RU2112035C1 - Method of preparing proteolytic enzymes from bacteria of genus bacillus - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves the use of antagonistically active strain Bacillus subtilis 3H (GISK N 248) producing neutral protease. Strain is cultured at sowing dose (3-5) x 10⁹ cells/ml, medium saturation with oxygen - at the range 70-90%. Cultural fluid is separated from biomass by microfiltration followed by sterilizing filtration of the end enzyme preparation. Obtained biomass is used for preparing the preparation-probiotic. Proteolytic activity of cultural fluid is 46 ± 6 PU/g and that of ready concentrate - 50 ± 5 PU/g. Invention can be used for production of bacterial and enzyme preparations. EFFECT: improved method of preparing. 1 tbl

Description

 The invention relates to biotechnology, the production of bacterial and enzyme preparations.

 Known methods for the cultivation of bacteria of the genus Bacillus are the main technology for obtaining a number of bacterial and enzyme preparations. Enzyme preparations with different activities are isolated from cultural supernatants obtained by homogeneous deep cultivation of various strains of bacteria of the genus Bacillus [1, 2]. Biomass is an unused production waste.

 At the same time, in known methods for producing bacterial preparations based on Bacillus subtilis biomass grown on liquid nutrient media under aeration and mixing conditions [3], the production waste is cell-free culture fluid containing various amounts of hydrolytic enzymes.

 Closest to the technical solution to the claimed is a method for producing proteolytic enzymes by co-cultivation of bacteria Bacillus thuringiensis v. galleriae 69-6 - producer of insecticide and Bacillus thuringiensis v. galleriae GFP-51 is a protease producer [4]. With this growing technology, two products are obtained for one fermentation - an enzyme preparation and a biological insecticide.

 In the known method, the obtained culture fluid containing two producers is separated by centrifugation into biomass and supernatant, from which the enzyme complex is isolated by subsequent acetone precipitation.

 The disadvantages of the described technology are the presence of ballast impurities of the biomass of the second strain in the insecticide preparation due to the simultaneous cultivation of two strains; low-tech, labor-intensive and energy-intensive centrifugation stage for separation of the culture fluid, as well as the acetone precipitation method of the target product, which requires the use of equipment in explosion-proof performance and unsafe for maintenance personnel.

 The present invention is the creation of technology that allows you to simultaneously obtain proteolytic enzymes and a probiotic drug.

 The problem is solved by the selection of a production strain, whose biomass is the main probiotic drug in its properties and is capable of producing a protease in the process of vital activity, working out the optimal cultivation conditions for it to accumulate antagonistically active biomass and a high level of proteolytic activity of the enzyme in the culture fluid, as well as developing a method for isolation and purification of the enzyme complex.

The essence of the invention lies in the use of an antagonistically active strain of Bacillus subtilis 3H (GISC N 248), which has the ability to produce a neutral protease [5]. The cultivation process of the strain is carried out at a sowing dose (3 - 5) • 10 ⁹ cells / ml, saturation of the medium with oxygen in the range of 70 - 90%. The supernatant is separated from the biomass by microfiltration followed by sterilizing filtration of the target enzyme preparation. The resulting biomass is the basis for obtaining the probiotic preparation.

 The solution to this problem is illustrated by the following examples.

Example 1. The strain Bacillus subtilis 3H is sown on 2 mattresses with an agarized synthetic nutrient medium containing peptone, maltose, mineral salts, trace elements and placed in a thermostat at 37 ^o C for 18 - 20 hours. Next, the microbial suspension washed with agar is seeded into a cultivation apparatus for growing on a liquid medium of the same composition in a volume of 5 l and grow 3-4 hours until the culture reaches the exponential growth phase.

Further, this culture in a dose of (1.0 ± 0.5) • 10 ⁹ cells / ml is used as seed for the main process in the Bior reactor with a capacity of 100 l. Cultivation is carried out at a temperature of 37 ^o C, saturation of the medium with oxygen of 70 - 80%, stirring at a rotational speed of the stirrer of 400 rpm, for 16 to 20 hours before the transition of the culture into a spore form and termination of the growth of proteolytic activity of the culture fluid.

In the culture fluid obtained by this method, the level of proteolytic activity ranges from 10 ± 3 PE / g, and the number of microbial cells is (20 ± 5) • 10 ⁹ cells / ml.

 The proteolytic activity of the culture fluid is determined by the Anson method at neutral pH values. The antagonistic activity of biomass is evaluated by the method of delayed antagonism to test strains of pathogenic microorganisms, and it is not less than 10 mm of growth inhibition.

 The enzyme-containing supernatant is separated from the biomass by microfiltration in flat-chamber apparatuses filled with Mifil membranes. Biomass is used to produce a probiotic bacterial preparation.

 After microfiltration, the proteolytic activity of the culture fluid drops to 2.0 - 4.0 PE / g due to non-specific sorption of the enzyme on the Mifil membranes.

 The enzyme-containing permeate purified from microbial cells is concentrated on a domestic AP-2.0 ultrafiltration unit made on the basis of VPU-15 fibers. To reduce nonspecific adsorption of the enzyme, the membranes of the installation are treated with a 0.5% albumin solution before ultrafiltration, and then washed with neutral phosphate buffer. The enzyme is concentrated to a volume of 3.8 ± 0.3 L and achieve activity (50 ± 5) PE / g. Then sterilizing filtration is carried out.

 Thus, the use in this example of a sowing dose sufficient to obtain biomass does not provide a high level of enzyme activity in the culture fluid.

Example 2. The strain Bacillus subtilis 3H is sown on 10 mattresses with agarized synthetic nutrient medium containing peptone, maltose, mineral salts, trace elements, and placed in a thermostat at 37 ^o C for 18 - 20 hours. Next, the culture washed with agar is seeded in the reactor " Bior "with a capacity of 100 l, containing 32 l of synthetic nutrient medium. Growing is carried out at a temperature of 37 ^o C, 80 - 90% saturation of the medium with oxygen, stirring at a speed of rotation of the mixer 430 rpm for 9 to 10 hours to achieve a concentration of microbial cells of 10 • 10 ⁹ . Then 40 l of the same medium are added to the reactor, while the sowing dose should be (4.2 ± 0.2) • 10 ⁹ cells / ml. The speed of rotation of the stirrer is increased to 450 rpm, the temperature regime and oxygenation are maintained unchanged for 14-16 hours until the culture passes into a spore form and the growth of proteolytic activity ceases.

 The level of proteolytic activity of the culture fluid obtained under these conditions ranges from 46 ± 6 PE / g. Next, the enzyme-containing supernatant is separated from the biomass by microfiltration using Mifil membranes, while its proteolytic activity remains at 12 ± 3 PE / g.

 The obtained permeate was concentrated by ultrafiltration, as in Example 1, to a volume of (10.5 ± 0.5) L of activity of 50 ± 5 PE / g and subjected to sterilizing filtration. Biomass is used to produce a probiotic preparation.

 Example 3. Cultivation is carried out as in example 2. But the enzyme is separated from the biomass by microfiltration using membranes "Millipore", "Pall", "Gelman Sciense", which allows you to save the proteolytic activity of the culture supernatant in the range of 46 ± 6 PE / g, and the final volume of the enzyme corresponds to 60 ± 1 l.

 Biomass is used to produce a probiotic preparation.

 Since the level of activity of the obtained enzyme corresponds to the level of its activity in the concentrates produced according to examples 1 and 2, in this case, the stage of further concentration of the target product can be excluded. Sterilizing filtration is carried out immediately after microfiltration using membranes from Millipore, Pall, Gelman Sciense. The resulting enzyme is used in liquid form or freeze-dried.

 Comparative characteristics of the production of the enzyme and biomass in examples 1 to 3 are presented in the table.

 From the table it follows that the method of producing biomass and the enzyme of example 3 provides a higher yield of the enzyme.

 Thus, when using the proposed method, the utilization rate of equipment is increased, since for one cultivation cycle from one reactor two products can be obtained — a proteolytic enzyme and a probiotic bacterial preparation; the consumption of the nutrient medium is halved, cultivation products are fully used, which offers the possibility of organizing waste-free production.

 Sources of information.

 1. A. p. USSR N 899646, class C 12 N 9/54, 01/23/82. A method of producing an alkaline protease.

 2. Patent DE N 3834550, cl. C 12 N 9/54, 04.19.90. Proteolytic enzyme, its preparation and use.

 3. RF patent N 2056855, cl. A 61 K 35/74, 03/27/96. The method of obtaining the drug Sporobacterin ".

 4. A.S. USSR N 1316239, class C 12 N 9/54, 07/30/91. A method of obtaining proteolytic enzymes.

 5. RF patent N 2067616, cl. C 12 N 1/20, 10/10/96. The bacterial strain Bacillus subtilis, bearing the property of antibiotic resistance, used to obtain the drug "Baktisporin".

Claims (1)

A method of obtaining proteolytic enzymes from bacteria of the genus Bacillus, including cultivating bacteria of the genus Bacillus on a liquid nutrient medium under aeration and mixing conditions, separating the enzyme-containing culture fluid and biomass, followed by isolation of the target product, characterized in that the Bacillus subtilis 3H strain is used for cultivation (GISK N 248), the culture process is conducted at seed dosage (3 - 5) • September ¹⁰ cells / mL, initial concentration of oxygen dissolved in the medium 0.8 - 1.8 g / l, the separation fermentosoderzhaschey kultural second liquid from the biomass is performed by microfiltration, followed by sterilization by filtration, the desired product.

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