L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Aktayeva Saniya Aidarbekovna on the topic «Isolation and study of biochemical characteristics of proteolytic enzymes from Bacillus licheniformis and Bacillus subtilis» by specialty «6D060700 – Biology».
The dissertation was carried out at the Department of «Department of General Biology and Genomics» of L.N. Gumilyov Eurasian National University.
The language of defense is russian
Official reviewers:
Zorov Ivan Nikitich – Candidate of Chemical Sciences, Senior Researcher at the Laboratory of Biotechnology of Enzymes at the Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences (Moscow, Russian Federation);
Li Konstantin Grigorievich – Candidate of Biological Sciences, Senior Researcher at the Biotechnology and eco-monitoring research park of the Karaganda State University named after Academician E.A. Buketov (Karagandy, Republic of Kazakhstan).
Temporary members of the Dissertation Council:
Bisenbaev Amangeldy Kuanbaevich – Doctor of Biological Sciences, Professor, Director of the Scientific Research Institute for Problems of Biology and Biotechnology of Al-Farabi Kazakh National University (Almaty, Republic of Kazakhstan);
Kozhakhmetov Samat Serikovich – Candidate of Biological Sciences, associate professor, leading researcher at the microbiome laboratory, Nazarbayev University (Astana, Republic of Kazakhstan);
Chulkin Andrey Mikhailovich – Candidate of Biological Sciences, Researcher at the Laboratory of Biotechnology of Enzymes at the Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences (Moscow, Russian Federation).
Scientific consultants:
Khassenov Bekbolat Baurzhanovich – Candidate of Chemical Sciences, Professor in the Biological Sciences, Head of the Laboratory of Genetics and Biochemistry of Microorganisms of the National Center for Biotechnology (Astana, Republic of Kazakhstan);
Saparbayev Murat Kalievich – Candidate of Biological Sciences, Head of the DNA Repair Laboratory of the Institute Gustave Roussy (Villejuif, France).
The defense will take place on March 12, 2025, at 12:00 in the Dissertation Council for the training direction «8D051 – Biological and related sciences» in the specialty «6D060700 – Biology» of L.N. Gumilyov Eurasian National University. Conducting a meeting of the dissertation council in a mixed (offline and online) format.
Link: https://clck.ru/3G6KFu
Address: Astana, Kazhymuqana str., 13, building №3, room №333.
Abstract (English): General description of the work. This thesis is devoted to the study of proteolytic enzymes secreted by the bacteria Bacillus licheniformis and Bacillus subtilis. Relevance. Proteolytic enzymes, or proteases, are enzymes that hydrolyse proteins by breaking peptide bonds. They are classified into four main families: serine, cysteine, aspartate, and metalloproteases. Proteases are found in all living organisms, including microbes, animals, and plants, however, in industrial biotechnology most enzymes are obtained through microbial synthesis. Among microbial sources, Bacillus species are considered promising due to their high secretion capacity and ability to produce proteases active at both neutral and alkaline pH levels. Bacillary protease-producing strains can grow on inexpensive nutrient media, which is important because the cost of media components can account for up to 40% of total the enzyme production cost. For commercial production, deep and solid-state fermentation methods are used. In deep fermentation, substrates such as molasses, fructose, malt extract, lactose, and skimmed milk serve as cost-effective media components. In solid-state fermentation, strains grow agricultural residues such as cereal straw, legume cake, and bagasse. Both fermentation methods are widely used in industrial biotechnology. Bacillary proteases have applications in the food industry, animal feed production, pharmaceuticals, stereoisomeric compound synthesis, cosmetics, in the leather processing, and wastewater treatment. Their tolerance to high temperatures, organic solvents, and detergents, makes them particularly valuable for detergent production. Several Bacillus-derived proteases have already been commercialized. However, the high industrial potential and broad diversity of Bacillus proteases remain subjects of ongoing research. Isolating Bacillus proteolytic strains from Kazakh biocenoses and investigating their enzymatic properties may lead to novel applications in protein hydrolysis. Purpose and objectives of the study. The aim of the study is to characterize the biochemical properties of proteolytic enzymes secreted by Bacillus subtilis and Bacillus licheniformis strains. In accordance with the objective, the following objectives were set: 1. To collect samples, screen, isolate and identify proteolytic microorganisms of the Bacillus genus. 2. To study the secretory proteome of Bacillus proteolytic strains using zymographic analysis, protein mass spectrometry, and quantitative enzymology. 3. To determine the biochemical properties of proteolytic enzymes from Bacillus subtilis and Bacillus licheniformis strains. 4. To assess the hydrolytic properties of these proteolytic enzymes on different protein substrates. 5. To perform a comparative analysis of proteolytic enzymes from both strains. 6. To conduct enzymatic hydrolysis of keratin-containing waste, such as chicken feathers, horns, hooves, hides, and cattle wool. This includes evaluating degree of hydrolysis, proteolytic and keratinolytic activities, and potential application as milk-coagulating agents. 7. To cultivate the producer strain in a bioreactor using submerged fermentation to obtain proteolytic enzyme preparations in both liquid and dry forms. Objects of study: Bacillus subtilis and Bacillus licheniformis strains. Subject: proteolytic enzymes of Bacillus subtilis and Bacillus licheniformis strains. Research methods: microbiology, molecular biology, biochemistry, mass spectrometry, proteomics, sequencing, genomics, biotechnology, immunology and scanning electron microscopy. Novelty of the topic. Scientific novelty consists in the fact that for the first time proteolytic and keratinolytic Bacillus subtilis and Bacillus licheniformis strains were isolated from the soil of Kazakhstan. Their secretory enzymes were identified and characterized. A cultivation method using feather-based medium was proposed, and the influence of various factors (temperature, pH, metal ions, detergents, inhibitors) on enzymatic activity was studied. The complete genome of Kazakhstani proteolytic strain Bacillus licheniformis T7 was sequenced for the first time and deposited in the NCBI GenBank database under accession number CP124861. Practical significance of the work. The results of this study provide insights into proteolytic enzyme properties, optimal production conditions, and their applications in enzymatic protein hydrolysis. This knowledge could contribute to the development of novel proteolytic enzyme-based bioproducts for the food, feed, and processing industries. Key Statements Defended: - Mesophilic microorganisms Bacillus subtilis A5.3 and Bacillus licheniformis T7 produce thermostable alkaline proteolytic enzymes related to serine proteases and metalloproteases; - proteolytic enzymes secreted by the bacteria Bacillus subtilis A5.3 and Bacillus licheniformis T7 have wide substrate specificity and are capable of hydrolysis of phosphoproteins (caseins), albumin (BSA, ovalbumin), fibrillar proteins (collagen, and keratin); - adhesion of proteolytic bacteria cells on the surface of the substrate is important for the efficient hydrolysis of solid water-soluble substrates; - The bacterial strains Bacillus subtilis A5.3 and Bacillus licheniformis T7 secrete a number of proteolytic enzymes with different structures and biochemical parameters, which together hydrolyse protein substrates; - Bacillus subtilis A5.3 and Bacillus licheniformis T7 strains isolated in Kazakhstan are effective producers of thermostable alkaline proteolytic enzymes. Main results of the study. 1. Bacterial isolates were isolated from soil samples, of which 2 strains were selected based on the results of proteolytic activity screening. The strains were identified as Bacillus subtilis A5.3 and Bacillus licheniformis T7. When cultured on feather medium, the strains produced proteolytic enzymes, with maximum production at 48-72 hours of fermentation. 2. The biochemical characteristics of the proteolytic enzymes of the isolated strains, Bacillus subtilis A5.3 and Bacillus licheniformis T7, were examined. According to the results obtained, the proteases belong to the classes of serine and metalloproteases, are alkaline and thermostable. The proteolytic enzymes produced by B. subtilis strain A5.3 have an activity of 158.8±2.5 U/ml and 109.3±4.0 U/ml for caseinolytic and β-keratinolytic activities, respectively, α-keratinolytic activity was not established for B. subtilis strain A5.3. The caseinolytic, β-keratinolytic and α-keratinolytic activities were 322.6±4.8 U/ml, 122.0±6.0 U/ml and 60.0±2.4 U/ml for enzymes of B. licheniformis strain T7, respectively. Additionally, the enzymes of B. licheniformis strain T7 had milk clotting activity at 10 U/ml. Comparison of the levels of keratinolytic and caseinolytic activities indicates that the B. subtilis A5.3 strain is more keratinolytic than B. licheniformis T7, which may be explained by the source of the strain being poultry farm feather waste. 3. Proteolytic enzymes of B. subtilis A5.3 strain were inhibited by metal ions with single, double or triple charge. Strong inhibition, threefold, was observed from Mn2+ ions; twofold when K+, Li+, Mg2+ and Cd2+ ions were added. Investigation of the effect of metal ions on the activity of proteolytic and keratinolytic enzymes of the extract of B. licheniformis T7 showed that single charge ions had no effect. Ni2+, Co2+, Mn2+, Mn2+ and Cd2+ (10 mM) have a strong inhibitory effect on proteolytic activity. Cu2+ and Cd2+ ions (10 mM) inhibit keratinolytic activity. Iron (III) ions (5-10 mM FeCl3) completely inactivate proteolytic and keratinolytic activities. The activity of proteolytic enzymes of B. subtilis A5.3 strains was inhibited by reducing agents and Tween 20 twofold, while Triton X-100 and SDS had no significant effect on the activity. For B. licheniformis T7 enzymes, the presence of SDS in the reaction reduced the values of both activities for more than 75%. 4. The study of protease species composition in enzymatic extracts of B. subtilis A5.3 and B. licheniformis T7 strains using methods of protein mass spectrometry, proteomics and genomics made it possible to identify 13 proteases and peptidases in the secretory proteome of B. subtilis A5. 3 with a molecular mass of 30.2-85.6 kDa. Seven proteolytic enzymes were identified in the secretory proteome of B. licheniformis strain T7: 4 of them belong to serine peptidases and 3 to metallopeptidases. 5. Bioinformatic analysis of the amino acid sequence of protein S8-378 showed that the protein contains a 29 a.o. signal peptide that ensures its secretion by B. licheniformis T7 cells. During secretory expression in B. licheniformis T7, due to autocatalytic action, the propeptide is removed and an enzymatically active variant of protein S8-378 is formed. 6. Proteolytic enzymes of B. subtilis A5.3 and B. licheniformis T7 strains have wide substrate specificity and are able to hydrolyze albumin (bovine serum albumin, ovalbumin), as well as fibrillar proteins (collagens, keratin). Casein has been found to be the most sensitive to the action of proteolytic enzymes: 1 mg of casein is completely hydrolysed in 1 minute. The most stable for hydrolysis is keratin. The keratinolytic activity of bacteria B. subtilis A5.3 and B. licheniformis T7 allows for complete hydrolysis of chicken feathers in 9 and 7 days, respectively. Adhesion of the bacterial cells to the feather surface plays a significant role in the efficiency and rate of feather substrates. Proteolytic enzymes secreted by the B. licheniformis T7, in contrast to the enzymes of the B. subtilis A5.3 strain, have a milk clotting activity of 10 U/ml in cow's milk and 23 U/ml in sheep's milk. Cow milk was coagulated using these enzymes and curds and cheese were produced. 7. The complete genomes of Bacillus licheniformis T7 and B. subtilis A5.3 strains were sequenced by nanopore sequencing (Oxford Nanopore technology). As a result, the complete nucleotide sequences of the strains were obtained and annotated. The genome of Bacillus licheniformis strain T7 has been deposited in the NCBI GenBank international database as CP124861. The information obtained on the strain genome will allow for further studies. Approbation of the work. The main provisions, results, conclusions and conclusion of the thesis have been published in 9 papers, including: 3 paper in a foreign scientific journals "PLoS ONE", "Scientific Reports" and "Biology (MDPI)", included into 1 and 2 quartiles of impact factor in Web of Science database; 3 papers in national scientific journals, recommended by the Committee on Quality Assurance in Science and Higher Education of the Ministry of Science and Higher Education of the Republic of Kazakhstan, 2 papers in the proceedings of international conferences. A patent of the Republic of Kazakhstan was received for the results of the dissertant's practical work. The personal contribution of the dissertant to the generalization of the results of scientific work recommended for protection. Literature review of the research work, definition of the goal and objectives, design and conduct of experiments, analysis and processing of the results of the practical work was carried out with the direct participation of the author. Relation of the work to the research programme. The work was aimed at obtaining and studying new enzymes of protease class. The work was carried out in Laboratory of Genetics and Biochemistry of Microorganisms, National Biotechnology Center, Limited Liability Company. Dissertation work was executed within the framework of projects AP14869708 "isolation and study of new proteolytic enzymes of bacterial origin", AP05135454 "Development of technology for producing strain-producer of recombinant keratinase for processing of low value feather and down raw materials into protein hydrolysate" and scientific-technical program BR10764944 "Development of methods of analytical control and food safety monitoring". The thesis volume and structure. The thesis is laid out on 134 pages; it contains 13 tables, 37 figures, 3 appendices. The list of references includes 331 references. Structurally, the thesis consists of a list of definitions, designations and abbreviations adopted in the thesis, introduction, literature review, materials and methods of experiments, results of own research, conclusion, list of references and appendices.
Conclusion of the Research Ethics Committee
Defense of the dissertation: https://youtu.be/wKV7DuCuo7s?si=0qEbOhzlhp5M--Hn
