Mesonia algae K4-1 from the Arctic secretes a novel cold-adapted and salt-tolerant protease EK4-1. It has the highest sequence similarity with Stearolysin, an M4 family protease from Geobacillus stearothermophilus, with only 45% sequence identity, and is a novel M4 family protease. Ek4-1 has a low optimal catalytic temperature (40 °C) and is stable at low temperatures. Moreover, EK4-1 is still active in 4 mol/L NaCl solution and is tolerant to surfactants, oxidizing agents and organic solvents; furthermore, it prefers the hydrolysis of peptide bonds at the P1’ position as the hydrophobic residues, such as Leu, Phe and Val, and amino acids with a long side chain, such as Phe and Tyr. Mn2+and Mg2+ significantly promoted enzyme activity, while Fe3+, Co+, Zn2+ and Cu2+ significantly inhibited enzyme activity. Amino acid composition analysis showed that EK4-1 had more small-side-chain amino acids and fewer large-side-chain amino acids. Compared with a thermophilic protease Stearolysin, the cold-adapted protease EK4-1 contains more random coils (48.07%) and a larger active pocket (727.42 Å3). In addition, the acidic amino acid content of protease EK4-1 was higher than that of the basic amino acid, which might be related to the salt tolerance of protease. Compared with the homologous proteases EB62 and E423, the cold-adapted protease EK4-1 was more efficient in the proteolysis of grass carp skin, salmon skin and casein at a low temperature, and produced a large number of antioxidant peptides, with DPPH, ·OH and ROO· scavenging activities. Therefore, cold-adapted and salt-tolerant protease EK4-1 offers wide application prospects in the cosmetic and detergent industries.
Crude enzymes produced by a marine bacterium Pseudoalteromonas sp. JS4-1 were used to hydrolyze phycobiliprotein. Enzymatic productions showed good performance on DPPH radical and hydroxyl radical scavenging activities (45.14 ± 0.43% and 65.11 ± 2.64%, respectively), especially small peptides with MWCO <3 kDa. Small peptides were fractioned to four fractions using size-exclusion chromatography and the second fraction (F2) had the highest activity in hydroxyl radical scavenging ability (62.61 ± 5.80%). The fraction F1 and F2 both exhibited good antioxidant activities in oxidative stress models in HUVECs and HaCaT cells. Among them, F2 could upregulate the activities of SOD and GSH-Px and reduce the lipid peroxidation degree to scavenge the ROS to protect Caenorhabditis elegans under adversity. Then, 25 peptides total were identified from F2 by LC-MS/MS, and the peptide with the new sequence of INSSDVQGKY as the most significant component was synthetized and the ORAC assay and cellular ROS scavenging assay both illustrated its excellent antioxidant property.
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