JP2003169665A - New method for producing saccharide containing koji oligosaccharide and nigerooligosaccharide and fungal cell and enzyme used threfor and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing saccharide containing koji oligosaccharide and nigerooligosaccharide in which new functionality is expected and which are α-glucooligosaccharides derived from starch and to provide an enzyme necessary for production of the oligosaccharide and a fungus derived from the genus Paecilomyces producing the enzyme and to provide a method for producing the enzyme and the fungus. <P>SOLUTION: This invention provides a method for efficiently producing saccharide containing koji oligosaccharide and nigerooligosaccharide by using at least one kind of substance selected from α-glucan, α-glucooligosaccharide and glucose as a raw material and provides the new α-glucosidase necessary for production of the saccharide and provides a method for producing the glucosidase and provides the fungus belonging to the genus Paecilomyces producing the α-glucosidase. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a sugar containing koji-oligosaccharide and nigerooligosaccharide, and more specifically, it contains at least one selected from α-glucan, α-glucooligosaccharide and glucose. A novel α-glucosidase showing the action of producing a sugar containing koji-oligosaccharide or nigerooligosaccharide by acting on a sugar solution, a method for producing the same, and a fungus belonging to the genus Paecilomyces that produces this α-glucosidase and its fungus. It relates to a manufacturing method.

[0002]

2. Description of the Related Art Maltooligosaccharides and isomaltooligosaccharides are known as oligosaccharides made from inexpensive starch.
Malto-oligosaccharides composed of α-1,4-glucoside bonds are widely used in foods such as sweeteners, various sauces and sauces because of their low sweetness and high moisturizing properties.

Isomaltooligosaccharides having an α-1,6-glucoside bond in the molecule have the characteristics of maltooligosaccharides such as low sweetness and moisturizing property, and also have a physiological effect such as selective growth activity of bifidobacteria and less prone to tooth decay. Is known, and it is widely applied to foods such as beverages and confectionery by taking advantage of its characteristics.

[0004] As described above, starch-derived oligosaccharides are widely used for the purpose of improving the physical properties of foods and imparting physiological functions, but koji-oligosaccharides having α-1,2-glucoside bonds and α-1, Regarding nigerooligosaccharides having a 3-glucoside bond, there have been few reports regarding their efficient preparation methods, and they have not been used as widely as foods as maltooligosaccharides and isomaltooligosaccharides.

On the other hand, it has recently been reported that koji-oligosaccharides and nigerooligosaccharides have various functionalities, and their potential as novel oligosaccharides derived from starch is drawing attention.

As a characteristic function of the nigerooligosaccharide,
As a food flavor improving agent (Patent Document 1) that alleviates the saltiness of salt-containing foods and improves the palatability under low salt, and a physical property improving agent such as a dye fading prevention effect for food (Patent Document 2) In addition to its use as a physiological function, a plant exposed to a pathogenic bacterium by giving an immunostimulating effect (Patent Document 3) that induces the production of interleukin 12 by using it in combination with a bacterium derived from the genus Lactobacillus and a nigerooligosaccharide But,
As an autoimmune action against pathogenic bacteria, a function of inducing and accumulating phytoalexin having an antibacterial action in a plant (Patent Document 4) and the like have been reported.

With respect to koji oligosaccharides, it has recently been reported that the reducing power is weak and the Maillard reactivity is weak, and that it is not acid-fermented by cariogenic bacteria (Non-patent Document 1).

[0008] As an efficient method for producing koji-oligosaccharides and nigerooligosaccharides having such various functions, koji-oligosaccharides are reacted with β-D-glucose 1-phosphate and glucose by kojibiose phosphorylase. A method for producing koji-oligosaccharide (Patent Document 5),
For nigerooligosaccharides, a substrate containing polysaccharides or oligosaccharides linked with α-1,4-glucoside, a transfer reaction of α-glucosidase obtained from a culture solution in which a fungus derived from the genus Acremonium ( Acremonium sp.) Is cultured, A method for producing a nigerooligosaccharide (Patent Document 6) is disclosed.

However, such a production method obtains either koji-oligosaccharide or nigerooligosaccharide, but it is not a method for producing both efficiently.

[0010]

[Patent Document 1] Japanese Patent Laid-Open No. 10-210949

[Patent Document 2] Japanese Patent Laid-Open No. 2000-189101

[Patent Document 3] Japanese Patent Laid-Open No. 11-228425

[Patent Document 4] Japanese Patent Laid-Open No. 10-36210

[Patent Document 5] Japanese Patent Laid-Open No. 10-304882

[Patent Document 6] JP-A-7-59559

[Non-patent document 1] Tomoyuki Nishimoto et al., Japan Society of Applied Glycoscience 20
Proceedings of the 2001 Conference, Vol. 48, p. 413 (20
2001)

[0011]

[Problems to be Solved by the Invention] As a method for simultaneously producing koji oligosaccharides and nigerooligosaccharides, a method using cyclodextrin glucanotransferase (Japanese Patent No. 1725186), a method using sucrose phosphorylase (No. 3073864), and buckwheat derived Α-Glucosidase (M. Takahashi et al., Agric. Biol. Ch
Em., Vol. 33. 1339-1410 (1969)) has been reported, but it requires a large amount of the enzyme to be used, or the production rate is as low as a few percent of the solid sugar content. There was a problem, and its application was unthinkable. From the above, an advantageous method for producing koji oligosaccharides and nigerooligosaccharides is required. The present invention addresses these needs.

[0012]

[Means for Solving the Problems] Therefore, the inventors of the present invention searched for a strain producing α-glucosidase that efficiently produces koji oligosaccharides and nigerooligosaccharides from starch and starch hydrolysates. A filamentous fungus, a strain belonging to the genus Paecilomyces, was found in this soil.

This strain is cultured under appropriate conditions, and α-glucosidase extracted from the recovered cells is allowed to coexist with a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose. It was found that cordierigosaccharides and nigerooligosaccharides accumulate as products in the reaction solution about 50 hours after the start of the reaction, and the present invention was completed based on this finding.

The present invention is thus described as α-glucan, α-
The present invention relates to a method for producing a sugar containing koji-oligosaccharide and nigerooligosaccharide, which are functional sugars derived from starch, from a sugar solution containing at least one selected from glucooligosaccharide and glucose.

More specifically, a novel α which acts on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose to produce a sugar containing koji-oligosaccharide or nigerooligosaccharide. The present invention relates to a glucosidase and a method for producing the same, a fungus belonging to the genus Paecilomyces that produces this α-glucosidase, and a method for producing the fungus.

In the present invention, koji-oligosaccharide means an oligosaccharide having one or more α-1,2-glucoside bonds in the molecule, and is an oligosaccharide consisting of only α-1,2-glucoside bonds. In addition, oligosaccharides composed of α-1,2-glucoside bonds and other bonds are also included. Specifically, a disaccharide kojibiose [O-.alpha.-D-glucopyranosyl - (1 → 2) - O -D- glucopyranose] In addition to, Koji Biot sill glucose trisaccharide [O-.alpha.-
D- glucopyranosyl - (1 → 2) - O -α-D- glucopyranosyl - (1 → 4) -D- glucopyranose], and others.

Further, in the present invention, a nigerooligosaccharide is
It means an oligosaccharide having one or more α-1,3-glucoside bonds in the molecule, and in addition to oligosaccharides consisting of only α-1,3-glucoside bonds, α-1,3-glucoside bonds and other It also includes an oligosaccharide consisting of a bond. In particular,
Disaccharide is nigerose [O -α-D- glucopyranosyl - (1 → 3) - O -D- glucopyranose] other, Escape sill glucose is a three sugars [O -α-D- glucopyranosyl - (1 → 3 ) - O -α-D- glucopyranosyl - (1
→ 4) -D-glucopyranose] and the like.

The α-glucosidase in the present invention is derived from a microorganism and is α-glucan, α-glucooligosaccharide,
Any enzyme may be used as long as it is an enzyme that acts on a sugar solution containing at least one selected from glucose to produce a sugar containing koji-oligosaccharide or nigerooligosaccharide, and in particular, a fungus belonging to the genus Paecilomyces, especially Paecilomyces lyrakinus HKS-124. (FERM P-1840
The α-glucosidase possessed by 6) and the like can be mentioned. The enzymatic chemistry of the enzyme is as follows.

This enzyme is an α-glucosidase that cleaves the α-glucoside bond at the non-reducing end of the substrate into an exo type.
In addition to the -1,4-glucoside bond, α-1,2-glucoside bond and α-1,3-glucoside bond are hydrolyzed, while the glucose residue from the sugar donor is transferred to the non-reducing terminal of the sugar acceptor. Glycosylation reaction of transferring to hydroxyl group at 2, 3, or 4 position of glucose residue is also performed. Further, in the presence of a high concentration of glucose, a sugar condensation reaction which is a reverse reaction of hydrolysis is also performed.

The transfer product obtained by transferring the α-glucosyl group to the hydroxyl group at the 4-position is decomposed again by this enzyme.
Finally, cordier oligosaccharides having an α-1,2-glucoside bond and nigerooligosaccharides having an α-1,3-glucoside bond accumulate as transfer products.

The substrate specificity of the hydrolysis reaction of this enzyme is shown in Table 1.
Shown in. It acts on maltose, disaccharides such as kojibiose and nigerose, malto-oligosaccharides such as maltotriose and maltopentaose, soluble starch with a high degree of polymerization and amylose to produce glucose.
Nigerose, which is a glucodisaccharide having an α-1,3-glucosidic bond, is the best substrate. It is difficult to act on glycogen, isomaltose, para-nitrophenyl α-glucoside, sucrose, etc. Further, it has no effect on α-cyclodextrin, oligosaccharides such as trehalose and panose.

[0022]

[Table 1]

As the sugar acceptor in the transfer reaction catalyzed by the present enzyme, not only glucose but also α-glucodisaccharides such as maltose, nigerose, kojibiose, trehalose and isomaltose, and α-glucose such as maltotriose and maltotetraose. -Gluco-oligosaccharide, cellobiose,
Suitable are β-glucodisaccharides such as laminaribiose, sophorose and gentiobiose, and oligosaccharides having a glucose residue at the non-reducing end, such as sucrose.

As shown in FIGS. 1 and 2, the optimum pH of this enzyme is around pH 5 (4 for both maltose degrading activity and transglycosylation activity).
0 ° C) and slightly acidic side, stable up to pH 4-8 (40 ° C, 1
Time incubation).

The optimum temperature of this enzyme is 6 as shown in FIG.
It has a residual activity of 50% or more after incubation for 30 minutes at 5 ° C (pH 5.5) up to 65 ° C.

Gel filtration chromatography (Superdex 2
The molecular weight of this enzyme was about 54,000 as a result of estimating the molecular weight from the relative elution time with the standard protein using 00HR (Pharmacia Biotech). Also, SDS
The value of the molecular weight obtained by gel electrophoresis from the relative mobility with respect to the standard protein was about 50,000. From these results, this enzyme is presumed to be a monomer protein.

Each metal ion (Na ⁺ is 50 mM, other ions are 1 mM) and EDTA (5 mM)
Table 2 shows the results of examining the effect on the maltose-degrading activity in the presence of the substance. The activity of this enzyme is inhibited by zinc ion and mercury ion. EDTA does not affect the maltose degrading activity of this enzyme.

[0028]

[Table 2]

The enzyme was subjected to isoelectric focusing (Fast System, Pharmacia Biotech Co., Ltd.) together with a standard protein having a known isoelectric point, and as a result, the isoelectric point of the enzyme was about 9.1.

The hydrolysis activity of this enzyme using maltose as a substrate was measured as follows.

2 for 0.1 mL of 5 mM maltose
Enzyme solution 0.15 containing 0 mM acetate buffer (pH 5.5)
After adding mL and reacting at 50 ° C. for 10 minutes, the amount of glucose produced was measured by Glucose B Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.). The amount of enzyme that decomposes 1 μmol of maltose in 1 minute was defined as 1U.

Further, the transfer reaction using maltose of the present enzyme as a substrate is carried out by a pre-column derivative for labeling the reducing end of the sugar with a sugar (kojibiose, kojibiosyl glucose, nigerose, nigerosyl glucose) produced by the transfer reaction. ABEE labeling method (S. Yasuno et a
l., Biosci. Biotechnol. Biochem., 61, 1944-1946, (1
997)). The specific analysis method was as follows.

50% for 0.5 mL of 2% maltose
An enzyme solution (0.5 mL) containing a mM ammonium acetate buffer (pH 5.5) was added, and the mixture was reacted at 50 ° C. for 1 hour. After deactivating the enzyme by treating it in a boiling bath for 10 minutes, 10 μL of the reaction solution was collected and labeled with a reducing sugar using an ABEE labeling kit (made by Honen Corporation). The reaction liquid containing the ABEE-labeled reducing sugar was subjected to HPLC analysis, and each sugar was identified by comparison with the elution time of each of the available standard sugars, which were analyzed in advance under the same conditions. HPLC measurement conditions: Column Hornenpack C18 Column temperature: 35 ° C. Mobile phase composition: 0.1M ammonium acetate buffer solution (pH 4.0) containing 10.5% acetonitrile Detection: UV detection flow rate: 1.0 mL / min

As the microorganism used for producing α-glucosidase in the present invention, a saccharide containing koji-oligosaccharide and nigerooligosaccharide using at least one saccharide selected from α-glucan, α-glucooligosaccharide and glucose as a substrate. It may be produced, and specifically, a fungus belonging to the genus Paecilomyces, particularly Paecilomyces lyrakinus, can be mentioned. Paecilomyces that can be used in the present invention
Examples of the Lyraquinas include NRRL895 strain, ATCC26839 strain, and D218 strain deposited in ATCC and the like. Particularly, those suitable for oligosaccharide production include Paecilomyces isolated from soil by the present inventors.
Lyraquinus HKS-124 (FERMP-1840
6) Examples include strains.

The mycological properties of the HKS-124 strain are as follows: Viable medium: potato dextrose agar (PDA),
Growth was slightly faster on malt agar and Czapek-yeast extract agar (CYA) medium.
The diameter reaches 60 mm or more at 5 ° C. for 2 weeks. Mycelium: Mycelium is initially white and rises upright. From one week after the start of culture, the color changes to purple with conidia formation. Morphological observation: Growth pH is 4-10, optimum pH is near neutral, growth temperature is 20-35 ° C, optimum growth temperature is 20.
It is -30 ° C. Morphological characteristics: Penicillus-like conidia formation structure was observed,
Furthermore, the penicillium-like structure is irregular, and in some conidia peduncles, branching is observed from the peduncle.

Based on the above morphological characteristics, this bacterium was identified as a kind of genus Paecilomyces.

Furthermore, as a result of determining the 28S rDNA nucleotide sequence of this strain and searching a similar nucleotide sequence from GenBank,
Paecilomyces lilaci
nus). From the above results, this strain was identified as Paecilomyces lyrakinus, and was named Paecilomyces lyrakinus HKS-124 strain.

The bacterium was FER to the Patent Biological Depository Center, National Institute of Advanced Industrial Science and Technology, on July 5, 2000.
Deposited as MP-18406. Microorganisms used in the present invention is not limited to wild strains, the above wild strains ultraviolet light,
X-ray, radiation, various chemicals [NTG (N-methyl-N '
-Nitro-N-nitrosoguanidine), EMS (ethyl methanesulfonate), etc.] can be used as long as they have the ability to produce sugars containing koji oligosaccharides and nigerooligosaccharides.

The medium used for culturing the strain of the present invention which produces the present enzyme may be any one so long as it can grow a microorganism and produces the present enzyme, and may be a synthetic medium or a natural medium.

The carbon source to be added to the medium may be any one that can be assimilated by microorganisms, such as glucose, starch and its decomposition products are most suitable. In addition to these, fructose, trehalose and lactose are also suitable. You can also use sucrose, molasses, etc. Examples of the nitrogen source include inorganic nitrogen compounds such as ammonium sulfate, ammonium acetate, and nitrates, and organic nitrogen-containing substances such as urea, yeast extract, soybean peptone, casein, polypeptone, malt extract, and corn steep liquor.
In addition, inorganic salts such as calcium salt, magnesium salt, potassium salt, sodium salt, phosphate salt, manganese salt and zinc salt are appropriately used.

Cultivation conditions may be such that microorganisms grow and the present enzyme is satisfactorily produced, and the temperature is usually in the range of 15 ° C to 35 ° C, preferably 20 ° C to 30 ° C. If the pH of the culture solution is between pH 3.0 and 9.0, it is not necessary to adjust it. During culture, it is desirable to perform aerobically by shaking, aeration and stirring. The culture time may be any time as long as the microorganism can grow, and is preferably 72 hours to 150 hours. The microorganism thus cultivated is separated and collected from the bacterial cells and the culture solution by centrifugation, filtration with a filter paper, a glass filter, or the like.

[0042]

BEST MODE FOR CARRYING OUT THE INVENTION In order to produce a sugar containing koji-oligosaccharide and nigerooligosaccharide in the present invention, the bacterial cells thus cultured are directly used as a substrate for α-glucan,
It may be allowed to act on at least one selected from α-glucooligosaccharide and glucose. In that case, the recovered bacterial cells may be used immediately after separation, or at -20 to -40 ° C.
It can also be used after thawed at the appropriate time after being frozen and stored.
In addition, the enzyme is extracted by the following procedure,
A purified enzyme may be used.

Α-Glucosidase, which has an activity of producing sugars containing koji oligosaccharides and nigerooligosaccharides, is confirmed to be active both in the extracellular culture solution and the intracellular extract in the latter stage of the culture of this strain. However, since the inside of the cells obtained by culturing for a shorter period of time retains sufficient activity to achieve this purpose, the enzyme in the cells is usually used.

The enzyme in the microbial cell may be extracted from the microbial cell by a conventional ultrasonic crushing method, a mechanical crushing method using glass beads or the like, a crushing method using a French press, or the like. The body was suspended in a weakly alkaline buffer solution having a pH of 6.0-10.0, preferably pH 7.0-9.0, and the suspension was left overnight at a low temperature of 5-10 ° C. to easily and contaminate the enzyme solution. It can be extracted outside the cells without containing substances (protein, pigment, etc.). After that, the intracellular enzyme crude extract obtained by removing the bacterial cell residue by filtration or centrifugation has a high specific activity as compared with each bacterial cell disruption method and can be sufficiently used as it is for carbohydrate synthesis, If necessary, this may be used as a starting material and concentrated by a conventional means such as salting out, alcohol precipitation, or membrane concentration to be used as a crude enzyme, or ion-exchange chromatography, hydrophobic chromatography, gel filtration chromatography. It may be used after purification using various chromatography such as chromatography. Koji oligosaccharide in the present invention,
Example 1 shows a specific example of isolation and purification of an enzyme that produces a sugar containing a nigerooligosaccharide.

Obtaining the oligosaccharide of the present invention by allowing the α-glucosidase thus obtained from the bacterial cell to act on at least one selected from the substrate α-glucan, α-glucooligosaccharide and glucose. You can

As a substrate capable of efficiently producing koji oligosaccharides and nigerooligosaccharides, maltooligosaccharides,
It is possible to use α-glucooligosaccharide such as maltopentaose, maltohexaose, maltotriose, nigerose, kojibiose and maltose, starch, amylose, amylopectin, glycogen, α-glucan such as starch syrup and glucose.

When glucose is used as a substrate, unlike other substrates, koji-oligosaccharides and nigerooligosaccharides can be produced by a condensation reaction, which is the reverse reaction of a hydrolysis reaction, but the yield is changed. Since it is lower than the reaction, it is preferable to use maltose from the viewpoints of economy and solubility in water.

The α-glucosyl group acceptor may have a glucose residue at the non-reducing end,
Specifically, glucose, maltotriose, maltose, isomaltose, isomaltotriose, panose, nigerose, nigerosylglucose, nigerotriose, kojibiose, kojibiosylglucose, kojitriose, cellobiose, laminaribiose, sophorose. , Gentiobiose, trehalose, sucrose and the like.

The concentration of the substrate may be any concentration as long as it dissolves in the reaction solution, and in the case of maltose, it may be in the range of 1-80%. In order to obtain sugar more efficiently, 10-50
It is desirable to carry out under the condition of% maltose. Specific methods for producing oligosaccharides are shown in Examples 2 and 3.

The temperature used for the reaction may be in the temperature range in which the enzyme is stable during the progress of the reaction, 30-80 ° C,
It is desirable to carry out at 40 to 70 ° C. The pH used in the reaction is usually pH 3.5-7.0, preferably pH 4.0-6.0. The reaction time varies depending on the reaction conditions, but since this enzyme produces a significant amount of nigerooligosaccharides having an α-1,3-glucoside bond in the early stage of the reaction, the reaction time is 3 for the purpose of efficiently obtaining nigerooligosaccharides. -12 hours to finish. It is usually 24-1 in order to produce a sufficient amount of koji-oligosaccharide.
A reaction time of 24 hours is required.

It is convenient that the enzyme concentration used in the reaction is high because the reaction time can be shortened. When the enzyme concentration is low, the yield of koji-oligosaccharide becomes poor. Specifically, 1 g maltose
On the other hand, a concentration of 10 U or more, preferably 15 U or more is suitable. Usually, under such conditions, 10-30% nigerooligosaccharides and 10-20% cordierigosaccharides accumulate in the sugar solids. The koji-oligosaccharide thus obtained and the sugar containing the nigerooligosaccharide can be concentrated as they are and used as a syrup. Exchange chromatography can also be used to obtain high concentrations of oligosaccharide syrup.

[0052]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[0053] The Example 1 Paecilomyces Rairakinasu HKS-124 strain, maltose (5%), polypeptone _{(1%), MgSO 4 ·} 7H 2 0 (0.01%),
Medium containing KCl (0.01%) and K ₂ HPO ₄ (0.05%) (150 mL / 500 mL flask x 30)
At 25 ° C, the cells were shake-cultured for 5 days. This culture solution is centrifuged, and the precipitated bacterial cell fraction is washed several times with 50 mM sodium acetate buffer (pH 5.5) and collected. 5
From the L culture solution, cells having a wet weight of about 200 g were obtained. Paecilomyces lyrakinus HK obtained by culture
The cell fraction of S-124 strain (200 g) was washed several times with 50 mM sodium acetate buffer (pH 5.5), and then 1000 mL of 50 mM phosphate buffer (pH
Suspend well in 8.0) and leave at 5 ° C overnight. Then, the supernatant is recovered by centrifugation, and the intracellular crude enzyme solution (0.2
(U / mL) 1000 mL was prepared. By this method, almost 90% of the enzyme activity in the cells could be extracted and collected outside the cells. This crude enzyme solution was applied to an SP-Toyopearl 650M column (30 mm × 180 mm, manufactured by Tosoh Corporation) equilibrated with 50 mM sodium acetate buffer (pH 5.5), and the NaCl concentration was increased linearly to elute the target protein. I went.

The enzyme active fraction thus eluted was collected (380 mL, 0.3 U / mL) and dialyzed against 50 mM sodium acetate buffer (pH 5.5). Next, ammonium sulphate was added to the collected eluate so that the eluate was 1.5 M, and the eluate containing 1.5 M ammonium sulphate was added.
Butyl Toyopearl column (18 mm x 250 m) buffered with 0 mM sodium acetate buffer (pH 5.5)
m, manufactured by Tosoh Co., Ltd.), the concentration of ammonium sulfate was linearly reduced from 1.5 M to 0 M, and the eluted maltose-degrading fraction (22 mL 1.6 U /
(mL) was collected. The collected active fraction was concentrated by ultrafiltration, and the eluted active fraction was subjected to gel filtration chromatography (30 mm x 100 cm) using Sephacryl S-100 (manufactured by Amersham Pharmacia Biotech). After collection, a single intracellular enzyme preparation was obtained by electrophoresis. The activity recovery rate was 12%, and the specific activity was improved 80 times.

Example 2 0.5 mL (20 U / mL) of intracellular purified enzyme prepared as in Example 1 was added to 45%.
Act on 1 mL of maltose (final concentration 30%), 50
The reaction started at ° C. After 3 hours from the start of the reaction, the enzyme was inactivated by heating in a boiling bath for 10 minutes. The reducing sugar in this reaction solution was labeled using an ABEE labeling kit, and the composition of the transfer product was analyzed by reverse phase HPLC. A standard reducing sugar of known concentration was similarly labeled with ABEE, and each component was quantified from its elution time and peak area. The results are shown in Table 3. 3 hours after the start of the reaction, 26.9% of nigerooligosaccharide and 7.6% of koji-oligosaccharide were obtained in the total solid content of the sugar. Nigerose, which is a disaccharide among nigerooligosaccharides, is 1
2.2%, the trisaccharide nigerosyl glucose is 14.
7%, and 2.5% of the disaccharide, kojibiose, and 5.1% of the trisaccharide, kojibiosylglucose, were obtained. The chromatogram of the oligosaccharides at this time is shown in FIG.

Example 3 0.5 mL (20 U / mL) of intracellular purified enzyme prepared in the same manner as in Example 1 was added to 45%.
Act on 1 mL of maltose (final concentration 30%), 50
The reaction started at ° C. 78 hours after the start of the reaction, the enzyme was inactivated by heating in a boiling bath for 10 minutes. The reducing sugar in this reaction solution was labeled using an ABEE labeling kit, and the composition of the transfer product was analyzed by reverse phase HPLC. A standard reducing sugar of known concentration was similarly labeled with ABEE, and each component was quantified from its elution time and peak area. The results are shown in Table 3. 78 hours after the start of the reaction, 13.2% of nigerooligosaccharide and 15.7% of koji-oligosaccharide were obtained in the solid content of total sugar. Of the nigerooligosaccharides, the disaccharide nigerose was 11.9% and the trisaccharide nigerosyl glucose was 1.
3%, and 13.9% of the disaccharide, kojibioose, and 1.8% of the trisaccharide, kojibiosylglucose, were obtained. The chromatogram of oligosaccharides at this time is shown in FIG.

[Example 4] The crude enzyme solution was prepared in the same manner as in Example 1, except that the strain to be used was replaced with Paecilomyces lyrakinus HKS-124 strain by Paecilomyces lyraquinas D218 strain. 45% of the crude enzyme solution (20 U / mL) was used in the same manner as in Example 3.
It acts on maltose, and the composition of the transition product is reversed phase HPL.
Analyzed in C. The results are shown in Table 3. 78 after the start of reaction
By time, 6.2% of nigerooligosaccharide and 5.3% of koji-oligosaccharide were obtained in the total solid content of sugar. Of the nigerooligosaccharides, the disaccharide nigerose is 5.8%, the trisaccharide nigerosyl glucose is 0.4%, and of the koji oligosaccharides, the disaccharide kojibiose is 4.5% and the trisaccharides are trisaccharides. 0.8% of kojibiosyl glucose was obtained.

[Example 5] 0.3 mL (20 U / mL) of the intracellular purified enzyme prepared as in Example 1 was added to 80
Act on 0.5 mL of% glucose (final concentration 50
%), And the reaction was started at 50 ° C. 72 hours after the start of the reaction, the enzyme was inactivated by heating in a boiling bath for 10 minutes. The reducing sugar in this reaction solution was labeled using an ABEE labeling kit, and the composition of the transfer product was analyzed by reverse phase HPLC. A standard reducing sugar of known concentration was similarly labeled with ABEE, and each component was quantified from its elution time and peak area. The results are shown in Table 3. Even in the condensation reaction using glucose as a substrate, nigerose and kojibiose can be obtained, although the yield is low as compared with the transfer reaction.

[0059]

[Table 3]

Example 6 1 mL of the enzyme solution prepared as in Example 1 was allowed to act on 2 mL of 45% maltose (final concentration 30%), and the reaction was started at 50 ° C. 100 μL was taken out over time and heated in a boiling bath for 10 minutes to inactivate the enzyme. The reducing sugar in this reaction solution was labeled with an ABEE labeling kit, and the composition of the transfer product was determined by reverse phase HP.
Analyzed by LC. A standard reducing sugar of known concentration was similarly used for ABE.
As a result of E-labeling and comparison with its elution time and peak area, as shown in FIG. 6, immediately after the start of the reaction, glucose was transferred to the substrate, maltose, through α-1,2, α-1,3 bonds. Trisaccharides such as kojibiosyl glucose and nigerosyl glucose are mainly produced, but these trisaccharides are rapidly reduced about 20 hours after the reaction is started.
Accordingly, glucose is used as a sugar receptor for α-1, 2, α
It can be seen that -1,3-bonded cordiose and nigerose accumulate as major transfer products.

[0061]

As described above, the novel α-glucosidase possessed by the bacterium belonging to the genus Paecilomyces disclosed by the present invention acts on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose. By doing so, it is possible to efficiently produce a sugar containing cordierigosaccharide and nigerooligosaccharide, which are oligosaccharides expected to have functionality.

[Brief description of drawings]

FIG. 1 is a graph showing the results of examining the effect of pH on the pH stability and maltose degrading activity of the present enzyme when maltose was used as a substrate.

FIG. 2 is a graph showing the results of examining the effect of pH on the transfer activity (amount of nigerosyl glucose produced) of this enzyme when maltose was used as a substrate.

FIG. 3 is a graph showing the results of examining the temperature stability of the present enzyme and the optimum temperature of maltose-degrading activity when maltose was used as a substrate. .

[Fig. 4] Fig. 4 shows H after the transfer product of 30% maltose solution and this enzyme were allowed to act at 50 ° C for 3 hours after ABEE labeling.
The chromatogram analyzed by PLC is shown.

FIG. 5: 30% maltose solution and this enzyme at 78 ° C. for 78
The chromatogram analyzed by HPLC after ABEE labeling of the transfer product at the time of making it act is shown.

FIG. 6 is a graph showing the time-dependent changes in the composition of transfer products when a 30% maltose solution and the present enzyme were allowed to act at 50 ° C., after the transfer products at each time were labeled with ABEE.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12R 1: 645) (72) Inventor Hirofumi Nakano 3-15-20 Hattori Nishimachi, Toyonaka City, Osaka Prefecture (72) Invention Person Hiroyuki Hashimoto 2-8-2-404 Minase, Shimamoto-cho, Mishima-gun, Osaka Prefecture (72) Inventor Sumio Kitahata 1-1-6 Mikumadai, Kumatori-cho, Sennan-gun, Osaka Prefecture (reference) 4B050 CC01 DD03 FF02 FF04 FF05 FF11 FF12 LL02 4B064 AF04 BH20 CA05 CD09 CD19 DA10 4B065 AA58X BA23 BB15 BB17 BB18 CA31 CA41

Claims (11)

[Claims] 1. Paecilomyces s characterized in that it acts on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose to produce a sugar containing koji-oligosaccharide and nigerooligosaccharide.
p.) Fungi belonging to the genus. 2. The fungus according to claim 1, which is Paecilomyces lilacinus. 3. The fungus of claim 1, wherein the fungus is Paecilomyces lyraquinas HKS-124 (FERM P-18
406) which is a fungus. 4. A method for producing bacterial cells, which comprises aerobically culturing the fungus according to claim 1 to recover bacterial cells in the culture. 5. A microorganism-derived α-characterized in that it acts on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose to produce a sugar containing koji-oligosaccharide and nigerooligosaccharide. Glucosidase. 6. The α-glucosidase according to claim 5, wherein the microorganism is a fungus belonging to the genus Paecilomyces. 7. The α-glucosidase according to claim 5, wherein the microorganism is Paecilomyces lyrakinus. 8. The α-glucosidase according to claim 5, wherein the microorganism is Paecilomyces lyrakinus HKS-124 (FERM P-18406). 9. The α-glucosidase according to claim 5, which has the following properties. (1) Substrate specificity Decomposes α-glucooligosaccharides such as maltose, nigerose, kojibiose and maltooligosaccharides and α-glucoside bonds on the non-reducing end side of α-glucans such as amylose, amylopectin and glycogen to release glucose. Further, it also has a transglycosylation activity of transposing the α-glucosyl group to the 2, 3 and 4 positions of the glucose residue at the non-reducing end of the sugar acceptor. It is difficult to act on glucosides such as para-nitrophenyl α-glucoside and methyl α-glucoside, and heterooligosaccharides such as sucrose. (2) Optimum pH 5.0 (40 ° C.) (3) Stable pH range 40 ° C. 1 hour treatment 4.0
It is -8.0. (4) Optimum temperature 65 ° C (pH 5.5). (5) Temperature stability The temperature at which 50% activity is obtained by treatment at pH 5.5 for 30 minutes is 65 ° C. (6) Molecular weight of about 54,000 (gel filtration chromatography), about 50,000 (SDS gel electrophoresis). (7) Isoelectric point is about 9.1. 10. The method according to any one of claims 5 to 9, wherein the bacterial cells according to claims 1 to 3 are aerobically cultivated and produced in the culture solution, or from the cultured cell extract. Α-
A method for producing α-glucosidase, which comprises collecting glucosidase. 11. A sugar containing koji-oligosaccharide and nigerooligosaccharide by allowing the α-glucosidase according to claim 5 to act on a sugar solution containing at least one selected from α-glucan, α-glucooligosaccharide and glucose. A method for producing a carbohydrate, which comprises obtaining quality.