RU2187554C1 - Method of extracellular inulinase preparing - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymes. SUBSTANCE: invention relates to preparing an extracellular inulinase used in food industry, medicine and in production of dietetic foodstuffs. Method of preparing an extracellular inulinase involves culturing Bacillus polymyxa VKMV-722 as a strain-producer of inulinase in nutrient medium containing molasses as a source of carbon and vitamins taken in the amount 8-10 wt.%, ammonium hydrophosphate as a source of nitrogen and phosphors taken in the amount 0.5-1.0 wt.%, pH value of medium is 7.0. Invention provides the increase of inulinase activity from 21 U/cm³ to 67.0-76.2 U/cm³ of cultural fluid as compared with the known one. EFFECT: increased activity of inulinase, decreased cost of enzyme.

Description

 The invention relates to biotechnology, and in particular to a method for producing extracellular inulinase used in the food industry, in medicine and in the production of dietary products.

The closest in technical essence and the achieved effect is a method for producing inulinase produced by Bacillus subtilis B-3-3. The relationship between the synthesis of inulinase and the conditions of cultivation of the strain (pH, substrate concentration, aeration) was studied. It was shown that Bacillus subtilis B-3-3 produces active inulinase (up to 21 units / cm ³ ) when cultured in an alkaline medium with a high concentration of carbon sources and good aeration. Sucrose can replace inulin as a carbon source in inulinase synthesis. Inulinase exhibits maximum activity at a pH of 6.5 and a temperature of 55 ^o C and remains stable at a pH of 6.0-8.0 and a temperature of 40-55 ^o C. The strain produces both intracellular and extracellular enzyme in a ratio of 1: 2, respectively (Study Inulinase production conditions Study and characterization of inulinase (produced) by strain B-3-3 / Xu Chunlan, Gu Tianchen // Shipin Yu Fajiao Tune = Food and Ferment. Ind. -1991. - Vol.1. 6-P. 16-21).

The disadvantages of this method are the use of inulin or sucrose as carbon sources, as well as their high concentration in a nutrient medium, which is economically unreasonable because of their high cost and is unjustified, as this produces produced inulinase with low activity (up to 21 units. / cm ³ ).

 The technical task of the invention is to increase the activity of extracellular inulinase and reduce its cost.

 The technical problem is achieved by the fact that in the method of producing extracellular inulinase, including the preparation of a nutrient medium, the cultivation of a producer strain, it is new that a strain of Bacillus polymyxa 722 is used as a producer strain of inulinase, and introduced into the nutrient medium as a source of carbon and vitamins molasses in an amount of 8-10 wt.%, as a source of amine nitrogen and phosphorus - ammonium hydrogen phosphate in an amount of 0.5-1.0 wt.%; pH of the medium is 7.0.

 The technical result of the invention is expressed in increasing the activity of extracellular inulinase, reducing its cost.

 The method is implemented as follows.

The strain Bacillus polymyxa 722 was obtained from the All-Russian Collection of Microorganisms of the Russian Academy of Sciences, where it is stored under VKM B-722. Pure culture is grown on meat-peptone agar for 3-4 days at a temperature of 31-33 ^o C. When cultivating the producer, molasses is used as a source of carbon and vitamins - sugar production waste in an amount of 8-10 wt.%, And as a source amine nitrogen and phosphorus - ammonium hydrogen phosphate in an amount of 0.5-1.0 wt.%.

A nutrient medium containing potassium dihydrogen phosphate, potassium chloride, magnesium sulfate, ferrous sulfate, ammonium hydrogen phosphate, molasses and water is inoculated with the Bacillus polymyxa 722 strain and grown under optimal conditions (pH 7.0; 37 ^° C; 72 hours) for the strain used . After completion of the cultivation, the culture fluid is separated from the biomass and inulinase activity is determined.

 In the present invention, a method based on the determination of reducing substances released during the hydrolysis of the substrate by the inulinase enzyme is used to determine inulinase activity. Inulin is used as a substrate by the company "ICN" (USA).

The amount of enzyme that catalyzes the formation of 1 μm of reducing substances (fructose) in 1 min is taken as a unit of inulinase activity. The analysis procedure is as follows. The reaction mixture, consisting of 2 cm ³ of inulin solution with a mass fraction of 5% in phosphate buffer pH 7.0 and 1 cm ³ of the enzyme solution, is incubated for 20 minutes at 40 ^o C. In parallel, a control experiment is prepared: 2 cm ³ of inulin solution with a mass fraction of 5% in phosphate buffer pH 7.0 add 3 cm ³ alkali (150 g NaOH and 200 g potassium sodium tartrate in 1 dm ³ distilled water) and 1 cm ³ culture liquid.

где А - активность инулиназы, ед./см³; А₀ - количество редуцирующих веществ, образующихся в результате гидролиза инулина, мкг; А_к - количество редуцирующих веществ в контрольной пробе, мкг; n - количество культуралъной жидкости, см³; τ - время гидролиза, мин; 180 - молекулярная масса фруктозы.In the experimental sample after 20 minutes the reaction is stopped by adding an alkali solution of 3 cm ³ . Then, 3 cm ³ of copper sulfate solution with a mass fraction of 4% is poured into the experimental and control samples. Then the tubes with the contents are shaken and immersed first in a boiling water bath for 6 min, then in a vessel with cold water and the amount of reducing substances is determined by the Bertrand semi-micrometer. Inulinase activity is calculated by the formula:

where a is the activity of inulinase, units / cm ³ ; And ₀ - the amount of reducing substances resulting from the hydrolysis of inulin, mcg; And _to - the amount of reducing substances in the control sample, mcg; n is the amount of culture fluid, cm ³ ; τ is the hydrolysis time, min; 180 - molecular weight of fructose.

 The composition of the nutrient medium provides an intensification of the accumulation of inulinase activity.

 The method is illustrated by examples.

Example 1. Prepare a nutrient medium of the following composition, wt.%:
Molasses - 6.0
(NH ₄ ) ₂ HPO ₄ - 0.25
KCl - 0.05
MgSO ₄ • 7H ₂ O - 0.05
KN ₂ PO ₄ - 0.1
FeSO ₄ • 7H ₂ O - 0.01
Water - Else
An aqueous-spore suspension of the Bacillus polymyxa 722 strain is prepared. For this, the 4-day culture of the strain is transferred from the bevel to a 250 cm ³ flask containing 50 cm ^{3 of} sterile water. In Erlenmeyer flasks with a capacity of 500 cm ³ , 120 cm ^{3 of} nutrient medium (pH 7.0) is added, inoculated with a spore suspension in an amount of 1% by volume of the medium and incubated on a rocking chair at a speed of 1.7-1.8 s ^-1 , temperature 37 ± 1 ^o C for 72 hours. At the end of cultivation, the bacteria are separated from the culture fluid by centrifugation (10000g, 10min).

After completion of the cultivation, the inulinase activity of the culture fluid is 52.6 units / cm ³ .

Example 2. Prepare a nutrient medium of the following composition, wt.%:
Molasses - 8.0
(NH ₄ ) ₂ HPO ₄ - 0.5
KCl - 0.05
MgSO ₄ • 7H ₂ O - 0.05
KH ₂ PO ₄ - 0.1
FeSO ₄ • 7H ₂ O - 0.01
Water - Else
Cultivation of biomass: inoculation of seed, compliance with the conditions of cultivation of the producer, the determination of activity is carried out analogously to example 1.

After completion of the cultivation, the inulinase activity of the culture fluid is 67.0 units / cm ³ .

Example 3. Prepare a nutrient medium of the following composition, wt.%:
Molasses - 10.0
(NH ₄ ) ₂ HPO ₄ - 1.0
KCl - 0.05
MgSO ₄ • 7H ₂ O - 0.05
KN ₂ PO ₄ - 0.1
FeSO ₄ • 7H ₂ O - 0.01
Water - Else
Cultivation of biomass: inoculation of seed, compliance with the conditions of cultivation of the producer, the determination of activity is carried out analogously to example 1.

After cultivation, the inulinase activity of the culture fluid is 76.2 units / cm ³ .

Example 4. Prepare a nutrient medium of the following composition, wt.%:
Molasses - 12.0
(NH ₄ ) ₂ HPO ₄ - 1.5
KCl - 0.05
MgSO ₄ • 7H ₂ O - 0.05
KN ₂ PO ₄ - 0.1
FeSO ₄ • 7H ₂ O - 0.01
Water - Else
Cultivation of biomass: inoculation of seed, compliance with the conditions of cultivation of the producer, the determination of activity is carried out analogously to example 1.

After completion of the cultivation, the inulinase activity of the culture fluid is 65.6 units / cm ³ .

As can be seen from the examples, during the cultivation of Bacillus polymyxa 722, the most effective is the introduction of molasses in the amount of 8-10 wt. % and ammonium hydrogen phosphate in an amount of 0.5-1.0 wt.%. The activity of extracellular inulinase increases from 21 units / cm ³ to 67.0-76.2 units / cm ^{3 of} culture fluid compared with the known method.

 Thus, the proposed method for producing extracellular inulinase makes it possible to obtain an enzyme with high activity and at the same time reduce its cost.

Claims (1)

 A method for producing extracellular inulinase, which includes preparing a nutrient medium, cultivating a producer strain, characterized in that the strain Bacillus polymyxa 722 is used as the producer strain of inulinase, and molasses is added in the amount of carbon and vitamins as a source of carbon and vitamins in the amount of 8-10 in the amount of 8-10 wt.%, as a source of amine nitrogen and phosphorus - ammonium hydrogen phosphate in an amount of 0.5-1.0 wt.%, pH of 7.0.