GEOLINKS International Conference 2021, Book 1
OBTAINING NUCLEIC ACID PREPARATIONS AND THEIR HYDROLYSATES FROM BIOMASS OF METHANE-OXIDIZING BACTERIA
Prof. Dr. Alla A. Krasnoshtanova, Elisaveta K. Borovkova
Due to the unfavourable environmental, social and economic situation, the need for the treatment of oncological diseases and diseases associated with impaired activity of the immune system is increasing. A lot of these drugs are made on the basis of nucleic acid components, the industrial production of which is practically non-existent in Russia. Therefore, a task of current interest is to develop the basis of the technology for obtaining components of nucleic acids, which can be widely used in medicine as immunomodulatory, wound-healing, antiviral, and diagnostic medicine, as well as for cancer treatment.
Most of the described in literature methods of isolating nucleic acid components from plant, animal and microbial raw materials are based on the use of toxic and expensive organic solvents, that’s why it is impossible to apply these methods outside of laboratory conditions. The most promising source of raw materials for nucleic acids is the biomass of microorganisms (yeast and bacteria) from biomass, since the use of such source makes it possible to quickly obtain a large enough amount of biomass, and, consequently, a larger amount of nucleic acids. This allows obtaining DNA in addition to RNA. RNA and DNA substances can be used to obtain nucleosides and nitrogenous bases, which are also widely used in medicine.
The purpose of these studies was to select the conditions for the extraction of RNA and DNA from the biomass of methane-oxidizing bacteria in one technological cycle, as well as to compare the efficiency of alkaline and acid hydrolysis of microbial RNA and DNA. The need for a two-stage extraction of nucleic acids from the biomass of methane-oxidizing bacteria in order to separately extract RNA and DNA was Substantiated. It was ascertained that at the first stage of extraction at a temperature of 90 ° C, pH 9.0 for 90 min, at least 85% of RNA is extracted. After the separation of the extract by centrifugation, the partially denuclearized biomass must be re-processed under the same conditions in order to extract DNA by at least 83%. The modes of concentration of RNA and DNA solutions by ultrafiltration were selected. It was found that in order to achieve effective deposition of nucleic acids at the isoelectric point, the concentration of the RNA solution must be carried out on the UPM-10 membrane at the concentration degree of 7, and the DNA solution on the UPM-100 membrane at the concentration degree 6. The dynamics of decomposition of nucleic-protein complexes in the medium of monoammonium phosphate was investigated. It was shown that the transition of NA into solution by at least 80% is achieved at a monoammonium phosphate concentration of 1.7 M, a temperature of 55 ° C for 90 min. The use of 5-fold washing of oligonucleotide substances with acidified water (pH 2.0) to remove excess mineral impurities was substantiated. А comparative assessment of acid and alkaline hydrolysis of RNA and DNA was carried out in order to obtain derivatives of nucleic acids.
methane-oxidizing bacteria, RNA, DNA, acid hydrolysis, alkaline hydrolysis