JP4683531B2 - Novel α-L-arabinofuranosidase and method of using the same - Google Patents

Description

  The present invention relates to α-L-arabinofuranosidase and a method for using the same, and more specifically, novel α-L-arabinofuranosidase derived from Acremonium microorganisms, DNA encoding the enzyme, and enzyme composition, and It relates to these uses.

  Cereal hulls contain a lot of fibers, and these fibers are composed of cellulose, hemicellulose, and the like. Among these, wheat bran, corn hull or rice husk hemicellulose is known to be a polysaccharide called arabinoxylan mainly composed of xylose and arabinose. In addition, soybean hulls contain arabinogalactan as hemicellulose. Furthermore, most of arabinose in pulp (beet pulp) discharged after recovering sucrose from beet exists as arabinan, which is a polysaccharide linked in a chain.

  Arabinoxylan, arabinogalactan, and arabinan are respectively β-1,4 linked xylose subunits, β-1,3 or β-1,4 linked galactose subunits, and α-1,5 linked L- The main chain is an arabinofuranose subunit, and an L-arabinofuranose residue as a side chain is bonded to the main chain in the form of α- (1 → 3) or α- (1 → 2). It has been known.

Enzymes that degrade arabinose-containing polysaccharides are collectively referred to as arabinanase, and are classified into endo-arabinanase and α-L-arabinofuranosidase according to their mode of action.
α-L-arabinofuranosidase acts on α-1,5,1,3 or 1,2-L-arabinoside bonds contained in polysaccharides such as arabinoxylan, arabinogalactan, arabinan, etc. In recent years, it has been attracting attention as a hemicellulose-degrading enzyme in plants or plant-derived materials, food processing enzymes, or wood pulp delignifying agents.
As an application example of α-L-arabinofuranosidase in each field, first, in feed applications, the increase in livestock and / or feed efficiency can be improved by adding to the feed. In the food field, it can be used to increase production yield in juice production, improve filterability and / or increase yield in wort production, increase aroma components in wine brewing, etc., increase food viscosity or gel strength . It is also disclosed that wood pulp can be used for delignification of pulp by treating with this enzyme (see Patent Document 1).

Regarding the enzymes produced by the filamentous fungus Acremonium cellulolyticus, there have been many reports on cellulase, pectinase (polygalacturonase), and xylanase (Patent Document 2, Non-Patent Document 1, 2 and 3), these enzymes have been shown to be effective in feed and silage applications (see Patent Document 3 and Patent Document 4).
However, there is no detailed report yet on α-L-arabinofuranosidase, and in order to further effectively use the host-derived enzyme or enzyme composition in the industry, detailed description of α-L-arabinofuranosidase is not provided. The elucidation of various enzymatic and protein properties and the isolation of the gene encoding the enzyme have been awaited.

  By the way, L-arabinose, which constitutes arabinoxylan and is contained in beet pulp, has a taste similar to that of sucrose, and is hardly absorbed from the intestinal mucosa. It is also known to inhibit the decomposition of sucrose by inhibiting the α-glucosidase activity present in the intestinal mucosa and suppress the increase in blood sugar. Thus, arabinose is attracting attention for its usefulness.

It has already been reported that this L-arabinose can be produced by alkaline extraction of hemicellulose contained in beet pulp and the like, and acid decomposition of this (see Patent Document 5, Patent Document 6, and Non-Patent Document 4). .
However, since these production methods require severe conditions such as strong acid and strong alkali, problems in the reaction apparatus and handling operation are inevitable. Furthermore, since these production methods are costly, it has been a problem in producing L-arabinose.

On the other hand, it has been reported that L-arabinose can also be produced by enzymatic action on beet pulp (see Patent Document 7 and Patent Document 8).
However, the production method has a problem in that it is difficult to efficiently obtain L-arabinose due to the complicated separation and purification work of the degradation products.

Japanese National Patent Publication No. 7-508162 WO98 / 39423 publication JP-A-4-117244 JP 7-236431 A Japanese Patent Laid-Open No. 9-299093 JP-A-11-313700 JP 2001-286294 A JP 2002-45194 A Agric. Biol. Chem. , 52, 2493-2501 (1988) Agric. Biol. Chem. , 53, 3359-3360 (1989) Agric. Biol. Chem. , 54, 309-317 (1990) Biotechnology and Bioengineering, Vol. 64, No. 6, 685-691, 1999

In order to use α-L-arabinofuranosidase in an industrially effective manner, it is essential to isolate and purify the enzyme and clarify its enzymatic and physical properties. In addition, in order to enable expression enhancement and mass production of the enzyme, it is necessary to isolate and analyze a gene encoding the enzyme.
Moreover, as an enzyme for feed addition, it must be stable at a low pH, preferably at pH 3 or less, more preferably at pH 2 or less.

The present invention relates to a novel α-L-arabinofuranosidase, a gene encoding the α-L-arabinofuranosidase, a method for expressing α-L-arabinofuranosidase using this gene, α-L- It is an object of the present invention to provide an enzyme composition comprising arabinofuranosidase and a method for treating plants and / or plant-derived materials with the α-L-arabinofuranosidase or enzyme composition.
Furthermore, as described above, while L-arabinose has high functionality, the conventional method for producing L-arabinose has a problem of high cost.
The object of the present invention is to provide a method for producing L-arabinose at low cost.

In order to solve the above-mentioned problems, the present inventors diligently studied and purified various enzymes of Acremonium cellulolyticus. As a result, a novel α-L-arabinofuranosidase was found from these enzyme groups, and a gene encoding the enzyme was isolated. The enzyme showed high stability at a relatively low pH around pH 2.
Furthermore, the effect of α-L-arabinofuranosidase on various plants or plant-derived products was examined, and the present invention was completed.

That is, the following invention is provided by the present invention.
Claim 1: α-L-arabinofuranosidase derived from Acremonium microorganism and having the following characteristics.
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: Stable in the range of pH 2 to 7.5, and high activity is maintained even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: stable in the range of 50 ° C. or less.
Claim 2: The α-L-arabinofuranosidase according to claim 1, wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus.

Claim 3: Protein represented by the following (a), (b) or (c).
(A) A protein consisting of residues 1 to 481 in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
(B) One to 481 residues in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing , and one of the -26 to -1 sequence portions in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing part or the whole, consisting of the protein.
(C) A modified protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence constituting the protein of (a) or (b), and has the following characteristics: A protein having (i) to (g) .
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: It is stable in the range of pH 2 to 7.5 and retains high activity even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: Stable within a range of 50 ° C. or less.
Claim 4: The protein according to claim 3, wherein the protein is derived from an Acremonium microorganism .
Claim 5: The protein according to claim 4 , wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus.

Claim 6 : A gene comprising DNA represented by the following (a) to (d) .
(A) DNA consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing.
(B) DNA encoding a protein consisting of residues 1 to 481 in the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing.
(C) Residues 1 to 481 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and one of the sequence portions from -26 to -1 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing DNA encoding a protein consisting of part or all.
(D) A modified protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence constituting the protein of (b) or (c) , and has the following characteristics: DNA encoding a protein having (i) to (g) .
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: It is stable in the range of pH 2 to 7.5 and retains high activity even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: Stable within a range of 50 ° C. or less.
Claim 7 : The gene according to claim 6 , wherein the gene is derived from an Acremonium microorganism .
Claim 8: The gene according to claim 7 , wherein the Acremonium microorganism is Acremonium cellulolyticus.

Claim 9 : A nucleic acid construct comprising a base sequence represented by the following (a) to (d) .
(A) The base sequence shown by sequence number 2 of a sequence table.
(B) A base sequence encoding a protein consisting of residues 1 to 481 in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
(C) Residues 1 to 481 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and one of the sequence portions from -26 to -1 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A base sequence encoding a protein consisting of part or all.
(D) A modified protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence constituting the protein of (b) or (c) , and has the following characteristics: A base sequence encoding a protein having (i) to (g) .
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: It is stable in the range of pH 2 to 7.5 and retains high activity even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: Stable within a range of 50 ° C. or less.
Claim 10 : An expression vector comprising the nucleic acid construct of claim 9 .

Claim 11 : A host cell transformed with the nucleic acid construct according to claim 9 or the expression vector according to claim 10 .
Claim 12 : The host cell according to claim 11 , wherein the host is Escherichia coli, yeast, actinomycetes or filamentous fungi.
Claim 13 : The host cell according to claim 12 , wherein the yeast is a microorganism belonging to the genus Saccharomyces, Hansenula or Pichia.
Claim 14 : The host cell according to claim 12 , wherein the yeast is Saccharomyces cerevisiae.
Claim 15 : The host cell according to claim 12 , wherein the filamentous fungus is a filamentous fungus belonging to the genus Acremonium, Humicola, Aspergillus, Trichoderma or Fusarium.
Claim 16 : The host cell according to claim 12 , wherein the filamentous fungus is Acremonium cellulolyticus, Humicola insolens, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, or Fusarium oxysporous.

Claim 17 : The host cell according to any one of claims 11 to 16 is cultured, and the α-L-arabi according to claim 1 or 2 is obtained from the cultured host cell and / or a culture solution thereof. The method for producing α-L-arabinofuranosidase according to claim 1 or 2, comprising a step of isolating nofuranosidase.
Claim 18 : The host cell according to any one of claims 11 to 16 is cultured, and the cultured host cell and / or the culture solution thereof is used as described in any one of claims 3 to 5. The method for producing a protein according to any one of claims 3 to 5 , comprising a step of isolating the protein.

Claim 19 : An enzyme composition comprising the α-L-arabinofuranosidase according to claim 1 or 2 or the protein according to any one of claims 3 to 5 .
Claim 20 : Any of cellulase, xylanase, protease, galactanase, galactosidase, arabinanase, mannanase, rhamnogalacturonase, polygalacturonase, pectin methylesterase, pectin lyase, and polygalacturonic acid lyase The enzyme composition according to claim 19 , further comprising one or more components.

Claim 21 : The α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. An enzyme agent for processing and / or improving utilization efficiency of a plant or plant-derived product containing a product.
Claim 22 : The α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. A feed additive containing food.
Claim 23 : A method for producing a feed having excellent digestibility, wherein the feed additive according to claim 22 is added to the feed.

Claim 24 : The α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. Enzyme for food processing containing foods.
Claim 25 : A method for producing a food using the food processing enzyme according to claim 24 .
Claim 26 : The α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. Wood pulp treatment agent containing products.
Claim 27 : A method for treating wood pulp, wherein the wood pulp treating agent according to claim 26 is used.

Claim 28 : α-L-arabinofuranosidase according to claim 1 or 2, protein according to any one of claims 3 to 5 , or enzyme composition according to claim 19 or 20. A method for producing L-arabinose, comprising using a product.
Claim 29 : The α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. Natural product selected from orange fiber containing α-L-arabinofuranosyl residue, orange juice koji, apple fiber, apple juice koji, beet fiber, beet pulp, rice sugar, soybean koji, peanut and corn koji A method for producing L-arabinose, characterized in that L-arabinose is obtained by acting on a product.

The present invention provides a novel α-L-arabinofuranosidase derived from an Acremonium microorganism, a gene encoding the enzyme, an expression vector containing the gene, and a host cell transformed with the expression vector. An efficient method for producing a novel α-L-arabinofuranosidase is also provided.
Furthermore, there is also provided a technique for utilizing this α-L-arabinofuranosidase or an enzyme composition containing the enzyme for processing of plants or plant-derived materials, and various methods such as feed field, food field, and production of L-arabinose. Use in various applications is expected.

  The enzyme according to the present invention is an enzyme exhibiting α-L-arabinofuranosidase activity, that is, α-L-arabinofuranoside arabinofuranohydrolase EC 3.2.1.55, specifically, α-L-arabinofuranoside. Or the enzyme which hydrolyzes (alpha) -L- arabinofuranosyl residues, such as arabinan, arabinoxylan, and arabinogalactan.

Examples of microorganisms that produce the α-L-arabinofuranosidase of the present invention include Acremonium microorganisms (Acremonium filamentous fungi). Examples of Acremonium microorganisms include Acremonium cellulolyticus Y-94 (deposit number FERM BP-5826) and Acremonium cellulolyticus TN strain (deposit number FERM BP-685).
Production of α-L-arabinofuranosidase using these microorganisms may be carried out according to a known method, for example, the method described in Japanese Patent Publication No. 60-43954 or Japanese Patent Publication No. 63-63197. Α-L-arabinofuranosidase can be collected from the culture after completion of the culture. Here, the supernatant obtained by removing the culture by centrifugation or the like can be used as a crude enzyme. Usually, however, the supernatant is concentrated by an ultrafiltration method or the like, and a preservative or the like is added. To concentrate enzyme, or after concentration to powder enzyme by spray drying.

The α-L-arabinofuranosidase of the present invention can be obtained by partially purifying or highly purifying these concentrated enzyme or powder enzyme as necessary.
Purification methods include conventional methods, that is, salting out using ammonium sulfate, organic solvent precipitation using alcohol, membrane separation, chromatographic separation using an ion exchanger, a carrier for hydrophobic chromatography, a carrier for gel filtration, and the like. These can be used alone or in appropriate combination.

(1) α-L-arabinofuranosidase of the present invention According to the first aspect of the present invention, α-L-arabinofuranosidase activity derived from the genus Acremonium and having the characteristics described in claim 1 above. A novel enzyme is provided. More detailed characteristics of the enzyme are as follows.
(A) Substrate specificity and action characteristics The α-L-arabinofuranosidase of the present invention hydrolyzes an α-L-arabinofuranosyl residue.
That is, as described above, the enzyme of the present invention is an enzyme exhibiting α-L-arabinofuranosidase activity, that is, α-L-Arabinofuranoside arabinofuranohydrolase EC3.2.55, specifically α-L -It hydrolyzes α-L-arabinofuranosyl residues such as arabinofuranoside or arabinan, arabinoxylan, arabinogalactan.
The α-L-arabinofuranosidase of the present invention shows high activity against arabinan and arabinoxylan, and relatively low activity against arabinogalactan. It also acts on linear arabinan having no side chain, and liberates L-arabinofuranose.
The α-L-arabinofuranosidase activity of the enzyme of the present invention can be examined by measuring the arabinose releasing activity against arabinan, arabinogalactan, arabinoxylan and the like by the DNS method (Borel et al, 1952). it can.

(B) Molecular Weight The molecular weight of the α-L-arabinofuranosidase of the present invention is 57,000 as a result of measurement by SDS-polyacrylamide gel electrophoresis.
(C) Isoelectric point The isoelectric point of the α-L-arabinofuranosidase of the present invention is 5.3 to 5.5 as a result of measurement by isoelectric point polyacrylamide gel electrophoresis.
(D) Optimum pH
The optimum pH of the α-L-arabinofuranosidase of the present invention is pH 3-4 in the arabinan degrading activity under the treatment condition at a temperature of 37 ° C., but has a high activity in the range of about pH 1.5-5.5. (Fig. 1).

(E) Stable pH range The α-L-arabinofuranosidase of the present invention is stable in the range of pH 2 to 7.5 in the treatment at room temperature (22 ° C.) for 24 hours, and in the range of pH 1.5 to 2. Retains high activity (FIG. 2).
(F) Optimum temperature of action The optimum temperature of action of the α-L-arabinofuranosidase of the present invention is about 50 ° C. in the arabinan decomposition activity under the treatment conditions of pH 3.5, but in the range of about 40-60 ° C. Also has high activity (FIG. 3).
(G) Temperature stability The α-L-arabinofuranosidase of the present invention is stable within a range of 50 ° C. or less in the treatment at pH 3.5 for 24 hours (FIG. 4).

(H) Specific activity The specific activity of the α-L-arabinofuranosidase of the present invention in the arabinan degradation activity is about 29 units / mg protein.
Here, the activity measurement method of the enzyme and the definition of one unit are as follows.
-Arabinane decomposition activity The enzyme amount was defined as 1 unit by producing an enzyme by reacting with a 5 mg / ml arabinan solution at pH 3.5 and 37 ° C to produce 1 µmol of reduced L-arabinofuranose per minute.

  The α-L-arabinofuranosidase of the first aspect of the present invention is an Acremonium microorganism, more preferably Acremonium cellulolyticus, even more preferably Acremonium cellulolyticus strain Y-94 (deposited). No. FERM BP-5826), Acremonium cellulolyticus TN strain (deposit number FERM BP-685).

  The α-L-arabinofuranosidase of the present invention can act on various products using the enzyme activity. As a method of use in that case, it can be carried out under conditions such as pH and temperature that are usually used in the above-mentioned fields of application, but a pH of 1.5 to 5 and a temperature of 50 ° C. or lower are appropriate due to the nature of the enzyme. . A sufficient effect can be obtained if the amount of α-L-arabinofuranosidase of the present invention is adjusted so that each purpose can be achieved with time.

(2) Protein of this invention According to the 2nd aspect of this invention, the protein represented by the following (a), (b) or (c) is provided.
(A) A protein comprising part or all of the first to 481-th sequence portion of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.
Here, “a part of the 1st to 481st sequence portion of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing” means, for example, a length that can be used as a probe, and a partial sequence thereof. Even so, it is intended to mean that partial sequence that still maintains α-L-arabinofuranosidase activity.

(B) part or all of the 1st to 481st sequence portion of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, and -26 of the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing A protein comprising a part or all of the first sequence portion on the N-terminal side.
In other words, the protein (b) means a protein having a part or all of the −26 to −1 sequence portion described in SEQ ID NO: 1 at the N-terminus of the protein in the protein (a). To do. Therefore, “part of the 1st to 481st sequence portion of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing” is the same as described for the protein (a).
Here, since “the 26th to −1st sequence portion of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing” is considered to be a signal peptide, “the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing” "A part of -26 to -1 sequence part" is a sequence that retains signal peptide activity, and is a sequence that remains at the N-terminus as a result of a difference in the processing position depending on the type of expression host. Also means.

(C) a protein having an altered amino acid sequence that constitutes the protein of (a) or (b) and having α-L-arabinofuranosidase activity.
In the present invention, the modified protein is a protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence of the above protein, and is still α-L-arabinofurano. It means a substance that retains the various characteristics described in the first aspect of the present invention, that is, the sidase activity.

  The protein of the second aspect of the present invention is a filamentous fungus, preferably a microorganism belonging to the genus Acremonium, more preferably Acremonium cellulolyticus, even more preferably Acremonium cellulolyticus strain Y-94 (deposit number FERM). BP-5826), Acremonium cellulolyticus TN strain (deposit number FERM BP-685).

  The protein of the present invention has α-L-arabinofuranosidase activity, that is, the various characteristics described in the first aspect of the present invention. Accordingly, the enzyme activity can be used to act on various products. As a method of use in that case, it can be carried out under conditions such as pH and temperature that are usually used in the above-mentioned fields of application, but a pH of 1.5 to 5 and a temperature of 50 ° C. or lower are appropriate due to the nature of the enzyme. . A sufficient effect can be obtained if the amount of the protein of the present invention is adjusted so that each purpose can be achieved with time.

(3) Gene of the present invention According to the second aspect of the present invention, since the amino acid sequence of the protein constituting α-L-arabinofuranosidase is given, the base sequence of the gene encoding it is easily determined. Various amino acid sequences encoding the amino acid sequence shown in SEQ ID NO: 1 and its modified protein can be selected.
Therefore, according to the third aspect of the present invention, the gene comprising the amino acid sequence shown in SEQ ID NO: 1 and the base sequence encoding the modified protein is represented by the following (a), (b) or (c): A gene comprising the DNA to be produced is provided.

(A) DNA consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing.
DNA consisting of the base sequence set forth in SEQ ID NO: 2 in the sequence listing is a typical sequence encoding the amino acid sequence of α-L-arabinofuranosidase. The base sequence described in SEQ ID NO: 2 in the sequence listing has an open reading frame that starts with the 1st to 3rd ATG and ends with the 1522 to 1524th TGA. The 79th to 81st base portion of the base sequence corresponds to the N-terminal amino acid of the mature protein consisting of 481 residues.

(B) DNA encoding a protein which is a modified sequence of the base sequence of (a) and has α-L-arabinofuranosidase activity.
In the present invention, the altered sequence means a base sequence that hybridizes with the base sequence of (a) above under stringent conditions and encodes a protein having α-L-arabinofuranosidase activity.
The stringent conditions are those having the full length of the DNA sequence set forth in SEQ ID NO: 2 labeled as a probe and pre-high for 1 hour according to the method of ECL direct DNA / RNA labeling detection system (Amersham). After hybridization (42 ° C.), the probe was added, hybridization was performed for 15 hours (42 ° C.), and then 0.4% SDS, 6 M urea added 1 × concentration SSC (SSC; 15 mM trisodium citrate, 150 The condition is such that washing at 42 ° C. for 20 minutes is repeated twice with mM sodium chloride, and then twice with 10 times washing at room temperature for 10 minutes at 5 times concentration SSC.

(C) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or a modified protein thereof and having α-L-arabinofuranosidase activity.
That is, the present DNA (c) is a DNA that encodes the protein of the second aspect of the present invention and includes all that are not included in the DNAs (a) and (b). Here, the modified protein is as described in the description of the protein (c) of the protein of the second aspect of the present invention.

The gene of the third aspect of the present invention is a filamentous fungus, preferably a microorganism belonging to the genus Acremonium, more preferably Acremonium cellulolyticus, even more preferably Acremonium cellulolyticus strain Y-94 (deposit number FERM). BP-5826), Acremonium cellulolyticus TN strain (deposit number FERM BP-685).
The gene (abf) encoding α-L-arabinofuranosidase that is abundantly expressed in the host Acremonium cellulolyticus can be isolated from Acremonium cellulolyticus by the following method, for example. .
That is, from a chromosome library or cDNA library derived from Acremonium cellulolyticus, a method commonly used in the field of genetic engineering, for example, using an appropriate DNA probe prepared based on partial amino acid sequence information Examples include screening methods.

The gene of the present invention encodes the protein of the second aspect of the present invention, and the protein has α-L-arabinofuranosidase activity, that is, the various activities described in the first aspect of the present invention. Therefore, it also encodes the α-L-arabinofuranosidase of the first aspect of the present invention.
Therefore, the gene of the present invention contributes to efficient production of the protein of the second aspect of the present invention and the α-L-arabinofuranosidase of the first aspect of the present invention.

(4) Nucleic acid composition of the present invention The gene shown as the third aspect of the present invention can also be used in various gene-related technologies as a nucleic acid composition such as a DNA composition or an RNA composition. It is the fourth aspect of the present invention to provide a simple nucleic acid construct.
That is, the fourth aspect of the present invention is a nucleic acid composition comprising a base sequence represented by the following (a), (b) or (c).
(A) The base sequence shown by sequence number 2 of a sequence table.
(B) A nucleotide sequence that is a modified sequence of the nucleotide sequence of (a) and encodes a protein having α-L-arabinofuranosidase activity.
(C) A base sequence encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, or a modified protein thereof, and a protein having α-L-arabinofuranosidase activity.
The details of (a), (b) and (c) above are as described for the base sequences constituting the DNAs of (a), (b) and (c) of the third aspect of the present invention. .

  The nucleic acid construct of the fourth aspect of the present invention may be naturally derived from the above-mentioned filamentous fungi and other organisms, or may be fully synthesized. Moreover, what synthesize | combined using a part of natural origin may be used.

The nucleic acid construct of the present invention encodes the protein of the second aspect of the present invention, and the protein exhibits α-L-arabinofuranosidase activity, that is, the various activities described in the first aspect of the present invention. Therefore, it also encodes the α-L-arabinofuranosidase of the first aspect of the present invention.
Therefore, the nucleic acid construct of the present invention contributes to efficient production of the protein of the second aspect of the present invention and the α-L-arabinofuranosidase of the first aspect of the present invention.
The nucleic acid construct of the present invention can be used in various gene-related technologies as a DNA construct or RNA construct, and can be introduced into a cell to suppress the expression of a target gene, for example. .

(5) Expression vector of the present invention According to the fifth aspect of the present invention, an expression vector comprising the nucleic acid construct according to the fourth aspect of the present invention is provided.
This expression vector of the present invention is capable of replicating in a host microorganism, and comprises the nucleic acid composition of the present invention in a state capable of expressing a protein encoded by the base sequence.

  The expression vector of the present invention can be constructed on the basis of a self-replicating vector, that is, as an extrachromosomal independent entity, and its replication does not depend on chromosomal replication, for example, a plasmid. Moreover, when the expression vector of the present invention is introduced into a host microorganism, it may be integrated into the genome of the host microorganism and replicated together with the chromosome into which it has been integrated.

In order to express a protein having a desired activity when actually introduced into a host cell, the expression vector of the present invention contains a DNA sequence (base) that controls the expression in addition to the nucleic acid composition of the present invention described above. Sequence) and genetic markers for selecting microorganisms. Examples of the DNA sequence that controls the expression include a DNA sequence that encodes each of a promoter, a terminator, and a signal peptide.
The promoter is not particularly limited as long as it exhibits transcriptional activity in the host microorganism, and can be obtained as a DNA sequence that controls the expression of a gene encoding a protein that is the same or different from the host microorganism. The signal peptide is not particularly limited as long as it contributes to protein secretion in the host microorganism, and can be obtained from a DNA sequence derived from a gene encoding a protein that is the same or different from the host microorganism. it can.
The gene marker in the present invention may be appropriately selected according to the method for selecting a transformant. For example, a gene encoding drug resistance and a gene complementary to auxotrophy can be used.

The procedures and methods for constructing the expression vector of the present invention can follow the methods commonly used in the field of genetic engineering.
For example, when Acremonium cellulolyticus, which is a type of filamentous fungus, is used as the host cell to be introduced, it can be performed by the method described in JP-A-2001-17180. That is, a recombinant vector for gene expression is prepared by linking the above-described nucleic acid construct of the present invention downstream of the promoter of the cbh1 gene that is most highly expressed in Acremonium cellulolyticus. By introducing this expression vector into Acremonium cellulolyticus, a large amount of α-L-arabinofuranosidase protein can be expressed.

(6) Host cell of the present invention Furthermore, according to the sixth aspect of the present invention, a host cell transformed with the expression vector is provided.
This host-vector system is not particularly limited, and for example, a system using Escherichia coli, yeast, actinomycetes, filamentous fungi, etc., and a fusion protein expression system with other proteins using them can be used. .
According to a preferred aspect of the present invention, by using yeast (yeast cells), the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect of the present invention is efficiently expressed. Can be. Examples of yeast cells include microorganisms belonging to the genus Saccharomyces, Hansenula, or Pichia, such as Saccharomyces cerevisiae. Furthermore, Saccharomyces cerevisiae can be mentioned as a preferable example thereof.

Furthermore, according to a preferred embodiment of the present invention, α-L-arabinofuranosidase or the protein of the second embodiment of the present invention can be efficiently expressed by using a filamentous fungus. Examples of host filamentous fungi include microorganisms belonging to the genus Acremonium, the genus Humicola, the genus Aspergillus, the genus Trichoderma, or the genus Fusarium. In addition, preferred examples thereof include Acremonium cellulolyticus, Humicola insolens, Aspergillus niger, Aspergillus Triger, Aspergillus orgels ), Fusarium oxysporus, and the like.
Procedures and methods for obtaining host cells transformed with the expression vector of the present invention can also be carried out according to methods commonly used in this field.

  Specific examples of such host cells of the present invention include Escherichia coli transformed with a plasmid (expression vector) comprising the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing as described in the Examples. Can do. This transformed Escherichia coli is deposited in the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan under the accession number of FERM P-18243.

(7) Method for producing α-L-arabinofuranosidase or protein of the present invention According to the present invention, the host cell of the sixth aspect of the present invention is cultured in an appropriate medium, and the cultured host cell and And / or the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect of the present invention can be isolated and obtained from the culture solution, and such a production method Is a seventh aspect of the present invention.
That is, in the seventh aspect of the present invention, the host cell of the sixth aspect of the present invention is cultured in an appropriate medium, and the host cell and / or the culture solution after the culture are used to produce the first aspect of the present invention. The present invention relates to a method for producing α-L-arabinofuranosidase or protein comprising the step of isolating the protein of the second embodiment of the present invention.

  The procedures and conditions for culturing host cells may be essentially equivalent to those of the host cells used (before transformation). As a method for isolating and recovering (collecting) the target protein after culturing the transformant, those commonly used in this field can be used.

(8) Enzyme composition of the present invention According to the eighth aspect of the present invention, the enzyme composition comprising the first aspect of the present invention α-L-arabinofuranosidase or the protein of the second aspect. Is provided.
Here, the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect is preferably produced by the production method of the seventh aspect of the present invention.
The enzyme composition of the present invention is generally included in the enzyme composition as long as it contains the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect. It may be produced by mixing with ingredients such as excipients (for example, lactose, sodium chloride, sorbitol, etc.), surfactants, preservatives and the like. In addition, the enzyme composition in the present invention can be prepared by molding into an appropriate shape, for example, powder or liquid.

  The enzyme composition of the present invention further includes cellulase, xylanase, protease, galactanase, galactosidase, arabinanase, mannanase, rhamnogalacturonase, polygalacturonase, pectin methylesterase, pectin lyase, and polygalacturonic acid lyase. It may further contain one or more components. By containing these enzymes, it can be expected that the utilization processing efficiency of plants or plant-derived materials can be further improved as compared to an enzyme composition containing α-L-arabinofuranosidase alone.

  The method of using the enzyme composition of the present invention can be performed under conditions such as pH and temperature that are usually used in the above-mentioned fields of application, but due to the nature of the enzyme, a pH range of 1.5 to 5 and a temperature of 50 ° C. or lower. Is appropriate. A sufficient effect can be obtained if the amount of the enzyme composition used is adjusted so as to achieve each purpose with time.

(9) Enzyme for improving processing and / or utilization efficiency of plant or plant-derived processed product of the present invention α-L-arabinofuranosidase of the first aspect, protein of the second aspect, and The enzyme composition of the aspect 8 is excellent in the degradation performance of α-L-arabinofuranosyl residues such as arabinan, arabinoxylan, arabinogalactin and the like, and therefore α-L such as arabinan, arabinoxylan, arabinogalatan, etc. -It is suitable for the improvement of processing and usability (utilization efficiency) of plants or plant-derived products containing arabinofuranosyl residues. It is a ninth aspect of the present invention to provide an enzyme agent for improving the processing and / or utilization efficiency of such a plant or plant-derived processed product.
That is, the ninth aspect of the present invention is the α-L-arabinofuranosidase obtained in the first aspect or the seventh aspect of the present invention, the protein of the second aspect, or the enzyme of the eighth aspect. Provided is an enzyme agent for processing and / or improving utilization efficiency of a plant or plant-derived material containing the composition.
Here, the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect is preferably produced by the production method of the seventh aspect of the present invention.
The plant of the present invention or the plant cultivar of the plant-derived material that is the subject of the plant-derived material is not particularly limited and can be applied to all plant tissues. Further, its use is not limited, and it can be widely applied to the feed field (for example, grass), the food field (for example, vegetable, fruit) and the like. As specific examples of plant-derived materials, the present invention can also be applied to processed fruit juice, puree, paste, squeezed koji, and extracted koji.

(10) Feed additive of the present invention The α-L-arabinofuranosidase of the first aspect of the present invention, the protein of the second aspect, and the enzyme composition of the eighth aspect are used in animal feed. By degrading α-L-arabinofuranosyl residues such as arabinan, arabinoxylan, arabinogalactan, etc. in the feed, or by synergizing with various enzymes contained in the enzyme composition, the fiber in the feed It is the tenth aspect of the present invention to provide such a feed additive that can efficiently decompose and improve the digestibility of the fibers in these feeds.
That is, the tenth aspect of the present invention is the α-L-arabinofuranosidase, the protein of the second aspect, or the eighth aspect of the first aspect of the present invention or obtained in the seventh aspect. A feed additive containing the enzyme composition is provided.
Here, the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect is preferably produced by the production method of the seventh aspect of the present invention.
When the feed additive of the present invention is added to the feed, α-L-arabinofuranosidase is contained in arabinan, arabinoxylan, arabinogalactan, etc. in animal feed. By hydrolyzing, the fiber in the feed is efficiently decomposed. In particular, when an enzyme composition containing various enzymes is used, the fiber in the feed is efficiently decomposed by the synergistic action of α-L-arabinofuranosidase and the various enzymes, so that the cells of the feed crop It is possible to further promote utilization of proteins accumulated in the inside.
In particular, α-L-arabinofuranosidase contained in the feed additive of the present invention is stable in the range of pH 2 to 7.5 and retains high activity in the range of pH 1.5 to 2. Therefore, it is considered that by adding to the feed as a feed additive (enzyme for feed addition), it is possible to maintain high activity without inactivation in the digestive tract of various livestocks and to act effectively.

(11) Enzyme for food processing of the present invention The α-L-arabinofuranosidase of the first aspect of the present invention, the protein of the second aspect, and the enzyme composition of the eighth aspect are used for food processing. By using it, it is possible to obtain effects such as an increase in aroma components such as juice and wine, clarification of fruit juice, viscosity reduction, color improvement, bitterness removal, brewing, and improvement in fermentation rate in breadmaking, such as this It is an eleventh aspect of the present invention to provide a simple food processing enzyme preparation.
That is, the eleventh aspect of the present invention is the α-L-arabinofuranosidase obtained in the first aspect or the seventh aspect of the present invention, the protein of the second aspect, or the enzyme of the eighth aspect. Provided is an enzyme agent for food processing containing the composition.
Here, the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect is preferably produced by the production method of the seventh aspect of the present invention.
The enzyme agent for food processing according to the eleventh aspect of the present invention can be used for the production of food to improve the production efficiency as described above, and juice, wine, bread, etc. with improved characteristics. Can be obtained efficiently.

(12) Wood pulp treating agent of the present invention The α-L-arabinofuranosidase of the first aspect of the present invention, the protein of the second aspect, and the enzyme composition of the eighth aspect are used for treating wood pulp. By being used, delignification can be performed without using a bleaching compound such as chlorine.
That is, the twelfth aspect of the present invention is the α-L-arabinofuranosidase obtained in the first aspect or the seventh aspect of the present invention, the protein of the second aspect, or the enzyme of the eighth aspect. Provided is a wood pulp treating agent containing the composition.
Here, the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect is preferably produced by the production method of the seventh aspect of the present invention.
The wood pulp which is the target of the wood pulp treating agent of the twelfth aspect of the present invention is not particularly limited, and includes all types of lignocellulosic materials.
Therefore, the wood pulp treating agent of the twelfth aspect of the present invention can be delignified without using a bleaching compound such as chlorine by using it for the treatment of wood pulp.

(13) Production method of L-arabinose of the present invention The α-L-arabinofuranosidase of the first aspect, the protein of the second aspect, and the enzyme composition of the eighth aspect of the present invention are beet pulp. It is the thirteenth aspect of the present invention to provide L-arabinose and a method for producing such L-arabinose.
That is, the thirteenth aspect of the present invention is the α-L-arabinofuranosidase obtained in the first aspect or the seventh aspect of the present invention, the protein of the second aspect, or the eighth aspect. Provided is a method for producing L-arabinose, which comprises using an enzyme composition.
Here, the α-L-arabinofuranosidase of the first aspect of the present invention or the protein of the second aspect is preferably produced by the production method of the seventh aspect of the present invention.
Examples of the target to which the α-L-arabinofuranosidase of the present invention acts include orange fiber containing α-L-arabinofuranosyl residue such as arabinan, arabinoxylan, arabinogalactan, mandarin orange juice, apple fiber And natural products such as apple juice lees, beet fiber, beet pulp, rice sugar, soybean koji, peanuts and corn koji. In particular, by acting on one or more natural products selected from the above-listed, L-arabinose, which is particularly useful for its usefulness among arabinoses, can be obtained.

The method for producing L-arabinose of the present invention will be described in detail as follows.
First, the amount of the enzyme that acts on various natural products in the present invention is not particularly limited as long as it is an amount necessary to liberate L-arabinose from the raw material. In the enzymatic degradation, beet pulp or the like is suspended in an aqueous medium, and the α-L-arabinofuranosidase of the first aspect, the protein of the second aspect, and the enzyme composition of the eighth aspect are added thereto. The reaction may be performed with stirring or standing. The reaction temperature is preferably 45 to 55 ° C, particularly 50 ° C. The pH of the reaction solution is preferably 1.5-5. The reaction time depends on the amount of beet pulp and the enzyme used and the amount of enzyme, but is usually preferably set to 5 to 48 hours.
The solution after completion of the reaction is subjected to heat treatment at 100 ° C. or more as it is, and the solution is subjected to solid-liquid separation (centrifugation, filtration, decantation, etc.) to obtain an L-arabinose solution. The obtained L-arabinose solution is subjected to diatomaceous earth filtration, filter paper filtration, microfiltration and other filtration treatments, either alone or in combination, and then concentrated according to conventional methods such as heating, reduced pressure, reduced pressure heating, ultrafiltration, etc. Obtain a liquid. The obtained concentrated liquid may be purified by urging ordinary methods using various chromatography using ion exchange resin, activated carbon or the like. Further, the purified L-arabinose solution may be concentrated in the same manner as described above, and the obtained concentrated liquid may be cooled (a seed crystal may be added) to obtain crystalline L-arabinose.

  EXAMPLES Next, although an Example demonstrates this invention in detail, the scope of the present invention is not limited to this.

Purification of α-L-arabinofuranosidase (1) Preparation of enzyme bulk powder In order to obtain α-L-arabinofuranosidase bulk powder derived from Acremonium microorganisms, microorganisms were cultured by the following method. As the medium, a medium having the following composition and heat-sterilized by a conventional method was used.

[Medium composition]
Cottonseed oil cake 2%, cellulose 2%, dipotassium hydrogen phosphate 1.2%, bactopeptone 1%, potassium nitrate 0.6%, urea 0.2%, potassium chloride 0.16%, magnesium sulfate heptahydrate 0 0.001%, copper sulfate / pentahydrate 0.001% (pH 4.0).

Acremonium cellulolyticus TN strain (FERM BP-685) was inoculated into 500 ml of the above medium and cultured at 30 ° C. with stirring for 48 hours. Next, the culture solution was scaled up to 15 L using the culture solution as a seed, and the scale-up was continued to finally make the culture solution amount in the 600 L tank 300 L, followed by aeration and agitation culture for 7 days.
The obtained culture broth was filtered with a filter press, concentrated to 15 L by ultrafiltration, added with 2 kg of lactose and powdered by spray drying. The α-L-arabinofuranosidase bulk powder obtained by this method was 5.0 kg.

(2) Purification of α-L-arabinofuranosidase The bulk powder obtained in (1) was dissolved in an acetate buffer (pH 4.5), and impurities were removed by high-speed cooling centrifugation. The obtained supernatant was purified by the following method as a starting material for enzyme purification.

i) Weakly basic anion exchange chromatography: The supernatant was adsorbed to DEAE Sepharose FF (Pharmacia) with Tris-HCl buffer (0.01 M, pH 7.5), and Tris-HCl buffer (0.01 M) was used. PH 7.5), each containing NaCl 0M, 0.1M, 0.15M, 0.2M, 0.5M, and 0.8M, and performing stepwise elution, 0.1 to 0.15M, preferably Fractionated α-L-arabinofuranosidase activity fraction eluted at 0.1M.

ii) Strong basic ion exchange chromatography: The α-L-arabinofuranosidase activity fraction obtained in i) above was added to Mono Q (10/10) using Tris-HCl buffer (0.05 M, pH 7.5). The fraction that showed α-L-arabinofuranosidase activity was obtained by performing linear gradient elution with 0 to 0.3 M NaCl in Tris-HCl buffer (0.05 M, pH 7.5). Sorted.

iii) Gel filtration chromatography: A phosphate buffer (0.05 M, pH 7.) containing 0.1M NaCl in the Superdex 75 (Pharmacia) α-L-arabinofuranosidase activity fraction obtained in ii) above. 0), and fractions showing α-L-arabinofuranosidase activity were collected.

Enzymatic and protein properties of α-L-arabinofuranosidase (1) Substrate specificity and action properties The activity of the purified α-L-arabinofuranosidase obtained in Example 1 against various substrates was examined. That is, the arabinose releasing activity for each of arabinan, arabinogalactan, and arabinoxylan (all manufactured by Megazyme) was examined by the DNS method (Borel et al, 1952).
As a result, the release from arabinan and then arabinoxylan was high, while the release activity from arabinogalactan was relatively low.
Further, as a result of comparing the effects on arabinan and linear arabinan, it was revealed that a considerable amount of arabinose (L-arabinofuranose) is also released from linear arabinan.

(2) Optimum pH
The optimum pH of α-L-arabinofuranosidase obtained in Example 1 was examined. That is, pH was adjusted with glycine-hydrochloric acid buffer (0.025M, pH 1 to 3.5), acetate buffer (0.025M, pH 3.5 to 6) and phosphate buffer (0.025M, pH 6 to 8). And the enzyme was treated with 5 mg / ml arabinan at 37 ° C. to measure the arabinan degradation activity, and the relative enzyme activity was calculated. The relationship between relative enzyme activity and pH is shown in FIG. In FIG. 1, ◇ indicates the results of the glycine-hydrochloric acid buffer solution, □ indicates the acetate buffer solution, and Δ indicates the phosphate buffer solution in each buffer solution.
As is apparent from FIG. 1, the present enzyme showed maximum activity at pH 3-4 and high activity at about pH 1.5-5.5.

(3) Stable pH Range The pH stability of α-L-arabinofuranosidase obtained in Example 1 was examined. That is, glycine-hydrochloric acid buffer (0.05 M, pH 1 to 3.5), acetate buffer (0.05 M, pH 3.5 to 6), phosphate buffer (0.05 M, pH 6 to 8), Tris- The pH was adjusted with HCl buffer (0.05 M, pH 8-9) and CHES buffer (0.05 M, pH 9-10), and the enzyme was treated at room temperature (22 ° C.) for 24 hours. Diluted and treated with 37 mg at 37 ° C. with 5 mg / ml arabinan in acetate buffer (0.05 M, pH 3.5), arabinan degrading activity was measured, and residual enzyme activity was calculated. The relationship between residual enzyme activity and pH is shown in FIG. In FIG. 2, ◇ indicates the results of the glycine-hydrochloric acid buffer solution, □ indicates the acetate buffer solution, Δ indicates the phosphate buffer solution, ○ indicates the Tris-HCl buffer solution, and X indicates the CHES buffer solution in each buffer solution.
As is apparent from FIG. 2, the enzyme was stable in the range of pH 2 to 7.5 and had high activity even in the range of pH 1.5 to 2.

(4) Optimal temperature (optimum temperature for action)
The optimum temperature (action optimum temperature) of α-L-arabinofuranosidase obtained in Example 1 was examined. That is, the enzyme was treated with 5 mg / ml arabinan at 20 to 60 ° C. in acetate buffer (0.05 M, pH 3.5), the arabinan degradation activity was measured, and the relative enzyme activity was calculated. The relationship between relative enzyme activity and temperature is shown in FIG.
As is apparent from FIG. 3, the optimum temperature of this enzyme was 50 ° C., but it had high activity in the range of about 40-60 ° C.

(5) Temperature stability The temperature stability of the α-L-arabinofuranosidase obtained in Example 1 was examined. That is, after the enzyme was treated in an acetate buffer (0.05 M, pH 3.5) for 24 hours at 20 to 70 ° C., these were diluted, and the acetate buffer (0.05 M, pH 3.5) was diluted. The arabinan degradation activity was measured by treating at 37 ° C. with 5 mg / ml arabinan, and the residual enzyme activity was calculated. The relationship between residual enzyme activity and temperature is shown in FIG.
As is apparent from FIG. 4, the enzyme was stable in the range of 50 ° C. or lower.

(6) Molecular weight In order to determine the molecular weight of α-L-arabinofuranosidase obtained in Example 1, SDS-polyacrylamide gel electrophoresis (12% gel) was performed.
As a result, the molecular weight of α-L-arabinofuranosidase was calculated to be about 57,000.
The molecular weight of the standard sample used in this test is as shown in Table 1 below.

(7) Isoelectric point In order to determine the isoelectric point (pI) of α-L-arabinofuranosidase obtained in Example 1, polyacrylamide gel isoelectric using an LKB amphoteric isoelectric focusing device. Point electrophoresis was performed. The isoelectric points of the standard samples used in this test are as shown in Table 2 below.
As a result, the isoelectric point of α-L-arabinofuranosidase was calculated to be approximately 5.3 to 5.5.

(8) Specific activity The specific activity of the α-L-arabinofuranosidase obtained in Example 1 in the arabinan degradation activity was examined. That is, an enzyme was allowed to act on a 5 mg / ml arabinan solution at pH 3.5 and 37 ° C., and the amount of enzyme that produced 1 μmol of reduced L-arabinofuranose per minute was defined as 1 unit.
As a result, the specific activity was about 29 units / mg protein.

Isolation of α-L-arabinofuranosidase gene derived from Acremonium cellulolyticus (1) Determination of partial amino acid sequence of α-L-arabinofuranosidase α-L-arabinofurano purified in Example 1 Sidase protein was electrophoresed (Sodium Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) using an electrophoresis system (Tefco) and 12% polyacrylamide gel. The protein in the polyacrylamide gel after SDS-PAGE was transferred to a polyvinylidene difluoride membrane (Immobilon- ^PSQ , manufactured by Millipore) using Multiphor II (manufactured by Pharmacia Biotech) according to the attached protocol. This membrane was stained with Coomassie Brilliant Blue R250 (manufactured by Nacalai Tesque), and then a portion where a protein of about 50 kDa was blotted was cut out.

  The N-terminal amino acid sequence was determined using a protein sequencer Model 492 (PE Biosystems) according to the attached protocol. As a result, it was revealed that the N-terminal amino acid sequence is as shown in SEQ ID NO: 3 in the sequence listing. When this sequence was subjected to homology search using BLAST, it showed high homology with Trichoderma reesei α-L-arabinofuranosidase.

On the other hand, the internal amino acid sequence was determined as follows.
That is, first, the purified protein was lyophilized and then dissolved in 0.1 M Tris-HCl buffer (pH 9.0). A 1/100 molar amount of lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the protein and reacted at 37 ° C. for 48 hours. The degradation product was subjected to column chromatography (column: C18 220 × 2.1 mm, 0.1% TFA, 5% acetonitrile to 0.085% TFA, 35) using a Model 172 μ preparative HPLC system (manufactured by PE Applied Biosystems). % Acetonitrile gradient), and two kinds of peptides were separated. The amino acid sequence of the obtained peptide fragment was determined by the aforementioned protein sequencer.
As a result, it was revealed that the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5 in the sequence listing were included as internal amino acid sequences.

(2) Isolation of genomic DNA Acremonium cellulolyticus strain Y-94 (FERM BP-5826) was heated to 30 ° C. with (S) medium (2% broth, 0.5% yeast extract, and 2% glucose). The cells were cultured for 2 days, and the cells were collected by centrifugation. The obtained microbial cells were frozen with liquid nitrogen and ground using a mortar and pestle. Genomic DNA was isolated from the ground cells by ISOPLANT (Nippon Gene) according to the attached protocol.

(3) Isolation of DNA fragment of α-L-arabinofuranosidase Based on the partial amino acid sequence determined in (1) above of purified α-L-arabinofuranosidase, Two primers having the base sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7 were synthesized. Using these primers, PCR was carried out using genomic DNA isolated from Acremonium cellulolyticus strain Y-94 as a template.
In PCR, 50 ng of genomic DNA was used as a template, 50 ng of genomic DNA as a template, 1.25 units of ExTaq DNA polymerase (Takara Shuzo), attached buffer, dNTP Mixture, and 10 μM of the above primers at 94 ° C. for 3 minutes (94 The reaction was performed under the conditions of 30 ° C. for 1 minute, 42 ° C. for 1 minute, 72 ° C. for 45 seconds) × 72 times for 3 minutes. By this reaction, a DNA fragment of about 560 bp was amplified, and this DNA fragment was inserted into a pT7 Blue (Stratagene) vector.
The base sequence of the DNA fragment thus cloned was determined using a DNA Sequencing Kit dRhodamine Terminator Cycle Sequencing Ready Reaction (manufactured by PerkinElmer) and an ABI PRISM 310 Genetic Analyzer (manufactured by PerkinElmer). Went according to.
As a result, the base sequence of the isolated DNA fragment is homologous to the α-L-arabinofuranosidase gene of the filamentous fungus, and is part of the gene encoding the desired α-L-arabinofuranosidase. It became clear that there was.

(4) Isolation of mRNA and preparation of cDNA library Acremonium cerulolyticus Y-94 strain was obtained from cellulase induction medium (4% cellulose, 1% bactopeptone, 0.6% potassium nitrate, 0.2% urea, 32 with 0.16% potassium chloride, 0.12% magnesium sulfate, 1.2% monopotassium phosphate, 0.001% zinc sulfate, 0.001% manganese sulfate and 0.001% copper sulfate (pH 4.0)) The cells were cultured at 4 ° C. for 4 days, and the cells were collected by centrifugation. The obtained microbial cells were frozen with liquid nitrogen and ground using a mortar and pestle. Total RNA was isolated from the ground cells by ISOGEN (Nippon Gene) according to the attached protocol.
Furthermore, mRNA was purified from the total RNA using mRNA Purification Kit (Pharmacia) according to the attached protocol.

From the thus obtained mRNA, cDNA was synthesized by Time Saver cDNA Synthesis Kit (Pharmacia) according to the attached protocol. This cDNA was inserted into a phage vector Lambda ZAP II (Stratagene).
The recombinant phage vector thus prepared was subjected to in vitro packaging using Gigapack III Gold Packaging Extract (Stratagene) according to the attached manual. Thereafter, this recombinant phage was infected with E. coli XL1-Blue MRF 'strain and cultured on a plate to form a plaque. The cDNA library thus prepared was 5.5 × 10 ⁵ plaque forming units.
Furthermore, this cDNA library was amplified according to the protocol attached to Lambda ZAP II. Recombinant phage in the amplified cDNA library was infected with E. coli XL1-Blue MRF 'strain and cultured on a plate to form plaques.

(5) Isolation of α-L-arabinofuranosidase gene A Hybond-N + membrane (manufactured by Amersham Pharmacia Biotech) was placed on the plaque-formed plate to attach the plaque. The membrane was treated with alkali, and the recombinant phage DNA on the membrane was denatured into a single strand and adsorbed on the membrane. Using the thus prepared membrane and the DNA fragment of the α-L-arabinofuranosidase gene amplified by PCR as a probe, screening was performed by ECL direct nucleic acid labeling and detection systems (manufactured by Amersham Pharmacia Biotech) according to the attached protocol. went. In this way, the selected plasmid vector pBluescript SK from the positive clones (-) in vivo excision of the was performed according to the protocol attached to the Lambda ZAP II.

Determination of the base sequence of α-L-arabinofuranosidase cDNA in the plasmid vector uses a primer for sequencing near the multiple cloning site of pBluescript SK (−), or a primer prepared based on the analyzed base sequence, DNA Sequencing Kit dRhodamine Terminator Cycle Sequencing Ready Reaction (PE Biosystems) and ABI PRISM 310 Genetic Analyzer (PE Biosystems) were used according to the attached protocol.
In this way, the base sequence of 1685 bp of the α-L-arabinofuranosidase cDNA of Acremonium cellulolyticus was determined. This cDNA contained one ORF consisting of a 1524 bp nucleotide sequence (SEQ ID NO: 2), and was found to encode a protein (SEQ ID NO: 1) of about 50 kDa.
When homology search was performed using BLAST for the protein predicted from this ORF, α-L-arabinofuranosidase of Acremonium cellulolyticus was found to be α-L-α-L-arabino of Trichoderma reesei. It showed the highest homology (79%) to furanosidase (Q92455).

Overexpression of α-L-arabinofuranosidase gene derived from Acremonium cellulolyticus (1) Preparation of recombinant vector for expression of α-L-arabinofuranosidase gene Insertion of α-L-arabinofuranosidase cDNA PCR was performed using the resulting plasmid as a template and two primers each having a base sequence described in SEQ ID NO: 8 and SEQ ID NO: 9 in the sequence listing. PCR was carried out at 94 ° C. for 3 minutes using 94 μl of ExTaq DNA polymerase (Takara Shuzo), attached buffer and dNTP Mixture, 100 ng plasmid DNA and 1 μM of the above primers in 50 μl of the reaction solution. The reaction was carried out under the conditions of 30 seconds, 48 ° C.-30 seconds, 72 ° C.-1 minute) × 30 times, 72 ° C.-3 minutes.
The DNA fragment thus obtained was inserted into pT7Blue (manufactured by Stratagene) to prepare pABF.

The thus obtained Escherichia coli (JM109) transformed with the plasmid vector pABF containing the coding region of arabinofuranosidase was the sixth in Tsukuba City, Ibaraki Prefecture, Japan under the accession number of FERM P-18243. Of the National Institute of Advanced Industrial Science and Technology (AIST).
pABF was cleaved with Stu I and Sph I, and this was inserted between Hpa I and Sph I of pCBHEX / DtR2 described in Japanese Patent Application Laid-Open No. 2001-17180, and a protein expression plasmid pCBHEX / DtR2 / ABF was inserted. Produced.

(2) Introduction of α-L-arabinofuranosidase gene into host Acremonium cellulolyticus strain Y-94 is cultured in (S) medium at 30 ° C. for 16 hours and centrifuged at 3500 rpm for 10 minutes. Bacteria were collected. The obtained bacterial cells were washed with 0.5 M sucrose and filtered through a 0.45 μm filter (10 mg / ml chitinase, 10 mg / ml zymolyase, 30 mg / ml β-glucuronidase and 0.5 M sucrose). ). The mycelium was protoplasted by shaking at 30 ° C. for 60 to 90 minutes. The suspension was filtered with absorbent cotton, and centrifuged at 2500 rpm for 10 minutes to recover protoplasts and washed with SUTC buffer (0.5 M sucrose, 10 mM calcium chloride and 10 mM Tris-HCl (pH 7.5)).
The protoplasts prepared as described above were suspended in 1 ml of SUTC buffer, 10 μg of DNA solution (10 μl) was added to 100 μl of this, and left on ice for 5 minutes. Next, 400 μl of a PEG solution (60% PEG 4000, 10 mM calcium chloride and 10 mM Tris-hydrochloric acid (pH 7.5)) was added and allowed to stand in ice for 20 minutes, 10 ml of SUTC buffer was added, and 2500 rpm, 10 minutes. Centrifuged. The centrifuged protoplast was suspended in 1 ml of SUTC buffer, centrifuged at 4000 rpm for 5 minutes, and finally suspended in 100 μl of SUTC buffer.

  Protoplasts treated as described above were added to (A) medium (3.9% potato dextrose agar medium (Nissui Pharmaceutical Co., Ltd., 17.1% sucrose)) supplemented with hygromycin B (500 μg / ml). ) Overlaid with soft agar and cultured at 30 ° C. for 5 to 9 days. The formed colony was used as a transformant.

(3) Expression of α-L-arabinofuranosidase and measurement of enzyme activity in Acremonium cellulolyticus transformants Two strains having high resistance to hygromycin B (transformant 1 (AA-1) and Transformant 2 (AA-5)) was cultured in a cellulase induction medium. The culture supernatant was analyzed by SDS-PAGE. The result is shown in FIG. In FIG. 5, the upper marker indicates the molecular weight marker, the result of the parent strain, the result of the transformant 1, the result of the transformant 2, and the molecular weight marker from the left side. The numbers on the right indicate the molecular weight, and the arrows on the left indicate the band portion of α-L-arabinofuranosidase.
As is apparent from FIG. 5, the transformant had a higher α-L-arabinofuranosidase secretion amount than the parent strain.
In addition, Table 3 shows α-L-arabinofuranosidase activity (u / ml) per 1 ml of culture supernatant.

  As is apparent from Table 3, the transformant showed higher activity than the parent strain, and in particular, transformant 1 showed a specific activity about 2.5 times that of the parent strain.

L-arabinose production method by decomposing beet pulp (1) Enzymatic decomposition of beet pulp After 750 g of beet pulp was suspended in 15 L of water, 7.5 g of enzyme composition was added and stirred at 50 ° C. for 24 hours. Reacted. After completion of the reaction, the reaction solution was separated with a filter cloth centrifuge (filter cloth 400 mesh) to obtain a water-soluble fraction. By filtering this water-soluble fraction, 12 L of a clear solution containing L-arabinose was obtained. The sugar in this solution was analyzed by high performance liquid chromatography. As analysis conditions, Shodex SUGAR SPO810 (two-linked) was used as an analytical column, the column temperature was 80 ° C., the flow rate was 0.8 mL / min, and elution was performed with ultrapure water. The sugar was detected by an evaporative light scattering detection method (SEDEX), and the content of L-arabinose was determined from the quantitative value of the standard product. As a result of analyzing the solution after the above reaction, 45.6 g of L-arabinose was accumulated in 12 L.

(2) Purification of L-arabinose fraction by chromatography 1.5 g of powdered activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd., trade name Hakuho FA) was added to the above solution and stirred at 55 ° C. for 1 hour. It filtered with 2 filter papers. The filtrate was heated and concentrated under reduced pressure to obtain a concentrated solution of Brix 37.6. This solution was separated and purified using chromatography to obtain an L-arabinose fraction. As chromatography conditions, an ion exchange resin (FX1080 manufactured by Organo Corporation, Ca ²⁺ type, bed volume 1.8 L) was used for elution with water. The purified L-arabinose fraction was 13.7 g L-arabinose (purity 73.7%, per solid).

(3) Crystallization of L-arabinose The L-arabinose fraction was concentrated by heating (70 ° C.) under reduced pressure until Brix60 was obtained. A small amount of crystalline L-arabinose was added to the concentrate and cooled to 70 to 4 ° C. By filtering this solution, 10.1 g of crystalline L-arabinose (purity 99.9% or more) was obtained.

The present invention provides a novel α-L-arabinofuranosidase derived from an Acremonium microorganism, a gene encoding the enzyme, an expression vector containing the gene, and a host cell transformed with the expression vector. An efficient method for producing a novel α-L-arabinofuranosidase is also provided.
Furthermore, there is also provided a technique for utilizing this α-L-arabinofuranosidase or an enzyme composition containing the enzyme for processing of plants or plant-derived materials, and various methods such as feed field, food field, and production of L-arabinose. Use in various applications is expected.

It is a figure which shows the optimal pH at 37 degreeC of (alpha) -L- arabinofuranosidase. It is a figure which shows the pH stability in the room temperature (22 degreeC) and a 24-hour process of (alpha) -L- arabinofuranosidase. It is a figure which shows the optimal temperature in pH3.5 of (alpha) -L-arabinofuranosidase. It is a figure which shows the temperature stability in pH3.5 and a 24-hour process of (alpha) -L- arabinofuranosidase. It is a figure which shows the result of the polyacrylamide gel electrophoresis which shows the expression of (alpha) -L- arabinofuranosidase in an Acremonium cellulolyticus transformant.

Explanation of symbols

  In FIG. 5, the upper row shows the meaning of each column, and shows the molecular weight marker, the parent strain result, the transformant 1 result, the transformant 2 result, and the molecular weight marker from the left side. The numbers on the right indicate the molecular weight, and the arrows on the left indicate the band portion of α-L-arabinofuranosidase.

Claims (29)

An α-L-arabinofuranosidase derived from an Acremonium microorganism and having the following characteristics.
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: Stable in the range of pH 2 to 7.5, and high activity is maintained even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: stable in the range of 50 ° C. or less.   The α-L-arabinofuranosidase according to claim 1, wherein the Acremonium microorganism is Acremonium cellulolyticus. A protein represented by the following (a), (b) or (c).
(A) A protein consisting of residues 1 to 481 in the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
(B) One to 481 residues in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing , and one of the -26 to -1 sequence portions in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing part or the whole, consisting of the protein.
(C) A modified protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence constituting the protein of the above (a) or (b), and the following characteristics A protein having (i) to (g) .
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: It is stable in the range of pH 2 to 7.5 and retains high activity even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: Stable within a range of 50 ° C. or less. The protein according to claim 3, wherein the protein is derived from an Acremonium microorganism . The protein according to claim 4 , wherein the Acremonium microorganism is Acremonium cellulolyticus. A gene comprising DNA represented by the following (a) to (d) .
(A) DNA consisting of the base sequence represented by SEQ ID NO: 2 in the sequence listing.
(B) DNA encoding a protein consisting of residues 1 to 481 in the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing.
(C) Residues 1 to 481 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and one of the sequence portions from -26 to -1 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing DNA encoding a protein consisting of part or all.
(D) A modified protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence constituting the protein of (b) or (c) , and has the following characteristics: DNA encoding a protein having (i) to (g) .
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: It is stable in the range of pH 2 to 7.5 and retains high activity even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: Stable within a range of 50 ° C. or less. The gene according to claim 6 , wherein the gene is derived from an Acremonium microorganism . The gene according to claim 7 , wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus. A nucleic acid construct comprising a base sequence represented by the following (a) to (d) .
(A) The base sequence shown by sequence number 2 of a sequence table.
(B) A base sequence encoding a protein consisting of residues 1 to 481 in the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
(C) Residues 1 to 481 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and one of the sequence portions from -26 to -1 in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A base sequence encoding a protein consisting of part or all.
(D) A modified protein in which addition, insertion, deletion, deletion, or substitution of one to several amino acids has occurred in the amino acid sequence constituting the protein of (b) or (c) , and has the following characteristics: A base sequence encoding a protein having (i) to (g) .
(A) Substrate specificity and action characteristics: Hydrolyze α-L-arabinofuranosyl residue.
(B) Molecular weight: 57,000 (by SDS-polyacrylamide gel electrophoresis)
(C) Isoelectric point: 5.3 to 5.5 (by isoelectric polyacrylamide gel electrophoresis)
(D) Optimal pH: pH 3-4.
(E) Stable pH range: It is stable in the range of pH 2 to 7.5 and retains high activity even in the range of pH 1.5 to 2.
(F) Optimum temperature of action: 50 ° C.
(G) Temperature stability: Stable within a range of 50 ° C. or less. An expression vector comprising the nucleic acid construct according to claim 9 . A host cell transformed with the nucleic acid construct according to claim 9 or the expression vector according to claim 10 . The host cell according to claim 11 , wherein the host is Escherichia coli, yeast, actinomycetes or filamentous fungi. The host cell according to claim 12 , wherein the yeast is a microorganism belonging to the genus Saccharomyces, Hansenula or Pichia. The host cell according to claim 12 , wherein the yeast is Saccharomyces cerevisiae. The host cell according to claim 12 , wherein the filamentous fungus is a filamentous fungus belonging to the genus Acremonium, Humicola, Aspergillus, Trichoderma or Fusarium. The host cell according to claim 12 , wherein the filamentous fungus is Acremonium cellulolyticus, Humicola insolens, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, or Fusarium oxysporous. The host cell according to any one of claims 11 to 16 is cultured, and the α-L-arabinofuranosidase according to claim 1 or 2 is isolated from the cultured host cell and / or culture medium thereof. The method for producing α-L-arabinofuranosidase according to claim 1 or 2, comprising a step. The process of culturing the host cell as described in any one of Claims 11-16 , and isolating the protein as described in any one of Claims 3-5 from the host cell and / or its culture solution after culture | cultivation. The method for producing a protein according to any one of claims 3 to 5 , comprising: An enzyme composition comprising the α-L-arabinofuranosidase according to claim 1 or 2 or the protein according to any one of claims 3 to 5 . One or more components of cellulase, xylanase, protease, galactanase, galactosidase, arabinanase, mannanase, rhamnogalacturonase, polygalacturonase, pectin methylesterase, pectin lyase, and polygalacturonic acid lyase Furthermore, it contains, The enzyme composition of Claim 19 characterized by the above-mentioned. A plant or plant containing the α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. Enzyme agent for processing derived products and / or improving utilization efficiency. The feed additive containing the α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20 . . A method for producing a feed having excellent digestibility, wherein the feed additive according to claim 22 is added to the feed. Food processing containing the α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20 . Enzyme agent. The manufacturing method of the foodstuff using the enzyme agent for food processing of Claim 24 . Wood pulp treatment containing the α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. Agent. A method for treating wood pulp, wherein the wood pulp treating agent according to claim 26 is used. Use of the α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20. To produce L-arabinose. The α-L-arabinofuranosidase according to claim 1 or 2, the protein according to any one of claims 3 to 5 , or the enzyme composition according to claim 19 or 20, L- by acting on a natural product selected from orange fiber containing arabinofuranosyl residue, orange juice koji, apple fiber, apple juice koji, beet fiber, beet pulp, rice sugar, soybean koji, peanut and corn koji A method for producing L-arabinose, comprising obtaining arabinose.

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