JP2987685B2 - Novel glucosyl (α1 → 6) branched hydrolase, method for producing and using the enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a glucosyl (α1 →
6) A method for producing glucosyl (α1 → 6) branched hydrolase using a branched hydrolase and a novel bacterium belonging to the genus Bacillus which produces the enzyme. The present invention relates to a method for producing glucosyl (α1 → 6) -branched sugars, characterized in that the sugars act on glucosyl (α1 → 6) branched sugars.

More specifically, a novel bacterium that produces an enzyme that cleaves a saccharide branch having an α-1,6-linked branch of glucose, production of the cleavable enzyme by culturing the bacterium, and reverse production of the produced cleavable enzyme The present invention relates to a method for producing a branched carbohydrate having a glucose branch by causing a mixture of glucose and various carbohydrates to act on the mixture.

Hereinafter, cyclodextrin is abbreviated as CD. The CD includes a normal ring CD, a macrocyclic CD, a branched C
There are various types such as D and CD derivatives.

[0004]

2. Description of the Related Art Amylo-1,6-glucosidase is conventionally known as a glucosyl (α1 → 6) branched hydrolase and has been found in rabbit muscle [J. Biol. Chem., 1].
88, 17-29 (1951)]. In addition, microorganisms have been found to be present in yeast [Arch. Biochem. Bioph.
ys., 121, 245-246 (1967)].

[0005] In the analysis of these enzymes, substrate B5 in which glucose is α-1,6-linked to maltotetraose is used, but it is difficult to use in an enzyme system in which α-amylase is mixed.

In 1966, Taylor, PM and Whela
n and WJ are glucosyl-α-CD (G ₁ -α-CD) and glucosyl-β-C as substrates capable of quantifying amylo-1,6-glucosidase even in an enzyme system mixed with α-amylase.
D (G ₁ -β-CD) was prepared.

[0007] These branched CDs have characteristics not found in unbranched CDs, such as very good solubility in water. In dextran, high functionality such as antitumor is found in a structure having one branch per 3 to 5 glucose units, and similarly, a polysaccharide derived from mushrooms has 3 to 5 functions.
Those having a branched structure such as one glucose in five glucose units have been found to have high functionality, and there has been a strong demand for a technique for branching and linking glucose to a saccharide.

Several methods for branching CDs of glucose have been proposed.

In a high concentration solution containing maltose and CD,
By the action of debranching enzyme such as pullulanase or isoamylase, maltosyl -CD by reverse synthesis reaction (G ₂ -
CD), and then reacting with glucoamylase to hydrolyze the maltosyl moiety to give G ₁ -CD (JP-A-61-197602 and JP-A-61-287901). In this method, even if a substrate other than maltose, for example, an oligosaccharide such as maltotriose, is used, a branched CD corresponding to the substrate can be obtained, and the glucoamylase is similarly acted upon.
Can be G ₁ -CD.

A method using amylo-1,6-glucosidase prepared from rabbit muscle or yeast cells (Japanese Patent Application Laid-Open No.
79491). In this method, amylo-1,6-glucosidase is allowed to act on a highly concentrated solution containing glucose and CD, and G1-CD is prepared by a reverse synthesis reaction.

[0011]

However, in the above method, a branching enzyme such as pullulanase or isoamylase is allowed to act to bind maltooligosaccharides of maltose or higher to a stem or a nucleus sugar. Because of the treatment, it is necessary that the carbohydrate as the trunk is not affected by the enzyme.

In the method using amylo-1,6-glucosidase, the enzyme preparation is limited to those derived from animal muscle or yeast.
There has been a strong demand for the development of bacterial enzymes.

However, amylo-1,6-glucosidase derived from bacteria or a similar enzyme agent has not been found so far.

[0014]

DISCLOSURE OF THE INVENTION The present inventors have widely and widely used natural sources to obtain bacterial amylo-1,6-glucosidase or a similar enzyme (hereinafter also referred to as glucosyl-branched hydrolase). We have been conducting intensive research on enzymes derived from various microorganisms. In this process, the target enzyme was obtained by culturing a strain belonging to the genus Bacillus newly isolated from soil, thereby completing the present invention.

That is, according to the present invention, a bacterium belonging to the genus Bacillus which produces a glucosyl-branched hydrolase, a method for producing the glucosyl-branched hydrolase using the bacterium belonging to the genus Bacillus, The present invention provides a method for producing a glucosyl-branched sugar, wherein the method acts on a mixture of saccharides.

The bacterium belonging to the genus Bacillus of the present invention can be screened, for example, as follows.
That is, using G ₁ -α-CD as a carbon source of the separation medium,
A strain that inoculates a suspension of soil on a flat plate agar medium, selects and picks up grown colonies, performs liquid culture, and produces glucose and α-CD in a reaction solution using G ₁ -α-CD as a substrate. , The activity of which is determined, and bacteria determined to be useful can be selected. The two strains thus selected (No. 1
4 and No. 17) were examined in detail.

The test for producing hydrogen sulfide used a TSI agar medium (Nissui), and the urease test used a Christensen urea medium (Eiken). The growth test under anaerobic conditions was performed by inoculating a plate with or without glucose (1%) on a normal agar medium (Eiken), culturing with a gas pack (BBL), and others with Gordon.
We followed these methods. The cultivation temperature was 30 ° C except for the growth temperature test. The results are shown below.

No. 14 No. 17 Cell shape Bacillus Bacillus No. 17 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Cell size 2.4-6.4 × 0.6-0.8μ 2.4-4.8 × 0.6-0.8μ Motility + (perihair) + (perihair) Spore swelling + + sporulation site Oval spore shape Oval to cylindrical Oval to cylindrical --------------------------------------------- −−

No. 17 formed spores well on a normal agar medium (Eiken), but No. 14 formed poorly, and spores were formed on a normal agar medium supplemented with yeast extract (0.1%). .

Cultural properties were determined using an ordinary agar medium "Eiken"
The results of observing colonies at 30 ° C. for 48 hours are shown below.

No. 14 No. 17-----------------------------------------------------------------shape- Surface Smooth surface Periphery Whole edge Whole edge Raised Convex convex convex Gloss / color Translucent, wet light, colorless Translucent, wet light, colorless ---------------- −−−−−−−−−−−−−−−−−−−−−

In both cases, when the medium surface is not well dried, the medium spreads thinly (No. 14 is remarkable). Longer culture (1
No. 14) is translucent, while No. 17 is opaque. FIGS. 1 to 6 show micrographs of No. 14 and No. 17 cells, sporangia, and pericytes.

The physiological properties were as follows:

No.14 No.17 Gram stainability − −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Catalase + + Anaerobic growth + + + VP test--pH (VP broth) pH 4.7-4.8 pH 4.8-4.9 Acid production: glucose + + arabinose--cellobiose + + galacto- ++ Lactose Weak positive Weak positive Mannose + + mannitol--Melibiose + + Merezitose ++ Raffinose ++ Xylose--Generation of gas from glucose--Casein Decomposition + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Formation of acetone − − Production of hydrogen sulfide + + Ureaase + + Decomposition of hippurate + + Growth at pH 6.8 + + Growth at pH 5.7--Sodium chloride tolerance: 2% + + 5% + + 7%-Weak positive 10%--Growth Temperature: 10 ℃--15 ℃ ＋ ＋ 30 ℃ ＋ ＋ 45 ℃ ＋＋ 50 ℃ Weak positive Weak positive 55 ℃--

From the above results, consideration will be made with reference to the literature described later. NO.14 and NO.17 are slightly different from each other in sporulation conditions, growth condition when culture is prolonged, sodium chloride tolerance (7%), etc. .

It grows under aerobic conditions. Form spores. Exercise by the circumference hair. It is classified into the genus Bacillus because it is positive for catalase.

Searching Bergey's manual (Reference 2) revealed that sporangia swelling, sporulation sites were semi-rigid to vertebral, spores were elliptical, and species with positive indole formation were Ba.
cillus alvei, Bacillus thiaminolyticus and Bacillu
s laterosporus.

However, the bacterium of the present invention is different from Bacillus alvei in the VP test, the production of dihydroxyacetone and the like.
It differs from llus thiaminolyticus in tyrosine degradability and citrate utilization, and differs from Bacillus laterosporus in tyrosine degradability, starch degradability and the like. These are summarized in Table 1 below.

[0029]

[Table 1] NO.14 NO.17 B.alvei B.thiamino- B.laterolyticus sporus VP test--+--Production of dihydroxyperoxyacetone--+--Production of indole +++++ dReduction of nitrate + +-+ + Decomposition of tyrosine--d + + Decomposition of starch + + + +-Acid production: arabinose---V-Mannite---V + Utilization of citrate---+-

D: Positive of 11-89% of strains V: Depends on strains Data of B. alvei and B. laterosporus are cited from Reference 2. B. thiaminolyticus data is cited from Reference 3.

Reference 1: RE Gordon, WC Haynes, and
CH Pang, 1973. The genus Bacillus. Agriculture
Handbook No.427.USDepartment of Agriculture, Was
hington.DC

Reference 2: D. Claus and RCW Berkley,
1986.Genus Bacillus Cohn 1872, 174 ^AL , p.1105-1139.
In PHA Sneath, NSMair, MESharpe and JGH
olt (ed.) Bergey's manual of systematic bacteriolog
y, vol.2.The williams & Wilkins Co., Baltimore.

Reference 3: LK Nakamura, 1990. Bacillus
thiaminolyticus sp.nov., nom.rev.lnt.J. Syst, B
acteriol, 40, 242-246.

Therefore, the strain of the present invention has no match among known strains, and No. 14 is designated as Bacillus sp.
lus sp.) KW-14, and No. 17 was named Bacillus sp. KW-17. The Bacillus SP KW-14 and Bacillus SP KW-17 were each submitted to the Ministry of International Trade and Industry by
-15314 and FERM P-15315.

Bacteria that can be used in the present invention can be similarly screened from nature in addition to the two strains belonging to the genus Bacillus described above.

As a method for producing a novel glucosyl-branched hydrolase using a strain belonging to the genus Bacillus, either a liquid culture or a solid culture may be used. More preferably, a liquid culture can be used. The liquid culture method can be performed, for example, as follows.

As a medium that can be used, any medium can be used as long as a microorganism capable of producing a novel glucosyl-branched hydrolase can grow. For example, glucose, sucrose, glycerin, dextrin, molasses, carbon sources such as organic acids, further ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or
Nitrogen sources such as peptone, yeast extract, corn steep liquor, casein hydrolyzate, meat extract, and inorganic salts such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt and zinc salt were added. Can be used. G ₁ -C having a glucosyl branch
D. A substrate such as isomaltose-based saccharide may be added to the medium.

The pH of the medium is adjusted, for example, to about 3 to 9, preferably about 7.0 to 8.0, and the culture temperature is usually about 10 to about 10.
-50 ° C, preferably about 25-37 ° C, 1-20
The cells are cultured under aerobic conditions for about 3 days, preferably about 3 to 12 days. For example, a shaking culture method or an aerobic submerged culture method using a jar fermenter can be used.

A novel glucosyl-branched hydrolase of the present invention can be obtained by isolating a novel glucosyl-branched hydrolase from the obtained culture solution by ordinary means.

For example, in order to isolate and purify glucosyl-branched hydrolase from a culture solution, a combination of ammonium sulfate precipitation, alcohol precipitation, various types of chromatography such as ion-exchange resin, etc., and treatment by a conventional method are used to obtain purified glucosyl-branched hydrolase Can be obtained.

Further, the present invention will be described more specifically. That is, Bacillus sp. KW-17 (FERM P-1531) described above as a strain producing a novel glucosyl branched hydrolase.
Using 5), the cells were cultured in a liquid medium, and production of the enzyme, purification of the enzyme, and various properties of the enzyme were examined.

From a fresh slant, remove one platinum loop fungus,
The following liquid medium was inoculated and cultured.

The liquid medium (100ml, pH8.0) Polypeptone 1.5 g NH 4 Cl 0.1 g MgSO 4 · 7H 2 O 0.01 g G 1 -α-CD 1.0 g 1M KH 2 PO 4 -K 2 HPO 4 buffer 20 ml

The culture conditions were agitation culture at 37 ° C., 145 rpm for 3 days on a rotary shaker. Under these conditions, the enzyme was produced outside the cells. It was added as a medium
It is considered that G ₁ -α-CD induced the production of the enzyme, and G ₁ -CD, a substrate having an α-1,6 bond such as isomaltose, and / or a mixture thereof, or a mixture containing them is also included. Enzyme production could be induced.

As shown in FIG. 7, after the growth of the cells reached the maximum in 24 hours, the production amount of the enzyme increased.
The maximum was at 42 hours.

When the enzyme was accumulated in the cells, the cells were cultured in a liquid medium in the same manner as in the case of extracellular production.

The liquid medium (100ml, pH7.5) Bacto Tryptone 0.5 g NH 4 Cl 0.1 g MgSO 4 · 7H 2 O 0.01 g Glucose 0.5 g 1M KH 2 PO 4 -K 2 HPO 4 buffer 20 ml

In the culture using a liquid medium, it is difficult for the bacteria to grow, and when a nitrogen source for improving the growth of the cells is sufficiently added, the amount of enzyme production decreases. It is necessary to culture at a controlled ratio.

As for the method of purifying the enzyme, for the extracellularly produced enzyme, after the completion of the culture, the culture was centrifuged (1000 rpm, 20 minutes), and the supernatant was obtained as a crude enzyme solution.
The enzyme was adsorbed through a column, and concentrated and partially purified. Thus, the purification of the extracellularly produced enzyme is easy.

In the case of intracellular enzymes, the cells were obtained by centrifuging the culture solution (1000 rpm, 20 minutes) after the completion of the culture. The obtained cells were suspended in physiological saline and centrifuged again to obtain washed cells. 50 mM phosphate buffe
r (pH 6.5, 2 mM EDTA, 1 mM 2-mercaptoethanol,
(Containing 0.1 mM Phenylmethylsulfonyl Fluoride), treated with an ultrasonic crusher for 5 minutes, centrifuged, and the supernatant was used as a crude enzyme extract.

Purification was performed as shown in Table 2 by ammonium sulfate salting out (30-70%
Ammonium sulfate), α-CD Sepharose chromatography, DEAE-
Performed using Toyopearl 650M. In particular, α-CD Seph
The purification effect on arosechromatography is large (Fig. 8),
The specific activity has increased more than 3,000 times.

[0052]

[Table 2] Total protein Total activity Specific activity Recovery rate mg UU / mg% Crude enzyme extract 1520 8.8 0.0058 100 Salting out ammonium sulfate 61 5.0 0.083 57 α-CD Sepharose 0.952 3.8 4.0 43 DEAE-Toyopearl 0.083 1.7 22.7 19

The results of examination of general enzymatic properties are as follows.

Activity measuring method: 5 containing 20 mM G ₁ -α-CD
50 μl of the enzyme solution was added to 50 μl of 0 mM phosphate buffer (pH 6.5), and the mixture was incubated at 40 ° C. for 1 hour. After heat inactivation, Glucose was quantified using a glucose CII test wako. One unit is the amount of enzyme that releases 1 μmol of glucose per minute.

Determination of optimal pH: 10 mM 100 mM G ₁ -α-CD
After adding 40 μl of various buffer solutions of 100 mM to the mixture and adding the enzyme solution, the mixture was incubated at 40 ° C. for 1 hour, heated and inactivated, and then Glucose was quantified by Glucose CII Test Wako. As a result, as shown in FIG. 9, the optimum pH was 6.5 and the activity could not be measured with the Tris-HCl buffer. It has been reported that amylo-1,6-glucosidase derived from rabbit muscle is also inhibited by Tris, and it is considered that the enzyme also has similar properties.

Measurement of optimum temperature: 50 μl of 50 mM phosphate buffer (pH 6.5) containing 20 mM G ₁ -α-CD was added to 50 μl of the enzyme solution.
After adding μl, incubating at each temperature for 1 hour and inactivating by heating, Glucose was quantified using Glucose CII Test Wako. As a result, the optimum temperature was 45 ° C. (Shown in FIG. 10)

Measurement of temperature stability: pH 6.5 50 mM phosph
After standing in an ate buffer for 10 minutes at each temperature, the enzyme activity was measured. As a result, the temperature stability was up to 50 ° C. (Shown in FIG. 11)

Substrate specificity: Various maltooligosaccharides, various branched CDs and various isomaltooligosaccharides [these were prepared as 20 mM solutions (50 mM phosphate buffer: pH 6.5)], and further, short-chain long-amylose, soluble starch, amylopectin , Glycogen, dextran and pullulan [0.5%
Solution], the amount of glucose released by a shaking reaction at 40 ° C. for 1 hour was quantified. As a result, G ₁ -α-CD
G ₁ -β-
The CD was about 58. Further, it only slightly acted on other substrates.

Next, a method for producing a glucosyl-branched sugar using the novel glucosyl-branched hydrolase of the present invention will be described in detail.

The glucosyl branched hydrolase of the present invention is allowed to act on a mixture of various sugars and glucose. Examples of various sugars include CD, maltose, isomaltose, cellobiose, trehalose, sucrose, lactose, starch, dextran, pullulan, cellulosic polysaccharides, polysaccharide degradation products, and the like.

The reaction can be carried out generally at a total concentration of various sugars and glucose of 10 to 70%, in which case the ratio of glucose to various sugars is 1: 1 to 100:
1 (g). The reaction temperature is 30 to
The reaction can be performed at 60 ° C. for a reaction time of 24 to 120 hours. In addition, these conditions can be appropriately changed according to the purity of the enzyme to be used, the purity of the substrate, and the like.

The analysis of glucosyl-branched saccharides produced under the above conditions can be examined under low-concentration substrates, mainly using amylo-1,6-glucosidase that cleaves the glucose branch. The details of the study are described below.

For α-CD, β-CD, γ-CD, soluble starch and SCA (Short Chain Amylose, average degree of polymerization = 23), 0.05 g of substrate and 0.2 g of glucose were added to 100 μl of 50 mM 50 mM.
After heating and dissolving in phosphate buffer (pH 6.5), add 100 μl of enzyme.
(0.02U) and reacted at 45 ° C for 3 days. After the reaction is completed, the enzyme is inactivated by heating, and the ODS column (CDs) or G30
Analyzed with 00PW XL.

The analysis conditions are as follows. (Dete
ction: RI) α-CD, γ-CD Column temperature: 20 ° C Column: Inertsile ODS-3 (4.7φ × 150 mm) Eluent, Flow rate: 2% EtOH, 0.9 ml / min. β-CD Column temperature: 20 ℃ Column: Inertsile ODS-3 (4.7φ × 150 mm) Eluent, Flow rate: 7% MeOH, 0.7 ml / min. Soluble starch and SCA Column temperature: 20 ℃ Column: TSK-GEL G3000PW XL Eluent, Flow rate: water , 0.5 ml / min Oligosaccharide Column temperature 85 ℃ Column: MCI GEL CK04S (10mmφ × 200mm) Pre-column: CK10SG (6mmφ × 50mm) Eluent, Flow rate: water, 0.4ml / min

The branching of various oligosaccharides was examined in the same manner and analyzed by MCI GEL. As a result, a product having a shorter HPLC retention time than the original oligosaccharide (region having a higher molecular weight than the original oligosaccharide)
was gotten. After separation by high performance liquid chromatography, amylo-
When 1,6-glucosidase was allowed to act, glucose and the original oligosaccharide were formed. Thus, it was confirmed that glucose was α-1,6 bonded to the glucose residue of the original oligosaccharide.

FIG. 12 shows an example of the production of a branched saccharide using CD as a substrate. As shown in the figure, when the ring is large, one having one or more branches is also generated. Similarly, in the case of a macrocyclic CD of δ or more, a CD having a plurality of branches was generated. Further, by allowing the branched CD to act as a substrate, further branching can be provided.

In the reaction between maltose and glucose, a small amount of panose or isopanose was produced, and even when cellobiose, trehalose, sucrose, or lactose was used instead of maltose, a slight amount of the product was obtained.

In the case of starch and pullulan-based polysaccharides, a mixture with glucose is allowed to act on the enzyme, followed by washing by organic solvent precipitation to remove glucose, and dissolving in water again.
Rabbit muscle was reacted with amylo-1,6-glucosidase to produce glucose, and thus a branching reaction was observed.

The cellulosic polysaccharide was reacted in suspension, and after completion of the reaction, the precipitate was collected and washed with water, and glucose was produced in the same manner. Thus, a branching reaction was recognized.

As described above, the glucosyl-branched hydrolase produced in the present invention is similar to the conventional amylo-1,6-glucosidase, and is composed of glucose and α-1,6-linked glucose and α-1,4 glucan. Can be cleaved, but in the presence of a large excess of glucose, glucosyl-branched saccharides can be produced more efficiently, and the substrate specificity is extremely wide.

The enzyme thus produced can produce glucosyl-CD having one or more glucose branches by acting on CD in the presence of high concentration of glucose. Is useful because it imparts resistance to the action of ordinary starch hydrolase.
In the case of D, it is possible to attach 1 to 4 branches.

[0072] Sucrose obtained by binding glucose with α-1,6 bonds is also referred to as the Andalose. Generally, carbohydrates having α-1,6 bonds, such as isomaltose, panose and palatinose, are anti-cariogenic. In addition, the enzyme of the present invention can also be used for the production of these oligosaccharides, since a bifidobacterial growth action is also observed.

When dextrin is branched, it is resistant to the action of ordinary starch-degrading enzymes, and the range of use is expanded. That is, a highly stable saccharide can be obtained by branching the saccharide which is easily affected by the amylolytic enzyme.

By branching the inner-branched CD, it is also possible to obtain a saccharide which is resistant to the action of the debranching enzyme. Recently developed cycloamylose and amylopectin cyclized saccharides having more than 10 glucoses or more are available. It is also thought that it can be made to act on the quality and to resist the action of the normal amylolytic enzyme.

In a reverse reaction of a debranching enzyme such as pullulanase,
It is also expected that maltooligosaccharides can form an inner-branched cyclic saccharide, but in this case, the generated inner-branched cyclic saccharide may be cleaved again by a forward reaction of the debranching enzyme.

Thus, by attaching a glucosyl branch to the generated inner-branched cyclic sugar, it is possible to suppress the cleavage and efficiently produce the inner-branched cyclic sugar. In addition, a method of mixing and reacting the glucosyl branched hydrolase of the present invention and a branching enzyme is also conceivable.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. Hereinafter, unless otherwise specified,% is shown by W / V%.

[0078]

【Example】

Example 1 Bacillus sp. KW-17 (FERM P-15315) was subjected to rotary shaker culture at 37 ° C., 145 rpm for 2 days in the above-mentioned liquid medium, and a culture solution of about 0.008 units / ml was obtained.

Example 2 In the same manner as in Example 1, Bacillus SP KW-14 (FER
When MP-15314) was cultured in liquid, it showed an enzyme activity of about 0.004 unit / ml.

Example 3 Using liquid medium 2, Bacillus sp. KW-17 (FERM
P-15315) was cultured in the same manner as in Example 1, and production of glucosyl-branched hydrolase was confirmed in the cells. Intracellular enzymes are obtained by sonication in ice water and centrifuged supernatant.
The production amount was about 1/10 compared to Example 1.

Example 4 The culture was carried out in the same manner as in Example 1 except that an equal mixture of G ₁ -β-CD and isomaltose was used instead of G ₁ -α-CD in the liquid medium. An enzyme activity of unit / ml was obtained.

Example 5 Cultivation was carried out in the same manner as in Example 3 except that an equal mixture of isomaltose and glucose was used instead of glucose in the liquid medium.
An enzymatic activity of 001 units / ml was obtained.

Example 6 The supernatant obtained by centrifuging the culture solution obtained in Example 1 was treated with α
-CD Sepharose column, concentrated and partially purified to give an enzyme solution of 0.21 units / ml. 0.5 g of α-CD and 2 g of glucose were dissolved in 1 ml of 50 mM phosphate buffer (pH 6.5), and the enzyme solution was dissolved. When 1 ml was added and reacted at 45 ° C. for 3 days, G ₁ -α-CD (in terms of α-CD) was obtained with a yield of 16%.

Example 7 The same operation as in Example 6 was carried out except that α-CD was replaced with β-CD. As a result, G ₁ -β-CD is _{12%, (G 1) 2} -
7% yield of β-CD was obtained. In addition, β-CD has a low solubility, and was reacted in a suspension state.However, the solubility was improved by branching, the dissolved portion was continuously taken out, and the enzyme was recovered by membrane separation. By returning to the reaction tank, glucosyl-branched β-CD can be produced continuously.

Example 8 In the same manner as in Example 7, the reaction was carried out using δ-CD.
As a result, (G ₁ ) _n -δ-CD (n = 1 to 4) was obtained with a yield of 16%. For a macrocyclic CD of ε or more, a similar procedure was performed using a mixture thereof to obtain (G ₁ ) _n -CD.

Example 9 A panose-like (having the same retention time of panose and HPLC and possibly a mixture of panose and isopanose) sugars in the same manner as in Example 6 except that maltose was used instead of α-CD. 3% quality was obtained. Maltose is degraded by the enzyme preparation, but its action can be suppressed by adding an excess amount of glucose.

Example 10 The procedure of Example 6 was repeated, except that sucrose was used instead of α-CD, to obtain 2% of a teandalose-like saccharide (having the same retention time as that of teandalose and HPLC). Lactose had a similar production rate.

Example 11 A saccharide having a higher degree of polymerization than trehalose was obtained in the same manner as in Example 6, except that α-CD was changed to trehalose.

Example 12 A reaction was carried out in the same manner as in Example 6 except that α-CD was changed to soluble starch. After the reaction was completed, glucose was removed by precipitation with 80% ethanol and dissolution in water, and the resulting saccharide was obtained. 0.5%
When dissolved in water to a certain concentration and reacted with amylo-1,6-glucosidase, glucose was produced, and molecules larger than the original substrate could be detected by HPLC. From this result, it was confirmed that the light was branched. Pullulan was similarly confirmed.

Example 13 The procedure of Example 12 was repeated, except that the soluble starch was replaced by cellulose and the 80% ethanol precipitation-water dissolution was replaced by washing with water.

[0091]

EFFECTS OF THE INVENTION As an enzyme for debranching α-1,6-linked glucosyl, it was first found in bacteria, and this enzyme has a wide substrate specificity. In addition, saccharides having various branches can be produced by utilizing the reverse synthesis reaction. Further, depending on the culture conditions, the enzyme can be produced inside and outside the cells, and purification is easy.

[Brief description of the drawings]

FIG. 1 is a photograph showing the form of an organism, showing cells of Bacillus sp. KW-14 (FERM P-15314) and sporangia. Conditions: Normal agar medium (Eiken) cultured at 30 ° C for 4 days in 0.1% yeast extract. Crystal violet stain, × 1250

FIG. 2 is a photograph showing a morphology of an organism, showing cells of Bacillus sp. KW-14 (FERM P-15314) and sporangia. Conditions: Normal agar medium (Eiken) cultured at 30 ° C for 4 days in 0.1% yeast extract. × 125
0.

FIG. 3 is a photograph showing the morphology of an organism, showing the hair around Bacillus sp. KW-14 (FERM P-15314). Conditions: 20 hours on ordinary agar medium (Eiken), 30 hours
Cultured at ℃ and stained by Nishizawa and Sugawara. × 1250

FIG. 4 is a photograph showing a morphology of an organism, showing cells of Bacillus sp. KW-17 (FERM P-15315) and sporangia. Conditions: Cultured at 30 ° C. for 3 days in 0.1% ordinary yeast extract (10% yeast extract, Eiken). Crystal violet stain, × 1250

FIG. 5 is a photograph showing a morphology of an organism, showing cells of Bacillus sp. KW-17 (FERM P-15315) and sporangia. Conditions: 3 days on ordinary agar medium (Eiken)
Cultured at 30 ° C. × 1250

FIG. 6 is a photograph showing the morphology of an organism, showing the hair around Bacillus sp. KW-17 (FERM P-15315). Conditions: 48 hours on ordinary agar medium (Eiken), 30 hours
Cultivated at ℃ and stained with Ryu. × 1250

FIG. 7: Bacillus sp. KW-17 (FERM)
3 shows changes over time in culture when the liquid medium (P-15315) was used.

[Explanation of symbols]

Solid squares indicate absorbance at 660 nm, solid circles indicate enzyme activity.

FIG. 8 shows an example of purification of glucosyl-branched hydrolase by α-CD Sepharose column chromatography.

[Explanation of symbols] The solid line indicates the ultraviolet absorption of the protein, and the solid circle indicates the enzyme activity.

FIG. 9 is a graph showing the optimum pH of glucosyl-branched hydrolase.

[Explanation of symbols]

Solid squares indicate the results using acetate buffer, solid circles indicate the phosphate buffer, solid triangles indicate the results using the tris-monohydrochloride buffer, solid diamonds indicate the results using the glycine-sodium hydroxide buffer.

FIG. 10 is a graph showing an optimum temperature of glucosyl-branched hydrolase.

FIG. 11 shows the temperature stability of glucosyl-branched hydrolase.

FIG. 12 shows the results of HPLC analysis showing the production of various glucosyl-branched CDs by glucosyl-branched hydrolase using glucose and various CDs.

[Explanation of symbols]

In the figure, the peaks 1, 2 and 3 are the original CD and G _{1 respectively.}
-CD and _{(G 1)} shows a 2 -CD.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masaaki Yokoe 4-161 Nagasaka, Kani-shi, Gifu (72) Inventor Ryuichi Oya 15 Inui, Nozaki, Nishiharu-cho, Nishikasugai-gun, Aichi Prefecture Examiner Midori Tominaga (56) References JP 62-25 (JP, A) U.S. Pat. No. 5,194,284 (US, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 9 / 44-9 / 46 BIOSIS (DIALOG) WPI (DIALOG)

Claims (5)

(57) [Claims] 1. A bacillus-derived, optimum pH of about 6.
5, and a temperature optimum of about 45 ° C., and the temperature stability up to about 50 ° C., the activity is inhibited by Tris buffer, grayed
Lucosyl-α-cyclodextrin and glucosyl-
Releases glucose by acting on β-cyclodextrin
But has a slight effect on dextran and pullulan
Novel glucosyl having the properties is a Runomi (α1 →
6) Branched hydrolase. 2. A novel glucosyl (a glucosyl (α1 → 6) -branched hydrolase according to claim 1) accumulated in a culture solution by culturing a bacterium belonging to the genus Bacillus. α1 → 6) Production method of branched hydrolase. 3. The bacterium belonging to the genus Bacillus is Bacillus sp. KW-14 (FERMP-15314) or Bacillus sp. KW-17 (FERMP-1531).
The method according to claim 2, which is selected from 5). 4. A method for producing a glucosyl (α1 → 6) -branched sugar, wherein the novel glucosyl (α1 → 6) -branched hydrolase according to claim 1 derived from a bacterium belonging to the genus Bacillus is allowed to act on a mixture of glucose and saccharide. Manufacturing method. 5. The carbohydrate is cyclodextrin, starch, dextran, pullulan, a cellulosic polysaccharide, a polysaccharide degradation product, trehalose, sucrose, or lactose.
Production method as described.