Evaluation of cross-linked aggregates from purified Bacillus subtilis levansucrase mutants for transfructosylation reactions

Background  

Increasing attention has been focused on inulin and levan-type oligosaccharides, including fructosyl-xylosides and other fructosides due to their nutraceutical properties. Bacillus subtilis levansucrase (LS) catalyzes the synthesis of levan from sucrose, but it may also transfer the fructosyl moiety from sucrose to acceptor molecules included in the reaction medium. To study transfructosylation reactions with highly active and robust derivatives, cross-linked enzyme aggregates (CLEAs) were prepared from wild LS and two mutants. CLEAs combine the catalytic features of pure protein preparations in terms of specific activity with the mechanical behavior of industrial biocatalysts.     

Synthesis and characterization of hydroquinone fructoside using <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> levansucrase

Hydroquinone (HQ) functions as a skin-whitening agent, but it has the potential to cause dermatitis. We synthesized a HQ fructoside (HQ-Fru) as a potential skin-whitening agent by reacting levansucrase from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a fructofuranose donor. The product was purified using 1-butanol partition and silica-gel column chromatography. The structure of the purified HQ-Fru was determined by ¹H and ¹³C nuclear magnetic resonance, and the molecular ion of the product was observed at m/z 295 (C12 H16 O7 Na)⁺. The HQ-Fru was identified as 4-hydroxyphenyl-β-d-fructofuranoside. The optimum condition for HQ-Fru synthesis was determined using a response surface method (RSM), and the final optimum condition was 350 mM HQ, 115 mM sucrose, and 0.70 U/ml levansucrase, and the final HQ-Fru produced was 1.09 g/l. HQ-Fru showed anti-oxidation activities and inhibition against tyrosinase. The median inhibition concentration (IC₅₀) of 1,1-diphenyl-2-picrylhydrazyl scavenging activity was 5.83 mM, showing higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). The K _i value of HQ-Fru (1.53 mM) against tyrosinase was smaller than that of β-arbutin (K _i  = 2.8 mM), indicating that it was 1.8-times better as an inhibitor. The inhibition of lipid peroxidation by HQ-Fru was 105.3% that of HQ (100%) and 118.9 times higher than that of β-arbutin (0.89% of HQ).     

<Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> levansucrase is involved in tolerance to NaCl,sucrose and desiccation,and in biofilm formation

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50–150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9–58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.     

Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-β-mannosidase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-β-mannosidase or 1,4-β- D -mannanase (EC 3.2.1.78), commonly named β-mannanase, is an enzyme that can catalyze random hydrolysis of β-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-β-mannosidase gene (manB) from B. licheniformis.     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Exposure to (1?→?3)-β-D-glucans in swine farms

The aim of this study was to assess the exposure to organic dust and (1 → 3)-β-D-glucans in the buildings where an intensive breeding of swine is going on and evaluation of the impact of the breeding technical conditions on the observed levels of bioaerosols. The study was carried out in 30 swine farms differentiated by the size of the herd and technical conditions of breeding. In 35 randomly selected buildings, air samples were collected by stationary measurements to determine the concentrations of organic dust and (1 → 3)-β-D-glucans in inhalable and respirable fractions. Furthermore, each of the investigated buildings was precisely characterized by means of a questionnaire for technical conditions and type of breeding. In each of the points, the microclimate parameters were measured, i.e., temperature, relative humidity, CO₂ concentration and air velocity. The analyzed levels of organic dust and (1 → 3)-β-D-glucans were characterized by a wide range of concentrations. For inhalable fraction, they reached respectively: organic dust (0.43–11.8 mg/m³), (1 → 3)-β-D-glucans (14–3,594 ng/m³). For respirable fraction, the results were as follows: organic dust (0.01–4.69 mg/m³), (1 → 3)-β-D-glucans (1–703 ng/m³). The concentrations of (1 → 3)-β-D-glucans were positively correlated with organic dust (r = 0.68; p < 0.001). The most significant factor increasing the concentrations of organic dust and (1 → 3)-β-D-glucans was the use of bedding in the form of cut straw. Additionally, the levels of (1 → 3)-β-D-glucans were affected by manual forage feeding, mechanical manure disposal and the lack of the liquid manure container in breeding buildings. In view of the hazardous effects of biological agents on the health of swine-breeding workers, the swine management systems without beddings should be used, along with automated dosing techniques.     

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K _m 1.66 mg mL⁻¹; V _max 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-d-glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K _i 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. ‘De novo’ sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Dynamics of ginsenoside biosynthesis in suspension culture of <Emphasis Type="Italic">Panax quinquefolium</Emphasis>

Biosynthesis of six saponins (ginsenosides) in suspension culture of P. quinquefolium Z₅ was investigated. Ginsenoside content in biomass reached the highest level, nearly 30 mg g⁻¹ d.w., between 25 and 30 days of the culture. Saponins were synthesized simultaneously with cell growth but their synthesis rate was not proportional to the growth rate. During the phase of rapid biomass multiplication, after which biomass reached 90% of its maximum yield, only half examined ginsenosides was produced. The second half of the final saponins yield was produced during the slow growth phase, in which only 10% of biomass was grown. During the intensive growth phase the productivity of six saponins examined per biomass (dry weight) unit was 3.4 μg mg⁻¹ d.w. day⁻¹, however, this parameter calculated for slow growth phase reached nearly 30 μg mg⁻¹ d.w. day⁻¹. There were differences in increase of the contents of six saponins determined in biomass, and it was the highest for saponins Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) and Rg₁ (20(S)-protopanaxatriol-6,20-di-O-β-d-glucoside).     

Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens

Although levan produced by Bacillus amyloliquefaciens is known to have efficient immunostimulant property which gives 100% survival of common carp when infected with Aeromonas hydrophila, no detailed reports are available describing kinetic studies of d-glucose production and levan formation. In this study, we cloned and characterized the enzymatic kinetics using levansucrase expressed in Escherichia coli. Optimum pH for d-glucose production and levan formation was 6.0 and 8.0, respectively, whereas optimum temperature was 30°C and 4°C, respectively. The K _m and V _max values for levansucrase were calculated to be 47.81 mM sucrose and 57.47 μmole/min mg protein, respectively. Prominent expression of levansucrase was obtained through xylose induction in Bacillus megaterium, where most of the His₆-tagged protein was secreted into the culture broth, giving levansucrase activity of 12,906 U/l. Response-surface methodology (RSM) was further employed to optimize the fermentation conditions and improve the level of levansucrase production. Maximum levansucrase activity of 20,251 U/l was obtained in 12 h of fermentation carried out at 28°C, starting induction with 0.735% xylose when A ₆₀₀ was 1.2, which was 1.6- and 62-fold higher than those obtained in the nonoptimized conditions for the recombinant strain and the native strain, respectively.     

Structural analysis of the exopolysaccharide produced by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ST1 solely by NMR spectroscopy

The use of lactic acid bacteria in fermentation of milk results in favorable physical and rheological properties due to in situ exopolysaccharide (EPS) production. The EPS from S. thermophilus ST1 produces highly viscous aqueous solutions and its structure has been investigated by NMR spectroscopy. Notably, all aspects of the elucidation of its primary structure including component analysis and absolute configuration of the constituent monosaccharides were carried out by NMR spectroscopy. An array of techniques was utilized including, inter alia, PANSY and NOESY-HSQC TILT experiments. The EPS is composed of hexasaccharide repeating units with the following structure: → 3)[α-d-Glcp-(1 → 4)]-β-d-Galp-(1 → 4)-β-d-Glcp-(1 → 4)[β-d-Galf-(1 → 6)]-β-d-Glcp-(1 → 6)-β-d-Glcp-(1 →, in which the residues in square brackets are terminal groups substituting backbone sugar residues that consequently are branch-points in the repeating unit of the polymer. Thus, the EPS consists of a backbone of four sugar residues with two terminal sugar residues making up two side-chains of the repeating unit. The molecular mass of the polymer was determined using translational diffusion experiments which resulted in M_w = 62 kDa, corresponding to 64 repeating units in the EPS.     

Adhesion forces in the self-recognition of oligosaccharide epitopes of the proteoglycan aggregation factor of the marine sponge <Emphasis Type="Italic">Microciona prolifera</Emphasis>

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell aggregation activity of MAF depends on two functional domains: (i) a Ca²⁺-independent cell-binding domain and (ii) a Ca²⁺-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly demonstrated self-recognition on the disaccharide level in the presence of Ca²⁺-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides, atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca²⁺-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the absence of Ca²⁺-ions. Cd²⁺-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH₂)₃S(CH₂)₆S- did not show a binding in the presence of Ca²⁺-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation in the presence of Ca²⁺-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between the pyruvated trisaccharide.     

Cloning, expression in Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4-β-mannosidase from Aspergillus niger BK01

Background  

Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-β-mannosidases (1,4-β-D-mannanases) catalyze the random hydrolysis of β-1,4-mannosidic linkages in the main chain of β-mannans. Biodegradation of β-mannans by the action of thermostable mannan endo-1,4-β-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).     

Analysis of cDNA libraries from developing seeds of guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>(L.) Taub)

Background  

Guar, Cyamopsis tetragonoloba (L.) Taub, is a member of the Leguminosae (Fabaceae) family and is economically the most important of the four species in the genus. The endosperm of guar seed is a rich source of mucilage or gum, which forms a viscous gel in cold water, and is used as an emulsifier, thickener and stabilizer in a wide range of foods and industrial applications. Guar gum is a galactomannan, consisting of a linear (1→4)-β-linked D-mannan backbone with single-unit, (1→6)-linked, α-D-galactopyranosyl side chains. To better understand regulation of guar seed development and galactomannan metabolism we created cDNA libraries and a resulting EST dataset from different developmental stages of guar seeds.     

β-Fructofuranosidase and sucrose phosphorylase of rumen bacterium <Emphasis Type="Italic">Pseudobutyrivibrio ruminis</Emphasis> strain 3

The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained β-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7), while the bacteria maintained on sucrose showed only phosphorylase. Partially purified β-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis–Menten kinetics with V_max 5.64 μM/mg/min and K_m 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis–Menten kinetics. The V_max were 0.599 and 0.584 μM/mg/min, while K_m were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The V_max were, however, 1.37 and 1.023 μM/mg/min, while K_m were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 μM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.     

Lactosucrose production by various microorganisms harboring levansucrase activity

Production of the artificial sweetener, lactosucrose, by various microorganisms containing levansucrase activity was investigated. Of the tested bacteria, Bacillus subtilis was the most effective producer using lactose as an acceptor and sucrose as a fructosyl donor. Lactosucrose production by this strain was optimal at pH 6.0 and 55 °C whereupon 181 g lactosucrose l^–1 was produced from 225 g lactose l^–1 and 225 g sucrose l^–1 in 10 h.     

Exobiopolymer production of <Emphasis Type="Italic">Ophiocordyceps dipterigena</Emphasis>BCC 2073: optimization,production in bioreactor and characterization

Background  

Biopolymers have various applications in medicine, food and petroleum industries. The ascomycetous fungus Ophiocordyceps dipterigena BCC 2073 produces an exobiopolymer, a (1→3)-β- D -glucan, in low quantity under screening conditions. Optimization of O. dipterigena BCC 2073 exobiopolymer production using experimental designs, a scale-up in 5 liter bioreactor, analysis of molecular weight at different cultivation times, and levels of induction of interleukin-8 synthesis are described in this study.     

Novel oligosaccharide has suppressive activity against human leukemia cell proliferation

Various oligosaccharides containing galactose(s) and one glucosamine (or N-acetylglucosamine) residues with β1–4, α1–6 and β1–6 glycosidic bond were synthesized; Galβ1–4GlcNH₂, Galα1–6GlcNH₂, Galα1–6GlcNAc, Galβ1–6GlcNH₂, Galβ1–4Galβ1–4GlcNH₂ and Galβ1–4Galβ1–4GlcNAc. Galα1–6GlcNH₂ (MelNH₂) and glucosamine (GlcNH₂) had a suppressive effect on the proliferation of K562 cells, but none of the other saccharides tested containing GlcNAc showed this effect. On the other hand, the proliferation of the human normal umbilical cord fibroblast was suppressed by none of the saccharides other than GlcNH₂. Adding Galα1–6GlcNH₂ or glucosamine to the culture of K562 cell, the cell number decreased strikingly after 72 h. Staining the remaining cells with Cellstain Hoechst 33258, chromatin aggregation was found in many cells, indicating the occurrence of cell death. Furthermore, all of the cells were stained with Galα1–6GlcNH-FITC (MelNH-FITC). Neither the control cells nor the cells incubated with glucosamine were stained. On the other hand, when GlcNH-FITC was also added to cell cultures, some of them incubated with Galα1–6GlcNH₂ were stained. The difference in the stainability of the K562 cells by Galα1–6GlcNH-FITC and GlcNH-FITC suggests that the intake of Galα1–6GlcNH₂ and the cell death induced by this saccharide is not same as those of glucosamine. The isolation of the Galα1–6GlcNH₂ binding protein was performed by affinity chromatography (melibiose-agarose) and LC-MS/MS, and we identified the human heterogeneous ribonucleoprotein (hnRNP) A1 (34.3 kDa) isoform protein (30.8 kDa). The hnRNP A1 protein was also detected from the eluate(s) of the MelNH-agarose column by the immunological method (anti-hnRNP-A1 and HRP-labeled anti-mouse IgG (γ) antibodies).     

Characterization of the catalytic domains of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endoglucanase I,II, and III,expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.