Lichenan (Icelandic Moss)

Marine macroalgae and aquaculture organisms have in common to form problematic biomass, either when washed ashore after extensive blooms or as processing remains, which accumulate in aquaculture facilities. Both sources of biomass are commonly regarded as waste. This study aimed to investigate whether both sources of waste can be combined in a beneficial way to yield value-added products. Crude extracts of shrimp (Penaeus vannamei) and crayfish (Cherax quadricarinatus) remains were analyzed for their catalytic potential and functional properties. Shrimp extracts showed a high potential for degrading β-1,3-glycosidic bonds (laminarin), while crayfish extracts showed a high potential for degrading β-1,4-glycosidic bonds (cellulose). The highest activities were observed at pH 4 to pH 6 and at 50 to 60°C, with an optimum range between 30 and 40°C. Pre-treated brown algae, Sargassum horridum, were incubated with the crude crustacean extracts. The extracts were capable of hydrolyzing brown algae biomass, thereby liberating glucose. Blends of shrimp and crayfish extracts were more efficient than shrimp extracts alone. The produced glucose was fermented by common yeast to bio-ethanol. This “proof of concept” showed that putative bio-waste can be utilized to extract active enzymes and suitable substrates for the production of value-added products such as bio-ethanol. This approach of combining two different sources of waste in a complementary process may contribute to the mitigation of marine bio-waste and be considered a valuable feedstock for biotechnological applications.

Bacteria and fungi are well known for efficient degradation of plant polysaccharides thanks to various enzymes involved in plant cell wall decomposition. However, little is known about the role of archaea in this process or the repertoire and features of their polysaccharide-degrading enzymes. In our previous work, we discovered an archaeal multidomain glycosidase (MDG) composed of three catalytic domains (GH5 and two GH12) and two cellulose-binding modules (CBM2). The recombinant MDG and individual GH5 catalytic domain were active against cellulose and a number of other polysaccharides at a wide range of temperatures, with optimum temperatures (T_opt) of 60°C and 80°C, respectively. The present study was focused on the characterization of two GH12 domains of the MDG. Purified recombinant TMDG_GH12-1 and TMDG_GH12-2 proteins were active as individual enzymes but exhibited distinct catalytic properties. Both enzymes were thermostable and active at extremely high temperatures: TMDG_GH12-1 was active at 40-130°C (T_opt 100°C), and its half-life (t_½) at 100°C was 42 h, which makes it one of the most thermostable glycosidases known so far, whereas TMDG_GH12-2 was active at 50-100°C (T_opt 90°C) with t_½ at 100°C being 30 min. Phylogenetic and structural analysis of both TMDG_GH12 proteins together with molecular docking and site-directed mutagenesis suggested that the presence of two disulfide bridges and the W → Q mutation in the active site contribute to the exceptional thermostability of TMDG_GH12-1. Further structural and mutational studies of the TMDG_GH12-1 domain will help to gain a better understanding of the molecular mechanisms of its extraordinary thermostability and substrate specificity.

Arabinoxylan is a prevalent hemicellulose type, notably heterogeneous and resistant to biodegradation. Arabinofuranosidases (Abfs) remove arabinofuranosyl decorations of arabinoxylan, thus enabling hydrolysis by xylanases. However, a variety of Abf and xylanase specificities have emerged in recent years, necessitating a deeper understanding of their role in biomass degradation. This work investigates the biochemical features of TtAbf43 from Thermothelomyces thermophila, which specifically removes the O-3-linked arabinofuranose moieties from di-substituted xylopyranoses. The enzyme also exhibited secondary hydrolytic activity on o-nitrophenyl-β-d-xylopyranoside and arabinan. The hydrolysis of pretreated wheat and corn bran substrates was assessed using TtAbf43 and AnAbf51, two enzymes with distinct catalytic specificities. The Abfs enhanced the performance of endo-xylanases TmXyn10 and AnXyn11, promoting the release of xylo-oligomers, while the xylanases, in turn, stimulated arabinose release by the Abfs. Additionally, the Abfs facilitated the endo- and exo-activities of the bifunctional xylobiohydrolase/glucuronoxylanase TtXyn30A for the release of xylobiose and short aldouronic acids from biomass. AnAbf51 also acted in synergy with the acetyl xylan esterase OCE6 and the exo-deacetylase TtCE16B in debranching enzymatically derived oligomers from lignocellulose, whereas TtAbf43 remained unaffected by the esterases. These diverse synergistic relationships among different hemicellulases could assist the development of new enzymatic approaches for efficient biomass valorization.

In this study, we report the molecular and enzymatic characterisation of Spg103, a novel bifunctional β-glucanase from the marine bacterium Streptomyces sp. J103. Recombinant Spg103 (rSpg103) functioned optimally at 60 °C and pH 6. Notably, Spg103 exhibited distinct stability properties, with increased activity in the presence of Na+ and EDTA. Spg103 displays both lichenase and cellobiohydrolase activity. Despite possessing a GH5 cellulase domain, FN3 and CBM3 domains characteristic of cellulases and CBHs, biochemical assays showed that rSpg103 exhibited higher activity towards mixed β-1,3-1,4-glucan such as barley β-glucan and lichenan than towards beta-1,4-linkages. The endolytic activity of the enzyme was confirmed by TLC and UPLC-MS analyses, which identified cellotriose as the main hydrolysis product. In addition, Spg103 exhibited an exo-type activity, selectively releasing cellobiose units from cellooligosaccharides, which is characteristic of cellobiohydrolases. These results demonstrate the potential of Spg103 for a variety of biotechnological applications, particularly those requiring tailor-made enzymatic degradation of mixed-linked β-glucans. This study provides a basis for further structural and functional investigations of the bifunctional enzyme and highlights Spg103 as a promising candidate for industrial applications.

Realising a fully circular bioeconomy requires the valorisation of lignocellulosic biomass. Cellulose is the most attractive component of lignocellulose but depolymerisation is inefficient, expensive and resource intensive requiring substantial volumes of potable water. Seawater is an attractive prospective replacement, however seawater tolerant enzymes are required for the development of seawater-based biorefineries. Here, we report a halophilic cellobiohydrolase SMECel6A, identified and isolated from a salt marsh meta-exo-proteome dataset with high sequence divergence to previously characterised cellobiohydrolases. SMECel6A contains a glycoside hydrolase family 6 (GH6) domain and a carbohydrate binding module family 2 (CBM2) domain. Characterisation of recombinant SMECel6A revealed SMECel6A to be active upon crystalline and amorphous cellulose. Mono- and oligosaccharide product profiles revealed cellobiose as the major hydrolysis product confirming SMECel6A as a cellobiohydrolase. We show SMECel6A to be halophilic with optimal activity achieved in 0.5X seawater displaying 80.6 ± 6.93% activity in 1 × seawater. Structural predictions revealed similarity to a characterised halophilic cellobiohydrolase despite sharing only 57% sequence identity. Sequential thermocycling revealed SMECel6A had the ability to partially reversibly denature exclusively in seawater retaining significant activity. Our study confirms that salt marsh ecosystems harbour enzymes with attractive traits with biotechnological potential for implementation in ionic solution based bioprocessing systems.

Viruses are ubiquitous in the oceans, exhibiting high abundance and diversity. Here, we systematically analyze existing genomic sequences of marine prokaryotes to compile a Marine Prokaryotic Genome Dataset (MPGD, consisting of over 12,000 bacterial and archaeal genomes) and a Marine Temperate Viral Genome Dataset (MTVGD). At least 40% of the MPGD genomes contain one or more proviral sequences, indicating that they are lysogens. The MTVGD includes over 12,900 viral contigs or putative proviruses, clustered into 10,897 viral genera. We show that lysogens and proviruses are abundant in marine ecosystems, particularly in the deep sea, and marine lysogens differ from non-lysogens in multiple genomic features and growth properties. We reveal several virus-host interaction networks of potential ecological relevance, and identify proviruses that appear to be able to infect (or to be transferred between) different bacterial classes and phyla. Auxiliary metabolic genes in the MTVGD are enriched in functions related to carbohydrate metabolism. Finally, we experimentally demonstrate the impact of a prophage on the transcriptome of a representative marine Shewanella bacterium. Our work contributes to a better understanding of the ecology of marine prokaryotes and their viruses.

Background: The combination of cellulase and lytic polysaccharide monooxygenase (LPMO) is known to boost enzymatic saccharification of cellulose. Although the synergy between cellulases (GH5, 6 or 7) and LPMOs (AA9) has been extensively studied, the interplay between other glycoside hydrolase and LPMO families remains poorly understood. Results: In this study, two cellulolytic enzyme-encoding genes SmBglu12A and SmLpmo10A from Streptomyces megaspores were identified and heterologously expressed in Escherichia coli. The recombinant SmBglu12A is a non-typical endo-β-1,4-glucanase that preferentially hydrolyzed β-1,3-1,4-glucans and slightly hydrolyzed β-1,4-glucans and belongs to GH12 family. The recombinant SmLpmo10A belongs to a C1-oxidizing cellulose-active LPMO that catalyzed the oxidation of phosphoric acid swollen cellulose to produce celloaldonic acids. Moreover, individual SmBglu12A and SmLpmo10A were both active on barley β-1,3-1,4-glucan, lichenan, sodium carboxymethyl cellulose, phosphoric acid swollen cellulose, as well as Avicel. Furthermore, the combination of SmBglu12A and SmLpmo10A enhanced enzymatic saccharification of phosphoric acid swollen cellulose by improving the native and oxidized cello-oligosaccharides yields. Conclusions: These results proved for the first time that the AA10 LPMO was able to boost the catalytic efficiency of GH12 glycoside hydrolases on cellulosic substrates, providing another novel combination of glycoside hydrolase and LPMO for cellulose enzymatic saccharification.

The β-glucans are structurally varied, naturally occurring components of the cell walls, and storage materials of a variety of plant and microbial species. In the human diet, mixed-linkage glucans [MLG - β-(1,3/4)-glucans] influence the gut microbiome and the host immune system. Although consumed daily, the molecular mechanism by which human gut Gram-positive bacteria utilize MLG largely remains unknown. In this study, we used Blautia producta ATCC 27340 as a model organism to develop an understanding of MLG utilization. B. producta encodes a gene locus comprising a multi-modular cell-anchored endo-glucanase (BpGH16_MLG), an ABC transporter, and a glycoside phosphorylase (BpGH94_MLG) for utilizing MLG, as evidenced by the upregulation of expression of the enzyme- and solute binding protein (SBP)-encoding genes in this cluster when the organism is grown on MLG. We determined that recombinant BpGH16_MLG cleaved various types of β-glucan, generating oligosaccharides suitable for cellular uptake by B. producta. Cytoplasmic digestion of these oligosaccharides is then performed by recombinant BpGH94_MLG and β-glucosidases (BpGH3-AR8_MLG and BpGH3-X62_MLG). Using targeted deletion, we demonstrated BpSBP_MLG is essential for B. producta growth on barley β-glucan. Furthermore, we revealed that beneficial bacteria, such as Roseburia faecis JCM 17581^T, Bifidobacterium pseudocatenulatum JCM 1200T, Bifidobacterium adolescentis JCM 1275T, and Bifidobacterium bifidum JCM 1254, can also utilize oligosaccharides resulting from the action of BpGH16_MLG. Disentangling the β-glucan utilizing the capability of B. producta provides a rational basis on which to consider the probiotic potential of this class of organism.

Strain LLG6346-3.1^T, isolated from the thallus of the brown alga Ericaria zosteroides collected in Mediterranean Sea near Bastia in Corsica, France, was characterized using a polyphasic method. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, motile by gliding, rod-shaped and grew optimally at 30-33°C, at pH 8-8.5 and with 4-5 % NaCl. Strain LLG6346-3.1^T used the seaweed polysaccharide alginic acid as sole carbon source which was vigorously liquefied. Phylogenetic analyses showed that the bacterium is affiliated to the genus Zobellia (family Flavobacteriaceae, class Flavobacteriia). Strain LLG6346-3.1^T exhibited 16S rRNA gene sequence similarity values of 98.5 and 98.3 % to the type strains of Zobellia russellii and Zobellia roscoffensis respectively, and of 97.4-98.2 % to other species of the genus Zobellia. The DNA G+C content of strain LLG6346-3.1^T was determined to be 38.28 mol%. Digital DNA-DNA hybridization predictions by the ANI and GGDC methods between strain LLG6346-3.1^T and other members of the genus Zobellia showed values of 76-88 %, and below 37 %, respectively. The phenotypic, phylogenetic and genomic analyses show that strain LLG6346-3.1^T is distinct from species of the genus Zobellia with validly published names and that it represents a novel species of the genus Zobellia, for which the name Zobellia alginoliquefaciens sp. nov. is proposed. The type strain is LLG6346-3.1^T (RCC 7657^T = LLG 32918^T).

Background: Lignocellulose biomass is the most abundant and renewable material in nature. The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them (1.2 mg TrepCel3, 0.8 mg TrepCel4) on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates (rice straw, RS; wheat straw, WS; leymus chinensis, LC) were evaluated for their in vitro digestibility, gas, NH₃-N and volatile fatty acid (VFA) production, and microbial protein (MCP) synthesis by adding enzymatic combination. Methods: Two endoglucanases’ genes were successfully expressed in Escherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability. Results: The maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40°C, while TrepCel4 was at pH 6.0 and 50°C. They were stable over the temperature range of 30 to 60°C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NH₃-N and VFA production, and MCP synthesis during in vitro rumen fermentation. Conclusions: The TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.