Exogenous cellulases of thermophilic micromycetes. II. Thermostability of enzyme preparations

The ability of a large number of higher fungi to form extracellular cellulases is investigated. Some representatives of these fungi grow at 40–50°C, and form extracellular cellulases exceeding cellulases of mesophilic fungi in thermostability. It is shown that cellulases of higher thermophilic fungi differ by their thermostability. The temperature optimum of cellulase action of higher fungi occurs within 60–62°C.     

Endoglucanase activities and growth of marine‐derived fungi isolated from the sponge Haliclona simulans

Aims: The conversion of cheap cellulosic biomass to more easily fermentable sugars requires the use of costly cellulases. We have isolated a series of marine sponge‐derived fungi and screened these for cellulolytic activity to determine the potential of this unique environmental niche as a source of novel cellulase activities. Methods and Results: Fungi were isolated from the marine sponge Haliclona simulans. Phylogenetic analysis of these and other fungi previously isolated from H. simulans showed fungi from three phyla with very few duplicate species. Cellulase activities were determined using plate‐based assays using different media and sea water concentrations while extracellular cellulase activities were determined using 3,5‐dinitrosalicylic acid (DNSA)‐based assays. Total and specific cellulase activities were determined using a range of incubation temperatures and compared to those for the cellulase overproducing mutant Hypocrea jecorina QM9414. Several of the strains assayed produced total or relative endoglucanase activities that were higher than H. jecorina, particularly at lower reaction temperatures. Conclusions: Marine sponges harbour diverse fungal species and these fungi are a good source of endoglucanase activities. Analysis of the extracellular endoglucanase activities revealed that some of the marine‐derived fungi produced high endoglucanase activities that were especially active at lower temperatures. Significance and Impact of the Study: Marine‐derived fungi associated with coastal marine sponges are a novel source of highly active endoglucanases with significant activity at low temperatures and could be a source of novel cellulase activities.     

Thermophilic Fungi: Their Physiology and Enzymes

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20°C and a maximum temperature of growth extending up to 60 to 62°C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45°C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62°C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

Thermophilic fungi: their physiology and enzymes.

Thermophilic fungi are a small assemblage in mycota that have a minimum temperature of growth at or above 20 degrees C and a maximum temperature of growth extending up to 60 to 62 degrees C. As the only representatives of eukaryotic organisms that can grow at temperatures above 45 degrees C, the thermophilic fungi are valuable experimental systems for investigations of mechanisms that allow growth at moderately high temperature yet limit their growth beyond 60 to 62 degrees C. Although widespread in terrestrial habitats, they have remained underexplored compared to thermophilic species of eubacteria and archaea. However, thermophilic fungi are potential sources of enzymes with scientific and commercial interests. This review, for the first time, compiles information on the physiology and enzymes of thermophilic fungi. Thermophilic fungi can be grown in minimal media with metabolic rates and growth yields comparable to those of mesophilic fungi. Studies of their growth kinetics, respiration, mixed-substrate utilization, nutrient uptake, and protein breakdown rate have provided some basic information not only on thermophilic fungi but also on filamentous fungi in general. Some species have the ability to grow at ambient temperatures if cultures are initiated with germinated spores or mycelial inoculum or if a nutritionally rich medium is used. Thermophilic fungi have a powerful ability to degrade polysaccharide constituents of biomass. The properties of their enzymes show differences not only among species but also among strains of the same species. Their extracellular enzymes display temperature optima for activity that are close to or above the optimum temperature for the growth of organism and, in general, are more heat stable than those of the mesophilic fungi. Some extracellular enzymes from thermophilic fungi are being produced commercially, and a few others have commercial prospects. Genes of thermophilic fungi encoding lipase, protease, xylanase, and cellulase have been cloned and overexpressed in heterologous fungi, and pure crystalline proteins have been obtained for elucidation of the mechanisms of their intrinsic thermostability and catalysis. By contrast, the thermal stability of the few intracellular enzymes that have been purified is comparable to or, in some cases, lower than that of enzymes from the mesophilic fungi. Although rigorous data are lacking, it appears that eukaryotic thermophily involves several mechanisms of stabilization of enzymes or optimization of their activity, with different mechanisms operating for different enzymes.     

Amylase and cellulase activities of thermophilic fungi causing deterioration of tobacco products in Nigeria

The conditions necessary for the production and activities of extracellular enzymes of thermophilic fungi obtained from tobacco products were determined. While amylases were produced in extractable amounts by all the fungi used in this study, extracellular cellulases were produced only by Chaetomium thermophile La Touche, Humicola insolens Cooney and Emerson, Malbranchea pulchella Sacc. et Penzig var. sulfurea (Miehe) Cooney and Emerson, and Mucor pusillus Lindt. A narrow range of pH 6–7 was best for the activities of the amylases of all the fungi used while a lower range of pH 5–6 was optimal for their cellulase activities. Peak activities were recorded for both enzymes at a temperature range of 45 °–50 °C. The possible use of the exo-enzymes of these fungi in industrial fermentation processes is suggested.     

Production of cellulases by thermophilic fungi grown on Leptochloa fusca straw

Seven indigenous thermophilic fungi were screened for cellulase and xylanase production when grown on Leptochloa fusca (kallar grass) straw. Aspergillus fumigatus produced the highest activities of 0.4, 2.5, 3.5 and 0.14 U/ml of filter paper cellulase, CM-cellulase, xylanase and -xylosidase, respectively. Sporotrichum thermophile produced 0.47 -glucosidase/ml. Chaetomium thermophile, Humicola grisea and Torula thermophila had lower activities than the other thermophilic fungi.The authors are with the National Institute for Biotechnology & Genetic Engineering, P.O. Box 577, Faisalabad, Pakistan.     

Characteristics of pullulanases from extremely thermophilic archaea isolated from deep-sea hydrothermal vents

Two out of three extremely thermophilic anaerobic archaea, isolated from deep-sea hydrothermal vents, produced pullulanase activity in the presence of maltose in the growth medium. Enzyme activities were mainly extracellular and characterized by optimum temperatures of 95°C and 80–95°C, optimum pH of 5.0–7.0 and a high degree of thermostability. One strain when grown in a fermenter with maltose as inducer produced pullulanase at 35 U/l. © Rapid Science Ltd. 1998     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

Characterization of hemicellulases from thermophilic fungi

The thermophilic fungi Thermomyces lanuginosus, Malbranchea cinnamomea, Myceliophthora fergusii and the thermotolerant Aspergillus terreus were cultivated on various carbon sources, and hemicellulolytic and cellulolytic enzyme profiles were evaluated. All fungi could grow on locust bean galactomannan (LBG), Solka floc, wheat bran and pectin, except T. lanuginosus, which failed to utilize LBG for growth. Different levels of cellulase and hemicellulase activities were produced by these fungal strains. Depending on the carbon source, variable ratios of thermostable hydrolytic enzymes were obtained, which may be useful in various applications. All strains were found to secrete xylanolytic and mannanolytic enzymes. Generally, LBG was the most efficient carbon source to induce mannanase activities, although T. lanuginosus was able to produce mannanase only on wheat bran as a carbon source. Xylanolytic activities were usually highest on wheat bran medium, but in contrast to other investigated fungi, xylanase production by M. fergusii was enhanced on pectin medium. Preliminary thermostability screening indicated that among the investigated species, thermotolerant glycosidases can be found. Some of the accessory activities, including the α-arabinosidase activity, were surprisingly high. The capability of the produced enzymes to improve the hydrolysis of lignocellulosic pretreated substrate was evaluated and revealed potential for these enzymes.     

Partial Purification,of Extracellular Cellulase from Pyricularia oryzae and Comparison of the Elution Patterns among Various Strains

In order to explain the difference in extracellular cellulase activities (C₁ and C_x enzyme activities) among various strains of P. oryzae, the elution patterns from the column were compared among various strains, following each step of the partial purification.

The crude enzymes, prepared by ammonium sulfate fractionation (0.2~0.8 sat.) from the culture filtrates, which were obtained from various strains of P. oryzae cultured on rice plant powder as the carbon source, were fractionated by DEAE-Sephadex A–50 chromatography into two components; the passing-through fraction (I) and the fraction (II) adsorbed and eluted from the column with 0.5 M NaCl The percentage of the enzyme activity (C_x enzyme activity) in fraction I to that of the crude extract was found to vary chracteristically according to the strain, and the variation was in a good correlation to that of the extracellular cellulase activities.

Fractions I and II were then separated by Sephadex G–100 into two (peaks a and b) and at least five (peaks c, d, e, f and g) components, respectively. The activities in peaks a, b and g were found to vary according to the strain, while those of peaks c and e were common among various strains.

The cell wall fraction prepared from C–3 strain, which was previously shown to be low in enzyme activity and thus out of the correlation between the degree of pathogenicity and extracellular cellulase activity, was found to exhibit higher cellulase activities (C₁ and C_x enzyme activities) than those of other strains examined. Thus, the low extracellular cellulase activity in the case of C–3 strain was suggested to be due to the abnormality in the mechanism of enzyme excretion.     

Crystal structure of histidine-containing phosphocarrier protein from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis> MB4 and the implications for thermostability

Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms, and therefore enables these organisms to survive at extreme temperatures. Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms, the structural basis of protein thermostability is not yet fully understood. The histidine-containing phosphocarrier (HPr) protein, a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species, is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors. In this study, the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified. Crystal structure with good quality has been determined at 2.3 Å resolution, which provides a firm foundation for exploring the thermostable mechanism. However, it shows that the crystal structure is conserved and no clue can be obtained from this single structure. Furthermore, detailed comparison of sequence and structure with the homologs from meso- or thermophilic bacteria shows no obvious rule for thermostability, but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr. Thus, mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria. Using site-directed mutations and the circular dichroism method, thermostability is evaluated, and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses, indicating that mutations cause instability in the HPrs. Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability, and is critical in engineering efficient enzymes that can work at a high temperature.     

Comparative molecular dynamics simulation studies for determining factors contributing to the thermostability of chemotaxis protein “CheY”

Comparative molecular dynamics simulations of chemotaxis protein “CheY” from thermophilic origin Thermotoga maritima and its mesophilic counterpart Salmonella enterica have been performed for 10?ns each at 300 and 350?K, and 20?ns each at 400 and 450?K. The trajectories were analyzed in terms of different factors like root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area, H-bonds, salt bridge content, and protein–solvent interactions which indicate distinct differences between the two of them. The two proteins also follow dissimilar unfolding pathways. The overall flexibility calculated by the trace of the diagonalized covariance matrix displays similar flexibility of both the proteins near their optimum growth temperatures. However, at higher temperatures mesophilic protein shows increased overall flexibility than its thermophilic counterpart. Principal component analysis also indicates that the essential subspaces explored by the simulations of two proteins at different temperatures are nonoverlapping and they show significantly different directions of motion. However, there are significant overlaps within the trajectories and similar direction of motions are observed for both proteins at 300?K. Overall, the mesophilic protein leads to increased conformational sampling of the phase space than its thermophilic counterpart. This is the first ever study of thermostability of CheY protein homologs by using protein dynamism as a main impact. Our study might be used as a model for studying the molecular basis of thermostability of two homologous proteins from two organisms living at different temperatures with less visible differences.     

Sporotrichum thermophile Growth, Cellulose Degradation, and Cellulase Activity

The activity of components of the extracellular cellulase system of the thermophilic fungus Sporotrichum thermophile showed appreciable differences between strains; β-glucosidase (EC 3.2.1.21) was the most variable component. Although its endoglucanase (EC 3.2.1.4) and exoglucanase (EC 3.2.1.91) activities were markedly lower, S. thermophile degraded cellulose faster than Trichoderma reesei. The production of β-glucosidase lagged behind that of endoglucanase and exoglucanase. The latter activities were produced during active growth. When growth was inhibited by cycloheximide treatment, the hydrolysis of cellulose was lower than in the control in spite of the presence of both endoglucanase and exoglucanase activities in the culture medium. Degradation of cellulose was a growth-associated process, with cellulase preparations hydrolyzing cellulose only to a limited extent. The growth rate and cell density of S. thermophile were similar in media containing cellulose or glucose. A distinctive feature of fungal development in media incorporating cellulose or lactose (inducers of cellulase activity) was the rapid differentiation of reproductive units and autolysis of hyphal cells to liberate propagules which were capable of renewing growth immediately.     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.     

Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation

Summary Six thermophilic fungi were examined for their ability to produce cellulolytic enzymes in liquid (LF) and solid-state fermentation (SSF). The best cellulase activities were achieved by Thermoascus aurantiacus and Sporotrichum thermophile. Taking into consideration that solid-state medium obtained from 100 g of dry sugar-beet pulp occupies about 11 of fermentor volume equivalent to 11 of LF, it was confirmed that enzyme productivity per unit volume from both fungi was greater in SSF than in LF. The cellulase system obtained by SSF with T. aurantiacus contained 1.322 IU/1 of exo--d-glucanase, 53.269 IU/1 of endo--d-glucanase and 8.974 IU/1 of -d-glucosidase. The thermal and pH characteristics of cellulases from solid-state fermentation of T. aurantiacus and S. thermophile are described.     

Differential responses of wood-rot fungi cellulases towards polyclonal antibodies against Trichoderma viride cellobiohydrolase I

Cellobiohydrolase (CBH) I, a main component of Trichoderma extracellular protein, was purified to an electrophoretically homogeneous state from a commercial cellulase preparation (Meicelase from T. viride) by column chromatography on anion and cation exchangers. The difference in the cross-reactivity of cellulolytic enzyme systems of brown-rot and white-rot fungi with the polyclonal antibodies to the CBH I was studied by enzyme-linked immunosorbent assay (ELISA). The antibodies were observed to react quantitatively and with great sensitivity with the antigen (CBH I), and at the same time to cross-react to some extent with T. viride cellulase components other than the CBH I. Nevertheless, the intensity of cross-reactivity of wood-rot fungi cellulases with the antibodies was parallel to the activity of exo-1,4-ß-glucanase. The cellulase system from brown-rot fungi, believed to lack exo-1,4-ß-glucanases, gave a negative response towards the antibodies. These results suggested the presence of some homologous sequences and structures with the T. viride CBH I in the enzymes of white-rot fungi and their absence in those of brown-rot fungi. Correspondence to: M. Ishihara     

Biochemical properties of an extracellular trehalase from <Emphasis Type="Italic">Malbranchea pulchella</Emphasis> var. Sulfurea

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0–5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K_M values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70±0.29 and 2.58±0.13 mM, respectively. Manganese ions affected only the apparent V_max, increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.     

Study of thermostable chitinases from Oerskovia xanthineolytica NCIM 2839

The mesophilic organism, Oerskovia xanthineolytica NCIM 2839, was adapted to grow at moderate thermophilic temperatures. At these elevated temperatures, it was found to produce two thermostable chitinases—C1 and C2. These were purified by ion exchange chromatography using DEAE cellulose. The chitinases C1 and C2 were found to be stable in a pH range from 3.0 to 9.0 with 7.5 and 8.0 being the optimum pH, respectively. The optimum temperatures of the activities of C1 and C2 were 50 and 55°C, respectively. These were activated by Mn⁺⁺ and Cu⁺⁺and inactivated by Hg⁺⁺. This is first report of an extracellular thermostable chitinase being produced by O. xanthineolytica NCIM 2839.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.