JP2009153516A - beta-L-ARABINOPYRANOSIDASE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide β-L-arabinopyranosidase mass producible by bacteria and the like. <P>SOLUTION: The β-L-arabinopyranosidase is a monomer, whose optimum PH of activity is ≥3 and ≤5 and optimum temperature of activity is ≥30°C and ≤50°C, and whose molecular weight by SDS-PAGE measurement is in a range of ≥40 kDa but ≤71 kDa. The β-L-arabinopyranosidase is mass producible by transformant and can produce arabinose and saccharides containing arabinose by acting to saccharides such as arabinogalactan. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to β-L-arabinopyranosidase.

Arabinose is a monosaccharide classified into pentose sugar and aldose, and exists as a constituent sugar of polysaccharides such as arabinan, arabinoxylan, arabinogalactan in hemicellulose constituting the cell wall of plants. For example, the arabinan is a polysaccharide in which arabinose is α1,5-linked, and is a constituent such as sugar beet pulp, which is a pomace of roots of sugar beet ( Beta vulgaris var. Saccharifera ). Further, the arabinogalactan is a polysaccharide arabinose and galactose are bonded, arabic rubber tree acacia gum which is taken from (Acacia senegal), larch arabinogalactan taken from trees larch (Larix) (Larch Arabinogalactan) Etc. The acacia gum and larch arabinogalactan are widely used as an emulsifier for foods such as confectionery and ice cream, and a stabilizer for pharmaceuticals, cosmetics and daily necessities. The sugar beet pulp is used for feed and the like.

  On the other hand, arabinose inhibits the sucrose activity of the human digestive tract, so that it has been known to alleviate the increase in blood sugar when taken together with sucrose. Therefore, it has been attracting attention as a blending raw material for health foods and sweeteners for alleviating blood sugar elevation. Therefore, a method for producing arabinose by causing an enzyme to act on a natural product containing arabinose (for example, Patent Document 1) and an enzyme for releasing arabinose from the natural product (for example, Patent Document 2) have been developed.

The enzyme that liberates arabinose may be α-L-arabinofuranosidase, α-L-arabinopyranosidase, β-L-arabinopyranosidase, α-L-arabinanase, α- It is classified as 1,5-L-arabinanase. Among the arabinan degrading enzymes, for example, the α-L-arabinofuranosidase has been reported to exist in many plants and bacteria (for example, Patent Document 3), and is used for delignification of wood pulp (for example, Patent Document 3) is applied to effective utilization of biomass. On the other hand, β-L-arabinopyranosidase is used as a useful enzyme in oligosaccharide production using the acacia gum or larch arabinogalactan as a raw material, or used in a method for detecting bacterial contamination in food (for example, patent documents). 4) has been reported. However, the β-L-arabinopyranosidase has been reported to have been reported from seeds of cajanus cajan and dry rot fungi ( Fusarium oxysporum ) (Non-Patent Documents 1 to 3), There are very few types, for example, those derived from prokaryotes have not been confirmed. Further, the amino acid sequence of β-L-arabinopyranosidase and the base sequence of the gene have not been clarified. Therefore, a technology for mass production of β-L-arabinopyranosidase by genetic engineering techniques. Is not established. Therefore, although β-L-arabinopyranosidase is expected to be industrially used, it is difficult to put it to practical use.

JP 2006-50996 A JP 2004-248630 A Japanese National Patent Publication No. 7-508162 Japanese National Patent Publication No. 11-506926 P. M.M. Dey, “β-L-Arabinoside from Cajanus indicus: a new enzyme”, Biochimica et Biophysica Acta (1973) 302, p. 393-398 P. M.M. Dey, “Further characterisation of β-L-Arabinosidease from Cajanus indicus”, Biochimica et Biophysica Acta (1983) 746, p. 8-13 Yuya Sugaya and three others, “Two types of β-L-arabinopyranosidases produced by Fusarium oxysporum 12s”, 2006, Annual Meeting of the Japanese Society of Applied Glycoscience, p. 37

  Therefore, an object of the present invention is to provide β-L-arabinopyranosidase capable of mass production.

  In order to achieve the object, the β-L-arabinopyranosidase of the present invention is a monomer, and has an optimum pH of activity in the range of 3 to 5 and the optimum temperature of the activity. Is in the range of 30 ° C. or more and 50 ° C. or less, and the molecular weight by SDS-PAGE measurement is in the range of 40 kDa or more and 71 kDa or less.

The β-L-arabinopyranosidase of the present invention may be the β-L-arabinopyranosidase described in any of (a) to (c) below.
(A) β-L-arabinopyranosidase consisting of the amino acid sequence set forth in SEQ ID NO: 1.
(B) β-L-arabinopyranosidase consisting of an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1.
(C) β-L-arabinopyranosidase consisting of an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1.

  According to the present invention, industrial use of arabinopyranosidase can be pioneered. Furthermore, since the β-L-arabinopyranosidase of the present invention is derived from a microorganism, it can be mass-produced from an industrial point of view, and is excellent in supply durability against mass use or consumption.

The β-L-arabinopyranosidase of the present invention may be derived from cells of the genus Streptomyces . Examples of the cells of the genus Streptomyces include cells of Streptomyces avermitilis . Examples of the cells of Streptomyces avermitilis include MA-4680 strain. The MA-4680 strain is deposited as Streptomyces avermitilis MA-4680 (NBRC number: 14893) with the National Institute of Biotechnology and Genetics (NBRC).

  In the β-L-arabinopyranosidase described in (b) of the present invention, the number of amino acid residues substituted, added, inserted or deleted is, for example, several, preferably 1 or more and 7 The number in the range of not more than 1, more preferably the number in the range of not less than 2 and not more than 4.

  In the β-L-arabinopyranosidase described in (c) of the present invention, the homology to SEQ ID NO: 1 is preferably 60% or more in one aspect, preferably 70% or more in the other aspect, In another aspect, preferably 75% or more, in another aspect, preferably 80% or more, in another aspect, preferably 85% or more, in yet another aspect, preferably 90% or more, or in another aspect Is 95% or more. Examples of homology algorithms include BLAST (hereinafter the same).

Next, the β-L-arabinopyranosidase gene of the present invention is the gene described in any of (d) to (f) below. The β-L-arabinopyranosidase gene of the present invention includes a gene consisting of a base sequence and a complementary sequence described in (d) to (f) below.
(D) A β-L-arabinopyranosidase gene consisting of the base sequence set forth in SEQ ID NO: 2.
(E) A β-L-arabinopyranosidase gene consisting of a base sequence in which one or several bases are substituted, added, inserted or deleted in the base sequence shown in SEQ ID NO: 2.
(F) A β-L-arabinopyranosidase gene comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence set forth in SEQ ID NO: 2.

  In the β-L-arabinopyranosidase gene described in (e) of the present invention, the number of bases substituted, added, inserted or deleted is, for example, in the range of several, preferably one or more and The range is 7 or less, more preferably 2 or more and 4 or less.

  In the β-L-arabinopyranosidase gene according to (f) of the present invention, the homology to SEQ ID NO: 2 is preferably 60% or more in one aspect, preferably 70% or more in the other aspect. In another aspect, preferably 75% or more, in another aspect, preferably 80% or more, in yet another aspect, preferably 85% or more, in yet another aspect, preferably 90% or more, or in yet another aspect. Preferably it is 95% or more.

  The vector of the present invention is a vector containing the β-L-arabinopyranosidase gene of the present invention.

  The transformant of the present invention is a transformant containing the vector of the present invention.

  The first method for producing β-L-arabinopyranosidase of the present invention is a production method including a step of culturing the transformant of the present invention.

The 2nd manufacturing method of (beta) -L- arabinopyranosidase of this invention is a manufacturing method including the process of culture | cultivating Streptomyces avermitilis ( Streptomyces avermitilis ) in the culture medium containing the saccharide | sugar containing an arabinose residue. In the present invention, the arabinose residue is not particularly limited, and examples thereof include an arabinopyranose residue and an arabinofuranose residue, and an arabinopyranose residue is preferable.

  The method for screening a cell producing β-L-arabinopyranosidase of the present invention comprises a step of culturing the cell in a medium containing a saccharide containing an arabinose residue, and β-L of a substance produced by the cell. -Measuring the arabinopyranosidase activity.

  The method for producing arabinose of the present invention is a production method in which β-L-arabinopyranosidase is allowed to act on a saccharide containing an arabinose residue to release arabinose, the β-L-arabinopyrano described above. The β-L-arabinopyranosidase of the present invention is used as a sidase.

  The sugar transfer method of the present invention is a sugar transfer method for transferring an arabinose residue from a saccharide containing an arabinose residue to a sugar receptor, and the β-L-arabi of the present invention is used as an enzyme that catalyzes the sugar transfer. It is characterized by using nopyranosidase. In the sugar transfer method of the present invention, the sugar receptor is preferably a saccharide or an alcohol. In the sugar transfer method of the present invention, the saccharide is preferably at least one selected from the group consisting of D-galactose, glucose, mannose, xylose and L-arabinose. In the sugar transfer method of the present invention, the alcohol is preferably at least one selected from the group consisting of methanol, ethanol, 1-propanol and 1-butanol.

  The method for producing a saccharide of the present invention is a method for producing a saccharide containing an arabinose residue, and is characterized by using the sugar transfer method of the present invention.

  The present invention is described in detail below.

(A) β-L-arabinopyranosidase (A-1) action characteristics of the present invention β-L-arabinopyranosidase of the present invention is an enzyme having β-L-arabinopyranosidase activity. Yes, specifically, an enzyme that hydrolyzes a β-L-arabinopyranose residue contained in a saccharide to release arabinose. Although it does not specifically limit as said saccharide, For example, acacia gum (Gum arabic), larch arabinogalactan (Larch Arabinogalactan), etc. are mentioned. The β-L-arabinopyranosidase of the present invention may have other enzyme activities. The other enzyme activity is not particularly limited, and examples thereof include glycosyltransferase activity for transferring sugar from a sugar donor to a sugar acceptor. The β-L-arabinopyranosidase of the present invention transfers arabinose from a saccharide (sugar donor) containing an arabinose residue to a sugar acceptor, for example, as described above, by the glycosyltransferase activity. Can do.

(A-2) Molecular weight The molecular weight range of the β-L-arabinopyranosidase of the present invention is, for example, 40 kDa or more, preferably 61 kDa or more, more preferably 64 kDa or more in the measurement using the SDS-PAGE method. And has an upper limit of 71 kDa or less, preferably 68 kDa or less, more preferably 66 kDa or less. Examples of specific ranges of the molecular weight are shown in Table 1 below.

  The β-L-arabinopyranosidase of the present invention may be either a natural protein or a recombinant protein. In the measurement using the SDS-PAGE method, the molecular weight of the natural protein of the present invention may be, for example, 64 kDa, and the molecular weight of the recombinant protein of the present invention may be, for example, 65 kDa. Further, the molecular weight of the β-L-arabinopyranosidase of the present invention may be, for example, 64265 Da as a theoretical value. The molecular weight of the natural type and recombinant β-L-arabinopyranosidase of the present invention is not limited to the molecular weight as long as it is within the above-mentioned molecular weight range.

(A-3) pH range showing enzyme activity The pH range in which β-L-arabinopyranosidase of the present invention shows arabinose releasing activity and transglycosylation activity is, for example, pH 2 or higher under the condition of a temperature of 37 ° C. It is a range of 9 or less, preferably a range of pH 2.5 or more and 7.5 or less. In addition, the pH range in which the β-L-arabinopyranosidase of the present invention exhibits an enzyme activity value of about 50% or more with respect to the arabinose releasing activity is, for example, pH 2 or more and 7 or less under the temperature condition. The range is preferably pH 2.5 or more and 6 or less. And, the optimum pH of the both enzyme activities of the β-L-arabinopyranosidase of the present invention is, for example, in the range of pH 3 or more and 5 or less, preferably pH 3.5 or more under the temperature condition. And it exists in the range of 4.5 or less. In addition, the optimum pH of the arabinose releasing activity of the β-L-arabinopyranosidase of the present invention is more preferably pH 4 under the temperature conditions. Furthermore, the pH range in which the β-L-arabinopyranosidase of the present invention exhibits a residual activity value of about 90% or more with respect to the arabinose releasing activity is, for example, pH 3 or more and 9 or 9 at a temperature of 30 ° C. It is the following range, Preferably, it is the range of pH 4 or more and 8 or less.

(A-4) Temperature range showing enzyme activity The temperature range in which β-L-arabinopyranosidase of the present invention shows both enzyme activities is, for example, 10 ° C. or more and 70 ° C. under the condition of pH 4.0. It is the following ranges, Preferably, it is the range of 20 degreeC or more and 60 degrees C or less. The temperature range in which the β-L-arabinopyranosidase of the present invention exhibits an enzyme activity value of about 40% or more with respect to the arabinose releasing activity is, for example, 10 ° C. or more and 60 ° C. under the pH condition. It is the following ranges, Preferably, it is the range of 20 degreeC or more and 50 degrees C or less. And the optimal temperature of the said both enzyme activities of (beta) -L-arabinopyranosidase of this invention exists in the range of 30 degreeC or more and 50 degrees C or less under the said pH conditions, Preferably, it is 35 degreeC. It is above and below 45 ° C. Moreover, the optimal temperature of the said arabinose release activity of (beta) -L- arabinopyranosidase of this invention is more preferably 40 degreeC on the said pH conditions. Furthermore, the temperature range in which the β-L-arabinopyranosidase of the present invention exhibits a residual activity value of about 80% or more with respect to the arabinose releasing activity is, for example, 60 ° C. or less under the condition of pH 4.0. Yes, preferably 45 ° C. or lower, more preferably in the range of 20 ° C. or higher and 45 ° C. or lower.

(A-5) Specific Activity Regarding the arabinose releasing activity, the specific activity of β-L-arabinopyranosidase of the present invention is not particularly limited. For example, in the case of a recombinant protein derived from the MA-4680 strain Is 18 units / mg. The specific activity is a numerical value representing an enzyme activity ratio, where the amount of enzyme that liberates 1 μmol of PNP per minute from PNP-β-L-arabinopyranoside is defined as 1 unit.

(A-6) Amino acid sequence The amino acid sequence of the β-L-arabinopyranosidase of the present invention is the sequence described in any of (a) to (c) above.

(A-7) Comparison of physical properties with known β-L-arabinopyranosidase As described above, β-L has been used so far in the seeds of pigeon bean ( Cajanus cajan ) and dry rot fungi ( Fusarium oxysporum ). -Production of arabinopyranosidase has been reported. Table 2 shows an example of β-L-arabinopyranosidase of the present invention, β-L-arabinopyranosidase derived from the bean seed and β-L-arabinopyrano derived from the dried rot of yam. This is a comparison of physical properties with a sidase. The stable pH in the table is a pH range showing a residual activity value of about 90% or more, and the stable temperature is a temperature range showing a residual activity value of about 90% or more. As illustrated in the table, the β-L-arabinopyranosidase of the present invention has two known β-L-arabinos with respect to molecular weight, specific activity, optimum pH, stable pH, and optimum temperature. It is a novel enzyme that is completely different from pyranosidase.

(A-8) Producing cell The β-L-arabinopyranosidase of the present invention may be produced from, for example, a cell or the like. The cells producing the β-L-arabinopyranosidase of the present invention are not particularly limited, and examples thereof include microorganisms such as Escherichia coli, yeast, filamentous fungi and actinomycetes, cells derived from plants and animals, and the like. It is done. Although it does not specifically limit as said microorganisms, For example, the microorganisms of the said Streptomyces genus ( Streptomyces ), etc. are mentioned. The cell may be, for example, a cell that produces natural β-L-arabinopyranosidase, or a host cell that produces recombinant β-L-arabinopyranosidase, which will be described later. There is no particular limitation.

(B) β-L-arabinopyranosidase gene of the present invention The β-L-arabinopyranosidase gene of the present invention is the gene described in any of (d) to (f) above. The β-L-arabinopyranosidase gene of the present invention includes a gene consisting of the base sequence described in (d) to (f) above and a complementary sequence. The β-L-arabinopyranosidase gene of the present invention may, for example, encode only the amino acid sequence of the β-L-arabinopyranosidase of the present invention, or may be fused with various proteins or peptides. It may be one that encodes a protein. The protein or peptide to be fused is not particularly limited. For example, a maltose binding protein, a histidine tag, a GST protein, etc. for the purpose of simplifying and reducing the cost of protein purification, improving stability and solubilizing, etc. But you can.

(C) Vector of the present invention The vector of the present invention contains the β-L-arabinopyranosidase gene of the present invention as described above. The vector for inserting the β-L-arabinopyranosidase gene of the present invention is not particularly limited as long as it can be replicated in a host cell. For example, a plasmid derived from Escherichia coli, a plasmid derived from actinomycetes, and hay. Examples include plasmids derived from fungi, plasmids derived from yeast, and λ phage. Specifically, for example, pIJ702, pACYC177, pACYC184, pBluescript, pBR322, pHSG367, pNUT4, pTrc99A, pUC19, pUB110, YEp13, λgt10 described later Examples include conjugated actinomycetes plasmid pTONA4. The method for inserting the β-L-arabinopyranosidase gene of the present invention into the vector is not particularly limited, and a conventionally known method can be appropriately employed. Specifically, the method for inserting the gene is, for example, a method in which the purified DNA of the gene is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site or a multicloning site of an appropriate vector, and ligated to the vector. Etc.

  Moreover, the vector of this invention should just be what can express the (beta) -L-arabinopyranosidase gene of this invention, and structures other than the said gene are not limited. Examples of the structure other than the β-L-arabinopyranosidase gene include a base sequence that controls expression and a gene marker for selecting a transformant. The base sequence for controlling the expression is not particularly limited, and examples thereof include cis elements such as promoters and enhancers, splicing signals, poly A addition signals, ribosome binding sequences (SD sequences) and the like. The gene marker is not particularly limited, and examples thereof include a dihydrofolate reductase gene, an ampicillin resistance gene, and a neomycin resistance gene.

(D) Transformant of the present invention The transformant of the present invention contains the vector of the present invention as described above. The transformant of the present invention can be obtained, for example, by introducing a vector capable of expressing the β-L-arabinopyranosidase gene of the present invention into a host cell. The host cell may be selected according to the vector to be introduced, and is not particularly limited. Examples thereof include cells such as actinomycetes, E. coli, yeast, filamentous fungi, yeast, COS cells, CHO cells, and Sf9 cells. Preferably, it is an actinomycete. Examples of the actinomycetes include, but are not limited to, for example, Streptomyces lividans, Streptomyces griseis, Streptomyces avermitilis R., Streptomyces avermiti Is mentioned. The method for introducing the vector into the transformant of the present invention is not particularly limited, and examples thereof include electroporation method, protoplast-PEG method, Agrobacterium method, Li method, Biolistic method, particle gun method and the like. Conventionally known methods can be appropriately used.

(E) Production method of β-L-arabinopyranosidase of the present invention As described above, the first production method of β-L-arabinopyranosidase of the present invention uses the transformant of the present invention. Culturing. In the first production method, the culturing step is not particularly limited, and for example, a conventionally known method can be appropriately employed depending on the transformant to be cultured. The first production method is not particularly limited other than including the culturing step, and may include other steps, for example. In the first production method, the β-L-arabinopyranosidase of the present invention can be purified and collected from the culture obtained by the culturing step. Although it does not restrict | limit especially as said culture, For example, a culture supernatant, a cultured cell, a cell extract etc. are mentioned. The culture may be a processed product such as the culture supernatant, cultured cells, cell extract and the like. The treated product is not particularly limited. For example, the culture concentrate, dried product, lyophilized product, solvent-treated product, surfactant-treated product, enzyme-treated product, protein fraction product, and sonicated product. , Milled products and the like. Further, the culture may be a mixture of the culture supernatant, the cultured cells, the cell extract, and the processed products thereof. The mixture can be mixed in any combination and ratio, and is not particularly limited. The purification method of the culture can be a conventionally known method such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography and the like, and is not particularly limited. Moreover, you may manufacture (beta) -L-arabinopyranosidase of this invention as a crude enzyme liquid which extract | collected the culture obtained by the said culture | cultivation process.

As described above, the second method for producing β-L-arabinopyranosidase of the present invention includes a step of culturing Streptomyces avermitilis in a medium containing a saccharide containing an arabinose residue. . The second production method is not particularly limited except that it includes the culture step, and may include other steps. In the second production method, the medium used in the culturing step is not particularly limited other than containing the saccharide containing the arabinose residue, and a medium suitable for culturing Streptomyces avermitilis is used. It can be adopted as appropriate. The saccharide is not particularly limited as long as it includes an arabinose residue, and examples thereof include gum acacia and larch arabinogalactan. The culture temperature and pH conditions in the culture step are not particularly limited, but for example, the above-described optimum temperature and optimum pH ranges are preferable. In the second production method, the β-L-arabinopyranosidase of the present invention can be purified and collected from the culture obtained by the culturing step. The culture is not particularly limited, and examples thereof include the aforementioned cultures. The method for purifying the culture is not particularly limited and can be a conventionally known method, as in the first production method. Moreover, you may manufacture (beta) -L- arabinopyranosidase of this invention as a crude enzyme liquid which extract | collected the culture obtained by the said culture | cultivation process similarly to said 1st manufacturing method.

(F) Screening Method for Cells Producing β-L-arabinopyranosidase of the Present Invention As described above, the screening method for cells producing β-L-arabinopyranosidase according to the present invention comprises the arabinose residue. A step of culturing the cell in a medium containing a saccharide containing a group; and a step of measuring β-L-arabinopyranosidase activity of a substance produced by the cell. The screening method is not particularly limited except that both the steps are included, and for example, other steps may be included. In the culturing step, the medium is not particularly limited except that it contains a saccharide containing an arabinose residue, and can be appropriately selected according to the cells to be cultured. The saccharide containing an arabinose residue is not particularly limited except that it contains an arabinose residue, and examples thereof include the aforementioned gum acacia and larch arabinogalactan. The cells to be screened are not particularly limited, and for example, cells of microorganisms, plants, animals, etc. can be used as appropriate. The cell culture can be performed using, for example, a conventionally known method according to the cells to be cultured. The step of measuring the β-L-arabinopyranosidase activity is not particularly limited. For example, a substance obtained by binding a chromophore to an arabinopyranoside group as a substrate, A culture supernatant of cells is allowed to act, and the color development amount of the liberated chromophore is measured, and the enzyme activity value may be calculated from the color development amount. The coloring group is not particularly limited, and examples thereof include a 4-nitrophenyl group (PNP group) and a 2-chloro-4-nitrophenyl group (CNP group).

(G) The method for producing arabinose of the present invention is the method for producing arabinose of the present invention, as described above, by causing the β-L-arabinopyranosidase of the present invention to act on a saccharide containing an arabinose residue. To release. The saccharide containing the arabinose residue is not particularly limited, and examples thereof include gum acacia, larch arabinogalactan, beet fiber, beet pulp, and orange fiber. In the method for producing arabinose of the present invention, for example, other glycolytic enzymes may be used in combination as appropriate. The other glycolytic enzyme is not particularly limited, and examples thereof include α-L-arabinofuranosidase, α-L-arabinanase, α-1,5-L-arabinanase, and α-L-arabinopyranosidase. Arabinan-degrading enzymes such as exo-β-1,3-galactanase, endo-β-1,6-galactanase, β-D-galactosidase, α-L-rhamnosidase and the like can be used. In the method for producing arabinose of the present invention, for example, when exo-β-1,3-galactanase is used in combination with a saccharide containing a type II arabinogalactan chain, for example, β- Galactooligosaccharides such as 1,6-galactobiose and β-1,6-galactotriose can be produced. Moreover, in the method for producing arabinose of the present invention, the step of releasing arabinose by allowing the β-L-arabinopyranosidase of the present invention to act is not particularly limited. For example, the saccharide containing arabinose is dissolved. You may add (beta) -L-arabinopyranosidase of this invention to a buffer solution, and it is made to react for predetermined time on predetermined pH and predetermined temperature conditions. The buffer is not particularly limited, and for example, McIlvine buffer, HEPES buffer, phosphate buffer, and the like can be used. The pH condition at the time of the reaction is not particularly limited. However, under the condition of a temperature of 37 ° C., for example, the pH range is 2 or more and 9 or less, preferably the pH range is 2.5 or more and 7.5 or less. More preferably, the pH is 4.0. In addition, the temperature condition during the reaction is not particularly limited. For example, under the condition of pH 4.0, the temperature range is, for example, 10 ° C. or more and 70 ° C. or less, preferably 20 ° C. or more and 60 ° C. or less. More preferably, it is 40 degreeC. Moreover, the manufacturing method of arabinose of this invention may have other processes other than the said reaction process, for example, and is not restrict | limited in particular. The other steps are not particularly limited, and for example, conventionally known steps can be appropriately employed.

(H) Glycosyltransferase method of the present invention As described above, the β-L-arabinopyranosidase of the present invention has glycosyltransferase activity as another enzyme activity in addition to the arabinopyranosidase activity. May be. Therefore, in the sugar transfer method of the present invention, the β-L-arabinopyranosidase of the present invention is used as the catalytic enzyme for the sugar transfer, and the sugar acceptor is converted from a saccharide (sugar donor) containing an arabinose residue. To transfer arabinose residues. The saccharide (sugar donor) containing the arabinose residue is not particularly limited, and examples thereof include arabinose, oligosaccharides containing arabinose residues, polysaccharides, sugar alcohols, amino sugars, glycopeptides, glycolipids, and the like. . In the sugar transfer method of the present invention, the sugar donor may be one or more, for example, two or more may be used in combination, and is not particularly limited. In addition, the sugar receptor is not particularly limited, and examples thereof include sugars, alcohols, amino acids, lipids, proteins, and the like. The saccharide that is the sugar receptor is not particularly limited, and examples thereof include monosaccharides, oligosaccharides, polysaccharides, sugar alcohols, amino sugars, glycopeptides, glycoproteins, glycolipids, and the like. Specifically as said saccharide | sugar which is the said sugar receptor, D-galactose, glucose, mannose, xylose, and L-arabinose are preferable, for example. The alcohol that is the sugar acceptor is not particularly limited, and examples thereof include primary alcohols, secondary alcohols, and tertiary alcohols. Specific examples of the alcohols include methanol, ethanol, 1-propanol, and 1-butanol. In the sugar transfer method of the present invention, the sugar receptor may be one or more, for example, two or more may be used in combination, and is not particularly limited. As the sugar transfer method of the present invention, for example, β-L-arabinopyranosidase of the present invention is added to a buffer solution in which the sugar donor and the sugar acceptor are dissolved, and the enzyme transfers the sugar. The reaction may be performed under temperature and pH conditions that exhibit enzyme activity, and is not particularly limited. The buffer solution is not particularly limited, and for example, the aforementioned buffer solution can be used. The pH condition showing the glycosyltransferase activity is not particularly limited. However, under the condition of a temperature of 37 ° C., for example, the pH range is 2 or more and 9 or less, preferably pH 2.5 or more and 7.5 or less. It is a range. In addition, the temperature condition showing the glycosyltransferase activity is not particularly limited. For example, under the condition of pH 4.0, the temperature range is, for example, 10 ° C. or more and 70 ° C. or less, preferably 20 ° C. or more and The range is 60 ° C. or lower.

(I) Method for producing saccharide containing arabinose residue of the present invention The method for producing a saccharide containing arabinose residue of the present invention uses the sugar transfer method of the present invention as described above. The saccharide containing an arabinose residue is not particularly limited except that it contains an arabinose residue, and examples thereof include oligosaccharides, polysaccharides, sugar alcohols, glycolipids, glycoproteins, glycosides, nucleosides, and nucleotides. Of these, oligosaccharides, polysaccharides, and sugar alcohols are preferable. The said manufacturing method is not restrict | limited especially except using the sugar transfer method of this invention, For example, you may combine another method. As said other method, the conventionally well-known method in the manufacturing method of saccharides can be employ | adopted, for example, Specifically, a hydrolysis method, an enzyme decomposition method, a heating method etc. are mentioned, for example, It does not restrict | limit in particular. .

  EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

(Production of natural β-L-arabinopyranosidase of the present invention)
The β-L-arabinopyranosidase of the present invention was cultured from the Streptomyces avermitilis MA-4680 (NBRC number: 14893) strain as follows and recovered from the culture supernatant. First, the cells of MA-4680 were streaked on an acacia gum-containing agar medium having the following composition, cultured at 30 ° C., inoculated into a liquid medium having the following composition, and pre-cultured (rotated culture) at 28 ° C. The cell suspension obtained by the pre-culture was inoculated into the liquid medium and rotated at 28 ° C. On the 6th day of culture, the culture solution was collected, filtered through filter paper to remove the cells, and the resulting culture supernatant was used as a crude enzyme solution.

(Acacia gum-containing agar medium)
Component Concentration (w / v%)
Gum arabic 1.0
KH ₂ PO ₄ 0.5
MgSO ₄ · 7 hydrate 0.05
Peptone 0.1
Yeast extract 0.1
agar 2.0
Purified water 96.25

(Liquid medium)
Component Concentration (w / v%)
Gum arabic 1.0
KH ₂ PO ₄ 0.5
MgSO ₄ · 7 hydrate 0.05
Peptone 0.1
Yeast extract 0.1
Purified water 98.25

(Measurement of activity of natural β-L-arabinopyranosidase of the present invention)
Using the enzyme substrate shown in Table 3 below, the PNP group released from the enzyme substrate was quantified as follows, and the enzyme activity of the crude enzyme solution was measured. First, 5 μL of the crude enzyme solution was added to 25 μL of each 2 mmol / L enzyme substrate and 20 μL of McIlvaine buffer (0.1 mol / L citric acid solution and 0.2 mol / L sodium dihydrogen phosphate mixed solution, pH 4.0), The reaction was performed at 37 ° C. for 10 minutes. Ten minutes after the start of the reaction, 50 μL of 0.2 mol / L sodium carbonate was further added to the reaction solution to stop the reaction, and the absorbance at 400 nm was measured. From the obtained absorbance, the specific activity (units / mg protein) was calculated with the amount of enzyme that liberates 1 μmol of PNP per minute as 1 unit.

  The graph of FIG. 1 shows the results of measuring various enzyme activities using the crude enzyme solution. In the graph, the horizontal axis represents the enzyme substrate used, and the vertical axis represents the specific activity (Units / L). In addition, in the left column of Table 3, each enzyme substrate indicated by an abbreviation is shown on the horizontal axis of the graph, and the Japanese name is shown in the right column of the same table. As shown in the figure, β-L-arabinopyranosidase activity was observed in the crude enzyme solution. The crude enzyme solution has a specific activity value lower than that of the β-L-arabinopyranosidase activity, but also has α-L-arabinofuranosidase activity and α-D-galactopyrano. Sidase activity was observed.

(Enzyme substrate)

(Purification of natural β-L-arabinopyranosidase of the present invention)
Next, the crude enzyme solution was purified as follows. First, the crude enzyme solution was ultrafiltered using Biomax (molecular weight of 10,000 or less, manufactured by Millipore) and concentrated 10 times. To the concentrated crude enzyme solution, ammonium sulfate was added to 70% saturation, and the mixture was allowed to stand at 4 ° C. overnight to precipitate proteins. The precipitate is recovered by centrifugation, and the recovered product is dissolved in 50 mmol / L acetate buffer (pH 4.0) containing 2 mmol / L CaCl _{2 and then} dialyzed at 4 ° C. for 1 day using the buffer. A crude enzyme solution after ammonium sulfate precipitation was obtained.

The crude enzyme solution after the ammonium sulfate precipitation was washed with 2 mmol / L CaCl ₂ -containing 50 mmol / L acetate buffer (pH 4.0), and then a “SP-Sepharose Fast Flow” column (manufactured by GE Healthcare) was used. The protein separated by ion exchange chromatography was eluted with sodium chloride having a concentration gradient of 0-1 mol / L, and active fractions were collected. The collected fraction was dialyzed with ₂ mmol / L CaCl ₂ -containing 50 mmol / L acetate buffer (pH 4.0) using a trade name “Seamless Cellulose Tube, Small Size 30” (manufactured by Wako Pure Chemical Industries, Ltd.). The dialyzed solution was washed with ₂ mmol / L CaCl ₂ -containing 50 mmol / L acetate buffer (pH 4.0) and then separated by ion exchange chromatography using a “Mono S” column (manufactured by GE Healthcare). The adsorbed protein was eluted with sodium chloride having a concentration gradient of 0-0.5 mol / L, and the active fraction was collected. In addition, the apparatus of the following brand name was used for the said ion exchange chromatography.

(Ion exchange chromatography equipment name)

  About the said fraction obtained in each said refinement | purification step, it carried out similarly to the activity measuring method of the said natural type | mold (beta) -L-arabinopyranosidase, and measured enzyme activity. Table 4 below shows the recovered liquid amount, total protein amount, total β-L-arabinopyranosidase activity, specific activity, degree of purification, and yield obtained in each purification step. As a result of purification, a natural β-L-arabinopyranosidase having 21 units / mg β-L-arabinopyranosidase activity was obtained.

(Molecular weight of the natural β-L-arabinopyranosidase of the present invention)
The active fraction obtained by ion exchange chromatography using the “Mono S” column was electrophoresed by SDS-PAGE to determine the molecular weight of the natural β-L-arabinopyranosidase of this example. FIG. 2 shows a photograph of the gel obtained by electrophoresis of the natural β-L-arabinopyranosidase by SDS-PAGE. Lane 1 shows the results of migration of molecular weight markers (BIO-RAD Low Marker, 1 μg each), and Lane 2 shows the purified β-L-arabinopyranosidase (active fraction after Mono S) obtained in this example. It is an electrophoresis result. As indicated by the arrow in the figure, the molecular weight of β-L-arabinopyranosidase of this example was 64 kDa.

(Determination of N-terminal amino acid sequence of natural β-L-arabinopyranosidase of the present invention)
The N-terminal amino acid sequence of the natural β-L-arabinopyranosidase of this example was determined as follows using a protein sequencer (trade name “protein sequencer G105A type”, manufactured by Hewlett-Packard Company). First, the protein in the gel after SDS-PAGE is transferred to a transfer membrane (trade name “Immobilon- ^PSQ ”, manufactured by Millipore) using a trade name “Transblot SD Cell” (manufactured by BIO-RAD). Blotted. The membrane was stained with 0.1% CBB staining solution (trade name “CBB-R250”), and then the portion on which the protein indicated by the arrow was blotted was cut out. Using the cut-out membrane, the N-terminal amino acid sequence of the natural β-L-arabinopyranosidase of this example was determined according to the protocol of the protein sequencer. As a result, it was revealed that the N-terminal amino acid sequence of the natural β-L-arabinopyranosidase of this example was the amino acid sequence represented by SEQ ID NO: 3.

(Determination of genomic base sequence of natural β-L-arabinopyranosidase of the present invention)
First, using the N-terminal amino acid sequence (SEQ ID NO: 3) of the natural β-L-arabinopyranosidase of this example, genomic information of a known Streptomyces avermitilis MA-4680 (NBRC number: 14893) strain is used. A BLAST search was performed. As a result, the N-terminal amino acid sequence corresponded to SAV2186 (SEQ ID NO: 4). Therefore, based on the information of SAV2186, a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6) were synthesized. In addition, genomic DNA was isolated from Streptomyces avermitilis MA-4680 (NBRC number: 14893) using the trade name “InstaGene (trademark) Matrix” (manufactured by BIO-RAD). Using the two primers, a PCR reaction was performed using the genomic DNA as a template. The PCR reaction was carried out using a trade name “Pusion DNA Polymerase” (manufactured by FINNZYMES), 35 cycles of 1 cycle (98 ° C. for 10 seconds, 58 ° C. for 10 seconds, 72 ° C. for 2 minutes). It was. As a result of electrophoresis of the PCR fragment obtained by the PCR reaction using an agarose gel, a band having a length of 1986 bp was obtained. The PCR fragment was cloned using a trade name “pGEM-T Easy Vector System” (manufactured by PROMEGA), analyzed with a DNA sequencer (trade name “ABI PRISM 310 Genetic Analyzer”, Applied Biosystems), and nucleotide sequence. It was determined. As a result, it was revealed that the genomic base sequence of β-L-arabinopyranosidase of this example consists of the base sequence shown in SEQ ID NO: 2. Moreover, from this sequence information, β-L-arabinopyranosidase of this example is a secretory signal sequence (amino acid numbers 1 to 44), a catalytic module belonging to carbohydrate hydrolase family 27 (GH27), a sugar bond It was revealed that it consists of a sugar-binding module belonging to module family 13 (CBM13). The SAV2186 (SEQ ID NO: 4) was a sequence portion that was conventionally estimated to be a secreted α-galactosidase. Therefore, it was revealed that the β-L-arabinopyranosidase of the present invention having β-L-arabinopyranosidase activity has specific enzyme characteristics that are different from conventional findings.

(Expression of recombinant β-L-arabinopyranosidase of the present invention)
In this example, a mating actinomycete plasmid pTONA4 is prepared as follows, the PCR fragment is introduced into the plasmid pTONA4, and expressed in Streptomyces lividans strain 1326 (NBRC number: 15675). Recombinant β-L-arabinopyranosidase was obtained.

(1) Preparation of plasmid pBSaphII First, pBluescript ll KS (1) (manufactured by TOYOBO) was used as a template, primer 1 (SEQ ID NO: 7), primer 2 (SEQ ID NO: 8) and KOD plus DNA polymerase (manufactured by TOYOBO). Was used to amplify E. coli JM109 ori (0.6 kb) by PCR. The PCR was performed by repeating 30 cycles of 1 cycle (94 ° C. for 2 minutes, 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 1 minute) and then cooling to 4 ° C. It was.

  Next, the Escherichia coli-actinomycete kanamycin resistance gene aphII (1.3 kb) was amplified by PCR. The PCR uses pTYM18 described in a reference document (Onaka et al., J. Antibiotics, 56, 950-956, 2003) as a template, and primer 3 (SEQ ID NO: 9) and primer 4 (SEQ ID NO: 9) as primers. The procedure was the same as that of ori except that 10) was used.

  The obtained fragments of ori and aphII were treated with restriction enzymes Sspl and Pstl, and the two fragments were ligated to each other and then introduced into E. coli JM109. The E. coli was cultured at 37 ° C. using LB medium (1% NaCl, 1% polypebutone and 0.5% yeast extract) containing 50 μg / mL kanamycin. A plasmid pBSaphII containing the ligated fragment was obtained from the E. coli using kanamycin as a selection marker.

(2) Preparation of plasmid pIJE702 The obtained pBSaphII and pIJ702 described in the reference (Molnar et al., J. Ferment. Bioeng., 72, 368-372, 1991) were each treated with the restriction enzyme Pstl, The two fragments were ligated and then introduced into E. coli JM109. The E. coli was cultured in the same manner as the E. coli expressing the pBSaphII. A plasmid pIJE702 containing the ligated fragment was obtained from the E. coli using kanamycin as a selection marker.

(3) Preparation of plasmid pIJEC'702 oriT (0.8 kB) was amplified by PCR. The PCR was performed in the same manner as ori except that pTYM18 was used as a template and primer 5 (SEQ ID NO: 11) and primer 6 (SEQ ID NO: 12) were used as primers. The obtained oriT fragment was treated with restriction enzymes HinclI and Smal. The pIJE702 was treated with the restriction enzyme sspI. The two fragments obtained were ligated together and then introduced into E. coli JM109. The E. coli was cultured in the same manner as the E. coli expressing the pBSaphII. A plasmid pIJEC702 containing the ligated fragment was obtained from the E. coli using kanamycin as a selection marker. Then, the Ndel site of pIJEC702 was deleted by silent mutation using QuikChange II Kit (manufactured by Stratagene), primer 7 (SEQ ID NO: 13) and primer 8 (SEQ ID NO: 14) to obtain pIJEC'702.

(4) Preparation of plasmid pTONA4
From the genomic DNA of Streptomyces cinnamoneu IFO12852 strain, the PLD promoter (PLDp, 0.3 kB) and PLD using the Saito Miura method (Saito et al., Biochem. Biophys. Acta., 72, 619-629, 1963). A terminator (PLDt, 0.2 kB) was prepared.

Amplified by PCR in the same manner as ori except that the PLD promoter was genomic DNA of Streptomyces cinnamoneu IFO12852 as a template and primers 9 (SEQ ID NO: 15) and 10 (SEQ ID NO: 16) were used as primers. I let you. The PLD terminator was amplified by PCR in the same manner as the PLD promoter except that primer 11 (SEQ ID NO: 17) and primer 12 (SEQ ID NO: 18) were used as primers.

  The obtained PLD promoter fragment was treated with restriction enzymes Asel and EcoRI, the PLD terminator fragment was treated with EcoRl and Bglll, and pIJEC'702 was treated with restriction enzymes AseI and BglII. The three fragments obtained were ligated, introduced into E. coli JM109, and cultured as described above. From E. coli JM109, plasmid pTONA4 containing the ligated fragment was obtained using kanamycin as a selection marker.

FIG. 3 shows the structure (restriction enzyme map) of the obtained plasmid pTONA4. As shown in the figure, the plasmid pTONA4 is an expression vector comprising a promoter sequence (PLDp) and a terminator sequence (PLDt) of the pld gene of the Streptomyces cinnamoneu IFO12852 strain.

(5) Preparation of transformant Streptomyces lividans 1326 strain (NBRC number: 15675) was transformed with the pTONA4 to obtain the transformant of this example. The transformant was inoculated into 100 mL of TSB medium placed in a 500 mL baffle flask, and rotationally cultured under conditions of 28 ° C. and 150 rpm. On the third day of culture, the culture solution was collected and filtered through filter paper to remove the cells, thereby obtaining a crude enzyme solution. The crude enzyme solution is precipitated by adding 70% saturated ammonium sulfate, and the resulting precipitate is dissolved in 50 mmol / L phosphate buffer (pH 7.2), dialyzed with the phosphate buffer, and then subjected to Lactosyl-Sepharose column. Separated by the affinity chromatography method used. The affinity chromatography method is described in the reference literature (S. Ito, A. Kuno, R. Suzuki, S. Kaneko, Y. Kawabata, I. Kusakabe & T. HASEGAWA, “Rational affinity purification of native 10 "Journal of biotechnology, 2004, 110, p.137-142). The enzyme solution after the dialysis was applied to a Lactosyl-Sepharose column, eluted with 50 mmol / L phosphate buffer (pH 7.2) at a flow rate of 0.5 ml / min, and separated into fractions. Β-L-arabinopyranosidase activity was measured for the collected fraction. And the fraction which has the said activity was collect | recovered in the said fraction, and it was set as the recombinant beta-L-arabinopyranosidase enzyme liquid after dialysis with a 50 mmol / L phosphate buffer (pH 7.0).

(Molecular weight of the recombinant β-L-arabinopyranosidase of the present invention)
The recombinant β-L-arabinopyranosidase enzyme solution was electrophoresed using SDS-PAGE. The result of the electrophoresis is shown in the photograph of FIG. In the photograph in the figure, lane 1 is a molecular weight marker (trade name “BIO-RAD Low Marker”, manufactured by BIO-RAD), and lane 2 is the recombinant β-L-arabinopyranosidase of this example. It is. The molecular weight of the recombinant β-L-arabinopyranosidase of this example was about 64000 Da, which was close to the molecular weight of the natural β-L-arabinopyranosidase of this example.

(Measurement of the recombinant β-L-arabinopyranosidase activity of the present invention)
The enzyme activity of the recombinant β-L-arabinopyranosidase of this example was confirmed as follows. First, 25 μL of 2 mmol / L PNP-β-L-arabinopyranoside and 20 μL of McIlvaine buffer (0.1 mol / L citric acid solution and 0.2 mol / L sodium dihydrogen phosphate aqueous solution, pH 4.0) 5 μL of the recombinant β-L-arabinopyranosidase enzyme solution was added and reacted at 37 ° C. for 10 minutes. Ten minutes after the start of the reaction, 50 μL of 0.2 mol / L sodium carbonate was further added to the reaction solution to stop the reaction, and the absorbance at 400 nm was measured. From the obtained absorbance, the specific activity (units / mg protein) was calculated with the amount of enzyme that liberates 1 μmol of PNP per minute as 1 unit. As a result, the recombinant β-L-arabinopyranosidase of this example acted on the β-L-bond of arabinose to release PNP, and the specific activity was 18 units / mg. Therefore, the recombinant β-L-arabinopyranosidase of this example has β-L-arabinopyranosidase enzyme activity, and the polynucleotide of this example is β-L-arabinopyranosidase. The gene sequence was confirmed to be encoded.

(Optimum pH of the recombinant β-L-arabinopyranosidase of the present invention)
The optimum pH range of the recombinant β-L-arabinopyranosidase of this example was confirmed as follows. First, 5 μL of the above recombinant β-L-arabinopyranosidase enzyme solution, 2 mmol / L PNP-β-L-arabinopyranoside 25 μL, predetermined pH (pH 2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 7.6) of 20 μL of McIlvine buffer were mixed and reacted at 37 ° C. for 10 minutes. Ten minutes after the start of the reaction, 50 μL of 0.2 mol / L sodium carbonate was further added to stop the reaction, and the absorbance at 400 nm was measured. From the absorbance, the enzyme activity value at each pH was calculated when the enzyme activity value at the pH at which β-L-arabinopyranosidase activity showed the maximum value was taken as 100. The graph of FIG. 5 shows the enzyme activity value. In the graph of the figure, the vertical axis is the enzyme activity value (%), and the horizontal axis is the pH. As shown in the figure, the recombinant β-L-arabinopyranosidase of this example has an optimum pH in the vicinity of pH 4.0, and has an enzyme activity value of 50% or more in the range of pH 2.5 or more and 6 or less. It showed high β-L-arabinopyranosidase activity.

(PH stability of the recombinant β-L-arabinopyranosidase of the present invention)
The pH stability of the recombinant β-L-arabinopyranosidase of this example was confirmed as follows. First, as a buffer solution, Glycine-HCl buffer having a predetermined pH (pH 1.0, 2.0, 2.6), Sodium Phosphate buffer having a predetermined pH (pH 7.5, 8.0), a predetermined pH (pH 8.0, 9.0) Tris-HCl buffer, predetermined pH (pH 9.5, 10.0, 11.0, 12.0, 13.0) Glycine-NaOH buffer and predetermined pH (pH 2.6, 3.0, 4.0, 5.0, 6.0, 7.0, 7.6) were prepared, respectively. Then, 5 μL of the recombinant β-L-arabinopyranosidase enzyme solution of this example, 40 μL of the buffer solution having the predetermined pH and 5 μL of 1 w / v% BSA were mixed and incubated at 30 ° C. for 1 hour. After completion of the incubation, the treated enzyme solution was mixed with McIlvaine buffer (0.1 mol / L citric acid solution and 0.2 mol / L sodium dihydrogen phosphate mixed solution, pH 4.0) and PNP-β-L-arabinopyra. Noside was added, and the remaining activity of β-L-arabinopyranosidase was measured in the same manner as described above. The measurement result is shown in the graph of FIG. In the graph of the figure, the vertical axis is the enzyme activity value (%), and the horizontal axis is the pH. As shown in the figure, the recombinant β-L-arabinopyranosidase of this example showed high pH stability with an enzyme activity value of 90% or more in the range of pH 4.0 or more and 8.0 or less. .

(Optimum temperature of the recombinant β-L-arabinopyranosidase of the present invention)
The optimum temperature of the recombinant β-L-arabinopyranosidase of this example was confirmed as follows. First, 5 μL of the above recombinant β-L-arabinopyranosidase enzyme solution, 2 mmol / L PNP-β-L-arabinopyranoside 25 μL, and 20 μL of McIlvaine buffer (pH 4.0) are mixed, and predetermined temperature conditions The reaction was allowed to proceed for 10 minutes under (20, 30, 40, 50, 60, 70, 80, 90 ° C.). Ten minutes after the start of the reaction, 50 μL of 0.2 mol / L sodium carbonate was further added to stop the reaction, and the absorbance at 400 nm was measured. The enzyme activity value at the temperature at which the β-L-arabinopyranosidase activity showed the maximum value was defined as 100, and the enzyme activity value at each temperature was calculated as a relative value. The graph of FIG. 7 shows the enzyme activity value. In the graph of the figure, the vertical axis is the enzyme activity value (%), and the horizontal axis is the temperature (° C.). As shown in the figure, the recombinant β-L-arabinopyranosidase of this example has an optimum temperature around 40 ° C., and an enzyme activity value of about 40% or more in the range of 20 ° C. or more and 50 ° C. It showed high β-L-arabinopyranosidase activity.

(Temperature stability of the recombinant β-L-arabinopyranosidase of the present invention)
The temperature stability of the recombinant β-L-arabinopyranosidase of this example was confirmed as follows. First, 5 μL of the above recombinant β-L-arabinopyranosidase enzyme solution, 40 μL of McIlvine buffer (pH 4.0) at each temperature (20 to 90 ° C.), and 5 μL of 1% BSA were mixed under a predetermined temperature condition ( 20, 30, 40, 45, 50, 60, 70, 80, 90 ° C.) for 1 hour. After completion of the incubation, the treated enzyme solution was mixed with McIlvaine buffer (0.1 mol / L citric acid solution and 0.2 mol / L sodium dihydrogen phosphate mixed solution, pH 4.0) and PNP-β-L-arabinopyra. Noside was added, and the remaining activity of β-L-arabinopyranosidase was measured in the same manner as described above. The measurement result is shown in the graph of FIG. In the graph of the figure, the vertical axis is the enzyme activity value (%), and the horizontal axis is the temperature (° C.). As shown in the figure, the recombinant β-L-arabinopyranosidase of this example showed high temperature stability with an enzyme activity value of 80% or more in the range of 20 ° C. or more and 45 ° C. or less.

(Comparison of enzyme properties with other β-L-arabinopyranosidases)
As described above, the above-described enzyme characteristics of the recombinant β-L-arabinopyranosidase of this example were compared with the previously reported β-L- derived from the bean ( C. indicus ) and the dry rot fungus ( F. oxysporum ). The enzyme properties of arabinopyranosidase were compared. Table 2 shows a comparison result of the enzyme activities. In addition, the measuring method of the specific activity of the bean-derived β-L-arabinopyranosidase described in the report is as follows. To 500 μL of 10 mmol / L PNP-β-L-arabinopyranoside and 2000 μL of McIlvaine buffer (pH 5.5), 300 μL of a bean-derived arabinopyranosidase enzyme solution was added and reacted at 30 ° C. for 15 minutes. 15 minutes after the start of the reaction, 5 mL of 0.1 mol / L sodium carbonate was further added to the reaction solution to stop the reaction, and the absorbance at 405 nm was measured. The amount of enzyme that liberates 1 μmol of PNP per minute was defined as 1 unit, and the specific activity (units / mg protein) of pigeon bean-derived β-L-arabinopyranosidase was calculated from the obtained absorbance. As shown in the table, the β-L-arabinopyranosidase of this example is different from any of β-L-arabinopyranosidase derived from pigeon pea and taro dry rot fungus (molecular weight, specific activity, (Appropriate pH / stable pH / optimum temperature). Moreover, as described above, the β-L-arabinopyranosidase of this example includes a catalyst module (GH27) and a sugar binding module (CBM13). In the enzyme group having this GH27 catalyst module, it has been revealed that the molecular weight of the catalyst module is about 40,000. From this, it is considered that the β-L-arabinopyranosidase derived from pigeonfish having a molecular weight of 25900 belongs to a family different from the β-L-arabinopyranosidase of this example. Therefore, the β-L-arabinopyranosidase of this example is a novel β-L-arabinopyranosidase that is different from the previously reported β-L-arabinopyranosidase from pigeon and taro dry rot fungi. is there.

(Substrate specificity of the recombinant β-L-arabinopyranosidase of the present invention)
The recombinant β-L-arabinopyranosidase of this example was allowed to act on 20 types of PNP substrates described in Table 5 below, and the specificity to the PNP substrate was confirmed. First, 5 μL of the recombinant β-L-arabinopyranosidase enzyme solution was added to 25 μL of 2 mmol / L PNP substrate and 20 μL of McIlvaine buffer (pH 4.0), and reacted at 37 ° C. for 10 minutes. Next, 50 μL of 0.2 mol / L sodium carbonate was further added to stop the reaction, and the absorbance of the reaction solution at 400 nm was measured. The average value of three measurement values was calculated, and the decomposition activity of each substrate when the decomposition activity for PNP-β-L-arabinopyranoside was taken as 100 was calculated as the decomposition rate (%). The table shows the decomposition rate. As shown in the same table, the recombinant β-L-arabinopyranosidase of this example shows a specific degradation activity against PNP-β-L-arabinopyranoside, Only showed a degradation rate of 1.5% with respect to PNP-α-D-galactopyranoside. From this result, it was shown that the recombinant β-L-arabinopyranosidase of this example has an activity specific to β-L-arabinopyranosidase.

(Production of arabinose and galactooligosaccharides)
The recombinant β-L-arabinopyranosidase of this example and exo-β-1,3-galactanase were allowed to act on larch arabinogalactan as follows, and the resulting degradation product was analyzed. did. First, an enzyme solution was added to a mixed solution of 0.5% larch arabinogalactan and McIlvine buffer (pH 5.0) and reacted at 37 ° C. for 30 minutes. As the enzyme solution, an enzyme solution containing only β-L-arabinopyranosidase (0.03 units / mL), β-L-arabinopyranosidase (0.03 units / mL) and exo- Two types of enzyme solutions containing β-1,3-galactanase (0.03 units / mL) were used. After 30 minutes from the start of the reaction, the reaction solution was heat-treated at 100 ° C. for 10 minutes to stop the reaction, and then high-performance anion-exchange chromatography (pulse permeation detection-pulsed amperometric detection): HPAEC-PA The degradation products were detected using a CarboPac ™ PA1 column (Dionex). FIG. 9 shows the detection result. In the figure, the upper panel is the result of reacting larch arabinogalactan only with the recombinant β-L-arabinopyranosidase (0.03 units / mL) of this example, and the lower panel is It is the result of reacting the recombinant β-L-arabinopyranosidase (0.03 units / mL) and exo-β-1,3-galactanase (0.03 units / mL) of this example. In both panels, the vertical axis represents the detection pulse (PAD, μC), and the horizontal axis represents the retention time (minutes). In addition, the peaks indicated by arrows in the figure are the respective degradation products (sugars) detected, Ara is arabinose, Gal is galactose, Gal2 is β-1,6-galactobiose, and Gal3 is β-1. , 6-galactotriose. As shown in the figure, when only the recombinant β-L-arabinopyranosidase of this example was allowed to act, the production of arabinose was confirmed, and the recombinant β-L-arabino of this example was confirmed. When pyranosidase and exo-β-1,3-galactanase were allowed to act, in addition to arabinose, production of oligosaccharides such as galactobiose and galactotriose could be confirmed.

(Glucosyl transfer reaction 1)
Using PNP-β-L-arabinopyranoside as a saccharide (sugar donor) containing an arabinose residue and D-galactose as a sugar acceptor, the recombinant β of this example was used as follows. Glucose transfer reaction with -L-arabinopyranosidase was performed.

First, 1 mg of PNP-β-L-arabinopyranoside and 4 mg of galactose were added to 15 μL of McIlvaine buffer (0.1 mol / L citric acid solution and 0.2 mol / L aqueous solution of sodium dihydrogen phosphate, pH 4.0). The suspension was suspended, 5 μL of the β-L-arabinopyranosidase enzyme solution of the present invention was added, and the mixture was reacted at 40 ° C. At 0, 27, 48, and 71 hours after the start of the reaction, 2 μL of the reaction solution was sampled, heat-treated at 100 ° C. for 10 minutes to stop the reaction, and 18 μL of water was further added to completely dissolve the insoluble matter. 2 μL of the lysate was applied to TLC (Silica gel 60 F ₂₅₄ aluminum sheets, manufactured by Merck), and developed twice using a developing solvent of ethyl acetate: acetic acid: water = 7: 2: 2. After two developments, TLC was sprayed with sulfuric acid and heated to 170 ° C. to detect sugar spots. In addition, D-galactose and L-arabinose were used as markers and developed twice as described above. The detection result is shown in the photograph of FIG. In the figure, lane 1 is D-galactose, lane 2 is L-arabinose, lane 3 is a reaction solution (0 hours after the start of the reaction), lane 4 is a reaction solution (after 27 hours), and lane 5 is a reaction solution (same as above). 48 hours later), Lane 6 shows the results of developing the reaction solution (after 71 hours). The black spots in the figure are, in order from the top, L-arabinose, D-galactose, and a sugar spot produced after the reaction. As shown in the figure, in the reaction solution (lanes 4 to 6) 27 hours after the start of the reaction, a new spot (a spot of sugar generated after the reaction) was detected below the D-galactose spot. That is, it was suggested that a new sugar was generated by the action of the β-L-arabinopyranosidase of the present invention, and that the β-L-arabinopyranosidase of the present invention has a sugar transfer catalytic action. . Moreover, it was estimated that the newly detected saccharide | sugar (sugar produced | generated after reaction) was a disaccharide which L-arabinose couple | bonded with D-galactose. And the spot of L-arabinose was also detected in the reaction liquid (lanes 4 to 6) after 27 hours from the start of the reaction. That is, the production of L-arabinose by the β-L-arabinopyranosidase of this example was confirmed.

(Glucosyl transfer reaction 2)
Using PNP-β-L-arabinopyranoside as a saccharide (sugar donor) containing an arabinose residue, using a monosaccharide and a disaccharide as a sugar acceptor, A transglycosylation reaction with β-L-arabinopyranosidase was performed. Table 6 below shows the monosaccharides and disaccharides used as sugar receptors.

First, the recombinant β-L-arabi was added to a phosphate buffer (pH 7.0) containing PNP-β-L-arabinopyranoside (50 mg / ml) and various sugar receptors (200 mg / ml). 1 unit of nopyranosidase enzyme solution was added and reacted at 40 ° C. As a control, a reaction solution containing no sugar receptor was reacted in the same manner. Six hours after the start of the reaction, each of the reaction solutions was sampled and heat-treated at 100 ° C. for 10 minutes to stop the reaction. This was diluted with water and subjected to high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (trademark PA 1 (trademark) manufactured by CarboPac (D) column, manufactured by CarboPac (D). The presence or absence of a glycosyl transfer product was detected. Further, from the detected peak area, the sugar transfer rate (%) of the various sugars was calculated using the following formula 1.
Glucose transfer rate (%) = B / (A + B) × 100 (1)
A = Arabinose peak area
B = peak area of glycosylated product

  FIG. 11 shows the detection results when glucose is used as the sugar receptor. In the figure, the upper panel is the result of the control (reaction solution not containing a sugar receptor), and the lower panel is the result of using glucose as a sugar receptor. In both panels, the vertical axis represents the detection pulse (PAD, μC), and the horizontal axis represents the retention time (minutes). Moreover, the peak indicated in the figure is each detected degradation product (sugar), Ara indicates arabinose, Glc indicates glucose, and an asterisk indicates a substance generated after the reaction. As shown in the figure, in addition to the peak of arabinose produced from a sugar donor by the recombinant β-L-arabinopyranosidase of this example and glucose added as a sugar acceptor, a new peak ( The peak of the substance produced after the reaction was detected. That is, the ability of the recombinant β-L-arabinopyranosidase of this example to transfer glucose to glucose was confirmed.

  Table 6 below shows the sugar transfer rates of various sugar receptors. As shown in the table, in the case of monosaccharides, when galactose, glucose, mannose, xylose and L-arabinose are used as sugar receptors, glycosyl transfer products are confirmed. In particular, galactose, glucose and xylose have high sugar transfer. Showed the rate. In addition, in the case of disaccharides, glycosyl transfer products were confirmed when lactose, maltose, and cellobiose were used as sugar receptors. In particular, lactose showed a high rate of sugar transfer. That is, it was confirmed that β-L-arabinopyranosidase of this example catalyzes a transglycosylation reaction using monosaccharide or disaccharide as a sugar acceptor.

(Glucosyl transfer reaction 3)
Using alcohols as the sugar acceptor and PNP-β-L-arabinopyranoside as the saccharide (sugar donor) containing an arabinose residue, the recombinant β-L of this example was used as follows. -Glucose transfer reaction with arabinopyranosidase was performed. In addition, methanol, ethanol, 1-propanol and 1-butanol were used as the alcohols.

First, the recombinant β-L-arabinopyra was added to a phosphate buffer (pH 7.0) containing 2 mmol / L PNP-β-L-arabinopyranoside and various alcohols (15 v / v%). 1 unit of nosidase enzyme solution was added and reacted at 40 ° C. As a control, the phosphate buffer containing no sugar receptor was similarly reacted. Three hours after the start of the reaction, 20 μL of the reaction solution was sampled. The reaction solution and the marker (L-arabinose) were subjected to TLC (Silica gel 60 F ₂₅₄ aluminum sheets, manufactured by Merck), and developed once using a developing solvent of chloroform: methanol: water = 30: 20: 4. went. After development once, sulfuric acid was sprayed on the TLC and heated to 170 ° C. to detect sugar spots. The photo of FIG. 12 shows the detection result. In this figure, lane 1 is a marker (L-arabinose), lane 2 is a control reaction solution, lane 3 is a methanol reaction solution, lane 4 is an ethanol reaction solution, lane 5 is a 1-propanol reaction solution, and lane 6 is 1-butanol. It is the result of developing each reaction solution. In the same figure, the spot indicated by the leftmost arrow is a spot of L-arabinose, and the spot indicated by the rightmost arrow is a spot of a substance (product) generated after the reaction. As shown in the figure, L-arabinose produced from a sugar donor by β-L-arabinopyranosidase of this example was used in reaction solutions (lanes 3 to 6) using various alcohols as sugar acceptors. In addition to these spots, a new spot (product spot) that was not found in the control reaction solution (lane 2) was detected. That is, it was confirmed that the β-L-arabinopyranosidase of this example catalyzes a transglycosylation reaction using alcohol as a sugar acceptor.

(Production of arabinose from glycosyl transfer products)
Production of arabinose was confirmed by allowing the β-L-arabinopyranosidase of the present invention to act on the glycosylated product obtained by the transglycosylation reaction 2 as follows. As the glycosyl transfer product, a glycosyl transfer product produced by a reaction using glucose as a sugar acceptor was used. That is, first, the reaction solution was subjected to chromatography using TOYOPEARL HW-40S to separate and remove PNP and β-L-arabinopyranosidase. The liquid after separation and removal was subjected to chromatography using activated carbon, monosaccharides were separated and removed, and fractions containing the glycosylated products were collected. Each collected fraction was concentrated by an evaporator and subjected to TLC (Silica gel 60 F ₂₅₄ aluminum sheets, manufactured by Merck) to purify the glycosylated product. 1 μL of the recombinant β-L-arabinopyranosidase enzyme solution was added to 4 μL of the aqueous solution containing the glycosyl transfer product, and reacted at 40 ° C. for a predetermined time (1 and 2 hours). The obtained reaction solution and marker (L-arabinose and D-glucose) were subjected to TLC (Silica gel 60 F ₂₅₄ aluminum sheets, manufactured by Merck), and a developing solvent of chloroform: methanol: water = 30: 20: 4 was used. And developed once. After development once, sulfuric acid was sprayed on the TLC and heated to 170 ° C. to detect sugar spots. The detection result is shown in the photograph of FIG. In the figure, Lane 1 is a marker (L-arabinose), Lane 2 is a marker (D-glucose), Lane 3 is a glycosylated product, Lane 4 is a reaction solution for 1 hour, and Lane 5 is a reaction solution for 2 hours. It is a result. Further, in the figure, spots indicated by arrows are spots of L-arabinose, D-glucose, and glycosyl transfer products in order from the top. As shown in the figure, the reaction solution (lanes 4 to 5) was produced by the β-L-arabinopyranosidase of this example using the glycosylated product as a substrate in addition to the glycosylated product spot. A spot of L-arabinose and a spot of degradation product D-glucose were detected. In this way, by causing the β-L-arabinopyranosidase of this example to act on the glycosylated product obtained by the glycosyltransferase activity of the β-L-arabinopyranosidase of this example, the arabino Production of L-arabinose by pyranosidase activity was confirmed.

  The present invention provides a novel β-L-arabinopyranosidase that can be mass-produced. When the novel β-L-arabinopyranosidase of the present invention is used, for example, by acting on the acacia gum or larch arabinogalactan used as an emulsifier, and modifying the sugar chain structure, It is possible to adjust the emulsification process during production or to produce a modified emulsifier. Moreover, since β-L-arabinopyranosidase of the present invention has glycosyltransferase activity of arabinose residues in addition to arabinopyranosidase activity, it contains not only arabinose residues but also arabinose residues. It can also be used to produce sugars. Therefore, the present invention is useful in a wide range of fields such as the food field and the medical field.

FIG. 1 is a graph showing the results of measuring various enzyme activities in a culture solution after cell culture in the purification step of an example of the β-L-arabinopyranosidase of the present invention. FIG. 2 is a photograph showing the results of electrophoresis of another example of the β-L-arabinopyranosidase of the present invention by SDS-PAGE. FIG. 3 is a diagram showing a configuration (restriction enzyme map) of an example of the vector of the present invention. FIG. 4 is a photograph showing the result of electrophoresis of yet another example of β-L-arabinopyranosidase of the present invention by SDS-PAGE. FIG. 5 is a graph showing changes in enzyme activity due to pH in yet another example of β-L-arabinopyranosidase of the present invention. FIG. 6 is a graph showing the pH stability of yet another example of β-L-arabinopyranosidase of the present invention. FIG. 7 is a graph showing changes in enzyme activity according to temperature in yet another example of β-L-arabinopyranosidase of the present invention. FIG. 8 is a graph showing the temperature stability of yet another example of the β-L-arabinopyranosidase of the present invention. FIG. 9 is a chart showing the analysis result of larch arabinogalactan degradation products by yet another example of β-L-arabinopyranosidase of the present invention and exo-β-1,3-galactanase. The upper panel is a degradation product when only β-L-arabinopyranosidase is allowed to act, and the lower panel is caused to act by β-L-arabinopyranosidase and exo-β-1,3-galactanase. The analysis results of the degradation products are shown. FIG. 10 is a TLC photograph showing the result of the glycosyltransferase reaction according to the example of the glycosyltransferase method of the present invention. FIG. 11 is a chart showing the result of the transglycosylation reaction according to another example of the transglycosylation method of the present invention. The upper panel shows the results of the control containing no sugar receptor, and the lower panel shows the results when glucose is used as the sugar receptor. FIG. 12 is a TLC photograph showing the result of the transglycosylation reaction according to still another example of the transglycosylation method of the present invention. FIG. 13 shows the analysis results of degradation products when a further example of β-L-arabinopyranosidase of the present invention is allowed to act on a glycosylated product obtained by another example of the glycosyl transfer method of the present invention. It is a TLC photograph which shows.

Claims (16)

β-L-arabinopyranosidase, which is a monomer, has an optimum pH of activity in the range of 3 to 5, and an optimum temperature of the activity of 30 ° C. to 50 ° C. Β-L-arabinopyranosidase, which has a molecular weight range of 40 kDa to 71 kDa as determined by SDS-PAGE. The β-L-arabinopyranosidase according to claim 1, which is derived from cells of the genus Streptomyces . The β-L-arabinopyranosidase according to claim 2, wherein the cells of the genus Streptomyces is Streptomyces avermitilis . The β-L-arabinopyranosidase according to any one of (a) to (c) below.
(A) β-L-arabinopyranosidase consisting of the amino acid sequence set forth in SEQ ID NO: 1.
(B) β-L-arabinopyranosidase consisting of an amino acid sequence in which one or several amino acid residues are substituted, added, inserted or deleted in the amino acid sequence shown in SEQ ID NO: 1.
(C) β-L-arabinopyranosidase consisting of an amino acid sequence having 60% or more homology with the amino acid sequence shown in SEQ ID NO: 1. The β-L-arabinopyranosidase gene according to any one of (d) to (f) below.
(D) A β-L-arabinopyranosidase gene consisting of the base sequence set forth in SEQ ID NO: 2.
(E) A β-L-arabinopyranosidase gene consisting of a base sequence in which one or several bases are substituted, added, inserted or deleted in the base sequence shown in SEQ ID NO: 2.
(F) A β-L-arabinopyranosidase gene comprising a nucleotide sequence having 60% or more homology with the nucleotide sequence set forth in SEQ ID NO: 2. A vector containing the β-L-arabinopyranosidase gene according to claim 5. A transformant comprising the vector according to claim 6. A method for producing β-L-arabinopyranosidase, comprising the step of culturing the transformant according to claim 7. A method for producing β-L-arabinopyranosidase, comprising a step of culturing Streptomyces avermitilis in a medium containing a saccharide containing an arabinose residue Manufacturing method. A method for screening a cell producing β-L-arabinopyranosidase, comprising:
Culturing the cells in a medium containing a saccharide comprising an arabinose residue;
Measuring the β-L-arabinopyranosidase activity of the substance produced by the cell. A method for producing arabinose, wherein β-L-arabinopyranosidase is allowed to act on a saccharide containing an arabinose residue to liberate arabinose, wherein the β-L-arabinopyranosidase is from A method for producing arabinose, characterized in that the β-L-arabinopyranosidase according to any one of 4 is used. A sugar transfer method for transferring an arabinose residue from a saccharide containing an arabinose residue to a sugar receptor,
A β-L-arabinopyranosidase according to any one of claims 1 to 4 is used as the enzyme that catalyzes the sugar transfer. The sugar transfer method according to claim 12, wherein the sugar receptor is a saccharide or an alcohol. The sugar transfer method according to claim 13, wherein the saccharide is at least one selected from the group consisting of D-galactose, glucose, mannose, xylose and L-arabinose. The sugar transfer method according to claim 13, wherein the alcohol is at least one selected from the group consisting of methanol, ethanol, 1-propanol, and 1-butanol. A method for producing a saccharide containing an arabinose residue,
A method for producing a saccharide comprising using the sugar transfer method according to any one of claims 12 to 15.

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