Circular bioeconomy in action: Upscaling cutlassfish waste for eco-friendly recombinant protein production.

The fish processing industry generates a significant amount of waste, and the recycling of this waste is an issue of global concern. We sought to utilize the heads of cutlassfish (Trichiurus lepturus), which are typically discarded during processing, to produce peptone, which is an important source of amino acids for microbial growth and recombinant protein production. Cutlassfish head muscle (CHM) were isolated, and the optimal protease and reaction conditions for peptone production were determined. The resulting peptone contained 12.22 % total nitrogen and 3.19 % amino nitrogen, with an average molecular weight of 609 Da, indicating efficient hydrolysis of CHM. Growth assays using Escherichia coli have shown that cutlassfish head peptone (CP) supports similar or superior growth compared to other commercial peptones. In addition, when recombinant chitosanase from Bacillus subtilis and human superoxide dismutase were produced in E. coli, CP gave the highest expression levels among six commercial peptones tested. In addition, the expression levels of chitosanase and superoxide dismutase were 20 % and 32 % higher, respectively, in CP medium compared to the commonly used Luria-Bertani (LB) medium. This study demonstrates the potential of using cuttlassfish waste in the production of microbial media, thereby adding significant value to fish waste. The results contribute to sustainable waste management practices and open avenues for innovative uses of fish processing by-products in biotechnological applications.

Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass.

BACKGROUND: Xylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the ß-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis. RESULTS: xynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50-55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold. CONCLUSION: The supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.

Optimising the DPPH Assay for Cell-Free Marine Microorganism Supernatants.

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.

A Novel Agarase, Gaa16B, Isolated from the Marine Bacterium Gilvimarinus agarilyticus JEA5, and the Moisturizing Effect of Its Partial Hydrolysis Products.

We recently identified a ß-agarase, Gaa16B, in the marine bacterium Gilvimarinus agarilyticus JEA5. Gaa16B, belonging to the glycoside hydrolase 16 family of ß-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 ∘C and pH 6-7, respectively, and the protein was highly stable at 55 ∘C for 90 min. Additionally, rGaa16Bc activity was strongly enhanced (2.3-fold) in the presence of 2.5 mM MnCl2. The Km and Vmax 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.

Environmental assessment of contaminated marine sediments treated with solidification agents: Directions for improving environmental assessment guidelines.

Treatment of dredged materials is a critical issue, since management and disposal of these products requires considerable investment of monetary resources, time, and space. The high concentration of pollutants in dredged materials, along with high water content and many fine particles make recycling these materials particularly difficult. In order to solve this problem, solidification/stabilization has been considered as a potentially viable solution for recycling dredged marine sediments. However, there are currently no guidelines that address potential biological and environmental impacts. To evaluate the stability of treated materials and their biological impacts, dredged marine sediments, which were polluted with heavy metals, were treated by solidification/stabilization using two different solidifying agents. To assess potential impacts, toxicity characteristic leaching procedures (TCLP, USEPA) and a bioassay (with the rotifer, Brachionus sp.) were performed with treated materials. In a TCLP test, we found that treatment with a solidification agent decreased the leaching concentration of heavy metals from sediment compared to the control. The rotifer bioassay showed no change in the survival rate during 24 h of exposure to both agents. However, survival differed between the two agents after 48 h of exposure. Screening physiological status using gene expression, showed that oxidative stress genes were significantly altered. These results suggest that more studies are needed to provide guidelines for deciding the usability of treated materials created by the solidification or stabilization of dredged materials.

Chronic adverse effects of oil dispersed sediments on growth, hatching, and reproduction of benthic copepods: Indirect exposure for long-term tests.

Laboratory-scale sediment exposure was conducted as a preliminary study to assess the long-term effects of sediment contaminated with crude oil. For this purpose, indirect exposure using a glass filter crucible was tested and compared with direct exposure by observing several parameters (e.g., mortality, growth, reproduction, hatching, and uptake) in the benthic copepod Tigriopus japonicus. In direct exposure, short-term exposure caused significant damages to the eggs of ovigerous females, and there were difficulties in observing small oil droplets. However, indirect exposure did not induce any mortality during a 96-h exposure in adults. A 10-day exposure was also possible in an indirect exposure method and caused a decrease in reproduction and consequently a reduction in the hatching rate. In fact, the water phase collected from indirect exposure indicated significant polycyclic aromatic hydrocarbon (PAH) concentrations, although only a few components were present. The components of PAHs were similar to water-accommodated fractions (WAFs) of crude oil that are associated with the water-soluble part, but the relative portion of high-molecular-weight of PAHs was higher than WAF. In this approach, exposure tests caused reduction in the uptake rate in copepods even in the 24-h exposure. In conclusion, the biological effects of oil droplets from direct exposure were excluded by using a glass filter in indirect exposures, and several parameters could be derived in the long-term exposure. These results indicate that the indirect method could likely assess the chronic effects of oil-contaminated sediments on individual level parameters for deriving the ultimate effects on the population and community.