KR20100109743A - Beta-agarase from pseudoalteromonas sp - Google Patents

Abstract

The present invention relates to a beta- agarase derived from Pseudoalteromonas sp strain having agar resolution at high temperature and to a recombinant biocatalyst prepared using the beta- agarase gene. By providing beta-agarase or the beta-agarase gene, it can be used in various industries where beta-agarase is applied.

Description

BETA-AGARASE FROM PSEUDOALTEROMONAS SP} Produced by Pseudoaltermonas Strains

The present invention provides a lipoprotein-forming signal sequence in a beta- agarase derived from Pseudoalteromonas sp strain having agar resolution at high temperature, a nucleotide sequence encoding the same, and a nucleotide sequence encoding the beta-agarase. Nucleotides included, the vector comprising the respective nucleotide sequence, the transformants transformed with the respective vectors, the respective transformants, cultures thereof, cell-free cultures thereof, concentrates of the cultures, the cell free The present invention relates to a biocatalyst having agar degradation activity comprising as an active ingredient at least one selected from the group consisting of a concentrated solution of a culture solution and a recombinant beta-agarase prepared by expressing the transformant.

Agar (agar) is a polysaccharide mainly found in the cell walls of red algae, a kind of dietary fiber. Agar is a polysaccharide that is difficult to be broken down by digestive enzymes, so it is not provided as a nutrient source, but it is known that it can reduce the absolute amount of cholesterol in the body by inhibiting blood cholesterol and adsorbing and excreted in the form of bile acids, widely used as a diet food. have. In addition, agar is mainly used as a solid medium for culturing microorganisms, as a stabilizer in confectionery and meat processing, and as a gelling agent in cosmetics and foods.

Agar is composed of about 70% agarose and about 30% agaropectin. The agarose is a neutral polysaccharide, and alternating α-1,3 and β-1,4 bonds between galactose are present in the agarose. On the other hand, agalopectin is an acidic polysaccharide, and the agalopectin is a sulfate, in particular, sulfate, gluconic acid or pyruvate, combined with agarose.

Agarase, an enzyme that decomposes agar, may be classified into alpha-agarase and beta-agarase depending on the site for decomposing the bond. The alpha-agarase decomposes α-1,3 bonds in the galactose polymer of agarose to prepare agaro-oligosaccharides. The agaro-oligosaccharides have apoptosis inducing activity, anticancer activity (Kato, 2000), and antiviral activity. , Antioxidant activity (Chen et al ., 2005; Kato, 2000), immunomodulatory activity (Yoshizawa et al ., 1995), have been reported to have antiallergic activity and anti-inflammatory activity. In addition, the beta-agarase decomposes β-1,4 bonds in the galactose polymer of agarose to prepare neoagaroligosaccharides, and the neoagaroligosaccharides inhibit bacterial growth (Kono et al ., 1989), Antioxidant activity, prevent starch aging, moisturizing effect (Kobayashi et al ., 1997), whitening effect (Kobayashi et. al ., 1997).

Since the effect of the agar oligosaccharides can be applied to various industries such as cosmetics, pharmaceuticals and functional foods, recently, research on the production of low value-added agar into high value-added agar oligosaccharides using agarase has been actively conducted. have. However, in the case of nucleotides encoding microorganism-derived agarases reported previously, a transformant such as E- coli is used because the produced agarase does not have a sequence such as a lipoprotein that can move out of the cell. There was a problem that the production of recombinant agarase is limited.

In addition, there is an increasing demand for the development of heat-resistant agarase that can exhibit activity in the temperature range in which agar exists in a sol phase at the temperature of 40 ° C. or lower.

Therefore, there is a signal sequence that encodes a heat-resistant agarase that maintains agar resolution even at a temperature of 40 ° C. or higher, and moves the agarase produced outside the cell, such as lipoproteins, together with a sequence encoding the agarase. There is a need for research on gene sequences encoding agarase that can effectively produce recombinant agarase.

In order to solve the problems of the prior art as described above, the present invention has a beta-agarase activity, the agar degrading enzyme that maintains agar resolution even at a temperature of 40 ℃ or more, which is the temperature at which the agar is changed from gel to sol phase, It is aimed at providing beta-agarase.

It is also an object of the present invention to provide an amino acid sequence of the beta-agarase and a polynucleotide sequence encoding the same.

In addition, an object of the present invention is to provide a polynucleotide sequence including a lipoprotein-forming signal sequence in a polynucleotide sequence encoding the beta-agarase.

Further, the vector comprising the respective polynucleotide sequence, the transformants transformed with the respective vectors, the respective transformants, the culture thereof, the cell-free culture medium, the concentrate of the culture, the cell-free culture solution It is an object of the present invention to provide a biocatalyst having an agar-degrading activity comprising as an active ingredient at least one selected from the group consisting of concentrated beta and recombinant beta-agarase prepared by expression in the transformant.

In order to achieve the above object, the present invention has a beta-agarase activity, the agar degrading enzyme, specifically beta-agar is maintained even at a temperature of 40 ℃ or more, the temperature at which the agar is changed from gel to sol phase Offer

The present invention also provides an amino acid sequence of the beta-agarase and a polynucleotide sequence encoding the same. The present invention also provides a polynucleotide sequence comprising a lipoprotein formation signal sequence in the polynucleotide sequence encoding the beta-agarase.

In addition, the present invention provides a vector comprising the respective polynucleotide sequence, transformants transformed with each of the vectors, each transformant, its culture, its cell-free culture, the concentrate of the culture, Provided is a biocatalyst having an agar-degrading activity comprising as an active ingredient at least one selected from the group consisting of a concentrate of a cell-free culture and a recombinant beta-agarase prepared by expression in the transformant.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention have been studying agarases that can produce low value-added agar from high value-added agar oligosaccharides, while the genes of heat-resistant beta-agarase having excellent activity at temperatures below 40 ° C in which agar is present as sol phase. By confirming the sequence, the present invention was completed based on this.

In the present invention, agar is a viscous, indigestible complex polysaccharide which is a cell wall component of red algae and is a kind of dietary fiber source. Agar is mainly used as a microbial solid medium, and as a stabilizer in confectionery and meat processing, as a gelling agent in cosmetics and foods, and as a colloid inhibitor in health foods, pharmaceuticals, dental impression materials, laboratory reagents and photographic emulsions. The agar is composed of agarose (agarose) and agaropectin, mainly composed of about 70% agarose and about 30% agalopectin.

In the present invention, agarase means an enzyme that degrades agarose. The agarase has two groups according to the decomposition pattern of the agarase, namely alpha-agarase that breaks down alpha-1,4 bond of agarose and beta-agar that breaks down beta-1,4 bond of agarose. It can be divided into beta-agarase.

In the present invention, culture refers to agarase derived from a cell, preferably a microorganism, more preferably Pseudoalteromonas sp, even more preferably Pseudoalteromonas sp AG52 strain. It means a culture medium in which a transformant transformed with a vector containing a nucleotide sequence is cultured, and the culture medium includes cultured microorganisms.

In the present invention, the cell-free culture means removing the cultured microorganisms in a culture medium in which cells or microorganisms are cultured. The culture medium means a solid composition or a liquid composition containing nutrients necessary for culturing microorganisms including animal cells, plant cells or bacteria, and preferably, may be a liquid composition. In the present invention, the concentrate means that the culture or the cell-free culture is concentrated.

The beta-agarase was cloned using Pseudoalteromonas sp AG52 strain using PCR technique and LA-PCR technique, and the cloned beta-agarase was synthesized by a total of 870 bp polynucleotides. Encoded, translated into 289 amino acids, having a size of about 33 kDa. In addition, the beta-agarase has 11 active sites and 7 calcium binding domains.

The beta-Niagara claim is the optimal reaction temperature of 45 to 55 ℃, and the optimum reaction pH pH 5.5, has a feature that significantly increased the agar decomposition activity by the addition of Fe ^{2 +.} In addition, the beta-agarase may produce neoagar oligosaccharides, the main product of neotetraose, through agar degradation activity.

Since the optimum reaction temperature is higher than 40 ℃ which is the temperature at which the agar is changed from gel to sol phase, agar can be decomposed agar at a temperature of 40 ℃ or more sol state to produce agar oligosaccharides, specifically neo-agar oligosaccharides, Since the efficiency of the enzyme that has been a problem can be solved, it may be applied to various industrial applications. In addition, the present invention can produce neoagar oligosaccharides containing neoagarotetraose and neoagarohexaose, which are known to have excellent physiological activities such as skin whitening effect, and therefore, are expensive from inexpensive agar. Neo-agar oligosaccharides can be produced, the economic effect will be very large.

The agarase of the present invention is a beta-agarase having the amino acid sequence set forth in SEQ ID NO: 5. The beta-agarase may preferably have an amino acid sequence encoded by the polynucleotide set forth in SEQ ID NO: 4. Specifically, the beta- agarase may be derived from Pseudoalteromonas sp strain.

The present invention also relates to polynucleotides encoding beta-agarases having the amino acid sequence set forth in SEQ ID NO: 5. Preferably the polynucleotide is a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 4.

In addition, the present invention relates to a nucleotide sequence comprising a lipoprotein-forming signal sequence in the nucleotide sequence encoding the beta-agarase. The nucleotide sequence may preferably be a polynucleotide set forth in SEQ ID NO: 6. A nucleotide sequence having a nucleotide sequence of SEQ ID NO: 6 in which a lipoprotein-forming signal sequence is linked to a nucleotide sequence encoding the beta-agarase may release the beta-agarase out of a cell by the lipoprotein-forming signal sequence. Therefore, the nucleotide sequence having the nucleotide sequence of SEQ ID NO: 6 can be efficiently produced beta-agarase through a transformant transformed with a recombinant vector containing the nucleotide sequence having the nucleotide sequence of SEQ ID NO: 6 In this respect, the industrial meaning is very large.

The present invention also relates to a biocatalyst having agar degradation activity. The biocatalyst is a vector comprising the respective nucleotide sequence, specifically, the nucleotide sequence of SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 6, transformants transformed with the respective vectors, the respective transformants, its At least one selected from the group consisting of a culture, its cell-free culture medium, the concentrate of the culture, the concentrate of the cell-free culture medium and recombinant beta-agarase prepared by expressing each transformant as an active ingredient It may be a biocatalyst having an agar decomposition activity.

The beta-agarase may be preferably composed of the amino acid sequence of SEQ ID NO: 5, more preferably may have an amino acid sequence encoded by the polynucleotide of SEQ ID NO: 4, the activity of the enzyme As long as it is maintained, the amino acid may be modified by deletion, substitution or insertion by a method known to those skilled in the art.

The polynucleotide encoding the beta-agarase according to the present invention may have a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5, preferably may have a nucleotide sequence set forth in SEQ ID NO: 4, As long as the activity of the enzyme is maintained, the nucleotide may be modified by deletion, substitution or insertion by a method known to those skilled in the art.

The polynucleotide containing the lipoprotein-forming signal sequence in the polynucleotide sequence encoding the beta-agarase according to the present invention may have a nucleotide sequence as set forth in SEQ ID NO: 6, and the activity and the lipoprotein-forming site of the enzyme As long as the beta-agarase having the amino acid sequence encoded by the polynucleotide sequence is retained extracellularly, the nucleotide is deleted and substituted by a method known to those skilled in the art. Or it may be modified by insertion.

In addition, the present invention, in order to solve the problem of increased cost and activity loss by the separation and purification process for the development of enzyme biocatalyst, polynucleotide encoding the beta-agarase or encoding the beta-agarase An expression vector comprising a polynucleotide having a lipoprotein-forming signal sequence in a polynucleotide sequence, a transformant transformed with the expression vector, and a recombinant beta-agarase using the transformant and a method for producing the same will be. Preparation of the vector and preparation of the transformant are prepared according to conventional genetic engineering methods.

The expression vector may be appropriately selected according to the host to be used, and all of the usual expression vectors capable of expressing the protein in the transformant may be applicable. For example, the protein expressed by the transformant may be purified. It may be an expression vector comprising a polynucleotide encoding a fusion partner (fusion-tag). Specifically, in the case of Escherichia coli, pET 11a, pET 16b, pET 21a (Novagen Co., Germany), pGEX 4T-1 (GE Healthcare, USA) or pHCE 19 (BioLeaders Co., South Korea), preferably pET 11a or pET 16b, in the case of yeast, the promoter of GAL1, a gene encoding galactose, can be used for expression of foreign genes. pYES2 (Invitrogen Co., USA) etc. can be used.

The transformant was prepared using the expression vector, and the host used for the production of the transformant was a prokaryotic cell or a yeast such as E. coli, Bacillus sp. Strain and Pseudomonas sp. Strain. Eukaryotic cells, such as fungi, and the like, but are not limited thereto.

Preferably, the expression vector is a polynucleotide having a gene sequence encoding beta-agarase of SEQ ID NO: 5 into a pET 11a vector, more preferably a polynucleotide having a sequence of SEQ ID NO: 4 or pET 16b The polynucleotide having the sequence of SEQ ID NO: 6 is inserted into the vector, and the host is preferably E. coli , for example, E. coli BL21 (DE3) or E. coli DE5α.

The method for producing beta-agarase using the transformant comprises culturing the transformant using a culture medium, the beta-agarase contained in the culture medium or beta present in the transformant. -Obtaining agarase.

The culture medium, culture conditions and culture methods may be appropriately selected according to the type of transformant, for example, inserting a polynucleotide having a gene sequence encoding beta-agarase of SEQ ID NO: 4 in pET 11a A recombinant vector prepared by introducing a recombinant vector prepared in E. coli BL21 (DE3) into a culture medium, or a polynucleotide having a gene sequence encoding beta-agarase of SEQ ID NO. After transforming the recombinant vector prepared by inserting the recombinant vector prepared into pET 16b into E. coli DE5α and culturing in a culture medium, IPTG (Isopropylthio-β-D-galactoside) can be added and cultured. Recombinant beta-agara prepared by the method expressed by the transformant can be purified by a conventionally known method, but can be used in the form of whole cells or cultures thereof or cell-free cultures thereof without purification.

In addition, the present invention relates to a recombinant beta-agarase prepared by expression in a transformant transformed with the expression vector.

The transformant may be preferably E. coli , for example E. coli BL21 (DE3) or E. coli DE5α. The expression vector may be a pET 11a vector or a pET 16b vector. For example, a recombinant beta-agarase prepared by a transformant prepared by introducing a pET 16b DE5α vector having a polynucleotide having the gene sequence of SEQ ID NO: 6 into E. coli may be used to provide a lipoprotein formation signal sequence. Can be released extracellularly.

In addition, the present invention is a living having a lipolytic activity comprising at least one selected from the group consisting of the transformant, the culture of the transformant, its cell-free culture, the concentrate of the culture and the concentrate of the cell-free culture. It relates to a catalyst.

The present invention also provides agar decomposing composition containing at least one selected from the group consisting of the beta-agarase, the recombinant beta-agarase and the biocatalyst.

As described above, the novel beta-agarase of the present invention has excellent heat resistance and has optimum activity at 45 to 55 ° C. higher than 40 ° C. in agar sol state, and can control decomposition activity using metal ions. In addition, it can decompose the agar to produce a neo-agar oligosaccharides containing expensive neotetraose, the main component of which is recognized as physiological activity such as whitening effect, the beta-agarase to the beta-agarase Since a signal sequence capable of dissociating a protein out of a cell is connected, efficient production of a biocatalyst is possible, and thus, a trait using a vector comprising a novel beta-agarase of the present invention and a polynucleotide encoding the agarase The converters and biocatalysts can be used in various fields, such as cosmetics and medical care, where neoagar oligosaccharides are used. Wu will be greater.

Hereinafter, the preparation examples and examples are presented in order to explain the present invention in more detail. However, the following Preparation Examples and Examples are merely illustrated to aid the understanding of the present invention, and may be modified in various other forms, and the scope of the present invention is not limited to the following Examples.

Example  1: Exploration and isolation of agar degrading enzyme-producing microorganisms

In order to clone the new beta-agarase of the present invention, samples were collected from seaweeds of Jeju coast and sampled, and the stock solution was plated in primary selection medium (1.5% agar / seawater) by 100 ul. , Primary culture was performed by incubating at 30 ° C. for 3 days. Strains showing agar degrading activity in the first screening were again plated on a secondary selection medium (Marine agar) and incubated at 30 ° C. for 3 days, and strains having excellent agar degradation were selected.

< Example  2> Genetic Identification of Agar Degrading Microorganisms

Identification of the strain showing agar resolution was performed by genetic identification. Genomic DNA was extracted from the strain showing agar resolution, and amplification of the 16s rRNA nucleotide sequence was performed by PCR using PCR primer pairs as shown in Table 1 below. Analyzed. As a result of the nucleotide sequence analysis, the nucleotide sequence of SEQ ID NO: 3 was analyzed and the total nucleotide sequence was 1398 bp. The nucleotide sequence for the National Center for Biotechnology Information (NCBI) Blast N program and the analysis of the similar sequences using the program DNassist, Pseudoalteromonas citrea CIP exhibits a strain of 105 339 16s rRNA sequence similarity of 99.8% (1396/1398 bp) Pseudomonas genus Alteromonas (Pseudoalteromonas sp) and the strain was named Pseudo-Altermonas AG52.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: 27F AGAGTTTGATCCTGG CTCAG One 1492R GGTTACCTTGTTACGACTT 2

< Example  3> Agar Raise  Active measurement

The following beta-agarase, recombinant protein and agarase activity of the biocatalyst were measured using a 3,5-dinitrosalicyclic acid assay (DNS assay), and a calibration curve was prepared using D-galactose. More specifically, the activity measurement is agarase solution and 1% edible agar mixed with a total volume of 200ul, and then reacted under a specific condition, 1 ml of DNS reagent is added and maintained for 10 minutes at 90 ℃ or more Cooled at room temperature and absorbance at 570 nm was measured, the activity value was calculated by applying the measured absorbance value to the calibration curve. The active unit was 1 unit of 1 μole of galactose produced in 1 minute.

< Example  4> Pseudoalteromonas  genus AG52 of Agar Raise  Gene sequencing

In order to analyze the agarase partial sequencing of the Pseudo-Altermonas genus AG52 strain, six primers of Table 2 were prepared, and PCR was performed using the primers.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: AGAF1 CWTCKTATATWAATGCTTGGC 7 8'-AGAR1 TGGYTGRTAATCTTGAAATGG 8 CyF3 YTNGARTAYTAYATHGAYGG 9 CyR3 TTRTANACNCKDATCCARTC 10 SaF1 TCNATHCAYYTNTAYGAYTTYCC 11 SaR1 CCAYTCNGCYTTNACNGG 12

More specifically, 5 µl of 10X Ex Taq polymerase buffer, 4 µl of 2.5 mM dNTP, 6 primers each of 100 pmole, genomic DNA 500ng, and 3 units of Ex Taq DNA polymerase were mixed in 50 µl of the total reaction solution. After denaturing at 94 ° C. for 5 minutes, it was repeated 30 times for 45 seconds at 94 ° C., 45 seconds at 45 ° C., 45 seconds at 72 ° C., and finally, the reaction was performed at 72 ° C. for 5 minutes.

The PCR product was purified and cloned into a pGEM easy T vector to analyze the nucleotide sequence. As a result, an agarase partial nucleotide sequence of 651 bp was confirmed. Analysis of the entire sequence using the base sequence was analyzed using a long and accurate (LA) PCR kit (Takara, Korea).

More specifically, 5 μg of genomic DNA was digested with restriction enzymes BamH I, EcoR I, Hind III, and Xho I, respectively, and purified by ethanol precipitation, and the respective DNAs were digested with BamH I, EcoR I, Hind III, Ligation of the Xho I cassette was performed. The Ligation reaction was performed by mixing 5 μl of cut DNA, 2.5 μl of cassette, 15 μl of ligation solution I, and 7.5 μl of ligation solution II, proceeding with reaction at 16 ° C. for 30 minutes to purify by ethanol precipitation, and used as a template for LA PCR. In order to amplify the front part of the agarase subsequence, the first PCR was performed using the primers of SEQ ID NO: 13 and the primers of SEQ ID NO: 14 among the primers of Table 3 below, and the primers of SEQ ID NO: 15 to amplify the rear part of the subsequences. And the first PCR was performed using the C1 cassette primer of SEQ ID NO: 14.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: LA52-F1 TCGTCGCTACGGTGTTCATTGGAA 13 C1 GTACATATTGTCGTTAGAACGCGTAATACGACTCA 14 LA52-R1 ACGTGCATACGTTGGTCAAACCAC 15

More specifically, the PCR reaction was performed by 50 μl of total reaction solution, 10 μl LA Taq polymerase buffer 5 μl, 2.5 mM dNTP 8 μl, MgCl 2 5 μl, each primer 10 pmole, template 1 μl, LA Taq DNA polymerase 5 units. After mixing, the reaction conditions were denatured at 94 ° C. for 105 minutes, then repeated 30 times at 94 ° C., 30 seconds at 55 ° C., 4 minutes at 72 ° C., and finally reacted at 72 ° C. for 5 minutes.

Secondary PCR was performed using the primary PCR product as a template, and the primer of SEQ ID NO: 16 in Table 4 below, the C2 cassette primer of SEQ ID NO: 17 for primer amplification, and the primer of SEQ ID NO: 18 for rear amplification and SEQ ID NO: Secondary PCR was performed using 17 C2 cassette primers under the same conditions as the first PCR.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: LA52-F2 TAGTTCGCAGCGTTTCAGGTCCTA 16 C2 CGTTAGAACGCGTAATACGACTCACTATAGGGAGA 17 LA52-R2 TGCCTCCATCGCATCAATTTCCTG 18

Each PCR product was purified by Gel purification kit (Bioneer, Korea) and cloned into pGEM easy T vector, and then sequenced. As a result of sequencing analysis, a 1234 bp nucleotide sequence of SEQ ID NO: 6 including an 870 bp agarase coding sequence of SEQ ID NO: 4 was obtained. The base sequence is shown in FIG. 2.

Said gene sequence is LipoP 1.0 Server (http://www.cbs.dtu.dk/services/LipoP/), ClustalW multiple alignment 1.8 program, SignalP program (http://www.cbs.dtu.dk/services/SignalP /), Nucleotide BLAST and Protein BLAST of the National Center for Biotechnology Information (NCBI). As a result, it showed 96.8% similarity with the nucleotide sequence of beta-agarase genus Eromonas (Accession number: U61972), and 76% similarity to beta-agarase (Accession number: M73783) with Pseudoalkerolomonas Atlantica. It was.

As shown in FIG. 2, the obtained nucleotide sequence of SEQ ID NO: 6 contained a lipoprotein-forming signal sequence unlike the signal sequence at the N-terminus and agarase sequences of other Eromonas genus and Pseudo-Altermonas genus, and GH16 beta Agarase domain (D ²² -K ²⁸⁷ aa), active sites (Y ⁶⁹ , N ⁷¹ , W ⁷² , W ¹³⁹ , S ¹⁴⁵ , D ¹⁵⁰ , E ¹⁵³ , F ¹⁷⁶ , R ¹⁷⁸ , E ²⁵⁷ , E ²⁵⁹ ) and It had calcium binding domains (Q ⁴⁷ , F ⁴⁸ , N ⁴⁹ , G ⁹¹ , A ⁹² , D ⁸² , W ⁸³ ).

< Example  5> Lipo  Protein-forming signal sequence Agar Raise  gene Cloning  And protein expression analysis in E. coli

The coding sequence including the signal sequence was PCR amplified using the primers of the genomic DNA of the genus Pseudoerteromonas genus AG52 as a template.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: 52SC-F GAGAGACATATGATGAATATATTAAAACTACTATCCTGTTCTAC 19 52SC-R GAGAGAGGATCCTTAGTTTGCTTTGTAGACACGT 20

The primers were designed to include Nde I site in 52SC-F and BamH I site in 52SC-R so that the amplified PCR product could be inserted into pET11a, an expression vector. The total amount used for PCR was 50 μl, and 5 μl of 10X Ex Taq polymerase buffer, 4 μl of 2.5 mM dNTP, 100 pmole of each primer, 500 ng of genomic DNA, and 3 units of Ex Taq DNA polymerase were prepared. After denaturation for minutes, 30 seconds at 94 ℃, 30 seconds at 45 ℃, 30 times was repeated for 1 minute at 72 ℃ and finally reacted for 5 minutes at 72 ℃.

The PCR product was purified and digested with Nde I and BamH I, restriction enzymes for cloning into the pET11a vector, respectively. First, BamH I was cut with BamH I 10 units in 5 ㎍ PCR product and 10ETg of pET16b vector, and then purified with Gel purification kit (Bioneer, Korea). The pET11a vector was treated with calf intestine phosphatase to remove 5 'phosphate. Each cleaved PCR product and pET11a vector were ligation with T4 DNA ligase (Takara, Korea) and transformed into E. coli DH5α. The cloned vector was collected using a Plasmid extraction kit (Bioneer, Korea) by culturing in LB broth containing transformed E. coli DH5α ampicillin. The recombinant gene is thus referred to as pET11a; AG52, and a cleavage map of the recombinant vector pET11a; AG52 is shown in FIG.

The pET11a; AG52 was transformed into E. coli BL21 (DE3), and after culturing at 37 ° C. until OD ₆₀₀ = 0.8, IPTG was added to a final concentration of 1 mM and induced expression at 25 ° C. for 24 hours. SDS-PAGE and activity measurements were performed by collecting intracellular proteins after culture supernatant and cell disruption to confirm expression proteins.

As a result of the SDS-PAGE analysis, a large number of rare codons were present in the signal sequence so that the expression level of the protein was low, and thus the protein expression could not be confirmed on the SDS-PAGE. The activity measurement resulted in 1 unit / ml.min activity in the culture supernatant. The protein showed 1.4unit / ml.min activity. From the results, it was confirmed that the recombinant protein can be extracellularly released from E. coli through the lipoprotein formation signal sequence.

< Example  6> no signal sequence Agar Raise  gene Cloning  And In E. coli  Protein expression analysis

The genomic DNA of Pseudoerteromonas genus AG52 strain as a template was PCR amplified a coding sequence containing no signal sequence using the primers of Table 6 below.

Primers Sequence (5 ′ → 3 ′) SEQ ID NO: 52C-F GAGAGACATATGGCAGATTGGGACGCATATAGTA 21 52C-R GAGAGAGGATCCTTAGTTTGCTTTGTAGACACGTATC 22

The primers were designed to include Nde I site in 52C-F and BamH I site in 52C-R to insert the amplified PCR product into pET16b, which is an expression vector. The total amount used for PCR was 50 μl, and 5 μl of 10X Ex Taq polymerase buffer, 4 μl of 2.5 mM dNTP, 100 pmole of each primer, 500 ng of genomic DNA, and 3 units of Ex Taq DNA polymerase were prepared. After denaturation for minutes, 30 seconds at 94 ℃, 30 seconds at 45 ℃, 30 times was repeated for 1 minute at 72 ℃ and finally reacted for 5 minutes at 72 ℃. The PCR product was purified to be a restriction enzyme for cloning into the pET16b vector. Nde I and BamH Each was cut with I. First, PCR products 5㎍ to cut into BamH I and pET16b vector for each 10㎍ BamH After I 10 units were cut and purified, Nde I 10 units were cut and purified using Gel purification kit (Bioneer, Korea). The pET16b vector was treated with calf intestine phosphatase to remove 5 'phosphate. Each of the cut PCR product and the pET16b vector were ligation with a T4 DNA ligase (Takara, Korea) this E. coli Transformed to DH5α. The cloned vector was collected using a Plasmid extraction kit (Bioneer, Korea) by culturing in LB broth containing transformed E. coli DH5α ampicillin. The recombinant gene is thus referred to as pET16b; AG52, and a cleavage map of the recombinant vector pET16b; AG52 is shown in FIG.

The pET11a; AG52 was transformed into E. coli BL21 (DE3), and after culturing at 37 ° C. until OD ₆₀₀ = 0.8, IPTG was added to a final concentration of 0.1 mM and induced expression at 10 ° C. for 24 hours. In order to confirm the expression protein, after cell disruption, intracellular proteins were collected, and protein expression was confirmed by SDS-PAGE. The results and the result of pure purification of his-tag-recombinant protein using nickel column are shown in FIG. 5. It was. As a result of measuring the specific activity of the purified protein shown in FIG. 5, the activity of 105 unit / mg when using edible agar as a substrate and 79.5 unit / mg when using agarose as a substrate.

< Example  7> recombination Agar Raise  Check for optimum conditions and decomposition patterns

In order to confirm the optimum reaction conditions of the agarase of the present invention, the agarase activity was measured by the method of Example 3 while varying the conditions of temperature, pH and metal ion addition.

7-1 Optimal Temperature Condition Measurement

In order to confirm the optimal temperature of the purified recombinant agarase, the difference was set at 5 ° C intervals in the range of 40 to 65 ° C. After incubating the recombinant agarase prepared in Example 6 for 30 minutes, the remaining activity was 1%. Measured by reacting with agar, the results are shown in FIG.

As shown in FIG. 6, the agarase of the present invention showed maximum activity at 55 ° C., and showed 90% and 92% activity at 45 ° C. and 50 ° C., respectively, compared to the maximum activity. From the above results, it was confirmed that the agarase of the present invention maintains excellent activity even at 40 ° C. or higher when the agar is in a sol state, and has a very high industrial value.

7-2 Optimal pH  Measure

To determine the optimal pH of the purified recombinant agarase, adjusted to pH 4.5, pH 5, pH 5.5, pH 6, pH 6.5, pH 7, pH 7.5, pH 8, pH 8.5 and pH 9 in which 1% agar was dissolved. The recombinant agarase prepared in Example 6 was mixed with the solution, and the activity was measured at 45 ° C. for 30 minutes, and the results are shown in FIG. 7.

As shown in FIG. 7, the agarase of the present invention exhibited maximum activity at pH 5.5.

7-3 Metal Ion Effect

In order to confirm the effect of metal ions on the activity of purified recombinant agarase, CaAg ₂ , CuSO ₄ , FeSO ₄ , KCl, MgSO ₄ , MnCl ₂ , NaCl, EDTA and ZnSO ₄ were each 2mM. After each addition to the% agar reaction solution was measured for 30 minutes at 45 ℃, the results are shown in FIG.

As shown in FIG. 8, it was confirmed that the activity of 160% was increased in 2 mM FeSO ₄ and the activity was completely inhibited on CuSO ₄ and ZnSO ₄ . From the above results, it was confirmed that Fe ²⁺ has an effect on increasing activity, and that the degree of reaction can be easily controlled using metal ions. Since the ease of regulation of the reaction in the enzymatic reaction is closely related to the industrialization, the agarase of the present invention was expected to be of great industrial value in this respect.

7-4 Agar Pattern Check

Confirmation of the agar decomposition pattern was performed using thin layer chromatography (TLC). N-butanol: acetic acid: Water (2: 1: 1, v / v) was used as the solvent of the thin layer chromatography, and the reaction was performed by mixing 180ul of 1% edible agar with 20ul of purified agarase at 45 ° C. The reaction was carried out for 30 minutes, 60 minutes and 120 minutes. The reactants were loaded by 2ul on a silica gel 60 TLC plate, and galactose, galactose disaccharide (NA2), tetrasaccharide (NA4), and hexasaccharide (NA6) were used as controls. TLC plate was developed on the above solvent and dried, and 10% sulfuric acid was sprayed and heated to confirm the sugar decomposition pattern. The results are shown in FIG. 9.

As shown in FIG. 9, the agarase was found to form mainly tetrasaccharide (neoagarotetraose) and hexasaccharide (neoagarohexaose) by decomposing edible agar.

Figure 1 is a diagram showing the 16s rRNA sequence of the genus Pseudoerteromonas AG52 strain according to an embodiment of the present invention.

Figure 2 is a diagram showing the agarase sequence of the genus Pseudoerteromonas AG52 strain according to an embodiment of the present invention, the underlined portion means the signal sequence, the area indicated by the thick square box is the active site of the agarase ( An active site, and a square marked area means a calcium binding module.

3 is a schematic diagram recombined into the pET11a vector of the agarase containing a lipoprotein-forming signal sequence according to an embodiment of the present invention.

4 is a schematic diagram recombined into the pET16b vector of agarase that does not include a lipoprotein-forming signal sequence according to an embodiment of the present invention.

5 is a photograph showing the result of the purification of the recombinant agarase produced in E. coli according to an embodiment of the present invention on the SDS-PAGE, wherein Lane M is a protein marker (size marker), the Lane 1 is Intracellular protein before expression induction, Lane 2 is a protein lysed after induction of expression, Lane 3 is a protein not lysed after induction of expression, Lane 4 represents a purified agarase protein do.

Figure 6 is a graph showing the optimum temperature measurement results of the purified recombinant agarase according to an embodiment of the present invention.

7 is a graph showing the results of the optimum pH measurement of the purified recombinant agarase according to an embodiment of the present invention.

8 is a graph showing the effect of metal ions on the activity of purified recombinant agarase according to an embodiment of the present invention.

9 is a photograph showing the results of decomposition of the purified edible agar according to the reaction time by recombinant agarase according to an embodiment of the present invention using thin layer chromatography, wherein G is D-galactose, and NA2 is neoagar. Robeos (disaccharide), NA4 is neoagarotetros (tetrasaccharide), NA6 is neoagarohexose (hexasaccharide), Lane 1 is a 1% edible agar and purified agarase The result of reacting for 30 minutes, Lane 2 is the result of 60 minutes of 1% edible agar and purified agarase, the lane 3 is the result of reacting 1% edible agar and purified agarase for 120 minutes.

<110> Cheju National University Industry-Academic Cooperation Foundation <120> BETA-AGARASE FROM PSEUDOALTEROMONAS SP <130> KP2009044 <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F Primer <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1429R Primer <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 1398 <212> DNA <213> AG52 16S rRNA <400> 3 caagtcgagc ggtaacagaa agtagcttgc tactttgctg acgagcggcg gacgggtgag 60 taatgcttgg gaacatgcct tgaggtgggg gacaacagtt ggaaacgact gctaataccg 120 cataatgtct acggaccaaa gggggcttcg gctctcgcct ttagattggc ccaagtggga 180 ttagctagtt ggtgaggtaa tggctcacca aggcgacgat ccctagctgg tttgagagga 240 tgatcagcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 300 atattgcaca atgggcgcaa gcctgatgca gccatgccgc gtgtgtgaag aaggccttcg 360 ggttgtaaag cactttcagt caggaggaaa ggttaatggt taatacccgt tagctgtgac 420 gttactgaca gaagaagcac cggctaactc cgtgccagca gccgcggtaa tacggagggt 480 gcgagcgtta atcggaatta ctgggcgtaa agcgtacgca ggcggtttgt taagcgagat 540 gtgaaagccc cgggctcaac ctgggaactg catttcgaac tggcaaacta gagtgtgata 600 gagggtggta gaatttcagg tgtagcggtg aaatgcgtag agatctgaag gaataccgat 660 ggcgaaggca gccacctggg tcaacactga cgctcatgta cgaaagcgtg gggagcaaac 720 gggattagat accccggtag tccacgccgt aaacgatgtc tactagaagc tcggagcctc 780 ggttctgttt ttcaaagcta acgcattaag tagaccgcct ggggagtacg gccgcaaggt 840 taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900 tgcaacgcga agaaccttac ctacacttga catacagaga acttaccaga gatggtttgg 960 tgccttcggg aactctgata caggtgctgc atggctgtcg tcagctcgtg ttgtgagatg 1020 ttgggttaag tcccgcaacg agcgcaaccc ctatccttag ttgctagcag gtaatgctga 1080 gaactctaag gagactgccg gtgataaacc ggaggaaggt ggggacgacg tcaagtcatc 1140 atggccctta cgtgtagggc tacacacgtg ctacaatggc gcatacagag tgctgcgaac 1200 ctgcgaaggt aagcgaatca cttaaagtgc gtcgtagtcc ggattggagt ctgcaactcg 1260 actccatgaa gtcggaatcg ctagtaatcg cgtatcagaa tgacgcggtg aatacgttcc 1320 cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgctccaga agtagatagt 1380 ctaaccctcg ggaggacg 1398 <210> 4 <211> 870 <212> DNA <213> Beta-Agarase AG52 <220> <221> CDS (222) (1) .. (870) <400> 4 atg aat ata tta aaa cta cta tcc tgt tct act tgc gca ata ctc tgc 48 Met Asn Ile Leu Lys Leu Leu Ser Cys Ser Thr Cys Ala Ile Leu Cys   1 5 10 15 aca gca aca cat gct gca gat tgg gac gca tat agt att ccg gct tct 96 Thr Ala Thr His Ala Ala Asp Trp Asp Ala Tyr Ser Ile Pro Ala Ser              20 25 30 gct gga tca ggt aaa aca tgg caa tta caa act gtt tcc gac caa ttt 144 Ala Gly Ser Gly Lys Thr Trp Gln Leu Gln Thr Val Ser Asp Gln Phe          35 40 45 aac tac caa gcc ggt act tca aat aaa ccg gca gca ttt acc aat cgt 192 Asn Tyr Gln Ala Gly Thr Ser Asn Lys Pro Ala Ala Phe Thr Asn Arg      50 55 60 tgg aat gct tcg tat att aat gct tgg ctt ggg cct ggt gat act gaa 240 Trp Asn Ala Ser Tyr Ile Asn Ala Trp Leu Gly Pro Gly Asp Thr Glu  65 70 75 80 ttc agt tca ggt cat tcc tac act act ggt ggt gcg tta ggc ctt cag 288 Phe Ser Ser Gly His Ser Tyr Thr Thr Gly Gly Ala Leu Gly Leu Gln                  85 90 95 gca act gaa aaa gca gga aca aat aaa gtg ctt gca gga att gtt tct 336 Ala Thr Glu Lys Ala Gly Thr Asn Lys Val Leu Ala Gly Ile Val Ser             100 105 110 tca aaa gca act ttt aca tac cca ctt tat ctt gag gca atg gta aaa 384 Ser Lys Ala Thr Phe Thr Tyr Pro Leu Tyr Leu Glu Ala Met Val Lys         115 120 125 ccg agt aat aac act atg gct aat ggt gta tgg atg ttg agc tct gat 432 Pro Ser Asn Asn Thr Met Ala Asn Gly Val Trp Met Leu Ser Ser Asp     130 135 140 tca act cag gaa att gat gcg atg gag gca tac ggc agt gat cgt gta 480 Ser Thr Gln Glu Ile Asp Ala Met Glu Ala Tyr Gly Ser Asp Arg Val 145 150 155 160 ggg caa gag tgg ttt gac caa cgt atg cac gta agt cac cat gtt ttt 528 Gly Gln Glu Trp Phe Asp Gln Arg Met His Val Ser His His Val Phe                 165 170 175 ata cgt gag cca ttt caa gat tac caa cca aaa gat gca ggc tct tgg 576 Ile Arg Glu Pro Phe Gln Asp Tyr Gln Pro Lys Asp Ala Gly Ser Trp             180 185 190 gta tac aat aac ggc gaa aca tac cga aat aaa ttt cgt cgc tac ggt 624 Val Tyr Asn Asn Gly Glu Thr Tyr Arg Asn Lys Phe Arg Arg Tyr Gly         195 200 205 gtt cat tgg aag gac gcg tgg aac cta gat tac tat att gat ggt gta 672 Val His Trp Lys Asp Ala Trp Asn Leu Asp Tyr Tyr Ile Asp Gly Val     210 215 220 tta gtt cgc agc gtt tca ggt cct aat ata att gat cct gaa ggc tat 720 Leu Val Arg Ser Val Ser Gly Pro Asn Ile Ile Asp Pro Glu Gly Tyr 225 230 235 240 acc ggt ggc aca ggg cta agt aaa cca atg cac atc ctt tta gat atg 768 Thr Gly Gly Thr Gly Leu Ser Lys Pro Met His Ile Leu Leu Asp Met                 245 250 255 gaa cat caa cct tgg cgt gat gta aaa cca aat tca gcc gag cta gct 816 Glu His Gln Pro Trp Arg Asp Val Lys Pro Asn Ser Ala Glu Leu Ala             260 265 270 gat tca aac aaa agt ata ttt tgg att gac tgg ata cgt gtc tac aaa 864 Asp Ser Asn Lys Ser Ile Phe Trp Ile Asp Trp Ile Arg Val Tyr Lys         275 280 285 gca aac 870 Ala asn     290 <210> 5 <211> 290 <212> PRT <213> Beta-Agarase AG52 <400> 5 Met Asn Ile Leu Lys Leu Leu Ser Cys Ser Thr Cys Ala Ile Leu Cys   1 5 10 15 Thr Ala Thr His Ala Ala Asp Trp Asp Ala Tyr Ser Ile Pro Ala Ser              20 25 30 Ala Gly Ser Gly Lys Thr Trp Gln Leu Gln Thr Val Ser Asp Gln Phe          35 40 45 Asn Tyr Gln Ala Gly Thr Ser Asn Lys Pro Ala Ala Phe Thr Asn Arg      50 55 60 Trp Asn Ala Ser Tyr Ile Asn Ala Trp Leu Gly Pro Gly Asp Thr Glu  65 70 75 80 Phe Ser Ser Gly His Ser Tyr Thr Thr Gly Gly Ala Leu Gly Leu Gln                  85 90 95 Ala Thr Glu Lys Ala Gly Thr Asn Lys Val Leu Ala Gly Ile Val Ser             100 105 110 Ser Lys Ala Thr Phe Thr Tyr Pro Leu Tyr Leu Glu Ala Met Val Lys         115 120 125 Pro Ser Asn Asn Thr Met Ala Asn Gly Val Trp Met Leu Ser Ser Asp     130 135 140 Ser Thr Gln Glu Ile Asp Ala Met Glu Ala Tyr Gly Ser Asp Arg Val 145 150 155 160 Gly Gln Glu Trp Phe Asp Gln Arg Met His Val Ser His His Val Phe                 165 170 175 Ile Arg Glu Pro Phe Gln Asp Tyr Gln Pro Lys Asp Ala Gly Ser Trp             180 185 190 Val Tyr Asn Asn Gly Glu Thr Tyr Arg Asn Lys Phe Arg Arg Tyr Gly         195 200 205 Val His Trp Lys Asp Ala Trp Asn Leu Asp Tyr Tyr Ile Asp Gly Val     210 215 220 Leu Val Arg Ser Val Ser Gly Pro Asn Ile Ile Asp Pro Glu Gly Tyr 225 230 235 240 Thr Gly Gly Thr Gly Leu Ser Lys Pro Met His Ile Leu Leu Asp Met                 245 250 255 Glu His Gln Pro Trp Arg Asp Val Lys Pro Asn Ser Ala Glu Leu Ala             260 265 270 Asp Ser Asn Lys Ser Ile Phe Trp Ile Asp Trp Ile Arg Val Tyr Lys         275 280 285 Ala asn     290 <210> 6 <211> 1234 <212> DNA <213> Beta-Agarase AG52 Lipo <400> 6 ggtattttca taagcttgag tttgaatatg gatacaaata atagaaggta cacacaaaag 60 agattgtttc atctagggcc tgtttatctt tcgatgatta aattcacaaa agtcactcgc 120 actagttaaa gaagcatatc tacattaatt tgcatggaga ttttatatga atatattaaa 180 actactatcc tgttctactt gcgcaatact ctgcacagca acacatgctg cagattggga 240 cgcatatagt attccggctt ctgctggatc aggtaaaaca tggcaattac aaactgtttc 300 cgaccaattt aactaccaag ccggtacttc aaataaaccg gcagcattta ccaatcgttg 360 gaatgcttcg tatattaatg cttggcttgg gcctggtgat actgaattca gttcaggtca 420 ttcctacact actggtggtg cgttaggcct tcaggcaact gaaaaagcag gaacaaataa 480 agtgcttgca ggaattgttt cttcaaaagc aacttttaca tacccacttt atcttgaggc 540 aatggtaaaa ccgagtaata acactatggc taatggtgta tggatgttga gctctgattc 600 aactcaggaa attgatgcga tggaggcata cggcagtgat cgtgtagggc aagagtggtt 660 tgaccaacgt atgcacgtaa gtcaccatgt ttttatacgt gagccatttc aagattacca 720 accaaaagat gcaggctctt gggtatacaa taacggcgaa acataccgaa ataaatttcg 780 tcgctacggt gttcattgga aggacgcgtg gaacctagat tactatattg atggtgtatt 840 agttcgcagc gtttcaggtc ctaatataat tgatcctgaa ggctataccg gtggcacagg 900 gctaagtaaa ccaatgcaca tccttttaga tatggaacat caaccttggc gtgatgtaaa 960 accaaattca gccgagctag ctgattcaaa caaaagtata ttttggattg actggatacg 1020 tgtctacaaa gcaaactaag tcattctaaa atatttgtaa tattaggttt tattgcttct 1080 cgttatacga cacggagcaa taaactttaa ggtccccaaa actacttaat gcggctatta 1140 cagccgcatt aagtataatt aacctgaact ctggatagta aatctatctc gagcagctat 1200 tgacgcgtga attctctccc tatagtgagt cgta 1234 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AGAF1 <400> 7 cwtcktatat waatgcttgg c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 8'-AGAR1 <400> 8 tggytgrtaa tcttgaaatg g 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CyF3 <400> 9 ytngartayt ayathgaygg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CyR3 <400> 10 ttrtanacnc kdatccartc 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SaF1 <400> 11 tcnathcayy tntaygaytt ycc 23 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> SaR1 <400> 12 ccaytcngcy ttnacngg 18 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LA52-F1 <400> 13 tcgtcgctac ggtgttcatt ggaa 24 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> C1 <400> 14 gtacatattg tcgttagaac gcgtaatacg actca 35 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LA52-R1 <400> 15 acgtgcatac gttggtcaaa ccac 24 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LA52-F2 <400> 16 tagttcgcag cgtttcaggt ccta 24 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> C2 <400> 17 cgttagaacg cgtaatacga ctcactatag ggaga 35 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LA52-R2 <400> 18 tgcctccatc gcatcaattt cctg 24 <210> 19 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> 52SC-F <400> 19 gagagacata tgatgaatat attaaaacta ctatcctgtt ctac 44 <210> 20 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 52SC-R <400> 20 gagagaggat ccttagtttg ctttgtagac acgt 34 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 52C-F <400> 21 gagagacata tggcagattg ggacgcatat agta 34 <210> 22 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> 52C-R <400> 22 gagagaggat ccttagtttg ctttgtagac acgtatc 37  

Claims (8)

A beta-agarase having the amino acid sequence set forth in SEQ ID NO: 5. The method of claim 1, The beta- agarase has an amino acid sequence encoded by the polynucleotide described in SEQ ID NO: 4, and is a beta- agarase derived from Pseudoalteromonas sp strain. A polynucleotide encoding beta-agarase having the amino acid sequence set forth in SEQ ID NO: 5. The method of claim 3, The polynucleotide has a nucleotide sequence of SEQ ID NO: 4. A polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 6. A vector comprising the nucleotide sequence of SEQ ID NO: 4 or the nucleotide sequence of SEQ ID NO: 6. A transformant transformed with the vector of claim 6. Claim 1 selected from the group consisting of the transformant of claim 7, its culture, its cell-free culture, the concentrate of the culture, the concentrate of the cell-free culture and recombinant beta-agarase prepared by expression in the transformant of claim 7. Biocatalysts having agar-degrading activity comprising at least one species as an active ingredient.

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