JP2006087404A - Alkali-resistant mannanase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new alkaline mannanase stable in an alkaline pH range and having an optimum pH in an alkaline pH region, to provide a gene of the same, to provide a method for producing the enzyme, and to provide use of the alkaline mannanase in various fields. <P>SOLUTION: This alkaline mannanase has physicochemical properties (a) to (h) as follows: (a) action: having endo-type mannanase activity so as to act on mannan or a mannooligosaccharide of a tetrasaccharide or higher and hydrolyze them into an oligosaccharide of a trisaccharide or lower; (b) the optimum pH: in a range of pH 9 to 10.5; (c) pH stability: having 70% or more of the mannanase activity under a processing condition of a temperature of 40°C, a pH of 6.5 to 10, and a time of 30 min; (d) an optimum temperature: having the optimum temperature in a range of about 55°C; (e) temperature stability: having 50% or more of the activity under a processing condition of a pH of 7.5, a temperature of 50°C, and a time of 110 min or a processing condition of a pH of 7.5, a temperature of 60°C, and a time of 60 min; (f) deactivation: being deactivated in the mannanase activity, when processed at a pH 7.5 and 40°C for 30 min in the presence of 1 mM N-bromosuccinimide; (g) inhibition by metal: being inhibited by Fe<SP>+3</SP>, Fe<SP>+2</SP>, Pb<SP>+2</SP>, Zn<SP>+2</SP>, Hg<SP>+2</SP>, Cd<SP>+2</SP>, and Sn<SP>+2</SP>; and (h) a molecular weight by SDS-PSGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis): being about 130 kDa. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a mannanase derived from a microorganism, more specifically, a novel alkaline mannanase which is stable in an alkaline pH range and has an optimum pH in the alkaline pH region, and a gene thereof. The present invention further relates to a method for producing the alkaline mannanase, and a vector and a transformant used for the production. Furthermore, the present invention relates to various uses of the alkaline mannanase.

  β-1,4-mannanase (EC.3.2.1.78, simply referred to herein as “mannanase”) is a homo- and hetero-β-D having β-1,4-D-mannopyranoside bonds in the molecule. -An enzyme that hydrolyzes β-1,4-D-mannopyranoside bonds, which are the main skeletons of mannan, galactomannan and glucomannan, to produce a series of manno-oligosaccharides. The various mannans that are decomposed by mannanase are components of hemicellulose, which is one of the main components of plant cell walls. Therefore, mannanase is a useful enzyme that is widely used in fields related to food, papermaking, fiber, and washing.

For this reason, mannase has been studied by many researchers, and has been isolated and purified from many microorganisms. For example, mannanases derived from filamentous fungi [Non-Patent Documents 1 to 3], mannanases derived from actinomycetes [Non-Patent Document 4 etc.], and mannanases derived from bacteria belonging to the genus Bacillus [(1) Non-Patent Document 5 (2) Non-patent document 6, (3) Patent document 1, (4) Patent document 2, (5) Patent document 3, (6) Patent document 4, and the like] are well studied. Here, regarding mannanases derived from bacteria belonging to the genus Bacillus , the above (1) is a mannanase having a molecular weight of 162 kDa and an optimum pH of 5.5 to 7.5 derived from Bacillus stearothermophilus ; (2) is derived from Bacillus subtilis A mannanase with a molecular weight of 38 kDa, an optimum pH of 5.5, an optimum temperature of 55 ° C. and an isoelectric point (p I ) of 4.8; (3) has a molecular weight of 37 ± 3 kDa derived from Bacillus sp., An optimum pH of 3-8, and p I mannanase 5.3-5.4 is;. in (4) alkalophilic Bacillus sp molecular weight 43 kDa derived from and 57 ± 3 kDa, and optimum mannanase pH 8-10 is ;; (5) the Bacillus sp. Mannanase having a molecular weight of 34 ± 10 kDa and an optimum pH of 7.5-10 derived from the above; mannanase derived from Bacillus agaradhaerens is described in (6), respectively.
Advances in Carbohydrate Chemistry and Biochemistry, 1976, 32, 299-316 Acta. Chem. Scand., 1968, 22, 1924 Nikko Kasei, 1969, 43, 317; Biochem. J., 1984, 219, 857 Agric. Biol. Chem., 1984, 48, 2189 Appl. Environ. Microbiol., 56, 11, 3505-3510 (1990) World J. Microbiol. Biotechnol., 10, 551-555 (1994); Japanese Patent Application No. 03-047076 Japanese Patent Application No. 08-51975 U.S. Patent No. 6,566,1143 U.S. Pat.No. 6,440,911

  An object of the present invention is to provide a novel alkaline mannanase having excellent stability and high mannanase activity in an alkaline pH range, and a gene thereof. Another object of the present invention is to provide a method for efficiently producing the alkaline mannanase, and an expression vector and a transformant used for producing the alkaline mannanase. Furthermore, an object of the present invention is to provide various uses of the alkaline mannanase.

In order to obtain a microorganism having the ability to produce an alkaline mannanase having the above characteristics, the present inventors have extensively searched the natural world, and as a result, certain new microorganisms belonging to the genus Bacillus have obtained a novel alkaline mannanase having the above characteristics. Furthermore, the present inventors have succeeded in developing a method for efficiently producing an alkaline mannanase produced by the microorganism by a genetic engineering technique, and completed the present invention.

That is, the present invention includes the following embodiments:
Item 1. Alkaline mannanase having the following physicochemical properties:
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺
(H) Molecular weight by SDS-PAGE: about 130 kDa.
Item 2. Alkaline mannanase comprising the protein described in the following (a) or (b):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein (a) action having an amino acid sequence in which one or a plurality of amino acid sequences is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having at least one physicochemical property listed below: : It has an endo-type mannanase activity that acts on manno-oligosaccharides having 4 or more sugars and hydrolyzing them to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ .
Item 3. The alkaline mannanase according to claim 2, wherein the protein shown in (b) has an amino acid sequence which is 40% or more identical to the amino acid sequence shown in SEQ ID NO: 1.
Item 4. Alkaline mannanase comprising a protein encoded by the DNA described in (c) or (d) below:
(C) DNA consisting of the base sequence shown in SEQ ID NO: 2,
(D) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 and encodes a protein having at least one of the following physicochemical properties:
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ .
Item 5. Item 5. The alkaline mannanase according to any one of Items 1 to 4, which is an enzyme derived from a microorganism belonging to the genus Bacillus of an alkalophilic mesophilic bacterium.
Item 6. Item 6. The alkaline mannanase according to Item 5, wherein the microorganism belonging to the genus Bacillus of an alkalophilic mesophilic bacterium is “Bacillus sp. Strain JAMB-750” (reception number FERM AP-20227).

Item 7. Alkaline mannanase gene encoding the protein described in the following (a) or (b):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein having an amino acid sequence obtained by deleting, substituting or adding one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1 and having at least one enzymatic property listed below (A) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze them to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ .
Item 8. Item 8. The alkaline mannanase gene according to Item 7, wherein the protein shown in (b) has an amino acid sequence that is 40% or more identical to the amino acid sequence shown in SEQ ID NO: 1.
Item 9. Alkaline mannanase gene comprising the DNA described in (c) or (d) below:
(C) DNA having the base sequence shown in SEQ ID NO: 2,
(D) Hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 2, and encodes a protein having at least one physicochemical property listed below or a precursor thereof. DNA
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ .
Item 10. Item 10. A recombinant vector containing the alkaline mannanase gene according to any one of Items 7 to 9.
Item 11. Item 11. A transformant obtained by transforming a host cell using the recombinant vector according to Item 10.
Item 12. Item 12. The transformant according to Item 11, wherein the host cell is Bacillus subtilis.
Item 13. Item 10. The alkaline mannanase according to any one of Items 1 to 6, wherein the transformant according to Item 11 or 12 is cultured in a medium, and alkaline mannanase having the following physicochemical properties is collected from the obtained culture. Production method:
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ .
Item 14. Microorganism “Bacillus sp. Strain JAMB-750” (reception number FERM AP-20227).
Item 15. Item 7. An enzyme composition containing the alkaline mannanase according to any one of Items 1 to 6.
Item 16. A cleaning composition containing at least the alkaline mannanase according to any one of Items 1 to 7 and a surfactant (excluding a textile product cleaning composition).
Item 17. Item 8. A bleaching composition comprising at least the alkaline mannanase according to any one of Items 1 to 7.
Item 18. A composition for fiber softening treatment, comprising at least the alkaline mannanase according to any one of Items 1 to 7.
Item 19. 8. A coffee extract treatment composition containing at least the alkaline mannanase according to any one of Items 1 to 7.
Item 20. Item 8. A method for producing a manno-oligosaccharide having a step of performing an enzyme treatment using the alkaline mannanase according to any one of items 1 to 7.
Item 21. Item 21. A composition containing mannooligosaccharides and biodegradable plastics produced by the method according to item 20.
Item 22. Item 15. A ground crushing agent comprising the alkaline mannanase according to any one of Items 1 to 7 or the enzyme composition according to Item 15.
Item 23. Item 23. A well, hot spring or oil drilling method using the ground crushing agent according to Item 22.
Item 24. Item 15. A white water scum inhibitor comprising the alkaline mannanase according to any one of Items 1 to 7 or the enzyme composition according to Item 15.

  In the present invention, “gene” or “DNA” is used to include not only double-stranded DNA but also single-stranded DNA such as a sense strand and an antisense strand constituting the DNA. The length is not particularly limited. Therefore, unless otherwise specified, “gene” or “DNA” as used herein means double-stranded DNA including genomic DNA, single-stranded DNA including cDNA (positive strand) and complementary to the positive strand. Single-stranded DNA having a specific sequence (complementary strand, reverse strand) and synthetic DNA are included, and “gene” also includes RNA. In addition to the coding region (including the signal peptide region), the “gene” or “DNA” can include, for example, an expression control region, a signal region, an exon, and an intron, as long as the effect of the present invention is retained.

  Unless otherwise specified, “gene” or “DNA” in the present invention includes not only “gene” or “DNA” represented by a specific nucleotide sequence, but also proteins encoded by these and physiological functions. Included are “genes” or “DNAs” that encode proteins that are equivalent (eg, homologues, variants or derivatives, also referred to herein as “functional equivalents”). In the present specification, such “gene” or “DNA” is also referred to as “homologue” of “gene” or “DNA” represented by a specific base sequence. Examples of such “homolog” include “gene” or “DNA” having a base sequence that hybridizes under stringent conditions to the complementary strand of “gene” or “DNA” represented by a specific base sequence. be able to. For example, as a gene (homolog) encoding a homologue, a corresponding gene derived from another microorganism other than Bacillus sp. Strain JAMB-750, which is an organism derived from the gene of the present invention, can be exemplified.

  The “enzyme (alkaline mannanase)” or “protein” as used in the present invention includes not only “enzyme (alkaline mannanase)” or “protein” represented by a specific amino acid sequence, but also “physiological functions equivalent to these”. “Enzyme (alkaline mannanase)” or “protein” (eg, “functional equivalents” such as homologues, variants or derivatives). For example, as a homologue (functional equivalent), a corresponding microorganism derived from a microorganism other than Bacillus sp. Strain JAMB-750, which is an organism derived from the “enzyme (alkaline mannanase)” or “protein” of the present invention, “Enzyme (alkaline mannanase)” or “protein” can be exemplified. Mutants (functional equivalents) include naturally occurring allelic variants, non-naturally occurring variants, and amino acid sequences modified by artificial deletion, substitution, addition, or insertion. Variants having are included. The “functional equivalents” such as homologues and mutants include an enzyme (alkaline mannanase) or protein specified by a specific amino acid sequence (SEQ ID NO: 1), and at least 40% or more in amino acid sequence, preferably May be those that are 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, and particularly preferably 90% or more.

  Hereinafter, the present invention will be described in more detail.

(1) Alkaline mannanase TECHNICAL FIELD This invention relates to a novel alkaline mannanase. Here, the mannanase is an enzyme having an activity to hydrolyze β-1,4-D-mannopyranoside bond which is a main skeleton of mannan, galactomannan, glucomannan, etc., and formally mannan endo-1,4 -β-mannosidase (EC3.2.1.78). Also known as β-mannanase, or β-1,4-mannanase.

  The presence or absence of such mannanase activity can be easily performed by visually judging the presence or absence of halo formation in a solid medium containing a mannan component. If mannanase is present in the test sample, mannan in the solid medium used for the culture is decomposed to form a halo on the solid medium. Although it does not restrict | limit in particular, it can carry out according to description of the reference example mentioned later. Here, as the mannan component, locust bean gum (main constituent component: [D-galactose: D-mannose (1: 4)]) can be preferably exemplified.

  The activity of mannanase is measured according to the 3,5-dinitrosalicylic acid method [GL Miller, Anal. Chem., 31, 426-428 (1959)]. Can be done. Although not particularly limited, for example, using a locust bean gum (main component; [D-galactose: D-mannose (1: 4)]) as a substrate, the substrate (locus bean) according to the 3,5-dinitrosalicylic acid method described above. Gum) A method for measuring the amount of reduced product produced can be mentioned. Details thereof are as described in a reference example described later.

  In the present invention, “alkaline mannanase” means a mannanase whose optimum pH of activity is in the alkaline region (pH 8 or more).

Examples of the alkaline mannanase targeted by the present invention include those having the following physicochemical properties (a) to (g):
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
Examples of the mannooligosaccharide having 4 or more sugars include locust bean gum, ivory nut, konjac mannan, and guar gum. The relative activity of the alkaline mannanase of the present invention for these manno-oligosaccharides is preferably about 60 ivory nuts, about 100 konjac mannans, and about 30 guar gums, where locust bean gum is 100. The alkaline mannanase of the present invention has endo-type mannanase activity, and does not exhibit hydrolytic ability for disaccharide or trisaccharide manno-oligosaccharide even when treated at pH 9 and 40 ° C. for 17 hours. Is preferred.
(B) Optimal pH: It has an optimal pH in the range of pH 9 to 10.5. The optimum pH is preferably in the range of pH 9.5 to 10.2, more preferably around pH 10.
(C) pH stability: It has a mannanase activity of 70% or more when treated for 30 minutes under conditions of pH 6.5 to 10 and 40 ° C. Preferably, it has a pH stability such that it has a mannanase activity of 80% or more when treated for 30 minutes under conditions of pH 6.5 to 10 and 40 ° C. Here, 70% or more, preferably 80% or more of mannanase activity means that the mannanase activity before treatment is 100%, the mannanase activity after treatment is 70% or more of the mannanase activity before treatment, Preferably, it means 80% or more. Furthermore, the alkaline mannanase of the present invention preferably has a mannanase activity of 80% or more, preferably 90% or more when treated for 30 minutes under conditions of pH 7 to 9.5 and 40 ° C.
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: It has an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes.

Here, having 50% or more mannanase activity means that the mannanase activity after the treatment is maintained at 50% or more of the mannanase activity before the treatment when the mannanase activity before the treatment is 100%. Say.
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N 2 -bromosuccinimide.

Here, “inactivation” means that the mannanase activity after treatment is 10% or less, preferably 5% or less of the mannanase activity before treatment, when the mannanase activity before treatment is 100%. To do. More preferably, it has the property that the mannanase activity disappears (0%) by the above treatment.
(G) Metal inhibition: in the presence of 1 mM concentrations of Fe ²⁺ , Fe ³⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , or Sn ²⁺ at pH 7.5 and 40 ° C, respectively. When treated for 30 minutes, the mannanase activity is inhibited, resulting in 50% or less of the mannanase activity (100%) before treatment.
(H) Molecular weight by SDS-PAGE: about 130 kDa.

  Furthermore, the alkaline mannanase of the present invention has the above physicochemical properties (a) to (g), and has an N-terminal Glu-Ser-Lys-Ile-Pro-Lys-Asp-Ser-Glu- Gly: a protein having an amino acid sequence consisting of SEQ ID NO: 5) is included.

  Preferred examples of the alkaline mannanase of the present invention having such characteristics include a protein having the amino acid sequence represented by SEQ ID NO: 1. Moreover, the alkaline mannanase of the present invention is not limited to one having such a specific amino acid sequence, and may be a functional equivalent thereof. Here, the functional equivalent has an amino acid sequence obtained by deleting, substituting, or adding one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1. Among the physicochemical properties described above, (a) The protein which has at least 1 of-(g) can be mentioned. Preferably, it has 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more of the physicochemical properties of (i) to (g), more preferably all 7 Is. The functional equivalent of the protein having the amino acid sequence represented by SEQ ID NO: 1 is at least one of the above-mentioned physicochemical properties (a) to (g) (preferably 2 or more, 3 or more, 4 or more) 5 or more, or 6 or more, more preferably 7), and an amino acid consisting of Glu-Ser-Lys-Ile-Pro-Lys-Asp-Ser-Glu-Gly (SEQ ID NO: 5) at the N-terminus Mention may be made of proteins having a sequence.

  More preferably, the above functional equivalent is 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably in the amino acid sequence and amino acid sequence shown in SEQ ID NO: 1. Mention may be made of proteins that have an identity of 80% or more, particularly preferably 90% or more, are stable in an alkaline pH range (pH 8 or more) and have an optimum pH in the alkaline pH range. In addition, the identity of the sequence when the two sequences are compared is indicated by the percentage of the number of amino acids that are identical between the sequences relative to the total number of amino acids.

  In accordance with the above principle, the sequence identity between two amino acid sequences is determined by the algorithm of Karlin and Altschul [Proc. Natl. Acad. Sci., USA, 87, 2264-2268 (1990) and Proc. Natl. Acad. Sci. , USA, 90, 5873-5877 (1993)]. As a program using such an algorithm, BLAST program [Altschul et al., J. Mol. Biol. 215, 403-410 (1990)], and sequence identity can be determined with higher sensitivity than the BLAST. Gapped BLAST program [Nucleic Acids Res., 25, 3389-3402 (1997)]. These programs are available, for example, on the Internet website of the National Center for Biotechnology Information.

  The alkaline mannanase of the present invention includes a protein encoded by DNA consisting of the base sequence shown in SEQ ID NO: 2 and functional equivalents thereof. Here, the functional equivalent is a protein encoded by a DNA (homolog) that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 2, Among the above-mentioned physicochemical properties, there may be mentioned proteins having at least one of (a) to (g). Preferably, it has 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more of the physicochemical properties of (a) to (g), more preferably all 7 It is.

Here, “stringent conditions” for hybridizing with DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 are Berger and Kimmel (Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego, CA, USA, 1987) can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe binds. For example, 6xssc (composition of 1xssc; 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5X Denhart and 100 mg / ml as washing conditions after hybridization (so-called “stringent conditions”) pro to a solution containing ml herring sperm DNA) - and 8-16 hours thermostated at 65 ° C. with blanking, conditions for hybridizing the like (Sambrook et al., Molecular Cloning-a Loboratory Mannual, 2 nd ed)..

  The alkaline mannanase of the present invention can be produced by chemical or genetic engineering techniques based on the amino acid sequence disclosed in the present invention. Details of the production method by genetic engineering will be described later. In addition, the modification in the amino acid sequence is a method already known in the art, for example, a site-directed mutagenesis method [Current Protocols I molecular biology, edit. Ausubel et al., John Wily & Sons, Section 8.1-8.5 (1987)]. Etc. can be used by introducing mutations such as substitution, deletion, insertion, addition and inversion as appropriate into the amino acid sequence to be modified.

The alkaline mannanase of the present invention is not particularly limited as long as it has the above-mentioned characteristics, but is preferably derived from a microorganism belonging to the genus Bacillus . Preferably, the microorganism is derived from an alkalophilic mesophilic bacterium belonging to the genus Bacillus having an optimum growth pH of around pH 9 and an optimum growth temperature of 33 to 35 ° C. An example of such a microorganism is not limited, but its 16S rDNA sequence is 98% or more and less than 100%, preferably in the range of 98.5-99.5%, more preferably 98.9% identical to the B. alcalophilus 16S rDNA sequence. Examples of such microorganisms are those that are 97% or more and less than 100%, preferably in the range of 97.5-99.5%, more preferably 97.9%, with the sequence of 16S rDNA of B. pseudalcalophilus .

  Here, the sequence identity between two sequences is expressed as a percentage of the total number of bases that are identical between these sequences. Similar to the amino acid sequence, the sequence identity between two nucleotide sequences is determined by the algorithm of Karlin and Altschul [Proc. Natl. Acad. Sci., USA, 87, 2264-2268 (1990) and Proc. Natl. Acad. Sci. USA, 90, 5873-5877 (1993)].

Preferred examples of the microorganism belonging to the alkalophilic Bacillus genus that produces the alkaline mannanase of the present invention include “Bacillus sp. Strain JAMB-750” (reception number FERM AP-20227) (JAMB-750 strain). . The characteristics of this microorganism are shown in Table 1.

当該菌体（JAMB-750株）は土壌から分離取得したものであり、pH 9前後で良く生育する好アルカリ性のBacillus属細菌である。さらに当該JAMB-750株は、16S rDNA塩基配列に基づく相同性解析の結果、Bacillus alcalophilus（DSM485^T）と98.9%の相同性を、またBacillus pseudoalcalophilus（DSM8725^T）と97.9％の相同性を有している。このことから本発明菌体（JAMB-750株）は、これらの菌体と近縁種のものではあるが、これらの菌とは異なるBacillus属に属する新種の微生物であると判断された。当該本発明の菌体は、「Bacillus sp. Strain JAMB-750」と命名されて、平成１６年９月２４日付けで、日本国茨城県つくば市東１丁目１番地１中央第６に住所を有する独立行政法人産業技術総合研究所、特許生物寄託センターに、寄託者が付した識別のための表示を「Bacillus sp. Strain JAMB-750」とし、受領番号を「ＦＥＲＭ ＡＰ−２０２２７」として寄託されている（通知番号：１６産生寄第２２７号）。

The microbial cells (JAMB-750 strain) are isolated and obtained from soil and are alkaliphilic Bacillus bacteria that grow well around pH 9. Furthermore the JAMB-750 strain as a result of homology analysis based on 16S rDNA base sequence, the homology Bacillus alcalophilus (DSM485 ^T) 98.9%, and have a homology of Bacillus pseudoalcalophilus (DSM8725 ^T) and 97.9% ing. Based on this, it was determined that the microbial cell of the present invention (JAMB-750 strain) is a new type of microorganism belonging to the genus Bacillus which is different from these microbial cells. The bacterial cell of the present invention is named “Bacillus sp. Strain JAMB-750” and has an address at 1st, 1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan, on September 24, 2004. Deposited at the National Institute of Advanced Industrial Science and Technology, the Patent Biological Depositary Center as “Bacillus sp. Strain JAMB-750” and the receipt number as “FERM AP-20227”. (Notification No .: 16 Production No. 227).

  The microorganism has the ability to produce the alkaline mannanase of the present invention and elute it outside the cells. Therefore, the alkaline mannanase of the present invention can be collected from the culture solution by culturing the above microorganism (JAMB-750 strain).

(2) Alkaline Mannanase Gene The present invention also provides the gene for alkaline mannanase. The gene referred to here includes genomic DNA, cDNA, synthetic DNA, and RNA.

  The said gene should just have a base sequence which codes the alkaline mannanase (protein) mentioned above. Specific examples include DNA comprising the base sequence shown in SEQ ID NO: 2 and homologues thereof. Here, examples of the homolog include a gene encoding a protein functionally equivalent to a protein (alkaline mannanase) encoded by a DNA having the base sequence represented by SEQ ID NO: 2. In addition, functionally equivalent means having a mannanase activity at least in the alkaline region, preferably the optimum pH of the activity, like the protein (alkaline mannanase) encoded by the DNA consisting of the base sequence shown in SEQ ID NO: 2. Is in the alkaline region (pH 8 or higher).

  Preferred homologs include proteins having at least one of (i) to (g) among the physicochemical properties described in (1) above. Preferably, it has 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more of the physicochemical properties of (a) to (g), more preferably all 7 It is.

  Specific examples of such a homolog include a gene having a base sequence that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 2. Examples of the stringent conditions include those described above. Specific examples of the homologue include a gene having the base sequence represented by SEQ ID NO: 4. The gene is DNA encoding a precursor of alkaline mannanase (SEQ ID NO: 3) having a signal peptide in the N-terminal region.

  The gene of the present invention can also be isolated and obtained from “Bacillus sp. Strain JAMB-750” as shown in the Examples. As another method, it can also be prepared by chemical synthesis.

(3) Recombinant vector This invention provides the recombinant vector containing the gene which codes the said alkaline mannanase. The recombinant vector contains a gene encoding alkaline mannanase in a state capable of being expressed in a desired host cell, and is used to transform the host cell. Therefore, the recombinant vector of the present invention may be any vector as long as it has such a form that transformation of such host cells can be achieved, for example, a plasmid, bacteriophage, or retrotransposon.

The recombinant vector of the present invention (expression vectors), when using E. coli (E. coli) and Bacillus subtilis (B subtilis.) Bacteria such as hosts, in general, at least a promoter - operator region (promoter, operator and ribosome binding region (Including the SD region)), a start codon, DNA encoding the alkaline mannanase of the present invention, a stop codon, a terminator region, and a replicable unit. When a fungal cell such as yeast or an animal cell is used as a host cell, it generally has at least a promoter, a start codon, a signal peptide and a DNA encoding the alkaline mannanase of the present invention, and a stop codon. In addition, the recombinant vector (expression vector) of the present invention, if necessary, a cis element such as an enhancer, a 5 ′ or 3 ′ untranslated region of DNA encoding the alkaline mannanase of the present invention, a splicing junction, It may contain polyadenylation sites, replicable units, homologous regions, selectable markers. These elements are not particularly limited as long as they correspond to the host used for the expression of the gene encoding the alkaline mannanase of the present invention, and can be selected based on common technical knowledge in the art.

  The selection marker is not particularly limited. For example, when the host used for gene expression is a bacterium, a drug resistance gene (for example, ampicillin resistance gene, neomycin resistance gene, cycloheximide resistance gene, tetramycin resistance gene, etc.) ) When the host is other than bacteria, such as yeast, various known selection markers including auxotrophic genes (for example, HIS4, URA3, LEU2, ARG4, etc.) can be used.

  The recombinant vector (expression vector) of the present invention can be conveniently prepared by introducing the DNA encoding the alkaline mannanase of the present invention into a known expression vector in a state in which the desired alkaline mannanase can be expressed. Specifically, it can be prepared by introducing it downstream of the promoter. Such introduction can be performed according to a general method of DNA recombination, for example, a method described in Molecular Cloning. (1989). (Cold Spring Harbor Lab.).

Examples of expression vectors include plasmid vectors such as pRS413, pRS415, pRS416, YCp50, pAUR112, and pAUR123. YCp-type E. coli- yeast shuttle vectors; pRS403, pRS404, pRS405, pRS406, pAUR101, and pAUR135 YIp type E. coli- yeast shuttle vector; E. coli derived plasmids (eg pBR322, pBR325, pUC18, pUC19, pUC119, pTV118N, pTV119N, pBluescript, pHSG298, pHSG396, pTrc99A or other ColE series plasmids) P1A-based plasmids such as pACYC177 or pACYC184; pSC101-based plasmids such as pMW118, pMW119, pMW218 or pMW219; plasmids derived from B. subtilis (eg pUB110, pTP5 etc.); E. coli such as pHY300PLK ( E. coli ) -B . subtilis shuttle vector. Examples of phage vectors include λ phage (Charon 4A, Charon21A, EMBL4, λgt100, gt11, zap), ψX174, M13mp18, and M13mp19. Examples of retrotransposons include Ty factor. In addition, expression vectors that are expressed as fusion proteins, such as pGEX series (Pharmacia) and pMAL series (Biolabs) can also be used.

In an embodiment of the present invention, the gene encoding the precursor of the alkaline mannanase of the present invention, pHY300PLK plasmid vector [E. (E coli.) - B. subtilis (B subtilis.) Shuttle vector, TaKaRa made:. E. (E coli The vector for expression of alkaline mannanase of the present invention is prepared by introducing the DNA into the multiple cloning site of the plasmid pACYC177 and the DNA derived from Streptococcus faecalis plasmid pAMα1]. In addition, pHY300PLK plasmid vectors include ampicillin resistance gene and tetramycin resistance gene; RNA primer gene (ori-177); plasmid replication gene (Rep-α-1 gene), replication origin [E. coli ( E. coli ) and Bacillus subtilis ( B. subtilis )]; polylinker and the like. According to the plasmid vector, Bacillus subtilis (B. Subtilis) and Escherichia coli (E. Coli) as a host, in which, to efficiently transcribed and translated genes alkaline mannanase of the invention linked as a foreign gene be able to.

(4) Transformant The present invention also provides a transformant obtained by introducing the above recombinant vector into a host cell and transforming it.

  The method of introducing (transforming) the recombinant vector into the host cell is not particularly limited, and it depends on the type of host cell to be introduced and the form of the recombinant vector, such as transformation method, transfection method, competent cell method, and electroporation. Depending on the situation, it can be appropriately selected.

The host cell, Escherichia coli (E. Coli) and Bacillus subtilis (B. Subtilis) bacteria such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces pombe (Saccharomyces pombe), Pichia pastoris (Pichia pastoris) yeasts such as, Examples include insect cells such as sf9 and sf21, animal cells such as COS cells, Chinese hamster ovary cells (CHO cells), and plant cells such as tobacco. Preferably, Escherichia coli (E. Coli) and Bacillus subtilis (B. Subtilis) bacteria and the like, and yeast.

  The mode of presence of the expression vector in the host cell is not particularly limited, and the expression vector may be inserted into the chromosome, replaced or integrated, or may exist in the form of a plasmid.

(5) Method for Producing Alkaline Mannanase The transformant thus obtained can be cultured in a suitable medium depending on the host, thereby producing the novel alkaline mannanase of the present invention. The present invention provides a method for producing a novel alkaline mannanase using such a transformant. Specifically, the method can be performed by culturing the above transformant in a medium and collecting alkaline mannanase from the obtained culture.

  The medium contains a carbon source, a nitrogen source, an inorganic salt, a vitamin, a drug and the like essential for the growth of the transformant. Carbon sources include mannan components, arabinose, cellobiose, fructose, galactose, glucose, glycerol, lactose, maltose, mannitol, mannose, sucrose, trehalose, and xylose; nitrogen sources include inorganic nitrogen such as ammonium sulfate and ammonium chloride, and casein decomposition Organic yeast sources such as food, yeast extract, polypeptone, bactotryptone and beef extract; inorganic salts such as sodium diphosphate or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride Etc .; various vitamins including vitamin B1 as vitamins; various antibiotics such as ampicillin, neomycin, cycloheximide, tetramycinThese are merely examples, and are not limited thereto.

As an example of the medium, when the host is a gram-negative bacterium such as E. coli or a gram-positive bacterium such as B. subtilis , LB medium (Nissui Pharmaceutical), M9 medium ( J. Exp. Mol. Genet., Cold Spring Harbor Laboratory, New York, p.431, 1972); and YPD medium (1 w / v% Bacto yeast extract, 2 w / v% Bacto peptone) when the host is yeast. , 2w / v% glucose), YPG medium (1w / v% Bacto yeast extract, 2w / v% Bacto peptone, 2w / v% glycerol), YP medium (1w / v% Bacto yeast extract, 2w / v% Bacto peptone) YPD medium (1 w / v% Bacto yeast extract, 2 w / v% Bacto peptone, 2 w / v% glucose), 0.7 w / v% Yeast Nitrogen Base (Difco), YP medium [1 w / v% Bacto yeast extract (Difco), 2w / v% Polypepton S (Nippon Pharmaceutical)], and the like.

  The culture is usually carried out in the temperature range of 10 to 40 ° C. for about several to 80 hours, and aeration and agitation can be added as necessary. The culture temperature can be set according to the host and is not particularly limited. However, the culture temperature is preferably 18 to 42 ° C, more preferably 25 to 38 ° C.

  The method for producing alkaline mannanase of the present invention is further carried out by collecting the desired alkaline mannanase (protein) from the culture thus obtained. The target protein can be collected by extracting the target protein accumulated in the culture supernatant or in the transformant (bacteria) by a known method after culture, and purifying as necessary. it can. Separation and purification can be performed by, for example, salting out, solvent precipitation, dialysis, ultrafiltration, gel electrophoresis, or gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography, etc. Can be combined.

(6) Use of alkaline mannanase The alkaline mannanase of the present invention can be used as a component of an enzyme composition, a cleaning composition, a bleaching composition, and a fiber softener. Hereinafter, each of these compositions will be described.

(6-1) Enzyme composition The enzyme composition of the present invention comprises an alkaline mannanase combined with one or more other enzymes. Enzymes used in combination with the alkaline mannanase of the present invention can include one or more enzymes selected from the group consisting of:
Protease, cellulase (endo-β-1,4-glucanase), β-glucanase (endo-β-1,3 (4) -glucanase), lipase, cutinase, peroxidase, laccase, amylase, glucoamylase, pectinase, reductase, Oxidase, phenol oxidase, ligninase, pullulanase, hemicellulase, xyloglunanase, xylanase, pectin acetylesterase, rhamnogalacturonan acetylesterase, polygalacturonase, rhamnogalacturonase, pectate lyase, pectin lyase, pectin Methyl esterase, cellobiohydrolase, transglutaminase, mannosidase, β-glucuronidase. Of these, protease, cellulase, lipase, amylase, pectinase, pullulanase and pectin lyase are preferable.

(6-2) Cleaning composition The cleaning composition of the present invention [however, the textile cleaning composition for textiles (including clothing) is excluded] contains the alkaline mannanase of the present invention. In addition to this, other cleaning ingredients can be included.

  Here, examples of the cleaning component include those having a cleaning action or those having a function of reinforcing this cleaning action, and can be appropriately selected according to the type of the object to be cleaned. Specifically, surfactants, builders, bleaching agents, foam inhibitors, foaming agents and the like can be exemplified.

  Here, the surfactant can be appropriately selected from nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. Of these, nonionic surfactants and / or anionic surfactants are preferred. Here, as the nonionic surfactant, for example, polyethylene, polypropylene, and polybutylene oxide condensates of alkylphenols; quaternary ammonium surfactants as cationic surfactants; and anionic surfactants Alkyl ester sulfonate surfactants; amphoteric surfactants include secondary and tertiary amine derivatives, heterocyclic secondary and tertiary amine derivatives, or quaternary ammonium, quaternary ammonium, quaternary Examples thereof include derivatives of phosphonium or tertiary phosphonium compounds. However, it is not limited to these, and surfactants usually used for cleaning agents can be widely used.

  Surfactants can be used at a rate of 0.1 to 60% by weight per cleaning composition. Although not limited, the ratio of each surfactant per cleaning composition is 0.001 to 50% by weight for nonionic surfactants, 0.2 to 25% by weight for cationic surfactants, Preferably 1 to 8 wt%, 1 to 40 wt%, preferably 3 to 20 wt% for anionic surfactants, 0.2 to 15 wt%, preferably 1 to 10 wt% for amphoteric surfactants Can be exemplified.

  Builders include aluminosilicate materials, silicates, and polycarboxylates. Preferred are inorganic compounds such as aluminosilicate materials (more specifically, hydrated synthetic zeolite) and silicates (including laminated silicates and crystalline silicates). However, the builder that is usually used for the cleaning agent can be widely used without being limited thereto. Builders can be used at a rate of 5 to 80% by weight per cleaning composition. When the cleaning composition is liquid, it is preferably used in a proportion of 5 to 30% by weight.

  As the bleaching agent, for example, oxygen bleaching agents, percarboxylic acid bleaching agents, and halogen bleaching agents such as bleaching agents that are usually used for cleaning agents can be widely used. The bleaching agent is not limited, but can be used in a proportion of 0 to 25% by weight per cleaning composition.

  As the foam inhibitor, for example, a foam inhibitor usually used in a cleaning agent such as silicone or silica-silicone can be widely used. The foam inhibitor is not limited, but can be used at a rate of 0 to 2% by weight per cleaning composition.

  Although not limited, the antalimanananase of the present invention is preferably used in a ratio of 0.0001 to 2% by weight, more preferably 0.0005 to 0.5% by weight, in the cleaning composition of the present invention. it can. Furthermore, the cleaning composition of the present invention can contain enzymes such as cellulase, amylase, pectin degrading enzyme, xyloglucanase and carbohydrase in addition to the alkaline mannanase. Here, cellulase, amylase, pectin degrading enzyme, xyloglucanase and carbohydrase preferably have a maximum activity in the range of pH 7 to 12, or 10% or more, preferably 25% or more, more preferably 40% of the maximum activity. % Alkaline enzyme or alkaline cellulase, alkaline amylase or alkaline amylase, alkaline pectin degrading enzyme or alkaline pectin degrading enzyme, alkaline xyloglucanase or alkaline xyloglucanase, and preferred Alkaline carbohydrase or alkali-resistant carbohydrase.

  Although not limited, the proportion of cellulase in the cleaning composition is 0.0001 to 2% by weight, preferably 0.00018 to 0.06% by weight; amylase is 0.0001 to 2% by weight, preferably 0.00018. A proportion of ˜0.06 wt%; a proportion of 0.0001 to 2 wt%, preferably 0.0005 to 0.5 wt% of pectin-degrading enzyme; 0.0001 to 2 wt% of xyloglucanase, preferably 0.000. The proportion of 0005 to 0.5% by weight; and the carbohydrase can be added in a proportion of 0.0001 to 2% by weight, preferably 0.0005 to 0.5% by weight.

  In addition to the above components, the cleaning composition of the present invention contains other components such as fluorescent brighteners, abrasives, bactericides, fogging inhibitors, anti-redeposition agents, softeners, colorants, and fragrances. You can also Those components that are usually used in detergents can also be widely used.

  The cleaning object of the cleaning composition of the present invention is not particularly limited as long as it is other than textile products (including clothing). Therefore, the cleaning composition of the present invention includes, for example, a dishwashing cleaning composition for dishes and the like; hair and skin cleaning compositions (hair shampoo, body shampoo) for hair and skin, etc .; Examples include hard surface cleaners for cleaning dirt adhering to the surface; contact lens cleaners; toothpaste and denture cleaners.

  The form of the cleaning composition of the present invention is not particularly limited, and has, for example, a liquid form, a paste form, a gel form, a stick form, a tablet form, a spray spray, a foam form, a powder form, a granular form, or a granular form. be able to. The particulate composition can also be present in compressed form, and the liquid composition can be in concentrated form.

(6-3) Composition for bleaching treatment The alkaline mannanase of the present invention enhances the bleaching effect in the bleaching process not containing chlorine in paper pulp (chemical pulp, semi-pulp, mechanical pulp, kraft pulp, etc.) It can be used effectively to reduce or eliminate the need for hydrogen. Therefore, the ant kalimannanase of the present invention can be used as one component of a composition for bleaching papermaking pulp that does not contain chlorine. Therefore, this invention provides the composition for the bleaching process of the papermaking pulp containing alkaline mannanase. In addition to the alkaline mannanase of the present invention, such a bleaching treatment composition can broadly contain various components generally used in the composition. In addition, although it is based on the specific activity as a compounding ratio of alkaline mannanase, it can be normally used in 0.001%-20% of range.

(6-4) Use in the textile industry and the cellulose fiber processing industry The alkaline mannanase of the present invention is used in the production of fibers or the processing of fibers (preferably cellulosic fibers), as required by other carbohydrate degrading enzymes (for example, xylo). Glucanase, xylanase, and various pectin-degrading enzymes can be used in combination. Here, the “cellulosic fiber” includes cotton, blended cotton, natural or artificial cellulosic material (for example, xylan-containing cellulose fiber such as wood pulp), or fibers, woven fabrics made from these blends, Nonwovens (including knits, wovens, denims, yarns, and toweling) are included. Examples of blends are cotton or rayon / viscose and wool, synthetic fibers (eg polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinylidone chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) And a blend of at least one selected from cellulose-containing fibers (for example, rayon / viscose, ramie, hemp, flax / linen, jute, cellulose acetate fiber, lyocell).

  The alkaline mannanase of the present invention can be used for the purpose of processing cellulosic fibers into a material that can be used for the production of clothing, or processing the cellulosic fibers into a material that can be suitably used for the production of clothing. The processing of cellulosic fibers in the textile industry includes a plurality of processes such as, for example, a process of spinning fibers into a yarn: a process of creating a woven fabric or a knit from the yarn; and a finishing process of subsequent dyeing.

  In such a process, a polymer paste (for example, mannan, starch, carboxymethylcellulose, polyvinyl alcohol, etc.) is added to the fiber, especially before weaving, and the material subjected to such treatment needs to be removed before processing. . The alkaline mannanase of the present invention can be used to remove such mannan-containing paste.

  Galactomannans such as guar gum and locust bean gum are widely used as thickeners for printing pastes used for textile printing. Therefore, the alkaline mannanase of the present invention or an enzyme composition containing the alkaline mannanase can be used to reduce the viscosity of the printing paste or to wash and remove the printing paste from the fiber after printing.

(6-5) Degradation and modification of plant-derived thickeners and gelling agents, etc.For thickeners, gelling agents or dispersants derived from plant components, mannan, galactomannan, glucomannan, or Some contain galactoglucomannan. Examples of such materials include gums such as locust bean gum and guar gum. The alkaline mannanase of the present invention or an enzyme composition containing the same decomposes a plant-derived substance containing a mannan component widely used in applications such as a thickener, a gelling agent or a dispersing agent, and its physicochemical properties It can be suitably used to modify (for example, viscosity). Specifically, it can be suitably used for reducing the viscosity of feed and food containing mannan and facilitating the processing of viscous mannan-containing substances (feed and food).

(6-6) Suppression of gel formation and precipitation generated during coffee liquor and processing thereof The alkaline mannanase of the present invention or an enzyme composition containing the same is used to hydrolyze mannans such as galactomannan present in the liquid coffee extract. It can be utilized for decomposition, and thereby it is possible to suppress the formation of gel and precipitation generated during coffee liquid and its processing. In particular, it can be effectively used for the suppression of gel formation during freezing drying of instant coffee, gel formation during hot storage such as hot bender, and precipitation formation.

(6-7) Use in underground crushing (oil drilling)
The alkaline mannanase of the present invention or an enzyme composition containing the alkaline mannanase can be used as an enzyme crushing agent as in US Pat. Nos. 5,865,597, 5,562,160, 5,2013,70 and 5,675,662. Specifically, it can be used as a crushing agent for crushing the ground and forming an underground part for the purpose of drilling wells or drilling oil.

  As the crushing agent, the alkaline mannanase of the present invention or the enzyme composition containing the same is mixed with a hydratable polymer and a crosslinking agent for crosslinking the hydratable polymer with the alkaline mannanase of the present invention, It is preferably used as a gel. The crosslinked polymer gel is pumped into the hole under the pressure sufficient to break the underground part, forming the desired hole in the underground part. The cross-linked polymer gel fed into the hole in the underground part is reduced in viscosity with time by the decomposition action of the alkaline mannanase of the present invention contained therein, and becomes a low-viscosity liquid. Therefore, the liquid accumulated in the underground part can be easily pumped back from the underground part to the hole surface. It is possible to form an underground part by pressing the cross-linked polymer gel, reducing the viscosity of the cross-linked polymer gel accumulated in the hole, and repeating the recovery operation.

(6-8) Production of Mannose and Oligosaccharide The alkaline mannanase of the present invention includes glucomannan such as konjac, locust bean gum, guar gum, galactomannan derived from soybean seed coat, galactomannan derived from coffee beans, galactoglucomannan derived from wood It can be used for the production of mannose, mannooligosaccharide, galactomannooligosaccharide and glucomannooligosaccharide by acting on seaweed-derived β-1,4 mannopolysaccharide and yeast-derived β-1,4 mannopolysaccharide. . Specifically, by causing the alkaline mannanase of the present invention to act on a plant polysaccharide having a β-1,4 mannopyranose ring, a product that does not contain glucose as a caloric component, such as a sweetener, an infusion component,飴, low calorie / non-calorie components, Salmonella colonization inhibitory components, biodegradable plastic accelerators can be produced.

(6-9) Feed additive The alkaline mannanase of the present invention can be used as a feed additive in the preparation of feed. Specifically, when the alkaline mannanase of the present invention is added as a feed additive to ruminant feed such as cattle, mannanoligosaccharides are decomposed by the action of the mannanase to decompose mannan contained in the grain in the weakly alkaline stomach. Can increase Bifidobacteria, and can bring about the effects of bowel regulation, weight gain promotion, improved feed efficiency, constipation and diarrhea reduction, shortened days of estrus, and stool odor improvement.

(6-10) Inhibitor of white water scum The alkaline mannanase of the present invention is useful as an inhibitor of white water scum that is circulated and used in the comb portion and paper making process in the paper making process. Unpulverized pulp and digested pulp from which solid impurities have been removed in a water bath through various combs are continuously wound up by a roll through a bleaching and drying process, but are reused in a comb tank and a papermaking tank. The water is alkaline and white and generates scum and scales due to polysaccharides derived from dissolved pulp, propagated microorganisms, microbial secretions, and pulp fiber waste sugar, and mixes scum into the pulp in combs and papermaking tanks. According to the alkaline mannanase of the present invention, generation of scum in white water can be suppressed by suppressing the growth of microorganisms due to cell wall damage or by promoting dissolution of the scum component. In addition, scum and scale can be decomposed or burned out in a bleaching or drying process. Such suppression of scum generation and decomposition by the alkaline mannanase of the present invention can be effectively used to prevent interruption of winding due to pulp cutting in the papermaking process.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these Examples. In the following examples, unless otherwise specified,% means w / v%.
Reference Example In the following examples, (1) the detection method of mannanase (confirmation of the presence of mannanase) and (2) the measurement of mannanase activity were carried out according to the following methods.
(1) Method for detecting mannanase Mannanase was detected by visually observing the presence or absence of halo formation in a solid medium containing a mannan component. That is, if mannanase is present in the test sample, mannan in the medium is decomposed to form a halo. Specifically, a solid medium containing 0.2% locust bean gum (Sigma, hereinafter the same), 0.5% Polypepton S (Nippon Pharmaceutical, the same hereinafter), 0.5% yeast extract (Difco, hereinafter the _{same), 0.1% K 2 HPO 4} , 0.02% MgSO 4. 7H 2 0,0.5% Na 2 CO 3, 1.5% agar) ( hereinafter, was applied to the referred) to as "mannan-containing solid medium", at 30 ° C. Incubate for several days. After colony formation, the solid medium is overlaid with 0.1% aqueous congolet solution and stained. Thereafter, the color is decolorized with a 1% aqueous sodium hydroxide solution, and the halo formed by the decomposition of mannan is judged visually to evaluate the presence or absence of mannanase in the test sample. Hereinafter, the method for detecting mannanase according to this method is also referred to as “halo method”.

(2) Measurement of mannanase activity Mannanase activity was measured by using 3,5-dinitrosalicylic acid method using locust bean gum (main constituent: [D-galactose: D-mannose (1: 4)]) as a substrate. GL Miller, Anal. Chem., 31, 426-428 (1959)] by measuring the amount of substrate (locust bean gum) reductate produced.

  Specifically, the enzyme solution to be measured is placed in a substrate solution [final concentration 0.3% locust bean gum, 50 mM glycine-NaCl-NaOH buffer, pH 9.0] (total volume 1 ml) and treated at 40 ° C for 30 minutes. And perform the enzyme reaction. After the reaction, add 1 ml of dinitrosalicylic acid solution, heat in boiling water for 5 minutes, and then cool with ice water. After cooling, add 4 ml of water and measure the absorbance at 535 nm. The amount of enzyme (protein amount) that produces a reducing sugar corresponding to 1 μmol of D-mannose per minute is defined as 1 unit (1 U). The amount of protein is measured using a protein assay kit (Bio-Rad) using bovine serum albumin as a standard protein.

  Hereinafter, the method for measuring mannanase activity according to this method is referred to as “dinitrosalicylic acid method”.

Example 1 Isolation of Alkaline Mannanase Producing Bacterium “Bacillus sp. Strain JAMB-750” Soil collected from Ohara Town, Chiba Prefecture was suspended in sterilized water, and the supernatant was applied to a mannan-containing solid medium at 30 ° C. for several days. Culture was performed. After colony formation, the solid medium was overlaid with a 0.1% aqueous solution of Congolet and stained, and then decolorized with a 1% aqueous sodium hydroxide solution to evaluate the presence or absence of halo formed by decomposition of mannan.

  Liquid culture was performed using colonies having halo-forming ability as mannanase production candidate strains. Specifically, the mannanase production candidate strain was inoculated in a liquid medium (mannan-containing solid medium excluding agar), and cultured with aerobic shaking at 30 ° C. for several days. Thereafter, the culture solution was separated into cells (precipitate) and supernatant by centrifugation, and the obtained supernatant was dialyzed against 5 mM phosphate buffer (pH 7.0). Using this as a crude enzyme solution, the mannanase activity at each pH was measured according to the dinitrosalicylic acid method described in Reference Examples. Thereby, the optimum pH of mannanase contained in the crude enzyme solution was determined. In this method, a mannanase production candidate strain (hereinafter referred to as “the present invention”) having an optimum pH in the alkaline region (pH 8 or higher) is isolated and subjected to the tests (1) to (8) shown below. Provided.

(A) Test content and method
(1) Morphological examination
0.5% Polypepton S, 0.5% yeast extract, 0.5% glucose, 0.1% K 2 HPO 4, and grown to the present invention cells using a medium containing 0.02% MgSO _4, cell morphology and size by an optical microscope, and flagella The presence or absence of motility was observed. Further, Gram staining (positive / negative), aerobic and anaerobic, were evaluated according to a conventional method.

(2) sporulation medium ^{[BBL TM Trypticace TM Soy Agar (1.5} % pancreatic digest of casein, 0.5% papaic digest of soybeen meal, 0.5% sodium chloride, 1.5% agar) and 0.01% MnCl ₂ · 4H ₂ O content] The bacterial cells of the present invention were cultured, and the presence or absence of spore formation was confirmed with a microscope.

(3) Growth condition test
Using a medium containing 0.5% Polypepton S, 0.5% yeast extract, 0.5% glucose, 0.1% K ₂ HPO ₄ , and 0.02% MgSO ₄ , the growth conditions (growth pH range, optimum growth pH, growth Temperature range, optimum growth temperature, growth salt concentration range). The pH test was performed at pH 1 intervals in the range of pH 6-11. The pH of the medium was adjusted using hydrochloric acid for 5, 6 and 7, NaHCO _{3 for} pH 8 and 9, and Na ₂ CO ₃ for pH 10 and above. A temperature gradient biophoto recorder (ADVANTEC) was used to measure the growth temperature range. The salt concentration test was performed using sodium chloride and adjusting the salt concentration of the medium to 3%, 5%, 7%, 10% and 15%.

(4) Physiological properties test
(4-1) Oxidase test and catalase test For the oxidase test, the filter paper for cytochrome oxidase test "Nissui" (manufacturer and vendor, Nissui Pharmaceutical Co., Ltd.) was used. For the catalase test, 3 w / v% H ₂ O ₂ was used. The test was conducted according to a conventional method.

(4-2) Hydrolysis of starch
BactoTryptic Soy Broth without Dextrose ^™ (1.7% pancreatic digest of casein, 0.3% soy bean peptone, 0.5% sodium chloride, 0.25% dipotassium phosphate) (Difco) and 0.5% starch azure [Na ₂ CO ₃ The cells of the present invention were inoculated to pH 9.5]], and after growth, the presence or absence of halo formation was confirmed. When a halo is formed, it is determined that the microbial cell of the present invention has starch degradability.

(4-3) Hydrolysis of casein
Solid medium [BBL Co. ^{Trypticace Soy Agar TM (1.5% pancreatic} digest of casein, 0.5% papaic digest of soybeen meal, 0.5% sodium chloride, 1.5% agar), and 0.5% skim milk-containing [pH 9.0 with Na ₂ CO ₃ The bacterial cells of the present invention were inoculated, and after the growth, the bacterial cells were removed, and a 30% trichloroacetic acid solution was dropped into the medium to confirm the presence or absence of halo formation. When a halo is formed, it is determined that the cell of the present invention has casein resolution.

(4-4) Hydrolysis of gelatin
The cells of the present invention were inoculated into a solid medium (containing Nutrient Broth (0.3% beef extract, 0.5% peptone) (Difco), 2% agar, and 1% gelatin [adjusted to pH 9.0 with Na ₂ CO ₃ ]). After growth, the cells were removed, and a 30% trichloroacetic acid solution was dropped into the medium to confirm the presence or absence of halo formation. When a halo is formed, it is determined that the cell of the present invention has casein resolution.

(4-5) Production of indole
Medium (SIM confirmation medium (0.3% beef extract, 2.8% peptone, 0.0025% sodium thiosulfate, 0.1% ammonium Fe (III) citrate, 0.3% agar) (manufactured by Nissui Pharmaceutical)) The cell of the invention was punctured and 1 ml of indole reagent ( p -dimethylaminobenzaldehyde, 5 g; amyl alcohol, 75 ml; concentrated hydrochloric acid, 25 ml) was added to confirm the presence or absence of discoloration. When discoloration is observed, it is determined that the microbial cell of the present invention has an indole-producing ability.

(4-6) Production of H ₂ S
Medium (SIM confirmation medium (0.3% beef extract, 2.8% peptone, 0.0025% sodium thiosulfate, 0.1% ammonium Fe (III) citrate, 0.3% agar) (manufactured by Nissui Pharmaceutical)) The microbial cells of the invention were punctured and confirmed to produce hydrogen sulfide. When the medium turns black, it is determined that the bacterial cells of the present invention have the ability to produce hydrogen sulfide.

(4-7) Reduction ability of nitrate and nitrite (KNO ₃ reduction, NaNO ₂ reduction) (medium 6 and 7, respectively)
Medium 6 (containing 1% beef extract, 1% peptone, 0.5% NaCl, and 0.1% KNO ₃ [adjusted to pH 9.5 with Na ₂ CO ₃ ]) or medium 7 (1% beef extract, 1% peptone, 0.5% After injecting NaCl and 0.001% NaNO ₂ (adjusted to pH 9.5 with Na ₂ CO ₃ ) into each test tube, the cells were inoculated to grow the cells of the present invention. After growth, 1 ml each of 0.33% sulfanilic acid and 0.5% dimethyl α-naphthylamine was added to medium 6 and the presence or absence of discoloration was observed. When the medium turns red, nitrite is present, and it is determined that the bacterial cells of the present invention have a reducing ability for nitrate. If the medium does not change color, add a small amount of zinc. At this time, if the culture medium turns red, nitrate remains in the culture medium, and it is determined that the cells of the present invention do not have the ability to reduce nitrate. On the other hand, when the medium does not change color when zinc is added, it is judged that the microbial cells of the present invention have the ability to reduce nitrite, and the microbial cells have reduced nitrite and have been denitrified. .

  In addition, after growing the cells of the present invention using the medium 7, when 1 ml each of 0.33% sulfanilic acid and 0.5% dimethyl α-naphthylamine is added to the medium 7, the medium does not change color. The invented microbial cells are judged to be capable of reducing only nitrite without reducing nitrate.

(4-8) DNase activity Inoculate the cell of the present invention in a solid medium (Bacto DNase TEST AGAR (2% Bacto Tryptose, 0.2% deoxyribonucleic acid, 0.5% sodium chloride, 1.5% Bacto ager) (Difco)), After growth, the cells were removed and 1 N hydrochloric acid was added dropwise to the medium to confirm the presence or absence of halo formation. When a halo is formed, it is determined that the bacterial cell of the present invention has DNase activity.

(4-9) Sugar assimilation Soft agar medium (0.3% agar, 0.7% K ₂ HPO ₄ , 0.2% KH ₂ PO ₄ , 0.01% MgSO ₄ , 0.1% (NH ₄ ) ₂ SO ₄ , 0.5% NaCl , 0.5% various carbon sources (L-arabinose, cellobiose, D-fructose, D-galactose, D-gulucose, grycerol, D-lactose, maltose, D-mannitol, D-mannose, sucrose, D-trehalose, xylose), And vitamin solution containing [adjusted to pH 9.9 with Na ₂ CO ₃ ], and cultivated by puncture culture of the present invention, and based on the presence or absence of growth, the ability of the present microbial cells to assimilate to various carbon sources (sugars) evaluated.

(5) Homology analysis using 16S rDNA nucleotide sequence Genomic DNA was extracted from the above mannanase production candidate strain (the present invention) having an optimum pH in the alkaline region according to a conventional method [H. Saito and K. Miura, Biochem Biophys. Acta, 72, 619-629 (1963)]. Using this as a template, PCR was performed using a universal primer capable of amplifying an approximately 1,500 bp 16S rRNA gene to amplify the 16S rRNA gene. After purifying the obtained PCR product, using this as a template, using a primer prepared from the internal common sequence of the 16S rRNA gene, a sequencing reaction was performed to determine the base sequence. Based on the obtained nucleotide sequence data, homology search was performed using the FASTA program (http://www.ddbj.nig.ac.jp).

(6) Measurement of GC content by HPLC
The GC content in the cells was measured according to the method of Tamaoka et al. [FEMS Microbiol Lett., 25, 125-128 (1984)]. DNA extracted and purified from cultured cells was ethanol precipitated, dried, and adjusted to a concentration of 0.5 μg / μl with distilled water. 10 μl of this DNA solution was kept in a boiling solution for 10 minutes, and then rapidly cooled with ice water to denature into single-stranded DNA. To this, 10 μl of nuclease P1 solution (0.1 mg / ml nuclease P1, 40 mM sodium acetate, 2 mM ZnSO ₄ , pH 5.3) was added and treated at 50 ° C. for 1 hour for hydrolysis to convert the DNA into nucleotides. Further, 10 μl of alkaline phosphatase (2.4 units / ml, 0.1M Tris-HCl, pH 8.1) was added to this, treated at 37 ° C. for 2 hours or longer, and the nucleoside was used as an analysis sample, and high performance liquid chromatography under the following conditions: (GC) was used to determine the GC content.

<GC content analysis conditions for high performance liquid chromatography>
Equipment: High performance liquid chromatograph LC Module 1 (Waters),
Detection wavelength: 260 nm,
Analytical column: Cosmosil 5C18 column (4.6 x 150 mm) (Nacalai Tesque),
Column temperature: room temperature (15-25 ° C),
Mobile phase: 0.02 M monoammonium phosphate-acetonitrile (19: 1, v / v),
Flow rate: 1 ml / min
Sample volume: 10 μl.

(7) Analysis of cell fatty acid composition
The fatty acid composition of the cells of the present invention was analyzed according to the method of Waino et al. [Int. Syst. Bacteriol., 49, 821-831 (1999)]. 20 mg of the lyophilized cell of the present invention was placed in a test tube of a heat-resistant screw cap with a Teflon (registered trademark) lining. To this was added 2 ml of 5% (v / v) anhydrous hydrochloric acid / methanol solution (domestic chemistry), and the cap was tightened. Heat treatment was performed at 100 ° C. for 3 hours. After heating, the mixture was cooled, 1 ml of distilled water was added, 3 ml of hexane was added, and the mixture was shaken vigorously for 2 minutes. The mixture was centrifuged at 3000 × g for 5 minutes, and the hexane layer was transferred to another screw cap. To the remaining hydrochloric acid / methanol solution, 3 ml of hexane was again added and the same operation was performed, followed by extraction three times. The recovered hexane layer was added with the same amount of distilled water and shaken vigorously for 2 minutes. This was centrifuged at 3000 × g for 5 minutes to remove hydrochloric acid from the hexane layer. The hexane layer was transferred to a new test tube containing 5 g of anhydrous sodium sulfate that had been washed with diethyl ether and dried beforehand, and the mixture was lightly stirred and allowed to stand for 3 hours. The hexane layer was transferred to a new test tube and concentrated to about 200 μl with a vacuum concentrator. This was subjected to gas chromatography / mass spectrometry (GC / MS) to analyze the cell fatty acid composition.

(8) Analysis of quinone composition
According to the method of Komagata et al. [Methods Microbiol., 19, 161-203 (1983)], the quinone composition of the bacterial cells of the present invention was analyzed. Place the lyophilized cell of the present invention (about 200 mg) into a screw-cap test tube, add 20 ml of chloroform-methanol (2: 1, v / v), and place in a cool dark place overnight to extract the cell components. . This chloroform-methanol suspension was filtered with a filter paper, and the filtrate was collected in an eggplant type flask and concentrated to dryness with a rotary evaporator. The bacterial cell components dried on the inner wall of the eggplant-shaped flask were extracted with a small amount of acetone. The acetone extract, silica gel TLC plates were spotted _{(Merck Kiesel-gel 60 F 254} , 0.2 mm thickness, 20 cm × 20 cm). Similarly, the following markers were spotted on the silica gel TLC plate. This TLC plate was developed with hexane-diethyl ether (4: 1, v / v) for 1 hour, dried, and then the spot of acetone extract (test sample) developed under an ultraviolet lamp (254 nm) The quinone band was confirmed.

  The confirmed quinone band fraction was scraped from the TLC plate, transferred to a test tube, added with 1 ml of acetone, and extracted by shaking lightly. The acetone extract was filtered through a 0.5 μm hydrophobic PTFE membrane filter (Sunprep FH4, Millipore) for high-performance liquid chromatography pretreatment, and then concentrated to about 100 μl using nitrogen gas. This was used as a test sample and subjected to high performance liquid chromatography under the following conditions to analyze the quinone composition.

<Quinone analysis conditions in high performance liquid chromatography>
Equipment: High performance liquid chromatograph LC Module 1 (Waters),
Detection wavelength: 270 nm,
Analytical column: m-Bondasphere (5 m, C18, 100 mm) (3.9 x 150 mm) (Waters),
Guard column: Delta-Pak (C18,100 mm) (Waters),
Column temperature: room temperature (15-25 ° C),
Mobile phase: methanol-isopropanol (2: 1, v / v),
Flow rate: 1 ml / min
Sample volume: 10 μl,
Standard samples: Menaquinone-6, Menaquinone-7, Ubiquinone-7, Ubiquinone-8, and Ubiquinone-10 are used.

(B) Test results The results are shown in Table 1.

表１に示す結果から、土壌から分離したマンナナーゼ生産候補株（本発明菌体）は、好アルカリ性のBacillus属細菌であることが分かった。さらに16S rDNA塩基配列の相同性解析の結果、B. alcalophilus（DSM485^T）と98.9%の相同性を、B. pseudoalcaliphilus（DSM8725^T）と97.3％の相同性を有していた。この結果から、本発明菌体は、B. alcalophilusに近縁する、Bacillus属に属する新種の菌株（微生物）であると考えられた。このため、当該本発明菌体を、「Bacillus sp. Strain JAMB-750」（略称：「JAMB-750株」)と命名して、平成１６年９月２４日付けで、日本国茨城県つくば市東１丁目１番地１中央第６に住所を有する独立行政法人産業技術総合研究所、特許生物寄託センターに、寄託者が付した識別のための表示を「Bacillus sp. Strain JAMB-750」とし、受領番号を「ＦＥＲＭ ＡＰ−２０２２７」として寄託した（通知番号：１６産生寄第２２７号）。

From the results shown in Table 1, it was found that the mannanase production candidate strain (the cell of the present invention) isolated from soil was an alkalophilic Bacillus genus bacterium. Further homology analysis of 16S rDNA base sequence result, the homology 98.9 percent B. alcalophilus (DSM485 ^T), had homology 97.3 percent B. pseudoalcaliphilus (DSM8725 ^T). From these results, it was considered that the bacterial cell of the present invention is a new strain (microorganism) belonging to the genus Bacillus , closely related to B. alcalophilus . For this reason, the cell of the present invention was named “Bacillus sp. Strain JAMB-750” (abbreviation: “JAMB-750 strain”) and dated September 24, 2004, East of Tsukuba City, Ibaraki Prefecture, Japan. 1-chome, 1-address, 1 center, 6th address, National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, “Bacillus sp. Strain JAMB-750” is displayed for identification. The number was deposited as “FERM AP-20227” (Notification Number: 16 Production No. 227).

Example 2 Purification of Mannanase (Native) The above Bacillus sp. Strain JAMB-750 strain (hereinafter simply referred to as “JAMB-750 strain”) was transformed into a liquid medium (0.2% locust bean gum, 5% (v / v) Mieki ( Ajinomoto), 0.2% yeast extract, 0.5% bonito meat extract (Wako Pure Chemicals), 0.1% KH ₂ PO ₄ , 0.1% methionine, 0.05% MgSO ₄ .7H ₂ O, 0.5% Na ₂ CO ₃ , pH 6.8) Inoculated and cultured at 30 ° C. for 2 days. After the culture, the culture solution was separated into cells and supernatant by centrifugation (10,000 × g , 10 minutes), and the supernatant was used as a crude enzyme solution, from which mannanase was purified. All purification steps were performed at 4 ° C. or lower.

  Specifically, first, ammonium sulfate was added to the culture supernatant so as to be 30-60% saturation and centrifuged, and then a small amount of 10 mM sodium phosphate buffer (pH 7.0) was added to the resulting precipitate. ) Was added and dissolved. This was dialyzed overnight against 10 mM sodium phosphate buffer (pH 7.0). After dialysis, the dialysate was adsorbed on a DEAE-Toyopearl 650M (Tosoh) column (2.5 × 20 cm) equilibrated with 10 mM sodium phosphate buffer (pH 7.0), and 10 mM containing 150 mM NaCl. Non-adsorbed protein was eluted by passing 200 ml of sodium phosphate buffer (pH 7.0). Subsequently, mannanase was eluted with a sodium chloride concentration gradient of 150 mM to 300 mM using a 10 mM sodium phosphate buffer (pH 7.0) containing sodium chloride. The mannanase active fractions were combined and adsorbed onto a hydroxyapatite (Nippon Chemical) column (2.5 × 20 cm) equilibrated with 10 mM sodium phosphate buffer (pH 7.0), and 50 mM sodium phosphate buffer (pH 7.0) After washing with 200 ml, mannanase was eluted using a phosphate buffer (pH 7.0) with a phosphate concentration gradient of 50 mM to 125 mM. The mannanase active fraction was concentrated by ultrafiltration (Amicon Ultra 10k; Millipore) and then gel filtration (Bio-Gel) equilibrated with 10 mM phosphate buffer (pH 7.0) containing 500 mM sodium chloride. A-1.5m Gel; Bio-Rad) column (2.5 × 120 cm) was used for molecular weight fractionation. The mannanase active fraction was concentrated by ultrafiltration (Millipore, Amicon Ultra PL-10), and the buffer was replaced with 5 mM Tris-HCl buffer (pH 7.5). The obtained mannanase activity fraction was subjected to SDS-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli [Nature, 227, 680-685 (1970)]. For electrophoresis, 10% acrylamide gel (80 × 100 mm, 1.0-mm thick) was used, and for protein staining, Coomassie Brilliant Blue (CBB) staining was used. As a result, it was confirmed that the purified protein showed a single band having a molecular weight of about 130 kDa (see the right lane in FIG. 1a).

  Next, the following experiment was performed on the purified protein in order to confirm the mannanase activity.

  After SDS-polyacrylamide gel electrophoresis, the gel was immersed in 50 mM glycine-NaCl-NaOH buffer (pH 9.0) and washed twice with gentle stirring to remove SDS. On this gel, an agar sheet containing 0.7% agar, 0.25% konjac mannan and 50 mM glycine-NaCl-NaOH buffer (pH 9.0) was laminated, and incubated at 40 ° C. for 1 hour. The agar gel was then immersed in a 0.1% Congo red solution at room temperature for 10 minutes and washed several times with a 1% sodium chloride solution. As a result, the purified protein having a molecular weight of about 130 kDa was confirmed to exhibit mannanase activity (see FIG. 1, b lane).

  Table 2 shows the degree of purification and yield of mannanase in each purification step. As shown in Table 2, mannanase was purified 459 times by the above purification operation, and showed a specific activity of 40 U / mg. The yield was 9%.

実施例３ アミノ末端領域のアミノ酸配列分析
実施例２で精製した酵素（アルカリ性マンナナーゼ）試料を、メタノールに浸潤させた２フッ化ポリビニリデン（polyvinylidene difluoride）膜（Applied Biosystems）上にプロットし、プロテインシークエンサー（model 476A, Applied Biosystems）を用いて、当該酵素のアミノ末端領域のアミノ酸配列を直接分析した。

Example 3 Amino acid sequence analysis of amino terminal region The enzyme (alkaline mannanase) sample purified in Example 2 was plotted on a polyvinylidene difluoride membrane (Applied Biosystems) infiltrated with methanol, and protein sequencer (Model 476A, Applied Biosystems) was used to directly analyze the amino acid sequence of the amino terminal region of the enzyme.

  As a result, the amino acid sequence of the amino terminal region of mannanase (Native) purified in Example 2 was found to be Glu-Ser-Lys-Ile-Pro-Lys-Asp-X-Glu-Gly (ESKIPKDXEG). . In addition, although X was unidentified among the said amino acid sequences, it was estimated from the base sequence of alkaline mannanase specified in Example 5 that it is serine (Ser).

Example 4 Enzymological properties of mannanase (Native) (1) Optimum pH
50 mM buffer (pH 4.1-pH 6.0: acetate buffer; pH 5.8-pH 8.5: phosphate buffer) with various pH (pH 4.1-11) containing substrate (final concentration 0.25% locust bean gum) pH 7.8-pH 11: glycine-NaCl-NaOH buffer) was added with mannanase (0.2 U / ml) purified in Example 2, and the enzyme reaction was carried out at 40 ° C. for 5 minutes. Other reaction conditions were the same as those for the dinitrosalicylic acid method described in Reference Examples. The results are shown in FIG. From this result, the mannanase derived from the JAMB-750 strain of the present invention has high activity on the alkali side, and the optimum pH is pH 9 to 10.5, preferably pH 9.5 to 10.2, particularly around pH 10. I found out. This optimum pH was the highest among other mannanases including mannanases belonging to B. alcalophilus [T. Akino et al., Appl. Microbiol. Biotechnol., 26, 323-327 (1987) Such].

(2) pH stability Mannanase (0.3 U / ml) purified in Example 2 was mixed with Britton & Robinson universal buffer (pH 3.5-pH 11.6) (concentration at the time of enzyme mixing 10 mM), and various pH conditions. After treatment for 30 minutes at 40 ° C. under pH 3-12, the remaining activity of mannanase in the reaction solution was measured using 0.1 ml thereof according to the dinitrosalicylic acid method described in the Reference Example. The results are shown in FIG. From this result, it was found that the mannanase was stable in the range of pH 6 to 10.5, particularly pH 6.5 to 10.

(3) Optimal temperature
Mannanase purified in Example 2 (0.2 U / ml) was added to a substrate solution (final concentration 0.25% locust bean gum, 50 mM glycine-NaCl-NaOH buffer, pH 9.0) heated to 20-70 ° C. The enzyme reaction was performed for 5 minutes (total volume 1 ml). Other reaction conditions were in accordance with the conditions of the dinitrosalicylic acid method described in Reference Examples. The results are shown in FIG. From this result, it was found that the optimum temperature was 50 to 60 ° C, particularly around 55 ° C.

(4) Temperature stability The temperature stability of the mannanase was examined. Specifically, mannanase (0.3 U / ml) purified in Example 2 was dissolved in 5 mM Tris-HCl buffer (pH 7.5), and 40 minutes, 50 degrees C, 60 degrees C, 10 minutes, 20 minutes, After treatment for 30 minutes, 40 minutes, 50 minutes and 60 minutes, 0.1 ml was used to measure the residual activity of mannanase according to the conditions of the dinitrosalicylic acid method described in the Reference Example. The results are shown in FIG. 3B (40 ° C .: black circle, 50 ° C .: black square, 60 ° C .: black triangle). As can be seen from the results, the mannanase of the present invention was stable even when treated at 40 ° C. (indicated by black circles in the figure) for 60 minutes. The initial rate constant (k) of irreversible thermal inactivation at 50-60 ° C. showed a linear regression line on semilogarithmic coordinates. The half-life at 50 ° C. was about 117 minutes, and the half-life at 60 ° C. was about 65 minutes. From this result, the mannanase of the present invention is a known alkaline mannanase [T. Akino et al., Appl. Microbiol. Biotechnol., 26, 323-327 (1987); T. Akino et al., Appl. Environ. Compared to Microbiol., 55, 3178-3183 (1989)], it was considered to be a widely available enzyme with this feature as an advantageous feature.

(5) Measurement of influence of metal ions and compounds Mannanase (0.2 U / ml) is mixed with various compounds (Table 3) or various metal salts (Table 4) (concentration at the time of enzyme mixing is 1 mM), 5 mM. Treated in Tris-HCl (pH 7.5) at 40 ° C. for 30 minutes. 0.1 ml was taken out of the solution, and the mannanase residual activity was measured according to the conditions of the dinitrosalicylic acid method described in the Reference Example. Table 3 shows the effects of various compounds on mannanase activity, and Table 4 shows the effects of metal salts.

この結果から、酢酸ヨード、ヨウ化アセトアミド、N-エチルマレイミド、ジチオスレイトール、1-エチル-3-(3-ジメチル-アミノプロピル)カーボネート、ジエチルピロカーボネート、EDTA、EGTA及び2-メルカプトエタノールは、実質的にマンナナーゼに影響しなかった。一方、N-ブロモスクシニミド（NBS）はマンナナーゼの活性を完全に阻害した。幾つかの酵素に対するNBSの阻害は、タンパク質中のトリプトファンの酸化であることが知られている[Methods Enzymol., 11, 498-506 (1967)]。このことから、マンナナーゼの触媒活性または構造の維持にトリプトファン残基が関わっていると考えられる。

From this result, acetate iodo, iodoacetamide, N -ethylmaleimide, dithiothreitol, 1-ethyl-3- (3-dimethyl-aminopropyl) carbonate, diethylpyrocarbonate, EDTA, EGTA and 2-mercaptoethanol are There was virtually no effect on mannanase. On the other hand, N- bromosuccinimide (NBS) completely inhibited mannanase activity. Inhibition of NBS on some enzymes is known to be tryptophan oxidation in proteins [Methods Enzymol., 11, 498-506 (1967)]. This suggests that tryptophan residues are involved in maintaining the catalytic activity or structure of mannanase.

On the other hand, for metals, metal ions (chlorides) such as Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Cu ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ are strong or moderate. (60-100%) inhibited mannanase activity. In contrast, under the same conditions, Na ⁺ , K ⁺ , Mn ²⁺ , Ca ²⁺ , Mg ²⁺ , Ni ²⁺ and Co ²⁺ did not substantially affect mannanase activity. Therefore, the mannanase of the present invention is a known alkaline mannanase [T. Akino et al., Appl. Microbiol. Biotechnol., 26, 323-327 (1987); T. Akino et al., Appl. Environ. Compared to Microbiol., 55,3 178-3183 (1989)], the enzyme was found to be relatively sensitive to heavy metals.

(6) TLC analysis and substrate specificity of hydrolyzate Using the mannanase purified in Example 2, ivory nut (mannose homopolymer; Megazyme), locust bean gum, guar gum, konjac mannan, and manno-oligosaccharide (disaccharide) ~ 6 sugars; Megazyme) were each hydrolyzed and the products produced over time were measured by TLC analysis. Specifically, 0.4% ivory nut, locust bean gum, guar gum, or konjac mannan, or manno-oligosaccharide (disaccharide [M2], trisaccharide [M3], tetrasaccharide [M4], pentasaccharide [M5], A solution (50 mM glycine-NaCl-NaOH buffer, pH 9.0) containing hexasaccharide [M6]) and mannanase (0.05 U / ml) was reacted at 40 ° C. (10 minutes, 1 hour, 17 hours). After a certain period of time, the reaction solution was heat-treated in boiling water for 5 minutes to stop the enzyme reaction. Spot 5 μl of each sample on a TLC plate (Silica gel 60, 20 × 10 cm; MERCK), including water, butanol and acetic acid water [water: butanol: acetic acid (1: 2: 1, v / v)] Double development was performed using a developing solvent. After development, 10% (v / v) sulfuric acid was sprayed on the TLC plate and heat-treated to develop spots. The results using ivory nuts, locust bean gum, guar gum, or konjac mannan as a substrate are shown in FIG. 4 (a), and the results using mannooligosaccharides (disaccharide to 6 sugars) as a substrate are shown in FIG. 4 (b).

  The relative activity of the mannanase of the present invention for locust bean gum, ivory nut, konjac mannan and guar gum was about 100: 60: 100: 30.

  In particular, from the results shown in FIG. 4B, the mannanase of the present invention has a strong hydrolytic ability for pentasaccharide and hexasaccharide manno-oligosaccharides, and also has a slight hydrolytic ability for tetrasaccharide manno-oligosaccharides. However, disaccharide and trisaccharide manno-oligosaccharides were found to have no hydrolytic ability.

  From this, it was shown that the mannanase of the present invention hydrolyzes manno-oligosaccharides larger than mannan and tetrasaccharide, and this hydrolysis pattern showed that the mannanase of the present invention is an endo-acting mannanase.

  As described above, mannanase derived from the JAMB-750 strain exhibits high activity at a high pH and is stable at high temperatures, and thus is considered useful in the fields of pulp bleaching, washing, and pharmaceuticals.

Example 5 Cloning of Mannanase Gene and Determination of Base Sequence Bacillus sp. Strain JAMB-750 (JAMB-750 strain) isolated in Example 1 was added to mannan-containing solid medium (0.2% locust bean gum, 0.5% Polypepton S). , 0.5% yeast extract, 0.1% K 2 HPO 4, extract ^{_{0.02% MgSO 4. 7H 2 0,0.5}} % Na 2 CO 3, 1.5% and cultured with agar), genomic according to a conventional method from the obtained cultured cells DNA [H. Saito and K. Miura, Biochim. Biophys. Acta, 72, 619-629 (1963)]. This genomic DNA was digested with Eco RI and purified using High Pure PCR Product Purification kit (Roche Diagnostics). This was digested with Eco RI and then ligated to the pUC18 plasmid vector previously treated with alkaline phosphatase (Roche Diagnostics) using DNA Ligation Kit Ver.2 (TaKaRa). After the ligated plasmid was introduced into competent cells ( E. coli HB101) and transformed [D. Hanahan, Mol. Gen. Genet., 166, 557-580 (1983)], this was transformed into ampicillin (100 It was applied to Luria-Bertani (LB) solid medium (Difco) containing μg / ml) and cultured at 37 ° C. overnight. After incubation, the above LB solid medium was overlaid with a gel containing 0.25% konjac mannan, 50 mM glycine-NaCl-NaOH buffer (pH 9.0), 0.7% agar and 1 mg / ml lysozyme, and incubated at 37 ° C. for 4 hours. Next, a 0.1% Congo red aqueous solution was poured onto the gel layer. Then, a clone having a clear zone (halo formation) around it was isolated as a clone having a mannanase activity, that is, a mannanase gene (positive clone).

  A plasmid was extracted from this positive clone (using High Pure Plasmid Isolation kit [Roche]), and the base sequence of the mannanase gene introduced into it was analyzed using the ABI Prism Big Dye Terminator Cycle Sequencing Kit and ABI 377 Sequencer. Decided. Since this plasmid lacked the downstream region of the mannanase gene, the downstream region of the mannanase gene was obtained using inverse PCR and LA PCR in vitro Cloning Kit (manufactured by Takara), and the entire nucleotide sequence of the mannanase gene was determined. FIG. 5 shows the determined base sequence of the mannanase gene and the amino acid sequence of mannanase deduced therefrom. The mannanase gene had a 2991 bp open reading frame (ORF) encoding a protein of 997 amino acids (109619 Da), which contained G + C at a rate of about 38.7 mol%.

  A potential ribosome binding site (5'-AAGGAG-3 ') is located 7 bp upstream of the start codon ATG, and the putative promoter sequences 5'-TTGATG-3' (-35 region) and 5'- TATCGT-3 ′ (-10 region) was located 205 bp upstream of the start codon and 19 bp apart.

  There was a potential cleavage site between Ala26 and Glu27, and the presence of a signal sequence consisting of 26 amino acids in the N-terminal region was observed. From this, the molecular weight of the mature enzyme (mature) was 106818 Da (971 amino acids), and the isoelectric point was calculated to be pI 4.12.

  The amino acid sequence estimated for the alkaline mannanase derived from JAMB-750 strain from the base sequence of the mannanase gene was analyzed by computer homology analysis using FASTA algorithm, and the amino acid sequence of other known mannanases [glycoside hydrolase (GH) family 26] And homology analysis (http://afmb.cnrs-mrs.fr/CAZY).

  The results are shown in Table 5. From these results, it was found that alkaline mannanase derived from the bacterial cell strain JAMB-750 of the present invention is a novel mannanase having very low homology in amino acid sequence with other mannanases derived from GH family 26.

実施例６ マンナナーゼの発現（組み換えマンナナーゼ）
JAMB-750株のゲノムDNAを鋳型として、LA Taq DNA polymerase（TaKaRa）を用いてPCRを行ない、マンナナーゼ遺伝子を増幅した。PCRに際して、マンナナーゼ遺伝子の5'及び3'末端領域の配列を有するオリゴヌクレオチドにBamHI制限酵素部位（下線部）を導入した下記の２つプライマーを作成し、これを使用した。
5'-TCTTGGATTCACAATGTCAAAATCGTAACGAGC-3'(配列番号６)
5'-CGTTTGGATCCAGTVAATGGAATAGAC-3' (配列番号７)。

Example 6 Expression of Mannanase (Recombinant Mannanase)
PCR was performed using LA Taq DNA polymerase (TaKaRa) with the genomic DNA of JAMB-750 strain as a template to amplify the mannanase gene. During PCR, the following two primers were prepared by using a Bam HI restriction enzyme site (underlined) in an oligonucleotide having sequences of the 5 'and 3' end regions of the mannanase gene and used.
5'-TCTT GGATTC ACAATGTCAAAATCGTAACGAGC-3 '(SEQ ID NO: 6)
5′-CGTTTGGATCCAGTVAATGGAATAGAC-3 ′ (SEQ ID NO: 7).

The amplified mannanase gene was treated with the restriction enzyme Bam HI and then ligated to the pHY300PLK ( E. coli - B . subtilis shuttle vector) (TaKaRa) digested with the same restriction enzyme and treated with alkaline phosphatase. did. The ligated plasmid was introduced into competent cells ( E. coli HB101). The obtained transformant ( E. coli HB101) was applied to an LB solid medium containing tetracycline (15 μg / ml) and cultured at 37 ° C. overnight. After culturing, a gel containing 0.25% konjac mannan, 50 mM glycine-NaCl-NaOH buffer (pH 9.0), 0.7% agar and 1 mg / ml lysozyme was overlaid on the LB solid medium, and incubated at 37 ° C. A halo was detected with a% Congo red aqueous solution, and the mannanase activity of the transformant ( E. coli HB101) was evaluated.

Example 7 Purification of Mannanase (Recombinant) A strain (transformant) having halo-forming ability in Example 6 was used as a mannanase gene-bearing strain, and a plasmid was extracted. Bacillus subtilis ISW1214 (TaKaRa) was transformed with the obtained plasmid [S. Chang et al., Mol. Gen. Genet., 168, 111-115 (1979)]. This was cultured in an LB regenerated solid medium (commercial product) containing ampicillin (100 μg / ml) at 30 ° C. for 1-2 days.

After culturing, the resulting transformant ( B. subtilis ISW1214) was added to a liquid medium (10% corn steep liquor (Wako Pure Chemical Industries), 0.1% yeast extract, 0.2% bonito meal extract, 0.1% KH ₂ PO ₄ , 0.02 % MgSO ₄ .7H ₂ O, 7% maltose, 0.05% CaCl ₂ , ampicillin (100 μg / ml), pH 6.8), and aerobically shake culture at 30 ° C. for 3 days. After culturing, the culture solution was separated into cells and supernatant by centrifugation (12,000 × g , 10 minutes), and the supernatant was used as a crude enzyme solution, from which mannanase was purified. All purification steps were performed at 4 ° C. or lower.

  Specifically, first, the culture supernatant was dialyzed overnight against 10 mM phosphate buffer (pH 7.0). After dialysis, the dialysate is adsorbed on a DEAE-650M (Tosoh) column (2.5 x 20 cm) equilibrated with 10 mM phosphate buffer (pH 7.0), and then adsorbed through the same buffer. Protein was eluted. Subsequently, mannanase was eluted by applying a sodium chloride concentration gradient from 0 mM to 500 mM using a 10 mM sodium phosphate buffer (pH 7.0) containing sodium chloride. The mannanase activity fraction was adsorbed on a hydroxyapatite (Nippon Chemical) column (2.5 x 20 cm) equilibrated with 10 mM phosphate buffer (pH 7.0), and then adsorbed on 50 mM phosphate buffer (pH 7.0). And the non-adsorbed protein was eluted. Next, mannanase was eluted using a phosphate buffer (pH 7.0) with a phosphate concentration gradient of 50 mM to 125 mM. The mannanase activity fraction was concentrated by ultrafiltration (Amicon Ultra 10k; Millipore), and the phosphate buffer was replaced with 10 mM Tris-HCl buffer (pH 7.5). The concentrated sample was adsorbed on a DEAE-650M column equilibrated with the same buffer, and passed through the same buffer to elute non-adsorbed protein. Subsequently, mannanase was eluted by applying a sodium chloride concentration gradient from 0 mM to 500 mM using a 10 mM sodium phosphate buffer (pH 7.0) containing sodium chloride. The mannanase activity fraction was concentrated by ultrafiltration, and the buffer was replaced with 5 mM Tris-HCl buffer (pH 7.5). The obtained mannanase activity fraction was subjected to SDS-polyacrylamide gel electrophoresis, and it was confirmed that the purified enzyme showed a single band having a molecular weight of about 130 kDA. The molecular weight of the mature enzyme predicted from the deduced amino acid sequence was 106818 Da, whereas the molecular weight by SDS-polyacrylamide gel electrophoresis was about 130 kDa for the recombinant enzyme as well as the Native enzyme. The mature enzyme is interpreted as not giving a true molecular weight by SDS-polyacrylamide gel electrophoresis, and the true molecular weight is estimated to be 100-110 kDa. Such a difference in molecular weight is a phenomenon often seen with alkaline enzymes.

  SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli [Nature, 2277, 680-685 (1970)]. For electrophoresis, 10% acrylamide gel was used, and for the staining, Coomassie brilliant blue (CBB) staining was used.

It is the electrophoresis image (SDS-PAGE) of the mannanase (Native) refine | purified in Example 2 (lane P in a). In the figure, lane M in a (CBB staining) is a migration image of a marker for indicating the molecular weight of the protein. b (activity staining) shows the result of confirming the mannanase activity for lane P of a. The single band observed in lane P is shown to have mannanase activity (arrow). In Example 4, it is a figure which shows the result of having measured the optimum pH (FIG. 2 (a)) and pH stability (FIG. 2 (a)) of the mannanase prepared and refine | purified in Example 2. FIG. In Example 4, it is a figure which shows the result of having measured the optimal temperature (FIG. 3 (a)) and temperature stability (FIG.3 (b)) of the mannanase prepared and refine | purified in Example 2. FIG. In FIG. B,-●-is the result of examining the stability at 40 ° C,-■-is at 50 ° C, and-▲-is the result at 60 ° C. FIG. 4 (a) shows the hydrolysis pattern of mannanase purified in Example 2 on ivory nut (mannose homopolymer; Megazyme), locust bean gum, guar gum, and konjac mannan (lane 0: unreacted, lane 1 / 6; reaction at pH 9, 40 ° C. for 10 minutes, lane 1; reaction at pH 9, 40 ° C. for 1 hour, lane 17; reaction at pH 9, 40 ° C. for 17 hours), FIG. Hydrolysis pattern of mannanase purified in 2 to mannooligosaccharide (disaccharide [M2], trisaccharide [M3], tetrasaccharide [M4], pentasaccharide [M5], hexasaccharide [M6]) (lane 0: unreacted) Lane 1/6; reaction at pH 9, 40 ° C. for 10 minutes, lane 1; reaction at pH 9, 40 ° C. for 1 hour, lane 17; reaction at pH 9, 40 ° C. for 17 hours). It is a figure which shows the gene sequence of the mannanase derived from the microbial cell (Bacillus sp. Strain JAMB-750) of this invention, and the amino acid sequence deduced from it.

Claims (24)

Alkaline mannanase having the following physicochemical properties:
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺
(H) Molecular weight by SDS-PAGE: about 130 kDa. Alkaline mannanase comprising the protein described in the following (a) or (b):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein (a) action having an amino acid sequence in which one or a plurality of amino acid sequences is deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and having at least one physicochemical property listed below: : It has an endo-type mannanase activity that acts on manno-oligosaccharides having 4 or more sugars and hydrolyzing them to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ . The alkaline mannanase according to claim 2, wherein the protein shown in (b) has an amino acid sequence which is 40% or more identical to the amino acid sequence shown in SEQ ID NO: 1. Alkaline mannanase comprising a protein encoded by the DNA described in (c) or (d) below:
(C) DNA consisting of the base sequence shown in SEQ ID NO: 2,
(D) a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 2 and encodes a protein having at least one of the following physicochemical properties:
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ . The alkaline mannanase according to any one of claims 1 to 4, which is an enzyme derived from a microorganism belonging to the genus Bacillus of an alkalophilic mesophilic bacterium. The alkaline mannanase according to claim 5, wherein the microorganism belonging to the genus Bacillus of an alkalophilic mesophilic bacterium is "Bacillus sp. Strain JAMB-750" (reception number FERM AP-20227). Alkaline mannanase gene encoding the protein described in the following (a) or (b):
(A) a protein having the amino acid sequence shown in SEQ ID NO: 1,
(B) a protein having an amino acid sequence obtained by deleting, substituting or adding one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO: 1 and having at least one enzymatic property listed below (A) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze them to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ . The alkaline mannanase gene according to claim 7, wherein the protein shown in (b) has an amino acid sequence which is 40% or more identical to the amino acid sequence shown in SEQ ID NO: 1. Alkaline mannanase gene comprising the DNA described in (c) or (d) below:
(C) DNA having the base sequence shown in SEQ ID NO: 2,
(D) Hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 2, and encodes a protein having at least one physicochemical property listed below or a precursor thereof. DNA
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ . A recombinant vector containing the alkaline mannanase gene according to any one of claims 7 to 9. A transformant obtained by transforming a host cell with the recombinant vector according to claim 10. The transformant according to claim 11, wherein the host cell is Bacillus subtilis. The alkaline transformant according to any one of claims 1 to 6, wherein the transformant according to claim 11 or 12 is cultured in a medium, and alkaline mannanase having the following physicochemical properties is collected from the obtained culture. Mannanase production method:
(I) Action: It has an endo-type mannanase activity that can act on mannanoligosaccharides having 4 or more sugars and hydrolyze to 3 or less sugars.
(B) Optimal pH: having an optimal pH in the range of pH 9 to 10.5,
(C) pH stability: having a mannanase activity of 70% or more under the treatment conditions of 40 ° C., pH 6.5-10, 30 minutes,
(D) Optimal temperature: It has an optimal temperature in the range of around 55 ° C.
(E) Temperature stability: having an activity of 50% or more under the treatment conditions of pH 7.5, 50 ° C. and 110 minutes, or the treatment conditions of pH 7.5, 60 ° C. and 60 minutes,
(F) Inactivation: Mannanase activity is inactivated by treatment at pH 7.5, 40 ° C. for 30 minutes in the presence of 1 mM N- bromosuccinimide.
(G) Metal inhibition: ^Inhibited by Fe ³⁺ , Fe ²⁺ , Pb ²⁺ , Zn ²⁺ , Hg ²⁺ , Cd ²⁺ , and Sn ²⁺ . Microorganism “Bacillus sp. Strain JAMB-750” (reception number FERM AP-20227). An enzyme composition comprising the alkaline mannanase according to any one of claims 1 to 6. A cleaning composition comprising at least the alkaline mannanase according to any one of claims 1 to 7 and a surfactant (excluding a textile product cleaning composition). A bleaching composition comprising at least the alkaline mannanase according to any one of claims 1 to 7. A composition for fiber softening treatment containing at least the alkaline mannanase according to any one of claims 1 to 7. A composition for treating a coffee extract containing at least the alkaline mannanase according to any one of claims 1 to 7. A method for producing a manno-oligosaccharide comprising a step of performing an enzyme treatment using the alkaline mannanase according to any one of claims 1 to 7. A composition comprising mannooligosaccharides produced by the method according to claim 20 and biodegradable plastics. A ground crushing agent comprising the alkaline mannanase according to any one of claims 1 to 7 or the enzyme composition according to claim 15. A method for drilling a well, hot spring or oil using the ground crushing agent according to claim 22. A white water scum inhibitor comprising the alkaline mannanase according to any one of claims 1 to 7 or the enzyme composition according to claim 15.

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