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.

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.

There has been an increasing consumer demand for cheese along with a search for products with new organoleptic parameters, which has resulted in extensive research on alternative milk coagulants. The ratio of proteolytic activity to milk-clotting activity determines the requirements for proteases used in the cheese making process. To date, plant enzymes have largely been used for this purpose, along with traditional enzymes of animal origin, chymosin and pepsin. The most popular types of proteases used in the food industry, especially in cheese making, are plant proteases belonging to the cysteine (papain, bromelain, ficin), aspartate (cinarase, cardosin), and serine (kukumizin, leucine) group of proteases. The aspartate proteases of microbial origin mucorpepsin and endotyapepsin have found wide application in cheese production due to low production costs and high organoleptic characteristics of the final product. The use of plant and microbial milk-clotting enzymes as an alternative to animal-derived enzymes allows not only to diversify the assortment of cheeses on the market but also to solve ethical and economic issues. In addition, vegetable and microbial preparations meet the requirements of vegetarianism, halal, and kosher food, thus further opening the market.

For the development of science-intensive technologies in biology, biotechnology and medicine, it is necessary to store valuable DNA samples: genes of enzymes, antigens, proteins for various purposes, and DNA loci in a depository. Storing DNA is inexpensive, and having such a depository will reduce the time spent on research in basic and applied science. The genetically engineered materials bank has been formed at the National center for biotechnology. Now, the bank has 88 samples of cloned genes of enzymes, antigens, proteins and diagnostically significant loci. Plasmid vectors with cloned genes are stored at a temperature -20°С. Besides competent cells of Escherichia coli strain DH5α were transformed with vectors and recombinant strains were isolated. Recombinant strains carrying the required plasmid vectors are stored for cryopreservation at a temperature -80°С.