# A halophilic metalloprotease from Salinivibrio sp. YH4 and its application in antioxidant peptide production

## TL;DR

A salt-loving bacteria produces a protease that converts fish collagen waste into antioxidant peptides, offering a sustainable solution for environmental and health needs.

## Contribution

A halophilic metalloprotease from Salinivibrio sp. YH4 is characterized and shown to produce antioxidant peptides from collagen waste.

## Key findings

- EYHIII is a thermostable and alkaliphilic metalloprotease with high activity under high salinity and specificity for hydrophobic residues.
- Fish collagen hydrolysates from EYHIII show strong antioxidant activity in vitro and reduce ROS in HUVECs under high glucose stress.
- The enzyme enables sustainable conversion of protein-rich by-products into functional antioxidants.

## Abstract

This study aimed to develop a sustainable strategy for valorizing protein-rich industrial by-products into functional antioxidants using halophilic biocatalysts, addressing environmental challenges and the demand for bioactive compounds.

A moderately halophilic bacterium, Salinivibrio sp. YH4, was isolated from Yuncheng Salt Lake and identified as S. costicola (99% 16S rRNA homology). The extracellular protease EYHIII? was purified and biochemically characterized for thermal/pH stability, halotolerance, and substrate specificity. Fish collagen hydrolysates generated by EYHIII were evaluated for antioxidant capacity via 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and peroxyl radical scavenging assays. Cellular bioactivity was validated in high glucose-stressed human umbilical vein endothelial cells (HUVECs), analyzing ROS levels and antioxidant enzyme activity.

EYHIII was a thermostable (5060°C) and alkaliphilic (pH 7.59.5) M4-family metalloprotease. The enzyme retained >80% activity under high salinity conditions (1 M NaCl) and exhibited strict substrate specificity for hydrophobic residues (Phe/Leu) at the P1’ position. It efficiently hydrolyzed both soluble and insoluble collagens. Fish collagen hydrolysates generated by EYHIII demonstrated potent antioxidant activity, scavenging 33.53 ± 3.30% of DPPH radicals and 45.55 ± 3.00% of hydroxyl radicals at 3 mg/mL, with a peroxyl radical absorbance capacity of 1.69 ± 0.07 mmol TE/g. In human umbilical vein endothelial cells (HUVECs), the hydrolysate reduced high glucose-induced reactive oxygen species (ROS) to baseline levels at 200 μg/mL. It also significantly upregulated antioxidant enzymes compared to damaged controls: superoxide dismutase (SOD, 103.55%), catalase (CAT, 110.96%), and glutathione peroxidase (GSH-Px, 135.79%) (all P < 0.05).

This study highlighted Salinivibrio sp. YH4 and its protease EYHIII as a sustainable platform for converting collagen waste into high-value antioxidants. These findings addressed both environmental pollution and the growing demand for functional bioactive compounds. The results underscored the potential of halophilic biocatalysts in advancing circular economy strategies for protein resource utilization.