RU2034923C1 - Strain of bacterium bacillus subtilis - a producer of saccharifying amylase splitting pullulan - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: strain Bacillus subtilis ВКПМ B-5166 shows increased capability to hydrolyze α-1,6 and a-1,4-glycosidic bonds in the starch. Under submerged cultivation of strain at aeration for 72 h the following levels of activity can be detected: amylolytic activity 246 U/ml; pullulanase activity 1.6 U/ml; b-glucanase activity 22 U/ml, and proteolytic activity 20 U/ml. EFFECT: increased enzyme activity of strain indicated above. 1 tbl

Description

 The invention relates to the microbiological industry, in particular the production of enzymes, and is a new strain of Bacillus subtilis T-2 producer of saccharifying amylase.

 The resulting preparation based on the claimed strain can be used in the starch industry for hydrolysis of starch in order to obtain crystalline glucose, maltose syrup, glucose-fructose syrups, as well as in the brewing and alcohol industries for the hydrolysis of starch-containing raw materials.

 The closest prototype to the claimed strain is a method for producing an enzyme preparation from Bacillus subtilis with α-amylase activity, cleaving both α-1,6-and α -1,4-glycosidic bonds.

The resulting enzyme having a pH optimum of 5.0-7.5 and a temperature optimum of 60-63 ^{° C} is capable of hydrolysing starch to give a syrup containing maltose and maltotriose in an amount (40-50)% by weight each of starch.

The producer was cultured on a medium of the following composition, soy flour 5.0; corn extract 0.5; meat extract 0.4; K ₂ HPO ₄ 0.3; MgSO ₄ ˙ 7H ₂ O 0.1; soluble starch 2.0; urea 0.5; CuSO ₄ 5˙10 ^-5; MnCl ₂ 1˙10 ^-4; CaCl ₂ 1˙10 ^-4; Zn _S O ₄ 1˙10 ^-4; FeSO ₄ 1˙10 ^-5, at ³⁰ ° C, pH 7.0 for 3 days.

As a seed used producer cells cultured on an agar medium, of the following composition, polypeptone 1.0,
soluble starch 1.0,
K ₂ HPO ₄ 0.3,
MgSO ₄ ˙ 7H ₂ O 0.1.

The inoculum was cultured at 30 ^{° C} for 72 hours.

 In the obtained culture fluid, the pullulanase activity is 9.6 units / ml, amylolytic activity is 45.2 units / ml.

A significant disadvantage of the strain of the prototype:
low amylolytic activity;
the absence of β-glucanase enzyme complex.

 The aim of the invention is to obtain a new strain of Bacillus subtilis T-2, with increased ability to hydrolyze α-1,6 and α-1,4 glycosidic bonds in starch.

 The proposed strain was isolated from Shatura peat bogs by the method of accumulative culture; 0.5% pullulan was used as a substrate.

 The resulting strain was deposited in VKPM under the number B-5166.

Cultural and morphological characteristics
The strain is a spore-forming rod, forming a chain of motile rod-shaped cells of 3 in pairs or single size (0.5-0.7) x (0.8-1.2) microns. The sticks are mobile, gram-positive. The spores are oval, with sporulation, sporangia does not change shape.

 Physiological and biochemical characteristics.

 On MPA it forms colonies, beige in color with smooth edges, on potato agar the growth is plentiful, the film is dry, wrinkled, thick cream color, with growth on the broth (MPB) forms a surface film, which subsequently sinks to the bottom.

Culture strict aerobe, with optimum growth temperature of 34 ^{° C-37 ° C} and pH 5.8-6.5.

 It utilizes glucose to form acid without gas evolution, as well as citrate, casein, glycerin. It utilizes starch, dextrins, glycogen, amylopectin, maltose, maltotriose, pullulan, isomaltose, glucose. It does not utilize xylose and arabinose.

 Nitrate does not recover.

 To determine the activity of enzymes, the culture fluid was centrifuged at 5000 rpm for 20 minutes and the supernatant was used.

 Pullulanase was determined by reducing sugars formed during the hydrolysis of pullulan (a linear polysaccharide consisting of maltose units connected by α-1,6-glycosidic bonds).

The unit of pullulanase activity accepted that amount of enzyme which under standard conditions (temperature ⁶⁰ ° C, pH 4.7) hydrolyzes pullulan at a rate corresponding to the formation of reducing groups equivalent to 1μ mol of glucose per minute, determined by the method of Somogyi-Nelson.

 Saccharifying amylase was determined by its ability to form reducing sugars formed during the hydrolysis of starch.

One unit of amylolytic activity is accepted that amount of enzyme which under standard conditions ⁽⁶⁰ ° C, pH 4.7) hydrolyzes starch at a rate corresponding to the formation of reducing groups equivalent to 1μ mol of glucose per minute, determined by the Nelson-Comodzhi.

Proteolytic activity was determined by the ability to catalyze the breakdown of the protein into peptides and amino acids. One unit of proteolytic activity (SS) receiving an amount of enzyme which, per 1 minute at pH 5.5 and ³⁰ ° C turns into the state nonprecipitating trichloroacetic acid sodium caseinate in an amount corresponding to 1 micromol of tyrosine (1 micromol of tyrosine is 0.181 mg) , activity is expressed in units / g (GOST 20264.2-88).

 β-glucanase activity was determined by the ability to form reducing sugars from lichenine or barley β-glucan, followed by the determination of sugars formed by the Somogy-Nelson method. Bioorganic chemistry, 1980, v. 6, p. 1225-1242.

One unit of β-glucanase activity defined as the amount of enzyme capable of forming per minute (at standard conditions at ³⁰ ° C and pH 5.5) in an amount of glucan oligosaccharides, which by reducing power equivalent to 1 micromol glucose.

 All experiments were performed in 3 replicates.

EXAMPLE EXAMPLE 1. producing Bacillus subtilis T-2 was grown under aerobic conditions in flasks with 0.75 ^dm3 fill factor of 0.4 for 72 hours at ^{30 °} C and pH _nach. 7.0 on a prototype medium having a composition, g / dm ³ : Soy flour 50.0 Meat extract 4.0 Corn extract 5.0 K ₂ HPO ₄ 3.0 MgSO ₄ ˙ 7H ₂ O 1.0 Soluble starch 2, 0 Urea 5.0 CuSO ₄ 5x10 ^-5 MnCl ₂ 2.5x10 ^-6 CaCl ₂ 1x10 ^-4 ZnSO ₄ 1x10 ^-4 FeSO ₄ 1x10 ^-5
As a seed, producer cells cultured on an agar medium of the following composition were used. Polypeptone 1.0. Soluble starch 1.0 K ₂ HPO ₄ 0.3 MgSO ₄ ˙ 7H ₂ O 0.1
The inoculum was cultured at 30 ^{° C} for 72 hours.

 Pullulanase ability of 0.9 u / ml, amylolytic ability of 140 u / ml, β-glucanase ability of 15 u / ml, proteolytic ability of 10.0 u / ml.

PRI me R 2. The producer of Bacillus subtilis T-2 was grown under aerobic conditions in a flask with a capacity of 0.75 DM ³ at a fill factor of 0.4 for 72 hours at 34 ^about C.

The seed was grown on an agar medium of the following composition, g / dm ³ : (NH ₄ ) ₂ SO ₄ 0.4 MgSO ₄ ˙ 7H ₂ O 1.0 CaCl ₂ : 2H ₂ O 0.5 KH ₂ PO ₄ 6.0 Tryptone 10 0 soluble Starch 5.0 pH (4.5-5.0) temperature 34 ^{° C} for 72 hours.

Composition for cultivation, g / dm ³ : MgSO ₄ 1,0 KH ₂ PO ₄ 2,0 (NH ₄ ) ₂ SO ₄ 1,0 MnCl ₂ 0,0025 CaCl ₂ 0,4 Soy flour 25,0 Soluble starch 60, 0 Corn extract 20.0 Initial pH of the medium (5.5-5.8).

 Pullulanase ability of 1.6 units / ml, amylolytic ability of 246.0 units / ml, β-glucanase ability of 22.0 units / ml, proteolytic ability of 20.0 units / ml.

PRI me R 3. The hydrolysis of 30% starch was carried out using the filtrate of the culture fluid obtained in example 2, at pH 4.7, a temperature of 60 ^about C for 24 hours at the rate of 0.1 units of pullulanase per 1 g of starch.

 The analysis of hydrolysis products by TLC is shown in the table.

The proposed strain of Bacillus subtilis T-2 has the following advantages compared to the prototype:
higher α-amylase activity;
the presence in the enzyme complex of thermostable β-glucanase with a pH optimum in the acidic zone;
thermostable neutral protease;
high glucose yield (64%) and the total yield of glucose and maltose (82%).

 These indicated advantages will make it possible to use the drug based on this strain in various areas of the food industry (starch, brewing, alcohol).

Claims (1)

 The bacterial strain Bacillus subtilis VKPM B-5166 is a producer of saccharifying amylase that breaks down pullulan.

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