JPS63116690A - Production of novel microbial species acremonium sp 15 and beta-d-1,2-glucanase - Google Patents

Abstract

PURPOSE:To efficiently obtain the titled enzyme reactive with a cyclic or straight-chain (1 2)-beta-D-glucan to produce sophorose in high yield, by cultivating a novel microorganism belonging to the genus Acremonium in a nutrient culture medium. CONSTITUTION:Sp 15DK2015 strain (FERM-9019) in novel species Acremonium Sp 15 separated from soil is preferably cultivated in a liquid culture medium containing a carbon source, nitrogen source, inorganic salt, etc., and added cyclic or straight-chain (1 2)-beta-D-glucan in an amount of 0.5 - 5wt% at pH 4.5 - 7.0 and 20 - 30 deg.C while aerobically stirring or shaking to afford the aimed beta-D-1,2-glucanase. The above-mentioned novel species have different mycological properties from Acremonium kiliense in that conidia are oval - elliptical form when cultivated in malt extract agar culture medium, potato.glucose agar culture medium or Czapek agar culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention provides new bacterial species Acremonium Sp15 and β
-D-1.2-Glucanase manufacturing method.

(Conventional technology) Recently, from the perspective of effective use of biomass.

Cellulase is emphasized. Among cellulases, cellulases produced by Trichoderma sp.

Sophorus (2-0-β-n-Glucopyran
Osy! -D glucose) [Biochemical Anne F Biophysical Research Communications;
tiochem.

Biopbys, Res, Comm,, 1.338 (
1959)). This sophorose is a type of 24-glucan in which two glucose units are linked by a β-1,2 bond. It is obtained by the action of D-1,2-glucanase. As β-D-1,2-glucanase.

Aspergillus fumigatus (Asper-H-j, l
-1, us-f um i -B a-t-i,), Fusarium oxysporum (formula! (-a,,, l':,
β-1) -1, 2- derived from filamentous fungi such as J.
Glucanases (Canadian Journal of Microbiology; Can, J., Microhiol,
, 7.312 (1961)). In addition, Cytophaga Alvenge Cola (8gΣ01p
Hand-Kyuho a arvensic, ol-aj 16M
β-D-1. from bacteria such as strain 12648. ．． 2-Glucanase is also known (Japanese Patent Application Laid-Open No. 59-15498
No. 5 Kokoyuki).

(Problems to be solved by the invention) Any of the above β-1)-1,,2-glucanases (1)
→2) The conversion ability of -β D-curcan to sofurose is low, and sofurose cannot be obtained in high yield.

(Object of the invention) The object of the present invention is to obtain a cyclic or linear (1-.2)-β-
A novel microorganism that prepares βD-1,2-glucanase that can act on D-glucan and produce sophot1-sose in high yield, and the production of β-rl-1,2-glucanase using the same It is about providing law.

(Means and effects for solving the problem) The new bacterial species Acremoniu J, Sp15 of the present invention is
belongs to the genus, and when cultured on malt extract agar, high-quality glucose agar, or Czapek agar,
The mycological properties differ from Acremonium chiliens in that the biconidia are oval to oval in shape. This bacterium is β
-D-1,2-glucanase is produced at a high rate.

The method for producing β-D-1.2-glucanase of the present invention involves culturing Acremonium bacteria and extracting β-D-1.2-glucanase from the culture.
- a step of obtaining glucanase, thereby -
The following objectives will be achieved.

Among the new bacterial species Acremonium Sp15 of the present invention.

In particular, the Acremonium Sp15 DK2015 strain (Feikoken Kyoiku No. 9019) is preferably used because of its high ability to produce β-D-1,2-glucanase. This strain is a new strain isolated and collected from soil in Higashi Ward, Osaka City by the inventors, and its mycological properties are shown below.

``Top J'' Hanyu - Quality - (1) Growth status on each medium ■ Malt extract agar medium Growth is good, and the diameter is 30 to 4 when cultured at 25°C and 110 mouths.
Naru to Qmm's colony. The colony surface is flat and has a fine saw-toothed edge. The colony surface is woolly and white in color. The underside of the colony is pale yellow.

Conidia are oval to oval in shape and are in good condition. Droplets are visible. Ascocarps and other sexual spore organs are not formed.

■Potato/glucose agar medium The growth is good and the diameter is 30~4 after culturing at 25°C for 110 days.
(The colony will be h + m. The colony is circular, flat, and has 1
The periphery has a fine sawtooth shape. The colony surface is woolly and white in color. The underside of the colony is pale yellow.

Conidia are oval to oval in shape and are in good condition. Droplets are visible. Ascocarps and other sexual spore organs are not formed.

■ Growth on Zapek agar medium is good, diameter 25-3 when cultured at 25°C with 110 mouths.
5 mm (becomes a D colony. The colony is round, flat, and 7
The periphery has a fine sawtooth shape. The colony surface is woolly and white in color. The underside of the colony is yellow to brown and forms several grooves. Produces a brown diffusible pigment. Conidia are oval to oval in shape and are in good condition. Droplets are visible.

Ascocarps and other sexual spore organs are not formed.

(2) Physiological properties It is an aerobic bacterium and has secondary physiological properties.

(3) Morphological properties In various media -■-, ascicarps and other reproductive organs were not observed, but formation of phialoid conidia in two clusters was observed. Formation of chlamydospores was also observed, many of them in chains. Hyphae are formed from various types of media -I-, branch in a complicated manner, and elongate horizontally and vertically with a hyphae width of 1 to 3 μm. Conidiophore 4; t ji'+. Perform pure techniques. The fraction 4F is transparent and oval to oval in shape, with a short axis of 1.0 to 1.6μ.
m, and the major axis is 2.3 to 3.2 μm. Chlamydospores are transparent and have a short axis of 3.0 to 10.0 μm and a long axis of 3.5 to 12.0 μm.

Reversal (Kawatoshi Inventors 1) The strain of the present invention
2015), based on the above-mentioned mycological properties.
Pure Culture (The Genera of F
ungiSporulatin in Pure C
u1ture ; Δ, R, GantncrVerla
g KG, ,1. ^, von Arx, 1.97
4) and Compen-di++m of 5oil
Fungi ; 8 academic I'r
ess+ 1980). This strain produces transparent conidia from the biconidiophore without having ascocarps or other sexual reproductive organs, and the produced conidia have phialo-type properties, as described in section 1. 2 It was identified as a bacterial species belonging to the genus Acremonium according to Ainworth's one-minute format. Moreover, Acremonium chiliens (Ac,
,,approximate to rc-yo4''. but.

When Acremonium chiliens is cultured in three types of media, all of the conidia formed are cylindrical, whereas the nine strains are oval to oval, so this species It turned out to be a new fungal species of the genus Acremonium, which does not belong to the genus Acremonium.

Jf1 □ Ugouto Aigyu - The culture medium for the above bacterial strain does not need to be special, and a normal culture medium can be used. Glucose is the carbon source.

Glycerin, maltose, starch, dextran.

Lactose 2 Sea JtL Molasses, powdered candy, etc. are used. Corn, potatoes, old potatoes, etc. can also be used.

Yeast extract, peptone, and dried yeast are nitrogen sources.

Soybean flour, cornstarch liquor, ammonia nitrogen,
Nitrate nitrogen etc. are used. As inorganic salts, K
11zOt+ K11zPOt+ CaC1z+ Mg
SO4, Na2HPO41(NH4)211P04, etc. are used. To advantageously produce β-D-1,2-glucanase in wood fungi.

Cyclic or linear (1-2) = 0 β-D-glucan
．． It is added at a rate of 5 to 5% by weight, preferably about 1% by weight. The above-mentioned cyclic (1-2)-β-D-glucan is 1, for example, as described in JP-A No. 59-71686.
~4 strains and Agrobacterium radiobacterium (μm rad) described in JP-A No. 59-82092.
It is produced by culturing the Al-5 strain in a normal medium containing glucose and the like.

Linear (1→2)-β-D-glucan is, for example,
Amemura et al. (Journal of Gene Microbiology; Journal of Gene
ral Micro-biolog)' 131,3
01 (1985)) by culturing Acetobacter bacteria in a conventional medium.

β-D-1. , 2-glucanase-producing wood fungus culture p11 is 2.0 to 10.0. Preferably 4.5-7.0
．． Culture temperature is 15-30°C, preferably 20-30°C
It is.

Cultivate aerobically in a liquid medium with stirring or shaking. Needless to say, culture can also be carried out on solid media. For example, when liquid culture is carried out at 25'C for 24 hours, the wood fungus grows sufficiently and β-D-1,2-glucanase is released outside the fungus.

β-D-1,2-glucanase is isolated from a culture of Acremonium bacteria by a conventional method. For example, the culture is centrifuged to remove bacterial cells and, if necessary, concentrated to obtain a crude enzyme solution.

Furthermore, for example, ammonium sulfate is added to the crude enzyme solution from which the bacterial cells have been removed, salted out, and the resulting solid content is dialyzed.Then, the dialyzed solution is purified using a Sephadex column, focused electrophoresis, etc. The purified enzyme (β-D-1
, 2-glucanase) is obtained.

-LP-2-L-+ 22 no [-L power go car = day λ month shin ■ action and W quality specificity: (1-2) -β
-Acts on D-glucan and produces sophorose.

■Optimal pH and stable p++ range: Optimum pH 4.0~
It is 4.5. Stable pH range is 4.5-6.0,
It is 100% stable when kept at 40°C for 1 hour (residual activity is 80% when the pH is set to 3.0 under the same conditions).
%, and the residual activity is 70% when the pH is set to 7-8).

■Temperature stability: Stable for 15 minutes at temperatures below 40°C (residual activity is 80% at 50°C, 65% at 55°C, and 20% at 60°C).

■Molecular weight: Molecular weight by SDS electrophoresis method is 35.0
It is 00.

■Isoelectric point: 9.6 p-=,,,,,,-D knee 1-2-gloss-a knee that is friendly to Nigno; Dissolve cyclic (1-2)-β-D-glucan in 6) to a depth of 2.5μ/l.
Add 2 ml and react at 40°C for 1 hour. Next, Somogi Copper? & 1 ml, to stop the reaction.

The reducing power is determined by the Somogyi-Nelson method. Known? A calibration curve is prepared in advance using Sophorus of degree A, and compared with this, the amount of Sophorous produced is calculated. 40℃
The amount of enzyme that produces 1++rmole of sophorose per hour is defined as 1 unit.

−Color−−−−−1−−□−−? Purified β-D-1,2-glucanase obtained by notation method or When a crude enzyme solution of β-D-1,2-glucanase is allowed to act on cyclic or linear (1-2)-β-D-glucan, sophorose is obtained.

For example, if 7-cyclic (1-2)-β-D-glucan is dissolved in an acetate buffer, β-Il-1,2-glucanase is added and the reaction is carried out at 40°C for 22 hours, one reaction solution contains sophorose. is generated. Sophorose can be obtained by purifying this using an activated carbon column or the like.

Sopho Town Nissu Taito As mentioned in the prior art section.

It can be effectively used as a cellulase induction substrate.

In addition, the inventors of Sophorus have discovered that it has a good sweet taste, and it can be used as a flavoring agent for foods in place of sucrose. especially.

Sophorose has almost no calories when ingested (animals do not have the enzyme to cleave the β-1,2 bond of glucose), so it is suitably used as a diet food.

(Example) The invention will be explained below with reference to examples.

Implementation - Practice anus - ■ U book q minute prisoner = Among the strains isolated from soil in Higashi Ward, Osaka City.

Bacterial strains that can grow on the medium shown in Table 1 were searched. Among the medium components in Table 1, cyclic (1-2)-β-D-glucan was published in the Journal of General Microbiology (J
, Gen, Microbiol, yo;2i3.
], 873 (1,982)) and purified.

Table 1 The obtained bacteria were cultured at 30° C. for 2 days in a liquid medium having the same composition. Vapor chroma I-graphing was performed on the culture solution on a filter paper using suboso hL and butanol-pyridine-water (6:4:3) as a developing solvent.

Bacteria growing in a particularly large culture solution of a Sophorus spot were taken out and cultured on a plate medium.

This bacterium is Acremonium Sp15 DK201 of the present invention.
It was confirmed that there were 5 strains based on their growth status, physiological properties, and morphological properties.

This bacterium was subcultured onto a malt extract agar medium once a week, and the bacterium was visually observed after 2M months, and it was confirmed that the form of the bacterium had not changed.

Rikifu j tanjishi” -9-1 sound j (-Pakyo-pisakefsofu■1
Prepare the medium shown in Table 2, dispense this medium into 10 500 mff Sakaro flasks each at 60 mE,
Sterilization was performed.

Table 2 One platinum loop of Acremonium Sp15 DK2015 strain obtained in Example 1 was inoculated into 10 of these sterilized medium, and 3
Shaking culture was performed at 0°C for 48 hours. Next, 1 portion of sterilized cyclic (1→2)-β-D-glucan was added to each medium.
% and cultured for an additional 24 hours.

Centrifuge the culture at 5000 rpm for 10 minutes).

The bacterial cells were removed. In this way a total of 500m 7! A crude enzyme solution was obtained. The enzyme activity of this crude enzyme solution is 10U.
/if.

Add ammonium sulfate to the crude enzyme solution at a saturation concentration of about 8
The precipitated solid content was collected by filtration. This was dissolved in 20mM acetate buffer (pH 5, 6 > 50mff) and dialyzed against the same buffer using cellulose film. O ~ 0.5M NaC in buffer (pH 5, 6)]?7
If by degree gradient method? I took care of a customer. Collect the eluting fractions.

Gel filtration was performed using a Sephadex G-75 packed column.
Further purification was performed using an SP-Sephadex packed column. This was subjected to focal electrophoresis to obtain β-D-1.2-glucanase 3nvz. The specific activity of this β-D-1,2-glucanase was 50 [1/■ protein.

20mM acetate buffer (pH 5,6) cyclic (
1-2) 2 g of -β-D-glucan and 135 U of purified β-D-1.2-glucanase obtained in Example 2 were added. After this was maintained at 40°C for 22 hours, it was adsorbed onto an activated carbon column. A 10% aqueous ethanol solution was passed through this column to elute the adsorbate. Purified Sophorose 960 ml was obtained from the eluate.

Instead of cyclic (1-2)-β-D-glucan, linear (
1-2) The same procedure as in Example 3 was performed except that 3 g of -β-D-glucan (prepared by the method of Δmemura et al.) was used, and 1400 μl of purified sophorose was obtained.

When cultured in the same manner as in Example 2, a total of 500
A crude enzyme solution of m1 β-1)-1,2-glucanase was obtained. Its enzyme activity was 3 U/me.

(Effects of the Invention) According to the present invention, a new bacterial species Acremonium Sp15, which is similar to Acremonium chiliens and has a different biconidia shape, can be obtained. This bacterium can produce 2β-D-1,2-glucanase at a high rate.

Since β-111-1,2-glucanase is released outside the bacterial body, it can be easily collected and purified. Obtained β-D-1
．． 2-glucanases are cyclic or linear (1→2)-β
-Acts on D-glucan and effectively produces Sophorose. Sophorose can be used as a cellulase induction substrate and as a sweetener for various foods.

2.

Claims (1)

[Scope of Claims] 1. Acremonium chiliens belongs to the genus Acremonium, and when cultured on malt extract agar, potato-glucose agar, or Czapek agar, conidia are oval to oval in shape. Acremonium Sp15 is a new bacterial species with different mycological properties. 2. The new bacterial species Acremonium Sp15 according to claim 1, which is Acremonium Sp15 DK2015 strain (Feikoken Bibori No. 9019). 3. Cultivate Acremonium bacteria and extract β-D- from the culture.
β-D-1 including the step of obtaining 1,2-glucanase
, 2-Glucanase production method. 4. The Acremonium genus bacteria is Acremonium Sp15.
The manufacturing method according to claim 3. 5. The Acremonium genus bacteria is Acremonium Sp15.
The production method according to claim 3, which is the DK2015 strain (Feikoken Bibori No. 9019). 6. The β-D-1,2-glucanase is (1→2)-
The manufacturing method according to claim 3, which has the ability to act on β-D-glucan to produce sophorose.