RU2196821C2 - Strain of mycelial fungus aspergillus awamori as producer of glucoamylase - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymes. SUBSTANCE: strain Aspergillus awamori N 400 VKM F-3689 D is obtained by multistage genetic selection of the parent strain Aspergillus awamori VKM F-758 using ultraviolet irradiation with simultaneous effect by chemical mutagens. Strain shows high glucoamylase activity. Glucoamylase shows activity in wide range of pH from 3.5 to 7.5 and temperature from 30 to 65 C. For achievement of high productivity of strain nutrient media used traditionally in industrial technologies in preparing such enzyme preparations can be used. Method does not require complex procedures in the producer strain culturing. Invention can be used for starch raw saccharification in different fields of food industry where high-active enzyme preparations are required. EFFECT: enhanced effectiveness of strain, high activity of enzyme. 3 ex

Description

 The invention relates to the microbiological industry, various fields of food biotechnology and can be used to obtain glucoamylase for the purpose of its further use for the enzymatic processing of starch-containing raw materials in the alcohol, starch, bakery and brewing industries.

 As producers of amylolytic enzymes, mold fungi of the genus Aspergillus of the species oryzae, usamii, awamori, batatae are most often used; the genus Rhizopus of the species delemar, fonkinsis, neveus, tonnensis, japonicum, topnineusis, as well as individual representatives of Neurospora crassa and Mucor. Yeast and yeast-like microorganisms of the genera Candida, Saccharomyces, Endomycopsis and Endomyces are also able to synthesize amylolytic enzymes.

 As a rule, natural microorganisms form a complex of amylolytic enzymes capable of hydrolyzing plant substrates based on starchy carbohydrates. This complex includes α-amylases and glucoamylases, hydrolyzing starch molecules to α-amylases and glucoamylases, hydrolyzing starch molecules to glucose and dextrins of various molecular weights; proteases that break down the protein of raw materials to amino acids, which are valuable nitrogenous nutrition for yeast; glucanases, which intensify the process of enzymatic processing of raw materials due to the hydrolysis of non-starchy polysaccharides; pectinases that destroy pectin, other enzymes of lytic action.

 Currently obtained multienzyme preparations have different enzyme composition and differ in the level of activity of individual enzymes. In general, well-known polyenzyme preparations with amylolytic activity need to improve their functional characteristics, which will increase the efficiency of their industrial use.

 Of particular interest are enzyme preparations with increased glucoamylase activity.

Glucoamylase - (α-1,4-glucan-glucanohydrolase, 3.2.1.3.) -Glucose-forming amylase, an exoenzyme, attacks starch from the non-reducing end of the polysaccharide chain, sequentially cleaving glucose residues and completely converting starch into glucose. Most isolated glucoamylases of microbial origin show some common properties. As a rule, these are acid-stable enzymes with an optimum pH of 4.5-5.0, an optimum temperature of 55-60 ^o C. In addition to specific α-1,4-glucan bonds, glucoamylases hydrolyze α-1,6 bonds both in polysaccharides and in low molecular weight oligosaccharides. Under their action, starch is completely hydrolyzed to glucose.

 The ability of glucoamylase to hydrolyze both α-1,4 and α-1,6 bonds with glucose cleavage puts this enzyme in first place in the efficiency of starch hydrolysis with the aim of further fermentation of the formed sugar.

In the alcohol, starch and syrup, baking industry requires a deep hydrolysis of starch. Hydrolysis is usually carried out in two stages: in the first stage, α-amylase is treated (starch liquefaction stage), then the gelatinized and liquefied suspension is treated at a temperature not exceeding 60 ^° C and pH 5.0-5.5 is treated with glucoamylase (starch saccharification stage). As a result of the joint action of enzymes, the degree of hydrolysis of starch reaches 98-99%.

 However, despite the high profitability of this technology, the efficiency of the enzymatic processing of starch can be increased through the use of highly active enzyme preparations. At the same time, a significant reduction in the cost of enzymatic processing of raw materials is achieved by reducing the dosage of enzymes and the duration of the hydrolysis process, a high degree of degradation of starch and, as a result, an increase in the yield of the final product are ensured.

Currently, in the industrial production of starch hydrolysis products - dextrose, glucose-fructose syrups, glucoamylases of producers belonging to the genus Aspergilllus: Asp are mainly used in the saccharification stage. niger. Asp. awamori, Asp. oryzae (1-5), the optimal conditions of action of which are pH 5.0, 55 ^o C.

 Recombinant and mutant strains of Aspergillus niger fungus glucoamylase producers are known. The strains described are: Asp. niger synthesizing 150 u / ml glucoamylase (1); Asp. niger V1-D, studies have been conducted on the effect of aeration conditions on microorganism growth and enzyme biosynthesis (2); Asp. niger N 402 In 1, obtained on the basis of a natural strain, contains 20 copies of the glucoamylase gene (3); Asp. niger B0 -1 (4). Asp. oryzae is a mutant that synthesizes both glucoamylase and amylase (5).

 The use of recombinant strains is associated with 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.

 Mold fungi of the Asp type are known and widely used for saccharification of starch-containing raw materials in the industrial production of dextrose, glucose-fructose syrups, ethanol. awamori.

 Active enough in relation to the synthesis of glucoamylase from known Asp. awamori are Asp. awamori 466, synthesizing when grown on a medium with corn flour using saccharification with malt milk and malt wort with diammonium phosphate for 184 h of growth of 183 u / ml of the enzyme (6), and Asp. awamori VUD T-2 F 203, when cultivated on a medium with corn flour during saccharification with malt milk or bacterial amylase, with special preparation of seed and a multi-stage process, it is possible to obtain glucoamylase activity of 200 units / ml for 130 hours of cultivation (7).

The closest analogue in terms of the combination of essential features and the achieved result is the strain Aspergillus awamori VNIIgenetics 120/177 CMPM F-166 (8). The strain grown on an agar medium with corn extract to cultivate the seed is cultivated on a medium containing starch, malt sprouts and mineral salts. The fermentation medium contains corn flour, corn extract, bacterial amylase. The cultivation is carried out at 28 ^{° C.} for 120 hours. The glucoamylase activity level is 240 units / ml. To increase the synthetic activity of the strain, a 2-stage preparation of seed is used, including first obtaining spore material on a medium with wheat bran, then germinating it on a liquid medium of a fermentation composition and subsequent cultivation on a medium containing corn flour, starch, IAC, amylosubtilin and diammonium phosphate, at 34 ^o C. The activity of glucoamylase for 120 hours of cultivation is 340 u / ml

 The disadvantage of the described strain is the need to obtain a sufficiently high activity of the culture fluid using multi-stage preparation of seed material and for the main fermentation process of the enriched nutrient medium.

 The problem to which the invention is directed, is the expansion of the arsenal of glucoamylase producing strains.

 The technical result consists in obtaining a new highly productive producer strain that does not require complex highly concentrated media and complex cultivation techniques for the synthesis of the product.

 The essence of the object of the invention is a new specially selected strain of Aspergillus awamori N 400 - producer of glucoamylase.

 The strain Aspergillus awamori is deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms named after G.K. Scriabin RAS under N VKM F- 3689 D.

 The strain is obtained using multistage genetic selection of the original strain Aspergillus awamori BKM F-758 using mutagenesis methods - ultraviolet radiation with simultaneous exposure to chemical mutagens.

The spore suspension of the initial strain (dilution titer of 10 ^-4 -10 ^-6 ) is irradiated with ultraviolet light in 20% glycerol for 4-5 minutes. Irradiated spores are plated on Petri dishes with selective media, with a survival rate of 1-2%. Inoculated plates are incubated for 7 days at 35 ^{° C.} and further incubated for 7 days at 22-23 ^° C. Each grown colony is subcultured into plates containing agarized media with the addition of starch as the sole carbon source.

 The intensity of the synthesis of glucoamylase mutant clones is tested by the ratio of the diameter of the starch bleach to the diameter of the colony (D = Dzpk / Dk). Clones with a D value of 2-4 times higher than in the control are tested for the effectiveness of the synthesis of glucoamylase during periodic cultivation on liquid media in flasks. The most productive option is again subjected to UV irradiation and selection, as described above.

Storage conditions: the strain can be stored in a lyophilized state for several years or on jambs with agarized Chapek or Maltz-agar medium at + 4 ^o C with mandatory reseeding at least once every 3 months.

Cultural and morphological characteristics of the strain
Macroscopic characteristics: colonies on agar Chapek with yeast extract (CYA), 25 ^o C, have a diameter of 70-71 mm / 7 days, radially grooved, the surface is velvety, the edge is thin, the conidial region is dark brown; exudate is absent, the reverse side is dull yellow.

Colonies on Chapek’s agar with yeast extract (CYA), 37 ^o С, have a diameter of 70-71 mm / 7 days. Cultural characteristics are similar to those at CYA, 25 ^o С.

Colonies on Chapek agar with yeast extract and 20% sucrose (CY20S), 25 ^o C, have a colony diameter of 68 - 70 mm / 7 days, slightly radially grooved, the surface is powdery, the edge is thin, the conidial region is dark brown; exudate is absent, the reverse side is dull yellow.

 Microscopic characteristics: conidial heads are spherical, then lohradial, decaying into separate columns, conidiophores are slightly colored in the terminal part, smooth-walled 250-1200 x 6 -12 microns, apical extensions are spherical 20-45 microns in diameter, covered with sterigma over the entire surface. Sterigma is predominantly two-tier, with formulas 6-16 x 3.5 -7 microns, phialids 5-8 x 2 - 4 microns. Conidia are spherical, 3.5-6 microns, smooth.

Physiological and biochemical properties of the strain
Sugar attitude
The strain culture assimilates glucose, sucrose, arabinose, raffinose, and weakly maltose, lactose, galactose, and rhamnose.

 Starch hydrolyzes to glucose.

 It assimilates ammonium salts of inorganic acids well. Consumes peptone, casein, amino acids. Peptonizes milk.

The temperature optimum of growth is 34-35 ^o C, pH 4.5-6.0. Aerobe.

 This type of mycelial fungus is not listed as pathogenic in the "Regulation on the procedure for recording, storing handling, dispensing and sending 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 intensity of sporulation, the color of spore pigment, colony coloration on the reverse side when grown on agarized media, a shorter growth cycle, the formation of more abundant biomass, and increased ability to glucoamylase biosynthesis during deep cultivation on liquid media.

 When the strain is screened on selective media, 3–6% of atypical colonies are cleaved, which indicates a high morphological stability of the strain and, therefore, its stability to glucoamylase production during prolonged cultivation.

The cultivation of the strain is carried out under aerobic conditions at a temperature of 35 ^o C for 120-144 hours, the pH of the medium is 5.2-5.5. For the growth of culture and biosynthesis of glucoamylase, starch, hydrolyzed starch, extrudate of cereal flour (corn, wheat, barley, rye), ammonium nitrogen, and corn extract can serve as a source of carbon and nitrogen.

 The strain has an increased ability for the synthesis of glucoamylase, and also produces small amounts of α-amylase, protease and β-glucanase.

 For the enzymatic treatment of starch-containing raw materials in the production of alcohol, the enzyme preparation can be used in the form of a culture fluid or in the form of concentrated preparations obtained by known biochemical methods, for example, ethanol precipitation from ultrafiltrate of a culture fluid.

Glucoamylase is active in a wide pH range - from 3.5 to 7.5 with an optimum at pH 4.5-5.0; in the temperature range from 30 to 65 ^o C with optimum at 60 - 62 ^o C. Glucoamylase is stable for 2 hours at 45 ^o C. Incubation at 55 ^o C for 2 hours led to a decrease in activity by 50%.

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

Example 1. Sowing material (inoculum) is obtained by growing the strain Aspergillus awamori BKM F-3689 D on a solid nutrient medium consisting of 50% wheat bran and 50% malt sprouts, moistened with tap water to 60%, the pH of the medium is 5.2-5 ,5. Spore seeds are grown in flasks containing 20-30 g of solid substrate under stationary conditions at a temperature of 35 ^o C for 5-6 days and at 22-23 ^o C for 7 days.

 The resulting seed in an amount of 0.08-0.1% by volume of the medium is introduced into rocking flasks with a volume of 750 ml, containing 100 ml of medium composition, g / l: corn flour - 240.0; tap water - the rest, the pH of the medium is 5.2-5.5.

The cultivation of the strain is carried out under aerobic conditions at a temperature of 35 ^o C on a shaker with 240 rpm for 120-144 hours. Every 24 hours, starting from 72 hours of growth, samples are taken in which the ability of the culture fluid to hydrolyze starch is determined.

 The method for determining the activity of glucoamylase is based on the determination of glucose formed during hydrolysis of soluble starch by an enzyme (9).

For a unit of glucoamylase activity, an amount of the enzyme is taken which, acting on soluble starch at 30 ^{° C.} and a pH of 4.7 for 1 minute, releases 1 μmol of glucose.

 The maximum glucoamylase activity in the culture fluid for 400 h of growth is 400 units / ml, for 144 h of growth - 450 units / ml. The specific glucoamylase activity in the culture fluid is 15.5 u / mg protein.

 Example 2. The method is carried out as in example 1, but in a fermentation medium instead of cornmeal, an extrudate of cornmeal in an amount of 250 g / l is used.

 The maximum glucoamylase activity in the culture fluid for 144 h of growth is 500 units / ml.

 Example 3. The method is carried out as in example 1, but the fermentation medium instead of cornmeal contains wheat flour in an amount of 180 g / L.

 The maximum glucoamylase activity in the culture fluid for 144 h of growth is 380 u / ml.

 Thus, the proposed strain Aspergillus awamori BKM F-3689 D can significantly increase glucoamylase activity in the culture fluid and, therefore, increase the efficiency of the use of enzyme preparations based on this strain in various fields of biotechnology.

 In addition, to obtain high productivity of the strain does not require the use of complex nutrient media and complex methods of cultivation of the producer. For cultivation, nutrient media traditionally used in industrial technologies for producing such enzyme preparations can be used.

LITERATURE
1. US patent N 4335208, CL 195-5, publ. 1982 g.

 2. Wongwicham A et. al .: Biotechnol. Bioeng, Nov 20; 65 (4), 1999.

 3. Withersy M et. al .: Biotechnol. Bioeng., 1998, Aug 20; 59 (4), 407-418.

 4. Pederson et. al. : Appl. Microbiol. Biotechnol., 2000, Mar 53 (3), 272-276.

 5. Bocring Sp .: Biotechnol. Bioeng, 1999, Dec 20; 65 (6), 638-648.

 6. USSR author's certificate N 800185, class C 12 D 13/10, publ. 1981

 7. Copyright certificate of the USSR N 1094346, class C 12 N 9/34, publ. 1985

 8. Copyright certificate of the USSR N 1271068, class C 12 N 9/34, publ. 1994

 9. GOST 20264-4.89.

Claims (1)

 The strain of mycelial fungus Aspergillus awamori BKMF-3689 D (All-Russian Collection of Microorganisms at the IBPM named after G.K. Scriabin RAS) is a producer of glucoamylase.