Feasibility of Enzymatic Protein Extraction from a Dehydrated Fish Biomass Obtained from Unsorted Canned Yellowfin Tuna Side Streams: Part II.

The enzymatic extraction of proteins from fish biomasses is being widely investigated. However, little or almost no research has paid attention to the exploitation of unsorted fishery biomasses. This work is part of a larger study, Part I of which has already been published, and focuses on an extensive characterization of two collagenous samples, namely gelatin (G) and hydrolyzed gelatin peptides (HGPs), extracted from a dehydrated fish biomass coming from unsorted canned yellowfin tuna side streams. The results indicate crude protein fractions of 90-93%, pH values between 3 and 5, white-yellow colors, collagen-like FTIR spectra, and 17% in terms of total amino acid content. Viscosity and the study of dynamic viscous-elastic behavior were analyzed. Thermo-gravimetric analysis was performed to assess the residual ashes. Both samples were investigated to determine their molecular weight distribution via size-exclusion chromatography, with a higher total average molecular weight for G compared to HGPs, with values of 17,265.5 Da and 2637.5 Da, respectively. G demonstrated technological properties similar to analogous marine gelatins. HGPs demonstrated antioxidant activity as per FRAP assay. All the results open up new perspectives for the potential use of these substances in biodegradable packaging, dietary supplements, and skin care cosmetics.

Current and Expected Trends for the Marine Chitin/Chitosan and Collagen Value Chains.

Chitin/chitosan and collagen are two of the most important bioactive compounds, with applications in the pharmaceutical, veterinary, nutraceutical, cosmetic, biomaterials, and other industries. When extracted from non-edible parts of fish and shellfish, by-catches, and invasive species, their use contributes to a more sustainable and circular economy. The present article reviews the scientific knowledge and publication trends along the marine chitin/chitosan and collagen value chains and assesses how researchers, industry players, and end-users can bridge the gap between scientific understanding and industrial applications. Overall, research on chitin/chitosan remains focused on the compound itself rather than its market applications. Still, chitin/chitosan use is expected to increase in food and biomedical applications, while that of collagen is expected to increase in biomedical, cosmetic, pharmaceutical, and nutritional applications. Sustainable practices, such as the reuse of waste materials, contribute to strengthen both value chains; the identified weaknesses include the lack of studies considering market trends, social sustainability, and profitability, as well as insufficient examination of intellectual property rights. Government regulations, market demand, consumer preferences, technological advancements, environmental challenges, and legal frameworks play significant roles in shaping both value chains. Addressing these factors is crucial for seizing opportunities, fostering sustainability, complying with regulations, and maintaining competitiveness in these constantly evolving value chains.

Mobile group I introns at nuclear rDNA position L2066 harbor sense and antisense homing endonuclease genes intervened by spliceosomal introns.

BACKGROUND: Mobile group I introns encode homing endonucleases that confer intron mobility initiated by a double-strand break in the intron-lacking allele at the site of insertion. Nuclear ribosomal DNA of some fungi and protists contain mobile group I introns harboring His-Cys homing endonuclease genes (HEGs). An intriguing question is how protein-coding genes embedded in nuclear ribosomal DNA become expressed. To address this gap of knowledge we analyzed nuclear L2066 group I introns from myxomycetes and ascomycetes. RESULTS: A total of 34 introns were investigated, including two identified mobile-type introns in myxomycetes with HEGs oriented in sense or antisense directions. Intriguingly, both HEGs are interrupted by spliceosomal introns. The intron in Didymium squamulosum, which harbors an antisense oriented HEG, was investigated in more detail. The group I intron RNA self-splices in vitro, thus generating ligated exons and full-length intron circles. The intron HEG is expressed in vivo in Didymium cells, which involves removal of a 47-nt spliceosomal intron (I-47) and 3' polyadenylation of the mRNA. The D. squamulosum HEG (lacking the I-47 intron) was over-expressed in E. coli, and the corresponding protein was purified and shown to confer endonuclease activity. The homing endonuclease was shown to cleave an intron-lacking DNA and to produce a pentanucleotide 3' overhang at the intron insertion site. CONCLUSIONS: The L2066 family of nuclear group I introns all belong to the group IE subclass. The D. squamulosum L2066 intron contains major hallmarks of a true mobile group I intron by encoding a His-Cys homing endonuclease that generates a double-strand break at the DNA insertion site. We propose a potential model to explain how an antisense HEG becomes expressed from a nuclear ribosomal DNA locus.

Bacteriophage origin of some minimal ATP-dependent DNA ligases: a new structure from Burkholderia pseudomallei with striking similarity to Chlorella virus ligase.

DNA ligases, the enzymes responsible for joining breaks in the phosphodiester backbone of DNA during replication and repair, vary considerably in size and structure. The smallest members of this enzyme class carry out their functions with pared-down protein scaffolds comprising only the core catalytic domains. Here we use sequence similarity network analysis of minimal DNA ligases from all biological super kingdoms, to investigate their evolutionary origins, with a particular focus on bacterial variants. This revealed that bacterial Lig C sequences cluster more closely with Eukaryote and Archaeal ligases, while bacterial Lig E sequences cluster most closely with viral sequences. Further refinement of the latter group delineates a cohesive cluster of canonical Lig E sequences that possess a leader peptide, an exclusively bacteriophage group of T7 DNA ligase homologs and a group with high similarity to the Chlorella virus DNA ligase which includes both bacterial and viral enzymes. The structure and function of the bacterially-encoded Chlorella virus homologs were further investigated by recombinantly producing and characterizing, the ATP-dependent DNA ligase from Burkholderia pseudomallei as well as determining its crystal structure in complex with DNA. This revealed that the enzyme has similar activity characteristics to other ATP-dependent DNA ligases, and significant structural similarity to the eukaryotic virus Chlorella virus including the positioning and DNA contacts of the binding latch region. Analysis of the genomic context of the B. pseudomallei ATP-dependent DNA ligase indicates it is part of a lysogenic bacteriophage present in the B. pseudomallei chromosome representing one likely entry point for the horizontal acquisition of ATP-dependent DNA ligases by bacteria.

Marine Bioactive Peptides in Supplements and Functional Foods - A Commercial Perspective.

Many bioactive peptides have been described from marine sources and much marine biomass is still not explored or utilized in products. Marine peptides can be developed into a variety of products, and there is a significant interest in the use of bioactive peptides from marine sources for nutraceuticals or functional foods. We present here a mini-review collecting the knowledge about the value chain of bioactive peptides from marine sources used in nutraceuticals and functional foods. Many reports describe bioactive peptides from marine sources, but in order to make these available to the consumers in commercial products, it is important to connect the bioactivities associated with these peptides to commercial opportunities and possibilities. In this mini-review, we present challenges and opportunities for the commercial use of bioactive peptides in nutraceuticals and functional food products. We start the paper by introducing approaches for isolation and identification of bioactive peptides and candidates for functional foods. We further discuss market-driven innovation targeted to ensure that isolated peptides and suggested products are marketable and acceptable by targeted consumers. To increase the commercial potential and ensure the sustainability of the identified bioactive peptides and products, we discuss scalability, regulatory frameworks, production possibilities and the shift towards greener technologies. Finally, we discuss some commercial products from marine peptides within the functional food market. We discuss the placement of these products in the larger picture of the commercial sphere of functional food products from bioactive peptides.

Isolation and complete genome sequence of the thermophilic Geobacillus sp. 12AMOR1 from an Arctic deep-sea hydrothermal vent site.

Members of the genus Geobacillus have been isolated from a wide variety of habitats worldwide and are the subject for targeted enzyme utilization in various industrial applications. Here we report the isolation and complete genome sequence of the thermophilic starch-degrading Geobacillus sp. 12AMOR1. The strain 12AMOR1 was isolated from deep-sea hot sediment at the Jan Mayen hydrothermal Vent Site. Geobacillus sp. 12AMOR1 consists of a 3,410,035 bp circular chromosome and a 32,689 bp plasmid with a G + C content of 52 % and 47 %, respectively. The genome comprises 3323 protein-coding genes, 88 tRNA species and 10 rRNA operons. The isolate grows on a suite of sugars, complex polysaccharides and proteinous carbon sources. Accordingly, a versatility of genes encoding carbohydrate-active enzymes (CAZy) and peptidases were identified in the genome. Expression, purification and characterization of an enzyme of the glycoside hydrolase family 13 revealed a starch-degrading capacity and high thermal stability with a melting temperature of 76.4 °C. Altogether, the data obtained point to a new isolate from a marine hydrothermal vent with a large bioprospecting potential.

Crystal structure of the DNA polymerase III ß subunit (ß-clamp) from the extremophile Deinococcus radiodurans.

BACKGROUND: Deinococcus radiodurans is an extremely radiation and desiccation resistant bacterium which can tolerate radiation doses up to 5,000 Grays without losing viability. We are studying the role of DNA repair and replication proteins for this unusual phenotype by a structural biology approach. The DNA polymerase III ß subunit (ß-clamp) acts as a sliding clamp on DNA, promoting the binding and processivity of many DNA-acting proteins, and here we report the crystal structure of D. radiodurans ß-clamp (Drß-clamp) at 2.0 Å resolution. RESULTS: The sequence verification process revealed that at the time of the study the gene encoding Drß-clamp was wrongly annotated in the genome database, encoding a protein of 393 instead of 362 amino acids. The short protein was successfully expressed, purified and used for crystallisation purposes in complex with Cy5-labeled DNA. The structure, which was obtained from blue crystals, shows a typical ring-shaped bacterial ß-clamp formed of two monomers, each with three domains of identical topology, but with no visible DNA in electron density. A visualisation of the electrostatic surface potential reveals a highly negatively charged outer surface while the inner surface and the dimer forming interface have a more even charge distribution. CONCLUSIONS: The structure of Drß-clamp was determined to 2.0 Å resolution and shows an evenly distributed electrostatic surface charge on the DNA interacting side. We hypothesise that this charge distribution may facilitate efficient movement on encircled DNA and help ensure efficient DNA metabolism in D. radiodurans upon exposure to high doses of ionizing irradiation or desiccation.

MutT from the fish pathogen Aliivibrio salmonicida is a cold-active nucleotide-pool sanitization enzyme with unexpectedly high thermostability.

Upon infection by pathogenic bacteria, production of reactive oxygen species (ROS) is part of the host organism's first line of defence. ROS damage a number of macromolecules, and in order to withstand such a harsh environment, the bacteria need to have well-functioning ROS scavenging and repair systems. Herein, MutT is an important nucleotide-pool sanitization enzyme, which degrades 8-oxo-dGTP and thus prevents it from being incorporated into DNA. In this context, we have performed a comparative biochemical and structural analysis of MutT from the fish pathogen Aliivibrio salmonicida (AsMutT) and the human pathogen Vibrio cholerae (VcMutT), in order to analyse their function as nucleotide sanitization enzymes and also determine possible cold-adapted properties of AsMutT. The biochemical characterisation revealed that both enzymes possess activity towards the 8-oxo-dGTP substrate, and that AsMutT has a higher catalytic efficiency than VcMutT at all temperatures studied. Calculations based on the biochemical data also revealed a lower activation energy (E a) for AsMutT compared to VcMutT, and differential scanning calorimetry experiments showed that AsMutT displayed an unexpected higher melting temperature (T m) value than VcMutT. A comparative analysis of the crystal structure of VcMutT, determined to 2.42 Å resolution, and homology models of AsMutT indicate that three unique Gly residues in loops of VcMutT, and additional long range ion-pairs in AsMutT could explain the difference in temperature stability of the two enzymes. We conclude that AsMutT is a stable, cold-active enzyme with high catalytic efficiency and reduced E a, compared to the mesophilic VcMutT.