WO1983001458A1

WO1983001458A1 - Process for the preparation of enzymes

Info

Publication number: WO1983001458A1
Application number: PCT/FI1981/000077
Authority: WO
Inventors: Oy Rintekno; Serlachius Oy G.A.
Original assignee: Lundell, Ralf
Priority date: 1981-10-26
Filing date: 1981-10-26
Publication date: 1983-04-28

Abstract

Procédé de préparation d'enzymes consistant à utiliser la masse de cellules de micromycètes formant du mycélium appartenant à la classe des Fungi imperfecti, la masse de cellules étant obtenue à partir d'une fermentation aérobie continue. Selon l'invention, la masse de cellules de micromycètes obtenue à partir d'une culture en continu est, après séparation de la couche utilisée dans la culture et après lavage de la masse de cellules, introduite dans un appareil à fermentation d'enzymes séparé, dans lequel une solution ou suspension de croissance contenant des substances inorganiques et/ou organiques qui induisent des enzymes ont été ou sont introduites, la fermentation des enzymes étant par la suite effectuée dans des conditions favorables à la formation d'enzymes, de préférence la solution ou suspension de croissance se trouvant à une température comprise entre 20 et 50oC, avec un pH compris entre 2,5 et 7,0, et l'air est introduit dans le procédé aérobie en une quantité égale à 10-50 % de la quantité de la solution ou suspension de croissance par minute, et la solution ou suspension de croissance ainsi que la masse de cellules et la couche de croissance restante et le produit enzymatique sont extraits de l'appareil à fermentation de manière discontinue après que la concentration ou les concentrations enzymatiques ont atteint un niveau favorable.Process for the preparation of enzymes consisting in using the cell mass of mycelium-forming micromycetes belonging to the class of Fungi imperfecti, the cell mass being obtained from a continuous aerobic fermentation. According to the invention, the mass of micromycete cells obtained from a continuous culture is, after separation of the layer used in the culture and after washing of the mass of cells, introduced into a separate enzyme fermentation apparatus , in which a growth solution or suspension containing inorganic and / or organic substances which induce enzymes has been or are introduced, the fermentation of the enzymes being subsequently carried out under conditions favorable to the formation of enzymes, preferably the growth solution or suspension at a temperature between 20 and 50oC, with a pH between 2.5 and 7.0, and air is introduced into the aerobic process in an amount equal to 10-50% of the amount of the growth solution or suspension per minute, and the growth solution or suspension as well as the mass of cells and the remaining growth layer and the enzyme product are extracts from the fermentation device discontinuously after the concentration or enzyme concentrations have reached a favorable level.

Description

Process for the Preparation of Enzymes

The present invention is concerned with a process for the preparation of enzymes by making use of 5 the cell mass of mycelium-forming microfungi belonging to the class Fungi imperfecti, the cell mass being obtained from continuous aerobic fermentation.

The production of enzymes by using mycelium- forming microfungi belonging to the class Fungi imper- 10 fecti is already known in prior art and in use in indus¬ try. The most important ones among microfungus-based enzymes of industrial production are enzymes that hydro- lyze polysaccharides and their derivatives, such as, for example, amylases, cellulases, hemicellulases, pectinases, 15 and lactases, as well as proteases which hydrolyse pro¬ teins and peptides.

The hydrolysing enzymes are formed during the stationary/dying stage following after the growth stage of microfungi. The composition of the growing substratum 20 has an essential effect on the formation of enzymes.

By optimizing the composition of thes substratum, it is possible to make a strain of microfungus produce sub¬ stantially one enzyme.

The industrial production of enzymes by means 25 of microfungi is based exclusively on the batch fermen¬ tation process. ^'A microfungus is first grown in a little fermenter, whereupon it is transplanted, usually via an intermediate fermenter, to the production fermenter, wherein the cell mass is grown to the ultimate quantity. 30. After the growth stage, the cells of microfungi produce enzymes by means of the same raw-materials.

The duration of the production fermentations is mostly several days, out of which time an essential part is taken by growing the cell mass. The process is 5 frequently modified so that part of the raw-materials are fed into the fermenter during the batch in order to maximize the quantity of cells in the substratum and,

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/,, VIPO thereby, the yield of enzymes. The fermentations are aseptic.

In prior art, continuous enzyme-fermentation processes of microfungi are known in which a growing broth containing cells of microfungus is passed as such from one continuous fermenter into another continuous fermenter in which the formation of the enzymes takes place. The enzyme yields have, however, been low because the stages of growth and of enzyme formation of the micro- fungi are separate from each other. Continuous fermen¬ tation requires that the cells increase in order that they should not be washed out of the fermenter. Slow growth of the cells means long delay times, which causes that an overwhelming proportion of the cells either form spores or die. A continuous process does not remain stable, and the formation of enzymes is reduced consi¬ derably. In practice, this had has the consequence that the microfungus-based enzyme-production processes are batch processes. In a continuous fermentation, the vital func¬ tions of the cells are directed at the production of new cell mass. For example, in. the single-cell protein process of U.S. Patent 3,809,614, wherein the microfungus Paecilomyces varioti is used, a cell mass rich in pro- teins is obtained from growing solutions containing carbohydrates and/or carbohydrate derivatives. In such a case the microfungus does not produce "extra" products of metabolism, such as enzymes, but it produces mainly cell mass out of the raw-materials. From the U.S.^* Patents 3_*288,683 and 3,652,400 it is known that the microfungus Paecilomyces varioti produces enzymes in batch cultures. The enzymes produ¬ ced by means of the said process, glucoamylase- and pro¬ tease, are surprisingly favourable as to their properties. The invention came out when the use of the microfungus Paecilomyces varioti as a producer of extra¬ cellular enzymes, especially of hydrolases, was studied.

O PI Batch culture tests with various raw-materials indicated that the said microfungus produces, e.g., glucoamylase and hemicellulases. well. Hereinafter attempts were made to develop an enzyme-production process suitable for industrial use by growing Paecilomyces. varioti as sub¬ merged cultures under aerobic conditions by means of various raw-materials that promote the formation of enzymes and by recovering enzyme-containing solutions after the fermentations. Now it has been noticed surprisingly that, when cells of the microfungus Paecilomyces varioti that were grown continuously are used and separated from the original growing solution and when the pure cells in this way obtained are grown in new nutrient solutions that promote the formation of enzymes, the cells produce enzymes rapidly, with the consequence that the 'Stage of production of enzymes remains short.

It is surprising and unexpected for a person skilled in the art -that the "pure" cells obtained in the above way produce enzymes immediately after the transplantation into the new nutrient solution and that the ability of the cells to produce enzymes remains unchanged for dozens of hours after the transplantation. This permits the production of enzymes in a revolutionary way in connection with continuous microfungus fermen¬ tations. Under these circumstances, an enzyme production unit can be connected to a single-cell protein process using the microfungus Paecilomyces varioti. In this case the cell mass of the microfungus is, after the continuous fermentation, used for the production of enzymes, whereupon the cell mass is returned to the single-cell protein unit. It is an advantage that the microfungus mass used for the production of enzymes does not constitute a waste problem, which is frequently the case in conventional production processes.

Moreover, it has been surprisingly found that by increasing the concentration of the microfungus in

OMPI _t. WIPO _* the nutrient solution of the enzyme fermentation, the formation of the enzymes is accelerated and enzyme concentrations are obtained that considerably exceed the concentrations that can be achieved by means of the batch processes representing the prior art technology. Advan¬ tages include a clear increase in the yield of enzymes and an essential facilitation of the after-treatment operations.

The process in accordance with the invention is mainly characterized in that the microfungus cell mass obtained from continuous culture is, after the substratum used in the culture has been separated and the cell mass washed, introduced into a separate enzyme fermenter, into which a growing solution or suspension containing inorganic and/or organic substances that induce enzymes have been or are introduced, and that the fermentation of enzymes is hereinafter performed under conditions favourable for the formation of enzymes, preferably so that the temperature of the growing solution or suspension is 20 to 50 C, pH 2.5 to 7.0, and that air is introduced into the aerobic process as a quantity of 10 to 50 per cent from the quantity of the growing solution or suspension per minute, and that growing solution or suspension together with the cell mass and with the remaining growing substratum and with the enzyme product are removed from the fermenter batchwise after the enzyme concentration or concentra¬ tions have reached a favourable level.

By means of thorough studies it has been possible to prove that, by using the process described above for^*the production of enzymes, by means of the microfungus Paecilomyces varioti it is possible to pro¬ duce all of the enzymes that are produced by the said microfungus in conventional batch fermentations. The microfungus Paecilomyces varioti belongs to the class Fungi imperfecti. The same class includes most of the microfungi, e.g. the microfungi belonging

OMPI to the genera Aspergillus, Penicillium and Trichoderma, by means of which hydrolysing enzymes intended for industrial use are produced by means of batch fermentatio processes, which represent the prior art technology. 5 It has been possible to cultivate species of the genera of microfungi concerned in temselves con¬ tinuously.

It is evident that, by means of the process described above and by using other microfungi belonging t ^* 10 the class Fungi imperfect!, producing enzymes, and that have been grown continuously, it is also possible to pro¬ duce large quantities of enzymes more rapidly than what can be achieved by means of batch cultures representing the prior art. 15 The substratum of continuous, aerobic culture of microfungi may consist of most various carbohydrates and/or suspensions, such as industrial waste, e.g. spent liquors from wood-processing industry.- In addition to this, other rawmaterials of cells are required, especi- 20 ally inorganic salts. The continuous fermentation of microfungi has an aseptic nature. In the process de¬ scribed above the separation of the microfungi from the substratum of the continuous culture is essential, especially if the nutrient solution consists of lignin- 25 containing spent liquors from wood-processing industry.

Lignin may bind proteins and thereby make the performance of the processes of recovery of enzymes more difficult. Washing is preferably performed by means of a washing liquid, which may be water, whose temperature 30 and pH do not differ essentially from those used in continuous fermentation. The washing liquid should be preferably sterile, in which case the further enzyme fermentation is protected from contaminating micro¬ organisms. The requirement of sterility of the washing 35 liquid. is not unconditional, for there are also other means of protection from contamination.

OMPI In the process, usually the entire cell mass that is needed for the production of the enzymes is batched right at the initial stage of the process into the enzyme fermenter. It is, however, possible to add to the cell mass during the enzyme fermentation, either during part of the time or throughout the entire duration of the batch.

The batching may also be continued after a partial emptying of the fermenter by adding new cell mass to replace the cell mass that was removed from the fermenter.

As raw-material for the process, it is possible to use inorganic and/or organic compounds that have pro¬ perties contributing to the maintenance of the cell mass and to the formation of enzymes. The raw-materials may be batched into the enzyme fermenter as a whole at the beginning of the production batch, or they may be added during the batch. If the fermenter is emptied at the beginning of the batch partly, the removed 'portion of - raw-material may be substituted by a new quantity of ra -material. As a rule, the ra -materials are added as sterilized, but this is not always necessary. As raw- materials, it is possible to use, e.g., various by-products of agricultural products, such as bran and bran extracts, or various by-products of wood-processing industry and extracts from same. Other organic raw-materials besides those mentioned here may also be used.

If necessary, inorganic compounds are also used. For a person skilled in the art it is evident that in a fermentation process, water is used, into which the raw-materials have been dissolved or suspended.

In the process the temperature of the growing/ enzyme-formation substratum is maintained at 20 to 50°C and the pH at 2.5 to 7.0. The temperature and/or the pH may be varied during the process, but most usually they are maintained at constant values. The fermentation process is aerobic, and the consumption of air per minute

OMPI during the process is about 10 to 50 per cent from the quantity of the fermentation substratum. In order to promote the transfer of material and heat, the fermen¬ tation broth is frequently stirred mechanically. It has been noticed that it is preferable to carry out the enzyme fermentation as aseptic, whereby the equipment and the raw-materials are sterilized by means of conventional processes before fermentation and the microfungus cells obtained from continuous fermen- tation are washed with water or with a nutrient solution. In all cases, aseptic conditions are, however, not necessary.

The procedure of the process in accordance with the invention is.the following. The starting point of the process is a conti¬ nuous culture of a microfungus performed by conventional means, from which the culture substratum that has been used and that contains cell mass of the microfungus is constantly available. The washed cell mass and the raw-materials needed for the formation of enzymes and for maintaining the vital functions of the cells are passed into an enzyme fermenter.

The fermentation is carried out as an aerobic batch process, which can be continued by removing enzyme- containing solution and by adding a compensating quantity of raw-materials and/or cell mass. The batch is finished by emptying the fermentor completely when it is consi¬ dered that the production capacity of the microfungus has been clearly reduced. The fermentation may also be discontinued earlier if other criteria so require. Microbe mass may also be added to the fermenter during the batch if the production of enzymes can be improved thereby. The raw-materials needed in the process and the cell mass can be transferred into the fermenter at the same time or separately. Air is supplied into the fermentation broth as a quantity of about 10 to 50 per

OMPI » W WIIPPOO cent from the volume of the fermenter broth per minute. If necessary, the fermenter broth can be stirred mecha¬ nically in order to improve the transfer of heat and material.. The temperature, 20 to 50 C, and the pH, 2.5 to 7.0, of the culture solution are either maintained as constant or controlled in accordance with a specified program, or they are allowed to adjust themselves freely during the fermentation process.

After the formation of enzyme, the microfungus mass and solid raw-material residues, if any, are sepa¬ rated from the enzyme-containing culture broth and washed, whereupon the solid fermentation residue can be used, e.g., as a protein-containing product for fodder or for techni¬ cal purposes. The enzyme-containing solution and the washing solution are treated by conventional means in order to recover the enzyme products.

The process for the production of enzymes described above has^{' "}a number of advantages as compared with the production processes representing the prior art technology.

By means of the process, an abundance of enzyme or enzymes is produced in a short time, because the pro¬ cess can always be started from maximum concentration of cells, and with a vigorous microfungus mass, containing few dead cells, it is always possible to achieve high enzyme concentrations and to maintain a most rapid formation of enzyme during the batch.

The process permits a highly flexible pro- duction, wherein

- it is easy to determine when the batch is to be started, for the inoculum is always ready,

- it is easy to control how many batches per year are to be carried out, - it is easy to choose what enzymes are to be produced and to apply the optimum growing substratum for the production of the enzyme concerned. The process is rapid, because the cell-growing step of a conventional batch process is omitted totally. This is why the process permits the use of a non-aseptic culture technique. The process is also suitable for the supply of additional raw-materials, as well as for the supply of additional microfungus, whereby the production capacity can be increased further.

The process may be connected to a single-cell protein factory using a microfungus, or it may be based on the cell quantity of a continuous production of micro¬ fungus constructed for enzyme production only.

If the enzyme process is integrated with a single-cell protein factory, an additional advantage is that the waste liquors and the solid residues, such as the cell mass and the solid raw-material, residues, can be returned to the single-cell protein factory, and there¬ by the environmental problems are reduced to none.

Owing to its high enzyme content, the after- treatment of the fermentation solution is more advan¬ tageous than the after-treatments of the enzyme-containing fermentation solutions from the processes representing the prior art technology.

Example 1

This reference example illustrates the produc¬ tion of enzyme by means of mycelium-forming microfungi in a way in itself known.

With the microfungus Paecilomyces varioti, an enzyme-production test was performed as a batch culture on a starch-bran substratum in a 2000 litre fermenter.

The composition of the pre-growing substratum in shaking flasks (1.5 1) was:

- glucose 20 g/1 - yeast extract 2 g/1

- (NH )₂S0₄ 10 g/1

- MgS0₄ - 7H₂0 1 g/ 1

^H

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The composition of the substratum in the trans¬ plantation fermenter (30 1) and in the main fermenter (1000 1) was: - wheat bran 40 g/1 - starch 25 g/1

- (NH_j.)₂S0₄ 5 g/1

- KH₂P0₄ 5 g/1

- MgS0₄-7H₂0 0.5 g/1

At the beginning of the growing, the lower limit of the pH had been adjusted at 4.5. The growing temperature was 37°C until the growth maximum of the microfungus, and 30°C at the enzyme-production stage. The microfungus reached its growth maximum about 24 hours after the beginning, at which time the content of cell mass in the growing broth was about 16 grams per kilogram of suspension. When the pH-value became higher than 6, it started being adjusted by means of phosphoric acid.'^** The pH was gradually lowered to 4.^"6. The batch fermentation took a total of 87 hours, i.e. 3-5 days. The activities obtained were: enzyme rate of activity enzyme production

- glucoamylase 1.0 nkat/ l 0.012 nkat/ml h - p-glucosidase 63 " " 0.72 " " "

- β-xylosidase 19 " " 0.22 " " "

The glucoamylase activity was determined by using 2mM p-nitrophenyl-<χ-_.-D-glucopyranoside as substrate at 45°C, at pH 4.5, the reaction time being 10 minutes. The quantity of p-nitrophenol liberated in the reaction indicates the enzyme activity unit (nkatal/ml) .

The 5-glucosidase activity was determined by using 1 mM 4-nitrophenyl-^-D-glucopyranoside as substrate at 50°C, at pH 4.8, the reaction time being 10 minutes. The quantity of 4-nitrophenol liberated in the reaction indicates the enzyme activity unit (nkatal/ml) .

OMPI The Λ-xylosidase activity was determined by using 2 mM o-nitrophenyl- ^-D-xylopyranoside as substrate at 45°C, at pH 4.5, the reaction time being 10 minutes. The quantity of o-nitrophenol liberated in the reaction indicates the enzyme activity unit (nkatal/ml) .

The growing solution was after-treated for the recovery of the enzymes. From it the bran residue and the yeast mass were separated. The solution was con¬ centrated by ultrafiltration to an almost 30-fold con- centration. As the final result, a yellowish brown, highly active enzyme concentrate was obtained.^'

By means of the following examples 2 to 4, production' of enzymes by means of microfungus cells that were obtained from continuous fermentation and washed is described.

In the examples 2 to 4, an 8-litre laboratory fermenter constructed for continuous, aseptic fermenta¬ tion was used for the production of the cell mass of the microfungus Paecilomyces varioti. The enzyme fer- mentations were carried out by means of a 4-litre labo¬ ratory fermenter which was provided with a filter (100 mesh) for the filtration and washing of the microfungus cell mass.

As the raw-material of the continuous fermen- tation was used acid calciumbisulfite spent liquor that had been stripped and pre-evaporated, obtained from a pulp factory. The composition of the spent liquor was:

- dry-substance content 19-3 % (w/w)

- reduction value 56.0.g/1 - density I.083 kg/1

- pH 3-55

- residual S0₂ 0.23 g/1

The spent liquor was, before use, diluted with water to a content of about 8 per cent (w/w) , and inor- ganic salts were added to it, whereupon it was sterilized.

The continuous fermentation was carried out under the following conditions:

OMPI ,. IIPPOO - - temperature 37°C

- pH 4.8

- aeration 9 1/min.

- ^* stirring 1050 rpm ^~— average delay time about 6 hours.

During continuous feeding, the cell concentra¬ tion of the microfungus remained at the level of 13 to 14 g/kg of suspension.

From the continuous fermentation of the micro- _, fungus Paecilomyces varioti, the waste liquor suspension was pumped straight into the enzyme, fermenter, whereupon the waste liquor was removed through a filter placed on the bottom of the fermenter. Hereupon the mycelium was washed with a phosphate solution (KH₂P0 , 10 g/1, pH 4.5). The washing was performed as 4 batches with a total of 4 litres of solution. The washing solutions had been sterilized and cooled to 30 C before the washing stage.

The filtration and washing of the mycelium were performed in the fermentation tests in about 30 minutes.

After washing, 4 litres of a substratum of the following composition were added to the enzyme fermenter:

- bran 40 g/1

- starch 20 g/1 - (NH₄)₂S0₄ 10 g/1

- KH₂S0₄-7H₂0 1 g/1

The substrate had been sterilized and cooled to 30°C.

The fermentation conditions are described separately in connection with each enzyme fermentation example.

For the purpose of measurement of enzyme acti¬ vity, samples were taken at the beginning of the enzyme fermentation at intervals of 0.5 hours (3 samples), where- upon the interval between samples was increased to 1 hour, and later to 2 hours. Example 2

Fermentation conditions (the mycelium content at the beginning of fermentation was estimated as 7.7 g/kg)

- aeration 4 1/min. - rate of stirring 900 rpm

- temperature 37- 0.4°C

- the pH was allowed to change freely from the starting value of 5.3.

Results: - The glucoamylase activity of the fermentation increased linearly right from the beginning of the fermentation. Over 24 hours 0.43 nkat/ml gluco¬ amylase was obtained, and the rate of production was 0.018 nkat/ml h.

Example 3

Fermentation conditions (the mycelium content at the beginning of fermentation was estimated as 8.7 g/kg)

- aeration "- 4 1/min. - rate of stirring 800 rpm

- temperature 30 1 0.4°C

- the pH was allowed to change freely from the starting value of 5-2.

Results: ~ In this fermentation, the glucoamylase activity of the fermentation also increased linearly, and gluco¬ amylase was obtained 0.55 nkat/ml over 24 hours. The rate of production was 0.023 nkat/ml h. By adding 20 g/1 of bran, the enzyme fermentation was continued by 20 hours and at the same rate of production, x-rtiereby the glucoamylase content increased to 1.0 nkat/ml. The formation of enzyme started immediate¬ ly on transplantation.

Example 4

Fermentation conditions:

The mycelium was introduced into the induction

OMPI IPO fermenter after 110 hours had elapsed from the starting of continuous fermentation of Paecilomyces varioti. The mycelium content In the continuous fermenter was 19-4 g/kg of suspension. About 5 kg of the suspension was transplanted. The fungus mass was filtered and washed. The mycelium content at the beginning of the test was estimated as about 15.^"5 g/kg of suspension, aeration 4 1/min.

- rate of stirring 1000 rpm - temperature 30 + 0.4°C

- the pH was allowed to change freely from the starting value of 5-5-

Results:

- The enzyme activities and corresponding rates of production of the ermentation were after 26 hours: enzyme rate of activity enzyme production

— glucoamylase 1.28 nkat/ml 0.049 nkat/ml h

— /3-gi_UCosidase 30 nkat/ml 1.15 nkat/ml h ^" — / -xylosidase 24 nkat/ml 0.92 nkat/ml h

All the enzymes were formed immediately after the transplantation almost linearly during the fermen-. tation.

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Claims

WHAT IS CLAIMED IS:

1. Process for the preparation of enzymes by making use of the cell mass of mycelium-forming micro- fungi belonging to the class Fungi imperfect!, the cell mass being obtained from continuous aerobic fermentation, c h a r a c t e r i z e d in that the microfungus cell mass obtained from continuous culture is, after the sub¬ stratum used in the culture has been separated and the cell mass washed, introduced into a separate enzyme fermenter, into which a growing solution or suspension containing inorganic and/or organic substances that induce enzymes have been or are introduced, and that the fermentation of enzymes is hereinafter performed under conditions favourable for the formation of enzymes, preferably so that the temperature of the growing solution or suspension is 20 to 50°C, pH 2.5 to 7.0, and that air is introduced into the aerobic process as a quantity of 10 to 50 per cent from .the quantity of the growing solu- tion or suspension per. minute, and that growing solution or suspension, together. ith the cell mass and with the remaining growing substratum and with the enzyme product are removed from the fermenter batchwise after the enzyme concentration or concentrations have reached a favourable level.

2. A process as claimed in claim 1, c h a r ¬ a c t e r i z e d in that the washing of the microfungus cell mass that was grown continuously is performed with water whose temperature is 20 to 50°C, pH 2.5 to 7-0, and which may contain Inorganic and/or organic compounds.

3. A process as claimed in.claim 1 or 2, c h a r a c t e r i z e d in that the washed cell mass is batched into the enzyme fermenter as a whole at the beginning of the fermentation.

4. A process as claimed in claim 1 or 2, c h a r a c t e r i z e d in that washed cell mass is added into the enzyme fermenter both at the beginning and during the fermentation.

5. A process as claimed in claim 3 or 4, c h a r a c t e r i z e d in that the cell mass is used in several subsequent batch fermentations.

6. A process as claimed in claims 1 or 2, c h a r a c t e r i z e d in that the cell mass is obtained from a microfungus-based single-cell protein process, to which the residual cell mass can be returned after the enzyme fermentation.

7- A process as claimed In claim 1, c h a r ¬ a c t e r i z e d in that grain brans and/or bran extracts and/or starch are used as enzyme-forming substances.

8. A process as claimed in claim 1' or 7, c h a r a c t e r i z e d in that the substances inducing formation of enzymes are batched into the enzyme.fermenter as a whole at. the beginning of the fermentation.

9. A process as claimed in claim 1 or 7, c h a r a c t e r i z e d in that the substances inducing formation of enzymes are added both at the beginning aria. during the fermentation, the temperature and pH-value of admixtures in solution or suspension form being not essentially different from the temperature and the pH prevailing in the growing broth.

10. A process as claimed in claim 1, c a r - a c t e r i z e d in that Paecilomyces varioti is used as the species of microfungus.

11. A process as claimed in claim 1, c h a r ¬ a c t e r i z e d in that the enzymes glucoamylase, -glucosidase, and -xylosidase are produced.

12. A process as claimed in claim 1, c h a ¬ a c t e r i z e d In that, at the end of a batch, part of the growing solution or suspension containing cell mass and residual materials is emptied from the enzyme fermen¬ ter and that new cell mass and new raw-materials are batched into the enzyme fermenter in order to add to the batch.

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