CS233604B1 - Trichoderma reesei strain CCF 1853 - Google Patents

Abstract

The subject of the invention is the preparation and obtaining of a new strain Trichoderma reesei 139, which is stored in the Collection of Fungal Cultures of the Department of Botany of Lower Plants of Charles University in Prague, Czechoslovakia, registration number CCF-1853 and in the Central Collection of Industrial Microorganisms of the All-Union Scientific Research Institute of Genetics and Selection of Industrial Microorganisms in Moscow, USSR, registration number C»PM F-170. i ; The main advantage of this new strain is the increased production of the cellulolytic system. The strain Trichoderma reesei 139 is applicable mainly in the food industry, for example in the degradation of various cellulose materials and in the feed industry

Description

The invention belongs to the field of microbiological industry, in particular it relates to enzyme production strains, namely the strain Trichoderma reesei (viride), which synthesizes enzymes of the cellulolytic complex. Cellulolytic enzymes produced on the basis of microbiological synthesis are widely used in agriculture, especially in animal husbandry for ensiling and improving the nutritional value of roughage and straw. The use of cellulases in the production of ethyl alcohol and technical glucose from cheap materials and agricultural waste containing cellulose is very promising. In the food industry, cellulase preparations are used to degrade the cellulose components of the processed raw material.

In well-known copyright certificates and patents, various types of bacteria, fungi and actinomycetes are used as cellulases producers. Among the fungal cultures, Geotrichum candidum, Thielavia terrestris, Myrothecium verrucaria, Trichoderma longibrachiatum, Penicillium iriensis and a whole range of strains of Trichoderma reesei (viride) and VNII genetics B7, 44, QM 9123 are known.

QM 9414, MCU 77 and others (USSR copyright certificates No. 368303, 577230, 591502, 745933, USA patents No. 3734831, 3764475,

3972775, German Patent No. 2133544, Great Britain Patent No. 1546544, International Application WO 80/01080.

In the USSR copyright certificate No. 591502, the production strain of cellulolytic enzymes Trichoderma viride VNII genetics B7 (according to the new nomenclature Trichoderma reesei) is described. The strain VNII genetics B7 has the following characteristic features: after 4 days of growth on agar media, it forms compact colonies with a diameter of 20-35 mm. Spore formation is very good. Conidia are yellow-beige in color, oval of two types - large (length 6.3 yu, width 4.3 yu) ^and small (length 4.54 yu, width 3.43 yu). Strain B7 completely liquefies from

233 B04 gelatin after 15-20 days of growth, peptonizes milk after 7-10 days of growth. Does not reduce nitrates even after 30 days of incubation. *

When cultivated on soil with 4% beet cuttings, the activity of eelulhs strain B7 based on hydrolysis of filter paper, dewaxed cotton and sodium carboxymethyl cellulose was:

hydrolysis of filter paper - 13-15 mg/ml (1.2-1.4 IU/ml), hydrolysis of dewaxed cotton - 200-250 mg/ml (0.8-0.9 IU/ml), hydrolysis of Na-KMC - 150-200 mg/ml (27.75-37.0 IU/ml).

The activity was determined according to Mandels-Weber Cellulases and their Applications”, Adv.Chem. Ser. 95,391,

1969. IU is the international unit of activity, equal to 1 uMol glucose/min.

Based on the largest number of characters (type of production strain, composition of the nutrient medium for enzyme biosynthesis) and the achieved effect, the Trichoderma reesei(viride) VNII genetics B7 strain, which is stored in the Central Collection of Industrial Microorganisms, with registration number CMBM F-79, can be considered a prototype.

The disadvantage of the prototype strain is the relatively low activity of the cellulolytic enzyme complex in the hydrolysis of Na-KMC, which limits the effectiveness of the use of preparations prepared by fermentation of the mentioned strain for silage of straw and roughage.

In the international application WO-80/01080, a new strain Trichoderma reesei MCU 77 is proposed, which, when cultivated on a medium containing 1% cellulose, peptone and urea, has cellulolytic activity: hydrolysis of filter paper - 1.75 IU/ml, hydrolysis of Na-KMC - 35.0 IU/ml.

Based on the achieved effect and the simultaneous high activity of the two types, strain MCU 77 can be considered a basic model. The disadvantage of this strain is the relatively low activity in the hydrolysis of Na-KMC.

The strain Trichoderma viride 44 is also known (author's research3

233 804 USSR No. 745933), in which the production of cellulases hydrolyzing filter paper is 22.0-25.0 mg/ml glucose (2.03-2.30 lU/ml). However, the description does not state the conditions under which the stated activities are achieved, nor does it state the activity of cellulases hydrolyzing Na-KMC. Due to this, it is not possible to compare this strain with our strain.

The aim of the proposed work is to obtain a new high-production strain of Trichoderma reesei, which has a higher activity of enzymes of the cellulolytic complex, especially cellulases hydrolyzing Na-KMC.

The set goal was achieved by creating a new strain of Trichoderma reesei, which is a mutant obtained by multi-stage selection using mutagenic factors (accelerated electrons) from the VNII genetics B7 strain.

The presented strain Trichoderma reesei 139 differs significantly from the prototype strain B7 both in activity and in a number of cultivation and morphological features.

Unlike the prototypes, strain 139 grows more slowly on agar media, colony growth and conidia formation in strain 139 are terminated after 7 days of incubation and in strain B7 after 4 days of incubation. The aerial mycelium is filamentous. Spore formation is scanty. Conidia are spherical or slightly oval, of one type in size and considerably smaller than in strain B7 (length - 3.7 Ái, width - 3.3 _v /u). Unlike strain B7, strain 139 does not coagulate milk and poorly assimilates nitrates.

With a high overall activity of cellulases of all types, the strain Trichoderma reesei differs in the targeted synthesis of cellulases actively hydrolyzing Na-KMC. When cultivated on nutrient medium with beet cuttings, the activity of cellulases produced by the strain is: hydrolysis of filter paper - 14-16 mg/ml (1.3-1.48 lU/ml)

233 804 Hydrolysis of Dewaxed Cotton - 200-250 mg/ml (0.8-0.9 IU/ml) Hydrolysis of Na-KMC :

according to the USSR methodology -200-250 mg/ml (37.0-46.3 IU/ml) according to the Czechoslovakia methodology - 140-160 mg/ml.

When cultivated on model soil with microcrystalline beech cellulose, the following cellulolytic activity was observed: hydrolysis of filter paper - 10-12 mg/ml (0.93-1.1 IU/ml) hydrolysis of dewaxed cotton - 50-60 mg/ml (0.19-0.23 IU/ml) hydrolysis of Na-KGM:

according to the USSR methodology - 240-250 mg/ml (44.4-46.3 IU/ml) according to the Czechoslovakia methodology - 120-150 mg/ml.

Under analogous conditions, the activity of the strain - prototype B7 (control on microcrystalline beech cellulose) is: hydrolysis of filter paper - 10-12 mg/ml (0.93-1.1 IU/ml) hydrolysis of dewaxed cotton - 45-50 mg/ml (0.17-0.21 IU/ml) hydrolysis of Na-KMC:

according to the USSR methodology - 220-250 mg/ml (40.7-46.3 IU/ml) according to the Czechoslovakia methodology - 100-130 mg/ml.

The Trichoderma reesei strain thus presented, when cultivated on fermentation media of various compositions, provides, in comparison with the starting strain B7, an increase in the activity of the cellulolytic enzyme complex in the hydrolysis of sodium carboxymethyl cellulose by an average of 20-30% and in the hydrolysis of insoluble substrates by 10-20%. cep 44^3

The use of the Trichoderma reesei strain in the industrial production of cellulolytic preparations allows for the intensification of the production process and increases the efficiency of the use of the said preparations in the feed and food industries in the degradation of cellulolytic components of a wide range of products.

233,804

Cer Ή583

Characteristics of the Trichoderma reesei strain, culti* sac morphological characteristics.

Synthetic soil Czapek-Dox is sucrose-dried in 7 days of growth, the diameter of the colony is 40-45 mm, the colony has an uneven edge. Colony flat. Aerial mycelium filamentous, dull white. Spore formation poorer. The soil is not colored.

Synthetic soil according to Pontecorvo: after 7 days of growth, the colony diameter is 30-35 mm. The colony is straight. The aerial mycelium is long-filamentous to filamentous, lemon-yellow in color. The conidia are light yellowish to beige in color, forming in the center of the colony. The soil is colored bright yellow.

Synthetic medium with starch: colonies with a flat surface, chem. Aerial mycelium filamentous. Conidia of light beige color, forming in the center of the colony. Medium colored yellow.

- Wort agar: after 7 days of growth, the average colony size is

50-55 mm. Colony raised, center of colony slightly sunken. Aerial mycelium filamentous to cottony. Conidia light beige nut-colored, spreading over the surface of the colony, usually also along its edges. Soil is light yellowish.

Raulin-Toma nutrient agar with the addition of 1% corn extract: after 7 days of growth, the colony diameter is 25-30 mm. The surface of the colony is smooth. The aerial mycelium is short-filamentous. Light beige spores are formed in the center of the colony. The soil is not colored.

Size and shape of conidia: when cultivated on agar media, conidia are spherical to oval in shape. One type is *-sized - length 3.6-3.8, and width 3.2-3.4 A.

Physiological-biochemical characteristics: gelatin liquefies in three weeks of growth. Does not coagulate milk. Hydrolyzes starch. Poorly assimilates nitrates. Well assimilates glucose, sucrose, maltose, lactose, cellobiose, mannose, xylose, arabinose, fructose, gly233 804 c-erin. Weakerly galactose, rhamnose.

Under submerged fermentation conditions, it synthesizes a complex of cellulolytic enzymes characterized by high activity in the hydrolysis of sodium carboxymethyl cellulose. ccp

Examples of the use of the Trichoderma reesei strain for the preparation of cellulolytic enzymes.

To perform and obtain the vaccine, strain 139 is cultivated on wort agar slants for 7 days at a temperature of 28-30°C,

Example I.

To perform submerged fermentation, a liquid nutrient medium of the following composition (wt%) is used:

beet pulp (dried) - 4.0 (NH ₄ ) ₂ S0 ₄ - 0.14

-kh ₂ after ₄ - 0.10

MgSO ₄ - 0.03

CaCl ₂ -0.03 urea - 0.03 microelements: (mg/ml soil)

PeS0 ₄ . 7H ₂ O -'1.0

ZnCl ₂ - 0.8

MnS0 ₄ . H ₂ 0 - 0.5

CoCl ₂ . 6H ₂ 0 - 0.5 (NH ₄ ) ₆ . 4H ₂ 0 . Mo ₇ 0 ₂₄ - 10.0 distilled water pH - 4.2-4.7

Submerged fermentation is carried out in 750 ml culture flasks, filled with 100 ml of nutrient medium, under aerobic conditions. Optimal incubation temperature 28-30°C.

The fermentation medium is inoculated with an aqueous suspension of spores in the amount of 6-8x10^ conidia per 100 ml of medium.

233 604, Cellulolytic activity of culture medium:

hydrolysis of filter paper - 14-16 mg/ml (1.3-1.48 IU/ml) hydrolysis of dewaxed cotton - 200-250 mg/ml (0.8-0.9 IU/ml) hydrolysis of Na-KMC:

according to the USSR methodology - 200-250 mg/ml (37.0-46.3 IU/ml) according to the Czechoslovakian methodology - 140-160 mg/ml Example II.

To perform submerged fermentation, a liquid medium with the following composition (wt%) is used:

microcrystalline beech cellulose .2.0 wheat bran extract 1.0 (NH ₄ ) ₂ S0 ₄ 0.3

NaON ₃ 0.3 kh ₂ po ₄ 1.0

MgS0 ₄ 0.05 distilled water. pH of nutrient medium 6.2

For inoculation of the nutrient medium, a water suspension of spores (10? conidia per 100 ml of medium) or vegetative mycelium grown submerged for 24 hours at a temperature of 28-30°C on medium of the following composition (wt. in %) is used:

sucrose 2.0 kh ₂ after ₄ 0.2 (nh ₄ ) ₂ so ₄ 0.2 MgSO ₄ 0.03 CaCl ₂ 0.03 ' microelements in quantity, given in example I. distilled water pH 4.5 - 4.9 Submerged fermentation is carried out in cultivation

500 ml bottles, filled with 100 ml of nutrient medium, for circulation and

233 604 shaker (240 rpm) at a temperature of 28 ax 50°C. Amount of vegetative inoculum - 10 ml per 100 ml of soil.

After six to seven days of submerged cultivation, the cellulolytic activity in the culture medium is:

hydrolysis of filter paper 10-12 mg/ml (0.95-1.1 HJ/ml) hydrolysis of dewaxed cotton 50-60 mg/ml (0.19-0^.25 IU/ml) hydrolysis Na KMC according to USSR methodology 240-250 mg/ml (44.4-46.5 IU/ml) according to Czechoslovakia methodology 120-150 mg/ml

The workflow by which the new strain Trichoderma reesei 159 was prepared consists of several operations:

a) Preparation of a basic sterile spore suspension of Trichoderma reesei.

A well-grown culture of the microorganism on wort agar is layered with 10 ml of sterile Tween 80 solution, mixed in a homogenizer and filtered under sterile conditions through a cotton filter. The filtered spore suspension is then further diluted with sterile Tween 80 solution so that the number of spores in 1 ml is approximately 1.2 - 1.6 x 1C^«

b) Irradiation of Trichoderma reesei spore suspension.

The basic spore suspension of the aforementioned microorganism, thus prepared, is pipetted sterilely into small sterile Petri dishes in an amount of 2.5 - 5.0 ml and inserted into the irradiation space.

A high-frequency linear accelerator of the Tesla VUVET type is used for irradiation. The irradiation dose is 50 Krad. The spore suspension in the Petri dishes is removed from the irradiation space immediately after irradiation.

c) Isolation of individual mutants of the said microorganism.

After irradiation, the basic spore suspension is further diluted with sterile O 7 / Tween 80 solution to approximately 10-10 spores per 1 ml. The spore suspension of the aforementioned microorganism, diluted in this way, is inoculated onto wort agar in Petri dishes, 0.1-0.2 ml for the virus and 0.1 ml for the control.

233 604 trolls by spreading them over the surface of the Petri dishes. The inoculated Petri dishes are then placed in a thermostat at 29 ex 50°C for 60 or 72 hours. After this time, individual monocolonies are isolated and re-inoculated onto wort agar slants. After seven days of cultivation, the cellulase activity and morphological properties of the individual isolated mutants are determined.

The strain Trichoderma reesei 139 prepared by successively quintuple irradiation with accelerated electrons is deposited in the Collection of Fungal Cultures of the Department of Botany of Charles University in Prague, Czechoslovakia, registration number CCF 1855 and in the Central Collection of Industrial Microorganisms of the All-Union Scientific Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, USSR, registration number CMPMF 170.

Claims (1)

SUBJECT NOW / CLIMB Trichoderma reesei strain, CCF 1853 ^ producer of cellulosic enzymes · Printed by Moravian Printing Works,