JP2012223189A - Isomaltase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an isomaltase acting on isomaltose and inactive to maltose.SOLUTION: There is provided the isomaltase having GFRMDVINF amino acid sequence at an active center, originated from filamentous fungi, and inactive to maltose.

Description

  The present invention relates to isomaltase useful for the production of glucose from starch.

  In industrial glucose production from starch, various enzymes are used for liquefaction with α-amylase, saccharification with glucoamylase, debranching with isoamylase, pullulanase, and the like. In this series of saccharification processes for glucose production, isomaltose is produced as a by-product due to side reactions such as sugar transfer and condensation reaction of glucoamylase, which is a saccharifying enzyme, and all starch cannot be converted into glucose. There is a problem in that glucose productivity is reduced.

Isomaltose is an α-1,6 glucoside-bonded glucose, and it is considered that an increase in glucose yield can be expected if it can be decomposed into glucose. In order to decompose isomaltose into glucose, the function of isomaltase (isomaltosidase, oligo-1,6-glucosidase), an enzyme that degrades α-1,6-glucoside bonds, is expected.
However, although the isomaltase reported so far can decompose isomaltose, various oligosaccharides are produced by the transglycosylation reaction and are not suitable for the above-mentioned purpose in order to produce glucose effectively. These isomaltases also act on maltose and eventually synthesize isomaltose via glucose. Therefore, a novel isomaltase having a property of acting on isomaltose but not on maltose and not synthesizing isomaltose from glucose has been desired.

Isoamylase and pullulanase, which are called starch branching enzymes, are enzymes that break down α-1,6-glucoside bonds, but do not act on low molecular weight isomaltose.
Aspergillus oryzae, a type of filamentous fungus, has been used since ancient times for the production of sake, miso, soy sauce and the like. The whole genome sequence has been analyzed so far (Non-Patent Documents 1 and 2), and research is currently being conducted for industrial use such as production of useful proteins and antibiotics (Non-Patent Document 3). A. Oryzae is known as a microorganism that produces a large amount of amylolytic enzymes such as amylase (Non-patent Document 4), and the presence of several α-amylase-like genes on the genome is presumed. -It is unknown whether it functions as an amylase.
Generally, α-amylase and isomaltase belong to the carbohydrate hydrolase family 13 (GH Family 13), and most of GH Family 13 acts on α-1,4 and α-1,6 glucoside bonds. It is said that. That is, it acts on both maltose and isomaltose. The enzyme belonging to GH Family 13 is said to have four highly conserved amino acid regions containing three catalytic residues (Non-patent Documents 5 and 6).

Macida, M .; Et al., “Genome sequencing and analysis of Aspergillus oryzae.” Nature, 438, 1157-61 (2005). Galagan, JE. Et al., "Sequencing of Aspergillus nidulans and comparable analysis with A. fumigatus and A. oryzae." Nature, 438, 1105-15 (2005). Abe, K .; Et al., “Impact of Aspergillus oryzae genomics on industrial production of metabolites.” Mypathology, 162, 143-53 (2006). Macida, M .; Et al. “Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of it's future.” DNA Res 15, 173-83 (2008). MacGregor EA et al. “Relationship of sequence and structure to the specificity in the alpha-amylase family of enzymes.” Biochim Biophys Act 2046. Nakajima, T .; “Comparison of amino acid sequences of eleven differential alpha-amylases.” Appl Microbiol Biotechnol 23 355-360 (1986). Keizo Y et al. “Val216 decades the substrate specificity of α-glucosidase in Saccharomyces cerevisiae.” Eur. J. et al. Biochem. 271, 3414-3420 (2004). Garcia, Bianca et al. “Molecular cloning of an α-glucosidase-like gene from Penicillium mini1um1 and structure1 of biotechnology1 of biotechnologies.

  An object of the present invention is to provide a novel isomaltase having the property of acting on isomaltose but not on maltose and not synthesizing isomaltose from glucose, and an efficient method for producing glucose from starch. It is to provide.

  Therefore, the present inventor paid attention to the filamentous fungus and conducted various studies to find isomaltase that acts on isomaltose but does not act on maltose. First, focusing on yeast that is completely different from filamentous fungi, we investigated isomaltase derived from Saccharomyces cerevisiae, which is one of the yeasts, and it has been reported that the 216th valine in the conserved region determines substrate specificity. (Non-patent document 7) Therefore, a filamentous fungus having a sequence having valine at the same position as this isomaltase was searched in the Aspergillus oryzae genome database (DOGAN). As a result, the present inventors have surprisingly found that a sequence having valine exists on the genome of Aspergillus, which is a filamentous fungus that is not known to produce isomaltase. Then, when a gene having the sequence was expressed in E. coli, it was found that isomaltase having an action of degrading isomaltose and not acting on maltose was obtained, and the present invention was completed. In addition, many enzymes (oligo-1,6-glycosidase, dextran glucosidase, neopullulanase, DexC protein, etc.) capable of degrading isomaltose have already been reported from bacteria, yeast, filamentous fungi and the like (Non-patent Document 8). Those not having the above-described structure are considered to act on both isomaltose and maltose.

  That is, the present invention provides an isomaltase having an amino acid sequence of GFRMDVINF (SEQ ID NO: 1) at the active center and not acting on maltose, which is derived from filamentous fungi.

The present invention also provides an isomaltase having the following amino acid sequence (a) or (b) and not acting on maltose.
(A) SEQ ID NO: 3 or SEQ ID NO: 5
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

  The present invention also provides a gene encoding the above isomaltase.

  Furthermore, the present invention provides a method for producing glucose, characterized by allowing starch to act on an enzyme selected from α-amylase, glucoamylase, isoamylase and pullulanase, and the above isomaltase.

  The isomaltase of the present invention has properties that it acts on isomaltose but not on maltose, and does not synthesize isomaltose from glucose. Therefore, if this isomaltase is used, glucose can be produced in high yield from starch.

The substrate specificity of commercially available glucoamylase 1/100 aqueous solution is shown. The substrate specificity of commercially available glucoamylase 1/1000 aqueous solution is shown. The SDS-PAGE result of the cell extract (soluble fraction) of a transformant (+ agl1) and a negative control strain (-agl1) is shown. (Gel concentration: 12%, A for CBB staining, B for His-Tag Monoclonal Antibody and Goat Anti-mouse HRP conjugate for secondary antibody) The western blotting result of the cell extract (soluble fraction) of a transformant (+ ag11) and a negative control strain (-ag11) is shown. (Gel concentration: 12%, A for CBB staining, B for His-Tag Monoclonal Antibody and Goat Anti-mouse HRP conjugate for secondary antibody) The reaction temperature range of this invention Aspergillus oryzae isomaltase (AGL1) is shown. The reaction pH range of this invention Aspergillus oryzae isomaltase (AGL1) is shown. The substrate specificity of this invention Aspergillus oryzae isomaltase (AGL1) is shown. The reaction temperature range of this invention Fusarium oxysporum isomaltase (FOAGL1) is shown. The reaction pH range of this invention Fusarium oxysporum isomaltase (FOAGL1) is shown. The substrate specificity of this invention Fusarium oxysporum isomaltase (FOAGL1) is shown. The saccharification result at the time of using together this invention Aspergillus oryzae isomaltase (AGL1) and a commercially available saccharification enzyme is shown. The saccharification result at the time of using together this invention Fusarium oxysporum isomaltase (FOAGL1) and a commercially available saccharifying enzyme is shown.

  The isomaltase of the present invention has an amino acid sequence of GFRMDVINF (SEQ ID NO: 1) at the active center. This amino acid sequence is considered to be an active center having the characteristic that the isomaltase of the present invention acts on isomaltose to decompose it into glucose but does not act on maltose. The isomaltase of the present invention does not synthesize oligosaccharides such as isomaltose from glucose. Therefore, the isomaltase of the present invention is characterized in that it is an enzyme with extremely high substrate specificity that produces glucose from isomaltose.

  The isomaltase of the present invention exhibits a relative activity of 50% or more at 25 to 40 ° C, and the optimum temperature is about 30 ° C. The isomaltase of the present invention exhibits a relative activity of 50% or more at pH 5 to 8, and the optimum pH is about 7.0. The substrate specificity of the isomaltase of the present invention is clear and acts on isomaltose, but not on maltose, nigerose, trehalose, dextran and starch. In addition, kojibiose acts about 20% of isomaltose. It also does not act on glucose and does not produce oligosaccharides.

The isomaltase of the present invention is characterized by having the above-mentioned amino acid sequence at the active center, and specific examples thereof have the following amino acid sequence (a) or (b) and do not act on maltose. Things.
(A) SEQ ID NO: 3 or SEQ ID NO: 5
(B) an amino acid sequence in which one or several amino acid sequences are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5;

  Here, the isomaltase having the amino acid sequence of SEQ ID NO: 3 is an isomaltase derived from Aspergillus oryzae, and the isomaltase having the amino acid sequence of SEQ ID NO: 5 is an isomaltase derived from Fusarium oxysporum. The homology (identity) between the amino acid sequence (b) and the amino acid sequence (a) may be 70% or more between SEQ ID NOs: 3 and 5 and their deletions, substitutions or additions. However, 80% or more is preferable, 90% or more is more preferable, and 95% or more is more preferable.

The gene of the present invention is a gene encoding the aforementioned isomaltase. The gene only needs to encode the amino acid sequence of the active center, but preferably encodes the amino acid sequence (a) or (b). Further preferred genes are those having the following base sequence (c) or (d).
(C) SEQ ID NO: 2 or SEQ ID NO: 4
(D) A base sequence of DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of SEQ ID NO: 2 or SEQ ID NO: 4.

  Here, the stringent condition refers to a condition of 42 ° C. in 700 mM NaCl in the presence of 50% formamide, or an equivalent condition. Further, the homology (identity) between the base sequence (c) and the base sequence (d) may be 70% or more with the base sequence of SEQ ID NO: 2 or 4, respectively, but preferably 80% or more, 90% or more is more preferable, and 95% or more is more preferable.

  The isomaltase of the present invention is prepared by, for example, cloning the isomaltase gene of the present invention derived from a filamentous fungus, incorporating the gene into an expression vector to prepare a recombinant plasmid, and transforming the resulting recombinant plasmid into E. coli or other host, It can be produced by culturing the transformant.

  The isomaltase gene of the present invention can be cloned by, for example, a PCR method from a cDNA library derived from a filamentous fungus or a method of removing an intron after PCR from a filamentous fungus genome. As an example, based on the amino acid sequence of isomaltase derived from Saccharomyces cerevisiae, a region similar to the region having the 216th valine residue in the conserved region was searched from the filamentous fungus database. As a result, a sequence of GFRMDVINF (SEQ ID NO: 1) was found from the genome database of Aspergillus oryzae. This sequence was assumed to be the active center of isomaltase. Primers were designed so that the gene having this active center was amplified, and the target gene was obtained by PCR using the Aspergillus oryzae cDNA library as a template.

  Examples of expression vectors for incorporating the isomaltase gene include plasmid vectors. Examples of the expression vector for E. coli include pET-14b and pBR322. Examples of Bacillus subtilis include pUB110. Examples of filamentous fungi include pPTRI. Moreover, pRS403 etc. are mentioned for yeast use.

  The obtained recombinant plasmid is transformed into Escherichia coli, Bacillus subtilis, filamentous fungi, yeast and the like, and the isomaltase of the present invention can be obtained by culturing the transformant.

  As described above, the isomaltase of the present invention decomposes isomaltose into glucose, does not act on maltose, and does not synthesize oligosaccharides from glucose. Therefore, glucose can be selectively produced by allowing an enzyme selected from α-amylase, glucoamylase, isoamylase and pullulanase and the isomaltase of the present invention to act on starch. Here, as α-amylase, for example, Christase T10 (Daiwa Kasei Co., Ltd.) can be used. As the glucoamylase, for example, GODO-ANGH (Godoshusei Co., Ltd.) can be used. As the isoamylase, for example, GODO-FIA (Godoshusei Co., Ltd.) can be used. As the pullulanase, for example, pullulanase “Amano” 3 (Amano Enzyme Inc.) can be used.

  The reaction is performed, for example, by adding the enzyme to a starch saccharifying enzyme such as starch or amylase, and mixing and stirring at a pH and temperature conditions at which the enzyme acts. According to the method of the present invention, since isomaltose is not by-produced, glucose can be produced in a high yield. Alternatively, after a general saccharification reaction, isomaltose and glucose are separated, the enzyme is added thereto, and the mixture is stirred under the pH and temperature conditions at which the enzyme acts. According to the method of the present invention, it is possible to convert by-produced isomaltose into glucose, and glucose can be produced in a high yield.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in detail, this invention is not limited to this at all.

Example 1 (degradation by glucoamylase)
15 μL of 1/100 or 1/1000 aqueous solution of commercially available glucoamylase (Godoshusei Co., Ltd .: GODO-ANGH) and 5 μL of 0.1 M acetate buffer pH 6 are mixed with 2% maltose or 2% isomaltose aqueous solution, It was allowed to act at 30 ° C. for 18 hours. The reaction was stopped in boiling water for 5 minutes, and the reaction solution was concentrated in vacuo and then subjected to silica gel TLC. The developing solvent was isopropanol / n-butanol / water (10/5/4), and after developing, air-dried, sprayed with an anthrone sulfuric acid solution, and heated at 105 ° C. to cause color development.

  As shown in FIG. 1 (result of commercially available glucoamylase 1/100 aqueous solution) and FIG. 2 (result of commercially available glucoamylase 1/1000 aqueous solution), commercially available glucoamylase worked well on maltose, but also decomposes isomaltose. I was able to. Further, it was found that maltose and isomaltose were decomposed, and that maltose produced isomaltose and maltose produced from isomaltose by transfer or recombination of glucose.

Example 2 (Agl1 gene discovery from database)
A putative α-amylase-like gene having valine at the homologous position with Saccharomyces cerevisiae isomaltase and high homology with other isomaltases was found on the Aspergillus oryzae genome database (DOGAN) and named agl1 (DOGAN ID: Ao090020000176). It was speculated that agl1 encodes isomaltase, not α-amylase. To date, there are no reports of isomaltase from filamentous fungi.
Moreover, the agl1 gene (foagl1) of Fusarium oxysporum was found by the same search.

Example 3 (Transformation of Escherichia coli with Aspergillus oryzae isomaltase gene)
PET-14b (Novagen) was used as an E. coli expression vector.
Primer sequences used for PCR for agl1 gene amplification are as follows.

glufa1.6F
5′-acgggccccatggccaccacc-3 ′ (SEQ ID NO: 10)
glufa1.6R
5′-ctgggggatcctaatcaggcctc-3 ′ (SEQ ID NO: 11)

  Using a cDNA library prepared from Aspergillus as a template, PCR was performed using primer glpha1.6F and primer glpha1.6R to obtain a fragment containing the agl1 gene. The obtained DNA fragment was recovered by ethanol precipitation or the like. A DNA fragment containing the agl1 gene was treated with BamHI and the DNA was recovered by ethanol precipitation or the like. Escherichia coli was transformed by ligation reaction with pET-14b which had been subjected to BamHI treatment and dephosphorylation treatment. A plasmid was prepared from the obtained colonies to obtain a plasmid pET-14b-agl1 containing the agl1 gene. The nucleotide sequence of the agl1 gene is shown in SEQ ID NO: 2, and the amino acid sequence of isomaltase encoded by the gene is shown in SEQ ID NO: 3.

Example 4 (Expression of Aspergillus oryzae isomaltase in E. coli)
E. E. coli BL21 (DE3), E. coli. E. coli AD494 (DE3), E. coli. coli Origami (DE3) pLysS and E. coli. E. coli Rosetta-gami (DE3) was transformed with plasmid pET-14b-agl1 to obtain a transformed strain. Moreover, the transformant was acquired using the pET-14b vector which does not have agl1 as a negative control. Colonies of each transformant were inoculated into LB medium and precultured. 40 mL of LB medium was inoculated with the preculture and cultured at 20 ° C. for 9 hours. Thereafter, IPTG was added to a final concentration of 0.1 mM, and induction culture was performed at 20 ° C. for 12 hours. After the expression induction, the cells were collected by centrifugation at 7,000 rpm for 20 minutes and suspended in 1 mL of 10 mM phosphate buffer (pH 7.0). The cells were crushed with an ultrasonic crusher. The disrupted bacterial cell solution was centrifuged at 12,000 rpm for 20 minutes, and the supernatant was recovered as a crude enzyme solution. The crude enzyme derived from each strain was subjected to SDS-PAGE and Western blotting at an acrylamide gel concentration of 12% to evaluate the expressed AGL1. The detection was performed by His-Tag Monoclonal Antibody and Goat Anti-Mouse HRP conjugate. The activity of AGL1 expressed in each transformant was evaluated using TLC. coli Origami (DE3) pLysS and E. coli. Although strongly observed in transformants using E. coli Rosetta-gami (DE3) as a host, maltose-degrading activity was observed in all transformants. From this, it was speculated that the observed maltose-degrading activity was derived from E. coli as the host, or that maltose-degrading activity derived from E. coli was added to the maltose-degrading activity of AGL1. E. coli-derived maltase has been reported. Therefore, E. coli, which is the optimal host. E. coli Origami (DE3) pLysS as a host strain (+ agl1) and E. coli. A comparison experiment with E. coli Origami (DE3) pLysS (-agl1, negative control) was performed. About the expression state of AGL1 protein, the transformed strain and the negative control strain were compared by SDS-PAGE and Western blotting (FIGS. 3 and 4). A band that could be estimated as AGL1 was confirmed in the transformed strain, but not in the negative control. That is, it was confirmed that host E. coli did not produce AGL1, but produced AGL1 by transformation.

Example 5 (Property of Aspergillus oryzae isomaltase expressed in E. coli: purification of AGL1)
After injecting ₄ mL of 200 mM NiSO ₄ into a TOYOPEARL AF-Chelate-650M column (φ10 × 45 mm), equilibration was performed with 40 mL of distilled water and 40 mL of 10 mM phosphate buffer (pH 7.0). 4 mL of AGL1 crude enzyme solution was applied to a nickel chelate TOYOPEARL AF-Chelate-650M column (φ10 × 45 mm), and the non-adsorbed fraction was eluted with 10 mM phosphate buffer (pH 7.0) containing 0.3 M NaCl. The adsorbed fraction was eluted with 10 mM phosphate buffer (pH 7.0) containing 0.3 M NaCl and 20 mM imidazole. The fraction in which a single band could be confirmed by SDS-PAGE was collected and dialyzed against 10 mM acetate buffer (pH 6.0).
The Bradford method was used for protein quantification.

  Prepared E. coli. 4 mL of a crude enzyme derived from a transformant using E. coli Origami (DE3) pLysS as a host was applied to a nickel chelate TOYOPEARL AF-Chalate-650M column (φ10 × 45 mm), and the adsorbed protein was eluted with 20 mM imidazole. When SDS-PAGE was performed on the non-adsorbed fraction and the adsorbed fraction, Fraction No. No. 11 confirmed the protein considered to be AGL1. A 20 mM phosphate buffer solution (pH 7.0) was added and mixed so that the final concentrations were 0.041% GOD, 0.01% POD, and 0.5% O-dianisidine to obtain a GOD reagent. 5 μl of the purified enzyme solution was added to a mixed solution of 10 μl of 20 mg / mL isomaltose and 5 μl of 200 mM phosphate buffer (pH 7.0) to prepare a reaction solution (total amount: 20 μl). In addition, a solution in which the purified enzyme solution was distilled water was used as a control. The enzyme reaction solution was incubated at 30 ° C. for 3 hours. After the enzyme reaction solution was treated at 100 ° C. for 3 minutes, 200 μl of GOD reagent was added and incubated at 30 ° C. for 10 minutes. After adding 4 μl of 4N HCl, the absorbance at 400 nm was measured. The principle of the GOD method is as follows.

  1 U of α-glucosidase activity was defined as an activity that produces 1 μmol of glucose per minute under these conditions, and the enzyme activity was calculated from the following equation.

  AGL1 was purified 27.6 times by Ni chelate column chromatography, and the yield was 76.6%. The purified enzyme showed a single band by SDS-PAGE.

Example 6 (Properties of Aspergillus oryzae isomaltase expressed in E. coli: optimum temperature)
5 μl of the purified enzyme solution was added to a mixed solution of 10 μl of 20 mg / mL isomaltose and 5 μl of 200 mM phosphate buffer (pH 7.0) to prepare a reaction solution (total amount: 20 μl). The reaction solution was incubated at a temperature of 20 to 60 ° C. for 3 hours, and the activity was measured by the GOD method.
Using the purified enzyme, the optimum temperature was evaluated by the GOD method (FIG. 5). The optimum temperature was 30 ° C.

Example 7 (Properties of Aspergillus oryzae isomaltase expressed in E. coli: optimum pH)
To a mixed solution of 10 mg of 20 mg / mL isomaltose and various buffer solutions of 200 mM, 5 μl of a purified enzyme solution was added to obtain a reaction solution (total amount: 20 μl). The reaction solution was incubated at 30 ° C. for 3 hours, and the activity was measured by the GOD method.
Using the purified enzyme, the optimum pH was evaluated by the GOD method (FIG. 6). The optimum pH was 7.0.

Example 8 (Properties of Aspergillus oryzae isomaltase expressed in E. coli: specificity)
5 μl of the purified enzyme solution was added to a mixed solution of 10 μl of each substrate at 5 mg / mL and 5 μl of 200 mM phosphate buffer (pH 7.0) to obtain a reaction solution (total amount 20 μl). The enzyme reaction solution was incubated at 30 ° C. for 3 hours, and the activity was measured by the GOD method.
Substrate specificity was evaluated by the GOD method using the purified enzyme (Table 2). AGL1 specifically acted on isomaltose, but also showed a slight activity against kojibiose.

Example 9 (degradation by filamentous fungus isomaltase)
To 16 μL of the filamentous fungus-derived isomaltase crude enzyme obtained in Example 5, 4 μL of glucose, maltose, and 0.1M acetic acid buffer pH 6 solution of isomaltose were added to a concentration of 10 mg / mL. The reaction was performed at 30 ° C. for 8 to 24 hours, and the reaction solution was subjected to TLC analysis in the same manner as in Example 1. As shown in FIG. 7, when glucose or maltose was used as a substrate, no decomposition or transfer product was detected. On the other hand, isomaltose decomposed and glucose spots increased. Further, no other transfer product was produced from isomaltose.

Example 10 (Fusalium oxysporum isomaltase gene cloning)
In the same manner as in Example 3, the isomaltase gene derived from Fusarium oxysporum was cloned. The base sequence of the gene is shown in SEQ ID NO: 4, and the amino acid sequence of isomaltase encoded by the gene is shown in SEQ ID NO: 5.
PCR was performed using a cDNA library prepared from Fusarium oxysporum as a template to obtain a DNA fragment containing the foagll1 sequence. The obtained fragment was incorporated into E. coli expression plasmid pET-14b to prepare expression plasmid pET-14b-foagl1.

Example 11 (Properties of enzyme obtained from Fusarium oxysporum isomaltase gene: optimum temperature, optimum pH, substrate specificity)
In the same manner as in Example 4, E. coli transformed with the expression plasmid pET-14b-foagl1 was cultured, and the cells were collected and subjected to ultrasonic disruption. As a result, the isomaltose-degrading activity was confirmed. The Fusarium oxysporum isomaltase enzyme (FOAGL1) obtained by purifying this cell disruption extract by the purification method of Example 5 was subjected to the following test.
The optimum temperature of the obtained Fusarium oxysporum isomaltase was examined by the same method as in Example 6, and the optimum pH was examined by the same method as in Example 7. As a result, the optimum temperature of Fusarium oxysporum isomaltase was 30 ° C. (FIG. 8). The optimum pH was 6.5 (FIG. 9).
Moreover, the substrate specificity was evaluated by the GOD method which is the same method as in Example 8 (Table 3). FOAGL1 specifically acted on isomaltose. It did not act on maltose or trehalose, and showed a slight activity against kojibiose and panose.

Example 12 (Degradation characteristics of Fusarium oxysporum isomaltase)
The decomposition of glucose, maltose and isomaltose of the Fusarium oxysporum isomaltase enzyme (FOAGL1) obtained in Example 11 was examined.
4 μL each of glucose, maltose, and isomaltose 0.1 M acetate buffer pH 6 solution was added to 16 μL of Fusarium oxysporum-derived isomaltase (FOAGL1) crude enzyme so that the concentration was 10 mg / mL. The reaction was performed at 30 ° C. for 8 to 24 hours, and the reaction solution was subjected to TLC analysis in the same manner as in Example 1. As shown in FIG. 10, when glucose or maltose was used as a substrate, no decomposition or transfer product was detected. On the other hand, isomaltose decomposed and glucose spots increased. Further, no other transfer product was produced from isomaltose.

Example 13 (Combination of the enzyme of the present invention and a commercially available saccharifying enzyme)
Soluble starch was dissolved by heating in 0.1 M acetic acid buffer (pH 4.2) to a concentration of 30% (w / w). 40 μL of commercially available saccharification enzyme OPTIMAX 4060 (glucoamylase / pullulanase mixed enzyme) was added to 50 mL of soluble starch solution, and the enzyme reaction was performed at 60 ° C. for 16 hours. After the reaction, the sample was heated at 100 ° C. to stop the enzymatic reaction (starch degradation product).
After the starch degradation product was diluted 10-fold with 0.1 M phosphate buffer (pH 6.8), Aspergillus oryzae isomaltase isomaltase (AGL1) obtained according to Example 4 or Example 5, or according to Example 10. Fusarium oxysporum-derived isomaltase (FOAGL1) was added and reacted at 30 ° C. for 5 hours. After the reaction, the reaction was stopped in boiling water for 5 minutes and subjected to silica gel TLC. The developing solvent was isopropanol / n-butanol / water (10/5/4), and after developing, air-dried, sprayed with an anthrone sulfuric acid solution, and heated at 105 ° C. to cause color development.

In each of the second lanes of FIGS. 11 and 12 where only OPTIMAX 4060 was applied, an isomaltose spot was confirmed. In contrast, in each of the third lanes of FIGS. 11 and 12 where isomaltase was allowed to act, the spot became thinner and it was found that isomaltose was decomposed.
From this result, it was found that the yield of glucose in the saccharification step can be improved by using this enzyme.

  The isomaltase of the present invention makes it possible to dramatically improve the yield of glucose in an industrial glucose production process from starch.

Claims (10)

  An isomaltase which has an amino acid sequence of GFRMDVINF at the active center and which does not act on maltose, derived from filamentous fungi.   The isomaltase according to claim 1, which does not synthesize oligosaccharide from glucose.   The isomaltase according to claim 1 or 2, wherein the filamentous fungus is Aspergillus oryzae or Fusarium oxysporum. An isomaltase having the following amino acid sequence (a) or (b) and not acting on maltose.
(A) SEQ ID NO: 3 or SEQ ID NO: 5
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.   The isomaltase according to claim 4, which is derived from a filamentous fungus.   The isomaltase according to claim 5, wherein the filamentous fungus is Aspergillus oryzae or Fusarium oxysporum.   The isomaltase according to any one of claims 4 to 6, wherein no oligosaccharide is synthesized from glucose.   A gene encoding the isomaltase according to any one of claims 1 to 7. The gene according to claim 8, which has the following base sequence (c) or (d).
(C) SEQ ID NO: 2 or SEQ ID NO: 4
(D) A base sequence of DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of SEQ ID NO: 2 or SEQ ID NO: 4.   A method for producing glucose, comprising reacting an enzyme selected from α-amylase, glucoamylase, isoamylase and pullulanase with the isomaltase according to any one of claims 1 to 7.