Cellulolytic Activity of Thermomonospora curvata: Nutritional Requirements for Cellulase Production

The use of a minimal medium for cellulase (C(1) and C(x)) production by Thermomonospora curvata increased extracellular C(1) activity (measured by rate of cotton fiber hydrolysis) 11-fold compared with the previously used yeast extract medium. Ground cotton fibers supported the highest cellulase production when compared to other soluble and insoluble carbohydrate sources. Maximal cellulase production occurred at 45 C, slightly less at 55 C, and was insignificant at 65 C (the highest temperature at which cellulase activity appeared stable). At a temperature of 55 C, an optimal pH of 8.0, and a cotton fiber concentration of 8 mg/ml, shake cultures of T. curvata degraded about 75% of the cellulose during the 10-day period.     

Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell-free broth from Clostridium thermocellum

The cellulase activity in cell-free broths from the thermophilic, ethanol-producing anaerobic bacterium Clostridium thermocellum is examined on both dilute-acid-pretreated mixed hardwood (90% maple, 10% birch) and Avicel. Experiments were conducted in vitro in order to distinguish properties of the cellulase from properties of the organism and to evaluate the effectiveness of C. thermocellum cellulase in the hydrolysis of a naturally occurring, lignin-containing substrate. The results obtained establish that essentially quantitative hydrolysis of cellulose from pretreated mixed hardwood is possible using this enzyme system. Pretreatment with 1% H(2)SO(4) and a 9-s residence time at 220, 210, 200, and 180 degrees C allowed yields after enzymatic hydrolysis (percentage of glucan solubilized/ glucan potentially solubilized) of 97.8, 86.1, 82.0, and 34.6%, respectively. Enzymatic hydrolysis of mixed hardwood with no pretreatment resulted in a yield of 10.1%. Hydrolysis yields of >95% were obtained from approximately 0.6 g/L mixed hardwood pretreated at 220 degrees C in 7 h at broth strengths of 60 and 80% (v/v) and in approximately 48 h with 33% broth. Hydrolysis of pretreated mixed hardwood is compared to hydrolysis of Avicel, a pure microcrystalline cellulose studied previously. The initial rate of Avicel hydrolysis saturates with respect to enzyme, whereas the initial rate of hydrolysis of pretreated wood is proportional to the amount of enzyme present. Initial hydrolysis rates for pretreated wood and Avicel at 0.6 g/L are greater for wood at low broth dilutions (1.25: 1 to 5 :1) by up to 2.7-fold and greater for Avicel at high broth dilutions (5 : 1 to 50 : 1) by up to 4.3-fold. Maximum rates of hydrolysis are achieved at <2 g substrate/L for both pretreated wood and Avicel. The substrate concentration at one-half the maximum observed rate for C. thermocellum broths is smaller for pretreated mixed hardwood than for Avicel and decreases with increasing broth dilution for both substrates. An initial activity per volume broth of approximately 11 mumol soluble glucose equivalent produced/L broth/min is observed for mixed hardwood pretreated at 220 degrees C and for Avicel at high broth dilutions; the initial activity per volume broth for Avicel is lower at low broth dilutions. The results indicate that pretreated wood is hydrolyzed at rates comparable to Avicel under many conditions and at rates significantly faster than Avicel under several conditions.     

Effect of cellulose physical characteristics, especially the water sorption value, on the efficiency of its hydrolysis catalyzed by free or immobilized cellulase

Cellulase, an enzymatic complex that synergically promotes the degradation of cellulose to glucose and cellobiose, free or adsorbed onto Si/SiO₂ wafers at 60 °C has been employed as catalyst in the hydrolysis of microcrystalline cellulose (Avicel), microcrystalline cellulose pre-treated with hot phosphoric acid (CP), cotton cellulose (CC) and eucalyptus cellulose (EC). The physical characteristics such as index of crystallinity (I_C), degree of polymerization (DP) and water sorption values were determined for all samples. The largest conversion rates of cellulose into the above-mentioned products using free cellulase were observed for samples with the largest water sorption values; conversion rates showed no correlation with either I_C or DP of the biopolymer. Cellulose with large water sorption value possesses large pore volumes, hence higher accessibility. The catalytic efficiency of immobilized cellulase could not be correlated with the physical characteristics of cellulose samples. The hydrolysis rates of the same cellulose samples with immobilized cellulase were lower than those by the free enzyme, due to the diffusion barrier (biopolymer chains approaching to the immobilized enzyme) and less effective contact between the enzyme active site and its substrate. Immobilized cellulase, unlike its free counterpart, can be recycled at least six times without loss of catalytic activity, leading to higher overall cellulose conversion.     

Optimization of cellulase production by <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>: application in the biosoftening of cotton fibers

The enhancement of the cellulase activity of Aspergillus nidulans by combinational optimization technique and the usage of cellulase for the biofinishing of cotton fibers were investigated in this study. The strain isolated from decayed, outer shell of Arachis hypogaea was compared for the first time for its ability to produce cellulolytic enzyme in shaken cultures using the optimized media formulated by combinational statistical approach using one factor at a time methodology (OFAT), Plackett Burmann Methodology (PB) and response surface methodology (RSM). A four-factor-five-level central composite design (CCD) was employed to determine the maximum activity of cellulase at optimum levels of carboxy methyl cellulose (CMC), ammonium nitrate and potassium dihydrogen phosphate at varying pH values. The cellulase activity is the best so far obtained with this strain of Aspergillus nidulans. The optimum values of the parameters studied were found to be 0.75 mg/l, 1.5 mg/l, 0.01 mg/l, and 2.15 g/l for KH₂PO₄, NH₄NO₃, Thiamine HCl and CMC, respectively at pH 6.0. This optimization led to the fine tuning of the cellulase production, thereby enhancing the cellulase activity from 4.91 to 60.54 U/ml. This cellulase of higher activity was employed in the biofinishing of the cotton fibers. The results of the scanning electron microscope (SEM) analysis after the treatment favored the fact that maximum surface finishing was achieved at a cotton fiber concentration of 15% (w/v) at 45°C and pH 5.0 using cellulase (60.54 U/ml) at 16th hour of the treatment. A probable mechanism of enzymatic finishing of cotton fibers has also been represented.     

Effect of pretreatment on the hydrolysis of cellulose by Penicillium funiculosum cellulase and recovery of enzyme

Penicillium funiculosum produces a complete cellulase which brings about 97% hydrolysis of cotton and has high beta-glucosidase, xylanase, laminarinase, and lichenase activities. This article deals with the effect of different pretreatments on the hydrolysis of sugarcane bagasse by P. funiculosum enzymes and the recovery of enzyme from the insoluble residues. Enzymic saccharification of bagasse pretreated with hot 1N NaOH followed by neutralization with HCI and steam treated under pressure (7 kg/cm(2)) gave 63 and 59% saccharification, respectively, in 48 h. Hemicellulose is not lost in these pretreatments. With a 30% slurry of steam-treated bagasse, a semisolid mass containing 14% sugar was obtained. A 90% recovery of CMCase, beta-glucosidase, and filter paper activity from the hydrolysates was obtained under the following conditions: (1) maintaining the ratio of enzyme to substrate high by stepwise addition of substrate, (2) brief grinding of the residual substrate with glass powder, and (3) addition of 0.4% Tween-80 to the eluting buffer. The high recovery of cellulolytic enzymes indicates that the adsorption of these enzymes on cellulose is not irreversible.     

Cellulase Hydrolysis of Cotton Cellulose: The Effects of Mechanical Action,Enzyme Concentration and Dyed Substrates

Experiments with dyed and undyed cotton, using different cellulose concentration and with or with out mechanical action showed a high effect of those factors. The process with mechanical action have a higher weight-loss. Dyed cotton with vat and sulfur dyes have almost die same weight-loss than undyed cotton, but on reactive dyed cotton the cellulase hydrolysis is shorter. It was also verified a decrease of hydrolysis extent with an increase of the reactive dye concentration on the cotton substrate. Some synergism due to the enzyme concentration was apparently observed on the changes of the length of the leaving sugars.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.     

Penicillium verruculosum WA 30 a new source of cellulase

Summary Of fungi 110 strains were screened for extracellular cellulase production in shake flask experiments. Twelve strains produced the enzyme in significant quantity. Since the enzyme activity was assayed by different methods (liberation of reducing sugar from cotton, filter paper, carboxymethylcellulose and cellobiose), the estimation of the productivity of the strains differed according to the substrate used. The best cotton degrading activity per fermentation volume as well as per mg of secreted soluble protein was achieved by Penicillium verruculosum WA 30, a wild-type strain, for which the cellulase productivity has not yet been published. The cotton degrading (so-called C₁) activity was successfully enhanced nearly threefold in medium experiments. Analyses of saccharification digests showed that glucose was the predominant product, with negligible amounts of cellobiose. The pH and temperature optima for WA 30 cellulase complex were pH 4.2 and 60°C.     

Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50 °C with Thermoanaerobacterium saccharolyticum ALK2

Studies were undertaken to understand phenomena operative during simultaneous saccharification and fermentation (SSF) of a model cellulosic substrate (Avicel) at 50°C with enzymatic hydrolysis mediated by a commercial cellulase preparation (Spezyme CP) and fermentation by a thermophilic bacterium engineered to produce ethanol at high yield, Thermoanaerobacterium saccharolyticum ALK2. Thermal inactivation at 50 °C, as shown by the loss of 50% of enzyme activity over 4 days in the absence of ethanol, was more severe than at 37 °C, where only 25% of enzyme activity was lost. In addition, at 50 °C ethanol more strongly influenced enzyme stability. Enzyme activity was moderately stabilized between ethanol concentrations of 0 and 40 g/L, but ethanol concentrations above 40 g/L accelerated enzyme inactivation, leading to 75% loss of enzymatic activity in 80 g/L ethanol after 4 days. At 37 °C, ethanol did not show a strong effect on the rate of enzyme inactivation. Inhibition of cellulase activity by ethanol, measured at both temperatures, was relatively similar, with the relative rate of hydrolysis inhibited 50% at ethanol concentrations of 56.4 and 58.7 g/L at 50 and 37 °C, respectively. A mathematical model was developed to test whether the measured phenomena were sufficient to quantitatively describe system behavior and was found to have good predictive capability at initial Avicel concentrations of 20 and 50 g/L.     

Enzyme regeneration during hydrolysis of steam-pretreated willow and requirement for cellulase complex composition

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willow Salix caprea by mixed cellulase of Trichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and permanent mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5:1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5-10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identified T. reesei cellobiohydrolase II as the predominant component, whereas clear domination of T. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to hydrolysis residual solids.     

Enzyme recovery in high-solids enzymatic hydrolysis of steam-pretreated willow: Requirements for the enzyme composition

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willowSalix caprea by mixed cellulase ofTrichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and constant mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading of 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5 : 1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5–10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identifiedT. reesei cellobiohydrolase II as the predominant component, whereas clear domination ofT. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to residual hydrolysis solids. Deceased     

The nature and mode of action of the cellulolytic component C1 of Trichoderma koningii on native cellulose

1. A purified cellulolytic component C(1) was isolated free from associated activities of the cellulase complex and shown to act as a beta-1,4-glucan cellobiohydrolase on both simple and complex forms of native cellulose. 2. The enzyme releases terminal cellobiose units from cellulose, its extent of action being determined principally by the product and by the nature of the substrate. 3. Component C(x) of the cellulase system is not required for the action of component C(1) (cellobiohydrolase). The enzyme synergizes extensively with cellobiase in extending the hydrolysis of native and of less-complex forms of cellulose to at least 70% with the liberation of glucose. 4. The cellobiohydrolase is relatively unstable, with an optimum at pH5 and a K(m) of 0.05mg/ml. The enzyme is inhibited by its product, from which it is released by cellobiase. 5. Of other compounds tested against the cellobiohydrolase the metal ions Cu(2+), Zn(2+), phenylmercuric and Fe(3+) are increasingly effective inhibitors. Glucose has no action at concentrations found inhibitory with cellobiose. 6. The relationship of the enzyme to the entire cellulase complex is discussed.     

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

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -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. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -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- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

The potential of enzyme recycling during the hydrolysis of a mixed softwood feedstock

Despite recent improvement in cellulase enzymes properties, the high cost associated with the hydrolysis step remains a major impediment to the commercialization of full-scale lignocellulose-to-ethanol bioconversion process. As part of a research effort to develop a commercial process for bioconversion of softwood residues, we have examined the potential for recycling enzymes during the hydrolysis of mixed softwood substrate pretreated by organosolv process. We have used response surface methodology to determine the optimal temperature, pH, ionic strength, and surfactant (Tween 80) concentration for maximizing the recovery of bound protein and enzyme activity from the residual substrates after hydrolysis. Data analysis showed that the temperature, pH and surfactant concentration were the major factors governing enzyme desorption from residual substrate. The optimized conditions were temperature 44.4 °C, pH 5.3 and 0.5% Tween 80. The optimal conditions significantly increased the hydrolysis yield by 25% after three rounds of hydrolysis. This bound enzyme desorption combining with free enzyme re-adsorption is a potential method to recover cellulase enzymes and reduce the cost of enzymatic hydrolysis.     

Purification and characterization of cellulolytic enzymes produced by Aspergillus nidulans

Three exo-glucanases, two endo-glucanases and two beta-glucosidases were separated and purified from the culture medium of Aspergillus nidulans. The optimal assay conditions for all forms of cellulase components ranged from pH 5.0 to 6.0 and 50 degrees C and 65 degrees C for exo-glucanases and endo-glucanases but 35 degrees C and 65 degrees C for beta-glucosidases. A close relation of enzyme stability to their optimal pH range was observed. All the cellulase components were stable for 10 min at 40-50 degrees C. Exo-II and Exo-III (Km, 38.46 and 37.71 mg/ml) had greater affinity for the substrate than Exo-I (Km, 50.00 mg/ml). The Km values of Endo-I and Endo-II (5.0 and 4.0 mg/ml) and their maximum reaction velocities (Vmax, 12.0 and 10.0 IU/mg protein) were comparable. beta-Glucosidases exhibited Km values of 0.24 and 0.12 mmol and Vmax values of 8.00 and 0.67 IU/mg protein. The molecular weights recorded for various enzyme forms were: Exo-I, 29,000; Exo-II, 72,500; Exo-III, 138,000; Endo-I, 25,000; Endo-II, 32,500; beta-Gluco-I, 14,000 and beta-Gluco-II, 26,000. Exo- and endo-glucanases were found to require some metal ions as co-factors for their catalytic activities whereas beta-glucosidases did not. Hg2+ inhibited the activity of all the cellulase components. The saccharification studies demonstrated a high degree of synergism among all the three cellulase components for hydrolysis of dewaxed cotton.     

嗜热毛壳菌多糖单加氧酶的氧化特性及协同作用

以嗜热毛壳菌Chaetomium thermophilum多糖单加氧酶CtPMO1为研究对象,利用薄层层析色谱法(TLC)、高效液相色谱-示差折光检测法(HPLC-RID)和飞行时间质谱(MALDI-TOF-MS)检测CtPMO1的酶活性,并根据定点突变的原理,将CtPMO1第1位的组氨酸(His1)和第166位的谷氨酰胺(Gln166)突变为H1A、Q166A和Q166E,研究两个突变位点是否参与CtPMO1的氧化作用;另外,采用3,5-二硝基水杨酸(DNS)法检测CtPMO1与纤维素酶(EGⅡ、BGLⅠ和CBHⅠ)的协同效应。研究发现CtPMO1在温度为50 ℃、pH为5.0的条件下降解磷酸膨胀纤维素(PASC),其酶解产物中不仅存在纤维二糖至纤维五糖,还存在C1氧化寡糖和C4氧化寡糖;此外,发现突变酶H1A完全丧失了酶活,Q166A丧失了C1和C4氧化活性,而Q166E保留了部分C1氧化活性;通过对CtPMO1与纤维素酶协同作用的探究,发现利用CtPMO1预处理磷酸膨胀纤维素(PASC),分别添加EGⅡ、BGLⅠ和CBHⅠ,使还原糖产量分别提高2.10倍、2.08倍和2.16倍,协同度分别是1.022、0.799和0.875。研究结果表明CtPMO1对底物具有C1和C4氧化的功能,其反应的最适温度为50 ℃、最适pH为5.0;CtPMO1活性中心氨基酸His1和平坦表面氨基酸Gln166均是关键性位点;CtPMO1预处理PASC,使纤维素酶的降解效率发生不同程度的提高。     

Studies on Cellulose Hydrolysis by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus extracellular cellulase extensively hydrolyzed crystalline celluloses such as Avicel (FMC Corp., Food and Pharmaceutical Products Div., Philadelphia, Pa.) but only if it was desalted and supplemented with Ca²⁺. The Ca²⁺ effect was one of increased enzyme stability in the presence of the ion. Although preincubation of the cellulase complex at 40°C for 5 h without added Ca²⁺ had a negligible effect on endoglucanase activity or on the subseqent hydrolysis of amorphous cellulose, the capacity of the enzyme to hydrolyze crystalline cellulose was almost completely lost. Adsorption studies showed that 90% of the Avicel-solubilizing component of the total enzyme preparation bound to 2% Avicel at 40°C. Under these conditions, only 15% of the endoglucanase and 25% of the protein present in the enzyme preparation adsorbed to the substrate. The protein profile of the bound enzyme, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was complex and distinctly different from the profile observed for total cellulase preparations. The specific activity of A. cellulolyticus cellulase with respect to Avicel hydrolysis was compared with that of commercially available Trichoderma reesei cellulase.     

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.