JP2003174892A - New galactanase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new galactanase, to provide a polynucleotide encoding the galactanase, to provide a method for expressing the galactanase with the polynucleotide, to provide an enzyme composition containing the galactanase, and to provide a method for treating a plant material with the enzyme composition. <P>SOLUTION: The galactanase is originated from a mold belonging to the genus Acremonium. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polypeptide having galactanase activity, that is, a novel galactanase.

[0002]

2. Description of the Related Art Galactans, which are abundant in fibers derived from plants such as citrus fruits, potatoes and soybeans, are β-1,4-
It is a polysaccharide containing a main chain having a galactoside bond. As an enzyme that decomposes these polysaccharides, β-1,4-galactanase (hereinafter sometimes simply referred to as “galactanase”) is known. So far, various fungi [eg,
Penicillium citrinum ["Agricultural and Biological Chemistry (Agricultural and Biological
cal Chemistry) ", 1985, No. 49.
Vol., P. 3445-3454 (Non-Patent Document 1)], Ba
cillus subtilis ["Agricultural and Biological Chemistry (Agri
cultural and Biological C
), 1972, Vol. 36, p. 1
945-1954 (Non-Patent Document 2)], Aspergi
illus niger , Aspergillus ac
uleatus ["Carbohydrate Polymers",
(Netherlands), Elsevier Science (Elsevi)
er Science), 1991, Volume 16, p.
167-187 (Non-Patent Document 3)], Myceliop
hthora thermophilum [ Table 200
0-505303 (Patent Document 1)], or Hum
icola insolens (Table 2000-505
No. 303) and the like] and exo-type galactanase produced by Bacillus subtilis [Patent No. 298921].
No. 7 specification (Patent Document 2)] has been reported.

As an example of application of galactanase to various fields, in the field of feed, the cause of indigestion of feed is eliminated by adding it to animal feed [Japanese Patent Publication No. 55-4023].
No. 8 (Patent Document 3)], or improvement of feed conversion ratio [Japanese Patent Publication No. 11-514528 (Patent Document 4)] and the like. Here, in order for the galactanase added to the feed to act effectively in the digestive tract of livestock, it must be stable at a relatively low pH. For example, in broilers, the pH in the glandular stomach and muscle stomach is known to drop to around 2 [Ryoji Onodera et al., "Livestock Nutrition", Kawashima Shoten,
1989, p. 73 (Non-Patent Document 4)], but a galactanase that is stable at such a low pH has not been known so far. For example, it is known to be stable at low pH, galactanase described in JP Kohyo 6-505154 (Aspergillus aculeatu
( derived from s .
In 5, the enzyme activity is reduced to 20% (when treated at room temperature for 1 hour). In the field of foods, improvement in turbidity stability of plant extracts [Japanese Patent Publication No. 10-501412 (Patent Document 5)], or depectinization and viscosity reduction during processing of wine or juice [Japanese Patent Publication No. 6-505154]. It is disclosed to be used for the publication (Patent Document 6)].

Further, galactanase can be obtained by reacting galactanase with galactans. Galactooligosaccharides have attracted attention for their intestinal regulating action and calcium absorption promoting action [JP-A-4-134031 (Patent Document 7)], and prevent or treat osteoporosis [JP-A-7-252].
No. 156 (Patent Document 8)], for the purpose of preventing or treating inflammatory bowel disease, it has been applied to the food and pharmaceutical fields. Further, the suppression of the occurrence of diarrhea or loose stools or the improvement of the weight gain rate [Japanese Patent Publication No. 2-579202 (Patent Document 9)], the intestinal flora improving effect [Japanese Patent Laid-Open No. 6-178654 (Patent Document 10)], Prevention or treatment of constipation for pigs [Patent No. 2571702 (Patent Document 11)], increase in milk fat percentage [Patent No. 2525638 (Patent Document 12)], suppression of accumulation of body fat [JP-A-6- 1786
54 (Patent Document 13)], quality improvement of eggs [Patent No. 3124409 (Patent Document 14)], or inhibition of growth of Salmonella [JP-A-5-208912].
Japanese Patent Publication (Patent Document 15)] for the purpose of application in the feed field.

Regarding the method for producing galactooligosaccharides,
Various reports have been made. Conventionally, many methods have been used in which lactose or a lactose-containing material is allowed to act with β-galactosidase or galactanase, and by glycosyl transfer, the formula: Gal- (Gal) n-Glc (wherein “Gal” represents galactose). "Gl
"c" represents glucose and "n" represents an integer of 0 or more) to obtain a galactooligosaccharide [Japanese Patent Publication No. 2-53033 (Patent Document 16), Japanese Patent Publication No. 3-54559]. (Patent Document 17), or Japanese Patent Laid-Open No. 9-117297 (Patent Document 18)]. Further, Japanese Patent No. 2615236 (Patent Document 19), Japanese Patent No. 2894292 (Patent Document 20), Japanese Patent No. 28
94293 specification (patent document 21), or patent 29
According to the specification of 89217 (Patent Document 22), galactanase is allowed to act on arabinogalactan derived from okara or soybean, and represented by the formula: (Gal) p (wherein “Gal” represents galactose and “p”). Represents an integer of 2 to 4) is disclosed. However, Japanese Patent No. 2615236
In each of the methods described in Japanese Patent No. 2894292 or Japanese Patent No. 2894292, galactotriose (Gal ₃ ) and galactotetraose (Gal ₄ ) are mainly produced to the same extent,
In the method described in Japanese Patent No. 2894293, galactotriose (Gal ₃ ) is mainly produced, and
According to the method described in Japanese Patent No. 989217, in addition to galactobiose (Gal ₂ ), galactotetraose (Ga
l ₄ ) is generated. Therefore, the galacto-oligosaccharide produced by the conventional methods is a mixture of various oligosaccharides and monosaccharides, and thus has a drawback that it is difficult to crystallize.
On the other hand, if galactobiase can generate galactobiose at a high concentration, it may be possible to easily crystallize. It is also known that the end products become galactose and galactobiose by the action of galactanase ["Agricultural and Biological Chemistry (Agricultural
l and Biological Chemistr
y) ”, 1985, Volume 49, p. 3445-345
4; "Carbohydrate Polymers (Carboh
hydrate Polymers ”, (Netherlands),
Elsevier Science
nce), 1991, Volume 15, p. 431-444
(Non-Patent Document 5); and "Carbohydrate Polymers (Carbohydrate Polymers).
s) ”, (Netherlands), Elsevier Science (El
sevier Science), 1991, 16th
Vol., P. 167-187] are the results after the reaction for a long time, and it is desired to improve the purity in a shorter time. A galactanase having such a reaction characteristic and a high specific activity has been desired.

[0006] Acremonium cellulolyticus , a type of filamentous fungus
Regarding the enzyme produced by lyticus, many reports have been made on cellulase, pectinase (polygalacturonase), and xylanase [[Agricultural and Biological Chemistry].
musical Chemistry) ", 1988, Vol. 52, p. 2493-2501 (Non-Patent Document 6);
"Agricultural and Biochemistry (Agricultural and Bio
logical Chemistry) ", 1989
53, p. 3359-3360 (Non-Patent Document 7); "Agricultural and Biological Chemistry (Agricultural and
Biological Chemistry) ", 19
1990, vol. 54, p. 309-317 (Non-patent document 8); and International Publication WO98 / 39423 pamphlet (Patent document 22)], it has been shown that these enzymes are effective in feed applications and silage applications [JP-A-4-117244]. Japanese Patent Publication (Patent Document 23) or Japanese Patent Laid-Open No. 7-236431 (Patent Document 24)]. However, there are no detailed reports on galactanase, and in order to further effectively utilize the enzyme or enzyme composition derived from Acremonium cellulolyticus in the industry, detailed enzymatic or protein properties of galactanase have been reported. The elucidation of the properties and the isolation of the gene encoding the enzyme have been long awaited.

[0007]

[Patent Document 1] Japanese Patent Publication No. 2000-505303

[Patent Document 2] Japanese Patent No. 2989217

[Patent Document 3] Japanese Patent Publication No. 55-40238

[Patent Document 4] Japanese Patent Publication No. 11-514528

[Patent Document 5] Japanese Patent Publication No. 6-505154

[Patent Document 6] Japanese Patent Publication No. 10-501412

[Patent Document 7] Japanese Unexamined Patent Application Publication No. 4-134031

[Patent Document 8] JP-A-7-252156

[Patent Document 9] Japanese Patent Publication No. 2-579202

[Patent Document 10] JP-A-6-178654

[Patent Document 11] Japanese Patent No. 2571702

[Patent Document 12] Japanese Patent No. 2525638

[Patent Document 13] JP-A-6-178654

[Patent Document 14] Japanese Patent No. 3124409

[Patent Document 15] JP-A-5-208912

[Patent Document 16] Japanese Patent Publication No. 2-53033

[Patent Document 17] Japanese Patent Publication No. 3-54559

[Patent Document 18] Japanese Patent Laid-Open No. 9-117297

[Patent Document 19] Japanese Patent No. 2615236

[Patent Document 20] Japanese Patent No. 2894292

[Patent Document 21] Japanese Patent No. 2894293

[Patent Document 22] International Publication WO98 / 39423 Pamphlet

[Patent Document 23] Japanese Patent Laid-Open No. 4-117244

[Patent Document 24] Japanese Patent Laid-Open No. 7-236431

[Non-Patent Document 1] “Agricultural and Biological Chemistry (Agricultural
and Biological Chemistr
y) ”, 1985, Volume 49, p. 3445-345
Four

[Non-Patent Document 2] "Agricultural and Biological Chemistry (Agricultural
and Biological Chemistr
y) ”, 1972, Vol. 36, p. 1945-195
Four

[Non-Patent Document 3] "Carbohydrate Polymers",
(Netherlands), Elsevier Science (Elsevi)
er Science), 1991, Volume 16, p.
167-187

[Non-Patent Document 4] Ryoji Onodera et al., "Livestock Nutrition",
(Yes) Kawashima Shoten, 1989, p. 73

[Non-Patent Document 5] "Carbohydrate Polymers",
(Netherlands), Elsevier Science (Elsevi)
er Science), 1991, Volume 15, p.
431-444

[Non-Patent Document 6] “Agricultural and Biological Chemistry (Agricultural
and Biological Chemistr
y) ", 1988, Vol. 52, p. 2493-250
1

[Non-Patent Document 7] “Agricultural and Biological Chemistry (Agricultural
and Biological Chemistr
y) ”, 1989, 53, p. 3359-336
0

[Non-Patent Document 8] “Agricultural and Biological Chemistry (Agricultural
and Biological Chemistr
y) ”, 1990, Vol. 54, p. 309-317

[0008]

In order to effectively utilize galactanase in industry, it is indispensable to isolate and purify an enzyme having a high specific activity and clarify its enzymatic and physical properties. . Further, in order to enhance the expression of the enzyme and enable mass production, it is necessary to isolate and analyze the gene encoding the enzyme. Further, the enzyme for feed addition must be stable at low pH (preferably pH 3 or lower, more preferably pH 2 or lower).
Furthermore, the enzyme for producing galactobiose needs to be an enzyme that produces galactobiose at a high concentration.

Under these circumstances, the inventors of the present invention have conducted intensive studies by fractionating and purifying various enzymes of Acremonium cellulolyticus. We have found a novel galactanase that exhibits a significantly higher specific activity than that of the enzyme and is stable over a wide pH range, and isolated the gene encoding the enzyme. Furthermore, when the action and effect of the above-mentioned galactanase on various plant materials was examined, it was found to be an enzyme that produces galactobiose at a high concentration. The present invention is based on such knowledge. Therefore,
An object of the present invention is to provide a novel galactanase, a polynucleotide encoding the galactanase, a method for expressing galactanase using the polynucleotide, an enzyme composition containing galactanase, and a treatment of plant material with the enzyme composition. To provide a method.

[0010]

The above problems can be solved by a polypeptide having the following physicochemical properties (a) and (b) and having galactanase activity according to the present invention: (a) Action and substrate specificity: The β-1,4-galactoside bond of a polysaccharide or oligosaccharide having a β-1,4-galactoside bond is hydrolyzed to an endo type. (B) Stable pH range: Stable in the range of pH 2-10.

The present invention also includes (a) a polypeptide comprising the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2; (b) the 21st amino acid sequence represented by SEQ ID NO: 2. ~
A polypeptide containing the 354th amino acid sequence and having galactanase activity; or (c) the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 to
The present invention relates to a polypeptide containing an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted at the 354th amino acid sequence at one or more positions, and which has galactanase activity.

The present invention also includes (a) a polynucleotide consisting of the 61st to 1065th base sequences of the base sequence represented by SEQ ID NO: 1; (b) the 21st position of the amino acid sequence represented by SEQ ID NO: 2. ~
A polynucleotide encoding a polypeptide consisting of the 354th amino acid sequence; (c) the 21st amino acid sequence represented by SEQ ID NO: 2 to
A polynucleotide containing a 354th amino acid sequence and encoding a polypeptide having galactanase activity; (d) 21st to 21st amino acid sequences represented by SEQ ID NO: 2
A polynucleotide containing an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted at the 354th amino acid sequence at one or more positions, and which encodes a polypeptide having galactanase activity Or (e) 61st to 10th of the base sequence represented by SEQ ID NO: 1
The present invention relates to a polynucleotide which hybridizes with a DNA consisting of the 65th nucleotide sequence under stringent conditions and which encodes a protein having a galactanase activity.

The present invention also relates to a polynucleotide construct containing the above polynucleotide. The present invention also relates to an expression vector containing the polynucleotide or the polynucleotide construct. The present invention also relates to a transformed cell transformed with the polynucleotide construct or the expression vector. The present invention also relates to a method for producing the above-mentioned polypeptide, which comprises a step of culturing the above-mentioned transformed cell; and a step of collecting the above-mentioned polypeptide from the transformed cell and / or the culture solution obtained in the above-mentioned culturing step. ..

The present invention also relates to an enzyme composition containing the above-mentioned polypeptide. The present invention also relates to a feed additive containing the above-mentioned polypeptide or enzyme composition. The present invention also relates to feed containing the feed additive.
The present invention also relates to a method for preparing the above feed, which comprises adding the above feed additive to a feed material. The present invention also relates to an enzyme agent for food processing and / or food additive, which comprises the above-mentioned polypeptide or enzyme composition. Further, the present invention is
The present invention relates to a food processing method using the enzyme for food processing. Furthermore, the present invention relates to a method for producing galactobiose, which comprises degrading galactans or galactooligosaccharides using the above-mentioned polypeptide or enzyme composition.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The polypeptide of the present invention has endo-β-1,4-galactanase activity. End-
An enzyme exhibiting β-1,4-galactanase activity (ie, Arabinogalactan 4-β-D-g
alactanohydrolase EC3.2.1.
89) is specifically an enzyme that hydrolyzes a β-1,4-galactoside bond of a polysaccharide or oligosaccharide having a β-1,4-galactoside bond (for example, galactans or galactooligosaccharides) to an endo type. Means end-β-
Also called 1,4-galactanase, galactanase, or arabinogalactanase.

The galactans or galactooligosaccharides are
It is a generic term for polysaccharides or oligosaccharides whose main chain is mainly galactose, and is not limited to the presence or absence of side chains and the type of side chain sugars. The polypeptide of the present invention (that is, galactanase) acts on a polysaccharide or oligosaccharide having a β-1,4-galactoside bond, for example, a galactan or a galactooligosaccharide having a β-1,4-galactoside bond in its main chain. To do. Examples of galactans having a β-1,4-galactoside bond in the main chain include galactan having arabinose, glucose, mannose, xylose, and / or uronic acid in the side chain, and specifically, for example, soybean The arabinogalactan contained can be mentioned.
The galactooligosaccharide having a β-1,4-galactoside bond in the main chain is, for example, represented by the formula: (Gal) m (wherein “Gal” represents galactose and “m” represents an integer of 2 or more). The represented galactooligosaccharide [eg, galactobiose (Gal ₂ ), galactotriose (Gal ₃ ), or galactotetraose (G
al ₄ )], formula: (Gal) n-Glc (wherein “Gal” represents galactose,
"c" represents glucose, and "n" represents an integer of 2 or more), arabinogalacto-oligosaccharide, or the like.

The polypeptide of the present invention has the following physicochemical properties and galactanase activity. (A) Action and Substrate Specificity Polysaccharides or oligosaccharides having β-1,4-galactoside bond, for example, β- of galactans or galactooligosaccharides
The 1,4-galactoside bond is hydrolyzed in an endo-type.
As a substrate, it shows high activity against galactans or galactooligosaccharides having a β-1,4-galactoside bond in the main chain. Further, arabinogalactan contained in soybean is known to consist of β-1,4 bonds,
The polypeptide of the present invention acts on soybean fiber and mainly produces galactobiose and galactose. (B) Molecular weight The molecular weight by SDS-polyacrylamide gel electrophoresis is about 40,000. (C) Isoelectric point The isoelectric point (pI) by polyacrylamide gel electrophoresis is about 4.5. (D) Optimum pH The optimum pH in the galactan-degrading activity is 5 to 6, particularly 5.5. (E) Optimum temperature The optimum temperature for galactan decomposition activity is about 50 ° C. (F) Stable pH range pH 2-1 in 5 hours treatment at room temperature (22 ° C)
It is stable in the range of 0. It should be noted that high activity is maintained even in the pH range of 1.5 to 2. In the present specification, “stable” at a certain pH means 22 ° C.
It means that the residual enzyme activity after the treatment for 5 hours was 80% or more. (G) Temperature stability In a 24-hour treatment at pH 5.5, it is stable at 50 ° C or lower. (H) Specific activity The specific activity in the galactan degrading activity is about 3000 units / mg protein.

The term "galactan degrading activity" as used herein means 0.5 under conditions of pH 5.5 and 37 ° C.
An enzyme is allowed to act on a galactan solution derived from% Lupin, and the amount of the enzyme that produces 1 μmol of the reduced galactan terminal in 1 minute in this measurement system is defined as “1 unit”.

The polypeptide of the present invention can be obtained, for example, from a filamentous fungus. As the filamentous fungus, a microorganism belonging to the genus Acremonium is preferable,
Cellulolyticus is more preferable, and Acremonium cellulolyticus Y-94 strain (FERM BP-582
6) or Acremonium cellulolyticus TN strain (F
ERM BP-685) is more preferred. In addition, Acremonium cellulolyticus Y-94 strain (FERM B
P-5826) is an Acremonium cellulolyticus TN strain (FERM BP-68) on January 12, 1983.
5) on October 13, 1984, respectively, National Institute of Advanced Industrial Science and Technology, Patent Biological Depository Center [(old)
Institute of Biotechnology, Industrial Technology Institute].

The polypeptide of the present invention can be obtained from a microorganism producing the polypeptide of the present invention by, for example, but not limited to, the following procedure. First, the microorganism is cultured by a culture method known per se. For example, Acremonium cellulolyticus Y-94 strain (FERM BP-58
26) or Acremonium cellulolyticus TN strain (FERM BP-685), for example, JP-B-60-43954 or JP-B-63-6.
Culturing can be performed according to the method described in Japanese Patent No. 3197. After the culturing of the microorganism is completed, the resulting culture is used as a crude enzyme for the polypeptide of the present invention as it is as a supernatant obtained by removing the bacterial cells by an appropriate separation means (for example, centrifugation). be able to. Usually,
The supernatant is concentrated by an appropriate concentration means (for example, ultrafiltration method), and if desired, a preservative or the like is added to it as a concentrated enzyme, or after the concentration, an appropriate powdering means (for example, The polypeptide of the present invention can be obtained as a powdery enzyme by a spray drying method). Furthermore,
By subjecting these concentrated enzymes or powdered enzymes to partial purification or highly purification, if necessary, the polypeptide of the present invention can be obtained in a more pure state. As the purification method, a conventional method for protein purification, for example, a salting-out method using ammonium sulfate or the like; an organic solvent precipitation method using alcohol or the like; a membrane separation method; or an ion exchanger, a carrier for hydrophobic chromatography, and / or The chromatographic separation method using a carrier for gel filtration and the like can be used alone or in appropriate combination.

The polypeptide of the present invention includes (a) the 21st position of the amino acid sequence represented by SEQ ID NO: 2 to
A polypeptide consisting of the 354th amino acid sequence; (b) the 21st amino acid sequence represented by SEQ ID NO: 2 to
A polypeptide containing the 354th amino acid sequence and having galactanase activity; (c) the 21st amino acid sequence represented by SEQ ID NO: 2 to
354th amino acid sequence 1 or more (preferably 1
Or several), a polypeptide containing an amino acid sequence in which one or more (preferably one or several) amino acids are substituted, deleted, and / or inserted, and having galactanase activity (hereinafter , A functionally equivalent variant); or (d) the 21st position of the amino acid sequence represented by SEQ ID NO: 2 to
A polypeptide containing an amino acid sequence having 90% or more homology with the 354th amino acid sequence and having galactanase activity (hereinafter referred to as a homologous polypeptide) is included. In the present specification, the term “galactanase activity” means a polysaccharide or oligosaccharide having a β-1,4-galactoside bond, for example, β-1,4-galactoside bond of a galactan or a galactooligosaccharide is hydrolyzed in an endo-type. It means the activity of decomposing.

The "polypeptide consisting of the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2", which is one of the polypeptides of the present invention, is an acremonium consisting of 334 amino acid residues. -A novel galactanase derived from Cellulolyticus. The "polypeptide consisting of the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2" means "the amino acid sequence represented by SEQ ID NO: 2 (that is, the amino acid sequence represented by SEQ ID NO: 2). 1st to 354th amino acid sequence)
From a precursor translated as "a polypeptide consisting of
It is considered to be a mature form in which the N-terminal side signal peptide (that is, the peptide consisting of the 1st to 20th amino acids in the amino acid sequence represented by SEQ ID NO: 2) has been cleaved off.

The "polypeptide containing the 21st to 354th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 and having galactanase activity" of the present invention includes, for example, (b1) SEQ ID NO: 2 A polypeptide comprising an amino acid sequence in which an appropriate marker sequence or the like is added to the N-terminal and / or C-terminal of the 21st to 354th amino acid sequence of the represented amino acid sequence, and having galactanase activity; (b2) A signal sequence (preferably 1 of the amino acid sequence represented by SEQ ID NO: 2 is added to the N-terminal of the 21st to 354th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2.
A polypeptide having a galactanase activity, which consists of the amino acid sequence of the 20th to 20th amino acid sequence, or a partial sequence thereof and which has an amino acid sequence having a function as a signal sequence) and which has galactanase activity; b3) In the amino acid sequence obtained by adding a signal sequence to the N-terminal of the 21st to 354th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2, further, at the N-terminal and / or C-terminal thereof, a suitable marker sequence Examples of the polypeptide include an amino acid sequence to which is added, and which has galactanase activity.

As the marker sequence that can be used in the present invention, a sequence for easily confirming the expression of the polypeptide, confirming intracellular localization, or purification can be used. For example, FLAG epitope , Hexa-histidine tag, hemagglutinin tag, or myc epitope.

The origin of the polypeptide of the present invention is not limited to Acremonium cellulolyticus. For example, a functionally equivalent variant of the present invention includes, for example, a naturally occurring variant in Acremonium cellulolyticus of a polypeptide consisting of the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2 ( However, in addition to containing galactanase activity, microorganisms other than Acremonium cellulolyticus [for example, filamentous fungi (preferably, excluding Acremonium cellulolyticus,
Microorganisms belonging to the genus Acremonium)], which are naturally occurring functionally equivalent variants (provided that they have galactanase activity). In addition, these natural polypeptides, particularly 21 of the amino acid sequence represented by SEQ ID NO: 2
Polypeptides (provided that they have galactanase activity) that are artificially modified by genetic engineering based on the amino acid sequence of the 1st to 354th amino acids are included.

The homologous polypeptide of the present invention is SEQ ID NO: 2.
Is not particularly limited as long as it is a polypeptide having a galactanase activity, which includes an amino acid sequence having 90% or more homology with the 21st to 354th amino acid sequences of the amino acid sequence represented by However, as the homologous polypeptide of the present invention, the homology with the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2 is more preferably 95% or more, further preferably 98% or more, particularly A polypeptide having an amino acid sequence of preferably 99% or more and having galactanase activity is preferable. In addition, in this specification, "homology" means BLAST (Basic local al).
igment search tool).

The polypeptide of the present invention includes "a polypeptide consisting of the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2" or "SEQ ID NO: 2".
A polypeptide consisting of the amino acid sequence represented by (i.e., the 1st to 354th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2) is preferable.

As long as the polynucleotide of the present invention is a polynucleotide encoding the polypeptide of the present invention,
It is not particularly limited. The term “polynucleotide” used herein includes both DNA and RNA. Specifically, the polynucleotide of the present invention includes: (a) 61st to 10th nucleotides of the base sequence represented by SEQ ID NO: 1;
A polynucleotide consisting of the 65th nucleotide sequence; (b) a polynucleotide encoding "a polypeptide consisting of the 21st to 354th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2"; (c) "SEQ ID NO: 2" A polynucleotide which includes the 21st to 354th amino acid sequence of the amino acid sequence represented by and which encodes a "polypeptide having galactanase activity"; (d) a polynucleotide which encodes a "functional variant"; or (E) a polynucleotide encoding a “homologous polypeptide” is included.

The polynucleotide of the present invention also comprises
"61st to 1065th of the base sequence represented by SEQ ID NO: 1
A polynucleotide which hybridizes with the DNA consisting of the nucleotide sequence at the second position under stringent conditions and which encodes a protein having galactanase activity is included. In the present specification, “stringent conditions” means that a probe having labeled DNA having the nucleotide sequence of the 61st to 1065th nucleotides of the nucleotide sequence represented by SEQ ID NO: 1 is used, and ECL direct DNA is used. / RNA labeling detection system (manufactured by Amersham) according to the protocol attached thereto, pre-hybridization (42 ° C.) for 1 hour, the probe was added, and hybridization was performed for 15 hours (42 ° C.). 4 by 1 × SSC containing 4% SDS and 6 mol / L urea (SSC; 15 mmol / L trisodium citrate, 150 mmol / L sodium chloride)
Repeat 20x wash at 2 ° C twice, then 5x SSC
It is a condition that the washing is performed twice at room temperature for 10 minutes.

The polynucleotide according to the present invention may be of natural origin, for example, or
It can also be a fully synthesized product. Further, it may be synthesized by utilizing a part of naturally occurring one. A typical method for obtaining the polynucleotide according to the present invention is, for example, a method commonly used in the field of genetic engineering from a chromosome library or a cDNA library derived from Acremonium cellulolyticus, for example, a partial amino acid sequence Examples include a method of screening using an appropriate DNA probe prepared based on information.

The polynucleotide of the present invention includes "a polynucleotide consisting of the 61st to 1065th base sequence of the base sequence represented by SEQ ID NO: 1" or "the base sequence represented by SEQ ID NO: 1 (that is, the sequence A polynucleotide consisting of the 1st to 1065th base sequence of the base sequence represented by No. 1) is preferred. The base sequence represented by SEQ ID NO: 1 begins with the 1st to 3rd ATG and is 10
It has an open reading frame that ends at the 63rd to 1065th TGAs. In addition, the 61st to 63rd base sequences are mature proteins consisting of 334 residues (that is, 21 of the amino acid sequence represented by SEQ ID NO: 2).
Corresponding to the N-terminal amino acid of the galactanase consisting of the amino acid sequence from the 1st to 354th amino acids.

The polynucleotide construct of the present invention expresses the polynucleotide of the present invention and a polynucleotide containing a base sequence necessary for expressing the polypeptide of the present invention encoded by the polynucleotide, by the polypeptide. There is no particular limitation as long as the order is included. The base sequence required to express the polypeptide is not particularly limited, for example,
Examples thereof include a promoter sequence located upstream of the polynucleotide to be expressed, a terminator sequence located downstream of the polynucleotide to be expressed, or a base sequence encoding a signal peptide.

The expression vector of the present invention contains the polynucleotide or the polynucleotide construct of the present invention, is replicable in the host cell, and is capable of expressing the galactanase encoded by the polynucleotide in the host cell. As long as it is not particularly limited. The expression vector of the present invention is, for example, a self-replicating vector, that is,
It is possible to construct a DNA (for example, a plasmid) which exists as an extrachromosomal independent entity and whose replication is independent of chromosomal replication. Alternatively, the expression vector of the present invention may be one which, when introduced into a host cell, is integrated into the genome of the host cell and is replicated together with the chromosome into which it is integrated. As the procedure and method for constructing a vector according to the present invention, those commonly used in the field of genetic engineering can be used.

The expression vector of the present invention comprises, in addition to the polynucleotide of the present invention, a nucleotide sequence for controlling the expression thereof, in order to actually introduce the vector into a host cell to express a protein having a desired activity, or Preferably, it contains a genetic marker for selecting a microorganism. Examples of the nucleotide sequence controlling expression include a promoter sequence, a terminator sequence, a nucleotide sequence encoding a signal peptide, and the like. The promoter sequence is not particularly limited as long as it shows transcription activity in the host cell, and can be appropriately selected according to the type of the host cell. The terminator sequence is
It is not particularly limited as long as it shows transcription termination activity in the host cell, and can be appropriately selected depending on the type of the host cell. Furthermore, the signal peptide is not particularly limited as long as it contributes to the secretion of the protein in the host cell, and can be appropriately selected according to the type of host cell. The gene marker can be appropriately selected according to the method of selecting transformants, and for example, a gene encoding drug resistance or a gene complementing auxotrophy can be used.

The transformed cell of the present invention can be obtained by transforming an appropriate host cell with the polynucleotide construct or expression vector of the present invention. The host-vector system that can be used in the present invention is not particularly limited, and for example, a system using Escherichia coli, actinomycetes, yeast, filamentous fungi, plant cells, or animal cells (for example, mammalian cells or insect cells) and the like. (Including fusion protein expression systems with other proteins) can be used. Transformation of a host cell with the expression vector of the present invention can also be performed according to a method commonly used in this field.

The yeast used as a host in the present invention is, for example, Saccharomyces.
ces) genus Hansenula (Hansenula) spp, or Pichia (Pichia) microorganism belonging to the genus, for example,
Saccharomyces
es cerevisiae ) and the like. Further, the filamentous fungus belonging to the incomplete fungus used as a host is, for example, Acremonium ( Acremo).
nium) genus Humicola (Humicola) genus Aspergillus (Aspergillus) genus Trichoderma (Trichoderma) genus, or Fusarium (Fusarium) can be mentioned microorganisms belonging to the genus, preferably, Acremonium cellulolyticus (Acremonium cellulolyticu
s ), Humicola Insolens ( Humicola)
insolens ), Aspergillus niger ( Asp
ergillus niger), Aspergillus oryzae (Aspergillusoryzae), Trichoderma viride (Trichoderma virid
e ), or Fusarium oxysporus ( Fusar)
ium oxysporus ) and the like.

In the method for producing the polypeptide of the present invention according to the present invention, the transformed cell of the present invention is cultured in an appropriate medium, and the polypeptide of the present invention is recovered from the obtained transformed cell and / or culture solution. By doing so, the polypeptide of the present invention can be obtained. Culture of transformed cells
It can be carried out by a method essentially equivalent to the culture of the microorganism used as a host. As a method for recovering the desired polypeptide from the transformed cells and / or the culture solution, a method commonly used in this field can be used.

For example, when the polypeptide of the present invention is produced using the filamentous fungus Acremonium cellulolyticus, it can be carried out according to the method described in JP 2001-17180 A, for example. That is, a recombinant vector for gene expression was prepared in which the gene encoding the polypeptide of the present invention was ligated to the downstream of the promoter of the CBH (cellobiohydrolase) 1 gene which is most highly expressed in Acremonium cellulolyticus Then, by introducing this vector into Acremonium cellulolyticus, a large amount of the above-mentioned polypeptide can be expressed.

The enzyme composition of the present invention comprises the polypeptide of the present invention and a carrier or diluent usually used for the enzyme composition. The enzyme composition of the present invention comprises, for example, a carrier or a diluent that can generally use the polypeptide of the present invention in the enzyme composition, such as an excipient (eg, lactose, sodium chloride, sorbitol, etc.). , A surfactant, an antiseptic, or the like can be mixed and produced. The shape of the enzyme composition in the present invention is arbitrary, and it can be prepared in an appropriate shape, for example, powder or liquid.

In addition to the polypeptide of the present invention, the enzyme composition of the present invention further comprises cellulase, xylanase, protease, galactosidase, arabinase, arabinofuranosidase, mannanase, rhamnogalacturonase, polygalacturonase, pectin. It may include at least one of methyl esterase, pectin lyase, or polygalacturonic acid lyase. By using an enzyme other than the polypeptide of the present invention (ie, galactanase) in combination, it is expected to further improve the utilization and / or processing efficiency of plant material, as compared with the enzyme composition containing galactanase alone. it can.

The polypeptide of the present invention is excellent in degrading polysaccharides or oligosaccharides having a β-1,4-galactoside bond, for example, galactans and galactooligosaccharides, and can be used in plant materials containing galactans or galactooligosaccharides. Suitable for processing and improving usability. That is, by treating a plant material with the polypeptide or enzyme composition of the present invention, galactans or galactooligosaccharides can be hydrolyzed, and the processing and / or utilization efficiency of the plant material can be improved.

Plant materials that can be treated with the polypeptide or enzyme composition of the present invention include harvested plants,
Alternatively, a treated product thereof (for example, a dried product, a cleaved product, an extract, a purified product, or the like) can be mentioned. Specifically, for example, all plants containing galactans or galactooligosaccharides, a cleaved product thereof, Examples thereof include plant tissues (for example, cell wall or epidermal tissues), plant tissue-derived products, cell contents, or extracted components. More specifically, in the field of feed or food, for example, grains, beans,
Targets are grasses, vegetables, fruits, potatoes, trees, and the like, and can also be applied to fruit juices, purees, pastes, pressed meals, or extracted meals obtained by processing these.

Further, the polypeptide or enzyme composition of the present invention may be added to an animal (for example, an animal such as the galactans at the same time, or the polypeptide or enzyme composition of the present invention and the galactan at the same time in the digestive organs). , An animal other than human) can decompose galactans that are substrates in the body of the animal to produce galactooligosaccharide. Galactooligosaccharides have been attracting attention for their intestinal flora improving effect, intestinal regulating action, calcium absorption promoting action, etc., and prevent diarrhea and / or loose stools by releasing an appropriate amount of galactooligosaccharide from food or feed. In addition, these effects can improve the growth rate and / or feed conversion rate of livestock. Further, it can be expected to improve bone density by promoting calcium absorption, prevent and treat osteoporosis, prevent bone fracture, or prevent leg weakness. Examples of target animals include humans, domestic animals (eg, cow, pig, horse, chicken, sheep, goat, etc.), or pet animals (eg, dog, cat, rabbit, etc.). .

The feed additive of the present invention comprises the polypeptide or enzyme composition of the present invention. The feed of the present invention also contains the feed additive of the present invention. By using the polypeptide or enzyme composition of the present invention in animal feed, galactans in the feed are decomposed, or fiber in the feed is produced by synergistic action with various enzymes contained in the enzyme composition. It can be efficiently decomposed and the digestive ability of the fiber in these feeds can be improved. Alternatively, by degrading galactans, or by synergistic action with various enzymes contained in the enzyme composition, the fiber in the feed can be efficiently degraded, and thus the intracellular content of the feed crop can be improved. It is possible to promote utilization of proteins and the like accumulated in the.

The feed additive of the present invention can improve the egg laying rate and / or the eggshell strength of poultry, and further can extend the egg laying period of poultry. That is, by adding the polypeptide or enzyme composition of the present invention to poultry feed, it is possible to improve the spawning rate and / or eggshell strength of poultry, and further to extend the spawning period of poultry. . Examples of the poultry include birds that eat eggs, such as chickens, quails, ducks, and ducks.

In the feed preparation method of the present invention,
The feed additive of the present invention is added. The feed material is not particularly limited as long as it is a feed material containing galactans or galactooligosaccharides, for example, soybean,
Corn, milo, wheat, oat, rye,
Mention may be made of barley or sugar beet.

The enzyme agent for food processing or food additive of the present invention contains the polypeptide or enzyme composition of the present invention. In the food processing method of the present invention, a food material is treated with the enzyme agent for food processing of the present invention. By using the polypeptide or enzyme composition of the present invention for processing foods, for example, increase in yield or aroma of juice or wine, clarification of fruit juice, improvement of color, improvement of fermentation rate in brewing, or, The effect of adjusting the viscosity of juice or jam can be obtained. Food raw materials that can be treated by the food processing method of the present invention, as long as a food raw material containing a polysaccharide or oligosaccharide having a β-1,4-galactoside bond (for example, galactans or galactooligosaccharides, or pectin), Although not particularly limited, for example, soybean,
Mention may be made of potatoes, citrus fruits, apples, sugar beets, grapes or berries.

In the method for producing galactobiose of the present invention,
Galactans or galactooligosaccharides are treated with the polypeptide or enzyme composition of the invention. When the polypeptide of the present invention is allowed to act on galactan derived from soybean fiber or lupine, galactobiose and galactose are mainly accumulated as a reaction product, the weight ratio of which is about 2: 1, and in the latter stage of the reaction. Hardly accumulates oligosaccharides such as galactotriose or galactotetraose.
Furthermore, the galactanase of the present invention exhibits a significantly higher specific activity than the conventionally known endo-type galactanase,
It is possible to efficiently produce galactobiose. From the reaction product obtained by treating a galactan or a galactooligosaccharide with the polypeptide or enzyme composition of the present invention, galactobiose can be purified by, for example, gel filtration chromatography or column chromatography using activated carbon. It can be easily performed by using, for example.

As the method of using the polypeptide or enzyme composition of the present invention, reaction conditions usually used in each application field (eg, pH, temperature, time, amount of enzyme used, etc.) are used.
Can be appropriately determined according to the raw material used, the type of galactanase, and the like.

[0050]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention.

Example 1 << Purification of galactanase enzyme >> (1) Preparation of enzyme bulk powder In order to obtain a galactanase bulk powder derived from an Acremonium microorganism, the microorganism was cultured according to the following procedure.
As the medium, 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 0.001%, and copper sulfate / pentahydrate 0.001% (pH 4.0), which was heat-sterilized by a conventional method.

Acremonium
Cellulolyticus Y-94 strain (FERM BP-582
6) was inoculated and cultured at 30 ° C. for 48 hours with stirring. Next, this culture solution was used as a seed and scaled up to 15 L, and further scaled up, and finally 600
The amount of the culture solution in the L-volume tank was set to 300 L, and aeration-agitation culture was performed for 7 days. The obtained culture broth was filtered with a filter press, concentrated to 15 L by ultrafiltration, 2 kg of lactose was added, and pulverized by spray drying. The bulk galactanase obtained by this method was 5.0 kg.

(2) Purification of galactanase enzyme The bulk powder obtained in Example 1 (1) was mixed with acetate buffer (pH 4.
It was dissolved in 5) and impurities were removed by high speed cooling centrifugation. The obtained supernatant was purified by the method shown below as a starting material for enzyme purification. First, the supernatant was subjected to weak basic anion exchange chromatography (DEAE Sepha).
Rose FF; Pharmacia Co., Ltd.) followed by adsorption with Tris-hydrochloric acid buffer solution (0.01 mol / L, pH 7.5), and then Tris-hydrochloric acid buffer solution (0.01 mol / L, pH).
7.5) each containing 0 mol / L and 0.1 mol of NaCl
/ L, 0.15 mol / L, 0.2 mol / L, 0.5
Stepwise elution was performed with each solution contained at a concentration of mol / L or 0.8 mol / L, and N
The galactanase active fraction eluted when the aCl concentration was 0.5 mol / L was collected. Subsequently, the obtained galactanase active fraction was subjected to a strongly basic ion exchange chromatography [Mono Q (10/10); Pharmacia] to tris-hydrochloric acid buffer solution (0.05 mol / L, p.
H 7.5) and then Tris-HCl buffer (0.
0.05 mol / L, pH 7.5)
/ L to 0.3 mol / L of the solution contained so as to have a concentration of linear gradient elution, and fractions showing galactanase activity were collected. The collected galactanase active fraction was subjected to gel filtration chromatography (S
upperdex 75; made by Pharmacia) 0.1 m
phosphate buffer containing ol / L-NaCl (0.05mo
1 / L, pH 7.0) and the fractions showing galactanase activity were collected to obtain purified galactanase enzyme.

[0053]

Example 2 << Enzymatic and protein properties of galactanase enzyme >> (1) Substrate specificity and action characteristics The purified galactanase enzyme obtained in Example 1 was applied to galactan (produced by Megazyme) derived from lupine. Then, the reaction product was examined over time. As a result, the formation of oligosaccharides having two or more sugars was observed at the early stage of the reaction, and it was revealed that the present enzyme acts in an endo mechanism. In the latter stage of the reaction, galactose and galactobiose were accumulated, but there was a tendency to accumulate more disaccharide galactobiose than monosaccharide galactose. In addition, soybean (arabinogalactan contained in soybean is β-
It is known to consist of 1,4 bonds), and the reaction product was examined over time,
It was confirmed that this was hydrolyzed by the endo mechanism. Furthermore, this enzyme had almost no activity to convert galactobiose into a monosaccharide, and had no effect on larch-derived arabinogalactan (manufactured by Sigma). Arabin-derived arabinogalactan consists of β-1,3 bond,
It was confirmed that it did not affect the -1,3 bond. From the above results, it was concluded that this enzyme is endo-β-1,4-D-galactanase. The specific activity of this enzyme in galactanase activity was about 3000 units / mg protein. The galactanase activity was measured at pH 5.5 and 37.
The enzyme was allowed to act on a 0.5% lupine-derived galactan solution under conditions of 0 ° C., and the amount of the enzyme that produced 1 μmol of the reduced galactan end in 1 minute was defined as 1 unit.

(2) Optimum pH The optimum pH of the galactanase enzyme purified in Example 1 at 37 ° C. is shown in FIG. In Figure 1, the symbol "□"
Is an acetate buffer (0.025 mol / L, pH 3.5-
6) shows the result when using, and the symbol “Δ” shows the result when using the phosphate buffer (0.025 mol / L, pH 6 to 8). This enzyme shows the maximum activity in the range of pH 5 to 6 in acetate buffer or phosphate buffer,
It had high activity in 3.5 to 7.

(3) Optimum temperature To investigate the optimum temperature of the action of the galactanase enzyme purified in Example 1, an acetate buffer solution (0.025 mol / L, p
H5.5), the result of measuring the galactan degrading activity is
As shown in FIG. The optimum temperature was 50 ° C, and high activity was obtained at 40 to 60 ° C. Further, it also had a relatively high activity at 35 to 60 ° C.

(4) Stable pH range Room temperature of the galactanase enzyme purified in Example 1 (22
FIG. 3 shows the pH stability when the treatment was performed at 5 ° C. for 5 hours. In FIG. 3, the symbol “⋄” indicates the result when a glycine-hydrochloric acid buffer solution (0.025 mol / L, pH 1 to 3.5) was used, and the symbol “□” indicates the acetate buffer solution (0.0
25 mol / L, pH 3.5 to 6) is used, and the symbol “Δ” indicates a phosphate buffer solution (0.025 mo).
1 / L, pH 6 to 8) is used, and the symbol "○" indicates Tris-HCl buffer (0.025 mol /
L, pH 8-9) is used, and the symbol "x" indicates CHES buffer (0.025 mol / L, p).
The results when H9 to 10) are used are shown. This enzyme has a pH
It was stable in the range of 2 to 10, but retained a high activity of 60% or more even in the range of pH 1.5 to 2.

(5) Temperature stability The temperature stability of the galactanase enzyme purified in Example 1 when treated with pH 5.5 for 24 hours is shown in FIG. This enzyme was stable at 50 ° C or lower.

(6) Molecular weight In order to determine the molecular weight of the galactanase enzyme purified in Example 1, SDS-polyacrylamide gel electrophoresis (12% gel; manufactured by Tefco) was performed. As a result, the molecular weight of galactanase was about 40,000 (under reducing conditions).
Was calculated. The molecular weight of the standard sample (manufactured by BIO-RAD) used in this test is shown in Table 1.

[0059]

(7) Isoelectric point The isoelectric point (p) of the galactanase enzyme purified in Example 1
Polyacrylamide gel isoelectric focusing was performed to determine I). As a result, the isoelectric point of galactanase was calculated to be about 4.5. Table 2 shows the isoelectric points of standard samples (manufactured by Sigma) used in this test.

[0061]

[0062]

[Example 3] << Isolation of galactanase gene from Acremonium cellulolyticus >> (1) Determination of partial amino acid sequence of purified galactanase Electrophoresis [Sodium Dodecyl]
sulphate-polyacrylamide ge
l electrophoresis (SDS-PAG
E)] did. The protein in the polyacrylamide gel after SDS-PAGE was transferred to a polyvinylidene difluoride membrane (Immob) using a transfer device (Multifour II; manufactured by Pharmacia Biotech) according to the attached protocol.
ilon-P ^SQ ; manufactured by Millipore). The membrane was stained with Coomassie Brilliant Blue R-250 (manufactured by Nacalai Tesque, Inc.), and then the portion where the protein of about 40 kDa was blotted was cut out. The determination of the N-terminal amino acid sequence is performed by using a protein sequencer (Mod
el492; manufactured by Perkin Elmer Co., Ltd.) according to the attached protocol. As a result, it was revealed that the N-terminal amino acid sequence is the amino acid sequence represented by SEQ ID NO: 3.

On the other hand, the internal amino acid sequence was determined as follows. That is, after the galactanase enzyme purified in Example 1 was freeze-dried, 0.1 mol / L
It was dissolved in Tris-HCl buffer (pH 9.0). 1/100 molar amount of lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the galactanase enzyme, and the mixture was incubated at 37 ° C. for 4 hours.
The reaction was carried out for 8 hours. The obtained degradation product was subjected to column chromatography [column = C18, column size = 220 m] using a preparative HPLC system (Model 172μ; manufactured by PE Applied Biosystems).
m × 2.1 mm, gradient = 0.1% trifluoroacetic acid (TFA) and 5% acetonitrile to 0.085%
TFA and 35% acetonitrile] were carried out to separate 3 kinds of peptides. The amino acid sequence of the obtained peptide fragment was determined by the protein sequencer.
As a result, as the internal amino acid sequence, the amino acid sequence represented by SEQ ID NO: 4, the amino acid sequence represented by SEQ ID NO: 5, and the amino acid sequence represented by SEQ ID NO: 6 were revealed.

(2) Isolation of genomic DNA Acremonium cellulolyticus strain Y-94 (FER
MBP-5826) in (S) medium (2% broth,
30% with 0.5% yeast extract and 2% glucose)
The cells were cultured at ℃ for 2 days, and the cells were collected by centrifugation.
The obtained cells were frozen in liquid nitrogen and then ground using a mortar and pestle. Commercially available DNA from the crushed cells
Preparation kit (ISOPLANT; manufactured by Nippon Gene Co., Ltd.)
Genomic DNA was isolated according to the attached protocol.

(3) Isolation of galactanase DNA fragment Based on the partial amino acid sequence of the galactanase enzyme obtained in Example 3 (1), one set of primers was synthesized.
Specifically, 22 of the amino acid sequence represented by SEQ ID NO: 3
A sense primer having the nucleotide sequence represented by SEQ ID NO: 7 was synthesized based on the amino acid sequence of the 28th to 28th amino acids.
Further, based on the amino acid sequence represented by SEQ ID NO: 6, an antisense primer having the base sequence represented by SEQ ID NO: 8 was synthesized. Using these primers, Example 3
Polymerase chain reaction (PCR) was performed using the genomic DNA isolated from Acremonium cellulolyticus strain Y-94 obtained in (2) as a template. PCR is
Using 50 ng of genomic DNA as a template in 50 μL of the reaction solution, 1.25 units of Taq DNA polymerase (E
xTaq DNA polymerase; manufactured by Takara Shuzo Co., Ltd.), attached buffer, dNTP mixture, and 10 μmol / L of the above primers were reacted at 94 ° C. (3 minutes), then at 94 ° C. (1 minute), 42 ° C. (1 Minutes), and 72
A cycle consisting of ° C (45 seconds) was repeated 30 times, and further reaction was carried out at 72 ° C (3 minutes).
This reaction amplifies a DNA fragment of about 500 bp,
This DNA fragment was inserted into pT7 Blue vector (Stratagene).

The nucleotide sequence of the DNA fragment thus cloned is determined by using a commercially available DNA sequencing kit (DNA Sequencing Kit dRho).
damine Terminator Cycle S
equating Ready Reaction;
Perkin Elmer Co., Ltd. and DNA sequencer (AB
I PRISM 310 Genetic Analysis
zer; manufactured by Perkin Elmer Co., Ltd.) according to the attached protocol. As a result, isolated DN
The base sequence of the A fragment is the Aspergillus acuritus galactanase gene (GenBank: L3459).
It showed homology to 9) and was revealed to be a part of the gene encoding the desired galactanase.

(4) Isolation of mRNA and preparation of cDNA library Acremonium cellulolyticus Y-94 strain was treated with cellulase induction medium [4% cellulose, 1% bactopeptone,
0.6% potassium nitrate, 0.2% urea, 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)]
After culturing at 32 ° C. for 4 days, the cells were recovered by centrifugation. The obtained cells were frozen in liquid nitrogen and then ground using a mortar and pestle. Commercial RN from ground cells
Total RNA was isolated using A preparation kit ISOGEN (manufactured by Nippon Gene Co., Ltd.) according to the attached protocol.
Furthermore, from the total RNA, a commercially available mRNA purification kit (mR
NA Purification Kit (manufactured by Pharmacia) according to the attached protocol.
Was purified.

From the thus obtained mRNA, cDNA
Synthesis Kit (Time Saver cDNA Synth
cDNA was synthesized according to the attached protocol according to the esit Kit (Pharmacia). This cdn
A was inserted into a phage vector (Lambda ZAP II; Stratagene). Regarding the recombinant phage vector thus prepared, a packaging kit (Gigapack III Gold Packa) was used.
ging Extract; manufactured by Stratagene, according to the attached manual, in vitro (in
Vitro) packaging was performed. Subsequently, this recombinant phage was infected with Escherichia coli XL1-Blue MRF ′ strain and cultured on a plate to form plaques. The cDNA library prepared in this way is
5.5 × 10 ⁵ pfu (plaque forming
units). Furthermore, this cDNA library was used as the phage vector (Lambda ZAP).
Amplification was performed according to the protocol attached to II). The recombinant phage in the amplified cDNA library was infected with Escherichia coli XL1-Blue MRF ′ strain and cultured on a plate to form plaques.

(5) Isolation of the galactanase gene The membrane (Hy
Bond-N + membrane; Amersham Pharmacia Biotech Co., Ltd.) was placed on the plaque. This membrane was treated with alkali to denature the recombinant phage DNA on the membrane into a single strand and adsorb it on the membrane. The membrane thus produced and Example 3 (3)
A DNA fragment of the galactanase gene amplified by PCR in PCR was used as a probe in a commercially available labeling and detection system (ECL direct nucleic acid
id labeling and detection
n systems; manufactured by Amersham Pharmacia Biotech) according to the attached protocol. For the positive clones selected by the screening, plasmid vector pBluescript SK was used according to the protocol attached to the phage vector (LambdaZAP II).
In-vivo extraction to (-)
The decision) was carried out.

Plasmid vector pBluescript
The nucleotide sequence of the galactanase cDNA in tSK (-) was determined by using a sequencing primer near the multiple cloning site of pBluescript SK (-) or a primer prepared based on the analyzed nucleotide sequence. Kit (DNA S
equating Kit dRhodamine
Terminator Cycle Sequencin
g Ready Reaction (manufactured by Perkin Elmer) and a DNA sequencer (ABI PRISM)
310 Genetic Analyzer (manufactured by Perkin Elmer) was used according to the attached protocol. As a result, a 1199 bp nucleotide sequence of the galactanase cDNA of Acremonium cellulolyticus was obtained. This cDNA contains one open reading frame (ORF) consisting of a 1065 bp base sequence (base sequence represented by SEQ ID NO: 1), and 38
It was revealed to encode a protein of kDa. Regarding the protein predicted from the ORF, B
LAST (Basic local alignment)
When homology search was performed with the tsearch tool), this galactanase showed the highest homology (68%) with the galactanase of Aspergillus acuritus (L34599). Escherichia coli (JM109) transformed with the plasmid vector pgal containing the thus obtained galactanase cDNA
Was deposited at the Patent Biological Depository Center of the National Institute of Advanced Industrial Science and Technology as of March 5, 2001, and the deposit number is FERM P-18242.

[0071]

Example 4 << Overexpression of the galactanase gene derived from Acremonium cellulolyticus >> (1) Preparation of recombinant vector for expressing galactanase gene The outline of the steps of this example is shown in FIG. Using the plasmid pgal having the galactanase cDNA inserted obtained in Example 3 as a template, a primer having the nucleotide sequence represented by SEQ ID NO: 9 and a primer having the nucleotide sequence represented by SEQ ID NO: 10 were prepared. Was used to perform PCR.
PCR was performed using 1.25 units Ta in 50 μL of reaction solution.
q DNA polymerase (LA Taq DNA polymerase; manufactured by Takara Shuzo), attached buffer, dNTP mixture, 20 ng plasmid DNA, and 1 μmol / L
After reacting at 94 ° C (3 minutes) using the above-mentioned primer, the cycle consisting of 94 ° C (30 seconds), 52 ° C (30 seconds), and 72 ° C (75 seconds) was repeated 30 times, and further 72 It was carried out by reacting at 0 ° C (3 minutes). The PCR reaction solution thus obtained was ethanol-precipitated, the PCR product was recovered, and then the restriction enzymes Hpa I and S were added.
cleaved with ph I, which contains the plasmid pCBHEX / DtR2 containing the Destomycin resistance cassette (DtR)
(Patent Document 1: JP 2001-17180 A) and Hpa I site
Inserted between Sph I site and plasmid pGAL-D
t was produced.

Plasmid pUC118 (Takara Shuzo)
The Bam HI site was cleaved, the cleaved site was blunted, and then ligated again to prepare a plasmid pUC118 lacking the Bam HI site. At the Xba I site of this plasmid, the plasmid pDHBAR (Watanabe,
M. Et al., Appl. Environ. Microbio
l. , 65, 1036-1044, 1999), and the bialaphos-resistant cassette excised with the restriction enzyme Xba I was inserted. This plasmid was digested with restriction enzyme Bam HI, the cleavage site religated after smoothing, Bam H
A plasmid was constructed containing the bialaphos resistance cassette with the I site deleted. From this plasmid, the restriction enzyme Xba
A bialaphos-resistant cassette was cut out by I and
A plasmid pGAL-bar for protein expression was prepared by replacing the previously prepared plasmid pGAL-Dt with the destamin resistance cassette at the Xba I site.

(2) Introduction of galactanase gene into host Acremonium cellulolyticus Y-94 strain (S)
Incubate for 16 hours at 30 ℃ in medium, 3500 rpm
The cells were collected by centrifugation at 10 minutes for 10 minutes. The obtained cells were washed with 0.5 mol / L sucrose to give 0.45
It was suspended in a protoplast-forming enzyme solution (10 mg / mL chitinase, 10 mg / mL zymolyase, 30 mg / mL β-glucuronidase, and 0.5 mol / L sucrose) filtered with a μm filter. 30 ° C
The mixture was shaken for 60 to 90 minutes at 37 ° C. for protoplast formation of mycelium. After filtering this suspension through absorbent cotton, 2500
Collect the protoplasts by centrifugation at rpm for 10 minutes,
UTC buffer [0.5 mol / L sucrose,
10 mmol / L calcium chloride, and 10 mmol / L
L tris-hydrochloric acid (pH 7.5)].

The protoplasts prepared as described above were suspended in 100 μL of SUTC buffer, 10 μg (10 μL) of the plasmid pGAL-bar solution prepared in Example 4 (1) was added thereto, and the mixture was allowed to stand on ice for 5 minutes. . Next, a PEG (polyethylene glycol) solution [6
0% PEG4000, 10 mmol / L calcium chloride, and 10 mmol / L tris-hydrochloric acid (pH 7.
5)] 400 μL was added, and the mixture was allowed to stand in ice for 20 minutes,
10 mL of SUTC buffer was added, and the mixture was centrifuged at 2500 rpm for 10 minutes. Centrifuge the protoplasts with SU
After suspending in 1 mL of TC buffer, centrifuge at 4000 rpm for 5 minutes, and finally 100 μC of SUTC buffer.
Suspended in L. The protoplasts treated as described above were placed on (A) agar medium [3.9% potato dextrose agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and 17.1% sucrose] on which (A) agar was added (A). ) After overlaying with soft agar medium [1.3% potato dextrose agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and 17.1% sucrose] and culturing at 30 ° C. for 5 to 9 days, the formed colonies were transformed. I made it a body.

(3) Expression of galactanase in Acremonium cellulolyticus transformants and measurement of enzyme activity The top 4 strains with high resistance to bialaphos were cultured in a cellulase induction medium. When the culture supernatant was analyzed by SDS-PAGE, the amount of galactanase enzyme secreted was higher than that of the parent strain, as shown in FIG. The galactanase activity was measured by the method described in Example 2 (1) and expressed as the activity (U / mL) per mL of the culture supernatant. The results are shown in Table 3. The transformant showed about 27 times the specific activity of the parent strain.

The "transformant 3" strain having the highest specific activity was cultured in the same manner as in Example 1 (1), and the enzyme bulk powder of the "transformant 3" strain (hereinafter referred to as galactanase-enhancing raw material) was cultured. Powder) was prepared.

[0077]

[Example 5] << Action of galactanase enzyme on chicken feed in vitro >> In order to imitate the action of galactanase in the digestive tract of chicken, the bulk galactanase prepared in Example 1 (1) was tested. (Hereinafter referred to as parent strain bulk powder), purified galactanase enzyme prepared in Example 1 (2) or galactanase-enhanced bulk powder prepared in Example 4 (3), soybean fiber, chicken feed The effect on fibrous and lupine-derived galactan was investigated. Soybean fiber and chicken feed fiber, soybean meal (okara) and chicken feed (whole planting type broiler late feed; manufactured by ITOCHU Feed Co., Ltd.)
Each was suspended in 8 mol / L urea solution, boiled for 5 minutes, washed with water and ethanol, and dried, and used as a substrate. The reaction solution is 20 mg / mL substrate, 25
μg / mL enzyme bulk powder (parent strain bulk powder or galactanase-enhanced bulk powder) or 0.5 μg / mL galactanase enzyme, and 25 mmol / L acetate buffer (pH 4.5),
After reacting at 37 ° C. for 1 hour, the reaction was stopped by boiling for 5 minutes. The reaction product was analyzed by TLC (thin layer chromatography).

The results are shown in FIG. In FIG. 7, test plots 1 to 3 are the results when soybean fiber was used as the substrate, and similarly, test plots 4 to 6 were chicken feed fiber as the substrate, and test plots 7 to 9 were the same. , Galactan as substrate,
These are the results when used respectively. In addition, test section 1,
4 and 7 are the results when the parent strain bulk powder was used as the enzyme, and similarly, test sections 2, 5, and 8 were the galactanase-enhanced bulk powder as the enzyme, test sections 3, 6, and 9
Shows the results when the galactanase enzyme was used as the enzyme. As shown in FIG. 7, galactobiose and galactose were produced from both substrates by the galactanase-enhanced bulk powder and the galactanase enzyme,
In the chicken digestive tract model, it was confirmed that galactobiose was produced from chicken feed by the action of the enzyme.

[0079]

Example 6 << Production of galactobiose by galactanase enzyme >> (1) Analysis of sugar composition of soybean fiber The sugar composition of soybean fiber used as a substrate for the production of galactobiose by galactanase enzyme was investigated. Soybean fiber was suspended in 12 mol / L or 9 mol / L sulfuric acid, and then diluted with pure water so that the sulfuric acid concentration became 0.36 mol / L. After replacing the inside of the reaction vessel with nitrogen, degassing,
Hydrolysis was performed by reacting at 121 ° C. for 30 minutes. After completion of the reaction, neutralized with barium carbonate and centrifuged,
The supernatant was subjected to HPLC (high performance liquid chromatography). The analysis results are shown in Table 4. In Table 4, the symbol “Glc” is glucose and the symbol “Gal” is
Galactose, the symbol "Xyl" is xylose, the symbol "Ara" is arabinose, and the symbol "UA" is uronic acid.

<< Table 4 >> Constituent sugar Glc Gal Xyl Ara UA % 11.9 51.7 6.2 25.7 4.5

(2) Production of galactobiose Using the galactanase enzyme purified in Example 1 (2) or the galactanase-enhanced bulk powder prepared in Example 4 (3), soybean fiber, or The galactan derived from lupine was allowed to act on each. The reaction was carried out in the same manner as in Example 5, and the reaction solution after the reaction was stopped was ultrafree-15 centrifugal filter unit Biomax-5.
After the protein was removed by applying it to a membrane-attached unit (Millipore), the reaction product was analyzed by HPLC. Table 5 shows yields of reaction products based on soybean fiber and galactan derived from lupine. In addition, Example 6
The yield (% by weight) based on the galactose residue in the soybean fiber examined in (1) is shown in parentheses. In Table 5,
The symbol "Glc2" is cellobiose and the symbol "Gl2"
“C” is glucose, the symbol “Glc2” is galactobiose, and the symbol “Gal” is galactose. << Table 5 >> Yield (% by weight) Substrate enzyme Glc2 Glc Gal2 Gal daisy fiber Galactanase enhanced bulk powder 0.6 0.5 27.0 (52.2) 16.6 (32.1) Galactanase enzyme − − 25.7 (49.7) 12.1 (23.4) Galactan galactanase Enhanced bulk powder − − 22.1 10.2 Galactanase enzyme − − 22.6 8.3

As shown in Table 5, the sugars released from soybean fiber or lupine-derived galactan by the action of the galactanase enzyme or the galactanase-enhancing bulk powder are mainly galactobiose and galactose, and their weight ratio is Was about 2: 1. Moreover, it was revealed that most of the galactose residues in soybean fiber were released as galactobiose. As a result of this analysis, monosaccharides other than glucose and galactose were not found. The reaction solution containing galactobiose and galactose thus obtained was purified by gel filtration chromatography (TSKgel Toyopearl HW40-S; manufactured by Tosoh Corporation). The purified galactobiose is T
It was confirmed to be single by LC.

[0083]

INDUSTRIAL APPLICABILITY The polypeptide of the present invention not only has a high specific activity but is stable even at low pH, and
It is an enzyme that produces galactobiose in high concentration. Therefore, according to the polypeptide or enzyme composition of the present invention, digestion promotion of a plant material containing a polysaccharide or oligosaccharide having a β-1,4-galactoside bond such as a galactan or a galactooligosaccharide, and its processing or utilization efficiency It is expected to be used in various fields such as food field, feed field, or medical field. Furthermore, according to the polypeptide or enzyme composition of the present invention, galactobiose can be produced at a high concentration.

[0084]

[Sequence listing free text] Numerical headings in the sequence listing
223>, "Artificial Sequence"
"ce" is described. Specifically, each base sequence represented by the sequences of SEQ ID NOs: 7 to 10 is a synthetic DNA.
It is an array.

[0085]

[Sequence list] <110> MEIJI SEIKA KAISHA, LTD. <110> NATIONAL INSTITUTE OF ADVANCED INDUSTRIAL SCIENCE AND TECHNOLOGY <120> Novel galactanase <130> MEJ020627P <150> JP 2001-275005 <151> 2001-09-11 <160> 10 <170> PatentIn Ver. 2.1 <210> 1 <211> 1065 <212> DNA <213> Acremonium cellulolyticus <220> <221> CDS <222> (1) .. (1065) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> mat_peptide <222> (61) .. (1062) <400> 1 atg agg gta ttc aac atc tta ctc tcg tct tta gca acc ttc acc acc 48 Met Arg Val Phe Asn Ile Leu Leu Ser Ser Leu Ala Thr Phe Thr Thr -20 -15 -10 -5 ctc acc tcc gca agc ctt caa tac cgt ggc gca gac ata tcc tcc ttg 96 Leu Thr Ser Ala Ser Leu Gln Tyr Arg Gly Ala Asp Ile Ser Ser Leu              -1 1 5 10 ctg gtc gaa gaa ggc gac gga ata tcc tac aag aat acg aat ggt gcc 144 Leu Val Glu Glu Gly Asp Gly Ile Ser Tyr Lys Asn Thr Asn Gly Ala          15 20 25 agc gca aaa ctc gag gcc att ctc gcc gct aac ggt gtg aat act atc 192 Ser Ala Lys Leu Glu Ala Ile Leu Ala Ala Asn Gly Val Asn Thr Ile      30 35 40 cga caa cga cta tgg gtt aat ccc agc gat ggc gtg tat ggt tta gat 240 Arg Gln Arg Leu Trp Val Asn Pro Ser Asp Gly Val Tyr Gly Leu Asp  45 50 55 60 tat aac ctg gaa ctc gca gcg agg atg gtg gat gca gga ttg aag att 288 Tyr Asn Leu Glu Leu Ala Ala Arg Met Val Asp Ala Gly Leu Lys Ile                  65 70 75 tat ctg gat atg cat ttt agt gat acg tgg gca gat ccg agt cac cag 336 Tyr Leu Asp Met His Phe Ser Asp Thr Trp Ala Asp Pro Ser His Gln              80 85 90 aca act cca tcc ggt tgg tca aca aca gac gtc ggt aca tta gca tgg 384 Thr Thr Pro Ser Gly Trp Ser Thr Thr Asp Val Gly Thr Leu Ala Trp          95 100 105 cag ctt tac aac tac aca atg gac gtc tgc aac agt ttc gct gag aaa 432 Gln Leu Tyr Asn Tyr Thr Met Asp Val Cys Asn Ser Phe Ala Glu Lys     110 115 120 aac atc cct ctc gaa atc gtg tct att ggc aac gaa att cgc gcc ggt 480 Asn Ile Pro Leu Glu Ile Val Ser Ile Gly Asn Glu Ile Arg Ala Gly 125 130 135 140 cta ctg tgg ccg ctc ggc tca acc agc agc tat tac aac atc gct aca 528 Leu Leu Trp Pro Leu Gly Ser Thr Ser Ser Tyr Tyr Asn Ile Ala Thr                 145 150 155 ctt ctc cac tcc gct gca tgg ggc ntc aag aat cca tat gac tta acg 576 Leu Leu His Ser Ala Ala Trp Gly Xaa Lys Asn Pro Tyr Asp Leu Thr             160 165 170 aag cct aag att atg att cat ctc gat aat ggc tgg tca tgg agt gaa 624 Lys Pro Lys Ile Met Ile His Leu Asp Asn Gly Trp Ser Trp Ser Glu         175 180 185 cag tca tac ttt tat gat acg gtg ttg gca gag ggt cct ttg ctc acg 672 Gln Ser Tyr Phe Tyr Asp Thr Val Leu Ala Glu Gly Pro Leu Leu Thr     190 195 200 acg gac ttc gat gtg atg ggt gtc agc tat tat ccg ttc tac agt agt 720 Thr Asp Phe Asp Val Met Gly Val Ser Tyr Tyr Pro Phe Tyr Ser Ser 205 210 215 220 tct gct aca ttg gcg tcc ctc aaa tcg aca ttg gct gaa atg gcg tct 768 Ser Ala Thr Leu Ala Ser Leu Lys Ser Thr Leu Ala Glu Met Ala Ser                 225 230 235 aca tgg ggc aaa ggg ttg att gtt gca gag aca aat tgg cca tat gct 816 Thr Trp Gly Lys Gly Leu Ile Val Ala Glu Thr Asn Trp Pro Tyr Ala             240 245 250 tgt ccc aat ccg gca tac tct ttc cca tct gat ctg aaa agt att ccg 864 Cys Pro Asn Pro Ala Tyr Ser Phe Pro Ser Asp Leu Lys Ser Ile Pro         255 260 265 ttc tcg gct gct ggt cag acg aca ttc tta aaa gat gtt gcg agc gtt 912 Phe Ser Ala Ala Gly Gln Thr Thr Phe Leu Lys Asp Val Ala Ser Val     270 275 280 gtg gcg ggg acg aaa aac ggg ttg gga atg ttc tat tgg gag cca gcg 960 Val Ala Gly Thr Lys Asn Gly Leu Gly Met Phe Tyr Trp Glu Pro Ala 285 290 295 300 tgg att ggg aat gcg gcg ctt ggt tct agt tgt agt gat aat ctg ttg 1008 Trp Ile Gly Asn Ala Ala Leu Gly Ser Ser Cys Ser Asp Asn Leu Leu                 305 310 315 gtc gat tct tat aca gat gtt ttt aga aca agt gtg gag gta ttt tcg 1056 Val Asp Ser Tyr Thr Asp Val Phe Arg Thr Ser Val Glu Val Phe Ser             320 325 330 agt att tga 1065 Ser Ile         335 <210> 2 <211> 354 <212> PRT <213> Acremonium cellulolyticus <400> 2 Met Arg Val Phe Asn Ile Leu Leu Ser Ser Leu Ala Thr Phe Thr Thr   1 5 10 15 Leu Thr Ser Ala Ser Leu Gln Tyr Arg Gly Ala Asp Ile Ser Ser Leu              20 25 30 Leu Val Glu Glu Gly Asp Gly Ile Ser Tyr Lys Asn Thr Asn Gly Ala          35 40 45 Ser Ala Lys Leu Glu Ala Ile Leu Ala Ala Asn Gly Val Asn Thr Ile      50 55 60 Arg Gln Arg Leu Trp Val Asn Pro Ser Asp Gly Val Tyr Gly Leu Asp  65 70 75 80 Tyr Asn Leu Glu Leu Ala Ala Arg Met Val Asp Ala Gly Leu Lys Ile                  85 90 95 Tyr Leu Asp Met His Phe Ser Asp Thr Trp Ala Asp Pro Ser His Gln             100 105 110 Thr Thr Pro Ser Gly Trp Ser Thr Thr Asp Val Gly Thr Leu Ala Trp         115 120 125 Gln Leu Tyr Asn Tyr Thr Met Asp Val Cys Asn Ser Phe Ala Glu Lys     130 135 140 Asn Ile Pro Leu Glu Ile Val Ser Ile Gly Asn Glu Ile Arg Ala Gly 145 150 155 160 Leu Leu Trp Pro Leu Gly Ser Thr Ser Ser Tyr Tyr Asn Ile Ala Thr                 165 170 175 Leu Leu His Ser Ala Ala Trp Gly Xaa Lys Asn Pro Tyr Asp Leu Thr             180 185 190 Lys Pro Lys Ile Met Ile His Leu Asp Asn Gly Trp Ser Trp Ser Glu         195 200 205 Gln Ser Tyr Phe Tyr Asp Thr Val Leu Ala Glu Gly Pro Leu Leu Thr     210 215 220 Thr Asp Phe Asp Val Met Gly Val Ser Tyr Tyr Pro Phe Tyr Ser Ser 225 230 235 240 Ser Ala Thr Leu Ala Ser Leu Lys Ser Thr Leu Ala Glu Met Ala Ser                 245 250 255 Thr Trp Gly Lys Gly Leu Ile Val Ala Glu Thr Asn Trp Pro Tyr Ala             260 265 270 Cys Pro Asn Pro Ala Tyr Ser Phe Pro Ser Asp Leu Lys Ser Ile Pro         275 280 285 Phe Ser Ala Ala Gly Gln Thr Thr Phe Leu Lys Asp Val Ala Ser Val     290 295 300 Val Ala Gly Thr Lys Asn Gly Leu Gly Met Phe Tyr Trp Glu Pro Ala 305 310 315 320 Trp Ile Gly Asn Ala Ala Leu Gly Ser Ser Cys Ser Asp Asn Leu Leu                 325 330 335 Val Asp Ser Tyr Thr Asp Val Phe Arg Thr Ser Val Glu Val Phe Ser             340 345 350 Ser Ile <210> 3 <211> 29 <212> PRT <213> Acremonium cellulolyticus <400> 3 Ser Leu Gln Tyr Arg Gly Ala Asp Ile Ser Ser Leu Leu Val Glu Glu   1 5 10 15 Gly Asp Gly Ile Ser Tyr Lys Asn Thr Asn Gly Ala Ser              20 25 <210> 4 <211> 5 <212> PRT <213> Acremonium cellulolyticus <400> 4 Ser Ile Pro Phe Leu   1 5 <210> 5 <211> 11 <212> PRT <213> Acremonium cellulolyticus <400> 5 Gly Thr Leu Ala Glu Met Ser Thr Trp Gly Lys   1 5 10 <210> 6 <211> 6 <212> PRT <213> Acremonium cellulolyticus <400> 6 Ala Ala Trp Gly Phe Lys   1 5 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 tayaaraaya cnaayggngc 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 yttraanccc cangcngc 18 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 ggggttaaca caatgagggt attcaacatc ttac 34 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 10 ggggcatgct caaatactcg aaaatacctc c 31

[Brief description of drawings]

FIG. 1 shows the optimum p of the polypeptide of the present invention at 37 ° C.
It is a graph which shows H.

FIG. 2 is a graph showing the optimum temperature of the polypeptide of the present invention at pH 5.5.

FIG. 3 is a graph showing pH stability of the polypeptide of the present invention after treatment at 22 ° C. for 5 hours.

FIG. 4 is a graph showing the temperature stability of the polypeptide of the present invention after treatment at pH 5.5 for 24 hours.

FIG. 5 is an explanatory view showing a method for producing the plasmid pGAL-bar of the present invention.

FIG. 6 is a photograph as a substitute for a drawing, which shows the result of electrophoresis showing the expression of galactanase in Acremonium cellulolyticus transformants.

FIG. 7 is a photograph as a substitute for a drawing, which shows the result of a thin-layer chromatograph showing the reaction product from various substrates by the polypeptide enzyme or galactanase-enhanced bulk powder of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12N 9/00 4B065 9/00 9/38 9/38 C12P 19/12 C12P 19/12 C12R 1: 645 // (C12N 9/38 1: 685 C12R 1: 645) C12R 1:69 (C12N 9/38 1: 885 C12R 1: 685) C12R 1:77 (C12N 9/38 C12N 15/00 ZNAA C12R 1:69) (C12N 9/38 C12R 1: 885) (C12N 9/38 C12R 1:77) (72) Inventor Asayuki Fukasawa 5-3-1 Chiyoda, Sakado City, Saitama Meiji Seika Co., Ltd. In-house (72) Inventor Kaoru Okakura 788 Meiji Confectionery Co., Ltd., Kajiyama, Odawara, Kanagawa Prefecture In-house Microbial Resource Research Institute (72) Inventor Naoki Mido 788 Meiji Seika Co., Ltd., Ojiwara, Kanagawa Resources Research Institute (72) Inventor, Rin Yamabe 1-1-1 East, Tsukuba, Ibaraki Prefecture DD21 DF13 DF14 4B024 AA03 AA05 AA10 BA12 CA04 DA11 EA04 GA11 GA21 HA01 4B050 CC03 DD03 EE02 FF11E FF12E JJ01 KK08 LL02 LL10 4B064 AF03 CA05 CA19 CC24 CD24 CE10 CE11 DA10 DA11 4B065 A63 A02 AA58AA AA58AA A58X BD18 CA20 CA31 CA41 CA43

Claims (37)

[Claims] 1. A polypeptide having the following physicochemical properties and having a galactanase activity. (A) Action and substrate specificity: The β-1,4-galactoside bond of a polysaccharide or oligosaccharide having a β-1,4-galactoside bond is hydrolyzed to an endo type. (B) Stable pH range: Stable in the range of pH 2-10. 2. The polypeptide according to claim 1, which further has the following physicochemical properties. (C) Molecular weight: 40,000 (by SDS-polyacrylamide gel electrophoresis) (d) Isoelectric point: 4.5 (by isoelectric point polyacrylamide gel electrophoresis) (e) Optimum pH: 5-6 is there. (F) Optimum action temperature: 50 ° C. 3. The polypeptide according to claim 1, which is derived from a filamentous fungus. 4. The polypeptide according to claim 3, wherein the filamentous fungus is a microorganism belonging to the genus Acremonium. 5. The polypeptide according to claim 4, wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus. 6. A polypeptide comprising (a) the 21st to 354th amino acid sequences of the amino acid sequence represented by SEQ ID NO: 2; (b) the 21st to 21st amino acid sequences represented by SEQ ID NO: 2 to
A polypeptide containing the 354th amino acid sequence and having galactanase activity; or (c) the 21st amino acid sequence of the amino acid sequence represented by SEQ ID NO: 2 to
A polypeptide containing an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted at the 354th amino acid sequence at one or more positions, and which has galactanase activity. 7. The polypeptide according to claim 6, which is derived from a filamentous fungus. 8. The polypeptide according to claim 7, wherein the filamentous fungus is a microorganism belonging to the genus Acremonium. 9. The polypeptide according to claim 8, wherein the microorganism belonging to the genus Acremonium is Acremonium cellulolyticus. 10. A polynucleotide comprising (a) the 61st to 1065th base sequences of the base sequence represented by SEQ ID NO: 1; (b) the 21st to the 21st amino acid sequence represented by SEQ ID NO: 2 to
A polynucleotide encoding a polypeptide consisting of the 354th amino acid sequence; (c) the 21st amino acid sequence represented by SEQ ID NO: 2 to
A polynucleotide containing a 354th amino acid sequence and encoding a polypeptide having galactanase activity; (d) 21st to 21st amino acid sequences represented by SEQ ID NO: 2
A polynucleotide containing an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted at the 354th amino acid sequence at one or more positions, and which encodes a polypeptide having galactanase activity Or (e) 61st to 10th of the base sequence represented by SEQ ID NO: 1
A polynucleotide which hybridizes with the DNA consisting of the 65th nucleotide sequence under stringent conditions and which encodes a protein having a galactanase activity. 11. The polynucleotide according to claim 10, which is derived from a filamentous fungus. 12. The polynucleotide according to claim 11, wherein the filamentous fungus is a microorganism belonging to the genus Acremonium. 13. A microorganism belonging to the genus Acremonium,
13. An Acremonium cellulolyticus.
The polynucleotide according to. 14. A polynucleotide construct comprising the polynucleotide of any one of claims 10-13. 15. An expression vector comprising the polynucleotide according to any one of claims 10 to 13 or the polynucleotide construct according to claim 14. 16. A transformed cell transformed with the polynucleotide construct according to claim 14 or the expression vector according to claim 15. 17. The transformed cell according to claim 16, wherein the host is Escherichia coli, yeast, actinomycete, or filamentous fungus. 18. The yeast is a microorganism belonging to the genus Saccharomyces, the genus Hansenula, or the genus Pichia.
The transformed cell according to. 19. The transformed cell according to claim 18, wherein the yeast is Saccharomyces cerevisiae. 20. The transformed cell according to claim 17, wherein the filamentous fungus is a microorganism belonging to the genus Acremonium, the genus Humicola, the genus Aspergillus, the genus Trichoderma, or the genus Fusarium. 21. The filamentous fungus according to claim 20, which is Acremonium cellulolyticus, Humicola insolens, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, Trichoderma reesei, or Fusarium oxysporus. Transformed cells. 22. The step of culturing the transformed cell according to any one of claims 16 to 21; and the transformed cell and / or culture solution obtained in the culturing step,
10. A method for producing the polypeptide, comprising a step of collecting the polypeptide according to any one of items 1 to 9. 23. A polypeptide produced by the method of claim 22. 24. An enzyme composition comprising the polypeptide according to any one of claims 1 to 9 or 23. 25. Cellulase, xylanase, protease, galactosidase, arabinase, arabinofuranosidase, mannanase, rhamnogalacturonase,
Polygalacturonase, pectin methyl esterase,
The enzyme composition according to claim 24, further comprising at least one of pectin lyase or polygalacturonic acid lyase. 26. A method according to claim 24 or 2 for treating plant material.
The enzyme composition according to 5. 27. A plant material is treated with the polypeptide according to any one of claims 1 to 9 or 23, or the enzyme composition according to claim 24 or 25, A method for improving processing efficiency and / or utilization efficiency of plant material. 28. The method according to claim 24 or 2 for administration to an animal.
The enzyme composition according to 5. 29. The polypeptide according to any one of claims 1 to 9 or 23, or 24 or 25.
A method of releasing galactooligosaccharide in the digestive organs of an animal, which comprises directly ingesting the enzyme composition according to 1. with galactans. 30. A feed additive comprising the polypeptide according to any one of claims 1 to 9 or 23 or the enzyme composition according to any one of claims 24 to 26 or 28. 31. The feed additive according to claim 30, for poultry. 32. The feed for poultry according to any one of claims 1 to 9 or 23.
Or a polypeptide according to any one of claims 24 to 26 or 28, wherein the enzyme composition according to any one of claims 24 to 26 or 28 is added. And / or a method for extending the spawning period. 33. A feed containing the feed additive according to claim 30 or 31. 34. The method for preparing a feed according to claim 33, wherein the feed additive according to claim 30 or 31 is added to the feed raw material. 35. A food processing product comprising the polypeptide according to any one of claims 1 to 9 or 23, or the enzyme composition according to any one of claims 24 to 26 or 28. / Or an enzyme agent for food addition. 36. A method of processing food, which uses the enzyme agent for food processing according to claim 35. 37. Use of the polypeptide according to any one of claims 1 to 9 or 23 or the enzyme composition according to any one of claims 24 to 26 or 28 for galactans or A method for producing galactobiose, which comprises decomposing galactooligosaccharide.