Production of cellulases and d-xylanase by some selected fungal isolates

Cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], β-d-glucosidase (β-d-glucoside glucanohydrolase, EC 3.2.1.21) and d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) production by Aspergillus ustus, Sporotrichum pulverulentum, Trichoderma sp. (a), Trichoderma sp. (b) and Botrytis sp. in solid state fermentation on different compounded media containing wheat bran (WB), rice straw (RS) and minerals was studied. Toyama's mineral solution mixed with RS was found to be a better substrate for cellulase and d-xylanase while with WB it induced higher β-d-glucosidase production. A ratio of substrate to mineral solution (w/v) of 1:4 or 1:5 supported high d-xylanase and cellulase production whereas a ratio of 1:2 gave the highest β-d-glucosidase activity. Among the fungal isolates, Aspergillus ustus gave the highest β-d-glucosidase activity of 60 U g⁻¹WB and the highest d-xylanase activity of 740 U g⁻¹was obtained with RS. A mixture of seven parts of RS and three of WB, mixed with 40 parts of Toyama's mineral solution yielded 6 U filter paper activity, 40 U β-d-glucosidase, 12 U carboxymethylcellulase and 650 U d-xylanase g⁻¹substrate.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Fungal and other β-d-glucosidases — Their properties and applications

β-d-Glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) has been described in a variety of fungi and bacteria. Its function — to catalyse the hydrolysis of cellobiose, and aryl and alkyl β-d-glucosides — depends upon the nature of its source. Recent interest in this enzyme centres on its role in the enzymatic hydrolysis of cellulose. The rate and extent of cellulose hydrolysis can be increased by supplementing commercial cellulases with immobilized β-d-glucosidase, which has high stability and can be recovered and reused. The current state of β-d-glucosidase biotechnology is described.     

β-d-Glucosidase stabilization in a polarized ultrafiltration membrane reactor

Cellobiose hydrolysis by β-d-glucosidase (β-d-glucoside glucohydrolase EC 3.2.1.21) can become the rate-limiting step in the hydrolysis of cellulosic wastes because of inhibition phenomena involving other enzymes of the cellulase complex. Enhancement of the overall rate can therefore be obtained by increasing the amount of β-d-glucosidase present in the reactor. Unfortunately, the thermal stability of β-d-glucosidase is rather poor compared to $\text{endo}\text{-(1 → 4)-β-}\text{d}\text{-}\text{glucanase}$ and cellobiohydrolase. A novel stabilization method is proposed that exploits the polarization phenomena that take place in an unstirred ultrafiltration membrane enzymatic reactor. As much as a 20-fold increase in half-life compared to the native enzyme is obtained by injecting small amounts of hydroxyethyl cellulose into the system. No reduction in enzyme activity levels is observed.     

Enhancement of amygdalin activated with β-d-glucosidase on HepG2 cells proliferation and apoptosis

The growth inhibition and induction of apoptosis brought by amygdalin and activated with β-d-glucosidase were tested for cytoactivity in HepG2 cells. The MTT viability assay showed that all samples had effects on HepG2 proliferation in dose and time response manners. IC₅₀ of stand-alone amygdalin and activation with β-d-glucosidase on the proliferation of HepG2 cells for 48 h were 458.10 mg/mL and 3.2 mg/mL, respectively. Moreover, apoptotic cells were determined by AO/EB (acridine orange/ethidium bromide) fluorescent staining method and Annexin V-FITC/PI staining flow cytometry cell cycle analysis. With increasing of amygdalin concentration and the incubation time, the apoptotic rate was heightened. Compared with the control, there was significant difference (p < 0.01). Together, these findings indicate that amygdalin had no strong anti-HepG2 activity; however the ingredients of amygdalin activated with β-d-glucosidase had a higher and efficient anti-HepG2 activity. It was therefore suggested that this combination strategy may be applicable for treating tumors with a higher activity.     

Simultaneous saccharification and fermentation of cellulose: effect of β-d-glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β-d-glucosidase to filter paper activity was ～1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ^?1, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

β-d-Glucosidase: multiplicity of activities and significance to enzymic saccharification of cellulose

Activity measurement of protein components resolved by polyacrylamide gel electrophoresis has indicated that the β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) system is composed of multiple enzymes, each differing in substrate specificity. The proportions of these enzymes varied in preparations obtained from Aspergillus phoenicis, almond emulsion and Trichoderma reesei. The enzyme component showing the highest cellobiase activity was most useful in improving cellulose saccharification by Trichoderma cellulases. The optimum ratio between filter paper and cellobiase activities, expressed in the appropriate units, was 1.0:0.9. The results indicate that for saccharification purposes, the β-d-glucosidase activity should be measured using cellobiose as a substrate, rather than salicin, esculin or 4-nitrophenyl-β-d-glucopyranoside.     

Acid-catalyzed and almond β-d-glucosidase-catalyzed hydrolysis of purin-6-yl 2-deoxy-1-thio-β-d-arabino-hexopyranoside

Hydrolysis of purin-6-yl 2-deoxy-1-thio-β-d-arabino-hexopyranoside (2) to 6-mercaptopurine and 2-deoxy-d-glucose is catalyzed by hydronium ion and almond β-d-glucosidase. The dependence of rate on acidity in water and deuterium oxide indicates that 2 and its conjugate acid undergo hydrolysis via a mechanism that involves a partially rate-limiting proton transfer. Although 2 is ≈103 more reactive than 6-purinyl β-d-glucothiopyranoside (1) in dilute aqueous acid, 1 is a better substrate for almond β-d-glucosidase.     

Enzyme stabilization towards thermal,chemical and proteolytic deactivation

An enzyme stabilization technique which consists of entrapping protein within a polymeric network has been discussed. The high macromolecular concentration levels which lead to formation of the network are produced as a consequence of polarization phenomena which take place within an unstirred ultrafiltration membrane reactor. Increases in enzyme half-life were generally produced in connection with simple and complex deactivation phenomena of widely different natures (thermal, chemical and proteolytic). Experimental tests have been carried out on the following enzymes: β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21), β-d-fructofuranosidase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26), acid phosphatase [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] and β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23).     

Optimization of the composition of the nutrient medium for cellulase and protein biosynthesis by thermophilic Aspergillus fumigatus NRC 272

An active strain of Aspergillus spp. has been selected for the production of cellulolytic enzymes and proteins when grown on peracetic acid-treated wheat straw. This strain produced a considerable amount of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] in the extracellular supernatant and exhibited good overall cellulolytic activity, as measured using filter paper and Avicel as substrates. Also, under the same conditions the strain showed high activities of β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37). The maximum enzyme yields (carboxymethylcellulose activity 26.4 units ml^?1, filter paper activity 2.26 units ml^?1 and Avicel activity 16.82 units ml^?1; β-d-glucosidase 9.09 units ml^?1 and β-d-xylosidase 1.92 units ml^?1) were obtained after 96 h incubation at 45°C.     

β-d-glucosidase activity in developing leaves of Nicotiana tabacum

Tobacco (Nicotiana tabacum L. cv. Burley 21) leaves were assayed for β-d-glucosidase activity, using esculin as substrate. The enzymatically produced esculetin was silylated and quantitatively measured by GLC, using a tritium foil electron-capture detector. In field-grown plants, the activity in mid-stalk leaves increased with plant maturation; conversely, the activity in the top leaves decreased.     

Immobilization of enzymes on chitin and chitosan

During the last few years, d-glucose isomerase, glucoamylase, β-d-galactosidase (lactase), β-d-glucosidase, d-glucose oxidase, AMP deaminase, urease, pronase, subtilisin, trypsin, papain, alkaline phosphatase, acid phosphatase, pepsin, chymotrypsin and lysozyme have been immobilized on chitin and on some of its derivatives, mainly with glutaraldehyde. The preparation and performances of the immobilized enzymes are described.     

Mixed inhibition of β-d-glucosidase from Stachybotrys atra by substrate analogues

The binding of series of alkyl and aryl β-d-glycopyranosides and their 1-thio analogues to the active site of β-d-glucosidase from Stachybotrys atra has been investigated. The binding constants for competitive and uncompetitive inhibition clearly demonstrated the existence of a hydrophobic aglycon-binding-site. The correlations found between competitive and uncompetitive inhibition suggest that the latter type of inhibition originates from the unspecific binding of the aglycon group to the aglycon binding-site of the intermediary enzyme-glycosyl complex.     

Enzyme activities of aerobic lignocellulolytic bacteria isolated from wet tropical forest soils

Lignocellulolytic bacteria have promised to be a fruitful source of new enzymes for next-generation lignocellulosic biofuel production. Puerto Rican tropical forest soils were targeted because the resident microbes decompose biomass quickly and to near-completion. Isolates were initially screened based on growth on cellulose or lignin in minimal media. 75 Isolates were further tested for the following lignocellulolytic enzyme activities: phenol oxidase, peroxidase, β-d-glucosidase, cellobiohydrolase, β-xylopyranosidase, chitinase, CMCase, and xylanase. Cellulose-derived isolates possessed elevated β-d-glucosidase, CMCase, and cellobiohydrolase activity but depressed phenol oxidase and peroxidase activity, while the contrary was true of lignin isolates, suggesting that these bacteria are specialized to subsist on cellulose or lignin. Cellobiohydrolase and phenol oxidase activity rates could classify lignin and cellulose isolates with 61% accuracy, which demonstrates the utility of model degradation assays. Based on 16S rRNA gene sequencing, all isolates belonged to phyla dominant in the Puerto Rican soils, Proteobacteria, Firmicutes, and Actinobacteria, suggesting that many dominant taxa are capable of the rapid lignocellulose degradation characteristic of these soils. The isolated genera Aquitalea, Bacillus, Burkholderia, Cupriavidus, Gordonia, and Paenibacillus represent rarely or never before studied lignolytic or cellulolytic species and were undetected by metagenomic analysis of the soils. The study revealed a relationship between phylogeny and lignocellulose-degrading potential, supported by Kruskal–Wallis statistics which showed that enzyme activities of cultivated phyla and genera were different enough to be considered representatives of distinct populations. This can better inform future experiments and enzyme discovery efforts.     

Solvent effect on enzyme-catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β-d-glucopyranosides

Almond β-d-glucosidase was used to catalyze alkyl-β-d-glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β-d-glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.     

A wall-bound exo-1,3-β-d-glucanase from Acacia cultured cells

Several wall-bound exo-1,3-β-d-glucanases have been solubilized by 4 M LiCl from suspension-cultured Acacia cells. One exhibits both exo-laminarinase (EC 3.2.1.39) and β-d-glucosidase (EC 3.2.1.21) activities and has been purified up to 30-fold by anion-exchange chromatography, gel filtration and flat-bed electrofocusing. This enzyme hydrolyses laminarin, laminaribiose and p-nitrophenyl-β-d-glucopyranoside. The enzyme, with a pI of 4.6, is apparently homogenous, since it behaves as a single protein with an apparent molecular weight of 62000 on SDS-polyacrylamide gel electrophoresis. Its K_m value in 0.1 M acetate buffer (pH 5.0) with p-nitrophenyl-β-d-glucopyranoside as substrate was 0.27 mM; with laminarin as substrate the K_m expressed in glucosyl residue concentration was 0.64 mM. Other kinetic experiments showed that exo-laminarinase and β-d-glucosidase activities correspond to two distinct catalytic sites in the same protein.     

β-d-glucosidase-catalysed transfer of the glycosyl group from aryl β-d-gluco- and β-d-xylo-pyranosides to phenols

The effect of phenols on the hydrolysis of substituted phenyl β-d-gluco- and β-d-xylo-pyranosides by β-d-glucosidase from Stachybotrys atra has been investigated. Depending on the glycon part of the substrate and on the phenol substituent, the hydrolysis is either inhibited or activated. With aryl β-d-xylopyranosides, transfer of the xylosyl residue to the phenol, with the formation of new phenyl β-d-xylopyranosides, is observed. With aryl β-d-glucopyranosides, such transfer does not occur when phenols are used as acceptors, but it does occur with anilines. A two-step mechanism, in which the first step is partially reversible, is proposed to explain these observations. A qualitative analysis of the various factors determining the overall effect of the phenol is given.     

Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β-d-glucosidase (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β-d-glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. d-Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. d-Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while d-fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β-d-glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Purification and characterisation of two endo-(1→3)-β-d-glucanases from Telescopium telescopium

The crystalline style of the gastropod Telescopium telescopium contains two (1→3)-β-d-glucanases and a β-d-glucosidase. The two glucanases (I and II) have been purified and shown to be endo-enzymes. Both enzymes attack laminarin, carboxymethylpachyman, and lichenin, but have no action towards carboxymethyl-cellulose. The main products of hydrolysis of laminarin are d-glucose and β-(1→3)-linked oligosaccharides of d.p. 2, 3, and 4. Glucanases I and II are similar to each other, although they differ in molecular weight and kinetic properties.     

Kinetic characterization of a crude β-d-glucosidase from Aspergillus wentii Pt 2804

The kinetic characteristics of β-d-glucosidase (cellobiase, β-d-glucosidase glucohydrolase, EC 3.2.1.21) from the filtered broth of a well grown culture of Aspergillus wentii have been studied. Both cellobiose and 4-nitrophenyl-β-d-glucoside (4NPG) were used as substrates and values of K_m, V_max for both the substrates were determined. Activity was maximum over a pH range of 4.5–5.5 but declined sharply beyond 5.5 for both substrates. The optimum temperature was between 60 and 65°C. Half-life of the cellobiase was ～38.0 h at 60°C and ～6.3 h at 65°C. However, the enzyme was found to be quite stable at 50°C. The activation and deactivation energies for 4NPG hydrolysis were 33.2 and 111.3 kJ mol^?1 K^?1, and 43.6 and 63.7 kJ mol K^?1 for cellobiose hydrolysis. Product inhibition was found to be of the competitive type. Preliminary experiments showed that marked synergistic activity exists between Trichoderma reesei and A. wentii cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] for cellulose hydrolysis.