The aim

The aim of the project is to obtain and study myo-inositol phosphohydrolase of bacillary origin and obtain a recombinant thermostable phytase based on it.

Relevance

Phosphorus is a component of high- and low-energy phosphates and nucleic acids and is necessary for protein synthesis, phospholipids, metabolic enzymes, and intracellular buffers that ensure acid-base balance and support skeletal mineralization.In plant fodder, most often the main source of proteins, vitamins, and minerals for farm animals, about 60–90% of phosphorus is contained in the form of phytic acid and its salts, phytates.Animals with one-chambered stomachs(birds, pigs, and fish)are not able to digest phytate.To avoid phosphorus deficiency in poultry and pigs, farmers use phosphorus-containing feed additives; however, this significantly increases the cost of feed and, consequently, the final product.At the same time, the excretion of phytic acid and phytates causes environmental pollution, in particular eutrophication.Phytic acid in feed formulations has anti-nutritional properties,as negatively charged phytates bind cations of calcium,iron,manganese,zinc,potassium,and magnesium, thereby reducing their bioavailability. Myo-inositol phosphohydrolases have phytase action by catalyzing the hydrolysis of phytic acid and phytates to monophosphate, which allows them to be used as an enzyme supplement in feeds.The scientific novelty of the project consists in obtaining and studying a new myo-inositol-phosphohydrolase; genetic engineering works on overexpression; and evaluation of its prospects for application as a phytate-hydrolyzing enzyme.

The practical significance of the project lies in the development of technology for obtaining recombinant thermostable enzyme preparations of phytase action for use in the feed industry and having a positive effect on the environment.

Expected  results

As a result, a collection of Bacillus strains with phytase activity will be created, and bacillary myo-inositol phosphohydrolases will be obtained and studied. Based on the results of the comparative analysis of biochemical properties, the most promising enzyme will be selected. Recombinant strain-producer of thermostable myo-inositol phosphohydrolase will be obtained by genetic engineering. Using biotechnology methods, a technology for the production of recombinant bacillary phytase will be developed using this strain-producer. The conditions for isolation, purification, and dry phytase production will be determined. As a result, on the basis of bacillary myo-inositol phosphohydrolase, a biopreparation of phytase action will be obtained for use in fodder production. The socio-economic effect is the development in Kazakhstan of technologies for obtaining recombinant enzymes, which have high practical significance in industry and have a positive effect on the environment.

Project leader

Baltin Kairat, candidate of biological sciences, senior researcher. ResearcherID: AAQ-9372-2020, ORCID: 0000-0002-6187-7223, Scopus Author ID: 55437315200

Research team members

Khassenov Bekbolat, full professor, candidate of chemical sciences, head of laboratory. ResearcherID: AAM-8657-2020, ORCID: 0000-0003-4572-948X, Scopus Author ID: 36096620800

Aktayeva Saniya, PhD, senior researcher. ResearcherID: AAR-5133-2020, ORCID: 0000-0001-6346-5866, Scopus Author ID: 57439359000

Maduakhasova Arina, Master’s student of the Faculty of Natural Sciences of the Eurasian National University named after L.N. Gumilyov, laboratory assistant, specialization – microbiology and biotechnology. ResearcherID: JNE-6827-2023, ORCID: 0009-0005-1460-0802

Astrakhanov Magzhan, laboratory assistant. ResearcherID: JMP-6059-2023 ORCID: 0009-0005-4471-6072

Publications and protection documents

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 3.7, Percentile 71.
2. 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 3.7, Percentile 71.
3. 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 5.5, Percentile 82.
4. Aktayeva S., Baltin K., Khassenov B. Isolation of Bacillus strains with eratinolytic activity // Eurasian Journal of Applied Biotechnology. 2020, No 2.  P.95-99. https://doi.org/10.11134/btp.2.2020.11. РИНЦ 0.117.
5. Barshevskaya L.V., Sotnikov D.V., Zherdev A.V., Khassenov B.B., Baltin K.K., Eskendirova S.Z., Mukanov K.K., Mukantayev K.K., Dzantiev B.B. Triple Immunochromatographic System for Simultaneous Serodiagnosis of Bovine Brucellosis, Tuberculosis, and Leukemia // Biosensors (Basel). 2019 Vol.9(4),115. Pp. 1-10. https://doi.org/10.3390/bios9040115. Q2, Impact Factor 5.972, Cite Score 5.6, Percentile 89.
6. 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 4.069, Cite Score 5.5, Percentile 87.
7. Кириллов С.О., Абельденов С.К., Балтин К.К., Мусахметов А.С., Ли П.К., Силаев Д.В., Раманкулов Е.М., Хасенов Б.Б. Способ получения ферментной кормовой добавки фитазы // Патент Республики Казахстан №34593 от 25.09.2020.
8. Кириллов С., Абельденов С., Силаев Д., Хасенов Б. Рекомбинантный штамм Pichia pastoris X-33/appa, продуцирующий бактериальную фитазу AppA // Патент Республики Казахстан №32368 от 16.08.2017 г.
9. Kirillov S., Silaev D., Abeldenov S., Khassenov B. Heterologous extracellular expression of bacterial phytase (AppA) in Pichia pastoris and its biochemical characterization // Eurasian Journal of Applied Biotechnology. 2016. Iss.4. pp. 36-44.
10.  Abeldenov S., Kirillov S., Kiribayeva A., Silayev D. Khassenov B. Expression, purification and biochemical characterization of recombinant phosphohydrolase AppA in Escherichia coli // Biotechnology. Theory and Practice. 2014. №3. Pp.61-65.
11. 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, IF 4.38, CiteScore 7.1, Percentile 93.
12. 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 4.069, Cite Score 5.5, Percentile 87.
13. Kalendar R., Khassenov B., Ramankulov Ye., Samuilova O., Ivanov K. Fast PCR: An in silico tool for fast primer and probe design and advanced sequence analysis // Genomics. 2017. Vol.109. pp. 312-319. https://doi.org/10.1016/j.ygeno.2017.05.005. Q2, Impact Factor 4.38, Cite Score 7.1, Percentile 58.
14. Abeldenov S., Talhaoui I., Zharkov D.O., Ishchenko A.A., Ramanculov E., Saparbaev M., Khassenov B. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis // DNA Repair. 2015, Vol. 33. Pp. 1-16. https://doi.org/10.1016/j.dnarep.2015.05.007. Q2, Impact Factor 4.821, Cite Score 7.0, Percentile 70.
15. Актаева С.А., Балтин К.К., Кирибаева А.К., Раманкулов Е.М., Хасенов Б.Б. Штамм бактерий Bacillus licheniformis T7 – продуцент протеаз и кератиназы Патент Республики Казахстан №35357 от 12.11.2021.
16. Aktayeva S., Baltin K., Ramankulov Ye., Khassenov B. Isolation and identification of the keratin degrading bacteria // Journal of Biotechnology. 2019. Vol.305S. P.S30. https://doi.org/10.1016/j.jbiotec.2019.05.112. Q2, Impact Factor 4.122, Cite Score 6.2, Percentile 71.
17. Akishev Zh., Abdullaeva A., Khassenov B., Ramankulov Ye. Pilot-scale production of recombinant Camelus bactrianus chymosin B in Pichia (Komagataella) pastoris under constitutive GAP promoter // Journal of Biotechnology. 2018. Vol. 280S. P.S61. https://doi.org/10.1016/j.jbiotec.2018.06.200. Q2, Impact Factor 4.122, Cite Score 6.2, Percentile 71.

Achieved results

2024

Bacteria of the genus Bacillus were isolated from soil, water and sludge samples. Based on the results of screening on selective media, isolates with phytase activity were selected. Species identification of strains with phytase activity was performed using molecular genetic and proteomic methods. Based on the data obtained, the strains were identified as Bacillus licheniformis, Bacillus subtilis and Bacillus paralicheniformis. Identification of bacillary myo-inositol phosphohydrolases was carried out using mass spectrometry and proteomics.