# Patent

## Title
Agarooligosaccharide hydrolase and method for producing 3,6-anhydro-L-galactose and galactose from agarose by using same

## Abstract

The present invention relates to agarooligosaccharide hydrolase and a method for producing 3,6-anhydro-L-galactose and galactose from agarose by using the same. More specifically, the production yield of 3,6-anhydro-L-galactose and galactose from agarose, that is, the saccharification yield, is improved by using β-agarooligosaccharide hydrolase having an agarotriose hydrolytic activity.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase of International Patent Application No.: PCT/KR2014/009470, filed Oct. 8, 2014, which claims priority to Korea Patent Application No. 10-2013-0121990, filed Oct. 14, 2013, the disclosures of which are incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to a novel β-agarooligosaccharide hydrolase having hydrolytic activity for agarooligosaccharides and to a method of producing 3,6-anhydro-L-galactose and galactose from agarose by using the same.
BACKGROUND ART
Compared to plant biomass currently used in many areas, algae have a lower content of recalcitrant components, such as lignin to use. Therefore, algae are advantageous in that they can be converted much more easily into a monosaccharide which is a substrate for producing bioenergy and biochemicals. Also, by not using food resources, algae are free from the problem of using food resources in producing energy. For these reasons, algae are receiving attention as important biomass in an aspect of the production of biochemicals including alternative energy.
Among algae, especially red algae (e.g., Gelidium amansii) is not only a raw material for the production of such bioenergy and biochemicals, but is also reported to contain agarooligosaccharides, which exhibit excellent physiological activity such as antioxidation, anti-inflammation, anti-cancer, anti-allergy, whitening, and moisturization, as a component, such that it is usefully employed in pharmaceutical and cosmetic fields (U.S. Pat. No. 7,662,2291 by Tomono et al. (2009); U.S. Patent No. 69/143,282 by Enoki et al. (2005)).
As a main constituent, red algae contain agarose which is a polymer having neoagarobiose (i.e. 3,6-anhydro-L-galactose and D-galactose bonded by an α-1,3-linkage) as a basic unit that is connected with another unit by a β-1,4-linkage. The present inventors empirically proved the fact that the physiological functionality of 3,6-anhydro-L-galactose (Yun et at (2013) Appl Microbiol Biotechnol. 97(7):2961-70). Also reported in relation to the production of the 3,6-anhydro-L-galactose is a preparation process of 3,6-anhydro-L-galactose and D-galactose by a mild chemical pretreatment and enzymatic saccharification to replace chemical treatment methods (Jol et at (1999) Anal Biochem. 268, 213-222; Kim et at (2010) Bull Korean Soc. 31(2) 511-514) that have problems of a very low yield and a high possibility of excessive hydrolysis.
Agarose is depolymerized by an exo-type Aga50D enzyme after a chemical pretreatment into neoagarobiose and agarotriose (D-galactose-β-1,4 linkage-3,6-anhydro-L-galactose-α-1,3 linkage-D-galactose) which are main reaction products. Then, the neoagarobiose is ultimately hydrolyzed into 3,6-anhydro-L-galactose and D-galactose by SdNABH (Korean Patent No. 10-1293668), which is an α-neoagarobiose hydrolase. During this process, a chemical pretreatment under mild conditions is essential to improve the reactivity of the Aga50D enzyme. However, agarotriose produced using a chemically pretreated substrate cannot be ultimately decomposed into 3,6-anhydro-L-galactose and D-galactose and remains as a reaction residue.
If the remaining agarotriose could be ultimately decomposed into 3,6-anhydro-L-galactose and D-galactose, a larger improvement in the production yield of 3,6-anhydro-L-galactose and D-galactose would be expected. Since endo-type and exo-type β-agarases cannot hydrolyze a substrate such as agarotriose, another approach of decomposing agarotriose into D-galactose and neoagarobiose using a β-galactosidase was tried, but commercially available β-galactosidases did not show hydrolytic activity for agarooligosaccharides.
DISCLOSURE
Technical Problem
The present invention is directed to providing a method of improving efficiency of agarose saccharification using a novel enzyme having hydrolytic activity for agarooligosaccharides.
The present invention is also directed to providing a method of producing galactose using a novel enzyme having hydrolytic activity for agarooligosaccharides.
Technical Solution
To achieve the above objectives, the present invention provides a composition for agarose saccharification comprising an agarase; a β-agarooligosaccharide hydrolase having hydrolytic activity for agarotriose and represented by an amino acid sequence of SEQ ID NO:1; and an α-neoagarobiose hydrolase.
The present invention also provides a method of saccharifying agarose by reacting a substrate and a composition for agarose saccharification according to the present invention, wherein the substrate is selected from the group consisting of agarose, agarotriose, and neoagarobiose.
The present invention also provides a composition for producing galactose comprising a β-agarooligosaccharide hydrolase having hydrolytic activity for agarotriose, agaropentaose, or agaroheptaose and represented by an amino acid sequence of SEQ ID NO:1.
In addition, the present invention provides a method of producing galactose by reacting a substrate and the β-agarooligosaccharide hydrolase of the present invention, wherein the substrate is selected from the group consisting of agarotriose, agaropentaose and agaroheptaose.
Advantageous Effects
The present invention is effective in improving the production yield of 3,6-anhydro-L-galactose and D-galactose from agarose, in other words, saccharification yield, by utilizing a β-agarooligosaccharide hydrolase showing activity toward agarotriose hydrolysis to galactose and neoagarobiose.



DESCRIPTION OF DRAWINGS
FIG. 1 is a TLC image of reaction products for various enzymes (PA: pretreated agarose, PAS: reaction products of Aga50D and SdNABH, with respect to the pretreated agarose, PASV: reaction products of Aga50D, SdNABH, and additionally a Vibrio sp. EJY3 crude enzyme with respect to the pretreated agarose).
FIG. 2 shows reducing equivalents of PAS and PASV of FIG. 1 measured by a DNS assay and summarized in a bar graph.
FIG. 3 is an SDS-PAGE image of a purified β-agarooligosaccharide hydrolase of the present invention.
FIGS. 4A and 4B show optimal temperature and pH results of the β-agarooligosaccharide hydrolase of the present invention. FIG. 4A shows the optimal pH of the β-agarooligosaccharide hydrolase with respect to an agarotriose substrate, and FIG. 4B shows the optimal temperature of the β-agarooligosaccharide hydrolase with respect to the agarotriose substrate (M: protein marker; Lane 1: the β-agarooligosaccharide hydrolase purified primarily using a HisTrap HP column; Lane 2: the β-agarooligosaccharide hydrolase purified secondarily using a HisTrap Q FF column).
FIGS. 5A and 5B illustrate the enzymatic activity of β-agarooligosaccharide hydrolase of the present invention on an agarotriose substrate. FIG. 5A is an HPLC result showing changes in the substrate and products in various reaction time between the β-agarooligosaccharide hydrolase and the agarotriose substrate, and FIG. 5B is an HPLC result showing changes in the substrate and products in various reaction time between the β-agarooligosaccharide hydrolase plus α-neoagarobiose hydrolase and the agarotriose substrate.
FIGS. 6A and 6B show the enzymatic activity of β-agarooligosaccharide hydrolase of the present invention on agarotriose, agaropentaose, and agaroheptaose substrates. FIG. 6A shows the reaction products of the β-agarooligosaccharide hydrolase on each substrate by TLC and FIG. 6B shows the reaction products of the β-agarooligosaccharide hydrolase and α-neoagarobiose hydrolase on each substrate by TLC.
FIG. 7 shows the enzymatic activity of β-agarooligosaccharide hydrolase of the present invention on various substrates (as standards, glucose, and galactose standards were used).
FIGS. 8A and 8B show the enzymatic activity of E. coli-derived β-galactosidase on various substrates (as standards, glucose, and galactose standards were used). FIG. 8A shows the results of an enzyme reaction on each of lactose, lacto-N-neotetraose, lactulose, 4-β-galactobiose, and α-1,3-β-1,4-galactotriose, and FIG. 8B shows the results of an enzyme reaction on each of agarotriose, agaropentaose, and agaroheptaose.
FIG. 9 is a schematic diagram for showing an agarose saccharification process using the β-agarooligosaccharide hydrolase (β-AOH) of the present invention.



BEST MODE
Hereinafter, the components of the present invention will be described in detail.
The present invention relates to a composition for agarose saccharification comprising an agarase; a β-agarooligosaccharide hydrolase having hydrolytic activity for agarotriose and represented by an amino acid sequence of SEQ ID NO:1; and a neoagarobiose hydrolase.
The composition for agarose saccharification of the present invention is characterized in that agarotriose, which used to remain unhydrolyzed in the conventional art, is effectively hydrolyzed such that the production efficiency of 3,6-anhydro-L-galactose and D-galactose from agarose (i.e. an efficiency of agarose saccharification) is significantly improved by utilizing a β-agarooligosaccharide hydrolase which hydrolyzes agarotriose that is a product of agarooligosaccharide hydrolysis into neoagarobiose and D-galactose for agarose saccharification.
According to one embodiment of the present invention, checked results of reducing equivalents from a DNS method show that the rate of saccharification is about 20% when an agarose is only pretreated, and that the rate increases up to 50% when a conventional enzymatic treatment (i.e. a treatment with agarase and neoagarobiose hydrolase) is employed. In contrast, when β-agarooligosaccharide hydrolase is additionally treated to hydrolyze agarotriose during the enzymatic treatment, the rate of saccharification is 70%.
The β-agarooligosaccharide hydrolase does not exhibit activity that a β-galactosidase reportedly shows. Instead, by acting on a non-reducing end of not just agarotriose but also of various agarooligosaccharides (n) such as agaropentaose and agaroheptaose, the β-agarooligosaccharide hydrolase can hydrolyze agarooligosaccharide into D-galactose and neoagarobiose (n−1).
The β-agarooligosaccharide hydrolase may originate from Vibrio sp. EJY3 but is not limited thereto.
The β-agarooligosaccharide hydrolase encompasses not only the amino acid sequence represented by SEQ ID NO: 1 but also a protein as a mutated protein of the enzyme with one or more of substitution, deletion, transposition, addition, etc. that has the hydrolytic activity for agarotriose in the scope of the enzyme of the present invention and, preferably, includes an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence disclosed in SEQ ID NO: 1 (also referred to as ‘AOH’).
In the present invention, a polypeptide having a certain percentage (e.g. 80%, 85%, 90%, 95%, or 99%) of sequence identity with respect to another sequence signifies that, when aligning the two sequences and comparing them, their amino acid residues are mutually the same by the above ratio. The alignment and percentage homology (or identity) may be determined using any suitable software program known in the art, such as those disclosed in the literature [CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. (eds) 1987 Supplement 30 section 7.7.18)]. Preferred programs include GCG Pileup program, FASTA (Pearson et al. 1988 Proc. Natl Acad. Sci USA 85:2444-2448), and BLAST (BLAST Manual, Altschul et al., Natl. Cent. Biotechnol. Inf., Natl Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al. 1997 NAR25:3389-3402). Another preferred alignment program is ALIGN Plus (Scientific and Educational Software, PA), which preferably uses default parameters. Another available sequence software program is TFASTA Data Searching Program that is usable in Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).
In the present specification, “protein” and “polypeptide” are used interchangeably herein. A conventional one-letter or three-letter code is used herein for amino acid residues.
In the present invention, when used with respect to cells, nucleic acids, proteins, or vectors, the term “recombinant” signifies that the cells, the nucleic acids, the proteins, or the vectors were modified by the introduction of heterologous nucleic acids or proteins, or the modification by alteration of the natural nucleic acid or proteins, or, alternatively, the term may refer to cells that originated from the modified cells. In other words, for example, a recombinant cell expresses a gene that is not seen in the inherent (non-recombinant) form of the cell, or, alternatively, it expresses an original gene that is expressed abnormally or is not expressed at all.
In the present specification “nucleic acid” encompasses a single-strand or double-strand DNA, RNA, and chemical variants thereof “Nucleic acid” and “polynucleotide” may be used interchangeably herein. Since the genetic code is degenerate, one or more codons may be used to encode a particular amino acid, and the present invention encompasses a polynucleotide encoding a particular amino acid sequence.
The term “introduction” used for inserting a nucleic acid sequence into a cell refers to “transfection,” “transformation,” or “transduction,” and includes a reference to an integration into eukaryotic or prokaryotic cells of the nucleic acid sequence. In this case, the nucleic acid sequence is integrated into the genome (e.g. chromosome, plasmid, plastid, or mitochondrial DNA) of the cell to be converted to an autonomous replicon or transiently expressed.
The β-agarooligosaccharide hydrolase may be transcribed or translated not just in a region in upstream or downstream of a coding region of an enzyme but also by a DNA segment, that is, a coding gene, associated with the production of a polypeptide and including intervening sequences between individual coding segments. For example, the transcription or translation may be achieved by the sequence of SEQ ID NO: 2 but not limited thereto.
The β-agarooligosaccharide hydrolase may be isolated from a supernatant of a Vibrio sp. EJY3 culture and purified, and, by utilizing recombinant genetic engineering techniques, the β-agarooligosaccharide hydrolase can be produced and isolated by stains other than Vibrio sp. EJY3, artificial chemical synthesis methods, or the like.
When using recombinant techniques, factors used for ease of expression of common recombinant proteins, such as genes resistant to antibiotics, and reporter proteins or peptides that can be used for affinity column chromatography may be used, and such techniques are in a scope in which it can be easily conducted by a person of ordinary skill in the technical field to which the present invention belongs. In addition, for example, a culture supernatant of a transformed E. coli may be used, instead of the β-agarooligosaccharide hydrolase of the present invention.
The β-agarooligosaccharide hydrolase may be purified using a HisTrap HP column and a HisTrap FF column. According to one embodiment, the molecular weight of the purified β-agarooligosaccharide hydrolase may be about 90 kDa, according to the results of SDS-PAGE analysis.
The β-agarooligosaccharide hydrolase is an enzyme that belongs to GH family 2, and when compared for activity with other β-galactosidases in the GH family 2, the reported conventional β-galactosidase does not exhibit the enzymatic activity of hydrolyzing agarotriose into D-galactose and neoagarobiose. However, only the β-agarooligosaccharide hydrolase of the present invention, which originates from Vibrio sp. EJY3, may show agarotriose hydrolysis activity.
The β-agarooligosaccharide hydrolase may exhibit optimal hydrolytic activity on agarotriose in a temperature range of about 30 to 40° C. and a pH range of about 5 to 9.6.
In addition, the composition for agarose saccharification of the present invention includes an agarase that hydrolyzes agarooligosaccharide to agarotriose and neoagarobiose, which is a disaccharide.
As the agarase, an enzyme (referred to as ‘Aga50D’) that breaks the β-1,4-glycosidic linkage between D-galactose and 3,6-anhydro-L-galactose of agarose may be used.
The agarase encompasses not only the amino acid sequence represented by SEQ ID NO: 3 but also a protein as a mutant protein of the enzyme with one or more of substitution, deletion, transposition, addition, etc. that has the agarooligosaccharide hydrolytic activity in the scope of the enzyme of the present invention and, preferably, includes an amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence represented by SEQ ID NO: 3.
The enzyme may have originated from Saccharophagus degradans 2-40T but is not limited thereto.
The agarase may be isolated from a culture supernatant of Saccharophagus degradans 2-40T and purified, and, by utilizing recombinant genetic engineering techniques, the agarase can be produced and isolated by strains other than Saccharophagus degradans, artificial chemical synthesis methods, or the like.
When using recombinant techniques, a culture supernatant of an edible strain, for example, transformed yeast may be used, instead of the agarase.
In addition, the composition for agarose saccharification of the present invention includes α-neoagarobiose hydrolase (referred to as ‘sdNABH’) that may hydrolyze neoagarobiose into 3,6-anhydro-L-galactose and D-galactose.
The α-neoagarobiose hydrolase encompasses not only the amino acid sequence represented by SEQ ID NO: 4 but also a protein as a mutant protein of the enzyme with one or more of substitution, deletion, transposition, addition, etc. that exhibits the neoagarobiose hydrolytic activity in the scope of the enzyme of the present invention and, preferably, includes an amino acid sequence having a sequence identify 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, and 99% or more with the amino acid sequence represented by SEQ ID NO: 4.
The enzyme may have originated from Saccharophagus degradans 2-40T but is not limited thereto.
The α-neoagarobiose hydrolase may be isolated from a culture supernatant of Saccharophagus degradans 2-40T and purified, and, by utilizing recombinant genetic engineering techniques, the α-neoagarobiose hydrolase can be produced and isolated by strains other than Saccharophagus degradans 2-40T, artificial chemical synthesis methods, or the like.
When using recombinant techniques, a culture supernatant of an edible strain, for example, transformed yeast may be replaced for use.
The composition for agarose saccharification of the present invention may produce 3,6-anhydro-L-galactose and D-galactose from agarose either by reacting pretreated agarose with an enzyme mixture of the agarase, the β-agarooligosaccharide hydrolase, and the α-neoagarobiose hydrolase, or by allowing the agarase, the β-agarooligosaccharide hydrolase, and the α-neoagarobiose hydrolase to react sequentially with the pretreated agarose.
The present invention also relates to a method of saccharifying agarose by reacting a substrate and the composition for agarose saccharification according to the present invention, wherein the substrate is selected from the group consisting of agarose, agarotriose, and neoagarobiose.
The method of saccharifying agarose of the present invention is characterized in that an agarase (which is an exo-type β-agarase), a β-agarooligosaccharide hydrolase, and an α-neoagarobiose hydrolase are allowed to react with a pretreated agarose to produce 3,6-anhydro-L-galactose and D-galactose.
The pretreated agarose refers to an agarose that is treated with a weak acid to produce agarooligosaccharides.
The weak acid may be one or two or more of acetic acid, formic acid, succinic acid, citric acid, malic acid, maleic acid, oxalic acid, and the like.
Considering production cost and separation of salt after neutralization by the weak acid, it is preferred that the weak acid is used at a concentration in a range of 0.5 to 60% (w/v). More specifically, it may be used at a concentration in a range of 20 to 40% (w/v).
The reaction between the agarose and the weak acid may be carried out in a temperature range of 40 to 150° C. and under conditions of 100 to 200 rpm for a duration ranging from 30 minutes to 6 hours. When the ranges are satisfied, it may be possible to minimize the products of excessive hydrolysis of agarose using the weak acid.
The resulted reaction products are agarooligosaccharides and may be obtained in a powder form by washing and drying to remove the remaining weak acid and the products of excessive hydrolysis.
The wash solvent may be a low-grade alcohol having 1 to 6 carbons but not particularly limited thereto.
For the enzymatic hydrolysis of the obtained agarooligosaccharides, an exo-type agarase, a β-agarooligosaccharide hydrolase, and an α-neoagarobiose hydrolase are used.
The agarase hydrolyzes agarooligosaccharides into agarotriose and neoagarobiose, the β-agarooligosaccharide hydrolase hydrolyzes agarotriose into D-galactose and neoagarobiose, and the α-neoagarobiose hydrolase hydrolyzes neoagarobiose into D-galactose and 3,6-anhydro-L-galactose.
Therefore, the agarotriose that used to remain during the conventional enzymatic hydrolysis of agarooligosaccharides is hydrolyzed by the β-agarooligosaccharide hydrolase, resulting in an improvement in the production yield of D-galactose and 3,6-anhydro-L-galactose.
3,6-Anhydro-L-galactose and D-galactose may be produced by reacting the pretreated agarose with an enzyme mixture of an agarase, a β-agarooligosaccharide hydrolase, and an α-neoagarobiose hydrolase or by allowing the agarase, the β-agarooligosaccharide hydrolase, and the α-neoagarobiose hydrolase to react sequentially with the pretreated agarose.
The reaction between agarooligosaccharides and the enzyme mixture or the sequential reaction between agarooligosaccharides and the enzymes may be carried out in a temperature range of 20 to 40° C. and under conditions ranging from 0 to 200 rpm for a duration ranging from 30 minutes to 7 days. More specifically, they may be carried out in a temperature range of 25 to 35° C. and under conditions ranging from 100 to 150 rpm for a duration ranging from 1 to 4 days.
When the agarooligosaccharides are in a powder form, it may be dissolved in a conventional buffer solution for use but not limited thereto.
The present invention also relates to a composition for the production of galactose comprising a β-agarooligosaccharide hydrolase having hydrolytic activity for agarotriose, agaropentaose or agaroheptaose and represented by the amino acid sequence of SEQ ID NO:1
Also, the present invention provides a method of producing galactose by reacting a substrate and the β-agarooligosaccharide hydrolase of the present invention, wherein the substrate is selected from the group consisting of agarotriose, agaropentaose, and agaroheptaose.
The β-agarooligosaccharide hydrolase may originate from Vibrio sp. EJY3 but is not limited thereto.
Since the β-agarooligosaccharide hydrolase produces D-galactose and neoagarobiose from agarotriose, agaropentaose, or agaroheptaose, it can be used for producing D-galactose.
The enzyme reactions may be carried out in a temperature range of 20 to 40° C. and a pH range of 5 to 9.6.
Hereinafter, the present invention will be described in detail with reference to exemplary embodiments. However, the following exemplary embodiments are intended merely to exemplify the present invention, and the scope of the present invention is not limited thereto.
MODES OF THE INVENTION
<Example 1> Determination of Enzymatic Activity of β-Agarooligosaccharide Hydrolase from the Crude Enzyme Extract of Vibrio sp. EJY3
To confirm that Vibrio sp. EJY3 has enzymatic activity towards the hydrolysis of agarotriose, a crude extract was obtained and assessed for the activity.
To obtain the crude extract, Vibrio sp. EJY3 was grown up to the mid-exponential phase in a medium including sea salt, then 40 mL of culture medium was centrifuged, cells were disrupted using a sonicator, and the crude extract was obtained. Using agarose as the substrate, the hydrolysis products by β agarase were observed by TLC (composition of a TLC solvent: n-butanol:ethanol:water=3:1:1 by volume).
The TLC results of reaction products for various enzymes were shown in FIG. 1 based on 3,6-anhydro-L-galactose, galactose, neoagarobiose standards. PA represents agarose pretreated by a reaction with 3% acetic acid at 130° C. for 30 minutes, PAS is a product from a reaction between the pretreated agarose with an exo-type β agarase (Aga50D) plus an α-neoagarobiose hydrolase (SdNABH), and PASV is a product obtained when Vibrio sp. EJY3 cruse extract crude extract (or crude enzyme) is added to the PAS.
As seen in FIG. 1, a decrease in agarotriose was observed when Vibrio sp. EJY3 crude enzyme was additionally engaged in the reaction. From this, it could be confirmed that the Vibrio sp. EJY3 crude enzyme contained an enzyme that hydrolyzes agarotriose.
In addition, agarotriose was completely hydrolyzed, and reducing equivalents were measured by a reducing sugar quantification method (a DNS method—50 μl of a DNS reagent is mixed with 100 μl of an enzyme reaction product that is diluted 25 times, treated at 95° C. for 5 minutes, and then absorbance is measured at 540 nm).
As shown in FIG. 2, an increase in the production yield of 3,6-anhydro-L-galactose and galactose was confirmed. The rate of saccharification when only a pretreatment was performed was about 20%, but the rate increased up to 50% when an enzymatic pretreatment was performed. To this, an additional treatment with Vibrio sp. EJY3 crude enzyme resulted in a rate of saccharification of 70% which was thought to be attributed to an ultimate the agarotriose hydrolysis.
<Example 2> Investigation into 3-Agarooligosaccharide Hydrolase from the Enzyme Candidates of Vibrio sp. EJY3
Among the enzymes that were reported in relation to Vibrio sp. EJY3, 8 enzymes that reportedly had activity for an agarase and a β-galactosidase were cloned to produce a recombinant strain, and then each crude enzyme activity for agarotriose was assessed. Among these, only one enzyme (VEJY3_09170) was found to have hydrolytic activity for agarotriose. As confirmed through UniProt (http://www.uniprot.org) and based on full genome sequence data of Vibrio sp. EJY3 that the present inventors revealed for the first time, VEJY3_09170 was identified as an enzyme that was classified as a β-galactosidase based on its genetic sequence and belonged to GH family 2 with many other β-galactosidases. Then, E. coli rosetta (DE3) was used as a host for the recombinant strain, and pET-21a was used as a vector. A colony that was inoculated on a solid medium having an ampicillin concentration of 50 μg/mL and obtained by transformation was again inoculated into an LB (Luria broth) containing 50 μg/mL ampicillin and then incubated for 12 hours under the conditions of 37° C. and 220 rpm (two 20 mL volumes). Then, it was inoculated into two 3 L Erlenmeyer flasks, each containing 1 L of LB broth, and shaking cultured under the same conditions for 3 hours (OD=0.8), cooled in ice for 1 hour, and added 0.1 mM IPTG to induce an expression under conditions of 16° C. and 120 rpm for 12 hours. The culture medium was centrifuged (6000 rpm, 4° C., 15 minutes) to recover cells, and the recovered cells were suspended in a 20 mM Tris buffer solution (Tris-HCl, 1 M NaCl pH 8), disrupted by sonicator, and then the suspension was centrifuged (16000 rpm, 4° C., 60 minutes) to separate β-agarooligosaccharide hydrolase from the supernatant and purify using a HisTrap HP column and a HisTrap Q FF column (GE Healthcare, Piscataway, N.J., USA), which was then observed through 8% SDS-PAGE.
FIG. 3 is an SDS-PAGE image of a purified β-agarooligosaccharide hydrolase. M represents a protein marker, Lane 1 represents a β-agarooligosaccharide hydrolase primarily purified by a HisTrap HP column, and Lane 2 represents the β-agarooligosaccharide hydrolase secondarily purified by a HisTrap Q FF column.
As shown in FIG. 3, the β-agarooligosaccharide hydrolase was found at a position corresponding to about 90 kDa.
<Example 3> Identification of Optimal Reaction Conditions of β-Agarooligosaccharide Hydrolase
The optimal conditions consisting only of temperature and pH of a β-agarooligosaccharide hydrolase were identified. Agarotriose (Aglyco, Beijing, China) was used as the substrate, and the results were determined at temperatures of 10, 20, 30, 35, 40, 45, and 50° C. and pH of 4, 5, 6, 7, 8, 9, and 10.
FIG. 4 shows the optimal temperature and pH results of the β-agarooligosaccharide hydrolase of the present invention. FIG. 4A shows the optimal pH of the β-agarooligosaccharide hydrolase with respect to an agarotriose substrate, and FIG. 4B shows the optimal temperature of the β-agarooligosaccharide hydrolase with respect to the agarotriose substrate
As shown in FIG. 4, the temperature at which the relative activity for agarotriose reached 50% ranged from about 30 to 40° C., and the pH ranged from 5 to 9.6. The optimal enzyme activity was attained at 35° C. and pH 7.
<Example 4> Determination of Biochemical Activity of Agarooligosaccharide Hydrolase Under the Optimal Conditions
β-Agarooligosaccharide hydrolase activity with respect to agarotriose, agaropentaose and agaroheptaose substrates was tested under conditions of 35° C. and pH 7, which were optimal conditions identified in the example 3.
When the changes in the substrate and products with respect to a time of reaction between the β-agarooligosaccharide hydrolase and the agarotriose substrate were observed through HPLC (KS-802 column) and TLC (conditions for the TLC solvent: n-Buthanol:EtOH:Water=3:1:1) analyses, it could be recognized that the β-agarooligosaccharide hydrolase breaks down one molecule of agarooligosaccharide into one molecule of neoagarooligosaccharide and one molecule of D-galactose by hydrolysis (FIG. 5A)
Also, when the changes in the substrate and products with respect to the time of reaction between the β-agarooligosaccharide hydrolase plus α-neoagarobiose hydrolase and the agarotriose substrate were analyzed by HPLC, it could be recognized that 3,6-anhydro-L-galactose and D-galactose were ultimately produced from agarooligosaccharide using the β-agarooligosaccharide hydrolase and α-neoagarobiose hydrolase in combination (FIG. 5B).
FIG. 6 shows the TLC results of β-agarooligosaccharide hydrolase activity of the present invention with respect to agarotriose, agaropentaose, and agaroheptaose substrates. As seen in FIG. 6A, β-agarooligosaccharide hydrolase hydrolyzed agaropentaose and agaroheptaose as well as agarotriose to produce D-galactose, and the ultimate production of 3,6-anhydro-L-galactose and D-galactose was observed when used in combination with β-agarooligosaccharide hydrolase and α-neoagarobiose hydrolase (FIG. 6B).
<Example 5> Comparison of Activity of β-Agarooligosaccharide Hydrolase and E. Coli-Derived β-Galactosidase
When compared with other β-galactosidases that belong to GH family 2 in terms of activity, the β-agarooligosaccharide hydrolase originated from Vibrio sp. EJY3 was found to be an enzyme that belongs to GH family 2 yet shows a new activity different from the previously reported β-galactosidases. The activity of an E. coli-derived β-galactosidase (Sigma-Aldrich, 3050 spruce street, USA) was tested with respect to each substrate, and the activity of a β-agarooligosaccharide hydrolase was tested for the same substrates.
FIG. 7 shows the activity results of β-agarooligosaccharide hydrolase with respect to various substrates. Enzyme reactivity was tested with respect to each of lactose, lacto-N-neotetraose, lactulose, 4-β-galactobiose, and α-1,3-β-1,4-galactotriose, and the results reveal that there was no reactivity with respect to any of the substrates.
FIG. 8 shows the results of E. coli-derived β-galactosidase hydrolase activity with respect to various substrates. Enzyme reactivity was tested with respect to each of lactose, lacto-N-neotetraose, lactulose, 4-β-galactobiose, and α-1,3-β-1,4-galactotriose, and the already known activity of β-galactosidase was confirmed (FIG. 8A). In addition, enzyme reactivity with respect to each of agarotriose, agaropentaose, and agaroheptaose was tested, and the results confirm that the E. coli-derived β-galactosidase cannot hydrolyze agarooligosaccharide (FIG. 8B).
The above results demonstrates that the present invention could improve the product yield and efficiency of agarose saccharification using β-agarooligosaccharide hydrolase, and the schematic view of the saccharification process is as shown in FIG. 9.
INDUSTRIAL APPLICABILITY
3,6-Anhydro-L-galactose and D-galactose of the present invention can be used as a useful material in food, cosmetic, and pharmaceutical fields.

## Claims

1. A method of saccharifying agarose to produce 3,6-anhydro-L-galactose and D-galactose comprising:
reacting acid-pretreated agarose with an enzyme mixture of an agarase, a β-agarooligosaccharide hydrolase, and an α-neoagarobiose hydrolase,
wherein the agarase comprises the amino acid sequence of SEQ ID NO: 3, the β-agarooligosaccharide hydrolase comprises the amino acid sequence of SEQ ID NO: 1, and α-neoagarobiose hydrolase comprises the amino acid sequence of SEQ ID NO: 4.

2. The method of claim 1, wherein the reaction is carried out in a temperature range of 20 to 40° C. under conditions of 0 to 200 rpm for a duration ranging from 30 minutes to 7 days.

3. A method of producing galactose comprising:
reacting a substrate with a β-agarooligosaccharide hydrolase to produce galactose,
wherein the substrate is selected from the group consisting of agarotriose, agaropentaose, and agaroheptaose, and
wherein the β-agarooligosaccharide hydrolase comprises the amino acid sequence of SEQ ID NO: 1.

4. The method of claim 3, wherein the reaction is carried out in a temperature range of 20 to 40° C. and pH range of 5 to 9.6.

5. A method of saccharifying agarose to produce 3,6-anhydro-L-galactose and D-galactose comprising
allowing an agarase, a β-agarooligosaccharide hydrolase, and an α-neoagarobiose hydrolase to react sequentially with acid-pretreated agarose,
wherein the agarase comprises the amino acid sequence of SEQ ID NO: 3, the β-agarooligosaccharide hydrolase comprises the amino acid sequence of SEQ ID NO: 1, and α-neoagarobiose hydrolase comprises the amino acid sequence of SEQ ID NO: 4.

6. The method of claim 5, wherein the reaction is carried out in a temperature range of 20 to 40° C. under conditions of 0 to 200 rpm for a duration ranging from 30 minutes to 7 days.