Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.
Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.
endo-1,4-β-Xylanase (EC 18.104.22.168) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.
Evidence for temporal regulation of the two Pseudomonas cellulosa xylanases belonging to glycoside hydrolase family 11.
Emami, K., Nagy, T., Fontes, C. M. G. A., Ferreira, L. M.A. & Gilbert, H. J. (2002). Journal of Bacteriology, 184(15), 4124-4133.
Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium. Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized. The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively. In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan. Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound. xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or β-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes. Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media. Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes. Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone. A transposon mutant of P. cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P. cellulosa. Based on the data presented here, we propose a model for xylan degradation by P. cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes.
Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases.
Cuyvers, S., Dornez, E., Rezaei, M. N., Pollet, A., Delcour, J. A. & Courtin, C. M. (2011). FEBS Journal, 278(7), 1098–1111.
The secondary substrate binding site (SBS) of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased Kd values by up to 70-fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates.
Comparison of prebiotic effects of arabinoxylan oligosaccharides and inulin in a simulator of the human intestinal microbial ecosystem.
Grootaert, C., Van den Abbeele, P., Marzorati, M., Broekaert, W. F., Courtin, C. M., Delcour, J. A., Verstraete, W. & Van de Wiele, T. (2009). FEMS Microbiology Ecology, 69(2), 231–242.
In this study, the prebiotic potential of arabinoxylan oligosaccharides (AXOS) was compared with inulin in two simulators of the human intestinal microbial ecosystem. Microbial breakdown of both oligosaccharides and short-chain fatty acid production was colon compartment specific, with ascending and transverse colon being the predominant site of inulin and AXOS degradation, respectively. Lactate levels (+5.5 mM) increased in the ascending colon during AXOS supplementation, while propionate levels (+5.1 mM) increased in the transverse colon. The concomitant decrease in lactate in the transverse colon suggests that propionate was partially formed over the acrylate pathway. Furthermore, AXOS supplementation strongly decreased butyrate in the ascending colon, this in parallel with a decrease in Roseburia spp. and Bacteroides/Prevotella/Porphyromonas (−1.4 and −2.0 log CFU) levels. Inulin treatment had moderate effects on lactate, propionate and butyrate levels. Denaturing gradient gel electrophoresis analysis revealed that inulin changed microbial metabolism by modulating the microbial community composition. In contrast, AXOS primarily affected microbial metabolism by ‘switching on’ AXOS-degrading enzymes (xylanase, arabinofuranosidase and xylosidase), without significantly affecting microbial community composition. Our results demonstrate that AXOS has a higher potency than inulin to shift part of the sugar fermentation toward the distal colon parts. Furthermore, due to its stronger propionate-stimulating effect, AXOS is a candidate prebiotic capable of lowering cholesterol and beneficially affecting fat metabolism of the host.
Arabinoxylan‐oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the simulator of human intestinal microbial ecosystem.
Sanchez, J. I., Marzorati, M., Grootaert, C., Baran, M., Van Craeyveld, V., Courtin, C. M., Broekaet, W. F. Declour, J. A.,Verstraete, W. & Van de Wiele, T. (2009). Microbial Biotechnology, 2(1), 101-113.
Arabinoxylan‐oligosaccharides (AXOS) are a recently newly discovered class of candidate prebiotics as – depending on their structure – they are fermented in different regions of gastrointestinal tract. This can have an impact on the protein/carbohydrate fermentation balance in the large intestine and, thus, affect the generation of potentially toxic metabolites in the colon originating from proteolytic activity. In this study, we screened different AXOS preparations for their impact on the in vitro intestinal fermentation activity and microbial community structure. Short‐term fermentation experiments with AXOS with an average degree of polymerization (avDP) of 29 allowed part of the oligosaccharides to reach the distal colon, and decreased the concentration of proteolytic markers, whereas AXOS with lower avDP were primarily fermented in the proximal colon. Additionally, prolonged supplementation of AXOS with avDP 29 to the Simulator of Human Intestinal Microbial Ecosystem (SHIME) reactor decreased levels of the toxic proteolytic markers phenol and p‐cresol in the two distal colon compartments and increased concentrations of beneficial short‐chain fatty acids (SCFA) in all colon vessels (25–48%). Denaturant gradient gel electrophoresis (DGGE) analysis indicated that AXOS supplementation only slightly modified the total microbial community, implying that the observed effects on fermentation markers are mainly caused by changes in fermentation activity. Finally, specific quantitative PCR (qPCR) analysis showed that AXOS supplementation significantly increased the amount of health‐promoting lactobacilli as well as of Bacteroides–Prevotella and Clostridium coccoides–Eubacterium rectale groups. These data allow concluding that AXOS are promising candidates to modulate the microbial metabolism in the distal colon.
Microbial community development in a dynamic gut model is reproducible, colon region specific, and selective for bacteroidetes and clostridium cluster IX.
Van den Abbeele, P., Grootaert, C., Marzorati, M., Possemiers, S., Verstraete, W., Gérard, P., Rabot, S., Bruneau, A., El Aidy, S., Derrien, M., Zoetendal, E., Kleerebezem, M., Smidt, H. & Van de Wiele, T. (2010). Applied and Environmental Microbiology, 76(15), 5237-5246.
Dynamic, multicompartment in vitro gastrointestinal simulators are often used to monitor gut microbial dynamics and activity. These reactors need to harbor a microbial community that is stable upon inoculation, colon region specific, and relevant to in vivo conditions. Together with the reproducibility of the colonization process, these criteria are often overlooked when the modulatory properties from different treatments are compared. We therefore investigated the microbial colonization process in two identical simulators of the human intestinal microbial ecosystem (SHIME), simultaneously inoculated with the same human fecal microbiota with a high-resolution phylogenetic microarray: the human intestinal tract chip (HITChip). Following inoculation of the in vitro colon compartments, microbial community composition reached steady state after 2 weeks, whereas 3 weeks were required to reach functional stability. This dynamic colonization process was reproducible in both SHIME units and resulted in highly diverse microbial communities which were colon region specific, with the proximal regions harboring saccharolytic microbes (e.g., Bacteroides spp. and Eubacterium spp.) and the distal regions harboring mucin-degrading microbes (e.g., Akkermansia spp.). Importantly, the shift from an in vivo to an in vitro environment resulted in an increased Bacteroidetes/Firmicutes ratio, whereas Clostridium cluster IX (propionate producers) was enriched compared to clusters IV and XIVa (butyrate producers). This was supported by proportionally higher in vitro propionate concentrations. In conclusion, high-resolution analysis of in vitro-cultured gut microbiota offers new insight on the microbial colonization process and indicates the importance of digestive parameters that may be crucial in the development of new in vitro models.
Glycoside Hydrolases from a targeted Compost Metagenome, activity-screening and functional characterization.
Dougherty, M. J., D’haeseleer P., Hazen, T. C., Simmons, B. A., Adams, P. D. & Hadi, M. Z. (2012). BMC Biotechnology, 12, 38.
Background: Metagenomics approaches provide access to environmental genetic diversity for biotechnology applications, enabling the discovery of new enzymes and pathways for numerous catalytic processes. Discovery of new glycoside hydrolases with improved biocatalytic properties for the efficient conversion of lignocellulosic material to biofuels is a critical challenge in the development of economically viable routes from biomass to fuels and chemicals. Results: Twenty-two putative ORFs (open reading frames) were identified from a switchgrass-adapted compost community based on sequence homology to related gene families. These ORFs were expressed in E. coli and assayed for predicted activities. Seven of the ORFs were demonstrated to encode active enzymes, encompassing five classes of hemicellulases. Four enzymes were over expressed in vivo, purified to homogeneity and subjected to detailed biochemical characterization. Their pH optima ranged between 5.5 - 7.5 and they exhibit moderate thermostability up to ~60-70°C. Conclusions: Seven active enzymes were identified from this set of ORFs comprising five different hemicellulose activities. These enzymes have been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering towards the goal of developing efficient enzyme cocktails for biomass degradation under diverse process conditions.
Hemicellulase production in Chrysosporium lucknowense C1.
Hinz, S. W. A., Pouvreau, L., Joosten, R., Bartels, J., Jonathan, M. C., Wery, J. & Schols, H. A. (2009). Journal of Cereal Science, 50(3), 318-323.
Filamentous fungi are widely used for enzyme production for the biofuel industry. The ascomycetous fungus Chrysosporium lucknowense C1 was isolated as a natural producer of neutral cellulases. It is at present an attractive alternative to well known fungi like Aspergillus sp. and Trichoderma reesei for protein production on a commercial scale. Besides many cellulases, a large number of hemicellulases (particularly xylanases and arabinofuranosidases) and esterases (acetyl xylan esterases and ferulic acid esterases) encoding genes have also been identified in the C1 genome. Many of these extracellular enzymes have been selectively expressed in C1 and then purified and characterized. Four arabinofuranosidases, two acetyl xylan esterases, two ferulic acid esterases, an α-glucuronidase and four xylanases have been purified and characterized. All these enzymes were found to be active towards arabinoxylans, demonstrating the high potential of C1 as a producer of hemicellulolytic enzymes.
Impact of cell wall‐degrading enzymes on water‐holding capacity and solubility of dietary fibre in rye and wheat bran.
Petersson, K., Nordlund, E., Tornberg, E., Eliasson, A. C., & Buchert, J. (2013). Journal of the Science of Food and Agriculture, 93(4), 882-889.
BACKGROUND: Rye and wheat bran were treated with several xylanases and endoglucanases, and the effects on physicochemical properties such as solubility, viscosity, water-holding capacity and particle size as well as the chemical composition of the soluble and insoluble fractions of the bran were studied. A large number of enzymes with well-defined activities were used. This enabled a comparison between enzymes of different origins and with different activities as well as a comparison between the effects of the enzymes on rye and wheat bran. RESULTS: The xylanases derived from Bacillus subtilis were the most effective in solubilising dietary fibre from wheat and rye bran. There was a tendency for a higher degree of degradation of the soluble or solubilised dietary fibre in rye bran than in wheat bran when treated with most of the enzymes. CONCLUSION: None of the enzymes increased the water-holding capacity of the bran or the viscosity of the aqueous phase. The content of insoluble material decreased as the dietary fibre was solubilised by the enzymes. The amount of material that may form a network to retain water in the system was thereby decreased.
Role of N-linked glycosylation in the enzymatic properties of a thermophilic GH 10 xylanase from Aspergillus fumigatus expressed in Pichia pastoris.
Chang, X., Xu, B., Bai, Y., Luo, H., Ma, R., Shi, P. & Yao, B. (2017). PloS One, 12(2), e0171111.
N-Glycosylation is a posttranslational modification commonly occurred in fungi and plays roles in a variety of enzyme functions. In this study, a xylanase (Af-XYNA) of glycoside hydrolase (GH) family 10 from Aspergillus fumigatus harboring three potential N-glycosylation sites (N87, N124 and N335) was heterologously produced in Pichia pastoris. The N-glycosylated Af-XYNA (WT) exhibited favorable temperature and pH optima (75°C and pH 5.0) and good thermostability (maintaining stable at 60°C). To reveal the role of N-glycosylation on Af-XYNA, the enzyme was deglycosylated by endo-β-N-acetylglucosaminidase H (DE) or modified by site-directed mutagenesis at N124 (N124T). The deglycosylated DE and mutant N124T showed narrower pH adaptation range, lower specific activity, and worse pH and thermal stability. Further thermodynamic analysis revealed that the enzyme with higher N-glycosylation degree was more thermostable. This study demonstrated that the effects of glycosylation at different degrees and sites were diverse, in which the glycan linked to N124 played a key role in pH and thermal stability of Af-XYNA.
Management of enzyme diversity in high-performance cellulolytic cocktails.
Reyes-Sosa, F. M., Morales, M. L., Gómez, A. I. P., Crespo, N. V., Zamorano, L. S., Rocha-Martín, J., Molina-Heredia, F. P. & García, B. D. (2017). Biotechnology for Biofuels, 10(1), 156.
Background: Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes. Results: One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC–MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail. Conclusions: These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.