RU2323254C2 - MYCELIAL FUNGUS PENICILLIUM FUNICULOSUM STRAIN AS PRODUCER OF CARBOHYDRASES COMPLEX CONTAINING CELLULASES, β-GLUCANASES, β-GLUCOSIDASES, XYLANASES AND XYLOGLUCANASES AND METHOD FOR PREPARING ENZYME PREPARATION OF CARBOHYDRASES COMPLEX FOR SACCHARIFICATION OF LIGNOCELULOSE MATERIALS - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymology.

SUBSTANCE: strain of mycelial fungus is prepared by the multistep mutagenesis and selection of the parent culture Penicillium funiculosum BKM F-3661. Prepared strain is cultured on liquid nutrient medium under conditions of continuous feeding with glucose. In 120 h after culturing cultural fluid is separated and concentrated by ultrafiltration and a liquid enzyme preparation or dried is obtained. Invention can be used for preparing fermented sugars designated for processing to ethanol, and as fodder supplements also.

EFFECT: valuable biological properties of fungus strain.

3 cl, 4 ex

Description

The invention relates to the field of biotechnology, in particular the field of biotechnological processing of lignocellulosic materials, and can be used to produce fermentable sugars intended for processing into ethanol, as well as as feed additives.

Many strains are known that produce individual carbohydrases, mainly cellulases, that are capable of enzymatically cleaving cellulose-containing materials, and methods for their cultivation under aerobic conditions.

Known (RU, RF patent 2057179, С12N 9/42, 1996) Aspergillus niger strains, when cultured on media with cellulolytic enzyme inducers, a complex of enzymes is obtained, including cellulase, xylanase, β-glucosidase, β-xylosidase, β-glucanase and laminarinase. The activity of enzymes in the culture fluid are, respectively: cellulase - 5.0 units / ml; xylanase - 125.0 units / ml; β-glucosidase - 20.0 units / ml; β-xylosidase - 36.0 units / ml; β-glucanase - 0.95 units / ml and laminarinase - 1.25 units / ml.

Although fungi of the genus Aspergillus form complexes with a wide spectrum of carbohydrases, the activity of most of the individual components of the complex is not high enough, which often makes their practical use impractical.

Cellulase producers are also strains of mycelial fungi of the genus Trichoderma.

In particular, the mutant Trichoderma. reesei MCG 80 (US patent 4472504, C12N 9/42, 1984), when continuously cultured on a nutrient medium with 8% cellulose and biotin, provides a cellulase activity of 17.2 units / ml APB (APB - cellulase complex activity, determined by filter paper and expressed in international units according to the IUPAC recommendation [T.K. Chose., Pure and Appl. Chem., vol. 59, 2, p. 257-268]).

Also known is a strain of mycelial fungus Tr.longibrachiatum VKM F-3634 D (RU, patent 2195490, C12N 9/42, 2002) - a producer of a complex of carbohydrases containing cellulases, beta-glucanases, xylanases, pectinases and mannanases. The activity of cellulases, β-glucanases, xylanases, pectinases and mannanases is about 6.5; 25.0; 20.0; 7.0 and 0.5 units / ml, respectively.

Known mutant Tr.reesei BCM 18.2 / QC (VGNKI-28) (RU, patent 2001949, С12N 9/42, 1993), obtained using parasexual processes from the original culture of Tr.reesei IMET 43803, which has increased productivity and allows in case of single-stage fermentation, ensure high enzyme activity (cellulases) per unit mass of the substrate used. The specified strain has enzyme systems that allow you to grow on a medium with cellulose, starch, chitin, pectin, xylan, laminarin, lichenin. When cultivated on a liquid nutrient medium based on beet pulp, an activity level of 3.5–4.2 units / ml of APB is achieved (cultivation time is 110 h), and when cultured in a fermenter on a medium with lactose, 18.2 units / ml ( 81 h), when cultured in a fermenter on whey - 12-14 units / ml (100-110 hours).

The disadvantage of these strains is the experimentally established data confirming that fungi of the genus Trichoderma, as a rule, have high productivity, mainly in relation to cellulases, but to a much lesser extent in relation to other carbohydrases (β-glucanases, xylanases, pectinases, mannanases, etc. .) necessary for the hydrolysis of various polysaccharides included (in addition to cellulose) in the composition of plant materials. All of the listed Trichoderma strains have low productivity for carbohydrases that are not cellulases (β-glucanases, xylanases, pectinases, mannanases, etc.).

Known (RU Patent 2167197, С12N 11/14, 2001) is a method for producing a biocatalyst for producing fermentable sugars from cellulose-containing material by producing an enzyme material, glucoamylase, followed by immobilization on a solid support, the immobilization being carried out by adsorption on the surface of a carbonized aluminosilicate having a surface of at least 2 m ² / g, with a carbon coating of at least 1.5 wt.% carbon, the carbon coating having the structure of fibrous catalytic carbon and mesopore an empty structure.

The disadvantage of this method should recognize its complexity, as well as low catalytic activity relative to the processing of cellulose-containing materials obtained biologically active catalyst.

The technical problem, the implementation of which the proposed technical solution is directed, is to develop a new biocatalyst for the production of fermentable sugars from cellulose-containing materials.

The technical result obtained by the implementation of the proposed technical solution is to increase the yield of fermentable sugars from cellulose-containing material.

When implementing the proposed technical solution, a new selection strain of the mycelial fungus Penicillium funiculosum S-2006 was used, producing a complex of carbohydrases containing cellulases, β-glucanase, β-glucosidase (cellobiase), xylanase and xyloglucanase.

The strain was obtained using multistage mutagenesis and selection from the original culture Pen.funiculosum BKM F-3661.

Mutagenesis and selection of Pen.funiculosum strains is carried out by suspending the spores of the fungus in distilled water with the addition of 0.1% Tween-80 and irradiating with ultraviolet light (irradiation with a light flux of 3 W / cm ² ) for 3 minutes. Then the control and irradiated spores are plated on plates with glucose-potato agar and incubated for 48 hours at 28 ° C. After that, the percentage of survival is calculated. Survival of 1-5% is considered sufficient for the selection of mutants. Each surviving clone was transferred to plates with selective agarized media containing 0.1% carboxymethyl cellulose (CMC) or 1% oat xylan (Sigma) and incubated for 48 hours at 28 ° C. Mutants with improved production are selected visually for the increased areas of enlightenment around the colonies. The most active mutants selected on the plates are tested for the productivity of the synthesis of cellulases, xylanases and β-glucosidases when cultured in a liquid medium in flasks. The most active variants selected during cultivation in flasks are again (repeatedly) subjected to irradiation and selection on plates and flasks, as described above.

Storage conditions: the strain can be stored in a lyophilized state for several years and / or on shoals with agarized Chapek's medium or wort-agar at + 4 ° С with obligatory reseeding at least once for 3-6 months.

Cultural and morphological characteristics of the obtained strain.

With growth on Maltz-agar, the diameter of the colonies reaches 36 mm on the 7th day with growth at 25 ° C. Colonies are dense, even, with a convex center. The mycelium is light, fluffy. The reverse side is fawn-reddish-orange. Conidiogenesis is weak, gray-greenish. Colonies on agar Chapek with yeast extract on the 7th day are 27-30 mm in diameter, folded, with a smooth edge, the surface is woolly. The mycelium is light, yellowish. The reverse side is fawn-orange. Conidiogenesis is weak.

The fungus does not grow on medium with glycerin and on апapek agar at 5 ° C. When cultured on Chapek’s agar at 37 ° C, the colonies are 18 mm in diameter, folded, medium density, the reverse side is brownish-reddish. -T

Conidiogenesis: biverthicillate brushes, pressed metulae, smooth (8-10 × 2.5-3.0). Acerous phialides (8-9 × 2.5-3.0), with a short neck. Conidia are elliptical, small (2.2 × 1.5-2.0), smooth.

When cultivated in deep conditions using soluble substrates (glucose, fructose, lactose), a loose branched mycelium with weak pelletization is formed, the specific initial growth rate of mycelium was 0.35 h ^-1 , at the end of cultivation 0.1 h ^-1 .

Physiological and biochemical characteristics of the strain.

Mesophilen. The optimum mycelium growth temperature is 32 ° C (29-34 ° C), the optimum for cellulase formation is 28 ° C (26-29 ° C). The optimal pH of growth and secretion of cellulases is 3.5-5.0. The growth of mycelium is also observed at pH 2.5, but there is no very weak formation of cellulases and other carbohydrases.

Nystatin resistance is good. With surface cultivation, it is resistant to a concentration of up to 0.5 μg / ml; at a concentration of 2.5 μg / ml, growth is inhibited. When digitonin (3.5-4.0 μg / ml) or Bengal pink (30-50 μg / ml) is added to the medium, the colony size decreases.

It is a prototroph. It is able to quickly assimilate glucose, lactose, glycerin, galactose, xylose, D-mannose, D-mannitol, trehalose, sorbose and sorbitol, slower - D-xylose, L- and D-arabinose, L-rhamnose and ribose. Weakly assimilates: D-glucosamine, deoxyribose, deoxygalactose, 2-deoxy-D-glucose and 5-thio-D-glucose.

Uses inorganic and organic nitrogen, assimilates the nitrate and ammonium forms of nitrogen well.

It forms enzyme systems that allow it to grow on the corresponding complex substrates: cellulose, starch, xylan, laminarin, β-glucan, lichenin, pectin and galactomannan. Able to utilize lactic acid at a concentration below inhibitory.

This type of mycelial fungus is not listed as pathogenic in the "Regulation on the registration, storage, handling, dispensing and transfer of cultures of bacteria, viruses, rickettsia, fungi, protozoa, mycoplasmas, bacterial toxins, poisons of biological origin."

The cultivation of the proposed strain Pen.funiculosum is carried out under aerobic conditions in a submerged state on a nutrient medium containing one or more substrates - carbon sources, which are the inducers of enzyme biosynthesis. Non-inductor substrates can also be used as substrates. The strain is capable of secreting a complex of enzymes — carbohydrases containing cellulases, β-glucanase, β-glucosidase (pellobiase), xylanase and xyloglucanase into the culture medium under the appropriate conditions of the cultivation process based on the use of soluble substrates, for example glucose or lactose. Glucose in the cultivation medium can be replaced by a cheaper product - starch hydrolyzate.

The activity of cellulases on filter paper (APB) was determined by the standard method [Ghose T.K. Measurement of cellulase activity. Pure appl. Chem., 1987, v. 59, p. 257-268] at 50 ° C and pH 4.8, using chromatographic paper No. 1 manufactured by Whatman (England) and the dinitrosalicylic method for the analysis of reducing sugars (BC). Enzymatic activities with respect to CMC, Avicel (microcrystalline cellulose), β-glucan, xylan and xyloglucan are determined by the initial Sun formation rates (within 5-10 minutes of the enzymatic reaction) by the Shomody-Nelson method [A.P. Sinitsyn, A.V. Gusakov, I.M. Chernoglazov. Bioconversion of lignocellulosic materials. Tutorial. - M .: Publishing House of Moscow State University, 1995, p.144-156] from the corresponding substrate (5 g / l) at a pH of 5.0 (0.1 M Na-acetate buffer) and 50 ° C (activity by avicel was determined at 40 ° C).

Activity against n-nitrophenyl-β-glucoside was determined at pH 5.0 and 40 ° C. An aliquot (0.05 ml) of the substrate stock solution (10 mM) was mixed with 0.85 ml of 0.1 M Na-acetate buffer, the mixture was preheated at 40 ° C for 5 min, and then the enzymatic reaction was started by adding 0.1 ml previously diluted and heated in the same way the enzyme solution. Exactly after 10 min of incubation of the solution at 40 ° C, the reaction was stopped by adding 0.5 ml of a 1 M solution of Na ₂ CO ₃ . Then, the absorbance of the solution was measured on a spectrophotometer at 400 nm against a comparison cuvette in which there was a control substrate solution prepared in the same way, but into which instead of an enzyme solution 0.1 ml of acetate buffer was added. The amount of released n-nitrophenol was calculated using its extinction coefficient (D ₄₀₀ = 18300 M ^-1 cm ^-1 ).

Enzyme preparations obtained using the proposed strain can be used in the form of a culture fluid, in the form of concentrated preparations obtained by ultrafiltration or vacuum evaporation, or in the form of dry preparations.

The possibility of using the invention is confirmed by examples that do not limit the scope and essence of the claims associated with them.

The proposed strain was obtained as follows.

Example 1. To obtain the inoculum (inoculum) culture of the claimed strain of the fungus Pen. funiculosum S-2006 is grown on wort or CM agar at 29 ° C for 7 days. and further at room temperature in the light for 5 days. Inoculation of flasks is carried out with 1 ml of a suspension of spores washed with agar with water containing 0.1% Tween 80. Cultivation of the strain is carried out under aerobic conditions in 750 ml Erlenmeyer rocking flasks containing 100 ml of an aqueous solution of the following medium in g / l: beet pulp - 40, malt sprouts - 14, (NH ₄ ) ₂ SO ₄ - 6, KH ₂ PO ₄ - 2, MgSO ₄ × H ₂ O - 0.6; pH 5.4. The flasks are incubated on a shaker at 30 ° C and 200 rpm for 120 hours

The activity of avicellase, carboxymethyl cellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and APB in the culture fluid for 120 hours of cultivation is 15, 130, 140, 10, 380, 100 and 12 units / ml, respectively.

Example 2. Cultivation is carried out in Erlenmeyer rocking flasks as described in example 1, using an aqueous nutrient medium of the following composition, in g / l: cellulose - 40, glucose - 10, (NH ₄ ) ₂ SO ₄ - 6, KH ₂ PO ₄ - 2, MgSO ₄ × 7H ₂ O - 0.6; pH 5.4.

The activity of avicellase, carboxymethyl cellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and APB in the culture fluid is 25, 260, 275, 15, 510, 140 and 18 units / ml, respectively, for 120 hours of cultivation.

Example 3. The enzyme preparation is obtained by culturing the strain Pen.funiculosum S-2006 in the ANKUM-2M fermenter (working volume 7 l) in an aqueous nutrient medium of the following composition, in g / l: cellulose - 50, glucose - 10, KH ₂ PO ₄ - 8, (NH ₄ ) ₂ SO ₄ - 3, MgSO ₄ × 7H ₂ O - 0.2, CaCl ₂ × 2H ₂ O - 0.2 at 28 ° C and maintaining the pH in the range of 4.5-5 by feeding HCl or NH ₄ OH. During cultivation, glucose is fed by continuously feeding it to the fermenter at a rate of 2-3 g / l / h. After 144 hours, a culture fluid containing a complex of highly active carbohydrases is separated and concentrated by ultrafiltration (on membranes with a transmission limit of 10 kDa) to obtain a liquid biocatalyst.

The activities of avicellase, carboxymethyl cellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase, and APB in the culture fluid are 45, 650, 690, 28, 1280, 260, and 42 units / ml, respectively, for 144 hours of cultivation. The activities of avicellase, carboxymethyl cellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and APB in the liquid enzyme preparation (UV concentrate) are 280, 3900, 4250, 170, 7580, 1570 and 260 units / ml, respectively.

Example 4. The enzyme preparation is obtained by culturing the Pen.funiculosum S-2006 strain in the ANKUM-2M fermenter (working volume 7 l) in an aqueous nutrient medium of the following composition, in g / l: cellulose - 60, glucose - 30 (enzyme is used as glucose starch hydrolyzate), KH ₂ PO ₄ - 12, (NH ₄ ) ₂ SO ₄ - 7, MgSO ₄ × 7H ₂ O - 0.4, CaCl ₂ × 2H ₂ O - 0.4 at 28 ° C and maintaining the pH in a range of 4.5-5 by feeding HCl or NH ₄ OH. During cultivation, they are fed with glucose (an enzymatic starch hydrolyzate is used as glucose) by continuously feeding it to the fermenter at a rate of 2-3 g / l / h. After 144 hours, the culture fluid containing the carbohydrase complex is separated, ultrafiltered, and the ultraconcentrate is freeze-dried to obtain a dry biocatalyst.

The activities of avicellase, carboxymethyl cellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase, and APB in the culture fluid are 58, 820, 870, 38, 1520, 360, and 55 units / ml, respectively, for 144 hours of cultivation. The activities of avicellase, carboxymethyl cellulase, β-glucanase, β-glucosidase, xylanase, xyloglucanase and APB in a dry enzyme preparation are 870, 13100, 13500, 610, 22900, 5760 and 870 units / g, respectively.

An experiment to determine the hydrolytic (saccharifying) ability of the obtained biocatalyst is carried out in a thermostatically controlled cell at 50 ° C, placed on a rocking chair. A substrate sample is applied to the cells (pre-treated with a steam explosion or hardwood or deciduous wood, pre-treated with a steam explosion of cereal straw, beet pulp, pulp and paper waste - short-fiber pulp, parchment scraps), 6.8 ml 0.1 M acetate buffer, pH 5.0, and 1 ml of a solution of a pre-diluted enzyme preparation. The reaction mixture is stirred on a rocking chair at a frequency of 250 vibrations / min. The concentration of the substrate in the reaction mixture is 50 g / l (in terms of dry matter), and the dilution of the enzyme preparation is selected so that the APB is 10 units. per 1 g of dry substrate. After 12 hours of hydrolysis, samples (1 ml) were taken from the reaction mixture, centrifuged for 1 min at 10,000 rpm and the concentration of BC and glucose was measured in the supernatant. The criterion for the hydrolytic ability of the preparations is glucose output for 12 hours of hydrolysis of an insoluble substrate at 50 ° C.

The glucose concentration is determined by the glucose oxidase-peroxidase method [Berezin I.V., Rabinovich M.L., Sinitsyn A.P. Investigation of the possibilities of the kinetic spectrophotometric method for determining glucose. Biochemistry, 1977, v. 42, No. 9, pp. 1631-1636].

According to the obtained experimental data, for any initial raw material (softwood or hardwood, straw of cereal plants, beet pulp, cellulose waste), the greatest amount of glucose fermented into alcohol in 12 hours was formed by the enzyme preparation Penicillium funiculosum S-2006, proposed for the implementation of the method. It provided a glucose yield of 15-50% glucose more than commercial or laboratory enzyme preparations Penicillium and commercial or laboratory enzyme preparations Trichoderma.

Thus, the proposed Penicillium funiculosum S-2006 strain has the ability to produce a complex of highly active carbohydrases, including cellulases, β-glucanases, β-glucosidases (cellobiases), xylanases and xyloglucanases, which makes it possible to obtain a complete complex of enzymes, as well as, if necessary, individual individual enzymes (components) of the complex.

Claims (2)

1. The strain of the mycelial fungus Penicillium funiculosum BKM F-3887D is a producer of a complex of carbohydrases containing cellulases, β-glucanase, β-glucosidase, xylanase and xyloglucanase. 2. A method of producing an enzyme preparation of a carbohydrase complex for saccharification of lignocellulosic materials, characterized in that the strain Penicillium funiculosum BKM F-3887D is cultured on a nutrient medium containing, g / l: cellulose 50-60, glucose 10-30, mineral salts - KH ₂ PO ₄ 8-12, (NH ₂ ) ₂ SO ₄ 3-7, MgSO ₄ · 7H ₂ O 0.2-0.4, CaCl ₂ · H ₂ O 0.2-0.4, with continuous feeding of glucose, at a temperature of 26-30 ° C and maintaining the pH in the range of 4.5-5.0, 120-144 hours after the start of cultivation, the culture fluid is separated and concentrated by ultrafiltration to obtain a liquid fe elementwise drug or dried to obtain a dry enzyme preparation.