JPS6022917B2 - Method for producing trehalase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for culturing trehalase-producing bacteria belonging to the genus Chaetomium and collecting trehalase from the culture.

In general, trehalose is a sugar in which two molecules of D-glucose have both reducing groups bonded to each other, and is known to exist in natural products such as ergot, molds, basidiomycete fruiting bodies, trehalamanna, yeast, and seaweed.

The trehalase targeted by the present invention is an enzyme that decomposes trehalose into two molecules of D-glucose, and has already been used in rat small intestine, insects, pseudopergillus, and yeast.
It is known to exist widely in

However, the content of trehalase in these is very low, and therefore purified trehalase enzyme has become extremely expensive. The present inventors conducted a wide search in nature for bacteria that produce large amounts of trehalase, and found that a strain recognized to belong to the genus Chaetomium produces large amounts of trehalase.

The ascocarps of this strain isolated here are superficial and angiosperms (
In addition, the shell wall of this angiogenus is gradated and has a pore and characteristic apical hairs.

In addition, the asci are efficacious, and when the ascospores mature, they become a sticky mass and are released from the pore □. From these points, it is clear that this strain belongs to the genus Chaetomimm.
Furthermore, when we examined the trehalase productivity of the ``0'' stock strain of the genus Chae mimm, we found that
etomiumaureumIF0 6544Chae
tomiumaureumIF0 6543Chaet
It was confirmed that raleIF○ 6572 also produced trehalase in omium trila.

The present invention was completed based on these findings, and is a method for producing trehalase, which is characterized by culturing trehalase-producing bacteria belonging to the genus Chaetomium, producing and accumulating trehalase, and collecting the same.

The strain used in the present invention is Cha isolated from nature.
The mycological properties of one strain of Etomi genus M are as follows.

Growth status of this strain in various media ■ Growth on oatmeal agar soil After culturing at 30°C for 60 days, the colony grew well, reaching a diameter of approximately 5 cm, and the colony was thin, flat, and fluffy.

The center part is clear green, and the middle part is grayish yellow-green.
The surrounding area is dull red. At this time, angiosperms are developing near the center of the colony. The underside is dark olive green, becoming lighter towards the periphery, and is smooth with no radial wrinkles or grooves. By continuing the culture, many angioparas with olive green apical hairs are formed on the surface of the colony, and the entire surface becomes olive green. After 9 days of culture at 30 qo, the diameter of the colony was approximately 7 qo.

■ Growth on potato glucose agar medium When cultured at 30 CC for 60 days, the colony grows well and reaches a diameter of approximately 5 cm.

It is almost fluffy and fan-flat. The outer periphery is white, but the rest of the body is a dull red. The underside is purplish-black, and the medium also changes from purplish-red to deep purplish-red as the culture progresses.
Also, a lot of mist-like cool or pink liquid is formed on the surface. After 9 days of culture, the diameter of the colony reached approximately 7 cm.
Angiospores with olive green apical hairs become scattered. ■ Growth on cellulose agar soil After 10 days of culture at 30 pm, the diameter of the colony reached approximately 70 mm, and numerous angiodia were formed in the center, scattered around the periphery, and the apical hairs were olive green in color. is remarkable.

Although the angiodia are well formed, the growth of the vegetative fungal system is extremely poor. The area around the colony appears as a small mist of pink horsefish exudate, giving it a pinkish tinge. At this time, there is no characteristic color tone on the back side, but as the culture progresses, it becomes gradated. ■ Growth on malt extract agar medium Growth on this medium is very similar to growth on potato glycose agar, but the formation of angiosperms is hardly observed.

■ Growth of colonies on YpSs agar medium is rather slow in spreading, reaching a diameter of approximately 35 skins after 30 days of culture, and is characterized by fluffy, colorless or white vegetative bacteria.

At this time, the underside of the colony is yellowish-white, and the color of the medium does not change in particular, but it gradually becomes tiered. Formation of the angiogeus is slow. In addition, the diameter of the colony reached approximately 5 mm after 12 days of culture. Physiological/Ecological Properties ■Temperature On oatmeal agar medium, no growth occurs at 40:00, but slight growth is observed at 8°C.

Around 30 qo is the best temperature for the growth of vegetative fungi and the formation of angiosperms.

At 37o0, immature angiosperms are observed, but 4
It does not grow at 0°C.

■ pH 30oo, pH 3.11 to pH 7.86 after 7 days of culture
It grows at pH 2.13 and PH 8. It does not grow on ropes.

Growth is good between pH 4.20 and pH 6.25.
Morphotrophytes of fruiting bodies are hyphal-like, branched, and have septate walls, and are usually colorless or white, but sometimes pink or clear-colored.

The Penthecimm are not embedded in the stroma and are superficial.

It is colorless when young, but turns black when mature. It has a large pore, is almost oval in shape, and most of its size is 100 mm.
-160 x 80 - 125 mountains. When it matures, it releases an elongated cylindrical mass of ascospores. The angiogel wall is made of polygonal cells and becomes more fragile as it ages. Usually "arcuate", sometimes with a more curved tip or one or two whorls, the olive-green top hairs are thinner and shorter than the top hairs, and have lateral hairs. The apical hairs are not branched, have septa, and have a rough surface.The tips are rounded, and the width at the center is 2.0 to 4.0 r.
, the base part is huge and becomes 4.0 to 5.0 Buddhas. The lateral hairs are very similar to the top hairs, except that they are shorter in length and 2.0 to 2.5 r wide. The asci are rod-shaped, and the ascospores are almost colorless when young, containing refractive grains, but turn olive green when mature. They are formed in double rows and are oval in shape, but sometimes one side is fan-flat or concave. Consists of one cell, size 9
．． It measures 5 to 12.0 x 5.0 to 6.0 French, and both ends are slightly pointed.

The above results are L. M. Ames MiA Monogmph
of ratio Chaetomiaceae, The Un
Ited States AJmyResearch
and Development Series,
No. 2 (1961), HariK. Seth: A.M.
ono scale aph of the C also nuS Chaet.

mium, Verlag v. n J. Cramer3
Identification using 301 Lehre (1970) etc. as a reference revealed that the angioplasty was oval and the size was 100-160 x 80.
1125 crests, the apical hairs are unbranched and usually arcuate;
This strain is Chaebmimamemm because the width is less than 5 mm, the tip is obtuse, and the ascospores are oval with pointed ends and 9.5-12 x 5.0-6.0 mm.
It was identified as belonging to Therefore, this strain was
Omiumaureum No. 9209. And the present strain, Chaetomiumamemm ship. 92
09 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology under the title Collected Bacteria No. 4516. The trehalase-producing bacteria belonging to the genus Chaetomium used in the present invention produce and accumulate trehalase in both aerobic liquid culture and solid culture using wheat skin.

As the carbon source in liquid culture, common carbon sources such as glucose, starch, soybean, etc. can be used. As a nitrogen source, polybeptone, meat extract, cornstarch liquor, defatted soybean, yeast extract, etc. can be used. Good results can be obtained by adding other inorganic salts. Further, as the solid culture medium, wheat hull, rice bran, steamed rice, soybean flour, etc. are suitable.
The culture temperature is no different from that of ordinary filamentous fungi,
25 to 3500 preferably (nearly 30oo gives good results). The initial pH is slightly acidic, and culture is usually started at pH 5.0.

Any number of days for culture can be selected as long as it is 3 days or more. In the case of liquid culture, the culture can be obtained in powder form by removing the bacterial cells by filtration or centrifugation, and then by a known method such as salting out or organic solvent precipitation.

In the case of solid culture, water or an appropriate buffer is added to the culture for extraction, and then the extract can be obtained as a powder in the same manner as the above-mentioned culture sterilization solution. The obtained enzyme acts on an aqueous trehalose solution to produce glucose. The trehalase phase enzyme powder thus obtained can be further purified using conventional enzyme purification methods such as desalting using molecular nodes, PH treatment, ammonium sulfate fractionation, organic solvent treatment, and various ion exchangers. The properties of the partially purified trehalase enzyme thus obtained are as follows.

m Substrate specificity Acts on trehalose and breaks it down into two molecules of glycose.

[2} Optimum temperature 5000'31 Temperature stability pH4.0, 45q at pH6.5
No deactivation occurs even after treatment for 0.03 minutes.

{4} Optimum pH FH4.0 '5} pH stability 3?
It is stable in the range of H3.5 to 9.0.

'6' Inhibitor HgH, FeH+, PCMB (1
×10-3M treatment at 40°C for 30 minutes).

[7} Molecular weight Calculated to be 400,000 by Sephadex G-200 gel filtration.

'8) Isodew point PH was 4.0 by ampholine isoelectric focusing.

Next, a method for measuring trehalase activity will be described below.

Trehalose in pH5.61/1M acetate buffer
is dissolved to a concentration of 2 skin to prepare a substrate solution. Add 0.5% of the sample to 0.5% of the substrate and react for 4000 minutes for 3 hours. The reducing power of the reaction solution is measured using the Somogyi-Nelson method. Enzyme activity is defined as 1 unit when it produces a reducing power equivalent to 1 M of glucose under the above conditions. Next, examples of the present invention will be shown. Example 1 A pH 5.0 medium containing 0.3% glycose, 0.5% voribeptone, 0.5% meat extract, 0.2% dipotassium phosphate, 0.05% magnesium sulfate, and 0.3% salt. After steam sterilizing for 1.1k920 minutes, Chaetomi
Umaureum side. 9209, FERM-P No.
4516 was inoculated and cultured under shaking for 3002 days, which was used as a seed culture.

3 m' of it was inoculated into the same medium and cultured on a shaker for 7 days at 30:00, resulting in a trehalase activity of 0.18 f/m. Example 2 ChaetomiumaureumIF065
No. 43 was seed cultured in the same manner as in Example 1.

As the main cultured soil, 100 parts of barley skin and 70 parts of water were added and mixed, then 1.1k92 powder was steam sterilized in 100 Erlenmeyer flasks for 10 nights, and inoculated with 1 part of the seed culture solution.
Cultured at 30qo for 7 days. The trehalase activity of the extract obtained by adding 100 ml of water to the whole culture and extracting at 30° C. for 3 hours was 0.63 F/secretion. Example 3 Chae's mimmtrilatera m0657
2 was seed cultured in the same manner as in Example 1.

Claims (1)

[Claims] 1. A method for producing trehalase, which comprises culturing a trehalase-producing bacterium belonging to the genus Chaetomium, producing and accumulating trehalase, and collecting the same.