An Enzymatic and Proteomic Analysis of Panus lecomtei during Biodegradation of Gossypol in Cottonseed.

Cotton is an important plant-based protein. Cottonseed cake, a byproduct of the biodiesel industry, offers potential in animal supplementation, although the presence of the antinutritional sesquiterpenoid gossypol limits utilization. The macrofungus Panus lecomtei offers potential in detoxification of antinutritional factors. Through an enzymatic and proteomic analysis of P. lecomtei strain BRM044603, grown on crushed whole cottonseed contrasting in the presence of free gossypol (FG), this study investigated FG biodegradation over a 15-day cultivation period. Fungal growth reduced FG to levels at 100 µg/g, with a complex adaptive response observed, involving primary metabolism and activation of oxidative enzymes for metabolism of xenobiotics. Increasing activity of secreted laccases correlated with a reduction in FG, with enzyme fractions degrading synthetic gossypol to trace levels. A total of 143 and 49 differentially abundant proteins were observed across the two contrasting growth conditions after 6 and 12 days of cultivation, respectively, revealing a dynamic protein profile during FG degradation, initially related to constitutive metabolism, then later associated with responses to oxidative stress. The findings advance our understanding of the mechanisms involved in gossypol degradation and highlight the potential of P. lecomtei BRM044603 in cotton waste biotreatment, relevant for animal supplementation, sustainable resource utilization, and bioremediation.

Evaluation of endoglucanase and xylanase production by Aspergillus tamarii cultivated in agro-industrial lignocellulosic biomasses.

To better understand the production of enzymes of industrial interest from microorganisms with biotechnological potential using lignocellulosic biomass, we evaluated the production of endoglucanase and xylanase from Aspergillus tamarii. CAZymes domains were evaluated in the genome, and a screening of the enzymatic potential of A. tamarii in various agricultural biomasses was done. The enzymatic profile could be associated with the biomass complexity, with increased biomass recalcitrance yielding higher activity. A time-course profile defined 48 h of cultivation as the best period for cultivating A. tamarii in sugarcane bagasse reached 12.05 IU/mg for endoglucanase and 74.86 IU/mg for xylanase. Using 0.1% (w/v) tryptone as the only nitrogen source and 12 µmol/L CuSO4 addition had an overall positive effect on the enzymatic activity and protein production. A 22 factorial central composite design was used then to investigate the simultaneous influence of tryptone and CuSO4 on enzyme activity. Tryptone strongly affected enzymatic activity, decreasing endoglucanase activity but increasing xylanase activity. CuSO4 supplementation was advantageous for endoglucanases, increasing their activity, and it had a negative effect on xylanases. But overall, the experimental design increased the enzymatic activity of all biomasses used. For the clean cotton residue, the experimental design was able to reach the highest enzyme activity for endoglucanase and xylanase, with 1.195 IU/mL and 6.353 IU/mL, respectively. More experimental studies are required to investigate how the biomass induction effect impacts enzyme production.

Sequencing and characterization of an L-asparaginase gene from a new species of Penicillium section Citrina isolated from Cerrado.

The enzyme L-asparaginase (L-ASNase) is used in the treatment of Acute Lymphoblastic Leukemia. The preparations of this enzyme for clinical use are derived from bacterial sources and its use is associated with serious adverse reactions. In this context, it is important to find new sources of L-ASNase. In this work, the Placket-Burman Experimental Design (PBD) was used to determine the influence of the variables on the L-ASNase production then it was followed by a 28-4 Factorial Fractional Design (FFD). The results obtained from PBD have shown a range of L-ASNase activity, from 0.47 to 1.77 U/gcell and the results obtained from FFD have showed a range of L-ASNase activity, from 1.10 to 2.36 U/gcell. L-proline and ammonium sulfate were identified as of significant positive variables on this production enzyme by Penicillium cerradense sp. nov. The precise identification of this new species was confirmed by morphological characteristics and sequence comparisons of the nuclear 18S-5.8S-28S partial nrDNA including the ITS1 and ITS2 regions, RNA polymerase II, ß-tubulin and calmodulin genomic regions. The genetic sequence coding for the L-ASNase was obtained after carrying out a full genome sequencing. The L-ASNase expressed by P. cerradense sp. nov may have promising antineoplastic properties.

An overview of Trichoderma reesei co-cultures for the production of lignocellulolytic enzymes.

Biorefineries are core facilities for implementing a sustainable circular bioeconomy. These facilities rely on microbial enzymes to hydrolyze lignocellulosic substrates into fermentable sugars. Fungal co-cultures mimic the process of natural biodegradation and have been shown to increase certain enzyme activities. Trichoderma reesei and its many mutant strains are major cellulase producers and are heavily utilized as a source of carbohydrate-active enzymes. Several reports have demonstrated that T. reesei co-cultures present higher enzyme activities compared with its monocultures, especially in the context of ß-glucosidase activity. The performance of T. reesei during co-culturing has been assessed with several fungal partners, including Aspergillus niger, one of the most recurrent partners. Various aspects of co-cultivation still need further investigation, especially regarding the molecular interactions between fungi in controlled environments and the optimization of the resulting enzyme cocktails. Since plenty of genetic and physiological data on T. reesei is available, the species is an outstanding candidate for future co-culture investigations. Co-cultures are still a developing field for industrial enzyme production, and many aspects of the technique need further improvement before real applications. KEY POINTS: • T. reesei co-cultures are an alternative for producing lignocellulolytic enzymes. • Several reports suggest an increase in certain enzyme activities in co-cultures. • More in-depth investigations of co-cultures are necessary for advancing this field.

Immobilization studies of a pectinase produced by Aspergillus terreus.

Aspergillus terreus can produce different holocellulose-degrading enzymes when grown in sugarcane bagasse, with predominant pectinase activity. Thus, pectinase was selected for purification and immobilization studies. Ion exchange and molecular exclusion chromatography studies were performed, after which it was possible to semipurify the enzyme with a yield of 80%. The crude extract pectinase (PECEB) and the partially purified enzyme (PEC2) were immobilized on monoamino-N-aminoethyl (MANAE)-agarose with pectinase activity yields of 66% and 98%, respectively. After immobilization in MANAE-agarose, the pectinase showed higher activity at acidic pH (pH 4.0) when compared to the nonimmobilized enzyme. It was also found that after the immobilization process, there was a threefold improvement in the enzyme's thermostability. Also, it was possible to reuse the immobilized enzyme for up to five cycles of hydrolysis with effective production of reducing sugars (0.196 mg/g of substrate). The industrial application test revealed a significant decrease in the viscosity of guava juice when the immobilized enzyme was used. PECEB, immobilized on MANAE-agarose, was the enzyme sample that generated the highest pulp viscosity reduction (approximately 47%). Although additional studies are needed for practical industrial application, the results obtained herein reveal the potential of application of immobilized pectinase in the industry.

Transcriptome Profiling-Based Analysis of Carbohydrate-Active Enzymes in Aspergillus terreus Involved in Plant Biomass Degradation.

Given the global abundance of plant biomass residues, potential exists in biorefinery-based applications with lignocellulolytic fungi. Frequently isolated from agricultural cellulosic materials, Aspergillus terreus is a fungus efficient in secretion of commercial enzymes such as cellulases, xylanases and phytases. In the context of biomass saccharification, lignocellulolytic enzyme secretion was analyzed in a strain of A. terreus following liquid culture with sugarcane bagasse (SB) (1% w/v) and soybean hulls (SH) (1% w/v) as sole carbon source, in comparison to glucose (G) (1% w/v). Analysis of the fungal secretome revealed a maximum of 1.017 UI.mL-1 xylanases after growth in minimal medium with SB, and 1.019 UI.mL-1 after incubation with SH as carbon source. The fungal transcriptome was characterized on SB and SH, with gene expression examined in comparison to equivalent growth on G as carbon source. Over 8000 genes were identified, including numerous encoding enzymes and transcription factors involved in the degradation of the plant cell wall, with significant expression modulation according to carbon source. Eighty-nine carbohydrate-active enzyme (CAZyme)-encoding genes were identified following growth on SB, of which 77 were differentially expressed. These comprised 78% glycoside hydrolases, 8% carbohydrate esterases, 2.5% polysaccharide lyases, and 11.5% auxiliary activities. Analysis of the glycoside hydrolase family revealed significant up-regulation for genes encoding 25 different GH family proteins, with predominance for families GH3, 5, 7, 10, and 43. For SH, from a total of 91 CAZyme-encoding genes, 83 were also significantly up-regulated in comparison to G. These comprised 80% glycoside hydrolases, 7% carbohydrate esterases, 5% polysaccharide lyases, 7% auxiliary activities (AA), and 1% glycosyltransferases. Similarly, within the glycoside hydrolases, significant up-regulation was observed for genes encoding 26 different GH family proteins, with predominance again for families GH3, 5, 10, 31, and 43. A. terreus is a promising species for production of enzymes involved in the degradation of plant biomass. Given that this fungus is also able to produce thermophilic enzymes, this first global analysis of the transcriptome following cultivation on lignocellulosic carbon sources offers considerable potential for the application of candidate genes in biorefinery applications.

Mild hydrothermal pretreatment of sugarcane bagasse enhances the production of holocellulases by Aspergillus niger.

Holocellulase production by Aspergillus niger using raw sugarcane bagasse (rSCB) as the enzyme-inducing substrate is hampered by the intrinsic recalcitrance of this material. Here we report that mild hydrothermal pretreatment of rSCB increases holocellulase secretion by A. niger. Quantitative proteomic analysis revealed that pretreated solids (PS) induced a pronounced up-regulation of endoglucanases and cellobiohydrolases compared to rSCB, which resulted in a 10.1-fold increase in glucose release during SCB saccharification. The combined use of PS and pretreatment liquor (PL), referred to as whole pretreated slurry (WPS), as carbon source induced a more balanced up-regulation of cellulases, hemicellulases and pectinases and resulted in the highest increase (4.8-fold) in the release of total reducing sugars from SCB. The use of PL as the sole carbon source induced the modulation of A. niger's secretome towards hemicellulose degradation. Mild pretreatment allowed the use of PL in downstream biological operations without the need for undesirable detoxification steps.

Optimization and partial purification of beta-galactosidase production by Aspergillus niger isolated from Brazilian soils using soybean residue.

ß-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular ß-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was optimized by a factorial design. Maximal ß-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R2 is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal ß-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin's presence, 80% of activity was preserved after 2 h. These results reveal a A. niger ß-galactosidase obtained from residue with favorable characteristics for food industries.

Microbial ß-mannosidases and their industrial applications.

Heteropolymers of mannan are polysaccharide components of the plant cell wall of gymnosperms and some angiosperms, including palm trees (Arecales and Monocot). Degradation of the complex structure of these polysaccharides requires the synergistic action of enzymes that disrupt the internal carbon skeleton of mannan and accessory enzymes that remove side chain substituents. However, complete degradation of these polysaccharides is carried out by an exo-hydrolase termed ß-mannosidase. Microbial ß-mannosidases belong to families 1, 2, and 5 of glycosyl hydrolases, and catalyze the hydrolysis of non-reducing ends of mannose oligomers. Besides, these enzymes are also involved in transglycosylation reactions. Because of their activity at different temperatures and pH values, these enzymes are used in a variety of industrial applications and the pharmaceutical, food, and biofuel industries.

Analysis of the Transcriptome in Aspergillus tamarii During Enzymatic Degradation of Sugarcane Bagasse.

The production of bioethanol from non-food agricultural residues represents an alternative energy source to fossil fuels for incorporation into the world's economy. Within the context of bioconversion of plant biomass into renewable energy using improved enzymatic cocktails, Illumina RNA-seq transcriptome profiling was conducted on a strain of Aspergillus tamarii, efficient in biomass polysaccharide degradation, in order to identify genes encoding proteins involved in plant biomass saccharification. Enzyme production and gene expression was compared following growth in liquid and semi-solid culture with steam-exploded sugarcane bagasse (SB) (1% w/v) and glucose (1% w/v) employed as contrasting sole carbon sources. Enzyme production following growth in liquid minimum medium supplemented with SB resulted in 0.626 and 0.711 UI.mL-1 xylanases after 24 and 48 h incubation, respectively. Transcriptome profiling revealed expression of over 7120 genes, with groups of genes modulated according to solid or semi-solid culture, as well as according to carbon source. Gene ontology analysis of genes expressed following SB hydrolysis revealed enrichment in xyloglucan metabolic process and xylan, pectin and glucan catabolic process, indicating up-regulation of genes involved in xylanase secretion. According to carbohydrate-active enzyme (CAZy) classification, 209 CAZyme-encoding genes were identified with significant differential expression on liquid or semi-solid SB, in comparison to equivalent growth on glucose as carbon source. Up-regulated CAZyme-encoding genes related to cellulases (CelA, CelB, CelC, CelD) and hemicellulases (XynG1, XynG2, XynF1, XylA, AxeA, arabinofuranosidase) showed up to a 10-fold log2FoldChange in expression levels. Five genes from the AA9 (GH61) family, related to lytic polysaccharide monooxygenase (LPMO), were also identified with significant expression up-regulation. The transcription factor gene XlnR, involved in induction of hemicellulases, showed up-regulation on liquid and semi-solid SB culture. Similarly, the gene ClrA, responsible for regulation of cellulases, showed increased expression on liquid SB culture. Over 150 potential transporter genes were also identified with increased expression on liquid and semi-solid SB culture. This first comprehensive analysis of the transcriptome of A. tamarii contributes to our understanding of genes and regulatory systems involved in cellulose and hemicellulose degradation in this fungus, offering potential for application in improved enzymatic cocktail development for plant biomass degradation in biorefinery applications.

Exploring Trichoderma and Aspergillus secretomes: Proteomics approaches for the identification of enzymes of biotechnological interest.

Filamentous fungal secretomes comprise highly dynamic sets of proteins, including multiple carbohydrate active enzymes (CAZymes) which are able to hydrolyze plant biomass polysaccharides into products of biotechnological interest such as fermentable sugars. In recent years, proteomics has been used to identify and quantify enzymatic and non-enzymatic polypeptides present in secretomes of several fungi species. The resulting data have widened the scientific understanding of the way filamentous fungi perform biomass degradation and offered novel perspectives for biotechnological applications. The present review discusses proteomics approaches that have been applied to the study of fungal secretomes, focusing on two of the most studied filamentous fungi genera: Trichoderma and Aspergillus.

Identification of multienzymatic complexes in the Clonostachys byssicola secretomes produced in response to different lignocellulosic carbon sources.

Multienzymatic complexes with plant lignocellulose-degrading activities have recently been identified in filamentous fungi secretomes. Such complexes have potential biotechnological applications in the degradation of agro-industrial residues. Fungal species from the Clonostachys genus have been intensively investigated as biocontrol agents; however so far their use as producers of lignocellulose-degrading enzymes has not been extensively explored. Secretomes of Clonostachys byssicola following growth on different carbon sources (passion fruit peel, soybean hulls, cotton gin trash, banana stalk, sugarcane bagasse, orange peel, and a composition of soybean hulls: cotton gin trash:orange peel) were subjected to enzymatic assays. Remarkable differences were observed among the samples, especially regarding levels of mannanase and pectinase activities. Secretomes were then subjected to Blue Native PAGE in order to resolve putative protein complexes which subsequently had their composition revealed by trypsin digestion followed by LC-MS/MS analysis. The protein bands (named I, II, III and IV) were shown to be composed by holocellulolytic enzymes, mainly cellulases and xylanases as well as proteins involved in biocontrol processes, such as chitinases and proteases. The high diversity of proteins found in these multicatalytic assemblies confirms C. byssicola as a novel source of plant biomass-degrading enzymes.

GH11 xylanase from Emericella nidulans with low sensitivity to inhibition by ethanol and lignocellulose-derived phenolic compounds.

An endo-ß-1,4-xylanase (X22) was purified from crude extract of Emericella nidulans when cultivated on submerged fermentation using sugarcane bagasse as the carbon source. The purified protein was identified by mass spectrometry and was most active at pH and temperature intervals of 5.0-6.5 and 50-60°C, respectively. The enzyme showed half-lives of 40, 10 and 7 min at 28, 50 and 55°C, respectively, and pH 5.0. Apparent Km and Vmax values on soluble oat spelt xylan were 3.39 mg/mL and 230.8 IU/mg, respectively, while Kcat and Kcat/Km were 84.6 s(-1) and 25.0 s(-1) mg(-1) mL. Incubation with phenolic compounds showed that tannic acid and cinnamic acid had an inhibitory effect on X22 but no time-dependent deactivation. On the other hand, ferulic acid, 4-hydroxybenzoic acid, vanillin and p-coumaric acid did not show any inhibitory effect on X22 activity, although they changed X22 apparent kinetic parameters. Ethanol remarkably increased enzyme thermostability and apparent Vmax and Kcat values, even though the affinity and catalytic efficiency for xylan were lowered.

Two ß-xylanases from Aspergillus terreus: characterization and influence of phenolic compounds on xylanase activity.

Sugarcane bagasse was used as an inexpensive alternative carbon source for production of ß-xylanases from Aspergillus terreus. The induction profile showed that the xylanase activity was detected from the 6th day of cultivation period. Two low molecular weight enzymes, named Xyl T1 and Xyl T2 were purified to apparent homogeneity by ultrafiltration, gel filtration and ion exchange chromatographies and presented molecular masses of 24.3and 23.60 kDa, as determined by SDS-PAGE, respectively. Xyl T1 showed highest activity at 50 °C and pH 6.0, while Xyl T2 was most active at 45 °C and pH 5.0. Mass spectrometry analysis of trypsin digested Xyl T1 and Xyl T2 showed two different fingerprinting spectra, indicating that they are distinct enzymes. Both enzymes were specific for xylan as substrate. Xyl T1 was inhibited in greater or lesser degree by phenolic compounds, while Xyl T2 was very resistant to the inhibitory effect of all phenolic compounds tested. The apparent km values of Xyl T2, using birchwood xylan as substrate, decreased in the presence of six phenolic compounds. Both enzymes were inhibited by N-bromosuccinimide and Hg(2+) and activated by Mn(2+). Incubation of Xyl T1 and Xyl T2 with L-cysteine increased their half-lives up to 14 and 24 h at 50 °C, respectively. Atomic force microscopy showed a bimodal size distribution of globular particles for both enzymes, indicating that Xyl T1 is larger than Xyl T2.

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes.

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

Holocellulase activity from Schizophyllum commune grown on bamboo: a comparison with different substrates.

The natural biodiversity that is found in tropical areas offers countless biotechnological opportunities; especially if we take in account that many biomolecules from several microorganisms have supported for many years, different industrial applications in areas such as pharmacology, agro-industry, bioprocess, environmental technology, and bioconversion. In order to find new lignocellulolytic enzymes and evaluate bamboo fibers as substrate, Schizophyllum commune a fungus with broad distribution was isolated and grown during 15 days in liquid culture medium containing 1% lignocellulosic fibers from bamboo, banana stem, and sugarcane bagasse. The enzymatic activity of xylanase, mannanase, polygalacturonase, CMCase, FPase, and avicelase were evaluated. Sugarcane bagasse and banana stem showed to induce higher hollocellulase activity when compared with bamboo as the main carbon source. The physical mechanism that the fungus uses to degrade bamboo was observed not only in fibers naturally infected but also in healthy fibers that were treated and untreated with enzyme solution. SEM analysis showed the structural disruption and invasion of the vascular bundles, parenchyma cells, and parenchymatous tissues as a consequence of the presence of this fungus and the catalytic action of its enzymes into the plant tissue.

Purification and characterization studies of a thermostable ß-xylanase from Aspergillus awamori.

This study presents data on the production, purification, and properties of a thermostable ß-xylanase produced by an Aspergillus awamori 2B.361 U2/1 submerged culture using wheat bran as carbon source. Fractionation of the culture filtrate by membrane ultrafiltration followed by Sephacryl S-200 and Q-Sepharose chromatography allowed for the isolation of a homogeneous xylanase (PXII-1), which was 32.87 kDa according to MS analysis. The enzyme-specific activity towards soluble oat spelt xylan, which was found to be 490 IU/mg under optimum reaction conditions (50°C and pH 5.0-5.5), was 17-fold higher than that measured in the culture supernatant. Xylan reaction products were identified as xylobiose, xylotriose, and xylotetraose. K (m) values (mg ml(-1)) for soluble oat spelt and birchwood xylan were 11.8 and 9.45, respectively. Although PXII-1 showed 85% activity retention upon incubation at 50 °C and pH 5.0 for 20 days, incubation at pH 7.0 resulted in 50% activity loss within 3 days. PXII-1 stability at pH 7.0 was improved in the presence of 20 mM cysteine, which allowed for 85% activity retention for 25 days. This study on the production in high yields of a remarkably thermostable xylanase is of significance due to the central role that this class of biocatalyst shares, along with cellulases, for the much needed enzymatic hydrolysis of biomass. Furthermore, stable xylanases are important for the manufacture of paper, animal feed, and xylooligosaccharides.

Evaluation of holocellulase production by plant-degrading fungi grown on agro-industrial residues.

Agaricus brasiliensis CS1, Pleurotus ostreatus H1 and Aspergillus flavus produced holocellulases when grown in solid and submerged liquid cultures containing agro-industrial residues, including sugar cane bagasse and dirty cotton residue, as substrates. These isolates proved to be efficient producers of holocellulases under the conditions used in this screening. Bromatological analysis of agro-industrial residues showed differences in protein, fiber, hemicellulose, cellulose and lignin content. Maximal holocellulase activity (hemicellulase, cellulase and pectinase) was obtained using solid-state cultivation with 10% substrate concentration. In this case, remarkably high levels of xylanase and polygalacturonase activity (4,008 and 4,548 IU/l, respectively) were produced by A. flavus when grown in media containing corn residue, followed by P. ostreatus H1 with IU/l values of 1,900 and 3,965 when cultivated on 5% and 10% sugar cane bagasse, respectively. A. brasiliensis CS1 showed the highest reducing sugar yield (11.640 mg/ml) when grown on medium containing sugar cane bagasse. A. brasiliensis was also the most efficient producer of protein, except when cultivated on dirty cotton residue, which induced maximal production in A. flavus. Comparison of enzymatic hydrolysis of sugar cane bagasse and dirty cotton residue by crude extracts of A. brasiliensis CS1, P. ostreatus H1 and A. flavus showed that the best reducing sugar yield was achieved using sugar cane bagasse as a substrate.

Production and characterization of hemicellulase activities from Trichoderma harzianum strain T4.

Xylan and mannan are the major constituent groups of hemicellulose in the cell wall of higher plants. The mesophilic fungus Trichoderma harzianum strain T4 produces extracellular xylanase and mannanase activities when grown in the presence of oat (Avena sativa)-spelt xylan and wheat bran as the carbon sources respectively. After the growth procedure, the crude extracts were submitted to ultrafiltration in an Amicon system fitted with a 10 kDa-cut-off membrane. Mannanase activity was only detected in the concentrated sample, whereas xylanase was also found in the permeate after ultrafiltration. Xylanase from the concentrated sample showed highest activity at 40 degrees C and pH 5.0. Mannanase activity was optimal at 65 degrees C and pH 2.6. Xylanase was stable in the temperature range 40-70 degrees C, presenting full stability for at least 48 h. Xylanase retained 100% of its original activity after incubation for 48 h at 70 degrees C. Xylanase was also stable at pH 5.0 and 6.0 for 48 h. However, mannanase activity was markedly less stable. The enzyme lost 50% of its activity at 55 degrees C after 45 min, whereas at 60 degrees C its half-life was 20 min. The Michaelis-Menten constant K(m) and V(max) for mannanase and xylanase activities were also calculated. Xylanase had more affinity for soluble xylan, with K(m) and V(max) values of 1.61 mg/ml and 10.03 units/ml respectively. The K(m) and V(max) values for crude mannanase were 6.0 mg/ml and 20.1 units/ml respectively. Xylanase and mannanase were activated by dithiothreitol, L-cysteine and L-tryptophan. Xylanase was partially purified by gel-filtration (Sephadex G-50) and hydrophobic-interaction (Phenyl-Sepharose) chromatographies. The partially purified enzyme was stable over the pH range 5-7 and temperature range of 40-60 degrees C. It was more active on soluble oat-spelt xylan and was activated by dithiothreitol, L-cysteine and L-tryptophan.

Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana.

A beta-xylanase (XynIII) of Acrophialophora nainiana was purified to homogeneity from the culture supernatant by ultrafiltration and a combination of ion exchange and gel filtration chromatographic methods. It was optimally active at 55 degrees C and pH 6.5. XynIII had molecular masses of 27.5 and 54 kDa, as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The purified enzyme hydrolyzed preferentially xylan as the substrate. The half-lives of XynIII at 50 and 60 degrees C were 96 and 1 h, respectively. It was activated by L-tryptophan, dithiothreitol, 5,5-dithio-bis(2-nitrobenzoic acid, L-cysteine and beta-mercaptoethanol and strongly inhibited by N-bromosuccinimide. The presence of carbohydrate was detected in the pure XynIII.