RU2354697C2 - STRAIN OF MYCELIAL FUNGUS ASPERGILLUS ORYZAE - ACIDIC α-AMYLASE PRODUCER - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: strain of mycelial fungus Aspergillus oryzae All Russian collection microorganisms F-3927 D - producer of acid α-amylase can be used in various areas of industry and agriculture for saccharifying starch-containing raw material.

EFFECT: increase of acid α-amylase output.

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Description

The invention relates to the field of biotechnology and can be used in the microbiological industry in the production of acidic α-amylase used in various fields of industry and agriculture for saccharification (hydrolysis) of starch-containing raw materials, namely in baking, alcohol, brewing, confectionery and starch-making industry (production molasses, syrups, glucose), in the production of cereals, vegetable products, fruit products (juices, syrups, extracts, jams, pectin), baby food , in the textile, paper industry in the production of detergents, as well as in medicine.

α-Amylase (α-1,4-glucan-glucanohydrolase, 3.2.1.1.) is an amylolytic action enzyme, belongs to the class of hydrolases, a subclass of glycosidases, hydrolyzes the internal α-1,4-glucosidic bonds in starch, glycogen and related poly - and oligosaccharides, leading to the formation of maltodextrins of various molecular weights, maltotriose, isomaltose and glucose.

According to the composition of the final products of starch hydrolysis, α-amylases are divided into two types: dextrinizing (thinning), leading to the formation of maltooligosaccharides (maltopentaoses and maltohexaoses), and sugar, leading to the formation of mainly maltose (and glucose).

The role of acidic α-amylase in bioconversion of starch-containing raw materials has now increased significantly in connection with the development and implementation of a technology for the production of fuel ethanol from starch-containing raw materials, which involves the use of low-temperature water-heat treatment using highly active and acid-resistant enzyme preparations of acidic α-amylase and The joint use of enzymes contributes to an increase in the speed and depth of degradation of starch; as a result, the degree of hydrolysis of starch reaches 98-99%, while the efficiency of the enzymatic treatment process can be significantly increased due to the use of highly active enzyme preparations.

Microorganisms of various taxonomic groups are known - producers of amylases and methods for their cultivation under aerobic conditions. The choice of producer strain depends on its ability to provide a high level of activity in the culture fluid, the rate of biosynthesis, the release of enzymes from a unit of the substrate used, and the cost of the substrate.

In world practice, for the industrial production of starch hydrolysis products, mainly amylases formed by fungi, belonging to the genus Aspergillus with yellow-green pigmentation, are mainly used: Asp.flavus Asp.oryzae, Asp.glauscus, Asp.ochraceusi, Asp.fumigatus, including the number of recombinant. The use of recombinant strains significantly complicates the process of obtaining enzyme preparations. This is primarily due to the need for ongoing research to maintain the strains in a stable and active state, the creation of special cultivation conditions that are not always available in industrial process control.

Currently, in Russia, preparations of acidic α-amylases are not produced, mainly due to the low activity of existing producers. Industrial-scale PA Sibbiopharm (Berdsk) Amilosubtilin G3x is an enzyme preparation of α-amylase based on the bacterial strain Bacillus subtilis, has slightly different properties than acidic α-amylase, being a liquefying drug that exhibits maximum activity in neutral and slightly alkaline zone.

A known strain of moldy fungus Asp.oryzae 740-A2 is an active producer of α-amylase (Kalunyants K.A. and Golger L.I. Microbial enzyme preparations. M: Food industry, 1979, pp. 8-9). The activity of the obtained α-amylase is 200.0 units / g of dry matter.

A disadvantage of the known strain should be recognized as the unstable activity of α-amylase during cultivation under industrial conditions, as well as increased susceptibility to infections.

The known (US patent 4,593,005) strain Asp.oryzae is a producer of acidic α-amylase capable of hydrolyzing granular starch. When cultured in flasks on a solid or liquid medium at a temperature of 45 ° C and pH from 4.0 to 7.0, it provides the maximum activity of the enzyme after 168 hours of growth - 12 units. in 1 ml of culture fluid. In nutrient media, starch, corn grits, wheat flour, molasses can be used as a carbon source, peptone, meat extract, yeast extract, casein, corn extract, malt extract, soybean oil, inorganic ammonium salts, nitrates can be used as a source of carbon. In addition, to ensure the normal growth of the producer and the biosynthesis of the enzyme, the presence of the following inorganic salts in the nutrient media is necessary: KH ₂ PO ₄ , FeSO ₄ , MgSO ₄ , KCl, CaCl ₂ , CoCl ₂ , MnSO ₄ .

Known (SU, copyright certificate 1158579) is a strain of the microscopic fungus Asp.oryzae НРВ-169 (collection of the Central Museum of Industrial Microorganisms "VNIIGenetika", collection number TsMPM F-257) - producer of α-amylase. Potato or corn starch may be used as a carbon source. The activity of α-amylase is 40-55 u / mg. Its disadvantage should be recognized as low activity relative to acidic α-amylase.

Also known (RU, patent 2070921) strain Asp.oryzae 387 (VKPM F-683) - producer of a complex of acidic and slightly acidic proteases, amylolytic and cytolytic enzymes. When cultivated on a nutrient medium containing,%: barley flour - 3.0; bran - 3.0; KH ₂ PO ₄ - 1.5; tap water, natural pH, under aerobic conditions at 28-30 ° C for 42-48 hours, the strain synthesizes a complex of acidic and slightly acidic proteases with a total proteolytic activity of 11.5 units PS / ml, acidic α-amylase with an activity of 12 units per 1 ml of QOL, as well as related enzymes of the cellulolytic complex. However, the claimed amylolytic activity of the strain Asp.oryzae 387 is very low, which makes its practical use from this point of view inappropriate.

Known (SU, copyright 1440922) strain Asp.oryzae VKPM F-369 - producer of amylolytic and proteolytic enzymes. When cultivated on a nutrient medium containing,%: barley flour - 14.0; soy flour - 0.5; KH ₂ PO ₄ - 0.5; tap water, natural pH, under aerobic conditions at 28 ° C for 96 hours, the strain synthesizes a complex of acidic and weakly acidic proteases, while the enzymatic activity is 20.7 in AC and 17.3 units / ml in PS. However, the claimed amylolytic activity of the strain Asp.oryzae VKPM-F-369 is very low, which makes its practical use from this point of view impractical.

The known strain Asp.awamori M-2002 (VKM F-3771D) (Semenova M.V. et al. Microbial biocatalysts for the processing industries of the agro-industrial complex - M .: VNIIIPBT, 2006, 304 p.). When cultivated in deep conditions for 192 h on a nutrient medium, when using cereal flour hydrolyzate as a carbon source, the strain synthesizes glucoamylase with an activity of 800-1080 units / ml. The introduction of CaCO ₃ , MgSO ₄ salts or a complex compound of calcium and magnesium carbonic salts (or magnesium oxide) into the nutrient medium helps to stabilize the pH of the medium during the entire fermentation at a level of 5.9 to 6.4, which leads to a change in the direction of the biosynthesis process in side of increased synthesis of acidic α-amylase. Thus, by changing the cultivation conditions of the strain Asp.awamori M-2002, it is possible to obtain highly active complex enzyme preparations with glucoamylase and α-amylase activity. However, being currently a highly active producer of glucoamylase, Asp.awamori M-2002 strain with respect to α-amylase synthesis is at a relatively low productivity level, providing a maximum α-amylase activity of 60-80 units / ml of culture fluid for 192 h of growth .

The disadvantages of all of the above producers are the low productivity of the strains in relation to acidic α-amylase and the complexity of the method of their cultivation. Highly active producers are not available for the organization of domestic production, since they are the property of foreign manufacturing companies.

The technical problem to which the present invention is directed is to obtain a new highly productive strain of mycelial fungi, a producer of acidic α-amylase, capable of providing a high level of activity and the release of the enzyme per unit mass of the used substrate when cultured on cheap and technologically advanced media.

The technical result that can be obtained by carrying out the invention is to obtain a new highly productive strain - a producer of acidic α-amylase, while expanding the range of substrates used (starch-containing substrates, corn extract, vegetable proteins).

To achieve the specified technical result, it is proposed to use a new breeding mutant strain of the mycelial fungus Asp.oryzae UV27R-14, a producer of acidic α-amylase.

The strain Asp.oryzae (Ahlburg) E. Cohn UV27R-14 was deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms named after G.K.Skryabin RAS under the number VKM F-3927 D.

Strain Asp.oryzae VKM F-3927D was obtained by multistage genetic selection using effective mutagenesis - ultraviolet irradiation with simultaneous exposure to chemical mutagens from strain Asp.oryzae VKM F-52 according to the following method. The spore suspension of the initial strain obtained in distilled water with the addition of 0.1% Tween-80 is irradiated with ultraviolet light (irradiation with a light flux of 3 mW / cm ² ) for 10 min. Irradiated spores are plated on Petri dishes with selective media, grown at 35 ° C for 5 days. Mutants with improved production of acidic α-amylase 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 acidic a - amylase when cultured in a liquid medium in flasks. The active clones selected during cultivation in flasks are again (repeatedly) irradiated and selected on dishes 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 ° C with obligatory reseeding at least once for 3-6 months.

Macroscopic characteristics. Colonies on Chapek agar with yeast extract (CYA) at 25 ° C have a diameter of 65-70 mm / 7 days, greenish-yellow, the surface is fluffy to flocculent, the edge is even; the mycelium is dense, white, exudate is absent, the reverse side is unpainted.

Colonies on Chapek agar with yeast extract (CYA) at 37 ° C have a diameter of 60-65 mm / 7 days, olive-yellowish, the surface is fluffy, the edge is even; the mycelium is dense, white, exudate is absent, the reverse side is unpainted.

Colonies on Chapek agar with yeast extract and 20% sucrose (CY20S) at 25 ° C have a colony diameter of 67-71 mm / 7 days, olive yellow, the surface is fluffy to ragged, the edge is even; the mycelium is dense, white, exudate is absent, the reverse side is unpainted.

Colonies on Maltz Agar (MEA) at 25 ° C have a diameter of 65-70 mm / 7 days, greenish-yellow, the surface is ragged, the edge is even; the mycelium is not very dense, white, there is no exudate, the reverse side is yellowish-gray.

Microscopic characteristics. Conidial heads spherical to loose columnar. Conidiophores are not colored, smooth-walled to slightly rough, 500-1000x10-25 microns. Vesicles (apical extensions) hemispherical to pear-shaped, 22-50 microns wide; single-tier and two-tier sterigms within the same colony, cover from 1/2 to 2/3 of the vesicle surface, metula - 8-12x4-6 microns, phialids - 8-12x3-5 microns. Conidia are spherical, 4.0-8.5 microns in diameter, smooth-walled to slightly rough during aging. It forms enzyme systems that allow it to grow on the corresponding complex substrates: starch, β-glucan, pectin and galactomannan.

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 morphological properties of the obtained mutant differ from the initial one by the character of sporulation, the color of spore pigment, a shorter growth cycle when grown on solid and liquid media, and increased synthesis of acidic α-amylase during deep cultivation.

When studying morphological variability during screening of the strain Asp.oryzae on selective media, atypical colonies were cleaved in the amount of 5-7%, which indicates the stability of the culture.

The cultivation of the strain Asp.oryzae VKM F-3927D is carried out under aerobic conditions in a submersible state at a temperature of 35 ° C for 120 hours, the pH of the medium is 5.8-6.2. For the growth of the culture and biosynthesis of acidic α-amylase, starch, hydrolyzed starch, soy, corn or other grain flour, or their extracts, ammonium nitrogen, and corn extract can serve as a source of carbon and nitrogen.

The activity of acidic α-amylase in the culture fluid is determined by the ability to catalyze the hydrolysis of starch to dextrins of various molecular weights. A unit of activity is taken to be such an amount of an enzyme that within 10 minutes at 30 ° C at pH 4.7 catalyzes the hydrolysis of 1 g of soluble starch, which is 30% of the reaction.

Enzyme preparations obtained using the proposed strain can be used in the form of culture fluid, concentrated liquid preparations obtained by ultrafiltration or evaporation of the culture fluid, or in the form of dry preparations (Amilorizin G10x, G20x) obtained by precipitation with ethanol or other organic solvents from ultrafiltrate of the culture fluid, as well as drying or granulation.

The method of hydrophobic chromatography of the enzyme preparation Amilorizin G10x yields a homogeneous acidic Asp.oryzae α-amylase with a molecular weight of 52 kDa, the specific amylolytic ability of which is 39 u / mg protein. Acidic α-amylase Asp.oryzae is active at temperatures from 30 to 70 ° C with optimum action at 58-60 ° C; stable in a wide range of pH values - from 3.5 to 8.0, with optimum action at pH 5.0-6.0; in a more acidic zone, characteristic of flour mixtures (pH 4.5), is 85-95% active; is a maltogenic acid α-amylase; the limiting degree of hydrolysis of soluble and insoluble starch was 38 and 34%, respectively.

Thus, the properties of the Asp.oryzae VKM F-3927D acid α-amylase enzyme ensure its effectiveness in saccharification of starch-containing raw materials. The possibility of using the invention is illustrated by examples, which do not limit the scope and essence of the claims associated with them.

Example 1. Sowing material (inoculum) is obtained by growing a strain on a liquid nutrient medium composition: wheat flour - 6%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0.3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, distilled water, pH 5.8-6.2, in flasks containing 50 ml of sterile medium, on a shaker with 240 rpm at 30 ° C for 48 hours .

The resulting seed in an amount of 2% by volume of the medium is introduced into rocking flasks with a volume of 750 ml containing 50 ml of medium of the following composition, wt.%: Wheat flour - 6%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0.3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, tap water, pH 5.8-6.2. The medium is pre-treated with a bacterial acidic α-amylase enzyme preparation at the rate of 2 u / g starch at 70 ° C for 20 minutes.

Cultivation is carried out under aerobic conditions on a rocking chair (240 rpm) at a temperature of 30 ° C.

The maximum enzymatic activity of acidic α-amylase (120 h of growth) in the culture fluid is 380 u / ml.

Example 2. Cultivation is carried out in rocking flasks, as described in example 1, using a liquid nutrient medium of the following composition, wt.%: Corn starch - 10%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0 , 3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, tap water, pH 5.8-6.2. The maximum enzymatic activity of acidic α-amylase (120 h of growth) in the culture fluid is 425 u / ml.

Example 3. Cultivation is carried out in rocking flasks, as described in example 1, using a liquid nutrient medium of the following composition, wt.%: Wheat flour - 10%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0 3%

KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, tap water, pH of the medium 5.8-6.2. The maximum enzymatic activity of acidic α-amylase per 120 h of growth in the culture fluid is 480 units / ml.

Example 4. The process of cultivation is carried out in laboratory fermenters of the company "Anglicon" (Great Britain), with a capacity of 10 l, equipped with automatic control systems for pH, pO _2, temperature and defoaming. Mass transfer is carried out using a bubbler and a two-tier mixer, providing aeration - up to 15 g O ₂ / l h.

Cultivation conditions: load factor of fermenters - 0.5; seed - a vegetative culture grown in flasks (according to example 1); the dose of inoculation of the crop in the fermenter is 2%. The temperature regime is differentiated: the first 48 hours of cultivation is 35 ° C, then 30 ° C.

The composition of the fermentation medium, g / l: wheat flour - 6%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0.3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1 , 2%, tap water, pH 5.8-6.2. For 24 and 30 hours of cultivation, make-up is made in the form of a 50% hydrolyzate of cornmeal starch in an amount of 30% of the volume of the fermentation medium.

The activity of acidic α-amylase per 120 hours of cultivation in the culture fluid is 700-800 u / ml.

The data obtained indicate an increase in amylase activity in the culture fluid of the claimed strain Asp.oryzae VKM F-3927D compared with the most famous of the mutant strains Asp.awamori M-2002 by 8-10 times.

To achieve high strain activity, the use of complex and expensive culture media is not required. For cultivation, nutrient media traditionally used in industrial technologies for producing such enzyme preparations can be used.

The high level of activity of acidic α-amylase allows us to consider the strain as a producer of a promising enzyme preparation for the treatment of starch or starch-containing raw materials with the aim of deep hydrolysis.

Claims (1)

The strain of the mycelial fungus Aspergillus oryzae VKM F-3927D is a producer of acidic α-amylase.