Novel GH117A α-neoagarobioses hydrolase derived from agar-degrading bacteria and method for producing L-AHG using the same

Abstract

The present invention relates to GH117A α-neoagarobiose hydrolase (α-NABH) derived from a novel agar-degrading bacterium Cellvibrio sp. KY-GH-1 deposited under accession number KCTC 13629BP, and a method for producing 3,6-anhydro-L-galactose (L-AHG) using said enzyme. The novel agar-degrading bacterium GH117A α-neoagarobiose hydrolase derived from Cellvibrio sp. KY-GH-1 of the present invention can produce L-AHG with high efficiency using neoagarobiose as a substrate without being significantly limited by temperature and pH ranges, and is therefore extremely effective in being widely used in the fermentation industry, food industry, pharmaceutical industry, and the like.

Description

[Technical Field]

[0001] This invention relates to GH117A α-neoagarobiose hydrolase (α-NABH) derived from a novel agar-degrading bacterium, Cellvibrio sp. KY-GH-1, deposited under deposit number KCTC 13629BP, and to a method for producing 3,6-anhydro-L-galactose (L-AHG) using the said enzyme. [Background technology]

[0002] Agar is a cell wall polysaccharide found in marine red algae and is a mixture of two components: agarose and agaropectin. Agarose is composed of cross-linked residues of α-1,3-linked 3,6-anhydro-L-galactose (L-AHG) and β-1,4-linked D-galactose. Agaropectin has the same repeating units, but some of the L-AHG residues are replaced with L-galactose sulfate, and some of the D-galactose residues are replaced with pyruvate acetal 4,6-O-(1-carboxyethylidene)-D-galactose. Because agar forms a stable gel matrix that is resistant to microbial degradation, it is widely used as a gelling material in the food industry and microbial culture media.

[0003] Approximately 15 genera of marine bacteria and 7 genera of non-marine bacteria possess agar-degrading properties, allowing them to utilize agar as their sole carbon source. To utilize agar as a carbon source, agar-degrading bacteria possess various combinations of agar-degrading enzymes, i.e., combinations of agarases, which hydrolyze agar into the monosaccharides L-AHG and D-galactose. Agarases are classified into two types according to their cleavage mechanism: α-agarase (EC 3.2.1.158) cleaves α-1,3-glycosidic bonds, while β-agarase (EC 3.2.1.81) cleaves β-1,4-glycosidic bonds.

[0004] Most agarases reported to date belong to the endo-type β-agarases that hydrolyze agarose to produce neoagarooligosaccharides (NAOS). In contrast, there are very few reports on α-agaras that hydrolyze agarose to produce agarooligosaccharides (AOS). Based on the similarity of amino acid sequences among bacterial agarases recorded in the Carbohydrate-Active Enzymes (CAZyme) database, agarases can be classified into different glycoside hydrolase (GH) families. Specifically, β-agaras are classified into various GH families such as GH16, GH50, GH86, and GH118, while α-agaras are classified into the GH96 and GH117 families. GH16, GH86, and GH118 β-agarases cleave agarose via endo-type hydrolysis to produce neoagarotetraose (NA4) / neoagarohexaose (NA6), NA6 / neoagarooctaose (NA8), and NA8 / neoagarodecaose (NA10), respectively. GH50 family β-agarases produce NA2, NA4, or NA2 / NA4 as end products through endo-type or exo-type agarose degradation activity. GH96 α-agarase primarily hydrolyzes agarose to A4. GH117 α-neoagarobiose hydrolase (α-NABH) hydrolyzes NA2 to L-AHG and D-galactose.

[0005] The various biological activities of agarose degradation products, such as antioxidant, antitumor, prebiotic, anti-inflammatory, anti-diabetic and anti-obesity, skin moisturizing and whitening, and anti-cariogenic activity, are presumed to be related to the presence of L-AHG, a monosaccharide that makes up agarose. However, because sufficient quantities of L-AHG are not yet commercially available for in vivo studies that can provide direct evidence of L-AHG's biological activity, there is a strong need for the development of mass production technologies that will enable the commercialization of L-AHG.

[0006] Recent enzymatic processes developed to produce L-AHG from agarose employ a method that involves the joint treatment of GH50 β-agarase and GH117 α-NABH to decompose agarose into NA2, and then ultimately decompose it into L-AHG and D-galactose. However, in order to saccharify agarose and produce L-AHG and D-galactose, it is still necessary to develop not only the exo-type GH50 β-agarase, which efficiently hydrolyzes agarose into NA2, but also GH117 α-NABH, which specifically acts on NA2 to hydrolyze it into the monosaccharides L-AHG and D-galactose.

[0007] In previous research, the inventors of the present invention analyzed the entire genome sequence of the Cervibrio strain KY-GH-1 (KCTC 13629BP) to search for genetic information that encodes agarase, which hydrolyzes agarose into L-AHG and D-galactose. It was confirmed that the KY-GH-1 strain possesses three GH50 family β-agarase genes (GH50A, GH50B, and GH50C) and two GH117 family α-NABH genes (GH117A and GH117B) within an agarase gene cluster spanning a ~77kb region. Through subsequent research, the inventors of the present invention revealed that GH50A β-agarase exhibits the highest exo-type β-agarase activity among the three isotopes (isozymes). However, it remains unclear which of the two GH117 α-NABH isotopes is more active in hydrolyzing NA2 to L-AHG and D-galactose. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention was devised to solve the problems of the conventional technology described above. The problem that the present invention aims to solve is to develop an efficient α-NABH necessary for the production of L-AHG from NA2, and to provide a method for producing L-AHG with high efficiency using this α-NABH. [Means for solving the problem]

[0009] To solve the above-mentioned problems, the present invention provides GH117A α-neoagarobiose hydrolase (α-NABH) containing Sequence ID No. 1.

[0010] The GH117A α-neoagarobiose hydrolase (α-NABH) is preferably derived from the Cellvibrio sp. KY-GH-1 strain, which has deposit number KCTC 13629BP.

[0011] The GH117A α-neoagarobiose hydrolase (α-NABH) is preferably obtained from a transformed E. coli organism into which the GH117A α-neoagarobiose hydrolase (α-NABH) gene has been incorporated.

[0012] The GH117A α-neoagarobiose hydrolase (α-NABH) gene is preferably cloned into an expression vector and incorporated into E. coli.

[0013] Furthermore, the present invention provides a method for producing 3,6-anhydro-L-galactose (L-AHG) by enzymatically decomposing the GH117A α-neoagarobiose hydrolase (α-NABH) by treating it with neoagarobiose (NA2).

[0014] Preferably, the treatment is carried out in a temperature range of 25 to 45 °C.

[0015] Preferably, the treatment is carried out in a pH range of 6.0 to 10.0.

[0016] Preferably, the treatment is further carried out by adding Mn 2+ Furthermore, the treatment is preferably carried out by adding Mn.

[0017] Preferably, the Mn 2+ is preferably in the form of MnCl2, MnSO4 or a mixture thereof.

[0018] Preferably, the treatment is further carried out by adding Tris(2-carboxyethyl)-phosphine (TCEP).

Advantages of the Invention

[0019] The GH117A α-neoagarobiose hydrolase derived from the novel agar-degrading bacterium Cellvibrio sp. KY-GH-1 of the present invention can produce L-AHG with high efficiency using neoagarobiose as a substrate without being greatly restricted by the temperature range and pH range, and thus has an excellent effect that it can be widely used in the fermentation industry, food industry, pharmaceutical industry, etc.

Brief Description of the Drawings

[0020] [Figure 1]This study compares the amino acid sequences of GH117A α-NABH and GH117B β-NABH from the bacterium *Vibrio ceruvianus* KY-GH-1. The amino acid sequences were arranged using Clusteral-X [Larkin et al. 2007] to maximize the appearance of identical sequences in an "open" reading frame. Each amino acid is represented by a single-letter abbreviation. Identical residues between the two sequences are marked with an asterisk at the top of the sequence. Each amino acid residue is highlighted using the Clusteral-X color scheme. Deletions are marked with a dash. [Figure 2] This report presents the results of testing the NA2-hydrolysis activity of recombinant His-tagged GH117A and GH117B α-NABH expressed in an E. coli expression system by SDS-polyacrylamide gel electrophoresis and thin-layer chromatography (TLC) analysis. (A) E. coli BL21(DE3) transformants were cultured in the presence of 0.125 mM IPTG to induce the synthesis of recombinant His-tagged GH117A or GH117B α-NABH enzyme proteins. The cells were then harvested and fractionated into total fraction, soluble fraction, and insoluble fraction. Equal amounts of each fraction were then subjected to electrophoresis. (B) SDS-PAGE of purified recombinant GH117A α-NABH and GH117B α-NABH. Electrophoresis lane; SM, pre-stained protein size markers; Crude GH117A α-NABH and Crude GH117A β-NABH, soluble fractions obtained from E. coli transformants expressing GH117A α-NABH and GH117B α-NABH, respectively; purified GH117A α-NABH and purified GH117B α-NABH, recombinant His-tagged enzymes purified by a nickel-nitrilotriacetic acid (Ni-NTA) purification system. (CE) As described in Materials and Methods, each purified recombinant enzyme protein was treated with 0.4% NA2 or 1.0% NAOS, and each enzyme reaction product was analyzed by TLC. Figure 2 shows representative results, which were nearly identical in two additional experiments. [Figure 3]The results of TLC analysis of hydrolysis products obtained by treating standards NA2, NA4, or NA6 with GH117A α-NABH enzyme are shown. NA2 (0.4%), 50 mM Tris-HCl (pH 7.5), and purified GH117A α-NABH (10 μg / mL) were mixed and reacted. Equal amounts of each individual enzyme reaction product were taken and analyzed by TLC. Standard D-galactose was purchased from Sigma-Aldrich, and standards NA2, NA4, NA6, etc., were purchased from Carbosynth (Berkshire, UK). L-AHG was obtained by hydrolyzing NA2 with GH117A β-NABH enzyme, and then purifying it. Figure 3 shows representative results, and nearly identical results were obtained in two additional experiments. [Figure 4] The results of TLC and liquid chromatography-mass spectrometry (LC-MS / MS) analysis of the hydrolysis products of the substrate NA2 treated with GH117A α-NABH at time intervals are shown. (A) NA2 (0.4%) was mixed with GH117A α-NABH (10 μg / mL) purified in 50 mM Tris-HCl (pH 7.5), and the mixture was reacted at 35°C for a specified time. Then, the same amount of each enzyme reaction product was analyzed by TLC. (B) The LC-MS / MS analysis of each enzyme reaction product was performed as described in the Materials and Methods section. Figure 4 shows representative results, and substantially the same results were obtained in two additional experiments. [Figure 5]This study compares the amino acid sequence of the Vibrio genus KY-GH-1GH117A α-NABH with the multiple amino acid sequences of nine GH117 family α-NABH strains obtained from the GenBank database. (A) The amino acids are aligned in an "open reading frame" using Clusteral-X to maximize identity, and each amino acid is represented by a single-letter abbreviation [Larkin et al. 2007]. Identical residues between all sequences are marked with an asterisk at the top of the sequence. Conservative substitutions are marked with a colon, and non-conservative substitutions with a dot, and each residue is highlighted in the Clusteral-X color scheme. Deletions are marked with a dash. (B) The phylogenetic relationships between Vibrio genus KY-GH-1GH117A α-NABH and the nine related GH117A α-NABH strains are based on the similarity of the amino acid sequences. The rooted phylogenetic tree was constructed using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) [Sneath and Sokal 1973]. The numbers on the nodes represent the bootstrap development level. Only values ​​above 50% are shown. The graduated bars indicate one substitution per 50 nucleotides. [Figure 6]The molecular chromatography analysis results of purified GH117A α-NABH are shown. (A) The molecular chromatography profile of purified GH117A α-NABH was performed using a Superdex™ 200 Increase 10 / 300 GL column equilibrated with 40 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. A protein solution containing 500 μg of GH117A α-NABH was injected into the column, and the column was eluted at a flow rate of 0.3 mL / min at 4°C. The protein elution profile was monitored by measuring the absorbance at 280 nm. (B) To measure the molecular weight of purified GH117A α-NABH, the following molecular weight size marker proteins were used under the same column elution conditions: position a, ferritin (440 kDa); position b, aldolase (158 kDa); position c, ovalbumin (44 kDa); position d, ribonuclease A (13.7 kDa). The elution peak position of GH117A α-NABH is marked with an arrow. [Figure 7] The biochemical properties of GH117A α-NABH are shown. (A,B) The effects of temperature and pH on enzyme activity were measured using 0.4% NA2 and 10 μg / mL purified GH117A α-NABH. Values ​​are shown as mean ± SEM (n=3, repeated 3 times per experiment). (C,D) The temperature and pH stability of purified GH117A α-NABH was examined after treating the enzyme for 4 hours at individual pH or temperature values. Values ​​are shown as mean ± SEM (n=3, repeated 3 times per experiment). (E) Lineweaver-Burk plots of Km and Vmax values ​​for purified GH117A α-NABH were examined using the indicated concentrations of enzyme and substrate NA2. Representative results are shown in Figure 7, and nearly identical results were obtained in two additional experiments. [Figure 8]This study demonstrates the effects of metal ions, reducing agents, and chelating agents on GH117A α-NABH activity. To measure the individual effects (A) of metal ions, the SH reducing agent TCEP (tris(2-carboxyethyl)phosphine), and the chelator EDTA (ethylenediaminetetraacetic acid) at 5 mM concentrations on GH117A α-NABH enzyme activity, as well as the combined effects of 5 mM MnSO4 / 5 mM TCEP or 5 mM MnSO4 / 10 mM TCEP, purified enzyme (10 μg / mL) was mixed with 20 mM Tris-HCl buffer (pH 7.5) and 0.4% NA2, and then treated at 35°C for 30 minutes. Enzyme activity measured without treatment with various ions and reagents was defined as 100%. Each value is shown as mean ± SD (n=3, measured 3 times per experiment). *P<0.05 and **P<0.01. [Figure 9] This paper describes the complete hydrolysis of NA2 to L-AHG / D-galactose catalyzed by the GH117A α-NABH enzyme and the purification of L-AHG from the hydrolysis product using Sephadex® G-10 column chromatography. (A) NA2 (2.0-5.0%) was treated with GH117A α-NABH (40 μg / ml) for 14 hours under optimal reaction conditions (5 mM MnSO4 and 10 mM TCEP, 35°C, pH 7.5), and then TLC analysis was performed to confirm the conversion of NA2 to L-AHG and D-galactose by hydrolysis. (B) To recover L-AHG from the hydrolysis product of NA2 catalyzed by the GH117A α-NABH enzyme, 4.0% NA2 (10 ml) was treated with GH117A α-NABH enzyme (20 μg / ml) for 14 hours under optimal conditions, and then lyophilized. The freeze-dried sample was dissolved in tertiary distilled water and then fractionated by molecular chromatography using a Sephadex (trademark) G-10 column. The presence or absence of L-AHG in each fraction was analyzed by TLC. Figure 9 shows representative results, and nearly identical results were obtained in two additional experiments. [Modes for carrying out the invention]

[0021] The present invention will be described in detail below.

[0022] In this invention, the enzymatic activity of recombinant proteins GH117A α-NABH and GH117B α-NABH obtained using an E. coli expression system and a pET-30a vector plasmid was compared. As a result, it was confirmed that GH117A α-NABH has significantly higher enzymatic activity in hydrolyzing the disaccharide NA2 to the monosaccharides L-AHG and D-galactose compared to GH117B α-NABH. Furthermore, the efficiency of the GH117A α-NABH enzyme (refer to the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2) in converting the NA2 substrate to L-AHG and D-galactose products under optimal reaction conditions was investigated.

[0023] Therefore, the present invention provides GH117A α-neoagarobiose hydrolase (α-NABH) containing Sequence ID No. 1.

[0024] The GH117A α-neoagarobiose hydrolase (α-NABH) is preferably derived from the Cellvibrio sp. KY-GH-1 strain, which has deposit number KCTC 13629BP.

[0025] The GH117A α-neoagarobiose hydrolase (α-NABH) is preferably obtained from a transformed E. coli organism into which the GH117A α-neoagarobiose hydrolase (α-NABH) gene has been incorporated.

[0026] The GH117A α-neoagarobiose hydrolase (α-NABH) gene is preferably cloned into an expression vector and incorporated into E. coli.

[0027] Furthermore, the present invention provides a method for producing 3,6-anhydro-L-galactose (L-AHG) by enzymatically degrading neoagarobiose (NA2) by treating it with GH117A α-neoagarobiose hydrolase (α-NABH).

[0028] The above process is preferably carried out in a temperature range of 25 to 45°C.

[0029] The aforementioned treatment is preferably carried out in a pH range of 6.0 to 10.0.

[0030] The above process is Mn 2+ It is preferable to further add the following.

[0031] The aforementioned Mn 2+ The substance is preferably in the form of MnCl2, MnSO4, or a mixture thereof. The concentration of the MnCl2, MnSO4, or a mixture thereof may be 1 to 10 mM, preferably 3 to 8 mM, more preferably 4 to 6 mM, and most preferably 5 mM.

[0032] The above treatment is preferably carried out with the addition of tris(2-carboxyethyl)-phosphine (TCEP). The concentration of TCEP may be 1 to 10 mM, preferably 3 to 8 mM, more preferably 4 to 6 mM, and most preferably 5 mM.

[0033] The present invention will be described in more detail below with reference to specific examples. The following examples describe one preferred specific example of the present invention, and it is clear that the scope of the rights of the present invention should not be limited by the matters described in the following examples. [Examples]

[0034] 1. Materials and Methods 1.1.Materials Restriction enzymes (NedI and XhoI) and T4 ligase were purchased from Roche (Basel, Switzerland). Naphthoresorcinol, D-galactose, isopropyl β-D-1-thiogalactopyranoside (IPTG), tris(2-carboxyethyl)phosphine (TCEP), 3,5-dinitrosalicylic acid (DNS), Coomassie Brilliant Blue (CBB) R-250, and kanamycin were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). The Micro BCA (trademark) kit was purchased from Pierce (Rockford, Illinois, USA), and the silica gel 60 aluminum thin-layer chromatography (TLC) plates coated with the F254 fluorescence indicator were purchased from Merck (Darmstadt, Germany). PageRuler® Pre-stained Protein Ladder and Ni-NTA resin were purchased from Thermo Fisher Scientific, Inc. (Rockford, Illinois, USA). The vector pET-30a for the expression of C-terminal 6xHis-tagged proteins was purchased from EMD Millipore, Inc. (Villerica, Massachusetts, USA), and E. coli BL21 (DE3) was purchased from Novagen, Inc. (Madison, Wisconsin, USA). Bio-Gel P-2 was purchased from Bio-Rad Laboratories, Inc. (Hercules, California, USA), and Sephadex® G-10 was purchased from GE Healthcare Biosciences AB, Sweden. The neoagarooligosaccharide (NAOS) mixture containing NA2-NA18 was provided by Dr. Lee Sang-hyun of Silla University [Lee et al., 2008]. As previously reported in a previous study, neoagarobiose (NA2) was purified from agarose hydrolysate produced by treatment with recombinant GH50A β-agarase [Kwon et al., 2020]. Specifically, the hydrolysate was freeze-dried, dissolved in tertiary distilled water, and molecular chromatography was performed using a Bio-Gel P-2 column.After eluting the column with tertiary distilled water (DI), each fraction was analyzed by TLC, and only the fraction containing NA2 was recovered. This was then freeze-dried to obtain NA2 powder. Standard samples NA2, NA4, and NA6 were purchased from Carbosynth (Berkshire, UK).

[0035] 1.2.E. Cloning and expression of GH117A and GH117B α-NABH genes using a coli expression system Two types of GH117 family α-NABH genes from the genus *Cervibrio* KY-GH-1 were amplified from hereditary DNA by PCR (polymerase chain reaction) using NdeI-forward primers (5'-CGCATATGGGTGATCTTCCAGAAAA-3' (SEQ ID NO: 3) for GH117A; 5'-GACAT-ATGAGCGACCAAGATTCTG-3' (SEQ ID NO: 4) for GH117B) and XhoI-reverse primers (5'-AACTCGAGGGAT-GCTACATTCTGAAAGG-3' (SEQ ID NO: 5) for GH117A; 5'-GGCTCGAGTGGATTGGATTTTCTAGCTT-3' (SEQ ID NO: 6) for GH117B). The amplified PCR products were purified, treated with NedI / XhoI, and then ligated to a pET-30a expression vector using T4 ligase. Recombinant pET-30a plasmid was used to transform E. coli BL21(DE3). Transformants containing either the GH117A α-NABH gene or the GH117B α-NABH gene were selected after being cultured overnight at 30°C on LB / kanamycin (50 μg / mL) agar plates. Induction of recombinant GH117A α-NABH or GH117B α-NABH expression was performed as previously described [Studier et al., 1990]. Specifically, each transformant was cultured in LB / kanamycin (50 μg / mL) medium at 25°C, followed by OD (Oral Dissociation). 600 When the concentration reached 0.5-0.6, 0.15 mM IPTG (isopropyl-β-thiogalactopyranoside) was added, and the mixture was further cultured at 25°C for 3 hours to induce the synthesis of recombinant GH117 α-NABH protein.

[0036] 1.3. Sequencing Analysis and Phylogenetic Tree Construction The amino acid sequences of the nine GH117 family α-NABHs were obtained by BLAST search using the NCBI database (http: / / www.ncbi.nlm.nih.gov). The amino acid sequences of individual open reading frames were obtained using Clusteral-X [Larkin et al., 2007]. Conserved amino acid residues were highlighted using the Clusteral-X color scheme. A rooted phylogenetic tree including individual GH117 family members was constructed using UPGMA based on amino acid sequence similarity [Sneath and Sokal, 1973].

[0037] 1.4. Quantification of cell lysates, proteins, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) To isolate and identify recombinant enzyme proteins produced by transformants, cells were suspended in 40 mM Tris-HCl buffer (pH 8.0), disrupted by sonication 20 times for 10 seconds, extracted at 4°C for 30 minutes, and then separated into three fractions: total fraction, soluble fraction, and insoluble fraction [Jun et al., 1996]. Protein quantification of cell lysates was performed using the Micro BCA (trademark) kit (Pierces, Inc., Rockford, Illinois, USA). The same amount of cell lysate (10 μg) was subjected to electrophoresis on an 8% SDS-polyacrylamide gel according to the Laemmli method [Laemmli, 1970]. After electrophoresis, the gel was stained with CBB R-250 (Coomsie Brilliant Blue R-250) and protein bands were detected. To quantify the concentration of a specific recombinant protein on an electrophoretic gel, the density of recombinant protein bands on the gel was measured, and the measured units were compared to a standard curve established as the unit for measuring the density of sequentially diluted bovine serum albumin (BSA) bands detected on the same gel. The density of protein bands on the gel was measured using ImageQuant™ TL software (Amersham, Arlington Heights, Illinois, USA) [Syrovy and Hodny, 1991].

[0038] 1.5. Purification of recombinant His-tagged α-NABH To purify recombinant His-tagged GH117A α-NABH or His-tagged GH117B α-NABH, a soluble fraction of cell lysates from E. coli transformants containing recombinant His-tagged GH117A α-NABH or recombinant His-tagged GH117B α-NABH in a soluble form was first secured. The recombinant enzyme proteins contained in these soluble fractions were purified by an immobilized metal ion affinity chromatography method using Ni-NTA resin [Spriestersbach et al., 2015].

[0039] 1.6. Measurement of the molecular weight of GH117A α-NABH by molecular chromatography Molecular chromatography was performed using a Superdex™ 200 Increase 10 / 300 GL column (GE Healthcare) equilibrated with 40 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Purified GH117A α-NABH (0.5 mg / 0.5 mL) was injected into the column and eluted under molecular chromatography conditions at 4°C at a flow rate of 0.3 mL / min. Protein elution was measured at a wavelength of 280 nm. A standard curve of retention capacity relative to protein molecular weight was established using size markers such as ferritin (440 kDa), aldolase (158 kDa), ovalbumin (44 kDa), and ribonuclease A (13.7 kDa), and the molecular weight of purified GH117A α-NABH was measured using this standard.

[0040] 1.7. Activity Analysis of GH117 α-NABH The activity of GH117 α-NABH was measured by detecting reducing sugars released from NA2 using the DNS method [Miller, 1959]. To measure the activity of GH117 α-NABH, a purified enzyme solution (~2 μg / 100 μl) was mixed with the same volume of 0.8% NA2 dissolved in 20 mM Tris-HCl buffer (pH 7.5). After reacting at 35°C for 30 minutes, the reducing sugars formed in the reaction mixture were measured by color development using the DNS reagent. One unit of enzyme activity was defined as the amount of enzyme that generates reducing power equivalent to 1 μmol of D-galactose per minute. The enzyme reaction rate constant of GH117A α-NABH was measured by adding a specified amount of enzyme to a substrate solution of NA2 (1~10 mg / ml, 20 mM Tris-HCl, pH 7.5). The Km and Vmax values ​​were calculated using the Lineweaver-Burk equation with the GraphPad Prism 8 statistical package (GraphPad Software, Inc., USA).

[0041] 1.8. Thin-layer chromatography (TLC)

[0042] TLC analysis of NA2 hydrolysates treated with GH117 α-NABH was performed on SilicaGel 60 aluminum plates and developed using n-butanol-ethanol-H2O[3:2:2(v / v)] solvent. To identify sugar substances on the TLC plate, a color-developing solution of ethanol, 0.2%(w / v) naphthresorcinol (Sigma-Aldrich), and 10%(v / v) H2SO4 was sprayed onto the TLC plate and then heated at 80°C for detection.

[0043] 1.9. Purification of L-AHG by size exclusion column chromatography To purify L-AHG from the NA2 hydrolysis product, NA2 was treated with GH117A α-NABH under optimal reaction conditions (5 mM MnSO4, 10 mM TCEP, 35°C, pH 7.5) for 14 hours, and the enzyme reaction product was freeze-dried. The freeze-dried sample was dissolved in tertiary distilled water (DI), and molecular chromatography was performed using a Sephadex™ G-10 column (ID 1.3 x 90 cm). Each fraction was collected in 2 mL portions while eluting the column with distilled water (DI). The fraction containing L-AHG was identified by TLC, collected, and freeze-dried.

[0044] 1.10. Confirmation of enzyme reaction products by liquid chromatography using Tandam mass spectrometry (liquid chromatography-mass spectrometry (LC-MS / MS)). LC-MS / MS analysis was performed using a Xevo TQ-S micromass spectrometer connected to an electrospray ionization (ESI) ion source with a Waters Acquity UPLC H-Class core system (Waters, Milford, Massachusetts, USA) [Zeng et al., 2016; Koti et al., 2013]. Sample elution was performed using a Waters Acquity UPLC Spherisorb amino column (2 mm × 100 mm, 3 μm particle size) maintained at a solvent flow rate of 200 μL / min and 40°C. The LC system consisted of (A) a 0.1% aqueous formic acid solution and (B) a 0.1% acetonitrile solution. To identify D-galactose and L-AHG in the enzymatic hydrolysate of substrate NA2, chromatography was first performed for 3 minutes starting with a solvent mixture of A:B in a 95:5 ratio. The A:B gradient was gradually changed to a 75:25 ratio at 13 minutes after the start of chromatography, and this ratio was maintained until 15 minutes. Subsequently, the A:B ratio was changed back to the initial 95:5 at 16 minutes after the start of chromatography, and this ratio was maintained until chromatography was stopped at 30 minutes after the start. The injection volume was 5 μL, and the sample manager temperature was set to 5°C. The 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; desolvation gas flow, 550 m / h; cone gas flow, 5 L / h; mass range, 100~800.

[0045] 1.11.Statistical analysis Unless otherwise noted, data were presented using a minimum of three independent experiments. All data are expressed as mean ± standard deviation (SD, for each group n ≤ 3). Statistical analysis involved evaluating the difference between two groups and the significance of one-way ANOVA using Student's t-test, followed by comparisons of three or more groups using Dunnett's multiple comparison post-hoc test. A p-value < 0.05 indicates statistical significance. Statistical analysis was performed using SPSS Statistics version 23 (IBM, Armonk, New York, USA).

[0046] 2.Results 2.1. Expression of GH117A α-NABHs and GH117B α-NABH enzymes from *Vibrio ceruvianus* KY-GH-1 as C-terminally His-tagged recombinant proteins using the *E. coli* and pET-30a vector system. The inventors of this invention confirmed the existence of two GH117 α-NABH genes (α-CvNabh117A and α-CvNabh117B) by analyzing the entire genome sequence of the bacterium *Vibrio cerevisiae* KY-GH-1 in prior research. As shown in Figure 1, α-CvNabh117A has an "open" reading frame (ORF) that encodes 364 amino acids constituting a 40.9 kDa protein (GH117A α-NABH), while α-CvNabh117B has an "open" reading frame (ORF) that encodes 392 amino acids constituting a 44.2 kDa protein (GH117B α-NABH). At this time, the ORF base sequence of α-CvNabh117A showed 53% similarity to that of α-CvNabh117B, and the amino acid sequences showed 35% similarity between them. This indicates that there is not much homology between the two GH117 α-NABHs present in the Vibrio cellulosus strain KY-GH-1. On the other hand, since neither the GH117A α-NABH nor the GH117B α-NABH enzyme proteins have an N-terminal signaling peptide sequence, this suggests that both enzymes can function inside the cells of Vibrio cellulosus strain KY-GH-1, i.e., in the cytoplasm.

[0047] In this invention, based on such data, two enzymes were overexpressed and purified as recombinant His-tagged enzyme proteins using an E. coli expression system, and it was investigated which of these purified recombinant GH117A α-NABH or recombinant GH117B α-NABH had higher enzymatic activity and more selective enzymatic activity towards NA2. After inducing the expression of enzyme proteins from each E. coli transformant expressing recombinant GH117A α-NABH or GH117B α-NABH by IPTG treatment, the cells were harvested and sonicated to obtain total fraction, soluble fraction, and insoluble fraction. Equal amounts of each fraction were taken and subjected to electrophoresis on an 8% SDS polyacrylamide gel, and the gel was stained with CBB to confirm the protein bands, thereby determining the soluble / insoluble ratio of each recombinant enzyme. As shown in Figure 2a, both GH117A α-NABH and GH117B α-NABH were predominantly detected in the soluble fraction of transformed cells, and the amount of each recombinant enzyme detected in the insoluble inclusion body fraction was not significant. When His-tagged GH117A and GH117B enzymes were purified in the cell soluble fraction using the Ni-NTA purification system, they were detected as a single protein band on 8% SDS-PAGE (Figure 2b). When the molecular weights (MW) of the purified recombinant GH117A and GH117B enzyme proteins were compared with size markers on the electrophoresis gel, the recombinant GH117A enzyme protein was found to be ~39.0 kDa, and the GH117B enzyme protein was found to be ~44.8 kDa.

[0048] When the NA2 hydrolysis activity of GH117A α-NABH and GH117B α-NABH was examined by TLC, GH117A α-NABH completely hydrolyzed NA2 to L-AHG and D-galactose, while GH117B α-NABH failed to hydrolyze NA2 (Figure 2c). To further investigate the difference in substrate specificity between GH117A α-NABH and GH117B α-NABH, the substrate (a mixture of NA2-NA18 NAOS) was treated with each enzyme at different time intervals, and the degradation products over time were analyzed by TLC. As a result, among the NA2-NA18 NAOS mixture, only NA2 was hydrolyzed by GH117A α-NABH treatment and converted to L-AHG and D-galactose, and NA2 hydrolysis began from the moment the enzymatic reaction started, with the amount of L-AHG produced continuously increasing during the 60-minute reaction period (Figure 2d). However, under the same conditions, NA4-NA18 were not degraded by GH117A α-NABH. This indicates that among NAOS (NA2-NA18), NA2 is the only substrate specifically targeted for GH117A α-NABH hydrolysis. On the other hand, it was shown that none of the NAOS (NA2-NA18) mixtures could hydrolyze GH117B α-NABH (Figure 2e). To confirm that NA2 is the sole substrate for GH117A α-NABH hydrolysis, standard samples NA2, NA4, and NA6 were treated with enzymes, and the individual hydrolysates were analyzed by TLC. The results showed that NA2 was completely degraded into L-AHG and D-galactose, while NA4 and NA6 were not, confirming that NA2 is the sole substrate for GH117A α-NABH catalysis (Figure 3).

[0049] Furthermore, TLC analysis revealed that when 0.4% NA2 was treated with purified recombinant GH117 α-NABH (10 μg / ml) in 50 mM Tris-HCl buffer (pH 7.5) at 35°C, the hydrolysis of NA2 increased in direct proportion to the reaction time, and it was confirmed that it was completely converted to monosaccharides after 60 minutes of reaction (Figure 4a). In addition, LC-MS / MS analysis further demonstrated that NA2 is converted to L-AHG and D-galactose in a time-dependent manner by the hydrolysis action of the enzyme (Figure 4b). These results support the conclusion that GH117A α-NABH is an essential enzyme indispensable for the hydrolysis of NA2 to L-AHG and D-galactose, which is the final enzymatic step in agarose saccharification, in the KY-GH-1 strain.

[0050] 2.2. Comparison of the amino acid sequence of GH117A α-NABH with the amino acid sequences of related enzymes from other bacterial strains. The amino acid sequence of GH117A α-NABH was compared and analyzed with the amino acid sequences of GH117A α-NABH from other agar-degrading bacteria using the ClusterX program [Larkin et al., 2007]. As shown in Figure 5a, the Vibrio genus KY-GH-1GH117A α-NABH (GenBank accession number WP_151030319.1) showed a 97.5% similarity to the Vibrio genus KY-YJ-3GH117A α-NABH (GenBank accession number WP_151057276.1). Furthermore, KY-GH-1GH117A α-NABH showed similarities of 97.3%, 97.3%, and 96.7% with *Vibrio celvirio* species *pealriver* (GenBank accession number WP_049629412.1) [Xie et al., 2017], *Vibrio celvirio* species *BR* (GenBank accession number WP_007640738.1), and *Vibrio celvirio* species *OA-2007* (GenBank accession number WP_062065015.1) [Syazni et al., 2015]. This indicates a high level of homology among *Vibrio celvirio* species GH117A α-NABH.

[0051] On the other hand, the amino acid sequence of GH117A α-NABH is the same as that of Agrobacterium haliotis (GenBank accession number WP_096085215.1), Gilvimarinus polysaccharolyticus (GenBank accession number WP_049720980.1), Gilvimarinus chinensis (GenBank accession number WP_020208680.1), Vibrio fluvialis (GenBank accession number WP_171934150.1), and Vibrio species EJY3 (GenBank accession number 014232194.1) GH117A The amino acid sequences of these molecules showed similarities of 77.5%, 77.4%, 77.2%, 76.8%, and 76.5% to those of α-NABH, respectively.

[0052] In the rooted phylogenetic trees of individual GH117A α-NABH strains, the genus *Cervibrio* KY-GH-1 GH117A α-NABH was more closely grouped with GH117A α-NABHs from *Cervibrio* KY-YJ-3, *Cervibrio* Pearl River, *Cervibrio* BR, and *Cervibrio* OA-2007 isolated in non-marine environments, compared to GH117A α-NABHs from marine *Agrobacterium haliotis*, *Gilvimarinus polysaccharolyticus*, *Gilvimarinus chinensis*, *Vibrio fluvialis*, and *Vibrio* EJY3 (Figure 5b).

[0053] Current analytical results showing that homologs of GH117A α-NABHs are present in all agar-degrading bacteria with a high level of amino acid sequence homology suggest that GH117A β-NABH, an enzyme that hydrolyzes the α-1,3-linkage of the NA2 substrate to produce the monosaccharides L-AHG and D-galactose, is an essential component enzyme in the agarose saccharification process carried out by agar-degrading bacteria.

[0054] 2.3. Enzymatic properties of GH117A α-NABH Several GH117 family α-neoagarooligosaccharide hydrolases (α-NAOSH) and α-NABH have been reported to perform enzymatic function in a dimeric form, whether located inside or outside the cytoplasm. In this study, to confirm whether recombinant GH117A α-NABH produced using an E. coli expression system hydrolyzes NA2 to L-AHG and D-galactose in a dimeric form, the molecular weight of purified recombinant GH117A α-NABH was measured by molecular chromatography using a Superdex™ 200 Increase 10 / 300 GL column.

[0055] The results showed that the measured molecular weight of GH117A α-NABH was 92.1 kDa (Figure 6). The calculated molecular weight of GH117A α-NABH, which is composed of 364 amino acids, is 40.9 kDa. This is consistent with the molecular weight of GH117A α-NABH estimated by SDS-PAGE analysis (~39.0 kDa), indicating that the molecular chromatography data shows that the purified GH117A α-NABH exists as a dimer.

[0056] In mass-producing L-AHG from NA2, the enzyme properties were examined to determine the effectiveness of recombinant GH117A α-NABH. When investigating the effect of temperature on the GH117A α-NABH enzyme activity, the highest activity was shown at 35 °C (a in Figure 7). Less than 50% of the maximum activity was retained at temperatures outside the range of 25 - 45 °C. The enzyme showed activity in a rather wide pH range of 6.0 - 10.0 (optimal pH 7.5) (b in Figure 7). The enzyme was stable up to 35 °C. However, after treatment at 40 °C for 4 hours, only 40% of the maximum activity was retained, and after treatment at temperatures above 45 °C for 4 hours, most of the enzyme activity was lost (c in Figure 7). Although GH117A α-NABH was stable in the pH range of 7.0 - 7.5, it quickly became inactive at acidic pH below 6.0 and retained 50 - 60% of its activity after treatment for 4 hours at pH 8.0 - 10.0. Such observation results indicate that GH117A α-NABH is more stable at alkaline pH than at acidic pH (d in Figure 7). The Km, Vmax, Kcat, and Kcat / Km values, which are the enzyme reaction rate constants of GH117A α-NABH for the substrate NA2, were 16.0 mM, 20.8 U / mg, 14.2 s -1 and 8.9×10 2 s -1 ·M -1 respectively (e in Figure 7).

[0057] The GH117A α-NABH activity was increased 1.4-fold, 1.5-fold, and 1.2-fold respectively when 5 mM MnCl2, 5 mM MnSO4, and 5 mM TCEP were present, but 85% of the enzyme activity was inhibited in the presence of 5 mM EDTA (a in Figure 8). The enhancing effects of Mn 2+ and TCEP on the enzyme activity were in a volume-dependent manner. 5 mM MnSO4 and 10 mM TCEP increased the enzyme activity to 1.5-fold and 1.7-fold of the maximum level respectively (b in Figure 8). Also, when 5 mM MnSO4 and 10 mM TCEP were present simultaneously, the enzyme activity increased significantly up to about 2.4-fold, and for the Mn 2+We also confirmed the synergistic effects of TCEP.

[0058] These results suggest that GH117A α-NABH may require manganese ions to exhibit proper enzymatic activity, but that the interaction with manganese ions may not be strong enough to resist the chelating effect of 5 mM EDTA.

[0059] 2.4. Yield of L-AHG produced by NA2 treatment with GH117A α-NABH under optimal conditions. To investigate whether GH117A α-NABH is an efficient enzyme for L-AHG production from NA2, the maximum concentration of NA2 at which GH117A α-NABH can completely hydrolyze to L-AHG and D-galactose was examined. NA2 at various concentrations (2.0%, 3.0%, 4.0%, and 5.0%) was reacted with GH117A α-NABH (40 μg / ml) for 14 hours under optimal reaction conditions (5 mM MnSO4 and 10 mM TCEP, 20 mM Tris-HCl, pH 7.5, 35°C), and the hydrolysis products were analyzed by TLC. The results showed that complete hydrolysis of the NA2 substrate to L-AHG and D-galactose by enzyme catalytic action was observed at NA2 concentrations from 2.0% to 5.0% (Figure 9a). However, incomplete hydrolysis was observed at NA2 concentrations above 6.0% (data omitted).

[0060] On the other hand, 5% NA2 (8 ml) was treated with GH117A α-NABH (40 μg / mL) under optimal reaction conditions for 14 hours, and the enzymatic reaction product was freeze-dried. The dried sample was dissolved in tertiary distilled water and fractionated by molecular chromatography using a Sephadex® G-10 column. TLC analysis of each fraction revealed D-galactose in fractions 30-31 and L-AHG in fractions 33-39 (Figure 9b). Fractions 33-39 were collected and freeze-dried to obtain L-AHG in powder form. 400 mg of the hydrolyzed NA2 obtained by enzymatic treatment was fractionated twice by Sephadex® G-10 column chromatography, resulting in the recovery of a total of approximately 192 mg of L-AHG, corresponding to approximately 92% of the theoretical maximum yield.

[0061] 3. Discussion In this invention, the GH117A β-NABH enzyme derived from the freshwater agar-degrading bacterium Cervibrio KY-GH-1 was produced as a soluble His-tagged recombinant protein using an E. coli expression system. The substrate specificity of the produced recombinant GH117A β-NABH enzyme was investigated, and it was confirmed that while the α-1,3-link of the disaccharide NA2 was hydrolyzed to the monosaccharides L-AHG and D-galactose, the enzyme was completely unable to hydrolyze the α-1,3-links present in NAOS of various DPs such as NA4 to NA18. Furthermore, 5.0% of the NA2 substrate was treated with recombinant GH117A β-NABH enzyme under optimal reaction conditions to completely hydrolyze it to L-AHG and D-galactose. After purifying the L-AHG from the hydrolysate by molecular chromatography using a Sephadex (trademark) G-10 column, approximately 192 mg of L-AHG was recovered, which corresponds to approximately 92% of the theoretical maximum yield of L-AHG that can be produced from 400 mg of NA2.

[0062] In previous research, the inventors of the present invention analyzed the base sequence of the entire genetic material of the agar-degrading bacterium Cervibrio KY-GH-1 strain and inferred that it contains two provisional GH117 α-NABH genes (α-CvNabh117A and α-CvNabh117B) that encode GH117A α-NABH (364 amino acids, 40.9 kDa) and GH117B α-NABH (392 amino acids, 44.2 kDa). The GH117 α-NABH enzyme is known to contribute to the final stage of enzymatic saccharification of agarose by hydrolyzing the disaccharide NA2 into the monosaccharides L-AHG and D-galactose. Even though both recombinant His-tagged GH117 α-NABH enzymes were expressed in a soluble form in E. coli transformants, only GH117A α-NABH exhibited the function of hydrolyzing NA2 to monosaccharides. This indicates that GH117A α-NABH is an essential enzyme indispensable to the final enzymatic step of agarose degradation in the Cervibrio strain KY-GH-1. Various research papers have reported that GH117 family α-NAOSH, including α-NABH, recognize NA2 as a substrate and hydrolyze it to L-AHG and D-galactose. However, it has also been reported that such α-NAOSH is not specific only to the NA2 substrate, but also acts on NA4 to produce L-AHG and agarotriose (A3), and also acts on NA6 to produce L-AHG and agaropentaose (A5), which is also retained. In particular, it has been reported that the α-NAOSH of Streptomyces coericolor A3, which has been studied recently, acts on NAOS of various degrees of polymerization (DPs) from 2 to 14, hydrolyzing only the first α-1,3-glycosidic bond at these non-reducing ends.In this invention, recombinant GH117A α-NABH enzyme protein was produced and purified using an E. coli expression system. The substrate specificity was then investigated using a NAOS mixture containing various DPs (diaphoretic proteins) with NA2-NA18 as substrates. The results showed that NA2 was hydrolyzed to L-AHG and D-galactose, while the remaining NA4-NA18 were completely unaffected by the enzymatic activity of GH117A α-NABH. Furthermore, additional investigations into the substrate specificity of GH117A α-NABH using standard NA2, NA4, or NA6 revealed that NA2 is the sole substrate of GH117A α-NABH and is completely hydrolyzed to L-AHG and D-galactose. These results indicate that GH117A α-NABH is an enzyme specialized in the hydrolysis of NA2, and this NA2 substrate specificity clearly distinguishes it from previously reported enzymes. On the other hand, although the amino acid sequence of GH117B α-NABH shares 35% similarity with that of GH117A α-NABH, it was unable to hydrolyze any of NA2-NA18, confirming that the enzymatic function of GH117B α-NABH does not contribute to the production of L-AHG from NAOS (NA2-NA18). The role of GH117B α-NABH in the Cervibrio strain KY-GH-1 remains unknown and is still unresolved.

[0063] When the amino acid sequence of GH117A α-NABH was aligned with the top nine congeners found in the NCBI GenBank database (http: / / www.ncbi.nlm.nih.gov / ) using the ClusterX program, it was confirmed that the GH117A α-NABH congener is present in all marine and non-marine agar-degrading bacteria and exhibits a high level of sequence homology (76.5-97.5%). This suggests that GH117A α-NABH is an essential component of the bacterial agar-degrading enzyme system. Furthermore, among these nine GH117 α-NABHs, the GH117A α-NABH from the Vibrio strain EJY3 is the only one whose enzymatic properties have been investigated and reported to degrade NA2 and NA4 as substrates. Therefore, the current data on GH117A α-NABH reported in this invention can provide insights into the GH117 family of α-NABHs that show considerable homology to GH117A α-NABH.

[0064] Furthermore, since none of these nine GH117 α-NABHs have been previously investigated for their enzymatic properties, the current data on GH117A α-NABH reported in this study can provide insights into GH117 family α-NABHs that show considerable homology to GH117A α-NABH.

[0065] The Km and Vmax values ​​(16.0 mM and 20.8 U / mg) of the enzymatic reaction rate constants of GH117A α-NABH against NA2 are comparable to those of other reported α-NABHs, including α-NAOSH from Vibrio species JT0107 (5.37 mM and 92 U / mg), namely α-NAOSH from Vibrio species OA-2007 (6 mM and 19 U / mg), α-NAOSH from Vibrio species WU-0601 (5.8 mM and 60 U / mg), α-NABH from Agarovorans gilvus WH0801 (6.45 mM and 6.98 U / mg), and Gayadomonas juubinieghe. The α-NABH of G7 (4.5 mM and 1.33 U / mg) and the α-NAOSH of Streptomyces coelicolor A3 (11.57 mM and no data available) should have been compared. Furthermore, the Vmax and Kcat values ​​of GH117A α-NABH relative to NA2 (20.8 U / mg and 14.2 s) were also relevant. -1 Regarding this enzyme, it was shown to be even more catalytic than other enzymes compared. Furthermore, it is noteworthy that none of the previously reported α-NAOSHs showed substrate specificity for NA2, in contrast to the NA2-specific enzymatic activity of GH117A α-NABH, which hydrolyzes only the α-1,3-glycosidic bond of NA2.

[0066] Generally, Mg 2+ and Na + Unlike marine β-agarases, which have been reported to depend on the presence of Mn, the GH117A α-NABH enzyme activity from the Cervibrio strain KY-GH-1 was not significantly affected by MgCl2 or NaCl. Instead, the GH117A α-NABH enzyme activity was affected by Mn 2+ Alternatively, it was significantly improved in a concentration-dependent manner in the presence of the reducing agent TCEP. Mn activity against GH117A α-NABH enzyme activity. 2+A synergistic effect of TCEP was observed, showing that enzyme activity increased 2.4 times when 5 mM MnSO4 and 10 mM TCEP were present together. Simultaneously, the enzyme reaction rate constants of GH117A α-NABH with respect to the NA2 substrate, Km, Vmax, Kcat, and Kcat / Km, were 5.8 mM, 33.7 U / mg, and 23.0 s, respectively. -1 and 4.0×10 3 s -1 ·M -1 This was shown (data omitted). Such research results indicate that GH117A α-NABH enzyme activity is related to Mn 2+ This demonstrates that the concentration-dependent enhancement of this enzyme in the presence of / TCEP is a unique enzymatic property.

[0067] Under optimal reaction conditions (5 mM MnSO4, 10 mM TCEP, 20 mM Tris-HCl, pH 7.5, 35°C), the GH117A α-NABH (40 μg / ml) enzyme was able to completely hydrolyze up to 5.0% of NA2 into the monosaccharides L-AHG and D-galactose. L-AHG, generated from the NA2 substrate by GH117A α-NABH enzymatic treatment, could be easily purified by fractionating the enzyme reaction product using molecular chromatography with a Sephadex™ G-10 column. Using this method, the recovery rate of L-AHG from the 5% NA2 hydrolysate obtained by GH117A enzymatic treatment was shown to be approximately 92% of the theoretical maximum yield.

[0068] Recently, in connection with the mass production of L-AHG, a constituent monosaccharide from the polysaccharide agarose, a two-step enzymatic process has been proposed, involving a NA2 production step using agarose degradation by exo-type β-agarase and a subsequent hydrolysis step of NA2 to L-AHG and D-galactose by GH117 α-NABH. Furthermore, a method has been reported for co-immobilizing β-agarase and α-NABH enzymes and utilizing them for hydrolysis of agarose with L-AHG. However, it is suspected that the enzymatic processing steps proposed in these prior studies have inefficient aspects that should be improved depending on the enzymatic characteristics of the enzymes used. In other words, the exo-type β-agarase used produced NAOS of various DPs in addition to NA2 from agarose, and the GH117 α-NABH used was not an enzyme specialized in NA2, but also acted on NA4 and NA6 to produce L-AHG / agarotriose (A3) and L-AHG / agalopentaose (A5), respectively, which had the drawback of reducing the efficiency of breaking down the polysaccharide agarose into the monosaccharides L-AHG and D-galactose.

[0069] Therefore, the development of not only an effective exo-type GH50β-agarase capable of producing NA2 as the main product from agarose, but also a GH117 α-NABH specifically capable of hydrolyzing the α-1,3-glycosidic bond only to the NA2 substrate, is currently highly desired for an efficient enzymatic process to produce monosaccharides using the enzymatic saccharification of agarose.

[0070] Based on the enzyme properties of GH117A β-NABH disclosed in this invention, it is expected that using GH117A β-NABH, which is specialized for the hydrolysis of NA2, will enable a one-step enzymatic process to produce L-AHG from NA2, which should become the standard for mass production of L-AHG, and will also significantly contribute to accelerating the mass production of L-AHG. Furthermore, we are confident that a method of combining GH117A α-NABH with a potent exo-type GH50A β-agarase, which can efficiently convert agarose to NA2, will also be useful as a process for saccharifying agarose for L-AHG production.

[0071] 4. Summary The results of this invention demonstrate that recombinant GH117A α-NABH derived from the agar-degradable Cervibrio strain KY-GH-1 can completely hydrolyze up to 5% of NA2 into the monosaccharides L-AHG and D-galactose. Since it was confirmed that none of NA4 to NA18, except for the substrate NA2, are hydrolyzed by GH117A α-NABH, it was revealed that the enzymatic activity of GH117A α-NABH is specialized for NA2. The optimal temperature and pH for the hydrolysis of substrate NA2 by the GH117A α-NABH enzyme were 35°C and 7.5, respectively. The GH117A α-NABH enzyme was stable up to 35°C and within the pH range of 7.0 to 7.5, but unstable above 35°C and beyond the pH range of 7.0 to 7.5. The GH117A α-NABH enzyme activity was increased 2.4-fold in the presence of 5 mM MnSO4 and 10 mM TCEP. The reaction rate constants of the GH117A α-NABH enzyme to NA2, i.e., Km, Vmax, Kcat, and Kcat / Km values, were 16.0 mM, 20.8 U / mg, and 14.2 s, respectively. -1 and 8.9 × 10 2 s -1 ·M -1 In particular, when 5 mM MnSO4 and 10 mM TCEP were present together, the Km, Vmax, Kcat, and Kcat / Km values ​​of GH117A α-NABH against the NA2 substrate were 5.8 mM, 33.7 U / mg, and 23.0 s, respectively.-1 and 4.0×10 3 s -1 ·M -1 This was demonstrated. The enzymatic properties of such recombinant α-NABH suggest that it may be useful for mass production of L-AHG using a one-step enzymatic process that decomposes the disaccharide NA2 into the monosaccharides L-AHG and D-galactose. As an alternative, it is suggested that a process combining such a saccharification process with a potent GH50 family β-agarase capable of efficiently decomposing agarose into NA2 via an exo-type hydrolysis method may also be useful.

[0072] Finally, among the nine α-NABH enzymes that share 76.5-97.5% similarity in amino acid sequence with GH117A α-NABH, the enzyme properties of GH117A α-NABH derived from the Vibrio strain EJY3, which shares 76.8% similarity, have not been investigated until now. Therefore, the research results on the GH117A α-NABH enzyme of the agar-degrading bacterium Vibrio KY-GH-1 disclosed in this invention can provide insights into α-NABH enzymes derived from other agar-degrading bacteria that share similarities with GH117A α-NABH. [Accession Number]

[0073] Depository name: Korea Institute of Biotechnology Accession number: KCTC 13629BP Entrustment date: August 27, 2018

Claims

1. A method for producing 3,6-anhydro-L-galactose, comprising enzymatically degrading neoagarobiose by treating it with GH117A α-neoagarobiose hydrolase having an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, by adding MnCl₂ and MnSO₄ to the neoagarobiose.

2. The method for producing 3,6-anhydro-L-galactose according to claim 1, characterized in that the process is carried out in a temperature range of 25 to 45°C.

3. The method for producing 3,6-anhydro-L-galactose according to claim 1, characterized in that the above treatment is carried out in a pH range of 6.0 to 10.

0.

4. The method for producing 3,6-anhydro-L-galactose according to claim 1, characterized in that the above treatment is further carried out by adding tris(2-carboxyethyl)phosphine.

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