Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Purification and characterization of an acidothermophilic cellulase enzyme produced by Bacillus subtilis strain LFS3

In the present investigation, a microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated and identified as Bacillus subtilis strain LFS3 by 16S rDNA sequence analysis. The carboxymethylcellulase (CMCase) enzyme produced by the B. subtilis strain LFS3 was purified by (NH?)?SO? precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 15 %. Native-PAGE analysis of purified CMCase revealed the molecular weight of enzyme to be about 185 kDa. The activity profile of CMCase enzyme showed the optimum activity at temperature 60 °C and pH 4.0, respectively. The enzyme activity was induced by Na?, Mg2?, NH??, and EDTA, whereas strongly inhibited by Hg2? and Fe3?. The purified enzyme hydrolyzed CMC, filter paper, and xylan, but not p-nitrophenyl β-D-glucopyranoside and cellulose. Kinetic analysis of purified enzyme showed the K(m) value of 2.2 mg/ml. Thus, acidophilic as well as thermophilic nature makes this cellulase a suitable candidate for current mainstream biomass conversion into fuel and other industrial processes.     

Alkaline xylanases from Bacillus mojavensis A21: Production and generation of xylooligosaccharides

Medium composition and culture conditions for the xylanases production by Bacillus mojavensis A21 were optimized using two statistical methods: Plackett-Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and Box-Behnken design used to optimize the value of the four significant variables: barley bran, NaCl, agitation, and cultivation time. The optimal conditions for higher production of xylanases were barley bran 18.66g/l, NaCl 1.04g/l, speed of agitation 176rpm and cultivation time 34.08h. Under these conditions, the xylanase experimental yield (7.45U/ml) closely matched the yield predicted by the statistical model (7.23U/ml) with R(2)=0.98. The medium optimization resulted in a 6.83-fold increase in xylanase production compared to that of the initial medium. Best xylanase activity was observed at the temperature of 50°C and at pH 8.0. The enzyme retained more 96% of its activity after 24h at pH ranges from 7.0 to 90.0. The enzyme preserved more 80% of its initial activity after 60min of pre-incubation from 30°C to 60°C. The main hydrolysis products yielded from corncob extracted xylan were xylobiose and xylotriose, suggesting the good potential of strain A21 in xylooligosaccharides production.     

Enhanced degradation of α-chitin materials prepared from shrimp processing byproduct and production of N-acetyl-D-glucosamine by thermoactive chitinases from soil mesophilic fungi

Soil isolates of mesophilic Penicillium monoverticillium CFR 2, Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 were cultivated in solid state fermentation (SSF) using wheat bran solid medium supplemented with α-chitin in order to produce chitinolytic enzyme. Under SSF cultivation, maximum enzymes (U/g IDS) production was 41.0 (endo-chitinase) and 195.4 (β-N-acetylhexosaminidase) by P. monoverticillium, 26.8 (endo-chitinase) and 222.1 (β-N-acetylhexosaminidase) by A. flavus and 13.3 (endo-chitinase) and 168.3 (β-N-acetylhexosaminidase) by F. oxysporum after 166?h of incubation. The crude endo-chitinase and β-N-acetylhexosaminidase derived from A. flavus and F. oxysporum revealed optimum temperature at 62?±?1°C, but the enzymes from P. monoverticillium showed optimum temperature at 52?±?1°C for maximum activity. Several fold increase in endo-chitinase and β-N-acetylhexosaminidase activities in the crude enzymes preparation was achieved after concentrating with polyethylene glycol. The concentrated crude chitinases from P. monoverticillium, A. flavus and F. oxysporum, respectively yielded 95.6, 96.6 and 96.1?mmol/l of N-acetyl-D: -glucosamine (GlcNAc) in 48?h of reaction from colloidal chitin. While, the crude enzyme preparations of P. monoverticillium, A. flavus and F. oxysporum produced 10.11, 6.85 and 10.7?mmol/l of GlcNAc respectively, in 48?h of reaction from crystalline α-chitin. HPLC analysis of colloidal chitin hydrolysates prepared with crude chitinases derived from P. monoverticillium, A. flavus and F. oxysporum revealed that the major reaction product was monomeric GlcNAc (~80%) and a small amount of (GlcNAc)(4) (~20%), indicating the potential of these enzymes for efficient production of GlcNAc from α-chitin.     

Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(?) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl? extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl? or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl?, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl?. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl? at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.     

Genetic surface-display of methyl parathion hydrolase on Yarrowia lipolytica for removal of methyl parathion in water

In this study, the mph gene encoding methyl parathion hydrolase from Pseudomonas sp. WBC-3 was expressed in Yarrowia lipolytica and the expressed methyl parathion hydrolase was displayed on cell surface of Y. lipolytica. The activity of methyl parathion hydrolase displayed on the yeast cells of the transformant Z51 was 59.5 U mg?1 of cell dry cells (450.6 U per mL of the culture) in the presence of 5.0 mM of Co2?. The displayed methyl parathion hydrolase had the optimal pH of 9.5 and the optimal temperature of 40 °C, respectively and was stable in the pH range of 4.5-11 and up to 40 °C. The displayed methyl parathion hydrolase was also stimulated by Co2?, Cu2?, Ni2? and Mn2?, and was not affected by Fe2?, Fe3?, Na?, K?, Ca2? and Zn2?, but was inhibited by other cations tested. Under the optimal conditions (OD(600 nm) = 2.6, the substrate concentration = 100 mg L?1 and 40 °C), 90.8 % of methyl parathion was hydrolyzed within 30 min. Under the similar conditions, 98.7, 97.0, 96.5 and 94.4 % of methyl parathion in tap water (pH 9.5), tap water (pH 6.8), seawater (pH 9.5) and natural seawater (pH 8.2) were hydrolyzed, respectively, suggesting that the methyl parathion hydrolase displayed on the yeast cells can effectively remove methyl parathion in water.     

Dilute phosphoric acid-catalysed hydrolysis of municipal bio-waste wood shavings using autoclave parr reactor system

The visibility of using municipal bio-waste, wood shavings, as a potential feedstock for ethanol production was investigated. Dilute acid hydrolysis of wood shavings with H?PO? was undertaken in autoclave parr reactor. A combined severity factor (CSF) was used to integrate the effects of hydrolysis times, temperature and acid concentration into a single variable. Xylose concentration reached a maximum value of 17 g/100 g dry mass corresponding to a yield of 100% at the best identified conditions of 2.5 wt.% H?PO?, 175 °C and 10 min reaction time corresponding to a CSF of 1.9. However, for glucose, an average yield of 30% was obtained at 5 wt.% H?PO?, 200 °C and 10 min. Xylose production increased with increasing temperature and acid concentration, but its transformation to the degradation product furfural was also catalysed by those factors. The maximum furfural formed was 3 g/100 g dry mass, corresponding to the 24% yield.     

Solid-state production of polygalacturonase by Aspergillus sojae ATCC 20235

The effect of solid substrates, inoculum and incubation time were studied using response surface methodology (RSM) for the production of polygalacturonase enzyme and spores in solid-state fermentation using Aspergillus sojae ATCC 20235. Two-stage optimization procedure was applied using D-optimal and face-centered central composite design (CCD). Crushed maize was chosen as the solid substrate, for maximum polygalacturonase enzyme activity based on D-optimal design. Inoculum and incubation time were determined to have significant effect on enzyme activity and total spore (p<0.01) based on the results of CCD. A second order polynomial regression model was fitted and was found adequate for individual responses. All two models provided an adequate R(2) of 0.9963 (polygalacturonase) and 0.9806 (spores) (p<0.001). The individual optimum values of inoculum and incubation time for maximum production of the two responses were 2 x 10(7) total spores and 5-6 days. The predicted enzyme activity (30.55 U/g solid) and spore count (2.23 x 10(7)spore/ml) were very close to the actual values obtained experimentally (29.093 U/g solid and 2.31 x 10(7)spore/ml, respectively). The overall optimum region considering the two responses together, overlayed with the individual optima. Solid-state fermentation provided 48% more polygalacturonase activity compared to submerged fermentation under individually optimized conditions.     

Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F

An extracellular halophilic α-amylase was purified from Nesterenkonia sp. strain F using 80 % ethanol precipitation and Q-Sepharose anion exchange chromatography. The enzyme showed a single band with an apparent molecular weight of 110 kDa by SDS-PAGE. The amylase exhibited maximal activity at pH 7-7.5, being relatively stable at pH 6.5-7.5. Optimal temperature for the amylase activity and stability was 45 °C. The purified enzyme was highly active in the broad range of NaCl concentrations (0-4 M) with optimal activity at 0.25 M NaCl. The amylase was highly stable in the presence of 3-4 M NaCl. Amylase activity was not influenced by Ca2?, Rb?, Li?, Cs?, Mg2? and Hg2?, whereas Fe3?, Cu2?, Zn2? and Al3?) strongly inhibited the enzyme activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. K(m) value of the amylase for soluble starch was 6.6 mg/ml. Amylolytic activity of the enzyme was enhanced not only by 20 % of water-immiscible organic solvents but also by acetone, ethanol and chloroform. Higher concentration (50 %) of the water-miscible organic solvents had no significant effect on the amylase activity. To the best of our knowledge, this is the first report on increased activity of a microbial α-amylase in the presence of organic solvents.     

Production of S-lactoylglutathione by organic-solvent-extracted glyoxalase I from Hansenula mrakii

Summary Glyoxalase I was extracted from Hansenula mrakii IFO 0895 by incubating the cells with buffer solution containing 50% acetone (enzyme activity 35 units/g cells) or 50% ethyl acetate (enzyme activity 28 units/g cells) at 30°C for 10 h. Glyoxalase II was also extracted from the cells, although the activity of the enzyme was lost during incubation with organic solvents, especially at higher temperature (30°C). By using the organic-solvent-extracted fraction of H. mrakii, enzymatic production of S-lactoylglutathione was studied, and approximately 82 mmol/l (30 g/l) of S-lactoylglutathione was produced from 120 mmol/l glutathione. Offprint requests to: A. Kimura     

Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850?min(-1) and a K (m) of 540?μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35?°C, 4% (v/v) ethanol, 2,500?U ml(-1) (8.3?mg?ml(-1)) of enzyme, and 40?g l(-1) oleic acid. Under these conditions, 40?g l(-1) (142?mM) oleic acid was converted into 40?g l(-1) (133?mM) 10-hydroxystearic acid for 150?min, with a molar yield of 94% and a productivity of 16?g l(-1)?h(-1), and olive oil hydrolyzate containing 40?g l(-1) oleic acid was converted into 40?g l(-1) 10-hydroxystearic acid for 300?min, with a productivity of 8?g l(-1)?h(-1).     

Improvement in raw sago starch degrading enzyme production from Acremonium sp. endophytic fungus using carbon and nitrogen sources

Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium.     

Polygalacturonase biosynthesis by Lactobacillus plantarum: effect of cultural conditions on enzyme production

Lactobacillus plantarum was found to produce extracellular polygalacturonase (EC 3.2.1.15.). Maximum enzyme production was obtained in a medium containing 0.5% glucose and 1.5% low methyl-pectin as inducer at 27°C at an initial pH of 6.8. Enzyme production was strongly inhibited by 5 μmol/l NiCl₂, 5 μmol/l CoCl₂, 5 μmol/l CuSO₄, and 10 μmol/l ZnCl₂. MnSO₄ and MgSO₄ at 200 μmol/l and 50 μmol/l respectively seemed to enhance enzyme biosynthesis. The optimal pH and temperature for enzyme activity were 4.5 and 30°C respectively. Enzyme production in batch culture accompanied growth.     

Inhibitory properties of endogenous subunit epsilon in the Escherichia coli F1 ATPase.

Homogeneous ? bound tightly to the purified Escherichia coli ATPase (ECF₁ from which ? had been removed and strongly inhibited its ATPase activity. ECF₁ containing ? had a lower specific activity than ECF₁ missing ?, provided that the ATPase assay was carried out at relatively high concentrations of enzyme. Antiserum specific for the ? subunit stimulated the ATPase, as did diluting the enzyme, apparently by dissociating ?. When the ATPase reaction was started by the addition of enzyme, the rate of ATP hydrolysis increased progressively during the first 3 min until a linear steady-state rate was reached. A prior incubation with ATP abolished the lag period and ADP prevented the ATP effect. ECF₁ missing ? gave a linear rate of ATP hydrolysis without a lag, unless ? was rebound to it before the assay. These results suggest that ECF₁ as purified is in an inhibited state due to the presence of the ? subunit, whose interaction with ECF₁ is governed by an equilibrium binding. ATP appears to convert ECF₁ to a form which more readily binds and releases ?.     

Development and testing of a novel lab-scale direct steam-injection apparatus to hydrolyse model and saline crop slurries

In this work, a novel laboratory-scale direct steam-injection apparatus (DSIA) was developed to overcome the main drawback of the conventional batch-driven lab rigs, namely the long time needed to heat fiber slurry from room to reaction temperatures greater than 150 °C. The novel apparatus mainly consisted of three units: (i) a mechanically-stirred bioreactor where saturated steam at 5-30 bar can be injected; (ii) an automatic on-off valve to flash suddenly the reaction medium after a prefixed reaction time; (iii) a cyclone separator to recover the reacted slurry. This system was tested using 0.75 dm3 of an aqueous solution of H?SO? (0.5%, v/v) enriched with 50 kg m?3 of either commercial particles of Avicel? and Larch xylan or 0.5 mm sieved particles of Tamarix jordanis. Each slurry was heated to about 200 °C by injecting steam at 28 bar for 90 s. The process efficiency was assessed by comparing the dissolution degree of suspended solid (Y(S)), as well as xylose (Y(X)), glucose (Y(G)), and furfural (Y(F)) yields, with those obtained in a conventional steam autoclave at 130 °C for 30 or 60 min. Treatment of T. jordanis particles in DSIA resulted in Y(S) and Y(G) values quite similar to those obtained in the steam autoclave at 130 °C for 60 min, but in a less efficient hemicellulose solubilization. A limited occurrence of pentose degradation products was observed in both equipments, suggesting that hydrolysis predominated over degradation reactions. The susceptibility of the residual solid fractions from DSIA treatment to a conventional 120 h long cellulolytic treatment using an enzyme loading of 5.4 FPU g?1 was markedly higher than that of samples hydrolysed in the steam autoclave, their corresponding glucose yields being equal to 0.94 and 0.22 g per gram of initial cellulose, respectively. Thus, T. jordanis resulted to be a valuable source of sugars for bioethanol production as proved by preliminary tests in the novel lab rig developed here.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

Pigeon pea waste as a novel, inexpensive, substrate for production of a thermostable alkaline protease from thermoalkalophilic Bacillus sp. JB-99

Thermoalkaliphilic Bacillus sp. JB-99 was grown in a 250 ml Erlenmeyer flask containing 50 ml medium containing (g/l) Pigeon pea waste 10; NaNO3, 5.0; K2HPO4, 5.0; MgSO4 x 2H2O, 0.2 and Na2CO3, 10.0. Incubations were carried out at 50 degrees C on a rotary incubator shaker for 15 h. A high level of extra cellular thermostable protease activity was observed after 24 h incubation. The optimum temperature and pH for activity were 70 degrees C and 11, respectively, so this enzyme showed stable activity at high temperature and under alkaline conditions.     

Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Hyper production of alkali stable xylanase in lesser duration by Bacillus pumilus SV-85S using wheat bran under solid state fermentation

High level production of an extracellular cellulase-poor alkali stable xylanase has been conceded from newly isolated Bacillus pumilus SV-85S under solid state fermentation using wheat bran as a substrate. Optimization of the fermentation conditions enhanced the enzyme production to 73,000 ± 1,000 IU/g dry substrate, which was 13.8-fold higher than unoptimized conditions (5,300 IU/g). The enzyme titre was highest after 48 h of incubation at 30°C with 1:3 ratios of substrate to moistening agent using wheat bran as a carbon source. The enzyme could be produced in significant levels by using either tap water or distilled water alone as a moistening agent. An elevated production of xylanase by B. pumilus SV-85S in the presence of wheat bran, a cheap and easily available agro-residue, in shorter duration would apparently reduce the enzyme cost substantially. The enzyme was completely stable over a broad pH (5-11) range and retained 52% of its activity at a temperature of 70°C for 30 min. The desired characteristics of this enzyme together with economic production would be important for its application in paper and pulp industry.     

Optimisation of culture medium and conditions for alpha-l-Arabinofuranosidase production by the extreme thermophilic eubacterium Rhodothermus marinus

The culture medium for Rhodothermus marinus was optimised on a shake-flask scale by using statistical factorial designs for enhanced production of a highly thermostable alpha-L-arabinofuranosidase (AFase). The medium containing 3.6 g/l birch wood xylan and 8.2 g/l yeast extract yielded a maximum of 110 nkat/ml AFase activity together with 125 nkat/ml xylanase and 65 nkat/ml beta-xylosidase activity. In addition, low levels of beta-mannanase (30 nkat/ml), alpha-galactosidase (0.2 nkat/ml), beta-galactosidase (0.3 nkat/ml), endoglucanase (5 nkat/ml) and beta-glucosidase (30 nkat/ml) were detected in the culture filtrate. Among the various carbon sources tested, birchwood xylan was most effective for the formation of AFase and xylanase activities, followed by oat spelt and beechwood xylans, and xylan-rich lignocelluoses (e.g., starch-free sugar beet pulp and wheat bran). Constitutive levels of enzyme activities were detected when the bacterium was grown on other polysaccharides and low-molecular-weight carbohydrates. A fermentation in a 5-l fermenter (3-l working volume) using the optimised medium yielded 60 nkat/ml AFase associated with 65 nkat/ml xylanase and 35 nkat/ml beta-xylosidase activities. The crude AFase displayed optimal activity between pH 5.5 and 7 and at 85 degrees C. It had half-lives of 8.3 h at 85 degrees C and 17 min at 90 degrees C. It showed high stability between pH 5 and 9 (24 h at 65 degrees C). The combined use of AFase-rich xylanase and mannanase from R. marinus in the prebleaching of softwood kraft pulp gave a brightness increase of 1.8% ISO. To our knowledge, this is the first report on the production of a high AFase activity by an extreme thermophilic bacterium and this enzyme is the most thermostable AFase reported so far.