Measurement of endo-1,4-β-glucanase.
McCleary, B. V., McKie, V. & Draga, A. (2012). “Methods in Enzymology”, Volume 510, (H. Gilbert, Ed.), Elsevier Inc., pp. 1-17.
Several procedures are available for the measurement of endo-1,4-β-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.
Measurement of polysaccharide-degrading enzymes in plants using chromogenic and colorimetric substrates.
McCleary, B. V. (1995). “New Diagnostics in Crop Sciences”, (J. R. Skerritt and R. Appels, Eds.), CAB International, pp. 277-301.
Enzymatic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well-defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material (Bamforth, 1982). In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall-degrading enzymes are used to destroy the three-dimensional structure and water-binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate-degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial processes.
New developments in the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase.
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.
Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.
McCleary, B. V. (1991). Chemistry in Australia, September, 398-401.
Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.
The reb1-1 mutation of Arabidopsis. Effect on the structure and localization of galactose-containing cell wall polysaccharides.
Nguema-Ona, E., Andème-Onzighi, C., Aboughe-Angone, S., Bardor, M., Ishii, T., Lerouge, P. & Driouich, A. (2006). Plant Physiology, 140(4), 1406-1417.
The Arabidopsis (Arabidopsis thaliana) root epidermal bulger1-1 (reb1-1) mutant (allelic to root hair defective1 [rhd1]) is characterized by a reduced root elongation rate and by bulging of trichoblast cells. The REB1/RHD1 gene belongs to a family of UDP-D-Glucose 4-epimerases involved in the synthesis of D-Galactose (Gal). Our previous study showed that certain arabinogalactan protein epitopes were not expressed in bulging trichoblasts of the mutant. In this study, using a combination of microscopical and biochemical methods, we have investigated the occurrence and the structure of three major Gal-containing polysaccharides, namely, xyloglucan (XyG), rhamnogalacturonan (RG)-I, and RG-II in the mutant root cell walls. Our immunocytochemical data show that swollen trichoblasts were not stained with the monoclonal antibody CCRC-M1 specific for α-L-Fucp-(1→2)-β-D-Galp side chains of XyG, whereas they were stained with anti-XyG antibodies specific for XyG backbone. In addition, analysis of a hemicellulosic fraction from roots demonstrates the presence of two structurally different XyGs in reb1-1. One is structurally similar to wild-type XyG and the other is devoid of fuco-galactosylated side chains and has the characteristic of being insoluble. Similar to anti-XyG antibodies, anti-bupleuran 2IIC, a polyclonal antibody specific for galactosyl epitopes associated with pectins, stained all root epidermal cells of both wild type and reb1-1. Similarly, anti-RG-II antibodies also stained swollen trichoblasts in the mutant. In addition, structural analysis of pectic polymers revealed no change in the galactosylation of RG-I and RG-II isolated from reb1-1 root cells. These findings demonstrate that the reb1-1 mutation affects XyG structure, but not that of pectic polysaccharides, thus lending support to the hypothesis that biosynthesis of Gal as well as galactosylation of complex polysaccharides is regulated at the polymer level.
Structural investigation of hemicellulosic polysaccharides from Argania spinosa: characterisation of a novel xyloglucan motif.
Ray, B., Loutelier-Bourhis, C., Lange, C., Condamine, E., Driouich, A. & Lerouge, P. (2004). Carbohydrate Research, 339(2), 201-208.
Hemicellulose polymers were isolated from Argania spinosa leaf cell walls by sequential extractions with alkali. The structure of the two main polymers, xylan and xyloglucan, was investigated by enzyme degradation with specific endoglycosidases followed by analysis of the resulting fragments by high performance anion exchange chromatography (HPAEC) and matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). The results show that A. spinosa xylan is composed of a β-(1 → 4)-linked-D-xylopyranose backbone substituted with 4-O-methyl-D-glucuronic acid residues. Xyloglucan oligosaccharide subunits were generated by treatment with an endo-(1 → 4)-β-D-glucanase of the xyloglucan-rich hemicellulosic fractions. MALDI-TOF mass spectra and HPAE-PAD chromatography of the pool of endoglucanase-generated xyloglucan oligomers indicated that A. spinosa cell wall contains a XXXG-type xyloglucan. In addition to XXXG, XXFG, XLXG/XXLG, XLFG fragments previously characterised in various plants, a second group of XXXG-type fragments was detected. The primary structure of the major subunit was determined by a combination of sugar analysis, methylation analysis, post-source decay (PSD) fragment analysis of MALDI-TOF MS and 1H NMR spectroscopy. This fragment, termed XUFG, contains a novel β-D-Xylp-(1 → 2)-α-D-Xylp side chain linked to C-6 of the second glucose unit from the nonreducing end of the cellotetraose sequence.
Dietary fibers from mushroom sclerotia: 3. In vitro fermentability using human fecal microflora.
Wong, K. H., Wong, K. Y., Kwan, H. S. & Cheung, P. C. K. (2005). Journal of Agricultural and Food Chemistry, 53(24), 9407-9412.
The in vitro fermentability of three novel dietary fibers (DFs) prepared from mushroom sclerotia, namely, Pleurotus tuber-regium, Polyporous rhinocerus, and Wolfiporia cocos, was investigated and compared with that of the cellulose control. All DF samples (0.5 g each) were fermented in vitro with a human fecal homogenate (10 mL) in a batch system (total volume, 50 mL) under strictly anaerobic conditions (using oxygen reducing enzyme and under argon atmosphere) at 37°C for 24 h. All three novel sclerotial DFs exhibited notably higher dry matter disappearance (P. tuber-regium, 8.56%; P. rhinocerus, 13.5%; and W. cocos, 53.4%) and organic matter disappearance (P. tuber-regium, 9.82%; P. rhinocerus, 14.6%; and W. cocos, 57.4%) when compared with those of the cellulose control. Nevertheless, only the W. cocos DF was remarkably degraded to produce considerable amounts of total short chain fatty acids (SCFAs) (5.23 mmol/g DF on organic matter basis, with a relatively higher molar ratio of propionate) that lowered the pH of its nonfermented residue to a slightly acidic level (5.89). Variations on the in vitro fermentability among the three sclerotial DFs might mainly be attributed to their different amounts of interwoven hyphae present (different amounts of enzyme inaccessible cell wall components) as well as the possible different structural arrangement (linkage and degree of branching) of their β-glucans.
α-Fucosidases with different substrate specificities from two species of Fusarium.
Paper, J. M., Scott-Craig, J. S., Cavalier, D., Faik, A., Wiemels, R. E., Borrusch, M. S., Bongers, M. & Walton, J. D. (2013). Applied Microbiology and Biotechnology, 97(12), 5371-5380.
Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on L-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene (FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ~1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily.
Biochemical analysis of expansin-like proteins from microbes.
Georgelis, N., Nikolaidis, N. & Cosgrove, D. J. (2014). Carbohydrate Polymers, 100, 17-23.
Expansins cause plant cell wall loosening and are present primarily in the plant kingdom. Gene sequence analysis suggests that expansins are present in several plant-colonizing or plant-pathogenic bacteria and fungi. However, experimental evidence of microbial expansin activity is largely lacking. Here we provide evidence that expansins from three plant pathogenic bacteria and one fungus cause extension of cell walls in vitro and weaken filter paper networks, without lytic activity. Since expansins were able to weaken cellulose networks, we tested whether they synergistically enhanced the activity of several cellulases in hydrolysis of cellulose. The microbial expansins did not show such synergism beyond the nonspecific effect of bovine serum albumin. Our results show that the expansins present in several pathogenic microbes have weak wall-loosening activity and we infer a role for these expansins in plant pathogenesis. Additionally, the convenient expression of several expansins in Escherichia coli makes a future comparative structure–function analysis among expansins possible in order to understand their activity at the molecular level.
KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis.
Pagant, S., Bichet, A., Sugimoto, K., Lerouxel, O., Desprez, T., McCann, M., Lerouge, P., Vernhettes, S. & Höfte, H. (2002). The Plant Cell, 14(9), 2001-2013.
The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein–KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.
Preparation of oligomeric β-glycosides from cellulose and hemicellulosic polysaccharides via the glycosyl transferase activity of a Trichoderma reesei cellulase.
York, W. S. & Hawkins, R. (2000). Glycobiology, 10(2), 193-201.
Oligoglycosyl (allyl, 2,3-dihydroxypropyl, ethyl, 2-hydroxyethyl, and methyl) β-glycosides were generated by endo-transglycosylation reactions catalyzed by commercially available Trichoderma reesei cellulase. A polymeric donor substrate (xyloglucan or cellulose) was incubated with the enzyme in an aqueous solution containing 20% of the acceptor alcohol (allyl alcohol, glycerol, ethanol, ethylene glycol, and methanol, respectively). The products of these reactions included oligomeric alkyl β-glycosides and reducing oligosaccharides. The high yield of alkyl β-glycosides may be explained by the resistance of the xyloglucan β-glycosides to cellulase-mediated hydrolysis. The resistance of the oligoxyloglucan β-glycosides to endoglucanase catalyzed hydrolysis supports the hypothesis that productive binding of the glycan substrate depends on its interaction with enzyme subsites on both sides of the cleavage point, leading to distortion of the ring geometry of the residue whose glycosidic bond is cleaved. Oligoxyloglucan β-glycosides were purified by a combination of gel-permeation and reversed-phase HPLC and were structurally characterized by MS and NMR spectroscopy. These results demonstrate that novel oligosaccharide β-glycosides can be efficiently produced by enzyme-catalyzed fragmentation/transglycosylation reactions starting with a polysaccharide donor substrate. This class of reactions may represent a convenient source of β-glycosides to be used as synthons for the rapid synthesis of complex glycans.
Saccharification of cellulose by recombinant Rhodococcus opacus PD630 strains.
Hetzler, S., Bröker, D. & Steinbüchela, A. (2013). Applied and Environmental Microbiology, 79(17), 5159-5166.
The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. Opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U ml-1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.
Regulation of the cellulose synthase-like gene family by light in the maize mesocotyl.
Van Erp, H. & Walton, J. D. (2009). Planta, 229(4), 885-897.
The cellulose synthase-like (ZmCSL) gene family of maize was annotated and its expression studied in the maize mesocotyl. A total of 28 full-length CSL genes and another 13 partial sequences were annotated; four are predicted to be pseudogenes. Maize has all of the CSL subfamilies that are present in rice, but the CSLC subfamily is expanded from 6 in rice to 12 in maize, and the CSLH subfamily might be reduced from 3 to 1. Unlike rice, maize has a gene in the CSLG subfamily, based on its sequence similarity to two genes annotated as CSLG in poplar. Light regulation of glycan synthase enzyme activities and CSL gene expression were analyzed in the mesocotyl. A Golgi-localized glucan synthase activity is reduced by ~50% 12 h after exposure to light. Β-1,4-Mannan synthase activity is reduced even more strongly (>85%), whereas Β-1,4-xylan synthase, callose synthase, and latent IDPase activity respond only slightly, if at all, to light. At least 17 of the CSL genes (42%) are expressed in the mesocotyl, of which four are up-regulated at least twofold, seven are down-regulated at least twofold, and six are not affected by light. The results contribute to our understanding of the structure of the CSL gene family in an important food and biofuel plant, show that a large percentage of the CSL genes are expressed in the specialized tissues of the mesocotyl, and demonstrate that members of the CSL gene family are differentially subject to photobiological regulation.
Cloning of a GH5 endoglucanase from genus Penicillium and its binding to different lignins.
Krogh, K. B. R. M., Kastberg, H., Jørgensen, C. I., Berlin, A., Harris, P. V. & Olsson, L. (2009). Enzyme and Microbial Technology, 44(6), 359-367.
The cel5C gene, coding for an endoglucanase (Cel5C) of Penicillium brasilianum was cloned and heterologously expressed in Aspergillus oryzae. This is only the second GH5 EG from the genus Penicillium reported in the CAZy database. The promoter region of the gene has putative binding sites for both the carbon catabolite repressor CreA and the activator XlnR. The pH optimum of Cel5C was found to be 4.0 and the temperature optimum was 70°C. At a typical temperature for lignocellulose hydrolysis Cel5C retained full residual activity after 20 h of incubation at pH 5.0 and 6.0. Adsorption to Avicel and steam pretreated spruce, was found to follow the Langmuir isotherm, and the maximum adsorption was similar for both substrates, 40 and 49 mg/g, respectively. The affinity for Avicel was 10 times higher than for steam pretreated spruce, 0.040 and 0.0035 L/mg, respectively. Non-productive binding of cellulolytic enzymes to lignin is an important obstacle to overcome for commercial biomass to ethanol production. Therefore, the adsorption on residual lignin produced from various biomass samples was investigated. Both substrate and pretreatment conditions resulted in different adsorptions of Cel5C to the residual lignin.
Functional genomic analysis supports conservation of function among cellulose synthase-like A gene family members and suggests diverse roles of mannans in plants.
Liepman, A. H., Nairn, C. J., Willats, W. G. T., Sørensen, I., Roberts, A. W. & Keegstra, K. (2007). Plant Physiology, 143(4), 1881-1893.
Mannan polysaccharides are widespread among plants, where they serve as structural elements in cell walls, as carbohydrate reserves, and potentially perform other important functions. Previous work has demonstrated that members of the cellulose synthase-like A (CslA) family of glycosyltransferases from Arabidopsis (Arabidopsis thaliana), guar (Cyamopsis tetragonolobus), and Populus trichocarpa catalyze β-1,4-mannan and glucomannan synthase reactions in vitro. Mannan polysaccharides and homologs of CslA genes appear to be present in all lineages of land plants analyzed to date. In many plants, the CslA genes are members of extended multigene families; however, it is not known whether all CslA proteins are glucomannan synthases. CslA proteins from diverse land plant species, including representatives of the mono- and dicotyledonous angiosperms, gymnosperms, and bryophytes, were produced in insect cells, and each CslA protein catalyzed mannan and glucomannan synthase reactions in vitro. Microarray mining and quantitative real-time reverse transcription-polymerase chain reaction analysis demonstrated that transcripts of Arabidopsis and loblolly pine (Pinus taeda) CslA genes display tissue-specific expression patterns in vegetative and floral tissues. Glycan microarray analysis of Arabidopsis indicated that mannans are present throughout the plant and are especially abundant in flowers, siliques, and stems. Mannans are also present in chloronemal and caulonemal filaments of Physcomitrella patens, where they are prevalent at cell junctions and in buds. Taken together, these results demonstrate that members of the CslA gene family from diverse plant species encode glucomannan synthases and support the hypothesis that mannans function in metabolic networks devoted to other cellular processes in addition to cell wall structure and carbohydrate storage.