# Patent

## Title
B-1,3-glucanase, polynucleotide, recombinant vector, transformant, production method for B-1,3- glucanase, enzyme preparation, and production method for paramylon having reduced molecular weight

## Abstract

A β-1,3-glucanase that exhibits decomposition activity with respect to paramylon derived from the genus Euglena. The β-1,3-glucanase is derived from the genus Euglena and exhibits the properties indicated below: (1) effect: hydrolyzing the β-1,3-bond of β-1,3-glucan; and (2) substrate specificity: decomposing at least paramylon. The β-1,3-glucanase additionally exhibits the properties indicated below: (3) decomposition activity: the ratio of paramylon decomposition activity with respect to laminarin decomposition activity is 20% or higher; (4) optimum pH: 3.7-7.0; and (5) optimum temperature: 30-70° C.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/JP2014/070459 filed Aug. 4, 2014, claiming priority based on Japanese Patent Application Nos. 2013-161588 filed Aug. 2, 2013 and 2013-184351 filed Sep. 5, 2013, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to a novel β-1,3-glucanase that decomposes paramylon, a polynucleotide, a recombinant vector, a transformant, a method for producing a β-1,3-glucanase, an enzyme preparation, and a method for producing low-molecular-weight paramylon.
BACKGROUND ART
A β-1,3-glucan is a polysaccharide having a β-1,3-bond of glucose as a main chain, and is present as a main structure in laminaran that is contained much in brown algae and the genus Laminaria, and in curdlan that is produced extracellularly by mutant strains of soil bacteria (Alcaligenes faecalis). Further, callose contained in cell walls of grains is also known.
β-1,3-glucans have a common characteristic of having a β-1,3-structure as a main chain, but depending on the origins thereof and the like, they are different from one another in terms of the presence/absence and the position of a branched side chain, the combination with a β-1,4-bond and a β-1,6-bond, the molecule size, and the like, thereby having different structures and properties, respectively.
A β-1,3-glucanase is an enzyme that hydrolyzes these β-1,3-glucans, and is used as an additive for fodders for improving body weight gains and feed demand ratios of domestic animals, as a physical property improving agent or a texture improving agent for confectionery, bread, and the like, and as an extraction efficiency improving agent for extracting yeast extract, and beer filtering efficiency improving agent, as well as for other various purposes.
There are β-1,3-glucanases that are derived from various origins, and have various substrate specificities, among which those exhibiting decomposition activity with respect to laminaran, curdlan, yeast cell walls, mycelium of lentinus edodes, pastoran, and the like, are known (Patent Documents 1 to 3).
CITATION LIST
Patent Document


    PATENT DOCUMENT 1: JP 2005-34146A
    PATENT DOCUMENT 2: JP 2005-224230A
    PATENT DOCUMENT 3: JP H10(1998)-507078A


No decomposition enzyme has been known that would exhibit decomposition activity with respect to paramylon derived from the genus Euglena, paramylon being one type of β-1,3-glucan.
Among the β-glucans, paramylon has a characteristic of being composed of only β-1,3-bonds. Further, paramylon exists in a granular form in euglena cells of all of species and varieties, and the number, the shape, and the uniformity of particles of paramylon are characterized depending on the species. As is the case with the other β-glucans, paramylon is expected to have functionality, but much remains unknown regarding the mechanism of action thereof.
Besides, neither any decomposition enzyme that decomposes paramylon nor any composition resulting from decomposition of paramylon is known.
Incidentally, an attempt to reinforce an energy-supply system that utilizes biomass and the like, which is a local unutilized resource, as inexhaustible resources that can substitute for exhaustible resources such as petroleum, has been promoted.
Biomass is defined as reproducible organic resources derived from living organisms, except for fossil resources. Biomass refers to resources that are synthesized by living organisms using solar energy, that are inexhaustible as long as life and the sun exist, and that do not increase carbon dioxide in the atmosphere even if burnt or the like, that is, that are carbon neutral resources.
Bioethanol is being developed as one of energies utilizing biomass. Bioethanol is produced by performing ethanol fermentation, distillation, and dehydration with respect to the following: saccharide of sugarcane, corn, or the like; a substance obtained by saccharifying, with enzyme, a starch-based raw material such as rice, wheat, corn, or the like; or a substance obtained by pretreating a cellulosic raw material such as thinnings, construction waste materials, rice straw, bagasse, or the like with pressurized hot water, acid, or alkali and saccharifying the same with a saccharification enzyme or the like (the Ministry of Agriculture, Forestry and Fisheries, the Biomass Industrialization Strategy (Sep. 6, 2012), Reference Materials: Summary of the Principal Techniques).
Fossil fuel such as petroleum and the like is abundant in reserves, and has been stably supplied globally. In contrast, regarding bioethanol, it is difficult to ensure the supply amount sufficient for supplying the same globally or over an entire country in the present circumstances, since waste materials or the like are used as raw materials in many cases in response to requests with view to sustainability, considering that such raw materials do not conflict with food and do not cause such a situation where arable lands for raw materials encroach arable lands for foods. Generally, therefore, attempts for energy supply utilizing biomass or the like are promoted in individual regions.
Bioethanol has been applied to practical use in a part of regions, but as compared with fossil fuel such as petroleum and the like, bioethanol has weaker price competitiveness, and has problems regarding stable supply and sustainability. In Japan, therefore, bioethanol has not yet been in sufficiently widespread use. Development of raw materials for bioethanol having practicability in terms of costs, stable supply, and sustainability is desired.
On the other hand, mass culture of euglena was considered difficult in the past, but in recent years, as a result of earnest studies by the inventors of the present invention, techniques for mass culture of the same have been established, and the way for supplying paramylon in large quantities has been opened. This leads to a desire for the development of functional substances derived from euglena, which now can be supplied in large quantities.
SUMMARY OF INVENTION
Technical Problem
The present invention was made in light of the above-described problems, and an object of the present invention is to provide a β-1,3-glucanase that exhibits decomposition activity of decomposing paramylon derived from the genus Euglena. 
Another object of the present invention is to provide a β-1,3-glucanase that is usable as a paramylon decomposition enzyme that converts paramylon derived from the genus Euglena into a raw material for bioethanol.
Still another object of the present invention is to develop a new functional substance derived from euglena, which now can be supplied in large quantities.
Solution to Problem
The inventors of the present invention, as a result of earnest studies, found that a novel β-1,3-glucanase having paramylon decomposition activity can be obtained from the genus Euglena, and arrived to the present invention.
As described above, mass culture of euglena, which accumulates paramylon, was considered difficult in the past, but in recent years, with earnest studies by the inventors of the present invention, techniques for mass culture of the same were established, and the way for supplying paramylon in large quantities was opened. Further, mass production of paramylon can be performed in a culture vessel of euglena, and does not require extended farm lands, unlike sugarcane, corn, and the like. Still further, since currently euglena is not food, there is no problem in terms of sustainability. Besides, since euglena has superior production efficiency, it is expected that supply stability can be ensured, which causes euglena to be considered as an expected candidate for a raw material of bioethanol.
Further, since paramylon is a linear polysaccharide composed of β-1,3-bonds alone, the saccharifying step can be simplified, as compared with cellulosic raw materials.
The above-described problems are solved by a β-1,3-glucanase that is derived from the genus Euglena and exhibits properties indicated below:
(1) Effect: hydrolyzing a β-1,3-bond of a β-1,3-glucan.
The β-1,3-glucanase may be a β-1,3-glucanase that additionally exhibits properties indicated below:
(2) substrate specificity: decomposing at least paramylon;
(3) decomposition activity: the ratio of paramylon decomposition activity with respect to laminarin decomposition activity is 20% or higher;
(4) optimum pH: 3.7 to 7.0;
(5) optimum temperature: 30° C. to 70° C.; and
(6) decomposition activity: the ratio of paramylon decomposition activity with respect to alkali-swollen paramylon decomposition activity is 25% or higher.
The studies by the inventors of the present invention in recent years enabled to find a novel β-1,3-glucanase derived from euglena, which now can be supplied in large quantities, thereby opening the way for novel utilization of euglena. It is known that properties of β-glucanase such as substrate specificity and the like vary with the origin thereof, and the finding of a β-1, 3-glucanase derived from euglena opens the way for providing a novel low-molecular-weight glucan and a method for producing the same, and further, opened the way for supplying a novel bioethanol raw material in which a novel low-molecular-weight glucan is used.
The β-1,3-glucanase may have a substrate specificity of decomposing alkali-swollen paramylon and laminarin, in addition to the substrate specificity of decomposing paramylon. Besides, the optimum temperature during a reaction time up to one hour may be 50° C. or higher, the optimum temperature during a reaction time from one hour up to two hours may be 40° C. or higher, and the optimum temperature during a reaction time of 20 hours or more may be 60° C. or lower.
Further, the above-described problems are solved by a β-1,3-glucanase made up of an amino acid sequence (a) or (b) shown below:
(a) an amino acid sequence set forth in SEQ ID NO. 2, 4, or 6; and
(b) an amino acid sequence that is obtained by deleting, substituting, or adding one or several amino acids with respect to an amino acid sequence set forth in SEQ ID NO. 2, 4, or 6, and that has hydrolysis activity of hydrolyzing a β-1,3-bond of a β-1,3-glucan.
Further, the above-described problems are solved by a polynucleotide made up of a base sequence (a) or (b) shown below:
(a) a base sequence set forth in SEQ ID NO. 1, 3 or 5; and
(b) a base sequence that is obtained by deleting, substituting, or adding one or several bases with respect to abase sequence set forth in SEQ ID NO. 1, 3 or 5, and that encodes a protein having hydrolysis activity of hydrolyzing a β-1,3-bond of a β-1,3-glucan.
Here, a recombinant vector including the above-described polynucleotide may be provided.
Further, a transformant including the above-described recombinant vector may be provided.
Further, there may be provided a method for producing a β-1,3-glucanase, the method including culturing the above-described transformant in a culture medium, generating and storing the β-1,3-glucanase in the culture product, and collecting the β-1,3-glucanase from the culture product.
An enzyme preparation for reducing the molecular weight of paramylon, containing the above-described β-1,3-glucanase, may be provided.
A method for producing low-molecular-weight paramylon, the method including allowing the β-1,3-glucanase to act on paramylon, to generate low-molecular-weight paramylon, may be provided.
Here, a glucosidase, together with the β-1,3-glucanase, may be allowed to act on the paramylon, so that glucose is generated as a main product generated from the low-molecular-weight paramylon.
Advantageous Effects of Invention
According to the present invention, by hydrolyzing paramylon, a novel low-molecular-weight paramylon can be obtained that is composed of straight-chain oligosaccharides and that has functionality.
Further, the present invention enables to hydrolyze a β-1,3-glucan containing paramylon. By utilizing the method for producing low-molecular-weight paramylon according to the present invention as a saccharifying step for bioethanol, a β-1,3-glucan such as paramylon or the like can be used as a raw material for bioethanol.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a graph showing separation of laminarin decomposition activity fractions of a solution of disrupted euglena by a hydrophobic column.
FIG. 2 is a graph showing separation of laminarin decomposition activity fractions of a solution of disrupted euglena by a gel filtration column.
FIG. 3 is a graph showing separation of laminarin decomposition activity fractions of a solution of disrupted euglena by an anion-exchange column.
FIG. 4 illustrates SDS-PAGE gel silver staining showing laminarin decomposition activity fractions of a solution of disrupted euglena. 
FIG. 5 illustrates a CBB stained image of SDS-PAGE gel of recombinant EgCel17A.
FIG. 6 is a graph showing substrate specificity of an enzyme EgCel17A, which is an example of the present invention.
FIG. 7 is a graph showing optimum temperature for the enzyme EgCel17A, which is an example of the present invention.
FIG. 8 is a graph showing laminarin decomposition activity in cases where the enzyme EgCel17A, which is an example of the present invention, was incubated at 30° C. to 70° C. for 1 to 20 hours.
FIG. 9 is a graph showing optimum pH for the enzyme EgCel17A, which is an example of the present invention.
FIG. 10 is a graph showing paramylon decomposition activity by Trichoderma cellulase preparation and the enzyme EgCel17A, which is an example of the present invention.
FIG. 11 is a graph showing paramylon decomposition activity of bacillus subtilis glucanase and the enzyme EgCel17A, which is an example of the present invention.
FIG. 12 is a graph showing influences exerted by the amount of added BSA on activity of the enzyme EgCel17A, which is an example of the present invention.
FIG. 13 is a graph showing influences exerted by the amount of added metal on activity of the enzyme EgCel17A, which is an example of the present invention.
FIG. 14 is a graph showing influences exerted by conditions of an alkali treatment of euglena on activity of the enzyme EgCel17A, which is an example of the present invention.
FIG. 15 is a graph showing influences exerted by the amount of added sodium chloride on activity of the enzyme EgCel17A, which is an example of the present invention.
FIG. 16 shows results of paper chromatography, showing decomposition activity and transglycosylation activity of the enzyme EgCel17A, which is an example of the present invention, with respect to laminarin and laminarioligosaccharides.
FIG. 17 is a graph showing results of HPLC of reaction products obtained by adding EgCel17A, and MoCel3A to alkali-swollen paramylon to cause reactions.
FIG. 18 shows results of thin-layer chromatography, showing decomposition activity and transfer activity of the enzyme EgCel17A, which is an example of the present invention, with respect to laminarioligosaccharides.
FIG. 19 is an image showing results of Western blotting of recombinant EgCel81A.
FIG. 20 is a graph showing results of hydrolysis activity measurement tests with respect to respective polysaccharides of the recombinant EgCel81A.



DESCRIPTION OF EMBODIMENTS
Hereinafter the present invention is described in detail.
The present invention relates to a β-1,3-glucanase derived from the genus Euglena. 
In the β-1,3-glucanase of the present invention, a protein having decomposition activity of decomposing paramylon produced by the genus Euglena is contained.
Paramylon is a macromolecule β-1,3-glucan) in which about 700 glucoses are polymerized with β-1,3-bonds, and is a storage polysaccharide that the genus Euglena contains. Paramylon particles are particles each of which is in a flat spheroidal shape and is formed with β-1,3-glucan chains spirally twining.
The paramylon particles are isolated from genus Euglena cultured, and purified into a microparticle form, by arbitrary appropriate methods, and are provided in a powder form usually.
For example, the paramylon particles can be obtained by the following: (1) culturing euglena cells in an arbitrary appropriate culture medium; (2) separating the euglena cells from the culture medium; (3) isolating paramylon from the separated euglena cells; (4) purifying the paramylon thus isolated; and, optionally, (5) cooling the same, and thereafter freeze-drying the same. As the euglena cells, all types of euglena cells can be used, for example, Euglena gracilis, Euglena intermedia, and Euglena piride, as well as other euglena types, for example, Astaia longa, can be used.
The culture of euglena cells can be performed by, for example, the supply batch method. The separation of euglena cells can be performed by, for example, centrifugation or simple sedimentation of culture solution. The isolation of paramylon can be performed by, for example, using a nonionic or anionic surfactant of a mostly biodegradable type. The purification of paramylon can be performed substantially simultaneously with the isolation.
More specifically, for example, the following procedure can be taken: Euglena gracilis powder (produced by Euglena Co., Ltd.) is put in distilled water, and is agitated at room temperature for two days. This is subjected to an ultrasonic treatment so that cell films are destroyed, and coarse paramylon particles are collected by centrifugation. The collected paramylon particles are dispersed in 1% aqueous solution of sodium dodecyl sulfate, are treated at 95° C. for two hours. The paramylon particles, collected by centrifugation again, are dispersed in 0.1% aqueous solution of sodium dodecyl sulfate, and are treated at 50° C. for 30 minutes. Lipids and proteins are removed by this operation, and thereafter, the particles are washed with acetone and ether and dried at 50° C., whereby purified paramylon particles can be obtained. The isolation and purification of paramylon from euglena is known, which is disclosed in, for example, E. Ziegler, “Die naturlichen and kunstlichen Aromen” Heidelberg, Germany, 1982, Chapter 4.3 “Gefriertrocken”, DE 43 28 329, and JP2003-529538A.
Examples of β-1,3-glucanase include β-1,3-glucanase derived from Euglena gracilis (E. gracilis), particularly, β-1,3-glucanase derived from Euglena gracilis (E. gracilis) Z strain.
Besides the above-described ones, the β-1,3-glucanase may be the following species: Euglena gracilis; Euglena gracilis Klebs; and Euglena gracilis var. bacillaris. Alternatively, the β-1,3-glucanase may be a SM-ZK strain as a mutant strain of Euglena gracilis (E. gracilis) Z strain (chloroplast-lacking strain), var. bacillaris as a variety thereof, β-1,3-glucanase derived from a gene mutation strain, such as chloroplast mutant strains of these species, etc.
The genus Euglena is widely distributed in fresh water in ponds and marshes, and Euglena separated from these may be used, or alternatively, arbitrary one of Euglena that is already isolated may be used.
The genus Euglena of the present invention encompasses all of the mutant strains. Further, those obtained by genetic approaches, for example, recombination, transduction, transformation, and the like are included in these mutant strains.
Further, other examples of β-1,3-glucanase of the present invention include a protein made up of an amino acid sequence set forth in SEQ ID NOS. 2, 4, or 6 in the sequence listing.
The amino acid sequence set forth in SEQ ID NO. 2 is named as “EgCel17A” in the present description, which was detected as the lowest band in a fluorescence imaging of a gel subjected to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Example 2, which is illustrated in FIG. 4. A nucleotide sequence including a gene that encodes the protein made up of the amino acid sequence set forth in SEQ ID NO. 2 is set forth in SEQ ID NO. 1.
The amino acid sequence set forth in SEQ ID NO. 4 is named as “EgCel81A” in the present description, which was detected as the second lowest band in the fluorescence imaging of a gel subjected to SDS-PAGE in Example 2, which is illustrated in FIG. 4. A nucleotide sequence including a gene that encodes the protein made up of the amino acid sequence set forth in SEQ ID NO. 4 is set forth in SEQ ID NO. 3.
The amino acid sequence set forth in SEQ ID NO. 6 is named as “EgCel81B” in the present description, which was detected as the third lowest band in the fluorescence imaging of a gel subjected to SDS-PAGE in Example 2, which is illustrated in FIG. 4. A nucleotide sequence including a gene that encodes the protein made up of the amino acid sequence set forth in SEQ ID NO. 6 is set forth in SEQ ID NO. 5.
Further, a protein that is obtained by substituting, deleting, inserting, and/or adding one or several amino acid residues with respect to one or several sites in a protein made up of the amino acid sequence set forth in SEQ ID NOS. 2, 4, or 6 is also encompassed by the present invention, as long as the protein has glyceroglycolipid decomposition activity. The term “several” signifies the numerical value of not more than 75, and preferably refers to the numerical value of not more than 50, further preferably the numerical value of not more than 25, and still further preferably the numerical value of not more than 10.
As another example of the protein, a protein having nucleotide sequence homology, identity or similarity of 70% or more, more preferably 80% or more, further preferably 90% or more, or particularly preferably 95% or more with respect to the protein made up of the amino acid sequence set forth in SEQ ID NOS. 2, 4, or 6, is encompassed by the present invention, as long as the protein has glyceroglycolipid decomposition activity.
The phrase “polynucleotide of the present invention” refers to a polynucleotide that encodes the β-1,3-glucanase of the present invention. The polynucleotide may have any morphology that is known presently, such as cDNA, genome DNA, artificially modified DNA, or chemically synthesized DNA.
Examples of a polynucleotide of the present invention include a DNA that has a nucleotide sequence set forth in nucleotide Nos. 1 to 1,611 in SEQ ID NO. 1, and that encodes a protein having paramylon decomposition activity; a DNA that has a nucleotide sequence set forth in nucleotide Nos. 1 to 3,119 in SEQ ID NO. 3, and that encodes a protein having paramylon decomposition activity; and a DNA that has a nucleotide sequence set forth in nucleotide Nos. 1 to 2,756 in SEQ ID NO. 5, and that encodes a protein having paramylon decomposition activity.
Other examples of the polynucleotide of the present invention include a DNA that has nucleotide sequence homology, identity or similarity of 70% or more, more preferably 80% or more, further preferably 90% or more, or particularly preferably 95% or more with respect to the nucleotide sequence set forth in nucleotide Nos. 1 to 1,611 in SEQ ID NO. 1, the nucleotide sequence set forth in nucleotide Nos. 1 to 3,119 in SEQ ID NO. 3, or the nucleotide sequence set forth in nucleotide Nos. 1 to 2,756 in SEQ ID NO. 5, and that encodes a protein having paramylon decomposition activity. Examples of such a DNA include variant DNAs discovered in the natural world, artificially modified variant DNAs, homologous DNAs derived from different species of organisms, identical DNAs, or similar DNAs.
Other examples of the polynucleotide of the present invention include DNAs that respectively hybridize under stringent conditions with the nucleotide sequence set forth in nucleotide Nos. 1 to 1,611 in SEQ ID NO. 1, the nucleotide sequence set forth in nucleotide Nos. 1 to 3,119 in SEQ ID NO. 3, and the nucleotide sequence set forth in nucleotide Nos. 1 to 2,756 in SEQ ID NO. 5, and that encodes a protein that has paramylon decomposition activity.
Further, a polynucleotide including a polynucleotide made up of the nucleotide sequence set forth in nucleotide Nos. 1 to 1,611 in SEQ ID NO. 1, the nucleotide sequence set forth in nucleotide Nos. 1 to 3,119 in SEQ ID NO. 3, or the nucleotide sequence set forth in nucleotide Nos. 1 to 2,756 in SEQ ID NO. 5 is also encompassed by the present invention, as long as the polynucleotide includes a region for encoding a protein having paramylon decomposition activity.
Further, examples of the β-1,3-glucanase of the present invention include proteins made up of amino acid sequences encoded by the polynucleotides of the present invention.
Further, examples of the β-1,3-glucanase of the present invention include modifications produced by modifying the polynucleotide of the present invention by a known method of shortening a DNA from an end thereof or by cassette mutation so that one or more arbitrary amino acids are deleted therefrom.
In this way, even a protein obtained based on the polynucleotide of the present invention by genetic engineering techniques is encompassed by the present invention as long as the protein has paramylon decomposition activity.
Such a β-1,3-glucanase does not necessarily have an entirety of the amino acid sequence set forth in SEQ ID NOS. 2, 4, or 6, but even a protein made up of, for example, a part of the sequence is encompassed by the β-1,3-glucanase of the present invention as long as the protein has paramylon decomposition activity. Further, a DNA encoding such a β-1,3-glucanase is also encompassed by the present invention.
As the culture solution for culturing the genus Euglena, for example, a culture solution to which nutrient salts such as a nitrogen source, a phosphorus source, minerals, and the like are added, can be used, which is, for example, a modified Cramer-Myers medium ((NH4)2HPO4: 1.0 g/L, KH2PO4: 1.0 g/L, MgSO4.7H2O: 0.2 g/L, CaCl2.2H2O: 0.02 g/L, Fe2(SO2)3.7H2O: 3 mg/L, MnCl2.4H2O: 1.8 mg/L, CoSO4.7H2O: 1.5 mg/L, ZnSO4.7H2O: 0.4 mg/L, Na2MoO4.2H2O: 0.2 mg/L, CuSO4.5H2O: 0.02 g/L, thiamine hydrochloride (vitamin B1): 0.1 mg/L, cyanocobalamin (vitamin B12), (pH3.5)). (NH4)2HPO4 may be replaced with (NH4)2SO4 or NH3aq. Further, other than the above-described ones, a known Hutner medium, or a known Koren-Hutner medium, prepared according to the descriptions in “Euglena physiology and biochemistry” edited by Shozaburo Kitaoka, Gakkai Shuppan Center, may be used.
The culture solution has pH of preferably 2 or more, and the upper limit of the pH is preferably 6 or less, and more preferably 4.5 or less. By setting pH on the acidic side, photosynthetic microorganisms are allowed to predominantly grow as compared with the other microorganisms, whereby contamination can be suppressed.
The culture temperature, the pH, and the aeration and agitation rate, however, can be appropriately selected to be suitable for the production of β-1,3-glucanase using euglena. 
Further, the culture of the genus Euglena may be performed by any liquid culture method such as flask culture, culture using a fermenter, the batch culture method, the semi-batch culture method (the fed-batch culture method), or the continuous culture method (the perfusion culture method).
The β-1,3-glucanase of the present invention may be a β-1,3-glucanase obtained by purifying or partially purifying disruption liquid of the genus Euglena disrupted.
A β-1, 3-glucanase also may be used, which is obtained by, after the culture of the genus Euglena is finished, obtaining a disruption solution containing the genus Euglena disrupted, and partially purifying, or purifying this disruption solution by subjecting the same to a normal reconstruction treatment, a treatment with a protein precipitant (the salting-out method), centrifugation, the osmotic shock method, the freeze-thaw method, ultrasonic disruption, ultrafiltration, gel filtration, any of various types of liquid chromatography such as adsorption chromatography, ion-exchange chromatography, affinity chromatography, and high-performance liquid chromatography (HPLC), the dialysis method, or a combination of these.
Further, the β-1,3-glucanase of the present invention can be obtained also by the following process: a host cell is transformed with a recombinant plasmid obtained by inserting the DNA of the present invention into a plasmid vector, and the β-1,3-glucanase is obtained from a culture product of this transformed cell. Such a recombinant plasmid obtained by inserting a DNA of the present invention into an appropriate vector is also encompassed by the present invention.
As the vector, a plasmid vector is suitably used, but various types of known vectors such as a cosmid vector, a bacteriophage, a virus vector, an artificial chromosome vector, and the like can be used.
With such a vector, host cells of other prokaryotes, or eukaryotes can be transformed. Further, by using a vector having an appropriate promoter sequence and/or a sequence relating to phenotypic expression, or alternatively by transfecting such a sequence to make the same an expression vector, a gene can be caused to express in each host.
By transfecting the vector into a host cell, the cell can be obtained. The host cell may be a prokaryotic cell or a eukaryotic cell as long as it is a cell in which the vector can be transfected.
As a host cell that is a prokaryotic cell, for example, koji mold (Aspergillus oryzae) can be suitably used, but other than the same, Escherichia coli, Bacillus subtilis, or the like also can be used.
Further, as koji mold, other than Aspergillus oryzae, the following types of koji mold belonging to the genus Aspergillus can be used: Aspergillus sojae; Aspergillus awamori; Aspergillus kawachii; Aspergillus usami; Aspergillus tamari; and Aspergillus Glaucus. 
As a host cell that is an eukaryotic cell, for example, cells of vertebrate animals, insects, and yeasts can be used.
In a case where Aspergillus oryzae (A. oryzae) is used as a host cell, it is preferable to use, as a plasmid vector, an Aspergillus oryzae expression vector pPPamyBSP utilizing an α-amylase gene promoter (amyBp).
The gene transfection to a cell can be suitably performed by preparing a protoplast of an Aspergillus oryzae host, and using a known protoplast PEG method (the polyethylene glycol method), but the same can be performed also by another known transfection technique such as the lipofection method, the electroporation method, the nucleofection method, the calcium phosphate method, the injection method, the microinjection method, or the like.
The transformant of the present invention, which is obtained by transfecting a vector into a host cell, can be cultured in the usual way, and by culturing the same, the β-1,3-glucanase of the present invention is intracellularly or extracellularly produced.
As a culture medium used for culturing the transformant, any can be appropriately selected from various types of commonly used media, depending on the host cell used.
In a case where Aspergillus oryzae is used as a host cell, a known culture medium such as YPM medium can be used. Further, other than this, a potato dextrose agar (PDA) medium, a potato dextrose broth (PDB) medium, a bran medium containing wheat bran, or the like may be used.
The β-1,3-glucanase of the present invention, which is produced as a recombinant protein inside or outside the cells of a transformant by culturing the transformant, can be separated and purified from a culture product by any of various types of separation operations utilizing physicochemical properties, chemical properties, biochemical properties (enzyme activity, etc.), and the like of the protein. For example, the following can be used: a normal reconstruction treatment; a treatment with a protein precipitant (the salting-out method); centrifugation; the osmotic shock method; the freeze-thaw method; ultrasonic disruption; ultrafiltration; gel filtration; any of various types of liquid chromatography such as adsorption chromatography, ion-exchange chromatography, affinity chromatography, and high-performance liquid chromatography (HPLC); the dialysis method; and combinations of these.
For example, in a case where the β-1,3-glucanase of the present invention, which is produced as a recombinant protein, is secreted to outside cells, distilled water is added to the culture medium and the culture medium is agitated, left to stand at room temperature for about three hours, and thereafter is filtered by filter paper, whereby the β-1,3-glucanase of the present invention can be extracted.
Further, in a case where the β-1,3-glucanase of the present invention produced as a recombinant protein is locally present inside cells, for example, a buffer solution is added to the culture medium, and the medium is disrupted using a disruption device driven intermittently while being iced, and the disruption liquid obtained is centrifuged so that supernatant is collected, whereby the β-1,3-glucanase of the present invention can be extracted.
Thus, by culturing the transformant of the present invention and separating and purifying the culture product and so on, the β-1,3-glucanase of the present invention can be produced at a high yield on an industrial scale.
Specific properties of the β-1,3-glucanase obtained from a transformant into which the polynucleotide of the present invention is transfected are indicated below, though the properties that the β-1,3-glucanase of the present invention has are not limited to these:
(1) effect: hydrolyzing a β-1,3-bond of a β-1,3-glucan;
(2) substrate specificity: decomposing paramylon;
(3) decomposition activity: the ratio of paramylon decomposition activity with respect to laminarin decomposition activity is 20% or higher;
(4) optimum pH: the optimum pH is 3.7 to 7.0;
(5) optimum temperature: the optimum temperature during a reaction time up to one hour is 50° C. or higher, the optimum temperature during a reaction time from one hour up to two hours is 40° C. or higher, and the optimum temperature during a reaction time of 20 hours or more is 60° C. or lower; and
(6) decomposition activity: the ratio of paramylon decomposition activity with respect to alkali-swollen paramylon decomposition activity is 25% or higher.
Further, a method for producing low-molecular-weight paramylon wherein the β-1,3-glucanase is caused to act on paramylon so that low-molecular-weight paramylon is generated is also encompassed by the present invention.
In the method for producing low-molecular-weight paramylon according to the present invention, the β-1,3-glucanase derived from the genus Euglena according to the present invention is added to paramylon suspension liquid that is obtained by suspending paramylon powder in a buffer such as water or phosphate buffer, and is incubated at pH 3.7 to 7.0, at a temperature of 30 to 70° C., for 15 minutes to 20 hours, so that the β-1,3-glucanase is caused to act on paramylon.
With this, β-1,3-bonds of paramylon are hydrolyzed by the β-1,3-glucanase, whereby low-molecular-weight paramylon is generated.
Here, “low-molecular-weight paramylon” refers to a saccharide generated by hydrolysis of the β-1,3-bonds of paramylon, and encompasses glucose, and oligosaccharides having a polymerization degree of 2 or more in which two or more glucoses are linked by β-1,3-bonds.
Further, in the method for producing low-molecular-weight paramylon according to the present invention, MoCel3A as a glucosidase derived from Magnaporthe oryzae, together with the β-1,3-glucanase derived from the genus Euglena according to the present invention, may be added to the paramylon suspension liquid.
Still further, the enzyme to be added together with the β-1,3-glucanase is not limited to this, and the enzyme may be another glucanase or glucosidase.
Still further, in the place of paramylon powder, alkali-swollen paramylon may be used. In a case where alkali-swollen paramylon is used and an alkali treatment and neutralization are performed, the same may be added to a buffer after the salt concentration is reduced preliminarily.
EXAMPLES
Hereinafter, the present invention is described in more detail by way of examples. The present invention, however, is not limited to these.
Example 1
Preparation of β-1,3-Glucanase from Euglena 
Euglena gracilis (E. gracilis) Z strain was suspended in a phosphate buffer (10 mM, pH 7.0), and thereafter, was disrupted by a sonicator, whereby a solution of disrupted euglena was obtained. The solution of disrupted euglena was centrifuged (22,000×g, 15 minutes), supernatant was collected therefrom, and ammonium sulfate (420 g/L) was added thereto.
This solution was left to stand at 4° C. for 30 minutes, and thereafter, centrifugation (22,000×g, 15 minutes) was performed, whereby precipitate was obtained.
The precipitate was dissolved with a phosphate buffer (10 mM, pH 7.0) containing 0.2 M ammonium sulfate, and was applied to a hydrophobic column (HiPrep phenyl, GE Healthcare) equilibrated with the same buffer.
The column was washed with a phosphate buffer containing 0.2 M ammonium sulfate (10 mM, pH 7.0), and thereafter, the concentration of ammonium sulfate was reduced, so that protein bound to the column was eluted.
In FIG. 1, the laminarin decomposition activity of each fraction herein is indicated by white circles, and the ammonium sulfate concentration upon elution is indicated by a dotted line. As illustrated in FIG. 1, fractions of fraction Nos. 26 to 36 exhibited high decomposition activity. These fractions of fraction Nos. 26 to 36 exhibiting high decomposition activity were collected.
Thereafter, the active fractions of fraction Nos. 26 to 36, which exhibited decomposition activity with respect to laminarin in the hydrophobic column, were applied to a gel filtering column (Superdex75, GE Healthcare) equilibrated with a phosphate buffer containing 0.2 M NaCl (10 mM, pH 7.0).
As illustrated in FIG. 2, fractions of fraction Nos. 28 to 36 exhibited high laminarin decomposition activity. These fractions of fraction Nos. 28 to 36 exhibiting high decomposition activity were collected.
Thereafter, the active fractions of fraction Nos. 28 to 36, which exhibited decomposition activity with respect to laminarin in the gel filtering column, were applied to an anion-exchange column (MonoQ, GE Healthcare) equilibrated with a phosphate buffer (10 mM, pH 7.5). After the column was washed with a phosphate buffer (10 mM, pH 7.5), the concentration of NaCl was increased, so that proteins bound to the column were eluted.
As illustrated in FIG. 3, fractions of fraction Nos. 28 to 34 exhibited high laminarin decomposition activity. These fractions of fraction Nos. 28 to 34, which exhibited high decomposition activity, were collected, whereby a partially purified β-1,3-glucanase was obtained.
Example 2
Determination of Amino Acid Sequence of β-1,3-Glucanase Derived from Euglena 
The active fractions of fraction Nos. 26 to 36, having proteins partially purified and exhibiting decomposition activity with respect to laminarin in the ion replacing column in Example 1, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12.5% gel, and were stained with Coomassie Brilliant Blue R-250 (CBB). FIG. 4 illustrates fluorescence imaging of gel obtained in electrophoresis. As illustrated in FIG. 4, four protein bands were detected.
Stained proteins were spliced out from the gel as gel pieces and digested with trypsin, whereby fragmented peptide mixture was obtained.
The fragmented peptide mixture having been treated with trypsin was separated and concentrated, using a hybrid-type mass analysis system (LTQ Orbitrap XL mass spectrometer; Thermo Fisher Scientific Inc.), according to the method suggested by Kawamura et al. (Kawamura, Y., and Uemura, M. (2003) Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. Plant J. 36, 141-154), and mass analysis of fragmented peptide was performed at the same time, whereby a mass value of the peptide and MS/MS spectrum of fragmented ions were acquired. Thereafter, the amino acid sequence was analyzed by MASCOT MS/MS ion search (Matrix Science Inc.) by the sequence tag method.
Consequently, among the four bands illustrated in FIG. 4, the three proteins other than the malate synthase, which is the largest, were estimated to be enzymes responsible for polysaccharide decomposition.
Band 1 was named as “EgCel17A”. Bands 2 and 3 were named as “EgCel81A” and “EgCel81B”, respectively.
Amino acid sequences of EgCel17A, EgCel81A, and EgCel81B are shown in SEQ ID NOS. 2, 4, and 6, respectively.
Example 3
Identification of Polynucleotides Encoding β-1,3-Glucanases EgCel17A, EgCel81A, and EgCel81B Derived from Euglena 
Euglena gracilis (E. gracilis) Z strain was cultured in a Koren-Hutner medium, 10 L, prepared according to the descriptions in Euglena physiology and biochemistry (edited by Shozaburo Kitaoka, Gakkai Shuppan Center), at 29° C. for 10 days. The medium was concentrated by centrifugation to about 6 times, and nitrogen gas was aerated through this medium until the dissolved oxygen concentration became 0.01 mg/L. This was hermetically sealed and was left to stand still for 24 hours, which were regarded anaerobically treated cells.
From euglena (100 mg) collected by centrifugation, total RNA was extract by using a RNA extraction kit (QIAGEN). From the total RNA thus prepared, with use of an oligo(dT) primer, a cDNA was synthesized by a reverse transcriptase (Transcriptase III, Invitrogen, Inc.). From partial gene sequences of EgCel17A, EgCel81A, and EgCel81B of the amino acid sequences of SEQ ID NOS. 2, 4, and 6 obtained in Example 2, the following were produced: as to EgCel17A, a 5′-Race primer and a 3′-Race primer set forth in SEQ ID NOS. 7 and 8, respectively; as to EgCel81A, a 5′-Race primer and a 3′-Race primer set forth in SEQ ID NOS. 9 and 10, respectively; as to EgCel81B, a 5′-Race primer and a 3′-Race primer set forth in SEQ ID NOS. 11 and 12, respectively.
Next, the gene sequence was read by the 5′-Race method and the 3′-Race method. GeneRacer (registered trademark) Kit (Invitrogen, Inc.) was used for synthesizing a cDNA. Further, using the DNA primers set forth in SEQ ID NOS. 7 to 12 and a DNA polymerase (GXL DNA polymerase, Takara Bio Inc.), PCR was performed.
All the gene sequences obtained were read by a DNA sequencer (Genome Analyzer IIx, Illumina, Inc.). The results of the gene sequence analysis are shown in SEQ ID NOS. 1, 3, and 5.
Example 4
Preparation of Recombinant Protein of EgCel17A
i) Gene Transfection to EgCel17A
A secretion signal of nucleotide Nos. 72 to 152 was removed from the isolated EgCel17A gene set forth in SEQ ID NO. 1, and in accordance with the method suggested by Takahashi et al. (Takahashi M, Takahashi H, Nakano Y, Konishi T, Terauchi R, Takeda T (2010), Characterization of a cellobiohydrolase (MoCel6A) produced from Magnaporthe oryzae. Appl. Environ. Microbiol., 76, 6583-6590.), a histidine tag 5′-TTAGTGATGGTGATGGTGGTGATGGCTAGG-3′ (SEQ ID NO: 13), composed of seven consecutive histidines, was added to the 3′-terminal. This DNA was inserted to an Aspergillus oryzae expression vector pPPamyBSP using an α-amylase gene promoter (amyBp).
The plasmid DNA thus prepared was transfected into an Aspergillus oryzae strain RIB40 by the PEG method, and according to the method suggested by Takahashi et al., the gene-transfected strain was selected in a Czapek-Dox agar medium containing 0.1 mg/ml of pyrithiamine and 1% of glucose.
ii) Purification of Recombinant EgCel17A
The EgCel17A gene-transfected strain of Aspergillus oryzae obtained by selection at i) was inoculated in a YPM liquid medium (1% Yeast extract, 2% Peptone, 2% Maltose), and was subjected to shaking culture at 25° C., at 120 rpm, for two days. The gene-transfected strain of Aspergillus oryzae was removed by double-layer gauze, and then, the medium was concentrated by ultrafiltration. The solution obtained was applied to a histidine tag binding resin (Talon metal affinity resin, Clontech Laboratories, Inc.) equilibrated with a buffer solution (50 mM phosphate buffer (pH 7.0), 50 mM NaCl), and thereafter, the resin was washed with the same buffer solution.
Further, the resin was washed with 0.2× elution liquid (50 mM phosphate buffer (pH 7.0), 50 mM NaCl, 40 mM imidazole). Proteins bound to the resin were eluted by 1× elution liquid (50 mM phosphate buffer (pH 7.0), 50 mM NaCl, 40 mM imidazole), and the replacement with phosphate buffer (10 mM, pH 7.0) and concentration was performed by ultrafiltration. Through the above-described process, recombinant EgCel17A using Aspergillus oryzae as the host was obtained.
Recombinant EgCel17A (5 μg) produced using Aspergillus oryzae as the host was separated by SDS-PAGE, by the same method as that in Example 2, and thereafter, CBB staining was performed. FIG. 5 illustrates the results of the electrophoresis. As illustrated in FIG. 5, a band was detected at a position similar to that of EgCel17A obtained in Example 2.
Test Example 1
Properties of β-1, 3-Glucanase Derived from the Genus Euglena 
i) Substrate Specificity
Using the recombinant EgCel17A (0.2 μg) obtained in Example 4, decomposition activity with respect to a plurality of polysaccharides was examined, so that substrate specificity of EgCel17A was studied.
First, alkali-swollen paramylon was prepared. Paramylon powder (availed from Euglena Co., Ltd.) was suspended in water, NaOH solution was added thereto, and the solution obtained was shaken for 15 minutes at room temperature. Thereafter, the solution was neutralized with acetic acid, whereby alkali-swollen paramylon was obtained. This alkali-swollen paramylon was washed with water.
Next, a substrate, a phosphate buffer (100 mM, pH5.5), and an enzyme preparation (enzyme reaction solution, 50 μl, containing column elution liquid or 0.2 μg recombinant EgCel17A) were mixed, whereby a reaction solution was prepared.
Here, as a substrate, the following were used: 1,3-1,4-β-glucan derived from barley (availed from Megazyme Inc.); xyloglucan (availed from Megazyme Inc.); carboxymethyl cellulose (availed from Sigma-Aldrich Co., LLC); hydroxyethyl cellulose (availed from Sigma-Aldrich Co., LLC); xylan (availed from Sigma-Aldrich Co., LLC); laminarin (availed from Sigma-Aldrich Co., LLC); cellulose derived from cotton (availed from Sigma-Aldrich Co., LLC); phosphate-swellable cellulose (produced from cellulose by the inventors of the present invention); paramylon (availed from Euglena Co., Ltd.); and alkali-swollen paramylon.
The substrate concentration in the reaction solution was set to be 0.1% in the case where the substrate was 1,3-1,4-β-glucan, xyloglucan, carboxymethyl cellulose, hydroxyethyl cellulose, xylan, or laminarin, or cellulose, and was set to 1% in the case where the substrate was phosphate-swellable cellulose, paramylon, or alkali-swollen paramylon.
In this way, phosphate-swellable cellulose, paramylon, and alkali-swollen paramylon, which are water-insoluble substrates, were added more to the reaction system, as compared with the water-soluble substrate.
The prepared reaction solution was incubated at 30° C. for one hour.
According to the method suggested by Lever, M. (Lever, M. (1972) A new reaction for colorimetric determination of carbohydrates. Anal. Biochem. 47, 273-279), 0.5N HCl solution (200 μl) containing 0.5% of 4-hydroxybenzhydrazide was added to the reaction solution, and thereafter, the solution was treated with boiling water for 5 minutes. The solution was cooled naturally, and then, a value at 410 nm was measured with a spectrophotometer, and an increase in the reducing power was calculated in terms of glucose, to be regarded as an activity value.
When cellulose, phosphate-swellable cellulose, paramylon, or alkali-swollen paramylon was used as a substrate, a reducing power of supernatant obtained by centrifugation after incubation was measured.
The measurement results are shown in FIG. 6. In FIG. 6, decomposition activity with respect to laminarin is assumed to be 100, and relative activity mean values±SE (n=3) are shown.
According to FIG. 6, EgCel17A exhibited high decomposition activity with respect to laminarin, paramylon, and alkali-swollen paramylon.
Further, EgCel17A also exhibited decomposition activity with respect to 1,3-1,4-β-glucan, hydroxyethyl cellulose, and phosphate-swellable cellulose.
On the other hand, EgCel17A did not exhibit decomposition activity with respect to xyloglucan, carboxymethyl cellulose, xylan, and cellulose.
The decomposition activity with respect to paramylon was a little more than 20% of the decomposition activity with respect to laminarin, and the decomposition activity with respect to alkali-swollen paramylon was a little more than 80% of the decomposition activity with respect to laminarin, which proves that EgCel17A exhibits high decomposition activity with respect to paramylon and alkali-swollen paramylon, which is at a much higher level than that of a conventional enzyme.
The levels of decomposition activity of EgCel17A were as follows in the height descending order: laminarin>alkali-swollen paramylon>paramylon>1,3-1,4-β-glucan≈phosphate-swellable cellulose>hydroxyethyl cellulose.
It is clear from this result that EgCel17A, which is an endo-1,3-β-glucanase, decomposes laminarin, paramylon, and alkali-swollen paramylon, and hardly decomposes the other polysaccharides.
ii) Optimum Temperature
At different temperatures, decomposition activity of EgCel17A with respect to laminarin was examined, so that optimum temperature for EgCel17A was studied.
The reaction solution containing 0.1% of laminarin, prepared in i) described above, was incubated at 30° C. and at 70° C. for 5 to 120 minutes. The results are shown in FIG. 7.
According to FIG. 7, in the decomposition of laminarin, in the case where the reaction was caused at 70° C., the maximum decomposition activity was exhibited 60 minutes later, which was about four times the decomposition activity in the reaction at 30° C.
Further, the reaction solution containing 0.1% of laminarin, prepared in i) described above, was incubated at 30° C., 40° C., 50° C., 60° C., and 70° C., for 1, 2, 3, 4, 5, 6, and 20 hours as to each temperature. As the activity values, mean values±SE (n=3) are indicated. The results are shown in FIG. 8.
It is clear from FIG. 8 that when a long-time enzyme reaction for 5 hours or longer was performed, the decomposition activity at 40° C. to 50° C. was higher than the decomposition activity at 70° C.
iii) Optimum pH
Laminarin decomposition activity of EgCel17A were examined at different pHs, so that optimum pH for EgCel17A was studied.
The reaction solution containing 0.1% of laminarin, prepared in i) described above, was incubated at pH 3.5 to 8.0. To adjust pH, sodium acetate (pH 3.5 to 5.5, white circles) and sodium phosphate (pH 5.5 to 8.0, black circles) were used. The results are shown in FIG. 9. As the activity values, mean values±SE (n=3) are indicated.
According to FIG. 9, high decomposition activity was observed at pH 3.7 to 7.0, in which particularly high decomposition activity was observed at pH 4.0 to 6.0.
iv) Comparison between Paramylon Decomposition Activity of EgCel17A and that of Trichoderma Cellulase Preparation
Cellulase preparation of Trichoderma (Trichoderma reesei cellulase, Sigma-Aldrich Co., LLC), EgCel17A, and mixture of cellulase preparation of Trichoderma and EgCel17A were used so that paramylon decomposition activities were compared.
As samples of the cellulase preparation of Trichoderma, the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that μg and 10 μg of cellulase preparation of Trichoderma, respectively, were contained therein in place of the enzyme preparation were used.
Further, as samples of EgCel17A, the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that 0.4 μg, 2 μg, and 4 μg of EgCel17A prepared in Example 2, respectively, were contained therein in place of the enzyme preparation were used.
As samples of the mixture of the cellulase preparation of Trichoderma and EgCel17A, the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that the following were contained therein in place of the enzyme preparation were used: 2 μg of the cellulase preparation of Trichoderma and 0.4 μg of EgCel17A prepared in Example 2; 2 μg of the cellulase preparation of Trichoderma and 2 μg of EgCel17A prepared in Example 2; 2 μg of the cellulase preparation of Trichoderma and 4 μg of EgCel17A prepared in Example 2; 10 μg of the cellulase preparation of Trichoderma and 0.4 μg of EgCel17A prepared in Example 2; 10 μg of the cellulase preparation of Trichoderma and 2 μg of EgCel17A prepared in Example 2; 2 μg of the cellulase preparation of Trichoderma and 4 μg of EgCel17A prepared in Example 2.
Each sample was incubated at pH 5.5 (sodium acetate buffer), at temperature of 40° C., for 18 hours.
The results are shown in FIG. 10. As the activity values, mean values±SE (n=3) are indicated.
Consequently, the cellulase preparation of Trichoderma exhibited slight paramylon decomposition activity, but EgCel17A exhibited decomposition activity of about 100 times that of the cellulase preparation of Trichoderma at the same concentration (2 μg). Besides, the sample of 2 μg of EgCel17A exhibited decomposition activity at the same level as the sample of 10 μg of the cellulase preparation of Trichoderma. 
iv) Comparison between Paramylon Decomposition Activity of EgCel17A and that of Bacillus Subtilis Glucanase
Using glucanase derived from Bacillus subtilis (availed from Megazyme Inc.) and EgCel17A, decomposition activities with respect to paramylon were compared.
As samples of bacillus subtilis glucanase, the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that 2 μg and μg of bacillus subtilis glucanase, respectively, were contained therein in place of the enzyme preparation were used.
Further, as samples of EgCel17A, the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that 0.2 μg, 0.4 μg, 1 μg, 2 μg, and 4 μg of EgCel17A prepared in Example 2, respectively, were contained therein in place of the enzyme preparation were used.
Each sample was incubated at pH 5.5 (sodium acetate buffer), at temperature of 40° C., for 18 hours.
The results are shown in FIG. 11. As activity values, mean values±SE (n=3) are indicated.
Consequently, glucanase derived from Bacillus subtilis hardly decomposed paramylon, and only EgCel17A exhibited paramylon decomposition activity.
v) Influences Exerted by Bovine Serum Albumin (BSA) on Paramylon Decomposition Activity of EgCel17A
Influences exerted by BSA on paramylon decomposition activity of EgCel17A were studied.
Samples were prepared that were obtained by using the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that 1.0 μg of EgCel17A prepared in Example 2 was added thereto in place of the enzyme preparation, and further by adding 0.2 μg, 5 μg, and 10 μg of BSA (Sigma-Aldrich Co., LLC) to the reaction solutions, respectively. These samples were incubated at pH 5.5 (sodium acetate buffer), at temperature of 40° C., for 18 hours.
The results are shown in FIG. 12. As activity values, mean values±SE (n=3) are indicated.
Consequently, the sample to which 5 μg of BSA was added exhibited paramylon decomposition activity improved by about 10%, as compared with the sample to which no BSA was added.
BSA is known to have an effect of stabilizing and activating enzyme, and the contribution of the same to the improvement of paramylon decomposition activity of EgCel17A is about 10%, which is found to be lower as compared with the contribution thereof to other enzymes. This therefore makes it clear that when paramylon is decomposed using EgCel17A so that low-molecular-weight paramylon is to be produced, the necessity of adding BSA is lower as compared with the case where another decomposition enzyme is used.
vii) Influences Exerted by Metal on Paramylon Decomposition Activity of EgCel17A
Influences exerted by metals on paramylon decomposition activity of EgCel17A were studied.
Metals were added to the same reaction solutions as the reaction solution prepared in i) described above containing 1% of paramylon except that 1.0 μg of EgCel17A prepared in Example 2 was added thereto in place of the enzyme preparation.
In other words, the following samples were prepared: no metal added sample; 10 mM NaCl added sample; 50 mM NaCl added sample; 100 mM NaCl added sample; 1 mM MgCl2 added sample; 1 mM KCl added sample; 1 mM CaCl2 added sample; 1 mM FeSO4 added sample; 1 mM MnCl2 added sample; 1 mM ZnSO4 added sample; 1 mM NiCl2 added sample; 1 mM CuSO4 added sample; and 1 mM CoCl2 added sample.
These samples were incubated at pH 5.5 (sodium acetate buffer), at temperature of 40° C., for 18 hours.
The results are shown in FIG. 13. As activity values, mean values±SE (n=3) are indicated.
Consequently, the 10 mM NaCl added sample, the 1 mM KCl added sample, and the 1 mM ZnSO4 added sample exhibited paramylon decomposition activities improved by about 10% as compared with the no metal added sample.
On the other hand, the 1 mM CaCl2 added sample, the 1 mM FeSO4 added sample, the 1 mM MnCl2 added sample, the 1 mM NiCl2 added sample, the 1 mM CuSO4 added sample, and the 1 mM CoCl2 added sample exhibited reduced paramylon decomposition activity as compared with the no metal added sample.
These results prove that in the case where metal ions of Ca, Fe, Mn, Ni, Cu, or Co are mixed in the reaction solution, the paramylon decomposition activity of EgCel17A decreases. It is clear that in the case where paramylon is to be decomposed by using EgCel17A so that low-molecular-weight paramylon is to be produced, a reaction solution or a culture medium in which the concentrations of these metal ions are reduced, or a reaction solution or a culture medium in which these metal ions are not mixed, may be used.
vii) Influences Exerted by Alkali Treatment to Euglena Containing Paramylon on Paramylon Decomposition Activity of EgCel17A
Regarding alkali-swollen paramylon decomposition activity of EgCel17A, influences on the decomposition activity exerted by the concentration of alkali solution for pretreating euglena were studied.
Euglena powder suspended in water was treated with NaOH solutions having different concentrations (not treated with NaOH; treated with 0.025 M NaOH; treated with 0.05 M NaOH; treated with 0.25M NaOH; treated with 0.5 M NaOH; treated with 1 M NaOH; treated with 2 M NaOH; treated with 3 M NaOH; treated with 5 M NaOH), and neutralized with acetic acid, whereby alkali treated euglena was obtained.
Used were the same reaction solutions as the reaction solution prepared in i) described above except that 1 mg of the alkali treated euglena at respective treatment concentrations was added thereto in place of 1% alkali-swollen paramylon, and further, 1 μg of EgCel17A prepared in Example 2 was added as the enzyme preparation. Each sample was incubated at pH 5.5 (sodium acetate buffer), at temperature of 40° C., for three hours.
The results are shown in FIG. 14. As the activity values, mean value±SE (n=3) are indicated.
Consequently, it is recognized that the treatment with NaOH of 0.5 M or more allowed EgCel17A to exhibit high decomposition activity. In particular, it is recognized that the treatment with 2 M NaOH resulted in the highest decomposition activity.
viii) Influences Exerted by NaCl on Paramylon Decomposition Activity of EgCel17A
Influences exerted by sodium chloride on a reaction of decomposition of alkali-swollen paramylon by EgCel17A.
Used were the same reaction solutions as the reaction solution prepared in i) described above containing 1% alkali-swollen paramylon except that 1 μg of EgCel17A was added thereto in place of the enzyme preparation, and further, 0 M, 0.1 M, 0.5 M, 1.0 M, and 2.0 M of NaCl were added, respectively.
Each sample was incubated at pH 5.5 (sodium acetate buffer), at temperature of 40° C., for three hours.
The results are shown in FIG. 15. With the decomposition activity in the case where no NaCl is added being assumed to be 100, relative activity mean values±SE (n=3) are shown.
In FIG. 15, a decrease in the decomposition activity of EgCel17A in the presence of NaCl of 0.5 M or more is recognized. It is clear from this result that when paramylon is decomposed using EgCel17A so that low-molecular-weight paramylon is produced, the efficiency of production of low-molecular-weight paramylon is reduced due to NaCl. In the case where alkali treatment and neutralization of paramylon or euglena is performed as a pretreatment for the decomposition of paramylon by using EgCel17A, it is necessary to consider the influences of produced salts. It is therefore made clear that in the case where alkali treatment and neutralization of paramylon or euglena is performed as a pretreatment for the decomposition of paramylon by using EgCel17A, the NaCl concentration may be reduced, or NaCl may be removed, before EgCel17A is added.
Test Example 2
Transglycosylation Activity of EgCel17A
In the present test example, transglycosylation activity of EgCel17A was studied.
First of all, laminarioligosaccharides (availed from Megazyme Inc.) having polymerization degrees of 4 to 7 was labeled at a glucose residue on the reductive end side with Sulphorhodamine, according to the method suggested by Fry et al. (Fry S. C. (2002) Novel ‘dot-blot’ assays for glycosyltransferases and glycosylhydrolases: optimization for xyloglucan endotransglycosylase (XET) activity. Plant J. 11, 1141-1150.).
The fluorescent-labeled laminarioligosaccharides were purified by paper chromatography (solvent; butanol:acetic acid:water=1:1:1).
Using the fluorescent-labeled laminarioligosaccharides, transglycosylation activity of EgCel17A was examined.
Used were the same reaction solutions as the reaction solution prepared in i) of the test example 1 except that 0.1% of the fluorescent-labeled laminarioligosaccharides, or mixture of 0.1% of the fluorescent-labeled laminarioligosaccharides and 0.1% of laminarin was used therein in place of the substrate, and 0.2 μg of EgCel17A obtained in Example 2 was added in place of the enzyme preparation.
The samples were incubated at 40° C., for 0 minute (no incubation), 15 minutes, 30 minutes, and 60 minutes, respectively.
The results are shown in FIG. 16.
As illustrated in FIG. 16, as time elapsed, fluorescence-labeled products having increased molecular weights were recognized in the reaction solutions containing laminarin as well as laminarihexaose (polymerization degree: 6) and laminariheptaose (polymerization degree: 7), respectively.
On the other hand, in the case where no laminarin was contained, as illustrated in FIG. 16, decomposition of laminarihexaose and laminariheptaose was recognized.
It is clear from these results that EgCel17A, after decomposing laminarin as a polymer, catalyzes a transfer reaction for transfer into fluorescence-labeled laminarihexaose and laminariheptaose.
Test Example 3
Method for Producing Glucose from Paramylon Using EgCel17A
Alkali-swollen paramylon and 100 mM phosphate buffer (pH5.5) were added to EgCel17A (1 μg) obtained in Example 2, which was followed by incubation at 40° C. for one hour. Then, a reaction product (1 μL) was applied to a HPLC column equilibrated with NaOH, and thereafter, the sodium acetate concentration (0 to 100 mM) was increased so that the reaction product was eluted.
Further, EgCel17A (1 μg) and MoCel3A (0.2 μg, (produced by the inventors of the present invention) Takahashi, M., Konishi T., Takeda T. (2011) Biochemical characterization of Magnaporthe oryzae β-glucosidases for efficient β-glucan hydrolysis. Appl. Microbiol. Biotechnol., 91, 1073-1082), which is a β-glucosidase derived from Magnaporthe oryzae, were mixed, and alkali-swollen paramylon and 100 mM phosphate buffer (pH5.5) were added thereto. The mixture was incubated at 40° C. for one hour, and thereafter, a reaction product was detected by HPLC.
The results are shown in FIG. 17.
As illustrated in FIG. 17, in the case where alkali-swollen paramylon was decomposed by EgCel17A alone, the peak of glucose was lower than the peaks of those having polymerization degrees of 3 and 4, but in the case where MoCel3A, which is β-glucosidase derived from Magnaporthe oryzae, was added to EgCel17A and alkali-swollen paramylon was decomposed with this, the peak of glucose was observed as indicating a main generation product.
What is described above makes it clear that mixing EgCel17A and MoCel3A enables to efficiently convert paramylon into glucose.
Test Example 4
Laminarioligosaccharide Decomposition and Transfer Reaction by EgCel17A
In the present test example, laminarioligosaccharide decomposition activity and transfer activity of EgCel17A were studied.
First of all, laminarioligosaccharides having polymerization degrees of 2 to 7 (100 μg), EgCel17A (0.1 μg), and a phosphate buffer (final concentration 100 mM, pH 5.5) were mixed, and were left to stand still at 40° C. After 0 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, and 18 hours of reaction, samples were collected, and reaction products were separated by thin-layer chromatography. Here, as a solvent, a solvent of 1-butanol:acetic acid:water=2:1:1 was used. After development, it was dipped in sulfuric acid/ethanol (5:95) liquid containing 0.5% of thymol, and thereafter, treated for 5 minutes at 110° C.
The results of separation of the reaction products by thin-layer chromatography are shown in FIG. 18.
As illustrated in FIG. 18, EgCel17A did not decompose laminaribiose (polymerization degree: 2), and decomposed laminarioligosaccharides having polymerization degrees of 3 to 7.
At the same time, as to laminarioligosaccharides having polymerization degrees of 3 to 7, and particularly those having polymerization degrees of 4 to 7, after 0.5 to 4 hours of reaction, laminarioligosaccharides having higher polymerization degrees were extracted, and a transfer reaction was caused to occur with respect to substrates of laminarioligosaccharides having polymerization degrees of 3 to 7, particularly those having polymerization degrees of 4 to 7.
As the reaction time elapsed to 18 hours, however, these substrates were decomposed to glucose, laminaribiose, and laminaritriose.
Example 5
Preparation of Recombinant Protein of EgCel81A and Determination of Activity Thereof
i) Plasmid Construction
A gene obtained by adding a secretion signal sequence of EGL2 (AB032830) derived from Pisum sativum to Egcel81A gene, and adding a histidine tag (His-tag) to a 3′ terminal thereof was transfected into a pCambia plasmid vector. The plasmid vector thus constructed was transfected into Agrobacterium by electroporation, and thereafter, transgenic Agrobacterium was selected on a YEB plate (yeast extract 1 g/L, peptone 5 g/L, beef extract 5 g/L, sucrose 5 g/L, MgSO27H2O 0.5 g/L) containing kanamycin.
ii) Gene Transfer to Rice Callus
Seeds of Oryza sativa (Sasanishiki) were sterilized, and inoculated in a C1 medium (N6-1-alanine 20 ml/L, N6-2-alanine 50 ml/L, N6-3-alanine 1 ml/L, N6-4-alanine 10 ml/L, N6-vitamin 1 ml/L, sucrose 3.75 g/L, casamino acid 0.3 g/L, proline 2.878 g/L, 2,4-dichloropfenoxyacetic acid (100 mg/L) 20 ml/L, gellan gum 3 g/L, (pH 5.8)), and was cultured at 30° C. for 5 days in a dark place, whereby callusing was induced. Rice calluses were mixed with Agrobacterium, spread over a K2 medium (N6-1-alanine ml/L, N6-2-alanine 50 ml/L, N6-3-alanine 1 ml/L, N6-4-alanine 10 ml/L, N6-vitamin 1 ml·L, sucrose 3.75 g, glucose 10 g/L, casamino acid 0.3 g/L, 2,4-dichloropfenoxyacetic acid (100 mg/L) 20 ml, gellan gum 3 g/L, (pH 5.2)), and was left to stand still for 3 days, at 25° C., in a dark place. After Agrobacterium was removed from collected rice calluses with a cleaning buffer, the rice calluses were inoculated in a K2 medium containing carbenicillin (400 μg/ml) and hygromycin (50 μg/ml), and cultured at 30° C. for 10 days. Thereafter, the rice calluses were inoculated in a K2 medium containing carbenicillin (300 μg/ml) and hygromycin (50 μg/ml), and gene transferred bodies were selected.
iii) Preparation of Protein
Regarding eight individual rice calluses, each rice callus (10 mg) was subjected to cell disruption in a buffer (50 mM sodium acetate (pH7.0), 300 mM sodium chloride), and thereafter, supernatant was collected by centrifugation (5,000 rpm, 5 min). The supernatant was concentrated and desalinized by ultrafiltration, and thereafter, the same was subjected to Western blotting and enzyme activity measurement.
iv) Western Blotting
Proteins contained in the supernatant, after being separated by SDS-PAGE, were transcribed into PVDF membranes. Western blotting was performed using an antibody against the His-tag. The results of Western blotting performed on the eight individual rice calluses are shown in FIG. 19. The numbers 1 to 8 in FIG. 19 indicate the eight individual rice calluses, respectively.
v) Enzyme Activity
Seven types of polysaccharide substrates (1,3-1,4-βglucan, xylan, xyloglucan, laminarin, glucomannan, PSC (phosphate-swellable cellulose), and paramylon), 0.1% each, and a buffer (100 mM acetic acid buffer (pH6.0)) were added to supernatant derived from rice callus of No. 1 in FIG. 19, whose signal was detected by Western blotting, and were left to stand still at 30° C. for 18 hours. To each reaction solution, p-hydroxybenzoic acid hydrazide was added, and the reaction solutions were boiled for 5 minutes. Then, absorbances at 410 nm were measured, whereby increased reducing powers were determined. The measurement results are shown in FIG. 20.
vi) Result
As described above, proteins were prepared from eight individual rice calluses, and Western blotting was performed by using an antibody against the His-tag. As a result, as illustrated in FIG. 19, five individual rice calluses (Nos. 1, 2, 4, 5, and 7), recognized to have produced EgCel81A, were obtained.
Further, using protein prepared from the rice callus (No. 1), hydrolysis activity was examined. As a result, as illustrated in FIG. 20, hydrolysis activity with respect to 1,3-1,4-β-glucan and laminarin was remarkably recognized. As β-1,3-bonds are included commonly in these polysaccharides, it was clarified that EgCel81A was an endo-1,3-β-glucanase.

## Claims

1. An isolated endo-1,3-β-glucanase comprising the amino acid having at least 90% identity to SEQ ID NO. 2 and contains at least one deletion, substitution, or addition relative to SEQ ID NO: 2, and having an endo-type hydrolysis activity of hydrolyzing a β-1,3-bond of a β-1,3-glucan.

2. The endo-1,3-β-glucanase according to claim 1, further exhibiting the following properties:
(1) substrate specificity: decomposing at least paramylon;
(2) decomposition activity: a ratio of paramylon decomposition activity with respect to laminarin decomposition activity is 20% or higher;
(3) optimum pH: 3.7 to 7.0;
(4) optimum temperature: 30° C. to 70° C.; and
(5) decomposition activity: a ratio of paramylon decomposition activity with respect to alkali-swollen paramylon decomposition activity is 25% or higher.

3. The endo-1,3-β-glucanase according to claim 2,
wherein the endo-1,3-β-glucanase additionally has a substrate specificity of decomposing alkali-swollen paramylon and laminarin, and
an optimum temperature during a reaction time up to one hour is 50° C. or higher, an optimum temperature during a reaction time from one hour up to two hours is 40° C. or higher, and an optimum temperature during a reaction time of 20 hours or more is 60° C. or lower.

4. An enzyme preparation for reducing a molecular weight of paramylon, the enzyme preparation containing the endo-1,3-β-glucanase according to claim 1.

5. A method for producing low-molecular-weight paramylon, the method comprising:
allowing the endo-1,3-β-glucanase according to claim 1 to act on paramylon, to generate the low-molecular-weight paramylon.

6. The method for producing the low-molecular-weight paramylon according to claim 5,
wherein a glucosidase, together with the endo-1,3-β-glucanase, is allowed to act on the paramylon, so that glucose is generated as a main product of the low-molecular-weight paramylon.

7. An isolated endo-1,3-β-glucanase comprising the amino acid having at least 90% identity to SEQ ID NO. 2 and contains at least one deletion, substitution, or addition relative to SEQ ID NO: 2.