| 1. |
- Aevarsson, Arnthór, et al.
(författare)
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Going to extremes - a metagenomic journey into the dark matter of life
- 2021
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968. ; 368:12
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Forskningsöversikt (refereegranskat)abstract
- The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.
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| 2. |
- Ara, Kazi Zubaida Gulshan, et al.
(författare)
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Engineering CGTase to improve synthesis of alkyl glycosides
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:5, s. 603-612
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Tidskriftsartikel (refereegranskat)abstract
- Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyse the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.
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| 3. |
- 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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| 4. |
- Khaleghipour, Leila, et al.
(författare)
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Extraction of sugarcane bagasse arabinoxylan, integrated with enzymatic production of xylo-oligosaccharides and separation of cellulose
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Sugarcane processing roughly generates 54 million tonnes sugarcane bagasse (SCB)/year, making SCB an important material for upgrading to value-added molecules. In this study, an integrated scheme was developed for separating xylan, lignin and cellulose, followed by production of xylo-oligosaccharides (XOS) from SCB. Xylan extraction conditions were screened in: (1) single extractions in NaOH (0.25, 0.5, or 1 M), 121 °C (1 bar), 30 and 60 min; (2) 3 × repeated extraction cycles in NaOH (1 or 2 M), 121 °C (1 bar), 30 and 60 min or (3) pressurized liquid extractions (PLE), 100 bar, at low alkalinity (0-0.1 M NaOH) in the time and temperature range 10-30 min and 50-150 °C. Higher concentration of alkali (2 M NaOH) increased the xylan yield and resulted in higher apparent molecular weight of the xylan polymer (212 kDa using 1 and 2 M NaOH, vs 47 kDa using 0.5 M NaOH), but decreased the substituent sugar content. Repeated extraction at 2 M NaOH, 121 °C, 60 min solubilized both xylan (85.6% of the SCB xylan), and lignin (84.1% of the lignin), and left cellulose of high purity (95.8%) in the residuals. Solubilized xylan was separated from lignin by precipitation, and a polymer with β-1,4-linked xylose backbone substituted by arabinose and glucuronic acids was confirmed by FT-IR and monosaccharide analysis. XOS yield in subsequent hydrolysis by endo-xylanases (from glycoside hydrolase family 10 or 11) was dependent on extraction conditions, and was highest using xylan extracted by 0.5 M NaOH, (42.3%, using Xyn10A from Bacillus halodurans), with xylobiose and xylotriose as main products. The present study shows successful separation of SCB xylan, lignin, and cellulose. High concentration of alkali, resulted in xylan with lower degree of substitution (especially reduced arabinosylation), while high pressure (using PLE), released more lignin than xylan. Enzymatic hydrolysis was more efficient using xylan extracted at lower alkaline strength and less efficient using xylan obtained by PLE and 2 M NaOH, which may be a consequence of polymer aggregation, via remaining lignin interactions.
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| 5. |
- Li, Xiaoya, et al.
(författare)
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Synthesis, Enzymatic Degradation, and Polymer-Miscibility Evaluation of Nonionic Antimicrobial Hyperbranched Polyesters with Indole or Isatin Functionalities
- 2021
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 22:5, s. 2256-2271
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Tidskriftsartikel (refereegranskat)abstract
- Most macromolecular antimicrobials are ionic and thus lack miscibility/compatibility with nonionic substrate materials. In this context, nonionic hyperbranched polyesters (HBPs) with indole or isatin functionality were rationally designed, synthesized, and characterized. Antimicrobial disk diffusion assay indicated that these HBPs showed significant antibacterial activity against 8 human pathogenic bacteria compared to small molecules with indole or isatin groups. According to DSC measurements, up to 20% indole-based HBP is miscible with biodegradable polyesters (polyhydroxybutyrate or polycaprolactone), which can be attributed to the favorable hydrogen bonding between the N-H moiety of indole and the C=O of polyesters. HBPs with isatin or methylindole were completely immiscible with the same matrices. None of the HBPs leaked out from plastic matrix after being immersed in water for 5 days. The incorporation of indole into HBPs as well as small molecules facilitated their enzymatic degradation with PETase from Ideonella sakaiensis, while isatin had a complex impact. Molecular docking simulations of monomeric molecules with PETase revealed different orientations of the molecules at the active site due to the presence of indole or isatin groups, which could be related to the observed different enzymatic degradation behavior. Finally, biocompatibility analysis with a mammalian cell line showed the negligible cytotoxic effect of the fabricated HBPs.
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| 6. |
- Linares-Pastén, Javier A., et al.
(författare)
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Modeled 3D-Structures of Proteobacterial Transglycosylases from Glycoside Hydrolase Family 17 Give Insight in Ligand Interactions Explaining Differences in Transglycosylation Products
- 2021
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Ingår i: Applied Sciences (Switzerland). - : MDPI AG. - 2076-3417. ; 11:9
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Tidskriftsartikel (refereegranskat)abstract
- The structures of glycoside hydrolase family 17 (GH17) catalytic modules from modular proteins in the ndvB loci in Pseudomonas aeruginosa (Glt1), P. putida (Glt3) and Bradyrhizobium diazoefficiens (previously B. japonicum) (Glt20) were modeled to shed light on reported differences between these homologous transglycosylases concerning substrate size, preferred cleavage site (from reducing end (Glt20: DP2 product) or non-reducing end (Glt1, Glt3: DP4 products)), branching (Glt20) and linkage formed (1,3-linkage in Glt1, Glt3 and 1,6-linkage in Glt20). Hybrid models were built and stability of the resulting TIM-barrel structures was supported by molecular dynamics simulations. Catalytic amino acids were identified by superimposition of GH17 structures, and function was verified by mutagenesis using Glt20 as template (i.e., E120 and E209). Ligand docking revealed six putative subsites (−4, −3, −2, −1, +1 and +2), and the conserved interacting residues suggest substrate binding in the same orientation in all three transglycosylases, despite release of the donor oligosaccharide product from either the reducing (Glt20) or non-reducing end (Glt1, Gl3). Subsites +1 and +2 are most conserved and the difference in release is likely due to changes in loop structures, leading to loss of hydrogen bonds in Glt20. Substrate docking in Glt20 indicate that presence of covalently bound donor in glycone subsites −4 to −1 creates space to accommodate acceptor oligosaccharide in alternative subsites in the catalytic cleft, promoting a branching point and formation of a 1,6-linkage. The minimum donor size of DP5, can be explained assuming preferred binding of DP4 substrates in subsite −4 to −1, preventing catalysis.
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| 7. |
- Linares-Pastén, Javier A, et al.
(författare)
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Novel xylan-degrading enzymes from polysaccharide utilizing loci of Prevotella copri DSM18205
- 2021
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423. ; 31:10, s. 1330-1349
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Tidskriftsartikel (refereegranskat)abstract
- Prevotella copri is a bacterium that can be found in the human gastrointestinal tract (GIT). The role of P. copri in the GIT is unclear, and elevated numbers of the microbe have been reported both in dietary fiber-induced improvement in glucose metabolism but also in conjunction with certain inflammatory conditions. These findings raised our interest in investigating the possibility of P. copri to grow on xylan, and identify the enzyme systems playing a role in digestion of xylan-based dietary fibers. Two xylan degrading polysaccharide utilizing loci (PUL10 and 15) were found in the genome, with three and eight glycoside hydrolase (GH) -encoding genes, respectively. Three of them were successfully produced in Escherichia coli: One extracellular enzyme from GH43 (subfamily 12, in PUL10, 60 kDa) and two enzymes from PUL15, one extracellular GH10 (41 kDa), and one intracellular GH43 (subfamily 137 kDa). Based on our results, we propose that in PUL15, GH10 (1) is an extracellular endo-1,4-β-xylanase, that hydrolazes mainly glucuronosylated xylan polymers to xylooligosaccharides (XOS); while, GH43-1 in the same PUL, is an intracellular β-xylosidase, catalyzing complete hydrolysis of the XOS to xylose. In PUL10, the characterized GH43-12 is an arabinofuranosidase, with a role in degradation of arabinoxylan, catalyzing removal of arabinose-residues on xylan.
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| 8. |
- Mobarec, Hugo, et al.
(författare)
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Microwave-assisted xylanase reaction : impact in the production of prebiotic xylooligosaccharides
- 2021
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Ingår i: RSC Advances. - 2046-2069. ; 11:20, s. 11882-11888
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Ngo, Ngoc T. N., et al.
(författare)
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Synthesis of novel oligomeric anionic alkyl glycosides using laccase/TEMPO oxidation and cyclodextrin glucanotransferase (CGTase)‐catalysed transglycosylation
- 2021
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 1097-0290 .- 0006-3592. ; 118:7, s. 2548-2558
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Tidskriftsartikel (refereegranskat)abstract
- Modification of alkyl glycosides, to alter their properties and widen the scope of potential applications, is of considerable interest. Here, we report the synthesis of new anionic alkyl glycosides with long carbohydrate chains, using two different approaches: laccase/TEMPO oxidation of a long‐carbohydrate‐chain alkyl glycoside, and cyclodextrin glucanotransferase (CGTase)‐catalysed elongation of anionic alkyl glycosides. The laccase/TEMPO oxidation of dodecyl β‐D‐maltooctaoside proceeded efficiently with the formation of aldehyde and acid products. However, depolymerization occurred to a large extent, limiting the product yield and purity. On the other hand, CGTase‐catalysed coupling/disproportionation reactions with α‐cyclodextrin and dodecyl β‐D‐maltoside diuronic acid (DDM‐2COOH) or octyl β‐D‐glucuronic acid (OG‐COOH) as substrates gave high conversions, especially when the CGTase Toruzyme was used. It was found that pH had a strong influence on both the enzyme activity and the acceptor specificity. With non‐ionic substrates (dodecyl β‐D‐maltoside and octyl β‐D‐glucoside), Toruzyme exhibited high catalytic activity at pH 5‐6, but for the acidic substrates (DDM‐2COOH and OG‐COOH) the activity was highest at pH 4. This is most likely due to the enzyme favoring the protonated forms of DDM‐2COOH and OG‐COOH, which exist at lower pH (pKa about 3).
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| 10. |
- Wagner-Egea, Paula, et al.
(författare)
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Assessment of IsPETase-Assisted Depolymerization of Terephthalate Aromatic Polyesters and the Effect of the Thioredoxin Fusion Domain
- 2021
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Ingår i: Applied Sciences (Switzerland). - : MDPI AG. - 2076-3417. ; 11:18
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Tidskriftsartikel (refereegranskat)abstract
- Terephthalate polyesters such as poly(ethylene terephthalate) (PET) have been massively produced over the last few decades due to their attractive properties in multiple applications. However, due to their limited biodegradability, they have accumulated in landfills and oceans, posing an environmental threat. Enzymatic recycling technologies are predicted to generate long-term socioeconomic benefits. In the present work, we compared the IsPETase (from Ideonella sakaiensis 201-F6) activity on a series of polyesters, including poly(butylene) terephthalate (PBT), poly(hexamethylene) terephthalate (PHT) and Akestra™, with PET. The IsPETase showed remarkable activity toward PET (39% degradation of the original polyester) that was higher than that toward Akestra™ (0.13%), PBT (0.25%) and PHT (0.13%) after 72 h. Thus, based on experimental data and computational analysis, we report insights into IsPETase activity on a series of terephthalate-based polyesters. Aside from that, the fusion domain (Trx) effect in the production and activity of a recombinant Trx-IsPETase is reported.
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