SU1738854A1 - Strain of fungum penicillium canescens - a producer of @@@-galactosidase - Google Patents

Abstract

Uses: biotechnologists. Summary of Invention: F-436 strain was obtained from Penlclllium canescens 20171 strain (VKPM F-178) by multistep selection using mutagenic factors and selection under selective conditions. Under subsurface cultivation on a nutrient medium containing 3% beet pulp, 5% protein-vitamin concentrate and 2.5% mono-potassium phosphate, in 120-144 hours of growth at 30 ° C, VKPM F-436 strain accumulates in the culture extracellular fluids of ft-galactosidase, whose activity is: 100-130 units / ml (hydrolysis of ONPH at 30 ° C), 140-160 units / ml (hydrolysis of ONPH at 37 ° C), 62-68 units / ml (hydrolysis of lactose at 30 ° C). The strain VKPM F-436 differs from the known producers of the same toxonomic group in a number of cultural, morphological, and physiological and biochemical characteristics. 2 tab. Yo

Description

The invention relates to the microbiological industry, in particular, to the production of enzymes, and interchangeably to a strain — producer of extracellular f, galactosidase.

The enzyme ft-galactosidase, obtained on the basis of microbiological synthesis, is used in a number of sectors of the food industry for the hydrolysis of lactose, and is also used in medicine and the pharmacological industry.

Various types of bacteria, actinomycetes, yeast and filamentous fungi are known as producers of ft-galactosidase. The last group of microorganisms is most promising for industrial use, since ft-galactosidase produced by fungi shows its activity at low pH values, which corresponds to the conditions of its use.

PaecMomyces varlotl mushroom producers are also known. Sclerotium sp. Cortlcium rolfsil, Chaetomella raphigera, Alternaria temnls, Culrularla Inaqualis. several types of aspergillus: A. oryzae, A. nlger, A. foetldus, A. awamorl, and also a number of types of penicilli: Penlclllium terel, P. cltrlnum, P. canescens, P. multicolor.

The disadvantage of these producers of ft-galactosidase is the low activity of the strains,

A known strain of Peniclllium canescens 20171 (VKPM F-178), isolated from VI

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in the course of growth on a liquid nutrient medium with soy flour and mineral salts to produce extracellular ft -galactosidase in the amount of 18-20 units / ml of culture fluid. The enzyme activity was determined by hydrolysis of 0-nitrophenyl-B-D-galactopyranoside (ONPH) at 30 ° C for 15 minutes.

Methods are known for increasing the productivity of strain 20171 when it is cultivated on media of a different composition, in particular, it proposes a medium containing beet pulp and protein-vitamin concentrate (B C K), on which the productivity of strain 20171 reaches 96 units / ml in 96 hours of growth. Replacing beet pulp with pumpkin or tomato pomace in the medium allows increasing the strain activity up to 65-70 units / ml.

The disadvantage of strain 20171 is its low level of activity.

The producer of ft-galactosidase Penictlllum multicolor KU-0-132 (PERM-P4375, ATCC 20539) is known, which, under conditions of deep fermentation, grows on a nutrient medium containing rice bran and flour from cotton seeds as the main components, as well as mineral salts, 140 units / ml of ft-galactosidase (enzyme activity was determined by hydrolysis of ONPH at 37 ° C).

The strain P. multicolor KU-0-132 among the known mushroom producers possesses the highest activity of ft -galactosidase, however, it is achieved by cultivating the strain on medium with cotton seed flour, which is difficult to obtain raw material, and appears higher reaction temperature determination (37 ° C).

Also known is the P. canescens strain TK-85, which is a mutant obtained under the action of ultraviolet rays from strain 20171. Strain TK-85 is characterized by rapid growth on agarium-vaine media and weak delayed conidia, m. When cultivated on a liquid nutrient medium with soybean flour and mineral salts for 72 hours, the activity of the extracellular / -galactosidase of strain TK-85 is 60 units / ml of culture fluid (hydrolysis of ONPH at 30 ° C).

The disadvantage of the strain P. canescens TK - 85 is the low level of synthesis of ft-ga-lachozidase.

Closest to the proposed set of features (type of producer

the common origin, the ability to produce the enzyme during growth on media, with available non-edible raw materials) is the strain Penlcllllum canescens 20171 (VKPM

F-178). The strain F-178, when cultivated on media with beet pulp and BVK, with pumpkin or tomato pomace and yeast, forms respectively 42 or 65-70 units / ml of ft -galactosidase.

A disadvantage of the known strain F-178 is the low level of activity.

The purpose of the invention is to increase the level of accumulation of ft-galactosidase in the culture fluid.

The goal is achieved by creating a strain of Penicllllum canescens DG-86/18. A new strain of Peniclllium canescens DG-86/18 with growth on liquid nutrient

the medium containing beet pulp and protein-vitamin concentrate, at 30 ° C for 120-144 h, accumulates in the culture fluid ft -galactosidase with an activity of 134 units / ml when determined by

hydrolyzing ONPH at 30 ° C, which is 160 units / ml when determined by the same method at 37 ° C.

The proposed strain Penicilllum canescens DG-86/18 obtained as a result

multi-stage selection using mutagenic factors and selection on media with 2-deoxyglucose from the natural isolate - strain Penlcllllum canescens 20171 (VKPM F-178). The strain of P. canescens DG-86/18 deposited in the All-Union Collection of Industrial Microorganisms of the Institute VNIIgenetika number VKPM F-436. The strain DG-86/18 differs from the known producers with a high level of accumulation of ft-galactosidase with growth on a liquid nutrient medium with non-food raw materials, as well as a number of cultural-morphological and physiological-biochemical characteristics.

Cultural and morphological features.

Wort agar (10 days of growth, 29 ° C). The diameter of the colonies is 7.5-7.8 cm. The surface and edge of the colonies are smooth. Aerial mycelium

white, tightly woven around the center, fluffy closer to the periphery of the colonies. Conidium formation in the center of the colonies is scarce and more abundant on the periphery of the colonies. Conidia are gray-blue. The reverse side of the colonies is dark brown.

Rowlen-Toma agar medium with sucrose and the addition of corn extract (10 days of growth, 29 ° C). The diameter of the colonies is 6.2-6.5 cm. The edge of the colonies is smooth. Aerial mycelium in the center of colonies fluffy,

further creeping. Conidiene formation is moderate. Conidia are light gray with a bluish tinge, blue along the edge of the colonies. Formed uniformly over the entire surface of the colonies. The reverse side of the colonies is folded, dark brown.

The agar medium of čapek with glucose and the addition of corn extract (10 days of growth. 29 ° C). The diameter of the colonies is 5.0-6.0 cm. The aerial mycelium is fluffy, sometimes rising. The edge of the colonies is smooth. Conidiene formation, except kra, is weak. Conidia are pale yellow in color, blue along the edge of the colonies. The reverse side of the colonies is weakly folded, dark brown.

Rowlen-Tom agar medium with sucrose (10 days of growth, 29 ° С). The diameter of the colonies is 4.0-4.5 cm. The edge of the colonies is smooth. Aerial mycelium felt. Conidiene scanty. Cream-colored conidia, blue at the edge of colonies. The reverse side of the colonies is weakly folded, light yellow.

Agar medium of čapek with glucose (10 days of growth, 29 ° С). The diameter of the colonies is 1.8-2.0 cm. The edge of the colonies is uneven, lobed. The surface of the colonies is pypukla, smooth. Aerial mycelium is white, tightly intertwined. Conidia are dark gray-blue in color and are formed mainly along the edge of the colonies. The reverse side of the colonies is not folded or has poorly marked large folds, not colored.

Physiological and biochemical signs.

Mesophilic, grows at 25-37 ° C, optimal growth temperature of 29 ° C. Gelatinu dilutes weakly, the milk does not completely coagulate up to 30 days of incubation. Nitrates restores completely by 10 days of growth.

Relation to carbon sources. Well absorbs mono-, di- and polysaccharides, including glycerin and starch. With growth on glucose, fructose, sucrose, maltose, mannose, glycerin forms a bright yellow or brown pigment.

Relation to nitrogen sources. Well absorbs ammonium nitrogen, peptone, urea, corn extract.

Genetic features (auxotrophicity) - prototroph. Strain non-pathogenic.

Comparison of cultural-morphological and physiological-biochemical characteristics of the new strain of Peniclllium canescens DG-86/18 and the well-known strain of Penlclllium canescens 20171 is given in Table. one.

When cultivated on a medium with beet pulp, BVK and KH2PQ4 in deep conditions, 120-144 h of growth, the strain

DG-86/18 accumulates in the culture fluid (3) galactosidase, the activity of which is 100-130 units / ml (hydrolysis of ONPH at 30 ° C) or 140-160 units / ml (hydrolysis of ONPH at 37 ° C).

Along with ft-galactosidase, the strain of R. canescens DH-86/18, when cultured on the above medium, synthesizes a number of other extracellular glycosidases with activities, units / ml:

Carboxymethylcellulase 32-35

Cellobiase 5-9

a-amylase2-3

Glucoamylase 0.7-1.2

Example. To obtain the initial seed, the culture of the strain DG-86/18 and the well-known strain 20171 is grown on Rowlen-Thom beveled agar medium with the addition of 1% corn extract W / V) at 28-30 ° C for 4-5 days, after which it is kept at room temperature for 2-3 days.

The sowing of the fermentation medium is carried out either with an aqueous suspension of conidia containing (2-5) xH6 conidia / ml, or with a 1 cm culture slice on a slanted agar.

The fermentation process is carried out in 750-ml Erlenmeyer flasks containing 50 ml of a liquid nutrient medium of the following composition, wt%:

beet pulp 3,0

Protein-Vitamin

concentrate5.0

Phosphate monosubstituted potassium2,5

Tap water

pH after

sterilization natural.

The nutrient medium is inoculated with one of the above methods and incubated at 30 ° C on a circular rocking chair rotating at a speed of 240-250 rpm.

The obtained culture liquid was centrifuged to separate the mycelium and solid residues of the medium. The activity of / 3-galactosidase is determined in the supernatant.

The evaluation of the activity of ft-galactosidase is carried out using a conventional method using ONFG as a substrate.

The reaction mixture, containing 1.5 mg of ONPH in 4 ml of 0.15 M citrate-phosphate buffer, pH 4.2, and 1 ml of the supernatant at the appropriate dilution, is incubated for 15 min at 30 or 37 ° C, then to 1 ml of the reaction 1 ml of 1M sodium carbonate solution and 1 ml of distilled water are added to the mixture.

The color intensity of the resulting 0-nitrophenol solution is determined at 420 nm.

The unit of activity of ft-galactosidase is the amount of enzyme that releases 1 µmol of 0-nitrophenol from the substrate per 1 minute under the given reaction conditions.

In tab. Table 2 shows the levels of activity of β-galactosidase after 120 and 144 hours of cultivation under equal conditions of the strains of Penlcillium canescens DG-86/18 and 20171 at a reaction temperature of determination of 30 ° C.

The mean activities of ft-galactosidase of the same culture fluids of the strains DG-86/18 and 20171 under the conditions of determination

at 30 ° C, respectively, 134.0

and 87.0 units / ml, and at 37 ° С 160.0 and 98.0 units / ml.

The above activity levels

/ -galactosidase of the proposed strain

Penlcilllum canescens DG-86/18 (VKPM F-436) and the well-known strain Penlcilllum canescens 20171 (VKPM F-178) show that F-436 strain is more active and more economical since it provides in one process the output of the enzyme at the stage cultivation is 1.5 times higher than the known strain F-178, which. in turn, determines the corresponding savings in raw materials, energy and

Claims (1)

labor resources in production. Claims of the invention The strain of the fungus Penlcilllum canescens VKPM F-436 is a producer of ft -galactosidase. Wednesday Strain DG-86/18 (F-436) Czapek with glucose growth with different ugvo Riven milk by 20% with sodium nitrate The diameter of the colonies 18-20 mm; colonies are convex, smooth; aerial mycelium white, tightly intertwined; the edge of the colonies is uneven; conidia of dark gray-blue color, located mainly along the edge of the colonies; the reverse side of the colonies is smooth or slightly folded, not colored Well absorbs mono-di - and polysaccharides, including glycerin, starch; during growth on glycerol, fructose, sucrose, glucose, maltose, mannose forms a yellow or brown pigment Does not completely coagulate up to 30 days of growth Thins weakly Nitrates restores completely by 10 days of growth Table 1 1 j Strain 20171 (F-178) The diameter of the colonies 36-38 mm; the aerial mycelium in the center is fluffy, then creeping; the edge of the colonies is smooth; conidiopathy abundant; conidia are gray, dark blue along the edge of colonies; the reverse side of the colonies is folded, not colored Well absorbs mono-di - and polysaccharides, including glycerin, starch, with growth on glycerin, fructose, sucrose forms a yellow pigment Coagulates completely by 12 days of growth Thins weakly Nitrates restores completely by 25 days of growth table 2