KR20180041377A - A Novel alpha-neoagarobiose hydrolase from Gayadomonas joobiniege G7 and use thereof - Google Patents

Abstract

The present invention relates to a novel alpha-neoagarobiose hydrolase derived from Gayadomonas joobiniege G7 and a use of the same. More specifically, the present invention relates to a novel alpha-neoagarobiose hydrolase which is identified from Gayadomonas joobiniege G7; can be overexpressed in a strain of different species; and decomposes anhydrous galactose at the non-reducing end of neoagarooligosaccharides to produce neoagarooligosaccharides composed of anhydrous galactose and an odd number of sugar molecules in a form in which anhydrous galactose molecules at the non-reducing end are eliminated. In addition, the present invention relates to a method of using the novel alpha-neoagarobiose hydrolase. The novel alpha-neoagarobiose hydrolase of the present invention is extremely effective in decomposing neoagarobiose into galactose and anhydrous galactose; can be mass produced through an overexpression system using host cells of different species; may be extremely beneficially used together with other enzymes in the industrial fields that produce biofuels and drug substances through saccharification from biomass. In addition, the novel alpha-neoagarobiose hydrolase acts on neoagarooligosaccharides formed by beta-agarase and composed of an even number of sugars (neoagarotetraose, neoagarohexaose, etc.) to eliminate only one anhydrous galactose molecule at the non-reducing end, and thus can be used in the production of novel functional neoagarooligosaccharides composed of an odd number of sugar molecules (neoagarotriose, neoagaropentaose, etc.).

Description

A Novel alpha-neoagarobiose hydrolase from Gayadomonas joobiniege G7 and use thereof, which is derived from G7,

The present invention relates to novel alpha-neoagarobiohydrolase derived from G7 to Kayadomonas juvinis and its use, and more specifically, it relates to a novel α-neoagarobiohydrolase which can be overexpressed in Lee, Neoagarobiose hydrolyzate capable of producing neoagarooligosaccharide composed of anhydrous galactose molecules and an odd sugar molecule in which anhydrogalactose at the non-reducing end is removed, by decomposing the anhydrous galactose of the non-reducing end of the And a method of using the same.

The seaweeds have the advantage that they produce much higher fuel yield than the commonly used energy grains, have excellent carbon dioxide absorbing ability that is the main cause of global warming, use only solar and non-drinking water, and grow very rapidly. The major constituent of seaweeds is polysaccharides. Recently, as interest in health has increased, it has been widely used as a material for multifunctional oligosaccharides having biological control functions. Especially, functional materials derived from seaweeds are known to have various physiological activities such as anti-viral, anti-viral, anti-blood coagulation and immunity enhancement. Among seaweeds, red algae are abundant in the sea, but they are composed of 20% of cellulose, 60% of agar, 10% of protein, 10% of other, and it is mainly used for edible and cosmetics , Purified and used as research agar or agarose.

Seaweeds can be harvested 4 ~ 6 times a year, so it is advantageous to secure raw materials and to exist in the sea infinitely so that production and harvesting costs are very cheap. Red algae is a rich source of agricultural and marine resources with a yield of 4,000 tons per year in Korea. However, less than 10% of the total production is processed by simple processing and used as inexpensive raw materials for industrial use or food. Therefore, it is necessary to study the development of new uses and value-added enhancement of Staphylococcus aureus, and it is necessary to develop a technology capable of producing high-value-added functional agar oligosaccharide from red algae, which is a vast agricultural and marine resource, by applying various molecular biological metabolic engineering methods . In order to apply it safely to human body, there is a need for a method of producing neoagarooligosaccharide by a new environmentally friendly bioconversion method which is not harmful such as hydrochloric acid and does not use an environmental pollutant source, and is applied to other raw materials It is necessary to develop a neoagarooligosaccharide product which is excellent in economy and efficacy. This kind of technology development can enhance the utilization rate of natural agricultural and marine resources that are collected in Korea, create high value-added products using agricultural and marine resources, contribute to the public health, and enable the next generation It is considered to be an absolutely necessary technology for green growth.

On the other hand, about 70% of the agar contained in the red algae is agarose, and the agarose contains galactose (D-galactose) and anhydrous galactose (3,6-anhydro-L-galactose) Agarobiose, which is a combined unit, is composed of a linear structure repeatedly connected by α-1,3 bonds. Agarose is hydrolyzed by β-1, 4 bond by beta-agarase to become the minimum unit neoagarobiose and decomposed into galactose and anhydrous galactose by alpha-neoagarose biosyme hydrate (See Fig. 1). In the case of anhydrous galactose constituting the neoagarose bios, the new material has been studied for its new physiological activity and its utility value is very high. And galactose is an expensive compound used as a raw material for sweetener Tagatose, and it can be used for producing bioethanol together with anhydrous galactose.

The present inventors have conducted various studies to develop a method for more effectively utilizing the above-described agarooligosaccharides derived from seaweeds. In particular, the present inventors have conducted various studies to develop a method for using agarooligosaccharides derived from algae such as microorganisms capable of producing useful neoagarooligosaccharides by specifically degrading such agarooligosaccharides And related enzymes. As a result, it is possible to identify novel alpha-neoagarobiose hydrolyzate and its gene from G7 (Gayadomonas jubiniege G7) strain and to produce recombinant enzyme thereof in large quantities to Gayadomasubinyi isolated from the coastal waters of Gaya Island, Korea And a neoagarooligosaccharide composed of an odd number of sugars in which galactose, anhydrous galactose, or non-reducing terminal anhydrogalactose is removed, is obtained by decomposing the neoagarooligosaccharide using the produced enzyme, And the present invention has been completed.

Korean Patent No. 10-1618765 Korean Patent No. 10-1615141

Ha SC, Lee S, Lee J, Kim HT, Ko HJ, Kim KH, Choi IG. Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40. Biochem Biophys Res Commun. 2011 Aug 26; 412 (2): 238-44. Hehemann JH, Smyth L, Yadava, Vocadlo DJ, Boraston AB. Analysis of keystone enzyme in agar hydrolysis provides insight into the degradation (of a polysaccharide from) red seaweeds. J Biol Chem. 2012 Apr 20; 287 (17): 13985-95. Ariga O, Okamoto N, Harimoto N, Nakasaki K. Purification and characterization of α-neoagarooligosaccharide hydrolase from Cellvibrio sp. OA-2007. J Microbiol Biotechnol. 2014 Jan; 24 (1): 48-51. Kwak MJ, Song JY, Kim BK, Chi WJ, Kwon SK, Choi S, Chang YK, Hong SK, Kim JF. Genome sequence of the agar-degrading marine bacterium Alteromonadaceae sp. strain G7. J Bacteriol. 2012 Dec; 194 (24): 6961-2. Chi WJ, Park JS, Kwak MJ, Kim JF, Chang YK, Hong SK. Isolation and characterization of a novel agar-degrading marine bacterium, Gayadomonas joobiniege gene, nov, sp. nov., from the Southern Sea, Korea. J Microbiol Biotechnol. 2013 Nov 28; 23 (11): 1509-18.

Accordingly, a primary object of the present invention is to provide a novel alpha-neoagarobiose hydrate derived from G7 (Gayadomonas jubiniege G7) strain to Gayadomus jubini.

It is another object of the present invention to provide a gene encoding the alpha-neoagarobiose hydrate.

It is still another object of the present invention to provide a recombinant vector for producing the alpha-neoagarobiose hydrate.

It is still another object of the present invention to provide a transformant for producing the alpha-neoagarobiohydrolase.

It is still another object of the present invention to provide a method for mass production of the alpha-neoagarobiose hydrate.

It is still another object of the present invention to provide a method for producing anhydrous galactose or galactose using the alpha-neoagarobiohydrolase.

It is still another object of the present invention to provide a method for producing neoagarooligosaccharide in which anhydrous galactose or non-reducing terminal anhydrous galactose is removed using the alpha-neoagarobiose hydrate.

According to one aspect of the present invention, there is provided an alpha-neoagarobiose hydrolase comprising the amino acid sequence of SEQ ID NO: 1.

According to another aspect of the present invention, there is provided an alpha-neoagarobiose hydrolase gene encoding the amino acid sequence of SEQ ID NO: 1. The alpha-neoagarobiohydrolase gene of the present invention may comprise the nucleotide sequence of SEQ ID NO: 2.

According to another aspect of the present invention, there is provided a recombinant vector for producing alpha-neoagarobiose hydrolase, which contains the alpha-neoagarobiose hydrolase gene, to provide.

According to another aspect of the present invention, there is provided a method for producing alpha-neoagarobiose hydrolase transformed with a recombinant vector for producing alpha-neoagarobiose hydrolase, A transformant for use in the present invention.

According to still another aspect of the present invention, there is provided an alpha-neoagarobiose hydratease characterized by culturing the transformant and overexpressing the alpha-neoagarobiose hydrolase gene, (alpha-neoagarobiose hydrolase) mass production method.

According to still another aspect of the present invention, there is provided a method for producing anhydrous galactose or galactose, which comprises reacting the alpha-neoagarobiose hydrolase with neoagarobiose. to provide. At this time, the enzyme reaction is preferably carried out at pH 6 to 8 and at 30 to 50 ° C.

According to another embodiment of the present invention, the present invention is characterized in that the alpha-neoagarobiose hydrolase is reacted with neoagarooligosaccharide except for neoagarobiose Galactose or a non-reducing terminal anhydrous galactose is removed. Preferably, the enzyme reaction is performed at a pH of 6 to 8 and at a temperature of 30 to 50 ° C, and the neoagarooligosaccharide is preferably neoagarotetraose or neoagarohexaose .

The alpha-neoagarose biosynthetic hydrate of the present invention is very effective in decomposing neoagarobiose into galactose and anhydrous galactose, and can be mass-produced through an over-expression system using a transgenic main stem cell, It can be very useful in combination with other enzymes in the industry producing fuel and pharmaceutical raw materials. In addition, by acting on neoagarooligosaccharide (neoagarotetraose, neoagarohexaose, etc.) formed by beta-agarase and composed of an even number of sugars to remove only one non-reducing terminal galactose anhydrous molecule, Can be used for the production of novel functional neoagarooligosaccharides (neoagarotriose, neoagaropentaose, etc.) composed of sugar molecules.

FIG. 1 is a diagram illustrating a process in which agarose is decomposed into galactose and anhydrogalactose by beta-agarase and alpha-neoagarobiose hydrolysis.
FIG. 2 is a graph showing that an alpha-neoagarobiose hydrate (hereinafter, referred to as 'NABH-1') of the present invention binds neoagarobiose to galactose and anhydrous galactose according to an embodiment of the present invention, A process for decomposing neoagarothelaose and neoagarohexaose into neoagarooligosaccharides composed of anhydrous galactose and an odd sugar molecule free from anhydrogalactose at the non-reducing end.
FIG. 3 is a result of agarose gel electrophoresis of a DNA fragment of NABH-1 gene obtained by PCR using the genomic DNA of a strain to a Gayadomonas jubini according to an embodiment of the present invention.
4 is a result of SDS-PAGE of NABH-1 purified using an NABH-1 gene transformant ( E. coli ER2566 / pET28a-NABH1) according to an embodiment of the present invention.
FIG. 5 is a graph showing the reaction products of NABH-1 and neoagarooligosaccharide purified using NABH-1 gene transformants ( E. coli ER2566 / pET28a-NABH1) according to an embodiment of the present invention by TLC chromatography).
6 is a graph showing the results of mass spectrometry of reaction products of NABH-1 and neoagarobiose purified using an NABH-1 gene transformant ( E. coli ER2566 / pET28a-NABH1) according to an embodiment of the present invention. The results are shown in Fig.
FIG. 7 is a graph showing the reaction products of NABH-1 and neoagarotetraose purified using NABH-1 gene transformants ( E. coli ER2566 / pET28a-NABH1) according to an embodiment of the present invention by mass spectrometry ), Respectively.
FIG. 8 is a graph showing the reaction products of NABH-1 and neoagarohexaose purified using an NABH-1 gene transformant ( E. coli ER2566 / pET28a-NABH1) according to an embodiment of the present invention by mass spectrometry ), Respectively.
FIG. 9 shows the results of analysis of the pH of the reaction of the present invention NABH-1.
10 shows the results of analysis of the reaction temperature of the present invention NABH-1.

NABH-1 of the present invention is an enzyme produced by G7 ( Gayadomonas joobiniege G7) to Gayadomonasubinyi , which consists of 359 amino acid sequences of SEQ ID NO: 1. In the microorganism-derived agarase, there are many cases in which the signal peptide is cleaved to be a mature form protein when expressed as a precursor protein containing the signal peptide and then secreted out of the cell by the following signal peptide program. The NABH -1 did not possess such a signal peptide.

Gaya is a strain isolated from the coastal waters of Gaya Island in Korea. KCTC23721, American Type Culture Collection (ATCC), KCTC23721, (ATCC BAA-2321) and DSM25250 (Deutsche Sammlung von Microorganismund Zellkulturen Gmb H, DSM).

The NABH-1 gene of the present invention encodes the amino acid sequence of SEQ ID NO: 1, and the original nucleotide sequence identified from the G7 strain by Gayadomonas jubini is the nucleotide sequence of SEQ ID NO: 2, but the type of host for expression It can be applied to a modified codon sequence.

The recombinant vector for producing NABH-1 of the present invention comprises the NABH-1 gene as described above. At this time, the vector may be selected from various kinds of known vectors in consideration of the type of host cell, promoter, selection marker, and the like. For example, when Escherichia coli is used as a host, vectors of the pET series can be used.

The transformant for producing NABH-1 of the present invention can be produced by introducing such a recombinant vector into a host organism. At this time, it is considered that various hosts can be used as a host, and it is more preferable to use microorganisms for stable expression or production efficiency of NABH-1. According to the present invention, it was confirmed that the NABH-1 gene of the present invention can be expressed very smoothly also in the expression system of E. coli. Therefore, when transformants are prepared using E. coli as a host, NABH-1 can be produced very easily. Transformation of the host organism can be accomplished by introducing the recombinant vector into the host organism by applying conventional transformation methods used in each host organism.

The mass production of NABH-1 of the present invention can be achieved by culturing the transformant and overexpressing the NABH-1 gene. At this time, conditions such as the type of culture medium, culture temperature, and incubation time can be selectively applied depending on the host and vector. For example, when the host is Escherichia coli and the vector of pET28 series is used, a method of culturing in LB (Luria Bertani) medium at about 15 to 40 DEG C for 12 hours to 5 days can be used.

The NABH-1 of the present invention has an activity of cleaving the α1-3 linkage of neoagarobiose to form 3,6-anhydro-α-L-galactose and galactose (β-D-galactose) (See FIG. 1). Therefore, anhydrous galactose or galactose can be produced by reacting NABH-1 of the present invention with neoagarobiozyme.

The NABH-1 of the present invention can perform an enzymatic reaction using a neoagarooligosaccharide as a substrate as well as neoagarobiose and a larger molecular weight. NABH-1 of the present invention specifically shows an activity of cleaving only the? 1-3 bond between the anhydrous galactose and the galactose molecule at the non-reducing end of the neoagarooligosaccharide. Therefore, when a neoagarooligosaccharide having a molecular weight larger than that of neoagarobiose is used as a substrate to react with NABH-1 of the present invention, neoagarooligosaccharide having anhydrous galactose and a non-reducing terminal anhydrogalactose can be produced. Using this, neoagarotetraose or neoagarohexaose can be used as a substrate to produce an oligosaccharide having a specific structure consisting of an odd number of sugar molecules and having both ends of the molecule composed of galactose molecules 2).

The NABH-1 of the present invention can exhibit enzyme activity over a pH of 4 to 11 and over 20 to 80 캜. Therefore, the enzyme reaction can be performed in the pH range and the temperature range. However, in order to increase the enzyme activity and increase the production efficiency of the reaction product, the enzyme reaction is preferably performed at pH 6 to 8 and 30 to 50 ° C , More preferably pH 6.5 to 7.5, and 35 to 45 캜.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1. Production of NABH-1 Encoding Gene Expression Vector and Transformants

Genomic DNA of G7 ( Gayadomonas joobiniege G7) (KCTC23721) was extracted from Ganoderma lucidum beanii and forward primer (5'- ATCACT CATATG TCTGAAAAAAAAATTAAGT-3 ', underlined portion of NdeI recognition site) (SEQ ID NO: 3) 5'-CTCCACA GGATCC TTTATTTTTAGTTGGAG-3 ' , underlined part is DNA containing the encrypted region (1,080bp) of NABH-1 which is by PCR using the BamHI recognition site) (SEQ ID NO: 4) composed of 359 amino acid fragment (SEQ ID NO: 5) was amplified (see Fig. 3). The amplified DNA fragment was double-digested with NdeI-BamHI restriction enzyme and ligated to the NdeI-BamHI restriction enzyme site of pET28a (+) to introduce pET28a-NABH1 ( NABH-1 encoding gene expression vector). E. coli DH5α was used for transformation and pET28a (+) produced an antibiotic substance, kanamycin resistant material. For the selection of transformed strains, pET28a-NABH1 was cultured in a final concentration of 50 μg / ml kanamycin Was added to the solid and liquid medium.

The NABH1 to transform E. coli ER2566 manufactured by pET28a-NABH1 was prepared a transformant (E. coli ER2566 / pET28a-NABH1 ) to produce.

Example 2. Production and purification of NABH-1

The transformant ( E. coli ER2566 / pET28a-NABH1) of Example 1 was cultured at 37 DEG C in 50 mL of LB medium supplemented with kanamycin at a final concentration of 50 mu g / mL until the OD600 value reached 0.6, wasopropyl β-d-1-thiogalactopyranoside (IPTG) was added at a final concentration of 1 mM to induce protein expression and further cultured at 28 ° C for 12 hours. The cells were recovered by centrifugation at 5,000 rpm for 10 minutes, suspended in 5 ml of PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ ), sonicated, disrupted and centrifuged at 6,000 rpm After centrifugation for 30 minutes, the supernatant was taken and used for purification.

For protein purification, the column was filled with 1.5 ml Ni ²⁺ -NTA resin (ClonTech, Japan), washed with 5 ml PBS buffer, and the supernatant was loaded. The column was washed with 10 ml of PBS buffer and 5 ml of 1 ml PBS buffer containing 200 mM imidazole was used in a total of 5 ml to elute the protein bound to the resin.

The recovered protein was confirmed to be a protein having a molecular weight of NABH-1 having a molecular weight of 40.8 kDa consisting of 359 amino acids using SDS-PAGE (see FIG. 4).

Example 3. Analysis of substrate decomposition products of NABH-1

Each of the neo-agarooligosaccharides (Neoagarobiose, neoagarotetraose, neoagarohexaose) was reacted with the NABH-1 obtained in Example 2, and the degradation products were analyzed.

Substrate degradation products were analyzed by TLC and MS (mass spectrometry).

For the analysis of substrate degradation products by TLC, 0.2 μl of each of neoagaroligosaccharide (neoagarobiose, neoagarotetraose, neoagarohexaose) and NABH-1 (6)) was prepared. The reaction was carried out at 40 ° C for 12 hours, and the decomposition products were analyzed by thin-layer chromatography (TLC). The reaction mixture (10 μl) was loaded on a Silica Gel 60 plate (EMD Merck AG, Darmstadt, Germany) and double-isolated on a solvent system of n-butanol, ethanol and water (2: 1: 1, v / v / v) Respectively. The separated product was visualized by spraying an ethanol solution containing 30% (w / v) sulfuric acid and the plate was heated to 120 ° C.

After the reaction, it was confirmed by TLC analysis that the reaction solutions containing the respective neoagarooligosaccharides (Neoagarobio, neoagarotetraose, neoagarohexaose) showed two types of degradation products (see FIG. 5 ).

For analysis of substrate degradation products using MS (mass spectrometry), 0.2 μl of a 0.2% neoagaroligosaccharide substrate (neoagarobiose, neoagarotetraose, neoagarohexaose) was added to 300 μl of PBS buffer (pH 7.4) And NABH-1 (36)) were prepared. After the reaction was carried out at 40 DEG C for 12 hours, the decomposed neoagarooligosaccharide was precipitated by adding 30 times methanol, and the supernatant was recovered by centrifugation at 14,000 rpm for 10 minutes. The supernatant was vacuum dried and analyzed by MS (mass spectrometry).

As a result, a peak was observed at the same position as the molecular weight of anhydrous galactose [(M + Na) ⁺ = 185m / z] and galactose [(M + Na) ⁺ = 203m / z] (See FIG. 6). Peaks were observed at the same molecular weight as the anhydrogalactose [(M + Na) ⁺ = 185m / z] and neoagarotriose [(M + Na) ⁺ = 509m / z] from the degradation products using neoagarotetraose (See FIG. 7). From the degradation products using neoagarohexaose, peaks at positions such as anhydrous galactose [(M + Na) ⁺ = 185m / z] and neoagaropentaose [(M + Na) ⁺ = 815m / (See FIG. 8).

From the MS analysis results, two kinds of degradation products from each of the neoagarooligosaccharides (Neoagarobiose, neoagarotetraose, neoagarohexaose) identified in the TLC analysis were obtained from the non-reducing terminal anhydrous galactose And its residues, galactose, neoagarotriose and neoagaropentaose, respectively.

Example 4. Activity assay of NABH-1

The enzyme biosynthesis of NABH-1 was determined by measuring the optimum pH and enzyme reaction temperature.

Enzyme Reaction of NABH-1 A buffer solution buffered at each pH was used for the appropriate pH measurement. For measurements at pH 4 ~ 6, 50 mM Sodium Citrate buffer was used. For measurement at pH 7 ~ 8, 50 mM Sodium Phosphate buffer was used. For measurements at pH 9-11, 50 mM Glycine-NaOH buffer was used. 500 占 퐇 of the reaction solution at each pH was composed so as to contain 100 占 퐂 of neoagarobio and 14 占 퐂 of NABH-1. The reaction was carried out at 40 ° C for 10 minutes, mixed with 500 μl of the DNS solution, boiled in a water bath for 10 minutes, and then cooled in ice water for 5 minutes. The absorbance of the reaction solution was measured by using a spectrophotometer. The enzyme activity was measured at 540 nm. From the measurement results, it was confirmed that the optimum pH of the enzyme reaction of NABH-1 was pH 7 (see FIG. 9).

Enzyme Reaction of NABH-1 For the appropriate temperature, a reaction solution containing 100 μg of neoagarobiose and 14 μg of NABH-1 was prepared in 500 μl of 50 mM sodium phosphate buffer (pH 7.0). The reaction was carried out at a temperature of 20 ° C to 80 ° C for 10 minutes, mixed with 500 μl of the DNS solution, boiled in a water bath for 10 minutes, and then cooled in ice water for 5 minutes. The absorbance of the reaction solution was measured by using a spectrophotometer. The enzyme activity was measured at 540 nm. From the measurement results, it was confirmed that the optimal temperature for enzyme reaction of NABH-1 was 40 ° C (see FIG. 10).

<110> Myongji University Industry and Academia Cooperation Foundation <120> A Novel alpha-neoagarobiose hydrolase from Gayadomonas joobiniege          G7 and use thereof <130> PA-D16462 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 359 <212> PRT <213> Unknown <220> <223> Gayadomonas jubiniege G7 <400> 1 Met Ser Glu Lys Lys Leu Ser Leu Ala Ser Lys Arg Ala Ile Glu Arg   1 5 10 15 Gly Tyr Asp Asn Lys Gly Pro Glu Trp Leu Ile Glu Phe Glu Glu Glu              20 25 30 Pro Leu Gln Gly Asp Phe Ala Tyr Glu Glu Gly Val Ile Arg Arg Asp          35 40 45 Pro Thr Ala Val Ile Gln Val Asp Gly Lys Tyr His Val Trp Tyr Thr      50 55 60 Lys Gly Thr Gly Glu Thr Val Gly Phe Gly Ser Thr Asn Pro Cys Asp  65 70 75 80 Lys Val Phe Pro Trp Asp Leu Thr Glu Val Trp His Ala Thr Ser Asp                  85 90 95 Asp Gly Leu Val Trp His Glu Glu Gly Cys Ala Ile Ala Arg Gly Glu             100 105 110 Ser Gly Arg Tyr Asp Asp Arg Ala Val Phe Thr Pro Glu Val Leu Ala         115 120 125 His Asp Gly Arg Tyr Tyr Leu Val Tyr Gln Thr Val Gln Tyr Pro Tyr     130 135 140 Thr Asn Arg Gln Tyr Glu Glu Ile Ala Ile Ala His Ala Asp Ser Pro 145 150 155 160 Tyr Gly Pro Trp Leu Lys Ser Glu Gln Pro Ile Leu Ser Pro Ser Lys                 165 170 175 Asp Gly Glu Trp Asp Gly Asp Glu Asp Asn Arg Phe Lys Val Lys Ser             180 185 190 Lys Gly Ser Phe Asp Ser His Lys Val His Asp Pro Cys Leu Met Phe         195 200 205 Phe Asn Gly Gln Phe Tyr Leu Tyr Tyr Lys Gly Glu Thr Met Gly Glu     210 215 220 Gly Met Asn Phe Gly Gly Arg Glu Ile Lys His Gly Val Ala Ile Ala 225 230 235 240 Asp Asp Ile Leu Gly Pro Tyr Thr Lys Ser Glu Tyr Asn Pro Ile Ser                 245 250 255 Asn Ser Gly His Glu Val Ala Val Trp His Tyr Asn Gly Gly Ile Ala             260 265 270 Ser Leu Ile Thr Thr Asp Gly Pro Glu Lys Asn Thr Ile Gln Trp Ala         275 280 285 Lys Asp Gly Ile Asn Phe Glu Ile Met Ser His Ile Lys Gly Ala Pro     290 295 300 Glu Ala Leu Gly Ile Phe Arg Asp Pro Gln Asp Arg Glu Ile Glu Ala 305 310 315 320 Pro Gly Leu Tyr Trp Gly Leu Cys His Lys Tyr Asp Asp Ser Trp Asn                 325 330 335 Trp Asn Tyr Ile Cys Arg Tyr Arg Val Lys Arg Gln Ile Leu Asp Ala             340 345 350 Gly Thr Phe Gln Asn Ser Asn         355 <210> 2 <211> 1080 <212> DNA <213> Unknown <220> <223> Gayadomonas jubiniege G7 <400> 2 atgtctgaaa aaaaattaag tcttgcaagt aaacgcgcca tcgaacgtgg ttatgataac 60 aaaggtcccg aatggttgat cgaatttgaa gaagaaccgt tacaaggtga ttttgcttac 120 gaagaaggtg ttatcagacg agatccgact gctgtcatac aggttgatgg aaaatatcat 180 gtttggtata caaaggggac aggcgaaact gttggttttg gttctaccaa tccttgtgat 240 aaagtattcc cgtgggattt gactgaagtt tggcatgcga cctcagatga tggtttagta 300 tggcatgaag aaggttgtgc aattgcccgt ggtgaaagtg gtcgttacga tgatagggct 360 gtttttacgc cagaggttct agctcacgat gggcgatatt atctggtcta tcagacagtt 420 cagtatccgt atactaaccg ccaatatgaa gaaatagcga tagcacatgc ggatagccct 480 tatggtccat ggttaaaatc tgaacagcct attttgagcc catcaaaaga tggtgaatgg 540 gacggtgatg aagataatcg ttttaaagtt aaatcaaaag gtagctttga tagtcacaaa 600 gttcacgacc cttgtttaat gttttttaac ggccagtttt atttatatta taaaggcgaa 660 actatgggcg agggtatgaa ttttggtggt cgagaaatta aacatggtgt tgccatcgca 720 gacgatattt taggtccgta tactaagtct gaatacaacc caattagtaa ttcgggccat 780 gaagttgcag tttggcacta taatggtggt atagcgtcat tgattacgac ggatgggcca 840 gaaaaaaata ccattcaatg ggcaaaagac ggtattaact ttgaaataat gtcgcatatt 900 aaaggtgcac cagaagcttt aggtatattc agagacccac aagatcgcga gatagaggca 960 cctggtttat actggggctt atgtcacaaa tatgatgatt cttggaactg gaattatatt 1020 tgccgatatc gggtaaagcg tcagatatta gatgcaggga catttcagaa ctccaactaa 1080                                                                         1080 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for NABH <400> 3 atcactcata tgtctgaaaa aaaattaagt 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for NABH <400> 4 ctccacagga tcctttattt ttagttggag 30 <210> 5 <211> 1109 <212> DNA <213> Artificial Sequence <220> <223> PCR product for cloning of NABH gene <400> 5 atcactcata tgtctgaaaa aaaattaagt cttgcaagta aacgcgccat cgaacgtggt 60 tatgataaca aaggtcccga atggttgatc gaatttgaag aagaaccgtt acaaggtgat 120 tttgcttacg aagaaggtgt tatcagacga gatccgactg ctgtcataca ggttgatgga 180 aaatatcatg tttggtatac aaaggggaca ggcgaaactg ttggttttgg ttctaccaat 240 ccttgtgata aagtattccc gtgggatttg actgaagttt ggcatgcgac ctcagatgat 300 ggtttagtat ggcatgaaga aggttgtgca attgcccgtg gtgaaagtgg tcgttacgat 360 gatagggctg tttttacgcc agaggttcta gctcacgatg ggcgatatta tctggtctat 420 cagacagttc agtatccgta tactaaccgc caatatgaag aaatagcgat agcacatgcg 480 gatagccctt atggtccatg gttaaaatct gaacagccta ttttgagccc atcaaaagat 540 ggtgaatggg acggtgatga agataatcgt tttaaagtta aatcaaaagg tagctttgat 600 agtcacaaag ttcacgaccc ttgtttaatg ttttttaacg gccagtttta tttatattat 660 aaaggcgaaa ctatgggcga gggtatgaat tttggtggtc gagaaattaa acatggtgtt 720 gccatcgcag acgatatttt aggtccgtat actaagtctg aatacaaccc aattagtaat 780 tcgggccatg aagttgcagt ttggcactat aatggtggta tagcgtcatt gattacgacg 840 gatgggccag aaaaaaatac cattcaatgg gcaaaagacg gtattaactt tgaaataatg 900 tcgcatatta aaggtgcacc agaagcttta ggtatattca gagacccaca agatcgcgag 960 atagaggcac ctggtttata ctggggctta tgtcacaaat atgatgattc ttggaactgg 1020 aattatattt gccgatatcg ggtaaagcgt cagatattag atgcagggac atttcagaac 1080 tccaactaaa aataaaggat cctgtggag 1109

Claims (11)

Alpha-neoagarobiose hydrolase consisting of the amino acid sequence of SEQ ID NO: 1. Alpha-neoagarobiose hydrolase gene encoding the amino acid sequence of SEQ ID NO: 1. 3. The method of claim 2,
An alpha-neoagarobiose hydrolase gene comprising the nucleotide sequence of SEQ ID NO: 2. A recombinant vector for producing alpha-neoagarobiose hydrolase containing the alpha-neoagarobiose hydrolase gene according to claim 2 or 3. A transformant for the production of alpha-neoagarobiose hydrolase transformed with the recombinant vector for producing alpha-neoagarobiose hydrolase according to claim 4. A method for mass production of alpha-neoagarobiose hydrolase characterized by culturing the transformant of claim 5 and overexpressing the alpha-neoagarobiose hydrolase gene. A method for producing anhydrous galactose or galactose, which comprises reacting the alpha-neoagarobiose hydrolase of claim 1 with neoagarobiose. 8. The method of claim 7,
Wherein the enzyme reaction is carried out at a pH of 6 to 8 and at a temperature of 30 to 50 占 폚. An anhydrous galactose or non-reducing terminal anhydrous polysaccharide characterized by reacting the alpha-neoagarobiose hydrolase of claim 1 with a neoagarooligosaccharide other than neoagarobiose. Production method of neoagarooligosaccharide from which galactose has been removed. 10. The method of claim 9,
Wherein the enzyme reaction is carried out at pH 6 to 8 and at 30 to 50 ° C. 10. The method of claim 9,
Wherein the neoagarooligosaccharide is neoagarotetraose or neoagarohexaose, wherein the anhydrogalactose or the non-reducing terminal anhydrogalactose is removed, wherein the neoagarooligosaccharide is neoagarotetraose or neoagarohexaose.

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