DE2011811C - Process for the production of a cell wall-disrupting enzyme - Google Patents

Description

Recently there have been large-scale processes Developed for yeast and chlorella production. Attempts have been made to use these microorganisms as food or to use feed. In the practical use of these microorganisms revealed however, many problems arise because of their difficult digestibility.

There are different methods of making Iytischer, enzymes produced by microorganisms are known. U.S. Patent 3,124,517 is a Process for the preparation of a lytic contained in the spores of Bacillus cereus ATCC 14893 Described enzyme that acts on certain types of bacteria. From British Patent 1,132,678 is a process for the production of enzymes from the culture broth of strains of the genus Flavobacterium known. These enzymes have a degrading effect on the cells of microorganisms. A method for Production of a lysing enzyme complex from the culture broth of Cytophaga NCIB 9497 is described in Belgian patent 664,444. Finally, from the French patent specification 1 333 739 a method for dissolving yeast cell walls by means of one of microorganisms such as B. by Trichoderma viridae produced extracellular / f-glucanase known. However, it is It is desirable to produce an enzyme with higher activity than that of the known enzymes, which is also stable Is able to effectively loosen cell walls of microorganisms.

The object of the invention is to create a method for the production of a cell wall-dissolving enzyme, in order to make the cell constituents more easily accessible through the breakdown of the cell walls by microorganisms close. The cell walls of z. B. Chlorella are particularly stable and can only be destroyed mechanically. The invention is based on the finding that the microorganism Micropolyspora sp. ATCC 21 489 in fermentation mash in aerobic breeding a cell wall-dissolving enzyme accumulates.

The invention accordingly provides a process for the production of a cell wall-dissolving enzyme by aerobic cultivation of a microorganism at pH ratios customary for this purpose and by obtaining the enzyme from the fermentation medium, which is characterized in that the microorganism Micropolyspora sp. ATCC 21 489 in a liquid nutrient medium of the usual composition, which, however, contains as the main carbon sources those with a relatively high degree of polymerization, starts at a temperature of 40 to 60 ⁰ C, separates the mycelium from the fermentation medium and the enzyme contained in the fermentation liquid is obtained by conventional methods.

The strain Micropolyspora sp. ATCC 21 489 has the following taxonomic properties:

ίο A. Morphological properties

1. aerial mycelium

Approximately 0.7 to 1.0 μ in diameter, branched and moderately adherent to the medium, white.

2. Substrate mycelium

Slightly thinner than the aerial mycelium, about 0.5 to 0.7 μ in diameter. It tends to invade the agai breeding ground.
3. Formation of domed bodies

Often an aggregation of small, kuppe! shaped bodies observed on the surface of the agar medium.
4. Spurs

Spherical or oval, approximately 0.8 to 1.0 μ in diameter, main formation directly on the side of the mycelium. Sometimes individual spores are bcobach-

tet. Mainly, however, chains of 2 to 10 spores occur. The chains are straight, spiral chains are not observed. The spore chains are relatively easy to separate into individual spores. When breeding no chains are observed in shaking culture

tet that contain more than 2 to 3 spores. When the spores germinate, the formation of 1 to 2 spore tubes observed from one spore.

5. Mycelial splitting

Not observed with plate growth, but mycelium fragments from 5 to 20 μ often occur during the preparation of the smear.

6. Composition of the cell wall

After hydrolysis the cell wall with 6 n-HCl was determined by paper chromatography that it contains mesorv-diaminopimelic acid, Contains arabinose and galactose. In the literature it is described that the Microorganisms of the genus Micropolyspora have the same composition as above.

B. Physiological properties

1. Sucrose Nitrate Agar No growth.

2. Glucose asparagine agar No growth.

3. Glycerin-asparagine agar

Moderate growth, formation of narrow layers on the surface of the culture medium, Subslratmyzcl pale brown, aerial mycelium white.

4. Broth agar

Good growth, air mycelium white, substrate mycelium pale brown, no soluble pigment, the nutrient medium has a powdery surface.

5. Gelatin agar

Slight growth on the surface of the culture medium after 4 days at 50 ° C, none on the 2nd day Liquefaction of the gelatin can be observed, however, moderate liquefaction is observed on the 4th day.

6. Bennet medium

Good growth, aerial mycelium white, substrate mycelium pale brown, soluble pigment pale brown.

7. Starch agar with inorganic salts

No growth after 4 days at 5O ⁰ C no starch liquefaction.

8. Starch beef peptone agar

Good growth, aerial mycelium white, substrate mycelium pale yellow-brown, soluble pigment pale brown, the starch is completely hydrolyzed after 2 days at 50 ° C.

9. Dextrin-Casein Agar

(good growth, aerial myceus! yellow-white, soluble Pigment pale yellow-brown.

10. Nitrate medium

Thin, film-like growth, aerial mycelium white, Ni rat is not reduced.

11. Litmus milk

Growth ring formation is observed Peptomsierung iv.vch 2 days at 50 ⁰ C.

12. Potato slices
No growth.·

13. Cellulose asparagine medium
No growth, cellulose is not broken down.
14. Cellulose-Dextrin-Casein Medium

Moderate and colony-like growth, aerial mycelium white, almost no decomposition of the cellulose is observed.

C. Growth temperature

On an agar culture medium is at 30 ⁰ C no growth, weak at 37 "C growth, of at 40 to 45 ° C Growth moderate, at 45 to 55 ° C good growth at 55 to 6O ⁰ C overgrowth with poor adhesion Aerial mycelium j and poor growth observed at 65 ° C.

D. Heat resistance of the spores

A 10-minute heat treatment at 100 ° C. does not destroy the spores. With extraction of the spores with hot 80% ethanol one determines the presence of 2,6-pyridinedicarboxylic acid. This is not found in known microorganisms of the genus Micropolyspora.

As described above, that is used in the present invention Microorganism mainly characterized by its morphology. Single spores or Spore chains are formed both on the air and on the substrate mycelium. The chains of spores become plentiful formed and are stable. Microscopic observation of the smear of the mycelia shows a clear one Destruction of the same. The composition of the cell wall of the microorganism is identical to that of the Microorganism Micropolyspora brcvicatena, which was described as a new species in 1961. the The microorganism used according to the invention is also similar to the microorganism in many physiological properties Micropolyspora brevicatena, but it differs in that Micropolyspora brt-vicatena shows poor wax on broth agar and also not at a temperature of 50 ° C grows.

The nutrient medium used in the method of the invention contains suitable carbon sources, nitrogen sources, inorganic salts and organic nutrients. The main carbon sources are polymers with a relatively high degree of polymerization, such as starch, dextran, dextrin or inulin. sugar with a low degree of polymerization, such as monosaccharides or disaccharides, are not suitable. Ammonium sulfate, ammonium chloride, Ammonium phosphate or ammonium nitrate can be used. The inorganic salts are added to the nutrient medium in order to achieve various to provide essential elements for the growth of the microorganism. It can be a suitable one Mixture of different phosphates. Potassium salts, magnesium salts, copper salts. Iron salts, manganese salts and zinc salts can be used. Organic nutrients are added to the medium in order to such as vitamins or amino acids that are necessary for the growth of the microorganism to be provided. For this purpose z. B. yeast or malt extract is used.

As described above, as a nutrient medium for carrying out the method of the invention, a the various mixtures of the mentioned nutrients can be used. The nutrient medium can also from the essential inorganic salts and cells or cell debris of certain yeasts, chlorella, fungi or bacteria which are sensitive to the cell wall-dissolving enzyme prepared according to the invention are exist. In the latter case, the cells or Cell debris as the main sources of carbon and nitrogen, and they provide organic nutrients. Further yeast or malt extract can be added to this nutrient medium as a further source of organic nutrients to add.

At the start of cultivation, the culture of Micropolyspora sp. ATCC 21489 generally by means of suspended in a platinum loop from an inclined tube in sterilized water. With this culture suspension a nutrient medium of the type described above is inoculated and usually 16 to 48 hours grown at a temperature of 40 to 60 C under aerobic conditions. According to the invention The cell wall-dissolving enzyme produced accumulates in the garmix during cultivation. After Removal of the mycelium, e.g. B. by centrifugation, the fermentation liquid wedge can be used directly to loosen cell winds, or it can be used for Obtaining a powdery enzyme preparation freeze-dried.

To obtain a purer enzyme preparation, the fermentation liquid is mixed with the raw enzyme salted out with ammonium sulfate or / ur precipitation of the enzyme treated with acetone. For salting out, the enzyme solution is added to a degree of saturation of 0.4 with ammonium sulfate. removes the η precipitate formed, sets more ammonium sulfate hi to a degree of saturation of 0.6 and wins the resulting enzyme precipitate.

The other option is to add ammonium sulfate to the enzyme solution all at once, in which case better results are obtained if the ammonium sulfate is added directly to a saturation level of 0.8. In acetone precipitation, acetone is added to the crude enzyme solution to form the enzyme precipitate. The best results are obtained with slow addition of acetone up to a concentration of 60%. After stirring for about one hour, the precipitate formed is isolated. The enzyme obtained in this way is dissolved in a suitable buffer (e.g. 0.01 m-KH ₂ PO ₄ buffer of pH 7.0) and then dialyzed against the same buffer for 20 hours. The dialyzed solution remaining in the dialysis membrane contains highly purified enzyme and can be freeze-dried in the same way as described above for the crude enzyme solution.

The substance thus obtained is a new enzyme that easily breaks down cell walls of various microorganisms, such as Yeast, bacteria, fungi and chlorella e.g. B. Candida rugosa, Candida krusei, Candida utilis, Candida pseudotropicalis, Candida parapsilosis, Candida lipolytica, Saccharomyces cerevisiae, Saccharomyces chevalieri, Pichia membranaefaciens, Hansenula anomala and Schizosaccharomyces pombe.

A preferred embodiment of the method of the invention is shown in the following scheme:

Nutrient medium

Culture suspension

Cultivation I for 20 hours at 50 ° C

Centrifuge

(5000 rpm, 10 minutes)

Cells

got over

Acetone treatment

(Raw
Enzyme solution)

Salting out with
Ammonium sulfate

Centrifuge

(10000 rpm, 10 minutes)

Precipitation survived

<Buffer solution

dialysis

Dialysis bath

Retentate

(Purified enzyme solution) Except for those in the examples below The cell wall-dissolving enzyme produced according to the invention has the following properties Features on:

a) It is stable in the pH range 5.0 to 9.0. Its pH optimum is in the range 7.0 to 8.0.

b It is) at a temperature of 40 to 70 ⁰ C, particularly from 50 to 6O ⁰ C, active.

c) It is essentially stable at low temperatures and is ^{not inactivated at a temperature of O 0} C •. In a lOminutigen treatment at 100 ⁰ C it is inactivated.

d) It is easily soluble in water and dilute salt solutions, but insoluble in acetone.

e) DurchAg ^+, Cu ^++, Hg ^++, Fe ^++, Zn ^++, Cd ⁺⁺ and Ni ⁺⁺ there will be inhibited excessively and by Mn ⁺⁺ and Co ^{+ +} while activated by Mg ^{+ +} will.

The cell wall-dissolving enzyme produced according to the invention enables valuable intracellular substances, z. B. proteins, nucleic acids, amino acids, vitamins and enzymes from microorganisms.

Carrying out comparative experiments showed that the enzyme from Micropolyspora sp. ATCC 21489 compared with various yeasts and against Chlorella, overall a considerably higher cell wall dissolving rate Has activity as enzymes by microorganisms

to previously from the aforementioned prior art known processes are produced. The examples illustrate the invention.

Example 1.

The nutrient medium of pH 7.4 containing 5 g of living cells from Candida rugosa JF-IO1, 1 g of K ₂ HPO ₄ , 0.5 g of MgSO _{4 .7H 2} O in 1000 ml of distilled water was treated with Micropolyspora sp. ATCC 21 489 inoculated and cultured with shaking at 50 to 55 ⁰ C for 24 hours. The living cells of Candida rugosa JF-IO1 were completely dissolved. The resulting fermentation mash was then centrifuged for 10 minutes at 5000 LJ min.

Candida rugosa JF-IOl is deposited and available at the Institute of Applied Microbiology, Tokyo University.

After removing the cells and spores, a crude enzyme solution of strong cell wall dissolver became Activity received.

A cell suspension containing the yeasts listed in Table I in a concentration of 10 ⁸ / ml and 0.1% K ₂ HPO ₄ and 0.05% MgSO ₄ - 7H ₂ O in distilled water was prepared and adjusted to the pH -Value 7.4 is set. The hoes used were living cells prepared by adding a _{pH 5.4 nutrient medium containing glucose, (NH 4 I 2} SO ₄ , KH ₂ PO ₄ , MgSO ₄ 7H ₂ O, malt extract and yeast extract! was inoculated with the respective Heien and cultured for 24 hours at 30 ° C with shaking.

The resulting crude enzyme solution was adjusted to pH 7.4, and 0.2 ml of the cell suspensions prepared above were added to each 3 ml of this solution. These mixtures held wurder with gentle agitation for 4 hours at 50 ⁰ C. The results reported in Table I show dai destroyed large amounts of Hefezellcn and digested WUR

The reduction in the turbidity of the reaction mixture, given in Table I was determined visually. The symbol "I- I H-" means a complete one Solution of the cells, the symbol »++« a moderate solution of the cells and the symbol > χπ · «a low solution.

The degree of dissolution of the cells was determined according to the following equation:

Degree of solution (%) = ^S '~ ^S " · KX).

where "Si" is the original number of cells and "S" "the number of cells remaining in the reaction mixture, means undissolved cells.

from pll-Werl 7.4 instead of the enzyme solution would.

Table Il

('hlorclhi / cllcn

Living cells

Living cells

Heatcbchandcltc cells
Illitzc-treated cells

Reaction 'early time. SIcI. Slancliird- solves! 3 0.78 19th 0.77 3 0.84 19th 0.80

Reaction * mixed

Table 1 Example 3

tribe

Candida rugosa .IF-IOl

Candida krusei IAM 4801

Candida krusei IAM 4489

Candida utilis IAM 4215

Candida utilis IAM 4295

Candida pscudolropicalis

IAM 4829

Candida parapsilosis IAM 4488. Candida lipolytica IAM 4947 ... Saccharomyccs ccrevisiac

IAM 4009

Saccharomyccs ccrevisiac

IAM 4308

Saccharomyccs chevalicri

IAM 4801

Pichia membranaefacicns

IAM 4025

Hanscnula anomala IAM 4668 .. Schizosaccharomyccs pombe

IAM 4879

Min.

iming _t ler TThungim KciikUous-

mixture

H H

H f

H H H-

H H- H-

H- H- H-

H- H- + + H H-

H +

H-

4 H-The crude enzyme solution obtained according to Example 1 ammonium sulfate was added to a degree of saturation of 0.4. The precipitate obtained was < I'm / cm 20 removed and more ammonium sulfal was added up to zi added to a degree of saturation of 0.6. The deslan dene precipitate was centrifuged and in de stillicrtcm suspended in water. The suspension was dialysicrl in a container made of regenerated cellulose. The purified enzyme was obtained as a retentate.

The purified enzyme was added to a liquid sample which had been heat-treated as the mycelium of Asper gillus oryzac IAM 2024. After the mixture had stood at 50 ° C. for 4 hours, the mcistcr The mycelium's central walls are destroyed.

2.0 ml of the enzyme solution obtained according to Example 3 were converted into 0.3 ml of a cell suspension (K) (ml) Escherichia coii IAM 1264 given the 3 minutes had been heat treated at 90 ° C. After setting the pH to 7.4, the Gcmiscl Left to stand at 50 ° C. for 4 hours. Approximately 70% of the cells were destroyed.

ZcII solution

81

90

KK)

> 90

86

83 92 93

42

61 81

77 86

65

Example 2

F. in 10 g of moistened cells from Chlorella cllipsoidea Tamiya and 1 g of K ₂ HPO ₄ . 0.5 g MgSO ₄ 7H ₂ O and 1 g HFC extract in KK) omI distilled water containing nutrient medium with pH 7.4 was sterilized for 3 minutes at 120 C, with Micropolyspora sp. Inoculated to ATCC 21489 and grown at 50 ° C for 24 hours. The mycelium of strain ATCC 21 489 and the undissolved chlorella cells were removed by centrifugation. A crude enzyme solution was obtained which had a strong activity against both living and heat-treated (at 98 ° C., 3 minutes) chlorella cells.

To 3.0 ml of the crude solution obtained in this way, 0.2 ml of a suspension of chlorine chloride (3 x 10 "ml) and the mixture was left at 50 ° C. for 18 hours shaken slightly. The results are shown in Table U, with haze as optical density (OD) is shown at 530 mii. Line standard solution was prepared by the Chlorcllasuspcnsion with the appropriate amount of phosphorus buffer

Example 4

Fin 20g strength. 2g (NII ₄ I ₂ SO ₄ , 2 g KH ₂ PO ₄ 5 g K ₂ HPO _4. 2 g MgSO ₄ -7H ₂ O. 0.006 g CuSO ₄ 5H ₂ O. 0.001 g IcSO ₄ -7 H, O, 0.008 g MnCl ₂ -4 H ₂ O 0.002 g ZnSO ₄ -7 H ₂ O und'lO g yeast extract in KKK) m nutrient medium containing distilled water pre-pil value 7.4 was with Micropolyspora sp. ATCC 21489 angcimpfl and grown for 24 hours at 50 C with shaking. The fermentation mash was centrifuged for 10 minutes at 5000 rpm and the mycelium and spores of the strain used were removed. A crude enzyme solution with a strong cell wall-dissolving activity was obtained.

The crude enzyme solution was mixed with ammonium sulfate up to a degree of saturation of 0.8, and the mixture was stirred at 5 ° C for 4 to 5 hours. The resulting brown precipitate was at Centrifuged off 10,000 rpm. dissolved in 9OmI 0.01 M phosphate buffer of pH 7.0 and 20 hours at 5 C against the same 0.01 m phosphate buffer dialyzed. A cleaned one remained in the dialysis container

Enzyme solution back. This solution was lyophilized. 195 mg of pure enzyme were obtained in powder form.

Example 5

I "> ic crude enzyme solution obtained according to Example 4 was at 5 C with stirring with ice-cold> \ acetone bis added to an Acctongchalt of 60%. The stirrer was continued for 4 to 5 hours. The educated nie

This was finally 10 minutes at K) OOO U; min. ab- / entril'ugicrl and in 90 ml of 0.01 M phosphite buffer from pH 7.0 dissolved. The resulting solution was buffered against the same 0.01 M phosphate for 20 hours Dialyzed at 5 C. The retentate consisted of purified Enzyme solution.

The technical form of the proper The procedure can be seen in the subsequent test report. The Micropolyspora sp. ATCC 21 489 The enzyme produced not only has a stronger lysicrend effect, but also builds the extremely stable one Chlorella cell walls are several times stronger than the known enzymes.

Comparison attempts
I. Microorganisms used

(a) Flavobaclcrium sp. ATCC 21 045
(British Patent 1,132,078).

(b) Trichodcrma viridae IFO 5836
(French patent 1,333,739).

(C) Cytophaga NCIB 9497

(Belgian patent 664 444).
(d) Micropolyspora sp. ATCC 21 489

(microorganism used according to the invention).

The microorganism used in U.S. Patent 3,124,517, Bacillus cereus No. 5832. ATCC 14893 is in the catalog of the American Type Culture Collection. 9th edition. 1970. not listed.

II. Substrate

Than to digest the hymn in question Yeast and Chlorella cells were selected as these are in practice the most important ones to be lysed Substrates are.

111. Carrying out the experiments

I. Preparation of the enzymes
(a) Enzyme produced by Cytophaga NClB 9497

40 ml one from 4.0% times loose. 2.4 ".. Medium consisting of malt extract, 1.0% peptone and distilled water (pH 7.0) in a 250 ml Erlcnmcyer flask are inoculated with Cytophaga NCIB 9497, the organism having previously been grown in a bouillon-agar medium. The inoculated nutrient medium is incubated at 26 C on a Rundsehü'Uelmaschine 2 days. The culture broth is then centrifuged for 10 minutes at 10 (XX) U min.. The Dbcrstand obtained is used as F.nzymlösung.

(b) from Flavobacterium sp. ATCC 21 045
produced enzyme

20 ml of one made from 2% glucose, 0.33% meat extract. 1.0% peptone, 0.33% Na (I and 0.33% K ₂ HPO ₄ (pH 7.0) existing nutrient medium in a 250 ml Erlenmeyer flask is inoculated with Flavobacterium sp. ATCC 21045 grown on broth agar Incubated on a rotary shaker for 2 days at a temperature of 30 ° C. The culture broth is then centrifuged for 10 minutes at 9000 rpm.

(c) from Trichoderma viridae IFO-5836
produced enzyme

80 ml of water are added to 100 g of wheat bran The mixture is sterilized for 30 minutes with steam under normal pressure. The nutrient medium thus obtained is inoculated with mycelium from Trichoderma viridae. The mycelium was on a glucose! lefeexlrakl malt extract medium bred. The inoculated ίο culture medium is 3 days at a temperature of 30 C grown in static culture. The solid culture medium obtained is then mixed with 500 ml of water added, and the enzyme I Slundo is extracted at 30 C from this mixture. After filtering the is mixture wirci the Filtral 10 minutes at K) OOOl) Centrifuged min. Is becomes a clear enzyme solution receive.

(d) from Micropolyspora sp. AlCC 21 489
produced enzyme

50ml of one made from 1.5 "starch, 1.0", llefeexlica 0.264% (NI I ₄ ), SO ₄ , 0.238 ", KH ₂ PO _4. 0.565", K ₂ HPO ₄ . 0.1% MgSO ₄ -7H ₂ O and 0.1% (VV) of the spiral element solution according to Pridham & Gottlieb (cf.

2s Wa k smaii, The Actinomycctes, Vol. 2. p. 332 (pH 7.4) existing nutrient medium in a 500 m shake flask were with the conidia from Mikropolyspora sp. A1 (C 2I4H9 inoculated, which were cultured in an agar medium obtained from K) "cells ml Candida rugosa .IE-IOl. 0.1""K ₁ IIPO ₄ . 0.05 "- '(i MgSO ₄ 7H ₂ O. 0.1""yeast extract and 1.5"., Agar and had a pH value of 7.4. The inoculated nutrient medium is shaken for 20 hours at M) C or K) hours at 55 C. incuhated. The one from dei

is culture broth obtained clear Π bet stand is used as an enzyme solution used.

2. Breeding of the eggs

A 500 ml shake flask contains K) O m of a liquid medium containing 5% glucose. OX ", (NIl ₁ I ₁ SO ₄₁ O.!""Na ₁ HPO _4. 0.1""KIl ₁ PO _4. 0.2", MgSi) ₄ -711 ₂ O. 0.2 ", KCI. 0.02% malt extract with 0.02 "- (i contains yeast extract. The pH value of the medium * is 5.2. This medium is inoculated with the yeasts listed below. The nutrient media thus introduced are incubated for 48 hours at 30 ° C. The grown yeast cells are centrifuged off. Washed twice with a () .2% aqueous KCI solution and subjected to the test.

The hydrocarbon assimilating yeasts are grown in the same manner as described above. with the exception that instead of 5 "" glucosi as the carbon source 2 ", kerosene or n-Dccan is used. When the cultivation is complete, a small amount of a 10% soap solution is added to the fermentation mash. The mixture is shaken thoroughly and centrifuged. The yeast cells are washed 4 times with a 0.2% aqueous K ('! Solution and subjected to the test.

yeast

Candida utilis IAM 4215 (feed yeast). Candida lipolytica IAM 4947 (Kohlemvasserstofi

assimilating yeast), Candida rugosa JF-IOI (hydrocarbon

assimilating yeast),.

Klocckcra japonica IFO 0151, Hansenula saturnus IAM 4776,

ΙΛΜ = Abbreviation for Institute of Applied Microbiology. University of Tokyo,

HO = Abbreviation for Institute of Fermentation, Osaka.

JF = Abbreviation for "Jet Fuel", refers to the isolation code there.

3. Cultivation of the Chlorella / rush

A nutrient medium containing 1.26g KNO, is placed in the flask. Contains 1.22g KIKIO ₄ , 0.46g MgSO ₄ -7H ₂ O. 0.0028 g IeSO ₄ - 711.0 and K) OOmI water. This medium is inoculated with Chloreila cllypsoidea lamiya. The organism is then cultured in a _S i ihermostatisierten fermenter for 5 days at 25 C at a i.ichteinslrahlung 10000-20000 Lux, wherein the medium is aerated oh by means of a compressor 5 "/ o carbon dioxide cnlhallender. 1 After completion of culturing are the chlorella cells centrifuged off and washed with water.

IV. Reaction Conditions

and determination met soil

I. Activity of the cell wall-dissolving enzyme against I Icle

In each case 0.1 ml of a suspension (10 "'cells ml) of the yeast grown above is added to 2 ml of the raw yeast produced by the test organisms. The mixture is then shaken for 3 hours at the optimum temperature (37 C for Flavobacterium sp. AT (C 21 045 and Cytophaga NCIH 9497.45 C for Trichoderma viridae IFO 5X36 and 50 C for Micropolyspora sp. ATCC 21 4X9) respond calmly. The reaction mixture is then made up to 10 ml with water. Fs will be the optical density (OD) of the mixture determined at 660 itw.

12th

Vl. Results

I. Activity of the cell wall-dissolving lin / .yms against I IeIe- ' zellen.jJie in a glucose source containing carbon dioxide Culture medium were grown

l'abelle

yeast Min the nt the Tcslo ^ unismu.s C. utilis IAM 4215 opli- C. lipolytica IAM 4947 ting C. rugosa JF-IOl density
"/ (I Flavohaclcriiim K. japonica IFO 0151 60 sp. ATCC 21 045 II. Saturnus IAM 4116 38 C. utilis IAM 4215 30th C. lipolytica IAM 4947 50 C. rugosaJ F-101 80 Cytophaga K. japonica IFO 0151 61 NCIH 9497 H. saturnus IAM 41 16 25th C. utilis IAM 4215 24 C. lipolytica IAM 4947 73 C. rugosa JF-IOI 35 rrichoderma K. japonica IFO 0151 80 viridae IFO 5836 11. Saturnus IAM 41 16 45 C. utilis IAM 4215 51 C. lipolytica IAM Ί947 XO C. rugosa JF-H) I. 82 Micropolyspora K. japonica IFO 0151 85 sp. ATCC 21 489 II. Saturnus IAM 4116 90 82 100 77

2. Activity of the cell wall dissolving enzyme
L'ogen ί 'hlorella / cllcn

2. Activity of the cell wall-dissolving enzyme against yeast cells, those contained in a kerosene as a source of coal Culture medium were grown

The enzyme solutions are mixed with chlorella ^{cells up to a concentration of K) 1} 'cells ml. After 5 seconds of reaction, the optical density (OD) of the reaction mixture is measured at 530 irw.

V. Hnzymaklivitäl

The enzyme activity is determined according to British patent specification 1 132 67S. Equation on p. 3, middle, determined.

fio

Decrease in optical density,%

OD ₀ -OD ₁
OD ₀

OD _n = optical density after 0 minutes.
OD, = optical density after t minutes.

100

Test org; inism

Flavobacterium

sp. ATCC 21 045
Trichoderma

viridae IFO 5836
Cytophaga

NCIB 9497
Micropolyspora

sp. ATCC 21 489

Table II

yeast

C. lipolytica IAM 4947

C. rugosa JF-IOl

C. lipolytica IAM 4947

C. rugosa JF-IOl

C. lipolytica IAM 4947

C. rngosa JF-IOl

C. lipolytica IAM 4947

C. rugosa JF-101

Decrease in optical density

13th

3. The activity of the wall-dissolving enzyme against Chlorella / .cllen

table

cslorpanism

Flavobactcrium sp.

ATCC 21045
Trichodcrma viridae

IR) 5836
Cytophaga

NCIB 9497

Optical density at 5.10 mn

Blank value 0.76 sample 0.73 blank value 0.85 sample 0.73 blank value 0.72 Sample 0.69

reduction

the

optical density

lortset / ung

Optical density at 5M

Micropolyspora sp.
ATCC 21 489

Blank value 0.78, sample 0.50

Vcrminderunj;

the

optical density

36

From the above data it can be seen that the Micropolyspora sp. ATCC 21 489 produced Knzym has a higher activity on yeast and chlorella cells than the other hnzymes. with the exception of the enzyme produced by Trichodcrma viridae. that on Hanscnula salurnus IAIvI 4116 a somewhat higher activity than that of Micropolyspora sp. ATC ^- C 2! 489 produced enzyme exercises.

Claims (1)

Claim: Process for the production of a cell wall dissolving Enzyme by aerobic cultivation of a microorganism under the usual pH value conditions and by recovering the enzyme from the fermentation medium, characterized in that that the microorganism Micropolyspora sp. ATCC 21489 in one liquid nutrient medium of usual composition, but this is the main carbon source contains those with a relatively high degree of polymerization, at a temperature of 40 to 60 ° C sets in, separates the mycelium from the fermentation medium and that in the fermentation liquid contained enzyme wins by conventional methods.