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.

Camel chymosin can be efficiently employed to produce cheese. Traditionally the rennet enzyme produced by the glands of the fourth stomach of ruminant animals (abomassum) is used in cheese making. Full-length Camelus bactrianus (Bactrian camel) prochymosin gene was synthesized and constitutively expressed in Pichia pastoris cells under glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. It was purified by sequential anion and cation exchange chromatography. SDS-PAGE analysis resulted in two bands, approximately 42 and 35 kDa. The 42 kDa band vanished when the sample was treated with endoglycosidase H, indicating that the recombinant protein is partially glycosylated. Optimal pH for the activity of the highest-purity recombinant chymosin was pH 4.5 for cow's milk and pH 4.0 for mare's milk. The range 45–50 °C and 70 °C for cow's and mare's milk types, respectively, was found to be the most appropriate for maximal relative milk-clotting activity. Concentration of CaCl2 that ensured the stability of the chymosin milk-clotting activity was between 20 and 50 mM with an optimum at 30 mM. Milk-clotting activity of camel recombinant chymosin and ability to make curd was successfully tested on fresh mare's milk. Pichia pastoris strain with integrated camel chymosin gene showed high productivity of submerged fermentation in bioreactor with milk-clotting activity 1412 U/mL and 80 mg/L enzyme yield. These results suggest that the constitutive expression of the camel chymosin Camelus bactrianus in the yeast Pichia pastoris has good prospects for practical applications.

Mutations in the human PARK7 gene that encodes protein DJ-1 lead to familial Parkinsonism due to loss of dopaminergic neurons. However, the molecular function of DJ-1 underpinning its cytoprotective effects are unclear. Recently, DJ-1 has been shown to prevent acylation of amino groups of proteins and metabolites by 1,3-bisphosphoglycerate. This acylation is indirect and thought to proceed via the formation of an unstable intermediate, presumably a cyclic 3-phosphoglyceric anhydride (cPGA). Several lines of evidence indicate that DJ-1 destroys cPGA, however this enzymatic activity has not been directly demonstrated. Here, we report simple and effective procedures for synthesis and quantitation of cPGA and present a comprehensive characterization of this highly reactive acylating electrophile. We demonstrate that DJ-1 is an efficient cPGA hydrolase with k_cat/K_m = 5.9 × 10⁶ M⁻¹s⁻¹. Experiments with DJ-1-null cells reveal that DJ-1 protects against accumulation of 3-phosphoglyceroyl-lysine residues in proteins. Our results establish a definitive cytoprotective function for DJ-1 that uses catalytic hydrolysis of cPGA to mitigate the damage from this glycolytic byproduct.

Esterases are ubiquitous enzymes found in all living organisms, including animals, plants, and microorganisms. They are involved in several biological processes, including the synthesis and breakdown of biomolecules, such as nucleic acids, lipids, and esters; phosphorus metabolism; detoxification of natural and artificial toxicants; polymer breakdown and synthesis; remodeling; and cell signaling. The present review focuses on the most industrially important esterases, namely lipases, phospholipases, cutinases, and polyethylene terephthalate hydrolases (PETases). Esterases are widely used in industrial and biotechnological applications. Notably, the biotechnological production of esters, including methyl acetate, ethyl acetate, vinyl acetate, polyvinyl acetate, and ethyl lactate, as an alternative to chemical production, represents a multi-billion-dollar industry. Currently, most enzymes (>75%) used in industrial processes are hydrolytic. Among them, lipases and phospholipases are primarily used for lipid modification. Lipases are the third most commercialized enzymes after proteases and carboxyhydrases, and their production is steadily increasing, currently representing over one-fifth of the global enzyme market. Esterases, particularly lipases, phospholipases, and cutinases, are employed in cosmetics, food, lubricants, pharmaceuticals, paints, detergents, paper, and biodiesel, among other industries. Overall, biotechnological production using enzymes is gaining global traction owing to its environmental benefits, high yields, and efficiency, aligning with green economy principles.

Esterases catalyze the hydrolysis and transesterification of short-chain fatty acid esters, and microbial esterases are used in the production of biofuels, cosmetics, food, and pharmaceuticals. The soil strain Bacillus paralicheniformis T7 secretes enzymes with esterase activity; however, many bacterial enzymes remain insufficiently studied. Therefore, this study aimed to identify and characterize novel GDSL esterases produced by B. paralicheniformis. Protein mass spectrometry, combined with proteomics and genomics, identified genes encoding two GDSL esterases, which were cloned into the pET-28c(+) vector. The resulting proteins were obtained in Escherichia coli BL21(DE3) as the recombinant esterases rEST-24 and rEST-28. These recombinant GDSL esterases showed maximum activity at 40 °C and pH 7.0. Moreover, Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ ions inhibited their activity, and rEST-28 was resistant to the detergents Tween-20, Tween-80, and Triton X-100. High-yield esterase activity was detected in bacteria cultured on feather medium and nutrient broth, and submerged fermentation of the B. paralicheniformis T7 strain on feather medium enabled the production of an esterase extract exhibiting activity of 17,618 ± 610 U/g. These results suggest that the B. paralicheniformis T7 strain can produce esterases and shows promising potential for application in technologies that degrade fatty acid esters using hydrolytic enzymes.