JP6074788B2 - 3,6-anhydro-L-galactose producing enzyme - Google Patents

Description

  The present invention relates to a 3,6-anhydro-L-galactose producing enzyme, a method for producing the enzyme, a method for producing 3,6-anhydro-L-galactose using the enzyme, and a product obtained by the method. It is related with the whitening agent which mixes.

  In red algae such as Tengu and Ogonori, there are polysaccharides composed of galactose or galactose derivatives, among which water-soluble mucilage exists. Examples of the water-soluble mucilage include agar containing agarose and agaropectin, porphyran, carrageenan, and some of them have a sulfate group. Various effects of these water-soluble mucilages have been studied. For example, in agar, constipation is improved, intestinal cholesterol absorption is inhibited, dioxins and other harmful substances are discharged, and the like. This agar agarose has a basic structure in which D-galactose and 3,6-anhydro-L-galactose are alternately arranged in a straight chain with β-1,4 bonds and α-1,3 bonds, respectively. Yes. Moreover, such a basic structure is a structure often found in polysaccharides derived from red algae such as porphyran and carrageenan.

And although many red algae exist as marine resources, they are only used for food and agar medium, and the fact is that these resources are not effectively utilized.
In recent years, attempts have been made to excise oligosaccharides from polysaccharides having 3,6-anhydro-L-galactose and D-galactose, such as agarose, and effective use of the excised oligosaccharides.
For example, an enzyme capable of producing neoagarobiose from a polysaccharide having a structure such as agar and the like, a method for producing an oligosaccharide such as neoagarobiose using the enzyme (Patent Documents 1 and 2), and neoagarobi A whitening external preparation containing ausose (Patent Document 3) has been proposed.

However, in reality, sufficient knowledge has not been obtained about 3,6-anhydro-L-galactose (hereinafter also referred to as “ALG”).
In order to produce 3,6-anhydro-L-galactose, generally, agar or agarose is heated in the presence of an acid to hydrolyze, but the anhydro structure of ALG may be lost by hydrolysis. Is expensive. In addition, there is a method of heating and hydrolyzing in the presence of an alkali, but similarly, there is a high possibility that the anhydro structure disappears, and it is also known that a monosaccharide undergoes a rearrangement reaction at room temperature in the presence of an alkali. As described above, in the acid / alkali treatment, it is difficult to obtain ALG efficiently.
For this reason, a means for obtaining 3,6-anhydro-L-galactose satisfactorily is desired.

Japanese Patent Application Laid-Open No. 8-149799 JP 2005-229889 A Japanese Patent Laid-Open No. 8-310937

  Therefore, in view of such a situation, the present invention provides an enzyme that favorably produces 3,6-anhydro-L-galactose, a method for producing the enzyme, and production of 3,6-anhydro-L-galactose using the enzyme. It is intended to provide a method.

  As a result of intensive studies on the search for a 3,6-anhydro-L-galactose (hereinafter also referred to as “ALG”)-producing enzyme, the present inventor has found that a novel non-marine microbial cell contains neoagarobiose hydrolyzed. It was found that there is activity of degrading enzyme (neoagarobiose hydrolase, NAH). There have been very few reports on neoagalobiose hydrolase, and it was quite surprising that an enzyme capable of degrading marine-derived polysaccharides was found from non-marine microorganisms. Furthermore, the enzyme of the present invention is a novel enzyme as described later, and 3,6-anhydro-L-galactose can be obtained from neoagarobiose and other oligosaccharides without loss of anhydro structure. I found it. Thus, the inventor of the present invention, 3,6-anhydro-L-galactose producing enzyme of the present invention, a method for producing the enzyme, a method for producing 3,6-anhydro-L-galactose using the enzyme, The invention of ALG application has been completed.

  That is, the present invention provides the inventions according to the following [1] to [14].

[1] A 3,6-anhydro-L-galactose producing enzyme comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity capacity (c) consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity capacity Protein

[2] The following (a) or the polynucleotides represented by (c).
(A) polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 3
( C) a polynucleotide comprising a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 or 3, and having 3,6-anhydro-L-galactose production activity

[3] having the following enzymological properties, cell Vibrio (Cellvibrio) derived from genus 3,6-anhydro -L- galactose forming enzyme.
1. Action: Neo agarobiose is used as a substrate, has affinity for it, and acts to produce 3,6-anhydro-L-galactose.
2. Substrate specificity: Acts with one or more selected from neoagarobiose, neoagarotetraose and neoagarohexaose to produce 3,6-anhydro-L-galactose.
3. Optimal pH: 6.0 .
4). Optimal temperature: 25 ° C. when neoagarobiose is used as a substrate.
5. Molecular weight: It is a homodimer showing a molecular weight of 42 kDa as measured by SDS-polyacrylamide gel electrophoresis and showing a molecular weight of 83 kDa as measured by gel filtration chromatography.
6). The N-terminal amino acid sequence is Gly-Asp-Leu-Pro-Glu-Lys-Lys-Leu-Ser-Lys-Ala-Ser-Leu-Arg-Ala-Ile-Glu (SEQ ID NO: 4).

[4] A polynucleotide encoding the protein described in [1] or [3] above, a polynucleotide consisting of the sequence ggt gat ctt cca gaa aaa aaa tta agt aaa gcc agt ttg cgc gct att gaa, or the above [2] A recombinant vector containing the polynucleotide of
[5] A transformant comprising the recombinant vector according to [4] above.

[6] A microorganism comprising a polynucleotide or gene encoding a 3,6-anhydro-L-galactose-producing enzyme comprising the following protein (a), (b) or (c) is cultured, and 3,6-anhydro: -A method for producing 3,6-anhydro-L-galactose producing enzyme, comprising collecting L-galactose producing enzyme.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity ability (c) consisting of an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity ability Protein

[7] Using a 3,6-anhydro-L-galactose-producing enzyme comprising the following protein (a), (b) or (c), 3,6-anhydro-α-L-galactose α (1- 3) A method for producing 3,6-anhydro-L-galactose characterized in that 3,6-anhydro-L-galactose is obtained from a saccharide having a basic structure of D-galactose as a raw material.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity ability (c) consisting of an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity ability Protein

[8] A microorganism containing a polynucleotide or gene encoding a 3,6-anhydro-L-galactose-producing enzyme comprising the following protein (a), (b) or (c) is cultured, and 3,6-anhydro -3,6-Anhydro-L-galactose characterized in that 3,6-anhydro-L-galactose is obtained from a carbohydrate having a basic structure of -L-galactose α (1-3) D-galactose Production method.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity ability (c) consisting of an amino acid sequence having 90 % or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity ability Protein

[9] (1) Hydrolysis of a β-1,4 bond of a carbohydrate having a basic structure of D-galactose β (1-4) 3,6-anhydro-L-galactose using a neo-agarobobiose-producing enzyme Disassembling,
(2) hydrolyzing a saccharide having the basic structure of 3,6-anhydro-L-galactose α (1-3) D-galactose using the 3,6-anhydro-L-galactose producing enzyme; ,
(3) A method for producing 3,6-anhydro-L-galactose, comprising obtaining 3,6-anhydro-L-galactose.

[10] Cell Vibrio sp (Cellvibrio sp.), Designated WU-0601, the microorganisms deposited as NITE P-1365.

[11] A method for producing a whitening material by the method for producing 3,6-anhydro-L-galactose as described above.
[12] A whitening agent containing a product obtained by the method for producing 3,6-anhydro-L-galactose as described above.
[13] A method for producing a whitening agent, wherein the product obtained by the method for producing 3,6-anhydro-L-galactose described above is blended.

[14] A polypeptide having the amino acid sequence of SEQ ID NO: 4, a polypeptide having an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, or inserted in the amino acid sequence, or ggt gat ctt cca gaa aaa aaa tta agt aaa gcc agt ttg cgc gct att gaa polynucleotide consisting of the sequence of.

  According to the present invention, there are provided an enzyme that favorably produces 3,6-anhydro-L-galactose, a method for producing the enzyme, a method for producing 3,6-anhydro-L-galactose using the enzyme, and the like. Can do. Thereby, 3,6-anhydro-L-galactose can be obtained favorably.

An example of the reaction for obtaining 3,6-anhydro-L-galactose from a polysaccharide or oligosaccharide using the ALG-generating enzyme of the present disclosure is shown. Purification of the ALG-producing enzyme of the present disclosure from a genus Cervibrio 2 shows the pH-dependent and temperature-dependent enzyme activity of the ALG-generating enzyme of the present disclosure. The homology relationship between each enzyme of GH32 and the ALG production enzyme of this indication is shown. An alignment between the ALG-generating enzyme of the present disclosure and a protein of unknown function showing high homology thereto is shown. 2 shows an SDS-PAGE electrophoretic analysis of an enzyme of the present disclosure. 2 shows an ALG-generating enzyme of the present disclosure and a gene encoding the same. 2 shows an ALG-generating enzyme of the present disclosure and a gene encoding the same. 2 shows an ALG-generating enzyme of the present disclosure and a gene encoding the same. FIG. 6 shows the ¹³ C NMR spectrum of ALG of the present disclosure and the structure of 3,6-anhydro-α-L-galactopyranose of the present disclosure.

1. 3,6-Anhydro-L-galactose synthase of the present disclosure (1) Enzymatic properties of the ALG synthase of the present disclosure (2) Amino acid sequence of the ALG synthase of the present disclosure and polynucleotide encoding the same (3) 1. Method for obtaining ALG-generating enzyme of the present disclosure Method for producing 3,6-anhydro-L-galactose of the present disclosure (1) Method for producing 3,6-anhydro-L-galactose by the ALG-generating enzyme of the present disclosure (2) Gene encoding the ALG-generating enzyme of the present disclosure 2. A method for producing 3,6-anhydro-L-galactose by a microorganism having Uses of 3,6-anhydro-L-galactose of the present disclosure (1) Pharmaceuticals, cosmetics, skin external preparations, foods, etc.

<1. 3,6-Anhydro-L-galactose producing enzyme of the present disclosure>
The present disclosure is a novel 3,6-anhydro-L-galactose producing enzyme, and the ALG producing enzyme has the following enzymatic properties and the amino acid sequence represented by SEQ ID NO: 1. The ALG-generating enzyme preferably forms a homodimer.
The polynucleotide of the present disclosure is a novel polynucleotide, and the polynucleotide has a polynucleotide encoding the amino acid sequence of the ALG-generating enzyme, the polynucleotide represented by SEQ ID NO: 2 or SEQ ID NO: 3. It is.

  The ALG-generating enzyme of the present disclosure has 3,6-anhydro-L-galactose generating activity, and preferably has a basic structure of 3,6-anhydro-L-galactose α (1-3) sugar residue. The α-1,3 bond of the carbohydrate possessed is hydrolyzed to produce 3,6-anhydro-α-L-galactose.

In general, when the reducing end of the sugar is an open ring of an aldehyde group or a ketone group, it easily reacts with other compounds. Therefore, it is conceivable to protect the portion by introducing a methyl group or the like at the reducing end during the recovery, but eventually it is necessary to return the protected portion to the hydroxyl group again or to remove the organic solvent. It becomes complicated and the recovery rate of ALG also decreases. For this reason, it is easier to recover ALG with high purity by using ALG with a closed ring, such as Neo Agarobiose, than with ALG with an open ring, such as Agarobiose. can do. However, there have been very few reports of neoagarobiose hydrolase, and it has been difficult to find such an enzyme.
However, by obtaining the ALG-generating enzyme of the present disclosure and using it for the production of ALG, there is little production of by-products due to structural changes caused by ring-opening at the reducing end of the carbohydrate, and the anhydro structure is not lost. Since the reaction is possible under the reaction conditions, 3,6-anhydro-L-galactose can be obtained satisfactorily.

(1) Enzymatic properties [action] of the ALG-generating enzyme of the present disclosure
The ALG producing enzyme of the present disclosure has 3,6-anhydro-L-galactose producing activity. Preferably, 3,6-anhydro-L-galactose is obtained by hydrolyzing the α-1,3 bond of a saccharide having a basic structure of 3,6-anhydro-L-galactose α (1-3) sugar residue. Is generated. Further, neoagalobiose is preferably used as a substrate and has a high affinity for it. Examples of the sugar residue include galactose and glucose, and galactose is preferable. A pyranose type is preferred as the sugar residue.
In addition to neoagarobiose, enzymes having high affinity for neoagarotetraose and neoagarohexaose are suitable. Of these substrates, an enzyme having higher affinity for neoagarobiose is preferred.

[Substrate specificity]
The substrate is not limited to neoagarobiose, but may be any substrate that can produce 3,6-anhydro-L-galactose with the ALG-generating enzyme of the present disclosure.
As a substrate, for example, an oligosaccharide having at least a basic structure of 3,6-anhydro-L-galactose α (1-3) D-galactose; and β- (1-4) -linked 3,6- An oligosaccharide having an anhydro-L-galactose basic structure; an oligosaccharide having an α (1-3) -linked D-galactose with this 3,6-anhydro-L-galactose; and an oligo having these repeating basic structures Examples include sugars.
The oligosaccharide preferably has 2 to 9 sugar residues, more preferably 2 to 6 sugar residues, and more preferably neoagarobiose, neoagarotetraose and neoagaro One or more selected from hexaose.

(Dynamic constant)
K _M = 4 to 5 × 10 (mM)
V _max = 6.5 to 7.5 × 10 ⁻³ (mM / sec)
k _cat = 3.5 to 4.5 × 10 (sec ⁻¹ )
[Reaction for 10 to 50 minutes at a concentration of 1 to 16 mM neoagarobiose]

[Optimum reaction pH]
The optimum pH (25 ° C.) is preferably around 5 to 7, and preferably around 5.5 to 6.0. In particular, the highest activity is obtained when a citrate buffer solution at pH 6.0 is used.

[Reaction temperature]
The optimum reaction temperature is preferably 10 to 40 ° C, more preferably 20 to 30 ° C.
[Thermal stability]
The activity is stable at 40 ° C. or lower, and the activity is maintained at 30 ° C. or lower. At 40 ° C., 45-55% activity remains. Above 50 ° C, the activity is almost 0%.

[Molecular weight]
As measured by SDS-PAGE electrophoresis (Lammli et al.), The ALG-generating enzyme of the present disclosure is almost single and exhibits a molecular weight of 35 to 45 kDa, particularly a molecular weight of 40 to 45 kDa.
Moreover, it shows a molecular weight of 70 to 90 kDa, particularly a molecular weight of 80 to 85 kDa as measured by gel filtration.
In the measurement and analysis of gel filtration at this time, the enzyme was equilibrated in advance with an elution buffer (10% glycerol, 1 mM dithiothreitol (DTT), 20 mM phosphate buffer, 0.15 mM NaCl, pH 7). Apply to a filtration column (Superose 6 10/300) and elute with 1 column volume of elution buffer.
Bovine serum albumin (MW 67,000), chymotrypsinogen (MW 25,000), α-amylase (MW 45,000), and β-amylase (MW 200,000) are used as standard proteins for gel filtration.

[Influence of metal ions, etc.]
One or more members selected from Ag ⁺ , Hg ²⁺ , Ni ²⁺ , Cu ²⁺ , Fe ³⁺ , and pCMB (parachloromercury benzoic acid) inhibit ALG generation activity. Among these, Ag ⁺ , Hg ²⁺ , Cu ²⁺ , and pCMB are about 85 to 100%, and ALG generation activity is inhibited. On the other hand, ALG generation activity is improved by one or more selected from Mn ²⁺ and Mg ²⁺ .

[N-terminal amino acid sequence]
The ALG-generating enzyme of the present disclosure preferably has an N-terminal amino acid sequence. As the N-terminal amino acid sequence (excluding the start codon Met), Gly-Asp-Leu-Pro-Glu-Lys-Lys-Leu -Ser-Lys-Ala-Ser-Leu-Arg-Ala-Ile-Glu (SEQ ID NO: 4). In this partial amino acid sequence, one or several amino acids may be substituted, deleted, or inserted.
The amino acid residue sequence on the N-terminal side may be obtained by a known method (Edman, P. (1950) Acta Chem. Scand. 4: 283-293). As an example, the enzyme is electrophoresed by SDS-polyacrylamide electrophoresis, and the resulting enzyme band is electrically transferred to a polyvinylidene fluoride (PVDF) membrane and then analyzed by a protein sequencer. Can be determined.
In addition, the N-terminal amino acid sequence (SEQ ID NO: 4), an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or inserted in the amino acid sequence and a polynucleotide encoding the amino acid sequence are the ALG-generating enzyme and ALG of the present disclosure. It can be used for screening, production, etc. of a production enzyme-producing microorganism. Furthermore, it is preferable to use a primer based on the polynucleotide and a conserved sequence (preferably a conserved sequence in glycosylhydrolase family 32), preferably a polynucleotide represented by SEQ ID NO: 5 and / or SEQ ID NO: 6.

(2) Amino acid sequence of ALG-generating enzyme of the present disclosure and polypeptide (gene) encoding the same

The ALG-generating enzyme of the present disclosure includes the following proteins (a), (b) and (c).
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 1.
(B) A protein having an activity of producing 3,6-anhydro-L-galactose, comprising an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence shown in SEQ ID NO: 1. Preferably, a protein having a high affinity for neoagarobiose.
(C) A protein comprising an amino acid sequence having 85% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having 3,6-anhydro-L-galactose production activity. Preferably, a protein having a high affinity for neoagarobiose.

The amino acid sequence of the ALG-producing enzyme shown in SEQ ID NO: 1 of this disclosure and the amino acid sequence of Sde_2657 derived from Saccharophagus degradans 2-40 showed 75% identity, but those showing 80% identity or more were found. There wasn't. Thus, the ALG-generating enzyme of the present disclosure is considered to be a novel enzyme that forms a new group in GH32 (see FIGS. 4 and 5). Since the gene encoding ALG-generating enzyme at the time of cloning does not have a signal peptide region required for secretion on the N-terminal side, the ALG-generating enzyme of the present disclosure is considered to be an enzyme present in the cell. . In addition, by clarifying the N-terminal amino acid sequence as described above, it is considered that searching for related enzymes having high identity with the enzyme of the present disclosure can be facilitated by using it for screening and the like based on this.
Further, the NAH gene of the present disclosure is a novel gene because it includes a polynucleotide encoding the ALG-generating enzyme of the present disclosure.

  In the present disclosure, the homology between the amino acid sequence and the base sequence is calculated by a known algorithm such as the Lipman-Person method (Science, 227, 1435, (1985)), and can be performed by comparing the sequences accordingly. it can. Specifically, the identity should be calculated using the search homology program or genetic matching program of genetic information processing software GENETYX (registered trademark) -ver 8.1 (Software Development: Genetics). Can do. For example, it is calculated by performing analysis with Unit size to compare (ktup) as 2. In the present disclosure, “homology” intends “identity”.

  In the present disclosure, the transcription initiation region is a region including a promoter and a transcription initiation site, and the ribosome binding site is a Shine-Dalgarno (SD) sequence (Proc. Natl. Acad. Sci. USA 74, 5463 (1974)).

  Here, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which one or several amino acids are substituted, deleted, or added means an amino acid sequence that is functionally equivalent to SEQ ID NO: 1, respectively. An amino acid sequence in which several, preferably 1 to 6, more preferably 1 to 3 amino acids are substituted, deleted or added, and still have 3,6-anhydro-L-galactose production activity An array. Preferably, 3,6-anhydro-L-galactose is converted to 3,6-anhydro-L-galactose by hydrolyzing the α-1,3 bond of a carbohydrate having a structure of α (1-3) sugar residue. Sequences having activity to generate are preferred. Furthermore, sequences that retain a high affinity for neoagarobiose are preferred. In addition, addition includes addition of 1 or several, preferably 1 to 6, more preferably 1 to 3 amino acids to both ends.

The functionally equivalent amino acid is obtained by hydrolyzing the α-1,3 bond of a carbohydrate having a structure of at least 3,6-anhydro-L-galactose α (1-3) sugar residue, and 3, It may be an enzyme having an activity ability to produce 6-anhydro-L-galactose, and may have additional properties. Furthermore, it is preferable to have a high affinity for neoagarobiose. Moreover, it is preferable to have substantially the same function as the protein encoded by the NAH gene shown in SEQ ID NO: 2 or SEQ ID NO: 3, specifically the function of the above-described ALG-generating enzyme of the present disclosure.

Further, in the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and further preferably 98% or more is suitable.
As shown in Examples below, since a novel group of enzymes was also discovered through the discovery of the ALG-generating enzyme of the present disclosure, the protein comprising the amino acid sequence shown in SEQ ID NO: 1 of the present disclosure and the activity of generating ALG as described above A novel enzyme that is functionally equivalent to having a high affinity for neoagarobiose and having an identity within the range of about 85% or more. Will be included in the group.

  The polynucleotide of the present disclosure has a polynucleotide encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or a protein consisting of an amino acid sequence functionally equivalent to the amino acid sequence. The following (a), The polynucleotides of (b) and (c) are also included. The gene of the present disclosure includes a gene comprising the polynucleotide of the present disclosure.

In the present disclosure, among these, the following (a) to (c) are preferable.
(A) A polynucleotide comprising the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3.
(B) encodes a protein that hybridizes with a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 under stringent conditions and has 3,6-anhydro-L-galactose production activity Polynucleotide. Preferably, a polynucleotide encoding a protein having a high affinity for neoagarobiose.
(C) A polynucleotide encoding a protein consisting of a base sequence having 85% or more identity with the base sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 and having 3,6-anhydro-L-galactose production activity. Preferably, a polynucleotide encoding a protein having a high affinity for neoagarobiose.
In the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, a base sequence having 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more is suitable.

The polynucleotide of the present disclosure includes a polynucleotide (DNA, etc.) represented by the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 in part by treatment with a mutagen, random mutation, specific site mutation, deletion or insertion, etc. Even if the nucleotide sequence is changed, these DNA variants hybridize under stringent conditions with the DNA represented by the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and at least 3,6-anhydro -The polynucleotide which codes the protein which has L-galactose production activity ability is included.
Preferably, the α-1,3 bond of a carbohydrate having a basic structure of 3,6-anhydro-L-galactose α (1-3) sugar residue is hydrolyzed to produce 3,6-anhydro-L- It has an activity to produce galactose. More preferably, it includes a polynucleotide encoding a protein having 3,6-anhydro-L-galactose production activity and having a high affinity for neoagarobiose.
For example, a base sequence in which one or several (for example, 2 to 3) base sequences are substituted, deleted, or added can be used. The addition includes addition to both ends. Here, “1 or several” means 1 to 6, preferably about 1 to 3.

Here, "stringent conditions" include conditions described in, for example, Molecular cloning-a laboratory manual, 2 nd edition (Sambrook et al, 1989). That is, 6XSSC (1XSSC composition: 0.15M sodium chloride, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / mL herring sperm DNA together with the probe at 65 ° C. Examples include conditions of constant temperature for 8 to 16 hours and hybridization.

  In addition, the gene represented by the polynucleotide represented by (b) or (c) described above or any one of these polynucleotides has, for example, an expression level of mRNA as compared to the polynucleotide represented by (a). Many may have additional properties such as high stability of the mRNA and excellent stability of the translated protein.

In addition, at least one of the transcription initiation control region, the translation initiation control region, and the secretion signal region is bound upstream of the polynucleotides (a) to (c) or any one of these polynucleotides. You may let them.
In the present disclosure, the transcription initiation region is a region including a promoter and a transcription initiation point, and the ribosome binding site is a Shine-Dalgarno (SD) sequence (Proc. Nalt. Acad) that forms a translation initiation control region together with the initiation codon. Sci. USA 74, 5463 (1974)).
Further, in the present disclosure, upstream or downstream of a gene is not a position from the replication start point, but upstream indicates a region continuing on the 5 ′ side of the gene or region regarded as a target, while downstream indicates a target A region following the 3 ′ side of the gene or region captured as.

(3) Method for Acquiring ALG Generating Enzyme of the Present Disclosure ALG producing enzyme of the present invention is a transformant in which a Cellvibrio genus (cell vibrio) bacterium or a mutant thereof, or a polynucleotide encoding the ALG producing enzyme or a fragment thereof is introduced. It can be produced and obtained by the body (preferably a microorganism).
As a method for obtaining the ALG producing enzyme of the present disclosure, a microorganism producing the ALG producing enzyme of the present disclosure is grown in a medium component, the ALG producing enzyme of the present disclosure is produced in the microorganism, and the produced ALG producing enzyme Is recovered.

As the carbon source of the medium component, agar, agarose, agarohexaose, agarotriose, etc. can be used alone or in combination. It is also possible to use other carbon sources such as glucose in combination.
As a nitrogen source of the medium components, inorganic nitrogen sources such as various inorganic ammonia salts, nitrates and urea, and organic nitrogen compounds such as casamino acids, yeast extract, beef extract, peptone, soybean flour, corn steep liquor, various amino acids, etc. In consideration of the assimilation and growth of the microorganisms used, one or more can be appropriately selected and used.
As an inorganic salt of the medium component, one or more of sodium, magnesium, calcium, iron, potassium phosphates, hydrochlorides, sulfates, carbonates, acetates and the like can be appropriately added. Furthermore, you may add antifoamers, such as a vegetable oil and surfactant, as needed.

The culture can be carried out in a liquid medium containing the above-mentioned medium components by using a normal culture method such as shaking culture, aeration stirring culture, or continuous culture.
The culture conditions may be appropriately selected depending on the type of culture medium and the culture method, and may be any conditions that allow the microorganism to grow and produce the ALG-producing enzyme. Usually, the culture may be performed by aeration and stirring at an initial culture pH of 6.0 to 8.0 and a temperature of 25 to 37 ° C., and the number of culture days is often about 1 to 2 days.

What is necessary is just to isolate | separate and collect | recover the ALG production | generation enzyme of this indication produced as mentioned above by a well-known method. An example will be described below.
When ALG-producing enzyme is present in the microbial cells, the microbial cells may be collected by centrifugation after completion of the culture, and may be physically and chemically treated and released to the outside of the microbial cells. For example, the recovered bacterial cells are washed with a buffer solution, the enzyme is released from the bacterial cells with a surfactant, and the bacterial cells are removed by centrifugation to obtain an enzyme extract (crude enzyme solution). It is done. Alternatively, the collected cells are washed with a buffer solution, frozen instantaneously with liquid nitrogen (about −80 ° C.) to damage cell membranes and cell walls, dispersed again in the buffer solution, mixed, and then centrifuged. By removing the microbial cells, an enzyme extract (crude enzyme solution) can be obtained.
Moreover, when there exists ALG production | generation enzyme outside a microbial cell, after completion | finish of culture | cultivation, a microbial cell is removed by filtration or centrifugation, and the culture filtrate containing the said enzyme is obtained.

The obtained enzyme-containing liquid is desalted by an operation such as gel filtration, and then subjected to one or more purification methods such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, and electrophoresis. Two or more types can be combined and purified with high purity. In addition, methods such as ammonium sulfate salting out, solvent precipitation, lyophilization, ultrafiltration, and chromatofocusing are also used.
As a specific example, the enzyme and microorganism can be separated from the culture solution, the microorganism can be removed therefrom, and the cell-free extract can be concentrated and recovered using a known enzyme separation and purification method. . Separation and purification methods include, for example, filtration methods such as gel filtration chromatography and ultrafiltration membrane, and enzyme precipitation methods by adding ammonium sulfate.

The Cellvibrio genus bacterium of the present disclosure is not particularly limited as long as it has a polynucleotide encoding the above-mentioned ALG-generating enzyme. -The thing which has the function to produce the galactose, and also the function to hydrolyze the (alpha) -1,3 coupling | bond part of a 3, 6- anhydro-L-galactose alpha (1-3) sugar residue is suitable.
Examples include Cellvibrio bacteria having a polynucleotide encoding the ALG-producing enzyme, bacteria equivalent thereto, and mutants thereof. Here, the mutant strain can be obtained from a wild strain by a known technique of treatment with ultraviolet rays, ionizing radiation, nitrous acid, nitrosoguanidine, ethylmethanesulfonate, and the like. Mutant strains include those obtained by further mutating mutant strains from wild strains.

The Cellvibrio sp. WU-0601 (NITE P-1365) strain is preferred. The mycological and physiological properties are shown below.
The WU-0601 strain is a gram-negative bacterium that grows under aerobic conditions. It is a koji mold having a size of (0.7 to 0.8) μm × (1.5 to 2.5) μm, has flagella, and has motility. It is oxidase positive and catalase positive, and has assimilability of glucose and sucrose.

The WU-0601 strain was obtained by the following procedure. A large number of microorganisms that can grow on an AD medium are obtained using soil, seawater, river water, or the like as a separation source. A cell-free extract is prepared by subjecting cells cultured in AD medium at 30 ° C. for 24 hours to physical and chemical treatment such as crushing and centrifugation. This cell-free extract is subjected to a detection test for activity of degrading neoagarobiose to obtain microorganisms having remarkable activity. In this manner, the WU-0601 strain was able to be collected from soil in the Kanto region of Japan.
Furthermore, the morphological and bacteriological properties of this strain WU-0601 were similar to those of the genus Cellvibrio or some of the genus Pseudomonas , but the 16S rDNA was not completely consistent. For this reason, this strain was presumed to be a novel microorganism belonging to the genus Cellvibrio , and this strain was named Cellvibrio sp. WU-0601 (NITE P-1365) strain. This Cellvibrio sp. WU-0601 (NITE P-1365) strain was identified as a novel microorganism from the above, on May 22, 2012, 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, 292-0818, Japan. Deposited as a Cellvibrio sp. WU-0601 (NITE P-1365) strain at the Patent Product Deposit Center (NPMD).

In order to produce the ALG-producing enzyme of the present disclosure from the Cellvibrio genus bacterium of the present disclosure, a medium for cultivating general soil bacteria (preferably a common medium used for culturing Cellvibrio genus bacteria) is used. Inoculate and culture at an appropriate temperature. Production of the ALG-producing enzyme of the present disclosure from the culture solution can be performed according to a conventional method. Specifically, after centrifuging the culture solution and removing the cells, it can be concentrated and recovered from the cell-free extract using a known enzyme separation and purification method. Separation and purification methods include, for example, filtration methods such as gel filtration chromatography and ultrafiltration membrane, and enzyme precipitation methods by adding ammonium sulfate.

The polynucleotide and gene of the present disclosure can be obtained from the natural world as described above, but the gene is cloned from the chromosomal DNA of the above-mentioned microorganism (preferably bacterium belonging to the genus Cervibrio). The disclosed ALG-generating enzyme can be produced and recovered in large quantities.
As a method for cloning the NAH gene, for example, the ALG-generating enzyme of the present disclosure can be obtained by ligating the polynucleotide and / or gene to a DNA vector capable of stably amplifying, or introducing it onto a chromosomal DNA that can be maintained in the gene. A method for producing the ALG-generating enzyme of the present disclosure by stably amplifying the DNA encoding the gene and introducing the gene into a host capable of expressing the gene stably and efficiently can be employed.

It should be noted that the polynucleotide and NAH gene of the present disclosure can be obtained by known methods (for example, “Sambrook, J., Fritch, EF, and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, Vol. 2, Cold (See Spring Harbor Laboratory Press, Cold Spring Harbor, NY). As an example, genomic DNA is extracted from an ALG-producing enzyme-producing strain, cleaved with an appropriate restriction enzyme, and then a phage vector is used to prepare a library consisting of the genomic DNA of the ALG-producing enzyme-producing strain. Alternatively, total RNA is extracted from an ALG-producing enzyme-producing strain, and a cDNA corresponding to mRNA is prepared by reverse transcriptase reaction using oligo dT as a primer, and then a phage vector is used to comprise the cDNA of the ALG-producing enzyme-producing strain. Create a library.
Based on the N-terminal amino acid sequence and conserved sequence as described above (preferably a conserved sequence in glycosyl hydrolase family 32), suitable primers are designed and synthesized, and used to generate genomic DNA derived from an ALG-producing enzyme-producing strain. Alternatively, a DNA chain of NAH gene is amplified by performing polymerase chain reaction (PCR) using cDNA as a template. Using this DNA fragment as a probe, a genomic library or a cDNA library is screened. In this way, the entire region of the NAH gene or a region necessary for expression can be isolated. After determining the nucleotide sequence of these DNA fragments, a restriction enzyme cleavage site is introduced upstream of the translation initiation codon and downstream of the translation termination codon by a technique such as PCR, and the polypeptide comprising only the NAH gene of the present disclosure It is possible to obtain a gene fragment containing

[Recombinant vector and its production method]
In addition, according to the present disclosure, it is possible to apply a recombinant vector containing a polynucleotide encoding the above-described ALG-generating enzyme, a gene encoding the enzyme, or a NAH gene. Thereby, a transformant can be obtained, and an ALG-producing enzyme can be produced by genetic engineering by culturing the transformant or the like.
The procedure and method for constructing the recombinant vector of the present disclosure may be those commonly used in the field of genetic engineering (Sambrook, J., Fritch, EF, and Maniatis, T. (1989) in Molecular Cloning : A Laboratory Manual, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Examples of vectors that can be used in the present disclosure include those that are incorporated into host chromosomal DNA and vectors that have a self-replicating autonomously replicating sequence present in the host cell in a plasmid state. Examples of the plasmid vector include pUC18 and pBR322 (Takara Bio) when Escherichia coli is used as a host. Note that the number of copies of the gene present in the host cell may be either one or multiple copies.

The recombinant vector of the present disclosure has, for example, a operably linked promoter sequence (control region) upstream of a polynucleotide sequence encoding the above-described ALG-generating enzyme, a gene encoding the enzyme, and a terminator downstream. In some cases, it can be made by operably linking genetic markers and / or other regulatory sequences.
Ligation of a promoter or terminator to the gene of the present disclosure and insertion of an expression unit into a vector can be performed by a known method (Sambrook, J., Fritch, EF, and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual, Vol. 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
Here, the promoter and terminator used in the present disclosure are not particularly limited. For example, a regulatory sequence of a glycolytic enzyme gene such as 3-phosphoglycerate kinase or glutaraldehyde-3-phosphate dehydrogenase; an amino acid such as tryptophan synthase Control sequences of synthetic enzyme genes; control sequences of hydrolase genes such as amylase, protease, lipase, cellulase; control of oxidoreductase genes such as nitrate reductase, orotidine-5'-phosphate dehydrase, alcohol dehydrogenase Examples include sequences. In addition, each control sequence means a polynucleotide that can exhibit a desired function in each control region that is usually present upstream or downstream of a gene (region encoding the amino acid sequence of a protein) on DNA.
The polypeptide or gene of the present disclosure may be linked to a foreign gene encoding a translation region of another protein and expressed as a fusion protein.

In addition, the gene marker is introduced into the recombinant vector by, for example, introducing an appropriate restriction enzyme cleavage site into the control sequence by PCR and inserting it into the plasmid vector, and then complementing the drug resistance gene and / or auxotrophic complementation. This can be done by linking a selectable marker gene such as a gene.
The selection marker can be appropriately selected according to the selection method of the transformant. For example, a gene encoding drug resistance and a gene complementary to auxotrophy can be used.
Examples of the drug resistance gene include genes for drugs such as destomycin, benomyl, oligomycin, hygromycin, G418, bleomycin, phosphinothricin, ampicin and kanamycin.
Examples of genes that complement this auxotrophy include argB gene, pyr4 gene, trpC gene, TRP1 gene, niaD gene, LEU2 gene, and URA3 gene.

[Transformant introduced with the recombinant vector of the present disclosure]
According to the present disclosure, a transformant can be obtained by transforming a host (preferably a microorganism) using the recombinant vector obtained above.
The host to be used is not particularly limited as long as it can be used as a host for gene recombination, preferably a microorganism. Examples of the host that can be used include microorganisms such as arbitrary bacteria and fungi. , Escherichia (Escherichia) genus, cell Vibrio (Cellvibrio) genus Pseudomonas (Pseudomonas) genus Flavobacterium (Flavobacterium) genus Bacillus (Bacillus) genus Serratia (Serratia) genus Corynebacterium (Corynebacterium) genus, Burebibakuteri Umm (Brevibacterium) genus, Agrobacterium (Agrobacterium) genus Acetobacter (Acetobacter) genus Gluconobacter (Gluconobacter) genus Lactobacillus Lactobacillus) genus Streptococcus (Streptococcus) genus or Streptomyces (Streptomyces) genus Saccharomyces ( Saccharomyces ), Schisaccharomyces Zosaccharomyces) genus Aspergillus (Aspergillus) genus Trichoderma (Trichoderma) sp, Nyuurosupora (Neurospora) genus, it is preferable to use a microorganism belonging and their variants such as. From the viewpoint of easy recombination, E. coli or a mutant thereof is more preferable.

  At this time, these microorganisms are recombinant microorganisms that have been subjected to mutations such as gene substitution, insertion, deletion, inactivation and the like so that 3,6-anhydro-L-galactose can be easily produced in the end. As this recombinant microorganism, a recombinant microorganism capable of producing neoagarobiose is preferable. The recombinant microorganism capable of producing neoagarobiose is preferably a microorganism whose gene has been mutated so that neoagagarobiose can be mass-produced using known techniques. Specifically, a microorganism having a gene encoding β-agarase capable of producing neoagarobiose may be used. For example, the amino acid sequence shown in SEQ ID NO: 7 as a β-agarase capable of producing neoagarobiose and the base sequence shown in SEQ ID NO: 8 as a gene encoding a β-agarase capable of producing neoagarobiose can be mentioned. Moreover, these microorganisms can be obtained by the methods described in Patent Documents 1 and 2.

  Examples of methods for causing mutations in the genes as described above include, for example, a recombinant PCR method (PCR Technology, Stockton press (1989)), a partial specific displacement method (Kramer, W. and Frits, H., J. Methods in Enzymology, 154,350 (1987)], SOE (splicing by overlap extension) -double crossover method using DNA fragments prepared by PCR [Gene, 77, 61, (1987)], chemical agent treatment (N-methyl- N′-nitrosoguanidine, nitrous acid, etc.) and a method of chemically synthesizing the target gene.

The transformant of the present disclosure can be obtained by introducing the recombinant vector for gene expression prepared as described above into the host according to a conventional method.
Examples of the introduction method include an electroporation method, a polyethylene glycol method, an Agrobacterium method, a competent method, a lithium acetate method, and a calcium chloride method. What is necessary is just to select suitably according to the host cell to be used.

<2.3,6-Anhydro-L-galactose production method>
The method for producing 3,6-anhydro-L-galactose according to the present disclosure can generate ALG using a carbohydrate having a basic structure of 3,6-anhydro-L-galactose α (1-3) sugar residue as a raw material. It suffices to have at least a production process (enzyme reaction system: for example, see the above-described scheme 1 and FIG. 1).
In this production process, the above-described ALG producing enzyme of the present disclosure, a microorganism having a gene encoding the ALG producing enzyme of the present disclosure, and the like are used.
More specifically, by using the ALG-generating enzyme of the present disclosure, a saccharide having a structure of 3,6-anhydro-L-galactose α (1-3) D-galactose as a raw material, 3,6-anhydro- It is preferable to produce L-galactose and collect it appropriately.
In addition, a microorganism containing a nucleotide encoding the ALG-producing enzyme of the present disclosure or a gene comprising the same is cultured, and a carbohydrate having a 3,6-anhydro-L-galactose α (1-3) D-galactose structure is used as a raw material 3,6-Anhydro-L-galactose is preferably produced and recovered.

Examples of the raw material include saccharides composed of galactose or galactose derivatives. Examples of the saccharide include polysaccharides and oligosaccharides.
As the oligosaccharide used as a raw material, the oligosaccharide described in the above [Substrate specificity] is preferably used. The oligosaccharide is preferably one or more selected from neoagarobiose, neoagarotetraose and neoagarohexaose, and among these, neoagarobiose is preferred.
Moreover, when using polysaccharide as a raw material, it is suitable to use the enzyme which can be decomposed | disassembled to the oligosaccharide as described in the above-mentioned [Substrate specificity]. Examples of the polysaccharide include agar containing agarose and agaropectin, porphyran, carrageenan and the like.

The reaction mode of the production method of 3,6-anhydro-L-galactose of the present disclosure is not particularly limited, and may be performed in a batch system or a continuous flow system.
Moreover, the enzyme and microorganism used in the production method of the present disclosure may be immobilized. The immobilization technique is not particularly limited as long as it is a known technique. For example, a carrier binding method in which a microorganism / enzyme is immobilized on a water-insoluble carrier via physical adsorption, ionic bond, or covalent bond; glutaraldehyde Examples of such a crosslinking method include cross-linking and immobilization with a reagent having a bivalent functional group such as a gel having a network structure and a comprehensive method in which microorganisms and enzymes are confined in a semipermeable membrane.
In addition, when the enzyme used in the production method of the present disclosure is present in a microorganism, ALG may be produced after the microorganism is treated such as crushing to release the enzyme to the outside of the cell. The solvent used for the reaction is preferably water.

The reaction temperature during the production of ALG is preferably 5 to 40 ° C, more preferably 10 to 40 ° C. The pH is preferably 5 to 8, more preferably 6 to 8.
With the ALG-generating enzyme of the present disclosure, ALG can be produced satisfactorily within such temperature and pH ranges. The ALG-generating enzyme of the present disclosure further has a strong acid / strong alkaline condition such as less than pH 2 or more than pH 8 or 85 ° C. It is desirable that the temperature is not high. Also from this point, it can be said that the ALG producing enzyme of the present disclosure is an enzyme suitable for producing ALG.
Furthermore, it is desirable to perform in the range of the said conditions also when using the beta-agarase which can produce neo agarobiose mentioned later. At this time, it is preferable to use an enzyme derived from the genus Alteromonas described in Patent Documents 1 and 2 described later. The β-agarase is desirably used together with the ALG producing enzyme of the present disclosure or used as a pretreatment reaction for the ALG producing enzyme reaction.

As a purification means of 3,6-anhydro-L-galactose of the present disclosure, a known purification means such as a chemical method and a physical method may be used. Activated carbon, gel filtration, molecular weight fractionation membrane, electric thin layer Fractionation methods by chromatography and the like; solvent extraction methods; crystal precipitation methods; various chromatographs using HPLC, gel filtration, silica gel, ion exchange resins, and the like. ALG or the like of the present disclosure can be recovered by combining the purification means alone or appropriately.
In addition, compounds such as ALG of the present disclosure can be used in known analysis methods such as nuclear magnetic resonance methods ( ¹ H-NMR, ¹³ C-NMR, etc.), mass spectrometry methods, ultraviolet absorption spectrum measurement methods, infrared absorption spectrum measurement methods, and the like. Can be analyzed.

  In the method for producing 3,6-anhydro-L-galactose of the present disclosure, a β-agarase capable of producing the above-described neo-agarobobiose, a polynucleotide encoding the enzyme, and a microorganism having a gene comprising the nucleotide are used. May be. As a result, carbohydrates having a basic structure of 3,6-anhydro-L-galactose α (1-3) D-galactose, such as polysaccharides and oligosaccharides derived from red algae can be efficiently and easily converted to 3,6-anhydro- L-galactose can be obtained.

Examples of the β-agarase include the following agar degrading enzymes and neo agarobiose producing enzymes.
An agar-degrading enzyme that is produced by a microorganism belonging to the genus Arteromonas and that selectively decomposes agarose to produce neo-agarobobiose and has the following properties.
(1) Action: Hydrolyzes β-1,4 bonds of agarose to produce neoagarobiose. Preferably the viscosity of the agarose solution is reduced.
(2) Substrate specificity: Acts on agarose, neoagarohexaose, neoagarotetraose, and rapidly decomposes into neoagarobiose. However, it does not act on neoagarobiose or lactose (lactose).
(3) pH stability and optimum pH: stable in the range of pH 6.0 to 9.0 under conditions where no substrate is present. When agar is used as a substrate, the optimum pH is 7.5, and high activity (95% of the maximum activity) is exhibited even at pH 8.0.
(4) Thermal stability and optimum temperature: Under conditions where no substrate is present, stable at 40 ° C. or lower, and about 3% of activity remains at 50 ° C.
When agar is used as a substrate, the optimum temperature is 20 to 50 ° C, particularly 40 ° C.
(5) Molecular weight: about 80 to 90 KDa (preferably about 84,000) (electrophoresis); about 170 to 190 KDa (preferably about 180,000) (gel filtration method)
(6) Influence of metal salt and the like: Inhibited by Cu ²⁺ , Mn ²⁺ , Fe ²⁺ , Hg ²⁺ and Zn ²⁺ . Moreover, it is somewhat inhibited by Ca ²⁺ , Mg ²⁺ , Ag ⁺ , and parachloromercury benzoic acid (PCMB). It is not inhibited by K ⁺ , Na ⁺ , or ethylenediaminetetraacetic acid (EDTA).

The agar-degrading enzyme belongs to Alteromonas and is deposited as FERM.P-14664 at the National Institute of Technology and Evaluation (NITE-IPOD) (formerly Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology) It is preferable that the microorganism is obtained from a microorganism (consignment date: November 25, 1994). The microorganism preferably possesses an agar-degrading enzyme that degrades agarose and selectively produces neoagarobiose.
In addition, as a method for producing neoagarobiose, it is preferable to obtain neoagarobiose by bringing the agar-degrading enzyme into contact with agar.
Furthermore, it is preferable to hydrolyze the β-1,4 bond of the agar to selectively obtain neoagarobiose.

Neoagarobiose producing enzyme comprising the following protein (a), (b) or (c):
(A) A protein comprising the amino acid sequence set forth in SEQ ID NO: 7.
(B) A protein having a neoagarobiose production activity ability, comprising an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 7. Preferably, it has a high affinity for agar, agarose or tetra-or higher neo-agaro-oligosaccharides.
(C) A protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 7, and having a neoagarobiose production activity. Preferably, it has a high affinity for agar, agarose or tetra-or higher neo-agaro-oligosaccharides.

  In addition, in the amino acid sequence shown in SEQ ID NO: 7, an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more is suitable.

Furthermore, it is preferable to have the following properties.
(1) Action: Hydrolyzes β-1,4 bond of agar, agarose or tetra- or more neo-agaro-oligosaccharide to produce neo-agarobiose.
(2) Substrate specificity: Acts on agarose, neoagarohexaose, neoagarotetraose, and rapidly degrades into neoagarobiose. However, it does not act on neoagarobiose or lactose.
(3) Optimal temperature: 40 ° C. Stable temperature: 40 ° C. or less.
(4) Molecular weight: about 170-190 kDa (preferably about 180 kDa) (by gel filtration chromatography); about 80-90 kDa (preferably about 82 kDa) (by SDS-PAGE).

In addition, the following polynucleotides that encode the neo agarobiose producing enzyme are preferable.
The following polynucleotides (a) to (g), which encode an amino acid sequence of a polypeptide that selectively degrades agar, agarose, or tetra- or more neo-agaro-oligosaccharides into neo-agarobiose.
(A) a polynucleotide encoding a polypeptide represented by the amino acid sequence of SEQ ID NO: 7;
(B) a polynucleotide encoding a polypeptide represented by an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence set forth in SEQ ID NO: 7.
(C) a polynucleotide encoding a polypeptide represented by the amino acid sequence represented by Nos. 32-798 of the amino acid sequence described in SEQ ID No. 7.
(D) A polynucleotide encoding a polypeptide represented by an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequences 32 to 798 of SEQ ID NO: 7.
(E) a polynucleotide represented by the nucleotide sequence set forth in SEQ ID NO: 8.
(F) A polynucleotide represented by Nos. 94 to 2394 of the nucleotide sequence set forth in SEQ ID NO: 8.
(G) A polynucleotide that hybridizes with the polynucleotide (a) to (f) under stringent conditions.

  Furthermore, it is preferable to use a vector containing a polynucleotide encoding the above-mentioned neoagarobiose producing enzyme. Furthermore, it is preferable to use a transformant introduced with the vector.

The method for producing neoagarobiose is a method for selectively treating neoagarobiose by causing the neoagarobiose producing enzyme to act on one or more selected from agar, agarose and tetra- or more neo-agaro-oligosaccharides. It is preferred to obtain robiose.
In addition, microorganisms such as the above-mentioned microorganisms of the genus Alteromonas or transformants that produce the above-mentioned neoagarobiose-producing enzyme are cultured on one or more selected from agar, agarose, and tetra- or more neo-agaro-oligosaccharides. It is preferable to selectively obtain neoagarobiose by allowing the resulting polypeptide to act.
In addition, it is preferable to use a product obtained by culturing the transformant and collecting the neoagarobiose-producing enzyme from the culture.

<3. Use of 3,6-anhydro-L-galactose of the present disclosure>
Furthermore, 3,6-anhydro-L-galactose has a conventional difficulty in that the anhydro portion is likely to disappear and the structural change caused by the open ring (for example, 3,6-anhydro-L-galacto-2-ketose). It was difficult to ensure a stable amount. [C. Rochas, P. Potin, and B. Kloareg, Carbohydrate Research, 253, 69-77 (1994)] describes agarooligosaccharides such as agarotetraose having ALG on the reducing end as NMR and the like. Although there is an example analyzed with an instrument, this analysis result shows the presence of a structure in which the C1 position of this terminal ALG is an open ring (structure of 3,6-anhydro-L-galacto-2-ketose). . On the other hand, in neo-agarotetraose containing ALG at a site that is not on the reducing end side, there is no structure in which the C1 position of ALG is opened, and whether or not ALG is present on the reducing end side is a result of NMR and the like. So the difference is clear.
As can be inferred from these results, the C1 position of ALG tends to be an open ring, and a keto group that easily undergoes a chemical change appears in the open ring structure. Therefore, in the presence of acid or alkali, the structure of ALG is susceptible to structural changes from the keto group, and it has been difficult to produce and isolate ALG by an easy method such as hydrolysis. For this reason, ALG's basic research and applied research are not progressing in various fields.

However, the ALG production method of the present disclosure provides 3,6-anhydro-L-galactopyranose [α, β type] and 3,6-anhydro-L-galactofuranose [α, β type] easily and efficiently. It becomes possible to do. Furthermore, various derivatives (for example, ALG derivatives such as 2-OMe-ALG, 3,6-anhydro-α or β-L-galactoside, etc.) according to demands are obtained by organic synthesis or the like based on the obtained ALG. Is also possible. For this reason, the products obtained by the ALG manufacturing method of the present disclosure can be expected to be used in a wide range of fields such as pharmaceuticals, cosmetics, topical skin preparations, and foods, and new applications can be provided. Become.
For example, as shown in Examples described later, it was confirmed that ALG obtained by the above-described method has a melanin production inhibitory action. Therefore, the product obtained by the method of the present disclosure can be used as a whitening material as it is or as a preparation such as a whitening agent containing it as an active ingredient.

  Moreover, what was obtained by the ALG manufacturing method of this indication is a substance which can be used in order to ingest, administer, or contact an animal including a human, and to aim at melanin production suppression, whitening, etc. For example, what was obtained by the ALG manufacturing method of this indication can be used for the purpose of prevention, improvement, or treatment, skin care, and beauty. Moreover, what was obtained by the ALG manufacturing method of this indication can be used for various preparations, such as a melanin production inhibitor and a whitening agent, and can be used in order to manufacture these various preparations.

  Therefore, what is obtained by the ALG production method of the present disclosure is a skin external preparation, cosmetic, pharmaceutical product, quasi-drug, food or functional food (for example, specified health care) for melanin production inhibitory action, whitening action, etc. The preparation of the present disclosure is useful as a topical skin preparation, cosmetic, functional food (eg, tablet, soft drink, etc.) and the like.

  In addition, you may use for the said formulation combining arbitrary components with the thing obtained by the manufacturing method of this indication as needed. Other preferable components may be pharmaceutically acceptable components, for example, cell activators, antioxidants, humectants, UV inhibitors, solvents (water, monovalent carbon number of 1 to 5 or many). Valent alcohols), water-soluble polymers, oil agents, surfactants, thickeners, powders, chelating agents, pH adjusters, emulsifiers, stabilizers, colorants, brighteners, flavoring agents, flavoring agents, Excipients, binders, disintegrants, lubricants, diluents, osmotic pressure adjusting agents, fragrances and the like can be mentioned, and these may be added according to the intended preparation.

The form of the preparation is not particularly limited, and may be any form such as liquid, paste, gel, solid, and powder.
ALG obtained by the production method of the present disclosure is particularly preferably used for external preparations for skin, cosmetics, and quasi-drugs, and preparations that are brought into contact with the skin for application or the like are suitable.
In addition, the dosage form of the preparation is not particularly limited, and skin care cosmetics such as lotion, milky lotion, cream, pack, cleansing, massage, and beauty essence; makeup cosmetics such as foundation, lipstick, and blusher; Cleaning agents such as shampoos and body soaps; hair cosmetics such as rinses and hair packs; and external preparations such as external preparations for skin such as ointments and dispersions, and various other agents. It can also be shaped.
The blending amount of ALG in the preparation is preferably 0.0001 to 30% by mass, more preferably 0.001 to 5% by mass.

Hereinafter, examples and comparative examples will be given to specifically describe the present invention (present technique), but the present invention (present technique) is not limited to the examples.

<Example 1: ALG-producing enzyme>
In the Kirimura laboratory, the activity of neoagarobiose hydrolase (also referred to as “NAH”) was detected in the cells of the non-marine bacterium Cellvibrio sp. WU-0601. Furthermore, NAH derived from Cellvibrio sp. WU-0601 strain was purified and its enzymatic properties were examined. Since it reacts with neoagarotetraose and neoagarohexaose to produce 3,6-anhydro-L-galactose (hereinafter also referred to as “ALG”), this “NAH” and its “ NAH gene” It was also found that it can be said to be “ALG-generating enzyme” and “gene encoding ALG-generating enzyme” that generate ALG.
There are very few reports on the purification and enzymatic properties of NAH, and no gene encoding NAH has been reported to date. Therefore, research on the enzymatic properties of NAH and the function of its gene is novel.
Then, the NAH of the present disclosure, for example, as shown in FIG. 1, finally ALG is obtained from a saccharide having “3,6-anhydro-L-galactose α (1 → 3) D-galactose” such as agarose. It is possible to generate efficiently. In addition, the gene encoding NAH of the present disclosure was cloned, and the gene was successfully expressed in E. coli. Moreover, since the produced | generated ALG suppresses melanin production, it can be utilized also as external compositions, pharmaceuticals, functional foods, etc., such as a whitening agent. Therefore, the present inventors also found that the ALG-generating enzyme of the present disclosure has very high industrial applicability.
Below, <1. Experimental method> and <2. Results> will be described.

<1. Experimental method>
<1.1. Strain used and culture method>
As a test bacterium, a non-marine bacterium Cellvibrio sp. WU-0601 strain was used. The strain is a novel microorganism obtained and identified as described below.
AD culture medium was used for culturing Cellvibrio sp. Strain WU-0601. AD medium is KCl 0.75 g, MgSO ₄・ 7H ₂ O 12.35 g, CaCl ₂・ 2H ₂ O 1.45 g, NH ₄ Cl 1.00 g, FeSO ₄・ 7H ₂ O 0.012 g, K ₂ HPO ₄ 0.075 g, peptone 1.25 g, yeast extract containing 1.25 g and Tris 1.21 g (Table 1), and the initial pH was adjusted to 8.0.
As pre-culture, the cells were cultured in 5 ml of AD medium at 30 ° C. and 120 rpm for 24 hours, and as main culture, they were cultured in 200 ml of AD medium at 30 ° C. and 120 rpm for 48 hours. Further, Escherichia coli JM109 was used as a host for gene cloning, E. coli BL21 (DE3) was used as a host for gene expression, and LB medium was mainly used for the culture.

<1.2. Purification method of NAH>
<1. The bacterial cells were collected from the culture solution of Cellvibrio sp. WU-0601 strain cultured according to the strain and culture method> by centrifugation (10000 × g, 10 min, 4 ° C.) 8.0). The cell suspension was crushed with an ultrasonic crusher, centrifuged (16000 × g, 30 min, 4 ° C.), and the supernatant was used as a cell-free extract. A 1% (w / v) streptomycin sulfate solution was added to the cell-free extract, and a solution obtained by removing nucleic acid was used as a crude enzyme solution.
The crude enzyme solution was fractionated with ammonium sulfate and passed through three columns to purify NAH. For the above purification, a chromatography system for biomolecule purification AKTA explorer 10S (Amersham Bioscience) was used. The purity of the target enzyme was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Each purification step is described below.
(Step 1) Fractionation with ammonium sulfate A 10 mM Tris-HCl buffer solution (pH 8.0) was added to the cell-free extract to 20 ml, and ammonium sulfate was gradually added. Centrifugation (4 ° C., 15,000 × g, 30 min) was performed at the point where saturation reached 40%, and the supernatant was recovered. Further, ammonium sulfate was gradually added, and the precipitate was recovered when the saturation reached 60%, and suspended in 10 mM Tris-HCl buffer (pH 8.0). The protein concentration was measured, and enzyme activity was measured by TLC analysis and HPLC.
(Step 1-2) Dialysis The obtained solution was dialyzed for 16 hours at 4 ° C. in 10 mM Tris-HCl buffer (pH 8.0) using Dialysis Membrane, Size 36 (Wako Pure Chemical Industries, Ltd.).

(Step 2) DEAE-Toyopearl 650S column chromatography An anion exchange column in which the obtained solution was passed through a Cellose acetate 0.20 μm filter (Whatman) and equilibrated with 50 mM Tris-HCl buffer (pH 8.0). The sample was applied to DEAE-Toyopearl 650S (Tosoh) at a flow rate of 2.0 ml / min, and the protein was eluted with a linear KCl gradient from 0 M to 0.30 M. The NAH activity of each obtained fraction was measured by TLC analysis, and the fractions in which the activity was detected were collected and concentrated (1500 × g, 15 min) with Centriprep YM-30 (Millipore). Thereafter, the protein concentration of the enzyme in the solution was measured, and the enzyme activity was measured.
Table 2 shows various setting conditions in Step 2.

(Step 3) HiLoad 16/60 Superdex 200 pg column chromatography HiLoad 16/60 Superdex, a gel filtration column in which the enzyme solution is equilibrated with 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl. Subjected to 200 pg (GE Healthcare). The same buffer was flowed at a flow rate of 0.5 ml / min to elute the enzyme. The ALG generation activity of each obtained fraction was measured by TLC analysis. The active fractions were combined, concentrated with Centriprep YM-30 (1500 × g, 15 min), and replaced with 50 mM Tris-HCl buffer. did. Thereafter, the protein concentration of the solution was measured, and the enzyme activity was measured.
Table 3 shows various setting conditions in Step 3.

(Step 4) Mono Q 5/50 GL Column Chromatography The enzyme solution was equilibrated with 50 mM Tris-HCl buffer (pH 8.0). Provided. After washing the column with 10 ml of the same buffer, the enzyme was eluted with a linear gradient of KCl (0 to 700 mM) in the same buffer. The protein concentration of the enzyme in each of the obtained fractions was measured, and the enzyme activity was measured by colorimetry and HPLC.
Table 4 shows various setting conditions in Step 4.

<1.3. Method for Measuring ALG Generation Activity>
A hydrolysis reaction of neoagarobiose was performed using NAH purified according to the above-described method. The unit of enzyme activity 1 unit (U) was defined as the activity to produce 1 μmol of D-galactose at 30 ° C. using neoagarobiose as a substrate. As standard reaction conditions, NAH 5 mU, neoagarobiose 2 mM, and 10 mM Tris-HCl buffer (pH 8.0) were mixed thoroughly so that the whole was uniform, and then 120 ° C. at a predetermined temperature. The reaction was performed while gently shaking at strokes / min. Thin layer chromatography (TLC) was used for qualitative detection of activity. High performance liquid chromatography (HPLC) was used for quantitative activity measurement.

(Thin layer chromatography (TLC))
The conditions for TLC analysis are shown below.
Stationary phase: Silicagel 60 (Merck)
Mobile phase: n-butanol / acetic acid / water (2/1/1 (v / v / v))
Color reagent: 0.2% (w / v) naphthoresorcinol ethanol / 20% (w / v) sulfuric acid aqueous solution: 1/1 (v / v)
Development was performed vertically upward, and after development, the color was developed by spraying the color reagent and heating at 100 ° C. for 5 min.
(High performance liquid chromatography (HPLC))
The HPLC analysis conditions are shown below.
System: HPLC L-7000 (Hitachi High Technology)
Column: SUGAR SP0810 x 2 + guard column x 1 connection type (Showa Denko)
Moving layer: H ₂ O
Flow rate: 0.8 ml / min
Column temperature: 80 ° C
Detection: RI
Sample: Prepared by passing the reaction solution through a 0.20 μm PTFE (Hydrophobic) membrane filter Measurement time: 30 min

<1.4. Methods for studying enzymatic properties of NAH>
Using purified NAH, the optimum temperature, temperature stability, optimum pH, influence of metal ions and chemical substances, and substrate specificity of the hydrolysis reaction were examined.
The standard conditions were as follows: NAH 5 mU / ml of reaction solution, neoagarobiose concentration 2 mM, temperature 30 ° C., 10 mM Tris-HCl buffer (pH 8.0). 1 ml of the reaction solution was reacted in a 1.5 ml microtube (Eppendorf) for 1 hour, then boiled for 10 minutes to stop the reaction, and the product in the reaction solution was analyzed by HPLC.
When determining the kinetic constants, each reaction solution with varying substrate concentration was allowed to react for a certain period of time under optimal reaction conditions, then boiled for 10 minutes to stop the reaction, and the product in the reaction solution was analyzed using HPLC. Each constant was calculated by Lineweaver-Burk plot.

The various examination methods regarding the enzymatic properties of NAH are shown below.
[Examination method of optimum reaction temperature and temperature stability]
Using an enzyme solution purified from NAH, the optimum temperature and temperature stability of the hydrolysis reaction of neoagarobiose were examined. Under standard conditions other than the reaction temperature, the temperature was changed to 20 to 50 ° C., and the amount of galactose produced under each condition in the reaction for 1 h was measured. In addition, regarding temperature stability, after remaining at a temperature of 4 to 60 ° C. for 30 minutes, the residual activity of the enzyme in the reaction at 30 ° C. for 1 h was examined.
[Examination method of optimum pH]
The optimum pH in the hydrolysis reaction of neoagarobiose was examined using the enzyme solution purified from NAH. Under standard conditions other than pH, the pH was changed to 4 to 9, and the amount of galactose produced under each condition was measured. At this time, 10 mM citrate / NaOH buffer (pH 4.0 to 7.0), 10 mM citrate / phosphate buffer (pH 4.5 to 7.0), 10 mM Tris / maleic acid / NaOH buffer (pH 5.0-7.5), 10 mM Tris / HCl buffer (pH 7.0-9.0) was used.
[Method for examining the effects of various metal ions]
Using purified NAH, the influence of metal ions and inhibitors in the hydrolysis reaction of neoagarobiose was examined. Under standard conditions, the amount of galactose produced was measured under the condition that appropriate amounts of EDTA and each metal ion were added. As the inhibitor, parachloromercury benzoic acid (pCMB) was used as an SH group inhibitor. pCMB was added to 0.2 mM, and other compounds to 2 mM.

[Method for examining substrate specificity]
Substrate specificity was examined using purified NAH. Under standard conditions, the reaction product after 24 h reaction was measured under the condition that each substrate was prepared in an amount of 20 mM each and added instead of neoagarobiose.

[Method for studying calculation of kinetic constants]
In order to analyze the properties of the enzyme more quantitatively, the reaction rate constant of NAH was calculated.
<8. Under the optimal reaction conditions judged in <Enzymatic properties of NAH>, the reaction was carried out at a neoagalobiose concentration of 1 to 16 mM for 10 to 50 min, and the measurement was compared. Based on this result, Km and Vmax were determined from the measured values of the substrate concentration (S: neoagarobiose) and the reaction rate (v: initial rate of product synthesis at each substrate concentration). Hanes-Woolf plot based on measured values (method of plotting [S] / v against [S]) and Lineweaver-Burk inverse plot (method of plotting 1 / v against 1 / [S]) Was used to calculate Km and Vmax. Furthermore, kcat (= Vmax / Km) was also calculated.

<1.5. Method for Cloning Gene Encoding NAH>
The N-terminal amino acid sequence of purified NAH was determined. Based on the N-terminal amino acid sequence of the obtained NAH and the conserved sequence in glycosyl hydrolase family 32 showing homology, sence primer (5'- CGA TCT GCC GGA AAA AAA -3 ') (SEQ ID NO: 5) and antisence primer (5′-GGR TCR TGN ACY TTR TG-3 ′) (SEQ ID NO: 6) was prepared, and PCR was performed using the whole DNA extracted from Cellvibrio sp. WU-0601 strain as a template.
The obtained amplified fragment was ligated to the pGEM-T Easy vector, and E. coli JM109 was transformed to obtain a partial base sequence of the enzyme gene. For gene analysis, GENETYX (registered trademark) was used in the laboratory, and analysis was carried out using public databases and software such as DDBJ.
The base sequence was determined by the dye terminator method using ABI PRISM 310 Genetic Analyzer. Furthermore, using the obtained nucleotide sequence information, inverse PCR was carried out using as a template the total DNA extracted from Cellvibrio sp. WU-0601 strain, digested with Sph I and Ssp I and then self-ligated. A gene region encoding NAH was found in the obtained PCR product, and the nucleotide sequence was determined.

<2. Result>
<2.1. Results of obtaining microorganisms exhibiting NAH activity>
A large number of microorganisms that can grow on AD media were obtained from soil, seawater, river water, and other sources. In particular, inoculate a solid AD medium supplemented with 30 g / l agar and incubate at 30 ° C for 5 days. agarase) was determined as a culture containing microorganisms. The culture containing agar-degrading enzyme-producing microorganisms was further subjected to pure separation, and 12 strains of agar-degrading enzyme-producing microorganisms were isolated. All the isolated microorganisms were bacteria, including the genus Alteromonas and Pseudomonas .
Cells cultured in AD medium at 30 ° C. for 24 hours were disrupted and centrifuged to prepare a cell-free extract. When this cell-free extract was subjected to a test for detecting the activity of degrading neoagarobiose, the most prominent activity was detected in one bacterium isolated from soil (WU-0601 strain).

The WU-0601 strain is a gram-negative bacterium that grows under aerobic conditions. (0.7-0.8) μm × (1.5-2.5) μm in size, with flagella and motility. It is oxidase positive and catalase positive, and has assimilability of glucose and sucrose.
The morphological and bacteriological properties of the strain WU-0601 were similar to those of the genus Cellvibrio or some of the genus Pseudomonas .
Furthermore, the base sequence of 16S rDNA of WU-0601 strain was determined and collated with a database. Known 16S rDNA and 98.2% homology Cellvibrio Fibrivorans is in bacteria, C. Ostraviensis the same 97.8% homology, C. Mixtus subsp. Mixtus the same 97.2% homology, C. Fulvus the same 97.1% Shows homology. On the other hand, it shows 92.1% homology with that of Pseudomonaskilonensis and 92.1% with P. umsongensis . Although none of the 16S rDNA base sequences completely matched, the WU-0601 strain is judged to belong to the genus Cellvibrio . However, since homology with the 16S rDNA of the closest C. fibrivorans is 98.2%, it is considered to be appropriate to judge as Cellvibrio sp.

Thus, to identify the test bacteria which produce NAH Cellvibrio sp. And was referred to as a Cellvibrio sp. WU-0601 strain. While many of the agar-degrading enzyme-producing bacteria are marine, Cellvibrio sp. WU-0601 does not require NaCl for growth and does not require desalting, so it is a non-marine bacterium. One feature is that the work efficiency when mass-producing NAH is good is advantageous.

<2.2. Purification results of NAH>
As shown in Table 5, NAH was purified from the crude enzyme extract of Cellvibrio sp. WU-0601 in a four-step purification with a yield of 2.14% and a purification factor of 24.3 times. This purified enzyme was almost single on SDS-PAGE (FIG. 6) and showed about 42 kDa. The molecular weight of the enzyme was calculated to be about 83 kDa by gel filtration chromatography. From this, this enzyme is considered to be a homodimeric enzyme.
In addition, NAH activity was not detected in the solution (extracellular fraction) obtained by centrifuging the culture solution (10000 × g, 10 min, 4 ° C.).

<2.3. Enzymatic properties of NAH>
The enzymatic properties of the purified NAH were examined. The optimum reaction conditions were a temperature of 25 ° C. and a pH of 6.0. NAH was inhibited by AgNO ₃ , HgCl ₂ , NiCl ₂ , CuCl ₂ , FeCl ₃ , and pCMB, and the activity increased by MnCl ₂ and MgCl ₂ . In addition, NAH acts best on neoagarobiose and reacts with neoagarotetraose and neoagarohexaose, so it can specifically cleave the α1,3 bond between ALG and D-galactose. It was suggested.

Details of each examination are shown below.
[Study results of optimum reaction temperature and temperature stability]
The results of examining the optimum temperature and temperature stability in the hydrolysis reaction of neoagarobiose are shown below. FIG. 3A shows the result of change in activity with temperature, and FIG. 3B shows the result of temperature stability. From FIG. 3 (A), the optimum temperature of the enzyme was determined to be 25 ° C. In Fig. 3 (B), the temperature stability was examined. After 30 minutes, the activity was maintained up to 30 ° C, but about 50% of the activity was detected at 40 ° C, and it was over 50 ° C. It was almost 0%.

[Examination result of optimum pH]
The result of examining the optimum pH in the hydrolysis reaction of neoagarobiose is shown in FIG. When the optimum pH in NAH was examined, it had high activity in the vicinity of pH 5.5 to 6.0. In particular, when the 10 mM citrate-phosphate buffer solution at pH 6.0 was used, the highest activity was shown. Therefore, the optimum pH for the enzyme was determined to be 6.0.

[Results of studying the effects of various metal ions]
The effects of metal ions and inhibitors on the hydrolysis of neoagarobiose were investigated. The results are shown in Table 6. It was inhibited by AgNO ₃ , HgCl ₂ , NiCl ₂ , CuCl ₂ , FeCl ₃ and pCMB, and the activity was increased by MnCl ₂ and MgCl ₂ . In general, as a SH reagent, [1] 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, Elman reagent), 2,2 ′ (4,4 ′)-dipyridyl disulfide, tetrathionic acid, 2,6 -Oxidizing agents such as dichlorophenol indophenol (DCIP) and oxidized glutathione, [2] mercaptide forming agents such as p-chloromercuribenzoic acid (pCMB), p-mercuribenzenesulfonic acid (PMBS), [3] iodoacetic acid And alkylating agents such as iodoacetamide and N-ethylmaleimide (NEM), and [4] metal ions such as Hg ²⁺ , Zn ²⁺ and Cu ²⁺ . Of these, it was inhibited by pCMB, Hg ²⁺ , and Cu ²⁺ this time. However, the activity was maintained in other SH group inhibitors, suggesting that the SH group (cysteine residue in the enzyme) is involved in the activity.

[Results of examination of substrate specificity]
Table 7 shows the results of examining the substrate specificity of purified NAH. NAH acts best on neoagarobiose and reacts slightly with neoagarotetraose and neoagarohexaose, thus specifically cleaving the α-1,3 bond between ALG and D-galactose. Was suggested.

[Results of study of kinetic constants]
In order to analyze the properties of NAH more quantitatively, reaction rate constants were calculated.
Reaction rate constant calculated this time is the maximum velocity V _max of the Michaelis constant K _m and the reaction is an equilibrium constant of complexation reaction between enzyme and substrate. K _m is an index indicating the affinity of the enzyme for the substrate. The smaller the value, the higher the affinity for the substrate. V _max is an index indicating the number of rotations of the enzyme, and is a value indicating how short a single reaction can be catalyzed.
Furthermore, k _cat (= V _max / K _m ) was also calculated. k _cat is molecular activity and indicates the number of substrate molecules that one enzyme molecule converts per unit time. The larger this value, the easier the substrate will react. By examining these values, the reaction characteristics of the enzyme can be estimated.
The kinetic constants of NAH were determined as K _M = 4.39 × 10 (mM), V _max = 7.07 × 10 ⁻³ (mM / sec), and k _cat = 4.13 × 10 (sec ⁻¹ ).

<2.4. Determination of N-terminal amino acid sequence>
The N-terminal 17 amino acid sequence of the purified enzyme was obtained. The sequence information is shown in Table 8.
In Table 8, the N-terminal amino acid sequence of WU-0601 strain is shown in SEQ ID NO: 4, the N-terminal amino acid sequence of Sde_2657 derived from Saccharophagusdegradans 2-40 is shown in SEQ ID NO: 9, and the N-terminal amino acid sequence of Vibrio sp. JT0107 strain is The N-terminal amino acid sequence of Bacillus sp. MK03 is shown in SEQ ID NO: 11.
It showed high homology with the above-mentioned N-terminal amino acid sequence of Sde_2657 (protein with unknown function) derived from Saccharophagus degradans 2-40. Furthermore, Vibrio sp. JT0107, a previously reported α-neoagalo-oligosaccharide hydrolase, showed homology. The N-terminal 17 amino acid sequences of NAH are Gly-Asp-Leu-Pro-Glu-Lys-Lys-Leu-Ser-Lys-Ala-Ser-Leu-Arg-Ala-Ile-Glu (SEQ ID NO: 4) The N-terminal amino acid sequence of Sde_2657 (protein of unknown function) derived from Saccharophagus degradans 2-40 belonging to glycosyl hydrolase family 32 was confirmed .
The properties of NAH were compared with the properties of the previously reported α-neoagaro-oligosaccharide hydrolase, and the novelty of this enzyme was examined. The enzymes of WU-0601 and JT0107 possessed similar properties in terms of substrate specificity, molecular weight, and N-terminal amino acid sequence. However, the N-terminal amino acid sequence has little homology. On the other hand, the enzyme of the MK03 strain decomposed neoagarohexaose best in substrate specificity, but showed a tendency different from that of the WU-0601 strain that decomposes neoagarobiose best. The N-terminal amino acid sequence is also completely different. From the above, NAH of WU-0601 strain was considered to be a highly novel enzyme. In addition, no gene encoding NAH has been cloned so far. Therefore, it was decided to clone the gene encoding NAH.

<2.5. Cloning of NAH-encoding gene>
<2.4. Determination of N-terminal amino acid sequence> DNA derived from WU-0601 strain was used as a template using primers (SEQ ID NO: 5 and SEQ ID NO: 6) prepared based on the N-terminal amino acid sequence described above and the conserved sequence of glycosyl hydrolase family 32 As a result of PCR, an amplified fragment of about 620 bp was obtained. The amino acid encoded by the base sequence of the obtained amplified fragment was completely identical to the N-terminal amino acid sequence, and showed 75% homology with the amino acid residue of Saccharophagus degradans 2-40-derived Sde_2657.
PCR primers were prepared based on the sequence information of the amplification products obtained, and inverse PCR was performed. The obtained PCR amplification product was subjected to direct sequencing. Sequence analysis revealed the presence of a 1092 bp ORF encoding 364 amino acids. The estimated molecular weight was about 41 kDa, which almost coincided with the results of SDS-PAGE of purified NAH from the WU-0601 strain. The amino acid sequence of NAH is the amino acid sequence described in SEQ ID NO: 1, and the base sequence encoding NAH is the base sequence described in SEQ ID NO: 2 (see FIGS. 7 to 9).

<2.6. Protein showing homology with the gene encoding NAH>
<2.5. NAH amino acid sequence obtained by encoding gene cloning> (SEQ ID NO: 1) was subjected to homology search by BLAST for, for unknown function protein, Saccharophagus degradans 2-40 from Sde_2657 (ABD81917) (74%) Pseudoalteromonas atlantica T6c-derived Patl_1968 (ABG40486) (72%), Mcroscilla sp. PRE1-derived MS110 (AAK62832) (59%), and Flavobactoriales bacterium HTCC2170 (EAR01928) (55%). On the other hand, as being known for function, Sphingomonas sp from SKA58 xylosidase, arabinosidase;.. Bacillus sp SG-1 from the sucrose-6-phosphate hydrolase (Sucrase ); Stigmatella aurantiaca DW4 / 3-1 from There are beta-xylosidase and the like, but all have lower homology than Sde_2657 (Fig. 4).
Based on the above, this enzyme is considered to be a novel enzyme that forms a new group in GH32.
FIG. 5 shows an alignment with a protein of unknown function that showed high homology.
In the cloned NAH gene, since there is no signal peptide region required for secretion on the N-terminal side, NAH was considered to be an enzyme present in cells. This is a result consistent with the absence of NAH activity in the extracellular fraction (3.1).

<2.7. High expression in E. coli of a gene encoding NAH>
<2.6. Cloning of the gene encoding NAH> The gene encoding NAH obtained in> was ligated to Escherichia coli high expression plasmid pet-21a to prepare pNAH. Furthermore recombinant E. coli E .coli BL21 (DE3) introduced the gene / PNAH was prepared. Using this strain, expression of the gene encoding the target NAH was induced, and it was confirmed by SDS-PAGE of the crude enzyme extract whether the enzyme was expressed in E. coli. FIG. 6 shows the results of SDS-PAGE electrophoresis.
From the results of SDS-PAGE, it was possible to confirm a strong band at the same position as the purified enzyme from the WU-0601 strain for the recombinant Escherichia coli. Moreover, since there was not much difference in Whole cell and Cell-free extract, it was also confirmed that there was no formation of inclusion bodies. Since the gene encoding the enzyme was normally expressed in recombinant Escherichia coli, the activity was measured using this crude enzyme extract (Table 9). However, the activity measurement in this item was performed under the following conditions, unlike the enzyme purification shown in Table 6. Reaction conditions: per 200 μl of reaction solution, protein amount 0.05 mg, neoagarobiose 10 mM, temperature 25 ° C., 10 mM citrate-phosphate buffer (pH 6.0).

From FIG. 6, it can be seen that ALG is generated from neo agarobiose. Further, pET21a (+) only product in the introduced E. coli was not detected in E .coli BL21 (DE3). This confirmed that NAH was expressed in E. coli.
In addition, quantitative analysis was performed by HPLC analysis, and the specific activity per protein (U / mg-protein) was measured. As a result, the specific activity (U / mg-protein) reached about 27 times that of the wild strain Cellvibrio sp. WU-0601. From the above, it can be said that the enzyme gene was successfully expressed in Escherichia coli.

In this disclosure, NAH derived from Cellvibrio sp. WU-0601 strain was purified and various properties were examined. NAH is a 83 kDa homodimeric enzyme consisting of the same subunit with a molecular weight of 42 kDa. It acts on neoagarobiose, neoagarotetraose, neoagarohexaose, and α-α of ALG and D-galactose. The activity of specifically cleaving 1,3 bonds was shown.
The optimum conditions for NAH were 25 ° C. and pH 6.0, which were inhibited by AgNO ₃ , HgCl ₂ , NiCl ₂ , CuCl ₂ , FeCl ₃ , and pCMB, and the activity increased by the addition of MnCl ₂ or MgCl ₂ . Furthermore, the kinetic constants were determined as K _M = 4.39 × 10 (mM), V _max = 7.07 × 10 ⁻³ (mM / sec), and k _cat = 4.13 × 10 (sec ⁻¹ ).
In addition, the N-terminal amino acid sequence of the enzyme was determined as Gly-Asp-Leu-Pro-Glu-Lys-Lys-Leu-Ser-Lys-Ala-Ser-Leu-Arg-Ala-Ile-Glu (SEQ ID NO: 4) did. Furthermore, the entire region of the gene encoding NAH was cloned, and the presence of a 1092 bp ORF encoding a polypeptide chain consisting of 364 amino acid residues and an estimated molecular weight of about 41 kDa was revealed. Moreover, NAH activity was successfully detected by highly expressing the ORF in E. coli.

<Example 2: β-agarase capable of producing neoagarobiose and ALG production method using ALG-producing enzyme of the present disclosure>
The β-agarase capable of producing neoagarobiose used in the present disclosure was prepared based on the content described in Patent Document 2 (Japanese Patent Application Laid-Open No. 2005-229889). No. 7 and SEQ ID No. 8). Moreover, the agar-degrading enzyme described in claim 1 of Japanese Patent No. 3529173 may be used as β-agarase capable of producing neoagarobiose.
A DNA fragment for ALG-generating enzyme was purified according to <1.5. Cloning method of gene encoding NAH> described above. This purified DNA fragment was ligated with restriction digests obtained by treating pET21-a (+) with NdeI and BamHI in the same manner, and the resulting hybrid DNA was used to transform E. coli BL21 (DE3) strain. Recombinant E. coli producing ALG-producing enzyme was obtained.
As a result, neoagarobiose producing enzyme and ALG producing enzyme were obtained, and ALG was produced using them.

To 100 ml of 10 mM Tris-HCl buffer solution (pH 7.8) in which 4 g / l agarose (manufactured by Nippon Gene) was dissolved by heating, 300 U of neoagarobiose-producing enzyme was added, and the reaction (40 ° C, 160 rpm, 2 After h), the reaction was stopped (100 ° C., 5 min) and concentrated to 5 ml with an evaporator.
Furthermore, after dissolving in 25 ml of 20 mM citrate phosphate buffer (pH 6.0), adding 20 U of ALG-producing enzyme, reacting (25 ° C., 160 rpm, 15 min), stopping the reaction (acetone equivalent), centrifuging ( After 15,000 × g, 30 min, 4 ° C.), the supernatant was concentrated to 1 ml with an evaporator to obtain a hydrolyzate.
As a result of performing thin layer chromatography on the standard products ALG and D-Gal, the Rf value of ALG was 0.46 and the Rf value of D-galactose was 0.10. Therefore, separation and purification by silica gel chromatography was considered possible.
In order to separate and purify ALG and D-galactose from the hydrolyzate, silica column chromatography was performed, and fractionation (1 ml / 1 tube) was performed. The column used had a diameter of 20 mm and a length of 300 mm, Wako Gel C-300 as a carrier, and acetonitrile / water = 12/1 (v / v) as a developing solution. Each fraction was confirmed by thin layer chromatography, and 9 to 16 fractions having spots near the Rf value of 0.46 and no other components were collected to obtain ALG.
[Thin layer chromatography for sugar analysis]
Development is performed vertically upward, and after development, the color is developed by spraying the color reagent and heating at 100 ° C for 5 min.
Stationary phase: Silica gel 60 (Merck)
Mobile phase: Acetonitrile / water (12/1 (v / v))
Color reagent: 0.2% (w / v) naphthoresorcinol ethanol / 20% (w / v) sulfuric acid aqueous solution (1/1 (v / v))

The recovered 3,6-anhydro-L-galactose was subjected to ¹³ C NMR analysis. As a result, a signal (δc: 90.23, 73.17, 84.21, 6 carbon atoms of 3,6-anhydro-α-galactopyranose having a closed ring structure was obtained. 78.35, 76.88, 73.09) were detected (FIG. 10).
Therefore, by using the ALG-generating enzyme of the present disclosure, 3,6-anhydro-galactose can be obtained efficiently without losing the anhydro structure.

<Example 3: Evaluation of melanin production inhibitory effect of 3,6-anhydro-L-galactose>
Using mouse-derived B16 melanoma cultured cells, the 3,6-L-anhydrogalactose obtained in <Example 1> described above was examined for melanin production inhibitory effect and cell viability.
Specifically, an appropriate amount of 10% FBS-containing MEN medium was taken in two 6-well plates, seeded with B16 melanoma cells, and allowed to stand at 37 ° C. in a carbon dioxide concentration of 5%. On the next day, the sample solution was added to the medium and mixed so that the final concentration of each sample solution was 0 (control), 10, 30, 100, 300, and 1000 μg / ml. On day 5 of the culture, the medium was changed, and each sample solution was added again. On the next day, the medium was removed and the cells were collected after washing with phosphate buffer on one plate, and the degree of whiteness of the cultured B16 melanoma cells was evaluated according to the following criteria. In addition, as a comparison target for verifying the effect of the present disclosure, a similar test was conducted for kojic acid, which is known to have a melanin production inhibitory action, with final concentrations of 0 (control), 12.5, 25, 50, 100. And 200 μg / ml (Table 10).

(Criteria)
++: white color equal to or higher than 200 μg / ml of kojic acid.
+: White color equivalent to 100 μg / ml of kojic acid.
±: White color equivalent to 50 μg / ml of kojic acid.
-: Kojic acid is white at 50 μg / ml or less.
The remaining plate was stained with formalin-fixed cells and added to a 1% crystal violet solution. The viable cell rate relative to the sample solution concentration was measured with a monocerator (manufactured by Olympus).

  This technology enables mass production of 3,6-anhydro-L-galactose. This makes it possible to conduct research and development for new uses of 3,6-anhydro-L-galactose. Therefore, the technology of the present disclosure can be used in a wide range of fields such as cosmetics, pharmaceuticals, and foods. The technology of the present disclosure is particularly applicable to external preparations for skin contact, cosmetics, quasi drugs, Contributes to the use of functional foods.

  The present technology can take the following configurations.

[1] A 3,6-anhydro-L-galactose producing enzyme comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, Protein having anhydro-L-galactose production activity (c) Consists of an amino acid sequence having 85% or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having 3,6-anhydro-L-galactose production activity protein

[2] A gene comprising the polynucleotide represented by the following (a), (b) or (c) and any one of the polynucleotides.
(A) a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 3 (b) hybridizing with a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 3 under stringent conditions, and 3, 6 A polynucleotide having an anhydro-L-galactose production activity (c) a nucleotide sequence having 85% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 or 3, and 3,6-anhydro-L-galactose Polynucleotide having ability to produce

[3] A 3,6-anhydro-L-galactose-producing enzyme derived from the genus Cellvibrio having the following enzymatic properties.
1. Action: Neo agarobiose is used as a substrate, has affinity for it, and acts to produce 3,6-anhydro-L-galactose.
2. Substrate specificity: Acts with one or more selected from neoagarobiose, neoagarotetraose and neoagarohexaose to produce 3,6-anhydro-L-galactose.
3. Optimum pH: 5-7.
4). Optimal temperature: 10 to 40 ° C. when neoagarobiose is used as a substrate.
5. Molecular weight: It is a homodimer showing a molecular weight of 35 to 45 kDa as measured by SDS-polyacrylamide gel electrophoresis and showing a molecular weight of 70 to 90 kDa as measured by gel filtration chromatography.
6). The N-terminal amino acid sequence is Gly-Asp-Leu-Pro-Glu-Lys-Lys-Leu-Ser-Lys-Ala-Ser-Leu-Arg-Ala-Ile-Glu (SEQ ID NO: 4).

[4] A recombinant vector containing the polynucleotide encoding the protein according to [1] or [3], the polynucleotide encoding the amino acid sequence according to SEQ ID NO: 4, or the polynucleotide according to [2].

[5] A transformant comprising the recombinant vector according to [4].
[6] The transformant according to [5], wherein the host is a microorganism.
[7] The transformant according to [6], wherein the microorganism is Escherichia coli or Bacillus subtilis.

[8] A microorganism comprising a polynucleotide or gene encoding a 3,6-anhydro-L-galactose synthase comprising the following protein (a), (b) or (c) is cultured, and 3,6-anhydro -A method for producing 3,6-anhydro-L-galactose producing enzyme, comprising collecting L-galactose producing enzyme.
A 3,6-anhydro-L-galactose producing enzyme comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity ability (c) consisting of an amino acid sequence having 85% or more identity with the amino acid sequence represented by SEQ ID NO: 1, Protein

[9] The microorganism according to the above [8], wherein the microorganism is a bacterium belonging to the genus Cellvibrio or a mutant strain thereof, or a transformant according to the above [5] or [6]. A method for producing an anhydro-L-galactose producing enzyme.

[10] 3,6-Anhydro-L-galactose α (1-3) using a 3,6-anhydro-L-galactose producing enzyme comprising the following protein (a), (b) or (c) A method for producing 3,6-anhydro-L-galactose, characterized in that 3,6-anhydro-L-galactose is obtained from a saccharide having a D-galactose structure as a raw material.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity ability (c) consisting of an amino acid sequence having 85% or more identity with the amino acid sequence represented by SEQ ID NO: 1, Protein

[11] The production of 3,6-anhydro-L-galactose as described in [10] above, wherein the reaction condition of the 3,6-anhydro-L-galactose producing enzyme is a reaction temperature of 10 to 40 ° C. Method.

[12] A microorganism comprising a polynucleotide or gene encoding a 3,6-anhydro-L-galactose-producing enzyme comprising the following protein (a), (b) or (c) is cultured, and 3,6-anhydro: Production of 3,6-anhydro-L-galactose characterized in that 3,6-anhydro-L-galactose is obtained from a saccharide having a structure of -L-galactose α (1-3) D-galactose Method.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity ability (c) consisting of an amino acid sequence having 85% or more identity with the amino acid sequence represented by SEQ ID NO: 1, Protein

[13] The 3,6- of [12], wherein the microorganism is a bacterium belonging to the genus Cellvibrio or a mutant strain thereof, or a transformant described in [5] or [6]. A method for producing anhydro-L-galactose.

[14] The method for producing 3,6-anhydro-L-galactose according to any one of the above [10] to [13], further using neoagarobiose producing enzyme.
[15] The above [14], wherein the neoagarobiose-producing enzyme hydrolyzes the β-1,4 bond of D-galactose β (1-4) 3,6-anhydro-L-galactose. A method for producing 3,6-anhydro-L-galactose.

[16] A microorganism named as Cellvibrio sp. WU-0601 and deposited as NITE P-1365.
[17] A method for producing a whitening material by the method for producing 3,6-anhydro-L-galactose according to any one of [10] to [15].
[18] A whitening agent containing the product obtained by the method for producing 3,6-anhydro-L-galactose according to any one of [10] to [15].
[19] A method for producing a whitening agent, comprising a product obtained by the method for producing 3,6-anhydro-L-galactose according to any one of [10] to [15].
[20] An amino acid sequence described in SEQ ID NO: 4, an amino acid sequence in which 1 to 3 amino acids are substituted, deleted or inserted in the amino acid sequence, and a polynucleotide encoding the same.
Use of the polynucleotide for screening the ALG-producing enzyme and the ALG-producing microorganism.

(1) Cellvibrio sp. WU-0601 (NITE P-1365) strain (contract date: May 22, 2012)
Deposited at: 2-5-8 Kazusa Kamashishi, Kisarazu City, Chiba Prefecture 292-0818, Japan Patent Evaluation Microorganism Deposit Center (NPMD), National Institute of Technology and Evaluation.
(2) Alteromonas sp. (FERM P-14664) strain (contract date: November 25, 1994)
Deposit place: 1-1-1 Higashi Tsukuba City, Ibaraki Prefecture 305-8566, Tsukuba Center Chuo No. 6, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (NITE-IPOD) [Formerly: National Institute of Advanced Industrial Science and Technology )

Claims (18)

A 3,6-anhydro-L-galactose producing enzyme comprising the following protein (a), (b) or (c):
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity capacity (c) consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity capacity Protein The polynucleotide represented by the following (a) or (c).
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or 3, (c) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by SEQ ID NO: 2 or 3, and 3,6-anhydro -Polynucleotide encoding a protein having L-galactose production activity A 3,6-anhydro-L-galactose-producing enzyme from the genus Cellvibrio having the following enzymological properties:
1. Action: Neo agarobiose is used as a substrate, has affinity for it, and acts to produce 3,6-anhydro-L-galactose.
2. Substrate specificity: Acts with one or more selected from neoagarobiose, neoagarotetraose and neoagarohexaose to produce 3,6-anhydro-L-galactose.
3. Optimum pH: 6.0.
4). Optimal temperature: 25 ° C. when neoagarobiose is used as a substrate.
5. Molecular weight: A homodimer having a molecular weight of 42 kDa as measured by SDS-polyacrylamide gel electrophoresis and a molecular weight of 83 kDa as measured by gel filtration chromatography.
6). N-terminal amino acid sequence is
Gly-Asp-Leu-Pro-Glu-Lys-Lys-Leu-Ser-Lys-Ala-Ser-Leu-Arg-Ala-Ile-Glu (SEQ ID NO: 4). Polynucleotides encoding claim 1 or 3 wherein the protein, ggt gat ctt cca gaa aaa aaa tta agt aaa gcc agt ttg cgc gct att gaa polynucleotide consisting of the sequence of, or set containing polynucleotide of Claim 2 Replacement vector.     A transformant comprising the recombinant vector according to claim 4.     The transformant according to claim 5, wherein the host is a microorganism.     The transformant according to claim 6, wherein the microorganism is Escherichia coli or Bacillus subtilis. A microorganism containing a polynucleotide or gene encoding a 3,6-anhydro-L-galactose-producing enzyme comprising the following protein (a), (b) or (c) is cultured, and 3,6-anhydro-L- A method for producing a 3,6-anhydro-L-galactose producing enzyme, comprising collecting the galactose producing enzyme.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity capacity (c) consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity capacity Protein     The 3,6-anhydro according to claim 8, wherein the microorganism is a bacterium belonging to the genus Cellvibrio or a mutant strain using the bacterium as a parent strain, or the transformant according to claim 5 or 6. -Method for producing L-galactose producing enzyme. 3,6-anhydro-L-galactose α (1-3) D-galactose using a 3,6-anhydro-L-galactose producing enzyme comprising the following protein (a), (b) or (c) A process for producing 3,6-anhydro-L-galactose, characterized in that 3,6-anhydro-L-galactose is obtained using a saccharide having the following structure as a raw material.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity capacity (c) consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity capacity Protein     The method for producing 3,6-anhydro-L-galactose according to claim 10, wherein a reaction condition of the 3,6-anhydro-L-galactose producing enzyme is a reaction temperature of 10 to 40 ° C. A microorganism containing a polynucleotide or gene encoding a 3,6-anhydro-L-galactose synthase comprising the following protein (a), (b) or (c) is cultured, and 3,6-anhydro-L- A method for producing 3,6-anhydro-L-galactose, characterized in that 3,6-anhydro-L-galactose is obtained from a saccharide having a galactose α (1-3) D-galactose structure as a raw material.
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, or inserted in the amino acid sequence set forth in SEQ ID NO: 1, (C) a protein having an anhydro-L-galactose production activity capacity (c) consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 1, comprising 3,6-anhydro-L-galactose production activity capacity Protein     13. The 3,6-anhydro of claim 12, wherein the microorganism is a bacterium belonging to the genus Cellvibrio or a mutant strain using the bacterium as a parent strain, or the transformant according to claim 5 or 6. -The manufacturing method of L-galactose.     Furthermore, the manufacturing method of 3, 6- anhydro-L- galactose of any one of Claims 10-13 using a neo agarobiose production | generation enzyme.     The 3,6-6 according to claim 14, wherein the neoagarobiose-generating enzyme hydrolyzes the β-1,4 bond of D-galactose β (1-4) 3,6-anhydro-L-galactose. A method for producing anhydro-L-galactose.     Cellvibrio sp. A microorganism named WU-0601 and deposited as NITE P-1365.     A method for producing a whitening material by the method for producing 3,6-anhydro-L-galactose according to any one of claims 10 to 15. ggt gat ctt cca gaa aaa aaa tta agt aaa gcc agt ttg cgc gct att gaa polynucleotide consisting of the sequences.