DE2408998A1 - PROCESS FOR PRODUCING A FRUCTAN-DEGRADING ENZYME - Google Patents

Description

Priority: February 26, 1973, Japan, No. 22 843/1973

The invention relates to the production of a fructan-degrading enzyme >

The invention relates in particular to a new process for the production of a fructan-degrading enzyme with the aid of a a fructan-degrading enzyme-forming microorganism of the genus Talaromyces, Eupenicillium, Sporotrichum, Sordaria, Chaetomium, -Gelasinospora, Stachybotris or Actinomyces.

It has recently been shown that dental and oral diseases such as Alveolar pyorrhea, periodonditis and the like, caused by the formation and deposition of dental plaque, the Läevan, a type of fructan produced by certain microorganisms found in the mouth. It was also found that a fructan-degrading enzyme is a suitable means of preventing these diseases (German Applicant's patent application P (corresponding to Japanese patent application 22842/1973).

It has previously been reported that a fructan degrading enzyme by

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Molds of the genera Aspergillus, Fusarium, Humichora, Penicillium and the like and bacteria of the genera Arthrobacter, Streptococcus, Streptomyces and the like 'is formed.

According to the invention it has now been found that microorganisms of the genus Talaromyces, Eupenicillium, Sporotrichum, Sordaria, Chaetomium, Gelasinospora, Stachybotris or Actinomyces can form a fructan-degrading enzyme, which by hydrolysing the ^ - (2- * 6) - and / i- (2- »1) -Fructo-furanoside bond can cleave, so that the viscosity of fructan decreases within a short * duration is, and that in particular a microorganism of the genus Talaromyces to a far greater extent for the production of a fructan degrading Enzyme is more capable than the previously known microorganisms. The invention is based on these findings Procedure.

The invention is based on the object of a new and effective process for the production of a fructan-degrading enzyme accessible through fermentation.

Other objects and advantages of the invention are from the following Description visible.

The invention relates to a process for the production of a fructan-degrading enzyme by cultivating a microorganism, which is characterized in that a microorganism of the genus Talaromyces, Eupenicillium, Sporotrichum, Sordaria, Chaetomium, Gelasinospora, Stachybotris or Actinomyces, which forms a fructan-degrading enzyme and the fructan-degrading enzyme formed is obtained from the culture broth.

Microorganisms which are suitable according to the invention and which form a fructan-degrading enzyme include, for example, the following listed microorganisms, some of which are available from the Technical Research Institute of Microbial Industry, Agency of Industrial

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Science & Technology, Ministry of International Trade and Industry, and from the Institute of Applied Microbiology, University of Tokyo, Japan, and the Institute for Fermentation, Osaka, Japan.

Deposit number of the microorganism

Talaromyces flavus var. Flavus Sordaria humana Chaetomium subspirale Gelasinospora longispora Stachybotris lobulata Eupenicillium javanicum Sporotrichum schenkii Talaromyces stipitatus Talaromyces luteus Sordaria fimicola Chaetomium aureum Chaetomium atrobrunneum Gelasinospora cerealis Stachybotris atra Actinomyces longisporus Actinomyces cyanoalbus Actionomyces lavendofoliae

It should be emphasized that, according to the invention, both natural and artificial Mutants and variants of the microorganisms enumerated above can be used.

The microorganisms given above are known strains, and their mycological properties are as follows References described:

F 223-8 1892 ■ IFO-12885 F 651-4 189o IFO-12857 F 216-4 1888 IFO-12882 F 652-2 1889 F 61-1 1891 F 11-6 IAM-8o67 F 61-7 IFO-5984 F 223-4 F 222-8 F 222-1 F 2o3-4 F 2o3-1 F 218-1 F 82-10

Talaromyces flavus var. Flavus

F 223-8

Stolk et al .: Studies in Micology, Vol. 2, p. 11 (1972)

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T. stipitatus

T. luteus

Sordaria humana

S. fimicola

Chaetomium subspirale

C. aureum

C. atrobrunneum

G. cerealis

Stachybotris lobulate

S. atra

F 223-4

F 222-8

F 651-4

F 222-1

F 216-4

F 2o3-4

F 2o3-1

Gelasinospora longispora F 652-2

F 218-1

F 61-1 F 82-1o Stolk et al .: Studies in Micology, Volume 2, p. 29 (1972) Stolk et al .: Studies in Micology, Vol. 2,

p. 23 (1972) Cain: Bibliotheca Mycologica, Vol. 9 » P. 18 (1934-) Cain: Bibliotheca Mycologica, Vol. 9 »

p. 17 (1934-) Udagawa: The Journal of General and Applied Microbiology, Vol. 6, p. 247 (1960) Ames: Bibliotheca Mycologica, Vol. 17, p. 14 (1961) Ames: Bibliotheca Mycologica, Vol. 17, S. 14 (1961) Udagawa et al .: Transactions of the Mycological Society of Japan, Vol. 8, pp.

Udagawa et al .: Bulletin of the National Science Museum, Tokyo, Vol. 16, P. 326 (1973) Tsubaki: Nagaoa, Vol. 4, p. 16 (195 * 0 Ellis: Dematiaceous Hyphomycetes, S.

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Eupenicillium javanicum F 11-6

Sporotrichum schenkii Actinomyces logisporus

A. cyanoalbus

Stolk et al .: Persoonia, Vol. 4-, p. 398 (1967)

F 61-7 Fukumi et al .: Byogenbi se ibut sugakusaikinhen (Japanese), S _t 956 (Igaku-Syoin, 1966)

IFO 12855 E.B. Shirling and D. Gottlieb: International Journal of Systematic Bacteriology, Vol. 18, p. 342 (1968)

IFO 12857 E.B. Shirling and D.

Gottlieb: International Journal of Systematic Bacteriology, Vol. 18, P. 314 (1968)

IFO 12882 E.B. Shirling and D. '

Gottlieb: International Journal of Systematic Bacteriology, Vol. 18, P. 339 (1968)

When carrying out the method according to the invention, a microorganism of the genus Talaromyces ,, Eupenicillium, Sporotrichum, Sordaria, Chaetomium, Gelasinospora, Stachybotris or Actinomyces, which is capable of producing a fructan-degrading enzyme Surface culture or submerged culture in a solid or liquid culture medium containing nutrient sources, of different Composition are bred, which is commonly called natural or liquid medium is used. Wheat bran, soybean meal, for example, can be used as nutrient sources "Pharmamedia" (manufactured by Traders Oil Mill Company, USA), Corn steep liquor, peptone, starch, glucose, sucrose, ammonium sulfate, Urea, various inorganic salts or a combination of these nutrients can be used. In particular

A. lavendofoliae

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If the cultivation takes place in a culture medium to which 0.5 to 2% fructan (for example Laevan, obtained from Aerobacter levaniucum or inulin) has been added, a tenfold or more increased formation of a fructan can occur, especially in a liquid culture degrading enzyme can be achieved than without the addition of a fructan.

The culture temperature and the pH of the culture medium are not particularly critical; however, it is preferred according to the invention from a practical point of view ^{to keep the temperature at about 28 °} C. and to adjust the pH value of the culture medium to 5 »5 to 7» o. The cultivation time depends on the cultivation conditions used; however, cultivation may be interrupted after determining the time at which maximum activity is achieved. The maximum activity can usually be obtained by growing for 3 to 5 days.

In order to obtain and purify the desired enzyme from a liquid culture broth or aqueous koji extract, it is optionally possible to use any conventional method already used for the recovery and purification of an enzyme from a culture broth can be used or koji extract containing this enzyme was known. So For example, it is possible to use some method of concentrating a culture broth or an aqueous koji extract under reduced pressure, a method of salting out with ammonium sulfate, sodium sulfate, common salt and the like, methods fractional precipitation with a solvent such as methanol, ethanol or acetone, adsorption and elution methods using a suitable adsorbent, precipitation methods using a protein precipitant, methods the precipitation at the isoelectric point, methods of separating impurities with the help of a heavy metal, electrical. dialysis methods and similar cleaning methods for themselves or to be used in combination.

The fructan-degrading enzyme obtainable with the aid of the process according to the invention has a higher activity for degradation and for

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faster liquefaction of fructan than the previously known enzymes. Its optimum activity it displays at a pH value of 4 to 5 », the optimum temperature is 4o to 4-5 ⁰ G. The pH value at which it is stable, is from 3 to 9» and it is stable at a temperature of not more than 55 ⁰ C

The invention is illustrated in more detail by the following examples, without that it should be restricted to these.

100 ml of a liquid culture medium (pH 6.0) containing 0.5% fructan (obtained from Aerobacter levanicum), 0.5% corn steep liquor, 0.5% Pharmamedia, containing 15% ammonium sulfate, o, o5% urea, o, 2% potassium acid phosphate, o, o3% magnesium sulfate and o.o3% calcium chloride. The medium was sterilized and inoculated with each of the indicated fructan-degrading enzyme-producing microorganisms. The cultivation was carried out by shaking culture at 28 ° C. for 72 hours. The activity of the fructan degrading enzyme was measured by the viscosity reducing method and the reducing sugar generation method. The results are shown in the table below. The following methods were used to measure activity:

Viscosity reduction method

To 2 ml of a 5% solution of fructan (from Aerobacter levanicum) in m / 2o citrate buffer (pH 5.4) was 0.2 ml of the culture broth and the mixture was kept at 40 ° C. After 3 hours and 19 hours, the specific viscosity of each mixture became measured using an Ostwald viscometer.

Method of the formation of reducing sugars Laevan from Aerobacter levanicum and inulin were chosen as representatives for a fructan with ß ~ (2- »6) bonds as main chain bond and for a fructan with ß- (2-> 1) bonds. To 1 ml of a 5% solution of each fructan in m / 2o citrate buffer (pH 5 * 4)

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one ml of the culture broth was added. The mixture became 3> o minutes leave at 40 ° C. The reducing sugar thus formed was then converted into fructose using the dinitrosalicylic acid method certainly. The amount of enzyme required to produce 1 mg of fructose in 60 minutes is defined as one unit.

tribe

the viscosity-reducing activity activity for the formation of reducing agents ^! Sugar units / ml

specific S viscosity after 3 hours

specific viscosity after 19 lbs. Laevan as J Inulin as, substrate! Substrate

Talaromyces flavus var. flavus F 223-4

T. stipitatus
F 223-4

T. luteus
F 222-8

Sordaria humana F 561-4

S-. fimicola
F 222-1

Chaetomium subspiral F 216-4

; C. aureum
j F 2o3-4

C. atrobrunneum! F 2o3-1

1.9

4.3

4.5

3.9

5.6

4.3

4.8

5.2

o, 2 18, o

2.6 1.6

1.6

1.7

4.6 o, 4 o, 6

2.2 2.4

3.3

3.6 2.0 2.4

14.2

33.6

3.6

o, 86

o, 4

9.8

7.4

1.6

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Gelasinospora 4.5 longispora 'F 652-2 5.5 G. cerealis
F 218-1 Stachybotris 5.2 lobulate F 61-1 5 5 S. atra F 82-10 ; Eupenicillium 4.1 javanicum F 11-6 Sporotrichum 2.6 schenkii F 61-7 Actinomyces 2.2 logisporus IFO 12885 5.5 A. cyanoalbus ^ # ^ IFO 12857 5.4 A. lavendofoliae
IFO 12882 5.6 comparison

2.9 o, 2: 8.4

4.2 o, 7 o, 5

5.8 o, 4 o, 4

4.1 0.4 o, 9

1.5 1.6 o, 2

1, o 5, o 9.4

ο, 5 5, ο 1.6

2.5 1, o o, 4

2.5 1.8: ο, 6

5.6!

In a fermenter with a capacity of 1oo 1, 5o 1 of a liquid culture medium (pH 6, o) was added, which was passed through. Addition of 0.5% fructan (from Aerobacter levanicum), 0.5% Pharmamedia, 0.1596 ammouin sulfate, 0.05% urea, 0.2% potassium acid phosphate, 0.05% magnesium sulfate to tap water. The medium was sterilized and treated with 2 l of a vaccine

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culture of Talaromyces flavus var. flavus inoculated that previously was obtained by shaking culture at 28 ° C for 2 days. was. Cultivation was carried out for 48 hours at 28 ° C at 23o rpm, one

2 aeration of 1 V / V / min. and carried out at an internal pressure of 1 kg / cm. The activity of fructan-degrading enzyme in the Culture broth (determined as units of activity of the formation of reducing sugar, according to the method of formation for reducing Sugar using Laevan from Aerobacter levanicum as substrate) was 20 units / ml.

The broth was concentrated to 10 1 at low temperature and three times the volume of acetone was added to the concentrate, to precipitate the fructan degrading enzyme, which is then recovered by filtration and dried under reduced pressure became. 126 g of an enzyme sample was obtained in the form of a white powder. The activity of this sample (given in the preceding Units) was 4,375 units / g.

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Claims (4)

(i) Process for the production of a fructan degrading enzyme by cultivating a microorganism capable of producing a fructan-degrading enzyme in a culture medium and obtaining a fructan-degrading enzyme from the culture broth, characterized in that one Microorganism of the genus Talaromyces, Eupenicillium, Sporotrichum, Sordaria, Chaetomium, Gelasinospora, Stachybotris or breeds Actinomyces. 2. The method according to claim 1, characterized in that the microorganism is Talaromyces flavus var. Flavus, Talaromyces stipitatus, Talaromyces luteus, Sordaria humana, Sordaria fumicola, - Chaetomium subspirale, Chaetomium aureum, Chaetomium atrobrunasulata Gelasinospora, longispora, Gelasinospora, , Stachybotris atra, Eupenicillium javanicum, Sporotrichum schenkii, Actinomyces longisporus, Actinomyces cyanoalbus or Actinomyces lavendofoliae are used. 3. Method according to claim 1 or 2, characterized in that that the microorganism Talaromyces flavus var. flavus F 223-8, Talaromyces stipitatus F 223-4 or Talaromyces luteus F 222-8 is used. 4. The method according to any one of claims 1 to 3, characterized in that o, 5 to 2 % of a fructan is added to the culture medium. 409836/1011 5. Process according to one of Claims 1 to 4, characterized in that the cultivation is carried out at a temperature of 28 ° C and a pH in the range from 5 »5 to 7. 409836/101 1