Production of γ-Decalactone by a Psychrophilic and a Mesophilic Strain of the Yeast <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis>

Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic strain Rhodotorula aurantiaca A19 was selected for its ability of growth and γ-decalactone production at low temperatures. The effects of temperature, initial pH, and castor oil concentration on the growth and γ-decalactone production by a psychrophilic and a mesophilic strain of R. aurantiaca were investigated. The highest γ-decalactone production in flasks (5.8 g/l) was obtained with the strain A19 at 14 °C and initial pH 7.0 in medium containing 20 g/l castor oil. On the other hand, these factors did not affect the production of γ-decalactone by the mesophilic strain. In fermentor, a γ-decalactone concentration of 6.6 g/l was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the mesophilic strain. Our results suggest that the ability to synthesize γ-decalactone is a particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of γ-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. Moreover, the amount of γ-decalactone obtained in fermentor with the strain 19 was on the order of concentrations usually described in patents.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Metabolic Flux and Nodes Control Analysis of Brewer’s Yeasts Under Different Fermentation Temperature During Beer Brewing

The aim of this work was to further investigate the glycolysis performance of lager and ale brewer??s yeasts under different fermentation temperature using a combined analysis of metabolic flux, glycolytic enzyme activities, and flux control. The results indicated that the fluxes through glycolytic pathway decreased with the change of the fermentation temperature from 15?°C to 10?°C, which resulted in the prolonged fermentation times. The maximum activities (V _max) of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) at key nodes of glycolytic pathway decreased with decreasing fermentation temperature, which was estimated to have different control extent (22?C84?%) on the glycolytic fluxes in exponential or flocculent phase. Moreover, the decrease of V _max of PFK or PK displayed the crucial role in down-regulation of flux in flocculent phase. In addition, the metabolic state of ale strain was more sensitive to the variation of temperature than that of lager strain. The results of the metabolic flux and nodes control analysis in brewer??s yeasts under different fermentation temperature may provide an alternative approach to regulate glycolytic flux by changing V _max and improve the production efficiency and beer quality.     

Production of ceramide with Saccharomyces cerevisiae

The possibility of producing the biologically active material of the skin, ceramide, was studied using yeasts. The yeast strain that produced the most ceramide, Saccharomyces cerevisiae (KCCM 50515), was selected, and the optimal conditions for ceramide production were determined using shakeflask culture and batch fermentation. By measuring the production rate of ceramide at various pH values and temperatures, the optimal conditions for ceramide production were found to be pH 6.0 and 30°C. When heat shock was applied to the cells for 1 h by increasing the culture temperature from 30 to 40°C after cell growth, the amount of ceramide produced was increased 5.9-fold. A cell growth and ceramide production model was developed with Monod kinetics and the Leudecking-Piret model. It showed that ceramide production was increased when the cells were in the stationary phase.     

Characterization of catechol 1,2-dioxygenase from cell extracts of <Emphasis Type="Italic">Sphingomonas xenophaga</Emphasis> QYY

Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C12O) in cell extracts. Characterization of the crude C12O showed that the maximum activity was obtained at 40–70°C and pH 7.8–8.8. Metal ions had different influences on the activity of crude C12O. It was suggested that strain QYY possessed an inducible and ferric-dependent C12O. Kinetic studies showed that the value of V _max and K _m was 0.25 μmol catechol/L/mg protein/min and 52.85 μmol/L, respectively. In addition, the partial purification of C12O was achieved by a HiTrap Q Sepharose column chromatography. Supported by the National Natural Science Foundation of China (Grant No. 50608011) and the 39th Postdoctoral Funds of China (Grant No. 20060390983)     

Thermotolerant yeast for simultaneous saccharification and fermentation of cellulose to ethanol

Ten promising microbial strains were screened for glucose fermentation over the temperature range of 37–47°C, and five temperature-tolerant yeasts (Saccharomyces cerevisiae SERI strain (D₅A),S. uvarum, andCandida generaacidothermophilium, brassicae, andlusitaniae), were chosen for SSF evaluation on Sigmacell-50 cellulose with Genencor 150 L cellulase enzyme.Brettanomyces clausenii (Y-1414) was included for comparison to previous studies both by itself and in mixed culture withS. cerevisiae (D₅A). Good conversion rates were achieved at temperatures as high as 43°C withC. brassicae andS. uvarum; mixed cultures of either of these yeasts with the thermotolerant cellobiose fermenting yeastC. lusitaniae achieved higher rates and yields than any of the three yeasts alone. However, the mixed culture ofB. clausenii andS. cerevisiae at 37°C achieved as high conversion rates and higher yields than any of the other yeasts tested.     

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi

Xylanases (EC3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97°C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 m in at 105°C) and active even at 110°C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and β-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of thi enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (μ_max=0.0195) at 90°C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.     

The Utilization of Gum Tragacanth to Improve the Growth of Rhodotorula aurantiaca and the Production of γ-Decalactone in Large Scale

The production of γ-decalactone and 4-hydroxydecanoic acid by the psychrophilic yeast R. aurantiaca was studied. The effect of both compounds on the growth of R. aurantiaca was also investigated and our results show that γ-decalactone must be one of the limiting factors for its production. The addition of gum tragacanth to the medium at concentrations of 3 and 4 g/l seems to be an adequate strategy to enhance γ-decalactone production and to reduce its toxicity towards the cell. The production of γ-decalactone and 4-hydroxydecanoic acid was significantly higher in 20-l bioreactor than in 100-l bioreactor. By using 20 g/l of castor oil, 6.5 and 4.5 g/l of γ-decalactone were extracted after acidification at pH 2.0 and distillation at 100 °C for 45 min in 20- and 100-l bioreactors, respectively. We propose a process at industrial scale using a psychrophilic yeast to produce naturally γ-decalactone from castor oil which acts also as a detoxifying agent; moreover the process was improved by adding a natural gum.     

Production, purification, and characterization of a polygalacturonase from a new strain of kluyveromyces marxianus isolated from coffee wet-processing wastewater

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M _r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.     

Phytase Production by a Marine Yeast <Emphasis Type="Italic">Kodamea ohmeri</Emphasis> BG3

The marine yeast strain Kodamea ohmeri BG3 isolated from the gut of a marine fish (Hexagrammes otakii) was found to secrete a large amount of phytase into the medium. The crude phytase produced by this marine yeast showed the highest activity at pH 5.0 and 65 °C. The optimal medium for phytase production contained oat 10.0 g/l, ammonium sulfate 15.0 g/l, glucose 30 g/l, and NaCl 20.0 g/l, while the optimal cultivation conditions for phytase production were pH 5.0, a temperature of 28 °C, and a shaking speed of 170 rpm. Under the optimal conditions, over 557.9 mU/ml of phytase activity was produced within 72 h of fermentation at the shake flask level. This is a very high level of phytase activity produced by yeasts. We think that the medium and process for phytase production by the marine yeast strain were very simple, and such marine yeast from the gut of natural marine fish may have a potential application in the maricultural industry and marine environmental protection. The results demonstrate that phytate was actively degraded by the crude phytase within a short period.     

Effect of Cultural Conditions and Media Constituents on Production of Penicillin V Acylase and CTAB Treatment to Enhance Whole-Cell Enzyme Activity of <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis> (NCIM 3425)

Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic β-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 °C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 μg/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.     

Microcalorimetric investigation of the effect of berberine alkaloids from <Emphasis Type="Italic">Coptis chinensis</Emphasis> Franch on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> growth

The inhibitory effects of three berberine alkaloids (BAs) from rhizome of Coptis chinensis Franch, a traditional Chinese medicinal (TCM) herb, on Staphylococcus aureus growth were investigated by microcalorimetry. The power-time curves of S. aureus with and without BAs were acquired; meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by studying the growth rate constant (k), half inhibitory ratio (IC₅₀), maximum heat-output power (P _max), peak time of maximum heat-output power (t _p) and total heat production (Q _t). The value of k of S. aureus in the presence of the three BAs decreased with the increasing concentrations of BAs. Moreover, P _max was reduced and the value of t _p increased with increasing concentrations of the three drugs. The inhibitory activity varied with different drugs. The values of IC₅₀ of the three BAs are respectively, 101.4 μg/mL for berberine, 241.0 μg/mL for palmatine and 792.3 μg/mL for jateorrhizine. The sequence of antimicrobial activity of the three BAs is: berberine > palmatine > jateorrhizine. It is suggested that the functional group methylenedioxy or methoxyl at C2 on the phenyl ring could possibly improve antimicrobial activity more strongly than hydroxyl at C2 on the phenyl ring. Supported by the National Natural Science Foundation of China (Grant No. 30625042)     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Drawing of poly(p-phenylene sulfide) by solid-state coextrusion

The effects of drawing temperature on the physical and mechanical properties of poly(p-phenylene sulfide) have been studied. A melt-quenched film was drawn by solid-state coextrusion both below (75°C) and above (95 and 110°C) the glass transition temperature T_g (85°C) of PPS. The maximum extrusion draw ratio (EDR_max) increased from 3.4 to 5.6 with increasing extrusion temperature T_e from 75 to 110°C. It was found that extrusion drawing just above the T_g of PPS (95°C) produced more stress-induced crystals. A high efficiency of draw in the amorphous region was achieved by extrusion at T_e-75°C. The tensile modulus at EDR_max decreased from 5.1 to 3.5 GPa with increasing T_e from 75 to 110°C. The low efficiency of draw for the samples extruded at 110°C is explained in terms of disentanglement and chain slippage during drawing due to a less effective network.     

Optimization of medium by orthogonal matrix method for submerged mycelial culture and exopolysaccharide production in Collybia maculata

Optimization of submerged culture conditions for the production of mycelial growth and exopolysaccharides (EPSs) by Collybia maculata was investigated. The optimum temperature and the initial pH for EPS production in a shake-flask culture of C. maculata were found to be 20°C and 5.5, respectively. Among the various medium’s constituents examined, glucose, Martone A-1, K₂HPO₄, and CaCl₂ were the most suitable carbon, nitrogen, and mineral sources for EPS production, respectively. The optimum concentration of the medium’s ingredients determined using the orthogonal matrix method was as follows: 30 g/L of glucose, 20 g/L of Martone A-1, 1g/L of K₂HPO₄, and 1g/L of CaCl₂. Under the optimized culture conditions, the maximum concentration of EPSs in a 5-L stirred-tank reactor was 2.4 g/L, which was approximately five times higher than that in the basal medium. A comparative fermentation result showed that the EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor despite the lower mycelial growth rate. The specific productivities and the yield coefficients in the airlift reactor were higher than those in the stirred-tank reactor even though the volumetric productivities were higher in the stirred-tank reactor than in the airlift reactor.     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

Maximizing the grain growth rate during the disorder‐to‐order transition in block copolymer melts

The factors controlling grain growth during the disorder‐to‐order transition in a polystyrene‐block‐polyisoprene copolymer melt were studied with time‐resolved depolarized light scattering. The ordered phase consisted of hexagonally packed polyisoprene cylinders, and the order–disorder‐transition temperature of the block copolymer (T_ODT) was 132 ± 1 °C. Our objective was to identify the temperature at which the grain growth rate was maximized (T_max) and compare it with theoretical predictions. We conducted seeded grain growth experiments, which comprised two steps. In the first step, which lasted for 43 min, the sample was cooled from the disordered state to 124 °C. This resulted in the formation of a small number of ordered grains or seeds. This was followed by a second step in which the sample was heated to temperatures between 124 and 132 °C and the seeds grew with time. Our objective was to study grain growth at different temperatures starting from the same initial condition. The value of T_max obtained experimentally was 128 °C. The theoretically predicted value of T_max, based entirely on the rheological properties of the disordered sample and T_ODT, was also 128 °C. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 2231–2242, 2001     

Effects of glucose,glutamine, and malate on the metabolism of spodoptera frugiperda clone 9 (sf9) cells

Optimal design and operation of bioreactors for insect cell culture is facilitated by functional relations providing quantitative information on cellular metabolite consumption kinetics, as well as on the specific cell growth rates (μ_G). Initial specific consumption rates of glucose, malate, and oxygen, and associated changes in μ_G, were measured forSpodoptera frugiperda clone 9 (Sf9) cells grown in batch suspension culture in medium containing 7–35 mM glucose, 0–16 mM malate, and 4–16 mM glutamine. The initial specific glucose consumption rate (q _G ) could be described by a modified Michaelis-Menten equation treating malate as a “competitive” inhibitorK ₁ = 6.5 mM) and glutamine as a “noncompetitive” inhibitorK _I = 14 mM) ofq _G , with aK _m of 7.1 mM for glucose. All three carbon sources were found to increase μ_G in a saturable manner, and a modified Monod equation was employed to describe this relationship (μ_Gmax = 0.047 h^-1). The initial specific oxygen consumption rate (q_O2) in Sf9 cells could be related to μ_G by the maintenance energy model, and it was calculated that, under typical culture conditions, about 15–20% of the cellular energy demand comes from functions not related to growth. Fitted parameters in mathematical expression for μg: K₄, Monod constant for glucose (mM); K₅, modified Monod constant for malate (mM); K₆, Monod constant for glutamine (mM); m_o2, specific consumption rate of oxygen by the cells under zero-growth conditions (nmol/cell/h); q_F, initial specific fumarate production rate (nmol/cell/ h);q _G , initial specific glucose consumption rate (nmol/cell/h); q_Gmax, maximum initial specific glucose consumption rate (nmol/cell/h);q _M , initial specific malate consumption rate (nmol/cell/h); q_o2, initial specific oxygen consumption rate (nmol/cell/h); Y_o2, cell yield on oxygen (cells/nmol); μ, initial specific cell growth rate (h^-1); μ_g, initial specific cell growth rate (h^-1); μG_max, maximum initial specific cell growth rate (h^-1).     

Production and Characterization of Cellobiohydrolase from a Novel Strain of <Emphasis Type="Italic">Penicillium purpurogenum</Emphasis> KJS506

A high cellobiohydrolase (CBH)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 according to the morphology and comparison of internal transcribed spacer rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, a maximum CBH activity of 2.6 U mg-protein⁻¹, one of the highest among CBH-producing microorganisms, was obtained. The optimum temperature and pH for CBH production were 30 °C and 4.0, respectively. The increased production of CBH in P. purpurogenum culture at 30 °C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. The internal amino acid sequences of P. purpurogenum CBH showed a significant homology with hydrolases from glycoside hydrolase family 7. The extracellular CBH was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast-protein liquid chromatography. The purified CBH was a monomeric protein with a molecular weight of 60 kDa and showed broad substrate specificity with maximum activity towards p-nitrophenyl β-d-cellobiopyranoside. P. purpurogenum CBH showed t _1/2 value of 4 h at 60 °C and V _max value of 11.9 μmol min⁻¹ mg-protein⁻¹ for p-nitrophenyl-d-cellobiopyranoside. Although CBHs have been reported, the high specific activity distinguishes P. purpurogenum CBH.