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Enzymes are versatile and ecological biocatalysts that are biodegradable, which leads to lower environmental impacts. Peptidases are enzymes that catalyze the hydrolysis reaction of peptide bonds in proteins. Protein hydrolysis is fundamental in food processing, cleaning products, and pharmaceuticals, among others. Microbial peptidases have been the preferred source for developing new enzymes, besides their advantages compared to plant or animal sources. This is owing to their characteristics, e.g., fast growth, wide diversity of microbial enzymes, longer shelf life, and potential for genetic manipulation of microorganisms. Enzymes catalyze biochemical reactions and are highly specific. They generally work in mild conditions and depend on their native conditions, such as pH, temperature, and solvent characteristics. However, the protease used must tolerate operational adversities such as extreme pH and temperature in industrial processes. For this, it is necessary to discover new peptidases and improve existing ones. Methods of metagenomic selection and genome mining or through extremophile diversity (e.g., thermophilic, cold-active, alkali tolerant, acidophilic, and halophilic microorganisms) have been used in the discovery of new enzymes. Protein engineering (by directed evolution, rational design, semi-rational design, and de novo design) and enzyme immobilization are strategies used to improve the catalytic properties, efficiency, and stability of enzymes. This review aimed to critically discuss the current state of the art and future trends of microbial peptidases, highlighting their applications in food processing. [ABSTRACT FROM AUTHOR]

Marine fishes have long been known due to their nutritional and pharmaceutical importance. In this study, marine crab (C. natator) body muscle (CBM) was utilized for the separation, purification, and identification of anti-inflammatory peptide using enzymatic hydrolysis. The amino acid composition of CBM depicted the presence of inflammation suppressing amino acids such as serine, aspartic acid, and glutamic acid. Furthermore, the CBM protein hydrolysates prepared using three enzymes (alcalase, trypsin, and papain) at different time intervals were analyzed for anti-inflammatory activity using albumin denaturation and membrane protection assays which exhibited 12th h papain with the highest denaturation inhibition (78.55 ± 1.36%) and membrane protection (69.02 ± 2.05%) activity. Moreover, separation of this active hydrolysate was performed by ultrafiltration using 10, 3 kDa cut-off membranes and gel chromatographic purification using Sephadex G-25 beads. The active peak (II) determined using anti-inflammatory assays was subjected to sequencing which revealed the peptide sequence of ESPVL (544 Da). Further, the functional aspects of the peptide were studied which showed increased stability and solubility of the peptide at varied pH range. Moreover, the effect of CBM derived peptide was studied on lipopolysaccharide-stimulated RAW264.7 cells. The purified peptide was found to be non-cytotoxic and suppressed cytokine expression which was estimated using enzyme immunoassay and western blot analysis. [ABSTRACT FROM AUTHOR]

In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese. [ABSTRACT FROM AUTHOR]