RU2312135C2 - Strain of bacterium paenibacillus campinasensis as producer of cyclodextrin glucanotransferase - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to a novel strain of microorganisms producing cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) with the enhanced specificity with respect to formation of beta-cyclodextrin. The strain of bacterium Paenibacillus campinasensis IB-10155 is deposited and stored in Collection of microorganisms in Biology Institute UNTS RAN. Activity of CGTase in cultural fluid is estimated to be 5.8 U/ml wherein 1 unit of activity is determined as amount of CGTase catalyzing formation of 1 mcM of ACB beta-cyclodextrin per 1 minute from 2% starch solution at pH = 6.0. Part of beta-cyclodextrin in sum of other cyclic products exceeds 70%. Invention provides high stability of CGTase catalyzing the transformation reaction of starch to beta-cyclodextrin at relative constant part of this product to sum of other cyclic dextrins in early steps of reaction.

EFFECT: valuable properties of strain.

2 tbl, 4 ex

Description

The invention relates to biotechnology, and in particular to a technology for the production of cyclodextringlucanotransferase preparations (TSGTaz K.F.2.4.1.19), an enzyme for producing cyclodextrins from starch.

In particular, the invention relates to the technology of beta-cyclodextringlucanotransferase (beta-CGTase), transforming starch mainly into beta-cyclodextrin. This cyclic oligosaccharide is most in demand in the food, pharmacological, textile industries, cosmetics, etc. [1-3]

CGTases catalyze the formation of three macrocyclic oligosaccharides (or cyclodextrins, differing in their molecular sizes and properties. Beta cyclodextrin consists of seven alpha-D-glucose residues, while alpha and gamma macrocyclic homologs have six and eight in their molecule rings glucopyranose units Due to the universal ability to form inclusion complexes with a whole range of organic and inorganic compounds, beta-cyclodextrin is in demand on the market and is commercially available in large volumes [4].

The raw material for the production of cyclodextrins is starch, and as a result of the reaction, a mixture of alpha, beta and gamma cyclodextrins is formed, as well as a mixture of linear and branched dextrins of various molecular sizes. The yield and ratio of the amount of synthesized individual cyclic oligosaccharides depends on the catalytic properties of the used CGTase.

The available information on the producers and catalytic properties of TsGTaz and, in particular, beta-TsGTaz, does not allow for their objective comparison because these analyzes were performed under non-identical conditions and in different laboratories. Meanwhile, the nature and depth of the reaction of the enzymatic transformation of starch into cyclodextrins significantly depends on many components: the nature and source of starch, the ratio of amylopectin and amylose in it, the mode of pretreatment of starch before the enzymatic reaction, pH, temperature, dosages of the enzyme, etc. Objective comparative information on the catalytic properties of various TSGTaz preparations can also not be obtained due to the limited availability of both enzyme samples and their producers, the lack of methodological x standards when measuring cyclizing activity. Nevertheless, among the main criteria that determine the effectiveness of beta-CGTase producers are: selectivity of CGTase in relation to the synthesis of beta-CD, the overall yield of beta-CD, and their thermal stability.

Bacterial strains of the species Paenibacillus macerans (obsolete name Bacillus macerans) and Bacillus sterothermophilus are known that form CGTases that catalyze the formation of, mainly, alpha-cyclodextrin [3]. In the processes of obtaining beta-cyclodextrins, these enzymes can only be used in technology using organic solvents, often very toxic, such as, for example, toluene [3, 5]. Beta-cyclodextrin obtained in contact with toluene does not meet modern environmental safety requirements of the final product.

Known strains of Bacillus sp., Bacillus circulans and Bacillus alkalophilus, [3, 5, 6], which are characterized by the formation of beta-specific TSHTaz, catalyzing mainly the synthesis of beta-cyclodextrin in the amount of three macrocyclic oligosaccharides. Beta-TSGTazy also differ in a deeper degree (compared with the TSTazy Paenibacillus macerans) trans-transformation of starch into cyclic products. The named cultures as producers of TSHT-az are acceptable for the processes of obtaining beta-cyclodextrin by the solventless technology.

The closest analogue in terms of the set of essential features and the achieved result is the Bacillus sp. Strain (deposited in the All-Russian collection of industrial microorganisms under the number VKM B-1793D), which forms extracellular beta-CGTase, the main product of which when exposed to starch is beta-cyclodextrin [7] .

The disadvantage of CGTase produced by the culture is its relatively low thermal stability.

Another disadvantage of the beta-CGTase strain of Bacillus sp VKM B-1793D is that the proportion of beta-cyclodextrin in the sum of other cyclic oligosaccharides continuously changes during the enzymatic reaction. The pronounced inconsistency in the ratio between cyclodextrins in the conversion mixture leads to technological instability at the stage of isolation and purification of the final product - crystalline beta-cyclodextrin.

The task to which the claimed invention was directed was to search for a natural strain that provides the synthesis of extracellular beta-CGTA, which has sufficient thermal stability, catalyzes the transformation of starch into beta-CD with a relatively constant proportion of this product in the sum of other cyclic dextrins at different stages of the reaction.

The technical result that can be obtained by carrying out the invention is to detect a culture of microorganisms with the ability to form CGTase, suitable for the synthesis of beta-cyclodextrin at a temperature of at least 55 ° C and catalyzing the reaction of transformation of starch to obtain a conversion mixture with a relative constancy of the proportion of beta -CD.

The essence of the invention is a strain of Paenibacillus campinasensis IB-10155.

The new IB-10155 strain was isolated from a soil sample taken in the Karmaskalinsky district (village of Staro-Musino) of the Republic of Bashkortostan by a microaerophilic accumulative culture on an elective nutrient medium containing mineral buffer components and cyclodextrin as the sole source of carbon and energy.

Morphological features of the strain. The cells are rod-shaped, 0.3-0.6 · 2-3.5 μm in size, after 24 hours of incubation at 37 ° C, mobile, gram-variable. Form endospores. Spore formation is terminal and subterminal, spores are elliptical, swelling sporangium.

The strain during growth on meat and peptone agar (MPA) every other day at 37 ° C forms flat translucent colonies with a diameter of 3-5 mm with a smooth edge and smooth surface, with the consistency of butter. On wet potato-glucose agar under the conditions mentioned above, the strain forms flat, irregular in shape and 5-10 mm in size, transparent, whitish-colored migrating colonies, most of which have a fuzzy center with radially migrating microcolonies. The described nature of the growth of the colonies is violated when using dried agar or at a temperature above 45 ° C. In liquid meat and peptone broth, the culture forms mucus. Both in a liquid and in a dense medium, the culture does not form pigments.

Biochemical characteristics of the strain

Aerob, but capable of anoxic growth in the presence of nitrates.

The Voges-Proskauer reaction (formation of acetylmethylcarbinol) is negative, the pH in the Voges-Proskauer broth after growth <6 (5.95).

The response to catalase daily culture is positive.

The strain forms acid (but not gas) from glucose, xylose, arabinose and mannitol. It is capable of fermenting these sugars when inoculated with an injection in an agar column.

Hydrolyzes (causes depolymerization) starch, casein and weakly gelatin.

Does not utilize citrate, tyrosine, and phenylalanine.

Urea does not decompose, indole does not form. Restores nitrates to nitrites.

The strain is capable of growth in a nutrient broth with 7% NaCl. It grows well in the pH range of 7.4-10.0 with an optimum of 9-9.5.

The temperature range for growth is 20-46 ° C with an optimum of 38-40 ° C.

The strain can be stored without loss of useful properties at a temperature of 5-10 ° C on potato-glucose (or potato) agar for at least 6 months with mandatory reseeding at least 1 time in 3 months.

Phylogenetic characteristics of the strain.

Comparison of the 1484 nucleotide sequence of the portion of the gene encoding 16S rRNA of IB-10155 strain revealed a 99.7% similarity to the portion of the same sequence (sequence number in Genebank AF021924), previously found in the type strain Paenibacillus campinasensis, deposited in the Korean collection of type CCST cultures under number 0364BP ^T.

Based on the study of the combination of morphological, biochemical and phylogenetic characteristics of the described culture, a new strain producing beta-CGTase was identified as Paenibacillus campinasensis IB-10155.

For the growth of culture and the biosynthesis of beta-cyclodextrin-glucanotransferase, starch, hydrolyzed starch, corn and other grain flour, corn wastes, potatoes, or their extracts can serve as a carbon source. Sources of nitrogen include peptone, yeast extract, corn extract, yeast cells, ammonia nitrogen. As mineral additives can be used disubstituted potassium phosphate, soda, calcium carbonate.

The cultivation of the strain is carried out under aerobic conditions in a fermenter or flasks at a temperature of 40 ° C for 64-72 hours, the pH of the medium is 8-9.5. In the process of cultivation, the activity of beta-CGTase in the culture fluid is monitored.

The possibility of using the invention is illustrated by examples, which do not limit the scope and essence of the claims associated with them.

Example 1. Illustrates the possibility of obtaining a liquid enzyme preparation of beta-CGTase by cultivating the patented strain in flasks on a rocking chair, followed by thickening of the fermentation liquid on the ultrafiltration module.

Inoculum is obtained by growing the strain on a solid nutrient medium composition: soluble starch 1%; peptone 0.4%, yeast extract 0.5%, potassium phosphate disubstituted 0.2, sodium carbonate 0.3% agar 1.6%, pH 8.6-9.0. Flasks with a liquid medium of the same composition but not containing agar were inoculated with the biomass of individual colonies. Cultivation is carried out in 250 ml flasks containing 60 ml of sterile medium, on a shaker with 240 rpm at 40 ° C for 64-72 hours.

The activity of beta-CGTase in the culture fluid and the number of enzyme units during dosing are determined by the ability of the test drug to catalyze the formation of beta-cyclodextrin from a 2% starch solution. The unit of activity is taken to be such an amount of enzyme that within 1 minute at 50 ° C catalyzes the formation of 1 μM beta-CD from 2% potato starch. The enzymatic reaction is carried out by mixing two volumes of 3% freshly prepared soluble potato starch in 0.1 M acetate buffer at pH 5.7 with one volume of the enzyme solution (QL) diluted with acetate buffer 50-400 times. The tube with the reaction mixture is thermostated for 60 minutes at 50 ° C in a solid state thermostat. A portion of the reaction mixture in a volume of 500 μl is added to 3000 μl of a measuring solution prepared on the basis of 0.1 M potassium carbonate and 0.4% alcohol phenolphthalein. The resulting solution was mixed and the absorption of the resulting mixture D _{1 was} determined at 553 nm in a glass cuvette with an optical path length of 1 cm. The concentration of phenolphthalein in the measuring solution was selected so that at the time of determining the activity, the optical density of this liquid was E ₅₅₃ = 1.00 ± 0.1. The control is a sample of a mixture of the enzyme with starch, identical to that which was used in the experiment, but which did not pass 1 hour heat treatment at 50 ° C. A portion of the liquid of the control sample is mixed with the measuring solution, similar to how it was done with the reaction mixture, after which the optical density of the obtained control mixture D ₂ is measured at 553 nm. The enzyme activity is determined by the formula (I):

where ΔD ₅₅₃ = (D ₂ -D ₁ ) is the difference in optical density between the measured and control solutions;

N is the degree of dilution of the enzyme.

The average beta-CHTase activity of the patented strain and other cyclodextrinogenic microorganisms used for comparison, measured in the culture fluid after 70 hours of growth, are given in table 1.

The average activity of beta-CGTase Paenibacillus campinasensis IB-10155 in the cell-free culture fluid is 5.8 ± 1.0 units / ml.

Example 2. Under comparable measurement conditions, the catalytic features of the beta-CGTase Paenibacillus campinasensis IB-10155 are demonstrated in comparison with the action of beta-cyclizing enzymes from other producers (table 1).

The experience is as follows. In 5 ml plastic tubes, 2 ml of 4% potato starch are placed in 50 mM acetate buffer pH 6.0, autoclaved for 30 minutes at 120 ° C. Portions of solutions of beta-CGTase obtained from various sources are metered into these tubes to achieve a ratio of 1, 2, 4, 8, 15, 30 units. beta-CGTase per 1 g of ACB starch in vitro. Portions of a preservative to prevent microbial growth are also introduced there. To equalize the concentration of substrate (starch) to 2.75% according to ASB, calculated portions of 50 mM acetate buffer pH 6.0 are added to all tubes. After 24 hours of exposure at 50 ° C, 900 μl of liquid are taken from the obtained solutions, which are mixed with 90 μl of 10 wt.% Xylose (internal standard), with 990 μl of acetonitrile. The precipitate formed is separated by centrifugation, and then the supernatant is analyzed by HPLC on an aminated silica gel column. A mixture of acetonitrile-water in a ratio of 60:40 is used as an eluent. The concentration of cyclodextrins in the samples is calculated by the internal standard method according to the peak areas using software and hardware. The yield of CD in experiments characterized by the maximum yield of the sum of cyclic products, as well as other design parameters defined in this example, are also presented in table 1.

Example 3. Demonstrates the resistance of CTG Paenibacillus campinasensis IB-10155 to thermal effects in comparison with enzymes of other producers.

The experiment is performed using samples of CGTaz preparations diluted with 50 mM acetate buffer at pH 6.0 to a specific activity of 1.0 u / ml, without the addition of stabilizers (calcium salts). Tubes with the same dilution volumes of the studied enzyme 1000 μl are placed in a solid state thermostat and incubated at 55 ° C. At certain time intervals, the heat-treated samples are removed and the residual beta-CGTase activity is measured in them according to the procedure described in Example 1. The inactivation time of 50% of the activity of enzyme preparations of various producers under the conditions of this experiment, as measured by graphical extrapolation and interpolation, is presented in table 1.

Example 4. The relative constancy of the proportion of beta-cyclodextrin at different stages of the enzymatic action during treatment of starch with an enzyme is demonstrated in comparison with the Bacillus sp. BKM B-1793D under conditions of excessive dosages of enzymes.

Two glass reactors with a volume of 200 ml, equipped with stirring and temperature control devices, add a solution of potato starch, subjected to 30 minutes processing at 120 ° C, and sodium acetate. Samples of lyophilized enzyme preparations beta-CAA Paenibacillus campinasensis IB-10155 and Bacillus sp. BKM B-1793D. The dosage of enzymes is selected according to the amount of substrate introduced into the reactor, while it is 30 units. beta-CGTase per 1 gram of ACB starch. 50 mM acetate buffer is also added to the reactor to achieve the required concentrations of ingredients, which are: 2.75 wt.% For starch (according to ASB), 50 mM for sodium acetate. The starting pH of the mixture should be 6.00 ± 0.05. At certain intervals, volumes of liquid are removed from the reactor, the enzymatic reaction is stopped in them by adding 50 vol.% Acetonitrile, and the concentration of cyclodextrins is measured by HPLC, as described in example 2. The determination results shown in table 2 indicate that when using TSHTazy of the patented strain Paenibacillus campinasensis IB-10155 (in comparison with the cyclizing enzyme of the prototype strain Bacillus sp. BKM B-1793D) there is a higher yield of the sum of cyclic products in relation to starch when The constant constancy of the proportion of beta cyclodextrin in the conversion products.

Table 1
The results of the conversion of a solution of 2.75% (ASV) of potato starch with beta-CGTaz preparations under comparable conditions. The strain number in the collection of IB UC RAS Title The activity of TSGTase VKZh U / ml % conversion to cyclic products Time loss of 50% activity of TSGTase at 55 ° C, min % beta in conversion products The output of the CD in relation to the ASV substrate, wt.% alpha CD beta CC gamma CD 10116 Bacillus sp. 1.0 51.0 9 71.0 10.0 36.0 5.0 10167 Bacillus sp. 3.9 90.0 19 60.0 23.0 54.0 13.0 10115 Paenibacillus sp. 0.35 50.0 6 31.3 30.0 15.7 4.3 10169 Bacillus sp. 1.29 74.5 eleven 71.6 11.4 53.4 9.8 10199 Bacillus sp. 0.44 73.2 6 57.9 23.7 42.4 7.2 10192 Bacillus sp. 0.61 62.0 12.4 68.9 10.9 42.7 8.4 10195 Bacillus sp. 3.9 86.7 46 61.0 18.1 52.9 15.8 10168 Bacillus sp. 5.8 94.4 19 62.4 20.3 58.9 15.2 10176 Paenibacillus sp. 0.43 77.5 161 65.0 16.3 50.4 10.8 10162 Paenibacillus sp. 0.39 79.6 89 67.7 14.5 53.8 11.2 10181 Paenibacillus sp. 0.67 68.8 120 75.0 7.5 51.6 9.7 10155 Paenibacillus campinasensis 5.8 74.6 325 73.9 8.5 55.1 11.0 663 Bacillus sp. VKMB B-1793D 2.9 68.0 9 67.4 14.5 45.8 7.7

table 2
Conversion of starch by the action of TSHTaza Paenibacillus campinasensis IB-10155 and Bacillus sp. BKM B-1793D in beta cyclodextrin. Time h TGTaza Bacillus sp. BKM B-1793D TSHTaza Paenibacillus campinasensis IB-10155 The proportion of beta CD in the cyclic reaction products, wt.% The output of beta-CD to starch, wt.% The conversion of starch in CD, wt.% The proportion of beta CD in the cyclic reaction products, wt.% The output of beta-CD to starch, wt.% The conversion of starch in CD, wt.% one 73 32 44 77 29th 38 2 71 39 55 75 41 53 four 69 45 65 74 52 70 8 67 45 68 74 55 75 fifteen 63 41 66 71 53 69 twenty 60 40 63 71 51 68 30.5 57 34 58 71 49 65

Information sources

1. Szente L., Szejtli J. Cyclodextrins as food ingredients. Trends in Food Science & Technology (2004), v15, p. 137-142.

2. Szeitli J. Cyclodextrins in the Textile Industry. Starch / Starke (2003) v. 55 p. 191-196.

3. Cyclodextrins // Ed. Egorova N.S. - The results of science and technology. VINITI. Microbiology Series. - 1988-1989. - T.20-21.

4. Abelyan V.A. Cyclodextrins: Preparation and use. Ed. House "Van Aryan", Yerevan, 2001, 519 p.

5. Szeitli J. Cyclodextrin technology. Kluwer academic publishers, 1989.450 p.

6. Patent of Russia N2244742, cl. C12N 1/20, 2000.

7. Copyright certificate SU 1738852 A1, cl. C12N 9/10, 1990.

Claims (1)

The bacterial strain Paenibacillus campinasensis, the Collection of microorganisms of the Institute of Biology of the Ufa Scientific Center of the Russian Academy of Sciences IB-10155 is a producer of beta-cyclodextringlucanotransferase.