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.

An indole-based fluorescent chemosensor targeting the autophagosome.

Autophagy is a process for the degradation and recycling of intracellular components and dysfunctional organelles. We developed an indole-embedded fluorescent naphthalimide for the selective imaging of autophagosomes in live cells. It was shown as intense puncta in the fluorescence confocal images and co-localizes with an autophagosome marker, LC3-RFP. In addition, it was applied to cellular autophagic models based on ER stress and starvation to verify its capability.

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.

Characterization of an acetyl xylan esterase from the marine bacterium Ochrovirga pacifica and its synergism with xylanase on beechwood xylan.

BACKGROUND: Acetyl xylan esterase plays an important role in the complete enzymatic hydrolysis of lignocellulosic materials. It hydrolyzes the ester linkages of acetic acid in xylan and supports and enhances the activity of xylanase. This study was conducted to identify and overexpress the acetyl xylan esterase (AXE) gene revealed by the genomic sequencing of the marine bacterium Ochrovirga pacifica. RESULTS: The AXE gene has an 864-bp open reading frame that encodes 287 aa and consists of an AXE domain from aa 60 to 274. Gene was cloned to pET-16b vector and expressed the recombinant AXE (rAXE) in Escherichia coli BL21 (DE3). The predicted molecular mass was 31.75 kDa. The maximum specific activity (40.08 U/mg) was recorded at the optimal temperature and pH which were 50 °C and pH 8.0, respectively. The thermal stability assay showed that AXE maintains its residual activity almost constantly throughout and after incubation at 45 °C for 120 min. The synergism of AXE with xylanase on beechwood xylan, increased the relative activity 1.41-fold. CONCLUSION: Resulted higher relative activity of rAXE with commercially available xylanase on beechwood xylan showed its potential for the use of rAXE in industrial purposes as a de-esterification enzyme to hydrolyze xylan and hemicellulose-like complex substrates.

A Novel Glycosyl Hydrolase Family 16 ß-Agarase from the Agar-Utilizing Marine Bacterium Gilvimarinus agarilyticus JEA5: the First Molecular and Biochemical Characterization of Agarase in Genus Gilvimarinus.

The agarase gene gaa16a was identified from a draft genome sequence of Gilvimarinus agarilyticus JEA5, an agar-utilizing marine bacterium. Recently, three agarase-producing bacteria, G. chinensis, G. polysaccharolyticus, and G. agarilyticus, in the genus Gilvimarinus were reported. However, there have been no reports of the molecular characteristics and biochemical properties of these agarases. In this study, we analyzed the molecular characteristics and biochemical properties of agarases in Gilvimarinus. Gaa16A comprised a 1,323-bp open reading frame encoding 441 amino acids. The predicted molecular mass and isoelectric point were 49 kDa and 4.9, respectively. The amino acid sequence of Gaa16A showed features typical of glycosyl hydrolase family 16 (GH16) ß-agarases, including a GH16 domain, carbohydrate-binding region (RICIN domain), and signal peptide. Recombinant Gaa16A (excluding the signal peptide and carbohydrate-binding region, rGaa16A) was expressed as a fused protein with maltose-binding protein at its N-terminus in Escherichia coli. rGaa16A had maximum activity at 55°C and pH 7.0 and 103 U/mg of specific activity in the presence of 2.5 mM CaCl2. The enzyme hydrolyzed agarose to yield neoagarotetraose as the main product. This enzyme may be useful for industrial production of functional neoagaro-oligosaccharides.

Corallibacterium pacifica gen. nov., sp. nov., a Novel Bacterium of the Family Vibrionaceae Isolated from Hard Coral.

A gram-negative, rod-shaped, motile, oxidase- and catalase-positive, non-pigmented marine bacterium, designated strain OS-11M-2T, was isolated from a coral sample collected from the Osakura coastal area in Micronesia. Phylogenetic analysis based on 16S ribosomal RNA (rRNA) gene sequences indicated that strain OS-11M-2T is a member of the family Vibrionaceae, its closest neighbors being Photobacterium damselae subsp. piscicida NCIMB 2058T (94.9%), Photobacterium damselae subsp. damselae CIP 102761T (94.75%), Grimontia marina IMCC5001T (94.5%), Enterovibrio coralii LMG 22228T (94.5%), and Grimontia celer 96-237T (94.5%). The major cellular fatty acids were summed feature 3 (21.4%), summed feature 8 (18.5%), iso-C16:0 (13.8%), and C16:0 (11.9%). The major respiratory quinone of the bacterium was ubiquinone-8 (Q-8) and its major polar lipid phosphatidylethanolamine. Six amino lipids, two phospholipids, and one polar lipid, all unidentified, were detected. The DNA G+C content was 49.7 mol%. The 16S rRNA gene sequence of OS-11M-2T was registered in GenBank under accession number MF359550. On the basis of phenotypic, genotypic, and phylogenetic analyses, strain OS-11M-2T represents a novel genus of the family Vibrionaceae, for which we propose the name Corallibacterium pacifica gen. nov., sp. nov., with the type strain of the type species being OS-11M-2T (= KCCM 43265T). The digital protologue database (DPD) taxon number for strain OS-11M-2T is GA00041.

Identification and molecular profiling of DC-SIGN-like from big belly seahorse (Hippocampus abdominalis) inferring its potential relevancy in host immunity.

Dendritic-cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a C-type lectin that functions as a pattern recognition receptor by recognizing pathogen-associated molecular patterns (PAMPs). It is also involved in various events of the dendritic cell (DC) life cycle, such as DC migration, antigen capture and presentation, and T cell priming. In this study, a DC-SIGN-like gene from the big belly seahorse Hippocampus abdominalis (designated as ShDCS-like) was identified and molecularly characterized. The putative, complete ORF was found to be 1368 bp in length, encoding a protein of 462 amino acids with a molecular mass of 52.6 kDa and a theoretical isoelectric point of 8.26. The deduced amino acid sequence contains a single carbohydrate recognition domain (CRD), in which six conserved cysteine residues and two Ca2+-binding site motifs (QPN, WND) were identified. Based on pairwise sequence analysis, ShDCS-like exhibits the highest amino acid identity (94.6%) and similarity (97.4%) with DC-SIGN-like counterpart from tiger tail seahorse Hippocampus comes. Quantitative real-time PCR revealed that ShDCS-like mRNA is transcribed universally in all tissues examined, but with abundance in kidney and gill tissues. The basal mRNA expression of ShDCS-like was modulated in blood cell, kidney, gill and liver tissues in response to the stimulation of healthy fish with lipopolysaccharides (LPS), Edwardsiella tarda, or Streptococcus iniae. Moreover, recombinant ShDCS-like-CRD domain exhibited detectable agglutination activity against different bacteria. Collectively, these results suggest that ShDCS-like may potentially involve in immune function in big belly seahorses.

A Rapid and Efficient Screening Method for Antibacterial Compound-Producing Bacteria.

Antibacterial compounds are widely used in the treatment of human and animal diseases. The overuse of antibiotics has led to a rapid rise in the prevalence of drug-resistant bacteria, making the development of new antibacterial compounds essential. This study focused on developing a fast and easy method for identifying marine bacteria that produce antibiotic compounds. Eight randomly selected marine target bacterial species (Agrococcus terreus, Bacillus algicola, Mesoflavibacter zeaxanthinifaciens, Pseudoalteromonas flavipulchra, P. peptidolytica, P. piscicida, P. rubra, and Zunongwangia atlantica) were tested for production of antibacterial compounds against four strains of test bacteria (B. cereus, B. subtilis, Halomonas smyrnensis, and Vibrio alginolyticus). Colony picking was used as the primary screening method. Clear zones were observed around colonies of P. flavipulchra, P. peptidolytica, P. piscicida, and P. rubra tested against B. cereus, B. subtilis, and H. smyrnensis. The efficiency of colony scraping and broth culture methods for antimicrobial compound extraction was also compared using a disk diffusion assay. P. peptidolytica, P. piscicida, and P. rubra showed antagonistic activity against H. smyrnensis, B. cereus, and B. subtilis, respectively, only in the colony scraping method. Our results show that colony picking and colony scraping are effective, quick, and easy methods of screening for antibacterial compound-producing bacteria.

First comparative characterization of three distinct ferritin subunits from a teleost: Evidence for immune-responsive mRNA expression and iron depriving activity of seahorse (Hippocampus abdominalis) ferritins.

Ferritins play an indispensable role in iron homeostasis through their iron-withholding function in living beings. In the current study, cDNA sequences of three distinct ferritin subunits, including a ferritin H, a ferritin M, and a ferritin L, were identified from big belly seahorse, Hippocampus abdominalis, and molecularly characterized. Complete coding sequences (CDS) of seahorse ferritin H (HaFerH), ferritin M (HaFerM), and ferritin L (HaFerL) subunits were comprised of 531, 528, and 522 base pairs (bp), respectively, which encode polypeptides of 177, 176, and 174 amino acids, respectively, with molecular masses of ∼20-21 kDa. Our in silico analyses demonstrate that these three ferritin subunits exhibit the typical characteristics of ferritin superfamily members including iron regulatory elements, domain signatures, and reactive centers. The coding sequences of HaFerH, M, and L were cloned and the corresponding proteins were overexpressed in a bacterial system. Recombinantly expressed HaFer proteins demonstrated detectable in vivo iron sequestrating (ferroxidase) activity, consistent with their putative iron binding capability. Quantification of the basal expression of these three HaFer sequences in selected tissues demonstrated a gene-specific ubiquitous spatial distribution pattern, with abundance of mRNA in HaFerM in the liver and predominant expression of HaFerH and HaFerL in blood. Interestingly, the basal expression of all three ferritin genes was found to be significantly modulated against pathogenic stress mounted by lipopolysaccharides (LPS), poly I:C, Streptococcus iniae, and Edwardsiella tarda. Collectively, our findings suggest that the three HaFer subunits may be involved in iron (II) homeostasis in big belly seahorse and that they are important in its host defense mechanisms.

Effect of Food Waste Compost on the Antler-Type Fruiting Body Yield of Ganoderma lucidum.

The effects of the composition of a mixture containing food waste compost (FWC), rice bran (RB), and oak sawdust (SD) on the antler-type fruiting body (FB) yield of Ganoderma lucidum were studied. Experiments were performed using 0 (control), 5, 10, 15, 20, 25, 30, 35, and 40% (w/w) FWC added to a basal growth medium consisting of 20% (w/w) RB and 80% (w/w) SD. The content of 15% FWC gave the highest FB yield (27.0 ± 1.3 g/bottle), which was 44% higher than the yield (18.6 ± 2.8 g/bottle) of the control treatment. However, FWC contents of 20~40% showed reduced yield (2.4~23.0 g/bottle), partly because FWC had a high Na concentration (0.6%). These results demonstrate the potential for use of FWC as a component of a growth medium for production of G. lucidum FBs.

Influence of Food Waste Compost on the Yield and Mineral Content of Ganoderma lucidum, Lentinula edodes, and Pholiota adipose Fruiting Bodies.

The objectives of this study were to evaluate applicability of food waste compost (FWC) as a substrate for cultivation of Ganoderma lucidum, Lentinula edodes, and Pholiota adipose, and to determine contents of Ca, Mg, Na, and K in fruiting bodies (FB). FB yield per substrate in FWC-free controls was 53 ± 4 g/kg for G. lucidum, 270 ± 90 g/kg for L. edodes, and 1,430 ± 355 g/kg for P. adipose. Substrates supplemented with FWC showed the highest FB production at FWC content of 10% for G. lucidum (64 ± 6 g/kg), and 13% for L. edodes (665 ± 110 g/kg) and P. adipose (2,345 ± 395 g/kg), which were 1.2~2.5 times higher than the values for the controls. P. adipose contained higher amounts of mineral elements than the other species. Ca, Mg, Na, and K content in FB did not show a significant relation to FWC content.

Piperine, a component of black pepper, inhibits adipogenesis by antagonizing PPARγ activity in 3T3-L1 cells.

This study investigated the antiadipogenic activity of black pepper extract and its constituent piperine in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. Both black pepper extract and piperine, without affecting cytotoxicity, strongly inhibited the adipocyte differentiation of 3T3-L1 cells. The mRNA expression of the master adipogenic transcription factors, PPARγ, SREBP-1c, and C/EBPß, was markedly decreased. Intriguingly, mRNA levels of PPARγ target genes were also down-regulated. Moreover, a luciferase reporter assay indicated that pipierine significantly represses the rosiglitazone-induced PPARγ transcriptional activity. Finally, GST-pull down assays demonstrated that piperine disrupts the rosiglitazone-dependent interaction between PPARγ and coactivator CBP. Genome-wide analysis using microarray further supports the role of piperine in regulating genes associated with lipid metabolism. Overall, these results suggest that piperine, a major component of black pepper, attenuates fat cell differentiation by down-regulating PPARγ activity as well as suppressing PPARγ expression, thus leading to potential treatment for obesity-related diseases.