Purification, properties, and industrial significance of transglucosidase from Aspergillus niger.
McCleary, B. V., Gibson, T. S., Sheehan, H., Casey, A., Horgan, L. & O’Flaherty, J. (1989). Carbohydrate Research, 185(1), 147-162.
A transglucosidase, an α-D-glucosidase with a high transferase activity, has been purified to homogeneity from culture broths of A. niger. The enzyme, which gave a single protein band on SDS-gel electrophoresis (mol. wt. 116,000) and two protein bands on isoelectric focusing (pI values 5.1 and 5.0), is a glycoprotein, containing 27.6% of carbohydrate, most of which is mannose, has an optimal pH of 4.0–4.5, and is stable in the pH range 4.0–6.0 and at <50°. The transglucosidase acts on various substrates to give transfer products and glucose. The Vmax and Km values (mm) for the following substrates were obtained: maltose (73.5, 1.7), isomaltose (31.9, 7.7), nigerose (63.6, 20.0), kojibiose (22.1, 3.6), phenyl α-D-glucopyranoside (2.5, 2.4), p-nitrophenyl α-D-glucopyranoside (2.1, 0.7), and methyl α-D-glucopyranoside (1.0, 26.3). The effect of the enzyme on the conversion, by amyloglucosidase, of wort saccharides into glucose has been quantified. A procedure, based on the use of phenyl α-D-glucopyranoside as substrate, is recommended for the assay of this enzyme in crude culture filtrates.
Weissella confusa Cab3 dextransucrase: Properties and in vitro synthesis of dextran and glucooligosaccharides.
Shukla, S., Shi, Q., Maina, N. H., Juvonen, M., Tenkanen, M. & Goyal, A. (2014). Carbohydrate Polymers, 101, 554-564.
Food-derived Weissella spp. have gained attention during recent years as efficient dextran producers. Weissella confusa Cab3 dextransucrase (WcCab3-DSR) was isolated applying PEG fractionation and used for in vitro synthesis of dextran and glucooligosaccharides. WcCab3-DSR had a molar mass of 178 kDa and was activated by Co2+ and Ca2+ ions. Glycerol and Tween 80 enhanced enzyme stability, and its half-life at 30°C increased from 10 h to 74 h and 59 h, respectively. The 1H and 13C NMR spectral analysis of the produced dextran confirmed the presence of main chain α-(1→6) linkages with only 3.0% of α-(1→3) branching, of which some were elongated. An HPSEC analysis in DMSO revealed a high molecular weight of 1.8 × 107 g/mol. Glucooligosaccarides produced through the acceptor reaction with maltose, were analyzed with HPAEC-PAD and ESI-MS/MS. They were a homologous series of isomaltooligosaccharides with reducing end maltose units. To the best of our knowledge, this is a first report on native W. confusa dextransucrase.
Structural analysis of enzyme-resistant isomaltooligosaccharides reveals the elongation of α-(1→ 3)-linked branches in Weissella confusa dextran.
Maina, N. H., Virkki, L., Pynnönen, H., Maaheimo, H. & Tenkanen, M. (2011). Biomacromolecules, 12(2), 409-418.
Weissella confusa VTT E-90392 is an efficient producer of a dextran that is mainly composed of α-(1→6)-linked D-glucosyl units and very few α-(1→3) branch linkages. A mixture of the Chaetomium erraticum endodextranase and the Aspergillus niger α-glucosidase was used to hydrolyze W. confusa dextran to glucose and a set of enzyme-resistant isomaltooligosaccharides. Two of the oligosaccharides (tetra- and hexasaccharide) were isolated in pure form and their structures elucidated. The tetrasaccharide had a nonreducing end terminal α-(1→3)-linked glucosyl unit (α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc), whereas the hexasaccharide had an α-(1→3)-linked isomaltosyl side group (α-D-Glcp-(1→6)[α-D-Glcp-(1→6)-α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). A mixture of two isomeric oligosaccharides was also obtained in the pentasaccharide fraction, which were identified as (α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc) and (α-D-Glcp-(1→6)[α-D-Glcp-(1→3)]-α-D-Glcp-(1→6)-α-D-Glcp-(1→6)-α-D-Glc). The structures of the oligosaccharides indicated that W. confusa dextran contains both terminal and elongated α-(1→3)-branches. This is the first report evidencing the presence of elongated branches in W. confusa dextran. The 1H and 13C NMR spectroscopic data on the enzyme-resistant isomaltooligosaccharides with α-(1→3)-linked glucosyl and isomaltosyl groups are published here for the first time.
New glycosidase substrates for droplet-based microfluidic screening.
Najah, M., Mayot, E., Mahendra-Wijaya, I. P., Griffiths, A. D., Ladame, S. & Drevelle, A. (2013). Analytical Chemistry, 85(20), 9807-9814.
Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30°C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.
Lactose- and cellobiose-derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase.
Shi, Q., Juvonen, M., Hou, Y., Kajala, I., Nyyssölä, A., Maina, N. H., Maaheimo, H., Virkki, L. & Tenkanen, M. (2016). Food Chemistry, 190, 226-236.
Dextran-producing Weissella have received significant attention. However, except for maltose, the acceptor reactions ofWeissella dextransucrases with different sugars have not been investigated. The action of recombinant Weissella confusa VTT E-90392 dextransucrase was tested with several potential acceptors, particularly, analogs lactose and cellobiose. The major acceptor products of both disaccharides were identified as branched trisaccharides, with a glucosyl residue α-(1→2)-linked to the acceptor’s reducing end. An additional product, isomelezitose (6Fru-α-Glcp-sucrose), was also produced when using lactose as an acceptor. This is the first report of the synthesis of isomelezitose by a dextransucrase. The NMR spectra of the three trisaccharides were fully assigned, and their structures were confirmed by selective enzymatic hydrolysis. The trisaccharides prepared from 13C6glc sucrose and lactose were analyzed by ESI-MSn, and the fragmentation patterns of these compounds were characterized.