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.     

Immobilization of cellulase through its carbohydrate side chains — A rationale for its recovery and reuse

The major types of components of cellulase [see 1,4-(1, 3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been adsorbed onto concanavalin A immobilized on Sepharose 4B, suggesting that they are glycoproteins. These components were covalently coupled to cyanogen bromide-activated Sepharose after aminoalkylation of their periodate-oxidized carbohydrate side chains to provide additional points of attachment of the enzyme to the support. Although there was only a 9% recovery of starting avicelase activity, the immobilized enzyme catalysed the hydrolysis of insoluble cellulose to glucose with greater efficiency than did free cellulase.     

Lignocellulose biotransformation with immobilized cellulase,d-glucose oxidase and fungal peroxidases

Three enzymes, cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], d-glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) immobilized on glass beads, have been incubated with lignocellulose. Fungal peroxidases from Trametes versicolor and Inonotus radiatus when mixed with cellulase and d-glucose oxidase were able to liberate phenolic compounds and d-glucose from lignocellulose. Three lignin monomers were identified. When the immobilized enzymes were incubated individually with lignocellulose they did not degrade lignin.     

Pretreatment of starch raw materials and their enzymatic hydrolysis by immobilized glucoamylase

The pretreatment of starch raw materials such as sweet potato, potato and cassava has been carried out using various types of crusher, viz juice mixer, homogenizer and high-speed planetary mill. The effect of pretreatment of the materials on their enzymatic hydrolysis was studied. High-speed planetary mill treatment was the most effective and comparable with heat treatment (pasting). Various crushing times were used to examine the effect of crushing by mill treatment on the enzymatic hydrolysis. In the enzymatic hydrolysis of cassava, the use of both cellulase [1,4-(1,3; 1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and glucoamylase [1,4-α-d-glucan glucohydrolase, EC 3.2.1.3] enhanced the d-glucose yield. The immobilization of glucoamylase was studied by radiation polymerization of hydrophilic monomers at low temperature, and it was found that enzymatic activity of the immobilized glucoamylase particles varied with monomer concentration and particle size. Starchy raw materials pretreated with the mill can be efficiently hydrolysed by immobilized glucoamylase.     

Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed.     

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.     

Application of cellulase and pectinase from fungal origin for the liquefaction and saccharification of biomass

Commercial cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma viride and pectinase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15] from Aspergillus niger have been applied to produce fermentation syrups from sugar-beet pulp and potato fibre. Cellulosic, hemicellulosic and pectic polysaccharides of these substrates were hydrolysed extensively. Recovery of enzymes has been investigated in a packed-column reactor, connected with a hollow-fibre ultrafiltration unit. Enzymes appeared to be stable in this type of reactor, although part of the enzyme activity was lost, especially by adsorption onto the substrate residue.     

Surface deactivation of cellulase and its prevention

When cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was exposed to air-liquid interface and subjected to shear, a significant deactivation was observed. The cellulase deactivation due to the interfacial effect combined with the shear effect was found to be far more severe and extensive than that due to the shear effect alone. Both increased cellulase concentration, and addition of surfactant (Zonyl or Triton) reduced the degree of deactivation. By using sufficient surfactant the cellulase deactivation can be prevented, and the cellulase can be stabilized and its use prolonged. The ratio of surface excess to the bulk protein is significantly reduced when the enzyme concentration is increased. The stabilizing effect of surfactant was attributed to the reduction in surface excess of cellulase.     

Effect of attached carbohydrates on the activity of Trichoderma viride cellulases

Trichoderma viride 1,4-β-d-glucan cellobiohydrolase (exo-cellobiohydrolase, 1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) purified from a commercial product to electrophoretic homogeneity by a procedure including affinity and DEAE-Sephadex chromatography, has attached carbohydrates in addition to the glycoprotein constituents. These carbohydrates are lost by consecutive gel filtration steps in Sephadex G-25 columns, whereupon there is a rapid increase in enzymatic activity. A single gel filtration step can eliminate d-glucose or cellobiose added to a solution of this enzyme, but not the carbohydrates attached during incubation with Avicel.After free carbohydrate elimination from crude cellulase complexes by Sephadex G-25 chromatography, liberation of d-glucose following incubation at 50°C and pH 4.8 was observed. This indicates that some carbohydrates remain bound after gel filtration. The elimination of carbohydrate from whole cellulase complex [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was favoured by a yeast treatment, with a simultaneous increase in activity, but the process is not reproducible, as a secondary inactivation process exists.     

Increases in endo-1,4-β-d-glucanase titres produced by Aspergillus fumigatus through application of simple statistical methods

Yields of endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] produced by Aspergillus fumigatus IMI 255091 in shake flask cultures have been improved through application of a form of evolutionary operation incorporating a standard factorial design. This approach gave considerable improvements in yield, up to the point at which the limitations of the shake flask technique were noticeable. Further improvements then resulted from use of a 5 litre disc-turbine agitated fermenter.     

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml^?1 at productivities >130 U l^?1 h^?1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.     

Studies on enzymic methods for extraction of inulin from Jerusalem artichokes

The release of inulin, d-fructose and protein from Jerusalem artichokes has been studied under diffusion and maceration conditions. The effects on release of added inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), protease and cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] enzymes have been investigated. The results indicate that added enzymes do not improve the efficiency of inulin or d-fructose release and that mechanical methods represent the most efficient means of carbohydrate solubilization. Treatment of plant extracts with inulinase is shown to have the disadvantage of increasing peptide solubilization. The potential for improved inulin solubilization by use of endo-inulinases is discussed.     

Action of Trichoderma reesei QM9414 and C30 cellulase on substrates with varying crystallinity

The action of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations from Trichoderma reesei QM9414 and C30 has been compared on Sigmacell, Solka Floc and alkali-treated bagasse in the presence and absence of added d-glucose and cellobiose. On the basis of equal filter paper activity the two preparations acted similarly on the two cellulosic substrates, while in the case of alkali-treated bagasse the C30 preparation gave greater d-glucose release. The relative levels of cellobiose produced from alkali-treated bagasse suggests that the non-cellobiose route was more important in d-glucose release by the C30 preparation compared to the QM9414 preparation.     

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.     

Major characteristics of the cellulolytic system of Clostridium thermocellum coincide with those of the purified cellulosome

The properties of the cellulosome (a cellulose-binding, multiple cellulase-containing protein complex isolated from Clostridium thermocellum) have been compared with the previously reported characteristics for crude cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations. Similar to the crude enzyme system, true cellulolytic activity was demonstrated for the purified cellulosome on the basis of extensive solubilization of microcrystalline cellulose. The cellulolytic activity of the purified cellulosome was enhanced both by calcium ions and by thiols, and was inhibited by cellobiose (the major end product of the cellulosome-mediated cellulose degradation). In addition, at low ionic strength, cellulose-adsorbed cellulosome was detached intact from the cellulose matrix. Using controlled conditions, maximum enzymatic activity was shown to correspond to suboptimal conditions of cellulosome adsorption to cellulose. The results suggest that previous data accumulated for the crude cellulase system in C. thermocellum essentially reflect the contribution of the cellulosome.     

Occurrence of a procellulase in the culture filtrates of Penicillium janthinellum

The endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3:1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] activity of two-day old culture filtrates of Penicillium janthinellum has been enhanced four-fold by incubating with a 10-day old culture filtrate of Penicillium funiculosum grown on the same medium. An inactive protein isolated by fractionation of two-day old culture filtrate of P. janthinellum using preparative isoelectric focusing, showed 30- to 50-fold enhancement of endo-1,4-β-d-glucanase activity. This fraction has been designated the ‘procellulase’ in the present paper. The purity of the procellulase was confirmed by analytical isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It had a molecular weight of 68 000 and an isoelectric point of pH 3.7.     

Improvements of the bioavailability of straw: acid and enzyme hydrolysis of wheat straw pretreated with saturated lithium chloride in hydrochloric acid

Wheat straw was pretreated with saturated lithium chloride in 4 m hydrochloric acid at 50°C for 1 h, then hydrolysed at 100°C for 1 min, to give 84% conversion to monosaccharides. Particle sizes, 150–355 mesh, were easily hydrolysed. Samples pretreated with saturated lithium chloride in 1 m hydrochloric acid at 27°C for 24 h were hydrolysed by Trichoderma viride cellulase (MVA 1284) [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] to give 20–23% monosaccharides for particle sizes of 150–250 mesh, and 82–95.4% for particle sizes of 250–355 mesh.     

Effect of peracetic acid, sodium hydroxide and phosphoric acid on cellulosic materials as a pretreatment for enzymatic hydrolysis

Crystalline cellulose and cellulosic wastes have been treated with various concentrations of peracetic acid and other reagents at 100°C for various times, washed with water, ethanol and air dried. For each treated cellulose, the degree of enzymatic solubilization was measured with Trichoderma viride cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4]. Cellulosic wastes such as sunflower stalks, wheat straw and sugar-cane bagasse were solubilized effectively by the enzyme. Delignification of wheat straw with 1% sodium hydroxide and treatment of this straw with peracetic acid enhanced the degree of enzymatic solubilization. Infrared spectra of the untreated and treated cellulosic wastes were recorded.     

Comparison of the 3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying for reducing sugars and determining cellulase activity

A comparison has been made between the 3,5-dinitrosalicylic acid (DNS) and alkaline copper methods of assaying for reducing sugars released during the enzymatic hydrolysis of cellulose by culture filtrates from Trichoderma harzianum E58. The DNS method was shown to be more readily influenced by the incubation conditions and by components derived from lignocellulosic substrates. The endo-1,4-β-d-glucanase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] values obtained with the DNS assay were always considerably higher than those obtained with the alkaline copper method and did not give reducing values that were proportional to the actual number of hemiacetal reducing groups. The alkaline copper assay was not affected by the degree of polymerization of the substrate. Although this latter method appeared to be superior to the DNS assay it was still affected by the incubation conditions, nature of the substrate and the influence of other cellulase components on each of the specific enzyme assays.     

Production of xylanolytic enzymes in association with the cellulolytic activities of Penicillium funiculosum

Penicillium funiculosum produced 16 and 0.4 units ml^?1 of d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37), respectively, in shake flasks. Both enzymes were 100% stable when heated at 50°C for 30 min and on prolonged heating d-xylanase and β-d-xylosidase showed 46 and 20% loss, respectively. Maximum hydrolysis (75%) of d-xylan was obtained when the end products were removed. The addition of β-d-xylosidase markedly influenced the degree of hydrolysis of d-xylan. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylose, d-xylobiose, d-xylotriose, d-xylotetraose, d-xylopentose and l-arabinose. The fractionation of culture filtrate of Penicillium funiculosum grown on cellulose powder or in a combination of cellulose powder and wheat bran indicated the presence of two d-xylanases. The role of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase on the overall hydrolysis of pure cellulose and lignocellulosic substrates is discussed.