RU2244742C2 - Method for isolation and selection of microorganisms as producers of cyclodextrin glucanotrasnferase, strain of microorganism bacillus circulans b-65 ncaim (p) 001277 (b-65) as producer of extracellular cyclodextrin transferase, cyclodextrin glucanotransferase obtained from its and its applying for preparing cyclodextrin - Google Patents

Abstract

FIELD: biotechnology, biochemistry, microbiology.

SUBSTANCE: the strain Bacillus circulans B-65 a producer of cyclodextrin glucanotransferase is isolated and selected form the soil sample by culturing in nutrient medium with amylolytic activity 12.17 U/ml and cyclodextrinogenic activity up to 0.530 U/ml. Cyclodextrin glucanotransferase isolated from B. circulans B-65 shows the high degree for conversion of starch to cyclodextrins and this enzyme is specific for formation of β-cyclodextrin. Invention can be used in food industry for preparing cyclodextrins and cyclodextrin glucanotransferase used in different branches of industry.

EFFECT: improved isolating method, valuable properties of strain and enzymes.

9 cl, 8 tbl, 2 ex

Description

Field of Invention

This invention relates to biotechnology, in particular to a method for producing cyclodextrin by exposure to cyclodextringlucanotransferase (also called CGT-ase) on starch and the production of CGT-ase by bacteria of the Bacillus circulans B-65 strain. According to the International Classification, the invention relates to the following classes: C 08 V 37/16; C 12 P 19/18; C 12 N 1/20; C 12 N 9/10.

OBJECTS OF THE INVENTION

This invention solves the problem of the biosynthesis of TSHT-basics, which is usually used to obtain cyclodextrin, as well as the problem of obtaining cyclodextrin by the action of TSHT-basics on starch. The biosynthesis of TSHT-basics is most often carried out using bacteria of the genus Bacillus, which usually produce TSHT-ase, acting on starch with the formation of a mixture of α-, β- and γ-cyclodextrins in a different ratio, while the yield of an individual cyclodextrin is not high, and its allocation increases the cost of production.

The biosynthesis of CGT-basics according to the invention is carried out by alkaliphilic bacteria of a new strain of Bacillus circulans B-65, characterized by the production of a large amount of CGT-basics, which is usually used to produce cyclodextrin. Cyclodextrins are obtained using CTG-basics from B. circulans bacteria, which is characterized by a high degree of conversion of starch to cyclodextrins and is specific for the formation of β-cyclodextrin.

State of the art

Cyclodextrins are obtained by the action of CTGase on starch. In the process of producing cyclodextrins, the most important step is to obtain CTGase. CTGase is produced by certain types of bacteria, among which bacteria belonging to the genus Bacillus are most often found: B. macerans, B. circulans, B. megaterium, B. coagulans, B.lentus, B. ohbensis, B. firmus, B. amyloliquefaciens, B. stearothermophilus, B. subtilis, B.cerus. CTGase is also produced by some other bacteria that do not belong to the genus Bacillus, for example: Klebsiella pneumoniae, Micrococcus luteus, Micrococcus varians. Most often, Bacillus macerans bacteria and alkaliphilic bacteria belonging to the genus Bacillus act as producers of TSGT-basics.

TSHT-ase is produced by growing bacteria in a liquid medium under aerobic conditions in a fermenter. This process is very expensive, and bacteria produce a certain amount of TST-basics. The expensive process of obtaining TSGT-basics significantly affects the high cost of the final product, i.e. cyclodextrin, which limits its widespread use in many industries.

Since cyclodextrins are widely used in industry and their cost is one of the significant factors limiting their use, scientists from all countries are looking for ways to increase the efficiency of the method for producing TSHT-basics. For this purpose, optimization of the composition of the nutrient medium and optimization of physico-chemical factors for bacterial growth are carried out, work is underway to increase the productivity of bacteria using various genetic methods (induced mutagenesis, genetic engineering), and work is underway to isolate new microorganisms that are characterized by high production of TSGT -azy.

In the method for producing cyclodextrin, the properties of the CTGase produced by bacteria are also very important. Properties TSGT-az produced by various types of bacteria belonging to the genus Bacillus, are presented in Table 1.

Table 1: Properties of CTG-az from Various Microorganisms Producer TSGT-basics The ratio of α-, β- and γ-CD Optim. pH pH stability Optim. temp-pa (° C) Thermostability (° C) without Ca ²⁺ with Ca ²⁺ Bacillus coagulans (thermophile) 2: 2,6: 1 6.5 6.0-9.0 65 fifty 60 Alkaliphilic Bacillus sp. (ATCC21783) 71% β-CD 6.5 and 8.5 - 65 Bacillus macerans (IF03490) 71% α-CD 5.5 - 55 - - Bacillus lentus (alkaliphilic) 1: 67: 1,6 6.5-7.5 6.5-8.5 45-55 - - Bacillus sp. (alkaliphilic) mainly β-CD 6.0 6.0-11.0 55 60 - Bacillus sp. (thermophile) - 6.5 5.0-10 fifty 55 - Bacillus stearothermophilus - 5.0-5.5 5.0-8.0 70-75 65 70 Bacillus macerans (IAM 1243) - 6.0 5.5-9.5 60 fifty - Bacillus macerans - 5.4-5.8 - 60 fifty - Producer TSGT-basics The ratio of α-, β- and γ-CD Optim. pH pH stability Optim. temp-pa (° C) Thermostability (° C) without Ca ²⁺ with sa ²⁴ Bacillus macerans (IFO 3490) 2.7: 1: 1 5.0-5.7 8.0-10 55 60 - Bacillus egaterium 1: 2.4: 1 5.0-5.7 7.0-10 55 55 - Bacillus circulans 3: 3: 1 5.5 6.0 9.0 60 45 60 Bacillus amyloliquefaciens 95% α-CD 6.0 - 70 60 65 Bacillus sp. 1.6: 4: 1 5.5 6.0-9.5 65-70 65 - Bacillus ohbensis mainly β-CD 5.5 6.5-9.5 60 55 - Bacillus macerans (IAM 1227) mainly α-CD 5.5 6.5 8.5 fifty fifty - Bacillus circulans (IF03329) β-> α-CD 6.0 6.0-9.5 55 55 - Bacillus coagulans (thermophile) 1: 0.9: 0.3 - - - - - Bacillus cereus (NCIMB 13123) 22: 23: 0 5,0 - 40 - - Bacillus firmus (alkaliphil) 0.08: 1: 0.2 - - - - - Acaliphilic Bacillus subtilis 0: 25.1: 4.7 - - fifty - - The alkaliphilic Bacillus sp. 5.7: 23.5:
4.7 - - 40 - -

From the data presented, it can be seen that TSG-basics from various microorganisms exhibit similar properties, but there are also certain differences. The greatest differences among TSHT-az are related to the quantities of specific α-, β- or γ-cyclodextrins formed, TSHTazy form mainly a mixture of α; β- and γ-cyclodextrins in various proportions. However, there are TST-basics that do not form α- or γ-cyclodextrins. Most TSHT-az form a mixture of cyclodextrins, in which β-cyclodextrin predominates, while γ-cyclodextrin is present in a minimal amount. Alkaliphilic microorganisms produce TST-basics, which give the highest content of β-cyclodextrin, compared with α- and γ-cyclodextrins. TSHT-basics from alkaliphilic microorganisms form β-cyclodextrin, which makes up more than 70% of the total number of cyclodextrins. From the data presented in Table 1, we can conclude that the TSG-basics produced by strains belonging to the species of Bacillus macerans give the highest yield of α-cyclodextrin compared to other cyclodextrins. In addition to B. macerans, the TSHTase produced by B. amyloliquefaciens AL 35 also forms α-cyclodextrin with a yield of up to 95%. Unlike most TSGTases from various producers, TSGTase produced by Bacillus subtilis No. 313 forms γ-cyclodextrin, but does not form either α- or β-cyclodextrins. TSGT-aze from Bacillus sp. AL-6 also gives the highest yield of γ-cyclodextrin and β-cyclodextrin, but does not form α-cyclodextrin.

In the method for producing cyclodextrins using TSHT-az, which at the end of the reaction give a mixture of cyclodextrins, α-, β- and γ-cyclodextrins are formed in different proportions, and the highest yield of one of any cyclodextrins leads to the lowest yield of other cyclodextrins. After the completion of the enzymatic reaction with the formation of cyclodextrins, they are isolated and purified. Since the isolation and purification of cyclodextrins increases the cost of the process for their preparation, it is desirable to isolate microorganisms that produce TST-basics specific for the formation of one of the cyclodextrins.

From the patent US 4477568 known TSGT-ase obtained by culturing the bacteria Bacillus circulans RIV No. 11115, which is used to obtain cyclodextrins. Using this TSG-ase, you can get up to 66% of β-cyclodextrins. This TSHTase is active at a temperature of 60 ° C, but its activity decreases significantly at 65 ° C, in the pH range from 4 to 7 (pH optimum is 6-6.5). There is a need for an enzyme that will produce more than 66% of β-cyclodextrins and will be active in a wider temperature range, as well as at higher pH, which will significantly reduce the risk of infections.

SUMMARY OF THE INVENTION

The biosynthesis of CTG-basics, which is then used to produce cyclodextrin, is carried out by alkaliphilic bacteria of the strain Bacillus circulans B-65. Biosynthesis is carried out by growing bacteria in an alkaline (pH 10) liquid medium, under aerobic conditions, in a fermenter. This nutrient medium contains: as a carbon source, from 0.5 to 3% starch, as a nitrogen source, from 0.5 to 6% of a substance selected from the group consisting of peptone, yeast extract, yeast autolysate, corn autohydrolyzate ( corn step liquid) or mixtures thereof, as a source of minerals: K ₂ HPO ₄ , MgSO ₄ × 7H ₂ O, Na ₂ CO ₃ .

After biosynthesis is complete, the bacterial cells are removed by centrifugation or filtration, and then the extracellularly produced CGTases are concentrated by ultrafiltration.

When exposed to thus TSGT-basics on starch, the concentration of which varies from 5% to 15%, get cyclodextrins. The enzymatic reaction is carried out for 24 hours in an alkaline medium (pH 8.0) at a temperature of 60 ° C.

The novelty of this invention lies in the fact that the biosynthesis of TSG-ase, which is used to obtain cyclodextrin, is carried out using alkaline bacteria B. circulars B-65. These bacteria were isolated from soil taken on the territory of Leskovac. B. circulans bacteria were isolated from approximately 2000 bacterial colonies, which were characterized by the production of TSGT-basics. The isolate, which was then identified and designated as B. circulans B-65, showed significantly greater activity in the production of TSHT-basics, and this property clearly distinguishes it from other isolates. Thus, the alkaliphilic bacteria B. circulans B-65 are a new strain, characterized in that it produces a significant amount of TST-basics, which can be used to produce cyclodextrins. The strain Bacillus circulans B-65 was deposited at the National Collection of Agricultural and Industrial Microorganisms, NCAIM in Budapest under the number NCAIM (P) 001277.

The advantage of using TSGT-basics, which is produced by this alkaliphilic strain of B. circulans B-65, lies in its use for producing β-cyclodextrin. The TSHTase of this strain is specific for the formation of β-cyclodextrin and is characterized by a high degree of conversion of starch to β-cyclodextrin. Under the reaction conditions described, at a starch concentration of 5%, TSHT-aza converts up to 45% of starch into cyclodextrins. Of the total number of cyclodextrins produced, the amount of β-cyclodextrin is about 95%, which is the highest value compared to the available literature. Unlike CTGase, known from US Pat. No. 4,477,768, the CTGase according to the invention is active at higher temperatures (up to 75 ° C), in a wider pH range (from 4 to 12).

Unlike the method of isolation and selection of bacteria, producers of CGT-ase according to the invention by known from patent US 4477568 also consists in that the medium composition for culturing bacteria as the base includes phenolphthalein or methyl orange not used FeSl ₃ × 6H ₂ O.

Isolation and selection of bacteria producing TSGT-basics

CGT-basics produced by bacteria were isolated from 54 different soil samples that were taken on the territory of Leskovac. A suspension of soil (0.5-1.0 g) in 10 ml of sterile distilled water was heated for 10 minutes at 80 ° C. After heating, 0.1 ml of the soil suspension was applied to agar plates with Parka medium containing: 1% soluble starch, 0.5% peptone, 0.5% yeast extract, 0.1% K ₂ NRA ₄ , 0.02% MgSO ₄ · 7H ₂ O, 1% Na ₂ CO ₃ , 0.03% phenolphthalein, 0.01% helianthine, 1.5% agar, pH 10.0 (Park et al., 1989). Incubation was carried out at 37 ° C. After 48 hours of incubation, colonies surrounded by the widest yellow areas on red-colored agar plates were isolated and purified.

The number of colonies obtained from various soil samples ranged from 0 to 200. The colonies that produced CGT-ase were surrounded by yellow zones of various sizes. Different soil samples had a different percentage of colonies producing TSGT-ase, which ranged from 0 to 90% of the total number of colonies. Taking into account the size of the zone around the colonies, 85 colonies were isolated and purified from 2,000 colonies producing CTG-basics. After purification in the Park environment, 45 colonies were selected for further work. The selected colonies were transferred to mown agar with Horikoshi II medium and stored at + 4 ° C after incubation.

45 selected alkaliphilic isolates producing CGT-ase were grown in liquid medium in Erlenmeyer bottles with stirring for subsequent selection of isolates. Incubation was carried out at 37 ° C for 48 hours with constant stirring at 200 rpm. Samples were taken on the third and fourth days, and after removal of the bacterial cells by centrifugation (10000 × g, 10 min, + 4 ° C), the amylolytic and cyclodextrinogenic activities of the supernatant were determined.

The amylolytic activity of the isolates ranged from 0.4 U / ml to 12.17 U / ml. The largest number of isolates showed amylolytic activity from 1 U / ml to 2 U / ml. The number of isolates that showed higher amylolytic activity gradually decreased, so that only 3 isolates showed amylolytic activity above 5 U / ml. Cyclodextrinogenic activity ranged from 0.019 U / ml to 0.530 U / ml. The cyclodextrinogenic activity of most isolates was up to 0.100 U / ml. Cyclodextrinogenic activity above 0.200 U / ml was demonstrated by a single isolate. Isolate B-65 showed amylolytic activity of 12.17 U / ml and cyclodextrinogenic activity of 0.530 U / ml, which were significantly higher compared to other isolates.

Morphological and taxonomic characteristics of a bacterial strain

For identification, the morphological and physiological-biochemical characteristics of the bacterial strain were investigated. The results of the study were analyzed using the “Bergey’s Manual Systematic Bacteriology” (Systematic Handbook of Bacteriology edited by Bergey, Sneath, 1968). Bacillus circulans (ATCC 4513) was used as a control strain.

The results of these studies showed that the bacteria of the new strain are sticks with a size of 0.6-0.7 × 2-4 microns, are motile, gram-positive, form elliptical endospores, grow under aerobic and anaerobic conditions, and produce catalase. These characteristics indicate that this bacterial strain belongs to the genus Bacillus. The results of these studies show that the bacterial strain: shows a negative reaction according to Voges-Proskauer; produces acids from glucose, arabinose, xylose and mannitol; does not produce gas from glucose; hydrolyzes gelatin and starch; does not use (does not metabolize) citrates; does not produce indole; does not grow in a medium with pH 6.8 and 5.7 and shows good growth in a medium with 2, 5, 7 and 10% NaCl.

The morphological and physiological and chemical characteristics of isolate B-65 basically coincide with those of Bacillus circulans. However, an essential feature of isolate B-65 is that it exhibits good growth in an alkaline environment (pH 10) and does not grow in a neutral environment, such as a nutrient medium with a pH of 6.8, which distinguishes it from the described strains of Bacillus circulans. Isolate B-65 also differs from Bacillus circulans strains in that it grows in a medium with 10% NaCl.

Based on these characteristics, isolate B-65 was identified as a strain of Bacillus circulans. Since its feature is good growth in an alkaline medium (pH 10) and lack of growth in a neutral medium, isolate B-65 was designated as an alkaliphilic strain of Bacillus circulans B-65. Characteristics of isolate B-65 are presented in Table 2.

Table 2: Characteristics of Bacillus circulans bacteria (ATCC 4513) and isolate B-65 Morphological and physiological-biochemical characteristics Bacillus circulans
(ATCC 4513) Isolate B-65 The form Sticks Sticks Size (μm) 0.5-0.7 × 2-5 0.6-0.7 × 2-4 Mobility + + Dispute form elliptical elliptical Gram stain + + Catalase + + Anaerobic growth + + Voges-Proskauer Analysis - - Acid production from carbohydrates:
D-glucose
L-arabinose
D-xylose
D-mannitol +
+
+
+ +
+
+
+ Glucose gas production - - Hydrolysis: Gelatin
starch +
+ +
+ The use of citrates - - Indole Products - - Growth at pH: 6.8 (culture medium)
5.7 +
+ -
- Growth in the presence of NaCl: 2%
5%
7%
10% +
+
+
- +
+
+
+ Growth at: 30 ° C
37 ° C
40 ° C +
+
+ +
+
+

The biosynthesis of CTG-ase strain B, circularis B-65

The biosynthesis of TSHT-basics is carried out by bacteria of the strain B. circulans B-65, growing in an alkaline (pH 10) liquid medium. Nutrient medium includes: 1% soluble starch, 0.5% peptone-1, 0.5% yeast extract, 0.1% K ₂ NRA ₄ , 0.02% MgSO ₄ × 7H ₂ O, 1% Na ₂ CO ₃ .

The inoculum for the method of producing TSGT-basics was prepared by inoculating a culture taken from mowed agar into a liquid medium. Incubation was carried out at 37 ° C with constant stirring (200 rpm) for 48 hours. 5% inoculum was added to the fermenter with fresh medium. The biosynthesis of CGT-basics was carried out for 96 hours at 37 ° C in a stream of sterile air at 0.5 vvm (volume of air per volume of medium min ^-1 , air volume per medium volume per minute) at a stirrer rotation speed of 300 ^-1 . After 96 hours of growth, the bacterial cells were removed by centrifugation (10,000 × g, 10 min, + 4 ° C) or by filtration, and then the concentrated extracellularly produced TSGTase was carried out by ultrafiltration. The CGT-ase thus obtained was then used to produce cyclodextrin. During the preparation process, the amylolytic and cyclodextrinogenic activities of the CTGase, determined extracellularly by the strain B. circulans B-65, were determined. The cell growth and activity of the CTG-ase produced by B. circulans B-65 strain growing under the described conditions are described in Table 3.

Table 3 The activity of CTG-basics, cell growth and pH changes during the growth of B. circulans B-65 strain Time (h) Amylolytic activity TSGT-basics (U / ml) Cyclodextrinogenic activity Cell growth (610 nm) 0 / / 0.125 24 7.30 0.20 1,677 48 9.70 0.24 1,489 72 14.85 0.38 1,497

TSHT-aza is produced in the process of cell growth. Until the completion of the growth phase, the cyclodextrinogenic activity reaches 0.20 U / ml, which is slightly more than 30%, compared with the activity obtained after a 96-hour growth period. After the completion of the growth phase, the production of CGT-basics continues, so that after 96 hours of growth the cyclodextrinogenic activity reaches 0.60 U / ml, and the amylolytic activity is 23.37 U / ml. The increase in cyclodextrinogenic and amylolytic activities after the completion of the growth phase is about 70% of the total value. Thus, isolate B-65 produces more TSHT-basics after the completion of the cell growth phase.

During the growth of B. circulans B-65, amylolytic and cyclodextrinogenic activities increase in proportion, which leads to the conclusion that the strain B. circulans B-65 produces CGT-ase and does not produce any other amylolytic enzymes.

This conclusion is confirmed by the determination of the content of cyclodextrins and reducing sugars during incubation of TSHT-basics from B, circulans B-65 with starch. These results showed that during the reaction, the formation of cyclodextrins was observed, while the number of reducing sugars did not increase, which would take place in the presence of other amylolytic enzymes.

The advantage of this method of obtaining TSGT-basics, which is confirmed by these results, is that the biosynthesis of TSHT-basics is carried out by a new alkaliphilic strain B. circulans B-65, which is characterized by the extracellular production of a large amount of TST-basics. Another, also important characteristic of the strain B. circulans B-65 is that in addition to the TSG-basics, this strain does not produce other amylolytic enzymes. The presence of these enzymes in the preparations of TSGT-basics in the process of producing cyclodextrins would lead to hydrolysis of starch, that is, to a decrease in the concentration of the substrate to obtain cyclodextrins, which would have a negative effect on the final yield of the product.

The advantage of the biosynthesis of TSGT-ase strain B. circulans B-65, compared with other producers of this enzyme is that the biosynthesis of TSHT-ase strain B. circulans B-65 is carried out in a very alkaline environment (pH 10), while reducing the risk infections by other microorganisms, which is extremely important for industrial production.

Properties TSGT-basics from B. circulans B-65

In addition to the fact that the B. circulans B-65 strain produces a large amount of CGT-ase, it is very important that this CGT-aze has good properties regarding thermal stability and the optimum temperature for its activity, as well as pH stability and optimal pH values.

TSGT-aze from the strain B. circulans B-65 is characterized in that it is stable at temperatures up to 55 ° C, and in the presence of Ca ²⁺ its stability increases to 60 ° C. The optimum temperature for its action is 60 ° C. Due to the high optimal temperature and high thermal stability of the TSG-basics from B. circulans B-65 strain, the method for producing cyclodextrins is carried out at a temperature of 60 ° C. The advantage of producing cyclodextrins at high temperature is that under these reaction conditions, the risk of infection by other microorganisms is reduced. At high temperatures, the enzymatic reaction rate is also higher, which reduces the duration of the cyclodextrin production process.

TSHT-ase from strain B. circulans B-65 is characterized by the fact that the pH value optimal for its activity is in the pH range from 5.0 to 6.0, however, TSHT-ase retains its activity in a rather wide range of pH values in alkaline medium . TSHT-aze from the strain B. circulans B-65 is stable at a pH of from 6.5 to 10.0. Due to these properties of TSGT-basics, cyclodextrins can be obtained in a wide range of pH values. The Michaelis constant for CTG-ase from B.circulans B-65 strain is from 3.016 mg / ml (calculated by the Lineweaver-Burck method) to 4.77 mg / ml (calculated by the Idi-Hofsti method). Inhibitors of this enzyme are NaN ₃ , CuSO ₄ , MnSO ₄ , FeSO ₄ , MgSO ₄ and glucose.

The results of the study of the properties of TSGT-basics from strain B. circulans B-65 are presented below.

The effect of pH on the activity of TST-basics

The effect of pH on the activity of TSGT-basics was investigated in the pH range from 4.0 to 10.0 at a temperature of 40 ° C. The following buffers were used to adjust the pH: 50 Mm acetate buffer (pH 4-5), 50 mM phosphate buffer (pH 5-8), 50 mM carbonate buffer (pH 8-10). The results of these studies, presented in Table 4, show that the optimal pH value for the activity of TSHT-basics is in the range of pH from 5.0 to 6.0, however, TSHT-aza retains its activity in a rather wide range of pH values in alkaline medium.

Table 4 Cyclodextrinogenic activity of TSHT-basics at various pH values. PH 50 mM buffer Cyclodextrinogenic activity (U / ml) Relative activity (%) ¹ 4 Acetate 0.18 19.8 4,5 0.74 80,4 5 Phosphate 0.92 100 5.5 0.91 98.9 6 0.89 96.7 6.5 0.76 82.6 7 0.66 71.7 8 0.56 60.3 nine Carbonate 0.42 45,2 10 0.26 28.6 ^{AL = L> 1} Relative activity is expressed as a percentage of the maximum activity obtained at pH 5.0.

The effect of pH on the stability of CTG-basics

The effect of pH on the stability of TSHT-basics was investigated in the pH range from 4.0 to 12.0. The following buffers were used to adjust the pH: 50 mM acetate buffer (pH 4-5), 50 mM phosphate buffer (pH 6-7), 50 mM Tris-HCl buffer (pH 8-10) and 50 mM glycine-NaOH buffer (pH 11-12). CGT-ase was incubated at various pH values for 1 hour at 40 ° C, and then the pH was adjusted to 6.0 and the residual enzymatic activity was determined. The results of these studies are presented in Table 5, showing that TSHT-aza was stable over a wide range of pH values from 6.5 to 10.0.

Table 5 Residual TSHTase activity after incubation at various pH values pH 50 mM buffer Cyclodextrinogenic activity (U / ml) Relative activity (%) 4 Acetate 0.18 19.8 5 0.74 80,4 6 Phosphate 0.92 100 6.5 0.91 98.9 7 0.89 96.7 8 Tris-HCl 0.76 82.6 nine 0.66 71.7 10 0.56 60.3 eleven Glycine-NaOH 0.42 45,2 12 0.26 28.6

The effect of temperature on the activity of CTG-basics

The effect of temperature on the activity of TSGT-basics in the temperature range from 30 ° C to 75 ° C at pH 6.0. The results of these studies showed that the optimum temperature for TSHT-asnoy activity was 60 ° C (table 6).

Table 6: Activity TSGT-basics at different temperatures Temperature (° C) Relative activity (%) thirty 23.1 40 36,2 45 49.8 fifty 71.7 55 85 60 100 65 94.7 70 45.3 75 15.9

The effect of temperature on the stability of TSGT-basics

The effect of temperature on the stability of TSHT-basics was studied in the temperature range from 30 ° C to 80 ° C. The study of thermal stability of TSGT-basics was carried out in the absence and in the presence of Ca ^{2+ ions} at a concentration of 10 mM. CGT-ase was incubated for 30 minutes at various temperatures at pH 8.5, and then the pH was adjusted to 6.0 and residual enzymatic activity was examined.

The results of the study (Table 7) showed that TSG-ase was stable in the temperature range up to 55 ° C and that in the presence of Ca ^{2+ ions} its stability increased to a temperature of 60 ° C.

Table 7 Residual TSHTase activity after incubation at various temperatures Temperature
(° C) Residual activity (%) without Ca ²⁺ Residual activity (%) with Ca ²⁺ thirty 97 97.3 40 98.3 96.8 fifty 96 96.8 55 96 98.2 60 52.8 96.8 65 17,2 81.7 70 3 19.0 75 - 0 80 - 0

Preparation of Iclodextrins

Cyclodextrins are obtained by exposure to starch in a concentration of 5% to 15% by exposure to CTGase from B. circulans B-65 strain. Due to the high optimal temperature of the enzyme and the high thermal stability of TSGTase from B. strain circulans B-65, the process of producing cyclodextrins can be carried out at temperatures up to 60 ° C. With regard to pH values, the production of cyclodextrins can be carried out in a wide range of pH values from 6.5 to 10.0.

The yield of cyclodextrins during the incubation of a 5% starch solution with various concentrations of TSHT-basics at 40 ° C and a pH of 6.5 is presented in Table 8.

Table 8 The yield of cyclodextrins at various concentrations of TST-basics ¹ Enzyme Concentration (U / g Starch) Incubation time
(h) CD
(g / l) CD output²
(%) Cyclodextrinogenic activity Amylolytic activity 0.6 23,4 24 10.8 21.6 1,2 46.8 24 11.2 22.3 1.8 70,2 24 11.7 23.3 2,4 93.6 24 12.1 24.2 3.0 117 24
48 14.8
19,2 29.5
38,4 4,5 175 24
48 17.8
19.7 35.6
39,4 6.0 234 24
48 21.7
22.5 43.3
44.9 7.5 293 24
48 21.1
20.3 42,2
40.6 ^{AL = L> 1} The reaction mixture, which contained 5% starch and the corresponding concentration of the enzyme, was incubated for 48 hours at 40 ° C.
² The yield of CD represents the degree of conversion of starch to CD.

The best results were obtained for TSGT-basics at a concentration of 6.0 U (cyclodextrinogenic activity) / g of starch. At this concentration of TSGTase, approximately 45% of the starch was converted to cyclodextrins.

After completion of the enzymatic reaction to obtain cyclodextrins, they can be removed from the reaction mixture by precipitation using, for example, trichlorethylene or toluene. From precipitated cyclodextrins, β-cyclodextrin can be removed by crystallization, due to the fact that it is much less soluble compared to other cyclodextrins. The cyclodextrins obtained by precipitation were dissolved in distilled water when heated at 100 ° C, and then β-cyclodextrin crystallized with a gradual decrease in temperature to 20-25 ° C with gentle stirring. The β-cyclodextrin crystals were then removed by filtration, washed with a small amount of cold water and dried at 70 ° C.

When analyzing the obtained cyclodextrins by spectrophotometric methods and HPLC, it was found that TSHT-aza from B. circulans B-65 strain, under the described conditions, converts up to 45% of starch into cyclodextrins and mainly forms β-cyclodextrin. In the resulting cyclodextrins, the amount of β-cyclodextrin is approximately 95%, while the amounts of α-cyclodextrin and γ-cyclodextrin are 2-3% and 0.6-3%, respectively.

TSHT-aze from strain B. circulans B-65 is characterized in that it is specific for the formation of p-cyclodextrin and converts a high percentage of starch into β-cyclodextrin. Therefore, the use of TST-basics from a new strain has significant advantages for obtaining β-cyclodextrin, compared with TST-basics from other known bacteria.

The following examples illustrate but do not limit the invention.

Example 1

Products TSGT-basics

TSHT-ase is produced by growing bacteria of strain B. circulans B-65 in a liquid medium containing: 1% soluble starch, 0.5% peptone, 0.5% yeast extract, 0.1% K ₂ NRA ₄ , 0.02% MgSO ₄ × 7H ₂ O, 1% Na ₂ CO ₃ . The inoculum for the method of obtaining TSGT-basics was obtained by inoculation in 450 ml of liquid medium Horikoshi (Horikoshi) II culture from mowed agar. Incubation was carried out for 48 hours at 37 ° C with constant stirring (200 rpm).

450 ml of inoculum was added to a fermenter containing 9 l of Horikoshi medium. Growth was carried out for 96 hours at 37 ° C in a stream of sterile air at 0.5 vvm and a stirrer speed of 300 min ^-1 . During the growth process, samples were taken and the amylolytic and cyclodextrinogenic activities of the supernatant were determined. After 96 hours of growth, the bacterial cells were removed by centrifugation (10,000 × g, 10 min, + 4 ° C), and then the extracellularly produced CGTases were concentrated by ultrafiltration.

Strain B. circulans B-65, growing in this way, after 96 hours produced extracellular TSGTase with an amylolytic activity of 23.4 U / ml and a cyclodextrinogenic activity of 0.6 U / ml. After concentrating the enzyme by ultrafiltration, an amylolytic activity of 450 U / ml and a cyclodextrinogenic activity of 12 U / ml were obtained.

Example 2

Preparation of Iclodextrins

Soluble starch (300 g) was diluted in 6 L of water with heating. The temperature was adjusted to 40 ° C. and the pH to 6.5. To a starch solution was added CGT-ase at a concentration of 6U (cyclodextrinogenic activity) per g of starch. Incubation was carried out for 30 hours at 40 ° C.

After 30 hours of incubation, the reaction was stopped by heating the reaction mixture for 10 minutes at 100 ° C. The reaction mixture was then decolorized with activated carbon. After removal of activated carbon by filtration, cyclodextrins were precipitated from the reaction mixture (6 L) with trichlorethylene (1.2 L). Precipitated cyclodextrins were then removed by filtration and dried at 70 ° C. The final yield was 107 g of cyclodextrins from 300 g of starch.

The resulting cyclodextrins were dissolved in 500 ml of distilled water while heating at 100 ° C. Then, with a gradual decrease in temperature to 20-25 ° C and with careful stirring, crystallization of β-cyclodextrin was carried out. After standing overnight at + 4 ° C, the crystals were removed by filtration, washed with a small amount of cold distilled water and dried at 70 ° C. Thus, 75.2 g of β-cyclodextrin were obtained as white crystals.

Claims (9)

1. The method of isolation and selection of bacteria-producers of cyclodextrin glucanotransferase, characterized in that the bacteria are isolated from a soil sample, prepare an aqueous suspension by heating to 80 ° C for 10-15 minutes and inoculate in agar plates with medium containing starch, peptone, yeast extract, K ₂ HPO ₄ , MgSO ₄ , Na ₂ CO ₃ , phenolphthalein, helianthin and agar, then incubated for 20 to 72 hours at 37 ° C, colonies are selected, selected colonies are cultivated in a liquid medium until production occurs enzymes, and carry out the selection of Bacillus circulans B-65 olates. 2. The bacterial strain Bacillus circulans B-65 NCAIM (P) 001277 (B-65) is a producer of extracellular cyclodextringlucanotransferase. 3. A method for producing cyclodextringlucanotransferase, characterized in that the alkaliphilic bacteria of the strain Bacillus circulans B-65 are cultured under aerobic conditions in a liquid medium for 24-120 hours at a temperature of 25 to 50 ° C, after cultivation, the bacterial cells are removed by centrifugation and filtration, and the concentration of the extracellularly produced TSHTase is carried out by ultrafiltration. 4. The method according to claim 3, characterized in that the nutrient medium contains - 0.5-3% starch as a carbon source, - 0.5-6% of a substance selected as a nitrogen source from the group consisting of peptone, yeast extract, yeast autolysate, corn autohydrolyzate, or a mixture thereof, - sources of minerals: K ₂ NRA ₄ , MgSO ₄ × 7Н ₂ О, Na ₂ СО ₃ . 5. Cyclodextringlucanotransferase catalyzing the formation of cyclodextrin from starch, characterized in that it is obtained from a strain of Bacillus circulans B-65 by the method described in claims 3 and 4, and has specificity for the formation of cyclodextrin, in particular β-cyclodextrin from starch, is active in temperatures up to 75 ° C, with optimum at 60 ° C, stable at temperatures up to 60 ° C, active at pH 4.0 to 12.0, and especially at pH 6.0 to 10.0. 6. A method of producing cyclodextrin, characterized in that the starch is subjected to cyclodextringlucanotransferase from a strain of Bacillus circulans B-65 at a temperature of from 40 to 70 ° C, at a solution pH of 6.0 to 12.0 for 20 to 72 hours, after completion reactions, the resulting cyclodextrins are removed by precipitation with trichlorethylene or toluene, and β-cyclodextrin is then recrystallized from distilled water. 7. The method according to claim 6, characterized in that a 5-15% starch solution is used. 8. The method according to claim 6, characterized in that the optimum temperature is 60 ° C. 9. β-Cyclodextrin, characterized in that it is obtained by the method described in claims 6-8.