Functional characteristics of two d-xylanases purified from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) were purified from Trichoderma harzianum culture filtrates. From kinetic analyses, apparent V_max and K_m values of 580 U mg^?1 protein and 0.16% d-xylan were obtained for the 20 000 dalton endo-1,4-β-d-xylanase, while values of 100 U mg^?1 protein and 0.066% d-xylan were obtained for the 29 000 dalton endo-1,4-β-d-xylanase. Substrate levels >1% (w/v) d-xylan were found to be inhibitory to both enzymes. Both d-xylanases were highly active against d-xylans obtained from various sources. Of the polymeric sugars tested, carboxymethyl cellulose was the only substrate which was hydrolysed to any extent. Little or no activity was observed against cellulose. Analyses by h.p.l.c. demonstrated the absence of hydrolytic activity by both d-xylanases on d-xylobiose. d-Xylotriose was cleaved to a limited extent by the 29 000 dalton d-xylanase only, while d-xylotetraose was hydrolysed by both. In the presence of d-xylotetraose, the 20 000 dalton d-xylanase had an associated transxylosidase activity which was not observed with the 29 000 dalton enzyme. When the solubilization assay was used, neither of the d-xylanases was inhibited by high concentrations of d-xylose and xylobiose.     

Extracellular β-d-xylosidase of Sclerotium rolfsii

Culture conditions are described for the production of extracellular β-d-xylosidase (xylobiase, exo-1,4-β-d-xylosidase, 1,4-β-d-xylan xylohydrolase, EC 3.2.1.37) in shake flasks by Sclerotium rolfsii. At the 1% cellulose level, a maximum activity of 0.82 U ml^?1is obtained in media containing either 1% corn steep liquor or 1% defatted coconut cake. The β-d-xylosidase has a molecular weight of 170 000 and catalyses the hydrolysis of 4-nitrophenyl-β-d-xylopyranoside optimally at pH 4.5 and 50°C. The energy of activation is 44 kJ mol^?1and the pI and K_mare 6.8 and 0.038 mm, respectively.     

Immobilization of cellulase and d-xylanase complexes from Aspergillus terreus F-413 on controlled porosity glasses

The major components of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase (see 1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. The final preparations contained over 20 mg protein g^?1 glass beads. The activity retained was 71.6–98.1% for cellulase complexes and 81–100% for d-xylanase complexes. The immobilization of the enzymes spread their optimum pH range. Cellulose and d-xylan were quantitatively hydrolysed by the immobilized enzymes. The major reaction products were identified as a d-glucose and d-xylose respectively.     

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.     

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.     

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 properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 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).     

Purification and characterization of two d-xylanases from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) from Trichoderma harzianum E58 have been purified by ultrafiltration and chromatography on carboxymethyl-Sepharose, phenyl-Sepharose and Sephadex G-75. The d-xylanases were shown to be homogeneous by the criteria of dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights were estimated to be 20 000 and 29 000, with pl values of 9.4 and 9.5, respectively. Typically, 456 mg of the 20 000 dalton and 1.9 mg of the 29 000 dalton d-xylanases were purified from 4.2 litre of culture filtrate with specific activities of 370 and 75 U mg^?1, respectively. Optimum d-xylanase activities were obtained when the enzymes were incubated at pH 5, 50°C, for the 20 000 dalton protein and pH 5, 60°C for the 29 000 dalton protein.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.     

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.     

Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp

Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the β-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-β-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce 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]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

Molecular cloning of Bacillus polymyxa (1→4)-β-d-xylanase gene in Escherichia coli

Escherichia coli has been transformed with recombinant plasmids carrying DNA from Bacillus polymyxa. A sensitive and simple immunoassay was used to screen for E. coli transformed with the chimeric plasmid carrying a (1→4)-β-d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) gene. One such clone was isolated. The enzyme present in extracts of these E. coli cells was active in catalysing the hydrolysis of (1→4)-β-d-xylo-oligosaccharides of identified structure from the mucilage of Plantago ovata Forsk, as indicated by the production of reducing sugars. This work holds promise for the large-scale production of xylanases as a new route to the economic and usable degradation of xylans and is a model for the large-scale production of enzymes.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

Studies on cellulolytic enzymes I: The use of 3,4-dinitrophenyl glycosides as substrates

The syntheses of 3,4-dinitrophenyl β-d-glucoside, β-cellobioside, β-cellotrioside, and β-cellotetraoside and their use to monitor the purification of two enzymes from a crude commercial cellulase preparation from Trichoderma viride are described. The enzymes isolated are an endo-β-1,4-d-glucan glucanohydrolase (EI) of molecular weight ca. 12 000 which catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenol-β-cellotetraoside, and an enzyme of molecular weight about 76 000 which catalysed the hydrolysis of 3,4-dinitrophenyl β-d-glucoside (EII) and is probably a cellobiase or exo-β-1,4-d-glucan glucohydrolase. Kinetic parameters are reported for the hydrolyses of 3,4-dinitrophenyl β-cellobioside, β-cellotrioside, and β-cellotetraoside catalysed by enzyme EI. In the presence of cellotriose, cellotetraose, or cellopentaose 3,4-dinitrophenyl β-d-glucoside underwent induced hydrolyses by EI. Similar but faster induced hydrolyses were shown by 3,4-dinitrophenyl β-d-xyloside and 3,4-dinitrophenyl β-d-6-deoxyglucoside; 3,4-dinitrophenyl 6-chloro-6-deoxy-β-d-glucoside and 3,4-dinitrophenyl 6-O-methyl-β-d-glucoside underwent slower induced hydrolyses than the glucoside. p-Nitrophenyl β-d-glucoside also underwent an induced hydrolysis in the presence of cellopentaose and the enzyme EI, but p-nitrophenyl 2-deoxy-β-d-glucoside did not. These results are discussed and compared with the results obtained previously on induced hydrolyses found with lysozyme. Kinetic parameters are reported for the hydrolysis of 3,4-dinitrophenyl and p-nitrophenyl β-d-glucosides catalysed by the enzyme EII. 3,4-Dinitrophenyl 6-deoxy-β-d-glucoside, β-d-xyloside, 6-chloro-6-deoxy-β-d-glucoside, 6-O-methyl-β-d-glucoside and p-nitrophenyl-β-d-galactopyranoside and 2-deoxy-β-d-glucopyranoside were hydrolysed 10² to 10³ times slower by EII than the corresponding glucosides, but 3,4-dinitrophenyl 2-acetamido-2-deoxy-β-d-glucoside was only hydrolysed about 25 times slower than 3,4-dinitrophenyl β-d-glucoside. The significance of these results is discussed. EII catalysed the release of 3,4-dinitrophenol from 3,4-dinitrophenyl β-cellobioside, β-cellobioside, and β-cellotetraoside, but these reactions showed induction periods which are consistent with stepwise removal of glucose residues from the oligosaccharide chains before release of the phenol.     

Purification and properties of cellulase enzymes from Robillarda sp. Y-20

One endo-β-1,4-glucanase (EC 3.2.1.4) and two unique β-glucosidases (EC 3.2.1.21) have been isolated from culture filtrates Robillarda sp. Y-20 by combinations of DEAE A-50 column chromatography and isoelectric focusing. These enzymes were homogeneous on gel filtration, isoelectric focusing and polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate (SDS). The molecular weights of endoglucanase, and the two β-glucosidases, I and II by SDS-polyacrylamide gel electrophoresis were 59000, 76000 and 54000, respectively. The pI values were 3.5, 7.5, and 3.8 for endoglucanase, β-glucosidase I and II, respectively. The major β-glucosidase I was a glycoprotein, but the endoglucanase and β-glucosidase II were not. The endoglucanase rapidly reduced the viscosity of carboxymethyl (CM) cellulose with concomitant production of reducing sugar. The enzyme had very low activity with crystalline cellulose such as insoluble acid treated cellulose, Avicel and filter paper. The endoglucanase attacked celloheptaose to cellotetraose more readily than cellotriose, but did not hydrolyze cellobiose. Both β-glucosidases attacked celloheptaose to cellotetraose more readily than cellotriose and cellobiose, but did not hydrolyze CM-cellulose and insoluble acid treated cellulose. Strong synergism was observed for hydrolysis of CM-cellulose by the endoglucanase and β-glucosidases.     

Hydrolysis of cellobiose using β-glucosidase from Penicillium funiculosum. Kinetic analysis

The kinetics of cellobiose hydrolysis was studied using β-glucosidase from Penicillium funiculosum, both free and immobilized on nylon powder, at different temperatures, pH values, enzymatic activities and initial cellobiose and glucose concentrations. The experimental results were fitted to a kinetic model by considering the substrate and product inhibitions as well as the thermal deactivation of β-glucosidase with a mean deviation of less than 10%. The immobilization of β-glucosidase led to an increase in the stability of the enzyme against changes in the pH value.     

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.     

Kinetics of the enzymatic hydrolysis of cellulose: 2. A mathematical model for the process in a plug-flow column reactor

The kinetics of enzymatic cellulose hydrolysis in a plug-flow column reactor catalysed by cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma longibrachiatum adsorbed on cellulose surface have been studied. The maximum substrate conversion achieved was 90–94%. The possibility of enzyme recovery for a reactor of this type is discussed. A mathematical model for enzymatic cellulose hydrolysis in a plug-flow column reactor has been developed. The model allows for the component composition of the cellulase complex, adsorption of cellulases on the substrate surface, inhibition by reaction products, changes in cellulose reactivity and the inactivation of enzymes in the course of hydrolysis. The model affords a reliable prediction of the kinetics of d-glucose and cellobiose formation from cellulose in a column reactor as well as the degree of substrate conversion and reactor productivity with various amounts of adsorbed enzymes and at various flow rates.