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.

Xylanase is one of industrial enzymes with diverse applications including the paper-bleaching industry and feed additives. Here, a strain having xylanolytic activity and identified as Bacillus sonorensis T6 was isolated from soil. A secretory enzyme was identified by mass-spectrometry as a xylanase of glycosyl hydrolase family 11, with a molecular weight of 23.3 kDa. The xylanase gene of Bacillus sonorensis T6 was cloned and expressed in Escherichia coli (yielding an enzyme designated as rXynT6-E) and in Pichia pastoris (yielding rXynT6-P). The recombinant xylanases were found to have optimal activity at 47–55°C and pH 6.0–7.0. The recombinant xylanase expressed in P. pastoris has 40% higher thermal stability than that expressed in E. coli. The recombinant xylanases retained 100% of activity after 10 h incubation in the pH range 3–11 and 68% of activity after 1 h at pH 2.0. The xylanase activities of rXynT6-E and rXynT6-P under optimal conditions were 1030.2 and 873.8 U/mg, respectively. The good stability in a wide range of pH and moderate temperatures may make the xylanase from Bacillus sonorensis T6 useful for various biotechnological applications, e.g., as an enzyme additive in the feed industry.

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.