L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Akishev Zhiger on the topic «Structure and functions of Camelus bactrianus and Bos taurus chymosins» by specialty «6D060700 – Biology».
The dissertation was carried out at the «Department of General Biology and Genomics» of L.N. Gumilyov Eurasian National University.
The language of defense is russian
Official reviewers:
Aleksandra Mikhaylovna Rozhkova – 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, Russia).
Zhigailov Andrey Viktorovich – Candidate of Biological Sciences, Head of the Laboratory of Protein and Nucleic Acids at the “M. Aitkhozhin Institute of Molecular Biology and Biochemistry” (Almaty, Kazakhstan).
Temporary members of the Dissertation Committee:
Shaikhin Serik Murzakhmetovich – Doctor of Biological Sciences, Head of the Laboratory of Genetics and Biochemistry of Microorganisms of the Republican Collection of Microorganisms (Astana, Kazakhstan).
Goncharova Alla Vladimirovna – Candidate of Biological Sciences, Associate Professor of the Department of Biotechnology, Biochemistry, Plant Physiology, Al-Farabi Kazakh National University (Almaty, Kazakhstan).
Almagul Kushugulova Rakhimberlieva – Doctor of Medical Sciences, Head of the Microbiome Laboratory of the Center for Life Sciences, Astana National Laboratory, Nazarbayev University (Astana, Kazakhstan).
Scientific advisors:
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, 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 February 25, 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. The defense meeting is planned to be held offline & online on the Microsoft Teams platform.
Link: https://clck.ru/3FkLBB
Address: 010008, Astana, Kazhymuqana str., 13, building №3, room №333
Abstract (English): General description of the work Examining the biochemical characteristics of recombinant chymosins from Bos taurus and Camelus bactrianus is the topic of the dissertation. Relevance of the topic Proteolysis is the hydrolysis of peptide bonds by proteolytic enzymes, proteinases, proteases, or peptidases, which results in the fragmentation of proteins and peptides into smaller peptides or amino acids. Asparagine proteases, the subject of this research, are present in mammals, birds, fish, plants, fungi, bacteria, and viruses. The extensive variety of tasks performed by asparagine proteases in nature is the impetus for their intense study and application in numerous economic sectors. Since the Neolithic Revolution, milk and its products have been among the principal sources of human nourishment. In 2023, worldwide milk production reached 965 million metric tons, an 18% increase from 2016 and more than 70% of the quantity produced in 2000. The most common type of milk is bovine milk. Approximately 96 percent of total cow and buffalo milk production remains stable (cow and buffalo milk accounted for 81 percent and 15.2 percent of world milk production, respectively). In 2021, goat, sheep, and camel milk accounted for 2.3%, 1.2%, and 0.4% of total global production, respectively. The agriculture of the Mediterranean, Western, and South-Eastern Asian regions rely heavily on this milk. Milk from deer, donkeys, and mares is not generally consumed, but it is culturally significant for some nations and ethnic groups. Other types of milk have recently been the subject of intense research due to their possible health benefits and prospective usage in infant formula due to their hypoallergenic characteristics, despite the prevalence of cow's milk. Currently, coagulants of animal, plant, and microbial origin are used to curdle milk in the cheese-making process, but traditionally, rennet, produced by the glandular cells of the fourth stomach section of ruminants, is utilized. The greatest amounts of rennet are produced during the early milking period of calves, goats, and lambs, and rennet is therefore only produced in young animals (3–10 days old). Rennet is a combination of the two endopeptidases: pepsin and chymosin, which are often known as renin. Chymosin content in renin ranges from 10% to 90%, and a preparation containing mostly or purely chymosin is of the highest use in cheesemaking since its composition has a major influence on the organoleptic and taste properties of cheese. Initially released in its inactivated prochymosin form, chymosin is later activated by autocatalytic proteolysis at low pH levels into a chymosin protein with a molecular weight of 36 kDa. Once activated, the enzyme breaks down the casein protein at a specified position, causing milk to curdle. The enzyme hydrolyzes the link between Phe105 and Met106 in the kappa-casein molecule, causing the casein micelle to destabilize and clot to form. The diminishing number of killed animals is a limiting factor in the global expansion in cheese production, which has prompted research to locate alternate sources of chymosin. Chymosins of fungal and plant origin are utilized in practice, but their utility in cheese-making is relatively low. The use of recombinant DNA technology to create the enzyme provides an alternate source of more value chymosin of animal origin. The filamentous fungi Aspergillus niger, Aspergillus awamori, and Trichoderma reesei are utilized to manufacture chymosin in fungi, however yeast offers a more promising alternative. The most effective expression system is based on Pichia (Komagataella) pastoris, which produces relatively low levels of its own secreted proteins, which greatly facilitates the purification of the recombinant protein and, as a result, has enabled the commercialization of a vast array of protein products for therapeutic and industrial applications. In addition to the known and utilized milk-clotting enzymes, additional enzymes are being sought, which may include a plant counterpart of pepsin from the Spanish artichoke (Cynara cardunculus), the yak (Bos mutus) or bison (Bison bison) chymosin, or the camel with one hump Camelus dromedarius chymosin. Obviously, microbial rennin is less expensive than animal chymosins, but the texture and organoleptic properties of cheese made with rennin from Rhizomucor miehei are inferior to those made with camel's chymosin, which can be produced not only in yeast cells, but also in cells of the fungus Aspergillus niger. The proposed research lies at the intersection of molecular biology, enzymology, biochemistry, industrial biotechnology, and food biotechnology. The molecular biochemistry part of the thesis will focus on the production and study of two recombinant chymosins from camelids and hornbills whose genomes are currently known: Camelus bactrianus and Bos taurus, utilizing a modern arsenal of molecular biology and biochemistry techniques: bioinformatic analysis, de novo gene assembly, site-directed mutagenesis, multilevel screening systems utilizing proteomics methods, and biochemical analysis techniques. In the biotechnological part of the dissertation, the method of microbial synthesis of target chymosin by deep fermentation in a bioreactor is utilized. The utilization of recombinant chymosin in cheesemaking and curd formation is one example of its use in food biotechnology. Scientific novelty and theoretical significance of the work The nucleotide sequence of the Camelus bactrianus prochymosin B gene was cloned for the first time. Using expression vectors pET28c(+), pGAPZαA and pPICZαA, plasmid constructs incorporating the nucleotide sequences of the prochymosin B genes of B. taurus and C. bactrianus were created. For the first time, the biochemical properties of recombinant calf and camel chymosins have been studied, including the determination of specific and total proteolytic activity of the enzymes. Milk coagulation activity in various milk kinds was investigated for the first time. Aims and objectives of the study. The aim of the study was to determine the biochemical characteristics of recombinant chymosins from Camelus bactrianus and Bos taurus with the determination of autocatalytic, proteolytic, and coagulation activities. To reach this purpose, the following objectives were pursued: 1. In silico calculation and synthesis of full-length preprohymosin B genes from Camelus bactrianus and Bos taurus. Cloning of the calf preprohymosin B gene in a bacterial vector, intracellular expression in Escherichia coli cells, and purification of recombinant protein by affinity chromatography. Preparation of polyclonal anti-chymosin B antibodies. 2. Cloning of calf and camel prochymosin B genes in yeast vectors, synthesis of recombinant proteins using the secretory method in Pichia pastoris, and purification by ion-exchange chromatography. 3. To investigate the biochemical parameters of specific milk-clotting and total proteolytic activity, as well as the substrate specificity of recombinant chymosins derived from Camelus bactrianus and Bos taurus. 4. Production of recombinant chymosins from Bos taurus and Camelus bactrianus by methods of deep fermentation in a bioreactor and coagulation testing on various milk kinds. 5. Development of a quantitative method to determine the biochemical and kinetic parameters of pepsin-like aspartic proteases. Objects of research: chymosin B aspartic proteases from Bos taurus and from Camelus bactrianus. Subject of research: biochemical properties of calf and camelus chymosins. Methods of research: methods of microbiology, molecular biology, biochemistry, genetic engineering, and biotechnology. Practical significance of the work. The recombinant producer strains, capable of secreting milk clotting enzymes, have been created. The recombinant analogs of calf and double humpback camel chymosins have been obtained. They have a set of technological properties that are attractive for cheese-making. The enzymes are of interest to milk processing companies. The technology of obtaining the biopreparation demanded in the cheese-making industry has been developed. The main points put forward for defense are The following scientific results are defended: 1. Cultivation of Pichia pastoris strains producing recombinant chymosin in growth medium containing: 1% yeast extract, 2% peptone, 100 mM citrate-phosphate buffer (pH 4.0), 10 mM ascorbic acid, 5% sorbitol, 1% beet molasses, and 4% glucose allows produce up to 20 mg per liter culture of the enzyme with milk-clotting activity. 2. Recombinant chymosins from B. taurus calf and C. bactrianus camel, obtained in a eukaryotic expression system, are acidic aspartic proteases with maximum activity at pH 4.5 and temperatures 0–55 °C. Chymosins are metal-dependent enzymes whose activity increases in the presence of calcium ions, with maximum activity at 30–40 mM CaCl2, and is inhibited by nickel ions by 80–100% for camel and calf chymosins, respectively. 3. The recombinant double-humped camel's chymosin has a higher milk clotting activity of 29%, 46%, 40%, and 31% for cow's, goat's, sheep's, and camel's milk, respectively, than that of calf's chymosin. On mare's milk, the specific activity of camel's chymosin is 15 times higher than that of calf's chymosin. 4. Recombinant camel chymosin, is a higher specific milk-coagulant protease than calf chymosin, as the ratio of specific milk-coagulant activity to total proteolytic activity in camel chymosin is 8.3 times higher than that of calf chymosin. 5. The method based on the use of a cloned fragment of κ-casein with a biotinylated tag allows to qualitative and quantitative determination of pepsin-like proteinase activity. Approbation of the work. The main provisions, results, conclusions and conclusions of the thesis were published in 12 papers, including 1 article in a foreign scientific journal "Biology (MDPI)", included in the databases of Thomson Reuters, Scopus and included in the 1st quartile on the impact factor in the base Web of Science; 1 article in a foreign scientific journal "Heliyon", included in the databases of Thomson Reuters, Scopus and included in the 2nd quartile on the impact factor in the base Web of Science; 6 articles in national scientific journals recommended by the Committee for Quality Assurance in Science and Higher Education of the Ministry of Science and Higher Education of the Republic of Kazakhstan; 1 paper in the proceedings of the international conference "European Biotechnology Congress -2017" (Dubrovnik, Croatia, 2017), published in the journal "Journal of Biotechnology", included in the 2nd quartile by impact factor in the Web of Science; 1 paper in the proceedings of the International Conference "Microbial Biotechnology: Fundamental and Applied Aspects" (Minsk, Belarus, 2017); 1 paper in the proceedings of the international conference "European Biotechnology Congress-2018" (Athens, Greece, 2018), published in Journal of Biotechnology, included in the 2nd quartile by impact factor in Web of Science database; 1 paper in the proceedings of Astana Biotech-2018 International Conference (Astana, Kazakhstan, 2018). Two patents of the Republic of Kazakhstan were received based on the results of the practical work. Relationship of the work to the scientific research program. This work was aimed at obtaining and studying the biochemical properties of recombinant chymosins from Bos taurus and Camelus bactrianus. The work was carried out at the Laboratory of Genetics and Biochemistry of Microorganisms at LLP “National Center for Biotechnology”. This dissertation work was carried out within projects 5134/GF "Production of genetically engineered enzyme preparations of chymosin and β-galactosidase for deep processing of milk", AP05133470 "Creation of strain-producer and development of production technology of recombinant camel's rennet enzyme, demanded at enterprises of the dairy industry" and scientific-technical program BR10764998 "Development of technologies using new strains of useful microorganisms, enzymes, nutrients, and other complexes in the production of special dietary foodstuffs". The volume and structure of the thesis. The thesis is 150 pages long and contains 16 tables, 63 figures, and 3 appendices. The list of references includes 360 references. Structurally, the thesis consists of a list of definitions, designations, and abbreviations adopted in the thesis, an introduction, three chapters including a literature review, materials and methods of experiments, results of own research, a conclusion, a list of references, and an appendix.
Conclusion of the Research Ethics Committee
Defense of the dissertation: https://youtu.be/GjcuPa5QTTM?si=DGNojhiyWl9UzwLJ
