115 assays (manual) / 1150 assays (microplate) / 1150 assays (auto-analyser)
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|Content:||115 assays (manual) / 1150 assays (microplate) / 1150 assays (auto-analyser)|
|Storage Temperature:|| Short term stability: 2-8oC, |
Long term stability: See individual component labels
|Stability:||> 2 years under recommended storage conditions|
|Assay Format:||Spectrophotometer, Microplate, Auto-analyser|
|Linear Range:||4 to 80 μg of L-arabinose or D-galactose per assay|
|Limit of Detection:|| 0.58 mg/L (L-arabinose), |
0.69 mg/L (D-galactose)
|Reaction Time (min):|| ~ 12 min (L-arabinose), |
~ 6 min (D-galactose)
|Application examples:||Analysis of hydrolysates of oligo- and polysaccharides (e.g. arabinan, arabinoxylan, galactan, arabinogalactan), milk, dairy products, foods containing milk (e.g. dietetic foods, bakery products, baby food, chocolate, sweets and ice-cream), food additives (e.g. sweeteners), cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).|
|Method recognition:||Novel method|
The L-Arabinose/D-Galactose test kit is a simple, reliable and accurate UV method for the measurement and analysis of L-arabinose and/or D-galactose in various materials including foods, feeds, beverages and plant products.
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).
See more of our monosaccharide assay kits.
- Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
- Very rapid reaction due to inclusion of galactose mutarotase (patented technology)
- Very cost effective
- All reagents stable for > 2 years after preparation
- Only enzymatic kit available
- 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
Measurement of available carbohydrates in cereal and cereal products, dairy products, vegetables, fruit and related food products and animal feeds: First Action 2020.07.
McCleary, B. V. & McLoughlin, C. (2021). Journal of AOAC International, qsab019.
Background: The level of available carbohydrates in our diet is directly linked to two major diseases; obesity and Type II diabetes. Despite this, to date there is no method available to allow direct and accurate measurement of available carbohydrates in human and animal foods. Objective: The aim of this research was to develop a method that would allow simple and accurate measurement of available carbohydrates, defined as non-resistant starch, maltodextrins, maltose, isomaltose, sucrose, lactose, glucose, fructose and galactose. Method: Non-resistant (digestible) starch is hydrolysed to glucose and maltose by pancreatic α-amylase and amyloglucosidase at pH 6.0 with shaking or stirring at 37°C for 4 h. Sucrose, lactose, maltose and isomaltose are completely hydrolyzed by specific enzymes to their constituent monosaccharides, which are then measured using pure enzymes in a single reaction cuvette. Results: A method has been developed that allows the accurate measurement of available carbohydrates in all cereal, vegetable, fruit, food, and feed products, including dairy products. Conclusions: A single-laboratory validation was performed on a wide range of food and feed products. The inter-day repeatability (%RSDr) was <3.58% (w/w) across a range of samples containing 44.1 to 88.9% available carbohydrates. The LOD and LOQ obtained were 0.054% (w/w) and 0.179% (w/w), respectively. The method is all inclusive, specific, robust and simple to use. Highlights: A unique method has been developed for the direct measurement of available carbohydrates, entailing separate measurement of glucose, fructose and galactose; information of value in determining the glycemic index of foods.Hide Abstract
McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.
A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X), 23-α-L-Araf-(1-4)-β-D-xylotriose (A2XX), 33-α-L-Araf-(1-4)-β-D-xylotriose (A3XX), 22-α-L-Araf-(1-4)-β-D-xylotriose (XA2X), 32-α-L-Araf (1-4)-β-D-xylotriose (XA3X), 23-α-L-Araf-(1-4)-β-D-xylotetraose (XA2XX), 33-α-L-Araf-(1-4)-β-D-xylotetraose (XA3XX), 23 ,33-di-α-L-Araf-(1-4)-β-D-xylotriose (A2+3XX), 23,33-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA2+3XX), 24,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA2+3XXX) and 33,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.Hide Abstract
Utilization of stalk waste separated during processing of sun-dried figs (Ficus carica) as a source of pectin: Extraction and determination of molecular and functional properties.
Çavdaroğlu, E. & Yemenicioğlu, A. (2021). LWT, 154, 112624.
This study aimed the utilization of fig stalk waste as an alternative pectin source. For this purpose, the characteristics of extracted stalk waste pectin (SP) were compared with those of citrus pectin (CP) and pectin extracted from defected substandard whole sun-dried figs (FP). The SP had a higher extraction yield (11.7%) than FP (9.4%). The galacturonic acid content and degree of esterification of SP (32.3 and 50.1%) were higher than those of FP (19.9 and 38.8%), but lower than those of CP (79.3 and 56.2%), respectively. The SP and CP had different sugar compositions (d-glucose, l-rhamnose, d-galactose and l-arabinose) and weight average molecular weights, but similar FTIR profiles. The SP showed almost 1.9 and 1.6-fold higher Trolox equivalent antioxidant capacity (TEAC), and 2.7 and 2.0-fold higher water absorption capacity than CP and FP, respectively. SP at 3% (w/w) showed the second highest viscosity after CP and the highest emulsion stability. Gels of SP and CP at 1.75–3% range had similar firmness, but SP formed more fracturable gels than CP. Sun-dried fig stalk waste is a better source of pectin than defected substandard whole sun-dried figs. The SP could be utilized to develop functional food with alternative textural and rheological properties.Hide Abstract
Escherichia coli AraJ boosts utilization of arabinose in metabolically engineered cyanobacterium Synechocystis sp. PCC 6803.
Ranade, S. & He, Q. (2021). AMB Express, 11(1), 1-14.
Lignocellulosic biomass can serve as an inexpensive and renewable source of carbon for the biosynthesis of commercially important compounds. L-arabinose is the second most abundant pentose sugar present in the plant materials. Model cyanobacterium Synechocystis sp. PCC 6803 is incapable of catabolism of L-arabinose as a source of carbon and energy. In this study, all the heterologous genes expressed in Synechocystis were derived from Escherichia coli K-12. Initially we constructed four Synechocystis strains that expressed AraBAD enzymes involved in L-arabinose catabolism, either in combination with or without one of the three arabinose transporters, AraE, AraFGH or AraJ. Among the recombinants, the strain possessing AraJ transporter was observed to be the most efficient in terms of dry biomass production and L-arabinose consumption. Later, an additional strain was generated by the expression of AraJ in the AraE-possessing strain. The resultant strain was shown to be advantageous over its parent. This study demonstrates that AraJ, a protein with hitherto unknown function plays a role in the uptake of L-arabinose to boost its catabolism in the transgenic Synechocystis strains. The work also contributes to the current knowledge regarding metabolic engineering of cyanobacteria for the utilization of pentose sugars.Hide Abstract
The protective effect of Scenedesmus dimorphus polysaccharide as an antioxidant and antiaging agent on aging rat model induced by D-galactose.
Armaini, A. & Imelda, I. (2021). Journal of Applied Pharmaceutical Science, 11(05), 054-063.
Premature skin aging occurs due to the increased formation of reactive oxygen species (ROS), which causes oxidative stress, DNA damage, and collagen degradation. This study investigates the protective effect of Scenedesmus dimorphus polysaccharides (SDP) as an antioxidant and anti-aging agent on an aging rat model induced by D-galactose (D-gal). This study used 48 male Wistar rats divided into six groups: (1) normal control, (2) polysaccharide control, (3) aging control induced by D-gal 0.25 mg/g bw/days, (4) drug control (vitamin E) treated twice with SDP in an aging rat model, (5) D-gal + SDP (0.2 mg/g bw), and (6) D-gal + SDP (0.8 mg/g bw) treated with oral treatment and observed for periods of 2, 4, and 8 weeks. Giving SDP at a dose of 0.8 mg/g bw can increase superoxide dismutases and catalase activity and reduce malondialdehyde after 8 weeks of observation, which is better than giving vitamin E. The treatment of SDP can stimulate collagen synthesis and reduce advanced glycation end products. Histopathology shows an increase in the area of fibrocollagen proportions and deposition from the matrix on giving SDP, which is better than vitamin E, since SDP can repair skin tissue. Thus, SDP can be useful as an antioxidant and anti-aging agent in an aging Wistar rats model.Hide Abstract
Monosaccharide constituents of potato root exudate influence hatching of the white potato cyst nematode.
Bell, C. A., Mobayed, W., Lilley, C. J. & Urwin, P. (2021). PhytoFrontiers, 1-26.
Plants secrete a large array of compounds into the rhizosphere to facilitate interactions with their biotic environment. Some of these exuded-compounds stimulate the hatching of obligate plant-parasitic nematodes, ultimately leading to a detrimental effect on the host plant. Determining these cues can help to provide new mechanisms for control and aid nematode management schemes. Here we show that glucose, fructose and arabinose, which are all present in potato root exudate (PRE), induce hatching of white potato cyst nematode (Globodera pallida) eggs whereas five other PRE-sugars had no effect. Although these monosaccharides resulted in significant hatching none induced the same level as PRE, suggesting that other components, possibly in combination, contribute to stimulation of nematode hatching. Glucose, but not arabinose or fructose, was also observed to attract juvenile G. pallida, indicating that these hatch-inducing components can have different roles in different stages of the life cycle. Applying a solution of these monosaccharides to G. pallida-infested soil pre-potato planting initiated hatching in the absence of a host. Host absence resulted in nematode mortality and a reduction in the G. pallida population. Therefore, subsequent invasion of the crop post-planting was also reduced, compared to untreated soil. Our data suggest that monosaccharide components of PRE play an important role in the hatching and attraction of G. pallida. As a result the hatch-inducing monosaccharides can be applied as a pre-planting treatment to induce hatching and reduce subsequent infection rates.Hide Abstract
A new, quick, and simple protocol to evaluate microalgae polysaccharide composition.
Decamp, A., Michelo, O., Rabbat, C., Laroche, C., Grizeau, D., Pruvost, J. & Gonçalves, O. (2021). Marine Drugs, 19(2), 101.
In this work, a new methodological approach, relying on the high specificity of enzymes in a complex mixture, was developed to estimate the composition of bioactive polysaccharides produced by microalgae, directly in algal cultures. The objective was to set up a protocol to target oligomers commonly known to be associated with exopolysaccharides’ (EPS) nutraceutical and pharmaceutical activities (i.e., rhamnose, fucose, acidic sugars, etc.) without the constraints classically associated with chromatographic methods, while maintaining a resolution sufficiently high to enable their monitoring in the culture system. Determination of the monosaccharide content required the application of acid hydrolysis (2 M trifluoroacetic acid) followed by NaOH (2 M) neutralization. Quantification was then carried out directly on the fresh hydrolysate using enzyme kits corresponding to the main monosaccharides in a pre-determined composition of the polysaccharides under analysis. Initial results showed that the enzymes were not sensitive to the presence of TFA and NaOH, so the methodology could be carried out on fresh hydrolysate. The limits of quantification of the method were estimated as being in the order of the log of nanograms of monosaccharides per well, thus positioning it among the chromatographic methods in terms of analytical performance. A comparative analysis of the results obtained by the enzymatic method with a reference method (high-performance anion-exchange chromatography) confirmed good recovery rates, thus validating the closeness of the protocol. Finally, analyses of raw culture media were carried out and compared to the results obtained in miliQ water; no differences were observed. The new approach is a quick, functional analysis method allowing routine monitoring of the quality of bioactive polysaccharides in algal cultures grown in photobioreactors.Hide Abstract
A novel glycosidase plate-based assay for the quantification of galactosylation and sialylation on human IgG.
Rebello, O. D., Gardner, R. A., Urbanowicz, P. A., Bolam, D. N., Crouch, L. I., Falck, D. & Spencer, D. I. (2020). Glycoconjugate Journal, 37(6), 691-702.
Changes in human IgG galactosylation and sialylation have been associated with several inflammatory diseases which are a major burden on the health care system. A large body of work on well-established glycomic and glycopeptidomic assays has repeatedly demonstrated inflammation-induced changes in IgG glycosylation. However, these assays are usually based on specialized analytical instrumentation which could be considered a technical barrier for uptake by some laboratories. Hence there is a growing demand for simple biochemical assays for analyzing these glycosylation changes. We have addressed this need by introducing a novel glycosidase plate-based assay for the absolute quantification of galactosylation and sialylation on IgG. IgG glycoproteins are treated with specific exoglycosidases to release the galactose and/or sialic acid residues. The released galactose monosaccharides are subsequently used in an enzymatic redox reaction that produces a fluorescence signal that is quantitative for the amount of galactosylation and, in-turn, sialylation on IgG. The glycosidase plate-based assay has the potential to be a simple, initial screening assay or an alternative assay to the usage of high-end analytical platforms such as HILIC-FLD-MSn when considering the analysis of galactosylation and sialylation on IgG. We have demonstrated this by comparing our assay to an industrial established HILIC-FLD-MSn glycomic analysis of 15 patient samples and obtained a Pearson’s r correlation coefficient of 0.8208 between the two methods.Hide Abstract
Structural analysis of β‐L‐arabinobiose‐binding protein in the metabolic pathway of hydroxyproline‐rich glycoproteins in Bifidobacterium longum.
Miyake, M., Terada, T., Shimokawa, M., Sugimoto, N., Arakawa, T., Shimizu, K., Igarashi, K., Fujita, K. & Fushinobu, S. (2020). The FEBS Journal, 287(23), 5114-5129.
Bifidobacterium longum is a symbiotic human gut bacterium that has a degradation system for β‐arabinooligosaccharides, which are present in the hydroxyproline‐rich glycoproteins of edible plants. Whereas microbial degradation systems for α‐linked arabinofuranosyl carbohydrates have been extensively studied, little is understood about the degradation systems targeting β‐linked arabinofuranosyl carbohydrates. We functionally and structurally analyzed a substrate‐binding protein (SBP) of a putative ABC transporter (BLLJ_0208) in the β‐arabinooligosaccharide degradation system. Thermal shift assays and isothermal titration calorimetry revealed that the SBP specifically bound Araf‐β1,2‐Araf (β‐Ara2) with a Kd of 0.150 μm, but did not bind L‐arabinose or methyl‐β‐Ara2. Therefore, the SBP was termed β‐arabinobiose‐binding protein (BABP). Crystal structures of BABP complexed with β‐Ara2 were determined at resolutions of up to 1.78 Å. The findings showed that β‐Ara2 was bound to BABP within a short tunnel between two lobes as an α‐anomeric form at its reducing end. BABP forms extensive interactions with β‐Ara2, and its binding mode was unique among SBPs. A molecular dynamics simulation revealed that the closed conformation of substrate‐bound BABP is stable, whereas substrate‐free form can adopt a fully open and two distinct semi‐open states. The importer system specific for β‐Ara2 may contribute to microbial survival in biological niches with limited amounts of digestible carbohydrates.Hide Abstract
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 Candida. Candida 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.Hide Abstract
Cross-linking of diluted alkali-soluble pectin from apple (Malus domestica fruit) in different acid-base conditions.
Gawkowska, D., Cieśla, J., Zdunek, A. & Cybulska, J. (2019). Food Hydrocolloids, 92, 285-292.
A diluted alkali-soluble pectin (DASP) fraction, extracted using sodium carbonate, is characterized by a low degree of methylesterification and has the ability to self-organize on mica. The aim of this study was to characterize the cross-linking process of this fraction, extracted from apples, over a wide pH range (3-11) and without the addition of salt. An FT-IR study showed an increase in the intensity of bands connected with νas and νs (COO−) and a decrease in the intensity of the band associated with ν (C=O) in the carboxyl group with increasing pH, which indicated the dissociation of the carboxyl groups of galacturonic acid units. An increase in the surface electrical charge of particles in the pH range of 3-7 confirmed this. The value of the average apparent dissociation constant (∼4.60) indicated the acidic character of the DASP fraction. An AFM study showed the morphological changes of the DASP fraction with increasing pH, which allowed for the evaluation of the cross-linking process. This fraction formed a network on mica at pH 4 and 9, while the aggregates were noted mainly at pH 11. For totally ionized carboxyl groups (pH 7), the pectin chains were separated from each other due to the electrostatic repulsion between them.Hide Abstract
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.Hide Abstract
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.Hide Abstract
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.Hide Abstract
Vilches, F., Ravanal, M. C., Bravo-Moraga, F., Gonzalez-Nilo, D. & Eyzaguirre, J. (2018). Carbohydrate Research, 455, 106-113.
Penicillium purpurogenum secretes numerous lignocellulose-degrading enzymes, including four arabinofuranosidases and an exo-arabinanase. In this work, the biochemical properties of an endo-arabinanase (ABN1) are presented. A gene, coding for a potential ABN was mined from the genome. It includes three introns. The cDNA is 975 bp long and codes for a mature protein of 324 residues. The cDNA was expressed in Pichia pastoris. The enzyme is active on debranched arabinan and arabinooligosaccharides. In contrast to other characterized ABNs, inactive on p-nitrophenyl-α-L-arabinofuranoside (pNPAra), ABN1 is active on this substrate. The enzyme has an optimal pH of 4.5 and an optimal temperature of 30-35°C. Calcium does not activate ABN1. ABN1 belongs to GH family 43 sub-family 6, and a Clustal alignment with sequences of characterized fungal ABNs shows highest identity (54.6%) with an ABN from Aspergillus aculeatus. A three-dimensional model of ABN1 was constructed and the docking with pNPAra was compared with similar models of an enzyme very active on this substrate and another lacking activity, both from GH family 43. Differences in the number of hydrogen bonds between enzyme and substrate, and distance between the substrate and the catalytic residues may explain the differences in activity shown by these enzymes.Hide Abstract
Stressler, T., Leisibach, D., Lutz-Wahl, S., Kuhn, A. & Fischer, L. (2016). Applied Microbiology and Biotechnology, 100(12), 5401-5414.
The industrial manufacturing process of lactose-free milk products depends on the application of commercial β-galactosidase (lactase) preparations. These preparations are often obtained from Kluyveromyces lactis. There is a gene present in the genome of K. lactis which should encode for an enzyme called arylsulfatase (EC 220.127.116.11). Therefore, this enzyme could also be present in β-galactosidase preparations. The arylsulfatase is suspected of being responsible for an unpleasant “cowshed-like” off-flavor resulting from the release of p-cresol from milk endogenous alkylphenol sulfuric esters. So far, no gene/functionality relationship is described. In addition, no study is available which has shown that arylsulfatase from K. lactis is truly responsible for the flavor generation. In this study, we cloned the putative arylsulfatase gene from K. lactis GG799 into the commercially available vector pKLAC2. The cloning strategy chosen resulted in a homologous, secretory expression of the arylsulfatase. We showed that the heretofore putative arylsulfatase has the desired activity with the synthetic substrate p-nitrophenyl sulfate and with the natural substrate p-cresol sulfate. The enzyme was biochemically characterized and showed an optimum temperature of 45-50 C and an optimum pH of 9-10. Additionally, the arylsulfatase was activated by Ca2+ ions and was inactivated by Zn2+ ions. Moreover, the arylsulfatase was inhibited by p-cresol and sulfate ions. Finally, the enzyme was added to ultra-heat treated (UHT) milk and a sensory triangle test verified that the arylsulfatase from K. lactis can cause an unpleasant “cowshed-like” off-flavor.Hide Abstract
de Souza, W. R., Maitan-Alfenas, G. P., de Gouvêa, P. F., Brown, N. A., Savoldi, M., Battaglia, E., Goldman, M. H. S., de Vries, R. P. & Goldman, G. H. (2013). Fungal Genetics and Biology, 60, 29-45.
The interest in the conversion of plant biomass to renewable fuels such as bioethanol has led to an increased investigation into the processes regulating biomass saccharification. The filamentous fungus Aspergillus niger is an important microorganism capable of producing a wide variety of plant biomass degrading enzymes. In A. niger the transcriptional activator XlnR and its close homolog, AraR, controls the main (hemi-)cellulolytic system responsible for plant polysaccharide degradation. Sugarcane is used worldwide as a feedstock for sugar and ethanol production, while the lignocellulosic residual bagasse can be used in different industrial applications, including ethanol production. The use of pentose sugars from hemicelluloses represents an opportunity to further increase production efficiencies. In the present study, we describe a global gene expression analysis of A. niger XlnR- and AraR-deficient mutant strains, grown on a D-xylose/L-arabinose monosaccharide mixture and steam-exploded sugarcane bagasse. Different gene sets of CAZy enzymes and sugar transporters were shown to be individually or dually regulated by XlnR and AraR, with XlnR appearing to be the major regulator on complex polysaccharides. Our study contributes to understanding of the complex regulatory mechanisms responsible for plant polysaccharide-degrading gene expression, and opens new possibilities for the engineering of fungi able to produce more efficient enzymatic cocktails to be used in biofuel production.Hide Abstract
Ravanal, M. C., Callegari, E. & Eyzaguirre, J. (2010). Applied and Environmental Microbiology, 76(15), 5247-5253.
The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl α-L-arabinofuranoside and p-nitrophenyl β-D-xylopyranoside with a Km of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both α-L-arabinofuranosidase and xylobiohydrolase functionalities.Hide Abstract