DE2261270C3 - Process for the production of enzyme complexes lysing cell walls, enzyme complexes obtainable in this way and their use - Google Patents

Description

The invention relates to a process for the preparation of cell wall lysing enzyme complexes, the following Have properties:

(a) They lyse living and dead cells at least from Aspergillus niger, Penicillium steckii, Saceharomyces cerevisiae, Candida utilis, Candida albicans, Candida lipolitica, Lentinus edodes. Bacillus subtilis, Lactobacillus lactis and Chlorella,

(b) their cell wall lysing activities are stable in the pH range from 3 to 9, being the optimal one pH is in the range of 5 to 7,

(c) their cell wall-lysing activities are optimal in a temperature range of 30-40 ⁰ C, the optimal temperature varying depending on the nature of the microorganisms whose cells are to be lysed,

(d) their cell wall lysing activities are stable in a low temperature range, but are quickly inactivated ^{at temperatures above 50 0 C,}

(e) their cell wall lysing activities are ^{inactivated by the presence of Mn +} +, Ni ⁺ + or Zn ⁺ +,

(f) they show the enzymatic activities of cellulase at an optimal temperature range of 40-50 ⁰ C and an optimum pH of 5.0, carboxymethyl cellulase, of j3-l, 3-glucanase at an optimal temperature range of 40-50 ⁰ C and an optimal pH value of 5.0, of chitinase at an optimal temperature of 35 ⁰ C and an optimal pH range of 4.0-5.5, of protease, hemi-cellulase and amylase, and

(g) They are composed essentially of enzyme components each having a molecular weight of at least 50,000,

the enzyme complexes obtainable in this way and their use.

It is known that yeast and Chlorella and the like are used in food and drink Forage bred on a large scale. Furthermore is

known that bacteria and fungi are grown to be they are used to extract valuable substances such as antibiotics, proteins, nucleic acids, amino acids, Vitamins and enzymes, to use. However, the cell walls of yeast and chlorella are so strong that they are difficult to digest and break down when used in food and feed. It has therefore been proposed to treat the yeasts and chlorella with an enzyme that can remove the Cell walls of these microorganisms. unlocks or breaks down in order to increase the solubility and digestibility to enhance. It has also been proposed to use bacterial cultures and fungal cultures with one of the cell walls lysing enzyme treat to the extraction of valuable intracellular substances from the cultured To facilitate microorganisms.

The invention was based on the object of providing enzymes which are able to cells and especially to lyse the cell walls of various microorganisms. During these examinations was found an enzyme complex or a complex consisting of different enzymes, which is caused by the known strains of the genus Pellicularia is produced and shows a high lysability, that is, he dissolves the cells of various microorganisms. The enzyme complex of the present invention can be cultivated known species of the genus Pellicularia can be obtained in a culture medium that can be assimilated with sources Contains carbon and assimilable nitrogen to produce this enzyme complex in the culture medium and assimilate and then isolate him from it.

The invention therefore relates to a process for the production of cell wall lysing enzyme complexes type mentioned at the beginning, which is characterized by the fact that a known strain of Pellicularia sasakii or a known strain of Pellicularia filamentosa in a nutrient medium that is assimilable carbon sources and contains assimilable nitrogen sources for the generation and enrichment of the enzyme complex in the Culture medium and then the enzyme complex is isolated from the nutrient medium.

The technical progress that can be achieved in this way is that the enzyme complex according to the invention is im Difference to the prior art contains endo- / M, 3-glucanase, which the microorganisms cell walls through statistical breakdown of their molecules into oligopolysaccharide units and is able to degrade through low protease activity, so that it has such cell walls able to lyse faster and more effectively.

Taxonomically the genus Pellicularia belongs to the family of the Corticiaceae of the order of the Aphyllophorales, which belongs to the subclass Homolasidiae of the class Basidomycetes.

A known strain of Pellicularia sasakii used according to the invention is known to be capable of the plant disease Cause leaf sheath blight in rice plants, a strain of the well-known Pellicularia species filamentosa the plant disease black rot in potato plants. Known strains of Pellicularia filamentosa are registered under ATCC No. 14 701.14 702.14 703 and 20,206, and a strain of Pellicularia sasakii under ATCC No. 13 289 as on Page 120 of the Catalog of Strains, 9th edition (1970) of the American Type Culture Collection.

In the present investigative work, a strain of Pellicularia sasakii from a Injury in a rice plant infected with leaf sheath burn isolated. This strain was named under F. R. I.

No. 1170 at the Japanese Public Deposit Institute, Fermentation Research Institute, Agency of Industrial Science & Technology of Japan, Inage, Chiba City, Japan "deposited on November 8, 1971. This strain was registered with American Type Culture Collection (ATCC), Rockville, Maryland, 12th IZ 1972 under ATCC No. 20 365 deposited without restriction A Pellicularia filamentosa strain was also found from an injury to a potato plant infected with black rot. This strain was isolated under the same date under the number F. R. I. No. 1171 deposited This strain was at IZ IZ 1972 under ATCC No. 20 366 unreservedly deposited F. R. I. No. 1170 and 1171 from the known tribes to differentiate between Pellicularia sasakii and Pellicularia filamentosa (see the Japanese publication "Microbiology Handbook" pages 376-377, published December 25, 1957 in Tokyo).

The morphological properties of the Pellicularia sasakii and Pellicularia filamentosa strains given above are briefly given below:

1. The plant mycelium is fibrous or has the shape of a mushroom cap, is initially colorless and changes its color into a yellowish-brown fawn after aging.

2. The mycelium generally has a diameter of 8 to 12 μm.

3. The sclerotium is formed

4. The basidia has the shape of a cylinder or egg-shaped and bears 4 to 8 branches.

5. Colorless spores with a smooth surface are formed

6. The hymenium is flat.

7. The fruit body forms a networked membrane.

Pellicularia sasakii and Pellicularia filamentosa can be differentiated from one another in that Pellicularia jasakii in rice plants and Pellicularia filamentosa in potato plants die Black rot and in aubergines the disease (of the seedlings) that causes it. Furthermore, Pellicularia forms sasakii a black-brown colored sclerotium from 5 to 13 mm, a basidia of 18χ9μΐη and spores of 9x7 μm. Pellicularia filamentosa forms a brown colored sclerotium from 1 to 3 mm, a basidia of 15x8 μίτι and spores of 9x7 μπι. However, some have Taxonomists have expressed the view that Pellicularia sasakii is merely a natural variant of Pellicularia filamentosa is.

For the production of the invention, the cell walls lysing enzyme complex can be any of the above-mentioned strains of Pellicularia sasakii or filamentosa can be used.

In the method of the invention, a known strain of Pellicularia sasakii or Pellicularia filamentosa grown in a nutrient medium containing assimilable carbon and nitrogen sources, to generate and accumulate the enzyme complex and then remove it from the medium isolate.

When carrying out the method according to the invention, a strain of Pellicularia sasakii or Pellicularia filamentosa either in a liquid nutrient medium or in a solid nutrient medium are grown using known fermentation methods. Preferably the Pellicularia strain at an incubation temperature

grown from 15 to 40 ° C for 48 to 240 hours, although the Breeding conditions may vary depending on the type of breeding process used.

The medium in which the Pellicukria strain is after a method according to the invention can be grown assimilable carbon and nitrogen sources contain. Carbohydrates such as starch, glycose, sucrose, Wheat bran, rice bran and the like can be used. As nitrogen sources «for example Ammonium salts, nitrates, peptones, meat extracts, yeast extracts, defatted soybean meal and the like be used. Also, an appropriate amount of a growth promoting substance such as for example vitamins, are added to the nutrient medium. A powder made from dry yeast, Chlorella and / or mushrooms can be added to promote enzyme formation. Furthermore, traces of organic salts, such as potassium salts, Calcium salts, magnesium salts, iron salts, Phosphates and the like are added to the nutrient medium in order to supply various types of trace elements, which are essential for the growth of the Pellicularia stem.

The cultivation of the Pellicularia strain is continued until a sufficient amount of the desired one is obtained Enzyme complex has been generated and accumulated in the nutrient medium. The Pellicularia strain will preferably grown under aerobic conditions.

Cultivation in a liquid nutrient medium is preferably carried out at a temperature of 20 to 33 ° C 48 up to 120 h under aerobic submerged conditions. The cultivation on solid nutrient medium is preferred at 20 to 35 ° C for 3 to 10 days.

The enzyme complex, which has a lysing effect on the cell walls of various microorganisms, is of cultivation in the Pellicularia cultivation culture and accumulates. The excess culture broth that by filtering off or centrifuging the solid material such as the mycelium and the solid Residues obtained from the nutrient medium can cause various cell walls to be lysed as such Microorganisms are used. This supernatant liquid can also be freeze-dried, to make a crude enzyme preparation. When cultivation is carried out in solid medium, the resulting solid culture can be washed with water or buffered water with a pH of 4.0 to 7.0 extracted. The resulting aqueous extract contains the enzyme complex, which is similar to the above-mentioned filtrate can be used.

In order to isolate the enzyme complex in the form of a purer enzyme preparation, the enzymes can do this containing nutrient medium freed from solids, for example by filtration or centrifugation or they can be extracted with water or buffered water. The one made in this way A crude aqueous solution of the enzyme complex can then be treated with known salting-out agents, such as Ammonium sulfate, sodium chloride and the like, or it is mixed with water Miscible organic solvents treated, as with the lower aliphatic alcohols methyl alcohol or ethyl alcohol, or with acetone to precipitate the enzyme complex from the crude solution. To the For further purification, the precipitated enzyme complex can be dissolved in a suitable buffer solution. One such buffer solution is 0.1 M acetate-buffered water. The enzyme solution can then be a Dialysis or gel filtration. The purified enzyme solution can then be freeze-dried and results in an enzyme preparation which essentially consists of the desired enzyme complex consists. The enzyme complexes prepared according to the invention from Pellicularia sasakii and from Pellicularia filamentosa are soluble in water, insoluble in acetone and ethanol and have the following properties:

a) You can get live and dead cells from bacteria, fungi, yeast, Basidiomycetes and Chlorella lyse,

b) the cell wall lysing activities are stable in a pH range from 3 to 9, the optimal being pH range is in the range from 5 to 7,

c) optimal activity and in particular cell wall lysing activity lies in a temperature range from 30 to 40 ° C, but the optimal temperature may vary depending on the type of microorganisms, whose cells are to be lysed vary somewhat,

d) the cell wall lysing activities are stable in the low temperature range, but they will quickly inactivated at temperatures above 50 ° C,

e) the cell wall-forming activities are inactivated by the presence of Mn ⁺ +, Ni ⁺ + or Zn ⁺ +,

f) the enzyme complex shows the enzymatic activities of cellulase, glucanase, chitinase, protease, Hemi-cellulase and carboxymethyl cellulase and

g) the enzyme complex is composed essentially of enzyme components, each at least have a molecular weight of 50,000.

It is believed that the enzyme complexes produced by Pellicularia sasakii and Pellicularia filamentosa a mixture of cellulase, glucanase, chitinase, protease, hemi-cellulase and carboxymethyl cellulase are in the form of a complex and their high activity in lysing cells of various microorganisms show due to a synergistic effect of the effects of the individual enzyme components, although it is also believed that these enzyme complexes produced by the various Pellicularia species have compositions which are more or less different from one another are. It has been observed that these enzyme complexes have more or less different properties and show potency for the individual enzymatic activities.

The cell wall lysing produced from a known strain of Pellicularia sasakii and Pellicularia filamentosa Enzyme complexes still have the following common properties:

a) You can get live and dead cells at least from Aspergillus niger, Penicillium steckii Saccharomyces ceirevisiae, Candida utilis, Candida albicans, Candida Iipolitica, Lentinus edodes, Bacillus subtilis, Lactobacillus lactis and Chlorella lyse:

b) their cell wall-lysing activities are stable in a pH range from 3 to 9, being the optimal one pH range is 5 to 7;

c) their cell wall lysing activities are optimal in a temperature range of 30 to 40 ° C, however, the optimal temperature may vary depending on the type of microorganism whose cells are being lysed should vary;

d) Their cell wall lysing activities are stable in low temperature range, but they will quickly inactivated at a temperature above 50 ° C;

e) zellwändelysierenden their activities are inactivated by the presence of Mn ++, Ni + + ⁺ + or Zn,

f) they show the enzymic activities of cellulase, carboxymethylcellulase, glucanase, chi tinase, protease, hemi-cellulase and amylase and

g) they are composed essentially of individual enzyme components that have a molecular weight of at least 50,000.

to

15th

The enzyme complex formed by Pellicularia sasakii also shows a cellulase activity, the one has an optimal temperature range of 40 to 50 ° C and an optimal pH value of 5.0, a | 3-1,3-glucanase activity that has an optimal temperature range from 40 to 50 ° C and an optimal pH value of 5.0, and a chitinase activity, which has an optimal temperature range of 35 ° C and an optimal pH value of 4.0 to 5.5. The from The enzyme complex formed by Pellicularia filamentosa also shows a cellulase activity, which is optimal Temperature range from 40 to 50 ° C and an optimal pH value of 5.0, a / M, 3-glucanase activity, which has an optimal temperature range of 40 to 50 ° C and an optimal pH value of 5.0 possesses and a chitinase activity that has an optimal temperature range of 35 ° C and an optimal shows pH from 4.0 to 5.5.

The cell wall lysing activities of the general properties b) -e) given above Enzyme complexes of the invention are as follows determined: the enzyme complex is mixed with 5 ml of a suspension of cells of Candida utilis IFO-0396 implemented, which initially has a haze that corresponds to an optical density (O. D.) value of 1.0, measured at a wavelength of 660 nm. The reaction is carried out at 35 ° C. for 60 min at a pH of 5.0 performed. After this reaction, the optical density of the cell suspension that lysed the Cells and the non-lysed cells, measured at 660 nm. When the reacted cell suspension a 1% reduction in haze (or O.D. value) compared to the initial one Cell suspension shows, then it can be assumed that this amount of the enzyme complex preparation The CeÜuiase activity, the J3-1,3-glucanase activity and the chitinase activity of the enzyme complex of the invention are measured as described in Example 13 later.

When the cell walls of microorganisms are lysed or with the help of the enzyme complex according to the invention are dissolved, the enzyme complex with the cells of the microorganism suspended in water This reaction can be carried out appropriately at a temperature of 20 to 60 ° C and preferably at 30 to 40 ° C and at a pH of 2 to 10 and preferably at a pH of 3.4 to 8 be performed. The reaction temperature and pH used can vary depending on the nature of the Microorganism are selected whose cells are lysed under the action of the enzyme complex sojjen. The cells of the microorganisms to be lysed can be living or dead cells when brought into contact with the enzyme. In any case the cells can be essentially completely dissolved, with the cell walls and the cellular Substances of the cell walls go into the aqueous phase of the cell suspension within 20 hours Start of reaction.

In order to carry out the reaction of the enzyme complex with the cells of the microorganisms, this can Enzyme preparation in the form of a solid powder or an aqueous solution or the nutrient broth or an extract the nutrient broth can be added to a suspension of the cells in water, adjusted to an optimal pH has been buffered to effect the enzymatic reaction. The cells can be used as an alternative to a Solution of the enzyme preparation in water or in a buffered solution, which is based on the optimal pH has been adjusted to effect the enzymatic reaction. When the reaction has expired, the cells are completely dissolved without leaving a solid residue in the solution remains behind. In some cases, however, a solid residue remains even after the reaction has ended. Whether the cells have been dissolved to the desired level can be determined by the Change in the turbidity of the cell suspension or the concentration of an aqueous component of the Cell protoplasm is determined, or by microscopically observing the conditions of the cells in the suspension. In this way, an aqueous cell solution can be obtained in which the material of the Cell membranes and the protoplasm of the cells together with the enzyme complex used in Water has been dissolved. If desired, the cell solution thus obtained can be filtered off in order to to remove possible solid residues. This solution can be heated to remove the leftover Deactivate enzymes if necessary. The cell solution can then be dehydrated in a vacuum or concentrated to obtain a dry product. The cell solution treated in this way can also be used in suitably extracted or chromatographies to determine the useful substances therefrom isolate.

The enzyme complex according to the invention can make the cells, more precisely the cell walls, of various types Lyse microorganisms. Such microorganisms are, for example, bacteria, fungi, yeast, Basidiomycetes as well as chlorella, one of the algae.

These properties of the enzyme complex according to the invention are significantly more advantageous in comparison z. B. with the known enzyme, which from Corticium rolfsii cultures by the method of published Japanese Patent Application No. 11,978/67 is isolated. The enzyme isolated from Corticium rolfsii has one optimal pH range of 2.0-2.5 and one for dissolving the cell wall of yeast and chlorella only limited ability to lyse The microorganism of the genus Corticium can be derived from the genus Pellicularia be differentiated in that the former produces a continuous hymenium in which the Basi, which is arranged side by side continuously, while the latter is a discontinuous hymenium produced wherein the base die is spaced from each other

The enzyme complex lysing the cell walls according to the Invention can be used in the extraction of valuable intracellular substances from the cells as well as for Used to produce a concentrated extract

030 208/153

which contains nutrients and other valuable substances that are different in the protoplasm of the cell Microorganisms, such as those from yeast, chlorella, bacteria, and fungi, are present. Be for this purpose the cells are treated with the enzyme complex in water to lyse the cell walls and the protoplasm to release, so that one contains a cell solution, which can then be partially dehydrated or completely in a concentrate or can be converted into a dry powder. The enzyme complex that lyses the cell walls the invention can also be used to prepare food or beverages before Preventing spoilage by adding an effective amount of the enzyme complex and thereby removing the Lines eventueii existing bacteria or Piize dissolves. Because of the beneficial properties the enzyme complexes according to the invention can be used in a wide variety of areas of the food industry, the pharmaceutical, animal feed and pesticides industries manufacturing industry.

The enzyme complex according to the invention and the process for the preparation thereof are described in the The following examples are explained in detail using preferred embodiments.

example 1

In a 300 ml Erlenmeyer flask were placed 19 g of wheat bran, 1 g of dried yeast and 18 ml of water. The flask was then heated and the contents of the flask were stirred to form a uniform pasty mixture. For sterilization, the nutrient medium was treated with steam ^{at 120 ° C. for 20 minutes.} A Pellicularia sasakii stock culture (FRI No. 1170) was inoculated onto the sterile nutrient medium. The solid soil culture of the Pellicularia strain was carried out at a constant temperature of 28 ° C. for 120 hours. The resulting culture was mixed with 80 ml of an acetate buffer solution of pH 4.0 and the mixture was agitated at room temperature for 1 hour to ensure that the enzymes were extracted into the liquid phase. The mixture was then filtered off under pressure and the centrifuged filtrate gave 70 ml of a clear solution which contained the enzyme complex formed by Pellicularia sasakii. 210 ml of acetone were added to this clear solution so that the enzyme complex precipitated. The precipitated enzyme complex was filtered off and dried, yielding 700 mg of an enzyme preparation in powder form. This enzyme preparation was excellent in lysing activity against various microorganisms including bacteria, fungi, yeast, basidiomycetes and chlorella.

Example 2

In a 300 ml Erlenmeyer flask were 10 g of wheat bran, 10 g of rice bran, 2 g of peptone and 20 ml Water brought in. The flask was heated and the contents of the flask were agitated at an even rate to obtain a pasty mixture. This culture medium was sterilized for 20 minutes at 120 ° C. with steam A Pellicularia filamentosa stock culture (F. R. L No. 1172) was placed on the sterile culture medium inoculated in. The solid-soil culture was carried out for 170 h at 28 ° C. The culture thus obtained was then mixed with 80 ml Water mixed, and this mixture was agitated well at room temperature to ensure that the enzymes were extracted into the aqueous phase. The mixture was then filtered under pressure and that The filtrate was centrifuged to give a clear solution that represented that produced by Pellicularia filamentosa Contained Encynic Complex. Ammonium sulfate was added to the clear solution up to 70% saturation, see above that the enzyme complex precipitated. The precipitate was filtered off and dried to give 1 g of one Enzyme preparation in powder form. This enzyme preparation turned out to be the cell walls of various microorganisms, as from bacteria, fungi, yeasts, Basidiomycetes and Chlorella, lysing active.

Example 3

In a 1 1 Erlenmeyer flask were 9 g of sucrose, b g of rice bran, 0.6 g of a powder of dried mushrooms (Cortinellus shiitake), 1.5 g of ammonium sulfate, 1.5 g of monopotassium phosphate, 0.3 g of potassium chloride, 0.4 g of magnesium sulfate and 0.003 g of iron (II) sulfate. To this, 300 ml of water was added to dissolve it.

The nutrient medium in the form of an aqueous solution was sterilized with steam at 120 ° C. for 20 minutes. One Stock culture of Pellicularia filamentosa (F. R. I. 1172) was inoculated onto the sterile liquid nutrient medium. The shaking culture of the Pellicularia strain was 120 h carried out at 23 ° C. The culture broth obtained was filtered to remove the To remove solids, and the resulting clear filtrate, which contained the enzymes, was one third of the original volume and then dialyzed through a dialysis membrane 400 ml of aqueous 99% ethyl alcohol were added to the concentrated solution, so that the enzyme complex precipitated. The precipitate was collected and dried to give 450 mg of an enzyme preparation in powder form. This enzyme preparation showed a good one Activity and lysed cell walls of various microorganisms, including bacteria, fungi, yeast, Basidiomycetes and Chlorella.

Example 4

In a 300 ml Erlenmeyer flask were 5 g of finely divided wheat bran, 0.5 g of ammonium sulfate, 0.5 g Monopotassium phosphate 0.1 g potassium chloride, 0.1 g magnesium sulfate, 0.001 g iron (II) sulfate and 100 ml water The contents of the flask were sterilized by means of steam for 20 min at 120.degree. C. A stock culture of Pellicularia sasakii (F. R. I. No. 1170) was based on the sterile liquid nutrient medium inoculated. The shaking incubation was carried out for 24 hours at 28 ° C. by means of a Rotary shaker performed to produce a seed culture. This was accompanied by 200 g of wheat bran, 5 g of a powder of dried mushrooms (Cortinellus shiitake) and 160 ml of a 5% aqueous ammonium sulfate solution placed in 5 wooden bowls. In each bowl the materials were mixed well around one to give a uniform mixture which was then sterilized with steam. 20 ml of the above Seed cultures were inoculated onto the nutrient medium in each dish. Station culture was carried out for 96 hours at 28 ° C. The cultures in these dishes were purified and the combined culture was mixed with 41 one aqueous solution extracted from an acetate buffer of a pH value of 4.0 The extract was taken under Pressure filtered and the centrifuged filtrate gave 3.61 of a clear solution similar to that of Pellicularia sasakii This clear solution contained up to 70% ammonium sulfate Saturation added so that the enzyme complex precipitated.

The precipitate was collected and taken up in 900 ml of water. The aqueous enzyme solution was lyophilized in vacuo and gave 32 g of an enzyme preparation in powder form. This enzyme preparation turned out to be cell walls of various microorganisms, such as bacteria, fungi, yeast, Basidiomycetes and Chlorella, lysing active.

Example 5

In three 300 ml Erlenmeyer flasks each 19 g Wheat bran, Ig dry yeast, and 18 ml of water were added, and the contents of each flask were added heated and stirred to a uniform pasty mass. This culture medium was then mixed with Steam sterilized at 120 ° C. Stock cultures of Pellicularia sasakii and Pellicularia filamentosa were made inoculated onto the sterile culture media in all three flasks. The solid soil cultivation was 120 hours for a long time at a constant temperature of 28 ° C. Thereafter, the obtained cultures were with 80 ml portions of a 0.1 η acetate buffer solution (pH 5.0) and agitated at room temperature for 1 hour to ensure that the enzyme complexes produced by the various Pellicularia species into the liquid Phases were extracted. The mixtures were pressure filtered and the filtrates gave after Centrifuge 70 ml portions of 2 clear solutions containing the enzyme complexes.

In parallel, cell suspensions of fungi, yeast, bacteria and chlorella were made in water from cultures of Aspergillus niger, Penicülium steckii, Saccharomyces cerevisiae IFO-0209, Candida utilis IFO-0396, Candida albicans, Candida lipolitica, and Lentinus edodes, each incubated in a malt medium; Cultures from Bacillus subtilis and Lactobacillus lactis were incubated in a broth medium; and the Chlorella pyrenoidosa culture was incubated in a Nakamura medium. Any of these cell suspensions was placed in 3 test tubes 20 ml portions of the above three clear solutions of the Enzyme complexes were then added to the 3 test tubes containing the cell suspensions. The content

ίο of each test tube was lightly in one for 20 h Water bath shaken at a constant temperature of 35 ° C so that the enzymes with the cell walls were reacted to dissolve the cells in the suspension. The cell suspensions were under the irl microscope observed to determine up to soften Degree the cells under the action of the enzyme complexes of Pellicularia sasakii and Pellicularia filamentosa disbanded or dismantled. The degree of dissolution of the cells was on the following scale judged:

+ + denotes complete dissolution of the cells,
+ denotes the dissolution of a larger part of the cells,
± denotes the dissolution of a smaller part of the cells,

- means that no dissolution of the cells could be determined.

The results thereby obtained are as follows Table I summarized. For comparison, the procedure was repeated adding 2 ml of the acetate buffer solution was added instead of the clear enzyme solutions mentioned above. In the comparison tests was no dissolution of the cells noted

Table I.

Type of characters to be resolved Enzyme addition Degree of resolution of the lines Enzyme from enzyme from Pellicularia Pellucularia sasakii filamentosa Aspergillus niger added ++ ++ not added - - Penicillium steckii added ++ ++ not added - - OHUlnMOl UlliJTUGS UCICVIMiIC added not added - - Candida utilis added ++ ++ not added - - Candida albicans added ++ ++ not added - - Candida lipolitica added ++ ++ not added - - Lentinus edodes added + + not added - - Bacillus subtilis added ± ± not added - - Lactobacillus lactis added + + not added - - Chlorella pyronoidosa added + + not added - -

Example 6

Cell suspensions in physiological saline solution were prepared from Candida utilis IFO-0396, Saccharomyces cerevisiae IFO-0209 and Hansenula anomala IFO-0122, which were each obtained by shaking the microorganisms in a liquid nutrient medium containing 3% glucose, 0.8% (NHi) ₂ SO ₄ , 0.5% KH ₂ PO ₄ , 0.2% NaCl, 0.2% MgSO ₄ · _{7H 2} O and 0.1% yeast extract (percent by weight) at a pH of 5 , 2 and over a period of 20 h. During this time it was shaken with a rotator (200 rpm). One drop of each cell suspension was placed on a slide and one drop of the clear solution of the enzyme complex of Pellicularia sasakü, as in Example 1, was added to this drop of the cell suspension on the slide Liquids of these drops were mixed quickly and then covered with the cover slip. The slides produced in this way were ^{stored in a room at a constant temperature of 30 °} C., and the change in the cells in the

liquid mass on the glass plate was observed under the microscope over a certain period of time found that the cells gradually dissolved over time. The degree of resolution of the observed cells was determined according to the following scale:

+ means that the cells have started to dissolve,

IU + + means that the dissolution of the cells continues,

+ + + means that the cells have stopped dissolving,

- means that no change was observed in the cells.

The test results are summarized in the following Table II. For comparison, a Experiment carried out in which a drop of the acetate buffer solution instead of the drop of the enzyme solution was added. No dissolution of the cells was observed in the comparative experiments.

Table II

Micro-enzyme organism additive to be dissolved

Degree of dissolution of the cells of the microorganisms with the passage of time (minutes) 1 3 5 10 15 30 60 90

Candida utilis Yes no Saccharomyces Yes cerevisiae no Hansenula anomala Yes no Example 7

In a 300 ml conical Erlenmeyer flask were placed 20 g of wheat bran, 0.5 g of a powder of dried mushrooms (Cortinellus shiitake) and 16 ml of an aqueous 5% ammonium sulfate solution, and the contents of the flask were heated and stirred to form a smooth, pasty mixture this broth was 20 min at 120 ° C with steam sterilized A stock culture of Pellicularia filamentosa was inoculated to the sterile culture medium and the hard soil culture was 144 hours conducted at 25 ⁰ C the Kulrur thus obtained was washed with 8Om! deionized water and agitated at room temperature for one hour to ensure that the enzyme complex produced by Pellicularia filamentosa was extracted into the liquid layer. The mixture was then filtered under pressure, the filtrate centrifuged, and 65 ml of a clear solution containing the enzyme complex were obtained. Ammonium sulfate was added to this clear solution up to 70% saturation, so that the enzyme complex precipitated. The precipitate was filtered off and dried, giving 1.7 g of an enzyme preparation in powder form.

This enzyme preparation was dissolved in an acetate buffer solution having a pH of 5.0 to prepare an enzyme solution containing 1% of the enzyme preparation given the L-tube. The dry yeast was prepared from a Candida species which was incubated by using a nutrient medium which contained a paraffinic petroleum-hydrocarbon fraction as a carbon source. The L-tube was ^{shaken at a constant temperature of 40 °} C. so that the enzymes with the Cells of the yeast or chlorella were reacted and caused the lysis of the cell walls. After a reaction time of 1 hour or 3 hours, 3 ml of an aqueous sodium hydroxide solution were added to the reaction mixture to bring the pH to about 7 ° C. D; c fviiSCuUng Was shaken again for another 15 min and centrifuged to remove the solids. The clear solution obtained in this way was analyzed for the content of sugar and proteins which had been released from the cells. The sugars were determined by a colorimetric method at 530 nm using 3,5-dinitrosalicylic acid as a color developing agent. The amount of sugar was expressed as the amount of glucose. The protein content was calculated by determining the nitrogen content of the proteins by the known Kjeldahl ninhydrin method and multiplying the nitrogen content found by 6.25. The results obtained are summarized in Table III below. For comparison purposes, an experiment was carried out in which 10 ml of an acetate buffer solution of pH 5.0 was used in place of the enzyme solution given above.

Table ΠΙ

Micro-enzyme to be dissolved

organisms addition

reaction time Content of from the cells Proteins released substances (in mg) 291.0 (Hours) sugar 368.2 1 70.2 95.1 3 75.4 95.4 1 5.7 162.2 3 5.6 198.9 1 30.3 40.1 3 34.1 30.6 1 0.4 3 0.4

Dried yeast

Dried
Chlorella

Yes
no

Yes
no

Example 8

This experiment should show that the enzyme complex according to the present invention also suitable for various foods and drinks such as tomato juice, potato juice and orange juice to keep from spoiling.

10 ml servings of tomato juice, potato juice and Orange juice was put in small bowls. The enzyme preparation prepared according to Example 1 was with cleaned with an ion exchange resin. 0.1 mg, 1 mg and 10 mg of the purified enzyme preparation were the Contents of the bowls added. Some bowls of the various beverages were not enzyme supplements added. The pans were then left to stand in the air at 28 ° C for a period of time, and it caused spoilage found that the discoloration, the appearance and the smell of the juices were examined. The test results are summarized in Table IV below. The symbol - indicates that the juice has not spoiled was.

Table IV Enzyme addition Lapse of time (days) 2 4th 7th material 1 iii + III + + + Il no addition
0.1 mg / 10 ml
1 mg / 10 ml
10 mg / 10 ml I I I I III + : + Tomato juice no addition
0.1 mg / 10 ml
1 mg / 10 ml
10 mg / 10 ml 111 + I I I I III + III + Potato juice no addition
0.1 mg / 10 ml
lmg / 10 ml
10 mg / 10 ml I I I I orange juice

Example 9

For 100 g of a dried cell cake one Candida lipolitica yeast was 1 l of an aqueous solution (pH 5.0) of a 0.5% strength, according to Example 2 produced enzyme preparation added. The mixture was held at 35 ° C. for 30 minutes to obtain the carry out enzymatic reaction. The reaction mixture was then 1 1 of an aqueous 0.2 η Sodium hydroxide solution added. The mixture was heated to 50 ° C for 30 minutes to ensure that the Protein of the yeast cells was extracted into the aqueous phase. The aqueous extract obtained was centrifuged to remove the cell cake, and the supernatant liquid was adjusted to pH 4.0 with 6 η-hydrochloric acid, so that the protein failed at the isoelectric point. The amount of precipitated protein corresponds to 76% of the total Protein content of the yeast cells. If this experiment was repeated without the enzyme preparation, the Amount of precipitated and isolated protein only 15% of the total protein content of the yeast cells.

Example 10

10 g of a feed consisting of dried Candida utilis cells became 100 ml of an aqueous one Solution (pH 6.0) was added which contained 0.5% of the enzyme preparation prepared according to Example 3. the The mixture was kept at 35 ° C. for 1 hour so that the enzymes reacted with the yeast cell walls. the The reaction mixture was then freeze-dried to give a powdered feed which was made from dsr with Enzyme treated Candida yeast existed. The experiment was repeated without adding the enzyme preparation, and a powdered feed was obtained which was made up only of the freeze-dried Candida yeast duration. The initial dried untreated Candida yeast, the enzyme-treated Candida yeast and the freeze-dried yeast only were treated with pepsin to determine digestibility. The one with it The results obtained are summarized in Table V below.

Table V

rehearse

Digestibility
by pepsin

Dried candida yeast
Untreated (comparison)

Only freeze-dried ones
Candida yeast (comparison)

Candida yeast, treated with the
enzyme complex according to the invention

58.4%
60.2%
97.7%

11th

example

A volume of a clear solution containing the enzyme prepared according to Example 5 with Pellicularia sasakii

030 208/153

complex, acetone was added twice so that the enzyme complex precipitated. The precipitate was collected and dried to give a powdery enzyme preparation. 20 g of a wet cake (solid content: 48% by weight) consisting of Candida utilis cells was added to 100 ml of an aqueous solution (pH 4.0) containing Percent of the powdered enzyme preparation contained. The mixture was then ^{gently shaken for 1 h at 35 °} C. in order to carry out the enzymatic reaction. After the reaction, the reaction mixture was centrifuged to remove the remaining solids. The supernatant liquid was lyophilized to obtain 7.8 g of a dry powdery extract of the soluble intracellular substance of Candida utilis cells. The analysis showed that the dry powdery extract contained 54.3% crude proteins, 4.0% crude fats, 1.7% crude fibrous materials, 6.4% ash and 33.6% soluble nitrogen-containing substances.

The procedure was repeated by adding 100 ml of acetate buffer solution with a pH of 4.0 Place of the enzyme solution were added, and only 1.7 g of a dry powder was obtained after Lyophilize the supernatant solution.

Example 12

A vaccine from the pathogenic yeast Candida albicans was prepared by inoculating a slant of this yeast on 100 ml of a potato glucose medium. This mixture was incubated at 30 ⁰ C 24, and the soil was then mixed with water to the volume of the original volume tenfold diluted 1 ml portions of the vaccine thus prepared (6 χ lO ^ -Zellenkolonien contained ml)

became 10 ml portions of a potato glucose medium (pH 6.0) added, the 0.5 mg, 1 mg, 2 mg, 4 mg, 8 mg, 16 mg or 32 mg / ml) of the enzyme preparation from Pellicularia sasakii according to Example 1. The inoculated nutrient medium was stationary at 35 ° C. for 24 hours incubated, and then the number of unlyysed Candida albicans cells was determined. The results are summarized in Table VI

Table VI

Content of enzyme preparation in the incubated culture medium

(mg / ml)

Number of cell colonies
per ml of the medium

0.5

16
32

2X10 ⁶

IXlO ⁵

7X10 ⁴

4X10 ⁴

2X10 ²

Example 13

In this example, the enzymatic activities of the enzyme complexes were investigated with Pellicularia sasakii and Pellicularia filamentosa according to Examples 4 and 2 were produced. The determination of enzymatic activities was carried out in the following way:

1) Cellulase activity: 5 ml of an enzyme solution (pH 5.0) was placed in an L-shaped tube, and 2 pieces of filter paper (1 cm 1 cm) were immersed in the enzyme solution in the tube shaken with a Monod shaker at 40 ^° C. The time was determined which was necessary until the pieces of filter paper had completely disintegrated. The strength of the cellulase activity was calculated according to the following equation:

Potency =

150

χ 1000

where (x) denotes the time in minutes that it took for the filter paper to disintegrate completely, and (y) denotes the volume in ml of the enzyme solution used.
2) /? - 1,3-glucanase activity: 0.5 ml of a 1% laminarin solution was placed in a test tube, and 0.5 ml of a dilute enzyme solution (pH 5.0) was added. The mixture was heated to 45 ° C. for 30 minutes. The amount of glucose present in the reaction mixture was then determined colorimetrically. 1 ml of the glucose formed in the reaction mixture was evaluated as one unit of the / MS glucanase activity per 0.5 ml of the diluted enzyme solution.

3) Chitinase activity: This activity was measured by adding 1 ml of a 1% suspension of a powdered poly-N-acetylglucosamine (0.147 mm) pH 5 was used and this was reacted with 1 ml of dilute enzyme solution at 45 ° C. for 2 h. the Formation of 1 μg / ml of N-acetylglucosamine in the Reaction mixture was found to be that corresponding to one unit of chitanase activity per ml of the enzyme solution Amount counted.

The optimal temperature and pH for the enzyme activities were determined for by Pellicularia sasakii and Pellicularia filamentosa produced enzyme preparations were measured. The received Results are summarized in Tables VII and VIII.

The enzyme activities were measured using an enzyme solution prepared according to Example 5.

Table VH

Properties of the enzyme complex produced by P. sasakii

Enzymatic activity Place of work Optimal Optimal temperature PH value (U / ml) CQ Cell wall lysing activity 210 30-40 5-7 Cellulase 500 40-50 5.0 jff-1,3-glucanase 17.0 40-50 5.0 Chitinase 12.0 35 4.0-5.5

Table VIII

Properties of the enzyme complex produced by P. filamentosa

Enzymatic activity Potency Optimal Optimal temperature PH value (U / ml) TQ Cell wall lysing activity 220 30-40 5-7 Cellulase 500 40-50 5 ^ -1,3-flucanase 17.2 40-50 5.0 Chitinase 12.0 35 4.0-5.5

Claims (7)

Patent claims: 1. A process for the preparation of cell wall-lysing enzyme complexes, having the following properties own: (a) they lyse living and dead cells of at least Aspergillus niger, Penicillium steckii, Saceharomyces cerevisiae, Candida utilis, Candida albicans, Candida lipolitica, Lentinus edodes, Bacillus subtilis, Lactobacillus lactis and Chlorella, (b) their cell wall lysing activities are stable in the pH range from 3 to 9, being the optimal one pH is in the range of 5 to 7, (c) their cell wall-lysing activities are optimal in a temperature range of 30-40 ⁰ C, the optimal temperature varying depending on the nature of the microorganisms whose cells are to be lysed, (d) their cell wall lysing activities are stable in a low temperature range, but are quickly inactivated ^{at temperatures above 50 0 C,} (e) their zellwandlysierenden activities by the presence of Mn ^++, Ni ⁺⁺ or Zn ^{+ +} inactivated (f) they show the enzymatic activities of cellulase at an optimal temperature range of 40-50 ⁰ C and an optimum pH of 5.0, carboxymethyl cellulase, of JS-l, 3-glucanase at an optimal temperature range of 40-50 ⁰ C and an optimal pH of 5.0, of chitinase at an optimal temperature of 35 ° C and an optimal pH range of 4.0-5.5, of protease, hemi-cellulase and amylase, and (g) They are composed essentially of enzyme components each having a molecular weight of at least 50,000, characterized in that a known strain of Pellicularia sasakii or a known strain of Pellicularia filamentosa in a nutrient medium with assimilable hydrocarbon sources and contains assimilable nitrogen sources for the generation and enrichment of the Enzyme complex grown in the nutrient medium and then the enzyme complex from the nutrient medium is isolated. 2. The method according to claim 1, characterized in that a known Pellicularia strain 48 up to 120 hours at a temperature of 20 to 35 "C under aerobic conditions in a liquid Medium is grown. 3. The method according to claim 1, characterized in that a known pellicularia strain 3–10 days at a temperature of 20–35 ° C in is grown in a solid medium. 4. The method according to claim 1, characterized in that as one of the assimilable carbon and sources of nitrogen bran used, the nutrient medium with water or buffered water extracted to an aqueous extract solution of the enzyme complex, the enzyme complex from the extract precipitated by salting out with ammonium sulfate or by adding acetone or ethyl alcohol and the precipitate is then dried. 5. The method according to claim 1, characterized in that in a liquid nutrient medium below cultured aerobic conditions, the resulting culture broth is filtered to remove the solids, the The clear filtrate obtained is concentrated dialytically through a dialysis membrane, the enzyme complex from the concentrated filtrate precipitated by salting out or by adding acetone or ethyl alcohol and the precipitate is then dried. 6. Zellwändelysierende enzyme complexes, characterized in that they according to the method according to claim 1-5 are obtainable. 7. Use of the cell wall lysing enzyme complexes according to claim 6 for dissolving Cells of microorganisms including bacteria, fungi, yeast, basidiomycetes and chlorella, wherein the suspended in an aqueous medium cells with the enzyme complexes or with the Enzyme complexes in the form of the corresponding broth or in the form of an extract from one appropriate solid culture medium are treated.