Study on the production of acetyl esterase and side-group cleaving glycosidases of ammonia fungi

Acetyl esterase was found to be widely distributed in ammonia fungi in a screen comprising 26 species (71 strains). No great differences appeared in enzyme production for acetyl esterase, beta-xylosidase, alpha-arabinosidase, or beta-glucosidase between different strains of the same species, but differences were detected between different genera. Acetyl esterase of Coprinus phlyctidosporus and Lyophyllum tylicolor may act cooperatively with beta-glucosidase. An increase in urea concentration significantly affected enzyme activity. It was supposed that urea used as 20 mg/g litter may solubilize leaf nutrients. At 20 mg urea added/g litter, a sizable increase in beta-glucosidase activity of C. phlyctidosporus and L. tylicolor was found, whereas a decrease in enzyme production of alpha-arabinosidase and beta-xylosidase was detected in some strains. Acetyl esterase and beta-glucosidase of C. phlyctidosporus, L. tylicolor, C. leucocephala 589, and C. rhombisperma 248 were most active in acidic conditions (pH 5.3-6.3), whereas acetyl esterase of L. nuda 561 and L. tarda 564 was most active in alkaline conditions (pH 8.3).     

In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential

AIMS: Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. beta-glucosidase, cellobiohydrolase and beta-xylosidase) and mycoparasitism (e.g. N-acetyl-beta-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential. METHODS AND RESULTS: Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0.997 and pH 4.0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0.950 for beta-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0.910 for cellobiohydrolase and at 0.993 for beta-xylosidase and N-acetyl-beta-glucosaminidase enzymes. Optimal pH values are suggested to be at 5.0 for beta-glucosidase, cellobiohydrolase and N-acetyl-beta-glucosaminidase, at 3.0 for beta-xylosidase, at 6.0 for trypsin-like protease and between 6.0 and 7.0 for chymotrypsin-like protease activities, respectively. CONCLUSIONS: Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.     

The impact of crop plant cultivation and peat amendment on soil microbial activity and structure

A rapid means for restoring soil fertility could be addition of peat to the plough layer. The impact of cultivation of eight different crops (the joint impact of plant and the management tailored for each plant), with and without soil amendment by peat treatment on soil microbiological, physical and chemical properties was assessed for two consecutive growing seasons. As a measure of the functional diversity of soil microbial community we estimated the activity of several different extracellular soil enzymes using the ZymProfiler® test kit. ATP content was measured to yield information on the amount of the active microbial biomass, and phospholipid fatty acid (PLFA) profiles were analysed to reveal the microbial community structure. The enzyme activity patterns of the soil samples indicated several differences due to the different crops and years but ATP content and PLFA profiles were rather stable. However, microbial biomass as total amount of PLFAs depended on the plant and peat treatment and ATP content varied between the years. The effects of the peat treatments were less clearly indicated by the biological parameters one or two years after the amendment, as only arylsulphatase and β-xylosidase activities were affected in both the years. Soil moisture, affecting enzyme activities, depended on the year and crop plant and peat addition increased it. Abbreviations: AMC – 7-amino-4-methylcoumarin; AP – aminopeptidase; ATP – adenosine triphosphate; C_mic– microbial biomass carbon; DNA – deoxyribonucleic acid; EC – electrical conductivity; FAME – fatty acid methyl ester; fw – fresh weight; MUF – 4-methylumbelliferyl; na – not added; N_mic– microbial biomass nitrogen; PDE – phosphodiesterase; PLFA – phospholipid fatty acid; PME – phosphomonoesterase; SOM – soil organic matter     

Pitfalls and problems in studies on the methylation of phosphatidylethanolamine

Recent articles have confused the steady state concentration of radioactivity in N-methylphosphatidylethanolamine (PME) and N,N-dimethylphosphatidylethanolamine (PDE) with the amount of these products formed during the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). This paper clarifies this problem and reports the apparent Km values for AdoMet and pH optima for the conversion of PE to PME, PDE, and PC by rat liver microsomes. We purified AdoMet and [methyl-3H]AdoMet and measured the transfer of tritium to PME, PDE, and PC as a function of time. There was an initial lag in the formation of [3H]PC followed by linear incorporation of isotope. In contrast, labeled PME and PDE reached and maintained steady state levels within 1 to 2 min. Hence, calculations of the rate of formation of PME, PDE, and PC must take into account the subsequent conversion of PME and PDE to PC. The PE N-methyltransferase was assayed at pH 6.6, 9.2, and 10.25 and the apparent Km for AdoMet for the three methylation reactions was calculated. The formation of PME was best estimated by the dpm in PME + 1/2 dpm in PDE + 1/3 dpm in PC. The synthesis of PDE from PME was estimated from 1/2 dpm in PDE and 1/3 dpm in PC, and the formation of PC from PDE estimated by 1/3 dpm in PC. The apparent Km for AdoMet at pH 10.25 for the conversion of PE to PME was 58 microM, PME to PDE was 65 microM, and PDE to PC was 96 microM. The pH optimum for each of these methylation reactions was 10.25. This high value was not due to alkaline degradation of AdoMet or denaturation of the enzyme. The apparent Km for AdoMet was also estimated for the conversion of exogenous PME to PDE (50 microM) and exogenous PDE to PC (45 microM). Since recent studies on the methylation of PE have not taken into account the conversion of newly formed PME and PDE to PC, the results and conclusions about apparent Km values for AdoMet, pH optima, and the number of enzymes involved must be re-evaluated.     

The copurification of beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase in two separate enzyme complexes isolated from Trichoderma harzianum E58

Two enzyme complexes, each with beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21), beta-xylosidase (beta-D-xylan xylohydrolase, EC 3.2.1.37), and 1,3-beta-glucanase (laminarinase, EC 3.2.1.39) activity, were purified to near homogeneity from the cellulolytic fungus Trichoderma harzianum E58. The two complexes had the same isoelectric point of pH 8.3 and identical subunit molecular masses of 75,400 daltons. The two complexes were also similar in that all activities were sensitive to inhibition by mercuric chloride (2 mM) and D-glucono-1,5-lactone (0.2% w/v). The activity ratios of the major and minor complexes were 1:1.7:4.3 and 1:1.6:3.1 for the beta-xylosidase, beta-glucosidase, and 1,3-beta-glucanase, respectively. Both complexes had approximately the same Km values for p-nitrophenyl beta-D-glucopyranoside and salicin. The pH optima of corresponding activities of the two complexes were also similar. The major and minor complexes differed in that the Km of the former for laminarin was almost threefold lower than that of the latter. Whereas all three activities of the minor complexes were inhibited by D-glucono-1,5-lactone with the same inhibition constant, the beta-glucosidase and 1,3-beta-glucanase of the major complex had inhibition constants which differed by more than 80,000 times. In addition, the inhibition on the 1,3-beta-glucanase in the major and minor complexes using D-glucono-1,5-lactone were noncompetitive and competitive, respectively. From the inhibition studies, the beta-glucosidase, beta-xylosidase, and 1,3-beta-glucanase activities in the minor complex were deduced to be more interdependent than the same activities in the major complex.     

Isolation, analysis, and expression of two genes from Thermoanaerobacterium sp. strain JW/SL YS485: a beta-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity.

The genes encoding acetyl xylan esterase 1 (axe1) and a beta-xylosidase (xylB) have been cloned and sequenced from Thermoanaerobacterium sp. strain JW/SL YS485. axe1 is located 22 nucleotides 3' of the xylB sequence. The identity of axe1 was confirmed by comparison of the deduced amino acid sequence to peptide sequence analysis data from purified acetyl xylan esterase 1. The xylB gene was identified by expression cloning and by sequence homology to known beta-xylosidases. Plasmids which independently expressed either acetyl xylan esterase 1 (pAct1BK) or beta-xylosidase (pXylo-1.1) were constructed in Escherichia coli. Plasmid pXylAct-1 contained both genes joined at a unique EcoRI site and expressed both activities. Substrate specificity, pH, and temperature optima were determined for partially purified recombinant acetyl xylan esterase 1 and for crude recombinant beta-xylosidase. Similarity searches showed that the axe1 and xylB genes were homologs of the ORF-1 and xynB genes, respectively, isolated from Thermoanaerobacterium saccharolyticum. Although the deduced sequence of the axe1 product had no significant amino acid sequence similarity to any reported acetyl xylan esterase sequence, it did have strong similarity to cephalosporin C deacetylase from Bacillus subtilis. Recombinant acetyl xylan esterase 1 was found to have thermostable deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid.     

Digestion in the soil predatory mite Pergamasus longicornis (Berlese) (Acari: Mesostigmata: Parasitidae)--detectable hydrolases

Nineteen hydrolytic enzymes were detected in individual adult Pergamasus longicornis (Berlese) mites--amylase, hide protease, alkali phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase, phosphoamidase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, alpha-glucosidase, beta-glucosidase, N-acetyl-beta-glucosaminidase, alpha-mannosidase, and alpha-fucosidase. All but the phosphatases were detected for the first time. Tryptic and chymotryptic activity were consistently not demonstrable. Comparisons are made with saprophagous mites. No clear enzymic specialization for predation was found.     

Ecophysiological variabilities in ectohydrolytic enzyme activities of some Pseudoalteromonas species,P. citrea,P. issachenkonii,and P. nigrifaciens

The ecophysiological variabilities in the ectohydrolytic enzyme profiles of the three species of Pseudoalteromonas, P. citrea, P. issachenkonii, and P. nigrifaciens, have been investigated. Forty-one bacteria isolated from several invertebrates, macroalgae, sea grass, and the surrounding water exhibited different patterns of hydrolytic enzyme activities measured as the hydrolysis of either native biopolymers or fluorogenic substrates. The activities of the following enzymes were assayed: proteinase, tyrosinase, lipase, amylase, chitinase, agarase, fucoidan hydrolase, laminaranase, alginase, pustulanase, cellulase, beta-glucosidase, alpha- and beta-galactosidases, beta-N-acetylglucosaminidase, beta-glucosaminidase, beta-xylosidase, and alpha-mannosidase. The occurrence and cell-specific activities of all enzymes varied over a broad range (from 0 to 44 micromol EU per hour) and depended not only on taxonomic affiliation of the strain, but also on the source/place of its isolation. This suggests 'specialization' of different species for different types of polymeric substrates as, for example, all strains of P. citrea and P. issachenkonii hydrolyzed alginate and laminaran, while strains of P. nigrifaciens were lacking the ability to hydrolyze most of the algal polysaccharides. The incidence of certain enzymes such as fucoidan hydrolases, alginate lyases, agarases, and alpha-galactosidases might be strain specific and reflect its particular ecological habitat.     

Differential stabilities of soil enzymes. Assay and properties of phosphatase and arylsulphatase.

Methods have been refined for the assay of phosphatase and arylsulphatase activities in soil, based on the chromogenic p-nitrophenyl ester substrates. Basic assay conditions have been defined, and pH optima and kinetic parameters have been determined. The enzymes follow Michaelis-Menten kinetics; this conclusion is based on three methods of analysis of data determined over a wide range of substrate concentrations. The enzyme activities are very stable to storage of wet soil for up to 4 weeks at soil temperatures and above. For example, phosphatase had a half-life of approximately 2 weeks at 50 degrees C; arylsulphatase was rather less stable. Both enzymes retained 80% of activity after incubation with pronase for 1 week at 25 degrees C. On the basis of this work and studies on other soil enzymes, it is concluded that remarkable stability is a general feature of soil enzymes.     

Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.     

Physiological differences among isolates of Phytophthora cinnamomi.

Significant differences in amylase, beta-glucosidase, and phosphatase activities were observed among four Phytophthora cinnamomi isolates grown in nutrient-amended sterilized soil for 20 days. Amylase pH optima for the four isolates were within a relatively narrow range; at pH 5.5 each isolate was within 90% of its peak activity. Isolates SB-216-1, 1-281, and C-39 each exhibited maximal beta-glucosidase activity at pH 5.0 and maximal phosphatase activity at pH 5.0-5.5. Maximal activity for these two enzymes of isolate A-7725 occurred at pH 3.5. In timed experiments, isolates 1-281 and A-7725 exhibited greater amylase activities than did the other two isolates. For beta-glucosidase, greatest activity was observed for SB-216-1; ACTivity of 1-281 was intermediate and least activity was observed for isolates A-7725 and C-39. Isolates SB-216-1 and 1-281 exhibited greatest phosphatase activities; isolate C-39 was intermediate in activity, and A-7725 was least active. Results indicate that significant differences exist among the isolates tested and that these differences can be quantitatively measured by the methods described.     

Bioconversion of hemicellulose: aspects of hemicellulase production by Trichoderma reesei QM 9414 and enzymic saccharification of hemicellulose

The growth of Trichoderma reesei QM9414 in shake flasks at 28 degrees C on hemicellulose substrates and bagasse resulted in rather low yields of hemicellulolytic enzymes (1.0-1.5 units/mL xylanase and 0.05-0.08 units/mL beta-xylosidase). The influence of pH on the synthesis of beta-xylosidase was greater than on the synthesis of xylanase. Both xylanase and beta-xylosidase showed optimal activity at pH 4-5 and 55-60 degrees C. Xylanase was stable at pH 2-10 but was heat labile and totally inactivated after 1 h at 65 degrees C. Enzyme stability towards heat could be increased in the presence of bovine serum albumin. The beta-xylosidase was more tolerant to heat, but stable over a pH range 2.5-6.0. The D-xylose inhibited both enzymes in a competitive manner. Hemicellulose (heteroxylan) was degraded to the extent of 30-40%within 24 h. The degree of hydrolysis decreased as the substrate concentration increased and increased with increased amounts of enzyme. Multiple enzyme doses resulted in increased saccharification in reduced times. The degree of hydrolysis was influenced by the amount of beta-xylosidase present in the hemicellulolytic enzyme preparation. The -;xylosidase was demonstrated to play an important role in the overall conversion of heteroxylan into xylose that is analogous to the role of beta-glucosidase in the saccharification of cellulose by cellulases.     

Extracellular enzyme activities associated with epiphytic microbiota on submerged stems of the reed Phragmites australis

Abstract Fluorogenic 4-methylumbelliferyl (MUF) compounds were used as analogue substrates for assay of extracellular enzyme activities associated with epiphytic microbiota at submerged Phragmites australis stem surfaces. Incubations at a range of MUF substrate concentrations indicated that saturation of enzyme activity was achieved at a MUF substrate concentration of about 200 μmol 1⁻¹. Later determinations at a single, saturation, concentration of MUF substrate were, therefore, carried out at about 200 μmol l⁻¹. Such determinations were undertaken using P. australis stems from eight gravel-pit ponds. The rate of enzymatic hydrolysis of MUF phosphate (analogue substrate for phosphatase activity) was > MUF β- d -glucopyranoside (β- d -glucosidase) > MUF β- d -galactopyranoside (β- d -galactosidase) > MUF sulphate (sulphatase) and MUF palmitate (lipase) on stems from all eight ponds. Thus the relative magnitude of the various components of total epiphyton extracellular enzyme activity might be a conservative feature.     

A lipase preparation with dual pH optima,wide temperature optima and broad substrate specificity for multiple applications

Summary Optimised procedures for the production and recovery of lipase at 25 litre scale by submerged fermentation using aRhizopus species isolated from petroleum-soaked soil are developed. These lipase preparations are characterized by dual pH optima (4.0 and 8.0), wide temperature optima (over 80% activity between 40–57°C), broad specificity for substrates, high degree of tolerance for common salts and excellent stability during usage as well as storage.     

Lipase of Mucor pusillus

Lipase of Mucor pusillus NRRL 2543 was recovered with ammonium sulfate precipitation, gel filtration on Sephadex G-75, and anion-exchange chromatography on diethylaminoethyl-Sephadex A-50. Maximal glycerol ester hydrolase (lipase) activity was observed at pH 5.0 to 5.5 and 50 C when trioctanoin and olive oil were used as substrates. The enzyme also showed esterase activity; it hydrolyzed, with the exception of methyl butyrate, all methyl esters tested. A minimum chain length of six carbons appeared to be a requirement for esterase activity, which was maximal at about pH 5.5 with methyl dodecanoate (C(12)) as the substrate. Neither the glycerol ester hydrolase (lipase) nor the esterase activity of the enzyme appeared to be affected by thiol group inhibitors, chelating agents, and reducing compounds. On the other hand, hydrolysis of triolein and methyl dodecanoate was arrested to the same extent in the presence of diisopropyl fluorophosphate, which suggested the involvement of serine in the active center of the enzyme. The enzyme remained stable during a 30-day storage at - 10 C.     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.     

Heterogonous expression and characterization of a plant class IV chitinase from the pitcher of the carnivorous plant Nepenthes alata

A class IV chitinase belonging to the glycoside hydrolase 19 family from Nepenthes alata (NaCHIT1) was expressed in Escherichia coli. The enzyme exhibited weak activity toward polymeric substrates and significant activity toward (GlcNAc)(n) [β-1,4-linked oligosaccharide of GlcNAc with a polymerization degree of n (n = 4-6)]. The enzyme hydrolyzed the third and fourth glycosidic linkages from the non-reducing end of (GlcNAc)(6). The pH optimum of the enzymatic reaction was 5.5 at 37°C. The optimal temperature for activity was 60°C in 50 mM sodium acetate buffer (pH 5.5). The anomeric form of the products indicated that it was an inverting enzyme. The k(cat)/K(m) of the (GlcNAc)(n) hydrolysis increased with an increase in the degree of polymerization. Amino acid sequence alignment analysis between NaCHIT1 and a class IV chitinase from a Picea abies (Norway spruce) suggested that the deletion of four loops likely led the enzyme to optimize the (GlcNAc)(n) hydrolytic reaction rather than the hydrolysis of polymeric substrates.     

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.     

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.     

alpha-L-Arabinofuranosidase from Streptomyces sp. PC22: purification, characterization and its synergistic action with xylanolytic enzymes in the degradation of xylan and agricultural residues

alpha-l-Arabinofuranosidase was purified from culture filtrates of the thermoalkaliphilic Streptomyces sp. PC22 to about 108-fold purity by (NH(4))(2)SO(4) precipitation followed by column chromatography. Its approximate molecular weight was 404kDa, with a subunit mass of approximately 79kDa. The evaluated K(m) and V(max) values with p-nitrophenyl-alpha-l-arabinofuranoside as substrate were 0.23mM and 124 U.mg(-1), respectively. The purified enzyme was optimally active at 65 degrees C and pH 6.0 and showed a mild but significant synergistic effect in combination with other xylanolytic enzymes, including xylanase, beta-xylosidase and acetyl esterase, on the degradation of oat-spelt xylan, corn cob and corn husk substrates with a 1.25, 1.32 and 1.21-fold increase in the amount of reducing sugar released, respectively, compared to the expected (additive) amounts for the individual enzymes acting alone. Sequential reactions using two xylan-backbone degrading enzymes (xylanase/beta-xylosidase) and two debranching enzymes (alpha-l-arabinofuranosidase/acetyl esterase) were also determined. The highest degree of synergy was obtained in sequential reactions with the debranching enzyme digestion preceding the xylan-backbone degrading enzymes.