The strain of filamentous fungus aspergillus awamori - producer of glucoamylase

 

(57) Abstract:

The invention relates to biotechnology, can be used for saccharification of starchy raw materials in various food industries that require highly active enzyme preparations that are resistant to acidic pH values. A strain of Aspergillus awamori N 400 VKM F-D 3689 received multistage genetic breeding of the original strain of Aspergillus awamori VKM F-758 with the use of ultraviolet irradiation with simultaneous exposure to chemical mutagens. The strain has a high glucoamylase activity. Glucoamylase active in the pH range of 3.5-7.5 and a temperature of 30-65oC. To obtain a high productivity of the strain can be used nutrient medium, traditionally used in industrial technologies for obtaining this kind of enzyme preparations that do not require complex methods of cultivation of the producer.

The invention relates to the microbiological industry, and various areas of food biotechnology and can be used to obtain glucoamylase with the purpose of its further use for the enzymatic treatment of starch-containing raw material in the alcohol, starch, baking and brewing industry.

As a rule, natural micro-organisms form a complex of amylolytic enzymes able to hydrolyze plant substrates based on starchy carbohydrates. This complex is composed of amylase and glucoamylase, gidrolizuemye molecules of starch to amylase and glucoamylase, gidrolizuemye molecules of starch to glucose and dextrins of different molecular weight; protease that destroys the protein of raw materials to amino acids, which are a valuable nitrogenous food for the yeast; glucanase, which intensify the process of enzymatic processing of raw materials due to the hydrolysis of non-starchy polysaccharides; pectinase, pectin-destroying, other enzymes political action.

Received currently multienzyme preparations have different enzyme composition and differ in the level of activity of individual enzymes. Generally known multienzyme preparations with amylolytic activity need to improve their funkcionalnuyu enzymes with increased activity of glucoamylase.

Glucoamylase - (-1,4-glucan-glucanohydrolase, 3.2.1.3.) -glucosuria amylase, assofermet, attacks the starch with nereguliruemaia the end of the polysaccharide chain, consistently arseplay glucose residues and completely transforming the starch into glucose. Most of the selected glucoamylase microbial origin they have some common properties. Typically, this cyclotosaurus enzymes with optimum pH of 4.5-5.0, the optimum temperature of 55-60oC. in Addition to the specific-1,4 - glucan links glucoamylase hydrolyzing -1,6 link as polysaccharides and low molecular weight oligosaccharides. Under their influence the starch is completely hydrolyzed to glucose.

The ability glucoamylase to hydrolyze as -1,4, and -1,6 connection with the removal of glucose puts this enzyme in first place on the efficiency of hydrolysis of starch to further fermentation of the resulting sugar.

In alcohol, starch and syrup, baking industry deep hydrolysis of starch. Typically, the hydrolysis is carried out in two stages: the first stage spend processing-amylase (stage liquefaction of starch), then klasterizovannykh and diluted suspension at a temperature not exceeding 60oC and pH 5.0-5.5 stump hydrolysis of starch reaches 98-99%.

However, despite the high profitability of this technology, the efficiency of the process of the enzymatic treatment of starch can be enhanced through the use of highly active enzyme preparations. When this is achieved a significant reduction in the cost of the enzymatic processing of raw materials by reducing the dosage of enzymes and duration of the hydrolysis process, provides a high degree of degradation of starch and as a consequence increase the yield of the final product.

Currently, when industrial production of the hydrolysis products of starch - dextrose, glucosefructose syrups, at the stage of saccharification mainly use glucoamylase producers belonging to the genus Aspergilllus: Asp. niger. Asp. awamori, Asp. oryzae (1-5), optimal conditions actions which pH 5.0, 55oC.

Known recombinant and mutant strains producers of glucoamylase of the fungus Aspergillus niger. Described strains: Asp. niger, synthesizing 150 u/ml glucoamylase (1); Asp. niger B1-D., studies on the influence of conditions of aeration on the growth of the microorganism and the biosynthesis of the enzyme (2); Asp. niger 402 N 1 derived from natural strain, contains 20 copies of the gene of glucoamylase (3); Asp. niger B0 -1 (4). Asp. oryzae mutant that synthesizes as Ogadenia constant research on the maintenance of the strains in a stable and active state, the establishment of a special culture conditions that are not always available for industrial process control.

Well-known and widely used for saccharification of starch-containing raw materials in industrial production of dextrose, glucosefructose syrups, ethanol fungi species Asp. awamori.

Quite active in the synthesis of glucoamylase of known fungi Asp. awamori is Asp. awamori 466 synthesizing when grown on medium with corn flour using saccharification malt milk and malt wort with diammonium phosphate 184 h growth 183 u/ml of enzyme (6), and Asp. awamori WOOD T-2 F 203, the cultivation of which in the environment with corn flour with malt saccharification milk or bacterial amylase with a special preparation of inoculum and mnogostadiinost process manages 130 h of cultivation to get the activity glucoamylase equal to 200 IU/ml (7).

The closest analogue of essential features and the achieved result is a strain of Aspergillus awamori, Vniigenetika 120/177 CMPM F-166 (8). The strain grown on agar medium with corn extract, to obtain the inoculum is cultivated on a medium containing KRA is CT, bacterial amylase. The cultivation is carried out at 28oWith over 120 hours Level of activity glucoamylase is 240 IU/ml For increasing synthetic activity of the strain using 2-stage preparation of inoculum comprising first receiving spore material in the medium with wheat bran, then germination in liquid fermentation medium composition and subsequent culturing in a medium containing corn flour, starch, BVK, aminocoumarin and DAP, when the 34oC. Activity glucoamylase 120 h of cultivation is 340 u/ml

The disadvantage of this strain is the necessity to obtain a sufficiently high activity in the culture fluid using a multistage preparation of inoculum for the main fermentation process enriched nutrient medium.

The problem to which the invention is directed, expanding Arsenal of strains-producers of glucoamylase.

The technical result consists in obtaining new high-yielding strain-producer, does not require for the synthesis of very concentrated product of complex environments and complex methods of cultivation PCs is docent glucoamylase.

A strain of Aspergillus awamori deposited in the all-Russian collection of microorganisms at the Institute of biochemistry and physiology of microorganisms them. G. K. Skryabin RAS under N VKM F - 3689 D.

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

A spore suspension of the original strain (titer dilution of 10-4-10-6) is irradiated with ultraviolet light in 20% glycerol for 4-5 minutes Irradiated spores plated on Petri dishes with selective environments, the survival rate is 1-2%. Seeded Cup incubated for 7 days at 35oWith and additionally incubated 7 days at 22-23oC. Each grown colony subcultured on cups containing agar medium with addition as the sole carbon source starch.

The intensity of synthesis of glucoamylase mutant clones are tested for the ratio of the diameter of the enlightenment starch to the diameter of the colony (D=D/D). Clones having the value of D is 2 to 4 times higher than in the control test on the efficiency of synthesis of glucoamylase with periodic cultivation on MS is E.

Storage conditions: strain can be stored in a lyophilized state for several years or jambs with agar medium of čapek or the best agar at +4oWith mandatory replanting at least once in 3 months.

Cultural and morphological characteristics of the strain

Macroscopic characteristics: colonies on agar of čapek with yeast extract (CYA), 25oWith, have a diameter 70-71 mm/cut, radially furrowed, velvety surface, edge thin, conidial region dark brown; bleed and have exudate absent, reverse dull yellow.

Colonies on agar of čapek with yeast extract (CYA), 37oWith, have a diameter 70-71 mm/sut., cultural characteristics similar to those on CYA, 25oC.

Colonies on agar of čapek with yeast extract and 20% sucrose (CY20S), 25oWith, have a diameter of colony 68 - 70 mm/sut., weak radial-grooved, surface porosity, edge thin, conidial region dark brown; bleed and have exudate absent, reverse dull yellow.

Microscopic characteristics: conidial heads globose, then richardella decaying into separate columns, conidiophores slightly coloured in terminalname over the entire surface. Sterigma mostly bunk, Metulla 6-16 x 3.5 -7 μm, Fieldy 5-8 x 2 - 4 microns. Conidia globose, 3.5 to 6 μm, smooth.

Physiological and biochemical properties of the strain

Relation to sugars

Culture strains metabolizes glucose, sucrose, arabinose, raffinose, and poorly - maltose, lactose, galactose and rhamnose.

Starch hydrolyses to glucose.

Well assimilates ammonium salts of inorganic acids. Consumes peptone, casein, amino acids. Peptonized milk.

Temperature optimum growth 34-35oWith a pH of 4.5-6.0. Aerobe.

This filamentous fungus is not listed as pathogenic in the "Regulations on the procedure for accounting, storage, handling, issuance and delivery of cultures of bacteria, viruses, Rickettsia, fungi, protozoa, Mycoplasma, bacterial toxins, poisons of biological origin".

The resulting mutant in their morphological properties different from the original intensity of sporulation, the color pigment dispute, the color of the colony on the reverse side when grown on agar media, a shorter growth cycle, the formation of the more abundant biomass, increased ability to biosynthesis of glucoamylase with globin is of 3-6% atypical colonies, that indicates a high morphological stability of the strain, and therefore, its stability in the products of glucoamylase during prolonged cultivation.

Cultivation of the strain is carried out in aerobic conditions at a temperature of 35oWith over 120-144 h, pH of 5.2 to 5.5. For culture growth and biosynthesis of glucoamylase source of carbon and nitrogen can serve as starch, hydrolyzed starch, flour extrudate cereals (corn, wheat, barley, rye), ammonia nitrogen, corn extract.

The strain has a high potential for the synthesis of glucoamylase, and also produces minor amounts of amylase, protease and glucanase.

For enzymatic treatment of starch-containing raw material in the production of alcohol enzyme preparation can be used in the form of cultural liquid or in the form of concentrated products known biochemical methods, such as precipitation with ethanol from ultrafiltrate cultural liquid.

Glucoamylase active in a wide range of pH from 3.5 to 7.5 with an optimum at pH 4.5 to 5.0; in the temperature range from 30 to 65oWith optimum at 60 - 62oC. Glucoamylase stable for 2P CLASS="ptx2">

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

Example 1. Seed material (AMF inoculum) is produced by cultivation of a strain of Aspergillus awamori BKM F-3689 D on solid nutrient medium consisting of 50% wheat bran and 50% malt sprouts, moistened with tap water up to 60%, pH of 5.2 to 5.5. Vyrashivayu spore inoculum is carried out in flasks containing 20-30 g of a solid substrate, in stationary conditions at a temperature of 35oC for 5-6 days and when 22-23oC for 7 days.

Received seed in the amount of 0.08 to 0.1% by volume environment contribute in Catalogne flask 750 ml containing 100 ml of medium composition, g/l: corn flour - 240,0; tap water - the rest, pH 5,2-5,5.

Cultivation of the strain is carried out in aerobic conditions at a temperature of 35oOn the rocking chair with 240 rpm for 120-144 hours Every 24 hours, starting from 72 h of growth, take samples, which determine the capacity of the culture fluid to hydrolyze starch.

Method for determining the activity of glucoamylase based on the determination of glucose formed by hydrolysis of soluble starch is acting on soluble starch at 30oC and a pH of 4.7 for 1 min, liberates 1 µmol of glucose.

Maximum glucoamylase activity in the culture fluid at 120 h of growth is 400 IU/ml at 144 h of growth - 450 u/ml Specific glucoamylase activity in the culture fluid equal to 15.5 IU/mg protein.

Example 2. The method is carried out as in example 1, but in a fermentation medium instead of corn flour used extrudate (corn flour) in the amount of 250 g/l

Maximum glucoamylase activity in the culture fluid at 144 h of growth 500 u/ml

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

Maximum glucoamylase activity in the culture fluid at 144 h of growth 380 IU/ml

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

In addition, to obtain high production strain does not require the use of complex nutrient media and complex p is used in industrial production technologies such enzyme preparations.

LITERATURE

1. U.S. patent N 4335208, CL 195-5, publ. 1982

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. Environ. 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, CL 12 D 13/10, publ. 1981

7. USSR author's certificate N 1094346, CL 12 N 9/34, publ. 1985

8. USSR author's certificate N 1271068, CL 12 N 9/34, publ. 1994

9. GOST 20264-4.89.

The strain of filamentous fungus Aspergillus awamori BKMF-D 3689 (all-Russian collection of microorganisms at ibfm them. G. K. Skryabin RAS) - producer of glucoamylase.

 

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