Cellulase (endo-1,4-β-D-glucanase)
(Trichoderma longibrachiatum)

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.

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.

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.

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.

Cell wall composition was studied during the development of apple cultivars (14–161/182 days after full bloom, DAA) maintaining firm fruit (Ariane) or evolving to mealy texture (Rome Beauty) when ripe and in sweet cherry cultivars (21/26–70/75 DAA) to assess their skin-cracking susceptibility (tolerant Regina and susceptible Garnet). Pectin sugar composition and hemicellulose fine structure assessed by enzymatic degradation coupled to MALDI-TOF MS analysis were shown to vary markedly between apples and cherries during fruit development. Apple showed decreasing rhamnogalacturonan I (RGI) and increasing homogalacturonan (HG) pectic domain proportions from young to mature fruit. Hemicellulose-cellulose (HC) sugars peaked at the beginning of fruit expansion corresponding to the maximum cell wall content of glucose and mannose. In contrast, HG peaked very early in the cell wall of young developing cherries and remained constant until ripening whereas RGI content continuously increased. HC content decreased very early and remained low in cell walls. Only the low content of mannose and to a lesser extent fucose increased and then slowly decreased from the beginning of the fruit expansion phase. Hemicellulose structural profiling showed strong varietal differences between cherry cultivars. Both apples and cherries demonstrated a peak of glucomannan oligomers produced by β-glucanase hydrolysis of the cell wall at the onset of cell expansion. The different glucomannan contents and related oligomers released from cell walls are discussed with regard to the contribution of glucomannan to cell wall mechanical properties. These hemicellulose features may prove to be early markers of apple mealiness and cherry skin-cracking susceptibility.

A modified TEMPO-catalyzed oxidation of the solvent-exposed glucosyl units of cellulose to uronic acids, followed by carboxyl reduction with NaBD₄ to 6-deutero- and 6,6-dideuteroglucosyl units, provided a robust method for determining relative proportions of disordered amorphous, ordered surface chains, and anhydrous core-crystalline residues of cellulose microfibrils inaccessible to TEMPO. Both glucosyl residues of cellobiose units, digested from amorphous chains of cellulose with a combination of cellulase and cellobiohydrolase, were deuterated, whereas those from anhydrous chains were undeuterated. By contrast, solvent-exposed and anhydrous residues alternate in surface chains, so only one of the two residues of cellobiosyl units was labeled. Although current estimates indicate that each cellulose microfibril comprises only 18 to 24 (1 → 4)-β-D-glucan chains, we show here that microfibrils of walls of Arabidopsis leaves and maize coleoptiles, and those of secondary wall cellulose of cotton fibers and poplar wood, bundle into much larger macrofibrils, with 67 to 86% of the glucan chains in the anhydrous domain. These results indicate extensive bundling of microfibrils into macrofibrils occurs during both primary and secondary wall formation. We discuss how, beyond lignin, the degree of bundling into macrofibrils contributes an additional recalcitrance factor to lignocellulosic biomass for enzymatic or chemical catalytic conversion to biofuel substrates.

One of the major limitations of lignocellulose conversion is the relatively low efficiency of cellulases. Expansins can act as an accessory protein to loosen the rigid cellulose structure and promote cellulose hydrolysis. However, the synergistic action of expansin is not well understood. In this study, we employed quartz crystal microbalance with dissipation to real-time monitor the adsorption of Bacillus subtilis expansin (BsEXLX1) and endoglucanase I (Cel7B) and the hydrolysis of cellulose. The effects of pH, temperature, and zinc ions on the initial adsorption rate and adsorption capacity of BsEXLX1 were examined. When 36.5 mM of zinc ions was added, the irreversible adsorption ratio of BsEXLX1 further increased to 4.63 times the value in the absence of zinc ions, whereas the initial adsorption rate and the hydrolysis rate constants of Cel7B could reach 2.16 times and 2.05 times the values in the absence of zinc ions, respectively.

EG1 from Volvariella volvacea is a processive endoglucanase belonging to glycoside hydrolase family 5. The impacts of cotton linter pulp characteristics, such as degree of polymerization (DP), crystallinity, and the initial cellulose-reducing ends, on the processivity of EG1 were investigated. Three commercial cotton linter pulp with different DP were used in present study. Ball milling was used to alter the crystallinity and DP of cellulose. The results indicate that the crystallinity has the most significant impact on enzyme processivity followed by initial cellulose-reducing ends. Whereas the DP indirectly affects the enzymatic hydrolysis and influenced by the pulp preparation method and conditions. The initial cellulose-reducing ends also affect enzyme adsorption but their impact is not obvious when the crystallinity is very low. These results also demonstrate the endo- and exo-action are both exist for EG1. The processive exo-action can start from the newly created cellulose-reducing ends by endo-action as well as the initial cellulose-reducing ends. The contribution of initial cellulose-reducing ends is affected by its quantity and cellulose crystallinity. A plausible action mode for EG1 is also proposed.

Transglycanases (endotransglycosylases) are enzymes that “cut and paste” polysaccharide chains. Several transglycanase activities have been discovered which can cut (i.e., use as donor substrate) each of the major hemicelluloses [xyloglucan, mannans, xylans, and mixed-linkage β-glucan (MLG)], and, as a recent addition, cellulose. These enzymes may play interesting roles in adjusting the wall’s physical properties, influencing cell expansion, stem strengthening, and fruit softening. Activities discussed include the homotransglycanases XET (xyloglucan endotransglucosylase, i.e., xyloglucan-xyloglucan endotransglycosylase), trans-β-mannanase (mannan-mannan endotransglycosylase), and trans-β-xylanase (xylan-xylan endotransglucosylase), plus the heterotransglycanases MXE (MLG-xyloglucan endotransglucosylase) and CXE (cellulose-xyloglucan endotransglucosylase). Transglycanases acting on polysaccharide donor substrates can utilize small, labeled oligosaccharides as acceptor substrates, generating easily recognizable polymeric labeled products. We present methods for extracting transglycanases from plant tissues and assaying them in vitro, either quantitatively in solution assays or by high-throughput dot-blot screens. Both radioactively and fluorescently labeled substrates are mentioned. A general procedure (glass-fiber blotting) is illustrated by which proposed novel transglycanase activities can be tested for. In addition, we describe strategies for detecting transglycanase action in vivo. These methods enable the quantification of, separately, XET and MXE action in Equisetum stems. Related methods enable the tissue distribution of transglycanase action to be visualized cytologically.

The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin‐embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved. Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin‐deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin‐embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross‐talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.