The idea of the project is to develop a technology for enzymatic hydrolysis of plastic waste using microbial enzymes – PET hydrolases and strains with PET-hydrolytic activity. The project includes searching for strains with PET-hydrolase activity, obtaining and studying enzymes hydrolysing polyethylene terephtholate. PET hydrolases will be studied and polyethylene terephtholate hydrolytic activity of microorganisms will be determined. The main problem the project is aimed at is the development of technology for biodegradation of PET plastic waste in landfills and conversion of polyethylene terephtholate.

Relevance

Polyethylene terephthalate (PET) is one of the most popular and demanded materials used in various fields. PET is produced mainly from crude oil and its production exceeds 30 million tonnes per year. This polymer consists of repeating units of terephthalic acid and ethylene glycol. The strength, chemical resistance, transparency and other qualities of PET have made it a convenient packaging material. However, the high chemical resistance and mechanical strength of PET causes difficulties in its disposal and/or reuse.

The scientific novelty of the project lies in the study of new enzymes – PET hydrolases, their modification in order to improve hydrolytic properties and stability. Practical significance of the project consists in obtaining new biopreparations oriented on the use in utilisation of plastic waste – their decomposition into simple substances safe for the environment. The results of the project can be used both for hydrolysis of PET waste into initial substances: terephthalic acid and ethylene glycol for subsequent synthesis of PET products and for complete biomutilisation of PET waste by hydrolytic microorganisms.

The aim

The aim of the project is to obtain, study and target modification of PET hydrolases of bacterial origin for biodegradation and conversion of PET plastic waste.

Expected results

Microorganisms will be collected from plastic waste accumulation sites: soil from landfill sites, solid domestic waste accumulation sites. Identification of isolates will be carried out on the basis of morphological, biochemical, molecular-genetic and proteomic analysis of strains. This will result in a collection of strains with PET-hydrolysing activity. PET hydrolases will be isolated. Their identification will be based on molecular weight, 2D-electrophoresis data and results of mass spectrometry experiment. Recombinant PET hydrolases will be obtained using genetic engineering techniques and purified.  The biochemical characteristics of native and modified PET hydrolases will be studied. Enzymatic activity values, the effect of pH and temperature on enzyme activity, temperature and pH stability of enzymes were determined, the effect of metal ions, solvents, detergents were studied; substrate specificity was determined and kinetic characteristics were established. Model experiments on the enzymatic hydrolysis of PET plastic waste will be carried out. The extent of hydrolysis of polyethylene terephtholate will be determined and hydrolysis products will be identified. Model experiments on hydrolysis and biodegradation of PET plastic by PET-hydrolysing microorganisms will be carried out. The extent of PET plastic degradation, microbial mass gain and the influence of associated factors on biodegradation will be evaluated.

Methods of microbiology, biochemistry, proteomics, molecular biology, and biotechnology will be used.

Project Leader

Dmitry Vitalievich Silaev (Scopus Author ID: 57219323485, ResearcherID: AAQ-8940-2020, ORCID: 0000-0001-6867-953X)

Research group members

Khasenov Bekbolat Baurzhanovich (Scopus Author ID: 36096620800, ResearcherID: AAM-8657-2020, ORCID: 0000-0003-4572-948X)

Kiribayeva Asel Kaliaskarova (Scopus Author ID: 57215499873, ResearcherID: N-6774-2017, ORCID: 0000-0002-8293-2340)

Musakhmetov Arman Sartambayevich (Scopus Author ID: 57203751227, ResearcherID: AAQ-9945-2020, ORCID: 0000-0002-6182-3487)

Sarsen Araylym (ResearcherID: AED-8089-2022 ORCID: 0000-0002-6071-430X)

Yulia Shamsieva (ResearcherID: AFX-3509-2022 ORCID: 0000-0002-3136-6000)

Publications and security documents of the project leader and members of the research team related to the project topic

1. Aktayeva S., Baltin K., Kiribayeva A., Akishev Zh., Silayev D., Ramankulov Ye., Khassenov B. Isolation of Bacillus sp. A5.3 Strain with Keratinolytic Activity // Biology (MDPI). 2022, Vol 11, Issue 2, e244. https://doi.org/10.3390/biology11020244. Q1, Impact Factor 5.168, Cite Score 3.3, Percentile 79.
2. Kiribayeva A., Mukanov B., Silayev D., Akishev Zh., Ramankulov Ye., Khassenov B. Cloning, expression, and characterization of a recombinant xylanase from Bacillus sp. T6 // PLoS One. 2022. Vol.17(3). e0202232. https://doi.org/10.1371/journal.pone.0202232. Q2, Impact Factor 3.24, Cite Score 5.3, Percentile 92.
3. Nurtaza A., Magzumova G., Yessimseitova A., Karimova V., Shevtsov A., Silayev D., Ramankulov Y., Kakimzhanova A. Micropropagation of the endangered species Malus niedzwetzkyana for conservation biodiversity in Kazakhstan // In Vitro Cellular and Developmental Biology – Plant, 2021, Vol.57(6), P. 965–976 https://doi.org.10.1007/s11627-021-10174-4. Q2, Impact Factor 2.252, CiteScore=3.1, Percentile 70.
4. Aipova R., Abdykadyrova A., Silayev D., Tazabekova E., Oshergina I., Ten E., Kurmanbayev A. The fabrication of the complex bio-fertilizer for wheat cultivation based on collection bacteria of the pgpr group // Biodiversitas, 2020, Vol.21(11), P. 5032–5039 https://doi.org/10.13057/biodiv/d211107. CiteScore 1.3, Percentile 40.
5. Kiribayeva A., Silayev D., Abdullayeva A., Shamsiyeva Yu., Ramankulov Ye., Khassenov B. Hydrolysis of plant biomass using recombinant alpha-amylase from Bacillus licheniformis and xylanase from Bacillus sonorensis // Eurasian Journal of Applied Biotechnology, 2022. No. 4. P.31-39 https://doi.org/10.11134/btp.4.2022.4. РИНЦ-0,117.
6. Kiribayeva A.K., Mukanov B.A., Baduanova S.D., Silayev D.V., Ramankulov Ye.M., Khassenov B.B. Isolation and study of a recombinant carbohydrase xylanase from Bacillus licheniformis // Eurasian Journal of Applied Biotechnology. 2019. Iss.1. pp.68-72. doi:10.11134/btp.1.2019.8. РИНЦ 0,117.
7. Akishev Zh., Aktayeva S., Kiribayeva A., Abdullaeva A., Baltin K., Mussakhmetov A., Tursunbekova A., Ramankulov Ye., Khassenov B. Obtaining of Recombinant
   Camel Chymosin and Testing Its Milk-Clotting Activity on Cow’s, Goat’s,
   Ewes’, Camel’s and Mare’s Milk // Biology (MDPI). 2022, Vol 11, Issue 11, e1545. https://doi.org/10.3390/biology11111545. Q1, Impact Factor 5.168, Cite Score 3.3, Percentile 79.
8. Akishev Zh., Kiribayeva A., Mussakhmetov A., Baltin K., Ramankulov Ye., Khassenov B. Constitutive expression of Camelus bactrianus prochymosin B in Pichia pastoris // Heliyon. 2021. Volume 7, Issue 5, e07137. https://doi.org/10.1016/j.heliyon.2021.e07137. Q2, Impact Factor 3.776, Cite Score 2.7, Percentile 75.
9. Matta E., Kiribayeva A., Khassenov B., Matkarimov B., Ishchenko A. Insight into DNA substrate specificity of PARP1-catalysed DNA poly(ADP-ribosyl)ation // Scientific Reports. 2020. Vol.10(1), 3699. pp.1-11. https://doi.org/10.1038/s41598-020-60631-0. Q1, Impact Factor 4.38, Cite Score 7.1, Percentile 93.
10. Mussakhmetov A., Shumilin I.A., Nugmanova R., Shabalin I.G., Baizhumanov T., Toibazar D., Khassenov B., Minor W., Utepbergenov D. A transient post-translational modification of active site cysteine alters binding properties of the parkinsonism protein DJ-1 // Biochemical and Biophysical Research communications. 2018. Vol.504(1). Pp. 328-333. https://doi.org/10.1016/j.bbrc.2018.08.190. Q2, Impact Factor 3.572, CiteScore 5.5, Percentile 62.
11. Turgimbayeva A, Abeldenov S, Zharkov D.O, Ishchenko A.A, Ramankulov Y, Saparbaev M, Khassenov B. Characterization of biochemical properties of an apurinic/apyrimidinic endonuclease from Helicobacter pylori // PLoS One. 2018. Vol.13(8). e0202232. https://doi.org/10.1371/journal.pone.0202232. Q2, Impact Factor 3.24, Cite Score 5.3, Percentile 92.

The findings

Soil samples were collected from the places of plastic waste accumulation: “ECO Polygon Astana” LLP and Almaty city dumps. The obtained samples were sown on solid nutrient medium with selective agents: Tween-20, calcium chloride and tributyrin. Thirteen isolates were isolated and screened for esterase activity. The isolated microorganisms were identified by MALDI-TOF BRUKER Biotyper proteomic analysis and by sequencing of 16S rRNA fragment. As a result, the following strains were identified: Bacillus megaterium, Yersinia intermedia, Serratia liquefaciens, Bacillus simplex, Bacillus cereus, Bacillus thuringiensis, Bacillus sonorensis, Bacillus licheniformis, Aeromonas veronii. The above strains were cultured on medium supplemented with tributyrin for induction of PET-hydrolytic enzymes. The secretory enzymes were separated from the cell mass by high-speed centrifugation and filtration. The native PET hydrolases were purified by ammonium sulfate precipitation and chromatographic separation. Identification of PET hydrolases was performed using PAGE-DSN coupled with protein mass spectrometry and proteomics. Based on mass spectrometric data, the isolated native PET hydrolases were found to belong to the esterase class with molecular masses ranging from 32 to 40 kDa.

The obtained PET hydrolase samples were analysed for esterase activity using specific p-nitrophenyl substrates (acetate, butyrate, octanoate). Cloning of PET hydrolase genes is currently underway.