# Patent

## Title
Alpha-neoagarobiose hydrolase and method for obtaining a monosaccharide using same

## Abstract

Provided are novel α-neoagarobiose hydrolase and a method for obtaining a monosaccharide using the same.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of PCT/KR2010/001816 filed Mar. 24, 2010, which claims the benefit of Korean Application No. 10-2009-0026300 filed Mar. 27, 2009, the entire contents of which applications are incorporated herein by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 1, 2014, is named U.S. Ser. No. 13/232,535_SequenceListing_ST25.txt and is 47,301 bytes in size.
TECHNICAL FIELD
The present invention relates a novel alpha (α)-neoagarobiose hydrolase and a method for obtaining a monosaccharide using the same.
BACKGROUND ART
In recent years, there has been an increasing demand for development of a new alternative energy for reducing carbon dioxide all over the world due to the exhaustion of raw petroleum and increase in their prices which will occur in the near future, as well as the global warming caused by an increased amount of carbon dioxide emitted into the air. Bioenergy produced from biomass, which is one of earth resources that are reusable and rich among a variety of alternative energies, has come into the spotlight as a main renewable energy, which meets the above-described requirements. Particularly, bioethanol has been considered to be an alternative to a transportation fuel which is currently on high demand. For example, the use of bioethanol has already been obliged by the law in developed countries including the United States. Since raw materials currently used to produce bioethanol are limited to sugar, starch and the like derived from maize or other food resources (first-generation bio-fuel), they compete with mankind food resources, which results in an increase in price of international grains. As an alternative to solve these problems, a new land biomass (land biomass or lignocellulosic biomass??) (second-generation bio-fuel) or marine algal biomass (third-generation bio-fuel), which does not compete with the food resources, has also come into the spotlight as a next-generation bioenergy resource, and technologies for generating bio energy using the biomass has been on active research.
Since the marine (algal)biomass has a high content of polysaccharide usable by microorganisms, compared to the land (lignocellulosic)biomass, and is free from lignin, the marine biomass may be relatively easily pretreated, and harvested several times a year. In particular, since the Korean peninsula is surrounded by the sea on three sides except for the northern part, seaweeds may be used as a bio-resource, and their total annual yield is over 13,754 tons in 2006. In this aspect, the Republic of Korea belong to global seaweeds-producing countries including China, Japan and North Korea, but the usability of seaweeds other than edible resources is in a poor state (Korean fishery production statistics in 2006 by the Agriculture and Fisheries Production Statistics Division of the Population and Social Statistics Bureau in the Korea National Statistical Office).
Recently, much research on production of bioenergy from seaweeds has been conducted in Japan and Korea. In the name of the “Ocean Sunrise Project,” the Tokyo Fisheries Promotion Foundation has made a plane to produce five billion liters of fuel ethanol by farming a large amount of seaweeds in 4.47 million km2 of the exclusive economic zone and unused sea areas in the sea belt of Japan (Aizawa M et al., Seaweed bioethanol production in Japan—The Ocean Sunrise Project, OCEANS Conference, Sep. 29-Oct. 4, 2007, Vancouver, Canada). In Korea, a great interest has been focused in production of seaweeds since an ocean bio-fuel was included in the field of novel renewable energy, which is one of the 17 New Growth-driving Industries finally issued by the Korean Government on January of 2009. According to the presentation of development and research for technical exploration and utilization of ocean biomass issued by the Ministry for Food, Agriculture, Forestry and Fisheries in 2009, when seaweeds were cultured in a sea area of a regular square with 71 km each side to produce bioethanol, the bioethanol may be produced at an amount of 3.774 billion liters a year, which will replace 31.4% of an expected consumption of volatile oils in Korea in 2030.
Among the seaweeds recently known in the art, there has been active research on the use of red seaweed biomass (for example, Gelidium amansii) as a source material. Red seaweeds accounts for at least 70% of the total dry weight of polysaccharides, which may be converted into fermentable sugars that are usable by microorganisms. In particular, a main component of the polysaccharide derived from the red seaweed biomass is agar which accounts for approximately 60% of the total dry weight. Therefore, the red seaweed biomass has been considered to be a main resource for production of bioenergy. Agar polysaccharide is a polymer obtained by binding D-galactose and 3,6-anhydro-L-galactose (hereafter, abbreviated as “AHG”), which are used as monomer units, through α-1,3-linkage or β-1,4-linkage (Duckworth, M. and W. Yaphe (1971) Carbohydrate Research 16, 435-445) (see FIG. 1).
It was known that microorganisms using the agar polysaccharide as a carbon source use β-agarase or α-agarase to cleave the agar polysaccharide into smaller oligosaccharides. In this case, the oligosaccharides are finally degraded into α-neoagarobiose (or, α-1,3-D-galactosyl-3,6-anhydro-L-galactose) by the β-agarase, and finally degraded into β-agarobiose (or, β-1,4-anhydro-L-galactosyl-D-galactose) by the α-agarase. It was known that, when a degradation product of the β-agarase is neoagarobiose, the neoagarobiose should be converted into galactose so as to be metabolized by a microorganism, and thus α-neoagarobiose hydrolase cleaving an α-1,3 linkage is necessarily required for convention of the neoagarobiose into the galactose (Ekborg, N. A. et al (2005) Int. J. Syst. Evol. Microbiol. 55, 1545-1549; Ekborg, N. A. et al., (2006) Appl. Environ. Microbiol. 72, 3396-3405). However, an enzyme cleaving the α-1,3 linkage of neoagarobiose in S. degradas was not found until now (Ekborg, N. A. et al. (2006) Appl. Environ. Microbiol. 72, 3396-3405).
It was reported that β-agarase that produces oligoagarosaccharides from agarose in a microorganism is produced by many microorganisms such as, for example, a Pseudomonas sp. strain (Ha, J. C. et al. (1997) Biotechnol. Appl. Biochem. 26:1-6), an Alteromonas sp. strain (Potin, P., et al. (1993) Eur. J. Biochem. 214:599-607), an Agarivorans sp. strain (Ohta, Y. et al. (2005) Biotechnol. Appl. Biochem. 41:183-191), a Pseudoalteromonas sp. strain (Belas, R. (1989) J. Bacteriol. 171:602-605), a Microsilla sp. strain (Zhong, Z. et al. (2001) Appl. Environ. Microbiol. 67:5771-5779), and a Vibrio sp. strain (Aoki, T. et al. (1990) Eur. J. Biochem. 187:461-465).
When agar polysaccharide derived from red seaweed was used as a resource for production of bioenergy, the agar polysaccharide is necessarily converted into fermentable sugars, which can be actually used by a microorganism through a multiple pretreatment procedures. The conversion of the agar polysaccharide into fermentable monosaccharides may be performed through two processes: chemical pretreatment and biological pretreatment. First, a chemical method using acid hydrolysis is a relatively simple process, but a biomass composed of polysaccharides is chemically pretreated at a high temperature to mass-produce toxic by-products such as furfural and hydroxymethylfurfural (HMF), and to yield a mixture of randomly cleaved monosaccharide and oliogosaccharide (Pickering et al., 1993, Journal of Applied Phycology 5: 85-91; Armis'en, 1995). On the contrary, the biological pretreatment and saccharification methods using an enzyme such as β-agarase has an advantage in that they are environment-friendly methods performed at a room temperature to obtain a fermentable sugar such as galactose, but a currently commercially available enzyme is limited to β-agarase and a final product of the β-agarase is disaccharide (neoagarobiose or agarobiose) which may not used by conventional microorganisms.
Neoagarobiose produced from a reaction of the β-agarase should be necessarily converted into a fermentable monosaccharide such as galactose for use in production of bioenergy. In this case, α-neoagarobiose hydrolase is required. Therefore, in a final step of the effective biological (enzymatic) pretreatment and saccharification processes to use a red algal biomass as a resource for production of bioenergy such as bioethanol, α-neoagarobiose hydrolase is necessarily required. Also, AHG which is produced with galactose as degradation products of neoagarobiose was not commercially available, and may be purchased only as D-AHG, which is, however, very expensive (200 pounds (G.B.)/100 mg as of 2009, Dextra Laboratories). Therefore, it is possible to mass-produce an expensive, rare monosaccharide, AHG, from agarose using the enzyme of the present invention.
The above information disclosed in this Background section is only for enhancement of understanding of the background of the invention and therefore it may contain information that does not form the prior art that is already known in this country to a person of ordinary skill in the art.
SUMMARY OF THE DISCLOSURE
The present invention is designed to solve the above-described problems, and therefore it is an object of the present invention to provide an enzyme which is necessarily required for conversion of fermentable monosaccharide, galactose, to produce a bioenergy.
Also, it is another object of the present invention to provide a method for converting neoagarobiose as a substrate into monosaccharides such as galactose and AHG.
In order to solve the above-described problems, the present invention provides α-neoagarobiose hydrolase (hereinafter, referred to as “α-NABH”) selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11.
In one preferred embodiment of the present invention, enzymes having the α-neoagarobiose hydrolase activity include proteins having the α-neoagarobiose hydrolase activity, such as mutant proteins produced by at least one mutation such as substitution, deletion, translocation or addition of the enzyme, as well as amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 11, all of which are included in the scope of the present invention. Preferably, the enzymes of the present invention include amino acid sequences 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 sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 11.
According to the present invention, a polypeptide having a sequence identity of certain percentage (for example, 80%, 85%, 90%, 95%, or 99%) with another sequence means that the two sequences has the same amino acid residues at any percentage when they are aligned to each other. The alignment and percentage homology or identity may be determined using any suitable software program known in the art, for example, those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al., (eds) 1987 Supplement 30 section 7.7.18). A preferred program includes GCG Pileup programs, for example 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 use basic parameters. Still another preferred sequence software program which may be used herein is a TFASTA Data Searching program available for Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).
According to one embodiment of the present invention, the enzyme may be obtained from Saccharophagus degradans (S. degradans) and the like, but the present invention is not limited thereto.
The S. degradans of the present invention may be typically purchased (S. degradans ATCC 43961), but the present invention is not limited thereto. Therefore, the S. degradans of the present invention may be available through various methods.
Also, the present invention provides a gene coding for the enzyme of the present invention.
According to one embodiment of the present invention, the gene may be set forth in SEQ ID NO: 12, but the present invention is not limited thereto.
According to one embodiment of the present invention, the gene may also be obtained from S. degradans, but the present invention is not limited thereto.
Also, the present invention provides a method for preparing the enzyme of the present invention. Here, the method includes cultivating S. degradans; and extracting the enzyme of the present invention from a culture solution.
In addition, the present invention provides a method for preparing galactose and AHG. Here, the method includes degrading α-neoagarobiose using the enzyme of the present invention; and extracting galactose and AHG from the degradation product.
Furthermore, the present invention provides α-neoagarobiose hydrolase including 13 motifs such as motifs 1, 2, 3, 4, 5, 6, 7, 14, 16, 17, 20, 34 and 40 among the motifs selected from the group consisting of motifs 1 to 50 (total 50 motifs) as shown in FIG. 8, and more preferably α-neoagarobiose hydrolase essentially including motifs 7 and 34 among the 50 motifs as shown in FIG. 8.
A protein motif may be represented by a regularly repeating pattern, and the expression of the pattern is typically represented by a regular expression; See, the World Wide Web (www) at expasy.ch/prosite/prosuser.html; for example:
PA[AC]−x−V−x(4)−{ED}.
This pattern is analyzed, as follows: [Ala or Cys]-any-Val-any-any-any-any-{any but Glu or Asp}
PA<A-x-[ST](2)-x(0,1)-V.
The pattern should be positioned at the N-terminus of a sequence (“<”), and is analyzed, as follows: Ala-any-[Ser or Thr]-[Ser or Thr]-(any or none)-Val; Sigrist C. J. A., Cerutti L., Hulo N., Gattiker A., Falquet L., Pagni M., Bairoch A., Bucher P. PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 3:265-274 (2002); Sigrist C. J. A., De Castro E., Langendijk-Genevaux P. S., Le Saux V., Bairoch A., Hulo N. ProRule: a new database containing functional and structural information on PROSITE profiles. Bioinformatics. 2005 Nov. 1; 21(21):4060-6. Epub 2005 Aug. 9; Timothy L. Bailey, Nadya Williams, Chris Misleh, and Wilfred W. Li, MEME: discovering and analyzing DNA and protein sequence motifs, Nucleic Acids Research, Vol. 34, pp. W369-W373, 2006).
According to the present invention, a base sequence and a protein sequence of novel α-neoagarobiose hydrolase are disclosed. In particular, since α-neoagarobiose hydrolase is necessarily used to produce AHG and galactose from agar polysaccharides during an enzymatic conversion process of a biomass into fermentable sugars in the recent bioenergy production using seaweed biomass as described above, reduction in costs and an increase in yield may be expected through the biomass pretreatment. Also, when an α-NABH gene is introduced into yeast or bacterium producing a bio-fuel, the bio-fuel may be expected to be directly produced from agar or neoagarobiose. Furthermore, since α-neoagarobiose hydrolase is used in a process of producing higher value-added monosaccharides, for example, galactose and AHG, from an ocean biomass, it is possible to produce a useful material.



BRIEF DESCRIPTION OF DRAWINGS
The above and other features of the present invention will now be described in detail with reference to certain exemplary embodiments thereof illustrated the accompanying drawings which are given hereinbelow by way of illustration only, and thus are not limitative of the present invention, and wherein:
FIG. 1 is a diagram showing a structure of agar polysaccharide.
FIG. 2 is a photograph showing reaction products by an endogenous crude enzyme obtained from S. degradans 2-40 using thin layer chromatography (TLC). Here, Lane A represents a galactose standard; Lane B represents a culture solution obtained by incubating 0.25% (w/v) agarose with an endogenous enzyme; and Lane C represents a culture solution obtained by incubating 0.3% (w/v) Geladium (red seaweed) powder with an endogenous enzyme. An enzymatic reaction was performed at 30° C. in 20 mM Tris-HCl (pH6.8) for 12 hours.
FIG. 3 shows a base sequence (SEQ ID NO: 4) and a protein sequence (SEQ ID NO: 12) of a gene screened according to one exemplary embodiment of the present invention.
FIG. 4 shows agarase which was expressed in E. coli and was purified; 12% SDS-polyacrylamide gel electrophoresis; Lane A represents a molecular size marker, and B: represents α-neoagarobiose hydrolase.
FIG. 5 is a TLC photograph showing reaction products which were produced from hydrolysis of agarose by α-neoagarobiose hydrolase and β-agarase. An enzymatic reaction was performed at 30° C. in 20 mM Tris-HCl (pH6.8), and a concentration of a substrate was 0.25% (w/v). (A): galactose, and (B and C): reaction mixtures. A: galactose standard; B: reaction product of α-neoagarobiose hydrolase (2-hour reaction); and C: neoagarobiose.
FIG. 6 shows reaction products which were produced from hydrolysis of agarose by α-neoagarobiose hydrolase and β-agarase being confirmed through liquid chromatography-mass spectrometry. From the mass spectrometric results of the reaction products using a mass spectrometer (Negatively inonized into formate (HCOO—, molecular weight: 45)), it was confirmed that a molecular weight of galactose was 225.1 (180+45), and a molecular weight of AHG was 207.1 (162+45), as shown in FIG. 6. Therefore, it was revealed that the reaction products of the α-neoagarobiose hydrolase were galactose and AHG. That is, the results obtained through mass spectrometry by the Seoul branch of the Korea Basic Science Institute were obtained from the mass spectrometric spectra as shown in FIG. 6, and we confirmed the hydrolysis products by the enzyme as a result of analysis of the results.
FIGS. 7A through 7D show analytic results of motifs of a homologous protein including α-neoagarobiose hydrolase.
FIG. 8 shows α-neoagarobiose hydrolase-specific motifs (SEQ ID NO: 13 to SEQ ID NO: 25).
FIG. 9 shows sequences of proteins (SEQ ID NO: 2 and SEQ ID NO: 5 to SEQ ID NO: 9) including all the α-neoagarobiose hydrolase-specific motifs confirmed in FIG. 8.
FIG. 10 shows sequences of proteins (SEQ ID NO: 3, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 1, and SEQ ID NO: 4) including the α-neoagarobiose hydrolase-specific motifs confirmed in FIG. 8.
FIG. 11 shows a multiple sequence alignment using the ten sequences obtained in FIGS. 9 and 10 and an amino acid sequence of the α-neoagarobiose hydrolase (SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 2, SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 10, and SEQ ID NO: 5).
FIG. 12 shows Pseudoalteromonas atlantica (P. atlantica)-derived agarase which was expressed in E. coli and was purified; 12% SDS-polyacrylamide gel electrophoresis; Lane A represents a molecular size marker; and Lane B represents α-neoagarobiose hydrolase.
FIG. 13 shows Streptomyces coelicolor (S. coelicolor)-derived agarase which was expressed in E. coli and was purified; 12% SDS-polyacrylamide gel electrophoresis; Lane A represents α-neoagarobiose hydrolase; and Lane B represents a molecular size marker.
FIG. 14 is a TLC photograph showing reaction products produced from hydrolysis of agarose by α-neoagarobiose hydrolase and β-agarase. An enzymatic reaction was performed at 30° C. in 20 mM Tris-HCl (pH6.8) for 2 hours, and a concentration of a substrate was 0.25% (w/v). (A): galactose, and (B to D): reaction mixtures. Lane A: galactose standard; Lane B: reaction product of S. degradans 2-40-derived α-neoagarobiose hydrolase; Lane C: reaction product of P. atlantica T6c-derived α-neoagarobiose hydrolase; and Lane D: reaction product of S. coelicolor A3-derived α-neoagarobiose hydrolase.



DETAILED DESCRIPTION OF THE DISCLOSURE
Hereinafter reference will now be made in detail to various embodiments of the present invention, examples of which are illustrated in the accompanying drawings and described below. While the invention will be described in conjunction with exemplary embodiments, it will be understood that present description is not intended to limit the invention to those exemplary embodiments. On the contrary, the invention is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalents and other embodiments, which may be included within the spirit and scope of the invention as defined by the appended claims.
As used herein, “a” or “an” may mean one or more than one of an item unless otherwise defined.
The term “recombination,” as used in connection with a cell, a nucleic acid, a protein or a vector, means that the cell, nucleic acid, protein or vector is modified by introduction of a heterogenous nucleic acid or protein or a change in innate nucleic acid or protein, or the cell is derived from such a modified cell. That is, for example, a recombination cell expresses a gene that has not been found in an innate (non-recombinant) form of the cell, or otherwise expresses an innate gene that is abnormally expressed or is not expressed at all.
The terms “protein” and “polypeptide” may be interchangeably used herein. In the present invention, a conventional one-letter code or three-letter code is used for amino acid residues.
The term “gene” refers to upstream and downstream regions of a coding region of the enzyme of the present invention, as well as DNA fragments associated with production of a polypeptide, including sequences interposed between respective coding fragments.
The term “nucleic acid” encompasses single-stranded or double-stranded DNA, RNA, and chemical variants thereof. The term “nucleic acid” and “polynucleotide” may be interchangeably used herein. Since the degeneracy may occur in the genetic codes, at least one codon may be used to encode a certain amino acid, and the present invention encompasses polynucleotides encoding any amino acid sequences.
The term “vector” refers to a polynucleotide sequence designed to introduce a nucleic acid sequence into at least one cell type. Examples of the vector include a cloning vector, an expression vector, a shuttle vector, plasmid, phage particle, cassette and the like.
The term “expression vector” used herein refers to a DNA construct having a DNA sequence operatively connected to a suitable control sequence to express target DNA in a suitable host. These control sequences may include a promoter initiating transcription, any operator sequences controlling transcription, a sequence encoding a suitable ribosomal binding site on mRNA, an enhancer and a sequence controlling initiation and termination of the transcription.
The term “promoter” means a regulatory sequence associated with linkage of RNA polymerase so as to initiate the transcription of a gene. The promoter may be an inducible promoter or constitutive promoter.
The term “derived” includes the terms “originating from,” “obtained from” or “obtainable from” and “isolated from.” When the term is used herein, it means that a polypeptide encoded by the nucleotide sequence is produced in a cell in which the nucleotide is innately present or the nucleotide sequence is inserted.
The term “culture,” “culturing,” “cultivation” or “cultivating” means that a microorganism cell colony is grown in a liquid or solid medium under suitable conditions. According to one preferred embodiment, the culturing or cultivating means a biological conversion of a substrate including agarose into a final product (typically in a container or reactor). The “fermentation” or “fermenting” means that an organic material is enzymatically and anaerobically degraded by a microorganism to produce simpler organic compounds. The fermentation generally occurs under the anaerobic conditions, but may also occur at the presence of oxygen. Therefore, the term is not intended to be limited to the strict anaerobic conditions.
The terms “recovering” or “recovery,” “isolating” or “isolation” and “separating” or “separation” used herein mean that a compound, a protein, a cell, a nucleic acid or an amino acid is removed from at least one component which is spontaneously bound thereto.
The terms “transforming” or “transformation,” “stably transforming” or “stable transformation” and “gene-transplanting” or “gene transplantation” used in connection with a cell used herein mean that a cell has a non-innate (for example, heterogenous) nucleic acid sequence which is present as an episome plasmid maintained through several generations, or is integrated into a genome of the cell.
The term “expression” or “expressing” used herein means a method of producing a polypeptide based on the nucleic acid sequence of a gene. Such a method includes transcription and translation.
The term “introduction” or “introducing” used when a nucleic acid sequence is inserted into a cell means “transfecting” or “transfection,” “transforming” or “transformation,” or “transducing” or “transduction,” and includes integrations of a nucleic acid sequence into eukaryote or prokaryote. In this case, the nucleic acid sequence is integrated into the genome (for example, chromosome, plasmid, chromatophore, or mitochondrial DNA) of a cell, and converted into an autonomous replicon, or temporarily expressed.
1. Enzymatic Process for Production of Fermentable Sugar, D-Galactose, from α-Neoagarobiose During Pretreatment Process of Marine Biomass Including Red Algae
Two pretreatment methods, for example, a chemical method and an enzymatic method, may be used to produce fermentable sugars from agar polysaccharide. First, the chemical method has a problem in that since complex polysaccharides are randomly degraded, it is difficult to selectively produce desired fermentable monosaccharides, and it may also generate by-products which inhibit the fermentation of the produced sugars. In addition, since a significant amount of a contaminant is discharged during the alkali treatment or acid treatment, lots of costs may incur to clean up the contaminant. Therefore, the chemical method is not suitable for the method for producing fermentable sugars which may be used by microorganisms. An enzyme which may be used in such an enzymatic method includes α-agarase or β-agarase. As the enzyme degrading the agar polysaccharide, α-agarase serves to finally produce γ-agarobiose disaccharides by hydrolyzing α-linkages among α-1,3 and β-1,4 linkages present in the agar polysaccharide, and β-agarase serves to produce α-neoagarobiose disaccharides by cleaving β-linkages. When the β-agarase is used during such an enzymatic pretreatment process, the final product is α-neoagarobiose, which is a non-fermentable sugar which may not be used by conventional microorganisms. In order to perform a pretreatment process of finally converting the α-neoagarobiose into fermentable sugars, α-neoagarobiose hydrolase is necessarily required.
2. Production of Galactose or AHG from α-Neoagarobiose which is an Enzymatic Degradation Product of Agar Polysaccharide
3,6-Anhydro-L-galactose (AHG) obtained by hydrolysis of α-neoagarobiose are not currently commercially available, and only D-AHG may be purchased. However, the D-AHG is very expensive (200 pounds (G.B.)/100 mg as of 2009, Dextra Laboratories). Therefore, it is possible to mass-produce expensive AHG from agarose using the enzyme of the present invention.
The present inventors have found that an ocean bacterium, S. degradans, has an α-neoagarobiose hydrolase activity, and screened a corresponding gene for the first time. Also, Escherichia coli (E. coli) was transformed to mass-produce a protein, from which the α-neoagarobiose hydrolase activity was confirmed. The enzymatic activity of a novel α-neoagarobiose hydrolase gene was used to apply to pretreatment of an ocean biomass including the agar polysaccharide, and used to obtain fermentable sugars including galactose from α-neoagarobiose obtained by the pretreatment process.
In recent years, an ocean bacterium, S. degradans, which can grow using the agar polysaccharide as a carbon source was found, and its genomic sequence was currently determined.
In order to screen a gene of the α-neoagarobiose hydrolase, the activity of α-neoagarobiose hydrolase was first confirmed in S. degradans. Next, the presence of an α-neoagarobiose hydrolase gene (protein database Uniprot database ID: Q21HB2) belonging to the glycosyl hydrolase family 32 among genes of S. degradans was estimated through the genomic sequence information analysis. In order to confirm whether a protein actually has an α-neoagarobiose hydrolase activity, a gene coding for the protein was cloned into an expression vector, and the protein was expressed in E. coli, separated and purified to finally confirm the activity of the enzyme.
EXAMPLES
Hereinafter, preferred embodiments of the present invention will be described in detail referring to the accompanying drawings. Prior to the description, it should be understood that the terms used in the specification and appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts corresponding to technical aspects of the present invention on the basis of the principle that the inventor is allowed to define terms appropriately for the best explanation. Therefore, the description proposed herein is just a preferable example for the purpose of illustrations only, not intended to limit the scope of the invention, so it should be understood that other equivalents and modifications could be made thereto without departing from the scope of the invention.
Example 1
Confirmation of Enzymatic Activity of α-Neoagarobiose Hydrolase from a Crude Extract of S. degradans 
In order to confirm whether or not S. degradans has an enzyme activity to produce monosaccharides, galactose and AHG, through hydrolysis by α-neoagarobiose, a crude extract was obtained to assay the enzyme activity using the following method. When S. degradans was grown to mid-log phase in a medium containing seawater, 40 ml of a culture solution was centrifuged, and cells were disrupted using ultrasonication, then a crude extract was obtained,
Agar polysaccharide was degraded with β-agarase to obtain product. The product which was degraded with β-agarase was used as a substrate to observe a reaction/degradation product through TLC. Lane A represents a galactose standard, Lane B represents a reaction product obtained by reaction of 0.25% (w/v) agarose with an endogenous crude enzyme solution of S. degradans, and Lane C represents a reaction product analyzed through TLC after the reaction of 0.3% (w/v) red seaweed, dry agar-agar powder, with an endogenous crude enzyme solution of S. degradans. From the reaction with the endogenous crude enzyme solution of S. degradans, it was seen that monosaccharides (for example AHG and galactose) and disaccharides were produced from both of the agarose and the agar-agar powder. Accordingly, it was revealed that S. degradans had enzymes to produce the monosaccharides such as galactose and AHG through the endogenous hydrolysis of α-neoagarobiose.
According to the present invention, a base sequence of the screened gene and an amino acid sequence of the corresponding protein are shown in FIG. 3.
Example 2
Biochemical Activity and Characterization of α-Neoagarobiose Hydrolase
The activity of purified α-neoagarobiose hydrolase was confirmed, as follows. First, an agar polysaccharide was treated with β-agarase to produce neoagarobiose as a final product of the enzymatic treatment. Then, the neoagarobiose was used as a substrate to confirm that it is a reaction product of the α-neoagarobiose hydrolase using TLC (TLC results shown in FIG. 5). It was confirmed that the substrate treated with the α-neoagarobiose hydrolase under the TLC solvent condition (n-buthanol:EtOH:water=3:2:2) was degraded into materials which are expected to be galactose and AHG. From the TLC results, it was revealed that a Rf value of galactose was approximately 0.46, and a Rf value of neoagarobiose was approximately 0.58.
In order to measure molecular weights of products obtained by the hydrolysis of the α-neoagarobiose hydrolase, the molecular weights of the products were determined through the liquid chromatography-mass spectrometry (LC-MS). The LC-MS results showed that the AHG has a molecular weight of 162, and the galactose has a molecular weight of 180. From the LC-MS results, it was seen that 207.1 m/z corresponds to a molecular weight of the AHG bound to formic acid, which indicates the presence of the AHG, and 179.1 m/z and 225.1 m/z correspond to molecular weights of the galactose and the galactose bound to formic acid, respectively, which indicates the presence of the galactose. Therefore, as shown through the LC-MS, it was confirmed that two monosaccharides, galactose and AHG, were the reaction products of the neoagarobiose hydrolase (see FIG. 6).
Example 3
α-Neoagarobiose Hydrolase-Specific Peptide Motif Sequence
In general, a protein motif refers to a short peptide sequence which appears like a kind of a pattern in proteins having the same molecular functions. Such a protein motif is evolutionarily highly conserved in the entire protein sequence, and is represented by a patterned amino acid sequence represented by a domain which is representative of the molecular functions and has an active region (Sigrist C. J. A., Cerutti L., Hulo N., Gattiker A., Falquet L., Pagni M., Bairoch A., Bucher P. PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 3:265-274 (2002); Sigrist C. J. A., De Castro E., Langendijk-Genevaux P. S., Le Saux V., Bairoch A., Hulo N. ProRule: a new database containing functional and structural information on PROSITE profiles. Bioinformatics. 2005 Nov. 1; 21(21):4060-6. Epub 2005 Aug. 9; Timothy L. Bailey, Nadya Williams, Chris Misleh, and Wilfred W. Li, MEME: discovering and analyzing DNA and protein sequence motifs, Nucleic Acids Research, Vol. 34, pp. W369-W373, 2006).
In order to confirm a sequence of the protein motif which can define the α-neoagarobiose hydrolase, first, an amino acid sequence of the α-neoagarobiose hydrolase was used as a template, and public database was searched through NCBI blast to collect 60 proteins (including α-neoagarobiose hydrolase) having a statistical significance (E-value <0.001). Theses sequences were used to search motifs specific to the α-neoagarobiose hydrolase using a protein motif search program, MEME; See the World Wide Web (www) at meme.sdsc.edu/meme4_1/intro.html; used parameter: mode=zero or one occurrence & nsites=50, mwin=8, and the other conditions uses default parameters; Timothy L. Bailey, Nadya Williams, Chris Misleh, and Wilfred W. Li, MEME: discovering and analyzing DNA and protein sequence motifs, Nucleic Acids Research, Vol. 34, pp. W369-W373, 2006). As a result, it was confirmed that the α-neoagarobiose hydrolase of S. degradans as shown in FIG. 7 has 13 specific motifs (motifs 1, 2, 3, 4, 5, 6, 7, 14, 16, 17, 20, 34 and 40) among the total 50 motifs obtained through analysis of the proteins having homologies. Therefore, it was able to confirm that the 13 specific motifs were representative of the activities of the α-neoagarobiose hydrolase. Among them, motifs 7 and 34 are two essential motifs which are inevitably present in the proteins representative of the α-neoagarobiose hydrolase activity, and shown in FIGS. 8 and 9, respectively (The protein motifs are generally represented according to a regular expression, and expressed in the same manner as in the drawings; Sigrist C. J. A., Cerutti L., Hulo N., Gattiker A., Falquet L., Pagni M., Bairoch A., Bucher P. PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 3:265-274(2002)). Therefore, the proteins including some of the 13 specific motifs and inevitably including motifs 7 and 34 among the 13 specific motifs may have the α-neoagarobiose hydrolase activity (See FIG. 8). Among the proteins having a sequence homology with the α-neoagarobiose hydrolase, we confirmed 10 proteins including some of the 13 specific motifs and inevitably including motifs 7 and 34 (See FIGS. 9 and 10).
The 10 proteins, which include some of the 13 specific motifs previously found in the α-neoagarobiose hydrolase and inevitably include motifs 7 and 34, have been found on the public database. The origins of the proteins were derived respectively from P. atlantica T6c, Microscilla sp. PRE1, Bacteroides plebeius DSM 17135, Gramella forsetii (strain KT0803), Flavobacteriales bacterium HTCC2170, Paenibacillus sp. oral taxon 786 str. D14, and Ruminococcus sp. 5_1_39BFAA, S. coelicolor A3. Their respective amino acid sequences are shown in FIG. 10.
After the ten proteins including some of the 13 specific motifs and inevitably including motifs 7 and 34 were selected from the proteins having a sequence homology with the α-neoagarobiose hydrolase as described above, their sequences as shown in FIG. 11 were listed in a multiple alignment manner. The sequences of the 10 proteins are shown in FIGS. 9 and 10.
Example 4
Confirmation of Protein Sequence Homology of α-Neoagarobiose Hydrolase Gene
Candidate genes, which are expected to have similar functions, were screened through the sequence homology search for the proteins of the S. degradans-derived hydrolases. The specific functions of the candidate genes were unknown in the art, but it was known that all the candidate genes belong to the glycoside hydrolase family 32 (GH32) according to the carbohydrate-related enzyme/protein database, CAZy See, the World Wide Web (www) at cazy.org. The known functions of the proteins belonging to the GH32 family include functions of enzymes, such as invertase (EC 3.2.1.26); endo-inulinase (EC 3.2.1.7); β-2,6-fructan 6-levanbiohydrolase (EC 3.2.1.64); endo-levanase (EC 3.2.1.65); exo-inulinase (EC 3.2.1.80); fructan β-(2,1)-fructosidase/1-exohydrolase (EC 3.2.1.153); fructan β-(2,6)-fructosidase/6-exohydrolase (EC 3.2.1.154); sucrose: sucrose 1-fructosyltransferase (EC 2.4.1.99); fructan:fructan 1-fructosyltransferase (EC2.4.1.100); sucrose:fructan 6-fructosyltransferase (EC 2.4.1.10); fructan:fructan 6G-fructosyltransferase (EC 2.4.1.243); and levan fructosyltransferase (EC 2.4.1.-). Therefore, it is disclosed in the present invention for the first time that some of the proteins belonging to the GH32 family have the same molecular function as the α-neoagarobiose hydrolase activity.
Among sequences screened from the homology search for the amino acid sequences of S. degradans 2-40-derived α-neoagarobiose hydrolases (hereinafter, referred to as “α-NABH”), protein sequences having at least 50% homology are listed according to the E-value from the blast search, as follows (Uniprot database numbers are described below, and original microorganisms and % identities are expressed in the parentheses).
1. Q15UF2 (P. atlantica (strain T6c/BAA-1087), 70%),
2. Q93PB3 (Microscilla sp. PRE1., 59%),
3. B4CY74 (Bacteroides plebeius DSM17135., 60%),
4. A0M245 (Gramella forsetii (strainKT0803)., 56%),
5. A4AR39 (Flavobacteriales bacterium HTCC2170., 57%),
6. C6J3P3 (Paenibacillus sp. oral taxon 786 str. D14., 58%),
7. C6JDD4 (Ruminococcus sp. 5_1_39BFAA., 58%),
8. C6J313 (Paenibacillus sp. oral taxon 786 str. D14., 57%),
9. Q15XP8 (P. atlantica (strainT6c/BAA-1087), 55%),
10. Q9RKF6 (S. coelicolor, 56%)
Example 5
Confirmation of Expression and Size in E. coli 
Among the previously screened proteins having at least 50% sequence homology, the P. atlantica T6c-derived protein (Q15UF2) having the highest sequence homology, and the S. coelicolor A3-derived protein (Q9RKF6) having the lowest sequence homology were cloned, respectively, to confirm whether or not the cloned proteins have α-NABH activity. First, base sequences of genes coding for these proteins were inserted respectively to an E. coli expression vector (pET21a, Novagen, U.S.) (hereinafter, an expression vector including the P. atlantica T6c-derived α-NABH gene was named “pPsAGAJ,” and an expression vector including the S. coelicolor A3-derived α-NABH gene was named “pScAGAJ”). In order to confirm whether the recombinant α-NABH is successfully expressed in E. coli, the expression vectors, pPsAGAJ and pScAGAJ, were transformed into E. coli BL21(DE3) used to express a target protein, and the transformants were plated on a solid medium supplemented with a 50 mg/L concentration of antibiotic ampicillin. A colony obtained through the above-described transformation was seeded in a Luria-Bertani (LB) medium supplemented with a 50 mg/L concentration of antibiotic ampicillin, and incubated at 37° C. for a day while stirring to obtain cells. In order to confirm the expression of the α-NABH gene, the transformants were then seeded in a LB medium supplemented with a 50 mg/L concentration of antibiotic ampicillin, and incubated at 37° C. while stirring until an OD600 value reached 0.5 to 1.0. A 0.5 mM/L concentration of IPTG was added to the LB medium to induce the expression of the α-NABH gene while stirring at 180 rpm for 4 hours. The resultant culture solution was centrifuged (at 12,000 rpm and 4° C. for 10 minutes) to recover cells. The recovered cell was then suspended in a 20 mM Tris buffer (Tris-HCl, pH 7.4), and disrupted using ultrasonication. Then, the size of the target protein was confirmed through 12% SDS-PAGE. The disrupted suspension in which the size of the target protein was confirmed was centrifuged for 15 minutes to obtain a supernatant, which was then used as a crude enzyme solution.
Example 6
Confirmation of Biochemical Activity of α-Neoagarobiose Hydrolase
The activity of purified α-NABH was confirmed, as follows. First, agar polysaccharide was treated with β-agarase to produce neoagarobiose which is a final product of the enzymatic treatment, and the neoagarobiose was then used as a substrate to confirm the presence of a reaction product of α-NABH using TLC. The confirmation through the TLC was performed by dropping 1 ul of a reaction solution on a silica gel 60 TLC plate and developing the silica gel 60 TLC plate under a TLC solvent condition (n-buthanol:EtOH:water=3:2:2). The developed TLC plate was treated with sulphuric acid (10% (v/v) H2SO4 in ethanol) that is a primary treatment solution, and dried. Then, the primarily treated plate was treated with naphthoresorcinol (0.2% (w/v) naphthoresorcinol in ethanol) that is a secondary treatment solution. The resultant TLC plate was dried, and heated.
The results from Example 4 to 6 are listed, as follows.
The expression and size of the P. atlantica- and S, coelicolor-derived α-NABH with which an expression strain, E. coli BL21(DE3), was transformed was confirmed through 12% SDS-PAGE. Expected molecular weights of the P. atlantica- and S, coelicolor-derived α-NABH were approximately 40.7 kDa and 41.1 kDa, respectively, which were confirmed to correspond to expected molecular weights (FIG. 12, FIG. 13).
Also, D-galactose was used as a reference material to confirm the presence of an expected degradation product. The α-NABH-treated disaccharide, neoagarobiose, was confirmed to be degraded into expected materials such as galactose and AHG whose Rf value is confirmed to be the same as that of D-galactose (FIG. 14).
The present invention has been described in detail. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

## Claims

1. A method for preparing galactose 3,6-anhydro-L-galactose (AHG), comprising steps of:
obtaining an α-neoagarobiose hydrolase from a culture of host cells transformed with a recombinant vector, wherein the recombinant vector comprises an isolated nucleic acid molecule encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2;
combining an α-neoagarobiose, a buffer and the α-neoagarobiose hydrolase to form a reaction solution;
heating the reaction solution at a temperature effective to cause a reaction between the α neoagarobiose and the α-neoagarobiose hydrolase and
obtaining galactose or AHG from the degradation product.