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D-Xylose Assay Kit

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0:05  Introduction
0:52  Principle
1:21    Reagent Preparation
2:04  Procedure
6:04  Calculation

D-Xylose Assay Kit K-XYLOSE Scheme
Product code: K-XYLOSE

100 assays (manual) / 1000 assays (microplate) / 1300 assays (auto-analyser)

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Content: 100 assays (manual) / 1000 assays (microplate) / 1300 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: D-Xylose
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 2 to 100 μg of D-xylose per assay
Limit of Detection: 0.7 mg/L
Reaction Time (min): ~ 6 min
Application examples: Analysis of D-xylose in fermentation broths and hydrolysates of plant material and polysaccharides.
Method recognition: Novel method

The D-Xylose test kit is a novel method for the specific, convenient and rapid measurement and analysis of D-xylose in plant extracts, culture media/supernatants and other materials.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

View our full range of monosaccharide assay kits.

Scheme-K-XYLOSE XYLOSE Megazyme

  • Very cost effective 
  • All reagents stable for > 2 years after preparation 
  • Only enzymatic kit available 
  • Rapid reaction (~ 6 min) 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included 
  • Suitable for manual, microplate and auto-analyser formats
Certificate of Analysis
Safety Data Sheet
FAQs Booklet Data Calculator Validation Report

Identifying transcription factors that reduce wood recalcitrance and improve enzymatic degradation of xylem cell wall in Populus.

Hori, C., Takata, N., Lam, P. Y., Tobimatsu, Y., Nagano, S., Mortimer, J. C. & Cullen, D. (2020). Scientific Reports, 10(1), 1-13.

Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.

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Prediction of bioactive compounds in barley by near-infrared reflective spectroscopy (NIRS).

Albanell, E., Martínez, M., De Marchi, M. & Manuelian, C. L. (2020). Journal of Food Composition and Analysis, 97, 103763.

Barley grains contain a variable amount of biologically active compounds such as non-starch polysaccharides and phenol compounds. These compounds are important in nutrition due to their significant health benefits and technological role in food. We developed predictive models for β-glucans (BG), arabinoxylans (AX), bound phenols (BP), free phenols (FP), and anthocyanins (AN) based on near-infrared spectroscopy (NIRS) using two different NIRS instruments with different spectral range and spectral steps. Regressions of modified partial least squares (MPLS) and several combinations of scattering correction and derivative treatments were tested. The optimal calibration models generated high coefficients of determination for BG and BP, but not for AN content. The instrument with the highest resolution only gave better results for BG prediction models, and the addition of the visible range did not prove to be ostensibly advantageous to the determination of any of the active compounds of study, not even in the case of AN analysis.

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Causes of vitamin K deficiency in patients on haemodialysis.

Wikstrøm, S., Aagaard Lentz, K., Hansen, D., Melholt Rasmussen, L., Jakobsen, J., Post Hansen, H. & Andersen, J. R. (2020). Nutrients, 12(9), 2513.

Background: A low vitamin K status is common in patients on haemodialysis, and this is considered one of the reasons for the accelerated atherosclerosis in these patients. The vitamin is essential in activation of the protein Matrix Gla Protein (MGP), and the inactive form, dp-ucMGP, is used to measure vitamin K status. The purpose of this study was to investigate possible underlying causes of low vitamin K status, which could potentially be low intake, washout during dialysis or inhibited absorption capacity. Moreover, the aim was to investigate whether the biomarker dp-ucMGP is affected in these patients. Method: Vitamin K intake was assessed by a Food Frequency Questionnaire (FFQ) and absorption capacity by means of D-xylose testing. dp-ucMGP was measured in plasma before and after dialysis, and phylloquinine (vitamin K1) and dp-ucMGP were measured in the dialysate. Changes in dp-ucMGP were measured after 14 days of protein supplementation. Results: All patients had plasma dp-ucMGP above 750 pmol/L, and a low intake of vitamin K. The absorption capacity was normal. The difference in dp-ucMGP before and after dialysis was −1022 pmol/L (p < 0.001). Vitamin K1 was not present in the dialysate but dp-ucMGP was at a high concentration. The change in dp-ucMGP before and after protein supplementation was −165 pmol/L (p = 0.06). Conclusion: All patients had vitamin K deficiency. The reason for the low vitamin K status is not due to removal of vitamin K during dialysis or decreased absorption but is plausibly due to a low intake of vitamin K in food. dp-ucMGP is washed out during dialysis, but not affected by protein intake to a clinically relevant degree.

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Identification of enzymatic genes with the potential to reduce biomass recalcitrance through lignin manipulation in Arabidopsis.

Sakamoto, S., Kamimura, N., Tokue, Y., Nakata, M. T., Yamamoto, M., Hu, S., Masai, E., Mitsuda, N. & Kajita, S. (2020). Biotechnology for Biofuels, 13(97), 1-16.

Background: During the chemical and biochemical decomposition of lignocellulosic biomasses, lignin is highly recalcitrant. Genetic transformation of plants to qualitatively and/or quantitatively modify lignin may reduce these recalcitrant properties. Efficient discovery of genes to achieve lignin manipulation is thus required. Results:To screen for new genes to reduce lignin recalcitrance, we heterologously expressed 50 enzymatic genes under the control of a cinnamate 4-hydroxylase (C4H) gene promoter, derived from a hybrid aspen, which is preferentially active in tissues with lignified cell walls in Arabidopsis plants. These genes encode enzymes that act on metabolites in shikimate, general phenylpropanoid, flavonoid, or monolignol biosynthetic pathways. Among these genes, 30, 18, and 2 originated from plants, bacteria, and fungi, respectively. In our first screening step, 296 independent transgenic plants (T1 generation) harboring single or multiple transgenes were generated from pools of seven Agrobacterium strains used for conventional floral-dip transformation. Wiesner and Mäule staining patterns in the stems of the resultant plants revealed seven and nine plants with apparent abnormalities in the two respective staining analyses. According to genomic PCR and subsequent direct sequencing, each of these 16 plants possessed a gene encoding either coniferaldehyde dehydrogenase (calB), feruloyl-CoA 6′-hydroxylase (F6H1), hydroxycinnamoyl-CoA hydratase/lyase (couA), or ferulate 5-hydroxylase (F5H), with one transgenic plant carrying both calB and F6H1. The effects of these genes on lignin manipulation were confirmed in individually re-created T1 transgenic Arabidopsis plants. While no difference in lignin content was detected in the transgenic lines compared with the wild type, lignin monomeric composition was changed in the transgenic lines. The observed compositional change in the transgenic plants carrying calB, couA, and F5H led to improved sugar release from cell walls after alkaline pretreatment. Conclusions:Simple colorimetric characterization of stem lignin is useful for simultaneous screening of many genes with the potential to reduce lignin recalcitrance. In addition to F5H, the positive control, we identified three enzyme-coding genes that can function as genetic tools for lignin manipulation. Two of these genes (calB and couA) accelerate sugar release from transgenic lignocelluloses.

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Determining different impact factors on the xylonic acid production using Gluconobacter oxydans DSM 2343.

Hahn, T., Torkler, S., van Der Bolt, R., Gammel, N., Hesse, M., Möller, A., Preylowski, B., Hubracht, v., patzsch, k. & Zibek, S. (2020). Process Biochemistry, 94, 172-179.

Xylonic acid is a promising compound for the substitution of gluconic acid. Gluconobacter oxydans DSM 2343 has proven to be a highly potent biocatalyst for the conversion of xylose to xylonic acid. In the present study, different nitrogen sources for the growth of G. oxydans and subsequent xylonic acid production were investigated for the first time with minimal medium. Application of 0.32 g/L glutamate supplemented with 0.15 g/L ammonium sulfate as a cheap nitrogen source enabled a xylonic acid productivity of 2.92 g/(Lh) which is similar to findings involving a complex medium (3.20 g/(Lh)). The study further investigated the impact of the xylose source on the growth and production of G. oxydans. Dose-response curves confirmed that G. oxydans is mainly insensitive towards the main inhibitory compounds, acetate and hydroxymethylfurfural, up to a concentration of 5 g/L and 2.5 g/L, respectively. However, batch investigations indicated that substitution of 25 % of the pure xylose with hemicellulosic xylose resulted in a xylonic acid yield of 90 % compared to the control approach without hemicellulosic xylose. The feeding of hemicellulosic xylose in a pulsed fed-batch mode even enabled the use of 50 g/L demonstrating that the proper selection of a feeding strategy for the hemicellulosic xylose greatly improves the production of xylonic acid.

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Isolation, characterization, and application of thermotolerant Streptomyces sp. K5 for efficient conversion of cellobiose to chitinase using pulse-feeding strategy.

2021-01-272021-01-27Mutturi, S., Ike, M., Yamagishi, K. & Tokuyasu, K. (2020). Process Biochemistry, 94, 58-65.

In this study, the selection of target sugars in lignocellulosic hydrolysate and the implementation of a bioprocess strategy led to efficient production of a catalyst for chitinous bioresource conversion. Streptomyces sp. K5, a chitinase producer in the presence of glucose, was isolated at 50 °C on an agar plate with glucose and colloidal chitin. The K5 was found to produce chitinase with a maximum activity of 70 U/L in a medium containing glucose and xylose as well as colloidal chitin as an inducer. The assimilation of glucose and xylose, however, was extremely slow, with significant residual being present even after 168 h of incubation. Assimilation tests of glucose, xylose, and cellobiose confirmed that K5 produces chitinase without chitinous inducer and assimilates cellobiose much more rapidly than either glucose or xylose. At the same time, the complete exhaustion of each sugar initiated chitinase inactivation. A pulse- feeding strategy was adopted for the cellobiose, taking its rapid assimilation, β-glucosidase activity, and chitinase inactivation into account, and a maximum chitinase activity of 235 U/L was achieved under pulse- feeding conditions that included four pulses.

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Application of an orcinol-ferric chloride colorimetric assay in barley and wheat accessions for water-extractable and total arabinoxylan.

Hu, G., Ellberg, S., Burton, C., Evans, C., Satterfield, K. & Bockelman, H. (2020). Journal of Cereal Science, 93, 102962.

Arabinoxylan is an important hemicellulose potentially affecting wheat baking qualities, barley malt quality, and may impart prebiotic benefits. Water-extractable (WEAX) and total arabinoxylan (TOAX) were characterized in 204 wheat and barley accessions, respectively, using an orcinol-ferric chloride assay. WEAX measurement accuracy was optimum when glucose concentration was greater than 12–13 times pentose concentration. The modified method removed enough excess glucose spectral absorbance to make the corrected and uncorrected lines different, but still significantly correlated (p = 0.009, R = 0.94). Mean WEAX (expressed as percentage WEAX of TOAX) for the wheat accessions was 15.90%, ranging from 8.82% to 24.87%, and for barley accessions WEAX the mean was 7.10%, and ranged from 2.98% to 13.86%. Conclusions are 1) the assay is useful for a breeding program because of its semi-high throughput design for the simultaneous analysis of 16 (WEAX) to 40 (TOAX) barley lines or 24 (WEAX) to 40 (TOAX) wheat lines, 2) the trichromatic measurement reduces the impact of glucose, and 3) the broad range of WEAX measured showed that barley and wheat accessions vary in their extractable and unextractable components.

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Tailored and synergistic enzyme-assisted extraction of carotenoid-containing chromoplasts from tomatoes.

Lombardelli, C., Liburdi, K., Benucci, I. & Esti, M. (2020). Food and Bioproducts Processing, 121, 43-53.

Despite significant efforts recently made to improve the recovery yield of food pigments from natural sources, the development of green and sustainable biotechnological approaches is currently under investigation. Within the context of circular economy, food wastes represent a cheap source for the recovery of valuable compounds including food ingredients. In this study, a conservative approach consisting in a tailored enzyme-assisted extraction protocol for the recovery of carotenoid-containing chromoplasts from unsold tomatoes in which lycopene is stable and protected against oxidation, has now been developed. A tailored enzymatic mix based on polygalacturonase, pectin lyase, cellulase and xylanase, has been designed taking into account the polysaccharide composition of the tomato cell wall. The optimal process conditions for enhancing the recovery of carotenoids from tomatoes i.e.; temperature, pH, enzymatic mix of total dosage and processing time, have been investigated. The suitable temperature and pH identified by the RSM analysis, were found to be: 45-55°C at pH 5-5.5. The treatment carried out using the total dosage of 25 U/g for 180 min was the most convenient for maximizing the recovery yield [about 4.30 ± 0.08 (mgLyc/Kgtomato)/U as carotenoid-containing chromoplasts and about 5.43 ± 0.04 (mgLyc/Kgtomato)/U as total carotenoids].

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Fed-batch enzymatic hydrolysis of alkaline organosolv-pretreated corn stover facilitating high concentrations and yields of fermentable sugars for microbial lipid production.

Gong, Z., Wang, X., Yuan, W., Wang, Y., Zhou, W., Wang, G. & Liu, Y. (2020). Biotechnology for Biofuels, 13(1), 13.

Background: Lignocellulosic biomass has been commonly regarded as a potential feedstock for the production of biofuels and biochemicals. High sugar yields and the complete bioconversion of all the lignocellulosic sugars into valuable products are attractive for the utilization of lignocelluloses. It is essential to pretreat and hydrolyze lignocelluloses at high solids loadings during industrial processes, which is more economical and environmentally friendly as capital cost, energy consumption, and water usage can be reduced. However, oligosaccharides are inevitably released during the high solids loading enzymatic hydrolysis and they are more recalcitrant than monosaccharides for microorganisms. Results: A fed-batch enzymatic hydrolysis of corn stover pretreated by the sodium hydroxide-methanol solution (SMs) at high solids loading was demonstrated to reach the high concentrations and yields of fermentable sugars. Glucose, xylose, cello-oligosaccharides, and xylo-oligosaccharides achieved 146.7 g/L, 58.7 g/L, 15.6 g/L, and 24.7 g/L, respectively, when the fed-batch hydrolysis was started at 12% (w/v) solids loading, and 7% fresh substrate and a standardized blend of cellulase, β-glucosidase, and hemicellulase were fed consecutively at 3, 6, 24, and 48 h to achieve a final solids loading of 40% (w/v). The total conversion of glucan and xylan reached 89.5% and 88.5%, respectively, when the oligosaccharides were taken into account. Then, a fed-batch culture on the hydrolysates was investigated for lipid production by Cutaneotrichosporon oleaginosum. Biomass, lipid content, and lipid yield were 50.7 g/L, 61.7%, and 0.18 g/g, respectively. The overall consumptions of cello-oligosaccharides and xylo-oligosaccharides reached 74.1% and 68.2%, respectively. Conclusions: High sugars concentrations and yields were achieved when the enzyme blend was supplemented simultaneously with the substrate at each time point of feeding during the fed-batch enzymatic hydrolysis. Oligosaccharides were co-utilized with monosaccharides during the fed-batch culture of C. oleaginosum. These results provide a promising strategy to hydrolyze alkaline organosolv-pretreated corn stover into fermentable sugars with high concentrations and yields for microbial lipid production.

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Vegetable wastes derived polysaccharides as natural eco-friendly plasticizers of sodium alginate.

Di Donato, P., Taurisano, V., Poli, A., d’Ayala, G. G., Nicolaus, B., Malinconinco, M. & Santagata, G. (2020). Carbohydrate polymers, 229, 115427.

In this paper, lemon and fennel wastes were recovered and used as secondary-raw polysaccharide sources. These polysaccharides were exploited as natural plasticizers of sodium alginate (A) based films, in order to improve sodium alginate performances, limited by its fragility, extending its potential application in a cost effective and eco-friendly way. Different green processes, such as maceration (MAC), ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE), were carried out for obtaining high yield of lemon and fennel polysaccharides (LP and FP). Actually, HPAE-PAD and TLC analyses evidenced the presence of xyslose, galactose, glucose and rhamnose monomers and galacturonic acid, typical of polysaccharides like pectin and xyloglucan chains. These findings were confirmed by NMR and FTIR spectroscopic analyses. Moreovers, gel filtration chromatography assessed the high molecular weight of recovered polysaccharides, particularly of FP waste fraction. The extracted polysaccharides were used as eco-friendly and cost-effective plasticizers of sodium alginate films (AFP and ALP). DSC analysis evidenced a significant decreasing of glass transition temperature of the polymer, tensile tests showed an enlightened rising of elongation at break and TGA analysis showed a faster degradation kinetics of AFP and ALP films, as expected in a plasticized system.

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Comparison of Japanese and Indian intestinal microbiota shows diet-dependent interaction between bacteria and fungi.

Pareek, S., Kurakawa, T., Das, B., Motooka, D., Nakaya, S., Rongsen-Chandola, T. et al. (2019). NPJ Biofilms and Microbiomes, 5(1), 1-13.

The bacterial species living in the gut mediate many aspects of biological processes such as nutrition and activation of adaptive immunity. In addition, commensal fungi residing in the intestine also influence host health. Although the interaction of bacterium and fungus has been shown, its precise mechanism during colonization of the human intestine remains largely unknown. Here, we show interaction between bacterial and fungal species for utilization of dietary components driving their efficient growth in the intestine. Next generation sequencing of fecal samples from Japanese and Indian adults revealed differential patterns of bacterial and fungal composition. In particular, Indians, who consume more plant polysaccharides than Japanese, harbored increased numbers of Prevotella and CandidaCandida spp. showed strong growth responses to the plant polysaccharide arabinoxylan in vitro. Furthermore, the culture supernatants of Candida spp. grown with arabinoxylan promoted rapid proliferation of Prevotella copri. Arabinose was identified as a potential growth-inducing factor in the Candida culture supernatants. Candida spp. exhibited a growth response to xylose, but not to arabinose, whereas P. copri proliferated in response to both xylose and arabinose. Candida spp., but not P. copri, colonized the intestine of germ-free mice. However, P. copri successfully colonized mouse intestine already harboring Candida. These findings demonstrate a proof of concept that fungal members of gut microbiota can facilitate a colonization of the intestine by their bacterial counterparts, potentially mediated by a dietary metabolite.

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Co-fermentation of xylose and glucose from ionic liquid pretreated sugar cane bagasse for bioethanol production using engineered xylose assimilating yeast.

Amoah, J., Ogura, K., Schmetz, Q., Kondo, A. & Ogino, C. (2019). Biomass and Bioenergy, 128, 105283.

An efficient method to co-ferment xylose and glucose in ionic liquid was developed via effective simultaneous saccharification and fermentation of sugar cane bagasse. Fermentation of pure xylose to ethanol in ionic liquids by two engineered xylose-assimilating Saccharomyces cerevisiae yeast strains, XR-XDH (Xylose Reductase-Xylitol Dehydrogenase pathway strain) and XI (Xylose isomerase pathway strain) resulted in lower conversion compared to ionic liquid-free medium. The ionic liquids however contributed to a higher saccharification efficiency of sugar cane bagasse. Among the five ionic liquids tested, 1-butyl-3-methylpyridinium chloride ([Bmpy][Cl]) yielded the highest saccharification efficiency resulting in 0.7 g/L xylose and 2.3 g/L glucose which are 5 and 2-folds, respectively, higher than the absence of ionic liquid. The high saccharification efficiency in [Bmpy][Cl] resulted in a more efficient assimilation of xylose from sugar cane bagasse in a simultaneous saccharification and fermentation process leading to 84.0% ethanol yield compared to 26.7% produced by the reference strain in the absence of ionic liquid. This efficient method for co-fermentation of xylose and glucose to ethanol will further enhance the effective utilization of biomass as a resource for bioethanol production.

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Progress in the production of biogas from Virginia mallow after alkaline-heat pretreatment.

Nowicka, A., Zieliński, M., Dębowski, M., Dudek, M. & Rusanowska, P. (2019). Biomass and Bioenergy, 126, 174-180.

This study aimed to analyze the coupled effect of electromagnetic microwave radiation and sodium hydroxide on the structure of a lignocellulosic complex of Virginia mallow and to determine the susceptibility of the pretreated substrate to anaerobic degradation. Effects of substrate digestion with conventional and microwave heating were compared as well. NaOH dose increase from 0.02 g·gt.s−1. to 0.2 g·gt.s−1. caused biogas production to increase in both heating variants. In turn, NaOH dose increase to 0.4 g·gt.s−1. resulted in diminished biogas production in variants with both microwave and conventional heating, i.e. by 15.3% and 20.8%, respectively. The highest biogas production effectiveness was achieved during fermentation of the substrate conditioned using microwave radiation coupled with the addition of NaOH in a dose of 0.2 g·gt.s−1., and the result obtained was by 10% higher compared to the conventionally-heated sample and by 39.4% higher compared to the microwave-heated and chemically-untreated sample. The coupled use of microwave heating and alkaline treatment allowed achieving a higher volume of biogas produced. Methane content in gaseous bacterial metabolites ranged from 58 to 61%, irrespective of NaOH dose and heating method.

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Selective fluorescence labeling: time-lapse enzyme visualization during sugarcane hydrolysis.

Imai, M., Mihashi, A., Imai, T., Kimura, S., Matsuzawa, T., Yaoi, K., Shibata, N., Kakeshita, H., Igarashi, K., Kobayashi, Y. & Sugiyama, J. (2019). Journal of Wood Science, 65(1), 1-10.

Enzymatic biomass saccharification is an important process for bioethanol production. Hitherto, numerous cellulase cocktails (crude enzyme) have been developed to improve enzymatic activity. For this purpose, the synergy of incorporating hydrolase functionality within a cellulase cocktail is a key function. However, such synergistic action, by potentially numerous different enzyme types, on biomass tissue has not been considered despite the importance toward the realistic case of biomass saccharification. This study aims to visualize the behavior of each of the key cellulase components on biomass tissue during saccharification. Time-lapse fluorescence microscopy observations were conducted during saccharification of a thin transverse sugarcane section to monitor enzymes modified with a fluorescence dye. Statistical image analysis successfully demonstrated a unique adsorption/desorption behavior of each enzyme component. Particularly, the behavior of endoxylanase10 (Xyn10), which was recently discovered from Penicillium sp. as a high-performance xylanase, displayed remarkable adsorption on tissues of sugarcane, which accounts for the superior activity of the cellulase mixture with Xyn10.

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Restriction-deficient mutants and marker-less genomic modification for metabolic engineering of the solvent producer Clostridium saccharobutylicum.

Huang, C. N., Liebl, W. & Ehrenreich, A. (2018). Biotechnology for Biofuels, 11(1), 264.

Background: Clostridium saccharobutylicum NCP 262 is a solventogenic bacterium that has been used for the industrial production of acetone, butanol, and ethanol. The lack of a genetic manipulation system for C. saccharobutylicum currently limits (i) the use of metabolic pathway engineering to improve the yield, titer, and productivity of n-butanol production by this microorganism, and (ii) functional genomics studies to better understand its physiology. Results: In this study, a marker-less deletion system was developed for C. saccharobutylicum using the codBA operon genes from Clostridium ljungdahlii as a counterselection marker. The codB gene encodes a cytosine permease, while codA encodes a cytosine deaminase that converts 5-fluorocytosine to 5-fluorouracil, which is toxic to the cell. To introduce a marker-less genomic modification, we constructed a suicide vector containing: the catP gene for thiamphenicol resistance; the codBA operon genes for counterselection; fused DNA segments both upstream and downstream of the chromosomal deletion target. This vector was introduced into C. saccharobutylicum by tri-parental conjugation. Single crossover integrants are selected on plates supplemented with thiamphenicol and colistin, and, subsequently, double-crossover mutants whose targeted chromosomal sequence has been deleted were identified by counterselection on plates containing 5-fluorocytosine. Using this marker-less deletion system, we constructed the restriction-deficient mutant C. saccharobutylicum ΔhsdR1ΔhsdR2ΔhsdR3, which we named C. saccharobutylicum Ch2. This triple mutant exhibits high transformation efficiency with unmethylated DNA. To demonstrate its applicability to metabolic engineering, the method was first used to delete the xylB gene to study its role in xylose and arabinose metabolism. Furthermore, we also deleted the ptb and buk genes to create a butyrate metabolism-negative mutant of C. saccharobutylicum that produces n-butanol at high yield. Conclusions: The plasmid vectors and the method introduced here, together with the restriction-deficient strains described in this work, for the first time, allow for efficient marker-less genomic modification of C. saccharobutylicum and, therefore, represent valuable tools for the genetic and metabolic engineering of this industrially important solvent-producing organism.

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A halotolerant bifunctional β-xylosidase/α-L-arabinofuranosidase from Colletotrichum graminicola: Purification and biochemical characterization.

de Carvalho, D. R., Carli, S., Meleiro, L. P., Rosa, J. C., de Oliveira, A. H. C., Jorge, J. A. & Furriel, R. P. M. (2018). International Journal of Biological Macromolecules, 114, 741-750.

A β-xylosidase from Colletotrichum graminicola (Bxcg) was purified. The enzyme showed high halotolerance, retaining about 63% of the control activity in the presence of 2.5 mol L−1 NaCl. The presence of NaCl has not affected the optimum reaction temperature (65°C), but the optimum pH was slightly altered (from 4.5 to 5.0) at high salt concentrations. Bxcg was fully stable at 50°C for 24 h and over a wide pH range even in the presence of NaCl. In the absence of salt Bxcg hydrolyzed p-nitrophenyl-β-d-xylopyranoside with maximum velocity of 348.8 ± 11.5 U mg−1 and high catalytic efficiency (1432.7 ± 47. L mmol−1 s−1). Bxcg revealed to be a bifunctional enzyme with both β-xylosidase and α-l-arabinofuranosidase activities, and hydrolyzed xylooligosaccharides containing up to six pentose residues. The enzyme showed high synergistic effect (3.1-fold) with an endo-xylanase for the hydrolysis of beechwood xylan, either in the absence or presence of 0.5 mol L−1 NaCl, and was tolerant to different organic solvents and surfactants. This is the first report of a halotolerant bifunctional β-xylosidase/α-l-arabinofuranosidase from C. graminicola, and the enzyme showed attractive properties for application in lignocellulose hydrolysis, particularly under high salinity and/or in the presence of residues of pretreatment steps.

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Yeast lipids from cardoon stalks, stranded driftwood and olive tree pruning residues as possible extra sources of oils for producing biofuels and biochemicals.

Tasselli, G., Filippucci, S., Borsella, E., D’Antonio, S., Gelosia, M., Cavalaglio, G., Turchetti, B., Sannino, C., Onofri, A., Mastrolitti, S., De Bari, I., Cotana, F. & Bari, I. (2018). Biotechnology for Biofuels, 11(1), 147.

Background: Some lignocellulosic biomass feedstocks occur in Mediterranean Countries. They are still largely unexploited and cause considerable problems due to the lack of cost-effective harvesting, storage and disposal technologies. Recent studies found that some basidiomycetous yeasts are able to accumulate high amount of intracellular lipids for biorefinery processes (i.e., biofuels and biochemicals). Accordingly, the above biomass feedstocks could be used as carbon sources (after their pre-treatment and hydrolysis) for lipid accumulation by oleaginous yeasts. Results: Cardoon stalks, stranded driftwood and olive tree pruning residues were pre-treated with steam-explosion and enzymatic hydrolysis for releasing free mono- and oligosaccharides. Lipid accumulation tests were performed at two temperatures (20 and 25°C) using Leucosporidium creatinivorum DBVPG 4794, Naganishia adeliensis DBVPG 5195 and Solicoccozyma terricola DBVPG 5870. S. terricola grown on cardoon stalks at 20°C exhibited the highest lipid production (13.20 g/l), a lipid yield (28.95%) close to the maximum theoretical value and a lipid composition similar to that found in palm oil. On the contrary, N. adeliensis grown on stranded driftwood and olive tree pruning residues exhibited a lipid composition similar to those of olive and almonds oils. A predictive evaluation of the physical properties of the potential biodiesel obtainable by lipids produced by tested yeast strains has been reported and discussed. Conclusions: Lipids produced by some basidiomycetous yeasts grown on Mediterranean lignocellulosic biomass feedstocks could be used as supplementary sources of oils for producing biofuels and biochemicals.

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Purification and characterisation of a thermostable β-xylosidase from Aspergillus niger van Tieghem of potential application in lignocellulosic bioethanol production.

Boyce, A. & Walsh, G. (2018). Applied Biochemistry and Biotechnology, 186(3), 712-730.

A locally isolated strain of Aspergillus niger van Tieghem was found to produce thermostable β-xylosidase activity. The enzyme was purified by cation and anion exchange and hydrophobic interaction chromatography. Maximum activity was observed at 70-75°C and pH 4.5. The enzyme was found to be thermostable retaining 91 and 87% of its original activity after incubation for 72 h at 60 and 65°C, respectively, with 52% residual activity detected after 18 h at 70°C. Available data indicates that the purified β-xylosidase is more thermostable over industrially relevant prolonged periods at high temperature than those reported from other A. niger strains. Maximum activity was observed on p-nitrophenyl-β-D-xylopyranoside and the enzyme also hydrolysed p-nitrophenyl β-d-glucopyranoside and p-nitrophenyl α-L-arabinofuranoside. The purified enzyme acted synergistically with A. niger endo-1,4-β-xylanase in the hydrolysis of beechwood xylan at 65°C. During hydrolysis of pretreated straw lignocellulose at 70°C using a commercial lignocellulosic enzyme cocktail, inclusion of the purified enzyme resulted in a 19-fold increase in the amount of xylose produced after 6 h. The results observed indicate potential suitability for industrial application in the production of lignocellulosic bioethanol where thermostable β-xylosidase activity is of growing interest to maximise the enzymatic hydrolysis of lignocellulose.

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Simplified sodium chlorite pretreatment for carbohydrates retention and efficient enzymatic saccharification of silvergrass.

Nan, Y., Jia, L., Yang, M., Xin, D., Qin, Y. & Zhang, J. (2018). Bioresource Technology, 261, 223-231.

In this work, a simplified and cost-effective chlorite pretreatment method to improve the hydrolysabiliy of biomass was developed. Compared to common used sodium chlorite-acetic acid (SCA) pretreatment (18.1%), sodium chlorite (SC) pretreatment resulted in less xylan loss (7.8%), thus led more carbohydrates retention. Moreover, the Chinese silvergrass pretreated by SC for 2 h achieved higher glucose yield (70.5%) than the substrate pretreated by SCA under the same pretreatment conditions did (58.7%), after 48 h enzymatic hydrolysis by cellulase. By synergistic action of cellulase and xylanase, the glucose yield of SC pretreated (12 h) samples reached to 93.5% with 808.7 mg/g DM total reducing sugars yields. In addition, without the usage of acetic acid could decrease the process cost and result in less inhibitor generation in pretreatment process.

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Mechanisms of utilisation of arabinoxylans by a porcine faecal inoculum: competition and co-operation.

Feng, G., Flanagan, B. M., Mikkelsen, D., Williams, B. A., Yu, W., Gilbert, R. G. & Gidley, M. J. (2018). Scientific Reports, 8(1), 4546.

Recent studies show that a single or small number of intestinal microbes can completely degrade complex carbohydrates. This suggests a drive towards competitive utilisation of dietary complex carbohydrates resulting in limited microbial diversity, at odds with the health benefits associated with a diverse microbiome. This study investigates the enzymatic metabolism of wheat and rye arabinoxylans (AX) using in vitro fermentation, with a porcine faecal inoculum. Through studying the activity of AX-degrading enzymes and the structural changes of residual AX during fermentation, we show that the AX-degrading enzymes are mainly cell-associated, which enables the microbes to utilise the AX competitively. However, potential for cross-feeding is also demonstrated to occur by two distinct mechanisms: (1) release of AX after partial degradation by cell-associated enzymes, and (2) release of enzymes during biomass turnover, indicative of co-operative AX degradation. This study provides a model for the combined competitive-co-operative utilisation of complex dietary carbohydrates by gut microorganisms.

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