To compare xylans from forestry with agricultural origins, hardwood xylan (birch) and cereal arabinoxylan (rye) were hydrolyzed using two variants of the xylanase RmXyn10A, full-length enzyme and ...catalytic module only, from Rhodothermus marinus. Cultivations of four selected bacterial species, using the xylooligosaccharide (XOS) containing hydrolysates as carbon source, showed selective growth of Lactobacillus brevis DSMZ 1264 and Bifidobacterium adolescentis ATCC 15703. Both strains were confirmed to utilize the XOS fraction (DP 2–5), whereas putative arabinoxylooligosaccharides from the rye arabinoxylan hydrolysate were utilized by only B. adolescentis. Escherichia coli did not grow, despite its capability to grow on the monosaccharides arabinose and xylose. It was also shown that Pediococcus parvulus strain 2.6 utilized neither xylose nor XOS for growth. In summary, RmXyn10A or its catalytic module proved suitable for high-temperature hydrolysis of hardwood xylan and cereal arabinoxylan, producing XOS that could qualify as prebiotics for use in functional food products.
We present here a series of thermoresponsive glycopolymers in the form of poly(N-isopropylacrylamide)-co-(2-β-mannooligosyloxy ethyl methacrylate)s. These copolymers were prepared from ...oligo-β-mannosyl ethyl methacrylates that were synthesized through enzymatic catalysis, and were subsequently investigated with respect to their aggregation and phase behavior in aqueous solution using a combination of 1H NMR spectroscopy, dynamic light scattering, cryogenic transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The thermoresponsive glycopolymers were prepared by conventional free radical copolymerization of different mixtures of 2-(β-mannooligosyloxy)ethyl methacrylates (with either one or two saccharide units) and N-isopropylacrylamide (NIPAm). The results showed that below the lower critical solution temperature (LCST) of poly(NIPAm), the glycopolymers readily aggregate into nanoscale structures, partly due to the presence of the saccharide moieties. Above the LCST of poly(NIPAm), the glycopolymers rearrange into a heterogeneous mixture of fractal and disc/globular aggregates. Cryo-TEM and SAXS data demonstrated that the presence of the pendant β-mannosyl moieties in the glycopolymers induces a gradual conformational change over a wide temperature range. Even though the onset of this transition is not different from the LCST of poly(NIPAm), the gradual conformational change offers a variation of the temperature-dependent properties in comparison to poly(NIPAm), which displays a sharp coil-to-globule transition. Importantly, the compacted form of the glycopolymers shows a larger colloidal stability compared to the unmodified poly(NIPAm). In addition, the thermoresponsiveness can be conveniently tuned by varying the sugar unit-length and the oligo-β-mannosyl ethyl methacrylate content.
The recent years have witnessed considerable developments in the interpretation of the three-dimensional structures of plant polysaccharide-degrading enzymes in the context of their functional ...specificity. A plethora of new structures of catalytic, carbohydrate-binding and protein-scaffolding modules involved in (hemi)cellulose catabolism has emerged in harness with sophisticated biochemical analysis. Despite significant advances, a full understanding of the intricacies of substrate recognition and catalysis by these diverse and specialised enzymes remains an important goal, especially if the application potential of these biocatalysts is to be fully realised.
Barley husks, rye bran, and a fiber residue from oat milk production were processed by heat pretreatment, various separation steps, and treatment with an endoxylanase in order to improve the ...prebiotic potential of these cereal byproducts. Metabolic functions were intended to improve along with improved microbial activity. The products obtained were included in a high-fat mouse diet so that all diets contained 5% dietary fiber. In addition, high-fat and low-fat controls as well as partially hydrolyzed guar gum were included in the study. The soluble fiber product obtained from rye bran caused a significant increase in the bifidobacteria (log copies of 16S rRNA genes; median (25–75 percentile): 6.38 (6.04–6.66) and 7.47 (7.30–7.74), respectively; p < 0.001) in parallel with a tendency of increased production of propionic acid and indications of improved metabolic function compared with high-fat fed control mice. The oat-derived product caused an increase in the pool of cecal propionic (from 0.62 ± 0.12 to 0.94 ± 0.08) and butyric acid (from 0.38 ± 0.04 to 0.60 ± 0.04) compared with the high-fat control, and it caused a significant increase in lactobacilli (log copies of 16S rRNA genes; median (25–75 percentile): 6.83 (6.65–7.53) and 8.04 (7.86–8.33), respectively; p < 0.01) in the cecal mucosa. However, no changes in measured metabolic parameters were observed by either oat or barley products.
Heat treatment of barley husks was performed to extract arabinoxylan with high yield and high weight-averaged molecular mass (
M
W). Microwave irradiation was employed for initial screening of ...suitable residence times (2–15
min), temperatures (120–210
°C) and initial pH (3–13) of the reaction slurry. Microwave irradiation was shown to be a good method for predicting the effects of heat treatment on a larger scale using steam pretreatment. A
M
W of about 40,000
Da was achieved without the addition of chemicals, by both microwave irradiation and steam pretreatment, with a yield of about 9%. The yield was significantly increased by slightly increasing the severity factor. However, the
M
W decreased below 20,000
Da at severity factors above 3.7. Arabinosyl side groups were enzymatically hydrolysed from the arabinoxylan by α-
l-arabinofuranosidase to a degree of 47%, demonstrating the ability to specifically alter the side group substitution of arabinoxylans with the use of enzymes.
Endo-β(1 → 4)-mannanases (endomannanases) catalyse degradation of β-mannans, an abundant class of plant polysaccharides. This study investigates structural features and substrate binding of ...YpenMan26A, a non-CBM carrying endomannanase from Yunnania penicillata. Structural and sequence comparisons to other fungal family GH26 endomannanases showed high sequence similarities and conserved binding residues, indicating that fungal GH26 endomannanases accommodate galactopyranosyl units in the -3 and -2 subsites. Two striking amino acid differences in the active site were found when the YpenMan26A structure was compared to a homology model of Wsp.Man26A from Westerdykella sp. and the sequences of nine other fungal GH26 endomannanases. Two YpenMan26A mutants, W110H and D37T, inspired by differences observed in Wsp.Man26A, produced a shift in how mannopentaose bound across the active site cleft and a decreased affinity for galactose in the -2 subsite, respectively, compared to YpenMan26A. YpenMan26A was moreover found to have a flexible surface loop in the position where PansMan26A from Podospora anserina has an α-helix (α9) which interacts with its family 35 CBM. Sequence alignment inferred that the core structure of fungal GH26 endomannanases differ depending on the natural presence of this type of CBM. These new findings have implications for selecting and optimising these enzymes for galactomannandegradation.
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/β‐pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.
Although glycosylation is common in Nature, it remains a challenge for organic chemistry. Herein, a method to obtain transglycosylases from retaining glycoside hydrolases is described. The approach pinpoints 6–12 single‐point mutants to assay. Invariably across six GH families, this generates enzyme variants that display increased transglycosylation yields, an improvement that is mostly due to a relative decrease in hydrolysis compared to transglycosylation. The method only requires sequence information, is fast and applicable to a broad range of glycosides.
To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 ...endo-β-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite −2 and Phe325Ala at subsite −3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite −2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 Å resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite −2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of 18O-labeled products obtained using H2 18O as a solvent. The results obtained with oligosacharide substrates show that although the catalytic efficiency (k cat/K m) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite −2 (due to the introduced Arg323) and demotion of it at subsite −3 (to which removal of Phe325 contributed). However, k cat/K m decreased ∼1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite −3 and beyond for the engineered enzyme.
Components of dissolved organic matter (DOM) span from sub-nm molecules to colloidal aggregates of several hundred nm. The colloidal fraction is important for the transport of organic matter and ...associated elements in the environment, and for the stability of DOM constituents with respect to microbial decomposition. This study focuses on the colloidal properties of DOM extracted from spruce forest soils of a chronosequence. The DOM samples were obtained by common water extraction procedures at 21 and 100°C, respectively. We applied an experimental approach combining chemical analysis with light and X-ray scattering techniques that informed on the colloidal size, charge, and structure of DOM. Results showed that two main types of colloids were present: semi-flexible cylinders and fractal aggregates. The cylinders consisted of carbohydrates, presumably hemicelluloses, while the aggregates were a composite material containing a large fraction of carbohydrates together with aliphatics and clay particles. These fractal aggregates dominated the cold-water extracts whereas the strong increase in total organic carbon by hot-water extraction caused a concomitantly strong increase of semi-flexible cylinders, which became the predominant species. Comparison between the chronosequence soils showed that with increasing forest age, the amount of carbon extracted per gram of soil declined and the concentration of the semi-flexible cylinders decreased. Thus, the distribution between the fractal aggregates and cylinders in the forest soil DOM samples depends on the composition of the soil organic matter and the leaching temperature. Changes in this distribution may have important implications for the reactivity and stability of DOM colloids.
PDF
