Antioxidants prevent ageing and are usually quantified and screened using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. However, this assay cannot be used for salt-containing samples, such as the cell-free supernatants of marine microorganisms that are aggregated under these conditions. Herein, the DPPH solvent (methanol or ethanol) and its water content were optimized to enable the analysis of salt-containing samples, aggregation was observed for alcohol contents of >70%. The water content of methanol influenced the activities of standard antioxidants but did not significantly affect that of the samples. Based on solution stability considerations, 70% aqueous methanol was chosen as the optimal DPPH solvent. The developed method was successfully applied to the cell-free supernatants of marine bacteria (Pseudoalteromonas rubra and Pseudoalteromonas xiamenensis), revealing their high antioxidant activities. Furthermore, it was concluded that this method would be useful for the screening of marine microorganism–derived antioxidants, which also has numerous potential applications, such as salt-fermented foods.

We recently identified a $\mathsf{\beta }$-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of $\mathsf{\beta }$-agarases, shows less than 70.9% amino acid similarity with previously characterized agarases. Recombinant Gaa16B lacking the carbohydrate-binding region (rGaa16Bc) was overexpressed in Escherichia coli and purified. Activity assays revealed the optimal temperature and pH of rGaa16Bc to be 55 ${}^{\circ }$C and pH 6–7, respectively, and the protein was highly stable at 55 ${}^{\circ }$C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl₂. The K_m and V_max of rGaa16Bc for agarose were 6.4 mg/mL and 953 U/mg, respectively. Thin-layer chromatography analysis revealed that rGaa16Bc can hydrolyze agarose into neoagarotetraose and neoagarobiose. Partial hydrolysis products (PHPs) of rGaa16Bc had an average molecular weight of 88–102 kDa and exhibited > 60% hyaluronidase inhibition activity at a concentration of 1 mg/mL, whereas the completely hydrolyzed product (CHP) showed no hyaluronidase at the same concentration. The biochemical properties of Gaa16B suggest that it could be useful for producing functional neoagaro-oligosaccharides. Additionally, the PHP of rGaa16Bc may be useful in promoting its utilization, which is limited due to the gel strength of agar.

Agarases produce agar oligosaccharides with various structures exhibiting diverse physiological activities. α-Neoagaro-oligosaccharide hydrolase (α-NAOSH) specifically cleaves even-numbered neoagaro-oligosaccharides, producing 3,6-anhydro-l-galactose (l-AHG) and odd-numbered agaro-oligosaccharides (OAOSs). In this study, α-NAOSH from the agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5 (Gaa117) was purified and characterized using an E. coli expression system to produce OAOSs and determine their bioactivity. Recombinant Gaa117 (rGaa117) showed maximum activity at pH 6.0 and 35 °C. rGaa117 retained >80% of its initial activity after 120 min at 30 °C. The activity was enhanced in the presence of Mn²⁺. K_m, V_max, and K_cat/K_m values of the enzyme were 22.64 mM, 246.3 U/mg, and 15 s⁻¹/mM, respectively. rGaa117 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose, producing OAOSs that commonly contained l-AHG. Neoagarobiose and neoagarotetraose mixtures, designated NAO24, and mixtures of l-AHG and agarotriose, designated AO13, were obtained using recombinant rGaa16B (β-agarase) and rGaa117, respectively, and their antioxidant activities were compared. AO13 showed higher hydrogen peroxide-scavenging activity than NAO24 in human dermal fibroblasts in vitro because of structural differences: AOSs have d-galactose at the non-reducing end, whereas NAOSs have l-AHG. In conclusion, OAOSs exhibited high ROS-scavenging activity in H₂O₂-induced human dermal fibroblasts. They may be applicable in cosmetics and pharmaceuticals for prevention of skin aging.

Green algae, particularly Ulva species, are rich in complex polysaccharides, such as ulvan, which have significant potential for biotechnological applications. However, the biochemical properties of ulvan depolymerised products remain underexplored. The enzymatic depolymerisation of ulvan has garnered attention owing to its cost advantages over alternative methods. Nevertheless, the biochemical characterisation of ulvan lyases, specifically those belonging to the polysaccharide lyase family 25 (PL25), is limited. In this study, we identified and biochemically characterised a novel PL25 ulvan lyase, PaUL25, which functions optimally at pH 10. Additionally, we explored the alpha (α)-glucosidase inhibitory properties of ulvan depolymerised products. PaUL25 exhibited optimum activity at 35 °C in Tris-HCl buffer (pH 10). Moreover, enzyme activity was enhanced by more than 150% in the presence of Mn²⁺ metal ions at and below concentrations of 10 mM. The endolytic action of PaUL25 produced ulvan oligosaccharides with degrees of polymerisation of 2 and 4 as its end products. Partially and completely hydrolysed ulvan oligosaccharides exhibited α-glucosidase inhibitory activity, with half inhibitory concentration IC₅₀ values of 3.21 ± 0.13 and 2.51 ± 0.19 mg/mL, respectively. These findings expand our understanding of PL25 and highlight the pharmaceutical potential of ulvan oligosaccharides, particularly as antidiabetic agents.

Fish waste disposal poses significant environmental and economic challenges, limiting sustainability in the marine food industry. Hence, sustainable valorization strategies are needed to enhance resource recovery while minimizing waste. As an approach, this study aimed to evaluate the potential of converting chum salmon (Oncorhynchus keta) head (CSH) waste into a high-value peptone for microbiological applications. Various proteolytic enzymes were screened for CSH hydrolysis, among which Protamex achieved the highest hydrolysis and recovery rates. The resulting chum salmon head peptone (CSHP) exhibited favorable characteristics, including a low average molecular mass (557 Da) and a high amino nitrogen content (4.9%), outperforming commercial animal (AP) and vegetable (VP) peptones. To assess its biotechnological potential, CSHP was evaluated as a nitrogen source for recombinant protein production and supported higher expression of human superoxide dismutase (hSOD) and human growth hormone (hGH) in Escherichia coli BL21(DE3), compared with AP, VP, and Luria–Bertani (LB) media. Furthermore, life cycle assessment revealed a substantially lower carbon footprint for CSHP production than that of conventional peptone sources. These findings suggest that CSHP is a reliable and sustainable alternative to traditional peptones, offering both therapeutic and industrial applications while contributing to marine waste reduction and circular bioeconomy strategies.

The production of recombinant proteins in Escherichia coli is often challenged by cytoplasmic expression due to proteolytic degradation and inclusion body formation. Extracellular expression can overcome these problems by simplifying downstream processing and improving protein yields. This study aims to compare the efficiency of two Bacillus subtilis chitosanase signal peptides in mediating extracellular secretion in E. coli. We identified a naturally occurring mutant signal peptide (mCsn2-SP) from B. subtilis CH2 chitosanase (CH2CSN), which is characterized by a deletion of six amino acids in the N-region relative to the signal peptide (Csn1-SP) from B. subtilis CH1 chitosanase (CH1CSN). The CH1CSN and CH2CSN genes were cloned into the pET-11a vector and protein secretion was evaluated in E. coli BL21(DE3) host cells. Expression was induced with 0.1 mM and 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C for one and three days. CH2CSN showed higher secretion levels compared to CH1CSN under all experimental conditions, especially with 0.1 mM IPTG induction for 3 days, which resulted in a 2.37-fold increase in secretion. Furthermore, it was demonstrated that mCsn2-SP is capable of secreting human Cu,Zn-superoxide dismutase (hSOD) in E. coli BL21(DE3) and successfully translocating it to the periplasmic region. This study represents the inaugural investigation into the utilisation of a naturally modified signal peptide, thereby corroborating the assertion that signal peptide deletion variants can influence protein secretion efficiency. Furthermore, the findings substantiate the proposition that such variants can serve as a viable alternative for the secretion of heterologous proteins in E. coli.