SU1509402A1 - Mushroom strain penicillium canescens - producer of beta-galactosidase - Google Patents

Abstract

The invention relates to biotechnology, in particular, to the preparation of a new producer of the β-galactosidase enzyme. The purpose of the invention is the detection of β-galactosidase strain producer, which accelerates the process of obtaining the enzyme and increases the biosynthesis of the enzyme. The invention allows to obtain β - galactosidase in the amount of 55-60 units / ml of culture fluid. From the in-depth culture of the proposed producer, the isolated enzyme preparation can be applied in the dairy and bakery industries to improve the quality of the finished product, as well as in medicine for intolerance to dairy sugar-lactose.

Description

The invention relates to the enzyme industry, in particular to the preparation of a new strain, the producer of the enzyme p-galactosidase, used to hydrolyze milk sugar, lactose, and is widely used in various fields of the food industry.

The purpose of the invention is to identify the producer strain of β-galactosidase, ensuring that the process of obtaining the enzyme is accelerated and the biosynthesis of 3-galactosidase is increased.

The proposed Penicilliuta canescens strain of VKPK F-376 (TK-85) is obtained by irradiation with ultraviolet rays at a dose of 200 Dk / m conidia of the original Penicillium strain.

canescens Sopp 20171 and has the specified properties.

Morphological signs. The strain is toxic ,. fast-growing, characterized by moderate sporulation on agarized media (much less than in the original culture, characterized as a rule, by abundant sporulation).

Conidia formation begins two days later than in the original culture. Objectives well developed, septic. .S.

Conidiophores 450–460 µm in length and 3.0–3.5 µm in diameter deviate from the felt mycelium; the shell is slightly rough. The tassels are double-haired, spread wide; they consist of twigs (metul) carrying the whorls finished, me

3150

thuli 13-15 X 2.0-2.8 microns, sterigms (fialida) 7-8 x 2.5 microns. Conidia 2.0-2.5 microns in diameter, spherical, formed in chains,

Cultural characteristics, the strain grows well on artificial media (mash agar, potato glucose agar, potato agar) and synthetic agar media (Czapek, Saburo medium, Rolen-Tom, agar medium with lactose),

On the апapek medium, colonies of a pale blue-gray color with moderate sporulation, on the reverse side of a yellowish-straw color, on days 12-15, acquire from light to dark brown.

Linear growth of colonies on agar media on days: Czapek’s medium: 5th day 48 mm, 10th day 70 mm, Wednesday Rolen Tom: 5th day 35 mm, 10th day 60 mm, potato-glucose Wednesday: 5th day 40 mm, 10th day 55 mm, Wednesday Saburo: 5th day 30 mm, 10th day 50mm, agarized - on Wednesday with lactose: 5th day 38 mm 10th day 60 mm

The cut of x grows well. Relation to carbon nutrition: it assimilates lactose, sucrose, glucose, galactor, maltose, fructose, arabinose, starch, inulin, sorbitol, mannitidr.

Relation to nitrogen nutrition. Well absorbs nitrate nitrogen, ammonium is better, from organic nitrogen sources assimilates peptone, urea, corn extract, etc.

Attitude to oxygen, Obligatny aerobes.

Relation to temperature, Mesophil, optimum growth temperature 28 -,

Relation to the acidity of the medium.

Deep cultivation of the fungus is carried out on a liquid nutrient medium, of the following composition, wt.%: Soya

It grows in a wide range of pH 2.0-9.0, 45 - у 3 3 3.0} Na, HPO. П2Н ,, 0. 1.5,

When growing PenicilUum satt (NH4) .SO .. OD; KS1 O., 05i I-IgSQ 0.015, nescens TK-85 on complex media, water the rest, initial pH 4; 2, containing soy flour, the activity of 5-galactosidase in the culture fluid reaches 55-60 u / ml ,.

For breeding the strain, a modified Czapek with lactose medium of the following composition is used, wt.%: Lactose - 3.0; 0.2. 0.1; 1-IgSO-0.05; KC1 0.05, corn extract 0.5; water the rest, the original pH of 5.8-6.0,

The cultivation is carried out in 750 ml flasks with a thermostatic 50 rocking chair, which makes 220 rpm (aeration level 1.4 g) for 72 hours.

Sowing the nutrient medium was carried out 2% suspension of conidia of the surface culture of the fungus grown in test tubes on beveled lactose agar for 12 days at

The nutrient medium for fermentation is the following medium.

Tava, wt.%: Soybean flour 3.0, X 1.5; (# 4) 2.30 0.2; KC1 0.05, llgSO 0.015, water the rest,

initial pH of 4.1-4.2,

The activity of the biosynthesized p-galactosidase strain was determined by the method of Kivu and Lardy,

In addition to highly active β-galactosidase, the proposed producer forms a protein-rich biomass that is not inferior. according to the activity of the known strain E, co11, the activity curve of / 3-galactosidase, equal to 60 units / mp (for lactose),

and specific act) dvosti 5.5 u / mg protein, mutant Penicillium canescens TK-85 in the culture filtrate contains. the activity of other glycosidases: invertases 12–14 units / ml, t (galactoscopy

3-4 U / MP, β-glucosidase 1.5-2 U / ml, K 1 each of these activities is rather high and indicates that mutant P, canescens TKT 85 is - with an active producer not only

p-galactosidase, but also of the glycosidase complex, which significantly expands the spectrum of enzyme use (saccharification of a large number of substrates, for example, in non-alcoholic

industrialization)

The proposed mutant is not toxic or pathogenic, cultivation and use experiments have been carried out on a semi-further scale.

Example 1, the Strain Penic.il lium canescens TK-85 support on agar medium with lactose, containing wt.%: Lactose 3.0, O ,, 0.1, KC1 0.1, -lIgS04j 0.05, Ky kurus extract 0.05, agar 2, water the rest, - the initial pH of the medium is 6.0,

Deep cultivation of the fungus is carried out on a liquid nutrient medium, of the following composition, wt.%: Soya

Ack 3.0} Na, HPO. П2Н ,, 0. 1.5,

(NH4) .SO .. OD; KC1 O., 05i I-IgSQ 0.015, water the rest, initial pH 4; 2,

The cultivation is carried out in 750 ml flasks with a thermostatted rocking chair, which makes 220 rpm (aeration level 1.4 g) for 72 hours.

Sowing the nutrient medium was carried out 2% suspension of conidia of the surface culture of the fungus grown in test tubes on beveled lactose agar for 12 days at

The biomass is separated by filtration and the activity is determined in the filtrate 5 -1509402

galactosidase, used as the activity of the biosynthesized strain of the substrate ortonitrophenyl-B-galactic p) -galactosidase is 60 units / ml, topyranoside. The reaction mixture, which is 10,000 units / g of the drug, is made up of 0.8 ml of phosphate-citrate s. Thus, it forms a buffer of pH 4.2, containing 125 mM, and has an activity of 60 units / ml. substrate and 0.02 ml of the enzyme solution. The proposed enzyme can be added (culture liquid), inc-. It is changed in food industry and boil at 30 ° C for 15 minutes, in medicine.

1 ml of 1 M is then added to the invention for stopping the reaction. The released strain of the fungus Penicillium canescens. o-NITROPHENOL is determined at L 420 nm. VKPM F-376 - producer of galactosyases.

Claims (1)

Claim The strain of the fungus Penicillium canescens VKPM F-376 - producer / 3-galactosidase.