 13380 aaagattgtg tagataacgg catttcaaca ttggggttta agatctttcc cttgctcagc 13440 cacgctgcat atcgatggaa tcagttgatg gggtcaggtc ttgaattttg caaagaaagc 13500 acagtttcga taaaaaccat gctttctttc ggtgcatcta cataatctta gatactaacc 13560 attgcaatga gtgttacacc tttagaagtt caacatgtca tgacttgcta ttacatggct 13620 gacaagtagg ctttatacgc ctttaaccgc actgtcgcag cgccgatgat atccataaag 13680 ccaagcattg ggtgcggttt ttccgcagac acccagccag agcctgtaga accaattgga 13740 atgttatacc cctgcggctc ttcaaattcg atgacaatgg tgcgcccgtg ggaattcata 13800 ttgttaccta cgtgctgtct aacatgttgg ctaccctgac gcacttgcac ttgcgcttca 13860 ccagtaccgg tagtcacact atgcactcgt ccgcggaaaa tctccccagg ataagcgtcc 13920 acatagaact cagcaaactg acctttttta atacctcgat aggtttgatc tgggattctc 13980 atctcaacaa actttttctc tgtggaatac aaatgcatgt taccggtgcg agtaccgctg 14040 gtaatgttag tgatcgacag ctgtccatca aacggcgcat gtacttcacg gcgctcatgt 14100 tcccatttgg cggtcgccat ttgtgcttca atattcatga tctgcgcatg tgcggcggcg 14160 atgtttgctt ctgcggtttc gatgcgcgtg cgagtatcgt ctctctccga ctcttgggca 14220 aaatctttta gcttttctaa acgctgtaga gtttttctat cgttttgttg ctggcgtctc 14280 aaagacaaaa tttcagcttc actggccttt ttctgtgcct gcaagctatc cattttggat 14340 tcggtatctt ctgagcgcag ggtgtaaata agatcgcctt tttttaccat ctggttatga 14400 gtgacataca cctcatccac ttcttggcca aataacgggc ttaataccgc atgtggcgct 14460 ttcaccgtgg tactatcggt taaatctgct ggtgaccaaa aacggtgcgc aacaaataac 14520 gcggtggcaa tcaacatacc tgtgcccacc attaaagtgg cattgcgtaa cgtccattta 14580 ataaagccta tcttgactaa aatccaacat ataagaatgt aaggcaacat aatctctttc 14640 attgcttact ctcctgactt ttcattttgc attgacgttt gtgcttgtgc ttgtgtttgc 14700 attgaagttt gcgcaggcat gttctttgag ctagccgtgc cgttacgaat aattttggaa 14760 atggattcac caatcacatc ccaacgggta aaggcgatga tgatagctaa tacccaccac 14820 gctttattga tgaacaaacc acaaagagaa agagcaaata cgatttgagt atgcgcgctc 14880 gacatattcg aggctttatg cactgcaatt tcatgcaatt gccacaataa atacagcacg 14940 taaatgatca cagagacagt gacgaaaaag acaaatccca taaacacttc tgagtctaat 15000 aatttgacta tgtttggaag cccattacta aaatattctg ctggtaagtc cgtaccatga 15060 atggcaccta gatgagacaa agcaaccgac gctccgccga taacggcaaa cacaataatg 15120 gctttaacaa tataagtaac aacagacaga gcgctccttg cgaattttct cccaagaccg 15180 tcactggtag gaggtaagga tgcatcagac attgattttc gcttcttagt tgtgagttct 15240 cagtcatctt aatagaagga ttcaatgatt gtggaatatg ccatattcct aaaaagtgct 15300 aggcaaaaaa aatcccttac ataagtaagg gatttgaatc tctaaaacga tattgaaaca 15360 tagttaaaac atcgttaaat cataaacata atcgctagtt gcgagttacg tttcgattag 15420 tgagttacgt taagcttgta gaaagttgct tgtgcgtagt cgtctgcatc gcctgttttg 15480 ttttggttgt aaacgcccgc tttaaagtac atgtattgac cgccttcacc gaagccactc 15540 tcagacatgt cgactgtttt aacaaggtct tttttaccat cacgcatgat agttacagaa 15600 agctcgttac ctttaacttg gatacggtag ctgaatttct cgttaagttt gataccgtct 15660 gtaggctctg ttggtgttgc atcttggtgc cagtaatcag gtagtgagtt accgatgatg 15720 ttgtgccaag tttctttacc aaacgcttta cgtggttcgt gcgcaaagta gatagaacct 15780 gtttcgtttt gtggcaattt acggtagtac aaacgaattg gttcgttgtt gtttgagtgg 15840 atttgaccga tgatcacacg accaacctgc cagtttttac ctgtagttgt tacgtggtta 15900 acggctagag ttgcttctag agtaccgtca acaccgccag ctactttctg gtctgcttga 15960 ggagccgttg agaataccca gttgttaccg ccaacacctt tcgttttgta ttgagtgtta 16020 ccacggcgaa tcatttcacg taattctgaa cgagtgtaag tggtgttttt agatgtttta 16080 gcacctttca ctggcgcttt aaataccatg ccgccatctt ctgccaagta gaagtagtta 16140 gcgtcagagt aaccagctgc taattctttt tctttaattt ggtcagattt gccatcgcca 16200 tcagaatcga taggcacact tactgtccaa cctagtaggt caaagttgtc tgaaggcgct 16260 ttagtttttg gcgcattgtt aggcgctaca tttgaacatg cagtcattgt cagcgcagcg 16320 gctaaggcaa cagcaccgaa aaccgctttc ttgcctttag aagtacccaa tgtttttaaa 16380 ctcatcattg catttacact ctatgtaata ttgtattacc aagaatgtta ccactgatgt 16440 caggctatga cctcttgtaa atctgtgacg cggatcatat tttgaagagt aaagctagct 16500 atcaaaccca tacacattca agttttaaat atttacattt caataaatca gtaatttgta 16560 tgattaatat gcacatgcgt tatttcatcg atttactcaa tattcgacat atttaacgtt 16620 aaatgtgttc caagggctat aaattagcca atatattgtc ggctttgatt atactattta 16680 gtttcttagg atcgggactg gatataaaaa tacaacagca ataaaaatcg ctataatacc 16740 agtcatcaac atcatgctta tcaagggctt ttatgacaca gtcaaacaca cctaatgttc 16800 caaatgatcc atcagaacgt tttaatcttt tttttaaagc ggctgcaacc gataaaaaaa 16860 tctggttgct aattgatgag cacggtagcg tgatgctcaa taccgaagat gaggaatgtg 16920 ttcctgtctg gccatccgaa gagcatgcat tgatgtgggc taccggtgaa tgggaaggtt 16980 tcaccgccga gcctatctct attgccaaat ggaaaagccg ttggacacat ggtttagaag 17040 atgacgagct ttcgttggtc gtttttcctg atcaagaagg tgaaggtatt gtgctctacc 17100 cagatgaatt tgaaatggaa ttgactaaac gcgaagctaa acttaaaaaa taacctcgcc 17160 tttcctctta tactaataca acatcattct ataaatgcgc ttcattatta ataatgaagc 17220 gcatttttat ttgtttcttt cttgcatcat gatctcattt ttaataacaa ctgcatgcat 17280 gagctcacag tttctatgac accaatttga cataaaatat gacaaatccg atgctttgct 17340 tttaatattt taattaattc aaagtaataa tgaataaaac actcttagtt ttagccttag 17400 ccgcaatcat tagcggatgt ggcagcagtg acgattcttc tcaaccaccc gcagtggacc 17460 caaaccctcc cggtgacgtt gatccgaccc caccagattc agatgatggt gatgataacg 17520 acgacggcaa cggcggtgat aacgacaacg gaagtggcga cgagggcgat gatgacagca 17580 gtgaagatca tatcgcgccc tatgacctca caaaattcca accaatcctg tctaactcag 17640 atctgcaaat gtccgatcct gaaggcgccg aatctaataa agagagcatt ataaaagacg 17700 gtaacttcca cggttacttt gacgactatt tttacgcaga gcaaagctcg caaaatttag 17760 tattcaaaat gtctaattac ctcatgcgca gtgaagttcg cgagctagaa aattttgacg 17820 ttgatgctga gggcattaaa cgcactttat atgcagaagt cagccttcct aatatagagc 17880 aagccatgtc taactcttct tcaagcagag accaaatgac attcttgcaa atccataaca 17940 aaggcaccgc aagtgatggc acaggctaca tccctcaccc attgctaaga gtggtgtacg 18000 aaaccgaacg tgatggtaac tatggccatt attgggcggt catcaaaaat aacgctgttg 18060 attgtaaagg cgacgctgcg ataccagatg acgctcgctg tgatgatgct tatgatcata 18120 tcgatctcgg tgaagccaat accgaccaat ttaccgaatt ccaattggcg atacaaagca 18180 gtcgtctttc tattcgagta aatggtattg aaaaagcgga tctggatatc gactattggg 18240 cgcacttact aagctacttt aaagccggcg tttataacca gtttgaaaac ggtgaatctg 18300 aagctcactt taaatctcta aaagtcattg aacagcaagt ggataccgac accgagacca 18360 cttaccaatg gaacatcgac gaatggaaaa tcaccattcc tgccagtaaa gacgattggt 18420 actgcgaagg tggcagtagc gcagcagagc tgttccctgc ccgttgtggt tattctgacc 18480 gggatgaact ggctaacgat gaagacattt atcacgcaga tcaggacatg acctacttct 18540 ccgtggaaaa cggtcgcatg cactttcgag cagatatggg ctacggttca acaacagaaa 18600 actcaagcta cattcgctcc gaattacgtg aactttatat gagtgatgaa gacaccactt 18660 gtagcagcag taatgaagat tctagctggt atcttgacga tgacagaact ggtgcatcta 18720 ctcacacact caaagcacta ttaagagttg aagagcatcc cgatttaagc caaccaaaag 18780 tcattttggg gcaaatacat ggctggaaaa tcagtcaggc tttagtcaag ttattgtggg 18840 aaggtgataa tcgaccagtg cgagtcattt taaatgatga ctatgaaaca aataactcaa 18900 gctgcggtca ttgtgaaccg tttagtgttg atttgggcac atataaaaca ggagaagaat 18960 ggggttacac cattcgcgcc aatcaagaag gtatctatct tgcaacctat gatgcagatg 19020 gcggtaattt gaagtcgcat accatccctt ggggcgaaga gtatgtcgat aatgacggtg 19080 ataaagtggt tttgtctgcc gattgggcat ctccagatat cgctttttac ttcaaagctg 19140 gcatataccc gcaattttca cctaattatt ctggtgaaat attcgacgtt agcttcagtt 19200 acttagacat agatcactat tagaaacact caagtcgatc ttgatcgcac ttattaatac 19260 atctttacat ttgtattatt aaattatgta ttatttacaa cacagaacga atgacgaaca 19320 tcgttctatc tagatttaaa agatatagaa atcatcaagt cgtattttct atatcaaaaa 19380 caaatgcagt tagtaactgt tttatattac aaataatatc gttggtttat acaacattag 19440 gtgaatggta aatgaaacat atatatctaa aaagcttgtt agcaacttct gtcttattag 19500 cagtaggctg tacttcagca ccttcagctc caaaatttga caacaacaaa gagacaggcg 19560 tagcacttct tactccagtt gctatcacag caagtagcca cgatggtaac ggtcctgacc 19620 gtttattcga ccaagactta actactcgtt ggtcttcatc tggtgacggc gagtcagcga 19680 cattggacta cggttcagtt caagaatttg acgcagttca agcagcattc agtaaaggta 19740 acgaacgtca aactcgcttt gatatccaag tatctgttga cggtgaaaac tggactactg 19800 tactaaaaga ccaaatcagt tcaggtgaag ccattggttt agagcgcttc cagttcgcac 19860 cagctgtcaa agcacgttac gtaaaatacg ttggccacgg taacactaaa aacaattgga 19920 acagtgtcac tgaactggct gctgttaact gtaacatcaa cgcatgtcct gcgagccaca 19980 tcattactcc agcagttgtt gctgcagaga agtcaatgat tgctgaaatg aaagcagcag 20040 agaaagcacg taaagaagca cgtaaagacc ttcgtaaagg taactttggc gtagaagcgg 20100 tttatccttg taaaactaca gtaacatgtc tacgtactga gcttccttca acaccagctc 20160 taccagcaac accacttgca ggtaatgcac cgggtgaaaa ctttgacatg tcgtactggt 20220 acctatctca accattcgac catgacaaag acggcaaacc tgacaacgtt tatgaatggg 20280 acctagcaaa cggttaccaa cacccagaag tattctacac agctgacgac ggcggcatgg 20340 tattccgcgc ttacgttaaa ggtgttcgta cttcaaaaaa cactaaatac gctcgtaccg 20400 agctacgtga aatgttgcgt cgtggtgact attctatccg tactcaaggc gtgaacaaaa 20460 acaactgggt attctcaagt gctccagaag ctgacctaaa agcagctggc ggcatcgatg 20520 gtgttctaaa cgcaacactg aaaatagatc acgctaccac aactggtaat gctaacgaag 20580 taggtcgctt tatcattggt caaatccacg accaaaacga tgagccaatt cgtctttact 20640 accgcaaact accaaaccaa gcaactggtg cggtgtactt tgcacacgaa agtcaaggcg 20700 caactaaaga aaaattctac tcgctagtag gtgatatgac cgctgaagtg ggtgatactg 20760 gtatcgctct aggcgaagtg ttcagctacc aaatcaaagt tgtgggtaac actatgactg 20820 taaccctatc tcgtgaaggt catgacgacg ttgttcaagt tgttgatatg agcgaaagtg 20880 gctacgatga aggcggccgt tacatgtact tcaaagccgg tgtttacaac caaaacatct 20940 caggtgacct agacgactat tctgaggcga cgttctacaa acttgacgca tctcacgacc 21000 aataccaagc taaataatat ttatcttcgt ataatctaaa atattaatgc cgctcacaat 21060 gtgagcggca tttttttatt ctatccacac taaataatat acaagtcttt ttacggccca 21120 aggtttaata ataaaacctc aaatcacaga taactattat gtggttatat acaggaatcg 21180 atattcatat ttcattggct atttacgtat tatatttctt acaattaacc gtgataacag 21240 ccatattata tctcaatccc aatatggcgg atttccataa aactgagcaa tataatcaat 21300 aaacacgcgc actttgggag ctaattgttt tgaactgggg taaacaaccc aaatagcaga 21360 gtcatccact aatggataat ccgctaacac ttgcactaga cgcccatcat ttaaatgttc 21420 atacgcaatc caagtggcgc acatggcaat ccccatgcca tttaccgcag catcacgcac 21480 cgcttcccca tgatccacct tgatttttcc ttttactttg acgctaacag gaccatcgct 21540 ttgttgaaaa acccaagatt ctagccctgc atgattgatg cacatatgtt ggtatagatc 21600 ggatggcgtt tgtggcgtgc catattcagc aatatagtca ggagatgcgc acataatgcg 21660 tttgtcggtg gcgatttttt tcgctatcaa tgttgagtct ttcaattcgg cattgcgaat 21720 ggctaaatca aaccctccat caatgagatc aatcatggaa tccgttagac gaatatcaac 21780 cgataagtca ggatactgtg ctaaaaaacc ttttaacggc gaaattaagt gcattcggcc 21840 aaaagacgct ggagctgcaa tcctcaatgt gccttttggg cacaccttgc ctgagccaac 21900 catcgcttgc gccgcatcga cattgtcgat aatttcttga gcataaggca gtaaggtttg 21960 cccttcttcg gttaaagaaa ctttacgagt ggtacgatga attagacgag cgcccatgct 22020 ttgttcaagt ttactgatgt gcatactagc aacaggtgcg gataaaccca actctttacc 22080 cgcaagactg atgttatggg tgaaagcaat ccgtacgaac aatctcagat gatcgatatt 22140 catttaaact aatctcttaa tttcgattta ttattagcag tatagtgacg aatactagtg 22200 ataagcacgc ttaggcgcaa atcagtttta agtttttatt aataatcaaa gtcatctatt 22260 gtttagtatg atagaacttc acacaaccta tattgcggag agtcgctcat gaaaaaaaac 22320 attctaataa caggttcaac cgatggcatt ggctttgaaa ccgctaaaaa gctagtggcg 22380 gatggacata atgtgttgat tcatggtcgt aacaagcaaa aagtgactaa agtacaagcg 22440 gcattatccg cattatcagg cgctggtaat gttgaaagtt atcttggcga tctttctaat 22500 cttgaagaca caaaggcgct cgcaaacgcc attaagcaaa aacatcaaca cctggacgtg 22560 ctcatcaaca atgctggcgt ttataacgta ccaacaatta ccacttcaga aaatttagac 22620 actcgttttg tggtaaacac catagcgcca tacctattaa ctcaatcact tttgaacctg 22680 tttgaccaaa cagggcgcat cgtaaacctc tcttctgccg cgcaagcacc tgttaatcta 22740 aacgctttag tcagtgctga cgcacacgaa agcgatggtg ctgtatacgc tcaaagcaaa 22800 ttagccatta ctatgtggtc agctgatctt gcgcttaaat taaaagagcg tgggcctagc 22860 gttgtcgcag tcaatccagc ctcattcctt ggcagtaaat tggtaaaaga agcgtatggc 22920 gttgcgggca atgatctgag tattggcgca gatattttgt atcgcgccgc cctatcagag 22980 gagtttgcta acgcatcagg tttgtatttt gacaatgata ttggtcgctt cgcatcacct 23040 catccacatg ccagtgacac tcaaaaaaat gcacaattgg tagcggtaat tgaaaaactt 23100 ttaaccgata gattaaagta atctcctact cttattacaa aaacaatgag cctagctttg 23160 ccccgcacct ttgccctgta tagctaggct ttttctatcc actgcatcat taataaacct 23220 tattattaat aaattcttaa ttctgattaa ggataaatcg ccattatcaa tcccgtgcca 23280 cacagttaag cttacccacc tcaacaatca ctaaaatcta aaaaggctta acatgcatta 23340 cgaaggtaaa atctatcgcc cttggatgga agctgacagt attctcattc aaaccaccct 23400 aggttgcagc aataatcaat gcaccttttg cactatgttc gatgacaaaa gattcaaagt 23460 tcgtgaactt gacgacatat tcaaagacat tgccgaagct cgtcgcctgt accctaaagt 23520 ggaatccatc tttctaattg atggtaatgt catggcaatg cgcaccgaaa agttaatcgc 23580 gattctcgat caaataaaag tgacttttcc tgagcttaaa aacttagctc tgtattcggg 23640 attcaatgac ttccgccgca agagtatttc tgaattaaaa gagatccgct ctgccggatt 23700 aactaccgcc tactctggcc ttgagtcggg tgaccctatt gtgctggaac gcattaaaaa 23760 aggaatgacc cgtgaacatg cgattaaagg catggaaatg gcaagagaag cagatattaa 23820 agtattagcg tcattcattt ttggtctagg tggccgtgag cgttcaaaag aacatgcgat 23880 caacaccacc agcttgctga atatcatgcg ccctgatgcg attgcaccta tggcgctggc 23940 cattcaaccg ggcagtgaat tagagcaaga actgcataat gggcaattta gtctacccag 24000 cccattacaa attttggaag aagaaaaata cctattggaa aatatggatg atttcccttg 24060 tttctattgg ggcgatcacg gcaacaatat cgctagcatg cgtggtgagt ggcctgccgc 24120 ccgccaaccg tttttggagc atattaataa gcacatcagc ttaaacccaa tgaccaagaa 24180 aaacgtcatt gagacctatg cttggtaaca tgctttagta aacgaaagca ataaacaaaa 24240 accgctttaa cactacaaag cggttttgtt ttcacactca acttacgaat aatctatccg 24300 cgcgtatctg tataagtaat agcagtgcgg gtttctagca ctcgaagctc atcccctact 24360 tcaatcacac cctcattgag tgcaatcaaa ttcatgccaa agaaaatcgc ccctttttcg 24420 ttggctctaa atttagctaa ggttgcagtg ggctcttggg cagccatttt ttcgcctgtc 24480 tttggatcaa ctgaggttaa tacgcagcgc tgacatgcct tgcctacttt aaattctacc 24540 tcgccaatgg cgattttttc ccagctgtct tcggcaaaag gtggtacacc ctctatcacc 24600 aagttggtgc gaaactgcga cattgattgt ggatgtgaag cactgcggtt taactcatcc 24660 agagaacctt gagagatcac caataacgga tagccgtcgg caaagctgac tttagttgcc 24720 agtttttcac gatagcgatt agattcttca ccgcaataca atagcaatgc tggcgtacct 24780 aatacgttac taaaccactg gttagcggtg tctgtggtgg tataagcgaa aaagcgatcg 24840 ctccagattt tacattcgaa ttcgttacgt tccagatctg atatagacaa atgcaggtct 24900 tgtgcattgg gagcactcag caaaatacca tctggcagta gcgtgctttt aatcgtcacc 24960 atcttagggt acttacgtgc ggtaatcatg cgacccgccg tatcagtcac cacaaaacga 25020 cggtcaaaac tgattccttc taaaccaaca taagctctgg attgtgcaat gccagatgtt 25080 gattttaccg gaaatatatt aatacccgat aattgagcta aatccttcac tttatacctc 25140 taactctctc aatgttaatg caatcttatt gcattttcgc tatcatttgc ccgcatttaa 25200 gtaatcgttt gcttggcgtg ataaccatac cacattctcg gtttcttctt ataccaatca 25260 cactaagtaa gtgatcagag ctagcgcagg aaaaatgctc gagaacaagg cagaattttc 25320 gataagtagt tattctacaa tcaaaaattc taacgccgtt atcgagcatt ttaacaagct 25380 agaatgagca gttatttagt acgattggta ttacaccttt agactttttt ggcgggagca 25440 ctactatgac caacttaacc attacacgcc cagatgattg gcatgtacac cttcgtgatg 25500 gtgatgtact aaaagataca gtaaaagaca ttagtcgcta caatggtcgc gctttagtta 25560 tgccaaatac cgttccgcca acgaccaaca ccgaacaagc tatcgcttac cttgagcgta 25620 tcaatgcaca caatcatagc gatactttta agccgttaat gtcgctttat ttgacagaca 25680 acaccagcaa agaagatatc caacaagcaa aagcatcagg tcaagttgtt gcatgtaaat 25740 tgtacccggc aggtgcaacc actaactcgg attctggcgt aactgacgct aagaaaatct 25800 accctgtgtt agaagagatg gaaaaacaag gcatcctgct gctgattcac ggtgaagtga 25860 ctacgcacga tgttgatatc tttgaccgcg aaaaacagtt cttagacaca gtattagcgc 25920 ctatcgtcaa cgatttccct aatctaaaaa ttgtcattga gcacattacg actgctgacg 25980 cggttgagtt tgtacaaaat gccaatgata acgttgccgc aaccattact gcgcaccact 26040 tgatgtataa ccgtaaccac atgttagtgg gtggcattcg tccgcacttc tactgcttgc 26100 caattctaaa gcgtaacagc catcaaaaag cattagttgc tgcagcaacc agcggtagta 26160 agaaattctt ccttggtacg gactctgcgc cacacgctaa aggtcgcaaa gaaaccgctt 26220 gtggttgtgc gggttcatac acagcgcacg catcattgga gctttacgca gaagtatttg 26280 aagccgaagg caaactagag cacctagaag cttttgctag ccataacggc ccagacttct 26340 acaatctgcc acgcaacagc gataccgtaa ctttagtgaa agaagagtgg gcagtagccg 26400 aaactatgcc atttggtagc gacatcgtcg tgccaattaa aggtggtgaa actatctctt 26460 ggaaagtaga agcttaatta actagtagct attaaattac aaagggttgc tgattaagca 26520 accctttttc gttttacact ctgcgttatt attcttcgtc ttgaagagag ctccacattt 26580 gcgcgcactt gatcgctcgt tgccagcctg cgtagcgttt ggtgcgtttt tcactgtcac 26640 aaattgggtg ataacgtgtt tctatttcag agcgagcttc cagttcggat aaatccttcc 26700 aatagccaac acttaacccc gctaagaaag cggcgcccaa tgccgtgact tcggtcaatt 26760 gcgggcgttg cacttcgcaa tttaacaagt ctgattgaaa ctgcattaag aaattatttc 26820 gcgccgctcc cccatccaca cgtaaactac ttagctctat acaagagtcg gcttgcatgg 26880 catgaatgac atcataagtt tgataagcaa tactttctaa agtagcgcgc acgatatgtt 26940 cggcactggt gccacgcgtg agccctacca tagtgccacg agcgtacggg tcccagtacg 27000 gtgcgcctag accagtgaaa gcaggcacaa cgtatacgcc atcagaggaa tccactttgt 27060 tggcgtaata ttcagaatct ttagattcat taagcagtcg cagttcgtct cttagccact 27120 gaatagaggc tcctgccata aatacggccc cttcaagggc ataacaaggc tttccttcgg 27180 cgtcgcaagc caaggtggta agcaagccgt gttgcgagtc caccttttgc gagccagtgt 27240 tcatcaatag gaaacagcca gtaccgtacg tgttttttac ctgacctgga ttgacacatc 27300 gatgcccaaa taatgaagct tgttggtcac ctgcgatgcc cgcaactgga atgcgaatct 27360 cgctattttc accaatgcag gtttctccaa acaaacttga cgagggttta acttctggaa 27420 gcatagattt gggaatatcc agcaattcga gaatttcctc atcccactgc atggtattga 27480 tattaaacat catagtgcga gaagcattgg tgtggtcggt ggcgtgcact ttaccgtgag 27540 tcagcttcca caataaccaa gtatccacag taccaaataa caacttgtcg tcgtcagcca 27600 gtgctcttgc acctggtaca ttatctaaaa tccatttgat tttactggcg gaaaaataag 27660 ggtctaatac caaacctgtt gcggttttta tgtactcttc ccagccttgt tcttttagct 27720 ctttacacaa ttgactggtg cggcgacatt gccaaacaat ggcattgtaa ataggtttgc 27780 cggtctcttt gtgccataca atggtggttt cacgttgatt ggtaatacca attcctgcga 27840 catcttctgg tgtaatagaa agcttgtcta gtacatcgat taacgttgaa cgttgcgatg 27900 aatagatttc catcggatcg tgttcaaccc aacctgcttg cggataatac tgggtaaatt 27960 ctcgttgtga ttgagcaaca atattggctt gtgagtcaaa cactaccgca cgagaactgg 28020 tagtgccttg gtctaaggcg acaatgtaag agcctgtggc catagttaat atcctactta 28080 attcgattca aagaaatcat actcaacatt atggcaatta cgtgagtgtc gagatcgagt 28140 tttgcactaa aaaaacgctt aaattgtcat ttgaggcgcg gtagctcata gtgtttattc 28200 cacttattgc actaaattag ttcaggtgca ccaaatagcg ccacatagtg gtgcaaaagc 28260 acgctacaca agagcaaaac ccctattaaa caatagattg gtttttggaa cagtttttgc 28320 ttattaaact tagtacccaa aaataacaag gaagtgtcgt catgcaagat acattaaccg 28380 tcattttagc cggaggcatg ggttctcgac ttgcgccctt aactcaagac cgtgccaagc 28440 cagcggttcc ctttggcgga aaataccgca ttatcgattt cacgctcagt aactgcctac 28500 attctgggct acgtcgctta ttggttctga ctcaatataa atctcactct ctacaaaaac 28560 atttgcgtga cggatggtcc attttcaatc cagagctggg tgagtacatc acagccgttc 28620 ccccacaaat gcgcaaaggt gacgcttggt acgaaggaac ggccgacgct atctatcaaa 28680 acctgtggct actatcgcgc agtgaagcta agcacgtggt ggtgctgtct ggtgaccata 28740 tttatcgtat ggactaccaa cctatgttaa agcaacataa gaaaaccaat gctgcgttaa 28800 cgatcgcttg catggaagtt cctgcccaag aagctagcgc ctttggtgtg gtaggtgcag 28860 aagcagacgg acgcattgta tcctttttgg aaaagcctgc cgatccgcca actcttgcgg 28920 ataacccaaa caaaagtttg gtttccatgg gcatttatat tttctcaatg gaagctttgg 28980 tagaggcgtt agaggcggac gctgatattg atacgtccag ccatgacttt ggacacgaca 29040 tcatccctaa gcttatcgat gctcaagggg tttacgccta tcgatttggc tctgaagaag 29100 gacgagtgtc gcaagacgta tattggcgtg atgtaggtac ggttgactcg ttttatcaag 29160 ccaatatgga tttacttgat ccggttccgc ctatcgatct ttatcaagat gattggggta 29220 ttcgctctta cgaacagcaa cgaccaccat caagaaccgt cccctctgtc acgggaaatc 29280 aaggtatctc tattaactcc atcgttgcca atggcgtggt gattagcggc ggctcggtac 29340 aacgttccat tttatcgtcc aacgttaaag tcagcgatgg cgccaccata atgaacagca 29400 tcttgtttga tgatgttaaa attggcgagc attgcgaact aagaaactgc attatcgata 29460 agcatgtcac cgtacctgat ggcaccaaaa ttggttatga catacaagct gacagagaac 29520 gatttactat ttccgagcaa ggtattgtcg tcgtcccgga aggctatcac ttcggataat 29580 tcggtgcatt aaacgcgcct taccaaatca aaccttgggg atcattctcc acctccgcaa 29640 tcagttggtg gagctggttt ccaagggttt cgattgctgg atgtgtatgc caaccttcaa 29700 acatgtcgtg ttgatctcta tgcgctatta ataacgtcca cgccttgtca cgaatgagaa 29760 ttttcgccac caaatagcga tgatcggctt gtttagaatc taacttaaat cgagtgtggt 29820 aaaactcaat acccgaatcg gacaattcaa attccgcctt actcaattca acacacacat 29880 tgtgattacg cgaacaacac gcatcactgg ccagtttgat tatttgccgt tgctccattt 29940 cagatagctt catggtgtac ctcataattt gacgttgcac acttgctgac aaaaacacca 30000 aacaatcgca taaaaatgtt acgcattggt catttagtgt cactaatttg taatacaatc 30060 gtcttactct gtgcaatatc tgtaattata gtttgaagaa ggatgaatat gtttagagta 30120 attatcacgg caatggtctg cctgatactg agtatagcga acgtacacgc agctcaagtg 30180 aatatcagtg gttctacctc ggtagcgcga gtgatggata ttttagccga gaattttaac 30240 aacacgcata aagatactta tatcgcagtg caaggtgttg gctctacagc agggatttct 30300 ttagttaaga ataatgtcgc tgaaattggt atgagctctc gactattaac cgaaagagag 30360 tatcaagacg atttagtggt cttgcctttt gcctacgacg gtttagccgt tgttgtgaac 30420 caacaaaaca gtgtcgataa tattacccgt gaacaactct ttaaaattta tacgggcaaa 30480 attaccaact ggaaagagat tggcggagcc gatcgtccca ttgctgtggt gactagagaa 30540 acgtcttcag gttctcgtta cgcatttgaa gaccaagttg gcctaactcg cgtgctaaac 30600 gaccgccgtg tttcaaccat tagtcaaacg agcttagtgg ttagtaccaa tagcatgatg 30660 aaaaccttga tcagctacaa cccgcaagcg atcggattta ttagcatggg gtctattgac 30720 gcttctgtta agccactcaa ctttgacggt gttgtgccaa attctgttaa cttactgaac 30780 aagagttacg aactagcgcg tccttttttg atcttccatc ataaagatct aaaagacgaa 30840 gcagcattgc agtttgccaa gtacgttacc tctaaagaag ccagcgctat tattgaaaat 30900 cagggctata ccgctcattt aaataaataa cgccaacagt attagtacat ataaaaatgg 30960 ccaaagctaa gctttggcca tttttgataa tctgttgtta cgctatcgat taaaagtata 31020 aacgaatcac cgattcagcc acacaagctg gacgacgtac gccttcaatc tccactttca 31080 gttcacgagt gatctcaaga cctttttgaa taggcgtcac tttcactaaa ctgctacgag 31140 cgcgaatgtt gctccctaca cgaacaggat aagggaagcg aacactgttc atccccatat 31200 taacggtcac tttagcggta ggaaacaagg gtttctcagg atcaacagca tctgacaaac 31260 gaggcagtag tgccaaagta agaaaaccat gcgcaatagt cgatttaaaa ggagactctt 31320 gctgtgcttt ttctggatcg gtatggatcc actgcatatc ttcagtcact cgaccaaacg 31380 catcgacacg ttcttgatca atgctaaacc agcttcccac gtgcaactct tcgccaattt 31440 tttgcgatag ctctaaatac agcgcttccg cctcaggctt catctcgatg atgtcttgag 31500 tgggattagc tacttcttca tttactgccg gcttttgaaa atcttttccc cacgcgcgaa 31560 aaatgtgctg tgcatgggca cggctcacta attcactcca atactctcta agagaagggg 31620 acatcttatc cataaactcc atatgatgtc tcgaaagaga tcctcgtttg tgtttaagaa 31680 tatcaacaac tttcatgcac gctccagtga agc 31713

Claims (3)

1280-3652th nucleotide sequence, 5025-6128th nucleotide sequence, 6365-7492th nucleotide sequence, 8687-9202th nucleotide sequence, 9206-9823th nucleotide sequence, 9823-11250th nucleotide sequence of SEQ ID NO: 1, 11377- 12189th nucleotide sequence, 12290-13306 nucleotide sequence, 13611-14642 nucleotide sequence, 14646-15212 nucleotide sequence, 15417-16388 nucleotide sequence, 17370-19223 nucleotide sequence, 19452-21017 nucleotide sequence, 21253-22143 2nd nucleotide sequence, 22309-23121 nucleotide sequence, 23333-24208 nucleotide sequence, 24294-24989 nucleotide sequence, 25446-26477 nucleotide sequence, 26549-28063 nucleotide sequence, 28362-29579 nucleotide sequence, 3010 A metagenomic library having alginate lyase activity comprising 22 open reading frames comprising 3-30930th nucleotide sequence and 31010-31696th nucleotide sequence.
[Claim 2] The metagnome library according to claim 1, wherein the metagnome library has a nucleotide sequence of SEQ ID NO: 1.
The metagenome library according to claim 1, wherein the metagenome library is constructed using genome DNA obtained from a microflora of the intestine of abalone.

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