Liu Dan, Xiao Yuyang, Wei Yingying, Xie Maojia, Huang Yu, Gan Chaoyu, He Hailun
Guangxi Colleges and Universities Key Laboratory of Biological Molecular Medicine Research, Department of Biochemistry and Molecular Biology, School of Pre-Clinical Medicine, Guangxi Medical University, Nanning, Guangxi, China.
School of Life Sciences, Central South University, Changsha, Hunan, China.
Front Microbiol. 2025 May 19;16:1595109. doi: 10.3389/fmicb.2025.1595109. eCollection 2025.
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, sp. YH4, was isolated from Yuncheng Salt Lake and identified as (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 < 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.
本研究旨在开发一种可持续策略，利用嗜盐生物催化剂将富含蛋白质的工业副产品转化为功能性抗氧化剂，以应对环境挑战并满足对生物活性化合物的需求。
从运城盐湖分离出一株中度嗜盐细菌盐弧菌属（Salinivibrio sp.）YH4，并鉴定为盐弧菌属（Salinivibrio sp.）（16S rRNA同源性为99%）。对胞外蛋白酶EYHIII进行了纯化，并对其热稳定性/pH稳定性、耐盐性和底物特异性进行了生化表征。通过1,1-二苯基-2-苦基肼（DPPH）、羟基和过氧自由基清除试验评估了EYHIII产生的鱼胶原蛋白水解产物的抗氧化能力。在高糖应激的人脐静脉内皮细胞（HUVECs）中验证了细胞生物活性，分析了活性氧水平和抗氧化酶活性。
EYHIII是一种热稳定（50-60°C）且嗜碱（pH 7.5-9.5）的M4家族金属蛋白酶。该酶在高盐条件（1 M NaCl）下保留了>80%的活性，并且对P1'位置的疏水残基（苯丙氨酸/亮氨酸）表现出严格的底物特异性。它能有效水解可溶性和不溶性胶原蛋白。EYHIII产生的鱼胶原蛋白水解产物表现出强大的抗氧化活性，在3 mg/mL时清除33.53±3.30%的DPPH自由基和45.55±3.00%的羟基自由基，过氧自由基吸收能力为1.69±0.07 mmol TE/g。在人脐静脉内皮细胞（HUVECs）中，水解产物在200 μg/mL时将高糖诱导的活性氧（ROS）降低至基线水平。与受损对照组相比，它还显著上调了抗氧化酶：超氧化物歧化酶（SOD，103.55%）、过氧化氢酶（CAT，110.96%）和谷胱甘肽过氧化物酶（GSH-Px，135.79%）（均P<0.05）。
本研究强调了盐弧菌属（Salinivibrio sp.）YH4及其蛋白酶EYHIII作为将胶原蛋白废物转化为高价值抗氧化剂的可持续平台。这些发现既解决了环境污染问题，又满足了对功能性生物活性化合物不断增长的需求。结果强调了嗜盐生物催化剂在推进蛋白质资源利用的循环经济策略方面的潜力。
