| 61. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
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A carbohydrate binding module as a diversity-carrying scaffold
- 2004
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
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| 62. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
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Engineered xyloglucan specificity in a carbohydrate-binding module
- 2006
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 16:12, s. 1171-1180
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Tidskriftsartikel (refereegranskat)abstract
- The field of plant cell wall biology is constantly growing and consequently so is the need for more sensitive and specific probes for individual wall components. Xyloglucan is a key polysaccharide widely distributed in the plant kingdom in both structural and storage tissues that exist in both fucosylated and non-fucosylated variants. Presently, the only xyloglucan marker available is the monoclonal antibody CCRC-M1 that is specific to terminal alpha-1,2-linked fucosyl residues on xyloglucan oligo- and polysaccharides. As a viable alternative to searches for natural binding proteins or creation of new monoclonal antibodies, an approach to select xyloglucan-specific binding proteins from a combinatorial library of the carbohydrate-binding module, CBM4-2, from xylanase Xyn10A of Rhodothermus marinus is described. Using phage display technology in combination with a chemoenzymatic method to anchor xyloglucan to solid supports, the selection of xyloglucan-binding modules with no detectable residual wild-type xylan and beta-glucan-binding ability was achieved.
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| 63. |
- Hassan, Noor, 1977-
(författare)
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Characterization and engineering of carbohydrate-active enzymes for biotechnological applications
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extremozymes are enzymes produced by microorganisms that live in extreme habitats. Due to their higher stability, extremozymes is attracting interest as biocatalysts in various industrial processes. In this context, carbohydrate-active extremozymes can be used in various processes relevant to the paper, food and feed industry.In this thesis, the crystal structure, biochemical characterization and the capacity to synthesize prebiotic galacto-oligosaccharides (GOS) were investigated for a β-glucosidase (HoBGLA) from the halothermophilic bacterium Halothermothrix orenii. The wild-type enzyme displays favorable characteristics for lactose hydrolysis and produces a range of prebiotic GOS, of which β-D-Galp-(1→6)-D-Lac and β-D-Galp-(1→3)-D-Lac are the major products (Paper I).To further improve GOS synthesis by HoBGLA, rational enzyme engineering was performed (Paper II). Six enzyme variants were generated by replacing strategically positioned active-site residues. Two HoBGLA variants were identified as potentially interesting, F417S and F417Y. The former appears to synthesize one particular GOS product in higher yield, whereas the latter produces a higher yield of total GOS.In Paper III, the high-resolution crystal structure and biochemical characterization of a hemicellulase (HoAraf43) from H. orenii is presented. HoAraf43 folds as a five-bladed β-propeller and displays α-Larabinofuranosidase activity. The melting temperature of HoAraf43 increases significantly in the presence of high salt and divalent cations, which is consistent with H. orenii being a halophile.Furthermore, the crystal structures of a thermostable tetrameric pyranose 2-oxidase from Phanerochaete chrysosporium (PcP2O) were determined to investigate the structural determinants of thermostability (Paper IV). PcP2O has an increased number of salt links between subunits, which may provide a mechanism for increased stability. The structures also imply that the N-terminal region acts as an intramolecular chaperone during homotetramer assembly.
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| 64. |
- Hero, Johan S., et al.
(författare)
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Endo-xylanases from Cohnella sp. AR92 aimed at xylan and arabinoxylan conversion into value-added products
- 2021
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 1432-0614 .- 0175-7598. ; 105:18, s. 6759-6778
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Tidskriftsartikel (refereegranskat)abstract
- 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-β-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.
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| 65. |
- Hreggvidsson, Gudmundur O, et al.
(författare)
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Biocatalytic refining of polysaccharides from brown seaweeds
- 2020. - 1
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Ingår i: Sustainable Seaweed Technologies : Cultivation, Biorefinery and Applications - Cultivation, Biorefinery and Applications. - 9780128179444 - 9780128179437 ; , s. 447-504
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Bokkapitel (refereegranskat)abstract
- Brown macroalgae constitute 40% of the global production of seaweed, corresponding to approximately 10 million tonnes annually. Traditionally, seaweeds have been the source of hydrocolloids, food, and feed products. Due to possibilities for large-scale farming, brown macroalgae are a biomass with considerable potential for increased utilization. The main constituent polysaccharides, being alginate, cellulose, laminaran, and fucoidan, are the components of greatest importance for biorefinery usage. The polysaccharides can be extracted and applied for their physical or bioactive properties or used as a carbon source for microbial conversions to biofuels and commodity chemicals. The structural complexity and heterogeneous sugar composition of the polysaccharides make them a challenging biorefinery feedstock. These challenges can be overcome by the increasingly innovative biocatalytic tools, enzymes and microbes, that are being developed and that can be expected to open new opportunities and expand the product portfolio. However, there are still knowledge gaps, and further understanding is required on the molecular level of these interesting polymers, the tools, the refining possibilities, as well as transforming this knowledge to innovations—processes and products.
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| 66. |
- Huang, Fang, et al.
(författare)
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A mixture of Nordic berries improves cognitive function, metabolic function and alters the gut microbiota in C57Bl/6J male mice
- 2023
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Ingår i: Frontiers in Nutrition. - 2296-861X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Our diets greatly influence our health. Multiple lines of research highlight the beneficial properties of eating berries and fruits. In this study, a berry mixture of Nordic berries previously identified as having the potential to improve memory was supplemented to young C57Bl/6J male mice to investigate effects on cognition function, metabolic health, markers of neuroinflammation, and gut microbiota composition. C57Bl/6J male mice at the age of 8 weeks were given standard chow, a high-fat diet (HF, 60%E fat), or a high-fat diet supplemented with freeze-dried powder (20% dwb) of a mixture of Nordic berries and red grape juice (HF + Berry) for 18 weeks ( n = 12 animals/diet group). The results show that supplementation with the berry mixture may have beneficial effects on spatial memory, as seen by enhanced performance in the T-maze and Barnes maze compared to the mice receiving the high-fat diet without berries. Additionally, berry intake may aid in counteracting high-fat diet induced weight gain and could influence neuroinflammatory status as suggested by the increased levels of the inflammation modifying IL-10 cytokine in hippocampal extracts from berry supplemented mice. Furthermore, the 4.5-month feeding with diet containing berries resulted in significant changes in cecal microbiota composition. Analysis of cecal bacterial 16S rRNA revealed that the chow group had significantly higher microbial diversity, as measured by the Shannon diversity index and total operational taxonomic unit richness, than the HF group. The HF diet supplemented with berries resulted in a strong trend of higher total OTU richness and significantly increased the relative abundance of Akkermansia muciniphila, which has been linked to protective effects on cognitive decline. In conclusion, the results of this study suggest that intake of a Nordic berry mixture is a valuable strategy for maintaining and improving cognitive function, to be further evaluated in clinical trials.
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| 67. |
- Huang, Fang, et al.
(författare)
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Cultivation of the gut bacterium Prevotella copri DSM 18205T using glucose and xylose as carbon sources
- 2021
-
Ingår i: MicrobiologyOpen. - : Wiley. - 2045-8827. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Prevotella copri DSM18205T is a human gut bacterium, suggested as a next-generation probiotic. To utilize it as such, it is, however, necessary to grow the species in a reproducible manner. Prevotella copri has previously been reported to be highly sensitive to oxygen, and hence difficult to isolate and cultivate. This study presents successful batch cultivation strategies for viable strain inoculations and growth in both serum bottles and a stirred tank bioreactor (STR), without the use of an anaerobic chamber, as long as the cells were kept in the exponential growth phase. A low headspace volume in the STR was important to reach high cell density. P. copri utilized xylose cultivated in Peptone Yeast Xylose medium (PYX medium), resulting in a comparable growth rate and metabolite production as in Peptone Yeast Glucose medium (PYG medium) in batch cultivations at pH 7.2.Up to 5 g/L of the carbon source was consumed, leading to the production of succinic acid, acetic acid, and formic acid, and cell densities (OD620 nm ) in the range 6-7.5. The highest yield of produced succinic acid was 0.63 ± 0.05 g/g glucose in PYG medium cultivations and 0.88 ± 0.06 g/g xylose in PYX medium cultivations.
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| 68. |
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| 69. |
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| 70. |
- Immerstrand, Tina, et al.
(författare)
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Characterization of the properties of Pediococcus parvulus for probiotic or protective culture use.
- 2010
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Ingår i: Journal of Food Protection. - 0362-028X. ; 73:5, s. 960-966
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Tidskriftsartikel (refereegranskat)abstract
- Pediococcus parvulus 2.6 (previously Pediococcus damnosus 2.6, here confirmed as P. parvulus by 16S DNA sequencing) displayed antibacterial activity toward several bacterial species, including isolates found as contaminants in oats, herein genetically identified as Bacillus cereus. No inhibition of Listeria monocytogenes was found under the conditions used. Antibacterial activity was retrieved after ammonium sulfate or acetone precipitation showed it to be peptide mediated. P. parvulus 2.6 has previously shown good technological properties in oat-based products. This, together with the currently found inhibition of food spoilage microorganisms like B. cereus, makes it suitable as a food protective culture. Survival trials of P. parvulus 2.6 at conditions mimicking the gastrointestinal tract were prompted by previously found cholesterol-lowering effects in humans after consumption of oat products cofermented by using P. parvulus 2.6 and Bifidobacterium spp. Viability was measured with in vitro, gutlike simulations at 37 degrees C. High survival was shown under two of three conditions (gastric juice, bile, and small intestine juice), defined as main obstacles of the gastrointestinal tract. The critical step was bile exposure. At a concentration of 20%, viability was low, but 0.3% bile (mean concentration in the intestine) did not have a major influence on growth. Viability of P. parvulus 2.6 was significantly decreased in gastric juice at pH 1.5 (with pepsin), but it was not significantly affected at pH 2.5, and was also improved at a lower pH in 20% oat milk. Viability was judged sufficient for colonization at gutlike conditions, qualifying the strain for further probiotic studies.
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