KR20240063092A - GH50A β-AGARASE DERIVED FROM A NOVEL AGAR-DEGRADING BACTERIUM AND PRODUCING METHOD OF NEOAGAROBIOSE USING THE SAME - Google Patents

Abstract

The present invention relates to the glycoside hydrolase GH50A β-agarase derived from the novel agar-degrading bacterium Cellvibrio sp. KY-GH-1 deposited under the deposit number KCTC 13629BP, and It relates to a method for producing neoagarobiose (NA2) using the enzyme. The glycoside hydrolase GH50A β-agarase derived from the novel agar-degrading bacterium Cellvibrio species KY-GH-1 of the present invention uses poorly degradable agar, agarose, agarooligosaccharide, and neoagaroligosaccharide as substrates. Therefore, neo-agarobiose can be produced with high efficiency without being greatly limited in temperature and pH ranges, so it has excellent effects that can be widely used in the fermentation industry, food industry, and pharmaceutical industry.

Description

Novel agar-decomposing bacteria-derived Ｇ５０Ａ β-agarase and method for producing neoagarobiose using the same {GH50A β-AGARASE DERIVED FROM A NOVEL AGAR-DEGRADING BACTERIUM AND PRODUCING METHOD OF NEOAGAROBIOSE USING THE SAME}

The present invention relates to the glycoside hydrolase GH50A β-agarase derived from the novel agar-degrading bacterium Cellvibrio sp. KY-GH-1 deposited under the deposit number KCTC 13629BP, and It relates to a method for producing neoagarobiose (NA2) using the enzyme.

Agar, a polysaccharide well known for the cell walls of marine red algae, is a mixture of two different ingredients. It consists of neutral agarose and negatively charged agaropectin. Agarose is composed of alternating residues of 3,6-anhydro-L-galactose (L-AHG) linked at α-1,3 and D-galactose linked at β-1,4 [1]. Agaropectin has the same structure, but the L-AHG residue is partially replaced by L-galactose sulfate and the D-galactose residue is partially replaced by pyruvic acid acetal 4,6-O-(1-carboxyethylidene)-D-galactose. Since the gel matrix of agar is generally resistant to microbial decomposition, it is widely used as a gelling material in the food industry and microbial culture media. At least 15 genera of marine bacteria and 7 genera of non-marine bacteria have been reported to produce agarase, which decomposes agar into the monomers D-galactose and L-AHG [2-6].

Agarases are divided into two types depending on the method of cutting the substrate. α-Agarase (EC 3.2.1.158) cleaves α-1,3-glycosidic linkage, and β-agarase (EC 3.2.1.81) cleaves β-1,4-glycosidic linkage. -Cleaves glycosidic linkage [2,3]. Most agarases reported to date are endo-type β-agarases that produce neoagarooligosaccharides (NAOS) by liquefying agar, and α-agarases that produce agarooligosaccharides (AOS). There are not many research reports. The Carbohydrate-Active Enzymes (CAZyme) database compares and analyzes amino acid sequence similarities between bacterial agarases and classifies them into several glycoside hydrolase (GH) families, namely β -Agarase includes the GH16, GH50, GH86, and GH118 families [7-9], and α-agarase includes the GH96 and GH117 families [10,11]. GH16, GH86 and GH118 β-agarases are made from agarose: neoagarosetetrose (NA4)/neoagarosehexose (NA6), neoagarosehexose (NA6)/neeoagaroseoctaose (NA8) and NA8/Neoagarodechaos (NA10). GH50 β-agarase produces NA2, NA4, or NA2/NA4 as the final product through exo- and endo-type agarose decomposition activity [2,12-15]. GH96 α-agarase mainly produces agarotetrose (A4) from agarose. GH117 α-neoagarobiose hydrolase (α-NABH) hydrolyzes NA2 to produce D-galactose and L-AHG [10,11].

Recently, NAOS and AOS, which are agar degradation products, have various biological activities such as antioxidant [16], antitumor [17,18], prebiotic [19], anti-inflammatory [20], anti-diabetic and anti-obesity activities. It was reported to have [21]. NA2 and L-AHG have been reported to have skin moisturizing, whitening, anti-inflammatory, and anti-caries effects [22-24]. Although the biological activity of these agar degradation products is presumed to be related to the presence of L-AHG, in vivo studies providing direct evidence for the biological function of L-AHG remain largely unconducted. -This is due to the difficulty of mass production for commercialization of AHG. Recently, agarose was treated with GH50 β-agarase and GH117 α-NABH to decompose into NA2, and then NA2 was decomposed into D-galactose and L-AHG to produce L-AHG from agarose. An enzymatic process has been proposed [25,26]. However, for a successful enzymatic process, the development of GH50 family β-agarase that can efficiently produce NA2 from agarose is urgently needed.

The present invention was developed to solve the problems of the prior art as described above. The problem to be solved by the present invention is to develop an efficient exo-type β-agarase required for NA2 production from agar, etc., and to use this to produce NA2 with high efficiency. The goal is to provide a method of producing .

In order to solve the above problems, the present invention provides glycoside hydrolase GH50 A β-agarase comprising SEQ ID NO: 1.

The glycoside hydrolase GH50A β-agarase is preferably derived from Cellvibrio species KY-GH-1 strain, accession number KCTC 13629BP.

The glycoside hydrolase GG50 A β-agarase is preferably obtained from an E. coli transformant into which the glycoside hydrolase GG50 A β-agarase gene has been introduced.

It is preferable that the glycoside hydrolase GG50A β-agarase gene is cloned into an expression vector and introduced into E. coli.

In addition, the present invention provides a method for producing neoagarobiose by treating the substrate with the glycoside hydrolase GH50 A β-agarase described in the present invention for enzymatic decomposition.

The substrate is preferably one or more selected from the group consisting of agar, agarose, agaro-oligosacchrides (AOS), and neoagaro-oligosacchrides (NAOS).

The treatment is preferably performed at a temperature range of 20 to 50°C.

The treatment is preferably performed in a pH range of pH 6.0 to 10.0.

The above treatment is preferably performed by further adding Mn ²⁺ .

The Mn ²⁺ is preferably in the form of MnCl ₂ , MnSO ₄ or a mixture thereof.

The treatment is preferably performed by further adding Tris(2-carboxyethyl)-phosphine (TCEP).

The glycoside hydrolase GH50A β-agarase derived from the novel agar-degrading bacterium Cellvibrio species KY-GH-1 of the present invention uses poorly degradable agar, agarose, agarooligosaccharide, and neoagaroligosaccharide as substrates. Therefore, neo-agarobiose can be produced with high efficiency without being greatly limited in temperature and pH ranges, so it has excellent effects that can be widely used in the fermentation industry, food industry, and pharmaceutical industry.

Figure 1: Experiment of detection of extracellular agarase activity on agarose plates using SDS-polyacrylamide gel electrophoresis and Lugol's iodine solution of recombinant His-tagged GH50A, GH50B and GH50C β-agarases expressed in E. coli. It is a result. (A) Individual E. coli BL21 (DE3) transformants were cultured in the presence of 0.075mM IPTG, and then cells were harvested and fractionated into total, soluble, and insoluble cell fractions. Equivalent amounts of each fraction were electrophoresed on an SDS-polyacrylamide gel. (B) Transformants expressing recombinant His-tagged GH50A, GH50B, or GH50C β-agarase were spotted on LB agarose plates containing kanamycin and IPTG and cultured at 30°C for 2 days. Culture plates were then stained with Lugol's iodine solution at RT. Representative results are shown in Figure 1, and two additional experiments showed similar results.
Figure 2a shows the results of multiple amino acid sequence alignment of nine GH50 family β-agarases and GH50A β-agarase available in the GeneBank database. Amino acids were expressed as single-letter abbreviations after aligning the open reading frame for maximum identity using Clustal X [29]. Identical residues in all aligned sequences are marked with an asterisk at the top of the alignment. Conservative substitutions are indicated with colons and semi-conservative substitutions are indicated with dots. Additionally, pseudo residues were highlighted using the Clustal X color scheme, and deletions were indicated with dashes.
Figure 2b shows a phylogenetic relationship diagram based on amino acid sequence similarity. An unrooted phylogenetic tree was constructed using UPGMA31 [30]. The number of nodes is the bootstrap occurrence level.
Figure 3 shows SDS-PAGE of purified recombinant his-tagged GH50A β-agarase. electrophoresis lane; SM, size marker; Crude GH50A, soluble fraction obtained from an E. coli transformant expressing GH50A β-agarase; purified GH50A, recombinant GH50A purified from the soluble fraction.
Figure 4 shows the biochemical properties of GH50A β-agarase. (A, B) The effects of temperature and pH on enzyme activity were measured using 0.4% agarose and 20 μg/mL purified GH50A β-agarase. (C, D) Temperature and pH stability of GH50A β-agarase was investigated after 4 hours of treatment at individual pH or temperature. (E) Lineweaver-Burk plot to obtain Km, Vmax, Kcat, and Kcat/Km values of GH50A β-agarase was investigated using the indicated concentrations of enzyme and agarose substrate. Each value is expressed as mean ± SD (n = 3, measured in 3 replicates for each individual experiment). Representative results are shown in Figure 4, and similar results were obtained in two additional experiments.
Figure 5 shows the effect of metal ions, reducing agents, and chelating agents on GH50A β-agarase activity. (A) Individual effects of metal ions, SH reducing agent TCEP and chelator EDTA at 5mM concentration on β-agarse activity and (B) joint effect of MnSO ₄ (5mM) and TCEP (5mM or 10mM) on enzymatic activity of GH50A. To measure the treatment effect, purified enzyme (20 μg/mL) was mixed with 20mM Tris-HCl buffer (pH 7.5) and 0.4% agarose and treated at 35°C for 30 minutes. At this time, the enzyme activity measured without treatment with various ions and reagents was defined as 100%. Each value was expressed as mean ± SD (n = 3, measured in 3 replicates for each individual experiment). *P < 0.05 and **P < 0.01.
Figure 6 shows the results of TLC analysis of hydrolysis products obtained from time-dependent treatment of GH50A β-agarose on agarose, NAOS, and AOS substrates. For hydrolysis of each substrate, 0.4% agarose (A), 1.0% NAOS (B), or 1.0% AOS (C) was incubated with 10 μg/mL of the indicated enzymes in 20 mM Tris-HCl (pH 7.5) at 35 °C. processed over time. An equivalent amount of each reaction mixture was spotted on a Silica Gel 60 aluminum plate and developed with n-butanol-ethanol-H ₂ O [3:2:2 (v/v)] solvent. Sugar substances on TLC were detected by spraying an ethanol solution containing 0.2% (w/v) naphthoresorcinol and 10% (v/v) H ₂ SO ₄ and then heating at 80°C. Representative results are shown in Figure 6, and similar results were obtained in two additional experiments.
Figure 7 shows the results of LC-MS/MS analysis of hydrolysis products obtained from time-dependent treatment of GH50A β-agarase for NAOS. For hydrolysis of NAOS, 1% NAOS was treated with 10 μg/mL of enzyme in 20 mM Tris-HCl (pH 7.5) at 35°C for separate times. LC-MS/MS analysis was performed as described in Materials and Methods.
Figure 8 shows the results of LC-MS/MS analysis of hydrolysis products obtained from time-dependent treatment of GH50A β-agarase for AOS. For hydrolysis of AOS, 1% AOS was treated with 10 μg/mL of enzyme in 20 mM Tris-HCl (pH 7.5) at 35°C for separate times. LC-MS/MS analysis was performed as described in Materials and Methods.
Figure 9 shows the hydrolysis pattern of agarose, NAOS or AOS by GH50A β-agarase. GH50A β-agarase produces NA2 through exo-type hydrolysis at the non-reducing end of agarose or NAOS, while AOS releases A3 from the non-reducing end and then produces NA2 in the same manner.
Figure 10 shows the effect of GH50A β-agarase enzyme concentration on exoform hydrolysis of agarose and TLC analysis of Bio-Gel P-2 column chromatography fractions of agarose hydrolyzate by GH50A β-agarase treatment. Shows the results. (A) Under optimal conditions (20mM Tris-HCl, pH 7.5, 5mM MnSO ₄ , 10mM TCEP), 0.4% agarose was treated with individual amounts of enzyme for 4 hours to induce a hydrolysis reaction, and the resulting reducing sugar was treated with DNS. It was measured by method. Each value was obtained as mean ± SD (n = 3, three replicates per independent experiment). *P < 0.05 and **P < 0.01. (B) 0.4% agarose was treated with GH50A β-agarase enzyme (20 μg/mL) for 4 hours under optimal conditions and then freeze-dried. The dried sample was dissolved in tertiary distilled water and fractionated using Bio-Gel P-2 column chromatography using tertiary distilled water as a mobile phase. Equal volumes of each fraction were spotted on Silica Gel 60 aluminum plates and developed with n-butanol-ethanol-H ₂ O [3:2:2 (v/v)]. The sugar material on the TLC plate was confirmed by color development as described in Figure 6. Representative results are shown in Figure 10, and similar results were obtained in two additional experiments.
Figure 11 shows the results of TLC analysis of Sephadex G-10 column chromatography fractions of AOS hydrolyzate by GH50A β-agarase treatment and LC-MS/MS analysis of NA2 and A3 obtained from column chromatography. (A) 1.0% AOS was treated with 20 μg/mL of GH50A β-agarase enzyme for 14 hours under optimal conditions and then freeze-dried. The dried sample was dissolved in tertiary distilled water and fractionated using Sephadex G-10 column chromatography using tertiary distilled water as a mobile phase. Equal volumes of each fraction were spotted on a Silica Gel 60 TLC aluminum plate and developed with n-butanol:ethanol:H ₂ O [3:2:2 (v/v)]. Sugar substances, A3 and NA2, on the TLC plate were confirmed by color development as described in Figure 6. (B) NA2 and A3 were confirmed by LC-MS/MS analysis as described in Materials and Methods. Representative results are shown in Figure 11, and similar results were obtained in two additional experiments.

Hereinafter, the present invention will be described in detail.

Recently, in order to search for genetic information encoding agarases that hydrolyze agarose into the final products L-AHG and D-galactose, the present inventors used Cellvibrio sp, a freshwater agar-degrading bacterium. The base sequence of the entire genome of strain KY-GH-1 (accession number: KCTC 13629BP) was analyzed [27]. As a result of genome sequence analysis, strain KY-GH-1 possesses genetic information for three sub-type GH50 β-agarases (GH50A, GH50B and GH50C) in the agarase gene cluster spanning approximately 77 kb on the genome. It was found that Therefore, in order to determine which of these three isozymes is the enzyme with the most prominent GH50 β-agarase activity for producing NA2 from agarose, the present invention uses Cellvibrio sp. Three GH50 β-agarases of KY-GH-1 were produced as recombinant His-tagged proteins using the E. coli expression system with pET-30a vector, and their enzymatic activities were compared. In addition, among these isoenzymes, GH50A β-agarase exhibits the highest exo-type β-agarase activity suitable for NA2 production through hydrolysis of agarose substrate (refer to the base sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2). ) was selected, and its enzymatic properties, enzymatic treatment conditions for NA2 production from agarose, and NA2 purification conditions and production yield from hydrolysis products were investigated to complete the present invention.

Therefore, the present invention provides Glycoside hydrolase GH50 A β-agarase comprising SEQ ID NO: 1.

The glycoside hydrolase GH50A β-agarase is preferably derived from Cellvibrio sp. KY-GH-1 strain, accession number KCTC 13629BP.

The glycoside hydrolase GG50 A β-agarase is preferably obtained from an E. coli transformant into which the glycoside hydrolase GG50 A β-agarase gene has been introduced.

It is preferable that the glycoside hydrolase GG50A β-agarase gene is cloned into an expression vector and introduced into E. coli.

In addition, the present invention provides a method for producing neoagarobiose by treating the substrate with the glycoside hydrolase GH50 A β-agarase described in the present invention for enzymatic decomposition.

The substrate is preferably one or more selected from the group consisting of agar, agarose, agaro-oligosacchrides (AOS), and neoagaro-oligosacchrides (NAOS).

The treatment is preferably performed at a temperature range of 20 to 50°C.

The treatment is preferably performed in a pH range of pH 6.0 to 10.0.

The above treatment is preferably performed by further adding Mn ²⁺ .

The Mn ²⁺ is preferably in the form of MnCl ₂ , MnSO ₄ or a mixture thereof. The concentration of MnCl ₂ , MnSO ₄ or a mixture thereof may be 1 to 10mM, preferably 3 to 8mM, more preferably 4 to 6mM, and most preferably 5mM.

The treatment is preferably performed by further adding Tris(2-carboxyethyl)-phosphine (TCEP). The concentration of TCEP may be 1 to 10mM, preferably 3 to 8mM, more preferably 4 to 6mM, and most preferably 5mM.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples describe a preferred embodiment of the present invention, and it is clear that the scope of the present invention is not to be construed as limited by the matters described in the following examples.

[Example]

1. Materials and Methods

1.1. E. coli Cloning and expression of GH50 β-agarase gene using expression system

Cellvibrio sp. Among the genes encoding the three β-agalases (GH50A, GH50B and GH50C) in the genomic DNA of strain KY-GH-1, Nde I-forward primer (5'-CCCGCATATGATGTGTTCGAGTTATAAGCTTG-3' for GH50A (SEQ ID NO: 3), 5'-GCGGCATATGAAAAACTCACAACATCTTAATC-3' (SEQ ID NO: 4) for GH50B), Bam HI-forward primer (5'-GGCGGGATCCATGAAAAAAAATCAACTACATTTA-3' (SEQ ID NO: 5) for GH50C) and Xho I-reverse primer (GH50A) For GH50B, use 5'-CGGGCTCGAGTTTTTTCGCGCGGCGAGTA-3' (SEQ ID NO: 6), for GH50B, use 5'-GCGCTCGAGTTTTCCAAAACGCTTAATGTAAAGG-3' (SEQ ID NO: 7), and for GH50C, use 5'-GAATCTCGAGTTGGATGGGAGGAATTTTA-3' (SEQ ID NO: 8). amplified. The PCR products of the GH50A and GH50B genes were purified and treated with Nde I/ Xho I, and the PCR products of the GH50C gene were purified and treated with Bam HI/ Xho I. In addition, these enzyme genes treated with restriction enzymes were linked to the pET-30a expression vector (Novagen, Madison, WI, USA) using T4 ligase (Roche, Basel, Switzerland) and added to the C-terminus of each expressed enzyme protein. 6x his-tag was connected. Each recombinant pET-30a plasmid was transformed into E. coli BL21(DE3) (Novagen) cells and then plated on an LB plate containing 50 μg/mL kanamycin (Sigma-Aldrich, St. Louis, MO, USA) for 30 days. Transformants were selected by culturing at ℃.

For expression of individual β-agarase genes inserted into the pET-30a plasmid in E. coli BL21(DE3), each transformant was shaken at 25°C in LB medium containing 50 μg/mL kanamycin (Sigma-Aldrich). The culture was cultured, and when the OD ₆₀₀ value of the culture medium reached 0.5~0.6, 0.075mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma-Aldrich) was added and the culture was further cultured at 25°C for 5 hours [28].

1.2. Sequence analysis and phylogenetic tree construction

The amino acid sequences of nine GH50 family β-agarases were obtained by BLAST search of the NCBI database (http://www.ncbi.nlm.nih.gov). The amino acid sequences of the open-reading frames of individual agarase genes were aligned using Clustal X [29]. At this time, conserved amino acid residues were indicated using the Clustal X color scheme. UPGMA was used to construct an unrooted phylogenetic tree of individual GH50 family members based on the amino acid sequence similarity of each GH50 family β-agarase [30].

1.3. Comparison of agarase activity by zymogram analysis on LB agarose plates

E. coli BL21(DE3) transformants carrying the pET-30a plasmid and each recombinant pET-30a plasmid containing the GH50A, GH50B, or GH50C β-agarase genes were cultured in 1.5% agarose (Invitrogen, Life Technology). , Grand Island NY, USA), and were spotted on LB plates containing 50 μg/mL kanamycin and 0.05 mM IPTG. After culturing at 30°C for 2 days, the culture plate was stained with Lugol's iodine solution at room temperature (RT) for zymogram analysis.

1.4. Cell lysates, protein quantification, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

To identify and locate the recombinant enzyme protein produced by the transformant, cells were suspended in 40mM Tris-HCl buffer (pH 8.0), lysed by sonication 20 times for 10 seconds, and incubated at 4°C for 30 minutes. After extraction, the lysate was fractionated into total fraction, soluble fraction, and non-soluble fraction [31].

The protein content of cell lysates was quantified using the Micro BCA kit (Pierce, Rockford, IL, USA). An equal amount of cell lysate (10 μg) was electrophoresed on an 8% SDS-polyacrylamide gel using the method of Laemmli [32]. The gel was stained with Coomassie brilliant blue (CBB) R-250 to detect protein bands. To quantify the concentration of a specific recombinant protein in the soluble fraction of a cell lysate, the density of the recombinant protein band on the gel is measured and the units of measurement are compared to the density of serially diluted bovine serum albumin (BSA) bands on the same gel. It was compared with the standard curve obtained by [33]. At this time, density measurement of protein bands on the gel was performed using ImageQuant TL software (Amersham, Arlington Heights, IL, USA).

1.5. Purification of recombinant His-Tagged GH50A β-agarase

To purify recombinant His-tagged GH50A β-agarase, soluble fractions from cell lysates of E. coli transformants containing the GH50A gene were purified using Ni-NTA resin (ThermoFisher Scientific, Rockford, IL USA). Immobilized metal ion affinity chromatography (IMAC) was performed [34].

1.6. Agarase activity assay

Quantitative analysis of GH50 β-agarase activity was performed using the DNS method [35], which detects reducing sugars released from agarose. The enzyme solution (100 μL) was mixed with an equal volume of 0.8% agarose liquefied in 20 mM Tris-HCl buffer (pH 7.5). After reacting at 35°C for 30 minutes, the reducing sugar formed in the reaction mixture was colorimetrically measured using DNS reagent. At this time, one unit of enzyme activity was defined as the amount of enzyme that generates reducing power equivalent to 1 μmol of D-galactose (Sigma-Aldrich) per minute.

1.7. Thin layer chromatography (TLC)

TLC analysis of GH50A β-agarase catalyzed hydrolysates of agarose, NAOS, or AOS was performed using Silica Gel 60 aluminum plates (F254 Merck, Darmstadt, Germany). TLC was developed with a solvent of n-butanol-ethanol-H ₂ O [3:2:2 (v/v)], and 0.2% (w/v) naphthoresorcinol in ethanol was used to confirm the sugar substance on TLC. (Sigma-Aldrich) and 10% (v/v) H ₂ SO ₄ were added to the color development solution, which was sprayed on a TLC plate and then heated at 80°C for detection. For the NAOS mixture containing D-galactose and NA2~NA18, the mixture [36] provided by Dr. Lee Sang-hyun of Silla University in Busan was used as a standard. To prepare an AOS mixture that can be obtained by hydrolyzing agarose with a weak acid, agarose was first added to a 3M acetic acid solution at a concentration of 1% and then treated at 80°C for 3 hours. After adjusting the pH to approximately 7.0 using 5M NaOH and freeze-drying, the obtained solid sample was washed three times with 95% (v/v) cold ethanol to remove sodium acetate salt and dried using a rotary evaporator to obtain AOS powder. got it

1.8. Molecular sieve chromatography using Bio-Gel P-2 or Sephadex G-10 columns

To purify NA2, agarose was treated with GH50A β-agarase under optimal reaction conditions, and then the NA2-containing hydrolyzate was freeze-dried, dissolved in tertiary distilled water (DI), and then purified on a Bio-Gel P-2 column. Chromatography was performed using (I.D. 1.3 × 60 cm, Bio-Rad Laboratories, Hercules, CA, USA). The column was eluted using tertiary DI as a mobile phase, and the fractions eluted at 3 mL each were analyzed by TLC to recover the fraction containing only NA2.

Meanwhile, to obtain AOS hydrolysis products containing neoagarobiose (NA2) and agarotriose (A3), 1% AOS substrate was mixed with GH50A β-agarase (20μg/mL) for 4 hours under optimal reaction conditions. After processing, it was freeze-dried. The prepared hydrolyzate was dissolved in distilled water (DI) and then chromatographed using a Sephadex G-10 column (ID 1.3 × 60 cm, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The column was eluted at 2 ml each using tertiary DI as a mobile phase, and each eluted fraction was analyzed by TLC to recover the fraction containing NA2 or A3.

1.9. Analysis of enzymatic reaction products using liquid chromatography and tandem mass spectrometry (LC-MS/MS)

LC-MS/MS analysis was performed using a Xevo TQ-S micro mass spectrometer with electrospray ionization on a multimode ESI ion source equipped with an Acquity UPLC H-Class core system (Waters Corporation, Milford, MA, USA). [37,38]. Sample elution was performed using a Waters Acquity UPLC Spherisorb amino column (2 mm × 100 mm, particle size 3 μm) maintained at 40°C with a solvent flow rate of 200 μL/min. The LC system consisted of (A) 0.1% aqueous formic acid and (B) 0.1% acetonitrile. To identify NA2~NA8 in the enzymatic reaction products of NAOS, chromatography was performed in a dual gradient solvent system with an A:B ratio of 35:65 for 5 min after initiation, and 12 min after initiation, the A:B ratio was This was gradually changed to reach 65:35. In addition, 13 minutes after starting chromatography, the ratio of A:B was changed to reach 35:65, and this ratio was maintained until chromatography was stopped 20 minutes after starting chromatography. Maintaining this ratio, the chromatography was stopped 20 minutes after start.

Chromatography to identify NA2 and A3 in the enzymatic hydrolyzate of AOS was performed in a dual gradient solvent system with an A:B ratio of 95:5 for 3 min after initiation, until A was reached at 13 min after initiation. The :B ratio was gradually changed to 75:25, and the A:B ratio of 75:25 was maintained until 15 minutes after initiation. Then, at 16 minutes after starting, the ratio of A:B was changed to 95:5, and this condition was maintained until the chromatography was terminated at 30 minutes. The injection volume was 5 μL and the sample storage temperature was set at 5°C. Mass spectrometer detector conditions were set as follows: capillary voltage, 2.0 kV; Cone voltage, 20 V; Source temperature, 150°C; Desolvation temperature, 250°C; Desolvent gas flow, 550 m/h; Cone gas flow, 5 L/h; and mass range 100 to 1500.

1.10. statistical analysis

Unless otherwise specified, data are presented from at least three independent experiments. All data were expressed as mean ± standard deviation (SD, for each group n ≤ 3). Statistical analysis was performed using Student's t-test to assess the significance of differences between two groups and one-way analysis of variance, followed by Dunnett's multiple comparison test for comparing three or more groups. P value <0.05 indicates statistical significance. Statistical analysis was performed using SPSS Statistics version 23 (IBM, Armonk, NY, USA).

2. Results and discussion

2.1. Cellvibrio sp. As an individual C-terminal His-Tagged protein of KY-GH-1, pET-30a vector of GH50 β-agarase and E. coli Production using expression

Cellvibrio sp. In our previous study on whole genome sequence analysis of KY-GH-1, Cellvibrio sp. The presence of three GH50 β-agarase genes (GH50A, GH50B, and GH50C) has been confirmed in the chromosomal DNA of KY-GH-1 [27]. Based on these data, we investigated which isozyme of GH50A, GH50B, and GH50C β-agarases had the most prominent exo-type β-agarase activity using a recombinant His-tagged enzyme expressed in an E. coli expression system.

After individual E. coli transformants were subjected to IPTG induction, cells were harvested and fractionated into total, soluble, and insoluble fractions. Recombinant GH50A, GH50B, and GH50C β-agarases obtained after electrophoresis of equal amounts of each fraction on an SDS-polyacrylamide gel were mainly detected in the cell lytic fraction. However, the levels of individual enzymes detected in the insoluble fraction were not significant (Figure 1A). The molecular masses (MWs) calculated from the amino acid composition containing 6x His-tag for GH50A, GH50B and GH50C are 90.3, 88.7 and 88.5 kDa, while the MWs of the recombinant His-tagged protein were determined by SDS-PAGE mobility using Size marker. When estimated using , it was found to be about 89.0, 90.1, and 94.0 kDa, respectively.

Transformants expressing GH50A, GH50B, and GH50C β-agarase were cultured for 2 days on LB agarose plates containing 0.05mM IPTG, and then stained by pouring Lugol's iodine solution on the LB agarose plates. At this time, the agarase enzyme activity of the colony was confirmed by the size of the transparent area around the colony. As a result, as shown in Figure 1B, agarose decomposition activity was most strongly observed in colonies of the GH50A β-agarase transformant. These results show that Cellvibrio sp. Among the individual isoenzymes of the GH50 family enzymes involved in the agarose decomposition process of KY-GH-1, GH50A β-agarase is the most dominant exo-type β-agarase.

2.2. Comparison of the amino acid sequence of GH50A β-agarase with that of related enzymes from other strains.

The amino acid sequence of GH50A β-agarase was compared and analyzed with other β-agarase sequences using the CLUSTALW program (Figure 2a). Cellvibrio sp. KY-GH-1 GH50A β-agarase was prepared from Alteromonas sp. It showed 97.6% similarity to E-1 β-agarase (GenBank accession number BAE97587.1) [39]. Additionally, Cellvibrio sp. OA-2007 (GenBank accession number WP_062065002.1) [40], Cellvibrio sp. pealriver (GenBank accession number WP_049629422.1) [41], Cellvibrio sp. KY-YJ-3 (GenBank accession number WP_151059417.1) and Cellvibrio sp. It showed 96.9%, 96.4%, 95.6%, and 91.7% similarity to the β-agarases of BR (GenBank accession number WP_157152532.1), respectively. These results suggest that Cellvibrio sp. It shows that there is a high level of homology between GH50 β-agarases.

Additionally, Cellvibrio sp. KY-GH-1 GH50A β-agarase is prepared from Catenovulum sp. RQJ05 (GenBank accession number WP_111979256.1), Catenovulum sediminis (GenBank accession number WP_143873817.1), Catenovulum agarivorans (GenBank accession number WP_035016017.1) [42], and Saccharophagus degradans (GenBank accession number WP_143710996) .1) β in [43] -It showed 66.9%, 64.3%, 63.2%, and 62.7% similarity with agarase. These β-agarases have a high level of homology (62.7–96.9%) in their translated amino acid sequences from the nucleotide sequences found in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/). , none of these have been investigated for enzymatic properties using purified or recombinant enzymes to date.

In an unrooted phylogenetic tree of individual β-agarases, Cellvibrio sp. KY-GH-1 GH50A β-agarase was obtained from Catenovulum sp., agar-degrading bacteria isolated from the marine environment. Compared to the β-agarases of RQJ05, Catenovulum sediminis , Catenovulum agarivorans and Saccharophagus degradans, Alteromonas sp, an agar-degrading bacterium isolated from a non-marine environment. E-1, Cellvibrio sp. OA-2007, Cellvibrio sp. pealriver, Cellvibrio sp. KY-YJ-3 and Cellvibrio sp. It was confirmed that it was more closely grouped with the β-agarases of BR (Figure 2b).

In several previous studies, GH50 series β-agarases cleave agarose through exo-type or exo-type/endo-type hydrolytic activity to produce NA2, NA4 or NA2/NA4 with a low degree of polymerization (DP). It has been reported to produce [2,12-15]. In addition, the current analysis results show that homologs of the GH50A gene are present in all agar-degrading bacteria, and GH50A β-agarase is an enzyme that hydrolyzes agarose into NA2 or NA2/NA4, Cellvibrio sp. This suggests that it is a constitutive enzyme essential for the agarose digestion process of strain KY-GH-1.

2.3. Enzymatic properties of GH50A β-agarase

To determine whether recombinant GH50A β-agarase could be used for mass production of NA2 from agarose, various enzymatic properties were investigated. Transformants expressing GH50A β-agarase were cultured with shaking in 100 ml LB medium containing 50 μg/mL kanamycin at 25°C until the OD 600 nm reached 0.5, and then further incubated at 25°C with the addition of 0.075mM IPTG. Cultured for 5 hours. After washing the cultured cells, they were suspended in 40mM Tris-HCl buffer (pH 8.0) and sonicated 20 times for 10 seconds. Then, they were left at 4°C for 30 minutes and then centrifuged at 16,000g for 20 minutes. The soluble fraction of the cell lysate was obtained as a centrifuged supernatant.

After purification of recombinant GH50A β-agarase from soluble cell lysate using the Ni-NTA purification system, the purified enzyme and soluble cell lysate (10 μg) were electrophoresed on an 8% SDS-polyacrylamide gel. As shown in Figure 3, purified GH50A β-agarase was detected as a single band on 8% SDS-PAGE, and the MW was confirmed to be ~90.3 kDa when compared to the pre-stained size maker. These results show that high purity recombinant GH50A β-agarase enzyme was obtained through purification process.

Under these conditions, when the density units of the GH50A β-agarase band contained in the soluble fraction were compared with a standard curve using a known concentration of BSA, it was confirmed that recombinant GH50A β-agarase accounts for approximately 32% of the total soluble protein. It has been done. This indicates that the GH50A β-agarase protein was successfully overexpressed as a soluble protein in the E. coli protein expression system.

When the enzyme activity of purified GH50A β-agarase was measured at various temperatures, the highest activity was observed at 35°C, and more than 80% of the maximum activity was maintained in the temperature range of 20 to 50°C (Figure 4A). Additionally, the enzyme showed maximum activity in a remarkably wide pH range of 6.0 to 10.0 (Figure 4B). The enzyme was stable up to 35°C. However, after treatment at 40°C for 4 hours, only 60% of the maximum activity was maintained, and after treatment at 50°C for 4 hours, enzyme activity was completely lost (Figure 4C). In addition, GH50A β-agarase was stable in the pH range of 7.0 to 9.0, but quickly lost its activity at acidic pH and retained only 30% of its maximum activity after 4 hours of treatment at pH 6.0, confirming that it was more stable at alkaline pH (Figure 4 D). Meanwhile, the Km, Vmax, Kcat, and Kcat/Km values of GH50A β-agarase were 26.6mg/ml, 16.9U/mg, 25.2S ^-1 , and 1.2×10 ⁵ s ^-1 ·M ^-1 , respectively ( Figure 4E).

GH50A β-agarase activity was improved by 1.7 to 1.8 times in the presence of 5mM MnCl ₂ or 5mM MnSO ₄ , suggesting that the enzyme activity is dependent on Mn ²⁺ (Figure 5A). However, enzyme activity was not affected by 5mM EDTA. Unlike marine bacterial β-agarases, which are generally reported to depend on the presence of Mg ²⁺ and Na ⁺ [14,44,45], the GH50A β-agarase activity investigated in this study was dependent on the presence of MgCl ₂ and NaCl. was not affected by , but rather, the activity appeared to be improved in a Mn ²⁺ dependent manner. In addition, when 5mM of tris(2-carboxyethyl)-phosphine (TCEP), known as an SH reducing agent, was added, the enzyme activity was improved by 1.2 times.

The enhancing effect of Mn ²⁺ and reducing agent TCEP on GH50A β-agarase enzyme activity was concentration-dependent. In particular, 10mM MnSO ₄ and 10mM TCEP increased agarase enzyme activity by 1.8-fold and 1.4-fold, respectively (Figure 5B). In addition, when 5mM MnSO ₄ and 10mM TCEP were added simultaneously, the enzyme activity increased by 2.5 times, confirming the synergistic effect of Mn ²⁺ and TCEP on GH50A β-agarase activity. This suggests that TCEP may contribute to promoting enzyme activity by preventing oxidation of the SH group of the enzyme.

As a result, it was confirmed that Mn ²⁺ ion and SH reducing agent TCEP are required for efficient enzymatic activity of GH50A β-agarase.

2.4. Substrate degradation mode of GH50A β-agarase

According to previous research reports, GH16, GH86, and GH118 β-agarases are endo-type hydrolases that decompose agarose into NAOS of various DPs of 4 to 10, while GH50 β-agarases decompose agarose into NAOS. The predominant end products are hydrolases that produce NA2, NA4, or NA2/NA4 through exo- or endo-type activity [2,12-15].

In order to determine the substrate degradation mode of GH50A β-agarase produced with recombinant enzyme in this study, hydrolysis was prepared by treating agarose or NAOS mixture of NA2 to NA18 as a substrate with GH50A β-agarase enzyme at different times. The product was analyzed by TLC. As a result of checking the hydrolysis pattern of the agarose substrate by GH50A β-agarose over time, NA2 was mainly produced from the start of the enzymatic reaction through exo-type hydrolysis of agarose, and the amount of NA2 was decreased during the reaction period of 60 minutes. It was confirmed that it continued to increase as the main product (A in Figure 6). In addition, it was confirmed that along with NA2, the main product, NA4 and some NAOS with higher DP were produced in much smaller amounts.

Meanwhile, when the enzymatic hydrolyzate obtained by treating the NAOS mixture containing NA2~NA18 with GH50A β-agarase was analyzed by TLC, NAOS with a DP larger than NA6 or NA6 was the preferred substrate for the enzymatic reaction to generate NA2. It was shown (B in Figure 6). In addition, TLC analysis results show that GH50A β-agarase can also hydrolyze AOS mixtures of several DPs obtained by liquefying agarose through weak acid treatment, ultimately using NA2 as the main product and the non-reducing end of AOS. It was shown that agarotriose (NA3), which is cleaved during the first hydrolysis, is produced as a by-product (C in Figure 6).

As a result of TLC analysis, in order to confirm the substrate preference of GH50A β-agarase, which prefers NA6~NA8 over NA4, the hydrolysis product of NAOS treated with the enzyme for a specified time was analyzed by LC-MS/MS. As shown in Figure 7, the LC-MS/MS results were consistent with the TLC data, showing that enzyme-catalyzed hydrolysis of NA6 to NA8 occurred before NA4. This confirms that NAOS, which has a larger DP than NA4, is the preferred substrate for NA2 production for GH50A β-agarase. In addition, LC-MS/MS analysis of the enzyme-catalyzed hydrolyzate of AOS showed that the main product NA2 and a small amount of by-product A3 were produced in direct proportion to the enzyme treatment time. These results demonstrate that exoform degradation of AOS by GH50A β-agarase to generate NA2 is initiated after cleavage and release of A3 from the non-reducing end of AOS (Figure 8).

Among the GH50 series β-agarases reported to date, there is no enzyme that releases A3 from the non-reducing end of AOS and then generates NA2 in exo form, so the GH50A β-agarase reported in the present invention cannot catalyze this reaction. It can be said to be the first enzyme to exist. In addition, most of the reported GH50 β-agarases were reported to generate NA4 or NA2/NA4 from agarose through their exo-type hydrolysis and endo-type hydrolysis actions [2,12-15], but GH50 β-agarase, which produces NA2 directly from agarose through small hydrolysis, is relatively rare. In this context, the freshwater bacterium Cellvibrio sp. Recombinant GH50A β-agarase derived from KY-GH-1 is a unique GH50 family β-agarase that can hydrolyze the substrate (agarose, NAOS or AOS) to its exoform to produce NA2 as the main product. It is expected to be a useful enzyme (Figure 9).

The inventors of the present invention used Cellvibrio sp, a freshwater-derived agar-degrading bacterium. Through previous research analyzing the genome sequence of KY-GH-1, Cellvibrio sp. The KY-GH-1 strain contains genes encoding four GH16 family β-agalases and two GH86 family β-agalases known to endo-hydrolyze agarose into NA4/NA6 and NA6/NA8, respectively. It was also revealed that it possesses genes encoding GH50 family β-agarases that generate NA2 through three exoform hydrolysis [27]. These previous and current research results show that, compared to treating agarose alone with GH50A β-agarase, agarose is treated with endo-type GH16/GH86 β-agarase and exo-type GH50A β-agarase. This suggests that combined treatment or a combination of pretreatment using weak acid and GH50A β-agarase treatment may be much more effective as a process for hydrolyzing agarose into NA2.

2.5. Yield of NA2 that can be produced by agarose or AOS treatment with GH50A β-agarase under optimal reaction conditions.

To investigate in more detail whether GH50A β-agarase is an efficient enzyme for NA2 production from agarose, 0.4 μg was used under optimal reaction conditions (5mM MnSO ₄ , 10mM TCEP, 35°C, 20mM Tris-HCl, pH 7.5). The NA2 yield of % agarose was investigated. After treatment for 4 hours under optimal reaction conditions, the concentration of GH50A β-agarase required to produce maximum reducing sugar from 0.4% agarose was measured using the DNS method and was found to be 20 μg/mL (Figure 10 A). When treated under the same conditions for 12 hours, the concentration of enzyme required for maximum production of reducing sugar was found to be 5 μg/mL (data omitted). 0.4% agarose (100 mL) was treated with GH50A β-agarase (20 μg/mL) under optimal conditions for 4 hours, the reaction product was freeze-dried, and the dried sample was dissolved in tertiary distilled water (6 mL). Molecular sieve chromatography was performed on 2 mL of 6 mL of sample using a Bio-Gel P-2 column. At this time, as a result of TLC analysis of individual fractions obtained from molecular sieve chromatography, it was confirmed that NA2 was mainly isolated in fractions 18 to 19. This shows that NA2 can be purified from the enzyme reaction product through molecular sieve chromatography using a Bio-Gel P-2 column (ID 1.3 × 60 cm, Bio-Rad Laboratories, Hercules, CA, USA) (Figure 10 B). Therefore, fractions 18 to 19 were recovered, and NA2 was recovered as a powder by freeze-drying. When the enzymatic hydrolyzate of agarose (400 mg) was fractionated by three rounds of molecular sieve chromatography, a total of 216 mg of NA2 was obtained, indicating a ~54% yield of NA2 from agarose.

To investigate whether GH50A β-agarase is an efficient enzyme for NA2 production from AOS, NA2 production from AOS under optimal reaction conditions (5mM MnSO ₄ , 10mM TCEP, 35°C, 20mM Tris-HC pH 7.5). The yield was investigated. 1.0% AOS (30 ml) was treated with 20 μg/ml GH50A β-agarase for 4 hours under optimal reaction conditions, and then the reaction product was freeze-dried. The dried sample was dissolved in 2 mL of distilled water and then molecular sieve chromatography was performed using a Sephadex G-10 column (ID 1.3 × 60 cm, GE Healthcare Bio-Sciences AB, Uppsala, Sweden). As a result of TLC analysis of each fraction obtained through molecular sieve chromatography, A3 was mainly identified in fractions 18 to 19, and NA2 was mainly identified in fractions 21 to 23 (A in Figure 11). In addition, the presence of NA2 and A3 in each obtained fraction was further tested by LC-MS/MS analysis (Figure 11B). These results show that NA2 and A3 can be purified from the enzymatic reaction products by Sephadex G-10 column chromatography. Fractions 21-23 were freeze-dried to recover NA2 powder (184 mg), which represents a ~61% NA2 yield from AOS.

3. Conclusion

Freshwater agar digestion Cellvibrio sp. Recombinant his-tagged GH50A β-agarase derived from KY-GH-1 acted on agarose substrate and produced NA2 as the main hydrolysis product. Enzyme activity showed more than 80% of maximum activity at pH 6.0~10.0 and 20~50℃, and was improved 2.5 times by the coexistence of MnSO ₄ and TCEP, but was not affected by 5mM EDTA. GH50A β-agarase hydrolyzes NAOS to produce NA2 regardless of DP, and preferred NA6 to NA18 over NA4 as a substrate for NA2 production. In addition, as a substrate for the production of NA2 by GH50A β-agarase, the NAOS mixture (NA6 to NA18) prepared by pre-treating agarose with endo-type β-agarase can be agarized with agarose or acetic acid, a weak acid. It was confirmed to be a more preferred substrate for GH50A β-agarase compared to the AOS mixture prepared by pre-treatment with rose. The enzymatic properties of GH50A β-agarase can be very useful in a single process for converting agarose to NA2 with ~54% yield or in a complex process with endo-type β-agarases such as GH16/GH86/GH118. showed that there is In addition, GH50A β-agarase is expected to be very useful in the saccharification process of agarose biomass to produce the monosaccharides L-AHG and D-galactose through complex treatment with GH117 family α-NABH. Meanwhile, none of the nine β-agarases showing a high level of amino acid sequence homology (62.7 to 96.9%) with the GH50 family β-agarases listed in the U.S. National Institutes of Health (NIH) Genbank database has been used to date. Since research on enzymatic properties has not been conducted, Cellvibrio sp. reported in the present invention. The results for GH50A β-agarase from strain KY-GH-1 may provide insight into other β-agarases that show high homology to GH50A β-agarase.

Korea Research Institute of Bioscience and Biotechnology KCTC13629BP 20180827

<110> KYUNGPOOK NATIONAL UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> GH50A BETA-AGARASE DERIVED FROM A NOVEL AGAR-DEGRADING BACTERIUM AND PRODUCING METHOD OF NEOAGAROBIOSE USING THE SAME <130> YP190039 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 2391 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50A beta-agarase <400> 1 atgtgttcga gttataagct tgatcggccc attactttgt ttctgaaaac cagcgtgctt 60 agtgtggctg cgctgttctt aatcgcctgt caggaagagg cggcagataa gggcgctgct 120 gtatccgaag ttaaatcaat gcctgcggat aaggtgttgt ttgattttga aggcgatcag 180 ttaccggcag aaattagttt ttttaacgct cagggaagtt tggttaaatc ctcagcgagt 240 gatacctccg catcgcaagc attaaaagta aagttcaact ctgttgatca tgaatacacc 300 tccttagtta tccagccaaa gtcagataca tggaattgga gtgatattgg cgatgctagt 360 cttgcctttg atattgccaa tgacggtgag cattccgtac agctattttt agatgtgtct 420 gatgcaaaag gtaatagctt tactcgcagc gtcagtgtgc cggtaggcaa gtcccgcgtt 480 tattattcca agttaagtgg ccacgatatg gtcagtgcaa atccggattc caaagtggag 540 ttgaatgcag cctcaggatt gcgcggcaac ccgccgacct ggtctggcga tgatgtgcag 600 ttcatctgga tgtggggcgt tatgaatttg gatttgtccg ctattaagcg catatcctta 660 agtgttcaat atgctttgca cgataaagaa atcaccctcg acaatattcg ggttattaaa 720 agccctgcaa tgaataaaga tttcctggtc aatctggtcg ataaatttgg ccagccagcc 780 aaagtggatt ttgccggtaa gattcattct gagacagagt tgcaagcagc aacccagagc 840 gagttgaaag agctaaataa cggtgcgcca ctagccgata gatctacatt tggcggttgg 900 aaaaatggtc cgaaacaacc tgcaacaggc tatttttatc ctaaaaaagt tgatggaaaa 960 tggtggttgg ttgatccaga agggtatctt tattttgcga caggattgga tatcatacgt 1020 ttagccaatg catacactat gactggctat gattacgatg cttcaactat tgagcagcgc 1080 agtgccgatg atttgactcc cgaagactcg aaaggaaaaa tccttatttc agaagaggcg 1140 caaaaaaccc gtcatttggt ttctaaaact cgcgccgaca tgtttgagtg gttgcccaag 1200 cacactgacc ctttgggtaa tcactatgat tacaatcgtg atgcccactc aggcccactg 1260 ctaaaaggcg aagcctttag tttctatagt gccaatcttg agcgtaaata cggtgaaaca 1320 gaacctgatt cctatttgcg ccaatgggaa aaagttactg tggatcgcat gttgaattgg 1380 ggtttcacct cgctaggaaa ctggactgat ccaaaattct acagcaatca acgcattcct 1440 tattttgcca atggctggat tattggcaac ttcaaaacag tttccagtgg caatgatttt 1500 tggggcggtt tgcctgatcc atttgatccg gtatttaaag agcgcgcact tgccacggcc 1560 aaggctattg ctgaagaaac taaaaatagc ccttggtgtg tcggcgtatt tattgataac 1620 gaaaaaagct ggggcgctc agaaagcaaa gagtctgaat atggcatagt gttaaataca 1680 ctgactcgcg atggcgcaga ctcgccaacg aaaaataaat ttacgcagtt gatgaaagaa 1740 aagtatgtgg atatagcagc attaaatacc gcatggggaa cctcagttga atcctgggat 1800 gcatttcaaa aaggtgtaaa aactggcatt aacaacgatg ttcaattgca agatttcagt 1860 ctgttattta cccagtatgc tgaagagtat ttcaaaattg ttgagggcgc cttaacacaa 1920 tatatgccta atcacctgta cttgggtgtg cgttttgcag attggggaat gccaaaagat 1980 gtagttaaag cagcggcaaa atacgccgat gttgttagct ataacttcta taaagaaggt 2040 ttaaccaaaa ataaatggac gtttttagcg gagctggata aacccagcat aatcggtgaa 2100 ttccatgtag gcacaacgga gtcaggtttg ttccacccag gcttggtgca tgcggccaat 2160 caggaagatc gtgcaaaaat gtataaagag tacatggaaa cggttgtcga taatccttat 2220 tttatggtg cacattggtt ccagtatatg gattcaccgg taactggccg ctcctatgat 2280 ggtgaaaact acaatgtagg ttttgtgtcg gtgactgata caccctatgc acctatggtg 2340 aaagccgcca aagagctgca cggtgaaatg tatactcgcc gcgcgaaaaa a 2391 <210> 2 <211> 797 <212> PRT <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50A beta-agarase <400> 2 Met Cys Ser Ser Tyr Lys Leu Asp Arg Pro Ile Thr Leu Phe Leu Lys 1 5 10 15 Thr Ser Val Leu Ser Val Ala Ala Leu Phe Leu Ile Ala Cys Gln Glu 20 25 30 Glu Ala Ala Asp Lys Gly Ala Ala Val Ser Glu Val Lys Ser Met Pro 35 40 45 Ala Asp Lys Val Leu Phe Asp Phe Glu Gly Asp Gln Leu Pro Ala Glu 50 55 60 Ile Ser Phe Phe Asn Ala Gln Gly Ser Leu Val Lys Ser Ser Ala Ser 65 70 75 80 Asp Thr Ser Ala Ser Gln Ala Leu Lys Val Lys Phe Asn Ser Val Asp 85 90 95 His Glu Tyr Thr Ser Leu Val Ile Gln Pro Lys Ser Asp Thr Trp Asn 100 105 110 Trp Ser Asp Ile Gly Asp Ala Ser Leu Ala Phe Asp Ile Ala Asn Asp 115 120 125 Gly Glu His Ser Val Gln Leu Phe Leu Asp Val Ser Asp Ala Lys Gly 130 135 140 Asn Ser Phe Thr Arg Ser Val Ser Val Pro Val Gly Lys Ser Arg Val 145 150 155 160 Tyr Tyr Ser Lys Leu Ser Gly His Asp Met Val Ser Ala Asn Pro Asp 165 170 175 Ser Lys Val Glu Leu Asn Ala Ala Ser Gly Leu Arg Gly Asn Pro Pro 180 185 190 Thr Trp Ser Gly Asp Asp Val Gln Phe Ile Trp Met Trp Gly Val Met 195 200 205 Asn Leu Asp Leu Ser Ala Ile Lys Arg Ile Ser Leu Ser Val Gln Tyr 210 215 220 Ala Leu His Asp Lys Glu Ile Thr Leu Asp Asn Ile Arg Val Ile Lys 225 230 235 240 Ser Pro Ala Met Asn Lys Asp Phe Leu Val Asn Leu Val Asp Lys Phe 245 250 255 Gly Gln Pro Ala Lys Val Asp Phe Ala Gly Lys Ile His Ser Glu Thr 260 265 270 Glu Leu Gln Ala Ala Thr Gln Ser Glu Leu Lys Glu Leu Asn Asn Gly 275 280 285 Ala Pro Leu Ala Asp Arg Ser Thr Phe Gly Gly Trp Lys Asn Gly Pro 290 295 300 Lys Gln Pro Ala Thr Gly Tyr Phe Tyr Pro Lys Lys Val Asp Gly Lys 305 310 315 320 Trp Trp Leu Val Asp Pro Glu Gly Tyr Leu Tyr Phe Ala Thr Gly Leu 325 330 335 Asp Ile Ile Arg Leu Ala Asn Ala Tyr Thr Met Thr Gly Tyr Asp Tyr 340 345 350 Asp Ala Ser Thr Ile Glu Gln Arg Ser Ala Asp Asp Leu Thr Pro Glu 355 360 365 Asp Ser Lys Gly Lys Ile Leu Ile Ser Glu Glu Ala Gln Lys Thr Arg 370 375 380 His Leu Val Ser Lys Thr Arg Ala Asp Met Phe Glu Trp Leu Pro Lys 385 390 395 400 His Thr Asp Pro Leu Gly Asn His Tyr Asp Tyr Asn Arg Asp Ala His 405 410 415 Ser Gly Pro Leu Leu Lys Gly Glu Ala Phe Ser Phe Tyr Ser Ala Asn 420 425 430 Leu Glu Arg Lys Tyr Gly Glu Thr Glu Pro Asp Ser Tyr Leu Arg Gln 435 440 445 Trp Glu Lys Val Thr Val Asp Arg Met Leu Asn Trp Gly Phe Thr Ser 450 455 460 Leu Gly Asn Trp Thr Asp Pro Lys Phe Tyr Ser Asn Gln Arg Ile Pro 465 470 475 480 Tyr Phe Ala Asn Gly Trp Ile Ile Gly Asn Phe Lys Thr Val Ser Ser 485 490 495 Gly Asn Asp Phe Trp Gly Gly Leu Pro Asp Pro Phe Asp Pro Val Phe 500 505 510 Lys Glu Arg Ala Leu Ala Thr Ala Lys Ala Ile Ala Glu Glu Thr Lys 515 520 525 Asn Ser Pro Trp Cys Val Gly Val Phe Ile Asp Asn Glu Lys Ser Trp 530 535 540 Gly Arg Ser Glu Ser Lys Glu Ser Glu Tyr Gly Ile Val Leu Asn Thr 545 550 555 560 Leu Thr Arg Asp Gly Ala Asp Ser Pro Thr Lys Asn Lys Phe Thr Gln 565 570 575 Leu Met Lys Glu Lys Tyr Val Asp Ile Ala Ala Leu Asn Thr Ala Trp 580 585 590 Gly Thr Ser Val Glu Ser Trp Asp Ala Phe Gln Lys Gly Val Lys Thr 595 600 605 Gly Ile Asn Asn Asp Val Gln Leu Gln Asp Phe Ser Leu Leu Phe Thr 610 615 620 Gln Tyr Ala Glu Glu Tyr Phe Lys Ile Val Glu Gly Ala Leu Thr Gln 625 630 635 640 Tyr Met Pro Asn His Leu Tyr Leu Gly Val Arg Phe Ala Asp Trp Gly 645 650 655 Met Pro Lys Asp Val Val Lys Ala Ala Ala Lys Tyr Ala Asp Val Val 660 665 670 Ser Tyr Asn Phe Tyr Lys Glu Gly Leu Thr Lys Asn Lys Trp Thr Phe 675 680 685 Leu Ala Glu Leu Asp Lys Pro Ser Ile Ile Gly Glu Phe His Val Gly 690 695 700 Thr Thr Glu Ser Gly Leu Phe His Pro Gly Leu Val His Ala Ala Asn 705 710 715 720 Gln Glu Asp Arg Ala Lys Met Tyr Lys Glu Tyr Met Glu Thr Val Val 725 730 735 Asp Asn Pro Tyr Phe Ile Gly Ala His Trp Phe Gln Tyr Met Asp Ser 740 745 750 Pro Val Thr Gly Arg Ser Tyr Asp Gly Glu Asn Tyr Asn Val Gly Phe 755 760 765 Val Ser Val Thr Asp Thr Pro Tyr Ala Pro Met Val Lys Ala Ala Lys 770 775 780 Glu Leu His Gly Glu Met Tyr Thr Arg Arg Ala Lys Lys 785 790 795 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50A beta-agarase Nde1 forward primer <400> 3 cccgcatatg atgtgttcga gttataagct tg 32 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50B beta-agarase Nde1 forward primer <400> 4 gcggcatatg aaaaactcac aacatcttaa tc 32 <210> 5 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50C beta-agarase BamH1 forward primer <400> 5 ggcgggatcc atgaaaaaaa atcaactaca ttta 34 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50A beta-agarase Xho1 reverse primer <400> 6 cgggctcgag ttttttcgcg cggcgagta 29 <210> 7 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50B beta-agarase Xho1 reverse primer <400> 7 gcgctcgagt tttccaaaac gcttaatgta aagg 34 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Cellvibrio sp. KY-GH-1 GH50C beta-agarase Xho1 reverse primer <400> 8 gaatctcgag ttggatggga ggaatttta 29

Claims (11)

Glycoside hydrolase GH50 A β-agarase comprising SEQ ID NO: 1. The glycoside hydrolase GH50 A β according to claim 1, which is derived from Cellvibrio sp. KY-GH-1 strain with accession number KCTC 13629BP. -agarase). The glycoside hydrolase GG50 A β-agarase according to claim 2, which is obtained from an E. coli transformant into which the glycoside hydrolase GG50 A β-agarase gene has been introduced. GH50 A β-agarase). The method of claim 3, wherein the glycoside hydrolase GH50 A β-agarase gene is cloned into an expression vector and introduced into E. coli. Glycoside hydrolase GH50 A β-agarase. Neoagarobiose, which is enzymatically degraded by treating a substrate with the glycoside hydrolase GH50 A β-agarase according to any one of claims 1 to 4. , NA2) production method. The method of claim 5, wherein the substrate is one or more types from the group consisting of agar, agarose, agaro-oligosacchrides (AOS), and neoagaro-oligosacchrides (NAOS). A method for producing neoagarobiose (NA2), characterized in that selected. The method for producing neoagarobiose (NA2) according to claim 5, wherein the treatment is performed in a temperature range of 20 to 50°C. The method for producing neoagarobiose (NA2) according to claim 5, wherein the treatment is performed in a pH range of pH 6.0 to 10.0. The method for producing neoagarobiose (NA2) according to claim 5, wherein the treatment is performed by further adding Mn ²⁺ . The method of claim 9, wherein the Mn ²⁺ is in the form of MnCl ₂ , MnSO ₄ or a mixture thereof. The method of claim 5, wherein the treatment is performed by further adding Tris(2-carboxyethyl)-phosphine (TCEP). Neoagarobiose (NA2) production method.