Modification of the carbohydrate composition of sulfite pulp by purified and characterized beta-xylanase and beta-xylosidase of Aureobasidium pullulans

Both beta-xylanase and beta-xylosidase were purified to homogeneity from a xylose-grown culture of Aureobasidium pullulans. Cellular distribution studies of enzyme activities revealed that beta-xylanase was an extracellular enzyme, during both the exponential and stationary phases, whereas beta-xylosidase was mostly periplasmic associated. The beta-xylanase exhibited very high specificity for xylan extracted from Eucalyptus grandis dissolving pulp, whereas the beta-xylosidase was only active on p-nitrophenyl xyloside and xylobiose. Comparison of kcat/Km ratios showed that the beta-xylanase hydrolyzed xylan from dissolving pulp 1.3, 2.1, and 2. 3 times more efficiently than Eucalyptus hemicellulose B, Eucalyptus hemicellulose A, and larchwood xylan, respectively. The beta-xylosidase exhibited a transxylosylation reaction during the hydrolysis of xylobiose. When applied on acid sulfite pulp, both enzymes released xylose and hydrolyzed xylan to a different extent. Although beta-xylosidase (0.4 U/g pulp) liberated more xylose from pulp than beta-xylanase (4.7 U/g pulp), it was responsible for only 3% of xylan solubilization. Treatment of pulp with beta-xylanase liberated 51.7 microgram of xylose/g and hydrolyzed 10% of xylan. The two enzymes acted additively on pulp and removed 12% of pulp xylan. A synergistic effect in terms of release of xylose from pulp was observed when the enzyme mixture of beta-xylanase and beta-xylosidase was supplemented with beta-mannanase. However, this did not result in further enzymatic degradation of pulp xylan. Both beta-xylanase and beta-xylosidase altered the carbohydrate composition of sulfite pulp by increasing the relative cellulose content at the expense of reduced hemicellulose content of pulp.     

Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.     

Production and properties of hemicellulases by a Thermomyces lanuginosus strain

Thermophilic fungi producing extremely high beta-xylanase and their associated hemicellulases have attracted considerable attention because of potential industrial applications. Thermomyces lanuginosus strain SSBP isolated from soil, produced beta-xylanase activity of 59 600 nkat ml-1 when cultivated on a medium containing corn cobs as substrate and yeast extract as nitrogen source. Lower beta-xylanase activities were produced after growth on other xylan substrates, sugars and soluble starch. Other hemicellulases were produced extracellularly at significantly lower levels than the beta-xylanase activity produced on corn cobs. No cellulase activity was observed. The optimal conditions for beta-xylanase production were 50 degrees C and pH 6.5, whereas 70 degrees C and between pH 5. 5 and 9.5 were optimal for beta-xylanase activity. The temperature optima for other hemicellulases were less than the xylanase with the exception of beta-mannosidase. The pH optima of the other hemicellulases were between 5.0 and 6.5. Xylanase was stable up to 70 degrees C and between pH 5.5 and 9.0 for 30 min whereas the other hemicellulase were less stable. These results suggest that the most suitable conditions for hydrolysis of hemicellulose by these enzymes would be at 50 degrees C and pH 6.0.     

Degradation of xylan to D-xylose by recombinant Saccharomyces cerevisiae coexpressing the Aspergillus niger beta-xylosidase (xlnD) and the Trichoderma reesei xylanase II (xyn2) genes.

The beta-xylosidase-encoding xlnD gene of Aspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor alpha1 signal peptide (MFalpha1(s)) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant beta-xylosidase showed optimum activity at 60 degrees C and pH 3.2 and optimum stability at 50 degrees C. The K(i(app)) value for D-xylose and xylobiose for the recombinant beta-xylosidase was determined to be 8.33 and 6.41 mM, respectively. The XLO2 fusion gene and the XYN2 beta-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of beta-xylanase activity when expressing only the beta-xylanase and 860 nkat/ml when coexpressing the beta-xylanase with the beta-xylosidase. The maximum beta-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the beta-xylanase. Coproduction of the beta-xylanase and beta-xylosidase enabled S. cerevisiae to degrade birchwood xylan to D-xylose.     

Optimization of culture conditions for the production of haloalkaliphilic thermostable protease from an extremely halophilic archaeon Halogeometricum sp. TSS101

AIMS: Isolation and screening of extreme halophilic archaeon producing extracellular haloalkaliphilic protease and optimization of culture conditions for its maximum production. METHODS AND RESULTS: Halogeometricum sp. TSS101 was isolated from salt samples and screened for the secretion of protease on gelatin and casein plates containing 20% NaCl. The archaeon was grown aerobically in a 250 ml flask containing 50 ml of (w/v) NaCl 20%; MgCl(2) 1%; KCl 0.5%; trisodium citrate 0.3%; and peptone 1%; pH 7.2 at 40 degrees C on rotary shaker. The production of enzyme was investigated at various pH, temperatures, NaCl concentrations, metal ions and different carbon and nitrogen sources. The partially purified protease had activity in a broad pH range (7.0-10.0) with optimum activity at pH 10.0 and a temperature (60 degrees C). The enzyme was thermostable and retained 70% initial activity at 80 degrees C. Maximum protease production occurred at 40 degrees C in a medium containing 20% NaCl (w/v) and 1% skim milk powder after 84 h in shaking culture. Enzyme secretion was observed at a broad pH range of 7.0-10.0. Addition of CaCl(2) (200 mmol) to the culture medium enhanced the production of protease. Protein rich flours proved to be cheap and good alternative source for enzyme production. Different osmolytes were tested for the growth and production of haloalkaliphilc protease and found that betaine and glycerol enhanced growth without secretion of the protease. Immobilization studies showed that whole cells immobilized in 2% alginate beads were stable up to 10 batches and able to secrete the protease, which attained maximum production within 60 h under shaking conditions. CONCLUSIONS: Halogeometricum sp. TSS101 secreted an extracellular haloalkaliphilic and thermostable protease. The optimum conditions required for maximum production are 20% NaCl, 1% skim milk powder and temperature at 40 degrees C. Addition of CaCl(2) (200 mmol) enhanced the enzyme production. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of haloalkaliphilic protease. SIGNIFICANCE AND IMPACT OF THE STudy: The low cost protein rich flours were used as an alternative carbon and nitrogen sources for enzyme production. Immobilization of halophilic cells in alginate beads can be used in continuous production of halophilic enzyme. The halophilic and thermostable protease from Halogeometricum sp. TSS101 is good source for industrial applications and can be a suitable source for preparation of fish sauce.     

Amino acid composition of bulk protein and salt relationships of selected enzymes of Salinibacter ruber,an extremely halophilic bacterium

The extremely halophilic bacterium Salinibacter ruber was previously shown to have a high intracellular potassium content, comparable to that of halophilic Archaea of the family Halobacteriaceae. The amino acid composition of its bulk protein showed a high content of acidic amino acids, a low abundance of basic amino acids, a low content of hydrophobic amino acids, and a high abundance of serine. We tested the level of four cytoplasmic enzymatic activities at different KCl and NaCl concentrations. Nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase functioned optimally at 0.5-2 M KCl, with rates of 60% of the optimum value at 3.3 M. NaCl provided less activation: 70% of the optimum rates in KCl were found at 0.2-1.2 M NaCl, and above 3 M NaCl, activity was low. We also detected nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate activity, which remained approximately constant between 0-3.2 M NaCl and increased with increasing KCl concentration. NAD-dependent malate dehydrogenase functioned best in the absence of salt, but rates as high as 25% of the optimal values were measured in 3-3.5 M KCl or NaCl. NAD-dependent glutamate dehydrogenase, assayed by the reductive amination of 2-oxoglutarate, showed low activity in the absence of salt. NaCl was stimulatory with optimum activity at 3-3.5 M. However, no activity was found above 2.5 M KCl. Although the four activities examined all function at high salt concentrations, the behavior of individual enzymes toward salt varied considerably. The results presented show that Salinibacter enzymes are adapted to function in the presence of high salt concentrations.     

Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme.

A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment. Therefore, the purification was carried out in 2 M NaCl. Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0. Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function. Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C. Peroxidase activity decreased rapidly above its maximum at 40 degrees C. An activation energy of 2.5 kcal (ca. 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca. 17 kJ)/mol was calculated for peroxidase. Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki [KiApp] of 50 and 67.5 microM, respectively). Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM). The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite. A heme content of 1.43 per tetramer was determined. The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.     

Purification and properties of amylase produced by a moderately halophilic Acinetobacter sp.

A moderately halophilic Acinetobacter sp., capable of producing dextrinogenic amylase, was isolated from sea-sands. Maximum enzyme production was obtained when the bacterium was cultivated aerobically in media containing 1 to 2M NaCl or 1M KCl. Two kinds of amylase, amylases I and II were purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, DEAE-Sephadex A-50 chromatography, and Sephadex G-200 gel filtration. Both enzymes had maximal activity at pH 7.0 in 0.2 to 0.6 M NaCl or KCl at 50 to 55 degrees C. The activities were lost by dialysis against distilled water. Molecular weights for amylases I and II were estimated to be 55 000 and 65 000 respectively by SDS-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose and maltotriose.     

Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31.

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1

An unidentified halophilic archaebacterium strain 172 P1 produced three extracellular proteases in media containing 15-27% salts. One component, F-II, was purified to homogeneity. It is a serine protease that can be inhibited by phenylmethylsulfonyl fluoride and chymostatin. A high concentration of NaCl was required for its stability; in the presence of 25% NaCl, only 4% of the activity was lost by incubating at 60 degrees C for 30 min, while complete inactivation occurred in the presence of 5% NaCl. F-II is a thermophilic and halophilic protease. High activity was obtained at 75-80 degrees C when F-II was assayed in the presence of 25% NaCl. The optimal concentration of NaCl required was 10-14% when assayed at 70 degrees C with azocasein as substrate, though a halophilic characteristic was not distinct at lower temperatures. Hydrolyses of the synthetic substrates succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-methylcoumaryl-7-amide or succinyl-alanyl-alanyl-alanyl-p-nitroanilide at 26 degrees C were maximal at 25 and 30% NaCl, respectively. F-II was most stable at pH 6-7, and its optimal pH was 10.7. Its molecular weight was estimated as 44,000-46,000 by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and by gel filtration--high-pressure liquid chromatography. The sequence of the 35 N-terminal amino acid residues was determined and compared with that of other serine proteases.     

Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

Characterization of extracellular esterase and lipase activities from five halophilic archaeal strains

A total of 118 halophilic archaeal collection of strains were screened for lipolytic activity and 18 of them were found positive on Rhodamine agar plates. The selected five isolates were further characterized to determine their optimum esterase and lipase activities at various ranges of salt, temperature and pH. The esterase and lipase activities were determined by the hydrolysis of pNPB and pNPP, respectively. The maximum hydrolytic activities were found in the supernatants of the isolates grown at complex medium with 25% NaCl and 1% gum Arabic. The highest esterase activity was obtained at pH 8-8.5, temperature 60-65 degrees C and NaCl 3-4.5 M. The same parameters for the highest lipase activities were found to be pH 8, temperature 45-65 degrees C and NaCl 3.5-4 M. These results indicate the presence of salt-dependent and temperature-tolerant lipolytic enzymes from halophilic archaeal strains. Kinetic parameters were determined according to Lineweaver-Burk plot. The KM and V (max) values were lower for pNPP hydrolysis than those for pNPB hydrolysis. The results point that the isolates have higher esterase activity comparing to lipase activity.     

Purification and biochemical characterization of a protease secreted by the <Emphasis Type="Italic">Salinivibrio</Emphasis> sp. strain AF-2004 and its behavior in organic solvents

A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.     

Killing of target cells by redirected granzyme B in the absence of perforin

We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 °C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.     

A role of xylanase,alpha-L-arabinofuranosidase,and xylosidase in xylan degradation

Renewable natural resources such as xylans are abundant in many agricultural wastes. Penicillium sp. AHT-1 is a strong producer of xylanolytic enzymes. The sequential activities of its xylanase, alpha-L-arabinofuranosidase, and beta-xylosidase on model hemicellulose oat-spelt xylan was investigated. Optimum production of the enzymes was found in culture containing oat-spelt xylan at 30 degrees C and initial pH 7.0 after 6 days. The enzymes were partially purified by ammonium sulphate fractionation and anion-exchange chromatography on DEAE-Toyopearl 650 S. The apparent molecular mass was 21 kDa, and the protein displayed an "endo" mode of action. The xylanase exhibited glycotansferase activity. It synthesized higher oligosaccharides from the initial substrates, and xylotriose was the shortest unit of substrate transglycosylated. Xylanolytic enzymes (enzyme mixture) produced by this Penicillium sp. interacted cooperatively and sequentially in the hydrolysis of oat-spelt xylan in the following order: alpha-L-arabinofuranosidase --> xylanase --> beta-xylosidase. All three enzymes exhibited optimal activity under the same conditions (temperature, pH, cultivation), indicating that they alone are sufficient to completely depolymerize the test xylan. Results indicate that the xylanolytic enzyme mixture of Penicillium sp. AHT-1 could be useful for bioconversion of xylan-rich plant wastes to value-added products.     

Xylanase production by Aspergillus awamori. Development of a medium and optimization of the fermentation parameters for the production of extracellular xylanase and beta-xylosidase while maintaining low protease production

A growth medium was developed for maximal production in batch culture of extracellular xylanase and beta-xylosidase by Aspergillus awamori CMI 142717 and a mutant (AANTG 43) derived from the wild-type strain. The optimum pH for the production of xylanase and beta-xylosidase was 4.0. The best temperature of xylanase production was 30 degrees C; 35 degrees C was optimal for beta-xylosidase. Protease production was never completely suppressed under any of the conditions tested. However, protease titre was 3.5-fold less than the control in medium in which proteose peptone and yeast extract were omitted: the level of xylanase was not affected (8.6 U mL(-1)) but beta-xylosidase titre was increased 4.7-fold to 1.5 U mL(-1). When corn steep liquor was used as the sole nitrogen source, xylanse and beta-xylosidase titres were further increased by 1.5- and 1.9-fold, respectively. Of the carbon sources investigated, ball-milled oat straw or oat spelt xylan produced the highest titres of xylanse and beta-xylosidase. None of the soluble carbon sources investigated produced the high titres of xylanase or beta-xylosidase induced by either oat straw for xylanse and beta-xylosidase was 2% and the optimum spore inoculum was between 10(6) and 10(7) spores/mL(-1) final concentration. The level of xylanse activity obtained in the culture filtrates of the mutant was a remarkable 820 U mL(-1) when the reducing sugar released was measured by the dinitrosalicylic acid method. This enzyme titre would appear to be the highest reported so far. The xylanases system contained the correct balance of enzymes to effect extensive hydrolysis of oat spelt xylan. The protease titre was very low.     

The beta-xylosidase of Thermomonospora

This study is concerned with characterizing cell-bound inducible beta-xylosidase produced by a strain of the thermophilic bacterial genus Thermomonospora. A crude preparation of this enzyme recovered from sonicated cells of this organism displayed high activity against paranitrophenyl-beta-xylopyranoside over a pH range of 5.5-7.7. The temperature optimum, based on a 30-min assay of activity, at pH 6.5 was 70 degrees C. The crude enzyme had a thermal half-life of approximately 1 week at 55 degrees C and pH 6.5. Xylose inhibited the enzyme. Values of K(m) and V(max) are estimated from the reaction rate data as 0.82 mM and 8 U/L, respectively.     

Purification and properties of thermostable xylanase and beta-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.

We isolated a thermophilic bacterium that produces both xylanase and beta-xylosidase. Based on taxonomical research, this bacterium was identified as Bacillus stearothermophilus. Each extracellular enzyme was separated by hydrophobic chromatography by using a Toyopearl HW-65 column, followed by gel filtration with a Sephacryl S-200 column. Each enzyme in the culture was further purified to homogeneity (62-fold for xylanase and 72-fold for beta-xylosidase) by using a fast protein liquid chromatography system with a Mono Q HR 5/5 column. The optimum temperatures were 60 degrees C for xylanase and 70 degrees C for beta-xylosidase. The isoelectric points and molecular masses were 5.1 and 39.5 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Heat treatment at 60 degrees C for 1 h did not cause inhibition of the activities of these enzymes. The action of the two enzymes on xylan gave only xylose.     

Xylan-hydrolyzing enzyme system from Bacillus pumilus CBMAI 0008 and its effects on Eucalyptus grandis kraft pulp for pulp bleaching improvement

The extracellular productions of beta-xylanase, beta-xylosidase, beta-glucosidase, beta-mannanase, arabinosidase, alpha-glucuronidase, alpha-galactosidase and Fpase from Bacillus pumilus CBMAI 0008 were investigated with three different xylan sources as substrate. The enzymatic profiles on birchwood, Eucalyptus grandis and oat were studied at alkaline and acidic pH conditions. B. pumilus CBMAI 0008 grown on the three carbon sources produced mainly beta-xylanase. At pH 10, the levels of xylanase were 328, 160 and 136 U/ml, for birch, oat and E. grandis, respectively. beta-Mannanase production was induced on E. grandis (5 U/ml) and arabinofuranosidase on oat (5 U/ml). Although small quantities of alpha-glucuronidase had been produced at pH 10, activity at pH 4.8 was 1.5 U/ml, higher than observed for Aspergillus sp. in literature reports. Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Celulose e Papel, Limeira, SP, Brazil) showed a reduction of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp.