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 220.127.116.11) 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.
A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.
McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.
The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.
Purification and Characterization of a Thermostable β-mannanase from Bacillus subtilis BE-91: Potential Application in Inflammatory Diseases.
Cheng, L., Duan, S., Feng, X., Zheng, K., Yang, Q. & Liu, Z. (2016). BioMed Research International, Article ID 6380147.
β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The β-mannanase activity was significantly enhanced in the presence of Mn+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+ and strongly inhibited by Ba2+, and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 μmol/min/mL versus 1.749 mg/mL and 33.45 µmol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.
Immobilization and stabilization of commercial β-1,4-endoxylanase DepolTM 333MDP by multipoint covalent attachment for xylan hydrolysis: Production of prebiotics (xylo-oligosaccharides).
Martins de Oliveira, S., Moreno-Perez, S., Romero-Fernández, M., Fernandez-Lorente, G., Rocha-Martin, J. & Guisan, J. M. (2017). Biocatalysis and Biotransformation, 1-10.
The commercial enzyme DepolTM 333MDP (D333MDP) was immobilized by multipoint covalent attachment onto 10% cross-linked agarose beads support highly activated with aldehyde groups. The enzyme immobilization process was very efficient, retaining 86% of its initial catalytic activity. Thermal stability of the immobilized D333MDP biocatalysts varied according to the incubation time of the enzyme-support. The optimal immobilized biocatalyst was produced after 24 h of incubation under alkaline conditions and longer incubation times resulted in a loss of stability. The optimal immobilized biocatalyst was 60- and 50-fold more stable at pH 5.5 and pH 7 at 50°C than the soluble enzyme, respectively. Activity and stability at pH 5.5 were enhanced when the optimal immobilized biocatalyst was modified by chemical amination of the enzyme surface. The chemical amination of the immobilized enzyme surface was 5-fold more stable at pH 5.5 and 50°C compared with the unmodified immobilized biocatalyst. The best immobilized biocatalysts (containing 100 UI/g of support) were evaluated in the beechwood xylan hydrolysis reaction at 50°C and pH 5.5. 80% of the reducing sugars were released after 6 h of hydrolysis with the aminated biocatalyst. Xylan hydrolysis reaction with the aminated biocatalyst was 80% faster than with the non-aminated one. The final composition of the xylooligosaccharides (XOS) obtained was identified and quantified by HPAEC-PAD which showed it was composed of 90% of xylobiose and 5% of xylotriose and xylose. The aminated immobilized-stabilized biocatalyst was used for four cycles of hydrolysis with no loss of catalytic activity, resulting in highly active and stable derivative suitable for industrial processes.
Improvement of the catalytic characteristics of a salt-tolerant GH10 xylanase from Streptomyce rochei L10904.
Li, Q., Sun, B., Li, X., Xiong, K., Xu, Y., Yang, R., Hou, J. & Teng, C. (2017). International Journal of Biological Macromolecules, 107, 1447-1455.
A GH10 xylanase Srxyn10 from Streptomyce rochei L10904, and its truncated derivative, Srxyn10M, were investigated. Both displayed great salt-tolerant ability, retaining more than 95% and 91% activity after incubation at 37°C for 1 h in 3.0 M and 5.0 M NaCl, respectively. They exhibited a special hydrolytic property of forming xylobiose as the major product and produced fewer xylose compounds when combined with a reported xylanase while digesting corncob xylans. The mutant, Srxyn10M, was constructed from Srxyn10 by deleting the C-terminal carbohydrate-binding module. It possessed a 3.26-fold higher specific activity on beechwood xylan than Srxyn10. Moreover, Srxyn10M showed greater substrate affinity and catalytic efficiency than Srxyn10 when beechwood xylan, birchwood xylan, and oat-spelt xylan were used as substrates. The thermostability was also greatly improved. Therefore, the application potential was markedly enhanced by the improvement of these properties.
Influence of viscosity on the growth of human gut microbiota.
Tamargo, A., Cueva, C., Álvarez, M. D., Herranz, B., Bartolomé, B., Moreno-Arribas, M. V. & Laguna, L. (2017). Food Hydrocolloids, In Press.
Numerous studies support the beneficial effects of dietary fibre. It is well known that fibre increases viscosity at intestinal level. Therefore, the effects of fibre on gut microbiota could be due not only by its intestinal bacteria fermentation but also to the increase in viscosity by itself. The aim of this study was to evaluate the effect of viscosity on the growth of gut microbiota at physiological conditions. For this purpose, four compartments from a gastrointestinal simulator (simgi®) were filled with Gut Nutrient Medium (GNM) plus different agar concentrations (0, 0.30, 0.45 and 0.60%), inoculated with faecal microbiota, and incubated 48 h under anaerobic conditions. Samples were collected at three time points (0, 24 h and 48 h) for representative intestinal bacterial enumeration and rheological characterization. Incubation of GNM gels with faecal microbiota changed the medium viscosity over time, even with constant conditions (temperature and pH). In such way that, in absence of agar (low viscosity), viscosity slightly increased over time; however, in viscous mediums, viscosity decreased over time. In relation to the growth of gut microbiota, results showed that viscosity favoured the growth of total anaerobes and Clostridium spp.; in contrast, total number of aerobes and members of the genus Enterococcus correlated negatively with viscosity increment. In conclusion, changes in intestinal viscosity seem to selectively modify microbiota composition. This is a pioneer work to understand the effect of food viscosity in the gastrointestinal system, showing that viscosity is an important factor itself to condition the growth of different bacteria’s groups.
Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-β-Xylanase from Caldicellulosiruptor owensensis.
Liu, X., Liu, T., Zhang, Y., Xin, F., Mi, S., Wen, B., Gu, T., Xinyuan Shi, X., Wang, F. & Sun, L. (2017). Journal of agricultural and food chemistry, 66(1), pp 187-193.
Xylanases (EC 18.104.22.168) are a kind of enzymes degrading xylan to xylooligosaccharides (XOS) and have been widely used in a variety of industrial applications. Among them, xylanases from thermophilic microorganisms have distinct advantages in industries that require high temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10 xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90°C with a half-life of about 1 h at 80°C and exhibited highest activity at pH 7.0. The activity of CoXynA activity was affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA was proposed to be due to the increased overall protein rigidity resulting from the reduced length and fluctuation of Loop 7.
Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.
Sporck, D., Reinoso, F. A. M., Rencoret, J., Gutiérrez, A., Rio, J. C., Ferraz, A. & Milagres, A. M. F. (2017). Biotechnology for Biofuels, 10(1), 296.
Background: New biorefnery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis. Results: Sugarcane bagasse was pretreated with an alkaline-sulfte chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of O-methyl-glucuronic acid and p-coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio. Conclusions: Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefning. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.
Expressing accessory proteins in cellulolytic Yarrowia lipolytica to improve the conversion yield of recalcitrant cellulose.
Guo, Z. P., Duquesne, S., Bozonnet, S., Nicaud, J. M., Marty, A. & O’Donohue, M. J. (2017). Biotechnology for Biofuels, 10(1), 298.
Background: A recently constructed cellulolytic Yarrowia lipolytica is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from Trichoderma reesei in order to enhance the degradation of recalcitrant substrate. Results: The production of EG I was enhanced using a promoter engineering strategy. This provided a new cellulolytic Y. lipolytica strain, which compared to the parent strain, exhibited higher hydrolytic activity on different cellulosic substrates. Furthermore, three accessory proteins, TrXYNII, TrLPMOA and TrSWO, were individually expressed in cellulolytic and non-cellulolytic Y. lipolytica. The amount of rhTrXYNII and rhTrLPMOA secreted by non-cellulolytic Y. lipolytica in YTD medium during batch cultivation in flasks was approximately 62 and 52 mg/L, respectively. The purified rhTrXYNII showed a specific activity of 532 U/mg-protein on beechwood xylan, while rhTrLPMOA exhibited a specific activity of 14.4 U/g-protein when using the Amplex Red/horseradish peroxidase assay. Characterization of rhTrLPMOA revealed that this protein displays broad specificity against β-(1,4)-linked glucans, but is inactive on xylan. Further studies showed that the presence of TrLPMOA synergistically enhanced enzymatic hydrolysis of cellulose by cellulases, while TrSWO1 boosted cellulose hydrolysis only when it was applied before the action of cellulases. The presence of rTrXYNII enhanced enzymatic hydrolysis of an industrial cellulose pulp and of wheat straw. Co-expressing TrXYNII and TrLPMOA in cellulolytic Y. lipolytica with enhanced EG I production procured a novel engineered Y. lipolytica strain that displayed enhanced ability to degrade both amorphous (CIMV-cellulose) and recalcitrant crystalline cellulose in complex biomass (wheat straw) by 16 and 90%, respectively. Conclusions: This study has provided a potent cellulose-degrading Y. lipolytica strain that co-expresses a core set of cellulolytic enzymes and some accessory proteins. Results reveal that the tuning of cellulase production and the production of accessory proteins leads to optimized performance. Accordingly, the beneficial effect of accessory proteins for cellulase-mediated degradation of cellulose is underlined, especially when crystalline cellulose and complex biomass are used as substrates. Findings specifically underline the benefits and specific properties of swollenin. Although in our study swollenin clearly promoted cellulase action, its use requires process redesign to accommodate its specific mode of action.
Analyzing Xyloglucan Endotransglycosylases by Incorporation of Synthetic Oligosaccharides into Plant Cell Walls.
Ruprecht, C., Dallabernardina, P., Smith, P. J., Urbanowicz, B. R. & Pfrengle, F. (2018). ChemBioChem, In Press.
The plant cell wall is a cellular exoskeleton consisting predominantly of a complex polysaccharide network that defines the shape of cells. During growth, this network can be loosened through the action of Xyloglucan Endo-Transglycosylases (XETs), glycoside hydrolases that 'cut and paste' xyloglucan polysaccharides through a transglycosylation process. We have analyzed cohorts of XETs in different plant species to evaluate xyloglucan acceptor substrate specificities using a set of synthetic oligosaccharides obtained by automated glycan assembly. The ability of XETs to incorporate the oligosaccharides into polysaccharides printed as microarrays and into stem sections of Arabidopsis thaliana, beans, and peas was assessed. We found that single xylose substitutions are sufficient for transfer, and xylosylation of the terminal glucose residue is not required by XETs, independently of plant species. To obtain some information on the potential xylosylation pattern of the natural acceptor of XETs, i.e. the non-reducing end of xyloglucan, we further tested the activity of xyloglucan xylosyl transferase (XXT) 2 on the synthetic xyloglucan oligosaccharides. This data sheds light on inconsistencies between previous studies towards determining the acceptor substrate specificities of XETs and have important implications for further understanding plant cell wall polysaccharide synthesis and remodeling.
Double blind microarray-based polysaccharide profiling enables parallel identification of uncharacterized polysaccharides and carbohydrate-binding proteins with unknown specificities.
Salmeán, A. A., Guillouzo, A., Duffieux, D., Jam, M., Matard-Mann, M., Larocque, R., Pedersen, H. L., Michel, G., Czjzek, M., Willats, W. G. T. & Hervé, C. (2018). Scientific Reports, 8(1), 2500.
Marine algae are one of the largest sources of carbon on the planet. The microbial degradation of algal polysaccharides to their constitutive sugars is a cornerstone in the global carbon cycle in oceans. Marine polysaccharides are highly complex and heterogeneous, and poorly understood. This is also true for marine microbial proteins that specifically degrade these substrates and when characterized, they are frequently ascribed to new protein families. Marine (meta)genomic datasets contain large numbers of genes with functions putatively assigned to carbohydrate processing, but for which empirical biochemical activity is lacking. There is a paucity of knowledge on both sides of this protein/carbohydrate relationship. Addressing this ‘double blind’ problem requires high throughput strategies that allow large scale screening of protein activities, and polysaccharide occurrence. Glycan microarrays, in particular the Comprehensive Microarray Polymer Profiling (CoMPP) method, are powerful in screening large collections of glycans and we described the integration of this technology to a medium throughput protein expression system focused on marine genes. This methodology (Double Blind CoMPP or DB-CoMPP) enables us to characterize novel polysaccharide-binding proteins and to relate their ligands to algal clades. This data further indicate the potential of the DB-CoMPP technique to accommodate samples of all biological sources.
The composition of accessory enzymes of Penicillium chrysogenum P33 revealed by secretome and synergistic effects with commercial cellulase on lignocellulose hydrolysis.
Yang, Y., Yang, J., Liu, J., Wang, R., Liu, L., Wang, F. & Yuan, H. (2018). Bioresource Technology, In Press.
Herein, we report the secretome of Penicillium chrysogenum P33 under induction of lignocellulose for the first time. A total of 356 proteins were identified, including complete cellulases and numerous hemicellulases. Supplementing a commercial cellulase with increasing dosage of P33 enzyme cocktail from 1-5 mg/g substrate increased the release of reducing sugars from delignified corn stover by 21.4% to 106.8%. When 50% cellulase was replaced by P33 enzyme cocktail, release of reducing sugars was 78.6% higher than with cellulase alone. Meanwhile, glucan and xylan conversion was increased by 37% and 106%, respectively. P33 enzyme cocktail also enhanced commercial cellulase hydrolysis against four different delignified lignocellulosic biomass. These findings demonstrate that mixing appropriate amount of P33 cocktail with cellulase improves polysaccharide hydrolysis, suggesting P33 enzymes have great potential for industrial applications.
Expression and characterization of the processive exo‐β‐1,4‐cellobiohydrolase SCO6546 from Streptomyces coelicolor A (3).
Lee, C. R., Chi, W. J., Lim, J. H., Dhakshnamoorthy, V. & Hong, S. K. (2018). Journal of Basic Microbiology, In Press.
The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50°C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters kcat and KM, for cellotetraose at pH 5.0 and 50°C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 22.214.171.124), acting on nonreducing end of cellulose.