WO2012157814A1 - Metagenome library having alginate lyase activity and novel enzyme alydw - Google Patents

Abstract

The present invention relates to a metagenome library having alginate lyase activity. The present invention provides a novel microorganism and a protein by analyzing an unknown metagenome which exist in a natural microorganism group. The present invention relates to alginate lyase and AlyDW having an amino acid sequence disclosed in the second sequence of the sequence listing. According to the present invention, provided is an enzyme for stably dissolving seaweeds by separating a microorganism having a superior ability in dissolving a polysaccharide in seaweeds. Also, the present invention provides a technique for reducing the amount of waste products derived during a seaweed processing process and enhancing resource utilization by using a technique in which a functional polysaccharide is mass-produced by means of bioengineering.

Description

 【Specification】

 [Name of invention]

 Metagenome Library and Novel Enzyme A 1 y DW with Alginate Lyase Activity

Technical Field

 The present invention relates to a metagenome library with alginate lyase activity and to the novel enzyme AlyDW. Background Art

 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 cellulose 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, abalone-derived microbial enzymes By using the technology of biotechnological mass production of functional polysaccharides by establishing reaction conditions that can produce low molecular weight alginic acid under mild reaction conditions without strong acid and strong alkali, There is a need to establish technologies that 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 determining the diversity, structure, and function of bacterial communities without microbial isolation and culture. Among them, the most frequently 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 studies are 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-degraded with agarase, laminase, cellulase and alginate lyase. To produce enzymes. ^In addition, alginate lyase alters the brown algae of microorganisms according to the β-elimination mechanism: a—Li guluronic acid (G) and β-D-mannuronic acid (M). There are many reports of degradation into MG (GM) and heteropolymeric MG (GM). ¹¹ abalone and Psuedoalteromonas sp. I AM 14594 (AF082561), Sphingomonas sp. AKAB120939), Klebsiella pneumonia subs. aerogenes (Ll9657), Vibrio sp. QY10KAY221030) and Psuedomonas sp. These enzymes are isolated from the gut of many marine bacteria, including OS-ALG-9 (AB003330). ² ' ⁴ ᅳ ¹³ ' ¹⁵ So far ， Alginate lyase reported in the Genebank database is around 23 RF. ²⁴ Previous studies have shown that alginate lyases of many different microorganisms include G block-specific polyguluronate lyase (EC 4.2.2.11) and M block-specific polymannuronate lyase (EC 4.2). .2.3), can be classified according to two kinds of substrate specificity. 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 lyase and alginate lolisaccharide are attracting attention from researchers in the food and pharmaceutical industry.

Independent culture studies using clone libraries of the 16s rRNA gene show the diversity of the entire bacteria in the viscera of the abalone Haliotis discus hannai, Alpha-, Gamma-, Epsi lonproteobacteria and mollicutes. ²¹ However, the information of the 16s rRNA gene does not provide effective information on the biological function of the microorganism. ¹⁷ to ^'23, in contrast, metadata genomic library using the phosphine imide (fosmid) cloning systems can be found a novel gene of the organism culture is not easy. The screening system using an insert size of about 40 kb isolated directly from samples of ¹ phosphide vector and various environments comprises chitinase, dehydrogenase, 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 the intestinal microflora of abalone using the metagenome library.

 Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

[Content of invention] [Problem to solve]

 We sought to develop a metagenome library and AlyDW with alginate lyase activity. As a result, the microorganisms derived from the abdominal intestine were isolated to identify metazyme libraries and novel enzymes having alginate lyase activity, and the present invention was completed by identifying the optimum activity conditions of these novel enzymes.

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

 Another object of the present invention is to provide a novel alginate lyase.

 Another object of the present invention is to provide a nucleic acid molecule encoding a novel alginate lyase.

 Another object of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.

 Another object of the present invention to provide a cell transformed by the recombinant vector.

 Another object of the present invention is to provide a method for decomposing alginate. The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

[Measures of problem]

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 9823-11250th nucleotide sequence, 11377-12189th nucleotide sequence, 12290-13306th nucleotide sequence, 13611-14642th nucleotide sequence, 14646-15212th nucleotide sequence, 15417-16388th nucleotide sequence, 17370-19223th nucleotide sequence, 19452—21017th nucleotide sequence, 21253-22143 nucleotide sequence, 22309-23121th nucleotide Sequence, 23333-24208 nucleotide sequence, 24294—24989 nucleotide sequence, 25446-26477 nucleotide sequence, 26549-28063 nucleotide sequence, 28362-29579 nucleotide sequence, 30103-30930 nucleotide sequence, and 31010-31696 nucleotide sequence Provided is a metagenome library having alginate lyase activity comprising 22 0RF comprising.

 As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide that exists in single- or double-stranded form and includes analogs of natural nucleotides unless otherwise specified (Scheit, Nucleotide Analogs, John Wiley). , New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

 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 a microflora of the intestine of abalone.

 As used herein, the term "metagenome" refers to the total microbial collection present in nature, and the DNA of microorganisms obtained from various natural environments such as soil, seawater, tidal flats, rivers, air, and the large intestine of animals according to their species. Metagenome is not separated 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 of nature, maintains and expresses it in host cells to make metagenome library to classify the lineage 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. Hal 1 (July 2004). "Predicting the evolution of antibiotic resistance genes. ". Nature Reviews Microbiology 2 (5). doi: l0.1038 / 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 lyase) and secondary metabolites. According to another aspect of the present invention, the present invention provides an alginate lyase having an amino acid sequence set forth in SEQ ID NO: 2.

 The present inventors, unlike the alginate decomposition method using a conventional microorganism, in order to solve the high process cost that must be discharged by neutralizing strong acid by acid-based low-molecular alginate production method by separating the microorganism with excellent polysaccharide degradability of seaweed to stably remove algae Efforts have been made to establish conditions for degradation. As a result, a novel alginate lyase enzyme was discovered, and accordingly, it was confirmed that the molecular weight of alginate could be reduced more efficiently and more stably.

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

 According to a preferred embodiment of the invention, the alginate lyase is derived from a microorganism. Most preferably the alginate lyase is derived from the intestinal microflora of abalone. According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the alginate lyase.

 Most preferably, the nucleic acid molecules of the invention comprise a sequence of the first sequence

Nucleotide sequence represented by the 15417-16388th nucleotide sequence.

As used herein, the term "nucleic acid molecule" is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusive, and the nucleotides that are the basic structural units in nucleic acid molecules are naturally modified nucleotides, as well as sugar or base sites modified. Analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

 Variations in nucleotides do not result in changes in proteins. Such nucleic acids include functionally equivalent codons or codons encoding the same amino acids (eg, due to the degeneracy of the codons, there are six codons for arginine or serine), or codons encoding biologically equivalent amino acids. And nucleic acid molecules.

 In addition, variations in nucleotides may result in changes in the alginate lyase itself. Even in the case of mutations that result in changes in the amino acids of the alginate lyases, those exhibiting almost the same activity as the alginate lyases of the present invention can be obtained.

 It will be apparent to those skilled in the art that biological function equivalents that may be included in the alginate lyase of the present invention will be limited to variations in amino acid sequences that exert biological activity equivalent to the alginate lyase of the present invention.

 Such amino acid variations are made based on the relative similarity of amino acid chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing variants, hydropathic idex of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); Plin (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-

3.5); lysine (-3.9); And arginine (-4.5).

 Hydrophobic amino acid indexes are very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing a mutation with reference to a hydrophobic index, substitutions are made between amino acids that exhibit a hydrophobic index difference of preferably within ± 2, more preferably within 1 and even more preferably within 0.5.

 On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0 士 1); Glutamate (+ 3.0 × 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-

0.4); Plin (-0.5 土 1); alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

 When introducing a mutation with reference to a hydrophilicity value, substitution is carried out between amino acids which exhibit a hydrophilicity value difference within the range of preferably within 2, more preferably within 1, and even more preferably within 0.5.

 Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins,

Academic Press, New York, 1979). The most commonly occurring exchanges include amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr,

Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile,

Exchange between Leu / V l, Ala / Glu, Asp / Gly.

Considering the above-described variations with biological agitation activity, the alginate lyase of the present invention or nucleic acid molecules encoding the same are also construed to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing. Substantial identity of the above, the present invention By aligning a sequence with any other sequence as much as possible, and analyzing the aligned sequence using algorithms commonly used in the art, for example, a sequence exhibiting homology of at least 99) is meant. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mo J. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al. , Meth. Mol. Biol. 24: 307-31 (1994). NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) can be accessed from the National Center for Biological Information (NCBI), etc. It can be used in conjunction with sequencing programs such as blastx, tblastn and tblastx. BLSAT is available at http: //www.ncbi. Accessible at nlm.nih.gov/BLAST/. Sequence homology comparison method using this program is http: // 丽. ncbi.nlm.nih.gov/BLAST/blast_help. You can check it in html. According to another aspect of the present invention, the present invention provides a recombinant vector comprising the above-described nucleic acid molecule of the present invention encoding an alginate lyase. The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference. Vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as a host. Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter capable of promoting transcription (for example, tac Promoter, lac promoter, / adJV5 promoter, lpp promoter, _¾ ^λ promoter, p _R promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosomal binding site for initiation of detoxification And transcription / detox termination sequences. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the leftward promoter of phage λ (p _L promoter, Herskowitz, I. and Hagen,

D., Ann. Rev. Genet., 14: 399-445 (1980)) can be used as regulatory sites. On the other hand, vectors that can be used in the present invention are plasmids often used in the art (eg pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (eg g.B, λ-Charon , λ ζ, and M13) or viruses (eg, SV40, etc.).

 Meanwhile, the vector of the present invention includes an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline. According to another aspect of the present invention, the present invention provides a transformant comprising the vector of the present invention described above.

 Host cells capable of stably and continuously cloning and expressing the vectors of the present invention are known in the art and can be used with any host cell, for example, E. coli JM109, E. coli BL2KDE3), E. coli RR1, E coli LE392,

Strains of the genus Bacillus, such as E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and enterobacteria and strains such as Salmonella typhimurium, Serratia marcensons and various Pseudomonas species. Etc.

The method of carrying the vector of the present invention into a host cell includes the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mo J. Biol., 166: 557- 580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like.

 Vectors injected into host cells can be expressed in host cells, in which case a large amount of alginate lyase is obtained. For example, when the expression vector includes a lac promoter, the host cell may be treated with IPTG to induce gene expression. According to another aspect of the present invention, the present invention provides a method of degrading alginate, comprising contacting the alginate lyase of claim 1 or the transformed cell of claim 5 with alginate.

 The present inventors have attempted to find enzymes having alginate resolution and microbial strains producing such enzymes and have tried to develop low molecular weight alginates using these enzymes or strains. As a result, the alginate lyase which can decompose alginate differently from the conventional synthesis method was discovered, and from this, the production of low molecular weight alginate was confirmed.

【Effects of the Invention】

 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 algae processing process and increasing the utilization of resources by utilizing the technology of biotechnological mass production of functional polysaccharides.

[Brief Description of Drawings]

1 is a result of the evaluation of alginate hydrolysis using the cetylpyridinium chloride (cetylpyridinium chloride) method. 1 confirms the activity Show plate screening. (A) Flavobacterium sp. Positive control group derived; (B) E. coli-derived negative control group and (C) alginolatic metagenome fragment (AlyDW).

 2 is a schematic diagram of an alginolatic metagenome fragment (Aly V). Differences in contrast shown in the figure indicate functional classification of putative genes. Diagonal stripes are 0RF related to cell processes, Brake arrows are 0RF related to metabolism, and light gray are 0RF related to 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. Left panel (a) shows the hydrolysis profiles of poly (pD-mannuronate, lane 1) and poly (α-L-guluronate, lane 2) after incubation at 40 ^° C. for 3 hours. The 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 (enzyme only, lane 6). M is G1 (glucose), G2 (cellobiose), G3 (cellotriose), G4 (cellotetraose), G5 (salopentaose), G6 (salonucleose), G7 (celloheptaose) and G8 (Salo Octaose) is a standard complex.

5-6 show the alignment results of putative alginate lyase and metagenome fragment (0RF-11) proteins derived from metagenome fragments. Amino acid sequences were aligned using the ClustalX program. The compared organisms were Klebsiella pneumoniae subsp. aerogenes, Saccharophagus degradans 2-40, Microbulbifer sp. 6532A, Vibrio harveyi aDA3, Cel lulophaga lytica DSM 7489, Vibrio sp. A9m, Vibrio alginolyticus 40B, Pseudoa I teromonas sp. CY2, Vibrio splendidus 12B01, Polaribacter sp. MED152 and Agar Ivor an s sp. JAM-Alra and the gene bank entry numbers of the organisms are Q59478, ABD81738, BAJ62034, ZP_0617515, YP_004263738, BAH79133, ZP_06182095, ACM89454, ΖΡ— 00990010, ΖΡ_01052859 and BAG70358, respectively. 7 is a result of the evaluation of alginate hydrolysis using the cetylpyridinium chloride (cetylpyridinium chloride) method. 7 shows plate screening confirming activity. (A) Flavobacterium sp. Positive control group derived; (B) negative control from E. coli; (C) Arginolatic Metagenome Fragment (AlyDW) and (D) Recombinant Alginic Acid Degrading Enzyme (AlyDWll).

 8 shows the results of SDS-PAGE analysis and zymogram active staining of recombinant alginic acid degrading enzyme (AlyDWll). (a) SDS-PAGE and staining using Coomassie Brilliant Blue R-250. M is the molecular weight marker, column 1 is the cell extract of pMAL-c2X-AlyDWl expressed using IPTG, column 2 is a washing process using an amylose affinity column, column 3 is the final eluted recombinant alginic acid degrading enzyme (AlyDWll). (b) shows the result of Zymogram activity staining, row 1 is recombinant alginic acid degrading enzyme, and row 2 is Flavobacterium sp. It is a positive control group of origin.

 Figure 9 shows the result of reducing sugar analysis confirming the effect of pH (A) and temperature (B) on the alginate lyase activity of the recombinant alginic acid degrading enzyme.

10 shows the alginate resolution of the recombinant alginic acid degrading enzyme through thin layer chromatography. (a) Hydrolysis result of reaction of recombinant alginic acid dehydrogenase with poly (β-D-mannuronate, row 1) or poly (α-L-gururonate, row 2) at 45 ^° C for 3 hours. Shows. Rows 3 and 4 are positive controls that reacted avobacteriuin sp. With poly? -D-mannuronate or poly aL-gururonate, lane 4), and columns 5 and 6 were poly ^ -D negative controls. -Reacted results with mannuronate or poly α-L-gururonate. (b) shows the result of degradation degree of alginic acid after reaction with recombinant alginic acid degrading enzyme with alginic acid at 45 ^° C for 3 hours (column 1). Row 2 was positive control, Flavobacterium sp. Alginate (3 rows) and enzyme (4 rows) were used as negative controls, respectively. M is G1 (glucose), G2 (cellobiose), G3 (cellotriose), G4 (cellotetraose), G5 (cellopentaose), G6 (cellonucleose), G7 (celloheptaose), Based on the combination of G8 (cellooctaose). DETAILED DESCRIPTION Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . EXAMPLES Experimental Materials and Methods

 Meta agenom i c library construction

In February 2009, abalone samples were obtained off the coast of 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 phosphid vector (pCClFOS ready vector, Epicentre, USA) with BamHI restriction enzyme and then ligated at 16 ^° C with DNA cleaved 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 lig / ml). Subsequently, Deck Escherichia coif) was transfected to construct a megnome library. Library Screening and Shotgun Sequence Analysis

Cetylpyridinium chloride (CPC) analysis was performed to evaluate the resolution of the active alginate lyase. Metagenome DNA was ligated into a phosphid vector (pCClFOS ready vector, Epicentre, USA) to pack into lambda phage and infected with Escherichia coli. 0.1% of the resulting infected cells It was incubated for one day in LB (Luria-Bertani) agar medium containing 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 Thermocyclers (Applied Biosys terns) and ABI PRISM ™ Bi Dye ™ Terminator Cycle Sequencing kit (Appl ied 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. Of: (0RF open reading frames) Estimated protein yijongsang Bi group were classified according to the category cluster ^{12 '22 23} eu CLUSTAL.X program phosphine imide 0RF clones obtained using a (01 "1 ± 010 003 groups , C0R). The complete nucleotide sequence was aligned with homologous genes obtained from the NCBI nucleotide database Determination of ⁶ S activity

The 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 carbonates by treating large amounts of reducing sugars. . ^{3 The} standard curve is 31.5 based on 525 nm absorbance measurements using a spectrophotometer (Mecassis, Korea).

Glucose was prepared in the β _& / ηί concentration range. Temperature using 0.1 Μ 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) Optimum enzyme activity for 2 hours at (15 ^° C-50 ^° C) and pH (4.0-10.0) was determined. Metal ions and nicotinamide adenine at optimal conditions (pH 8.0 and 40 ^° C) Inhibition and enhancement of enzyme activity in the presence of dinucleotide (nicotinamide adinine dinucleotide, NAD ⁺ ) was determined. Determination of alginate lyase activity

LB (Luria-) at 40 ^° C for 16 hours for the production of alginate lyase

Bertani) clones were cultured. Culture medium was concentrated using a protein concentration kit (Rapid-Con ™ Protein Concentration Kit, Elpis, Seoul, Korea). AlyDW was treated with a 2-mercaptoethanol-free sample buffer according to the method described by Lae 隱 Π and treated with SDS-PAGE (sodium dodecyl sul fate-polyaery 1 amide gel electrophoresis) with 10% acrylamide gel. ) Was analyzed. ^{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 / t casein, 2 mM EDTA, 0.01% NaN ₃ ) with 25% methanol. And regenerated for 30 minutes. The polyacrylamide gel was washed with 100 mM phosphate buffer 1 (ρΗ 8) and then covered with a 1.5% agarose gel consisting of 1 mg / sodium alginate, 10 gM NaCl and 100 mM phosphate buffer KpH 8). 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. Analysis of Banung Products Using Thin Layer Chromatography

Standard combinations of eel lool igosaccharides (cellobiose, eel lotriose ᅳ eel lotetraose, eel lopentaose, and cell ohexaose ), Eel loheptaose and cellooctaose) were purchased from Sigma-Aldrich. To determine the alginate lyase activeol possessed by the test clone, 0.1% alginate was reacted with AlyDW (2.19 mg / m £) 50 ^ in 37 ^° C., 100 mM phosphate buffer KpH 8). Asahipak GS-310 column (21.5 隱 ID x 500 隨, Showa Denko Kogyo Co.Ltd., Tokyo) on fractions of alginate oligomers by gel filtration chromatography (GFC) , Japan (Shimaju) System, LC-6AD pump, RID-10A detector, SPD-M20A detector: Shimazu, Toko, Japan). Watering 5

The sample was separated by pouring. Aliquot (2) of the digested alginate solution was injected to give a fraction. The reaction product was reacted in 1-propanol, nitromethane and water mixture (5: 3: 2 v / v / v) for 2 hours before silica gel folate (silica gel plate, Merck KGaA). Hydrolysis products were isolated from thin layer chromatography using (64271 Darmstadt, Germany). After separation, in sugar 1 ml of sulfuric acid and 10 m stock solution (1 g diphenylamine, 1 ml aniline, 100 mi acetone) The mixture was sprayed and visualized. ²⁶ Results and discussions

 Screening and 0RF Analysis of Meta-Gnome Fragments

 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 abalone (FIG. 1). Analysis of the shot-gun sequence data shows that the positive metagnome fragments have genes with an average G + C content of 43.3% and a length of 31.7 kb of 22 expected 0RFs. a was prepared based on TMHMM survey, b indicates that it does not belong to the C0G group (FIG. 2 and Table 1).

 Table 1

. / Ga aproteobacter ia

 Vibrio sp

 Hypothet ical protein 3.00E-258 1-NA

 . / Gammaproteobacter ia

 Vibrio coral liilyticu

 Hypothet ical protein 1.00E-73 1-NA

 s / Gan aproteobacteria

 Vibrio parahaemolyt icus

 Transcr ipt ional egulator 2.00E 一 79 0 C0G0583

 Gammaproteobacter i a

 Photobacteriu sp.

 Hypothet ical protein 2.00E-61 1-NA

 Gammaproteobacter i a

 Mult idrug resistance Vibrio vulnificus

 2.00E-166 2 Q C0G1566 ef f lux pump / Gammaproteobacter j a

 Vibrio vulnificus

 Hypothet ical protein 3.00E-55 3-NA

 Gammaproteobacter i a

 Alginate lyase (= Poly Klebsiella pneumoniae

6.00E-117 0-NA ji? -Dmannuronate lyase) subsp. aerogenes / Gammaproteobactern ^''

 Vibrio splendidus

 L-yase, putat ive 0.00E + 00--NA

 Gammaproteobacter j a

 Vibrio sp.

 Alginate lyase 0.00E + 00 0-NA

 / Gammaproteobacter ί a

 Transcr ipt ional Vibrio splendidus

 9.00E-122 0 K C0G0583 regulator, LysR fami ly / Gammaproteobacter

 Vibrio sp.

³ robable oxidoreductase 1.00E-95 0 QR C0G1028

 Gammaproteobacter i a

 Oxygen- i ndependent

 Vibrio splendidus

coproporphyrinogen I II 6.00E-138 0 C C0G1032

 / Gammaproteobacter

oxidase, putat ive

^"Lavodoxin reductase Vibrio parahaemolyt icus

 4.00E-72 0 R C0G3217 fami ly 1 protein / Gammaproteobacter i a

Vibrio cor a lli ^' i lyt icus

 ihydroorotase 2.00E-171 0 F C0G0418

/ Gammaproteobacter ia Vibrio cholera

 19 Glycerol kinase 0.00E + 00 0 C C0G0554

 / Ga maproteobacteria

 G 1 ucose- 1-phospha t e Vibrio shiloni i

 20 0.00E + 00 0 0 C0G0448 adeny lyltr ans fer ase / Gammaproteobacteria

 ABC transporter,

 Vibrionales bacterium

 21 periplasmic 2.00E-88 0 P C0G0226

 / Gammaproteobacteria

 substratebinding protein

 Vibrio oriental is

 22 ^ IodN 一 related protein 6.00E-80 0 I C0G32030

 / Gammaproteobacteria The 17 ORFs in the ORFs show significant similarities with the genes of the functions known in the NCBI nucleotide database, and 50 RF is a hypothetical protein in the genome database of NCBI bacteria. Six 0RFs are involved in cell processing and information storage, and 10 0RFs are involved in metabolism. 3 0RF (11, 12 and 13) are Klebsiella pneumonia subspecies. The aerogenes, Vibrio splendidus and Vibrio species showed similarity with the protein sequence of the alginate lyase gene of the Gammaproteobacteria door. AJyW features

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, showing the characteristics of the low temperature active enzyme (Fig. 3). It showed maximum alginolitic 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. The negative control group was represented by T as a relative value, and the standard deviation value was derived from three independent experiments.

 Table 2

Chemical fold activity Single Combination (Cation + NAD +)

None 1.00 + -0.05-

NAD + (0 0.94 ± 0.01-

EDTA (1 mM) 0.80 ± 0.01-

CaCh 0.94 ± 0.02 1.5 0.20

 MgCl 0.93 ± 0.01 1.29 ± 0.10

 KC1 (1 mM) 0.90 ± 0.01 1.40 ± 0.36

 MgS0 0.90 ± 0.15 1.68 ± 0.05

FeSC ⁽ l 0.86 ± 0.03 1.11 ± 0.04

 CoC 1.13 ± 0.02 1.36 ± 0.24

 MllC 0.81 ± 0.01 1.11 ± 0.14

 CuCl2 0.98 ± 0.01 1.17 ± 0.10

 NaCl (1 mM) 0.94 ± 0.02 1.16 ± 0.05

AgNC 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 effects on alginate lyase activity compared to controls without metal cations 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 moles / min / mg protein at optimal conditions. Haliotis spp. , Lit tor in spp. And alginate lyases obtained from marine mollusks including Turbo cornutus are endo-poly (M) and exo-poly (G) lyases having optimal values at 25-501 and pH 4.0-9.6. Enzyme activity increased in the presence of divalent cations such as Ca ²⁺ or Mg ²⁺ . 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 (AlyE). After 3 hours, the alginate hydrolysis end product of AlyDW was subjected to thin-layer chromatography. Analyzes (FIG. 4). 4, AlyDW has endolitic activity and degrades poly (-1) -1113 皿 111 ^" 011 ^ 6) over poly (α-L-guluronate). Alginate lyase isolated from the most preferred endo-poly (M) lyase ²⁷

 High performance liquid chromatography:

HPLC) and three of the 20 fractions were purified by Flavobacteri 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 make AlyDW a useful enzyme to make products of lower molecular weight than commercial enzymes. Arginolitic 0RF-11 features

 AlyDW has a molecular mass of 35, 67 and 57 kDa, which is theoretically 30 RF (0RF11, 12 and 13). 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 and activated staining under regeneration conditions.

Speculative amino acid sequence of 0RF 11 (AlyDW) and Ae_2Al & Saccharophagus degradans 2-40) and d \ k {Klebsiel la pneumoniae subsp. homology with the aeri e / 2es) genes was 63% and 65%, respectively. AlgispiMjcrobulbifer sp. 6532A), VME_15370 (Vibrio harveyi 1DA3), Cel \ y_305 (CeJluIophaga lytica DSM 7489), alg (Vibrio sp. A9m), VMC_35250 (Vibrio alginolyticus 40B), alyPI (Pseudoalteromonas sp. CY2), V12B01_24259 (Vibrio splendidus 12B01), W 52_06195 {PoJarjbactor sp. MED152) and AlglA Aga / voraus sp. JAM-A1) gene was 54¾-60% (Figures 5-6). Polysaccharide lyase (PL) family 7 alginate lyases are believed to act as substrate binding and catalytic sites (R / E) (S / T / N) EL, Q (I / V) H and It has three kinds of highly conserved amino acid sequences of YF AG (V / I) YNQ. The 0RF 11 of ²⁴ AlyDW metagenome fragments are G and M specific lyase (AlyA) of K. pneumonia, M-specific lyase (AlxM) of marine bacteria ATCC 433367, and G-specific lyase (ALY) of Cor nebactenwn ¾σ. -1) also has RSEL, QIH and YFKAGVYNQ identified. The amino acid sequence is It is judged to be an essential part of maintaining the stable three-dimensional structure and function of alginate lyase. ¹³¹⁴

Manufacturing and Analysis of AlyDWll

After extracting the DNA of the microorganisms in the abalone digestive system, the extracted genomic DNA was made into 32 kb DNA fragments, end repaired and size-selected, and then the CopyControl Cloning-Ready back frame pCClFOS (POSMID). After ligating to a library preparation kit (Epicentre) and transforming E. coli, plated and cultured in a plate medium containing antibiotics, transformants exhibiting antibiotic resistance were selected to produce a phosphid library. After screening clones having an alginate degrading activity from the library, clones having an endolytic alginate lyase activity that hydrolyzed alginic acid to low molecules were obtained (FIG. 7). Alginate lyase gene 0RF-11 was amplified using primers with Ban l and Hind cleavage sites. After ligating into a pMAL-c2X vector (NEW ENGLAND BioLabs, UK) and placing it in E. coli BL2KDE3), PMTG-c2X-AlyDWll expressed by IPTGGs using 3ropyl-3-D-thiogalactopyranoside) was subjected to amylose affinity column. To elute the recombinant protein. 10% SDS-PAGE was used to identify AlyDWll overexpressed at about 76 kDa (FIG. 8). AlyDWll showed the highest activity at 45 ^° C as a result of searching for the proper pH and temperature of alginic acid degrading enzyme through reducing sugar assay. Optimum pH appeared at pH 7 and decreased activity at pH values above pH 8 (FIG. 9). Cofactors such as metal ions confirmed that the combination of NAD +, Mg ²⁴ or Ag + was 1.6-2.1 times higher for alginate lyase activity than conditions without metal cations and NAD +, respectively (Table 3).

 [Table 3]

EDTA (1 mM) 0.75 ± 0.25-

CaCl ₂ (1 mM) 0.95 ± 0.03 1.53 ± 0.20

MgCl ₂ (1 mM) 0.91 ± 0.03 1.27 ± 0.12

 KC1 (1 mM) 0.92 ± 0.01 1.43 ± 0.13

MgS0 ₄ (1 mM) 0.93 ± 0.15 1.65 ± 0.05

FeS0 ₄ (1 mM) 0.90 ± 0.14 1.20 ± 0.04

CoCl ₂ (1 mM) 1.08 ± 0.05 1.30 ± 0.14

MnCl ₂ (1 mM) 0.85 ± 0.01 U4 ± 0.16

CuCl ₂ (1 mM) 1.01 ± 0.08 1.10 ± 0.10

 NaCl (1 mM) 0.96 ± 0.06 1.20 ± 0.05

AgN0 ₃ (1 mM) 1.31 ± 0.03 2.10 ± 0.21

 The final product of alginate hydrolysis with alginate lyase (AlyDWll) was observed 3 hours later by Thin-layer Chromatography (TLC). AlyDWll has endortic activity and shows better activity of poly (^-D-mannuronate) than poly (α-L-gururonate), which is endo-poly (M) in most marine molluscs. It is synonymous with lyase observed. Among the products obtained by reacting with alginic acid, alginate low-molecular oligosaccharides ranging from 180 to 342 Da, 540 to 720 Da, and 1,080 to 1,260 Da were obtained, which shows that the enzyme can produce a lower molecular weight than commercial enzymes (FIG. 10). The AlyDWll DNA sequence was registered with GeneBank Accession Number, JN392921. Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof. Reference

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Iwamoto Y, Muramatsu T (2005) Structure-act ivi ty relationship of alginate oligosaccharides in the induct ion of cytokine production from RAW264.7 cells. FEBS Lett 579: 4423-4429

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9.Larsen B, Hooen K, Ostgaard K (1993) Kinetics 254 and specificity of alginate lyases. Hydrobiologia 2 30aoett577 Mr B _il L 3221 _: —

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Claims

[Patent Claims]

 [Claim 11

 1280-3652th nucleotide sequence of SEQ ID NO: 1, 5025-6128 nucleotide sequence, 6365-7492 nucleotide sequence, 8687-9202 nucleotide sequence, 9206-9823 nucleotide sequence 9823-11250 nucleotide sequence, 11377-12189 1st nucleotide sequence, 12290-13306 nucleotide sequence, 13611-14642 nucleotide sequence, 14646-15212 nucleotide sequence, 154Γ7-16388 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 Leo Tide sequences 30103-30930 second nucleotide sequence and meta genome library with a second nucleotide sequence 31010-31696 alginate rayiah activity containing 22 open reading frames, including.

[Claim 2]

 [Claim 2] The metagnome library according to claim 1, wherein the metagnome library has a nucleotide sequence of SEQ ID NO: 1.

[Claim 3]

 The meta-genome library according to claim 1, wherein the meta-genome library is constructed using genome DNA obtained from a microflora of intestines of abalone.

[Claim 4]

Alginate lyase comprising the amino acid sequence set forth in SEQ ID NO: 2 sequence.

[Claim 5]

 A nucleic acid molecule encoding an alginate lyase comprising the amino acid sequence set forth in SEQ ID NO: 2.

[Claim 6]

 The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the 15417-16388th nucleotide sequence of SEQ ID NO: 1.

[Claim 7]

 A recombinant vector comprising the nucleic acid molecule of claim 4.

[Claim 8]

 Cell transformed by the recombinant vector of claim 7. [Claim 9]

 A method of degrading alginate, comprising contacting the alginate lyase of claim 4 or the transformed cells of claim 8 with alginate.