RU2806183C1 - Symbiotic mixture of bacteria for xanthan destruction - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention concerns a new symbiotic mixture of bacteria that produces a xanthanase enzyme complex. The basis of the symbiotic mixture is made up of strains identified as a result of metagenomic analysis: Paenibacillus sp.FSL R7-277 — 83%, Cellulosimicrobium cellulans — 17%. Percentages reported at taxonomic levels are percentages for mapped reads only. Metagenomic reads have been deposited in the NCBI Sequence Read Archive (SRA) database (BioProject database number PRJNA849974). The composition of the proposed xanthanase complex was determined by measuring the specific activities of the following enzymes: 27.5 units/g of beta-glucosidase, 25.2 units/g of alpha-mannosidase, 6.3 units/g of beta-mannosidase, 7.9 units/g of endoglucanase, 183 units/g of xanthan lyase.

EFFECT: invention makes it possible to expand the range of microorganisms producing enzymes that degrade xanthan, and can also be used in biotechnology to produce new highly specific enzymes capable of complete destruction of the main and side chains of xanthan.

1 cl, 1 tbl, 3 ex

Description

The invention relates to the field of microbiology and biotechnology and concerns a new symbiotic mixture of bacteria that produces a xanthanase enzyme complex. The invention can find application in the field of oil production, food and pharmacological (medical) industries.

A large volume of xanthan polysaccharide is used in the processes of intensification of oil and gas wells as a proppant, due to the ability of xanthan to increase the viscosity of drilling fluids. Products based on xanthan gum are produced under such trademarks as Rhodopol-23P, Zibozan, Flowzan, Flo-Vis, etc.: Siberian Federal University Journal. Technics and techology. 2013. T. 6(1). pp. 95-106 [1]. From the point of view of ecology and environmental management, it is important that upon completion of the work, the viscosity of drilling solutions (proppant) is significantly reduced, for which it is necessary to carry out their destruction.

There are methods for chemical destruction of the complex xanthan polymer that require exposure to strong acids and high temperatures: Macromolecules. 1993. T. 26, No. 22, pp. 6111-6120 [2]. Biological methods of destruction of xanthan gums through enzymes synthesized by microorganisms are recognized as more environmentally friendly and less energy-consuming methods.

Xanthan is an extracellular polysaccharide produced by the microorganism Xanthamonas campestris. The main chain of xanthan consists of D-glucopyranose residues connected to each other by a P~1,4-glycosidic bond. The side chains are formed by D-mannose (β-1,4), D-glucuronic acid (β-1,2) and D-mannose, which is linked to the glucose molecule by an α-1,3 bond: Plant biopolymers. 2021. No. 4. pp. 95-104 [3]; J. Environ. Manage. 2014. T. 144. pp. 1-25 [4]. Xanthan side chains are modified with pyruvate and/or acetyl groups, their amount in the structure is related to the viscosity and stability of the biopolymer: Biotechnol. Res. Innov. 2022, T. 6, No. 1. P.e202204 [5].

The enzymes used in the destruction of xanthan are collectively termed “xanthanase” or “xanthanase complex.” The term “xanthanase” is used to refer to the entire complex of xanthanolytic enzymes that catalyze xanthan degradation reactions. The lack of information on the use of individual enzymes included in this complex, their characteristics and contribution to the process of xanthan degradation, as well as on the producers of such enzymes, limits the creation of an effective tool for the complete destruction of xanthan main and side chains. Based on the structure of the xanthan molecule, for its complete degradation, including its side chains, at least the following enzymes are required: endo-glucanase, xanthan lyase, beta-glucosidase or glucuronidase, mannosidase (alpha and/or beta): Diss . for academic competition Ph.D. degrees biol. Sciences, Moscow, 2022 [6].

There are known methods for the destruction of xanthan using enzymes produced by bacteria of the genus Paenibacillus: Iran J Biotechnol. 2017. T. 15, No. 2. pp. 120-127 [7], as well as through xanthanase from the bacterial strain Corynebacterium 20/122: Patent EP 0030393 B1, 1983 [8]. Patent RU2754795 registered the bacterial strain Paenibacillus taichungensis 15, which has the ability to decompose xanthan and use it as the only carbon source: Patent RU 2754795 [9]. When the strain is cultivated for 96 hours, it is capable of producing cultural fluid with an activity of 26 units/ml towards xanthan. Patent WO 9839397, “Methods and materials for xanthan degradation,” describes the use of a mixed bacterial culture deposited in the microorganism collection under ATCC number 55941, demonstrating a total xanthanase activity of 10.4 units/g [10].

A common disadvantage of the listed inventions is the limited information on the composition of enzymes designated by the general term “xanthanase”, which leads to uncertainty in the assessment of xanthanase activity, including due to different measurement techniques.

The closest analogue (prototype) is the bacterial consortium NRRL B-18445, which produces the xanthanase enzyme complex. During the cultivation of a mixture of bacteria for 8 days in the culture liquid, a total xanthanase activity of 195 units/mg for xanthan was detected: patent US 4996153 [11]. The main disadvantages of this invention, as well as the above analogues, are the lack of information about the produced enzymes that catalyze the destruction of the main and side chains of xanthan, which does not allow the identification of new specific enzymes as tools for xanthan degradation.

The technical result is achieved by identifying a symbiotic mixture of bacteria containing, according to metagenomic analysis, microorganisms Paenibacillus sp. - 83% and Cellulosimicrobium cellulans - 17%, which has the property of degrading xanthan.

It was experimentally determined that the claimed symbiotic mixture containing microorganisms Paenibacillus sp., Cellulosimicrobium cellulans, which, when cultivated in a medium containing 0.3% (w/v) xanthan at a temperature of 28°C, produces the following enzymes: beta-glucosidase, alpha -mannosidase, beta-mannosidase, endoglucanase and xanthan lyase, catalyzing the destruction of the main and side chains of xanthan.

The specific implementation is as follows:

1. On a liquid nutrient medium of the composition, % (w/v): xanthan, 0.3; peptone, 0.1; YNB, 0.34 at a temperature of 28°C, the growth of a symbiotic mixture of bacteria for the destruction of xanthan was detected.

2. Identification of the bacterial mixture was performed by metagenomic analysis of total DNA sequences isolated from the mixed bacterial culture. Isolation of total metagenomic DNA, library preparation, and sequencing on a MiSeq® System (Illumina, USA) using MiSeq Reagent kit v2 (Illumina; USA) was carried out according to the protocol described in detail previously [12]. Sequencing was performed on a Hiseq 2500 platform (Illumina) in paired-end read mode with a read length of 160 bp. Metagenomic analysis was carried out using the Kaiju software package (https://github.com/bioinformatics-centre/kaiju) based on the NCBI BLAST databases, screening out sequences with a classification length of less than 50 base pairs. To compare the obtained data, the JSpeciesWS service was used (https://jspecies.ribohost.eom/jspeciesws/#home).

Identification of bacterial cultures in a mixture isolated on a medium containing xanthan was carried out by metagenomic shotgun sequencing based on the content of marker genes specific to organisms identified from 100,000 reference genomes (99,500 bacteria and archaea and 500 eukaryotes) and comparative analysis. The coincidence of genomes by no less than 85% allowed us to talk about generic identity.

The results of metagenomic analysis suggest the possible proportions of the following bacteria homologous to Paenibacillus sp. - 83%, Cellulosimicrobium cellulans - 17%. Percentages reported at taxonomic levels are percentages for mapped reads only. Metagenomic reads have been deposited in the NCBI Sequence Read Archive (SRA) database (BioProject database number PRJNA849974).

3. Morphological analysis of the bacteria included in the symbiotic mixture revealed the following features of each of the isolated microorganisms:

- Paenibacillus spp.

Vegetative cells are rods, 0.5-1.0 (3.0-4.0 µm, found singly or in short chains. The cells produce oval spores located terminally in swelling sporangia and are gram-negative. Colonies at 30°C at TSA medium is cream-colored, smooth, with regular, intact edges.On media containing 2% glucose, colonies form mucus.

- Cellulosimicrobium cellulans

Gram-positive motile rods of irregular shape 0.5-0.6 (2.0-5.0 µm, straight or slightly curved, some in pairs of V-shaped configuration. After 24 hours of incubation at 37°C on 5% agar with the addition of horse blood, the colonies were slightly yellow, convex and smooth, and with further incubation they penetrated into the agar, forming substrate mycelium.Cells grown on a medium with blood agar are long and thin gram-positive rods, which with further incubation become coccobacillary.

4. Testing of the total xanthanase activity of the symbiotic mixture of bacteria was carried out during deep cultivation at a temperature of 28°C for 36-38 hours on a nutrient medium of the following composition, % (wt./vol.): xanthan, 0.3; peptone, 0.1; ΥΝΒ, 0.34, followed by measurement of viscosity and the amount of reducing sugars (see Example 1).

The standard temperature for bacterial growth in laboratory conditions is 37°C. However, as experiments have shown, when a symbiotic mixture of bacteria containing Paenibacillus sp., Cellulosimicrobium cellulans grows at a temperature of 37°C, xanthanase activity does not appear, which is confirmed by data on the absence of a decrease in the viscosity of xanathan. Reliable results are achieved at a temperature of 28°C.

5. The symbiotic mixture of bacteria was stored in a refrigerator at a temperature of 4-8°C on Petri dishes on LB agar (pH 6.5-7.0). Long-term storage of the symbiotic mixture was carried out in a freeze-dried state; 10% sucrose and 1% gelatin were used as a cryoprotectant; the ampoules were stored at 4-8°C. The declared symbiotic mixture of xanthanolytic bacteria is stored in the collection of microorganisms of the biotechnology laboratory of the Scientific Research Center "KI" PNPI.

It was experimentally shown that the bacterial mixture demonstrated the presence of both individual enzymatic activities and general xanthanase activity, leading to the degradation of the main and side chains of xanthan. A mixture of bacteria artificially composed of microorganisms individually isolated from the original mixture does not lead to the manifestation of xanthanase activity.

The invention is illustrated by the following examples, which do not limit its application:

Example 1. Determination of the total xanthanase activity of a symbiotic mixture of bacteria on a medium with a xanthan concentration of 0.3%.

To obtain cultural fluid, a liquid medium of the following composition was prepared, % (wt./vol.): xanthan, 0.3; peptone, 0.1; YNB, 0.34. The medium was poured into 100 ml volumes into 500 ml flasks and sterilized. After 36-38 hours of cultivating the mixture of bacteria at 28°C on a shaker (120 rpm), the specific activity of the xanthanase complex was assessed in selected samples of cultural fluid. One unit of activity was taken to be the amount of enzyme preparation that, within 1 minute at 40°C and pH 6.5, releases 1 µmol of reducing sugars, equivalent to 1 µmol of glucose. The total amount of reducing sugars was determined by the iora-chlorohydrobenzoic acid reagent test according to the described method: J. Biol. Eng. 2017, T. 11, No. 1 [13]. The specific xanthanase activity at the end point of cultivation after 36-38 hours was 19.6 units/g.

After 36-38 hours of cultivation, the viscosity of cultural fluids was 1.05 cP (1 rpm), the viscosity of the control solution was 230 cP (1 rpm). During cultivation, the viscosity of the liquid nutrient medium containing xanthan decreased by 230 times compared to the control solution. The difference with the prototype in assessing the value of total xanthanase activity is associated with different approaches to measuring enzyme activities. The determination of activity in the present invention is carried out in accordance with international standards for calculating specific enzymatic activity: Dawson R., Elliott D., et al. Biochemist's Handbook. 1991. M.: Mir. 544 pp. [14].

Example 2. Testing the total xanthanase activity of a symbiotic mixture of bacteria when growing on a medium with a xanthan concentration of 1% (w/v).

The cells of the symbiotic mixture were introduced into a sterile liquid medium as described in example 1. The initial concentration of xanthan in the medium was 1% (w/v). After 74 hours of culture at 28°C on a shaker (120 rpm), the viscosity of the liquid culture medium containing 1% (w/v) xanthan decreased from 6.0 to 1.2 cP (0.1 rpm).

Example 3. Determination of the activities of the enzymes of the xanthanase complex of a symbiotic mixture of bacteria capable of complete degradation of the main and side chains of xanthan.

The cells of the symbiotic mixture were cultured in the same liquid medium and under the same conditions as described in Example 1. After 36-38 hours of cultivation of the symbiotic mixture in selected samples of the culture liquid, the specific activities of beta-glucosidase, alpha-mannosidase, beta-mannosidase and endoglucanases. A reaction mixture containing 100 μl of culture liquid, 5 mmol of the appropriate substrate (iara-nitrophenyl-β-D-glucopyranoside, para-nitrophenyl-α-L-mannopyranoside, para-nitrophenyl-β-D-mannopyranoside, para-nitrophenyl-β- D-cellopyranoside) and 50 mM phosphate-citrate buffer (pH 6.5) were incubated for 120 minutes at 40°C. The reaction was stopped by adding 0.5 ml of 10% soda solution. The absorbance of free para-nitrophenol was measured at a wavelength of 400 nm on a JascoV-560 UV/Vis spectrophotometer (Jasco, Analytical Instruments, Japan) relative to the control. One unit of activity was defined as the activity required to produce 1 μmol of para-nitrophenol in 1 minute under given conditions.

Xanthan lyase activity was determined in 1 ml of a reaction mixture containing 0.05% xanthan and 50 mmol phosphate-citrate buffer, pH 6.5, monitoring the increase in absorbance at 235 nm. One unit of enzyme activity was defined as the amount of enzyme required to increase the absorbance at 235 nm by 1.0 per minute.

The protein content in the samples was determined by the Lowry method (calibration standard - bovine serum albumin, photocolorimetry - at 750 nm): J. Biol. Chem. 1951. T. 1, pp. 265-275 [15].

The results of measurements of the specific activities of the enzymes included in the xanthanase complex of the symbiotic mixture are given in Table 1.

In comparison with the prototype, the problem of determining the main enzymatic activities necessary for the degradation of the main and side chains of xanthan has been solved.

Conclusions: a symbiotic mixture of bacteria for the destruction of xanthan was identified, containing, according to the results of metagenomic analysis, microorganisms Paenibacillus spp., Cellulosimicrobium celluloliticum, which, when cultivated in a medium containing 0.3% (wt./vol.) xanthan at a temperature of 28°C , produces enzymes: beta-glucosidase, alpha-mannosidase, beta-mannosidase, endoglucanase, xanthan lyase, which catalyze the destruction of the main and side chains of xanthan. A symbiotic mixture of xanthanolytic bacteria has a total xanthanase activity of 19.6 U/g when cultured for 36 hours at 28°C in a medium containing 0.3% (wt/vol) xanthan. The specific activities of the proposed xanthanase complex are: beta-glucosidase 27.5 units/g, alpha-mannosidase 25.2 units/g, beta-mannosidase 6.3 units/g, endoglucanase 7.9 units/g, xanthan lyase 183 units /G.

Thus, the possibility of expanding the range of enzymes, such as beta-glucosidase, alpha-mannosidase, beta-mannosidase, endoglucanase and xanthan lyase, catalyzing the destruction of the main and side chains of xanthan, has been proven.

Funding: The work was carried out with the financial support of the “Kurchatov Genome Center - PNPI” by the program for the development of world-class genetic research centers, Agreement No. 075-15-2019-1663.

Bibliography

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Claims (1)

A symbiotic mixture of bacteria containing strains of microorganisms Paenibacillus sp. - 83%, Cellulosimicrobium cellulans - 17%, which has the property of degrading xanthan.