JPH0761260B2 - New Bacillus microorganism - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel microorganism, more specifically to a novel microorganism belonging to the genus Bacillus that produces amylase that produces maltotetraose from starch or starch-based sugars. .

[Conventional technology]

Conventionally, α-amylase, β-amylase, glucoamylase, etc. have been known as amylase acting on starch, and have been used for the production of glucose and maltose. However, in recent years, sugars called oligosaccharides having a large molecular weight of about 3 to 10 glucose residues have attracted attention, and their applications in the fields of food and pharmaceutical industries are being developed.

For example maltotriose (G _3), maltotetraose (G _4), maltopentaose (G _5), oligosaccharides such as maltohexaose (G ₆₎ is a sweetener for food and pharmaceutical industry, an excipient , It can be widely used as a raw material for clathrates or diagnostic agents for clinical tests.

To date, regarding maltotriose, the N-A468 enzyme produced by Streptomayces griceus (JP-A-50-52278, 51-9739 and 5)
1-110049) and maltotetraose, amylase produced by Pseudomonus stuzeri [Arch. Biochem. Biophys., 145 , 105 (197
1)], maltodextrin for maltopentaose, amylase (N.Saito Produced by Bacillus licheniformis (Bacillus licheniformis);. Arch.Biochem.Biophys, 155, 290 (1
973)) and maltohexaose, the amylase G ₆ enzyme of Bacillus circulans (JP-A-57-146590) and the like are known.

[Problems to be solved by the invention]

The present inventors have conducted a search for microorganisms from the soil for the purpose of producing microorganisms having a strong malto-oligosaccharide-forming enzyme result. As a result, a strain belonging to the genus Bacillus decomposes starch into saccharified products containing maltotetraose as a main component. The present invention has been completed by discovering that amylases are secreted and produced in large quantities outside the cells.
That is, the present invention relates to a novel microorganism which belongs to the genus Bacillus and exocrine-produces amylase produced from starch or starch-based sugars with maltotetraose as the main component in the culture solution.

Regarding the production of oligosaccharides by an enzyme produced by a microorganism belonging to the genus Bacillus, Bacillus circulans (Baci
illus circulans) amylase G ₆ produces maltohexaose as a main component, and examples in which maltocyclodextrin glucosyltransferase such as Bacillus macerans produces cyclodextrin are known.

However, a bacterium of the genus Bacillus that produces an enzyme that produces a saccharified product containing maltotetraose as a main component from starch is not known.

The microorganisms that produce maltotetraose are Pseudomonus sp. (P.stuzeri) and P. saccharophila (P. saccharophila) (Naoki Okemoto et al., Summary of Japan Agricultural Chemistry in 1985). , Streptomyces H.359.USY.
6 (Streptomyces H.359.USY.6) (K.Kondo et al; J.Ant
ibiot., 28 , 157 (1975)) is known, but it is known that a microorganism belonging to the genus Bacillus produces an enzyme that produces maltotetraose-based saccharified products. Absent.

On the other hand, examples of the amylase productivity of alkaline bacterial microorganisms Bacillus No.171 (Bacillus No.171) belonging to the genus Bacillus which produces an enzyme that produces a cyclodextrin (K.Horikoshi, T.Akiba, Alkalophilic Microorganism
s ″ (1982), Academic Publishing Center, Tokyo), Airthrobacter N, which produces an enzyme that produces maltohetasauose
o.YA1-9-3 ( Arthrobacter No. YAl-9-3) (Masao Nomoto et al., Summary of the Japan Agricultural Chemistry Conference 1984) and Bacillus H- that produces an enzyme that produces maltohexaose
167 ( Bacillus H-167) strain (Takaya Hayashi, Hiroki Horikoshi, Showa
Three cases of the 61st Annual Meeting of the Japanese Agricultural Chemistry) are related to maltooligosaccharides, and no alkaline bacterium that produces the enzyme that produces maltotetraose is known.

[Means for solving problems]

The microorganism used in the present invention is a microorganism belonging to the genus Bacillus, for example, a microorganism belonging to the genus Bacillus represented by Bacillus sp.

Bacillus sp. # 707 is a microorganism isolated from soil collected in Ichihara City, Chiba Prefecture, and has the following mycological properties. In addition, the test and classification method of mycological properties,
"Aerobic Sporehoming Bacteria"
["Aerobic Sporeforming Bacteria" (United State D
epartment of Agriculture, Nov. 1952 by NRSmith, R.
E.Gordon & FEClark)] and "Bergey's Manual of Determnative Bacteriolo"
gy, 1957)].

(Mycological properties of Bacillus sp. # 707) a. Form 1 15g starch, 10g polypeptone, yeast extract
_{5g, K 2 HPO 4 1g,} MgSO 4 · 7H 2 O0.2g, sodium carbonate 10 g,
Table 1 shows the morphology observed on a plate agar medium having a composition (pH 10.0) containing 15 g of agar (however, sodium carbonate is heat-sterilized separately from other components and then added to the medium).

B. Growth state in each medium Table 2 shows the growth state of # 707 bacteria in each medium.

In addition, since there was almost no growth in medium pH 7.0 and no change was observed, 1% sodium carbonate was added to each medium.
In addition, the pH was set to 10.0.

C. Physiological Properties Table 3-1 to 3-2 show the results of the test on the # 707 bacterium by adding 1% of sodium carbonate to the physiological test medium.

From the mycological properties of the above-mentioned microorganisms, a comparison was made with known bacterial species belonging to the genus Bacillus according to the classification method of the above-mentioned literature, and it was found that Bacillus sp.
No.), the endospores are oval in shape, and the endospores are located at the ends, and spores of vegetative cells are not observed, and Bacillus stearothermophilus ( Bacillus s)
It is appropriate to compare with tearothermophilus ). However, as shown in Table 4, the Bacillus sp. # 707 strain was positive in the Phosphorus proscalael test (VP test) in physiological properties, whereas the Bacillus stearothermophilus (B. stearothermophilus) was positive. Is negative, and the growth pH range of # 707 strain is 9-12, while that of Bacillus stearothermophilus (B. stear
othermophilus) is different in that it is in the weakly acidic neutral range.

From the above results, it is clear that Bacillus sp. # 707 (Microtechnology Research Institute No. 8792) is a microorganism belonging to the genus Bacillus because it is an aerobic spore bacterium. It was concluded that it was appropriate to identify it as a new species of alkalophilic bacterium. The method for culturing a microorganism according to the present invention may be a liquid medium or a solid medium, but a liquid medium is preferable and starch or starch-based sugar is required as a carbon source. As the nitrogen source, broth extract, soybean powder, corn steep liquor, corn gluten, etc. can also be used.

An example of a liquid medium is shown. g 1 per medium composition Starch 15 (pH 10.0) peptone 10 yeast extract _{5 K 2 HPO 4 1 MgSO 4} · 7H 2 O 0 · 2 sodium carbonate 10 (however, sodium carbonate, after sterilized separately from the other components It is added to the medium.) Any of static culture, shaking culture, and aeration and agitation culture is possible, but shaking culture or aeration and agitation culture is preferable. The medium pH is 9-12, preferably pH 10,
The culture temperature is 10 to 45 ° C., preferably 37 to 40 ° C., and the microorganism secretes and produces a large amount of amylase in the culture medium after the culture time of 1 to 4 days.

Therefore, after filtering or centrifuging the culture solution to remove bacteria,
Amylase is separated and collected from the culture supernatant. This amylase can be collected by a known separation and purification means.
Examples of the method for separating and purifying amylase include salting out with a salt such as ammonium sulfate, fractional precipitation with a hydrophilic organic solvent such as alcohol or acetone, adsorption with an ion exchange resin, and molecular sieve chromatography.

The novel amylase produced by the novel microorganism according to the present invention is
It has the following features.

(A) A saccharified product containing maltotetraose as a main component is produced from starch or a starch-based saccharide. FIG. 1 shows an example of the results of liquid chromatography analysis of saccharified products produced when soluble starch is used as a substrate. In this example, a sugar-decomposed product was chromatographed using a silica-based reversed-phase column ODS-NH ₂ (5Φ × 250 mm) column with a solution of acetonitrile: water = 65: 35 as an eluent to obtain a differential refractometer (Showa Denko). (Manufactured by K.K.) to detect oligosaccharides. In FIG. 1, G ₁ represents glucose, G ₂ represents maltose, G ₃ represents maltotriose, and G ₄ represents maltotetraose.

(B) The optimum action pH of the novel enzyme is on the alkaline side of 7 to 9, and Fig. 2 shows the relative activity (%) at the reaction temperature of 50 ° C.

(C) The optimum action temperature of the novel enzyme is 50 ° C, and Fig. 3 shows the relative activity (%) at the reaction pH 8.0.

For these properties, the amylase activity was measured by the method based on the following iodine starch reaction.

300 μl of 0.2% starch solution (0.1 M glycine / caustic soda buffer pH 8.0) was added to 20 μl of enzyme solution (appropriately diluted so that the absorbance at 700 nm was reduced by 10 to 20%) and reacted at 40 ° C. for 10 minutes. , A solution of 2 × 10 ^-4 N iodine in 0.15N hydrochloric acid
The reaction is stopped by adding 4.5 ml and the absorbance at 700 nm is measured. One unit means the amount of enzyme that reduces the absorbance by 10% under the above conditions.

For pH 3.8-5.8, citrate buffer, pH 5.8
Phosphate buffer for -7.0, Tris-hydrochloride buffer for pH 7.0-9.0 and carbonate buffer for pH 9.0-1.0.

〔Example〕

Starch 15g, peptone 10g, yeast extract 5g, K ₂ HP
Contains 1 g of O ₄ and 0.2 g of MgSO ₄ .7H ₂ and pH of 1 with 10 g of sodium carbonate
Put the medium adjusted to 0.0 into a Sakaguchi flask and
Inoculated with SP # 707 bacterium (Microtechnology Research Institute, No. 8792), 40
The cells were cultivated with shaking at ℃ and the amylase activity was measured over time.
The peak of the enzyme activity is on the 4th day, and the change in the enzyme activity is the 4th day.
As shown in the figure.

[Reference example]

The cells were removed from the culture broth obtained by the method of Example by centrifugation and 25 ml of the supernatant was subjected to an enzymatic reaction. Gelatinized 5
% Corn starch aqueous solution (200 ml, pH 8.0), 25 ml of supernatant as a crude enzyme solution was added and reacted at 50 ° C for 3 hours. The resulting saccharified product was analyzed by liquid chromatography to find that the sugar composition was glucose (G ₁ ). 13%, maltose (G ₂ ) 18%, maltotriose (G ₃ ) 23%, maltotetraose (G ₄ )
It was 43%, and maltooligosaccharides having maltopentaose (G ₅ ) or more was 3%.

In Fig. 5, the glycated product during the reaction is sampled with time,
The results of thin layer chromatography analysis of changes in sugar composition during the reaction are shown. The analytical method was to spot the saccharified product on a silica gel thin layer plate, and chloroform: methanol: acetic acid = 2: 3:
After developing with the solvent of No. 1, sugar was sprayed to detect sugar.

In Fig. 5, M ₁ is corn syrup, and M ₂ is glucose (G ₁ ), maltose (G ₂ ), maltotriose (G ₃ ), maltopentaose (G ₅ ) and maltononaose (G ₉ ). , And 0 to 300 are saccharified products at each enzyme reaction time.

〔The invention's effect〕

A novel microorganism belonging to the genus Bacillus of the present invention efficiently produces an amylase that decomposes starch or starch-based sugars to produce maltotetraose, and is a microorganism for providing the enzyme in a large amount and at low cost. Available as

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a liquid chromatogram of a saccharified product obtained by allowing an amylase produced by the novel microorganism of the present invention to act on starch, and FIG. 2 is a graph showing the action pH of the amylase, FIG. Is a graph showing the action temperature of the amylase, FIG. 4 is a graph showing the change of enzyme activity in the shaking culture test of the microorganism, and FIG. 5 is a thin-layer chromatogram of the sugar composition change during the enzymatic reaction of the amylase. An example is shown.

Claims (1)

[Claims] 1. An amylase-forming ability which is positive for the Forges Proscalael test (VP test), grows at pH 9 to 12, and can decompose malttetraose by degrading starch or starch-based sugars. Bacillus sp.