RU2324734C1 - BACTERIA STRAIN BACILLUS LICHENIFORMIS- "BKM B-2396 D" - PRODUCENT OF THERMOSTABLE α -AMYLASE - Google Patents

Abstract

FIELD: chemistry, organic.

SUBSTANCE: invention relates to biotechnology and may be used for production of enzyme preparations of thermostable α-amylase. The invention enables one to increase output of thermostable α-amylase.

EFFECT: increase of thermostable α-amylase output.

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Description

The invention relates to the field of biotechnology and can be used in the microbiological industry for the production of thermostable α-amylase enzyme preparations used in various industries.

Amylolytic enzymes are widely used to liquefy or convert starch and starch-containing plant materials into products such as maltodextrins, sugar syrups, dextrose, maltose, glucose, etc.

In most industrial processes, the use of thermostable amylolytic enzymes is necessary, due to their ability to hydrolyze raw materials at high temperatures - 80-100 ° C. The advantage of the high-temperature process is the combination of the gelatinization process of starch and its enzymatic hydrolysis, a reduction in the dosage of enzymes, a reduction in the duration of the conversion of starch, an increase in the yield of the final product, and a reduction in the cost of the process.

In world practice, bacteria of the genus Bacillus are most widely used as producers of commercial preparations of α-amylases: B.subtilis, B.licheniformis, B.amyloliquefaciens, B.stearothermophilus, etc. (1-9).

In natural strains, the ability to biosynthesize thermostable amylolytic enzymes is rarely detected (2-3, 7, 9). As a rule, the level of amylolytic activity of these producers is significantly inferior to the productivity of mesophilic strains. Recombinant strains producing thermostable α-amylase are known, studies have been conducted to increase the thermostability of mesophilic α-amylases by introducing disulfide bonds into the amino acid sequence of the enzyme molecule (10-14). However, the use of recombinant strains significantly complicates the process of obtaining industrial enzyme preparations based on them and, accordingly, increases the cost of the technology and the cost of the final product.

In Russia, the preparation of bacterial α-amylase based on B.subtilis - Amylosubtilin is currently being produced on an industrial scale. However, this α-amylase does not belong to the category of thermostable; its optimum temperature is 65-70 ° С.

Known (SU, copyright certificate 1788966 C 12 N 1/21, 1993), the bacterial strain Bacillus amylollquefaciens is a producer of Bacillus licheniformic α-amylase, which has a registration number VKPM B-5208 in the All-Union Collection of Industrial Microorganisms and secrets 220-250 units. enzyme activity per 1 ml of culture fluid. The activity of α-amylase was determined in accordance with the method of Gosstandart GOST 20264.4-74.

Known (SU, copyright certificate 1344247, C12N 9/32, 1987) a strain of Bacillus licheniformis ATCC 39326 - producer of thermostable α-amylase. When cultivating the strain on a medium containing lactose, a nitrogen source, mineral components and water at a pH of 5.5-8.0, thermostable α-amylase with an activity of 1500-2000 Lycone-ml is obtained after 72 hours of cultivation.

Known (RU, patent No. 2001103, C12N 9/26, 1993) is the aerobic thermophile B.licheniformis, which, when cultivated on a medium with cornmeal, is optimal for the preparation of the product synthesizes a complex of extracellular amylolytic enzymes: α-amylase, α-glucosidase and pullulanase. A known strain isolated from a natural soil association, produced under specified conditions, at least three thermostable amylolytic enzymes active in the pH range of 5.0-10.0, active at 30-100 ° C with maximum enzyme activity in the culture fluid at 90-95 ° FROM. The strain is thermostable in aqueous solutions of pH 6.0-8.5 for 40 minutes. The activity of α-amylase is 1.78 units / ml / min, α-glucosidase is 3.49 units / ml / min and pullulanase is 0.68 units / ml / min.

The closest in technical essence to the proposed invention is a strain of B.licheniformis-78 (deposited in the All-Russian collection of microorganisms under the number VKM B-2184 D). The cultivation of the strain is carried out at 40 ° C for 80-100 hours on a flour medium composition, g / l: corn flour - 160.0; feed yeast - 30.0; NaH ₂ PO ₄ - 3.0; MgSO ₄ - 2.0; CaCO ₃ - 2.5; pH 7.2-7.4. Under these conditions, the strain produces a complex of thermostable enzymes, including α-amylase and pullulanase, and has the concomitant proteolytic and cellulolytic activities, providing a deeper and more efficient hydrolysis of complex raw materials. The maximum activity of α-amylase is 200-230 units / ml.

α-Amylase synthesized by the producer is characterized by a wide pH range of activity of pH 5.0-10.0 and the ability to hydrolyze the substrate at high temperatures of 90-105 ° C.

The disadvantage of the strain is the relatively low level of amylolytic activity, which prevents the creation of a cost-effective technology for the production of thermostable α-amylase enzyme preparation.

The technical problem to which the claimed invention is directed is to obtain a new highly active producer that provides a high level of activity of thermostable α-amylase.

The technical result that can be obtained by carrying out the invention is to provide a high level of activity of thermostable α-amylase in a fermentation medium.

The essence of the object of the invention is a new specially selected strain of bacteria Bacillus licheniformis 103 - producer of a complex of thermostable amylolytic enzymes.

The bacterial strain B.licheniformis 103 is 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-B-2396-D.

The bacterial strain B.licheniformis 103 was isolated by selection in the study of the natural variability of the strain B.licheniformis BKM B-2184 D using methods of effective mutagenesis by ultraviolet irradiation and treatment with nitrosoguanidine.

Storage conditions. The strain can be stored without loss of useful properties at room temperature on the jambs with an agar medium of the following composition: meat and peptone broth and malt wort (7 ° B) in a ratio of 1: 1; soluble starch 1.0%; agar agar 2.0%; pH of 7.5, at least 6 months with mandatory reseeding at least once every 2 months.

The main method for long-term storage of the strain is the lyophilization method.

Cultural and morphological characteristics of the strain.

Cells are gram-positive, single, motile rods of size 0.6-0.8 and 0.2-0.3 microns, spore-forming. In the first hours of growth (logarithmic phase), chains of 2-4 cells of a more elongated shape form, by 48-56 hours of growth (stationary phase), the chains disintegrate, the cells thicken, spores appearing in a central position and oval in shape.

On meat and peptone agar (40 ° C, 24 h), the strain gives abundant growth, irregularly shaped colonies, flat, mucous, smooth, opaque, 3-6 mm in diameter with even edges, creamy at the beginning of growth, then gradually turning pink.

When grown on meat and peptone broth (40 ° C, 24 hours) - abundant cell growth, the solution becomes cloudy, a creamy pink tint appears by 24 hours of growth.

Abundant growth and the formation of hydrolysis zones around colonies are observed on an agar medium with starch. Colonies of irregular shape, mucous, smooth, opaque, 2-4 mm in diameter, with smooth edges. By 72 hours a brownish pink color appears.

Physiological and biochemical properties.

Aerobe. The temperature range of growth is 18-45 ° C. The optimum growth temperature is 40 ° C. It grows at pH values of 5.0-10.0. The optimum pH of growth is 6.5-8.0.

Starch quickly hydrolyzes, gelatin dilutes. Restores litmus milk. Nitrates reduces to nitrites. Synthesizes tyrosinase, forms hydrogen sulfide. It ferments with the formation of acid arabinose, xylose, fructose, sucrose, trehalose, maltose, raffinose, mannose, mannitol, sorbitol, glycerin, dextrin, glycogen without gas evolution, ferments glucose anaerobically, utilizes citric acid.

The strain is able to grow on environments with ammonium salts as the sole source of nitrogen.

Based on these properties, the strain BKM-B-2396-D was identified by the Bergi Determinator (15) as a strain of Bacillus licheniformis.

The species B.licheniformis, to which the strain B.licheniformis BKM-B-2396-D is assigned, 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 ”, is not in the lists of pathogenic bacteria according to the Regulation of the Ministry of Health and in the lists of pathogenic bacteria in Official Journal of the European Communities NC 217/3237, 08.24.92.

The obtained B.licheniformis 103 BKM-B-2396-D mutant differs from the closest B.licheniformis BKM B-2184 D mutant with a flatter colony shape and a denser mucous consistency. Strain BKM-B-2396-D, like BKM B-2184 D, retains antibiotic streptomycin resistance, indicating a 30S mutation of the ribosome subunit, and the ability to biosynthesis of concomitant enzymes: pullulanase, protease, β-glucanase, β-laminarinase.

For the growth of culture and biosynthesis of thermostable α-amylase, starch, hydrolyzed starch, maltose, corn and other grain flour can serve as a carbon source. The source of nitrogen is soy flour, yeast cells.

The cultivation of the strain is carried out under aerobic conditions at a temperature of 40 ° C for 80-120 hours, a pH of 7.0-8.5. Every 24 hours, starting from 48 hours of growth, samples are taken in which the amylolytic activity and, if necessary, the activity of concomitant enzymes are determined.

The activity of α-amylase in the culture fluid is determined by the ability to catalyze the hydrolysis of starch to dextrins of various molecular weights (16). The unit of activity is the amount of enzyme that catalyzes the hydrolysis of 1 g of soluble starch for 10 minutes at 90 ° C at pH 7.5, which makes up 30% of the reaction.

If necessary, determine the activity of concomitant enzymes according to the following methods.

The activity of pullulanase is determined by the reducing sugars formed during the hydrolysis of pullulan (a linear polysaccharide consisting of maltotriose units connected by α-1,6-glycosidic bonds). The unit of activity is the amount of the enzyme that, at 60 ° С, pH 6.0 hydrolyzes pullulan at a rate corresponding to the formation of reducing groups equivalent to 1 μmol of glucose per minute, determined by the Somogy-Nelson method (17).

Proteolytic activity is determined according to the Kaverzneva method using a 2% casein solution as a substrate (18). A unit of proteolytic activity (PE) is such an amount of an enzyme that in 1 minute at 30 ° C catalyzes the transition to the non-precipitated TCA state of such an amount of casein that contains 1 μmol tyrosine.

β-Glucanase activity is determined by the ability to form reducing sugars from barley β-glucan, followed by the determination of sugars formed by the Somogy-Nelson method (17). The unit of activity is taken as the amount of enzyme capable of producing standard amounts of oligosaccharides of β-glucan per minute under standard conditions at a temperature of 50 ° C and pH 5.5 from β-glucan, which corresponds to 1 μmol of glucose in terms of reducing ability. The activity of β-laminarinase is determined in a similar way, but laminarine is used as a substrate.

The presence of concomitant pullulanase activity allows hydrolysis of not only α-1,4-, but also α-1,6-glycosidic bonds in the starch molecule to dextrins with the formation of glucose and low molecular weight maltooligosaccharides.

Enzyme preparations obtained using the proposed strain can be used in the form of a culture fluid, in the form of concentrated concentrated preparations obtained by ultrafiltration or evaporation of a culture fluid, or in the form of dry preparations obtained by precipitation of ethanol or other organic solvents from an ultrafiltrate of a culture fluid, as well as drying or granulating.

α-amylase is active in the range of pH 5.0-10.0 with two noticeable activity peaks at pH 7.5 and 8.5; at a temperature of 30-105 ° C with optimum activity at 90-95 ° C. The enzyme is able to maintain activity unchanged in aqueous solutions at a temperature of 90 ° C and pH 6.0-8.5 for 40 minutes, in the presence of calcium ions (0.005M) - 2 hours; at a temperature of 100 ° C in the presence of calcium ions, the activity does not change for 20 minutes.

Thus, the properties of the enzyme α-amylase and the presence of concomitant activities ensure the effectiveness of the use of the enzyme preparation in the bioconversion of plant 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. Inoculum (inoculum) is obtained by growing a strain on a liquid nutrient medium composition: soluble starch - 1%; meat and peptone broth and malt wort in a ratio of 1: 1, pH 7.5. Cultivation is carried out in flasks containing 50 ml of sterile medium, on a shaker with 240 rpm at 38-40 ° C for 48 hours.

The resulting seed in an amount of 1% by volume of the medium is introduced into rocking flasks with a volume of 750 ml, containing 50 ml of medium composition, g / l: corn flour - 150.0; soy flour - 30.0; CaCO ₃ - 2.5; MgSO ₄ × 7H ₂ O - 2.0, pH of the medium 7.0-7.5.

The medium is pre-treated with an enzymatic preparation of bacterial α-amylase 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 40-42 ° C.

The maximum enzymatic activity of thermostable α-amylase per 120 h of growth is 187 units / ml.

The activities of concomitant enzymes are: pullulanase - 50 units / l, protease - 40 units / ml, β-glucanase - 42 units / ml, β-laminarinase - 18 units / ml (Table 1).

Table 1.
The dynamics of the accumulation of enzyme activity during the cultivation of a strain of Bacillus licheniformis BKM-B-2396-D. Age of culture, h The activity of the culture fluid α-amylase, units / ml pullulanase, u / l protease, units / ml β-glucanase, units / ml β-laminarinase, units / ml 48 42 2 26 3 0 72 87 eighteen 80 eleven 5 96 134 32 73 38 12 120 187 fifty 40 42 eighteen

Example 2. Cultivation is carried out in rocking flasks as described in example 1, using a liquid nutrient medium of the following composition, g / l: corn flour - 80.0; wheat flour - 80.0; soy flour - 30.0; CaCO ₃ - 2.5; MgSO ₄ × 7H ₂ O - 2.0, pH of the medium 7.0-7.5.

The maximum enzymatic activity of thermostable α-amylase at 120 h of growth is 245 units / ml.

Example 3. The cultivation is carried out in rocking flasks as described in example 1, using a liquid nutrient medium of the following composition, g / l: corn flour extrudate - 80.0; wheat flour extrudate - 80.0; soy flour - 30.0; CaCO ₃ - 2.5; MgSO ₄ × 7H ₂ O - 2.0, pH of the medium 7.0-7.5.

The maximum enzymatic activity of thermostable α-amylase at 120 h of growth is 250 u / ml.

Example 4. The process of cultivation is carried out in laboratory fermenters of the company "Anglican" (UK), with a capacity of 3 l, equipped with automatic control systems for pH, pO ₂ , temperature and defoaming. Mass transfer is carried out using a bubbler and a 3-tier mixer, providing aeration - up to 15 g O ₂ / l · h.

Cultivation conditions: loading coefficient 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 41 ° C, then 38 ° C.

The composition of the fermentation medium, g / l: corn flour extrudate - 150.0; soy flour - 20.0; CaCO ₃ - 2.5; MgSO ₄ × 7H ₂ O - 2.0, pH of the medium 7.0-7.5.

At 24 and 30 hours of cultivation, make-up is made in the form of a 50% hydrolyzate of cornmeal extrudate in an amount of 5% of the volume of the fermentation medium.

The activity of thermostable α-amylase for 120 hours of cultivation is 260 units / ml.

Thus, the inventive strain of Bacillus licheniformis VKM-B-2396-D has an increased ability to synthesize thermostable α-amylase.

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 thermostable α-amylase and the presence of concomitant enzymes allow us to consider the strain as a producer of an enzymatic complex, promising for the processing of starch or starch-containing raw materials with the aim of deep hydrolysis.

Claims (1)

The bacterial strain Bacillus licheniformis BKM B-2396 D is a producer of thermostable α-amylase.