SU1068477A1 - Method for preparing complex of enzymes - Google Patents

Description

The invention relates to the enzyme industry of microbiological prohottOimeHHOCTjH, namely to methods for producing enzymes, and can be used in the production of enzymes, and can be used in the production of enzymes and other biologically active substances used in the wine-making, canning, pharmaceutical, chemical industry , as well as in rural households ..

Known methods are the production of enzymes, in particular cellulose, pectinases of monophenol monooxygenases and peroxidases, which provide for the cultivation of their producers, for example, mold fungi, yeast, higher basidiomycetes, etc., in a nutrient medium containing water, mineral salts and organic additives, followed by organic fungi, yeast, higher basidiomycetes and others in a nutrient medium containing water, mineral salts and organic additives the culture fluid from the mycelium and the remnants of a solid substrate. Organic additives can include carbon sources, nitrogen sources, compounds that stimulate the biosynthesis of individual enzymes, for example, straw, sawdust, cotton, filter paper, beet pulp, beet pulp, extracts from beet pulp, grape marc, malt. out sprouts, etc. 1.

A disadvantage of the known methods is that they allow us to utilize the biosynthesis of individual enzymes, however, for effective hydrolysis of complex substrates, for example, in the food industry, in the processing of forest wastes, woodworking, industry, and agriculture. Others require the use of enzyme complexes, including simultaneously high-activity celluloses, pectinases, monophenol monooxygenase and peroxidase.

The closest to the technical essence and the achieved positive effect to the proposed is a method of obtaining a complex of enzymes by cultivating higher basidiomycetes on a nutrient medium containing a carbon source, mineral salts: single potassium phosphate, dvuzame, wheezed potassium phosphate, one unsubstituted; ammonium phosphate, magnesium sulfate, and water, followed by separation of the filtrate of the culture fluid from the mycelium and residues of the solid substrate, according to which, along with the higher basidiomycete, it is cool. A lower mushroom is eaten, for example, Pleurotus astreatus and .Fusarium tulmorum 2.

The disadvantages of this method are the high cost and technological complexity of growing

at the same time two cultures, hence the high consumption of inoculum, as well as insufficiently wide range of enzymes.

The aim of the invention is expansion. assortment of enzymes in the complex, an increase in the yield of cellulose, pectinase, peroxidase, monophenol oxygen, and a decrease in the cost of the product.

The goal is achieved by the fact that according to the method of obtaining the complex of enzymes by cultivating higher basidiomycetes on a nutrient chain containing carbon source, mineral salts, monosodium phosphate, monosodium potassium phosphate, dibasic potassium phosphate, magnesium sulfate, and water followed by mercury filter. from the mycelium and residues of the hard substrate, a complex environment is used, in which cellulose is used as a source of carbon as waste. of paper or straw, extract of beet pulp and hydrol, and also add thiamine in the following ratio of components, wt.%:

Cellulose 1.3-1.5

Extract from beet pulp 8,8-9,0

Oh, 4-0.5 Hydrol

 Thiamine 0.00015-0,00020

Single substituted

.0.17-0.20

ammonium phosphate

One Substituted Oh, 05-0) 06

phos

Disubstituted

0.03-0.04

potassium phosphate 0.04-0.05

Magnesium sulfate Up to 100

tap water

After separation of the culture liquid from the solid phase, additional extraction of enzymes immobilized on the solid components of the nutrient medium with 0.5 n is carried out with acetate buffer at pH 4.0 for one hour at 10 ° C.

Example 1. Used Coriolus hirsutus 0-69, which was grown at 26-2o With the method of surface film on a complex nutrient medium of the following composition, wt.%:

Wednesday 1

1.31

Filter paper

Beet extract8, 80. saccharum 0.43

Hydrol 0.00015

Thiamine 0.17

NH4HgP04 0.052

K Ilf04 0.035

0.043

MgS04 Up to 100

Water plumbing

Nutrient media were also used as controls, which include components that stimulate the biosynthesis of individual cellulose enzymes (medium 2-f, pectinase (cpu, yes 3), monofiol monooxygenase (medium 4) May.%:. Wednesday 2

Filter paper 1/47 Tiamin0,00015

NdNGRO 0.19

КН2.р4, 0.059

K2HP040,039

MgSOji0.049

Plumbing Up to 100 Wednesday 3

Extract from beet pulp 9.0 Thiamin0,00015

NH4H2P040,18

KK2RO4-0,054

K2NRO4.0.036

. Mgs040,045

 Plumbing Up to 100 Wednesday 4

Hydrol0.49

Thiamine 0.00015 KH4H2PQJ.0.20

KH2P040,059

KjHPOjj. 0.039 MgSO40.049

Piped water Up to 100, During the cultivation of Corilus hirsutus, on the third, sixth and tenth days, the ability to hydrolyze insoluble cellulose and endo-1, 4-J-glucavase, exo-1,4- / -glyukiaaee, polygalacturrnase exopoly-galacto-suprocent activity was determined. , pectinexterase, monofeiolmonoox genase (polyphenol oxidase, laccase) and peroxidase in the filtrates of the culture fluid, obtained by separating the mycelium and residues of the solid substrate. The measurement results are presented in Table. 1, from which it follows that the use of a complex medium containing a compound at the same time, stimulating the biosynthesis of these enzymes, allows intensifying the process from 1.5 to 4.0 times and expanding the range of enzymes of the complex.

Extraction of O, 5N acetic acid buffer pH 4, O from residues of solid Substrate Enzyme Filtration Pulp and paper hydrolysis capacity, lulose mg% glucose Exo-1,4-) 3- {glu-Carboximecanase) .Tyl cellulose, mg% glucose

substrate for 1 h at 10C allowed us to further increase the yield of endo-1,4-β-glucanase, exo1, 4-p-glucanase, polygalacturonase, exopolygalacturonase, and pectin esterase (Table 2).

Example 2. p. As a producer, Coriolus versicolor. (Fr) was used. Buel, nei 087

, Composition of medium 1, wt.% Filter paper 1.40 Extract from beet pulp. 9.00 Hydrol 0.50

Thiamin0,00015

NHi H2P040.20

КН2Р040,06

. KjHPQ, Oh, .045

Water Up to 100

The results are presented in Table. 3 (levels of extracellular enzyme activities when grown on complex media with cellulose in the form of filter paper waste (1) or straw (2). In control, the environment where filter paper waste (3) and straw (4) were used as the sole carbon source. , extract from beet pulp (5) or hydrol ((5-day culture).

Example 3. Composition of media and cultivation conditions are similar to Example 1. Producer Pleurotus astreat ue (Fr) Kujnm PC. K-69, Cultivation time 10 days. ,

In tab. 4 shows the activity of Pleurotus astreatus K-69 on various media.

PRI me R 4. CqcTaB media as in example 1. Producer Irpex lacteus Fr. 0187.

The results are presented in Table. five.

The invention allows to reduce the cost of the finished product by about 30%.

Thus, the implementation of the invention provides for an expansion of the range of enzymes in the complex, an increase in the yield of cellulose, pectinase, peroxidase, myofenol monooxygenase, and reduce the cost of the resulting product. Table 1 Full culture | 6-day culture Nutrient medium options g-l-gt 1T1: g zT: O 56.7 56.7 25.2- 83.7 O 34.5

Continuation of table 1

Carboxime Endo-1, 4- / Tylcelluloglucanene,% Depot10, 6 4.6 Limerization Polygalactu Pectin, sugarcane, 31.221 units / ml Pectov Exopolygalacurna Acid, 5.61, High-ranking Pectin Exectyl Citrate, I have been a member of a member of a member of a member of a member of a member of a way, he was a member of a member of a way, he was a member of a member of a way, he was a member of a member of a way, he worked as a member of a day-to-day, he was a member of a woman, he was a member of the Citizen Acid, 5.61, High-melancholy Pectin Exactic, Pectin Exactic Acid, 5.61 monooxygenase unit / ml 0,6 O Konophenol-Pyrocatechin, monboxygene A unit / ml Peroxidase Benzidine, 1,4 O

The ability to hydrolyze cellulose

Exo-1,4- $ glucanase

59.0

Endo-1,4- / glucanase

4.0

Polygalacturonase

500.0

Exopolygalacturonase

7.2

Continued Tab. one

102.0

5.7

2.7

37.0

208.3

125.0200.0

4.1

6.2

3.4 6.8 2.6 13.4 7.7 6.8 4.9 7.0 33.3 250.0 100.0 142.8 111.1 3.7 3.0 1.5 1, 0 1.5 2.6 0.1 0.3 0.1 0/2 0.1 O 0O6.1 O0.7 6.0 OO 1.0 OO0.6 OO5.4 0.6 0.9 5 , 6

Pectinexerase

Monophenol monooxygenase

Monophenol monooxygenasePheroxidase

NOTE. Note.

Continued tabl,

0.1

0.1 4.2

about

1.3 0.6

3.7 0.3

complex nutrient medium according to the invention O - medium with filtering paper t L - medium with extract from steampie pulp; W - medium with hydrol.,

Table 2 is a complex nutrient medium according to the invention; {} - Wednesday with filter paper.

Pectinex High-methoxylated teraea

pectin, u / ml 0,27

Benzidia

Peroxidase

u / ml 0.27

MonophenolBenzidine,

u / ml 25.4

monooxygenase

Eck 30-1,4-: Carboximetl ilcellulose a, glucanase mg%

Endo-1,4-0

Carboxymethylcellulose; a, glucanase% depolymerization

Pectin beet Endylyllovichny,

galactoroyed / ml

Naza

Pektova

Exopoli

city of galactic acid, units / ml

Naza

PeroxyBenzidine, u / ml dase

Benzidine,

Monophenol / ml monoxyganase

Continuation of table 4

Traces

About 18.0

0.9

Traces

T d 6 l ntsa 5

322.3

35.5

ABOUT

87.7

51.1

48.7 250.0

0.92

2.64

2D 0.6 9.2

1.7

Traces

Claims (1)

2. Afanasyeva M .. M. th Kadyrov R.M. Microrology and phytopathology. 1980., v. 14, ”5, p. 410-418 (prototype). (54) (57 11. METHOD FOR PRODUCING THE ENZYME COMPLEX by cultivating higher basidiomycetes in a nutrient medium containing a carbon source, mineral salts: monosubstituted ammonium phosphate, monosubstituted potassium phosphate, disubstituted potassium phosphate, magnesium sulfate, and water, followed by separation of the culture filtrate liquids from mycelium and solid substrate residues, characterized in that, in order to expand the assortment of enzymes in the complex, increase the yield of celluloses, pectinases, peroxidase, monophenol monooxygenase and reduce ebestoimosti resulting product, used a complex medium, which is used as a source uglerodatsellyulozu filter paper waste or straw extract from sugar beet pulp, and the hydrological and additional thiamine make the following component ratio, mass%.: Cellulose 1.30-1.50 Beet pulp extract Hydrol • Thiamine Univariate ammonium phosphate; Single substituted potassium phosphate; Disubstituted potassium phosphate. Magnesium sulfate. Tap water. 2. Method according to claim u and i. the fact that after separation of the culture fluid from the solid phase, an additional extraction of enzymes immobilized on solid components of the nutrient medium of 0.5 and. acetate buffer at pH 4.0 for one hour at 10 ° C. in, wie, ai 0.40-0.50 0.00015-0.00020 0.17-0.20 Up to 100.0 lich g lo0.05-0.06 Sg 0.03-0.04 0.04-0.05