A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

Metabolic engineering of Thermoanaerobacterium AK17 for increased ethanol production in seaweed hydrolysate.

Sustainably produced renewable biomass has the potential to replace fossil-based feedstocks, for generation of biobased fuels and chemicals of industrial interest, in biorefineries. In this context, seaweeds contain a large fraction of carbohydrates that are a promising source for enzymatic and/or microbial biorefinery conversions. The thermoanaerobe Thermoanaerobacterium AK17 is a versatile fermentative bacterium producing ethanol, acetate and lactate from various sugars. In this study, strain AK17 was engineered for more efficient production of ethanol by knocking out the lactate and acetate side-product pathways. This was successfully achieved, but the strain reverted to acetate production by recruiting enzymes from the butyrate pathway. Subsequently this pathway was knocked out and the resultant strain AK17_M6 could produce ethanol close to the maximum theoretical yield (90%), leading to a 1.5-fold increase in production compared to the wild-type strain. Strain AK17 was also shown to successfully ferment brown seaweed hydrolysate from Laminaria digitata to ethanol in a comparatively high yield of 0.45 g/g substrate, with the primary carbon sources for the fermentations being mannitol, laminarin-derived glucose and short laminari-oligosaccharides. As strain AK17 was successfully engineered and has a wide carbohydrate utilization range that includes mannitol from brown seaweed, as well as hexoses and pentoses found in both seaweeds and lignocellulose, the new strain AK17_M6 obtained in this study is an interesting candidate for production of ethanol from both second and third generations biomass.

Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system.

Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys-), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).

Sialidases and fucosidases of Akkermansia muciniphila are crucial for growth on mucin and nutrient sharing with mucus-associated gut bacteria.

The mucolytic human gut microbiota specialist Akkermansia muciniphila is proposed to boost mucin-secretion by the host, thereby being a key player in mucus turnover. Mucin glycan utilization requires the removal of protective caps, notably fucose and sialic acid, but the enzymatic details of this process remain largely unknown. Here, we describe the specificities of ten A. muciniphila glycoside hydrolases, which collectively remove all known sialyl and fucosyl mucin caps including those on double-sulfated epitopes. Structural analyses revealed an unprecedented fucosidase modular arrangement and explained the sialyl T-antigen specificity of a sialidase of a previously unknown family. Cell-attached sialidases and fucosidases displayed mucin-binding and their inhibition abolished growth of A. muciniphila on mucin. Remarkably, neither the sialic acid nor fucose contributed to A. muciniphila growth, but instead promoted butyrate production by co-cultured Clostridia. This study brings unprecedented mechanistic insight into the initiation of mucin O-glycan degradation by A. muciniphila and nutrient sharing between mucus-associated bacteria.

AmiP from hyperthermophilic Thermus parvatiensis prophage is a thermoactive and ultrathermostable peptidoglycan lytic amidase.

Bacteriophages encode a wide variety of cell wall disrupting enzymes that aid the viral escape in the final stages of infection. These lytic enzymes have accumulated notable interest due to their potential as novel antibacterials for infection treatment caused by multiple-drug resistant bacteria. Here, the detailed functional and structural characterization of Thermus parvatiensis prophage peptidoglycan lytic amidase AmiP, a globular Amidase_3 type lytic enzyme adapted to high temperatures is presented. The sequence and structure comparison with homologous lytic amidases reveals the key adaptation traits that ensure the activity and stability of AmiP at high temperatures. The crystal structure determined at a resolution of 1.8 Å displays a compact α/ß-fold with multiple secondary structure elements omitted or shortened compared with protein structures of similar proteins. The functional characterization of AmiP demonstrates high efficiency of catalytic activity and broad substrate specificity toward thermophilic and mesophilic bacteria strains containing Orn-type or DAP-type peptidoglycan. The here presented AmiP constitutes the most thermoactive and ultrathermostable Amidase_3 type lytic enzyme biochemically characterized with a temperature optimum at 85°C. The extraordinary high melting temperature Tm 102.6°C confirms fold stability up to approximately 100°C. Furthermore, AmiP is shown to be more active over the alkaline pH range with pH optimum at pH 8.5 and tolerates NaCl up to 300 mM with the activity optimum at 25 mM NaCl. This set of beneficial characteristics suggests that AmiP can be further exploited in biotechnology.

Novel Function of CtXyn5A from Acetivibrio thermocellus: Dual Arabinoxylanase and Feruloyl Esterase Activity in the Same Active Site.

Enzymes' uncharacterised side activities can have significant effects on reaction products and yields. Hence, their identification and characterisation are crucial for the development of successful reaction systems. Here, we report the presence of feruloyl esterase activity in CtXyn5A from Acetivibrio thermocellus, besides its well-known arabinoxylanase activity, for the first time. Activity analysis of enzyme variants mutated in the catalytic nucleophile, Glu279, confirmed removal of all activity for E279A and E279L, and increased esterase activity while removing xylanase activity for E279S, thus allowing the proposal that both reaction types are catalysed in the same active site in two subsequential steps. The ferulic acid substituent is cleaved off first, followed by hydrolysis of the xylan backbone. The esterase activity on complex carbohydrates was found to be higher than that of a designated ferulic acid esterase (E-FAERU). Therefore, we conclude that the enzyme exhibits a dual function rather than an esterase side activity.

Crystal structure of DNA polymerase I from Thermus phage G20c.

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

A novel membraneless ß-glucan/O2 enzymatic fuel cell based on ß-glucosidase (RmBgl3B)/pyranose dehydrogenase (AmPDH) co-immobilized onto buckypaper electrode.

A novel membraneless ß-glucan/O2 enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus ß-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction. The fuel for the bioanode was produced by enzymatic hydrolysis of ß-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel. The maximum power density of the assembled ß-glucan/O2 biofuel cell reached 26.3 ±â€¯4.6 µWcm-2 at 0.36 V in phosphate buffer containing 0.5 % (w/v) ß-glucan at 40 °C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that ß-glucan can be employed as fuel in an enzymatic biofuel cell.

Investigation of Structural Features of Two Related Lipases and the Impact on Fatty Acid Specificity in Vegetable Fats.

One of the indispensable applications of lipases in modification of oils and fats is the possibility to tailor the fatty acid content of triacylglycerols (TAGs), to meet specific requirements from various applications in food, nutrition, and cosmetic industries. Oleic acid (C18:1) and stearic acid (C18:0) are two common long fatty acids in the side chain of triglycerides in plant fats and oils that have similar chemical composition and structures, except for an unsaturated bond between C9 and C10 in oleic acid. Two lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL), show activity in reactions involving oleate and stearate, and share high sequence and structural identity. In this research, the preference for one of these two similar fatty acid side chains was investigated for the two lipases and was related to the respective enzyme structure. From transesterification reactions with 1:1 (molar ratio) mixed ethyl stearate (ES) and ethyl oleate (EO), both RML and ROL showed a higher activity towards EO than ES, but RML showed around 10% higher preference for ES compared with ROL. In silico results showed that stearate has a less stable interaction with the substrate binding crevice in both RML and ROL and higher tendency to freely move out of the substrate binding region, compared with oleate whose structure is more rigid due to the existence of the double bond. However, Trp88 from RML which is an Ala at the identical position in ROL shows a significant stabilization effect in the substrate interaction in RML, especially with stearate as a ligand.

Crystal structure and initial characterization of a novel archaeal-like Holliday junction-resolving enzyme from Thermus thermophilus phage Tth15-6.

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Šresolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.

Lignocellulose degradation for the bioeconomy: The potential of enzyme synergies between xylanases, ferulic acid esterase and laccase for the production of arabinoxylo-oligosaccharides.

The success of establishing bioeconomies replacing current economies based on fossil resources largely depends on our ability to degrade recalcitrant lignocellulosic biomass. This study explores the potential of employing various enzymes acting synergistically on previously pretreated agricultural side streams (corn bran, oat hull, soluble and insoluble oat bran). Degrees of synergy (oligosaccharide yield obtained with the enzyme combination divided by the sum of yields obtained with individual enzymes) of up to 88 were obtained. Combinations of a ferulic acid esterase and xylanases resulted in synergy on all substrates, while a laccase and xylanases only acted synergistically on the more recalcitrant substrates. Synergy between different xylanases (glycoside hydrolase (GH) families 5 and 11) was observed particularly on oat hulls, producing a yield of 57%. The synergistic ability of the enzymes was found to be partly due to the increased enzyme stability when in combination with the substrates.

Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products.

The genus Cohnella belongs to a group of Gram-positive endospore-forming bacteria within the Paenibacillaceae family. Although most species were described as xylanolytic bacteria, the literature still lacks some key information regarding their repertoire of xylan-degrading enzymes. The whole genome sequence of an isolated xylan-degrading bacterium Cohnella sp. strain AR92 was found to contain five genes encoding putative endo-1,4-ß-xylanases, of which four were cloned, expressed, and characterized to better understand the contribution of the individual endo-xylanases to the overall xylanolytic properties of strain AR92. Three of the enzymes, CoXyn10A, CoXyn10C, and CoXyn11A, were shown to be effective at hydrolyzing xylans-derived from agro-industrial, producing oligosaccharides with substrate conversion values of 32.5%, 24.7%, and 10.6%, respectively, using sugarcane bagasse glucuronoarabinoxylan and of 29.9%, 19.1%, and 8.0%, respectively, using wheat bran-derived arabinoxylan. The main reaction products from GH10 enzymes were xylobiose and xylotriose, whereas CoXyn11A produced mostly xylooligosaccharides (XOS) with 2 to 5 units of xylose, often substituted, resulting in potentially prebiotic arabinoxylooligosaccharides (AXOS). The endo-xylanases assay displayed operational features (temperature optima from 49.9 to 50.4 °C and pH optima from 6.01 to 6.31) fitting simultaneous xylan utilization. Homology modeling confirmed the typical folds of the GH10 and GH11 enzymes, substrate docking studies allowed the prediction of subsites (- 2 to + 1 in GH10 and - 3 to + 1 in GH11) and identification of residues involved in ligand interactions, supporting the experimental data. Overall, the Cohnella sp. AR92 endo-xylanases presented significant potential for enzymatic conversion of agro-industrial by-products into high-value products.Key points• Cohnella sp. AR92 genome encoded five potential endo-xylanases.• Cohnella sp. AR92 enzymes produced xylooligosaccharides from xylan, with high yields.• GH10 enzymes from Cohnella sp. AR92 are responsible for the production of X2 and X3 oligosaccharides.• GH11 from Cohnella sp. AR92 contributes to the overall xylan degradation by producing substituted oligosaccharides.

Exploring Codon Adjustment Strategies towards Escherichia coli-Based Production of Viral Proteins Encoded by HTH1, a Novel Prophage of the Marine Bacterium Hypnocyclicus thermotrophus.

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.

Chemical and biochemical bleaching of oat hulls: The effect of hydrogen peroxide, laccase, xylanase and sonication on optical properties and chemical composition.

Oat hulls are an excellent dietary fibre source for food supplements due to their rich lignocellulose composition as well as their great abundance as low-value agricultural side stream. For the production of white fibre supplements, a mild, but effective bleaching of the hulls is required. Chemical bleaching with hydrogen peroxide and sodium hydroxide was here found to be a suitable method increasing the CIE L* value (corresponds to a lightness value) above 85. The developed method is mild, retaining the hull's chemical composition. Only a minor decrease in coniferaldehyde structures upon bleaching was detected. Colour and chemical variabilities of oat hulls from different growth seasons did not influence the required bleaching conditions to achieve the desired optical properties. The inclusion of biochemical bleaching steps utilizing the xylanase Pentopan Mono BG, the laccase NS51003 and sonication was industrially not feasible as they could not reduce the required amount of subsequently applied bleaching chemicals significantly.

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases.

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d/l-, α/ß-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.

Xylooligosaccharides Increase Bifidobacteria and Lachnospiraceae in Mice on a High-Fat Diet, with a Concomitant Increase in Short-Chain Fatty Acids, Especially Butyric Acid.

Effects of xylooligosaccharides (XOSs) as well as a mixture of XOS, inulin, oligofructose, and partially hydrolyzed guar gum (MIX) in mice fed a high-fat diet (HFD) were studied. Control groups were fed an HFD or a low-fat diet. Special attention was paid to the cecal composition of the gut microbiota and formation of short-chain fatty acids, but metabolic parameters were also documented. The XOS group had significantly higher cecum levels of acetic, propionic, and butyric acids than the HFD group, and the butyric acid content was higher in the XOS than in the MIX group. The cecum microbiota of the XOS group contained more Bifidobacteria, Lachnospiraceae, and S24-7 bacteria than the HFD group. A tendency of lower body weight gain was observed on comparing the XOS and HFD groups. In conclusion, the XOS was shown to be a promising prebiotic candidate. The fiber diversity in the MIX diet did not provide any advantages compared to the XOS diet.

Microwave-assisted xylanase reaction: impact in the production of prebiotic xylooligosaccharides.

The enzymatic production of prebiotic xylooligosaccharides (XOS) has become an attractive way to valorise lignocellulosic biomass. However, despite numerous xylanases reported for potential use in the production of XOS, most of the family GH10 also produce xylose. This monosaccharide can negatively affect the selectivity to stimulate the growth of intestinal microorganisms beneficial to human health. In this work, thermostable alkali-tolerant xylanase (BhXyn10A) from Bacillus halodurans S7 has been used to produce XOS under conventional convective heat transfer and microwave radiation. The microwave-assisted reaction markedly decreases the xylose content in the hydrolysates and significantly increases the yield of XOS, compared to conventional heating. Molecular dynamics simulations of BhXyn10A have shown increased fluctuations of the amino acids of the aglycone subsites suggesting that these subsites can determine the production of xylose. Thus, microwave heating could affect the amino acid fluctuations in the aglycone subsites reducing the xylose formation. These findings open up new avenues in enzyme technology for the production of XOS.

Identification of Phlorotannins in the Brown Algae, Saccharina latissima and Ascophyllum nodosum by Ultra-High-Performance Liquid Chromatography Coupled to High-Resolution Tandem Mass Spectrometry.

Phlorotannins are bioactive polyphenols in brown macroalgae that make these algae interesting as healthy food. Specific phlorotannins are, however, seldom identified, and extracts from different species are often only analysed for total phenolic content (TPC). In this study, our focus was to identify phlorotannin molecules from Saccharina latissima and Ascophyllum nodosum (a species rich in these compounds) using ultra-high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS2). Water and ethanol (30 and 80% v/v) were used at solid:liquid ratios, extraction times and temperatures, proposed to result in high TPC in extracts from other species. The S. latissima extracts, however, did not allow phlorotannin detection by either UHPLC-UV/Vis or UHPLC-HRMS2, despite a TPC response by the Folin-Ciocalteu assay, pinpointing a problem with interference by non-phenolic compounds. Purification by solid phase extraction (SPE) led to purer, more concentrated fractions and identification of four phlorotannin species in A. nodosum and one in S. latissima by UHPLC-HRMS2, using extracts in ethanol 80% v/v at a solid:liquid ratio of 1:10 for 20 h at 25 °C with an added 10 h at 65 °C incubation of remaining solids. The phlorotannin with the formula C12H10O7 (corresponding to bifuhalol) is the first identified in S. latissima.

Taxogenomic assessment and genomic characterisation of Weissella cibaria strain 92 able to metabolise oligosaccharides derived from dietary fibres.

The importance of the gut microbiota in human health has led to an increased interest to study probiotic bacteria. Fermented food is a source of already established probiotics, but it also offers an opportunity to discover new taxa. Four strains of Weissella sp. isolated from Indian fermented food have been genome sequenced and classified into the species W. cibaria based on whole-genome phylogeny. The genome of W. cibaria strain 92, known to utilise xylooligosaccharides and produce lactate and acetate, was analysed to identify genes for oligosaccharide utilisation. Clusters including genes involved in transportation, hydrolysis and metabolism of xylooligosaccharides, arabinooligosaccharides and ß-glucosides were identified. Growth on arabinobiose and laminaribiose was detected. A 6-phospho-ß-glucosidase clustered with a phosphotransferase system was found upregulated during growth on laminaribiose, indicating a mechanism for laminaribiose utilisation. The genome of W. cibaria strain 92 harbours genes for utilising the phosphoketolase pathway for the production of both acetate and lactate from pentose and hexose sugars but lacks two genes necessary for utilising the pentose phosphate pathway. The ability of W. cibaria strain 92 to utilise several types of oligosaccharides derived from dietary fibres, and produce lactate and acetate makes it interesting as a probiotic candidate for further evaluation.

Extraction and Modification of Macroalgal Polysaccharides for Current and Next-Generation Applications.

Marine macroalgal (seaweed) polysaccharides are highly promising for next-generation applications in several industries. However, despite the reported comprehensive potential of these polysaccharides, commercial products are scarce on the market. Seaweed cultivations are increasing in number and production quantity, owing to an elevated global trend of utilization interest in seaweed. The extraction of polysaccharides from seaweed generally generates low yields, but novel methods are being developed to facilitate and improve the extraction processes. Current areas of applications for seaweed polysaccharides mainly take advantage of the physicochemical properties of certain polysaccharides, such as gelling, thickening and emulsifying. However, many of the numerous bioactivities reported are still only at research level and lack clinical evidence for commercialization. It has been suggested the construction of smaller units may generate better defined molecules that are more suitable for biomedical applications. Enzymatic modification is a promising tool for the generation of more defined, targeted biomolecules. This review covers; structural differences between the most predominant marine algal polysaccharides, extraction processes, modification alternatives, as well as a summary of current and potential next-generation application areas.