CS261811B1 - Process for preparing cellulose enzymes and/or enzymes for the degradation of cell wall - Google Patents

Description

The present invention relates to a process for producing cellulase enzymes and / or cell membrane degrading enzymes by aerobic submerged culture of fungal mutants. The complex of enzymes formed can be used for the partial or total degradation of cellulose as well as hemicelluloses in industrial products, waste, waste water or in the removal of living or dead plant cell walls.

It is known that cellulase complex enzymes are produced by aerobic submerged cultivation of fungi. The known processes differ mainly in the fungal strains used, in the substrates used in the fermentation process and in the process control process. The cost-effectiveness of cellulase recovery is essentially defined by the enzymatic activity achieved per unit volume of fermentation medium, the amount of enzyme produced per unit of time (enzymatic productivity) as well as the amount of enzyme formed and based on the substrate used (enzyme yield). known fungi strains of the genera Aspergillus, Penicillium and Trichoderma.

E.g. according to the patent DD-WP 225 712, it is used as a cellulase producer of Penicillium janthinellum, the cellulose being used as a substrate in combination with a pre-prepared green chop. In batch culture, using a total of 6% cellulose added to the green chop, after 8 days, the cellulase activity of 6 International Units / cm ^{3 was obtained} on a filter paper test, corresponding to a yield of 100 International Units / g cellulose. In another method, according to JP 59-151188, the Trichoderma koningii strain is used as a cellulase producer: microcrystalline cellulose (Avicel) at a concentration of 3% / o is used as the substrate. The resulting enzymatic activities after 7 days were 11.5 International Units / cm ³ (IU / cm ³ ) for endo-beta-glucanase activity and 0.85 IU / cm ³ for exo-cellobiohydrolase corresponding to the exocelobiohydrolase yield required for degradation microcrystalline cellulose in the range of 28.3 IU per gram of cellulose; the corresponding productivity is 5 IU exocelobiohydrolase per dm ³ and hour. From the patent and scientific literature known cellulase producers with the highest known cellulase production belong to the species Trichoderme reesei (formerly Trichoderma viride). Various mutants of Trichoderma reesei were grown to improve cellulase production.

DD-WO 80/01080 discloses cellulase formation using the Trichoderma reesei MCG 77 mutant. Cellulase formation is inhibited by glucose but not glycerol; lactose induces cellulase formation. The cellulase activity in the nutrient medium containing 1% cellulose is 1 to 1.8 IU / cm ³ FPA, in the nutrient medium with 1% lactose 1.35 IU / cm ³ FPA and in the nutrient medium with 1% lactose and 1% cellulose 1.75 IU / cm ³ FPA. U.S. Pat. No. 4,472,504 discloses cellulase formation using a Trichoderma reesei MCG 80 mutant that has been produced from a Trichoderma reesei Rut C 30 mutant. IU / cm ³ FPA, which corresponds to a yield of 215 IU FPA / g cellulose. Cultivation of the same strain on a culture medium with 2% lactose yields a cellulose activity of 1.7 IU / cm ^{3 of} FPA, corresponding to a yield of 85 IU FPA / g lactose. These Trichoderma reesei mutants produce relatively high cellulase activity based on cellulose, in particular microcrystalline cellulose and cotton cellulose, but not on soluble substrates such as naipr. glucose. This implies, in addition to the high cost of the substrate, the process deficiencies.

Mutants of Trichoderma reesei that are capable of producing glucose-based cellulases as a carbon source have also been bred. The production of cellulases by T. reesei mutants using glucose as a substrate has been reported at the 3rd European Congress on Biotechnology (Munich, Germany, September 10-14, 1984; M. Bailey: "Cellulase production by mutants of Trichoderma reesei on a non-cellulose pod"). The highest cellulase activity described was obtained with a T. reesei mutant VTT-D-79125 based on 4% glucose -b 4% distillate spent and was 6.4 IU / cm ³ FPA. With cellulose instead of glucose, approximately the same cellulase activity was achieved in this strain.

All known processes for obtaining cellulases have the disadvantage that the cellulase yields relative to the substrate used are relatively low or that they require expensive substrates such as microcrystalline cellulose or cotton cellulose, or that the cellulase activities achieved in the fermentation medium are low, or that by increasing the concentration The increase in cellulase activity is not proportional to the substrate.

It is an object of the invention to achieve high activity cellulase erism complex using cost-effective substrates while at the same time achieving high enzyme yields per substrate to ensure efficient utilization of fermenter volume and substrate utilization. ~

It is an object of the present invention to provide improved cellulase mutants and use them in a culture process, achieving high cellulase activity in a single fermentation step at a uniformly high cellulase yield relative to the substrate and using cost-effective substrates.

According to the invention, the object of the present invention is to provide mutants of Trichoderma reesei ZIMET 43 803 and / or Trichoderma reesei ZIMET 43 804 by culturing aerobically on a submerged manner in a culture medium containing one or more substrates which alone or in combination with a single or multiple non-inducing substrates induce cellulase formation and / or cellular degradation of enzymes,

It has been found that these mutants are characterized by cellulase production with partially suppressed catabolite formation also in relation to glucose and that these mutants secrete high concentrations of cellulase complex enzymes on soluble substrates, such as e.g. glucose or lactose. Both strains were obtained by mutation from Trichoderina reesei CCI and were deposited in the deposit of the ZIMET culture collection at the Central Institute for Microbiology and Exiperimental Therapy of the GDR Academy and registered under ZIMET 4'3 80i3, coated ZIMET 43 804.

The strains are characterized by the following features.

Description of the strain (morphology, physiology:

Both strains are characterized by morphological features typical of the genus Trichoderma and differ in physiological properties in cellulase formation.

Maintenance of the strains is possible on nutrient media suitable for Trichoderma strains. Suitable is e.g. potato-glucose agar or sporulating agar (ammonium sulfate 0.2%; primary potassium phosphate 0.5 per cent; glucose 2%; yeast extract 0.7%; agar 2%). A little suitable for maintaining the strain is Czapek-Dox agar as it is; did not find that the strains described were capable of assimilating nitrites as a nitrogen source. A suitable culture temperature is 28-30 ° C, a culture time of about 5-6 days and an additional 5-7 days at room temperature until the strain is fully developed. Vaccination is done through conidia; the color of conidia is green, initially yellow-green.

Keeping the strain in lyophilized form or above the coated v. liquid nitrogen is possible by generally practiced methods, without a decrease in the ability to synthesize cellulose so far.

Both strains have the ability to produce cellulose at partial. suppression of the repressive effects of catabolites, i. j. in the presence of easily assimilable substrates, e.g. various sugars or glycerol, cellular enzymes are formed up to a certain substrate concentration. The ability to form cellulase differs in both deposited strains in terms of the concentration of easily assimilable substrates in which cellulase formation is not yet suppressed.

The ability to form a cellulase by suppressing catabolite formation can be tested using the method of B. S. Montecourt and D. E. Eveleigh (Applied and Environmental Microbiology 33, 1977, 178-183).

The acid-swollen cellulose in combination with the appropriate repressor of interest, in particular glucose, fructose, xylose, lactose, coated with glycerol, serves as the substrate on the agar medium. In order to limit the colony size, Bengal red is added to the agar broth at a concentration of 30 mg / dm ^{1 * 3} . The thickness of the agar layer was adjusted to 4 mm. With a dish diameter of 9 cm, it is expedient to bake 100 to 200 conidia per dish. According to the method, clarified zones are formed by cultivation and incubation at 45 ° C for 20 hours, while maintaining the viability of the conidia.

Therefore, clonal selection can also be applied in this manner. The shape and size of the individual colonies differ in the strains described, depending on the substrate used. On cellulose agar, large colonies are formed with an adherent thin mycelium, while colonies on glucose, coated fructose are smaller, similar to colonial mutants, have a thicker layer of mycelium and spore more strongly. Generally, ibengal red agar is stained around the saliva developed colonies. Testing of the ability to produce cellulase by suppressing catabolite formation in the strains described is also possible in tubes. E.g. The conidia were inoculated on agar broth with acid-swollen cellulose in combination with the respective repressor under investigation, but without the addition of Bengal red. The diameter of the tubes should be 9 mm. The depth of clarification serves as a measure of cellulase-forming ability; co-cultivation and incubation at 45 is done as in the thatched test.

The strain ZIMET 43 804 shows, with respect to glucose and fructose, a greater ability to produce cellulase with partially suppressed catabolite production:

substrate

ZIMET 43 804

ZIMET 43 804 ° / o cellulose 2 mm% cellulose · 1-5% lactose 8 mm ° / o cellulose -j- 5% glucose 0 mm% cellulose 5% fructose 0 mm ° / o cellulose 5% glycerol, 2 mm mm ' 5 mm mm mm mm (acid-swollen cellulose) (depth of clarification in a test tube)

281 Sil

The temperature of the T. reesei ZIMET 43 803 and T. reesei ZIMET 43 804 mutant cultures is 22 to 36 ° C, preferably 30 to 34 ° C for mycelium growth and 25 to 30 ° C for enzyme formation. The pH for culture is in the range of 2 to 7, preferably 2.5 to 5.5 for mycelium growth and 3.2 to 6.0 for enzyme formation. Suitable fermentation vessels are all fermenters, operative with the exclusion of foreign infection, which guarantee a sufficient supply of oxygen and in which there are no permanent and extremely high shear forces to which a considerable part of the fermentation medium is constantly exposed.

The starting points for cultivation are conidia of surface cultures, in particular slant agar cultures on potato-dextrose soil. As inoculum for the fermentation medium conidia are used, preferably in a concentration of 10 ⁷ to 10 ⁹ per liter of fermentation broth, or a suspension of vegetative mycelium, preferably at 1 to 15 vol. % of the pod to be vaccinated. For inoculation of large ferments, it is expedient to add the inoculum in successive stages. The process of culturing T. reesei ZIMET 43 803 or T. reesei ZIMET 43 804 mutants to obtain cellulase or cell wall degrading enzymes is different depending on the substrates or substrate combinations used, as well as the enzyme composition to be used. doslahnuť. To form a cellulase enzyme complex without an increased proportion of side activities such as e.g. xylanase activity and glucanase activity can be used as an inducing substrate of pure cellulose, but it is preferable to adjust the pH and temperature profile of the fermentation medium according to Example 1. Up to a concentration of about 3% cellulose, an amount of cellulase enzyme is formed proportionally to the cellulose used. Although higher cellulose concentrations lead to higher cellulase concentrates, the increase in cellulase concentration is no longer proportional to the amount of cellulose used, so that the yield of cellulases relative to the amount of substrate decreases. Increased cellulase enzyme concentrations, proportional to the amount of cellulose used, are possible according to the described fed-batch technique, wherein the cellulose supply is controlled by a control device or a computer-controlled process control depending on the carbon dioxide content of CO2 in the fermentation waste gas so The carbon dioxide, based on mycelium proteins, remains at approximately the same process parameters during the administration of the substrate. The use of wheat bran or other component of the complex nutrient medium can increase the development of side activities such as cellobiase and xylanase. For the production of enzymes for degrading the fungal cell wall, the fungal cell walls are preferably used as an inducing substrate in the discontinuous process. The enzymatic complex formed has a composition which makes it suitable, for example, for the production of living fungal protoplasts.

The recovery of cellulase enzymes based on soluble inducing substrate or soluble non-inducing substrates in combination with inducing substrates is carried out according to the present invention using a fed-hatch technique, wherein soluble inducing substrates or soluble non-inducing substrates or combinations of soluble inducing substrates and combinations of soluble inducing substrates medium such that the actual concentration of soluble substrates in the fermentation broth is adjusted to be at least not suppressed in the region in which cellulase formation is induced. The upper limit of this region is determined by the degree of cellulase production ability by partially suppressing the formation of catabolites, which degree corresponds to the particular substrate used.

The fermentation process of the fed-batch technique consists of a double-contiguous section (backpack) and a phase of refilling the substrate (fed-batch section). The discontinuous phase serves for the cultivation of the mycelium, whereas in the substrate replenishment phase the enzyme is predominantly produced. The substrate supplied is controlled by the concentration of carbon dioxide in the fermentation waste gases. If non-inducing substrates are not added, the basic nutrient medium must comprise at least one inducing substrate, such as e.g. cellulose, wheat bran or distillery stillage. If soluble inducing substrates are added, the nutrient medium need not contain other inducing substances; in this case, it is also possible to cultivate the mycelium in a discontinuous fermentation section based on non-inducing substrates, such as e.g. glucose, sucrose or glycerol.

It is also possible to start with a supply of soluble indukujúclch substrate ¹ immediately after seeding, when the vaccination is done with vegetative mycelium most like in a concentration of 2-8 g / ^dm3, calculated on a dry weight basis. When using soluble inducing substrates, e.g. lactose, it is also possible to continuously separate the secreted enzymes from the fermentation medium by means of known membrane separation processes. The solids (fungal mycelium) are separated from the enzymes by microfiltration and the low molecular weight soluble components of the nutrient broth are ultrafiltrated. It is expedient to return the solids to the nutrient medium immediately, while part or all of the ultrafiltrate is introduced into the inducing substrate to be added and returned to the fermentation medium.

The separation and purification of the enzymes obtained can be carried out by known methods. Depending on the application, unprocessed fermentation medium, ending in culture, dried crude enzymes or refined enzymes may be used.

The specific activity of the crude cellulase enzymes fermented on the basis of wood pulp or lactose is in the range of 1.4 to 1.9 m. j. (FPA) / <mg protein.

Example 1:

The Trichoderma reesei ZIMET 43 803 strain is cultivated in a 30 dm ³ fermenter to obtain cellulase enzymes. He.weten 10 Hz wood pulp is used as a substrate; the fermentation is next to in the batch process.

The fermenter is provided with a generally used six-blade propeller stirrer, the total diameter of which is about a third of the internal diameter of the fermenter-α. The height of incorporation of the stirrer in the fermenter is about one-fifth of the total height of the internal space of the fermenter. The fermentor is supplied with gas through a ring located below the stirrer, the ring having a diameter approximately equal to that of the stirrer and having 1 mm wide openings 8 mm apart. The fermenter is equipped with commonly used measuring and control technology, in particular a temperature measuring and control device, a pH measuring and controlling device, a dissolved oxygen measuring and controlling device, a carbon dioxide concentration measuring device in waste fermentation gases, with a control signal, e.g. control of metering pumps, which ensures protection against excessive addition of the deflector. Particularly suitable is a fermenter which allows a computer controlled process.

The nutrient medium has the following composition:

cellulose He.weten 10 Hz 30.0 g / dm ³ ammonium sulfate 4.8 g / dm ³ dihydrogen potassium 3.0 g / dm ³ , magnesium sulphate, heptahydrate 0.4 g / dm ³ calcium chloride 0.3 g / dm ³ peptone 1.5 g / dm ³ Tweon 80, polyoxyethylene- 1.0 g / dm ³ sorbitan monooleate trace salt solution 10,0 cm ³ / dm ³ Composition of trace salt solution: sulfate heptahydrate ferrous 0.50 g / dm ³ sulfate heptahydrate manganese 0.16 g / din ³ sulfate heptahydrate 0.14 g / dm ³ zinc cobalt chloride 0.20 g / dm ³

The components of the nutrient medium required for the preparation of 16 dm ³ are dissolved in 16 dm ^{3 of} water in a suitable manner, they are suspended, and introduced into the sterilized fermenter, excluding the foreign germs. Finally, 1 dm ^{3 of} fresh mycelium suspension is inoculated into the fermenter as inoculum.

The inoculum is prepared in a shake culture. The same nutrient medium components are used as for the 30-liter fermenter, but the KH2PO4 concentration is increased to 15 g / dm ³ and additionally 0.9 g / dm ^{3 of} urea is added to the medium; the concentration of the other ingredients is the same as in the nutrient medium in the form, ntori.

Ten flasks of 100 cm ^{3 of} this nutrient medium are prepared and, with the exclusion of foreign germs, each flask is inoculated with about 10 ⁷ conidially deposited T. reesei ZIMET 43 803 strain. The length of the culture is 36 to 60 h. until the fermenter is inoculated.

The temperature of the culture at the beginning of the fermentation is adjusted to 30 ^c C; the pH control limits are set to 3.5 (lower limit) and 5.5 (upper limit of regulation) and the stirrer speed is set to 500 / min. and a gas intensity of 30 standard dm ^{3 of} air per hour and 1 dm ^{3 of} fermentation medium corresponding to a total of 4'50 normal dm ^{3 of} air per hour. Silicone antifoam is used to prevent foam formation. Depending on the condition and age of the inoculum, the pH at the start of the fermentation is in the desired range of about 4.0 to 5.4. During the fermentation, the pH drops, while reaching the lower control limit, it is kept constant at 3.5 by means of a 12% ammonia solution. Occasionally, a temporary pH rise is observed in the initial phase, which is stopped and maintained at 5.5 with 10% sulfuric acid when the upper regulatory limit is reached.

When the mycelium growth, which corresponds to the logarithmic phase of the growth of the unicellular organisms, is reached, the fermentation temperature is set to 11 and 28 ° Ό. This moment is reached after about 35 to 60 hours and is recognizable by increasing the concentration of carbon dioxide in the fermenter waste gas. In the present example, when the CO2 concentration in the dried exhaust gas is reached, it is 0.2% by volume. fermentation temperature is set at ^{28 0} C. The concentration of CO2 increases further after exceeding the maximum fall again. The fermentation is terminated after 120 to 150 hours when the CO2 concentration in the waste gas from the fermenter drops to 0.05% by volume. or lower and a pH of the degree.

The cellulase activity in the culture filtrate represents 9.0 to 9.4 IU FPA / cm ³ on filter paper at the end of the fermentation, which corresponds to a yield of 300 to 310 IU FPA per gram of Heweten 10 Hz wood pulp. Using a known microfiltration and ultrafiltration procedure, the enzyme complex is separated from the other nutrient medium and lyophilized. The lyophilized crude enzyme has the following composition and activity:

protein content 44.5% activity on filter paper (FPA) (IU / mg protein) 1.52 cellobiase (IU / mg protein) 0.034 alpha-amylase (IU / mg protein, 30 ° C) 0.54 xylanase (IU / mg protein) mg protein) 5.8 protease not detectable

The solids separated from the fermentation broth, which consist predominantly of the cell walls of the mold used, are subjected to spray-drying or lyophilization and finally piilverized. Thiocell walls are suitable e.g. as a substrate for the production of cell wall degrading enzymes.

The determination of cellulase activity as well as protein content is made according to methods recommended by ШРАС (T. K. Ghose: "Recommendations on Hie Measurement of Cellulase Activities", prepared by IUPAC, Commission for Biochemistry; July 1983). Determination of amylase activity was made at 30 degrees Celsius for a year; all other enzymatic activities were determined! at 50 ° C; 1 International Unit corresponds to the release of 1 micromole of glucose per minute from the substrate used.

P a 1 .a d 2

T. reesei ZIMET 43 803 is cultured in a fermentor as described in Example 1 to obtain cellulase enzymes. Heweten 10 Hz wood pulp is used as substrate. Fermentation is carried out by the fed-batch technique, in which the additional feeding of the substrate is controlled by the metabolic activity of the fungal mycelium.

The composition of the basic nutrient medium is as follows:

cellulose Heweten 10 Hz 300.0g ammonium sulphate 72.0g potassium dihydrogen phosphate 00.0g magnesium sulphate iheptahydrate 7.5g calcium chloride 7.5g peiptone 45.0g

Tween 80, polyoxyethylsorbate monooleate 15.0 g trace salt solution 150 cm ³ [as in example 1).

These components are dissolved in water, coated, suspended, adjusted to a volume of 10.5 dm ³ and introduced into the fermenter. 1 is used as an inoculum suspension of mycelial tuft of ^3, as described in Example 1. The medium to the addition of substrate was prepared as follows: 1 350 g of cellulose Heweten 10 Hz, and 45 g of carboxymethyl cellulose were dry mixed, and uniformly stirring was suspended in water, with water make up to a total volume of 4,5 dm ³ and sterilize.

fermentation:

Adjustment of the temperature profile and stirrer speed as well as pH control and defoaming are done as in Example 1. The air is adjusted to 420 normal dm ³ air per hour. The feed of the cellulose suspension is started as soon as the concentration of carbon dioxide in the fermentation off-gas exceeds the maximum and drops to about 80% of the maximum value, under the same process parameters. The maximum concentration of carbon dioxide is about 0.7 to 0.9 by volume in the dried fermentation waste gas; the occasional slightly significant maximum concentration of carbon dioxide in the fermenter waste gas does not need to be taken into account in the process control process.

This concentration of carbon dioxide of 80% of the maximum value is set as the control limit at the beginning of the substrate replenishment; when it falls below the control limit, s-a switches on the feed pump, which is fed behind the cellulosic suspension into the fermentation medium until the carbon dioxide concentration exceeds the control limit. The pump feed rate is adjusted to 0.5% by weight of the fermentation medium per hour, at the start of substrate addition, to 53 g / h of cellulosic suspension. The concentration of mycelium proteins in the fermentation broth is determined at the start of substrate addition; the regulatory limit of carbon dioxide adapts to the varying concentration of mycelium proteins, thus increasing proportionally as the concentration of mycelium proteins increases.

Together 4.5 dm ^{3 of a} 30% cellulosic suspension is added to the fermentation medium. The fermentation is terminated after 320 to 360 h when the carbon dioxide concentration in the waste gas decreases to 0.2% or less and the pH rises.

The cellulase activity in the culture filtrate at the end of the fermentation is 32 IU FPA / cm ³ , which corresponds to a yield of 290 IU FPA per gram of Heweten 10 Hz cellulose.

Example 3

For the production of beech wall degrading enzymes, T. reesei ZIMET 43 803 is fermented in a shake culture. The fungal cell walls obtained, for example, are used as a substrate. as described in Example 1. The nutrient medium has the following composition:

dried fungal cell walls 25.0 g / dm ³ potassium dihydrogen phosphate 15.0 g / dm ³ ammonium sulfate 4.8 g / dm ³ magnesium sulfate heptahydrate 0.4 g / dm ³ calcium chloride 0.3 g / dm ³

281811 urea 0.6g / dm peptone 1.5g / dm

Tween 80, polyoxyethylene sorbitan monooleate 1.0g / dm trace salt solution (as in example 1) 10.0cm ³ / dm ³

Shake flasks, e.g. round flasks with a gross volume of 500 cm ³ are filled with 100 cm ^{3 of} nutrient medium and inoculated with conidia at a concentration of 5 x 10 ⁸ / dm ^{3 of} nutrient medium. The cultivation temperature is 30 ° C, the cultivation time is 8 days. The culture filtrate obtained is concentrated by ultrafiltration or dialysis in a ratio of 10: 1. The culture concentrate which can be processed into a dried enzyme product is suitable e.g. for the production of live mold protoplasts.

Example 4

T. reesei ZIMET 43 804 is cultured in a stirrer fermenter as described in Example 1 to obtain cellulase enzymes. The nutrient medium comprises wheat bran and cellulose as inducing substrates in combination with glucose as a non-inducing substrate. Fermentation is performed by a fed-batch technique, wherein the additional addition of the substrate from a regulatory perspective is solved as described in Example 2.

The composition of the basic nutrient medium is as follows:

wheat bran 140.0 g cellulose Heweten 10 Hz 140.0 g glucose 210.0 g ammonium sulfate 67.0 g potassium dihydrogen phosphate 45.0 g magnesium sulfate heptahydrate 5.6 g calcium chloride 5.6 g peptone 21.0 g Tween 80 polyoxyethylene sorbitan monooleate 14.0 g trace salt solution (as in example 1) 140 cm ³

These components are added to the fermenter in 12.6 dm ^{3 of} water. As the inoculum, 2 dm ³ mycelium suspensions are used, which are cultured on a nutrient medium of the same composition as the basic nutrient medium for 24 to 36 hours in a pre-stage (shaker culture or fermenter); the preculture is inoculated with conidia. A 50% glucose solution is used as substrate replenishment medium.

fermentation:

The air is set at 490 normal dm ³ per hour. Moreover, the process parameters are the same as those given in Example 1, coated 2. The glucose solution supply is started as soon as the carbon dioxide concentration drops to about 70 ° / about the maximum value after the maximum is exceeded. The pump feed rate is set to 75 cm ³ / h. The pump, as described in Example 2, is started when the CO2 concentration drops to a value below the control limit, and switches off again when the control limit is exceeded. The control limit of carbon dioxide concentration, as described in Example 2, is adapted to varying concentrations of mycelial proteins.

The concentration of carbon dioxide in the fermentation waste gases has an oscillating course during the refilling of the substrate. Together, 1.4 dm ^{3 of a} 50% glucose solution is added to the fermentation medium. Fermentation is terminated after: 120 to 140 hours when the concentration of the SO 2 decreases to 0% percent or less and the pH value is degrees. The cellulase activity in the culture filtrate at the end of the fermentation was 12.5 IU FPA / cm ³ .

Example 5

T. reesei ZIMET 43 804 is cultured to obtain lactose-based cellulase enzymes 11a. The process is conducted using the fed-batch technique as described in Examples 2 and 4. The composition of the basic nutrient medium is as follows:

lactose 140.0 g ammonium sulfate 67.0 g potassium dihydrogen phosphate 45.0 g calcium chloride 5,6 g magnesium sulfate heptahydrate 5,6 g peptone 21,0 g Tween 80, polyoxyethylene sorbitan monooleate 14,0 g trace salt solution (as in example 1) 140 cm ³

These components are placed in a fermenter in a 12.8 'decimeter ³ water. 1 dm ^{3 of a} mycelium suspension, which is cultured for 35 to 40 hours on nutrient medium of the same composition in shake flasks, is used as the inoculum material; shake culture is inoculated with conidia. A 20% lactose solution is used to replenish the substrate.

fermentation:

The air is adjusted to 420 normal dm ³ air per hour; moreover, the process parameters are set as in Example 1, coating 2. The feeding of the lactose solution is started as soon as the carbon dioxide concentration drops to about 80% of the maximum value. The pump feed rate is set to 100 cm ³ / h. The fermentation was terminated after about 100 to 110 hours when a total of 2.8 dm ^{3 of a} 20% lactose solution was added to the fermentation medium. The cellulase activity in the culture filtrate is 10.5 IU FPA per cm ³ . By means of membrane separation processes, the crude en13 separated and finally lyophilized from the fermentation medium has the following composition, coating activities:

protein content 45.7 o / _o filter paper (FPA) activity (IU / mg protein) and cellobiose (IU / mg protein) 0.035 alpha-amylase (IU / mg protein, 0.50 ° C) 5.6 xylanase body protease

Example 6

The ability of T. reesei ZIMET 43 803 and T. reesei ZIMET 43 804 to produce cellulases is examined by cultivation on different substrates and compared with the parent T. reesei CCI mutant. The nutrient medium has the following composition:

281811

16 dihydrogen potassium 15.0 g / dm ^{1 2 3} ammonium sulfate 1.4 g / dm ³ calcium chloride 0.3 g / dm ³ sulfate heptahydrate magnesium 0.3 g / dm ³ urea 0.3 g / dm ³ peptone 1.5 g / dm ³ Tween 80, polyoxyethylene sorbitan monooleate 1.0 g / dm ³ trace salt solution (as in example 1) 10,0 cm ³ / dm ³ substrate 10.0 g / dm ³

The shake flasks are filled with 100 cm ^{3 of} test nutrient medium and inoculated with 10 ⁷ conidia of one and the same suspension of the respective strain. The cultivation time is 7 days, the cultivation temperature is 30 ° C. Obtaining cellulase activities are obtained:

substrate ZIMET 43 804 ZIMET 43 804 CCI 0,5% wheat bran Ц- 0.5% wood pulp 3.0 2.9 1.5 0.5% glucose -ΊΟ, 5% wood pulp 2.0 2.1 1.0 0.5% lactose + 0.5% wood pulp 1.4 1.8 1.2 5.5% glucose + distillery stillage 1.0 1.3 0.6

(50 o / o)

Cellulase activity is reported in IU FPA / cm ³ .

Possible areas for use relate, for example, to the fruit and vegetable processing industry, the distillery and brewing industries, the extraction of plant substances containing pulp, as well as improving the digestibility of bulk feed and silage feed in agriculture. The enzyme complexes obtained can also be used as biochemicals, for example for the production of protoplasts and for the analytical evaluation of feedstuffs.

Claims (4)

Process for the preparation of cellulase enzymes and / or enzymes for cell wall degradation by fungi in a submerged process, characterized in that the fungal mutants Trichoderma reesei, ZIMET 43 803 and / or Trichoderma reesei ZIMET 43 804 are cultivated at a temperature of 22 to 36 ° C at pH 2 to 7, under conditions known for the cultivation of Trichoderma strains, in a nutrient medium containing one or more substrates inducing the production of cellulase enzymes and / or cell wall breakdown enzymes, either singly or in combination with one or more thereof, non-inducing substrates. 2. The process according to claim 1, wherein the inducing substrates are either soluble, such as lactose or whey, or insoluble, such as cellulose, cellulosic waste, or fungal cell walls. 3. A process according to Claims 1 and 2, characterized in that soluble inducing substrates and / or soluble non-inducing substrates are used which are added to the medium by the fed-batch technique such that the added substrates induce formation in the ferrnentation medium. enzymes or at least do not suppress it. _A * 4. A process according to claims 1 to 3, characterized in that bran or distillery stillage is used as an inducing substrate in combination with soluble carbohydrates such as glucose, xylose, lactose or whey.