In the cheese-making industry, commonly chymosin is used as the main milk-clotting enzyme. Bactrian camel (Camelus bactrianus) chymosin (BacChym) has a milk-clotting activity higher than that of calf chymosin for cow’s, goat’s, ewes’, mare’s and camel’s milk. A procedure for obtaining milk-clotting reagent based on recombinant camel chymosin is proposed here. Submerged fermentation by a recombinant yeast (Pichia pastoris GS115/pGAPZαA/ProchymCB) was implemented in a 50 L bioreactor, and the recombinant camel chymosin was prepared successfully. The activity of BacChym in yeast culture was 174.5 U/mL. The chymosin was concentrated 5.6-fold by cross-flow ultrafiltration and was purified by ion exchange chromatography. The activity of the purified BacChym was 4700 U/mL. By sublimation-drying with casein peptone, the BacChym powder was obtained with an activity of 36,000 U/g. By means of this chymosin, cheese was prepared from cow’s, goat’s, ewes’, camel’s and mare’s milk with a yield of 18%, 17.3%, 15.9%, 10.4% and 3%, respectively. Thus, the proposed procedure for obtaining a milk-clotting reagent based on BacChym via submerged fermentation by a recombinant yeast has some prospects for biotechnological applications. BacChym could be a prospective milk-clotting enzyme for different types of milk and their mixtures.

Environmental safety and economic factors necessitate a search for new ways of processing poultry farm feathers, which are 90% β-keratin and can be used as a cheap source of amino acids and peptones. In this study, feather-decomposing bacteria were isolated from a site of accumulation of rotten feathers and identified as Bacillus. Among them, the Bacillus sp. A5.3 isolate showed the best keratinolytic properties. Scanning electron microscopy indicated that Bacillus sp. A5.3 cells closely adhere to the feather surface while degrading the feather. It was found that Bacillus sp. A5.3 secretes thermostable alkaline proteolytic and keratinolytic enzymes. Zymographic analysis of the enzymatic extract toward bovine serum albumin, casein, gelatin, and β-keratin revealed the presence of proteases and keratinases with molecular weights 20–250 kDa. The proteolytic and keratinolytic enzymes predominantly belong to the serine protease family. Proteome analysis of the secreted proteins by nano-HPLC coupled with Q-TOF mass spectrometry identified 154 proteins, 13 of which are proteases and peptidases. Thus, strain Bacillus sp. A5.3 holds great promise for use in feather-processing technologies and as a source of proteases and keratinases.

Amylases are enzymes that are known to hydrolyze starch. High efficiency of amylolytic enzymes allows them to compete in the industry with the technology of chemical hydrolysis of starch. A Bacillus licheniformis strain with high amylolytic activity was isolated from soil and designated as T5. The gene encoding α-amylase from B. licheniformis T5 was successfully expressed in both Escherichia coli (rAmyT5-E) and Pichia pastoris (as rAmyT5-P). According to the study, the recombinant α-amylases rAmyT5-E and rAmyT5-P exhibited the highest activity at pH 6.0 and temperatures of 70 and 80 °C, respectively. Over 80% of the rAmyT5-E enzyme activity was preserved following incubation within the pH range of 5–9; the same was true for rAmyT5-P after incubation at pH 6–9. N-glycosylation reduced the thermal and pH stability of the enzyme. The specific activity and catalytic efficiency of the recombinant AmyT5 α-amylase were also diminished by N-glycosylation.

Barcodes are conserved sequences of genomic, plastid and mitochondrial DNA that can be utilized to uniquely identify an unidentified specimen to its species when conventional identification methods are inapplicable. Among prokaryotic and eukaryotic species, nuclear ribosomal internal transcribed spacer (ITS) sections are one of the most often utilized DNA markers in DNA barcoding and phylogenetic research. In addition to the ribosomal genes, the plastid genes are the most suitable for identifying plant species. The Aksu-Zhabagly Nature Reserve is the oldest nature reserve in Central Asia and is home to 1,312 vascular plant species, 44 of which are categorized as threatened or endangered in Kazakhstan's red data book. In this study, a collection of specimens of uncommon tulip species was compiled, along with their morphological identification and DNA barcoding. The ITS region and parts of the matK and ycf1b genes of tulip plastid DNA were sequenced. The evolutionary link between species of tulips was investigated. Phylogenetic study predicted two Tulipa subclades. Tulipa species have substantially preserved MatK genes. Tulips' ycf1b gene has evolved more slowly than other Liliaceae family members. Nuclear and plastid DNA sequences investigated Tulipa species evolutionary relationships. The findings about the ITS region of nuclear DNA were more definite. Overall, our work shows that genetic data will be important in determining species concepts in this genus, however, even with a molecular perspective pulling apart closely related taxa can be extremely challenging.

Phytases are widely used as feed additives because they improve phosphorus utilization from plant-derived feed ingredients and reduce phosphorus excretion into the environment. Most recombinant AppA phytases produced in Komagataella phaffii have been obtained using methanol-inducible expression systems, whereas information on constitutive methanol-free production remains limited. In this study, Escherichia coli AppA phytase was expressed in K. phaffii under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Cultivation temperature and initial glucose concentration were evaluated in shake-flask cultures, while the selected conditions were assessed in a 2 L stirred-tank bioreactor. Extracellular phytase activity reached 114.17 ± 2.71 U/mL in shake flasks at 27 °C with 3% initial glucose. The K. phaffii-produced enzyme was N-glycosylated, with an apparent molecular weight of approximately 54 kDa compared with approximately 45 kDa after deglycosylation. Compared with purified rAppA-E, the rAppA-P preparation showed a lower temperature optimum, a narrower pH–activity profile, and lower apparent thermostability. In a single bioreactor run, extracellular phytase activity reached 186.81 ± 0.25 U/mL, approximately 1.6-fold higher than the maximum mean shake-flask value. These findings demonstrate constitutive, methanol-free extracellular production of AppA in K. phaffii and support further development of controlled cultivation strategies.

Исследование посвящено потенциалу Bacillus subtilis A5.3 и Bacillus licheniformis T7 в качестве источников протеолитических ферментов с коллагеназной активностью для биотехнологической переработки коллагенсодержащих отходов сельского хозяйства. Были проведены выделение, идентификация и характеризация этих бактериальных штаммов с использованием морфологических, биохимических и молекулярно-генетических методов. Протеолитическая активность оценивалась на казеине и желатине в качестве субстратов. Оптимальные условия для активности ферментов были определены как 70°C и pH 10,0 для B. subtilis A5.3 и 60°C и pH 9,0 для B. licheniformis T7. Исследование влияния ионов металлов, детергентов и ингибиторов на активность ферментов выявило значительные различия между двумя штаммами: B. subtilis A5.3 продемонстрировал более высокую чувствительность к ионам Mn²⁺, Cd²⁺ и Mg²⁺, тогда как B. licheniformis T7 показал большую устойчивость к этим металлам. Зимографический анализ подтвердил наличие нескольких активных протеаз в ферментных экстрактах обоих штаммов с молекулярными массами от 20 до 150 кДа. Наблюдаемая активность гидролиза желатина свидетельствует о наличии коллагеназоподобных протеаз, что подчеркивает промышленный потенциал этих штаммов в гидролизе коллагенсодержащих отходов. Полученные результаты подтверждают пригодность B. subtilis A5.3 и B. licheniformis T7 в качестве перспективных кандидатов для разработки устойчивых методов утилизации коллагенсодержащих отходов.