RU2177995C2 - Strain of bacterium bacillus licheniformis as producer of thermostable amylolytic and proteolytic enzymes - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry. SUBSTANCE: invention relates to the strain of bacterium Bacillus licheniformis VKM B-2184D isolated by selection method in study of natural variability of the strain VKPM B-6508 using methods of effective mutagenesis. The strain produces complex consisting of five thermostable amylolytic and proteolytic enzymes, not less. The strain shows activity in the range of pH 5.0-11.0 and temperature 30-105 C with maximal activity of alpha-amylase, pullulanase and protease at 90-95 C, 75 C and 60 C, respectively. The strain shows promise for simultaneous treatment of starch or starch-containing raw to perform its profound hydrolysis and as a producer of alkaline proteases for profound digestion of proteins to amino acids. The strain shows the enhanced ability to synthesize the complex of thermostable amylolytic and proteolytic enzymes and eliciting activity of beta-glucanase and laminarinase also. Invention can be used in alcoholic, starch-syrup, brewing, baking, confectionary, fruit-berry and textile productions where thermostable enzymes are required. EFFECT: strain indicated above, valuable properties. 1 tbl, 2 ex

Description

 The invention relates to the field of biotechnology, namely to obtain a complex of amylolytic and proteolytic enzymes, and can be used in the microbiological industry.

 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 the industrial use of amylolytic enzymes, thermostable enzymes are necessary, due to their ability to hydrolyze the feed at high temperatures - 80-100 ^o C, which is very important from the point of view of starch gelatinization. In this case, two processes are combined: gelatinization of starch and its enzymatic hydrolysis. The cost of the enzymatic process is significantly reduced due to a reduction in the dosage of the enzyme and the duration of starch hydrolysis, the preparation of raw materials for boiling is better done - a high degree of starch hydrolysis is ensured, and, as a result, the yield of the target product increases.

Deep hydrolysis of starch is necessary in the alcohol, starch and syrup, textile and other branches of biotechnology. Typically, hydrolysis is carried out in two stages: in the first stage, alpha-amylase is treated at a temperature of 65-70 ^o C and a pH of 6.0-6.5, then the solution is cooled to 58 ^o C, acidified to pH 4.7 and glucoamylase or beta amylase. As a result of the combined action of the enzymes alpha-amylase and glucoamylase, the degree of hydrolysis of starch reaches 97-98%. However, despite the high degree of degradation of starch, the process of enzymatic treatment still needs to be improved.

 The disadvantage of the described amylolysis process is the incompleteness of the conversion of raw materials, since a part of the glycosidic bonds, namely the alpha-1,6-bond, in the starch molecule remains inaccessible to the action of these enzymes. In addition, a decrease in the temperature of the hydrolyzable material and an increase in the ion concentration with a change in pH, which are necessary before the second stage of the enzymatic treatment, lead to unproductive energy costs and difficulties in cleaning the hydrolysis product, respectively.

 To eliminate these drawbacks, it is necessary to use preparations containing a complex of hydrolytic enzymes of various specificity (natural or artificially created), working with the same parameters, while the complex should contain one of the branching enzymes (pullulanase or beta-amylase) capable of hydrolyzing alpha-1, 6-glycosidic bonds in the molecule of starch and maltooligodextrins.

 The efficiency of the bioconversion process: plant materials increases significantly when using an enzymatic complex containing, in addition to amylolytic enzymes, active cellulases and proteases. Thus, in the processing of starchy raw materials, it is advisable to hydrolyze not only starch, but also the cell walls of plant materials, as well as shells and films, which will provide better contact between starch and amylolytic enzymes, increase the utilization of dry matter of raw materials and thereby increase the yield of the final product. The presence of proteolytic action in the complex of enzymes allows hydrolyzing the proteins, peptones and polypeptides of the feed to amino acids, which are valuable nitrogenous nutrition for yeast.

 The implementation of biotechnology for the use of proteolytic enzymes together with amylolytic enzymes in the production of ethanol can reduce the duration of alcoholic fermentation by 40% with a simultaneous increase in ethanol yield by 2.8%, process highly concentrated grain wort (1).

 The presence of glucanase in enzyme preparations is useful, which intensifies the process of hydrolysis of plant materials and helps to increase the yield of alcohol by hydrolysis of other polysaccharides of the raw materials.

 The vast majority of producers of amylolytic enzymes are characterized by the preferred biosynthesis of one of the enzymes. The ability to form a complex of extracellular amylolytic enzymes, especially thermostable, is very rare. In this regard, a number of thermostable anaerobes from the genus Clostridium can be called.

 A known strain of Clostridium thermohidrosulfuricum synthesizing a complex containing thermostable pullulanase and glucoamylase (2).

 The literature describes several anaerobic thermophiles from the genus Clostridium, which carry out the biosynthesis of amylase and pullulanase (3) or alpha-amylase and glucoamylase active at high temperature (4).

 However, the complexity of the cultivation of anaerobic bacteria in the factory makes this method of obtaining a preparation of amylolytic enzymes practically unsuitable.

 From literature data it is known that the most thermostable hydrolytic enzymes, such as alpha-amylases, pullulanases, and in some cases proteases, are enzymes produced by the thermophilic bacteria Bacillus licheniformis.

 The aerobic thermophile Bacillus licheniformis is known, which, when cultivated on a medium with cornmeal, has the optimal composition for synthesizing a product, it synthesizes a complex of extracellular amylolytic enzymes - alpha-amylase and pullulanase in quantities of 2 units / ml and 0.68 units / l, respectively (5). The disadvantage of this strain is the low activity of the produced enzymes, a narrow spectrum of the complex and the duration of the cultivation process (144 hours).

The closest analogue in terms of the set of essential features and the achieved result is the Bacillus licheniformis strain (deposited in the All-Union Collection of Industrial Microorganisms under the number VKPM B-6508), which forms a complex of amylolytic enzymes on a composition medium, g / l: corn flour -160.0; feed yeast - 30.0; NaH ₂ PO ₄ - 3.0; MgSO ₄ - 20.0; CaCO ₃ - 2.5; pH 7.2-7.4 sequentially treated with alpha-amylase and glucoamylase (6). The complex contains thermostable alpha-amylase (up to 69.0 units / ml), and also, as we have determined, has pullulanase (0.9 units / l) and protease (4.7 units / ml) activities.

 The disadvantages of the strain are the relatively low level of activity, as well as the high cost of cultivation due to the use of a highly concentrated nutrient medium and the duration of the process.

 The problem to which the invention is directed, is the creation of a thermostable strain that provides the synthesis of a highly active complex of hydrolytic enzymes of different specificity, active at high temperature and working in close conditions.

 The technical result that can be obtained by carrying out the invention is to obtain a strain having an increased ability to synthesize a complex of thermostable amylolytic and proteolytic enzymes, which also has beta-glucanase and laminarinase activities.

 The essence of the object of the invention is a strain of Bacillus licheniformis VKM B-2184 D - producer of a complex of thermostable amylolytic and proteolytic enzymes.

 A new strain of Bacillus licheniformis - 78 was isolated by selection by studying the natural variability of the VKPM - 6508 strain using methods of effective mutagenesis by ultraviolet irradiation and treatment with nitrosoguanidine.

 The resulting bacterial strain Bacillus licheniformis-78 was deposited in the All-Russian collection of microorganisms under N В-2184 D.

Strain characteristics
Cultural and morphological characters. 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 2-3 cells of a more elongated shape are formed, by 48-56 hours of growth (stationary phase), the chains disintegrate, cells thicken, spores appear having a central position and an oval shape.

Meat peptone agar (t 40 ^o C, 24 hours). The strain gives abundant growth, colonies of irregular shape with a raised center, mucous, smooth, opaque, 2-5 mm in diameter with smooth edges, at the beginning of growth - cream, then gradually turning pink.

Meat peptone broth (t 40 ^o C, 24 hours). Abundant cell growth, the solution becomes cloudy, a creamy pink hue appears by 24 hours of growth.

 Agar medium with casein or microbial cells. Abundant growth and the formation of hydrolysis zones around the colonies. Colonies of irregular shape, mucous, smooth, opaque, with a raised center, 2-6 mm in diameter, with smooth edges. By 72 hours of growth, the colonies have a brownish-burgundy color.

 Physiological and biochemical properties.

 Aerobe.

The temperature range of growth is 18-45 ^o C.

The optimum growth temperature is 40 ^o C.

 It grows at pH values of 5.0 - 10.0.

The optimum pH of growth is 40 ^o C.

 Gelatin liquefies. Starch and casein quickly hydrolyze. Restores litmus milk.

 Nitrates reduces to nitrites. Forms tyrosinase (catalase-positive), forms hydrogen sulfide. Causes hemolysis.

 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, as well as on a medium containing yeast cells (pressed baker's yeast, fodder yeast or IAC), as a source of carbon and nitrogen nutrition.

 The main method for storing the strain is lyophilization.

 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-peptone broth and malt wort (7 B) in a ratio of 1: 1; soluble starch 1.0%; agar-agar - 2.5%; pH of 7.5, at least 6 months with mandatory reseeding at least once every 2 months.

 Based on these properties, the VKM strain B-2184 was identified by the Bergi Identifier (7) as a strain of Bacillus licheniformis.

 The species Bacillus licheniformis, to which the strain is assigned, is not listed 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 strain of Bacillus licheniformis VKM B-2184 D differs from the strain of Bacillus licheniformis VKPM B-6508 closest to it by the greater pigmentation of the colonies (from creamy pink to brownish burgundy) with growth on agarized growth media. Modifications resulting from mutations of the 3 OS ribosome strain of you. licheniformis VKPM B-6508 led to a change in the level of synthesis of the main enzymes - alpha-amylase, pullulanase and protease, and also contributed to the production of concomitant enzymes - beta-glucanase and beta-laminarinase.

 The strain Bacillus licheniformis BKM B-2184 produces at least five extracellular enzymes, the main of which are thermostable alpha-amylase, pullulanase, protease, as well as the concomitant enzymes beta-glucanase and beta-laminarinase. Enzymes are active at close pH and temperature.

 For the growth of culture and the biosynthesis of hydrolytic enzymes, starch, hydrolyzed starch, maltose, corn and other grain flour, or their extracts, yeast cells, can serve as a carbon source. Sources of nitrogen are peptone, yeast extract, yeast cells, urea, ammonia nitrogen.

The cultivation of the strain is carried out under aerobic conditions at a temperature of 40 ^o C for 80-100 hours, a pH of 7.5-7.8. Every 24 hours, starting from 48 hours of growth, samples are taken in which the ability of the culture fluid is determined to hydrolyze starch, pullulan, casein, beta-glucan, laminarin.

Alpha amylase activity in the culture fluid is determined by the ability to catalyze the hydrolysis of starch to dextrins of various molecular weights (8). The unit of activity is taken as such an amount of enzymes that within 10 minutes at 90 ^° C and pH 7.5 catalyzes the hydrolysis of 1 g of soluble starch, which is 30% of the reaction.

The activity of pullulanase is also determined by the reducing sugars formed during the hydrolysis of pullallan (a linear polysaccharide consisting of maltotriose units connected by alpha-1,6-glycosidic bonds). The unit of activity is the amount of the enzyme which, at 60 ^° C, pH 6.0 hydrolyzes pullallan at a rate corresponding to the formation of reducing groups equivalent to 1 μmol of glucose per minute, determined by the Somogy-Nelson method (9).

Proteolytic activity is determined according to the Kaverzneva method using a 2% casein solution as a substrate (10). 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.

 Beta-glucanase activity is determined by the ability to form reducing sugars from barley beta-glucan, followed by the determination of sugars formed by the Somogy-Nelson method (9). The unit of activity is the amount of enzyme capable of per minute under standard conditions at a temperature of 30 degrees. and pH 5.5 to obtain from glucan such an amount of oligosaccharides that according to its reducing ability corresponds to 1 μmol of glucose.

 The activity of beta-laminarinase is determined in a similar way, but laminarine is used as a substrate.

 The enzymatic complex can be used in the form of a culture fluid when used in enzymatic processing in the production of alcohol or isolated by known biochemical methods, for example, ethanol precipitation from ultrafiltrate of a culture fluid. If necessary, individual enzymes can be isolated from the complex by traditional methods.

 Due to the presence of pullulanase activity, the complex hydrolyzes not only alpha-1,4-, but also alpha-1,6-glycosidic bonds in the starch and dextrin molecule with the formation of glucose and low molecular weight maltooligosaccharides.

Alpha-amylase complex 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 ^o С with an optimum activity at 90-95 ^o С. The enzyme is able to maintain activity unchanged in aqueous solutions at temperatures of 90 ^o С and pH 6.0-8.5 for 40 minutes, in the presence of calcium ions ( 0.005 M) - 2 hours; at a temperature of 100 ^o C in the presence of calcium ions, the activity does not change for 20 minutes.

Pullulanase is active in the range of pH 5.0-9.5 with optimum activity at pH 6.0-6.5; in the temperature range 30-80 ^o With activity optimum at 75 ^o C. The period of complete stability at 70 ^o C - 15 minutes

Protease is active over a wide pH range from 6.5 to 11.0, with optimum at pH 9.0; in the temperature range of 30-70 ^o C with an optimum activity of 60 ^o C. The period of complete stability of the enzyme at 70 ^o C is 10 minutes, at pH 11 and a temperature of 60 ^o C retains 60% of the activity.

 Thus, the main enzymes of the complex are active at close pH and temperature, which ensures the efficient use of the complex in bioconversion of plant materials.

 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. Inoculum (inoculum) is obtained by growing a strain on a liquid nutrient medium composition: soluble starch - 1%; meat-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 rocking chair with 240 rpm at 40 ^o C for 36-48 hours.

The resulting seed in an amount of 0.3% 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 - 120.0; dry fodder yeast - 20.0; CaCO ₃ - 2.5; MgSO ₄ • 7H ₂ 0 - 2.5, pH of the medium 7.5-7.8. The medium is pretreated with amylosubtilin Gzx at the rate of 2 u / g starch at 50 ^{° C.} for 20 minutes. Then, the liquefied mass is saccharified by glucavamorin G 3X at the rate of 6 units / g of starch until the blue color of the iodine sample disappears and sterilized in the usual way.

 The maximum enzymatic activity of the culture fluid at 120 hours of growth is: alpha-amylase - 148.7 units / ml; pullulanase - 29.2 u / l; protease - 40.1 u / ml; - beta-glucanase - 41.5 u / ml, beta-laminarinase - 17.8 u / ml (table).

 Example 2. The method is carried out as in example 1, but instead of cornmeal, the medium contains an extrudate of cornmeal in an amount of 100 g / l.

 The maximum enzymatic activity of the culture fluid at 96 hours of growth is: alpha-amylase - 160.0 units / ml; pullulanase - 32.1 units / l; protease - 44.0 u / ml; - beta-glucanase - 45.1 units / ml, beta-laminarinase - 19.6 units / ml.

 Thus, the inventive strain of Bacillus licheniformis BKM B-2184 D has an increased ability to synthesize a complex of thermostable amylolytic and proteolytic enzymes, which has a wide range of enzymes of various specificity, including beta-glucanase and beta-laminarinase activities.

 The main among the produced enzymes are thermostable - amylase, pullulanase and protease, which are active at close to the same pH and temperature, which allows us to consider this strain as a producer of enzymes that make up the natural complex, promising for the simultaneous processing of starch or starch-containing raw materials with the aim of its deep hydrolysis, on the one hand, on the other hand, is used as a producer of alkaline proteases for the deep breakdown of proteins to amino acids.

 In addition, the use of the proposed strain allows you to improve the method of obtaining a complex of thermostable amylolytic enzymes by reducing the duration of growing the producer, reducing the concentration of the substrate in the nutrient medium.

SOURCES OF INFORMATION
1. Rimareva LV. Biotechnology of complex preparations of acidic proteases. Abstract of the doctor. dis. Moscow, 1997.

 2. Appl. Environ. Microbiol, 1985 .-- 49. P. 1168-1173.

 3. Appl. Microbiol. Biotechnol. - 1987.27. - P. 192-1988.

 4. UK patent N 2145094, CL C 12 N 9/34, 1985.

 5. Patent of Russia N 2001103, class C 12 N 9/26, 1993.

 6. Nefedova L. I., B. A. Ustinnikov, N. V. Tsurikova, G. N. Ermakova, N. A. Kichakova. Cultivation of the producer of thermostable alpha-amylase. Theoretical journal "Storage and processing of agricultural raw materials." V. 5, p. 38, 1996.

 7. A brief identifier of the Bergi bacteria. "World", M., ed. J. Howlta, 1980.

 8. Enzyme preparations. GOST 20264.4. -74.

 9. Klesov A. A., M. L. Rabinovich, A. P. Sinitsyn and others. Enzymatic hydrolysis of cellulose. Bioorganic chemistry. 1980.T 6.P. 1225-1242.

 10. Kaverzneva E. D. Standard method for determining proteolytic activity for complex preparations of proteases. Applied Biochem. and microbiol. , T 7., no. 2, p. 225-228.

Claims (1)

 The bacterial strain Bacillus licheniformis VKM B-2184 D is a producer of a complex of thermostable amylolytic and proteolytic enzymes.

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