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

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

 

The scope of the invention

This invention relates to biotechnology, in particular the method of producing cyclodextrin by exposure tsiklodekstringlyukanotransferazy (also called TSGT-Aza) for starch and products TSGT-basics bacteria strain Bacillus circulans B-65. According to the International classification, the invention relates to the following classes: 08 In 37/16; 12 P 19/18; C 12 N 1/20 C 12 N 9/10.

The purpose of the invention

This invention solves the problem of biosynthesis TSGT-the basics, which is typically used to obtain a cyclodextrin, as well as the problem of obtaining cyclodextrin by exposure TSGT-the basics on starch. Biosynthesis TSGT-basics most often carried out using bacteria of the genus Bacillus, which usually produce TSGT-ABC affecting starch with the formation of the mixture α-, β- and γ-cyclodextrin in different terms, at the same time, the output of the individual cyclodextrin is not high, and its secretion increases the cost of production.

Biosynthesis TSGT-elements according to the invention is carried out alkaliphile bacteria of a new strain of Bacillus circulans B-65, characterized by the production of a large number of TSGT-the basics, which is typically used to obtain a cyclodextrin. Cyclodextrins are obtained by TSGT-the basics of bacteria Century circulans, which is characterized by a high degree of conversion of starch into cyclodextrins, and specific to education, the project β -cyclodextrin.

The level of technology

Cyclodextrins are produced by the impact CTG-the basics on starch. In the process of getting cyclodextrins most important stage is to obtain CTG-the basics. CTG-Aza is produced by some species of bacteria, most of which are bacteria belonging to the genus Bacillus: B. macerans, B. circulans, B. B. megaterium, B. coagulans, B.lentus, Century ohbensis, B. firmus, B. amyloliquefaciens, B. stearothermophilus, B. subtilis, B.cerus. CTG-Aza is produced and some other bacteria belonging to the genus Bacillus, for example: Klebsiella pneumoniae, Micrococcus luteus, Micrococcus varians. Often as producers TSGT-the basics are the bacteria Bacillus macerans and alkaliphile bacteria belonging to the genus Bacillus.

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

Since cyclodextrins are widely used in industry and their cost is one of the major factors limiting their application, the scientists of all countries are looking for ways to increase efficiency of the method of obtaining TSGT-the basics. For this is what the goals are optimization of nutrient medium composition and optimization of physico-chemical factors for the growth of bacteria, work is underway to increase the productivity of bacteria using genetic methods (induced mutagenesis, genetic engineering), work is also underway to identify new organisms that are characterized by high production TSGT-the basics.

In the method of producing cyclodextrin is also very important properties TSGT-basics produced by bacteria. Properties TSGT-AZ, produced by various species of bacteria belonging to the genus Bacillus, are presented in Table 1.

Table 1: Properties TSGT-AZ from various microorganisms
Producer TSGT-the basicsRatio α-, β- and γ-CDOptim. pHpH high resistanceOptim. pace-pa (°)Thermostability (°)
without Sa2+CA2+
Bacillus coagulans (thermofill)2:2,6:16,56,0-9,0655060
Alkaliphilic Bacillus sp. (ATSS)71% β -CD6,5 and 8,5-65  
Bacillus macerans (IF03490)71% α-CD5,5-55--
Bacillus lentus (alkaliphile-tion)1:67:1,66,5-7,56,5-8,545-55--
Bacillus sp. (alkaliphile-tion)mainly β-CD6,06,0-11,05560-
Bacillus sp. (thermofill)-6,55,0-105055-
Bacillus stearothermophilus-5,0-5,55,0-8,070-756570
Bacillus macerans (IAM 1243) -6,05,5-9,56050-
Bacillus macerans-5,4-5,8-6050-
Producer TSGT-the basicsRatio α-, β- and γ-CDOptim. pHpH stabilityOptim. pace-pa (°)Thermostability (°)
without Sa2+CA24
Bacillus macerans (IFO 3490)2,7:1:15,0-5,78,0-105560-
Bacillus egaterium1:2,4:15,0-5,77,0-105555-
Bacillus circulans3:3:15,5 6,0 9,0604560
Bacillus amyloliquefaciens95% α-CD6,0-706065
Bacillus sp.1,6:4:15,56,0-9,565-7065-
Bacillus ohbensismainly β-CD5,56,5-9,56055-
Bacillus macerans (IAM 1227)mainly α-CD5,56,5 8,55050-
Bacillus circulans (IF03329)β->α-CD6,06,0-9,55555-
Bacillus coagulans (thermofill)1:0,9:0,3- ----
Bacillus cereus (NCIMB 13123)22:23:05,0-40--
Bacillus firmus (alkalife)0,08:1:0,2-----
Califolnai Bacillus subtilis0:25,1:4,7--50--
Califolnai Bacillus sp.5,7:23,5:

the 4.7
--40--

From the presented data we can see that TSGT-the basics from different organisms show similar properties, but there are some differences. The greatest differences among TSGT-AZ relate to quantities produced specific α-, βor γ-cyclodextrin, TSGT-basics consists of a mixture of α; β- and γ-cyclodextrin in various soo is wearing. There are, however, TSGT-elements that do not form αor γ-cyclodextrins. Most TSGT-AZ form a mixture of cyclodextrins, in which prevails β-cyclodextrin, whereas γ-cyclodextrin is present in a minimum quantity. Alkaliphile microorganisms produce TSGT-the basics that give the greatest content β-cyclodextrin, compared to αand γ-cyclodextrins. TSGT-the basics of alkaliphilic microorganisms form β-cyclodextrin, which is more than 70% of the total number of cyclodextrins. From the data presented in Table 1, we can conclude that TSGT-basics produced by strains belonging to the species Bacillus macerans, give the greatest output α-cyclodextrin, compared with other cyclodextrins. In addition to C. macerans, TSGT-Aza produced Century. amyloliquefaciens AL 35, forms a α-cyclodextrin with the yield up to 95%. Unlike most TSGT-AZ from different producers, TSGT-Aza produced by Bacillus subtilis No. 313, forms γ-cyclodextrin, but does not form any αor β-cyclodextrins. TSGT-Aza from Bacillus sp. AL-6 also gives the greatest output γ-cyclodextrin and β-cyclodextrin, but does not form α-cyclodextrin.

In the method of producing cyclodextrins using TSGT-AZ, which at the end of the reaction gives a mixture of cyclodextrins, α-, β- and γ-cyclodextrin the s are formed in different ratio, and the greatest output of any one or cyclodextrin leads to lowest output of other cyclodextrins. After completion of the enzymatic reaction with the formation of cyclodextrins, carry out their separation and purification. Since the isolation and purification of cyclodextrins increase the cost of the retrieval method, it is desirable to select microorganisms that produce TSGT-basics-specific education one of cyclodextrins.

From patent US 4477568 known TSGT-Aza obtained by kultivirovanija bacteria Bacillus circulans RIV No. 11115, which is used to obtain cyclodextrin. With this TSGT-the basics you can get up to 66% β-cyclodextrin. This TSGT-Aza active at a temperature of 60°but its activity decreases significantly at 65°With, in the pH range from 4 to 7 (the pH optimum - 6-6 .5). There is a need for an enzyme that will produce more than 66% β-cyclodextrin and will be active in a wider range of temperatures, and at higher pH, which will greatly reduce the danger of infections.

Summary of invention

Biosynthesis TSGT-the basics, which is then used to obtain cyclodextrin is alkaliphile bacteria strain Bacillus circulans B-65. Biosynthesis is carried out by growing the bacteria in an alkaline (pH 10) a liquid medium, under aerobic conditions, Fe is Manticore. The culture medium contains: as a source of carbon, from 0.5 to 3% starch as a source of nitrogen is from 0.5 to 6% of a substance selected from the group consisting of peptone, yeast extract, yeast autolysate, corn autohydrolysis (corn step liquid), or a mixture thereof, as a source of minerals: K2NRA4, MgSO4× 7H2O, Na2CO3.

After completion of the biosynthesis of the bacterial cells are removed by centrifugation or filtration, and then produced extracellular TSGT-basics concentrated using ultrafiltration.

When exposed to the thus obtained TSGT-the 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 environment (pH 8.0) at a temperature of 60°C.

The novelty of this invention lies in the fact that the biosynthesis TSGT-the basics, which is used to obtain cyclodextrin carried out using alkaliphilic bacteria Century circulars B-65. These bacteria were isolated from soil taken on-site, Leskovac. Bacteria Century circulans were isolated from approximately 2000 of bacterial colonies, which was characterized by the production TSGT-the basics. The isolate, which was then identified and marked as Century circulans, 65, showed significantly greater activity in produc the programming TSGT-the basics, and this property is clearly distinguishes it from other isolates. Thus, alkaliphile bacteria Century circulans B-65 represent a new strain, characterized in that it produces a significant amount of TSGT-the basics, which can be used to obtain a cyclodextrin. The strain Bacillus circulans B-65 deposited in the National collection of agricultural and industrial microorganisms (National Collection of Agricultural and Industrial Microorganisms, NCAIM) in Budapest under the number NCAIM (P) 001277.

The advantage of using TSGT-the basics, which is produced by the data alkaliphiles strain Century circulans B-65 is in its use to obtain β-cyclodextrin. TSGT-Aza this strain-specific education β-cyclodextrin and is characterized by a high degree of conversion of the starch in β-cyclodextrin. In the described reaction conditions when the concentration of the starch 5%, TSGT-Aza performs the transformation to 45% starch into cyclodextrins. Of the total number produced cyclodextrins number β-cyclodextrin is about 95%, which is the highest value compared with available literature data. Unlike TSGT-basics, is known from patent US 4477568, TSGT-Aza according to the invention are active at higher temperatures (up to 75° (C)in a wider range of pH (4 to 12).

The difference allocation method and villages of the Ktsia bacteria-producers TSGT-elements according to the invention from known from patent US 4477568 also is in a medium composition for culturing bacteria as grounds include phenolphthalein or color indicators methyl orange and not used Fel3× 6N2O.

Selection and breeding of bacteria-producers TSGT-the basics

TSGT-basics produced by bacteria, were isolated from 54 different soil samples, which were taken on-site, Leskovac. 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 were applied to the agar Cup with the environment of the Park, containing: 1% soluble starch, 0.5% peptone, 0.5% of yeast extract, 0.1% of K2NRA4, 0,02% MgSO4· 7H2O, 1% Na2CO3, 0.03% phenolphthalein, 0.01% gelantine, 1.5% agar, pH of 10.0 (Park et al., 1989). Incubation was performed at 37°C. After 48 hours of incubation, colonies, surrounded by the most wide yellow areas colored red agar plates, were isolated and purified.

The number of colonies obtained from various soil samples, ranged from 0 to 200. Colonies that were produced TSGT-ABC, were surrounded by yellow zones of various sizes. Different soil samples had different percentage of colonies producing TSGT-ABC, which varied from 0 to 90% of the total number of colonies. Taking into account the size of the zone around the colonies was isolated and purified 85 colonies is C 2000 colonies, producing TSGT-the basics. After cleaning the environment of the Park were selected 45 colonies for further work. Selected colonies were transferred to oblique agar with environment Horikoshi (Horikoshi) II and after incubation were stored at +4°C.

45 selected alkaliphilic isolates producing TSGT-ABC, were grown in liquid medium in Erlenmeyer flasks with stirring for further breeding isolates. Incubation was performed at 37°C for 48 hours with constant stirring at 200 rpm On the third and fourth day were sampled, and after removal of the bacterial cells by centrifugation (10000 × g, 10 min, +4° (C) was determined amylolytic and cyclodextrines activity of the supernatant.

The amylolytic activity of the isolates ranged from 0.4 U/ml to 12,17 U/ml Highest number of isolates demonstrated the amylolytic activity of 1 U/ml to 2 U/ml Number of isolates, which showed higher amylolytic activity was gradually decreased, so that the amylolytic activity of greater than 5 U/ml showed only 3 isolates. Cyclodextrines activity ranged from 0,019 U/ml to 0,530 U/ml Cyclodextrines activity of most isolates was 0,100 U/ml Cyclodextrines activity above 0,200 U/ml was demonstrated only in isolation. solat 65 showed amylase activity 12,17 U/ml and cyclodextrines activity 0,530 U/ml, which were significantly higher compared with other isolates.

Morphological and taxonomic characteristics of the bacterial strain

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

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

Morphological, physiological and chemical characteristics of the isolate is 65 essentially the same characteristics of Bacillus circulans. However, a significant feature of the isolate 65 is that it shows good growth in alkaline medium (pH 10) and does not grow in a neutral environment, such as nutrient medium with a pH of 6.8, which distinguishes it from the described strains of Bacillus circulans. Isolate 65 is also different from the strains of Bacillus circulans the fact that it grows in an environment with 10% NaCl.

Based on these characteristics the isolate 65 was identified as a strain of Bacillus circulans. Because its feature is good growth in alkaline medium (pH 10) and the lack of growth in a neutral environment, isolate 65 identified as alkaliphilic strain of Bacillus circulans, 65. Of this isolate 65 are presented in Table 2.

Table 2: characteristics of the bacteria Bacillus circulans (ATCC 4513) and isolate 65
Morphological and physiological-biochemical characteristicsBacillus circulans

(ATCC 4513)
Isolate 65
FormWandWand
Size (µm)0,5-0,7×2-50,6-0,7×2-4
Mobility++
The dispute formellipticalelliptical
Staining gram++
Catalase ++
Anaerobic growth++
Analysis Voges-Proskauer--
Production of acid from carbohydrates:

D-glucose

L-arabinose

D-xylose

D-mannitol
+

+

+

+
+

+

+

+
Gas production from glucose--
Hydrolysis of: gelatin

starch
+

+
+

+
The use of citrate--
Production of indole--
Growth at pH: 6.8 cm (medium)

the 5.7
+

+
-

-
Growth in the presence of NaCl: 2%

5%

7%

10%
+

+

+

-
+

+

+

+
Growth: the 30°

37°

40°
+

+

+
+

+

+

Biosynthesis TSGT-basics strain, circularis B-65

Biosynthesis TSGT-basics is a bacteria strain Century circulans B-65, growing in alkaline (pH 10) liquid medium. Nutrient medium includes: 1% soluble starch, 0.5% of peptone-1, 0.5% of yeast extract, 0.1% of K2NRA4, 0,02% MgS4× 7H2O, 1% Na2CO3

The inoculum for a method of obtaining TSGT-the basics were prepared by inoculation of culture, taken from the slanted agar, in a liquid environment. Incubation was carried out at 37°with constant stirring (200 rpm) for 48 hours. In the fermenter with fresh environment contributed 5% inoculum. Biosynthesis TSGT-basics was carried out for 96 hours at 37°With the flow of sterile air at 0.5 vvm (volume of air per volume of medium min-1the volume of air per volume of medium per minute) when the rotation speed of the mixer 300-1. After 96 hours of growth of bacterial cells were removed by centrifugation (10000 × g, 10 min, +4° (C) or by filtration, and then carried out the concentrated extracellular produced TSGT-the basics by ultrafiltration. Thus obtained TSGT-ABC then used to obtain a cyclodextrin. In the process of obtaining determined amylolytic and cyclodextrines activity TSGT-basics, extracellular produced by strain Century circulans B-65. Cell growth and activity TSGT-basics produced by strain Century circulans B-65, growing in the described conditions described in Table 3.

Table 3
Activity TSGT-basics, cell growth and pH changes during growth of strain Century circulans B-65
Time (h)Activity AMI the political activity TSGT-the basics (U/ml) Cyclodextrines activityCell growth (610 nm)
0//0,125
247,300,201,677
489,700,241,489
7214,850,381,497

TSGT-Aza is produced in the process of cell growth. Prior to the completion of the growth phase of the value cyclodextrines activity reaches of 0.20 U/ml, which is slightly more than 30%, compared with the activity obtained after 96-hour growth period. After completion of the growth phase of the product TSGT-basics continues, so after 96 hours of growth cyclodextrines activity reaches a value of 0.60 U/ml, and the amylolytic activity 23,37 U/ml Increase cyclodextrines and amylolytic activities after completion of the growth phase is about 70% of the total. Thus, the isolate B-65 produces more TSGT-basics upon completion of the phase of cell growth.

In the process of growth Century. circulans B-65 amylolytic and cyclodextrines activity increases proportionally, which leads to the conclusion that the strain Century circulans B-65 produces TSGT-ABC and does not produce any other amylolytic enzymes.

This conclusion is confirmed by the op is edelenyi content cyclodextrins and reducing sugars during incubation, TSGT-basics of In, circulans B-65 with starch. These results showed that during the reaction was observed the formation of cyclodextrins, while the amount of reducing sugars did not increase that would have occurred in the presence of other amylolytic enzymes.

The advantage of this method of obtaining TSGT-the basics, which is confirmed by these results is that the biosynthesis TSGT-basics is the new alkaliphiles strain Century circulans B-65, which is characterized by extracellular products of a large number of TSGT-the basics. Another important characteristic of the strain Century circulans B-65 is the fact that in addition to TSGT-the basics this strain does not produce other amylolytic enzymes. The presence of these enzymes in the drugs TSGT-the basics in the process of getting cyclodextrins would lead to the hydrolysis of starch, i.e. to reduce the concentration of the substrate to obtain a cyclodextrin, which would have a negative impact on the final yield of the product.

The advantage of biosynthesis TSGT-basics strain Century circulans B-65, compared with other producers of this enzyme is that the biosynthesis TSGT-basics strain Century circulans B-65 is in a very alkaline environment (pH 10), thus reducing the risk of infection by other microorganisms, which is extremely important for the production in an industrial environment.

Properties TSGT-the basics of the Century circulans B-65

Besides that shtam the Century circulans B-65 produces TSGT-ABC in large quantities, it is very important that this TSGT-Aza characterized by good properties regarding thermal stability and optimal for its activity, temperature and pH-stability and optimal pH values.

TSGT-Aza from strain Century circulans B-65 is characterized by the fact that it is stable at temperatures up to 55°and in the presence of CA2+its stability is increased to 60°C. the Optimum temperature for its validity is 60°C. Due to the high optimum temperature of action and high thermal stability TSGT-the basics of strain Century circulans B-65 method of producing cyclodextrin is carried out at a temperature of 60°C. the Advantage of the cyclodextrin at a high temperature is that under these conditions the reaction decreases the risk of infection by other microorganisms. At high temperatures well above the rate of the enzymatic reaction, which reduces the duration of the production of cyclodextrins.

TSGT-Aza from strain Century circulans B-65 is characterized by the fact that the optimum for the activity of the pH value is in the range of pH from 5.0 to 6.0, however, TSGT-Aza retains its activity in a fairly wide range of pH values in the alkaline environment. TSGT-Aza from strain Century circulans B-65 stable at pH from 6.5 to 10.0. Because of these properties TSGT-the basics of getting cyclodextrins can is to be implemented in a wide range of pH values. The Michaelis constant for TSGT-the basics of strain B.circulans B-65 is 3,016 mg/ml (calculated by the method of Lineweaver-Burck) to 4.77 mg/ml (calculated by the method of Idi-Hofsti). Inhibitors of this enzyme are NaN3, CuSO4, MnSO4, FeSO4, MgSO4and glucose.

The results of the study of the properties TSGT-the basics of strain Century circulans B-65 below.

The effect of pH on the activity of TSGT-the basics

The effect of pH on the activity of TSGT-basics investigated in the pH range from 4.0 to 10.0 at a temperature of 40°C. To summarize the pH used the following buffers: 50 Mm sodium acetate buffer (pH 4-5), 50 mm phosphate buffer (pH 5-8), 50 mm carbonate buffer (pH 8-10). The results of these studies are presented in Table 4 show that the optimal pH for activity TSGT-ASE is in the range of pH from 5.0 to 6.0, however, TSGT-Aza retains its activity in a fairly wide range of pH values in the alkaline environment.

Table 4
Cyclodextrines activity TSGT-basics at different pH values.
PH50 mm bufferCyclodextrines activity (U/ml)Relative activity (%)1
4Acetate0,1819,8
4,50,4 80,4
5Phosphate0,92100
5,50,91the 98.9
60.8996,7
6,50,7682,6
70,6671,7
80,5660,3
9Carbonate0,4245,2
100,2628,6
28,6Relative activity is expressed as a percentage of the maximum activity obtained at pH 5.0.

The effect of pH on the stability TSGT-the basics

The effect of pH on the stability TSGT-basics investigated in the pH range from 4.0 to 12.0. To summarize the pH used the following buffers: 50 mm sodium 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). TSGT-ABC incubated at different pH values for 1 h at 40°and then the pH is brought to 6.0 and was determined residual enzymatic activity. The results of these studies are presented in Table 5, showing that TSGT-Aza was stable in a wide range of pH values from 6.5 to 10.0.

Table
Residual TSGT-asna activity after incubation at various pH values
pH50 mm bufferCyclodextrines activity (U/ml)Relative activity (%)
4Acetate0,1819,8
50,7480,4
6Phosphate0,92100
6,50,91the 98.9
70,8996,7
8Tris-HCl0,7682,6
90,6671,7
100,5660,3
11Glycine-NaOH0,4245,2
12 0,2628,6

The effect of temperature on the activity TSGT-the basics

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

Table 6: Activity TSGT-the basics when FS is cnyh temperatures
Temperature (°)Relative activity (%)
3023,1
4036,2
4549,8
5071,7
5585
60100
6594,7
7045,3
7515,9

The effect of temperature on the stability TSGT-the basics

The effect of temperature on the stability TSGT-basics investigated in the temperature range from 30°C to 80°S. a Study of thermal stability TSGT-basics conducted in the absence and in the presence of ions of CA2+at a concentration of 10 mm. TSGT-ABC were incubated for 30 minutes at various temperatures at pH 8.5, and then the pH is brought to 6.0 and investigated residual enzymatic activity.

The results of the study (table 7) showed that TSGT-Aza was stable in the temperature range up to 55°and that in the presence of ions of CA2+its stability was increased to a temperature of 60°C.

Table 7
Residual TSGT-asna activity after incubation at various temperatures
Temperature

(°)
About the adequate activity (%) without Sa 2+Residual activity (%) and CA2+
309797,3
4098,396,8
509696,8
5596of 98.2
6052,896,8
6517,281,7
70319,0
75-0
80-0

Getting Icodextrin

Cyclodextrins get, affecting TSGT-asoi from strain Century circulans B-65 for starch in a concentration of from 5% to 15%. Due to the high optimum temperature of enzyme and high thermal stability TSGT-the basics of strain Century circulans B-65, the process of obtaining cyclodextrins can be performed at temperatures up to 60°C. with regard to pH, obtaining cyclodextrins can be performed in a wide range of pH values from 6.5 to 10.0.

The output of cyclodextrins in the incubation of a 5%solution of starch with different concentrations TSGT-basics at 40°C and pH 6.5 is shown in Table 8.

Table 8
The output of cyclodextrins with different conc the operations TSGT-the basics 1
The concentration of enzyme (U/g of starch)The incubation period

(h)
CD

(g/l)
Output CD2

(%)
Cyclodextrines activityAmylase activity
0,623,42410,821,6
1,246,82411,222,3
1,8to 70.22411,723,3
2,493,62412,124,2
3,011724

48
14,8

19,2
29,5

38,4
4,517524

48
17,8

19,7
35,6

39,4
6,023424

48
21,7

22,5
43,3

44,9
7,529324

48
21,1

20,3
42,2

40,6
40,6The reaction mixture, which contained 5% starch and the appropriate concentration of enzyme, incubated for 48 hours at 40°C.

2The output of the CSD represents the degree into which I starch in the CSD.

The best results were obtained for TSGT-the basics at a concentration of 6.0 U (cyclodextrines activity)/g of starch. With this concentration, TSGT-the basics about 45% of starch into cyclodextrins.

After completion of the enzymatic reaction get cyclodextrins can be removed from the reaction mixture by precipitation, using, for example, trichloroethylene or toluene. From the besieged cyclodextrins β-cyclodextrin can be removed by crystallization, due to the fact that it is much less soluble compared with other cyclodextrins. Cyclodextrins obtained by precipitation, dissolved in distilled water by heating at 100°and then was led β-cyclodextrin with a gradual lowering of the temperature to 20-25°With careful stirring. Crystals β-cyclodextrin was then removed by filtration, washed with small amount of cold water and dried at 70°C.

The analysis of the obtained cyclodextrins spectrophotometric methods and HPLC, it was found that TSGT-Aza from strain Century circulans B-65, in the described conditions, makes up 45% of starch into cyclodextrins and mainly produces β-cyclodextrin. The resulting cyclodextrin number β-cyclodextrin is approximately 95%, while the number αqi is latextra and γ -cyclodextrin is 2-3% and 0.6-3%, respectively.

TSGT-Aza from strain Century circulans B-65 is characterized by the fact that it is specific for the formation of p-cyclodextrin and converts a high percentage of starch in β-cyclodextrin. Consequently, the use TSGT-the basics of the new strain has significant advantages for obtaining β-cyclodextrin, compared with TSGT-Azami from other known bacteria.

The following examples illustrate, but not limit the invention.

Example 1

Products TSGT-the basics

TSGT-Aza is produced by growing bacteria strain Century circulans B-65 in a liquid medium, containing 1% soluble starch, 0.5% peptone, 0.5% of yeast extract, 0.1% of K2NRA4, 0,02% MgS4× 7H2O, 1% PA2CO3. The inoculum for a method of obtaining TSGT-basics was obtained by inoculation in 450 ml of a liquid medium, Horikoshi (Horikoshi) II culture of the slanted agar. Incubation was carried out for 48 hours at 37°with constant stirring (200 rpm).

In the fermenter, containing 9 l environment Horikoshi, made 450 ml of inoculum. The growth was carried out for 96 hours at 37°With the flow of sterile air at 0.5 vvm and the rotation speed of the mixer 300 min-1. In the process of growth was sampled and determined amylolytic and cyclodextrines activity of the supernatant. After 96 hours of bacterial growth the e cells were removed by centrifugation (10000 × g, 10 min, +4° (C), and then produced extracellular TSGT-basics concentrated by ultrafiltration.

Strain Century circulans B-65, growing thus, after 96 hours was produced extracellular TSGT-ABC with amylolytic activity of 23.4 U/ml and cyclodextrines activity of 0.6 U/ml After concentration of the enzyme by ultrafiltration received the amylolytic activity of 450 U/ml and cyclodextrines activity 12 U/ml

Example 2

Getting Icodextrin

Soluble starch (300 g) was dissolved in 6 l of water when heated. The temperature was brought to 40°and pH to 6.5. The starch solution was added TSGT-ABC in concentration 6U (cyclodextrines 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 charcoal. After removal by filtration of activated carbon cyclodextrins besieged from the reaction mixture (6 l) trichloroethylene (1.2 l). Besieged cyclodextrin was then removed by filtration and dried at 70°C. the Final yield was 107 g of cyclodextrin from 300 g of starch.

The resulting cyclodextrin was dissolved in 500 ml of distilled water by heating at 100°C. Then, when the gradual peniteniary to 20-25° And with careful stirring was carried out by crystallization β-cyclodextrin. After keeping overnight at +4°the crystals were removed by filtration, washed with small amount of cold distilled water and dried at 70°C. Thus received 75.2 g β-cyclodextrin in the form of white crystals.

1. The method of selection and breeding bacteria-producers tsiklodekstringlyukanotransferazy, characterized in that the bacteria isolated from the soil sample, prepared in aqueous suspension by heating up to 80°C for 10-15 min and inoculant in agar Cup with medium containing starch, peptone, yeast extract, K2HPO4, MgSO4, Na2CO3, phenolphthalein, gelatin and agar, then incubated for 20 to 72 h at 37°To carry out the breeding colonies, selected colonies were cultured in liquid medium until such time as is the production of enzymes, and perform the selection of the isolate Bacillus circulans, 65.

2. The bacterial strain of Bacillus circulans, 65 NCAIM (P) 001277 (65) - producer of extracellular tsiklodekstringlyukanotransferazy.

3. The method of obtaining tsiklodekstringlyukanotransferazy, characterized in that alkaliphile bacteria strain Bacillus circulans, 65 cultivated under aerobic conditions in a liquid medium within 24-120 hours at a temperature of from 25 to 50°after culturing bacterial cells and is removed by centrifugation and filtration, and concentration of extracellular produced TSGT-basics is carried out by ultrafiltration.

4. The method according to claim 3, wherein the nutrient medium contains

- 0,5-3% starch as a carbon source,

- 0.5 to 6% of a substance selected as the nitrogen source from the group consisting of peptone, yeast extract, yeast autolysate, corn autohydrolysis or mixtures thereof,

sources of minerals: K2NRA4, MgSO4× 7H2Oh, PA2CO3.

5. Tsiklodekstringlyukanotransferazy catalyzing the formation of cyclodextrin from starch, characterized in that it is derived from a strain of Bacillus circulans, 65 method, characterized in PP and 4, and has specificity for the formation of a cyclodextrin, in particular β-cyclodextrin, starch, active at temperatures up to 75°C, with an optimum at 60°S, stable at temperatures up to 60°active at pH 4.0 to 12.0, and especially at a pH of from 6.0 to 10.0.

6. The method of producing cyclodextrin, characterized in that the starch is subjected to the action tsiklodekstringlyukanotransferazy from a strain of Bacillus circulans, 65 at a temperature of from 40 to 70°when the solution pH from 6.0 to 12.0 for 20 to 72 hours, after completion of the reaction the resulting cyclodextrin is removed by deposition of trichloroethylene or toluene and β-cyclodextrin then cross stilizovala of distilled water.

7. The method according to claim 6, characterized in that use 5-15%solution of starch.

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 p-8.



 

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